GENOMIC TOOLS TO MONITOR AND EXPLOIT MICROBIAL DIVERSITY FOR AGRICULTURAL DISEASE AND ARIDLAND MANAGEMENT

Microbes play important roles in natural and human managed ecosystems. Objective 1 focuses on understanding how microbial communities are integral to the development and promotion of healthy soils. Their overall role in arid soils is less well understood requires study of specific fungi and bacteria which may be indicators or regulators of soil health. Tools to monitor the microbial composition is important for efficient land management practices. In arid regions biological crusts are results of cooperation between bacteria and fungi and their presence can improve soil moisture, promote nutrient exchange, and reduce dust through aggregation of soils. This project will explore the microbial community makeup of these crusts to better catalog processes that support their formation, restoration after damage, and overall impact on the health of soils. This work will take place in managed public lands to study the microbial makeup in undisturbed, health biological crusts. Measurements of plant health in fields with adjacent crusts will be compared to those without to test whether promotion of crusts within agriculture fields can benefit soil moisture and nutrient availability. Objective 2 focuses on microbes associated with the shot hole borer beetle which carries fungi that are causing disease in avocado and a multitude of trees in Southern California. The work will focus on native microbial communities of trees and how they respond to the introduction of the Fusarium fungus, identification of antagonistic bacteria or fungi that can impede growth of Fusarium, and genetic studies of the beetle-carried fungi to track spread and possibility of sexual reproduction among isolates. Objective 3 focuses on acquisition of fungicide resistance in post-harvest associated fungi. Fungicide application can reduce crop loss but leads to resistance in microbes. The acquisition of fungicide resistance among post-harvest disease fungi in particular is less well studied and requires improved monitoring and development of genomic techniques for rapid diagnostics to trace the common source and spread of resistance mechanisms. The project will help to understand if resistance is acquired locally in farms or is a result of spores dispersed across regions which can inform mitigation and prevention strategies.

Objective 1. Study of Biological crust microbial diversity to understand native microbiomes in arid soils. Field sampling locations. Field trips to Sheephole Wilderness, Bureau of Land Management (collecting permit to N. Pietrasiak with Stajich added as collaborator), Joshua Tree National Park (permit to graduate students N. Pombubpa, J. Adams), and the UC Reserves at Mott Rimrock, Oasis de los Osis, Sweeney Granite Mountain, and Boyd Deep Canyon will be taken to sample BSCs. Undergraduate students working in Stajich labs will be given opportunities to learn field work techniques and participate in sampling. Additional sampling will be performed in Temecula in areas adjacent to vineyards and olive groves to capture soil microbial diversity.Samples for metabarcoding and metagenomics: We will collect three representative 2 cm2 samples at a site for each type of biological crust. Samples will be collected from systematic locations to spatially represent the 25m2 transect area (roughly one sample each 1/3 of the areas). Samples will be stored in sterile sample containers on dry ice until laboratory storage at -80°C. In the laboratory, a subsample of equal volume from each of the three cores will be made into a composite sample for metagenomic sequencing. DNA will be extracted fromsoil samples with MoBio Power Soil kits. DNA libraries for metagenomics will be prepared from these extractions followed by sequencing on 2x150 bp Illumina NovoSeq platform. Analysis of these data will involved metagemomics pipelines to assemble the data,identify bacteria and fungal contigs, and annotate these data with gene prediction pipelines.The DNA for metabarcoding will be processed with amplification primers for ITS1 and 16S ampilfication follwing the Earth Microbiome Project protocol. These data will be processe with QIIME and amptk pipelines to identify patterns of microbial diversity among the crust types.Sampling site characterization and environmental metadata. At each sampling site we will designate an area of 25-50 m2 that is representative of the habitat. Air temperature, days since measurable rainfall, soil surface temperature, and soil moisture will be noted for each sampling location at representative spots. We will establish two diagonal 25 m transects. Each end of the transect will be precision GPSed using a Trimble GPS unit. Along both transects we will record % ground cover of abiotic and biotic land surface categories using the line point intercept method following (Herrick et al. 2005). Land surface categories may include rocks, gravel, vascular plants to functional groups, and BSC community types following the classification by Pietrasiak et al. (2013). These data will be incorporated as metadata.Plant ecophysiology linking to BSC health. In identified grape vineyards, with permission from growers and land owners, soils 10-20 meters outside the planting area will be collected. Areas with and without BSCs will be profiled. Preliminary visits to Temecula wine growing region identified several examples of undisturbed lands with BSCs adjacent to vineyards and many examples of vineyards with only disturbed soils. Ecophysiological profiling of plants during moderate and extreme heat will be performed for plants located at a range of distances from the BSCs and from plants in vineyards with or without disturbed soil profiles nearby. These data will be compared to both the soil microbiome makeup and plant profile to test for correlations and establish a trend in how soil health, focusing on the presence and makeup of the microbiome of biological crusts, are informative to plant responses to stress.Objective 2. Genomic profiling of Fusarium dieback across Southern California. DNA from isolated of F. kuroshium and F. euwallacea along with associated fungi Graphium kuroshium will be obtained from collaborator Akif Eskalen (UC Davis) and sequenced with short read Illumina to achieve 10-20x coverage. Sequence variants will be identified with pipelines developed in the Stajich lab using best practices for SNP and INDEL calling. Reference genomes will be constructed by sequencing single strains with long sequence read technology to support gap closure. The identified sequence variants will be examined in the context of the larger population of strains sequenced to identify geographic and host association patterns. These data will be analyzed in to examine population structure using population genetics methods. The key questions surround compare evidence of gene and allele exchange through recombination.Microbiome profiling of affected and naive trees. Core samples from avocado trees, fruit with and without Fusarium dieback infections will be profiled for microbiome composition. This will be performed in collaboration with Eskalen lab at UC Davis to obtain wood from infected and unexposed trees and fruit from regions in San Diego and Orange Counties. These samples will be processed to extract total DNA following standard protocols established in our labs, followed by amplicon sequencing and processing to explore using primers to amplify 16S rRNA and ITS regions to profile bacteria and fungi respectively. We will compare these profiles to better understand the effects of these fungal and beetle associations on phytobiomes. In addition, for uninfected or asymptomatic trees which still have beetle galleries we will investigate if there is evidence for any protective effect of native microbes by exploring some of the primary differences in microbiome communities in exposed but unaffected trees to exposed and infected trees.Community and stakeholder engagement. This work involves engagement with the California Invasive Species Council - Shot Hole Borer Research Committee (http://iscc.ca.gov/ishb.html). We are actively engaging with this working group to develop research plans and questions that will be established to outline funding opportunities and goals around mitigating Fusarium dieback and Shot Hole Borer transmitted diseases. This working group also enables communication and engagement with multiple stakeholders surrounding protection of native ecosystems, agriculture, and ornamental and urban landscapes which will be impacted by this expanding tree disease threat.Objective 3. Profiling phenotypic and genetic changes in fungicide resistance. To evaluate changes in resistance and tolerance of fungi to fungicides we will explore sensitivities of Rhizopus stolonifer fungi isolated from soils, fruits, and nuts. Isolation will be performed by observing growth on post-harvest rot or from soil "sprinkle plate" methods. Extended sporangia, a hallmark of Rhizopus growth, will be used to identify candidate isolates. Single sporangia will be taken by forceps to inoculate a sterile PDA or MEYE media plate. Subculturing to ensure a single isolate will be performed at least 2 times. The isolate ITS will be amplified and sequenced by Sanger sequencing to confirm species. Isolates will be banked as sterile plug cultures in triplicate and isolation source The Relative sensitivities expressed in minimum inhibitory concentration to standard in-field fungicides used including flutolanil and azole fungicides used in clinical setting. Isolates will be stored in local culture collection and contributed to USDA NRRL collection as appropriate.Whole genome sequencing of these isolates will be peformed in order to test hypotheses about population genetic structure, exchange, recombination, and endemic patterns. Together these will be used to form haplotype networks and undestand the patterns of how these fungi disperse. Study of fungicide resistance will be attempted to be correlated to genetic differences to evaluate the relative frequency of alleles that may be contributing or linked to resistance.