Increasing Problems with Erwinia's

Increasing problems with Erwinia’s - the ecology of blackleg pathogens Outline

The pathogen Blackleg situation in Europe Ecology: introduction and dissemination Control Wilting symptoms Basal stem rot (blackleg) Browning of vascular system, maceration of pith tissue Tuber symptoms

Infections on wounded tubers Stolon end infections Browning of vascular and parachymatic tissue Blackleg causal organisms

E. chrysanthemi Æ Dickeya paradisiaca, D. dianthicola, D. chrysanthemi, D. zeae, D. dieffenbachia, D. dadantii

E. carotovora subsp. atroseptica Æ Pectobacterium atrosepticum

Erwinia carotovora subsp. carotovora Æ Pectobacterium carotovorum subsp. carotovorum

E. c. subsp. brasiliensis (South America+South Africa) Æ Pectobacterium carotovorum subsp. brasiliensis Survey Erwinia in blackleg-diseased plants (NL)

Source: NAK Declassified + rejected seed lots in the Netherlands

15 10

% declassified 5

5 8 1 4 7 9 9 0 0 0 9 997 9 0 003 0 0 1994 1 1996 1 1 1999 2000 2 2002 2 2 2005 2006 2 2008 Year

Source: NAK - EAPR Meeting , Hämeenlinna ; 2-6 July 2007 Survey Erwinia in blackleg-diseased plants (Fr) 2003 2004 2005 • 59 fields • 63 fields • 49 fields •117 plants •91 plants • 79 plants •30 varieties •27 varieties •27 varieties

60 Pca fields % fields 40 Dck Pcc No 20

0 2003 2004 2005

Source : V. Hélias

Research program funded by potato growers and fresh potato industry : UMR 1099 Biologie des Organismes et des Populations appliquée à la Protection des Plantes (Bio3P) Is the increase due to global warming?

http://www.cru.uea.ac.uk Growth temperature characteristics Erwinia’s

Growth E. carotovora E. c. subsp. E. chrysanthemi temperature subsp. carotovora atroseptica (in oC) Minimum 6 3 6

Optimum 28-30 27 34-37

Maximum 37-42 35 ≥ 37

Revised after Pérombelon & Kelman, 1980 Temperature versus Ech infections

20 April May 15 June oC 10 July 5 August

0 2001 2003 2005 Normal Year

High average temperatures in June associated with Dickeya infections Recent potato strains (Biovar 3): - Israel - Poland - Finland - The Netherlands

European potato strains ≤ 1995 DnaX-sequence analysis Pectinolysis at 37 oC for Dickeya species

Janse & Ruissen, 1988 Introduction Erwinia’s during seed multiplication

Cultivar 1st generation 2nd generation

Nr. seed Eca Ech Nr. seed Eca Ech lots (E-ELISA) (E-PCR) lots (E-ELISA) (E-PCR) Desiree 13 0 0 13 1 1

Kondor 23 0 0 20 2 2

Spunta 17 1 0 18 3 4

5x10 tubers/seed lot were analysed Field generations versus % degradation in 2006

10 Clonal selection, 8 no composite Clonal selection, 6 composite 4 In vitro

% degradation % 2 0 1-yr 2-yrs 3-yrs 4-yrs generations

Source: NAK/NAK-Agro Low densities of Ech can give blackleg Ech Eca

100 100 90 90 80 80 70 70 4 60 60 50 low, 10 50 4 40 40 low, 10 cells/ml 30 30 % diseasedplants % diseased plants 20 20 cells/ml 10 10 0

3-jun 10-jun 17-jun 24-jun 1-jul 8-jul 15-jul 22-jul 0 3-jun 10-jun 17-jun 24-jun 1-jul 8-jul 15-jul 22-jul

C4, sympt field C4, Sympt lab A4, sympt field A4, sympt lab C4, Elisa Ech A4, Elisa Eca

100 100 90 90 80 80 7 70 70 high, 10 7 60 60 high, 10 50 50 cells/ml 40 40 30 30 % diseased plants

cells/ml plants diseased % 20 20 10 10 0 10-jun 17-jun 24-jun 22-jul 15-jul 1-jul 3-jun 8-jul 0 3-jun 10-jun 17-jun 24-jun 1-jul 8-jul 15-jul 22-jul

C7, sympt field C7, sympt lab A7, sympt field A7, sympt lab C7, Elisa Ech A7, Elisa Eca Low densities of Ech can give blackleg

10 0 100 90 90 80 80 70 70 60 60 2006, low 2005, low 50 40 50

% plants 30 40 % plants 20 30 10 20 0

3-jun 10-jun 17-jun 24-jun 1-jul 8-jul 15-jul 22-jul 10 0 7-jul 9-jun 14-jul 21-jul 16-jun 23-jun 30-jun

Symptoms EL ISA Symptoms ELISA

100 100 90 90 80 80 70 70 60 60 50 50 40 40 % plants % plants 30 30 20 20 10 10 2006, high 2005, high 0 0 3-jun 10-jun 17-jun 24-jun 1-jul 8-jul 15-jul 22-jul 7-jul 9-jun 14-jul 21-jul 16-jun 23-jun 30-jun

Symptoms ELISA Symptoms ELISA Erwinia cannot overwinter in soil ...

8 7 6 Ecc1990 5 Eca1987 4 Ech2019 3 Ech1991 log cfu/g log 2 Ech980 1 0 017234284 dagen

Bron: J.M. van der Wolf, J. van Doorn ... even not in crop debris

1000000000 100000000 10000000 1000000 0 days 100000 36 days 10000 64 days 1000 112 days 100 10 1 log cfu+1/g plant material plant log cfu+1/g MHMH

Dickeya Ecc

Stem fragments were still present at day 112 Soil M = peaty soil, Soil H = sandy soil Erwinia survive for less than two days on materials

8 7 6 5 Ecc 4 Ech 3 Eca log cfu/ml 2 1 0 036243048 urenhours

PVC Erwinia can disseminate in soil

100

80 s

40 % Ech positive plant % positive Ech 20

0 Control Treated plants Neighbouring plants Plants adjacent row

six weeks after treating plants Can root infections result in an infected progeny?

?? Colonization of plants after soil infestation

Soil of 12 plants was inoculated 2 weeks after planting when stolons were formed Pots were dipped in trays containing GFP-tagged Dickeya solani suspensions and left for 40 min Populations dynamics in roots, seed potato, stolons, progeny tubers and stems were analysed 1, 15 and 30 d.p.i. Can stem infections result in an infected progeny?

?? Distribution after stem inoculation: set up

10 plants were inoculated 2 weeks after planting when stolons were formed Suspensions, of Dickeya sp., were injected into stems, always 3 stems per plant For decreasing the risk of cross contamination of soil inoculation point and soil were covered with plastic foil, plant were watered from the bottom Symptom development

plant Symptoms inside stems Blackleg symptoms 1 no symptoms no symptoms 2 browning typical blackleg 3 browning no symptoms 4 browning no symptoms 5 browning typical blackleg 6 browning typical blackleg 7 no symptoms no symptoms 8 browning typical blackleg, plant decomposed

9 browning no symptoms 10 browning no symptoms 4 water control plants were negative Stem infections can result in an infected progeny!

Inoculation point: 100%

Stem base: 100% Stolons: 60% Roots (upper): 90% Progeny tubers: 70%/15%

Roots (intermediate): 60%

Roots (down): 30% Spread of contamination during harvest

A strip with rotten Erwinia-tubers was harvested, thereafter tubers from Erwinia-free plants Contamination levels of tubers were analysed at different distances from the contaminated strip using enrichment-PCR Harvesting procedures z Control z Two-phase system, hand harvesting of lifted tubers z Two-phase system, harvesting of lifted tubers with a machine z Potato harvester with axial roller set z Potato harvester with a sieve band

Avoid spread of contamination via machines

Average smearing (m) 2

0 Control two-phase, hand Machines

Variation high No significant differences between machines Rainfall harvest time versus blackleg incidence

10 Declassification

% declassified % 5

0 2000 2001 2002 2003 2004 2005 2006 Year

180 160 140 120 July 100 80 August 60 Rainfall Rainfall (mm) Rainfall 40 20 0 2000 2001 2002 2003 2004 2005 Normal year Concluding remarks

Blackleg incidences fluctuate largely, but last years more problems are found Incidences of last three-years are more connected to Dickeya infections Dickeya’s can induce symptoms at low densities Dickeya is not a better survivor Avoid spread of contamination during harvest to control Erwinia’s Contributors

HZPC Research z Henk Velvis Plant Research International FNPPPT/INRA-Rennes z Patricia van der Zouwen z Valérie Helias z Robert Czajkowski z Jan van der Wolf NAK z Gé van de Bovenkamp z Eisse de Haan