Interaction of endophytic microbes with legumes.

Large numbers of bacterial and fungal endophytes have been reported from different plant tissues: roots, nodules, leaves, flowers and sprouts of legumes, with numbers ranging from few to more than 150. Endophytes can accelerate seedling emergence, promote plant establishment under adverse conditions and enhance plant growth. Endophytic microbes promote plant growth by helping plants in acquiring nutrients, e.g. via nitrogen fixation, phosphate solubilization or iron chelation, by preventing pathogen infections via antifungal or antibacterial agents, by outcompeting pathogens for nutrients by siderophore production, or by establishing the plant's systemic resistance. Further growth promotion is affected by producing phytohormones such as auxin or cytokinin, or by producing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which lowers plant ethylene levels. For establishment of endophytes in different tissues, endophytic microbes must be compatible with the host plants and able to colonize the tissues of the host plants without being recognized as pathogens. A particular bacterium or fungus may affect plant growth and development using one or more of these mechanisms, and they may use different mechanisms at various times. The population density of endophytes is highly variable, depending mainly on the microbial species and host genotypes, developmental stage and environmental conditions. Genotypic and cultivar specific endophytes have also been reported. The quantum benefit derived by plants from an endophyte and vice versa is still not clear. It seems that the endophytic genus or species best adapted for living inside a plant is naturally selected. Here, we concentrate on soil or rhizosphere-derived endophytes recruited out of a large pool of soil or rhizospheric microbes. Some endophytes are more aggressive colonizers and displace others, but seeming lack of strict specificity has been observed. However, the processes of host-microbe signaling and colonization and the mechanisms leading to mutual benefits are less-well characterized. It is still not clear which population of microorganisms (endophytes or rhizospheric) promotes plant growth and the way the interactions among endophytes influence plant productivity. Though attempts to know the molecular ecology and interactions are underway, a high amount of progress is required to fully understand the mechanism of establishment, the way interactions take place in planta, between different microbes and plants and exlusive benefits by endophytes and plants.

Reactive oxygen species measurement in neat and washed semen: comparative analysis and its significance in male infertility assessment.

PURPOSE: Oxidative stress (OS) is a major concern in idiopathic male infertility as elevated levels of reactive oxygen species (ROS) in the semen is believed to adversely affect sperm functional competence and damage both nuclear and mitochondrial DNA. Therefore, identifying infertile men with seminal OS may be used as a valuable tool in the assessment of male infertility. Semen is a complex mixture of spermatozoa and seminal plasma which is rich in enzymatic and non-enzymatic antioxidants. However, the measurement of ROS in the semen and its application in male infertility assessment is poorly understood. METHODS: The aim of the present study was to compare the significance of ROS measurement in washed and neat semen. The study included 65 infertile men with abnormal semen (SA) parameters, 17 infertile men with normal semen (NS) parameters and 43 fertile controls (FC). ROS levels in both washed and neat semen were measured by luminol-dependent chemiluminescence assay and the values were expressed as 10(4 )RLU per minute per 20 million spermatozoa. RESULTS: The levels of ROS in both washed and neat semen were found to be significantly higher (P < 0.0001) in infertile men with abnormal and normal semen parameters as compared with FC (P < 0.0001). The lowest median level of ROS was found in FC (neat semen 0.03, washed semen 0.24), whereas infertile men with SA group had the highest median ROS level (neat semen 3.44, washed semen 27.42). In all subjects, ROS levels were found to be higher in washed semen than in neat semen. A strong positive correlation (r = 0.847, P < 0.0001) of ROS levels between neat and washed semen was observed. CONCLUSION: Measurement of ROS in neat semen reflects the original oxidative status in the actual sperm microenvironment and is an easy, simple, inexpensive and rapid method compared with ROS measurement from washed semen. ROS measurement in washed semen is done to assess ROS levels following sperm processing as in cases opting for assisted conception. As both ROS values in neat and washed show a positive correlation, neat semen may be used as a valuable tool for identifying infertile men with seminal OS. ROS levels are elevated in nearly 70% men with idiopathic infertility. Hence, ROS analysis in neat semen has both good diagnostic and prognostic value in male infertility assessment.

Molecular diversity of native rhizobia trapped by five field pea genotypes in Indian soils.

Five pea cultivars; HFP 4, HVP 3-5, HFP 9426, Jayanti and Hariyal, being grown in CCS Haryana Agricultural University farm were used to isolate native rhizobia. Selected 54 rhizobia, from all cultivars, were authenticated as rhizobia by plant infectivity test. Along with nodulation, symbiotic effectiveness in terms of symbiotic ratios showed wide range of effectiveness of pea rhizobia from 1.11 to 5.0. DNA of all the 54 rhizobia was extracted and amplified by PCR, using ERIC and 16S rDNA primers. Dendrogram based on ERIC profiles of these 54 rhizobia showed the formation of 13 subclusters at 80% level of similarity. Dendrogram based on RFLP of 16S rDNA by three restriction endonucleases; Msp I, Csp 6I and Rsa I; also formed 13 subclusters at 80% level of similarity. However, positioning of subclusters was different from that of ERIC based dendrogram. Majority of the isolates i.e. 64.8% by ERIC profiles and 44.4% by RFLP of 16S rDNA formed one cluster. Isolates from same nodule were not 100% similar. Considering each cluster representing a rhizobial genotype, both techniques used to assess molecular diversity indicated the presence of 13 genotypes of field pea rhizobia in CCS Haryana Agricultural University farm soil. Two pea rhizobial genotypes were able to nodulate all the five pea cultivars. Furthermore, high strain richness index (0.43-0.5) of field pea rhizobia was observed by both the techniques.

Molecular diversity of a native mesorhizobial population of nodulating chickpea (Cicer arietinum L.) in Indian soils.

Chickpea plants with nodules were collected from 32 different farmers' fields of eight districts of Haryana state. In total, 137 mesorhizobial isolations were made from these nodules and authenticated. Finally, 50 mesorhizobia were selected based on nodulation test, growth characteristics, and site of sampling. The molecular diversity of the mesorhizobial population was assessed by PCR-amplified ERIC profiles as well as RFLP of 16S rDNA. Considerable molecular diversity in Haryana soils was observed. Chickpea rhizobia were grouped into six different clusters at the 70% similarity level by both methodologies, but clustering of the strains was different. Considering that each cluster represented a mesorhizobial genotype, Haryana soils showed a high richness index (0.46), and RFLP analysis showed that the mesorhizobial genotype (MG) I was present in 38% of nodules, followed by MG III which was detected in 34% of the nodules. The distribution of different MG in Haryana soils showed that all six types of rhizobia were never present in any of the districts; however, a maximum of five types were present in the Bhiwani district. Rhizobial genotype III was invariably present in all the nodule samples tested.

Molecular diversity, effectiveness and competitiveness of indigenous rhizobial population infecting mungbean Vigna radiata (L. Wilczek) under semi-arid conditions.

Nodules from mungbean crop raised for the first time at Ram Dhan Singh (RDS) farm of Chaudhary Charan Singh (CCS) Haryana Agricultural University, Hisar were collected from 17 different locations. Twenty-five mungbean rhizobia were isolated and authenticated by plant infection test. DNA of all these rhizobia was extracted purified and amplified using enterobacterial repetitive intergenic consensus (ERIC) primers. All the mungbean rhizobial isolates were clustered into 4 groups at 65% of similarity and were further divided into 17 subclusters at 80% of similarity. All the 4 types of rhizobia were not present at any of the location and group 2 or 4 rhizobia were invariably present. Efficacy of these rhizobia in terms of nodulation, nitrogen uptake and chlorophyll a fluorescence was determined under pot culture conditions. Strain MB 307 showed maximum nitrogen uptake of 31.9 mg N plant(-1) followed by strain MB 1205, MB 1206(2), MB 308, MB 1524 and strain MB 1521 was found to be the least efficient in terms of N 2 fixation. Nodule occupancy by different rhizobia ranged from 5.5 to 40.3%. Most of the strains belonging to the 2nd group which clustered maximum number of strains were comparatively better competitors and formed 19.5-40.3% of the nodules and were also effective. Isolate MB 307, the most efficient strain, was found to have nodule occupancy of 31.5%. Such type of predominant, efficient and better competitor strains should be selected for enhancing nodule competitiveness.

Effectivity of host-Rhizobium leguminosarum symbiosis in soils receiving sewage water containing heavy metals.

Disposal of sewage water in cultivated soils often containing considerable amount of potentially toxic metals such as Cu, Zn, Ni, Cd, Pb and Cr can be beneficial or harmful to plant growth, rhizobial survival, nodulation and nitrogen fixation. Soil samples from 14 such locations were collected. Symbiotic effectivity of host-Rhizobium leguminosarum symbiosis in these soils was assessed. The total metal contents of Cd, Cu, Zn and Ni in all the 14 samples collected from farmer's fields receiving sewage water ranged between 1.3 and 6.7, 55.8-353.2, 356.0-1028.0 and 90.0-199.7 mg kg(-1) of soil, respectively. In Rohtak 1 soil, levels of Cd, Cu and Zn were highest while Ni was highest in Sonipat 2 soil. The content of available Cd, Cu, Zn and Ni in these soils ranged from 1.0-29.3; 6.2-47.0; 2.4-13.5, respectively, and was 2-9 percent of their total metal contents. All the N2 fixing parameters in pea and Egyptian clover were adversely affected by the presence of heavy metals. Available Cd and Cu contents significantly affected the N contents of pea and Egyptian clover plants, whereas Ni contents were negatively correlated with the plant biomass of pea and Egyptian clover.

Effect of temperature of thawing and diluent on the post--thaw physiological changes of buffalo frozen semen.

The effect of thawing was studied in buffalo semen diluted in three diluents (Tris egg-yolk, Egg-yolk citrate and Citric Acid whey) at three temperatures (5 degrees C, 35 degrees C and 75 degrees C) on motility, eosin staining, morphological and acrosomal changes, hyaluronidase, glutamic oxalacetic transaminase and glutamic pyruvic transaminase activities. The motility and lack of staining of sperm by eosin were maximum on thawing at 35 degrees C and in tris egg-yolk diluent followed by egg-yolk citrate and citric acid whey. Hyaluronidase, glutamic oxalacetic transaminase and glutamic pyruvic transaminase increased significantly in the extra-cellular fluid on thawing of semen diluted with all the three diluents. The buffalo semen diluted in tris egg-yolk and thawed at 35 degrees C for 30 seconds gave the best results.