Arbuscular Mycorrhizal Fungal Associations in Tea Under Sustainable Production Systems in Florida

Progress report for GS21-243

Project Type: Graduate Student
Funds awarded in 2021: $16,444.00
Projected End Date: 08/31/2023
Grant Recipient: University of Florida
Region: Southern
State: Florida
Graduate Student:
Major Professor:
Dr. Bala Rathinasabapathi
University of Florida
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Project Information

Summary:

This project is an exploration of arbuscular mycorrhizal fungal (AMF) species associating with tea (Camellia sinensis) roots under various soil, environmental, and management conditions in Florida. Species in the genus Camellia are known to associate with AMF in their native and historical ranges, but the community profile of AMF on tea roots in the United States has not yet been described (Bag et al., 2020). Given the growing interest in cultivating tea as a specialty crop in the United States, and especially in the Southeast, we are undertaking research to assess AMF-tea associations in Florida. This research will describe the AMF communities associating with tea, tea plant health, and tea leaf quality in different soil types, environmental conditions, and management conditions. The project addresses these questions with two different approaches: In the first, AMF communities are described from across a wide geographical area within Florida and the greater Southeast; in the second, an experimental plot at the University of Florida (UF) Plant Science Research and Education Unit (PSREU) in Citra, FL is used to test AMF fungal communities on early-stage establishing tea plants under different cover crop treatments. The results of these efforts will inform management practices for new tea growers in Florida and the Southeast. This report describes the work completed during the first 6 months of this 2-year project.

Project Objectives:

1) Study and characterize AMF communities in association with tea roots in Central Florida, North Florida, and in the Southeast. This objective casts a wide net to capture the diversity of AMF-tea community composition according to geographical occurrence, including considerations of climate, soil physical properties, and local vegetative communities. AMF species and community profiles determined from this objective will inform Objective 2.

2) Evaluate AMF colonization and community profile in association with tea roots under several ground cover strategies for tea farms, including biotic ground cover and WBC. Correlations between AMF and soil physical properties, soil nutrient levels, tea plant health, and product quality metrics will be investigated.

3) Report the results of this research to inform BMPs for ground cover management in tea growing systems. In particular, the work is intended to address issues of cover crop species selection, effect on soil nutrients and soil physical properties. In addition, symbiotic interactions between cover crop, tea plant, and soil fungal communities, will be studied and described with relevance to tea plant health and management recommendations. This objective includes dissemination of findings through webinars, extension outreach, conference presentations, and research publications.

 

Research

Materials and methods:

Planting Materials:

This project will focus on a landrace tea variety that has shown good establishment and growth in the Gulf states. ‘Fairhope’ originated from three plants rescued from a defunct Lipton research farm in Fairhope, AL in the 1970s. These three plants, chosen for distinct morphological features from one another, were transplanted and allowed to cross breed. The resulting progeny were both numerous and vigorous; today they comprise all the plants at the Fairhope Tea Plantation in Fairhope, AL, as well as a great number of nursery plants available for retail sale throughout the Southeast (personal communication).

Field Sites

The proposed research will occur on 10 sites distributed throughout Florida, described below (Figure 3).

Regional: There are 6 regional sites, consisting of 3 farmer/private grower sites, 3 sites administered by Florida Agricultural and Mechanical University (FAMU) Extension service. The FAMU sites house between 2-4 plants that were 2-year-old seedlings when established in October, 2019.

Citra: The site is maintained by UF’s Plant Science Research and Education Unit (PSREU) in Citra, FL (Figure 3). It houses 240 ‘Fairhope’ tea plants arranged in a randomized block design. The plants were installed in September, 2020 and will be established for 1 year at the time of this proposal’s start. The plants were 3 year-old seedlings at the time of installation. This site is managed with a summer/winter annual cover crop rotation (Figure 4). Each rep contains 12 tea plants, spaced 60 cm apart in beds 4’ wide. Tea plants are mulched with pine straw 12-18” wide on center, to prevent competition from cover crops. Ground cover height is managed by mowing as needed to maintain 4-6” tall. For this proposal, the site would continue to be managed as described.

The “RDG” site is a SARE partner grower farm (SARE LS18-297), Rain Drop Gardens in Reddick, FL, with 72 ‘Fairhope’ tea plants established in 2018 as 1 y/o seedlings at the same spacing and bed size as the Citra site. There are two living ground cover treatments with 6 plants x 6 replicates in each treatment: winter annual legume (crimson clover) and perennial legume (perennial peanut). The control plot is 3 WBC replicates. Tea plants are mulched and mowed as at Citra. This site also contains grower-established mature tea hedgerows, which will be included in the statewide survey.

All sites included in this study will be subjected to environmental tests and monitoring to characterize the soils and climate (Table 1).

Objective 1

Objective 1 is to characterize AMF colonization and diversity. This phase of the project will be performed in Year 1 at the 8 Statewide sites, plus SARE Partner site, RDG (SARE Project Number P0086519), for a total of 9 sites. Percent colonization, AMF community profiling, plant health, and product quality analyses will be assessed for each site (Table 2). This objective’s expected result is a survey of AMF species and community profiles that associate with tea roots under Florida environmental and management conditions.

Because AMF activity is highest in mid-to-late-summer in most climates, all root sampling for colonization percentage and community profiling will occur then (20). Tea leaf nutrient quantification is timed to coincide with this period of high AMF activity. A detailed and standardized protocol for sampling procedures will be prepared and distributed to project partners prior to any sample collection.

Colonization rates will be determined by root staining and arbuscule counting using the gridline intersect method, which estimates colonization by examining roots with a gridded microscope objective and counting arbuscules at the gridline intersections (21). AMF species identification and community profiling will be performed by root DNA extraction and PCR amplification. Two rounds of amplification using universal fungal-specific primers first, and AMF-specific primers second, will be used for AMF species discovery (22). The ITS region will be sequenced for species identification. Primers for phylogenetic analysis will be based on a literature search of phylogenies of the fungal genera identified in the discovery phase. Plant characteristics for Objective 1 include leaf nutrients, quality indicators for made tea (total polyphenols and caffeine) (23), and disease ratings. Plant size index (PSI) will be estimated by measuring three dimensions to calculate volume. Environmental conditions will be measured as described in Table 1.

Objective 2

Research for Objective 2 will occur at Citra and RDG. These sites will be subjected to all sampling protocols listed in Table 1. These will be performed in Years 1 and 2, excluding Fungal Diversity, which will occur in Year 2. Ground cover analysis and pruned biomass will be included in this objective. (Table 3). Ground cover density and diversity will be measured every 3 months (once a season). Analysis of variance (ANOVA) testing will be used to estimate relationships between tea, AMF, ground cover, and environmental conditions. Post-hoc tests will be used to further evaluate significant associations. The expected result of this objective is the evaluation of different ground cover strategies and their effects on tea plant growth and quality, and soil health.

Objective 3

The results will be further interpreted during Year 2 and culminate in written and verbal communications directed at two audiences: horticultural recommendations for growers, and academic deliverables for the scientific community.

Grower Recommendations: Recommendations will be communicated during Year 2. To improve accessibility, communications will leverage electronic technologies and comprise one webinar and one virtual field day. After delivery, recommendations will be written as fact sheets and published on UF’s Institute of Food and Agriculture Science (IFAS) Electronic Data Information Source (EDIS) website.

Academic Deliverables: Results will be presented at one conference in 2023. Candidate conferences include the American Society of Horticultural Sciences conference (ASHS, August 2023) and the North American Agroforestry Conference (NAAC, June 2023). Additionally, a refereed publication will be submitted to a peer-reviewed journal prominent in the field of horticulture or sustainable agriculture.

Progress as of 4/1/2022:

Objective 1: Tea plants from 5 sites in Florida were sampled for roots to determine community profile of AMF. A root staining method to quantify AMF colonization rate was performed on tea roots collected in Gainesville, FL in February of 2022 (method modified after Moukarzel et al, 2020). Presence of AMF structures were visually confirmed using microscopy (Figure 1).

arbuscule and hypha of am fungi in plant root cells

Figure 1. AMF structures observed on tea roots collected in Gainesville, FL in February of 2022. Roots were stained with Trypan blue and visualized under 400x magnification. The white circle indicates an arbuscule formed within a tea root cell. The white arrows indicate a stained hyaline fungal hypha running parallel to the plant cell walls, a morphological feature indicative of AMF species.

A literature search was performed to determine 1) optimal DNA extraction method from mycorrhized roots, and 2) primers for PCR amplification of fungal DNA to identify fungal species. Root tissue DNA extractions were performed on stained roots confirmed to have AMF colonization (method modified after Diedhiou et al., 2014). Primers have been identified and obtained for nested PCR amplification of taxonomically significant regions (Table 1). DNA extractions of single hyphal DNA extractions and amplifications will be performed, and product sequences confirmed with Sanger sequencing.

Table 1. PCR primers for nested amplification of fungal barcoding regions.

 

PCR Amplification 1(modified after Binder and Hibbet, 2003)

PCR Amplification 2 (Kohout et al., 2014)

Forward Primer

ITS1F

ITSo7

Reverse Primer

LR5

ITS4

Region

ITS12 and LSU

ITS2

Product Size

±1.8kb

±300bp

 

We will perform seasonal comparisons of AMF root colonization rates on a site-by-site basis between spring and early autumn. Initial root sampling is to take place according to plant phenology, i.e., at first flushing of new spring leaves. Plant tissue samples, soil samples, and plant characterization data have been collected, and soil temperature monitoring loggers have been deployed, at 2 of 5 sites in Florida.

Objective 2: The experimental tea field has been maintained in a rotation of winter and summer cover crops as outlined in the proposal (Figure 2). Pine bark mulch was added to the treatments without weed barrier cloth. Cover crop establishment measurements were taken in March of 2022.

Works referenced:

Bag, S., Mondal, A., & Banik, A. (2022). Exploring tea (Camellia sinensis) microbiome: Insights into the functional characteristics and their impact on tea growth promotion. Microbiological Research, 254, 126890. https://doi.org/10.1016/j.micres.2021.126890

Binder, M., & Hibbett, D. (n.d.). Resuspending freeze-dried oligonucleotides. 7. Retrieved from: https://www2.clarku.edu/faculty/dhibbett/Protocols_Folder/Primers/Primers.pdf

Diédhiou, A. G., Borges, W. L., Sadio, O., & De Faria, S. M. (2014). Assessment of DNA extraction methods for detection of arbuscular mycorrhizal fungi in plant roots by nested-PCR. Acta Scientiarum. Biological Sciences, 36(4), 433. https://doi.org/10.4025/actascibiolsci.v36i4.21689

Kohout, P., Sudová, R., Janoušková, M., Čtvrtlíková, M., Hejda, M., Pánková, H., Slavíková, R., Štajerová, K., Vosátka, M., & Sýkorová, Z. (2014). Comparison of commonly used primer sets for evaluating arbuscular mycorrhizal fungal communities: Is there a universal solution? Soil Biology and Biochemistry, 68, 482–493. https://doi.org/10.1016/j.soilbio.2013.08.027

Moukarzel, R., Ridgway, H. J., Guerin-Laguette, A., & Jones, E. E. (2020). An improved clearing and staining protocol for evaluation of arbuscular mycorrhizal colonisation in darkly pigmented woody roots. New Zealand Plant Protection, 73, 33–39. https://doi.org/10.30843/nzpp.2020.73.11711

Research results and discussion:

Root tip DNA extraction of C.sinensis resulted in a yield of 6,000 ng of genomic DNA from a 50mg root sample (120 ng DNA/1 mg fresh weight root). A sample of lemongrass roots included in the same extraction yielded 2,300 ng of genomic DNA (46 ng DNA/ 1mg fresh weight root). The roots were previously stained using the method referenced above and presence of AMF structures was confirmed prior to extraction (after Mourkazel et al., 2020). The extraction method used was the TE buffer and activated charcoal method described in Diedhou et al. (2014). This method is optimized for woody roots with high amounts of secondary metabolites. This yield is comparable to the results reported by Diedhou et al., however, the method is nonspecific to fungi. Therefore, this extraction result likely includes both plant and fungal genomic DNA. Presence of fungal DNA is to be confirmed by nested PCR, described above.

Participation Summary
4 Farmers participating in research

Educational & Outreach Activities

4 Consultations
1 Webinars / talks / presentations

Participation Summary:

60 Farmers
20 Ag professionals participated
Education/outreach description:

Research progress is reported weekly to doctoral advisors and laboratory colleagues.

Seminar talk: “Tea Research Update: Camellia sinensis in North Central Florida.” October 1, 2021. US League of Tea Growers Annual Conference, Amite, Louisiana. Farmers and agricultural professionals present: 80

Consultations with growers: 4

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.