Blastospores of Entomopathogenic Fungi Emerge as the Promising Active Ingredients in Biocontrol Agents: Progressive View on Its Industrial Production and Biogenesis Mechanisms

Fungal entomopathogens represent a large group of filamentous fungi that infect a plethora of arthropods in natural eco-system and have great potential in pest management programs as biocontrol reagents [1]. The host infection caused by entomopathogenic fungi (EPF) features the direct penetration of the exoskeleton by infectious hypha developing from conidia that adhere to the host cuticle [2]. After invading the host body cavity, filamentous cells develop into the single thin-walled yeast-like blastospores via dimorphic transition for rapid proliferation by efficiently assimilation of various nutrients in the host hemocoel, which ultimately causes the host’s death. Therefore, the fungus’s ability to perform dimorphism is essential for the pathogen colonization and virulence [3]. In addition to being in the hemocoel, blastospores can also be produced in submerged liquid cultivation and have great potential as the main active ingredient in practical formulations [4]. The advantages of submerged fermentation (SMF) over solid-state fermentation (SSF) mainly involve better compatibility with industrial production equipment, easier control of the whole process, and simpler procedure for product recovery [5]. In comparison with aerial conidia produced via SSF [6], the SmF-produced blastospores are more hydrophilic and virulent [7,8,9,10]. In addition, SmF for blastospore production exhibits higher productive efficiency because it takes less time and space than conidiation in SSF [11,12,13]. Thus, SmF has been attracting more and more attention in production of EPF infectious cells such as Beauveria bassiana, Metarhizium anisopliae, and Isaria (formerly Paecilomyces) fumosoroseus [14,15], and blastospores were developed into biological control agents for field application (Figure 1). Here, we provide an overview of the mechanisms underlying blastospore biogenesis and technological advances in fermentation bioprocess, which will enable researchers to explore suitable parameters and tools for a productive and efficient production of blastospores.

Commercial use of fungal biocontrol agents is highly dependent on the low-cost production of infectious propagula. To efficiently produce blastospores with high activity, a plethora of physicochemical factors are recognized and utilized to optimize fermentation process control.

The carbon (C) and nitrogen (N) sources have been considered as important factors influencing fungal blastospore yield, and their concentrations should be optimized according to fungal species and isolates (Supplementary Table S1). Kleespies and Zimmermann (1992) tested the effects of various carbohydrates and corn-steep products (nitrogen source) on blastospore yield in three strains of M. anisopliae and found that the blastospore yields of the tested strains displayed different responses to nutrient types [16]. In general, preferred carbohydrates are glucose and fructose; however, different corn steep brands yield different blastospore yields. M. flavoviride (IMI 330189) displays the maximal production on sucrose and yeast extract with a C/N ratio of 1.6 [17]. However, M. anisopliae (EAMa 01/58-Su) produces high blastospore concentration on different C/N ratios, which is dependent on the types of carbon and nitrogen sources. This strain produced the greatest amount of blastospores in a broth consisting of glucose (30 g/L) and peptone (30 g/L) at a C/N ratio of 10.66. [18]. Glucose-enriched cultures (140 g glucose/L) improve blastospore yield of M. robertsii ESALQ1426 under optimal growth conditions, with production as high as 5.9 × 10⁸ blastospores/mL within 2 days [19]. B. bassiana and I. fumosorosea produce higher blastospore concentrations in media containing cottonseed flour as a nitrogen source than acid hydrolyzed casein [11]. Combinational nitrogen source of corn steep powder and yeast extract (YE) gives M. anisopliae 416.96 a higher blastospore yield than the combination of cottonseed flour and YE [20]. Cliquet and Jackson (1999) tested the effects of thirteen amino acids on blastospore yield of Isaria (formerly Paecilomyces) fumosoroseus (Mycotech strain 612) and found that glutamate, aspartate, and glycine resulted in higher blastospore yields (6.6–11.0 × 10⁸ blastospores/mL) than the nitrogen source of casamino acids (6.0 × 10⁸ blastospores/mL) [21]. When sucrose is used as a carbon source, the B. bassiana GHA strain prefers corn steep liquor as a nitrogen source to casaminoacids or peptone in blastospore production [22]. As for B. bassiana KK5, an optimized nutrient complex with plant substrates results in a maximum spore concentration of 8.5 × 10⁸/mL which is approximately 3 times that of pre-optimized nutrient [23]. M. flavoviride (IMI 330189) displays different blastospore productions in various synthetic and semi-synthetic broths, with higher concentrations of blastospores in richer media [24]. When using a high casamino acid concentration, the blastospore yield for Isaria (formerly Paecilomyces) fumosoroseus strain Mycotech 612 is significantly higher when compared to that in medium containing a lower casamino acid concentration [25].

In addition to carbon and nitrogen sources, nutritional additives have attracted more attention due to their important roles in blastospore production. When producing the blastospores of M. anisopliae var. anisopliae strain 97, its yield was significantly increased by the addition of 5% lecithin (10 fold), 3% collagen (3.7 fold), and 1.5% lactic acid (2 fold) compared to that in the standard medium [26]. Zinc ion is required for fungal growth and blastospore production of Isaria (formerly Paecilomyces) fumosoroseus (Mycotech strain 612); however, addition of vitamins does not result in a significant change in blastospore yield [21]. When producing the blastospores of Cordyceps javanica IF-1106, the maximum sporulation yield is obtained by the addition of an optimal complex of metal ions containing Mn²⁺, Cu²⁺, Fe²⁺, and Fe³⁺ [27]. As for Akanthomyces attenuatus JEF-147, magnesium sulfate (1%) significantly increases the blastospore production in a glucose-based liquid culture [28].

Entomopathogenic fungi exhibit diversified patterns for blastospore biogenesis [29]. Fungal strains of the same genus and species can behave differently in response to various nutritional components in culture media. These findings suggest that various nutrients have great potential for regulating blastospore production, but most of the information provided cannot be extrapolated to all strains within the genus and species. Therefore, when producing blastospores on a large scale, suitable strain-specific nutrients must be evaluated to achieve maximal productivity.

Blastospores are produced in liquid culture fermentation, either in shake flasks or in deep-tank bioreactors for different scales of production [30]. Initial inoculation size has an important influence on blastospore production rate. As for Isaria (formerly Paecilomyces) strain 612, when blastospores are used as initial inocula, and a blastospore concentration of 10⁶ spores/mL results in maximum concentrations of blastospores within 2 days. However, the concentration of 10⁵ spores/mL causes one-day delay in presence of maximum yield [31]. Physicochemical properties of liquid medium have a great influence on optimal blastospore production, in which pH value and water availability (a_w) of liquid media are considered as prior physicochemical factors. To obtain maximum blastospore yield of M. anisopliae (strain 416.96), the pH of the liquid medium is controlled at 6.8–8.0, and polyethylene glycol (molecular weight 200) (PEG 200) is used as the a_w-modifying agent [20]. Similarly, PEG 200 greatly suppresses the generation of mycelial pellet and increases blastospore yield of M. anisopliae and B. bassiana [26,32]. In addition, PEG 200 decreases the blastospore size of B. bassiana [32]. However, M. anisopliae strains display different optimal pH for blastospore production [16]. Adoption of low-pH (pH 4) medium and relatively large initial inoculation size results in very low levels of bacterial contamination [30]. Téllez-Martínez et al. (2016) scaled up fermentation production of M. acridum blastospores in a stirred tank bioreactor with optimal nutrients and production parameters (constant stirring at 635 rpm, a temperature of 26 °C, and pH 3.3), which resulted in a yield of 1.25 × 10⁸ blastospores/mL [33].

The dissolved oxygen level is critical for blastospore formation under the submerged conditions [34]. Dissolved oxygen > 40% is beneficial for blastospore production by M. anisopliae strain MaQ01 [8]. The maximum blastospore yield of M. flavoviride Mf189 was obtained with pH and pO₂ regulated to 7 and 100%, respectively [17]. Adequate dissolved oxygen levels combined with high glucose concentrations enhanced the blastospore yield of B. bassiana [35]. To increase dissolved oxygen level, constant agitation and aeration are explored in blastospore production of Isaria (formerly Paecilomyces) (strain 612) [22,36]. In the 6-L bioreactor, the filtered atmospheric air is initially pumped into B. bassiana culture at 1.5 L/min, and the dissolved oxygen level is maintained above 10% in the subsequent cultivation stage [13]. These findings suggest that fungal strains require different optimal physicochemical conditions, which must be optimized in the fermentation process. Particularly, the physiological and genetic bases for blastospore formation under various physicochemical conditions remain largely unknown, which deserves further investigation.

Blastospores are produced in a liquid environment and must be harvested and dried for further formulation process. Usually, blastospores are harvested via centrifugation and filtration [23]. Isotonic solutions (e.g., PEG 200) are suitable for washing the harvested blastospores to increase the spore viability [37]. In application-oriented process design, the harvesting process is connected to the drying process. During blastospore production of B. bassiana and I. fumosorosea, the resultant culture is mixed with diatomaceous earth and vacuum-filtered. The resulting filter cake is ground and dried in an air drying chamber under a programed relative humidity [11,38,39]. In addition to diatomaceous earth, other powder are also used as support matrix in air drying of blastospores, including silica gel, sand, calcined kaolin clay, talc powder, and the like [40,41]. In blastospore production of Cordyceps species, liquid cultures are dried using a spray dryer equipped with a spinning disk atomizer. The parameters for spray drying are set as follows: inlet temperature at ~85 °C, outlet temperature at ~50 °C, and 5.7 bar at wheel atomizer [12]. Spray drying of blastospore with the addition of ascorbic acid improves product stability during storage [42]. The vacuum freeze-drying method is also used to dry EPF blastospores (e.g., Isaria (formerly Paecilomyces) ARSEF 4491 and Mycotech strain 612). Typically, the harvested blastospores are freeze dried in a lyophilizer with a eutectic-automatic program [21,43]. The rotary vacuum drum dryer is used to dry fungal biomass (e.g., blastospores) mixed with inert carrier (e.g., diatomaceous earth) and other additives to prepare a formulation [15]. Therefore, it is recommended to dry fungal biomass under vacuum conditions, which increases drying efficacy and fungal viability.

Dried blastospores are sealed in polypropylene plastic packages on vacuum-packaging machine and stored at a conventional refrigerator at 4 °C [4]. After a storage of 5 months at 4 °C, the survival rate for the lyophilized Isaria (formerly Paecilomyces) (strain ARSEF 4491) blastospores still exceeds 65%, and less than 25% of the air-dried blastospores are viable after 90-d storage. When stored at 22 °C, the lyophilized blastospores lose most viability within 30 d, none of the air-dried blastospores are alive [43]. When producing another Isaria (formerly Paecilomyces) (ARSEF 3581), the addition of proteinous substrates (e.g., whole milk) to starch-oil formulations significantly improves the storage stability of freeze-dried blastospores at 4 °C [44]. This indicates that further investigations are required to optimize the drying method and improve the storage stability of blastospores.

As summarized in Table 1, a list of entomopathogenic fungi has been developed into blastospore-based formulations to control various arthropods. Blastospores are easily formulated into water or Tween solution due to their hydrophilic surface [7]. In many occasions, this type of formulation is only suitable for laboratory work (e.g., virulence test). Bead formulation is an emerging one, when compared with the traditional types (e.g., wettable powder and emulsifiable oil), but it could be considered as a variant of granule formulation. Obviously, given the physical states of existing formulations, the room for designing a new formulation type is rather limited. Kaiser et al., (2020) used a single substance or co-formulation of B. bassiana blastospore to control pollen beetles. For co-formulation, B. bassiana blastospores were combined with colza oil containing 2% surfactant. Single use of blastospores resulted in the mortality of 20%, and the co-formulation led to the mortality beyond 60%, which indicates a strong synergistic effect between blastospore and vegetable oil [45]. The air-dried blastospores of B. bassiana cultured on corn steep liquor are formulated with diatomaceous earth, and the formulation remains virulent against three-instar larvae of Plutella xylostella after 6-month storage at 4 °C [22]. Thus, many more investigations are needed to test different types of substrates and optimize the formula to improve blastospore stability. Blastospores are generally considered more virulent than conidia as they generate germ tubes and penetrate the host cuticle at a faster rate. However, the blastospores of M. brunneum ARSEF 4556 are less virulent against Culex quinquefasciatus larvae than conidia due to the lower adhesion rate of blastospores [46]. This indicates that the interaction of blastospores with the host cuticle must be seriously considered in designing the formula of the blastospore-based mycoinsecticides.

As discussed above, blastospore production is an essential prerequisite for large-scale application of blastospore-based formulations. Meanwhile, it is important to keep blastospore stability/viability in the subsequent biotechnological process from fermentation, to drying, to formulation, and finally to product storage. Drying blastospores is a desiccation process that imposes stressful challenges on fungal cells, including denaturation of macromolecules, loss of cytomembrane integrity, stoppage of cellular self-protection mechanisms, and so on. Therefore, desiccation is considered as an essential process in formulation development [14]. Dietsch et al. (2021) have summarized a slew of approaches to increase the blastospore tolerance to desiccation. In fermentation, in addition to nutrients, physicochemical conditions (e.g., osmotic stress) are optimized to produce desiccation-tolerant blastospores with an increased survival rate after drying. In addition to additives, formulation types (e.g., powder and granule) and processing method (e.g., encapsulation) are also considered in the fabrication of the final preparation [14]. There are only a limited number of options for formulation types and production methods. Thus, exploring various additives could create opportunities to improve the desiccation tolerance or storage stability of blastospore formulations, which is critical for commercial stability and viability.

As for conidial formulation, many antioxidant enzymes are revealed to be involved in conidial resistance to environmental stresses, including UV irradiation, high temperature, and various agrichemical agents [59]. In addition, mannitol biosynthesis-related enzymes have been functionally characterized in B. bassiana, and their losses greatly weaken conidial multi-stress tolerances [60]. Polyols (e.g., glycerol and mannitol) have been considered as the important index of blastospore quality, and the increased level of polyols enhances blastospore viability under stressful conditions. Accumulation of polyols is modified by nutrients and cultural conditions [20]. However, very few investigations provided the direct mechanisms on the environmental stability of blastospores.

Spore viability investigations indicate that conidia retained their stability for a longer period than blastospores [61]. Therefore, the current main challenges are to increase blastospore production and formulation stability. In depth-exploration of molecular mechanisms is very necessary for blastospore biogenesis and stress tolerance, which will enhance blastospore physiology via genetic modification.

In the past decade, genetic dissection of functional genes has been greatly cumulated with the publication of the genomic sequences for a series of insect pathogenic fungi [62]. A recent review has summarized the understanding of sub-cellular structures (e.g., organelles and cell wall) and their associated proteins in asexual development (i.e., conidiation and blastospore generation) in entomopathogenic fungi [63]. Here, we focus on the molecular insights known about functional genes, signal transduction, and transcriptional regulation involved in fungal development into blastospores. Gene functions are analyzed through gene disruption with a strategy of homologous recombination [64]. However, most investigations are limited to blastospore formation under flask-scale assay conditions.

G-protein signaling pathways are characterized by the recognition of environmental stimuli by the membrane-bound G-protein coupled receptor (Gpcr) and play essential roles in cell development and differentiation [64]. In B. bassiana, GPCR3 contributes to fungal development and stress resistance. Blastospore production is dramatically attenuated in the gene disruption mutant when cultured in minimal broth with glucose as a single carbon source. This phenotypic defect is repressed when using the trehalose-based minimal broth. BbGpcr3 mediates the glucose-induced blastospore production, and trehalose regulates blastospore formation via an alternative signal-reception pathway [65]. This finding suggests that in in vitro production of blastospores, BbGpcr3 mediates fungal response to different carbon sources. Glucose and trehalose are prevalent in the insect hemolymph [66]. Filamentous fungi preferentially utilize glucose as the favorite carbon source [67]. These findings imply that BbGpcr3 is involved in the early stage of in vivo dimorphism, when the fungus first reaches the host hemocoel, and ultimately contributes to fungal virulence [65]. Regulators of G protein signaling (RGS proteins) constitute the key elements of GPCR complexes and play important roles in conidiation of B. bassiana [68]. Unraveling their roles in blastospore generation will improve understanding of the G-protein signaling pathway involved in fungal development under submerged condition.

SNF1/AMPK protein kinase is highly conserved in eukaryotic organisms and regulates carbohydrate metabolism and energy generation [69]. In filamentous fungi, SNF1 is critical for fungal assimilation of complex carbon sources (e.g., cellulose and pectin) and asexual conidiation [70]. In addition to its well-known roles, SNF1 contributes to blastospore production and size in B. bassiana. Furthermore, the SNF1-mediated transcriptome is associated with metabolism, cell cycle, and substrate transport during blastospore formation [71].

Autophagy is a conserved cellular self-degradation mechanism for damaged and superfluous substrates [72]. More than forty genes have been characterized as autophagy-related genes (ATGs). In B. bassiana, autophagy and its related genes are required for fungal development into blastospores [73]. Atg1 is a serine/threonine protein kinase indispensable for autophagy initiation. B. bassiana Atg1 (BbAtg1) mediates a comprehensive proteome and phosphoproteome during conidiation, and this phosphoproteome is associated with various physiological terms, including metabolism, signal transduction, cell cycle, autophagy, and the like [74]. BbAtg1 plays an important role in blastospore production [75]. Presumably, Atg1 might also phosphorylate a slew of targets for blastospore formation in B. bassiana.

Mitogen activated protein kinase (MAPK) cascade is a prevalent signaling transduction pathway by which eukaryotic cells respond to environmental changes [76]. In B. bassiana, the homolog of yeast High-osmolarity glycerol 1 (Hog1) is a functional MAPK, and its loss has no significant effects on the formation of in vivo blastospores [77]. However, the MAPK kinase Ste7 is involved in blastospore formation and its activity is regulated by four C-terminal Ser/Thr residues [78]. ΔBbgpcr3 mutant strain restores the blastospore production in the trehalose-based broth, and most genes in MAPK signaling pathways appear in the trehalose-induced transcriptome [65]. Many MAPK components are critical for aerial development of entomopathogenic fungi, e.g., Mpk1 (Fus3/Kss1 pathway) and Slt2 (cell wall integrity pathway) [79]. In M. acridum, a FUS3/KSS1-type mitogen activated protein kinase is required for appressorium formation, cuticle penetration, and virulence of the entomopathogenic fungus [80]. Thus, the roles of MAPK members in blastospore formation are yet to be revealed in future investigations.

Protein phosphatase type 1 (PP1) dephosphorylates serine and threonine residues of phosphoproteins and plays an important role in fungal metabolism and development. A yeast ortholog of Glc8 contributes to fungal development into blastospores in B. bassiana, and genetic analysis indicates that Glc8 mediates the transcription of OSMC2 (a member of OsmC protein family) during blastospore genesis [81]. Overall, the phosphorylation and its converse process are associated with a variety of target proteins during blastospore generation, and these processes are balanced by a complex regulatory network.

As summarized above, understanding of signal transduction remains fairly limited in blastospore formation; particularly, cross-talk among different pathways. SNF1/AMPK pathway acts as the energy-sensing one, and the mechanistic target-of-rapamycin complex 1 (mTORC1) functions as the key sensor of cellular amino acids and glucose. SNF1/AMPK acts as a secondary modulator for dissociation of mTORC1 form the lysosome/vacuole when cellular responding to nutrient availability [69]. What the induction signals for blastospore formation are, and how these signaling pathways interplay, remain important and active areas of investigation.

Cell-division control is essential for fungal differentiation, and the cell-division cycle includes four stages of genome duplication (S-phase), nuclear division (M-phase), and two gap phases (G1 and G2). At the checkpoint of G1/S transition, transcription processes are regulated by two transcription-factor complexes (i.e., SBF and MBF), in which Swi6 acts as a common transcriptional activator, generating the SBF and MBF complexes with DNA-binding proteins Swi4 and Mbp1, respectively [82]. In B. bassiana, both Swi6 and Mbp1 localize in the nucleus and exhibit a direct interaction relationship. As for biological functions, both factors contribute to fungal dimorphism into blastospores. BbMbp1 contributes to blastospore formation by regulating the genes associated with metabolism, cell transport, and development; whereas, BbSwi6 regulates genes involved in metabolism and energy generation [83,84]. These findings suggest that, in addition to their roles in the MBF complex, Swi6 and Mbp1 function in blastospore production via dissimilar genetic pathways in B. bassiana. Forkhead (Fkh) transcription factors are conserved in eukaryotic organisms and regulate the cell cycle via activation of the cyclin-B gene cluster regulatory elements in the G2 phase. B. bassiana has a homolog of Fkh2, and its loss leads to a great increase in blastospore yield but a reduction in cell size [85]. Thus, transcriptional activation of cell cycle dependent activities is essential for blastospore generation.

Carbon catabolite repression is prevalent in fungi. In yeast, three transcription factors (Mig1, 2, and 3) contain Cys₂His₂ zinc-finger motif with DNA-binding activity and are required for transcriptional activation of the glucose-repressed genes [86]. In filamentous fungi, CreA is the homolog of yeast Mig1. B. bassiana CreA plays important roles in conidiation and blastospore production, in addition to its function as a carbon metabolism repressor [87]. As mentioned above, B. bassiana Gpcr3 couples carbon sensing to fungal development into blastospores [53]. However, more genetic evidence is still needed to elucidate the connection between signal sensing and transcriptional regulation pathways.

Zn(II)₂Cys₆ transcription factors are fungal specific transcription regulators. Tpc1 (Transcription factor for Polarity Control 1) belongs to this family and regulates chitin synthesis during blastospore generation [88]. In yeast, the transcription factor Wor1 is a master regulator of cell morphological transformation. B. bassiana Wor1 is required for blastospore production via transcriptional activation of cell-wall-protein genes (e.g., allergen-like protein gene) [89].

Multiprotein bridging factor 1 (Mbf1) is a highly conserved transcriptional co-activator. In general, Mbf1 mediates the connection between the basal transcription machinery and the transcription factor. In B. bassiana, Mbf1 contributes to the blastospore production in minimal media with saccharide as the sole carbon source; however, this impaired phenotype is not present on the rich medium [90]. This finding implies that Mbf1 mediates assimilation of saccharides during blastospore formation; however, the transcription factor recruited by Mbf1 remains to be elucidated.

Comparative transcriptomics indicates that M. anisopliae activates a series of physiological processes to produce blastospores, including oxidative stress, amino acid metabolism, respiration process, transmembrane transport, and secondary metabolism [91]. Blastospore production of B. bassiana is dramatically increased by combinational application of high glucose concentrations and aeration. Transcriptomic analysis reveals that the up-regulated genes by high glucose concentrations are related to cellular antioxidant, calcium transport, conidiation, and osmotic sensor [92].

Current researches have coupled transcription of the cell cycle and metabolism with fungal dimorphism. Accumulated transcriptomic data will facilitate the discovery of transcriptional regulation hubs during blastospore genesis.

Cell cycle is finely tuned for cell proliferation and development. Cyclin-dependent kinase 1 (Cdk1) determines the checkpoint of G2/M transition, and its phosphorylation and dephosphorylation processes are regulated by the nuclear kinase Wee1 and cell division cycle 25 (Cdc25, a dual-specificity phosphatase). In B. bassiana, the Cdk1 phosphorylation state is also controlled by the homologs of Wee1 and Cdc25, and single disruption of Wee1 and Cdc25 results in significant decrease in blastospore production and alteration in blastospore size [93]. Tudor domain-containing protein (Tdp1) contributes to Cdk1 activity and regulates the S, G2/M, and G0/G1 phases in the cell cycle involved in blastospore formation [94]. In yeast, Cdc14 inactivates CDK activity by dephosphorylation. Its homolog controls cytokinesis and fungal differentiation into blastospores in B. bassiana [95]. Cdc42, a small GTPase, functions as a switch molecule in cell cycle and polarized growth, and participates in blastospore generation via controlling pyruvate metabolism [96]. Cell division cycle factors participate in blastospore formation through potential multiple pathways.

Epigenetics refers to the study of changes in gene expression that are not ascribed to alterations in the DNA sequence in response to intrinsic and extrinsic stimulations. Epigenetic regulatory mechanisms encompass DNA methylation, histone modification, chromatin remodeling, and microRNA expression [97]. The histone acetyltransferase (HAT) Gcn5 acts as a core subunit of the HAT complex. In B. bassiana, Gcn5 directly interacts with Ada2, which binds to Ada3, generating the HAT complex [98], and this complex is essential for the fungus to produce blastospores [99]. Histone deacetylase (HDA) represents a superfamily of enzymes that catalyze the reverse reaction of histone acetylation. Rpd3 and Sir2 belong to class I and class III HAD, respectively, and these HADs significantly contribute to blastospore production [100,101]. So, histone acetylation is greatly involved in comprehensive physiologies during fungal development, and the downstream targets remain largely unknown. Currently, only histone acetylation has been evaluated in entomopathogenic fungi, and other epigenetic mechanisms are highly anticipated in future studies on blastospore formation.

The central developmental pathway (CDP), which involves three major transcription factors, BrlA, AbaA, and WetA, regulates the temporal and spatial expression of conidiation-related genes. In B. bassiana and M. robertsii, the CDP transcription factors have additional roles in blastospore formation [102,103,104]. The E3 ubiquitin ligase Ubr1 plays a key role in protein ubiquitylation as revealed in yeast. The Ubr1-mediated ubiquitylation regulates fungal dimorphic change in B. bassiana via transcriptional activation of genes involved in the central developmental pathway (BrlA, AbaA, and WetA) [105]. In filamentous fungi, the upstream developmental activation pathway (UDP) is essential for activation of BrlA gene when conidiation is initiated, which involves the key activator FluG (fluffy gene). FluG constitutes three transcriptional regulatory pathways with FlbA, FlbC, and FlbE-FlbD-FlbB axis [106]. However, FluG and other FluG-like proteins have a limited contribution to conidiation in B. bassiana, and negatively regulates blastospore formation under in vitro condition [107,108]. In addition, Zhang et al. (2019) summarized that the transcription of CDP genes are mediated by other pathways (e.g., histone post-translational modification and MAPK cascade) during conidiation [102]. However, the details underlying the interaction of CDP with other pathways remain largely unknown in blastospore biogenesis.

As revealed in C. javanica, exogenous metal ions increase the blastospore production [27]. Siderophores represent a group of small molecule iron chelators and are critical for iron acquisition in filamentous fungi. B. bassiana ARSEF 2860 holds a general siderophore biosynthesis pathway to produce a slew of siderophores. Ablation of siderophore synthesis results in a significant reduction in blastospore formation under the iron-limited condition [109]. B. bassiana ARSEF 3153 is a strain that does not produce siderophores at the early stage of growth. Overexpression of N⁵-hydroxyornithine acetylase gene (SidL) activates the siderophore production in early growth and increases the blastospore yield [110]. This implicates that the pathways for nutrient assimilation are the potential targets for the strain improvement in blastospore production. However, to date, pathways for acquiring micronutrients are limited in filamentous entomopathogenic fungi.

14-3-3 protein represents a family of structurally similar phospho-binding proteins that regulate multiple cellular functions. In B. bassiana, Bmh1 and Bmh2 contribute equally to physiologies, and their single mutants show similar development defects in blastospore biogenesis, with a reduction of 60% in production and a decrease in cell size [111]. Mas5 and Mdj1 belong to the heat shock protein 40 (Hsp40) family, and their homologs contribute to about 60% and 80% of blastospore production in B. bassiana, respectively [112,113]. Gelsolin represents a superfamily of actin-binding proteins that contain three or six gelsolin-like domains. B. bassiana has a unique CapG protein (designated as BbGel1) with three gelsolin-like domains. BbGel1 mediates a comprehensive proteome during blastospore formation, and at least regulates the cellular level of a hydrophobin-like protein (Hyd4) [114]. Cyclophilin B is one of the prototypical proteins of the cyclophilin family. In B. bassiana, the homolog of cyclophilin B is localized in the cell wall and plays an essential role in blastospore production [115]. Class II lysyl-tRNA synthetase (KRS) is involved in cytosolic protein synthesis in budding yeast. Ablation of its homolog in B. bassiana results in an approximate 70% reduction in blastospore yield [116].

More and more functional genes have been linked to fungal development into blastospores under the flask-scale conditions, but their connections to large-scale production remain to be completely explored.

In the past decade, great efforts have been made to explore the EPF blastospore as a new infectious inoculum in biocontrol of insect pests. For the large-scale production, utilization of low-cost materials and optimal physiochemical conditions are highly desirable in the process of fermentation, and optimization of ingredients and additives is greatly demanded in the formulation process. Advances in the molecular mechanisms of blastospore biogenesis will facilitate genetic engineering of pathways and functional genes, but more efforts are needed to explore the regulatory networks and interplay mechanisms among different pathways, particularly those involved in blastospore formation under large-scale production conditions. Furthermore, it is a main challenge to enhance blastospore viability/vitality during formulation and storage, in which the mechanisms remain to be revealed. Molecular understandings of blastospore biogenesis and bioactivity will open opportunities for prevision cell design, facilitating the production of the blastospores with economical productivity coupled with quality and effectiveness.

The following supporting information can be found at: https://www.sciepublish.com/article/pii/1191, Table S1: Medium composition for blastospore production of various entomopathogenic fungi.

N.H.: Formal analysis, Investigation, Writing—original draft. H.-R.X.: Formal analysis, Investigation. Z.-K.Z.: Formal analysis, Investigation. M.-G.F.: Writing—review & editing. S.-H.Y.: Conceptualization, Funding acquisition, Supervision, Writing—review & editing.

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This work was supported by National Natural Science Foundation of China (32572913) and Natural Science Foundation of Zhejiang Province (LZ24C140002).

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.