Transfer of Downy Mildew Resistance from Wild Basil (Ocimum americanum) to Sweet Basil (O. basilicum).

Sweet basil (Ocimum basilicum) is susceptible to downy mildew caused by the oomycete foliar pathogen Peronospora belbahrii. No resistant varieties of sweet basil are commercially available. Here, we report on the transfer of resistance gene Pb1 from the highly resistant tetraploid wild basil O. americanum var. americanum (PI 500945, 2n = 4x = 48) to the tetraploid susceptible O. basilicum 'Sweet basil' (2n = 4x = 48). F1 progeny plants derived from the interspecific hybridization PI 500945 × Sweet basil were resistant, indicating that the gene controlling resistance (Pb1) is dominant, but sterile due to the genetic distance between the parents. Despite their sterility, F1 plants were pollinated with the susceptible parent and 115 first backcross generation to the susceptible parent (BCs1) embryos were rescued in vitro. The emerging BCs1 plants segregated, upon inoculation, 5:1 resistant/susceptible, suggesting that resistance in F1 was controlled by a pair of dominant genes (Pb1A and Pb1A'). Thirty-one partially fertile BCs1 plants were self-pollinated to obtain BCs1-F2 or were backcrossed to Sweet basil to obtain the second backcross generation to the susceptible parent (BCs2). In total, 1 BCs1-F2 and 22 BCs2 progenies were obtained. The BCs1-F2 progeny segregated 35:1 resistant/susceptible, as expected from a tetraploid parent with two dominant resistant genes. The 22 BCs2 progenies segregated 1:1 resistant/susceptible (for a BCs1 parent that carried one dominant gene for resistance) or 5:1 (for a BCs1 parent that carried two dominant genes for resistance) at a ratio of 4:1. The data suggest that a pair of dominant genes (Pb1A and Pb1A') residing on a two homeologous chromosomes is responsible for resistance of PI 500945 against P. belbahrii.

Fungal Endophyte Diversity and Bioactivity in the Indian Medicinal Plant Ocimum sanctum Linn.

Endophytic mycopopulation isolated from India's Queen of herbs Tulsi (Ocimum sanctum) were explored and investigated for their diversity and antiphytopathogenic activity against widespread plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum. 90 fungal isolates, representing 17 genera were recovered from 313 disease-free and surface sterilised plant segments (leaf and stem tissues) from three different geographic locations (Delhi, Hyderabad and Mukteshwar) during distinct sampling times in consequent years 2010 and 2011 in India. Fungal endophytes were subjected to molecular identification based on rDNA ITS sequence analysis. Plant pathogens such as F. verticillioides, B. maydis, C. coarctatum, R. bataticola, Hypoxylon sp., Diaporthe phaseolorum, Alternaria tenuissima and A. alternata have occurred as endophyte only during second sampling (second sampling in 2011) in the present study. Bi-plot generated by principal component analysis suggested tissue specificity of certain fungal endophytes. Dendrogram revealed species abundance as a function of mean temperature of the location at the time of sampling. Shannon diversity in the first collection is highest in Hyderabad leaf tissues (H' = 1.907) whereas in second collection it was highest from leaf tissues of Delhi (H' = 1.846). Mukteshwar (altitude: 7500 feet) reported least isolation rate in second collection. Nearly 23% of the total fungal isolates were considered as potent biocontrol agent. Hexane extract of M. phaseolina recovered from Hyderabad in first collection demonstrated highest activity against S. sclerotiorum with IC50 value of 0.38 mg/ml. Additionally, its components 2H-pyran-2-one, 5,6-dihydro-6-pentyl and palmitic acid, methyl ester as reported by GC-MS Chromatogram upon evaluation for their antiphytopathogenic activity exhibited IC50 value of 1.002 and 0.662 against respectively S. sclerotiorum indicating their significant role in antiphytopathogenic activity of hexane extract. The production of 2H-pyran-2-one, 5,6-dihydro-6-pentyl from M. phaseolina, an endophytic fungus is being reported for the first time.

Resistance Against Basil Downy Mildew in Ocimum Species.

Downy mildew, caused by the oomycete Peronospora belbahrii, is a devastating disease of sweet basil. In this study, 113 accessions of Ocimum species (83 Plant Introduction entries and 30 commercial entries) were tested for resistance against downy mildew at the seedling stage in growth chambers, and during three seasons, in the field. Most entries belonging to O. basilicum were highly susceptible whereas most entries belonging to O. americanum, O. kilimanadascharicum, O. gratissimum, O. campechianum, or O. tenuiflorum were highly resistant at both the seedling stage and the field. Twenty-seven highly resistant individual plants were each crossed with the susceptible sweet basil 'Peri', and the F1 progeny plants were examined for disease resistance. The F1 plants of two crosses were highly resistant, F1 plants of 24 crosses were moderately resistant, and F1 plants of one cross were susceptible, suggesting full, partial, or no dominance of the resistance gene(s), respectively. These data confirm the feasibility of producing downy mildew-resistant cultivars of sweet basil by crossing with wild Ocimum species.

Bioactive polyketides and alkaloids from Penicillium citrinum, a fungal endophyte isolated from Ocimum tenuiflorum.

Chemical investigation of the endophytic fungus Penicillium citrinum cultured on white beans or on rice led to the isolation of two new alkaloids (1 and 2), along with fourteen known polyketides (6-12, 14-20) and four known alkaloids (3-5, and 13). The structures of the isolated compounds were determined by extensive analysis of the 1D, 2D NMR, and MS data, and by comparison with the literature. Compound 13, which had been previously obtained only by chemical synthesis, was isolated as a natural product for the first time, while compound 6 was firstly reported as a fungal metabolite. A re-isolation of sclerotinin A (14) revealed it to be a diastereoisomeric mixture (14a and 14b), whose stereochemistry was proposed for the first time based on ROESY experiment. All isolated compounds were evaluated for their cytotoxic and antibacterial activities. Compounds 12 and 17 showed significant cytotoxicity against the murine lymphoma cell line L5178Y with IC50 values of 1.0, and 0.78 µg/ml, respectively, while compounds 5, 11, and 15 were moderately active against Staphylococcus aureus ATCC 29213 (MIC 64 µg/ml).

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.

Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion.

Molecular and functional characterization of endophytic fungi from traditional medicinal plants.

This study reports the isolation of 63 endophytic fungal isolates from two traditional medicinal plants, Ocimum sanctum and Sapindus detergens from different locations of Amritsar, India. The functional characterization of the fungi for their ability to produce anti bacterial and anti cancer agent was carried out. Sixteen strains were characterized at molecular level by sequencing the amplified ITSI-5.8-ITSII region of rDNA. The phylogenetic tree resolved the endophytic fungi into different clades. The fungal endophytes belonging to order Pleosporales (Alternaria sp., Phoma sojicola and Exserohilum sp.) were functionally versatile as they produced diverse biomolecules including antibacterial agent active against Mycobacterium smegmatis, as well as cytotoxic activity against different human cancer cell lines of lung, ovary, breast, prostrate, neuroblastoma and colon.

Endophytic bacteria from Ocimum sanctum and their yield enhancing capabilities.

Endophytes are beneficial microbes that reside intercellularly inside the plants. Interaction of endophytes with the host plants and their function within their host are important to address ecological relevance of endophyte. Four endophytic bacteria OS-9, OS-10, OS-11, and OS-12 were isolated from healthy leaves of Ocimum sanctum. These isolated microbes were screened in dual culture against various phytopathogenic fungi viz. Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani, Alternaria solani, and Colletotrichum lindemuthianum. Of these, strain OS-9 was found to be antagonistic to R. solani, A. solani, F. solani, and C. lindemuthianum while OS-11 was found antagonistic against A. solani only. The growth-promoting benefits of the endophytes were initially evaluated in the glasshouse by inoculated seeds of O. sanctum. Treatment with endophytes OS-10 and OS-11 resulted in significant enhancement of growth as revealed by increase in fresh as well as dry weight. Further, field trials involving two genotypes OS Purple and CIM-Angana were conducted with strains OS-10 and OS-11. The growth-promoting effect was visible on both the genotypes tested as the endophytes significantly enhanced fresh herbage yield (t/ha). Interestingly, these endophytes increased the content of essential oil particularly in cultivar OS Purple and thereby increasing the total oil yields. Molecular characterization of strain OS-11 indicated the strain to be highly related to the type strain of Bacillus subtilis.

Packed with Salmonella--investigation of an international outbreak of Salmonella Senftenberg infection linked to contamination of prepacked basil in 2007.

Salmonella Senftenberg is uncommon in the United Kingdom. In January-June 2007, the Health Protection Agency reported on 55 primary human cases of Salmonella Senftenberg in England and Wales. In May 2007, fresh basil sold in the United Kingdom was found to be contaminated with Salmonella Senftenberg. We launched an investigation to elucidate the cause of this outbreak. Isolates were examined using plasmid profiling and pulsed-field gel electrophoresis, and the outbreak strain (SSFTXB.0014) was identified. We enquired via Enter-net whether other countries had isolated the outbreak strain, analyzed samples of fresh herbs from U.K. retailers, and interviewed patients on food history. Thirty-two patient-cases were referred to this outbreak in England and Wales. Onsets of illness occurred between 5 March and 6 June 2007. Fifty-six percent of patient-cases were females and 90% adults (>20 years old); three were admitted to hospital as a result of Salmonella infection. Scotland, Denmark, the Netherlands, and the United States reported on 19 cases of Salmonella Senftenberg infection presenting with the outbreak strain since January 2007. Eight samples of prepacked fresh basil imported from Israel tested positive with the same strain. A minority of patients could recall the consumption of basil before illness, and some reported consumption of products where basil was a likely ingredient. Environmental investigations in Israel did not identify the contamination source. Microbiological evidence suggested an association between contamination of fresh basil and the cases of Salmonella Senftenberg infection, leading to withdrawal of basil from all potentially affected batches from the U.K. market.

Studies on rhizosphere microbial diversity of some commercially important medicinal plants.

A preliminary study was made on four medicinal plants viz., Ocimum sanctum L., Coleus forskholii Briq, Catharanthus roseus (L.) G. Don. and Aloe vera in order to identify and enumerate the rhizosphere, non-rhizosphere and diazotrophic microorganisms in soil. The diazotrophic bacterial population studied includes Azospirillum, Azotobacter and Pseudomonas. The rhizosphere bacterial populations were 23.33 x 10(6)g(-1) in O. sanctum followed by C. roseus (20.46 x 10(6)g(-1)), A. vera (18.44 x 10(6)g(-1)) and C. forskholii (16.64 x 10(6)g(-1)). The fungi populations were 19.44 x 10(4)g(-1) in C. roseus, 18.66 x 10(4)g(-1) in O. sanctum, 16.44 x 10(4)g(-1) in A. vera and 14.22 x 10(4)g(-1) in C. forskholii. The actinomycetes population was 12.22 x 10(5)g(-1) in O. sanctum, 10.44 x 10(5)g(-1) in C. roseus, 8.44 x 10(5)g(-1) in A. vera and 6.22 x 10(5)g(-1) in C. forskholii. The diazotrophic bacterial population of Azospirillum, Azotobacter and Pseudomonas is 8.2 x 10(4)g(-1), 12 x 10(4)g(-1), 6 x 10(4)g(-1) in the rhizosphere soil. In all the four medicinal plants the microbial population is more in the rhizosphere soil, when compared to non-rhizosphere soil. These results are helpful in developing a biofertilizer consortium for these commercially grown medicinal plants.

Isolation and identification of a novel strain of Pseudomonas chlororaphis capable of transforming isoeugenol to vanillin.

Vanillin is undoubtedly one of the most popular and widely used flavoring agents in the world. Taking into consideration the worldwide demand for natural vanillin and its limited supply, alternative routes for its production including biotransformation are being constantly explored. In this regard, a novel soil bacterium capable of converting isoeugenol to vanillin was isolated by conventional enrichment process from soils of Ocimum field. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolate was identified as Pseudomonas chlororaphis CDAE5 (EMBL # AM158279). Vanillin formation was analyzed by gas chromatography (GC), and its structure was confirmed by GC-mass spectrometry and nuclear magnetic resonance. After 24-h reaction, the vanillin concentration reached 1.2 g L(-1) from 10 g L(-1) isoeugenol in 20-mL reaction solution at 25 degrees C and 180 rpm. The strain showed potential to be a good candidate for biotechnological production of vanillin from isoeugenol. Further studies for standardization and optimization for higher yield of vanillin production needs to be investigated.