植物病原棒形杆菌属分类最新进展

植物病原棒形杆菌属Clavibacter的分类随着研究的深入和鉴定技术水平的不断进步一直在发生着变化,之前普遍认可的观点为该属仅含有1个种,即密执安棒形杆菌C. michiganensis,种下又分为5个亚种,分别为密执安亚种C. michiganensis subsp. michiganensis、诡橘亚种C. michiganensis subsp. insidiosus、尼布拉斯加亚种C. michiganensis subsp. nebraskensis、环腐亚种C. michiganensis subsp. sepedonicus和花叶亚种C. michiganensis subsp. tessellarius。最近几年又有4个新亚种陆续被报道,分别为菜豆亚种C. michiganensis subsp. phaseoli、辣椒亚种C. michiganensis subsp. capsici、加利福尼亚亚种C. michiganensis subsp. californiensis和智利亚种C. michiganensis subsp. chilensis。随着全基因组分析和多基因分析技术在细菌分类上的应用,之前被广泛认可的4个亚种(诡橘亚种、尼布拉斯加亚种、环腐亚种和花叶亚种)及新亚种辣椒亚种也曾被建议定义为种,即C. insidiosus、C. nebraskensis、C. sepedonicus、C. tessellarius和C. capsici。为便于研究人员了解该属最新的分类研究现状,本文对植物病原棒形杆菌属的分类历史及最新分类现状进行了系统梳理,期望为该属病原菌的风险评估、检测鉴定等研究提供分类学参考。     

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.     

Polymorphism analysis of housekeeping genes for identification and differentiation of <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subspecies

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.     

Repetitive Sequence-derived PCR Profiling Using the BOX-A1R Primer for Rapid Identification of the Plant Pathogen Clavibacter Michiganensis Subspecies Sepedonicus

Repetitive sequence-derived PCR using the BOX-A1R primer was used to generate genomic fingerprints of Clavibacter michiganensis subspecies sepedonicus, the causal agent of bacterial ring rot disease of potato. A total of 35 C. michiganensis subsp. sepedonicus strains were selected for study in order to represent the widest possible historical, morphological and geographical diversity of the organism. Comparison was made with genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. insidiosus, C. michiganensis subsp. tessellarius, C. michiganensis subsp. nebraskensis as well as other related Gram positive plant pathogens. The resultant genomic fingerprints and subsequent cluster analysis show C. michiganensis subsp. sepedonicus to form a remarkably homogeneous group with approximately 84% similarity between all of the strains tested. There was no evidence to suggest that fingerprints varied with historic, morphological or geographic diversity. In addition, C. michiganensis subsp. sepedonicus isolated from asymptomatic sugar beet had the same fingerprint as those which were isolated as potato pathogens. This group was easily distinguished from the clusters formed by the other subspecies of C. michiganensis and Gram positive plant pathogens. The potential for this technique to be used as a relatively rapid method to replace the time consuming and sometimes inconclusive eggplant bioassay test is discussed.     

Analysis of the toxicity of purothionins and hordothionins for plant pathogenic bacteria

Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.     

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.     

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

Development of a tomato plant resistant to Clavibacter michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene

The bacteriophage CMP1 endolysin gene (lys), encoding, a peptidase that was shown to effectively reduce Clavibacter michiganensis by specifically hydrolysing its murein, was transferred into tomato plants by Agrobacterium‐mediated transformation. The presence of the gene was verified by PCR and the gene product was confirmed in immunoblots and stably expressed over three generations. Transgenic tomato plants did not show disease symptoms after infection with C. michiganensis subsp. michiganensis, despite the fact that small amounts of bacteria could still be identified in xylem sap and leaf extracts, although in significantly reduced amounts.     

Clonal populations of Clavibacter michiganensis subsp. michiganensis are responsible for the outbreaks of bacterial canker in greenhouse tomatoes in Italy

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected in greenhouses from 17 farms during tomato bacterial canker outbreaks occurring between 2005 and 2008 in Sicily, were analysed by a multiphasic approach. Population studies were conducted to investigate the possible sources of inocula. Cmm strains were characterized by PCR assays targeting virulence genes, fingerprinting techniques, metabolic profiles and virulence. These strains were comparatively analysed with Cmm strains isolated in other parts of Italy over a period of 15 years. Chromosomal genes encoding virulence determinants tomA, ppaA, chpC, and the plasmid‐encoded genes pat‐1 and celA were detected by PCR in all tested strains, except for four Sicilian Cmm strains where the pat‐1 gene was not amplified. Using BOX‐PCR, Cmm strains were differentiated into 13 haplotypes and clonal populations were identified. Cmm strains isolated from different farms in 2008 showed the same BOX‐PCR haplotype. A distinct BOX‐PCR haplotype was obtained from atypical Cmm strains lacking pat‐1 and isolated in 2006/7 from three farms. Cmm strains with two different haplotypes were detected in one farm, whereas the other farms contained strains with only a single haplotype. A new fAFLP protocol based on the amplification of ApaI/MseI fragments was developed and was able to differentiate C. michiganensis subspecies. Different populations were delineated for the multiple outbreaks occurring in Sicily, whereas similar populations were recorded in other Italian regions over a period of 12 years. The results are consistent with previous studies that demonstrate that Cmm outbreaks are associated with propagation material.     

Effect of phytotoxic compounds produced by Clavibacter michiganensis subsp. michiganensis on resistant and susceptible tomato plants

A phytotoxic fraction of high molecular weight was isolated from the culture filtrate ofClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker of tomato, and partly purified. This high molecular weight fraction consists of sugars and a minor protein moiety and is therefore probably of similar nature to that of the toxin fromC. michiganensis subsp.michiganensis reported earlier in literature.The high molecular weight fraction was albe to induce wilting, the predominant symptom of the disease, as shown in a bioassay with tomato cuttings. However, this wilting reaction turned out to be non-specific in the bioassay, since (partially) resistant and susceptible genotypes responded similarly. No correlation could be found between the degree of virulence of fiveC. michiganensis subsp.michiganensis strains and the amount of the phytotoxic high molecular weight fraction produced in vitro.As the isolated high molecular weight fraction showed a phytotoxic effect on tomato plants it is worthwhile to test its potential for use as a selective agent in in vitro selection.Samenvatting Een fytotoxische fractie werd geïsoleerd uit cultuurfiltraat vanClavibacter michiganensis subsp.michiganensis, de veroorzaker van de bacterieverwelkingsziekte bij tomaat. Een eerste karakterisering toonde aan dat deze toxische fractie hoog-moleculaire component(en) bevat, bestaande uit polysacchariden en een gering percentage eiwit. Dit is in overeenstemming met toxines vanC. michiganensis subsp.michiganensis die al eerder beschreven zijn.Deze hoogmoleculaire toxische fractie was in staat verwelking te induceren van stengeltoppen van verschillendeLycopersicon esculentum enL. peruvianum genotypen in een bioassay. Gewichtsverandering van de stengeltoppen, uitgedrukt als percentage ten opzichte van het begingewicht, werd gebruikt als parameter voor verwelking. De toxische fractie reageerde niet-specifiek in de bioassay, want er werd geen verschil gevonden in respons van (partieel) resistente en gevoelige genotypen. Er bleek geen correlatie te zijn tussen de mate van virulentie van verschillende isolaten vanC. michiganensis subsp.michiganensis en de hoeveelheid van de toxische fractie geproduceerd in vitro.Het mogelijke gebruik van deze hoogmoleculaire toxische fractie als selectief agens bij in vitro selectie zal nader onderzocht worden.     

Internal methods comparison study and inter‐laboratory study on Clavibacter michiganensis subsp. michiganensis in tomato seeds1

Bacterial canker of tomato is a disease caused by Clavibacter michiganensis subsp. michiganensis, a quarantine bacterium, the spread of which has not been completely controlled in spite of the phytosanitary measures taken within the EPPO region. Since 2008 the French National Laboratory for Plant Health (LNPV) has been working on the assessment of the methods used in laboratories to detect the presence of Clavibacter michiganensis subsp. michiganensis in tomato seeds i.e. dilution plating on semi‐selective media and immunofluorescence. In the 1st stage of the assessment, a methods comparison study was performed with reference strains to determine the performance criteria of the tests in optimal conditions. In the 2nd stage, an inter‐laboratory study on naturally and artificially contaminated seeds was performed with 8 laboratories from 6 European countries. This study demonstrated the strengths and weaknesses of the tests currently in use. Two laboratories took the opportunity the collaborative study offered to evaluate alternative tests: BIO‐PCR and IMS‐plating. These could offer interesting alternatives to optimise the detection procedure for Clavibacter michiganensis subsp. michiganensis on tomato seeds.     

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.     

In Planta - Complementation of Clavibacter Michiganensis Subsp. sepedonicus Strains Deficient in Cellulase Production or HR Induction Restores Virulence

Clavibacter michiganensis subsp. sepedonicus, a Gram positive bacterium that causes bacterial ring rot of potato, was studied in eggplant, an alternate host, using strains that differed in phenotype. Two factors affecting virulence, the ability to induce a hypersensitive response (HR) and cellulase production, were studied. A plasmid-free isolate of C. michiganensis subsp. sepedonicus that causes HR on tobacco but is unable to produce cellulase multiplied efficiently in planta, but caused only weak symptoms. In contrast, a strain that is unable to induce HR on tobacco but produces cellulase was impaired in the ability to multiply in the host and caused no symptoms. When the two non-virulent strains were coinoculated into eggplants, typical disease symptoms developed. This enhancement was not due to formation of a new phenotype or significant increases in population density of either of the strains. Our results suggest that both cellulase production and the ability to induce HR are required for a successful infection process and disease induction by C. michiganensis subsp. sepedonicus. Our results additionally suggest that the ability to induce HR on non-host plants is required for multiplication in the host plant, whereas cellulase expression is necessary for induction of disease symptoms.     

Multilocus sequence analysis reveals a novel phylogroup of Xanthomonas euvesicatoria pv. perforans causing bacterial spot of tomato in Iran

A multilocus sequence analysis (MLSA) was performed on five housekeeping genes (fusA, gapA, gltA, lacF and lepA) of 22 Xanthomonas euvesicatoria strains recently isolated from alfalfa, pepper and tomato plants in Iran. In addition, 161 strains isolated worldwide from pepper, poinsettia, rose and tomato plants were included in the analysis. All X. euvesicatoria pv. perforans isolates from tomato plants in Iran clustered in a monophyletic group, although five MLSA haplotypes were detected among them. The Iranian tomato strains presented 10 nucleotide differences in the lepA gene sequences compared to the known worldwide population of X. euvesicatoria pv. perforans. Statistical analyses revealed a recombination event that had occurred in the lepA gene of the strains isolated from tomato in Iran. BOX‐PCR analysis confirmed the inclusion of Iranian tomato strains within X. euvesicatoria pv. perforans. Furthermore, X. euvesicatoria pv. euvesicatoria strains isolated from pepper in Iran differed in one nucleotide in the lepA gene sequence from the known worldwide population of the pathovar, and clustered in a group containing strains isolated in Nigeria. The strains isolated from alfalfa in Iran clustered with the type strain of X. euvesicatoria pv. alfalfae. Altogether, the results reveal the existence of a phylogenetically novel population of X. euvesicatoria pv. perforans in Iran which needs further in‐depth analysis to pinpoint the epidemiological impact of these strains.     

Temperature at the early stages of Clavibacter michiganensis subsp. michiganensis infection affects bacterial canker development and virulence gene expression

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial canker and wilt, causes severe economic losses in tomato net‐houses and greenhouses worldwide. In this study, seedlings which were transplanted and inoculated monthly over 2 years wilted and died earlier in the spring (21–24°C) and autumn (18–23°C) than in the winter (15–18°C) and summer (28–31°C): T₅₀ (the time taken for 50% of the plants to wilt or die) was 2 and 3–4 months after inoculation, respectively. A highly significant correlation was found between the average temperatures during the first month after inoculation and T₅₀; the shortest T₅₀ mortality (70 days) was observed for an average temperature of 26°C. Expression of virulence genes (pat‐1, celA, chpC and ppaA) by Cmm was higher in plants inoculated in the spring than in those inoculated in the summer. In another set of experiments, seedlings were inoculated and maintained in controlled‐environment growth chambers for 2 weeks. Subsequently, they were transplanted and maintained in commercial‐type greenhouses for 4–5 months. The temperatures prevailing in the first 48 h after inoculation were found to affect Cmm population size and virulence gene expression and to have season‐long effects on bacterial canker development.     

Effects of plant age on disease development and virulence of Clavibacter michiganensis subsp. michiganensis on tomato

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during which inoculations led to wilt and death of tomato plants was defined. This period, designated ‘window of vulnerability’, ranged from transplanting to the 17‐ to 18‐leaf stage. Plants inoculated after this period expressed disease symptoms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase, and the serine protease‐encoding pat‐1, chpC and ppaA, was induced during the early stages after inoculation in plants inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and outside of this period were insignificant after the first week post‐inoculation, indicating that differences in disease severity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm will have a season‐long effect on plant mortality and may minimize, or even prevent, yield losses.     

Application of Trichoderma harzianum Strain KABOFT4 for Management of Tomato Bacterial Wilt Under Greenhouse Conditions

Clavibacter michiganensis subsp. michiganensis is a very important pathogen that causes bacterial wilt of tomato (BWT). Biological control of plant diseases is a critical tool for protecting the environment from chemical pollution. Twenty-five isolates of the genus Trichoderma were obtained from a healthy tomato root. Of the 25 isolates, KABOFT4 showed highly antagonistic activity that controlled the growth of C. michiganensis subsp. michiganensis (Cmm7) under in vitro conditions. The 5.8S ribosomal RNA gene and internal transcribed spacer identified the isolate as Trichoderma harzianum KABOFT4. The effect of this isolate as a soil drench and/or foliar application on bacterial wilt under greenhouse conditions was studied. The germination percentage of tomato seed treated with KABOFT4 increased by 36.7% compared to infected seed treated with only the pathogen Cmm7. Under greenhouse conditions, tomato seedlings treated with KABOFT4 as a soil drench, foliar and soil treatment, and foliar treatment had a 61.3, 26.7, and 40% reduced disease severity relative to the infected control, respectively. All treatments had a positive effect on tomato plants that presented as greater vegetative growth and accumulation of dry matter. The best fresh and dry weight was recorded when plants were treated with KABOFT4 as a soil and foliar application. Tomato plants treated with KABOFT4 also had increased total phenol and flavonoid contents in inoculated and non-inoculated plants compared to untreated plants. Under greenhouse conditions, T. harzianum strains can be used as an environmentally friendly way to manage the most economically important tomato disease. The results showed that a native endophytic strain of T. harzianum was a potent biocontrol agent against C. michiganensis subsp. michiganensis. Application of this strain to tomatoes in the greenhouse resulted in a decrease in disease severity and an increase in crop biomass.

     

Monitoring the occurrence of tomato bacterial spot and range of the causal agent Xanthomonas perforans in Iran

A 2‐year comprehensive field survey was conducted across major tomato‐growing areas of Iran. Two hundred and thirty‐four tomato fields and six tomato‐producing greenhouses were surveyed for the potential presence of bacterial spot disease. Five hundred and ninety‐six tomato samples with and without symptoms were analysed. While Xanthomonas spp. were found in association with tomato plants both with and without symptoms from five surveyed counties, the bacterial spot disease was observed only in plants from three of them. Only strains isolated from plants with symptoms induced disease symptoms on tomato, while those isolated from symptomless plants caused symptoms only on cabbage and common bean. None of the isolates caused disease symptoms on pepper and eggplant. Phylogenetic analysis showed that X. perforans is the causal agent of tomato bacterial spot in Iran, although X. campestris and X. axonopodis were also associated with symptomless tomato plants. All X. perforans isolates in this study were sensitive to streptomycin, copper sulphate and copper oxychloride at concentrations of 50 mg L^?1, 200 mg L^?1 and 0.8 g L^?1, respectively. Unlike the type strain of X. perforans, isolates in this study did not produce bacteriocin against other Xanthomonas spp., nor were they detected using the usual species‐specific primer pair Bs‐XpF/Bs‐XpR. This suggests an atypical nature of X. perforans strains in Iran, which leads to the hypothesis that X. perforans strains in Iran may have a separate origin to those causing disease epidemics elsewhere. The aggregated dispersal pattern of the diseased tomato fields signifies the seedborne introduction of the pathogen into the country.     

Phylogenetic analyses of plant-growth-promoting rhizobacteria isolated from tomato,lettuce, and Japanese pepper plants in Hyogo Prefecture,Japan

Approximately 30,000 fluorescent bacterial strains isolated from tomato, lettuce, eggplant, Chinese cabbage, and Japanese pepper plants at seven different locations in Hyogo Prefecture, were screened for plant-growth-promoting (PGP) activity to induce disease resistance against bacterial wilt in tomato. The 37 strains that had higher PGP activity were subjected to molecular phylogenetic analyses using the sequences of the 16S rRNA, gyrB and rpoD genes. Most of the strains were identified as Pseudomonas fluorescens or its close relative, P. putida, while a few strains were grouped with more distantly related bacterial species such as Enterobacter and Stenotrophomonas. The phylogenetic relationships among tomato and lettuce isolates mostly coincided with the source locality and host plants, with a few exceptions. In contrast, isolates from Japanese pepper plants did not form their own cluster but represented several different bacterial species.     

Characterization and pathogenicity assessment of Plectosphaerella species associated with stunting disease on tomato and pepper crops in Italy

This study follows a survey carried out in 2012 and 2013 on tomato and pepper crops in the Foggia province (southern Italy), for morphological, molecular and pathogenicity analyses of Plectosphaerella fungi. The Plectosphaerella genus includes several species that are pathogens of horticultural plants. The survey identified tomato and pepper crops that showed abundant wilt, leaf yellowing, and discolouration and necrosis of roots, plus collar and stem symptoms. Different fungi including Plectosphaerella spp. were isolated from tissues with and without symptoms. Subsequent molecular and morphological studies identified first records of P. citrulli infecting tomato and pepper, and P. pauciseptata and P. ramiseptata infecting pepper. Pathogenicity testing confirmed that most isolated species of Plectosphaerella caused symptoms on tomato and pepper, with P. ramiseptata the most aggressive. On the basis of these data, it is considered that Plectosphaerella species may cause stunting disease in tomato and pepper.