· Suppression of bacterial wilt in Eucalyptus and bacterial speck in Arabidopsis by fluorescent...

Suppress ion of bacter ia l wi l t

in Euca lyptus and bacter ia l speck

in Arab idops i s by f luorescent

Pseudomonas spp . s t ra ins :

cond i t ions and mechan isms

O n d e r d r u k k i n g v a n b a c t e r i e l e v e r w e l k i n g i n E u c a l y p t u s e n

b l a d v l e k k e n z i e k t e i n A r a b i d o p s i s d o o r f l u o r e s c e r e n d e

P s e u d o m o n a s s p p . : o p t r e d e n e n m e c h a n i s m e n

(met een samenvatting in het nederlands)

P R O E F S C H R I F T

ter verkrijging van de graad van doctor aan de Universiteit Utrechtop gezag van de Rector Magnificus, Prof. dr.W.H. Gispen, ingevolge hetbesluit van het College voor Promoties in het openbaar te verdedigen op

maandag 30 september 2002 des middags te 12.45 uur

door

L o n g x i a n R a n

geboren op 21 November 1962 te Sinan,Guizhou, P. R. China

he more a man learns, the more he sees his ignorance.T

From ‘Book of Rites. Study’

Confucius, 551-479 BC, the great philosopher of China

Dedicated tomy parents and parents-in-law

my wife and daughter

Promotor: Prof. dr. ir. L. C. van LoonCo-promotores: Dr. P. A. H. M. Bakker

Section Phytopathology, Faculty of Biology,Utrecht UniversityProf.Wu GuangjinCentral South Forestry CollegeZhuzhou, Hunan, P.R. China

The studies described in this thesis were performed at the SectionPhytopathology, Faculty of Biology, Utrecht University (Sorbonnelaan, 16,3584 CA Utrecht, The Netherlands) and the Section of Forest Pathology,Central South Forestry College (Zhuzhou, 412006, Hunan, P. R. China).This research was supported by grant no. WB 83-169 from the NetherlandsFoundation for the Advancement of Tropical Research (WOTRO) and theNatural Science Foundation of China (grant no. 39970615).

Cover/ layout: Femke Bulten, Beeldverwerking & VormgevingPrint: PrintPartners Ipskamp, EnschedeISBN: 90-393-3118-9

Contents

C h a p t e r 1 7

General introduction

C h a p t e r 2 2 9

Suppression of bacterial wilt in Eucalyptus urophylla by fluorescent Pseudomonas spp. in China

C h a p t e r 3 4 9

Induction of systemic resistance against bacterial wiltin Eucalyptus urophylla by fluorescent Pseudomonas spp.

C h a p t e r 4 6 9

No role for bacterially-produced salicylic acid in inductionof systemic resistance in Arabidopsis

C h a p t e r 5 8 3

Mutants of Pseudomonas putida WCS358 unable to catabolizephenolic acids are less competitive in the rhizosphere but retainthe ability to induce systemic resistance in Arabidopsis

C h a p t e r 6 9 9

General discussion

R e f e r e n c e s 1 0 9

S u m m a r y 1 2 3

S a m e n v a t t i n g 1 2 7

1 3 1

A c k n o w l e d g e m e n t s 1 3 5

C u r r i c u l u m v i t a e 1 3 9

L i s t o f p u b l i c a t i o n s 1 4 1

G e n e r a l I n t r o d u c t i o n • 7

Genera l In t roduct ion

1 . T h e g e n u s E u c a l y p t u s

The eucalypts belong to plant family Myrtaceae (Myrtles) and are nativemainly to Australia.A few species also occur naturally in the Philippines, PapuaNew Guinea, and Indonesia, only two of which are not found in Australia.One of those, Eucalyptus deglupta, is the only eucalypt found in the northernhemisphere, occurring in the southern Phillipines, as well as in New Guineaand parts of Indonesia (Jacobs, 1979; Qi, 1989). Another species, growing intropical and subtropical regions, is E. urophyl la , which dominates as purevegetations in Gunung Mutis, West Timor (Pedersen and Arneberg, 1999).There are ninety genera in the family Myrtaceae, and in the genus Eucalyptusthere have been over 600 species described. The total number can only beestimated, because there are numerous separate varieties that are grouped underthe same species name.The reason is that trees within an eucalypt grove growclosely together and produce many hybrids (Santos, 1997).

Australia has about 26 million ha of Eucalyptus forests, and theseconstitute 85 % of the native flora (Turnbull, 1995). Even though eucalyptsoccur naturally from 7 ˚N to 43 ˚S (Jacobs, 1979), they can grow in most ofthe tropical and temperate climatic regions of the world between latitudes 40 ˚Nand 45 ˚S (Eldridge e t a l . , 1993). Many species have been introduced intoother parts of the world from the early 19th century onward, and eucalyptplantations can be found in more than 90 countries, with a total area ofapproximately 20 million ha. India (8 million ha), Brazil (3 million ha) andChina (1.5 million ha) are the main eucalypt growing countries (FAO, 2001a).Eucalypts continue to be the second most commonly planted trees in forestplantations, accounting for 10 % of global forest plantations, surpassed onlyby pine trees, which occupy 20 % (FAO, 2001a, b).The most important species,in terms of growth potential, are E. grandis, E. camaldulensis, E. tereticornis,E. globulus , E. urophyl la , E. viminal is , E. sal igna , E. deglupta , E. exser ta ,

C h a p t e r 1

E. citr iodora, E. paniculata, and E. robusta (Eldridge et al., 1993).In Australia, eucalypts have been used in the manufacture of ships, bridges,

railroad ties, railroad vehicles, wagons, furniture, agricultural implements, pavingblocks, barrels, poles, piles, and posts (Santos, 1997).They furnish gum, resin, oil,and nectar, and its oils extracted from leaves are used in medicine and in fragrances.

In the regions into which eucalypts were introduced, further uses have beenexploited, as shade trees, as windbreaks to protect crops, farm animals, and buildings(Qi, 1989; Santos, 1997), as biomass fuel (Brito, 1997), and as alcohol to fuelengines.

However, Eucalyptus would not have become so popular, if it had not beensuccessfully developed for producing paper. In Australia, the first eucalypt paperwas made in 1914, but the disadvantage of its short fibres hampered its widerutilization for over 60 years.A breakthrough was achieved when paper industriesswitched from utilization of long to short fibres (Santos, 1997). Since the 1970s,India, Brazil, China, and other countries in tropical, subtropical and temperateregions started to introduce different provenances from eucalypts' native countries.Short rotation (5-7 years) of eucalypts for paper pulp is now widely applied inindustrial eucalypt plantations (Santos, 1997;Tamale et al., 1995).

2 . E u c a l y p t c u l t u r e i n C h i n a

Eucalyptus species were first introduced into south China from Italy andFrance in 1890 (Qi, 1989). Since then, more introductions of eucalypt specieshave been made, principally into Guangdong, Guangxi and Fujian provinces(Fig. 1A,Table 1, zone 1), and south west China, such as Yunnan (zone 4) andSichuan (zone 5) provinces. In the early days, eucalypts were plantedsporadically for ornamental and landscape purposes. In the mid 1960s,eucalypts were grown on larger scales along railroads to increase the supply ofcrosstie wood. A scientific research cooperation on Eucalyptus was establishedin south China in 1972.This greatly promoted establishment of the industrialplantations of eucalypts, and a journal entitled "Eucalyptus Science andTechnology" was published periodically since then (Qi, 1989). By 1987, aEucalypt Research Center was established in Zhanjing, Guangdong province,which launched a comprehensive research program on eucalypt ecology,silviculture, pest and disease protection, and utilization. Since the late 1980s,clonal propagations were widely applied to support the production of eucalyptwoodchips for paper pulp. Because of their exceptional characteristics of fastgrowth, high yield and strong adaptability, selected clones are now grownmassively on an industrial scale in short rotation periods (5-7 years) for biomassfuel and wood pulp for paper in south China.

8 • C h a p t e r 1

G e n e r a l I n t r o d u c t i o n • 9

HAINAN

TAIWAN

TIBET

XINJIANG

QINHAI

SICHUAN

YUNNAN

GUANGXI GUANGDONG

HONGKONG

HUNAN

HUBEI

GANSU

SHAANXI HENAN JIANGSU

SHANDONG

BEIJING

ANHUI

ZHEJIANGJIANGXI

FUJIANQUIZHOU

INNER MONGOLIA

JILIN

HEILONGJIANG

LIAONING

Map of China

HAINAN

TAIWANGUANGXI

GUANDONG

GUIZHOU

YUNNAN

SICHUAN

SHAANXI

HUBEI JIANGSU

HUNAN JIANGXI

South China

5

34

2

1

A

B

Fig. 1 . Geographical distribution of eucalypt plantations and species in south China (Jiang et al.,

2000). A: indicates the five main zones of Eucalyptus plantations in south China.

B: Map of China.

1 0 • C h a p t e r 1

3 . E u c a l y p t b a c t e r i a l w i l t a n d o t h e r d i s e a s e s

Massive growth and overexploitation of eucalypt trees has caused severeroot and leaf diseases. Among them, eucalypt bacterial wilt is the most seriousproblem. In China, it was first reported in E. grandis and E. sal igna inGuangxi autonomous region (Cao, 1982). Since then, more reports on theoccurrence of eucalypt bacterial wilt in other provinces in south China werepublished (He, 1995; Lai, 1990; Li, 1992; Liang, 1986;Wang, 1992).The diseaseis becoming epidemic and causes a lot of damage in coastal areas, such asGuangdong, Hainan province and Guangxi. This disease is caused by thesoilborne bacterial pathogen Ralstonia solanacearum (Coutinho et al., 2000;Lin et al., 1993;Wu and Liang, 1988a). It infects young plants, especially undertwo-year-old of E. urophylla, E. grandis, E. saligna, and hybrids of E. grandisx urophyl la or E. urophyl la x grandis , which are all fast growing species ofhigh quality for making paper pulp (Luo et al., 1998).The average percentage

Area

1. South China area

2. Central area

3. Northern edge area

4. Yunnan plateau area

5. Sichuan basinarea

Region

Hainan, Guangdong,Guangxi, and part of Fujian

Northern parts ofGuangxi, south ofJiangxi, south-east of Hunan and southof Zhejiang

Hubei, northern partof Jiangxi, Jiangsuand Guizhou plateau

Yunnan, part ofSichuan

Sichuan

Climate

High temperatureand abundant rainfall

Cold in winter andhot in summer

Cold in winter

Warm in winter andcool in summer,rainfall well distributed

Abundant rainfall

Main species

Eucalyptus urophylla , E. grandis , E. maculata , E. tereticornis , E.citr iodora ,E. camaldulensis , E. grandisx urophylla , and E. ABL12

E. terecticornis , E. botry-oides , E. camaldulensis , andE. amplifol ia

E. amplifol ia, E. camaldu-lensis and E. c inerea

E. globulus, E.maideni, E. nitens,E. viminalis , E. bicostata, E. camphoraand E. smithii

E. botryoides, E. camaldu-lensis , E. viminalis , E. bicostata, and E. globulusx robusta

Table 1 . Zones of Eucalyptus plantat ions in South China (after J iang et a l . , 2000) .

of wilted trees can reach 30-40 % (Li and Wu, 1996; Lin et al. , 1996), but insome regions, mortality of young trees can be more than 90 % (Li and Wu,1996; Zhang e t a l . , 1996). For instance, in 1993 out of 561 ha of eucalyptplantations on the Qinlian forest farm of Guangxi, 209 ha were infected, andin severely infected areas 82 % of the young trees died (Meng et al., 1997).

Bacterial wilt has also been reported from other countries. In Brazil, lossesof up to 17 % occurred for 6 to 15-month-old transplants established forplantations on recently cleared areas of rainforest (Dianese e t a l . , 1990).Clonally propagated E. grandis x camaldulensis hybrids were reported to beinfected by R. solanacearum in KwaZulu/ Natal province of South Africa(Coutinho e t a l . , 2000). Investigations were also carried out in Congo andUganda, and showed that plantations of less than one-year-old in Kissoko andLivuiti in Congo and less than two-year-old around Entebbe and Kampala inUganda were frequently infected by R. solanacearum (Roux e t a l . , 2000,2001). The disease has been reported from Australia as well (Akiew andTrevorrow, 1994).

This disease can occur on trees of up to three or four years but is mostsevere on plants younger than 18 months (Ciesla et al., 1996).The bacteriuminfects the plants through the roots, gains access to and is transported in thevascular system, and colonizes most of the vascular elements, dramaticallylimiting water uptake and solute flow.Typical symptoms are discoloration ofpart or the entire crown of the trees, leaf drooping, dying off of branches, andfinally wilting. Diseased trees show black or dark brown stripes along the stems,and the roots are rotten (Roux et a l . , 2000).The xylem of all affected treesshows extensive discoloration. Bacterial slime may ooze out within severalminutes from the surface of a cross section through an infected branch. Mostof the infected trees die in the same year. Only few, slightly affected trees canrecover, but they remain smaller than non-infected trees.

Other diseases of Eucalyptus of lesser importance are caused by fungi andare listed in Table 2.

4 . T h e p a t h o g e n , R a l s t o n i a s o l a n a c e a r u m

Ralstonia solanacearum (formerly known as Burkholder ia solanacearumor Pseudomonas solanacearum) (Coutinho et al. , 2000; Lin et al. , 1993;Wuand Liang, 1988a) occurs in several physiological races. Eucalypt-infectingstrains in Australia, China and South Africa belong to race 1/biovar III (Akiewand Trevorrow, 1994; Coutinho et al. , 2000; Lin et al. , 1993), whereas thosein South America are race 1/biovar I (Dianese et al. , 1990). In south China,Lin et al. (1996) collected 55 isolates from the epidemic regions of eucalypt

G e n e r a l I n t r o d u c t i o n • 1 1

1 2 • C h a p t e r 1

bacterial wilt, and most of them were identified as race 1, biovar III, confirmingprevious results (Wu and Liang, 1988a).

According to field investigations and inoculation experiments, the highlysusceptible eucalypt species in China are E. urophylla, E. grandis x urophylla,E. urophylla x grandis , E. grandis , and E. saligna (He, 1995; Li, 1992; Lin eta l . , 1996; Meng e t a l . , 1997). Wu and Liang (1988b) found differences insusceptibility to eucalypt bacterial wilt within species and provenances.Theydemonstrated that E. grandis x urophylla , E. leizhou No. 1 (a natural hybridof E. exser ta x robusta (Qi, 1989)) and E. saligna (clones No. 7451, 7651) areresistant to bacterial wilt, and E. ABL12 (Congo No.12), E. camaldulens i s(clone No.13923) and E. urophylla were susceptible. However, E. ABL12 andE. camaldulens i s were the most resistant species according to field

Pathogen

Botryosphaeriadothidea

Botrytis c inerea

Ceratocystis f imbriata

Cryphonectriacubensis

Cylindrocladiumquinqueseptatum ;C. scoparium

Mycosphaerellamoleniana

M. cryptica

Puccinia psidi i

Geography

Australia, Colombia,South Africa, USA,Venezuela

China

Brazil, Congo, Uganda,USA

Australia, Congo,India, South America,Vietnam

Australia, Brazil, China,India, South Africa,Vietnam, Venezuela

South Africa

Chile

Argentina, Australia,Brazil, Paraguay, SouthAfrica, Taiwan

Symptoms

Dieback and cankers ofstems and branches

Shoot or leaf decay ofseedlings

Superficial cracking ofouter bark, or sunkencankers on stems

Basal canker, or longitu-dinal cracks on maintrunk

Leaf spot and shootblight

Straw-coloured necrosis

Straw-coloured necrosis

Golden-yellow uredinialpustules on branches,shoots and leaves

Reference

Ciesla et al . , 1996;Kliejunas et al . , 2001

Cen et al . , 1998; Menget al . , 1997

Ciesla et al . , 1996;Kliejunas et al . , 2001;Roux et al . , 2000,2001

Ciesla et al . , 1996;Kliejunas et al . , 2001;Roux et al . , 2000

Booth et al . , 2000a;Ciesla et al . , 1996;Deng et al . , 1997

Ciesla et al . , 1996

Kliejunas et al . , 2001

Booth et al . , 2000b;Ciesla et al . , 1996;Kliejunas et al . , 2001;Wang, 1992

Table 2 . Fungal diseases of Eucalyptus.

investigations in endemic regions of eucalypt bacterial wilt (Lin et al., 1996).Probably different provenances of those species were used.

R. solanacearum can not only infect Eucalyptus but also other woodyplants, i.e. casuar ina , mulberry, olive (He, 1997; Wang e t a l . , 1997), andsolanaceous plants. Bacterial wilt is widely distributed in the tropical,subtropical and some warm temperate regions (Hayward, 1991).The host rangeof R. solanacearum is exceedingly wide and except for Agrobac te r iumtumefaciens, no other bacterial plant pathogen infects as many different speciesof plants as does R. solanacearum . Representatives of more than 50 familiesof plants are hosts (Hayward, 1995), and about 300 species have been reportedto be infected (Lu, 1998). Potato, tobacco and tomato are the most severelyaffected hosts in China. The race pathogenic to eucalypt appears to be thesame as the one affecting tobacco and tomato (Huang et al. , 1995; Lu, 1998;Wang e t a l . , 1996a). Bacterial wilt was first reported in Indonesia in 1864,where it caused destructive damages to tobacco. The pathogen was namedPseudomonas solanacearum by an American plant pathologist, Erwin F. Smithin 1896 (Lu, 1998). It was transferred to the genus Burkholder ia in 1992(Saddler, 1994;Yabuuchi e t a l . , 1996). Three years later, it was moved againand named Rals tonia so lanacearum , based on phenotypic characterization,cellular lipid and fatty acid analysis, and phylogenetic analysis of 16S rDNA(Yabuuchi e t a l . , 1995). The latter name was accepted and validated in theInternational Journal of Systematic Bacteriology (Yabuuchi et al., 1996).

Since much variation exists in pathogenicity, physiological andbiochemical characters and host ranges among isolates of R. solanacearum ,race and biovar or biotype classifications are commonly used to distinguishthem. Based on in vitro and in vivo properties, R. solanacearum was classifiedinto three races. Race 1 affects tobacco, tomato, many other solanaceous hostsand some diploid bananas. Race 2 causes bacterial wilt of bananas (Mokodisease) and Heliconia. Race 3 infects potatoes and tomatoes, but is not highlyvirulent on other solanaceous hosts (Buddenhagen and Kelman, 1964; Coutinhoet al., 2000; Hayward, 1983). Some strains do not fit within this scheme, as Heet al. (1983) showed that three isolates infecting mulberry were different fromraces 1-3.These isolates were designated as a new race, i.e. race 4.

Four biovars were accepted to differentiate Ralstonia strains accordingto their ability to utilize and/or oxidize three hexose alcohols (mannitol,sorbitol and dulcitol) and three disaccharides (lactose, maltose and cellobiose)(Hayward, 1964). Biovar I cannot utilize any of these six chemicals; biovar IIutilizes the hexose alcohols, but not the disaccharides; biovar III utilizes boththe hexose alcohols and the disaccharides and IV utilizes only thedisaccharides.As more strains of R. solanacearum were isolated with time fromdifferent hosts, this biochemical classification scheme could no longer


1 4 • C h a p t e r 1

Reference

Buddenhagen and Kelman, 1964

He, 1997; He et al . , 1983

Ren et al . , 1981

He et al . , 1983; Ren et al . , 1981

Prior and Steva, 1990; Prior et al . , 1990

Ciesla et al . , 1996; Dianese et al . , 1990

Akiew and Trevorrow, 1994; Coutinho et

al. , 2000; Lin et al . , 1993; Wu and Liang,

1988a

He et al . , 1983; Ren et al . , 1981

He et al . , 1983; Ren et al . , 1981

He, 1997; He et al . , 1983;

Ren et al . , 1981

He et al . , 1983; Ren et al . , 1981

He et al . , 1983; Zeng and Dong, 1995

He et al . , 1983; Prior and Steva , 1990;

Zeng and Dong, 1995

Hsu et al . , 1993

Prior and Steva, 1990; Prior et al . , 1990

Abo-EI-Dahab et al . , 1978; Buddenhagen

and Kelman, 1964; Hayward, 1991; He et

al. , 1983; Ren et al . , 1981; Van Elsas et

al. , 2000

Martin et al . , 1981

He et al . , 1983

He et al . , 1983

Prior and Steva, 1990

He et al . , 1983; Liu et al . , 1999a; Liu and

Zhang, 2001; Prior and Steva, 1990; Shuai

et al . , 1997

Prior and Steva, 1990

Grimault and Prior, 1993; Prior and Steva,

1990; Prior et al . , 1990

Adhikari and Basnyat, 1998; Hanudin,

1997; He et al . , 1983; Ren et al ., 1981

Host

Banana

Casuarina

Eggplant

Eucalyptus

Ginger

Mulberry

Olive

Peanut

Pepper

Peri l la

Potato

Sesame

Sweet potato

Tobacco

Tomato

Race

2

1

1

1

1

1

1

4

1

1

1

1

1

3

1, 2, 3

1

1

1

1

1

1

1

Biovar

I, III

III

IV

III

I, II, III, IV

I

III

IV

I

III

III

IV

III

III

I, II, III

II

I, II, III

n.d.*

IV

II

III

II

I, III

III

Region

Central America

China

China

China

France

Brazil

Australia, China,

South Africa

China

China

China

China

China

China, France

Taiwan

France

America, Australia,

China, Egypt,

The Netherlands

Peru

China

China

Florida, USA

China, Colombia

North Carolina, USA

France

China, Indonesia,

Nepal

Table 3 . Races and biovars of R. solanacearum on different host plants.

*n.d = not determined.

accommodate all strains (Ren e t a l . , 1981; Zeng and Dong, 1995).Furthermore, since in most instances there is no direct correlation betweenthese physiological characteristics and pathogenicity (Hayward, 1964), biovardesignation in the laboratory is not particularly useful in determining thepotential host range of a strain (He et al., 1983). Only race 3, the potato race,can be equated with biovar II (Hayward, 1983,1991).

In many countries of southern Europe and the Mediterranean area, andin south America (Argentina, Chile, and Uruguay), biovar II is the sole biovarpresent. Recently, this biovar was reported to cause severe damage ("brownrot") to potato in the Netherlands (Van Elsas et al. , 2000, 2001). In general,biovar I is predominant in the America and biovar III in Asia (Hayward, 1991).

A summary of the races and biovars of important crops is given in Table 3.

5 . E p i d e m i o l o g y o f e u c a l y p t b a c t e r i a l w i l t

As stated by Hayward (1991), there are several apparent anomalies in thedistribution of bacterial wilt on certain hosts. One of the hosts is Eucalyptus,which originally was affected by bacterial wilt only in Brazil and China, andnot in Australia. Although this disease was reported later in Australia (Akiewand Trevorrow, 1994), South Africa (Coutinho et al. , 2000), Congo (Roux etal., 2000) and Uganda (Roux et al., 2001), it by no means reached a level likein south China, where hundreds of ha of eucalypt plantations are destroyedevery year (Shi et al., 2000).

Prerequisites for a disease to become epidemic are: a virulent pathogenwith a large amount of inoculant; abundant presence of susceptible hosts; andfavorable environmental conditions for disease development.As shown in Table3, the predominant Rals tonia strains in China belong to race 1, biovar III,which is the same as the pathogen of tobacco, tomato and eggplant. Thosecrops are widely grown in south China, implying that the pathogen can infecteucalypts cultivated on infected fields formerly grown with solanaceous hosts.Moreover, Ralstonia is a soilborne pathogen, that is able to survive very wellin wilted and dead trees. Even after two or three years, the pathogen can berecovered from dead trees and is pathogenic to eucalypt seedlings (Lin et al.,1996).The population density of R. solanacearum in soil of a severely epidemicregion of eucalypt bacterial wilt was 2-3 times higher than that in a soil wherethis disease occurred sporadically, and 8.5 times higher than where diseaseoccurred for the first time (Lin e t a l . , 1996). Apparently, the pathogenaccumulates in infected soil by surviving on the debris of dead plants or humicsubstances in the soil.

R. solanacearum infects eucalypts through wounded roots. It can be


transmitted by splashing water, and transported by water flow after heavy rain(Zhang et al. , 1996). It can also be transferred through infected seedlings topreviously uninfected regions (Meng et al., 1996). For example, several youngplantations were severely damaged in Guangxi autonomous region in 1993,because infected seedlings from Qinzhou Forest Research Center were usedfor transplanting (Meng et al., 1996).

The species and provenances used are major factors in disease occurrence,development and epidemiology. In China, most of the currently grown speciesare the highly susceptible E. urophylla , E. grandis , and their two hybrids, E.grandis x urophyl la , and urophyl la x grandis , and E. sal igna (Huang, 1996;Shen and Huang, 2000). About 60,000 ha of new plantations of these specieswas established every year (Shi e t a l . , 2000). These species are the fastestgrowing trees and provide the best quality for paper pulp (Luo et al., 1998).

The development of disease is closely correlated with rainfall andtemperature. In 1991, on Wuchuan and Doumen forest farms in Guangdongprovince, eucalypt bacterial wilt started to occur in June (140 mmprecipitation, 25 ˚C average temperature), with a slow development from Julyto mid August (80 mm, 27-28 ˚C). However, from the last half of August toSeptember (190 mm, 28-30 ˚C), the percentage of diseased plants increaseddramatically, and the epidemic slowed down with dropping temperature andless rainfall in October (Lin e t a l . , 1996). Eucalypt bacterial wilt becameepidemic on Leizhou forest farm because of heavy rainfall and hurricanes inAugust 1997. In contrast, disease only occurred sporadically in the same regionin 1998, when rainfall was much less and no hurricanes occurred (Wei et al.,1998). Rainfall in both May and August appears to be a key factor in whethereucalypt bacterial wilt will be epidemic or not. For instance, in Guangxiautonomous region, the rainfall in May and August was 255 mm and 61mm,respectively, in 1992, and eucalypt bacterial wilt was very slight. However, in1993, the rainfall in those two months reached 363 mm and 358 mm,respectively, and disease was severe (Meng et al., 1996).

6 . C o n t r o l o f e u c a l y p t b a c t e r i a l w i l t

Eucalypt trees are valuable, but growing them seems a gamble since for along time no comprehensive control measures were taken and no effectivecontrol methods were available. During the last decade, eucalypt bacterial wiltbecame prevalent in south China and losses are increasing. Many attempts aremade to control this devastating disease, but so far none is sufficiently effective.The following approaches have been taken.

1 6 • C h a p t e r 1

H o s t r e s i s t a n c e

Various levels of resistance to eucalypt bacterial wilt exist among speciesor provenances. Under field conditions E. grandis x urophyl la , E. sal igna(clone No. 7451, 7651), E. le izhou No.1, E. ci t r iodora and E. exser ta werethe most resistant species and provenances, and the most susceptible ones wereE. urophyl la , E. ABL12 (Congo No.12) and E. camaldulens i s (clone No.13923) (Wu and Liang, 1988b). However, according to other reports, E. grandisx urophyl la did not show resistance to bacterial wilt (Li, 1992; Lin e t a l . ,1996; Meng e t a l . , 1996; Shi e t a l . , 2000). It is not clear whether thisdiscrepancy resulted from changes in the resistance of E. grandis x urophyllaor differences in pathogenicity of the Ralstonia isolates used. Some resistantspecies and provenances had been identified and selected by using a largecollection of eucalypt species, in which E. citr iodora and E. saligna x exser taremained completely free of bacterial wilt infection, and E. leizhou No.1 wasalso highly resistant (Huang, 1996). Other reports agree that E. exser ta is aresistant species, and did not get infected by R. solanacearum in Guangxi andHainan province (Gan et al., 1998; Li, 1992; Meng et al., 1996). It is unclearwhether this eucalypt species is adequate for paper pulp production, andwhether it is universally resistant to bacterial wilt in any situation tested.

In agricultural crops, selection or development of resistant cultivars hasbeen an important strategy in the control of bacterial wilt, and some successwas achieved in the case of tobacco and peanut (Hayward, 1991). In tobacco,somaclonal variation was exploited to develop clones with increased resistanceto bacterial wilt (Daub and Jenns, 1989). For potato, it was demonstrated thatintroduction of wild, resistant material from Solanum stenotomum intocommercial cultivars by somatic hybridization significantly increased theresistance of somatic hybrids to bacterial wilt (Fock et al., 2001).

It is generally agreed that breeding for resistance is not completelyeffective, producing only modest gains and often lacking stability or durability,and the variability in strains of R. solanacearum combined with the influenceof environmental factors often restricts the expression of resistance to specificregions (Prior et al., 1996).

C r o p r o t a t i o n s a n d i n t e r c r o p p i n g

Systems of rotation cropping and intercropping have been practiced byChinese farmers to maintain soil fertility, and to eradicate deleteriousorganisms. In eucalypt plantations, rotation with other plants has not beendocumented, probably because eucalypt growers consider this traditionalcropping measure unprofitable. However, because of continuous growing ofeucalypt serious ecological problems have arisen (Santos, 1997; Xiang, 2000).Some efforts have been made to plant other species, such as the leguminous


1 8 • C h a p t e r 1

Acac ia or conifer trees after harvest of eucalypt trees. It was reported thatAcacia allows nitrogen fixation and alleviation of soil toxicity due to growthof eucalypt trees (Wang et al., 1998; Xiang, 2000). No infection of Acacia orpine tree by R. solanacearum has been reported.

In agricultural cultivation systems, rotation or intercropping seems morepromising and applicable. In some developing countries, control of bacterialwilt of potato by intercropping has been practiced as a means of reducingpathogen populations in soil, and transmission of the pathogen by root contact.Corn, bean and cowpea were used as intercrops to effectively reduce incidenceof bacterial wilt in potato (Hayward, 1991). Rotation of tomato with non-host plants such as rice or cowpea can significantly reduce disease severity ofbacterial wilt, but not rotation with the host crop, eggplant. Bacterialpopulations declined after cropping of rice and cowpea, but not eggplant(Michel e t a l . , 1996). Later, it was demonstrated that intercropping tomatowith cowpea, soybean or Welsh onion planted within the row, reduced bacterialwilt only slightly or not at all (Michel et al., 1997).The onset of bacterial wiltwas delayed by 1-3 weeks, and wilt severity was reduced by 20-26 %, whensusceptible tomato was grown after corn, or lady's fingers (Abelmoschuses culentus ) (Adhikari and Basnyat, 1998). In Fiji, sugar cane was used as anintercrop to control bacterial wilt of tomato (Hayward, 1991). Sugarcane isalso cultivated widely in south China and might also be beneficial as anintercrop or a rotation crop to eucalypt growers. Intercropping and croprotations should be studied further for their potential applications to controlbacterial wilt in agriculture and forestry.

C h e m i c a l c o n t r o l

Chemical control of bacterial diseases is not always effective. Severalantibiotics have been tested in v i t ro against R. solanacearum isolated frominfected eucalypts. Among those, aureomycin and validacin had no effect atall. Penicillin, terramycin and tetracycline inhibited growth of the pathogenfor a few days only. Streptomycin was the most effective agent and inhibitedbacterial growth for more than 15 days (Huang, 1998). The effectiveness ofstreptomycin in controlling eucalypt bacterial wilt in vivo is not known yet.

Nonomura e t a l . (2001) demonstrated that 3-(3-indolyl)butanoic acidcan be used to protect tomato against bacterial wilt in hydroponic systems.Twelve organic chemicals produced in China were tested for control ofbacterial wilt in tobacco fields. Results showed that only one agent, calledYishuangqing, was persistently effective, and reduced disease by 54-78 % (Chenand Huang, 1996). However, because of environmental safety, chemical controlis being discouraged.


E c t o m y c o r r h i z a l f u n g i

Nine species of ectomycorrhizal fungi were found on six eucalypt species(Gong and Chen, 1991), and stimulated growth of E. urophyl la seedlings(Aggangan et al., 1996). Such fungi are also able to control eucalypt bacterialwilt. Upon treatment with ectomycorrhizal fungi the percentage of diseasedplants was reduced by 40-73 % in nurseries and by 20-39 % in the field (Gonget al. , 1999).These control effects and abilities to form a mycorrhizal sheathon the root surface in different eucalypt species and provenances need furtherinvestigation as mycorrhizal fungi are reported to be species specific (Molinaet al., 1992).

A v i r u l e n t s t r a i n s

Since the beginning of the 1980s, much research has been carried out toattempt controlling bacterial wilt by use of avirulent strains. Luo and Wang(1983) reported that UV- and gamma-ray-induced avirulent mutants of R.solanacearum had some control effects on tomato and peanut bacterial wilt.Kempe and Sequeira (1983) also demonstrated that use of avirulent andincompatible strains resulted in a significant reduction in the severity of potatobacterial wilt.They speculated that induction of resistance was the mechanisminvolved. Later results indicated that application of avirulent strains can delaydisease development in tobacco and tomato by 10-30 days and reduce diseaseseverity (Chen and Echandi, 1984; Dong et al., 1999; Ren et al., 1993; Zhenge t a l . , 1994). However, control was reduced with time because of poorcompetitiveness of the avirulent strains with the virulent pathogen.Spontaneously occurring avirulent mutant strains were able to induce diseaseresistance in peanut when injected into the stem (Kang and He, 1994).

Avirulent mutants were also obtained by Tn5 transposon mutagenesis.Significant protection was obtained in tomato by simultaneous or sequentialroot inoculations, when the relative inoculum ratio of avirulent to virulentcells was equal to, or higher than, 10. Most of the Tn5-induced mutants showedreduced colonization and multiplication within the host compared with thevirulent strain (Trigalet and Trigalet-Demery, 1990). One of the mutants, HrcV-,was chosen to investigate the mechanisms involved. Through microscopicstudies of the colonization of the vascular tissues by the HrcV- mutant, itbecame clear that competition for space in the xylem vessels is one of thepossibilities for the protective activity of this mutant strain (Etchebar et al. ,1998). In China, Kang et al. (1995) used a mutant defective in the productionof extracellular polysaccharide to control bacterial wilt in tomato. Results fromgreenhouse experiments were promising, but the strain was less effective infield tests.

However, none of these approaches to biological control of bacterial wilt

has reached a point of commercial application and much more work is needed(Hayward, 1991).

A n t a g o n i s t i c b a c t e r i a l s t r a i n s

Some preliminary results were reported on control of bacterial wilt usingantagonistic bacteria (Anuratha and Gnanamanickam, 1990; Dong et al., 1996;Luo and Wang, 1983), but most of those were not well identified. Luo andWang (1983) isolated 606 Pseudomonas spp. strains from the rhizosphere oftomato, tobacco and Casuar ina, and tested them for control of bacterial wilt.Only two strains, 94a and 22a, had control effects in tomato, tobacco andpeanut. A collection of 250 bacterial strains from the rhizosphere of tobacco,tomato, carrot, mango and citrus plants was tested; five strains could delaydevelopment of bacterial wilt in tobacco for up to 7-10 days (Dong e t a l . ,1996). In India, Anuratha and Gnanamanickam (1990) found only one strain(Pfcp) out of 125 fluorescent Pseudomonas spp., and two (B33 and B36,Baci l lus spp.) out of 52 nonfluorescent bacteria that could protect banana,eggplant and tomato against bacterial wilt. In another report from India, abacterial strain with strong inhibitory activity in vi t ro and under glasshouseconditions was not effective against bacterial wilt in the field (Sunaina et al.,1997).

Actinomycetes are famous for their antibiotic production. Among the12,000 known antibiotics, more than half are produced by Streptomyces spp.(Nie and Tan, 2000). Their abilities to control bacterial wilt have also beeninvestigated. For tests on tomato, three application methods were compared,i.e., 1) soaking tomato seeds in a culture filtrate of the antagonist prior tosowing, 2) inoculation of the soil with the antagonist 7 days before sowing,and 3) coating of the tomato seeds with spores of the antagonist before sowing.The first treatment proved to be the least effective, the third was the mosteffective and the second had only moderate effects (Elabyad e t a l . , 1993).Moura et al. (1998) demonstrated that 18 strains of 190 actinomycetes isolatedfrom the rhizosphere, rhizoplane and root tissue (endophytes) of tomato inBrazil showed 100 % control of bacterial wilt in tomato by seed dipping in aspore suspension.This confirmed the results obtained by Elabyad et al.(1993).

Tr a n s g e n i c p l a n t s

During the last decade, genetic engineering techniques have been usedfor the introduction of potent antibacterial proteins derived from insects, suchas lysozyme and cecropins, into host plants, as a way of augmenting resistanceto bacterial diseases (Hayward, 1991).

Transgenic tobacco plants expressing the lytic peptide cecropin B fromthe giant silk moth, Hyalophora ce c ropia , exhibited delayed symptoms and

2 0 • C h a p t e r 1

reduced disease severity and mortality after inoculation with a highly virulentstrain of R. solanacearum (Jaynes e t a l . , 1993). However, another reportshowed that similar tobacco plants containing a cecropin B transgene had noresistance to R. solanacearum, even though the pathogen was highly sensitiveto cecropin B in vitro (Florack et al., 1995). No cecropin B protein could bedetected in the transgenic tobacco plants that failed to show resistance tobacterial infections.The most likely explanation is degradation of the proteinby endogenous tobacco proteases.

In China, transgenic potato plants harboring either the single antibacterialShiva A or cecropin B peptide genes, or their combination were obtained (Jiae t a l . , 1998). Only three out of 1,050 transgenic lines showed enhancedresistance to bacterial wilt in the greenhouse and in the field, implying thatthe rate of successful transformation was very low. Similar transformation oftobacco did not result in plants that had gained resistance against bacterial wilt(Li et al., 1998).

Several papers report on antagonistic activities of antibacterial peptidesagainst R. solanacearum in vitro (Li et al., 1994; Zhang et al., 1995; Zhao etal., 1998). Li et al. (1994) and Zhang et al. (1995) found that an antibacterialpeptide from the Chinese oak silkworm, Antheraea pernyi , strongly inhibitsthe growth of R. solanacearum isolates from eucalypt, patchouli andCasuar ina . However, other reports indicated that the same peptide did notinhibit the growth of R. solanacearum isolated from tomato and potato (Wanget al., 1995; Zhao et al., 1998).Therefore, further work is required to evaluatethe potential of antibacterial peptides for plant transformation.

Transformation of eucalypt with antibacterial peptides is being performedin the Eucalypt Research Centre in Zhanjiang, China, but so far withoutsuccess (Xie et al. , personal communication).There have been some reportsabout the regeneration of eucalypt from leaf disks or hypocotyls (Mullins eta l . , 1997; Tibok e t a l . , 1995; Wang e t a l . , 1996b). However, successfultransformation of eucalypt with Agrobacter ium tumefaciens or other methodsis still in its infancy.

7 . M e c h a n i s m s o f r h i z o b a c t e r i a l b i o c o n t r o l

As described above, thus far no well-studied bacterial antagonists havebeen used to control bacterial wilt caused by R. solanacearum . Attempts tocontrol bacterial wilt with antagonistic bacterial strains in the field failedbecause of a lack of understanding of the nature of the bacterial determinantsand mechanisms involved in biocontrol. The strategies through whichbiocontrol bacteria can protect plants against soilborne diseases are generally


2 2 • C h a p t e r 1

classified as: competition for nutrients, siderophore-mediated competition foriron, antibiosis, and induced systemic resistance.

C o m p e t i t i o n f o r n u t r i e n t s

A bacterial strain merely surviving under starvation conditions in the soilcannot be expected to actively inhibit infection by a pathogen.Whether it cansuccessfully control soilborne diseases is primarily dependent on the availabilityand utilization of nutrients exuded from plant roots. Organic compoundsreleased from the roots are considered to be the main nutrient source formicroorganisms in the rhizosphere (Bakker et al., 1991). Competition for thesecompounds between beneficial and pathogenic microorganisms could limitgrowth of, and infection by, pathogens (Bakker, 1989). Introduction ofpseudomonads into the rhizosphere reduces the activity of pathogenicFusar ium spp. and Pythium spp. through competition for carbon (Elad andBaker, 1985; Elad and Chet, 1987). Parke (1991) also demonstrated thatPseudomonas spp. are capable of competing for nutrients with soilborne plantpathogens through utilization of carbon sources from the root exudates.

S i d e r o p h o r e - m e d i a t e d c o m p e t i t i o n f o r i r o n .

In neutral or alkaline soils, the availability of soluble iron is extremelylow (10-17 M), whereas a minimum concentration of 10-6 M is commonlyneeded for microorganisms to grow (Neilands et al. , 1987).To acquire iron,most microorganisms produce siderophores, low-molecular-weight metaboliteswith a high affinity for Fe3+, under conditions of low iron availability (Bakkere t a l . , 1991). These siderophores chelate Fe3+ from the environment andtransport it into the microbial cells (Leong, 1986; Neilands, 1981).

Pseudomonas spp. strains typically produce fluorescent siderophores(synonymically called pseudobactins or pyoverdins) under low iron conditions(Kloepper et al., 1980). Most fluorescent Pseudomonas spp. strains can inhibitthe growth of other microorganisms in v i t ro at low iron availability bysiderophore-mediated competition for ferric iron (Buyer and Leong, 1986;Geels and Schippers, 1983; Kloepper e t a l . , 1980). In vivo , the fluorescentsiderophores of Pseudomonas spp. can effectively suppress disease bycompetition for iron with pathogens that cannot utilize the siderophores ofthese bacterial strains (Bakker e t a l . , 1987, 1991; Schippers e t a l . , 1987).Suppression by P. putida WCS358 of fusarium wilt of carnation and radish,caused by F. oxysporum f. sp. dianthi (Fod) and F. oxysporum f. sp. raphani(For), respectively, was demonstrated to involve competition for iron. In bothcarnation (Duijff et al. , 1994) and radish (Leeman et al. , 1996b), applicationof WCS358 strongly suppressed fusarium wilt under iron-limited conditions,whereas a pseudobactin-negative Tn5 transposon mutant did not show any


protective effects. Siderophore-mediated competition for iron has also beendemonstrated to be involved in the biocontrol of Pythium-induced dampingoff in tomato (Buysens et al., 1996).

A n t i b i o s i s

The involvement of antibiosis in control of plant diseases has beenreviewed (Fravel, 1988; Glick et al., 1999; Handelsman and Stabb, 1996;Weller,1988). Antibiosis is the antagonism mediated by specific or non-specific, low-molecular-weight compounds produced by microbes (Fravel, 1988).Antibioticsproduced by fluorescent Pseudomonas spp. include hydrogen cyanide (HCN),2,4-diacetylphloroglucinol (DAPG), phenazine-1-carboxylic acid (PCA),pyoluteorin (Plt) and pyrrolnitrin (Glick et al., 1999; Keel et al., 1990; Ligonet al. , 2000; Maurhofer et al. , 1994b).The biocontrol abilities of antibiotic-producing strains have been well studied through comparison of mutantsdefective in the synthesis of specific antibiotic with their wild type strains(Chin-A-Woeng et a l . , 1998; Cronin et a l . , 1997; Fenton et a l . , 1992; Keele t a l . , 1992; Maurhofer e t a l . , 1994b; Thomashow and Weller, 1988), bygenetically modifying strains to overproduce one or more antibiotics (Delanyet al., 2001; Girlanda et al., 2001; Maurhofer et al., 1992, 1995), or by transferof genes encoding antibiotic production to non-antibiotic-producing strains(Fenton et al., 1992).

P. fluorescens CHA0 is one of the best studied disease-suppressive strains.It can secrete at least four secondary metabolites with antibiotic activity: HCN,DAPG, Plt and pyrrolnitrin.These compounds have been shown to be involvedin the suppression of black root rot of tobacco caused by Thielaviopsis basicola(Keel et al., 1990; Stutz et al., 1986), Erwinia soft rot in potato tubers (Wanget a l. , 2000), Gaeumannomyces graminis var. t r i t i c i-induced take-all diseasein wheat (Keel et al., 1992), and Pythium damping-off of cress (Maurhofer eta l . , 1994b). Moreover, a genetically modified derivative, CHA0-Rif(pME3424), that overproduced DAPG and pyoluteorin, displayed improvedbiocontrol of Pythium damping-off in cucumber compared with wild-typeCHA0-Rif (Girlanda et al. , 2001). By producing 2,4-diacetylphloroglucinol,the wild-type strain was reported to moderately suppress crown and root rotof tomato caused by F. oxysporum f. sp. radicis-lycopersici (Duffy and Défago,1997). However, it was found recently that the phytotoxic pathogenicity factorfusaric acid represses the production of DAPG (Notz e t a l . , 2002). Thus,antibiotic activity may be counteracted by certain pathogens through specificpathogenicity factors.

Other well-documented P. fluorescens strains are 2-79 and Q2-87, whichproduce PCA and DAPG, respectively. Both strains have proved to be effectivein suppressing take-all disease in wheat (Bangera and Thomashow, 1996; Bull

et al., 1991; Hamdan et al., 1991;Thomashow and Weller, 1990). P. fluorescensstrain F113 was reported to be suppressive to damping-off of sugar beetseedlings caused by P. ultimum (Delany et al., 2001), as well as to potato cystnematodes (Cronin et al., 1997) through DAPG production.

It was recently demonstrated that DAPG produced by fluorescentPseudomonas spp. is the key compound responsible for natural suppression oftake-all in wheat and barley (Raaijmakers et al., 1997, 1999; Raaijmakers andWeller, 1998). Disease suppression was lost when DAPG-producingPseudomonas spp. were eliminated. Conversely, conducive soils gainedsuppressiveness when DAPG-producing Pseudomonas strains were introducedby adding small amounts of a suppressive soil from a field with a history ofcontinuous monoculture of wheat, known as take-all decline soil (Raaijmakersand Weller, 1998).

Strain 2-79 can also inhibit teliospore germination of Til le t ia laevi s ,whereas a spontaneous mutant deficient in PCA production had no effect(McManus e t a l . , 1993). Incidence of common bunt (smut) caused by thispathogen was reduced by 65 % and 50 %, respectively, during consecutiveseasons when wheat seeds and 2-week-old seedlings were treated with strain2-79. However, similar treatment of seeds and seedlings with its PCA-deficientmutant afforded no protection against this disease (McManus et al., 1993).

Biocontrol of tomato foot and root rot by P. chlororaphis strain PCL1391was also based on phenazine-1-carboxamide production, because a phenazinebiosynthetic mutant of PCL1391 had substantially decreased biocontrol activity(Chin-A-Woeng e t a l . , 1998). Fusarium wilt of chickpea, caused by F.oxysporum f. sp. c i c e r i s , was suppressed through the production of PCA byPseudomonas aeruginosa strain PNA1 (Anjaiah et al., 1998).

I n d u c t i o n o f s y s t e m i c r e s i s t a n c e

Induction of systemic resistance (ISR) (Kloepper et al., 1992; Pieterse etal., 1996) as a mechanism by which non-pathogenic rhizobacteria (Van Loonet a l . , 1998) suppress plant diseases is receiving increasing attention and hasbeen widely investigated during the last decade. Rhizobacteria-mediated ISRhas been reported to be effective against fungi, bacteria and viruses inArabidops i s , bean, carnation, cucumber, radish, tobacco, and tomato(Hammerschmidt et al., 2001;Van Loon et al., 1998). Recently, it was shownthat Rhizobium et l i strain G12 can induce systemic resistance in potato tothe potato cyst nematode Globodera pallida (Reitz et al., 2001).

Van Peer et al. (1991) were the first to convincingly demonstrate that P.fluorescens strain WCS417 can induce systemic resistance against fusarium wiltin carnation, caused by F. oxysporum f. sp. dianthi (Fod). Heat-killed cells ofWCS417r and its purified lipopolysaccharide (LPS) were also active as

2 4 • C h a p t e r 1


inducing stimuli (Van Peer and Schippers, 1992).WCS417 can also antagonizeFod through siderophore-mediated competition for iron, but its siderophore-negative mutant still induces resistance. Hence, WCS417 can act by at leasttwo independent mechanisms in suppressing fusarium wilt in carnation (Duijffet al., 1993).

In radish, strain WCS417 and P. f luores cens WCS374 were capable ofinducing systemic resistance against For, but WCS358 was not (Leeman et al.,1995a, b). Strain WCS374 gave greater disease control in the induced systemicresistance bioassay when iron availability in the nutrient solution was low(Leeman et al. , 1996a).WCS417r can also induce ISR against fusarium wiltin tomato (Duijff et al., 1998).To further analyze rhizobacteria-mediated ISR,Arabidops i s-P. syr ingae pv. tomato DC3000 (Pst ) was adopted as a modelpathosystem. In this system,WCS417 and WCS358 were able to trigger ISR,but WCS374 was not (Van Wees et al. , 1997).These results demonstrate thattriggering ISR is plant species- and Pseudomonas spp.-specific.

Rhizobacteria-mediated ISR also reduced the severity of anthracnose incucumber caused by Colletotr ichum orbiculare (Wei et al. , 1991), whereas P.f luores cens CHA0 is able to induce systemic resistance in tobacco againsttobacco necrosis virus (Maurhofer et al., 1994a) and black root rot, caused byThielaviopsis basicola (Troxler et al., 1997).

A possible role of bacterially produced salicylic acid (SA) in inducingsystemic resistance has received much attention. In radish, induction ofsystemic resistance by WCS374 and WCS417 was associated with the capacityof these strains to produce SA (Leeman et al., 1996a). However, in Arabidopsis,strain WCS374, capable of producing large amounts of SA in vit ro , does notinduce resistance (Van Wees et al., 1997). Strain WCS417 induces ISR in bothwild-type and transgenic NahG plants. The latter convert SA into catecholand are non-responsive to applied SA, clearly suggesting that SA is not involvedin ISR by WCS417 in Arabidopsis (Pieterse et al., 1996).

P. aeruginosa strain 7NSK2 (De Meyer and Höfte, 1997) and Ser rat iamarces cens strain 90-166 (Press e t a l . , 1997) have also been reported toeffectively suppress diseases through rhizobacteria mediated ISR. In the caseof 7NSK2, SA appears to be responsible for ISR against tobacco mosaic virusin tobacco (De Meyer et al., 1999a). However, strain 90-166, able to produceSA, induced systemic resistance against C. orbi cu lare in cucumber and P.syr ingae pv. tabaci in tobacco, and a mutant unable to produce SA was similarlyactive (Press et al., 1997).

8 . A i m s a n d o u t l i n e o f t h e t h e s i s

The main aim of this study was to investigate the possible roles ofsiderosphere and bacterially-produced SA in antagonism of fluorescentpseudomonads against R. solanacearum and in induction of systemic resistance(ISR) against eucalypt bacterial wilt.The mechanisms were studied in a modelsystem: A. thal iana challenged with P. syr ingae pv. tomato DC3000. Inaddition, the importance of plant-derived phenolic acids for bacterial rootcolonization and induction of ISR were evaluated.

Eucalyptus-R. solanacearum was used as a plant/pathogen model systembecause of the huge damage of eucalypt bacterial wilt on young eucalypt treesin south China (Lin et al., 1995, 1996). SA-producing strains, i.e. P. fluorescensWCS374,WCS417, CHA0, and P. aeruginosa 7NSK2 were chosen since theyhave been well studied and disease suppression has been documented indifferent plant-pathogen systems. However, the role of SA secreted by thesebacteria in the induction of systemic resistance against different pathogens isnot clear.Therefore, it would be helpful to compare these strains in one plant-pathogen model, such as in Eucalyptus urophyl la-R. solanacearum with apractical importance, and in the Arabidops i s-P. syr ingae pv. tomato modelsystem, which was developed during the last decade to study the molecular-genetic mechanisms of rhizobacterially-mediated ISR. Furthermore, none ofthe SA-producing fluorescent Pseudomonas spp. mentioned above was testedagainst vascular bacterial wilt caused by R. solanacearum before.

The non SA-producing P. put ida strain WCS358 was included in thisstudy because it can effectively compete for iron with other bacteria(Raaijmakers et al., 1995b) and induces ISR in A. thaliana (Van Wees et al.,1997).We also investigated the possible involvement of phenolic acids in theactivation of ISR by WCS358 by using mutants defective in the utilization ofspecific phenolic acids.

In chapter 2, suppression of eucalypt bacterial wilt by several fluorescentPseudomonas spp. and by a genetically modified strain constitutively expressingeither 2,4-diacetylphloroglucinol or phenazine-1-carboxylic acid biosyntheticgenes is examined. Mixing the biocontrol bacteria through soil or root dippingof the seedlings in a suspension of protective bacteria before transplanting wereused to investigate the protective effects of the strains.

In chapter 3, pathogenicity of R. solanacearum to 3-month-old seedlingswas tested by applying the pathogen to wounded shoot tips. This artificialinoculation method was used to test for induced systemic resistance byfluorescent Pseudomonas spp. by keeping the microorganisms spatiallyseparated.

In vitro determination of SA production by Pseudomonas spp. strains and

2 6 • C h a p t e r 1

the effects of incubation temperature and iron availability in the growthmedium on SA production are described in chapter 4. The ISR-inducingabilities of SA-producing bacteria were studied in the Arabidopsis-P. syr ingaepv. tomato model system. The involvement of bacterially produced SA wasinvestigated further by using the NahG transformant and the ein2 and npr1mutants of Arabidopsis.

In chapter 5, phenolic acids in root exudates of Arabidops i s weredetermined by gas chromatography. Mutants of WCS358 defective in theutilization of specific phenolic acids were evaluated for the effect of thesemutations on Arabidopsis root colonization and elicitation of ISR.

Finally, in the last chapter, the bacterial determinants and mechanismsinvolved in suppression of eucalypt bacterial wilt, and bacterial speck inArabidopsis are discussed.


2 8 • C h a p t e r 1

Suppress ion of bacter ia l wi l t in

Euca lyptus urophy l la by f luorescent

Pseudomonas spp . in Ch ina

L . X . R a n 1 .2 , C . Y. L i u 2 , G . J . W u 2 , P. A . H . M . B a k k e r 1 a n d

L . C . v a n L o o n 1

1 Faculty of Biology, Section Phytopathology, Utrecht University,P.O. Box 80084, 3508 TB Utrecht,The Netherlands.

2 Central South Forestry College, 412006, Zhuzhou, Hunan, P. R. China

C h a p t e r 2

Suppress ion of bacter ia l wi l t in

Euca lyptus urophy l la by f luorescent

Pseudomonas spp . in Ch ina

A b s t r a c t

Bacterial wilt caused by Rals tonia so lanacearum race 1, biovar III hasbecome a severe impediment in Eucalyptus plantations in south China.Thedisease mainly attacks young eucalypt trees, and no effective control measuresare available yet. In this study, strains of fluorescent Pseudomonas spp. that areeffective in suppressing plant diseases by known mechanisms, were tested fortheir potential to control bacterial wilt in Eucalyptus. P. putida WCS358r, P.fluorescens WCS374r, P. fluorescens WCS417r and P. aeruginosa 7NSK2 antagonizeR. solanacearum in vitro by competition for iron, whereas growth inhibition byP. fluorescens CHA0r is antibiosis-based. No correlations were found betweenantagonistic abilities of these Pseudomonas spp. in vitro and biocontrol effectsagainst bacterial wilt in Eucalyptus in vivo. None of the strains suppressed diseasewhen mixed together with the pathogen in the soil or when seeds or seedlingswere treated with the strains before transfer of seedlings into soil infested withR. solanacearum. However, when seedlings were dipped with their roots in abacterial suspension before transplanting into infested soil,WCS417r suppressedbacterial wilt by 30-45 %. WCS358r was marginally effective, whereas itssiderophore-minus mutant had no effect at all, indicating that siderophore-mediated competition for iron can contribute but is not effective enough tosuppress bacterial wilt in Eucalyptus. A derivative of WCS358r, constitutivelyproducing 2,4-diacetylphloroglucinol (WCS358::phl) reduced disease.Combined treatment with WCS417r and WCS358::phl did not improvesuppression of bacterial wilt over the effects of each strain alone.

S u p p r e s s i o n o f e u c a l y p t b a c t e r i a l w i l t b y P s e u d o m o n a s s p p • 3 1

I n t r o d u c t i o n

Eucalypt was introduced into China for landscape purposes more than110 years ago (Qi, 1989). Now, Eucalyptus is grown mainly for pulpwood,flakeboard, plywood, poles for construction, and crossbeams for railroad tracks.In 1982 bacterial wilt in Eucalyptus was reported in the Guangxi AutonomousRegion (Cao, 1982). Since then, it has spread to other provinces in the south(He, 1995; Lai, 1990; Li, 1992; Liang, 1986; Wang, 1992). Bacterial wilt isbecoming epidemic and is causing severe problems in Guangdong, Hainan andGuangxi, leading to great losses in eucalypt plantations with an averagepercentage of about 30-40 % wilted trees (Li and Wu, 1996; Lin et al., 1996).Trees under two years old of fast growing Eucalyptus urophylla , E. grandis ,E. sal igna , and hybrids of E. grandis x urophyl la or E. urophyl la x grandis ,are most vulnerable (Li and Wu, 1996). At Leizhou forest farm (the largest inChina), in two plantations of 1.5-year-old E. grandis x urophylla, the averagemortality reached 70 % in 2001 (L. X. Ran, unpublished observation).

Eucalyptus bacterial wilt is a soil-borne, vascular disease caused byRalstonia solanacearum (Coutinho et al., 2000; Lin et al., 1993;Wu and Liang,1988a). The bacterium enters the plant through the roots, is transportedthrough the vascular system to other tissues, and colonizes most of the vasculartissues.As a result, water uptake and solute flow become severely limited.Veinaltissues degenerate, usually resulting in brown or black stripes inside the stem,finally resulting in rapid foliar drooping and a general and irreversible wilt.When infected branches are cut, bacterial ooze exudes from the wound site.The disease has been reported from other countries as well, such as Brazil(Dianese et al. , 1990), Australia (Akiew and Trevorrow, 1994), Congo (Rouxet al., 2000), South Africa (Coutinho et al., 2000) and Uganda (Roux et al.,2001). R. solanacearum has been classified as a European Plant ProtectionOrganization (EPPO) A2 quarantine pathogen and is considered likewise bythe Asian and Pacific Plant Protection Committee (APPPC) and the InterAfrican Phytosanitary Council (IAPSC) (Ciesla et a l . , 1996; Saddler, 1994).The pathogen has been identified as R. solanacearum race 1, biovar III(Coutinho e t a l . , 2000; Lin e t a l . , 1993; Wu and Liang, 1988a), which isinfective to e.g., casuar ina , olive, tomato, tobacco and eggplant (He, 1997;Huang et al., 1995;Wang et al., 1996a;Wang et al., 1997)

In the past two decades, great efforts have been made to control bacterialwilt in Eucalyptus . Selection of resistant species, provenances and clones isconsidered the most effective approach to reduce disease severity. Fieldinvestigations in China showed that the most resistant species are E. grandis x

urophylla, E. saligna (clones No. 7451, 7651), E. leizhou No. 1, E. citr iodoraand E. exser ta (Wu and Liang, 1988b). However, recent reports agree that the

3 2 • C h a p t e r 2

most widely used hybrid, E. grandis x urophyl la , is highly susceptible tobacterial wilt (Lin et al., 1996; Meng et al., 1996; Shen and Huang, 2000; Shiet al., 2000). It is not clear whether the discrepancy must be attributed to theuse of different clones of E. grandis x urophyl la for testing, or whether theresistance of this hybrid had decreased. Some resistant clones becamesusceptible when introduced into a different environment (Chen and Wu,1995). Moreover, disease resistance decreased when plants were propagated bytissue culture consecutively for three years (Shi et al., 2000).

Inhibitory effects on growth of R. solanacearum by several antibioticsindicated that streptomycin can suppress growth of R. solanacearum in vitrofor more than 15 days (Huang, 1998). However, in field tests streptomycin hadonly short-term effects in solanaceous vegetables. It is expensive and thepathogen readily develops resistance against the antibiotic.Therefore, it is notsuitable for practical applications to control bacterial wilt (Liu and Zeng,1999).

Antibacterial peptides from the Chinese oak silkworm were tested fortheir bactericidal action against R. solanacearum , and found to have variableeffects on growth of R. solanacearum (Li et al., 1994;Wang et al., 1995; Zhanget al., 1995; Zhao et al., 1998).Transgenic tomato, tobacco and potato plantsexpressing antibacterial peptides were variably found to have enhanced or noincreased resistance to bacterial wilt (Florack et al., 1995; Jaynes et al., 1993;Jia et al. , 1998; Montanelli et al. , 1995;Tian et al. , 2000). In 1997, a projectwas initiated in the Eucalypt Research Centre of China to transformEucalyptus species (Zhang et al., 1998), but progress is hampered, apparentlyby difficulties to regenerate transformants.

In the last few years experiments on biocontrol were reported. Forexample, ectomycorrhizal fungi were found to suppress eucalypt bacterial wilt(Gong et al. , 1999). Antagonistic Streptomyces spp. were reported to controlbacterial wilt of tomato, but it is not clear whether these strains have sustainablebiocontrol effects (Elabyad et al., 1993).

UV- and gamma ray-induced avirulent mutants of R. solanacearum hadsome control effects on bacterial wilt in tomato and peanut (Luo and Wang,1983). Tn5-induced avirulent mutants suppress bacterial wilt in tomato bycolonizing the roots and preventing subsequent colonization by thecorresponding wild type strain (Trigalet and Trigalet-Demery, 1990). Kang etal. (1995) demonstrated that an extracellular polysaccharide defective mutanthad suppressive effects on tomato bacterial wilt, suggesting a potential forcontrolling the disease. However, the efficacy of the avirulent mutants is largelydependent on competition for space with the virulent pathogen, and theavirulent mutants persist for less than a month.Application of avirulent mutantsto control bacterial wilt on a large scale is unlikely to be successful because


they are not sufficiently competitive in the rhizosphere and constitute apotential danger to other related hosts.

Antagonistic Pseudomonas spp. were tested to suppress bacterial wilt intobacco (Dong et al., 1996; Liu et al., 1999b; Luo and Wang, 1983; Zhang eta l . , 1999). Most of these antagonistic bacteria are not well defined. Somestrains show promising results, but the mechanisms involved are not known.In eucalypts, no data on the use of fluorescent pseudomonads for controllingbacterial wilt have been reported so far.

Here we report the effects of several Pseudomonas spp. strains with knownbiocontrol properties on bacterial wilt in E. urophylla seedlings. Mechanismssuch as siderophore-mediated competition for iron (Duijff et al., 1993; Leemane t a l . , 1996b), antibiotic production (Keel e t a l . , 1990, 1992), and salicylicacid-mediated induced resistance (De Meyer and Höfte, 1997; Leeman et al.,1996a) were considered as possible antagonistic activities.

M a t e r i a l s a n d m e t h o d s

I s o l a t i o n a n d m a i n t e n a n c e o f R a l s t o n i a s o l a n a c e a r u m

Branches from wilted trees were debarked and the brown or blackenedvascular strands were cut into small pieces with a sterilized razor blade.Thepieces were soaked for 10 min in 50 ml sterile distilled water, during which amilky exudate oozed out of the plant tissue. Upon dilution, the suspensionwas plated on modified Kelman agar plates [in g.l-1: proteose peptone (Oxoid)10, casamino acids (Oxoid) 5, glucose 10, bacto agar (Difco) 10] (Kelman,1954), and incubated for 48 h at 30 ˚C. Single colonies were streaked ontomodified Kelman agar plates containing tetrazolium chloride (50 mg.l-1). R.solanacearum was identified as forming slimy, milky colonies with a pink centeron this medium (Fahy and Hayward, 1983). Moreover, strains were resistantto ampicillin (40 µg.ml-1), cycloheximide (100 µg.ml-1) and chloramphenicol(13 µg.ml-1). Primary pathogenicity tests were performed by dipping the rootsof 4-week-old seedlings of E. urophylla in a bacterial suspension containing108 cfu.ml-1.

The identification of the R. solanacearum isolates was verified on thebasis of colony and cell morphology, Gram staining and staining of flagella.The biovar was determined by testing the ability to grow on three hexosealcohols (mannitol, sorbitol and dulcitol) and three disaccharides (lactose,maltose and cellobiose).The race was specified by inoculation of 4-week-oldtobacco plants through injection of the basal stem with 50 µl of a bacterialsuspension at 108 cfu.ml-1, as well as by inoculation of 15 seedlings of 4-week-old tomato and eggplant by transplanting them into infested soil containing 5

3 4 • C h a p t e r 2

x 107 cfu.g-1. All inoculated plants were kept at 28-30 ˚C at day and a relativehumidity of 85 % for disease development.

Two strains of R. solanacearum , which both cause typical wiltingsymptoms on E. urophylla seedlings were selected for the experiments in vitroand bioassays in vivo . Strain Rlz was isolated from 1.5-year-old diseased E.urophylla , and collected by Xian Shenghua in the Leizhou Forest Bureau onDecember 22, 1999. Strain Rrp was originally isolated from an infected E.urophylla tree in Raoping, China and provided by Prof. Zhang Jingning andDr. Luo Huanliang in July, 1999.These isolates were routinely maintained insterile distilled water at 4-8 ˚C for preservation up to one year (Sly, 1983).Fresh 24 h cultures from modified Kelman agar plates were suspended indistilled water. The bacterial concentration was determined spectrophoto-metrically at 660 nm and adjusted to 108 cfu.ml-1. Aliquots of 1.5 ml werestored. For long term preservation, bacteria were cultured for 48 h in liquidKelman medium at 30 ˚C. Aliquots of 1 ml bacterial culture were transferredaseptically to cryogenic vials, mixed with 0.5 ml 50 % sterilized glycerol (Sly,1983), and stored at -80 ˚C.

Inoculum of the pathogen was prepared by growth on modified Kelmanagar plates for 48 h at 30 ˚C, collecting the bacterial cells in sterile distilledwater, and centrifuging the suspension twice at 12,000 x g for 10 min. Thepellet was resuspended in distilled water and bacterial density was measuredby spectrophotometry at 660 nm.

S e l e c t i o n o f s p o n t a n e o u s r i f a m p i n - r e s i s t a n t m u t a n t s

R. solanacearum strains Rlz and Rrp (Table 1) were transferred stepwiseto modified Kelman's agar plates containing increasing concentrations (25,50,100, 150, 200, 250 µg.ml-1) of rifampin (Glandorf et al., 1992).The stabilityof the antibiotic resistance in two selected mutants (Rlzr, Rrpr) was checkedby subculturing the mutants 10 times at 30 ˚C on Kelman agar plates withoutrifampin and comparing the numbers of colony forming units on plates withand without rifampin (100 µg.ml-1) after each subculture.

C u l t i v a t i o n o f P s e u d o m o n a s s p p .

The sources and relevant characteristics of the bacterial strains used arelisted in Table 1. Pseudomonas spp. were routinely cultured on King's mediumB (KB) agar plates (King e t a l . , 1954) at 28 ˚C. Media were solidified with1.2 % agar (Difco Laboratories, Detroit, MI). Ampicillin (40 µg.ml-1),cycloheximide (100 µg.ml-1), chloramphenicol (13 µg.ml-1), kanamycin (50µg.ml-1), and rifampin (100 µg.ml-1) were added for antibiotic selection, whenapplicable.

For bioassays, fluorescent Pseudomonas spp. were grown on KB plates for


3 6 • C h a p t e r 2

Strain

PathogensR. solanacearum Rlz

Rlzr

R.solanacearum Rrp

Rrpr

Pseudomonas strainsP. aeruginosa 7NSK2

KMPCH

MPFM1

MPFM1-569

P. f luorescens CHA0r

P. f luorescens WCS374

WCS374rWCS374sid- (JM374)P. f luorescens WCS417

WCS417r

WCS417sid- (S680)P. putida WCS358

WCS358r

JM218WCS358::phl

WCS358::phz

Relevant characteristics*

isolated from wilted branch of Eucalyptusurophylla in Leizhou, China; ampr, chlr; wildtype spontaneous rifr mutant of Rlz; ampr, chlr, rifr

isolated from wilted seedling of E. urophyllain Raoping, China; ampr, chlr; wild type spontaneous rifr mutant of Rrp; ampr, chlr,rifr

wild type; Pch+, Pvd+, SA+; competes for iron,induces systemic resistance; amp+, chl+

chemical mutant of MPFM1; Pch-, Pvd-, SA+;ampr, chlr, Kmr

Tn5 mutant of 7NSK2; Pch-, Pvd-, SA+; ampr,chlr, Kmr

pchA replacement mutant of MPFM1; Pch-,Pvd-, SA-; ampr, chlr, Kmr

rifr strain of wild-type CHA0, isolated fromtobacco rhizosphere; ampr, chlr, rifr; produces2,4-diacetylphloroglucinol, pyoluteorin,pyrrolnitrin and HCN; induces systemic resistanceisolated from potato rhizosphere, wild type;ampr, chlr; competes for iron, induces systemic resistance (ISR) rifr strain of WCS374; ampr, chlr, rifr

Tn5 mutant of WCS374, sid-; ampr, chlr, Kmr

isolated from wheat rhizosphere, wild type;ampr, chlr; competes for iron, induces ISRrifr strain of WCS417; ampr, chlr, rifr


isolated from potato rhizosphere, wild type;ampr, chlr; competes for iron, induces ISRrifr strain of WCS358; ampr, chlr, rifr


transformant of WCS358 consitutively pro-ducing 2,4-diacetylphloroglucinol; ampr, chlr,rifr

transformant of WCS358 consitutively pro-ducing phenazine-1-carboxylic acid; ampr,chlr, rifr

Reference or Source

This study

""

"

Buysens et al . , 1996;De Meyer and Höfte,1997"

"

"

Keel et al . , 1992;Maurhofer et al . ,1994a; Stutz et al . ,1986

Geels and Schippers,1983; Leeman et al . ,1995b, 1995c

Weisbeek et al . , 1986Duijff et al . , 1993;Lamers et al . , 1988;Leeman et al . ,1995b; Van Wees etal. , 1997Duijff et al . , 1993Duijff et al . , 1994;Geels and Schippers,1983; Leeman et al . ,1996b; Van Wees etal. , 1997Marugg et al . , 1985Glandorf et al., 2001

"

*Abbreviations: Pch = pyochelin; Pvd = pyoverdin; SA = salicylic acid, sid = pseudobactin siderophore;ampr, chlr, Kmr, rifr = resistant to ampicillin, chloramphenicol, kanamycin, and rifampin, respectively.

Table1. Microorganisms used in this study.

24-30 h at 28 ˚C, and suspended in 10 mM MgSO4. The suspension wascentrifuged twice at 7,600 x g for 10 min.The bacterial pellet was resuspendedin 10 mM MgSO4, and the concentration adjusted as described above.

I n v i t r o a n t a g o n i s m b e t w e e n f l u o r e s c e n t p s e u d o m o n a d s a n d

R . s o l a n a c e a r u m

In vi t ro antagonism between Pseudomonas spp. and the pathogen wasstudied on KB agar plates. A drop of 100 µl suspension of R. solanacearumcontaining 108 cfu.ml-1 was spread on the plate. Immediately thereupon, a 7mm diameter agar disk cut from a plate on which the biocontrol strain hadbeen grown for 24 h at 28 ˚C, was placed on the centre of the plate. Zonesof growth inhibition of R. solanacearum were measured after incubation for48 h at 28 ˚C. Effects of temperature and iron availability on in v i t roantagonism between pseudomonads and R. solanacearum (Rrpr) were studiedon KB plates with or without 200 µM FeCl3 at 28 ˚C, 31 ˚C or 34 ˚C.

C u l t i v a t i o n o f p l a n t s

A loamy soil rich in humus from Liuyang city, China, was collected andused for all bioassays. Per gram this soil contained: organic matter 3.1 mg, N1.9 mg, P 1.2 mg, K 16.5 mg, Ca 3.2 mg, Mg 3.9 mg and Fe 71.7 mg; the pHwas 6.92.The soil was mixed with river sand at a ratio of 12 to 5 (v/v).Themixed soil was packaged in 2 kg quantities, which were autoclaved twice for1 h on alternate days.

Seeds of Eucalyptus urophyl la were sown in trays on top of the wettedsoil mixture and covered with a thin layer of river sand.The trays were placedin an incubator kept at a temperature of 30 ˚C during the day (12 h fluorescentlight) and 25 ˚C at night, and a relative humidity of 90 %. After one week, thegerminated seedlings were transferred to a growth cabinet kept at 25-28 ˚Cduring the light period and at 20-23 ˚C during the night, and at 70 % relativehumidity.The seedlings were routinely supplied once a week with half-strengthHoagland nutrient solution (Hoagland and Arnon, 1938), containing 10 µMFeEDDHA (Fe-ethylenediamine di-o-hydroxyphenylacetic acid; CIBA-Geigy,Basel, Switzerland), and watered with tap water when necessary.

Arabidopsis thaliana accession Col-0 plants were grown as described byPieterse et al. (1996).

P a t h o g e n i c i t y o f R . s o l a n a c e a r u m t o E u c a l y p t u s u r o p h y l l a

The R. solanacearum strains, Rrp and Rlz, and their rifampin-resistantmutants, Rrpr and Rlzr were tested for their pathogenicities to E. urophyl laby mixing suspensions of 100 ml of the pathogen at different concentrationswith 1 kg of potting soil. Four-week-old E. urophylla seedlings, about 1.5 cm


in height, were transplanted into pots (5.5 cm diameter) containing theinoculated soil, with 5 plants per pot and 10 pots per treatment. As controltreatment, 100 ml of sterile distilled water was added to the soil.The transferredseedlings were kept in containers at a temperature of 24-25 ˚C during the dayand 20-22 ˚C at night, and a relative humidity over 90 % for 5 days, thenplaced in another growth cabinet at a higher temperature of 30 ˚C in the lightand 25 ˚C during darkness, and a relative humidity of 85 % for diseasedevelopment. Plants were scored at intervals for symptoms of bacterial wiltand the percentage of wilted plants was calculated.

B i o a s s a y s

The biocontrol strains were applied in different ways to assess theirabilities to control bacterial wilt. In several experiments, both the biocontrolstrain and the pathogen were mixed into the soil at a ratio of 1 : 1(each 5 x107 cfu.g-1 soil) or 10 : 1 (fluorescent pseudomonads at 5 x 107 cfu.g-1 soil andR. solanacearum at 5 x 106 cfu.g-1 soil), prior to the transfer of 4-week-oldseedlings to the soil. Since the height of the eucalypt seedlings differed,seedlings were selected for height and a similar set of plants was allotted toeach treatment. For bacterization at the seed stage, 40 ml of a suspension ofPseudomonas spp. containing 109 cfu.ml-1, were poured onto 200 g of pot soilin a germination tray. Eucalyptus seeds were sown on the soil surface andcovered with a thin layer of river sand. Four weeks later, the seedlings weretransplanted in soil containing R. solanacearum at 5 x 107cfu.g-1. Forbacterization at the seedling stage, 40 ml of Pseudomonas spp. suspension waspoured onto the soil around the seedlings 1 week before transfer. Another wayto apply the bacteria was to dip roots of 4-week-old seedlings for 10-15 minin suspensions of Pseudomonas spp. at 109 cfu.ml-1, and transferring them tosoil containing the pathogen at 5 x 107 cfu.g-1.

D i s e a s e a s s e s s m e n t a n d d a t a a n a l y s i s

Root infection resulted in rapid wilting and damping off. The roots ofdead seedling were completely rotted.The percentage of diseased plants wasscored at different time points on the basis of wilting symptoms.Wilted plantswere collected, rinsed in sterile distilled water, and ground in a mortar insterile distilled water (10 ml.g-1 tissue). Dilutions of the homogenate wereplated on modified Kelman agar plates containing ampicillin (40 µg.ml-1),cycloheximide (100 µg.ml-1), chloramphenicol (13 µg.ml-1) and, in case rifr

strains had been used for inoculation, rifampin (150 µg.ml-1), to verify if diseasewas caused by R. solanacearum . All experiments were repeated two or threetimes. For statistical analyses of disease development, the area under the diseaseprogress curves (AUDPC) was calculated according to the midpoint rule

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(Garrett and Mundt, 2000). Percentages of wilted seedlings in all otherbioassays were compared by using one-way analysis of variance (ANOVA) withSPSS-software (SPSS for Windows, release 8.0), followed by Fisher's test forleast significant differences at α = 0.05.

R e s u l t s

C h a r a c t e r i s t i c s o f R . s o l a n a c e a r u m a n d i t s s p o n t a n e o u s

r i f a m p i n - r e s i s t a n t m u t a n t s

Strains Rlz and Rrp of R. solanacearum are Gram-negative, rod-shaped,0.4-0.7 x 1.3-1.9 µM in size with one or two polar flagella. Both form rough,butyrous, slimy and milky white colonies on KB and modified Kelmanmedium.The optimum temperature for growth is 30-35 ˚C. No fluorescentpigments are formed on KB medium.According to the standard to differentiateraces of R. solanacearum set by Buddenhagen (1962), both Rlz and Rrpbelong to race 1. As both strains were capable of acidifying three hexosealcohol and three disaccharides, they were identified as biovar III. Except toEucalyptus, the isolates were pathogenic on tobacco, tomato and eggplant, butnot on Arabidopsis (data not shown).

In order to selectively detect R. solanacearum in bioassays, spontaneousrifampin-resistant mutants were isolated. The morphology of the mutantsselected was identical to that of the wild type, and rifampin resistance wasstable upon repeated subculture in vitro under non-selective conditions.

P a t h o g e n i c i t y o f R . s o l a n a c e a r u m t o E . u r o p h y l l a

In order to select a proper inoculum density of R. solanacearum fortesting the biocontrol efficacy of Pseudomonas spp., the pathogenicity of strainRrp was determined by constructing disease progress curves of plants grownin soil containing different densities of the pathogen. Results are presented inFig. 1. From the 7th day onward, a clear relationship between inoculum densityand disease development was apparent. The percentage of wilted seedlingsincreased sharply in the treatments with higher inoculation densities of 107

and 108 cfu.g-1 soil compared with those below. A density of 107 cfu.g-1 soilled to about 50 % wilted seedlings by 21 days.

Table 2 shows the values of the area under the disease progress curves(AUDPC) for both the wild-type strains and their rifampin-resistant mutantsapplied at different inoculum densities.The wild-type strain Rlz was marginallymore pathogenic than Rrp, whereas the rifampin-resistant mutants wereslightly less pathogenic, particularly at lower densities.


I n v i t r o i n h i b i t i o n o f R . s o l a n a c e a r u m b y s t r a i n s o f

P s e u d o m o n a s s p p .

P. putida strain WCS358 and P. fluorescens strains WCS374 and WCS417,and their rifampin-resistant derivatives have been used extensively in studieson biocontrol of fusarium wilt of carnation, radish and tomato, and of bacterialspeck in Arabidops i s . All these strains and their corresponding rifampin-resistant mutants inhibited growth of R. solanacearum on agar plates, withoutany major differences in the level of inhibition achieved (Table 3).As evidencedby the complete loss of inhibitory activity of the pseudobactin-minus mutantsof strain WCS358 and WCS417, inhibition by these strains can be fullyexplained by siderophore-mediated competition for iron. In contrast, apseudobactin-minus mutant of WCS374,WCS374sid-, maintained the ability

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Table 2 . Pathogenicity of R. solanacearum strains Rlz and Rrp, and their rifampin-resistant mutants

Rlzr and Rrpr to E. urophylla. Values given represent the areas under the disease progress curves

(AUDPC). A higher value means more disease.

Population density of inocula (cfu.g-1 soil)

Strain 105 106 107 5x107 108

Rrp 267 343 595 ND* 761

Rrpr 240 339 578 ND 736

Rlz 322 424 676 688 793

Rlzr 231 269 598 649 712

* ND = Not Determined.

0

10

20

30

40

50

60

70

5 10 15 20 25

days after seedling transfer

CK

105

106

107

108

% w

ilted

see

dlin

gs log cfu.g-1 soil

F ig . 1 . Development of bacterial wilt in Eucalyptus urophylla growing in soil containing different

densities of R. solanacearum strain Rrp. CK = treatment with seedlings transplanted into potting soil

without pathogen.

to inhibit growth of R. solanacearum. In no case did R. solanacearum strainsinhibit growth of any of the Pseudomonas strains.

If siderophore-mediated competition for iron is the main mechanism bywhich Pseudomonas spp. strains antagonize R. solanacearum, inhibition shoulddisappear when the agar medium is supplemented with iron. Table 4 showsthat the inhibitory activities of WCS358r, WCS374r and WCS417r againstRrpr were lost completely in the presence of FeCl3, indicating thatsiderophore-mediated competition for iron is responsible for the inhibition ofgrowth of Rrpr in vit ro by these strains. For strain WCS374 production of asecond siderophore, pseudomonine (Mercado-Blanco et al., 2001) may explaingrowth inhibition by the pseudobactin mutant.The constitutive phloroglucinolproducing derivative of WCS358, WCS358::phl was strongly suppressive toRrpr in either the absence or presence of iron. The phenazine producingderivative was less effective than WCS358::phl. In the presence of iron, noclear inhibition zone was observed, although growth of R. solanacearum wasvisibly reduced.

Both P. fluorescens strain CHA0 and P. aeruginosa strain 7NSK2 produceSA in vitro under iron-limited conditions (De Meyer et al. , 1999a; Meyer etal. , 1992). Bacterially-produced SA has been implicated in the induction ofdisease resistance in tobacco (Maurhofer et a l . , 1994a) and bean (De Meyere t a l . , 1999b), respectively. However, CHA0 is known to also produce 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt), pyrrolnitrin, and hydrogencyanide (HCN) (Duffy and Défago, 1999; Keel et al., 1990; Maurhofer et al.,1994b). Inhibition of growth of Rrpr by CHA0r was independent of ironavailability, indicating that antibiotics were the most important factors ininhibiting Rrpr. 7NSK2 and its pyoverdin-minus mutant MPFM1 suppressed


Table 3 . In vitro growth inhibition of R. solanacearum Rlz and Rrp, and their rifampin-resistant

mutants Rlzr and Rrpr by fluorescent Pseudomonas spp. The inhibition zone is given in mm.

Strain Rlz Rlzr Rrp Rrpr

WCS358 22 23 21 22

WCS358r 20 24 21 23

JM218 0 0 0 0

WCS374 17 18 17 17

WCS374r 16 17 15 16

WCS374sid- 13 14 13 13

WCS417 22 22 22 24

WCS417r 21 21 21 22

WCS417sid- 0 0 0 0

the growth of Rrpr only in the absence of iron. MPFM1 was less inhibitorythan the wild type, demonstrating that pyoverdin produced by 7NSK2contributes to the suppression of Rrpr. Mutants KMPCH, not producingpyochelin and pyoverdin, and MPFM1-569, producing none of thosemetabolites, did not inhibit at all.These results demonstrate that both pyoverdinand pyochelin are responsible for the inhibition of Rrpr, and SA is not toxicto the growth of R. solanacearum . Indeed, R. solanacearum grew up to themargin of paper disks that had been dipped in 10 mM SA (data not shown).In the presence of iron, 7NSK2 and all its mutants fully lost their inhibitoryabilities, indicating that 7NSK2 and its pyoverdin-negative mutant suppressedthe growth of Rrpr by competing for iron.

B i o c o n t r o l o f E u c a l y p t u s b a c t e r i a l w i l t b y f l u o r e s c e n t

p s e u d o m o n a d s

When biocontrol bacteria were mixed together with the pathogenthrough the soil in which eucalypt seedlings were transplanted, neither strainWCS358r or its sid- derivative JM218, nor WCS374r or WCS417r protectedthe seedlings from infection by Ralstonia solanacearum (Fig. 2). Also CHA0rand 7NSK2 or its derivatives did not significantly suppress bacterial wilt (datanot shown). Even when population densities of the biocontrol strains were 10times higher than that of the pathogen, no control effects were detected (datanot shown). Prior bacterization of plants would give the biocontrol agent acompetitive advantage over the pathogen. Therefore, plants were bacterizedeither at the germination or the seedling stage, four and one week, respectively,

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Table 4 . In vitro growth inhibition of R. solanacearum strain Rrpr by fluorescent Pseudomonas

spp. in the absence or presence of iron. The zone of growth inhibition is given in mm.

Strain Fe- Fe+

WCS358r 22 0

WCS358::phl 28 28

WCS358::phz 24 a*

WCS374r 18 0

WCS417r 25 0

CHA0r 28 30

7NSK2 30 0

MPFM1 15 0

KMPCH 0 0

MPFM1-569 0 0

*a = no clear inhibition zone observed, but growth reduction apparent.

before transfer of the seedlings to soil containing the pathogen. In severalrepeated experiments none of these treatments provided control effects againstbacterial wilt in E. urophylla (data not shown).Alternatively, the roots of four-week-old seedlings were dipped in a suspension of Pseudomonas spp., and theroot-dipped seedlings were subsequently transferred to pathogen-containingsoil. Results from a typical experiment are shown in Fig. 3. Strain WCS374rwas not effective at any time point. Strain WCS358r tended to reduce disease,although never significantly.Yet, the siderophore-minus mutant of WCS358,JM218 had no suppressive effect at all. In contrast, strain WCS417r reducedbacterial wilt by 45 %, 42 %, 37 % and 30 % at 7, 10, 15 and 21 days aftertransfer, respectively. In a repeated experiment,WCS417r suppressed diseaseby 45 % at 21 days.

Increasing the dipping time in the suspension of WCS417r from 10 to 60


20

0

40

60

80

100

CK WCS358r JM218 WCS374r WCS417r

% w

ilted

see

dlin

gs

F ig . 2 . Disease incidence in eucalypt seedlings 3 weeks after transfer into soil containing 5 × 107

cfu of R. solanacearum strain Rrpr and Pseudomonas spp. per gram soil. CK = treatment with

seedlings transferred into soil containing the same density of pathogen and 10 mM MgSO4.

0

10

20

30

40

50

60

70

80

90

100

days after seedling transfer

% w

ilted

see

dlin

gs

CK WCS374r WCS358r JM218 WCS417r

7 10 15 21

a a

b

a

aa

a ab

bab

ab

a a

b

b

ab ab

a a

ab

F ig . 3 .Disease incidence in eucalypt seedlings at different times after transfer into soil containing

R. solanacearum Rrpr (5 × 107 cfu.g-1). Prior to transfer seedling roots were dipped in suspension of

10 mM MgSO4 (CK) or 109 cfu Pseudomonas spp. ml-1.

4 4 • C h a p t e r 2

Table 5 . Development of bacterial wilt of E. urophylla grown in soil containing 5 × 107 cfu.g-1 of

R. solanacearum strain Rrpr after root dip in suspension of antagonistic Pseudomonas spp. Values

given represent area under the disease progress curves (AUDPC).

CK: treatment with root dip in 10 mM MgSO4. A higher value means more disease.

Strain Exp. 1 Exp. 2 Exp. 3

CK 699 648 905

WCS358r 767 606 ND

WCS358::phl 504 561 785

WCS358::phz 787 615 ND

WCS417r 402 320 431

417r+358::phl ND* 533 632

* ND = Not Determined.

min did not increase protection. As described in chapter 4, WCS374r caninduce resistance when grown at 34 ˚C. However, cultivation of WCS374r at34 ˚C in the presence or absence of Fe3+ did not result in protective activity.Also strains CHA0r and 7NSK2 and derivatives of the latter were all inactivein suppressing bacterial wilt in the root-dipping assay (data not shown).

Thus, in all experiments performed with root dipping,WCS417r was theonly strain able to consistently suppress bacterial wilt of E. urophylla seedlings.

S u p p r e s s i o n o f b a c t e r i a l w i l t b y g e n e t i c a l l y m o d i f i e d

W C S 3 5 8

Because of their effectiveness in antagonizing R. solanacearum in vitro ,the genetically modified derivatives of WCS358, WCS358::phl andWCS358::phz, were also tested, alone or in combination with WCS417r.Whereas WCS358 did not suppress disease in two repeated experiments,WCS358::phl reduced disease by 28 and 24 % in experiments 1 and 3,respectively. In contrast, WCS358::phz proved fully ineffective (Table 5). Incombination,WCS358::phl and WCS417r performed less well than WCS417ralone, suggesting that the two strains antagonize each other in the rhizosphere(Table 5, Exp. 2, 3).

D i s c u s s i o n

Under iron-limited conditions, all wild-type strains tested inhibitedgrowth of R. solanacearum in vitro.When iron was supplied to the medium,all strains, except CHA0r, lost their suppressive activity. This indicates thatstrains WCS358r,WCS374r,WCS417r and 7NSK2 inhibit R. solanacearum

by competition for iron.This conclusion is supported by the observation thatsiderophore-minus mutants of these strains did not inhibit R. solanacearum ,or inhibition was at least reduced compared to the parental strain.The abilityof the pseudobactin-minus mutant of WCS374 to inhibit growth of thepathogen may be explained by the production of the siderophorepseudomonine (Mercado-Blanco et al., 2001). Strain CHA0 is able to produceseveral antibiotics, including 2,4-diacetylphloroglucinol, pyoluteorin,pyrrolnitrin, and HCN (Duffy and Défago, 1999; Keel et al., 1990; Maurhofere t a l . , 1994b). Because its suppressive activity was not iron-dependent, it islikely that one or more of these antibiotics are responsible for its antagonisticactivity towards R. solanacearum .The strong growth inhibition observed forWCS358::phl suggests that 2,4-diacetylphloroglucinol is the metabolite thatinhibits R. solanacearum.

In biocontrol assays, only strain WCS417r consistently suppressed bacterialwilt in E. urophylla .This strain was previously shown to be effective againstfusarium wilt in carnation (Van Peer e t a l . , 1990, 1991), radish (Leeman e ta l . , 1996a), Arabidops i s (Van Loon e t a l . , 1998;Van Wees e t a l . , 1997) andtomato (Duijff e t a l . , 1998). Both pseudobactin siderophore-mediatedcompetion for iron and ISR are mechanisms implicated in the suppression offusarium wilt by WCS417r and both may also be involved in the control ofbacterial wilt. If competion for iron reduces the activity of the pathogenicbacterium and at the same time WCS417r can induce ISR in the seedlings ofE. urophyl la , this combination of mechanisms could consistently suppressbacterial wilt. The mechanisms involved will be further investigated bycomparing effects of WCS417r and its pseudobactin minus mutants, as well asby testing the effect of WCS417r under conditions where the antagonist andthe pathogen remain spatially separated (chapter 3).

Strains WCS358r, WCS374r and 7NSK2 effectively antagonized thepathogen in vitro by competition for iron, but did not suppress disease in vivo.This was not due to insufficient root colonization, because these strains survivewell in the rhizosphere of E. urophyl la seedlings (data not shown). It couldbe that they do not produce siderophores in the rhizosphere of eucalyptseedlings or that competition for iron alone is not effective enough to suppressbacterial wilt. WCS358r, previously shown to control fusarium wilt incarnation and radish by competition for iron (Duijff et al., 1994; Raaijmakerset a l . , 1995a), tended to suppress bacterial wilt in some bioassays, but neversignificantly reduced disease severity. However, its siderophore-minus mutantJM218 was not effective at all in any of the experiments. This suggests thatsiderophore-mediated competition for iron by WCS358r can contribute tothe suppression of bacterial wilt.

In spite of its ability to produce antibiotics that appear effective against


R. solanacearum in vi t ro , strain CHA0r did not suppress bacterial wilt ineucalypt. Possibly, antibiosis alone is not effective enough to control bacterialwilt because the pathogen quickly infects the plant tissues, or the antibioticsare not produced by CHA0r in the rhizosphere of E. urophyl la . Regulationof the production of secondary metabolites in CHA0 is complex andinfluenced by many biotic and abiotic factors (Notz et al., 2001, 2002).

The effectiveness of WCS417r depended on the type of assay used.Whenit was mixed through soil containing the pathogen, no protection againstbacterial wilt was observed, apparently because the pathogen is too fast ininfecting the plant. ISR is a plant-mediated mechanism that requires time todevelop and, in these bioassays, may have become active too late to controlthe pathogen. Bacterizing seeds or seedlings before transplanting intopathogen-infested soil did not protect eucalypt seedlings either, because thepathogen can readily enter the root tissues through the wounds generated bythe transfer. Yet, WCS417r was effective when applied by root dip prior totransfer of the seedlings into pathogen-infested soil.

The derivative of WCS358 constitutively producing the antibiotic 2,4-diacetylphloroglucinol was active in reducing bacterial wilt. WCS358::phlinhibited R. solanacearum in vitro equally in the absence and in the presenceof iron, indicating that the antibiotic was active against the pathogen. Incontrast, the phenazine-1-carboxylic acid producing strain WCS358::phzantagonized R. solanacearum in vitro only significantly in the absence of ironand did not control the disease.

Of all strains tested, WCS417r and WCS358::phl possess potential forcontrolling bacterial wilt.As previously demonstrated by De Boer et al. (1999),enhanced suppression of fusarium wilt of radish can be gained by combiningPseudomonas spp. strains. A combination of WCS417r and WCS358::phl forroot dip was likewise tested for additive control effects. However, noimprovement was obtained, and the control effectiveness was even in betweenthat of each strain alone. Possibly 2,4-diacetylphloroglucinol secreted byWCS358::phl in the rhizosphere of Eucalyptus is toxic to WCS417r, causingreduced control by WCS417r. Results from in v i t ro antagonism betweenWCS358::phl and WCS417r did not indicate obvious inhibition of eitherstrain.Thus, how WCS358::phl and WCS417r interact is not known yet.

Successful control of bacterial wilt can be obtained if the Pseudomonasstrains used can first weaken the pathogen in the rhizosphere and then stop itby activation of the plants' intrinsic defence system. This may explain whyWCS417r was effective when applied by root dipping, but failed to protectthe plant by mixing through the soil or bacterization in the seedling stage.Strains with only one suppressive mechanism, such as antibiosis, competitionfor iron, or ISR do not appear to be effective enough to control bacterial wilt.

4 6 • C h a p t e r 2

The antibiotic producers, such as CHA0r and WCS358::phl, may weaken thepathogen to some extent, but once the pathogen succeeds in eluding theprotection by the biocontrol strains, it may readily enter the cortex of rootand multiply in the vascular system, leading to failure of protection.

A c k n o w l e d g e m e n t

Grateful thanks are due to Drs. Défago and Höfte for providing P.f luores cens strain CHA0r, and P. aeruginosa strain 7NSK2 and its mutants,respectively. Seeds and wilted branches of Eucalyptus urophylla were kindlyprovided by Mr. Xian Shenghuan. Strain Rrp of Ralstonia solanacearum wasobtained from Prof. Zhang Jingning and Dr. Luo Huanliang.This research wassupported by the Netherlands Foundation for the Advancement of TropicalResearch (WOTRO) and the Natural Science Foundation of China (NSFC).


4 8 • C h a p t e r 2

Induct ion of sys temic res i s tance

aga inst bacter ia l wi l t in Euca lyptus

urophy l la by f luorescent

Pseudomonas spp .

L . X . R a n 1 , 2 , Z . N . L i 2 , G . J . W u 2 , L . C . v a n L o o n 1 a n d

P. A . H . M . B a k k e r 1


2 Central South Forestry College, 412006, Zhuzhou, Hunan, P. R. China

C h a p t e r 3

I n d u c t i o n o f s y s t e m i c r e s i s t a n c e i n E u c a l y p t u s • 5 1

I nduct ion of sys temic res i s tance

aga inst bacter ia l wi l t in

Euca lyptus urophy l la by

f luorescent Pseudomonas spp .

A b s t r a c t

Shoot tip inoculation of Eucalyptus urophyl la with Rals toniaso lanacearum was developed to investigate the ability of specific strains offluorescent Pseudomonas spp. to induce systemic resistance (ISR) in thispathosystem. Most of the strains used can produce salicylic acid (SA) in vitroand, therefore, chemical SA was used as a reference treatment.Whereas a soildrench with SA did induce systemic resistance in E. urophylla, infiltration ofSA into the leaves did not trigger systemic resistance in this plant species.None of the fluorescent Pseudomonas spp. strains could induce ISR againstbacterial wilt when applied to the soil, but two strains,WCS358r and WCS374rtriggered ISR when they were infiltrated into two lower leaves 3-7 days beforechallenge inoculation. A mutant of strain WCS358r defective in biosynthesisof the fluorescent siderophore pseudobactin, did not induce ISR, suggestingthat pseudobactin358 is the ISR-inducing determinant of WCS358.The ISRinducing determinant(s) of WCS374r are as yet unknown. However, SAproduction does not seem to be involved.Transformation of the siderophore-minus mutant of WCS358 with the SA biosynthetic genes from WCS374, didnot enable this transformant to induce ISR in E. urophylla.

5 2 • C h a p t e r 3


Selected non-pathogenic plant growth-promoting rhizobacteria (PGPR)are known to induce systemic resistance (ISR) (Kloepper e t a l . , 1992;VanLoon e t a l . , 1998;Van Peer e t a l . , 1991; Wei e t a l . , 1991). During the pasttwo decades, bacterial determinants responsible for this induction have beenidentified and shown to be differentially effective in different plants. Thisdifferential effectiveness can be clearly demonstrated with results of threePGPR strains, Pseudomonas put ida WCS358, Pseudomonas f luores censWCS374 and P. f luores cens WCS417, in different plant-pathogen systems.Strain WCS358 can induce ISR in Arabidopsis thaliana against Pseudomonassyr ingae pv. tomato (Pst ) (Van Wees e t a l . , 1997), but neither in carnationagainst Fusar ium oxysporum f. sp. dianthi (Fod) (Duijff et al. , 1993), nor inradish against F. oxysporum f. sp. raphani (For) (Leeman et al., 1995b). StrainWCS374 can induce resistance in radish against For (Leeman et al. , 1995b),but not in Arabidopsis against Pst (Van Wees et al., 1997). It has been reportedfor strain WCS417 that it induces ISR in carnation (Van Peer et a l . , 1991),radish (Leeman e t a l . , 1995b), Arabidops i s (Pieterse e t a l . , 1996), tomato(Duijff e t a l . , 1998), and bean (Bigirimana and Höfte, 2002) against severalpathogens. Bacterial determinants involved in the elicitation of ISR by thesestrains are the fluorescent siderophore pseudobactin, lipopolysaccharide (LPS),and flagella of WCS358 (P.A.H.M. Bakker, I. van der Sluis and L.C. van Loon,unpublished results); pseudobactin, other iron-regulated metabolites and LPSof WCS374 (Leeman e t a l . , 1995b, 1996a), and LPS and iron-regulatedmetabolites other than pseudobactin for WCS417 (Leeman e t a l . , 1995a,1996a). Interestingly, strains WCS374 and WCS417 produce salicylic acid (SA)under conditions of iron limitation (Leeman et al. , 1996a), and SA is knownto induce systemic acquired resistance in a wide range of plant species (Sticheret al., 1997). However, ISR elicited by WCS417 in A. thaliana is independentof SA accumulation in the plant (Pieterse et al., 1996).

Another plant growth-promoting rhizobacterium with the ability toinduce systemic resistance is P. aeruginosa 7NSK2. It produces as siderophores,the fluorescent pyoverdin, pyochelin, and SA under iron-limiting conditions(Buysens e t a l . , 1996). The production of SA by 7NSK2 is required for theinduction of systemic resistance in tobacco against tobacco mosaic virus(TMV) (De Meyer et al., 1999a) and has also been implicated in the systemicresistance induced by this strain in bean against Botryt i s c inerea (De Meyerand Höfte, 1997). P. fluorescens strain CHA0 is a biocontrol agent of varioussoilborne diseases (Défago e t a l . , 1990), and induces systemic resistance intobacco against tobacco necrosis virus (TNV) (Maurhofer et al., 1994a). CHA0also has the ability to produce SA. However, the metabolite of this strain that

was suggested to be involved in ISR is pyoverdin (Maurhofer et al. , 1994a).Therefore, the extent to which bacterially produced SA is involved in ISRremains unclear.

Eucalypt bacterial wilt has been a severe problem in clonally propagatedtrees on commercial plantations in south China since the 1980s (Gan et al. ,1998; Lin et al. , 1996; Lu and Pan, 1995), and no effective control measuresare available yet. As shown in chapter 2,WCS417r is able to suppress bacterialwilt when applied by root dip. Because WCS417r is effective in suppressingdiseases in several plant species through induction of systemic resistance, wewondered whether WCS417r also induces systemic resistance in Eucalyptusand whether bacterially-produced SA could play a role. Other strains knownto induce systemic resistance in other host-pathogen systems were tested forcomparison, as was the effect of application of SA.


C u l t i v a t i o n o f E u c a l y p t u s u r o p h y l l a

Eucalyptus urophyl la is highly susceptible to bacterial wilt caused byRalstonia solanacearum (Lin et al. , 1996;Wu and Liang, 1988b). Seeds werepurchased from the Leizhou Forestry Bureau (Leizhou, P. R. China) andgerminated as described in chapter 2. Four-week-old seedlings (average height1.5 cm) were transferred to pots containing 100 g of a sand/loam soil mixturethat had been autoclaved twice for 1 h on alternate days, with 4 plants per potand 5 pots for each treatment.The plants were grown in a growth cabinet foranother 7-8 weeks with a 12 h light and 12 h dark cycle at 25-28 ˚C and 20-23 ˚C, respectively, and at a relative humidity of 70 %. Once a week each potreceived 10 ml of half-strength Hoagland nutrient solution (Hoagland andArnon, 1938), supplemented with 10 µM FeEDDHA (Fe-ethylenediamine di-o-hydroxyphenylacetic acid; CIBA-Geigy, Basel, Switzerland).Tap water wasprovided whenever necessary.When the plants were grown in soil containingbiocontrol strains, the seedlings received half-strength Hoagland nutrientsolution without FeEDDHA, and sterile water.

B a c t e r i a l s t r a i n s

The sources and relevant characteristics of the bacterial strains used arelisted in Table 1.

Pseudomonas spp. were routinely cultured on King's medium B (KB) agarplates (King e t a l . , 1954) at 28 ˚C. Media were solidified with 1.2 % agar(Difco Laboratories, Detroit, MI).Ampicillin (40 µ g.ml-1), cycloheximide (100µg.ml-1 ), chloramphenicol (13 µg.ml-1), kanamycin (50 µg.ml-1), and rifampin


5 4 • C h a p t e r 3

Strains

Pathogens

R. solanacearum Rlz

Rlzr

R.solanacearum Rrp

Rrpr

Pseudomonas strains

P. aeruginosa 7NSK2

KMPCH

MPFM1

MPFM1-569

P. f luorescens CHA0r

P. f luorescens WCS374r

P. f luorescens WCS417r

P. putida WCS358r

JM218

pMB374-07

Relevant characteristics*

isolated from wilted branch ofEucalyptus urophylla in Leizhou,China; ampr, chlr; wild type

spontaneous rifr mutant of Rlz; ampr,chlr, rifr

isolated from wilted seedling of E.urophylla in Raoping China; ampr,chlr; wild type

spontaneous rifr mutant of Rrp; ampr,chlr, rif

wild type; Pch+, Pvd+, SA+; competesfor iron, induces systemic resistance

chemical mutant of MPFM1; Pch-, Pvd-,SA+; ampr, chlr, Kmr

Tn5 mutant of 7NSK2; Pch+, Pvd-, SA+;ampr, chlr, Kmr

pchA replacement mutant of MPFM1;Pch-, Pvd-,SA-; ampr, chlr, Kmr

isolated from tobacco rhizosphere;HCN+, Phl+, Plt+, Pvd+, SA+; ampr, chlr,rifr; suppresses disease through antibi-otics, induces systemic resistance

isolated from potato rhizosphere;Pvd+, SA+; ampr, chlr, rifr; competes foriron, induces systemic resistance

isolated from wheat rhizosphere; Pvd+,SA+; ampr, chlr, rifr; competes for iron,induces systemic resistance

isolated from potato rhizosphere;Pvd+, SA-; ampr, chlr, rifr; competes foriron, induces systemic resistance

Tn5 mutant of WCS358; sid-; ampr, chlr,Kmr

transformant of JM218 with cosmidpMB374-07; produces SA andpseudomonine; ampr, chlr, Kmr

Reference or Source

This study

“

“

“

Buysens et al . , 1996; De Meyer and Höfte, 1997

“

“

“

Keel et al . , 1992;Maurhofer et al . , 1994a;Stutz et al . , 1986

Geels and Schippers, 1983;Leeman et al . , 1995b,Leeman et al . , 1995c

Duijff et al . , 1993;Lamers et al . ,1988;Leeman et al . , 1995b;Van Wees et al . , 1997

Duijff et al . , 1994; Geelsand Schippers, 1983;Leeman et al . , 1996b Van Wees et al . , 1997

Marugg et al . , 1985

Mercado-Blanco et al . ,2001

*Abbreviations: HCN = hydrogen cyanide, Pch = pyochelin, Phl = 2,4-diacetylphloroglucinol, Plt = pyoluteorin, Pvd = pyoverdin, SA = salicylic acid, sid = pseudobactin siderophore; ampr,chlr, Kmr, rifr = resistant to ampicillin, chloramphenicol, kanamycin, and rifampin, respectively.

Table 1. Microorganisms used in this study.


(100 µg.ml-1) were added for antibiotic selection, when applicable. Culturesgrown for 24-30 h were scraped off the agar plates, and suspended in 10 mMMgSO4. The suspension was centrifuged twice at 7,600 x g for 10 min. Thebacterial pellet was resuspended in 10 mM MgSO4, and the concentrationadjusted to 109 cfu.ml-1 based on the absorbance at 660 nm.

For preparation of inoculum of the pathogen, bacterial cells were streakedonto modified Kelman agar plates [in g.l-1: proteose peptone (Oxoid) 10,casamino acids (Oxoid) 5, glucose 10, bacto agar (Difco) 10] (Kelman, 1954)containing tetrazolium chloride (50 mg.l-1), and incubated for 48 h at 30 ˚C.Single slimy, milky colonies with a pink center (Fahy and Hayward, 1983) weretransferred to modified Kelman agar plates without tetrazolium, and grown for48 h at 30 ˚C. Bacterial cells were collected in sterile distilled water, andcentrifuged twice at 12,000 x g for 10 min.The pellet was suspended in distilledwater and bacterial density measured by spectrophotometry at 660 nm.

B i o a s s a y f o r i n d u c e d s y s t e m i c r e s i s t a n c e u p o n a p p l i c a t i o n o f

s a l i c y l i c a c i d

Two strains of R. solanacearum, Rrp and Rlz, and their rifampin-resistantderivatives, Rrpr and Rlzr were tested for their pathogenicity in a bioassaydeveloped to study induction of systemic resistance. Shoot tips of 12-week-oldeucalypt seedlings were cut off and 0.2-0.3 µl of bacterial suspensions containing102-109 cfu.ml-1 were applied to the wound site using a micropipette. Theinoculated plants were moved to a growth cabinet with a 12 h day at 30 ˚C and12 h night at 25 ˚C cycle, and a relative humidity of over 93 %. Disease wasscored 10 days later. For each treatment, 20 plants were used.

To test whether SA is active in inducing systemic resistance in Eucalyptus,10 ml of solution containing different concentrations of SA was poured on thesoil of each pot containing 4 seedlings, at different times with respect to challengeinoculation with the pathogen. Challenge inoculation was performed bydecapitating the seedlings and putting a 0.2-0.3 µl droplet containing 105 cfuRrpr.ml-1 on the wound. Alternatively, SA solutions at different concentrationswere pressure-infiltrated into two lower leaves (approximately 5-10 µl per 10-20mg leaf) of 12-week-old seedlings 7 days before challenge inoculation.

To study possible direct effects of SA on growth of R. solanacearum, amodification of methods described earlier (Johnson and Curl, 1972; Loo, 1945)was used. One hundred µl of a suspension of Rrpr (108 cfu.ml-1) was evenly spreadonto modified Kelman agar medium (Kelman, 1954). Sterile filter paper discs (7mm diameter) were dipped in 1, 5, 10 or 20 mM of sterile SA solution, andplaced in the center of the agar plates seeded with Rrpr. After incubation for 48h at 30 ˚C, plates were inspected for zones of growth inhibition around the paperdiscs.

5 6 • C h a p t e r 3

B i o a s s a y f o r i n d u c e d s y s t e m i c r e s i s t a n c e u p o n s o i l

b a c t e r i z a t i o n

The bioassay was performed by adding 50 ml solution containingbiocontrol bacteria at 109 cfu.ml-1 to 1 kg of soil, mixing evenly, and planting4-week-old seedlings, 4 plants per pot and 5 pots for each treatment. Theseedlings were allowed to grow for 7-8 weeks to an average height of 10-15cm before shoot tip inoculation with Rrpr at 105 cfu.ml-1. Alternatively, 10 mlof bacterial suspension was poured onto the soil of each pot containing threeor four (7- to 8-week-old) seedlings 4 or 7 days before challenge inoculation.Bacterial colonization of the roots was checked one week later. In a third typeof experiment, seedlings were grown in bacterized soil but received a boosterdose of 10 ml as a soil drench when 7-8 weeks old. Forty plants per treatmentwere used. Five plants per treatment were sampled at three time points fordetermination of root colonization: 1. just before application of the boosterdose, 2. one week later or at the time of challenge inoculation, and 3. twoweeks after challenge inoculation or at the end of the experiment.

B i o a s s a y f o r i n d u c e d s y s t e m i c r e s i s t a n c e u p o n b a c t e r i z a t i o n

b y l e a f i n f i l t r a t i o n

In another type of bioassay, biocontrol bacteria were infiltrated into thefourth pair of leaves from the top of 11- to 12-week-old seedlings by pressureinfiltration. Eight to 10 h before infiltration, pots with 4 seedlings were placedin containers and covered with transparent plastic film to increase relativehumidity to more than 93 %. Aliquots of 5-10 µl of bacterial suspension wereinfiltrated into the abaxial surface of the leaves using a syringe without aneedle.Thereupon, seedlings were kept at high humidity for 24 h.

B a c t e r i a l c o l o n i z a t i o n o f p l a n t t i s s u e s

Roots of 5 plants per treatment were collected, weighed, and shakenvigorously for 1 min in glass tubes containing 10 vol (v/w) of 10 mM MgSO4.Aliquots of 100 µl of appropriate dilutions from the resulting suspensions weretransferred to 24-well plates, followed by the addition of 400 µl of KB agarsupplemented with cycloheximide (100 µg.ml-1), ampicillin (40 µg.ml-1),chloramphenicol (13 µg.ml-1), and rifampin (150 µg.ml-1) for rifampin-resistantWCS strains and CHA0r. For 7NSK2 and its mutants, selective antibiotics wereused as decsribed by De Meyer and Höfte (1997). After incubation at 28 ˚C for20-36 h the numbers of cfu.g-1 of fresh weight root were determined bycounting colonies under a microscope.

To determine in how far biocontrol bacteria are transported in thevascular system of the plants, 5 shoot tips, infiltrated leaves, and two stemsegments of 2 cm length above and below the infiltrated leaf pair were


harvested at the time of challenge inoculation.Tissues were rinsed with sterilewater containing 0.01 % household detergent for 5 min, washed in sterilizedwater 3 times, and homogenized in sterile 10 mM MgSO4. The number ofcfu.g-1 fresh weight of tissue was determined as described above.

D i s e a s e r a t i n g s a n d d a t a a n a l y s i s

Bacterial ooze was apparent on the cut surface of the decapitated shoots5 days after challenge inoculation, and the shoot below the tip became brownor black with time. Diseased seedlings were scored at different time points ona scale based on the length of the blackened shoot tip zone:

0--- no symptoms;1--- blackened zone shorter than 0.5 cm;2--- blackened zone 0.5 to 2.0 cm;3--- blackened zone over 2.0 cm or seedling completely wilted and dead.The disease index (DI) was calculated as DI= 100 x Σ (Ni x Xi) / 3 x Σ

Ni, where Ni indicates the number of seedlings in each class, and Xi is the classnumber. When the disease index in the control treatment was over 80, theexperiment was terminated. Disease indices were statistically analyzed forsignificant differences using one-way analysis of variance (ANOVA), followedby Fisher's least significant difference test (α = 0.05), using SPSS 8.0 (SPSSfor Windows). Normal distribution and homogeneity of variances were testedbeforehand.

R e s u l t s

P a t h o g e n i c i t y o f R . s o l a n a c e a r u m t o E . u r o p h y l l a

When inoculated onto decapitated shoots, R. solanacearum strains Rlzand Rrp, and their spontaneous rifampin-resistant mutants, Rlzr and Rrpr,induced disease in a clear density-dependent manner (Fig. 1). No significantdifference in pathogenicity was observed between the two wild-type strains,or between strain Rrp and its rifampin-resistant mutant Rrpr. In contrast, therifampin-resistant mutant Rlzr was significantly less pathogenic, particularly atlower inoculum densities. At densities exceeding 108 cfu.ml-1, most of theinoculated plants were completely wilted one week later, whereas if theinoculum density was lower than 104 cfu.ml-1, some seedlings were not diseasedeven after 10 days. An inoculation density of 105 cfu.ml-1 was adopted forfurther experiments, using the equally pathogenic strain Rrp and its rifampin-resistant mutant, Rrpr.

5 8 • C h a p t e r 3

0

10

20

30

40

50

60

70

80

90

5 6 7 8 9 10 11 12 13 14 15

days after challenge inoculation

5 mM

1 mM

0.1 mM

CK

dis

ease

index

F ig . 2 . Induction of systemic acquired resistance against bacterial wilt in E. urophylla by pouring sal-

icylic acid on the soil. Plants were challenge inoculated on day 7 by applying 20-30 cfu of strain Rrpr

to the cut surface of decapitated seedlings. CK = water-treated control.

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9

log cfu.ml-1

Rlz

Rlzr

Rrp

Rrpr

dis

ease

index

F ig . 1 . Pathogenicity of R. solanacearum wild-type strains, Rlz and Rrp, and their rifampicin-resist-

ant derivatives, Rlzr and Rrpr, to 10-15 cm high E. urophylla seedlings when inoculated at different

densities onto the shoot tips. Disease was scored 10 days after inoculation.


I n d u c t i o n o f s y s t e m i c a c q u i r e d r e s i s t a n c e b y s a l i c y l i c a c i d

Strain Rrp rapidly colonized decapitated seedling, causing a disease indexof 76 by day 10 and 85 by day 15. SA reduced disease development in aconcentration-dependent manner. In plants treated with 5 mM of SA as a soildrench shoot tip blackening developed relatively slowly and remainedlocalized. Disease indices were reduced by 15-28 % by day 15 (Fig. 2). Higherconcentrations of SA (10 and 20 mM), while strongly suppressing diseasedevelopment after challenge inoculation, proved toxic and at least half of theplants died within 2 to 5 days (data not shown).

To determine the optimum interval for SAR to develop, the time betweenapplication of SA and challenge inoculation was varied. As can be seen fromFig. 3, disease index was significantly reduced already at an interval of 1 day(α = 0.05), and decreased further when the time interval increased up to 7days.Thereafter, disease severity increased again (Fig. 3).These results indicatethat a time interval of 7 days between a soil drench of SA and challengeinoculation of the shoot tip was optimal for disease suppression. Infiltrationof solutions of SA at concentrations ranging from 1 µM to 5 mM into thefourth pair of leaves, did not induce systemic resistance. In several repeatedexperiments no reduction of disease caused by tip-inoculated Rrpr was evident(data not shown). Moreover, all leaves infiltrated with 5 mM SA and about30 % of the leaves infiltrated with 1 mM SA became curled and necrotic.

At concentrations up to 20 mM, SA did not have any inhibitory effecton the growth of R. solanacearum in vi t ro . Thus, disease suppression byapplication of SA to the soil cannot be due to direct inhibitory effect on thepathogen, and induced resistance appears to be involved.

0

20

40

60

80

100

CK 0 1 3 5 7 10

time interval (days)

a ab

bcd cd d

dis

ease

index cd

bc

F ig . 3 . Induction of SAR against bacterial wilt in E. urophylla by applying SA (5 mM) to the soil at

different time intervals before challenge inoculation with R. solanacearum Rrpr, applied at 20-30 cfu

to the cut surface of decapitated seedlings. CK = water-treated control.

6 0 • C h a p t e r 3

0

10

20

30

40

50

60

70

80

5 6 7 8 9 10 11 12 13 14 15


CK

WCS358r

WCS374r

WCS417r

dis

ease

index

F ig . 4 . Induction of systemic resistance against bacterial wilt in E. urophylla by applying

Pseudomonas strains to the rhizosphere. Seedlings were inoculated on day 7 by applying 20-30 cfu of

R. solanacearum Rrpr to the cut surface of decapitated seedlings. CK = treated with 10 mM MgSO4.

5

5.2

5.4

5.6

5.8

6

6.2

6.4

6.6

6.8

7 8 10

weeks after seedling transplant

log c

fu.g

-1 r

oot

WCS358r

WCS3

WCS417r

WCS358r

WCS374r

WCS417r

F ig . 5 . Population densities of Pseudomonas spp. strains in the rhizosphere of E. urophylla, one

week before, at the time of, and two weeks after challenge inoculation with R. solanacearum.

Bacteria were mixed in the soil at a density of 5 x 107 cfu.g-1 at the time of transplant of the seedlings.

A booster dose (10 ml at 109 cfu.ml-1) was given to each pot at the 7th week after seedling transfer.

0

20

40

60

80

7 10


dis

ease

index

CK

28 °C, Fe-

28 °C, Fe+

31 °C, Fe-

31 °C, Fe+

34 °C, Fe-

34 °C, Fe+

F ig . 6 . Induction of systemic resistance in E. urophylla by WCS374r grown at different temperature

and iron availability. Bacteria were mixed in the soil at a density of 5 x 107 cfu.g-1 at the time of trans-

plant of the seedlings.


E f f e c t s o f f l u o r e s c e n t P s e u d o m o n a s s p p . o n b a c t e r i a l w i l t

w h e n a p p l i e d t o s o i l

Several ISR-inducing fluorescent Pseudomonas spp. strains were testedfor their ability to induce resistance when mixed through soil. Uponinoculation of seedling shoot tips with the pathogen, strains WCS358r,WCS374r and WCS417r had no effect on disease development (Fig. 4).Thislack of disease suppression was not due to poor colonization of the Eucalyptusroots by the bacteria, because their population densities were well above 105

per gram (Fig. 5), a level sufficient for triggering ISR in radish (Raaijmakerse t a l . , 1995a). In A. thal iana strain WCS374r did not trigger ISR whengrown at 28 ˚C, but it did when grown at elevated temperatures (chapter 4).When grown at low iron availability WCS374r produces SA in vi t ro , and atelevated temperatures SA production is increased in this strain. However,WCS374r grown at different temperatures and iron availabilities, never inducedresistance in Eucalyptus (Fig. 6), although rhizosphere population densitieswere above 105 cfu.g-1 root (data not shown). Likewise, strains 7NSK2 andCHA0r did not reduce bacterial wilt in Eucalyptus , either by mixing themthrough soil before transplant of the seedlings, or by pouring suspensions onthe soil at 4 or 7 days before challenge inoculation. A combined treatment wassimilarly ineffective.

E f f e c t s o f f l u o r e s c e n t P s e u d o m o n a s s p p . o n b a c t e r i a l w i l t

w h e n a p p l i e d b y l e a f i n f i l t r a t i o n

Effects of the five strains were further tested by infiltrating them into thefourth pair of leaves and challenging the decapitated shoot tips 7 days later.Compared to the water control, strain CHA0r was ineffective in systemicdisease suppression in two repeated experiments (Fig. 7 A, B).Variation wasseen in that 7NSK2 appeared effective in the second experiment, but not inthe first, and WCS417r was effective in the first, but not in the secondexperiment. In several further repetitions, no significant disease reduction wasobserved for either strain (data not shown). In contrast, both WCS358r andWCS374r consistently decreased bacterial wilt by 15-25 % in these (Fig. 7A,B) and other experiments (cf.Table 2).Whereas 7NSK2 occasionally reduceddisease, its pyoverdin- and pyochelin- mutants, MPFM1 and KMPCH, neverachieved significant disease control, and the pyoverdin-, pyochelin- and SA-negative mutant, MPFM1-569, never reduced disease incidence (data notshown).

The possible involvement of the fluorescent siderophore pseudobactin358of WCS358r in the induction of resistance was investigated by usingpseudobactin-minus mutant JM218 of this strain. As can be seen in Table 2,JM218 did not reduce disease, whereas the parental strain WCS358r did.These

results suggest that pseudobactin358 is the determinant of WCS358rresponsible for induction of systemic resistance in E. urophyl la . Again,WCS374r effectively suppressed disease in this bioassay (Table 2).The possibleinvolvement of SA and the SA-containing siderophore pseudomonine, thatare both produced by WCS374 under conditions of iron limitation (Mercado-Blanco e t a l . , 2001), was studied using JM218 transformed with cosmidpMB374-07. Transformant JM218-pMB374-07 produces both SA andpseudomonine in vitro. However, like JM218 it did not reduce disease in thistype of bioassay (Table 2).

Different time intervals between infiltration of the bacterial cells into theleaves and challenge inoculation of the shoot tips with the pathogen were usedto test for the time required for systemically induced resistance to develop. As

6 2 • C h a p t e r 3

0

20

40

60

80

100

5 6 7 8 9 10 11 12 13 14 15


CK

WCS358r

WCS374r

WCS417r

CHA0r

7NSK2

0

20

40

60

80

100

5 6 7 8 9 10 11 12


CK

WCS358r

WCS374r

WCS417r

CHA0r

7NSK2

dis

ease

index

20

0

40

60

80

100

5 6 7 8 9 10 11 12 13 14 15


dis

ease

index

F ig .7 . Induction of systemic resistance against eucalypt bacterial wilt by infiltration of suspensions

of fluorescent pseudomonads at a density of 109 cfu.ml-1 into the fourth pair of leaves 7 days before

challenging decapitated shoot tips with 20-30 cfu of R. solanacearum strain Rrpr. CK = infiltrated with

10 mM MgSO4.

A

B


Treatment CK WCS358r JM218 JM218-pMB374-07 WCS374r

Disease index 77.1 50 70.9 67.4 47.9

Significance (α=0.05) a bc ab abc c

Table 2 . Induction of systemic resistance in Eucalyptus by infiltration of bacterial suspensions at 109

cfu.ml-1 into the fourth pair of leaves 7 days before challenge inoculation of decapitated shoot tips

with 105 cfu.ml-1 of R. solanacearum Rrpr. Disease was scored 12 days after the challenge inoculation.

0

20

40

60

80

100


dis

ease

index CK

3 days

5 days

7 days

9 days

5 7 10

a

bbb

ab

ab

a

b cc

aab

bb

b

time intervals

time intervals

0

20

40

60

80

100


dis

ease

index

CK

3 days

5 days

7 days

9 days

5 7 10 12 15

a

a

a

aa

a a

aa

a a aa

aa

bb

b b

ababab ab

CK

3 days

5 days

7 days

9 days

CK

3 days

5 days

7 days

9 days

a a

F ig 8 . Effects of time intervals between infiltration of 109 cfu.ml-1 of WCS358r (A ) and WCS374r (B )

into the fourth pair of leaves and challenge inoculation of decapitated shoot tips with 20-30 cfu of R.

solanacearum Rrpr on induction of systemic resistance against Eucalyptus bacterial wilt. CK = infil-

trated with 10 mM MgSO4.

A

B

6 4 • C h a p t e r 3

0

20

40

60

80

100


dis

ease

index

CK 6 7 8 9

5 7 10 12 15

aba a

ab b

ab ab

a

bc

a a

a

b

ab

abab

ab

a

b

ab abab

a

b

c

0

20

40

60

80

100


dis

ease

index

CK 6 7 8 9

5 7 8 10

a

a

aa

a

aa

a

b

bb

abab

ab

bc c

c

ab

abab

(log cfu.ml-1)

-1(log cfu.ml )

F ig . 9 . Effects of population density of (A ) WCS358r and (B ) WCS374r on the induction of systemic

resistance when infiltrated into the fourth pair of leaves. Decapitated seedlings were challenge

inoculated with 20-30 cfu of R. solanacearum Rrpr 7 days later. CK = infiltrated with 10 mM MgSO4.

A

B

seen in Fig. 8A, an interval of 3 days was not sufficient to reach significantprotection by strain WCS358, whereas 5 days did. Five days was also sufficientto reach systemic protection by strain WCS374, although it took longer for asignificant protection to be manifested (Fig. 8B).Thus, for both strains 3 to 5days between induction and challenge seems to be required for ISR to beinduced.

To determine the dose of bacteria required to significantly suppress diseasedevelopment in the leaf infiltration assay, WCS358r and WCS374r wereinfiltrated at different population densities. For strain WCS358 populationdensities of 109 cfu.ml-1 significantly suppressed disease, whereas lower densitieswere not effective (Fig. 9A). Strain WCS374r was effective at 108 and 109

cfu.ml-1, but not at 106 and not consistently at 107 cfu.ml-1 (Fig. 9B).

S p a t i a l s e p a r a t i o n b e t w e e n i n f i l t r a t e d f l u o r e s c e n t

P s e u d o m o n a s s p p . a n d c h a l l e n g e - i n o c u l a t e d R . s o l a n a c e a r u m

To see whether the disease suppression is plant-mediated and, therefore,can be ascribed to induced resistance, it was tested whether the infiltratedPseudomonas strains remained spatially separated from the challenge-inoculated R. solanacearum . At the time of challenge inoculation around 107

cfu.g-1 tissue were present in the infiltrated leaves (Table 3). The two 2-cmstem segments below the infiltrated leaf pair were colonized to substantiallevels, but colonization of the stem above the infiltrated leaves was confinedto the first 2 cm. No Pseudomonas spp. cells could be recovered from the shoottip (Table 3).The observations indicate that the infiltrated bacterial cells movemainly downward through the vascular tissues.


0-2 cm 2-4 cm 0-2 cm 2-4 cm shoot tips

WCS358r 7.59 4.81 3.39 2.40 0 0

WCS374r 6.72 4.25 2.92 2.17 0 0

a: detection limit is 100 cfu.g-1 tissue;

b: stem segments below or above the infiltrated leaf pair.

Strains

belowb aboveb

Ta b l e 3 . Transport of bacterial cells in E. urophylla seedlings one week after infiltration of the

fourth pair of leaves with 5 x 108 cfu.g-1 tissue. Values are given in log cfu per gram tissuea.

D i s c u s s i o n

Bacterial wilt of Eucalyptus , caused by R. solanacearum , is a seriousproblem in production plantations in south China. Possibilities to employfluorescent Pseudomonas spp. strains for control of this disease are describedin chapter 2. One strain in particular, P. f luores cens WCS417r, showed

Infiltrated leaves

promising results. In several plant-pathogen systems,WCS417r can induce asystemic resistance (Bigirimana and Höfte, 2002; Duijff et al. , 1998; Leemanet al., 1995b; Pieterse et al., 1996;Van Peer et al., 1991).Therefore possibilitiesto control bacterial wilt of Eucalyptus by induction of systemic resistancewere investigated in this study.

Application of SA induces systemic resistance in plants (Sticher e t a l . ,1997) and for root colonizing P. aeruginosa 7NSK2 production of SA seemsto be a prerequisite for induction of resistance in bean and tobacco (De Meyerand Höfte, 1997; De Meyer et al., 1999a). In this study application of SA as asoil drench induced resistance in Eucalyptus against R. solanacearum .However, when pressure infiltrated into lower leaves, SA did not inducesystemic resistance against bacterial wilt, confirming that the effect of SA isdependent on the site of application. Application of SA on plant roots resultsin triggering of systemic acquired resistance in tobacco (Van Loon andAntoniw, 1982), radish (Leeman et al., 1996a), Arabidopsis (Van Wees et al.,1997), and bean (De Meyer e t a l . , 1999b). In contrast, infiltration of leaveswith SA only occasionally resulted in systemic resistance in tobacco (Van Loonand Antoniw, 1982), whereas it did induce systemic resistance in Arabidopsis(Pieterse et al., 1996).

Application of Pseudomonas spp. strains, that have been reported to triggerISR in different plant-pathogen systems, to roots of Eucalyptus , were noteffective against bacterial wilt, whereas strains 7NSK2, CHA0,WCS374r, andWCS417r all produce SA in vi t ro . Either the strains did not produce SA onEucalyptus roots, or the amounts produced were not sufficient for triggeringsystemic resistance. For both WCS358r and WCS417r, ISR in Arabidopsis isindependent of SA signaling (Van Wees et al. , 1997).These strains were alsounable to trigger ISR when applied to Eucalyptus roots. However, wheninfiltrated into the leaf,WCS358r and WCS374r did induce systemic resistancein Eucalyptus, whereas WCS417r, 7NSK2 and CHA0r were not effective. Sinceno bacterial cells of either WCS358r or WCS374r were detected in the shoottips of seedlings that had been infiltrated with these strains in the fourth pairof leaves, the protective effects are plant-mediated and, hence, have to beascribed to ISR (Van Loon et al., 1998).

For WCS358r, the fluorescent siderophore pseudobactin358 is thedeterminant that triggers ISR, because a transposon insertion mutant defectivein the biosynthesis of this siderophore, JM218, did not induce ISR. Thedeterminant(s) of WCS374r that trigger(s) ISR in Eucalyptus are as yetunknown. In radish the pseudobactin siderophore of WCS374r can triggerISR (Leeman et al., 1996a), but also the 0-antigen of the LPS is an importantdeterminant (Leeman et al., 1995a). Expression of the SA and pseudomoninebiosynthetic genes of WCS374 (Mercado-Blanco et al. , 2001) in JM218 did

6 6 • C h a p t e r 3

not lead to induction of ISR by the pseudobactin mutant of WCS358r.Whereas WCS417r induces systemic resistance in various plant species, it

can not trigger ISR in Eucalyptus . Because WCS417r did colonize the rootsof E. urophylla as well as WCS358r or WCS374r, the bacterial determinantsinvolved in the induction of resistance by WCS417r in other plant species, areapparently not perceived by E. urophylla .Whether this lack of perception isrestricted to E. urophylla or Eucalyptus in general remains to be investigated.However, these results again illustrate the host specificity in the elicitation ofISR by fluorescent Pseudomonas spp. The control of bacterial wilt byWCS417r described in chapter 2 is probably not due to induced resistance,but rather to a direct antagonism between WCS417r and the pathogen.

The ability of Eucalyptus to express ISR induced by fluorescentPseudomonas spp. strains is intriguing and deserves further exploration.


P. f luores cens strain CHA0r, and P. aeruginosa strain 7NSK2 and itsmutants were kindly provided by Drs. Défago and Höfte, respectively. Gratefulthanks are due to Mr. Zhang Yungang, Li Xiangdong and Xiao Guowang forpreparing the soil for all experiments performed in China. Zeng lingcai andLiu Chaoyang are thanked for taking care of the eucalypt seedlings in theForest Pathology lab of Central South Forestry College. This research wassupported by the Netherlands Foundation for the Advancement of TropicalResearch (WOTRO) and the Natural Science Foundation of China (NSFC).


6 8 • C h a p t e r 3

No ro le for bacter ia l ly -produced

sa l i cy l i c ac id in induct ion of

sys temic res i s tance in Arab idops i s

L . X . R a n 1 , L . C . v a n L o o n a n d P. A . H . M . B a k k e r

Faculty of Biology, Section Phytopathology, Utrecht University,P.O. Box 80084, 3508 TB Utrecht,The Netherlands.

1 Permanent address: Central South Forestry College, 412006, Zhuzhou,Hunan, P. R. China

C h a p t e r 4

I S R b y S A - p r o d u c i n g P s e u d o m o n a s s p p . i n A r a b i d o p s i s • 7 1

No ro le for bacter ia l ly -produced

sa l i cy l i c ac id in induct ion of

sys temic res i s tance in

Arab idops i s

A b s t r a c t

The role of bacterially-produced salicylic acid (SA) in the induction ofsystemic resistance in plants by rhizobacteria is far from clear.We comparedfour SA-producing fluorescent Pseudomonas spp. strains, P. f luores censWCS374r,WCS417r and CHA0r, and P. aeruginosa 7NSK2, for their abilitiesto produce SA under different growth conditions and to induce systemicresistance in Arabidopsis thal iana against Pseudomonas syr ingae pv. tomato(Pst). All strains produced SA, varying from 5 fg.cell-1 for WCS417r to over25 fg.cell-1 for WCS374r. Addition of 200 µM FeCl3 to standard succinatemedium (SSM) abolished SA production in all strains.Whereas the incubationtemperature did not affect SA production in WCS417r and 7NSK2, strainsWCS374r and CHA0r produced more SA if grown at 33 ˚C rather than at28 ˚C.WCS417r, CHA0r and 7NSK2 induced systemic resistance apparentlyassociated with their ability to produce SA, but WCS374r did not. Conversely,a mutant of 7NSK2, unable to produce SA, still induced systemic resistancein Arabidopsis.The possible involvement of SA in the induction of resistancewas evaluated using SA-non-accumulating transgenic NahG plants. StrainsWCS417, CHA0 and 7NSK2 induced resistance in NahG Arabidopsis . AlsoWCS374r, when grown at 33 ˚C or 36 ˚C, induced ISR in these plants, butnot in ethylene-insensitive ein2 and in non-PR-expressing npr1 mutant plants,

7 2 • C h a p t e r 4

irrespective of the growth temperature of the bacteria. These resultsdemonstrate that in A. thal iana SA is not the primary determinant of thebacterial strains in the induction of systemic resistance.


Specific strains of non-pathogenic rhizobacteria induce a systemicresistance in plants that phenotypically resembles the systemic acquiredresistance (SAR) that is induced by pathogens. When challenge-inoculatedwith a pathogen, induced plants allow less proliferation of the pathogen anddevelop less severe symptoms of disease. Rhizobacteria-mediated inducedsystemic resistance (ISR) is studied under conditions in which the inducingbacteria and the challenging pathogen are spatially separated on the plant,thereby excluding direct interactions between the two populations (Van Loonet al. , 1998). SAR is characterized by an accumulation of salicylic acid (SA)and pathogenesis-related proteins (PRs) (Sticher e t a l . , 1997). For ISR,accumulation of SA and PRs does not seem to be required. In radish, inductionof systemic resistance against fusarium wilt by Pseudomonas fluorescens strainWCS417 was not accompanied by accumulation of PRs (Hoffland e t a l . ,1995). Likewise, in Arabidops i s thal iana ISR induced by WCS417 isindependent of SA accumulation and PR gene expression (Pieterse e t a l . ,1996), but instead requires an intact response to ethylene and jasmonic acid(Pieterse et al., 1998).

Exogenous application of SA induces systemic resistance in a wide rangeof plant species (Métraux, 2001; Van Loon, 1997). Several ISR-inducingrhizobacteria are known to be capable of producing SA in vit ro under iron-limiting conditions and, therefore, might be able to activate the SAR pathway.Leeman et al. (1996a) found that P. fluorescens WCS374 induces ISR in radishagainst fusarium wilt at low iron availability, associated with its ability toproduce SA in v i t ro . However, WCS374 did not induce resistance in A.thaliana against P. syr ingae pv. tomato (Pst), despite the fact that SA suppliedto roots induced resistance in this pathosystem (Van Wees et a l . , 1997).Thecurrent hypothesis to explain these findings is that in the rhizospherebacterially-produced SA is incorporated into pseudomonine, a non-fluorescentsiderophore of WCS374 that contains a SA group (Mercado-Blanco e t a l . ,2001).

The SA-producing Ser rat ia marcescens strain 90-166 induces resistancein cucumber and tobacco to Colle to t r i c hum orbi cu lare and P. syr ingae pv.tabac i , respectively. Mutants defective in SA biosynthesis still inducedresistance, and a mutant that had lost the ability to induce ISR could stillproduce SA.Thus, SA produced by S. marcescens 90-166 is not the primary

determinant of induced systemic resistance in cucumber or tobacco, eventhough a siderophore may be involved (Press et al., 1997).

Maurhofer e t a l . (1994a) demonstrated that pyoverdin production isimportant for induction of systemic resistance in tobacco to tobacco necrosisvirus (TNV) by P. fluorescens strain CHA0. Colonization of tobacco roots byCHA0 caused induction of PR-proteins and elevated levels of SA in the leaves.A possible role of translocation of SA produced by the bacteria to the leavescould not be ruled out. Indeed, induction of systemic resistance by bacterially-produced SA was demonstrated by expression of the SA biosynthetic genes ofP. aeruginosa in P. f luores cens strain P3.The derivative of P3 that producesSA showed improved induction of systemic resistance in tobacco against TNV(Maurhofer et al., 1998).

SA produced by P. aeruginosa strain 7NSK2 is a decisive bacterialdeterminant in the induction of systemic resistance against Botrytis cinerea inbean, since mutants defective in the production of SA were not able to induceISR (De Meyer and Höfte, 1997). Moreover, nanogram amounts of SAproduced by 7NSK2 could still activate systemic resistance in bean (De Meyere t a l . , 1999b). By making use of transgenic tobacco plants expressing theNahG gene, in which SA is converted into catechol (Delaney et a l . , 1994),De Meyer e t a l . (1999a) showed that 7NSK2 lost its ability to induce ISRagainst tobacco mosaic virus. They concluded that the disease resistanceinduced by P. aeruginosa 7NSK2 depended exclusively on the production ofSA by the bacterium (De Meyer et al., 1999a).

In the present study different SA-producing bacterial strains werecompared for their ability to induce ISR against Pst in A. thaliana . BecauseSA production in vitro occurs only under low-iron conditions, and De Meyeret al. (1999a, b) cultivated strain 7NSK2 at 37 ˚C, effects of iron availabilityand incubation temperature of the bacteria on SA production and ability toinduce ISR were investigated. To further evaluate the role of bacterially-produced SA and discriminate between different signaling pathways inArabidopsis, ethylene-insensitive mutant ein2 (Knoester et al., 1999) and non-PR-expressing mutant npr1 (Cao et al., 1997) plants were used.

M a t e r i a l s a n d M e t h o d s

B a c t e r i a l c u l t u r e s

Bacterial strains used are listed in Table 1. All fluorescent Pseudomonasspp. strains were grown on King's medium B (KB) agar plates (King e t a l . ,1954) with or without 200 µM FeCl3 at 28 ˚C or 33 ˚C for 20-24 h.Additionally, strain WCS374r was grown at 31 ˚C and 36 ˚C for 24 h and 48


7 4 • C h a p t e r 4

h, respectively. Bacterial cells were scraped off the plates, suspended in sterile10 mM MgSO4, washed twice by centrifugation at 7,600 x g for 10 min, andresuspended in 10 mM MgSO4. Suspensions were adjusted to a density of 109

colony-forming-units (cfu).ml-1 based on their optical density at 660 nm.The pathogen P. syr ingae pv. tomato DC3000 (Pst;Whalen et al., 1991)

was cultured overnight in liquid KB medium at 28 ˚C and 240 rpm. Thesuspension was washed twice with 10 mM MgSO4 by centrifugation at 7,600x g for 10 min and density was adjusted to 109 cfu.ml-1.

A n a l y s i s o f i n v i t r o p r o d u c t i o n o f s a l i c y l i c a c i d b y

f l u o r e s c e n t P s e u d o m o n a s s p p . s t r a i n s

Strains were cultured in 25 ml of liquid standard succinate medium (SSM)(Meyer and Abdallah, 1978) for 24 h at 28 ˚C and 240 rpm.Then, 50 µl wastransferred into a 100 ml flask containing 25 ml SSM (three flasks pertreatment) with or without 200 µM FeCl3, and incubated for 48 h at 28 ˚Cand 240 rpm. After determining the population density at 660 nm, the culturewas centrifuged at 12,000 x g for 10 min, and the supernatant acidified to pH

Table 1. Bacterial strains used in this study.

Strain Characteristics* Reference

P. aeruginosa 7NSK2 wild type; Pch+, Pvd+, SA+; ampr, chlr De Meyer and Höfte,1997

KMPCH chemical mutant of MPFM1; Pch-, Pvd-, “

SA+; ampr, chlr, Kmr

MPFM1 Tn5 mutant of 7NSK2; Pch+, Pvd-, SA+; “

ampr, chlr, Kmr

MPFM1-569 pchA replacement mutant of MPFM1; “

Pch-, Pvd-, SA-; ampr, chlr, Kmr

P. f luorescens CHA0r isolated from tobacco rhizosphere; HCN+, Maurhofer et al ., 1994a;

Phl+, Plt+, Pvd+, SA+; ampr, chlr, rifr Stutz et al ., 1986

P. fluorescens WCS374r isolated from potato rhizosphere; Geels and Schippers, 1983

SA+, ampr, chlr, rifr

P. fluorescens WCS417r isolated from wheat rhizosphere; Lamers et al ., 1988

SA+, ampr, chlr, rifr

P. putida WCS358r isolated from potato rhizosphere; Geels and Schippers, 1983

SA-, ampr, chlr, rifr

P. syringae pv. tomato causal agent of bacterial speck; rifr Whalen et al ., 1991DC3000 (Pst )

*Abbreviations: HCN = hydrogen cyanide, Pch = pyochelin, Phl = 2,4-diacetylphloroglucinol, Plt = pyoluteorin, Pvd = pyoverdin; SA = salicylic acid; ampr, chlr, Kmr, rifr = resistant to ampicillin, chloramphenicol, kanamycin, and rifampin, respectively.


1.5-2 with 2 M HCl. SA in the acidified supernatant was determined accordingto Meyer et al. (1992), and expressed as fg per cell.

P l a n t c u l t i v a t i o n a n d i n o c u l a t i o n

Seeds of wild-type Arabidopsis thaliana ecotype Col-0, transgenic NahGplants, and mutants npr1 (Cao et al. , 1997) and ein2 (Knoester et al. , 1999)were germinated in quartz sand, supplemented with 100 ml half-strengthHoagland nutrient solution (Hoagland and Arnon, 1938) containing 10 µMFeEDDHA (Fe-ethylenediamine di-o-hydroxyphenylacetic acid; CIBA-Geigy,Basel, Switzerland) per kg sand. Two-week-old seedlings were transplantedinto pots containing 100 g of a potting soil-sand mixture (autoclaved twicefor 1 h on alternate days), supplemented with 5 ml 10 mM MgSO4 containingfluorescent Pseudomonas spp. to a final density of 5 x 107 cfu.g-1 soil, or anequal volume of 10 mM MgSO4 as a control. Seedlings were grown for anotherthree weeks in a climate chamber with a 8 h day and 16 h night cycle at 24 ˚Cand 20 ˚C, respectively, and at 70 % relative humidity. Half-strength Hoaglandsolution without FeEDDHA was applied to the plants once a week, and tapwater was given when needed.

When five weeks old, 25 plants per treatment were challenge inoculatedwith Pst by dipping the rosette leaves into a suspension containing 2.5 x107

cfu.ml-1 (Col-0 wild-type, npr1 and ein2 mutants), or 2.5 x 106 cfu.ml-1 (NahGtransformant) and 0.01 % (v/v) Silwet L-77 (Van Meeuwen Chemicals BV,Weesp,The Netherlands) as detergent.The inoculated seedlings were held at100 % relative humidity for disease to develop. The percentage of leavesshowing symptoms of chlorosis and necrosis was scored 3 to 4 days later.

R o o t c o l o n i z a t i o n b y f l u o r e s c e n t P s e u d o m o n a s s p p .

At the end of each experiment, roots of five plants per treatment wereharvested separately and shaken for 1 min in 10 vol (g/v) of 10 mM MgSO4

with 0.5 g of glass beads (0.6 mm diameter). Aliquots of 100 µl at properdilutions were pipetted into 24-well plates and 400 µl of KB agar supplementedwith ampicillin (40 µg.ml-1), cycloheximide (100 µg.ml-1) and chloramphenicol(13 µg.ml-1) (KB+), at 45-50 ˚C, was added to each well. For strains WCS358r,WCS374r,WCS417r, and CHA0r, KB+ agar was supplemented with rifampin(150 µg.ml-1). For mutants KMPCH, MPFM1 and MPFM1-569, KB+ wassupplemented with kanamycin (200 µg.ml-1). Plates were incubated at 28 ˚Cfor 24-36 h. P. aeruginosa 7NSK2 was grown in KB+ only and incubated at37 ˚C for 16-20 h. After incubation the number of cfu per gram of fresh rootwas determined microscopically.

7 6 • C h a p t e r 4

D a t a a n a l y s i s

Leaves showing necrotic or water-soaked lesions surrounded by chlorosiswere scored as diseased (Pieterse e t a l . , 1996). The percentages of diseasedleaves were statistically analyzed for significance using one-way analysis ofvariance (ANOVA), followed by Fisher's least significant differences test atα = 0.05, using SPSS software (SPSS for Windows, version 8.0).

R e s u l t s

S A p r o d u c t i o n i n v i t r o b y f l u o r e s c e n t P s e u d o m o n a s s p p .

To compare the capacity of fluorescent Pseudomonas spp. to produce SAunder nutrient- and iron-limited conditions, strains WCS374r, WCS417r,CHA0r and 7NSK2 were tested at different growth temperatures. StrainWCS374r hardly grew at 36 ˚C and analysis of SA production at thistemperature could not be performed. As seen in Table 2,WCS374r producedthe highest level of SA, amounting to about 20 fg.cell-1 at 28 ˚C and increasingto about 25 fg.cell-1 at 31-33 ˚C.Temperature had no significant effect on SAproduction by WCS417r and 7NSK2. In contrast, the amount produced byCHA0r doubled when the bacterium was grown at 33 ˚C rather than 28 ˚C.

Supplementing the medium with 200 µM FeCl3 strongly suppressed SAproduction in all strains (data not shown), confirming that SA production bythe bacteria is iron-regulated.

E f f e c t s o f t e m p e r a t u r e a n d i r o n a v a i l a b i l i t y d u r i n g b a c t e r i a l

g r o w t h o n i n d u c t i o n o f s y s t e m i c r e s i s t a n c e

Strains WCS417r, CHA0r and 7NSK2, grown under iron-limitedconditions, were equally effective in inducing ISR in Col-0 wild-typeArabidopsis, whether cultivated at 28 ˚C or at 33 ˚C (Fig. 1). Strain WCS374rdid not induce resistance when grown at 28 ˚C, in accordance with previousresults by Van Wees e t a l . (1997). However, this strain did induce ISR in

Table 2 . Salicylic acid production by fluorescent Pseudomonas spp. growing at different tempera-

tures in liquid standard succinate medium (SSM) without iron. SA production is expressed as fg.cell-1

(means ± standard error).

Strain 28 °C 31 °C 33 °C

WCS374r 18.8 ± 0.46 27.2 ± 0.87 26.0 ± 0.61

WCS417r 4.5 ± 0.17 n.d.* 5.4 ± 0.47

CHA0r 6.0 ± 0.55 n.d. 13.7 ± 0.09

7NSK2 8.4 ± 0.20 n.d. 8.2 ± 0.48

* n.d. = not determined.


Arabidops i s when grown at 33 ˚C (Fig. 1). Addition of iron to the growthmedium did not influence the abilities the strains to induce resistance at28 ˚C and/or 33 ˚C (Fig. 2). Because SA production was found only in theabsence of added iron, the resistance induced is unlikely to be the result ofbacterially-produced SA. Similarly, the non-SA producing strain WCS358rinduced ISR independent of the cultivation temperature or the iron availability

0

10

20

30

40

50

60

CK WCS374r WCS417r CHA0r 7NSK2

% le

aves

with

sym

ptom

s a a

b

c c cbc

c c

28 °C 33 °C

F ig . 1 . Induction of systemic resistance in A. thaliana by fluorescent Pseudomonas spp. strains

grown on KB agar plates at 28 ˚C or 33 ˚C. Two-week-old seedlings were transplanted to soil that

was either mixed with 10 mM MgSO4 (CK) or inducing bacterial cells in 10 mM MgSO4. Plants were

grown for another three weeks and challenge-inoculated by dipping the rosette in a suspension of

P. syringae pv. tomato. Disease severity is presented by the mean percentage of leaves with necrot-

ic or/and water-soaked symptoms. Different letters indicate statistically significant differences

between treatments (Fischer's LSD test; α = 0.05).

28 °C 33 °C

0

10

20

30

40

50

60

70

CK WCS374r WCS417r CHA0r 7NSK2

% le

aves

with

sym

ptom

s a a

b b b

b b b

b

F ig . 2 . Induction of systemic resistance in A. thaliana by Pseudomonas spp. strains grown on KB

agar plates supplemented with 200 µM FeCl3 at 28 ˚C or 33 ˚C. Experimental details were the same

as described in Fig. 1.

7 8 • C h a p t e r 4

in the growth medium (Fig. 3). Population densities of the introduced strainsin the rhizosphere of Arabidopsis varied between 6.5 and 7 log cfu.g-1 freshroot. Incubation temperature and iron availability did not significantlyinfluence the population densities in the rhizosphere (data not shown).Thus,the inability of strain WCS374r to induce resistance when grown at 28 ˚C,cannot be ascribed to insufficient root colonization.

To investigate the involvement of siderophores, including SA, producedby strain 7NSK2, three selected mutants were tested for their ability to induceISR. A significant reduction in the percentage of diseased leaves was observedin all bacterial treatments (Fig. 4), including the non-SA producing mutant

Fe- Fe+

0

10

20

30

40

50

60

70

CK WCS358r, 28 °C WCS358r, 33 °C

% le

aves

with

sym

ptom

s a

b b b b

F ig . 3 . Induction of systemic resistance in A. thaliana by P. putida strain WCS358r grown on KB

agar plates with or without 200 µM FeCl3 at 28 ˚C or 33 ˚C. See Fig. 1 for experimental details.

0

10

20

30

40

50

60

70

CK 7NSK2 MPFM1 KMPCH MPFM1-

569

% l

eaves

wit

h s

ym

pto

ms a

bb b

b

F ig . 4 . Induction of systemic resistance in A. thaliana by P. aeruginosa strain 7NSK2 and its

mutants defective in pyoverdin (MPFM1), pyoverdin and pyochelin (KMPCH), and pyoverdin,

pyochelin and SA (MPFM1-569) biosynthesis. See Fig. 1 for experimental details.


MPFM1-569.These results demonstrate that pyoverdin, pyochelin and SA arenot necessary for the induction of ISR by 7NSK2 in Arabidopsis . Moreover,all strains tested appear to induce resistance independent of SA production.

I n d u c e d r e s i s t a n c e s i g n a l i n g

To exclude the involvement of SA in the induction of systemic resistanceby the strains tested, the experiments were repeated using SA-non-accumulating NahG plants. Strains WCS417r, CHA0r, and 7NSK2 all induceda systemic resistance in this transformant (Fig. 5) similar to that in wild-typeCol-0 plants (Fig. 1). Therefore, ISR by these strains is independent of SAsignaling.

Because induction of systemic resistance by strain WCS374r wastemperature dependent (Fig. 1),WCS374r grown at different temperatures wastested for ISR in wild type, NahG, e in2 , and npr1 plants. As shown in Fig.6A, the ability of WCS374r to induce resistance increased gradually when thegrowth temperature of the bacteria was increased from 28 ˚C to 36 ˚C.Essentially similar results were obtained in NahG plants (Fig. 6B), confirmingthat SA produced by WCS374r grown at higher temperatures was not the mainfactor to induce resistance. Similar tests in mutant e in2 plants demonstratedthat WCS374r grown at any temperature did not induce resistance (Fig. 6C),clearly indicating that induction of systemic resistance by WCS374r inArabidopsis requires sensitivity to ethylene. Likewise, no systemic resistancewas induced in npr1 mutant plants (Fig. 7). Thus, resistance induced byWCS374r is dependent on NPR1.The lack of resistance induction was notdue to defective root colonization, as no significant differences in colonizationlevels of WCS374r in the rhizosphere of the wild-type, transgenic and mutantplants were observed (data not shown).

20

0

40

60

80

CK WCS417r CHA0r 7NSK2

a

b b c

% l

eav

es w

ith

sy

mp

tom

s

F ig . 5 . Induction of systemic resistance in the NahG transformant of A. thaliana by fluorescent

Pseudomonas spp. strains WCS417r, CHA0r and 7NSK2. See Fig. 1 for experimental details.

8 0 • C h a p t e r 4

0

10

20

30

40

50

60

CK 28 °C 31 °C 33 °C 36 °C

% l

eaves

wit

h s

ym

pto

ms

a

ab abb

c

0

20

40

60

80

100

C K 28 °C 31 °C 33 °C 36 °C% l

eav

es w

ith

sy

mp

tom

s

ab

bc c d

a

0

10

20

30

40

50

60

70

80

CK 28 °C 31 °C 33 °C 36 °C% l

eaves

wit

h s

ym

pto

ms

a a a aa

F ig . 6 . Effect of growth temperature of P. fluorescens strain WCS374r grown on KB agar plates on

induction of systemic resistance in Arabidopsis A) wild-type Col-0, B) transgenic NahG and C) ein2

mutant plants. See Fig. 1 for experimental details.

0

20

40

60

CK 28 °C 33 °C

% le

aves

with

sym

ptom

s a a a

F ig . 7 . Effect of growth temperature of P. fluorescens strain WCS374r grown on KB agar plates on

induction of systemic resistance in npr1 mutant plants of A. thaliana. See Fig. 1 for experimental

details.

A

B

C


D i s c u s s i o n

Production of SA in bacteria is often linked to the production of specificsiderophores. For example, in P. aeruginosa it is linked to the production ofpyochelin (De Meyer and Höfte, 1997), and in P. f luores cens WCS374 topseudomonine (Mercado-Blanco e t a l . , 2001). The production ofpseudomonine is regulated by iron availability, but in addition this siderophoreis produced at elevated temperatures, whereas the production of the fluorescentsiderophore pseudobactin is shut down at these temperatures (Mercado-Blancoet al. , 2001).Therefore, effects of iron availability and temperature on the invi t ro SA production by different fluorescent Pseudomonas spp. strains werestudied. For all strains tested, SA production in SSM was completely suppressedby the addition of 200 µM FeCl3. This is consistent with earlier reports(Leeman et a l . , 1996a; Mercado-Blanco et a l . , 2001; Meyer et a l . , 1992). P.fluorescens strains WCS374r and CHA0r produced more SA when the growthtemperature was increased from 28 ˚C to 33 ˚C, whereas for strains WCS417rand 7NSK2 incubation temperature did not affect SA production.To study apossible role of bacterially-produced SA in the induction of systemic resistancein vivo, the bacteria were grown at different temperatures and iron availabilitybefore applying them onto the plant roots.

In bioassays, growth temperature of the bacteria had diverse effects withthe different strains. For strains WCS417r, CHA0r and 7NSK2, their resistance-inducing abilities were not affected by growth temperature. Apparently, thestrongly increased SA production of CHA0r grown in v i t ro at highertemperature (33 ˚C), had no effect on its ability to induce systemic resistanceagainst Pst in A. thal iana . For WCS374r, incubation temperature did affectthe ability of this strain to induce systemic resistance. When incubated at28 ˚C or 31 ˚C WCS374r did not induce ISR, but this strain reduced thepercentage of diseased leaves consistently in Col-0 wild type and NahG plantswhen the incubation temperature was raised to 33 ˚C or 36 ˚C. WCS374rproduced comparable amounts of SA in vitro when grown at 31 ˚C or 33 ˚C.One might have expected, therefore, that when grown at either of theseincubation temperatures, it would have had the same effect.We thus concludethat the increased disease resistance observed upon application of WCS374rgrown at higher temperatures is not due to SA.This conclusion is supportedby the results obtained with the ethylene-insensitive mutant e in2 . In thismutant application of SA induces resistance (Van Wees et al., 2000). However,WCS374r did not induce ISR in e in2 , irrespective of the incubationtemperature. Also in mutant npr1 WCS374r was not effective, confirmingresults obtained by Pieterse e t a l . (1998). As far as iron availability isconcerned, growing bacteria in the absence or presence of FeCl3 did not affect

their resistance-inducing abilities. For strain 7NSK2, this result is at variancewith that of De Meyer and Höfte (1997) who observed induction of resistanceto gray mold in bean only when the bacterium was prepared from iron-limitedKB, but not when grown on iron-rich LB medium.

De Meyer e t a l . (1997, 1999a) showed that SA produced by 7NSK2 isrequired for the induction of systemic resistance in bean against B. c inereaand in tobacco against TMV, respectively. In our A. thaliana-Pst model system,SA production by 7NSK2 or its mutants defective in different iron-chelatingmetabolites, is not necessary for the induction of systemic resistance. In wild-type Arabidops i s the SA-negative mutant MPFM1-569 decreased diseaseseverity, significantly, indicating that SA is not the decisive bacterialdeterminant for induction of resistance in Arabidops i s . Moreover, in NahGplants 7NSK2 did induce resistance against Pst.

Strains WCS417r and CHA0r were likewise able to induce resistance inNahG plants, confirming earlier results reported for WCS417 (Pieterse et al.,1996), and indicating that also for CHA0 ISR in Arabidopsis is independentof SA accumulation. In tobacco, induction of systemic resistance by CHA0seems to be dependent on accumulation of SA (Maurhofer e t a l . , 1998). InArabidopsis, however, there is no evidence for the involvement of SA in ISRelicited by rhizosphere-inhabiting Pseudomonas spp.These findings suggestthat plant species can differ in their reaction to resistance-inductingrhizobacteria by activating different signaling pathways.


Grateful thanks are due to Drs. Défago and Höfte for providing P.f luores cens strain CHA0r, and P. aeruginosa strain 7NSK2 and its mutants,respectively. Ientse van der Sluis and Johan A. van Pelt are thanked for providinggenerous help in experiments performed in Section Phytopathology of UtrechtUniversity. This research was supported by the Netherlands Foundation forthe Advancement of Tropical Research (WOTRO).

8 2 • C h a p t e r 4

Mutants of Pseudomonas put ida

WCS358 unab le to catabo l i ze

phenol i c ac ids a re less compet i t ive

in the rh i zosphere but re ta in the

ab i l i ty to induce sys temic

res i s tance in Arab idops i s

L . X . R a n 1 , 2 , V. Ve n t u r i 3 , H . W. G r o e n e v e l d 4 , L . C . v a n L o o n 1 a n d

P. A . H . M . B a k k e r 1


2 Permanent address: Central South Forestry College, 412006, Zhuzhou,Hunan, P. R. China

3 Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy

4 Section Ecophysiology, Utrecht University, P.O. Box 80084,3508 TB Utrecht,The Netherlands.

C h a p t e r 5

P h e n o l i c a c i d s a n d r o o t c o l o n i z a t i o n i n A r a b i d o p s i s • 8 5

Mutants of Pseudomonas putida

WCS358 unable to catabolize

phenolic acids are less competit ive

in the rhizosphere but retain the

abil i ty to induce systemic

resistance in Arabidopsis

A b s t r a c t

The abilities of mutants of Pseudomonas putida strain WCS358 impaired inthe utilization of specific phenolic acids to colonize roots of Arabidopsis thalianaand induce systemic resistance (ISR) were investigated. In a gnotobiotic system,bacterial numbers in the presence of plant roots were 10-fold higher than in theabsence of plants, indicating that root exudates secreted by Arabidopsis areimportant for survival of the bacteria. Several phenolic acids, i.e. p-coumaric, p-hydroxybenzoic, protocatechuic, and vanillic acid were detected in the rootexudates of A. thaliana, but in relatively low amounts. No differences wereobserved in the population densities of the wild-type strain, its mutants and thecomplemented strains, indicating that phenolic acids were not a limiting factorfor growth of the bacteria under gnotobiotic conditions. However, in pottingsoil bioassays, mutants FAI1, FAI15 and VBHB, impaired in the utilization ofcoumaric, vanillic and hydroxybenzoic acid, respectively, did not colonizeArabidopsis roots as well as the parental strain. Nevertheless, their ability to inducesystemic resistance against Pseudomonas syringae pv. tomato was unimpaired.

8 6 • C h a p t e r 5


Phenolic acids and their derivatives are abundant soil constituents,resulting from the biodegradation of lignin and accumulating in humicsubstances, and of great significance in plant-soil systems (Siqueira et al., 1991).Phenolic monomers are present in root exudates, where they can be readilydegraded by many rhizosphere bacteria, which can use them as the sole carbonsource.They also function in the sequestering of metal ions, particularly Fe,for uptake by plant roots.

The concentration of phenolic acids as percentage of soil organic matterranges from 2.1 to 4.4 % from grass roots and from 0.1 to 0.6 % from rootsof dicotyledonous plants. Ferulic, p-coumaric and p-hydroxybenzoic acids arethe predominant phenolic acids found in root exudates (Siqueira et al., 1991).Several phenolic compounds can act as signals in the communication betweenplants and rhizobacteria. The crown gall bacterium, Agrobac te r iumtumefac iens , is attracted by phenolic acids that are released from woundedplant cells.

The possible roles of phenolic acids in different plant-microbe systemshave been investigated during the last two decades. Seven phenolic compoundsthat are widely distributed among dicotyledonous plants, catechol, gallic acid,pyrogallic acid, p-hydroxybenzoic acid, protocatechuic acid, β-resorcylic acid,and vanillin, induce expression of the virulence loci on the Ti-plasmid of A.tumefac iens (Bolton et al. , 1986). As a result, transfer of T-DNA is initiated.There are several reports that such phenolic compounds also serve as molecularsignals in plant interactions with parasitic angiosperms (Lynn and Chang, 1990)and pathogenic, as well as mutualistic, microorganisms (Peters and Verma,1990). Le Strange et al. (1990) showed that the nodulation gene nodD1 ofRhizobium sp. strain NGR234 was activated by such simple phenoliccompounds as vanillin and isovanillin in extracts from wheat seedlings. Morerecently, it was demonstrated that salicylic acid was involved in theestablishment of the Rhizobium meli lot i-alfalfa symbiosis (Martinez-Abarcae t a l . , 1998). These compounds may also be expected to have a role in theestablishment and activities of plant growth-promoting bacteria in therhizosphere of plants.

Pseudomonas put ida strain WCS358 was originally isolated from therhizosphere of potato (Geels and Schippers, 1983), and has since been shownto suppress fusarium wilt in carnation (Duijff et a l . , 1993, 1994) and radish(Leeman et al., 1996b) by siderophore-mediated competition for iron , as wellas to induce systemic resistance in Arabidops i s against several fungi andbacteria (Van Loon et al. , 1998;Van Wees et al. , 1997).The outer membranelipopolysaccharide (LPS), the pseudobactin siderophore, and flagella have all

been implicated in the induction of resistance in Arabidops i s againstPseudomonas syr ingae pv. tomato (Pst ) (P.A.H.M. Bakker, I.Van Der Sluis,and L. C. van Loon, unpublished). However, little information is available onplant factors that are present in root exudates and may play a role in bacterialroot colonization as a prerequisite for resistance induction.

Venturi et al. (1998) showed that WCS358 can utilize coumaric, ferulic,and vanillic acids as the sole carbon source in vitro. By Tn5 insertion, mutantswere obtained that can no longer utilize ferulic and coumaric (FAI1), ferulicand vanillic (FAI15), or hydroxybenzoic acid (VBHB) (Bertani et a l . , 2001;Venturi et al., 1998) (Fig. 1).

In the present study, these three mutants were compared to the parentalstrain,WCS358, for their colonization of Arabidopsis roots and their abilityto induce systemic resistance against Pst.


B a c t e r i a l s t r a i n s a n d c u l t u r e c o n d i t i o n

The strains used in this study are listed in Table 1. Pseudomonas put idastrain WCS358r and its mutants, FAI1, FAI15, and VBHB defective in growth


OH

OCH3

CH

HC

COOH

OH

CH

HC

COOH

trans-ferulic acid

FAI1

fca/vdh

(fca::Tn5)

p-coumaric acid

FAI1

fca/vdh

(fca::Tn5)

OH

OCH3

COOH

OH

COOH

vanillic acid

FAI15

vanA/vanB

(vanA/vanB::Tn5)

p-hydroxybenzoic acid

pobA

OH

OCH

COOH

protocatechnic acid

VBHG

pcaHG

VBHB(pobC::Tn5)

RINGFISSION

(pcaH::Tn5)

F ig . 1 . Biodegradation pathway of trans-ferulic, p-coumaric and p-hydroxybenzoic acids to proto-

catechuic acid by Pseudomonas putida WCS358 (From Bertani et al. , 2001).

8 8 • C h a p t e r 5

on specific phenolic acid monomers, and the complemented strains FAI1C,FAI15C and VBHBC were routinely cultured in liquid King's medium B (KB)(King et al. , 1954) at 28 ˚C for 20-24 h. Cells were collected, washed twicewith 10 mM MgSO4 by centrifugation for 10 min at 7,600 x g , andresuspended in 10 mM MgSO4. Pseudomonas syr ingae pv. tomato DC 3000(Pst) was cultured similarly and resuspended in 10 mM MgSO4, containing0.01 % (v/v) of the surfactant Silwet L-77 (Van Meeuwen Chemicals BV,Weesp,The Netherlands), to a final density of 2.5 x 107 cfu.ml-1.

To check the ability of WCS358r, its mutants and their complementedstrains to grow on phenolic acids as the sole carbon source, bacteria weregrown in M9 liquid medium and on M9 agar plates (Sambrook et al., 1989),supplemented with 0.15 % of the relevant compounds, for 24-96 h at 28 ˚C(Venturi et al., 1998).The results described earlier (Bertani et al., 2001;Venturiet al., 1998) were confirmed (data not shown).

Strain

Pseudomonas

putida WCS358r

FAI1

FAI1C

FAI15

FAI15C

VBHB

VBHBC

P. syringae pv.

tomato DC 3000

(Pst )

Characteristics*

isolated from the rhizosphere of potato; ampr,

chlr, rifr

WCS358 fca ::Tn5; fer-, cou-; ampr, chlr, kanr, strr

complemented strain of FAI1 containing

pCOSI1; ampr, chlr, kanr, strr, tetrr

WCS358 vanA/vanB ::Tn5; fer-, van-; ampr, chlr,

kanr, strr

complemented strain of FAI15 containing

pCOSE1; ampr, chlr, kanr, strr, tetrr

WCS358 pobC ::Tn5; hyd-; ampr, chlr, kanr, strr

complemented strain of VBHB containing

pCOSHB; ampr, chlr, kanr, strr, tetrr

causal agent of bacterial speck, rifr

Reference

Geels and Schippers, 1983

Venturi et al . , 1998

“

“

“

Bertani et al . , 2001

“

Whalen et al . , 1991

Table 1 . Bacterial strains used in this study.

* Abbreviations: cou-, fer-, hyd- and van- = inability to use coumaric, ferulic, hydroxybenzoic andvanillic acid, respectively; ampr, chlr, kanr, rifr, and strr = resistant to ampicillin, chloramphenicol,kanamycin, rifampin, and streptomycin, respectively.


H y d r o p o n i c g r o w t h o f A r a b i d o p s i s t h a l i a n a

Seeds of Arabidopsis thaliana ecotype Columbia (Col-0) were disinfectedby immersion in a 1 : 1 mixture (v/v) of alcohol (96 %) and 1 % hypochloritefor 5 min. Subsequently, the seeds were washed three times for 1 min withalcohol (96 %), and allowed to dry overnight in a flow cabinet.The seeds werethen sown on Murashige and Skoog (1962) agar medium supplemented with10 g.l-1 sucrose. Plates were incubated in a growth cabinet with a day/nightcycle of 8/16 h at 23 ˚C /20 ˚C for 5 days.

Individual seedlings of about 1 cm in length were transferred asepticallyto Eppendorf tubes, of which the bottom tip was cut off and the hole wasplugged with cotton wool (Fig. 2).The seedlings were held in place by glassbeads (0.6 mm diameter). Each tube was inserted into a plastic holder (28 mmdiameter, 38 mm high), that was placed in a 250 ml erlenmeyer flask containing30 ml autoclaved half-strength Hoagland nutrient solution (Hoagland andArnon, 1938), containing 10 µM FeEDDHA (Fe-ethylenediamine di-o-hydroxyphenylacetic acid; CIBA-Geigy, Basel, Switzerland).The flasks weresealed with a cotton wool plug. One week later bacteria were introduced intothe nutrient solution to a final population density of 5 x 107 cfu.ml-1. Nutrientsolution only, nutrient solution with bacteria and without plants, and nutrientsolution with plants and without bacteria were used as controls. The

roots

Hoagland solution

cotton wool

Eppendorfvial

plastic cap

glass beads

A. thaliana

cotton woolplug

F ig . 2 . Gnotobiotic system for collecting root exudates of Arabidopsis thaliana.

9 0 • C h a p t e r 5

experiment was performed three times with 5 plants per treatment each.To detect possible contamination of the system, nutrient solutions were

streaked onto KB agar plates at the time root exudates were collected, and theplates were incubated for 24-36 h at 28 ˚C.

A n a l y s i s o f p h e n o l i c a c i d s b y g a s c h r o m a t o g r a p h y

Six to eight weeks after seedling transfer, the remaining nutrient solutionwas centrifuged for 10 min at 7,600 x g to remove bacterial cells. For eachtreatment, the supernatants were pooled, acidified with 1 M HCl to pH 2, andextracted with 10 ml ethyl acetate, followed by 10 ml of a 1 : 1 (v/v) mixtureof ethyl acetate : cyclopentane containing 1 % (v/v) isopropanol (Raskin et al.,1989).The organic phases were pooled and dehydrated by addition of 1.0-1.5g anhydrous Na2SO4.The sample was centrifuged for 2 min at 1500 x g, andthe supernatant was concentrated to dryness under a stream of nitrogen.

Because in preliminary experiments, no cinnamic acid was found in theroot exudate of Arabidopsis, cinnamic acid was used as an internal standard.Cinnamic acid (5 µg) was added to each sample before acidification to serveas an internal reference for yield. As standards, 15 mg of vanillic acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid, salicylic acid andcinnamic acid were dissolved either separately or as a mixture in 5 ml ethylacetate. Twenty µl of each solution or mixture was dried under a stream ofnitrogen, and silylated with 100 µl of a mixture of pyridine :hexamethyldisilazane : trimethylchlorosilane, 5 : 1 : 1 (v/v/v) (Sweeley et al.,1963). Root exudate samples were silylated with 50 µl of the silylation reagent.The mixture was allowed to stand for 30 min before injection of 1.5 µl intothe gas chromatograph.

GC analysis was performed on a Hewlett Packard 5890 gas chromatographwith integrator 3390 A and a Wcot silica chrompack capillary column of CP-Sil-5 CB (length: 25 m; inside diameter: 0.32 mm) (Chrompack NederlandB.V., Middelburg,The Netherlands).The oven temperature was increased from140 ˚C to 185 ˚C with 3 ˚C.min-1.The injection and detector temperatureswere 250 ˚C and 300 ˚C, respectively. Nitrogen was used as the carrier gas.The column was flushed at 240 ˚C for 1 h before runs.

A n a l y s i s o f b a c t e r i a l p o p u l a t i o n d e n s i t y i n n u t r i e n t s o l u t i o n

To determine the population density of the introduced bacteriaremaining in the nutrient solution by the time of phenolic acid analysis,appropriate dilutions were plated on KB agar. The stability of the plasmidsin the complemented strains was determined by transferring 50 individualcolonies from KB agar plates to the same medium supplemented withtetracycline (50 µg.ml-1).


B i o a s s a y f o r i n d u c e d s y s t e m i c r e s i s t a n c e i n A r a b i d o p s i s

Seeds of A. thal iana were sown in river sand supplemented with half-strength Hoagland nutrient solution containing 10 µM FeEDDHA. Seedlingswere grown in a growth chamber with a 8 h day and 16 h night cycle at24 ˚C and 20 ˚C, respectively, and at 70 % relative humidity. After two weeks,the seedlings were transferred to potting soil mixed with river sand at a ratioof 12 : 5 (v/v), which had been autoclaved twice for 1 h on alternate days,and bacterized with WCS358r or its mutants to a final density of 5 x 107 cfu.g-1

soil. Plants were grown individually in pots (5.5 cm diameter) with 25 potsper treatment. Controls without bacteria were supplemented with an equalamount of sterile 10 mM MgSO4. Plants were given half-strength Hoaglandsolution without 10 µM FeEDDHA once a week, and tap water whennecessary.

Bacteria- and control-treated plants were challenged when five weeks oldby dipping the leaves in a suspension of virulent Pst (Whalen et al., 1991) at2.5 x 107 cfu.ml-1. Five hours before challenge inoculation, the plants wereplaced at 100 % relative humidity.Three or four days later, the percentage ofleaves with symptoms was determined per plant and expressed as relativedisease index (Van Wees et al., 1997). Leaves showing necrotic or water-soakedlesions surrounded by chlorosis were scored as diseased (Pieterse et al., 1996).Percentages of diseased leaves were statistically analyzed for significance usingone-way analysis of variance (ANOVA), followed by Fisher's least significantdifferences test (α = 0.05). Data were analyzed with SPSS for Windows (version8.0).

D e t e r m i n a t i o n o f A r a b i d o p s i s r o o t c o l o n i z a t i o n

Populations of P. putida strain WCS358r and its mutants on Arabidopsisroots were investigated 1, 2, and 3 weeks after seedling transfer, using 5 plantsat each time point. Roots were harvested, weighed, and shaken vigorously for1 min in sterile 10 mM MgSO4 (10 ml.g-1 fresh roots), containing 0.5 g ofglass beads (0.6 mm diameter). Proper dilutions were plated on KB agarsupplemented with the appropriate antibiotics (cf.Table 1) in 24-well plates.The number of colony forming units (cfu) per gram of root fresh weight wasdetermined after incubation for 24-36 h at 28 ˚C.

To check for stability of the plasmids used for complementation of themutants, colonies were transferred to KB plates containing tetracycline (50µg.ml-1). Colonization levels were log-transformed before statistically analyzingfor significance using one-way analysis of variance (ANOVA), followed byFisher's least significant differences test at α = 0.05, using SPSS software (SPSSfor Windows, version 8.0).

9 2 • C h a p t e r 5

R e s u l t s

P h e n o l i c a c i d s i n t h e r o o t e x u d a t e o f A . t h a l i a n a

To analyze the phenolic acids exuded by Arabidopsis roots in the absenceand the presence of rhizobacteria, a gnotobiotic system was developed. Gaschromatographic analysis of the root exudates resulted in complex peakpatterns (Fig. 3). Retention times of a standard mixture of pure phenolic acids(Fig. 3A) were used to identify the relevant compounds (shown in arrows) inthe root exudates (Fig. 3B) In the first experiment, results showed that A.thal iana can secrete coumaric, vanillic, hydroxybenzoic, and protocatechuicacid (Table 2). However, in the second experiment only hydroxybenzoic acidwas present. In both experiments, no ferulic acid was detected. In Exp. 1 (Table2), when WCS358r or its mutants were added to the roots of the seedlings,the amounts of phenolic acids found reflected the characteristics of the bacteriato metabolize specific phenolic acids. WCS358r metabolized all of theprotocatechuic and vanillic acid, and degraded about 80 % and 90 % ofhydroxybenzoic and coumaric acid, respectively. Mutant FAI1, which cannotutilize coumaric acid as a sole carbon source, was not able to metabolize anyof the compounds secreted by Arabidopsis . In contrast, coumaric acid couldnot be detected in the nutrient solution when its complemented strain, FAI1Cwas added. Moreover, also hydroxybenzoic and protocatechuic acid wereundetectable in this case, whereas vanillic acid was only partially degraded.The levels of the phenolic acids in the nutrient solution with FAI1 were higherthan in the presence of plants alone, indicating that the presence of bacteriastimulates the roots to release more phenolics. In the nutrient solution withmutant FAI15, only vanillic acid remained, which cannot be metabolized. Incontrast, the complemented strain, FAI15C, completely degraded this chemical(Table 2, Exp. 1). Except for FAI1, coumaric, hydroxybenzoic andprotocatechuic acids were completely or mostly utilized by all strains.Thesein vivo results corroborate the findings by Venturi et a l . (1998) and ours invitro. Mutant VBHB, which is supposed not to utilize hydroxybenzoic acid invi t ro , did reduce the level in the nutrient solution about 60-fold, essentiallysimilar to the complemented strain VBHBC (Table 2, Exp. 2).Thus, these invivo results are at variance with those of Bertani et a l . (2001) in v i t ro .Theabsence or presence of phenolic acids in the nutrient solutions was not incomplete agreement with the phenotypes of the bacterial strain and mutantsadded. However, the bacteria will not only utilize specific compounds but alsoinfluence their exudation by the roots. Therefore these results should beinterpreted with care.


Retention time of peaks in F ig . 3A:

3 .63 min = salicylic acid;

3.82 min = cinnamic acid (internal

standard in B);

5.06 min = p-hydroxybenzoic acid;

7.41 min = vanillic acid;

8.96 min = protocatechuic acid;

11.31 min = p-coumaric acid;

15.28 min = trans-ferulic acid.

F ig . 3 . Gas chromatograms of a standard mixture of phenolic acids (A) , and root exudates of

Arabidopsis thaliana Col-0 ecotype grown in half-strength Hoagland solution containing 10 µM

FeEDDHA (B) .

1 Peaks that appeared in root exudates

were identified by their retention

times. Peaks indicated with arrows

represent (F ig . 3B ):

1 salicylic acid;

2 internal standard;

3 p-hydroxybenzoic acid;

4 vanillic acid;

5 protocatechuic acid;

6 p-coumaric acid.

2

3

4

5

6

A

B

9 4 • C h a p t e r 5

B a c t e r i a l p o p u l a t i o n d e n s i t i e s i n t h e g n o t o b i o t i c s y s t e m

In the experiments in which phenolic acids were quantified, bacterialdensities were also determined.WCS358r and its mutants were inoculated ata density of 5 x 107 cfu.ml-1.After 6-8 weeks without plants levels had dropped100-fold, in the presence of plants 10-fold in several experiments. Thus,without root exudates, bacteria could still survive in the nutrient solution, butthe presence of plant roots was highly beneficial. No differences were observedin the population levels of WCS358r, its mutants and complemented strainsin either the absence or the presence of plants (Fig. 4). No contaminatingmicroorganisms were found in the treatments, nutrient solution alone, or plantswithout inoculated bacteria.

I n d u c t i o n o f s y s t e m i c r e s i s t a n c e a g a i n s t P. s y r i n g a e p v .

t o m a t o

We determined whether the inability of the mutants of WCS358r to usecertain phenolic acids impairs their root colonization of Arabidops i s plantsgrown in soil and thereby influences their abilities to induce systemicresistance. The colonization levels of the mutants and their complementedstrains were determined, and compared to colonization by the parental strainWCS358r.The population density of WCS358r decreased slowly with time inthe rhizosphere of Arabidopsis (Table 3). Population densities of mutants FAI1,FAI15, and VBHB were significantly lower than those of the parental strain,especially after 2 and 3 weeks (Table 3). Unexpectedly, the complemented

Table 2 . Phenolic acids (µg.l-1) present in nutrient solution in which Arabidopsis seedlings had

grown alone (CK) or in the presence of bacteria.

Exp.1 Exp.2

Treatmentcou* hyd pro van hyd

CK 19 27 30 8 418

WCS358r 2 5 b.d. b.d. 15

FAI1 37 37 54 24 7

FAI1C b.d. b.d. b.d. 6 24

FAI15 b.d. b.d. b.d. 8 10

FAI15C b.d. 2 b.d. b.d. b.d.

VBHB 7

VBHBC 11

*Abbreviation: cou = coumaric acid, hyd = hydroxybenzoic acid, pro = protocatechuic acid, van =vanillic acid, b.d. = below level of detection.


4

4,5

5

5,5

6

6,5

7

WCS358r FAI1 FAI1C FAI15 FAI15C VBHB VBHBC

log

cfu.

ml-1

no plant with plant

Fig. 4. Bacterial population density in the nutrient solution 8 weeks after inoculation of WCS358r,

its mutants and their complemented strains at 5 x 107 cfu.ml-1 in the absence or presence of

Arabidopsis seedlings.

Table 3 . Root colonization of Arabidopsis by WCS358r, its mutants and their complemented strains

after bacterization with 5 × 107 cfu.g-1 root. Samples were collected 1, 2 and 3 weeks after seedling

transfer, respectively.

1st week 2nd week 3rd weekTreatment log cfu.g-1 root log cfu.g-1 root log cfu.g-1 root

WCS358r Ax 7.51 ± 0.12 a* 6.96 ± 0.07 a 6.82 ± 0.07 a

WCS358r B 7.31 ± 0.13 a 7.02 ± 0.08 a 6.93 ± 0.04 a

FAI1 6.71 ± 0.20 b 6.19 ± 0.08 b 6.19 ± 0.08 b

FAI1C 7.04 ± 0.07 ab 6.27 ± 0.10 b 6.24 ± 0.12 b

FAI15 7.08 ± 0.17 ab 6.28 ± 0.08 b 6.06 ± 0.09 b

FAI15C 7.24 ± 0.22 a 6.42 ± 0.05 b 6.29 ± 0.10 b

VBHB 6.93 ± 0.10 b 6.47 ± 0.06 c 6.09 ± 0.08 b

VBHBC 7.05 ± 0.08 b 6.74 ± 0.07 b 6.22 ± 0.07 b

* Within each column different letters indicate significant difference. x: A refers to experimentA in Fig. 5 and B to experiment B in the same figure.

strains were also significantly lower, and at each of three time points, thepopulation densities did not differ from that of the mutants.The plasmids usedfor complementation of the mutants were stably maintained in the rhizosphere(data not shown). Although the population densities of the mutants and theircomplemented derivatives were significantly lower than that of the wild-typestrain, their colonization levels are sufficient for induction of resistance, unlessthe mutation impairs induction itself.

After three weeks, plants were challenge inoculated with P. syr ingae pv.tomato to test for ISR. Results from typical experiments are shown in Fig. 5.

All mutants were able to significantly decrease the percentage of diseasedleaves to the same extent as the parental strain WCS358r.Thus, the inabilityof the mutants to use specific phenolic acids as carbon source did not affecttheir resistance-inducing ability.

D i s c u s s i o n

In the gnotobiotic system developed, population densities of WCS358r,its mutants and the complemented strains were 10-fold higher in the presencethan in the absence of plants.This indicates that root exudates are importantfor survival of the bacteria. No major differences were found between thedensities of WCS358r, its mutants and the complemented strains in either thepresence or the absence of plants.Thus, the inability to utilize specific phenolicacids did not affect the population densities of the mutants. However, in rootexudates various compounds are present (Lugtenberg e t a l . , 2001) andphenolic acids are probably not a limiting factor. Although amino acids andsugars alone were demonstrated not to be the main nutrients supportinggrowth of P. fluorescens strain WCS365 on tomato roots (Lugtenberg et al. ,1999; Simons et al., 1997), we cannot exclude that the total amount of aminoacids, sugars, and especially organic acids (Lugtenberg et al. , 2001) provided

9 6 • C h a p t e r 5

0

20

40

60

80

100

120 a a

b b b b

b b b b

A B

CK

WCS358r

VBHB

VBHBC

rela

tive

dis

ease

ind

ex

CK

WCS358r

FAI1FAI15

FAI1C

FAI15C

F ig . 5 . Induction of systemic resistance by WCS358r, its mutants and their complemented strains.

Plants were cultivated in soil either mixed with 10 mM MgSO4 (CK) or bacterized with WCS358r, its

mutants and the complemented strains. The relative disease index is the mean (n = 25) of the pro-

portion of leaves with symptoms per plant compared to that of control plants (set at 100 %). The

absolute proportions of diseased leaves of the controls (CK) depicted in A and B were 59.2 % and

66.2 %, respectively. Within each frame, different letters indicate statistically significant differences

between treatments (Fischer's LSD test; n = 25, α = 0.05). The data presented are from representa-

tive experiments that were repeated at least twice with similar results.

ample nutrition for the growth of the bacteria. Investigations on the role ofspecific compounds in root colonization may be hampered by a shift in uptakeand metabolism of other compounds by bacterial mutants.

Nevertheless, in potting soil bioassays, population densities of all themutants on Arabidopsis roots were lower compared to that of the wild typestrain WCS358r (Table 3). Unexpectedly, population densities of thecomplemented strains were also lower than that of WCS358r. In vitro, noobvious differences in growth were found between the wild type, its mutantsor the complemented strains.What causes the lesser competitiveness is not clear.In the case of the complemented strains, the presence of the plasmid may havecontributed to lesser growth. In spite of the lesser root colonization of themutants and complemented strains, no effects on the induction of resistanceagainst Pst were found, apparently because bacterial numbers were still highenough for resistance to be induced. Raaijmakers et al. (1995a) demonstratedthat a threshold bacterial population density of approximately 105 cfu per gramon the roots is required for significant suppression of fusarium wilt of radish.

Whether phenolic acids serve a signaling role in P. putida remainsenigmatic. In one experiment, several phenolic acids, including coumaric,hydroxybenzoic, protocatechuic and vanillic acid, were detected in the rootexudates of A. thaliana. No ferulic acid was detected. The amounts ofhydroxybenzoic and protocatechuic acid are much higher than those ofcoumaric or vanillic acid. Only utilization of the latter was blocked in FAI1and FAI15, respectively, and hydroxybenzoic and protocatechuic acid were stillavailable as carbon sources. Results from the other experiment showed that, ofthe phenolic acids identified, only hydroxybenzoic acid was detected in theroot exudates, and the amount was much higher than that of the other test(Table 2).The average fresh weight of the plants in the control from the secondexperiment reached 298 mg (harvested 8 weeks after seedling transfer), but wasonly 34 mg (harvested 6 weeks after seedling transfer) in the first experiment.Hydroxybenzoic acid was expected to accumulate in the nutrient solution withmutant VBHB, but only a trace amount was left. Strain VBHB grew very slowlyon hydroxybenzoic acid as sole carbon source compared with its complementedstrain VBHBC and the reasons for the disappearance of hydroxybenzoic acidin the nutrient solution with VBHB are not clear.

Protocatechuic acid has been described as an activator of the induction ofthe vir genes of A. tumefaciens (Bolton et al., 1986). All three mutants used werestill able to utilize protocatechuic acid, which was secreted in relatively highamount by Arabidopsis roots.Therefore, we cannot exclude that protocatechuicacid has a signaling role in the interaction of the bacterium with plant roots.However, the presence of several phenolic compounds may cause redundancy,in that different phenolics may substitute for each other.To avoid a shift in the


utilization of phenolic acids, mutants that have lost their ability to metabolizeseveral phenolic compounds are needed.


This research was supported by the Netherlands Foundation for theAdvancement of Tropical Research (WOTRO).

9 8 • C h a p t e r 5

G e n e r a l d i s c u s s i o n • 9 9

Genera l d i s cuss ion

R o o t c o l o n i z a t i o n a s a p r e r e q u i s i t e f o r d i s e a s e s u p p r e s s i o n

b y r h i z o b a c t e r i a

Several definitions have been given to characterize root colonization,depending on different criteria (Glandorf, 1992), but unanimously emphasizingactive growth and proliferation in, on, or around roots. Root exudates rich inamino acids, monosaccharides, and organic acids are consistently secreted intorhizosphere soil, providing nutrients for the growth of rhizobacteria(Lugtenberg et al. , 2001). As an effective biocontrol agent, a bacterium musthave one or more disease suppressive mechanisms, and an effective competitivecolonization potential with respect to the resident microflora.

In bioassays or field experiments with antagonistic rhizobacteria, rootcolonization is a prerequisite for biocontrol effectiveness (Bull et a l . , 1991;Raaijmakers et al. , 1995a; Schippers et al. , 1987). A minimum of 105 cfu.g-1

fresh root appeared to be required for biocontrol of fusarium wilt of radishby either Pseudomonas putida WCS358 or Pseudomonas fluorescens WCS374.When rhizosphere population densities of both these strains dropped belowthis threshold level, a relatively small decline in population density had a majornegative effect on their efficacy to suppress fusarium wilt (Raaijmakers et al.,1995a). Obviously, colonization of large parts of the root system will facilitatedisease suppression, because colonization is necessary for delivery of activebacterial metabolites. In this study, mutants of P. put ida strain WCS358defective in catabolizing specific phenolic acids were less apt in rootcolonization but still capable of inducing systemic resistance, because theymaintained a sufficient population density on the roots (chapter 5). In contrast,colonization-defective mutants of P. chlororaphis PCL1391 lost their abilityto control tomato foot and root rot caused by Fusar ium oxysporum f. sp.radi c i s- lycopers i c i , whereas they produced wild-type levels of phenazine-l-carboxamide, the antifungal metabolite responsible for control of this disease

C h a p t e r 6

1 0 0 • C h a p t e r 6

(Chin-A-Woeng e t a l . , 2000). Suppression of tomato foot and root by P.f luorescens WCS365 was similarly affected in colonization mutants (Dekkerset al., 2000).

P. put ida strain 89B-27 and Ser rat ia marces cens strain 90-166, bothinduced resistance in cucumber against anthracnose caused by Colletotr ichumorbi cu lare , but there was no relationship between the level of ISR inducedand the population densities of the two strains on the roots. In fact, the levelof ISR increased over time, while the populations of the bacteria decreased(Liu et a l . , 1995).This suggests that colonization plays a less important rolein ISR-induced biocontrol than in direct disease suppression e.g. competitionfor nutrients, or antibiosis. A temporary or local presence of ISR-inducingcells seems sufficient to cause long-lasting resistance of the whole root system(Lugtenberg e t a l . , 2001). For elicitation of ISR in radish by WCS374,however, a threshold level of 105 cfu.g-1 root was still necessary (Raaijmakerse t a l . , 1995a), indicating that a minimum number of cells is needed forinduction.

In this study, colonization levels of the fluorescent pseudomonads used inboth the Eucalyptus-R. solanacearum and Arabidopsis-P. syr ingae pv. tomato(Pst) model systems were always higher than 105 cfu.g-1 fresh root.Thus, failureto suppress bacterial wilt in eucalypt, or to induce ISR in Arabidops i s , isunlikely to be due to poor root colonization (chapters 3, 4). In vi t ro P.f luorescens strain CHA0r was the most antagonistic against R. solanacearum ,but it was not able to protect E. urophyl la seedlings against bacterial wilt(chapter 2).Whether CHA0r spread evenly over the root surface, and produced2,4-diacetylphloroglucinol (DAPG) in the rhizosphere could not bedetermined. Bacterial cells tend to congregate in grooves and large parts ofcell surfaces are left uncolonized (Bowen e t a l . , 1976). Thus, the pathogenmay circumvent the presence of the antagonist and readily attach to and invadethe roots.

B a c t e r i a l d e t e r m i n a n t s i n v o l v e d i n m i c r o b i a l a n t a g o n i s m

Several rhizobacterial determinants have been shown to be responsiblefor suppression of diseases.The production of siderophores and antibiotics hasbeen recognized as important for the inhibition of soilborne pathogens(O'sullivan and O'Gara, 1992). Siderophore-mediated disease suppressionoccurs through competition for the limited amount of iron available in thesoil, depriving pathogens and other deleterious microbes of iron. Siderophoresof different rhizobacteria vary in their effectiveness in controlling diseases.The fluorescent pseudobactin siderophore (pyoverdin) of P. put ida strainWCS358 was demonstrated to be effective in the control of fusarium wilt ofcarnation (Duijff et al., 1994) and radish (Leeman et al., 1996b; Raaijmakers

G e n e r a l d i s c u s s i o n • 1 0 1

et al., 1995a), as well as in promotion of potato yield in short rotations (Bakkere t a l . , 1986, 1987). When tested against bacterial wilt in eucalypt, WCS358suppressed disease in some bioassays, but not in all. However, seedlings dippedin a suspension of the siderophore-negative mutant JM218 never showed anyreduction in disease severity, indicating that WCS358 can suppress R.solanacearum through siderophore-mediated competition for iron (chapter 2).However, for controlling eucalypt bacterial wilt WCS358r is not as effectiveas in the suppression of fusarium wilt of carnation or radish. Alternatively thelesser efficacy of WCS358r in the suppression of eucalypt bacterial wilt maybe due to the high disease incidence in control plants. Treatment withWCS358r significantly reduced fusarium wilt of carnation only if diseaseincidence was moderate, and not if it was high (Duijff et a l . , 1994). Similarobservations were made in the control of fusarium wilt in radish (Raaijmakerset a l . , 1995a). Production of either pyoverdin or pyochelin by P. aeruginosastrain 7NSK2 proved to be necessary to achieve wild-type levels of protectionagainst Pythium damping-off in tomato. Mutant KMPCH, deficient in theproduction of pyoverdin and pyochelin, but SA positive, still inhibited thisdisease, though it was less active than the parental strain.Thus, SA producedby this strain provided some suppression of Pythium damping-off (Buysens eta l . , 1996). Pyoverdin, pyochelin and SA have all been implicated in thesuppression by P. f luores cens CHA0 of Pythium damping-off and take-alldisease caused by Gaeumannomyces graminis var. t r i t i c i in wheat (Schmidli-Sacherer et al., 1997). However, both 7NSK2 and CHA0 failed to protect E.urophylla seedlings from invasion by R. solanacearum (chapter 2).

Antibiotic production by rhizobacteria has often been associated withcontrol of plant diseases (Bakker et al., 1991; Fravel, 1988; Glick et al., 1999;Handelsman and Stabb, 1996;Weller, 1988). Antibiotic compounds producedby fluorescent Pseudomonas spp. include hydrogen cyanide (HCN), DAPG,phenazine-1-carboxylic acid (PCA), pyoluteorin (Plt) and pyrrolnitrin. In thesuppression of take-all of wheat, a mutant of P. fluorescens strain 2-79 defectivein PCA production but producing the siderophore conferred little protection(Hamdan e t a l . , 1991; Thomashow and Weller, 1990). Indeed, pyoverdin-deficient mutant derivatives controlled take-all as effectively as the parentalstrains (Hamdan et al., 1991). Synthesis of phenazine-1-carboxamide has alsoproved a decisive factor in the suppression of other diseases, such as tomatofoot and root rot by P. chlororaphis PCL1391 (Chin-A-Woeng et al. , 1998),and root rot of cocoyam (Xanthosoma sagi t t i fo l ium ), caused by Pythiummyr iotylum by P. aeruginosa PNA1 (Tambong and Höfte, 2001).

P. f luores cens strain CHA0 can produce several antibiotics, includingHCN (Blumer and Haas, 2000; Laville et al., 1992), DAPG, Plt (Maurhofer eta l . , 1994b; Schmidli-Sacherer e t a l . , 1997), and pyrrolnitrin (Duffy and

1 0 2 • C h a p t e r 6

Défago, 1999). Each of these metabolites has been demonstrated to suppressplant diseases. A gacA mutant of CHA0 in which the production of DAPG,HCN, and Plt was pleiotropically blocked had a drastically reduced ability tosuppress black root rot of tobacco, caused by Thielaviopsis basicola , undergnotobiotic conditions (Laville et al., 1992), supporting previous observationsthat the antibiotics DAPG and HCN individually contribute to the suppressionof black root rot (Keel e t a l . , 1990; Voisard e t a l . , 1989). Pyoluteorinproduction of CHA0 is involved in the suppression of Pythium damping-offof cress but not of cucumber (Maurhofer et al., 1994b). CHA0 has also beenreported to suppress black root rot of tobacco caused by T. basicola throughproduction of DAPG (Keel et al., 1990), P. ultimum-induced damping-off insweet corn (Maurhofer et al., 1992), G. graminis var. tr i t ic-induced take-allof wheat, and F. oxysporum f. sp. cucumer inum and Phomopsis s c l e ro t io idesin cucumber (Maurhofer et al., 1995).

In this study, CHA0r and two genetically modified derivatives ofWCS358,WCS358::Phl and WCS358::phz, constitutively producing DAPGand PCA, respectively (Bakker e t a l . , 2002; Glandorf e t a l . , 2001) stronglyinhibited growth of R. solanacearum in vi t ro . However, CHA0r andWCS358::phz were not able to control eucalypt bacterial wilt at all, whetherapplied by root dipping, soil bacterization or seed soaking (chapter 2).WCS358::Phl provided limited protection, but never reduced diseasesignificantly compared with non-treated controls (chapter 2). These resultssuggest that antibiotics are not effective enough against bacterial wilt causedby R. solanacearum. It may be that antibiotics produced by these strains werephytotoxic, and caused some damage to the roots of E. urophylla, since in thetreatments with CHA0 or WCS358::phz higher percentages of wilted seedlingswere observed always (chapter 2).

B a c t e r i a l d e t e r m i n a n t s i n v o l v e d i n i n d u c t i o n o f s y s t e m i c

r e s i s t a n c e

Several rhizobacterial strains can induce systemic resistance in a numberof plant diseases (Van Loon et al., 1998).Three kinds of bacterial determinantshave been implicated in the elicitation of ISR (Van Loon e t a l . , 1998;VanWees, 1999). For some strains, lipopolysaccharide (LPS) and siderophores areinvolved. A number of strains produce SA in v i t ro under iron-limitingconditions, suggesting that they can induce systemic acquired resistance (SAR).In studies by Leeman e t a l . (1996a) it was found that P. f luores cens strainsWCS374 and WCS417 produced SA at about 50 and 10 µg.ml-1, respectively,in standard succinate medium (SSM), whereas P. put ida WCS358 did notproduce SA.This production is comparable to the amounts determined in ourstudy (chapter 4). Under iron-limited conditions, the former two, SA


producing, strains induced systemic resistance in radish against fusarium wilt,but WCS358 did not.The chemical SA can induce resistance in the same plant-pathogen system when trace amount of SA (100 fg.g-1 talcum emulsion) areapplied to radish roots (Leeman et al., 1996a).These results suggest that SA isinvolved in ISR against fusarium wilt in radish. However, in the A. thalianamodel system, WCS374 did not induce systemic resistance, even thoughchemical SA (1 mM) induces resistance (Van Wees et al. , 2000). In contrast,the non-SA producing WCS358 suppressed Pst in Arabidopsis through ISR.WCS417 elicited ISR in Arabidopsis, as it does in radish, but it did so also inNahG-transformed plants (Van Wees e t a l . , 2000), indicating that SA is notinvolved in the elicitation of ISR in Arabidops i s by this strain. Because noPR proteins were induced associated with ISR in radish (Hoffland e t a l . ,1995), the role of SA produced by WCS374 and WCS417 in radish needs tobe further investigated.

P. f luores cens CHA0, producing SA under iron-starvation conditions(Meyer et al. , 1992), induced systemic resistance against TNV (Maurhofer etal., 1994a). CHA400, a pyoverdin-negative, but SA-positive mutant of CHA0partially maintained the ability to induce resistance against TNV. Furthermore,introduction of the SA biosynthetic genes pchBA from P. aeruginosa PAO1(Serino et al., 1997) into P. fluorescens strain P3, which does not produce SA,rendered this strain capable of SA production in v i t ro and significantlyimproved its ability to induce systemic resistance in tobacco against TNV(Maurhofer e t a l . , 1998). These results implicate SA in the induction ofsystemic resistance against the virus.

P. aeruginosa strain 7NSK2 produces several siderophores under ironlimitation: pyoverdin, pyochelin, and SA. Both a pyoverdin-negative mutant(MPFM1) and a mutant lacking pyoverdin and pyochelin (KMPCH) inducedresistance in bean against gray mold (De Meyer and Höfte, 1997) and intobacco against tobacco mosaic virus (TMV) (De Meyer et al., 1999a), whereasa mutant deficient in SA production (MPFM1-569) did not. In NahG tobaccoplants (Gaffney et al., 1993), 7NSK2 and its mutants were unable to elicit ISRagainst TMV (De Meyer et a l . , 1999a). Furthermore, nanogram amounts ofSA produced by 7NSK2 and its SA-positive mutant KMPCH appeared to besufficient to activate the systemic resistance against Botrytis c inerea in bean(De Meyer et al., 1999b).Therefore, the resistances induced by 7NSK2 in beanand tobacco seem dependent on bacterially produced SA (Van Loon e t a l . ,1998)

In contrast, the SA-producing rhizobacterial strain S. marces cens 90-166 induced resistance against P. syr ingae pv. tabaci in both wild-type andNahG-transformed tobacco. Moreover, a SA negative mutant retained ISR-eliciting activity in cucumber against C. orbiculare, and an ISR- mutant (90-

1 0 4 • C h a p t e r 6

166-2882) still produced SA. Based on these results, bacterial SA does notappear to be involved in the resistance induced by this strain in tobacco andcucumber.

Whether SA is involved in the induction of resistance may depend onthe plant-pathogen system and on the conditions in the rhizosphere. Basedon the results published so far, the sensitivity of the plant to chemical SA maybe an important factor. Radish, bean and tobacco are relatively sensitive tochemical SA. In radish, as little as 100 fg.g-1 talcum emulsion significantlyreduced fusarium wilt (Leeman et al. , 1996a). Hydroponic feeding of 1 nMSA to bean plants induced phenylalanine ammonia-lyase activity in the roots,increased free SA levels in the leaves, and led to a significant reduction ofspreading lesions of B. cinerea (De Meyer et al., 1999b). In tobacco grownon nutrient solution, addition of 25 µM SA caused a 45 % reduction in averagelesion area resulting from TMV infection (Enyedi et al., 1992). Probably lowerconcentrations might have sufficed, because transient wilting of the leaves wasobserved. Thus, in SA-sensitive plants, bacterially produced SA may inducesystemic resistance against their pathogens.

In Arabidopsis , 1 mM SA was reported to induce resistance against Pst(Van Wees et al. , 1997), which is 40 times more than needed in tobacco. Nodisease reduction was observed by applying SA at concentrations lower than1 mM (L. X. Ran, unpublished results). In cucumber, exogenously applied SAinduced only localized resistance against Cladospor ium cucumer inum anddid not function as a mobile, systemic resistance-inducing signal (Narusaka etal., 1999). Likewise, exogenously applied SA did not induce systemic resistanceto cucumber root rot caused by P. aphanidermatum (Chen e t a l . , 1999),suggesting that cucumber is rather insensitive to SA. SA produced by S.marces cens 90-166 is not the primary determinant of induced systemicresistance in cucumber (Press et al. , 1997), which may be related to the factthat this strain produces only 3 µg.ml-1 SA in v i t ro (Press e t a l . , 1997), toolittle to stimulate cucumber plants to increase their level of resistance.

In our study, several SA-producing strains (WCS374r,WCS417r, 7NSK2,CHA0r) did not induce systemic resistance in E. urophyl la against R.solanacearum, whether applied by pouring bacterial suspensions onto the soilor mixing them with potting soil before seedling transfer (chapter 3). Thisindicated that bacterial SA is not involved in ISR against bacterial wilt causedby R. solanacearum in eucalypt. SA may have been produced in therhizosphere but not in quantities sufficient to trigger the plant, becauseexogenously applied SA was effective only at a concentration of 5 mM.Concentrations lower than 5 mM did not effectively protect E. urophyl lafrom infection by R. solanacearum (chapter 3). In Arabidopsis , bacterial SAappears not to be involved in inducing systemic resistance against Pst either


(chapter 4), because all SA-producing bacteria,WCS417r, CHA0r and 7NSK2induced resistance against Pst in NahG-transformed plants, in which theconstitutively expressed salicylate hydroxylase converts SA to inactive catechol(Gaffney et al., 1993) (chapter 4). Moreover, the SA-producing strain WCS374grown at 28 ˚C did not elicit ISR at all in Arabidopsis (Van Wees et al., 1997).

M e c h a n i s m s i n v o l v e d i n s u p p r e s s i o n o f b a c t e r i a l w i l t i n E .

u r o p h y l l a b y P s e u d o m o n a s s p p .

P. f luores cens WCS417r was the only strain that suppressed eucalyptbacterial wilt in a root dip bioassay. In in vitro inhibition tests, it was observedthat this strain lost its inhibitory activity against R. solanacearum when 200µM of FeCl3 was supplied. A pseudobactin-minus mutant, S680 (417PSB-)(Leeman et al., 1996a), did not inhibit the growth of the pathogen under iron-limited conditions (chapter 2). Thus, siderophore production contributessubstantially to the in vitro antagonism of WCS417 against R. solanacearum .Pseudobactin may act in in vivo disease suppression by siderophore-mediatedcompetition for iron (chapter 4), or induced resistance. WCS417r did notinduce systemic resistance against R. solanacearum inoculated on woundedshoot tips (chapter 4). However, it has to be noted that under natural conditionsR. solanacearum infects plants through the roots, not the shoot tip. Based onthe present results the mode of action of WCS417r in the suppression ofbacterial wilt remains unclear.

WCS358r tended to suppress bacterial wilt, whereas its siderophore-negative mutant, JM218, was inactive.WCS358r inhibited Ralstonia in vitroin the absence, but not in the presence of iron. JM218 was inactive under bothconditions (chapter 2). Therefore, the weak controlling activity of WCS358appears to be based on siderophore-mediated competition for iron, and wasmuch lower than WCS417r-mediated suppression.This situation is similar towhat was observed in the control of fusarium wilt of carnation by these twostrains (Duijff et al., 1993).

In a modification of the bioassay for ISR, bacterial cells of eachPseudomonas strain were infiltrated into lower leaves of E. urophyl la , afterwhich the shoot tips were challenge-inoculated with R. solanacearum. In thesebioassays, WCS358r and WCS374r induced systemic resistance, whereasWCS417r did not. For strain WCS358r, it is obvious that this inducedenhancement of resistance is siderophore-mediated, since injection with itssiderophore-minus mutant, JM218, did not induce ISR (chapter 3). StrainsCHA0r and 7NSK2 were not effective in disease suppression through ISR inany of the situations tested.

1 0 6 • C h a p t e r 6

M e c h a n i s m s i n v o l v e d i n s u p p r e s s i o n o f b a c t e r i a l s p e c k i n

A r a b i d o p s i s b y P s e u d o m o n a s s p p .

WCS374 induces systemic resistance in radish against fusarium wilt andthis induced resistance is associated with its ability to produce SA in v i t ro(Leeman et al., 1995b; 1996a). However, in Arabidopsis,WCS374 did not induceresistance (Van Wees et al., 1997). When WCS374r was grown at 33 ˚C instandard succinate medium (SSM), it produced more SA, 26 fg.cell-1, versus19 fg.cell-1 at 28 ˚C. Bacteria grown at 33 ˚C and 36 ˚C induced systemicresistance against Pst in Arabidops i s , but also in NahG-transformed plants(chapter 4), indicating that the ISR induced by WCS374r when grown athigher temperature is not due to the increased amount of SA produced.

7NSK2 induced ISR against gray mold in bean only when it was grownunder iron limitation (De Meyer and Höfte, 1997). However, whether 7NSK2was grown at 28 ˚C or 33 ˚C on KB medium with or without addition of 200µM FeCl3 did not affect its ISR-eliciting activity in Arabidopsis (chapter 4).The factor(s) responsible for the induction of systemic resistance in Arabidopsisby 7NSK2 remain unknown, because wild-type as well as the mutants lackingpyoverdin and/or pyochelin and /or SA still induced resistance (chapter 4).

C o n c l u d i n g r e m a r k s

Studying the bacterial determinants responsible for disease suppressionby Pseudomonas strains is important for both theoretical and practical reasons.On the one hand, understanding how bacterial determinants act in vivo willcertainly facilitate the practical use of rhizobacteria for biocontrol of diseases,such as eucalypt bacterial wilt. On the other hand, defined mutants ofArabidopsis can aid in clarifying the role of each determinant or cooperativeeffects of several ones in biocontrol.

Several fluorescent Pseudomonas strains have been tested to controlbacterial wilt (Kempe and Sequeira, 1983; Kim and Misaghi, 1996; Luo andWang, 1983;Van Overbeek et al. , 2002), and some of those showed transientsuppressive effects. However, this is the first time that well characterizedPseudomonas strains, including two genetically modified derivatives ofWCS358 producing DAPG and PCA, respectively, were analyzed for controlof bacterial wilt. Our results demonstrate that effective strains which act bymicrobial antagonism or by inducing resistance are few, and there is nocorrelation between in v i t ro antibiosis and biocontrol efficacy in v ivo . Aspreviously demonstrated for WCS358r-mediated suppression of fusarium wiltin carnation and radish (Duijff et al., 1994; Raaijmakers et al., 1995a), bacterialtreatment was effective only if disease incidence was moderate, and not ifdisease incidence was high.

Because the DAPG-producing strains CHA0r and WCS358::phl showed


strong antibiosis against R. solanacearum in vi t ro , it was expected that theywould also effectively suppress bacterial wilt in eucalypt. However, this wasnot the case.There may be several reasons which could explain these results:1) Did the antibiotic-producing strains colonize the root parts sensitive to

invasion by R. solanacearum to sufficient densities? Overall colonization bythe strains was above the level required for effective protection againstfusarium wilt in radish and at least WCS358::phl is known to produce DAPGin the rhizosphere of wheat. Thus, neither overall colonization norproduction of DAPG seems to have been a limiting factor.

2) Are antibiotics phytotoxic to young seedlings of E. urophylla? It is knownthat seedlings of cress, tobacco and sweet corn are strongly reduced ingrowth when colonized by CHA0 or CHA0/pME3090 overproducingDAPG and Plt (Maurhofer et al., 1992; Maurhofer et al., 1995). DAPG andPlt may also have some toxicity for E. urophyl la , making the plants morevulnerable to infection by Ralstonia.

3) Can bacterial cells of these strains form a biofilm-like sheet to preventinvasion by the pathogen? It was reported that treatment with apolysaccharide flocculant prepared from Klebs ie l la pneumoniae H12,promoted plant stem growth by flocculation of the antibiotic-producingbacterium, P. f luores cens S272, on the roots of radish and Gomphrenaglobosa (Nakata e t a l . , 2000). Whether addition of a flocculant, such as,methylcellulose or alginate, to the bacterial suspension of our strains canenhance suppression needs to be investigated further.

Since the R. solanacearum strains isolated from wilted E. urophylla werenot pathogenic to A. thal iana (L. X. Ran, unpublished data), mechanismsinvolved in reduction of bacterial wilt could not be checked in Arabidopsis .In preliminary tests, the pathogen was able to infect tobacco and tomato plants(L. X. Ran, unpublished data), and suitable mutants to study the involvementof different signaling pathways are available in both plants. Thus, furtherinvestigations can be carried out in tobacco or tomato to clarify if SA couldbe responsible for disease reduction by WCS374r in E. urophylla.

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Summary

Bacterial wilt caused by Rals tonia so lanacearum in Eucalyptus hasbecome a serious problem in clonal plantations in south China since the 1980s.Currently, in short-rotation clonal plantations, fast-growing species or cloneswith high quality for paper pulping are widely cultivated. These include E.grandis, E. urophyl la , and their hybrids, E. grandis x urophyl la and E.urophyl la x grandis . Most of those are highly susceptible to bacterial wilt.Moreover, some resistant clones obtained by selection and breeding lost theirresistance when grown in a different climate, or the resistance decreased whenclones were propagated consecutively by tissue culture or cuttage for morethan three years. No effective control measures against bacterial wilt areavailable.

With the financial support of the Netherlands Foundation for theAdvancement of Tropical Research (WOTRO) and the Natural ScienceFoundation of China, research was initiated to suppress bacterial wilt ineucalypt by fluorescent Pseudomonas spp. strains. These non-pathogenicrhizobacteria are known to suppress soilborne diseases in differentpathosystems, and some of them are effective against fungal, bacterial and viraldiseases by known mechanisms, such as competition for nutrients (especiallyfor iron), antibiosis, or induction of systemic resistance (ISR).The prerequisitesgenerally considered for successful suppression of soilborne diseases byrhizobacteria are: active and long-term colonization of the root surface of theplant, and effective expression of disease-suppressive mechanisms.

In this work, biological control was studied in two plant-pathogen systemsusing several well-defined Pseudomonas strains.The abilities of strains P. putidaWCS358r, P. fluorescens WCS374r,WCS417r and CHA0r, and P. aeruginosa7NSK2 to suppress bacterial wilt in eucalypt and bacterial speck inArabidops i s , caused by Pseudomonas syr ingae pv. tomato (Pst ), wereinvestigated.The strains were first tested for their abilities to suppress bacterialwilt in eucalypt and the possible involvement of ISR in disease suppression

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was investigated.The same rhizobacterial strains were studied further in themodel pathosystem Arabidops i s-Pst , to investigate in how far bacterially-produced salicylic acid (SA) plays a role in biocontrol.

To assess antagonistic interactions between the rhizobacterial strains andR. solanacearum , in vitro interactions between these bacteria were analysed.WCS358r,WCS374r,WCS417r and 7NSK2 antagonized R. solanacearum invi t ro by competition for iron, whereas growth inhibition by P. f luores censCHA0r was antibiosis-based. Notably, a pseudobactin siderophore-minusmutant of WCS374 inhibited growth of R. solanacearum under iron-limitedconditions, suggesting that another siderophore, pseudomonine, can also inhibitgrowth of the pathogen.

None of the strains suppressed disease when mixed together with thepathogen into the soil, or when seeds or seedlings were treated with the strainsbefore transfer of seedlings into soil infested with R. solanacearum. However,when seedlings were dipped with their roots in a bacterial suspension beforetransplanting into infested soil,WCS417r suppressed bacterial wilt by 30-45 %.P. putida strain WCS358r was marginally effective, whereas its pseudobactin-minus mutant had no effect at all, indicating that siderophore-mediatedcompetition for iron can contribute, but is not effective enough to suppressbacterial wilt in Eucalyptus . A derivative of WCS358r that constitutivelyproduces 2,4-diacetylphloroglucinol (WCS358::phl) reduced disease. However,the combination of WCS417r and WCS358::phl did not improve suppressionof bacterial wilt over the effects of each strain alone (chapter 2), suggestingthat the strains interfere with each other in the rhizosphere. No correlationswere found between the antagonistic abilities of the Pseudomonas spp. in vitroand biocontrol effects against bacterial wilt in Eucalyptus in vivo.

To study the possible involvement of ISR in control of bacterial wilt, abioassay was developed in which the rhizobacteria and the pathogen were keptspatially separated. Because exogenously applied SA induces systemicresistance in many plant species, SA was also tested in Eucalyptus .Whereas asoil drench with SA did induce systemic resistance in E. urophylla , injectionof SA into the leaves did not trigger systemic resistance. Several bacterial strainscapable of producing SA in v i t ro , i.e. WCS374r, WCS417r, CHA0r and7NSK2, as well as a non-SA-producing strain,WCS358r, were tested. Noneof the rhizobacterial strains could induce ISR against bacterial wilt whenapplied to the soil, but two strains, WCS374r and WCS358r, triggered ISRagainst R. solanacearum in the upper part of the stem when they were injectedinto lower leaves before challenge inoculation. A mutant of strain WCS358rdefective in biosynthesis of pseudobactin 358, the fluorescent siderophore ofWCS358, was not able to induce ISR when injected into the leaves, suggestingthat pseudobactin 358 is the ISR-inducing determinant of WCS358 (chapter

3). The ISR-inducing determinant(s) of WCS374r in this system are as yetunknown. SA production does not seem to be involved, because a siderophore-minus mutant of WCS358 transformed with the SA biosynthetic gene clusterfrom WCS374, as well as several other SA-producing strains, did not inducesystemic resistance.

In how far bacterially-produced SA plays a role in the induction ofsystemic resistance by rhizobacteria was investigated in the model systemArabidops i s-Pst . The same strains as those used in the Eucalyptus-R.solanacearum system were tested. Strains WCS374r, WCS417r, CHA0r and7NSK2, grown in standard succinate medium (SSM), all produced SA in vitro,varying from 5 fg.cell-1 for WCS417r to over 25 fg.cell-1 for WCS374r.Addition of 200 µM FeCl3 to SSM abolished SA production in all strains.Whereas the incubation temperature did not affect SA production in WCS417rand 7NSK2, strains WCS374r and CHA0r produced more SA when grown at33 ˚C than at 28 ˚C.WCS417r, CHA0r and 7NSK2 induced systemic resistancein Arabidops i s apparently associated with their ability to produce SA, butWCS374r did not. Conversely, a mutant of 7NSK2, unable to produce SA,still induced systemic resistance. The possible involvement of SA in theinduction of resistance was evaluated using SA-non-accumulating transgenicNahG plants. Strains WCS417r, CHA0r and 7NSK2 induced resistance inNahG Arabidopsis . Also WCS374r, when grown at 33 ˚C or 36 ˚C, inducedISR in these plants, but not in ethylene-insensitive e in2 or in non-PR-expressing npr1 mutant plants, irrespective of the growth temperature of thebacteria.These results demonstrate that in Arabidopsis SA is not the primarydeterminant of the bacterial strains in the induction of systemic resistance(chapter 4).

We also studied the effects of phenolic acids on root colonization andISR. Mutants of WCS358 unable to catabolize specific phenolic acids wereused to investigate their abilities to colonize the roots and to induce systemicresistance in Arabidopsis. In a gnotobiotic system, Arabidopsis roots exuded acomplex mixture of phenolic compounds, among which small amounts of p-coumaric, p-hydroxybenzoic, protocatechuic, and vanillic acid were identified.No differences were observed between population densities of the wild-typestrain and its mutants in the nutrient solution of the gnotobiotic system,indicating that these phenolic acids were not a limiting factor for growth ofthe bacteria under these conditions. However, in potting soil bioassays, mutantsFAI1, FAI15 and VBHB, impaired in the utilization of coumaric, vanillic, andhydroxybenzoic acid, respectively, colonized Arabidopsis roots less well thanthe parental strain. Nevertheless, their ability to induce systemic resistanceagainst Pst was unimpaired (chapter 5).

The roles of bacterial determinants in suppression of plant diseases seem

S u m m a r y • 1 2 5

species-specific. The pseudobactin siderophore of WCS358 can suppressbacterial wilt in Eucalyptus through both competition and induction ofsystemic resistance. For none of the strains bacterially-produced SA appearsto be involved in the induction of systemic resistance in either the E.urophyl la-R. solanacearum or the Arabidopsis-Pst system. Determinants ofWCS374 involved in ISR in Eucalyptus need further investigation. Productionof 2,4-diacetylphloroglucinol by CHA0r and a derivative of WCS358,WCS358::phl, consistently inhibited growth of R. solanacearum in vi t ro .However, only WCS358::phl reduced disease severity of bacterial wilt in vivo.Whether the antibiotic was actually produced by these strains in therhizosphere of Eucalyptus also needs to be investigated further.

The observed suppression of bacterial wilt by both direct interactionsbetween the rhizobacterium and the pathogen and by ISR in the plant offersopportunities for the development of effective control of the disease. Thiswarrants further research on the modes of action involved.

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S a m e n v a t t i n g • 1 2 7

Samenvatt ing

Bacteriële verwelkingziekte in Eucalyptus , veroorzaakt door Ralstoniasolanacearum, is sinds de 80-er jaren een ernstig probleem in plantages in ZuidChina. Momenteel worden op deze plantages vrijwel uitsluitend snel groeiendesoorten en klonen met een hoge kwaliteit voor de productie van papiergeteeld.Tot deze soorten behoren E. grandis , E. urophylla , en hun hybridenE. grandis x urophyl la en E. urophyl la x grandis , waarvan de meeste zeervatbaar zijn voor bacteriële verwelking. Bovendien verliezen sommigeresistente klonen hun resistentie bij teelt onder andere klimatologischeomstandigheden of neemt de resistentie af bij het gedurende meer dan driejaar vermeerderen van klonen door middel van weefselkweek of stekken. Erzijn geen effectieve beheersmaatregelen beschikbaar tegen bacteriëleverwelking.

Met financiële steun van de Stichting voor Wetenschappelijk Onderzoekvan de Tropen (WOTRO) en de Natural Science Foundation van China werdeen onderzoek geïnitieerd naar onderdrukking van bacteriële verwelking inEucalyptus door stammen van fluorescerende Pseudomonas spp. Deze nietpathogene, wortelbewonende bacteriën kunnen diverse bodemgebondenziekten onderdrukken. Sommige Pseudomonas stammen zijn effectief tegendoor schimmels, bacteriën en virussen veroorzaakte ziekten, waarbijverschillende mechanismen, zoals concurrentie om nutriënten (met nameijzer), antibiose, of inductie van systemische resistentie (ISR) een rol spelen.Algemeen beschouwde voorwaarden voor succesvolle onderdrukking vanbodemgebonden ziekten door wortelbewonende bacteriën zijn: actieve enlangdurige kolonisatie van het worteloppervlak van de plant en effectieve expressie van ziekteonderdrukkende mechanismen.

In dit onderzoek werd biologische bescherming door verschillende, goedgedefinieerde Pseudomonas stammen in twee plant - pathogeen systemenbestudeerd. Het vermogen van de stammen P. putida WCS358r, P. fluorescensWCS374r, WCS417r en CHA0r en P. aeruginosa 7NSK2 om bacteriële

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verwelking in Eucalyptus en bacteriële bladvlekkenziekte in Arabidops i sthaliana, veroorzaakt door P. syr ingae pv. tomato (Pst), te onderdrukken werdonderzocht. De bacteriestammen werden eerst getest op hun vermogenbacteriële verwelking in Eucalyptus te onderdrukken. Nagegaan werd of ISRhierbij een rol speelt. Dezelfde stammen werden verder in het modelsysteemArabidopsis –Pst bestudeerd om te onderzoeken in hoeverre salicylzuur (SA)een rol speelt bij ziekteonderdrukking.

Antagonistische interacties tussen de wortelbewonende pseudomonadenen R. solanacearum werden in v i t ro geanalyseerd. De stammen WCS358r,WCS374r,WCS417r en 7NSK2 antagoneren R. solanacearum door middelvan concurrentie om ijzer, terwijl groeiremming door stam CHA0r gebaseerdis op antibiose. Een mutant van WCS374r die gestoord is in de productie van de ijzerbindende siderofoor pseudobactine, remde groei van R. solanacearumnog steeds onder ijzerarme omstandigheden. Dit suggereert dat een tweedesiderofoor van WCS374r, pseudomonine, ook in staat is groei van hetpathogeen te remmen.

Geen van de geteste stammen was in staat ziekte te onderdrukken wanneerze samen met het pathogeen door de grond werden gemengd, of wanneerzaden of zaailingen met de bacteriën werden behandeld voordat de zaailingenwerden overgeplant in grond geinoculeerd met R. solanacearum .Onderdrukking van de ziekte werd wel waargenomen bij behandeling van dewortels van zaailingen door dompelen in een bacteriesuspensie vóór hetoverplanten van de zaailingen in met het pathogeen besmette grond. Onderdeze omstandigheden onderdrukte WCS417r bacteriële verwelking met 30 –45 %. Behandeling van wortels met P. putida stam WCS358r verminderde deziekte in lichte mate, terwijl een pseudobactine mutant van deze stam geenenkel effect had. Dit suggereert dat concurrentie om ijzer door middel vansideroforen een bijdrage levert aan, maar niet voldoende is voor effectieveonderdrukking van bacteriële verwelking in Eucalyptus . Een genetischgemodificeerd derivaat van WCS358r dat in staat is constitutief 2,4-diacetylphloroglucinol te produceren (WCS358::phl) onderdrukte de ziektesignificant. Een combinatie van WCS417r en WCS358::phl verbeterde deziekteonderdrukking echter niet in vergelijking met de effecten van de enkelestammen (hoofdstuk 2). Dit suggereert dat de stammen met elkaar interfererenin het wortelmilieu van de plant. Er werd geen correlatie gevonden tussen hetvermogen van de Pseudomonas spp. stammen om in v i t ro groei van hetpathogeen te remmen en in vivo bacteriële verwelking te onderdrukken.

Om de mogelijke betrokkenheid van ISR bij de onderdrukking vanbacteriële verwelking te bestuderen, werd een biotoets ontwikkeld waarin de Pseudomonas bacteriën en het pathogeen ruimtelijk gescheiden werdengehouden. Aangezien het toedienen van exogeen SA systemische resistentie

kan induceren in veel plantensoorten, werd ook SA getest in Eucalyptus .Toediening van SA aan grond induceerde ISR in E. urophylla, maar infiltratievan bladeren met een SA bevattende oplossing niet. Pseudomonas spp. stammenWCS374r,WCS417r, CHA0r en 7NSK2, die in vitro SA kunnen produceren,alsmede de niet SA producerende stam WCS358r, werden getest op hunvermogen ISR te induceren in Eucalyptus . Geen van deze stammen was instaat ISR te induceren indien ze waren toegediend aan de grond. Tweestammen, WCS358r en WCS374r, induceerden ISR tegen R. solanacearumwanneer ze werden geinfiltreerd in bladeren onderaan de stengel. Eenpseudobactine mutant van WCS358r was niet in staat ISR te induceren. Ditsuggereert dat pseudobactine de ISR inducerende determinant van WCS358ris in dit systeem (hoofdstuk 3). De ISR inducerende determinant(en) vanWCS374r in dit systeem zijn vooralsnog onbekend. Productie van SA lijktgeen rol te spelen, aangezien een pseudobactine mutant van WCS358rgetransformeerd met het gencluster uit WCS374r dat synthese van SAbewerkstelligt, alsmede andere SA producerende stammen, niet in staat blekenresistentie te induceren.

In hoeverre door de bacterie geproduceerd SA een rol speelt bij inductievan resistentie door de Pseudomonas spp. stammen werd onderzocht in hetmodelsysteem Arabidopsis-Pst . In dit systeem werden dezelfde stammen diegebruikt waren in het Eucalyptus-R. solanacearum model, verder onderzocht.De stammen WCS374r, WCS417r, CHA0r en 7NSK2 produceerden SA instandaard succinaat medium (SSM) variërend van 5 fg/cel voor WCS417r totmeer dan 25 fg/cel voor WCS374r. Het toevoegen van 200 µM FeCl3 aan SSMonderdrukte in alle stammen de productie van SA volledig. Deincubatietemperatuur had geen effect op de SA productie door WCS417r en7NSK2. De stammen WCS374r en CHA0r produceerden meer SA wanneerze bij 33 ˚C in plaats van bij 28 ˚C werden gekweekt. De door WCS417r,CHA0r en 7NSK2 geïnduceerde resistentie in Arabidopsis leek geassocieerdmet hun vermogen SA te produceren, terwijl dat voor WCS374r niet het gevalwas. Echter, een mutant van 7NSK2 die geen SA meer kan produceren, wasnog steeds in staat ISR te induceren. De mogelijke betrokkenheid van SA bijISR werd verder onderzocht door gebruik te maken van transgene NahGplanten, die niet in staat zijn SA te accumuleren. In deze NahG Arabidopsisplanten induceerden WCS417r, CHA0r en 7NSK2 resistentie. Ook WCS374r,indien opgekweekt bij 33 ˚C of 36 ˚C, induceerde resistentie in NahG planten,maar niet in ethyleen-ongevoelige ein2 of de in de expressie van PR-eiwittengestoorde npr1 mutanten van Arabidopsis . Deze resultaten tonen aan dat SAniet de primaire determinant is van de inductie van ISR door dezebacteriestammen in Arabidopsis (hoofdstuk 4).

De invloed van fenolische zuren op wortelkolonisatie en inductie van

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ISR werd eveneens onderzocht. Mutanten van WCS358r die niet in staat zijnspecifieke fenolische zuren als substraat te gebruiken, werden met de ouderstamvergeleken met betrekking tot hun vermogen wortels te koloniseren enresistentie te induceren in Arabidopsis. In een gnotobiotisch systeem scheiddenwortels van Arabidops i s een complex mengsel van fenolische verbindingenuit. In dit mengsel werden kleine hoeveelheden p-coumaar-, p-hydroxybenzoë-, protecathechu- en vanillinezuur geïdentificeerd. Populatie-dichtheden van de ouderstam en de mutanten in de voedingsoplossing van hetgnotobiotische systeem waren vergelijkbaar. Dit geeft aan dat onder dezeomstandigheden fenolische zuren geen beperkende factor zijn voor groei vande bacteriën. Echter, in een potgrond systeem koloniseerden de mutantenFAI1, FAI15 en VBHB, die gestoord zijn in het gebruik van respectievelijk p-coumaar-, vanilline- en p-hydroxybenzoëzuur, de wortels van Arabidops i sslechter dan de ouderstam. Ondanks de verminderde kolonisatie was hetvermogen van de mutanten om resistentie te induceren tegen Pst onaangetast(hoofdstuk 5).

De rol van bacteriële determinanten bij de onderdrukking vanplantenziekten lijkt afhankelijk te zijn van de plantensoort. Het pseudobactinesiderofoor van WCS358r onderdrukt bacteriële verwelking in Eucalyptuszowel door concurrentie om ijzer als door inductie van ISR.Voor geen vande Pseudomonas stammen lijkt bacterieel geproduceerd SA een rol te spelenbij de inductie van ISR, noch in het E. urophylla-R. solanacearum , noch inhet Arabidops i s-Pst systeem. Determinanten van stam WCS374r, diebetrokken zijn bij inductie van resistentie in Eucalyptus , vergen naderonderzoek. Productie van 2,4-diacetylphloroglucinol door CHA0r en eenderivaat van WCS358r,WCS358::phl, remde de groei van R. solanacearum invi t ro sterk en consistent. Echter, alleen stam WCS358::phl onderdruktebacteriële verwelking in v ivo . De vraag of de stammen het antibioticumdaadwerkelijk produceren in het wortelmilieu van Eucalyptus dient naderonderzocht te worden.

De in dit onderzoek waargenomen onderdrukking van bacteriëleverwelking door zowel directe interacties tussen de Pseudomonas bacteriënen het pathogeen als door ISR, opent mogelijkheden voor effectievebeheersing van de ziekte. Nader onderzoek naar de betrokken mechanismenis hiervoor onmisbaar.

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A c k n o w l e d g e m e n t s • 1 3 5

Acknowledgements

At the moment of the finalization of this thesis, I would like to give myheartfelt thanks to the people who provided me with their help during thepast five years.

Firstly, grateful thanks are due to my promotor, Kees van Loon. From myliving in Utrecht to scientific research in the Phytopathology lab of UtrechtUniversity, and the setup of chapters to the correction of them, you paidpainstaking care. Five years ago, when I came to the Netherlands for the firsttime, it is you who picked me up from the Airport, and took care of all thespecific details for me. Despite the fact that you are always busy with the affairsof the whole group, you and your wife Ada tried to make use of all thepossibilities to let me feel at home. I still remember a party at the eve of"Sinterklaas" organized by Ada, a happy night we spent together and watchthe exciting program of China Acrobatic Group, the first aid food you boughtfor me upon my arrival in Holland at the second time, and the "lekker"traditional Dutch dishes prepared by Ada. For helping me to accomplish theWOTRO project, you paid a visit to China and guided me through thedifficulties I encountered. For this thesis, you made your largest efforts forcorrections from the structure of a chapter to the use of a word. I cannot sayI have learnt all what you have taught me, but as your student I hope that Ican still receive your guidance in the future. It would be impossible to list allthe details here, just let them be in my mind deeply.

Secondly, I would like to express my gratitude to my co-promotor, PeterBakker, for his initiation of the project, the theoretical and practical guidance,and for his cares of my life both in and outside Holland. I will never forgetyour every invitation of me to your cosy house, where I felt completelyrelaxed. I still clearly remember the scene in which I talked with your kindmother and took photos with your two lovely nieces. I also understood thatyou had tried your best to prepare the Indonesian food for me, but I shouldsay that you can cook more delicious typical Chinese food now. We had so

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much fun in Holland, China and Argentina.The "water-proof watch", "froggysky", and the story of suntan lotion, etc., will never fade away.The nice melody,"een, nul, nul; nul, een, nul;......" when we scored the disease symptom ofArabidops i s together in the greenhouse, will never be boring. At the sametime, I wish to express my great thanks to your faithful wife, Boet. All thefunny stories I shared with Peter are also shared with you. In Amsterdam,Floriade, Beijing, and Zhuzhou, everywhere we went to, joy and delight alwaysaccompany us.

I shall always remember with gratitude my supervisor in China, Prof.WuGuangjin, for his basic work on the epidemiology of eucalypt bacterial wilt,and Prof. Lin Xuejian for her encouragement to finish this work.

Bob Schippers, please accept my profound appreciation for your help. Itis you who transferred my first letter to Kees van Loon, then I got a chanceto study in Utrecht University. Thank you from the bottom of my heart,Anneke Schippers, for your nice invitations to your house and a boat trip inAmsterdam. Most impressively, I wish I could sit beside your fireplace, smellthe light smoke and enjoy the delicious cakes you prepared yourself.

Corné Pieterse, you are thanked for providing me practical help, especiallyfor your telling me the truth of fricandel. I am also grateful to your wife,Lisette, for her kind entertaining and hospitality in your house. I am sincerelywishing your two lovely daughters all the best.

Grateful thanks to my friends, Ientse van der Sluis and Hans van Pelt, foryour help in the lab and greenhouse. It is you who told me how to choosethe right Dutch cheese. Thanks are sent to Bas Valstar and Fred Siesling forautoclaving potting soil for me; to John Nieuwenhuis for your joyfulmerriment.

My gratitudes are sincerely presented to my roommates, Bart Geraats, BasVerhagen, and Martin de Vos, for creating a unusual quiet atmosphere to letme concentrate on writing this thesis, and helping me to deal with all theletters in Dutch I received; Especially to Bart and Saskia for introducing methe delicious candy, "Mars" and other food; to Bas and Madelinde for providingme a chance to taste the typical Dutch cake; to Martin de Vos and Inge forgiving me an opportunity to play a joyful soccer game with the melody "wijare the suckers"; to Mareike Viebahn and James ter Beek for your kind mannerand hospitality, and to Vivian van Oosten for showing your dear "worm"around and teaching me how to serve the nice food you prepared. At the sametime, thanks to Bart and Mareike for your great job to be my paranimfen.

Thanks are also given to former Ph.D students in Section Phytopathology,Marjan de Boer for your composition of a lyric for a farewell party on theoccasion of my return to China in 1998, Saskia van Wees for your joyful BeijingOpera-like shout, and Jurriaan Ton for your useful advice at the last stage of

writing the thesis. I also thank the students, Leon Commandeur and Ronaldvan Doorn for providing me useful information.

I would like to express my sincere thanks to the administrative staff inboth the Netherlands and China who helped me in various ways. They areBertus Ebbenhorst, Bor Duplica, Hans de Nooijer, Helene Eijkelenboom,Henny Blom, Maartje van Stiphout, Margriet Dekker, Natasja Miller, Petravan den Beemt, René Kwant, and Theo Mastwijk at the Faculty of Biology,Utrecht University; and Chen Haili, Gu Wenzhong, Han Xuezhong, He Lixin,He Naxin, Li Jiping, Li Zhihui, Li Xiangdong, Lin Qinzhong, Liu Junang, LiuYouquan, Song Xiaojing, Shu Guizhen, Tan Xiaofeng, Tang Daisheng, WeiMeicai, Wen Shizhi, Wu Xiaofu, Xiao Guowang,Yu Feng, Zhang Huaiyun,Zhang Yungang, Zhao Kun, Zhao Zechang, Zhou Guoying at Central SouthForestry College.

Special thanks to Femke Bulten for her designing the cover and layout ofthe thesis; to Maaike van Kilsdonk for her advice about how to transfer theword file to Macintosh system; Henri Groeneveld for his guidance on thetechniques of gas chromatography; Gerard Niemann, Piet Wolswinkel andMaria Vliegen for your hospitality; Feng Jianjun and Sheng Jiafen,Tang Shiyao,Xie Yaojian, Xian Shenghua for their help to collect diseased Eucalyptusurophylla infected by Ralstonia solanacearum and seeds of E. urophylla.

I gratefully acknowledge the Netherlands Foundation for theAdvancement of Tropical Research (WOTRO) and the Natural ScienceFoundation of China, for their financial support for the research.

Thanks are also presented to several of my students in China, Dai Xiaoli,Liang Jiecheng, Liu Chaoyang, Xiang Miaolian, Zeng lingcai for their takingcare of the seedlings of E. urophylla and preparation of potting soil.

The Chinese friends are thanked for making my stay in Utrecht mucheasier.They are Huang Yunxin, Ji Yaying, Li Guangfen, Li Rui, Li Yunyan, SunPengyue,Tian Yixin,Wang Peilian,Yuan Zhaorui, Zhang Kecheng, Zhang Yihuaand Zhao Chunyan.

From the deep bottom of my heart, I am grateful to my wife, Li Zhengnanand daughter, Ran Xin for their understanding, support and love, and for allthe consistent help from my parents, parents-in-law, my brother and sister-in-law, Ma Xiaoheng and Li Yan.

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C u r r i c u l u m v i t a e • 1 3 9

Cur r i cu lum v i tae

Longxian Ran was born on 21 November 1962 in Sinan, GuizhouProvince, P. R. China. In July 1981 he graduated from Sinan senior high school.Since September 1981, he studied in the Department of Plant Protection inSouth-West Agricultural University, Chongqing, Sichuan Province, andreceived his Bachelor's degree of Agriculture in 1985. From July 1985 toNovember 1988 he worked as a librarian in Central South Forestry College,Zhuzhou. From December 1988 onward, he moved to the Faculty ofEnvironment and Resource and worked as a teacher of Forest Pathology inthe same College. From September 1993 to June 1996 he worked on a keyresearch project titled "Control of bamboo moulds" financed by the Scientificand Technological Committee of Hunan, and received his Master's degree onForest Protection under the guidance of Prof.Wu Guangjin and Lin Xuejian.From October 1997 to September 1998, he worked in the SectionPhytopathology, Utrecht University as a guest researcher with financial supportfrom the Netherlands organization for international cooperation in highereducation (NUFFIC) and the China Educational Committee. In June 1998,he enrolled as a PhD student in Utrecht University under the guidance ofProf. dr. L. C. van Loon, Dr. P. A. H. M. Bakker and Prof.Wu Guangjin, withsupport of the Netherlands Foundation for the Advancement of TropicalResearch (WOTRO) and the Natural Science Foundation of China. He willbe back to the Faculty of Environment and Resource, Central South ForestryCollege in Zhuzhou, Hunan, and work as an associate professor.

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L i s t o f p u b l i c a t i o n • 1 4 11 4 1

L i s t o f pub l i ca t ion

Ran, L. X. 1992. Cultivation of Flammulina ve lut ipes using rice straw and garden soil.

Edible Fungi 14: 25-26.

Wu, G. J., Lin, X. J., and Ran, L. X. 1992. Control of rust disease in fishscale bamboo.

Economic Forest Researches 10: 63-67.

Wu, G. J., Lin, X. J., and Ran, L. X. 1994. Identification of bamboo moulds and

formulation of anti-mould agents. Economic Forest Researches 12: 50-55.

Ran, L. X. 1996. Advances in bamboo mould control. Economic Forest Researches 14: 56-58.

Ran, L. X., Wu, G. J., and Lin, X. J. 1997. Physiological characteristics of bamboo

moulds and mould control. Journal of Central South Forestry University 17: 14-19.

Ran, L. X., Lin, X. J., Wen, S. Z., and Xie, Y. R. 1998. Studies of tissue culture in

Arundo donax. Journal of Central South Forestry University 18: 49-53.

Ran, L. X., Wen, S. Z., and Xie, Y. R. 1998. Fast propagation in Arundo donax .

Economic Forest Researches 16: 13-16.

Ran, L. X., Wu, G. J., and Lin, X. J. 1998. Identification and control of soft rot in oil

Camellia. China Forest Science and Technology 1: 37-39.

Ran, L., Van Loon, L. C., and Bakker, P. A. H. M. 2000. Effects of temperature and

iron availability on induction of systemic resistance by salicylic acid producing fluorescent

Pseudomonas bacteria. Auburn University website, available:

http://www.ag.auburn.edu/argentina/pdfmanuscripts/ran.pdf [Accessed, 08/01/2002]

Ran, L., Liu, C., Wu, G., Van Loon, L. C. en Bakker, P. A. H. M. 2002.

Onderdrukking van Ralstonia solanacearum op Eucalyptus urophylla door fluorescerende

Pseudomonas spp. in China. Gewasbescherming 33: 23.

Bakker, P. A. H. M., Mercado-Blanco, J., Ran, L., Van der Sluis, I., and Van

Loon, L. C. 2002. Production of salicylic acid and pseudomonine and suppression of

disease by Pseudomonas f luorescens WCS374. In: Influence of a-biotic and biotic factors

on biocontrol agents (Y. Elad, J. Köhl and D. Shtienberg, eds). IOBC/WPRS Bulletin. In

press.

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