Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Microbiology & Molecular Genetics
Non Technical Summary
Alternative pest 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
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
For the past several years we have been working under the hypothesis that myxobacteria growth and development both influence and are influenced by the local microbial environment. This includes a variety of plant pathogens in the soil and rhizosphere. These interactions, between myxobacteria and plant pathogens, can then lead to alterations in the progression of the disease to the plants. 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. This current proposal is a continuation of my previous ARS proposals.(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) Identify novel predation dependent pathways using RNAseq methodologies.
Project Methods
1. Develop methods for measuring interactions between M. xanthus DK1622 and the fungus P. ultimum strain S-11 and S. minor in soil microcosms. As we demonstrated from our last grant, P. ultimum strain S-11 is used to generate inoculum in soil mixed with sterile beet leaves using standard methods (20). The concentration of strain S-11 is estimated by plating dilutions of the inoculum or soil samples on Pink medium. Two days prior to planting, twice-pasteurized soil either mixed with the inoculum (200 ppg) or with no inoculum is used to fill growth pots. Strain DK1622 is applied (25 ml) as a suspension in water (1 X 107 CFU/ml) to the soil and water is applied as a control. We use four replications per treatment with each replication consisting of 30 seedling cells. Cucumber seeds are planted two days later, and are grown in a growth chamber at 22°C (12/12 h light and dark cycle). The numbers of plants emerging at 10 and 14 days are counted. Preliminary data indicate that oospores and hyphal swellings, the two basic forms of inoculum of P. ultimum, may be differentially sensitive to biocontrol by strain DK1622 (21). In a separate experiment we will test this hypothesis by comparing inocula from strains S-11 (producing both hyphal swellings and oospores), S-8 (producing only hyphal swellings) and S-44 (producing primarily oospores). The strain most amenable to biocontrol will be used thereafter. The strength of our approach is that it is not intimately tied to just one pathosystem because many plant pathogens are inhibited by myxobacteria.2. Develop methods for measuring interactions between M. xanthus DK1622 and P. syingae pv. alisalensis (Psa) in soil microcosms. We will use established methods for studying interactions between myxobacteria and bacterial plant pathogens in soil microcosms. A sandy silt loam soil collected from an agricultural field in Watsonville, Calif., will be sifted with a #12 sieve, weighed to 10 g, placed into a large test tube and covered with a cap. Soil samples will be used without sterilization or alternatively will be autoclaved twice. Few toxic phenolics will be produced on autoclaving due to the very low organic matter content of the soil we are using. Based on preliminary data, soil samples will be inoculated with: 1) DK1622 at 1 x 105 CFU/g soil, 2) Psa at 1 x 109 CFU/g soil, 3) a mixture of the two bacteria at the same final soil concentration, or 4) a buffer control. The soil moisture among treatments will be identical throughout the experiment (2). Tubes will be incubated at 27°C in the dark throughout the experiment. One-gram samples will be removed immediately after inoculation and periodically over seven days to evaluate the changes in populations of both myxobacteria and plant pathogenic bacteria. In all studies, DK1622 and Psa will be enumerated on appropriate semi-selective media. Populations of vegetative cells versus resistant myxospores will be distinguished by sensitivity to heating using published and standard laboratory methods. Five replicate microcosms for each treatment will be established. The treatments will be applied to the microcosms in a completely randomized design and population changes will be monitored over time.3. Define the role of known developmental mutants in predation and biocontrol. We propose to begin testing on a variety of known regulatory genes that effect both development and vegetative growth. These genes include but will not be limited to: bsgA, ASGA, rpoS, SDEK, nla18, nla4, and nla24;, the last three are NtrC like activators that have dramatic effects on development and vegetative growth. We propose to construct, null and overproducing strains as described below. Each mutant will be characterized for predation in our lab, and then sent to Salinas to be tested as biocontrol agents.Null mutations will be constructed by one of two means, both used successfully in my lab. First, if the potential gene identified is greater than 1-kb, we will use insertional mutagenesis by cloning a 400 to 500-bp fragment into a suicide plasmid in M. xanthus that encodes kanamycin resistance. If the gene is smaller than 1-kb, a two-step deletion process will be used. In this scenario, we will construct a deletion of the gene, and clone this fragment into a suicide plasmid that contains a kanamycin resistance gene and the E. coli galK, gene. All deletion and insertion strains will be confirmed by Southern analysis or PCR to confirm the deletion or insertion. Lastly, all null mutants generated will be complemented with a wild-type copy of the mutated gene. Merodiploid strains will be constructed using a vector that allows for site- directed recombination at the Mx8att site in the M. xanthus chromosome. Using this system, isogenic complemented pairs can now be constructed for the purposes of analysis.Construction of over-expression strains is difficult because currently we lack a good exogenously regulatable promoter system. Therefore we will take two approaches. First we will clone the gene in question behind the mgl promoter, a highly expressed constitutive promoter. An alternative approach will be to construct a tandem duplication of the gene in question including all regulatory regions. While this approach only provides a 2-fold effect we have been successful with such tandem duplications providing an increase in gene expression. As described above all constructs will be verified by southern or PCR analysis. The ability of constructs to over-produce the gene product in question will be analyzed by quantitative PCR, to detect any increase in message, and by protein gels, to detect an increase in protein level. Next null and over-expression mutants will be compared for predation in plate bioassays and in bioassays. In addition we will assay for motility, vegetative growth defects and development.4. Identification of predation-dependent pathway using RNAseq. RNAseq provides a rapid and relatively inexpensive means to monitor large gene sets (M. xanthus encodes ~7600 genes) in both end-point and in temporal experiments. We have been successful in using RNAseq in M. xanthus in characterizing steady-state exponential growth in CTM broth, 3-day colonies grown on CTM agar plates, and biofilms. These studies demonstrate the feasibility of performing RNAseq and our ability to generate reproducible and high quality data. Therefore, RNAseq is the method of choice to identify and compare gene sets that respond to different growth conditions, in this case under predation conditions or general growth. Gene sets will be compared to correlate expression profiles to specific predation or non-predation conditions,; thereby allowing for the identification of specific and common gene sets to both growth conditions: Predation and non-predation.We propose to use differential RNAseq analysis to compare cells grown on standard media to those cells actively growing by predation on different prey bacteria and fungi. M. XANTHUS cells will be grown in the presence or absence of prey for 2 days, then the RNA harvested for analysis. The major problem using RNAseq in bacteria is that over 95% of the bulk RNA isolated is 16S and 23S rRNA, which means only about 5% of the reads are mRNA. Our current RNAseq protocols allow us to deplete over 90% of the rRNA such that we currently get over 85% uniquely mapping sequences. With reads typically at 32 - 20 million reads/sample our depth of coverage for this analysis is more than sufficient to do these experiments. During the analysis phase, contamination from the prey bacteria will be ignored since the reads will only be mapped to the M. XANTHUS genome. The only other potential problem is the rRNA from the prey, and to eliminate this problem we will construct oligos specific to the prey to remove them from the amplification step as described below.