Evaluation of a guardian plant system to suppress Frankliniella occidentalis (Thysanoptera: Thripidae) in greenhouse ornamentals.

BACKGROUND: Western flower thrips (WFT), Franklinella occidentalis (Pergande), is an economically damaging pest of greenhouse ornamentals. A 'guardian plant system' (GPS) that targeted WFT was evaluated under controlled and commercial greenhouse conditions. This system used mycotized millet grains with the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin applied to soil of potted marigolds, Tagetes patula (L.), combined with the foliar-dwelling predatory mite Neoseiulus (=Amblyseius) cucumeris (Oudemans) in slow-release sachets under controlled greenhouse conditions, and with the addition of a pheromone lure under commercial settings. RESULTS: Significantly fewer WFT and less foliar damage on GPS was observed over the 10 and 12 weeks experimental periods compared to the untreated controls. Predatory mites were maintained up to 10 weeks with one release under controlled greenhouse conditions and 12 weeks with two releases in commercial greenhouses. In commercial greenhouses, greater numbers of WFT were found on marigolds than on crop plants within 1 m of the system. Fungal granules persisted for 12 weeks up to 2.5 × 105 CFU g-1 in the GPS soil. CONCLUSION: The use of biological control agents to suppress WFT within a GPS could be a useful IPM strategy for greenhouse production. The marigold GPS attracted WFT which were suppressed primarily through predation by foliar-dwelling predatory mites and to a lesser extent, infection from conidia produced by the granular fungal formulation in the soil. Further investigations into system deployment and fungal granular application rates and new fungal formulations are suggested to improve system efficacy. © 2023 Society of Chemical Industry.

Evaluation of the entomopathogenic fungus Metarhizium brunneum and the predatory mite Stratiolaelaps scimitus against Rhizoglyphus robini under laboratory conditions.

Rhizoglyphus robini Claparède (Acari: Acaridae) is a pest of bulbs, corms and tubers of several economically important crops. The biological control of R. robini has yet to be fully explored as an alternative to chemical pesticides. Entomopathogenic fungi in the genera Metarhizium (Hypocreales: Clavicipitaceae) are used for the biological control of several agricultural pests. The soil-dwelling predatory mite, Stratiolaelaps scimitus (Womersley) (Acari: Acaridae) is also frequently used alone or in combination with other biological control agents. There are few reports on the use of M. brunneum or S. scimutus against R. robini. The objectives of this research were to investigate the in vitro effect of different predatory mite ratios of S. scimitus on R. robini mortality and the combined use of a M. brunneum-based granule with S. scimitus as potential strategies to manage this pest. Mortality of R. robini in Petri dishes containing predators was significantly higher than without predators. When soil-filled containers containing R. robini were treated with both M. brunneum granules and S. scimitus, the lower densities of the bulb mite were obtained with the highest ratio of predator/prey mites. The number of bulb mites in the containers treated with only M. brunneum was significantly lower than the untreated control. These results demonstrate the potential for releasing of S. scimitus alone and in combination with M. brunneum granules to manage R. robini.

A Review of Commercial Metarhizium- and Beauveria-Based Biopesticides for the Biological Control of Ticks in the USA.

There is a need for the development of sustainable, nonchemical tick management strategies. Mycoacaricide and mycoinsecticide product development worldwide has focused primarily on fungi in the genera Beauveria (Hypocreales: Cordycipitaceae) and Metarhizium (Hypocreales: Clavicipitaceae). Microbial biopesticides containing entomopathogenic fungi have potential in tick management. However, despite considerable progress in the development of fungal biopesticides over the past 20 years, the establishment of commercial products available for use against ticks continues to be slow. We reviewed published scientific literature and compiled a comprehensive list of reports of the effectiveness of commercial biopesticides based on the fungal genera Metarhizium and Beauveria and registered for use in the USA against ixodid ticks under laboratory and field conditions. We also report on results when these biopesticides were used as a part of integrated tick management. Until efficacious fungus-based products become more available, tick management will rely primarily on synthetic chemical acaricides, with natural-product acaricides as the alternative.

First Report of the Bat Tick Carios kelleyi (Acari: Ixodida: Argasidae) From Vermont, United States.

The soft tick Carios kelleyi (Cooley and Kohls, 1941) is an ectoparasite of bats that can harbor bacteria known to cause disease in humans, such as Rickettsia spp., Bartonella spp., and relapsing fever Borrelia spp. Human-tick encounters may occur when bats occupy attics or similar dwellings with access points to human-inhabited areas. During May 2021, a partially engorged adult female C. kelleyi was collected from a Vermont home with an attic that was being used as a roost by big brown bats, Eptesicus fuscus (Chiroptera: Vespertilionidae). The source of the blood in the tick was the domestic dog, Canis lupus familiaris. Subsequently, eight C. kelleyi larvae were collected from a rescued E. fuscus adult. This is the first report of a soft tick species from Vermont and it is unknown how long C. kelleyi has been present in this state. Reports of C. kelleyi are on the rise across the northeastern United States but the implications for the health of humans, domestic animals, and bats in northern New England remain unclear. Bat management plans should consider the importance of bat exclusion in preventing tick encounters with members of the household and should include a tick monitoring component if bats are evicted.

Biological control of Western flower thrips, Frankliniella occidentalis using a self-sustaining granular fungal treatment.

Western flower thrips (WFT), Frankliniella occidentalis, is one of the most destructive pests of vegetables, fruits and ornamental crops worldwide, causing extensive damage by direct feeding of the crop and transmitting economically important viruses. Despite the successes of biocontrol agents to control WFT, more efficient and cost-effective ways must be found to encourage grower adoption of integrated pest management. A sustainable fungal treatment was developed to preserve fungal inoculum in potting soil and reduce thrips populations. Combining cooked, oven-dried millet with BotaniGard® (a commercial form of Beauveria bassiana strain GHA) to potting soil increased spore production and persistence of the fungus in the soil. In treated pots with millet, spore concentrations were 3-4 times greater after 30 days compared with spore yields at 10 days. The number of WFT adults was significantly lower in the marigold pots treated with GHA mix + millet than untreated controls, 12% and 10% in treated pots and 70% and 68% in untreated pots in sterile and non-sterile soil, respectively. Incorporation of millet in the potting mix enhanced the effect of the fungal treatments by providing a nutritive substrate on which the fungus could become established. This method is relatively inexpensive and easy for growers to use in greenhouses because granular formulations of B. bassiana are not commercially available.

Gene diversity explains variation in biological features of insect killing fungus, Beauveria bassiana.

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Evaluation of spray applications of Metarhizium anisopliae, Metarhizium brunneum and Beauveria bassiana against larval winter ticks, Dermacentor albipictus.

Dermacentor albipictus (Acari: Ixodidae), the winter tick, is a one-host tick that parasitizes large ungulates. They can dramatically affect moose, Alces alces (Artiodactyla: Cervidae), causing significant physiological and metabolic stress and mortality among heavily parasitized individuals. Entomopathogenic fungi in the genera Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria (Hypocreales: Cordycipitaceae) are promising tick biological control agents. We examined the pathogenicity of experimental and commercially formulated isolates of M. anisopliae, M. brunneum and B. bassiana sprayed at concentrations of 106, 107 and 108 conidia/mL against the larval stage of D. albipictus and assessed the efficacy of spraying the commercial product Met52®EC, containing M. brunneum, strain F52, under laboratory conditions. Results showed larval D. albipictus mortality was significantly higher and occurred earlier when treated with M. anisopliae and M. brunneum isolates compared to B. bassiana at 106, 107 and 108 conidia/mL. Mortality was observed as early as 3 days in the M. anisopliae and M. brunneum treatments and after 6 days in the B. bassiana treatments. After 21 days, larval mortality ranged from 74-99% when ticks were treated with M. anisopliae and M. brunneum isolates at 106, 107 and 108 and conidia/mL. In contrast, mortality of ticks treated with B. bassiana ranged from 30 to 64%. When larvae were treated with the commercial product Met52, mortality was ~ 45% after 3 days and ~ 96% after 9 days. These results demonstrate the effectiveness of M. anisopliae and M. brunneum against D. albipictus.

Virulence of BotaniGard(®) to Second Instar Brown Marmorated Stink Bug, Halyomorpha halys (Stål) (Heteroptera: Pentatomidae).

The brown marmorated stink bug, Halyomorpha halys (Stål) (BMSB) is an exotic invasive insect originating in East Asia, currently causing significant damage to fruits, vegetables and other crops throughout most of the Mid-Atlantic states of the U.S. It also is a nuisance pest, entering homes in the fall in search of suitable overwintering sites. Two formulations of BotaniGard(®) with a strain of Beauveria bassiana (GHA) as the active ingredient were tested against second instar BMSB. Both the wettable powder and the emulsifiable suspension formulations were efficacious at 1 × 107 conidia mL(-1), causing 67%-80% mortality 9 days post treatment and 95%-100% after 12 days. The wettable powder formulation was slightly more efficacious.

Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170.

Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.

A novel approach: Beauveria bassiana granules applied to nursery soil for management of rice water weevils in paddy fields.

BACKGROUND: Spraying of entomopathogenic fungi formulated as wettable powders and suspension concentrates based on oils or water has been used for the management of crop pests. However, the use of fungal granules to control paddy field pests has not been fully explored. Herein, several Beauveria bassiana isolates (ERL1170, ERL1578 and ERL836) were produced as granules using a millet-based solid culture. The granules were applied to the rice nursery 3 days before transplanting, and their control efficacy against rice water weevils was determined in paddy fields. RESULTS: The solid cultures produced ∼1 × 10(8) conidia g(-1) of millet grains 10 days after the inoculation. The granules were applied to the soil in the rice nursery 3 days before the rice seedlings were transplanted in the paddy fields. Rice in plots with granules of ERL1578 had 17.3% leaf damage (74% control efficacy) 14 days post-application, whereas rice plants in the untreated control had 66.5% damage. Rice plants treated in the nursery with ERL1170 and ERL836 had 52-54% damage. In the rice plots previously treated with ERL1578, the smallest numbers of larvae and adults were observed 38 days post-application. In laboratory conditions, ERL1578-treated larvae turned pink and were covered with mycelial mass. CONCLUSION: Applications of millet-based B. bassiana granules on rice nursery soil can be an effective and efficient biological control strategy for the management of rice water weevils. This method can be relatively inexpensive and requires less labor than practices involving the spraying of fungi directly on rice in paddy fields.

Transformation of Beauveria bassiana to produce EGFP in Tenebrio molitor for use as animal feed additives.

Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

Bumblebee venom serine protease increases fungal insecticidal virulence by inducing insect melanization.

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.

An oil-based formulation of Isaria fumosorosea blastospores for management of greenhouse whitefly Trialeurodes vaporariorum (Homoptera: Aleyrodidae).

BACKGROUND: To date, emphasis has been placed on studying the virulence of Isaria fumosorosea conidia rather than the virulence of their blastospores. In the present study, a comparison was made of the efficacy of blastospores of I. fumosorosea (SFP-198) versus conidia against greenhouse whitefly (GWF) Trialeurodes vaporarioum nymphs and thermotolerance at 50 °C. To improve thermotolerance and potency, the blastospores were suspended in corn oil with a wetting and an insect-filming agent. RESULTS: Blastospores had similar efficacy against GWF nymphs to conidia in glasshouse conditions but had less thermotolerance than conidia. However, blastospores in corn oil had increased thermotolerance, comparable with that of conidia in corn oil. Application of SFP-198 blastospore oil-based formulation containing isotridecyl alcohol ethoxylated-3EO (TDE-3) and sodium alginate (SA) as adjuvants showed enhanced efficacy (95.7% mortality), compared with the SA-free oil formulation (72.8%) in 10 days. CONCLUSION: SFP-198 blastospores can be used as an effective biological control agent, given their virulence against GWF, the enhanced thermotolerance in corn oil and the oil-based formulation studies to enhance their efficacy. Blastospores can be easily produced in liquid cultures in less than 4 days, which is significantly shorter than using traditional solid cultures for conidial production.

Entomopathogenic Fungi Associated with Exotic Invasive Insect Pests in Northeastern Forests of the USA.

Mycopathogens of economically important exotic invasive insects in forests of northeastern USA have been the subject of research at the Entomology Research Laboratory, University of Vermont, for the last 20 years. Elongate hemlock scale, European fruit lecanium, hemlock woolly adelgid and pear thrips were analyzed for the presence of mycopathogens, in order to consider the potential for managing these pests with biological control. Fungal cultures isolated from insects with signs of fungal infection were identified based on morphological characters and DNA profiling. Mycopathogens recovered from infected insects were subdivided into three groups, i.e., specialized entomopathogenic; facultative entomopathogens; ubiquitous opportunistic contaminants. Epizootics were caused by fungi in the specialized group with the exception of M. microspora, P. marquandii and I. farinosa. Inoculation of insects in laboratory and field conditions with B. bassiana, L. muscarium and Myriangium sp. caused insect mortality of 45 to 95%. Although pest populations in the field seemed severely compromised after treatment, the remnant populations re-established themselves after the winter. Although capable of inducing high mortality, a single localized aerial application of a soil-dwelling fungus does not maintain long-time suppression of pests. However, it can halt their range expansion and maintain populations below the economic threshold level without the use of expensive insecticides which have a negative impact on the environment.

Virulence of select entomopathogenic fungi to the brown marmorated stink bug, Halyomorpha halys (Stål) (Heteroptera: Pentatomidae).

The brown marmorated stink bug. Halyomorpha halys, (BMSB) is an exotic pest causing damage to fruits and vegetables. They also transmit diseases. Integrated pest management strategies are lacking for this invasive. For the first time, information is presented on the virulence of several entomopathogenic fungi against BMSB. Three Beauveria bassiana and two Metarhizium anispoliae isolates were bioassayed against adult BMSB. One B. bassiana isolate was the active ingredient in BotaniGard® which produced 85 and 100% mortality in 9 and 12 days post treatment, respectively. Experimental isolates ERL 1170 and ERL 1540 were also efficacious. Metarhizium anispoliae isolates produced lower mortalities than the B. bassiana isolates. Data presented showed the potential for management of BMSB with entomopathogenic fungi.

Generating thermotolerant colonies by pairing Beauveria bassiana isolates.

Low thermotolerance in entomopathogenic fungi is the main impediment to their industrialization. This research, for the first time, describes the generation of a thermotolerant colony by pairing and subculturing (cycling) two Beauveria bassiana isolates without sexual reproduction. A mixture of B. bassiana ERL1578 and ERL1576 was inoculated on quarter-strength Sabouraud dextrose agar with yeast extract (»SDAY). The paired culture (ERL1578 + 1576) was cycled three times to increase the frequency of possible hyphal fusion at the first cycle (c. 5/5 × 10(5) conidia), followed by a heat treatment as a selection pressure. Two non-paired isolates served as controls. Two morphologically different colonies (BbHet1 and BbHet2) were isolated from the pairing. BbHet1 colony had the highest conidial yield. BbHet2 had the most rapid mycelial growth and produced sponge-like mycelial masses (the others were flat), and its conidia were darker than the non-paired colonies under a microscope (400×). BbHet2 conidia had 60.7% germination after exposure to 45 °C for 60 min (the others had < 15%) without significant loss of virulence against Western flower thrips, Frankliniella occidentalis; however, there was a slight decrease in conidial yield. The new phenotypes formed suggested that a genetic variation happened as a result of heterokaryosis and/or recombination, more than environmental adaptation, when mixing different conidia. This methodology seems to be very useful for enhancing thermotolerance in fungi.

Production of thermotolerant entomopathogenic fungal conidia on millet grain.

Thermotolerance of entomopathogenic (insect-killing) fungi should be seriously considered before industrialization. This work describes the feasibility of millet grain as a substrate for production of thermotolerant Beauveria bassiana (Bb) GHA and ERL1170 and Metarhizium anisopliae (Ma) ERL1171 and ERL1540 conidia. First, conidial suspensions of the Bb isolates, produced on millet grain in polyethylene bags, were exposed to five temperatures (43-47 °C) at 15-min intervals for up to 120 min (experiment I). Agar-based quarter-strength (») Sabouraud dextrose agar supplemented with yeast extract (SDAY) and whey permeate media served as controls. Millet-grain-based culture was superior in producing the most thermotolerant Bb conidia, followed by whey permeate agar and »SDAY-based cultures. Secondly, to compare the thermotolerance of conidia produced at the same conditions, the Bb isolates were then produced on agar-based millet powder medium, with »SDAY and whey permeate agar media as controls, and the two Ma isolates were added (experiment II). They were then exposed to the same temperatures as above. More thermotolerant Bb and Ma conidia were produced on millet powder agar than on whey permeate agar and »SDAY overall. These results suggest that millet grain can be used as a substrate to produce thermotolerant conidia in a mass production system.

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay.

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and »SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45°C for 45min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14d were more hydrophobic and exhibited greater thermotolerance than those produced on »SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.

Entomopathogenic activity of a variety of the fungus, Colletotrichum acutatum, recovered from the elongate hemlock scale, Fiorinia externa.

A fungal epizootic in populations of Fiorinia externa Ferris (Hemiptera: Diaspididae) infesting hemlock trees, Tsuga canadensis (L.) Carrière (Pinales: Pinaceae) in forests of the Northeastern US has been recently detected. The current known distribution of the epizootic spans 36 sites in New York, Pennsylvania, New Jersey and Connecticut. Colletotrichum acutatum Simmonds var. fioriniae Marcelino and Gouli var. nov. inedit. (Phyllachorales: Phyllachoraceae) was the most prevalent fungus recovered from infected scales. Bioassays indicated that this C. acutatum variety is highly pathogenic to F. externa. Mortality rates of >90 and >55% were obtained for F. externa crawlers and settlers, respectively. Significantly lower mortality levels,

Host plant associations of an entomopathogenic variety of the fungus, Colletotrichum acutatum, recovered from the elongate hemlock scale, Fiorinia externa.

A fungal epizootic has been detected in populations of the scale Fiorinia externa Ferris (Hemiptera: Diaspididae) in the eastern hemlock, Tsuga canadensis (L.) Carrière (Pinales: Pinaceae), of several northeastern states. Colletotrichum acutatum Simmonds var. fioriniae Marcelino and Gouli var. nov. inedit (Phyllachorales: Phyllachoraceae), a well-known plant pathogen, was the most commonly recovered fungus from these infected scales. This is the second report of a Colletotrichum sp. infecting scale insects. In Brazil C. gloeosporioides f. sp. ortheziidae recovered from Orthezia praelonga is under development as a biopesticide for citrus production. C. acutatum was detected growing endophytically in 28 species of plants within the epizootic areas. DNA sequences of the High Mobility Box at the MAT 1-2, mating type gene indicate that Colletotrichum sp. isolates recovered from scale insects and plants within epizootic areas were identical. Results from plant bioassays showed that this entomopathogenic Colletotrichum variety grew endophytically in all of the plants tested without causing external symptoms or signs of infection, with the exception of strawberry plants where mild symptoms of infection were observed. The implications of these findings with respect to the use of this fungus as a biological control agent are discussed.