Development and Assessment of Potential Diagnostics and Biocontrol Measures Against Soft Rot in Vegetables

Development and assessment of potential diagnostics and biocontrol measures against soft rot in vegetables

Maja Zaczek-Moczydłowska

Dr Katrina Campbell, Dr Gillian Young, Dr Colin Fleming & Dr Richard O’Hanlon

Postgraduate symposium 2018 Department of Agriculture, Environment and Rural Affairs Stormont, Belfast Bacteriophages as microbial pests control agent (MPCA)

• The biological control agents need to be safety for human, animals and environment

(Regulation (EU) N0. 1107/2009: Directive 2009/128/EC, Regulation (EC) No 1185/2009, Regulation (EC) No 396/2005, Regulation (EC) No 1272/2008)

• Lytic bacteriophages as MPCAs

-High specify to infect only target bacteria -Lysis (death) of bacterial cells -Most abundant entity in biosphere -Non-toxic to eukaryotic hosts Bacteriophages as biocontrol

• AgriPhage® (Omnilytics™), US Xantomonas campestris pv. vesicatoria Pseudomonas syringae pv. tomato

• ErwiPhage® (Enviroinvest), Hungary Erwinia amylovora

• Biolyse® (APS Biocontrol Ltd.), UK Pectobacterium and Dickeya spp.

• Bafasal® and Bafador® (Proteon Pharmaceuticals), Poland Salmonella

• PhageGuard Listex™ P100 and PhageGuard S (Micreos), Netherlands Listeria and Salmonella Soft rot Enterobacteriaceae (SRE)

Erwinia

Samsonia Erwinia Pectobacterium Dickeya Pantoea Brenneria

ECONOMY IMPACT FOOD SAFETY

Loses 30% - 50% of harvested crops Aim

Hypothesis

• Bacteriophages can be used as efficient biocontrol in field conditions

Develop and evaluate biocontrol formulation against soft rot in vegetables (potato and onion) using bacteriophages through bioassays and field trials Project

Part 1 Aim : Isolation and characterisation of Pectobacterium and Dickeya spp. in Northern Ireland

Part 2 Aim: Isolation and characterisation of potential MPCAs (bacteriophages)

Part 3 Aim: Testing formulated cocktail in vitro and in vivo Methodology

Isolation of bacteriophages

Isolation of Screening bacteria against SRE

Bioassays and Formulation of field trials ‘cocktail’ Pectobacterium and Dickeya spp. detected in vegetables in Northern Ireland

• Statutory samples submitted to 38 AFBI • Field survey 2017 (DAERA Plant Health Inspection Branch) 56 15 • Vegetables collected from local growers and shops

• 202 samples tested on CVP 15 media • Analysis of statutory samples 93 20 data submitted to AFBI from 2005-2017 (3450 samples tested) Number of isolated bacterial strains in each county

European Journal of Plant Pathology Zaczek et al., (in review) Pectobacterium and Dickeya spp. detected in vegetables in Northern Ireland

Phylogenetic attribution of Northern Irish strains based on recA gene sequences

• P. atrosepticum predominant species causing blackleg detected in Northern Ireland

• P. c. subsp. carotovorum isolated from carrots

. European Journal of Plant Pathology Zaczek et al., (in review) Pectobacterium and Dickeya spp. detected in vegetables in Northern Ireland

• First record for Northern Ireland for detection Dickeya spp. confirmed by DNA sequences

• First record for detection Dickeya aquatica from sources other than water

European Journal of Plant Pathology Zaczek et al., (in review) Bacteriophage characterisation

Order: Caudovirales Family: Myoviridae

Head: 61.7-70 nm x 80-125 nm Tail: 110-113.9 nm

ΦMA7 ΦMA1 ΦMA15

100 nm 100 nm 100 nm Bacteriophage characterisation

Order: Caudovirales Family: Siphoviridae

Head: 50,1-58,7 x 42,1-48,78 nm Tail: 223-227 nm

ΦMA11 ΦMA12

100 nm 100 nm Bacteriophage characterisation

Order: Caudovirales Family: Podoviridae

Head: 45.8-59.5 x 47.2-72.2 nm Tail: short, non-contractile - 17.2nm

ΦMA2 ΦMA1A ΦMA1B ΦMA7.1 ΦMA5 ΦMA13

100 nm 100 nm 100 nm 100 nm 100 nm 100 nm

ΦMA8 ΦMA9 ΦMA19 ΦMA6 ΦMA16 ΦMA18

100 nm 100 nm 100 nm 100 nm 100 nm 100 nm

ΦMA17 ΦMA20 ΦMA14

100 nm 100 nm 100 nm Bacteriophages characterisation

Host range of selected bacteriophages for cocktail formulation 16 Spotting assay 15 P. atrosepticum P.carotovorum 14

10 10 9

8 Overlay assay 6 Number of of isolates Number 6 5 5

4 4 4 4 3

2 2 2

0 MA1 MA1A MA2 MA5 MA6 MA7

Bacteriophages Bacterial suppression test in vitro

* *

• *p<0.05- p - value<0.0001 (ANOVA) • Fisher’s least significant difference (LSD) – 3.45 Bacterial suppression test in vitro

Positive Phage Sterile control ‘cocktail’ + bacteria water

Phage ‘cocktail’ + bacteria Bacteriophages activity and stability in vitro

• Bacteriophages are active against bacteria under wide range of temperatures and UV radiation over the time Use of bacteriophages as MPCA in potato

Potato seeds British Queen, Dunbar Standard, Maris Piper and Amora (Pectobacterium ca.108 cfu/mL)

16 Crossnacreevy 14 Greenmount 12

6 Blackleg symptoms Blackleg [%] 4 * * 2

Treatments

• *p<0.05- p - value<0.005 (ANOVA) • Fisher’s least significant difference (LSD) – 0.5 Use of bacteriophages as MPCA in potato

Blackleg of potato Phage treatment Use of bacteriophages as MPCA in potato

Potato seeds British Queen, Dunbar Standard, Maris Piper and Amora (Pectobacterium ca.108 cfu/mL)

Belfast 2016

* *

Treatment • *p<0.05- p - value<0.005 (ANOVA) • Fisher’s least significant difference (LSD) – 1.99 Use of bacteriophages as MPCA in potato

Post - harvest experiment on potato collected in field trial 2016 British Queen, Dunbar Standard, Maris Piper and Amora (Pectobacterium ca.108 cfu/mL)

* *

NC VI SD PC

Fisher’s least significant difference (LSD) – 1.99

*p<0.05- p - value<0.005 (ANOVA) Use of bacteriophages as potential MPCA in onions

Onions bulbs Stuttgarter (SRE low natural inoculum)

2016 25 2017

* * * Soft rot [%] rotSoft

10 * * * 5

0 Untreated Negative control Foliage application Soil drench Liquid immersion Treatment Fisher’s least significant difference (LSD) – 2.33 *p<0.05- p - value<0.005 (ANOVA) Conclusions

• Three species related to soft rot symptoms of vegetables are present in Northern Ireland

• The species P. atrosepticum and P. c. subsp. carotovorum were confirmed to be present in potato. Predominant species causing blackleg of potatoes is P. atrosepticum

• Bacteriophages suppress bacteria in vitro

• Bacteriophages formulation is stable in wide range of temperatures and UV radiation in vitro

• Isolated bacteriophages suppress bacteria in vivo

• Bacteriophages cocktail applied on onions in the field causes increased yields

. References

Czajkowski R., Pérombelon M.C.M., van Veen., van der Wolf J.M., (2011). Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review. Plant Pathology,60,999-1013 Czajkowski R., Pérombelon M.C.M., Jafra S., Lojkowska E.,Potrykus M.,van der Wolf., Sledż W., (2015). Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review. Annals of Applied Biology,166,18-38 De Boer S.H., Ward L.J., (1995). PCR detection of Erwinia carotovora subsp. atroseptica associated with potato tissue. Phytopathology, 85, 854-858 Buttimer C., McAuliffe O., Ross R.P., Hill C., O’Mahony J., Coffey A. (2017). Bacteriophages and Bacterial Plant Diseases. Front Microbiol, 8: 34. Gardan, L., Gouy, C., Christen, R. & Samson, R. (2003). Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. Int J Syst Evol Microbiol 53, 381–391. Pérombelon M.C.M., (2002). Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathology,51,1-12 Hauben, L., Moore, E. R., Vauterin, L., Steenackers, M., Mergaert, J.,Verdonck, L. & Swings, J. (1998). Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst Appl Microbiol 21, 384–397 Khayi S., Cigna J., Chong T., Laurent A., Chan K., Helias J., Faure D. (2016).Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov. Int J of Systematic and Evolutionary Microbiology 66: 5379-5383 Nabhan, S., De Boer, S. H., Maiss, E. & Wydra, K. (2012a).Taxonomic relatedness between Pectobacterium carotovorum subsp.carotovorum, Pectobacterium carotovorum subsp. Odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov. J Appl Microbiol 113, 904–913 Samson R., Legendre J. B., Christen R., Fischer-Le Saux M., Achouak W., Gardan L. ( 2005 ). Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int J Syst Evol Microbiol 55, 1415–1427 Toth I. K., van der Wolf J. M., Saddler G., Lojkowska E., Helias V., Pirhonen M., Tsror (Lakhim) L., Elphinstone J. G. ( 2011 ). Dickeya species: an emerging problem for potato production in Europe. Plant Pathol 60, 385–399 Waleron M., Waleron K., Podhajska A.J., Lojkowska E., (2002). Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of recA gene treatment. Microbiology, 148, 583-595 Waleron M.,Waleron K., Lojkowska E., (2013). Occurrence of Pectobacterium wasabiae in potato field samples. European Journal of Plant Pathology,137,149-158 Waleron M., Waleron K., Lojkowska E., (2014). Characterisation of Pectobacterium carotovorum subsp. odoriferum causing soft rot of stored vegetables. Eurpean Journal of Plant Pathology,139,457-469 Zaczek-Moczydlowska M., Fleming C.C., Young K.G., Campbell K., O’Hanlon R. (in review) Pectobacterium and Dickeya species detected in vegetables in Northern Ireland. European Journal of Plant Pathology Acknowledgements

The author thanks Supervisors Dr Katrina Campbell Dr Gillian Young Dr Colin Fleming Dr Richard O’Hanlon

Agri-Food and Bioscience Institute- AFBI staff Mr John Brazil Mr Brendan Moreland Mrs Rhonda Swan Ms Deborah Moffett Mrs Julie Morgan Mr Joseph Larkin Ms Fiona McHardy Miss Karina O’Neill Mr Trevor Martin Mr John Pollock Mr James Trudgett

Queen’s University Belfast staff and students Miss Cali Plahe Mr Ciaran Carlin Miss Julia Wilson Dr Stephen McFarland

DAERA For funding of the PhD project, provision of samples and assistance with field trials