Inhibition of plant pathogenic fungi by endophytic Trichoderma spp. through mycoparasitism and volatile organic compounds.

Antagonism of plant pathogenic fungi by endophytic fungi is a well-known phenomenon. In plate assays, the antagonism could be due to mycoparasitism, competition for space or antibiosis, involving a chemical diffusate, or a volatile organic compound (VOC). In this study, we demonstrate that besides mycoparasitism, VOCs play a major role in antagonism of pathogenic fungi by four endophytic fungi belonging to the genus Trichoderma. Using a double-plate assay, we show that all the four endophytic Trichoderma species significantly inhibited mycelial growth of three of the four pathogens, (Sclerotinia sclerotiorum-TSS, Sclerotium rolfsii-CSR and Fusarium oxysporum-CFO), while that of Macrophomina phaseolina-CMP was not affected. GC-MS analysis of the pure cultures of one of the endophytic fungi studied, namely, Trichoderma longibrachiatum strain 2 (Acc. No. MK751758) and the pathogens, F. oxysporum-CFO and M. phaseolina-CMP revealed the presence of several VOCs including hydrocarbons, alcohols, ketones, aldehydes, esters, acids, ethers and different classes of terpenes. In mixed double plates, where the endophyte was grown along with either of the two plant pathogens, F. oxysporum-CFO or M. phaseolina-CMP, there was an induction of a number of new VOCs that were not detected in the pure cultures of either the endophyte or the pathogens. Several of these new VOCs are reported to possess antifungal and cytotoxic activity. We discuss these results and highlight the importance of such interactions in endophyte-pathogen interactions.

An endophyte from salt-adapted Pokkali rice confers salt-tolerance to a salt-sensitive rice variety and targets a unique pattern of genes in its new host.

Endophytes, both of bacterial and fungal origin, are ubiquitously present in all plants. While their origin and evolution are enigmatic, there is burgeoning literature on their role in promoting growth and stress responses in their hosts. We demonstrate that a salt-tolerant endophyte isolated from salt-adapted Pokkali rice, a Fusarium sp., colonizes the salt-sensitive rice variety IR-64, promotes its growth under salt stress and confers salinity stress tolerance to its host. Physiological parameters, such as assimilation rate and chlorophyll stability index were higher in the colonized plants. Comparative transcriptome analysis revealed 1348 up-regulated and 1078 down-regulated genes in plants colonized by the endophyte. Analysis of the regulated genes by MapMan and interaction network programs showed that they are involved in both abiotic and biotic stress tolerance, and code for proteins involved in signal perception (leucine-rich repeat proteins, receptor-like kinases) and transduction (Ca2+ and calmodulin-binding proteins), transcription factors, secondary metabolism and oxidative stress scavenging. For nine genes, the data were validated by qPCR analysis in both roots and shoots. Taken together, these results show that salt-adapted Pokkali rice varieties are powerful sources for the identification of novel endophytes, which can be used to confer salinity tolerance to agriculturally important, but salt-sensitive rice varieties.

UPLC and ESI-MS analysis of metabolites of Rauvolfia tetraphylla L. and their spatial localization using desorption electrospray ionization (DESI) mass spectrometric imaging.

Rauvolfia tetraphylla L. (family Apocynaceae), often referred to as the wild snakeroot plant, is an important medicinal plant and produces a number of indole alkaloids in its seeds and roots. The plant is often used as a substitute for Ravuolfia serpentine (L.) Benth. ex Kurz known commonly as the Indian snakeroot plant or sarphagandha in the preparation of Ayurvedic formulations for a range of diseases including hypertension. In this study, we examine the spatial localization of the various indole alkaloids in developing fruits and plants of R. tetraphylla using desorption electrospray ionization mass spectrometry imaging (DESI-MSI). A semi-quantitative analysis of the various indole alkaloids was performed using UPLC-ESI/MS. DESI-MS images showed that the distribution of ajmalcine, yohimbine, demethyl serpentine and mitoridine are largely localized in the fruit coat while that for ajmaline is restricted to mesocarp of the fruit. At a whole plant level, the ESI-MS intensities of many of the ions were highest in the roots and lesser in the shoot region. Within the root tissue, except sarpagine and ajmalcine, all other indole alkaloids occurred in the epidermal and cortex tissues. In leaves, only serpentine, ajmalcine, reserpiline and yohimbine were present. Serpentine was restricted to the petiolar region of leaves. Principal component analysis based on the presence of the indole alkaloids, clearly separated the four tissues (stem, leaves, root and fruits) into distinct clusters. In summary, the DESI-MSI results indicated a clear tissue localization of the various indole alkaloids, in fruits, leaves and roots of R. tetraphylla. While it is not clear of how such localization is attained, we discuss the possible pathways of indole alkaloid biosynthesis and translocation during fruit and seedling development in R. tetraphylla. We also briefly discuss the functional significance of the spatial patterns in distribution of metabolites.

Mechanism of Resistance to Camptothecin, a Cytotoxic Plant Secondary Metabolite, by Lymantria sp. Larvae.

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of eukaryotic topoisomerase I (Top 1). Because of this property, several derivatives of CPT are widely used as chemotherapeutic agents. The compound is produced by several plant species, including Nothapodytes nimmoniana (Family: Icacinaceae) presumably as a deterrent to insect pests. Here, we report, a lepidopteran larva, Lymantria sp. of Lymantriidae family which feeds voraciously on the leaves of N. nimmoniana, without any adverse consequences. Larval body weight and molting period were unaffected despite captive feeding of the larva with CPT enriched leaves. Mass spectrometric analysis indicated that nearly 46% of the ingested CPT was excreted while the rest was sequestered predominantly in the exuviae and setae (~35%). Although most of the CPT was in the parental form as found in the plant, traces of inactive, sulfated forms of CPT were recovered from the larva. Compared to that in plant, there were no critical mutations at the CPT binding domain of the insect's Top 1. The gut pH of the larva was alkaline (pH 10.0). The alkaline gut environment converts CPT from its active, lactone form to inactive, carboxylate form. It is likely that such conversion might help the larva to reduce the overall burden of CPT in its gut. We discuss the results in the context of the mechanisms of resistance adapted by insects to plant toxins.

Camptothecin-producing endophytic bacteria from Pyrenacantha volubilis Hook. (Icacinaceae): A possible role of a plasmid in the production of camptothecin.

BACKGROUND: Camptothecin (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this property, several derivatives of CPT are used as chemotherapeutic agents. CPT is produced by several plant species belonging to the Asterid clade as well as by a number of endophytic fungal associates of these plants. In this study, we report the production of CPT by four bacterial endophytes and show the possible role of a plasmid in the biosynthesis of CPT. METHODS: Endophytic bacteria were isolated from leaves, stems and fruits of Pyrenacantha volubilis Hook. (Icacinanceae). The bacterial isolates were purified and analyzed for production of CPT by ESI-MS/MS and NMR analysis. Bacterial identity was established based on the morphology and 16s rRNA sequence analysis. Crude extracts of the bacterial endophytes were evaluated for their cytotoxicity using colon cancer cell lines. The role of plasmid in the production of CPT was studied by purging the plasmid, using acriflavine, as well as reconstituting the bacteria with the plasmid. RESULTS: Four bacterial isolates, Bacillus sp. (KP125955 and KP125956), Bacillus subtilis (KY741853) and Bacillus amyloliquefaciens (KY741854) were found to produce CPT in culture. Both based on ESI-MS/MS and NMR analysis, the identity of CPT was found to be similar to that produced by the host plant. The CPT was biologically active as evident by its cytotoxicity against colon cancer cell line. The production of CPT by the endophyte (Bacillus subtilis, KY741853) attenuated with sub-culture. A likely role of a plasmid in the production of CPT was established. A 5 kbp plasmid was recovered from the bacteria. Bacterial isolate cured of plasmid failed to produce CPT. CONCLUSION: Our study implies a possible role of a plasmid in the production of CPT by the endophytic bacteria and opens up further work to unravel the exact mechanisms that might be involved.

Species Adulteration in the Herbal Trade: Causes, Consequences and Mitigation.

The global economy of the international trade of herbal products has been increasing by 15% annually, with the raw material for most herbal products being sourced from South and Southeast Asian countries. In India, of the 8000 species of medicinal plants harvested from the wild, approximately 960 are in the active trade. With increasing international trade in herbal medicinal products, there is also increasing concern about the widespread adulteration and species admixtures in the raw herbal trade. The adverse consequences of such species adulteration on the health and safety of consumers have only recently begun to be recognised and documented. We provide a comprehensive review of the nature and magnitude of species adulteration in the raw herbal trade, and identify the underlying drivers that might lead to such adulteration. We also discuss the possible biological and chemical equivalence of species that are used as adulterants and substitutes, and the consequences thereof to consumer health and safety, and propose a framework for the development of a herbal trade authentication service that can help regulate the herbal trade market.

An endophytic fungus, Gibberella moniliformis from Lawsonia inermis L. produces lawsone, an orange-red pigment.

Lawsone (2-hydroxy-1, 4-napthoquinone), also known as hennotannic acid, is an orange red dye used as a popular skin and hair colorant. The dye is produced in the leaves of Lawsonia inermis L, often referred to as the "henna" tree. In this study, we report the production of lawsone by an endophytic fungus, Gibberella moniliformis isolated from the leaf tissues of Lawsonia inermis. The fungus produced the orange-red dye in potato dextrose agar and broth, independent of the host tissue. Presence of lawsone was confirmed spectrometrically using HPLC and ESI-MS/MS analysis. The fragmentation pattern of lawsone was identical to both standard lawsone and that extracted from plant tissue. This is a first report of lawsone being produced by an endophytic fungus, independent of the host tissue. The study opens up interesting questions on the possible biosynthetic pathway through which lawsone is produced by the fungus.

Restoration of camptothecine production in attenuated endophytic fungus on re-inoculation into host plant and treatment with DNA methyltransferase inhibitor.

Fungal endophytes inhabit living tissues of plants without any apparent symptoms and in many cases are known to produce secondary metabolites similar to those produced by their respective host plants. However on sub-culture, the endophytic fungi gradually attenuate their ability to produce the metabolites. Attenuation has been a major constraint in realizing the potential of endophytic fungi as an alternative source of plant secondary metabolites. In this study, we report attempts to restore camptothecine (CPT) production in attenuated endophytic fungi isolated from CPT producing plants, Nothapodytes nimmoniana and Miquelia dentata when they are passed through their host plant or plants that produce CPT and when treated with a DNA methyl transferase inhibitor. Attenuated endophytic fungi that traversed through their host tissue or plants capable of synthesizing CPT, produced significantly higher CPT compared to the attenuated fungi. Attenuated fungus cultured in the presence of 5-azacytidine, a DNA methyltransferase inhibitor, had an enhanced CPT content compared to untreated attenuated fungus. These results indicate that the attenuation of CPT production in endophytic fungi could in principle be reversed by eliciting some signals from plant tissue, most likely that which prevents the methylation or silencing of the genes responsible for CPT biosynthesis.

Ambient ionization mass spectrometry imaging of rohitukine, a chromone anti-cancer alkaloid, during seed development in Dysoxylum binectariferum Hook.f (Meliaceae).

Rohitukine, a chromone alkaloid, possesses anti-inflammatory, anti-cancer and immuno-modulatory properties. It has been reported from four species, belonging to the families, Meliaceae and Rubiaceae. Stem bark of Dysoxylum binectariferum (Meliaceae) accumulates the highest amount of rohitukine (3-7% by dry weight). In this study, we examine the spatial and temporal distribution of rohitukine and related compounds during various stages of seed development in D. binectariferum using desorption electrospray ionization mass spectrometry imaging (DESI MSI). Rohitukine (m/z 306.2) accumulation increased from early seed development to seed maturity stage. The spatial distribution of rohitukine was largely restricted to the cotyledonary tissue followed by the embryo and least in the seed coat. Besides rohitukine, rohitukine acetate (m/z 348.2) and glycosylated rohitukine (m/z 468.2) were also detected, both through mass fragmentation and exact mass analysis through Orbitrap mass spectrometry. These results indicate a dynamic pattern of chromane alkaloid accumulation through seed development in D. binectariferum.

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: bioactivity-guided isolation and LCMS-based quantification.

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.

Isolation of endophytic bacteria producing the anti-cancer alkaloid camptothecine from Miquelia dentata Bedd. (Icacinaceae).

Camptothecine (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this activity, several semi-synthetic derivatives of CPT are in clinical use against ovarian and small lung cancers. Together with its derivatives, CPT is the third largest anti-cancer drug in the world market. CPT is produced by several plant species belonging to the Asterid clade. In the recent past, several studies have reported the production of CPT by endophytic fungal associates of some of these plant species. In this paper, we report the production of CPT by endophytic bacteria isolated from Miquelia dentata Bedd. (Icacinaceae). Besides CPT, the bacteria also produced 9-methoxy CPT (9-MeO-CPT), in culture, independent of the host tissue. The chemical nature of CPT and 9-MeO-CPT was determined by LC-MS and ESI-MS/MS analysis, and was shown to be similar to that produced by the host tissue. One of the bacterial isolates examined, showed indications of attenuation of CPT production through sub-culture. This is the first report of production of CPT by endophytic bacteria. The identity of the bacteria was ascertained by Gram staining and 16s rRNA sequencing. We discuss the possible mechanisms that might be involved in the synthesis of CPT by endophytic bacteria.

Biodiversity and chemodiversity: future perspectives in bioprospecting.

Biological diversity and its constituent chemical diversity have served as one of the richest sources of bioprospecting leading to the discovery of some of the most important bioactive molecules for mankind. Despite this excellent record, in the recent past, however, bioprospecting of biological resources has met with little success; there has been a perceptible decline in the discovery of novel bioactive compounds. Several arguments have been proposed to explain the current poor success in bioprospecting. Among them, it has been argued that to bioprospect more biodiversity may not necessarily be productive, considering that chemical and functional diversity might not scale with biological diversity. In this paper, we offer a critique on the current perception of biodiversity and chemodiversity and ask to what extent it is relevant in the context of bioprospecting. First, using simple models, we analyze the relation among biodiversity, chemodiversity and functional redundancies in chemical plans of plants and argue that the biological space for exploration might still be wide open. Second, in the context of future bioprospecting, we argue that brute-force high throughput screening approaches alone are insufficient and cost ineffective in realizing bioprospecting success. Therefore, intelligent or non-random approaches to bioprospecting need to be adopted. We review here few examples of such approaches and show how these could be further developed and used in the future to accelerate the pace of discovery.

Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn (Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin.

Camptothecin and 10-hydroxycamptothecin are two important precursors for the synthesis of the clinically useful anticancer drugs, topotecan and irinotecan. In recent years, efforts have been made to identify novel plant and endophytic fungal sources of camptothecin and 10-hydroxycamptothecin. In this study we have isolated endophytic fungi strains from Apodytes dimidiata (Icacinaceae), a medium sized tree from the Western Ghats, India. The fungi were identified as Fusarium solani using both ITS rDNA sequencing and spore morphology. Two strains, MTCC 9667 and MTCC 9668 were isolated, both of which produced camptothecin and 9-methoxycamptothecin in their mycelia; one of the strains, MTCC 9668 also produced 10-hydroxycamptothecin, though in small amounts. The yields of camptothecin in MTCC 9667 and MTCC 9668 were 37 and 53 microg/100g, respectively, after 4 days of incubation in broth culture. The yields of 10-hydroxycamptothecin and 9-methoxycamptothecin in MTCC 9668 were 8.2 and 44.9 microg/100g, respectively. Further research in optimizing the culture conditions of these fungal strains might permit their application for the production of camptothecin and 10-hydroxycamptothecin.

Development of polymorphic microsatellite loci in Nothapodytes nimmoniana, a medicinally important tree from the Western Ghats, India.

Nothapodytes nimmoniana is a medicinally important tree species that occur in the Western Ghats, a megadiversity hotspot in southern India. Inner stem bark of the tree contains an important anti-cancer alkaloid, camptothecin for which the natural population of the tree is heavily harvested. In this paper, we report the isolation and characterization of eight polymorphic microsatellite loci using enrichment hybridization protocol. Analysis of 36 individuals representing two populations revealed three to 12 alleles per locus. Observed heterozygosity ranged from 0.21 to 0.94 for the two populations. None of the loci tested showed linkage disequilibrium. These markers are invaluable for evaluating the genetic structure and assessing the genetic impacts of harvesting of N. nimmoniana in the Western Ghats to formulate strategies for conservation of the species.

Prospecting for Camptothecines from Nothapodytes nimmoniana in the Western Ghats, South India: identification of high-yielding sources of camptothecin and new families of camptothecines.

Camptothecin (CPT), a monoterpene alkaloid, is an important anti-cancer compound obtained from several plant sources including Camptotheca acuminta (from China) and Nothapodytes nimmoniana (from India). Currently, by far the highest levels of CPT (approximately 0.3% w/w) are reported from Nothapodytes nimmoniana, a small tree distributed in the Western Ghats, India. In recent years because of the heavy demand, there has been a serious threat of extinction of the populations of the tree in the Western Ghats forest of south India. Several studies have chemically profiled populations of the species in the Western Ghats to identify sources of high yield and therefore to enable the sustainable production and harvesting of CPT. In this study, using both high-performance liquid chromatography and liquid chromatography-mass spectrometry, we report for the first time the identification of trees that produce at least 5- to 8-fold more CPT than hitherto reported. Furthermore, we show for the first time the production of a few minor camptothecines, including 10-hydroxy camptothecin, in the stem and root bark extracts of the tree. These results have important implications for not only harnessing the high-yielding individuals for clonal multiplication but also for exploiting some of the minor camptothecines, which also have been shown to have important anti-cancer and anti-viral activity.

Patterns of species discovery in the Western Ghats, a megadiversity hot spot in India.

Even since Linnaeus,naturalists and taxonomists have been systematically describing species new to science. Besides indicating gaps in taxonomic effort, understanding the temporal patterns of species discovery could help in identifying drivers that determine discovery. In this study we report the patterns of discovery of eight taxa--birds, butterflies, frogs, tiger beetles, grasses, asters, ferns and orchids--in the Western Ghats, a megadiversity centre in India. Our results indicate that the discovery curves for birds and butter flies have been saturated while those for frogs and grasses continue to increase. Within each taxon, the major drivers of discovery were commonness of the species and their size. The average years taken for discovery across taxa were directly related to the per cent endemicity and species richness of the taxa. We discuss the trajectories of discovery with respect to rarity or endemicity of the species and life history features, and the implications these might have for strategizing the discovery process in India.

Genetic structure of the rattan Calamus thwaitesii in core, buffer and peripheral regions of three protected areas in central Western Ghats, India: do protected areas serve as refugia for genetic resources of economically important plants?

Given the increasing anthropogenic pressures on forests, the various protected areas--national parks, sanctuaries, and biosphere reserves--serve as the last footholds for conserving biological diversity. However, because protected areas are often targeted for the conservation of selected species, particularly charismatic animals, concerns have been raised about their effectiveness in conserving nontarget taxa and their genetic resources. In this paper, we evaluate whether protected areas can serve as refugia for genetic resources of economically important plants that are threatened due to extraction pressures. We examine the population structure and genetic diversity of an economically important rattan, Calamus thwaitesii, in the core, buffer and peripheral regions of three protected areas in the central Western Ghats, southern India. Our results indicate that in all the three protected areas, the core and buffer regions maintain a better population structure, as well as higher genetic diversity, than the peripheral regions of the protected area. Thus, despite the escalating pressures of extraction, the protected areas are effective in conserving the genetic resources of rattan. These results underscore the importance of protected areas in conservation of nontarget species and emphasize the need to further strengthen the protected-area network to offer refugia for economically important plant species.