Source: UNIVERSITY OF GEORGIA submitted to
COMMUNITY DEVELOPMENT AND BIOLOGICAL CONTROL ACTIVITY OF PHYLLOPLANE YEASTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0203279
Grant No.
(N/A)
Project No.
GEO-001659
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 1, 2005
Project End Date
Apr 30, 2012
Grant Year
(N/A)
Project Director
Buck, J. W.
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Biological control of foliar plant diseases with epiphytic yeasts provides an environmentally friendly alternative to traditional chemical control measures. The purpose of this study is to determine the effects of long-term exposure of biocontrol yeast and fungus pathogen on biocontrol efficacy and the development of fungicide resistance. The project also examines the effect of various ecological disturbances on natural yeast communities on leaf surfaces.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2152121110250%
2152130107050%
Goals / Objectives
The production of ornamental plants, including nursery and floriculture crops, is a thriving and quickly expanding industry. Over a six-year span, the value of this industry in the United States increased over 23% to $14.3 billion in 2002. Botrytis cinerea is a ubiquitous fungal pathogen that infects stems, leaves and flowers of many greenhouse-grown crops. Effective management of Botrytis blight requires an integrated approach combining sanitation, manipulation of environmental conditions, and the use of fungicides. Although new fungicides have been developed in recent years, non-chemical alternatives to fungicides, which would fit into an integrated management strategy for the disease, are needed. One promising alternative to chemical fungicides is the use of biological control with epiphytic yeasts. Epiphytic yeasts are active colonizers of plant surfaces and have received considerable attention as potential biocontrol agents for fungus pathogens. The yeast phylloplane community is thought to provide a natural buffer against infection by some foliar plant pathogens; however, maintaining high communities of introduced yeasts on plant surfaces in the field is a major hurdle for biological control programs. Obtaining a better understanding of the effects of ecological disturbances (e.g. disease, pesticides) on yeast communities on leaves could lead to better biocontrol strategies. A combination of yeast and fungicides can reduce variability in biocontrol efficacy. However, it is unknown what effect long term exposure of yeast to fungal pathogen has on the stability of biocontrol efficacy (will the yeast become less effective over time?) or fungicide resistance in the fungus (due to exposure to the chemical). Assessing both the stability of yeast biocontrol and the effect of yeast combined with a fungicide on managing the development of fungicide resistance in the fungus will further validate the use of biocontrol yeasts for disease management on floriculture crops. The objectives of this work are to: 1. determine if a yeast/fungicide mixture affects the development of fungicide resistance in sensitive isolates of B. cinerea. 2. determine if yeast biocontrol activity is stable through repeated exposures to the pathogen (will the yeast fail?). 3. determine the effects of ecological disturbance (e.g. pollution, disease) on the epiphytic yeast community on leaf surfaces.
Project Methods
Objective 1: Three isolates of B. cinerea sensitive to vinclozolin will each be inoculated onto geranium seedlings alone or combined with either vinclozolin, or yeast mixed with vinclozolin. Seedlings will be incubated until sporulating lesions are present. After two to three weeks, conidia will be collected and bulked from all sporulating leaves within each treatment (one round of infection). Individual conidia collected from the different treatments will be used to start new cultures on PDA while the remainder will be stored. These new cultures will provide the inoculum for the next round of infection. Nine strains of the fungus will be obtained over ten rounds of infection on seedlings. Relative pathogenicity of all strains will be compared in seedlings assays. Sensitivity to vinclozolin will be determined by assessing radial growth of each strain on vinclozolin-amended agar. Objective 2: Four isolates of B. cinerea will be inoculated onto geranium seedlings alone, as a four-isolate mixture, as a four-isolate mixture with yeast (with and without vinclozolin) or each isolate alone combined with yeast, or yeast mixed with vinclozolin. Seedlings will be incubated until lesion formation and sporulation occurs. Ten rounds of infection will be performed with 12 strains obtained at the end of the trials. The biocontrol efficacy of the yeast will be evaluated against the 12 strains using a seedling assay. Objective 3: Yeast communities will be assessed on turfgrass leaves by dilution plating. Leaves will be collected from five areas within 1 x 1 m plots. Samples will consist of six to eight bentgrass leaves or four to six tall fescue leaf blades per treatment. Leaf washate will be incubated on agar at 25 C in the dark for 48 hours and yeast colonies will be enumerated. Plates will then be incubated at 4 C for 48 hours and any additional yeast colonies quantified. Data will be converted to colony forming units/cm2 of leaf tissue. Abiotic disturbances will be investigated by treating turfgrass with various pesticides (e.g. fungicides) and following community size and composition through time. The biodiversity of the communities would be assessed by determining number of species and population sizes of the species in question. Novel yeast species will be characterized by physiological tests and ribosomal sequence data and subsequently named.

Progress 05/01/05 to 04/30/12

Outputs
OUTPUTS: Yeast populations are quantitatively and qualitatively affected by the presence of disease lesions on plant leaves. Infection of leaf tissues by rust fungi - obligate parasites - should cause similar modifications to the yeast community. Observations of rust lesions on plants suggest that the presence of non-rust yeast and fungi can affect lesion development and urediniospore production. PARTICIPANTS: Dr. Shannon Nix (former post-doctoral researcher) Dr. Leon Burpee (Collaborator) TARGET AUDIENCES: Target audiences include academic researchers and industry professionals. PROJECT MODIFICATIONS: No major changes in approach were incorporated into this research.

Impacts
Our data continue to support the hypothesis that the presence of yeast and fungi in disease lesions can affect subsequent disease progression.

Publications

  • No publications reported this period


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: In tall fescue (Festuca arundinacea), brown patch caused by the fungal pathogen Rhizoctonia solani, is characterized by irregular-shaped lesions that often coalesce resulting in extensive leaf necrosis. Presumably, nutrients released from the wounding and death of foliar cells influences the microbial carrying capacity of these leaf surfaces. In a previous study, we observed significant increases in the yeast carrying capacity of tall fescue leaves infected with R. solani compared to non-infected leaves. We have also observed that the yeast carrying capacity of tall fescue can be increased by applying exogenous nutrients to the leaf surface. In the present study we evaluated the impact that select yeasts had on lesion size and disease severity caused by R. solani on tall fescue. These studies have been presented at the annual meeting of the American Phytopathological Society and published in peer-reviewed journals. PARTICIPANTS: Dr. Shannon Nix (Post-doctoral researcher) Dr. Leon Burpee (Collaborator) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our data suggest that epiphytic yeast populations on leaves is influenced by nutrient availability and wounding and fluctuates during the day with highest populations observed in late evening and early morning. These factors combined with emigration and immigration from leaf surfaces will shape community composition.

Publications

  • Nix, S.S., Burpee, L.L., and Buck, J.W. 2009. Responses of two epiphytic yeasts to foliar infection by Rhizoctonia solani or mechanical wounding on the phylloplane of tall fescue. Canadian Journal of Microbiology. 55:1160-1165.
  • Nix-Stohr, S., Burpee, L, and Buck, J.W. 2007. The effects of wounding and fungal infection on two phylloplane yeast populations in tall fescue. Phytopathology 97:S85


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The objective of our current work was to determine if two common turf phylloplane yeasts (Cryptococcus laurentii and R. glutinis) differentially colonize the phylloplane in response to physical disturbance of the leaf surface. We hypothesized that disrupting the leaf surface by mechanically bruising or infecting the leaves with R. solani would increase yeast carrying capacity of the leaves as a result of nutrients released to the surface from damaged tissues.A growth chamber experiment was conducted to determine how foliar disease or wounding affects the ability of two phylloplane yeasts (Rhodotorula glutinis and Cryptococcus laurentii) to colonize leaves of tall fescue (Festuca arundinacea). Yeasts were applied separately and together onto healthy leaves, leaves infected with Rhizoctonia solani (diseased) and mechanically bruised (wounded) leaves. In all three trials, the leaf disturbance treatment significantly affected the abundance of yeast on the phylloplane of tall fescue. Yeast abundance on the diseased or wounded treatments was significantly greater than on the non-treated, healthy leaves. In two of the three trials, the yeast species applied also had a significant affect on yeast abundance. Typically, R. glutinis was significantly more abundant than C. laurentii when applied individually but not significantly greater than the yeast cfu of the co-inoculated treatment. When the two yeasts were co-inoculated onto the leaves, R. glutinis comprised 89.7%, 75.4%, and 67.6% of the recovered yeast cfu on healthy, diseased and wounded leaves, respectfully. Our data suggest that these two species of yeasts will differentially colonize compromised leaf tissue with disease or wounds favoring populations of R. glutinis over C. laurentii. PARTICIPANTS: Dr. Shannon Nix (Post-doctoral researcher) Dr. Leon Burpee (Collaborator) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our data indicates that isolates of Cryptococcus laurentii may actually be more responsive to the changes that occur in the habitat as a result of physical disruption of the leaf surface and may explain why C. laurentii are the dominant yeasts of bentgrass and tall fescue on both diseased (Rhizoctonia solani or Sclerotinia homoeocarpa) and healthy tissues under field conditions. Understanding how yeast species interact on the phylloplane under various biotic and abiotic disturbances will facilitate using these organisms as biological control agents.

Publications

  • No publications reported this period


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Objective: To assess the short-term (24-hour) dynamics of yeast abundance on the phylloplane of tall fescue (Festuca arundinacea). We hypothesized that phylloplane yeast abundance would significantly change over short temporal periods, that abundance would be greatest in the early morning and least in the afternoon, and that abundance would be greater on the nutrient treated leaves than on the non-nutrient treated leaves. In a subsequent investigation, we assessed atmospheric yeast abundance above the canopy of tall fescue in the morning and in the afternoon. We hypothesized that as the leaf surface dries, yeast cells would become air borne and emigrate from the leaf surface into the atmosphere. Consequently, yeasts would be more prevalent in atmospheric samples in the afternoon than morning. A significant effect of sampling time was observed on yeast abundance of tall fescue leaves (P<0.0001). Yeast populations were significantly more numerous at 0000 hours and significantly less numerous at 1800 hours. There was no statistical difference in abundance between 0600 and 1200 hours but abundance at both these times was significantly different from abundance at 1800 and 0000 hours, respectively. The most notable change in yeast density occurred between the hours of 1800 and 0000 hours where culturable yeast abundance increased by over 44%. Nutrient treatment resulted in an average 50% increase in yeast populations compared to the non-nutrient control (P=0.0809). Atmospheric yeast abundance was significantly greater in the morning than in the afternoon in 5 of the 6 experimental trials. Yeast populations in the morning samples were on average between 2-fold (trial 1) and 19-fold (trial 5) greater than samples taken in the afternoon. In summary, we observed a diurnal pattern of yeast abundance on the turf phylloplane where populations declined over the course of the day and increased in the evening. Our results suggest the following colonization model: phylloplane yeasts on tall fescue reproduce during the late evening and early morning, stabilize during the late morning and early afternoon through exchange of immigrants and emigrants and decline during the late afternoon/early evening. PARTICIPANTS: Dr. Shannon Nix (Post-doctoral Researcher) Dr. Leon Burpee (Collaborator)

Impacts
Understanding how yeast communities on leaf surfaces change over short time periods is integral to developing viable biocontrol methods for foliar plant diseases. Yeast populations increase during the late evening and early morning suggesting that biocontrol activity would be highest at these times.

Publications

  • Nix, S.S., Burpee, L.L., Jackson, K.L., and Buck, J.W. 2008. Short-term temporal dynamics of yeast abundance on the tall fescue phylloplane. Can. J. Microbiol. In press.


Progress 01/01/06 to 12/31/06

Outputs
Objective: Do biotic or abiotic disturbances affect yeast community size and composition on the turfgrass phylloplane? A more comprehensive understanding of the environmental and nutritional factors affecting yeast populations on leaves would facilitate the use of these microorganisms for biological control of fungal plant pathogens. In this study we test the hypothesis that the phylloplane yeast community of tall fescue (Fescue arundinacea) is nutrient limited and that nutrient availability, abundance and content is a significant factor in determining population size. As a test of this hypothesis we investigated the effects of applying various solutions of sucrose and or yeast extract, and different nitrogen based solutions on the abundance of culturable yeasts colonizing the phylloplane of tall fescue. Four experiments were conducted from 5 June to 9 August 2006. Experiments 1-3 were designed to evaluate the effect of foliar applications of sucrose and or yeast extract on phylloplane yeast populations of tall fescue. Treatments consisted of three nutrient solutions applied at five concentrations plus a non nutritive control (de-ionized H2O). Experiment 4 was designed to investigate the response of the phylloplane yeast community to foliar applications of five different nitrogen based solutions and a non nutritive control (de-ionized H2O). A day after foliar application of nutrient treatments, two replicate samples per treatment plot were collected and total yeast colony forming units from the 36 plots were determined by dilution plating. In both trials of the sucrose + yeast extract experiment, significant positive linear relationships were detected between yeast abundance in the phylloplane and total nutrients applied to leaves of tall fescue. When the yeast abundance of non-nutrient controls was compared to the treatment with the highest concentration of sucrose and yeast extract (125g/L total nutrient) we observed a 225% and 75% increase in total population size in Trials 1 and 2, respectively. In the sucrose experiment, a significant linear relationship between yeast abundance and nutrients was not observed. In contrast, when yeast extract without sucrose was sprayed on tall fescue a similar pattern to the combined sucrose and yeast extract experiment emerged and a significant linear relationship was observed. The 25g/L yeast extract treatment resulted in a 203% increase in yeast cfu in Trial 1 and 150% in Trial 2. In the nitrogen experiment, only the tryptone and yeast extract treatments significantly increased the number of yeast cfu on tall fescue. The yeast population increased an average of 179% with tryptone and 162% with yeast extract compared to the non-nutritive control. Our results clearly demonstrate that microbial population dynamics can be manipulated through the application of nutrients. It is however, critical to determine which nutrients and concentrations result in statistically significant population increases.

Impacts
The use of antagonistic yeasts for biological control on ornamental plants would provide an alternative to traditional chemicals. Our work demonstrates that the phylloplane yeast community is significantly altered by nutrient availability, abundance and content and that these factors are significant in determining population size. The ability to maintain large populations of antagonistic yeasts on plant surfaces should enhance biological control of foliar pathogens.

Publications

  • Stohr, S.N., Burpee, L.L., and Buck, J.W. 2007. The influence of exogenous nutrients on the abundance of yeasts on the phylloplane of turfgrass. Microbial Ecology. In press.


Progress 01/01/05 to 12/31/05

Outputs
The long-term objective of our research is to incorporate natural populations of yeasts into a disease control strategy for foliar diseases of turfgrass. The phylloplane of creeping bentgrass (Agrostis palustris), a species grown throughout the temperate zone on golf putting greens, is a niche for dynamic communities of yeasts which are significantly affected by fungicides and the presence of foliar disease. Because bentgrass golf greens are typically irrigated daily in the southeastern USA, we hypothesized that natural populations of yeasts on the phylloplane of turfgrass would be easily dislodged by this shear force. A better understanding of the processes affecting yeast population sizes on leaves could allow for better management of yeasts (e.g. the ability to maintain large populations on leaves) for the purpose of biological control of foliar pathogens of turfgrass. Irrigation and an in vitro agitation assay were used to determine the percentage of the epiphytic yeast community adhering to the phylloplane of creeping bentgrass. Colony forming units (cfu) of total yeast populations (adherent and non-adherent cells) and of adherent populations (cells not removed by agitation) were determined by leaf washing and dilution plating. In an in vitro assay, 40.0 and 57.1% of the yeast were adhering to the leaves while in initial field trials the percentage of adherent yeasts ranged from 40.0 to 71.9% of the total population. Adherent yeast cfu on leaves in the morning were significantly lower on bentgrass (8,000 to 31,000 cfu/cm2) compared to total yeast cfu (14,000 to 47,000 cfu/cm2) on the non-irrigated control. No differences in yeast populations were observed between irrigated and non-irrigated plots 2 h after the 9 a.m treatments. Yeast populations followed a diurnal pattern with larger cfu recovered from bentgrass leaves in the morning and significantly lower populations recovered in the afternoon. At 2 p.m the adherent yeast were 83.1 to 100% of the total yeast population recovered from the leaves. The relative adhesiveness of the epiphytic yeast community on bentgrass leaves is dynamic with non-adherent cells making up a larger percentage of the population in the mornings compared to afternoons.

Impacts
Biological control of foliar diseases of turfgrass would provide an environmentally friendly disease management option.

Publications

  • No publications reported this period