Endophytic , Insect Pathogenic Fungi Research Articles

Endophytic insect pathogenic fungi-host plant-herbivore mutualism: elucidating the mechanisms involved in the tripartite interactions.

Various techniques used by crop plants to evade insect pests and pathogen attacks have been documented. Among these, plant defense strategies induced by endophytic insect pathogenic fungi are arguably one of the most discussed. Endophytic fungi frequently colonize plants and inhabit their internal tissues for a portion of their lifespan without producing visible symptoms of the disease. This phenomenon is widespread and diverse in both natural and agricultural ecosystems, and is present in almost all plant organs. Many fungi can obtain nutrients by infecting and killing insects, and this ability has been developed numerous times in different fungal lineages. These species mainly consist of those in the order Hypocreales (Ascomycota), where the generalist insect pathogens, Beauveria sp. (Cordycipitaceae) and Metarhizium sp. (Clavicipitaceae) are two of the most studied endophytic entomopathogenic fungal genera. However, most fungi that kill insects do not survive in the tissues of living plants. The data published thus far show a high degree of variability and do not provide consistent explanations for the underlying mechanisms that may be responsible for these effects. This implies that available knowledge regarding the colonization of plant tissues by endophytic insect pathogenic fungi, the effects of colonization on plant metabolism, and how this contributes to a decrease in herbivore and pathogens damage is limited. To adequately utilize fungal-based products as biological control agents, these products must be effective and the reduction of pests and infection must be consistent and similar to that of chemical insecticides after application. This article discusses this possibility and highlights the benefits and the specific techniques utilized by endophytically challenged plants in invading insect pests and disease pathogens.

Entomopathogenic Fungi-Mediated Solubilization and Induction of Fe Related Genes in Melon and Cucumber Plants

Endophytic insect pathogenic fungi have a multifunctional lifestyle; in addition to its well-known function as biocontrol agents, it may also help plants respond to other biotic and abiotic stresses, such as iron (Fe) deficiency. This study explores M. brunneum EAMa 01/58-Su strain attributes for Fe acquisition. Firstly, direct attributes include siderophore exudation (in vitro assay) and Fe content in shoots and in the substrate (in vivo assay) were evaluated for three strains of Beauveria bassiana and Metarhizium bruneum. The M. brunneum EAMa 01/58-Su strain showed a great ability to exudate iron siderophores (58.4% surface siderophores exudation) and provided higher Fe content in both dry matter and substrate compared to the control and was therefore selected for further research to unravel the possible induction of Fe deficiency responses, Ferric Reductase Activity (FRA), and relative expression of Fe acquisition genes by qRT-PCR in melon and cucumber plants.. In addition, root priming by M. brunneum EAMa 01/58-Su strain elicited Fe deficiency responses at transcriptional level. Our results show an early up-regulation (24, 48 or 72 h post inoculation) of the Fe acquisition genes FRO1, FRO2, IRT1, HA1, and FIT as well as the FRA. These results highlight the mechanisms involved in the Fe acquisition as mediated by IPF M. brunneum EAMa 01/58-Su strain.

Three Methods Assessing the Association of the Endophytic Entomopathogenic Fungus Metarhizium robertsii with Non-Grafted Grapevine Vitis vinifera.

Characterizing the association of endophytic insect pathogenic fungi (EIPF) with plants is an important step in order to understand their ecology before using them in biological control programs. Since several methods are available, it is challenging to identify the most appropriate for such investigations. Here, we used two strains of Metarhizium robertsii: EF3.5(2) native to the French vineyard environment and ARSEF-2575-GFP a laboratory strain expressing a green fluorescent protein, to compare their potential of association with non-grafted grapevine Vitis vinifera. Three methods were used to evaluate the kinetics of rhizosphere and grapevine endospheric colonization: (i) Droplet Digital (ddPCR), a sensitive molecular method of M. robertsii DNA quantification in different plant parts, (ii) culture-based method to detect the live fungal propagules from plant tissues that grew on the medium, (iii) confocal imaging to observe roots segments. Both strains showed evidence of establishment in the rhizosphere of grapevines according to the culture-based and ddPCR methods, with a significantly higher establishment of strain EF3.5(2) (40% positive plants and quantified median of exp(4.61) c/μL) compared to strain ARSEF-2575-GFP (13% positive plants and quantified median of exp(2.25) c/μL) at 96-98 days post-inoculation. A low incidence of association of both strains in the grapevine root endosphere was found with no significant differences between strains and evaluation methods (15% positive plants inoculated with strain EF3.5(2) and 5% with strain ARSEF-2575-GFP according to culture-based method). ddPCR should be used more extensively to investigate the association between plants and EIPF but always accompanied with at least one method such as culture-based method or confocal microscopy.

Effect of the Entomopathogenic Fungus Beauveria bassiana on the Development of Faba Bean (Vicia faba) Diseases in the Field Conditions

Several ascomycetous entomopathogenic fungi, including species in the genera Beauveria, are plant symbionts/endophytes and are termed as endophytic insect-pathogenic fungi. It was shown that the fungus Beauveria bassiana (BBK-1 strain) successfully colonized Vicia faba bean plants in laboratory and field conditions of Western Siberia. The B. bassiana reisolate passed through the plants had significantly higher antagonistic activity against phytopathogens in comparison with the primary stem of entomopahogenic fungi. Pre-sowing faba bean seeds treatment reduced the level of infection of the seed material with phytopathogens, significantly decrease the development and prevalence of root rot disease. A decrease in the disease development index (chocolate spot, powdery mildew, fusariosis and other spots diseases) was found as a result of the use of B. bassiana. The effectiveness and prolonged action of B. bassiana on plants opens up new opportunities both in the creation of biological products and in molecular-genetic research and selection of certain pairs of plants and fungi based on the principle of the greatest synergy.

The multifunctional lifestyles of Metarhizium: evolution and applications.

The genus Metarhizium is comprised of a diverse group of common soil fungi that exhibit multifunctional lifestyles with varying degrees of saprotrophic, endophytic, and insect pathogenic modes of nutrient acquisition. The transcriptome of these species is modulated to reflect immediate needs of the fungus and availability of resources-a form of transcriptional plasticity that allows for physiological adaptation to environments with diverse and dynamic exploitable nutrient sources. In this review, we discuss the endophytic, insect pathogenic lifestyles of Metarhizium spp., including their symbiotic interface, origins, and evolution, and agricultural applications. Isotope labeling experiments have demonstrated that a mutually beneficial exchange of limiting nutrients occurs between the fungus and its host plant, with nitrogen derived via insect pathogenesis being translocated from Metarhizium to host plants in exchange for fixed carbon in the form of photosynthate. Thus, the endophytic and entomopathogenic abilities of Metarhizium spp. are not exclusive of one another, but rather are interdependent and reciprocal in nature. Although endophytic, insect pathogenic fungi (EIPF) could certainly have evolved from insect pathogenic fungi, phylogenomic evidence indicates that this genus is more closely related to plant-associated fungi than animal pathogens, suggesting that Metarhizium evolved from a lineage of plant symbionts, which subsequently acquired genes for insect pathogenesis. Entomopathogenicity may have been an adaptive trait, allowing for procurement of insect-derived nitrogen that could be translocated to host plants and bartered for fixed carbon, thereby improving the stability of fungal-plant symbioses. Given their ability to simultaneously parasitize soil insects, including a number of pests of agriculturally important crops, as well as promote plant health, growth, and productivity, Metarhizium spp. are considered promising alternatives to the chemical pesticides and fertilizers that have wreaked havoc on the health and integrity of ecosystems. KEY POINTS: • Metarhizium is a fungus that is an insect pathogen as well as a plant symbiont. • The genus Metarhizium has specialist and generalist insect pathogens. • Metarhizium is phylogenetically most closely related to plant endophytes.

The entomophagous caterpillar fungus Ophiocordyceps sinensis is consumed by its lepidopteran host as a plant endophyte

Abstract Endophytic insect pathogenic fungi of the order Hypocreales reside in plant tissues as mutualistic partners that protect plants against insect herbivores. Caterpillar fungus Ophiocordyceps sinensis (order: Hypocreales, family: Ophiocordycipitaceae) is often described as an entomophagous fungus that parasitizes lepidopteran larvae of the ghost moth genus Thitarodes (family: Hepialidae), but recent evidence suggests involvement of a plant-endophytic stage in O. sinensis infection of insect larvae. In this study, we screen sample plant species in a caterpillar fungus collection site at Mt. Gongga, Sichuan, China, for endophytic O. sinensis and analyze the diet of Thitarodes larvae living in the soil of the same habitat. We show that the entomophagous caterpillar fungus O. sinensis is a prevalent plant endophyte (present in 52.6% of all plant genera and 66.7% of plant families) and that the presence of endophytic O. sinensis in plant leaf tissue is significantly correlated with its presence in plant root tissue. Our analysis of larval diet content reveals a high diversity generalist diet and confirms that the plant families harboring endophytic O. sinensis are consumed by Thitarodes larvae, although the composition and diversity of an individual's diet do not predict its level of fungal infection. These results suggest a reproductive strategy of O. sinensis involving a plant endophytic stage that facilitates infection of root-eating host larvae. Our study highlights the role of resident plant communities as a third participating partner in this complex symbiosis.

Root colonization by endophytic insect-pathogenic fungi.

Several ascomycetous insect-pathogenic fungi, including species in the genera Beauveria and Metarhizium, are plant root symbionts/endophytes and are termed as endophytic insect-pathogenic fungi (EIPF). The endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of insect-derived nitrogen transfer to plant hosts via fungal mycelia. In exchange for the insect-derived nitrogen, the plant provides photosynthate to the fungus. This symbiotic interaction offers other benefits to the plant-EIPF can improve plant growth, they are antagonistic to plant pathogens and herbivores and can enhance the plant tolerance to abiotic stresses. The mechanisms and underlying biochemical and genetic features of insect pathogenesis are generally well-established. However, there is a paucity of information regarding the underlying mechanisms in this plant-symbiotic association. Here we review five aspects of EIPF interactions with host plant roots: (i) rhizosphere colonization, (ii) signalling factors from the plant and EIPF, (iii) modulation of plant defence responses, (iv) nutrient exchange and (v) tripartite interactions with insects and other micro-organisms. The elucidation of these interactions is fundamental to understanding this symbiotic association for effective application of EIPF in an agricultural setting.

Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplanecolonization and modulates the transfer of insect derived nitrogen to plants.

The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

Biology and applications of endophytic insect-pathogenic fungi.

Biology and applications of endophytic insect-pathogenic fungi.

Insect Pathogenic Fungi asEndophytes.

In this chapter, we explore some of the evolutionary, ecological, molecular genetics, and applied aspects of a subset of insect pathogenic fungi that also have a lifestyle as endophytes and we term endophyticinsect pathogenic fungi (EIPF). We focus particularly on Metarhizium spp. and Beauveria bassiana as EIPF. The discussion of the evolution of EIPF challenges a view that these fungi were first and foremost insect pathogens that eventually evolved to colonize plants. Phylogenetic evidence shows that the lineages of EIPF are most closely related to grass endophytes that diverged c. 100MYA. We discuss the relationship between genes involved in "insect pathogenesis" and those involved in "endophytism" and provide examples of genes with potential importance in lifestyle transitions towardinsect pathogenicity. That is, some genes for insect pathogenesis may have been coopted from genes involved in endophytic colonization. Other genes may be multifunctional and serve in both lifestyle capacities. The interactions of EIPF with their host plants are discussed in some detail. The genetic basis for rhizospheric competence, plant communication, and nutrient exchange is examined and we highlight, with examples, the benefits of EIPF to plants, and the potential reservoir of secondary metabolites hidden within these beneficial symbioses.

Fungi with multifunctional lifestyles: endophytic insect pathogenic fungi.

This review examines the symbiotic, evolutionary, proteomic and genetic basis for a group of fungi that occupy a specialized niche as insect pathogens as well as endophytes. We focus primarily on species in the genera Metarhizium and Beauveria, traditionally recognized as insect pathogenic fungi but are also found as plant symbionts. Phylogenetic evidence suggests that these fungi are more closely related to grass endophytes and diverged from that lineage ca. 100 MYA. We explore how the dual life cycles of these fungi as insect pathogens and endophytes are coupled. We discuss the evolution of insect pathogenesis while maintaining an endophytic lifestyle and provide examples of genes that may be involved in the transition toward insect pathogenicity. That is, some genes for insect pathogenesis may have been co-opted from genes involved in endophytic colonization. Other genes may be multifunctional and serve in both lifestyle capacities. We suggest that their evolution as insect pathogens allowed them to effectively barter a specialized nitrogen source (i.e. insects) with host plants for photosynthate. These ubiquitous fungi may play an important role as plant growth promoters and have a potential reservoir of secondary metabolites.

Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria

Abstract Endophytic fungi may display preferential tissue colonization within their plant hosts. Here we tested if the endophytic, insect pathogenic fungi (EIPF) Metarhizium and Beauveria showed preferential localization within plant tissues, in the field and under laboratory conditions. In the field, plants were sampled from three separate sites (Brock University, St. Catharines, Ontario; Pelham, Ontario; and Torngat Mountains National Park, Newfoundland, Canada) and EIPF were isolated from plant roots, the hypocotyl, and stem and leaves. Two genera of EIPF, Metarhizium spp. and Beauveria bassiana, were isolated from plants sampled, as well as the nematophagous fungus, Pochonia chlamydosporium. Metarhizium spp. were almost exclusively found in roots, whereas B. bassiana and P. chlamydosporium were found throughout the plant. The Metarhizium species were identified by RFLP and 95 % were Metarhizium robertsii, 4.3 % were M. brunneum, and 0.7 % were M. guizhouense. Lab studies with M. robertsii and B. bassiana reflected observations found in the field, that is, Metarhizium was restricted to the roots of plants while B. bassiana was found throughout the plant. Insect infection by these EIPF is preferential with respect to above and below ground insects, and the present study correlates above and below ground insect infections with endophytic colonization by these EIPF.

Ubiquity of Insect-Derived Nitrogen Transfer to Plants by Endophytic Insect-Pathogenic Fungi: an Additional Branch of the Soil Nitrogen Cycle

The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with (15)N-labeled nitrogen, and we tracked the incorporation of (15)N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.

Insects as a Nitrogen Source for Plants.

Many plants have evolved adaptations in order to survive in low nitrogen environments. One of the best-known adaptations is that of plant symbiosis with nitrogen-fixing bacteria; this is the major route by which nitrogen is incorporated into plant biomass. A portion of this plant-associated nitrogen is then lost to insects through herbivory, and insects represent a nitrogen reservoir that is generally overlooked in nitrogen cycles. In this review we show three specialized plant adaptations that allow for the recovery of insect nitrogen; that is, plants gaining nitrogen from insects. First, we show specialized adaptations by carnivorous plants in low nitrogen habitats. Insect carnivorous plants such as pitcher plants and sundews (Nepenthaceae/Sarraceniaceae and Drosera respectively) are able to obtain substantial amounts of nitrogen from the insects that they capture. Secondly, numerous plants form associations with mycorrhizal fungi that can provide soluble nitrogen from the soil, some of which may be insect-derived nitrogen, obtained from decaying insects or insect frass. Finally, a specialized group of endophytic, insect-pathogenic fungi (EIPF) provide host plants with insect-derived nitrogen. These soil-inhabiting fungi form a remarkable symbiosis with certain plant species. They can infect a wide range of insect hosts and also form endophytic associations in which they transfer insect-derived nitrogen to the plant. Root colonizing fungi are found in disparate fungal phylogenetic lineages, indicating possible convergent evolutionary strategies between taxa, evolution potentially driven by access to carbon-containing root exudates.