N2-fixation and growth promotion in cedar colonized by an endophytic strain of Paenibacillus polymyxa

Inoculation of western red cedar with Paenibacillus polymyxa P2b-2R, an endophytic diazotroph of a pine, was previously shown to result in biological nitrogen fixation (BNF) in seedlings grown under N-limited conditions, but biomass accumulation was reduced after a 9-month growth period. To determine if the seedling growth reduction was temporary, we inoculated cedar seed with strain P2b-2R and grew seedlings for up to 13 months in a N-limited soil mix containing 0.7 mM of available N labeled as Ca(¹⁵NO₃)₂. P2b-2R developed a persistent endophytic population comprising 10²–10⁶?cfu g^?1 plant tissue inside pine roots, stems, and needles. At the end of the growth period, P2b-2R had reduced ¹⁵N foliar N abundance by 36 % and increased shoot biomass by 46 % compared to controls. Our results indicate that inoculated seedlings derived 36 % of foliar N from the atmosphere and suggest that BNF by P. polymyxa can significantly enhance growth of cedar in a N-limited soil if seedlings are grown for a sufficient amount of time. These findings support the hypothesis that endophytic diazotrophs may facilitate regeneration and growth of western red cedar at N-poor sites.     

Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues

Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling stem tissue and fixes nitrogen (N) in association with pine and western red cedar (Thuja plicata Donn.). To evaluate endophytic colonization by this microorganism, we generated P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, and grew pine seedlings that were inoculated with the marked strain in a N-limited soil. Tissue disintegration during sample preparation precluded examination of needles for the GFP-labeled endophyte but GFP was detected on roots and in stems of 2- to 14-week-old pine seedlings using confocal laser scanning microscopy. Due to excessive autofluorescence of seedling tissues, labeled bacteria were clearly discernible only in stem tissues of 4- and 6-week-old seedlings. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. Some labeled bacteria appeared to contain endospores and none were detected in vascular tissues. We conclude that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include the stem cortex but that GFP labeling is of limited value for localization of endophytic bacteria in pine seedling tissues.     

nif gene sequence and arrangement in the endophytic diazotroph Paenibacillus polymyxa strain P2b-2R

We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer–polymerase chain reaction (SSP-PCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus.     

Assessment of genetic relatedness among mango cultivars of India using RAPD markers

Summary

DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic relationships in a number of fruit crops. A wide genetic diversity exists in the mango fruit in India. Present day commercial cultivars originated mainly from this subcontinent. In this study, 18 commercial mango cultivars, traditionally grown in western, southern, northern and eastern parts of India, were selected to assess genetic relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, 27 primers amplified mango genomic DNA. None of these primers produced unique band pattern for each cultivar. RAPD data were used to calculate a squared Euclidean distance matrix, and based on this cluster analysis was done using a minimum variance algorithm. Cluster analysis clearly showed two groups—the first consisting of western, northern and eastern mango cultivars and the second group consisting of southern cultivars. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later.     

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.     

Sustaining biodiversity conservation in human-modified landscapes in the Western Ghats: Remnant forests matter

Human-modified tropical landscapes under semi-natural or agro-ecosystems often harbor biodiversity of significant conservation value. In the Western Ghats of India, these ecosystems also provide connectivity between protected areas and other remnant forests. We investigated the conservation value of these landscapes and agro-ecosystems using results from 35 studies covering 14 taxonomic groups. Large, conspicuous taxonomic groups and tree-covered land-use types have received much focus in this area of research in the Western Ghats. We computed a response ratio defined as the log ratio of species richness in human land use to species richness in forest control site from 17 studies. In a meta-analysis, we investigated variation of this ratio across studies with respect to three variables: taxonomic group, the land-use type sampled and the extent of forest cover within the study landscape. Higher forest cover within the landscape emerged as a major positive influence on biodiversity in human-modified landscapes for vertebrates and vegetation while no patterns emerged for invertebrates. Our results suggest that loss of remnant forest patches from these landscapes is likely to reduce biodiversity within agro-ecosystems and exacerbate overall biodiversity loss across the Western Ghats. Conservation of these remnant forest patches through protection and restoration of habitat and connectivity to larger forest patches needs to be prioritized. In the densely populated Western Ghats, this can only be achieved by building partnerships with local land owners and stakeholders through innovative land-use policy and incentive schemes for conservation.     

Techno-economic evaluation of Asian seabass (Lates calcarifer) nursery rearing in small net cages (hapas) under different coastal salinities

Nursery rearing is the critical interim phase in farming of Asian seabass fish (Lates calcarifer), which produce fish fingerlings as an input for grow-out farming. The present study evaluated the techno-economic performance of seabass nursery rearing in low and high saline coastal waters. The results indicated that seabass nursery rearing is technically efficient with a mean technical efficiency of 99.83% and 92.45%, respectively under low and high saline conditions. The mean survival of young fishes was 63.50% and 42.50% with a mean daily weight gain of 0.08 g and 0.15 g, respectively in low and high saline waters. While the benefit cost ratio (BCR) was 2.76 and 1.9, the internal rate of return (IRR) was calculated to be 300% and 130%, respectively, indicating the economic viability of nursery rearing under different salinity regimes. Furthermore, it was observed that nursery rearing in low saline waters was more efficient and highly remunerative. In the Indian socio-economic scenario, a mean monthly income per person spending 2 h per day was found to be 129 USD and 317 USD respectively in high and low saline nursery systems which is a considerable earning. The results explicitly stated that nursery rearing itself is an exclusively livelihood development activity for the coastal fisher families with an active participation of fisher women. Establishment of finfish hatcheries to ensure continued supply of seabass seeds is the key factor in facilitating wider adoption of nursery rearing as a sustainable farming activity.

     

Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.     

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA₃). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.This revised version was published online in November 2005 with corrections to the Cover Date.