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.

First Report of Blight Caused by Sclerotium rolfsii on the Invasive Exotic Weed, Vincetoxicum rossicum (Pale Swallow-Wort), in Western New York.

Pale (Vincetoxicum rossicum) and black swallow-wort (V. nigrum) are perennial, twining vines that are increasingly invasive in natural and managed ecosystems in the northeastern United States and southeastern Canada. Both species, introduced from Europe in the 1800s, are listed as noxious weeds or banned invasive species by the USDA-Natural Resource Conservation Service. Observations by C. Southby, a local naturalist, over several years at a meadow populated by pale swallow-wort in Powder Mill Park, Monroe County, NY, revealed a gradual disappearance of pale swallow-wort with restoration of native grasses and some dicotyledonous plant species, in a 6.7-m-diameter area. Diseased swallow-wort plants had extensive yellowing and wilting of foliage, likely due to splitting of the basal stem, with white mycelium throughout the stem and crown; small, reddish brown sclerotia were evident, but roots were not affected. Stem tissue sections from 20 symptomatic plants were vacuum infiltrated with 2% NaOCl for 20 min, then plated onto malt yeast agar and potato dextrose agar amended with 60 mg/liter of penicillin and 80 mg/liter of streptomycin, resulting in development of fast-growing, white mycelium which then formed numerous, irregularly shaped (2 to 4 mm diameter), reddish brown sclerotia at the plate edges. Two individual cultures were identified as S. rolfsii (1) based on size, shape, and color of the sclerotia and presence of characteristic clamp connections in the mycelium. The isolate was suspected to be S. rolfsii var. delphinii due to the reported inability of S. rolfsii to persist in regions with extremely low winter temperatures (4), but molecular data showed otherwise. Sequences of the 18S gene (GenBank JN543690), internal transcribed spacer region (JN543691), and 28S gene (JN543692) of the ribosomal DNA identified the isolate, VrNY, as S. rolfsii (2,3). Pathogenicity tests were conducted with individual 2-month-old seedlings of V. rossicum and V. nigrum grown in steam-sterilized Metromix 360 in SC10 polypropylene conetainers in a growth chamber with a diurnal cycle of 25/20°C, a photoperiod of 14-h light/10-h dark, and fertilized at 3 week intervals. Two independent replications of 12 plants of each species were each inoculated at the stem base with a 4-mm-diameter mycelial agar plug from the growing edge of a colonized plate. The agar plug was held in place with 5 g of sterile sand. Control plants (12 of each species per replication) were treated with sterile agar plugs. Plants for each treatment were placed within a clear plastic bag to maintain 90% relative humidity for 72 h, and then removed from the bags. Disease symptoms developed over 21 days, with >90% of inoculated plants showing symptoms within 2 weeks. Control plants were symptomless. Incidence of mortality was 66 and 60% for V. rossicum and V. nigrum, respectively, by 3 weeks. The fungus reisolated from diseased stem and crown tissue produced characteristic mycelium with irregular sclerotia, consistent with those of S. rolfsii. Since spread of this fungus is based on movement of soilborne sclerotia, this isolate may offer potential as a bio-herbicide for control of swallow-wort in natural ecosystems if the isolate can be demonstrated to have a host range restricted to this invasive weed. References: (1) B. A. Edmunds and M. L. Gleason. Plant Dis. 87:313, 2003. (2) C. E. Harlton et al. Phytopathology 85:1269, 1995. (3) I. Okabe and N. Matsumoto. Mycol. Res. 107:164, 2003. (4) Z. Xu et al. Plant Dis. 92:719, 2008.

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.

Sensitivity of partially purified ice nucleation activity of Fusarium acuminatum SRSF 616.

Factors that affect bacterial ice nucleation, including growth medium, growth phase, nutrient deprivation, and cold-temperature exposure, were investigated in the ice nucleation active (INA) fungus Fusarium acuminatum SRSF 616. Ice nucleation activity remained relatively constant throughout the growth cycle, and the cell-free culture supernatant consistently displayed higher ice nucleation activity than the hyphal pellet. Although nutrient starvation and low-temperature exposure enhance bacterial ice nucleation activity, reducing the concentration of C, N, or P in synthetischer nährstoffarmer broth (SNB) did not increase fungal ice nucleation activity, nor did exposure to 4 degrees C or 15 degrees C. From the SNB supernatant, selected INA chromatography fractions were obtained that demonstrated increased sensitivity to proteinase K and heat compared with culture supernatant. We propose that partial purification of the fungal ice nuclei resulted in removal of low-molecular-weight stabilizing factors.

Identification of ice-nucleating active Pseudomonas fluorescens strains for biological control of overwintering Colorado potato beetles (Coleoptera: Chrysomelidae).

Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to elevate the supercooling point, the temperature at which freezing is initiated in body fluids, of Colorado potato beetles, Leptinotarsa decemlineata (Say), and to persist in their gut. Adult beetles fed ice-nucleating active strains of Pseudomonas fluorescens, P. putida, or P. syringae at 10(6) or 10(3) bacterial cells per beetle had significantly elevated supercooling points, from -4.5 to -5.7 degrees C and from -5.2 to -6.6 degrees C, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was -9.2 degrees C. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88-335 still had significantly elevated supercooling points, indicating that these strains of bacteria were retained. Furthermore, beetle supercooling points were comparable to those observed immediately after ingestion, suggesting that beetle gut conditions were favorable not only for colonization but also for expression of ice-nucleating activity by these two strains. The results obtained from exposure to a single, low dose of either bacterial strain also show that a minimum amount of inoculum is sufficient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also confirmed using a polymerase chain reaction technique that detected ice-nucleating active bacteria by virtue of their ina genes. Application of these ice-nucleating active bacteria to elevate the supercooling point of this freeze-intolerant insect pest could significantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.

Long-term retention of ice-nucleating active Pseudomonas fluorescens by overwintering colorado potato beetles.

Ice nucleating-active Pseudomonas fluorescens F264C was fed to Colorado potato beetles to determine bacterial retentioin in the beetle gut and its effect on the cold hardiness of this insect pest. The bacrterium was present in beetles recovered after overwintering in the field, seven months after their exposure to P. fluorescens. Retention was evident not only in the detection of the P. fluorescens ice nucleating gene, inaW, in bacterial cultures from beetle guts but also in the elevated supercooling points of some treated beetles.

Genetic variation in Beauveria bassiana populations associated with the darkling beetle, Alphitobius diaperinus.

A study was conducted to assess genetic variation within and among populations of Beauveria bassiana (Deuteromycotina: Hyphomycetes) associated with the darkling beetle, Alphitobius diaperinus (Coleoptera: Tenebrionidae), using RAPD markers. A hierarchical collection of samples (strains from the same insect specimen, from insects from the same location, and from insects from different locations) was obtained from infected beetles from North Carolina (NC) and West Virginia (WV), USA. Ten primers resolved 81 strains into 80 distinct multiband phenotypes reflecting the substantial amount of variation that was present. Variation present within populations was evident not only in the separation of each strain as a distinct multiband phenotype but also in the separation of strains within a population into separate clusters. Among populations, a group sharing more than 89% similarity was observed among all the strains from Martin Co. and Greene Co., NC and 61% of the strains collected from WV. Some genetic differentiation was present among the other populations but the separation was not distinct with a few strains from some populations showing greater affinity to strains from other collection sites.