Conidial Yield Research Articles

VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae.

The ergosterol biosynthesis pathway plays an important role in model pathogenic bacteria Saccharomyces cerevisiae, but little is known about the biosynthesis of ergosterol in the pathogenic fungus Verticillium dahliae. In this study, we identified the VdERG2 gene encoding sterol C-8 isomerase from V. dahliae and investigated its function in virulence by generating gene deletion mutants (ΔVdERG2) and complemented mutants (C-ΔVdERG2). Knockout of VdERG2 reduced ergosterol content. The conidial germination rate and conidial yield of ΔVdERG2 significantly decreased and abnormal conidia were produced. In spite of VdERG2 did not affect the utilization of carbon sources by V. dahliae, but the melanin production of ΔVdERG2 was decreased in cellulose and pectin were used as the sole carbon sources. Furthermore, the ΔVdERG2 mutants produced less microsclerotia and melanin with a significant decrease in the expression of microsclerotia and melanin-related genes VaflM, Vayg1, VDH1, VdLAC, VdSCD and VT4HR. In addition, mutants ΔVdERG2 were very sensitive to congo red (CR), sodium dodecyl sulfate (SDS) and hydrogen peroxide (H2O2) stresses, indicating that VdERG2 was involved in the cell wall and oxidative stress response. The absence of VdERG2 weakened the penetration ability of mycelium on cellophane and affected the growth of mycelium. Although ΔVdERG2 could infect cotton, its pathogenicity was significantly impaired. These phenotypic defects in ΔVdERG2 could be complemented by the reintroduction of a full-length VdERG2 gene. In summary, as a single conservative secretory protein, VdERG2 played a crucial role in ergosterol biosynthesis, nutritional differentiation and virulence in V. dahliae.

The C2H2 Zinc Finger Protein MaNCP1 Contributes to Conidiation through Governing the Nitrate Assimilation Pathway in the Entomopathogenic Fungus Metarhizium acridum.

Zinc finger proteins are an important class of multifunctional regulators. Here, the roles of a C2H2 zinc finger protein MaNCP1 (Metarhizium acridum nitrate-related conidiation pattern shift regulatory factor 1) in nitrogen utilization and conidiation were explored in the entomopathogenic fungus M. acridum. The results showed that MaNCP1-disruption mutant (ΔMaNCP1) impaired the ability to utilize nitrate, ammonium and glutamine and reduced the expression of nitrate assimilation-related genes, suggesting that MaNCP1 was involved in governing nitrogen utilization. In addition, the conidial yield of the ΔMaNCP1 strain, cultured on the microcycle conidiation medium (SYA), was significantly decreased, which could be restored or even enhanced than that of the WT strain through increasing the nitrate content in SYA medium. Further study showed that MaAreA, a core regulator in the nitrogen catabolism repression (NCR) pathway, was a downstream target gene of MaNCP1. Screening the differential expression genes between WT and ΔMaNCP1 strains revealed that the conidial yield of M. acridum regulated by nitrate might be related to NCR pathway on SYA medium. It could be concluded that MaNCP1 contributes to the nitrate assimilation and conidiation, which will provide further insights into the relationship between the nitrogen utilization and conidiation in fungi.

Development of Cordyceps javanica BE01 with enhanced virulence against Hyphantria cunea using polyethylene glycol-mediated protoplast transformation.

Cordyceps javanica has promising application prospects as an entomopathogenic fungus with a wide range of hosts. To enhance the virulence of C. javanica, a polyethylene glycol (PEG)-mediated protoplast genetic transformation system was constructed. Strains overexpressing the subtilisin-like protease genes CJPRB and CJPRB1 and the tripeptidyl peptidase gene CJCLN2-1 were constructed with this system, and the effects of these strains on Hyphantria cunea were tested. The aminoglycoside G418 was used at 800 μg ml−1 to screen the transformants. C. javanica hyphae were degraded with an enzyme mixture to obtain protoplasts at 1.31 × 107 protoplasts ml−1. The transformation of 2 μg of DNA into 1,000 protoplasts was achieved with 20% PEG2000, and after 6 h of recovery, the transformation efficiency was 12.33 ± 1.42 transformants μg−1 plasmid. The LT50 values of CJPRB, CJPRB1, and CJCLN2-1-overexpressing C. javanica strains were 1.32-fold, 2.21-fold, and 2.14-fold higher than that of the wild-type (WT) strain, respectively. The three overexpression strains showed no significant differences from the WT strain in terms of colony growth, conidial yield, and conidial germination rate. However, the infection rate of the CJPRB1 strain was faster than that of the WT strain, with infection occurring within 4–5 days. The CJCLN2-1 strain had a significantly higher mortality rate than the WT strain within 4–10 days after infection. A C. javanica genetic transformation system was successfully constructed for the first time, and an overexpression strain exhibited enhanced virulence to H. cunea compared with the WT strain.

Effects of Macroautophagy and Mitophagy on the Pathogenicity of Fusarium graminearum.

Fusarium graminearum is the main pathogen of Fusarium head blight (FHB), which causes huge economic losses every year. In this study, an attempt was made to control FHB from the point of view of the physiological behavior of the pathogen itself. Autophagic inhibitors and activators were used, and the pathogenicity-related indices of F.graminearum were measured. The results showed that under nitrogen-rich conditions, macroautophagy inhibition and activation greatly reduced the mycelium weight to 0.28 and 0.25 g/ml at 24 h, which were 17.82 and 24.77% lower than that of the control treatment, respectively. Mitophagy inhibition also significantly decreased the mycelium weight (P < 0.05). Conidial yield was found to be affected by factors related to autophagy occurrence. It was found that both autophagy inhibition and activation could reduce the conidiation of F.graminearum. The toxin contents in wheat medium of macroautophagy activation treatments were 0.678, 0.190, 0.402, and 0.195 μg/g when cultured for 8 and 24h under 0% N and 100% N conditions, respectively, which were significantly higher than those of the control treatments (P < 0.05). The infection length was measured to characterize the infectivity of F.graminearum, and we found that the length was short under macroautophagy activation conditions. However, mitophagy did not seem to affect the infectivity of F.graminearum. In summary, the above results indicate that macroautophagy and mitophagy inhibition could reduce the pathogenicity of F.graminearum, which may provide a new perspective for management of plant fungal diseases.

The role of WSC domain-containing protein encoding gene AOL_s00043g401 in the growth and nematode trapping of Arthrobotrys oligospora.

Arthrobotrys oligospora is a model nematode-trapping fungus that has been extensively investigated to understand the interactions between fungi and nematodes. Nematode capture by A. oligospora is a complex process in which recognition of nematodes is generally believed to be mediated by lectins from the fungi. Lectins are a group of carbohydrate-binding proteins that widely exist in microorganisms and contain specific glycosylation recognition domains. In this work, we report the effect of a putative WSC domain-containing protein encoding gene AOL_s00043g401 (g401) on the growth and nematode-trapping of A. oligospora. The g401 gene was knocked out using the homologous recombination approach, and the △g401 mutant strain was then evaluated for its growth rate, conidial yield and germination rate, adaptation to environmental stresses, and nematocidal activity. Interestingly, the deletion of the putative lectin gene g401 had no significant effect on saprophytic growth, conidial yield and germination rate, responses to high salt, surfactant, and strong oxidative environments, as well as nematode-trapping efficiency of A. oligospora. We speculate that this phenomenon might have been caused by an intrinsic genetic compensation of the g401 in this fungus. For instance, more copies of WSC domain encoding genes or PQQ-DH domain encoding genes were found in the genome of A. oligospora. These findings provide further experimental evidence on the effect of lectin-related functional proteins in A. oligospora on nematode capture and will help further analyze their potential roles in the biological control of nematodes in the future.

N-terminal zinc fingers of MaNCP1 contribute to growth, stress tolerance, and virulence in Metarhizium acridum

C2H2 zinc finger proteins (ZFPs) are a class of important transcriptional regulators in eukaryotes involved in multiple biological regulation processes. Here, MaNCP1, a C2H2 ZFP, was functionally characterized in the model entomopathogenic fungus Metarhizium acridum. Deletion of MaNCP1 delayed conidial germination and hyphal growth, decreased the conidial yield and reduced the tolerances to UV-B irradiation and heat-shock. The N-terminal zinc fingers (ZFs) of MaNCP1 made the main contributions to these traits. In addition, disruption of MaNCP1 altered the conidial surface structure and decreased the conidial hydrophobicity. Bioassays showed that the virulence of the MaNCP1-disruption strain (ΔMaNCP1) was reduced in topical inoculation compared to the WT or the mutant complemented strain (CP), and the N-terminal C2H2 ZFs made a major contribution to virulence. Furthermore, the ΔMaNCP1 and C2H2 ZFs deletion mutants (MaNCP1∆N and MaNCP1∆N+C) impaired cuticular penetration. RNA-seq showed that several cuticle-degrading genes were down-regulated in the ΔMaNCP1 background, suggesting that MaNCP1 plays vital roles in regulating insect cuticle penetration. In summary, MaNCP1 affected the growth, stress tolerances and virulence of M. acridum, and the N-terminal C2H2 ZFs played indispensable roles in these important biocontrol traits. These results provide further insights into the functions of C2H2 ZFPs in entomopathogenic fungi.

MaOpy2, a Transmembrane Protein, Is Involved in Stress Tolerances and Pathogenicity and Negatively Regulates Conidial Yield by Shifting the Conidiation Pattern in Metarhizium acridum.

Opy2 is an important membrane-anchored protein upstream of the HOG-MAPK signaling pathway and plays important roles in both the HOG-MAPK and Fus3/Kss1 MAPK. In this study, the roles of MaOpy2 in Metarhizium acridum were systematically elucidated. The results showed that the MaOpy2 disruption significantly reduced fungal tolerances to UV, heat shock and cell-wall-disrupting agents. Bioassays showed that the decreased fungal pathogenicity by topical inoculation mainly resulted from the impaired penetration ability. However, the growth ability of ∆MaOpy2 was enhanced in insect hemolymph. Importantly, MaOpy2 deletion could significantly increase the conidial yield of M. acridum by shifting the conidiation pattern from normal conidiation to microcycle conidiation on the 1/4SDAY medium. Sixty-two differentially expressed genes (DEGs) during the conidiation pattern shift, including 37 up-regulated genes and 25 down-regulated genes in ∆MaOpy2, were identified by RNA-seq. Further analysis revealed that some DEGs were related to conidiation and hyphal development. This study will provide not only the theoretical basis for elucidating the regulation mechanism for improving the conidial yield and quality in M. acridum but also theoretical guidance for the molecular improvement of entomopathogenic fungi.

The Deubiquitinating Enzyme MrUbp14 Is Involved in Conidiation, Stress Response, and Pathogenicity in Metarhizium robertsii.

Protein ubiquitination, which is involved in various biological processes in eukaryotic cells, is a reversible modification of proteins. Deubiquitinases can maintain ubiquitin homeostasis by removing ubiquitin or modulating protein degradation via the ubiquitin-proteasome system (UPS). Metarhizium robertsii, an entomopathogenic fungus, has become a model fungus for investigating the interactions between insects and fungal pathogens. To explore the possible effects of the deubiquitination process on the development, stress response, and virulence of M. robertsii, disruption of MrUbp14 (an ortholog of the yeast ubiquitin-specific protease gene, Ubp14) was performed. The results of this study showed that the deletion of MrUbp14 led to accelerated conidial germination, reduced conidial yields, and decreased expression levels of some genes involved in conidiation. Furthermore, the MrUbp14 mutant (ΔMrUbp14) exhibited decreased tolerance to cell wall-damaging stressors (Congo red and SDS) and heat stress. Importantly, the results of the bioassay demonstrated that the fungal virulence of the ΔMrUbp14 strain was largely reduced in cuticle infection, but not in direct injection, which was accompanied by a significant decline in appressorium formation and cuticle penetration. Moreover, our results demonstrated that the disruption of MrUbp14 resulted in significantly increased ubiquitination levels of total protein, suggesting that MrUbp14 acts as a deubiquitinating enzyme in M. robertsii. In summary, our phenotypic changes in the gene disruption mutants suggest that MrUbp14 is important for conidiation, stress response, and fungal virulence in M. robertsii.

The Arf-GAP Proteins AoGcs1 and AoGts1 Regulate Mycelial Development, Endocytosis, and Pathogenicity in Arthrobotrys oligospora.

Small GTPases from the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development, endocytosis, and virulence in fungi. Here, we identified two orthologous Arf-GAP proteins, AoGcs1 and AoGts1, in a typical nematode-trapping fungus Arthrobotrys oligospora. The transcription of Aogcs1 and Aogts1 was highly expressed in the sporulation stage. The deletion of Aogcs1 and Aogts1 caused defects in DNA damage, endocytosis, scavenging of reactive oxygen species, lipid droplet storage, mitochondrial activity, autophagy, serine protease activity, and the response to endoplasmic reticulum stress. The combined effects resulted in slow growth, decreased sporulation capacity, increased susceptibility to chemical stressors and heat shock, and decreased pathogenicity of the mutants compared with the wild-type (WT) strain. Although deletion of Aogcs1 and Aogts1 produced similar phenotfypic traits, their roles varied in conidiation and proteolytic activity. The ΔAogts1 mutant showed a remarkable reduction in conidial yield compared with the WT strain but not in proteolytic activity; in contrast, the ΔAogcs1 mutant showed an increase in proteolytic activity but not in sporulation. In addition, the growth of ΔAogcs1 and ΔAogts1 mutants was promoted by rapamycin, and the ΔAogts1 mutant was sensitive to H-89. Collectively, the ΔAogts1 mutant showed a more remarkable difference compared with the WT strain than the ΔAogcs1 mutant. Our study further illustrates the importance of Arf-GAPs in the growth, development, and pathogenicity of nematode-trapping fungi.

Production of Microsclerotia by Metarhizium sp., and Factors Affecting Their Survival, Germination, and Conidial Yield.

Microsclerotia (MS) produced by some species of Metarhizium can be used as active ingredients in mycoinsecticides for the control of soil-dwelling stages of geophilic pests. In this study, the MS production potential of two Metarhizium brunneum strains and one M. robertsii strain was evaluated. The three strains were able to produce MS in liquid fermentation, yielding between 4.0 × 106 (M. robertsii EAMa 01/158-Su strain) and 1.0 × 107 (M. brunneum EAMa 01/58-Su strain) infective propagules (CFU) per gram of MS. The EAMa 01/58-Su strain was selected for further investigation into the effects of key abiotic factors on their survival and conidial yield. The MS were demonstrated to be stable at different storage temperatures (−80, −18, and 4 °C), with a shelf-life up to one year. The best temperature for MS storage was −80 °C, ensuring good viability of MS for up to one year (4.9 × 1010 CFU/g MS). Moreover, soil texture significantly affected CFU production by MS; sandy soils were the best driver of infective propagule production. Finally, the best combination of soil temperature and humidity for MS germination was 22.7 °C and 7.3% (wt./wt.), with no significant effect of UV-B exposure time on MS viability. These results provide key insights into the handling and storage of MS, and for decision making on MS dosage and timing of application.

An inactivating mutation in the vacuolar arginine exporter gene Vae results in culture degeneration in the fungus Metarhizium robertsii

Culture degeneration usually results in great commercial losses in the economically important filamentous fungi, but the genetic causes of the degeneration remain elusive. In the fungus Metarhizium robertsii, we found that deletion of the vacuolar arginine exporter gene Vae caused culture degeneration. Compared to the WT strain, the mutant showed increased apoptosis, reactive oxygen species (ROS) level and mitochondrial membrane potential collapse, reduced conidial yield and abnormal lipid droplet formation. The extent of the degeneration in the mutant gradually increased over the successive subculturing, which eventually became irreversible; compared to the third subculture of the mutant, the seventh subculture showed a lower conidial yield and pathogenicity to insects, stronger apoptosis, higher ROS level and a smaller number of conidial lipid droplets. Incorporation of the genomic clone of Vae could not restore the WT phenotypes in the seventh subculture, but could in the third one. Loss-of-function in Vae resulted in vacuolar arginine accumulation and reduction in the cytosolic arginine. This downregulated the expression of the regulator CAG9 of G protein signalling pathway, which accounted for most of the phenotypic changes associated with the degeneration of the mutant. We identified a deleterious mutation that causes culture degeneration in a filamentous fugus.

MaSln1, a Conserved Histidine Protein Kinase, Contributes to Conidiation Pattern Shift Independent of the MAPK Pathway in Metarhizium acridum.

ABSTRACTAs a conserved sensor kinase in the HOG-MAPK pathway, Sln1 plays distinct functions in different fungi. In this study, the roles of MaSln1 in Metarhizium acridum were analyzed using gene knockout and rescue strategies. Deletion of MaSln1 did not affect conidial germination, conidial yield, or resistance to chemical agents. However, fungal tolerance to heat shock and UV-B were significantly reduced after deletion of MaSln1. Insect bioassays showed that fungal pathogenicity was significantly impaired when MaSln1 was deleted. Further studies showed that MaSln1 did not affect either germination or appressorium formation of M. acridum on locust wings, but it significantly increased appressorium turgor pressure. In addition, disruption of MaSln1 resulted in a conidiation pattern shift in M. acridum. Microscopic observation revealed, however, that some genes located in the MAPK signaling pathway, including MaSho1, MaHog1, MaMk1, and MaSlt2, were not involved in the conidiation pattern shift on SYA medium (microcycle medium). Meanwhile, of the 143 differently expressed genes (DEGs) identified by RNA-seq, no genes related to the MAPK pathway were found, suggesting that MaSln1 regulation of the conidiation pattern shift was probably independent of the conserved MAPK signaling pathway. It was found that 22 of the 98 known DEGs regulated by MaSln1 were involved in mycelial growth, cell division, and cytoskeleton formation, indicating that MaSln1 likely regulates the expression of genes related to cell division and morphogenesis, thus regulating the conidiation pattern shift in M. acridum.IMPORTANCE The productivity and quality of conidia are both crucial for mycopesticides. In this study, we systematically analyzed the roles of MaSln1 in fungal pathogens. Most importantly, our results revealed that deletion of MaSln1 resulted in a conidiation pattern shift in M. acridum. However, some other genes, located in the MAPK signaling pathway, were not involved in the conidiation pattern shift. RNA-seq revealed no genes related to the MAPK pathway, suggesting that the regulation of the conidiation pattern shift by MaSln1 was probably independent of the conserved MAPK signaling pathway. This study provided a new insight into the functions of Sln1 and laid a foundation for exploring the mechanisms of conidiation pattern shifts in M. acridum.

Selection and characterization of thermotolerant Beauveria bassiana isolates and with insecticidal activity against the cotton-melon aphid Aphis gossypii (Glover) (Hemiptera: Aphididae).

Cotton-melon aphid Aphis gossypii (Glover) causes severe damage mainly to cucurbits. Twenty-two Beauveria sp. isolates were simultaneously assessed for their pathogenicity and heat tolerance. The selected isolates were identified molecularly and characterized in terms of conidial germination rate, mycelial growth, conidial yield and endophytic activity. Screening bioassays showed that the B. bassiana isolates B3, B7, B9 and B12 were the most toxic, inducing mortality equal to or slightly higher than the commercialized strain B. bassiana BNat (70.7%). Median lethal concentration (LC50 ) bioassays revealed that only isolate B12 had a significantly lower LC50 value (5.4× 105 conidia ml-1 ) than strain BNat (5× 106 conidia ml-1 ). The heat tolerance screening test (1h of exposure to 45°C) allowed us to select isolates B3, B7, B9 and B12 with germination rates of 57.5% to 80.1% after 24 h incubation at 25°C, all significantly higher than strain BNat (22.1%). The germination rates of all isolates decreased significantly after 2h of exposure to 45°C, with the exception of isolate B12 which displayed the highest thermotolerance (72% germination). The four selected isolates were able to endophytically colonize cucumber leaves when applied to the foliage. Inoculation of cucumber plants with isolate B12 did not affect cucumber plant growth. However, several plant growth parameters were improved 5 weeks after root inoculation. On the basis of its potent toxicity and thermotolerance, isolate B12 is a good candidate for further development as a biopesticide for use in integrated pest management strategies for aphid control. © 2022 Society of Chemical Industry.

MaAts, an Alkylsulfatase, Contributes to Fungal Tolerances against UV-B Irradiation and Heat-Shock in Metarhizium acridum

Sulfatases are commonly divided into three classes: type I, type II, and type III sulfatases. The type III sulfatase, alkylsulfatase, could hydrolyze the primary alkyl sulfates, such as sodium dodecyl sulfate (SDS) and sodium octyl sulfate. Thus, it has the potential application of SDS biodegradation. However, the roles of alkylsulfatase in biological control fungus remain unclear. In this study, an alkylsulfatase gene MaAts was identified from Metarhizium acridum. The deletion strain (ΔMaAts) and the complemented strain (CP) were constructed to reveal their functions in M. acridum. The activity of alkylsulfatase in ΔMaAts was dramatically reduced compared to the wild-type (WT) strain. The loss of MaAts delayed conidial germination, conidiation, and significantly declined the fungal tolerances to UV-B irradiation and heat-shock, while the fungal conidial yield and virulence were unaffected in M. acridum. The transcription levels of stress resistance-related genes were significantly changed after MaAts inactivation. Furthermore, digital gene expression profiling showed that 512 differential expression genes (DEGs), including 177 up-regulated genes and 335 down-regulated genes in ΔMaAts, were identified. Of these DEGs, some genes were involved in melanin synthesis, cell wall integrity, and tolerances to various stresses. These results indicate that MaAts and the DEGs involved in fungal stress tolerances may be candidate genes to be adopted to improve the stress tolerances of mycopesticides.

AoSsk1, a Response Regulator Required for Mycelial Growth and Development, Stress Responses, Trap Formation, and the Secondary Metabolism in Arthrobotrys oligospora.

Ssk1, a response regulator of the two-component signaling system, plays an important role in the cellular response to hyperosmotic stress in fungi. Herein, an ortholog of ssk1 (Aossk1) was characterized in the nematode-trapping fungus Arthrobotrys oligospora using gene disruption and multi-phenotypic comparison. The deletion of Aossk1 resulted in defective growth, deformed and swollen hyphal cells, an increased hyphal septum, and a shrunken nucleus. Compared to the wild-type (WT) strain, the number of autophagosomes and lipid droplets in the hyphal cells of the ΔAossk1 mutant decreased, whereas their volumes considerably increased. Aossk1 disruption caused a 95% reduction in conidial yield and remarkable defects in tolerance to osmotic and oxidative stress. Meanwhile, the transcript levels of several sporulation-related genes were significantly decreased in the ΔAossk1 mutant compared to the WT strain, including abaA, brlA, flbC, fluG, and rodA. Moreover, the loss of Aossk1 resulted in a remarkable increase in trap formation and predation efficiency. In addition, many metabolites were markedly downregulated in the ΔAossk1 mutant compared to the WT strain. Our results highlight that AoSsk1 is a crucial regulator of asexual development, stress responses, the secondary metabolism, and pathogenicity, and can be useful in probing the regulatory mechanism underlying the trap formation and lifestyle switching of nematode-trapping fungi.

Enhancing the Biocontrol Potential of the Entomopathogenic Fungus in Multiple Respects via the Overexpression of a Transcription Factor Gene MaSom1.

Entomopathogenic fungi play important roles in the control of populations of agricultural and disease vector pests in nature. The shortcomings of mycoinsecticides for pest management in the field cannot be completely overcome by improving single biocontrol properties of fungi. Therefore, enhancing the biocontrol potential of entomopathogenic fungi in multiple respects by genetic engineering is desirable. Transcription factors are usually involved in various important processes during fungal growth and pathogenesis via regulating a series of genes, and are important candidates for fungal improvement via genetic engineering. Herein, overexpression of MaSom1, a key transcription factor gene in the cAMP/PKA pathway, improves the biocontrol traits of Metarhizium acridum in multiple respects. When compared with WT, the MaSom1-overexpression strains exhibit enhanced tolerances to UV-B and heat shock, with increased mean 50% inhibition times by 66.9% and 155.2%, respectively. Advanced conidiation emerged accompanied by increased conidial yield up to 3.89 times after 3-day incubation for the MaSom1-overexpression strains compared to WT. Furthermore, when compared with WT, the virulence of the MaSom1-overexpression strains was also increased with the mean 50% lethality times reduced by 21.8% to 23.8%. Taken together, the MaSom1-overexpression improved the biocontrol potential of M. acridum in multiple respects. Our results provide insights into the application of key transcription factors for genetic engineering and offer a credible way to further improve the biocontrol potential of entomopathogenic fungi.

A Simple and Low-Cost Strategy to Improve Conidial Yield and Stress Resistance of Trichoderma guizhouense through Optimizing Illumination Conditions.

Light is perceived by photoreceptors in fungi and further integrated into the stress-activated MAPK HOG pathway, and thereby potentially activates the expression of genes for stress responses. This indicates that the precise control of light conditions can likely improve the conidial yield and stress resistance to guarantee the low cost and long shelf life of Trichoderma-based biocontrol agents and biofertilizers. In this study, effects of wavelengths and intensities of light on conidial yield and stress tolerance to osmotic, oxidative and pH stresses in Trichoderma guizhouense were investigated. We found that 2 μmol photons/(m2 × s) of blue light increased the conidial yield more than 1000 folds as compared to dark condition and simultaneously enhanced conidial stress resistance. The enhanced conidial stress resistance is probably due to the upregulated stress-related genes in blue light, which is under the control of the blue light receptor BLR1 and the MAP kinase HOG1.

Effectiveness of several nutritional sources on the virulence ofBeauveria bassianas.l. CEP147 against the planthopperDelphacodes kuscheli

AbstractDelphacodes kuscheliFennah (Hemiptera: Delphacidae) is the main planthopper vector ofMal de Río Cuarto virus(MRCV), aFijivirusthat severely affects maize production in Argentina. The effect of several nutritional sources on the virulence ofBeauveria bassianas.l. CEP147 (Balsamo‐Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) against this planthopper was evaluated with the aim to select some of them to be incorporated in future mass production studies. Ten agar‐agar 2% media were used. Media were supplemented with 10 g of sucrose and 4% flour of either amaranth, chia, flax, oat bran, parboiled rice, poppy, quinoa, or wheat germ. Sabouraud dextrose agar with 1% yeast extract (SDAY) was used as control, and sucrose agar medium supplemented with 2% chitin was used as a possible inductor of enzymatic activity. We evaluated the effect of each medium on colony growth, conidial yield, viability, size, adherence, hydrophobicity, protease activity, cumulative mortality, and median survival time (MST). Principal component analysis indicated that conidial viability and adherence explained the most of the total variance, indicating that these variables can be used as indicators of fungal virulence. Medium supplemented with chia flour not only increased conidial viability (100%) and adherence (0.09 A550) with respect to the control medium, but also produced highD. kuschelimortality (91.7%), with the lowest MST (5.2 days). We suggest that chia flour, which is rich in carbohydrates, proteins, vitamins, and minerals, might be incorporated as a nutritional supplement to solid substrates in order to increase the virulence ofB. bassianas.l. CEP147 against this and other pests. However, future studies are necessary to evaluate the effectiveness of this nutritional source on solid‐state fermentation.

A peroxisomal sterol carrier protein 2 (Scp2) contributes to lipid trafficking in differentiation and virulence of the insect pathogenic fungus Beauveria bassiana

Sterol carrier protein 2 (SCP2) represents a family of proteins binding a variety of lipids and plays essential roles in cellular physiology. However, its physiological roles are largely unknown in filamentous fungi. In this study, we functionally characterized an orthologous Scp2 gene in the filamentous insect pathogenic fungus Beauveria bassiana (BbScp2). BbScp2 was verified to be a peroxisomal protein and displayed different affinities to various lipids, with strong affinity to palmitic acid (PA) and ergosterol (ES). No significant binding activity was detected between protein and oleic acid (OA) or linoleic acid (LA). Ablation of BbScp2 did not cause significant effects on fungal growth on various carbon sources, but resulted in a modest reduction in conidial (49%) and blastospore yield (45%). In addition, exogenous lipids could recover the defectives in conidiation of ΔBbScp2 mutant strain. BbScp2 was required for the cytomembrane functionality in germlings, and its loss resulted in a more significant decrease in virulence indicated by cuticle infection assay than intrahemocoel injection assay. Our findings indicate that Scp2 links the lipid trafficking to the asexual differentiation and virulence of B. bassiana.

Temperature-dependent modelling and spatial prediction reveal suitable geographical areas for deployment of two Metarhizium anisopliae isolates for Tuta absoluta management

Tuta absoluta is one of the most devastating pests of Solanaceae crops in Africa. We previously demonstrated the efficacy of Metarhizium anisopliae isolates ICIPE 18, ICIPE 20 and ICIPE 665 against adult T. absoluta. However, adequate strain selection and accurate spatial prediction are fundamental to optimize their efficacy and formulations before field deployment. This study therefore assessed the thermotolerance, conidial yield and virulence (between 15 and 35 °C) of these potent isolates. Over 90% of conidia germinated at 20, 25 and 30 °C while no germination occurred at 15 °C. Growth of the three isolates occurred at all temperatures, but was slower at 15, 33 and 35 °C as compared to 20, 25 and 30 °C. Optimum temperatures for mycelial growth and spore production were 30 and 25 °C, respectively. Furthermore, ICIPE 18 produced higher amount of spores than ICIPE 20 and ICIPE 665. The highest mortality occurred at 30 °C for all the three isolates, while the LT50 values of ICIPE 18 and ICIPE 20 were significantly lower at 25 and 30 °C compared to those of ICIPE 665. Subsequently, several nonlinear equations were fitted to the mortality data to model the virulence of ICIPE 18 and ICIPE 20 against adult T. absoluta using the Entomopathogenic Fungi Application (EPFA) software. Spatial prediction revealed suitable locations for ICIPE 18 and ICIPE 20 deployment against T. absoluta in Kenya, Tanzania and Uganda. Our findings suggest that ICIPE 18 and ICIPE 20 could be considered as effective candidate biopesticides for an improved T. absoluta management based on temperature and location-specific approach.