Influence of root-knot nematode, Meloidogyne incognita (Kofoid & White) Chitwood infection on different plant growth parameters in Mungbean, Vigna radiata (L.) Wilczek

Root-knot nematode, Meloidogyne incognita (Kofoid & White) Chitwood, is a major threat to mungbean cultivation. The pest causes a significant reduction in plant growth parameters that ultimately results in loss of grain yield. The present study was carried out under glass house condition to study the effect of different inoculum load of root-knot nematode M. incognita on plant growth, nodulation and nematode development and nutrients status of Mungbean. The results revealed a progressive decline in plant growth parameters viz., fresh and dry shoot weight and shoot length with respect to increase in inoculum level. However, fresh and dry root weight showed the opposite trend. The fresh and dry shoot weight was decreased by 44% and 66%, respectively at 4 J2s/g soil. The chlorophyll content in the leaves also decreased with the increase of inoculum level from 100-6000 J2s/pot. Nutrients contents of the plant viz. N, P, K, Ca and Mg were significantly reduced in shoots while in roots these was increased with an increase of inoculum levels. Nodulation was affected by 80% at the highest inoculum level i.e. 6000 J2s/pot. Also leghaemoglobin, bacteroid content and nitrogenase activity was reduced progressively with increased levels of nematode inoculum. Thus, the root-knot nematode, M. incognita interferes with the process of symbiotic nitrogen fixation between mungbean host and rhizobium and that can affect the quality of produce.

Horizontal gene transfer among nitrogen-fixing bacteria in the guts of termite Coptotermes gestroi

Aims@#Arthropods guts, such as termite harbor diverse microorganisms including those that are capable of fixing atmospheric nitrogen (N2). Nitrogen-fixing bacteria can help termite to overcome their shortage of dietary N by providing fixed N2. Nitrogenase enzyme is responsible for this trait and encoded by nif genes which are highly conserved and are primarily used in the identification of N2-fixing microorganisms. Here, we characterized N2-fixing bacteria isolated from the hindguts of termite Coptotermes gestroi.@*Methodology and results@#A total of 46 bacterial isolates were obtained after a primary screening based on their ability to grow on Burk’s media. Subsequently, the nifH gene from two of these isolates, namely S7 and S20, were successfully amplified and sequenced. Molecular phylogenetic analysis of 16S rRNA gene sequence revealed that isolate S7 is closely related to Ralstonia pickettii ATCC27511 (99.34% similarity, 1059 bp), whereas isolate S20 is closely related to Microbacterium sp. NCCP-451 (LC488936) (99.06% similarity, 948 bp). Besides that, the recA gene of isolate S7 is closely related to Ralstonia pickettii 12D (CP001644) (100% similarity, 442 bp) and the type strain of Ralstonia pickettii (ATCC 27511) (NZ KN050646) (98.97% similarity, 438 bp). Meanwhile, nifH gene of isolate S7 showed highest similarity to the uncultured bacterium NR1606 (AF035490) (99.93% similarity, 277 bp). Moreover, the nifH gene of isolate S20 is clearly separated from Azoarcus sp. and distantly related to Microbacterium sp. The incongruence between the partial 16S rRNA and nifH gene sequences could indicate the possibility of horizontal transfer of nif genes.@*Conclusion, significance and impact of study@#The phylogenetic incongruence between housekeeping genes (16S rRNA and RecA) and nifH gene in these bacteria provides new insight on potential horizontal gene transfer (HGT) activity taking place in bacterial communities particularly in the guts of arthropods. The finding of this study on potential HGT can also aid in the prediction of origins and evolution of gene transfer among bacteria.

Bacillus spp: una alternativa para la promoción vegetal por dos caminos enzimáticos / Bacillus spp: An alternative for plant promotion by two enzymatic pathways

Resumen Objetivo. Se realizó una revisión sobre las características de las fitasas y nitrogenasas de Bacillus spp. y sus opciones de uso como alternativa biofertilizante. El género Bacillus es secretor de proteínas y metabolitos eficientes para el control de plagas y enfermedades, promueve el crecimiento vegetal a través de la solubilización de fósforo y la producción de reguladores de crecimiento como el ácido indol acético; así mismo participa en la fijación de nitrógeno cuando hace parte de consorcios microbianos. Como biofertilizante es una opción amigable para el suelo y el ambiente que da respuesta a la necesidad de implementar la agricultura sostenible.

Abstract Objective. We conducted a review of the characteristics of the phytase and nitrogenase of Bacillus sp. and their potential use as an alternative biological fertilizer. The genus Bacillus is an efficient secretor of proteins and metabolites; to control pests and diseases, promote plant growth through the solubilization of phosphorus and production of growth regulators as acetic indole. Likewise it is involved in nitrogen fixation when it is part of microbial consortia. As biological fertilizer is a friendly option for the soil and the environment that responds to the need to implement sustainable agriculture.

Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia.

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under freeliving conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).

Composition andffunction of the microbial commuity related with the nitrogen ccycling on the potato rhizosphere / Composición y función de la comunidad microbiana relacionada con el ciclaje de nitrógeno en la rizosfera de Solanum tuberosum (BAHUIN) grupo phureja

In the S. tuberosum group phureja crops, mineral fertilizer and organic amendments are applied to meet the plants´ nutritional demands, however the effect of such practices on the associated rizospheric microbial communities are still unknown. Nitrogen plays an important role in agricultural production, and a great diversity of microorganisms regulates its transformation in the soil, affecting its availability for the plant. The aim of this study was to assess the structure of microbial communities related with the N cycle of S. tuberosum group phureja rizospheric soil samples, with contrasting physical-chemical properties and fertilization strategy. Few significant differences between the community composition at the phylum level were found, only Planctomycetes phylum was different between samples of different soil type and fertilization strategy. However, the analysis of nitrogen-associated functional groups made by ribotyping characterization, grouped soils in terms of such variables in a similar way to the physical-chemical properties. Major differences between soil samples were typified by higher percentages of the ribotypes from nitrite oxidation, nitrogen fixation and denitrification on organic amendment soils. Our results suggest that, the dominant rhizosphere microbial composition is very similar between soils, possibly as a result of population´s selection mediated by the rhizosphere effect. However, agricultural management practices in addition to edaphic properties of sampled areas, appear to affect some functional groups associated with the nitrogen cycling, due to differences found on soil´s physicalchemical properties, like the concentration of ammonium that seems to have an effect regulating the distribution and activity of nitrogen related functional groups in the S. tuberosum rhizosphere.

Fertilización mineral y enmiendas orgánicas son aplicadas para satisfacer las demandas nutricionales de los cultivos de S. tuberosum grupo phureja . Sin embargo, el efecto de esas prácticas sobre la comunidad microbiana asociada a la rizósfera aún no se conocen. El nitrógeno juega un papel importante en la producción agrícola y una gran diversidad de microorganismos regulan su transformación en el suelo, afectando su disponibilidad para la planta. El objeto de este estudio fue determinar la composición de la comunidad microbiana de la rizósfera de S. tuberosum grupo phureja , asociada con el ciclo del nitrógeno, en muestras de suelo contrastantes en sus propiedades fisicoquímicas y estrategia de fertilización. Pocas diferencias significativas entre la composición de la comunidad microbiana a nivel de phylum fueron encontradas, Í°nicamente el phylum Planctomycetes fue diferente entre las muestras de suelos con estrategias de fertilización diferentes. Sin embargo, el análisis de grupos funcionales asociados al nitrógeno llevado a cabo por la caracterización de ribotipificación, agrupó los suelos en términos de esas variables en una forma similar a las propiedades fisicoquímicas del suelo. Diferencias mayores entre las muestras de suelo fueron tipificadas por los altos porcentajes de ribotipos asociados a la oxidación de nitrito, fijación de nitrógeno y denitrificación sobre los suelos con enmiendas orgánicas. Nuestros resultados sugieren que la composición microbiana dominante es muy similar entre suelos, posiblemente como resultado de la selección de poblaciones mediada por el efecto rizosférico. Sin embargo, las prácticas del manejo agrícola en conjunto con las propiedades del suelo en las áreas muestreadas, parecen afectar algunos grupos funcionales asociados con el ciclo de nitrógeno, debido a las diferencias encontradas en las propiedades fisicoquímicas del suelo, como la concentración de amonio que parece tener un efecto regulando la distribución y actividad de los grupos funcionales relacionados con el ciclo del nitrógeno en la rizosfera de S. tuberosum.

Estimation of Nitrogenase Enzyme Activities and Plant Growth of Legume and Non-legume Inoculated with Diazotrophic Bacteria

Biological Nitrogen Fixation (BNF) process benefits the agriculture sector especially for reducing cost of nitrogen fertilizer. In the process, the diazotrophs convert N2 into ammonia (NH3) which is useable by plants. The BNF process is catalysed by nitrogenase enzyme that involved protons and electrons together with evolution of H2 therefore, the assessment of N2 fixation is also available via H2 production and electron allocation analysis. Thus, the aims of this experiment were to estimate the nitrogenase enzyme activities and observe the influence of diazothrophs on growth of legume (soybean) and non legume (rice) plants. Host plants were inoculated with respective inocula; Bradyrhizobium japonicum (strain 532C) for soybean while Azospirillum brasilense (Sp7) and locally isolated diazotroph (isolate 5) for rice. At harvest, the plants were observed for plant growth parameters, H2 evolution, N2 fixation and electron allocation coefficient (EAC) values. The experiment recorded N2 fixation activities of inoculated soybean plants at 141.2 μmol N2 h-1 g-1 dry weight nodule, and the evolution of H2 at 144.4 μmol H2 h-1 g-1 dry weight nodule. The electron allocation coefficient (EAC) of soybean was recorded at 0.982. For inoculated rice plants, none of the observations was successfully recorded. However, results for chlorophyll contents and plant dry weight of both plants inoculated with respective inocula were similar to the control treatments supplied with full nitrogen fertilization (+N). The experiment clearly showed that inoculation of diazotrophic bacteria could enhance growth of the host plants similar to plants treated with nitrogenous fertilizer due to efficient N2 fixation process

Caracterización fisiológica de la comunidad microbiana endófita de la caña de azúcar / Physiological characterization of sugarcane’s endophytic microbial community

El uso desmedido de fertilizantes químicos nitrogenados y pesticidas ha traído graves consecuencias ambientales, por lo que se ha prestado gran atención al estudio de la microbiota nativa de los cultivos y sus beneficios a la planta, incluyendo la caña de azúcar. Este trabajo se realizó con el objetivo de caracterizar la microbiota nativa de la caña de azúcar. Se utilizaron 5 cepas bacterianas y 50 aislados provenientes del interior de este cultivo. Se determinó la actividad nitrogenasa y la influencia de la fuente de carbono, nitrógeno y el pH en la misma, mediante cromatografía gaseosa. Se detectó la producción de ácido indolacético por Dot-Immunobinding y el método de Salkowski. Del total de cepas y aislados, 19 mostraron actividad nitrogenasa, con valores entre 100 y 5000 //g/mL, y 6, Gluconacetobacter diazotrophicus PAl-5, Gluconacetobacter diazotrophicus 1-05, Gluconacetobacter diazotrophicus 4-02, aislado 17, aislado 30 y aislado 305; además, tienen la capacidad de producir AIA (valores entre 1,7 y 2,5 JMg/mL). Se demostró que las fuentes nutricionales y el pH del medio de cultivo influyen sobre la actividad nitrogenasa de las cepas representativas de la comunidad endófita.

Excessive application of chemical nitrogen fertilisers and pesticides has badly affected the environment. This has led to great interest being shown in studying a crop’s native microbial community and its benefit for plants. This paper was thus aimed at characterising sugarcane’s endophytic microbial community. 5 sugar cane strains and 50 isolates were used. Gas chromatography was used for measuring nitrogenase activity and the influence of carbon and nitrogen sources and pH on cultures. Indol acetic (IAA) production was detected by Dot-Immunobinding and Salkowski’s method. These results show that 19 strains and isolates had nitrogenase activity, values ranging from 100 to 5000 /zg/mL; 6 of them produced IAA (values ranging from 1,7 to 2,5 //g/mL): Gluconacetobacter iazotrophicus PAl-5, Gluconacetobacter diazotrophicus 1-05, Gluconacetobacter diazotrophicus 4-02, 17, 30 and 305. It was demonstrated that culture medium nutrient sources and pH affectedthe nitrogenase activity of the strains representing the endophytic community.

Cloning of ferredoxin I gene from Azotobacter vinelandii using synthetic oligonucleotide probes.

Two synthetic oligonucleotide probe mixtures, whose sequences were inferred from two separate stretches of amino acids, one closer to the carboxy terminal and the other closer to the amino terminal, of ferredoxin I protein of Azotobacter vinelandii, were used to select ferredoxin I gene clones from a cosmid gene library of Azotobacter vinelandii. Restriction analysis revealed that 7 out of 10 selected clones were of the same type. All these clones were found to hybridize with fixABCX genes of Rhizobium meliloti.

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany.

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup+ (S24 and CT2014) and one Hup– (M11) Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H2 uptake rates were higher in 1 and 2% H2 in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H2 uptake were observed both, due to host species as well as due to Rhizobium strains. However, no host dependent complete repression of the expression of H2 uptake activity was observed in nodules of any of the host species formed with Hup+ strains.

Nitrification and simultaneous denitrification by Azospirillum brasilense 12S.

Azospirillum brasilense, an associative diazotrophs from sorghum roots grows autotrophically on NH + 4 and CaCO3. NH + 4 a is also oxidized to NO – 2 and then denitrified. Addition of malate to the autotrophic medium enhances both NH + 4 oxidation as well as NO – 2 dissimilation. The incomplete nitrification linked denitrification results in a rapid loss of nitrogen from the growth medium. The bacterium also shows assimilatory NO3− and NO 2 reductases and fixes nitrogen at < 50 μg N/ml of NH + 4 , NO−3 or NO-2.

In vitro and in vivo nitrogenase activity of Rhizobium mutants and their symbiotic effectivity.

A slow growing nitrogen-fixing strain of Vigna radiata var. aureus (mung bean) Rhizobium which expressed nitrogenase activity in a synthetic medium was isolated from its native population. Mutants with decreased and increased nitrogenase activity were derived from this strain by treatment with acridine orange and ethidium bromide. These mutants were tested for symbiotic effectivity in vivo. The effectivity of mutants with decreased nitrogenase activity in the culture medium was lower than the parent strain; however, the effectivity of mutants with higher nitrogenase activity did not increase above that of the parent. This suggests that the plant is perhaps a limiting factor in the full expression of rhizobial nitrogenase in the nodules.