Recent Advancements in Pathogenic Mechanisms, Applications and Strategies for Entomopathogenic Fungi in Mosquito Biocontrol.

Fungal diseases are widespread among insects and play a crucial role in naturally regulating insect populations. Mosquitoes, known as vectors for numerous infectious diseases, pose a significant threat to human health. Entomopathogenic fungi (EPF) have emerged as highly promising alternative agents to chemical mosquitocides for controlling mosquitoes at all stages of their life cycle due to their unique infection pathway through direct contact with the insect's cuticle. In recent years, significant advancements have been made in understanding the infection pathways and pathogenic mechanisms of EPF against mosquitoes. Various strategies involving the use of EPF alone or combinations with other approaches have been employed to target mosquitoes at various developmental stages. Moreover, the application of genetic technologies in fungi has opened up new avenues for enhancing the mosquitocidal efficacy of EPF. This review presents a comprehensive summary of recent advancements in our understanding the pathogenic mechanisms of EPF, their applications in mosquito management, and the combination of EPF with other approaches and employment of transgenic technologies. The biosafety concerns associated with their use and the corresponding approaches are also discussed. The recent progress suggests that EPF have the potential to serve as a future biorational tool for controlling mosquito vectors.

The carbon catabolite repressor CreA is an essential virulence factor of Metarhizium acridum against Locusta migratoria.

BACKGROUND: CreA has been proved to be a core gene in asexual conidiation in Metarhizium acridum, which regulates the shift of normal conidiation and microcycle conidiation. At present, research on CreA in fungi has focused on carbon source metabolism. There is a lack of research on the effect of CreA in virulence of pathogenic fungi. RESULTS: The virulence of the MaCreA disrupted strain (ΔMaCreA) for Locusta migratoria was lost by topical inoculation bioassay. The formation rate and turgor pressure of the appressoria decreased. Growth of ΔMaCreA in host hemolymph was delayed, and the number of hyphal bodies was significantly reduced. The conidial cell wall of ΔMaCreA became thicker, the mannan content decreased, and the chitin content increased significantly, and it was more sensitive to calcofluor white and Congo Red. α-1,3-Glucan and ß-1,3-glucan are more exposed on the surface of ΔMaCreA conidia than on the wild type. Lmspätzle and Lmcactus, the immune response genes in the host Toll pathway, showed stronger transcriptional activities at the early stage of ΔMaCreA invasion. The phenoloxidase activity assay also showed stronger immunostimulation by ΔMaCreA in vitro. CONCLUSION: The main reasons for the loss of virulence of ΔMaCreA in the topical inoculation were the reduced penetration ability of appressoria, limited growth in hemolymph and stronger insect immunostimulation of ΔMaCreA. © 2022 Society of Chemical Industry.

De novo genome assembly of a foxtail millet cultivar Huagu11 uncovered the genetic difference to the cultivar Yugu1, and the genetic mechanism of imazethapyr tolerance.

BACKGROUND: Setaria italica is the second-most widely planted species of millets in the world and an important model grain crop for the research of C4 photosynthesis and abiotic stress tolerance. Through three genomes assembly and annotation efforts, all genomes were based on next generation sequencing technology, which limited the genome continuity. RESULTS: Here we report a high-quality whole-genome of new cultivar Huagu11, using single-molecule real-time sequencing and High-throughput chromosome conformation capture (Hi-C) mapping technologies. The total assembly size of the Huagu11 genome was 408.37 Mb with a scaffold N50 size of 45.89 Mb. Compared with the other three reported millet genomes based on the next generation sequencing technology, the Huagu11 genome had the highest genomic continuity. Intraspecies comparison showed about 94.97 and 94.66% of the Yugu1 and Huagu11 genomes, respectively, were able to be aligned as one-to-one blocks with four chromosome inversion. The Huagu11 genome contained approximately 19.43 Mb Presence/absence Variation (PAV) with 627 protein-coding transcripts, while Yugu1 genomes had 20.53 Mb PAV sequences encoding 737 proteins. Overall, 969,596 Single-nucleotide polymorphism (SNPs) and 156,282 insertion-deletion (InDels) were identified between these two genomes. The genome comparison between Huagu11 and Yugu1 should reflect the genetic identity and variation between the cultivars of foxtail millet to a certain extent. The Ser-626-Aln substitution in acetohydroxy acid synthase (AHAS) was found to be relative to the imazethapyr tolerance in Huagu11. CONCLUSIONS: A new improved high-quality reference genome sequence of Setaria italica was assembled, and intraspecies genome comparison determined the genetic identity and variation between the cultivars of foxtail millet. Based on the genome sequence, it was inferred that the Ser-626-Aln substitution in AHAS was responsible for the imazethapyr tolerance in Huagu11. The new improved reference genome of Setaria italica will promote the genic and genomic studies of this species and be beneficial for cultivar improvement.

Discovery of broad-spectrum fungicides that block septin-dependent infection processes of pathogenic fungi.

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.

Colony heating protects honey bee populations from a risk of contact with wide-spectrum Beauveria bassiana insecticides applied in the field.

BACKGROUND: The safety of fungal insecticides to apiculture is a public concern but remains poorly understood. This study seeks to evaluate whether, how and why wide-spectrum Beauveria bassiana insecticides are safe to honey bees in a novel assessment system. RESULTS: Mesonotum dipping with a 108 conidia ml-1 suspension and body contact with conidial suspension in sucrose solution caused high mortalities of adult forager bees at 25 °C optimal for conidial germination and hyphal invasion. Intriguingly, colony sizes in the hives contaminated by the forager bees contacting viable and inactivated conidia at two sites (1.2 km in distance), respectively, showed similar increase percentages (31.7% versus 29.2%) during a 4-week summer period of exposure to environment. No sign of fungal infection was found within each of the monitored colonies. Neither was fungal outgrowth observed on surfaces of bee cadavers cleaned from each hive at either site. Hourly counts of cleaned cadavers from videotapes presented no significant difference in colony-cleaning behavior between the two sites. During the period, in-hive temperatures at both sites were persistently stabilized at approximately 35 °C, which abolished conidial germination and were far above the out-hive temperature range. CONCLUSION: It is colony heating that protects honey bee populations from a risk of forager bees' contact with formulated conidia applied for arthropod pest control. No role was detected for colony self-cleaning behavior in protecting the bee colonies from the risk. © 2020 Society of Chemical Industry.

Disruption of an adenylate-forming reductase required for conidiation, increases virulence of the insect pathogenic fungus Metarhizium acridum by enhancing cuticle invasion.

BACKGROUND: Metarhizium acridum, is a specific acridid pathogen developed for use against the migratory locust (Locusta migratoria manilensis). Adenylate-forming reductases (AFRs) include enzymes that are involved in natural product biosynthesis. Here, we genetically characterize the functions of a class IV AFR in M. acridum (MaAfrIV ) on fungal development and virulence. RESULTS: Gene expression analyses indicated MaAfrIV was induced on locust wings early during the infection process. Surprisingly, loss of MaAfrIV increased virulence (25.20% decrease in the median lethal time) against the locust in topical bioassays but was no different than the wild type when the cuticle was bypassed by direct infection of conidia into the insect hemocoel. Virulence markers including protease (Pr1) expression and appressorial turgor pressure were higher in the mutant than the parent strain. No difference was seen in the expression of host immune genes (Toll pathway) or in polyphenol oxidase (PPO) activity in locusts infected by the ΔMaAfrIV or wild type strains. However, the ΔMaAfrIV strain was unable to successfully sporulate on dead cadavers. CONCLUSION: Disruption of MaAfrIV increased fungal virulence by promoting insect cuticle invasion without altering host immune response or fungal immune evasion. Although loss of MaAfrIV conferred an apparent benefit to the fungus in terms of enhanced virulence, a significant trade-off was seen in the inability of the fungus to sporulate on the cadaver. As conidiation on the cadaver is essential for subsequent propagation in the environment, loss of MaAfrIV can reduce the engineering strains survivability in the field and improve the safety. © 2019 Society of Chemical Industry.

The MaCreA Gene Regulates Normal Conidiation and Microcycle Conidiation in Metarhizium acridum.

As a C2H2 type zinc finger transcription factor, CreA is the key in Carbon Catabolism Repression (CCR) pathway, which negatively regulates the genes in carbon sources utilization. As conidiation in filamentous fungi is affected by nutritional conditions, CreA may contribute to fungal conidiation, which has been well studied in filamentous fungi, especially Aspergillus spp., but researches on entomopathogenic fungi are not enough. In this study, we found a homologous gene MaCreA in Metarhizium acridum, and the MaCreA deletion strain showed delayed conidiation, significant decrease in conidial yield, and 96.88% lower conidial production, when compared with the wild-type strain, and the normal conidiation and microcycle conidiation pattern shift was blocked. RT-qPCR showed that the transcription levels of the genes FlbD and LaeA (related to asexual development) were significantly altered, and those of most of the conidiation-related genes were higher in ΔMaCreA strain. The results of RNA-Seq revealed that MaCreA regulated the two conidiation patterns by mediating genes related to cell cycle, cell division, cell wall, and cell polarity. In conclusion, CreA, as a core regulatory gene in conidiation, provides new insight into the mechanism of conidiation in entomopathogenic fungi.

The protein phosphatase gene MaPpt1 acts as a programmer of microcycle conidiation and a negative regulator of UV-B tolerance in Metarhizium acridum.

The Ser/Thr protein phosphatase Ppt1 (yeast)/PP5 (humans) has been implicated in signal transduction-mediated growth and differentiation, DNA damage/repair, cell cycle progression, and heat shock responses. Little, however, is known concerning the functions of Ppt1/PP5 in filamentous fungi. In this study, the Ppt1 gene MaPpt1 was characterized in the insect pathogenic fungus, Metarhizium acridum. The MaPpt1 protein features a three-tandem tetratricopeptide repeat (TPR) domain and a peptidyl-prolyl cis-trans isomerase-like (PP2Ac) domain. Subcellular localization using an MaPpt1::eGFP fusion protein revealed that MaPpt1 was localized in the cytoplasm of spores, but gathered at the septa in growing hyphae. Targeted gene inactivation of MaPpt1 in M. acridum resulted in unexpected reprogramming of normal aerial conidiation to microcycle conidiation. Although overall vegetative growth was unaffected, a significant increase in conidial yield was noted in ΔMaPpt1. Stress-responsive phenotypes and virulence were largely unaffected in ΔMaPpt1. Exceptionally, ΔMaPpt1 displayed increased UV tolerance compared to wild type. Digital gene expression data revealed that MaPpt1 mediates transcription of sets of genes involved in conidiation, polarized growth, cell cycle, cell proliferation, DNA replication and repair, and some important signaling pathways. These data indicate a unique role for Ppt1 in filamentous fungal development and differentiation.

A bifunctional catalase-peroxidase, MakatG1, contributes to virulence of Metarhizium acridum by overcoming oxidative stress on the host insect cuticle.

Microbial pathogens are exposed to damaging reactive oxygen species (ROS) produced from a variety of sources including chemical reactions due to exposure to stress (UV, heat) or by hosts as a defense response. Here, we demonstrate that a bifunctional catalase-peroxidase, MakatG1, in the locust-specific fungal pathogen, Metarhizium acridum, functions as a ROS detoxification mechanism during host cuticle penetration. MakatG1 expression was highly induced during on-cuticle appressoria development as compared to vegetative (mycelia) growth or during in vivo growth in the insect hemocoel. A MakatG1 deletion mutant strain (ΔMakatG1) showed decreased catalase and peroxidase activities and significantly increased susceptibility to oxidative (H2 O2 and menadione) and UV stress as compared to wild-type and complemented strains. Insect bioassays revealed significantly reduced virulence of the ΔMakatG1 mutant when topically inoculated, but no impairment when the insect cuticle was bypassed. Germination and appressoria formation rates for the ΔMakatG1 mutant were decreased on locust wings and quinone/phenolic compounds derived from locust wings, but were not affected on plastic surfaces compared with the wild-type strain. These data indicate that MakatG1 plays a pivotal role in penetration, reacting to and detoxifying specific cuticular compounds present on the host cuticle during the early stages of fungal infection.

Application of the entomogenous fungus, Metarhizium anisopliae, for leafroller (Cnaphalocrocis medinalis) control and its effect on rice phyllosphere microbial diversity.

Microbial pesticides form critical components of integrated pest management (IPM) practices. Little, however, is known regarding the impacts of these organisms on the indigenous microbial community. We show that Metarhizium anisopliae strain CQMa421 was highly effective in controlling the rice leafroller, Cnaphalocrocis medinalis Guenee. In addition, M. anisopliae distribution and its effects on phyllosphere microbial diversity after application in field trials were investigated. Phylloplane specific distribution of the fungus was observed over time, with more rapid declines of M. anisopliae CFUs (colony-forming units) seen in the top leaf layer as compared to lower layers. Application of the fungus resulted in transient changes in the endogenous microbial diversity with variations seen in the bacterial observed species and Shannon index. Notable increases in both parameters were seen at 6-day post-application of M. anisopliae, although significant variation within sample replicates for bacteria and fungi were noted. Application of M. anisopliae increased the relative distribution of bacterial species implicated in plant growth promotion and organic pollutant degradation, e.g., Methylobacterium, Sphingobium, and Deinococcus. These data show minimal impact of M. anisopliae on endogenous microbial diversity with transient changes in bacterial abundance/diversity that may result in added benefits to crops.

An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na(+)-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance.

In fungi, ENA ATPases play key roles in osmotic and alkaline pH tolerance, although their functions in thermo- and UV-tolerances have not been explored. Entomopathogenic fungi are naturally widespread and have considerable potential in pest control. An ENA ATPase gene, MaENA1, from the entomopathogenic fungus Metarhizium acridum was functionally analyzed by deletion. MaENA1-disruption strain (ΔMaENA1) was less tolerant to NaCl, heat, and UV radiation than a wild-type strain (WT). Digital Gene Expression profiling of conidial RNAs resulted in 281 differentially expressed genes (DEGs) between the WT and ΔMaENA1 strains. Eighty-five DEGs, 56 of which were down-regulated in the ΔMaENA1 strain, were shown to be associated with heat/UV tolerance, including six cytochrome P450 superfamily genes, 35 oxidoreductase genes, 24 ion-binding genes, seven DNA repair genes, and five other genes. In addition, eight genes were components of stress responsive pathways, including the Ras-cAMP PKA pathway, the RIM101 pathway, the Ca(2+)/calmodulin pathway, the TOR pathway, and the HOG/Spc1/Sty1/JNK pathway. These results demonstrated that MaENA1 influences fungal tolerances to Na(+), heat, and UV radiation in M. acridum, and is involved in multiple mechanisms of stress tolerance. Therefore, MaENA1 is required for the adaptation and survival of entomopathogenic fungi in stressful conditions in the environment and in their hosts.

Enhancing the utilization of host trehalose by fungal trehalase improves the virulence of fungal insecticide.

Entomopathogenic fungi proliferate in insect hemolymph by using host nutrients after penetrating the cuticle. To improve the virulence of the locust specific fungus, Metarhizium acridum, we genetically modified the fungus to overexpress ATM1, an endogenous hydrolase of trehalose, which is the main carbon source in insect hemolymph. Compared with the wild-type strain, Metarhizium acridum overexpressing ATM1 gene secreted more acid trehalase into locust hemolymph. The trehalose concentrations in locusts infected with the ATM1-overexpressing strain were 5.5 and 6.1 mmol/l, lower than that in locusts infected with the wild-type strain at 3 and 5 days post-inoculation, representing 44.5 and 60.7 % reduction, respectively. Correspondingly, overexpressing ATM1 accelerated the growth of Metarhizium acridum in host hemolymph, and the dose causing 50 % mortality (LD50) of the ATM1-overexpressing strain was reduced by 8.3-fold compared with the wild-type strain, suggesting that increasing the utilization of host nutrients by pathogens could be a promising way to improve the virulence of biopesticides based on parasites of pests.

The acid trehalase, ATM1, contributes to the in vivo growth and virulence of the entomopathogenic fungus, Metarhizium acridum.

For pathogens, the ability to acquire available nutrients in a host is a key to their survival and replication. Entomopathogenic fungi of the genus Metarhizium secrete trehalase, which enables them to use trehalose, the predominant sugar in insects. Here, the roles of the acid trehalase gene (ATM1) in the in vivo growth and virulence of Metarhizium acridum were investigated. Phenotypic analysis showed that disruption of ATM1 severely reduced fungal growth on exogenous trehalose as the sole carbon source. Bioassays showed that ATM1 disruption impaired the virulence of M. acridum against the host insect Locusta migratoria. The ATM1-disruption strain (ΔATM1) grown more slowly than the wild-type strain (WT) and complemented transformant (CP) in locust blood, which was consistent with the activity of acid trehalase in the hemolymph of infected locusts. Correspondingly, the trehalose concentration in locusts infected by ΔATM1 was significantly higher than in those infected by WT or CP. Thus, ATM1 disruption led to a significant reduction in virulence by adversely affecting the fungal growth in insect hemolymph, which resulted from the inability of the mutant strain to use trehalose.

Integration of an insecticidal scorpion toxin (BjαIT) gene into Metarhizium acridum enhances fungal virulence towards Locusta migratoria manilensis.

BACKGROUND: Entomopathogenic fungi have been developed as biopesticides, but poor efficacy has blocked their application. One approach to improving virulence is by genetic manipulation. BjαIT from the venom of Buthotus judaicus is an insect-selective neurotoxin. To clarify the insecticidal potency of BjαIT as a virulence candidate in microbial biocontrol agents, the entomopathogenic fungus Metarhizium acridum was genetically modified with BjαIT, and its resulting activity against locusts (Locusta migratoria manilensis) was assessed. RESULT: In comparison with the wild-type strain, the engineered isolate BjαIT-102 grew significantly quicker in locust haemolymph. Correspondingly, the median lethal dose (LC50 ) for BjαIT-102 was 18.2-fold lower, and the median lethal times (LT50 ) for BjαIT-102 were reduced by 28.1 and 30.4%, respectively, after topical inoculation and injection. BjαIT-102 formed conidia on dead locusts, although the conidial yield was reduced 1.58-fold. Moreover, there were no significant differences in germination and appressorium formation between the BjαIT-102 and wild-type strains. CONCLUSION: Expression of BjαIT in M. acridum significantly increased virulence against locusts by shortening the in vivo infection period without affecting conidium formation on the carcasses. This study demonstrated that engineering entomopathogenic fungi to incorporate BjαIT offers great potential for increasing their virulence.

Expression of scorpion toxin LqhIT2 increases the virulence of Metarhizium acridum towards Locusta migratoria manilensis.

LqhIT2 is an insect-specific neurotoxin from the venom of scorpion. In this study, the LqhIT2 gene was introduced into the entomopathogenic fungus, Metarhizium acridum. The virulence of the genetically modified strain MaLqhIT2 was then evaluated against locusts (Locusta migratoria manilensis). Compared with the wild-type strain, the median lethal cell density (LC50) for MaLqhIT2 was a 22.6-fold lower, and the median times to death (LT50) for MaLqhIT2 were reduced by 30.3 and 29.6 %, respectively, after topical inoculation and injection. MaLqhIT2 also grew significantly faster in the hemolymph than wild-type strain. There were no significant differences in germination, appressorium formation and sporulation in locust carcasses between the MaLqhIT2 and wild-type strain. These results indicate that LqhIT2 increased the virulence of M. acridum towards locusts by shortening the in vivo infection period, without affecting cuticle penetration or conidia formation in the carcasses. LqhIT2 thus shows considerable potential for increasing fungal virulence against locusts.

The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum.

BACKGROUND: The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors. RESULTS: Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium), Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. CONCLUSION: MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.

[Cloning and expression of Ser/Thr protein phosphatase type 5 during microcycle conidiation in Metarhizium ansopliae].

OBJECTIVE: To clone Ser/Thr protein phosphatase type 5 gene (PP5) from Metarhizium anisopliae, analyze the structure of PP5 gene with its encoding protein and expression profile during two conidiation program (microcycle conidiation and normal conidiation). METHODS: The DNA sequence of PP5 was isolated by blasting the expressed sequence tags (EST) of PP5 in subtracted library with genomic data of M. anisopliae. Primers were designed based on the DNA sequence to clone the full length cDNA of PP5 by PCR, and the characteristics of the encoded protein was analyzed by online tools and biological softwares. The PP5 expression profile was quantified by real time PCR at different stages of microcycle conidiation and hyphal stage of normal conidiation in M. anisopliae. RESULTS: The genomic DNA, which was interrupted by six introns, was 2100 bp long. The cDNA, encoding 325 amino acid residues, is 1428 bp. Analysis to Ser/ Thr protein phosphatase type 5 in M. anisopliae show a conserved structure features. Quantitative real time PCR analysis showed that PP5 expression varied obviously in different stages of microcycle conidiation. Expression was sharply up-regulated after 16 h, with the highest transcript levels at 24 h in microcycle conidiation, but lowly expressed in normal conidiation. CONCLUSION: This work presents the first report about the detailed sequence and structure of PP5 from entomopathogenic fungi. Comparison of expression profile of microcycle conidiation and normal conidiation reveals that PP5 is principally involved in microcycle conidiation in M. anisopliae, and it provides ideal candidate for further studies to PP5 and its molecular regulation.

Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum.

BACKGROUND: The entomopathogenic fungus Metarhizium acridum has been used as an important biocontrol agent instead of insecticides for controlling crop pests throughout the world. However, its virulence varies with environmental factors, especially temperature. Neutral trehalase (Ntl) hydrolyzes trehalose, which plays a role in environmental stress response in many organisms, including M. acridum. Demonstration of a relationship between Ntl and thermotolerance or virulence may offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi through genetic engineering. RESULTS: We selected four Ntl over-expression and four Ntl RNA interference (RNAi) transformations in which Ntl expression is different. Compared to the wild-type, Ntl mRNA expression was reduced to 35-66% in the RNAi mutants and increased by 2.5-3.5-fold in the over-expression mutants. The RNAi conidiospores exhibited less trehalase activity, accumulated more trehalose, and were much more tolerant of heat stress than the wild-type. The opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Furthermore, virulence was not altered in the two types of mutants compared to the wild type. CONCLUSIONS: Ntl controlled trehalose accumulation in M. acridum by degrading trehalose, and thus affected conidiospore thermotolerance. These results offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi without affecting virulence.

Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum.

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ~30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ~16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogeneous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

Trehalose-6-phosphate synthase 1 from Metarhizium anisopliae: clone, expression and properties of the recombinant.

Trehalose, an important component in fungal spores, is a disaccharide which protects against several environmental stresses, such as heat, desiccation, salt. Trehalose-6-phosphate synthase 1 (TPS1) is a subunit of trehalose synthase complex in fungi; it plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Metarhizium anisopliae encoding TPS1 (designated as MaTPS1) was isolated. The MaTPS1 cDNA is composed of 1836 nucleotides encoding a protein of 517 amino acids with a molecular mass of 58 kDa. The amino acid sequence has a relatively high homology with the TPS1s in several other filamentous fungi. Southern blot analysis showed that MaTPS1 gene occurs as a single copy in the M. anisopliae genome. And MaTPS1 was cloned into Pichia pastoris KM71 and secretively expressed with a histamine tag to facilitate a rapid purification of recombinant MaTPS1 (designated reTPS1). The properties of reTPS1 were examined. The K(m) value of reTPS1 for UDP-glucose and glucose-6-phosphate was 9.6 mM and 3.9 mM, respectively, and the optimal pH and temperature were about 6.5 and 40 degrees C. The enzyme was highly specific to glucose-6-phosphate for glucosyl acceptor, and its activity decreased rapidly as the concentrations of phosphate increased. The expression system will provide sufficient amounts of reTPS1 for future structural characterization of the protein and use for further investigation of MaTPS1's function.