Pseudomonas Viridiflava Research Articles

First report on Pseudomonas viridiflava causing bacterial leaf spot of curly lettuce in Turkey

During the winter growing season of 2016, bacterial leaf spot disease, characterized as water-soaked,dark-brown and irregular spots 2-8 mm in diameter, was observed on curly type lettuce (Lactuca sativa var. crispa) in greenhouses of Samsun province (Carsamba town) in the Black Sea Region of Turkey. Fluorescent colonies were further purified on KB to perform further tests with the reference isolate. Pathogenicity of isolates was confirmed on 30-day-old curly type lettuce plants, and they were identified as Pseudomonas viridiflava based on biochemical test results confirmed using PCR-amplification of the 16S rDNA gene and sequencing. This is the first report of P. viridiflava, the causal agent of curly type lettuce in Turkey.

Comparative genome analysis of the vineyard weed endophyte Pseudomonas viridiflava CDRTc14 showing selective herbicidal activity

Microbes produce a variety of secondary metabolites to be explored for herbicidal activities. We investigated an endophyte Pseudomonas viridiflava CDRTc14, which impacted growth of its host Lepidium draba L., to better understand the possible genetic determinants for herbicidal and host-interaction traits. Inoculation tests with a variety of target plants revealed that CDRTc14 shows plant-specific effects ranging from beneficial to negative. Its herbicidal effect appeared to be dose-dependent and resembled phenotypically the germination arrest factor of Pseudomonas fluorescens WH6. CDRTc14 shares 183 genes with the herbicidal strain WH6 but the formylaminooxyvinylglycine (FVG) biosynthetic genes responsible for germination arrest of WH6 was not detected. CDRTc14 showed phosphate solubilizing ability, indole acetic acid and siderophores production in vitro and harbors genes for these functions. Moreover, genes for quorum sensing, hydrogen cyanide and ACC deaminase production were also found in this strain. Although, CDRTc14 is related to plant pathogens, we neither found a complete pathogenicity island in the genome, nor pathogenicity symptoms on susceptible plant species upon CDRTc14 inoculation. Comparison with other related genomes showed several unique genes involved in abiotic stress tolerance in CDRTc14 like genes responsible for heavy metal and herbicide resistance indicating recent adaptation to plant protection measures applied in vineyards.

Pseudomonas floridensis sp. nov., a bacterial pathogen isolated from tomato.

An unusual fluorescent pseudomonad was isolated from tomato exhibiting leaf spot symptoms similar to bacterial speck. Strains were fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco and produced a soft rot on potato slices. However, the strains produced an unusual yellow, mucoid growth on media containing 5 % sucrose that is not typical of levan. Based on multilocus sequence analysis using 16S rRNA, gap1, gltA, gyrB and rpoD, these strains formed a distinct phylogenetic group in the genus Pseudomonas and were most closely related to Pseudomonas viridiflava within the Pseudomonassyringae complex. Whole-genome comparisons, using average nucleotide identity based on blast, of representative strain GEV388T and publicly available genomes representing the genus Pseudomonas revealed phylogroup 7 P. viridiflava strain UASW0038 and P. viridiflava type strain ICMP 2848T as the closest relatives with 86.59 and 86.56 % nucleotide identity, respectively. In silico DNA-DNA hybridization using the genome-to-genome distance calculation method estimated 31.1 % DNA relatedness between GEV388T and P. viridiflava ATCC 13223T, strongly suggesting the strains are representatives of different species. These results together with Biolog GEN III tests, fatty acid methyl ester profiles and phylogenetic analysis using 16S rRNA and multiple housekeeping gene sequences demonstrated that this group represents a novel species member of the genus Pseudomonas. The name Pseudomonas floridensis sp. nov. is proposed with GEV388T (=LMG 30013T=ATCC TSD-90T) as the type strain.

FIRST REPORT OF PSEUDOMONAS VIRIDIFLAVA CAUSING CABBAGE BACTERIAL LEAF SPOT IN TURKEY

In the autumn of 2016, white head cabbage (Brassica oleracea var. capitata f. alba) showing circular to irregular lesions 2–3 mm in size surrounded by a yellow halo and necrotic areas on the leaves, were observed in the Samsun province of Turkey. Extract from symptomatic tissue macerated in plastic bags (Bioreba AG, Switzerland), plated on King’s medium B (King et al., 1954) and incubated at 26°C for 48 h yielded colonies with the same LOPAT profile of Pseudomonas viridiflava strain M66 from tomato. They were levan-, oxidase-, arginine-negative, caused hypersensitive reactions on tobacco leaves (Nicotiana tabacum cv. Xanthi) and macerated potato slices. Molecular characterization using ERIC-PCR (Rademaker et al., 1998) yielded the same band pattern for strains BT5X and M66. Blastn analysis of a 1400 bp 16S rDNA and a 835 bp gyrB gene sequence (GenBank accession Nos. KY769809, MF314821) showed 99% and 100% similarity to the sequences of the type strain ATCC13223T =CFBP 2107 (NR_114482- HM190239) of Ps. viridiflava deposited in GenBank. Multilocus sequence analysis (MLSA) produced by maximum likelihood method using concatenated sequences of 16S rDNA and gyrB allowed clustering of our strain in the same clade together with the type strain ATCC13223T and other representive P. viridiflava strains retrieved from GenBank. Cabbage plants (cv. Beyaz Bursa) were spray-inoculated with a suspension of 108 CFU ml-1 of an overnight culture grown in Luria-Bertani broth reacted six days post inoculation with spots similar to those observed in the field. Reisolated colonies were fluorescent under UV light and had the same morphology on King’s medium B as the original culture and positive strain M66. To our knowledge, this is the first report of P. viridiflava causing a leaf spot disease of head cabbage in Turkey.

Microparticles containing gallic and ellagic acids for the biological control of bacterial diseases of kiwifruit plants

Over the last decades, kiwifruit cultivation has gained increasing importance all over the world, but some bacterial diseases seriously threaten its cultivation. The most dangerous one is the bacterial canker caused by Pseudomonas syringae pv. actinidiae Takikawa et al., but also floral bud necrosis by Pseudomonas syringae pv. syringae van Hall and bacterial blight by Pseudomonas viridiflava (Burkholder) Dowson affect kiwifruit plants. Their control relies mainly on antibiotics, where allowed, and copper, although this adversely affects the environment, so that alternative control measures are needed. Here we investigate gallic acid and ellagic acid, substances easily obtainable from some plant tissues, for their antimicrobial activity aiming to use them as support in the biocontrol of kiwifruit bacterial diseases. These active principles demonstrated their effectiveness as pure substances in both in vitro and in vivo tests. Moreover, their encapsulation in methacrylate polymeric microparticles showed to improve their usefulness and to prolong remarkably their activity up to 14 days after the treatment, when applied in greenhouse or in field on artificially and naturally infected plants, respectively. The encouraging results obtained by this type of microformulation point the way to future alternative biological control strategies against kiwifruit bacterial diseases.

An ELISA method to identify the phytotoxic Pseudomonas syringae pv. actinidiae exopolysaccharides: A tool for rapid immunochemical detection of kiwifruit bacterial canker

The phytotoxic exopolysaccharides produced by Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit, were isolated and partially identified. Their phytotoxic activity was evaluated on host and non-host plants and their role in the complex mechanisms of host-pathogen interaction was also discussed. The phytotoxic exopolysaccharides, which are natural antigens, were used to arise specific antibodies by rat immunization. The antibodies were used to develop a rapid and specific method to unambiguously detect P.s. pv. actinidiae exopolysaccharides isolated from bacterial culture and infected plant samples. Indeed, the antibodies recognized the exopolysaccharides produced by other two strains of P. s. pv. actinidiae but did not cross reacted with those isolated from P. s. pv. syringae and Pseudomonas viridiflava culture filtrates. Finally, the same antibodies significantly recognized the exopolysaccharides extracted from infected kiwi leaves.

The Spectrum of Weed Phytopathogens in Wheat Agrophytocenosis

Bacterial diseases, pests and weeds are the main factors that do not allow generating high yields of agricultural crops. The article presents the results of a study of agents of weed diseases. It was shown that the affected weeds in wheat agrophytocenosis present an ecological niche for phytopathogenic bacteria, belonging to the species Pseudomonas syringae, Pseudomonas viridiflava, Pseudomonas fluorescens, Pectobacterium carotovorum, Pantoea agglomerans, Xanthomonas translucens and Pseudomonas sp. In addition to these pathogens, phytopathogenic yeasts Rhodosporidium diobovatum and Rhodotorula sp. were isolated and identified. Most of the phytopathogens, isolated from weeds, are widely specialized and in experiment affect various species of weeds and cereals. Fatty acid composition of the total cellular lipids of these plant pathogens was identified and proved to be characteristic for each species of bacteria. P. syringae was determined as the main agent of bacterial diseases of weeds, its strains have a high degree of serological relationship to the agent of basal bacteriosis of cereals – P. syringae pv. atrofaciens and belong to the same serogroups. The results of RAPD-PCR analysis revealed that P. syringae strains isolated from weeds and disease agents of cereals present a genetically homogeneous group. The results obtained indicate that wheat and weeds are affected by the same agents of bacterial diseases.

Pyrene biodegradation enhancement potential of lipopeptide biosurfactant produced by Paenibacillus dendritiformis CN5 strain

Effect of biosurfactant on biodegradation of pyrene was studied using a microbial consortium predominantly composed of Pseudomonas viridiflava (49.5%) and Pseudomonas nitroreducens (32.5%) in a batch experiment containing lipopeptidic biosurfactant, produced by Paenibacillus dendritiformis CN5 strain, and mineral salt medium. The results showed that the lipopeptide at 600 and 300mgL−1 enhanced pyrene degradation to 83.5% and 67% respectively in 24days compared to 16% degradation in its absence. However degradation of pyrene was reduced to 57% as the lipopeptide supplementation was raised to 900mgL−1. This demonstrates that the biodegradation of pyrene was found to increase with an increase in the lipopeptide concentration up to a threshold level. The experimental data were fitted to the logistic kinetic model which provided best fit with a coefficient of determination (R2) values≥0.97. Maximum specific growth rate, μmax of 0.97 and 0.69d−1 were achieved in the 600 and 300mgL−1 lipopeptide amendments in comparison to 0.54d−1 in the unamended one. The carrying capacity, Xmax increased 4.4-fold in 600mgL−1 lipopeptide supplemented samples in comparison to its absence. Generally the lipopeptide showed potential application in improving bioremediation of polycyclic aromatic hydrocarbons contaminated environmental media.

Efficacy of lactoferricin B in controlling ready-to-eat vegetable spoilage caused by Pseudomonas spp.

The microbial content of plant tissues has been reported to cause the spoilage of ca. 30% of chlorine-disinfected fresh vegetables during cold storage. The aim of this work was to evaluate the efficacy of antimicrobial peptides in controlling microbial vegetable spoilage under cold storage conditions. A total of 48 bacterial isolates were collected from ready-to-eat (RTE) vegetables and identified as belonging to Acinetobacter calcoaceticus, Aeromonas media, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonas putida, Pseudomonas simiae and Pseudomonas viridiflava species. Reddish or brownish pigmentation was found when Pseudomonas strains were inoculated in wounds on leaves of Iceberg and Trocadero lettuce and escarole chicory throughout cold storage. Bovine lactoferrin (BLF) and its hydrolysates (LFHs) produced by pepsin, papain and rennin, were assayed in vitro against four Pseudomonas spp. strains selected for their heavy spoiling ability. As the pepsin-LFH showed the strongest antimicrobial effect, subsequent experiments were carried out using the peptide lactoferricin B (LfcinB), well known to be responsible for its antimicrobial activity. LfcinB significantly reduced (P≤0.05) spoilage by a mean of 36% caused by three out of four inoculated spoiler pseudomonads on RTE lettuce leaves after six days of cold storage. The reduction in the extent of spoilage was unrelated to viable cell density in the inoculated wounds. This is the first paper providing direct evidence regarding the application of an antimicrobial peptide to control microbial spoilage affecting RTE leafy vegetables during cold storage.

Deteksi dan Identifikasi Dickeya sp. sebagai Organisme Pengganggu Tumbuhan Karantina A2 pada Tanaman Kentang di Jawa

Erwinia chrysanthemi (currently Dickeya sp.) is one of the A2 quarantine pest that must be concerned of its distribution on potato in Indonesia. The aim of this study is to detect and identify E. chrysanthemi from potato in Java. A total of 400 samples of potato plants showing symptoms of soft rot were obtained from several potato areas in Pangalengan and Garut (West Java), Dieng (Central Java), and Batu-Malang (East Java). Disease incidence was determined by indirect enzyme-linked immunosorbent assay (I-ELISA) using polyclonal antiserum. E.chrysanthemi was isolated from plant samples with positive ELISA results. Furthermore, bacterial isolates were characterized by GEN III OmniLog ID System and PCR using specific primers Ec3F/Ec4R, as well as the universal 16S rRNA primer pair of 27F/1429R. The incidence of E. chrysanthemi based on ELISA was obtained. Based on physiological characters; Gram, catalase, oxidase, and oxidation-fermentation, there were 4 isolates similar to the genus of Erwinia . However, the results of Gen III OmniLog System, PCR, and nucleotide sequences analysis of 16S rRNA confirmed that none of the isolates were identified as E.chrysanthemi . Otherwise, those 4 isolates were identified as Pseudomonas oryzihabitans , Pantoea agglomerans , and Pseudomonas viridiflava . The result of this study indicated that the existence of E. chrysanthemi as an A2 quarantine pest on potato in Java can not be confirmed and remains as an A1 quarantine pest.

A multimodal machine vision system for quality inspection of onions

A multimodal machine vision system was developed to evaluate quality factors of onions holistically and nondestructively. The system integrated hyperspectral, 3D, and X-ray imaging sensors. A LabVIEW program was developed to acquire color images, spectral images, depth images, X-ray images of onions, and measure the weight of onions. With the multimodal data collected, algorithms were developed to calculate the maximum diameter, volume, density, and detect latent defects of onions. Three groups of sweet onions (regular, inoculated with Burkholderia cepacia, and inoculated with Pseudomonas viridiflava) were tested. Results showed that the system accurately measured the weight (RMSE=3.6g), diameter (RMSE=1.7mm), volume (RMSE=16.5cm3), and density (RMSE=0.03g/cm3) of onions, and correctly classified 88.9% healthy and defective onions. This work demonstrated a promising approach to evaluate both external and internal quality parameters of onions, which is applicable to onion packinghouses. The proposed system and methods are also potentially applicable to quality inspection of other agricultural products.

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity.

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild-type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild-types attained similar population densities. Given the importance of the methyl-directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild-type phenotype.

Role of Arginine decarboxylase (ADC) in Arabidopsis thaliana defence against the pathogenic bacterium Pseudomonas viridiflava.

Polyamine biosynthesis starts with putrescine production through the decarboxylation of arginine or ornithine. In Arabidopsis thaliana, putrescine is synthesised exclusively by arginine decarboxylase (ADC), which exists as two isoforms (ADC1 and 2) that are differentially regulated by abiotic stimuli, but their role in defence against pathogens has not been studied in depth. This work analysed the participation of ADC in Arabidopsis defence against Pseudomonas viridiflava. ADC activity and expression, polyamine levels and bacterial resistance were analysed in null mutants of each ADC isoform. In non-infected wild-type (WT) plants, ADC2 expression was much higher than ADC1. Analysis of adc mutants demonstrated that ADC2 contributes to a much higher extent than ADC1 to basal ADC activity and putrescine biosynthesis. In addition, adc2 mutants showed increased basal expression of salicylic acid- and jasmonic acid-dependent PR genes. Bacterial infection induced putrescine accumulation and ADC1 expression in WT plants, but pathogen-induced putrescine accumulation was blocked in adc1 mutants. Results suggest a specific participation of ADC1 in defence, although basal resistance was not decreased by dysfunction of either of the two ADC genes. In addition, and as opposed to WT plants, bacterial infection increased ADC2 expression and ADC activity in adc1 mutants, which could counterbalance the lack of ADC1. Results demonstrate a major contribution of ADC2 to total ADC activity and the specific induction of ADC1 in response to infection. A certain degree of functional redundancy between the two isoforms in relation to their contribution to basal resistance is also evident.

Evaluation of antibacterial activity of Aspergillus and Penicillium spp

Bacteria have known as one of the main important limitation factors of many agricultural products. The purpose of the present study was to evaluate the antibacterial activities of different species of Penicillium and Aspergillus on some important bacteria. For this intention 12 and 21 species of Aspergillus and Penicillium, were applied, respectively. Gram-negative bacteria conducted in this research were Pseudomonas viridiflava, Xanthomonas campestrispv. Campestris and Escherichia coli and the only gram-positive bacterium was Rhatayibacter iranicus. Fungal and bacterial isolates were dual cultured simultaneously on Potato Dextrose Broth (PDB) and were shaked with 120 rpm. The samples were investigated after a week. The results of this study demonstrated that none of fungal species could inhibit E. coli and the most of the studied species couldn’t suppress the growth of P. viridiflava except isolates of (5033, 5037, and 5071) from P. crysogenum. On the other hand, A. awamori, A. ferreus, A. ostiatus, A. niger, A. foetidus, P. mali, P. crustaseum and P. citrinumcould lead to inhibition of X. campestris pv. campestris Gram positive bacterium, Rhatayibacter iranicus, was not able to grow in the presence of all species of Aspergillus. On the contrary, species of P. piscarium, P. corylophilum, P. griseofulvum and P.chrysogenum couldn’t prevent its growth.

Occurrence, genetic diversity and pathogenicity characteristics of Pseudomonas viridiflava inducing alfalfa bacterial wilt and crown root rot disease in Iran

Alfalfa (Medicago sativa), the world’s most influential forage crop, is infected by many diseases such as alfalfa bacterial wilt disease. The causal agent of bacterial crown and root rot and wilt disease is Pseudomonas viridiflava, which is a substantial pathogen of alfalfa worldwide. This pathogen spreads through the xylem and under field conditions, plants show growth stunting, chlorosis and wilting symptoms not previously reported. In this study- the first on Pseudomonas viridiflava on alfalfa from Iran, we have investigated the pathogenicity and genetic diversity of Pseudomonas viridiflava in some parts of Iran. To survey the causal agent of the disease, symptomatic plants were collected from the main alfalfa growing area. Pathogenicity of the collected strains was confirmed on alfalfa plants under green-house conditions using a completely randomized design. Determination of bacterial strains was done based on standard bacteriological methods and PCR assay using specific primers. Effects of bacterial strains on wet weight, dry weight, stem length and root length of infected plants were measured and the data were analyzed by SAS software and Duncan’s assessment. The diversity of liquid cellular proteins of bacterial strains was examined on Polyacrylamide gel. To delineate of genetic diversity the total DNA was drawn out. Fourteen random primers were used in a RAPD test. To sketch the dendrogram, RAPD fragments were used to calculate genetic diversity with NTSYS software. This data showed pathogenicity and genetic diversity of Pseudomonas viridiflava in Iran.

Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava

This work investigated the roles of the tetraamine thermospermine (TSpm) by analysing its contribution to Arabidopsis basal defence against the biotrophic bacterium Pseudomonas viridiflava. The participation of polyamine oxidases (PAOs) in TSpm homeostasis and TSpm-mediated defence was also investigated. Exogenous supply of TSpm, as well as ectopic expression of the TSpm biosynthetic gene ACL5, increased Arabidopsis Col-0 resistance to P. viridiflava, while null acl5 mutants were less resistant than Col-0 plants. The above-mentioned increase in resistance was blocked by the PAO inhibitor SL-11061, thus demonstrating the participation of TSpm oxidation. Analysis of PAO genes expression in transgenic 35S::ACL5 and Col-0 plants supplied with TSpm suggests that PAO 1, 3, and 5 are the main PAOs involved in TSpm catabolism. In summary, TSpm exhibited the potential to perform defensive functions previously reported for its structural isomer Spm, and the relevance of these findings is discussed in the context of ACL5 expression and TSpm concentration in planta. Moreover, this work demonstrates that manipulation of TSpm metabolism modifies plant resistance to pathogens.

Pseudomonas viridiflava in imported carrot seeds

In 2005, a sample of carrot seeds var. Kikuyo imported from Japan was submitted to the Instituto Biológico for bacteriological phytosanitary certification. Circular, convex and creamy colored colonies were obtained. These isolates produced a green fluorescent pigment on King’s B medium and incited a hypersensitivity reaction in tobacco leaves. They were oxidase and arginine dihydrolase negative, did not produce levan but caused soft rot in potato slices and were identified as Pseudomonas viridiflava. Artificial inoculations by infiltration of bacterial suspensions at the crown area or by placing a drop of bacterial suspension on this area and pricking with a sterile needle, produced light to dark brown areas around the injured points 5 days after inoculation. The bacterium was then re-isolated. Although this bacterium could be an opportunistic pathogen and was isolated from asymptomatic samples, eventual losses in the field and post-harvest storage is possible.

Sleeping hibiscus a new host for Pseudomonas viridiflava.

Since 2008, a disease consisting of brown to black angular to irregular spots, 2-4 mm in diameter and surrounded by a chlorot- ic halo, was observed in northern Iran on the leaves of sleeping hibiscus (Malvaviscus penduliflorus), a widely cultivated orna- mental shrub. Bacterial colonies were isolated from symptomatic leaves on nutrient agar containing 5% sucrose. Twenty represen- tative isolates were Gram-negative and negative for oxidase and arginine dehydrolase, positive for fluorescent pigment produc- tion, potato soft rot, induction of a hypersensitive response on to- bacco, acid production from xylose and mannose, and utilization of D-tartrate. The isolates reacted negatively in tests for urease and nitrate reduction, acid production from cellobiose and rham- nose and utilization of L-tartrate (Lelliott and Stead, 1988). Path- ogenicity was ascertained by spraying a bacterial suspension cali- brated at 107 CFU/ml on two young sleeping hibiscus plants, which reproduced the field syndrome A 16S ribosomal RNA fragment of 1550 bp was amplified from 10 bacterial isolates by PCR using the primer A1/B6 (Manceau and Horvais, 1997). A partial 16S rRNA gene sequence was deposited in GenBank data- base under the accession No. JF836019. Homology searches with the 16S rRNA sequence in database showed that the isolates un- der study were most similar (100%) to pathogenic Pseudomonas viridiflava isolates ICMP 2848, LMG 2352. Based on phenotypic, pathogenicity and genomic properties, the bacterium that causes leaf spot of sleeping hibiscus was therefore identified as Ps. viridi- flava. To the best of our knowledge, this is first report of the oc- currence of Ps. viridiflava on sleeping hibiscus and the first record of its presence in Iran.

Assessment of rhizobacteria from grapevine for their suppressive effect on the parasitic nematode Xiphinema index

The capacity of rhizobacteria isolated from grape plants to protect grapevine roots from damage by the nematode Xiphinema index was assessed in trials carried out in the growing season 2008–2009 and 2009–2010. A total of two groups of bacteria were evaluated, one comprising 49 isolates, of which 37 were assessed in previous studies, and one comprising 90 isolates not previously evaluated. Two assays were performed on the first group, one per season, and one on the second group, lasting two growing seasons. Pots of field soil (3 L) planted with two-month old Thompson Seedless vines were inoculated with 200 or 400 specimens of X. index after inoculation with 106 cfu/ml of test rhizobacteria; the posts were maintained in a shaded greenhouse. Seven isolates from the first group of bacteria (Bacillus brevis, Bacillus megaterium, Cytophaga johnsonae, Pseudomonas fluorescens, Rahnella aquatilis and Stenotrophomonas maltophilia) were effective in decreasing damage in both of the assays (P < 0.05). Ten isolates from the second group (Variovorax paradoxus, Pseudomonas pseudoalcaligenes, Bacillus mycoides, Bacillus sphaericus, Bacillus thuringiensis, Curtobacterium flaccumfaciens, Pseudomonas putida, Pseudomonas alcaligenes and Pseudomonas viridiflava) resulted in the lowest number of galls per gram root. B. brevis and B. megaterium also demonstrated good activity in previous studies on grape plants and are thus promising candidates for further research.

Comparison of pathogenicity of Clavibacter michiganensis subsp. insidiosus and Pseudomonas viridiflava strains on alfalfa

Hamedan province of Iran is a suitable niche for alfalfa growth but many diseases including alfalfa bacterial wilt, bacterial crown and root rot diseases cause economic crop losses. Bacterial wilt is caused by Clavibacter michiganensis subsp. insidiosus, and bacterial crown and root rot diseases are caused by Pseudomonas viridiflava. In this study, we investigated the pathogenicity of C. michiganensis subsp. insidiosus and P. viridiflava strains collected from the main alfalfa growing areas of Hamedan province. Pathogenicity of the virulent strains was tested on alfalfa and the bacterial strains caused symptoms, and data were collected about stem length, root length, wet weight and dry weight of the infected plantlets. The data about the pathogenicity of C. michiganensis subsp. insidiosus and P. viridiflava on alfalfa were compared with each other and were analysed by SAS software and Dunkan's test. Resulted data showed more pathogenicity of C. michiganensis subsp. insidiosus than P. viridiflava on alfalfa. These data also showed that both of these bacteria produced the most losses on wet weight and dry weight of alfalfa plantlets.