Cost-benefit analysis of inoculating blueberry bushes with ericoid mycorrhizae

Cost-benefit analysis of inoculating blueberry bushes with ericoid mycorrhizae

Summary

We were able to arrive at primary conclusions for the project in 2014:
1. There was no significant difference in Ericoid mycorrhizae colonization levels between treated bushes (inoculated with commercial product) and controls. In fact, many controls exhibited very high colonization levels, implying that Ericoid was present before planting either in the nursery media or the native field soil.
2. We did not have enough data to draw clear conclusions on the effect of colonization on yield. Our four-year-old blueberry bushes were still too small to produce reliable fruit yield, or at least of quantities sufficient to analyze for row-by-row comparisons of treated vs controls or observe correlations between fruit yield and colonization levels.
3. After many hours of trial and error, we were able to refine an efficient, low-cost, non-toxic “farm lab” method for clearing and staining blueberry roots for Ericoid mycorrhizae observation and quantification under the microscope.

While researchers have found Ericaceous plants colonized with Ericoid mycorrhizae (EM) can grow bigger and use nutrients more efficiently compared to the same plants without mycorrizae colonization (Scagel 2005, Yang et al 2002, Zinati et al 2011), very little research has been done to quantify the economic impact of EM in commercial blueberry fields. Commercial EM inoculant products have recently become available. We sought to answer the question, is the cost of buying and using inoculation products justified? We also intended to investigate whether it is practical for a farmer with basic scientific background and equipment to measure mycorrhizae colonization at the farm.

Our research was structured into two basic steps: 1. To verify whether roots became colonized after inoculation compared to roots that we did not inoculate. 2. To determine if bushes with colonized roots yielded more fruit than bushes with less colonization. Throughout the study we tinkered with methods for setting up a “farm lab” and observing Ericoid mycorrhizae. Our goal was to develop mycorrhizae observation methods that are practical for other growers who might want to assess mycorrhizae colonization as a diagnostic tool on their own farms.

Mark Starrett from UVM Plant and Soil Science Department served as the project adviser. Dr. Starrett has studied Ericoid mycorrhizae, and found that many peat-based nursery media products contain Ericoid (Gorman and Starrett 2003). His research implied we might find Ericoid colonization in controls that were not inoculated with commercial product, i.e. Ericoid might be present in the nursery potting mix for all 1000 bushes (treated and controls) that we bought in and outplanted. Our hypothesis was that inoculated bushes would receive a higher dose of inoculant and Ericoid would colonize roots to a greater degree than in the controls.  Our results indicate otherwise, that inoculation had no effect on boosting colonization levels.  Furthermore, some uninoculated bushes scored very high in Ericoid colonization, indicating they already contained Ericoid, likely from nursery potting media, prior to the project.  Ericoid could also be present in our native field soil, but with no Ericaceous plants growing there or in the vicinity for hundreds of years (or perhaps ever), it is highly unlikely that the source of Ericoid was the native field soil.

Many thanks to Alison Brody and Jon Gonzales of the UVM Plant and Soil Science Department for their assistance with developing clearing and staining methods and their overall enthusiasm and encouragement with this project.  

Objectives/Performance Targets

In 2014 we hosted one workshop on the farm, developed mycorrhizae observation methods, collected root samples, collected bush canopy volume data, processed root samples, quantified mycorrhizae colonization and analyzed data.

We hosted a workshop on the farm in August attended by twelve aspiring and current blueberry growers and representatives from UVM Extension and Northeast Organic Farming Association. We didn’t have results to share at the time, but we did cover the topic of on-farm mycorrhizae assessment methods. Growers were introduced to the process involved in clearing and staining roots and learned how to identify Ericoid mycorrhizae under the microscope. We integrated these topics into a broader discussion of best practices for blueberry stand establishment and management.

We developed a low-cost, non-toxic method for clearing, staining and observing Ericoid mycorrhizae on blueberry roots in a “farm lab.” Researchers typically observe mycorrhizae in university labs equipped with autoclaves (devices that supply high pressure and temperature) and a full line of glassware and other specialized, calibrated equipment. However, we believed that while the methods might seem complicated, they can in fact be performed with basic chemicals, glassware and equipment, basic lab skills and locally available materials.

Our farm lab was nothing more than a cleared off workshop table in our welding shop where we stored reagents and glassware, and conducted all experiments on the roots. Our method was adapted from Vierheilig et al. “Ink and Vinegar, a Simple Staining Technique for Arbuscular Mycorrhizae Fungi” (Vierheilig et al. 1998). After various experiments and trial and error, we were able to refine a method using a crock pot as a hot water bath. While it is not perfect, the method worked adequately for clearing, staining and observing over 100 samples in a standardized manner. We will be publishing the farm lab method in a “how-to” booklet with photos to accompany the final report for this project in February 2015.

We collected root samples in early October from all 40 rows of bushes in the study. This was not as easy of a process as we had imagined. Initially we had imagined using a soil probe (3/4” diameter and ~2 feet long) to do four borings around each bush being sampled. Our four-year-old bushes did not have sufficiently developed root systems for this; the borings resulted in too few roots in each sample. The only way we could extract adequate roots was to dig down with a hand fork (three-pronged claw) in several places under the canopy of each sampled bush. We dug down 8” deep until blueberry roots were visible, then clipped a sampling of various roots to make up a composite sample for each sampled bush. We sampled three bushes per row. The three samples per row were taken from low, medium and high vigor bushes based on a brief visual scan of all 25 bushes in each row. Aside from this, we randomly selected bushes to be sampled.

We tagged every bush that we sampled for roots so we could also collect bush canopy volume data. We took two canopy width measurements, on the axis parallel to the row and perpendicular to the row. We averaged these two data points to get an average canopy diameter. We also measured canopy height from the ground to the tip of highest healthy cane. We calculated canopy volume as the volume of a cylinder.

We were not able to compare yields of berries between inoculated and non-inoculated bushes. The bushes, at ~four years of age, were simply still too small. Some varieties did yield a small amount of fruit, but there was not enough for us to confidently weigh yields considering the random pressure we had from wild turkey.

In November we processed 120 root samples in preparation for quantification of root cell colonization. Samples were held in loosely closed plastic bags in field soil in the refrigerator for about 3-4 weeks prior to washing and moving to the farm lab for the chemical procedure and slides preparation.

Most of the 120 samples made it through the clearing and staining and slide preparation steps. Unfortunately we had one or two sample identifications wash off during heating in the hot water bath. A few other slides got cracked due to glycerin sticking to the underside of the slide and in effect gluing them to the slide notebook. These mistakes reflect our skill level as amateur lab scientists and are to be expected in these kinds of procedures.  We look forward to sharing what we have learned to help others avoid our mistakes and common pitfalls in our forthcoming publication at the close of this project in February 2015.

Viewing almost 120 samples under the microscope was tedious but fascinating to observe mycorrhizal structures, hyphae and arbuscules. Aside from collecting data for our comparisons, we also gained a better understanding of how the mycorrhizae tends to associate with blueberry roots.  To quantify samples, slides were randomly toggled on 200X magnification, then scrolled to the nearest feeder root for viewing on 400X magnification to count % of root cells containing arbuscules.  This was done three times for every sample to arrive at a mean % root cell colonization for every sample. 

Attached are pictures of Ericoid mycorrhizae taken with a microscope camera.

• Good shot of Ericoid hyphae surrounding roots
• What a blueberry grower welcomes
• Nicely cleared and stained root, but little colonization

Accomplishments/Milestones

There was no significant difference in Ericoid mycorrhizae colonization levels between treated bushes (inoculated with commercial product) and controls in any of the four blocks of the study. In fact, many controls (not inoculated with commercial inoculant) exhibited very high colonization levels. Our data are attached.

Based on these results and our doubt that Ericoid was present in native field soil prior to planting blueberry bushes, we believe Ericoid mycorrhizae were present in the potting media from the 1000 bushes we bought in from a nursery in Michigan. This would be consistent with results from Starrett and Gorman, who found Ericoid in the majority of peat-based potting media tested in their study (Gorman and Starrett, 2003). We hypothesized that inoculating with additional commercially available inoculant at the time of outplanting would significantly increase Ericoid colonization levels, but our results indicate otherwise.

It was not a stated objective of our study to confirm a relationship between mycorrhizae colonization levels (regardless of inoculation method) and bush vigor in the field. However, it was very easy to tag bushes and take a quick canopy volume measurement while sampling roots, hence we decided to see what we could find with data analysis.  Also, bush vigor can be regarded as a general proxy for future fruit yield (although small bushes can certainly yield more in some cases than larger bushes). We observed very weak correlation between Ericoid colonization levels and bush canopy volume in some varieties (Nelson and Bonus), but weak inverse correlation for other varieties (Bluejay). Additional data is needed to corroborate these results.

Any analysis of the relationship between mycorrhizae colonization and bush characteristics (biomass, yield, flowering characteristics) is incomplete without standardizing for soil variables.  We know that biomass, flowering and yield are influenced by the chemical and physical characteristics of soils.  When examining how soil biology, such as mycorrhizae, effects bush characteristics, it will be difficult to affirm any direct cause and effect unless soil chemical and physical characteristics are constant across all treatments or samples.  Moreover, various studies confirm that the ability of arbuscular mycorrhizae to function depends on baseline soil conditions, such as pH, nutrient availability and composition of soil organic matter.  Any analysis of the effectiveness of mycorrhizal colonization practices across treatments is incomplete without first confirming that soil conditions are conducive to such practices.     

While we did take baseline soil tests for all four blocks in the study and confirmed no difference in soil pH, we did not have the capacity to do a detailed account for all variables — or even baseline soil measurements for every bush sampled for roots– in our study.

We assumed going into our study that we did not have significant variation in baseline soil pH, nutrient availability and organic matter composition across treatments within the same block in the field. After completing this study, we have changed our minds.  We think it is not safe to assume, for purposes of assessing mycorrhizal activity, that baseline soil conditions are constant across rows in a block or up and down a row in a field.

Our bushes over the three-season course of this project exhibited noticeable variation in growth all over the field. These were subtle variations, but lead us to believe there was significant variation throughout the field in soil conditions.  Up and down the same row, regardless of inoculation treatment, some bushes grew very vigorously while some grew very slowly. We kept to a strict standard throughout the study of conducting management identically across all bushes in a row and row by row in a block, so there must be another explanation for the variation in bush characteristics.

What explains the variability in growth of our bushes? It’s possible that the genetics of bushes vary within the same variety. It is possible that during the 2 years bushes were propagated and managed in the nursery prior to arriving at our farm, there were differences in how each bush was managed, giving some bushes a head start and holding some bushes back. Aside from this we can only look to our field’s soils. We suspect there is significant variability of soil indicators within a row or across treatment rows. We would not even be surprised if there is significant variability in soil variables in the root zone of a single bush. If this is true, theoretically one root sample from one side of a bush’s root zone could display an entirely different level of Ericoid mycorrhizae root cell colonization than another root sample from another side of the root zone.

Theoretically, two bushes of the same variety, planted the same way, inoculated or not inoculated in the same manner and managed in the same way could display different levels of Ericoid colonization if soil conditions vary enough across or within root zones to alter Ericoid colonization and function. We suspect the variability in field soil factors explains the extremely wide range of Ericoid colonization levels we observed in bushes in all blocks and treatments, regardless of whether or not bushes were inoculated.

In summary, our project revealed more questions than answers. An important research question is to what degree do soil factors, such as pH, nutrient availability and organic matter composition (both % and type of organic matter) vary from one rhizosphere to another within close areas of proximity in an average farm field or commercial blueberry stand? Another important research question is to what degree will variability of each of these soil factors effect Ericoid colonization and function for the benefit of the blueberry bush and producer?  Another way to phrase this is: What was it about the soil conditions in the rhizosphere that explain why we found such high colonization levels in some roots and such low colonization levels in others, regardless of inoculation practices?  Then the obvious question is what can a producer do to manage rhizospheres in a particular way to foster symbiotic associations between Ericoid mycorrhizae and blueberry bushes? We suspect it is not enough for a blueberry producer to simply inoculate roots with Ericoid mycorrhizae or confirm that inoculant is present in nursery or field soil. Soil around every root and under every bush needs to be right for the mycorrhizae to function optimally.

Finally, we can report a few results from our trials with comparing and developing appropriate Ericoid observation methods. We tested a variety of methods for clearing and staining Ericoid. We will save the details for a publication that will accompany the final report for the project, but here are some basic lessons learned:

• Clearing in 10% KOH works much better than 15% KOH, which can be too powerful and destroys the roots.
• We tested many different time periods for clearing in KOH to achieve adequate clearing while not destroying roots from oversoaking. We tested 12 hours, 24 hours, 2 days, 3 days, 4 days and one week. We also tested various temperature regimes. We tested at room temperature, “low” setting and “high” setting on a crock pot hot water bath. We found that 24 hours of clearing in a hot water bath on “high” setting in the crock pot was optimum.
• We tested the same variables of time and temperature with staining roots. We found that a 12 hour stain on “high” setting in the crock pot hot water bath was optimum.
• Ink and vinegar is a safe, non-toxic stain that can be used, but it must be mixed fresh before each staining.

References:

Gorman NR, Starrett MC. 2003. Screening Commercial Peat and Peat-based Products for
the Presence of Ericoid Mycorrhizae. J. Environ. Hort. 21(1):30–33.

Scagel CF. 2005. Inoculation with Ericoid Mycorrhizal Fungi Alters Fertilizer Use of Highbush Blueberry Cultivars. HortScience 40(3):786-794.

Vierheilig H, Coughlan AP, Wyss U, and Piche Y. 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Applied and Environmental Microbiology 64: 5004–5007.

Yang WQ, Goulart BL, Demchak K, Li YD . 2002. Interactive effects of mycorrhizal inoculation and organic soil amendments on nitrogen acquisition and growth of highbush blueberry. J. Amer. Soc. Hort. Sci. 127:742-748.

Zinati G, Dighton J, Both A. 2011. Fertilizer, Irrigation, and Natural Ericaceous Root and Soil Inoculum (NERS): Effects on Container-grown Ericaceous Nursery Crop Biomass, Tissue Nutrient Concentration, and Leachate Nutrient Quality. Hortsci. 46(5):799-807.

• % root cell colonization data

Collaborators: