Source: UNIVERSITY OF CALIFORNIA submitted to
BIOLOGY AND CONTROL OF SOIL-BORNE PATHOGENS IN GREENHOUSE PRODUCTION
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0403701
Grant No.
58-5310-0-214
Project No.
5310-21000-008-05S
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 15, 2000
Project End Date
Jun 30, 2005
Grant Year
2000
Project Director
KRUEGER R
Recipient Organization
UNIVERSITY OF CALIFORNIA
(N/A)
RIVERSIDE,CA 92521
Performing Department
PLANT PATHOLOGY
Non Technical Summary
(N/A)
Animal Health Component
50%
Research Effort Categories
Basic
20%
Applied
50%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2051461116030%
2052121116015%
2052122116015%
2054010116010%
2054020116030%
Goals / Objectives
Control soil-borne pathogens in recycled irrigation water using biosurfactants.
Project Methods
Evaluate efficiency of naturally-occurring, biosurfactant producing bacteria (as well as the purified biosurfactant) for the control of zoosporic pathogens in recycled irrigation water. Evaluate a microbial fermentation device as an onsite fermentation/injection unit for application of biosurfactant-producing bacteria (or the purified biosurfactant) into the recycled irrigation water. Evaluate the significance of insect vectors in the spread of root-infecting pathogens in commercial greenhouses and strategies for their control. Documents SCA with UC-Riverside. Formerly 5310-21000-004-07S (8/02) and 5310-21000-005-06S.

Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Greenhouse growers are facing stiffer governmental regulations concerning the discharge of spent nutrient solution in order to abate ground water pollution resulting from nutrient salts and agricultural chemicals. At the same time the use of recycled irrigation water by greenhouse growers is increasing due to increased pressure by water purveyors and governmental agencies to reduce water usage. When used as a completely closed recirculating system, the above would seem to be a solution the problems of pollution and water conservation. However, serious grower concerns exist regarding the spread of soil-borne, root-infecting plant pathogens (particularly the zoosporic species) in recycled irrigation water. These pathogens are spread primarily in recycled irrigation water. Additionally, certain insects have been implicated as vectors of some of these soil borne pathogens. In agricultural cultural systems employing recycled irrigation water, zoosporic plant pathogens constitute the most serious threat to economic production. Control of zoosporic pathogens would benefit all producers and users of plant material for landscape and interiorscape purposes. A major benefactor of this technology would be the $3,290,000,000.00 nursery industry in California. Our overall goal is to develop biologically sound and nonpolluting strategies for the efficacious control of zoosporic root-infecting pathogens in recirculating hydroponic systems. Our strategies include (i) the use of naturally-occurring, biosurfactant-producing bacteria (or the purified biosurfactant(s)) for the control of zoosporic pathogens in recycled irrigation water, (ii) to identify the role of insects in the spread of root-infecting pathogens and (iii) to develop strategies for the control of insects that vector these pathogens. 2. List the milestones (indicators of progress) from your Project Plan. 1. To have refined the technology for commercial implementation of a purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. 2. Explore nitrogen stabilizers as an amendment to the recycled irrigation water to enhance the population of microorganisms employed as biocontrol agents. 3. Evaluate fungus gnat and moth fly control with Beauveria-colonized bait under greenhouse conditions. 4. Identify the role of greenhouse insect pests as potential vectors of bacterial plant pathogens. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. To have refined the technology for commercial implementation of a purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. Milestone Fully Met 2. Explore nitrogen stabilizers as an amendment to the recycled irrigation water to enhance the population of microorganisms employed as biocontrol agents. Milestone Substantially Met 3. Evaluate fungus gnat and moth fly control with Beauveria-colonized bait under greenhouse conditions. Milestone Substantially Met 4. Identify the role of greenhouse insect pests as potential vectors of bacterial plant pathogens. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? (FY2006) 1. Verify and refine the efficacy of biosurfactants in controlling the spread of Phytophthora, as well as other zoosporic pathogens, when it is injected into the irrigation line. 2. Quantitatively and qualitatively assess changes in the resident bacterial population in recycled nutrient solutions amended with specific chemicals. 3. Evaluate the efficacy of new bait formulations of Beauveria bassiana for sustained control of shore flies, fungus gnats and moth flies in greenhouse trials. (FY2007) 1. Verify and refine efficacy of biosurfactants in controlling the spread of bacterial pathogens when it is injected into the irrigation line. 2. Quantitatively and qualitatively assess changes in plant growth and control of root disease cause by zoosporic plant pathogens and provide dose and application frequency data for commercial application of nitrogen stabilizer. 3. Assess insect transmission of Phytophthora species and bacterial plant pathogens by shore flies and fungus gnats at different life stages. (FY2008) 1. To identify the mechanism of disease suppression following the application of nitrogen stabilizers to the recycled irrigation water. 2. Provide dose and application frequency data for commercial application in greenhouses of bait formulation of Beauveria bassiana for sustained control of shore flies, fungus gnats, and moth flies. 3. Evaluate pathogen dissemination and transmission of Phytophthora species to susceptible healthy plants (or plant parts) by life stages of shore flies and fungus gnats that were shown to acquire the pathogen. 4a What was the single most significant accomplishment this past year? Root rot is a major problem in greenhouses using recirculating nutrient irrigation systems. We were able to demonstrate the efficacy of biosurfactants in the control of root rot was demonstrated in vivo using the pepper - Phytophthora capsici pathosystem. Amending the recirculating nutrient solution with either a rhamnolipid or saponin biosurfactant (150 or 200 mg a.i. /L, respectively) completely suppressed disease development in both ebb-and-flow and top-irrigated cultural systems, with either an organic potting mix or rockwool as the planting medium. while in the absence of either biosurfactant, all plants died within 5 to 7 weeks following introduction of the pathogen into the system. While plants from systems treated with either biosurfactant weighed less than those in untreated control systems, indicating that there may have been some phytotoxicity, treatment of the reservoir solution with biosurfactants significantly increased bacterial populations, and it cannot be concluded at this point whether or not the decrease in plant weight can be attributed to the biosurfactant itself or to the increased bacterial populations and the metabolites they produce. Our results provide conclusive evidence that biosurfactants (either a rhamnolipid or a saponin) can provide efficacious disease control of root rot caused by zoospores of Phytophthora capsici in a recirculating cultural system. 4d Progress report. 5310-21000-005-06S (Biology and Control of Soil-Borne Pathogens in Greenhouse Production) SCA ) SCA 58-5310-0-214. This report serves to document research conducted under a reimbursable agreement between ARS and the University of California. Objective 1. To refine the technology for commercial implementation of a purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. Accomplishments: Investigations initiated last year utilizing both ebb- and-flow and top-irrigated recirculating cultural systems were completed. In top-irrigated and ebb-and-flow cultures, the amendment of the reservoir solution with either of two biosurfactants proved efficacious is controlling the spread of Phytophthora by the recirculating nutrient solution. The two biosurfactants tested were a rhamnolipid and a saponin. Biosurfactant concentration was measured daily by taking surface tension measurements, and more product was added when the concentration in the nutrient solution decreased to levels below that which provides acceptable lytic activity. This level was set at the surface tension that causes lysis of zoospores in fewer than 5 min (i.e., 35 dynes/cm of rhamnolipid, or 50 dynes/cm of saponin. These concentrations were verified by determining whether or not the solution would prevent the colonization of pepper leaf baits by zoospores. As a result of these measurements, the rhamnolipid product was added every 1 to 7 days (average 2.5), whereas the saponin product was added every 3 to 10 days (average 5). The efficacy of biosurfactants in the control of root rot was demonstrated in vivo using the pepper - Phytophthora capsici pathosystem. Amending the recirculating nutrient solution with either the rhamnolipid or saponin biosurfactant (150 or 200 mg a.i./L, respectively) completely suppressed disease development in both ebb-and- flow and top-irrigated cultural systems, with either an organic potting mix or rockwool as the planting medium. In the absence of either biosurfactant, all plants died within 5 to 7 weeks following introduction of the pathogen into the system. Plants from systems treated with either biosurfactant weighed less than those in untreated control systems, indicating that there may have been some phytotoxicity. However, treatment of the reservoir solution with biosurfactant significantly increased bacterial populations, and it cannot be concluded at this point whether or not the decrease in plant weight can be attributed to the biosurfactant itself, or to the increased bacterial populations and the metabolites they produce. Objective 2: Explore nitrogen stabilizers as an amendment to the recycled irrigation water to enhance the population of microorganisms employed as biocontrol agents. Accomplishment: Amending the recirculating nutrient solution of hydroponically-grown pepper with a nitrogen stabilizer at 12.5 mg a.i. /L resulted in the selective enhancement of the population of indigenous fluorescent Pseudomonas spp. by two orders of magnitude. Similar results were obtained with other nitrogen stabilizers, or their active ingredients (i.e., xylene and 1, 2, 4-trimethylbnezene). Most importantly, these amendments significantly reduced sporangial production on pepper roots and controlled disease development caused by Phytophthora capsici. Objective 3. . Evaluate fungus gnat and moth fly control with Beauveria-colonized bait under greenhouse conditions Accomplishment: We significantly reduced the formulation time of the bait (reduction from two week to one week) and demonstrated that bait can be stored, fully viable, under refrigeration (4C) for two years. Objective 4. Identify the role of greenhouse insect pests as potential vectors of bacterial plant pathogens. Accomplishment: Our original objective was to identify the role of greenhouse insect pests as potent vectors of bacterial plant pathogens. However, we changed the target pathogen from bacterial pathogens to Phytophthora ramorum. Phytophthora ramorum, the causal agent of sudden oak death, is a soil borne pathogen with both an above- and below- ground life stages (i.e., sporangia and chlamydospores) in commercial greenhouse and nursery operations as well as in native plant stands. To our knowledge, the potential role of an insect vector for P. ramorum in greenhouse production systems has not been investigated. However, our studies showed that larvae of fungus gnats and shore flies, two of the most common insect pests in greenhouses and nurseries, can ingest and excrete both sporangia and chlamydospores of Phytophthora ramorum. Whether these spores were alive and could function as inoculum has not been determined. However, there is a distinct probability that these insects could be involved in transmission of this pathogen in a manner similar to that which we have documented for Fusarium avenaceum, Thielaviopsis basicola, and Pythium aphanidermatum. The latter pathogen is closely related to P. ramorum. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A. We established that shore flies, fungus gnats and moth flies can serve as aerial vectors of certain soil borne, root-infecting plant pathogenic fungi (i.e.,Fusarium, Verticillium and Theilaviopsis). The above findings have prompted further investigations into the role of these common greenhouse insect pests, which were previously considered merely as a nuisance, as vectors of species of Phytophthora (including P. ramorum), Pythium , and certain bacterial pathogens of greenhouse plants. B. We isolated and identified a strain of Beauveria bassiana as an entomopathogen of shore flies, fungus gnats, and moth flies and developed it as a biopesticidal bait for delivering the entomopathogen to insect infested sites in greenhouses. The above findings have prompted investigations on application frequency and efficacy of the biopesticide bait for insect control in greenhouses. C. We discovered and provided data on the efficacy of biosurfactants (rhamnolipids and saponins) for the control of zoosporic plant pathogens in recycled irrigation water. Further experiments refining the timing, dose and frequency of surfactant application are currently under investigation. D. We identified a chemical (i.e., a nitrogen stabilizer) which, when added to recycled irrigation water, selectively enhances the population of resident fluorescent Pseudomonas species, which, in turn (either directly or indirectly), significantly reduced sporangial production on pepper roots and controlled disease development caused by Phytophthora capsici. These results prompted further investigations into the specific mechanism involved in disease control and refinement of the timing, dose, and frequency of application of the chemical for management of diseases caused by Phytophthora and Pythium spp. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A. In 1998, we patented a naturally-produced biosurfactant produced by Pseudomonas spp. (i.e., rhamnolipids) for use as a biopesticide for control of zoosporic pathogens. U.S. Patent No. 5,767,090: Microbially- produced rhamnolipids (biosurfactants) for the control of plant pathogenic zoosporic fungi. As a result of our discovery, a commercial company licensed our invention, obtained (May 2004) an EPA registration for the biosurfactant as a biopesticide, and was awarded The 2004 Presidential Green Chemistry Challenge Award, at a reception held on June 28, 2004, in Washington, DC, National Academy of Sciences Auditorium. B. On March 17, 2004, we submitted a Disclosure and Record of Invention, UC (Case No. 2004-457-1) - Formulation of Beauveria bassiana as a biopesticidal bait for the control of dipterous pests of greenhouse crops. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Invitational seminar (45 min.), Department of Plant Pathology, Insect vectors of soil borne plant pathogenic fungi in greenhouses and their control with an entomopathogenic fungus University .of California, Riverside, Dec. 4,2003. Invitational seminar (60 min.), Rotary Club., Insects as vectors of soil borne plant pathogenic microorganisms, Dec. 16, 2003, Cal Baptist, Riverside. Invitational seminar (60 min.), Insect vectors of soil borne plant pathogenic fungi in greenhouses and their control with an entomopathogenic fungus, University of California, Department of Plant Pathology, Davis, May 17, 2004. Invitational presentation, (15 min.), Insect vectors of soil borne fungi and their control with Beauveria bassiana, Soil Fungus Conference, Reno Nevada, March 24, 2004. Nielsen, C.J., Stanghellini, M.E., and Ferrin, D.M. 2003. Phytophthora in recirculating cultural systems: The influence of different irrigation regimes on disease development. Phytopathology 94: S153. Stanghellini, M.E., Ferrin, D. M., and Radewald, K.K. 2003. Effect of fumigation depth on root rot of melon caused by Monosporascus cannonballus. Phytopathology 94: S154. El-Hamalawi, Z.A., and Stanghellini, M.E. 2003. Acquisition and aerial dissemination of Fusarium and Verticillium by adult shore flies. Phytopathology 94: S151. Stanghellini, M.E., and El-Hamalawi, Z.A. 2004. Efficacy of Beauveria bassiana on colonized millet seed as a biopesticide for the control of shore flies in greenhouses. Phytopathology 94: S98. El-Hamalawi, Z.A., and Stanghellini, M.E. 2004. Disease development on lisianthus (Eustoma grandiflorum) following aerial transmission of Fusarium avenaceum by adult shore flies, fungus gnats, and moth flies. Phytopathology 94: S27. Pagliaccia, D., Merhaut, D., and Stanghellini, M.E. 2004. Enhancement of the fluorescent pseudomonad population after amending the recirculating nutrient solution of hydroponically-grown plants with a nitrogen stabilizer. Phytopathology 94:S80. Nielsen, C.J., Stanghellini, M.E., and Ferrin, D.M. 2004. Cyclic production of sporangia and zoospores by Phytophthora capsici on pepper roots in hydroponic culture. Phytopathology 94: S76. El-Hamalawi, Z.A., and Stanghellini, M.E. 2005. Disease development on Lisianthus following aerial transmission of Fusarium avenaceum by adult shore flies, fungus gnats, and moth flies. Plant Disease 89:619-623.

Impacts
(N/A)

Publications

  • Stanghellini, M.E., Nielsen, C.J., Kim, D.H., Rasmussen, S.L., Rorbaugh, P.A. Influence of sub- versus top-irrigation and surfactants in a recirculating system on disease incidence caused by Phytophthora spp. in potted pepper plants. Plant Disease. 2000. v. 84. p. 1147-1150.


Progress 09/15/00 to 06/30/05

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Greenhouse growers are facing stiffer governmental regulations concerning the discharge of spent nutrient solution in order to abate ground water pollution resulting from nutrient salts and agricultural chemicals. At the same time the use of recycled irrigation water by greenhouse growers is increasing due to increased pressure by water purveyors and governmental agencies to reduce water usage. When used as a completely closed recirculating system, the above would seem to be a solution to the problems of pollution and water conservation. However, serious grower concerns exist regarding the spread of soil-borne, root-infecting plant pathogens (particularly the zoosporic species) in recycled irrigation water. These pathogens are spread primarily in recycled irrigation water. Additionally, certain insects have been implicated as vectors of some of these soil borne pathogens. In agricultural cultural systems employing recycled irrigation water, zoosporic plant pathogens constitute the most serious threat to economic production. Control of zoosporic pathogens would benefit all producers and users of plant material for landscape and interiorscape purposes. A major benefactor of this technology would be the $3,290,000,000.00 nursery industry in California. Our overall goal is to develop biologically sound and nonpolluting strategies for the efficacious control of zoosporic root-infecting pathogens in recirculating hydroponic systems. Our strategies include (i) the use of naturally-occurring, biosurfactant-producing bacteria (or the purified biosurfactant(s)) for the control of zoosporic pathogens in recycled irrigation water, (ii) to identify the role of insects in the spread of root-infecting pathogens and (iii) to develop strategies for the control of insects that vector these pathogens. This project contributes to National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement. 2. List by year the currently approved milestones (indicators of research progress) (FY2006): 1. To verify and refine the efficacy of biosurfactants in controlling the spread of Phytophthora, as well as other zoosporic pathogens, when it is injected into the irrigation line. 2. To quantitatively and qualitatively assess changes in the resident bacterial population, including the fluorescent Pseudomonas population, in recycled nutrient solutions amended with specific chemicals. 3. Evaluate the efficacy of new bait formulations for sustained control of shore flies, fungus gnats and moth flies in expanded greenhouse trials. 4. Evaluate pathogen dissemination and transmission of various species of Phytophthora and Pythium by various life stages of shore flies and fungus gnats. (FY2007): 1. Search for chemical substrates which selectively enhance resident populations of fluorescent pseudomonads in amended recycled irrigation water. Anticipated accomplishment: Discovery of new and more efficacious substrates that enhance specific bacterial populations and suppress disease development. 2. Establish the relationship between enhanced populations of fluorescent pseudomonads and suppression of root disease caused by zoosporic pathogens. Anticipated accomplishment: There will be a relationship between population densities of specific fluorescent pseudomonads and disease suppression which can be manipulated to optimize commercial application of the technology. 3. Continue studies on the acquisition and transmission of various species of Phytophthora and Pythium by shore flies and fungus gnats. Anticipated accomplishment: Specific insect life stages will be identified as probable vectors for management. 4. Evaluate acquisition and transmission of Phytophthora ramorum by land and water snails. Anticipated accomplishment: The role of snails in the acquisition and transmission will be verified and targeted for further research. (FY2008): 1. To identify the mechanism of disease suppression following the application of chemicals which stimulate resident fluorescent pseudomonads in recycled irrigation water. Anticipate accomplishment: Identification of the specific mechanism will enable us to select specific substrates for maximum enhancement of that mechanism. 2. Continue identification of chemical substrates which selectively enhance resident populations of fluorescent pseudomonads in amended recycled irrigation water. Anticipated accomplishment: Discovery of new and more efficacious substrates that will enhance specific bacterial populations and suppress disease development. 3. Evaluate the epidemiological significance of insect and snail transmission of Phytophthora and Pythium species in greenhouse production systems. Anticipated accomplishments: Establish the necessity of management strategies for insect and snail eradication/abatement. (FY2009): 1. Establish dose and application frequency data for commercial application of specific chemical substrates to recycled irrigation water which suppress root disease. Anticipated accomplishment: Supply industry partners with efficacy data on chemical substrate application technology for disease management. 2. Continue evaluation of the epidemiological significance of insect and snail transmission of Phytophthora and Pythium species in greenhouse production systems. Anticipated accomplishment: Establish the necessity and refinement of management strategies for insect and snail eradication/abatement. 4a List the single most significant research accomplishment during FY 2006. Control of root rot by use of nitrogen stabilizers to enhance the resident fluorescent Pseudomonas species populations. Root rot is a major problem in greenhouses using recirculating nutrient irrigation systems. In our research, our goal is to develop biologically sound and nonpolluting strategies for the efficacious control of zoosporic root-infecting pathogens in recirculating hydroponic systems. Research conducted by University of California, Riverside, Department of Plant Pathology in cooperation with the National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA was able to provide conclusive evidence that certain chemical substrates, (i.e., nitrogen stabilizers) which, when added to recycled irrigation water, selectively enhance the population of resident fluorescent Pseudomonas species. These species, in turn, significantly suppress, either directly or indirectly, disease development caused by Phytophthora capsici on peppers and Pythium aphanidermatum on cucumbers. These research results should eventually benefit the nursery plants due to root-infecting pathogens. This research addresses Problem Statement 1A, efficiently and effectively manage plant and microbial genetic resources, under National Program 301 Plant Genetic Resources, Genomics, and Genetics Improvement. 4b List other significant research accomplishment(s), if any. Recognizing fungus gnats as an aerial vector of Phytophthora ramorum. Shore flies, fungus gnats and moth flies are normally viewed as a nuisance; this research shows they are vectors of pathogens normally considered to be soilborne which can kill healthy plants under normal greenhouse conditions. Research conducted by the University of California, Riverside, Department of Plant Pathology in cooperation with the National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA was able to show that these common greenhouse insect pests, such as fungus gnats, may serve as a vector for Phytophthora ramorum. Recognition of fungus gnats as aerial vectors of a disease that is normally soilborne is a significant finding. This new information can help reduce losses in commercial greenhouses due to P. Ramorum. This research addresses Problem Statement 1A, efficiently and effectively manage plant and microbial genetic resources, under National Program 301 Plant Genetic Resources, Genomics, and Genetics Improvement. Development of a new delivery system of Beauveria bassiana for control of shore flies, the aerial vector of Fusarium avenaceum. Shore flies have been shown to be aerial vectors of Fusarium avenaceum, a fungal disease normally considered to be soilborne which caused economic damage in commercial greenhouses. Research conducted in the University of California, Riverside, Department of Plant Pathology in cooperation with the National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, have isolated a strain of Beauveria bassiana (Bb) from a shore fly cadaver which is effective as a biopesticide for the control of shore flies. A new method of delivery was developed for the B. bassiana (i. e., B. bassiana on colonized millet seed) and the potential efficacy of the delivery system has been demonstrated. This research may provide an environmentally friendly method for the control of F. avenaceum in commercial greenhouses. This research address the problem statement 1A, efficiently and effectively manage plant and microbial genetic resources, under National Program 301 Plant Genetic Resources, Genomics, and Genetics Improvement. 4d Progress report. Evaluation of the efficacy of naturally-occurring, biosurfactant for the control root rot of pepper caused by zoosporic pathogens in recycled irrigation water: Accomplishments: Twenty recirculating hydroponic cultural systems (both ebb-and flow and top irrigation) were constructed in a greenhouse to assess the above objective. Two methods of application were investigated: injecting concentrated biosurfactant into the irrigation line, and treating the reservoir itself. In top-irrigated pot culture, in which the biosurfactant was injected into the irrigation line, the biosurfactant must be present in the irrigation water with every irrigation cycle. When plants are irrigated twice a day, if the biosurfactant was injected during only one of the cycles, there was not enough residual activity to inhibit the production of zoospores during the irrigation cycle in which no chemical was applied. The pathogen spread through the system, though at a reduced rate from the untreated system. If the biosurfactant was injected during both irrigation cycles, zoospore production was not detected, and the non-inoculated plants in the system remained healthy. In top-irrigated and ebb-and-flow rockwool cultures, the reservoir water itself was brought up to an effective concentration of biosurfactant, and this was maintained by adding chemical every 2-5 days (average 3.5). Biosurfactant concentration was ascertained daily by taking surface tension measurements. Minimum effective concentrations were based on bioassays performed in the lab. Several systems were treated with surfactant only every other day (surfactant was added to the reservoir), even when concentration of surfactant in the reservoir was not found to be at what is considered to be the minimum effective concentration. There was no spread of the pathogen in any of the treated systems, whether treated every other day or based on reservoir concentration. Further analysis would be necessary to determine if this is consistent, if the length of time between reservoir treatments could be increased, and to determine why the pathogen is not moving even though surfactant could not be detected. Additionally, production of sporangia and zoospores by Phytophthora capsici on roots of hydroponically-grown peppers was shown to be cyclic in nature. Production of sporangia on peppers grown with a 12-hr photo period was diurnal; the greatest number of full sporangia were present at hours 8-10 of the light cycle, while empty sporangia were most abundant at hours 4-6 of the dark cycle. Zoospore production was primarily nocturnal; populations began to increase at the start of the dark cycle and, in general, peaked sharply at hour 4 of the dark cycle. Zoospore populations at this time ranged from 9-65 times (mean of 33) greater than when populations were at their lowest (hours 6-8 of the light cycle). Altogether, 83% of the total zoospore population, sampled every two hours over a 24-hr period, was isolated from the hydroponic solution during the dark cycle. When peppers were grown under continuous light, a cyclic pattern of sporangium and zoospore production did not occur. These results may be used in selection of optimum sampling time for determination of the presence of zoosporic pathogens in recycled irrigation water and timing of pesticide application directed specifically towards the motile zoospore stage. Further, fluorescent Pseudomonas spp. have historically been associated, via diverse mechanisms, with suppression of root disease caused by a numerous fungal and fungal-like pathogens. However, inconsistency in performance in disease abatement following their employment has been a problem. This has been attributed, in part, to the inability of the biocontrol bacterium to maintain a critical threshold population necessary for sustained biocontrol activity. Numerous methods, which include the addition of various food sources, have been used to selectively enhance and maintain desired populations of the biological control bacterium. Recently, we discovered that a nitrogen stabilizer (N- Serve, Dow Agrosciences) exhibited in vitro fungicidal activity towards zoospores Phytophthora and Pythium spp., and selectively enhanced the resident population of fluorescent pseudomonads in the amended and recycled nutrient solution used in the cultivation of hydroponically- grown gerbera and pepper plants. Additionally, the chemical had a positive effect on plant growth, flower productivity (gerbera), and a stabilizing effect on the pH and electrical conductivity of the nutrient solution. The population dynamics of bacteria in recirculating nutrient solutions used to irrigate hydroponically-grown gerbera plants after amendment with N-ServeRG were compared to those in non-amended nutrient solutions. Significant bacterial population increases (total bacterial populations which included the fluorescent Pseudomonas spp.) were consistently recorded in N-ServeRG- amended compared to nonamended nutrient solutions. These increases, which were 2 or more log units higher that their respective controls, occurred within 48 to 72 hrs after each consecutive addition of N-ServeRG to the nutrient solution. The fluorescent Pseudomonas spp. population accounted for 0.7 % of the total aerobic heterotrophic bacterial population in unamended and 40% in N-Serve- amended nutrient solutions. Similar and significant increases in the resident fluorescent Pseudomonas spp. population were also recorded following the addition of N-serve to the nutrient solution used to irrigate pepper plants. Identification and evaluation of strategies for the control of insect vectors of soilborne plant pathogens in commercial greenhouses: Accomplishments: Shore flies (Scatella spp.), fungus gnats (Bradysia spp.) , and moth flies (Psychoda spp.)are the most common and abundant insects in commercial greenhouse facilities. Various life stages of these insects, which have historically been considered merely a nuisance, have been implicated as potential sources of plant pathogen introduction into and spread within a commercial facilities. Our current studies provide conclusive evidence that adult shore flies, fungus gnats and moth flies are attracted to and readily acquire (either externally and/or internally) macroconidia of F. avenaceum produced on naturally-infected stems of a host plant (i.e., lisianthus) and then disseminate acquired macroconidia to healthy plants, which subsequently died. A high percentage of transmission, as evidenced by the number of plants infected (75% within 4 days), reflects the efficiency of these insects as vectors. Fusarium crown and stem rot of lisianthus (Eustoma grandiflorum), caused by Fusarium avenaceum, is a destructive disease in California. The pathogen produces large masses of orange-colored macroconidia on stem lesions that extend up to 35 cm in length from the soil surface. Populations of macroconidia (97% viability) range from 1.1 x 108 to 1.9 x 108 per cm of infected stem tissue. These results provide strong evidence that an above- ground life stage for a soilborne pathogen can serve as a source of inoculum for acquisition and aerial dissemination by adult shore flies, fungus gnats, and moth flies and that control of these insects, which previously were considered merely as a nuisance, should receive serious consideration as a strategy in disease management. Additionally, a strain of Beauveria bassiana (Bb), isolated from a shore fly cadaver, was evaluated as a biopesticide for the control of shore flies. We developed a new method of delivery of the potential biological control agent. Our results demonstrate the potential efficacy of B. bassiana on colonized millet seed as a biopesticide for the control of shore flies. Bb was grown on autoclaved millet seed for 2 weeks, air- dried at 24DGC for 7 days and stored in paper bags. Dried Bb-colonized millet seeds were broadcast on the surface of pots containing potting medium naturally-infested with larvae and pupae or pots infested only with adult flies. Controls consisted of insect-infested pots amended with autoclaved millet seeds. Pots of the same treatment were placed in insect- proof screen cages. At daily intervals for 15 days, adult fly populations were recorded in each cage. In cages initially containing only larvae and pupae, adult fly populations in the control (no Bb) treatment reached a final population of 70 adults on day 15. In contrast only two adult flies were observed in the Bb-treatment on day 15. In cages containing only adult shore flies, the entire population was dead within 10 to 12 days in the Bb-treatment. In the absence of Bb, 65% of the initial population of adults was still alive on day 12. 5. Describe the major accomplishments to date and their predicted or actual impact. All accomplishments address National Program 301 (Plant Genetic Resources, Genomics, and Genetic Improvement), Component I (Plant and Microbial Genetic Resource Management), Problem Statement a (Efficiently and Effectively Manage Plant and Microbial Genetic Resources). We established that shore flies, fungus gnats and moth flies can serve as aerial vectors of certain , root-infecting plant pathogenic fungi (i. e., Fusarium, Verticillium and Theilaviopsis). We isolated and identified a strain of Beauveria bassiana as an entomopathogen of shore flies, fungus gnats, and moth flies and developed it as biospesticidal bait for delivering the entomopathogen to insect- infested sites in greenhouses. We discovered and provided data on the efficacy of biosurfactants (rhamnolipids and saponins) for the control of zoosporic plant pathogens in recycled irrigation water. We identified chemical substrates (i.e., nitrogen stabilizers) which, when added to recycled irrigation water, selectively enhances the population of resident fluorescent Pseudomonas species, which, in turn (either directly or indirectly), significantly disease development caused by Phytophthora capsici on pepper and Pythium aphanidermatum on cucumber. We demonstrated that fungus gnat larvae can ingest and excrete viable chlamydospores of Phytophthora ramorum, as well as hyphal swellings of several species of Pythium, and that infested larvae can transmit P. ramorum to healthy rhododendron leaves which subsequently became infected with the latter pathogen. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? In 1998, we patented a naturally-produced biosurfactant produced by Pseudomonas spp. (i.e., rhamnolipids) for use as a biopesticide for control of zoosporic pathogens. U.S. Patent No. 5,767,090: Microbially- produced rhamnolipids (biosurfactants) for the control of plant pathogenic zoosporic fungi. As a result of our discovery, a commercial company licensed our invention, obtained (May 2004) an EPA registration for the biosurfactant as a biopesticide, and was awarded The 2004 Presidential Green Chemistry Challenge Award, at a reception held on June 28, 2004, in Washington, DC, National Academy of Sciences Auditorium. On March 17, 2004, we submitted a Disclosure and Record of Invention, UC (Case No. 2004-457-1) - Formulation of Beauveria bassiana as a biopesticide bait for the control of dipterous pests of greenhouse crops. Beauveria bassiana- colonized millet seed were supplied to a commercial grower for testing. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Invitational seminar (45 min.), Department of Plant Pathology, Insect vectors of soil borne plant pathogenic fungi in greenhouses and their control with an entomopathogenic fungus University .of California, Riverside, Dec. 4,2003. Invitational seminar (60 min.), Rotary Club., Insects as vectors of soil borne plant pathogenic microorganisms, Dec. 16, 2003, Cal Baptist, Riverside. Invitational seminar (60 min.), Insect vectors of soil borne plant pathogenic fungi in greenhouses and their control with an entomopathogenic fungus, University of California, Department of Plant Pathology, Davis, May 17, 2004. Invitational presentation, (15 min.), Insect vectors of soil borne fungi and their control with Beauveria bassiana, Soil Fungus Conference, Reno Nevada, March 24, 2004. Invitational presentation, (15 min.), Phytophthora ramorum and fungus gnat larvae. Soil Fungus Conference, Reno Nevada, March 23, 2006 (presented by Ms. N. Hyder., PhD student). Invitational presentation, (15 min.), Biological control of Pythium and Phytophthora in recirculating cultural systems. Soil Fungus Conference, Reno Nevada, March 23, 2006. (presented by Ms. Deborah Pagliaccia, PhD student). Invitational presentation, (30 min.), Update on the Biology and Control of Soil- Borne Pathogens in Greenhouse Production, 2nd Floral and Nursery Crops Researchers Workshop, Portland, Oregon, June 12-15, 2006, (presented by M. E. Stanghellini, project PI). Poster presentation: Hyder, N., Stanghellini, M.E., and Coffey, M. 2006. Role of fungus gnat larvae in the acquisition and transmission of oomycete propagules. American Phytopathological Society, Annual Meeting, Quebec, Canada. July 28-Aug.2, 2006. Scientific Publications: Nielsen, C.J., Stanghellini, M.E., and Ferrin, D.M. 2003. Phytophthora in recirculating cultural systems: The influence of different irrigation regimes on disease development. Phytopathology 94: S153. Stanghellini, M.E., Ferrin, D. M., and Radewald, K.K. 2003. Effect of fumigation depth on root rot of melon caused by Monosporascus cannonballus. Phytopathology 94: S154. El-Hamalawi, Z.A., and Stanghellini, M.E. 2003. Acquisition and aerial dissemination of Fusarium and Verticillium by adult shore flies. Phytopathology 94: S151. Stanghellini, M.E., and El-Hamalawi, Z.A. 2004. Efficacy of Beauveria bassiana on colonized millet seed as a biopesticide for the control of shore flies in greenhouses. Phytopathology 94: S98. El-Hamalawi, Z.A., and Stanghellini, M.E. 2004. Disease development on lisianthus (Eustoma grandiflorum) following aerial transmission of Fusarium avenaceum by adult shore flies, fungus gnats, and moth flies. Phytopathology 94: S27. Pagliaccia, D., Merhaut, D., and Stanghellini, M.E. 2004. Enhancement of the fluorescent pseudomonad population after amending the recirculating nutrient solution of hydroponically-grown plants with a nitrogen stabilizer. Phytopathology 94:S80. Nielsen, C.J., Stanghellini, M.E., and Ferrin, D.M. 2004. Cyclic production of sporangia and zoospores by Phytophthora capsici on pepper roots in hydroponic culture. Phytopathology 94: S76. Ferrin, D.M., and Stanghellini, M.E. 2004. The effect of two melanin biosynthesis inhibitors on growth and reproduction of Monosporascus cannonballus. Phytopathology 94: S29. Ferrin, D.M., and Stanghellini, M.E. 2004. The effect of osmotic water potential on growth and reproduction of Monosporascus cannonballus. Phytopathology 94: S29. Nielsen, C.J., Ferrin, D.M., and Stanghellini, M.E. 2005. Efficacy of rhamnolipid and saponin biosurfactants in the management of Phytophthora root rot of pepper in a recirculating cultural system. Phytopathology 95: S75. Pagliaccia, D., Stanghellini, M.E., and Saccardo, F. 2005. Induction of disease resistance in pepper by fluorescent pseudomonads via chemical selection: an integrated approach to management of root diseases. Phytopathology 95: S79. Roberts, B.W., Bruton, B.D., Edelson, J.V., Lu, W., Perkins Veazie, P.M., Shrefler, J.W., and Stanghellini, M.E. 2005. Watermelon seedling mortality associated with Pythium aphanidermatum. HortScience 40:873. El-Hamalawi, Z.A., and M. E. Stanghellini. 2005. Disease development on lisianthus following acquisition and aerial transmission of Fusarium avenaceum by adult shore flies, fungus gnats, and moth flies. Plant Dis. 89:619-623. Stanghellini, M.E., and Z.A. El-Hamalawi. 2005. Efficacy of Beauveria bassiana on colonized millet seed as a biopesticide for the control of shore flies. Hort. Sci.40:1384-1388. Waugh, M.M., D. M. Ferrin, and M.E. Stanghellini. 2005. Colonization of cantaloupe roots by Monosporascus cannonballus. Mycological Research 109:1297-1301. Ferrin, D.M., and M. E. Stanghellini. 2005. Effect of osmotic water potential on mycelial growth and perithecial production of Monosporascus cannonballus in vitro. Plant Pathology 55:421-426. Hyder, N., Stanghellini, M.E., and Coffey, M. 2006. Role of fungus gnat larvae in the acquisition and transmission of oomycete propagules. Inoculum 57 (4).

Impacts
(N/A)

Publications

  • Stanghellini, M.E., Nielsen, C.J., Kim, D.H., Rasmussen, S.L., Rorbaugh, P.A. Influence of sub- versus top-irrigation and surfactants in a recirculating system on disease incidence caused by Phytophthora spp. in potted pepper plants. Plant Disease. 2000. v. 84. p. 1147-1150.


Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Greenhouse growers are facing stiffer governmental regulations concerning the discharge of spent nutrient solution in order to abate ground water pollution resulting from nutrient salts and agricultural chemicals. At the same time the use of recycled irrigation water by greenhouse growers is increasing due to increased pressure by water purveyors and governmental agencies to reduce water usage. When used as a completely closed recirculating system, the above would seem to be a solution the problems of pollution and water conservation. However, serious grower concerns exist regarding the spread of soil-borne, root- infecting plant pathogens (particularly the zoosporic species) in recycled irrigation water. These pathogens are spread primarily in recycled irrigation water. Additionally, certain insects have been implicated as vectors of some of these soilborne pathogens. In agricultural cultural systems employing recycled irrigation water, zoosporic plant pathogens constitute the most serious threat to economic production. Control of zoosporic pathogens would benefit all producers and users of plant material for landscape and interiorscape purposes. A major benefactor of this technology would be the $3,290,000,000.00 nursery industry in California. 2. List the milestones (indicators of progress) from your Project Plan. Our overall goal is to develop biologically sound and nonpolluting strategies for the efficacious control of zoosporic root-infecting pathogens in recirculating hydroponic systems. Our strategies include (i) the use of naturally-occurring, biosurfactant-producing bacteria (or the purified biosurfactant(s)) for the control of zoosporic pathogens in recycled irrigation water, (ii) to identify the role of insects in the spread of root-infecting pathogens and to develop strategies for the control of insects that vector these pathogens. 3. Milestones: A. Anticipated accomplishments by end of year 3 (2004). 1. To have refined the technology for commercial implementation of a purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. Results: i) In top-irrigated pot culture, the biosurfactant must be present in the irrigation water with every irrigation cycle. When plants are irrigated twice a day, if the biosurfactant was injected into the irrigation water only once a day, there was not enough residual activity to inhibit the production of zoospores during the irrigation cycle in which no chemical was applied. The pathogen spread through the system, though at a reduced rate from the untreated system. If the biosurfactant was injected during both irrigation cycles, zoospore production was not detected, and the non-inoculated plants in the system remained healthy, (ii) in top-irrigated and ebb-and-flow rockwool cultures, the biosurfactant proved efficacious is controlling the spread of Phytophthora by the recirculating nutrient solution. In this case, the reservoir water itself was brought up to an effective concentration of biosurfactant, and this was maintained by adding chemical every 2-5 days (average 3.5). Biosurfactant concentration was measured daily by taking surface tension measurements. Minimum effective concentrations were based on bioassays performed in the lab. 2. Most recently, we discovered a chemical which, when used as an amendment to the recycled irrigation water, may enhance the population of microorganisms (in particular- the fluorescent Pseudomonas population) employed as biocontrol agents. 3. Identification of the onset of zoospore production in recirculating cultural systems. Results: Production of sporangia and zoospores by Phytophthora capsici on roots of hydroponically-grown peppers was shown to be cyclic in nature. Production of sporangia on peppers grown with a 12-hr photoperiod was diurnal; the greatest number of full sporangia was present at hours 8-10 of the light cycle, while empty sporangia were most abundant at hours 4-6 of the dark cycle. Zoospore production was primarily nocturnal; populations began to increase at the start of the dark cycle and, in general, peaked sharply at hour 4 of the dark cycle. Zoospore populations at this time ranged from 9-65 times (mean of 33) greater than when populations were at their lowest (hours 6-8 of the light cycle). Altogether, 83% of the total zoospore population, which were estimated every two hours over a 24-hr period, was recovered from the hydroponic solution during the dark cycle. When peppers were grown under continuous light, a cyclic pattern of sporangium and zoospore production did not occur. 4. To document the method of pathogen acquisition by insect vectors and identify strategies for the control of insect vectors of soilborne plant pathogens in greenhouse cultural systems. Results: Briefly, we documented that soilborne plant pathogens which sporulate above-ground on infected stem tissue can be externally acquired by shore flies, fungus gnats and moth flies, but only the shore fly can ingest and excrete viable population of these pathogens. Additionally, we developed bait, which consists of millet seed colonized by Beauveria bassiana, which attract the insects to the colonized bait, where the insects then acquire the entomopathogen which subsequently kill the insect pests. B. Milestone for 2005, 2006, 2007. 2005: 1. Rhamnolipids. In a recent experiment, the biosurfactant was tested in top-irrigated recirculating units, with plants growing in organic media or in rockwool. Several systems were treated with surfactant only every other day (surfactant was added to the reservoir), even when concentration of surfactant in the reservoir was not found to be at what is considered to be the minimum effective concentration based on bioassays in the lab. However, there was no spread of the pathogen in these systems. Further analysis is necessary to determine if this is consistent, if the length of time between reservoir treatments could be increased, and to determine why the pathogen is not moving even though surfactant could not be detected. Additionally, we plan to expand the study to include other classes of biosurfactants which may be more resistant to biodegradation, and determine the microorganisms that are degrading the biosurfactant. Perhaps if a specific group(s) can be identified as responsible, it may be possible to alter reservoir conditions in an attempt to control their populations. 2. N-Serve: Adjustment and evaluation of the frequencies of amendment addition (N-Serve) to the recirculating nutrient solutions in greenhouse studies to facilitate economic control of zoosporic pathogens. Identification of the specific fluorescent Pseudomonas sp. stimulated by N-Serve and assessment, and nature of, its in vitro and in vivo antagonism to Phytophthora and Pythium spp. 3. Insect vs. Beauveria bassiana: Evaluate fungus gnat and moth fly control with Beauveria-colonized bait under greenhouse conditions. Refine and improve bait formulation production. 4. New Project: To identify the role of greenhouse insect pests as potential vectors of bacterial plant pathogens. Studies to include method of acquisition and transmission to susceptible hosts. 2006: 1. Develop a new model system using chrysanthemum and P. aphanidermatum for assessment of the efficacy of N-Serve for the enhancement and maintenance of beneficial bacteria in the recycled irrigation water. Evaluate the efficacy of known biological control agents (BCA), i.e., Pseudomonas aureofaciens and P. fluorescens, for control. 2. Evaluate the efficacy of new bait formulations for sustained control of shore flies, fungus gnats and moth flies in expanded greenhouse trials. 2007: 1. Increase greenhouse testing of top-performing bacterial isolates in disease control. 2. Identify the mechanism of disease suppression (i.e., antibiotic production by the introduced BCA strains, induced host resistance, etc.) following the application of N-Serve to the recycled irrigation water. 3. Evaluate Beauveria-bait formulations in commercial nurseries for insect control efficacy. 4. What were the most significant accomplishments this past year? A. Shore flies, fungus gnats and moth flies are normally viewed as a nuisance; this research shows they are vectors of pathogens normally considered to be soilborne which can kill healthy plants under normal greenhouse conditions. Research conducted in the University of California, Riverside, Department of Plant Pathology in cooperation with the National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, provided conclusive evidence that adult shore flies, fungus gnats and moth flies are attracted to and readily acquire (either externally and/or internally) macroconidia of F. avenaceum (Fusarium crown and stem rot) produced on naturally-infected stems of a host plant (i.e., lisianthus) and then disseminate macroconidia to healthy plants, causing their deaths. Under experimental conditions a 75 percent transmission rate of F. avenaceum by the macroconidia carried by the flies and the gnats occurred in healthy plants in four days from naturally infected lisianthus source plants. Recognition of shore flies, fungus gnats and moth flies as aerial vectors of a disease that is normally soilborne can help reduce losses in commercial greenhouses due to F. avenaceum. B. Shore flies have been shown to be aerial vectors of Fusarium avenaceum, a fungal disease normally considered to be soilborne which causes economic damage in commercial greenhouses. Research conducted in the University of California, Riverside, Department of Plant Pathology in cooperation with the National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, have isolated a strain of Beauveria bassiana from a shore fly cadaver which may be useful for biological control of shore flies in commercial greenhouses. A new method of delivery was developed for B. bassiana as a biocontrol agent for shore flies which resulted in entire populations of shore flies being killed in 10-12 days while without B. bassiana, 65 percent of the populations remained. This research may provide an environmentally friendly method for the control of F. avenaceum in commercial greenhouses. C. None D. This report serves to document research conducted under a reimbursable agreement between ARS and the University of California. Additional details of research can be found in the report for the parent project 5310-21000-005-00D National Clonal Citrus and Date Germplasm Repository. Objective 1: Evaluation of the efficacy of naturally-occurring, biosurfactant for the control root rot of pepper caused by zoosporic pathogens in recycled irrigation water. Accomplishments: Twenty recirculating hydroponic cultural systems (both ebb-and flow and top irrigation) were constructed in a greenhouse to assess the above objective. Two methods of application were investigated: injecting concentrated biosurfactant into the irrigation line, and treating the reservoir itself. In top-irrigated pot culture, in which the biosurfactant was injected into the irrigation line, the biosurfactant must be present in the irrigation water with every irrigation cycle. When plants are irrigated twice a day, if the biosurfactant was injected during only one of the cycles, there was not enough residual activity to inhibit the production of zoospores during the irrigation cycle in which no chemical was applied. The pathogen spread through the system, though at a reduced rate from the untreated system. If the biosurfactant was injected during both irrigation cycles, zoospore production was not detected, and the non-inoculated plants in the system remained healthy. In top-irrigated and ebb-and-flow rockwool cultures, the reservoir water itself was brought up to an effective concentration of biosurfactant, and this was maintained by adding chemical every 2-5 days (average 3.5). Biosurfactant concentration was ascertained daily by taking surface tension measurements. Minimum effective concentrations were based on bioassays performed in the lab. Several systems were treated with surfactant only every other day (surfactant was added to the reservoir), even when concentration of surfactant in the reservoir was not found to be at what is considered to be the minimum effective concentration. There was no spread of the pathogen in any of the treated systems, whether treated every other day or based on reservoir concentration. Further analysis would be necessary to determine if this is consistent, if the length of time between reservoir treatments could be increased, and to determine why the pathogen is not moving even though surfactant could not be detected. Additionally, production of sporangia and zoospores by Phytophthora capsici on roots of hydroponically-grown peppers was shown to be cyclic in nature. Production of sporangia on peppers grown with a 12-hr photoperiod was diurnal; the greatest number of full sporangia were present at hours 8-10 of the light cycle, while empty sporangia were most abundant at hours 4-6 of the dark cycle. Zoospore production was primarily nocturnal; populations began to increase at the start of the dark cycle and, in general, peaked sharply at hour 4 of the dark cycle. Zoospore populations at this time ranged from 9-65 times (mean of 33) greater than when populations were at their lowest (hours 6-8 of the light cycle). Altogether, 83% of the total zoospore population, sampled every two hours over a 24-hr period, was isolated from the hydroponic solution during the dark cycle. When peppers were grown under continuous light, a cyclic pattern of sporangium and zoospore production did not occur. These result may be used in selection of optimum sampling time for determination of the presence of zoosporic pathogens in recycled irrigation water and timing of pesticide application directed specifically towards the motile zoospore stage. Further, fluorescent Pseudomonas spp. have historically been associated, via diverse mechanisms, with suppression of root disease caused by a numerous fungal and fungal-like pathogens. However, inconsistency in performance in disease abatement following their employment has been a problem. This has been attributed, in part, to the inability of the biocontrol bacterium to maintain a critical threshold population necessary for sustained biocontrol activity. Numerous methods, which include the addition of various food sources, have been used to selectively enhance and maintain desired populations of the biological control bacterium. Recently, we discovered that a nitrogen stabilizer (N- Serve, Dow Agrosciences) exhibited in vitro fungicidal activity towards zoospores Phytophthora and Pythium spp., and selectively enhanced the resident population of fluorescent pseudomonads in the amended and recycled nutrient solution used in the cultivation of hydroponically- grown gerbera and pepper plants. Additionally, the chemical had a positive effect on plant growth, flower productivity (gerbera), and a stabilizing effect on the pH and electrical conductivity of the nutrient solution. The population dynamics of bacteria in recirculating nutrient solutions used to irrigate hydroponically-grown gerbera plants after amendment with N-ServeRG were compared to those in non-amended nutrient solutions. Significant bacterial population increases (total bacterial populations which included the fluorescent Pseudomonas spp.) were consistently recorded in N-ServeRG- amended compared to nonamended nutrient solutions. These increases, which were 2 or more log units higher that their respective controls, occurred within 48 to 72 hrs after each consecutive addition of N-ServeRG to the nutrient solution. The fluorescent Pseudomonas spp. population accounted for 0.7 % of the total aerobic heterotrophic bacterial population in unamended and 40% in N-Serve- amended nutrient solutions. Similar and significant increases in the resident fluorescent Pseudomonas spp. population were also recorded following the addition of N-serve to the nutrient solution used to irrigate pepper plants. Objective 2: Identification and evaluation of strategies for the control of insect vectors of soilborne plant pathogens in commercial greenhouses. Accomplishments: Shore flies (Scatella spp.), fungus gnats (Bradysia spp.) , and moth flies (Psychoda spp.)are the most common and abundant insects in commercial greenhouse facilities. Various life stages of these insects, which have historically been considered merely a nuisance, have been implicated as potential sources of plant pathogen introduction into and spread within a commercial facilities. Our current studies provide conclusive evidence that adult shore flies, fungus gnats and moth flies are attracted to and readily acquire (either externally and/or internally) macroconidia of F. avenaceum produced on naturally-infected stems of a host plant (i.e., lisianthus) and then disseminate acquired macroconidia to healthy plants, which subsequently died. A high percentage of transmission, as evidenced by the number of plants infected (75% within 4 days), reflects the efficiency of these insects as vectors. Fusarium crown and stem rot of lisianthus (Eustoma grandiflorum), caused by Fusarium avenaceum, is a destructive disease in California. The pathogen produces large masses of orange-colored macroconidia on stem lesions that extend up to 35 cm in length from the soil surface. Populations of macroconidia (97% viability) range from 1.1 x 108 to 1.9 x 108 per cm of infected stem tissue. These results provide strong evidence that an above- ground life stage for a soilborne pathogen can serve as a source of inoculum for acquisition and aerial dissemination by adult shore flies, fungus gnats, and moth flies and that control of these insects, which previously were considered merely as a nuisance, should receive serious consideration as a strategy in disease management. Additionally, a strain of Beauveria bassiana (Bb), isolated from a shore fly cadaver, was evaluated as a biopesticide for the control of shore flies. We developed a new method of delivery of the potential biological control agent. Our results demonstrate the potential efficacy of B. bassiana on colonized millet seed as a biopesticide for the control of shore flies. Bb was grown on autoclaved millet seed for 2 weeks, air- dried at 24DGC for 7 days and stored in paper bags. Dried Bb-colonized millet seeds were broadcast on the surface of pots containing potting medium naturally-infested with larvae and pupae or pots infested only with adult flies. Controls consisted of insect-infested pots amended with autoclaved millet seeds. Pots of the same treatment were placed in insect- proof screen cages. At daily intervals for 15 days, adult fly populations were recorded in each cage. In cages initially containing only larvae and pupae, adult fly populations in the control (no Bb) treatment reached a final population of 70 adults on day 15. In contrast only two adult flies were observed in the Bb-treatment on day 15. In cages containing only adult shore flies, the entire population was dead within 10 to 12 days in the Bb-treatment. In the absence of Bb, 65% of the initial population of adults was still alive on day 12. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A. In 1998, we patented a naturally-produced biosurfactant produced by Pseudomonas spp. (i.e., rhamnolipids) for use as a biopesticide for control of zoosporic pathogens. U.S. Patent No. 5,767,090: "Microbially- produced rhamnolipids (biosurfactants) for the control of plant pathogenic zoosporic fungi". As a result of our discovery, Jeneil Biosurfactant Co. licensed our invention, obtained (May 2004) an EPA registration for the biosurfactant as a biopesticide which will be marketed as "Zonix", and was awarded The 2004 Presidential Green Chemistry Challenge Award, at a reception held on June 28, 2004, in Washington, DC, National Academy of Sciences Auditorium. B. On March 17, 2004, we submitted a "Disclosure and Record of Invention", UC (Case No. 2004-457-1) - "Formulation of Beauveria bassiana as a biopesticidal bait for the control of dipterous pests of greenhouse crops." 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Invitational seminar (45 min.), Department of Plant Pathology, "Insect vectors of soilborne plant pathogenic fungi in greenhouses and their control with an entomopathogenic fungus" University of California, Riverside, Dec. 4,2003. Invitational seminar (60 min.), Rotary Club., "Insects as vectors of soilborne plant pathogenic microorganisms", Dec. 16, 2003, Cal Baptist, Riverside. Invitational seminar (60 min.), "Insect vectors of soilborne plant pathogenic fungi in greenhouses and their control with an entomopathogenic fungus", University of California, Department of Plant Pathology, Davis, May 17, 2004. Invitational presentation, (15 min.), Insect vectors of soilborne fungi and their control with Beauveria bassiana", Soil Fungus Conference, Reno Nevada, March 24, 2004. Nielsen, C.J., Stanghellini, M.E., and Ferrin, D.M. 2003. Phytophthora in recirculating cultural systems: The influence of different irrigation regimes on disease development. Phytopathology 94: S153. Stanghellini, M.E., Ferrin, D. M., and Radewald, K.K. 2003. Effect of fumigation depth on root rot of melon caused by Monosporascus cannonballus. Phytopathology 94: S154. El-Hamalawi, Z.A., and Stanghellini, M.E. 2003. Acquisition and aerial dissemination of Fusarium and Verticillium by adult shore flies. Phytopathology 94: S151. Stanghellini, M.E., and El-Hamalawi, Z.A. 2004. Efficacy of Beauveria bassiana on colonized millet seed as a biopesticide for the control of shore flies in greenhouses. Phytopathology 94: S98. El-Hamalawi, Z.A., and Stanghellini, M.E. 2004. Disease development on lisianthus (Eustoma grandiflorum) following aerial transmission of Fusarium avenaceum by adult shore flies, fungus gnats, and moth flies. Phytopathology 94: S27. Pagliaccia, D., Merhaut, D., and Stanghellini, M.E. 2004. Enhancement of the fluorescent pseudomonad population after amending the recirculating nutrient solution of hydroponically- grown plants with a nitrogen stabilizer. Phytopathology 94:S80. Nielsen, C.J., Stanghellini, M.E., and Ferrin, D.M. 2004. Cyclic production of sporangia and zoospores by Phytophthora capsici on pepper roots in hydroponic culture. Phytopathology 94: S76.

Impacts
(N/A)

Publications

  • Stanghellini, M.E., Nielsen, C.J., Kim, D.H., Rasmussen, S.L., Rorbaugh, P.A. Influence of sub- versus top-irrigation and surfactants in a recirculating system on disease incidence caused by Phytophthora spp. in potted pepper plants. Plant Disease. 2000. v. 84. p. 1147-1150.


Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Greenhouse growers are facing stiffer governmental regulations concerning the discharge of spent nutrient solution in order to abate ground water pollution resulting from nutrient salts and agricultural chemicals. At the same time the use of recycled irrigation water by greenhouse growers is increasing due to increased pressure by water purveyors and governmental agencies to reduce water usage. When used as a completely closed recirculating system, the above would seem to be a solution the problems of pollution and water conservation. However, serious grower concerns exist regarding the spread of soil-borne, root- infecting plant pathogens (particularly the zoosporic species) in recycled irrigation water. These pathogens are spread primarily in recycled irrigation water, as well as by certain insect vectors. Our major objective(s) are to: develop biologically sound and non-polluting strategies for the efficacious control of these pathogens. The latter strategies include (1) the use of naturally-occurring, biosurfactant-producing bacteria (or the purified biosurfactant) for the control of zoosporic pathogens in recycled irrigation water and (2) development of strategies for the control of insects that vector these soil-borne plant pathogens. 2. How serious is the problem? Why does it matter? In agricultural cultural systems employing recycled irrigation water, zoosporic plant pathogens constitute the most serious threat to economic production. Control of zoosporic pathogens would benefit all producers and users of plant material for landscape and interiorscape purposes. A major benefactor of this technology would be the $2,000,000,000.00 nursery industry in California. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Our research supports the National Program in Plant Disease (303) in the following manner. We have identified zoosporic pathogens as a principle constraint to economic production of greenhouse plants in cultural systems employing recycled irrigation water and demonstrated that these particular pathogens are spread in the recirculating irrigation water, in addition to being vectored by certain insect pests (shore flies) in the greenhouse. Disease management strategies are focused on the use of biological methods and exclusion. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003: Our objective was to evaluate the significance of various greenhouse insect pests as potential vectors of plant pathogenic, root-infecting fungi. This work was conducted by Dr. Zeinab El- Hamalawi and Dr. M.E. Stanghellini in the Department of Plant Pathology at the University of California. Results of current studies demonstrated the epidemiological significance of adult shore flies in the acquisition and dissemination of soilborne fungal plant pathogens; additionally, we identified an entomotpathogenic fungus which is currently being evaluated as a biological control agent for control of the adult shore flies in enclosed agricultural environments. This information will contribute towards devising control strategies for soil-borne fungal pathogens. B. Other Significant Accomplishment(s), if any: C. Significant Accomplishments/Activities that Support Special Target Populations: None D. Progress Report This report serves to document research conducted under a specific cooperative agreement between ARS and the University of California, Riverside. Additional details of research can be found in the report for the parent project 5310-21000-005-00D National Clonal Citrus and Date Germplasm Repository. Objective 1: Evaluation of the efficacy of naturally-occurring, biosurfactant-producing bacteria (as well as the purified biosurfactant) for the control root rot of pepper caused by a zoosporic pathogen (Phytophthora capsici) in recycled irrigation water. In attempting to refine the method of biosurfactant application, specifically concerning the timing of application, we examined the influence of irrigation duration and frequency on inoculum production and disease progression. Studies in the laboratory indicated that zoospores, the life stage that is targeted by the biosurfactant, are not produced by the pathogen until at least 15 minutes after they are exposed to a water stimulus. We needed to investigate whether this is the case in a natural system, as a suitable concentration of biosurfactant would only need to be applied at the time when inoculum would be produced. In the duration study, disease onset occurred 2 weeks earlier and spread throughout the system about 3 times faster with two, 30-min irrigations than with two, 5- min irrigations per day. Various irrigation frequencies were also compared. Disease progresses throughout a system about 3 times faster when irrigated twice a day, and 30 times faster when irrigated 7 times a day, than when only irrigated once a day. A second study examined the influence of irrigation timing (day or night) , as it has been shown that some Oomycetes have a cyclic pattern of sporangia and zoospore production. In the timing study, disease onset occurred about 3 weeks earlier and spread throughout the system about 7 times faster, when irrigated at night rather than during the day. Inoculum densities over a 30- min irrigation cycle show a pattern in which high numbers of zoospores are released early in the cycle, then taper off, and begin to increase again after 20 min. Inoculum produced during a 5-min irrigation cycle range from 7-25% of the total inoculum produced in a 30-min irrigation cycle. This indicates that the biosurfactant must be present during the entire irrigation cycle, and that decreasing the length of irrigation does not negate the need for biosurfactant use, as our laboratory studies would have suggested. However, this information may be very useful in the development of an integrated disease management program. New lab studies have focused on the effect of the biosurfactant on other life stages of the pathogen. It has thus far been determined that a low concentration of biosurfactant (25-ppm) can inhibit hyphal growth and zoospore cyst germination by 50%. The activity shown against the mycelium is fungistatic, and the pathogen can recover if removed from the presence of the surfactant. Future work will include the effect of the biosurfactant on sporangia production and germination. It has also been determined that the biosurfactant can persist at an efficacious level in an organic potting media from 2-3 weeks. As the organic matter essentially filters out the biosurfactant from the solution, the material binds to the media, yet remain active against zoospores of the pathogen. We are hypothesizing that the biosurfactant may not need to be applied to the irrigation water with every cycle if there is a suitable concentration binding to the potting media. Future studies will include a pathosystem consisting of snapdragon and Phytophthora cinnamomi, in the potting media that may affect the dynamics and influence the persistence of the biosurfactant in the media. Objective 2: Identification and evaluation of strategies for the control of insect vectors of soil-borne plant pathogens in commercial greenhouses. Shore flies (Scatella spp.), fungus gnats (Bradysia spp.), and moth flies (Psychoda spp.) are the most common and abundant insects in commercial greenhouse facilities. Various life stages of these insects, which have historically been considered merely a nuisance, have been implicated as potential sources of plant pathogen introduction into and spread within commercial facilities. Results of our current investigations have demonstrated that: (a) Adult shore flies are preferentially attracted to fungal cultures of Thielaviopsis, Verticillium and Fusarium growing on agar medium compared to the agar medium alone. (b) Subsequent to pathogen acquisition (which was external and internal), adult shore flies were demonstrated to transmit the acquired fungal pathogen (Verticillium, Thielaviopsis, and Fusarium) to their respective hosts (geranium, carrot and basil) which subsequently became infected. These results support our hypothesis that adult shore field can serves as an aerial vector for soil-borne plant pathogenic fungi. (c) The rate of dispersal of Thielaviopsis (used in a model system) by pathogen-infested adult shore was evaluated in an enclosed chamber in which 56 small Petri dishes containing carrot agar were placed. Within 18 hrs, 84% of the Petri dishes were infested with the pathogen. These results demonstrated conclusively the epidemiological significance of adult shore flies as an aerial vector. d) We isolated a fungus (tentatively identifies as Beauveria bassiana) which, in preliminary studies, were shown to be highly pathogenic to adult shore flies. 6. What do you expect to accomplish, year by year, over the next 3 years? FY04: Refine the technology for commercial implementation of the purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. FY05: Document the significance of shore flies, fungus gnats, and moth flies as aerial vectors of root-infecting fungi. FY06: Evaluate the efficacy of an entomopathogenic fungus (Beauvaria bassiana) as a biological control agent for these insect vectors. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Oral presentations at several meetings have resulted in the dissemination of our results to other researchers and the lay audience. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Stanghellini, M.E. Insect Transmission of Root-infecting Pathogens. Invited seminar presentation to Graduate Program in Microbiology, February 26, 2003, University of California, Riverside. Stanghellini, M.E. Biology and Control of Soil-Borne Pathogens in Greenhouse Production: Significance of insects as aerial vectors of root- infecting pathogens. Oral presentation at the Floriculture and Nursery Research Initiative Researchers Meeting, March 24-27, 2003, Raleigh, North Carolina. Stanghellini, M.E. Biology and Control of Soil-Borne Pathogens in Greenhouse Production: Biological surfactants for the control of zoosporic pathogens. Oral presentation at the Floriculture and Nursery Research Initiative Researchers Meeting, March 24-27, 2003, Raleigh, North Carolina. Stanghellini, M.E. Biology and Control of Soil-Borne Root- infecting Pathogens: Insects and Surfactants. Invited oral presentation to North Cyprus IPM Workshop Program, May 23, 2003, University of California, Riverside, CA.

Impacts
(N/A)

Publications

  • Stanghellini, M.E., Nielsen, C.J., Kim, D.H., Rasmussen, S.L., Rorbaugh, P.A. Influence of sub- versus top-irrigation and surfactants in a recirculating system on disease incidence caused by Phytophthora spp. in potted pepper plants. Plant Disease. 2000. v. 84. p. 1147-1150.


Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Greenhouse growers are facing stiffer governmental regulations concerning the discharge of spent nutrient solution in order to abate ground water pollution resulting from nutrient salts and agricultural chemicals. At the same time the use of recycled irrigation water by greenhouse growers is increasing due to increased pressure by water purveyors and governmental agencies to reduce water usage. When used as a completely closed recirculating system, the above would seem to be a solution the problems of pollution and water conservation. However, serious grower concerns exist regarding the spread of soil-borne, root-infecting plant pathogens (particularly the zoosporic species) in recycled irrigation water. These pathogens are spread primarily in recycled irrigation water, as well as by certain insect vectors. Our major objective(s) are to: develop biologically sound and nonpolluting strategies for the efficacious control of these pathogens. The latter strategies include (i) the use of naturally-occurring, biosurfactant-producing bacteria (or the purified biosurfactant) for the control of zoosporic pathogens in recycled irrigation water and (ii) development of strategies for the control of insects that vector these soilborne plant pathogens. 2. How serious is the problem? Why does it matter? In agricultural cultural systems employing recycled irrigation water, zoosporic plant pathogens constitute the most serious threat to economic production. Control of zoosporic pathogens would benefit all producers and users of plant material for landscape and interiorscape purposes. A major benefactor of this technology would be the $2,000,000,000.00 nursery industry in California. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? Our research supports the National Program in Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement (301) in the following manner. We have identified zoosporic pathogens as a principle constraint to economic production of greenhouse plants in cultural systems employing recycled irrigation water and demonstrated that these particular pathogens are spread in the recirculating irrigation water, in addition to being vectored by certain insect pests (shore flies) in the greenhouse. Disease management strategies are focused on the use of biological methods and exclusion. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002 year: Our objective was to evaluate the significance of various greenhouse insect pests as potential vectors of plant pathogenic, root-infecting fungi. This work was conducted by Dr. Zeinab El-Hamalawi and Dr. M. E. Stanghellini in the Department of Plant Pathology at the University of California. Results of acquisition studies demonstrated that various life stages of each of three common greenhouse insects (i.e., shore flies, fungus gnats and moth flies), subsequent to exposure to cultures of three different root-infecting fungi (i.e., Verticillium, Fusarium, and Thielaviopsis), were internally and/or externally infested with high populations of viable spores of each of the plant pathogenic fungi tested. These results indicate that all three insect species are capable of acquiring and serving as potential vectors of these plant pathogenic, root-infecting fungi, and that control of these insects, which previously were considered merely as a nuisance, should receive serious consideration as a strategy in disease management. B. Other Significant Accomplishment(s), if any: None C. Significant accomplishments/Activities that Support Special Target Populations: None D. Progress Report This report serves to document research conducted under a reimbursable agreement between ARS and the University of California. Additional details of research can be found in the report for the parent project 5310-21000-005-00D National Clonal Citrus and Date Germplasm Repository. Objective 1: Evaluation of the efficacy of naturally-occurring, biosurfactant-producing bacteria (as well as the purified biosurfactant) for the control root rot of pepper caused by a zoosporic pathogen (Phytophthora capsici) in recycled irrigation water. Accomplishments: We designed and established an appropriate experimental system for evaluation of the above objective in two separate greenhouse units. A purified biological surfactant (i.e., a rhamnolipid produced by a bacterium know as Psuedomonas spp.) was evaluated in our cultural system. Results showed that amending the reservoir with the biosurfactants was not economically feasible because the biosurfactants was rapidly biodegraded (within 24 hrs) by resident microorganisms in the reservoir. Thus, we (graduate student, Carrieann Nielsen and Dr. M. Stanghellini) changed the method of application of the biosurfactants from incorporation into the reservoir to direct injection into the incoming irrigation water thereby reducing the amount of surfactant needed. Current research is focused on refinement of the optimal dosages, timing and application frequencies of the biosurfactant for efficacious control of disease. Determination of the precise timing of application of the biosurfactant will be based upon knowledge of the life cycle of the pathogen, particularly the onset of the production of the motile stage (i. e., zoospores). The latter life stage is the "weak link" in the life cycle of the pathogen and the zoospore is destroyed upon contact with a biosurfactant. Current studies indicate that the highest population of zoospores is produced within 30 min. following onset of an irrigation cycle. Objective 2: Identification and evaluation of strategies for the control of insect vectors of soilborne plant pathogens in commercial greenhouses. Accomplishments: Shore flies (Scatella spp.), fungus gnats (Bradysia spp.) , and moth flies (Psychoda spp.) are the most common and abundant insects in commercial greenhouse facilities. Various life stages of these insects, which have historically been considered merely a nuisance, have been implicated as potential sources of plant pathogen introduction into and spread within commercial facilities. Results of our investigations have demonstrated that: (a.) All three insects were capable of completing their entire life cycle (egg to egg) solely on a diet of each of the following root-infecting plant pathogenic fungi: Fusarium acuminatum, Thielaviopsis basicola, and Verticillium dahaliae. These three fungi, which are destructive pathogens of ornamental and nursery crops, were used throughout our investigation. (b.) Larvae of all three insect genera were shown to ingest and excrete large numbers (1-3 million/larva) of viable (>93% germination) conidia of each of the three plant pathogenic fungi. Additionally, all three fungi were consistently recovered from pupal cases following emergence of the respective adults of the three insect genera. (c.) All three plant pathogenic fungi were externally observed (and documented via scanning electron microscopy) on various body parts of adult life stages of all three insects following expose of the adults to cultures of the three fungi. Additionally, numerous colonies (20 to 70) of each of the three fungi developed on agar medium following placement of the externally-infested adults onto a fungal growth agar medium. (d.) No recognizable (via microscopy) fungal structures were observed in frass deposits from the adult life stages of the adult fungus gnats or moth flies. (e.) Only the adult shore fly was documented to ingest and excrete viable spores of all three fungal plant pathogens. Adult shore flies placed on agar cultures of the three fungi were shown to acquire the pathogens within 20 minutes. After a 24 hr acquisition time period, all frass deposits (38-43 deposits per insect) were shown to contain high populations (70,000 to 630,000 per frass deposit) of conidia, whose viability was consistently greater than 90%. 6. What do you expect to accomplish, year by year, over the next 3 years? Anticipated accomplishments by end of year 3. (a) To have refined the technology for commercial implementation of the purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. (b) To have documented the method of pathogen acquisition by insect vectors and identified strategies for the control of insect vectors of soilborne plant pathogens in greenhouse cultural systems. Adult shore flies, based upon the above results, have been chosen for further investigation on the epidemiology and transmission involving the various plant pathogenic fungi and their respective host plants. Studies are in progress to determine if shore flies are preferentially attracted to specific fungi and, if so, to identify the nature of the attractant. Preliminary data indicate the there is attractant involved. Approximately 86% of adult population of shore flies were attracted to fungi on agar medium compared to agar medium alone. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Based upon our published results on the efficacy of synthetic surfactants for the control of zoosporic pathogens, coupled with discussions and requests for technical assistance, a major commercial nursery operation in California has implemented our strategy in a successful disease management program. We are now establishing a collaborative study with the same major commercial nursery operation in California to assess the efficacy of a biological surfactant, which we discovered, for the control of zoosporic pathogens. Registration of the latter biosurfactant for use as a biopesticide is in progress by a commercial manufacturer of that biological surfactant. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Presentations: "Control and Insect Transmission of Root-infecting Pathogens", presentation by M.E. Stanghellini to CAN Research Advisory Committee Meeting , Nov. 14. 2001 at the Riverside Botanic Gardens Conference Room. "Acquisition and dissemination of root-infecting pathogens by shore flies", presentation by M.E. Stanghellini at the Soil Fungus Conference, March 20-23, 2002, Albuquerque, New Mexico.

Impacts
(N/A)

Publications

  • Stanghellini, M.E., Nielsen, C.J., Kim, D.H., Rasmussen, S.L., Rorbaugh, P.A. Influence of sub- versus top-irrigation and surfactants in a recirculating system on disease incidence caused by Phytophthora spp. in potted pepper plants. Plant Disease. 2000. v. 84. p. 1147-1150.


Progress 10/01/00 to 09/30/01

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Greenhouse growers are facing stiffer governmental regulations concerning the discharge of spent nutrient solution in order to abate ground water pollution resulting from nutrient salts and agricultural chemicals. At the same time the use of recycled irrigation water by greenhouse growers is increasing due to increased pressure by water purveyors and governmental agencies to reduce water usage. When used as a completely closed recirculating system, the above would seem to be a solution the problems of pollution and water conservation. However, serious grower concerns exist regarding the spread of soil-borne, root-infecting plant pathogens (particularly the zoosporic species) in recycled irrigation water. These pathogens are spread primarily in recycled irrigation water, as well as by certain insect vectors. Our major objective(s) are to: develop biologically sound and nonpolluting strategies for the efficacious control of these pathogens. The latter strategies include (i) the use of naturally-occurring, biosurfactant-producing bacteria (or the purified biosurfactant) for the control of zoosporic pathogens in recycled irrigation water and (ii) development of strategies for the control of insects that vector these soilborne plant pathogens. 2. How serious is the problem? Why does it matter? In agricultural cultural systems employing recycled irrigation water, zoosporic plant pathogens constitute the most serious threat to economic production. Control of zoosporic pathogens would benefit all producers and users of plant material for landscape and interiorscape purposes. A major benefactor of this technology would be the $2,000,000,000.00 nursery industry in California. 3. How does it relate to the National Program(s) and National Component(s)? Our research supports the National Program in Plant Disease (303) in the following manner. We have identified zoosporic pathogens as a principle constraint to economic production of greenhouse plants in cultural systems employing recycled irrigation water and demonstrated that these particular pathogens are spread in the recirculating irrigation water, in addition to being vectored by certain insect pests (shore flies) in the greenhouse. Disease management strategies are focused on the use of biological methods and exclusion. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2000 year: Serious grower concerns exist regarding the spread of zoosporic pathogens in ebb-and-flow cultural systems. Therefore, we (M.E. Stanghellini, C.J. Nielsen, D.H. Kim, S.L.Rasmussen, and P.A. Rorbaugh) designed a series of experiments to assess the ability of a zoosporic pathogen to spread in such a cultural system and to compare the relative danger of pathogen spread in an ebb-and-flow system of irrigation to a top-irrigation system. Our results indicate that the use of recycled irrigation water in an ebb- and -flow cultural system will contribute to spread of zoosporic pathogens but is less conducive to spread of zoosporic pathogens than its use in a top-irrigated cultural system. Our results will alleviate grower concerns regarding the perceived increased danger in the use of ebb-and-flow irrigation systems compared to top-irrigation systems. B. Other Significant Accomplishment(s), if any: C. Significant accomplishments/Activities that Support Special Target Populations: D. Progress Report This report serves to document research conducted under a reimbursable agreement between ARS and the University of California. Additional details of research can be found in the report for the parent project 5310-21000- 004-00D National Clonal Citrus and Date Germplasm Repository. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Major accomplishments over the life of the project: Objective: Evaluation of the efficacy of naturally-occurring, biosurfactant-producing bacteria (as well as the purified biosurfactant) for the control root rot of pepper caused by a zoosporic pathogen (Phytophthora capsici) in recycled irrigation water. Accomplishments: We have designed and established an appropriate experimental system for evaluation of the above objective in two separate greenhouse units. A purified biological surfactant (i.e., a rhamnolipid produced by a bacterium know as Psuedomonas spp.) was evaluated in our cultural system. Various levels of disease control were obtained. Current research is focused on refinement of the optimal dosages, timing and application frequencies of the biosurfactant for efficacious control of disease. Determination of the precise timing of application of the biosurfactant will be based upon knowledge of the life cycle of the pathogen, particularly the onset of the production of the motile stage (i.e., zoospores). The latter life stage is the "weak link" in the life cycle of the pathogen and the zoospore is destroyed upon contact with a biosurfactant. Objective: Identification and evaluation of strategies for the control of insect vectors of soilborne plant pathogens in commercial greenhouses. Accomplishments: We have identified shore flies as a major insect pest in greenhouses that are capable of vectoring soilborne plant pathogenic fungi. Current research is focused on insect acquisition of the pathogen. 6. What do you expect to accomplish, year by year, over the next 3 years? Anticipated accomplishments by end of year 2: (a) To have refined the biosurfactant application timing, frequency, and dosage for efficacious control of zoosporic pathogens in cultural systems employing recycled irrigation water. The anticipated use of a fermentation device as an onsite fermentation/infection unit for application of the biosurfactant- producing bacterium was not deemed feasible and therefore abandoned as an objective. (b) To have established the epidemiological significance of insect vectors in the spread of soilborne plant pathogens in greenhouses and identified the method of pathogen acquisition. Anticipated accomplishments by end of year 3. (a) To have refined the technology for commercial implementation of the purified biosurfactant (rhamnolipid) for management of zoosporic pathogens in recycled irrigation water. (b) To have identified strategies for the control of insect vectors of soilborne plant pathogens in greenhouse cultural systems. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? We presented our research data in a poster session at the "Ornamental Horticulture Conference" held at the University of California, Riverside, September 14, 2000 and hosted a greenhouse tour of our research facilities to conference attendees. We presented an invitational paper on "Zoosporic pathogens in recirculating systems: research update" at the Horticulture Research and Education Conference, hosted by the California Association of Nurserymen, Park Plaza Hotel, Oakland, CA, September 12, 2001. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)

Impacts
(N/A)

Publications

  • Stanghellini, M.E., Nielsen, C.J., Kim, D.H., Rasmussen, S.L., Rorbaugh, P.A. Influence of sub- versus top-irrigation and surfactants in a recirculating system on disease incidence caused by Phytophthora spp. in potted pepper plants. Plant Disease. 2000. v. 84. p. 1147-1150.