Fungal Endophytes: Beyond Herbivore Management

1Endophytes are ubiquitous, forming associations with a diverse group of organisms throughout the plant kingdom and provide indirect defense for plants against herbivores. Endophytes can exist in a host plant in the form of mutualistic root endophytes or plant-associated endophytes. They are plant-associated microorganisms that colonize and live part of their life cycle within a plant without causing harm or disease (such as lesions, retardation in growth, discoloration or chlorosis, etc.) to their host.

2The tissues and organs of the host plants such as leaves, branches, stems, fruits, flowers, and roots are often colonized by fungal endophytes without showing visible symptoms. Some fungal endophytes can also act as insect pathogenic agents by infecting lepidopterous larvae, aphids, thrips, and other cosmopolitan insects, which are of great concern in agriculture worldwide. They are known to infect specific hosts and pose little or no risk to non-target organisms or beneficial insects.

3There are various reports of possible artificial inoculation of plants with fungal entomopathogens to establish as plant endophytes artificially. Some previous reviews were focused on the role of endophytic fungi in plant herbivore management. Herein, we indicated that fungal endophytes such as Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) not only protect host plants from arthropod pests, but also protect its hosts from diseases and plant parasitic nematodes, as well as enhance plant growth. Our review further summarized other effects of fungal endophytes on their host plants and primary pests. Furthermore, we highlighted the major sub-classes in which fungal endophytes can be classified based on criteria such as: their mode of reproduction, the source of nutrition, mode of transmission, symptoms expression in the hosts, the colonized plant parts, and their general morphology. We are of the opinion that the general knowledge of these findings would help to improve the application and overall adoption of fungal endophytes for pests and diseases management programs.

4Fungal endophytes have been reported as naturally occurring in several host plants. A single plant part (leaf, stem, or root) can contain different endophyte species. Higher vascular plants have been found hosting endophytic fungi in a symbiotic plant-fungus interaction. The interaction is termed symbiotic, as endophytic fungi, in exchange for the nutrients derived from the host plants, provide benefits to their hosts. These fungal endophytes existing symbiotically within the colonized host plants are utilized as an indirect defense against herbivores.

5Many fungi traditionally known as insect pathogens such as Beauveria bassiana, Clonostachys rosea, Isaria farinosa, and Acremonium sp. (now known as Neotyphodium) have been isolated as naturally occurring endophytes from asymptomatic plant tissues. In addition to the aforementioned, some many more endophytic entomopathogenic fungi were reported to have been re-isolated from colonized host plants after artificial inoculation. These include Metarhizium anisopliae; B. bassiana; Fusarium oxysporum, Hypocrea lixii, Gibberella moniliformis, and Trichoderma asperellum. According to the authors, artificially inoculated entomopathogenic fungi were successfully colonized at various degrees of endophytic colonization.

6So far, using various artificial inoculation methods, successful colonization of several endophytic entomopathogenic fungi have been reported in wheat (Triticum aestivum), common bean (Phaseolus vulgaris), corn (Zea mays), tomato (Lycopersicon esculentum), soybeans (Glycine max), coffee (Coffea spp.), opium poppy (Papaver somniferum) cassava (Manihot esculenta), sorghum (Sorghum bicolor), cotton (Gossypium hirsutum), and in some other economically important crops.

7Generally, fungal endophytes are known to be beneficial to crop plants, while only few species can be pathogenic by causing diseases to the host after an incubation or latency period. Some species are neutral without offering benefit or posing harm to their hosts. opined that some endophytic fungal species may exist as latent or inactive pathogens, but become active and reproduce under certain environmental conditions or when their host plants are stressed or grow old. This was supported by the findings of. on the other hand, described latent infections as the condition in which the host is infected without showing any symptoms. The pathogen is inactive or in latent state until symptoms are induced as a result of environmental changes, nutritional conditions of the host plant or the stage of maturity of the pathogen or the host.

8There are speculations that, many thousands of endophytes useful to mankind are currently exiting but still unexplored due to limited research attention in this related field. However, with environmental contamination, deforestation, habitat fragmentation and biodiversity losses, many of these endophytes might be permanently lost before their value is explored. There is, therefore, need to explore these biological agents reservoir not only for the management of arthropod pests and diseases, but also to elaborate on their diversity and other functions under different agro-ecological zones.

9Endophytes are diverse in taxonomy, but only few species have been isolated, identified and characterized to date. Fungal endophytes can be classified broadly into ecological categories or otherwise, in terms of their diversity or functional roles. Based on these categories, they have been grouped into two major groups as clavicipitaceous and non-clavicipitaceous fungal endophytes. Clavicipitaceous fungal endophytes are mostly common in grasses, while the non-clavicipitaceous are predominant with vascular and non-vascular plant species.

10However, various authors have indicated the need to further classify these fungal endophytes to sub-classes based on different criteria such as: the host range, the mode of reproduction, the part of plant colonized, the mode of transmission, source of nutrition, and ability to express symptoms in the host plant (see;;;; ).

11Fungal endophytes are classified based on the mode of reproduction as: sexual or asexual. For instance, the Epichloë endophytes have been divided into the genera Epichloë and Neotyphodium (formally Acremonium) that reproduces sexually and asexually respectively.

12They can as well be classified based on the mode of transmission in the host as: vertically–transmitted and horizontally–transmitted endophytes. Vertically-transmitted endophytes are transferred directly from the host plants (parents) to their progenies. True endophytes (such as the species belonging to the genus Neotyphodium) are mostly vertically transmitted through seeds from one plant to another. When transmission is vertical through the host seeds, they are referred to as seed-transmitted endophytes. Most Epichloë endophytes are seed-transmitted endophytes. In addition, the paper of also reported vertical transmission of B. bassiana through seeds in opium poppy plants inoculated artificially via seed soaking.

13On the other hand, horizontally-transmitted endophytes are transferred between different individuals in a given population. This mode of transmission is common with fungal endophytes that infect plants via airborne spores. In this case, endophytes are usually multiplied via vegetative propagules, or transmission by spores in the case of spore-transmitted endophytes. Most woody and herbaceous plants harbor different species of unspecialized endophytic fungi. These fungal species which generally exhibit weak pathogenicity against insect herbivores are mostly transmitted horizontally.

14Another classification is based on the source of nutrition, that is, whether nutrients are derived by the fungus from living or dead matter. Based on this, endophytes can be classified as necrotrophs, or as biotrophs. Biotrophic fungi are the types that develop and obtain nutrients within the tissue of a living host, while, necrotrophic fungi are the species that mortify the host cells in order to grow on the dead tissues. All endophytes are heterotrophs, unlike green plants that utilize CO2 directly for photosynthesis, they obtain carbon from the plants in the form of organic compounds. These fungi-plant interactions occur when endophytic fungi obtain their carbon supply from their hosts, and in exchange for the energy resources they derived from the host plants, provide benefits to the plant. However, as a result of periodic evolutionary and ecological changes, there is possibility of some fungal endophytes switching between the two lifestyles. That is, from biotrophic to necrotrophic lifestyle. For instance, Leptosphaeria maculans occurring asymptomatic in healthy Arabidopsis thaliana plants became a necrotrophic pathogen when the plant was stressed.

15Based on the expression of infection, endophytes are classified as symptomatic (expressing symptoms) and asymptomatic (symptomless). A good number of endophytic fungi infect above ground internal plant tissues without showing symptoms. Great attention is focused on these species of endophytes because they are ubiquitous and have vast diversity and many roles. For instance, Fusarium spp. was identified as asymptomatic as it was confirmed to express no symptoms in cord roots of banana cultivar (Pisang Awak – Musa ABB). Although in some rare cases, symptomatic endophytes can be categorized as asymptomatic when the host plant is resistant to the fungi. However, as earlier stated, a change in environmental conditions could cause a sudden switch in the behavior of asymptomatic endophytic fungi. A clear example is the case of fungal species that were isolated as symptomless endophytes, yet, became pathogenic under changed environmental conditions. To this end, we can reach a conclusion that, the age of the host plant harboring the fungus and the environmental conditions have a larger role to play in determining whether an endophytic fungus acts as a symptomless endophyte or otherwise as a symptom-producing plant pathogen.

16On the basis of the host plant part that is affected by the fungal endophytes, they can be classified as root and foliar endophytes. The possibility of endophytic fungi to exhibit preferential tissue colonization within their colonized hosts has widely been reported. For instance, in a study conducted by, B. bassiana and Pochonia chlamydosporia were reportedly localized within the stems and leaves, while Metarhizium spp. was mostly found within the plant roots. Several other previous findings have also reported the potential of endophytic fungi exhibiting localized endophytic colonization rather than colonizing the host plants systemically. Thus, fungal endophytes that infect plant roots (as in the case of Fusarium spp., Metarhizium spp., Piriformospora indica, and Glomus spp.) are known as root endophytes. These groups of endophytes infect plant tissues from the rhizosphere. Other endophytes that invade stems and leaves of plants or species that are primarily localized to the foliar part of the plant are known as foliar endophytes.

17Fungal endophytes are generally identified based on their morphological features, they can be isolated from their host plant tissues and cultured in the most suitable growth media for morphological identification and classification. However, aside morphological characterization, there are suggestions that, isolation and molecular characterization of entomopathogenic endophytes are very imperative in order to expand the current data on entomopathogenic fungi. Molecular phylogenetic classification to identify endophytes can be carried out through amplifying and sequencing a small fragment of fungi DNA. This practice involves the analysis of nucleic acids and proteins to study the evolutionary relationships of fungal endophytes. A molecular phylogenetic relationship of Epichloë typhina with other clavicipitaceous endophytes also confirms the suggestion that fungal endophytes may coevolve with their host plants.

18Fungal endophytes have been confirmed to produce several beneficial effects to their host plants. The potential of entomopathogenic fungal endophytes exerting detrimental effects on the insect pests feeding on the plants colonized by these endophytic pathogens has been widely reported in several recent studies. Most of these findings provided results that indicate that plants colonized by endophytes are protected from substantial damage, and plant pests feeding on such plants are less productive. The mechanism through which endophytic fungi reduce insect herbivore damage are numerous, some of the common measures include: reduction in the insect developmental rate, causing feeding deterrence, retardation of insect growth, reducing survival and oviposition.

19Reduction in plant damage caused by many insect pests has been reported in several crop plants following treatment with endophytic entomopathogenic fungi. For instance, reduction in poppy stem gall wasp (Iraella luteipes) (Hymenoptera: Cynipidae) damage in opium poppy treated with B. bassiana was found by, reduced tunneling by lepidopteran larvae of European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae) and Sesamia calamistis Hampson (Lepidoptera; Noctuidae) was also reported in maize. See also.

20reported a reduction in the damage caused by Helicoverpa zea (Lepidoptera; Noctuidae) in tomato following treatment with B. bassiana (see also ). H. zea was also reported to be controlled in cotton using B. bassiana and Purpureocillium lilacinum. 50% mortality of all larval instars and reduced longevity of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) larvae fed with B. bassiana colonized tomato leaves was also recorded by. also found a reduction in Helicoverpa armigera damage of tomato plants. recorded a similar result with Coffee berry borer (Hypothenemus hampei) in coffee plant treated with B. bassiana. found a similar reduction in larval survival and overall reduction in plant damage by banana weevil (Cosmopolites sordidus) (Coleoptera: Curculionidae). See also.

21Reduction in damage caused by the cotton aphid Aphis gossypii Glover (Hemiptera: Aphididae) and white jute stem weevil (Apion corchori) in cotton and white jute respectively was reported by and. In addition, in our previous study, we also found a similar reduction in damage caused by Liriomyza huidobrensis (Diptera: Agromyzidae) in Vicia faba and P. vulgaris (see ). Some other reports on insect pests damage reduction following treatment of crops with entomopathogenic fungi are available (see,;;;;; ). Most of these studies have attributed the reduction in the damage by insect pests to the accumulation of mycotoxins in plant tissues. opined that the harmful effects of endophytic fungi on insect herbivores are due to the production of fungal metabolites.

22Some previous studies have also indicated the possibility of using fungal endophytes and natural enemies such as parasitoids in combination for suppressing insect herbivore population and damage in plant. For instance, reported the possibility of using endophytic fungal entomopathogens, B. bassiana and Metarhizium brunneum in combination with the aphid endoparasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae) for the management of the green peach aphid Myzus persicae Sulzer (Homoptera: Aphididae) in sweet pepper Capsicum annum L. (Solanaceae). Similarly, in one of our previous studies, we also reported the possibility of utilizing endophytic entomopathogenic fungi and either of the two leafminer parasitoids Phaedrotoma scabriventris (Hymenoptera: Braconidae) and Diglyphus isaea (Hymenoptera: Eulophidae) in combination for the management of pea leafminer L. huidobrensis in V. faba (see also,;; ).

23However, some of the previous studies reported negative effect of fungal endophytes on the natural enemies parasitizing insects feeding on fungi-colonized plants. The negative effects reported include reductions in growth, fecundity and adult survival of natural enemies. The primary cause of these adverse effects is the transmission of mycotoxins across the food chain from the colonized plants through the insect pests to the parasitoids. A typical example is the case of Neotyphodium coenophialum which produces loline alkaloids that reduced the survival of the parasitoid Euplectrus comstokii introduced in tall fescue for the management of fall armyworm (Spodoptera frugiperda). also suggested that the negative effects of endophytic fungi on natural enemies may be due to the transfer of endophyte-produced toxins. There are also suggestions that the reduction in the size of insect herbivores feeding on endophyte-infected plants and possible reduction in nutritional value of the insects due to infection by endophytic entomopathogenic fungi may indirectly affect the natural enemies.

24Aside herbivore management, the potential of endophytic entomopathogenic fungi serving dual purpose biological control of both insects and plant pathogens has been reported. reported that endophytic colonization of tomato and cotton seedlings through seed soaking in B. bassiana conidia protected the seedlings against plant pathogenic Rhizoctonia solani and Pythium myriotylum. In another study by, basal rot of onion (a disease caused by Fusarium oxysporum f. sp. cepae) was significantly reduced following treatment of onion bulbs with B. bassiana. Similarly, disease incidence and severity of downy mildew – caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. was also significantly reduced in grapevine following colonization of leave tissues by B. bassiana. The evidence of B. bassiana offering protection against plant viral pathogens is also available. found evidence of reduction in disease incidence and severity of Zucchini yellow mosaic virus (ZYMV) in B. bassiana inoculated squash plants.

25In addition to pests and diseases management, fungi occurring in the host plants as endophytes provide other benefits to the colonized host. Established beneficial effects include: increasing plant growth, plant development and nutrients (nitrogen and phosphorus) uptake into plants and improvement in overall plant hardiness, as well as, preventing colonization of the host by foreign parasitic organisms.

26Endophytes colonize the host plant tissue hence creating a barrier that prevents foreign pathogenic organisms from colonizing the same host plant and consequently control phytopathogenic diseases. Endophytes are considered as primary sources of bioactive compounds, that not only serve as storehouse of unique bioactive secondary metabolites, such as alkaloids, saponins, tannins, phenolic acids, steroids, quinones and terpenoids, but also act as insect antagonist, antimicrobial, anticancer and many other important properties. They can as well be referred to as biofertilizers because they serve as plant growth promoters that facilitate nutrient uptake not only through plant root system, but also through the transfer of the insect-derived nitrogen to plants. For example, Metarhizium robertsii infects and kills soil-born insects, produces fungal mycelia from the dead insects and thereafter, forms an endophytic association with the plant roots, hence enhancing nitrogen translocation. The findings from the study conducted by indicated that each of the crop plants examined - haricot bean (P. vulgaris), wheat (T. aestivum), soybean (G. max), and switchgrass (Panicum virgatum), derived a substantial amount of nitrogen from the soil insects infected with entomopathogenic fungi. They opined that M. robertsii possibly supplied nitrogen to the crop plants in exchange for carbon. There is evidence that nitrogen uptake by plants in this plant-fungi-soil interaction may play a larger role in soil nitrogen cycling and insect pests’ infection.

27Fungal endophytes also improve the colonized plant height, weight and other growth parameters are also influenced. reported an improvement in the height, fresh weight of shoots and roots of V. faba plants following artificial inoculation of Beauveria brongniartii, B. bassiana and M. brunneum. In another study, B. bassiana and P. lilacinum also increased the growth and dry biomass of colonized cotton plants. Several other previous studies have also related improved plant growth with endophytic entomopathogenic fungi. See.

28Endophytes also induce chemicals that impede the growth and development of other competitors, including pathogenic organisms, help plants not only to tolerate biotic stresses such as below-ground herbivory by nematodes and other root-feeding insects, but also, abiotic stresses, including salt, drought or heat stresses. Endophytic fungi also indirectly enhance seed dispersal by ants. In a study by, seeds of Fescue (Festuca arundinacea L. Schreb) infected with Acremonium coenophialum (now known as Neotyphodium coenophialum) were protected against two primary seed harvesting ants – Pogonomyrmex rugosus and Pogonomyrmex occidentalis. The infected seeds were discarded after being collected by the ants and this periodic dispersal by the insects indirectly improves seed distribution.

29However, not all endophytic fungi-plant associations protect plants from insect herbivores, only few species associations act as defense mutualisms. In some cases, the presence of fungal endophytes in a plant can result in higher rates of water loss in leaves and it has been recorded that some endomycorrhizae may increase pest damage by making their host plants more susceptible to the pest.

30The beneficial effects of fungal endophytes on the host plants and primary pests are numerous and not limited to those highlighted so far in this review. The suggestion that the subject cannot be discussed exhaustively cannot be far from the truth. Our opinion is, there might be other roles of fungal endophytes yet uncovered. However, it is of note that, few reports on certain harmful effects on the host plants are also available (see Table 1 and Supplementary Figure S1). In the light of this, it is of note that, the relationship that exists between host plants and endophytes can be likened to a balanced antagonism, as the host derives both positive and negative effects depending on the environmental conditions. Hence, the environmental conditions could be said to distinguish a mutualistic endophyte from a pathogenic endophyte.

31Fungal endophytes provide protection for crop plants against insects attack. The ability to minimize attack from all kinds of insect pests; lepidopterous larvae, aphids and thrips, and other cosmopolitan insects has widely been reported. This has also been the primary aim of artificial inoculation of entomopathogenic fungi into economic crops to establish as endophytes.

32Aside insect management, the potential of fungal endophytes in providing protection for plants against plant-parasitic nematodes and plant disease pathogens, enhancing host growth, promoting nutrient acquisition and improving tolerance to abiotic stresses, as well as enhancing resistance to mammalian herbivores have all been reported and clearly emphasized in this review.

33Since fungal endophytes promote phosphorus, nitrogen and other essential nutrients uptake in the host plants, deep knowledge on this would assist organic and inorganic fertilizer users to ensure optimum usage. Moreover, the ability of fungal endophytes to improve the plant ability to tolerate heat stress, salt, drought and other abiotic stresses adds a new dimension to host plants–endophytes interactions, and could significantly be explored or used in agriculture not only to mitigate pests and diseases under climate change conditions, but also as an alternative approach to entomopathogenic fungi autodissemination in inundating application. That is, fungal endophytes could be a suitable replacement for entomopathogenic fungi which are normally applied as inundative sprays to offer short-term pest control. These entomopathogenic fungi when successfully established as endophytes in plants can offer long-term pests and diseases control.

34The recent discoveries indicating that fungal endophytes provide other beneficial effects to their host plants aside mere protection against pests will go a long way in defining the huge importance of fungal endophytes in crop production for human sustainability. Also, we hope that this information would increase the effective use of fungal endophytes by exploring all their functional attributes as an integral part of the integrated pest management programs throughout the different agroecological zones worldwide.

35LW designed the review outline. The manuscript was written by BB, KA, and CD. KA and RK reviewed the manuscript.

36The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.