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

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.