Progress report for LNE23-472R
Project Information
Orchardists increasingly plant orchards using dwarf trees with limited root systems. Roots are injured when trees are dug at the nursery and subsequently planted. Rising land costs and the need to return land to production reduces growers’ abilities to rotate orchard land into cover crops for more than a season. This lack of rotation increases the risk of concentrating apple pathogens in the soil. Many growers establish orchards with irrigation and regular fertilization, but these approaches are costly, requiring synthetic or high levels of organic fertilizers, and large quantities of water in dry years.
Many fungi species form associations with apple roots, and there are commercially available formulations that growers can apply to the roots at planting. Previous research found trees inoculated with fungi showed improved survival, growth, and nutrient uptake, and may make trees more resilient to drought. However, few trials have been conducted in commercial northeast orchards.
We will investigate if the addition of commercial mycorrhizal products to newly planted apple orchards, under varying levels of soil phosphorus, improves tree growth, survival, nutrient uptake, and water use efficiency, compared to naturally occurring fungi under various soil, climate, and management conditions found in the northeast. We will assess if fungal colonization, and therefore any subsequent benefits to tree growth, survival, nutrient uptake, and water use efficiency, vary between different apple varieties and rootstocks.
Three research sites will be conducted on commercial orchards in New York, New Hampshire, and New Jersey. We will inoculate 50 trees each with four commercial fungi products, with 50 trees serving as a control. During each of the three years, we will collect data on tree growth, survival, root colonization, root microbial community distributions, nutrient uptake, and water use. Two greenhouse studies will also be conducted. One study will determine the effects mycorrhizae have on drought stress on two varieties and four rootstocks. The second study will look at the effects mycorrhizae and three levels of P fertilization have on nutrient uptake.
A survey conducted in September 2022 showed grower interest in this work. When asked “Would you be interested in using mycorrhizal fungi when planting your new orchards/nurseries?”, 20 responded “Yes”, 1 responded “No”, and 32 responded “Yes, if there was more research documenting clear benefits”. This shows growers are interested in incorporating these materials into their plantings, but more data are needed before they implement these materials.
Growers will play key roles in this project. Field research is taking place at commercial orchards. Growers will apply treatments, maintain sites, and host field meetings. They will play key roles in developing outreach materials. Our advisory committee includes five commercial growers, who will be providing input throughout the course of the project through bi-annual meetings.
While the general benefits of mycorrhizal fungi have previously been documented, more data is needed from field trials on commercial farms in the northeast before growers can be confident in applying these materials on their farms. The knowledge generated from our multi-state on-farm field trials and greenhouse work will provide growers the additional data needed to determine if inoculating trees prior to planting their orchard will provide tangible benefits to their commercial plantings. Positive results may allow them to decrease their fertilizer and irrigation inputs when establishing their future plantings while improving orchard productivity and profitability.
Orchardists increasingly plant orchards using dwarf trees with limited root systems. Roots are injured when trees are dug at the nursery and subsequently planted. Rising land costs and the need to return land to production reduces growers’ abilities to rotate orchard land into cover crops for more than a season. This lack of rotation increases the risk of concentrating apple pathogens in the soil. Many growers establish orchards with irrigation and regular fertilization, but these approaches are costly, requiring synthetic or high levels of organic fertilizers, and large quantities of water in dry years.
Many species of mycorrhizal fungi form symbiotic associations with apple roots. In exchange for carbohydrates, fungi greatly increase the surface area and absorptive capacity of the tree roots. One genus of fungi commonly studied in apple is Glomus, which has been found to form very strong associations with apple roots (Reich, 1988). There are commercially available blends of these Glomus fungi that growers can apply directly to the tree roots when planting the orchard.
Previous studies have found trees inoculated with these fungi showed improved growth and increased nutrient uptake compared to non-inoculated trees (Reich, 1988; Bokszczanin et al. 2021). These studies suggest trees inoculated with fungi may require less nutrient inputs during the establishment phase. High levels of soil phosphorous may also inhibit fungal growth (Van Geel et al, 2015), however the effects of soil phosphorous on the root colonization and subsequent benefits appears inconsistent in the literature, and it is unclear what the effects of pre-plant P incorporation may be on the fungi in northeastern orchards (Miller et al., 1985; Morin et al., 1994). Trees inoculated with these fungi showed improved root development and increased drought tolerance (Huang et al., 2020). This suggests trees inoculated with these materials may be more efficient in their water use, requiring less water during the establishment phase. An unpublished field trial from Pennsylvania found tree survival in a new orchard was improved in trees inoculated with a fungi blend (Racsko, personal communication). These studies suggest mycorrhizal fungi inoculations may improve the survival and productivity of newly established orchards.
Apples are grown on approximately 95,000 acres across the Northeast. While New York, Pennsylvania, and Virginia are large producers, every Northeast state has commercial apple orchards. Growers replace about 5% of their orchards on an annual basis, representing roughly 4,750 acres per year. Growers we discussed this work with are interested in growing healthier trees with fewer inputs, and see these inoculants as an opportunity to improve their tree heath, soil health, and overall farm productivity and sustainability.
Inoculating tree roots with beneficial fungi at orchard planting may allow farmers to establish healthier, more productive trees with fewer fertilizer and irrigation inputs. This would allow them to increase their sustainability and resilience in the long-term. This is particularly critical with climate change and the projected increases in the frequency and severity of droughts across the northeast, as we observed throughout much of the northeast in 2022.
Research
Will the addition of commercial mycorrhizal fungi products to newly planted apple orchards, under varying levels of soil phosphorus, improve tree growth, survival, nutrient uptake, and water use efficiency, compared to naturally occurring fungi under the various soil, climate, and management conditions found in northeast orchards?
Does fungal colonization, and therefore any subsequent benefits to tree growth, survival, nutrient uptake, and water use efficiency, vary between different apple varieties and rootstocks?
Orchard Field Trials
We will establish three orchard field trials to determine if four commercial mycorrhizal products improve root colonization rates, tree survival, tree growth, nutrient uptake, and water uptake under two soil phosphorous levels.
Field sites will be hosted at the following locations, to represent a diversity of site and management conditions from across the northeast.
- Northern Orchards in Peru, NY, Barnsby Pink Lady on M.9 Nic 29 Rootstock, Schroon Fine Sandy Loam, Replant Site
- Apple Hill Farm, Concord, NH, Premier Honeycrisp on G.935 Rootstock, Canterbury Fine Sandy Loam, Replant Site
- Melick's Town Farm in Califon, NJ, Evercrisp on G.935 Rootstock, Washington Loam, Virgin Ground
Treatments: Trees will be treated with one of four mycorrhizal fungi treatments (Mycoapply, MycoBloom, Mykos Gold, BioOrganics) and an uninoculated control). Trees will be treated with one of two levels of phosphorous fertilizer (0 pounds per acre, 200 pounds per acre).
Methods: 3 trees will make up the fungi x P application treatments experimental unit, replicated 5 times down the row in a completely randomized block design for a total of 150 trees per planting. We will apply each mycorrhizal treatment as a root dip or broadcast directly to the roots, as per the manufactures’ recommendation. We will apply P by dissolving triple superphosphate in water, and then applying a gallon of material in a ring around each of the P treatment trees shortly after the trees have settled following planting.
Data Collection:
- Tree growth and survival: at planting, we will measure trunk circumference of each tree at 30cm above the graft union. We will repeat these measurements in October of 2023 through 2025. We will then convert measurements to cumulative annual growth. At each October measurement date, we will rate each tree as alive (0) or dead (1).
- Root Colonization: in years 1 and 3 of the project, fine (absorptive) roots will be collected from one tree per treatment and preserved in 50% ethanol prior to staining and evaluation. At the laboratory, roots will be stained with trypan blue, stored in acidic glycerol for 72 hours, and installed on microscope slides. To increase throughput, we plan to utilize the newly available AMFinder software (Evangelisti et al., 2021) to identify AMF colonization parameters correlated with increased plant performance (e.g., arbuscules, hyphae, vesicles, and root length colonization). If the AMFinder method fails, we will quantify the percentage of roots colonized by AMF with a compound microscope using the intersection method (McGonigle et al., 1990).
- Soil Community Profiles: To understand how commercial mycorrhizae formulations impact soil microbial community composition, we will collect one soil sample from each field trial prior to planting, and 10 samples (one from each fungi x P treatment) in July of the third year of the trial. Samples will be sent to the Missouri soil health lab for phospholipid fatty acid analysis (PFLA). PLFA analysis is used to quantify total microbial biomass and shifts in microbial community structure by differentiating microbial groups (Gram Positive; Gram Negative; Anaerobe; Actinomycetes; Methanobacter; Fungi; and AMF) to provide estimates of microbial biomass for each group (Frostegard and Baath, 1996).
- Water Use Efficiency: To assess water use efficiency, we will take carbon isotope samples from each treatment replicate around June 15th and October 15th of every year of the study. Three leaves from each treatment replicate will be dried, ground, and submitted to the Cornell Stable Isotope Lab for analysis. Results will give us the concentrations of different carbon isotopes within the plant, which correlates well to seasonal water use efficiency within the plant (Farquhar et al., 1989).
- Nutrient Uptake: In late July of years 2 and 3 of the project, we will collect foliar samples from each treatment replicate for nutrient analysis. Nutrient analysis will be performed by the Cornell Nutrient Analysis Laboratory in Ithaca, NY.
Data Analysis and Presentation of Results: Tree growth, root colonization, water stress, and nutrient uptake data will be analyzed through JMP using the fit model function for each field site. Tree survival data will be analyzed through SAS using the GLIMMIX function. Our analysis will be presented in both tabular form, and through appropriate graphs organized by treatment. All results will be formatted and prepared for both extension style and peer reviewed journal format.
Greenhouse Trials
The benefits of mycorrhizal fungi on young apple trees and the interaction between rootstocks, scions, and abiotic stresses will be detailed through complimentary greenhouse experiments. Grafted, potted trees will be grown in the greenhouse for 3.5-4 months at 24°C with ambient light supplemented using LED lights with a 12-h photoperiod. Pots will be watered to full saturation every 3 days without fertilizer addition. Standard pesticide applications will be applied. Physiological and plant performance traits will be measured throughout as detailed below.
Treatments: Experiment 1 will evaluate the effect of fungi type, soil type, variety, rootstock, and drought treatment on tree growth and physiological performance. Experiment 2 will evaluate the effect of fungi type, phosphorus availability, and rootstock on tree growth and physiological performance.
Methods: Experimental design for experiment 1 is 5 replicate trees x 4 rootstocks (B.9, M.9, G.11, and G.41) x 2 scions (Honeycrisp, Gala) x 4 fungi treatments (no fungi control, 3 commercial products) x 2 water treatments (Control, Drought). Trees will be grown under full watered conditions for 2.5-3 months. Then trees will be exposed to a simulated drought treatment of 14 days with no irrigation. Physiology measures will be collected prior to (every week) and throughout (daily) the drought stress treatment. On day 15, all trees will be fully watered to allow recovery. Growth resumption and recovery metrics will be monitored for an additional 2 weeks before terminating the experiment.
Experimental design for experiment 2 is 5 replicate trees x 1 scion (Honeycrisp) x 4 rootstocks (B.9, M.9, G.11, and G.41) x 4 fungi treatments (no fungi control, 3 commercial products) x 3 P-availability types (None, low: 12 mg/kg, high: 50 mg/kg). Trees will be grown under full watered conditions with no additional fertilizer besides P additions for 2.5-3 months.
Data Collection: Physiological measurements will track growth rate, stomatal conductance, photosynthetic rate, and water potential. Leaf samples will be taken from experiment 1 at the conclusion of the experiment to determine water use efficiency differences through carbon isotope analysis. From experiment 2, leaf ion analysis will be conducted with the idea that fungi inoculations will alter the pattern and concentration of ions. At the end of the study, all potted trees will be destructively sampled to measure root, trunk, branch, and leaf dry matter accumulation. Trunk diameter increment will be calculated by deducting the trunk diameter at the end of the experiment from the initial trunk measurement taken 5 inches above the grafted union. Fine root tissue will be harvested from all experimental trees in both experiments and processed for AMF quantification.
Data Analysis and Presentation of Results: Data will be examined for treatment factors using MANOVA and linear regression methods for single factors and all interaction effects. Analysis will be conducted in the R programming language and results presented as graphics and table formats. All results will be formatted and prepared for both extension style and peer reviewed journal format.
2023 Activities
Field Trials: In 2023, we successfully established our three field research sites at three commercial orchards:
- Northern Orchard in Peru, NY, Barnsby Pink Lady on M.9 Nic 29 Rootstock, Schroon Fine Sandy Loam, Replant Site
- Apple Hill Farm, Concord, NH, Premier Honeycrisp on G.935 Rootstock, Canterbury Fine Sandy Loam, Replant Site
- Riamede Farm in Chester, NJ, Golden Delicious on Bud 9 Rootstock, Parker Gravely Sandy Loam, Replant Site
Prior to planting, soil samples were collected from each field site. Soil samples were analyzed by the University of Missouri Soil Health Assessment Center for Phospholipid Fatty Acid Analysis to estimate the biomass of key microbial functional groups within the soil, and estimated relative percentages of these key groups, including AMF. (See results section for details).
At planting, five mycorrhizal treatments were applied to the trees within the new orchard plantings (1. Symvado, 2. MycoBloom, 3. Mykos Gold, 4. Promate 5. Control). All mycorrhizal treatments were applied at label recommended rates, and were applied according to the manufacturers suggestions for newly planted tree fruit. In NY, trees were planted with a furrow tree planter. Trees were planted into pre dug holes at the NJ and NH field sites.
A few weeks following orchard planting, trees at the NY and NJ field sites also received one of two fertilizer treatments (1. High P and N, 2. Low P and N). Rates were determined based on soil tests taken by the farmers in the previous season. We used polyphosphate and Urea Ammonium Nitrate (UAN) as our fertilizers, as they were both liquid based fertilizers that could quickly make their way to the root zone. Fertilizers were applied by hand, in 1 gallon of water per tree applied directly to the herbicide strip. The NH field site received the fertility treatments of (1. Lime and 2. Control) prior to planting. The rate was determined by the amount of lime required to raise the soil pH of the site to 7.0. We did not adjust P at this site, as the soil test determined that this site was already in the correct range for P.
All treatments were managed similarly, according to each of the growers standard management practices for first year trees. Trees at the NJ field site were irrigated shortly after planting in May. No irrigation occurred at the NY or NH field site.
Shortly after planting, tree circumferences were measured at 30cm above the graft union. The same measurements were done in late October, along with an assessment of tree survival. Growth and survival data were analyzed using the Fit Model feature of JMP Statistical Software. (See results section for details).
In July and October, we collected two leaves from each tree to conduct carbon isotope analysis to estimate tree water use efficiency between treatments. Leaves are currently in line to be analyzed by the Cornell Stable Isotope Lab in Ithaca, NY.
In August, root samples were collected from each treatment using a soil corer near the base of each tree, and were stored in ethanol to be stained for future colonization assessments. Colonization estimates are currently ongoing by the Londo lab in Geneva NY, and we expect to have full 2023 results back by June 2024.
Greenhouse Trials:
In year 1, our experimental objective was to test the potential interaction effects of AMF inoculum on apple growth and development. Specifically, our task was to evaluate the impact of different rootstock-scion combinations, different AMF treatments, and different stress treatments with the goal of identifying optimized combinations for stress adaptation. Initially, we proposed to study stomatal function and photosynthetic response, biomass changes, and root colonization metrics as evidence of differential effects. The experiment design thus included 5 biological replicate trees x 3 stress treatments (Control, Drought, Low Phosphorus) x 4 AMF treatments (None, MycoApply, MykoBloom, MykosGold) x 2 scions (Honeycrisp, Aztec Fuji) x 4 rootstocks (B.9, M.9, G.41, G.935) for a total of 480 trees.
Several factors have impacted the initial experiment. One was the late start of the grant in the tree availability season. By the time funds were available, tree selection was minimal. As a result, the trees ordered for the experiment were much larger than anticipated, arriving not as 2-3 foot starts, but as 6ft finished and feathered trees. The first modification to the study was to increase the pot size to accommodate the large trees, and changing from a standard greenhouse to a large polyhouse where the trees could be kept on the floor. While this change facilitated working with the large
trees, it also introduced a lower light level due to the use of shade netting to help keep climate control in the polyhouse.
The experiment was initiated on May 15th where all pots were inoculated with AMF treatments and randomized within the polyhouse and all trunk diameters were recorded with calipers, 6 inches above the graft union. Licor measurements of stomatal conductance and photosynthetic efficiency were taken on June 5th and 6th . Drought and Low phosphorus treatments were initiated July 7th (Low P) and July 10th (Drought). Carbon isotope samples were collected from all trees from control and drought treatments on July 10th . Wilting was observed in drought treatments on July 19th and water volumes were adjusted to compensate for the increasing temperatures in the polyhouse. Carbon isotope samples were collected again on August 8th . Drought treatments were concluded on August 25th (7 weeks of treatment) and trunk diameters were remeasured. The experiment was terminated on September 18th as pest pressure had reached a significant level in the polyhouse. Root tissue was sampled from all trees for AMF quantification on October 2nd . On October 11th , “healthy” trees were transplanted to the field in a randomized design to track tree performance under field conditions in subsequent years. Due to loss of some replicates of rootstock-scion combinations, the field trees represented a balanced design of 400 trees, rather than the full 480 trees.
Data analysis is underway for stomatal conductance and photosynthetic efficiency metrics.
Leaf samples for carbon isotope were collected to examine water use differences between the AMF treatments in control and drought treatments. Those samples have been dried, powdered, and are waiting for analysis at the Cornell COIL facility. The data analysis queue is currently quite long due to repair and personnel issues at COIL. Root staining and analysis of AMF metrics are ongoing. Staining duration is being optimized, initial attempts have identified some instances of AMF vesicles and hyphae.
Our approach going forward is going to change slightly from the original design. The results of the large tree polyhouse experiment were inconclusive. We believe this is in part due to the reduced light and climate control in the polyhouse. We clearly observed plant stress in drought and low Phosphorus treatments, but these observations were not born out in any of the measured metrics. Our plan is to rerun this experiment in year 2 by purchasing smaller, more manageable sized trees and conducting the experiment in greenhouses. In year 2 we are planning to do rooted rootstocks and will verify that AMF colonization has occurred. Then we plan to bud graft scion wood onto the rootstocks for year 3 assessments of scion performance.
Field Studies
Tree Survival
First year tree survival was assessed in October 2023. Trees were rated either alive (1) or dead (0). All trees survived in NY. In NH, one tree was lost in the Symvado x no lime treatment. In NJ, one tree each was lost in the Mykos Gold x low fert treatment, 1 tree was lost in the Mykos Gold x high fert treatment, and 1 tree was lost in the Symvado x low fert treatment. Neither mycorrhizal nor fertilizer treatment had a significant impact on tree survival at any of our sites in 2023.
Tree Growth
Tree growth was measured in May and October 2023, and the cumulative tree growth between the different mycorrhizal and fertility treatments are presented in Table 1. Treatment differences were assessed using the Fit model feature on JMP Statistical Software. Dead trees were removed from the growth analysis.
Table 1. 2023 cumulative tree growth by fungi and fertility treatment at our NY, NJ, and NH field sites. Treatments with differing letters significantly differ (p=.05) according to Tukey's HSD test.
| 2023 Field Trial Trunk Circumference Growth (cm) | ||||
| NY | NJ | NH | ||
| Fungi | Symvado | 0.6 B | 0.9 | 1.4 |
| Mycobloom | 0.7 AB | 0.9 | 1.5 | |
| Mykos Gold | 0.6 B | 0.9 | 1.5 | |
| Promate | 0.9 A | 1.0 | 1.5 | |
| Control | 0.7 AB | 1.0 | 1.4 | |
| P-value | 0.00382 | 0.6076 | 0.87525 | |
| Fertility | Low | 0.73 | 1.0 | 1.5 |
| High | 0.67 | 0.9 | 1.4 | |
| P-Value | 0.1382 | 0.2512 | 0.7481 | |
| Fungi x Fertility | P-Value | 0.6361 | 0.1098 | 0.4124 |
Soil PLFA Analysis
Pre-plant soil Phospholipid fatty acid analysis provided us estimates of the initial amounts and ratio of mycorrhizal fungi present at each of our three field sites prior to the incorporation of commercial fungi products. Please note that this test is only a rough estimate, as it does not test for exact numbers.
In NY, NH, and NJ, total phospholipid fatty acid content (pmols/g) was analyzed from soil samples from each field site. The NY, NH, and NJ field sites contained approximately 75947, 220211, and 139731 pmols of PLFA per gram of soil, respectively.
The estimated percentage of AM Fungi associated with the PLFA extracted from the soil samples analyzed for our NY, NH, and NJ field sites were 6, 5, and 6% respectively. These estimates were not from exact counts, but rather from the analysis of different fatty acids present within each soil sample. Other microbial functional groups evaluated in this test included gram negative bacteria, methanotrophs, eukaryotes, non-AM fungi, gram positive bacteria, and actinomycetes. We are curious to see how our commercial fungi blends will impact these percentages over the next two years, so this data simply serves as our "baseline" prior to orchard planting.
Greenhouse Studies
Stomatal Conductance and Photosynthetic Efficiency
Initial interpretation does not suggest that AMF treatments had any meaningful impacts on these performance metrics, with the large caveat that the movement of the experiment to the polyhouse resulted in a much lower light level than is optimal. As a result, physiological measurements were very close to the baseline. Differences could be seen between scions in these traits, and both stomatal conductance and photosynthesis was lower in drought and low phosphorus treatments.
Trunk Diameter
Trunk diameter measures across the experiment were similarly underwhelming. No significant effect of AMF treatment, or stress treatment, were detected. Significant effects of scion and rootstock were detected, with Aztec Fuji demonstrating increased growth on G.935 relative to Honeycrisp, and the opposite response observed on M.9.
Root Colonization
Most samples examined to date do not have strong indication of AMF colonization, either from the greenhouse experiment or from field collaborators in NH, NJ, and NY. It is possible that colonization under these conditions is taking longer than anticipated. Our plan is to continue processing root samples for the first year of the study so we can compare with the 2nd and 3rd years of collection.
