<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Characterization of fertile amphidiploid between <Emphasis Type="Italic">Raphanus sativus</Emphasis> and <Emphasis Type="Italic">Brassica alboglabra</Emphasis> and the crossability with <Emphasis Type="Italic">Brassica</Emphasis> species

A fertile amphidiploid × Brassicoraphanus (RRCC, 2n = 36) between Raphanus sativus cv. HQ-04 (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) was synthesized and successive selections for seed fertility were made from F₄ to F₁₀. F₁₀ plants exhibited good fertility with 14.9 seeds per siliqua and 32.3 g seeds per plant. Cytological observation revealed that frequent secondary pairing occurred among 3 chromosome pairs in pollen mother cells of plants (F₄) with lower fertility, but not of plants with high fertility (F₁₀). GISH analysis indicated that these F₁₀ plants included the expected 18 chromosomes from R. sativus and B. alboglabra, respectively, but they lost approximately 27.6% R. sativus and 35.6% B. alboglabra AFLP (amplified fragment length polymorphism) bands. The crossability of the Raphanobrassica with R. sativus and 5 Brassica species (13 cultivars) were investigated. Seeds or F₁ seedlings were easy to be produced from crosses × Brassicoraphanus × R. sativus, and B. napus, B. juncea and B. carinata × Brassicoraphanus. Fewer seeds or seedlings were obtained from crosses × Brassicoraphanus × B. napus, B. juncea and B. carinata. However, few seeds were harvested in the reciprocals of × Brassicoraphanus with B. rapa and B. oleracea. The possible cause of fertility improvements and the potential of the present × Brassicoraphanus for breeding were discussed.     

Responses of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> to exogenously added sinapic acid in vitro

To assess the influence of phenolic acids from plant root exudates on soil pathogens, we studied the effect of sinapic acid added to chemically defined media on the growth and virulence factors of Fusarium oxysporum f. sp. niveum. Sinapic acid inhibited the growth and conidial formation and germination of F. oxysporum f. sp. niveum by 6.7–8.8% and 11.2–37.3%, respectively. Mycotoxin production by F. oxysporum f. sp. niveum was stimulated by 81.6–230.7%. Pectinase, proteinase, cellulase, and amylase activities were stimulated at a lower concentration of sinapic acid, while they were inhibited at a higher concentration. It is concluded that sinapic acid inhibited the growth and conidial germination of F. oxysporum f. sp. niveum and decreased the pathogenic enzymes’ activity at higher doses.     

A significant proportion of indigenous rhizobia from India associated with soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) distinctly belong to <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Ensifer</Emphasis> genera

The diversity among 269 rhizobia isolated from naturally occurring root nodules of soybean collected from two different agro-ecological regions of India, based on RFLP and sequences of the intergenic spacer (IGS) between the 16S and 23S rRNA genes, growth rate, and indole acetic acid production, revealed their significant, site-dependent genomic diversity. Among these bacteria, nine IGS genotypes were identified with two endonucleases. They were distributed into five divergent lineages by sequence analysis of each IGS representative strain, i.e., (1) comprising IGS genotypes I, II, III, and reference Bradyrhizobium yuanmingense; (2) with genotype IV and strains of unclassified bradyrhizobia genomic species; (3) including genotypes V, VI, and Bradyrhizobium liaoningense; (4) with IGS genotype VII and Bradyrhizobium elkanii strains; and (5) comprising IGS genotypes VIII, IX, and different Ensifer genus bacteria. Host-specificity test revealed that all rhizobia-nodulated soybean and cowpea and only part of them formed nodules on Arachis hypogeae and Cajanus cajan. The great diversity of soybean nodulators observed in this study emphasises that Indian soil is an important reservoir of nitrogen-fixing rhizobia.     

Inoculation of soil by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Y-IVI improves plant growth and colonization of the rhizosphere and interior tissues of muskmelon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Laboratory tests and greenhouse experiments were carried out to investigate the abilities of Bacillus subtilis Y-IVI to promote plant growth and to colonize the rhizosphere and interior tissues of muskmelon. Laboratory tests showed that B. subtilis Y-IVI can produce indole acetic acid, siderophores, and ammonia. The inoculation of soil with green fluorescent protein-tagged Y-IVI (GY-IVI) significantly increased plant shoot and root dry weights as compared with the non-inoculated soils. The inoculation of soil with B. subtilis GY-IVI maintained approximately 10⁸ colony-forming units (cfu) of GY-IVI per gram of dry rhizosphere soil for 1 month. The GY-IVI recovered from the interior of crowns and roots in the inoculated soil were 10⁶ and 10⁷ cfu g⁻¹ dry weight, respectively, suggesting that GY-IVI acted as an endophyte. In the present study, we combined the two important growth promotion ingredients, colonization ability and growth promotion metabolites produced by biological agents, to investigate B. subtilis Y-IVI’s promotion effects on muskmelon growth.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> SQR 9 can control <Emphasis Type="Italic">Fusarium</Emphasis> wilt in cucumber by colonizing plant roots

Fusarium wilt is one of the major constraints on cucumber production worldwide. Several strategies have been used to control the causative pathogen, Fusarium oxysporum f. sp. cucumerinum J. H. Owen, including soil solarization, fungicide seed treatment and biological control. In this study, F. oxysporum f. sp. cucumerinum was successfully controlled by a newly isolated strain, Bacillus subtilis SQR 9, in vitro and in vivo. Greenhouse experiments were carried out to evaluate the effect of inoculation and solid fermentation of organic fertilizer with B. subtilis SQR 9, hereby defined as bio-organic fertilizer (BIO), on the control of Fusarium wilt. In comparison with the control, the wilt incidence was significantly reduced (49–61% reduction) by application of BIO. The rhizosphere population of F. oxysporum f. sp. cucumerinum, as detected both by selective plating and realtime PCR, was significantly lower in BIO-treated plants than the control. The localization of bacterial cells, pattern of colonization and survival of B. subtilis SQR 9 in the rhizsosphere of cucumber, was examined by fluorescent microscopy and explored following recovery of the green fluorescent protein (gfp)-labeled SQR 9 with the new gfp-marked shuttle vector pHAPII through selective plating. The preferential sites of the labeled strain were the differentiation and elongation zone, root hair and the lateral root junctions. The population of the strain was 10⁶ cfu/g root in rhizoplane. These results indicate that the strain was able to survive well in the rhizosphere of cucumber, suppressed growth of F. oxysporum in the rhizosphere of cucumber and protected the host from the pathogen.     

Effect of the arbuscular mycorrhizal fungus <Emphasis Type="BoldItalic">Glomus intraradices</Emphasis> on the false root-knot nematode <Emphasis Type="BoldItalic">Nacobbus aberrans</Emphasis> in tomato plants

Information about the interaction between arbuscular mycorrhizal fungi (AMF) and the false root-knot nematode Nacobbus aberrans (Thorne, 1935) Thorne & Allen, 1944 is scarce. The effect of Glomus intraradices Schenk & Smith on tomato (Lycopersicon esculentum L.) cv. Platense inoculated with nematode juveniles from Lisandro Olmos (Argentina) was studied under greenhouse conditions. Six treatments with five replications were performed. After 80 days, nematode reproduction and percentage of AMF colonization in roots were estimated. Some plant growth parameters were also measured. In general, plants with AMF and AMF plus nematodes grew as well as the control without AMF and without nematodes. Furthermore, G. intraradices was beneficial in reducing nematode-induced damage in roots (lower number of galls) as well as in having a suppressive effect on parasite reproduction. This is the first study on the use of G. intraradices as a possible strategy in the control of N. aberrans in tomato.     

Enhanced dissipation of chrysene in planted soil: the impact of a rhizobial inoculum

Results from an innovative approach to improve remediation in the rhizosphere by encouraging healthy plant growth and thus enhancing microbial activity are reported. Mixed grass-legume systems, together with microbial inoculants, were used to remediate a polycyclic aromatic hydrocarbon (chrysene) spiked agricultural soil. Inoculants were symbiotic rhizobia, which may play an important role in rhizoremediation by increasing plant and root growth. An inoculum of an isolate of Rhizobium leguminosarum bv. trifolii, selected for PAH tolerance, was produced using a peat carrier. The inoculum and white clover (Trifolium repens L.), were planted into soils with ryegrass (Lolium perenne L.). The soils spiked with chrysene (500 mg kg⁻¹) then aged for 4 weeks. Shoot- and root-biomass of plants, and the amount of root nodulation, were determined. Rhizobial populations, soil pH and soil nitrogen were also monitored throughout the trial. In addition, the ability of the inoculated rhizobial strain to utilise chrysene as a sole carbon source was assessed. Direct uptake and/or degradation of chrysene by the clover and ryegrass did not occur to a significant degree. Enhanced losses of chrysene were seen in planted, non-sterile soils that contained a rhizobial inoculum. No direct degradation of chrysene by R. leguminosarum bv. trifolii was observed and no enhanced losses of PAHs were detected in sterile soils after inoculation with rhizobia. Results suggest that the enhanced dissipation of chrysene, observed in the non-sterile planted inoculated pots, was not a result of degradation of chrysene by R. leguminosarum bv. trifolii. The symbiotic association with R. leguminosarum bv. trifolii improved plant vigour and growth in inoculated planted treatments. This may have stimulated the rhizospheric microflora to degrade chrysene.     

Identification of <Emphasis Type="Italic">SUB1A</Emphasis> alleles from wild rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.

Submergence stress is a major constraint to rice production in South and Southeast Asia. Most rice (Oryza sativa L.) cultivars die within a week of complete submergence, while a small number of accessions are submergence-tolerant for up to 2 weeks or more. These cultivars have the tolerant allele of the SUB1A gene, one of three ERF genes at this locus on rice chromosome 9. In all O. sativa varieties studied, the SUB1A gene is limited to a subset of indica accessions of O. sativa. Thus far, there has been no published report of the SUB1A gene in wild rice species. Here we report evidence of the SUB1A gene found in wild species of O. rufipogon Griff. accessions by the use of degenerate primers corresponding to the most highly conserved regions of the SUB1 locus. The results indicated that two SUB1A-like alleles, e.g. OrSub1A-1 and OrSub1A-2, were identified from two O. rufipogon accessions. Submergence treatment shows that both of the accessions with SUB1A-like genes were submergence-intolerant. This preliminary study provides insight into the origin and allelic variation of SUB1A, an agronomically important gene that is rapidly being introduced into widely-grown rice cultivars.     

Mitigation of salt stress in wheat seedlings by a <Emphasis Type="Italic">gfp</Emphasis>-tagged <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis>

Root colonization and mitigation of NaCl stress on wheat seedlings were studied by inoculating seeds with Azospirillum lipoferum JA4ngfp15 tagged with the green fluorescent protein gene (gfp). Colonization of wheat roots under 80 and 160 mM NaCl stress was similar to root colonization with this bacterial species under non-saline conditions, that is, single cells and small aggregates were mainly located in the root hair zone. These salt concentrations had significant inhibitory effects on development of seedlings, but not on growth in culture of gfp-A. lipoferum JA4ngfp15. Reduced plant growth (height and dry weight of leaves and roots) under continuous irrigation with 160 mM NaCl was ameliorated by bacterial inoculation with gfp-A. lipoferum JA4ngfp15. Inoculation of plants subjected to continuous irrigation with 80 mM NaCl or to a single application of either NaCl concentration (80 or 160 mM NaCl) did not mitigate salt stress. This study indicates that, under high NaCl concentration, inoculation with modified A. lipoferum reduced the deleterious effects of NaCl; colonization patterns on roots were unaffected and the genetic marker did not induce undesirable effects on the interaction between the bacterium and the plants.     

The flavonoid naringenin enhances intercellular colonization of rice roots by<Emphasis Type="Italic"> Azorhizobium caulinodans</Emphasis>

The intercellular colonization of rice roots by Azorhizobium caulinodans and other diazotrophic bacteria has been studied using strains marked with the lacZ reporter gene. A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and this was observed by light microscopy. After colonization of lateral roots, bacteria moved into intercellular space within the cortical cell layer of roots. Naringenin at 1×10^-5 and 5×10^-5 M concentration significantly enhanced root colonization. The role of nodABC and regulatory nodD genes was also studied; lateral root crack (LRC) colonization of rice was shown to be Nod factor and NodD independent. Lateral root crack colonization of rice was also observed with similar frequency following inoculation with Azospirillum brasilense and the colonization by A. brasilense was stimulated by naringenin and other flavonoid molecules.     

Characterization of the gibberellic acid response of the <Emphasis Type="Italic">Brassica napus</Emphasis> L. em. Metzg. dwarf mutant <Emphasis Type="Italic">NDF</Emphasis>-<Emphasis Type="Italic">1</Emphasis>

A novel dwarf mutant of Brassica napus L. em. Metzg., named NDF-1, was derived from a high doubled haploid line ‘3529’ of which seeds were jointly treated with chemical inducers and fast neutron bombardment. The germination results showed that the germination of NDF-1 was insensitive in response to exogenous gibberellic acid 3 (GA₃). The studies on growth response to exogenous GA₃ showed that NDF-1 seeding has at least 10-fold insensitivity than the wild-type. Moreover, no matter what concentrations of GA₃ were added to the seedlings and adult plants, the NDF-1 could not restore the wild type phenotype. These results indicated that the B. napus dwarf mutant NDF-1 was GA-insensitive mutant. The histological observations showed that the key reason of leading NDF-1 to dwarf was the reduction of hypocotyls and stems cell numbers.     

Selective interactions between arbuscular mycorrhizal fungi and<Emphasis Type="Italic"> Rhizobium leguminosarum</Emphasis> bv.<Emphasis Type="Italic"> viceae</Emphasis> enhance pea yield and nutrition

This study evaluated the response of pea (Pisum sativum cv. Trapper) to arbuscular mycorrhizal fungi (AMF) and Rhizobium leguminosarum bv. viceae strains varying in their effectiveness on pea. Plants were inoculated with the AMF species Glomus clarum NT4 or G. mosseae NT6 and/or ten Rhizobium strains, and grown for 90 days in soil containing indigenous AMF and rhizobia. The effectiveness of the Rhizobium strains on the growth (P <0.046; r =0.64) and N nutrition (P <0.04; r =0.65) of 6-week-old pea grown under gnotobiotic conditions was correlated with the growth and N nutrition of 90-day-old pea grown in natural soil for all strains except LX48. The growth and yield response of pea to co-inoculation with AMF and Rhizobium strains depended on the particular AMF-Rhizobium strain combination. In some cases, the yield and N nutrition of pea inoculated with a superior Rhizobium strain was significantly (P <0.05) enhanced by an apparently compatible AMF species compared to the Rhizobium treatment. On the other hand, an apparently incompatible AMF species significantly (P <0.05) reduced the performance of an effective Rhizobium strain. In general, treatments with effective Rhizobium strains or co-inoculation treatments with effective Rhizobium strains and a compatible AMF species produced the best results. Changes in total shoot dry matter production was significantly (P <0.05) correlated with the total shoot N (P <0.0001; r =0.95) and P content (P <0.0001; r =0.87), indicating that this response was mediated by enhanced N and P nutrition. Growth, yield and nutrition of pea were not related to AMF colonization of roots. Our results suggest that careful co-selection of AMF species and Rhizobium strains can enhance pea yield and nutrition.     

Comparative analysis of <Emphasis Type="Italic">Argonaute</Emphasis> gene sequences in bananas (<Emphasis Type="Italic">Musa</Emphasis> sp.) shows conserved species-specific Ago-7 PIWI domains

The Argonaute proteins are key components in the effector complex of RNA silencing pathways which interact with small RNAs to mediate sequence specific silencing of nucleic acid targets. In plants, these proteins are involved in a diversity of biological roles such as antiviral defence, heterochromatin regulation and in the regulation of growth and development. This report describes the study of Argonaute gene sequences in bananas which are an important food staple for many developing nations. This study successfully isolated AGO7-specific PIWI domain genomic sequences from 12 diploid Musa species of three Musa sections and also from Ensete. The Musa AGO7-specific PIWI domain sequences showed the highest similarity to rice AGO7/SHOOTLESS4 with an average amino acid identity of 78%. Phylogenetic analysis of the Musa sequences revealed phylogenetic grouping that agrees fairly well with the present knowledge of the taxonomic classification of Musa species. In addition, this study estimated that there are at least 15 Argonaute genes or loci containing PIWI domain sequences in the genome of Musa acuminata ssp. malaccensis.     

Pre-inoculation with arbuscular mycorrhizal fungi suppresses root knot nematode (<Emphasis Type="Italic">Meloidogyne incognita</Emphasis>) on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

A pot experiment was conducted to evaluate the influence of pre-inoculation of cucumber plants with each of the three arbuscular mycorrhizal (AM) fungi Glomus intraradices, Glomus mosseae, and Glomus versiforme on reproduction of the root knot nematode Meloidogyne incognita. All three AM fungi tested significantly reduced the root galling index, which is the percentage of total roots forming galls. Numbers of galls per root system were significantly reduced only in the G. intraradices + M. incognita treatment. The number of eggs per root system was significantly decreased by AM fungus inoculation, no significant difference among the three AM fungal isolates. AM inoculation substantially decreased the number of females, the number of eggs g⁻¹ root and of the number of eggs per egg mass. The number of egg masses g⁻¹ root was greatly reduced by inoculation with G. mosseae or G. versiforme. By considering plant growth, nutrient uptake, and the suppression of M. incognita together, G. mosseae and G. versiforme were more effective than G. intraradices.     

Effects of conventionally bred and <Emphasis Type="BoldItalic">Bacillus thuringiensis</Emphasis> (<Emphasis Type="BoldItalic">Bt</Emphasis>) maize varieties on soil microbial biomass and activity

Genetically modified (GM) maize containing genes from the soil bacterium Bacillus thuringiensis (Bt) was cultivated on 29% of the total maize production area worldwide in 2009. Most studies to date compare Bt-maize varieties with their near isogenic lines; however, there is little information on the variability of conventional maize breeding lines and how the effects of Bt varieties are ranked within. In our study on the potential risks of Bt-maize varieties, we analyzed tissue quality and compared the effects of ten conventional and GM maize varieties on soil microbiological properties in a replicated climate chamber experiment. All maize varieties were cultivated twice in the same soil microcosm. Shoot yields and soluble C in leaf tissue of Bt varieties were higher than the ones of non-Bt. Soil dehydrogenase activity was reduced by 5% under Bt varieties compared to non-Bt, while most of the other soil microbial properties (soil microbial biomass, basal respiration) showed no significant differences between Bt and non-Bt varieties. The leaves and roots of one Bt variety were decomposed to a greater extent than the ones of its near isogenic line; the conventional breeding lines also showed higher values. Changes in crop and soil parameters were found when comparing the first and the second crops, but the effects of repeated cropping were the same for all tested varieties. For the studied parameters, the variation among non-Bt-maize varieties was similar to the difference between Bt and non-Bt varieties.     

Cytological and RAPD data revealed genetic relationships among nine selected populations of the wild bramble species, <Emphasis Type="Italic">Rubus</Emphasis><Emphasis Type="Italic">parvifolius</Emphasis> and <Emphasis Type="Italic">R. coreanus</Emphasis> (Rosaceae)

Rubus parvifolius L. and R. coreanus Miq. are the two morphologically distinct, endemic wild brambles in East Asia that may be useful in raspberry breeding and cultivar development. In this study, genetic relationships among a total of nine populations of these two species, collected from Sichuan Province in southwestern China, were investigated by cytological and RAPD analyzing. With the exception of ploidy levels, R. parvifolius and R. coreanus had similar cytological features. Six R. parvifolius populations included diploids, triploid, tetraploid, and mixed diploid-tetraploid cytotype plants; among them, the population that produced the fewest seeds was the triploid, while the three R. coreanus populations were all diploid. But obvious genetic differentiation between R. parvifolius and R. coreanus was revealed by RAPD markers, the Euclidean’s GS coefficient generated by UPGMA cluster analysis between R. parvifolius and R. coreanus was only 0.442, about half of that observed within species. Both genetic relationships among these populations and the origin of polyploidy in R. parvifolius populations are also discussed.     

Soil nematode community varies between rice cultivars but is not affected by transgenic Bt rice expressing <Emphasis Type="Italic">Cry1Ab</Emphasis> or <Emphasis Type="Italic">Cry1Ab</Emphasis>/<Emphasis Type="Italic">Cry1Ac</Emphasis>

Insecticidal crystal (Cry) proteins produced by transgenic Bacillus thuringiensis (Bt) rice that enter the soil via root exudation and plant residues may be harmful to non-target soil organisms. We conducted a 3-year field investigation to determine if soil nematode abundance and diversity were affected by exposure to two transgenic Bt rice cultivars, compared to their non-transgenic near isolines. Near isolines were Kemingdao (KMD-Bt) expressing the single Cry1Ab gene and its non-Bt near isoline Xiushui-11 (XSD), as well as Huahui-1 (HH-Bt) expressing the fused Cry1Ab/Cry1Ac gene and its non-Bt near isoline Minghui-63 (MH). Nematode variables including community composition, abundance, trophic groups, and most of the common genera differed significantly between the rice cultivars. However, these nematode variables were similar under transgenic Bt rice and its non-Bt near isoline, although higher Shannon’s index value and Pielou’s index value were found in soils planted with Bt rice than the non-Bt near isoline. During this 3-year field study, gene modification (single Cry1Ab gene and fused Cry1Ab/Cry1Ac gene) supports a more uniform distribution of nematode species but had no effect on soil nematode abundance and community composition. We conclude that continuous cultivation of KMD-Bt and HH-Bt rice varieties for 3 years is not detrimental to soil nematode communities under field conditions.     

A new mycorrhizal helper bacterium, <Emphasis Type="Italic">Ralstonia</Emphasis> species,in the ectomycorrhizal symbiosis between <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">Suillus granulatus</Emphasis>

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.     

Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth

The aim of this study was to identifiy the endophytic bacteria recovered from the foliage, tap roots and nodules of red clover plants (Trifolium pratense L.); and to assess the effects of the nodule bacteria, alone and in combination with Rhizobium spp., on the growth and development of red clover seedlings. Thirty-one bacteria species from 14 different genera were recovered from within the foliage, roots and nodules of red clover plants cv. AC Charlie. Genera diversity and species number were greatest in foliage tissues. Pantoea agglomerans (59.6%) was the most frequent species recovered in foliage tissues, Agrobacterium rhizogenes A in the tap root (49.2%) and Rhizobium leguminosarum BV phaseoli and R. loti B in the nodules (27.2% each). Recovery of Rhizobium species was not restricted to the nodules, and species of this genus were systemic throughout the plant. Clover root nodules were host to 12 bacteria species other than rhizobia, of which 8 were specific to this tissue. Using non-selective media, R. leguminosarum BV trifolii constituted only 8.8% of all the root nodule bacteria recovered. In root bacterization experiments, species of nodule bacteria promoted growth of red clover more often when applied in combination with R. leguminosarum BV trifolii than when applied singly. However, Bacillus megaterium, Bordetella avium and Curtobacterium luteum consistently promoted growth either individually or in combination with R. leguminosarum BV trifolii. Nodulation was promoted when R. leguminosarum BV trifolii was coinoculated with Bacillus insolitus, B. brevis or A. rhizogenes A. Single isolate applications of Rhizobium species to roots always led to the depression of clover growth, but mixtures of R. leguminosarum BV trifolii and R. leguminosarum BV phaseoli resulted in growth promotion. The latter is considered further evidence of the beneficial allelopathic side effect of strain competition for the same ecological niche. Received: 27 June 1996