Effects of ericoid mycorrhizal fungi on performance of V. macrocarpon and V. oxycoccos under abiotic stresses related to climate change

Final report for GNC20-302

Project Type: Graduate Student
Funds awarded in 2020: $14,879.00
Projected End Date: 12/31/2023
Grant Recipients: University of Wisconsin Madison; The Board of Regents of the University of Wisconsin System Research and Sponsored Programs
Region: North Central
State: Wisconsin
Graduate Student:
Faculty Advisor:
Dr. Amaya Atucha
University of Wisconsin Madison
Faculty Advisor:
Dr. Juan Zalapa
University of Wisconsin-Madison, Department of Horticulture
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Project Information


This study, “Effects of Ericoid mycorrhizal fungi (ErMF) on performance of V. macrocarpon under abiotic stresses related to climate change & assessment of ErMF diversity in cultivated and wild cranberry settings,” endeavors not only to encourage sustainable agricultural practices but to expand stress tolerance of cranberry (Vaccinium macrocarpon Ait.), thereby easing challenges faced by growers. Cranberry growers are under increasing pressure from climate effects, which impacts cranberry production through repeated cycles of water and heat stress. These climatic events are well-established and predicted to intensify. Cranberry vines have evolved in a unique bog ecosystem  and are highly susceptible to water stress. With the aim to ameliorate the effects of these abiotic stressors and reduce the negative effects on yield and fruit size, growers increase the frequency and quantity of fertilizer and irrigation. However, these practices have raised concern regarding impacts on water quality of the surrounding wetlands with potentially limited benefit on plant performance. Multiple studies conducted in blueberry have shown that inoculation with ErMF have a reductive effect of abiotic stressors, improving plant vigor. ErMF are ubiquitous, with local strains already present and active in agricultural soils, such as the cranberry production system. A comparison of ErMF strains in commercial cranberry farms to those in association in wild cranberry (which grow under more stressful conditions than cultivated), might provide opportunities for selection of strains that could be used for inoculation in commercial production settings, with the potential to provide stress mitigation benefits. In addition, the role of ericoid mycorrhizae in nitrogen (N) uptake in cranberry production is well known. However, the potential role ErMF play in phosphorus (P) uptake is not well understood. Although cranberries are not heavily fertilized with P, contamination from P agricultural runoff poses risk to the quality of surrounding waterways. Inoculation with ErMF may stretch cranberry’s range of tolerance, allowing growers to lessen water and fertilizer usage in response to climate change impacts.

Project Objectives:

The objectives of this study are: (1) to evaluate the ErMF biodiversity in commercially cultivated cranberry farms and wild cranberry bogs across Wisconsin; (2) establish the role of ErMF on organic P uptake by cranberry vines; and (3) evaluate if cranberry vines inoculated with ErMF have higher tolerance to water stress.


Materials and methods:

Objective 1: Evaluation of ErMF biodiversity in commercially cultivated and wild cranberry sites across Wisconsin

In order to establish a baseline for the diversity of ErMF in both cultivated cranberry beds and wild bogs, in fall 2020, we sampled soil cores from five commercial cranberry marshes of varied soil conditions and textures across the state of Wisconsin. From these soil core samples, we surface-sterilized cranberry roots and plated select segments onto agar. This provided us with fungal growth from the inside of the cranberry root, where ErMF and other endophytes reside. This process resulted in 76 fungal isolates, 71 of which survived the isolation process. We performed DNA extractions on these isolates, and initial Sanger sequencing data has indicated several candidates for ErMF status. All isolates were transferred into long term storage for any future use. In 2022, we finalized protocols to sample soil and root tissue from wild and commercially cultivated locations (five commercial grower sites and seven wild locations), as well as their post-collection processing in the lab. With grower cooperation and after obtaining permits from the Wisconsin Department of Natural Resources to collect plant tissue samples from sites with recorded wild cranberry plants, we collected cranberry root samples from all five commercial grower sites and seven wild locations. Processing samples included an intensive cleaning system, staining, and microscopy to verify root colonization. Roots were cleaned of debris via submersion in DI water, with gentle agitation and forceps to loosen entanglements of the root system. Because ErMF prefer to colonize first and second order roots, those were preferentially selected for surface sterilization in a bleach solution, lightly dried, and stored at -80 °C until all samples had been processed and stored. All samples were then simultaneously freeze dried, to preserve genetic material for sequencing. Portions of the samples were then weighed out and prepared for shipping and sent to a sequencing company for analysis of the entire fungal genome inside the cranberry root tissue. The metagenomic sequencing results are under ongoing analysis with publications forthcoming. 

Objective 2: Establish the role of ericoid mycorrhizal fungi (ErMF) on organic Phosphorus uptake

Fungal mediation of P in isolation: To verify the ability of our fungal strain ATCC 32985 to break down organic P independent of a plant host, we employed a Phytase Screening Media (PSM) as an indicator of organic P processing. This agar media recipe incorporates organic P in the form of phytin; if the fungal tissue culture can produce the proper enzyme to break it down (phytase), the petri plate will exhibit a clear visual indicator of success in the form of a “clearing zone” of transparency in the agar surrounding the fungal tissue. 

Fungal mediation of P in association: To assess the impact of ErMF inoculation on cranberry’s P uptake, we also developed a system of axenic root growth in cranberry cuttings using hydroponics. Cranberry cuttings were made from runner tissue (lateral vegetative growth), surface sterilized, and trimmed of excess leaf tissue. The tops of the cuttings were coated in petroleum jelly to minimize transpiration. Sets of 40 cuttings were suspended in 10 L tubs of microfiltered water via foam holders. Each tub was supplemented with an oxygen pump and weekly maintenance of water changes and added macro- and micronutrients. The hydroponic system was kept at an ambient temperature of approximately 29.4 °C to encourage growth. We typically saw cuttings begin to root within one week and ready for nutrient experiments by day 18.

For the inoculation and nutrient experimentation, we also designed and implemented a hydroponic “pod” system to quickly inoculate rooted cranberry cuttings with ATCC 32985. The system encourages connection of developing roots and mycelia by maintaining close proximity and minimizing disruption, without the need to remove the plant from a hydroponic environment. Upon root development in the hydroponics system described previously, cuttings were transferred into treatment group pods and inoculated with mycelial tissue from liquid culture of ATCC 32985. Colonization took approximately six days to occur after inoculation and confirmed visually as well as through staining and microscopy.

We calculated the chemistry for various levels of organic and inorganic P input in the aqueous nutrient supplements injected into the hydroponic system, using aqueous phytic acid and a solution of KH2PO4. This allowed us to compare uptake of organic and inorganic P in inoculated and non-inoculated cranberry plants based on equivalent P molecules available across the different nutrient treatments. We employed a Malachite Green Phosphate Assay kit to assess the phosphate content in old and newly generated plant tissues from each treatment group, a necessary comparison due to high mobility of phosphorus within plant tissue. 

Objective 3: Assessment of ErMF’s impact on water stress response in cranberry plants

Plant culture: Our third objective investigates how inoculation with ErMF affects drought stress responses in cranberry plants. In January of 2022, we established the protocol for growing plants in solid media using an autoclaved 50/50 peat and perlite mixture, and rooting hormone. A humidity tent was constructed in the greenhouse and approximately 1000 cranberry plants were grown for use in our drought testing. The same liquid inoculum of ATCC 32985 was used to inoculate our greenhouse plants, and once again we confirmed colonization with staining and microscopy. Plants were wrapped in thin plastic at the base of the stem, exposing only the shoot tissue to the environment; this step limits transpiration to the leaf and shoot tissue, and plants do not lose water from the soil surface. A plastic tube was fitted in the pots while packing to facilitate water addition. Initial transpiration data was used to assign pots to their treatments. Two watering treatments were implemented: severe stress (SS) and well-watered (WW) conditions with 10 replicates each, 5 mycorrhizal (ErM) and 5 non-mycorrhizal (NM) per watering treatment. This resulted in four treatment groups: SS-ErM (Stressed with ErM inoculation), SS-NM (Stressed with no inoculation), WW-ErM (Well-watered with ErM inoculation), and WW-NM (Well-watered with no inoculation). SS plants received no additional water over the course of the study, and WW plants were watered daily at 80% replenishment of the water they transpired. Plants were assigned to a treatment at the time of treatment imposition, based on their transpiration rates in the 24-hour period prior to the study’s start, so that plants having similar transpiration could be allocated equally in each treatment. Plant weights were collected daily in the evening around the same time and used to calculate that day’s transpiration, water to add that evening in the WW condition, and all plants’ transpiration rates. Greenhouse conditions were maintained with a temperature of approximately 29.4 °C and 16 hours of supplemental overhead lighting.

Chlorophyll fluorescence: Once plants were established in the drought experiment, both light and dark adapted leaf chlorophyll fluorometric data were collected using the Opti-Sciences PSK “Plant Stress Kit”, which includes both the Y(II) meter and the FV/FM meter to evaluate photosynthetic performance in the plants. This data was collected weekly, and then daily once the plants began to exhibit symptoms of stress. 

Osmotic potential: We also collected leaf tissue samples from all plants to assess osmotic potential in the leaves as a proxy for the plant’s water potential. This helps us to understand how plants in various treatments adjust their cellular water retention and solute concentration, which are critical factors in their ability to tolerate drought conditions through maintaining hydration and nutrient uptake under water stress. Samples of 6 mm tissue discs were collected from mature, established leaves on a plant’s oldest available runner tissue. All collected leaves had a minimum width of 6 mm to accommodate the tissue punch. Samples were stored immediately upon cutting in a -80 °C freezer, and then analyzed with a VAPRO Model 5600 Vapor Pressure Osmometer under a 10-minute equilibration. 

Soil aggregation: Mycorrhizal fungi tend to play a crucial role in improving soil structure and stability, which can affect the resilience of plant communities to drought by enhancing water retention and nutrient availability in the soil. In order to assess soil structure and stability, at the conclusion of the study, all plants were left to dry out for 10 days for a rehydration slake test to assess soil aggregation across treatments. 

Research results and discussion:

Objective 1: Evaluation of ErMF biodiversity in commercially cultivated and wild cranberry sites across Wisconsin

All fungal communities: Looking first at the complete fungal communities, we saw striking differences in the commercially cultivated cranberry roots versus cranberry roots collected from sites of wild cranberry growth. While we had hypothesized there would be more diversity in the wild sites, the cultivated cranberry roots demonstrated 1613 unique fungal species, as opposed to 572 unique fungal species found in the wild cranberry roots. Sixty-nine of those species were seen in both wild and cultivated locations. We used a Principal Coordinates Analysis (PCoA) to assess similarities or differences among samples based on their distance matrices. The PCoA demonstrated clustering of the wild samples and a separate clustering of the cultivated samples, indicating that the cultivated fungal communities are overall more similar to each other than they are to the wild fungal communities. 

ErM fungal communities: When only the detectable ErMF communities across the wild and cultivated sites are considered, there was an overlap in the ErM fungi  present. Due to the dynamic and developing taxonomy of ErM fungi as our understanding expands, we worked with this data at the genus level to be as inclusive as possible. Working with a list of six genera confirmed to house species of ErM fungi (Hyaloscypha, Oidiodendron, Meliniomyces, Hymenoscyphus, Rhizoscyphus, and Serendipita), we found that five genera are ubiquitous across all sites: Hyaloscypha, Oidiodendron, Meliniomyces, Hymenoscyphus, and Rhizoscyphus. Serendipita also appeared in three out of the five cultivated collection sites, and six out of the seven wild collection sites. The samples demonstrate high overlap of the ErMF genera present, but the abundance of these genera varies from site to site. While all cultivated samples had much higher ratios of the Rhizoscyphus genus, many of the wild sites were dominated by the Serendipita genus. 

Objective 2: Establish the role of ErMF on organic Phosphorus uptake

Fungal mediation of P in isolation

We successfully verified the function of our fungal strain independent of a plant host by employing a Phytase Screening Media (PSM) as an indicator of organic P processing by the ATCC 32985 strain. In every plate, the fungi inoculated into the media produced enough of the phytase enzyme to break down the organic P content, creating the transparent clearing zone we were looking for in the agar surrounding the fungal tissue. We showed that, at least in isolation, ErMF can break down organic P.

Fungal mediation of P in association with cranberry plants

Effects of nutrient treatment and inoculation on phosphate content in leaf tissue: We conducted various statistical tests, but found no significant statistical differences across the inoculation treatments, indicating that mycorrhizal status alone does not significantly affect phosphate content in sampled leaf tissue. There was also no significant interaction effect between mycorrhizal status and nutrient level on phosphate content. However, the nutrient level has a statistically significant effect (p=0.006) on phosphate content, suggesting that different levels of nutrient availability can influence these outcomes, and in our various measurements of other plant growth metrics discussed below, inoculation confers other significant effects on plant growth and morphology. 

Shoot biomass accumulation across treatment groups: We conducted a two-way ANOVA analysis on nutrient level and ErM status on production of various types of cranberry tissue across the study. While nutrient treatments had less of an impact overall, ErM plants had greater upright biomass production (p=0.039). While the inoculation treatment (ErM status) shows a significant effect on the change in the number of uprights, the nutrient levels alone do not, and there is no significant interaction effect between these two factors. This indicates that the impact of ErM status on the change in the number of uprights is consistent across different nutrient levels.

Root mass across treatments:  ErMF inoculation also has a significant impact on the dry weights of the plants’ root mass (p=0.0198), which is not surprising given the expansion of the root system by the hyphae of ErMF. For the mycorrhizal treatment group, the average root+fungi mass dry weight is approximately 0.515 grams, while the non-mycorrhizal treatment group demonstrates an average root-only mass dry weight of approximately 0.443 grams. Neither the main effect of nutrient treatment groups (p=0.1618), nor the interaction between "ErM status" and "Nutrient level" (p=0.3470) demonstrated significance, suggesting once again that the different nutrient levels do not significantly affect the root mass dry weight across the levels tested and that inoculation with ErMF plays a significant role in the root mass accumulation. 

Objective 3: Assessment of ErMF’s impact on water stress response in cranberry plants

Mortality times across treatments: The average survival time for each inoculation treatment group under the stress condition (SS) suggests that on average, plants in the mycorrhizal group (ErM) lived longer than those in the non-mycorrhizal group (NM).

ErM group: 24.0 days

NM group: 20.2 days

Light-adapted chlorophyll data: Inoculated plants, whether under severe stress (ErM-SS) or well-watered (ErM-WW) conditions showed markedly higher averages of ETR_Fv/Fm measurements (electron transport rate, maximum possible efficiency of PSII) throughout the study than their non-inoculated counterparts (NM-SS and NM-WW), suggesting that inoculation with ErMF may improve photosynthetic efficiency. The average Y_Fm ∆Fv/Fm measurements (effective efficiency of PSII) revealed the same trend: inoculated plants, especially ErM-SS plants, exhibited higher readings than NM plants. The heightened response by ErM-SS plants in particular suggests a possible mitigating effect of mycorrhizal inoculation on the adverse impacts of drought related to photosynthetic capacity.

Dark-adapted chlorophyll data: The inoculated group once again had a slightly higher mean Fv/Fm (maximum quantum yield of PSII) value across most measurement dates compared to the non-inoculated group, suggesting a beneficial effect of the inoculation under the conditions tested. Similar trends are observed for Fv/Fo (potential quantum efficiency of PSII), with the inoculated group generally displaying higher mean values than the non-inoculated group. 

Leaf osmotic potential across time: From leaf tissue collected as each SS plant reached the cusp of its mortality, we can assess the average final osmotic potential values for each treatment group.

SS-ErM: 4645 mmol/kg

SS-NM: 4794 mmol/kg

WW-ErM: 1116.8 mmol/kg

WW-NM: 1059.6 mmol/kg

These averages highlight significant differences between stressed and well-watered groups, with stressed plants showing much higher osmotic potentials, indicating a higher solute concentration, which is a common response to drought stress. The differences between ErM inoculated and NM plants within the same water treatment group also suggest variations in how plants respond to drought stress with different inoculation treatments, although the differences are less pronounced than between water treatments. 

Soil aggregation: In a qualitative analysis, mycorrhizal soil samples held together far better than their non-mycorrhizal counterparts over a 90-minute rehydration period under slake test conditions. In a visual assessment, there was less debris and discoloration in the water of the mycorrhizal slake tests, as well less accumulation of media on the bottom of the slake test jars. This result is as expected, given the tendency of other types of mycorrhizal fungi to improve soil aggregation, and may be a contributing factor to the relative longevity of mycorrhizal plants under the severe stress treatment.  

Participation Summary
5 Farmers participating in research

Educational & Outreach Activities

1 Published press articles, newsletters
1 Tours
5 Webinars / talks / presentations
2 Workshop field days

Participation Summary:

950 Farmers participated
400 Ag professionals participated
Education/outreach description:

Five cranberry growers have collaborated with our team in this study, allowing us to collect soil and root samples from their commercial cranberry farms for the survey of diversity in Ericoid mycorrhizal fungi: Cranberry Creek in Minocqua, WI; Bartling’s Manitowish Cranberry Co. in Manitowish Waters, WI; Cutler Cranberry in City Point, WI; Gottschalk Cranberry, Inc. in Wisconsin Rapids, WI; and Cranberry Lake in Phillips, WI. We selected these growers because of their geographical distribution and the types of soils in their respective farms. In addition, some of these commercial grower locations have wild cranberries nearby which we used to test sampling protocols for our subsequent wild cranberry sample collection. The article published in the Cranberry Crop Management Journal in 2021 reached an audience of 850 growers across the growing region. My presentation to the Wisconsin Cranberry Growers Association in January 2022 reached an audience of 300 attendees, and each poster session resulted in discourse with approximately 30 community members per event. In addition, 350 attendees participated in the 2022 Wisconsin State Cranberry Growers’ Association Field Day. I also gave a tour of my lab work and research to a visiting group of leading growers in spring 2022 as a part of a day-long visit with university researchers. I presented to the Wisconsin Cranberry Growers Association once again in January 2024 reached an audience of 300 attendees. 65 growers responded to a survey that followed the January 2024 presentation, and results are included in project outcomes. Additional papers and journal articles are forthcoming. 

Published press/articles/newsletters

  1. Honeyball, B. and Atucha, A. 2021. Ericoid Mycorrhizal Fungi & Cranberry: Mutualisms with Potential. Cranberry Crop Management Journal. Vol. 34, Issue 4.


  1. Cranberry School 2022. Honeyball, B. Zalapa, J. and Atucha. A. 2022. Super roots for super fruits? Ericoid mycorrhizal fungi in association with Vaccinium macrocarpon. Wisconsin Cranberry School (Virtual). January 19, 2022.
  2.  2022 Beneficial Microbes Conference. Honeyball, B., Atucha, A., Zalapa, J. Evaluation of Ericoid Mycorrhizal Fungi Biodiversity in Commercial Cranberry Farms across WI. 8th Conference on Beneficial Microbes. Oral Presentation. July 13, 2022.
  3. 2022 Kenneth B. Raper Symposium: Showcasing Microbiology in Wisconsin. Honeyball, B., Atucha, A., Zalapa, J. Evaluation of Ericoid Mycorrhizal Fungi in Cultivated Cranberry across WI. Oral Presentation. September 6, 2022.
  4. Firmly Rooted: Perseverance through challenges. Honeyball, B. Atucha, A., Zalapa, J. Phosphorus mediation by Hyaloscypha hepaticicola, explored in isolation and in association with Vaccinium macrocarpon. 2023 Plant Sciences Symposium. Oral Presentation. November 3, 2023. 
  5. Cranberry School 2024 Honeyball, B. Zalapa, J. and Atucha. A. 2022. Exploring ericoid mycorrhizal fungi: fungal biodiversity in wild and cultivated cranberry. Wisconsin Cranberry School. January 25, 2024.

Workshops/Field days

  1. Ericoid mycorrhizal potential role in cranberry fertilization programs. Wisconsin State Cranberry Growers’ Association Field Day. Cranberry Research Station Black River Falls, WI (350 attendees). August 11, 2022.
  2. Wisconsin State Cranberry Growers’ Association Research Roundtable. Wisconsin Cranberry Research Station, Black River Falls, WI (40 attendees). November 15, 2023.

Project Outcomes

62 Farmers reporting change in knowledge, attitudes, skills and/or awareness
Project outcomes:

Project outcomes include increased researcher understanding of how ErMF affect vine performance in cranberry and evaluation through the presentation of findings. 65 growers responded to a survey after a presentation on this project to gauge their knowledge of the direct and indirect impacts of ErMF on crops and the environment, with 62 reporting an increase to their knowledge of ErMF and 59 expressing interest in these fungi as a new tool for field implementation. This project supports future research endeavors through extensive work in the development of novel protocols, particularly towards the potential benefits for growers and environmental conservation, providing a foundation for the next levels of ErMF research in Wisconsin cranberry.

Knowledge Gained:

Over the course of this project, my skills and experience have increased dramatically– with such a wide-ranging project and set of experiments, how could they not? Beginning graduate school, I  never anticipated the sheer bulk of protocols I’d need to learn, update, or even create. Aside from the science itself, I’ve had the opportunity to talk with a lot of different types of plant people, be they growers, botanists, agronomists, pathologists, or specialists in water quality. What I hear about most often from people in Extension, who mediate that line between growers and academics, is the need to reach a balance and reciprocity in the communication of academically produced data and grower knowledge/insight. I think there is a lot of room for intentionality there, especially in a project like mine that aims to set the stage for future collaborative research. When I conducted my final survey earlier this year, 95.4% of growers surveyed reported knowledge gained and 90.7% of growers expressed interest in participating in field trials; that feels like a significant success to me, even if we are years away from agricultural implementation.

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.