INTEGRATING COVER CROPS AND SHEEP GRAZING IN ALMOND ORCHARDS

Research objectives

1. Test for the prevalence and die-off of fecal pathogens in the soil of almond orchards grazed by sheep to understand the food safety risk of ground-harvested almonds in a grazed system.
2. Complete a cost analysis comparing the costs and returns of conventional almond production to the costs and returns from a livestock and crop integrated almond production system.

Project sites

We sampled soil from two California almond orchards, one in Kern County and one in Merced County. The soil on the Kern farm is a clay loam, Calflax and Panoche series, and the main soils on the Merced farm are sandy loam soils of the Montpelier and Pents series. During the study both farms utilized cover crops and grazing as orchard floor management practices.

Research design, Data collection, and Analysis Methods

Treatments

All almond orchard acreage we include in our study was planted with cover crops. Within these orchard systems we established a grazed and ungrazed treatment. The ungrazed treatment was about one-quarter the size of the grazed treatment. Ungrazed treatment areas were mowed.

Randomization of sample locations within each orchard

Pathogen persistence and population size in soil is affected by several environmental factors including weather; soil temperature, moisture, and texture; organic matter; etc. (Sharma et al., 2019, Topp et al., 2003, and Underthun et al, 2018). In an almond orchard there are two micro-climate zones where these factors differ significantly. The wetting zone under the trees is a moist-temperate zone where soil remains moist due to irrigation, and temperature fluctuations may be more minimal due to shading from the canopy. Outside the wetting zone is a dry-arid zone where the soil will remain drier through the growing season and temperature fluctuations may be more severe due to higher sun exposure. These zones also represent two primary locations for potential contamination during harvest. The wetting zone is where almonds will first contact the soil when shaken from the trees, and may remain as the crop dries. The almonds are then blown and swept into the dry-arid zone in between the trees (row middle) where they will be picked up from the ground and mixed with the soil in this area.

The Produce Safety Rule of the Food Safety Modernization Act (FSMA) addresses concerns about the feasibility of compliance for farms that rely on grazing animals, but does not require establishing waiting periods between grazing and harvest. However, farmers are encouraged to voluntarily consider applying intervals appropriate for the farm’s commodities and practices. Organic farmers follow the National Organic Program standards for raw animal manure, applying a 120 day interval between incorporating raw manure into the soil and harvest. Moreover, third-party food safety auditors have directed attention toward these integrated crop-livestock farms due to the possible contamination risk of nuts. As a result, the standard practice in almond orchards is to remove all grazing animals at least 120 days before harvest.

We characterized the longitudinal profile of the occurrence of bacterial pathogens and indicator species in orchard soils from cessation of sheep grazing until almond harvest. Seven pairs of sample locations were selected in the grazed treatment and three pairs of sample locations were selected in the ungrazed treatment. For each paired sample, one of the paired sample locations was within the wetting zone of irrigation; the second was outside the wetting zone. A random number generator in Excel identified the tree row to be sampled and the tree to be sampled. Tree rows on the external edge of an orchard were excluded from the randomization to reduce edge effects. Samples were collected at 0, 7, 14, 30, 60, 90, and 120 days post-grazing in both treatments. In the grazed treatment seven trees were selected at each sampling interval (different trees at every sampling) (7 trees = 14 paired samples; 14 x 7 sampling dates = 98 samples/year/orchard). In the ungrazed treatment three trees were selected at each sampling interval (3 trees = 6 paired samples; 6 x 7 sampling dates = 42 samples/year/orchard). Post-grazing samples totaled to 560 samples over two years. Forty pre-grazing samples were collected in the same manner, 20 in Year 1 and 20 in Year 2 (10 paired samples in each orchard per year). Two paired samples in the ungrazed treatment (2 trees = 4 paired samples) and 3 paired samples (3 trees = 6 paired samples) in the grazed treatment.

Soil sampling

Trees were divided into quadrants based on cardinal directions - northwest, northeast, southeast, and southwest - and soil sub-samples were taken under each tree from each of the four quadrants. Soil sub-samples from under each tree were placed in resealable plastic bags and mixed. Samples from the row middles were collected from either the south or west side of the tree. Actual sampling depended on which direction the rows ran. For example, if the almond trees were in rows that ran north to south, the row middles fell on the west and east sides of the trees. In that case, soil sub-samples were collected from the west side of the tree. Similar to the soil samples from under the trees, three sub-samples were mixed together. Using the example above in which tree rows ran north to south, three soil sub-samples were collected from north to south, at least one meter apart.

At each sample location within the orchard, a sterile sampling scoop (Spectrum, New Jersey, USA) was used to collect three or four soil subsamples of about 50 grams each from the upper 2 inches of soil, soil samples were placed into resealable plastic sample bags and shipped overnight on ice to the University of California, Davis.

Bacterial analyses

UC Davis provided analyses on pathogens of interest for food safety as well as indicator bacteria that sheep fecal matter may host.

Detection of Pathogens (Atwill Lab)

For each sample location and sampling date, 25 grams of mixed soil were placed into a 710 ml Whirl-Pak(R) Homogenizer Blender Filter Bag (MilliporeSigma, Darmstadt, Germany) and incubated in 225 ml tryptic soy broth (TSB; Difco, San Jose, CA) on a shaking incubator (50 rpm) at 25° C for 2 hr followed by 42° C for 8 hrs. Then, 0.5 ml of the TSB enrichment were transferred into 4.5 ml of TSB and modified Enterohemorrhagic E. coli broth (mEHEC). These secondary TSB and mEHEC (BioControl Systems, Inc., Bellevue, WA) enrichments were screened for E. coli O157, stx 1/2 genes, and Salmonella using quantitative-PCR (qPCR) (Atwill et al., 2015; Baker et al., 2019; Suo et al., 2010). All suspect positives from the qPCR screen were plated onto their respective selective agar and suspect colonies qPCR-confirmed for E. coli O157 and Salmonella as previously described (Atwill et al., 2015; Gorski et al., 2011). Suspect shigatoxin producing E. coli (STEC) colonies were confirmed using multiplex conventional PCR to identify O26, O45, O103, O111, O121, O145 and O157 serogroups of E. coli (Paddock et al., 2012).

Generic E. coli Most Probable Number (MPN) (Pires Lab)

A subset of soil samples were cultured for indicators of contamination and generic E. coli quantification. To quantify E. coli in soil, we used a standard tube method with serial dilutions up to 10-6 in quadruplicate followed by streaking onto CHROMagar ECC to quantify E. coli as described previously (Patterson et al., 2018). At least one presumptive positive isolate per sample was purified and confirmed using a standard PCR method (Chen and Griffiths, 1998). MPN series cell densities were calculated based on dilution to extinction using an MPN Calculator (Curiale, 2004). Fecal coliforms were assayed using the USEPA 1680 protocol (Reynnells et al., 2014).

Whole Genome Sequencing

A subset of isolates (soil and fecal) representative of orchard type, treatment, time post-grazing and year were selected for whole genome sequencing analysis (WGS;~50 isolates). We will conduct WGS for these isolates to identify potential genomic elements, (i.e., genus/species, serotype, virulence genes and antimicrobial resistance genes). DNA extraction and purification will be conducted using previously described methods (Carroll et al., 2017). DNA will be quantified using a Nanodrop One (ThermoFisher). Library preparation and WGS will be conducted at the Weimer Lab using Illumina sequencing methods as commonly used (Bandoy et al, 2020; Miller et al.,2021). These results are pending as the WGS analysis is still being conducted.

Almond yield assessment

Yield per acre for the different treatments was assessed in August after trees were shaken. For each replicate at the Kern farm, almonds were collected from the ground following standard on-ground commercial harvesting techniques. Subsamples of a minimum of five pounds of harvested nuts from each replicate were collected to run a turnout analysis to separate almond kernels from other materials such as hulls, shells, dried leaves, wood sticks, soil particles, etc. This allowed us to determine the percentage of almond kernels in the gross weights and extrapolate the values to yield per acre based on the harvested area. Yield from each orchard was compared to historical average almond yields per orchard. At the Merced farm, yield assessment was conducted by a private-party.

Quantifying economic impacts

We used partial budget analysis methods to evaluate the changes in economic costs and benefits from integrating livestock into almond orchards in comparison to using conventional methods. We gathered information for cost and benefit estimates through conversations with participating growers and fellow Co-PIs and collaborators on this proposal. As a baseline for the conventional methods, we used the most recent Almond Cost and Returns Studies, updated in 2024. Potential increased costs associated with integrating livestock include: contract grazing fees and repairs to irrigation equipment. Potential benefits include: decreased herbicide use and decreased labor for mowing and/or navel orangeworm sanitation. Quantifying these benefits and costs helps growers evaluate their current practices in comparison to integrating livestock. 

Manure pellet counts

Just prior to harvest, after the orchard floor was cleaned, we surveyed for manure pellets under 50 trees in each orchard. We used the same Excel program as mentioned above to randomly identify the trees sampled. Under each tree sampled a one meter square quadrat was placed three times: 1) under the tree, right next to the trunk; 2) under the tree canopy, near the dripline, and; 3) in the row middle. Each time the quadrat was placed a visual survey for manure pellets was conducted and manure pellets were counted.