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

Progress report for FW22-399

Project Type: Farmer/Rancher
Funds awarded in 2022: $24,974.00
Projected End Date: 06/01/2025
Host Institution Award ID: G347-22-W9210
Grant Recipient: JAL Farms
Region: Western
State: New Mexico
Principal Investigator:
Mary Lucero
JAL Farms
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Project Information


     This project aims to develop assessment protocols that guide biostimulant use practices to improve crop and soil health or remediate crops  damaged by herbicide drift. Experiments will be conducted on a seedling fruit orchard that was contaminated by drift in 2021.

Questions to be answered by investigation include: 

  • How closely do rapid and affordable, site-based assessments of leaf color patterns, microscopically detected bacterial:fungal ratios and changes in refractometer based Brix readings correlate with laboratory assessments of plant, soil, or compost nutritional status and microbial diversity?
  • Can biostimulant additions (including bioactive composts) improve plant and soil function, as measured by seedling germination bioassays and substrate induced respiration (SIR), in herbicide-treated plants and soils?
  • How long will herbicide contamination and detectable plant symptoms persist in plants and soils following biostimulant treatments?

     Results will provide farmers, gardeners, and crop advisors with empirical data demonstrating use of biostimulants for improving plant and soil health in a seedling fruit orchard recovering from herbicide damage due to drift. 

     More broadly, the project will demonstrate the effectiveness of popular on-site and laboratory assessments for guiding decisions that effectively address routine or triage-based plant and soil health demands.   

     Findings will be disseminated in underserved rural, urban, and indigenous communities through a combination of podcasting and personal communication with growers at farmer's markets, community meetings, and presentations to nonprofit organizations that support grower education. 

Project Objectives:


  • Evaluate utility of site-based and laboratory assessments of plant and soil biological health in fruit orchards for distinguishing between healthy (ie: nutritionally complete and biologically functional) and less healthy soils, leaves, and composts.
  • Evaluate effectiveness of biostimulants (compost, compost teas, and fulvic acid for improving detectable plant and soil health responses. 
  • Evaluate plant and soil responses to various biostimulant treatments in the presence of herbicides.


  • Use a combination of live presentations, workshops, and blog articles to:
    • describe effective visual, site-based, and laboratory monitoring techniques that support sustainable use of composts and other biostimulants or biological practices.
    • illustrate mitigation strategies that can reduce losses associated with herbicide drift or other factors that damage plant and soil health.
    • help growers, crop advisors, and general audiences gain appreciation for biological approaches to crop production. 
  • Help growers in underserved rural, urban, and indigenous communities optimize use of composts and affordable, on-site testing strategies to increase local, sustainable food production. 


Click linked name(s) to expand/collapse or show everyone's info
  • Dr. David Johnson - Technical Advisor
  • David Lucero - Producer
  • Dr. Mary Lucero - Producer (Educator and Researcher)


Materials and methods:

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 4oC 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 85oC 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 MicroRespTM 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.


Table 1.  Promising chemical substrates for use as soil health restoration indicators




Biological function(s)


Acetyl-glucosamine (N-)



chitin component

Saul-Tcherkas(2); Bongiorno(3)

Alanine (L-)

amino acid



Sassi(4); Dennis(5)

aspartic acid (D,L)

amino acid


binds to auxin to mark for storage, degradation


Cysteine (L-)

amino acid


protein, glutathione precursor, nitrogenase precursor, bind metal centers in many proteins, including metallothionein.



amino acid


auxin precursor

Stepanova (6)


amino acid


dessication tolerance (osmolyte), simplest stable amino acid


D + Cellobiose

carbohydrate (C12)


cellulose degradation product


Trehalose (dihydrate)

carbohydrate (C12)

C12H22O11 · 2H2O

cellular energy,dessication tolerance (osmolyte)

Saul-Tcherkas (2); Sassi(4)

Glucose (D-)

carbohydrate (C6)


cellular energy, somewhat universal carbon source, most common SIR substrate

Saul-Tcherkas(2); Sassi(4); Dennis(5); Tahtamouni(7); Campbell(8)

galacturonic acid

carboxylic  acid/sugar

C6H10O7 · H2O

pectin component

Gomez (9)

Citric acid

carboxylic acid


TCA cycle, root exudate

Saul-Tcherkas(2); Dennis(5); Gazdag (10)

Malic Acid (L-)

carboxylic acid (di)


Root exudate, dicarboxylic acid

Saul-Tcherkas, 2009(2), Sassi(4), 2012

Oxalic acid

carboxylic acid


secreted by soil fungi, perhaps to increase metal cation availablility

Saul-Tcherkas (2); Dennis(5)

Tween 80

carrier, fatty acid (analog)


cell membrane components


Protocatechuic acid



lignin, humus degradation product

Saul-Tcherkas(2); Dennis(5)



  1. J. DC, S.-J. HC. (2010), https://youtu.be/DxUGk161Ly8 -.
  2. V. Saul-Tcherkas, Y. Steinberger, Substrate utilization patterns of desert soil microbial communities in response to xeric and mesic conditions. Soil Biology and Biochemistry 41, 1882-1893 (2009).
  3. G. Bongiorno et al., Soil management intensity shifts microbial catabolic profiles across a range of European long-term field experiments. Applied soil ecology : a section of Agriculture, ecosystems & environment 154, (2020).
  4. M. B. Sassi, J. Dollinger, P. Renault, A. Tlili, A. Bérard, The FungiResp method: An application of the MicroResp™ method to assess fungi in microbial communities as soil biological indicators. Ecological indicators 23, 482-490 (2012).
  5. P. G. Dennis, T. Kukulies, C. Forstner, T. G. Orton, A. B. Pattison, The effects of glyphosate, glufosinate, paraquat and paraquat-diquat on soil microbial activity and bacterial, archaeal and nematode diversity. Sci Rep 8, 2119 (2018).
  6. A. N. Stepanova, J. M. Alonso, Auxin catabolism unplugged: Role of IAA oxidation in auxin homeostasis. Proc Natl Acad Sci U S A 113, 10742-10744 (2016).
  7. T. Mohammad Emad, K. Sa’eb, L. Mary, S. Jesus, U. Adrian, Soil community catabolic profiles for a semiarid reclaimed surface coalmine. International Journal of Mining, Reclamation and Environment, 1-17 (2023).
  8. C. D. Campbell, S. J. Chapman, C. M. Cameron, M. S. Davidson, J. M. Potts, A Rapid Microtiter Plate Method To Measure Carbon Dioxide Evolved from Carbon Substrate Amendments so as To Determine the Physiological Profiles of Soil Microbial Communities by Using Whole Soil. Appl. Environ. Microbiol. 69, 3593-3599 (2003).
  9. M. J. Fernández-Gómez, R. Nogales, H. Insam, E. Romero, M. Goberna, Role of vermicompost chemical composition, microbial functional diversity, and fungal community structure in their microbial respiratory response to three pesticides. Bioresour Technol 102, 9638-9645 (2011).
  10. O. Gazdag et al., Density and Diversity of Microbial Symbionts under Organic and Conventional Agricultural Management. Microbes and environments 34, 234-243 (2019).


Research results and discussion:

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

Leaf Sap Extraction

There was not enough free sap available to conduct refractometry. The onset of seasonal leaf senescence prevented further trials.

It is believed that the thicker, more ligneous nature of tree leaves compared to typical garden vegetables promotes immediate adsorption of sap by leaf carbohydrates, as it is extracted from the vascular tissue.  Experimentally testing this hypothesis would fall beyond the scope of this project.


Reliability Assessment of Microbial Direct Counts and Fungal:Bacterial Ratios

A reliability analysis of the direct counts and fungal bacterial ratios revealed that the direct counts were 98% reliable for the log transformed averages of the bacterial counts for a given sample, and 92% reliable for log transformed averages of the fungal counts for a given sample.  The log transformed averages of the fungal:bacterial ratios were only 72% reliable.  

A subsequent analysis of unrelated soil and compost samples (not shown) revealed that the fungal counts can reach 98% reliability when only spores are counted.  Decayed plant material can sometimes resemble certain fungal fruiting bodies viewed under the microscope.  It is thought that eliminating fruiting bodies, hyphae, and other non-spore structures improves reliability by reducing the opportunity for false positives during examination.

The low reliability of the fungal:bacterial ratios was surprising, since fungal:bacterial ratios are widely regarded as a useful soil health indicator.  It is worth noting, however that a reliability assessment simply refers to the consistency of the measure.   While these results suggest we can be confident that the same sample will give the same result when the same method is applied, it tells us nothing about the value of the measurement for predicting soil health.  The correlations between microbial counts and fungal to bacterial ratios should become more clear when the values obtained from field samples are compared against soil chemical and nutritional parameters.  

Participation Summary
2 Producers participating in research

Research Outcomes

Recommendations for sustainable agricultural production and future research:

Leaf Sap Extraction

Although Brix values taken from leaf sap of most annuals and many forage crops can provide valuable indicators of general plant health, extracting leaf sap from fruit trees is less feasible for routine monitoring on orchards.  This is because the leaf sap is simply too difficult to extract.  Conceivably, leaf extracts could measured by preparing an aqueous suspension of the minced leaves, filtering off suspended solids, and measuring the Brix values of the remaining solution.  However, obtaining meaningful values in this manner would require numerous additional steps, including an accompanying leaf moisture analysis in order to correct for varying moisture concentrations.  The added analysis time would eliminate the convenience that makes leaf Brix monitoring attractive. 

It should be noted that Brix monitoring of fresh fruit is a well proven technique for assessing sugars.  As such, it remains a valuable tool for monitoring fruit quality. 

Microbial Direct Counts and Assessment of Fungal:Bacterial Ratios

Direct counts of soil microorganisms can provide a highly reliable and cost effective method to monitor soil microbial populations.  However, the method does require some initial investment, including purchase of a suitable compound microscope and hemocytometer.  It also takes a commitment to learning how to distinguish fungi from bacteria under the microscope.

For the best quantitative results, samples should be diluted to a concentration of < 300 bacterial cells per 0.2 mm grid, and fungal cell counts should only include spores.  Tap water used to suspend microbes should be free of chlorine (carbon filtration is sufficient).  


Education and Outreach

3 Consultations
2 Webinars / talks / presentations

Participation Summary:

59 Farmers participated
25 Ag professionals participated
Education and outreach methods and analyses:

     Educational outreach objectives were modified in year 1 included two invited presentations.  On March 3, 2023, Mary Lucero delivered a presentation entitled Cover Crops and Low Till Management at the 2023 NM Fruit Growers Workshop in Abiquiu, NM.  Approximately 50 growers and agriculture professionals were present.   On March 9, she presented Maintaining Healthy Soils in an Unhealthy Ecosystem to the New Mexico Farmers' Marketing Association in Santa Fe, NM.  Approximately 45 of the enrolled participants attended this section.  Both presentations were delivered in communities with diverse and  historically underserved populations. 


Education and outreach results:

The initial objective of recording 20 podcast episodes was modified due to technical difficulties associated with recording quality and with making the recordings and transcriptions ADA compliant. After consulting with SARE staff, it was decided to drop the podcasts altogether and look for alternative outreach opportunities.   Outreach has since been carried out through the two live presentations discussed above.  A third presentation has been scheduled for August of 2023. 

In both completed presentations, the benefits of biologically based agricultural approaches, laboratory testing, and on-site monitoring were highlighted.   For Cover Crops and No Till Management, the audience consisted of experienced fruit growers, so the presentation used soil test data to illustrate the loss in soil quality following deep tillage and laser leveling, followed by the restoration which occurred over the course of two years without tillage, when using cover crops and biostimulants in place of synthetic fertilzers. Participant survey data from this presentation was gathered by the Rio Arriba County Cooperative Extension Service, and will be made available at a later date. 

In Maintaining Healthy Soils in an Unhealthy Ecosystem, the audience consisted of less experienced farmers market gardeners, including less

Charts illustrating survey responses.
Figure 1. Survey responses from the presentation entitled Healthy Soils in an Unhealthy Ecosystem.

traditional urban and first time growers.  This time, the topic centered on basic soil health principles, the benefits of biological practices, and elementary monitoring steps relevant to small operations.  Examples of environmental and anthropogenic factors that can interfere with soil health were discussed.  Program feedback was assessed using the Western Region Sustainable Agriculture Research and Education Program Outreach Survey.  Survey results are illustrated in Figure 1,  to the right.  A smaller group was anticipated, so only 30 surveys were available to distribute.  Twenty-two of these surveys were returned.  

     The four questions addressed to all respondents (Figure 1A) indicated the presentation was effective in providing new awareness (100% of respondents), new knowledge (95% of respondents), and skills (81% of respondents) that impacted opinions or attitudes. 

     Responses to questions presented to producers (Figure 1B) revealed unanimous willingness to adopt new practices and diversify production.  Curiously, all responses also indicated willingness to use the project to increase networking with other producers.   Ninety-four percent of respondents planned to use some aspect of the presented information to reduce purchased off-farm inputs. An additional 94% were willing to adopt one or more of the practices shown. 

     Fifteen agricultural professionals responded to the survey.  Professional responses were overwhelmingly positive, as all answers were either yes (83%), or not applicable to their situation (17%).  


16 Farmers intend/plan to change their practice(s)
3 Farmers changed or adopted a practice

Education and Outreach Outcomes

Recommendations for education and outreach:

Projects for federal grants that include podcasting or other audio-visual media production need to include budget for transcription and other ADA compliance factors. 

Information about sustainable agriculture that illustrates, not only the long term global benefits, but the immediate benefits to the producer has been well received in live presentations. 

45 Producers reported gaining knowledge, attitude, skills and/or awareness as a result of the project
Non-producer stakeholders reported changes in knowledge, attitudes, skills and/or awareness as a result of project outreach
53 General public
6 Students
13 Ag Service Providers

Information Products

    Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.