Humic Substances Isolated from Recycled Biomass Trigger Jasmonic Acid Biosynthesis and Signalling.

Intensive agriculture maintains high crop yields through chemical inputs, which are well known for their adverse effects on environmental quality and human health. Innovative technologies are required to reduce the risk generated by the extensive and harmful use of pesticides. The plant biostimulants made from humic substances isolated from recyclable biomass offer an alternative approach to address the need for replacing conventional agrochemicals without compromising the crop yield. The stimulatory effects of humic substances are commonly associated with plant hormones, particularly auxins. However, jasmonic acid (JA) is crucial metabolite in mediating the defence responses and governing plant growth and development. This work aimed to evaluate the changes in the biosynthesis and signalling pathway of JA in tomato seedlings treated with humic acids (HA) isolated from vermicompost. We use the tomato model system cultivar Micro-Tom (MT) harbouring a reporter gene fused to a synthetic promoter that responds to jasmonic acid (JERE::GUS). The transcript levels of genes involved in JA generation and activity were also determined using qRT-PCR. The application of HA promoted plant growth and altered the JA status, as revealed by both GUS and qRT-PCR assays. Both JA enzymatic synthesis (LOX, OPR3) and JA signalling genes (JAZ and JAR) were found in higher transcription levels in plants treated with HA. In addition, ethylene (ETR4) and auxin (ARF6) signalling components were positively modulated by HA, revealing a hormonal cross-talk. Our results prove that the plant defence system linked to JA can be emulated by HA application without growth inhibition.

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus.

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

Genome sequencing of the vermicompost strain Stenotrophomonas maltophilia UENF-4GII and population structure analysis of the S. maltophilia Sm3 genogroup.

The Stenotrophomonas maltophilia complex (Smc) is a cosmopolitan bacterial group that has been proposed an emergent multidrug-resistant pathogen. Taxonomic studies support the genomic heterogeneity of Smc, which comprises genogroups exhibiting a range of phenotypically distinct strains from different sources. Here, we report the genome sequencing and in-depth analysis of S. maltophilia UENF-4GII, isolated from vermicompost. This genome harbors a unique region encoding a penicillin-binding protein (pbpX) that was carried by a transposon, as well as horizontally-transferred genomic islands involved in anti-phage defense via DNA modification, and pili glycosylation. We also analyzed all available Smc genomes to investigate genes associated with resistance and virulence, niche occupation, and population structure. S. maltophilia UENF-4GII belongs to genogroup 3 (Sm3), which comprises three phylogenetic clusters (PC). Pan-GWAS analysis uncovered 471 environment-associated and 791 PC-associated genes, including antimicrobial resistance (e.g. blaL1 and blaR1) and virulence determinants (e.g. treS and katG) that provide insights on the resistance and virulence potential of Sm3 strains. Together, the results presented here provide the grounds for more detailed clinical and ecological investigations of S. maltophilia.

Inoculation with the endophytic bacterium Herbaspirillum seropedicae promotes growth, nutrient uptake and photosynthetic efficiency in rice.

MAIN CONCLUSION: Higher vacuolar proton pump activity may increase plant energy and nutrient use efficiency and provide the nexus between plant inoculation with Herbaspirillum seropedicae and growth promotion. Global change and growing human population are exhausting arable land and resources, including water and fertilizers. We present inoculation with the endophytic plant-growth promoting bacterium (PGPB) Herbaspirillum seropedicae as a strategy for promoting growth, nutrient uptake and photosynthetic efficiency in rice (Oryza sativa L.). Because plant nutrient acquisition is coordinated with photosynthesis and the plant carbon status, we hypothesize that inoculation with H. seropedicae will stimulate proton (H+) pumps, increasing plant growth nutrient uptake and photosynthetic efficiency at low nutrient levels. Plants were inoculated and grown in pots with sterile soil for 90 days. Herbaspirillum seropedicae endophytic colonization was successful and, as hypothesized, inoculation (1) stimulated root vacuolar H+ pumps (vacuolar H+-ATPase and vacuolar H+-PPase), and (2) increased plant growth, nutrient contents and photosynthetic efficiency. The results showed that inoculation with the endophytic bacterium H. seropedicae can promote plant growth, nutrient uptake and photosynthetic efficiency, which will likely result in a more efficient use of resources (nutrients and water) and higher production of nutrient-rich food at reduced economic and environmental costs.

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies.

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum ß-lactamases, including blaCTX-M-55 and blaNDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of blaNDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.

Genome sequencing and assessment of plant growth-promoting properties of a Serratia marcescens strain isolated from vermicompost.

BACKGROUND: Plant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in a wide range of environments, including soil. RESULTS: Here we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria. CONCLUSIONS: Collectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.

Changes in metabolic profiling of sugarcane leaves induced by endophytic diazotrophic bacteria and humic acids.

Plant growth-promoting bacteria (PGPB) and humic acids (HA) have been used as biostimulants in field conditions. The complete genomic and proteomic transcription of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus is available but interpreting and utilizing this information in the field to increase crop performance is challenging. The identification and characterization of metabolites that are induced by genomic changes may be used to improve plant responses to inoculation. The objective of this study was to describe changes in sugarcane metabolic profile that occur when HA and PGPB are used as biostimulants. Inoculum was applied to soil containing 45-day old sugarcane stalks. One week after inoculation, the methanolic extracts from leaves were obtained and analyzed by gas chromatography coupled to time-of-flight mass spectrometry; a total of 1,880 compounds were observed and 280 were identified in all samples. The application of HA significantly decreased the concentration of 15 metabolites, which generally included amino acids. HA increased the levels of 40 compounds, and these included metabolites linked to the stress response (shikimic, caffeic, hydroxycinnamic acids, putrescine, behenic acid, quinoline xylulose, galactose, lactose proline, oxyproline and valeric acid) and cellular growth (adenine and adenosine derivatives, ribose, ribonic acid and citric acid). Similarly, PGPB enhanced the level of metabolites identified in HA-treated soils; e.g., 48 metabolites were elevated and included amino acids, nucleic acids, organic acids, and lipids. Co-inoculation (HA+PGPB) boosted the level of 110 metabolites with respect to non-inoculated controls; these included amino acids, lipids and nitrogenous compounds. Changes in the metabolic profile induced by HA+PGPB influenced both glucose and pentose pathways and resulted in the accumulation of heptuloses and riboses, which are substrates in the nucleoside biosynthesis and shikimic acid pathways. The mevalonate pathway was also activated, thus increasing phytosterol synthesis. The improvement in cellular metabolism observed with PGPB+HA was compatible with high levels of vitamins. Glucuronate and amino sugars were stimulated in addition to the products and intermediary compounds of tricarboxylic acid metabolism. Lipids and amino acids were the main compounds induced by co-inoculation in addition to antioxidants, stress-related metabolites, and compounds involved in cellular redox. The primary compounds observed in each treatment were identified, and the effect of co-inoculation (HA+PGPB) on metabolite levels was discussed.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae.

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Compost biofortification with diazotrophic and P-solubilizing bacteria improves maturation process and P availability.

BACKGROUND: Phosphorus-containing fertilizers play an important role in tropical agriculture owing to the well documented shortage of plant-available P in soils. Traditional P fertilizer production is based on chemical processing of insoluble rock phosphate (RP), which includes an acid treatment at high temperature. Processing the RP increases fertilizer costs, making it unavailable for undercapitalized and typically family-based farmers. Biotechnological methods have been proposed as an alternative to increase phosphate availability in RP. In this study, Burkholderia silvatlantica and Herbaspirillum seropedicae were co-inoculated into an RP-enriched compost with the aim of determining the effects of this technology on the levels of phosphatase activities and release of plant-available P. RESULTS: Inoculation of both microorganisms resulted in higher organic matter decomposition and higher humic acid formation in composting. Herbaspirillum seropedicae was the most promising microorganism for the production of acid and alkaline phosphatase enzymes. Both microorganisms presented potential to increase the supply of P from poorly soluble sources owing to increased levels of water-soluble P and citric acid P. CONCLUSION: Burkholderia silvatlantica and H. seropedicae in RP-enriched compost may represent an important biotechnological tool to reduce the overall time required for composting and increase the supply of P from poorly soluble sources. © 2016 Society of Chemical Industry.

Differential effects of salinity and osmotic stress on the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

Plant growth-promoting bacteria (PGPB) represent a promising alternative to the massive use of industrial fertilizers in agriculture. Gluconacetobacter diazotrophicus is a PGPB that colonizes several plant species. Although this bacterium is able to grow at high sucrose concentrations, its response to environmental stresses is poorly understood. The present study evaluated G. diazotrophicus PAL5 response to stresses caused by sucrose, PEG 400, NaCl, KCl, Na2SO4 and K2SO4. Morphological, ultrastructural and cell growth analysis revealed that G. diazotrophicus PAL5 is more sensitive to salt than osmotic stress. Growth inhibition and strong morphological changes were caused by salinity, in consequence of Cl ion-specific toxic effect. Interestingly, low osmotic stress levels were beneficial for bacterial multiplication, which was able to tolerate high sucrose concentrations, Na2SO4 and K2SO4. Our data show that G. diazotrophicus PAL5 has differential response to osmotic and salinity stress, which may influence its use as inoculant in saline environments.

Initial pH of medium affects organic acids production but do not affect phosphate solubilization.

The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

Initial pH of medium affects organic acids production but do not affect phosphate solubilization

The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae.

BACKGROUND: Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. RESULTS: A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. CONCLUSIONS: Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.

Changes in labile phosphorus forms during maturation of vermicompost enriched with phosphorus-solubilizing and diazotrophic bacteria.

The aim of this study was to assess the effect of N(2)-fixing and P-solubilizing bacteria during maturation of vermicompost on phosphorus availability. A bacterial suspension containing Burkholderia silvatlantica, Burkholderia spp. and Herbaspirillum seropedicae was applied at the initial stage of vermicomposting. At the end of the incubation time (120days), the nitrogen content had increased by18% compared to uninoculated vermicompost. Water-soluble P was 106% higher in inoculated vermicompost while resin-extractable P increased during the initial vermicomposting stage and was 21% higher at 60days, but was the same in inoculated and uninoculated mature compost. The activity of acid phosphatase was 43% higher in inoculated than uninoculated vermicompost. These data suggest that the introduction of the mixed culture had beneficial effects on vermicompost maturation.

Nitric oxide mediates humic acids-induced root development and plasma membrane H+-ATPase activation.

It is widely reported that some humic substances behave as exogenous auxins influencing root growth by mechanisms that are not yet completely understood. This study explores the hypothesis that the humic acids' effects on root development involve a nitric oxide signaling. Maize seedlings were treated with HA 20 mg C L(-1), IAA 0.1 nM, and NO donors (SNP or GSNO), in combination with either the auxin-signaling inhibitor PCIB, the auxin efflux inhibitor TIBA, or the NO scavenger PTIO. H(+)-transport-competent plasma membrane vesicles were isolated from roots to investigate a possible link between NO-induced H(+)-pump and HA bioactivity. Plants treated with either HA or SNP stimulated similarly the lateral roots emergence even in the presence of the auxin inhibitors, whereas NO scavenger diminished this effect. These treatments induced H(+)-ATPase stimulation by threefold, which was abolished by PTIO and decreased by auxin inhibitors. HA-induced NO synthesis was also detected in the sites of lateral roots emergence. These data depict a new scenario where the root development stimulation and the H(+)-ATPase activation elicited by either HA or exogenous IAA depend essentially on mechanisms that use NO as a messenger induced site-specifically in the early stages of lateral root development.

Chemical composition and bioactivity properties of size-fractions separated from a vermicompost humic acid.

Preparative high performance size-exclusion chromatography (HPSEC) was applied to humic acids (HA) extracted from vermicompost in order to separate humic matter of different molecular dimension and evaluate the relationship between chemical properties of size-fractions (SF) and their effects on plant root growth. Molecular dimensions of components in humic SF was further achieved by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) based on diffusion coefficients (D), while carbon distribution was evaluated by solid state (CP/MAS) (13)C NMR. Seedlings of maize and Arabidopsis were treated with different concentrations of SF to evaluate root growth. Six different SF were obtained and their carbohydrate-like content and alkyl chain length decreased with decreasing molecular size. Progressive reduction of aromatic carbon was also observed with decreasing molecular size of separated fractions. Diffusion-ordered spectroscopy (DOSY) spectra showed that SF were composed of complex mixtures of aliphatic, aromatic and carbohydrates constituents that could be separated on the basis of their diffusion. All SF promoted root growth in Arabidopsis and maize seedlings but the effects differed according to molecular size and plant species. In Arabidopsis seedlings, the bulk HA and its SF revealed a classical large auxin-like exogenous response, i.e.: shortened the principal root axis and induced lateral roots, while the effects in maize corresponded to low auxin-like levels, as suggested by enhanced principal axis length and induction of lateral roots. The reduction of humic heterogeneity obtained in HPSEC separated size-fractions suggested that their physiological influence on root growth and architecture was less an effect of their size than their content of specific bioactive molecules. However, these molecules may be dynamically released from humic superstructures and exert their bioactivity when weaker is the humic conformational stability as that obtained in the separated size-fractions.

Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc.

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca(5)(PO(4))(3)OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus.

Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67.

A beta-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs. IR42 and IR72. Internal populations peaked at over 10(6) log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 10(3) to 10(4) log CFU per gram of fresh weight by 28 DAI. GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots. Bacteria entered the roots via cracks at the points of lateral root emergence, with cv. IR72 appearing to be more aggressively infected than cv. IR42. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed. Dense colonies of H. seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI. Epiphytic bacteria were also seen. Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased. Only cv. IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.