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.

•Krs is a lysyl-tRNA synthetase localized in cytoplasm of Beauveria bassiana.•Krs is required for conidial germination and dimorphic transition of B. bassiana.•Krs is vital for sustaining virulence and stress tolerance of B. bassiana.Krs is a class II lysyl-tRNA synthetase (KRS) that is involved in cytosolic protein synthesis in budding yeast but functionally has not been explored in filamentous fungi. Previous transcriptomic analysis has revealed that a Krs-coding gene is likely involved in pathogenesis of Beauveria bassiana, a classic insect pathogen as a global source of fungal insecticides. Here, we show that Krs is localized in the cytoplasm of hyphal cells and acts as a substantial virulence factor in B. bassiana. Deletion of krs resulted in 10-h delayed germination, decreased (15 %) thermotolerance, and lowered (46 %) UV-B resistance of aerial conidia despite limited impact on conidiation capacity and slight or inconspicuous influence on radial growth on rich and minimal media with different carbon (10 sugars/polyols) and nitrogen (17 amino acids) sources. The deletion mutant suffered 58 % reduction in submerged blastospore yield (an index of in vitro dimorphic transition rate) in a minimal medium, and the reduction increased to 71 % in another trehalose-based medium mimic to insect haemolymph. In standardized bioassays, median lethal actions of Δkrs against Galleria mellonella larvae through the infections passing and bypassing the insect cuticle were prolonged to 192 and 153 h from wild-type median lethal time (LT50) estimates of 119 and 109 h, respectively. Microscopic examination revealed 2-d delayed presence of in vivo formed hyphal bodies in the haemolymph of the larvae infected by Δkrs in either mode. These findings unveil a vital role of Krs in conidial germination and dimorphic transition and its contribution to the fungal potential against arthropod pests.

•CypB is localized on hyphal cell periphery and septa of Beauveria bassiana.•CypB is required for B. bassiana growth, conidiation and dimorphic transition.•CypB contributes significantly to B. bassiana virulence via normal infection.•CypB is essential for the in vitro and in vivo asexual cycles of B. bassiana.Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16–42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10 h longer to germinate 50% at optimal 25 °C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4 d from a wild-type estimate of 4.7 d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H2O2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana.

Mdj1 is a member of the Hsp40 family containing a DnaJ or J domain. Here, we have examined the functions of an Mdj1 orthologue (56.68 kDa) in Beauveria bassiana, a filamentous fungal insect pathogen widely applied in biological control of insect pests. Deletion of mdj1 in B. bassiana resulted in significant growth defects on a variety of complex and minimal media. The Δmdj1 mutant exhibited not only a drastic reduction (92 %) in aerial conidiation during optimal cultivation but also a remarkable decrease (77 %) in submerged blastospore production. Compared to wild-type, the deletion mutant was significantly more sensitive to the stresses of cell wall perturbation, high osmolarity, oxidation, carbendazim fungicide, several metal ions, and acidic/alkaline pH during conidial germination and/or colony growth. In the mutant, conidial thermotolerance and UV-B resistance decreased by 61 and 25 %, respectively, and virulence to Galleria mellonella larvae was largely attenuated. Transcript levels of many phenotype-related genes were drastically suppressed in the absence of mdj1, accompanied with impaired cell walls and reduced intracellular anti-stress molecules, including superoxide dismutases, catalases, glycerol, trehalose, and mannitol. These data indicate that Mdj1 plays a vital role in normal fungal development and contributes significantly to the biological control potential of B. bassiana against insect pests.

Two thioredoxin (Trx) reductases (Trr1/2) are known to play overlapping roles in the yeast Trx-Trr redox system but are generally unexplored in filamentous fungi, which possess multiple Trx homologues. This study seeks to characterize the functions of Trr1 and Trr2 in Beauveria bassiana, a filamentous fungal insect pathogen, and to probe their Trx partners. Both Trr1 and Trr2 were evidently localized in the cytoplasm of B. bassiana, unlike the two yeast homologues that have been reported to localize in the cytoplasm and mitochondria, respectively. Most of the six trx genes were greatly upregulated at the transcriptional level in the absence of trr1 instead of trr2 in B. bassiana, in which the trr1/2 double deletion failed in many attempts. Deletion of trr1 resulted in increased Trx activity, severe cysteine auxotrophy, and drastically reduced activities of peroxidases and superoxide dismutases under normal or oxidative conditions despite little change in catalase activity. Such changes disappeared in the absence of trr2 and were completely restored by complementation of trr1/2 or overexpression of trx1/6 in the Δtrr1 mutant, but were not restored at all by overexpression of trx2/3/4/5 or trr2 in the same mutant. All of these mutants exhibited similar trends of changes in the antioxidant response, conidiation, germination, thermotolerance, UV-B resistance, and virulence. Taken together, the findings indicate that Trr1 could reduce Trx2–5 and hence dominate the intracellular redox state, profoundly affecting the potential of B. bassiana against arthropod pests. Trr2 could reduce Trx1/6 but function only in the absence of Trr1.

The Pal/Rim pathway essential for fungal adaptation to ambient pH has been unexplored in Beauveria bassiana, a classic fungal entomopathogen. Here, we show the characterized Pal pathway comprising transcription factor PacC and upstream six Pal partners (PalA/B/C/F/H/I) in B. bassiana. Their coding genes were all transcribed most abundantly in standard wild-type culture under the alkaline condition of pH 9. Deletion of pacC or each pal gene resulted in a significant delay of culture acidification in a minimal broth (initial pH = 7.3). This delay concurred with altered accumulation levels of intra/extracellular organic acids and drastically depressed expression of some enzyme genes required for the syntheses of oxalic and lactic acids. Our deletion mutants except ΔpalI showed growth defects and maximal sensitivity to NaCl, KCl, LiCl, or sorbitol at pH 9, an alkaline condition leading to fragmented vacuoles in their hyphal cells exposed to osmotic stress. In these mutants, conidiation was significantly facilitated at pH 3 more than at pH 7 but suppressed slightly at pH 9. Mild virulence defects also occurred in the absence of pacC or any pal gene. These changes were restored by targeted gene complementation. Taken together, PacC and Pal partners regulate the growth, conidiation, and osmotolerance of B. bassiana in a pH-dependent manner, highlighting their vitality for the fungal pH response.

The phytochemical camptothecin (CPT) from Camptotheca acuminate (Nyssaceae) and the byproduct bamboo tar (BT) from bamboo charcoal making are insoluble in water. Here, we prepared water-soluble CPT-Na and BT-Na by alkalization of CPT and BT and a mixture of both salts (CPT/BT-Na), and evaluated their insecticidal activities against brown planthopper Nilaparvata lugens and Asiatic rice striped borer Chilo suppressalis under laboratory and field conditions. The modeling analysis of time-concentration-mortality responses indicated that the lethal concentrations (LC50 and LC90) of CPT-Na and BT-Na sprayed together against the two rice pests were reduced, respectively, by 3.5–14.8-fold and 3.3–29.1-fold in comparison with the estimates of the two salts sprayed separately. The reduction magnitude varied with post-spray days. These highlight a strong synergism of the two salts in insecticidal activity. In two field trials, a spray of CPT/BT-Na (CPT-Na + BT-Na: 1.5–3.0 + 135–270 g ha−1) resulted in more efficacious and persistent rice pest controls than not only a doubled spray of either CPT-Na or BT-Na, but also a recommended spray of imidacloprid against N. lugens or fipronil against C. suppressalis. All together, the mixture is a promising botanical insecticide for effective management of different rice insect pests.

Many filamentous fungi produce only conidia for dispersal and survival in vitro or in vivo. Here, we show that the developmental regulator WetA and the velvet protein VosA are not only required for conidial maturation but indispensable for conidiation in Beauveria bassiana, a filamentous entomopathogen. Deletion of wetA or vosA resulted in more than 90 % transcriptional depression of brlA and abaA, two activator genes in the central developmental pathway, during the critical period of conidiophore development and conidiation. Consequently, ΔwetA and ΔvosA strains lost 98 % in and 88 % of their conidiation capacities under optimal culture conditions, respectively. The conidia of ΔwetA showed more defective features than those of ΔvosA, including smaller size, lesser density, lower hydrophobicity, and impaired cell walls although intracellular trehalose content decreased more in the aging culture of ΔvosA than of ΔwetA. As a result, conidial sensitivity to cell wall perturbation was elevated in ΔwetA but unaffected in ΔvosA, which produced conidia more sensitive to the oxidant menadione and the wet-heat stress at 45 °C. Both deletion mutants showed similar defects in conidial tolerance to high osmolarity or UV-B irradiation but no change in conidial sensitivity to the other oxidant H2O2 or the fungicide carbendazim. Moreover, ΔwetA lost more virulence to Galleria mellonella larvae than ΔvosA. All these phenotypical changes were restored by either wetA or vosA complementation. Taken together, WetA and VosA are indispensable for asexual development and contribute differentially to conidial quality and hence the biological control potential of B. bassiana against insect pests.

Ecm33 is one of several glycosylphosphatidylinositol (GPI)-anchored proteins. This protein is known to be involved in fungal cell wall integrity, but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33 orthologues, i.e., Bbecm33 in Beauveria bassiana and Mrecm33 in Metarhizium robertsii. Bbecm33 and Mrecm33 were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33 and Mrecm33 caused slight growth defects, but conidial yield decreased much more in ΔBbecm33 (76 %) than in ΔMrecm33 (42 %), accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell walls in ΔBbecm33 only. Consequently, ΔBbecm33 was far more sensitive to the cell wall-perturbating agents Congo red and sodium dodecyl sulfate (SDS) than ΔMrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants (menadione and H2O2), two fungicides (carbendazim and ethirimol), osmotic salt NaCl, and Ca2+ during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by 55 % in ΔBbecm33 but was unaffected in ΔMrecm33, unlike a similar decrease (25–28 %) of conidial thermotolerance in both. All the changes were restored to wild-type levels by gene complementation through ectopic gene integration in each fungus. However, neither ΔBbecm33 nor ΔMrecm33 showed a significant change in virulence to a susceptible insect host. Our results indicate that Bbecm33 and Mrecm33 contribute differentially to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r2 = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT50s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-β-d-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic efficiency (kcat/Km) of 1.31 × 104 mM−1 s−1 in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH 7.0 and was much more sensitive to Cu2+ and Zn2+ than to Li+ and Mn2+. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.

To clarify the potential use of hydrophobicity-related traits of aerial conidia in formulation design of fungal biocontrol agents, hydrophobicity rates (Hr) and surface areas (Sa) of aerial conidia were assessed with 48 strains of Beauveria bassiana,Isaria fumosorosea and Metarhizium spp. Inter- or intra-specific variation was large in Hr (59.7–92.2%) and Sa (7.9–25.3 μm2 conidium−1) measurements, which were significantly correlated (r2 = 0.55). Six isolates of the three fungi with distinguished Hr and Sa were further studied. Conidial wall proteins of these isolates were sequentially extracted with sodium dodecyl sulfate (SDS), formic acid (FA) and trifluoroacetic acid (TFA). Their Hr values were significantly correlated to the contents (Pc) of TFA-soluble, but FA-insoluble, proteins (2.7–44.8 μg per 107 conidia; r2 = 0.79) and reduced drastically by the FA/TFA treatments, which eliminated the hydrophobin-based rodlet layers of conidial surfaces. However, the SDS treatments had no effect on either Hr or rodlet layers. The dispersancy of a tested emulsifier to oil formulations of the six isolates in water was adversely correlated to their Hr (r2 = 0.94). The results indicate that both Pc and Sa are inherent hydrophobicity-related traits and can be utilized to select fungal biocontrol candidates for improved formulation and application.

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.

Conidia of Beauveria bassiana and Metarhizium spp. smeared on glass slides were assayed for their responses to irradiation with weighted 312-nm UV-B and 365-nm UV-A at gradient doses of 0.005–1.1 and 1.0–18.0 J cm−2, respectively. All inverted, sigmoid dose–survival trends showed good fit to a survival model (r2 ≥ 0.97), yielding respective UV-B LD50s of 0.23–0.59 and 0.05–0.65 J cm−2 for 24 B. bassiana and 36 Metarhizium isolates, and UV-A LD50s of 2.78–10.46 J cm−2 for 24 Metarhizium isolates. Myzus persicae apterae on detached leaves were sprayed with a concentrated spore suspension of B. bassiana or M. anisopliae, followed by exposure to the UV-B doses to cause 10–90% viability losses. These doses caused aphid mortality reductions as expected but affected neither spray-to-death period nor fungal growth on cadavers. The results highlight the merits of using UV-tolerant candidates and photoprotection measures in fungal formulations for pest control.

To explore possible role of intracellular trehalose accumulation in fungal tolerance to summer-like thermal stress, 3-day colonies of Beauveria bassiana grown on a glucose-free medium at 25°C were separately exposed to 35, 37.5 and 40°C for 1–18 h, respectively. Trehalose accumulation in stressed mycelia increased from initial 4.2 to 88.3, 74.7 and 65.5 mg g−1 biomass after 6-h stress at 35, 37.5 and 40°C, respectively, while intracellular mannitol level generally declined with higher temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to decreased trehalase activity (r2 = 0.73) and mannitol content (r2 = 0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r2 = 0.41) or mannitol 1-phosphate dehydrogenase (r2 = 0.30) under the stresses. All stressed cultures were successfully recovered at 25°C but their vigor depended on stressful temperature, time length and the interaction of both (r2 = 0.98). The highest level of 6-h trehalose accumulation at 35°C was found enhancing the tolerance of the stressed cultures to the greater stress of 48°C. The results suggest that the trehalose accumulation result partially from metabolized mannitol and contribute to the fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1–1.6 J cm−2 (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0–2.61 mW cm−2. Irradiated conidia were then incubated for 24 h at 25°C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (Is). For all isolates, the Is − D observations fit well with the survival model Is = 1/[1 + exp(a + bD)] (0.94 ≤ r2 ≤ 0.99) generated widely spanned lethal doses of 0.154–0.928, 0.240–1.139, and 0.383–1.493 J cm−2 for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm−2 measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.

The effect of fungal infection on the reproductive potential of two-spotted spider mite, Tetranychus urticae, was evaluated as part of the full biocontrol potential of three entomopathogenic fungi by modeling of fecundity probability. Female mites (≤2-day-old) on leaves were exposed to the sprays of Beauveria bassiana, Paecilomyces fumosoroseus and Metarhizium anisopliae at the concentrations of 1.13 × 103, 1.55 × 103 and 0.95 × 103 deposited conidia mm−2 and then individually reared at 25°C and 12:12 L:D for oviposition. Mite mortalities 10 days after spraying were 73.1, 75.4 and 67.9% in the fungal treatments versus 15.5% in control. On average, females infected by the three fungal species survived 5.8, 6.2 and 6.3 days, and laid 3.1, 4.0 and 4.0 eggs per capita, respectively. These were 3–4 fold lower than the control fecundity at 12.3. The cumulative probabilities [P(m ≤ N)] for the counts of infected and non-infected (control) females laying m eggs per capita (m ≤ N) during 10 days fit very well the equation P(m ≤ N) = 1/[1 + exp(a + bm)] (r2 ≥ 0.98), yielding a solution to the probability for the female mites to achieve a specific fecundity {P(m ≤ N)−P[m ≤ (N − 1)]}. Consequently, the infected mites had 71–78% chance to lay ≤5 eggs per capita but only 5–8% to deposit >10 eggs despite some variation among the tested fungi. In contrast, the chances for the non-infected mites to achieve the low and high fecundities were 23 and 55%. The fitted probabilities provide a full coverage of the fecundity potential of infected versus non-infected mites and are more informative than the mean fecundities.

The flexible peptides (GGGGS)n (n ≤ 3), the α-helical peptides (EAAAK)n (n ≤ 3) and two other peptides were used as linkers to construct bifunctional fusions of β-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both β-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304–426% and 82–143% of the parental ones, respectively. The peptide linker (GGGGS)2 resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)3 also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide linkers to separate the two moieties at a reasonable distance for beneficial interaction.

•Beauveria bassiana has a novel Ras GTPase (Ras3) localized to plasma membrane.•Mutant Δras3 suffers phenotypic defects reducing the fungal biocontrol potential.•Sensitivities to high osmolarity and menadione oxidation are greatly increased in Δras3.•These changes are concurrent with blocked Hog1 phosphorylation and reduced SOD activity.Two Ras ATPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. Here we show the significance of a novel Ras homolog (Ras3) for Beauveria bassiana. Ras3 possesses five domains and two GTP/GDP switches typical for Ras family and was proven to localize to plasma membrane despite the position change of a membrane-targeting cysteine in C-terminal CAAX motif. Deletion of ras3 altered temporal transcription pattern of ras1 instead of ras2. Compared with wild-type, Δras3 grew significantly faster in a rich medium but slower in some minimal media, and produced far fewer conidia with impaired quality, which was evident with slower germination, attenuated virulence, reduced thermotolerance and decreased UV-B resistance. Moreover, Δras3 was much more sensitive to the oxidative stress of menadione than of H2O2 and to the stress of high osmolarity than of cell wall perturbation during growth. The high sensitivity of Δras3 to menadione was concurrent with reductions in both gene transcripts and total activity of superoxide dismutases. Intriguingly, the high osmosensitivity was concurrent with not only reduced transcripts of a critical transcription factor (Msn2) and most signaling proteins in the high-osmolarity-glycerol pathway of Δras3 but nearly undetectable phosphorylation signal of Hog1 hallmarking the pathway. All the changes were restored by ras3 complementation. Taken together, Ras3 is involved in the Hog1 pathway required for osmoregulation and hence can positively regulate conidiation, germination, multi-stress tolerance and virulence linked to the biological control potential of the filamentous insect pathogen.

•Ktr1, Ktr4 and Kre2 are three α-1,2-mannosyltransferase orthologues in B. bassiana.•Ktr4 and Kre2 are indispensible for conidiation and nutritional utilization.•Ktr4 and Kre2 contribute more to cell wall integrity than Ktr1.•Ktr4 and Kre2 contribute more to multistress tolerance and virulence than Ktr1.Members of α-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mnt1 in Beauveria bassiana, a filamentous enotmopathogen, and found that they were positive, but differential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on substrates with altered carbon or nitrogen sources and availability, accompanied with reduced conidial size and complexity. This contrasts to the dispensability of Ktr1 for fungal growth and conidiation. More cell wall damage occurred in Δktr4 and Δkre2 than in Δktr1, including altered contents of the cell wall components mannoproteins, α-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Δktr4 and Δkre2 became more sensitive to oxidation and cell wall perturbation than Δktr1 during colony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Δktr4 and Δkre2 but only the thermotolerance decreased in Δktr1. All the phenotypical changes were well restored to wild-type levels by the complementation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktr1 although all of them are significant contributors.

•Ktr1, Ktr4 and Kre2 are three α-1,2-mannosyltransferase orthologues in B. bassiana.•Ktr4 and Kre2 are indispensible for conidiation and nutritional utilization.•Ktr4 and Kre2 contribute more to cell wall integrity than Ktr1.•Ktr4 and Kre2 contribute more to multistress tolerance and virulence than Ktr1.Members of α-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mnt1 in Beauveria bassiana, a filamentous enotmopathogen, and found that they were positive, but differential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on substrates with altered carbon or nitrogen sources and availability, accompanied with reduced conidial size and complexity. This contrasts to the dispensability of Ktr1 for fungal growth and conidiation. More cell wall damage occurred in Δktr4 and Δkre2 than in Δktr1, including altered contents of the cell wall components mannoproteins, α-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Δktr4 and Δkre2 became more sensitive to oxidation and cell wall perturbation than Δktr1 during colony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Δktr4 and Δkre2 but only the thermotolerance decreased in Δktr1. All the phenotypical changes were well restored to wild-type levels by the complementation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktr1 although all of them are significant contributors.

Msn2/4 transcription factors in some fungi have null effects on virulence and cellular stress responses. Here we found that the transcriptional regulation of Msn2 orthologs is vital for the conidiation, virulence and multi-stress responses of Beauveria bassiana (Bb) and Metarhizium robertsii (Mr), which lack Msn4 orthologs. Compared to wild-type and complemented strains of each fungus with all similar phenotypes, ΔBbmsn2 and ΔMrmsn2 showed remarkable defects in conidial yield (∼40% decrease) and virulence (∼25% decrease). Both delta mutants lost 20–65% of their tolerances to hyperosmolarity, oxidation, carbendazim, cell wall perturbing and high temperature at 34 °C during colony growth. Their conidia were also significantly (18–41%) less tolerant to oxidation, hyperosmolarity, wet-heat stress at 45 °C and UV-B irradiation. Accompanied with the defective phenotypes, several conidiation- and virulence-associated genes were greatly repressed in ΔBbmsn2 and ΔMrmsn2. Moreover, differentially expressed genes in the transcriptomes of ΔBbmsn2 versus wild type were ∼3% more under oxidative stress, but ∼12% fewer under heat shock, than those in the ΔMrmsn2 counterparts. Many stress-responsive effector genes and cellular signaling factors were remarkably downregulated. Taken together, the two entomopathogens could have evolved somewhat distinct stress-responsive mechanisms finely tuned by Msn2, highlighting the biological significance of Msn2 orthologs for filamentous fungi.Graphical abstractDownload high-res image (122KB)Download full-size imageHighlights► Msn2 inactivation caused similar severe multi-phenotypic defections of Beauveria bassiana and Metarhizium robertsii. ► ΔBbmsn2 and ΔMrmsn2 had different numbers of genes differentially expressed under different stresses. ► Msn2 transcription regulation is vital for conidiation, virulence and multi-stress responses of both fungi.

•Beauveria bassiana has six thioredoxin (Trx) homologs localized in different parts of cells.•All six Δtrx mutants are not responsive to several oxidants except menadione.•Only Δtrx2 shows changes in menadione sensitivity, Trx/SOD activity and redox homeostasis.•One or more phenotypes relating to biological control are changed in all Δtrx mutants.Thioredoxins (Trx) can detoxify sulfide or act as electron donors in the reduction of disulfide and dithiol to protect yeast cells from ROS damage but remain poorly explored in filamentous fungi. Here we show more Trx homologs in Beauveria bassiana than in many other fungi and examine their functions. This filamentous entomopathogen has six Trx homologs, including four (Txr1–4) evidently localized in cytoplasm, one (Trx5) in nuclear membrane and another (Trx6) in mitochondria. Deletion of each trx had no effect on radial growth on rich or minimal medium but resulted in remarkable transcriptional up-regulation of other partners for compensation. Compared with wild-type, only Δtrx2 was significantly more sensitive to menadione whereas none of six Δtrx mutants was responsive to other oxidants including H2O2. Intriguingly, Δtrx2 showed uniquely a significant increase in total Trx activity in normal cultures but a remarkable decrease in total SOD activity in the cultures grown normally or co-cultivated with menadione. The ratio of reduced/oxidized glutathione accumulated in hyphal cells stressed with menadione decreased to only 0.4 in Δtrx2 from ∼1.0 observed in wild-type and other mutants. The six Δtrx mutants displayed one or more phenotypic changes associated with the fungal biocontrol potential, including conidiation, and germination, thermotolerance, UV-B resistance and virulence of their conidia. All the changes were restored by trx complementation. Taken together, the greater Trx diversity evolutionarily gained by B. bassiana could help it to maintain cellular redox homeostasis and infect insect hosts in diverse habitats.

Carbohydrate transporters are critical players mediating nutrient uptake during saprophytic and pathogenic growth for most filamentous fungi. For entomopathogenic fungi, such as Beauveria bassiana, assimilation of α-glucosides, in particular, trehalose, the major carbohydrate constituent of the insect haemolymph, has been hypothesized to represent an important ability for infectious growth within the insect hemocoel. In this study, a B. bassiana α-glucoside transporter homolog was identified and genetically characterized via generation of a targeted gene disruption mutant. Trehalose utilization was compromised in the mutant strain. In addition, inactivation of the α-glucoside transporter resulted in decreased conidial germination, growth, and yield on various carbohydrates (α-glucosides, monosaccharides and polyols) as compared to the wild-type strain. Insect bioassays revealed decreased mean lethal mortality time using both topical and intrahemocoel injection assays, although final mortality levels were comparable in both the mutant and wild type. Gene expression profiles showed altered expression of other putative transporters in the knockout mutant as compared to the wild type. These results highlighted complex sugar utilization and responsiveness in B. bassiana and the potential role for trehalose assimilation during fungal pathogenesis of insects.

The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of fungal biocontrol agent Beauveria bassiana for higher frequency. The blastospores from ≤ 24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1 μg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar in 360 μL reaction system containing 100 U HindIII or XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV cm− 1. The optimized REMI and EP generated averagely 39 and 53 transformants μg− 1 plasmid DNA whereas polyethylene glycol (PEG) integration yielded only 22. All transformants grew well on Czapek's agar containing 400 μg PPT mL− 1 after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting. Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively.

•BbVmaH is vacuolar ATPase subunit H in Beauveria bassiana.•BbVmaH can sustain intra/extracellular pH and cellular sensitivity to metal ions.•BbVmaH is involved in in vitro asexual cycle, virulence and stress tolerance.•BbVmaH plays a vital role in sustaining the fungal potential against insect pests.Vacuolar ATPase (V-ATPase) is a conserved multi-subunit protein complex that mediates intracellular acidification in fungi. Here we show functional diversity of V-ATPase subunit H (BbVmaH) in Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of BbvmaH resulted in elevated vacuolar pH, increased Ca2+ level in cytosol but not in vacuoles, accelerated culture acidification and reduced accumulation of extracellular ammonia. Aerial conidiation and submerged blastospore production were largely delayed and reduced in the deletion mutant, respectively, accompanied with a significant delay in conidial germination, alterations of conidia and blastospores in morphology, size and/or density, and severe growth defects in minimal media with different carbon and nitrogen sources. Despite null responses to osmotic, oxidative and cell wall perturbing stresses, the deletion mutant showed increased sensitivity to Ca2+, Zn2+ and Cu2+ during growth while its conidia were less tolerant to a wet-heat stress at 45 °C and UV-B irradiation. Intracellular glycerol and mannitol contents also decreased significantly. Its virulence to Galleria mellonella larvae was significantly attenuated when conidia were topically applied for normal cuticle infection or injected into haemocoel for cuticle-bypassing infection. All phenotypic changes were restored by targeted gene complementation. Our results indicate that BbVmaH plays an important role in sustaining not only vacuolar acidification but also cytosolic calcium accumulation, ambient pH homeostasis, in vitro asexual cycle and virulence in B. bassiana.

Conidial tolerance to the upper thermal limits of summer is important for fungal biocontrol agents, whose conidia are formulated into mycoinsecticides for field application. To develop an efficient assay system, aerial conidia of eight Metarhizium anisopliae, four M. anisopliae var. anisopliae, and six M. anisopliae var. acridum isolates with different host and geographic origins were wet-stressed for ≤180 min at 48 °C or incubated for 14 d colony growths at 10–35 °C. The survival ratios (relative to unstressed conidia) of each isolate, examined at 15-min intervals, fit a logistic equation (r2 ≥ 0.975), yielding median lethal times (LT50s) of 14.3–150.3 min for the 18 isolates stressed at 48 °C. Seven grasshopper isolates from Africa had a mean LT50 of 110 (73–150) min, but could not grow at 10 or 15 °C. The mean LT50 of five non-grasshopper isolates capable of growing at 10–35 °C was 16 (10–26) min only. Three isolates with typically low (type I), medium (type II), and high (type III) levels of tolerance to 48 °C were further assayed for ≤4-d tolerance of their conidia to the wet stress at 38, 40, 42, or 45 °C. The resultant LT50s decreased to 20, 53 and 167 min at 48 °C from 507, 1612, and 8256 min at 38 °C for types I, II and III, respectively. For the distinguished types, the logarithms of the LT50s were significantly correlated to the temperatures of 38–48 °C with an inverse linearity (r2 ≥ 0.88). The method developed to assay quantitatively fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.

•B. bassiana has five vacuolar Ca2+ exchangers (VCXs) homologous to yeast Vcx1 ortholog.•All VCXs are vital for tolerance to Ca2+ and endoplasmic reticulum stresses.•Two or three VCXs involve in cell responses to Mn2+, oxidative and cell wall stresses.•Four of them contribute significantly to the fungal virulence.Multiple Vcx1 (vacuolar calcium exchanger) paralogues exist in many filamentous fungi but are functionally unexplored unlike a single Vcx1 ortholog well characterized in yeasts. Here we show that five Vcx1 paralogues (Vcx1A–E) in Beauveria bassiana are conditionally functional for intracellular Ca2+ homeostasis and contribute differentially to multistress tolerance and virulence in the filamentous entomopathogen. Each vcx1 deletion drastically upregulated transcriptional expressions of four other partners and six P-type Ca2+-ATPases, resulting in elevated or lowered intracellular Ca2+ concentration in some deletion mutants treated with Ca2+ stress or untreated at 25 and 30 °C. When calcineurin was inactivated by cyclosporine A, Ca2+ tolerance decreased by 11–17% in five Δvcx1 mutants, but Mn2+ sensitivity increased only in Δvcx1A and Δvcx1D, at optimal 25 °C. These two mutants were also more sensitive to Ca2+ stress at 30 °C when calcineurin was active, and showed minor growth defect at 25 and 30 °C when calcineurin was inactive. Moreover, all the Δvcx1 mutants were more sensitive to dithiothreitol (stress-response trigger to endoplasmic reticulum) and Congo red (cell wall stressor); three of them were consistently less tolerant to the oxidants menadione and H2O2. The fungal virulence to Galleria mellonella larvae decreased by 15–40% in four Δvcx1 mutants excluding Δvcx1E, which was uniquely defective in conidial thermotolerance. All the changes were restored by each vcx1 complementation. Our findings indicate that the five Vcx1 paralogues in B. bassiana contribute differentially to calcineurin-dependent Ca2+/Mn2+ tolerance, multistress responses and virulence, and recall attention to multifunctional Vcx1 paralogues in filamentous fungi.

•Beauveria bassiana Miro (bMiro) is localized on mitochondrial outer membrane.•Deletion of bmiro alters mitochondrial distribution and mobility rather than morphology.•Deletion of bmiro reduces mitochondrial mass and ATP content.•Deletion of bmiro increases cellular tolerance to several metal ions.•The bMiro contributes significantly to the fungal potential against insect pests.Miro homologues are small mitochondrial Rho GTPases belonging to the Ras superfamily across organisms and are generally unexplored in filamentous fungi. Here we identified a Miro orthologue (bMiro) in Beauveria bassiana, a filamentous fungal insect pathogen as a classic biological control agent of insect pests. This orthologue was proven to anchor on mitochondrial outer membrane in a manner depending completely upon a short C-terminal transmembrane domain. As a result of bmiro deletion, mitochondria in hyphal cells were largely aggregated, and their mass and mobility were reduced, accompanied with a remarkable decrease in ATP content but little change in mitochondrial morphology. The deletion mutant became 42%, 37%, 19% and 10% more tolerant to Ca2+, Mn2+, Zn2+ and Mg2+ than wild-type, respectively, during cultivation in a minimal medium under normal conditions. The deletion mutant also showed mild defects in conidial germination, vegetative growth, thermotolerance, UV-B resistance and virulence despite null response to oxidative and osmotic stresses. All these phenotypic changes were restored by targeted gene complementation. Our results indicate that bMiro can control mitochondrial distribution and movement required for the transport of ATP-form energy and metal ions and contributes significantly to the fungal potential against insect pests through the control.

•Cytosolic Cu/ZnSOD, mitochondrial FeSOD and cell wall Cu/ZnSOD exist in B. bassiana.•Single sod deletion or knockdown exerts minor or little effect on total SOD activity.•All sod mutants show altered transcripts and activities of SOD partners and catalases.•Three SODs play distinct roles in antioxidation, UV tolerance and virulence.Beauveria bassiana, a filamentous entomopathogen, has five distinct superoxide dismutases (SODs), including cytosolic and mitochondrial MnSODs (Sod2/3) which have proved contributing primarily to intracelluar SOD activity and additively to antioxidation and virulence. Here we characterized cytosolic Cu/ZnSOD (Sod1), mitochondrial FeSOD (Sod4) and cell wall-anchored Cu/ZnSOD (Sod5). The latter two are unexplored despite existence in many filamentous fungi, and their subcellular localization was well confirmed with specifically stained cells expressing Sod4::eGFP or Sod5::eGFP fusion. Total SOD activity decreased by ∼15% in Δsod1 but increased by 11–20% in three sod4 knockdown mutants (Δsod4 was lethal) when co-cultivated with menadone and H2O2. Surprisingly, total catalase activity decreased much more in the sod4 mutants (69–75%) than in Δsod1 (27–33%) under normal and oxidative conditions. However, Δsod5 showed little change in either SOD or catalase activity. Transcript levels of SOD partners and five catalases also changed more dramatically in the sod4 mutants than in Δsod1 and Δsod5. As a consequence of global effect, intracellular ROS levels induced by both oxidants were higher in Δsod1 than in the sod4 mutants and Δsod5. All the mutants were differentially more sensitive to the two oxidants and UV-A/UV-B irradiations and less virulent to Galleria mellonella larvae but not responsive to high osmolarity, cell wall stress and high temperature. Taken together with previously characterized Sod2 and Sod3, our results provide full insight into the SOD family, unveiling the interactions of each SOD with other partners and catalases in the antioxidant reaction associated with the fungal biocontrol potential.

•Sole adenylate cyclase (AC) exists in B. bassiana (Bb) and M. robertstii (Mr).•BbAC and MrAC disruptions caused severe growth defects on rich and minimal media.•Two deletion mutants showed antagonistic conidiation changes during cultivation.•Their virulence and multistress tolerances changed differentially or reversely.•BbAC and MrAC contribute differentially to the biocontrol potential of two fungi.Adenylate cyclase (AC) is a core element of cAMP signalling network. Here we show functional diversity and differentiation of Beauveria bassiana AC (BbAC) and Metarhizium robertsii AC (MrAC). Severe growth defects occurred in ΔBbAC and ΔMrAC grown on nutrition-rich SDAY and several minimal media but were largely alleviated by adding cAMP to SDAY. Conidial yield increased greatly in ΔBbAC but decreased in ΔMrAC. During colony growth, ΔBbAC was highly sensitive to oxidation, high osmolarity, cell wall perturbation, carbendazim fungicide, Mn2+, Zn2+, Fe3+, and EDTA but more tolerant to Cu2+ while ΔMrAC showed higher osmotolerance, decreased sensitivity to Fe3+, and null response to carbendazim or cell wall stress despite similar responses to oxidation and other metal ions. Conidial UV-B resistance decreased by 32 % in ΔBbAC and 22 % in ΔMrAC despite little change in their theromotolerance. Median lethal time (LT50) estimates of ΔBbAC and ΔMrAC against susceptible insects were 10.9 and 1.4 d longer than those from wild-type strains respectively. All the phenotypic changes were restored to wild-type levels by each gene complementation. Taken together, BbAC and MrAC regulated differentially conidiation, pathogenicity, and multistress responses in B. bassiana and M. robertsii, thereby making different contributions to their biocontrol potential.

A Cu/Zn-superoxide dismutase (SOD) was characterized for the first time from Beauveria bassiana by gene cloning, heterogeneous expression and function analysis. This 154-aa SOD (BbSod1) was deduced from a 465-bp gene cloned, showing 49–96% sequence identity to Cu/Zn-SODs from other 57 fungi. BbSod1 and its form engineered with two site-directed mutations P143S and P145L (BbSod1-Mut) or a fused copper chaperon Lys7 (BbSod1-Lys7) were expressed well in Escherichia coli. Crude extracts and purified BbSod1-Mut from cell cultures exhibited much higher antioxidation activities than the counterparts of BbSod1-Lys7 whereas BbSod1 showed no substantial activity. The engineered enzymes were best induced by overnight incubation at 20 °C in Luria-Bertani medium including 2.5 mM Cu2+, 0.5 mM Zn2+ and 0.5 mM isopropyl-d-thiogalactopyranoside after 5-h growth to log-phase at 37 °C. Our results highlight alternative means to producing highly active fungal Cu/Zn-SOD in E. coli by making use of the two site-directed mutations without chaperon.