Draft Genome Sequence of the Biocontrol Strain Serratia plymuthica A30, Isolated from Rotting Potato Tuber Tissue

Serratia plymuthica A30 is a Gram-negative bacterium expressing antagonistic activity toward blackleg- and soft rot-causing Dickeya sp. biovar 3 (“Dickeya solani”). Here, we present the draft genome sequence of strain A30, which has been isolated from rotten potato tuber tissu

Ecology and control of Dickeya spp. in potato

Potato blackleg caused by pectinolytic Pectobacterium and Dickeya species is a bacterial disease creating serious economic losses in (seed)potato production worldwide. Effective management to control blackleg is absent and validated, cost-effective detection protocols for blackleg bacteria do not exist. This situation results partially from the lack of knowledge of the ecology of blackleg pathogens in general, and Dickeya spp. in particular. Since 2000 an increasing frequency of Dickeya spp. in seed potatoes in Europe has been observed which is connected to emergence of a new species belonging to biovar 3 but unclassified inside known and described six Dickeya species. This species was provisionally named __D. solani__ and isolated from potato in The Netherlands, Finland, Poland, Germany, Belgium, France, United Kingdom and Israel. The objectives of the study described in this thesis were to acquire knowledge on the ecology (i. e. distribution of Dickeya spp. in seed potato tubers, role of the soil-borne inoculum in disease incidence, role of the aerial haulm infection for the blackleg incidence in progeny tubers) of a new genetic clade of biovar 3 Dickeya spp.; and to find and characterize in detail the valuable biocontrol agent(s) (isolated from rotten potato tissue) to cure seed potato tubers from blackleg caused by biovar 3 Dickeya spp. (__D. solani__). Direct isolation of viable bacterial cells using (selective) plating techniques combined with molecular (16S rDNA and Rep-PCR), serological (DAS-ELISA) and biochemical (biovar determination) characterization of the isolates were used to evaluate the presence of Dickeya and Pectobacterium spp. in different plant parts and tissues. Bacterial population dynamics in planta was studied in greenhouse experiments. For the studies on colonization of potato tissues by biovar 3 Dickeya spp. (__D. solani__) and Serratia plymuthica A30, bacterial strains were transformed with plasmid-based genes coding for red or green fluorescent proteins (DsRed and GFP, respectively) that were constitutively expressed in bacterial cells. Epifluorescence stereo microscopy and confocal laser scanning microscopy were used to visualize bacterial presence in different tissues of potato tubers and plants. Studies on the distribution of Dickeya ( and __D. solani__) and Pectobacterium species in seed potatoes revealed that the pathogens were distributed unevenly inside tubers and that the stolon end tuber part harbored the highest bacterial inoculum, whereas deeper located tissues were usually not colonized. Studies on the importance of soil-borne biovar 3 Dickeya sp. (__D. solani__) inoculum for systemic colonization of potato plants and latent infection of progeny tubers unveiled that D. solani is able to systemically colonize progeny tubers from soil via the roots and to cause true blackleg symptoms in infected plants after soil infestation. Studies on the role of haulm infections for latent contamination of progeny tubers by D. solani presented that the injection of bacteria into stems can result in the downward vascular translocation and a colonization of underground plant parts and finally, latent contamination of progeny tubers. Potato leaves inoculation with a biovar 3 Dickeya sp., showed degradation of the inoculated plant material and spreading of the internal inoculum to the petiole and axil and finally to the main stem but bacteria were not detected in the underground plant parts. Studies on characterization of the bacteria antagonistic to biovar 3 Dickeya sp. (__D. solani__) allowed to obtain knowledge of the presence of such strains in rotten potato tissue and on possibility of their used in the biocontrol of Dickeya spp. in potato. Selection of a candidate strain (Serratia plymuthica A30) that was effective in protecting potato plant tissue from blackleg was performed on the basis of in vitro and greenhouse tests. In vitro tests proved that S. plymuthica A30 possess different mechanisms by which it can control Dickeya spp. (i. e. production of antibiotics and biosurfactants). The interaction of D. solani and S. plymuthica A30 under greenhouse conditions was investigated in detail using artificially co-inoculated tubers and by application of A30 to the soil. It was showed that both micro-organisms interact in situ and that S. plymuthica A30 is able to decrease or eradicate Dickeya sp. inoculum from infected potato plants by competition un the same niches. In conclusion, a new biovar 3 Dickeya spp. strain was present in high densities in stolon end of naturally infected progeny tubers. The bacterium efficiently colonized various plant tissues after artificial inoculation of soil or haulms and was able to systemically colonize entire potato plant in relatively short time. For biocontrol of biovar 3 Dickeya spp. in potato, a bacterial strain characterized as S. plymuthica A30 tested in in vitro and under greenhouse conditions proved to have great potential for controlling blackleg in conditions favorable for disease development and when Dickeya sp. was applied in the high inoculum. The A30 strain effectively protected potato plants against blackleg in greenhouse experiments, both in vacuum infiltrated tuber and when applied as superficial treatment during planting. Treatment with A30 reduced the disease incidence to 0%. S. plymuthica A30 was able to colonize the plants internally, and combat internal infections with D. solani.STW Technologiestichting via grant 10306: Curing seed potatoes from blackleg bacteriaUBL - phd migration 201

Bacterie zorgt voor doorbraak in erwiniabestrijding

Wageningen – Wetenschappers hebben een bacterie gevonden die de aardappel- en bollenziekte erwinia kan bestrijden. Tot nu toe is erwinia niet te bestrijden. Er zijn ook geen resistente rassen tegen de ziekte. Erwinia wordt veroorzaakt door de dickeya-bacterie en leidt tot zwartbenigheid en natrot in aardappelen en bloembollen. De ziekte veroorzaakt alleen in Nederland jaarlijks enkele tientallen miljoenen euro’s schade. De bacterie die de dickeya-bacterie bestrijdt is gevonden door onderzoeker Robert Czajkowski van Plant Research International (PRI-WUR). Hij ontdekte in kasproeven dat de bacterie Serratia plymuthica de dickeya-bacteriën effectief kan bestrijden. De antagonist (tegenstander) dringt net als dickeya de wortels binnen en verspreidt een antibioticum dat de dickeya-bacteriën doodt. Daarna was de dickeya-bacterie niet of nauwelijks terug te vinden in een besmette plant. Het PRI heeft de bacterie gepatenteerd en onderzoekt de mogelijkheden die te vermarkten. PRI-onderzoeker Jan van der Wolf noemt het onderzoek van strategische waarde. ”Op langere termijn kan het leiden tot een product waarmee je dickeya kunt bestrijden. Het werkt goed in kasproeven. We gaan het volgend jaar testen in veldproeven

Draft Genome Sequence of the Biocontrol Strain Serratia plymuthica A30, Isolated from Rotting Potato Tuber Tissue

Serratia plymuthica A30 is a Gram-negative bacterium expressing antagonistic activity toward blackleg- and soft rot-causing Dickeya sp. biovar 3 (“Dickeya solani”). Here, we present the draft genome sequence of strain A30, which has been isolated from rotten potato tuber tissu

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan(A (R)) assay

A Serratia plymuthica-specific TaqManA (R) assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqManA (R) assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqManA (R) assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100 cfu ml(-1)) in vitro, down to 50 bacterial cells per reaction (1,000 cfu ml(-1)) in spiked potato root extracts and down to 5 bacterial cells per reaction (100 cfu ml(-1)) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqManA (R) assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan® assay

A Serratia plymuthica-specific TaqMan® assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan® assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan® assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100 cfu ml-1) in vitro, down to 50 bacterial cells per reaction (1,000 cfu ml-1) in spiked potato root extracts and down to 5 bacterial cells per reaction (100 cfu ml-1) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan® assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrate

Distribution of Dickeya spp. and Pectobacterium carotovorum subsp carotovorum in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed

Effectieve kolonisatie van aardappelplanten door Dickeya-soorten (Erwinia chrysanthemi) : themanummer fytobacteriologie

De bacterieziekten zwartbenigheid en stengelnatrot, veroorzaakt door Pectobacterium en Dickeya (Erwinia)- soorten, berokkenen grote schade aan de pootaardappelteelt. Bij PRI en HZPC wordt onderzoek verricht naar de verspreiding van deze pathogenen tijdens teelt- en (na)oogst. Het was al bekend dat er bij aanwezigheid van rotte knollen, tijdens teelt en (na)oogst, versmering van pootgoed kan plaatsvinden. Getracht is de vraag te beantwoorden hoe verspreiding tijdens de teelt kan plaatsvinden, zowel boven- als via ondergrondse delen van de plan

Chemical disinfectants can reduce potato blackleg caused by ‘Dickeya solani’

Treatments of tubers with chemical disinfectants (70 % ethanol, 1 % sodium hypochlorite, 2 % copper sulphate, 5 % peracetic acid, 10 % hydrogen peroxide, 1 % MennoClean (benzoic acid), 5 % trisodium phosphate and 0.2 % caffeine) were evaluated for control of blackleg caused by ‘D. solani’. All disinfectants effectively killed bacteria in axenic cultures within 5 min and all, except hydrogen peroxide and trisodium phosphate, were able to kill ‘D. solani’ in spiked potato extracts. Treatments with all disinfectants except trisodium phosphate, reduced the pathogen populations on the periderm of tubers previously inoculated by dipping in a suspension of ‘D. solani’, when the disinfectant was applied for at least 15 min. However, in replicated greenhouse experiments, treatments with all disinfectants except hydrogen peroxide and caffeine resulted in phytotoxicity, reducing sprouting of tubers with 10 – 100 %. Sodium hypochlorite and MennoClean were selected for further studies. Treatments of inoculated tubers with these disinfectants prevented developing of soft rot symptoms on tubers when subjected to favourable rotting conditions. In replicated greenhouse experiments, inoculated tubers treated with sodium hypochlorite and MennoClean reduced the blackleg disease incidences from 50 % to 0 % and to 5 %, respectively, and symptomless infection of potato stems from 93 % to 0 % and to 5 %, respectively. A combination of both compounds was highly phytotoxic. The potential use of sodium hypochlorite and MennoClean in an integrated management strategy for potato blackleg is discusse