Research Project: Microbial and Arthropod Biological Control Agents for Management of Insect Pests of Greenhouse Crops and Trees

Location: Emerging Pests and Pathogens Research

2017 Annual Report

Objectives
Objective 1: Integrate mycoinsecticides to manage selected insect pests of greenhouse crops, including, but not limited to, soil and foliar treatments for thrips control and for optimizing abiotic conditions. (NP304, Component 3, Problem Statement 3A2) Subobjectives: 1a. Characterize and quantify the effects of biotic and abiotic factors on efficacy of fungal pathogens applied against western flower thrips. 1b. Assess the potential of combining soil and foliar applications of fungi against western flower thrips as a strategy to achieve acceptable levels of efficacy at reduced moisture levels. Objective 2: Characterize pest microbial associates and determine the efficacy of microbial control agents to manage insect pests, including, but not limited to, Asian ambrosia beetles, walnut twig beetle, and coffee berry borer. Subobjectives: 2a. Determine effects of commercially available fungal biocontrol agents on Asian ambrosia beetles and their symbionts. 2b. Assess efficacy of commercially available entomopathogenic fungi for the walnut twig beetle. 2c. Assess efficacy of Beauveria bassiana against coffee berry borer in the Kona coffee-growing region on the island of Hawaii; determine best practices for effective deployment of this pathogen as one component of an area wide IPM program. 2d. Characterize the indigenous isolates of Beauveria infecting coffee berry borer in Hawaii and determine the roles of these pathogens in natural suppression of this insect pest. Objective 3: Determine the impact of natural enemies, such as parasitic wasps and microbial biopesticides, introduced for management of emerald ash borer. Subobjectives: 3a. Quantify EAB and parasitoid densities to determine whether establishment of EAB parasitoids has significant effects on EAB populations. 3b. Determine impact of natural enemies released against emerald ash borer on ash health and survival.

Approach
The goals of this project are to integrate entomopathogenic fungi into management systems for insect pests of greenhouse ornamental and vegetable crops and insect pests of trees in nursery, field, and natural settings and to track fungal strains in these environments. The work comprises fundamental laboratory studies as well as applied field and greenhouse research. This project will develop basic information on the biology of fungal pathogens associated with insects, their genetic and phenotypic variability, and their activity and persistence in field and greenhouse environments. Integration of fungal pathogens will be accomplished for management of key pests. Studies will identify minimal ambient moisture requirements for effective deployment of fungal pathogens against western flower thrips infesting greenhouse crops. Microbial control agents will be developed for management of Asian ambrosia beetles and walnut twig beetle. Application methods and strategies will be developed for optimal use of fungi against coffee berry borer in the Kona coffee districts of Hawaii. Parasitoid releases for management of emerald ash borer will be evaluated for their impact on their host and the resulting impacts on ash health and regeneration. Biological control agents used in the ways developed in this project will provide safe, effective biological alternatives to synthetic chemical insecticides or as rotational partners for insecticide resistance management.

Progress Report
Collaborating scientists of ARS-Ithaca, New York, ARS-Hilo, Hawaii, and the University of Hawaii-Manoa are conducting field studies aimed at assessing efficacy of spray applications of Beauveria bassiana (Bb strain GHA formulated as BotaniGard® ES) applied for areawide control of the coffee berry borer (CBB). A fourth season of detailed evaluations of efficacy of Bb sprays applied by backpack mist blower in high- vs. low-elevation Kona coffee fields is in progress. Molecular genetic studies aimed at characterizing the diverse naturally-occurring strains of Bb found infecting CBB in Hawaii are also nearing completion. These studies have also identified morphological differences between the wild strains and commercial strain GHA that have enabled rapid differentiation of these pathogens in field-collected CBB. Our capacity to identify infections caused by the wild vs. commercial strains of Bb (verified by molecular methods) has resulted in more precise estimates of efficacy of the commercial spray applications. Most recent analyses indicate that each BotaniGard spray applied by backpack mist blower during the early months of the season (March–May) results in ca. 15–20% infection of CBB in green berries (detectable in berry samples collected within 2–3 days after application). Data from subsequent samples at days 7–10 after application indicate that horizontal transmission of the pathogen (from Bb-killed to healthy CBB) results in an additional 8–12% infection. Total impact of a spray thus averages ca. 25 to 30% mortality of CBB in the green coffee berries. Cumulative mortality from multiple sprays typically reaches 50–70% and reduces overall damage to less than 10%. Two manuscripts are in preparation. New treatments initiated in 2016, involving application of both BotaniGard and an added surfactant at ½ the recommended rates, are continuing. Thus far, results from the 2017 tests are in agreement with those from 2016, with efficacy of the half rate being nearly equal to the full rate at high elevation but substantially lower under drier conditions at low elevation. Investigations were initiated to determine the efficacy of Bb applications against CBB in the Ka’u coffee growing region of Hawaii Island. The Ka’u coffee production system differs markedly from the Kona system, with trees planted in dense rows and pesticide applications made via tractor-driven orchard sprayers. The Ka'u plantings generally contain approximately 940 trees per acre compared to 540 in the Kona-style plantings, and in order to provide coverage of the denser foliage, the Ka’u growers typically apply greater spray volumes than the Kona growers employing backpack sprayers (ca. 100 vs. 32 gallons/acre). In applying the recommended rate of 1 quart BotaniGard per acre, the concentration of active ingredient in the Ka’u sprays is therefore ca. one-third that of the Kona sprays. Sampling of spray depositions from the two systems has revealed that the Ka’u sprayers apply ca. one-half the amount of active ingredient (fungal spores) per square mm of target surface (plastic coverslips pinned to the distal ends of coffee berries). We have recorded markedly lower efficacy of the BotaniGard sprays in Ka’u compared to Kona, and this is likely a contributing factor, especially considering the dry conditions in our test fields. However, a number of other practices, including tank-mixing of BotaniGard with different agrochemicals, including foliar fertilizers and associated spreader stickers, may be involved and warrant investigation. The ambrosia beetle Xylosandrus germanus is an invasive ambrosia beetle attacking a wide range of host trees, including economically important orchard and nursery trees. The beetle is hard to control because of its cryptic habits, with only adult females found outside of beetle galleries or tunnels under the bark during flights to search for new breeding sites. Research efforts to control this beetle conducted by ARS scientists in Ithaca, New York, in collaboration with researchers from Cornell University, have shown that entomopathogenic fungi can be used to kill the females, and that infected females produce fungal conidia than can also kill her progeny, sometimes up to 100% in the gallery. Further, mycoparasitic fungus can also be used to kill or suppress the associated fungal symbiont the beetle grows in their galleries for food. Based on these results, strategies implementing the use of either entomopathogenic or mycoparasitic fungi against this beetle are being tested in apple orchards in upstate New York. During the first field trial a methodology was developed using 2-year old potted apple trees, with the pots bagged and flooded to attract beetles. Flooding stresses the trees and results in increased ethanol production that incites beetle attacks. These potted trees were laid at the edge of wooded areas adjacent apple orchards, which are the main sources of beetle populations moving into orchards. Additional studies are underway to test different dosages of either insect pathogenic or mycoparasitic fungi to determine an optimal dose that is both effective in controlling the beetle and economical. Additional field tests in apple were attempted in 2017, but wet weather during late spring/early summer, when peak beetle flight activity is normally observed, resulted in too few beetle attacks to allow treatment comparisons. Thousand cankers disease (TCD), caused by the walnut twig beetle (WTB), Pityophthorus juglandis, and its associated fungal symbiont, Geosmithia morbida, is a deadly disease of the eastern black walnut, Juglans nigra. Few management options are available for preventing or reducing impact of TCD on black walnut trees. In collaboration with scientists at the University of Tennessee, the USDA Forest Service, and Cornell University, we are evaluating the use of commercial strains of Metarhizium brunneum and Beauveria bassiana against WTB. Laboratory and field studies have shown that WTB adults are readily susceptible to both fungi. In assays using small log sections, both fungal treatments resulted in lower number of WTB galleries per log, but only Metarhizium-treated logs had significantly fewer brood compared to controls. Fungus-sprayed 1-meter logs were attacked significantly less frequently than control logs, and beetle reproduction, as measured by emergence of next-generation adults, was reduced by more than 80% compared to controls. Fungal infection of larvae within galleries was also observed indicating that inoculated or infected adults can transmit the fungus to their offspring. This results in the death of adults from fungal infection and, subsequently, of their brood from fungal inocula introduced to the gallery or produced by infected parents. These results show the potential use of entomopathogenic fungi in the integrated management of TCD. Additional field tests (third field season) were conducted in Tennessee in 2017 but yielded insufficient data for analysis because of low pest populations. Laboratory studies aimed at defining the optimal periods of high-humidity necessary to support high rates of infection by insect pathogenic fungi applied against western flower thrips (WFT) over a range of temperature conditions initiated in 2016 in collaboration with a former visiting Fulbright Scholar from the University of Agriculture, Faisalabad, Pakistan, are continuing. Numerous additional tests with Bb commercial strain GHA were conducted at constant 15, 20, 25, and 30 ºC, with all thrips exposed to the same dose of the pathogen. At constant 25ºC, infection plateaued at approximately 70% within 32 hours of incubation under near-moisture-saturated conditions (ca.100% RH). Maximum infection was achieved also within 32 hours at 30ºC, but total mortality was lower: ca. 55%. At 20ºC, infection was similar to that at 25ºC (ca. 70%), but required 48 hour of high humidity, and at 15ºC, infection reached only 33% within 56 hours. These results indicate potential limitations to use of Bb for thrips control at low and high temperatures. Tests are planned to investigate impacts of fluctuating temperatures (simulating more natural conditions of warm daytime/cool nighttime temperatures in spring bedding plant cultures in northeastern greenhouses). Soil drench applications of Bb strain GHA and another insect pathogenic fungus Metarhizium brunneum (Mb) commercial strain F-52 (active ingredient of the Met52® biopesticide) are under investigation by ARS-Ithaca scientists as alternatives/complements to foliar applications for control of western flower thrips in greenhouse ornamentals. Processing of thrips samples collected from flowering impatiens in 10 previously conducted tests (six tests of Bb and four of Mb) was completed in 2017. In one Bb test and one Mb test, the treatments were ineffective, and possible causes of the control failures are under investigation. In the other tests, treatments by both pathogens provided equivalent levels of control, reducing total thrips populations on flowers by 32% and on foliage by 40% relative to spray carrier controls; respective reductions in populations of adult thrips were 40 and 52%. Though thrips control from these treatments is not adequate from the perspective of greenhouse pest management, the findings are encouraging in that the results were obtained under very low-humidity conditions found to render foliar applications almost completely ineffective. Drench or sprench applications of these insect-pathogenic fungi can thus be recommended as components of thrips IPM programs.

Accomplishments
1. Naturally-occurring strains of the insect pathogenic fungus Beauveria bassiana (Bb) are highly virulent against the coffee berry borer (CBB). The commercial strain of Bb sold under the trade names BotaniGard and Mycotrol does not persist for more than a few weeks in CBB populations following spray applications, after which it is displaced by naturally occurring strains of Bb. Hawaiian coffee growers have expressed interest in the use of these naturally-occurring strains as biocontrol agents. However, development of these fungi as biopesticides requires knowledge of their innate virulence (infectiousness and speed of kill). ARS researchers in Ithaca, New York, conducted bioassays of naturally occurring Bb strains against laboratory-reared CBB, and found that many are highly infectious and rapidly lethal against this pest. The results indicate that these fungi have potential to be more effective biocontrol agents than commercially available strains of Bb.

2. Negative interactions between insect-pathogenic or mycoparasitic fungi and symbionts of ambrosia beetles. Ambrosia beetles are among the most important insect pests in orchards and nurseries and are difficult to control using conventional insecticides because of their cryptic habits. Beetles rely on their fungal symbiont, which they grow in tunnels or galleries in host trees, as their primary food source. In collaboration with researchers from Cornell University, ARS scientists in Ithaca, New York, conducted competition studies between fungal symbionts associated with these beetles and the insect pathogenic fungi Beauveria bassiana and Metarhizium brunneum or the mycoparasitic fungus Trichoderma harzianum. Laboratory assays showed that the symbionts were outcompeted by these fungi. Complementary assays using beech logs also showed that less than 50% of the beetles produced progeny due to sparse growth or absence of the symbiont in those treated with the mycoparasitic fungus and due to death of female adults prior to laying eggs in those treated with insect pathogenic fungi, indicating multiple ways to target these beetles in the field.

3. Efficacy of insect-pathogenic fungi against the walnut twig beetle. The walnut twig beetle and its associated fungal symbiont cause thousand-cankers disease in walnuts, an economically important source of lumber and nuts. The use of chemical insecticides is not feasible because of regulations restricting applications on nut crops. Laboratory studies conducted by researchers from Cornell University, the University of Tennessee, USDA Forest Service in Asheville, North Carolina, and ARS in Ithaca, New York, showed that up to 90% of walnut twig beetles die of fungal infection 4 days after exposure to insect-pathogenic fungi. Field studies using commercially available products based on these fungi also showed more than 80% reduction in numbers of next generation beetles emerging from sprayed logs because of fewer beetle attacks and mortality among those that tunneled into the logs. These findings are the first to show an environmentally friendly option to target beetle populations and control thousand cankers disease in walnut trees.

Review Publications
Jandricic, S.E., Wraight, S.P., Gillespie, D.R., Sanderson, J.P. 2016. Biological control outcomes using the aphidophagous predator Aphidoletes aphidimyza (Diptera: Cecidomyiidae) under multi-prey conditions: Effect of crop stage, within-plant aphid distribution and apparent competition. Insects. 7(4):15.
Wraight, S.P., Ramos, M.E. 2017. Effects of mode of inoculation on efficacy of wettable powder and oil dispersion formulations of Beauveria bassiana applied against Colorado potato beetle larvae under low-humidity greenhouse conditions. Biocontrol Science and Technology. 27:348-363.
Wraight, S.P., Ramos, M.E. 2017. Characterization of the synergistic interaction between Beauveria bassiana strain GHA and Bacillus thuringiensis morrisoni strain tenebrionis applied against Colorado potato beetle. Journal of Invertebrate Pathology. 144:47-57.
Castrillo, L.A., Griggs, M., Vandenberg, J.D. 2016. Competition between biological control fungi and fungal symbionts of ambrosia beetles Xylosandrus crassiusculus and X. germanus (Coleoptera:Cuculionidae): mycelial interactions and impact on beetle brood production. Biological Control. 103:138-146.
Parisio, M.S., Gould, J.R., Vandenberg, J.D., Bauer, L.S., Fierke, M.K. 2016. Evaluation of recovery and monitoring methods for parasitoids released against Emerald Ash Borer. Biological Control. 106:45-53.