Characterization of Spanish isolates of Pseudomonas corrugata from tomato and pepper

Pseudomonas was isolated from tomato plants with pith necrosis collected from different areas in Spain and from pepper plants with pith necrosis in Tenerife (Canary Islands). Cultural, biochemical and physiological characteristics of isolates from both hosts were similar to those of the type strain of P. corrugata. Serological heterogeneity was observed among isolates of P. corrugata in immunofluorescence tests. A plasmid of more than 273 MDa was detected in all the isolates. Inoculation of pepper and tomato plants with isolates from both hosts caused similar pith necrosis. This is apparently the first report of P. corrugata on pepper.     

A new genomovar of Pseudomonas cichorii, a causal agent of tomato pith necrosis

Recent taxonomic advances, based on biochemical and genotypic processes demonstrate that the plant pathogenic species Pseudomonas cichorii consists of a cluster of closely related genomic groups. Prior to this study, three morphotype groups had been described (C1-C3), all sharing various phenotypic and biochemical characters but partially differing in their DNA content. All entities of the complex could cause disease among a variety of hosts, including lettuce, celery, chrysanthemum and others. In this study, we present the biochemical and molecular characterization of P. cichorii isolates as the causal agent of pith necrosis of tomato plants. A detailed characterization of the genetic variability among strains belonging to P. cichorii was achieved using BOX-PCR and Multi Locus Sequence Analysis utilizing three housekeeping genes (gyrB, rpoD, rpoB). In addition, a number of biochemical and physiological tests were also used for the identification of the tomato P. cichorii isolates. To our knowledge, this is the first complete biochemical, molecular and phylogenetic study of P. cichorii strains isolated from tomato plants affected by pith necrosis disease. Our findings demonstrate the emergence of a new genomovar of P. cichorii, yet another indication for the genetic heterogeneity of the 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.     

Pseudomonas populations causing pith necrosis of tomato and pepper in Argentina are highly diverse

Pseudomonas species causing pith necrosis symptoms on tomato and pepper collected in different areas of Argentina were identified as Pseudomonas corrugata , P. viridiflava and Pseudomonas spp. Their diversity was analysed and compared with reference strains on the basis of their phenotypic characteristics, copper and antibiotic sensitivity tests, serology, pathogenicity, DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis of a 16S rRNA gene fragment. All P. corrugata strains tested were copper-resistant while P. viridiflava strains were more variable. Numerical analysis of phenotypic data showed that all P. corrugata strains formed a single phenon that clustered at a level of about 93%, while all the P. viridiflava strains clustered in a separated phenon at a level of 94%. Genomic analysis by repetitive (rep)-PCR and 16S rRNA-RFLP fingerprinting and serological analysis showed that the two species contained considerable genetic diversity. Inoculations of tomato and pepper plants with strains from both hosts caused similar pith necrosis symptoms. Strains of both P. corrugata and P. viridiflava were grouped according to their geographical origin and not according to the original host. This is the first report of Pseudomonas viridiflava causing pith necrosis on pepper.     

Identification of <Emphasis Type="Italic">Xanthomonas</Emphasis> species associated with bacterial leaf spot of tomato,capsicum and chilli crops in eastern Australia

Several species of Xanthomonas cause bacterial leaf spot, a disease that affects solanaceous crops worldwide. The diversity of 64 Australian isolates of Xanthomonas spp. associated with bacterial leaf spot in tomato, capsicum and chilli crops in eastern Australia was determined using multi-locus sequence analysis of atpD, dnaK, efp and gyrB genes, species-specific PCR assays and biochemical analyses. At least five species of Xanthomonas associated with bacterial leaf spot were identified in Australian tomato, capsicum and chilli crops and their pathogenicity assessed. Phylogenetic and biochemical analyses identified X. euvesicatoria, X. perforans and X. vesicatoria as the most frequently recovered pathogenic species. Non-pathogenic and weakly pathogenic species were also identified. The suitability of the identification methods used and the implications of the detection of these species will be discussed.     

Rutin promoted resistance of tomato against <Emphasis Type="Italic">Xanthomonas perforans</Emphasis>

Xanthomonas perforans is the causal agent of bacterial spot, one of the most devastating diseases of tomato that results in considerable yield losses worldwide. Rutin, as a polyphenolic substance, was used to induce resistance in tomato against X. perforans. Rutin at concentration of 2 mM had ability to reduce the disease severity of bacterial spot. On the other hand, 2 mM rutin had no antibacterial activity in vitro. Expression profiling of pathogenesis-related gene 5 (PR-5), Phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX) was probed during the enhanced resistance by rutin. Pretreatment with rutin (rutin/ X. perforans) led to induction of PR-5, PAL and LOX compared to controls (water/ X. perforans). Our results suggest that rutin-induced resistance against X. perforans in tomato might be mediated through stimulation of some defense genes such as PR-5, PAL and LOX.     

Bacterial diseases of tomato in southern Spain: application of a detached tissue assay to evaluate bacterial pathogenicity

Tomatoes are one of the most important crops in southern Spain, especially during the cold season. As a preliminary step in the design of an integrated disease management programme for tomato, a study on the occurrence of bacterial diseases in tomato houses of Almería (ES) was carried out during the 1993/1997 growing seasons. Sixty-four bacterial strains were isolated from tomato plants showing symptoms of bacterial diseases and 41 of them (64%) were characterized as pathogenic. The bacterial tomato pathogens most frequently isolated were Erwinia carotovora subsp. carotovora, Pseudomonas corrugata, Pseudomonas syringae pv. tomato and Xanthomonas vesicatoria. The pathogenicity of the bacterial isolates on tomato was tested by the conventional seedling assay and by an in vitro assay using detached tissues developed in our laboratory. Close correspondence between the two assays was observed. An in vitro detached tissue assay is proposed for determining the pathogenicity of bacterial isolates on tomato.     

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.     

Identification of<Emphasis Type="Italic">Pseudomonas viridiflava</Emphasis> on tomato by traditional methods and enzyme-linked immunosorbent assay

Pseudomonas viridiflava is one of the causal agents of tomato stem necrosis in the eastern Mediterranean region of Turkey. The bacterium causes general wilting, yellowing of tomato plants, dark blotches on the pruning sites of the stem, browning, and hollowing of the pith.P. viridiflava strains, isolated from Antakya and Mersin, were identified by traditional methods and indirect enzyme-linked immunosorbent assay (ELISA). For indirect-ELISA, polyclonal antisera were produced against a regional isolate ofP. viridiflava (AD-OZ 3a). Using indirect-ELISA, the pathogenic bacterium was identified rapidly and safely from both pure culture and inoculated plants in 2 days. There was no cross reaction with other stem necrosis pathogens. With indirect-ELISA, the lower limit forP. viridiflava detection in pure culture was 10³ colony-forming units per milliliter. http://www.phytoparasitica.org posting Feb. 11, 2004.     

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.     

Characterization of Phoma adonidicola causing a spot blight on Adonis palaestina

Adonis palaestina Boiss. is one of the top three natural sources of red pigment astaxanthin, which has been used as a valuable antioxidant nutraceutical and a feed additive for salmonid fish raising. Since 2004, a blight disease causing significant damage to plants of A. palaestina in Inner Mongolia, China has occurred. The disease caused small, brown lesions on petioles and stems of the host plants. The disease initially appeared in the field as a brown necrosis on lower parts of the plant, and eventually expanded to the whole plant resulting in complete defoliation. The fungus consistently isolated from symptomatic tissues was identified as Phoma adonidicola based on morphological characteristics. Phylogenetic analysis based on LSU (Large Subunit - 28S), ITS (ITS1-5.8S-ITS2 region), and TUB (β-tubulin) showed that the P. adonidicola isolates fall in the same well-supported clade, which is closely related to Stagonosporopsis ajacis. All isolates of P. adonidicola also caused typical spots on inoculated plants, and were successfully reisolated from the symptomatic tissues. The disease of A. palaestina was proposed as spot blight.     

Isolation and characterization of <Emphasis Type="Italic">Serratia rubidaea</Emphasis> from dark brown spots of tomato fruits

Bacterial contamination of fresh tomato fruits is of great concern. From naturally infected tomato fruits showing dark brown irregularly shaped spots, 36 bacterial isolates were recovered and identified on phenotypic characteristics and sequences of the gene encoding the 16S rRNA. Five isolates showing spots on tomato fruits in the pathogenicity test with healthy tomato fruits belong to the genus Serratia on the basis of phenotypic characteristics. One representative isolate of these has been further identified as a Serratia rubidaea by sequencing of the 16S rRNA gene. This is the first evidence showing that a S. rubidaea strain can cause spots on tomato fruits. Virulence of the S. rubidaea was also confirmed by the production and secretion of a large variety of enzymes capable of degrading the complex polysaccharides of the plant cell wall and membrane constituents. Nineteen bacterial isolates of the 36 did not induce any spot symptoms in a pathogenicity test on artificially infected tomato fruits although these are known as phytopathogenic bacteria. Five of these 19 bacterial isolates were identified as Ralstonia species on the basis of biochemical tests. Sequencing of the 16S ribosomal gene of one representative isolate revealed that the isolate is closely related to Ralstonia solanacearum. Six isolates of the 19 were related to Xanthomonas vesicatoria on the basis of biochemical tests and eight were related to the Enterobacteriaceae. One representative isolate of the Enterobacteriaceae could be identified by the 16S rRNA gene as Enterobacter cloacae subsp. dissolvens. The 12 other strains were related to Proteus mirabilis based on the 16S RNA gene sequence of one representative isolate. The isolates related to P. mirabilis did not produce any symptoms on artificially infected tomato fruits. The nucleotide sequences of S. rubidaea strain E9, E. cloacae strain E23, P. mirabilis strain E11, and R. solanacearum strain E15 have been deposited in the GenBank nucleotide sequence database under accession numbers HM585373 to HM585376.     

Diversity among Pseudomonas corrugata and Pseudomonas mediterranea isolated from tomato and pepper showing symptoms of pith necrosis in Greece

Bacterial isolates from tomato plants with symptoms revealing pith necrosis collected over a 20‐year period were screened and characterized using biochemical and pathogenicity tests, as well as molecular approaches. The examined isolates were categorized into two main Pseudomonas species: P. corrugata and P. mediterranea. The diversity of these isolates was characterized through a variety of biochemical, serological, pathogenicity, DNA fingerprinting and multi‐locus sequence analysis features. Of the total Pseudomonas isolates causing pith necrosis, the biochemical profile of the selected non‐fluorescent pseudomonads consisting of P. corrugata and P. mediterranea revealed differentiation in only three characters among 43 examined. Serological analysis managed to identify the P. corrugata isolates using the laboratory prepared antiserum anti‐PC14. Genomic analyses using rep‐PCR fingerprinting and multilocus sequence analysis (MLSA) revealed considerable inter‐ and intraspecies genetic diversity. However, artificial inoculations of several plant species revealed similar pathogenicity patterns for both P. corrugata and P. mediterranea isolates. These results provide the basis for a more comprehensive understanding of the biology, sources and shift in genetic diversity and evolution of both P. corrugata and P. mediterranea populations and could support the development of molecular identification tools and epidemiological studies in diseases caused by these species. Moreover, this is the first report of P. mediterranea in Greece on tomato as well as on pepper plants.     

新疆加工番茄腐霉根腐病病原鉴定及其rDNA的ITS区段分析

为了明确新疆加工番茄根腐病的病原种类,从成苗期到果实成熟期对各主栽区的病原物进行分离,并进行形态学鉴定和ITS序列分析.结果表明,从162个根腐病样中获得120个分离物,其中腐霉菌93株,占总分离物的77.5%.腐霉菌分离物鉴定为3个种,瓜果腐霉Pythium aphani-dermatum(Edson)Fitzp,56株,占腐霉分离物的60.22%;简囊腐霉P.monospermum Pringsheim,6株,占6.45%;棘腐霉P.acanthicum Drechsler,2株,占2.15%;另有29个分离物因未诱发出雄器和藏卵器而无法鉴定到种.     

Occurrence of <Emphasis Type="Italic">Phaeoacremonium</Emphasis> spp. and <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis> in grape propagation materials and young grapevines

During a 6-year study, grapevine propagation materials and young grapevines were analysed to evaluate the presence of internal wood discolouration and the occurrence of fungal species involved in Petri disease. The intensity of wood discolouration increased with the ageing of the plants. The maximum incidence of dark streaks was observed in the rootstock while necrosis originating from buds or nodes were notably present in the trunk and cordon of older vines. In contrast, the highest levels of brown-red halo symptoms, defined as discoloured areas around the pith, were recorded in the early growth stages. Phaeoacremonium spp. and Phaeomoniella chlamydospora were usually isolated from the rooted-grafts and the 3-year old plants, respectively. The number of infected grapevines increased with age. Most of the P. chlamydospora strains were isolated from dark streaks or dots, while Phaeoacremonium spp. were detected in brown-red halo symptoms and other symptomatic or asymptomatic wood. The greatest incidence of the two fungal taxa was recorded in the lower parts of the grapevine, including the roots and rootstock.     

Differentiation of the closely related species,Alternaria solani and A. tomatophila,by molecular and morphological features and aggressiveness

The main causal agent of early blight, the noxious disease of solanaceous crops, is generally considered to be Alternaria solani Sorauer (in a broad sense). However, heterogeneity in many morphological features of this pathogen has been noted suggesting that the disease may not be caused by a single species. Recent research has revealed that several large-spored Alternaria species may cause disease of potato and tomato including A. solani sensu stricto and A. tomatophila. The goal of our research was to compare Russian large-spored Alternaria isolates from tomato and potato to test the hypothesis that early blight of tomato and potato are caused by different species. Cluster analysis of genetic distances estimated from 12 polymorphic molecular markers (universally primed-polymerase chain reaction and randomly amplified polymorphic DNA) revealed two groups of isolates accepted here as A. solani and A. tomatophila that were supported by morphology and host plant association. Differentiation of species was supported by phylogeny derived from the DNA sequences of a portion of the Alt a1, gpd and calmodulin genes. Species-specific primers based on the Alt a1 and calmodulin gene sequences for both species were designed. Under laboratory conditions, A. solani isolates were equally aggressive on both tomato and potato, whereas A. tomatophila was highly aggressive to tomato but only weakly aggressive to potato. In the field, A. solani was isolated from potato, tomato and from several wild potato species including S. schickii, S. papita and S. kurtzianum. The majority (90 %) of A. solani isolates carried the mating type locus 1 (MAT1) idiomorph MAT1-1 while the majority (88 %) of A. tomatophila isolates carried the MAT1-2 idiomorph.     

Molecular characterization ofPseudomonas syringae pv.tomato isolates from Tanzania

Bacterial speck caused byPseudomonas syringae pv.tomato is an emerging disease of tomato in Tanzania. Following reports of outbreaks of the disease in many locations in Tanzania, 56 isolates ofP. syringae pv.tomato were collected from four tomato- producing areas and characterized using pathogenicity assays on tomato, carbon source utilization by the Biolog Microplate system, polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis. All theP. syringae pv.tomato isolates produced bacterial speck symptoms on susceptible tomato (cv. ‘Tanya’) seedlings. Metabolic fingerprinting profiles revealed diversity among the isolates, forming several clusters. Some geographic differentiation was observed in principal component analysis, with isolates from Arusha region being more diverse than those from Iringa and Morogoro regions. The Biolog system was efficient in the identification of the isolates to the species level, as 53 of the 56 (94.6%) isolates ofP. syringae pv.tomato were identified asPseudomonas syringae. However, only 23 isolates out of the 56 (41.1%) were identified asPseudomonas syringae pv.tomato. The results of this work indicate the existence ofP. syringae pv.tomato isolates in Tanzania that differ significantly from those used to create the Biolog database. RFLP analysis showed that the isolates were highly conserved in theirhrpZ gene. The low level of genomic diversity within the pathogen in Tanzania shows that there is a possibility to use resistant tomato varieties as part of an effective integrated bacterial speck management plan. http://www.phytoparasitica.org posting August 8, 2008.     

Widespread distribution of Xanthomonas perforans and limited presence of X. gardneri in Brazil

Tomato bacterial spot is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri. In order to determine the distribution, frequency of occurrence, and diversity of these species in the Brazilian commercial tomato fields, a survey was conducted between 2009 and 2012. In this period, 204 strains were obtained from 33 counties (22 with processing tomatoes and 11 with fresh‐market tomatoes). Pathogenicity tests, BOX‐PCR, PCR with species‐specific primers, and sequence analysis of the avirulence gene avrXv3 were performed in order to identify the strains at species and race level. Xanthomonas perforans predominated among the strains (92%) and was present in most counties. In addition, this species was prevalent in most areas of both fresh‐market tomatoes (63.6% of counties surveyed) and processing tomatoes (95.4% of counties surveyed). Fifteen strains (7.5%) were identified as X. gardneri, which was found mostly in fresh‐market fields located at regions with altitude higher than 900 m, and only one strain of X. euvesicatoria (0.5%) was found in a processing tomato field. High genetic diversity was observed within X. perforans, with 137 BOX‐PCR haplotypes. Race T3 prevailed (97.5%), but reported here for the first time is the occurrence of five strains identified as race T4 in fresh‐market fields in the state of São Paulo. The race T4 phenotype of these strains resulted from the presence of an 859 bp insertion in the avirulence gene avrXv3. This insertion is related to amino acid sequences of a transposase found in X. gardneri, and to amino acid sequences of X. campestris.     

番茄细菌性髓部坏死病病原的鉴定

2015年,在广东省广州市番茄种植区发生番茄髓部坏死病,从番茄病样中分离到一种细菌。随机选取5株细菌进行致病性测定,结果显示,这5株细菌均可侵染番茄植株产生典型的髓部坏死症状,与田间病株的症状相同。5株细菌的16S rDNA序列间同源性为99.9%~100%,且与菊苣假单胞菌(Pseudomonas cichori)MAFF211996、TIs01和IP1-05菌株的16S rDNA序列同源性最高,为99%。利用菊苣假单胞菌hrcR基因的特异引物Hrp1a/Hrp2a进行PCR,从这5株细菌的DNA中均能扩增出大小约897 bp的特异片段;这些片段序列间同源性为99%~99.9%,且与P.cichori 83-1菌株hrcR基因的序列同源性为99.9%。这些结果表明,引起广东番茄髓部坏死病的病原为菊苣假单胞菌(P.cichorii)。生理生化试验显示,该病原菌与文献报道的菊苣假单胞菌的生理生化特征均相吻合,进一步证实引起广东番茄髓部坏死病的病原为菊苣假单胞菌。