Date of Award

Spring 5-3-2024

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Botany and Plant Pathology

Advisor

Seanna Annis

Second Committee Member

Ek Han Tan

Third Committee Member

Shawkat Ali

Abstract

Sphaerulina vaccinii is a fungal leaf spot pathogen of lowbush blueberry, Vaccinium angustifolium, causing Sphaerulina leaf spot. Infection by this pathogen results in symptoms of dark lesions on the leaves and stems in either of the crop cycle years. Severe infection by this pathogen is thought to result in decreased fruit retention and yield. As wild blueberries are central to Maine’s economy, reliable disease identification is essential for using suitable management strategies to control disease. Molecular-based methods for pathogen differentiation have been used efficiently to identify fungal pathogens in economically important crops. To develop a molecular-based method to identify Sphaerulina vaccinii, samples of late-stage foliar lesions were collected in six lowbush blueberry fields in 2022. Fungal isolates were retrieved from these lesions and the DNA of their ITS regions in the ribosomal gene cassette and B-tubulin gene was sequenced. A S. vaccinii-specific molecular assay was developed using the B-tubulin gene sequences. The primers developed (SphB and SphNS) matched uniquely to S. vaccinii DNA and could reliably detect 10 pg of S. vaccinii DNA or more. This primer set was used to determine the temporal spore dispersal of S. vaccinii. Spore traps made of microscope slides coated in petroleum jelly were placed near stems with lesions in eight Downeast and Midcoast crop fields in Maine in 2022 and 2023, respectively. Fields were sampled over a six-week period, from bloom break in early May to when late-stage lesions were observed in June. DNA was extracted from the spore trap petroleum jelly and tested for presence of S. vaccinii DNA using the developed PCR assay. The specific primer set, SphB/SphNS, could detect DNA from 500,000 spores or more. Sphaerulina vaccinii was detected in at least one field in every week sampled during both years, indicating the spore release window may be longer than originally thought. Spores were found in Downeast fields at least one week before fields in the Midcoast region. Primers specific for S. vaccinii (B-tubulin) and Thekopsora minima (ITS) were used to detect their respective fungal DNA in samples from whole leaves with suspected late-stage S. vaccinii lesions. Sphaerulina vaccinii was detected in 87% of the leaves, and T. minima was detected in

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