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.

Sample Collection

Funding for grant activities was not accessible until August of 2022.   Necessary supplies were also severely back-ordered.  This made sample collection impossible so late in the growing season.  Therefore, it was decided to delay sampling until 2023. 

Method Optimization

Activities between fall, 2022 and early spring of 2023 centered on method optimization for site-based monitoring, and a review of recent findings related to substrate-induced respiration using Microresp™ methods.  The site-based methods tested included leaf sap extraction for measuring leaf Brix in the orchard, and microscopy aimed at quantifying fungal and bacterial communities in the soil. 

Leaf Sap Extraction

Fully expanded young leaves of mature size were collected from 3 varieties of apple and 3 varieties of peach trees in mid-morning on September 12, 2022.     Initial attempts to extract sap utilized a handheld garlic press. This method has worked well for ripe fruit, vegetable plant leaves, and green forage.  However, no sap could be extracted using from any of the fruit tree varieties tested. 

Next, a screw press (Figure 1) was constructed.  The screw press was capable of holding 200 mL volume of diced apple leaves.  New leaves were collected as before, on October 14.  Leaves were diced to approximately 5 mm², and placed in the holding cup of the screw press. When pressure was applied, the crushed leaves emerged from the screw press as a damp paste.  

Microbial Direct Counts and Assessment of Fungal:Bacterial Ratios

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).

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.

The proposed approach to substrate induced respiration offers tremendous flexibility to the end user, since respiration can be measured in the presence of almost any 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 was reviewed to identify compounds that have already proven useful as universal respiration indicators, were involved in auxin metabolism, or had otherwise proven valuable as soil health restoration indicators.  The compounds that reported the highest potential to inform soil responses relevant to herbicide remediation are provided below (Table 1).  These compounds will be evaluated against herbicide treated and untreated soils in Year 2.

References

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