Modeling Agrilus planipennis (Coleoptera: Buprestidae) within-tree colonization patterns and development of a subsampling technique.

Emerald ash borer, Agrilus planipennis Fairmaire, an insect native to central Asia, was first detected in southeast Michigan in 2002, and has since killed millions of ash trees (Fraxinus spp.) in North America. The objectives of this study were to 1) determine an optimal sampling location on girdled ash trap trees for detection of A. planipennis larvae based on measurements of tree characteristics, and 2) develop a whole-tree estimation method for extrapolating larval densities from subsampled heights. We conducted sampling at 1-m increments, recording larval presence, height on tree bole, bolt diameter, and bark roughness for 58 infested ash trees. Analyzing height and diameter separately, generalized linear mixed models indicated the probability of A. planipennis detection was maximized at 17.2 cm for diameter and increased linearly as vertical height increased. There was also a positive relationship between intermediate bark roughness and A. planipennis presence. Stepwise regression indicated the optimal bolts for extrapolating whole tree larval densities were, in order of importance, at heights of 1-2 m, 4-5 m, 7-8 m, and 0-1 m. Subsampling with just one or two bolts explained 70% and 86%, respectively, of the variance in A. planipennis densities. Our results can be used by resource managers to improve efficiency of detection efforts and estimate infestations of A. planipennis.

Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops.

Forty-three isolates of the entomopathogenic fungus Beauveria bassiana were screened for virulence against second-instar larvae of diamondback moth (Plutella xylostella) (DBM), European corn borer (Ostrinia nubilalis) (ECB), corn earworm (Helicoverpa zea) (CEW), and fall armyworm (Spodoptera frugiperda) (FAW); 30 of these isolates were tested against beet armyworm (Spodoptera exigua) (BAW). Highly virulent isolates were also tested against black cutworm (Agrotis ipsilon) (BCW), and the most virulent isolate was also assayed against imported cabbage worm (Pieris rapae) (ICW) and cabbage looper (Trichoplusia ni) (CL). All lepidopteran species tested were susceptible to B. bassiana. Corn earworm and beet armyworm were most susceptible to fungal infection, and fall armyworm was least susceptible. Limited testing suggested low susceptibility of black cutworm and cabbage looper. B. bassiana isolate 1200 exhibited virulence against all pest species greater than or equal to commercial strain GHA of B. bassiana currently registered in the USA as BotaniGard. In assays in which larvae were topically sprayed and maintained on the treated substrate for 24h at 100% relative humidity, 6-day (25 degrees C) median lethal concentrations (LC(50)s) of this isolate against CEW, BAW, DBM, FAW, ICW, ECB, CL, and BCW were 4, 5, 7, 11, 12, 98, 125, and 273 conidia/mm(2), respectively. The respective LC(50)s of commercial strain GHA against these pest species were 9, 67, 97, 1213, 29, 1668, 541, and 3504 conidia/mm(2). Use of LC(50) versus median lethal concentration ratios (comparing LC(50)s of each isolate to a "standard" strain) generated similar rankings of isolate virulence. Results from parametric ANOVAs of log LC(50) values followed by Tukey HSD multiple comparisons tests and those from Kruskal-Wallis nonparametric analyses followed by sequential Bonferroni tests for means comparisons were nearly identical.

Vegetative compatibility groups in indigenous and mass-released strains of the entomopathogenic fungus Beauveria bassiana: likelihood of recombination in the field.

Using nitrate non-utilizing (nit) mutants, we determined vegetative compatibility groups (VCG) among strains of Beauveria bassiana representing strains indigenous to North America, isolated from diverse insect hosts, and strains that have been mass released for insect control. Genetic similarity among these strains was analyzed using random amplified polymorphic DNA (RAPD) markers. Our data revealed 23 VCGs among the 34 strains tested, with most of these groups comprised of only a single strain. We also observed a VCG comprised of eight genetically similar strains isolated from Colorado potato beetles (CPB). Co-inoculation studies of CPB larvae with complementary nit mutants from the same or from different VCGs revealed heterokaryosis in four out of five same-VCG pairs, with only 5-17% of the sporulating cadavers generating few parasexual recombinants. In contrast, none of the infected beetles treated with non-compatible pairs generated recombinants. The large number of VCGs observed and the low frequency of in vivo recombination limited to vegetatively compatible strains indicate that this self/non-self recognition system may be an effective barrier preventing genetic exchange between dissimilar strains in the field.

Strain-specific detection of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized amplified region markers.

Field studies on the efficacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to differentiate the non-native strain from indigenous populations. In this study we developed strain-specific molecular markers based on polymerase chain reaction amplification of sequence-characterized amplified regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate density of propagules of a commercial strain of B. bassiana (strain GHA) in field samples. Using random amplified polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-14(0.44), OPA-15(0.44), and OPB-9(0.67), generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F/R(445), OPA15 F/R(441), and OPB9 F/R(677), respectively. All three SCAR primers were highly sensitive, capable of detecting 100pg B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the field.

Factors influencing the infectivity of isolates of Paecilomyces fumosoroseus against diamondback moth, Plutella xylostella.

Paecilomyces fumosoroseus isolate 1576 was isolated from an insect, but is avirulent against larvae of diamondback moth, Plutella xylostella, and several other species. Isolate 1576 grew faster and produced more conidia than isolate 4461 on potato dextrose agar. Pregermination of conidia failed to increase the infectivity of isolate 1576, but the procedure did increase the infectivity of isolates 3682, 4461, and 4482. Isolates 1576 and 4461 were both more infective when moisture was high during incubation of inoculated larvae. Starved Pl. xylostella larvae were more susceptible than fed larvae to isolate 1576 (40 and 10% mortality, respectively), but starved and fed larvae were similar in susceptibility to isolate 4461. These results show that isolate 1576 grows vigorously in aerial culture and is capable of infecting stressed Pl. xylostella larvae. Further tests are needed to characterize its pathogenicity toward its original host or closely related species.

The effect of mode of exposure to Beauveria bassiana on conidia acquisition and host mortality of Colorado potato beetle, Leptinotarsa decemlineata.

The effects of the mode of exposure of second instar Colorado potato beetles to Beauveria bassiana on conidia acquisition and resulting mortality were investigated in laboratory studies. Larvae sprayed directly with a B. bassiana condial suspension, larvae exposed to B. bassiana-treated foliage, and larvae both sprayed and exposed to treated foliage experienced 76, 34, and 77% mortality, respectively. The total number of conidia and the proportion of germinating conidia were measured over time for four sections of the insect body: the ventral surface of the head (consisting mostly of ventral mouth parts), the ventral abdominal surface, the dorsal abdominal surface, and the legs. From observations at 24 and 36 h posttreatment, mean totals of 161.1 conidia per insect were found on sprayed larvae, 256.1 conidia on larvae exposed only to treated foliage, and 408.3 conidia on larvae both sprayed and exposed to treated foliage. On sprayed larvae, the majority of conidia were found on the dorsal abdominal surface, whereas conidia were predominantly found in the ventral abdominal surface and mouth parts on larvae exposed to treated foliage. Between 24 and 36 h postinoculation the percentage of conidia germinating on sprayed larvae increased slightly from 80 to 84%). On the treated foliage, the percentage of germinated conidia on larvae increased from 35% at 24 h to 50% at 36 h posttreatment. Conidia germination on sprayed larvae on treated foliage was 65% at 24 h and 75% at 36 h posttreatment. It is likely that the gradual acquisition of conidia derived from the continuous exposure to B. bassiana inoculum on the foliar surface was responsible for the increase in germination over time on larvae exposed to treated foliage. The density and germination of conidia were observed 0, 4, 8, 12, 16, 20, and 24 h after being sprayed with or dipped in conidia suspensions or exposing insects to contaminated foliage. Conidia germinated twice as fast on sprayed insects as with any other treatment within the first 12 h. This faster germination may be due to the pressure of the sprayer enhancing conidial lodging on cuticular surfaces.

Penetration of cuticle and proliferation in hemolymph by Paecilomyces fumosoroseus isolates that differ in virulence against lepidopteran larvae.

Frequencies of cuticular penetration and speed of proliferation in hemolymph were demonstrated for two isolates of Paecilomyces fumosoroseus that differ in virulence against diamondback moth, Plutella xylostella. Penetrant hyphae of virulent isolate 4461 were visible in larval cuticle cross-sections of diamondback moth and fall armyworm, Spodoptera frugiperda, within 22 h after inoculation. Virtually no penetration was observed for isolate 1576 for up to 52 h after inoculation. Isolate 4461 also proliferated more quickly than isolate 1576 in the hemolymph of fall armyworm when the isolates were injected as blastospores.

Comparison of blastospores of two Paecilomyces fumosoroseus isolates: in vitro traits and virulence when injected into fall armyworm, Spodoptera frugiperda.

Blastospores of two isolates of Paecilomyces fumosoroseus were compared to determine their productivity in vitro and their relative pathogenicity after injection of fall armyworm, Spodoptera frugiperda. Blastospores of less virulent P. fumosoroseus isolate 1576 are smaller than those of isolate 4461, and they germinate and proliferate more slowly in vitro. The pathogenicity of injected blastospores of isolate 1576 against S. frugiperda varies with larval size. In small larvae, percentage mortality was lower among those injected with isolate 1576 than among those injected with isolate 4461. Large larvae were equally susceptible to both isolates. Survival times were higher for isolate 1576 and were higher at lower doses for both isolates. Those larvae injected late in development that ultimately died, regardless of treatment, did not lose weight typical of developing pupae. Injection of large larvae with isolate 4461 resulted in a significantly longer time to pupation among apparently uninfected larvae.

Use of the green fluorescent protein for investigations of Paecilomyces fumosoroseus in insect hosts.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been expressed in a variety of prokaryotic and eukaryotic organisms and has been used extensively as a marker in the study of host-pathogen interactions. We have expressed GFP in the entomopathogenic fungus Paecilomyces fumosoroseus through co-transformation with a vector that confers resistance to glufosinate ammonium. All cell types express GFP and were readily detected by fluorescence microscopy. No correlation was observed between the amount of fluorescence and the pattern of vector integration as observed by Southern analysis. Fluorescent hyphae and conidia were easily distinguished on two insect hosts, the Russian wheat aphid, Diuraphis noxia, and the diamondback moth, Plutella xylostella, and blastospores were also detected in the hemolymph of the diamondback moth. GFP-tagged strains of P. fumosoroseus can be used to study the developmental fate of the fungus within its insect hosts.

Effect of relative humidity on viability of primary conidia of Zoophthora radicans.

Four isolates of the entomopathogenic fungus Zoophthora radicans were compared in a laboratory study to evaluate the effect of relative humidity (RH) on duration of primary conidial viability. Primary conidia were showered onto agar-coated glass microscope slides within an enclosed chamber equilibrated to one of five test RH levels (60, 75, 80, 95, or 100%). Target RH levels were achieved by recirculating air through a glycerin/water solution, of controlled specific gravity, contained in a reservoir within the chamber. Conidial samples of each isolate incubated for 5, 10, 30, 60, 120, 180, or 240 min at each RH were removed and inspected using a technique of simultaneous vital fluorochrome staining to determine percentage conidial viability. At 60% RH, isolates did not differ significantly and average viability dropped to less than 10% within the first 60 min. At 75% RH, viability did not change significantly over 4 h. However, average viabilities at 75% RH differed significantly for the four isolates and ranged from 24 to 63%. At 80% RH, viability differed significantly among isolates and declined slowly over time, remaining above 80% for 2 h and above 50% for 4 h. At 95 and 100% RH, average viability was near 95% and did not vary significantly with time or isolate. These data can be used to assist selection of appropriate isolates for biological control.

Pathogenicity of Paecilomyces fumosoroseus isolates to diamondback moth, Plutella xylostella: correlation with spore size, germination speed, and attachment to cuticle.

Infectivity to larvae of the diamondback moth, Plutella xylostella, was compared among eight Paecilomyces fumosoroseus isolates. Isolate infectivity was assessed for correlation with spore length and germination speed. Four isolates applied to P. xylostella cuticle were also compared for number of spores remaining on the cuticle after washing and for percentage germination after 36 h. Infection of larvae inoculated with the different isolates at an average dosage of 4000 conidia/cm2 ranged from 2 to 47%. The correlation of infectivity with spore length and germination speed in broth was highly significant. Fewer spores of the least infective isolate, ARSEF 1576, attached to larval cuticle compared to spores of the more infective isolates ARSEF 3682, 4461, and 4482 (P < 0.05). After 36 h on larval cuticle, the percentage of spores germinated for isolates 1576 and 3682 was 3 and 95%, respectively. Spores of isolate 1576 were smaller, germinated more slowly, and attached to cuticle in smaller numbers than spores of the more infective isolates. Further research will expand our understanding of the mechanisms of virulence among isolates of P. fumosoroseus.

Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundata.

The spore load of Ascosphaera species spores on larval chalkbrood cadavers and newly emergent adults of the alfalfa leafcutting bee, Megachile rotundata, was determined. The spore content of chalkbrood cadavers ranged from 3 x 10 to 5 x 10. Adults emerging through zero to nine cadavers carried spores on all body parts examined by scanning electron microscopy. Estimates of the total number of spores obtained from a series of adult washes ranged from 9 x 10 to 8 x 10. Some adult males which emerged through no cadavers carried 10 to 10 spores, indicating that nesting materials might also have been contaminated. However, the control of chalkbrood in commercial bee populations may not be accomplished simply by providing clean nesting materials as adults may still emerge through diseased larvae.