Variability in conidial thermotolerance of Metarhizium anisopliae isolates from different geographic origins

Notable variability in thermotolerance was found among conidia of 16 isolates of the insect-pathogenic fungi Metarhizium anisopliae var. anisopliae and one M. anisopliae var. acridum isolated from latitudes 61 degrees N to 54 degrees S. Conidial suspensions were exposed to 40 or 45 degrees C for 2, 4, 8, and 12 h. Most of the isolates tolerated 40 degrees C very well, with relative germination (germination relative to unheated controls) above 90% after 12 h of exposure. Exceptions were three isolates originating from high latitude, viz., ARSEF 2038 (38 degrees N, South Korea), 4295 (54.4 degrees S, Australia), and 5626 (61.2 degrees N, Finland) that had approximately 80% germination. High variability, however, was observed among isolates at 45 degrees C; viz., after 2 h exposure, relative germination was above 80% for six isolates, between 50 and 70% for three isolates, and between 0 and 30% for eight isolates. After 8 and 12 h at 45 degrees C, only two M. anisopliae isolates pathogenic to grasshoppers, viz., ARSEF 324 (latitude 19 degrees S, Australia) and 3609 (15 degrees N, Thailand), had high relative germination (91.6 and 79.4%, respectively, for 8 h exposures; and 90 and 47.1%, respectively, for 12 h). These isolates also were the most tolerant to UV-B radiation [J. Invertebr. Pathol. 78 (2001) 98-108]. The median lethal dose (LD50) for isolate ARSEF 324 was 49.4 and 47.9 degrees C, for 2 and 4 h of exposures, respectively. Exposure of conidia to wet-heat greatly delayed germination of some isolates. In general, isolates from higher latitudes demonstrated greater heat susceptibility than isolates from nearer the equator. Dry conidia tolerated 50 degrees C better than 45 degrees C in aqueous suspension.     

Variability in azygospore production among Entomophaga maimaiga isolates

This study describes in vitro and in vivo azygospore production by nine isolates of Entomophaga maimaiga, a fungal pathogen of the gypsy moth, Lymantria dispar. The three E. maimaiga isolates that consistently produced azygospores in vitro were also strong producers of azygospores in vivo. However, two additional isolates that were strong azygospore producers in vivo did not produce azygospores in vitro. Isolates that produced azygospores in vitro produced both conidia and azygospores more frequently in vivo than isolates not producing azygospores in vitro. In vitro azygospore production varied over time as well as by isolate. After >2 years of cold storage, while three isolates continued in vitro azygospore production, three isolates no longer produced azygospores in vitro.     

Studies on Entomopathogenic Fungi Isolated from Dead Pine Caterpillars, Dendrolimus spectabilis

ABSTRACT ABSTRACT Entomopathogenic fungi isolated from pine caterpillars, as biological control agents were evaluated against the 5th instar larvae of silkworms, bombyx mori . The body weights of silkworm larvae were reduced by entomopathogenic fungi. Virulence of the spores isolated from the insects was more stronger than that of the spores isolated from media. LD₅₀ value of the pathogen was 6,464x10³ spores/ml. Entomopathogenic fungi, Beauveria , isolated from dead pine caterpillars was possible agents for use in microbiological control.     

Foliar quality influences tree-herbivore-parasitoid interactions: effects of elevated CO<Subscript>2</Subscript>, O<Subscript>3</Subscript>, and plant genotype

This study examined the effects of carbon dioxide (CO₂)-, ozone (O₃)-, and genotype-mediated changes in quaking aspen (Populus tremuloides) chemistry on performance of the forest tent caterpillar (Malacosoma disstria) and its dipteran parasitoid (Compsilura concinnata) at the Aspen Free-Air CO₂ Enrichment (FACE) site. Parasitized and non-parasitized forest tent caterpillars were reared on two aspen genotypes under elevated levels of CO₂ and O₃, alone and in combination. Foliage was collected for determination of the chemical composition of leaves fed upon by forest tent caterpillars during the period of endoparasitoid larval development. Elevated CO₂ decreased nitrogen levels but had no effect on concentrations of carbon-based compounds. In contrast, elevated O₃ decreased nitrogen and phenolic glycoside levels, but increased concentrations of starch and condensed tannins. Foliar chemistry also differed between aspen genotypes. CO₂, O₃, genotype, and their interactions altered forest tent caterpillar performance, and differentially so between sexes. In general, enriched CO₂ had little effect on forest tent caterpillar performance under ambient O₃, but reduced performance (for insects on one aspen genotype) under elevated O₃. Conversely, elevated O₃ improved forest tent caterpillar performance under ambient, but not elevated, CO₂. Parasitoid larval survivorship decreased under elevated O₃, depending upon levels of CO₂ and aspen genotype. Additionally, larval performance and masses of mature female parasitoids differed between aspen genotypes. These results suggest that host-parasitoid interactions in forest systems may be altered by atmospheric conditions anticipated for the future, and that the degree of change may be influenced by plant genotype.     

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.     

Fluctuating asymmetry and survival ability in the forest tent caterpillar mothMalacosoma disstria: implications for pest management

Fluctuating asymmetry of the first tarsal segment of the proleg of the forest tent caterpiller mothMalacosoma disstria Hbn. (Lepidoptera: Lasiocampidae) was significantly inversely related to survival ability in the lab. The monitoring of population levels of fluctuating asymmetry could have important implications in pest management of this and other species by providing an indication of the health of a population.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

Selection of Beauveria bassiana isolates to control Alphitobius diaperinus

This paper presents results on isolates of the entomopathogenic fungus Beauveria bassiana for Alphitobius diaperinus (Coleoptera: Tenebrionidae) control. Of the 30 isolates tested, four (CG 71, CG 152, UNIOESTE 4, and UNIOESTE 40) resulted in mortality rates >or= 40% within 10 days. These mortality rates could be considered high because of the resistance of these species to B. bassiana. Tests were conducted using these isolates to estimate LC(50), mortality rate over time, vegetative growth, and conidial production on artificial medium and on insects. Isolates CG 71, CG 152, and UNIOESTE 4 showed the best performance and great potential to be used in an integrated management program in poultry farms to control A. diaperinus. Also, the molecular profiles of 12 isolates were analyzed using the RAPD technique. The high-virulence isolates presented a more homogeneous RAPD pattern than the others. Genetic sequencing of the ITS region was performed for one of the virulent isolates (UNIOESTE 4) and compared with sequences deposited at the NCBI database, confirming its taxonomical position as belonging to B. bassiana Clade A.     

Species confirmation of fungal isolates by molecular analysis

Traditional taxonomy of hyphomycetes has been based on conidial morphology and development. In order to confirm species level for the detection and identification of the entomopathogenic fungus, we analysed the species-specific fingerprints to investigate molecular characteristics within isolates of six species and to resolve morphologically atypical isolates. The extent of fingerprint profile observed by RAPD was sufficient to confirm the species level of all the isolates. The genetic similarity among morphologically identified isolates of each species was considerably higher, allowing us to conclude that all the isolates are of same species. These results establish a molecular framework for further taxonomic, phylogenetic and comparative biological investigations.     

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (=Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2h of weighted UV-B irradiance at 978mWm(-2) (providing a total dose of 7.04kJm(-2)) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0 degrees to 22 degrees latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22 degrees , however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.     

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

Hydrated conidia of Metarhizium anisopliae release a family of metalloproteases

Metarhizium anisopliae spores release isoforms of metalloprotease during hydration over a 4-day incubation period. The isoforms were identified and characterized by using one-dimensional native PAGE (1-DE nPAGE) and one-dimensional SDS non-dissociating (1-DE nSDS-PAGE) zymography. The ability of these isozymes to degrade gelatin varied as revealed by 2-D spot densitometry. 1-DE nPAGE zymography revealed five isoforms of gelatinase from Tween wash of conidia. Where as, one to three activities with different intensities appeared on gel from washing of conidia to incubation in water till day 4. The relative migrations of these activities on 1-DE nPAGE zymograms appeared as fast, medium and slow on gel. The 2-D spot densitometry of zymograms indicated isoforms have different proteolytic activity as quantified by pixel intensities. SDS-PAGE zymography indicated the release of two isozymes of Mr 103 and 12 kDa during Tween treatment of conidia. However, during the first washing step with water and incubation of spores at day 2 and 3, respectively, only 12 kDa protein was evident. Majority of these proteases were inhibited by EDTA, but stimulated by CaCl(2), and MgCl(2). The presence of isozymes in conidia and their release during hydration must have functional significance for fungi and in this case it should provide advantages to M. anisopliae in its saprobic or pathogenic modalities. To our knowledge this is the first report describing release of metalloprotease isozymes from conidia.     

Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT₅₀, without a reduction in LC₅₀. The LT₅₀ of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC₅₀, more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.     

Food consumption by Chilo partellus (Lepidoptera: Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding natural versus artificial diets on mortality and mycosis

Second and third instar Chilo partellus larvae were infected with Beauveria bassiana and Metarhizium anisopliae (both at 1x10(8)conidia/ml) and daily consumption of maize leaves was measured. Infection by the fungi was associated with reduced mean daily food consumption. Reduction in food consumption became evident 3-4 days after treatment with the fungi for second instar larvae and 4-5 days for third instar larvae. Four conidial concentrations, 1x10(5), 1x10(6), 1x10(7), and 1x10(8)conidia/ml, were tested against second instar larvae. Food consumption dropped by 70-85% when the second instar larvae were treated with the fungi at 1x10(8)conidia/ml. Reduction in food consumption by C. partellus larvae infected with B. bassiana and M. anisopliae may offset the slow speed of kill of the fungi. The effect of artificial versus natural diets on mortality and mycoses of second instar larvae treated with the fungi at 1x10(8)conidia/ml was determined. Larvae provided with artificial diet suffered little mortality and mycoses than larvae provided with maize leaves. The LT(50) was longer for larvae provided with artificial diet.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

A comparison of the thermal bleaching responses of the zoanthid Palythoa caribaeorum from three geographically different regions in south Florida

Coral bleaching involves the loss of symbiotic algae (zooxanthellae) from reef corals and other cnidarians during periods of environmental stress, particularly elevated temperature. In this study we compared the thermal bleaching responses of the zoanthid Palythoa caribaeorum from three populations along the southeast coast of Florida. Winter (2002-2003) and summer (2003) samples from three geographically separate sites were experimentally exposed to increased temperatures and the loss of zooxanthellae was measured. Population densities of zooxanthellae were analyzed and their genetic identity determined using PCR-DGGE analysis of the internal transcribed spacer region 2. The results showed that samples of P. caribaeorum from reefs that experienced the smallest range in annual seawater temperature released the most zooxanthellae. Seasonal comparisons revealed that winter samples lost more zooxanthellae than summer samples. P. caribaeorum harbored two genetic types of zooxanthellae, C1 and D1a. Individual colonies contained populations of only C1 or D1a, or combinations of C1 and D1a. However, these genotypic patterns did not relate latitudinal distribution nor to differences in experimental thermal tolerance.