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.     

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).     

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。     

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.     

Characterization of Pectobacterium species from Iran using biochemical and molecular methods

Characteristics of forty strains from macerated potato tubers and water-soaked lesions of some ornamental plants were studied in north parts of Iran. The causal organisms isolated from infected tissues were identified as Pectobacterium spp. based on their physiological and biochemical assays and confirmed by species and subspecies specific PCR and RFLP analysis of 16S–23S intergenic transcribed spacer region. Artificial inoculation of isolates to their related hosts generated the same symptoms on potato and ornamental plants, from which the same bacteria were isolated and identified. We detected two groups of atypical isolates in this study. The first group from potato classified as Pectobacterium carotovorum subsp. carotovorum by phenotypic tests but was unable to elicit HR on tobacco leaves, to grow at 37°C and to amplify the pel gene relevant to this subspecies. The second one from ornamental plants which was again characterized as Pectobacterium carotovorum subsp. carotovorum in biochemical assays, produced a unique ITS-RFLP profile different from all of known Pectobacterium species and subspecies. Our findings based on phylogenetic analysis using concatenated partial sequences of housekeeping genes mdh and gapA, indicated the occurrence of P. wasabiae as a novel species in potato storage in Iran. Furthermore we detected a distinct clade of Pectobacterium spp. from some ornamental plants including Schlumbergera bridgesii, Syngonium podophyllum and Iris spp.     

Rapid Identification of Clavibacter michiganensis Subspecies sepedonicus Using Two Primers Random Amplified Polymorphic DNA (TP-RAPD) Fingerprints

Twenty strains of Clavibacter michiganensis subsp. sepedonicus from different geographic origins and other reference strains of the same and different species, including other potato pathogens, were analysed with a new procedure named TP-RAPD that originates fingerprints of bacterial species. This procedure uses two primers to amplify the 16S rDNA gene. At 45 °C of annealing, the PCR product electrophoresed in agarose gels produced a band pattern that was different in all bacterial species studied as well as in the subspecies of C. michiganensis. All strains of C. michiganensis subsp. sepedonicus displayed the same TP-RAPD number of pattern. Unlike Gram negative bacteria, Gram positives of high G + C content, such as Clavibacter, produced low bands in TP-RAPD. By using a different set of two primers also based in the 16S rDNA sequence from Escherichia coli a more adequate amplification of Gram positives of high G + C including a greater number of bands was obtained. TP-RAPD patterns using the new set of primers described in this work is a reliable and fast method to identify C. michiganensis subsp. sepedonicus.     

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.     

Genotypic and phenotypic variability of <Emphasis Type="Italic">Pectobacterium</Emphasis> strains causing blackleg and soft rot on potato in Turkey

Pectinolytic bacteria were isolated from 48 potato plants showing the symptoms of blackleg and collected in different fields of commercial potato production areas at Samsun, Amasya, Corum and Yozgat provinces in Turkey in 2015. The survey resulted in the isolation of 26 pectinolytic strains that belonged to P. atrosepticum, P. carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum and P. parmentieri species based on molecular identification with species-specific PCR and phenotypic characterization. The identified strains indicated typical biochemical characteristics of the assigned species. For 16 representative Pectobacterium isolates 10 unique rep-PCR band patterns were obtained. The 16S rRNA and recA and gapA gene fragment sequencing confirmed the species identity of the isolates. The phenotypic characterization of the strains revealed that for all assays but one (cellulase, protease activity, swimming but not swarming), the tested Pectobacterium species were significantly different from each other proving the diversity of the strains belonging to these genera. Recent outbreaks of blackleg and/or soft rot in potato production areas in Turkey may pose a threat on other crops, as tomato, pepper, cucumber, onion, cabbage, broccoli and sugar beet are cultivated in the same provinces.     

Rapid Identification of Clavibacter michiganensis Subspecies Sepedonicus based on the Stable Low Molecular Weight RNA (LMW RNA) Profiles

Clavibacter michiganensis subsp. sepedonicus causes potato ring rot disease. The identification process for this bacterium is complex and long. This work demonstrates that the stable low-molecular-weight (LMW) RNA profiles allow their rapid identification. Staircase electrophoresis in polyacrylamide gels was used to analyze the LMW RNA profiles of 54 strains of C. michiganensis subsp. sepedonicus from different geographic origins. The profiles of several strains of other subspecies of C. michiganensis and other pathogens of potatoes were also analyzed. All the strains of C. michiganensis subsp. sepedonicus had the same LMW RNA profile. They had a band in class 2 of tRNA that was absent in the other subspecies of the species C. michiganensis. Also, the LMW RNA of C. michiganensis subsp. sepedonicus was different with respect to the LMW RNA profiles of other pathogens of potato. The results indicate the possible utilization of LMW RNA profiles in identification of the bacteria causing potato ring rot disease.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">brasiliensis</Emphasis> causing blackleg on potatoes in South Africa

In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg occurred, causing severe economic losses in commercial potato production fields. Symptoms were initially observed on only one stem per plant, on which the top leaves rolled upwards, wilted and became necrotic. As symptoms progressed to the lower leaves with subsequent leaf desiccation, a light to dark brown discolouration of the vascular system at the stem base developed, followed by external darkening. Under prevailing wet and humid conditions stems became slimy and pale. In the stems, the pith became necrotic and hollow. These symptoms were similar to those described in Brazil, where the causal agent was identified as a new subspecies, Pectobacterium carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical blackleg symptoms were made on CVP medium. Sequences and phylogenetic analysis of the partial 16S–23S rDNA intergenic spacer region indicated that the isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb in South Africa and it appears to be the most important causal agent of blackleg in South Africa. The disease poses a major potential threat to the South African potato industry especially in terms of seed exports, tuber quality and yield.     

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.     

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。     

Occurrence of Pectobacterium wasabiae in potato field samples

During the growing seasons of 1996 and 1997, samples of potato stems and tubers with symptoms of blackleg and soft rot were collected in different regions in Poland. After growing to pure cultures on crystal violet pectate (CVP) medium, isolates of bacteria were identified as Pectobacterium spp. on the basis of their ability to degrade pectate and with the use of biochemical tests. About 43 % strains isolated from 122 different plant samples were identified as Pectobacterium carotovorum subsp. carotovorum, whereas the rest of the pectinolytic bacteria was identified as Pectobacterium atrosepticum. A recent screening of these isolates with recA PCR-RFLP allowed identification of 18 different RFLP groups within the tested P. c. subsp. carotovorum strains. The third largest group of the tested P. c. subsp. carotovorum strains (14 %), which were assigned to the profile 3 recA PCR-RFLP, was re-identified as Pectobacterium wasabiae (formerly Erwinia carotovora subsp. wasabiae) on the basis of recA and 16S rRNA genes sequences. About 50 % of P. wasabiae isolated from potato, in contrast to horseradish isolates of P. wasabiae, have an ability to grow at 37°C and some of them grow on media containing 5 % of NaCl. In a pathogenicity test with 11 strains of P. wasabiae these strains showed a high capacity to rot potato tubers.     

Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.     

马铃薯环腐病生防菌株P1的鉴定、防病效果及促生作用研究

 在马铃薯环腐病区采集健康的马铃薯Solanum tuberosum块茎,从中分离到1株对马铃薯环腐病菌Clavibacter michiganense subsp. sepedonicum具有强拮抗作用的内生细菌P1。经形态观察、生理生化鉴定,菌株P1归属为Bacillus sp.,进一步经16SrDNA序列对比分析,确定为巨大芽孢杆菌B. megatherium。经证明,P1菌株的培养液抗菌粗提物属于蛋白类,该抗菌蛋白对紫外光不敏感,pH为7.0时抑菌活性最强,温度高于80℃时抑菌活性明显下降。温室试验表明,P1菌株能显著提高马铃薯植株的株高、茎粗、产量及大薯率,其对马铃薯环腐病防效达53.4%。     

Inoculating plant material by jet injection

Both tubers and plants of potato were successfully inoculated with Erwinia carotovora subsp. itroseptica by means of a needle-less medical jet injector. The instrument has also been used to infect potato tubers with Phorna exigua var. foveata, Fusarium solani var. coeruleum and F. sulphureum. It is suggested that high-pressure injection is a potentially useful tool for introducing pathogens into plant materials.     

中生菌素的抗生作用

 本文研究了中生菌素对革兰氏阳性菌、革兰氏阴性菌、丝状真菌的抗生作用。结果表明,中生菌素是一种抗菌谱比较广的农用抗生素。其对菌体细胞膜无明显的影响。100 ppm时引起部分菌体原生质凝聚。同位素标记前体物掺入实验表明,中生菌素15.6 ppm对白菜软腐病菌DNA、RNA的合成影响很小,但强烈抑制菌体蛋白质的合成。用化学测定法分析了中生菌素对枯草杆菌细胞内大分子物质含量的影响,得到了和前体物掺入法相似的结果。说明了中生菌素的作用机制是抑制菌体蛋白质的合成。     

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.     

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.