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.     

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.     

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.     

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.     

Resistance to bacterial wilt in tomato as discerned by spread of Pseudomonas (Burholderia) solanacearum in the stem tissues

Different criteria were compared for assessing bacterial wilt resistance in 13 tomato genotypes varying in disease susceptibility. Wilt severity and bacterial invasiveness at collar and midstem were compared in the field under cooler (March to May, 20–28°C) and warmer months (June to August, 23–29°C), which were unfavourable and favourable to wilt symptom expression, respectively. A model was proposed for determining resistance regardless of climatic conditions prevalent during field experimentation. This model was based on an estimate of bacterial invasiveness termed the colonization index. Using a qualitative imprint method we confirmed that the more resistant the genotype, the lower the bacterial colonization of the stem. The colonization index accounted both for wilted plants and for infected asymptomatic plants in which Pseudomonas solanacearum populations failed to produce wilt. The colonization index at midstem was the more useful indicator of resistance under favourable conditions. When environmental conditions were unfavourable to wilt, colonization index at collar level discerned resistant genotypes more clearly. The results formed the basis for a model for predicting the degree and stability of resistance in tomato.     

The effects of soil solarization and soil fumigation on Fusarium wilt of watermelon grown in plastic house in south-eastern Spain

The effects of pre-planting solarization or fumigation with metham-sodium of sand-mulched soil on fusarium wilt of watermelon in plastic house culture were investigated at Almeria, south-eastern Spain. In two trials, 2 months' solarization increased the average maximum soil temperature by c. 5°C to 44-48° C at 10 cm depth and by 4-5° C to 40-42° C at 20-30 cm. The amount of Fusarium oxysporum in the upper 15 cm of a naturally infested soil was reduced by solarization and by fumigation. During the 9 months following treatment, the F. oxysporum population stabilized at a low level in soil solarized for 2 months, but fluctuated in soil solarized for 1 month and increased in fumigated soil. The amount of wilt in watermelon sown into this soil after treatment was generally low; plants growing in solarized or fumigated soil suffered less wilt than plants in untreated soil but the differences were not significant. In a soil artificially infested with the highly pathogenic race 2 of F. oxysporum f. sp. niveum, F. oxysporum populations were greatly reduced following solarization or fumigation, and fluctuated erratically thereafter. Solarization for 2 months completely controlled wilt in watermelon and gave a fruit yield almost five times that of plants in untreated soil. Solarization for 1 month only slowed disease development slightly but gave a yield more than twice that in untreated soil. Fumigation with metham-sodium retarded disease development considerably and tripled fruit yield. Plant performance was significantly better in soil solarized for 2 months than in uninfested control soil, suggesting beneficial effects of this treatment additional to wilt control.     

Phenotypic and genetic characterization of Erwinia carotovora from mulberry (Morus spp.)

Phenotypic and genetic characteristics of nine bacterial strains isolated from mulberry ( Morus spp.), which were originally described as Erwinia carotovora ssp. carotovora (Ecc), were investigated. Based on the results of biochemical tests, these bacterial strains were divided into two different types, type 1 and type 2. Two strains of type 1 were similar to Ecc, whereas seven strains of type 2 were distinct from Ecc. A polyphasic study that included serological assay, specific PCR assay for E. carotovora ssp. atroseptica (Eca), PCR-RFLP of a pectate lyase ( pel ) gene and RAPD-PCR was performed on the type 2 strains, and the data were compared with those of related E. carotovora subspecies. The results of serological and specific PCR assays for Eca showed that the type 2 strains were distinct from Eca. In RFLP analysis of the pel gene using Sau 3AI, the type 2 strains showed a unique RFLP pattern. On the basis of RAPD analysis, similarity of RAPD patterns within the type 2 strains was very high. A unique RAPD fragment was isolated from the type 2 strains and used as a probe for Southern hybridization. This probe hybridized only with PCR products from the type 2 strains. Based on phenotypic, serological and genetic characteristics, the type 2 strains isolated from mulberry may belong to a distinct E. carotovora subspecies other than Eca or Ecc.     

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.     

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。     

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.     

彩色马蹄莲欧文氏菌PCR检测

采用聚合酶链式反应(PCR)技术检测彩色马蹄莲欧文氏菌纯培养和彩色马蹄莲样品,并用分离培养技术加以验证。结果表明,PCR能特异地检测出所有10个彩色马蹄莲欧文氏菌菌株,并证实了昆明地区侵染彩色马蹄莲的细菌为胡萝卜软腐欧文氏菌(Erwiniacarotovora subsp.carotovora)。PCR与分离培养技术检测结果基本一致,但总体上PCR检测阳性率稍高于分离培养检测的发病率。接种马铃薯、大白菜24h后接种点处均出现明显软腐症状。该项技术具有更高的灵敏度,适用于彩色马蹄莲种苗的检测和病害流行学研究。     

Infection of plant material derived from Solanum acaule with Clavibacter michiganensis ssp. sepedonicus: temperature as a determining factor in immunity of S. acaule to bacterial ring rot

The development of potato ( Solanum tuberosum ) cultivars immune (completely free of infective agents) to bacterial ring rot disease, caused by Clavibacter michiganensis ssp. sepedonicus , would be a significant step toward eradication of the disease. Earlier results suggested that the wild potato species Solanum acaule , acc. PI472655 (genotype 7-8), was immune to C. michiganensis ssp. sepedonicus . The goal of the present study was to investigate the possibility of transferring this desirable trait to cultivated potato through interspecific somatic hybridization. Eight different somatic hybrids between S. acaule and S. tuberosum , with three different genome ratios, were tested for susceptibility to infection in the glasshouse using two C. michiganensis ssp. sepedonicus strains. All the somatic hybrids expressed symptoms of ring rot and were susceptible to infection as determined by IFAS (indirect immunofluorescent antibody staining) tests. The genome compositions of the hybrids influenced bacterial titres. Most of the hybrids with a higher proportion of the S. acaule genome contained lower numbers of C. michiganensis ssp. sepedonicus cells than hybrids with lower proportions of the S. acaule genome. In growth chamber tests, temperature was found to be a determining factor in the expression of immunity in the tetraploid S . acaule 7-8 line. At 21°C, S . acaule 7-8 was immune to infection by C. michiganensis ssp. sepedonicus , but at 15°C, plants supported large populations of the pathogen. However, none of the S. acaule plants expressed disease symptoms. Thus, S. acaule exhibits temperature-dependent immunity to infection by C. michiganensis ssp. sepedonicus and should be considered tolerant rather than immune.     

Low temperatures enhance winter wilt of pepper plants caused by Pythium sp.

Pepper is the main vegetable crop grown in the Arava region of southern Israel. It is grown in the winter in nethouses and greenhouses. Low temperature wilt of mature pepper plants has been known for years in this region. The incidence of plant wilting was usually low when the soil was pretreated with methyl bromide. In recent years methyl bromide usage has been banned and disease incidence has increased. The causal agent of this phenomenon was unknown until the current study. Pythium sp. was the most common microorganism genus isolated from wilted plant roots. Young pepper plants were artificially inoculated with Pythium isolated from wilted plants and maintained at temperatures of 20°, 14°, 10.5° and 8.6°C. Significant wilting was observed in plants grown at 8.6°C, with symptoms starting 2?weeks after inoculation. At 10.5°C wilting developed more slowly and inoculated plants maintained at 14° and 20°C did not exhibit any wilting symptoms. The unique variation in sporangium morphology and the sequence of the ribosomal internal transcribed spacer (ITS) suggest that a new species of Pythium is involved. The fungicide metalaxyl-M was found effective in controlling the disease in pot experiments. The relationship between low temperatures and high disease incidence can explain the high disease incidence in the Arava Valley of Israel during the cold winters of 1999?C2000, 2004?C2005 and 2006?C2007.     

Selection methods for determining resistance of carnation cultivars to Fusarium oxysporum f.sp. dianthi

The level of resistance of carnation ( Dianthus caryophyllus ) cultivars to wilt caused by Fusarium oxysporum f.sp. dianthi was compared in root-dip-inoculated plants grown in pots (filled with tuff or sandy soil) in a greenhouse and plants grown in a field where the soil was artificially infested with the fungus. In the field, wilt symptoms appeared first in susceptible and subsequently in resistant cultivars; none was immune. Variations in the level of resistance were expressed either by different percentages of wilted plants (i.e. disease incidence) or by delayed disease progress as compared to a susceptible cultivar. The range of disease severity in the field, ranked on a scale from 0 to 4, was highly and significantly correlated with the percentage of diseased plants. The greenhouse test was unreliable as a predictor of the degree of resistance observed in the field. Similar wilt levels in the greenhouse and the field were found only in susceptible cultivars.     

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.     

Characterisation of Erwinia carotovora subspecies and detection of Erwinia carotovora subsp. atroseptica in potato plants, soil and water extracts with PCR-based methods

A PCR-RFLP test based on a pectate-lyase encoding gene permits the detection of several Erwinia carotovora subspecies, but requires complete DNA extraction. This paper reports on the suitability of a simplified PCR-RFLP protocol to characterise E. carotovora strains and on the performance of PCR, using the same primers, to detect the atroseptica subspecies in substrates of epidemiological significance. A collection of 140 strains from various hosts and geographical origins was characterised for biochemical traits and PCR-RFLPs. PCR performed on boiled bacterial suspensions yielded an amplification product of 434 bp in 109 of the 140 strains. None of the E. carotovora subsp. betavasculorum strains was amplified, even after complete DNA extraction. RFLPs of the PCR product yielded 24 groups, 3 of which were new. Twenty one groups were specific to one subspecies. Several strains biochemically similar to E. carotovora subsp. atroseptica, but growing at 37 °C, showed PCR-RFLP profiles characteristic of E. carotovora subsp. carotovora. Phenetic and cladistic analyses gave three main domains, not strictly related to hosts or geographical origins. The atroseptica (RFLP groups 1 and 2) and wasabiae (group 21) subspecies constituted one of the domains, despite clustering distantly from one another. Host specialisation and molecular homogeneity suggest a clonal structure within these subspecies. Conversely, E. carotovora subsp. odorifera, despite its limited host range and geographical distribution, and E. carotovora subsp. carotovora showed great molecular diversity, spreading respectively across five and 19 RFLP groups. These two subspecies shared RFLP groups 4, 5 and 6. The tree nodes in the phenograms showed a low robustness when bootstrapping the data matrix. PCR coupled with a 48h enrichment step in a polypectate-rich medium improved detection thresholds of E. carotovora subsp. atroseptica (1.5.10²- 1.5.10³ bacteria/ml in leaves, stems, and tuber peel extracts to 4.10⁷ bacteria/ml in wash water) relative to either immunomagnetic separation coupled with PCR or DAS-ELISA (2.10⁵ in plant samples to 2.10⁷ bacteria/ml in wash water).     

空心菜青枯病病原菌的鉴定

 空心菜青枯病是近年来在广东空心菜产区新发生的病害,病株率一般为5%~20%,严重达50%以上。症状初始表现为植株上部1~2片叶萎蔫、下垂,病叶失去光泽呈灰绿色;随着病情的发展,病叶不断增多,最终整株萎蔫,甚至倒伏。经细菌学鉴定、室内致病性测定、16S rDNA序列比较及系统进化分析,结果表明引起该病害的病原是茄科雷尔氏菌(Ralstonia solanacearum);致病性和碳水化合物利用试验结果表明,该病原菌属茄科雷尔氏菌1号生理小种,生化变种4。     

Bacterial Wilt of China Aster Caused by Erwinia chrysanthemi

In July 2000, a bacterial wilt was found on China aster (Callistephus chinensis Nees) on Hokkaido, Japan. Bacteriological characteristics of the isolated bacteria were identical to Erwinia chrysanthemi. This is the first report on bacterial wilt of China aster caused by E. chrysanthemi. Received 4 June 2001/ Accepted in revised form 6 September 2001     

Effect of temperature and leaf wetness duration on infection of sweet oranges by Asiatic citrus canker

Asiatic citrus canker, caused by Xanthomonas smithii ssp. citri , formerly X. axonopodis pv. citri , is one of the most serious phytosanitary problems in Brazilian citrus crops. Experiments were conducted under controlled conditions to assess the influence of temperature and leaf wetness duration on infection and subsequent symptom development of citrus canker in sweet orange cvs Hamlin, Natal, Pera and Valencia. The quantified variables were incubation period, disease incidence, disease severity, mean lesion density and mean lesion size at temperatures of 12, 15, 20, 25, 30, 35, 40 and 42°C, and leaf wetness durations of 0, 4, 8, 12, 16, 20 and 24 h. Symptoms did not develop at 42°C. A generalized beta function showed a good fit to the temperature data, severity being highest in the range 30–35°C. The relationship between citrus canker severity and leaf wetness duration was explained by a monomolecular model, with the greatest severity occurring at 24 h of leaf wetness, with 4 h of wetness being the minimum duration sufficient to cause 100% incidence at optimal temperatures of 25–35°C. Mean lesion density behaved similarly to disease severity in relation to temperature variation and leaf wetness duration. A combined monomolecular-beta generalized model fitted disease severity, mean lesion density or lesion size as a function of both temperature and duration of leaf wetness. The estimated minimum and maximum temperatures for the occurrence of disease were 12°C and 40°C, respectively.     

Aetiology and biological control of Fusarium wilt of pinks (Dianthus caryophyllus) using Trichoderma aureoviride

Fusarium oxysporum f.sp. dianthi (F.o.d.) was the species isolated most frequently from wilted glasshouse-grown pinks in south-west England, whereas F. avenaceum was less prevalent and F. culmorum was not found. Although infections by F. avenaceum , via the roots or through a cut-stem wound, caused symptoms identical to those characterizing mild infections due to F.o.d., the former was always pathogenically weak relative to the latter. Of several organisms screened for potential antagonistic capacity towards F.o.d., using an in-vitro dual culture plate technique, Trichoderma aureoviride , isolated as an endophyte from healthy pinks, proved most effective. Elevated temperatures substantially reduced the antagonistic capacity of micro-organisms in both in-vitro and in-vivo screens, except in the case of T. aureoviride where maximum antagonism occurred at 28°C, which was near the optimum for wilt disease in pinks. The tolerance shown by T. aureoviride to a wide range of pesticides used in commercial production of pinks under cover indicated its potential in a proposed integrated disease control programme.