Showing papers by "Richard A. Humber published in 2001"

Polyketide synthase genes in insect- and nematode-associated fungi.

Abstract: Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (~ 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

Characterization of aphid pathogenic species in the genus Pandora by PCR techniques and digital image analysis

Abstract: Twenty-eight isolates of the insect patho- genic fungus genus Pandora Humber derived from various geographical locations and insect hosts were studied in order to examine both genotypic as well as phenotypic variation. We included sixteen Pandora neoaphidis, two Pandora nouryi, three Pandora kon- doiensis, one Pandora bullata and six Pandora del- phacis isolates in the study. The sizes of ITS bands and RAPD-PCR patterns were used to investigate the genotypic variations. For phenotypic characterization the length and width of the primary conidia were measured and the size and shape of the conidia were quantified in digital images. The size of ITS divided Pandora isolates from Aphididae into the following four groups: (i) all P neoaphidis isolates, (ii) two iso- lates of P kondoiesis including the ex-neotype, (iii) one isolate of P. nouryi and (iv) one isolate of P. kon- doiesis and one isolate of P. nouryi. The analysis of RAPD data fully supported the groupings obtained for the ITS lengths. The RAPD technique was able to distinguish between all isolates except those iso- lated from the same epizootic. It was shown that, among the P. neoaphidis isolates, a correlation be- tween RAPD-PCR profile and geographical origin of the isolate was present while no correlation between host species and profile was seen. The analysis of dig- ital images was here employed for the first time for entomopathogenic fungi. The digital image analysis supported at a quantitative level the original descrip- tions of P. neoaphidis and P delphacis primary conidia as ovoid to ellipsoidal whereas the primary conidia of P. nouryi and P kondoiensis are ovoid. Within these

Effects of two cryopreservation techniques on viability and pathogenicity of entomophthoralean fungi

Abstract: The difficulties in long-term storage of cultures of Entomophthorales create a barrier to working with these entomopathogenic fungi. Relatively few laboratories have access to controlled cooling apparatus and storage in liquid nitrogen, but a simpler, more affordable technique to store cultures at -80°C is available. We compared viability among three entomophthoraleans and pathogenicity for one species for both storage techniques over 10 months. Fluorescent staining for viability demonstrated that there was no statistically significant difference by storage treatment for all three fungi. Although cells of Entomophaga aulicae (Reichardt in Bail) Humber decreased in viability after 8 and 10 months of storage, similar declines were not seen with Entomophaga maimaigaHumber, Shimazu & Soper or Zoophthora radicans (Brefeld) Batko. Bioassays of E. maimaiga against gypsy moth larvae, Lymantria dispar (L.), demonstrated no differences in time to death or percent mortality after 10 months of storage by either method. However, after 10 months, fewer cadavers of larvae injected with cultures stored at -80°C abundantly produced conidia. Our findings sug- gest that for these isolates from three species of Entomophthorales, storage at -80°C after a simple freezing protocol had a minor effect compared with storage at -196°C, but some cultures were more sensitive to prolonged freezing than others. Resume : La difficulte de conserver a long terme les cultures d'Entomophthorales freine l'utilisation de ces champi- gnons entomopathogenes. Relativement peu de laboratoires disposent d'appareils a refroidissement controle et de conservation en azote liquide, mais il existe une methode plus simple et plus accessible de conservation a -80°C. Les auteurs ont compare la viabilite de trois entomophthorales et la pathogenicite d'une espece, soumises aux deux metho- des de conservation pendant 10 mois. La coloration en fluorescence pour la viabilite demontre qu'il n'y a pas de dif- ference significative entre les deux methodes de conservation pour les trois champignons. Bien que la viabilite des cellules de l'Entomophaga aulicae diminue apres 8-10 mois de conservation, on n'observe pas ces declin avec les Entomophaga maimaigaet Zoophthora radicans. Des essais biologiques contre la spongieuse, le Lymantria dispar ,n e montrent aucune difference quant au delai de mortalite ou de pourcentage de mortalite, apres 10 mois de conservation avec chacune des methodes. Cependant, apres 10 mois de conservation, moins de cadavres de larves injectees avec des cultures conservees a -80°C ont produit des conidies en abondance. On suggere que pour ces isolats de ces trois espe- ces d'Entomophthorales, la conservation a -80°C, apres un protocole simple de congelation, a un effet mineur compa- rativement a une conservation a -196°C, mais certaines cultures sont plus sensibles que d'autres a la congelation. Mots cles : Entomophthora aulicae, Entomophaga maimaiga, Zoophthora radicans, conservation, culture in vitro, champignons entomopathogenes. (Traduit par la Redaction) Note 864