G. Riba

Genetic diversity of Beauveria bassiana and relatedness to host insect range

Thirty-eight strains of the entomopathogenic Beauveria bassiana , isolated from diverse species of Lepidoptera (Pyralidae) or Coleoptera (Curculionidae, Chrysomelidae, and Scolytidae) from various geographical sites, were examined by RFLP and RAPD analyses. Similar groupings were recovered from both approaches and these showed clear relationships between the population structure of B. bassiana and some defined host species. Strains isolated from members of the Pyralidae were recovered as two main groups, one group consisted of all strains isolated from Ostrinia irrespective of their origin. The second group consisted primarily of strains isolated from Diatraea in Cuba. All strains isolated from the curculionid genus Sitona clustered as a single distinct group, separated from strains from other curculionids. In contrast, strains isolated from the pyralid genus Maliarpha , and the coleopteran Chrysomelidae, gave heterogenous patterns and were not recovered as distinct groups. Groups from cluster analysis and non- hierarchic ordination methods were compared and the relative merits of the different grouping strategies are discussed.

Comparative analysis of molecular and biological characteristics of strains of Beauveria brongniartii isolated from insects

Variations within the ribosomal DNA gene of the entomopathogenic filamentous genus Beauveria were examined in 28 strains of B. brongniartii and 2 strains of B. bassiana . These strains were isolated from diverse geographical and biological origins, more especially from the white grub Hoplochelus marginalis . Internal transcribed spacer regions (ITS1 and ITS2) were analysed by polymerase chainreaction amplification and restriction fragment-length polymorphism. The analysis revealed a high degree of polymorphism within B. brongniartii species and allows separation of the strains into seven distinct groups. One of these groups contains specifically all strains isolated from H. marginalis . The genetic separation of these strains is supported by their biological origin, since they are the only strains virulent against H. marginalis . Sequence data from the ITSs confirm the conclusions of PCR-RFLP analysis and allow estimation of the genetic distances between the different groups. Intraspecific variability due to point mutation is 0·70–14·67% in ITS1 and 1·80–16·67% in ITS2. This surprisingly high level of polymorphism is discussed.

28s rDNA group‐I introns: a powerful tool for identifying strains of Beauveria brongniartii

The nuclear ribosomal DNA of the entomopathogenic fungus Beauveria brongniartii is polymorphic in terms of both restriction site and length. Insertions of 350–450 bp long, identified as group‐I introns, were detected in the 28s rDNA. A panel of 47 strains of B. brongniartii, two B. bassiana and one Metarhizium anisopliae of various geographical and biological origins were found to contain 14 variant forms of intron differing in size and restriction pattern, at four different positions. Twelve types of ribosomal large subunit were defined on the basis of variant distribution and compared with strain clustering based on internal transcribed spacers analysis. There was a correlation between the characteristic introns and isolates collected from the sugar cane pest Hoplochelus marginalis. Primers for polymerase chain reaction amplification were chosen from these variants, and used to develop a specific method for detecting strains pathogenic towards Hoplochelus.

Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus, Nomuraea rileyi

Abstract High levels of both endo - and exo -chitinase activity were detected in two virulent isolates but not in one avirulent isolate of Nomuraea rileyi . The greatest difference in chitinolytic activity occurred at germination when endo -chitinase activity in the virulent isolates was 10 to 17 times higher than that in the avirulent isolate, and exo -chitinase activity of the virulent isolates was 15 to 18 times higher than that of the avirulent isolate. Both endo - and exo -chitinase activity were simultaneously expressed in all isolates.

Alterations of insect epicuticular hydrocarbons during infection with Beauveria bassiana or B. brongniartii

Abstract Scanning electron microscopy allowed us to distinguish the successive phases induced on the integument by virulent and nonvirulent strains. By chemical analyses, the epicuticular hydrocarbons alterations were observed on Ostrinia nubilalis and Melolontha melolontha larval integument. More precisely, only within the first 6 hr or so, even before the spore germination, several monomethylalkanes completely disappeared, even if the strain was not able to specifically penetrate in the host. Finally, between 32 and 51% of the cuticular hydrocarbons of M. melolontha and 77 and 86% of those of O. nubilalis might be affected 96 hr after a cuticular application of conidia of Beauveria bassiana or B. brongniartii.

Phylogenetic relationships within the genus Metarhizium based on 28S rRNA sequences and isozyme comparison

A ribosomal RNA sequencing technique was used to sequence partially two rapidly evolving domains of the 28S-like rRNA molecule from 42 isolates of Metarhizium . Phylogenetic analysis underlined the separation between M. flavoviride and M. anisopliae and the distinction of M. anisopliae var. majus strains was confirmed. The divergence of two so-called M. anisopliae strains from New Zealand was also demonstrated. Secondly, starch gel electrophoresis was used to identify isoenzyme polymorphism among a limited number of strains. Seven enzyme systems were analyzed: MDH, PGD, GPDH, G6PDH, GOT, PGM and α-esterases. Phylogenetic relationship studies were performed by parsimony analysis. The isoenzyme polymorphism data confirmed results of rRNA sequence analysis.

A glycoprotein highly toxic for Galleria mellonella larvae secreted by the entomopathogenic fungus Beauveria sulfurescens

The culture filtrate of the hyphomycete fungus Beauveria sulfurescens contains several types of toxin. They cause different symptoms when injected into the hemocoel of Galleria mellonella larvae and display different chromatographic properties. One of these toxins was partially purified by a three-step fractionation procedure. The toxic fraction contained only 0.075% of the protein of the crude extract, indicating that the toxic component was present at very low concentration in the culture filtrate of B. sulfurescens .The apparent molecular weight of the toxin was between 10 and 29 × 10 4 . The toxin is a N-linked glycoprotein with oligomannosidic-type sugar chains. It displayed no proteolytic activity and appeared to be extremely potent on G. mellonella larvae: its LD 50 was less than 10 μg/kg of larvae. The injection of the toxin into larvae was accompanied by variable insect cuticle melanization.

Comparative studies of eleven isolates of the fungal entomopathogen Tolypocladium cylindrosporum and two isolates of Tolypocladium extinguens

Abstract The virulence of 11 isolates of Tolypocladium cylindrosporum and 2 isolates of Tolypocladium extinguens was evaluated against mosquito larvae. Second-instar Aedes aegypti larvae were more susceptible to infection by blastospores than were second-instar Anopheles stephensi larvae. Only the T. extinguens isolates were not infectious for A. aegypti or A. stephensi. Nine of the remaining strains killed 100% of larvae at the highest dose tested (106 blastospores/ml). In vitro sporulation and conidiospore size were also studied. Significant differences were observed for both characteristics. Characterization of strains according to in vitro sporulation and virulence made it possible to select the most promising strains for future laboratory and field tests against mosquitoes.

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

Abstract A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.

Infection of Aedes albopictus by Tolypocladium cylindrosporum

The infection process of Tolypocladium cylindrosporum in Aedes albopictus is discussed. The integument is a common site of infection. Spores of T. cylindrosporum are able to adhere to the exoskeleton and penetrate it. During its early stages of development the fungus is always surrounded by a thick bacterial muff. However these bacteria did not invade the host, and no bacterial cell was observed on the intact cuticle. Conidia filtered by larvae rapidly filled the gut and most of them were hydrolyzed in the midgut. Even if germination occurred at a low level in the digestive tract, the peritrophic membrane and the mesenteral cells were not penetrated by fungus.