Specific primers for Xanthomonas vesicatoria, a tomato bacterial spot causal agent

Xanthomonas vesicatoria is a member of the species complex associated with tomato bacterial spot. New and specific primers for X. vesicatoria were developed and validated. The primers were highly specific and detection was positive using purified bacterial DNA, bacterial suspensions and foliar lesions. These primers represent an additional tool for detection and identification of one of the species involved in this important disease complex.     

辣椒细菌性疮痂病病原菌分类、检测及综合防治研究进展

辣椒细菌性疮痂病是一种世界性分布的细菌性病害,该病能引起辣椒严重的产量损失和品质下降。国外特别是美国对该病害研究较早且较深入,国内相关研究几乎是空白。本文主要围绕病原菌的分类、检测和病害综合防治等研究进展做一概述。     

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.     

PM 7/110 (1) Xanthomonas spp. (Xanthomonas euvesicatoria,Xanthomonas gardneri,Xanthomonas perforans,Xanthomonas vesicatoria) causing bacterial spot of tomato and sweet pepper

Specific scope

This standard describes a diagnostic protocol for Xanthomonas spp. causing bacterial spot of tomato and sweet pepper (Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas vesicatoria) ¹ .

Specific approval and amendment

Approved in 2012–09.     

Complementary bacterial enrichment techniques for the detection ofPseudomonas syringae pv.tomato andXanthomonas campestris pv.vesicatoria in infested tomato and pepper seeds

A scheme for routine seed testing forXanthomonas campestris pv.vesicatoria andPseudomonas syringae pv.tomato in pepper and tomato seeds was developed. The scheme is based on different bacterial enrichment techniques. As few as 1000 and 10–100 colony forming units per gram of seeds were detected using a liquid enrichment technique or leaf enrichment technique, respectively. Relatively large amounts of saprophytes on the seed surfaces did not interfere with the detection of the pathogens.     

Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field

A total of 404 isolates of Xanthomonas campestris pv. vesicatoria , obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2 , shared taxonomic group B strain characteristics, including the presence of the β-protein band, positive amylolytic activity and inability to oxidize cis -aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot-susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.     

Impact of bacterial spot outbreaks on the phytosanitary quality of tomato and pepper seeds

The impact of disease outbreaks on the phytosanitary quality of seeds was investigated for two pathosystems: tomato–Xanthomonas vesicatoria and pepper–Xanthomonas euvesicatoria. This study, which was performed in Italy and Serbia, aimed to evaluate the season‐to‐season transmission of phytopathogenic regulated bacteria associated with phytosanitary risks posed by seeds produced in areas where bacterial infections are possible. For each pathosystem, field plots were experimentally inoculated to simulate an initial infection rate of 1%, 5% and 15%. The area under the disease progress curve (AUDPC) was calculated for each field plot, the seeds produced were analysed to determine the contamination level and rate, and the plant‐to‐seed transmission was evaluated by a seedling grow‐out (SGO) assay. To investigate transmission under field conditions, a second‐year experiment was performed, wherein seeds collected from the first year were used to establish new field plots. During the first growing season, AUDPC values were positively correlated with the percentages of initial infection for each pathosystem. Seed contamination levels in pepper ranged from 34 to 100 CFU g^?1, and the contamination rate ranged from 1.50% up to 3.17% for X. euvesicatoria, whereas processing and fresh market tomato seeds produced both in Italy and Serbia were not infected by X. vesicatoria. During SGO assays and the second cropping year, no symptoms were observed in either tomato or pepper plants. Therefore, the calculated pepper seed contamination rate for X. euvesicatoria appeared to be less than the threshold necessary to initiate a disease outbreak. Finally, all seeds obtained during the second cropping year were uninfected.     

新疆加工型辣椒细菌性斑点病的发生和病原鉴定

 新疆加工型辣椒主要产区巴音郭楞蒙古自治州发生了一种严重危害辣椒的细菌性病害。从发病辣椒叶片中分离细菌,通过烟草过敏性反应、马铃薯软腐试验和接种辣椒等致病性测定,确定了13个致病菌株,各菌株之间致病力无明显差异。通过菌体形态、培养性状观察、生理生化反应、寄主范围测定,结合16S rDNA和rpoD基因扩增、序列测定和系统发育分析,将病原菌鉴定为丁香假单胞菌丁香致病变种(Pseudomonas syringae pv. syringae)。病原菌人工接种还能侵染番茄、茄子、马铃薯及黄瓜、四季豆、白菜、萝卜、芹菜等植物。P. syringae pv. syringae引起加工型辣椒细菌性斑点病在国内属首次报道。     

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.     

Multiphasic analysis of xanthomonads causing bacterial spot disease on tomato and pepper in the Caribbean and central america: evidence for common lineages within and between countries

ABSTRACT Four hundred thirty-three xanthomonad strains isolated from tomato or pepper plants from 32 different fields in four Caribbean and Central American countries were screened for the ability to hydrolyze starch and sodium polypectate and for resistance to copper and streptomycin. Of these, 95 representative strains were further characterized by various phnetic tests, and 63 of these strains were then analyzed by genomic fingerprinting. Most of the strains (>90%) were tolerant to copper. However, there was much more variability in sensitivity to streptomycin. All strains in Guadeloupe and 93% of the strains in Barbados were sensitive to streptomycin. The majority of strains were typical Xanthomonas campestris pv. vesicatoria group A strains. In Barbados, however, a unique group of strains was identified that was serologically similar to group A strains but was amylolytic. These strains were designated A1. The occurrence of X. campestris pv. vesicatoria group B strains in Central America was found to be limited to two fields in Costa Rica and one in Guatemala. No group B strains were identified in the Caribbean, in contrast to common occurrence in the central United States and in South America. T3 strains were not found in this study, despite the recent increase of such strains in Florida and Mexico. Unique strains from Costa Rica belonging to the X. gardneri group were identified. Little linkage was found among phenotypic and rep-polymerase chain reaction (rep-PCR) genomic fingerprinting profiles of the pathogens except at the species/pathovar level; strains displaying virtually identical fingerprint profiles were found to correspond to distinct races and vice versa. The rep-PCR genomic fingerprinting analyses suggest that certain lineages may have evolved or predominated in specific regions or specific countries.     

Simultaneous detection and genetic variability of stone fruit viroids in the Czech Republic

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.     

DNA probes and PCR primers for the detection of a phytoplasma associated with peanut witches'-broom

EcoRI restriction fragments of genomic DNA from the phytoplasma associated with peanut witches'-broom (PNWB) were cloned in plasmid pGEM-3Zf(+). Cloned inserts from seven PNWB-phytoplasma-specific recombinant plasmids and two subcloned plasmids were excised with restriction enzymes, labeled with digoxigenin, and used as probes. Probe PNWB281 and its derivative subclones PNWB281-4 and PNWB281-5 hybridized with DNA from PNWB-phytoplasma infected peanut and periwinkle specifically but not with DNA from healthy plants or plants infected with phytoplasmas associated with sweetpotato witches'-broom (SPWB), loofah, Ipomoea obscura, and paulownia witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Six other probes hybridized with DNA derived from PNWB and SPWB-phytoplasma-affected periwinkle but not with DNA from healthy plants or plants infected with other phytoplasmas mentioned. In Southern hybridizations, four of the nine cloned and subcloned probes could differentiate the PNWB-phytoplasma from SPWB-phytoplasma. Three primer pairs for PCR were synthesized according to the partial sequences at both ends of the cloned inserts and were able to distinguish PNWB-phytoplasma from SPWB-phytoplasma by using PCR for the first time. A minimum of 1 pg and 10 pg of total DNA from diseased periwinkle and peanut, respectively, was sufficient to amplify the specific PNWB-phytoplasma PCR fragments, allowing the detection of PNWB-phytoplasma DNA from healthy-looking periwinkle plants two weeks after graft inoculation.     

Involvement of lipoxygenase,lipoxygenase pathway volatiles,and lipid peroxidation during the hypersensitive reaction of pepper leaves to Xanthomonas campestris pv. vesicatoria

Lipoxygenase activity and protein, production of lipid-derived volatiles, and lipid peroxidation levels were determined in pepper (Capsicum annuum L., cv. Early Calwonder-10R) leaves during the hypersensitive reaction induced by avirulent race 2 of Xanthomonas campestris pv. vesicatoria. Lipoxygenase activity increased during the collapse phase of the hypersensitive reaction (8 to 12 h after inoculation), and an increase in electrolyte leakage occurred. However, Western blot analysis revealed that lipoxygenase proteins decreased during the same period. When only one longitudinal half of a pepper leaf was inoculated with the avirulent bacterium race, a significant increase in lipoxygenase activity was observed in both inoculated and noninoculated leaf halves, 10 h after inoculation. In addition, lipoxygenase protein decreased in inoculated leaf halves, but remained unchanged in noninoculated ones. The evolution of some volatile compounds derived from the lipoxygenase pathway [(E,E)-2,4-hexadienal, 1-hexanol, 3-hexen-1-ol, 2,4-hexadienal and 2,4-eptadienal] and carotenoid degradation (α- and β-ionone) increased in the incompatible interaction during the collapse phase of the hypersensitive reaction. The level of the oxidative index (A₂₃₅/A₂₀₅) of leaf lipid extracts, determined to estimate lipid peroxidation, significantly increased in the advanced stage of the hypersensitive reaction. Furthermore, determination of the oxidative index in neutral lipid, glycolipid and phospholipid fractions showed that the oxidative index was significantly increased only in the glycolipid fraction. Lipoxygenase activity and protein, electrolyte leakage, volatiles and lipid peroxidation were not changed in pepper leaves inoculated with the virulent race 1 of X. campestris pv.vesicatoria during the time interval considered (2–12 h after inoculations). The hypothesis that a lipoxygenase with chloroplastic location is induced in the incompatible interaction, and which is responsible for the increase in lipid peroxidation is advanced.     

A method for detection of seedborne bacterial diseases in tomato seeds

A method for detection and quantitative estimation of tomato seedborne pathogenic bacteria has been developed. It enables detection in a 7 g tomato seed sample of as few as ten colony-forming units per gram tomato seeds of the following seedborne pathogens of tomato:Pseudomonas syringae pv. tomato,Pseudomonas corrugata, Xanthomonas campestris pv.vesicatoria, andClavibacter michiganense subsp.michiganense. With representative seed samples, the method employs dry grinding, weighing, bacterial extraction and quantitative calculation on selective or semi-selective medium. The efficiency of this method was tested by diluting pathogen-free seed lots with naturally or artificially infested tomato seeds. This procedure enables one to determine the minimal threshold of pathogen which can be detected by this method on media, in comparison with the percentage of diseased seedlings developed from the same seed lots in the growth chamber or in the greenhouse.     

Evidence for the Preemptive Nature of Tomato Race 3 of Xanthomonas campestris pv. vesicatoria in Florida

ABSTRACT Until recently, tomato race 1 (T1) of Xanthomonas campestris pv. vesicatoria was the only race causing bacterial spot of tomato in Florida. In 1991, tomato race 3 (T3) was first identified in 3 of 13 tomato production fields surveyed. By 1994, T3 was observed in 21 of 28 fields and was the only race identified in 14 fields. In field studies, tomato genotypes with resistance to either T1 or T3 or susceptibility to both were co-inoculated with strains of both races. Lesions on 10 plants in each of three replications for each genotype were sampled three times during the experiment; bacterial isolations were made from each lesion, and tomato race identifications were made for each strain. At the third sampling date, T3 was isolated from 97% of the lesions on the susceptible genotype Walter and the T1-resistant genotype Hawaii 7998, while T3 was isolated from 23% of the lesions and T1 from the remaining 77% on the T3-resistant genotypes PI 128216 and PI 126932. In surface population studies done in growth rooms, suspensions of T1 and T3 were applied alone and in combination to the leaf surfaces of susceptible and resistant genotypes. T1 populations were reduced more than 10-fold when applied in combination with T3, compared with populations that developed when T1 was applied alone. T3 populations were not affected when applied in combination with a T1 strain. In greenhouse studies with the T3-resistant genotype Hawaii 7981, disease was significantly reduced in plants inoculated with T3 in combination with T1, compared with plants inoculated with T1 alone. These results clearly demonstrate the competitive nature of T3 in the presence of T1 and help explain the emergence of T3 as a prevalent race in Florida.