The rhizosphere microbiome is a critical regulator of nutrient acquisition and plant growth in citrus. Here, we evaluated the effects of the entomopathogenic fungus Metarhizium anisopliae CQMa421 on soil nutrient status, rhizosphere bacterial community structure, and fruit quality in citrus using soil physicochemical assays, plant physiological measurements, and 16S rRNA amplicon high-throughput sequencing. CQMa421 application markedly reshaped soil properties, increasing available potassium by 128.50% and organic matter by 75.05%. In addition, total nitrogen, alkali-hydrolyzable nitrogen, and available phosphorus increased by 112.68%, 155.30%, 305.74% respectively, while soil pH decreased by 0.4 units. CQMa421 treatment significantly increased leaf total nitrogen content and elevated fruit vitamin C by 12.00%. Microbial community profiling showed an enrichment of putatively beneficial taxa, including Proteobacteria and Firmicutes, in treated soils. Functional prediction suggested enhanced nutrient cycling potential, with increased representation of genes associated with carbohydrate metabolism and inorganic ion transport. Collectively, these results indicate that M. anisopliae CQMa421 acts as a plant growth-promoting fungus by enhancing soil nutrient availability and restructuring the rhizosphere microbiome, thereby improving the overall nutrient status of the soil and enhancing citrus fruit quality.

Entomopathogenic microorganisms, including bacteria, fungi, and viruses, present a promising approach to mosquito control due to their high efficacy, species specificity, and environmental safety. Targeting larval stages is a strategic component of integrated mosquito management, leveraging the accessibility and concentration of immature mosquitoes in aquatic habitats. This review comprehensively examines diverse entomopathogenic larvicides, encompassing their larvicidal molecules, biochemical mechanisms of action, current application strategies, and prevailing challenges. Bacterial agents, notably Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lbs), remain the most widely deployed. Entomopathogenic fungi and viral pathogens demonstrate potent larvicidal activity, highlighting their significant potential for the development of novel bio-larvicides. Innovative formulation strategies and synergistic combinations of microorganisms with chemical pesticides offer promising avenues for efficacy enhancement. Concurrently, the ongoing isolation and characterization of novel larvicidal microorganisms are crucial for expanding the repertoire of biological control agents. These approaches enhance long-term control sustainability by mitigating resistance development. While challenges persist for large-scale implementation, entomopathogenic microorganisms present a compelling and sustainable pathway for effective mosquito management.

BACKGROUND:Mosquitoes are major vectors of disease and pose a significant threat to public health. Chemical insecticides face resistance and environmental issues, prompting the need for eco-friendly alternatives such as entomopathogenic fungi. However, their potential across mosquito life stages remains underexplored. As a primary malaria vector in East Asia, Anopheles sinensis remains understudied in terms of biological control approaches. RESULTS:Four effective fungal strains were initially screened from 22 fungal isolates based on their pathogenicity against An. sinensis larvae. Among them, strain Metarhizium anisopliae (Ma)421 demonstrated the best overall performance when further assessed for its mosquito-killing activity at the egg, pupa and adult stages, as well as its sporulation capacity on mosquito cadavers. Fungal infection reduced hatching rates, impaired larval pupation and adult emergence, and caused morphological deformities in newly emerged adults-such as malformed wings and legs-ultimately leading to mortality. Further analysis of Ma421 revealed a significant suppression of blood-feeding behavior, reduced egg-laying, and lower hatching, pupation and adult emergence rates in the next generation. These findings indicated that Ma421 induced transgenerational effects by decreasing reproductive success in subsequent generation, which is an important finding with promising implications for sustainable mosquito control. CONCLUSION:This study fills a significant gap in the fungal-based biological control of An. sinensis, with the multistage efficacy of Ma421 highlighting its potential for use in integrated vector management. Its transgenerational impact further underscores its potential as an eco-friendly alternative to chemical insecticides, supporting its incorporation into sustainable mosquito management strategies. © 2025 Society of Chemical Industry.

Metarhizium acridum is a typical filamentous fungus that has been widely used to control grasshoppers, locusts, and crickets. Genetic engineering is a common strategy to enhance its virulence, conidiation, and stress tolerance. Here, we report that the M. acridum Asp f2-like protein (MaAL) plays key roles in virulence, conidiation, and stress tolerance. Disruption of MaAL (ΔMaAL) resulted in faster conidial germination and significantly increased conidial yield. The conidial yield of the ΔMaAL strain was 113.33 % higher than that of the wild-type (WT) strain. The ΔMaAL strain exhibited significantly enhanced tolerance to UV-B irradiation, heat shock, and high osmotic stress. Furthermore, both topical inoculation and intra-hemocoel injection demonstrated significantly increased virulence against Locusta migratoria. Specifically, the LT50 of ΔMaAL was reduced by 1.54 days for topical inoculation and 1.08 days for intra-hemocoel injection compare to WT. Further investigation revealed that the deletion of MaAL led to increased turgor pressure in appressoria, reduced immune responses in locusts, and faster hyphal growth in the hemolymph. This study explores the function of Asp f2-like proteins in entomopathogenic fungi, providing theoretical support and genetic resources for the development of high-virulence strains.

Ergot alkaloids (EAs) are a class of secondary metabolites produced by fungi. These compounds are predominantly synthesized by Ascomycota, with variations in types and biosynthetic pathways among different fungal species. The EA synthesis has minimal impact on the normal growth and development of most EA-producing fungi, but serves as a virulence factor that influences the biocontrol functions of entomopathogenic fungi and symbiotic fungi in plants. In the medical field, EAs have been widely used for treating neurological disorders such as Parkinson's disease. However, the biosynthetic pathways of EAs are highly complex and significantly influenced by environmental factors, resulting in low yields from field production or chemical synthesis. To address the global demand for EAs, various strategies have been developed to reprogram the biosynthetic pathways in some chassis strains, aiming to simplify the process and increase EA production. This review summarizes the biosynthetic pathways and regulatory mechanisms of EAs in fungi, their biological functions, and recent advances in strategies for synthetic reprogramming.

BACKGROUND The entomopathogenic fungus (EPF) Metarhizium acridum, a typical filamentous fungus, has been utilized for the biological control of migratory locusts (Locusta migratoria manilensis). Fungal-specific transcription factors (TFs) play a crucial role in governing various cellular processes in fungi, although TFs with only the Fungal_trans domain remain poorly understood. RESULTS In this study, we identified a unique fungal-specific TF in M. acridum, named MaFTF1, which contains only a Fungal_trans domain and functions as a negative regulator of M. acridum virulence by influencing cuticle penetration. The virulence of the MaFTF1 knockout strain (Delta MaFTF1) against L. migratoria was increased, with a median lethal time (LT50) similar to 0.91 days shorter than that of the wild-type (WT) strain when inoculated topically, mimicking natural infection conditions. Correspondingly, Delta MaFTF1 penetrated the cuticle earlier than did the WT strain. Our investigation revealed that the development of appressoria was accelerated in Delta MaFTF1 compared with the WT strain. Furthermore, the appressoria of the Delta MaFTF1 displayed higher turgor pressure and an upregulated expression of fungal hydrolases active toward the insect cuticle. RNA sequencing analysis indicated that the differences in appressorium behavior between the strains were due to MaFTF1 regulating a complex metabolism pathway. CONCLUSION This study revealed that MaFTF1 acts as a negative regulator of virulence, impacting the process of cuticle penetration by slowing the formation of appressoria, decreasing their turgor pressure, and reducing the expression of hydrolases in appressoria, revealing an unexpected strategy in the EPFs. (c) 2024 Society of Chemical Industry.

Conidiation and stress tolerance are pivotal traits in entomopathogenic fungi, critically influencing their production costs and environmental tolerance. While the transcription factor high-mobility group protein (HMG), characterized by a conserved HMG-box domain, has been extensively studied for its role in sexual development, its functions in entomopathogenic fungi remain largely unexplored. This study employed gene knockout to investigate the role of MaHMG in Metarhizium acridum. The deletion of MaHMG delayed conidiation initiation and caused a highly significant 58% reduction in conidial yield versus that of the wild type (WT) after 15 days. Furthermore, the conidiation pattern on microcycle induction medium (SYA) shifted from microcycle to normal conidiation. The ΔMaHMG mutant exhibited decreased conidial germination rates and markedly reduced tolerance following UV-B irradiation and heat-shock treatments, alongside increased sensitivity to the cell wall perturbant calcofluor white (CFW). RNA-seq analysis during this conidiation shift identified 88 differentially expressed genes (DEGs), with functional annotation implicating their predominant association with hyphal development, cell wall biogenesis, cell cycle progression, and conidiation. In conclusion, MaHMG functions as a critical positive regulator governing both conidiation and stress tolerance in M. acridum, underscoring its fundamental role in fungal biology and potential as a target for enhancing biocontrol agent performance.

Spodoptera frugiperda, a globally invasive pest, has developed severe resistance to chemical insecticides, necessitating sustainable control strategies. This study investigated the synergistic interaction between emamectin benzoate (EB) and the entomopathogenic fungus Metarhizium anisopliae CQMa421 against fourth-instar larvae. Bioassays revealed a synergistic ratio (SR) of 4.19 when EB was combined with CQMa421 spores (2 × 107 spores/mL), reducing EB usage by 76.1 %. CQMa421 did not cause significant effects on S. frugiperda, whereas emamectin benzoate (EB) significantly disrupted feeding, development, and molting, particularly delaying fourth-instar molting by 4.65 days. Compared to EB alone, the combined treatment delayed larval molting by 1.2 days, suppressed feeding (18.3 % reduction in frass weight and 11.1 % weight loss), reduced adult emergence by 12.3 %, shortened oviposition duration by 1.5 days, and decreased total fecundity by 42.5 %. Additionally, the combined treatment increased pupal deformity by 4.4 %, collectively inhibiting population recovery. Mechanistic studies demonstrated that EB accelerated fungal spore germination (GT₅₀ reduced by 3.4 h), appressorium formation (AT₂₅ advanced by 4.5 h), and penetration of the cuticle of Spodoptera frugiperda. Furthermore, the combined treatment significantly downregulated immune-related genes (Attacin: 86.6 %; Cecropin: 61.2 %; MyD88: 50.5 %). This indicates that EB enhances the pathogenicity of the M. anisopliae synergistically by disrupting the host's immune barrier and delaying larval molting. This research provides a strategy and theoretical basis for controlling the resistance of Spodoptera frugiperda through reduced pesticide use and increased effectiveness.

The entomopathogenic fungus (EPF) Metarhizium acridum is a typical filamentous fungus and has been used to control migratory locusts (Locusta migratoria manilensis). This study examines the impact of the Zn(II)2Cys6 transcription factor, MaAzaR, in the virulence of M. acridum. Disruption of MaAzaR (ΔMaAzaR) diminished the fungus’s ability to penetrate the insect cuticle, thereby decreasing its virulence. The median lethal time (LT50) for the ΔMaAzaR strain increased by approximately 1.5 d compared to the wild-type (WT) strain when topically inoculated, simulating natural infection conditions. ΔMaAzaR compromises the formation, turgor pressure, and secretion of extracellular hydrolytic enzymes in appressoria. However, the growth ability of ΔMaAzaR within the hemolymph is not impaired; in fact, it grows better than the WT strain. Moreover, RNA-sequencing (RNA-Seq) analysis of ΔMaAzaR and WT strains grown for 20 h on locust hindwings revealed 87 upregulated and 37 downregulated differentially expressed genes (DEGs) in the mutant strain. Pathogen–host interaction database (PHI) analysis showed that about 40% of the total DEGs were associated with virulence, suggesting that MaAzaR is a crucial transcription factor that directly regulates the expression of downstream genes. This study identifies a new transcription factor involved in EPF cuticle penetration, providing theoretical support and genetic resources for the developing highly virulent strains.

Entomopathogenic fungi are valuable sources of biological pesticides, with conidial yield and quality being pivotal factors determining their broad applications. AzaR, a fungus-specific zinc-cluster transcription factor, is known to regulate the biosynthesis of polyketone secondary metabolites in Aspergillus niger; however, its role in pathogenic fungi remains unclear. This study investigated the role of MaAzaR in the growth, development, and environmental tolerance of Metarhizium acridum. MaAzaR deletion slowed down conidial germination rate, caused reduction in conidial yield, lowered fungal tolerance to UV radiation, did not affect fungal heat-shock tolerance, and increased fungal sensitivity to the cell-wall-destructive agent calcofluor white. Furthermore, MaAzaR deletion transformed microcycle conidiation to normal conidiation on the microcycle conidiation medium. Transcription profile analysis demonstrated that MaAzaR could regulate transformation of the conidiation pattern by controlling the expression of genes related to cell division, mycelium growth and development, and cell wall integrity. Thus, this study identified a new gene related to fungal conidiation and environmental tolerance, enriching our understanding of the molecular mechanism of microcycle conidiation and providing theoretical support and genetic resources for the development of high-yielding strains.

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.

柑橘全爪螨是我国柑橘的重要害虫/螨,已对多种杀螨剂产生抗药性.本文研究了微生物杀虫/螨剂球孢白僵菌ZJU435与杀螨剂乙唑螨腈对柑橘全爪螨的室内联合毒力及田间防效.室内毒力测定结果表明,100亿孢子/mL球孢白僵菌ZJU435 OD(可分散油悬浮剂)与30％乙唑螨腈SC(悬浮剂)按制剂体积10:1配比组合时,其共毒系数最高,达到249.90,具有明显的增效作用.田间试验结果表明,100亿孢子/mL球孢白僵菌ZJU435 OD(20 mL/15 L)与30％乙唑螨腈SC(2 mL/15 L)桶混,药后3,7,14 d的校正防效分别为84.41％,90.02％及92.86％,桶混后速效性显著优于球孢白僵菌ZJU435单剂(40 mL/15 L),且与乙唑螨腈单剂(4 mL/15 L)差异无统计学意义,持效性与球孢白僵菌ZJU435单剂差异也无统计学意义.综上,球孢白僵菌ZJU435与乙唑螨腈按10:1比例联合使用时的速效性增效均表现优异,因此推荐在实际生产应用中加以推广.

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 (Delta MaCreA) for Locusta migratoria was lost by topical inoculation bioassay. The formation rate and turgor pressure of the appressoria decreased. Growth of Delta MaCreA in host hemolymph was delayed, and the number of hyphal bodies was significantly reduced. The conidial cell wall of Delta MaCreA became thicker, the mannan content decreased, and the chitin content increased significantly, and it was more sensitive to calcofluor white and Congo Red. alpha-1,3-Glucan and beta-1,3-glucan are more exposed on the surface of Delta MaCreA conidia than on the wild type. Lmspatzle and Lmcactus, the immune response genes in the host Toll pathway, showed stronger transcriptional activities at the early stage of Delta MaCreA invasion. The phenoloxidase activity assay also showed stronger immunostimulation by Delta MaCreA in vitro. CONCLUSION The main reasons for the loss of virulence of Delta MaCreA in the topical inoculation were the reduced penetration ability of appressoria, limited growth in hemolymph and stronger insect immunostimulation of Delta MaCreA. (c) 2022 Society of Chemical Industry.

目的 测试金龟子绿僵菌CQMa421饵剂室内和现场防治德国小蠊效果,为真菌杀虫剂在防治卫生害虫上的应用提供参考.方法 配制不同含量的金龟子绿僵菌CQMa421饵剂,分别在实验室和现场环境研究其对德国小蠊种群数量的影响,采用SPSS 25.0软件对死亡率、半数致死时间(LT50)、半数致死浓度(LC50)和90％致死浓度(LC90)进行分析,评价金龟子绿僵菌CQMa421饵剂杀蟑效果;采用x2检验分析不同组别间死亡率的差异.结果 室内实验结果表明,1×108孢子/g金龟子绿僵菌CQMa421饵剂喂食德国小蠊后12d,其LT50均值为(6.85±0.24)d;现场实验中,采用每2.5 m2放置1盒1×108孢子/g的金龟子绿僵菌CQMa421饵剂,药剂投放30 d后,相对密度下降率为93.22％.结论 1×108孢子/g的金龟子绿僵菌CQMa421饵剂可有效控制德国小蠊种群密度.

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.

Annual economic losses of eggplant due to western flower thrips ( Frankliniella occidentalis ) infestations are considerable. F. occidentalis is difficult to control due to rapid proliferation rates and resistance to chemical insecticides. A more effective biological control strategy is urgently required. To assess the potential efficacy of M. anisopliae CQMa421 in the biological control of F. occidentalis on eggplant, we evaluated its virulence and insecticidal activity in the laboratory and field. The laboratory results indicated that the LT 50 of M. anisopliae CQMa421 against F. occidentalis at 2 × 10 7 conidia/ml was 5.5 days. The results from the experimental field trial showed that a single spray of M. anisopliae CQMa421 reduced thrips by 50–70% compared to the control; this was significantly lower than for the chemical insecticide imidacloprid. However, the results from demonstration field trials where consecutive sprays were made indicated that M. anisopliae CQMa421 controlled the pests at a level equivalent to that obtained with a chemical insecticide. The findings clearly demonstrated the feasibility of using M. anisopliae CQMa421 as an alternative to chemical insecticides in the control of F. occidentalis under field conditions.

金龟子绿僵菌Metarhizium anisopliae是一种重要的生防真菌,具有安全、易在害虫种群中流行、环境友好、不易产生抗药性等优点,已在中国、美国、欧盟等多个国家或地区登记和应用.本文对我国广谱金龟子绿僵菌CQMa421农药的菌株选育、产品登记、应用技术及产品应用情况等方面进行了概述.

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.

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. This article is protected by copyright. All rights reserved.

BACKGROUND The safety of fungal insecticides to apiculture is publically concerned 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 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 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 ~35°C, which abolished conidial germination and were far above 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. This article is protected by copyright. All rights reserved.