The discovery of the potential of endophytic entomopathogenic fungi: a historical overview

Insects, plants, and fungi have shared similar environmental conditions throughout their evolution (^{Khare et al., 2018}). In the Metarhizium genera, its pathogenicity against insects is recognized since 1879 when E. Metchnikoff discovered that the fungus was the cause of death of some arthropods (^{Stone and Bidochka, 2020}). However, it was not long ago that mycologists and entomologists observed in detail the influence that fungi have on insects, and motivated by the interest to understand these interactions, a great opportunity arose for their study and understanding. Thus, Quentin Wheeler, as a proponent of phylogenetic analysis, hoped to encourage entomologists and mycologists to work together, so he organized a Symposium with the participation of the Entomological Society of America, held in Syracuse and Ithaca, New York, USA in 1981 (^{Vega and Blackwell, 2005}). This allowed the foundations to elucidate the mechanisms of these prominent biocontrolling agents. Later, ^{Bing and Lewis (1992)} reported the endophytic growth and colonization of B. bassiana in maize, in addition to observing for the first time that colonization of the plant by the fungus caused the mortality of the European corn borer Ostrinia nubilalis (Hübner 1796) (Lepidoptera: Crambidae). The findings documented by ^{Bing and Lewis (1992)}, are now common to practically all plant species that have been analyzed for endophytic fungi and show that something common in EEF, is that they can colonize plant tissues without damaging the plant, while on the contrary, they bring benefits such as protection against pests and phytopathogens. In addition, it is currently known that EEF naturally colonize cocoa plants, beans, sorghum and wheat, among others (^{Behie et al., 2017}; ^{Sánchez-Rodríguez et al., 2018}; ^{Ambele et al., 2020}; ^{Mantzoukas and Grammatikopoulos, 2020}).

Since then, more than 7000 scientific articles have dealed with a better understanding of the impact of environmental factors (e. g. humidity, temperature, presence or limitation of nutrients) in the colonization of plants by fungi, as well as their entomopathogenic role (^{Vega et al., 2009}). However, much remains to be understood, which is why it is necessary to lead scientific research towards an approach based on a new paradigm, where EEF can be used massively under field conditions. Therefore, it is still necessary to unravel the full potential of these entomopathogenic agents, including investigations for their use as endophytes, antagonists of plant diseases, colonizers of the rhizosphere and plant growth promoting fungi. This will allow the use of EEF on a large scale as a healthy and sustainable alternative to conventional agrochemicals (^{Khare et al., 2018}).

Potential of endophytic entomopathogenic fungi as promoter of plant growth and protection

The term endophytic refers to any organism that establishes a non-obstructive, asymptomatic, and transient cost-benefit relationship within the living tissues of the host plant (^{Jaber and Ownley, 2017}). Endophytic microorganisms constitute a diverse group and can be found in a wide range of plants, including algae, mosses, grasses and other vascular plants (^{Bamisile et al., 2018b}). In the case of EEF, they mainly belong to the Phylum Ascomycota, although those belonging to the Phylum Basidiomycota, Oomycota and Zygomycota are also important. An example as EEF use is the fungus belonging to the Basidiomycota phylum, Leucocalocybe mongolica (S. Imai) X.D. Yu & Y.J. Yao, 2011 (Agaricales: Clitocybaceae); ^{Wang et al. (2022)} documented that this fungus was able to promote the growth of Arabidopsis thaliana (Brassicaceae), wheat and cotton, showing that in these plants, when in the presence of L. mongolica, the fungus allowed the development of the bacterium Bacillus pumilus Meyer and Gottheil 1901 (Bacillales: Bacillaceae), which was able to solubilize phosphorus when the plants were placed under saline stress, thus promoting their growth.

Another more recent example was described by ^{Andersen et al. (2024)}, who applied 10 mL of Pythium oligandrum (Pythiales: Pythiaceae) (fungus belonging to the phylum Oomycota) by foliar spray on table potato crops where they included four varieties (Kuras, Desirée, King Edward and Kuras Field) under field conditions and greenhouse. The results obtained revealed that the Kuras genotype responded to P. oligandrum treatment with significantly greater plant height and fresh weight of both shoots and roots compared to the untreated treatments, respectively.

Historically, the classification has grouped EEF both inside (colonize grasses), and outside (colonize angiosperms, conifers, ferns, and non-vascular plants) the family Clavicipitaceae (^{Bamisile et al., 2018b}). One of the most important ecological functions of EEF as endophytes is the capacity that they confer on the host plant to take up nutrients. This is because the mechanism used by EEF to increase the plant’s ability to absorb nutrients, is the plant-fungus symbiosis, which allows the plant to improve nutrient absorption when these fungi increase the effective areas of root absorption through the formation of dextramatrical hyphal length (EMH) (^{Chen et al., 2020}; ^{Ai-Tian et al., 2021}). These hyphae constitute extensions of the fungal mycelium and extend beyond the plant root surface, thereby increasing significantly the volume of soil explored resulting in a greater capacity to absorb water and nutrients, including nitrogen and phosphorus (^{Mohamed et al., 2022}; ^{Zeng et al., 2024}). The response of the plant is an increase in its growth, greater tolerance to stress and production of secondary metabolites, which protect the plant against insects and pathogens (^{Yun et al., 2017}; ^{Clifton et al., 2018}; ^{Ahmad et al., 2020}).

It has recently been documented that, while some species of fungi function as natural endophytes in plants, others can be deliberately introduced into them using different inoculation methods. For the application of EEF various methods are available, but two of them constitute the majority of scientific studies developed by the scientific community, namely the promotion of plant growth and their impact as biological control agents of plant pathogens (^{Fontana et al., 2021}). In this regard, pioneering studies document different application strategies: through known conidia suspensions (^{Garrido-Jurado et al., 2017}; ^{Jaber and Enkerli, 2017}), direct inoculation of seeds (^{Ramakuwela et al., 2020}), injection of inoculum in stems (^{Mantzoukas and Eliopoulos, 2020}), inoculation of roots by immersion (^{Russo et al., 2015}), and through irrigation with conidia suspensions (^{Sánchez-Rodríguez et al., 2018}).

There are other mechanisms involved in promoting plant growth mediated by EEF; probably one of the most important is the absorption of nutrients (e. g. iron). This has been demonstrated in cabbage crops (Brassica oleracea L.) colonized by B. bassiana and M. brunneum, which promoted significantly longer shoot length (123 % for B. bassiana and 53.3 % cm for M. brunneum) in comparison with the control treatments (^{Dara et al., 2017}). ^{Behie et al. (2017)}, verified the ability of the fungus M. robertsii to fix nitrogen derived from corpses of Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) insects and transfer it to bean plants (Phaseolus vulgaris L.), observing a significant increase in root growth (up to 31.8 %). These results have generated greater acceptance and popularity in the use of EEF in agricultural systems, with a significant reduction in agrochemicals, and with it, less damage to the environment and to human and animal health (^{Dara, 2019}; ^{Quesada-Moraga, 2020}).

Recent studies conclude that the endophytism carried out by EEF and the close beneficial interactions with plants are a process like that of mycorrhizal fungi (^{Jaber, 2018}). In addition, the EEF confer abiotic resistance to the plant against water stress (drought and waterlogging), salinity and mineral toxicity (^{Martinez-Medina et al., 2016}; ^{Wei et al., 2020}). EEF has now been reported to have the ability to act as antagonists against phytopathogens. For instance, a study carried out with cotton plants (Gossypium hirsutum L.) under greenhouse conditions showed that soil inoculation with conidia of B. bassiana and M. brunneum, induced resistance in plants from 15 % to 57 % against the pathogen Fusarium oxysporum Schltdl., 1824 (Hypocreales: Nectriaceae), indicating the potential of EEF in plant protection (^{Dara et al., 2016}).

Several capacities of EEF have also been verified (Table 1). Examples of this are observed in the potential of B. bassiana to be transmitted inside the soil by endophytically colonizing the roots of various plant species: cacao (Theobroma cacao L.) (^{Ambele et al., 2020}), barley (Hordeum vulgare L.) (^{Veloz-Badillo et al., 2019}), strawberry (Fragaria x ananassa Weston) (^{Canassa et al., 2020}), beans (Phaseolus vulgaris L.) (^{Behie et al., 2017}), corn (Zea mays L.) (^{Ahmad et al., 2020}), cassava (Manihot esculenta Crantz) (^{Greenfield et al., 2016}), soybean (Glycine max L.) (^{Russo et al., 2019}), tobacco (Nicotiana tabacum L.) (^{Lee and Kim, 2019}) and wheat (Triticum durum Desf., and Triticum aestivum L.) (^{Sánchez-Rodríguez et al., 2018}; ^{González-Guzmán et al., 2020}). The above shows that EEF in natural environments colonize and promote plants development, being able to survive in or around them (even in the absence of their host insects) by obtaining nutrients directly from the plant without negatively affecting it. An important consideration within the potential of EF as endophytic is that they indirectly affect pest populations through non-entomopathogenic mechanisms, such as antibiosis and antixenosis (^{Dara, 2019}), and induced systemic resistance (^{Wei et al., 2020}). The suggested explanation is that secondary metabolites produced by fungi generate greater resistance to the plant against the attack of pest insects, reducing their appetite, fecundity, fertility, and longevity. Furthermore, the studies confirm that EEF not only affected the development of pests but were also able to promote a significant increase in plant growth (^{Sánchez-Rodríguez et al., 2018}; ^{Sun et al., 2020}; ^{Wei et al., 2020}).

Effect of pesticides and genetic modified plants

Modern agriculture is based on crop production systems that require the use of agrochemicals to improve and protect yields. Production of these substances increased by 11 % annually, from 0.2 million in the 1950 to more than 5 million tons in 2000. Herbicides, insecticides, and fungicides are the groups with the highest sales worldwide, with carbamates, organophosphates, pyrethroids, neonicotinoids and growth regulators being some of the most widely used substances (^{Carvalho, 2017}).

The close association between endophytic fungi and their host plants suggests an important effect of agrochemicals in the former. However, there is still little knowledge in this regard (^{Zhou et al., 2016}; ^{da Costa Stuart et al., 2018}). While several authors report that some pesticides did not affect the diversity of endophytic fungi (^{Zhou et al., 2016}; ^{Win et al., 2021}), others have registered a reduction in diversity in treated crops (^{da Costa Stuart et al., 2018}). However, there is consensus in the fact that pesticides can alter the composition of endophytic fungi, because where pesticides were applied, species that were not registered in the untreated crops usually appeared and predominated. It is argued that these changes could negatively influence the physiology of the plant, and probably the quality of the food we consume (^{Zhou et al., 2016}; ^{da Costa Stuart et al., 2018}; ^{Win et al., 2021}). It is clearly appreciated that the previous studies were developed in different regions, crops and pesticides. Given the wide variety of pesticides (many with systemic activity), crops and varieties, and the complex interactions with the environment, the knowledge about the effect of chemical pesticides on endophytic fungi appears to be still incipient. Additionally, the aforementioned studies have been focused on endophytic fungi in general, and only sporadically some species of entomopathogens are recorded. Although there is abundant knowledge on the impact of pesticides on entomopathogenic fungi in vitro (^{Pérez-González and Sánchez-Peña, 2017}; ^{Wari et al., 2020}), there is a lack of knowledge about their effect endophytically.

Regarding the potential impacts of genetic engineering technologies on the well-being of farmers and the environment, there is much to analyze, highlighting that the economic effects of genetically modified crops are highly understood (^{Ganesan et al., 2017}; ^{Kumar et al., 2018}; ^{Araus et al., 2019}). Cotton Gossypium spp (Malvaceae) is one of the most important crops worldwide that serves as raw material for textile industries, and has a high economic impact of approximately 600 billion dollars annually (^{Khan et al., 2020}). In this sense, genetically modified cotton is in high demand and in its genome is the gene that encodes the Cry1Ac protein from Bacillus thuringiensis Berliner 1915 (Bacillales: Bacillaceae), which acts as a bioinsecticide against lepidopteran pests. This protein is expressed in these plants throughout their life cycle, and can interact directly with microorganisms associated with the crop, which could negatively affect the environmental functions of the endophytic fungal community (^{Bruinsma et al., 2003}). Based on the above, ^{De Souza Vieira et al. (2011)} report in Brazil the evaluation of a transgenic Bt cotton (Acala 90B) and a natural non-Bt cotton (Acala 90) during different stages of crop development (pre-flowering, flowering, capsule formation and opening), to investigate the possible effects non-target in endophytic fungal communities. The results obtained revealed that the expression of the Bt Cry1Ac protein of the genetically modified cotton plants did not affect the degree of fungal colonization, which allowed the obtaining of a total of 17 genera of endophytic fungi, including the species Acremonium spp., Cladosporium spp., Colletotrichum spp., Curvularia spp., Fusarium spp., Glomerella spp., Guignardia spp., Lecanicillium spp., Nigrospora spp., Pestalotiopsis spp., Phoma spp., Phomopsis spp., Rhizopus spp., Rhodotorula spp., Talaromyces spp., Tritirachium spp. and Xylaria spp., respectively. In the case of EEF, in a study carried out by ^{Morjan et al. (2002)}, is documented that herbicides used in genetically modified crops of soybeans and corn had negative effects on the fungi B. bassiana, M. anisopliae, Nomuraea rileyi (Farlow, 1974) (Hypocreales: Clavicipitaceae) and Neozygites floridana Weiser & Muma, 1966 (Neozygitales: Neozygitaceae), when exposed to seven standardized 0.96 % formulations (the lowest recommended dose for field applications) that include glyphosate as an active ingredient. In the study, the mycelial growth area and the spore density were estimated, finding that the four analyzed fungi were susceptible to glyphosate. These results are alarming, since spraying these herbicides in large areas and with 100 % coverage in transgenic crops can be detrimental to EEF and thus, negatively impact the natural epizootics necessary for the suppression of pest insects. Meanwhile, other studies mention that the flow of genes transferred horizontally in genetically modified crops is considered an important evolutionary force that alters gene frequencies through mutations, genetic drift and selection (^{Lu and Yang, 2009}). In this sense, gene flow can negatively affect the environment by creating a reduction in the genetic variability of native populations, the alteration of biodiversity and the alteration of the soil microenvironment. For these reasons, the introduction of genetically modified plants in agroecosystems poses potential risks for the species that inhabit it, including EEF, with the premise that it is difficult to predict their consequences in the future, possibly leading to the development of superweeds, the evolution of new viral pathogens and the evolution of pest insects with resistance to new compounds (^{Tsatsakis et al., 2017}). For these reasons, it is important to analyze and discuss the long term and large-scale impact of genetically modified crops on other non-target organisms in agricultural ecosystems before carrying out their approval and release into the environment. It is also necessary to make efforts that favor the design of new products, as well as contribute to the improvement of application devices. Besides, the growing public concern about the excessive use of agrochemicals and genetically modified organisms has triggered a social pressure for a transition towards agroecological pest management. Given this scenario, ^{Lechenet et al. (2017)} highlights that it is possible to achieve a significant decrease in the use of herbicides, fungicides, and insecticides (37, 47 and 60 %, respectively) through the adoption of new production strategies where biological control is of fundamental importance.

Considerations in the application of endophytic entomopathogenic fungi: the elucidation of its potential in sustainable agriculture

For its implementation in agricultural systems, EEF must be applied to plants, at least through three possible strategies: to the seed, to the foliage or to the roots. It is important to note that as research with EEF progresses, it has been discovered that their success on the capacity of colonization and growth promotion of host plants depends on the methodology used for inoculation (^{Jaber and Enkerli, 2016}; ^{Bamisile et al., 2018a}) and some of the main EEF mechanisms of action involved in generating enhanced pest control or disease resistance, include the following: A) Toxin and enzyme production, an example of this is the fungus Aspergillus sojae Sakaguchi & K. Yamada ex Murakami, 1971, which was isolated from the oregano plant Coleus amboinicus Lour, 1790 (Lamiaceae) and determined the production of toxic enzymes such as 2-furancarboxaldehyde and levoglucosenone on the cotton leaf worm Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae), where when applied on L₃ larvae of S. litura by dipping, 57 % mortality was recorded (^{Elango et al., 2020}); B) Competition for nutrients, this is due to the fact that EEF colonize the interior of plants and compete with phytophagous insects for available nutrients. Thus, Acremonium alternatum Link, 1809 caused L₁ larvae of Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) feeding on leaves of previously inoculated cabbage plants to show mortality during the first 10 days of development (^{Raps and Vidal, 1998}); C) Induction of resistance in the host plant, EEF has been shown to be able to biosynthesize “phytochemicals” originally thought to be produced only by their host plants, which induce defense responses in the host plant against diseases (^{Ancheeva et al., 2020}). For example, in a study by ^{Russo et al. (2015)}, it was shown that the foliar spraying of B. bassiana turned out to be more successful than the inoculation of seeds in tobacco, corn, wheat, and soybean plants which promoted the growth of these crops. In any case, it is important to standardize the methodologies for its inoculation, as well as to consider the concentration of conidia to be applied (^{Jaber and Enkerli, 2017}; ^{Javed et al., 2019}). Besides, the implementation of EEF in agro-food production requires to a great extent, the understanding of the biotic and abiotic factors that interact in the plant-fungus relationship. Understanding the effect of these factors and inoculation methods, becomes a challenge for the development of sustainable agricultural production systems (^{Tall and Meyling, 2018}; ^{Vega, 2018}). A possible explanation for the limited success obtained with EEF inoculation in seeds and roots, is focused on the competition with other soil-dwelling microorganisms that are transmitted in the same way as EEF: through water currents and wind (^{Bamisile et al., 2018a}).

According to the above, it is necessary to identify and understand the effect of other microorganisms, and the soil characteristics that favors or limit the activity of EEF. For instance, ^{Mayerhofer et al. (2017)} investigated through ribosomal marker sequencing, the possible effects of Ascomycota, Zygomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Blastocladiomycota, Proteobacteria, Actinobacteria, Chloroflexi and Acidobacteria naturally present in soils on the fungus M. brunneum. The study was carried out under field conditions for the biological control of Agriotes spp. in potato. The results revealed an efficiency of 77 % referring to decrease in tubers damaged by the insect compared to the control, as well as a successful establishment of the fungus in the crop, without negatively affecting the soil microbial communities described above. The foregoing will facilitate its manipulation and effectiveness for the development of technologies with a socially, economically, and ecologically viable approach (^{Jaber and Enkerli, 2016}; ^{Bamisile et al., 2018a}; ^{Vega, 2018}). This approach must consider the ecology of EEF, enhancing their development and the availability of a greater number of products on a commercial scale for use in current agriculture (^{Jaber and Ownley, 2017}). It should also be considered that among the desired characteristics of EEF strains, are a broad spectrum towards pest species, greater persistence, stimulation of plant growth, and the compatibility with other IPM (included chemical pesticides) strategies under greenhouse and field conditions (^{Gathage et al., 2016}; ^{Clifton et al., 2018}; ^{Mantzoukas and Grammatikopoulos, 2020}).

The successful positioning of EEF as biological control agents widely used worldwide requires developing standard application methodologies under field conditions. In this regard, the factors that influence the development of fungi should be investigated (e. g. UV radiation and humidity). This will make possible to exploit all the beneficial capacities of such important microorganisms (^{Jaber and Ownley, 2017}; ^{Vega, 2018}).

An important aspect to highlight is the compatibility of EEF with entomophagous insects (^{Gathage et al., 2016}; ^{González-Mas et al., 2019}), which expands the possibilities in their use in IPM. For instance, the survival of the predator Rhynocoris marginatus (Fabricius, 1794) (Hemiptera: Reduviidae) was not affected when it fed on larvae of Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae) infected with the fungi I. fumosorosea and B. bassiana (^{Ullah et al., 2019}). These results suggest that EEF are compatible to be used together with the predator to promote the biological control of S. litura.

On the other hand, studies are needed to document the persistence of EEF in natural environments inside and outside their host plant. In one of them, it was documented that cotton seeds inoculated with the fungi B. bassiana and Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones and Samson 2011 (Hypocreales: Ophiocordycipitaceae), showed a successful endophytic colonization in the seedlings; its persistence fluctuated from 7 to 14 days after application under greenhouse conditions, and its presence limited the incidence of the aphid Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) (^{Lopez et al., 2014}). It has been documented that in some cases the persistence of the fungi in endophytic form can be reduced with the age of the plant. For example, B. bassiana as endophyte of corn plants can persist up to 60 days in the culture. However, its persistence was reduced (44.44 ± 11.11 %) with the increase in the age of the plant, documenting that upon reaching 75-90 days of age, the persistence of the fungus was null. This was manifested in the protection against the corn stem borer Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae) (^{Renuka et al., 2016}). Therefore, knowing in detail the persistence of EEF colonization within the plant will allow better results to be obtained.

Another important criterion to consider is the maintenance of shelf viability and pathogenicity. It is known that subcultures of entomopathogenic fungi in an artificial medium tend to reduce their pathogenicity (^{Jaber and Ownley, 2017}). Given this fact, it is necessary to carry out continuous maintenance of the EEF isolates and to avoid the loss of these capacities, being recommendable to keep the isolates in tubes with sterile distilled water (^{Richter et al., 2016}). For its part, current research on the potential of EEF as endophytic indicates the need to study the effects of the interactions between EEF strains genotype and plant genotype, with the aim of selecting the most compatible isolates to obtain higher levels of success in the plant colonization, considering the environmental conditions surrounding the fungus and its host, as well as the need to carry out studies under greenhouse and field conditions (^{Busby et al., 2016}; ^{Kumar et al., 2018}).