Influence of genetic diversity of seventeen Beauveria bassiana isolates from different hosts on virulence by comparative genomics

Laurent B., Moinard M., Spataro C., Ponts N., Barreau C., Foulongne-Oriol M.

Fusarium graminearum is one of the main causal agents of the Fusarium Head Blight, a worldwide disease affecting cereal cultures, whose presence can lead to contaminated grains with chemically stable and harmful mycotoxins. Resistant cultivars and fungicides are frequently used to control this pathogen, and several observations suggest an adaptation of F. graminearum that raises concerns regarding the future of current plant disease management strategies. To understand the genetic basis as well as the extent of its adaptive potential, we investigated the landscape of genomic diversity among six French isolates of F. graminearum, at single-nucleotide resolution using whole-genome re-sequencing. A total of 242,756 high-confidence genetic variants were detected when compared to the reference genome, among which 96% are single nucleotides polymorphisms. One third of these variants were observed in all isolates. Seventy-seven percent of the total polymorphism is located in 32% of the total length of the genome, comprising telomeric/subtelomeric regions as well as discrete interstitial sections, delineating clear variant enriched genomic regions- 7.5 times in average. About 80% of all the F. graminearum protein-coding genes were found polymorphic. Biological functions are not equally affected: genes potentially involved in host adaptation are preferentially located within polymorphic islands and show greater diversification rate than genes fulfilling basal functions. We further identified 29 putative effector genes enriched with non-synonymous effect mutation. Our results highlight a remarkable level of polymorphism in the genome of F. graminearum distributed in a specific pattern. Indeed, the landscape of genomic diversity follows a bi-partite organization of the genome according to polymorphism and biological functions. We measured, for the first time, the level of sequence diversity for the entire gene repertoire of F. graminearum and revealed that the majority are polymorphic. Those assumed to play a role in host-pathogen interaction are discussed, in the light of the subsequent consequences for host adaptation. The annotated genetic variants discovered for this major pathogen are valuable resources for further genetic and genomic studies.

Fan Y., Liu X., Keyhani N.O., Tang G., Pei Y., Zhang W., Tong S.

Significance Since the discovery of oosporein more than 70 years ago, there have been conflicting reports on its potential antimicrobial and insecticidal activities. Our results indicate that oosporein is unlikely to function as an insect toxin or to be involved in early to mid-infection processes, including penetration and immune evasion. Instead, oosporein most likely functions after death of the host to thwart bacterial competition on a host cadaver, allowing the fungus to maximally use host nutrients and complete its life cycle. Our data also reveal that oosporein production is regulated by a cascade of transcription factors, with BbSmr1 acting as an upstream negative regulator, targeting the expression of OpS3, which in turn acts as a positive regulator of the oosporein biosynthetic gene cluster.

Valero-Jiménez C.A., Faino L., Spring in’t Veld D., Smit S., Zwaan B.J., van Kan J.A.

Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence. A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85–16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1–1–1 or the MAT1–2–1 gene. Moreover, a putative new MAT gene (MAT1-2–8) was detected in the MAT1–2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented. Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.

Dionisio G., Kryger P., Steenberg T.

Ortiz-Urquiza A., Keyhani N.O.

Research on the insect pathogenic filamentous fungus, Beauveria bassiana has witnessed significant growth in recent years from mainly physiological studies related to its insect biological control potential, to addressing fundamental questions regarding the underlying molecular mechanisms of fungal development and virulence. This has been in part due to a confluence of robust genetic tools and genomic resources for the fungus, and recognition of expanded ecological interactions with which the fungus engages. Beauveria bassiana is a broad host range insect pathogen that has the ability to form intimate symbiotic relationships with plants. Indeed, there is an increasing realization that the latter may be the predominant environmental interaction in which the fungus participates, and that insect parasitism may be an opportunist lifestyle evolved due to the carbon- and nitrogen-rich resources present in insect bodies. Here, we will review progress on the molecular genetics of B. bassiana, which has largely been directed toward identifying genetic pathways involved in stress response and virulence assumed to have practical applications in improving the insect control potential of the fungus. Important strides have also been made in understanding aspects of B. bassiana development. Finally, although increasingly apparent in a number of studies, there is a need for progressing beyond phenotypic mutant characterization to sufficiently investigate the molecular mechanisms underlying B. bassiana's unique and diverse lifestyles as saprophyte, insect pathogen, and plant mutualist.

Atwell S., Corwin J.A., Soltis N.E., Subedy A., Denby K.J., Kliebenstein D.J.

How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from 4 independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e. polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative mating assays showing that these loci control vegetative incompatibility. This suggests that the vegetative mating loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over ten years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen’s broad host range.

Lamb S.A., Rahman M.M., McFadden G.

Although the production of single gene knockout viruses is a useful strategy to study viral gene functions, the redundancy of many host interactive genes within a complex viral genome can obscure their collective functions. In this study, a rabbit-specific poxvirus, myxoma virus (MYXV), was genetically altered to disrupt multiple members of the poxviral ankyrin-repeat (ANK-R) protein superfamily, M-T5, M148, M149 and M150. A particularly robust activation of the NF-κB pathway was observed in A549 cells following infection with the complete ANK-R knockout (vMyx-ANKsKO). Also, an increased release of IL-6 was only observed upon infection with vMyx-ANKsKO. In virus-infected rabbit studies, vMyx-ANKsKO was the most extensively attenuated and produced the smallest primary lesion of all ANK-R mutant constructs. This study provides the first insights into the shared functions of the poxviral ANK-R protein superfamily in vitro and in vivo.

Boomsma J.J., Jensen A.B., Meyling N.V., Eilenberg J.

Lineages of insect pathogenic fungi are concentrated in three major clades: Hypocreales (several genera), Entomophthoromycota (orders Entomophthorales and Neozygitales), and Onygenales (genus Ascosphaera). Our review focuses on aspects of the evolutionary biology of these fungi that have remained underemphasized in previous reviews. To ensure integration with the better-known domains of insect pathology research, we followed a conceptual framework formulated by Tinbergen, asking complementary questions on mechanism, ontogeny, phylogeny, and adaptation. We aim to provide an introduction to the merits of evolutionary approaches for readers with a background in invertebrate pathology research and to make the insect pathogenic fungi more accessible as model systems for evolutionary biologists. We identify a number of questions in which fundamental research can offer novel insights into the evolutionary forces that have shaped host specialization and life-history traits such as spore number and size, somatic growth rate, toxin production, and interactions with host immune systems.

Finn R.D., Bateman A., Clements J., Coggill P., Eberhardt R.Y., Eddy S.R., Heger A., Hetherington K., Holm L., Mistry J., Sonnhammer E.L., Tate J., Punta M.

Pfam, available via servers in the UK (http://pfam.sanger.ac.uk/) and the USA (http://pfam.janelia.org/), is a widely used database of protein families, containing 14 831 manually curated entries in the current release, version 27.0. Since the last update article 2 years ago, we have generated 1182 new families and maintained sequence coverage of the UniProt Knowledgebase (UniProtKB) at nearly 80%, despite a 50% increase in the size of the underlying sequence database. Since our 2012 article describing Pfam, we have also undertaken a comprehensive review of the features that are provided by Pfam over and above the basic family data. For each feature, we determined the relevance, computational burden, usage statistics and the functionality of the feature in a website context. As a consequence of this review, we have removed some features, enhanced others and developed new ones to meet the changing demands of computational biology. Here, we describe the changes to Pfam content. Notably, we now provide family alignments based on four different representative proteome sequence data sets and a new interactive DNA search interface. We also discuss the mapping between Pfam and known 3D structures.

Wilkening S., Tekkedil M.M., Lin G., Fritsch E.S., Wei W., Gagneur J., Lazinski D.W., Camilli A., Steinmetz L.M.

The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups. Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes. Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.

Weischenfeldt J., Symmons O., Spitz F., Korbel J.O.

With the increased cataloguing of human structural variants, our understanding of their influence on phenotype is ever improving. Here, the influence of structural variants on phenotypes including disease is discussed, and strategies for further characterization are presented. Genomic structural variants have long been implicated in phenotypic diversity and human disease, but dissecting the mechanisms by which they exert their functional impact has proven elusive. Recently however, developments in high-throughput DNA sequencing and chromosomal engineering technology have facilitated the analysis of structural variants in human populations and model systems in unprecedented detail. In this Review, we describe how structural variants can affect molecular and cellular processes, leading to complex organismal phenotypes, including human disease. We further present advances in delineating disease-causing elements that are affected by structural variants, and we discuss future directions for research on the functional consequences of structural variants.

Mburu D.M., Maniania N.K., Hassanali A.

Isolates of the fungus Beauveria bassiana have different levels of virulence and repellency against the termite Macrotermes michaelseni. In the present study, we compared the volatile profiles and gene sequences of two isolates of the fungus with different levels of virulence and repellence to the termite. Gas chromatography–mass spectrometric analyses showed quantitative and qualitative differences in the composition of volatiles of the two isolates. The repellencies of synthetic blends of 10 prominent components that mimicked the volatiles of each of the two isolates were significantly different. Subtractive bioassays showed that the repellency of each isolate was due to synergistic effects of a few constituents. As previously reported for isolates of Metarhizium anisopliae, some differences also were found in the nucleotide sequences of the two isolates of B. bassiana, suggesting a genetic basis for the observed intra-specific differences in their repellency and virulence against the termite.

Xiao G., Ying S., Zheng P., Wang Z., Zhang S., Xie X., Shang Y., St. Leger R.J., Zhao G., Wang C., Feng M.

The ascomycete fungus Beauveria bassiana is a pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. We sequenced the genome of B. bassiana and a phylogenomic analysis confirmed that ascomycete entomopathogenicity is polyphyletic, but also revealed convergent evolution to insect pathogenicity. We also found many species-specific virulence genes and gene family expansions and contractions that correlate with host ranges and pathogenic strategies. These include B. bassiana having many more bacterial-like toxins (suggesting an unsuspected potential for oral toxicity) and effector-type proteins. The genome also revealed that B. bassiana resembles the closely related Cordyceps militaris in being heterothallic, although its sexual stage is rarely observed. A high throughput RNA-seq transcriptomic analysis revealed that B. bassiana could sense and adapt to different environmental niches by activating well-defined gene sets. The information from this study will facilitate further development of B. bassiana as a cost-effective mycoinsecticide.

Cingolani P., Platts A., Wang L.L., Coon M., Nguyen T., Wang L., Land S.J., Lu X., Ruden D.M.

Fan Y., Borovsky D., Hawkings C., Ortiz-Urquiza A., Keyhani N.O.

Sui L., Lu Y., Cheng K., Tian Y., Liu Z., Xie Z., Zhang Z., Li Q.

Oberti H., Sessa L., Oliveira‐Rizzo C., Di Paolo A., Sanchez‐Vallet A., Seidl M.F., Abreo E.

AbstractBACKGROUNDBiological control methods involving entomopathogenic fungi like Beauveria bassiana have been shown to be a valuable approach in integrated pest management as an environmentally friendly alternative to control pests and pathogens. Identifying genetic determinants of pathogenicity in B. bassiana is instrumental for enhancing its virulence against insects like the resistant soybean pest Piezodorus guildinii. This study focused on comparative genomics of different B. bassiana strains and gene expression analyses to identify virulence genes in the hypervirulent strain ILB308, especially in response to infection of P. guildinii and growth on hydrocarbon HC15, a known virulence enhancer.RESULTSStrain ILB308 showed the highest number of virulence‐related features, such as candidate virulence proteins, effectors, small secreted proteins and biosynthetic gene clusters. ILB308 also had a high percentage of unique DNA sequences, including six accessory scaffolds. Gene expression analysis at 4 days post inoculation revealed upregulation of known virulence factors, including Tudor domain proteins, LysM motif‐containing proteins, subtilisin‐like proteases and novel genes encoding secreted effectors and heat‐labile enterotoxins. Growth on HC15 led to the upregulation of genes associated with oxidoreductase activity related to cuticular alkane degradation and fermentation metabolism/antioxidant responses in the hemolymph. The low number of known B. bassiana virulence genes points to novel or unknown mechanisms acting on the interaction between P. guildinii and strain ILB308.CONCLUSIONThe presence of accessory genomic regions and unique virulence genes in ILB308 may contribute to its higher virulence. These genes could be considered as potential targets for enhancing fungal virulence through genetic manipulation. © 2025 Society of Chemical Industry.

Lakshita N., Yulani R.A., Wijonarko A., Indarti S.

Abstract Background Control techniques using biological control agents such as Beauveria bassiana (Balsamo) Vuillemin have the advantage of not showing any negative impacts on environmental health and safety issues. This study used isolates from B. bassiana collection from Laboratory of Pest Monitoring belonging to Indonesian Ministry of Agriculture (LPHP) in Central Java which showed potential in controlling target pest. The problems that still occur are the lack of facilities and infrastructure and the lack of quality testing of collection isolates in LPHP, so that the isolate identification process is still carried out in simple method, and bioassay testing on the fall armyworm (FAW), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) as a target pest, is not commonly conducted. The results of bioassay testing can be used to determine the potential of a biological agent to control target pest. Result The two DNA extraction methods showed different results regarding DNA concentration and purity values, but both methods were good and could be used to amplify DNA using PCR. The DNA band was amplified at 500–600 bp using primers ITS 1 and ITS 4. The results of molecular analysis showed that the four isolates of B. bassiana from Central Java were found in the same clade as B. bassiana from South Sumatra, Dhaka, and Oromia, where these isolates showed similar similarities descended from a common ancestor. Genetically, B. bassiana isolates from Central Java show more genetic similarities to B. bassiana isolates from South Sumatra, Indonesia. Quality testing was carried out by calculating the density and germination ability values for LPHP isolates from Sukoharjo (Sukoharjo isolate), Temanggung (Purworejo isolate), and Banyumas (Banyumas and Cilacap isolate), which showed varying results. The bioassay test used three isolates, namely B. bassiana from Sukoharjo, Banyumas, and Cilacap, which were selected based on density values, germination ability, and molecular analysis. The ability to cause death of the three isolates against S. frugiperda showed different results where the isolate from Sukoharjo, Banyumas, and Cilacap caused mortality of 60, 40, and 60%, and the LC50 value of each isolate was 3.3 × 106, 1.3 × 107, and 3.5 × 107 conidia ml−1, respectively. Conclusion Morphological identification by macroscopic, microscopic, and molecular analysis showed that the isolate from the LPHP collection in Central Java, Indonesia, was B. bassiana. Genetically, the four isolates showed similar characteristics to isolates from South Sumatra, Indonesia. B. bassiana isolates from collections from Central Java showed potentials as a biological control agent against S. frugiperda.

Segura-Vega J., González-Herrera A., Molina-Bravo R., Solano-González S.

BackgroundThe fungus Beauveria bassiana is widely used for agronomical applications, mainly in biological control. B. bassiana uses chitinase enzymes to degrade chitin, a major chemical component found in insect exoskeletons and fungal cell walls. However, until recently, genomic information on neotropical isolates, as well as their metabolic and biotechnological potential, has been limited.MethodsEight complete B. bassiana genomes of Neotropical origin and three references were studied to identify chitinase genes and its corresponding proteins, which were curated and characterized using manual curation and computational tools. We conducted a computational study to highlight functional differences and similarities for chitinase proteins in these Neotropical isolates.ResultsEleven chitinase 1 genes were identified, categorized as chitinase 1.1 and chitinase 1.2. Five chitinase 2 genes were identified but presented a higher sequence conservation across all sequences. Interestingly, physicochemical parameters were more similar between chitinase 1.1 and chitinase 2 than between chitinase 1.1 and 1.2.ConclusionChitinases 1 and 2 demonstrated variations, especially within chitinase 1, which presented a potential paralog. These differences were observed in their physical parameters. Additionally, CHIT2 completely lacks a signal peptide. This implies that CHIT1 might be associated with infection processes, while CHIT2 could be involved in morphogenesis and cellular growth. Therefore, our work highlights the importance of computational studies on local isolates, providing valuable resources for further experimental validation. Intrinsic changes within local species can significantly impact our understanding of complex pathogen-host interactions and offer practical applications, such as biological control.

Ranesi M., Vitale S., Staropoli A., Di Lelio I., Izzo L.G., De Luca M.G., Becchimanzi A., Pennacchio F., Lorito M., Woo S.L., Vinale F., Turrà D.

Beauveria bassiana (Bb) is a widespread entomopathogenic fungus widely used in agriculture for crop protection. Other than pest control, fungi belonging to the B. bassiana complex represent an important microbial resource in agroecosystems, considering their multiple interactions with other microorganisms as antagonists of phytopathogens, or with plants as endophytic colonizers and growth promoters. Here, we characterised field collected or commercial isolates of B. bassiana relative to the environmental factors that affect their growth. We further compared the metabolome, the entomopathogenic potential and biocontrol activity of the tested isolates respectively on the insect pest Spodoptera littoralis or against the fungal plant pathogen Fusarium oxysporum. Our analysis revealed that the B. bassiana complex is characterised by a high level of inter-isolate heterogeneity in terms of nutritional requirements, establishment of intra- or inter-kingdom interactions, and the nature of metabolites produced. Interestingly, certain B. bassiana isolates demonstrated a preference for low nutrient plant-derived media, which hints at their adaptation towards an endophytic lifestyle over a saprophytic one. In addition, there was a noticeable variation among different B. bassiana isolates in their capacity to kill S. littoralis larvae in a contact infection test, but not in an intrahaemocoelic injection experiment, suggesting a unique level of adaptability specific to the host. On the other hand, most B. bassiana isolates exhibited similar biocontrol efficacy against the soil-dwelling ascomycete F. oxysporum f. sp. lycopersici, a pathogen responsible for vascular wilt disease in tomato plants, effectively averting wilting. Overall, we show that the effectiveness of B. bassiana isolates can greatly vary, emphasising the importance of isolate selection and nutritional adaptability consideration for their use in sustainable agriculture.

Zamora-Avilés N., Orozco-Flores A.A., Cavazos-Vallejo T., Romo-Sáenz C.I., Cuevas-García D.A., Gomez-Flores R., Tamez-Guerra P.

Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain’s geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL50) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification.

Morda W., Nuvoli M.T., Ruiu L.

The need to reduce the impact of plant protection products on agroecosystems fosters the use of augmentative biological control involving the release of beneficial species into the field, the employment of entomopathogenic microbials, and the protection of naturally occurring biocontrol agents. This study aimed to investigate the compatibility of the entomopathogenic fungus Beauveria bassiana with the generalist insect predator Chrysoperla lucasina, in comparative experiments involving a laboratory-reared and a wild chrysopid strain. The larvae of the predators were exposed to different concentrations of fungal conidia up to a concentration of 107 conidia/mL by contact and ingestion. The treated insects showed only slight differences in terms of survival and immature development time compared to the untreated control insects. A significant decrease in the proportion of the male adults of C. lucasina that emerged from the laboratory-reared larvae that were exposed to higher concentrations of the fungus suggested a potentially different susceptibility between the sexes. A slightly lower adult emergence rate was observed in the wild strain, while no significant differences were recorded in the adult reproductive performance. These findings indicate that the B. bassiana strain ATCC 74040, at concentrations commonly used in the field, did not pose a significant risk to C. lucasina and can be safely used in combination with this predator for sustainable pest management.

Zhang Z., Tian Y., Sui L., Lu Y., Cheng K., Zhao Y., Li Q., Shi W.

IntroductionAspergillus nomiae is known as a pathogenic fungus that infects humans and plants but has never been reported as an entomophagous fungus (EPF) that can provide other functions as an endotype.MethodsA strain of EPF was isolated and identified from diseased larvae of Spodoptera litura in a soybean field and designated AnS1Gzl-1. Pathogenicity of the strain toward various insect pests was evaluated, especially the ability to colonize plants and induce resistance against phytopathogens and insect pests.ResultsThe isolated EPF strain AnS1Gzl-1 was identified as A. nomiae; it showed strong pathogenicity toward five insect pests belonging to Lepidoptera and Hemiptera. Furthermore, the strain inhibited the growth of Sclerotinia sclerotiorum in vitro, a causal agent of soil-borne plant disease. It colonized plants as an endophyte via root irrigation with a high colonization rate of 90%, thereby inducing plant resistance against phytopathogen infection, and disrupting the feeding selectivity of S. litura larvae.DiscussionThis is the first record of a natural infection of A. nomiae on insects. A. nomiae has the potential to be used as a dual biocontrol EPF because of its ability to not only kill a broad spectrum of insect pests directly but also induce resistance against phytopathogens via plant colonization.

Zhang Z., Guo W., Lu Y., Kang Q., Sui L., Liu H., Zhao Y., Zou X., Li Q.

Abstract Background The entomogenous fungus Beauveria bassiana is used as a biological insecticide worldwide, wild B. bassiana strains with high pathogenicity in the field play an important role in controlling insect pests via not only screening of highly virulent strains but also natural infection, but the pathogenicity degeneration of wild strains severely affected aforementioned effects. Previous studies have showed that multiple factors contributed to this phenomenon. It has been extensively proved that the mycovirus infection caused hypovirulence of phytopathogenic fungi, which has been used for plant disease biocontrol. However, it remains unknown whether the mycovirus epidemics is a key factor causing hypovirulence of B. bassiana naturally in the field. Methods Wild strains of B. bassiana were collected from different geographic locations in Jilin Province, China, to clarify the epidemic and diversity of the mycoviruses. A mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) we have previously identified was employed to clarify its impact on the pathogenicity of host fungi B. bassiana against the larvae of insect pest Ostrinia furnacalis. The serological analysis was conducted by preparing polyclonal antibody against a BbCV2 coat protein, to determine whether it can dissociate outside the host fungal cells and subsequently infect new hosts. Transcriptome analysis was used to reveal the interactions between viruses and hosts. Results We surprisingly found that the mycovirus BbCV2 was prevalent in the field as a core virus in wild B. bassiana strains, without obvious genetic differentiation, this virus possessed efficient and stable horizontal and vertical transmission capabilities. The serological results showed that the virus could not only replicate within but also dissociate outside the host cells, and the purified virions could infect B. bassiana by co-incubation. The virus infection causes B. bassiana hypovirulence. Transcriptome analysis revealed decreased expression of genes related to insect epidermis penetration, hypha growth and toxin metabolism in B. bassiana caused by mycovirus infection. Conclusion Beauveria bassiana infected by hypovirulence-associated mycovirus can spread the virus to new host strains after infecting insects, and cause the virus epidemics in the field. The findings confirmed that mycovirus infection may be an important factor affecting the pathogenicity degradation of B. bassiana in the field.

Parker D., Meyling N.V., De Fine Licht H.H.

Abstract Intraspecific pathogen diversity is crucial for understanding the evolution and maintenance of adaptation in host–pathogen interactions. Traits associated with virulence are often a significant source of variation directly impacted by local selection pressures. The specialist fungal entomopathogen, Metarhizium acridum, has been widely implemented as a biological control agent of locust pests in tropical regions of the world. However, few studies have accounted for natural intraspecific phenotypic and genetic variation. Here, we examine the diversity of nine isolates of M. acridum spanning the known geographic distribution, in terms of (1) virulence towards two locust species, (2) growth rates on three diverse nutrient sources, and (3) comparative genomics to uncover genomic variability. Significant variability in patterns of virulence and growth was shown among the isolates, suggesting intraspecific ecological specialization. Different patterns of virulence were shown between the two locust species, indicative of potential host preference. Additionally, a high level of diversity among M. acridum isolates was observed, revealing increased variation in subtilisin-like proteases from the Pr1 family. These results culminate in the first in-depth analysis regarding multiple facets of natural variation in M. acridum, offering opportunities to understand critical evolutionary drivers of intraspecific diversity in pathogens. Abstract Exploring the intricate world of host–pathogen interactions, our study delves into the diverse landscape of Metarhizium acridum, a specialist fungal entomopathogen. By dissecting patterns of virulence, growth dynamics, and genomic variability across its geographic distribution, we unveil compelling evidence of intraspecific diversity. Our findings highlight varying virulence patterns among distinct locust species and a notable abundance of gene diversity, particularly in subtilisin-like proteases. These insights shed light on potential evolutionary drivers shaping natural variation within M. acridum.

Zhang Z., Guo W., Lu Y., Kang Q., Sui L., Liu H., Zhao Y., Zou X., Li Q.

Abstract Background The entomogenous fungus Beauveria bassiana is used as a biological insecticide worldwide, but its application is affected by pathogenicity degeneration in the field. Previous studies showed that multiple factors contribute to this phenomenon. Mycovirus infection causes hypovirulence of phytopathogenic fungi and mycoviruses have been used for plant disease biocontrol. However, it remains unknown whether mycovirus epidemics are key factors causing hypovirulence of B. bassiana naturally in the field. Methods A mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) we have previously identified was employed to clarify its impact on the pathogenicity of host fungi Beauveria bassiana against the larvae of insect pest Ostrinia furnacalis. Wild strains of B. bassiana were collected from different geographic locations in Jilin Province, China, to clarify the epidemic and genetic diversity of the mycovirus BbCV2. The serological analysis was conducted by preparing polyclonal antibody against a BbCV2 coat protein, to determine whether the it can dissociate outside the host fungi cells and subsequently infect new hosts. Transcriptome analysis was used to reveal the interactions between viruses and hosts. Results We found that themycovirus BbCV2 possessed efficient and stable horizontal and vertical transmission capabilities and caused B. bassiana hypovirulence. Surprisingly, this virus was prevalent in the field as a core virus in wild B. bassiana strains, without obvious genetic differentiation. The serological results showed that the virus could not only replicate within the host cell, but also dissociate outside to infect new hosts. Transcriptome analysis revealed decreased expression of genes related to insect epidermis penetration and toxin metabolism in B. bassianacaused by mycovirus infection. Conclusion B.bassiana infected by hypovirulence-associated mycovirus can spread the virus to new host strainsafter infecting insects, and causethe virus epidemics in the field. The findings confirmed that mycovirus infection may be an important factor affecting the pathogenicity degradation of B. bassiana in the field.

Gu Z., Chen L., Zhang W., Su P., Zhang D., Du X., Peng Q., Liu Z., Liao X., Liu Y.

Beauveria bassiana is a well-known insecticidal biocontrol agent. Despite its broad field applications, its survival, colonization, and stability under field conditions remained unclear, mainly due to the lack of a quick and reliable detection method. In this study, we developed a quantitative real-time PCR technology to monitor the stability and population dynamics of B. bassiana in different substrates (water, soil, and on the cotton leaves surface), different spores of B. bassiana applied on Chinese cabbage leaves surface, and the lethality of Pieris rapae spraying with different spores of B. bassiana. Our results showed a decreased concentration of B. bassiana DNA in all three substrates from the 1st day till 9th day of post inoculation (dpi) period, possibly due to the death of B. bassiana. After this decrease, a quick and significant rebound of B. bassiana DNA concentration was observed, starting from the 11th dpi in all three substrates. The B. bassiana DNA concentration reached the plateau at about 13th dpi in water and 17th dpi in the soil. On cotton leaves surface, the B. bassiana DNA concentration reached the highest level at the 17th dpi followed by a small decline and then stabilized. This increase of DNA concentration suggested recovery of B. bassiana growth in all three substrates. We found that the most suitable killing effectiveness of P. rapae was the 1.0 × 107 spores/mL of B. bassiana. In summary, we have established a detection technology that allows a fast and reliable monitoring for the concentration and stability of B. bassiana under different conditions. This technology can benefit and help us in the development of proper management strategies for the application of this biocontrol agent in the field.

Tu C., Zhang Y., Zhu P., Sun L., Xu P., Wang T., Luo J., Yu J., Xu L.

The entomopathogenic fungus is recognized as an ideal alternative to chemical pesticides, nonetheless, its efficacy is often limited by insect's innate immune system. The suppression of the host immunity may overcome the obstacle and promote the toxicity of the fungi. Here, by using an entomopathogenic fungus Beauveria bassiana and immune genes dsRNA-expressing bacteria, we explored the potentially synergistic toxicity of the two agents on a leaf beetle Plagiodera versicolora (Coleoptera: Chrysomelidae). We first determined the susceptibilities of P. versicolora to a B. bassiana 476 strain (hereafter referred to Bb476). And the immune genes were identified based on the transcriptome of Bb476 challenged beetles. Subsequently, five immune genes (PGRP1, Toll1, Domeless，SPN1，and Lysozyme) were targeted by feeding dsRNA-expressing bacteria, which produced a 71.4, 39.0, 72.0, 49.0, and 68.7% gene silencing effect, respectively. Furthermore, we found a significantly increased mortality of P. versicolora when combined the Bb476 and the immune suppressive dsRNAs. Taking together, this study highlights the importance of insect immunity in the defense of entomopathogens and also paves the way toward the development of a more efficient pest management strategy that integrates both entomopathogens and immune suppressive dsRNAs.

Francis J.R., Manchegowda H.K.

Molecular phylogenetic species recognition is essential in precisely differentiating the morphologically cryptic species of Metarhizium and Beauveria. These fungal entomopathogens can be an alternative to conventional synthetic insecticides and are emerging as effective biocontrol agents against sucking insect pests. In the present study, we identified Metarhizium and Beauveria species in diverse agricultural and semi-natural ecosystems of south Karnataka State of India using phylogenetic markers and evaluated their pathogenicity against Scirtothrips dorsalis. Based on morphological characters, 34 Metarhizium and 15 Beauveria isolates were identified in agricultural and semi-natural ecosystems. Phylogeny of Metarhizium was resolved using markers, TEF1-α, MzIGS3, and ITS rDNA to two species, M. pingshaense and M. guizhouense and Beauveria was resolved using markers, TEF1-α, Bloc, and ITS rDNA to a single species, B. bassiana based on phylogenetic optimality criteria. Isolates of M. pingshaense and B. bassiana resolved three phylogenetic clades each and M. guizhouense resolved one phylogenetic clade. Phylogenetic clades of M. pingshaense and B. bassiana exhibit intraclade variation and are not associated with crop plant or host insect. Laboratory bio-efficacy studies of 49 isolates of Metarhizium and Beauveria against adult S. dorsalis showed that pathogenicity varied significantly among isolates. Greenhouse bioassays revealed the high virulence of B. bassiana isolates over M. pingshaense isolates against S. dorsalis. Low LC50 value of 2.8 × 104 conidia/ml was obtained for B. bassiana GKVK 01_06 isolate against S. dorsalis. Our results indicate that indigenous B. bassiana isolates have strong potential in the management of S. dorsalis.

Castro-Vásquez R., Solano-González S., Molina-Bravo R., Montero-Astúa M.

Beauveria bassiana , a well-known entomopathogenic fungus, has a worldwide distribution; however, genomes of isolates from the Neotropical region are scarce. Here, we report the draft genome sequences of eight B. bassiana isolates from Costa Rica, Puerto Rico, and Honduras.