Progress 09/01/02 to 08/31/06
Outputs
Beauveria bassiana isolate 11-98 (Bb 11-98) was evaluated as a dual biological control for plant pathogens and insect pests. Application of Bb 11-98 conidia to tomato and cotton seed protected seedlings from damping-off caused by Rhizoctonia solani. Seed treatments with Bb 11-98 conidia were more effective than mycelia or alginate formulations against damping-off in tomato. In tomato and cotton, disease control was dependent upon population density of conidia on seed. Conidial rates of log 6 to 7 colony forming units (CFU)/seed were most effective in tomato. Rates of log 7 to 9 CFU/seed reduced disease and increased seedling growth in cotton; log 7 was most effective in sandy soil and log 9 was more effective in high organic matter mixes. Endophytic Bb 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings, grown from seed treated with Bb 11-98 using standard plating techniques onto selective medium. As the rate
of conidia applied to seed increased, the percent of plant tissues from which Bb 11-98 was recovered increased. We used ITS 1 and 4 primers, polymerase chain reaction (PCR), and electrophoresis of PCR products to detect Bb 11-98 in tomato and cotton. However, these primers produced PCR products for the plants. We developed new ITS primers to detect a single 421-bp band for Bb 11-98, and no PCR products for cotton. In samples containing DNA from both Bb 11-98 and cotton, Bb 11-98 was detected at ratios of 1:1000 Bb 11-98 to cotton DNA; Bb 11-98 was detected in seedlings grown from Bb 11-98 treated seed. Using scanning electron microscopy, hyphae of Bb 11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and spongy mesophyll tissues. Bb 11-98 and isolate GHA (BotaniGard) increased growth of cotton seedlings in soil infested with Pythium myriotylum. In parasitism assays, hyphae of Bb 11-98 were observed coiling around hyphae of P.
myriotylum. Assays were conducted to determine the efficacy of Bb 11-98 against Macrosiphum euphorbiae and Helicoverpa zea. When M. euphorbiae were introduced to tomato foliage treated with Bb 11-98 conidia, mortality was 40% 10 days after treatment (DAT); following direct exposure to Bb 11-98 conidia, mortality was 34% 10 DAT. Reproduction of treated and untreated adults was not different, and at log 4 CFU/ml of Bb 11-98, M. euphorbiae produced an average of 81 nymphs 10 DAT. Application of log 7 CFU/ml Bb 11-98 to the epidermis of H. zea larvae yielded 17% mortality 30 DAT. But H. zea consumption of tomato foliage treated with Bb 11-98 conidia produced higher rates and faster mortality in larvae. In choice and no-choice larval feeding assays, using plants with endophytic Bb 11-98 against third instar H. zea larvae, larval feeding was not affected. In no-choice assays, H. zea larvae consumed slightly less foliage from plants treated with Bb 11-98 after 120 h, but feeding by
individual larvae was highly variable and treatments did not differ. Larvae that fed for 120 h on tomato foliage grown from seeds treated with Bb 11-98 had 17% mortality, compared to 0% mortality on plants grown from untreated seed.
Impacts
Annual losses due to soilborne plant pathogens are estimated at 5 to 10% for individual crops. In 2005, the fresh market tomato crop in Tennessee was valued at $42 million; with losses due to root diseases in the range of $2 to $4 million. The crop value of cotton lint and cottonseed in Tennessee was approximately $233 million; with losses from root diseases in the range of $12 to $23 million. These losses do not include the immediate cost of agrichemicals applied to reduce additional losses, or the long-term cost of clean-up from pesticide run-off from agricultural soils into water supplies, use of petroleum resources to produce most pesticides, and human health costs due to pesticide exposure. Production of pesticide-free or organic tomatoes can potentially add 10 to 30% in increased crop value. In addition, integrating multiple strategies into disease control programs will reduce development of pathogen and pest resistance to existing chemical pesticides. Our
discovery that Beauveria bassiana, known for its ability to control insect pests, can also effectively protect tomato and cotton seedlings against disease caused by Rhizoctonia and Pythium will broaden applications of this unique biopesticide and provide growers with new management options for root diseases of tomato and cotton.
Publications
- 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. Phytopathology 96:S42.
- Powell, Wesley A. 2005. Potential of Beauveria bassiana 11-98 as a biological control agent against tomato pests; and detection of the mycotoxic metabolite beauvericin in tomato plants using HPLC. M.S. thesis. The University of Tennessee, Knoxville. 85 pp.
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Progress 01/01/05 to 12/31/05
Outputs
Beauveria bassiana is an entomopathogenic fungus with an extensive host range of insect pests. B. bassiana isolate 11-98 is endophytic in tomato and applied as a seed treatment, can provide significant protection against Rhizoctonia damping off. Initial experiments on control of R. solani in cotton (log 6 B. bassiana 11-98 spores per seed) in a high organic matter soil were variable. Efficacy was improved significantly and variability in biocontrol was reduced by adapting a strain of 11-98 with enhanced endophytic ability in cotton and increasing the rate of Beauveria spores on cotton seed to log 9. In subsequent disease assays, in soil with less organic matter and clay, and higher percent sand, biocontrol of Rhizoctonia was achieved with log 7 spores per seed, suggesting that soil texture or cation exchange capacity play a role in biocontrol efficacy with seed treatments of Beauveria. Biocontrol of Pythium myriotylum, with seed treatments of Beauveria, were confirmed
in cotton and tomato. In both cases, the ability to colonize plant tissues by subcultures of B. bassiana was established prior to biocontrol tests with Pythium. Control of Thielaviopsis black root rot in cotton has not been achieved with Beauveria seed treatments. Endophytic colonization of cotton seedling by B. bassiana, grown from Beauveria-treated seed was confirmed using standard dilution plating and electron microscopy. The cotton-adapted strain of 11-98 was isolated from roots, stems and leaves of endophytically colonized cotton. The lethal effects of different rates of B. bassiana 11-98 against the potato aphid (Macrosiphum euphorbiae) on tomato foliage were evaluated. Sprays were applied topically to insects and to tomato foliage prior to aphid infestation. The highest conidial rate (log 7) resulted in 22% aphid mortality. Potato aphids probably molt too quickly for Beauveria spores to germinate and successfully penetrate the epidermis. Regardless of treatment, after 10 days,
female fecundity resulted in five times the original number of aphids treated. To address the question of even dispersal of B. bassiana spores in aqueous solutions, surfactants and dispersing agents (Suffusion, vegetable oil, Triton X 100, Tween 20, Tween 80 and Tween 85 at different rates, and kaolin at different ratios) were added to aqueous suspensions of B. bassiana spores. All surfactants except Tween 85 were phytotoxic to tomato. There was not benefit for suspensions using kaolin. Lethal effects of two rates of Beauveria (log 6 and log 7 spores per ml water) were screened against third-instar tomato fruitworm (Helicoverpa zea) larvae, applied topically and to tomato foliage prior to introduction of larvae and subsequent feeding. Seventeen percent mortality occurred following topical exposure to the highest conidial rate. However, for insects that fed on foliage treated with 11-98, mortality was 75% at the highest conidial rate with no mortality in controls.
Impacts
Annual losses due to soilborne plant pathogens are estimated at 5 to 10% for individual crops. In 2004, the fresh market tomato crop in Tennessee was valued at $42 million; with losses due to root diseases in the range of $2 to $4 million. The crop value of cotton lint and cottonseed in Tennessee was approximately $274 million; with losses from root diseases in the range of $14 to $27 million. These losses do not include the immediate cost of agrichemicals applied to reduce additional losses, or the long-term cost of clean-up from pesticide run-off from agricultural soils into water supplies, use of petroleum resources to produce most pesticides, and human health costs due to pesticide exposure. Production of pesticide-free or organic tomatoes can potentially add 10 to 30% in increased crop value. In addition, integrating multiple strategies into disease control programs will reduce development of pathogen and pest resistance to existing chemical pesticides. Our
discovery that Beauveria bassiana, known for its ability to control insect pests, can also protect tomato and cotton seedlings against disease caused by multiple soilborne plant pathogens will broaden applications of this unique biopesticide and provide growers with new management options for root diseases of tomato and cotton.
Publications
- Griffin, M., Ownley, B., Klingeman, W., and Pereira, R. 2005. Biocontrol of Rhizoctonia damping-off of cotton with endophytic Beauveria bassiana. Phytopathology 95:S36.
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Progress 01/01/04 to 12/31/04
Outputs
Beauveria bassiana is an entomopathogenic fungus with an extensive host range of insect pests. Isolates of this fungus can colonize plants endophytically, and colonization has been linked to its ability to control insect pests. Our studies are focused on determining the potential of B. bassiana to control disease in plants caused by soilborne pathogens that cause significant economic damage and crop loss in seedlings. We established that B. bassiana can control Rhizoctonia damping-off disease of tomato, and can provide some protection to cotton against a seedling disease complex (plant pathogens: Rhizoctonia, Pythium, Fusarium, and Thielaviopsis) in field soils. Seed treatment with B. bassiana resulted in endophytic colonization of tomato seedlings. Our research is focused on optimizing biocontrol activity of B. bassiana for management of plant diseases and insect pests in greenhouse-grown tomatoes, identifying abiotic and biotic factors that influence biocontrol by
B. bassiana against damping-off of cotton seedlings in field soil, and determining the extent of endophytic colonization by B. bassiana in tomato, cotton, and snap bean and whether colonization is correlated with biocontrol activity against R. solani. We confirmed that seed treatments with higher rates of B. bassiana are more effective than low rates for control of Rhizoctonia disease in tomato seedlings. Pre-emergence damping-off of cotton caused by Pythium was significantly reduced and growth of plants from seed treated with a Tennessee isolate of B. bassiana, or a commercially available isolate was significantly greater than plants from untreated seed. The growth enhancement effect on cotton seedlings from seed treated with B. bassiana was observed also in plants grown in soil without Pythium. Endophytic colonization by B. bassiana in the commercial transgenic cotton cultivar Delta Pine 436 was confirmed. Cotton seeds were coated with B. bassiana and the fungus was detected in
internal leaf and stem tissues of seedlings. Studies are underway to determine if differences in cotton cultivar affect endophytic colonization, and subsequent biocontrol. In addition to standard plating techniques, we use ITS primers for detection of B. bassiana in plant tissues with PCR. These primers are unsuitable for detection of B. bassiana from plants with Real-Time PCR, a more sensitive quantitative detection method; we are currently developing and evaluating primers to optimize detection of B. bassiana in plant tissues using Real-Time PCR. Studies continue on aphid management on tomato with B. bassiana. In a no-choice assay, designed to quantify aphid mortality and reproductive rate following exposure to different rates of the Tennessee isolate of B. bassiana in aqueous solution, mortality was suboptimal. Conidia of B. bassiana resist wetting and aqueous treatment rates exceeding log 5 colony forming units per ml are difficult to achieve reliably. Studies are underway to
determine optimal application methods for control of aphids on tomato, degree of spore suspension and viability, and tomato phytotoxicity with formulations that include talc, kaolin clay, oils, and various surfactants.
Impacts
Microbial control of plant pathogens and insect pests is an important component of biologically-based efforts to reduce our reliance on chemical pesticides and increase sustainability of U.S. agriculture. Development of biopesticides that can control both plant pathogens and insect pests will have enormous value for plant protection in U.S. agriculture. Such biopesticidal organisms would be similar to wide spectrum pesticides with their potential for a significant reduction in pest management costs. In addition, plant disease and pest control with microbial agents offers the environmental advantages associated with biological control. From these studies we expect that formulations of B. bassiana or application techniques will be developed for dual control of damping-off disease and aphids in tomato. By identifying the biotic and abiotic soil factors that influence biocontrol of seedling disease in cotton with B. bassiana, and the relationship between endophytic
colonization by B. bassiana and biocontrol of disease we will be able to optimize the effectiveness of this organism as a biopesticide.
Publications
- Ownley, B.H., R.M. Pereira, W.E. Klingeman, III, N.B. Quigley, and B.M. Leckie. 2004. Beauveria bassiana, a dual purpose biological control with activity against insect pests and plant pathogens. Pages 255-269 in: Emerging Concepts in Plant Health Management. R.T. Lartey and A.J. Caesar, eds. Research Signposts, T.C., Kerala, India.
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Progress 01/01/03 to 12/31/03
Outputs
Beauveria bassiana is a well-known fungal pathogen of insect pests. In previous studies we determined that B. bassiana can protect tomato seedlings from damping-off disease caused by the soilborne pathogen Rhizoctonia solani. Based on our work on formulations of B. bassiana isolate 11-98 (Bb 11-98) preparations of conidia are more consistent than mycelia for effective control of Rhizoctonia damping-off in tomato. In addition, seed treatments provide more consistent disease control than preparations of the fungus added to potting soil. Disease control is dependent upon population density of conidia on the seed with 1 x 107 colony forming units (CFU) per seed, being the most effective. Spores of Bb 11-98 are compatible with traditional seed treatment fungicides, which had minimal effect on the population density of Bb 11-98 after 4 months cold storage. Preliminary microscopic observations confirm that Bb-98 conidia germinate and grow in an artificial diet solution for
aphids when maintained at 23 C. Several rates of Bb-98 conidia were suspended in artificial diet and presented to feeding aphids in two trials to test the ability of conidia to directly affect mortality and reduce feeding and reproduction of tomato aphids (Macrosiphum euphorbiae). In the first trial, 15 aphids per CFU-rate treatment were presented with 150-ul diet solutions containing 1 x 103; 1 x 104; 1 x 105 and 1 x 106 CFU per ml diet solution, and compared to an untreated control with diet solution only. In a second trial, rates of 1 x 105; 1 x 106; 1 x 107; 1 x 108 and 1 x 109 CFU per ml diet solution were evaluated. Aphid mortality, number of aphids feeding or walking around the arena, and the number of newborn aphids was recorded on 12 h intervals for 120 h. Based on preliminary results, conidia growing within the diet solution do not directly affect aphid mortality after 120 h feeding. Conidia are larger than the oral aperture of aphids, thus effects would be attributed to the
production of secondary metabolites during fungal growth. Higher rates of conidial suspensions acted as antifeedants and aphid reproduction was reduced as conidial concentrations increased. In the second trial, mortality was high when diet contained 1 x 109 CFU per ml diet, however conidia were too dense to remain in solution and aliquots could not be managed reliably.
Impacts
Soilborne plant pathogens cause an estimated $4 billion annual loss to crop production in the USA. To reduce development of pathogen resistance and our reliance on chemical pesticides, research is underway on biological controls, however, relatively little research has been done on the integration of individual microbial biological controls for management of both plant diseases and insect pests. Our discovery that Beauveria bassiana, known for its ability to control insect pests, can also protect tomato seedlings against disease caused by the soilborne pathogen Rhizoctonia solani, will broaden the applications of this unique biopesticide and lead to new management options for seedling diseases.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Beauveria bassiana is a well-known fungal pathogen of insect pests. In previous studies we determined that application of conidia of B. bassiana to tomato seed could protect the seedlings from damping-off disease caused by the soilborne pathogen Rhizoctonia solani. Using ITS primers and polymerase chain reaction techniques, we determined that B. bassiana is an endophyte of tomato seedlings when grown from seed treated with B. bassiana conidia. In recent studies on comparisons of different formulations (conidia, mycelia, or alginate prills), rates, and applications methods (seed treatment, or soilless potting mix) of B. bassiana to determine the most effect treatment for control of R. solani, plant stands of several different types of B. bassiana treatment were significantly greater than the untreated, infested control (only 40% plant stand). At three weeks after seeding, seed treatment with B. bassiana conidia resulted in the highest percentage plant stand with 86%.
This was comparable to the untreated healthy control, without R. solani, which had 80% plant stand.
Impacts
Soilborne plant pathogens cause an estimated $4 billion annual loss to crop production in the USA. To reduce development of pathogen resistance and our reliance on chemical pesticides, research is underway on biological controls, however, relatively little research has been done on the integration of individual microbial biological controls for management of both plant diseases and insect pests. Our discovery that Beauveria bassiana, known for its ability to control insect pests, can also protect tomato seedlings against disease caused by the soilborne pathogen Rhizoctonia solani, will broaden the applications of this unique biopesticide and lead to new management options for seedling diseases.
Publications
- Leckie, Brian M. 2002. Effects of Orally Administered Beauveria bassiana Mycelia and Metabolites on Helicoverpa zea; and Detection of Endophytic B. bassiana in Tomato Plants using ITS Primers. M.S. Thesis. The University of Tennessee, Knoxville.
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