Bioprospecting for microbial endophytes and their natural products.

Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships, ranging from symbiotic to slightly pathogenic. Because of what appears to be their contribution to the host plant, the endophytes may produce a plethora of substances of potential use to modern medicine, agriculture, and industry. Novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation, culture, purification, and characterization of some choice endophytes in the recent past. The potential prospects of finding new drugs that may be effective candidates for treating newly developing diseases in humans, plants, and animals are great.     

Systematics, distribution, and host specificity of grass endophytes.

Clavicipitaceous endophytes (Ascomycetes) are distributed worldwide in many grasses and sedges forming a perennial and often mutualistic association with their hosts. Most endophytes appear to produce alkaloid toxins in infected plants. The high frequency of infection in many grasses and in certain grassland communities may indicate a selective advantage of infected over non-infected host plants due to their toxic effects on grazing animals and insects. Field observations and artificial inoculations of seedlings have demonstrated a high degree of specificity of most endophytes to their host plant, particularly in asexual, seed-borne endophytes. Specific isozyme genotypes found on several host species suggest that host-specific physiological races may occur. Knowledge of host range and host specificity is vital for potential applications of endophytes in pest control.     

Bacterial endophytes: recent developments and applications.

Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes appear to originate from the rhizosphere or phyllosphere; however, some may be transmitted through the seed. Endophytic bacteria can promote plant growth and yield and can act as biocontrol agents. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. There is increasing interest in developing the potential biotechnological applications of endophytes for improving phytoremediation and the sustainable production of nonfood crops for biomass and biofuel production.     

Maize seed endophytes

Maize is a vital global crop, and each seed (kernel) hosts an ecosystem of microbes living inside it. However, we know very little about these endophytes and what their role is in plant production and physiology. In this Microreview, I summarize the major questions around maize seed endophytes, including what organisms are present, how they get there, whether and how they transmit across generations, and how they and the plant affect each other. Although several studies touch on each of these areas, ultimately there are far more questions than answers. Future priorities for research on maize seed endophytes should include understanding what adaptations allow microbes to be seed endophytes, how the host genetics and the environment affect these communities, and how maize seed endophytes ultimately contribute to the next generation of plants.     

Unraveling the role of fungal symbionts in plant abiotic stress tolerance

Fungal symbionts have been found to be associated with every plant studied in the natural ecosystem, where they colonize and reside entirely or partially in the internal tissues of their host plant. Fungal endophytes can express/form a range of different lifestyle/relationships with different host including symbiotic, mutualistic, commensalistic and parasitic in response to host genotype and environmental factors. In mutualistic association fungal endophyte can enhance growth, increase reproductive success and confer biotic and abiotic stress tolerance to its host plant. Since abiotic stress such as, drought, high soil salinity, heat, cold, oxidative stress and heavy metal toxicity is the common adverse environmental conditions that affect and limit crop productivity worldwide. It may be a promising alternative strategy to exploit fungal endophytes to overcome the limitations to crop production brought by abiotic stress. There is an increasing interest in developing the potential biotechnological applications of fungal endophytes for improving plant stress tolerance and sustainable production of food crops. Here we have described the fungal symbioses, fungal symbionts and their role in abiotic stress tolerance. A putative mechanism of stress tolerance by symbionts has also been covered.Key words: abiotic stress, endophytes, fungal symbiont, mycorrhizal fungus, Piriformospora indica, stress tolerance, symbiosis     

Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)

Burkholderia endophytes were identified within the leaves of non-nodulated members of the genus Psychotria. In contrast to leaf-nodulated Psychotria species, which are known to accommodate their endosymbionts into specialized endosymbiont-housing structures, non-nodulated species lack bacterial leaf nodules and harbor endosymbionts intercellularly between mesophyll cells. Based on molecular data (rps16, trnG, and trnLF), the phylogenetic reconstruction of the host plants revealed a separate origin of leaf-nodulated and non-nodulated Psychotria species. Despite a distinct phylogenetic position of the two host clades, the endophytes of the non-nodulated plants were not placed into a single monophyletic group but were found to be closely related to the leaf-nodulated endosymbionts. The observation of genetically similar endophytes in both nodulated and non-nodulated Psychotria lineages suggests that the host plant is playing a crucial role in the induction of leaf nodule formation. Moreover, the concentration of endosymbionts into specialized leaf nodules may be considered as a more derived evolutionary adaptation of the host plant, serving as an interface structure to facilitate metabolic exchange between plant and endosymbiont.     

Fungal endophytes: common host plant symbionts but uncommon mutualists

Fungal endophytes are extremely common and highly diverse microorganismsthat live within plant tissues, but usually remain asymptomatic.Endophytes traditionally have been considered plant mutualists,mainly by reducing herbivory via production of mycotoxins, suchas alkaloids. However, the vast majority of endophytes, especiallyhorizontally-transmitted ones commonly found in woody plants,apparently have little or no effect on herbivores. For the systemic,vertically-transmitted endophytes of grasses, mutualistic interactionsvia increased resistance to herbivores and pathogens are morecommon, as predicted by evolutionary theory. However, even inthese obligate symbioses, endophytes are often neutral or evenpathogenic to the host grass, depending on endophyte and plantgenotype and environmental conditions. We present a graphical model based upon variation in nitrogenflux in the host plant. Nitrogen is a common currency in endophyte/hostand plant/herbivore interactions in terms of limitations tohost plant growth, enhanced uptake by endophytes, demand forsynthesis of nitrogen-rich alkaloids, and herbivore preferenceand performance. Our graphical model predicts that low alkaloid-producingendophytes should persist in populations when soil nutrientsand herbivory are low. Alternatively, high alkaloid endophytesare favored under increasing herbivory and increasing soil nitrogen,at least to some point. At very high soil nitrogen levels, uninfectedplants may be favored over either type of infected plants. Thesepredictions are supported by patterns of infection and alkaloidproduction in nature, as well by a manipulative field experiment.However, plant genotype and other environmental factors, suchas available water, interact with the presence of the endophyteto influence host plant performance.     

Endophytic fungal communities in woody perennials of three tropical forest types of the Western Ghats,southern India

Fungal endophytes of tropical trees are expected to be exceptionally species rich as a consequence of high tree diversity in the tropics and the purported host restriction among the endophytes. Based on this premise, endophytes have been regarded as a focal group for estimating fungal numbers because their possible hyperdiverse nature would reflect significantly global fungal diversity. We present our consolidated ten-year work on 75 dicotyledonous tree hosts belonging to 33 families and growing in three different types of tropical forests of the NBR in the Western Ghats, southern India. We conclude that endophyte diversity in these forests is limited due to loose host affiliations among endophytes. Some endophytes have a wide host range and colonize taxonomically disparate hosts suggesting adaptations in them to counter a variety of defense chemicals in their hosts. Furthermore, such polyphagous endophytes dominate the endophyte assemblages of different tree hosts. Individual leaves may be densely colonized but only by a few endophyte species. It appears that the environment (the type of forest in this case) has a larger role in determining the endophyte assemblage of a plant host than the taxonomy of the host plant. Thus, different tropical plant communities have to be studied for their endophyte diversity to test the generalization that endophytes are hyperdiverse in the tropics, estimate their true species richness, and use them as a predictor group for more accurate assessment of global fungal diversity.     

Are tropical fungal endophytes hyperdiverse?

Fungal endophytes are ubiquitous fungi that inhabit healthy plant tissues without causing disease. Endophytes have been found in every plant species examined to date and may be important, but often overlooked, components of fungal biodiversity. In two sites in a lowland, moist tropical forest of central Panama, we quantified endophyte colonization patterns, richness, host preference, and spatial variation in healthy leaves of two co-occurring, understory tree species [ Heisteria concinna (Olacaceae) and Ouratea lucens (Ochnaceae)]. From 83 leaves, all of which were colonized by endophytes, we isolated 418 endophyte morphospecies (estimated 347 genetically distinct taxa), most of which were represented by only a single isolate (59%). Among morphospecies encountered in more than one leaf (nonsingletons), we found evidence of host preference and spatial heterogeneity using both morphospecies frequencies and presence/absence records. Based on these data, we postulate that tropical endophytes themselves may be hyperdiverse and suggest that extrapolative estimates that exclude them will markedly underestimate fungal species diversity.     

Exploring the potentialities of beneficial endophytes for improved plant growth

Pathogen affects plant growth, host health and productivity. Endophytes, presumed to live inside the plant tissues, might be helpful in sustaining the future of agriculture. Although recent studies have proven that endophytes can be pathogenic, commensal, non-pathogenic, and/or beneficial, this review will focus on the beneficial category only. Beneficial endophytes produce a number of compounds which are useful for protecting plants from environmental conditions, enhancing plant growth and sustainability, while living conveniently inside the hosts. The population of endophytes is majorly controlled by location, and climatic conditions where the host plant grows. Often the most frequently isolated endophytes from the tissues of the plant are fungi, but sometimes greater numbers of bacteria are isolated. Beneficial endophytes stand a chance to replace the synthetic chemicals currently being used for plant growth promotion if carefully explored by researchers and embraced by policymakers. However, the roles of endophytes in plant growth improvement and their behavior in the host plant have not been fully understood. This review presents the current development of research into beneficial endophytes and their effect in improving plant growth.     

The interactive effects of plant microbial symbionts: a review and meta-analysis

In nature, plants often associate with multiple symbionts concurrently, yet the effects of tripartite symbioses are not well understood. We expected synergistic growth responses from plants associating with functionally distinct symbionts. In contrast, symbionts providing similar benefits to a host may reduce host plant growth. We reviewed studies investigating the effect of multiple interactions on host plant performance. Additionally, we conducted a meta-analysis on the studies that performed controlled manipulations of the presence of two microbial symbionts. Using response ratios, we investigated the effects on plants of pairs of symbionts (mycorrhizal fungi, fungal endophytes, and nitrogen-fixers). The results did not support the view that arbuscular mycorrhizal (AM) fungi and rhizobia should interact synergistically. In contrast, we found the joint effects of fungal endophytes and arbuscular mycorrhizal fungi to be greater than expected given their independent effects. This increase in plant performance only held for antagonistic endophytes, whose negative effects were alleviated when in association with AM fungi, while the impact of beneficial endophytes was not altered by infection with AM fungi. Generalizations from the meta-analysis were limited by the substantial variation within types of interactions and the data available, highlighting the need for more research on a range of plant systems.     

Evolutionary origins and ecological consequences of endophyte symbiosis with grasses

Over the past 20 yr much has been learned about a unique symbiotic interaction between fungal endophytes and grasses. The fungi (Clavicipitaceae, Ascomycota) grow intercellularly and systemically in aboveground plant parts. Vertically transmitted asexual endophytes forming asymptomatic infections of cool-season grasses have been repeatedly derived from sexual species that abort host inflorescences. The phylogenetic distribution of seed-transmitted endophytes is strongly suggestive of cocladogenesis with their hosts. Molecular evidence indicates that many seed-transmitted endophytes are interspecific hybrids. Superinfection may result in hyphal fusion and parasexual recombination. Most endophytes produce one or more alkaloid classes that likely play some role in defending the host plant against pests. Hybridization may have led to the proliferation of alkaloid-production genes among asexual endophytes, favoring hybrids. The ergot alkaloid ergovaline, lolitrems, and lolines are produced by only a single sexual species, Epichlo? festucae, but they are common in seed-transmitted endophytes, suggesting that E. festucae contributed genes for their synthesis. Asexual hybrids may also be favored by the counteracting of the accumulation of deleterious mutations (Muller's rachet). Endophyte infection can provide other benefits, such as enhanced drought tolerance, photosynthetic rate, and growth. Estimates of infection frequency have revealed variable levels of infection with especially high prevalence in the subfamily Pooideae. Longitudinal studies suggest that the prevalence of seed-transmitted endophytes can increase rapidly over time. In field experiments, infected tall fescue suppressed other grasses and forbs relative to uninfected fescue and supported lower consumer populations. Unlike other widespread plant/microbial symbioses based on the acquisition of mineral resources, grass/endophyte associations are based primarily on protection of the host from biotic and abiotic stresses.     

A potential antioxidant resource: Endophytic fungi from medicinal plants

Medicinal plants and their endophytes are important resources for discovery of natural products. Several previous studies have found a positive correlation between total antioxidant capacity (TAC) and total phenolic content (TPC) of many medicinal plant extracts. However, no information is available on whether such a relationship also exists in their endophytic fungal metabolites. We investigated the relationship between TAC and TPC for 292 morphologically distinct endophytic fungi isolated from 29 traditional Chinese medicinal plants. The antioxidant capacities of the endophytic fungal cultures were significantly correlated with their total phenolic contents, suggesting that phenolics were also the major antioxidant constituents of the endophytes. Some of the endophytes were found to produce metabolites possessing strong antioxidant activities. Several bioactive constituents from the fungal cultures and host plant extracts were identified. This investigation reveals that the metabolites produced by a wide diversity of endophytic fungi in culture can be a potential source of novel natural antioxidants.     

植物内生菌及其防治植物病害的研究进展

综述了植物内生菌及其防治植物病害的研究进展.植物内生菌分布广,种类多,几乎存在于所有目前已研究过的陆生及水生植物中,目前全世界至少已在80个属290多种禾本科植物中发现有内生真菌,在各种农作物及经济作物中发现的内生细菌已超过120种.感染内生菌的植物宿主往往具有生长快速、抗逆境、抗病害、抗动物危害等优势,比未感染内生菌的植株更具生存竞争力.植物内生菌的防病机理主要表现在通过产生抗生素类,水解酶类,植物生长调节剂和生物碱类物质,与病原菌竞争营养物质,增强宿主植物的抵抗力以及诱导植物产生系统抗性等途径抑制病原菌生长.另外,对植物内生真菌和内生细菌的分离、筛选和检测方法;利用植物内生菌控制植物病害的途径如人工接种内生菌,利用内生菌代谢产生的抗生素以及将内生菌作为基因工程的载体菌等进行了综述.同时,对植物内生菌作为生物防治因子未来发展前景及存在的问题进行了讨论.利用植物内生菌作为生物防治因子进行大田防病,需要考虑它的病理学、生态学和形态学等方面的影响.     

Community structure,spatial distribution,diversity and functional characterization of culturable endophytic fungi associated with Glycyrrhiza glabra L.

A total of 266 endophytic fungal isolates were recovered from 1019 tissue segments of Glycyrrhiza glabra collected from four different locations in the North-Western Himalayas. The endophytes grouped into 21 genera and 38 different taxa. The host had strong affinity for the genus Phoma, followed by Fusarium. The species richness was highest at the sub-tropical location, followed by the sub-temperate location and the temperate locations, respectively. The tissue specificity of endophytes was also evident. Some endophytes showed potential antimicrobial activity against phyto-pathogens indicating that they may be helpful to the host in evading pathogens. All the endophytic taxa produced the plant growth promoting hormone, indole acetic acid (IAA), though in varying concentrations. None of these endophytes caused any symptoms of disease in co-cultivation with the tissue cultured plants. Further, all the endophytes had a positive influence on the phenolic and flavonoid content of the host. Three endophytes, Stagonosporopsis cucurbitacearum, Bionectria sp. and Aspergillus terreus also increased the host root (rhizome) and shoot growth visibly. Such endophytes are potential candidates for developing endophyte-based technologies for sustainable cultivation and enhanced productivity of G. glabra. This is the first report of community structure and biological properties of fungal endophytes associated with G. glabra.     

Forest habitat characteristics affect balance between sexual reproduction and clonal propagation of the ectomycorrhizal mushroom Hebeloma cylindrosporum

Endophytic fungi, especially asexual, systemic endophytes in grasses, are generally viewed as plant mutualists, mainly through the action of mycotoxins, such as alkaloids in infected grasses, which protect the host plant from herbivores. Most of the evidence for the defensive mutualism concept is derived from studies of agronomic grass cultivars, which may be atypical of many endophyte-host interactions. I argue that endophytes in native plants, even asexual, seed-borne ones, rarely act as defensive mutualists. In contrast to domesticated grasses where infection frequencies of highly toxic plants often approach 100%, natural grass populations are usually mosaics of uninfected and infected plants. The latter, however, usually vary enormously in alkaloid levels, from none to levels that may affect herbivores. This variation may result from diverse endophyte and host genotypic combinations that are maintained by changing selective pressures, such as competition, herbivory and abiotic factors. Other processes, such as spatial structuring of host populations and endophytes that act as reproductive parasites of their hosts, may maintain infection levels of seed-borne endophytes in natural populations, without the endophyte acting as a mutualist.     

The need to study the holobiome for gainful uses of endophytes

It is established that endophytes (bacteria and fungi) affect positively several traits of their host plants including growth, nutrient acquisition, and tolerance to abiotic and biotic stressors. Owing to this, endophytes are envisaged as a route to improve sustainable agricultural production. However, such effects of endophytes on crops although evident under lab conditions, are devoid of consistency under field conditions. The main reason for this could be the lack of our understanding of the functioning of the endophytes as a consortium within the plant (holobiome). It is essential to study the complex web of interactions which operate in the inner tissues of plants harboring endophytes. I highlight here, alluding to the work on foliar fungal endophytes, the importance of studying endophytes as a consortium rather than merely as one species in isolation to better appreciate their role in the plant holobiome. Such an approach should aid in harnessing the technological potential of endophytes.     

A suite of rare microbes interacts with a dominant,heritable, fungal endophyte to influence plant trait expression

Endophytes are microbes that live, for at least a portion of their life history, within plant tissues. Endophyte assemblages are often composed of a few abundant taxa and many infrequently observed, low-biomass taxa that are, in a word, rare. The ways in which most endophytes affect host phenotype are unknown; however, certain dominant endophytes can influence plants in ecologically meaningful ways—including by affecting growth and immune system functioning. In contrast, the effects of rare endophytes on their hosts have been unexplored, including how rare endophytes might interact with abundant endophytes to shape plant phenotype. Here, we manipulate both the suite of rare foliar endophytes (including both fungi and bacteria) and Alternaria fulva–a vertically transmitted and usually abundant fungus–within the fabaceous forb Astragalus lentiginosus. We report that rare, low-biomass endophytes affected host size and foliar %N, but only when the heritable fungal endophyte (A. fulva) was not present. A. fulva also reduced plant size and %N, but these deleterious effects on the host could be offset by a negative association we observed between this heritable fungus and a foliar pathogen. These results demonstrate how interactions among endophytic taxa determine the net effects on host plants and suggest that the myriad rare endophytes within plant leaves may be more than a collection of uninfluential, commensal organisms, but instead have meaningful ecological roles.Subject terms: Microbial ecology, Community ecology, Microbial ecology     

Role of Diverse Non-Systemic Fungal Endophytes in Plant Performance and Response to Stress: Progress and Approaches

Plant–fungal symbiotic associations are ubiquitously distributed in natural plant communities. Besides the well-studied mycorrhizal symbiosis and grass systemic clavicipitaceous endophytes, recently, nonsystemic and horizontally transmitted fungal endophytes serving as plant symbionts have been increasingly recognized. Pure culture isolation and culture-independent molecular methods indicate that all parts of healthy plant tissues potentially harbor diverse and previously unknown fungal lineages. Limited evidence also supports a hypothesis that endophytic mycobiota dynamics may have a role in evolution of plants. High variability or “balanced antagonism” can be generally characterized with host–endophyte interactions, which implies that the outcome of symbiotic interactions can fall within a continuum ranging from mutualism to commensalism, and ultimately pathogenicity. Despite this complicated system, admittedly, fungal endophytes really endow the host with an extended phenotype. Accumulating facts illustrate that plant nutrition acquisition, metabolism, and stress tolerance may be strengthened or modulated via fungal symbionts. Piriformospora indica, a member of the order Sebacinales, simultaneously confers host resistance to biotic and abiotic stress. The ecological relevance of other fungal groups, including foliar endophytes, root dark septate endophytes (DSEs), some opportunistic and avirulent microsymbionts (for example, Trichoderma and Fusarium), and even uncultured fungi structurally and physiologically integrated with host tissues, are also being deeply exploited. Production of bioactive metabolites by fungi, overexpression of stress-related enzymes, and induced resistance in hosts upon fungal colonization are responsible for direct or indirect beneficial effects to hosts. More knowledge of endophyte-mediated enhancement of host performance and fitness will offer alternatively valuable strategies for plant cultivation and breeding. Meanwhile, with unprecedented loss of biodiversity, discovery of indigenously novel symbiotic endophytes from natural habitats is urgently needed. In addition, we present some approaches and suggestions for studying host–endophyte interactions.     

Divergent rainforest tree microbiomes between phases of the monsoon cycle,host plants and tissues

• The Australian Monsoon Tropics (AMT) contain some of the most biodiverse forests on the continent. Little is known about the dynamics of rainforest plant microbiomes in general, and there have been no community-level studies on Australian rainforest endophytes, their seasonality, tissue and host specificity.
• We tested whether community composition of tropical tree endophytes (fungi and bacteria) differs: (i) at different points during a monsoon cycle, (ii) between leaf and stem tissues, (iii) between forest microclimates (gully/ridge), and between (iv) host plant species, and (v) host plant clade, using amplicon sequencing of the bacterial 16S and fungal ITS2 gene regions.
• Results indicated that the composition of rainforest plant microbiomes differs between wet and dry seasons, which may be explained by physiological shifts in host plants due to annual climate fluctuations from mesic to xeric. Endophyte microbiomes differed between leaves and stems. Distinct fungal communities were associated with host species and clades, with some trees enriched in a number of fungal taxa compared to host plants in other clades. Diversity of bacterial endophytes in plant stems increased in the dry season.
• We conclude that the microbiomes of tropical plants are responsive to monsoonal climate variation, are highly compartmentalised between plant tissues, and may be partly shaped by the relatedness of their host plants.