Botrytis fruit rot or gray mold, caused by Botrytis cinerea, is common fruit disease in California strawberries (Koike et al. 2018). Botrytis cinerea has a wide host range infecting several commercially important crops including blueberry (Saito et al. 2016), grapes (Saito et al., 2019), and tomato (Breeze, 2019). Fungal infection can cause flower or fruit rot. Fruit can be infected directly or through a latent infection in the flowers. Moist and cool conditions favor fungal infections and increased sugar content in the ripening fruit can also contribute to the disease development. Initial symptoms of infection appear as brown lesions and a thick mat of gray conidia is characteristic symptom in the later stages of infection. As chemical fungicides are primarily used for gray mold control, fungicide resistance is a common problem around the world (Panebianco et al., 2015; Liu et al., 2016; Stockwell et al., 2018; Weber and Hahn, 2019). In strawberry, cultural control options such as removing diseased plant material or using cultivars with traits that can reduce gray mold infections may not be practical when the disease is widespread in the field or cultivar choice is made based on other factors. Non-chemical control options are necessary to help reduce the risk of chemical fungicide resistance, prolong the life of available chemical fungicides, achieve desired disease control, and to maintain environmental health. Although there are several botanical and microbial fungicides available for gray mold control, limited information is available on their efficacy in California strawberries. A study was conducted in the spring of 2019 to evaluate the efficacy of several chemical, botanical, and microbial fungicides in certain combinations and rotations to help identify effective options for an integrated disease management strategy.
Strawberry cultivar San Andreas was planted late November, 2018 and the study was conducted in April and May, 2019. Each treatment had a 20' long strawberry plot with two rows of plants replicated in a randomized complete block design. Plots were maintained without any fungicidal applications until the study was initiated. Table 1 contains the list of treatments, application rates and dates of application, and Table 2 contains the type of fungicide used and their mode of action. Beauveria bassiana and Metarhizium anisopliae s.l. are California isolates of entomopathogenic fungi, isolated from an insect and a soil sample, respectively. These fungi are pathogenic to a variety of arthropods and some strains are formulated as biopesticides for arthropod control. However, earlier studies in California demonstrated that these fungi are also known to antagonize plant pathogens such as Fusarium oxysporum f.sp. vasinfectum Race 4 (Dara et al., 2016) and Macrophomina phaseolina (Dara et al., 2018) and reduce the disease severity. To further evaluate their efficacy against B. cinerea, these two fungi were also included in this study alternating with two chemical fungicides.
Treatments were applied with a CO2-pressurized backpack sprayer using 66.5 gpa spray volume. Five days before the first spray application and 3 days after each application, all ripe fruit were harvested from each plot and incubated at the room temperature in vented plastic containers. The level of gray mold on fruit from each plot was rated using a 0 to 4 scale (where 0=no disease, 1=1-25% fruit with fungal infection, 2=26-50% infection, 3=51-75%, and 4=76-100%) 3 and 5 days after each harvest (DAH). Due to the rains, fruit could not be harvested after the 3rd spray application for disease rating, but was harvested and discarded after the rains to avoid cross infection for the following week's harvest. Data were analyzed using analysis of variance using Statistix software and significant means were separated using Least Significant Difference separation test.
Gray mold occurred at low to moderate levels during the study period. Along with B. cinerea, there were a few instances of minor fungal infections from Rhizopus spp. (Rhizopus fruit rot) and Mucor spp. (Mucor fruit rot). Pre-treatment disease ratings were statistically not significant (P = 0.6197 and 0.5741) 3 and 5 DAH. While the chemical standard treatment with the rotation of Captan, Merivon, Switch, and Pristine (treatment 2) appeared to result in the lowest disease rating throughout the observation period, treatments 3 and 5 after the 1st spray application, treatments 5 and 11 along with 3, 4 and 6 after the 2nd spray application, and treatments 3 and 5 along with 11 after the 4th spray application also had similar disease control at 3 DAH. When disease at 5 DAH was compared, the lowest rating was seen in treatment 2 after the 1st and 2nd spray applications, and treatments 2, 3, and 11 after the 4th application. Several other treatments also provided statistically similar control during these days.
When the average disease rating for the three post-treatment observation events was considered, treatment 2, 3, 5, and 11 had the lowest disease at both 3 and 5 DAH. Treatments 4 and 12 at 3 DAH also had a statistically similar level of disease control to treatment 2.
In general, most of the treatments provided moderate to high control compared to the disease in untreated control when the post-treatment averages were considered. Only treatment 7 and 13 had lower control at 3 DAH.
This study compared a variety of registered and developmental products along with two entomopathogenic fungi in managing B. cinerea. Considering the fungicide resistance problem in B. cinerea in multiple crops, having multiple non-chemical control options is very important to achieve desirable control with integrated disease management strategies. Since the active ingredients in the botanical and bacterial fungicides used in this study are not public, discuss will be limited on their modes of action and efficacy at this point. Similarly, the active ingredient of WXF-17001 is also not known, however, an earlier study by Calvo-Garrido et al. (2014) demonstrated that a fatty acid-based natural product reduced B. cinerea conidial germination by 54% and disease severity in grapes by 96% compared to untreated control. The product used by Calvo-Garrido et al. (2014) is thought to be fungistatic and reduce the postharvest respiratory activity and ethylene production in fruits.
While chemical fungicides have a specific mode of action, biological and other products act in multiple manners either directly antagonizing the plant pathogen or by triggering the plant defenses. For example, amending the potting medium with biochar resulted in induced systemic resistance in tomato and reduced B. cinerea severity by 50% (Mehari et al., 2015). Luna et al. (2016) also showed that application of β-aminobutyric acid and jasmonic acid promoted seed germination and long-term resistance to B. cinerea in tomato. Burkholderia phytofirmans, beneficial endophytic bacterium, offered protection against B. cinerea in grapes by mobilizing carbon resources (callose deposition), triggering plant immune system (hydrogen peroxide production and priming of defense genese), and through antifungal activity (Miotto-Vilanova et al. 2016). Similarly, entomopathogenic fungi such as B. bassiana are also known to induce systemic resistance against plant pathogens (Griffin et al. 2006). Compared to other options evaluated in the study, entomopathogenic fungi have an advantage of controlling both arthropod pests and diseases, while also having plant growth promoting effect (Dara et al. 2017).
Rotating fungicides with different mode of actions reduces the risk of resistance development and using some combinations will also maintain control efficacy. This study provided the efficacy of multiple control options and their combinations and rotations for B. cinerea. This is also the first study demonstrating the efficacy of entomopathogenic fungi against B. cinerea in strawberry.
Acknowledgements: Thanks to Sipcam Agro and Westbridge for funding the study, technical assistance of Hamza Khairi for data collection, and the field staff at the Shafter Research Station for the crop maintenance.
Breeze, E. 2019. 97 Shades of gray: genetic interactions of the gray mold, Botrytis cinerea, with wild and domesticated tomato. The Plant Cell 31: 280-281. https://doi.org/10.1105/tpc.19.00030
Calvo-Garrido, C., A.A.G. Elmer, F. J. Parry, I. Viñas, J. Usall, R. Torres, R.H. Agnew, and N. Teixidó. 2014. Mode of action of a fatty acid-based natural product to control Botrytis cinerea in grapes. J. Appl. Microbiol. 116: 967-979. https://doi.org/10.1111/jam.12430
Dara, S. K., S. S. Dara, S.S.R. Dara, and T. Anderson. 2016. First report of three entomopathogenic fungi offering protection against the plant pathogen, Fusarium oxysporum f.sp. vasinfectum. UC ANR eJournal of Entomology and Biologicals https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=22199
Dara, S. K., S.S.R. Dara, and S. S. Dara. 2017. Impact of entomopathogenic fungi on the growth, development, and health of cabbage growing under water stress. Amer. J. Plant Sci. 8: 1224-1233. https://doi.org/10.4236/ajps.2017.86081
Dara, S.S.R., S. S. Dara, and S. K. Dara. 2018. Preliminary report on the potential of Beauveria bassiana and Metarhizium anisopliae s.l. in antagonizing the charcoal rot causing fungus Macrophomina phaseolina in strawberry. UC ANR eJournal of Entomology and Biologicals https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=28274
Griffin, M. R., B. H. Ownley, W. E. Klingeman, and R. M. Pereira. 2006. Evidence of induced systemic resistance with Beauveria bassiana against Xanthomonas in cotton. Phytopathol. 96.
Koike, S. T., G. T. Browne, T. R. Gordon, and M. P. Bolda. 2018. UC IPM pest management guidelines: strawberry (diseases). UC ANR Publication 3468. https://www2.ipm.ucanr.edu/agriculture/strawberry/Botrytis-Fruit-Rot/
Liu, S., Z. Che, and G. Chen. 2016. Multiple-fungicide resistance to carbendazim, diethofencardb, procymidone, and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province, China. Crop Protection 84: 56-61.
Luna, E., E. Beardon, S. Ravnskov, J. Scholes, and J. Ton. 2016. Optimizing chemically induced resistance in tomato against Botrytis cinerea. Plant Dis. 100: 704-710. https://doi.org/10.1094/PDIS-03-15-0347-RE
Mehari, Z. H., Y. Elad, D. Rav-David, E. R. Graber, and Y. M. Harel. 2015. Induced systemic resistance in tomato (Solanum lycopersicum) against Botrytis cinerea by biochar amendment involves jasmonic acid signaling. Plant and Soil 395: 31-44.
Miotto-Vilanova, L., C. Jacquard, B. Courteaux, L. Wortham, J. Michel, C. Clément, E. A. Barka, and L. Sanchez. 2016. Burkholderia phytofirmans PsJN confers grapevine resistance against Botrytis cinerea via a direct antimicrobial effect combined with a better resource mobilization. Front. Plant Sci. 7: 1236. https://doi.org/10.3389/fpls.2016.01236
Panebianco, A., I. Castello, G. Cirvilleri, G. Perrone, F. Epifani, M. Ferrarra, G. Polizzi, D. R. Walters, and A. Vitale. 2015. Detection of Botrytis cinerea field isolates with multiple fungicide resistance from table grape in Sicily. Crop Protection 77: 65-73.
Saito, S., T. J. Michailides, and C. L. Xiao. 2016. Fungicide resistance profiling in Botrytis cinerea populations from blueberry in California and Washington and their impact on control of gray mold. Plant Dis. 100: 2087-2093. https://doi.org/10.1094/PDIS-02-16-0229-RE
Saito, S., T. J. Michailides, and C. L. Xiao. 2019. Fungicide-resistant phenotypes in Botrytis cinerea populations and their impact on control of gray mold on stored table grapes in California. European J. Plant Pathol. 154: 203-213.
Stockwell, V. O., B. T> Shaffer, L. A. Jones, and J. W. Pscheidt. 2018. Fungicide resistance profiles of Botrytis cinerea isolated from berry crops in Oregon. Abstract for International Congress of Plant Pathology: Plant Health in A Global Economy; 2018 July 29-Aug 3; Boston, MA.
Weber, R.W.S. and M. Hahn. 2019. Grey mould disease of strawberry in northern Germany: causal agents, fungicide resistance and management strategies. Appl. Microbiol. Biotechnol. 103: 1589-1597.
- Author: Surendra K. Dara
- Author: Suchitra S. Dara
- Author: Stefan Jaronski, USDA-ARS
The western Grapeleaf skeletonizer (WGLS), Harrisina metallica Stretch (Lepidoptera: Zygaenidae), previously known to cause severe defoliation to vineyards and backyard grapevines appears to be re-emerging in California. Since its first detection in San Diego in 1941, WGLS spread through commercial vineyards and backyard grapes becoming a serious problem. Although two biological control agents from Arizona and Mexico were introduced in California for WGLS control, a naturally occurring granulovirus (Harrisina brillians granulovirus) nearly eradicated WGLS populations and kept them under control. WGLS has not been a problem especially in conventional vineyards. However, based on some unpublished observations, WGLS populations are emerging in organic vineyards and backyard grapevines.
WGLS lives up to its name by skeletonizing and defoliating grape leaves. Organic vineyards are especially at risk and uncontrolled populations can destroy vineyards resulting in significant losses. Metallic bluish or greenish black moths lay barrel shaped yellowish eggs on the lower side of the leaves. There are five larval instars. Early instars are cream colored and develop black and purple bands in later stages. Pupation occurs in a whitish cocoon. Upon hatching, larvae start feeding side by side in a row on the lower side of leaf. Damage by younger larvae appears as whitish leaf area containing veins and the upper cuticle, which eventually turn brown. Older larvae skeletonize leaves leaving larger veins. Larvae may also feed on fruit leading to bunch rot. Severe damage can cause defoliation and sunburn of the exposed fruit.
A study was conducted to evaluate the efficacy of six non-chemical control options that included formulations of spinosad, two subspecies of Bacillus thuringiensis, and a botanical insecticide/growth regulator along with two unformulated entomopathogenic fungal isolates native to California. Larvae were collected from an infested, untreated backyard grapevine and maintained in one gallon plastic tubs with screened lids on infested leaves. Fresh, untreated grape leaves from uninfested vines were provided daily for 3 days before starting the assay. For each treatment, five 4-5 instar larvae were placed on a grape leaf disc (rinsed in water and dried) in a Petri plate (100 mm dia) with a moist filter paper. Larvae were treated by spraying 1 ml of the treatment solution (containing Dyne-Amic as a surfactant at 0.125% vol/vol). Application rates for commercial formulations were determined based on label recommendations for 100 gallons of spray volume. Entomopathogenic fungal concentrations were also determined based on the label rates for similar commercial products. Treatments were replicated four times and the assay was conducted twice. Larval mortality was observed daily and dead larvae were removed and incubated separately. Fresh leaf discs were provided as needed to the remaining larvae. Actual and corrected (for control mortality) total mortality were calculated.Data were arcsine-transformed for statistical analysis and significant means were separated using Tukey's HSD test.
Both cumulative daily mortality and total mortality significantly (P < 0.0001) differed among treatments. Entrust and M. anisopliae resulted in the highest mortality followed by B. bassiana, Neemix, and Agree. In general, feeding reduced or ceased in all larvae following treatment and could have contributed to a lower mortality in B. thuringiensis treatments. Entomopathogenic fungi emerged from all the cadavers from respective treatments. Microbial and botanical options provided good control of WGLS. These non-chemical alternatives can be effectively used in both organic and conventional vineyards. California isolates of B. bassiana and M. anisopliae demonstrated good control efficacy and the potential to be developed as microbial pesticides.
Acknowledgements: Thanks to the technical assistance of Alor Sahoo in carrying out these assays, and Certis and Corteva for providing the pesticide formulations.
Federici, B. A. and V. M. Stern. 1990. Replication and occlusion of a granulosis virus in larval and adult midgut epithlium of the western grapeleaf skeletonizer, Harrisina brillians. J. Invertebr. Pathol. 56: 401-414.
- Author: Sumanth S. R. Dara
- Author: Suchitra S. Dara
- Author: Surendra K. Dara
Charcoal rot, caused by Macrophomina phaseolina, is one of the important fungal diseases of strawberry in California. Macrophomina phaseolina is a soilborne fungus and has a wide host range, including alfalfa, cabbage, corn, pepper, and potato, some of which are cultivated in the strawberry production areas in California. The fungus infects the vascular system of the plant roots, obstructing the nutrient and water supply and ultimately resulting in stunted growth, wilting, and death of the plant. The fungus survives in the soil and infected plant debris as microsclerotia (resting structures made of hyphal bodies) and can persist for up to three years. Microslerotia germinate and penetrate the root system to initiate infection. Plants are more vulnerable to fungal infection when they are experiencing environmental (extreme weather or drought conditions) and physiological (heavy fruit bearing) stress.
Soil fumigation is the primary management option for addressing charcoal rot in strawberry. Crop rotation with broccoli can also reduce the risk of charcoal rot due to glucosinolates and isothiocyanates in broccoli crop residue that have fungicidal properties. Beneficial microorganisms such as Bacillus spp. and Trichoderma spp. are also considered, especially in organic strawberries, to antagonize M. phaseolina and other soilborne pathogens and provide some protection. The role of beneficial microbes in disease management or improving crop growth and health is gaining popularity in the recent years with the commercial availability of biofungicide, biostimulant, and soil amendment products. In a couple of recent strawberry field studies in Santa Maria, some of the beneficial microbial products improved fruit yield or crop health. These treatments can be administered by inoculating the transplants prior to planting, immediately after planting or periodically applying to the plants and or the soil. Adding beneficial microbes can help improve the soil microbiome especially after chemical or bio-fumigation and anaerobic soil disinfestation.
Similar to the benefits of traditionally used bacteria (e.g., Bacillus spp. and Pseudomonas spp.) and fungi (e.g., Glomus spp. and Trichoderma spp.), studies with entomopathogenic fungi such as Beauveria bassiana, Isaria fumosorosea, and Metarhizium spp. also demonstrated their role in improving water and nutrient absorption or antagonizing plant pathogens. The advantage of entomopathogenic fungi is that they are already used for arthropod pest management in multiple crops, including strawberry; now, there are the additional benefits of promoting crop growth and antagonizing plant pathogens. In light of some promising recent studies exploring these roles, a study was conducted using potted strawberry plants to evaluate the efficacy of two California isolates of Beauveria bassiana and Metarhizium anisopliae s.l. and their application strategies against M. phaseolina.
About 6 week old strawberry plants (cultivar Albion) from a strawberry field at the Shafter Research Station were transplanted into 1.6-gallon pots with Miracle-Gro All Purpose Garden Soil (0.09-0.05-0.07 N-P-K) and maintained in an outdoor environment. They were regularly watered, and their health was monitored for about 5 months prior to the commencement of the study. Conidial suspensions of the California isolates of B. bassiana and M. anisopliae s.l. were applied one week before, after, or at the time of applying microsclerotia of M. phaseolina to the potting mix. The following treatments were evaluated in the study:
- Untreated control
- Soil inoculated with M. phaseolina
- Soil inoculated with B. bassiana 1 week prior to M. phaseolina inoculation
- Soil inoculated with M. anisopliae s.l. 1 week prior to M. phaseolina inoculation
- Soil inoculated with B. bassiana at the time of M. phaseolina inoculation
- Soil inoculated with M. anisopliae s.l. at the time of M. phaseolina inoculation
- Soil inoculated with B. bassiana 1 week after to M. phaseolina inoculation
- Soil inoculated with M. anisopliae s.l. 1 week after to M. phaseolina inoculation
Entomopathogenic fungi were applied as 1X1010 viable conidia in 100 ml of 0.01% Dyne-Amic (surfactant) solution distributed around the plant base. To apply M. phaseolina, 5 grams of infested cornmeal-sand inoculum containing 2,500 CFU/gram was added to four 5 cm deep holes around the base of the plant. Each treatment had six pots each planted with a single strawberry plant representing a replication. Treatments were randomly arranged within each replication. The study was repeated once a few days after the initiation of the first experiment.
Plant health was monitored starting from the first week after the M. phaseolina inoculation and continued for seven weeks. Plant health was rated on a scale of 0 to 5 where 0=dead and 5=very healthy and the rest of the ratings in between depending on the extent of wilting. Data from both experiments were combined and analyzed by ANOVA using Statistix software and significant means were separated using LSD test. The influence of entomopathogenic fungal treatments applied at different times as well as the combined effect of different applications within each fungus were compared for seven weeks. Ratings for some plants that were scorched from hot summer temperatures and died abruptly were removed from the analyses.
Untreated control plants maintained good health throughout the observation period varying between the rating of 4.3 and 4.9. In general, plant health declined considerably from the 5th week after M. phaseolina inoculation. Plant health appeared to be slightly better in plants treated with entomopathogenic fungi, but there was no statistically significant difference in any except one instance. Plants treated with M. anisopliae one week prior to the application of M. phaseolina had a rating of 3.0 compared to 1.6 rating of plants inoculated with M. phaseolina alone.
When data from different treatments for each entomopathogenic fungus were compared, both B. bassiana and M. anisopliae s.l. appeared to reduce the wilting, but the plant health rating was not significantly different from the M. phaseolina treatment alone.
This is the first report of the impact of entomopathogenic fungi on M. phaseolina with some promise. Additional studies under more uniform environmental conditions and with more treatment options would shed more light on this approach of using entomopathogenic fungi against M. phaseolina. The current study evaluated single application of the entomopathogenic fungi and we plan to conduct additional studies with multiple applications.
Acknowledgements: We thank Dr. Kelly Ivors (previously at Cal Poly San Luis Obispo) for the pathogen inoculum and Dr. Stefan Jaronski, USDA-ARS, Sidney, MT for multiplying the entomopathogenic fungal inocula.
Dara, S. K. and D. Peck. 2017. Evaluating beneficial microbe-based products for their impact on strawberry plant growth, health, and fruit yield. UC ANR eJournal Strawberries and Vegetables. https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=25122
Dara, S. K. and D. Peck. 2018. Evaluation of additive, soil amendment, and biostimulant products in Santa Maria strawberry. CAPCA Adviser, 21(5): 44-50.
Dara, S. K., S.S.R. Dara, and S. S. Dara. 2017. Impact of entomopathogenic fungi on the growth, development, and health of cabbage growing under water stress. Amer. J. Plant Sci. 8: 1224-1233. http://file.scirp.org/pdf/AJPS_2017051714172937.pdf
Dara, S. K., S. S. Dara, S.S.R. Dara, and T. Anderson. 2016. First report of three entomopathogenic fungi offering protection against the plant pathogen, Fusarium oxysporum f.sp. vasinfectum. UC ANR eJournal Strawberries and Vegetables. https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=22199
Koike, S. T., G. T. Browne, and T. R. Gordon. 2013. UC IPM pest management guidelines: Strawberry diseases. UC ANR Publication 3468. http://ipm.ucanr.edu/PMG/r734101511.html
Partridge, D. 2003. Macrophomina phaseolina. PP728 Pathogen Profiles, Department of Plant Pathology, North Carolina State University. https://projects.ncsu.edu/cals/course/pp728/Macrophomina/macrophominia_phaseolinia.HTM
Vasebi, Y., N. Safaie, and A. Alizadeh. 2013. Biological control of soybean charcoal root rot disease using bacterial and fungal antagonists in vitro and greenhouse condition. J. Crop Prot. 2(2): 139-150.
Entomopathogens are microorganisms that are pathogenic to arthropods such as insects, mites, and ticks. Several species of naturally occurring bacteria, fungi, nematodes, and viruses infect a variety of arthropod pests and play an important role in their management. Some entomopathogens are mass-produced in vitro (bacteria, fungi, and nematodes) or in vivo (nematodes and viruses) and sold commercially. In some cases, they are also produced on small scale for non-commercial local use. Using entomopathogens as biopesticides in pest management is called microbial control, which can be a critical part of integrated pest management (IPM) against several pests.
Some entomopathogens have been or are being used in a classical microbial control approach where exotic microorganisms are imported and released for managing invasive pests for long-term control. The release of exotic microorganisms is highly regulated and is done by government agencies only after extensive and rigorous tests. In contrast, commercially available entomopathogens are released through inundative application methods as biopesticides and are commonly used by farmers, government agencies, and homeowners. Understanding the mode of action, ecological adaptations, host range, and dynamics of pathogen-arthropod-plant interactions is essential for successfully utilizing entomopathogen-based biopesticides for pest management in agriculture, horticulture, orchard, landscape, turf grass, and urban environments.
Important entomopathogen groups and the modes of their infection process are described below.
There are spore-forming bacterial entomopathogens such as Bacillus spp., Paenibacillus spp., and Clostridium spp, and non-spore-forming ones that belong to the genera Pseudomonas, Serratia, Yersinia, Photorhabdus, and Xenorhabdus. Infection occurs when bacteria are ingested by susceptible insect hosts. Pseudomonas, Serratia and Yersinia are not registered in the USA for insect control.Several species of the soilborne bacteria, Bacillus and Paenibacillus are pathogenic to coleopteran, dipteran, and lepidopteran insects. Bacillus thuringiensis subsp. aizawai, Bt subsp. kurstaki, Bt subsp. israelensis, Bt subsp. sphaericus, and Bt subsp. tenebrionis are effectively used for controlling different groups of target insects. For example, Bt subsp. aizawai and Bt subsp. kurstaki are effective against caterpillars, Bt subsp. israelensis and Bt subsp. sphaericus target mosquito larvae, and Bt subsp. tenebrionis is effective against some coleopterans.
When Bt is ingested, alkaline conditions in the insect gut (pH 8-11) activate the toxic protein (delta-endotoxin) that attaches to the receptors sites in the midgut and creates pore in midgut cells. This leads to the loss of osmoregulation, midgut paralysis, and cell lysis. Contents of the gut leak into insect's body cavity (hemocoel) and the blood (hemolymph) leaks into the gut disrupting the pH balance. Bacteria that enter body cavity cause septicemia and eventual death of the host insect. Insects show different kinds of responses to Bt toxins depending on the crystal proteins (delta-endotoxin), receptor sites, production of other toxins (exotoxins), and requirement of spore. The type responses below are based on the susceptibility of caterpillars to Bt toxins.
Type I response – Midgut paralysis occurs within a few minutes after delta-endotoxin is ingested. Symptoms include cessation of feeding, increase in hemolymph pH, vomiting, diarrhea, and sluggishness. General paralysis and septicemia occur in 24-48 hours resulting in the death of the insect. Examples of insects that show Type I response include silkworm, tomato hornworm, and tobacco hornworm.
Type II response – Midgut paralysis occurs within a few minutes after the ingestion of delta-endotoxin, but there will be no general paralysis. Septicemia occurs within 24-72 hours. Examples include inchworms, alfalfa caterpillar, and cabbage butterfly.
Type III response – Midgut paralysis occurs after delta-endotoxin is ingested followed by cessation of feeding. Insect may move actively as there will be no general paralysis. Mortality occurs in 48-96 hours. Higher mortality occurs if spores are ingested. Insect examples include Mediterranean flour moth, corn earworm, gypsy moth, spruce budworm.
Type IV response – Insects are naturally resistant to infection and older instars are less susceptible than the younger ones. Midgut paralysis occurs after delta-endotoxin is ingested followed by cessation of feeding. Insect may move actively as there will be no general paralysis. Mortality occurs in 72-96 or more hours. Higher mortality occurs if spores are ingested. Cutworms and armyworms are examples for this category.
Unlike caterpillars, the response in mosquitoes is different where upon ingestion of Bt subsp. israelensis delta-endotoxin, the mosquito larva is killed within 20-30 min.
While Bt with its toxic proteins is very effective as a biopesticide against several pests, excessive use can lead to resistance development. Corn earworm, diamondback moth, and tobacco budworm are some of the insects that developed resistance to Bt toxins. Genetic engineering allowed genes that express Bt toxins to be inserted into plants such as corn, cotton, eggplant, potato, and soybean and reduced the need to spray pesticides. However, appropriate management strategies are necessary to reduce insect resistant to Bt toxins in transgenic plants.
Paenibacillus popilliae is commonly used against Japanese beetle larvae and known to cause the milky spore disease. Although Serratia is not registered for use in the USA, a species is registered for use against a pasture insect in New Zealand. In the case of Photorhabdus spp. and Xenorhabdus spp., which live in entomopathogenic nematodes symbiotically, bacteria gain entry into the insect host through nematodes. Biopesticides based on heat-killed Chromobacterium subtsugae and Burkholderia rinojensis are reported to have multiple modes of action and target mite and insect pests of different orders.
Entomopathogenic fungi typically cause infection when spores come in contact with the arthropod host. Under ideal conditions of moderate temperatures and high relative humidity, fungal spores germinate and breach the insect cuticle through enzymatic degradation and mechanical pressure to gain entry into the insect body. Once inside the body, the fungi multiply, invade the insect tissues, emerge from the dead insect, and produce more spores. Natural epizootics of entomophthoralean fungi such as Entomophaga maimaiga (in gypsy moth), Entomophthora muscae (in flies), Neozygites fresenii (in aphids), N. floridana (in mites), and Pandora neoaphidis (in aphids) are known to cause significant reductions in host populations. Although these fastidious fungi are difficult to culture in artificial media and do not have the potential to be sold as biopesticides they are still important in natural control of some pest species. Hypoclealean fungi such as Beauveria bassiana, Isaria fumosorosea, Hirsutella thompsonii, Lecanicillium lecanii, Metarhizium acridum, M. anisopliae, and M. brunneum, on the other hand, are commercially sold as biopesticides in multiple formulations around the world. Fungal pathogens have a broad host range and are especially suitable for controlling pests that have piercing and sucking mouthparts because spores do not have to be ingested. However, entomopathogenic fungi are also effective against a variety of pests such as wireworms and borers that have chewing mouthparts.
Related to fungi, the spore-forming microsporidium, Paranosema (Nosema) locustae is a pathogen that has been used for controlling locusts, grasshoppers, and some crickets. When P. locustae is ingested, the midgut tissues become infected, followed by infection in the fat body tissues. The disease weakens and eventually kills the orthopteran host within a few weeks.
Various insects killed by different species of entomopathogenic fungi
Entomopathogenic nematodes are microscopic, soil-dwelling worms that are parasitic to insects. Several species of Heterorhabditis and Steinernema are available in multiple commercial formulations, primarily for managing soil insect pests. Infective juveniles of entomopathogenic nematodes actively seek out their hosts and enter through natural openings such as the mouth, spiracles, and anus or the intersegmental membrane. Once inside the host body, the nematodes release symbiotic bacteria that kill the host through bacterial septicemia. Heterorhabditis spp. carry Photorhabdus spp. bacteria and Steinernema spp. carry Xenorhabdus spp. bacteria. Phasmarhabditis hermaphrodita is also available for controlling slugs in Europe, but not in the USA.
Infective juvenile of Steinernema carpocapsae entering the first instar larva of a leafminer through its anus.
Nematodes in beet armyworm pupa (left) and termite worker (right).
Similar to bacteria, entomopathogenic viruses need to be ingested by the insect host and therefore are ideal for controlling pests that have chewing mouthparts. Several lepidopteran pests are important hosts of baculoviruses including nucleopolyhedroviruses (NPV) and granuloviruses (GV). These related viruses have different types of inclusion bodies in which the virus particles (virions) are embedded. Virus particles invade the nucleus of the midgut, fat body or other tissue cells, compromising the integrity of the tissues and liquefying the cadavers. Before death, infected larvae climb higher in the plant canopy, which aids in the dissemination of virus particles from the cadavers to the lower parts of the canopy. This behavior aids in the spread of the virus to cause infection in healthy larvae. Viruses are very host specific and can cause significant reduction of host populations. Examples of some commercially available viruses include Helicoverpa zea single-enveloped nucleopolyhedrovirus (HzSNVP), Spodoptera exigua multi-enveloped nucleopolyhedrovirus (SeMNPV), and Cydia pomonella granulovirus (CpGV).
Most entomopathogens typically take 2-3 days to infect or kill their host except for viruses and P. locustae which take longer. Compared to viruses (highly host specific) and bacteria (moderately host specific), fungi generally have a broader host range and can infect both underground and aboveground pests. Because of the soil-dwelling nature, nematodes are more suitable for managing soil pests or those that have soil inhabiting life stages.
Biopesticides based on various entomopathogenic microorganisms and their target pests
Microbial control and Integrated Pest Management
There are several examples of entomopathogen-based biopesticides that have played a critical role in pest management. Significant reduction in tomato leaf miner, Tuta absoluta, numbers and associated yield loss was achieved by Bt formulations in Spain (Gonzalez-Cabrera et al, 2011). Bt formulations are also recommended for managing a variety of lepidopteran pests on blueberry, grape, and strawberry (Haviland, 2014; Zalom et al, 2014; Bolda and Bettiga, 2014; Varela et al, 2015).
Lecanicellium muscarium-based formulation reducedgreenhouse whitefly (Trialeurodes vaporariorum) populations by 76-96% in Mediterranean greenhouse tomato (Fargues et al, 2005). In other studies, B. bassiana applications resulted in a 93% control of twospotted spider mite (Tetranychus urticae) populations in greenhouse tomato (Chandler et al, 2005) and 60-86% control on different vegetables (Gatarayiha et al, 2010). The combination of B. bassiana and azadirachtin reduced rice root aphid (Rhopalosiphum rufiabdominale) and honeysuckle aphid (Hyadaphis foeniculi) populations by 62% in organic celery in California (Dara, 2015a). Chromobacterium subtsugae and B. rinojensis caused a 29 and 24% reduction, respectively, in the same study. IPM studies in California strawberries also demonstrated the potential of entomopathogenic fungi for managing the western tarnished plant bug (Lygus hesperus) and other insect pests (Dara, 2015b, 2016). Entomopathogenic fungi also have a positive effect on promoting drought tolerance or plant growth as seen in cabbage (Dara et al, 2016) and strawberry (Dara, 2013) and antagonizing plant pathogens (Dara et al, 2017)
Application of SeMNPV was as efficacious as methomyl and permithrin in reducing beet armyworms (S. exigua) in head lettuce in California (Gelernter et al, 1986). Several studies demonstrated PhopGV as an important tool for managing the potato tubermoth (Phthorimaea operculella) (Lacey and Kroschel, 2009).
The entomopathogenic nematode, S. feltiae,reduced raspberry crown borer (Pennisetia marginata) populations by 33-67% (Capinera et al, 1986). For managing the branch and twig borer (Melagus confertus) in California grapes, S. carpocapsae is one of the recommended options (Valera et al, 2015).
Entomopathogens can be important tools in IPM strategies in both organic and conventional production systems. Depending on the crop, pest, and environmental conditions, entomopathogens can be used alone or in combination with chemical, botanical pesticides or other entomopathogens.
Acknowledgements: Thanks to Dr. Harry Kaya for reviewing this article.
Bolda, M. P. and L. J. Bettiga. 2015. UC IPM Pest Management Guidelines: Caneberries. UC ANR Pub. 3437.
Capinera, J. L., W. S. Cranshaw, and H. G. Hughes. 1986. Suppression of raspberry crown borer Pennisetia marginata (Harris) (Lepidoptera: Sesiidae) with soil applications of Steinernema feltiae (Rhabditida:Steinernematidae). J. Invertebr. Pathol. 48: 257-258.
Chanlder, D., G. Davidson, and R. J. Jacobson. 2005. Laboratory and glasshouse evaluation of entomopathogenic fungi angainst the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), on tomato, Lycopersicon esculentum. Biocon. Sci. Tech. 15: 37-54.
Dara, S. K. 2013. Entomopathogenic fungus Beauveria bassiana promotes strawberry plant growth and health. UCANR eJournal Strawberries and Vegetables, 30 September, 2013. (http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=11624)
Dara, S. K. 2015a. Reporting the occurrence of rice root aphid and honeysuckle aphid and their management in organic celery. UCANR eJournal Strawberries and Vegetables, 21 August, 2015. (http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=18740)
Dara, S. K. 2015b. Integrating chemical and non-chemical solutions for managing lygus bug in California strawberries. CAPCA Adviser 18 (1) 40-44.
Dara, S. K. 2016. IPM solutions for insect pests in California strawberries: efficacy of botanical, chemical, mechanical, and microbial options. CAPCA Adviser 19 (2): 40-46.
Dara, S. K., S.S.R. Dara, and S.S. Dara. 2016. First report of entomopathogenic fungi, Beauveria bassiana, Isaria fumosorosea, and Metarhizium brunneum promoting the growth and health of cabbage plants growing under water stress. UCANR eJournal Strawberries and Vegetables, 19 September, 2016. (http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=22131)
Dara, S.S.R., S. S. Dara, S. K. Dara, and T. Anderson. 2017. Fighting plant pathogenic fungi with entomopathogenic fungi and other biologicals. CAPCA Adviser 20 (1): 40-44.
Fargues, J., N. Smits, M. Rougier, T. Boulard, G. Rdray, J. Lagier, B. Jeannequin, H. Fatnassi, and M. Mermier. 2005. Effect of microclimate heterogeneity and ventilation system on entomopathogenic hyphomycete infectiton of Trialeurodes vaporariorum (Homoptera: Aleyrodidae) in Mediterranean greenhouse tomato. Biological Control 32: 461-472.
Gatarayiha, M. C., M. D. Laing, and M. Ray. 2010. Effects of adjuvant and conidial concentration on the efficacy of Beauveria bassiana for the control of the two-spotted spider mite, Tetranychus urticae. Exp. Appl. Acarol. 50: 217-229.
Gelernter, W. D., N. C. Toscano, K. Kido, and B. A. Federici. 1986. Comparison of a nuclear polyhedrosis virus and chemical insecticides for control of the beet armyworm (Lepidopter: Noctuidae) on head lettuce. J. Econ. Entomol. 79: 714-717.
González-Cabrera, J., J. Mollá, H. Monton, A. Urbaneja. 2011. Efficacy of Bacillus thuringiensis (Berliner) in controlling the tomato borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). BioControl 56: 71–80.
Haviland, D. R. 2014. UC IPM Pest Management Guidelines: Blueberry. UC ANR Pub. 3542.
Lacey, L. A. and J. Kroschel. 2009. Microbial control of the potato tuber moth (Lepidoptera: Gelechiidae). Fruit Veg. Cereal Sci. Biotechnol. 3: 46-54.
Varela, L. G., D. R. Haviland, W. J., Bentley, F. G. Zalom, L. J. Bettiga, R. J. Smith, and K. M. Daane. 2015. UC IPM Pest Management Guidelines: Grape. UC ANR Pub. 3448.
Zalom, F. G., M. P. Bolda, S. K. Dara, and S. Joseph. 2014. UC IPM Pest Management Guidelines: Strawberry. UC ANR Pub. 3468.
Strawberry is an important commodity in California with a crop value of $2 billion (NASS, 2013). Lygus bug or western tarnished plant bug (Lygus hesperus), twospotted spider mite (Tetranychus urticae), greenhouse whitefly (Trialeurodes vaporariorum), and western flower thrips (Frankliniella occidentalis) are considered as important arthropod pests of strawberries which can cause significant yield losses. According to the Pesticide Use Report of California Department of Pesticide Regulation (2014), more than 200,000 lb of chemical insecticide and miticide active ingredients were used in strawberries in 2012. Among the 50,000 lb of biorational active ingredients that were additionally applied, 97% were Bacillus thuringiensis products used against lepidopteran pests. Apart from the release of various species of predatory mites against twospotted spider mites, pest management in strawberries is mainly dependent on chemical pesticides and IPM is generally limited to the rotation of pesticides in different modes of action groups.
In an effort to develop an effective IPM program with a particular emphasis on lygus bug management, research has been conducted for the past few years in Santa Maria to evaluate the role of various non-chemical alternatives. Field studies in 2013 showed that botanical and microbial pesticides can be effectively used in combination and rotation with chemical pesticides (Dara, 2014). Additional studies were conducted in 2014 to evaluate the efficacy of various combinations and rotations of new and existing chemical pesticides along with botanical, earth-based, and microbial pesticides.
A large scale field study was conducted during June and July, 2014 in a conventional strawberry field of variety Del Rey at Goodwin Berry Farms, Santa Maria. Chemical pesticides included those from IRAC mode of action groups 3A (sodium channel modulators) 4A (neonicotinoids), 4C (sulfoximines), 6 (chloride channel activators), 9C (selective homopteran feeding blockers), and 15 (inhibitors of chitin biosynthesis). Additionally, diatomaceous earth, azadirachtin, and two entomopathogenic fungi, Beauveria bassiana and Metarhizium brunneum (formerly known as M. anisopliae) were also used. Diatomaceous earth is a powder form of fossilized remains of diatoms and contains silicon dioxide as an active ingredient. Silicon dioxide interferes with the integrity of the cuticle by absorbing the waxy layer and causes mortality due to desiccation. Both B. bassiana and M. brunneum are soilborne entomopathogenic fungi which cause infection when a conidiospore comes in contact with an insect or a mite. Azadirachtin, a secondary metabolite present in neem seed, is a limonoid compound which interferes with the synthesis of various proteins and thus affects molting, mating, sexual communication, and reproductive ability. It also has insecticidal properties and acts as an antifeedant and repellent. Using these alternatives can help pest management which is sometimes difficult to achieve with chemical pesticides alone.
Treatments included an untreated control, a wettable powder formulation of acetamiprid as the grower standard, and other materials in different combinations and rotations (Tables 1 and 2). Each plot had seven 75' long and 64” wide beds and treatments were replicated four times in a randomized complete block design. Treatments were administered late afternoon or in the evening using a tractor-mounted sprayer except for diatomaceous earth dust which was applied by a backpack dust applicator. Three applications were made at 7-8 day intervals and observations were made once before the first application and 5-6 days after each application. On each observation date, 20 plants were randomly sampled from the middle three beds of each plot by gently beating the plant to dislodge insects into a container. The number of aphids, lygus bugs (young and mature nymphs and adults), thrips, whitefly adults, and various species of natural enemies were counted from each plant. Natural enemy complex included bigeyed bug (Geocoris spp.), minute pirate bug (Orius spp.), lacewing (Chrysoperla spp. and Chrysopa spp.), damsel bug (Nabis spp.), lady beetle (multiple species), parasitoids (multiple species), and spiders (multiple species). Data were analyzed using statistical procedures.
Table 1. List of treatments used in this study and their application rates per acre – Active ingredients
*3A Sodium channel modulators 4A Neonecotinoids, 4C Sulfoxamines, 6 Chloride channel activators, 9C Selective homopteran feeding blockers, and 15 Inhibitors of chitin biosynthesis.
Table 2. List of treatments used in this study and their application rates per acre – Trade names
*3A Sodium channel modulators 4A Neonecotinoids, 4C Sulfoxamines, 6 Chloride channel activators, 9C Selective homopteran feeding blockers, and 15 Inhibitors of chitin biosynthesis.
Results and Discussion
Actual numbers of various pests and natural enemies are presented in Table 3 and percent change post-treatment compared to pre-treatment is presented in different figures.
Table 3. Pest and natural enemy populations from various treatments before and after treatment per 20 sample plants. Post-treatment counts include averages for three spray applications. Refer to Tables 1 and 2 for the list of treatments.
Aphid: Negligible number of aphids was seen only in few treatments and data are not presented.
Lygus bug: Lygus numbers increased in all treatments after treatment and there were no statistical differences (P > 0.05). However, when the percent change, compared to pre-treatment counts, was considered, some treatments appeared to be more effective than others in preventing population buildup. The high rate of Sequoia (treatment 8) limited the increase to 14% followed by the rotation of Diafil high rate-BotaniGard low rate+Assail 70 WP-Met 52+Molt-X (treatment 12) indicating the potential of non-chemical alternatives for lygus bug management (Table 4). When BotaniGard+Molt-X combination was applied twice after Rimon+Brigade combination (treatment 4), it appeared to be the fourth best rotation limiting the population build up to 54%. Untreated control and Assail 70 WP had the highest lygus numbers with 383% and 1083% increase, respectively.
Percent change in all stages of lygus bugs after three spray applications.
Treatments ranked according to their efficacy as expressed by the percent change/control of lygus bugs after three spray applications.
Thrips:There was a general reduction in thrips numbers post-treatment. There was a 48% reduction in their post-treatment numbers in untreated control while it varied from 35% treatment 2 to 68% in treatment 12.
Percent change in western flower thrips populations after three spray applications.
Whitefly adult:Most of the treatments reduced whitefly populations except for one treatment where there was a 20% increase (treatment 11 – Diafil low rate followed by Sequoia low rate+Molt-X, and Met 52) compared to a 13% in untreated control. There was a 7 to 78% reduction in whitefly populations in all other treatments.
Percent change in greenhouse whitefly adult populations after three spray applications.
Natural enemy complex:The number of big-eyed bug, parasitoids, and spiders significantly varied among various treatments post-treatment (P < 0.05, data not shown). When the percent change was considered for the entire natural enemy complex, there was a reduction in all treatments with 41% reduction in untreated control and 53-86% reduction in various treatments.
Diafil application left a white deposit on strawberry plants for several days making the berries unmarketable. It may not be practical for managing lygus bug, which usually appears after fruit production starts.
These results support last year's data in demonstrating the potential of non-chemical alternatives such as microbial and botanical pesticides. These tools are essential for sustainable pest management and can make a significant reduction in chemical pesticide use without compromising the control efficacy.
California Department of Pesticide Regulation. 2014. Summary of pesticide use report data 2012: Indexed by commodity.
Dara, S. K. 2014. New strawberry IPM studies with chemical, botanical, and microbial solutions. CAPCA Adviser 17: 35-37.
National Agricultural Statistics Service (NASS) 2013. California agricultural statistics: 2012 crop year.