Pantothenate auxotrophy of Methylobacterium spp. isolated from living plants

A number of pink-pigmented facultative methylotrophs (PPFMs) belonging to Methylobacterium spp. isolated from living plant samples were found to require B vitamins for their growth in minimal medium, and most B vitamin-auxotrophic PPFMs required pantothenate (vitamin B_⁵). Further investigation of pantothenate auxotrophy using the representative strain Methylobacterium sp. OR01 demonstrated that this strain cannot synthesize β-alanine, one of the precursors of pantothenate. β-alanine and several precursors of pantothenate restored the growth of Methylobacterium sp. OR01 in minimal medium. Furthermore, this strain could colonize leaves of Arabidopsis thaliana cultivated in medium without pantothenate or its precursors. Pantothenate, β-alanine and several precursors were detected in the suspension of A. thaliana leaves. These results suggest that pantothenate-auxotrophic PPFMs can symbiotically colonize the surface of plant leaves by acquiring β-alanine and other precursors, in addition to pantothenate. Finally, the fitness advantage of B vitamin auxotrophy of PPFMs in the phyllosphere environment is discussed.     

Distribution of pink-pigmented facultative methylotrophs on leaves of vegetables

The distribution of pink-pigmented facultative methylotrophs (PPFMs) on the leaves of various vegetables was studied. All kinds of vegetable leaves tested gave pink-pigmented colonies on agar plates containing methanol as sole carbon source. The numbers of PPFMs on the leaves, colony-forming units (CFU)/g of fresh leaves, differed among the plants, although they were planted and grown at the same farm. Commercial green perilla, Perilla frutescens viridis (Makino) Makino, gave the highest counts of PPFMs (2.0-4.1×10(7) CFU/g) of all the commercial vegetable leaves tested, amounting to 15% of total microbes on the leaves. The PPFMs isolated from seeds of two varieties of perilla, the red and green varieties, exhibited high sequence similarity as to the 16S rRNA gene to two different Methylobacterium species, M. fujisawaense DSM5686(T) and M. radiotolerans JCM2831(T) respectively, suggesting that there is specific interaction between perilla and the PPFMs.     

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria     

Prevention of pink-pigmented methylotrophic bacteria (Methylohacterium mesophilicum) contamination of plant tissue cultures

Pink-pigmented facultative methylotrophic bacteria (PPFMs) have been found on the surfaces of leaves of most plants tested. We found PPFMs on the leaf surfaces of all 40 plants (38 species) tested and on soybean pods by pressing onto AMS medium with methanol as the sole carbon source. The abundance ranged from 0.5 colony forming unit (cfu) /cm² to 69.4 cfu/cm² on the leaf surfaces. PPFMs were found in homogenized leaf tissues of only 4 of the species after surface disinfestation with 1.05% sodium hypochlorite and were rarely found in cultures initiated from surface disinfested Datura innoxia leaves or inside surface disinfested soybean pods. Of 20 antibiotics tested for PPFM growth inhibition, rifampicin was the most effective and of seven others which also inhibited PPFM growth, cefotaxime should be the most useful due to the expected low plant cell toxicity. These antibiotics could be used in concert with common surface sterilization procedures to prevent the introduction or to eliminate PPFM bacteria in tissue cultures. Thus, while PPFMs are present on the surfaces of most plant tissues, surface disinfestation alone can effectively remove them so that uncontaminated tissue cultures can be initiated in most cases.     

Methylobacterium bullatum sp. nov., a methylotrophic bacterium isolated from Funaria hygrometrica

A novel, pink-pigmented aerobic, facultatively methylotrophic bacterial strain (F3.2^T) isolated from the phyllosphere of Funaria hygrometrica, was analyzed using a polyphasic approach. Cells were Gram-negative, motile rods, strictly aerobic and non-spore-forming and exhibited surface structures varying in quantity, distribution and morphology. The isolate grew at 10–33 °C over a pH range of 5.5–8.0 and in the presence of less than 1.0% NaCl. Strain F3.2^T shared less than 70% DNA–DNA binding to the next type strain of the genus Methylobacterium (M. adhaesivum DSM 17169^T). In addition to the major cellular fatty acid C_18:1ω7c (81.7%), present in all Methylobacterium species (and also members of the genus Alphaproteobacteria), a high value (11.7%) of the fatty acids (summed feature) C_16:1ω7c and/or iso-C_15:02OH was determined. Phylogenetic analyses based on 16S rDNA and methanol dehydrogenase gene sequences, DNA–DNA hybridization values, chemotaxonomic and phenotypic characteristics indicate that the strain F3.2^T represents a novel species within the genus Methylobacterium. We propose the name Methylobacterium bullatum sp. nov. for this species. The type strain is the strain F3.2^T (DSM 21893^T = LMG 24788^T).     

Competitiveness of Diverse Methylobacterium Strains in the Phyllosphere of Arabidopsis thaliana and Identification of Representative Models, Including M. extorquens PA1

Facultative methylotrophic bacteria of the genus Methylobacterium are consistently found in association with plants, particularly in the phyllosphere. To gain a better understanding of the mechanisms underlying the dispersal and occurrence of Methylobacterium on plants, diverse strains were isolated, identified, and studied with regard to their competitiveness on the model plant Arabidopsis thaliana. As a basis for this study a comprehensive collection of Methylobacterium isolates was established. Isolates were obtained from five different naturally grown A. thaliana populations and diverse other plant genera at these and further sites. They were classified using automated ribosomal internal spacer analysis (ARISA) and a representative subset was identified based on 16S rRNA gene sequence analysis. A comparison of their ARISA patterns with those generated based on a cultivation-independent approach from the same sampling material confirmed that the isolates were abundant colonizers of the studied plants. In competition experiments, colonization efficiency of the strains was found to be linked to phylogeny, rather than to the geographical origin or plant genus from which they were isolated. The most competitive colonizers were related to the species Methylobacterium tardum and Methylobacterium extorquens. Higher cell numbers were observed in the phyllosphere of A. thaliana when a mixture of different strains was applied relative to inoculation with only one strain, suggesting partial niche heterogeneity. Based on the results of the competition experiments, representative strains with different colonization efficiencies were selected, which will serve as models in future studies aiming at a better understanding of plant colonization by this bacterial genus. Among them is the meanwhile genome-sequenced strain M. extorquens PA1, which represents a competitive species of plant colonizers with a broad dispersal. This strain was characterized in more detail including physiological, morphological, and chemotaxonomical properties.     

Methylobacterium Species Promoting Rice and Barley Growth and Interaction Specificity Revealed with Whole-Cell Matrix-Assisted Laser Desorption / Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) Analysis

Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant–microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization- time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion.     

Intergeneric conjugal transfer ofEscherichia coli/Methylobacterium sp. shuttle vector

To develop a host-vector system forMethylobacterium sp. using a construct based on a small indigenous methylotrophic plasmid, theE. coli —Methylobacterium sp. shuttle vector pWUBR (12.7 kb, Ap^r, Tc^r) was constructed by joining theE. coli plasmid pBR328 and the cryptic plasmid pWU7 (7.8 kb), isolated from the soil facultative methylotrophic bacterium,Methylobacterium sp. strain M17.Via mobilization by the pDPT51 R plasmid, belonging to the IncP-1 incompatibility group, plasmid pWUBR was transferred into the original host of cryptic plasmid pWU7, strain M17, where a competition between the introduced hybrid plasmid and the indigenous cryptic plasmid took place, and into the plasmidlessMethylobacterium sp. strain R2b. The stability of pWUBR in Tc^r methylotrophic transconjugants after 25 generations of growth under nonselective conditions was more than 90 % in both hosts. The ability to replicate in R2b strain demonstrates that the host spectrum of pWUBR is not restricted to the original host of pWU7 and indicates the possibility to use the present system for other methylotrophs.     

The abundance of pink-pigmented facultative methylotrophs in the root zone of plant species in invaded coastal sage scrub habitat

Pink-pigmented facultative methylotrophic bacteria (PPFMs) are associated with the roots, leaves and seeds of most terrestrial plants and utilize volatile C(1) compounds such as methanol generated by growing plants during cell division. PPFMs have been well studied in agricultural systems due to their importance in crop seed germination, yield, pathogen resistance and drought stress tolerance. In contrast, little is known about the PPFM abundance and diversity in natural ecosystems, let alone their interactions with non-crop species. Here we surveyed PPFM abundance in the root zone soil of 5 native and 5 invasive plant species along ten invasion gradients in Southern California coastal sage scrub habitat. PPFMs were present in every soil sample and ranged in abundance from 10(2) to 10(5) CFU/g dry soil. This abundance varied significantly among plant species. PPFM abundance was 50% higher in the root zones of annual or biennial species (many invasives) than perennial species (all natives). Further, PPFM abundance appears to be influenced by the plant community beyond the root zone; pure stands of either native or invasive species had 50% more PPFMs than mixed species stands. In sum, PPFM abundance in the root zone of coastal sage scrub plants is influenced by both the immediate and surrounding plant communities. The results also suggest that PPFMs are a good target for future work on plant-microorganism feedbacks in natural ecosystems.     

Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense

We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in the tissues of seeds treated with M. fujisawaense strains than in control seeds treated with MgSO₄. Though the increased activities of ACC synthase in the tissue extracts of the treated seedlings might be due to bacterial indole-3-acetic acid, the amount of ACC was reduced due to bacterial ACC deaminase activity. The activities of ACC oxidase, the enzyme catalyzing conversion of ACC to ethylene remained lower in M. fujisawaense treated seedlings. This consequently lowered the ethylene in plants and prevented ethylene inhibition of root elongation. Our results collectively suggest that Methylobacterium commonly found in soils, as well as on the surfaces of leaves, seeds, and in the rhizosphere of a wide variety of plants could be better exploited to promote plant growth.     

Oviposition and development response of perilla seed bugs (Nysius sp.) (Heteroptera: Lygaeidae) to five crop seeds

The perilla seed bugs, Nysius plebeius Distant and Nysius hidakai Nakatani, are emerging pests of perilla crop in Korea. Here, we investigated the life-history variables of N. plebeius and N. hidakai on five commonly grown crop seeds: perilla [Perilla frutescens (L.) Britt.], sesame (Sesamum indicum L.), sorghum [Sorghum bicolor (L.)], proso millet (Panicum miliaceum L.) and foxtail millet (Setaria italic L.). Bug oviposition, total developmental period, adult emergence, adult longevity and adult weight of both Nysius species were determined in both choice and no-choice tests. The oviposition and development periods of N. plebeius and N. hidakai varied significantly among seed species. The most preferred seed species for oviposition were perilla seeds, followed by sesame, sorghum and foxtail millet, whereas proso millet seeds were the least preferred. The bugs development period was faster in perilla seeds, with higher adult emergence, greater adult longevity and heavier adult weight. The higher oviposition preference and faster development with longer longevity of both Nysius species on perilla seeds is probably due to the physicochemical and nutritional attributes of the seeds. This information may enable the exploration of resistant genetic materials and chemical traits associated with seeds for breeding programme. Further, the outcomes of this study are discussed in terms of bug's behavioural response to crop seeds and management strategy of Nysius species as alternative to chemical management.     

The Ways of Plant Colonization by MethylobacteriumStrains and Properties of These Bacteria

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacteriumare indispensible inhabitants of the plant phyllosphere. Using maize Zea maysas a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicumAPR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicumAPR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicumcells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicumor M. extorquensdid exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas campestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense,andAgrobacterium tumifaciens). Neither epiphytic nor soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, andM. fujisawaensecatalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacteriumcan protect plants against adverse environmental factors.     

Structure and Colonization Dynamics of Epiphytic Bacterial Communities and of Selected Component Strains on Tomato (Lycopersicon esculentum) Leaves

The sizes and compositions of bacterial populations found on leaves of greenhouse and field grown tomato plants were studied by dilution plating, fatty acid methyl ester analysis (FAME), and BIOLOG plates of isolates in pure cultures. In the greenhouse, overhead-irrigated plants sustained higher microbial populations (up to 10⁵ cfu g⁻¹) than soil-irrigated plants (10³ cfu g⁻¹). Strains isolated from overhead-irrigated plants grown in a vegetable garden (n=216) and from greenhouse-grown plants (n=114) were subjected to FAME analysis. Similarly, strains from soil-irrigated field-grown plants (n=83) were identified using BIOLOG plates. In each case, populations were dominated by a few genera. When concentrated phyllosphere washes (CPW) were sprayed on greenhouse-grown, soil-irrigated plants, leaf bacterial populations of more than 10⁵ CFU g⁻¹ were sustained for 4 days; sterile buffer-sprayed leaves sustained less than 10⁴ CFU g⁻¹. No significant enrichment of any strain isolated from the sprayed leaves could be detected by FAME identification of randomly selected colonies. However, when recurring leaf saprophytic species (both Gram-positive and Gram-negative) isolated from these experiments and from plants grown outdoors were tested for epiphytic colonization under stressful conditions, all could still be detected at various levels up to 4 days after inoculation, indicating differential epiphytic fitness. The non-epiphytic bacteriaEscherichia coli andAzospirillum brasilense disappeared from the leaf surface within the same experimental period.     

Extensive host range of an endophytic fungus, <Emphasis Type="Italic">Guignardia endophyllicola</Emphasis> (anamorph: <Emphasis Type="Italic">Phyllosticta capitalensis</Emphasis>)

Isolation of endophytic species of Guignardia (anamorph: Phyllosticta) from healthy leaves of 94 plants (91 species and 3 varieties) in 69 genera, 42 families, was carried out in a test site (Kyoto Herbal Garden) to investigate the host range of Guignardia endophyllicola (anamorph: Phyllosticta capitalensis). Species of Guignardia and Phyllosticta were isolated from the leaves of 67 plants (66 species and 1 variety) belonging to 54 genera, 38 families. Among them, 53 isolates from different plants belonging to 43 genera in 36 families were similar in morphology, and sequence analysis of internal transcribed spacer (ITS) regions of ribosome DNA revealed these isolates to be conspecific with G. endophyllicola. In addition, this fungus was isolated from leaves of various plants collected in different places in Japan and Thailand. Thus, this endophytic fungus has been revealed to live within various vascular plants, angiosperms, gymnosperms, and pteridophytes.     

Competition between two similar plant varieties,green shrunk perilla and red shrunk perilla,in mixed cultures

Influences of plant density and time after seeding on the growth of two horticultural forms of perilla (Perilla frutescens var.crispa), green shrunk perilla (f.viridi-crispa) and red shrunk perilla (f.crispa), were examined in a mixed culture experiment. Relationships between mean individual plant weight and plant density in mixed populations were approximated by Ogawa's non-interaction type (NI-type) reciprocal equation. The density conversion factors in the equation for green and red perillas were always, respectively, smaller and larger than unity, suggesting that effects of a green perilla on the other individuals were always stronger than those of a red one in a mixed population. All coefficients in the NI-type reciprocal equation were expressed as functions of time after seeding. As a result, time trends of mean individual plant weights for both species in mixed populations could be reasonably estimated for different plant densities and mixed proportions. The results were also applied to Lotka-Volterra's equation. Time trends of Lotka-Volterra's competition coefficients for both plants could be calculated and were compared with those of density conversion factors.     

Induced Response of Tomato Plants to Injury by Green and Red Strains of Tetranychus Urticae

We studied the induced response of tomato plants to the green strain and the red strain of the spider mite Tetranychus urticae. We focused on the olfactory response of the predatory mite Phytoseiulus persimilis to volatiles from T. urticae-infested tomato leaves in a Y-tube olfactometer. Tomato leaves attracted the predatory mites when slightly infested with the red strain, or moderately or heavily infested with the green strain. In contrast, neither leaves that were slightly infested with green-strain mites, nor leaves that were moderately or heavily infested with the red strain attracted the predators. We discuss the specific defensive responses of tomato plants to each of the two strains.     

Endophytic bacterium,Bacillus amyloliquefaciens,enhances ornamental hosta resistance to diseases and insect pests

A total of 84 bacterial endophytes were isolated from seeds of 6 cultivars of ornamental hostas, and they were identified to 5 species based on morphological characteristics and 16S rDNA sequence analysis. Among them, the strain ‘Blu-v2’, which was isolated from the seeds of cultivar ‘Blue Umbrella’ and identified to be Bacillus amyloliquefaciens, showed highest antifungal activity and capacity to deter feeding by Fall armyworms (Spodoptera fruigiperda). Lipopeptides in cultures of Blu-v2 were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its antifungal activities were verified. However, the lipopeptide preparation did not show toxicity to larvae of Fall armyworms. In a greenhouse experiment, Blu-v2 was inoculated into small plantlets of hosta (cultivar ‘Rainforest Sunrise’). The leaves of plants with bacteria (endophyte-infected?=?E+) and without bacteria (endophyte-free?=?E?) were used in seven-day feeding experiments employing fourth-instar larvae of Fall armyworms. We found that there was a significant decrease in the weights of larvae fed with E+ compared to E? plants; and the mortality rate of larvae fed with E? leaves was lower (3.33%) compared to that of larvae fed with E+ leaves (30%). Based on our studies, we suggest that endophytic B. amyloliquefaciens strain Blu-v2 has potential value as a biocontrol agent to reduce damage from fungal diseases and insect pests of hosta cultivars.     

First report of seed dispersal by ants in Dicentra peregrina (Papaveraceae), an alpine plant in the Japanese Alps

Seed dispersal by ants, known as myrmecochory, is commonly observed among various plant taxa. The seeds of these plants have an elaiosome to attract ants. In Japan, myrmecochory has been well studied in several lowland plant species, but not in highland plant species that grow above the tree line. We investigated whether the seeds of Dicentra peregrina, known as the “queen of the alpine plants” in Japan, are carried by Formica gagatoides ants at 2510 m a.s.l. on Mt Norikura, Kita‐Alps, Japan. We observed F. gagatoides workers picking up D. peregrina seeds by grasping the elaiosome and carrying the seeds into their nests. We inferred from the observed ant behavior and the seed morphology that D. peregrina is a myrmecochorous species.     

Changes in the population of seed bacteria of transgenerationally Cd‐exposed Arabidopsis thaliana

Plant‐associated bacteria can have beneficial effects on the growth and health of their host. Nevertheless, the role of endophytic bacteria present in seeds has not been investigated in depth. In this study, the cultivable endophytic population of seeds from Arabidopsis thaliana exposed to 2 μm cadmium for several generations (Cd seeds) was compared with a population isolated from seeds of plants that were never exposed to Cd (control seeds). We observed obvious differences between the two types of seed concerning genera present and phenotypic characteristics of the different isolates. Sinorhizobium sp. and Micrococcus sp. were only found in control seeds, while Pseudomonas sp., Bosea sp. and Paenibacillus sp. were only found in Cd seeds. Sphingomonas sp., Rhizobium sp., Acidovorax sp., Variovorax sp., Methylobacterium sp., Bacillus sp. and Staphylococcus sp. occurred in varying numbers in both types of seed. Metal tolerance and 1‐aminocyclopropane‐1‐carboxylate deaminase activity were predominantly found in strains isolated from Cd seeds, while the production of siderophores, indole‐3‐acetic acid and organic acids was more prevalent in endophytes isolated from control seeds. These data support the hypothesis that certain endophytes are selected for transfer to the next generation and that their presence might be important for subsequent germination and early seedling development.