First Report of Strawberry Anthracnose Crown Rot Caused by Colletotrichum siamense in New England.

Abstract

In the summer of 2023, the Connecticut Agricultural Experiment Station was contacted by a farm in southern Connecticut due to reports of strawberry (Fragaria × ananassa) plants showing signs of severe wilting and crown rot across multiple fields, covering ~20 hectares. Cut crowns from diseased plants had marbled red and white lesions typically associated with anthracnose crown rot (ACR). Symptomatic plants were collected from five June-bearing cultivars (cvs. AC Valley Sunset, Lyla, Dickens, and Allstar) spanning four non-adjacent fields with incidence ranging from 5-90% and severity ranging mild wilting in low incidence fields to severe wilting/mortality in high incidence fields. Internal tissue from diseased crowns was surface sterilized in 0.6% NaOCL for 3 minutes, rinsed with sterile water, and plated on potato dextrose agar. After one-week, hyphal tips of fungi were transferred to fresh plates which formed dense mycelial mats of fluffy, greyish-white hyphae. Orange spore masses formed near the center of the colonies, each of which contained numerous cylindrical and fusiform straight conidia, matching spores within the genus Colletotrichum (De Silva et al. 2019). Average conidia (n=192) length was 15.7 ± 1.6 µm and width was 5.4 ± 0.7 µm. Fungi matching this morphology were isolated from 83% of the collected symptomatic crowns and hyphae were collected from two isolates, CT5-1 and CT23-1, for DNA extraction using the GeneJET Plant Genomic DNA Purification Kit. PCR was performed using primers targeting actin (ACT), calmodulin (CAL), β-tubulin (TUB2), GAPDH (gpdA), and ITS, followed by Sanger sequencing, which yielded identical sequences for both isolates (CT5-1 Accessions numbers: PP002078-81, OR999066)(Carbone and Kohn 1999; Hassan et al. 2018; Templeton et al. 1992). These were combined with sequences from fourteen Colletotrichum genomes, all of which were aligned, trimmed, and concatenated using Mega11 (Tamura, Stecher, and Kumar 2021). Model selection was conducted using IQ-TREE and selected parameters were used to generate maximum-likelihood trees from all five loci individually and the concatenated sequence, all of which placed the isolates in a high confidence cluster with Colletotrichum siamense (Nguyen et al. 2015). To confirm the pathogenicity of the pathogen, strawberry plants (cv. Jewel) (n=5) five weeks after bare root transplant were infected. The base of each crown was penetrated 5 mm deep with a sterile 20 µL pipette tip and then inoculated with 10 µL of spores at a concentration of 106 spores/mL. Control plants (n=5) were inoculated with 10 µL of sterile water. Plants were maintained at 30°C day (16-hour)/20°C night (8-hour) in a growth chamber and assessed after 14-days. Four of the five inoculated plants had visible wilt symptoms and bisected crowns revealed the marbled red and white lesions typifying ACR. Control plants had no clear wilting and bisected crowns were visibly healthy. C. siamense re-isolated from infected tissue presented with identical hyphal /spore morphology and ITS/Tub2 were re-amplified and sequenced, yielding identical sequences to CT5-1. Plant inoculations with the same variety were repeated, yielding identical symptom development and crown lesions. C. siamense has been a dominant source of ACR throughout the southeastern US but has not previously been a major problem in the Northeast. Given the extent of the field infection, it is likely that these isolates can survive the colder winter temperatures of New England, but further experimentation is necessary to determine the extent of the pathogen's winter hardiness.