Final Report for GS10-095
Project Information
Funds from SARE were used in 2011 for my dissertation research project to examine the efficacy of biopesticides for potential organic pest management of cucumber beetles on melon and pumpkin crops in the Southeastern U.S. Cucumber beetles are an annual pest on cucurbit crops and can damage pumpkin, cantaloupe/muskmelon, summer squash, cucumbers and watermelon. In addition to vectoring bacterial wilt, beetles chew stems, leaves and feed directly on fruit, reducing both marketable yield and quality. Organic growers are limited in what they can use for cucumber beetle management and need alternatives, and consumers increasingly demand vegetables that are grown chemical-free. Biopesticides made from naturally occurring microorganisms that kill the insect pest while preserving beneficial insects are worth exploring. This project evaluated the efficacy of three different mycoinsecticides in reducing cucumber beetle damage in an attempt to offer sustainable IPM guidelines suitable for small to medium sized growers in our region. The biopesticides tested were: 1. MBI-203, an early stage product under registration by Marrone Bio Innovations (Davis, CA), comprised of secondary metabolites isolated from a bacteria, Chromobacterium substugae; 2. Mycotrol O, a commercially available strain of Beauveria bassiana strain GHA (Laverlam International Corporation, Butte, MT) labeled for use on cucumber beetles; 3. Beauveria bassiana strain 11-98; 4. Isaria fumosorosea 3581 (a fungal strain known to infect cucumber beetles but has not been tested). These substances were tested for efficacy in the lab, and compounds 1, 2 and 4 were used in field testing.
Introduction
Cucumber beetles are annual pests on cucurbits and are difficult to manage due to alternative hosts of larval and adult stages and are highly mobile. The options for organic growers are limited and include crop rotation, use of reflective mulches, kaolin clay, nematodes, Beauveria bassiana and pyrethrins to manage these beetles. The efficacy of some of these methods is questionable. Conventional chemicals are effective but can have negative effects on the applicator, can pollute groundwater and may impact aquatic organisms as well as beneficial insects and pollinators. Consumer demand is moving increasingly toward chemical-free produce, and growers need to use a variety of strategies to minimize pesticide use to avoid environmental effects and pest resistance.
Both spotted (Diabrotica undecimpunctata) and striped cucumber beetles (Acalymma vittata) vector bacterial wilt. The spotted cucumber beetle, Diabrotica undecimpunctata howardi, is also serious pest of field crops. Larvae are known as southern corn rootworm and feed on roots of corn in the soil. Adults may defoliate plants of the Curcurbitaceae such as cantaloupe, cucumber, squash and watermelon; other host plants include corn, soybean, beans and sweet potato. The striped cucumber beetle, Acalymma vittata, has similar host preferences in the field to the spotted cucumber beetle, described above, however it is considered a cucurbit specialist (Bach 1980; Ellers-Kirk and Fleischer 2006), and larvae only develop on cucurbit roots (Bach 1980). Adults are long-lived; surviving up to 125 days, and females may lay up to 4 eggs per day (Ellers-Kirk and Fleischer 2006). Similar to the spotted cucumber beetle, there are two generations per year of A. vittata in the southern U.S. (Ellers-Kirk and Fleischer 2006).
The inundative use of entomopathogenic fungi should not be thought of as a therapeutic control like a typical chemical pesticide, but rather, as a form of biological control that should be used in tandem with other management practices, such as using insect predators and parasitoids and other cultural techniques (Jaronski 2009). Successful control can only be achieved by “winning the numbers game,” where infective propagules are introduced in sufficient numbers to reduce pest populations, a feat that is easier accomplished in a controlled greenhouse setting rather than in the field (Jaronski 2009). Therefore it is practical to begin research on biopesticides in a laboratory setting before moving to the field. Environmental factors can constrain efficacy of biopesticides and applied research to investigate the practicality of using these pathogens in agricultural pest management strategies focuses on the effective formulation and dispersal of infective propagules in the field and whether this is economically feasible (Jackson et al. 2010). This graduate student grant helped fund laboratory and field efficacy studies on 1. MBI-203,comprised of secondary metabolites isolated from a bacteria, Chromobacterium substugae; 2.Mycotrol O, a commercially available strain of Beauveria bassiana strain GHA; and 3.Isaria fumosorosea 3581.
1.Determine the efficacy of of early stage biopesticide MBI-203 on cucumber beetles using a laboratory bioassay.
2.Determine the efficacy and virulence of Beauveria bassiana (Mycotrol-O and Bb11-98) and two strains of Isaria fumosorosea (3581 and 1506) on cucumber beetles using a laboratory bioassay.
3.Examine the efficacy of early stage biopesticide MBI-203 in managing two species of diabroticite beetles (Diabrotica undecimpunctata and Acalymma vittata) and effects on yield of melon and pumpkin plants in the field.
4.Compare two different rates of the novel biopesticide MBI-203 with the label rate of Beauveria bassiana (Mycotrol O) and Carbaryl (Sevin 80 S).
5.Determine if beetle densities are reduced by alternating commonly used plant protectants with MBI-203 in an integrated pest management program for sustainable melon and pumpkin production.
6.Examine the efficacy of entomopathogenic fungi Isaria fumosorosea strain 3581 in managing two species of diabroticite beetles (Diabrotica undecimpunctata and Acalymma vittata) and effects on yield of melon and pumpkin plants in the field.
7.Examine the potential of row covers to reduce cucumber beetle populations in melon and pumpkin plants before flowering, and effects on yield.
Cooperators
Research
Mycotrol-O was purchased from Arbico Organics (Oro Valley, AZ). The bioassay was started on 2 Dec 2010. The methods were as follows: melon leaves were harvested from the greenhouse and cut into leaf disks (5.5 cm in diameter). Leaf disks were sterilized in 10% Clorox/sterile water solution for 2 minutes and put on sterile filter paper (wetted with sterile water) in a Petri plate (standard size, 15 mm x 100 mm). Striped cucumber beetles (Acalymma vittata) were obtained from French Agricultural Research Inc. (Lamberton, MN). Two cucumber beetles were added to each plate, 35 sterile water control plates and 35 Mycotrol-O plates were used (5 reps of 7). The rate of Mycotrol-O was 500 uL in 50 mL sterile water, sprayed three times (to runoff) with an atomizer. Plates were parafilmed and incubated at 23°C. Beetle mortality and estimated leaf damage was counted daily until day 4, when photos were taken of each leaf disk and beetles were given a new leaf and sprayed again as described, and monitored daily for another 4 days. Area of the leaf disk was analyzed using WinFOLIA (2006a, Regent Instruments Inc., Zurich, Switzerland). A mixed model ANOVA was performed on percent leaf area consumed (SAS 9.3, Cary, IN). Dead beetles were plated on Sabouraud dextrose agar (SDA) to determine presence of Beauveria bassiana.
Dry spores of Beauveria bassiana 11-98 were obtained from cultures maintained by Mary Dee in Dr. Bonnie Ownley’s lab. The bioassay was started on 8 Dec 2010. Two beetles were added to each plate, 50 sterile water control plates and 50 Bb 11-98 plates were used (5 reps of 10). The Bb 11-98 spray was prepared by adding 0.8 g of dry spores to 100 mL sterile water with a drop of Tween surfactant and shaken vigorously (spore count determined to be 7.6 x 106). All else was as described above.
The MBI-203 product was obtained from Marrone Bio Innovations (Davis, CA). The bioassay was started on 24 Feb 2011. Two beetles were added to each plate, 50 sterile water control plates, 50 MBI (1X) and 50 MBI (2X) plates were used (5 reps of 10). The MBI-203 sprays were prepared by adding 5 mL of product to 100 mL sterile water (1X) and 10 mL of spray to 100 mL sterile water (2X). The assay ran until day 4, when photos were taken. Dead beetles were not plated out (MBI-203 will not result in mycosis).
Dry spores of Isaria fumosorosea strain 3581 were obtained from Dr. Mark Jackson, microbiologist from the USDA-ARS (Peoria, IL). The bioassay was started on 24 Mar 2011. Two beetles were added to each plate, 50 sterile water control plates and 50 Ifr 3581 plates (5 reps of 10). The Ifr 3581 spray was prepared similarly to the Bb 11-98 spray, with 0.5 g of spores added to 25 mL of sterile water with a drop of Tween.
Field trial 2011: Materials & Methods
On 13-May-2011, 350 each of pumpkin (‘Cannon Ball’ F1) (Osborne International Seed Co., Mount Vernon, WA) and melon (Galia ‘Diplomat’ F1) (Johnny’s Selected Seeds, Winslow, ME) were started in 50-cell plug trays in the greenhouse. The growing media was McEnroe’s Premium Lite Growing Mix (McEnroe Organic Farm, Millerton, NY). The media contains compost and nutrients sufficient for germination and no additional fertilizers were used. Greenhouse conditions were the same as described in 2010. On 23-May, a field planted in winter rye and crimson clover at the OCU was flailed mowed (Alamo SH74, Alamo Industrial, Seguin, TX) and spaded (Imants, Reusel, The Netherlands) to prepare for planting.
On 17-Jun, plastic (biodegradable) and dripline irrigation (T-tape) were laid in rows and transplants were set by hand. Pumpkin plots were 20’ long with 18’ in between rows. Melon plots were 10’ long with 18’ in between rows. Pumpkin plants were set 4’ apart in-row, and melon plants were set 2’ apart in-row (5 plants per row). A 4’ wide strip of buckwheat was seeded on either side of pumpkin/melon plots. There were four replicates of each crop, with 10 plants in each plot. At planting, melon and pumpkin plots were side-dressed with 2 and 4 lbs of soybean meal (7-2-1, TN Farmers Co-op), respectively, at the rate of 80 lbs on N per acre.
Spray treatments consisted of Beauveria bassiana strain GHA (Mycotrol-O, AI 10.9%, Laverlam International, Butte, MT) was used in field rates of 1X (1 qt/acre) and 2X (2 qt/acre).
A novel biopesticide, MBI 203 EP, comprised of a Chromobacteria substugae (94.5% AI) developed by Marrone Bio Innovations (Davis, CA). The field rates of MBI 203 EP were 1X (5% v/v) and 3X (15% v/v).
Carbaryl (Sevin concentrate, 22.5% AI, TechPac LLC, Lexington, KY) was used at the 1X label rate (1.5 fl oz per 1000 ft2).
Isaria fumosorosea Strain 3581 from Mark Jackson (USDA ARS) was used at the rate of 1 g per 100 mL water (equal to 1 x 107 field rate).
The surfactant used was Nu Film P (Pinene polymers, Miller Chemical, Hanover, PA) at the rate of 1 pint per 100 gallons water.
The treatments were:
1. Mycotrol-O label rate, B1X
2. Mycotrol-O double rate, B2X
3. unsprayed control, N
4. MBI 203 label rate, M1X
5. MBI 203 triple rate, M3X
6. MBI 203 label rate alternated with Mycotrol-O label rate, MB
7. MBI 203 label rate alternated with Sevin label rate, MSV
8. Sevin label rate, SV
9. IFR 3581 with surfactant, IFR+S
10. Row cover, removed at flowering, RC
11. Row cover, removed at flowering plus weekly sprays of IFR+S, RC+IFR+S
12. water control, W
13. water plus surfactant, W+S
The spray treatments began on 28 Jun and were repeated weekly for 8 weeks (5 Jul, 12 Jul, 19 Jul, 26 Jul, 2 Aug, 9 Aug and 16 Aug), as described in 2010. Insect scouting was performed weekly before spraying for 9 weeks. Row covers were removed at flowering on 11 Jul to allow for pollination.
Melons were harvested on 5 Aug, 8 Aug, 10 Aug, 15 Aug, 20 Aug, 22 Aug, and 25 Aug. Pumpkins were harvested on 31 Aug.
Percent leaf area consumed was measured using visual estimation and leaf analysis software (winFOLIA) using uploaded digital images. Regression analysis was performed to see how well these values were correlated. Correlation was not strong (R-square <.50), so only the measures obtained from the software were used to analyze leaf area consumption. MBI-203 was the only treatment that showed differences in percent leaf area consumed, indicating that beetles consumed less of the treated leaf disks than the untreated leaf disks. However, the double rate (2X) did not show a response. Beetles in the 2X treatment had higher mortality rates than the 1X and untreated groups. Perhaps at the lower rate, MBI-203 acts as a feeding inhibitor and at the higher rate beetles consume the leaves and have increased mortality. By day 4, 54% of beetles were dead in the 2X group compared to 24% and 28% for the 1X and untreated group, respectively.
The Mycotrol-O treated leaves were consumed equally as the sterile water treated leaves, and beetles in the treated group this experienced higher mortality than the control group, but not until day 6, with 41% and 17% mortality for the treated versus untreated groups, respectively. Mycosis was confirmed in 19% of the beetles in the treated group.
The Beauveria bassiana 11-98 treated leaves were consumed equally as the sterile water treated leaves, and 16% of beetles in the treated group died by day 5, compared to 8% of the control beetles. “Sluggish” behavior was observed in 20 beetles from the treated group, characterized by lack of activity and unresponsiveness when probed. Of the 20 “sluggish” beetles, 19 were visually confirmed infected by Bb 11-98 when plated onto selective media. In total, 29% of beetles exposed to Bb 11-98 spores in Petri dishes were confirmed infected, showing that Bb 11-98 is pathogenic on striped cucumber beetles.
The Isaria fumosorosea strain 3581 treated leaves were consumed equally as the sterile water treated leaves. Beetles from both groups had similar survival rates. Of the beetles exposed, 39% were confirmed infected.
Field Study Conclusions:
Because cucumber beetles and squash bugs are both common and occur simultaneously, we monitored for both in the field studies. Cucumber beetles were found more often on melons than squash bugs. Squash bug egg clusters were not found on melons. Significantly fewer cucumber beetles were found in the SV treatment (Sevin label rate) and RC+IFR+S (row cover, Isaria and surfactant) than in the BB2X (Mycotrol-O double label rate) treatment. Sevin is a broad spectrum, insecticide that kills on contact, and fewer insects were expected in this treatment. The row covers may have prevented contact between the cucumber beetles and the melon plants early on, although no differences were seen in the RC (row cover only) treatment. The higher rate of Mycotrol-O was not effective in managing cucumber beetles on melons in the field. Overall, cucumber beetle populations were right around economic threshold levels of 1 beetle per plant.
Squash bugs were found more often on pumpkins than cucumber beetles. Significantly fewer squash bugs were found on the MB (MBI-203 rotated with Mycotrol-O) treatment and the row cover (RC) treatment than the unsprayed control treatment (N). The MB treatment may be repelling squash bugs on pumpkins, and row covers may prevent contact before flowering. Egg clusters were not different between treatments, and were above the economic threshold levels of 1 egg mass per plant.
Melon plants experienced low mortality through the course of the field season. Bacterial wilt vectored by cucumber beetles was low. There was no difference in melon yield between treatments. Marketable yields were low, mostly due to cracking and splitting. Other reasons for unmarketable fruit included poor pollination/misshapen fruit, bacterial spots and soft rots, punctures caused by rodents and/or birds, and rootworm/cucumber beetle damage.
Pumpkin plants experienced high mortality early on due to rootworm feeding on young roots. Plants under the row covers wilted. Though lightweight, row covers may not provide enough ventilation. There was no difference in pumpkin yield between treatments. Pumpkin yields were low due to poor plant survival. The main reason for unmarketable fruit was due to vine decline (shriveled fruit). Other reasons for unmarketable fruit included scarring due to squash bug feeding, poor pollination and bacterial spot.
Lab Study Conclusions:
Percent leaf area consumed was measured using leaf analysis software. MBI-203 was the only treatment that showed differences in percent leaf area consumed, indicating that beetles consumed less of the treated leaf disks than the untreated leaf disks. However, the double rate (2X) did not show a response. Beetles in the 2X treatment had higher mortality rates than the 1X and untreated groups. Perhaps at the lower rate, MBI-203 acts as a feeding inhibitor and at the higher rate beetles consume the leaves and have increased mortality. By day 4, 54% of beetles were dead in the 2X group compared to 24% and 28% for the 1X and untreated group, respectively.
The Mycotrol-O treated leaves were consumed equally as the sterile water treated leaves, and beetles in the treated group this experienced higher mortality than the control group, but not until day 6, with 41% and 17% mortality for the treated versus untreated groups, respectively. Mycosis was confirmed in 19% of the beetles in the treated group.
The Isaria fumosorosea strain 3581 treated leaves were consumed equally as the sterile water treated leaves. Beetles from both groups had similar survival rates. Of the beetles exposed, 39% were confirmed infected, which is promising.
Project Outcomes
The MBI-203 formulation shows some promise in managing cucumber beetles, but may work more as an anti-feedant than an insecticide. Future studies could use this product in a trap crop scenario, where it may be applied to cash crops in a push-pull strategy. In the field, MBI-203 likely has a low residual due to environmental degradation. Creating covered bait stations using cucumber beetle bait may work as an attract-and-kill strategy. This warrants future testing.
The Mycotrol-O product is labeled for management of cucumber beetles and did not show satisfactory results. We would not recommend this product for organic growers as it is expensive and the efficacy is poor.
More research needs to be done on Isaria fumosorosea. Lab results show promising infection, but field results are variable, most likely due to decreases in spore viability due to UV light or other environmental factors. A formulation using sunscreens and other surfactants may increase viability and infection in the field.
Areas needing additional study
This project helped identify many aspects of using biopesticides that warrant further study. These include:
1. Creating OMRI approved surfactants that increase field life of biopesticides.
2. Using MBI-203 for the protection of the cash crop in a trap-crop scenario, using a push-pull strategy.
3. Using MBI-203 with cucumber beetle feeding stimulant in bait stations in an attract-and-kill stategy.
4. Testing all of these compounds on cucumber beetle larvae (rootworms) in laboratory bioassays in the soil environment. If effective, this would reduce damage on corn and cucurbit roots, and reduce overall populations of adults. A new pesticide delivery system would be needed for field studies on larvae(soil drenches?)