Progress report for GNC20-302
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 crops, thereby easing challenges faced by growers. Cranberry (Vaccinium macrocarpon Ait.) growers are under increasing pressure as a result of climate change, which is affecting 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 ecosystem (bog) and are highly susceptible to water stress. Hoping 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 ericoid mycorrhizal fungi (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.
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.
- 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 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 only 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, which were processed at the lab. Processing samples includes 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 an second order roots, those were preferentially selected for surface sterilization in a bleach solution, lightly dried, and stored at -80C until all samples had been processed and thus 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 arrived in late December 2022.
- Establish the role of ericoid mycorrhizal fungi (ErMF) on organic Phosphorus uptake
- We developed a system of axenic root growth in cranberry cuttings using hydroponics and are currently trialing methods of inoculation. This system of root development differs from standard cranberry cutting methods in that it provides us with roots free of media entanglement, an ongoing issue with the extremely fine ericoid roots. Cranberry cuttings are made from runner tissue (lateral vegetative growth), surface sterilized, and trimmed of excess leaf tissue. The tops of the cuttings are coated in petroleum jelly to minimize transpiration. Sets of 40 cuttings are suspended in 10L tubs of microfiltered water via foam holders. Each tub is supplemented with an oxygen pump and weekly maintenance of water changes and added macro- and micronutrients. The hydroponic system is kept at an ambient temperature of approximately 85F to encourage growth. We typically see cuttings begin to root within one week.
- We also designed and implemented a hydroponic pod system to quickly and efficiently inoculate sample groups of 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, cuttings in each condition are inoculated with mycelial tissue from liquid culture of ATCC 32985. Colonization takes approximately six days to occur after inoculation and is confirmed via 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, allowing us to compare uptake of organic and inorganic P in inoculated and non-inoculated cranberry plants. Also explored were many potential protocols for P analysis, further laying groundwork for our P uptake study. Ultimately we decided on the malachite green colorimetric assay.
- Possibly the most significant step was the use of a Phytase Screening Media (PSM) as an indicator of organic P processing by the fungi. We had great success using PSM with the ATCC 32985 strain. This agar media recipe incorporates organic P in the form of phytin; if the fungi on the plate produce the proper enzyme to break it down (phytase), there is a clear visual indicator of success in the form of a “clearing zone” of transparency in the agar surrounding the fungal tissue. We showed that, at least in isolation, ErMF can break down organic P.
- Assessment of ErMF’s impact on water stress response in cranberry plants
- Soil moisture variables will be evaluated via tensiometer in a greenhouse setting. In 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.
Educational & Outreach Activities
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 in the 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.
- Honeyball, B. and Atucha, A. 2021. Ericoid Mycorrhizal Fungi & Cranberry: Mutualisms with Potential. Cranberry Crop Management Journal. Vol. 34, Issue 4.
- 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.
- 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.
- 2022 Kenneth B. Raper Symposium Honeyball,B., Atucha,A., Zalapa,J. Evaluation of Ericoid Mycorrhizal Fungi in Cultivated Cranberry across WI. 2022 Kenneth B. Raper Symposium: Showcasing Microbiology in Wisconsin. Oral Presentation. September 6, 2022.
- 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.
Project outcomes will include increased researcher understanding of how ErMF affect vine performance in cranberry and evaluation through the presentation of findings. Growers will be surveyed before and after presentations to gauge their knowledge of the direct and indirect impacts of ErMF on crops and the environment; we expect to significantly increase grower interest in this new tool for sustainability, particularly given concrete benefits for growers and the environment, lowering requisite water and fertilizer usage. In addition, this project will support future research endeavors through extensive work in the development of novel protocols. Our metabarcoding analysis of fungal biodiversity in cultivated and wild sample sites will also provide a foundation for the next levels of ErMF research in Wisconsin cranberry, perhaps especially for growers interested in organic cultivation.