Multiplex PCR for the identification of Agrobacterium biovar 3 strains

A biovar 3-specific primer set Ab3-F3/Ab3-R4 was designed based on the comparison of sequences of the 16S rDNA region of agrobacteria and related rhizobia for rapid identification of Agrobacterium biovar 3 strains. A 570-bp 16S rDNA fragment was amplified from cell lysates of Agrobacterium biovar 3 strains by polymerase chain reaction (PCR) using Ab3-F3/Ab3-R4 primers. Discrimination of Agrobacterium tumefaciens biovar 3 from Agrobacterium radiobacter biovar 3 and of Agrobacterium biovar 3 strains from other Agrobacterium strains was done simultaneously using multiplex PCR with a mixture of two primer sets (Ab3-F3/Ab3-R4 and VCF3/VCR3) previously designed for the virC region of Ti-plasmid and Ri-plasmid.     

The presence of crown gall of grape incited byagrobacterium tumefaciens biovar 3 in Israel

Crown gall was previously reported on grape in Israel but the pathogen was not isolated and characterized. The three recognized biovars ofAgrobacterium tumefaciens can be tumorigenic on grape, but biovar 3 is the most important world wide. A single occurrence of tumors in a vineyard yielded bacteria which incited galls on grape,Nicotiana glauca and tomato, but not on bryophyllum. The bacteria were confirmed asA. tumefaciens because they contained DNA which hybridized with T-DNA from a Ti plasmid. Biochemical and physiological tests, octopine production and utilization, and agrocin 84 insensitivity conformed with those of bv. 3. Subsequent occurrences of the grape disease have not been found, but the presence ofA. tumefaciens bv. 3 in Israel is a potential threat to nurseries and vineyards.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

Chromosomal and Plasmid Diversity of Agrobacterium Strains Isolated from Ficus benjamina Tumors

Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR–RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation as a tool for random mutagenesis of <Emphasis Type="Italic">Colletotrichum trifolii</Emphasis>

We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene (hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into the fungal genome. More significantly, about 75% of transformants had a single copy of the T-DNA. The results demonstrate that insertional mutagenesis via A. tumefaciens is a useful tool for studying the function of C. trifolii genes.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Efficacy of olive mill waste water and its derivatives in the suppression of crown gall disease of bitter almond

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens. Thabet Yangui and Ali Rhouma contributed equally to this work and are regarded as joint first authors.     

Three evolutionary lineages of tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici, based on sequences of IGS, MAT1, and pg1, are each composed of isolates of a single mating type and a single or closely related vegetative compatibility group

Three evolutionary lineages of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici were found among a worldwide sample of isolates based on phylogenetic analysis of the ribosomal DNA intergenic spacer region. Each lineage consisted of isolates mainly belonging to a single or closely related vegetative compatibility group (VCG) and a single mating type (MAT). The first lineage (A1) was composed of isolates VCG 0031 and MAT1-1; the second (A2) included VCG 0030 and/or 0032 and MAT1-1; and the third (A3) included VCG 0033 and MAT1-2. Race 1 and race 2 isolates belonged to the A1 or A2 lineages, and race 3 belonged to A2 or A3 lineages, suggesting that there is no correlation between race and lineage. However, for the isolates from Japan, race 1 (with one exception), race 2, and race 3 isolates belonged to A2, A1, and A3 lineages, respectively. These results suggest that the races could have evolved independently in each lineage; and in Japan the present races were likely to have been introduced independently after they had evolved in other locations.     

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.     

Molecular characterization of A2 and D strains of Soybean mosaic virus, which caused a recent virus outbreak in soybean cultivar Sachiyutaka in Chugoku and Shikoku regions of Japan

Coat protein sequences of two isolates in strain A₂ and five isolates in strain D of Soybean mosaic virus (SMV), which caused a recent mosaic outbreak in soybeans (cv. Sachiyutaka) in Chugoku and Shikoku in Japan, were compared to published data on 15 other Asian-origin isolates. Sequence comparison and cluster analysis showed that SMV isolates of strain A₂ from these districts were closely related, as were those of strain D, but strains A₂ and D were not. Thus, the two strains may have different origins and be carried through seed transmission.     

Genetic Structure and Variation in Aggressiveness in European and Australian Populations of the Grapevine Dieback Fungus, Eutypa lata

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.     

Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

Biological control of grapevine crown gall: purification and partial characterisation of an antibacterial substance produced by <Emphasis Type="Italic">Rahnella aquatilis</Emphasis> strain HX2

Strain HX2 of Rahnella aquatilis has been previously reported as a potential biological control agent (BCA) of grapevine crown gall. The production of an antibacterial substance (ABS) was suggested to be an important factor in the biocontrol process. This study was undertaken to determine the antibacterial properties and mode of action of ABS. Isolation and purification of ABS from culture broth of strain HX2 was achieved by methanol exaction, chromatography on macroporous resin and silica gel, and high-performance liquid chromatography (HPLC). Physical and chemical characteristic analysis revealed that ABS may be a thermostable and alkali-sensitive substance containing sugar(s) and an unknown 285-nm absorbed substance. Antibacterial activity assays revealed that ABS displayed a broad activity spectrum against all the 13 test isolates of phytopathogenic bacteria including the genera of Agrobacterium, Clavibacter, Pectobacterium, Pseudomonas, and Xanthomonas. Agrobacterium spp. strains were more sensitive to ABS than other tested strains, with larger inhibition zones and lower minimal inhibitory concentration (MIC). ABS exhibited a bactericidal effect against A. vitis both in vitro and in vivo. This compound did not cause bacterial cell lysis, as determined by morphological observation with an electron microscope. Also, no leakage of cytoplasmic materials from cells of A. vitis occurred after treatment with ABS at concentrations equivalent to the MIC. However, an inhibition of the incorporation of radiolabelled precursors into RNA and protein was observed after treatment with ABS. These results suggest that ABS inhibits RNA and protein syntheses in tumorigenic A. vitis.     

Differentiation of citrus bacterial canker strains in Korea by host range,rep-PCR fingerprinting and 16S rDNA analysis

Soil-borne cereal mosaic virus (SBCMV) causes a severe disease in susceptible cultivars of winter wheat. The virus is vectored by the soil-borne protist Polymyxa graminis. Experiments were conducted to investigate whether SBCMV RNA2 could persist in seed from SBCMV-infected susceptible cultivars of winter wheat. Over 7,000 seedlings were generated from seed collected from two cultivars of SBCMV-infected winter wheat. Seedlings were grown in a glasshouse compartment and batch tested for the presence of SBCMV using real-time RT-PCR. The majority of batches tested positive for SBCMV, indicating an RNA2 transmission rate of 1.8–9.4% in wheat. The presence of the virus was confirmed by amplifying and sequencing a larger (400 bp) fragment of viral RNA2 in a sub-set of the seedlings testing positive by real-time RT-PCR. Root extracts from this sub-set tested negative for P. graminis using real-time PCR. The implications for disease epidemiology of this virus are discussed. The authors are British Civil Servants and as such their work is subject to British Crown Copyright. This means the exclusive copyright for the article cannot be transferred.     

The Use of ARMS PCR in Detection and Identification of Xanthomonads Associated with Pistachio Dieback in Australia

Pistachio dieback occurs in the main pistachio growing areas of Australia. Xanthomonas strains belonging to the translucens group have been identified as the causal agent of the disease and two distinct groups, A and B, have been recognised within the pathogen population. In this study, specific primers for amplification of DNA of the pathogen were developed by sequencing the Internal Transcribed Spacer (ITS) region of rDNA from strains representing groups A and B, as well as from X. translucens isolated from wheat in Australia and one Xanthomonas translucens strain from orchard floor grasses. Primers were designed for amplification of DNA sequences specific to each group and a multiplex PCR test was developed that identified and differentiated strains of each group in a single PCR assay. To determine the specificity of the primers, PCR was carried out with DNA from 65 strains of the pistachio pathogen, 31 type and reference strains of Xanthomonas, and from 191 phytobacteria commonly found in and around pistachio orchards. In the multiplex PCR, a 331 bp fragment was amplified from all strains belonging to group A and a 120 bp fragment from all strains in group B. No PCR products were obtained from the other bacteria tested except for the type strain of X. translucens pv. cerealis, which has not been found in Australia. The assay was used to detect strains from both groups of the pathogen in pistachio plant material.     

Evaluation ofAmaranthus sp. andVernonia amygdalina, and soil amendments with poultry manure for the management of root-knot nematodes on eggplant

The suppressive effect of vernonia (Vernonia amygdalina), amaranth (Amarathus sp.) and poultry manure on root-knot nematodes (RKNs) (Meloidogyne spp.) infecting eggplant (Solanum macrocarpon) was studied at two sites in southern Benin naturally infested with these nematodes. After 3 months, soil and root-inhabiting RKN populations were significantly less (P≦0.05) in the plots cropped with vernonia, amaranth, and eggplant amended with poultry manure (PM) at the rate of 40 t ha⁻¹ as compared with the rate of 20 t ha⁻¹ and with the control. Poultry manure was more effective after 2 months than after 3 months. Overall, vernonia was the most effective treatment affecting RKN populations in the roots and the soil. The use of these treatments in nematode management through rotation and co-planted crops is discussed. http://www.phytoparasitica.org posting August 6, 2008.