Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.     

PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 10² and 1 × 10³ colony forming units (CFU) mL⁻¹ and detection sensitivity was increased to as few as 2–4 CFU mL⁻¹ after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.     

Simple and sensitive BIO-PCR detection of potato blackleg pathogens from stem,tuber, and soil samples

Journal of General Plant Pathology - A BIO-PCR was developed for the detection of potato blackleg pathogens, including Pectobacterium wasabiae (Pw), P. atrosepticum (Pa), P. carotovorum subsp....     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

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.     

Genetic characterisation of <Emphasis Type="Italic">Pectobacterium wasabiae</Emphasis> causing soft rot disease of potato in New Zealand

Pectobacterium wasabiae has a narrow host range, having previously only been associated with Japanese horseradish. However, recent characterisation of Pectobacterium causing soft rotting in New Zealand has identified putative P. wasabiae isolates pathogenic to potato. In this study, phylogenetic reconstruction of acnA and mdh DNA sequences and fluorescent amplified fragment length polymorphisms (fAFLP) were used to confirm the identity of the putative P. wasabiae isolates. Both methods clustered the potato isolates closely with the type strain for P. wasabiae, ICMP9121, and also differentiated them from other plant pathogenic enterobacteria. PCR, DNA hybridisation and hypersensitive response (HR) assays were subsequently used to investigate the presence in P. wasabiae of the type III secretion system (T3SS) as well as other virulence factors known to be involved in development of disease by enterobacteria. Although all P. wasabiae strains appeared to elicit a type III-dependent HR in tobacco, genes associated with the T3SS and the putative virulence factors HecB and DspE could not be detected. Thus, genetic characterisation of P. wasabiae confirmed that it is a naturally occurring pathogen on potato, which does not possess the same suite of virulence factors that are involved in the pathogenicity of other enterobacteria on this host.     

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.     

内蒙古马铃薯黑胫病病原菌的分离和鉴定

 马铃薯是我国的第四大主粮。内蒙古是我国最大的马铃薯生产基地,马铃薯产业是当地农民收入的主要来源。2017年8月,内蒙古锡林郭勒盟部分马铃薯田块发生了一种细菌性黑胫病,发病面积约为200亩,发病率约为20%。发病初期,植株的茎基部变色、发黑,伴有臭味,严重时茎秆腐烂,植株死亡。为明确该地区马铃薯细菌性黑胫病的病原菌种类,本研究对该病原菌进行了分离,并利用生理生化测定,Biolog分析和分子生物学手段将该病原菌鉴定为胡萝卜软腐果胶杆菌巴西亚种(Pectobacterium carotovorum subsp. brasiliense)。据以往报道,我国马铃薯黑胫病主要由黑腐果胶杆菌(P. atrosepticum)和胡萝卜软腐果胶杆菌胡萝卜亚种(P. carotovorum subsp. carotovorum)两种病原菌引起,本研究是P. carotovorum subsp. brasiliense引起马铃薯黑胫病在国内的首次报道。     

Evaluation of a diagnostic microarray for the detection of major bacterial pathogens of potato from tuber samples

Potato can be infected with many bacterial pathogens, the detection of which is necessary in seed certification. In this study, a diagnostic microarray previously tested for specificity of probes for detecting the potato bacteria causing blackleg and soft rot (Pectobacterium atrosepticum, Pectobacterium carotovorum, and Dickeya spp.), ring rot (Clavibacter. michiganensis subsp. sepedonicus), scab (Streptomyces scabies and Streptomyces turgidiscabies) and brown rot (Ralstonia solanacearum) from pure culture was evaluated for analytical sensitivity when testing directly from tuber samples. The microarray readily detected all the bacterial species when 100 ng of the target bacterial DNA from pure culture was mixed with DNA from soil microbes and potato. However, detection was inconsistent when total DNA isolated directly from infected tubers or enriched bacterial culture was used. While the high specificity of the probes could be confirmed from the results of the DNA cocktail experiment used as a control, the study demonstrated that the level of analytical sensitivity of the microarray under the tested condition was not sufficient to detect bacteria directly from tubers. Therefore, in addition to the cost and organizational complexities, the low analytical sensitivity and limited reproducibility of the microarray are constraints for establishing the platform for routine detection of potato bacterial pathogens from tuber samples.     

Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland

Biochemical characterisation of Dickeya strains isolated from potato plants and river water samples in Finland showed that the majority of the strains were biovar 3. They thus resembled the strains recently isolated from potato in the Netherlands, Poland and Israel and form a new clade within the Dickeya genus. About half of the Finnish isolates resembling strains within this new clade were virulent and caused wilting, necrotic lesions and rotting of leaves and stems. Similar symptoms were caused by D. dianthicola strains isolated from one potato sample and from several river water samples. Frequently, the rotting caused by the Dickeya strains was visible in the upper parts of the stem, while the stem base was necrotic from the pith but hard and green on the outside, resulting in symptoms quite different from the blackleg caused by Pectobacterium atrosepticum. The presence of Dickeya in the symptomatic plants in the field assay was verified with a conventional PCR and with a real-time PCR test developed for the purpose. The virulent Dickeya strains reduced the yield of individual plants by up to 50% and caused rotting of the daughter tubers in the field and in storage. Management of Dickeya spp. in the potato production chain requires awareness of the symptoms and extensive knowledge about the epidemiology of the disease.     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

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.     

Characterisation of pectobacterium wasabiae causing blackleg and soft rot diseases in South Africa

Pectolytic bacteria were isolated from potato tubers and stems showing tuber soft rot and blackleg symptoms. Approximately half (52 %) of the isolates could grow at both 27 and 37 °C while another half (48 %) failed to grow at 37 °C. All isolates could be amplified with primers specific to the pectate lyase (pel) gene. Carbon utilisation profiles could not conclusively identify these isolates. PCR amplification using primers specific for Pectobacterium carotovorum subsp. brasiliensis was positive for all isolates that grew at 37 °C. However, the group that did not grow at 37 °C failed to amplify with P. atrosepticum specific primers. To characterise this group of isolates, the intergenic transcribed spacer region (ITS) was amplified and PCR products digested with two restriction enzymes (RsaI and CfoI) to generate ITS-PCR-RFLP profiles. The profiles of these new isolates were compared to those of the type strains of other pectolytic bacteria. Profiles of five of the selected atypical strains generated with the enzyme CfoI appeared to be most similar to those of P. wasabiae type strain. Phylogenetic analysis using concatenated partial gene sequences of housekeeping genes mdh and gapA clustered these isolates together with those of P. wasabiae reference strains thus confirming their identity. These strains were virulent on potato tubers and stems but did not elicit hypersensitive response on tobacco plants. This is the first report of P. wasabiae causing soft rot and blackleg of potatoes in South Africa.     

A culture filtrate of Phytophthora infestans primes defense reaction in potato cell suspensions

Priming of defense reactions by an elicitor results in an enhanced ability of the plant to respond to subsequent pathogen challenges. We previously showed that application of lipopolysaccharides (LPS) to potato cell suspensions causes apoplastic acidification, but does not stimulate lipoxygenase (LOX) activity. Here, we tested the ability of various elicitors to prime and elicit defense reactions in potato cell suspensions. Adding 20 microg ml(1) LPS, laminarin, harpin N, or a concentrated culture filtrate (CCF) of Phytophthora infestans to cell cultures 18 h before a second elicitation with LPS did not alter the intensity of apoplastic acidification compared with a single LPS application. Conversely, high concentrations (200 or 400 microg ml(1)) of LPS, laminarin, and harpin N activated LOX in cells pretreated with 1 microg ml(1) CCF, but not in cells pretreated with LPS, laminarin, or harpin N. LOX response was maximal in pretreated cells of potato cv. Bintje when the second elicitation occurred 18 to 24 h after CCF application. These results showed that LOX activation is primed in potato cells by CCF, but not by LPS, harpin N, or laminarin. Finally, bioassays showed a slightly greater reduction of rot weight in half tubers treated with CCF followed by LPS before inoculation with Pectobacterium atrosepticum than in half tubers treated with either preparation alone, indicating a priming effect of CCF on both LOX induction and disease suppression.     

Putative new species of the genus Dickeya as major soft rot pathogens in Phalaenopsis orchid production

Bacterial soft rots are a serious limitation to the production of orchids and other horticultural plants. Here, the characterization of causative bacteria isolated from Phalaenopsis orchids showing symptoms, from a commercial production site, is reported. The most commonly isolated bacteria were identified as Dickeya spp. Partial sequencing of 16S rDNA, fliC and dnaX showed diversity among the isolates and divided the isolates into two groups, with greatest similarity to previously reported undefined Dickeya lineages from orchids (UDL‐3 and UDL‐4). Two isolates (B16, S1) were sequenced using next‐generation sequencing, which has provided draft genomes of these two isolates for further studies (Ali? et al., 2015 ). Newly developed fliC‐based lineage‐specific quantitative real‐time PCR assays were used to distinguish among the lineages and to assess their relative abundances in diseased tissues. Virulence and aggressiveness comparison tests in vivo on Phalaenopsis orchids, potato plants and witloof chicory leaves indicated high virulence and extreme maceration potential of these novel Dickeya isolates, compared to a reference panel of other Dickeya spp. Pantoea cypripedii (formerly Pectobacterium cypripedii), which has previously been reported as a soft rot pathogen of orchids, was not detected, and isolates obtained from culture collections did not cause symptoms on artificially infected Phalaenopsis orchids.     

朝鲜蓟细菌性根茎腐烂病病原的分离与鉴定

 在湖南省常德市西洞庭管区种植的朝鲜蓟（Cynara scolumus  L.）上发现了一种新的病害,其症状表现为地上部分从外层叶片开始逐步枯萎,随后根部和主茎杆的髓部腐烂变褐,最后整株枯萎。从田间感病朝鲜蓟茎杆的病健交界处用NA培养基分离,获得10个菌株,分别指定为HNXDT001~010,并进行了致病性测定、形态观察和细菌学特征分析,同时对HNXDT002菌株进行分子生物学鉴定。结果表明,该系列菌株在NA培养基上均形成灰白色圆形菌落,稍突起,有光泽,半透明。在显微镜下菌体呈短杆状,两端钝圆,具有2～8根周生鞭毛,革兰氏阴性。10个菌株通过针刺法接种均可导致朝鲜蓟茎杆、胡萝卜、辣椒、白菜、土豆、番茄和莴苣茎杆软腐,经科赫法则验证为致病病原菌。该菌株的16S rDNA序列和果胶酶基因片段测序(分别用16S rDNA通用引物16SF/16SR和果胶酶基因引物Y1/Y2扩增)与系统发育学分析表明,其16S rDNA序列(GenBank Accession No. JF721958)与胡萝卜软腐果胶杆菌胡萝卜亚种（Pectobacterium carotovorum  subsp.  carotovorum）菌株ATCC15713 (GenBank Accession No. U80197)序列同源性高达99％;果胶酶基因序列(GenBank Accession No. JF721960)与胡萝卜软腐果胶杆菌胡萝卜亚种（Pectobacterium carotovorum subsp. carotovorum）PC1菌株(GenBank Accession No. CP001657)序列同源性为93%。结果表明：朝鲜蓟细菌性根茎腐烂病病原为胡萝卜软腐果胶杆菌胡萝卜亚种。     

Characterization of Pectobacterium brasiliense strains from potato and vegetables in Israel

Pectobacterium brasiliense (Pbr) infects a wide range of crops worldwide, causing potato blackleg and soft rot and vegetable soft rots. This study aimed to characterize the genetic diversity and virulence variability among 68 Pbr strains isolated from either symptomless potato progeny tubers, diseased potato plants, ware potatoes wash water, or vegetables grown in Israel, as well as strains isolated from symptomless seed tubers grown in Europe, or diseased potato plants grown in France. The collection was typed using PCR and TaqMan real-time PCR analyses, dnaX sequence analysis, pulsed-field gel electrophoresis (PFGE), and pectolytic activity. dnaX phylogeny grouped almost all strains in a common genetic clade related to Pbr, which was distinct from the other Pectobacterium species. PFGE analysis identified two main clusters, including one major group of 47 strains with 95%–100% similarity. Maceration assays on two potato cultivars showed significant differences between strains but with no correlations with the source of the strains nor the status of the host (with/without symptoms). Molecular (dnaX sequences and PFGE profiles) and phenotypic analyses (tuber maceration tests) showed that the tested Pbr strains are not a homogeneous group. Analysis of the tested Pbr strains isolated from potato and vegetables grown in fields with a history of potato cultivation suggests that seed tubers imported from Europe may be the main source for Pbr in Israel. To the best of our knowledge, this is the first study that describes biodiversity and population structure of P. brasiliense isolated from potato and vegetables under hot climate conditions.     

Pectinolytic bacteria associated with potato soft rot and blackleg in South Africa and Zimbabwe

Soft rot and blackleg can cause severe economic losses in potato production in South Africa and Zimbabwe depending on climatic conditions. The aim of the study was to identify the predominant bacteria causing potato soft rot and blackleg in these countries. Samples, comprising of stems and tubers from potato plants with blackleg and soft rot symptoms were collected from 2006?C2009 from potato production areas where disease outbreaks occurred. The isolates from these plants and tubers yielded Gram negative, pectinolytic bacteria on crystal violet pectate and inoculated tubers. Identification was based on biochemical and phenotypic characteristics, rep-PCR, Amplified Fragment Length Polymorphisms and sequences of gyrB and recA genes. Isolates from Zimbabwe were identified as Pectobacterium carotovorum subsp. brasiliensis (Pcb) (21 isolates), Dickeya dadantii subsp. dadantii (Dd) (20 isolates), P. c. subsp. carotovorum (Pcc) (16 isolates) and P. atrosepticum (Pa) (4 isolates). Pcb, Pcc and Dd subsp. dadantii were isolated from samples collected from all the regions, while Pa was isolated from Nyanga the coolest region in Zimbabwe. In South Africa, however, Pcb was the most common causal agent of soft rot and blackleg. P. atrosepticum was the only pathogen isolated from samples collected in Nyanga, Zimbabwe, and was not isolated from any South African samples. AFLP analysis separated the Pcb strains into 12 clusters, reflecting subdivision in terms of geographic origin, and Pcc isolates were clearly differentiated from Pcb isolates. A large degree of DNA polymorphism was evident among these 12 clusters. The study identified all the pathogens associated with the blackleg/soft rot disease complex.     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。