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Organic and Regenerative Farms are a Gold Mine for Potentially Novel Species of Soil Bacteria
* 1 , 2 , 3 , 3 , 4 , 4 , 1 , 1 , 5 , 1 , 1 , 1 , 1 , 1 , 1 , 1 , 1 , 1 , 1 , 6
1  Los Angeles Pierce College
2  Cold Spring Harbor Laboratory
3  James Madison University
4  Rodale Organic Institute
5  Oregon State University
6  California State University, Northridge
Academic Editor: Nico Jehmlich


Between Fall 2021- Spring 2023 soil metabarcoding samples and soil bacterial isolates were collected from Los Angeles Pierce College farm and local farms, and sequenced on Illumina MiSeq and Sanger platforms. Soil physical properties from the sites were also measured including pH, EC, TDS, organic matter by dry combustion, texture by touch, and nitrogen, phosphorus and potassium by spectrophotometry. These data were compared with reference measurements from the USDA NRCS soil surveys. Comparisons between fields were carried out in the R stats package.

Preliminary data showed that the conditions between the fields differed significantly in values for organic matter, N, K, and pH (p<0.05); this offered a diverse panel of substrates for discovery. The cultivated bacteria were isolated on Nutrient Agar, Luria Broth Agar, or ISP-6 medium. Barcoding isolate DNA was extracted using 10% chelex solution heated for 10 minutes at 100 C. Quality control was achieved through electrophoresis using the E-gel system. DNA extraction from soil samples used the Qiagen DNEasy Power Soil Kit. Quality control was achieved with spectrophotometry using the ThermoFisher MultiSkan SkyHigh Microplate Spectrophotometer. Barcoding data analysis was performed on the DNA Subway Blue Line and EZBioCloud; metabarcoding data analysis was carried out using the DNA Subway Purple Line.

Initial metabarcoding results from Tulare fall 2021 hemp fields versus recently harvested fields or fallow ground suggested a difference in alpha diversity between hemp ground and the two other fields. In spring 2021 and fall 2022, 137 bacterial isolates from Pierce College, Rodale Institute for Organic Agriculture in Camarillo, and Tulare, CA were screened; this investigation revealed up to 30 potentially novel species based on the partial 16S sequences. The soil samples from fall 2022 were taken from pasture, fallow, cover crop, and hemp plantings. The purpose of the cover crop trial was to test replacement of plastic mulch with living mulch and cover crops in strawberry and artichoke production.

Some of the more notable genera discovered from the isolates were Lysobacter, Streptomyces, and Bacillus, which include antibiotic producers. Some of the bacterial isolates were challenged with crude ethanolic extracts of tea tree and lemon balm. Percent transmittance was measured at 600 nm. The growth was similar between replicated treatments for putative Bacillus mojavensis, Curvibacter lanceolatus, Streptomyces bobili. Both tea tree and lemon balm were effective at controlling Bacillus pumilus compared with the control, whereas putative Pseudomonas segetis grew more when treated with either lemon balm or tea tree extracts, and Paracoccus marcusii grew more when treated with tea tree oil versus the control.

Additional work is needed to characterize the plant terpenes and soil properties. More investigation into the taxonomic composition and predicted functions of the soil bacteria is necessary. Further research will focus on identification of the main terpene components of the plant extracts by gas chromatography.

Keywords: soil microbiome, cannabis sativa, organic agriculture, cover crops