ROLE OF MYXOCOCCUS XANTHUS IN AGRICULTURAL SOILS AND THEIR POTENTIAL USE AS BIOLOGICAL CONTROL AGENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0205359
• Project No.: CA-D-MMG-7473-H
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015

Project Director
Singer, M.

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
Alternative pet control management approaches are important to prevent the rampant use of toxic chemical compounds from contaminating ecologically sensitive areas. This study will provide information as to how MYXOCOCCUS XANTHUS can be used to control many microbial agricultural pests. This study will also examine how M.XANTHUS interacts with other microbes in natural habitats, including agricultural pathogens and non-pathogens in agricultural soils. We will also learn about the effectiveness of M. XANTHUS as a potential biological control organism, providing alternatives to chemical pesticides. Finally, this work will provide information on how antibiotics produced by M. XANTHUS to attack agricultural pests, are regulated and produced, potentially leading to the development of alternative antimicrobial agents.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	4010	1100	100%

Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

myxococcus xanthus

predation

secondary metabolites

biological control

Goals / Objectives
OBJECTIVES: We hypothesize that myxobacteria influence and are influenced by plant pathogens in the soil and rhizosphere, which results in an alteration in the disease outcome. The specific objectives of this research are to develop tools to rapidly quantify and identify myxobacteria from agricultural soils and to understand the influence of plants and plant pathogens on the cell cycle and gene expression in myxobacteria. We propose to use these tools to define the nature of the predator-prey relationship between myxobacteria and plant pathogens. The information and tools developed as a result of this research are needed for our long-term goal of manipulating the interactions between myxobacteria and plant pathogens to mitigate plant disease. This project is in collaboration between my lab and the laboratory of C. Bull at the USDA/ARS in Salinas CA. All work with plant pathogens will be done at the USDA/ARS facility in Salinas, while all of the molecular and non-pathogenic work will be performed in my lab at UC DAVIS. Therefore the specific objectives of this project are: (1) Develop quantitative methods for measuring predation ability of M. XANTHUS against both fungal and bacterial plant pathogens both in the lab as well as with bioassays in conjunction with the plant. (2) Define the role of secondary metabolites and developmental regulators in biocontrol and/or predation of plant pathogens in agricultural soils

Project Methods
We are taking a two prong approach to look at the use of MYXOCOCCUS XANTHUS as a possible biological control agent. First, we have developed two independent predation assays to date, the streak assay and the plate assay, (21) that will be used to determine the effectiveness of M. XANTHUs as a biological control agent on a variety plant bacterial pathogens. I propose to continue these studies with fungal pathogens under laboratory conditions as well as to develop bioassays to be used in the green house and field. These studies will be done in collaboration with C. Bull at the USDA/ARS at Salinas California. All greenhouse and field tests will be done at the Salinas facility. Second, using the genome sequence M. XANTHUS and DNA microarrays we have identified candidate genes that we predicted are involved in the production of secondary metabolites and/or secreted degradative enzymes, such as lipases, proteases, cellulases and chitinases. Our approach is to make null mutations in each of these genes and then determine their phenotype with respect to predation, vegetative growth and development using standard laboratory protocols and the predation assays that we developed. The phenotypic analysis of these mutants, specifically with respect to predation, will allow us to quantify the importance of each identified gene product on predation. In addition, we will be able to determine which gene sets are required for which type of pathogen, bacterial and/or fungal. Finally, we propose to determine the expression patterns for each of these genes, using DNA microarrays and quantitative PCR. These data will allow us to define their regulatory circuitry in order to better engineer more efficient biocontrol agents.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Agricultural communities using natural products and K-6th grade elementary students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I have been collaborating with the USDS/ARS lab in Salinas on this project, providing strains and support to researchers at that site. This project has primarily been accomplished with 2 undergraduates and a graduate student, who is African-American. All of the students participated in our weekly laboratory research group meetings. The undergraduates were trained in a variety of laboratory techniques and assays, are taught to analyze data, and present results in group meeting. One high school student was part of the Davis High School Biotechnology program and worked in the lab for two quarters. Lastly a visiting graduate student from Brazil was been working on a joint project in the lab. How have the results been disseminated to communities of interest?Work from this project has been presented in the Undergraduate Research Conference on campus by 2 of the undergraduates. Work has been brought to a local elementary school where students from the lab presented a short hands-on-demonstration during the school's science fair, which I co-chaired. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have continued our work with the 5 grn mutants in M, xanthus. We have shown that all 5 genes are necessary for wild type predation and that none are required for development. We have demonstrated that all five loci are expressed during early development and in conjunction with Dr. C. Bull at the USDA/ARS in Salinas CA using qPCR to examine expression. We have also shown that these genes are activated by nutrient stress and we have begun a series of experiments to determine their ability to kill known important agricultural plant pathogens.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Smaldone, G.T., Y. Jin, D.L. Whitfield, A.Y. Mu, E.C. Wong, S. Wuertz, and M. Singer. Growth of Myxococcus xanthus in continuous-Flow-Cell Bioreactors as a method of studying development. 2014. Applied and Environ. Miocrobiol. 80:(8):2461-2467
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Skotnicka, D., Smaldone, G. T., Petters, T., Liang, J., Kaever, V., Singer, M., S�gaard-Andersen, L. A minimal threshold of c-di-GMP is essential for fruiting body formation and sporulation in Myxococcus xanthus.
• Type: Book Chapters Status: Published Year Published: 2014 Citation: A. Treuner-Lange, L. S�gaard-Andersen, and M. Singer. Chapter 4. Cell cycle regulation in Myxoccocus xanthus during vegetative growth and development: Regulatory links between DNA replication and cell division; in "Myxobacteria: Genomics, Cellular and Molecular Biology," ed. Z. Yang and P.I Higgs, Caister Academic Press

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Agricultural communities using natural products and K-6th grade elementary students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I have been collaborating with the USDS/ARS lab in Salinas on this project, providing strains and support to researchers at that site. This project has primarily been accomplished with 2 undergraduates and a graduate student, who is African-American. All of the students participated in our weekly laboratory research group meetings. The undergraduates were trained in a variety of laboratory techniques and assays, are taught to analyze data, and present results in group meeting. One high school student was part of the Davis High School Biotechnology program and worked in the lab for two quarters. Lastly a visiting graduate student from Brazil was been working on a joint project in the lab. How have the results been disseminated to communities of interest? Work from this project has been presented in the Undergraduate Research Conference on campus by 2 of the undergraduates. Work has been brought to a local elementary school where students from the lab presented a short hands-on-demonstration during the school's science fair, which I co-chaired. What do you plan to do during the next reporting period to accomplish the goals? We will continue our collaboration with the Salinas USDA/ARS lab to look at the effect of these mutants on plant pathogens. We will also focus on under what conditions cells express these genes using RNA HiSeq protocols we have developed.

Impacts
What was accomplished under these goals? We have continued our work with the 5 grn mutants in M. xanthus and have shown that all 5 genes are necessary for wild type predation and that none are required for development. We have demonstrated that all five loci are expressed during early development using RNAseq. We have also shown that these genes are activated by nutrient stress and we have begun a series of experiments to determine their ability to kill known important agricultural plant pathogens.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Smaldone, G.T., Y. Jin, D.L. Whitfield, A.Y. Mu, E.C. Wong, S. Wuertz, and M. Singer. Growth of Myxococcus xanthus in continuous-Flow-Cell Bioreactors as a method of studying development. 2014. Applied and Environ. Miocrobiol. 80:(8):2461-2467

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audiences included the general public (provided scientific education contributing to science literacy), high school students (provided microbiology research opportunities), and elementary school students (providing hands on school presentations) . I am also continuing our collaboration with the USDA/ARS lab in Salinas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I have been collaborating with the USDS/ARS lab in Salinas on this project, providing strains and support to researchers at that site. This project has primarily been accomplished with 2 undergraduates and a graduate student, who is African-American. All of the students participated in our weekly laboratory research group meetings. The undergraduates were trained in a variety of laboratory techniques and assays, are taught to analyze data, and present results in group meeting. One high school student was part of the Davis High School Biotechnology program and worked in the lab for two quarters. Lastly a visiting graduate student from Brazil was been working on a joint project in the lab. How have the results been disseminated to communities of interest? Work from this project has been presented in the Undergraduate Research Conference on campus by 2 of the undergraduates. Work has been brought to a local elementary school where students from the lab presented a short hands-on-demonstration during the school's science fair, which I co-chaired. What do you plan to do during the next reporting period to accomplish the goals? We will continue our collaboration with the Salinas USDA/ARS lab to look at the effect of these mutants on plant pathogens. We will also focus on under what conditions cells express these genes using RNA HiSeq protocols we have developed.

Impacts
What was accomplished under these goals? We have continued our work with the 5 grn mutants in M, xanthus. We have shown that all 5 genes are necessary for wild type predation and that none are required for development. We have demonstrated that all five loci are expressed during early development and in conjunction with Dr. C. Bull at the USDA/ARS in Salinas CA using qPCR to examine expression. We have also shown that these genes are activated by nutrient stress and we have begun a series of experiments to determine their ability to kill known important agricultural plant pathogens.

Publications

• Type: Book Chapters Status: Published Year Published: 2014 Citation: A. Treuner-Lange, L. S�gaard-Andersen, and M. Singer. Chapter 4. Cell cycle regulation in Myxoccocus xanthus during vegetative growth and development: Regulatory links between DNA replication and cell division; in "Myxobacteria: Genomics, Cellular and Molecular Biology," ed. Z. Yang and P.I Higgs, Caister Academic Press

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

Outputs
OUTPUTS: We have continued our work with the 5 grn mutants in M, xanthus determining their role in predation and development. We have shown that all 5 genes are necessary for wild type predation and that none are required for development. We have demonstrated that all five loci are expressed during early development and in conjunction with Dr. C. Bull at the USDA/ARS in Salinas CA we have begun a series of experiments to determine their ability to kill known important agricultural plant pathogens. We presume that expression of each of these genes is dependent upon starvation and other nutritional stressors but limited to these responses and not required for development. We are also looking at expression of the grn mutants in various developmental mutant backgrounds using qPCR. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The use of natural microbial predators against agriculturally important plant pathogens provides an ecologically and environmentally sound strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases and the data provide by this research will be useful in understanding these diseases based on a novel strategy for reducing populations of the pathogen while in a transient sole state. Our work takes advantage of predatory microbes already in the soil systems to attack a variety of plant pathogens. We are currently writing this work up to be submitted to J. Bacteriology.

Publications

• Bragg, J.H., A. Rajkovic, C.R. Anderson, R. Curtis, J.M. Van Houten, B.N. Begres, C.J. Naples, M.J Snider, D. Fraga, and M. Singer. 2012. Identification and characterization of an arginine kinase from Myxococcus xqnthus required for fruiting body formation and cell differentiation. J. Bacteriol. 194(10):2668-76

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We have continued our work with the 5 grn mutants in M, xanthus determining their role in predation and development. We have demonstrated that all five loci are expressed during early development and in conjunction with Dr. C. Bull at the USDA/ARS in Salinas CA we have begun a series of experiments to determine their ability to kill known important agricultural plant pathogens. We are also looking at expression of the grn mutants in various developmental mutant backgrounds using qPCR. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The use of natural microbial predators against agriculturally important plant pathogens provides an ecologically and environmentally sound strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases and the data provide by this research will be useful in understanding these diseases based on a novel strategy for reducing populations of the pathogen while in a transient sole state. Our work takes advantage of predatory microbes already in the soil systems to attack a variety of plant pathogens.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: For the past year we have continued our work with the grn mutants in M. xanthus and their role in both predation and early developmental gene expression. We have continued to characterize these mutants in their ability to prey on a variety of important agricultural plant pathogens, both bacterial and fungal. This project continues to be done in collaboration with Dr. C. Bull at the ARS in Salinas CA. We are also looking at expression of the grn mutants in various developmental mutant backgrounds using qPCR. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The use of natural predators against agriculturally important plant pathogens provides an ecologically and environmentally sounds strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases and the data provide by this research will be useful in understanding these diseases based on a novel strategy for reducing populations of the pathogen while in a transient sole state. Our work takes advantage of predatory microbes already in the soil systems to attack a variety of plant pathogens.

Publications

• No publications reported this period

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

Outputs
OUTPUTS: We are continuing our characterization of the grn mutants and their role in predation. We have been examining the role of the grn mutants and other early developmental mutants on their ability to prey on a variety of plant pathogens, both bacterial and fungal. This is being done in collaboration with Dr. C. Bull at the ARS in Salinas CA. We are also looking at expression of the grn mutants in various backgrounds using qPCR. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The use of natural predators of plant pathogens already present in the agroeco-system provides and ecologically and environmentally sounds strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases. The data provide by this research will be useful in understanding these diseases based on a novel strategy for reducing populations of the pathogen while in a transient sole state.

Publications

• No publications reported this period

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

Outputs
OUTPUTS: We are continuing our characterization of the grn mutants and their role in predation. We have also been using DNA microarrays to examine effects on gene expression in the grn mutants and several others, including dks. This work has been done primarily with undergraduates performing independent study projects in my lab. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The use of natural predators of plant pathogens already present in the agroeco-system provides and ecologically and environmentally sounds strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases. The data provide by this research will be useful in understanding these diseases based on a novel strategy for reducing populations of the pathogen while in a transient sole state.

Publications

• No publications reported this period

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

Outputs
We are continuing our characterization of the grn mutants and their role in predation. We have also been using DNA microarrays to examine effects on gene expression in the grn mutants and several others, including dks. This work has been done primarily with undergraduates performing independent study projects in my lab.

Impacts
The use of natural predators of plant pathogens already present in the agroeco-system provides and ecologically and environmentally sounds strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases. The data provide by this research will be useful in understanding these diseases based on a novel strategy for reducing populations of the pathogen while in a transient sole state.

Publications

• No publications reported this period

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

Outputs
The Myxobacteria represent a rich source of bioactive secondary metabolites. Antibiotic compounds have been isolated from 55 % and 95% of the proteolytic and cellulolytic myxobacteria, respectively. During the last year we have made significant progress in identifying the control elements of the antibiotic producing genes from M. xanthus. In addition we have been able to demonstrate that antimicrobial production from the GRN genes are coupled to nutrient limitation.

Impacts
The use of natural predators of plant pathogens already present in the agroeco-system provides an ecologically and environmentally sound strategy for disease control. There are very few methods available to mitigate foliar bacterial and fungal diseases. The data provided by this research will be useful in mitigating these diseases based on a novel strategy for reducing populations of the pathogen while in a transient soil state.

Publications

• No publications reported this period