UNDERSTANDING MICROBIOME IN ORDER TO DEVELOP EFFECTIVE DISEASE CONTROLS FOR A CHEMICAL PESTICIDE-FREE PEONY FARMING SYSTEM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020008
• Project Start Date: Jul 17, 2019
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ALASKA
(N/A)
FAIRBANKS,AK 99775

Performing Department
Agriculture and Horticulture

Non Technical Summary
The peony (Paeonia spp), a flowering perennial plant, is one of two new, non-traditional agricultural crops in Alaska. The first peony farm was established in 1996. Establishing an agricultural industry in Alaska is very difficult and the peony cut-flower industry is no exception. In the past decade, diseases have become increasingly common. In a preliminary survey conducted recently, the PI learned that diseases, especially on some farms, are a serious threat to peony cut-flower production. Peonies, the tree and herbaceous species, are famed for their large, colorful and showy flowers. In the past decade, peonies of the herbaceous species attracted the attention of the cut-flower industry in the U.S. and the world. Growing market demand propelled peonies sales into a multimillion dollar industry. Because the flowering period of peonies is fairly short, with a limited shelf-life under current technology, to provide a continuous year-round supply of peonies requires tight coordination in production globally. Peonies are available from countries in the Northern Hemisphere (the Netherlands, Israel and the contiguous US states) from approximately mid-February through mid-July. Peonies from the Southern Hemisphere (New Zealand, Chile) are available from late-September through mid-January (Garfinkel, 2017). A production schedule gap--from mid-July through late-September--is left in the global supply chain. Because this is the time when Alaska peonies come to bloom (Holloway et al, 2005), the potential niche market and the reputed sale value of $10 per stem attracted many investors (some of whom had no previous experience in farming). Peony farms, many built on virgin soil, started by using rhizomes/root stocks imported primarily from the Netherlands. Presently, at $4 per stem, approximately 160 small peony farms are in production (down from 200), but the situation is decades away from market saturation (Auer, 2008). More than 25 Peony diseases were reported from the U.S. and the world, (Garfinkel and Chastagner, 2016). In a study on Botrytis and other peony diseases, Garfinkel found 6 species of Botrytis and S. sclerotiorum, Phoma spp and M. acerina. in Alaska (Garfinkel, 2017, Garfinkel and Chastagner, 2018). The recommended control for grey mold/Botrytis blight is frequent 17 application of chemical fungicides. A few plants displaying the symptoms of tobacco rattle virus were found on one farm (Chastagner, 2018, personal communication). Tobacco rattle virus (TRV) is invasive to Alaska (McBeath, unpublished data). The pathway of introduction of TRV and other pathogens to Alaska needs to be investigated. Other diseases, such as those of crown and roots remain unknown. Growers frequently attribute the death of some peony plants to winter kill. However, results of the preliminary survey indicated that peony growers consider the most devastating disease to be bud blast. Because this disease is not well understood in Alaska environmental condition, and systematic research on the cause of the disease is much needed. Conventional agricultural practices that rely heavily on the use of chemicals could have especially serious consequences for the environment and human health in the far North. This is because of the persistence of pesticides in cold soils and subsequent bioaccumulation in plant tissues. To protect the ecosystem and human health, environmentally-responsible disease management measures for grey mold/Botrytis blight, bud blast and other diseases using biological and nutritional measures should be studied, and biological control and nutrient therapy practices should be developed.

Animal Health Component

(N/A)

Research Effort Categories

Basic

10%

Applied

(N/A)

Developmental

90%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	2129	1103	90%
216	2129	1103	10%

Knowledge Area
215 - Biological Control of Pests Affecting Plants; 216 - Integrated Pest Management Systems;

Subject Of Investigation
2129 - Florist crops, other;

Field Of Science
1103 - Other microbiology;

Keywords

metagenome

disease resistance

biological control

nutrient therapy

trichoderma atroviride

Goals / Objectives
The ultimate goal of this project is to assist peony farmers in Alaska to grow healthy plants, which will produce high quality cut-flowers using environmentally-responsible means of disease control. Specific objectives of this proposed project are as follows:a. Establishment of baseline information of peony farms,b. Diagnoses of diseases found during yearly disease surveys conducted on the farm,c. Evaluations of the nutrient levels in peony tissues, using ICP-OES technology,d. Evaluation of the performance of Plant Helper and Cu-I-PO3 on Botrytis blight control,e. Initiation of studies on microbial communities in the rhizosphere and phyllosphere of peonies and vegetation in and around the peony farms and,f. Discovery, identification and characterization of beneficial microbe in Alaska. We hypothesize that: (i) microbes within the microbial communities can be detected at the strain level and quantified, using reduced representation sequencing of microbial genomes method; (ii) the diversity and abundance of the microbial community will be mostly host-genotype dependent; (iii) the microbiome influences host diseases' resistance by acting as a host secondary genome; (iv) that plant nutrient plays a significant role in plant-microbe interaction and biocontrol performance; and (v) that the ability for strain-level profiling of the microbiome will lead to high-throughput detection of beneficial microbes and biocontrols.

Project Methods
There are both applied research and basic research components of this proposed project. The first action of this proposed project is to conduct a questionnaire survey to acquire information from each participant peony farm (see Section 4.A.). This information provides the starting point for the subsequent field survey and disease diagnosis activities (see Section 4.B). The disease situation will be monitored through yearly field disease survey and diagnostic activities (see Section 4. B.). This project will enlist the participation of peony farmers, who serve as "citizen scientists" are frontline observers of the project. The first alert sent by these citizen scientists will enable the PI to make rapid responses. Another applied research action is to use knowledge and commercially available products in disease control. Field testing of Plant Helper (formulated cold tolerant Trichoderma atroviride) and Copper phosphite and Copper-halo-phosphite, will yield information on the suppression of Botrytis blight/grey mold and other diseases, on improvement of nutrient uptake, and on enhancement of the quality and quantities of peony flower production, etc. (see Section 4.D.) To acquire accurate evaluation of efficacies of these products on peony plants, nutrients in the tissues of peony will be used as an indicator. Nutrient data of peony tissues, acquired using microwave assisted acid digestion and ICP-OES (Section 4.C.), will be evaluated along with traditional evaluation by visual observation. This is the first project to apply cutting edge technologies in the study of the interactions of plants (peony), diseases (grey mold/Botrytis blight, bud blast, white mold), microbial communities and agricultural practices in Alaska. Protocols used in this research are detailed in Section 4.E. The PI has more than 30 years of experience in the isolation, identification, selection, fermentation, formulation and commercialization of biological control agents. It is highly likely that a large number of beneficial microorganisms will be produced in this project, through conventional methods and advanced technologies (see Section 4.D.). These beneficial microbes will be identified and characterized. These isolates displaying novel abilities will be evaluated for their commercialization potential. This is also the first project to combine nutrient therapy with biocontrols in the development of environmentally-responsible disease control strategies. The novel copper phosphate and copperhalo-phosphite, which is compatible with T. atroviride in tests conducted in the laboratory, has high mobility in plant tissues and theoretically should add additional benefits to disease control (see Section 4.D).

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Peony farmersin Alaska specifically but also in other states. Scientists interested in the microbiome, the general public interested in the peony, specifically, Extension agents, government officials(eg. Division of Ag.), Legislators who are interested in the peony crops in Alaska. Changes/Problems:This is the first full year of a five-year Hatch project, entitled "Understanding microbiome in order to develop effective disease controls for a chemical pesticide-free peony farming system. In 2020, this project was seriously impacted by the COVID-19 pandemic. Rules and restrictions published by the State and University in March limited the progress of this project, especially field work. Fortunately, research of this project was able to continue after the Plant Pathology and Biotechnology Lab received from UAF an "essential function" statues. What opportunities for training and professional development has the project provided?Training farmers in zone meetings. Question and answer sessions for the farmers. Peony Farm Surveys: This study was designed to gain a thorough understanding of the peony farms, especially the health of peony plants and controls. Questionnaires were designed to gather information on: 1) general information (geographic location, crop history, and source of peony rhizomes/root stocks, cultivation and cultural practices), 2) environmental information, 3) disease information and management methods used. The survey was a success: out of the 77 peony farms, 72 farmers (94%) responded. Citizen scientists were instructed on peony diseases before the growing season, so they might serve as advance scouts for early disease detection. In this way, the PI and her research team can provide rapid response to the diseases. In early 2020, communications with the peony farmers were conducted by the PI, and her associates through zoom meetings, emails, telephone calls and mails. Understanding of peony farmers about this research project was further enhanced through face-face interaction in a receiption (07/06/20) and farm visits (July 7th and 8th). Our efforts were richly rewarded. Peony farmers we met were very gracious and appreciative. They also demonstrated a greater interest in the project by their enthusiasm. To date, the collaborators of this project have increased to a total of 6. How have the results been disseminated to communities of interest?Preliminarily, in early 2020, communications with the peony farmers were conducted by the PI, and her associates through zoom meetings, emails, telephone calls and mails. Understanding of peony farmers about this research project was further enhanced through face-face interaction in a reception (07/06/20) and farm visits (July 7th and 8th). Informal information sharing occurs through PI's outreach efforts. What do you plan to do during the next reporting period to accomplish the goals?Summer 2021, incomingPhD student to conduct field research.

Impacts
What was accomplished under these goals? Peony Farm Surveys: This study was designed to gain a thorough understanding of the peony farms, especially the health of peony plants and controls. Questionnaires were designed to gather information on: 1) general information (geographic location, crop history, and source of peony rhizomes/root stocks, cultivation and cultural practices), 2) environmental information, 3) disease information and management methods used. The survey was a success: out of the 77 peony farms, 72 farmers (94%) responded. Field treatment trials: This part of the research involves peony farm collaborators in the design and establishing of field experiements on their farms. The purpose is to test the efficacies of Plant Helper™ and Cu-I-PO3(fast action) in the control of soilborne diseases and Botrytis blosson rot, and in promotion of plant growth and development. The COVID-19 pandemic caused a delay of Cu-I-PO3treatment. In June 4, 2020, 7 peony cultivars (Elsa Sass, Red Charm, Duchess, Dr. Alex, Henry Sass, Sarah and Festiva) were treated with Plant Helper. Plants treated with Plant Helper werer significantly larger--plants taller, leaes larger, stems thicker. In late-September, untreated peony plants moved into sceneses. Peony plants treated with Plant Helper stayed green and continued to produce photosynthates which were stored in the root system (crown, root). These findings were furthre confirmed by biomass analysis conducted in the laboratory. Nutrient analysis: Leaves and soils (from rootzones) were harvested from 7 cultivars treated with Plant Helper and their untreated controls. Variations in responses to treatment were found among different cultivars, however all treated varieties showed elevated potassium and iron. Both elements play significant roles in plant defense system which means Plant Helper treatment enhances plant disease resistance. Microbiome Studies: High molecular weight DNA were extracted from isolates obtained from the rhizospheres of peony plants. These DNAs will be sent to Dr. Bode Olukolu, University of Tennessee, for next generation sequencing and further analysis. One of the primary goals of this project is in the discovery and identification of cold adapted beneficial bacilli. Target plant pathogens are Botrytis spp. (grey mold, most prevalent disease on peony in Alaska) and other economically important plant pathogens. Interactions were also studied among the bacteria isolates and Trichoderma atroviride (the primary active ingredient of Plant Helper). In the fall of 2019, soil samples were taken from the roots of peony plants in the UAF Georganson Botanical Garden and processed. In July 2020, samples of peony leaves and soils (from root zones) were obtained from four peony farms, in Homer (3) and Wasilla (1). These samples were stored at -80 C freezer awaiting for processing. To date, a total of 163 bacteria isolates (primarily Bacillus spp.) were obtained. Among them, more than 53 are adapted to cold temperature (can grow at 7C) and 18 isolates demonstrates different degrees of antagonism against Botrytis spp., Rhizoctonia solani, Fusarium spp., and Penicillium spp. However, warm temperature Bacillus sp. isolates showed stronger ability to suppress the growth of plant pathogens. Based on their BLAST scores, (homology of DNA sequences with known species), efficacious isolates were identified as Bacillus subtilis, and B. amyloliquefaciens. A cold adapted isolate was identified as B. mycoides, but it was ineffective in the suppression of target pathogens. One of the most interesting discoveries was the cold-adapted Bacillus Isolate #129, which under laboratory conditions, can suppress the growth of Trichoderma atroviride. This discovery provides an explanation regarding the discrepancies of efficacies between lab test results and field treatments of biological control of plant diseases. Glycerol stocks of the isolates were stored for studies in the future. They will also be compared with data obtained from the microbiome studies. Nutrient Recycling Studies: The hypothesis is that in Alaska, plant diseases can facilitate the degradation of plant tissues and hence, impact positively on the recycling of nutrients. In the fall of 2019, fallen leaves from aspen trees were collected and divided into two groups depending on the numbers of sori of Melampsora rust disease--mesh bags contained: 1) leaves with large numbers of Melampsora rust sori, and 2) leaves with no sori or a limited number of sori. Due to the COVID-19, retrieving of the mesh bags in the spring 2020, after breakup was postponed. Hopefully, mesh bags can be excavated in the Spring of 2021. Plant tissues in the mesh bags will then be evaluated and degradation evaluated by the loss of weight and minerals. Objective 4. Provide outreach, education, extension and technology transfer to our clients and stakeholders--growers, biocontrol industry, graduate and undergraduate students, K-12 students and other scientists. In 2019, questionnaires were sent to peony farmers in Alaska. Included in the survey to peony farmers were also invitations to serve as "citizen scientists" and "research collaborators". Among the survey responses received, 75% of peony farmers expressed strong interest in serving as "citizen scientists". One peony farmer also indicated interest in participating in this project as a collaborator. ?

Publications

Progress 07/17/19 to 09/30/19

Outputs
Target Audience:Questionnaire has been designedand distributed to 77 Peony farm owners/managers in theFairbanks area. Responses received from 72 (94%). With the survey, invitations to particpate as "citizen scientists" and "research collaborators" were also sent. Among the survey responses, 75% of peony farmers expressed strong interest in serving as "citizen scientists". Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Citizen scientists will be instructed on peony diseases before the growing season, so they may serve as advance scouts for early disease detection. In this way, the PI and her research team can provide rapid responds to the diseases. Field treatment trials: This part of the research willinvolve peony farm collaborators in the design and establishment of field experiements on their farms. The purpose is to test the efficacies of Plant Helper™ and Cu-I-PO3 (fast action) in the control of soilborne diseases and Botrytis blosson rot, and in promotion of plant growth and development. Microbiome Studies: High molecular weight DNAwere extracted from isolates obtained from the rhizospheres of peony plants. These DNAs will be sent to Dr. Bode Olukolu, University of Tennessee, for next generation sequencing and further analysis. In spring 2020, after breakup, mesh bags collected in fall 2019 for theNutrient RecyclingStudieswill be excavated. Plant tissues in the mesh bags will be evaluated. Degradation will be evaluated by the loss of weight and minerals.

Impacts
What was accomplished under these goals? Goal accomplishments: a.Initial steps toward the collection of baseline informationhas begun with the distribution of questionnaires and invitations to peony growers. Seventy-two farmers (94%) responded with informationfor ourinitial database: 1) general information including geographic location, crop history, and source of peony rhizomes/root stocks, cultivation and cultural practices; 2)environmental information; 3) disease information and management methods used. b. Disease surveys on farms will be initiated in summer 2020. Peony farmers responded to the PI's invitations to participate as "citizen scientists" and to serve as advance scouts for early disease detection on their farms. c. Initial steps were completed, to establish communication with farmers. d. Farmers at local peony farms are being identified and communication established.This part of the research involves peony farm collaborators in the design and establishment of field experiements on their farms. e. and f. Glycerol stocks of isolates stored from previous efforts for the control of Botrytis and other pathogens in Alaska, for future biological control studies such as the present study, will be compared with data obtained from the earliermicrobiome studies. High molecular weight DNA were extracted from isolates obtained from the rhizospheres of peony plants. These DNAs will be sent to Dr. Bode Olukolu, University of Tennessee, for next generation sequencing and further analysis. Nutrient Recycling Studies: The hypothesis is that in Alaska, plant diseases can facilitate the degradation of plant tissues and hence, impact positively on the recycling of nutrients. In the fall of 2019, fallen leaves from aspen trees were collected and divided into two groups depending on the numbers of sori of Melampsora rust disease--mesh bags contained: 1) leaves with large number of Melampsora rust sori, and 2) leaves with no sori or a limited number of sori. In the spring 2020, after breakup, mesh bags will be excavated. Plant tissues in the mesh bags will be evaluated. Degradation will be evaluated by the loss of weight and minerals.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cheng, M.Y., McBeath, J.H., Dong, J.H., Han, C.G., Zhang, Z.K., 2019. First report of Phytoplasma Candidatus Phytoplansma australiense associated with purple top diseased potatoes (Solanum tuberosum) in Guangdong. Plant Disease 103 (5): 1015.