Genome Comparisons between Botrytis fabae and the Closely Related Gray Mold Fungus Botrytis cinerea Reveal Possible Explanations for Their Contrasting Host Ranges.

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility.

Deciphering the Monilinia fructicola Genome to Discover Effector Genes Possibly Involved in Virulence.

Brown rot is the most economically important fungal disease of stone fruits and is primarily caused by Monilinia laxa and Monlinia fructicola. Both species co-occur in European orchards although M. fructicola is considered to cause the most severe yield losses in stone fruit. This study aimed to generate a high-quality genome of M. fructicola and to exploit it to identify genes that may contribute to pathogen virulence. PacBio sequencing technology was used to assemble the genome of M. fructicola. Manual structural curation of gene models, supported by RNA-Seq, and functional annotation of the proteome yielded 10,086 trustworthy gene models. The genome was examined for the presence of genes that encode secreted proteins and more specifically effector proteins. A set of 134 putative effectors was defined. Several effector genes were cloned into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana plants, and some of them triggered necrotic lesions. Studying effectors and their biological properties will help to better understand the interaction between M. fructicola and its stone fruit host plants.

Dynamics in Secondary Metabolite Gene Clusters in Otherwise Highly Syntenic and Stable Genomes in the Fungal Genus Botrytis.

Fungi of the genus Botrytis infect >1,400 plant species and cause losses in many crops. Besides the broad host range pathogen Botrytis cinerea, most other species are restricted to a single host. Long-read technology was used to sequence genomes of eight Botrytis species, mostly pathogenic on Allium species, and the related onion white rot fungus, Sclerotium cepivorum. Most assemblies contained <100 contigs, with the Botrytis aclada genome assembled in 16 gapless chromosomes. The core genome and pan-genome of 16 Botrytis species were defined and the secretome, effector, and secondary metabolite repertoires analyzed. Among those genes, none is shared among all Allium pathogens and absent from non-Allium pathogens. The genome of each of the Allium pathogens contains 8-39 predicted effector genes that are unique for that single species, none stood out as potential determinant for host specificity. Chromosome configurations of common ancestors of the genus Botrytis and family Sclerotiniaceae were reconstructed. The genomes of B. cinerea and B. aclada were highly syntenic with only 19 rearrangements between them. Genomes of Allium pathogens were compared with ten other Botrytis species (nonpathogenic on Allium) and with 25 Leotiomycetes for their repertoire of secondary metabolite gene clusters. The pattern was complex, with several clusters displaying patchy distribution. Two clusters involved in the synthesis of phytotoxic metabolites are at distinct genomic locations in different Botrytis species. We provide evidence that the clusters for botcinic acid production in B. cinerea and Botrytis sinoallii were acquired by horizontal transfer from taxa within the same genus.

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.

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.

Experimental evolution to increase the efficacy of the entomopathogenic fungus Beauveria bassiana against malaria mosquitoes: Effects on mycelial growth and virulence.

Entomopathogenic fungi such as Beauveria bassiana are currently considered as a potential control agent for malaria mosquitoes. The success of such strategies depends among others on the efficacy of the fungus to kill its hosts. As B. bassiana can use various resources for growth and reproduction, increasing the dependency on mosquitoes as a nutritional source may be instrumental for reaching this goal. Passage of entomopathogenic fungi through an insect host has been shown to increase its virulence. We evaluated the virulence, fungal outgrowth, mycelial growth rate, and sporulation rate of two B. bassiana isolates (Bb1520 and Bb8028) that underwent 10 consecutive selection cycles through malaria mosquitoes (Anopheles coluzzii) using an experimental evolution approach. This cycling resulted in an altered capacity of evolved B. Bassiana lineages to grow on different substrates while maintaining the ability to kill insects. Notably, however, there were no significant changes in virulence or speed of outgrowth when comparing the evolved lineages against their unevolved ancestors. These results suggest that fungal growth and sporulation evolved through successive and exclusive use of an insect host as a nutritional resource. We discuss the results in light of biocontrol and provide suggestions to increase fungal virulence.

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.

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.

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.