The systemic activity of mutualistic endophytic fungi in...

Institut für Nutzpflanzenwissenschaften und Ressourcenshutz

der

Rheinischen Friedrich-Wilhelms-Universität Bonn

___________________________________________________________________________

The systemic activity of mutualistic endophytic fungi in Solanaceae and Cucurbitaceae plants on the behaviour of the phloem-feeding insects Trialeurodes

vaporariorum, Aphis gossypii and Myzus persicae

Inaugural – Dissertation

Zur

Erlangung des Grades

Doktor der Agrarwissenschaften

(Dr. agr.)

der Hohen Landwirtschaftlichen Fakultät

der

Rheinischen Friedrich-Wilhelms-Universität

zu Bonn

vorgelegt am 13. 04. 2010

von

Roy Donald Menjivar Barahona

aus

Tegucigalpa, Honduras

Referent: Professor Dr. Richard A. Sikora Korreferent: Professor Dr. Heiner E. Goldbach Tag der mündlichen Prüfung: 04 Juni 2010

Dedicated to my family that has always been my main support

The systemic activity of mutualistic endophytic fungi in Solanaceae and Cucurbitaceae plants on the behaviour of the phloem-feeding insects

Trialeurodes vaporariorum, Aphis gossypii and Myzus persicae

The biocontrol activity of mutualistic endophytic fungi inoculated into the rhizosphere on the root-knot nematode Meloidogyne incognita and the phloem-feeding insects: Trialeurodes vaporariorum (Westwood) the green house whitefly (GHWF), Aphis gossypii (Glover) the melon aphid, and Myzus persicae (Sulzer) the green peach aphid were investigated on host plants in two plant families. Fusarium oxysporum strain 162 (Fo162) was successfully re-isolated from the roots of squash, melon and pepper, 27.8, 27.4 and 28.8 percent respectively, following endophyte pre-inoculation at sowing. The results showed for the first time that Fo162 was able to effectively colonize these three new host plants. Early root penetration of M. incognita was reduced 83, 70 and 73 percent in the three plants, respectively. In the absence of Fo162, the level of nematode galling was significantly higher in all crops compared to the rate of galling on endophyte inoculated plants. Organic matter amendments did not affect the biological control activity of Fo162. Strain Fo162 as well as five other endophytic isolates with known biological control activity toward plant-parasitic nematodes, were investigated for their potential to induce systemic effects against the GHWF on tomato. The endophytic isolates of Trichoderma atroviride MT-20 and S-2 as well as Fo162 added to the soil at sowing significantly reduced the number of GHWF on the leaves of tomato over the controls in a choice-test as measured over a 10 day period after insect release. The highest level of biocontrol activity was obtained with Fo162. The endophytic fungi did not alter chlorophyll content of the tomato leaves, which was thought to have influenced host selection behaviour. The choice-test also demonstrated that Fo162 negatively affected GHWF behaviour on tomato, squash, and melon at the time of host selection and oviposition. 79 and 74 percent of the adults and eggs detected on the tomato leaves were found on untreated plants, whereas on squash and melon plants the presence of Fo162 reduced host preference of the GHWF equally 80 percent. Significant negative effects of Fo162 on T. vaporariorum reproduction in tomato, A. gossypii in melon and squash as well as on M. persicae in pepper were also detected. Tomato plants treated with Fo162 had a reduced number of 2nd and 3rd nymphal stages and total eggs that were able to complete their life cycle. In squash there was a significant negative effect on the reproductive rate of the melon aphid and in Fo162 treated plants as compared to the control plants. Pepper plants inoculated with Fo162 also had a negative effect on the reproduction of the green peach aphid. Final populations only reached 15% on treated plants of that attained on the untreated plants. RP-HPLC analysis demonstrated that tomato, squash, melon and pepper plants colonized by Fo162 had altered concentrations of metabolite accumulation both in the presence and in the absence of the phloem-feeding insects. Metabolite accumulation in the presence of the phloem-feeding insect populations increased and was negatively correlated with different developmental stages of T. vaporariorum. The results of the present studies demonstrated that the mutualistic fungal endophytes are able to induce systematic resistance in host plants when applied to the soil at sowing. Endophytic root colonization enhanced systemically plant defence mechanisms simultaneously against the root-knot nematode M. incognita in the roots and toward phloem-feeding insects in the leaves.

Systemische Aktivität von mutualistisch endophytischen Pilzen in Solanaceaen und Cucurbitaceaen Pflanzen auf das Verhalten der Phloem-

ernährenden Insekten Trialeurodes vaporariorum, Aphis gossypii und Myzus persicae

Die biologische Kontrollaktivität von mutualistischen endophytischen Pilzen, inokuliert in die Rhizosphäre, gegen den Wurzelgallennematoden Meloidogyne incognita und die Phloem-ernährenden Insekten, Weiße Fliege (GHWG) Trialeurodes vaporariorum (Westwood), die Blattlaus Aphis gossypii (Glover) und die grüne Pfirsich Blattlaus Myzus persicae (Sulzer) wurde an Pflanzen aus zwei Familien untersucht. Fusarium oxysporum Stamm 162 (Fo162) wurde erfolgreich von Gurke, Melone und Paprika re-isoliert, mit Kolonisationsraten von jeweils 27,8%, 27,41% und 28,84%. Diese Ergebnisse zeigten zum ersten mal, dass Fo162 effektiv die Wurzeln von diesen Kulturpflanzen kolonisieren konnte. Die frühe Wurzeleindringung von M. incognita wurde im Vergleich zur Kontrollvariante jeweils in jeder Kulturpflanze um 83%, 70% und 73% reduziert. In Abwesenheit von Fo162 war die Gallenbildung durch die Nematoden signifikant höher als in inokulierten Pflanzen. Die Stärke der biologischen Kontrolle wurde nicht durch organische Bestandteile im Boden beeinflusst. Stamm Fo162 und fünf weitere endophytische Isolate mit bekannter biologischer Kontrollaktivität gegen pflanzenparasitäre Nematoden wurden auf die die Bildung einer systemischen Resistenz gegen GHWG an Tomaten getestet. Die endophytischen Isolate von Trichoderma atroviride MT-20 und S-2 als auch Fo162 inokuliert während der Saat, reduzierten die Anzahl von GHWG an den Pflanzen zehn Tage nach Freilassung signifikant. Das höchste Level der Kontrollaktivität wurde mit Fo162 erziehlt. Ein Einfluss der Endophythen auf den Chlorophyll Gehalt von Blättern der Tomate konnte nicht nachgewiesen werden. Fo162 hatte einen negativen Einfluss auf Auswahl und Eiablage der weißen Fliege an Tomate, Gurke und Melone. 79 und 74 Prozent der Adulten und Eier an Tomatenpflanzen wurden auf unbehandelten Pflanzen gefunden, wobei an Gurke und Melone die Präsenz von Fo162 die Wirtspräferenz um 80% senkte. Signifikante negative Effekte von Fo162 auf die Vermehrung der GHWG an Tomate, A. gossypii an Melone und Gurke, als auch von M. persicae an Paprika wurden nachgewiesen. An Tomatenpflanzen behandelt mit Fo162, wurde die Anzahl des zweiten und dritten Larvenstadiums und die Anzahl der Eier, welche den Lebenszyklus beendeten, reduziert. Die Kolonistation der Melonenblattlaus auf Fo162 inokulierten Gurkenpflanzen zeigte einen negativen Effekt im Vergleich zu den Kontrollpflanzen. An Paprika reduzierte Fo162 die Vermehrungsrate der grünen Pfirsischblattlaus um 85% im Vergleich zu den unbehandelten Pflanzen. RP-HPLC Analysen der mit Fo162 behandelten Tomaten, Gurken und Paprika Pflanzen ergaben eine Veränderung der Metabolitenproduktion in den Blättern sowohl wenn die Insekten präsent waren als auch ohne Präsenz der Insekten. Wenn die verschiedenen Insektenpopulationen vorhanden waren, war die Metabolitbildung ebenfalls erhöht und negativ korreliert mit T. vaporariorum Entwicklungsstadien. Die Ergebnisse der vorliegenden Studie demonstrierten, dass die mutualistischen Endophyten in der Lage waren eine induzierte Resitenz hervorzurufen wenn sie während der Saat inokuliert wurden. Wurzelbesiedlung durch Endophyten erhöhte die systemischen Pflanzen-Abwehrmechanismen, gleichzeitig gegen die Wurzelgallennematode M. incognita in den Wurzeln, als auch gegen Phloem saugende Insekten an den Blättern.

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CONTENTS GENERAL INTRODUCTION...................................................................................... 1

1. IMPORTANCE OF VEGETABLES ............................................................................. 1 1.1. Solanaceae crops .................................................................................... 2 1.2. Cucurbitaceae crops ................................................................................ 3

2. IMPORTANCE OF THE PLANT PARASITIC NEMATODES .............................................. 5 3. IMPORTANCE OF THE PHLOEM-FEEDING INSECTS ................................................... 6 4. MUTUALISTIC ENDOPHYTIC FUNGI AS BIOLOGICAL CONTROL AGENTS....................... 8 5. PLANT DEFENSE MECHANISMS ........................................................................... 11 6. SCOPE OF THE STUDY....................................................................................... 13

GENERAL MATERIALS AND METHODS............................................................... 14 1. FUNGAL ISOLATES ............................................................................................ 14

1.1. Origin of the mutualistic endophytic fungus ........................................... 14 1.2. Culture of the isolates ............................................................................ 14

2. NEMATODE INOCULUM ...................................................................................... 15 3. PHLOEM-FEEDING INSECT PRODUCTION.............................................................. 15

3.1. Trialeurodes vaporariorum (Westwood)................................................. 15 3.2. Aphis gossypii (Glover) and Myzus persicae (Sulzer)............................ 15

4. PLANT PRODUCTION ......................................................................................... 16 5. SOIL AND SUBSTRATE ....................................................................................... 16 6. HIGH PRESSURE LIQUID CHROMATOGRAPHY (PR-HPLC ANALYSIS) ...................... 17 7. CULTURE MEDIA AND REAGENTS ........................................................................ 18 8. STATISTICAL ANALYSIS...................................................................................... 18

BIOLOGICAL CONTROL OF MELOIDOGYNE INCOGNITA ON CUCURBITACEAE AND SOLANACEAE CROPS BY THE NON-PATHOGENIC ENDOPHYTIC FUNGUS FUSARIUM OXYSPORUM STRAIN 162 ................................................. 19

1. INTRODUCTION................................................................................................. 19 2. EXPERIMENTAL DESIGNS................................................................................... 21

2.1. Endophyte colonization.......................................................................... 21 2.2. Nematode biocontrol efficacy and growth promotion ............................. 22 2.3. Influence of organic matter on Fo162 biocontrol activity ........................ 22

3. RESULTS ......................................................................................................... 24 3.1. Endophyte colonization........................................................................ 24 3.2. Nematode biocontrol efficacy and growth promotion ............................. 25 3.3. Influence of organic matter on Fo162 biocontrol activity ........................ 26

4. DISCUSSION..................................................................................................... 28 4.1. Endophyte colonization.......................................................................... 28 4.2. Nematode biocontrol efficacy and growth promotion ............................. 28 4.3. Influence of organic matter on Fo162 biocontrol activity ........................ 29

5. CONCLUSION ................................................................................................... 31

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SYSTEMIC BIOLOGICAL CONTROL ACTIVITY OF MUTUALISTIC ENDOPHYTIC FUNGI TOWARD THE GREENHOUSE WHITEFLY TRIALEURODES VAPORARIORUM (WESTWOOD) IN TOMATO ..................................................... 32


2.1. Efficacy of endophytic fungi toward T. vaporariorum on tomato ............. 35 2.2. Metabolite production analysis............................................................... 37 2.3. Relationship between Fo162 and the maximum temperature difference (MTD) on tomato leaves .................................................................................... 37

3. RESULTS ......................................................................................................... 39 3.1. Efficacy of endophytic fungi toward T. vaporariorum on tomato ............ 39 3.2. Metabolite production analysis ............................................................... 43 3.3. Relationship between Fo162 and MTD.................................................. 44

4. DISCUSSION..................................................................................................... 46 4.1. Efficacy of endophytic fungi toward T. vaporariorum on tomato ............ 46 4.2. Metabolite production analysis............................................................... 47 4.3. Termographic measurements ................................................................ 48

5. CONCLUSION ................................................................................................... 49 SYSTEMIC RESISTANCE INDUCED BY THE MUTUALISTIC FUNGUS FUSARIUM OXYSPORUM STRAIN 162 ON TOMATO PLANTS TOWARD THE PHLOEM-FEEDING TRIALEURODES VAPORARIORUM ..................................................... 50


2.1. Host preference ...................................................................................... 53 2.2. Reproduction experiment ....................................................................... 53 2.3. Metabolite accumulation in tomato leaves .............................................. 53 2.4. Relationship between the accumulation of compounds in the leaf and T. vaporariorum development................................................................................ 54

3. RESULTS ......................................................................................................... 55 3.1. Host preference ..................................................................................... 55 3.2. Reproduction experiment ....................................................................... 56 3.3. Metabolite accumulation in tomato leaves .............................................. 57 3.4. Relationship between the accumulation of compounds in the leaf and T. vaporariorum development................................................................................ 59

4. DISCUSSION..................................................................................................... 62 4.1. Host preference ..................................................................................... 62 4.2. Reproduction experiment ....................................................................... 62 4.3. Metabolite accumulation and relationship in tomato leaves toward T. vaporariorum ..................................................................................................... 63

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MECHANISMS AND REACTION OF PHLOEM-FEEDING INSECTS TO INDUCED SYSTEMIC RESPONCES OF SOLANACEAE AND CUCURBITACEAE HOST PLANTS INCREASED BY THE MUTUALISTEIC ENDOPHYTE FUSARIUM OXYSPORUM STRAIN 162..................................................................................... 66

1. INTRODUCTION................................................................................................. 66 2. EXPERIMENTAL DESIGN..................................................................................... 69

2.1. Influence of ISR on host preference ...................................................... 69 2.2. Reproduction ......................................................................................... 69 2.3. Antibiosis test......................................................................................... 70 2.4. ISR effect on metabolites production in leaves....................................... 71

3. RESULTS ......................................................................................................... 73 3.1. Influence of ISR on host preference ...................................................... 73 3.2. Reproduction ......................................................................................... 75 3.3. Antibiosis test......................................................................................... 79 3.4. ISR effect on metabolites production in leaves ...................................... 81

4. DISCUSSION..................................................................................................... 86 4.1. Influence of ISR on host preference ...................................................... 86 4.2. Reproduction ......................................................................................... 86 4.3. Antibiosis test......................................................................................... 87 4.4. ISR effect on metabolites production in leaves ...................................... 88

5. CONCLUSION....................................................................................................... 90 REFERENCES ......................................................................................................... 92

Chapter 1 General Introduction

1

GENERAL INTRODUCTION

1. Importance of vegetables

The goals of agricultural production according to Goldman et al. (2009) have

traditionally been to try to accommodate needs for adequate and reliable yields to

provide a sufficient food supply in a growing world. Vegetables represent one of the

most important components in our daily diet and are rich in protein vitamins and

essential minerals. They provide fibre, contain little fat and are low in calories (Craig

and Beck, 1999; Wargovich, 2000; Sikora and Fernandez, 2005). Vegetables are a

high value cash crop for small-scale producers as well as an important to larger-scale

commercial farmers. The main producers are found in the tropical regions and

subtropical regions of the world. The biggest producers, in order of importance are

found in Asia, Africa, South America and Central America. Production is on the

increase and yield levels improved because of important advances in production and

processing technology. Modern breeding methods supported by new molecular

techniques have been developed that are making major improvements in all kindes

of vegetables and that shorten the development time for new cultivars (Sikora and

Fernandez, 2005). The production level of various vegetable crops in these tropical

regions is shown in table 1.

Table1. Area in 1000 ha, yields in metric tonnes and total production in 1000 metric tons for select vegetables in regions with tropical and sub tropical climates. a

Africa C. America S. America Asia Vegetable Area Yield Product. Area Yield Product. Area Yield Product. Area Yield Product.

Cabbages 86 17 1,485 22 14 327 59 9 498 2,260 20 44,909 Lettuce 15 20 299 13 21 271 15 13 193 587 19 11,144 Tomato 609 20 12,452 82 28 2,336 149 44 6,628 2,323 25 57,330 Cauliflower 13 20 241 22 12 253 5 16 75 632 19 12,117 Squash 227 8 1,788 39 12 473 5 137 7 858 18 26,469 Cucumbers and gherkin

4 16 72 1,729 18 3,167 94 13 717 857 14 11,557

Aubergines 46 19 940 3 24 60 1 19 9 1,506 17 26,000 Spinach 4 17 58 2 11 20 1 17 13 702 14 9,869 Chillie and pepper

268 8 1,989 146 13 1,814 29 14 397 970 15 14,059

Green onion and shallots

38 13 466 45 25 1,131 22 5 113 106 20 2,126

Dry onion 281 14 4,012 19 14 260 160 21 3,416 1,971 17 32,575 Garlic 32 12 367 6 8 47 45 8 346 902 12 10,722 Carrots 74 13 952 18 25 432 46 21 935 507 19 9,749 Maizes 375 4 1,413 19 10 186 86 8 704 133 6 790 a From Sikora and Fernandez (2005) FOASTAT databases at: appjs.fao.org/faostat


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1.1. Solanaceae crops

The Solanaceae represent the third most economically important plant taxon, and the

most valuable in terms of vegetable crops with agricultural utility (Mueller et al.,

2005), represented for more than 3000 species, including the tuber-bearing potato, a

number of fruit-bearing vegetables (tomato, eggplant, peppers), ornamental plants

(petunias, Nicotiana), plants with edible leaves and medicinal plants (Knaps, 2002)

Tomato (Lycopersicon esculentum Mill.) probably the most important vegetable

produced worldwide and it has increased greatly in popularly over the last century. It

is grown in practically every country of the world and is one of the most widely

produced and consumed fruits for human consumtion worldwide (Giaconi and Escaff

1995; Wener, 2000). The global production of tomatoes, both fresh and processed,

has increased by about 300% in the last four decades. The annual worldwide

production of tomatoes in 2004 has been estimated at 100 million tons fresh fruit

produced on 3.7 million hectares (FAOSTAT 2004). China, the United States,

Turkey, Italy, and India are the countries that produce the most tomato worldwide.

Pepper (Capsicum annuum L.) originated from the tropical Americas. Fresh pepper

is important because it also has high vitamin C content (Vanderslice et al., 1990).

Peppers have also increase in popularity recently due to the availability of a wide

number of new varieties for human consumption (Marín et al., 2004). The importance

of the carotenoid compounds contained in pepper has been recognized, not only as

precursors of vitamin A but also as antioxidants for cell protection and in the

prevention of degenerative diseases (Stahl and Sies, 2003). Peppers are also rich in

vitamins such as ascorbic acid, which is found in quantities even higher than in

lemon. The typical hot taste of some varieties is due to the alkaloid capsaicin, which

is also used in medicine for its hyperaemia promoting ability. The worldwide

production of chillies and peppers increased with the factor 2.6 within the last 20

years to about 26 million tons in 2007 (FAOSTAT, 2007). The countries with the

highest level of production are China, Mexico, United States, Turkey and Spain.

Pests of Solanaceae crops

Cultivated tomato is a natural host to over 100 arthropod herbivores that feed on

roots, leaves or fruits (Lange and Bronson, 1981). Included among the major pests of


3

tomato are adult and larval stages of Coleoptera (beetles), Lepidoptera (moths),

Diptera (flies), Thysanoptera (thrips), Heteroptera (true bugs), Acari (spider mites)

and the damage caused by whiteflies (Trialeurodes vaporariorum (Westwood) and

Bemisia tabaci (Gennadius); Homoptera: Aleyrodidae, are the most common and

important pest on commercial greenhouse tomatoes production (Brown and Bird,

1992; Moriones et al., 1993; Bedford et al., 1994; Blau and Toscano, 1994; Jiang et

al, 1999; Avilla et al., 2005).

Root-knot nematodes of the genus Meloidogyne are among the main pathogens of

tomato (Williamson and Hussey, 1996; Jacquet et al., 2005) and pepper (Peixoto,

1995; de Souza-Sobrinho et al., 2002) crops. Resistance has been found in wild

tomato species and has been bread into commercial cultivars. Grafting of resistant

root stocks from these wild relatives onto the shoots of commercial cultivars is now a

common practice (Lee, 1994).

Cultivated peppers also are hosts at least 23 families of arthropod herbivores that

feed on roots, leaves or fruits including among the major pests of pepper are adult

and/o larval of Lepidoptera (Noctuidae), Diptera (Tephritidae), Hemiptera

(Cicadelidae), Coleoptera (Cucurlionidae and Crisomelidae) (Prado, 1991) and

Homoptera (Aphididae and Aleirodidae) ((Prado, 1991; Li et al., 2002). These last

two families have been reported also affecting under greenhouse condition to pepper

crop (Yankova et al., 2009). Resistances has recently been breed into sweet pepper

cultivars and has demonstrated to be resistant to the root-knot nematode

Meloidogyne javanica but are usually susceptible to the Southern root-knot nematode

M. incognita (Thies et al., 1997).

1.2. Cucurbitaceae crops

Crops in the family Cucurbitaceae are produced and consumed worldwide and

includes many economically important vegetables such as cucurbits, cucumber,

melon and watermelon (Lira and Caballero 2002; Wehner and Maynar, 2003).

According to Jeffrey, (1990), this family includes 118 genera and 825 species and is

used as fruit, food and decoration.


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Melon (Cucumis melon L.), Among Cucurbitaceae, melon is one of the most

important cultivated cucurbits. They are grown primarily for their fruit, which generally

have a sweet aromatic flavour, with great diversity in size (50 g to 15 kg), flesh colour

(orange, green, white, and pink), rind colour (green, yellow, white, orange, red, and

gray), form (round, flat, and elongated), and dimension (4 to 200 cm) (Nuez-Palenius

et al., 2008). Melon is important also for its richness in vitamin A and C content

(Wehner and Maynar, 2003). India is considered the center of domestication and is

where the highest genetic diversity of melon can be found. With regards to yield in

the crops of the Cucurbitaceae family, melon occupies third place in importance both

for consumption as fresh fruit and in area cultivated (Chavez, 2001). The global

production of melon has doubled within the last two decades to 26 million tons in

2007 (FAOSTAT 2007). The countries with the highest levels of production worldwide

are: Brazil (41%), Costa Rica (22%), Israel (13.5%) and Morocco (11.1%), (FAO,

2008).

Sqaush (Cucurbita pepo L.) is an annual herbaceous climbing plant with

undeterminate growth. The plant is distinguished for its richness in β-carotine, vitamin

B1 and minerals. Squash is produced worldwide in most climates and production has

reached 20 million tons in 2007 (FAOSTAT, 2009). The most important producer

countries in million ton per year reported by FAO, (2008) are: China (4.1), India (3.5),

Ukraine (0.915) and Egypt (0.7) (FAO 2008).

Pests of Cucurbitaceae crops

Pests affect the yield and quality of all Cucurbitaceae crops. Melons are damaged by:

the aphid (Aphis gossypii), the mite (Tetranychus spp), whiteflies (Bemisia tabaci and

Trialeurodes yaporariorum), and soil grubs (Agrotis ypsilon) (Wang and Liu, 2000).

Squash pests include the melon aphid, A. gossypii Glover (Ullman et al., 1991),

silverleaf whiteflies and squash silverleaf disorder associated with silverleaf whitefly

feeding (Yokomi et al., 1990; Summers and Stapleton, 2002; Costa et al., 1994).

Root-knot nematodes, Meloidogyne spp., are important limiting factors in the

production of most Cucurbitaceae crops such as squash and melon (Netscher and

Sikora, 1990) and Squash (Di Vito et al., 1992; Thies et al., 1997; Webster et al.,

2001).


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2. Importance of the plant parasitic nematodes

Plant parasitic nematodes are responsible for over $ 100 billion dollars in economic

losses worldwide to a variety of crops. The root-knot nematodes are the most

economically important group of plant parasitic nematodes worldwide and a known to

parasitize nearly every crop grown, reducing both yield and crop quality (Sasser and

Freckman, 1987).

The sedentary endoparasitic root-knot nematodes are among the most successful

parasites in nature. They parasitize over 2000 plants species and have a highly

specialized and complex feeding relationship with their host (Hussey and Janssen,

2002). Plant roots injured by nematodes are, in addition, susceptible to soil borne

pathogens. This interaction results in increased crop losses due to the resulting

synergistic disease complexes (Sikora and Carter, 1987). Four species, Meloidogyne

incognita, M. javanica, M. arenaria and M. hapla, account for 95% of all root-knot

nematode infestations in agriculture.

M. incognita (Figure 1) the most economically important species, causing an

estimated average crop loss of 5% worldwide is a main obstacle to production of

adequate supplies of food in many developing countries (Hussey and Jansen, 2002;

Luc et al., 2005). Four pathogenic races have been identified for M. incognita by their

host spectrum (Trudgill, 1997; Manzanilla-Lopez, 2004; Sikora and Fernandez,

2005). The life cycle begins when the infective second stage juveniles (J2) hatch and

migrate to the root where they penetrate the epidermis and move intercellulary while

feeding in the cortical tissue. They then injecting secretory proteins produced in their

oesophageal gland cells through the stylet into five to seven undifferentiated

procambial cells and transform these root cells into very specialized feeding cells

called giant cells (Figure 1). The nematode becomes sedentary and these giant cells

become the permanent feeding sites for the parasites throughout their life cycle

(Trudgill, 1997; Hussey and Janssen, 2002; Karssen and Moens, 2006).

Chemical nematicides are one of the primary means of control for plant-parasitic

nematodes. However, potential negative impacts on the environment, high human

toxicity and the loss of effectiveness after prolonged use due to biodegradation have

led to either their banning, removal from the market or restricted use on many crops.


There is an urgent need for new, safe and effective options for nematode

management (Zuckerman and Esnard, 1994). Several options are currently being

assessed around the world to identify and develop ecologically sustainable

management options for controlling nematode damage to plants.

6

A B

Figure 1. Galling symptoms in tomato root (A) induced after root penetration by the second stage juvenile of root-knot nematode Meloidogyne incognita (B).

3. Importance of the phloem-feeding insects

Phloem-feeding insects are highly specialized in their mode of feeding and present a

unique stress on plant fitness (Brown and Czosnec, 2002; Jones, 2003). While many

herbivores cause extensive damage, phloem-feeding insects, such as aphids and

whiteflies, cause modest to barely perceptible damage (Walling, 2008). Phloem-

feeding insects provide additional challenges to plants as they deplete

photosynthates (sucrose and amino acids) produced in the leaf mesophyll cells that

are normally transported to other areas of the shoot and to the roots and seeds

(Winter et al. (1992). They are often vectors of viruses, and can introduce chemical

and/or protein effectors that alter plant defense signalling, infestation symptoms, and

plant development (Kaloshian and Walling, 2005). This relationship is more

analogous to a plant-biotrophic pathogen interaction, where the pathogen is

sustained in a localized area and is dependent on living plant cells (Puterka and

Burton, 1999; Kempema et al., 2007; Zarate et al., 2007). When these attributes are

combined with broad host ranges, breeding strategies that promote invasiveness,

highly evolved feeding strategies, the ability to adapt to a wide range of plant

habitats, and the emergence of insecticide-resistant strains, it is not surprising that


phloem-feeding insects cause heavy losses in agriculture and horticulture (Goggin,

2007).

Although whiteflies and aphids are members of Hemiptera (Figure 2), plant selection,

their life cycles, and feeding activities are distinct (Baumann, 2005; Kaloshian and

Walling, 2005). Whiteflies use colors, while aphids use both visual and olfactory cues

to direct flight responses to host plants (Gerling, 1990; Powell et al., 2006). Upon

landing, adults evaluate the tactile and chemical cues of the plant surface to

determine the suitability of a plant as shelter or as a feeding and/or oviposition host.

For insects that have sessile instars, like whiteflies, the plant chosen for egg

deposition is a crucial maternal decision. On a good host, the next generation will

thrive; on a poor host, insect populations will decline (Walling, 2008).

7

A B C

Figure 2. Phenotypic characteristic of Green House Whitefly Trialeurodes vaporariorum Westwood: (A) female laying eggs, (B) wingless parthenogenetic adult female of the melon aphid, A. gossypii Glover, and (C) wingless parthenogenesis progeny of Green Peach Aphid Myzus persicae Sulzer.

The life cycle of aphids is complex and involves sexual and asexual

(parthenogenetic) modes of reproduction (Moran and Thompson, 2001; Tagu et al.,

2008). A major form of aphid polyphenism is the switch between viviparous

parthenogenesis and sexual reproduction with eggs, depending on environmental

cues. Typically, aphid species reproduce most of the year by viviparous

parthenogenesis, a major factor in their destructive potential. A parthenogenetic

female can produce up to 120 genetically identical embryos in approximately 10 days

(Tagu et al., 2008). Both winged (alate) and wingless (apterous) individuals and

multiple generations exist. Some aphids require regular alternation of plant hosts

where sexual morphs mate and lay eggs on the primary host for several generations

and give rise to a generation of alate morphs that move to the secondary host, which


8

is usually a distinct plant species. On the secondary host, the parthenogenic mode of

reproduction is initiated and used for subsequent generations (Kaloshian and

Walling, 2005).

In contrast, only two whitefly developmental stages are mobile (the first instar and

adult), and whitefly development resembles complete metamorphosis (Byrne and

Bellows, 1991). Upon emergence from an egg, the first instar (the crawler)

establishes a feeding site that is used nearly continuously for over 28 days (Gill,

1990; Byrne and Bellows, 1991; Johnson and Walker, 1999; Freeman et al., 2001; da

Cunha et al., 2007). The immobility of nymphs, longer life cycles, and prolonged

nymph feeding are features that distinguish the whitefly-plant and aphid-plant

interactions that take advantage of their adept feeding strategies to avoid or deter

many plant defenses (da Cunha et al., 2007).

4. Mutualistic endophytic fungi as biological control agents

The term endophyte was coined by the German scientist Heinrich Anton De Bary in

1884 (Wilson, 1995), and is used to define fungi or bacteria occurring inside plant

tissues of their host without causing any apparent symptoms in the host (Petrini,

1991; Wilson, 1995). Fungal endophytes have been detected in hundreds of plants,

including many important agricultural crops such as wheat (Larran et al., 2002a),

bananas (Pocasangre et al., 2000; Cao et al., 2002), soybeans (Larran et al., 2002b),

and tomatoes (Hallman and Sikora 1994a; Larran et al., 2001). Extensive research

has been conducted on the use of mutualistic endophytes for the biological control of

plant-parasitic nematodes (Hallmann and Sikora, 1994a,b; Dababat and Sikora,

2007a,b; Sikora et al., 2008). These studies have been carried out on vegetables,

legumes, potato, banana, cotton and rice, with most of the work directed at high

value horticultural crops. A wide range of plant-parasitic nematodes have been

targeted for biological control with endophytes, for example, the root-knot nematode,

Meloidogyne incognita, the reniform nematode, Rotylenchulus reniformis, the cyst

nematode, Globodera pallida and the burrowing nematode, Radopholus similes

(Sikora et al., 2008). The mechanisms of action responsible for biocontrol vary

greatly between endophyte and include: antibiosis, predation, pathogenesis,

competition, repellence and induced resistance (Stirling, 1991; Schuster et al., 1995;


9

Hallmann and Sikora, 1996; Hallmann et al., 2001; Clay and Schardl, 2002; Vu,

2005; Sikora et al., 2007).

Several reports have also shown that fungal endophytes provide protection against

herbivorous insects (Breen, 1994; Clement et al., 1997) when colonizing within

healthy leaves and stems (Siegel et al. 1985; Carroll, 1988; Clay, 1991a; Clay,

1991b; Müller and Krauss, 2005; Meister et al., 2006).The mode of action of these

endophytes antagonistic towards insects and even livestock feeding on tall fescue

was considered to be due to toxic metabolites of the fungus Neotyphodium

coenophialum (reclassification, Acremonium coenophialum) (Glenn et al., 1996;

Bacon et al., 1977). This type of mechanism has only been demonstrated for the

seed-borne endophytes of certain cool season grasses (Funk et al., 1983; Latch et

al,. 1985; Clay 1988; Clay 1991a;).

Neotyphodium spp, are endophytic clavicipitalean fungi (Ascomycota: Hypocreales:

Clavicipitaceae), that infect mostly grasses (Clay, 1989). They have been found

associated with perennial ryegrass and tall fescue with a capacity to improve plant

resistance towards at least of 23 species of insects in 10 families and 5 orders

(Breen, 1994; Meister et al. 2006; Rudgers and Clay, 2007). These hyphae of these

endophytes are found predominantly in the intercellular spaces of the leaf and stem

tissue (Breen, 1994) being vertically transmitted maternally through seeds (Latch and

Christensen, 1982; Siegel et al., 1984; Bacon, et al., 1986; Knoeh et al., 1993;

Schardl et al., 2004). The negative effects toward insect herbivores have been

generally ascribed to toxins or fungal metabolites produced by the antagonistic

endophytes (Funk et al., 1983; Johnson et al., 1985; Clay, 1988; Bush et al., 1997;

Brem and Leuchtmann, 2001; Clay and Schardl., 2002; Kucht et al., 2004) who have

the ability to produce both antibiotic and antixenotic responses in their insect

herbivores (Dahlman et al., 1991), growth inhibition (Miller et al., 1985; Bacon et al.,

1986; Pederson et al., 1988; Strongman et al., 1988; West et al., 1988; Calhoun et

al., 1992) and to affect the fecundity (Cheplick and Clay 1988, Clay 1991b). Fungal

alkaloids have been implicated as the mechanism for these effects (Cheplick and

Clay 1988; Powell and Petroski, 1992; Petroski et al., 1992; Ball et al. 2006; Gonthier

et al., 2008). Their distribution and concentration in the plant tissue are correlated

with the location of mycelia in the plant (Davies et al., 1993; Ball et al., 1995).


10

However, although Acremonium endophytes are not found in roots, they have been

found to enhanced resistance toward some root-feeding insects (Breen, 1994).

Other genera of nonpathogenic mutualistic endophytic fungi, such as Trichoderma

spp (Altomare et al., 1999; Howell et al., 2000), and F. oxysporum, (Hallman and

Sikora, 1996; Fuchs et al., 1997; Menjivar, 2005; Pocasangre et al., 2006; Dababat,

2006; Dababat and Sikora, 2007abc), have been shown to be effective biocontrol

agents inducing resistance in host plants, which enhances the ability of the plant to

defend it self (Stirling, 1991; Vidal, 1996; Alabouvette et al., 1998; Larkin and Fravel

1999; Pereira et al. 1999; Trouvelot et al., 2002; Vu et al., 2006; Sikora et al., 2007

Sikora et al., 2008). In some instances the plants infected with these endophytes are

able to convey enhanced defence proprieties to the next host generation as seen in

banana (zum felde, 2002).

Only a few investigations have been carried out to evaluate the possible effects of

endorhiza endophytes that enhance resistance to nematodes, for efficacy toward

insects on the same or other host plants. For example, F. oxyporum strains V2w2

and III4w1 that was shown to reduce the population of the burrowing nematodes R.

similis on banana, were evaluated by Paparu et al., (2008) for pathogenic effects on

the eggs of the banana weevil eggs Cosmopolites sordidus (Coleoptera:

Curculionidae), the most important insect pest of bananas and plantains (Musa spp.)

(Koppenhöfer et al., 1994; Gold et al., 2001; Gold et al., 2004). Using conidial

suspensions they obtained 50.3 and 43.7% mortality of banana weevil eggs,

respectively. This effect was similar with to results found by Griesbach (1999) who

showed that spore suspensions of non-pathogenic Fusarium isolates isolated from

banana corms, caused 80-100% mortality in weevil eggs and 32% in larvae. He also

reported in banana field trials a reduction in damage by C. sordidus after inoculation

with F. oxysporum. The control was considered due to anti-feeding effects in

endophyte inoculated plants resulting in retarded development of weevil larva. The

prevalent mode of action appears to be through the production of metabolites that act

as either oviposition repellents or as antifeedant toxins (Gold et al., 2001).


11

The endorhiza endophyte F. oxysporum that is limited to the roots of plants and able

to reduced nematode development in the root has not been studied for systemic

effects on insects feeding on the leaves of the same host. The capacity of these fungi

to induce systemic resistance (ISR) toward pathogen suggests movement of either

resistance induction signals or toxic metabolites systemically up into the leaves from

the roots. This systemic resistance could also improve plant defenses against

herbivorous insects and comprise plant traits that negatively affect insect preference

(host plant selection, oviposition and feeding behavior) or performance (growth rate,

development, reproductive success) resulting in increased plant fitness (Ajlan and

Potter, 1991).

5. Plant defense mechanisms

Plants are exposed to attack by pathogens and insect pests with different attackers

evoking varied plant responses to protect itself against these biotic stresses. Plants

have evolved defense strategies to counteract potential invaders. These strategies

require a broad range of mechanisms to effectively combat invasion by microbial

pathogens or attack by herbivorous insects (Choudhary, 2008). The plants utilize

both constitutive and induced defences for protection against a wide range of biotic

threats. Constitutive defences according to Walling, (2000) include physical barriers

such as the leaf cuticle, cell walls, and stored metabolites that inhibit the feeding,

growth, and development of herbivores, while induced plant defences include the

activation of both direct and indirect mechanisms (Walling, 2000; Kessler and

Baldwin, 2002). Direct defenses according to Baldwin et al. (2002) and Kliebenstein,

(2004) involve the synthesis of secondary metabolites that influence insect

attraction/deterrence and inhibit insect growth and development. In addition, induced

proteins, such as protein inhibitors, polyphenol oxydases, arginase, and threonine

deaminase, inhibit insect digestive enzymes and/or decreased the nutritive value of

the plant tissue (Ryan, 2000; Chen et al., 2005). Indirect defenses include the

release of volatiles that signal the location of insects on infested plants to parasitoids

and predators of the herbivore (Baldwin et al., 2002; Dicke et al., 2003).

Plants have developed efficient surveillance systems that rapidly detect biotic

intruders including viruses, bacteria, fungi, nematodes, mites, and insects. Like plant-

pathogen interactions, both compatible and incompatible plant-herbivore interactions


12

occur (Kaloshian and Walling, 2005). Plant responses to hemipteran insects have

substantial overlap with responses mounted against microbial pathogens, as seen in

changes in RNA profiles and emission of volatiles. Responses to known defense

signals and characterization of the signaling pathways controlled by the first cloned

insect R gene (Mi-1) indicate according to Kempema et al. (2007) that perception and

signal transduction leading to resistance may be similar to plant-pathogen

interactions.

Dababat (2006) and Dababat and Sikora (2007a) found that inoculations of the

endophytic fungus F. oxysporum 162 induced systemic resistance (ISR) in tomato

plants toward M. incognita. The results demonstrated that the presence of the

endophyte caused ISR and reduced nematode infection by over 40% in the

responder side of the split-root plants. Vu et al (2006) in investigations with the same

isolate but with the migratory endoparasitic nematode R. similis on banana also

detected ISR that led to changes in root attractiveness in the responder side of the

root system. This means that ISR is not the mechanism responsible for reduced

nematode attraction or penetration in the responder root, but only initiates systemic

changes in the plant that reacts by altered exudate secretion into the rhizosphere.

Nevertheless, emerging research has shown that plants utilize induced defense

mechanisms that are dependent on the attacker, and, in certain interactions, a subset

of responses are species specific (McDowell and Dangl, 2000; Walling, 2000;

Kaloshian and Walling, 2005). Hence, those changes in root exudates induced

systemically by the endophytic F. oxsporum strain 162 could be causing leaf-level

changes in the accumulation of compounds affecting negatively the behavioural and

sensory mechanisms by which insects recognize their hosts and reject unsuitable

plants.


13

6. Scope of the study

The overall goal of the present study was to investigate the influence of the

mutualistic-endophyte Fusarium oxysporum strain 162 to enhance defence

mechanisms in Solancea and Cucurbitacea plants toward the root-knot nematode

Meloidogyne incognita and phoem-feeding insects. The objectives of these

investigations were to determine:

1- The efficacy of the mutualistic fungus Fo162 as a potential biological control

agent against M. incognita in melon, squash and pepper plants.

2- The biocontrol activity of 6 endophytic fungi against the phloem-feeding insect

Trialeurodes vaporariorum (Westwood) in tomato.

3- The mechanisms of action of F. oxysporum strain 162 in tomato plants toward the

phloem-feeding insect T. vaporariorum.

4- The capacity of F. oxysporum as a biocontrol agent against different Hemipteran

phloem-feeding insects in squash, melon and pepper plants.

Chapter 2 General Materials and Methods

14

GENERAL MATERIALS AND METHODS

Material and methods generally used in this study are described in this chapter.

Additional techniques and procedures applied in individual experiments are

described within the respective chapter.

1. Fungal isolates

1.1. Origin of the mutualistic endophytic fungus

Trichoderma atroviride strain MT-20 was isolated from surface sterilized banana

roots Musa AAA in Guatemala (zum Felde, 2002) and the strain S-2 in Costa Rica

(Cañizares, 2003). Fusarium oxysporum strain P-12 was isolated from banana roots

in Costa Rica (Meneses, 2003) and the strain 162 from the cortical tissue of surface

sterilized tomato root, Lycorsicom esculentum Mill. cv. Moneymarker in Kenya

(Hallmann and Sikora, 1994a). Fusarium sp. strain F14 was isolated from surface

disinfected rice roots grown in alluvial soil in Mekong Delta, Vietnam (LE et al., 2009).

Fusarium sp. strain Bonn-7 was isolated from cortical tissue of surface sterilized

radish roots in Bonn, Germany (Xiaonin Yan, 2009, personal communication). All

these isolates were previously shown to have biocontrol activity against plant-

parasitic nematodes (Hallmann and Sikora 1994 a,b; Pocasngre et al., 2006;

Dababat and Sikora, 2007 a,b, and c; VU et al., 2006; Mendoza and Sikora, 2008).

1.2. Culture of the isolates

Spores of all the endophytic fungi were stored in Cryobank storage vials

(CRYOBANKTM, MASTE Group Ltd., Merseyside, UK) at -80ºC. Fungal propagation

for the experiments were initiated with spores from these stocks on 100% Potato

Dextrose Agar plates (PDA; Difco, Sparks, MD, USA), amended with 150 mg L-1 of

streptomycin sulphate (AppliChem) and 150 mg L-1 of Chloramphenicol (AppliChem)

to avoid bacterial contamination. The fungal culture was incubated for 3 weeks in the

dark at 24ºC. After incubation, the mycelium and conidia were scraped from the

media surface with a spatula and suspended in tap water. Spores were separated

from the mycelium by sieving the suspension through four layers of cheese-cloth.


15

Spore density was measured using a hemacytometer (Thomas Scientific,

Philadelphia PA) and adjusted to desired concentrations with tap water.

2. Nematode inoculum

Meloidogyne incognita race 3 with a broad spectrum of vegetables crops (Taylor and

Sasser, 1978) was originally isolated from an infested field in Florida, USA (Supplied

by Dr. D. Dickson, University of Florida, Gainsville) and was multiplied in a

permanent cultivation system using tomato plants cv. Furore grown in a large

container (150 x 80 x 40 cm) filled with an autoclaved sand:soil substrate (2:1, v:v)

mixture in the glasshouse at 27 ± 5°C. Eight weeks old tomato plants were uprooted

and nematode eggs were extracted from galled roots using 1.5% NaOCl solution as

described by Hussey and Barker (1973). Roots were gently washed with tap water,

cut in 1-2 cm pieces and macerated 2 times for 10 s each time in a Warring blender

(Bender and Hohbein) with tap water. Every 500 ml of the macerated solution was

mixed with 258 ml of 4% NaOCl (AppliChem) and manually shaken for 3 min. This

suspension was poured over four nested sieves; 250 μm on the top, followed by 100

μm, 45 μm and 25 μm aperture sieve. Eggs remaining in the 25 μm sieve were rinsed

with tap water, collected in a beaker and aerated in tap water for 10-12 days at room

temperature in the dark using an aquarium pump to facilitate hatching. Freshly

hatched second stage juveniles (J2) were collected by a modified Bearmann

technique. The juveniles in tap water suspension were used as inoculum.

3. Phloem-feeding insect production

3.1. Trialeurodes vaporariorum (Westwood)

The greenhouse whitefly T. vaporariorum (Westwood) (Homoptera: Aleyrodidae) was

obtained from the INRES population. The insects were multiplied on tomato

(Lycopersicon esculentum Mill) cv. Hellfrucht/JW Frühstamm and tobacco (Nicotiana

rustica) in the greenhouse. The insect were held with the host plant in mesh cages of

50 X 50 X 50 cm under glasshouse condition at 27 ± 5ºC.

3.2. Aphis gossypii (Glover) and Myzus persicae (Sulzer)

The start up population of the melon aphid A. gossypii (Glover) and the Green peach

aphid M. persicae (Sulzer), (Homoptera: Aphidide), were obtained from BAYER


16

CROP SCIENCE (Bayercode: APHIGO, Bayer CropSeience Deutschland GmbH,

Langenfeld) in January 2000. Untreated cotton Gossypium hirsutum L. cv. “Çukurva

1518” was obtained from Ege University Bornova-Izmir, Turkey. Savoy cabbage

Brassica oleraceae L. convar. capitata (L.) Alef. Var. sabauda cv. “Vertus2 spät” was

obtained from Kiepenkerl Aurich, Germany. These crops were used as host plants to

establish the colony of A. gossypii and M. persicae respectively. The plants were

grown in an incubator at 25 ± 2ºC with at a relative humidity of 60%, a light intensity

of 2000 Lux and a light/dark photoperiod of 16 h/8h. For all experiments apterous

adults were randomly selected from the forage plant, and the development stage

verified using a binocular at a magnification of 40x.

4. Plant production

Seeds of tomato (Lycopersicon esculentum Mill) cultivar Hellfrucht/JW Frühstamm

(Enza Zaden Holland) and pepper (Capsicun annum L) cultivar Yolo

Wonder/Heidelb. Riesen (Enza Zaden Germany), were sown in 96-well multi-pot

trays (50.5 x 30 x 5 cm) for seedling production. Squash seeds (Cucúrbita pepo) cv

Eight Ball (HILD Samen GmbH, Germany) and Melon (Cucumis melon L) seeds cv

Mehari (Syngenta Seeds B.V., Holand) were sown in 70-plug commercial seedling

trays containing autoclaved sand passed through a 2 mm mesh sieve. The trays

were maintained in a climate chamber at 27 ± 5ºC with 13 h supplemental artificial

light day-1 until transplant day. The seedlings were fertilized with 5 ml of a suspension

containing 2% commercial fertilizer (14-10-14:N-P-K, AGLUKON, Düsseldorf,

Germany) once per week. After 4 weeks, the tomato seedlings were transplanted

and used in the experiments. Squash and melon, the seedlings were grown in the

greenhouse as above for 2 weeks before use. The plants were then transplanted into

300 g of an autoclaved sand:soil mixture (2:1,v/v) in 10 cm diameter pots and

fertilized with 20 ml of the suspension described above.

5. Soil and substrate

A field soil containing (14.6% clay, 77.6% silt and 7.8% sand, pH 5.7 mixed with sand

(1:2 v/v) was passed through a 2 mm mesh screen and autoclaved (121ºC for 60 m),

hereafter referred to as soil, was used in all the tests. One week after autoclaving,


17

plastic bags containing the substrate were opened to allow the substrate to air dry

and stabilize at room temperature for 48 hours before being used in experiments.

6. High pressure liquid chromatography (PR-HPLC analysis)

To separate and quantify the active compounds produced in the tomato leaves of

endophyte treated and untreated plants, reversed phase high liquid chromatography

(RP-HPLC) analysis was performed. For this, the first and second young leaves of

each plant were harvested together. Five hundred milligrams of leaf material was

transferred to 12 ml polyethylene round bottom tubes (Sarstedt, Germany) and

frozen in liquid nitrogen. The leaves were pulverized by placing a metal spatula into

the tube, followed by vortexing for 2 minutes. After this, a 1 ml solution of 50% (v/v)

ethanol (HPLC grade; Sigma, Germany) and 50% (v/v) methanol (HPLC grade;

Sigma, Germany), supplemented with 0.75% (w/v) of butylated hydroxytoluene

(Sigma, Germany), was added per sample. After additional vortexing, the samples

were incubated in liquid nitrogen for 10 min. Then the samples were incubated at

room temperature for 15 min. and centrifuged at 4000×g at 25°C for 10 min. After

centrifugation, the suspension was transferred to Eppendorf tubes and centrifuged at

14000×g at 25°C for 5 min. From each sample the upper 600 μL was filter sterilized

(0.45 μm) and transfer to HPLC vials. RP-HPLC was performed on a HEWLETT

PACKARD (HP) system using a LiChrospher®100 C18 reversed phase column (250

by 4.0 mm, 5 µm), preceded by a LiChrospher® C18 reversed phase guard column

(4.0 by 4.0 mm, 5 µm). The HPLC system consisted of a HP 1050 pump unit, HP

1050 diode array detector, HP 1046A fluorescence array detector, and 1050

autosampler which were controlled by ChemStation for LC 3D systems. Before

samples were injected, the column was equilibrated with 90% water, supplemented

with 0.1% (v/v) trifluoroacetic acid, (solvent A) and 10% acetonitril (solvent B). After

injection the samples were eluted at a flow rate of 1.0 ml/min using an isocratic flow

of 90% solvent A and 10% solvent B for 2 min, a linear gradient to 10% solvent A and

90% solvent B for 28 min, followed by a isocratic flow for 5 min with 10% solvent A

and 90% solvent B. Before the next sample was injected the column was re-

equilibrated by a 1 min linear gradient to 90 % solvent A and 10% solvent B, followed

by a 4 min isocratic flow of 90 % solvent A and 10% solvent B.


18

7. Culture media and reagents

Potato Dextrose Agar (PDA) Potato Dextrose Broth (Oxoid LTD) 18 g

Agar (AppliChem GmbH) 14 g

Deionized water 1 l

Chloramphenicol 150 ppm

Streptomycin sulphate 150 ppm

Fuchsine acid (MERCK) 2 g fuchsine acid powder + 198 ml water (1% fuchsine acid)

Lactic acid solution:

Lactic acid 1750 ml

Glycerine 126 ml

Tap water 124 ml

8. Statistical analysis

Data from all experiments were tested for homogeneity of variances and subjected to

T-test (p ≤ 0.05) to compare 2 treatments or one-way analysis of variance (ANOVA).

When the overall F-test was significant, the treatment mean values were compared

using the Fisher’s Least Significant Difference Test (LSD) at p ≤ 0.05. To compare

the gall index caused by M. incognita and populations of A. gossypii, a nonparametric

analysis of variance Kruskal-Wallis was used since it allows comparisons of the

expectations of 2 or more distributions without making the assumption that the error

terms are normally distributed. To conform to assumption of normality, the GHWF

population was transformed using a Log(n+1) transformation before statistical

analysis was performed with the exception of the reproduction experiment that was

analysed in percent relationship. All peaks from the accumulation compounds

provided by the RP-HPLC analysis in the 250 nm wave length were analysed and

only those with statistically different areas were compared with the different

development stages of the insect through Pearson regression analysis using InfoStat

for Windows Version 2008 (Grupo InfoStat, FCA, Universidad Nacional de Córdoba,

Argentina).

Chapter 3 Fo162 as biocontrol agent toward M. incognita

19

BIOLOGICAL CONTROL OF Meloidogyne incognita ON

CUCURBITACEAE AND SOLANACEAE CROPS BY THE NON-

PATHOGENIC ENDOPHYTIC FUNGUS Fusarium oxysporum STRAIN

162

1. Introduction

Meloidogyne incognita is a sedentary endoparasitic nematode that can severely

decrease yield in most vegetable crops, especially those of the Cucurbitaceae and

Solanaceae families (Sikora and Fernandez, 2005). Currently one of the main tools

used to control root-knot nematodes is the use of fumigant and non-fumigant

nematicides (Desaeger and Csinos, 2006).

In melon, the fumigant nematicide methyl bromide was used until recently as the

main nematode control tool (Webster et al., 2001).The removal of methyl bromide

from the world market due to it is negative effect on atmospheric ozone has had

major impact on nematode control in this crop. Non-fumigant nematicides which are

also used in melon have microbial biodegradation problems due to repetitive use in

the same fields (Cabrera et al., 2010). Their use also has been strictly limited due to

environmental side-effects and human health risks (Karpouzas and Giannakou,

2002; Santos et al., 2006). For these reasons increased interest has been place on

developing other strategies for nematode management.

Fusarium oxysporum strain 162 (Fo162) is an endophytic fungus which is found in

the endorhiza of plant root tissues (Hallmann and Sikora, 1994a) and is able to

colonize roots of a number of other crop plants (Sikora et al., 2008). Most studies to

determine the efficacy of the strain Fo162 toward plant parasitic nematodes have

been conducted only on tomato and banana (Dababat and Sikora, 2007a, b, and c;

Mendoza and Sikora, 2008; Vu, et al., 2006). Penetration of Radopholus similis was

reduced up to 47% in banana roots treated with Fo162 compared to untreated control

(Vu et al., 2006). The number of galls on tomato roots caused by M. incognita was

reduced up to 36% by endophyte treatment with Fo162 (Dababat and Sikora, 2007c).


20

Additionally, single and combined applications of Fo162 with other mutualistic fungi

provided enhanced nematode control in banana (Pocasangre et al., 2006, zum Felde

et al., 2006; Mendoza and Sikora, 2008). Research dealing with the colonization and

biocontrol potential of Fo162 on other crops has not been carried out.

Vänninen et al. (2000) and Rumbos (2005) demonstrated that the persistence of

fungal conidia of some fungal antagonists of nematodes is influenced by physical,

chemical and biological properties of the substrate. The use of organic amendments

can influence these properties giving to the soil characteristics of good aeration,

structure, drainage, moisture-holding capacity, buffering capacity, and nutrient

availability to support the growth of antagonistic fungi (Rodriguez-Kabana, 1985).

Mendoza (2008) demonstrated that by increasing the content of organic matter in

clay soil, the longevity of the fungal conidia of the nematode antagonist,

Paecilomyces lilacinus strain PL 251, was prolonged.

The effects of Fo162 colonization of the plant root system on the antagonistic

potential and on the mode of action, has been clearly demonstrated against M.

incognita in tomato (Hallman and Sikora, 1996; Dababat, 2006) and toward R. similis

in banana (Vu et al., 2006; Mendoza and Sikora, 2008). The effect of Fo162

colonization on M. incognita control in other crops has not been investigated.

Therefore, the aims of this study were to:

1. Evaluate the capacity of Fo162 to colonize the root tissue of hosts in the

Solanaceae and Cucurbitaceae plant families.

2. Determine Fo162 biocontrol activity toward to root-knot nematode.

3. Elucidate endophyte influence on plant growth parameters.

4. Investigate interactions between soil organic matter content and Fo162 as it

impacts nematode biocontrol.


21

2. Experimental designs

2.1. Endophyte colonization

Cucúrbita pepo (squash) seeds cv Eight Ball and Cucumis melon (melon) seeds cv

Mehari, were sown in 20 g of autoclaved sand in 4 commercial 70-plug seedling trays

(4.0 x 4.5 x 2.5 cm). Pepper (Capsicun annum L) seeds of the cultivar Yolo Wonder

were sown in 96-well multi-pot trays (50.5 x 30 x 5 cm). Seedling of squash and

melon were incubated for 2 weeks and pepper for 4 weeks in a climatic chamber at

27 ± 5ºC with 13 h additional artificial light. Plants were then transplanted into 7 x 7 x

8 cm plastic pots containing 300 g of autoclaved soil (described chapter 2). Plants

were fertilized with 2% Aglikon (N-P-K:14-10-14) once per week and watered as

needed.

The mutualistic endophytic fungus F. oxysporum strain 162 was originally isolated

from the cortical tissue of surface sterilized tomato roots (Hallmann and Sikora,

1994). The endophyte, produced as outlined in (chapter 2), was applied twice at a

rate of 1x106 cfu g soil-1 to the respective endophyte-inoculated treatment. The first

inoculation was performed at sowing by injecting the endophyte into the planting

hole, and the second inoculation was made by injection 2 cm deep into the

rhizosphere of the seedlings using 3 holes made around the stem base with a plastic

rod one day after transplanting. Pots inoculated with tap water acted as controls.

Pots for the experiments were arranged in a randomized complete block design,

each treatment replicated 7 times and the experiments were repeated once.

To verify successful conlonization, the endophyte was re-isolated from the endorhiza

of squash, melon and pepper plants 10 days after the second inoculation. After

harvesting, the roots were surface-sterilized by soaking them in a solution of 0.5%

NaOCl for 3 min, followed by three rinses in sterile deionized water. The root system

was divided onto 3 zones; zone-1 the upper third close to the stem; zone-2 the

central area and zone-3 represented by the bottom third.

Seven root pieces approx. 0.5 cm in length from each zone were used to determine

the percentage root colonized per plant and the zone of fungal colonization. After the

root pieces were sterilized they were first pressed onto 10% PDA media in separate


22

Petri dishes to check the success of the sterilization process and then mounted onto

a new PDA plates for Fo162 colonization determination. Surface sterilization was

considered successful when no fungal colonies developed on the first plates used for

the imprint. If no fungal growth occurred, the roots in the second set of plates were

incubated for 14 days at 24ºC and examined for the presence of Fo162. Growth

characteristics and fungal morphology as described by Leslie and Summerell (2006)

were used to determine that the colonies growing out of the root pieces resembled

Fusarium oxysporum Fo162 and not other contaminents.

2.2. Nematode biocontrol efficacy and growth promotion

This test was designed to determine the ability of Fo162 to reduce the penetration of

M. incognita J2 into squash, melon and pepper roots. Plant production and Fo162

inoculation was conducted as outlined in experiment 2.1. Second stage juveniles (J2)

collected by the modified Bearmann technique described in (chapter 2) and

suspended in the tap water were used as inoculum. One day after the second Fo162

inoculation, squash, melon and pepper seedlings were inoculated with 1000 J2.

Nematodes were injected into 3 holes made with a plastic rod around the stem base

at a 2 cm depth. All pots were arranged in a completely randomized design on a

greenhouse bench at 27 ± 3ºC with 13 h/day supplemental artificial light.

Plants were harvested 10 days after nematode inoculation and the shoot and root

parts were separated and fresh weights were recorded. Roots were gently washed

with tap water and nematode penetration determined. Nematode penetration was

determined by staining the root system in a 0.1% acid fuchsine solution (Ferris, 1985)

and macerating them in water using an Ultra-Turrax (IKA-WERKE, Staufen,

Germany) at 11,000 U min-1 for 2 minutes. Stained nematodes were counted under a

stereomicroscope and the total number of nematodes per gram root was determined.

2.3. Influence of organic matter on Fo162 biocontrol activity

The effect of soil amended with organic matter (OM) on the saprophytic endophyte

and it’s ability to control M. incognita root damage was evaluated using the root gall

index proposed by Bridge and page (1980). Galling has a linear relationship to yield

loss (Barker et al., 1981). Autoclaved soil passed through a 2 mm sieve, was

amended with 5, 10, 15 or 20% (v/v) non-sterile OM from three years old composted


23

plant residues which was first sieved through a 5 mm mesh to remove large particles

and debris. The two substrates were mixed mechanically and then pleaced into 7 x 7

x 8 cm plastic pots before planting. Plant and Fo162 inoculum production is

described in 2.2, and J2 inoculation in 2.3. The experiment consisted of 6 treatments,

each with or without OM as follow: 1) autoclaved soil without Fo162, 2) autoclaved

soil with Fo162, 3) autoclaved soil with Fo162 amended with 5% OM, 4) autoclaved

soil with Fo162 amended with 10% OM, 5) autoclaved soil with Fo162 amended with

15% OM and 6) autoclaved soil with Fo162 amended with 20% OM. The pots were

arranged in a randomized block design on a greenhouse bench and incubated for 6

weeks at daily temperature of 27 ± 4ºC and with 13 h/day supplemental artificial light.

The gall index was determined 6 weeks after J2 inoculation. The plants were then

harvested, the roots separated from the soil by careful washing and the 10 category

gall index of each root was carried out visually.


3. Results


Fo162 was re-isolated from both the Cucurbitaceae and Solanaceae plants 10 days

after the last fungal inoculation (Figure 1). The overall level of colonization was

similar in both experiments (A and B). Total colonization in squash was of 27.8%,

melon 27.4% and pepper 28.8%. In squash no colonization preference among the

three zones investigated was detected. However, there was a non-significant

tendency toward colonization of the middle level. In contrast Fo162 colonization in

melon and pepper was higher in the upper root zone with 68.9 and 58.7%

respectively, than the other two zones of the root system.

Root zones

0

5

10

15

20

25

30

35

Squash Melon Pepper

Col

oniz

atio

n in

%

lower Middle upper

A

NS

0

5

10

15

20

25

30

35

Squash Melon Pepper

Col

oniz

atio

n in

%

B

NS

Root zones

0

5

10

15

20

25

30

35

Squash Melon Pepper

Col

oniz

atio

n in

%

lower Middle upper

A

NS

0

5

10

15

20

25

30

35

Squash Melon Pepper

Col

oniz

atio

n in

%

B

NS

Figure 1. Endophytic root colonization and zone preference of Fusarium oxysporum strain 162 in squash (Cucúrbita pepo L.) cv Eight Ball, melon (Cucumis melon) cv Mehari and pepper (Capsicun annum L) cv Yolo Wonder, ten days after second Fo162 inoculation (A: n=21) and (B: n=24). NS= not significant according to the LSD test (p ≤ 0.05). Vertical bars represent the standard error of the mean (SEM).

24



M. incognita early root penetration was reduced significantly when treated with Fo162

on both the Cucurbitaceae and Solanaceae crops compared to the untreated control

(Figure 2). Nematode penetration on squash, melon and pepper was reduced

significantly 83, 70 and 73% in the Fo162 treated plants when compare to the

untreated control respectively. These results show clearly the impact of Fo162 as a

biocontrol agent toward M. incognita in both crops families.

0

50

100

150

200

250

300

350

Squash Melon Pepper

Pen

etra

tion

Fo162 Untreated

* **

A

0

50

100

150

200

250

300

350

Squash Melon Pepper

Pen

etra

tion

***

B

0

50

100

150

200

250

300

350

Squash Melon Pepper

Pen

etra

tion

Fo162 Untreated

* **

A

0

50

100

150

200

250

300

350

Squash Melon Pepper

Pen

etra

tion

***

B

Figure 2. Effect of Fusarium oxysporum strain 162 in squash (Cucúrbita pepo L.) cv Eight Ball and melon (Cucumis melon) cv Mehari (Capsicun annum L) and pepper cv Yolo Wonder on Meloidogyne incognita early root penetration after two fungal inoculations under glasshouse conditions. Means with (*) are significantly different based on T-test (p ≤ 0.05). Vertical bars represent the standard error of the mean (SEM).

25


26

Inoculation of Fo162 at the sowing and to the seedling had no significant effects on

root fresh weight of squash, melon and pepper when compared to the untreated-

control plants (Table 1). Shoot fresh weight of squash treated with Fo162 was,

however significantly higher than the untreated control. In contrast melon and pepper

inoculated with Fo162 did not show a significant effect on these growth parameters

when compared with their respective controls.

Table 1. Effect of Fusarium oxysporum strain 162 on root and shoot fresh weight (mean ± S.D.) of squash (Cucúrbita pepo L.) cv Eight Ball, melon (Cucumis melon) cv Mehari and pepper (Capsicun annum L) cv Yolo Wonder under glasshouse conditions at 27 ± 3 ºC. T-test (p ≤ 0.05).

Shoot fresh weight (g) Root fresh weight (g) Treatments 1° essay 2° essay 1° essay 2° essay

Squash + Fo162 17.1 ± 1.6 21.1 ± 2.8 1.5 ± 0.2 3.7 ± 0.9

Squash untreated 12.6 ± 1.9 15.7 ± 3.0 1.5 ± 0.4 2.9 ± 1.0

P-value 0.0001 0.7583

Melon + Fo162 12.3 ± 3.2 6.9 ± 2.2 3.2 ± 0.8 3.4 ± 0.8 Melon untreated 12.4 ± 3.4 4.8 ± 1.0 3.1 ± 0.9 2.0 ± 0.7 P-value 0.5788 0.1321

Pepper + Fo162 4.5 ± 0.8 8.4 ±1.7 4.0 ± 0.5 3.2 ± 0.9

Pepper untreated 4.8 ± 1.4 6.3 ± 1.2 5.0 ± 0.8 2.5 ± 0.7

P-value 0.1380 0.7811


The gall index was significantly reduced in all 3 crops in the inoculated treatments

when compared to the un-inoculated control. This confirms the ability of Fo162 as

biocontrol agent of M. incognita. There was no increase in Fo162 biocontrol on

squash when treated with OM. The gall index was similar in the 5 and 10% OM

treatments when compared to the control. However increasing OM to 15 and 20%

had a negative significant effect on Fo162 efficacy on squash (Figure 3). The gall

index increased 0.9 and 0.6 over the treatment with 5% OM respectively. In melon,

all treatments amended with OM, showed a slight decrease in the gall index when

the percentage of OM was was increased. No negative effect of OM on biocontrol

was not observed on pepper plants according to LSD test (p ≤ 0.05). Stimulation of


other microorganisms present in the rhizosphere through organic matter

amendments did not significantly affect Fo162 growth.

R2 = 0,9636

0

1

2

3

4

5

6

Control 0% OM 05% OM 10% OM 15% OM 20% OM

Organic Matter (OM) levels

Pep

per g

all i

ndex

b

a a a a

a

C

R2 = 0,9991

0

1

2

3

4

5

6

Control 0% OM 05% OM 10% OM 15% OM 20% OG

Mel

on g

all i

ndex

c

a a

a

b

a

B

R2 = 0,9511

0

1

2

3

4

5

6


Squ

ash

gall

inde

x

c

b b

aab ab

A

R2 = 0,9636

0

1

2

3

4

5

6



Pep

per g

all i

ndex

b

a a a a

a

C

R2 = 0,9991

0

1

2

3

4

5

6


Mel

on g

all i

ndex

c

a a

a

b

a

B

R2 = 0,9636

0

1

2

3

4

5

6



Pep

per g

all i

ndex

b

a a a a

a

C

R2 = 0,9991

0

1

2

3

4

5

6


Mel

on g

all i

ndex

c

a a

a

b

a

B

R2 = 0,9511

0

1

2

3

4

5

6


Squ

ash

gall

inde

x

c

b b

aab ab

A

Figure 3. Organic matter effects on the biological control activity of Fusarium oxysporum strain 162 on Meloidogyne incognita galling intensity on squash (Cucúrbita pepo L.) cv Eight Ball (A; n=15) melon (Cucumis melon) cv Mehari (B; n=15) and pepper (Capsicun annum L) cv Yolo Wonder (C; n=22) evaluated 6 weeks after J2 inoculation. Means with same letter are not significantly different according to the LSD test (p ≤ 0.05). Vertical bars represent the standard error of the mean (SEM).

27


28

4. Discussion


Root colonization of squash, melon and pepper by Fo162 were attained 27% in both

Cucurbitaceae crops and 28% in the Solanaceous crop pepper in the short time

period available for colonization of eleven days. These results on colonization are

similar to those reported in other crops. Mendoza and Sikora (2008) reported that

Fo162 colonized banana roots to a level of 25% in a 3 week period under similar

growing conditions. In melon and pepper Fo162 colonization was highest in the

upper root zone than in the other lower zones. Olivain and Alabouvette (1997)

reported that as soon as non-pathogenic Fusarium oxysporum hyphae penetrate into

the root either inside the cells or in the intercellular spaces, the cells showed defense

reactions that limits intensive colonization. At the root apex, the defense reactions

were rapid and intense, leading to cell death, and therefore the colonization was

always limited to a very few root tip cells, with the meristematic zone never being

colonized. In the differentiated zone of the root, wall appositions and thickenings,

intercellular plugging, intracellular deposits and hypertrophied cells corresponding to

defense reactions limited the extension of the fungus, both longitudinally and

centripetally. This phenomenon, described as compartmentalization (Baayen et al.,

1996 and Ouellette et al., 2002) has been reported in carnation infected by F.

oxysporum f. sp. dianthi and could explain why the Fo162 colonization in the upper

root zone of melon and pepper plants was higher than in the other two levels.


The results obtained on both the Solanaceae and Cucurbitaceae crops demonstrated

that Fo162 reduced early root penetration of M. incognita in root tissue significantly

over the non-inoculated control plants. The high levels of biocontrol found in this

investigation are probably the result of the multiple applications that led to higher and

quicker Fo162 colonization (Dababat and Sikora, 2007b). Dababat (2006) also

demonstrated that Fo162 reduced the attractiveness of the tomato host to M.

incognita and reported that fungal inoculation performed at the seedling stage induce

systemic resistance, a process subsequently detected in tomato (Dababat and

Sikora, 2007c). The high level of effectiveness in the present tests could be attributed

to the additional endophyte inoculation performed into the soil around the seedling


29

one day before nematode inoculation, which may have caused induce resistance

responses. Dababat (2006) also reported that multiple inoculations with Fo162 led to

slightly higher levels of control of M. incognita compared with a single inoculation.

Hallmann and Sikora (1996) reported that toxic metabolites produced by Fo162

reduced M. incognita mobility within 10 min of exposure and Diedhiou et al. (2003)

showed that strains of non-pathogenic F. oxysporum reduced root-knot nematode

infestation by preventing juveniles from invading the roots and by interfering with

juvenile development within the root tissue. This effect could have been attained by

the seedling inoculation directly prior to nematode inoculation in the present tests.

In this investigation, the Fo162 treatment did not affect root and shoot fresh weight

significantly on tomato, melon nor pepper. An increase was detected only on squash.

Similar results were reported by Dabatat and Sikora (2007a) on tomato. Vu (2005)

also found that Fo162 did not significantly affect banana growth two weeks after

inoculation. However, she reported that after a 14 weeks period Fo162 caused a

significant increase in banana growth. In addition, Niere et al. (1999), Menjivar (2005)

and Pocasangre et al. (2000) documented that other non-pathogenic Fusarium

strains stimulate plant growth of banana cultivars one and three months after

endophytic inoculation. Fo162 might have exhibited plant growth enhancement in the

plants tested in the present tests if evaluation was made later in the growth phase to

give the endophyte-plant interaction more time to establish.


The evaluation with the interaction between Fo162 and OM content on biological

control activity on Solanaceae and Cucurbitacea plants showed that OM did not

positively nor negatively influence of biocontrol of M. incognita on these crops. The

incorporation of OM into the pathozone did not have the desired effect of increasing

Fo162 saprophytic growth and therefore the biocontrol of the nematode in the host.

Some studies have demonstrated that soil supplemented with different amounts of

OM affect the persistence and density of some biocontrol agents thereby reducing

the speed of fungal density decline to maintain biocontrol activity (Paulitz, 2000;

Kiewnick et al., 2005; Mendoza, 2008). In the present experiments, OM did not cause

an increase in the biocontrol potential of Fo162 in squash and pepper. Conversely, in

melon an increase in biocontrol was observed. The interaction with OM is still poorly


30

understood with regards to fungi that control nematodes in the endorhiza. In the

presents test the unexpected negative effect on biocontrol detected on squash

amended with 15 and 20% OM was probably due to the increase in root growth that

could have stimulated more root exudates production and a corresponding increase

in rhizosphere competent microorganisms. The presence of higher numbers of

microorganisms in the rhizosphere would have reduced Fo162 colonisation through

competition.


31

5. Conclusion

1. The mutualistic endophytic fungus Fo162 is able to effectively colonize plants in

other crops than banana and tomato. Pepper, melon and squash were colonized

up to 27% after two inoculations of Fo162.

2. High levels of biocontrol activity of Fo162 towards the root-knot nematode

Meloidogyne incognita on both Solanaceae and Cucurbitaceae plants was

detected and demonstrated that nematode biocontrol potential is not limited to

certain plants in specific families.

3. Fo162 treatment did not generally affect root and shoot fresh weight in the plants

except for squash shoot fresh weight.

4. The capacity of Fo162 as a biocontrol agent toward the root-knot nematode M.

incognita was not significantly enhanced in squash and pepper plants by the

amount of organic matter found in the soil.

Chapter 4 Endophytes as biocontrol agent toward whitefly

32

SYSTEMIC BIOLOGICAL CONTROL ACTIVITY OF MUTUALISTIC

ENDOPHYTIC FUNGI TOWARD THE GREENHOUSE WHITEFLY

Trialeurodes vaporariorum (Westwood) IN TOMATO

1. Introduction

Whiteflies (Homoptera:Aleyrodidae) are insect pests common to greenhouse

vegetable production worldwide that affect negatively the biochemistry, physiology,

anatomy, and development of infested plants. These insects may deplete plant

reserves, reduce primary production, and cause direct phytotoxic effects and act as

vectors of important plant viruses (Yee et al., 1996; Henneberry et al., 2000; Chen el

al., 2004; Cohen and Antignus, 1982). Whiteflies may cause secondary damage

through honeydew excretion that enables sooty mould development which blocks

sunlight and reduces photosynthesis ultimately reducing yield quantity and quality

(Perkins, 1986; Henneberry et al., 1997; Yee et al., 1996; Yee et al., 1998). The

greenhouse whitefly (GHWF) T. vaporariorum (Westwood) is one of the most

important pests of ornamental and vegetable crops worldwide (Jauset et al., 1998).

As a result of the multi-component damage caused by this phloem-feeding insect

crop yield is reduced and cosmetic damage devalues fruit and ornamental qualities

(Byrne et al., 1990).

The most common measure of whitefly control in intensive agriculture is fumigation

with chemical insecticides (van Lenteren, 2000). However, the repetitive use of

chemical control against whiteflies has raised two major constraints. Firstly, the

development of resistant strains to insecticides and secondly, environmental pollution

(Fransen, 1990; van Lenteren, 2000). These limitations have encouraged the

development of alternative methods of insect control.

Biocontrol of insects is an alternative to chemical pesticide use. Mutualistic

endophytic fungi, such as Fusarium oxysporum, (Hallmann and Sikora 1996; Fuchs

et al., 1997) and Trichoderma spp (Benhamou and Chet, 1997) have been reported

to be effective biocontrol agents towards important soil borne and air borne plant


33

diseases. These investigations have increased attention concerning the potential

value of fungal antagonists in promoting plant disease resistance (Vidal, 1996).

Agents that induce resistance could also directly affect plant defence traits that

negatively affect insect preference (host plant selection, oviposition, feeding

behaviour) or performance (growth rate, development, reproductive success)

resulting in increased plant fitness in a hostile environment (Ajlan and Potter, 1991;

Gregg, 2008).

Plant resistance to arthropod herbivores is often mediated by phytochemicals that

negatively affect the feeding, growth or reproduction of the attacking pest (Karban

and Baldwin, 1997; Walling, 2000). These compounds including alkanes, alkenes,

alcohols, ketones, aldehydes, ethers, esters, and carboxylic acids (Dudareva et al.,

2004; Niinemets et al., 2004). Some of these herbivore-induced plant compounds are

known to play an important role in the interactions between plants and arthropods

(Dicke et al., 2003; Turlings and Wackers, 2004; van Poecke and Dicke, 2004;

Arimura et al., 2005). For example, metabolites may act either directly by deterring

oviposition and repelling lepidopteran herbivores (De Moraes et al., 2001; Kessler

and Baldwin, 2001) or intoxicating (Vancanneyt et al., 2001; Andersen et al., 1994)

as well as indirectly attracting natural enemies of herbivores (Turlings et al., 1990).

The induction and release of such compounds is dependent on the interaction of

abiotic factors such as wounding (Schmelz et al., 2001; Howe, 2004; Mithofer et al.,

2005) and biotic factors such as plant hormones (de Bruxelles and Roberts, 2001;

Thaler et al., 2002; Farmer et al., 2003; Rojo et al., 2003; Schmelz et al., 2003;

Ament et al., 2004; van Poecke and Dicke, 2004), herbivore-derived elicitors

(Mattiacci et al., 1995; Alborn et al., 1997; Halitschke et al., 2001; Spiteller and

Boland, 2003; Merkx-Jacques and Bede, 2004) and associated microorganisms

(Cardoza et al., 2002).


34

The existence of soil microorganisms that can induce the release of specific

compounds which can affect insect host preference in the foliage thereby diminishing

plant damage is currently unknown. This process could be of importance as an

alternative to insecticides in integrated pest management systems. The goals of the

following investigations were to:

1. Evaluate the effect of six soilborne endophytic fungi as biocontrol agents in

tomato plants toward T. vaporariorum.

2. Study the effect of these endophytes on the chlorophyll concentration index

(CCI) in the tomato leaves.

3. Determine the effect of the endophytic fungi on plant growth promotion.

4. Examine the metabolites produced by tomato leaves when colonized by

Fo162.

5. Evaluate the relationship between Fo162 and the maximum temperature

difference (MTD) on tomato leaves.


35


2.1. Efficacy of endophytic fungi toward T. vaporariorum on tomato

2.1.1. Choice test

To evaluate the effect of endophytic fungi on host choice behaviour of the GHWF T.

vaporariorum, six strains of mutualistic endophytic fungi were used (Table 1); four

from the genus Fusarium and two from Trichoderma.

Each endophytic fungus was applied with a pipette twice at a rate of 1 x 106 colony

forming units (cfu) g soil-1 to the soil used for tomato transplanting. The first

inoculation was performed in the seeding trays at sowing. The second inoculation

was performed at the day of seedling transplanting into 3 holes made around the

stem base with a plastic rod, 2 cm deep and close to the tomato roots. Tomato plants

treated with tap water were used as untreated controls. Ten days after the second

fungal inoculation one thousand 3 days old whiteflies (mixed sex) were aspirated

from the reproduction cages using an exhauster tube and released in a glasshouse

cabin over the randomized tomato plants treated with one of the six fungi or

untreated. To determine host plant choice of the whiteflies, the total number of

insects present on each plant was counted daily for 10 days after insect release. At

the end of each count, the insects were separated from the host plants by agitating

the leaves by hand and the plants were again randomized to ensure that each day

the insect had to make a new host plant selection. The endophytes, insects and

tomato plants were produced for this experiment as outlined in (chapter 2).


36

Table 1. Soilborne endophytic fungal isolates belonging to two different genera used to determine their activity as biocontrol agents toward the GHWF T. vaporariorum in tomato plants.

Endophyte ID code Origin Host plant Reference

Fusarium oxysporum

Fo162 Kenia Lycopersicon

esculentum

Hallmann & Sikora,

1994

Fusarium oxysporum P-12 Costa Rica Musa AAA Meneases 2003

Fusarium verticiloides F-14 Vietnam Oryza sativa LE et al. 2009

Fusarium sp. Bonn 7 Germany Raphanus

satibus

Xiaonin Yan personal

communication 2009

Trichoderma atroviride MT-20 Guatemala Musa AAA zum Felde 2002

Trichoderma atroviride S-2 Costa Rica Musa AAA Cañizares 2003

Absolute control

2.1.2. Chlorophyll concentration index (CCI)

Twenty days after the second inoculation or ten days after the GHWF release to

allow insect host choice, the second leaf from the top of each plant treated and

untreated with the fungal isolates and the control was examined for its chlorophyll

content index (CCI). CCI was measured 10 times per experiemtal unit using a

portable chlorophyll meter (Minolta SPAD-502) in wavebands ranging from 650 to

940 nm to determine if the isolates caused changes in CCI that influenced insect

behaviour.

2.1.3. Plant growth promotion

The experiment was terminated 21 days after the second inoculation with the

endophytes. Shoot fresh weight (g), plant height from the base of the stem to the

growing point (cm), stem diameter (mm) and root fresh weight from washed and

dried roots were recorded. Every treatment was replicated 8 times and each

experiment was conducted twice. All treatments were arranged in a completely

randomized design on a glasshouse bench at 27 ± 3ºC with 13 h/day supplemental

artificial light. Plants were watered as needed and fertilized once per week with 2%

AGLUKON.


37

2.2. Metabolite production analysis

The choice test was conducted a second time as described above with only two

treatments. One treatment had the endophyte F. oxysporum strain 162 and the other

was treated with tap water and used as an untreated control to confirm the effects

shown in the bioassay with Fo162. After the second fungal inoculation and before the

whiteflies were released for the choice test, a total of 20 plants were randomly

selected from each treatment and placed inside meshed cages (50 x 50 x 50 cm). In

each cage 5 plants were treated similarly and there were two treatments per cage,

one with fungi and one without. Ten whiteflies were released inside two of the four

cages. The plants were harvested 48 hours after releasing the insects or 12 days

after the second fungal inoculation in the respective treatment.

The first and second leaves from the top of each plant were harvested. The leaves of

each sample were surface sterilized with methanol (75%) to avoid contamination

between sub-samples and treatments. 500 mg of the mixture of first and second leaf

were transferred to polyethylene tubes in liquid nitrogen for 10 minutes to extract the

metabolites. The methodologies used for metabolites extraction and for high

pressure liquid chromatography (PR-HPLC) analysis were described in chapter 2.

2.3. Relationship between Fo162 and the maximum temperature difference (MTD) on tomato leaves

A digital infrared thermography camera was used to determine if Fo162 colonization

of the roots cause changes in the temperature in the leaf tissue as a stress response.

The plants cultivar Hellfrucht/JW Frühstamm was grown as outlined in chapter 2.

Twelve tomato plants were inoculated twice with Fo162 at rate of 1 x 106 cfu/g soil-1.

Twelve tomato plants also were treated with tap water and used as controls. All

plants were arranged in a completely randomized design on a greenhouse bench at

27 ± 3ºC with 13 h/day diurnal light.

Ten days after the second inoculation 4 treatments were prepared: 1) treated plants

under the effect of Fo162, 2) untreated plants, 3) treated plants under the effect of

Fo162 and T. vaporariorum and 4) untreated plants in the presence of T.

vaporariorum. To obtain treatments 3 and 4, six plants from each set of plants treated


38

and untreated with Fo162 were randomly selected and placed separately inside

meshed cages (50 x 50 x 50 cm). Then 100 GHWF adults were released into each

cage. Twelve days after the second inoculation and 2 days after release of GHWF,

the plants were transferred to the laboratory for thermographic meaurements.

2.3.1. Thermographic measurements

The plants were equilibrated in the laboratory for 1 hour before thermal images were

recorded between 10 and 12 am. Digital thermo images were obtained using a

VARIOSCAN 3201 ST (Jenoptic laser, jena, Germany) sterling-cooled infrared

scanning camera with a spectral sensitivity from 8 to 10 μm and a geometric

resolution of 1.5 mrad (240 x 360 pixels focal plane array and a 30ºx20º field of view

lens with a minimum focus distance of ~0.2 m) Thermal resolution is 0.03 K, and

accuracy of absolute temperature measurement less than ± 2 K. Digital thermograms

were analysed with the software package IRBIS plus V 2.2 (Infratec, Dresden,

Germany) which allowed for correction of object emissivity after images had been

recorded.


39

3. Results


3.1.1. Choice test

The two glasshouse tests showed that the inoculation of mutualistic endophytic fungi

into the soil reduced significantly the number of T. vaporariorum on tomato leaves

compared to the untreated control as seen in the significant linear regression (Figure

1). The treatment with the highest biocontrol activity was F. oxysporum strain 162.

The presence of this fungus in the roots decreased the number of T. vaporariorum on

the treated plants over that of the other fungal treatments up until the end of the

evaluation period. T. atroviride strain MT-20 and strain S-2 diminished the number of

greenhouse whiteflies at a similar level, reducing insect numbers 50% over the

control. However, at the end of the evaluation period strain MT-20 exerted a slightly

higher level of biocontrol activity than strain S-2. F. oxysporum strain P-12 and

Fusarium strain Bonn-7 reduced significantly the number of T. vaporariorum on the

treated plants one day after insects release. The biocontrol activity remained

constant until the end of the evaluation period. Nevertheless, at the end of the

experiment the mean number of insects per plant inoculated with these two strains

was not greatly affected over the non-treated control. Fusarium sp. strain F-14 was

the endophyte with the least amount of biocontrol activity. Tomato plants treated with

this fungus had more insects than the untreated control one day after releasing the

insects and this effect was constant during all the entire sampling period.


020406080

100120140160180

1 2 3 4 5 6 7 8 9 10Days after releasing 1000 GHWF adults

Num

ber o

f T. v

apor

ario

rum

Fo162 Fo. P-12 F-14 Bonn-7 Ta. S-2 Ta. MT-20 Comtrol

B R² Control = 0.51 R² Bonn-7 = 0.32R² F-14 = 0.61 R² Fo162 = 0.83R² P-12 = 0.42 R² MT-20 = 0.73

R² S-2 = 0.45

020406080

100120140160180


Num

ber o

f T. v

apor

ario

rum



R² S-2 = 0.45

020406080

100120140160180

1 2 3 4 5 6 7 8 9 10

Num

ber o

f T. v

apor

ario

rum

A R² Control = 0.58 R² Bonn-7 = 0.60R² F-14 = 0.83 R² Fo162 = 0.77R² P-12 = 0.23 R² MT-20 = 0.22 R² S-2 = 0.65

020406080

100120140160180


Num

ber o

f T. v

apor

ario

rum



R² S-2 = 0.45

020406080

100120140160180


Num

ber o

f T. v

apor

ario

rum



R² S-2 = 0.45

020406080

100120140160180

1 2 3 4 5 6 7 8 9 10

Num

ber o

f T. v

apor

ario

rum

A R² Control = 0.58 R² Bonn-7 = 0.60R² F-14 = 0.83 R² Fo162 = 0.77R² P-12 = 0.23 R² MT-20 = 0.22 R² S-2 = 0.65

Figure 1. Lineal regression of the number of Trialeurodes vaporariorum in a choice test during 10 days of sampling on tomato treated with Trichoderma atroviride strain MT-20, T. atroviride strain S-2, Fusarium oxysporum strain P-12, F. oxysporum strain 162, Fusarium sp. strain F-14, Fusarium sp. strain Bonn-7 or untreated.

3.1.2. Chlorophyll concentration index (CCI) in tomato leaves

The chlorophyll content in tomato leaves evaluated after fungal treatment was

significantly different among the seven treatments in the first experiment (Figure 2A).

The CCI in the untreated control was 43% whereas in the endophyte treatments it

ranged between 39.4 and 44.4%. However, only the treatments with Fo162 (39.8%)

and Bonn-7 (39.4%) were statistically different when compared the untreated control.

In the second experiment the CCI (Figure 2B), was not statistically different between

the treatments.

40


0

10

20

30

40

50

60

Fo162 P-12 F-14 Bonn-7 MT-20 S-2 Control

Chl

orop

hyll

conc

entra

tion

inde

x (%

) B NS

0

10

20

30

40

50

60


Chl

orop

hyll

conc

entra

tion

inde

x (%

) Aab

cd dabc a

bcdbcd

0

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40

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60


Chl

orop

hyll

conc

entra

tion

inde

x (%

) B NS

0

10

20

30

40

50

60


Chl

orop

hyll

conc

entra

tion

inde

x (%

) Aab

cd dabc a

bcdbcd

Figure 2. Chlorophyll concentration index (CCI) in tomato leaves of plants treated with one of six different endophytic fungi or untreated after endophyte inoculation. Mean with same letter are not significantly difference according to the LSD test (p ≤ 0.05; n=16). Vertical bars represent the standard error of the mean (SEM).


Plant growth promotion effects are shown in Table 2. Plant height and shoot fresh

weight increased significantly 10.4 cm and 2.2 g in the first experiment Fusarium sp.

strain Bonn-7 over the untreated control. The other endophytes treatments did not

affected plant growth significantly compared to the untreated control according to

DLS test (p ≤ 0.05). Stem diameter and root fresh weight also showed no effects in

all the treatments. In experiment II the results plant height where the Fusarium

strains Bonn-7, Trichoderma strain S-2, Fo162 with F-14 increased significantly.

Stem diameter as experiment 1, was not affected by endophytes treatments. Growth

promotion also Fusarium strain Bonn-7 and F-14 influenced positively the shoot and

root fresh weights. Both strains caused an increase in shoot weight (17 and 11%) 41


42

and root weight (15 and 30 %) respectively over the untreated control. Strain Bonn-7

induced statistically higher growth in both parameters whereas strain F-14 only

affected root weight positively. The results demonstrated a clear trend in Fusarium

strain Bonn-7 as a growth promotion agent in tomato plants over all parameters.

Table 2. Plant growth parameters of tomato cv. Hellfrucht harvested twenty days after the second inoculation of six mutualistic endophytic fungi.

Treatments Plant Height

(cm)

Stem Diameter

(mm)

Shoot fresh

weight (g)

Root fresh weight

(g)

Experiment I F. oxysporum 162 30.3 ± 0.4 ab 5.3 ± 0.1 16.3 ± 0.3 a 5.4 ± 0.3

F. oxysporum P-12 26.9 ± 2.5 a 5.6 ± 0.0 15.6 ± 0.1 a 4.9 ± 0.1

Fusarium F-14 36.6 ± 0.5 bc 5.6 ± 0.1 17.0 ± 0.4 a 7.7 ± 0.1

Fusarium Bonn-7 40.9 ± 1.2 c 5.5 ± 0.1 19.1 ± 0.5 b 4.8 ± 0.1

T. atroviride MT-20 30.3 ± 0.9 ab 5.1 ± 0.1 13.7 ± 0.4 a 4.6 ± 0.2

T. atroviride S-2 34.8 ± 1.1 bc 5.2 ± 0.1 15.8 ± 0.4 a 4.6 ± 0.1

Untreated control 30.5 ± 0.7 ab 5.6 ± 0.1 16.9 ± 0.6 a 4.6 ± 0.1

P-value 0.0042 0.1225 0.0045 0.6611 LSD 7.93408 N.S. 2.64739 N.S.

Experiment II F. oxysporum 162 26.4 ± 0.4 bc 5.1 ± 0.1 14.1 ± 0.1 bcd 4.7 ± 0.1 a

F. oxysporum P-12 24.9 ± 0.7 ab 5.3 ± 0.1 12.4 ± 0.3 a 4.1 ± 0.1 a

Fusarium F-14 26.0 ± 0.6 bc 5.4 ± 0.1 14.1 ± 0.2 cd 6.0 ± 0.1 c

Fusarium Bonn-7 28.1 ± 0.6 c 5.6 ± 0.1 14.9 ± 0.3 d 5.3 ± 0.2 b

T. atroviride MT-20 25.2 ± 0.5 ab 5.1 ± 0.1 12. 5 ± 0.3 a 4.4 ± 0.1 a

T. atroviride S-2 26.6 ± 0.4 bc 5.3 ± 0.1 12.7 ± 1.3 ab 4.2 ± 0.1 a

Untreated control 23.1 ± 0.3 a 5.3 ± 0.1 12.7 ± 1.3 abc 4.6 ± 0.1 a

P-value 0.0352 0.5547 0.0012 0.0001 LSD 2.858 N.S. 1.41422 0.64724

Means followed by different letters in the same column indicate significant differences according to the LSD test (p ≤ 0.05; n = 16).



The white fly choice experiment confirmed that treatment with Fo162 significantly

decreases insect host preference. From the first day after releasing the whitefly up

until experimental termination there were about 50% less insects in the Fo162

treated plant compared to untreated control. This difference increased over time with

a final population of 16 and 30 insects per plant in the treatments with Fo162 in both

experiments compared to 75 and 89 insects per plant in untreated control,

respectively. Pool data in lineal regression is shown in Figure 3.

0

10

20

30

40

50

60

70

80

90


Nun

ber o

f T. v

apor

ario

rum

Fo162 Untreated control R² Fo162 = 0.34 R² Control = 0.92

Figure 3. Mean number Trialeurodes vaporariorum in tomato plants treated with Fusarium oxysporum strain 162 in a choice test (○) or untreated (▲). The arrow indicates the day when the tomato plants were removed from the cages, harvested and processed for the HPLC analysis. n = 20.

The metabolites accumulation in leaves from the tomato plants treated and untreated

with Fo162 was determined by HPLC analysis. Sixty peaks per treatment were

detected by HPLC analysis of tomato leaves from the plants taken before the

whiteflies were released in the cages. Five of these metabolites showed

concentrations (areas measured in mAU,s) statistically different (p ≤ 0.05) when

compared to treatments in absence of insects (Figure 4A). These peaks had the

retention time of 7.4 (a1), 11.1 (a2), 11.6 (a3), 12.1 (a4) and 26.8 minutes (a5). While

the area of nine peaks in the treatments in presence of whiteflies had significant 43


differences between the Fo162 treated and untreated control (Figure 4B). The

retention time of these peaks were 2.46 (b1), 12 (b2), 15.9 (b3), 16.8 (b4), 24.1 (b5),

26.8 (b6), 27.2 (b7), 27.4 (b8) and 29.6 minutes (b9).

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

A

a1

a2

a3

a4

a5

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

A

a1

a2

a3

a4

a5

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

B

b1 b2

b3b4

b5 b6

b7 b8

b9

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

B

b1 b2

b3b4

b5 b6

b7 b8

b9

min5 10 15 20 25 30 35

mAU

0

100

200

300

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500

600

700

A

a1

a2

a3

a4

a5

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

A

a1

a2

a3

a4

a5

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

B

b1 b2

b3b4

b5 b6

b7 b8

b9

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

B

b1 b2

b3b4

b5 b6

b7 b8

b9

Figure 4. Peaks detected by HPLC analysis represent metabolites accumulated (areas measured in mAU,s) from tomato plants treated with Fusarium oxysporum strain 162 (----) or untreated (──) in the absence (A) or presence of Trialeurodes vaporariorum (B). Arrows indicate peaks which area were significant different according to T-test (p ≤ 0.05).

3.3. Relationship between Fo162 and MTD

Digital infrared thermography is used to measure leaf temperature to monitor

infections by microorganisms. Changes in temperature emitted by the leaves allow

discrimination between healthy and diseased plant. Four treatments were analyzed

to determine if Fo162 is able to induce this response in tomato plants in the presence

or the absence of the phloem feeding insect T. vaporariorum.

44


Differences in leaf temperature between plants treated and untreated with Fo162

were not detected through the digital infrared thermography (Figure 5). The presence

of GHWF on the plant however caused an increase in leaf temperature of 0.47, 0.34

and 0.16 degrees in the Control+GHWF, Fo162 and Fo162+GHWF treatments

respectively compared with the absolute control. Although these changes are not

statistically different they show a distinct trend in the response of the plant in the

presence of the endophyte.

45

20,2

20,4

20,6

20,8

21

21,2

Control + GHWF Fo162 Fo162 + GHWF Control

Deg

rees

NS21,4

Figure 5. Leaf temperature thermograms of tomato leaves treated and untreated with Fo162, in presence or absence of GHWF. NS: Mean are not significantly difference according to ANOVA (p ≤ 0.05; n = 6). Vertical bars represent the standard error of the mean (SEM).


46

4. Discussion


4.1.1. Choice test

This investigation revealed that the application of endophytic fungi to tomato

seedlings resulted in biocontrol activity against whiteflies. The effects of any

particular plant defence trait on arthropods depend on the specific attributes of the

plant trait and the degree of physical, biochemical or behavioural interaction between

the arthropod and the plant (Duffey, 1996; Baldwin et al., 2001). An increase in these

factors that leads to an improvement in the plant defence system against T.

vaporariorum is positively induced with the inoculation of endophytes. This was

shown in the present glasshouse investigation for Fusarium Fo162 and Trichoderma

MT-20 and S-2. The ability of these nonpathogenic mutualistic endophytic fungi

triggered plant defence reactions in the plant and negatively affected insect host

preference. The result was consistently observed with Fo162.

4.1.2. Chlorophyll concentration index (CCI)

Like many other insects, T. vaporariorum has been found to be attracted to light

transmitted and/or reflected in the blue-green-yellow region (500–600 nm) of the

spectrum (MacDowall 1972; Coombe, 1981, Coombe, 1982; Vernon and Gillespie,

1990) to green leaves (Vaishampayan et al., 1975). Variation in the perception of

plant quality and quantity of light could mean changes in the dietary nitrogen

concentration of plants for some phloem feeding insects affecting their population

density, growth and behaviour. Bentz et al. (1995) showed this result in B. tabaci on

poinsettia and Jauset et al. (1998) on the greenhouse whitefly T. vaporariorum

Westwood on tomato. Besides affecting plant morphology and growth, light also

influence the production and concentration of biochemical compounds that may be

important regulatory enzymes in the biosynthesis of compounds that allow the plant

to structure his defence system (Hahlbrock and Griesebach, 1979; Chapell and

Hahlbrock, 1984; Tegelberg et al., 2004). None of the isolates tested in the present

study demonstrated effect on the content of chlorophyll in the leaves that might be

linked in the abnormal behaviour displayed by T. vaporariorum. Hence, the effect of

lack of attraction exhibited by GHWF adults to tomato leaves colonized by Fo162 is


47

not associated with changes in chlorophyll content in the leaves but is due to other

factors.


This investigation confirmed that two mutualistic endophytic fungi, Fusarium sp.

strain F-14 and Bonn-7, were capable of enhancing plant growth. The results are in

accordance with Le et al. (2009) who reported that Fusarium sp. strain F-14

increased over 25 percent rice root weight. Xiaonin Yan, (2009) (personal

communication) also observed plant growth promotion by Fusarium strain Bonn-7 on

cucumber and squash. This plant enhancement could be explained by the fact that

these fungi have very low biocontrol activity. This may lead the non-activation of plant

response mechanisms needed for biocontrol which requires a high consumption of

plant nutrients and energy (Walters and Heil, 2007). In addition the metabolic cost of

these phytochemicals production can also be high. In particular, terpenoids are more

expensive for the plant to manufacture than other primary and secondary metabolites

due to the need for extensive chemical reduction (Gershenzon, 1994). Furthermore,

it was confirmed that the isolates F-14 and Bonn-7 fungi have the ability to stimulate

growth promotion in tomato plants but are not able to induce plant resistance.


It is known that some species of Trichoderma and Fusarium enhance the production

of potent secondary metabolites, such as trichodimerol (Evidente et al., 2009),

trichothecenes (Marasas et al., 1984), fumonisins (Nelson et al., 1992) and/or

beauvericin (Gupta et al., 1991; Logrieco et al., 1998). These compounds have been

reported to have lethal activity or repellent effects towards some sucking arthropods

(Ganassi et al., 2000; Ganassi et al., 2007). The present results of the experiment

demonstrated that isolated of Fusarium (Fo162) are also capable of induce in the

plant the production of specific metabolites which could be responsible for reduce

host whitefly preference.

Herbivore-induced plant based metabolites are known to play an important role in the

interactions between plants and arthropods (Dicke et al., 2003; Turlings and

Wackers, 2004; van Poecke and Dicke, 2004; Arimura et al., 2005). Leaves normally

release small quantities of volatile chemicals, but when a plant is damaged by

herbivorous insects, many more volatiles are released (Pare´and Tumlinson 1999).


48

These highly detectable metabolites may act by deterring oviposition by lepidopteran

herbivores (De Moraes et al., 2001; Kessler and Baldwin, 2001) or by attracting

natural enemies of herbivores (Turlings et al., 1990). Pare´ and Tumlinson (1999)

and Vourinen (2004) also report that total compounds emission from herbivore-

damaged plants could be 2.5-fold higher than that from intact plants. This suggests

that the host plant were signalled to defend themselves through the rapid stimulation

of a general cascade of non-specific defence responses (Benhamou, 1996;

Benhamou and Garand, 2001). The result of the present experiment show that root

colonization by the endophyte Fo162 stimulated the production of plant metabolites

emission, triggering changes in the blend of these organic compounds in the

absence of the phloem-feeding insect. This effect is directly related to Fo162

colonization stimulating metabolites that are released by the leaves. These changes

in metabolites concentration enable the host plant to maintain a protective barrier to

initial and over time attack. Important is the fact that induction occur without the need

for elicitors released by insects when feeding. This behaviour has been described by

Karban and Baldwin, (1997) as induced resistance due to the negative effect of the

herbivore that can result from increased levels of putative defensive primary and

secondary plant metabolites.

4.3. Termographic measurements

Considering that the maximum temperature difference (MTD) measured by

termography allows the quantitative analysis of spatial and dynamic physiological

information on plant status (Jones, 2004) as is negatively correlated with

transpiration rate and leaf temperature. Oerke et al. (2006) studded that leaf or

canopy temperatures can be used as an indicator of plant stress and stomatal

closure as shown by (Jones, 2004; Cohen et al., 2005; Grant et al., 2007). The MTD

results obtained here demonstrated that the mutualistic endophy Fo162 is able to

induce plant resistance without an increase in the transpiration rate. It furthermore

demonstrated that the mutualistic endophyte does not adversely stress the plant at

this level of detection.


49

5. Conclusion

1. Induced plant resistance to T. vaporariorum stimulated by endophytic fungal

colonization of the root system by the isolates Fo162 and T. atroviride S-2 and

MT-20 provided high levels of plant defence.

2. The lack attraction of T. vaporariorum to the host on plants colonized by Fo162

and T. atroviride S-2 and MT-20 was not related to changes in the chlorophyll

concentration index.

3. It demonstrated that only the mutualistic endophytic fungi Fusarium sp. F-14 and

Bonn-7 enhanced plant shoot and root weights, probably due to the low level of

biocontrol. The isolates Fo162, S-2 and MT-20 that produced high level of

biocontrol were not able to induce plant growth promotion.

4. The isolate Fusarium Fo162 was shown for the first time to have the capacity to

induce plant resistance in tomato plants toward the phloem-feeding insect T. vaporariorum. Using HPLC analysis distinet changes in the metabolites

production within the leaves in the absence of the insects was detected and could

be the factor whereby insect host choice was negatively affected.

5. Using digital infrared thermography it was determined that induced plant

resistance by Fo162 does not affect the transpiration rate of tomato leaves. This

demonstrated that capacity of the plants to induce defence mechanisms has a

low energy cost.

Chapter 5 Induced systemic resistance by Fo162

50

SYSTEMIC RESISTANCE INDUCED BY THE MUTUALISTIC FUNGUS

Fusarium oxysporum STRAIN 162 ON TOMATO PLANTS TOWARD

THE PHLOEM-FEEDING Trialeurodes vaporariorum .

1. Introduction

Plants have evolved various strategies to defend themselves against herbivores and

pathogens (Farag and Peré 2002; Frost et al., 2008). Although some of these

strategies are constitutive, i.e. present at all times, others are induced only in

response to herbivore feeding or pathogen infection. Constitutive defences include

physical barriers such as the leaf cuticle, cell walls, and stored metabolites known as

phytoanticipins (van Etten et al.,1994), which can negatively interfere with feeding,

growth and negatively affect development of herbivores (Walling, 2000). A wide array

of studies have documented induced defenses in plants (Schultz and Baldwin, 1982;

Haruta et al., 2001; Kessler and Baldwin, 2001/2002; van Dam et al., 2004; Miranda

et al., 2007; Frost, 2008), which include the activation of both direct and indirect

mechanisms to deter herbivores (Walling, 2000; Kessler and Baldwin, 2002). Direct

defence involves the synthesis of secondary metabolites known as phytoalexins.

These compounds are both synthesized by and accumulate in plants after exposure

to attackers (Paxton, 1980; Van Peer et al., 1991; van Etten et al., 1994; Dakora and

Phillips, 1996) and produced in both susceptible and resistant responses (van Etten

et al., 2001) thus altering insect attraction or deterrence and inhibiting insect growth

and development (Baldwin et al., 2001, Baldwin et al., 2002; Kliebenstein, 2004).

Plants can produce a remarkably diverse array of low-molecular-mass compounds

also known as secondary metabolites, of which approximately 100,000 have been

detected (Dixon, 2001). Many of these can serve as airborne semiochemicals,

stimulating or deterring interactions between plants and insect herbivores (Paré and

Tumlinson, 1999). Undamaged plants maintain a certain baseline level of metabolites

that are released from the leaf surface and/or from accumulated storage sites within

the leaf (Paré and Tumlinson, 1999). When attacked, the plant already has the

means to deter or to kill the herbivore (Gatehouse, 2002). After mechanical injuries or


51

feeding by insects, the plant can then further increase the quantity of such

compounds and in addition, systemically produce other relevant compounds

(Mattiacci et al., 1995; Alborn et al., 1997).

Non-pathogenic microorganisms like fungi and bacteria isolated from healthy plants

were demonstrated to trigger defense responses. Some of them led to induced

systemic resistance in the host plants enhancing the plant’s ability to defend itself

from pathogen and pest attack (Stirling, 1991; Alabouvette et al., 1998; Larkin and

Fravel, 1999; Pereira et al., 1999; Trouvelot et al., 2002; Vu et al. 2006; Sikora et al.,

2007, Sikora et al., 2008). It also was shown that plant roots, colonized by the

endophytic Fusarium oxysporum strain 162, induce systemic resistance responses in

tomato against the root-knot nematode, Meloidogyne incognita. It was suggested that

the direct endophyte-plant interaction systemically alters root exudate patterns in the

root tissue system (Vu et al 2006; Sikora et al., 2007; Sikora et al., 2008).

Whiteflies (Hemiptera : Aleyrodidae) are generally highly polyphagous pest insects

and cause significant damage in subtropical and temperate agricultural systems by

feeding on phloem sap of the plant. In addition to this direct damage, whiteflies can

transmit destructive pathogenic plant viruses (Gerling, 1990; Gerling and Mayer,

1995) and cause fungal growth on plant surfaces due to the excretion of honey dew

(Gelman et al., 2002). Whiteflies attack of more than 600 different species of plants in

both field and greenhouse settings, including food, fiber and ornamental species

(Gelman et al., 2005) and cause billions of dollars of crops looses each year (Heinz,

1996; Henneberry et al., 1997; Chu and Henneberry, 1998). Of all species of

whiteflies, the greenhouse whitefly (GHWF) Trialeurodes vaporariorum (Westwood)

is a very serious pest of plants especially vegetables and ornamental crops grown in

greenhouses (van Lenteren and Martin, 2000). GHWF is a polyphagous species able

to attack 249 genera in 84 angiosperm plant families (Russell, 1977).


52

In the present study the aims were to evaluate whether or not the endophytic fungus

F. oxysporum strain 162 is able to:

1. Trigger antixenosis conditions in the host against the phloem-feeding insect T.

vaporariorum.

2. Affect the reproduction and development of the GHWF and

3. Induce chemical alterations in the plant leaves that might affect insect behaviour.


53


General methodology used for the preparation of soil, production of tomato plants,

reproduction of the GHWF T. vaporariorum as well as the reproduction and

inoculation of the endophyte Fo162 is described in chapter (2).

2.1. Host preference

A choice test was carried out to assess the GHWF feeding and egg laying preference

between an endophyte treated and untreated host. Ten days after the second fungal

inoculation with Fo162 the plants were placed in meshed cages (50 X 50 X 50 cm).

Each cage contained 8 tomato plants, four inoculated with Fo162 and four control

plants, in a random design. Five hundred 3 days old adult whiteflies (mixed sex) were

collected from the reproduction boxes by using an exhauster tube and released in

each cage. The cages were placed in a glasshouse set at 27 ± 7°C with a diurnal

cycle of 13 hrs. light to 11 hrs. darkness. Two days after insect release, the total

number of insects and deposited eggs on each plant were counted. This experiment

was performed with 8 replicates and conducted twice.

2.2. Reproduction experiment

To evaluate the effect of the presence of the endophyte in tomato plants on the life

cycle of the GHWF, five hundred 3 days old adult whiteflies (mixed sex) were

released as outlined above. Two days after their release, the adults were removed

and the number of eggs deposited on the second leaf from the shoot tip counted.

Then the development of the eggs and emerging nymphal stages were followed over

time. The experiment was performed with 15 replicates and conducted twice.

2.3. Metabolite accumulation in tomato leaves

To determine whether Fo162 treated plants alter the accumulation of compounds

within tomato leaves and whether these compounds are associated with the

presence of different densities of GHWF, a total of 20 Fo162 treated and 20

untreated control tomato plants were placed inside 4 meshed cages, each cage

containing 10 plants (5 plants per treatment) in a random design. The first meshed

cage did not contain insects. Inside the second, third and fourth meshed cages, 10,

20 and 50 whiteflies per plant were released, respectively. The leaves were collected

13 days after the second fungal inoculation, which corresponded with 72 hours after


54

insect release. The presence of soluble compounds in the first and second young

leaves of each plant was evaluated through RP-HPLC analysis as described in

chapter (2).

2.4. Relationship between the accumulation of compounds in the leaf and T. vaporariorum development

To detect a possible correlation between the different stages of the insect and

specific soluble leaf compounds, the development of the GHWF population on the

host and the accumulation of the compounds produced in Fo162 treated and

untreated control plants were evaluated at four different time intervals. Ten days after

of the second inoculation with Fo162, treated and untreated plants were placed in a

random design in the glasshouse. Approximately 1500 GHWF adults were released

and after 5, 10, 14 and 18 days, 6 plants of each treatment were collected. The

number of individuals per developmental stage were counted and removed from the

first and second leaves. These leaves were then processed for RP-HPLC analysis as

described in chapter (2). The insect counting and RP-HPLC data were combined and

statistically correlated.


55

3. Results


A choice test was performed to identify the insect preference on Fo162 treated or

untreated plants. Therefore 500 GHWF were released in meshed cages containing 4

Fo162 inoculated and 4 control plants. Two days after insect release, the average

number of insects and eggs per plant were determined. The numbers of GHWF

adults and eggs found on the control plants were three and two times higher than

those found on the Fo162 inoculated plants, respectively (Figure 1). According to T-

test (p ≤ 0.05), these results show a clear and significant host preference of the

GHWF for the untreated plants. They also layed more eggs on the control plants

when compared to the Fo162 treated plants. However, more eggs per adult were

deposited on the Fo162 inoculated plants when compared to the control plants.


0

10

20

30

40

50

60

70

80

Fo162 Control

Mea

n nu

mbe

r of T

. vap

orar

ioru

m e

ggs B

0

5

10

15

20

25

30M

ean

num

ber o

f T. v

apor

ario

rum

adu

lts A

0

10

20

30

40

50

60

70

80

Fo162 Control

Mea

n nu

mbe

r of T

. vap

orar

ioru

m e

ggs B

0

5

10

15

20

25

30M

ean

num

ber o

f T. v

apor

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rum

adu

lts A

*

*

Figure 1. Choice assay indicating the mean number of adults (A) and eggs (B) of Trialeurodes vaporariorum on tomato plants treated and untreated with the non-pathogenic endophytic fungus Fusarium oxysporum strain 162, two days after releasing the insects. Columns in the same group with (*) are significantly different based on T-test (p ≤ 0.05; n = 16). Vertical bars indicate the standard error of the mean (SEM).


Twelve days after releasing five hundred insects into meshed cages, containing

either Fo162 inoculated or untreated control plants, the different development stages

on the second leaf from the shoot tip of each plant were determined. On the Fo162

inoculated plants the number of insects in the various developmental stages was

greatly reduced. The number of the 2nd (57 versus 76%) and 3rd (50 versus 75%)

nymphal stages and total eggs (48 versus 72%) that were able to complete their life

cycle and reach the adult stage were lower and statistically different according to T-

test (p ≤ 0.05) in Fo162 treated compared to untreated plants respectively as shown

in the pool data (Figure 2). The other stages showed no significant differences

56


between the treatments. The results indicated that the development of the GHWF is

delayed when the endophyte is present in the root system.

0102030405060708090

100Eg

gs

Cra

wle

r

Nyn

phal

2°

Nyn

phal

3°

Nyn

phal

4° Adul

ts

Egg

s to

Adul

ts

Eggs

Cra

wle

r

Nyn

phal

2°

Nyn

phal

3°

Nyn

phal

4° Adul

ts

Egg

s to

Adul

ts

Treated Fo162 Untreated control

Rep

rodu

ctio

n pe

rcen

t (%

) of

Tria

leur

odes

vap

orar

ioru

m

***

Figure 2. The presence of different developmental stages of T.vaporariorum on Fo162 inoculated tomato plants compared with untreated control plants, 24 days after releasing 1500 adult insects. Columns indicated with (*) are significantly different from the columns of the control, based on a T-test (p ≤ 0.05; n = 30). Vertical bars indicate the standard error of the mean (SEM).

3.3. Metabolite accumulation in tomato leaves

The accumulation of metabolic compounds within leaves of Fo162 inoculated and

control tomato plants, both in the absence and presence of different GHWF densities,

was evaluated. The plants were harvested 72 h after insect release and analyzed by

HPLC. The number of compounds which could be detected in leaves exposed and

not exposed to different insect densities was not statistically different according to

ANOVA (p ≤ 0.05). However plants treated with Fo162 that were not exposed to

insects showed 10 individual peaks that accumulated to levels which were

significantly higher compared to the control plants (Fig. 3A). When 10 GHWF were

released on to Fo162 treated plants, twelve peaks differing in accumulation when

compared to the control plants were detected (Fig. 3B). Only the peaks with retention

times of 4.2, 8.7, 9.4, 9.8, 10.4, 11.2, and 11.9 minutes were simultaneously found in

the absence insects. When 20 insects were released, nine individual peaks were

detected (Fig. 3C). The peaks with retention times of 8.7, 11.3, 11.9 and 12.6 were

found both in the absence and or presence of 10 GHWF. The 8.2 and 12.7 minute

57


peaks differed with insect density. After 50 insects were released, fifteen peaks

showed a difference in height between Fo162 treated and untreated plants (Fig. 3D).

Ten of those peaks were present in at least one of the other treatments. These

results indicated that both inoculation with Fo162 and the insect are able to change

the accumulation of metabolic compounds in the tomato leaves.

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

Fo 162 Control A

* *

*

* * *

* *

*

*

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

B

**

**

*

*

*

*

*

* **

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

C

****

*

* *

* *

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Metabolites at specific time retention (min)

Pea

k ar

ea (m

AU

,s)

D

* ***

*

*

*

*

*

* *

* * * *

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

Fo 162 Control A

* *

*

* * *

* *

*

*

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

B

**

**

*

*

*

*

*

* **

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

Fo 162 Control A

* *

*

* * *

* *

*

*

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

B

**

**

*

*

*

*

*

* **

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

C

****

*

* *

* *

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3


Pea

k ar

ea (m

AU

,s)

D

* ***

*

*

*

*

*

* *

* * * *

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3

Pea

k ar

ea (m

AU

,s)

C

****

*

* *

* *

0

1000

2000

3000

4000

5000

6000

2 2,16 2,25 3,44 4,28 5,54 6,7 7,71 8,29 8,72 9,44 9,83 10,2 10,5 11,3 11,9 12 12,7 13,4 16,2 23,5 27,4 29,8 32,3


Pea

k ar

ea (m

AU

,s)

D

* ***

*

*

*

*

*

* *

* * * *

Figure 3. Accumulation of metabolites from young tomato leaves, treated and untreated with F. oxysporum strain 162, 72 hours after releasing the GHWF. (A) plants that were not exposed to GHWF (B, C and D )were exposed to 10, 20 and 50 insects per plant, respectively. Peak area that are statistically different from the control (T-test: p ≤ 0.05 and n = 6) are indicated by an asterisk. Vertical bars indicate the standard error of the mean (SEM).

58


3.4. Relationship between the accumulation of compounds in the leaf and T. vaporariorum development

To determine whereas or not a correleation exists between GHWF developmental

and the accumulation of compounds produced within the leaves of Fo162 treated and

untreated tomato, plants were evaluated at 4 different time intervals. The results

showed that the number of adults and eggs was significantly higher on the untreated

control plants when compared to the Fo162 treated plants at all time points (Fig. 4).

Ten days after insect release, the number of crawlers found on untreated plants was

higher than on Fo162 treated tomato plants. Nymph 2 numbers after 14 and 18 days

and nymph 3 numbers after 18 days were significantly decreased in Fo162 treated

plants when compared to the controls. The results demostrated that root treatment

with Fo162 reduces the reproduction rate of the insec in the shoot by disrupting the

speed of development of the various GHWF life stages.

050

100150200250300350400

Adu

lts

Egg

s

Adu

lts

Egg

s

Cra

wle

r

Adu

lts

Egg

s

Cra

wle

r

Nyn

phal

2° Adu

lts

Egg

s

Cro

wle

r

Nyn

phal

2°N

ynph

al3°

5 days 10 days 14 days 18 daysDays sampled after released 1500 GHWF

Mea

n nu

mbe

r of s

tage

san

d in

star

s co

unte

d

Treated Fo 162 Untreated

*

*

*

*

*

*

* *

*

*

**

Figure 4. Presence of T. vaporariorum life stages on Fusarium oxysporum strain 162 treated and untreated tomato leaves, evaluated 5, 10, 14 and 18 days after releasing 1,500 GHWFs. Columns in the same group indicated with (*) are significantly different based on T-test (p ≤ 0.05; n = 6). Vertical bars indicate the standard error of the mean (SEM).

To elucidate whether the accumulation of certain compounds in the leaves concurred

with the delayed insect development, a correlation analysis with both parameters was

performed. Five days after releasing the insects, eight peaks were identified that

differed when the Fo162 treated plants were compared with the control plants (Fig.

5A). Ten, fourteen and eighteen days after insect release 10, 3 and 4 peaks with a

59


60

significant difference in area were identified, respectively(Fig. 5B-D). Of those, nine

peaks were correlated with the presence of the adults in at least one of the time

point. Twelve were correlated with egg stage and only four to crawler and nymph 3

stages. The compounds with a retention time of 2.2 and 2.3 minutes showed the

most consistent effect with regard to the presence of the different life stages of the

insect. The first was negatively correlated to the presence of adults, crawlers and

nymph 3 stages, whereas the second was positively correlated with adults and eggs

stages and negatively correlated to both the crawler and nymph 3 stages.


0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

*

***

*

*

*

* *

B

*

02000400060008000

100001200014000160001800020000

Pea

k ar

ea (m

AU

,s)

Fo 162 Control

* **

*

*

*

* *

A

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

* *

*

C

02000400060008000

100001200014000160001800020000

1,9 2,2 2,3 5,4 6,6 7,2 8,3 8,7 9,4 9,9 10,8 11,2 11,6 15 27,6 29,8


Pea

k ar

ea (m

AU

,s)

*

*

**

D

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

*

***

*

*

*

* *

B

*

02000400060008000

100001200014000160001800020000

Pea

k ar

ea (m

AU

,s)

Fo 162 Control

* **

*

*

*

* *

A

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

* *

*

C

02000400060008000

100001200014000160001800020000

1,9 2,2 2,3 5,4 6,6 7,2 8,3 8,7 9,4 9,9 10,8 11,2 11,6 15 27,6 29,8


Pea

k ar

ea (m

AU

,s)

*

*

**

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

*

***

*

*

*

* *

B

*

02000400060008000

100001200014000160001800020000

Pea

k ar

ea (m

AU

,s)

Fo 162 Control

* **

*

*

*

* *

A

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

*

***

*

*

*

* *

B

*

02000400060008000

100001200014000160001800020000

Pea

k ar

ea (m

AU

,s)

Fo 162 Control

* **

*

*

*

* *

A

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

* *

*

C

02000400060008000

100001200014000160001800020000

1,9 2,2 2,3 5,4 6,6 7,2 8,3 8,7 9,4 9,9 10,8 11,2 11,6 15 27,6 29,8


Pea

k ar

ea (m

AU

,s)

*

*

**

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Pea

k ar

ea (m

AU

,s)

* *

*

C

02000400060008000

100001200014000160001800020000

1,9 2,2 2,3 5,4 6,6 7,2 8,3 8,7 9,4 9,9 10,8 11,2 11,6 15 27,6 29,8


Pea

k ar

ea (m

AU

,s)

*

*

**

D

Figure 5. Peaks area (mAU,s) detected by HPLC analysis from tomato leaves sampled in 4 different times (A, 5 days; B, 10 days; C, 14 days and D, 18 days) after releasing 1,500 GHWF on control plants or plants inoculated with Fusarium oxysporum strain Fo162. Columns indicate the time retention of the compounds identified at A (2.19, 2.26, 7.20, 9.88, 10.75, 11.24, 11.57 and 27.59); B (2.19, 2.26, 5.44, 6.64, 7.20, 8.27, 8.70, 9.88, 15.01 and 29.76); C (1.95, 8.69 and 27.59) and D (2.26, 8.23, 9.44 and 10.751), Peaks marker with an asterisk are significantly different when compared to the control, based on a T-test (p ≤ 0.05; n = 6). Vertical bars indicate the standard error of the mean (SEM).

61


62

4. Discussion


It is well established that direct and indirect plant defences are involved in response

to herbivory and other biotic stresses (Karban et al., 2000; Thaler et al., 2001;

Schultz and Baldwin, 1982; Haruta et al., 2001; van Dam et al., 2004; Miranda et al.,

2007). However, most of these previous studies have focused on plant-derived cues

associated with abiotic factors such as wounding, where the presence of herbivores

feeding can conceivably serve as a signal to induce priming (Takabayashi and Dicke,

1996; Schmelz et al., 2001; Howe, 2004; Mithöfer et al., 2005; Frost et al., 2008).

Nothing, however, is known about induced systemic resistance due to the presence

of no-pathogenic fungi growing endophytically in the endorhiza of a host on the

accumulation of compounds within leaves and their influence on the behaviour of

phloem-feeding insects. The results of the present choice test showed that the

introduction of the endophytic fungus, Fusarium oxysporum strain Fo162, through

soil inoculation induced systemic biocontrol activity against whiteflies in tomato

leaves. Host preference of the flies and oviposition were 79% and 74 % higher on

control plants when compared to the Fo162 inoculated plants, respectively.


It has been documented that chemical changes in the cues emitted by the plant, are

induced by the interaction with an endophyte (Cardoza et al., 2002; Cardoza et al.,

2003) and that these changes are perceived by the insect and could play an

important role during insect recognition of a host (Masson and Mustaparta, 1990;

Schütz et al., 1997; Dicke et al., 2003; Bruce et al., 2005). The insect detects these

olfactory cues during orientation, host selection, landing and at ovoposition (Landolt,

1993; De Moraes et al., 2001). Bleeker et al. (2009) established that B. tabaci

preference behaviour is indeed influenced by compounds in the shoot of tomato

plants. The effects of metabolites on decreasing oviposition rates and increased egg

predation on the emitting plant has also been demonstrated (De Moraes, 2001;

Pichersky and Gershenzon, 2002; Pöykkö et al., 2005). Both visual and olfactory

cues play a predominant role. In the case of whiteflies the olfactory stimuli associated

with the host plant initiate host targeting, whereas visual cues improve the accuracy

of landing (Visser, 1988). These are reasons for this tipe of interaction. The immature


63

stages of whiteflies, for exymple, have limited dispersal abilities as compared to the

adults. In fact, only the crawler stage has very limited dispersal capabilities (Walker

and Zareh, 1990). The choice of an oviposition site by an adult female can thus be

critical for developmental speed and survival of the offspring. In the present study

inoculation of tomato plants with Fo162 reduced the speed of development, which

may well be due to antixenosis that is induced by the presence of the endophyte in

the root (Reitz and Sikora, 2001; Sikora, 1992; Waceke et al., 2001; Sikora et al.,

2003).

In a previous study working with several commercial varieties of tomato containing

the Mi-1.2 gene which provides resistance to nematodes (Meloidogyne spp.) and to

the potato aphid Macrosiphum euphorbiae, Rossi et al. (1998), Nombela et al. (2001)

and Nombela et al. (2003) indicated that the Mi gene may also be involved in partial

resistance to the Q-biotype of B. tabaci due to the existence of antixenosis and

antibiosis responses against this insect. Using transcriptome analysis after Bemissia

tabaci (SLWF) feeding in Arabidopsis, Zarate et al. (2007) and Kempema et al.

(2007) found a similarity between plant responses to phloem-feeding whiteflies and

pathogens. The lack of attractiveness of plant parasitic nematodes (Vu et al 2006;

Sikora et al., 2007 and Sikora et al., 2008) and herbivores to Fo162 treated plants, as

seen in the present study, is clearly related to physiological changes in the root

(exudates-nematodes) and in the leaves (metabolite accumulation-insects) that

affects systemically pest behaviour to the host plant.

4.3. Metabolite accumulation and relationship in tomato leaves toward T. vaporariorum

Natural resistance of tomato to many herbivores is attributed to the synthesis of both

constitutive and inducible defensive compounds (Farrar and Kennedy, 1992) (Ryan

1987; Rosenthal and Berenbaum, 1992; Takabayashi et al., 1994, Peré and

Tumlinson, 1997a; Peré and Tumlinson, 1997b, Agelopoulos and Pickett, 1998;

Kessler and Baldwin, 2001; Pohnert, 2002). Changes in the behaviour and

concentration of compounds within leaves in the presence of the insect were also

reported by Pohnert (2002) and Gatehouse (2002). The effect of a particular

compound on arthropods depends on the specific activity of the compound and the

physical, biochemical, and behavioural interaction between the arthropod and the


64

plant (Duffey and Stout, 1996; Baldwin et al., 2001). In the present experiments with

the plant-endophyte association in the roots the result showed accumulation of

compounds within the leaves. The result indicated possible activated systemic

defence mechanisms in the absence of the herbivore and which is further increased

in the presence of the GHWF. Although, the plant defence system against T.

vaporariorum is positively affected by inoculation with the endophyte and Fo162

induced alterations in the accumulation of specific compounds, it is difficult to

determine the role of these compounds in insect behaviour at this time. More work is

needed on this interaction that might allow a better understanding of how Fo162

treated plants improve systemic chemical defences that negatively affect the phloem-

feeding GHWF T. vaporariorum.


65

5 Conclusions

1. The mutualistic non-pathogenic fungus F. oxysporum strain 162 has showen to

be able to stimulate changes in tomato leaves that are detrimental toward the

phloem-feeding insect T. vaporariorum reducing both host selection and

oviposition.

2. The mutualistic endophyte Fo162 influenced negatively the reproduction rate of T.

vaporariom on tomato by disrupting different stages of the insect life cycle

especially the nymphal instars 2th, 3th and total eggs that were able to complete

their life cycle and reach the adult stage.

3. Fo162 Inoculation in tomato plants did not produce lead to accumulation of new

chemical compounds within leaves but was able to trigger changes in the

concentration of 10 metabolites in the absence of the insects. After release of

different densities of whiteflies the number of metabolites affected increased to 15

as possible respond to the insect feeding alone.

4. The metabolites affected by inoculation of the isolate Fo162 showed a strong

correlation between concentration and reduction in the reproduction rate of all

stages of T. vaporariorum.

5. The mutualistic endophyte was shown to enhance the mechanisms of defense of

tomato, increasing the biological control capacity toward the phloem-feeding

insect T. vaporariorum.

Chapter 6 Mechanisms of resistance induced by Fo162

66

MECHANISMS AND REACTION OF PHLOEM-FEEDING INSECTS TO

INDUCED SYSTEMIC RESPONCES OF SOLANACEAE AND

CUCURBITACEAE HOST PLANTS INCREASED BY THE MUTUALISTEIC

ENDOPHYTE Fusarium oxysporum STRAIN 162

1. Introduction

The relationship between phloem-feeding insects and plants has been studied and

offers an intriguing example of a highly specialized biotic interaction (Gerling, 1990;

Walling, 2000; Martines et al., 2003; Kaloshian and Walling, 2005; Powell et al.,

2006; Kempema et al., 2007; Walling, 2008). These insects according to

Schoonhoven et al. (2007) have evolved to survive on a nutritionally unbalanced diet

of phloem sap, and to minimize wound responses in their host plants due to precise

and selective feeding habits allowing them to avoid allelochemicals and indigestible

compounds that are more abundant in other plant tissues. As a consequence, plant

perception of, and responses to phloem-feeding insects differ from plant interactions

with other insect-feeding guilds (Brown and Czosnek, 2002; Jones, 2003; Thompson

and Goggin, 2006).

During compatible interactions, plants perceive the amount of tissue damage, the

quality and quantity of salivary signals (effectors), and the magnitude of electrical

and/or hydraulic signals caused by hemipteran attack (Walling, 2000). Thompson

and Goggin (2006) mentions that after integration of this suite of signals, plants

deploy signal transduction pathways to regulate large cohorts of genes to provide the

best defense response to its intruder. Many induced genes appear to address the

changes in physiological status imposed by hemipteran feeding and defense-

response genes are activated or suppressed Almost without exception,

pathogenesis-response (PR) genes, RNAs, proteins, and/or activities of these are

elevated after phloem-feeding insect attack (Walling, 2000). A good example is the

Mi gene in tomato (Lycopersicon esculentum), which confers resistance to root-knot

nematodes (Meloidogyne spp.), potato aphid Macrosiphum euphorbiae), and

sweetpotato whitefly biotypes B and Q (Bemisia tabaci; Milligan et al., 1998; Rossi et


67

al., 1998; Vos et al., 1998; Nombela et al., 2003). The Mi gene encodes a classical

resistance (R) protein homologous to proteins conferring resistance against viruses,

bacteria, and fungi (Milligan et al., 1998).

Herbivore damage is limited by a wide variety of constitutive or induced plant

defences. Constitutive traits such as preformed chemical defences are expressed

prior to insect damage and frequently have deterrent effects on insect settling or

feeding behaviours (Thompson and Goggin, 2006). These defenses can influence

herbivore settling, feeding, oviposition, growth and development, fecundity, and/or

fertility (Walling 2000). Plants can display phenotypic plasticity even in preformed

defences. Trichome densities, for example, may increase in response to prior

herbivory (Traw and Bergelson, 2003). Other forms of insect resistance depend upon

far more rapid defence responses that are expressed only under herbivore pressure

(Koornneef and Pieterse 2008). A good example is the case of race-specific innate

resistance, where plants carry a particular resistance gene (R gene) and recognize a

corresponding avirulence gene product in the pest, resulting in an incompatible

interaction (Flor, 1971; Lapitan et al., 2007).

The mutualistic endophyte Fusarium oxysporum strain 162 (Fo162) has been shown

to be able to induce systemic resistance (ISR) by initiating systemic changes in

banana and tomato plants affecting root exudates that reduced the attraction or

penetration of the migratory endoparasitic nematode Radopholus similes and

Meloidogyne incognita (Vu, 2005; Vu et al 2006; Dababat and Sikora, 2007a).

Decreased mobility and hatching as well as antibiosis through toxic metabolites have

also been attributed to this isolate (Hallmann and Sikora, 1996; Amin, 1994; zum

Felde, 2002; Meyer, 2004; Dababat 2006; Sikora et al., 2007).


68

Considering that the relationship between plant-hemiptera is more analogous to a

plant-biotrophic pathogen interaction (Walling 2000; Klingler et al., 2005), as well as

the ability of Fo162 to produce toxic metabolites and induce systemic resistance

against pathogen, the aims of this study were to:

1. Evaluate the capacity of Fo162 to induce antixenosis conditions toward T.

vaporariorum.

2. Investigate the influence of Fo162 on the population dynamics of the Aphids

Aphis gossypii and Myzus persicae.

3. Test the toxic effect of metabolites produced by the isolate Fo162 toward the

Aphis gossypii.

4. Investigate the systemic activity of Fo162 toward T. vaporariorum, Aphis gossypii

and M. persicae.


69

2. Experimental design

General methodology used for the preparation of soil, production of squash

(Cucurbita pepo) cv. Eight Ball, melon (Cucumis melon) cv. Mehari and pepper

(Capsicum annuum) cv. Yolo Wonder plants, and reproduction of the phloem-feeding

insects T. vaporariorum, A. gossypii and M. persicae as well as the endophyte Fo162

as described in chapter (2).

2.1. Influence of ISR on host preference

To evaluate the influence of the endophyte on the behaviour of T. vaporariorum on a

squash and melon, the soil substrate was treated with Fo162 inoculated twice at a

rate of 1 x 106 colony forming units (cfu) g soil-1, as described in chapter (2). Squash

and melon plants not treated with Fo162 but treated with tap water were used as

untreated controls. Ten days after the second fungal inoculation two thousand 3 days

old whiteflies (mixed sex) were aspirated from the reproduction boxes using an

exhauster tube and one thousand insects were released per host in separated

glasshouse cabins over the randomized squash and melon plants respectively. To

determine host preference, the total number of insects present on each plant was

counted daily for nine days beginning two days after released. At the end of each

count, the insects were separated from the host plants by agitating the leaves by

hand and the plants were again randomized to ensure that each day the insect had a

new selection of host plants. The experiment was conducted in screened-cages on a

glasshouse bench at 27 ± 7°C with 13 h per day supplemental artificial light. The

treatments were replicated 8 times and each experiment was conducted twice.

2.2. Reproduction

To evaluate the effect of the endophyte Fo162 on A. gossypii in squash and melon

and on M. persicae in pepper host, three experiments separately were designed. Ten

days after the second inoculation of Fo162, ten wingless aphid adults per plant in

clip-cages were attached to the abaxial side of the 4th leaf. After 48 h exposure to the

insects, the clip-cages were removed carefully to avoid damage to the leaft tissue.

The host preference of A. gossypii on squash and melon was evaluated 2 weeks

after release of the ten wingless aphid adults on the abaxial side of the 4th leaf. The

leaf surface was categorized using the following scale: zero ≤ than 10 individuals;


70

one = population > 10 and up to 25 % of the leaf surface colonized; two = 26% to

50% of the leaf surface area colonized and; three > 50% of the leaf surface area

colonized.

To effect of Fo162 toward M. persicae on pepper plants was measured 24 days after

release of ten wingless aphid adults on the abaxial side of the 4th leaf. The total

number of aphids was counted and the fundamental net reproductive rate (growth

factor) as proposed by Begon et al. (1990) was calculated. The equation Nt = N0 × Rt,

was used where: N0 is the initial number of aphids and Nt the number of aphids after

t-days. A growth factor with the value one represents a treatment that did not affect

the insect within the period of time evaluated. If the factor is smaller than one, the

treatment applied reduced the population growth parameter, and if the factor is

higher than one, the treatment has a positive affect on the growth factor. Each

treatment was replicated 6 times and the experiment was repeated once.

2.3. Antibiosis test

2.3.1. Metabolite production

To determine the effect of metabolites produced by Fo162 toward A. gossypii, the

reproduction index (RI) used by Chaman et al. (2003) was evaluated. The equation

used for determinations the RI of aphids was calculated as (RI = Pf / Pi), where Pf

represent final population and Pi the initial population. The endophyte was first

cultured on PDA in an incubator at 25°C for two weeks. Five 1 cm plugs of PDA with

fungal mycelia were added to flasks containing 200 ml of PDB. The flasks were

shaken at 100rpm in darkness at 25°C for two weeks. Fungal mycelia were removed

from the broth by filtration through 3 layers of cheese cloth. The suspension was then

centrifuged at 5000 rpm for 20 min at 20°C to remove mycelia and the supernatant

then filtered through a combination of micro-filters of 0.45µm and 0.20 µm pore size.

Streptomycin sulphate (0.3 g) was added to the solution so that the final antibiotic

concentration was 150ppm to prevent microbial growth. Fo162 used was produced

as described in chapter (2).


71

2.3.2. Bioassay

The experiment was conducted under sterile conditions. Squash leaf discs (60 mm

diameter) were treated with 10 wingless adults of A. gossypii and placed in Petri

dishes (60 x 15 mm) on water agar 7 g L-1 (Merich Microbiology Agar-Agar). Five

treatments were evaluated: 1) 1 ml of Fo162 metabolite suspension, 2) 2 ml of Fo162

metabolite suspension, 3) 4 ml of Fo162 metabolite suspension 4) sterile water and

5) control filtrate from un-inoculated PDA and a filtrate from PDB medium as a

second control. The liquid suspension of each treatment was sprayed using a fine

(Eco Spray) vaporizer. To ensure that each leaf disk was treated with the same

amount of solution the disks were placed in a cylinder with a volume of 2.8 L before

the application of the suspension and then removed after 6 minutes exposure to the

spray. The squash leaf dishes were sprayed, and then stored for 3 and 24 hours in

an incubator at 25°C ± 2 with at a relative humidity of 60%, and at a light intensity of

2000 Lux with a light/dark photoperiod of 16 h/8h. The growth factor described above

for the reproduction experiment was calculated. Each treatment was replicated 8

times and the experiment was repeated once.

2.4. ISR effect on metabolites production in leaves

The experiment was carried out separately for each of the following treatments: 1)

plants treated with Fo162, 2) untreated plants, 3) treated with Fo162 and respective

phloem-feeding insect and 4) untreated plants and the presence of the respective

phloem-feeding insect. Insects were settled in the respective treatment 10 days after

the second Fo162 inoculation. Soil treatment and plant production is as described in

chapter (2).

2.4.1. HPLC analysis

To separate and measure (mAU,s) metabolite production in the leaves of squash and

pepper, a HPLC was conducted 48 h after the placement phloem-feeding insect on

the plants. The first and second squash leaves from the top of the shoot were

evaluated in the experiment with T. vaporariorum. In the study with aphids, the fourth

leaf of squash and pepper from the top of the shoot after 48 h exposed to A. gossypii

and M. persicae respectively were evaluated. The methodology used to analyze the

accumulation of the metabolites within the leaves was described in chapter (2).


72

T. vaporariorum on squash plants

Ten days after the second fungal inoculation with Fo162 the squash plants were

placed in meshed cages (50 x 50 x 50 cm). Each cage contained 3 plants of the

same treatment and there were two treatments per cage; one with Fo162 and the

other without. Ten GHWF per cage were released into two of the four cages.

Approximately 48 hours after the insects had been released the top two leaves were

harvested for a HPLC analysis.

Aphids on squash and pepper plants

To assess response of squash and pepper plants treated and untreated with Fo162

and with or without the phloem-feeding insects A. gossypii and M. persicae, 10

wingless aphids were settled on their respective host in clip-cages on the abaxial

side of the 4th leaf in messed cages in the greenhouse. Twenty four hours later the

clip-cages were removed to avoid damage to the foliar tissue. After 48 h of exposed

the aphids to the respective host, the 4th leaf of each plant was harvested for a HPLC

analysis.


3. Results


The first glasshouse preference experiment on squash plants with Fo162 soil

treatment led to a significant reduction (T- test p ≤ 0.05) in host selection of T.

vaporariorum or a 65% reduction in the number of insects over the untreated control

for 9 consecutive days (Figure 1A). After release the GHWF the population

decreased steadily and strongly. Similar behaviour was obtained in the second

experiment (Figure 1B) where approximately 80% of the GHWF remained on

untreated plants when compared to plants treated with Fo162 over the 9 of sampling

dates.

0

50

100

150

200

1 2 3 4 5 6 7 8 9

Num

ber o

f T. v

apor

ario

rum Fo 162 ControlA R² Fo162 = 0.16

R² Control = 0.20

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9

Days after releasing 1000 GHWF adults

Num

ber o

f T. v

apor

ario

rum

BR² Fo162 = 0.42 R² Control = 0.88

0

50

100

150

200

1 2 3 4 5 6 7 8 9

Num

ber o

f T. v

apor

ario

rum Fo 162 ControlA R² Fo162 = 0.16

R² Control = 0.20

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9


Num

ber o

f T. v

apor

ario

rum

BR² Fo162 = 0.42 R² Control = 0.88

Figure 1. Lineal regression evaluating the effect of Fusarium oxysporum strain 162 and untreated plants on the number of Trialeurodes vaporariorum (westwood) during 9 days of sampling on squash plants (Cucurbita pepo) cv. Eight Ball in two experiments (A and B; n = 8).

73


In the host selection test with T. vaporariorum on melon, the untreated melon plants

contained 73 and 80% of the population when compared to the Fo162 treated plants

during the first 5 sampling dates. The results, therefore, were similar in both the

melon and squash experiments. However after 5 days there was an unaccountable

decline in the number on the control plant. This was not observed on the Fo162

treated host which remained at a constant low population during the 9 samplings

(Figure 2).

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9


Num

ber o

f T. v

apor

ario

rum

BR² Fo162 = 0.22 R² Control = 0.32

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9

Num

ber T

. vap

orar

ioru

m

Fo162 ControlA R² Fo162 = 0.13 R² Control = 0.53

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9


Num

ber o

f T. v

apor

ario

rum

BR² Fo162 = 0.22 R² Control = 0.32

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9

Num

ber T

. vap

orar

ioru

m

Fo162 ControlA R² Fo162 = 0.13 R² Control = 0.53

Figure 2. Lineal regression evaluating the effect of Fusarium oxysporum strain 162 and untreated plants on the number of Trialeurodes vaporariorum (westwood) during 9 days of sampling on melon plants (Cucumis melon) cv. Mehari, in two experiments (A and B; n = 12).

74


75

3.2. Reproduction

To evaluate the effect of the endophyte on the reproduction of A. gossypii on squash

and melon and M. persicae on pepper, ten wingless adult aphids per plant were

placed on the abaxial side of the 4th leaf and allowed to multiply for 14 and 24 days

for A. gossypii and M. persicae respectively.

After two weeks, the reproduction capacity of A. gossypii on the leaves of squash

plants inoculated with Fo162 was statistically lower than the control in both

experiments according to Kruskal Wallis no parametric analysis (A, p = 0.0269 and B,

p = 0.0305) (Figure 3). Approximately 80 and 90% of the untreated leaves showed a

colonization of A. gossypii exeeding 50% of leaf area, the colonozation leaves by the

melon aphid on treated plants had 60% of the samples below 25% of leaf area in

both bioassays respectively.


0

10

20

30

40

50

60

70

80A

. gos

sypi

i le

af c

olon

izat

ion

(%) zero one two threeA

CATEGORIES

0

10

20

30

40

50

60

70

80

Fo 162 Untreated

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

B*

*

0

10

20

30

40

50

60

70

80A

. gos

sypi

i le

af c

olon

izat

ion


CATEGORIES

0

10

20

30

40

50

60

70

80

Fo 162 Untreated

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

B

0

10

20

30

40

50

60

70

80A

. gos

sypi

i le

af c

olon

izat

ion


CATEGORIES

0

10

20

30

40

50

60

70

80

Fo 162 Untreated

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

B*

*

Figure 3. Colonization capacity of Aphis gossypii (Glover) in two experiments (A and B) on squash plants (Cucurbita pepo) cv. Eight Ball, 14 days after release of 10 wingless aphids. Categories zero ≤ than 10 individual; one = population > 10 and up to 25 % of the leaf surface colonized; two = 26% to 50% of the leaf surface area colonized and; three > 50% of the leaf surface area colonized NS = no significant difference according to Kruskal Wallis no parametric analysis (p ≤ 0.05; n = 12).

There was no significant difference in the behaviour of the melon aphid with regards

to colonization of the melon leaf surface (figure 4 A, p = 0.1435 and B, p = 0.2462). In

the first experiment there was a trend towards higher colonization of the foliage on

untreated plants with 100% of the samples in the categories two and three, whereas

on the endophyte treated leaves only 74% of the aphids were detected. However in

the second experiment colonization behaviour between treated and untreated was

76


similar with 100% and 87% of the samples among categories zero and one

respectively.

0

10

20

30

40

50

60

70

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

zero one two threeA CATEGORIES

0

10

20

30

40

50

60

70

Fo 162 Untreated

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

B

NS

NS

0

10

20

30

40

50

60

70

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)


0

10

20

30

40

50

60

70

Fo 162 Untreated

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

B

0

10

20

30

40

50

60

70

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)


0

10

20

30

40

50

60

70

Fo 162 Untreated

A. g

ossy

pii

leaf

col

oniz

atio

n (%

)

B

NS

NS

Figure 4. Colonization capacity of Aphis gossypii (Glover) in two experiments (A and B) on melon plants (Cucumis melon) cv. Mehari, 14 days after release of 10 wingless aphids. Categories zero ≤ than 10 individual; 1 = population > 10 and up to 25 % of the leaf surface colonized; 2 = 26% to 50% of the leaf surface area colonized and; 3 > 50% of the leaf surface area colonized NS = no significant difference according to Kruskal Wallis no parametric analysis (p ≤ 0.05; n = 12).

The fundamental net reproductive rate was evaluated four weeks after 10 M.

persicae wingless adults were placed on the pepper leaves. The results of the first

experiment (Figure 5.A) demonstrated that plants colonized by the mutualistic

endophyte Fo162 were no longer a suitable host for reproduction of the green peach

aphid. The aphid reproductive rate was statistically lower (t28=1.02) on the

77


endophyte plants compared to the untreated plants (t28= 1.10) according to T-test (p

≤ 0.05, n = 6). This represented a reduction in the M. persicae population on Fo162

treated plants of 73% over the untreated controls. In experiment 2 (Figure 5.B)

similar results with regards to fundamental net reproductive rate were obtained

between the untreated (t28=1.14) compared with (t28=1.04) rate of Fo162 treated

plants. This represented a reduction due to the presence of the endophyte Fo162 of

85% over the untreated plants.

0,92

0,96

1

1,04

1,08

1,12

1,16

Net

repr

oduc

tive

rate

of

Miz

us p

ersi

cae

*

A

0,92

0,96

1

1,04

1,08

1,12

1,16

Fo162 Untreated

Net

repr

oduc

tive

rate

of

Miz

us p

ersi

cae

B

*

0,92

0,96

1

1,04

1,08

1,12

1,16

Net

repr

oduc

tive

rate

of

Miz

us p

ersi

cae

*

A

0,92

0,96

1

1,04

1,08

1,12

1,16

Fo162 Untreated

Net

repr

oduc

tive

rate

of

Miz

us p

ersi

cae

B

*

Figure 5. Fundamental net reproduction rate for Myzus persicae (Sulzer) on pepper (Capsicun annum L) cv Yolo Wonder, under glasshouse conditions in two experiments (A and B) 4 weeks after releasing of 10 wingless aphids. Means with (*) are significantly different based on T-test (p ≤ 0.05; n = 6). Vertical bars represent the standard error of the mean (SEM).

78



The effect of three concentrations of metabolites produced in liquid fermentation of

Fo162 on the melon aphid A. gossypii was studied. The reproduction index after 3

and 24 hours feeding on squash leaf discs was evaluated.

The results 3 hours after leaf discs with aphid treated with the metabolites there was

no statistical reduction in the insect population among the metabolites treatments ot

to the respective controls in either of the two experiments (Figure 6).

0,0

0,5

1,0

1,5

Rep

rodu

ctio

n in

dex

Water PDB Fo162A NS

0,0

0,5

1,0

1,5

1 ml 2 ml 4 ml

Rep

rodu

ctio

n in

dex

B NS

0,0

0,5

1,0

1,5

Rep

rodu

ctio

n in

dex

Water PDB Fo162A NS

0,0

0,5

1,0

1,5

1 ml 2 ml 4 ml

Rep

rodu

ctio

n in

dex

B NS

Figure 6. Reproduction index of Aphis gossypii (Glover) in two experiments (A and B) three hours after of exposure to 1, 2 and 4 ml of metabolites produced of liquid fermentation of Fo162 and aplied to leaf discs. NS = no significant difference based on ANOVA (p ≤ 0.05; n = 8). Vertical bars represent the standard error of the mean (SEM).

79


The results of the effect of metabolite treatments on the reproduction index twenty-

four hours after exposure were similar to those after three hours of exposure for all

the three amounts of metabolites evaluated in both experiments (Figure 7). The

population of the melon aphid was not reduced nor increased significantly.

0,0

0,5

1,0

1,5

2,0

Rep

rodu

ctio

n in

dex

Water PDB Fo162

A NS

0,0

0,5

1,0

1,5

1 ml 2 ml 4 ml

Rep

rodu

ctio

n in

dex

NSB

0,0

0,5

1,0

1,5

2,0

Rep

rodu

ctio

n in

dex

Water PDB Fo162

A NS

0,0

0,5

1,0

1,5

1 ml 2 ml 4 ml

Rep

rodu

ctio

n in

dex

NSB

Figure 7. Reproduction index of Aphis gossypii (Glover) in two experiments (A and B) twenty-four hours after of exposure to 1, 2 and 4 ml of metabolites produced of liquid fermentation of Fo162 and aplied to leaf discs. NS = no significant difference based on ANOVA (p ≤ 0.05; n = 8). Vertical bars represent the standard error of the mean (SEM).

80


81


T. vaporariorum on squash plants

Fo162 colonization of roots stimulated changes in the organic compounds within

leaves. HPLC analysis detected 50 peaks per leaf on the average.

The compounds detected in Fo162 treated and untreated leaf extracts in the

absences of GHWF were not different in spectrum when compared to the respective

control. However, when the concentration (peak area in mAU,s) under each

compound in each respective time retention (min) was compared between the Fo162

treated and untreated control (Figure 8), 6 metabolites were statistically different (p ≤

0.05). These peak areas with significant differences were detected in the retention

times of: 2.22 (a), 2.47 (b), 10.89 (c), 26.76 (d), 27.17 (e) and 29.56 minutes (f). The

first 3 peaks were higher for the Fo162 treated plants, while the second three

compounds were in untreated plants. The total numbers of peaks detected were not

different in the presence of the GHWF. Nevertheless, when the concentration of the

metabolites at the same time retention was compared, the compound at retention

time of 2.35 was statistically higher in untreated plants with insects. The results show

that Fo162 stimulates a detectable change in squash physiology within the leaves

and that in the presence of T. vaporariorum this change is always significantly

different compared to the untreated and Fo162 treated plants.


min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400B

Retention time (min) of each metabolite

Pea

k ar

ea (m

AU

,s)

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400B


Pea

k ar

ea (m

AU

,s)

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400B

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400B


Pea

k ar

ea (m

AU

,s)

a

b

c

d

e

f

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400A

a

c

b

e

df

Peak

are

a (m

AU

,s)

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400A

a

c

b

e

df

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400A

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400

min5 10 15 20 25 30 35

mAU

0

200

400

600

800

1000

1200

1400A

a

c

b

e

df

Peak

are

a (m

AU

,s)

Figure 8. Peaks areas (mAU,s) detected by HPLC analysis at 250 nm wavelength from young leaves of squash 12 days after transplanting. (A) Fusarium oxysporum strain 162 treated and (B) untreated plants in the absence of Trialeurodes vaporariorum (Westwood). Arrows indicate retention time of the compounds at 2.22 (a), 2.47 (b), 10.89 (c), 26.76 (d), 27.17 (e) and 29.56 (f) minutes that were statistically different between the treatments without insects according to T-test (p ≤ 0.05; n = 6).

A. gossypii on squash plants

The number of compounds detected by HPLC analysis in older leaves of squash

from Fo162 treated and untreated plants in the absence and presence of A. gossypii

were not different when compared. Nevertheless when the respective time retention

of the peak areas were compared between the control and Fo162 treatments in

82


absence of insects three compounds (Figure 9) were statistically different according

to T. test (p ≤ 0.05). They were detected at the retention times of: 2.350 (a), 5.757

(b), and 27.237 minute (c). The first and the second compound had a higher

concentration in the Fo162 treated plants, while the third metabolite (27.337 time

retention) had a higher concentration in the leaves from the untreated plants.

0

2000

4000

6000

8000

10000

12000

14000

16000

2.350 5.757 27,237

Times retention (minute)

Pea

ks a

rea

mA

U*s

Squ

ash

leaf Fo162 Untreated

*

*

*

Figure 9. Peaks area (concentration in mAU,s) detected by HPLC analysis at 250 nm wavelength from mature leaves of squash 12 days after transplanting treated or untreated with Fusarium oxysporum strain 162 plants in the absence of the melon aphid Aphis gossypii (Glover). Means with (*) are significantly different in the respective time retention based on T-test (p ≤ 0.05; n = 6). Vertical bars represent the standard error of the mean (SEM). Seven compounds detected by HPLC analysis were significantly higher in

concentration (Figure 10) in mature leaves of squash 2 days after they were exposed

to 10 wingless adults of A. gossypii (T-test p ≤ 0.05). Five compounds in the retention

times of: 7.665, 8.710, 10.424, 11.691 and 27.039 minute had higher concentrations

in leaves from untreated plants, while 2 compounds at the retention times of: 26.750

and 27.152 minute, had higher concentrations in leaves from Fo162 treated plants.

These results demonstrate the capacity of Fo162 to trigger changes in the physiology

of the plant in the absence and presence of A. gossypii which may allow the plants to

respond defensively to aphid infestation as shown in the present experiments.

83


0

2000

4000

6000

8000

10000

12000

14000

16000

7,665 8,710 10,424 11,691 26,750 27,039 27,152

Time retention (minute)

Pea

ks a

rea

mA

U*s

Squ

ash

leaf Fo162 untreated

* * * * *

*

*

Figure 10. Peaks area (concentration in mAU,s) detected by HPLC analysis at 250 nm wavelength from mature leaves of squash 12 days after transplanting treated or untreated with Fusarium oxysporum strain 162 plants in the presence of 10 wingless adults of Aphis gossypii (Glover). Means with (*) are significantly different in the respective time retention based on T-test (p ≤ 0.05; n = 6). Vertical bars represent the standard error of the mean (SEM).

M. persicae on pepper plants

The number of compounds detected by HPLC analysis in the mature leaves from

Fo162 treated and untreated pepper plants in the absence or presence of M.

persicae, did not different when compared to the respective spectrum in the controls.

The results were similar to that observed with GHWF. Nevertheless when the peak

areas (mAU,s) were compared between both treatments in the respective time

retention with or without the presence of the green peach aphid, two compounds

could be identified at the retention times of 2.238 minute in treatments without aphids

and at 18.335 minute treatments with M. persicae that were statistically different

according to T. test (p ≤ 0.05). The first compound had a higher concentration when

the green peach aphid was not present on untreated plants while the second

compound showed a higher concentration in the leaves from Fo162 treated plants

(Figure 11).

84


0

1000

2000

3000

4000

5000

6000

2,238 with out aphids 18,335 with aphids

Time retention (minute)

Pea

k ar

ea (m

AU

,s) P

eppe

r lea

f Fo162 Untreated**

Figure 11. Peaks area (concentration in mAU,s) detected by HPLC analysis at 250 nm wavelength from mature leaves of pepper 12 days after transplanting treated or untreated with Fusarium oxysporum strain 162 plants with (at time retention of 2.238 minute) and without (at time retention of 18.335 minute) the presence of 10 wingless adults of green peach aphid Myzus persiceae (Sulzer). Means with (*) are significantly different in the respective time retention based on T-test (p ≤ 0.05; n = 6). Vertical bars represent the standard error of the mean (SEM).

85


86

4. Discussion


The results obtained in the present glasshouse preference experiments on the effect

of the mutualistic endophyte F. oxysporum strain 162 on squash and melon plants

toward GHWF, showed that soil aplication resulted in significant biocontrol activity on

both plants. Host Preference was reduced in the presence of the endophyte on both

hosts. This change of preference by GHWF is provably related to the activation of

plant response mechanisms (Walling, 2000; Kessler and Baldwin, 2002) trigger by

Fo162 (Dababat, 2006; Dababat and Sikora 2007a) that alter normal insect

behaviour. Defenses developed within the plant involve the synthesis of secondary

metabolites that have beeb shown to play an important roles in the interaction

between plants and arthropods (Dicke et al., 2003; Turlings and Wackers, 2004; van

Poecke and Dicke, 2004; Arimura et al., 2005). Adults GHWF evaluate the tactile and

chemical cues of the plant surface after landing to determine the suitability of a plant

as shelter or as a feeding and/or oviposition host (Walling, 2008). Changes in the

synthesis of these cues stimulated by Fo162 also influence the behaviour of the

insect on the Cucurbitacea plants hosts. The resulting effect increased plant fitness

in a hostile environment as reported by Ajlan and Potter, (1991) and Gregg, (2008).

4.2. Reproduction

Plant resistance to arthropod herbivores is often mediated by phytochemicals that

negatively affect the feeding, growth, and / or reproduction of the attacking pest

(Karban and Baldwin, 1997; Walling, 2000). Reduction in the capacity of A. gossypii

to colonize squash and M. persicae to pepper leaves consequently led to a negative

effect on reproduction in the experiments. The plants colonized by the endophyte

Fo162 suppressed population growth of the melon aphid for two weeks and the

green peach aphid for four weeks on the leaf surface.

Insects on the leaft surface are exposed to compounds imbedded in the hydrophobic

cuticular waxes, including non-volatile secondary metabolites such as alkaloids (Cai

et al., 2004), terpenoids (Aharoni et al., 2003) and C-6 aldehydes (Vancanneyt et al.,

2001), as well as volatile and semivolatile compounds (i.e. monoterpenes and

glucosinolate-derivedvolatiles), which influence directly herbivore performance


87

(Karban and Baldwin, 1998; Pare´ and Tumlinson, 1999; Müller and Riederer, 2005).

The quality and / or quantity of these phytochemical compounds were altered by the

presence of the endophyte Fo162, causing the negative changes in behavior and in

population growth observed for both aphids on leaves from treated plants when

compared with controls. This endophyte has been shown to be responsible for

induced systemic resistance (ISR) related to systemic changes in the physiology of

tomato and banana plants the leads to altered root exudates and reduced attraction

and penetration of the root-knot nematode M. incognita and the migratory

endoparasitic nematode Radopholus similis ( Dababat and Sikora, 2007a; Vu et al.,

2006). Therefore it is not surprising to find that plants treated with Fo162 also show a

similar defense-signaling pathway toward phloem-feeding insects that have

prolonged stylet interactions with the phloem sap (Walling, 2000; Klingler et al.,

2005). Agrawal (1998), demonstrated that by increasing the concentrations of the

defensive mustard oil glycosides (glucosinolates) in wild radish (Raphanus sativus L),

which is a resistance trait completely regulated by SA, JA and ET, he was able to

reduce plant colonization of the green peach aphids (Myzus persicae) by 30%. Kim

and Jander (2007) found that one indolic glucosinolate, 4M13M, is synthesized at

elevated levels after aphids infestation and is a potent aphid deterrent. More

defensive compounds have been identified from diverse plant species, however

relatively little is known about the underlying genetic mechanisms that control their

biosynthesis in response to developmental and environmental cues (Li et al., 2002).

However these environmental cues can provide a reliable indication of the presence

of herbivores and can conceivably serve as a signal to induce priming (Frost et al.,

2008). The non-pathogenic endophyte Fo162 also seems to have priming activity

enhancing or triggering the production of a chemical response in the plant during

phloem-feeding on both Solanaceae and Cucurbitaceae plants.


The capacity to 1) decrease mobility 2) increase mortality through disintegration of

internal tissue and 3) inhibit hatching of plant parasites nematode through the activity

of secondary metabolites produced by isolates of F. oxysporum have been

repeatedly demonstrated (Hallmann and Sikora 1996; Amin, 1994; zum Felde, 2002;

Meyer, 2004). Traditionally endophytes have been considered plant mutualists that

reduce herbivory via production of toxins, such as alkaloids (Faeth and Fagan, 2002).


88

In previous studies with filtrates with Fo162, metabolites were shown to be highly

toxic towards sedentary nematodes and less towards migratory plant parasites

(Hallmann and Sikora, 1996). The in vitro tests conducted in the present studies with

fungal filtrates of Fo162 collected after liquid fermentation, demonstrated that the

endophyte did not have metabolite based antibiosis activity toward the melon aphid

A. gossypii after 24 hours of expositure to three different amounts. The reproduction

index of the phloem-feeding insect also was not affected.


Symbiotic fungi usually have compatible or beneficial interactions with their host

plants, which contribute to growth or health promotion due to secondary metabolite

accumulation, for example, of alkaloids and terpenoid (Zhi-lin et al., 2007). The

results of analysis of squash and pepper leaves in the presence or absence of

phloem-feeding insects showed that the non-pathogenic endophyte Fo162 does not

produce new metabolites in the leaves, but is able to stimulate changes in their

concentration within leaves as has been described by Hasegawa et al. (2006) and

Zhi-lin et al. (2007). This effect on plant physiology could have been responsible for

the reduction in attraction of GHWF to squash (antixenosis). Such changes were

responsible for reduce A. gissypii and M. persicae survival and reproduction on

squash and pepper plants in other experiments reported upon here. These fungal

induced changes in metabolite concentrations even in the absence of the insects

showed that Fo162, an endophyte fungus restricted to the root system of the host

plant was able to stimulate systemic plant responses in squash and pepper plants

that move acropedal up into the leaves and then alter insect behaviour. Choudhary et

al. (2008) described this process as induced resistance due to increased de novo

production of secondary compounds.

Changes in the behaviour of the insect due to the presence of an endophyte in a

plant have been described by other authors. For example the endophyte

Acremonium strictum inoculated to tomato seedling altered host-plant preference of

whitefly adults (Vidal, 1996). Epichlöe endophytes in grasses that grow in the true

stem and leaf primordia are able to produce alkaloids as a feeding deterrent toward

sap-sucking insects (Siegel et al., 1990; Clement et al., 1997; Schardl et al., 2004).

However, the impact of root colonizing endophyte Fo162 could go beyond the affect


89

the pure changes in metabolite concentrations. The endophyte may stimulate

defense signaling pathways such as Salicylic Acid (SA), required for systemic

acquired resistance (SAR) (Rairdan and Delaney, 2002; Durrant and Dong, 2004);

Jasmonic Acid (JA), and related oxylipins that mediate induced resistance to chewing

insects and cell-content feeders (Halitschke and Baldwin, 2004; Pozo et al., 2004);

and/or JA and Ethylene (ET) responsible of wound-response, that are required for

the elicitation of systemic induced disease resistance (Pozo et al., 2004).

Experiments, however, are still needed to determine the concentration of these

signalling compounds that modulate phloem-feeding insects in the presence of

Fo162.


90

5. Conclusion

1. Inoculation with the mutualistic endophyte F. oxysporum strain 162 to Cucurbita

pepo and Cucumis melon reduced significantly 80% host-plant suitability toward

the GHWF T. vaporariorum. A significant effect of antixenosis was developed

under the influence of Fo162 on both squash and in melon plants over nine daily

samplings.

2. Colonization of the melon aphid A. gossypii over a 2 weeks period was negatively

affected on squash in the presence of the endophyte isolate Fo162. Less than

25% of the leaft area was colonized on 60% of the treated leaves versus on 50%

colonization on 90% of the untreated plants leaft area. These results confirm that

antibiosis properties could have been acquired by the plants due to the influence

of the non-pathogenic fungus F. oxysporum strain 162 that affected insect

reproduction.

3. Fo162 in melon plants did not alter A. gossypii colonization over untreated plants

despite belonging to the same family with squash. Hence a negative effect of

Fo162 toward A. gossypii colonization on one host can not be generalized to all

plants in the Cucurbitaceae family.

4. The mutualistic endophyte Fo162 negatively influenced the fundamental net

reproductive rate of the green peach aphid M. persicae on pepper 4 weeks after

release of the aphids. The insect population dispured itself over 27% of the

treated plants versus 73% on untreated plants. The results were similar in squash

toward A. gossypii.

5. Biocontrol activity expressed by Fo162 in squash and pepper plants towards

aphids is not influenced directly by endophyte metabolites production. Direct

exposure of the melon aphid A. gossypii for up to 24 hours to three different

doses of endophyte metabolites had no negative affects on population growth.


91

6. The plants treated with Fo162 do not produce different metabolites than untreated

plants. However the concentrations of specific metabolites were affected.

7. The presence of the endophytic fungus probably triggers physiological changes

within the plant and therefore acts like an elicitor that deploys signal transduction

pathways to induce the defenses mechanisms in the host before insects attack.

8. This phenomenon commonly referred in ecological literature as delayed induced

resistance seems to quickly activate the mechanisms of resistance in the plants

and has been demonstrated clearly in the different experiments presented here.

9. The changes in metabolite concentrations were detected by HPLC analysis and

these compounds alone or together could be responsible for the antixenosis and

antibiosis activity shown in both Solanaceae and Cucurbitacea plants toward the

phloem-feeding whitefly and aphids.

Chapter 7 References

92

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Acknowledgements Firstly, I want to express my sincere and eternal gratitude and thankfulness to my

graduate advisor Prof. Dr. Richard Sikora for supporting and guiding me through the

entire process of the doctoral research in Germany.

I would like to thanks Prof. Dr. Heiner Goldbach for accepting to be my second supervisor to evaluate this thesis. My special thanks to Dr. Alexander Schouten and Dr. Joachim for their guidance and

orientation in this research as well as for their comments on this manuscript.

I am grateful with Dr. Luis Pocasangre for his timely advice, guidance and motivation

during the study time.

My special thanks to all colleagues of INRES-Phytomedizin Böden Ökosystem Team

2006-2010 for their outstanding support, cooperation and useful discussions.

I would like to express my special gratitude with the German Academic Exchange

Service (DAAD) for funding this research throughout a scholarship.

Finally, I am very grateful with all my family members, especially my mother that I

owe everything I am, brothers and sisters as well as my wife and my son Roy

Ernesto, for their encouragement and support to reach this goal in my professional

life.

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CURRICULUM VITAE

Name: Roy Donald Menjivar Barahona

EDUCATION: 2007 - 2010 Ph.D at the University of Bonn

Institute of Crop Science and Resource Conservation (INRES). Research Unit: Phytopathology and Nematology in Soil Ecosystems. Rheinische Friedrich-Wilhems-University of Bonn. Bonn, Germany.

2004 – 2005 Master of Science (M.Sc.) ECOLOGICAL AGRICULTURE, School of Post degree, CATIE. Turrialba, Costa Rica.

1991 - 1994 Engineer in Agronomic Science Universidad Nacional Autónoma de Honduras (UNAH/CURLA), Centro Universitario Regional del Litoral Atlántico, La Ceiba Atlántida, Honduras.

1986 – 1990 Bachelor in sciences and letters High School 18 de Noviembre, Catacamas, Olancho, Honduras

WORK EXPERIENCE 2001-2006 University Lecturer

Agriculture National University (UNAG); Honduras.

2003-2004 Vice Dean of DPV Vegetable Production Department. National University of Agriculture (UNAG), Honduras.

2000 University Lecturer Universidad Nacional Autónoma de Honduras (UNAH/CURNO), Centro Universitario Regional nort oriental. Juticalpa, Honduras.

2000 General Manager Nutrientes Concentrados (NUTRICON). Honduras.

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1999 University Lecturer Private University Jose Cecilio del Valle. Tegucigalpa, Honduras. 1998-1999 Manager of Technical Operations

Nutrientes Concentrados (NUTRICON), Honduras.

1997-1998 Technical Adviser for the validation of biopesticides and sales Nutrientes Concentrados (NUTRICON), Honduras.

1997 - 1998 Private Consultant • System organization of Transference of Agricultural Basic

Technology in the Municipality of Lepaterique, Francisco Morazán. (DICTA, SAG), Honduras.

• Validation of technologies IPM for the Brassica oleracia vr. capitata (cabbage) and B. oleracia vr. italic (broccoli) crops.

1995-1997 Coordinator of Program

Program: Integrated pest Management in vegetables (IPM-Lepaterique), FEPROH-GTZ, Tegucigalpa, M.D.C.

1995 Credit Adviser Department of Financial Appraisements, COBIRNO, Catacamas, Olancho, Honduras.

1994 Instructor of Chemistry laboratory Department of Chemistry, University, Universidad Nacional Autónoma de Honduras (UNAH/CURLA), Centro Universitario Regional del Litoral Atlántico, La Ceiba Atlántida, Honduras.

PUBLICATION 2010 Menjivar, R.D., Hagemann, M.H, Kranz, J., Cabrera J. A.,

Dababat, A. A. and Sikora, R. A. 2010. Biological control of Meloidogyne incognita on cucurbitaceae crops by the non-pathogenic endophytic fungus Fusarium oxysporum strain 162. Int. J. of Pest Management (in review process).

2010 Menjivar, R.D., Pocasangre, L.E., zum Felde, A., Cabrera,

J.A., Quezada M. and Sikoraa. R.A. 2010. Effect of mutualistic endophytic fungi on the biocontrol of Radopholus similis, plant growth and yield in commercial banana production. Crop Protection (in review process).

2009 Newcombe, G., Shipunov, A., Eigenbrode, S.D., Raghavendra,

A.K.H., Ding, H., Anderson, C. L., Menjivar, R., Crawford, M. and Schwarzländer, M. 2009. Endophytes influence protection and growth of an invasive plant. Communicative and Integrative Biology 2:1, pp 1-3.

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2008 Menjivar, R.D., Kanz, J. and Sikora, R.A. 2008. Impact of an endophytic endorhiza fungus on leaf-sucking insects. Poster presented at the “Deutscher Pflanzenschutztagung” University of Kiel, Germany. September 22-25, 2008.

2008 Menjivar, R.D., Kanz, J. and Sikora, R.A. 2008. Effect of non-

pathogenic Fusarium oxysporum strain 162 in Solanaceae and Cucurbitaceae crops on Trialeurodes vaporariorum. Poster presented at the “Deutscher Tropentag 2008” Hohenhein University in Stuttgat, Germany. October 7-9, 2008.

2007 zum Felde, A., R. A. Sikora, L. E. Pocasangre and Menjivar,

R.D. 2007. Establishing and Detecting In Planta Suppression of Radopholus similis in Successive Generations of Banana Plants Protected with Mutualistic Fungal Endophytes. Poster presented at 35. Tagung des Arbeitskreises Nematologie, 13-14 March 2007, Potsdam-Golm, Germany.

2006 Pocasangre, L.E., Menjivar, R.D., zum Felde, A., Riveros, A.E.

Rosales, F.E. and Sikora, R.A. 2006. Endopytic Fungi as Biological Control Agents of Plant Parasitic Nematodes in Banana. Pp. 249-254. In Publicación especial – XVII Reunion Internacional ACORBAT 2006, Santa Catarina, Brasil.

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