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

Location: Emerging Pests and Pathogens Research

2016 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, Cornell University, and the University of Hawaii-Manoa are conducting field studies aimed at assessing efficacy of spray applications of Beauveria bassiana (Bb) applied for areawide control of the coffee berry borer Hypothenemus hampei (see accomplishments). In support of field-efficacy evaluations, molecular genetic studies aimed at characterization of the diverse naturally-occurring strains of Bb found infecting Coffee Berry Borer (CBB) in Hawaii are continuing. To date we have extracted >2000 Bb isolates from foundress CBB infesting coffee berries collected from our study fields in South Kona and at survey sites in the districts of North and South Kona, Ka'u, South Hilo, and Hamakua. Sampling was expanded in 2016 to include soils from coffee fields in South Kona and Ka’u. Preliminary findings indicate that Bb commercial strain GHA (Bb GHA formulated as BotaniGard®) is no more persistent in the soil than it is in the crop canopy. Bb populations are dominated by GHA following spray applications, but rapidly decrease to levels below those of the feral strains, which exhibit much greater natural epizootic potential and persistence in CBB populations. A new treatment, application of both BotaniGard and an added surfactant at ½ the recommended rates, was included in our 2016 field studies. In terms of post-spray percent infection, we observed comparable efficacy of the full- vs. ½ -rate treatments at high elevation, but reduced efficacy of the low rate under the much drier conditions at low elevation. These treatments will require replication over multiple seasons before any recommendations can be formulated. Studies investigating interactions between Bb GHA and various agrochemicals used to control plant pests and diseases in ornamental greenhouse crops are continuing. A summary of our findings on interactions between Bb and the biorational chemical insecticide Entrust (based on spinosoid bacterial toxins) applied against western flower thrips (WFT) is presented below. Another biorational insecticide based on a bacterial toxin (Grandevo®) was included in these studies, but proved ineffective against WFT in our laboratory bioassays. Serving as a positive control, the broad-spectrum benzimidazole fungicide Benomyl applied to bean leaf disks strongly inhibited activity of Bb sprayed on the leaf disks after the fungicide residue was allowed to dry. As a result of this antagonism, few Bb infections were observed among larval thrips exposed to the treated leaf disks (75% reduction in Bb activity). Studies of interactions between Bb GHA and Trichoderma-based fungicides (Rootshield®/Plantshield®) are in progress. Studies aimed at defining the optimal periods of high-humidity necessary to support high rates of infection by entomopathogenic fungi applied against WFT over a range of temperature conditions were initiated in collaboration with a visiting Fulbright Scholar from the University of Agriculture, Faisalabad, Pakistan. At constant 25ºC, maximum infection of larval thrips resulted from maintenance of near-moisture-saturated (ca. 100% RH) conditions for a period of 48 h following initial exposure to Bb GHA conidia on bean leaves. Only low levels of infection (< 43%) resulted from high-humidity periods of < 24 h. In the case of Metarhizium brunneum strain F52, maximum infection was observed after exposure to high humidity for 40 h, with low rates of infection (< 50%) resulting from high-humidity periods < 16 h. These observations suggest, as hypothesized, that Mb is infectious at somewhat lower levels of moisture than Bb; however, in greenhouse tests, this positive attribute of Mb has not translated to greater control of WFT infesting flowering impatiens. 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 entomopathogenic fungi Beauveria bassiana and Metarhizium brunneum 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. Asian ambrosia beetles Xylosandrus crassiusculus and X. germanus are among the most important exotic pests of orchards and nurseries in the U.S. and are difficult to control using conventional insecticides because of their cryptic habits. The use of biological control agents may prove effective by targeting both beetles and fungal symbionts inside tree galleries: entomopathogenic fungi could be used to target adult female beetles and their brood, or mycoparasitic fungi, e.g., Trichoderma harzianum, could be used to target their associated fungal symbionts. In collaboration with researchers at Cornell University, we used a combination of fungal and insect assays to examine competition between beetle-symbiotic fungi and biological control fungi. We also examined the impact of biological control fungi on beetle brood production. Laboratory assays showed T. harzianum outcompeted different strains of Ambrosiella roeperi and A. grosmanniae associated with X. crassiusculus and X. germanus, respectively, whether in primary or secondary resource capture assays. In contrast, entomopathogenic fungi Beauveria bassiana and Metarhizium brunneum blocked the spread of symbionts only in primary competition assays. Complementary assays showed that beetle galleries in T. harzianum-treated beech stems had sparse symbiont growth, many of which contained no, or only a small number, of beetle offspring. The number of offspring produced by foundresses in T. harzianum-treated stems were reduced compared to controls and comparable to those in stems treated with either entomopathogenic fungus at the higher dosages. In these galleries, reduced brood production was likely due to adult female mortality prior to laying eggs or after laying only a few eggs. These results show the potential of using biological control fungi in targeting ambrosia beetle populations either directly by killing foundresses and reducing brood production or indirectly by suppressing symbiont growth in their galleries. Field studies of these fungi were done in collaboration with Tennessee State and Cornell Universities against three invasive Asian ambrosia beetles, Xylosandrus crassiusculus and Cnestus mutilatus in Tennessee and X. germanus in New York. Results from the second year of field tests showed that all three of the fungal products tested resulted in fewer beetle attacks. The effect was most pronounced for Metarhizium brunneum. This repellency was evident in field studies both in New York, where more than 90% of the attacks were from X. germanus, and in Tennessee, where mixed ambrosia beetle attacks from X. crassiusculus and Cnestus mutilatus were observed. Examination of infested logs showed infected foundresses that produced galleries with shorter tunnels and smaller brood, some of which also showed fungal infection. A third season of trials are underway.

Accomplishments
1. Effectiveness of insect pathogenic fungi against western flower thrips. Efficacy of Beauveria- and Metarhizium-based biopesticides has proven inconsistent under commercial-greenhouse conditions. ARS researchers in Ithaca, New York conducted studies to identify environmental conditions, particularly levels of moisture, required to promote sufficient activity of these pathogens to control western flower thrips on ornamental crops. A program of twice-weekly sprays, applied during the late afternoon at 3 to 4-day intervals, provided more than 70% control (sufficient to prevent pest population increases) when ambient greenhouse relative humidity was maintained at more than 90% for approximately 40 hours following each spray application. These findings provide greenhouse growers with information necessary for the successful use of these microbial biocontrol agents.

2. Negative interaction between insect-pathogenic fungi and an insecticide used against western flower thrips. Previous studies have demonstrated enhanced (synergistic) activity of fungal pathogens applied in combination with various insecticides. Such activity has potential to improve the pest control ability of these microbial biocontrol agents. In collaboration with a visiting Fulbright scholar from the University of Agriculture, Faisalabad, Pakistan, ARS researchers in Ithaca, New York, investigated interactions between Beauveria bassiana and Metarhizium brunneum and the biorational insecticide Entrust®. Exposing thrips to mixtures of fungal spores and Entrust resulted in 8–10% lower-than-expected mortality. The negative interactions were mitigated under extended high-humidity conditions (optimal for fungal infection). Results suggest these control agents might best be used in rotation rather than in combination; however, considering the low level of negative interaction, efficiencies gained from tank mixing would likely outweigh the consequent minor losses in efficacy.

3. Efficacy of Beauveria bassiana for areawide control of coffee berry borer (CBB). Following an invasive outbreak in 2010, the entomopathogenic fungus Beauveria bassiana (Bb) has been applied for CBB management in Hawaii; however, efficacy of these applications has not been thoroughly evaluated. ARS researchers from Ithaca, New York, in collaboration with researchers from ARS in Hilo, Hawaii, the University of Hawaii at Manoa, and Cornell University, conducted extensive field studies in the Kona coffee-producing districts of Hawaii. Genetic characterizations of Bb isolates from CBB enabled detailed assessments taking into account the diverse naturally-occurring strains of this pathogen also active against CBB in Hawaii. Individual spray applications of Bb resulted in infection of 25–40% of CBB. Cumulative rates of infection from monthly sprays reached 50–70%, with highest levels observed in areas where sound sanitation practices were employed (removal of all berries from trees between field seasons). These findings are informing economic analyses of the CBB Areawide Control Program and enabling growers to successfully integrate this pathogen with other control practices.

4. Inexpensive traps for efficient monitoring of emerald ash borer parasitoids. The emerald ash borer (EAB), Agrilus planipennis, is a destructive invasive insect pest and the target of an extensive biological control campaign designed to mitigate EAB-caused ash tree mortality. Three wasp parasitoids of EAB have been released in North America to establish them as biocontrol agents. ARS researchers in Ithaca, New York, in collaboration with researchers at the State University of New York, USDA APHIS, and USDA Forest Service, evaluated alternative methods to document establishment and monitor dispersal of these parasitoids in the field. Simple, liquid-filled yellow pan traps were as effective as more labor-intensive methods for detecting parasitoids, but tree felling and bark peeling would still be required for quantification of parasitism within EAB populations. As a result of our studies, we recommend that yellow pan traps, the least expensive and most convenient monitoring tool, be used as the preferred method for parasitoid recovery as the scale of the EAB biological control program continues to expand and monitoring efforts correspondingly increase.

None.

Review Publications
Wraight, S.P., Ugine, T.A., Ramos, M.E., Sanderson, J.P. 2016. Efficacy of spray applications of entomopathogenic fungi against western flower thrips infesting greenhouse impatiens under variable moisture conditions. Biological Control. 97:31-47.
Wraight, S.P., Filotas, M.J., Sanderson, J.P. 2016. Comparative efficacy of emulsifiable-oil, wettable-powder, and unformulated-powder preparations of Beauveria bassiana against the melon aphid Aphis gossypii. Biocontrol Science and Technology. 26:894-914.