Contamination and subsequent multiplication of soft rot erwinias on healthy potato leaves and debris after haulm destruction

Small plots of potatoes were inoculated with a mixture of Erwinia carotovora (E. c.) subsp. carotovora and E. carotovora subsp, atroseptica strains resistant to rifampicin. Subsequently the population off, c. subsp, carotovora and E. c. subsp, atroseptica (rifampicin-resistant and wild types) present as epiphytes on the surface of potato leaves was assessed using three methods, qualitative, semi-quantitative and quantitative, during 1986 and 1987. The population was generally low (< 10² colony forming units (> 10⁴cfu/g leaves) but reached higher levels (> 10⁴ cfu/g) on occasions later in the growing season, Rifampicin-resistant erwinias were reisolated only infrequently throughout this study. Different methods of haulm destruction (herbicide, pulverization, sulphuric acid treatment and natural senescence) greatly influenced the number of erwinias present in the resulting plant debris. Pulverization resulted in the highest population (10⁶-10⁷ wild-type cfu/g) in both seasons. In 1987. the wettest of the two seasons of this study, herbicide treatment resulted in similarly high populations. The results suggest that the high numbers of erwinias found in the haulm debris were probably derived from the generally low populations of epiphytic bacteria previously present on healthy leaves, E. c. subsp, carotovora was the most frequent subspecies in the rotting plant debris; E. c. subsp, atroseptica was more commonly found on healthy leaves. The implications of the results are discussed in relation to the production of seed potatoes with a low risk of blackleg.     

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 10³ culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.     

A simplified slice method for assessing tuber susceptibility of potato cultivars to Erwinia carotovora subsp atroseptica

The susceptibility of 24 potato cultivars to soft rot caused by Erwinia carotovora subsp. atroseptica was assessed in a simplified form of a tuber slice test described previously. Freshly made wounds on tuber slices were inoculated with drops (0–02 ml) containing 10⁸, 10⁷ or 10⁶ cells/ml and incubated aerobically for 3 days at 15° or 20°C. At 20°C differences between cultivars were clear at all concentrations but at 15°C less so at the lowest concentration unless incubation time was extended. The rank order of cultivar susceptibility was similar to that found in previous seasons.     

Potato blackleg: Epidemiology,host-pathogen interaction and control

Blackleg is caused byErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Echr) in cool and hot climates respectively. The bacteria are opportunistic pathogens and rely on their strong pectolytic character to infect plants when conditions favor their multiplication. Blackleg is a seedborne disease and the bacteria can survive in a quiescent form in lenticels and wounds during storage. The contaminated mother tuber and not the blackleg plant is the main source of progeny tuber contamination. Other sources of the pathogen are airborne (insects and aerosols) erwinias deposited on leaves and from there to the soil and progeny tubers, and erwinias in rotting tubers smeared into wounds incurred during mechanical crop handling.Most seed tubers are contaminated but blackleg incidence is related to seed contamination level modulated by soil water status. Competitiveness of the erwinias in the rotting mother tuber is affected by temperature, Eca is favored at <25°C and Echr at higher temperatures. The ubiquitousE. carotovora subsp.carotovora apparently fails to compete successfully with the other erwinias and saprophytic pectolytic bacteria in mother tubers and therefore does not cause blackleg.Disease control measures are based on avoiding tuber contamination by cultural means (early harvesting), reducing tuber contamination level (dry storage and hot water treatment) and planting ‘clean’ seed identified by quantifying tuber contamination rather than by visual crop inspection. Finally, recently identified highly resistant, even under anaerobic conditions, wildSolanum spp. could be used to breed for resistant cultivars by conventional methods or by genetic engineering.     

Potato diseases caused by soft rot erwinias: an overview of pathogenesis

Three soft rot erwinias, Erwinia carotovora ssp. carotovora , E. carotovora ssp. atroseptica and E. chrysanthemi are associated with potatoes causing tuber soft rot and blackleg (stem rot). Latent infection of tubers and stems is widespread. As opportunistic pathogens, the bacteria tend to cause disease when potato resistance is impaired. Pathogenesis or disease development in potato tubers and stems is discussed in terms of the interaction between pathogen, host and environment, microbial competition and recent findings on the molecular basis of pathogenicity. Emphasis is placed on the role of free water and anaerobiosis in weakening tuber resistance and in providing nutrient for erwinias to multiply. Blackleg symptoms are expressed when erwinias predominate in rotting mother tubers, invade the stems and multiply in xylem vessels under favourable weather conditions. Soft rot erwinias tend to out-compete other bacteria in tuber rots because of their ability to produce larger quantities of a wider range of cell wall-degrading enzymes. However, despite extensive studies on their induction, regulation and secretion, little is known about the precise role of the different enzymes in pathogenesis. The putative role of quorum-sensing regulation of these enzymes in disease development is evaluated. The role certain pathogenicity-related characters, including motility, adhesion, siderophores, detoxifying systems and the hrp gene complex, common to most bacteria including symbionts and saprophytes, could play in latent and active infections is also discussed.     

Microbiological, immunological and molecular methods suitable for commercial detection and quantification of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, on seed potato tubers: a review

Contamination of seed potato tubers by Erwinia carotovora subsp. atroseptica is widespread with the bacteria usually sited superficially in lenticels and suberized wounds. As seed contamination level is related to blackleg incidence, seed health is best assessed by determining the number of cells of E. c. atroseptica per mL of tuber-peel extract. The relative specificity, sensitivity and ease of use of four recently developed microbiological, immunological and molecular methods to detect and/or quantify tuber contamination are discussed in relation to the testing of commercial seed stock. Sensitivities of all four methods are at or below the threshold level for blackleg development (< 10³ cells mL^-1), but there are differences regarding their specificity and ease of use. Three of them allow enumeration of most live cells of the bacterium, using specific monoclonal and polyclonal antibodies against the predominant serogroup I: (a) immunomagnetic separation of E. c. atroseptica before viable count on a selective-diagnostic growth medium, crystal violet pectate, (b) immunofluorescence staining and counting of colonies in pour-plate medium in tissue culture plates and (c) enrichment of the bacterium in peel-extract dilutions directly in microtitre plates prior to DAS-ELISA. In the fourth method, both live and dead cells are detected, but not quantified, by PCR amplification of target sequences using specific primers for E. c. atroseptica regardless of serogroup.     

A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specificity

A detection method is described for latent infections of Pseudomonas solanacearum race 3 in potato. This method is based on extraction of 200 heel ends of potato, followed by screening with an indirect antibody stain (IFAS) and (in IFAS-positive cases) a pathogenicity test on tomato (PT). Using the method in some sensitivity and specificity tests with more than 600 samples it appears that: (a) its detection level is 10⁴ cells ml^-1 in IFAS and 10²— 10⁴ cells ml^-1 in PT; (b) per cent recovery is 0.1–10% (10% at lower contamination levels); (c) false-positive samples due to cross-reacting bacteria in IFAS were limited to only 2–3%, using two polyclonal antisera against whole cells of P. solanacearum. The merits of the method in comparison with others (ELISA, selective media) are discussed.     

Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel-end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 10⁶–7.4 × 10⁷ colony-forming units per ml. Following concentration by high-speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi-selective media, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre-enrichment of samples in semi-selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi-selective medium and tomato bioassay could detect fewer than 10⁴ cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.     

Methods of testing potato cultivars for resistance to soft rot of tubers caused by Erwinia carotovora subsp. atroseptica

The resistance of eight potato cultivars to tuber soft rot caused by E. carotovora subsp. atroseptica was assessed in 2 years using three different test methods. Similar cultivar resistance rankings were obtained for any one method within a year and between years for two methods (single site, infectivity titration), but not for the third (vacuum infiltration). However, the ranking of cultivars differed for the three methods. Ranking was not affected by inoculating the cortex or the more susceptible medullary tissue, or by assessing rotting in terms of infection frequency or lesion size, but it was affected by oxygen concentration during incubation. Differences among cultivars were greater when inoculated tubers were incubated anaerobically than when incubated with 5% oxygen. There was no relationship between the relative susceptibilities of cultivars to tuber soft rot in storage in January/February and those of mother tubers after planting.     

Internal colonization pathways of potato plants by Erwinia carotovora ssp. atroseptica

Transmission of pectinolytic Erwinia species from infected mother tubers to daughter tubers has been studied mainly through detection tests, carried out at harvest, on limited samples of tubers produced by plants grown from artificially inoculated mother tubers. However, detection has not been performed on samples collected at different stages of crop development, in order to follow the contamination progress in different organs through the plants to the progeny tubers. In this study the bacterial contamination of progeny tubers was investigated by detecting Erwinia carotovora ssp. atroseptica in different symptomless plant organs (stolons, stems, progeny tubers) and in the parts with or without symptoms of diseased stems, collected at various stages of crop development. Infection levels in below- and above-ground organs of plants of two cultivars differing in their resistance to Erwinia, infected by either vacuum infiltration or sand wounding, were monitored throughout the growing season and at harvest using DAS-ELISA and PCR. Detection tests showed that healthy organs from symptomless plants were less frequently contaminated than symptomless organs from diseased plants, and that stolons were precociously and more frequently contaminated than stems and daughter tubers, irrespective of the health of the plant. Stem infections were shown to progress latently in the stem, bacteria usually being recovered 10–15 cm past visible lesions. In many cases, typical aerial stem-rot symptoms could be related to this upward movement of bacteria from the infected mother tuber. Daughter tubers without symptoms were shown to be frequently contaminated, usually at heel ends, suggesting internal contamination from mother tuber to progeny.     

Novel in vivo use of a polyvalent Streptomyces phage to disinfest Streptomyces scabies-infected seed potatoes

A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1 × 10⁹ plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly ( P  < 0·05) reduced levels of surface lesions of scab (1·2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced ( P  < 0·05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers.     

Methods for assessing the susceptibility of potato tubers of different cultivars to rotting by Erwinia carotovora subspecies atroseptica and carotovora

The susceptibility of tubers of different potato cultivars to soft rot by Erwinia carotovora subspp. uroseptica and carotovora was assessed in 3 years by two methods. In one method, whole tubers inoculated at wounds with either bacterium were incubated under anaerobic conditions for 5 or o days at 15°C. In the other method, wounds made in tuber slices were allowed to heal or not, before inoculation with different concentrations of each bacterium and were then incubated under aerobic conditions for 3 days at 15°C. Most cultivars gave consistent reactions in repeated experiments using the same method, but there was some seasonal variation. A few cultivars were consistently susceptible (Klondyke and Manna) or resistant (Drayton) in both methods but others gave completely contrasting results (Record). In both methods and with all cultivars more rotting was caused by subsp. atroseptica than by subsp. carotovora because of the temperature of Incubation.     

Development of symptoms caused by Erwinia carotovora ssp. atroseptica under field conditions and their effects on the yield of individual potato plants

Infection of seed tubers by pectinolytic Erwinia species can lead to the development of various symptoms during vegetative growth of potato crops, including non-emergence of plants, chlorosis, wilting, haulm desiccation and typical blackleg. The relationships between types of symptoms and yield are poorly documented, and are investigated by following the development of symptoms in potato plants grown under field conditions from seed tubers artificially inoculated with E. carotovora ssp. atroseptica ( Eca ) , and measuring the yield of each plant. Symptoms were classified into five main types (non-emergence, wilting/chlorosis, blackleg, haulm desiccation and plant death). Each plant was scored for types of symptom on four successive dates; plants without visible symptoms were scored as healthy. The method of inoculation and inoculum concentration proved major factors for the subsequent development of symptoms. Disease development was more severe after vacuum infiltration of bacteria into seed tubers than after shaking tubers in contaminated sand. Disease usually progressed from chlorosis and/or wilting to partial or total desiccation on a given plant. Yield losses varied according to symptom type, but the relationship between symptoms recorded and yield also depended on scoring dates. The data suggest that the beginning of tuber growth might be the most suitable stage for predicting yield losses from symptom observations. In both cultivars studied (Bintje, highly susceptible, and Désirée, moderately resistant), the yield of symptomless plants growing from inoculated seed tubers was significantly less than that of control plants, indicating that the presence of bacteria on the seed tuber was detrimental, even in the absence of visible symptoms. Differences in symptom expression in the field between cultivars matched the level of visible infection of tubers at harvest, as Bintje tubers showed a higher incidence of rot than Désirée tubers.     

Mixed rots of potato tubers in Ukraine

The pathogens involved in rotting potato tubers at harvest or in store have been identified in the forest and forest-steppe zones of Ukraine. Fusarium spp., Phytophthora infestons, Erwinia carotovora and Ditylenchus destructor are the most important.     

Heat treatment of seed tubers for control of potato blackleg (Erwinia carotovora subsp. atroseptica) and other diseases

The thermal death points of Erwinia carotovora subsp. atroseptica and subsp. carotovora were determined in relation to duration of heat treatment, age of culture and culture medium. No isolates cultured in liquid media survived heating at 53°C for 5 min while those on solid media were killed by heating at 54°C for 10 min. After immersing naturally contaminated potato tubers for 10 min in water at 55°C, Erwinia could not be detected. The same treatment of naturally or artificially contaminated seed tubers gave complete absence of blackleg infection in the field and decreased the amounts of powdery scah(Spongospora subterranea) and black scurf (Rhizoctonia solani) on progeny tubers.     

Effect of temperature of production of Botrytis allii conidia on their pathogenicity to harvested white onion bulbs

Botrytis allii colonies incubated at low temperatures have been reported to produce larger conidia that germinate faster and give rise to longer germ-tubes than those grown at room temperature. The present study compared the effect of conidia produced at 20°C and at 0 and –2°C on their pathogenicity to artificially inoculated white onion bulbs, and the effect of conidial concentration (5×10³ and 5×10⁴ conidia/mL) on disease incidence, lesion area, incubation and latent period during storage at 20, 5 and 0°C. At all storage temperatures and periods tested conidia produced at −2°C caused a higher disease incidence and larger areas of rot than those produced at higher temperatures. When the conidial production temperature was raised to 20°C, the duration of incubation on the bulbs inoculated with 5×10⁴ conidia/mL was more than doubled during storage at 0°C, tripled at 5°C, and took 50% longer at 20°C. The incubation period was not significantly affected by conidial concentration at 20°C, and only slightly at 5 and 0°C, but at low temperatures the latent period was longer because of the delay induced in sporulation. These data are consistent with the packers' opinion that cross-infection of spring onions by long-term refrigerated onions in grading lines caused earlier and heavier rotting.     

Studies of a wilt disease of the potato plant in Israel caused by Erwinia chrysanthemi

In Israel field infections of potato plants by Erwinia chrysanthemi are characterized by wilting of the leaves followed by total desiccation of the plants. These symptoms are indistinguishable from those caused by Verticillium dahliae or those that develop during the normal process of plant senescence. Diagnosis of E. chrysanthemi in the spring-sown (February) crop in Israel is difficult because all three conditions often appear at approximately the same time, late in the growing season in May when the air temperature exceeds 25°C. The symptoms of E. chrysanthemi infection were reproduced in the field when potato seed tubers, tested and found to be contaminated at a low level with E. carotovora pv. carotovora , were inoculated with a strain of E. chrysanthemi isolated from a diseased potato plant. When plants in a growth cabinet at 30°C were stem-inoculated with E. chrysanthemi , similar symptoms developed when the relative humidity was low ( c . 80%). Presence of the disease only on plants grown from seed contaminated with E. chrysanthemi and not from uncontaminated seed suggests that the bacterium is seed borne, as is E. carotovora pv. atroseptica , the blackleg pathogen.     

Highly sensitive detection of Ralstonia solanacearum in latently infected potato tubers by post-enrichment enzyme-linked immunosorbent assay on nitrocellulose membrane

A post-enrichment enzyme-linked immunosorbent assay (ELISA) on nitrocellulose membrane (NCM-ELISA) is described for the detection of Ralstonia solanacearum in latently infected potato tubers. The polyclonal antiserum specificity was significantly improved by adsorption with cross-reacting bacteria. The detection efficiency after enrichment was compared with those of nucleic acid spot hybridization (NASH), double-antibody-sandwich immunoassay (DAS-ELISA) and plating on modified Kelman's medium. After 48 h of incubation of the tuber extracts with modified SMSA broth at 30°C, sensitivities of post-enrichment NCM-ELISA, DAS-ELISA and NASH were similar. As few as 10cells mL⁻¹ were detected in either inoculated or naturally infected tuber extracts. Of 255 field samples, no cross-reactivity of NCM-ELISA was observed. Post-enrichment NCM-ELISA thus provides a reliable and sensitive low-cost method that is rapid and easy to use, making it suitable for assessing susceptibility of breeding lines to bacterial wilt, ecological studies and seed quality control in developing countries.     

The interaction between bactericidal chemicals and the wound healing process of potato tuber tissue

In-vitro tests of dichlorophen against Erwinia carotovora, vars. atroseptica and carotovora, have shown good antibacterial activity; in-vivo tests on potato tubers, however, showed poor control of bacterial soft rotting. Under aerobic or near anaerobic conditions, wounded potato tubers dipped in dichlorophen showed significant increases in soft rotting over water-treated tubers. Under near anaerobic conditions, neither dichlorophen-dipped nor water-dipped tubers showed evidence of wound healing. However, when incubated aerobically, a distinct wound barrier was evident in the water-dipped but not the dichlorophen-dipped tubers. To investigate the possibility that dichlorophen was inhibitory to the initiation of wound healing, an image analysing computer was used to measure the accumulation of fluorescent products in the walls of sectioned tuber tissues treated with dichlorophen. Dichlorophen treatment resulted in very low levels of fluorescence, indicating little phenolic accumulation in the walls. Other bactericides also decreased fluorescence to some extent.     

硫酸铜防治马铃薯软腐病及其对薯块有关酶类的影响

 用0.05% CuSO₄·5H₂O浸泡马铃薯薯块半小时,几乎完全防治了贮藏期的细菌性软腐病。田间试验中,叶面喷洒0.1% CuSO₄·5H₂O能显著减轻黑胫病的发生。叶面喷铜后叶片,茎杆和子薯的含铜量提高,子薯对伤口接种和皮孔接种的抗性增强。叶面喷铜对子薯的多酚氧化酶、过氧化物酶活性没有明显影响。生长期内喷25次和10次铜使薯块组织的苯丙氨酸(PAL)活性升高。用CuSO₄·5H₂O浸泡薯块或向薯块的酶抽提液中直接加入Cu²⁺,对多酚氧化酶和过氧化物酶活性均影响不大,而PAL活性显著降低。文中讨论了Cu²⁺在薯块抗性中的作用。