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.     

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.     

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.     

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.     

Effect of temperature on the transmission of Potato mop-top virus from seed tuber and by its vector, Spongospora subterranea

Potato mop-top virus (PMTV) causes disease in both the growing plant and tubers (spraing) of potato and is transmitted by the plasmodiophorid Spongospora subterranea , the cause of powdery scab. The effect of temperature during plant growth on the transmission of PMTV from infected seed tubers and from infested growing media was investigated in a series of glasshouse experiments. Symptoms developed on foliage of plants derived from infected seed tubers but none developed when PMTV was transmitted by S. subterranea in soil. The incidence of foliar symptoms was greatest on plants grown at 12°C, less at 16°C, few at 20°C and absent at 24°C. The transmission of PMTV from infected seed tubers was not significantly affected by temperatures between 12 and 24°C, but when the virus was transmitted by S. subterranea , minimal tuber infection occurred at 24°C and no differences were recorded at temperatures between 12 and 20°C. The incidence of powdery scab on tubers was greatest at 12 and 16°C and very low at 20 and 24°C. However, the incidence and severity of root galling caused by S. subterranea , was greatest at 20 and very low at 24°C. The incidence of powdery scab was greater on tubers of plants derived from infected seed tubers grown in a fluctuating temperature regime of 12 h at 20°C followed by 24 h at 12°C than on those grown at a constant 20°C, whereas the incidence of tuber infection by PMTV and spraing was similar for both regimes. This demonstrates that infection of roots can occur at a higher temperature than that for powdery scab on tubers and that this root infection can enable the transmission of PMTV into the potato plant.     

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.     

Blackleg development and tuber yield in relation to numbers of Erwinia carotovora subsp. atroseptica on seed potatoes

The relationship between number of viable cells of Erwinia carotovora subsp. atroseptica on inoculated potato seed tubers and blackleg development was investigated in 2 years for five cultivars grown in the contrasting climates of Scotland and Israel. Blackleg, and to a lesser extent non-emergence, increased with higher numbers of bacteria on the seed tubers at planting. This relationship was also found for several commercial seed stocks of one cultivar naturally contaminated with different numbers of E. carotovora subsp. atroseptica.The threshold number of bacteria necessary for the development of blackleg declined during the growing season and was also higher for the cultivar Pentland Crown in comparison with the others. In general, yield declined linearly with blackleg incidence and there was a 0.8% reduction in yield for every 1 % blackleg at 13 weeks after planting. Yield loss was positively related to the incidence of blackleg late in the season, whereas the relationship between yield loss and the incidence of non-emergence was poor.     

Detection,quantification and classification of soft rot erwinias associated with potato tubers

An improved method for detection, quantification and classification of soft rot bacteria associated with potato seed tubers, plant material, soil and water has been developed. The method is based on the use of a modified version of the crystal violet pectate selective medium (CVP), enrichment cultures under anaerobic conditions using pectate as the sole carbon source for recovery improvement, and the quantitative estimation ofErwinia spp. by employing a new solid medium - most probable number (MPN) method. The use of this method enabled an improvement in the recovery and identification of specificErwinia spp. in mixed populations. This was done by incubating CVP plates — used for the MPN counting — at three different temperatures (15, 28 and 39°C). These combined techniques were used for estimating low level populations at less than one cell per gram or ml tested ofErwinia carotovora subsp.carotovora, E. carotovora subsp.atroseptica, andE. chrysanthemi.     

Effect of growth media, storage environment, soil temperature and delivery to soil on binucleate Rhizoctonia AG-G for protection of potato from Rhizoctonia canker

Binucleate Rhizoctonia (BNR) isolates propagated for 20 days at 24°C on oat kernels and for 30 days on vermiculite amended with potato broth were recovered from an average of 62% of whole kernels, 100% of chopped kernels and 71 % of vermiculite particles within the cultures, respectively. Viability of BNR isolates 232-CG and JF-3S4-3 was higher when stored at 5 than at 24°C, and was slightly affected by the vacuum used to reduce the O₂ level. After 17 weeks of storage at 5°C in air, BNR isolates 232-CG and JF-3S4-3 maintained similar viability (75% viability on whole oat kernels and 100% viability on chopped oat kernels), but in vermiculite amended with potato broth, viability of isolate 232-CG remained at 100% while that of JF-3S4-3 was 28%. In the glasshouse, BNR isolates 232-CG and JF-3S4-3 protected potato plants from Rhizoctonia canker caused by R. solani in soil maintained at 11, 17 and 23°C. Protection from Rhizoctonia canker was greater when BNR was delivered to soil than when placed on seed pieces. BNR-colonized-whole oat kernels placed in soil (15 g m of row) gave the greatest protection from Rhizoctonia canker in all experiments. In two field experiments in soil naturally infested with R. solani AG-3. the amount of BNR-colonized oat kernels was reduced from 15 g/m of row to 1-9 g m of row without affecting protection of potato plants from Rhizoctonia canker.     

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.     

Escherichia coli carrying single pectate lyase genes from the phytopathogenicErwinia Chrysanthemi causes disease symptoms in potato

Introduction of the clonedErwinia chrysanthemi pelB orpelE genes intoEscherichia coli enabled the latter to cause blackleg disease symptoms in potato similar to those observed with the pathogenErwinia carotovora. TheE. coli cells multiplied in the roots and moved systemically in the potato stems, concomitant with the appearance of disease symptoms.E. coli cells expressing the clonedpel genes also interacted with the fungal pathogen,Verticillium dahliae, as observed previously withErwinia spp. The results indicate that either of two clonedpel genes permitsE. coli to cause relatively sophisticated diseases in potato plantlets.     

Characterization of Erwinia chrysanthemi, the soft-rot pathogen of white-flowered calla lily, based on pathogenicity and PCR-RFLP and PFGE analyses

Isolates of Erwinia chrysanthemi from Zantedeschia aethiopica (white-flowered calla lily) induced symptoms of soft rot on inoculated cv. Innocence flower-stem segments. Isolates from Phalaenopsis aphrodite and potato caused mild symptoms, while those from green onion and celery produced no symptoms. In addition to pathogenicity, the isolates were further characterized at the molecular level. A specific oligonucleotide primer set was designed for the detection of the pelZ gene of E. chrysanthemi . All E. chrysanthemi isolates tested contained pelZ as determined by PCR amplification. No amplification was observed with other Erwinia spp. The pelZ of E. chrysanthemi isolate S3-1 from Z. aethiopica was cloned, sequenced and compared with the nucleotide sequence of pelZ in GenBank. A point mutation produced an Ahd I restriction site, leading to the development of a PCR-RFLP assay to discriminate white-flowered calla lily isolates from others of E. chrysanthemi . Furthermore, macrorestriction analysis by modifying a pulsed-field gel electrophoresis (PFGE) protocol used by PulseNet revealed the genomic variation within E. chrysanthemi . After digestion with the restriction enzyme Xba I, white-flowered calla lily E. chrysanthemi isolates could be easily distinguished from other isolates. Differences in virulence, combined with PCR-RFLP and PFGE analyses, suggest that white-flowered calla lily E. chrysanthemi isolates are a new strain or pathotype in Taiwan.     

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.     

不同种类的细菌对大白菜软腐欧氏杆菌根表吸附的竞争作用

用吸附竞争试验,测定了8种细菌共10个菌株对大白菜软腐欧氏杆菌(Erwinia carotovorasubsp.carotovora)菌株 RL4在大白菜根表吸附的竞争作用。竞争菌的吸附竞争作用与它们对大白菜根表的吸附能力相一致,在等量混合接种时,只有能对大白菜根表发生吸附的7个菌株表现出程度不同的吸附竞争能力,其中以菌株 BCE 4(RL 4是从它筛选出的抗利福平菌株)竞争最强,它也是对寄主根表吸附能力最强的菌株,其吸附竞争作用最高可达98%,不表现1∶1的数量关系。RL4和 BCE4的热杀死菌体不发生吸附竞争。发生吸附竞争的有效时间为竞争菌对根表预吸附15分钟以后,或病菌(RL4)吸附15分钟以前。     

抑制植物病原细菌的植物筛选

以3种重要的植物病原细菌—辣椒青枯病菌(Ralstonia solanacearum)、柑橘溃疡病菌(Xanthomonas citri)和大白菜软腐病菌(Erwinia carotovorapv.carotovora)为供试菌,对43种植物甲醇提取物进行离体抑菌活性测定。研究结果表明,漆树对辣椒青枯病菌抑菌活性最强,其次是金秀清明茶;抑制柑橘溃疡病菌活性最高的植物有漆树和十大功劳;对大白菜软腐病菌没有筛选到抑菌活性较好的植物;垫状卷柏、七叶一枝花、少花龙葵、水半夏和豆瓣菜粗提物对3种植物病原细菌均无抑制作用。     

菊欧文氏菌分子检测技术的研究

 蝴蝶兰细菌性软腐病对蝴蝶兰的生长危害严重, Erwinia chrysanthemi(菊欧文氏菌)、Erwinia carotovora subsp. carotovora(胡萝卜软腐欧文氏菌胡萝卜软腐亚种)是引起蝴蝶兰软腐病的主要病原细菌, 其中E.chrysanthemi被列入我国三类检疫性有害生物。本文对菊欧文氏菌分子检测技术进行了研究, 设计出针对该病原细菌的特异性引物, 应用实时荧光PCR方法检测样品中存在的菊欧文氏菌, 检测灵敏度达到10²cfu/mL。     

Detection of soft rot Erwinia spp. on seed potatoes: conductimetry in comparison with dilution plating, PCR and serological assays

Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 10⁴ colony forming units (cfu) ml^-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 10⁵ cfu ml^-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 10⁴ cfu ml^-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 10⁴ cfu ml^-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection.     

细菌凝集素与菌体脂多糖在大白菜与软腐欧氏杆菌接触识别中的作用

 用3种试验方法测定了大白菜细菌凝集素(Agin-SD60)和软腐欧氏杆菌脂多精(LPS),在双方接触识别中的作用。在吸附抑制试验中,来自大白菜和马铃曾的Agin-SD60显示约98%的吸附抑制效应,另3种植物的Agin-SD60及大白菜外源凝集素(lectin)和细咆壁蛋白质(CWP)无明显作用;同时用不同种类的7个菌侏的LPS作测定,只有病菌的LPS吸附抑制作用明显(93.37%),其胞外多糖(EPS)也无作用。在菌体凝集试验中,也只有大白菜和马铃薯的Agin-SD60表现50%~100%的凝集活性。在琼脂双扩散试验中,寄主Agin-SD60可与病菌菌体及其LPS发生免疫沉淀。这些结果说明,Agin-SD60和菌体LPS在大白菜与软幅欧氏杆菌接触识别中分别作为植物识别子(cognor)和细菌识别子(cognon)起作用。     

Modelling the spread of Xanthomonas campestris pv. campestris in module-raised brassica transplants

A series of experiments was performed to quantify the rate of dispersal of Xanthomonas campestris pv. campestris in module-raised brassica transplants, in a simulated commercial plant-raising system. Seeds were sown in '308' module seed trays and set out in blocks in the glasshouse. Primary inoculum was introduced as inoculated seeds sown in one or more cells. Trays were watered via an overhead-gantry irrigation system, hand-watered or capillary-watered. Disease symptoms were monitored visually and the presence of the pathogen on samples of plants was monitored by leaf washing, dilution and plating on selective medium. Spread of symptoms was greatest in the gantry-watered trays, was very limited in hand-watered trays and was almost non-existent in capillary-watered trays. Dispersal of bacteria followed a similar pattern, but the proportion of plants contaminated was much greater than the proportion showing symptoms, and approached 100% after six weeks in the gantry-watered trays within 50 plants distance from a single primary infector. Models relating the proportion of plants with symptoms, or contaminated, to the distance from primary infector and time since sowing were fitted to the data. Predictions of the proportions of plants contaminated in commercial-scale blocks of transplants suggested that high levels of disease in the field could be explained by rapid rates of pathogen spread during plant-raising, and that the widely-used tolerance standard for seed health testing (0·01%) should be revised to 0·004%. In addition to seed health testing, control should focus on raising transplants under conditions that minimise the rates of disease spread and pathogen dispersal.     

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.