Novel Plant-Microbe Rhizosphere Interaction Involving Streptomyces lydicus WYEC108 and the Pea Plant (Pisum sativum)

A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-β-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.     

Characterization of streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots.

The actinomycete Streptomyces lydicus WYEC108 showed strong in vitro antagonism against various fungal plant pathogens in plate assays by producing extracellular antifungal metabolites. When Pythium ultimum or Rhizoctonia solani was grown in liquid medium with S. lydicus WYEC108, inhibition of growth of the fungi was observed. When WYEC108 spores or mycelia were used to coat pea seeds, the seeds were protected from invasion by P. ultimum in an oospore-enriched soil. While 100% of uncoated control seeds were infected by P. ultimum within 48 h after planting, less than 40% of coated seeds were infected. When the coated seeds were planted in soil 24 h prior to introduction of the pathogen, 96 h later, less than 30% of the germinating seeds were infected. Plant growth chamber studies were also carried out to test for plant growth effects and for suppression by S. lydicus WYEC108 of Pythium seed rot and root rot. When WYEC108 was applied as a spore-peat moss-sand formulation (10(8) CFU/g) to P. ultimum-infested sterile or nonsterile soil planted with pea and cotton seeds, significant increases in average plant stand, plant length, and plant weight were observed in both cases compared with untreated control plants grown in similar soils. WYEC108 hyphae colonized and were able to migrate downward with the root as it elongated. Over a period of 30 days, the population of WYEC108 colonized emerging roots of germinating seeds and remained stable (10(5) CFU/g) in the rhizosphere, whereas the nonrhizosphere population of WYEC108 declined at least 100-fold (from 10(5) to 10(3) or fewer CFU/g). The stability of the WYEC108 population incubated at 25 degrees C in the formulation, in sterile soil, and in nonsterile soil was also evaluated. In all three environments, the population of WYEC108 maintained its size for 90 days or more. When pea, cotton, and sweet corn seeds were placed into sterile and nonsterile soils containing 10(6) or more CFU of WYEC108 per g, it colonized the emerging roots. After a 1-week growing period, WYEC108 populations of 10(5) CFU/g (wet weight) of root were found on pea roots in the amended sterile soil environment versus 10(4) CFU/g in amended nonsterile soil. To further study the in vitro interaction between the streptomycete and P. ultimum, mycelia of WYEC108 were mixed with oospores of P. ultimum in agar, which was then used as a film to coat slide coverslips.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.     

Use of Randomly Amplified Polymorphic DNA as a Means of Developing Genus- and Strain-Specific Streptomyces DNA Probes

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.     

Plant phenology and genetic variability in root and nodule development strongly influence genetic structuring of Rhizobium leguminosarum biovar viciae populations nodulating pea

The symbiotic relationships between legumes and their nitrogen (N(2))-fixing bacterial partners (rhizobia) vary in effectiveness to promote plant growth according to both bacterial and legume genotype. To assess the selective effect of host plant on its microsymbionts, the influence of the pea (Pisum sativum) genotype on the relative nodulation success of Rhizobium leguminosarum biovar viciae (Rlv) genotypes from the soil populations during plant development has been investigated. Five pea lines were chosen for their genetic variability in root and nodule development. Genetic structure and diversity of Rlv populations sampled from nodules were estimated by molecular typing with a marker of the genomic background (rDNA intergenic spacer) and a nodulation gene marker (nodD region). Differences were found among Rlv populations related to pea genetic background but also to modification of plant development caused by single gene mutation. The growth stage of the host plant also influenced structuring of populations. A particular nodulation genotype formed the majority of nodules during the reproductive stage. Overall, modification in root and nodule development appears to strongly influence the capacity of particular rhizobial genotypes to form nodules.     

Streptomyces lydicus A_(01) affects soil microbial diversity,improving growth and resilience in tomato

The actinomycete Streptomyces lydicus A_(01) promotes tomato seedling growth; however, the underlying mechanism is unclear. In this study, we investigated whether changes in soil microbial diversity, following Streptomyces lydicus A_(01) treatment, were responsible for the increased tomato seedling growth. Eukaryotic 18 S ribosomal DNA(rDNA) sequencing showed that S. lydicus A_(01)-treated and untreated soil shared 193 operational taxonomic units(OTUs), whereas bacterial 16 S rDNA sequencing identified 1,219 shared OTUs between the treated and untreated soil. Of the 42 dominant eukaryotic OTUs, eight were significantly increased and six were significantly decreased after A_(01) treatment. Of the 25 dominant bacterial OTUs, 12 were significantly increased and eight were significantly decreased after A_(01) treatment.Most of the eukaryotes and bacteria that increased in abundance exhibited growth promoting characteristics,which were mainly predicted to be associated with mineralization of nitrogen and phosphorus, phosphate solubilization, nutrient accumulation, and secretion of auxin, whereas some were related to plant protection,such as the degradation of toxic and hazardous substances. Soil composition tests showed that S. lydicus A_(01) treatment enhanced the utilization of nitrogen, phosphorus, and potassium in tomato seedlings. Thus, microbial fertilizers based on S. lydicus A_(01) may improve plant growth, without the detriment effects of chemical fertilizers.     

Polyamines in Nodules from Various Plant-Microbe Symbiotic Associations

Polyamine compositions of root or stem nodules collected fromvarieties of nitrogen-fixing leguminous (22 species) and non-leguminous(5 species) plants were investigated. Relatively high concentrationsof homospermidine were observed in root or stem nodules of allthe leguminous plants. Based on the ratio of homospermidineto spermidine, legume nodules were generally characterized intotwo major groupes; one containing almost equal amounts of homospermidineand spermidine, and the other a high homospermidine/spermidineratio. Root nodules from pigeon pea (Cajanus cajan L. Millsp)was the only exception which exhibited very low homospermidine/spermidineratio. Amongst the legumes, nodules of adzuki bean (Vigna angularis),siratro (Macroptilium atropurpureum DC. Urb.), pea (Pisum sativumL.), and hairly vetch (Vicia hirsuta S.F. Gray) were rich indiamine putrescine. Such characters of nodule polyamine compositionwere inherent characteristics of each legume species, and notrelated to the type of infected rhizobia (Rhizobium or Bradyrhizobium).In contrast to herbaceous leguminous plants, nonleguminous woodyplants, which symbiotically associate with actinomycete Frankiaspecies, contained little polyamines in their root nodules.Root nodules of non-leguminous Parasponia andersonii infectedby bradyrhizobia were found to contain large quantities of putrescineand homospermidine. No significant differences in polyaminecomposition were observed between root and stem nodules bothin Aeschynomene indica and Sesbania rostrata. (Received June 13, 1994; Accepted August 17, 1994)     

Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia

A study was conducted to determine whether colonization of legume roots and nodulation byRhizobium meliloti andBradyrhizobium japonicum could be enhanced by using inocula containing microorganisms that produce antibiotics suppressing soil or rhizosphere inhabitants but not the root-nodule bacteria. An antibiotic-producing strain of Pseudomonas and one of Bacillus were isolated, and mutants ofR. meliloti andB. japonicum sp. resistant to the antibiotics were used. The colonization of the alfalfa rhizosphere and nodulation byR. meliloti were enhanced by inoculation of soil withPseudomonas sp. in soil initially containing 2.7×10⁵ R. meliloti per g. The colonization of soybean roots byB. japonicum was enhanced by inoculating soil with three cell densities ofBacillus sp., and nodulation was stimulated byBacillus sp. added at two cell densities. In some tests, the dry weights of soybeans and seed yield increased as a result of these treatments, and co-inoculation with Bacillus also increased pod formation. Inoculation of seeds withBacillus sp. and the root-nodule bacterium enhanced nodulation of soybeans and alfalfa, but colonization byB. japonicum andR. meliloti was stimulated only during the early period of plant growth. Studies were also conducted withStreptomyces griseus and isolates ofR. meliloti andB. japonicum resistant to products of the actinomycete. Nodulation of alfalfa byR. meliloti was little or not affected by the actinomycete alone; however, both nodulation and colonization were enhanced if the soil was initially amended with chitin andS. griseus was also added. Chitin itself did not affectR. meliloti. Treatments of seeds with chitin orS. griseus alone did not enhance colonization of alfalfa roots byR. meliloti or soybean roots byB. japonicum, but the early colonization of the roots by both bacterial species was promoted if the seeds received both chitin andS. griseus; this treatment also increased nodulation and dry weights of alfalfa and soybeans and the N content of alfalfa. It is suggested that co-inoculation of legumes with antibiotic-producing microorganisms and root-nodule bacteria resistant to those antibiotics is a promising means of promoting nodulation and possibly nitrogen fixation.     

Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats.     

A new root-nodulating symbiont of the tropical legume Sesbania, Rhizobium sp SIN-1, is closely related to R. galegae, a species that nodulates temperate legumes

Abstract Rhizobium sp. SIN-1, isolated in India from root nodules on the tropical legume Sesbania aculeata , also induces nitrogen-fixing nodules on roots of S. macrocarpa, S. speciosa, S. procumbens, S. punicea, S. rostrata , and Vigna unguiculata . Unlike Azorhizobium caulinodans , SIN-1 does not induce stem nodules on S. rostrata . The nodules induced by SIN-1 develop exclusively at the bases of secondary roots. Electron microscopic studies of mature nodule sections revealed rhizobia within intercellular spaces, indicating a 'crack entry' mechanism of root infection. SIN-1 is a fast-growing, acid-producing, salt-tolerant Rhizobium that utilizes a wide variety of carbon sources. The nodulation ( nod ) genes of this strain are located on a 300-MDa symbiosis ( sym ) plasmid. Fatty acid profile and sequence comparison of a 260-bp conserved region of the 16S rRNA gene demonstrated that SIN-1 is phylogenetically closely related to R. galegae , a species that nodulates temperate legumes.     

Plant mediated interactions between Pseudomonas fluorescens, Rhizobium leguminosarum and arbuscular mycorrhizae on pea

Using undisturbed sandy loam soil cores heavily infested with mycorrhizae, the effects of the antibiotic-producing Pseudomonas fluorescens strain F113 and its non-antibiotic derivative Ps. fluorescens F113G22 on nodulation by introduced and indigenous Rhizobium were studied. Furthermore, the effects of the different microbial inocula on the colonization of the pea roots by mycorrhizae were studied. It was found that Ps. fluorescens F113 enhanced nodulation by Rhizobium fourfold, while the nodules produced were much larger and strongly pigmented (pink) compared with those in other treatments. The proportion of roots colonized by arbuscular mycorrhizae was not significantly affected by the different treatments.     

Nodulation phenotypes of gibberellin and brassinosteroid mutants of pea

The initiation and development of legume nodules induced by compatible Rhizobium species requires a complex signal exchange involving both plant and bacterial compounds. Phytohormones have been implicated in this process, although in many cases direct evidence is lacking. Here, we characterize the root and nodulation phenotypes of various mutant lines of pea (Pisum sativum) that display alterations in their phytohormone levels and/or perception. Mutants possessing root systems deficient in gibberellins (GAs) or brassinosteroids (BRs) exhibited a reduction in nodule organogenesis. The question of whether these reductions represent direct or indirect effects of the hormone deficiency is addressed. For example, the application of GA to the roots of a GA-deficient mutant completely restored its number of nodules to that of the wild type. Grafting studies revealed that a wild-type shoot or root also restored the nodule number of a GA-deficient mutant. These findings suggest that GAs are required for nodulation. In contrast, the shoot controlled the number of nodules that formed in graft combinations of a BR-deficient mutant and its wild type. The root levels of auxin and GA were similar among these latter graft combinations. These results suggest that BRs influence a shoot mechanism that controls nodulation and that the root levels of auxin and GA are not part of this process. Interestingly, a strong correlation between nodule and lateral root numbers was observed in all lines assessed, consistent with a possible overlap in the early developmental pathways of the two organs.     

Assessing Genotypic Diversity and Symbiotic Efficiency of Five Rhizobial Legume Interactions Under Cadium Stress for Soil Phytoremediation

In the framework of soil phytoremediation using local legume plants coupled with their native root-nodulating bacteria to increase forage yields and preserve contaminated soils in arid regions of Tunisia, we investigated the diversity of bacteria from root nodules of Lathyrus sativus, Lens culinaris, Medicago marina, M. truncatula, and M. minima and the symbiotic efficiency of these five legume symbiosis under Cadmium stress. Fifty bacterial strains were characterized using physiological and biochemical features such heavy metals resistant, and PCR-RFLP of 16S rDNA. Taxonomically, the isolates nodulating L. sativus, and L. culinaris are species within the genera Rhizobium and the ones associated to Medicago sp, within the genera Sinorhizobium. The results revealed also that the cadmium tolerance of the different legumes-rhizobia interaction was as follows: M. minima<M. truncatula<M. marina<L. sativus<L. culinaris indicating that the effect of Cadmium on root nodulation and biomass production is more deleterious on M. minima-S. meliloti and M. truncatula-S. meliloti than in other symbiosis. Knowledge on genetic and functional diversity of M. marina, L. sativus and L. culinaris microsymbiotes is very useful for inoculant strain selection and can be selected to develop inoculants for soil phytoremediation.     

Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

Legume nodulation: The host controls the party

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen‐fixing rhizobia bacteria is central to their advantage. This plant–microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.     

Legumes tolerance to rhizobia is not always observed and not always deserved

Rhizobia display dual lifestyle. These bacteria are soil inhabitants but can also elicit the formation of a special niche on the root of legume plants, the nodules. In such organs, rhizobia can promote the growth of their host by providing them nitrogen they captured from atmosphere. All along the infection process, the plant innate immunity has to be controlled to maintain compatible interaction. However, nodulation does not always result in profit for the plant as compatible interactions include both nitrogen‐fixing and non‐fixing associations. In recent years, our knowledge on the mechanisms involved in the control of plant innate immunity during rhizobia‐legume interactions has greatly improved notably by the identification of bacterial and plant genes activating or suppressing the plant defences. Surprisingly, results also demonstrated that in some cases, plant defence reactions result in abortion of the nodulation process despite that the rhizobial strain has all the genetic potential to establish mutualism. In such situation, experimental evolution approaches highlighted possible rapid switches of incompatible rhizobia either to mutualistic or parasitic behaviour. Here, we review this recent literature.     

Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.