Baking bad: plants in a toasty world with necrotrophs.

Rising global temperatures pose a threat to plant immunity, making them more susceptible to diseases. The impact of temperature on plant immunity against biotrophic and hemi-biotrophic pathogens is well documented, while its effect on necrotrophs remains poorly understood. We venture into the uncharted territory of necrotrophic fungal pathogens in the face of rising temperatures. We discuss the role of the plant hormones salicylic acid (SA) and jasmonic acid (JA) in providing resistance to necrotrophs and delve into the temperature sensitivity of the SA pathway. Additionally, we explore the repercussions of increased temperatures on plant susceptibility to necrotrophs. We put forward a research agenda with an experimental framework aimed at providing a comprehensive understanding of how plants and pathogens adapt to increasing temperatures.

Botrytis cinerea infection accelerates ripening and cell wall disassembly to promote disease in tomato fruit.

Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.

Cytotoxic activity of Nep1-like proteins on monocots.

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are found throughout several plant-associated microbial taxa and are typically considered to possess cytolytic activity exclusively on dicot plant species. However, cytolytic NLPs are also produced by pathogens of monocot plants such as the onion (Allium cepa) pathogen Botrytis squamosa. We determined the cytotoxic activity of B. squamosa BsNep1, as well as other previously characterized NLPs, on various monocot plant species and assessed the plant plasma membrane components required for NLP sensitivity. Leaf infiltration of NLPs showed that onion cultivars are differentially sensitive to NLPs, and analysis of their sphingolipid content revealed that the GIPC series A : series B ratio did not correlate to NLP sensitivity. A tri-hybrid population derived from a cross between onion and two wild relatives showed variation in NLP sensitivity within the population. We identified a quantitative trait locus (QTL) for NLP insensitivity that colocalized with a previously identified QTL for B. squamosa resistance and the segregating trait of NLP insensitivity correlated with the sphingolipid content. Our results demonstrate the cytotoxic activity of NLPs on several monocot plant species and legitimize their presence in monocot-specific plant pathogens.

Bitter and sweet make tomato hard to (b)eat.

The glycoalkaloid saponin α-tomatine is a tomato-specific secondary metabolite that accumulates to millimolar levels in vegetative tissues and has antimicrobial and antinutritional activity that kills microbial pathogens and deters herbivorous insects. We describe recent insights into the biosynthetic pathway of α-tomatine synthesis and its regulation. We discuss the mode of action of α-tomatine by physically interacting with sterols, thereby disrupting membranes, and how tomato protects itself from its toxic action. Tomato pathogenic microbes can enzymatically hydrolyze, and thereby inactivate, α-tomatine using either of three distinct types of glycosyl hydrolases. We also describe findings that extend well beyond the simple concept of plants producing toxins and pathogens inactivating them. There are reports that toxicity of α-tomatine is modulated by external pH, that α-tomatine can trigger programmed cell death in fungi, that cellular localization matters for the impact of α-tomatine on invading microbes, and that α-tomatine breakdown products generated by microbial hydrolytic enzymes can modulate plant immune responses. Finally, we address a number of outstanding questions that deserve attention in the future.

Peeling the Onion: Towards a Better Understanding of Botrytis Diseases of Onion.

Onion is cultivated worldwide for its bulbs, but production is threatened by pathogens and pests. Three distinct diseases of onion are caused by species that belong to the fungal genus Botrytis. Leaf blight is a well-known foliar disease caused by B. squamosa that can cause serious yield losses. Neck rot is a postharvest disease that manifests in bulbs after storage and is associated with three species: B. aclada, B. allii, and B. byssoidea. The symptomless infection of onion plants in the field makes it difficult to predict the incidence of neck rot in storage, although progress on the detection of latent infection has been made. In onion cultivation for seed production, blighting of the inflorescence is caused by all four onion-specific Botrytis species plus the broad host range pathogen B. cinerea. Flower blight can reduce seed yield and contaminate seed. In this review, the long history of Botrytis diseases of onion is discussed, as well as recent and future approaches to acquire a better understanding of the biology and ecology of Botrytis spp. pathogenic on onion. New fundamental insights in the genetic, biochemical, and physiological aspects of Botrytis-onion interactions are essential to improve the breeding of Botrytis-resistant onion cultivars.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Visualization of Three Sclerotiniaceae Species Pathogenic on Onion Reveals Distinct Biology and Infection Strategies.

Botrytis squamosa, Botrytis aclada, and Sclerotium cepivorum are three fungal species of the family Sclerotiniaceae that are pathogenic on onion. Despite their close relatedness, these fungi cause very distinct diseases, respectively called leaf blight, neck rot, and white rot, which pose serious threats to onion cultivation. The infection biology of neck rot and white rot in particular is poorly understood. In this study, we used GFP-expressing transformants of all three fungi to visualize the early phases of infection. B. squamosa entered onion leaves by growing either through stomata or into anticlinal walls of onion epidermal cells. B. aclada, known to cause post-harvest rot and spoilage of onion bulbs, did not penetrate the leaf surface but instead formed superficial colonies which produced new conidia. S. cepivorum entered onion roots via infection cushions and appressorium-like structures. In the non-host tomato, S. cepivorum also produced appressorium-like structures and infection cushions, but upon prolonged contact with the non-host the infection structures died. With this study, we have gained understanding in the infection biology and strategy of each of these onion pathogens. Moreover, by comparing the infection mechanisms we were able to increase insight into how these closely related fungi can cause such different diseases.

Comparative genomics of plant pathogenic Botrytis species with distinct host specificity.

BACKGROUND: Fungi of the genus Botrytis (presently containing ~ 35 species) are able to infect more than 1400 different plant species and cause losses in a wide range of crops of economic importance. The best studied species is B. cinerea, which has a broad host range and is one of the best studied necrotrophic plant pathogenic fungi. Most other Botrytis spp. have a narrow host range and have been studied in less detail. To characterize genomic variation among different representatives of Botrytis spp., we sequenced and annotated the draft genomes of nine Botrytis species: B. calthae, B. convoluta, B. elliptica, B. galanthina, B. hyacinthi, B. narcissicola, B. paeoniae, B. porri and B. tulipae. RESULTS: Bioinformatics and comparative genomics tools were applied to determine a core of 7668 shared protein families in all Botrytis species, which grouped them in two distinct phylogenetic clades. The secretome of all nine Botrytis spp. was similar in number (ranging from 716 to 784 predicted proteins). A detailed analysis of the molecular functions of the secretome revealed that shared activities were highly similar. Orthologs to effectors functionally studied in B. cinerea were also present in the other Botrytis species. A complex pattern of presence/absence of secondary metabolite biosynthetic key enzymes was observed. CONCLUSIONS: Comparative genomics of Botrytis show that overall, species share the main signatures and protein families in the secreted proteins, and of known effectors. Our study provides leads to study host range determinants in the genus Botrytis and provides a stepping stone to elucidate the roles of effector candidates in the infection process of these species.

Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1-dependent differences.

Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine-rich repeat receptor kinases (LRR-RK) FLS2 and EFR, and the LRR receptor protein (LRR-RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR-RK and LRR-RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP-mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23-regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2-signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP-type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.

The obligate alkalophilic soda-lake fungus Sodiomyces alkalinus has shifted to a protein diet.

Sodiomyces alkalinus is one of the very few alkalophilic fungi, adapted to grow optimally at high pH. It is widely distributed at the plant-deprived edges of extremely alkaline lakes and locally abundant. We sequenced the genome of S. alkalinus and reconstructed evolution of catabolic enzymes, using a phylogenomic comparison. We found that the genome of S. alkalinus is larger, but its predicted proteome is smaller and heavily depleted of both plant-degrading enzymes and proteinases, when compared to its closest plant-pathogenic relatives. Interestingly, despite overall losses, S. alkalinus has retained many proteinases families and acquired bacterial cell wall-degrading enzymes, some of them via horizontal gene transfer from bacteria. This fungus has very potent proteolytic activity at high pH values, but slowly induced low activity of cellulases and hemicellulases. Our experimental and in silico data suggest that plant biomass, a common food source for most fungi, is not a preferred substrate for S. alkalinus in its natural environment. We conclude that the fungus has abandoned the ancestral plant-based diet and has become specialized in a more protein-rich food, abundantly available in soda lakes in the form of prokaryotes and small crustaceans.

A novel Zn2 Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea.

D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2 Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.

Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea.

BACKGROUND: Botrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis. RESULTS: In this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered. CONCLUSIONS: This is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.

Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against mosquitoes.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Mind the gap; seven reasons to close fragmented genome assemblies.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions.

Genes involved in virulence of the entomopathogenic fungus Beauveria bassiana.

Pest insects cause severe damage to global crop production and pose a threat to human health by transmitting diseases. Traditionally, chemical pesticides (insecticides) have been used to control such pests and have proven to be effective only for a limited amount of time because of the rapid spread of genetic insecticide resistance. The basis of this resistance is mostly caused by (co)dominant mutations in single genes, which explains why insecticide use alone is an unsustainable solution. Therefore, robust solutions for insect pest control need to be sought in alternative methods such as biological control agents for which single-gene resistance is less likely to evolve. The entomopathogenic fungus Beauveria bassiana has shown potential as a biological control agent of insects, and insight into the mechanisms of virulence is essential to show the robustness of its use. With the recent availability of the whole genome sequence of B. bassiana, progress in understanding the genetics that constitute virulence toward insects can be made more quickly. In this review we divide the infection process into distinct steps and provide an overview of what is currently known about genes and mechanisms influencing virulence in B. bassiana. We also discuss the need for novel strategies and experimental methods to better understand the infection mechanisms deployed by entomopathogenic fungi. Such knowledge can help improve biocontrol agents, not only by selecting the most virulent genotypes, but also by selecting the genotypes that use combinations of virulence mechanisms for which resistance in the insect host is least likely to develop.

Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1.

Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.

The endo-arabinanase BcAra1 is a novel host-specific virulence factor of the necrotic fungal phytopathogen Botrytis cinerea.

The plant cell wall is one of the first physical interfaces encountered by plant pathogens and consists of polysaccharides, of which arabinan is an important constituent. During infection, the necrotrophic plant pathogen Botrytis cinerea secretes a cocktail of plant cell-wall-degrading enzymes, including endo-arabinanase activity, which carries out the breakdown of arabinan. The roles of arabinan and endo-arabinanases during microbial infection were thus far elusive. In this study, the gene Bcara1 encoding for a novel α-1,5-L-endo-arabinanase was identified and the heterologously expressed BcAra1 protein was shown to hydrolyze linear arabinan with high efficiency whereas little or no activity was observed against the other oligo- and polysaccharides tested. The Bcara1 knockout mutants displayed reduced arabinanase activity in vitro and severe retardation in secondary lesion formation during infection of Arabidopsis leaves. These results indicate that BcAra1 is a novel endo-arabinanase and plays an important role during the infection of Arabidopsis. Interestingly, the level of Bcara1 transcript was considerably lower during the infection of Nicotiana benthamiana compared with Arabidopsis and, consequently, the ΔBcara1 mutants showed the wild-type level of virulence on N. benthamiana leaves. These results support the conclusion that the expression of Bcara1 is host dependent and is a key determinant of the disease outcome.

Functional analysis of hydrophobin genes in sexual development of Botrytis cinerea.

Hydrophobins are small secreted fungal proteins that play roles in growth and development of filamentous fungi, i.e. in the formation of aerial structures and the attachment of hyphae to hydrophobic surfaces. In Botrytis cinerea, three hydrophobin genes have been identified. Studies by Mosbach et al. (2011) showed that hydrophobins are neither involved in conferring surface hydrophobicity to conidia and aerial hyphae of B. cinerea, nor are they required for virulence. The present study investigated the role of hydrophobins in sclerotium and apothecium development. Expression analysis revealed high expression of the Bhp1 gene during different stages of apothecium development. Two Bhp1 splice variants were detected that differ by an internal stretch of 13 amino acid residues. Seven different mutants in which either a single, two or three hydrophobin genes were knocked out, as well as two wild type strains of opposite mating types, were characterized for sclerotium and apothecium development. No aberrant morphology was observed in sclerotium development when single deletion mutants in hydrophobin genes were analyzed. Sclerotia of double knock out mutant ΔBhp1/ΔBhp3 and the triple knock out mutant, however, showed easily wettable phenotypes. For analyzing apothecium development, a reciprocal crossing scheme was setup. Morphological aberrations were observed in crosses with two hydrophobin mutants. When the double knock out mutant ΔBhp1/ΔBhp2 and the triple knock out mutant were used as the maternal parent (sclerotia), and fertilized with wild type microconidia, the resulting apothecia were swollen, dark brown in color and had a blotched surface. After initially growing upwards toward the light source, the apothecia in many cases collapsed due to loss of structural integrity. Aberrant apothecium development was not observed in the reciprocal cross, when these same mutants were used as the paternal parent (microconidia). These results indicate that the presence of hydrophobins in maternal tissue is important for normal development of apothecia of B. cinerea.

Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative d-galacturonate transporters.

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.

Natural variation in virulence of the entomopathogenic fungus Beauveria bassiana against malaria mosquitoes.

BACKGROUND: Insecticide resistance is greatly hampering current efforts to control malaria and therefore alternative methods are needed. Entomopathogenic fungi have been proposed as an alternative with a special focus on the cosmopolitan species Beauveria bassiana. However, few studies have analysed the effects of natural variation within fungal isolates on mosquito survival, and the implications and possible exploitation for malaria control. METHODS: Laboratory bioassays were performed on adult female mosquitoes (Anopheles coluzzii) with spores from 29 isolates of B. bassiana, originating from different parts of the world. In addition, phenotypic characteristics of the fungal isolates such as sporulation, spore size and growth rate were studied to explore their relationship with virulence. RESULTS: All tested isolates of B. bassiana killed An. coluzzii mosquitoes, and the rate at which this happened differed significantly among the isolates. The risk of mosquitoes dying was around ten times higher when they were exposed to the most virulent as compared to the least virulent isolate. There was significant variation among isolates in spore size, growth rate and sporulation, but none of these morphological characteristics were correlated, and thus predictive, for the ability of the fungal isolate to kill malaria mosquitoes. CONCLUSIONS: This study shows that there is a wide natural variation in virulence of isolates of B. bassiana, and that selecting an appropriate fungal isolate is highly relevant in killing and thus controlling malaria mosquitoes, particularly if used as part of an integrated vector management strategy. Also, the wide variation observed in virulence offers the opportunity to better understand the molecular and genetic mechanisms that drive this variation and thus to address the potential development of resistance against entomopathogenic fungi.