Blackleg – disease biology and control

measures

Jan van der Wolf

Lay-out presentation

� The disease and its causative agents

� Initial infection and spread of the pathogen

� Control

● Hygiene

● Cultivation practices

● Seed testing

● Resistance breeding

● Seed treatments

Lay-out presentation

�The disease and its causative agents

� Initial infection and spread of the pathogen

� Control

● Seed testing

● Hygiene

● Cultivation practices

● Resistance breeding

● Seed treatments

Symptoms Dickeya/Pectobacterium potato

Important features causative agent

�Enterobacteriaceae (not spore forming) � killed at 53 oC

� Pectinolytic � cause maceration of plant tissue

� Facultative anaerobes � multiplies at low oxygen conditions (wet tubers)

�Motile �can move in free water to tubers

0.8 �m

Disease development field experiment

2014 (averages)

Distribution of Dickeya solani

Dickeya solani in Northern Ireland

Distribution of P. c. subsp. brasiliense

• Broad host: potato, chinese cabbage, tomato, egg plant and nepenthes (carnivorous plant)

Conclusions: Blackleg causing pathogens

� Various Pectobacterium and Dickeya species, but increasingly P. c. subsp. brasiliense causes blackleg

� Strains of P. c. subsp. brasiliense (and D. solani) are highly homogeneous indicating a recent introduction and rapid spread within seed potato production area’s in the Netherlands

� P. atrosepticum and P. c. subsp. brasiliensis were most aggressive under Dutch climate conditions in two-years field experiments with vacuum-infiltrated seed tubers

� Under high temperature conditions, Dickeya solani can be more destructive than other blackleg pathogens

� Surveys are important to recognize new variants of a pathogen timely

Lay-out presentation

� The disease and its causative agents

�Initial infection and spread of the pathogen

� Control

● Seed testing

● Hygiene

● Cultivation practices

● Resistance breeding

● Seed treatments

Initial infections

� Mini tubers and clonal selection are basically free of Erwinia’s

� In the 2nd year of multiplication ca. 20% of the seed lots are infected(symptomless crop)

Yr 0: start minitubers(11 growers)

Potential sources of introduction: machines

Planting Spraying (tyres):

Infections of haulms,

roots and tubers

Flailing (haulm topper):

Infections of haulms (via flails),

roots and tubers (via tyres or

indirectly via infected haulms)

Harvesting Sorting

+ +

+

Data 2006. Source: NAK/NAK-Agro

Mechanization plays a role!?

Potential sources initial infection: “air –

and soilborne”

+?

Insects Rain/aerosols Irrigation water

Weeds/potato volunteers Soil

+/-?

Pectobacterium can be spread over long

distances via aerosols after flailing

� Pectobacterium can survive in aerosols 2 h at 65% RH and 18 oC

� Calculated nrs Pectobacterium cells deposited per m2 from aerosols:

● Distance to source 50 m : 1000

● Distance to source 100 m: 100

● Distance to source 1000 m: 3

Nr of Pectobacterium cells released during flailing

Experiment Pectobacterium, “disease free” crop

Pectobacterium in syptomatic crop

1 5x108/ha 108/ha

2 7x107/ha 8x107/ha

Source Scotland, Perombelon et al. 1979

Haulm infections rarely happen?

(19 fields, 100 leaves/field, 15 July 2012, E-TaqMan)

Haulm infections can result in infected

progeny at 42 dpi (D. solani, 106 cfu/ml)

Inoculation point: 100%

Stem top: 40%

Progeny: 20%

Stolons: 40%Stem base: 20%

Leaf inoculation results in higher infection

rates for wounded than for not-wounded

leaves

Leaf inoculation results in highter infection

rates for D. solani than for P. wasabiae

Risks transmission from haulms to soil

Mechanical (flailing) Chemical (reglone,

10 ml/L diquat)

overhead sprayline with Micro sprinklers0 - 5 DPH: in 4 intervals of 6 hrs in total 4 L/m2

6 - 10 DPH: in 4 intervals of 6 hrs in total 1 L/m2

In addition at 8 DPH extra application of 4 L/m2

Insects can transmit Erwinia’s

� Pectobacterium be transmitted by various species of flies

� Pectobacterium is found in densities up to 2 million bacterial cells per fly

� Pectobacterium can survive up to 72 h on flies

� Infected insects are found from May till September, but not in October

� Flies can be contaminated with Pectobacterium present in potato dump piles and carry them up to 200 m after which they can infect (damaged) plants

(Source: VS, Kloepper et al., 1979, 1981)

Contaminated insects can be found in the

entire potato ecosystem

Source Percentage infected

1975 1976

Drainage wells, storage rooms potato

0.3 0.9

Dump piles 1.3 3.3

Potato crop 0 1.1

Lettuce crop 3.5 0.4

Barley, spinach, carrot crop

nd 0

(Ca. 10.000 sampled insects in the USA (Kloepper et al., 1979)

Infected weeds are mainly found during

the growing season

� In Schotland and the USA, in 71 of 130 weed species Erwinia was found

�Mainly Pcca (67 -98%) was found

�Bacteria were mainly found during the growing season

In the Netherlands, infected crops are

rarely found one year after harvest of a

heavily infected potato crop (%)

Year Crop Weeds(roots+rhizosphere soil)

Potato volunteers

Pa Ds Pw Pa Ds Pw

2009 Grass 0 0 0

Maize 0 0 0 0 0 0

2010 Grass 0 0 0

Cabbage 0 0 0

Wheat 0 2 0 0 35 5

2011 Maize 0 0 0

Cabbage 0 0 0

Sugerbeet 0 0 0 0 0 0

In all cases: crop and soil were

negative

Risks transmission from haulms to soil

Mechanical (flailing) Chemical (reglone,

10 ml/L diquat)

overhead sprayline with Micro sprinklers0 - 5 DPH: in 4 intervals of 6 hrs in total 4 L/m2

6 - 10 DPH: in 4 intervals of 6 hrs in total 1 L/m2

In addition at 8 DPH extra application of 4 L/m2

Mechanical haulm killing results in higher

infection rates than chemical haulm killing

(days)

Tubers can become infected from bacteria

washed into soil

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0 4 8 11

log

(cf

u+

1)/

g

days after haulm killing

Pulverisation

Water D. sol P. was

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0 4 8 11

log

(cf

u+

1)/

g

days after haulm killing

Chemical

Water D. sol P. was

(2013)

Effect haulm destruction

Spread via machines

Planting Spraying (tyres):

Infections of haulms,

roots and tubers

Flailing (haulm topper):

Infections of haulms (via flails),

roots and tubers (via tyres or

indirectly via infected haulms)

Harvesting Sorting

++

+

+

++

Contact infection

� Row of tubers from a downgraded seed lot planted adjacent to mini-tubers

� Grower or machines were moving from left to right

� Haulms were tested at different distances from infected plants at the end of the season

Spread from rotten tubers during water

logging results in tuber- and root infections

control

Roots, 1 dpi

D. solani

Dickeya released from rotten tubers moves

with free water in soil to neighbouring plants

Peel infections

Short term survival of suspended bacteria

on materials

0

1

2

3

4

5

6

7

8

0 3 6 24 30 48

uren

log

cfu

/ml

Ecc

Ech

Eca

hoursPVC

J. van Doorn (PPO-Lisse)

Pcca

Ddia

Patr

Long term survival of Dickeya in decay on

tubers

Decay brought on tubers and air-dried

Long term survival of Erwinia in hyacinth

bulb material on material (wood)

Overleving van Erwinia in hars op hout

1.00E-01

1.00E+01

1.00E+03

1.00E+05

1.00E+07

1.00E+09

0 2 4 6 8 10 12 14 16 18 20 22 24 26

tijd in dagen

cfu

/ml

Ecc

Ech

Eca

J. van Doorn (PPO-Lisse)

Days after inoculation

Pcca

Ddia

Patr

0

1

2

3

4

5

6

7

8

0 1 7 23 42 84

dagen

log

cfu

/g

Ecc1990

Eca1987

Ech2019

Ech1991

Ech980

Bron: J.M. van der Wolf, J. van Doorn

Survival free in soil up to 3 months at 50%

FC and 6-8 oC in peat soil

Days

Pcca

Patr

Dsol

Ddia(1)

Ddia(2)

Survival up to 112 days at 6-8 oC and 50%

Field Capacity in stem fragments soil

110

1001000

10000100000

100000010000000

1000000001000000000

veen zand veen zand

Ech Ecc

log

cfu+

1/g

plan

t mat

eria

l

0 dagen

36 dagen

64 dagen

112 dagen

After 112 days stem fragments were still visible

0 days

36 days

63 days

112 days

peat sand peat sand

PccaDdia

Survival in stem fragments buried in the field

Afname van Ech in begraven stengelfragmenten. Analyse met PCR. In

blauw: telling met IFC

0

1

2

3

4

5

6

7

8

30-jul

27-a

ug

24-se

p

22-o

kt

19-n

ov

17-d

ec

14-jan

11-feb

10-m

rt

7-apr

Log

n pe

r m

l ext

ract

After 8 months no living bacteria could be detected anymore (blue dots)

Ventilation during storage has little effect

on survival of D. solani

Tubers were dipped in suspensions with rotten tubers

3 months of storage, 40 pools of 4 peelings

Lay-out presentation

� The disease and its causative agents

� Initial infection and spread of the pathogen

�Control

● Hygiene

● Cultivation practices

● Seed testing

● Resistance breeding

● Seed treatments

� Concluding remarks

Control during cultivation

� Use of clean seed

● Minitubers

● Certified seed � field inspections and laboratory testing

� Wash and disinfect machines and materials

� Avoid water logging of soil � drainage

� Avoid damage of your crop � air-borne inoculum

� No surface water for irrigation � can be contaminated

� Selection and roguing: only useful early in season?!

� Full field spraying followed by flailing (after 5 days) is preferred above flailing followed by spraying

Control during cultivation

� Plant a high grade seed lot at a distance of at least 100 meter from a low grade seed lot

� Plant a low grade seed lot downwind not upwind of a high grade seed lot

� Flailing should be done preferably during dry weatherconditions, sunshine and little wind

� Full field spraying is preferred above flailing

� Avoid the presence of haulm debris on soil above tubers

� Clean and disinfect harvesters in case you move from a lower to a higher grade seed lot

During harvesting, grading and storage

�Harvest manually as long as possible

� Postpone harvest of tubers till rotten mother tubers are gone

�Remove rotten tubers from harvesters and graders

�Avoid wounding of tubers

�Harvest under dry conditions

�After harvest: Dry, dry, dry ........

�Store tubers in well-ventilated rooms at low temperatures

�Use less susceptible cultivars

Disease expression

� No difference in disease incidence and disease expression between large (35/60) and small (28/35) tubers

� More disease expression on sand than on clay

� No effect N2 fertilisation (0, 50, 100, 150% of recommended dosage) on disease expression

Seed testing in the Netherlands: laboratory testing

� 200 tubers in 4 composite samples of 50 tubers to calculate the disease incidence:

● I = {1 – [(N – p)/N]1/n}*100 � I = incidence, N= number of subsamples, p =

number positives, n = number of tubers per subsample (Plant Disease 86, 960

(2002))

� Test heel ends of tubers

� Incubate in Pectate Enrichment Broth to multiply Erwiniaprior to testing (72 h, low oxygen conditions)

� Use TaqMan assays for final testing

TaqMan detection Dickeya/Pectobacterium

Generic Pectobacterium/Dickeya

(JHI, PRI)

Generic Dickeya

(FERA, PRI)

P. atrosepticum

(FERA, PRI)

P. wasabiae (PRI)

D. solani (JHI)

D. dianthicola (JHI, PRI)

D. dadantii (PRI)

D. dieffenbachiae (PRI)

D. zeae (PRI)

D. chrysanthemi (PRI)

D. paradisiaca (PRI)

+

Internal amplification control

P. c. subsp. brasiliense (PRI)

Resistance breeding: petiole test

� Test conditions

● 25 oC

● 16 h L/8 h D

● Ca. 100% RH

� Bacterial strains

● Pcc 1948 (107 cfu/ml)

● Pw 1957 (107 cfu/ml)

● Ds 2222 (105 cfu/ml)

340 Accessions

532 genotypes

Nearly all potato species were included

The CBSG collection

Results petiole assay

* Yungasensa, tolerance known from literature

Pcc Pw Ds Pcc Pw Ds

< 40% maceration

49 141 23 6 24 1*

40-80% maceration

182 209 188 48 128 18

> 80% maceration

301 182 321 178 380 513

2 DPI 3 DPI

Preliminary conclusions

� Tolerance against stem maceration (but also susceptibility) is present in Yungasensa (S. chacoense)

Disinfection of tubers

Excellent results with older tubers

Disinfection of tubers

With freshly harvested tubers, effects are limited

Conclusions: Options for control

� Use of pathogen-free seed in combination with hygiene and cultivation practices are still the most important management tools

� Sources of resistance (tolerance) have been identified

� There are perspectives for tuber treatments (biocontrolagents, disinfectants) able to reduce disease incidence

Acknowledgement

� Colleagues from PRI (P. Kastelein, L. de Haas, P. van der

Zouwen, M. Krijger, H. Rietman, R. Visser, T. Been, L. Leite)

� Collaborators from NIOO (R. Czajkowski, H. van Veen)

� Collaborators in the Erwinia Deltaplan (H. Velvis, K. Kristelijn,

D. Boomsma)

� Collaborators from the EU Euphresco project

� Collaborators in Canada and USA (S. de Boer, S. Li, A.

Charkowski)

� Dutch potato growers (organizations)

� Dutch Ministry of Economic Affairs

� Dutch foundation for Technical Science (STW)