Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field populations of Plutella xylostella and, therefore, are likely to interact during the infection process. We have investigated the possible outcomes of these interactions in the laboratory. Using four isolates, two of each fungal species, inter-specific interaction experiments were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoculated directly using sporulating mats of mycelium, both species had the same opportunity to infect and only the relative concentration of conidia of each pathogen species applied was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both species of fungi depended on larval activity and proximity to inoculum. In the Petri dish experiment, the species with the largest concentration of conidia out-competed the other regardless of virulence, and results were similar in the intact plant experiment. The ecological implications for competition or co-existence of these two pathogens in the field are discussed.     

Taxonomic and phylogenetic analysis of the Oomycota mosquito larvae pathogen Crypticola clavulifera

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.     

Host range of Neozygites floridana isolates (Zygomycetes: Entomophthorales) to spider mites

Neozygites floridana (Weiser & Muma) (Zygomycetes: Entomophthorales) has been reported infecting naturally at least 18 species of tetranychids worldwide. However, the host range of N. floridana is unknown. Epizootics caused by this pathogen to tetranychid populations indicate that N. floridana has the potential to be used as a biological control agent. However, the virulence and specificity of species and strains of Neozygites need to be assessed in the laboratory to reveal its potential as a biological control agent. N. floridana isolates are currently been investigated in Brazil as biological control agents against the tomato red mite, Tetranychus evansi Baker & Pritchard, and the two-spotted spider mite, Tetranychus urticae Koch. The pathogenicity of five strains of N. floridana obtained from T. urticae, T. evansi and T. ludeni Zacher was assessed against populations of Mononychellus tanajoa (Bondar), Schizotetranychus sacharum Flechtmann & Baker, Tetranychus abacae Baker & Pritchard and Tetranychus armipenis Flechtmann & Baker, in addition to the species from which the fungus was obtained. Mummified mites were placed on leaf discs of the host plant of each tetranychid to promote fungal sporulation, and after 24 h the mites were transferred to the leaf discs. Contamination, infection and mummification were evaluated daily for seven days after confinement. Each isolate was pathogenic to three or four out of the six spider mite species tested. However, except for isolate ESALQ1421, all isolates caused higher levels of infection and significant mummification only to the tetranychid species from which they were collected. None of the isolates was pathogenic to S. sacharum and only one isolate infected T. abacae. Alternative hosts may be important for N. floridana survival in tropical regions where resting spores are rarely found.     

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between ?12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (?12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (?12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C–40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales)

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.     

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation,oxidative stress,and cell surface alterations

The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.     

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

Biological and molecular characteristics of Beauveria bassiana isolates from California Lygus hesperus (Hemiptera: Miridae) populations

Lygus hesperus is an important pest of many crops grown in the Western US. In addition, other species of Lygus cause damage in other parts of the world. To date, no selective pesticide exists for the control of Lygus spp. and broad spectrum pesticides that also kill natural enemies may lead to secondary pests. Entomopathogenic fungi may offer an alternative to chemical pesticides. Isolates of Beauveria bassiana collected from San Joaquin Valley of California (SJV) L. hesperus populations were screened for their ability to grow at high temperatures and for their ability to infect and kill L. hesperus adults and nymphs under laboratory conditions. No isolate grew at 37 or 35 °C but most isolates were able to grow at 32 °C. In addition, one L. hesperus isolate was more efficacious at higher doses than the commercial isolate. Microsatellite markers were used to determine that selected isolates could be distinguished from other isolates. Preliminary information suggested 82 SJV isolates of B. bassiana were closely related to each other but distantly related to the commercial isolate.     

The Paxillus involutus (Boletales, Paxillaceae) complex in Europe: Genetic diversity and morphological description of the new species Paxillus cuprinus, typification of P. involutus s.s., and synthesis of species boundaries

Paxillus involutus is a model species for ecological or physiological studies of ectomycorrhizal agaricomycetes. Three to six groups or species linked to it have been ecologically and morphologically distinguished. Phylogenetic studies have revealed the existence of four species in Europe: Paxillus ammoniavirescens, Paxillus obscurisporus, P. involutus, and a fourth as yet not described species. We studied 47 collections from 24 French and Italian locations, supplemented with GenBank data, in order to genetically and taxonomically delineate these species. Phylogenetic analyses of three nuclear DNA regions (rDNA internal transcribed spacer (ITS), tef1-α, and gpd) confirmed the four European species. Morphology, culture, and ecology features allowed us to delineate species boundaries and to describe the fourth species we named Paxillus cuprinus since it turns coppery with age. As there is no existing original herbarium specimen for P. involutus, one of our collections was chosen as the epitype. The low genetic diversity found in P. cuprinus correlates with stable morphological traits (basidiome colour, ovoid–amygdaliform spores with an apical constriction) and with ecological preferences (association with Betulaceae in open and temperate areas). In contrast, P. ammoniavirescens is characterized by a high genetic diversity and a high variation of its morphological and ecological features.     

Transmission and infectivity of spores of Burenella dimorpha (Microsporida: Burenellidae)

Burenella dimorpha infects the tropical fire ant, Solenopsis geminata, producing two morphologically distinct types of spores. A binucleate, nonpansporoblast membrane-bounded (NPMB) spore develops in and destroys the hypodermis, rupturing the cuticle in the pupal stage. A uninucleate, pansporoblast membrane-bounded (PMB) spore develops in the fat body. Adult ants cannibalize ruptured pupae but do not ingest spores. Instead, the spores and particulate foods are diverted to the infrabuccal cavity, formed into an infrabuccal pellet, and fed to fourth-instar larvae only. This larval instar is the only stage in the life cycle of S. geminata that is vulnerable to infection. NPMB spores are infective, but PMB spores do not extrude their polar filaments in the larval gut and are expelled in the meconium upon pupation.     

Mass production,infectivity, and field application studies with the entomogenous fungus Paecilomyces farinosus

Hyphal bodies (HB) of the entomogenous fungus Paecilomyces farinosus mass produced in shaken liquid cultures are infective per os and topically to Spodoptera exigua larvae. Pathogenicity of HB was greatest at pH 5.5. It was reduced by exposure for more than one-half hour to temperatures above 30°C. P. farinosus infected, killed, and produced mycelia on the surface of first-instar S. exigua larvae at a temperature—relative humidity combination as low at 15°C and 70% RH. Using two types of microdroplet applicators (droplet in the size range 1–30 and 30–90 μm, respectively) under field conditions, HB were delivered to distances of 76 m from the site of spraying. The number of HB deposited on the leaf surfaces of treated crops caused mortality of 70 to 90% in bioassayed larvae.     

Susceptibility of the filbertworm (Cydia latiferreana, Lepidoptera: Tortricidae) and filbert weevil (Curculio occidentalis, Coleoptera: Curculionidae) to entomopathogenic nematodes

The objective of this study was to determine the susceptibility of the two primary direct insect pests of hazelnuts in Oregon to three species of entomopathogenic nematodes. The entomopathogenic nematodes (Heterorhabditis marelatus Pt. Reyes, Steinernema carpocapsae All and Steinernema kraussei L137) were used in laboratory soil bioassays to determine their virulence against filbertworm, Cydia latiferreana (Walsingham) (Lepidoptera: Tortricidae) and filbert weevil, Curculio occidentalis (Casey) (Coleoptera: Curculionidae). All three nematode species were infective in laboratory bioassays. Infectivity ranged from 73-100% and 23-85% for filbertworm and filbert weevil, respectively. Field results were similar to those found in the laboratory with filbertworm larvae being more susceptible to nematode infection.     

The effects of photoperiod and light intensity on the sporulation of Brazilian and Norwegian isolates of Neozygites floridana

The objective of this study was to determine the effects of light intensity and duration (photoperiod) on the sporulation (discharge of primary conidia) and conidia germination (from non-infective primary conidia to infective capilliconidia) of Neozygites floridana isolates from Tetranychus urticae originating from Norway and Brazil. Two light intensities (40 and 208 μmol m⁻² s⁻¹), three photoperiods (24 h of continuous light (24 h D), 12 h of darkness followed by 12 h of light (12 h D: 12 h L) and 24 h of continuous darkness (24 h D)) and two temperatures (18 °C and 23 °C) were tested. The fungus produced similar amounts of primary conidia and capilliconidia at 12 h D:12 h and 24 h D, indicating that the fungus discharges almost all of its conidia during the first 12 h of darkness. Light had less of an effect on the production of primary conidia than on capilliconidia formation. At 24 h L, capilliconidia formation was significantly lower for all tested light intensities, temperatures and isolates compared to 12 h D:12 h L and 24 h D. At both light intensities, 24 h L resulted in a significantly lower capilliconidia formation for the Norwegian isolate compared to the Brazilian isolate. Our data suggest that, even though 24 h L reduced sporulation, some capilliconidia formation may occur at the low light intensities found on the underside of strawberry leaves during parts of the day as well as the top of a non-shaded strawberry leaf during the dim evening and morning hours in the tropics and during the dim, long summer days in temperate regions.     

Further observations on the ecology of Chthamalus depressus (Poli)

An account is given of the forms of Chthamalus depressus (Poli). An extreme hypobiotic form common in caves is described; it is shown that all forms may be found in very restricted localities (even on a single boulder) according to different micro-habitats. Some biometrical data on the species and regressions of the several variables are given; they indicate that thickness tends to increase with exposure.The effect of exposure on growth-form is considered and compared with similar data on the laying down of the shell in molluscs. Thickening of the shell is believed to be due to withdrawal of the mantle tissue under wave action. The effect of light on the penetration of algae is discussed; animals growing in darkness have no endolithic algae. Erosion is considered to be due to mechanical forces acting on shells much weakened by endolithic algae.The changes on transplantation to Scotland have been observed, and measurements of the growth rate made; growth is slow, but somewhat greater under conditions of total immersion.The reproductive and moulting cycles, fertility, and general behaviour have been determined and the results compared with similar data on C. stellatus (Poli).The results are discussed in relation to the ecology of C. depressus and, in particular, relative to possible competition with C. stellatus.     

Further observations on Coelomomyces (Blastocladiales,Coelomomycetaceae) parasitic in Chironomidae (Diptera) in Marion Lake,British Columbia

A report is given of the infection levels of Coelomomyces beirnei and of Coelomomyces chironomi var. canadense in chironomids in Marion Lake, British Columbia. The former species has a wide host range and infections appear to occur at random.Issued as Canadian Contribution to the International Biological Programme No. 271.     

Degradation of the isoflavone biochanin a by isolates of Nectria haematococca (Fusarium solani)

Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for their virulence on pea plants and their ability to degrade the phytoalexin pisatin were assayed for the catabolism of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxyisoflavone). Eleven isolates catabolized the isoflavone along the pathway: biochanin A → dihydrobiochanin A → 3-(p-methoxyphenyl)-6-hydroxy-γ-pyrone → p-methoxyphenylacetic acid → p-hydroxyphenylacetic acid → 3,4-dihydroxyphenylacetic acid.     

Effect of copper,nutrient nitrogen,and wood-supplement on the production of lignin-modifying enzymes by the white-rot fungus Phlebia radiata

Production of the oxidoreductive lignin-modifying enzymes – lignin and manganese peroxidases (MnPs), and laccase – of the white-rot basidiomycete Phlebia radiata was investigated in semi-solid cultures supplemented with milled grey alder or Norway spruce and charcoal. Concentrations of nutrient nitrogen and Cu-supplement varied also in the cultures. According to extracellular activities, production of both lignin peroxidase (LiP) and MnP was significantly promoted with wood as carbon source, with milled alder (MA) and low nitrogen (LN) resulting with the maximal LiP activities (550 nkat l⁻¹) and noticeable levels of MnP (3 μkat l⁻¹). Activities of LiP and MnP were also elevated on high nitrogen (HN) complex medium when supplemented with spruce and charcoal. Maximal laccase activities (22 and 29 μkat l⁻¹) were obtained in extra high nitrogen (eHN) containing defined and complex media supplemented with 1.5 mM Cu²⁺. However, the nitrogen source, either peptone or ammonium nitrate and asparagine, caused no stimulation on laccase production without Cu-supplement. This is also the first report to demonstrate a new, on high Cu²⁺ amended medium produced extracellular laccase of P. radiata with pI value of 4.9, thereby complementing our previous findings on gene expression, and cloning of a second laccase of this fungus.