Laboratory studies assessing the microbial biocontrol potential of diverse strains of Beauveria bassiana isolated from coffee berry borer, with emphasis on strains from Hawai'i Island and comparisons to commercial strain GHA.

A series of experiments investigated genetically diverse strains of Beauveria bassiana (Bb) isolated from coffee berry borer (CBB). Objectives included assessment of their biocontrol potential, particularly in comparison to Bb commercial strain GHA currently applied for CBB control, and identification of various attributes potentially contributing to their comparatively greater epizootic potential in CBB populations. Bioassays identified one strain from Hawai'i Island and one from Puerto Rico with virulence greater than GHA based on equal weights of unformulated conidial powder (CP); however, the greater potency of the CPs was ultimately explained by their 2.4-fold greater conidial densities (ca. 3.1 vs 1.3 × 1011 viable conidia/g CP). Density was explained, in large part, by conidial size, but not by size alone. Bb-inoculated CBB held on moist filter paper were more susceptible to infection than those held on cooked green coffee bean (CGCB). A Bb strain representative of the most common Hawaiian haplotype produced 2.6x more conidia after death of CGCB-held beetles than GHA (19.1 vs 7.3 x106 conidia/cadaver). Following host death, no difference was observed in time to emergence and initial conidial production by GHA and a selected group of Hawaiian strains; however, mass sporulation was initiated within 2 days by strain GHA compared to 4-5 days by the Hawaiian strains. In a preliminary evaluation of conidial mass-production potential, CP yields of several strains were comparable to GHA on a weight basis and significantly greater than GHA on a conidial basis (1.3-1.6 vs 0.7 × 1013 viable conidia/kg barley substrate).

Collection, isolation, in vitro culture, and laboratory transmission of Hirsutella eleutheratorum (Hypocreales: Ophiocordycipitaceae) from coffee berry borer on Hawai'i Island.

The insect pathogenic fungus Hirsutella eleutheratorum was first reported as a pathogen of coffee berry borer (CBB) Hypothenemus hampei in Colombia in 1993. A similar CBB pathogen identified as Hirsutella sp. was reported also from Colombia in 2007; attempts at isolation and in vitro culture of this fungus were unsuccessful. During 2016 and 2017 on the island of Hawai'i, extensive sampling of CBB populations was conducted in coffee fields treated with Beauveria bassiana-based biopesticides and in untreated fields. Among the samples collected from two high-elevation sites in the district of South Kona were rare findings of adult foundress CBB infected with a species of Hirsutella fitting the description of H. eleutheratorum. Prevalence of the pathogen was, in all cases, very low (<1%), having no significant impact on pest populations, even under conditions supporting epizootics of B. bassiana. The fungus was readily isolated from freshly-killed CBB and cultured on potato dextrose agar (PDA). Molecular characterization identified the fungus as a member of the Hirsutella citriformis clade, which includes species recently placed in the genus Ophiocordyceps. Adult CBB exposed to fungus-killed beetles or to PDA cultures of the fungus succumbed to infection within 10-14 days. Under high-humidity laboratory conditions, the fungus emerged from the killed host and produced long, conidia-bearing synnemata characteristic of the species. To our knowledge, this is the first record of H. eleutheratorum from CBB in Hawai'i and the first account of isolation, in vitro culture, genetic characterization, host-to-host transfer, and culture-to-host transfer of this fungal pathogen.

Prevalence of naturally-occurring strains of Beauveria bassiana in populations of coffee berry borer Hypothenemus hampei on Hawai'i Island, with observations on coffee plant-H. hampei-B. bassiana interactions.

Beauveria bassiana (Bb) strain GHA is a major component of an areawide pest management program for coffee berry borer (CBB) in Hawai'i. Recent studies have aimed to provide comprehensive assessments of the efficacy of the Bb-spray component of these programs for economic analyses; however, evaluations have been complicated by activity of naturally-occurring strains of this pathogen infecting CBB. Investigations were therefore undertaken to characterize these strains, assess their natural epizootic potential, and account for their contribution to CBB population suppression. A number of field sites were encountered with no history of significant use of commercial Bb-based biopesticides and where strain GHA was not detectable. Sampling of these sites was conducted early in the coffee season. Greatest activity of wild-type Bb strains was observed on high-elevation farms (>500 m), where 24-42% of foundress beetles in green coffee berries were infected. In contrast, infection rates did not exceed 4% on farms at low elevations (<300 m). Rates of 23-29% infection, comparable to those on high-elevation farms, were recorded in a stand of feral coffee at 293 m elevation, but the coffee was completely shaded and ventilation restricted by a dense overstory of vegetation. Despite high activity of naturally-occurring Bb at some sites (primarily sites at high elevations with humid, moderate-temperature environments and dense pest populations), these fungi did not prevent CBB from exceeding the economic threshold for commercial spray applications. Nevertheless, the high natural epizootic potential of these fungal strains suggests strong potential for development as microbial biocontrol agents.

Characterization of the synergistic interaction between Beauveria bassiana strain GHA and Bacillus thuringiensis morrisoni strain tenebrionis applied against Colorado potato beetle larvae.

Studies were undertaken to further characterize the previously identified synergistic activity of Bacillus thuringiensis- and Beauveria bassiana-based biopesticides against Colorado potato beetle (CPB). A flowable concentrate of B. thuringiensis morrisoni strain tenebrionis (Bt) (Novodor® FC) and a wettable powder of B. bassiana strain GHA (Bb) (Mycotrol® 22WP) were applied against CPB larval populations infesting potato in field plots. Novodor FC and an oil-dispersion formulation of Bb (Mycotrol ES) were applied against second-instar CPB larvae on potted potato plants in greenhouse tests under low relative humidity (RH), variable-temperature conditions. Each pathogen was applied alone and in combination (tank-mixed) with the other pathogen. In the field tests, each biopesticide was also combined with the spray-carrier (formulation without active ingredient) of the other pathogen. Results from the greenhouse tests showed that under warm, dry conditions, low activity of Mycotrol was counterbalanced by high activity of the Novodor, and under cool, somewhat more humid conditions, low Novodor activity was balanced by high activity of Mycotrol, with the result being a constant level of synergism (CPB mortality ca. 20 percentage points higher than predicted by independent action). Similar levels of synergism were observed under the markedly different conditions of the field and greenhouse environments, and the synergism was confirmed as arising from interaction of the two micobes, as the Bt spray carrier had no significant effect on efficacy of the Mycotrol product and the Bb spray carrier had no effect on the efficacy of Novodor. The great capacity of these two control agents to act in concert to control CPB is well documented (the fast-acting, toxic Bt acting to protect potato crops from defoliation and the slow-acting Bb reducing survival to the adult stage). These finding further underscore the strong complementary action of these agents applied jointly against CPB.

Within-plant distribution of Aulacorthum solani (Hemiptera: Aphididae), on various greenhouse plants with implications for control.

Foxglove aphid, Aulacorthum solani (Kaltenbach) (Hemiptera: Aphididae), has recently undergone a status change from an occasional pest to a serious pest in greenhouses of North America and the United Kingdom. Little nonanecdotal information exists on the ecology of this insect in greenhouse crops. To help improve integrated pest management decisions for A. solani, the within-plant distribution of this pest was explored on a variety of common greenhouse plants in both the vegetative and flowering stage. This aphid generally was found on lower leaves of vegetative plants, but was found higher in the canopy on reproductive plants (on flowers, flower buds, or upper leaves). Aphid numbers were not consistently positively correlated with total leaf surface areas within plant strata across plant species. Thus, the observed differences in preferred feeding sites on vegetative versus flowering plants are possibly a response to differences in nutritional quality of the various host-plant tissues. Despite being anecdotally described as a "stem-feeding aphid," A. solani was rarely found feeding on stems at the population densities established in our tests, with the exception of racemes of scarlet sage (Salvia splendans). Although some previous reports suggested that A. solani prefers to feed on new growth of plants, our results indicate that mature leaves are preferred over growing tips and young leaves. The implications of the within-plant feeding preferences of A. solani populations with respect to both biological and chemical control are discussed.

Pathogenicity of conidia-based preparations of entomopathogenic fungi against the greenhouse pest aphids Myzus persicae, Aphis gossypii, and Aulacorthum solani (Hemiptera: Aphididae).

Seeking new isolates of entomopathogenic fungi with greater virulence against greenhouse aphid pests than those currently registered in North America for control of these insects, single-dose screening assays of 44 selected fungal isolates and 4 commercially available strains were conducted against first-instar nymphs of Myzus persicae and Aphis gossypii. The assays identified a number of Beauveria and Metarhizium isolates with virulence equal to or greater than that of the commercial strains against the nymphal aphids, but none exhibited exceptionally high virulence. Virulence of Isaria isolates was unexpectedly low (<31% mortality at doses>1000conidia/mm(2)). In dose-response assays, Beauveria ARSEF 5493 proved most virulent against M. persicae and A. gossypii; however, LC50s of this isolate did not differ significantly from those of B. bassiana commercial strain JW-1. Dose-response assays were also conducted with Aulacorthum solani, the first reported evaluations of Beauveria and Metarhizium against this pest. The novel isolate Metarhizium 5471 showed virulence⩾that of Beauveria 5493 in terms of LC25 and LC50, but 5493 produced a steeper dose response (slope). Additional tests showed that adult aphids are more susceptible than nymphs to fungal infection but confirmed that infection has a limited pre-mortem effect on aphid reproduction. Effects of assay techniques and the potential of fungal pathogens as aphid-control agents are discussed.

Larval Bradysia impatiens (Diptera: Sciaridae) potential for vectoring Pythium root rot pathogens.

A series of laboratory experiments were conducted to investigate the capacity of Bradysia impatiens (Johannsen) larvae to ingest propagules from two strains each of Pythium aphanidermatum (Edson) Fitzp. and P. ultimum Trow and transmit the pathogens to healthy geranium seedlings on a filter-paper substrate in petri dishes. The capacity of fungus gnat larvae to transmit P. aphanidermatum to seedlings rooted in a commercial peat-based potting mix and germination of Pythium oospores and hyphal swellings before and after passage through the guts of larval fungus gnats were also examined. Assays revealed that Pythium spp. transmission by larval fungus gnats varied greatly with the assay substrate and also with the number and nature of ingested propagules. Transmission was highest (65%) in the petri dish assays testing larvae fed P. aphanidermatum K-13, a strain that produced abundant oospores. Transmission of strain K-13 was much lower (<6%) in plug cells with potting mix. Larvae were less efficient at vectoring P. ultimum strain PSN-1, which produced few oospores, and no transmission was observed with two non-oospore-producing strains: P. aphanidermatum Pa58 and P. ultimum P4. Passage of P. aphanidermatum K-13 through larval guts significantly increased oospore germination. However, decreased germination of hyphal swellings was observed following larval gut passage for strains of P. ultimum. These results expand previous studies suggesting that larval fungus gnats may vector Pythium spp.

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.

Developmental times and life table statistics of Aulacorthum solani (Hemiptera: Aphididae) at six constant temperatures, with recommendations on the application of temperature-dependent development models.

Aulacorthum solani (Kaltenbach) (known as foxglove aphid or glasshouse potato aphid) is a pest of increasing economic importance in several agricultural crops worldwide, including greenhouse vegetables and ornamentals. Developmental rates and age-specific life tables for a North American population of A. solani on pansy (Viola × wittrockiana) (Gams.) were determined at six constant temperatures, and comparisons were made to previous studies of A. solani from differing geographic regions and host crops. On pansy, A. solani developed fastest at 25°C, passing through the four nymphal instars in an average of 6.9 d. The highest intrinsic rates of population increase (0.410 and 0.445) and shortest population doubling times (1.69 and 1.56 d) were recorded at 20 and 25°C, respectively. Average total fecundity remained high from 10 to 20°C (74-68 nymphs/adult); a significant decrease to 39 nymphs/adult occurred at 25°C. For calculating developmental thresholds, we present here a method of adjusting the lower developmental threshold (t(min)) using estimates from nonlinear models to provide an improved estimate of the thermal constant (K, in degree-days). We also call attention to the necessity of using a simulation method to estimate the true upper developmental threshold (t(max)) and optimum developmental temperature (t(opt)) from the Lactin-2 model of temperature-dependent development.

Fungus gnat feeding and mechanical wounding inhibit Pythium aphanidermatum infection of geranium seedlings.

ABSTRACT A series of laboratory tests were conducted to investigate potential effects of fungus gnat (Bradysia impatiens) feeding damage on susceptibility of geranium seedlings (Pelargonium x hortorum) to infection by the root rot pathogen Pythium aphanidermatum. Effects were compared with those from similar tests in which the seedlings were mechanically wounded by severing the root tip with a scalpel. Assays of geranium seedlings in petri dishes revealed a pronounced negative fungus gnat-Pythium interaction, with exposure to fungus gnat larvae 24 h prior to inoculation with P. aphanidermatum zoospores resulting in up to 47% fewer seedling deaths than would have been expected if the two agents had acted independently. Similar results were observed when seedlings were subjected to mechanical wounding 24 h prior to zoospore inoculation. In contrast, no interaction occurred when seedlings were mechanically wounded immediately prior to inoculation. The degree of plant damage inflicted by the feeding activities of the larval fungus gnats had no significant effect on the combined damage from fungus gnats and Pythium in petri dishes. Ancillary studies showed that Pythium development on V8 agar was not inhibited by the presence of fungus gnat-associated microorganisms, nor were seedlings inoculated with these microbes less susceptible to Pythium infection. The precise mechaism or mechanisms underlying the observed interactions were not elucidated; however, the results strongly suggest that both fungus gnat feeding and mechanical wounding activated systemic defenses that made the seedlings more resistant to Pythium infection.

Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae.

Commercial biopesticides based on the fungal pathogen Beauveria bassiana strain GHA and the bacterial pathogen Bacillus thuringiensis tenebrionis were applied alone and in combination (tank mixed) against larval populations of the Colorado potato beetle, Leptinotarsa decemlineata, in small plots of potatoes over three field seasons. Interactions between the two products were evaluated in terms of pest-control efficacy. B. bassiana (formulated as Mycotrol) was applied at low and medium label rates of 1.25 and 2.5 x 10(13) conidia/ha, and B. thuringienis (formulated as Novodor) was applied at low and high label rates of 40.3 and 120.8 x 10(6) Leptinotarsa units/ha. Two weekly applications of the bacterial pesticide alone provided 50-85% control of beetle larvae within 14 days after the initial application, while applications of the mycopesticide alone produced no greater than 25% control. Maximum control, in nearly all tests, was produced by the combination of the two products. The combined treatments produced a statistically significant 6-35% greater reduction in larval populations than would have been predicted had the two biopesticides acted independently. This low-level synergistic interaction was observed during all field seasons and resulted from combinations at all rates, including, in one of two tests, the low rates of each product. These results indicate that B. thuringiensis and B. bassiana have strong potential for integrated biologically based management of Colorado potato beetle.

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.

Pathogenicity of the entomopathogenic fungi paecilomyces spp. and Beauveria bassiana against the silverleaf whitefly, Bemisia argentifolii.

Pathogenicities of three species of entomopathogenic fungi against preimaginal Bemisia argentifolii were measured and compared. Third-instar nymphs on excised leaves of Hibiscus rosa-sinensis were exposed to spray applications of 14 isolates of Beauveria bassiana, 22 isolates of Paecilomyces fumosoroseus, and five isolates of Paecilomyces farinosus. B. bassiana and P. fumosoroseus isolates of diverse origins were highly pathogenic to the whitefly nymphs; median lethal doses of 14 of the 22 P. fumosoroseus and four of the 13 B. bassiana isolates ranged between 50 and 150 conidia/mm2. Five isolates of P. farinosus were also pathogenic; however, LC50s were relatively high, ranging between 350 and 4000 conidia/mm2. Nymphs infected with all but one isolate of B. bassiana displayed a pronounced red pigmentation. Postmortem hyphal growth and sporulation of B. bassiana was relatively slow and usually confined to the region immediately surrounding the dead host. Whitefly nymphs patently infected with P. fumosoroseus and P. farinosus were lightly pigmented yellow or orange. Postmortem hyphal growth and sporulation of P. fumosoroseus rapidly covered the dead host and extended several millimeters onto the surrounding leaf surface. The results indicate that highly virulent strains of P. fumosoroseus and B. bassiana with considerable whitefly control potential are widespread and numerous.