Optimizing Monitoring and Biostimulant Practices for Sustainability in Orchards Contaminated by Herbicide Drift

Research Objective 1: Evaluating the correspondence between site-based plant and soil health indicators and lab-based soil, compost, and plant health tests.

Plant Leaf Analysis

Leaf samples for refractometer analysis were collected from 3 varieties of apple and 3 varieties of peach in mid-morning on Sep. 12, 2022.  Attempts to extract sap with either a handheld garlic press, vice grips, or a constructed screw press were each unsuccessful. Sap sufficient for refractometer analysis was readily collected from clover, alfalfa, and pasture grass species in the surrounding cover crop.  

Leaf and soil samples were collected in July, 2023 from mixed varieties of apple trees located on a seedling orchard at J.A.L. Farms in Fort Sumner, NM. Varieties included equal numbers of Honeycrisp, Gibson Golden, Aztec Fuji, and Arkansas Black. 

Leaf samples for tissue analysis consisted of fully developed leaves collected from south-facing branches of apple trees that were located > 6 m away from the edge of each row.  Leaves were collected and stored in a cold ice chest without directly contacting the ice.  Trees on the southeast portion of the orchard were visibly chlorotic, and made up the bulk of the symptomatic leaf samples described below. 

Leaf samples for sap analysis were carried out as above, except that old leaves located on the same branch as each sampled new leaf were also sampled.  New and old leaves were placed in separate sample bags.

Harvested leaves were divided into categories based on visual symptoms as follows:

• Asymptomatic- Dark green, leathery textured leaves free of mechanical damage or other deformities.
• Light Green and soft- lighter green leaves which may or may not have a soft texture, free from mechanical damage or deformities 
• Chlorotic-very light green or yellow leaves with varied patterns of chlorosis.
• Mechanical damage-includes insect damage, breaks from wind and rain, etc.

Symptomatic leaves were visualized with stereomicroscopy and photographed, before pooling all symptomatic leaves so that asymptomatic and symptomatic samples were sent to laboratories for analysis.  

Triplicate samples of new, mature leaves from the southeast, and remaining orchard were submitted to Ward Laboratories in Kearny, Nebraska for leaf tissue nutrient analysis. 

Pairs of old and new leaf samples were sent to New Age Laboratories in South Haven, MI for sap nutrient analysis. Due to the precise selection of south facing, newly mature leaves and the small size of the trees that is required for the sap analysis protocol, it was not possible to collect enough leaves for repeat samples. 

Site Based Microscopic Analysis of Fungal and Bacterial Abundance

Four soil samples representing the top 4 inches of soil were collected from the orchard, garden, and pasture on JAL Farms, in Fort Sumner, NM and from the garden at Spirit Farm, in Vanderwagon, NM.  Each sample represented a composite of 12 cores collected from randomly distributed locations within the sample area.  

Two compost samples were also collected.  One sample came from Spirit Farm and one from JAL Farms.   Both composts were made using  Johnson Su Bioreactors(1).  However, the compost at Spirit Farms, which operates on harvested rainwater,  is irrigated irregularly by hand.  The compost at JAL Farms was immature at the time of sampling.  Each sample represented a composite of 12 small spades filled with compost which was removed from different areas within the bioreactor. 

Soil and compost samples were each strained through a 3 mm sieve and blended to ensure uniformity.  Samples were stored at 4^oC and analyzed within one week of sampling. 

Each soil or compost sample was analyzed by diluting 0.5 g of soil or compost in 50 mL of carbon filtered tap water.  The diluted sample was rocked gently for 60 seconds.  Aliquots of suspension were removed from the unsettled suspension using a transfer pipette, and placed in the counting chamber of a Neubauer hemocytometer (Figure 2) and examined at 400X magnification on a Motic type 102M compound microscope.   

Bacteria cells present in each of the 5 diagonal squares in the center of the hemocytometer were counted.  Fungal cells present in each of the four corner squares (one is outlined in blue), and in the center square of the same size were counted.  If more than 300 bacteria were present in a single 0.2 mm grid, the sample was diluted and reanalyzed.

Three replicates of each sample were analyzed, providing a total of 15 bacterial, and 15 fungal values for each sample. 

To convert the number of cells/ml of solution to cells per gram of dry soil or compost, soil and compost moisture content was determined gravimetrically.  Fresh weights of 40 to 50 grams soil or compost were dried at 85^oC to a constant weight (36 hours).  Soil moisture (M) was recorded as (fresh weight – dry weight) / dry weight.  

The number of cells/ml was converted to cells/gram dry soil by multiplying counted cells/mL in each square by the dilution factor (df) of 100 to get the total number of cells in the solution.  This value was then divided by the dry weight (dw) of soil, which was calculated as dw = fw/(1+M) where fw=the fresh weight of soil (1 g).

Site Based SIR using the MicroResp^TM  system.

Substrate induced respiration via the MicroResp^TM  system offers tremendous flexibility to the end user, since respiration can be measured in the presence of almost any carbon source (ie: "substrate"). Typical applications select a variety of amino acids, carbohydrates, and carboxylic acids.  Environmental contaminants of interest are also evaluated.  To assist in identification of microbial responses indicative of soil health and response to pesticides, research articles in which MicroResp^TM was utilized were reviewed to identify substrates that have already proven useful as universal respiration indicators, were involved in auxin metabolism, or have other potential as soil health restoration indicators.  Substrates that reported the highest potential to inform soil responses relevant to herbicide remediation were selected.  In cases where substrate availability or ease of handling were problematic, analog substances were substituted.  The compounds used are listed in Table 1 by compound class and biological function.

Table 1.  Identification codes for aqueous control and eleven carbon substrates used in substrate reduced respiration. 

SIR Analysis of Orchard Soil and Compost

Composted manure was initiated in fall of 2022 by transferring manure and straw collected from cattle pens to one of three Johnson-Su style composting bioreactors.  Samples of bioreactor compost were drawn in July of 2024.  One sample, drawn from 12 random points within the interior regions of the bioreactor was gathered from each bioreactor.  An additional bioreactor, established on Spirit Farm in Northwestern NM, also provided a sample of compost. 

Soil samples were collected from the J.A.L farms orchard (3), or from the vegetable garden at Sprit Farm.  Each sample represented soil from 12 four inch deep cores gathered from random points throughout the sample area. 

Each compost and soil sample was manually homogenized and past through a 3 mm sieve.  One 60 g aliquot of the homogenized sample was subjected to gravimetric moisture analysis to determine water content.  The remaining sample was subjected to substrate induced respiration testing (SIR) using a modified MicroResp^TM protocol and the substrates described above. Modification of the protocol was necessary due to supply chain failures.  Flat bottom detection plates recommended for Microresp^TM _{could not be obtained at the time of analysis.  It was necessary to use round bottom detection plates. However, corresponding round-bottom strip well plates could not be found for CO2 Calibration assays.  Therefore, accurate calibration of CO2 emission rates was not possible.  However, a calibration curve developed using flat bottom strip well plates confirmed the anticipated inverse relationship between absorbance readings at 570 nm and CO2 emission.} 

Microbial activity across substrates was calculated based on normalized absorbance at 570 nm as described in the MicroResp^TM Technical Manual (2022). 

Research Objective 2: Evaluate the effectiveness of biostimulants alone, and in the presence of added nutrients for improving plant and soil function, including function in the presence of herbicide contamination.

Herbicide Residue Testing

Leaf and soil samples were collected for herbicide residue testing according to laboratory recommendations, and submitted to South Dakota Agricultural Laboratories in Brookings, South Dakota for  analysis.  

SIR Analysis of Herbicide Remediation

A bulk sample of soil will be collected from JAL Farms orchard in April of 2024 by collecting 20 cores from each row of trees, mixing thoroughly, and sieving soil as described above.  Subsamples of the bulk mix will be combined with biostimulant at nutrient blends at 1X and  2X, the recommended application amounts. Samples will then be subjected to MicroResp^TM _{analysis as described above.} 

A  linear mixed effects model with field as a random effect is being developed in  R software (lme4 package) to compare microbial response across herbicide and biostimulant treatments.  Microbial diversity will also be calculated using the Shannon-Weaver Index (H=- Σp_i * ln(p_i)), so that microbial diversity may be used as an indicator of soil remediation

Seed Germination Bioassays Herbicide Remediation

Seed germination bioassays will be carried out in summer 2024 as described in the proposal.