Classification of plant associated bacteria using RIF, a computationally derived DNA marker

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.     

Partial Proteome of the Corynetoxin‐Producing Gram‐Positive Bacterium,Rathayibacter toxicus

Rathayibacter toxicus is a Gram‐positive bacterium that is the causative agent of annual ryegrass toxicity (ARGT), a disease that causes devastating losses in the Australian livestock industry. R. toxicus exhibits a complex life cycle, using the nematode Anguina funesta as a physical vector to carry it up to the seed head of the host plant. ARGT is caused by a tunicamycin‐like corynetoxin that is produced in R. toxicus‐infected seed galls. We analyzed protein expression in R. toxicus under stationary growth phase conditions to obtain a more complete understanding of the biology of this organism and identify potential targets for immunoassay development. A total of 323 unique proteins were identified, including those with putative roles in secondary metabolism and pathogenicity. The proteome analysis for this complex phytopathogenic Gram‐positive bacterium will facilitate in the characterization of proteins necessary for host colonization and toxin production, and assist in the development of diagnostic assays. Data are available via ProteomeXchange with identifier PXD004238.     

Grass species and their fungal symbionts affect subsequent forage growth

Persistence of forage grasses is enhanced through the deliberate and selective use of symbiotic fungal endophytes that confer benefits, particularly pest resistance. However, they have also been implicated in reduced plant community diversity as a result of directly or indirectly enhancing competitive ability. A relatively underexplored mechanism by which endophytes might influence pasture plant composition is by altering the biotic or abiotic soil conditions. To examine the soil conditioning effects of forage grass species and their fungal symbionts we tested the responses of three pasture plants, perennial ryegrass, prairie grass, and white clover in nine different soils that had been conditioned by monocultures of endophyte-containing (E+), or endophyte-free (E?), perennial ryegrass, tall fescue, or meadow fescue. Conditioning grass species had little effect on the responses of perennial ryegrass and prairie grass regardless of E+ or E? treatments. In contrast, conditioning species had a strong effect on the response of white clover, resulting in reduced biomass when grown in perennial ryegrass conditioned soils. The presence of endophyte also had significant growth consequences for white clover, but was either positive or negative depending on the conditioning grass species. In comparison to their respective E? treatments, E+ tall and meadow fescue conditioned soils resulted in reduced biomass of white clover, whereas E+ perennial ryegrass conditioned soils resulted in increased biomass of white clover. Among the conditioning strains (AR1, AR37, NEA2, WE) of E+ perennial ryegrass, white clover showed significantly different responses, but all responses were positive in comparison to the E? treatment. By examining the effects of several grass species and endophyte strains, we were able to determine the relative importance of grass species vs. fungal symbiont on soil conditioning. Overall, the conditioning effect of grass species was stronger than the effects associated with endophyte, particularly with regard to the response of white clover. We conclude that both grass species and their fungal endophytes can influence pasture plant community composition through plant–soil feedback.     

白星花金龟成虫肠道细菌的分离鉴定

目的自星花金龟是我国北方常见腐食性昆虫,为了利用其腐食性行为转化处理有机垃圾、畜禽粪便及农作物秸杆等农业有机废弃物资源,并探索其中肠消化的微生态机制,而对其肠道细菌进行系统研究。方法从白星花金龟成虫肠道中无菌操作分离获得5株细菌,对其菌体形态、培养性状、染色反应、生理生化反应等进行鉴定及数量测定。结果白星花金龟成虫肠道的5个细菌菌株分别属于气球菌属（Aerococcus）、短状杆菌属（Brachybacterium）、棍状杆菌属（Clavibacter）、李斯特菌属（Listeria）、皮杆菌属（Dermabacter）,以皮杆菌属和棍状杆菌属数量最多,分别为1．3×10^7和1．0×10^7;其次为短状杆菌属和气球菌属,分别为8×10^6和5×10^6;李斯特菌属数量最少,为7×10^5。结论各菌株之间细菌数量存在明显差异,以皮杆菌属细菌数量最多,分离未获得真菌和放线菌。     

Determination of genetic differences between fluid and nonfluid variants of<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp.<Emphasis Type="Italic">sepedonicus</Emphasis> using rep-PCR technique

Testing of 23 isolates of Clavibacter michiganensis subsp. sepedonicus for analysis by rep-PCR (using BOX, ERIC, REP primer sets) was used for the purpose of localization of genetic markers for fluid and/or nonfluid strains. None of the primer sets was successful in detecting genetic differences between the isolates and no polymorphism was generated.     

Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus

Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.     

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.     

Purification and characterization of a collagenolytic enzyme produced by Rathayibacter sp. strains isolated from cultures of Clavibacter michiganensis subsp. michiganensis

We show in this work that collagenolytic Rathayibacter sp. are isolated with phytopathogenic Clavibacter michiganensis subsp. michiganensis strains. The Rathayibacter strains isolated all produced collagenases. One of these collagenases (from the strain 1715) was purified by ammonium sulphate precipitation, DEAE cellulose and Sephadex G 200 chromatography. Characterization of the enzyme showed that it is a true collagenase which is able to degrade both native collagen, gelatin and probably other proteins from plants sharing sequence homologies with collagen.     

番茄茎内生细菌的分离鉴定及青枯病拮抗菌的筛选

采用化学法进行表面灭菌处理,运用平板涂布法及平板划线法从番茄茎内得到17株内生细菌.通过形态观察和生理生化指标鉴定,17株内生细菌分属于6个属,即葡萄球菌属(Staphylococcus)、短芽孢杆菌属(Brevi-bacillus)、芽孢杆菌属(Bacillus)、棍状杆菌属(Clavibacter)、欧文氏菌属(Erwinia)和乳杆菌属(Lactobacillus).采用滤纸片法从17株内生菌中筛选出1株对青枯菌(Pseudomonas solanacearum)有拮抗作用的菌株,编号为TS-06,属于芽孢杆菌属(Bacillus).其抑菌圈半径为2.5 mm.     

黄粉虫不同虫态肠道细菌分离及鉴定

目的研究黄粉虫肠道细菌的类群与其取食的关系,为进一步开发利用提供理论依据。方法从人工饲养的黄粉虫幼虫、蛹和成虫肠道环境中分离纯化获得9个细菌菌株,对其菌体形态、染色反应、培养性状、生理生化反应进行系统研究。结果研究结果表明,上述9个细菌菌株分别属于放线杆菌属（Actunobacillus）、丙酸杆菌属（Propionibacterium）、柠檬酸杆菌属（Citrobacter）、沙雷菌属（Serratia）、芽胞杆菌属（Bacillus）、皮杆菌属（Dermabacter）、短状杆菌属（Brachybacterium）、棍状杆菌属（Clavibacter）和微小杆菌属（Exiguobcacterium）。结论通过对黄粉虫不同虫态即幼虫、蛹和成虫肠道细菌分离,其细菌种类存在一定差别。     

Development of a PCR test for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens

A chromosomal DNA library of the bacterial pathogen of bean, Curtobacterium flaccumfaciens pv. flaccumfaciens NCPPB 559 was constructed in the plasmid pGEM-7Zf(+). Several clones were identified that hybridised to all Curtobacterium flaccumfaciens pathovars including: C. f. betae, C. f. flaccumfaciens, C. f. oortii, C. f. poinsettiae and, in addition, to some strains of Clavibacter michiganensis subsp. insidiosus and Clavibacter michiganensis subsp. One of these clones (pPMP-26), after subsequent digestion with restriction endonucleases EcoRI/SacI, yielded a fragment of approximately 0.2 Kb (pPMP-26D) that hybridised specifically to C. f. flaccumfaciens and not to any of the other plant pathogenic members of the order Actinomycetales or any of the other prokaryotic bean pathogens tested. This fragment was subcloned and sequenced, analysis of the resultant 198 bp sequence showed that no significant homology existed with any other sequence currently deposited in public databases. Further analysis of these data facilitated the design of PCR primers which were subsequently tested against a wide range of plant pathogenic actinomycetes and other prokaryotic bean pathogens. Results show that these primers are highly specific for all strains of C. f. flaccumfaciens with no cross-reaction to strains from any other bacterial taxa tested.     

Isolate-dependent impacts of fungal endophytes in a multitrophic interaction

Neotyphodium lolii , an endophytic fungus of perennial ryegrass, deters Argentine stem weevil, Listronotus bonariensis , an important insect pest of pastures in New Zealand. Deterrence is apparently due to several alkaloids the fungus produces. We asked if the fungus also affects the third trophic level. Specifically, we tested if several different isolates of the fungus altered the growth and survival of the parasitoid, Microctonus hyperodae . Adult weevils were collected from paddocks near Lincoln, New Zealand and maintained in an environmental chamber. Weevils were assorted into treatment groups and fed perennial ryegrass (cv. Nui) lacking endophyte infection (nil) or containing one of the following endophyte strains: ARW, AR1, AR6, AR37. All endophyte strains differed from one another with respect to the profile of alkaloids they produce. Following two weeks of acclimation, weevils were placed with M. hyperodae to allow for parasitization. Weevils were then placed into Petri dishes (10 per dish) and fed grass clippings from the appropriate treatment group. We collected weevils as they died and dissected them to assess the development of parasitoids. Emergence of prepupae from hosts and survival to adulthood were also recorded. Fungal isolates did not differ in their influence on weevil feeding or survival. In contrast, the effect of the endophyte on the parasitoid varied among isolates of the fungus. Isolates ARW and AR6 reduced parasitoid adult emergence relative to nil endophyte. In contrast, AR37 had no negative effect on survival of the parasitoid. Furthermore, an index of developmental rate showed that parasitoids developed more slowly when reared from hosts fed grass containing any of the strains of N. lolii , except AR37, compared to endophyte-free grass. Negative effects of the endophyte on parasitoid survival were associated with the presence of ergovaline while effects on parasitoid development rate were associated with the presence of any alkaloid.     

Diversity of 746 heterotrophic bacteria isolated from microbial mats from ten Antarctic lakes

Microbial mats, growing in Antarctic lakes constitute unique and very diverse habitats. In these mats microorganisms are confronted with extreme life conditions. We isolated 746 bacterial strains from mats collected from ten lakes in the Dry Valleys (lakes Hoare and Fryxell), the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts) and the Larsemann Hills (lake Reid), using heterotrophic growth conditions. These strains were investigated by fatty acid analysis, and by numerical analysis, 41 clusters, containing 2 to 77 strains, could be delineated, whereas 31 strains formed single branches. Several fatty acid groups consisted of strains from different lakes from the same region, or from different regions. The 16S rRNA genes from 40 strains, representing 35 different fatty acid groups were sequenced. The strains belonged to the alpha, beta and gamma subclasses of the Proteobacteria, the high and low percent G+C Gram-positives, and to the Cytophaga-Flavobacterium-Bacteroides branch. For strains representing 16 fatty acid clusters, validly named nearest phylogenetic neighbours showed pairwise sequence similarities of less than 97%. This indicates that the clusters they represent, belong to taxa that have not been sequenced yet or as yet unnamed new taxa, related to Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavobacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomonas, Saligentibacter, Sphingomonas and Sulfitobacter.     

Genetic diversity and population structure of Clavibacter michiganensis subsp. nebraskensis

Clavibacter michiganensis subsp. nebraskensis (CMN) is a gram-positive bacterium and an incitant of Goss's bacterial wilt and leaf blight or "leaf freckles" in corn. A population structure of a wide temporal and geographic collection of CMN strains (n = 131), originating between 1969 and 2009, was determined using amplified fragment length polymorphism (AFLP) analysis and repetitive DNA sequence-based BOX-PCR. Analysis of the composite data set of AFLP and BOX-PCR fingerprints revealed two groups with a 60% cutoff similarity: a major group A (n = 118 strains) and a minor group B (n = 13 strains). The clustering in both groups was not correlated with strain pathogenicity. Group A contained two clusters, A1 (n = 78) and A2 (n = 40), with a linkage of 75%. Group A strains did not show any correlation with historical, geographical, morphological, or physiological properties of the strains. Group B was very heterogeneous and eight out of nine clusters were represented by a single strain. The mean similarity between clusters in group B varied from 13% to 63%. All strains in group B were isolated after 1999. The percentage of group B strains among all strains isolated after 1999 (n = 69) was 18.8%. Implications of the findings are discussed.     

Comparison of genetic variability between Czech and foreign isolates of phytopathogenic bacteria Clavibacter michiganensis subsp. sepedonicus by Rep-PCR technique

Repetitive-sequence-based polymerase chain reaction (Rep-PCR) method was used for analysis of genetic variability among bacterial populations from different world locations. Collection of 26 Czech and 13 foreign strains ofClavibacter michiganensis subsp.sepedonicus was amplified using BOX primer targeting to repetitive motif occurring in eubacterial genomes. Genetic fingerprints were visually compared and statistically evaluated by cluster analysis. Genetic similarity was estimated to be approximately 80% among all tested strains. Populations of these bacteria seem to be highly homogeneous; potential influence of geographic origin was not confirmed.     

Prevalence of ergot derivatives in natural ryegrass pastures: detection and pathogenicity in the horse

In the present study, we determined the incidence and effects of season and weather on clinical manifestations of endophyte-infected ryegrass toxicity, performed chemical detection and pharmacological bioassays on ryegrass extracts, and conducted trials on: (i) effects of domperidone or metochlopramide on ovarian inactivity induced by endophyte-infected ryegrass; (ii) efficacy of buspirone or dihydrochloro phenyl piperazine (m-CPP) for preventing suppressed milk production induced by endophyte-infected ryegrass; and (iii) efficacy of domperidone to induce ovulation during winter anestrus. Mares with toxicosis had prolonged gestation, embryonic losses, dystocia, poor mammary gland development, low milk production, prolonged uterine involution, and suppressed ovarian activity. Foals had respiratory failure, abnormalities of the skin, umbilicus, bone, and muscle, failure to thrive, blindness, testicular atrophy, and decreased serum total immunoglobulin concentrations. Endophyte-infected ryegrass and the incidence of toxicosis were correlated (r=0.861, P=0.03). Ergot alkaloids were not detected in extracts of endophyte-infected ryegrass by either thin-layer chromatography or spectrophotometry, but their presence was inferred in bioassays of extracts (dose-related increases in the contractile response of rat uterus). Mares given metoclopropamide (0.6 mg/kg/d), given orally every 8h for up to 7d) ovulated earlier (4-7d vs. 15-18d, P<0.001) than those given domperidone (1.1mg/kg/d) orally for up to 18d). Although both metoclopropamide and domperidone induced milk production, the latter did not induce ovarian cyclicity in healthy mares during seasonal anestrus. Based on these findings, we inferred that endophyte-infected ryegrass is associated with ergot alkaloid intoxication in horse.     

Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus

A lateral flow immunoassay for the rapid detection of Clavibacter michiganensis subsp. sepedonicus bacteria causing potato ring rot was developed. Multimembrane composites (test strips) containing polyclonal antibodies against the bacteria and gold nanoparticle-antibody conjugates were used for the analysis. The test strips are suitable for the analysis of potato tuber and leaf extracts within 10 min; the detection limit of bacteria is 4 × 10⁵ cells/mL. No cross-reactivity with strains of Clavibacter michiganensis subsp. michiganensis, Pectobacterium carotovorum subsp. carotovorum and saprophytes of healthy potato plants was detected. The results of analysis of 26 potato samples by the developed tests were compared with those obtained by the PCR method and using the commercial enzyme immunoassay kits. The results of lateral flow immunoassay were confirmed in 96.2% of cases, which supports the high correlation with other analytical approaches. The developed immunoassay may be considered as a promising means of phytosanitary control.     

Reproductive barriers between populations of the cereal rust mite <Emphasis Type="Italic">Abacarus hystrix</Emphasis> confirm their host specialization

Recent studies have shown that certain host populations of the cereal rust mite Abacarus hystrix are highly specialized in their host use and it is likely that reproductive isolation exists between them. Here I verified this expectation by testing for reproductive barriers between ryegrass and quackgrass populations of A. hystrix. I performed reciprocal crosses between individuals from both populations and observed results of crosses. Leaves of the three grass species, ryegrass, quackgrass and wheat, were used as mating arenas. I used two criteria to determine reproductive barriers between strains: the proportion of female progeny and viability of progeny. If studied populations of this haplodiploid species are reproductively isolated a male-biased sex ratio and/or hybrid progeny of reduced viability would be expected. I found that in the presence of quackgrass and ryegrass pre-zygotic barriers between studied populations exists. On wheat I observed asymmetry in reproductive barriers. Between females from quackgrass and males from ryegrass a pre-zygotic barrier existed (only males obtained). However, the opposite reciprocal cross (females from ryegrass and males from quackgrass) produced progeny of both sexes. A male-biased sex ratio and low adult emergence suggests that post-zygotic mechanisms acted here. Low viability of progeny obtained from crosses in which females from ryegrass were engaged suggests that the origin of the female nymph acted as a predictor of hybrid inviability. The pattern of sterility observed in the cereal rust mite indicates that in the presence of three hosts (ryegrass, quackgrass and wheat) pre- and post-zygotic reproductive barriers between quackgrass and ryegrass populations of this mite exist. In addition to host fidelity (which acts as pre-zygotic barrier) there are post-zygotic barriers to gene flow.     

Studies on siderophore exchange properties between staphylococci and various species of gram-positive and gram-negative bacteria

The ability of iron utilizing by means of staphylococcal siderophores by bacteria belonging to genera: Acinetobacter, Corynebacterium, Curtobacterium, Clavibacter, Bacillus and Mycobacterium was investigated. The staphylococcal donor strains (18 species) used in these experiments were characterized by the ability to utilize siderophores produced by various strains belonging to aforenamed genera. The utilization of staphylococcal siderophores was studied on agar media in which minimally effective concentrations of ethylenediaminedi-ortho-hydroxyphenylacetic acid (EDDA) were used to inhibit indicator strains. Test colonies (staphylococcal) were applied to the surface of the media to determine whether the indicator organisms could obtain the required iron for growth by utilizing chelators from the test colony. The growth inhibition by EDDA of most strains from the Acinetobacter rods and from the coryneform-organisms (plant pathogen) genera, and strains from the species: B. subtilis, M. phlei, M. smegmatis, M. fortuitum was reversed by staphylococcal siderophores. None of the staphylococcal strains investigated, had the ability to exchange siderophores with strains from the species: C. pseudodiphtheriticum, Corynebacterium ANF group, B. megaterium, M. vaccae, M. chitae and M. parafortuitum.     

Selection of perennial and Italian ryegrass plants resistant to ryegrass mosaic virus

Perennial ryegrass plants collected from fields and Italian ryegrass plants grown from seed were selected for resistance to infection by ryegrass mosaic virus (RMV) by repeated manual inoculation. Two of 108 perennial ryegrass plants and one of 150 Italian ryegrass plants were symptomless after seven and nine inoculations respectively. These three plants were propagated vegetatively. Plants of the two perennial ryegrass clones showed no symptoms after further manual inoculations with the initial isolate of RMV, or with an inoculum from infected plants collected from several fields, or after inoculation by viruliferous mites. Electron microscopy and back tests indicated that the plants were virus free. Some plants of the selected Italian ryegrass clone became infected after a further inoculation with mites or sap, but fewer than similarly inoculated unselected plants.