Contamination of potatoes by Erwinia carotovora during grading

Considerable tuber contamination by soft rot erwinias in rotting tubers can occur when grading potato stocks. Erwinia carotovora pv. carotovora , from a single rotting tuber, contaminated c. 100 kg of potatoes during mechanical grading, c. 50% being contaminated with 10⁴–10⁵ bacteria per tuber. Survival of the bacteria during storage was related to the degree of damage sustained by the tubers during grading which in turn depended on the model of oscillating riddle grader used and the cultivar of potato. Chemical disinfection of the grader immediately before use and of tubers immediately after grading reduced contamination.     

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.     

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.     

A rapid method to identify and quantify soft rot erwinias on seed potato tubers1

Potato tubers are usually contaminated by more than one species or pathovar of soft rot erwinia and, because blackleg incidence is related to the contamination level of seed tubers, the disease potential of seed stocks may be assessed by determining seed-tuber contamination level. A method is described for identifying and quantifying directly from tubers the three soft rot erwinias commonly associated with potatoes. Replicate lots of 10–15 tubers are peeled by dry abrasion in a commercial potato peeler and the peel weight determined by weighing the tubers before and after peeling. Sap is expressed from the peel, an antioxidant (0.075% dithiothreitol) added, and the sap is dilution-plated on a diagnostic selective medium (crystal violet pectate [CVP]). After incubating for 24 h at 20°C, the plates are velvet-replicated onto fresh plates of CVP with or without 35 μg ml^-1 erythromycin and incubated for 48 h at 27°C and 24 h at 33.5 or 37°C. Soft-rot erwinias typically form deep cup-like cavities on CVP and they can be identified and enumerated according to the pattern of cavity formation. Cavities are formed by Erwinia carotovora pv. atroseptica only at 27°C, by E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C, whereas E. chrysanthemi forms cavities at all temperatures but fails to grow in the presence of erythromycin. Contamination levels can be expressed as the number of different erwinias per tuber or per g peel.     

QTLs for potato tuber resistance to Dickeya solani are located on chromosomes II and IV

The goal of this research was to identify quantitative trait loci (QTLs) for potato tuber resistance to the soil- and seedborne bacterium Dickeya solani and for tuber starch content, to study the relationship between these traits. A resistant diploid hybrid of potato, DG 00-270, was crossed with a susceptible hybrid, DG 08-305, to generate the F₁ mapping population. Tubers that were wound-inoculated with bacteria were evaluated for disease severity, expressed as the mean weight of rotted tubers, and disease incidence, measured as the proportion of rotten tubers. Diversity array technology (DArTseq) was used for genetic map construction and QTL analysis. The most prominent QTLs for disease severity and incidence were identified in overlapping regions on potato chromosome IV and explained 22.4% and 22.9% of the phenotypic variance, respectively. The second QTL for disease severity was mapped to chromosome II and explained 16.5% of the variance. QTLs for starch content were detected on chromosomes III, V, VI, VII, VIII, IX, XI, and XII in regions different from the QTLs for soft rot resistance. Two strong and reproducible QTLs for resistance to D. solani on potato chromosomes IV and II might be useful for further study of candidate genes and marker development in potato breeding programmes. The relationship between tuber resistance to bacteria and the starch content in potato tubers was not confirmed by QTL mapping, which makes the selection of genotypes highly resistant to soft rot with a desirable starch content feasible.     

Induction of defence responses against Erwinia soft rot by an endogenous pectate lyase in potatoes

Pectate lyase (PL) enzymes are major virulence factors of Erwinia carotovora (Ec) bacteria. They degrade plant cell wall pectin into unsaturated oligogalacturonates (OG) known to elicit plant defence responses. Therefore, a gene encoding the isoenzyme PL3 of Ec ssp. atroseptica was transformed by means of Agrobacterium into potatoes of cv. Désirée. Four PL-transgenic potato plant lines selected on the basis of greenhouse experiments were grown over a period of 4 years (1997–2000) in the field. It is shown that the heterologous PL enzyme mediated an enhanced resistance to Erwinia soft rot in field grown tubers. Thus compared to the non-transgenic counterpart extension of rotting was significantly reduced (P < 0.001) on the wound surface of PL-expressing tubers. Moreover, the threshold density ofEc -bacteria causing a progressive soft rot was up to 19-fold higher on tuber tissue containing the PL enzyme. An induction of plant defence responses in PL-transgenic potatoes may be indicated by an enhanced resistance of tuber tissue cell walls to Erwinia -derived enzymes, an increased PPO- and PAL-activity in tuber tissue as well as by a strengthened formation of necrosis on the wound surface of tubers after infection with Ec -bacteria.     

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.     

软腐欧氏杆菌在离体条件下产生胞外酶的比较研究

 3种软腐细菌在21,26和31℃下及6种培养液中的生长量和产生胞外酶的能力各不相同。Ecc在26℃生长最好,Eca为21℃,Ech在三种温度下的生长量差别不大。与其它2种细菌比较,Ech的生长及产生果胶裂解酶(PL)的能力总是最强的。随着温度的升高,各种细菌的PL活性都下降。果胶水解酶(PG)变化不大,但Ech在高温下产PG有减少趋势,且总没有另二种细菌的活性强。Eca以26℃时产PG最适宜。Ech在较高温度下蛋白酶活性大大下降,而Ecc,变化不大,Eca的蛋白酶活性则随温度升高而加强。培养液的无细胞滤液对马铃薯块茎组织的浸离力与其中PL的活性有密切关系,但没有或只有很低果胶酶活性,而蛋白酶活性正常的这种滤液也能使小薯碟浸离。Eca在31℃培养后果胶酶活性很低,蛋白酶活性很高,其无细胞滤液对马铃薯组织也表现较强浸离力。     

A potato wilt disease in South Africa caused by Erwinia carotovora subspecies carotovora and E. chrysanthemi

In South Africa during the 1988 season, a wilt disease appeared in seed-potato crops. The disease was initially characterized by a one-sided wilt of the upper leaves, and in its advanced stage was indistinguishable from verticillium, fusarium and pseudomonas wilts. A basal stem rot was occasionally associated with wilted plants when wet, humid conditions prevailed. Erwinia carotovora ssp. carotovora and E. chrysanthemi were isolated from wilted plants, but E. carotovora ssp. carotovora was the predominant pathogen. Stem inoculations in the glasshouse showed that both pathogens were capable of causing wilt symptoms. An indication was found that E. chrysanthemi was more virulent at a higher temperature range (28-32°C) and E. carotovora ssp. carotovora at a lower temperature range (20-25°C). It was found that the potting compost, irrigation water and some parent tubers used for the propagation of plantlets at the Foundation Seed Scheme, Eastern Transvaal, were contaminated with E. carotovora ssp. carotovora.     

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.     

A methodology to detect and quantify five pathogens causing potato tuber decay using real-time quantitative polymerase chain reaction

ABSTRACT Late blight (Phytophthora infestans), pink rot (Phytophthora erythroseptica), leak (Pythium ultimum), dry rot (Fusarium sambucinum), and soft rot (Erwinia carotovora subsp. carotovora and subsp. atroseptica) are particularly damaging diseases of stored potato tubers worldwide. In this study, we present a methodology to detect and quantify the causal agents of the five aforementioned diseases from whole potato tubers, using real-time quantitative-polymerase chain reaction. Six primer pairs were designed to amplify targets smaller than 150-bp DNA in single copy protein-coding gene targets of each of the pathogens and the potato host. Using a large collection of pure culture DNA samples, all primer pairs specifically detected the DNA target in the intended pathogenic species. Amplification efficiencies over a five-log dilution series ranged between 95 and 100% and were unaffected by the presence of large amounts of host DNA. The detection level of the primers reached 0.5 pg of target DNA. Pathogens were detected in 100 pg of total DNA extracted from 170 to 250 g of tubers, 4 days after inoculation, regardless of the presence of symptoms. The presence of P. erythroseptica, Pythium ultimum, or E. carotovora was also detected in 1 ng of DNA extracted from potato tubers collected from a commercial storage facility. This study provides the first step in a methodology to predict the storability of potato tubers following harvest.     

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。     

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.     

Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low-temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real-time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.     

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.     

软腐欧氏杆菌的蛋白酶与致病作用的关系

 胡罗卜软腐欧氏杆菌(Erwinia carotovora var.carotovora Dyc)在合成培养基MS中产生两种蛋白酶,但在无细胞滤液中测不到果胶裂解酶和果胶水解酶的活性。用等电聚焦电泳测得这两种蛋白酶都是碱性蛋白酶,等电点分别为8.3和8.9。变性温度为49-50℃,对酸或碱具有较高的稳定性。纯化的蛋白酶单独作用马铃薯块、能使其变软,切片观察表明细胞壁被明显降解。通过滤膜结合法,将供体菌1830/pJB4J1::Tn5的转座子转入到野生型StEcc-12中,从2000个接合子中获得了11个缺少产蛋白酶能力的突变体。这些突变体的分泌性蛋白质等电聚焦图谱与野生型菌株明显不同,其中一个变突体蛋白酶带完全缺失,另外一个则出现8条新的蛋白带,其他5个属部分缺失。这些突变体对马铃薯块组织离析、腐烂的能力都有不同程度的减弱。研究结果认为在由胡罗卜软腐欧氏杆菌引起的软腐病程中蛋白酶和果胶酶是协同作用的,果胶酶降解植物细胞间和胞壁中的果胶质,而蛋白酶则降解植物细胞壁和膜中的蛋白质。     

Stem and bulb nematode, Ditylenchus dipsaci, associated with a dry rot of potato tubers

About 10% of the ware-sized tubers from a 1990 potato crop were found to have dry rot. The affected tubers had well defined circular patches of dry rot, either with the tuber surface depressed and cracked, or with deep, open cavities where the tuber surface had disintegrated. The rotted tissues contained large numbers of the stem and bulb nematode, Ditylenchus dipsaci. This is the first recorded case in the UK; dry rot of potato tubers is usually caused by the potato tuber nematode D. destructor.     

腐烂茎线虫在马铃薯不同组织中的数量动态

腐烂茎线虫病是近年来我国马铃薯生产中的一种新病害, 了解腐烂茎线虫的侵染规律对该病害的预防和控制及对马铃薯产量和品质提高具有重要意义。本研究采用盆栽法, 在苗期接种腐烂茎线虫, 研究该线虫在马铃薯不同组织中的数量动态及危害程度。结果显示, 在发芽-幼苗期(播种0 ~ 75 d), 母薯先受到腐烂茎线虫侵染; 在发棵期(播种75 ~ 90 d), 母薯和匍匐茎均检测到腐烂茎线虫; 在结薯期到成熟期(播种90 ~ 105 d), 线虫开始危害新生块茎, 随着块茎膨大危害逐渐加重。在马铃薯成熟期, 症状表现最明显, 新生块茎中线虫数量最多, 可达767.67条/g。不同时期的接种试验表明, 在匍匐茎形成期(播种后70 d)接种, 马铃薯受害最为严重, 发病率达100%, 块茎中线虫密度最高, 达到99.32条/g, 且块茎鲜重最低, 为14.02 g/个。本研究结果可为马铃薯腐烂茎线虫的适时有效防治提供参考。     

Biochemical and molecular diversity among Erwinia isolates from potato in Algeria

The variability within a collection of 100 isolates of Erwinia collected from various potato cultivars and locations in Algeria was studied using physiological, biochemical and molecular tests. The comparison of their biochemical characteristics with those of the type isolates CFBP 1526 ( E. carotovora ssp. atroseptica ), CFBP 2046 ( E. carotovora ssp. carotovora ) and CFBP 2048 ( E. chrysanthemi ) indicated that all the isolates collected in Algeria belonged to the species E . carotovora . They included 40 typical E. carotovora ssp. carotovora and 14 E. carotovora ssp. atroseptica ; the remaining 46 isolates could not be classified as E. carotovora ssp. atroseptica or ssp. carotovora , even though they were true Erwinia. Amplification of total genomic DNA with the primers Y1 and Y2, specific for E. carotovora , yielded an amplified fragment of the expected size in 99 isolates. The primers Y45 and Y46 specifically amplified a 439-bp DNA fragment in all E. carotovora ssp. atroseptica isolates tested, but not in isolates of the other E. carotovora subspecies or in atypical isolates, as expected from the characteristics of these primers . The digestion patterns of the 99 amplified products with the restriction enzymes Alu I, Hae II, Hpa II and Sau3A I yielded 12 RFLP groups, three of which were undescribed. The 14 isolates of E. carotovora ssp. atroseptica shared a single restriction pattern (RFLP group 1), while the typical isolates of E. carotovora ssp. carotovora and the atypical isolates composed the remaining groups (3, 4, 8–10, 12, 14, 22 and 25–27), reflecting the heterogeneity among these isolates.     

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.