Potato Blackleg Disease Research Articles

Complete Genome Sequence of Dickeya dianthicola ME23, a Pathogen Causing Blackleg and Soft Rot Diseases of Potato.

In 2014, an outbreak of potato blackleg and soft rot disease emerged in North America and continues to impact potato production. Here, we report the annotated genome sequence of Dickeya dianthicola ME23, a strain hypothesized to be representative of the bacterial population responsible for this disease outbreak.

Biochemical, physiological, and molecular characterization of Dickeya dianthicola (formerly named Erwinia chrysanthemi) causing potato blackleg disease in Japan

The taxonomic assignment of Japanese potato blackleg isolates of Dickeya spp. has not been confirmed after the changes in their former name, Erwinia chrysanthemi. Therefore, we investigated and identified 23 representative isolates of Dickeya spp. from symptomatic stems of potatoes in Japan, with biochemical tests and phylogenetic sequence analysis using recA, dnaX, rpoD, gyrB, and 16S rDNA sequences. Results of our biochemical tests showed that all isolates can be assigned to phenon 5 and biovar 1, which are associated with D. dianthicola. Based on the recA, dnaX, rpoD, gyrB, and 16S rDNA sequences, all isolates are in the same clade with D. dianthicola and were clearly distinguished from D. chrysanthemi, D. dadantii, D. dadantii subsp. dieffenbachiae, D. solani, D. zeae, and D. paradisiaca. Therefore, we conclude that Dickeya spp. isolated from potatoes with blackleg symptoms in Japan are D. dianthicola.

Combination of Different Antagonistic Bacteria to Control of Potato Blackleg Disease Caused by Pectobacterium atrosepticum under Greenhouse and Field Conditions

Three different antagonistic bacterial isolates, Pseudomonas fluoresces (Pf2), Bacillus subtilis(Bs3) and Rahnella acquatilis (Ra39) restricted the growth of Pectobacterium atrosepticum, the causal agent of black leg disease of potato, in vitro. Under greenhouse and field conditions, potato plants pre-treated with the three antagonistic bacterial isolates, individually or in combination, showed reduced disease severity relative to non-treated control plants. All isolates produced siderophores in different degrees but did not produce indole acetic acid (IAA) or hydrogen cyanide (HCN).The obtained results indicated that combination of Pseudomonas fluoresces (Pf2), Bacillus subtilis (Bs3) and Rahnella acquatilis (Ra39) is beneficial in controlling black leg disease of potato caused by pectobacterium atrosepticum.

Pectobacterium atrosepticum and Pectobacterium carotovorum Harbor Distinct, Independently Acquired Integrative and Conjugative Elements Encoding Coronafacic Acid that Enhance Virulence on Potato Stems.

Integrative and conjugative elements (ICEs) play a central role in the evolution of bacterial virulence, their transmission between bacteria often leading to the acquisition of virulence factors that alter host range or aggressiveness. Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell. In this study, the importance of horizontally acquired island 2 (HAI2), an ICE in the genome of Pectobacterium atrosepticum SCRI1043, was studied using a strain in which the entire ICE had been removed by CRISPR-Cas-mediated genome editing. HAI2 encodes coronafacic acid, a virulence factor that enhances blackleg disease of potato stems caused by P. atrosepticum SCRI1043. As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems. A subsequent screen for HAI2-related ICEs in other strains of the Pectobacterium genus revealed their ubiquitous nature in P. atrosepticum. Yet, HAI2-related ICEs were only detected in the genomes of a few P. carotovorum strains. These strains were notable as blackleg causing strains belonging to two different subspecies of P. carotovorum. Sequence analysis of the ICEs in different strains of both P. atrosepticum and P. carotovorum confirmed that they were diverse and were present in different locations on the genomes of their bacterial host, suggesting that the cfa cluster was probably acquired independently on a number of occasions via chromosomal insertion of related ICEs. Excision assays also demonstrated that the ICEs in both P. atrosepticum and P. carotovorum are mobilized from the host chromosome. Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum.

First Report of Potato Blackleg Disease Caused by Pectobacterium atrosepticum in Guangdong China.

Potato (Solanum tuberosum L.) is an important crop in China. In 2013, diseased potatoes exhibiting blackleg and soft rot symptoms were found in the winter potato growing areas of Huizhou city, Guangdong Province, China, with an incidence of approximately 20%. Initially, the stem bases of infected plants blackened and this symptom spread upward. Later, foliage of the diseased plants became yellow and the stem rotted with vascular discoloration. Twenty diseased plants with typical black leg symptoms were collected from a 10-ha potato field with approximately 60,000 potato plants per hectare. A bacterium with small, irregular, round, fluidal, white colonies was isolated from the vascular tissue of all diseased plants on nutrient agar at 26°C for 2 days. Ten strains were randomly selected for pathogenicity assays. Potato plants (cv. Favorita) at the five- to six-leaf stage were inoculated by injecting their stems with 1 ml of each strain in a bacterial suspension (3 × 108 CFU/ml). The inoculated potato plants were incubated at 16 to 21°C and 65 to 85% humidity, and exhibited the same symptoms as the diseased potato plants in the field by 3 to 5 days post inoculation (dpi). The bacterium was reisolated from the diseased tissue (stem) of the inoculated potato plants and produced characteristic pits on crystal violet pectate medium (1). The bacterium utilized a-methyl glucoside, glucose, lactose, maltose, cellobiose, raffinose, melibiose, and citrate, but not d-arabitol, sorbitol, or malonate. The bacteria also gave a positive reaction for catalase and production of reducing substances from sucrose, but gave a negative reaction for oxidase, production of phosphatase, and indole. Using the universal bacterial 16S rDNA primer set, 27f/1541R (4), 1,400-bp fragments were amplified from the 10 strains. The sequences of the 10 fragments (GenBank Accessions KC695819 to KC695828) were identical and had 100% sequence identity with 16S rDNA of Pectobacterium atrosepticum CFBP 1526 (JN600332). Further, the 438-bp and 690-bp fragments were respectively amplified from all 10 strains with the P. atrosepticum-specific primers Y45/Y46 (3) and ECA1f/ECA2r (2). To our knowledge, this is the first report of potato blackleg disease caused by P. atrosepticum (formerly named as Erwinia carotovora subsp. atroseptica) in Guangdong Province, China.

Detection and Identification of Erwinia carotovora Subsp. Atroseptica ( Van Hall , 1902 ) the Causal Agent of Potato Blackleg by RFLP-PCR

Potato blackleg disease is surveyed for spreading out in different potato growing areas in Jordan including; Jordan Valley (Al-Karama, Ashshuna Al-Janubiyya, Muthallath Al-Misri, Mu’addi, Deir Alla, Dirar, and Kurayyima), Amman (Al-Yadoda), Madaba (Om Al-Amad), Jerash (Tawahin Al-Udwan), Ma’an (Rum, and Al-Mudawwara). Forty six bacterial isolates of Erwinia carotovora subsp. atroseptica (Eca), the causal agent of the disease, were isolated and identified by different biochemical, physiological and nutritional tests from 145 diseased potato samples collected. Polymerase chain reaction (PCR)-based amplification of a 690-bp band was used to confirm the presence of Eca in the isolated bacteria estimated by the reference culture (ATCC 15713). PCR amplification was followed in isolation, identification and detection of Eca in infected and symptomless potato-stem and- tuber samples. The results showed that Eca was isolated from 30% of naturally infected potato samples with blackleg, while the 690-bp band was detected in 88% of DNA extracts from the naturally infected potato samples. In the other hand Eca was not isolated from the symptomless potato stem samples, while the 690-bp band was detected in 8% of the DNA extract from these stem samples. However; using symptomless-tuber samples have improved isolation efficiency. Eca was isolated directly from 4% of the samples when their suspension was streaked on Logan’s medium plates however 690-bp band was detected from 32% of their DNA-extract amplified with PCR. Genetic variability among the Eca isolates has been able to detect 690-bp PCR products for 46 different isolates those were digested with the restriction enzymes i.e. AluI, HaeII, HpaII and SauAI. Polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) analyses were able to cluster the Eca isolates into five PCR-RFLP groups with low genetic variability.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection and identification of Pectobacterium atrosepticum

Through exploration of available genomic sequence data for pectolytic bacterial plant pathogens, we identified genomic fragments best suited for developing specific diagnostic assays. A gene cluster encoding a pathogenicity-related phytotoxin similar to coronafacic acid of Pseudomonas syringae was identified as suitable for developing a loop-mediated isothermal amplification (LAMP) assay for rapid identification and detection of Pectobacterium atrosepticum, the causal agent of potato blackleg disease. The LAMP assay differentiates P. atrosepticum from other pectolytic potato pathogens and other bacteria and has a detection sensitivity of 2.5 × 102 CFU/mL−1. Together with a simplified DNA extraction method, the LAMP assay could serve as an easy and rapid method with potential application for on-site disease diagnosis and field surveys.

제주지역에서 분리한 감자 줄기검은병균의 유전적 특성

제주지역의 감자 줄기검은병징으로부터 분리한 12균주의 유전적 특성을 분석하기 위해 Eca-specific PCR, PCR-RFLP, ERIC-PCR을 실시하여 그 결과를 E. carotovora대조균들과 비교하였다. Eca-specific PCR 결과 Eca 대조균들은 특이적 밴드를 형성한 반면, 줄기검은병균은 특이적 밴드를 형성하지 않았다. 또한, pel유전자의 RFLP분석 결과 줄기검은병균은 pattern 2를 나타내었으나, Eca 균주는 pattern 3을 나타내어 Eca와는 다른 특성을 보여주었다. 16S rDNA의 RFLP분석결과 이번 실험에 이용된 대부분의 균주가 pattern 1을 나타냈지만, 12개의 줄기검은병균 중 11개의 균이 pattern 2을 나타내어 Ecc와도 다른 특성을 보여주었다. 제주지역의 무름병징과 줄기검은병징을 나타내는 균주들의 유전적 관계를 분석하기 위해 ERIC-PCR을 실시한 결과 줄기검은병균들은 특이적 밴드를 형성하였으며, 서로 높은 유연관계를 보여주었다. 따라서 제주지역의 줄기검은병징으로부터 분리한 균주들은 Eca, Ecc균들과는 다른 특성을 가지고 있음을 알 수 있었다. A collection of 12 Erwinia carotovora strains from blackleg diseased potato in Jeju island was characterized genetic diversity by 5. cayotovora subsp. atposeptica (Eca)-specific PCR, PCR-RFLP of the two genes (16S rRNA and pel) and repetitive sequence PCR (ERIC-PCR). The results were compared with those of the other E. carotovora representative strains. None of the blackleg strains produced PCR amplicons with Eca-specific primers in contrast to the single 690 bp amplicon obtained with Eca strains. In addition, on the basis of pel gene RFLP with Sau3AI, the blackleg strains belonged to the pattern 2 whereas Eca strains belonged to the other one (pattern 3). By analysis of 16S rDNA RELP generated with HinfI, the most strains including the E. carotovera subsp. carotovora (Ecc) representative strains used in this study belonged to the pattern 1 whereas the blackleg strains belonged to the pattern 2 except for one strain. Moreover, ERIC-PCR analysis showed that the blackleg strains were closely related to each other and had an unique DNA band. Based on these molecular approaches, we have confirmed that the blackleg disease of potato is caused by a different E. carotovora from Eca and Ecc in Jeju island.

Involvement of N-acylhomoserine Lactones Throughout Plant Infection by Erwinia carotovora subsp. atroseptica (Pectobacterium atrosepticum)

Erwinia carotovora subsp. atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage. The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step. Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL). The role of HSL throughout plant infection was analyzed. To this purpose, HSL produced by a specific E. carotovora subsp. atroseptica wild-type strain, which was particularly virulent on potato, were identified. A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated. The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development. Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated. Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants. Therefore, the use of QS quenching strategies for biological control in E. carotovora subsp. atroseptica cannot prevent initial infection and multiplication of this pathogen.

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil

To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Report of the International Conference on Potato Blackleg Disease available

Report of the International Conference on Potato Blackleg Disease available

Diagnose und Vorkommen bakterieller Naßfäuleerreger in Kartoffellagerbeständen

Summary From 1971 to 1976 soft-rotting potato tubers out of 6 districts of the GDR were studied upon the occurrence of bacterial soft rot pathogens. For this purpose bacterial strains were isolated from diseased tissue after enrichment on potato slices and identified (1971–1974) or bacteria isolated out of soft-rotted tissue were directly differentiated on various selective media without pre-enrichment (1975–1976). Tuber samples from 39 different sites were analyzed and more than 2,400 pathogenic bacterial strains were isolated thereout. By both methods in the most years 30–40% of the isolates obtained could be identified as Erwinia carotovora subsp. atroseptica , the causal agent of the black-leg disease of potato. Further E. carotovora subsp. carotovora (3–33%), pectolytic Pseudomonas (2–29%) and Bacillus spp. (9–82%) frequently were found. In contrast, Clostridium strains could be isolated continuously but in a lower frequency (2%). Bacteria from other genera ( Enterobacter, Klebsiella, Alcaligenes and others) occurred only very seldom. The results we obtained show, that out of the 26 possible combinations and 5 single variants of soft rot pathogens, only 18 in the samples studied were realized. In all combinations frequently found, the strains of both subspecies of Erwinia carotovora participated as a component. In the extreme dry year 1976 a considerable alteration of the normal pathogen population was to observe. Under these conditions Bacillus strains occupied 80 per cent of the pectolytic isolates. The importance of the results obtained for successful control of tuber soft rot is discussed, concerning the fact that all soft rot pathogens found can infect the tubers only pathing wounds or lenticells.

The role ofErwinia carotovora in the epidemiology of potato blackleg. II. The effect of soil temperature on disease severity

Relative disease severity (seedpiece decay or post emergence blackleg) incited byErwinia carotovora var.carotovora (Ecc) andErwinia carotovora var.atroseptica (Eca) was studied in the field and the greenhouse. When inoculated tubers were planted in cool soils in the field or greenhouse (7.0–18.5°C average minimum and 16–26°C average maximum temperature during the first 30 days after planting)Eca caused significantly more disease thanEcc. Under these experimental conditionsEcc did not cause significantly more disease than that found in non-inoculated controls. When soil temperatures were high at planting time (21.4–24.0°C average minimum and 29.6–35.0°C average maximum temperature for the first 30 days after planting), inoculation withEcc resulted in severe disease whileEca produced no more infection than that found in uninoculated controls. At intermediate soil temperatures both were infective. Seedpiece decay by both organisms was correlated with soil temperatures during the first 30 days after planting. As the soil temperature increased, inoculation withEcc resulted in more disease than inoculation withEca. At lower soil temperatures the opposite result was obtained. Field and greenhouse studies showed that bothEca andEcc were able to cause typical post emergence blackleg infection, the former at low soil temperature and the latter at high temperature conditions. The data suggest that bothEcc andEca are components of the potato blackleg and soft rot disease complex. Environmental conditions, especially soil temperature, determine which organism is causative under a particular set of conditions.

Effect of inorganic fertilizers on the Incidence of potato blackleg disease

In assessing disease levels in a number of potato fertilizer trials in south-east Scotland it was found that an increase in the rate of application of complete fertilizer or nitrogen alone resulted in a decrease in the proportion of stems affected by potato blackleg.

Experiments on the control of black-leg disease of potato by disinfection of seed tubers with mercury compounds and streptomycin

Black-leg disease of potato, caused by the bacteriumPectobacterium carotovorum var.atrosepticum (Erwinia atroseptica) is very widespread in potato stocks in Scotland, but under the prevailing environmental conditions the disease rarely reaches epidemic proportions and is not usually considered to be a serious problem. But when seed tubers are exported from Scotland and grown in warmer countries such as Israel, epidemics of black-leg are liable to develop, and attempts have been made to control these outbreaks by surface disinfection of the whole seed with solutions of several chemical substances. Disinfectants used included mercuric chloride, ethoxyethylmercury chloride (EEMC), streptomycin and a mixture of streptomycin and EEMC. The solutions also contained a surface active agent to increase their wetting and penetrating action. Tubers taken at random from black-leg infected crops were disinfected soon after lifting, transported from Scotland to Israel by rail and cargo boat, and then further, as yet untreated samples were disinfected. Tubers washed in water only were used as controls. The seed was planted in replicated plots, given overhead irrigation when necessary, and the black-leg content of the subsequent crop was determined. The results showed that in the case of one experiment where about one-fifth of the control plants was affected, disinfection reduced the amount of black-leg considerably, but did not eliminate the disease. In another experiment, disinfection appeared to increase the amount of black-leg, while in the remaining three experiments the treatment had no significant effect. It is concluded that treatment of whole tubers with mercurials, streptomycin preparations or mixtures of the two cannot be said to control potato black-leg disease. The failure of surface disinfection is probably caused partly by some of the bacterial infection being systemic in the tuber and partly by the poor penetration of the disinfectants through the suberized surface layers of the mature tuber.

A Selective Medium for the Isolation of Coliform Soft Rot Bacteria from Plant Tissue

DURING further studies on black leg disease of potato, difficulty was experienced in isolating the appropriate pathogens, which were identified as Erwinia atroseptica (van Hall) Jennison syn. Bacterium atrosepticum (van Hall) Burgwitz. Even using the screening methods of Noble and Marshall1, namely, seeding plates of meat infusion agar with contaminated material from infected stems and tubers, selection of the organisms was difficult owing to the growth of many other types of bacteria. MacConkey's lactose bile-salt neutral red agar was found to be more selective, but Pseudomonas spp. could not now be identified by fluorescence in ultraviolet light, although a number still grew on the plates. The selective pectate gels of Rudd-Jones2 and others were also tried, but proved to be difficult to prepare especially with limited facilities, and were often too soft to use for plating.