Physiological mechanisms driving virulence in insect-pathogenic fungi.

Entomopathogenic fungi are myco-biocontrol, potentially the most versatile biological control agents with a wide host range and are an environmentally sound and effective means of reducing insect-pests. The use of microbial control agents particularly entomopathogenic fungi, have been investigated for the control of a wide range of orchard and field crop pests and are a widespread component of most terrestrial ecosystems. Entomopathogenic fungi are a major component of integrated pest management techniques as biological control agents against insect pests and other arthropods in horticulture, forestry and agriculture and are found in the divisions of Zygomycota, Ascomycota, Deuteromycota, Chytridiomycota and Oomycota, which were previously classified within fungi. Insect control using entomopathogenic fungi is achieved when sufficient infective propagules, conidia contact a susceptible host and conditions are suitable for a lethal mycosis to develop. A wide range of fungi occur in the soil environment and they have various ecological functions. Most of these fungi, along with a range of bacteria, can grow on artificial media in vitro . Several methods have been used to describe the variation within a species of entomopathogenic and mycoparasitic fungi including morphological characteristics of spores and colonies, extracellular protein profiles, pathogenecity, growth and nutrient requirements. Furthermore, immune taxonomic and chemotaxonomic methods have been used, though only with limited success. Taxonomic procedures are becoming more and more complex and it is generally accepted that some forms of molecular identification techniques are needed in addition to the traditional morphological characteristics formally used to classify fungal species. During the last four decades, over 80 companies worldwide have developed 171 mycoinsecticides and myco-acaricides. Use of mycoinsecticdes is likely to rise if research is focus on; improving its performance under challenging environmental conditions, formulations that will increase persistence, longer shelf life, ease of application, pathogen virulence and wider spectrum of action. Keywords: Eco-friendliness, fungi , insect pest , myco-acaricides, mycoinsecticdes DOI: 10.7176/JEES/10-3-03 Publication date: March 31 st 2020

Intracellular trehalose accumulation is relevant to fungal life and pathogenicity. Trehalose-6-phosphate synthase (TPS) is known to control the first step of trehalose synthesis, but functions of multiple TPS genes in some filamentous fungi are variable. Here, we examined the functions of two TPS genes (tpsA and tpsB) in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest control. Intracellular TPS activity and trehalose content decreased by 71-75 and 72-80% in ΔtpsA, and 21-30 and 15-45% in ΔtpsB, respectively, and to undetectable levels in ΔtpsAΔtpsB, under normal and stressful conditions. The three mutants lost 33, 50, and 98% of conidiation capacity in standard cultures. Conidial quality indicated by viability, density, intracellular trehalose content, cell wall integrity, and hydrophobicity was more impaired in ΔtpsA than in ΔtpsB and mostly in ΔtpsAΔtpsB, which was also most sensitive to nutritional, chemical, and environmental stresses and least virulent to Galleria mellonella larvae. Almost all of phenotypic defects in ΔtpsAΔtpsB approached to the sums of those observed in ΔtpsA and ΔtpsB and were restored by targeted gene complementation. Altogether, TpsA and TpsB play complementary roles in sustaining trehalose synthesis, conidiation capacity, conidial quality, multiple stress tolerance, and virulence, highlighting a significance of both for the fungal adaptation to environment and host.

Conidiation under illumination enhances conidial tolerance of insect-pathogenic fungi to environmental stresses

Chapter 29 - Metarhizium

Biological control of Drosophila suzukii: Efficacy of parasitoids, entomopathogenic fungi, nematodes and deterrents of oviposition in laboratory assays

Fungal entomopathogens are widespread in nature and contribute to the natural regulation of insects. They can be exploited for pest management as biological control agents of pests in attempts to improve the sustainability of crop protection. Four types of biological control are recognized: classical, inoculation, inundation, and conservation biological control. Classical biological control is the intentional introduction and permanent establishment of an exotic biological agent for long-term pest management. Inoculation biological control is the intentional release of a living organism as a biological control agent with the expectation that it will multiply and control the pest for an extended period, but not permanently. Inundation biological control is the release of large numbers of mass-produced biological control agents to reduce a pest population without necessarily achieving continuing impact or establishment. Conservation biological control is a modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests. The traditional and the most popular approach in biological control with entomopathogenic fungi has been to apply the fungal material to the cropping system (as biopesticide), using an inundation biological control strategy. The term biopesticide is used for microbial biological pest control agents that are applied in a similar manner to chemical pesticides. The use of biopesticides can substitute for some (but not all) chemicals and provide environmentally safe and sustainable control of pests but EU legislation and prohibitive registration costs are discouraging the development and commercialization of many promising new products.

Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus

The oriental fruit moth, Grapholita molesta (Busck) is a key pest of tree fruit of Europe, Asia, America, Africa, Australia, and New Zealand, which makes a big damage to apple trees, pear tree and the stone fruit of the peach, plum, apricot, nectarine, cherry and so on. It is difficult to control these pests with traditional chemical methods meanwhile with the increasing demand for food safety, biological control method to this pest has attracted more and more people's attentions. Beauveria bassiana is one of the most studied and applied entomopathogenic fungi, can infected and kill the oriental fruit moth as a biological control agent. The entomopathogenic fungi with a wide range hosts and they are harmless to the environment, human and animal. Using entomopathogenic fungi to control pests has many advantages and they have been an important part in biological control of pests, although it still has some natural defects, such as long effective time and easy to be affected by environmental conditions. In order to make good use of it in the future, it is necessary to deeply understand their living conditions and infection mechanism to insects. Entomopathogenic fungi can invades the insects from the body wall through contact directly, but also can through the digestive tract, stomata and wounds and other ways into the insect body. But insects have evolved a strong innate immune system to protect themselves from infection by the pathogens and adverse conditions. When insects are infected by entomopathogenic fungi, their innate immune system will firstly be activated. And the insects will resist the infection by their immune response, which will lead to the reduction of infection efficiency and the control effect. So, it is necessary to study the immune response of insects introduced by entomopathogenic fungi, and it is a hotspot in pest control. This article summarized the occurrence and control technologies of oriental fruit moth, and the research status of entomopathogenic fungus (B. bassiana), finally it summarized the insect immune response induced by entomopathogenic fungi. This will provide a significantly deepened the understanding on mechanisms of insect and entomopathogenic fungi. And it prospected the improvement of effective on biological control of oriental fruit moth by B. bassiana, which provide a theoretical basis for supply better services to plant protection in the future.

Tea mosquito bug, Helopeltis theivora Waterhouse, is the major sucking pest of tea plant. It mainly attacks the young shoots essential for tea production. In lacunae of proper management, the pest remains active throughout the year resulting in almost 100% crop loss. To manage the population of this pest below Economic Threshold Level (ETL) of 5%, several rounds of chemical pesticides are applied during the season. Tea, being a consumable product; the unwanted pesticide residue cause a major concern for the tea industry. Thus, incorporation of alternate strategies like Biological Control Agents (BCA) in pest management programme is important to overcome the problems besides prevent the pest from developing resistance. The BCAs like entomopathogenic bacteria, fungi and virus are effective in an eco-friendly management of the pest population. The entomopathogenic fungi, Beauveria bassiana has been found to be effective pest control agent in several agro ecosystems including tea. Commercial B. bassiana formulations are available but the local strains are reported to be more efficient in managing the pest population. The present study was aimed to analyze the potential of a new strain of B. bassiana named as BPA/B7 (I.D. No. 10,928.8) isolated from tea soils of Tinsukia (Assam) against H. theivora. The efficacy of six concentrations (5 mlL-1, 10 mlL-1, 15 mlL-1, 20 mlL-1 and 25 mlL-1 ) of powder formulation of BPA/B7 was compared with a commercial formulation to estimate the LC50 of the same. The BPA/B7 (B. bassiana with a spore density of 1.68X106 spores/ml was found to kill 50% of H. theivora at a concentration of 21.87 mlL-1 within 96hrs. Further studies on the standardization of both liquid/powder formulations, shelf life studies, followed by their field evlauation, will ensure the possibility of utilizing this strain as a potential componemt of intergrated management of H. theivora.

The Gti1/Pac2 protein family, which is highly conserved across fungi, is pivotal in processes such as fungal development, spore formation, protein export, toxin production, and virulence. Despite its importance, the precise functions of Pac2 within entomopathogenic fungi have yet to be fully understood. In our study, the MaPac2 gene from M. acridum was identified, and its functions were explored. Studying the domain of the protein showed that MaPac2 comprises 422 amino acids with a characteristic Gti1/Pac2 family domain (Pfam09729). Additionally, MaPac2 is predicted to have an N-terminal protein kinase A phosphorylation site and a potential cyclin-dependent kinase phosphorylation site, highlighting its potential regulatory roles in the fungus. Our findings indicate that the inactivation of MaPac2 resulted in faster germination of conidia and a marked reduction in conidial production. Furthermore, stress tolerance tests revealed that the absence of MaPac2 significantly bolstered the fungal resilience to UV-B radiation, heat shock, SDS exposure, and stresses induced by hyperosmotic conditions and oxidative challenges. Virulence assessments through bioassays indicated no substantial differences among the WT, MaPac2-disrupted strain, and CP strains in the topical inoculation trials. Interestingly, deletion of MaPac2 increased the fungal virulence by intrahemocoel injection. Furthermore, we found that disruption of MaPac2 impaired fungal cuticle penetration due to the diminished appressorium formation but increased the fungal growth in locust hemolymph. These findings provide further insights into the roles played by Gti1/Pac2 in insect pathogenic fungi.

Earthworms influence soil ecosystem functions such as decomposition and regulation of nutrient cycles by burrowing, feeding, or secreting their cutaneous excreta. Earthworms can also modulate soil biodiversity and their interactions. Recent research has revealed that earthworm feeding activity or excretions can alter the performance of entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPFs), known biological control agents. Still, the impact of earthworm cadavers on the growth and reproduction of other biological control agents such as nematophagous fungus (NFs) and EPFs has been poorly explored. We suggest that earthworm body tissue could be used as a food supply by certain fungal species. We hypothesize that fungi with a high adaptive capacity as pathobionts such as NF will use earthworm cadavers to a certain extent, while the growth and reproduction of fungi with limited or specific nutrition needs, such as EPFs, will be restricted. In this study, we evaluated two NF species: a nematode-trapping fungus Arthrobotrys musiformis Drechsler (Orbiliales: Orbiliaceae) and a nematode egg or cyst parasite Purpureocillium lilacinum (Thom) (Hypocreales: Ophiocordycipitaceae), and one EPF species: Beauveria bassiana (Balsamo). We exposed each fungus to two different Aporrectodea molleri extract media at six concentrations in agar medium (n = 3 per treatment) and liquid medium (n = 5): (i) earthworms devoid of intestinal contents (EDG) (C1...C6), and (ii) fresh earthworms (FE) (C1...C6). The use of FE and EDG was designed to discriminate the effect of earthworms? intestinal content on fungal growth. These treatments were also compared to alternative conventional media potato dextrose agar (PDA) and brain heart infusion (BHI) as the positive controls. We evaluated the vegetative growth capacity daily for 18 days, registering the reproduction at the end of the experiment. We observed that EPF B. bassiana did not grow in FE and EDG solid medium but showed germination in their liquid media. However, both NF species could grow and reproduce in both media but following a species-specific pattern. In all cases, FE and EDG reduced fungal growth compared to PDA and BHI. Our results suggest that the use of earthworm as a resource in natural conditions might depend on the expected specialization, with EPF B. bassiana with the lowest use, NF A. musiformis moderately, and P. lilacinum widely used. The study illustrates the complexity of the soil organisms? interactions and highlights the necessity to advance the understanding of the contribution of all the beneficial soil organisms in a comprehensive manner.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

The viral, bacterial, fungal and nematode pathogens of arthropod pests of apple and pear in northern and central Europe and their use as biocontrol agents are reviewed. Baculoviruses are important viral pathogens of several lepidopterous pests of apple and pear but other viral pathogens have not been investigated in depth and are little known. The granuloviruses of codling moth, Cydia pomonella (CpGV), and to a lesser extent, of the summer fruit tortrix moth, Adoxophyes orana (AoGV), have been researched extensively and are exploited as biological control agents. Commercial development and use has been limited because of their high costs, slow action, short persistence and specificity relative to broad-spectrum pesticides. The widespread development of strains of codling moth multi-resistant to insecticides and the desire to reduce dependence on pesticides have improved the commercial prospects of CpGV and use is likely to increase. The development of a genetically improved egt-strain of CpGV (lacking the ecdysteroid-UDP glucosyl transferase gene) in the UK is a significant breakthrough, though commercialization in the UK may be difficult due to adverse public attitudes to the release of genetically-modified microorganisms. Future research and development approaches include further genetic manipulation of CpGV and AoGV to improve potency, speed of kill and/or persistence, improvement of formulation (to reduce UV light sensitivity) and development of cheaper mass production techniques and possibly in vitro production. A systematic search for baculoviruses and other viruses of apple and pear pests is likely to reveal important new opportunities. The most important bacterial pathogen used as a biological control agent is Bacillus thuringiensis (Bt). However, Bt products currently available have limited effectiveness against many orchard pests due to the pests' cryptic life habits. The HD-1 Bt strain has been investigated and used extensively for control of leaf-rolling tortricid larvae and is widely used, but efficacy is moderate. Advances in biotechnology and genetic engineering provide opportunity for development of Bt strains designed specifically to control orchard pests, but this has not yet been done for commercial reasons. Other research approaches include the evaluation of new Bt products developed for other markets worldwide and the bioassay of strains from Bt collections against specific apple or pear pests. Entomopathogenic fungi provide good opportunity for development as biological control agents of apple and pear pests. The main factor limiting their effectiveness is the requirement for high humidities and moderate temperatures for spore germination and development. For foliar pests, a useful starting point for research might be the control of sucking pests which excrete honeydew (e.g. Cacopsylla sp. or aphids) or those that inhabit protected microenvironments (e.g. Dasineura sp.). Key areas for research are improved formulation, the selection of low temperature-active strains, field evaluation and avoiding possible adverse effects of fungicides. An alternative approach is to examine the exploitation of entomopathogenic fungi in soil, to which many species of entomopathogenic fungi are adapted ecologically. Apple and pear orchards provide long-term stable habitats where populations of entomopathogenic fungi in soil are likely to be large. There are few important soil pests of apple or pear. However, many species spend part of their life in soil, mainly to pupate or overwinter, where they may be targeted by fungal entomopathogenic biocontrol agents. Entomopathogenic nematodes have many attributes which favour them as biological control agents. However, their requirement for surface moisture for survival and movement means there are only limited prospects for using them as biological control agents for foliar pests. As with entomopathogenic fungi, there are better prospects for control of pests that occur in soil. Microbial pathogens and entomopathogenic nematodes are important components of the natural enemy complex of apple and pear orchards and more effort needs to be devoted to fostering them and exploiting them as biocontrol agents in sustainable, biologically-based Integrated Pest Management programmes. They can in many cases be mass produced at low cost by bulk fermentation processes and applied as sprays (as 'biopesticides') and are, at least potentially, ideal biological control agents for many apple or pear pests. Important general characteristics are their comparative environmental and human safety, compatibility with other control strategies in Integrated Pest Management programmes and reproductive capacity. They tend to be effective in a narrower range of environmental conditions than pesticides, but there is considerable potential to improve their effectiveness by improved formulation, strain selection and genetic manipulation. They are often host-specific and thus, offer restricted marketing opportunities, which is a significant barrier to development and commercialisation. Registration procedures and associated fees for microbial agents are a further significant barrier. Such requirements do not apply currently to nematodes.

False codling moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae) is an important pest of various fruit crops in South Africa. Current FCM control strategies include the use of chemical insecticides. However, FCM has developed resistance to some of the insecticides, and stringent chemical residue restrictions have been imposed by some foreign markets. Thus, the demand for high-quality fruit has translated into a need for new, efficient and effective integrated pest management (IPM) strategies. One such strategy is the control of the soil-dwelling life stages of FCM, using entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPF). Both of the biocontrol agents concerned have individually been shown to be effective against FCM. However, it is possible that, if they are applied simultaneously, a synergistic relationship might be observed between EPNs and EPF that could serve to enhance their efficacy against the target pest. In addition to reviewing previous and current control options against FCM in South African fruit crops, this study investigates the potential for using EPNs and EPF individually, and in combination, as biological control agents against FCM within an IPM system.

The behavioural response of an insect to a fungal pathogen will have a direct effect on the efficacy of the fungus as a biological control agent. In this paper we describe two processes that have a significant effect on the interactions between insects and entomopathogenic fungi: (a) the ability of target insects to detect and avoid fungal pathogens and (b) the transmission of fungal pathogens between host insects. The behavioural interactions between insects and entomopathogenic fungi are described for a variety of fungal pathogens ranging from commercially available bio-pesticides to non-formulated naturally occurring pathogens. The artificial manipulation of insect behaviour using dissemination devices to contaminate insects with entomopathogenic fungi is then described. The implications of insect behaviour on the use of fungal pathogens as biological control agents are discussed.