Genetic diversity among naturally-occurring strains of Beauveria bassiana associated with the introduced coffee berry borer, Hypothenemus hampei, (Coleoptera: Curculionidae) on Hawai'i Island.

The coffee berry borer (CBB), Hypothenemus hampei, is considered the most important insect pest of coffee worldwide. CBB was discovered on Hawai'i Island in 2010 and soon thereafter on the islands of O'ahu (2014) and Maui (2016). As part of an areawide effort to manage CBB in Hawai'i, we conducted a survey of naturally-occurring Beauveria associated with the beetle to complement field efficacy studies of the commercial B. bassiana strain GHA. Sampling of CBB from coffee farms or unmanaged sites in various districts on the islands of Hawai'i and O'ahu, and also from Puerto Rico, resulted in >1800 Beauveria isolates. These were initially characterized using colony morphology to differentiate strain GHA, registered for use in Hawai'i, from indigenous congenerics. A total of 114 isolates representative of these indigenous morphotypes were selected for further characterization. Sequencing of the intergenic regions B locus and EFutr identified all as Beauveria bassiana sensu stricto. Sixteen haplotypes were observed, with one more common haplotype present in 12 of 16 sites sampled on Hawai'i Island. This B locus-EFutr haplotype, designated Bb1, was the only haplotype observed in 2016 epizootics on two high-elevation coffee farms on Hawai'i Island with no history of GHA application. Many of the haplotypes showed genetic similarity to those collected from CBB from other countries, including Brazil, Columbia, Nicaragua, and Kenya, but a few were identical to those from other insect species collected in Hawai'i before 2010. This diversity suggests a mixed lineage among B. bassiana strains associated with CBB in the three Hawaiian islands.

Biological Control Outcomes Using the Generalist Aphid Predator Aphidoletes aphidimyza under Multi-Prey Conditions.

The aphidophagous midge Aphidoletes aphidimyza (Diptera: Cecidomyiidae) is used in biological control programs against aphids in many crops. Short-term trials with this natural enemy demonstrated that that females prefer to oviposit among aphids colonizing the new growth of plants, leading to differential attack rates for aphid species that differ in their within-plant distributions. Thus, we hypothesized that biological control efficacy could be compromised when more than one aphid species is present. We further hypothesized that control outcomes may be different at different crop stages if aphid species shift their preferred feeding locations. Here, we used greenhouse trials to determine biological control outcomes using A. aphidimyza under multi-prey conditions and at different crop stages. At all plant stages, aphid species had a significant effect on the number of predator eggs laid. More eggs were found on M. persicae versus A. solani-infested plants, since M. persicae consistently colonized plant meristems across plant growth stages. This translated to higher numbers of predatory larvae on M. periscae-infested plants in two out of our three experiments, and more consistent control of this pest (78%-95% control across all stages of plant growth). In contrast, control of A. solani was inconsistent in the presence of M. persicae, with 36%-80% control achieved. An additional experiment demonstrated control of A. solani by A. aphidimyza was significantly greater in the absence of M. persicae than in its presence. Our study illustrates that suitability of a natural enemy for pest control may change over a crop cycle as the position of prey on the plant changes, and that prey preference based on within-plant prey location can negatively influence biological control programs in systems with pest complexes. Careful monitoring of the less-preferred pest and its relative position on the plant is suggested.

Conidial vigor vs. viability as predictors of virulence of entomopathogenic fungi.

We tested the hypothesis that debilitated conidia exhibiting slow-germination (requiring>16h to germinate) are less virulent than vigorous conidia exhibiting fast germination (requiring⩽16h to germinate). Preparations of Beauveria bassiana s.l. strain CG 1027 with variable ratios of vigorous to debilitated conidia were assayed against third-instar larvae of Spodoptera frugiperda. As the proportion of debilitated conidia in test preparations increased, LC50 expressed in terms of total viable conidia increased, while LC50 expressed solely in terms of vigorous conidia remained constant, indicating that vigorous conidia were responsible for nearly all mortality observed in the assays. Larvae treated with conidia from low-quality batches (with high proportions of debilitated conidia) survived consistently longer than those treated with comparable doses of conidia from high-quality batches. These results confirm our previous hypotheses that inclusion of debilitated conidia in viability assessments can lead to overestimation of the quality (potency) of mycoinsecticide preparations and support our recommendation for use of short incubation periods for assessing viability whenever viability is relied upon as an indicator of product quality.

Biology and feeding requirements of larval hunter flies Coenosia attenuata (Diptera: Muscidae) reared on larvae of the fungus gnat Bradysia impatiens (Diptera: Sciaridae).

The larval feeding requirements and biology of the generalist predatory muscid hunter fly Coenosia attenuata Stein 1903 (Diptera: Muscidae) were investigated at 25 degrees C. Larval C. attenuata were fed second-, third, and fourth-instar (L2, L3, and L4) larvae of the fungus gnat Bradysia impatiens (Johannsen) (Diptera: Sciaridae) at variable rates to determine minimum and optimum numbers of these prey required for normal development. The proportion of C. attenuata larvae surviving to pupation differed significantly as a function of L2 and L3 prey numbers. When the number of prey/d was increased from 10 to 15 L2 and from 5 to 7 L3 per day, the respective percentages of pupation increased from 0 to 77% and from 0 to 48%. In contrast, all numbers of L4 prey (1-7 prey per d) supported pupation, and the pupation rate did not vary with prey number. At the highest prey numbers tested, mortalities of C. attenuata larvae fed L2, L3, and L4 fungus gnat larvae were 7, 30, and 75%, respectively. The higher mortality of larvae fed L4 prey was clearly the result of lethal wounds inflicted by the fungus gnat larvae in defensive strikes against the predators. At prey numbers supporting maximum rates of adult emergence, larval development required 12-14 d, and duration of the pupal stage was approximately 10 d. C. attenuata larvae killed large numbers of prey during their development (means of up to 232 L2, 144 L3, or 87 L4 fungus gnats), and larvae provided with marginally inadequate numbers of prey survived for long periods (mean 14-22 d, maximum 34 d) before succumbing to apparent starvation. These are favorable attributes with respect to use of C. attenuata as a biological control agent, suggesting a strong potential to substantially impact high-density pest populations and to survive in low-density pest populations.

Transstadial transmission of Pythium in Bradysia impatiens and lack of adult vectoring capacity.

Fungus gnats have been shown to transmit a variety of plant-pathogenic fungi that produce aerial dispersal stages. However, few studies have examined potential interactions between fungus gnats and oomycetes, including Pythium spp. A series of laboratory experiments were conducted to determine whether fungus gnat adults are vectors of several common greenhouse Pythium spp., including Pythium aphanidermatum, P. irregulare, and P. ultimum. An additional objective was to determine whether P. aphanidermatum can be maintained transstadially in the gut of a fungus gnat larva through the pupal stadium to be transmitted by the subsequent adult. Adult fungus gnats did not pick up infectious Pythium propagules from diseased plants and transmit them to healthy plants in any experiment. Species-specific primers and a probe for real-time polymerase chain reaction were developed to detect the presence of P. aphanidermatum DNA in fungus gnat tissue samples. P. aphanidermatum DNA was detectable in the larval and pupal stages; however, none was detected in adult fungus gnats. These results are in agreement with previous studies that have suggested that adult fungus gnats are unlikely vectors of Pythium spp.

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments.

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a(w), temperature, and O(2) concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 degrees C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O(2) concentrations and stored at 50 degrees C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O(2) (100% N(2)) to as high as 63-93% when treated with 21-22% O(2). For conidia stored over a broad range of moderate to high temperatures in the absence of O(2), SR-FR differences were

A tritrophic effect of host plant on susceptibility of western flower thrips to the entomopathogenic fungus Beauveria bassiana.

Adult female western flower thrips (Frankliniella occidentalis) were exposed 12-24h to bean (Phaseolus vulgaris) and impatiens (Impatiens wallerana) leaf disks treated with Beauveria bassiana conidia and then transferred to clean bean or impatiens at various times post-treatment. Significantly greater levels of fungal infection were observed when thrips were treated on bean versus impatiens, but exposure to impatiens following treatment had no effect on fungal infection (percent mortality). This result, combined with observations of no inhibition of germination of conidia exposed to intact or macerated impatiens foliage, indicated that the negative effect of the impatiens host plant was not due to plant chemical compounds (antibiosis). Further observations revealed that insects acquired (picked-up) 75% more conidia from treated bean disks than from treated impatiens disks. This difference in dose acquisition was determined to account for the observed difference in percent mortality (15%) following treatment on the two host plants. Median lethal doses (LD(50)) of B. bassiana were not significantly different on the two host plants, but median lethal concentrations were nearly 7-fold greater on impatiens. This difference was explained by disproportionate rates of conidial acquisition at measured rates of conidial deposition (an inverse relationship was observed between application rate expressed as conidia/mm(2) and the number of conidia acquired). The mechanism underlying the differential rates of conidial acquisition from bean versus impatiens was not determined.

Developmental times and life tables for shore flies, Scatella tenuicosta (Diptera: Ephydridae), at three temperatures.

Development times and survivorship of immature shore flies and longevity and reproduction of adult shore flies, Scatella tenuicosta Collin, reared on algae-infested filter paper, were studied at three temperatures (constant 20, 26, and 28.5 degrees C) through life table analysis. The development time for each individual life stage and the total time from egg to adult decreased with increasing temperature. Duration of the third (ultimate) larval instar ranged from 3.3 +/- 0.09 d at 20 degrees C to 1.4 +/- 0.04 d at 28.5 degrees C and was 1.7-1.9 times longer than the approximately equal first and second instars. Development of male and female shore flies from egg to adult needed an average of 14.5 +/- 0.13, 8.2 +/- 0.05, and 7.0 +/- 0.04 d at 20, 26, and 28.5 degrees C, respectively, and needed an estimated 154.4 +/- 1.2 thermal units (degree days). At these respective temperatures, adult females lived 21.8 +/- 2.2, 19.9 +/- 2.4, and 15.0 +/- 1.4 d and produced 379 +/- 62, 710 +/- 119, and 477 +/- 83 eggs during oviposition periods of 14.3 +/- 2.1, 15.0 +/- 2.2, and 10.8 +/- 1.4 d; daily lifetime egg production averaged 16.3 +/- 2.3, 33.5 +/- 3.8, and 29.7 +/- 3.5. Developmental stage-specific mortality was relatively low for all life stages at all temperatures, with maximum percent mortalities of 5.7% occurring in both the egg stage and in the third instar. The highest net reproductive rate (R(o)) was obtained for insects reared at 26 degrees C and was 329.6. The intrinsic rate of natural increase (r(m)) was highest at 28.5 degrees C and was 0.430. Generation time and doubling time of the population were shortest at 28.5 degrees C and were 12.4 and 1.6 d, respectively. Results suggested that 26 degrees C was near optimum for reproduction.

Acquisition of lethal doses of Beauveria bassiana conidia by western flower thrips, Frankliniella occidentalis, exposed to foliar spray residues of formulated and unformulated conidia.

Secondary acquisition of Beauveria bassiana conidia was recorded on the whole bodies and selected body parts of second-instar nymphs and adult female western flower thrips exposed to foliar spray residues of three differently formulated conidial preparations, for 24 h. Conidia were formulated in emulsifiable oil or with clay (wettable powder), or were essentially unformulated conidia (technical grade powder suspended in water with a surfactant). Formulation had no significant effect on dose acquisition and no effect on virulence of acquired conidia. The mean nymphal LC50/LD50 was 116 conidia/mm2 and 52conidia/insect, respectively; the values for adults were 19 conidia/mm2 and 5conidia/insect. Greatest numbers of conidia were recorded on the legs and abdomens of nymphs and on the legs, wings, and thoraces of adults. As would be expected, numbers of conidia acquired increased with residue concentration (application rate). However, an inverse relationship was noted between acquisition rate (conidia acquired/total conidia applied) and residue concentration. The mechanism underlying this response was not determined. However, there was no indication that any body parts (e.g., tarsi) became saturated with spores, which suggests that either the thrips were repelled by the conidial residues or that as the concentrations of conidia on the substrate increased, conidia somehow became more difficult to acquire. Slopes of the LC probit regressions were lower than those of the LD regressions (mean 1.14 vs 1.78), suggesting that the low slopes often obtained in fungal pathogen assays could be partly an artifact of unequal rates of dose acquisition at low vs high application rates.

Development of a novel bioassay for estimation of median lethal concentrations (LC50) and doses (LD50) of the entomopathogenic fungus Beauveria bassiana, against western flower thrips, Frankliniella occidentalis.

To conduct laboratory experiments aimed at quantifying secondary acquisition of fungal conidia by western flower thrips (Frankliniella occidentalis), an efficient assay technique using Beauveria bassiana as the model fungus was developed. Various application protocols were tested and it was determined that the percent mortality did not vary among protocols. Peak mortality of second-instar nymphs, under constant exposure to conidia, occurred 5 days post-inoculation. Second-instar thrips that were exposed to conidia within 24 h of the molt to second instar were more susceptible to Beauveria bassiana than thrips exposed after times greater than 24 h post-molt. Conidia efficacy, which was monitored at 24 h intervals, did not differ significantly within 72 h. A test of the final bioassay system was conducted in a series of assays aimed at determining the LD50 of B. bassiana technical powder against second-instar western flower thrips. It was determined that B. bassiana (strain GHA) is highly effective at very low doses (LD50 of 33-66 conidia/insect).