Non Technical Summary
Fire blight, caused by the bacterium Erwinia amylovora, is top on the list of the most concerned diseases of apple and pear by organic growers. Among all organic approved control measures of fire blight, Blossom Protect, a biological control with a yeast-like fungus Aureobasidium pullulans as the active ingredient, is the most effective and widely used material. Integrated disease management protocols centered around Blossom Protect have been developed and widely adopted by organic growers in the Pacific Northwest. However, applying Blossom Protect under humid conditions can cause of fruit russeting, a disorder of fruit skin that reduce the value of fresh market crops, therefore cannot be adopted by growers in the east.We hypothesize that there are other yeasts from apple and pear flowers that can effectively suppress fire blight similar to A. pullulans in Blossom Protect, without causing significant russeting. Prior to this research, we determined that A. pullulans in Blossom Protect induces plant defense response which is the mechanism for its disease suppression. In this project, we aim to perform an extensive search of flower/fruit colonizing yeasts for their ability to 1. induce plant defense, 2. suppress fire blight, and 3. cause russeting. Isolates with desirable fire blight suppression efficacy with minimal russeting will be selected for a multi-year, multi-location field trial. Extension and education activities include disseminating research findings to growers, educating fruit consumers about the cause of fruit russeting and other skin disorders, and training the next generation organic growers in the Northeast through undergraduate education.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1110	1102	25%
212	1110	1100	25%
215	1110	1102	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
1110 - Apple;

Field Of Science
1100 - Bacteriology; 1102 - Mycology;

Goals / Objectives
Isolate yeast-like fungi from apple and pear flowers collected from different cultivars and at different geographical locations.Screen the yeast culture collection for their potential to induce plant defense response and to cause fruit russeting.Test the fire blight biocontrol efficacy and fruit safety of selected yeasts in field trials.Disseminate research findings, increase the marketability of organic fruits, and prepare future organic growers through extension and education

Project Methods
Objective 1.Isolate yeast-like fungi from apple and pear flowers and fruits collected from different cultivars and at different locations.1.1 Sample collectionApple and pear flowers at full bloom and 2-5 week-old immature fruits will be collected for yeast isolation. Flower and fruit samples will be collected from 25 different apple cultivars and 5 pear cultivars at 4 locations (CT, NY, NH, and OR). Additional sampling will be performed in experimental orchardsat Oregon State University (OR), participating farms in CT, NH, and NY with 5 cultivars sampled at each site. All the farms listed are either certified organic or in the process of being transitioned into organic.1.2 Isolation of microorganism within plant cuticleCuticle portion of the collected flower hypanthium and immature fruits will be dissected with a sterile razor blade, placed in Eppendorf tubes containing 0.5 ml of sterile potassium phosphate buffer (pH 7.0), vortexed for 10 seconds and placed in a water bath sonicator for 3 minutes to release the attached microorganisms present on the surface and internal in the cuticle.Buffer with cuticle microbes will be serial diluted and plated on three different media for yeast isolation: Potato Dextrose Agar (PDA), Yeast extract-Peptone-Dextrose (YPD), and Sabouraud Agar (SA). Chloramphenicol, streptomycin sulfate, and tetracycline will be supplemented into the media at the concentration of 50 µg/liter to suppress bacterial growth. Plates will be incubated at 28 °C for 48-96 hours.1.3 Establish culture collection and identificationAfter the microbial colonies develop, 15 yeast-like fungal colonies representing various morphology and growth characteristics from each biological sample will be selected, purified by streaking on a fresh media, and preserved in 20% glycerol in a -80 °C freezer. Filamentous fungi will not be included in this study. The selected yeast strains will be further identified by amplifying and sequencing the fungal ITS1 gene. Relevant information of the culture collection, including colony morphology and growth characteristics, ITS sequences, and strain identification will be documented in a strain database and managed as described in the data management plan.Objective 2. Screen the yeast culture collection for their potential to induce plant defense response and to cause fruit russeting.2.1 Screen the yeast collection for their ability to induce plant defense response.Yeast strains obtained from Objective 1 will be cultured on the corresponding media.The mycelium on agar surface will be collected and resuspended in sterile water, adjusted to OD600=0.1(cell optical density), and spray-inoculated onto apple flowers on the day of petal open, with two flower clusters (a total of 12 individual flowers as biological replicates) per yeast isolate. Water treatment, Blossom Protect treatment, and acibenzolar-S-methyl treatment (a chemical inducer of SAR) will be used as controls (Johnson et al. 2016).Three days post yeast treatment, hypanthium portion of the yeast-treated flowers will be dissected from the rest of the flower parts (sepal, filament, style, and the base of ovary) for RNA isolation. RNA will be isolated using RNeasy Plant Mini Kit (Qiagen), and used as input in a quantitative reverse transcription PCR (qRT-PCR) analysis. Expression ofPR2(plant defense gene) andTublin(a gene the expression of which is consistent, used as an internal control) will be measured, using aDDCtmethod. Statistical analyses will be performed using the one-way analysis of variance (ANOVA) model in the 'stats' package in R. The screening will be performed in a high-throughput manner using 96-well plates.2.2 Screen the yeast collection for their ability to cause fruit russetingWe will perform russeting evaluation using the in orchard attached fruit assay as developed in our preliminary study (Fig. 8A). The russeting evaluation will be performed on 'Golden smoothie' apple, a cultivar highly susceptible to russeting, located at the Connecticut Agricultural Experiment Station Lockwood Farm (Hamden, CT). Flowers will be treated with Oxidate 5.0 (1.0% v/v) to clean off the indigenous yeasts.In vitrocultures of yeasts will be spray-inoculated to the flowers / fruitlets twice, once at full bloom and again one week after petal fall. Water treatment and Blossom Protect treatment will be used as controls. 20 biological replicates will be included in each treatment. Russeting will be evaluated one week prior to harvest.The in orchard russeting assay is limited by the availability of growing fruits during the growing season. To mitigate this limitation, we will also perform a cutinase production assay on Czapek-Dox medium (Dickman et al. 1986) in the laboratory. Cutinase degrades cutin, the major component of plant cuticle, therefore, yeasts that produce cutinase are more likely to cause russeting than yeasts that don't.Objective 3. Test the fire blight biocontrol efficacy and fruit safety of selected yeasts in the field.We expect to identify 6-10 yeast isolate that induce a plant defense response without causing significant russeting from Objective 2. In Objective 3, we will further test whether these yeast isolates can effectively and consistently suppress fire blight, and evaluate the fruit finish through a two-year field trial. The field experiment will be conducted on three apple cultivars with different susceptibility to fire blight and russeting: 'Golden Smoothie': moderately tolerant to fire blight and highly susceptible to russeting; 'Gala': highly susceptible to fire blight and tolerant to russeting; and 'Red Delicious": tolerant to both fire blight and russeting. These trees are located at two locations, 1. Lockwood farm at the Connecticut Agricultural Experiment Station, Hamden, CT, and 2. Griswold Research Center, Griswold, CT. Both locations are currently under transition to organic production. This experiment will be repeated for three years.Objective 4. Disseminate research findings, increase the marketability of organic fruits, and prepare future organic growers through extension and educationActivity 1.We will disseminate the research findings (Objective 1-3) through a multi-channel approach. We aim to target apple and pear growers, both the existing organic growers and conventional growers that have interests in transitioning into organic. We will also reach out to agricultural companies that develop and market biological controls, such as Certis Biologicals, Corteva Agriscience, Dora Agri, Bayer Crop Science et al., which may be interested in collaborating as industry partners for future commercialization of the discovered yeasts. Activity 2.We will educate the general public and fruit consumers about fruit finish cosmetic problems.Disseminate knowledge through social media.We will produce 10-20 short videos introducing 1. Benefits of organic fruits, 2. Different types of fruit cosmetic issues, including common ones caused by diseases, pests, and nutrient deficiency, and stresses.These short videos will be posted on Youtube Channel "Each an Organic Apple A Day".Disseminate knowledge at local grocery stores, farmer's markets, public schools (K-12).We will produce flyers introducing the common types of fruit finish issues. We will partner with local grocery stores (Big Y, Stop and Shop), and farmer's markets to disseminate such flyers to promote the acceptance of fruit with minor cosmetic problems.Activity 3.We will provide guest lectures to undergraduate students on topics of organic fruit production and pest management in the Northeast.We will integrate the organic production in the existing curriculum of fruit production courses at two universities in the northeast, University of Connecticut and University of Massachusetts-Amherst.