TY - BOOK
T1 - Fungal virulence in host-pathogen interactions
T2 - Disentangling the infection process in an environmental context
AU - Maistrou, Sevasti
PY - 2019
Y1 - 2019
N2 - Entomopathogenic fungi are among the most widespread natural enemies of insects. The system of entomopathogenic fungus-insect is very appealing for studies of host pathogen interactions. Both organisms share millions of years of co-evolution and additionally they have relatively short life cycles; thus much information about the virulence of the pathogen and the immune reactions of the host are now clarified. Understanding the interactions of the fungal pathogen-insect system is fundamental for the success of biological control and furthermore the study of these interactions provides us with more insight knowledge on fungal virulence evolution and immunology. Fungal virulence, the disease producing power of the pathogen on the host, defines significantly the outcome of these interactions. It is a quantitative trait and as such virulence will be variable, depending on the pathogen, the host and the abiotic and biotic environmental conditions. Although many aspects about virulence, fungal Virulence Factors and the immune response of the host against fungal pathogens have been elucidated, several elements of the host-pathogen interactions remain elusive. Particularly, elements of the insect immune responses, the mechanism underlying the expression of important Virulence Factors by the fungus during the infection progression or the interplay between environmental and virulence traits, for example the importance of high or low virulence for adaptation of the fungal pathogen to its natural environment are not yet fully clarified. The aim of this PhD project was to study the virulence of the entomopathogenic fungus Beauveria bassiana in an ecologically relevant way; how virulence is relevant for the ecological fitness of the fungus, how coinfections by different strains of Beauveria spp. alter virulence and elucidate the importance of an antifungal peptide produced by the host for the virulence of B. bassiana. To make the studies ecologically relevant, all Beauveria strains included in the present study originated from a population of Beauveria spp. in an agricultural field and its neighboring hedgerow in Denmark with known abundance and distribution patterns within the habitat. Virulence is a central trait of the pathogenic phase of the entomopathogenic fungal life cycle and it seems feasible to assume that high virulence would benefit the fungus strain or genotype to maintain its population size during its free living stage. However, major variations in virulence among different Beauveria strains are well documented in the literature and such variations were also found among the Beauveria strains studied here. The specific population was found to include Beauveria strains with different levels of virulence against adults of the beetle Tenebrio molitor, measured as host mortality and sporulation on the cadaver at the final stage of the infection process, germination speed and relative radial growth in vitro. Interestingly, these differences in virulence traits could be linked to the ecological success of the strains based on abundance and distribution within the habitat. The Beauveria strains of the two most abundant clades in the ecosystem showed to be most superiorly adapted in selected virulence traits expressed as the effective doses necessary for induction of similar host mortality, the sporulation frequency for production of new infective propagules and the time needed for conidia germination, which all could provide a likely reason for the domination of the phylogenetic clade represented by these strains which was the only one clade represented in the agricultural field habitat. However, variations in virulence didnot appear to be important for strains representing clades with low abundance and scarce distribution in the agroecosystem. Next, it was hypothesized that since these Beauveria strains co-exist in sympatry it is likely that more than one strains could infect the same host individual simultaneously, potentially affecting virulence compared to an infection caused by only one strain. Moreover, the genetic relatedness between the co-infecting strains could have an impact on the resulting virulence. Mixed infections are considered to be common in nature and due to the limited insect resources represented by the host, co-infections may be expected to result in an increase in virulence. However, it has been hypothesized that closely related strains or spiteful behavior can have the opposite effect leading to reduced virulence. Indeed, based on the present results, intra-specific strains of Beauveria bassiana developed competing relationships leading to reduced virulence, when co-infections of distantly related strains were considered, even when hosts were exposed to at very small dosages. Closelyrelated strains, on the other hand, seemed to have an additive effect when combined suggesting no interactions between the strains. The mechanism behind the competing relations is yet unknown and in vitro growth of combinations of the strains didn’t reveal any indications of direct interference competition on nutrient media. Finally, the focus on the third study was shifted to the insect host and its immune responses towards B. bassiana infection. The beetle Tenebrio molitor expresses an antifungal peptide, tenecin3, among various antimicrobial peptides which are part of the insect’s innate immune defenses. However, tenecin3 is constitutively expressed, and constitutive defenses are expected to provide a fitness benefit only when the pathogen they target is common and widespread in the environment of the host. It was therefore expected that the peptide tenecin3 should be important for the survival of T. molitor against fungal entomopathogens. Antifungal peptides of insects are in general understudied, especially when it comes to their importance against entomopathogenic fungi. Through an RNAi approach, where gene expression for the peptide was silenced in adult beetles, the insects were infected by a strain of B. bassiana by topical application of conidia suspensions. The results showed that tenecin3 has a fungicidal effect in the host since the pathogen development inside the hemolymph was delayed in control beetles compared to gene silenced beetles. When tenecin 3 was studied against B. bassiana in vitro the peptide also slowed down the growth of the fungus in a dose-dependent manner. The peptide tenecin3 therefore plays an important role for T. molitor when infected by the fungus. The present PhD research has revealed new information on the plasticity of virulence traits within populations of the entomopathogenic fungal genus Beauveria. Fungal strains representing the most widespread clade within the studied agroecosystem exhibited a relatively high level of virulence for all traits measured, indicating that high virulence is an important trait for successful habitat colonization. However, adaptations to endure abiotic factors in the ecosystem remain also important. The virulence of the strains is, however, affected by co-infections by other strains which could facilitate the occurrence of low virulent strains within the ecosystem. It is new knowledge that an anti-fungal peptide can affect disease progression of B. bassiana in T. molitor which improves the understanding of host-pathogen interactions within insect pathology. Overall, the presented research has contributed in clarifying fundamental aspects of the ecological adaptations of entomopathogenic fungi, which could be implemented in development of biological control strategies.
AB - Entomopathogenic fungi are among the most widespread natural enemies of insects. The system of entomopathogenic fungus-insect is very appealing for studies of host pathogen interactions. Both organisms share millions of years of co-evolution and additionally they have relatively short life cycles; thus much information about the virulence of the pathogen and the immune reactions of the host are now clarified. Understanding the interactions of the fungal pathogen-insect system is fundamental for the success of biological control and furthermore the study of these interactions provides us with more insight knowledge on fungal virulence evolution and immunology. Fungal virulence, the disease producing power of the pathogen on the host, defines significantly the outcome of these interactions. It is a quantitative trait and as such virulence will be variable, depending on the pathogen, the host and the abiotic and biotic environmental conditions. Although many aspects about virulence, fungal Virulence Factors and the immune response of the host against fungal pathogens have been elucidated, several elements of the host-pathogen interactions remain elusive. Particularly, elements of the insect immune responses, the mechanism underlying the expression of important Virulence Factors by the fungus during the infection progression or the interplay between environmental and virulence traits, for example the importance of high or low virulence for adaptation of the fungal pathogen to its natural environment are not yet fully clarified. The aim of this PhD project was to study the virulence of the entomopathogenic fungus Beauveria bassiana in an ecologically relevant way; how virulence is relevant for the ecological fitness of the fungus, how coinfections by different strains of Beauveria spp. alter virulence and elucidate the importance of an antifungal peptide produced by the host for the virulence of B. bassiana. To make the studies ecologically relevant, all Beauveria strains included in the present study originated from a population of Beauveria spp. in an agricultural field and its neighboring hedgerow in Denmark with known abundance and distribution patterns within the habitat. Virulence is a central trait of the pathogenic phase of the entomopathogenic fungal life cycle and it seems feasible to assume that high virulence would benefit the fungus strain or genotype to maintain its population size during its free living stage. However, major variations in virulence among different Beauveria strains are well documented in the literature and such variations were also found among the Beauveria strains studied here. The specific population was found to include Beauveria strains with different levels of virulence against adults of the beetle Tenebrio molitor, measured as host mortality and sporulation on the cadaver at the final stage of the infection process, germination speed and relative radial growth in vitro. Interestingly, these differences in virulence traits could be linked to the ecological success of the strains based on abundance and distribution within the habitat. The Beauveria strains of the two most abundant clades in the ecosystem showed to be most superiorly adapted in selected virulence traits expressed as the effective doses necessary for induction of similar host mortality, the sporulation frequency for production of new infective propagules and the time needed for conidia germination, which all could provide a likely reason for the domination of the phylogenetic clade represented by these strains which was the only one clade represented in the agricultural field habitat. However, variations in virulence didnot appear to be important for strains representing clades with low abundance and scarce distribution in the agroecosystem. Next, it was hypothesized that since these Beauveria strains co-exist in sympatry it is likely that more than one strains could infect the same host individual simultaneously, potentially affecting virulence compared to an infection caused by only one strain. Moreover, the genetic relatedness between the co-infecting strains could have an impact on the resulting virulence. Mixed infections are considered to be common in nature and due to the limited insect resources represented by the host, co-infections may be expected to result in an increase in virulence. However, it has been hypothesized that closely related strains or spiteful behavior can have the opposite effect leading to reduced virulence. Indeed, based on the present results, intra-specific strains of Beauveria bassiana developed competing relationships leading to reduced virulence, when co-infections of distantly related strains were considered, even when hosts were exposed to at very small dosages. Closelyrelated strains, on the other hand, seemed to have an additive effect when combined suggesting no interactions between the strains. The mechanism behind the competing relations is yet unknown and in vitro growth of combinations of the strains didn’t reveal any indications of direct interference competition on nutrient media. Finally, the focus on the third study was shifted to the insect host and its immune responses towards B. bassiana infection. The beetle Tenebrio molitor expresses an antifungal peptide, tenecin3, among various antimicrobial peptides which are part of the insect’s innate immune defenses. However, tenecin3 is constitutively expressed, and constitutive defenses are expected to provide a fitness benefit only when the pathogen they target is common and widespread in the environment of the host. It was therefore expected that the peptide tenecin3 should be important for the survival of T. molitor against fungal entomopathogens. Antifungal peptides of insects are in general understudied, especially when it comes to their importance against entomopathogenic fungi. Through an RNAi approach, where gene expression for the peptide was silenced in adult beetles, the insects were infected by a strain of B. bassiana by topical application of conidia suspensions. The results showed that tenecin3 has a fungicidal effect in the host since the pathogen development inside the hemolymph was delayed in control beetles compared to gene silenced beetles. When tenecin 3 was studied against B. bassiana in vitro the peptide also slowed down the growth of the fungus in a dose-dependent manner. The peptide tenecin3 therefore plays an important role for T. molitor when infected by the fungus. The present PhD research has revealed new information on the plasticity of virulence traits within populations of the entomopathogenic fungal genus Beauveria. Fungal strains representing the most widespread clade within the studied agroecosystem exhibited a relatively high level of virulence for all traits measured, indicating that high virulence is an important trait for successful habitat colonization. However, adaptations to endure abiotic factors in the ecosystem remain also important. The virulence of the strains is, however, affected by co-infections by other strains which could facilitate the occurrence of low virulent strains within the ecosystem. It is new knowledge that an anti-fungal peptide can affect disease progression of B. bassiana in T. molitor which improves the understanding of host-pathogen interactions within insect pathology. Overall, the presented research has contributed in clarifying fundamental aspects of the ecological adaptations of entomopathogenic fungi, which could be implemented in development of biological control strategies.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01012003788&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - Fungal virulence in host-pathogen interactions
PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
ER -