Intraspecific variation and host specificity of Entomophthora muscae sensu stricto isolates revealed by random amplified polymorphic DNA,universal primed PCR,PCR-restriction fragment length polymorphism,and conidial morphology

The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily.     

Entomophthora muscae resting spore formation in vivo in the host Delia radicum

The formation in vivo of Entomophthora muscae resting spores was investigated in the host, Delia radicum (cabbage root fly), by analysis of field data on the seasonal occurrence of E. muscae resting spores over 4 years. E. muscae resting spores in D. radicum were spherical with an average diameter of 39.4 microm, and the average numbers produced were estimated at 5.7 x 10(4) resting spores/female cadaver. Resting spores were found only after midsummer in D. radicum and almost exclusively in females. The proportion of infected females with resting spores was negatively correlated with average weekly day length after midsummer. We did not detect any significant year effect; thus, the results support the hypothesis that the photoperiod is the most important abiotic factor controlling E. muscae resting spore formation in D. radicum.     

Entomophthora species with E. muscae-like primary spores on two new insect orders, Coleoptera and Hymenoptera

Adults of Cantharis livlda (Coleoptera: Cantharidae) and of Torymus druparum (Hymenoptera: Torymidae) were found naturally infected by fungi from the Entomophthorales in Denmark. The morphology of the primary spores of the two fungi clearly showed that they belong to the genus Entomophthora s.str. No species from this genus has been reported so far from these insect orders. With respect to spore size and number of nuclei per spore, the fungi fall within the range of species from the E. muscae complex, known only from Diptera. A transfer of the fungus from T. druparum to Psila rosae (Diptera: Psilidae) was however possible. The findings thus confirm a significant widening of the host range of species within the E. muscae complex.     

Entomopathogenic fungi in flies associated with pastured cattle in Denmark

Cattle flies, including Musca autumnalis, Haematobia irritans, and Hydrotaea irritans, are pests of pastured cattle. A 2-year study of the natural occurrence of entomopathogenic fungi in adult cattle flies and other flies associated with pastures showed that the four species included in the Entomophthora muscae species complex (E. muscae sensu lato) caused high infection levels in several species of flies. However, only a few specimens of cattle flies were infected by E. muscae sensu stricto despite the fact that cattle flies were observed to perch on spear thistles, which acted as transmission site for all four Entomophthora species. Transmission experiments with E. muscae s.l. supported the field data. Of the two species considered host specific, E. syrphi caused substantial infection in a muscid, and E. scatophagae likewise could be transmitted to a muscid. This emphasizes the need for a revision of the two species. Low prevalences were recorded of another entomophthoralean, Furia americana, and of the hyphomycetes Beauveria bassiana and Verticillium lecanii.     

Natural host range and prevalence of the genus strongwellsea (Zygomycota: entomophthorales) in denmark

The natural occurrence and host range of species of the insect pathogenic fungal genus Strongwellsea [Zygomycota: Entomophthorales] were studied by extensive sampling and examination of adult Diptera [Cyclorrhapha]. The host range for Strongwellsea spp. was significantly enlarged. Three families were documented as new hosts: Muscidae (three species), Calliphoridae (one species), and Sarcophagidae (one species). Further, within the family Anthomyiidae six new host species were recorded and three new host species were documented in the Fanniidae. Strongwellsea castrans was identified as the pathogen in the Anthomyiids, while records from Fanniidae belonged to S. magna. The records of S. magna were the first outside the type locality (California). Primary conidia morphology indicated that muscid and calliphorid species were infected by three undescribed species of Strongwellsea. For the sarcophagid fly, no conidia were encountered, so the Strongwellsea species could not be identified. The tested sampling methods had each different advantages. Sweep netting and diagnosis in situ gave the best opportunity to sample a high number of infected dipterans per time unit spend, while sweep netting followed by incubation in the laboratory was the only method for the documentation of resting spores. The prevalence of S. castrans in the cabbage root fly Delia radicum was obtained by two methods: Samples collected by sweep net and incubated and water trap samples. Water trap captures gave higher prevalences of conidial infections than sweep-net captures. Measured prevalences of Strongwellsea spp. infections are therefore highly dependent on sampling method. The occurrence of resting spores of S. castrans in D. radicum was almost exclusively restricted to females and varied during the season. In samples from 1988 through 1993, no infected females in June contained resting spores, while 43.0% of the S. castrans-infected females from samples in August contained resting spores. During September and October, a decreasing proportion of S. castrans-infected D. radicum contained resting spores. The results document that species from the genus Strongwellsea are common fungal pathogens of adult flies from different families, occasionally with high prevalences. It also appears that the two described species of Strongwellsea, S. castrans and S. magna, have a range of dipterous host species that may always belong to a single family, Anthomyiidae and Fanniidae, respectively. Our data shows also that the host family Muscidae may be exploited by two new species of Strongwellsea. Copyright 1999 Academic Press.     

Entomophthora infecting wheat bulb fly at Rothamsted, Hertfordshire, 1967–71

Entomophthora dipterigena, E. hylemyiae and, most commonly, E. muscae infected wheat bulb flies at Harpenden, Hertfordshire, between 1967 and 1971. The mean annual percentages of infected flies caught were 19, 0, 1, 29 and 16 respectively. These showed an increase with increasing host density. In 1970, two-thirds of the female flies were killed by E. muscae before they laid any eggs.
Conidiophores developed in most flies infected with E. muscae during the 2 weeks after peak emergence of flies. Subsequently, resting spores developed in a progressively increasing proportion of infected flies. Resting spores developed in only six of 130 infected males compared with 103 of 244 infected females in 1970 and 1971. The physiological age of infected flies probably determined whether conidiophores or resting spores developed.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

MORPHOLOGY AND THERMAL DEATH POINT OF OLPIDIUM BRASSICAE

The morphology of single-sporangial isolates of lettuce, tomato, mustard, and oat Olpidium brassicae (Wor.) Dang. growing in their respective hosts as well as in cowpea were compared in situ and after extraction from the roots. The sporangia, zoospores, and resting spores of all isolates were within the established limits of the species. Single exit tubes or pores predominated which means that these isolates should not be transferred to the genus Pleotrachelus. A satisfactory assay for the presence of resting spores was developed by air-drying of the roots for a week or longer. This treatment killed zoospores and vegetative sporangia, but not resting spores. Factors affecting resting spore formation were investigated unsuccessfully. The thermal death point of zoospores of mustard isolates that did not form resting spores was between 40 and 45 C for 10 min.     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999), using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16-100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.     

Echinococcus granulosus from Mexican pigs is the same strain as that in Polish pigs

Samples of Echinococcus granulosus from seven pigs from Mexico were compared with isolates of the parasite from pigs in Poland and representative strains and species of Echinococcus. Isolates from pigs in Mexico were found to be genetically identical to E. granulosus from Polish pigs and distinct from other major genotypes by sequencing part of the mitochondrial cytochrome c oxidase I (COI) mtDNA locus, restriction fragment length polymorphism (RFLP) of the polymerase chain reaction (PCR) amplified rDNA internal transcribed spacer (ITS) 1 using five different enzymes, and random amplified polymorphic DNA (RAPD) analysis. These results were complemented by data on hook morphology and together strengthen the view that Echinococcus maintained in a cycle involving pigs and dogs is a distinct strain that is conserved genetically in different geographical areas. The present study supports the close relationship of the cervid, camel and pig strains and raises the question of their taxonomic status.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Observations on chytridiaceous parasites of Phanerogams

Summary A reexamination of Physoderma maculare Wallroth the type species of the genus, on Alisma, confirms Clinton's (1902) account of the production of an epibiotic stage from resting spore zoospores. The latter on mature host tissue may also give rise to the endobiotic stage which bears resting spores. On seedlings, however, resting spore zoospores produce only epibiotic sporangia. The fungus could not successfully infect Sagittaria, a closely related host.Contribution No. 1152     

Laboratory bioassays of entomopathogenic fungi for control of Delia radicum (L.) larvae

Laboratory soil bioassays were performed at economic field rates for in-furrow (3.85 x 10(6)spores/g dry soil) and broadcast (3.85 x 10(5)spores/g dry soil) applications with three isolates of Metarhizium anisopliae (F52, ATCC62176, and ARSEF5520) and one isolate of Beauveria bassiana (GHA). All isolates tested were infective to second instar Delia radicum (L.). The conditionally registered M. anisopliae isolate (F52) performed best killing an average of 85 and 72% of D. radicum larvae at the high and low concentration, respectively. The mean LC50 and LC95 of F52 against second instar D. radicum was 2.7 x 10(6) and 1.8 x 10(8)spores/g dry soil, respectively. The use of F52 in an integrated management program is discussed.     

Species recognition, geographic distribution and host-pathogen relationships: a case study in a group of lignicolous basidiomycetes, Phellinus s.l

Morphological, phylogenetic (sequencing of the ribosomal ITS region) and, if applicable, biological (pairings of single-spore testers) species recognition have been used to resolve relationships among 69 collections belonging to the Hymenochaetales genera Phellinus s.str. and Fomitiporia. The isolates originate from a variety of host plants in Europe, North America and Asia. Separate application of recognition modes led to differing results concerning the number of species, geographic distribution and host range. Sole application of morphological criteria was of limited value, especially in taxa exhibiting a wide distribution, both in terms of geographic origin and ecological niche. Relationships of putatively con-specific collections originating from different continents preferably should be resolved by using an integrative approach. In this study, application of a strict morphological approach led to the recognition of seven species. When using molecular and pairing test data, at least 12 species were detectable. Two of them, F. hesleri and F. polymorpha, are described as new. The number of Phellinus s.str. and Fomitiporia species supposed to have Northern Hemispheric or cosmopolitan distribution, when morphological characters are applied for species recognition, has been reduced significantly. As firm tendencies within morphological species, genetic divergence was more distinct in uniparental than in biparental taxa. In the latter, a strong correlation was observed between phylogenetic and biological species recognition. Overall length of the ribosomal ITS region clearly separated Phellinus s.str. and Fomitiporia but was of limited value as a diagnostic tool at species level. The level of innerspecific morphological plasticity of fruit bodies differs widely between even closely related species, suggesting that morphological transitions occur quite frequently in this fungal group. Considerable instability of the reproduction mode was evident in strains belonging to Phellinus tremulae and among closely related species of Fomitiporia.     

Relationship between physiochemical properties, aggregation and u.v. inactivation of isolated indigenous spores in water

AIMS: The objective of the study was to compare ultraviolet (u.v.) inactivation kinetics of indigenous aerobic spores in surface water with their laboratory-cultured spore isolates and to investigate the relationship between physicochemical characteristics and u.v. inactivation kinetics of spore isolates. METHODS AND RESULTS: Lake water samples were analysed for the presence of indigenous aerobic spores. Different bacterial isolates from the heterogeneous indigenous population were genetically characterized, resporulated and examined for hydrophobicity, surface charge, particle size distribution and survival at different u.v. 254 nm fluence levels. Cultured isolated spores exhibited a three-stage inactivation curve consisting of shoulder, first order and tailing regions whereas indigenous spores exhibited only one stage of linear kinetics. Hydrophobicity of the Bacillus spore isolates was inversely related to the extent of u.v. inactivation before tailing occurred. CONCLUSIONS: Tailing in the u.v. inactivation curves results from aggregation of a portion of the spore population because of hydrophobic interactions, supporting the link between aggregation of spores, hydrophobicity and u.v. inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of the link between spore physicochemical parameters and u.v. disinfection performance furthers the understanding of factors that affect inactivation of microbes in natural waters supplied to drinking water treatment plants.     

Morphological and molecular analyses in Scleroderma species associated with some Caesalpinioid legumes, Dipterocarpaceae and Phyllanthaceae trees in southern Burkina Faso

A combination of morphotypes, polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses and internal transcribed spacer (ITS) sequencing was used to investigate Scleroderma species that were collected from woodlands in Burkina Faso. We harvested 52 specimens from 20 sites during rainy seasons between 1997 and 2000. According to their morphological features, these specimens were initially characterised, and we then identified six species of Scleroderma. Two of the species were clearly determined as Scleroderma dictyosporum Pat. and S. verrucosum Pers. The four remaining species were characteristically described as (1) displaying big spores with spines up to 2 μm (Scleroderma sp1), (2) producing spores without ornamentation (Scleroderma sp2), (3) spores with very small spines (Scleroderma sp3) and (4) with yellow sporocarps and sub-spherical spores (Scleroderma sp4). The specimens were then analysed using PCR/RFLP of the intergenic regions of rDNA, ITS and IGS1 and ITS sequencing. The restriction fragments obtained with two endonucleases, HinfI and MboI on ITS and IGS1 regions, showed that some isolates of S. dictyosporum had the same patterns as isolates and basidiocarps of Scleroderma sp4 (IR265, IR408, SP4-2903). Isolates of Scleroderma sp3 (IR252) had common restriction fragments as isolates of S. verrucosum (IR500, IR600). Intraspecific differences were observed in the two previously determined species, as well as in Scleroderma sp2. The ITS sequencing and phylogenetic analyses showed that the ribotypes identified by PCR/RFLP within these species might be phylogenetic species. Combining these molecular results allowed regrouping the six morphological species in three sets of cryptic species: a first set with two species including S. dictyosporum Pat., a second set with four species, including both S. verrucosum Pers. and Scleroderma sp1 and a third set with two species, including Scleroderma sp2. These investigations and the combined morphological and molecular analyses used to sort out species paved the way for identifying larger populations of Scleroderma species in Burkina Faso and other tropical zones.     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999),using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16–100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.This revised version was published online in October 2005 with corrections to the Cover Date.     

Spore germination and isozyme patterns in the apomictic fern Cyrtotnium falcatum

Progeny from a plant of the apomictic species Cyrtomium falcatum were examined by protein electrophoresis. These progeny were produced from spores originating in sporangia with differing spore numbers. Analysis of zymograms indicates that variant banding patterns were exhibited in 16.1% of the progeny sampled. These anomalous phenotypes suggest that some apomictically reproducing plants can produce genetically variable progeny.