First Report of Black Dot Root Rot, Caused by Colletotrichum coccodes, on Tomato in Kentucky High Tunnels

As the most profitable vegetable crop grown in Kentucky (KY) high tunnels, tomato (Solanum lycopersicum L.) is often produced without crop rotation. In November 2016, ‘Mountain Fresh Plus’ tomato samples were received by the University of Kentucky Plant Disease Diagnostic Lab from Nelson County, KY. The high tunnel had an extended history of tomato production, and 100% of plants were affected. Common late-season symptoms of plant dieback and fruit decay were reported. Brown discoloration with dry, cracked cankers and microsclerotia were observed on primary and secondary sample roots, respectively. Symptomatic root sections (4 to 6 mm) were sterilized with 1% NaOCl and then placed on acidified potato dextrose agar (aPDA). Hyphal tips of the cultured fungus were aseptically transferred to fresh aPDA, and then mycelium was aseptically transferred to potato dextrose broth. DNA was extracted from 3-day-old broth culture (Li et al. 2008). Polymerase chain reaction (PCR) amplicons using ITS1/ITS4 (White et al. 1990) were generated, using protocols modified from Gardes and Bruns (1993) and Matsuda et al. (2005). Modifications included 35 total cycles, 30 s annealing step, and final extension for 10 min. Additional PCR products were amplified using partial actin (ACT; Prihastuti et al. 2009) and glyceraldehyde-3-phosphate dehydrogenase primers (GPDH; Berbee et al. 1999). ExoSap-IT (Affymetrix, Cleveland, OH) was used for PCR product cleanup, and then bidirectional Sanger sequence (Eurofin Genomics, Louisville, KY) was generated for each housekeeping gene. The internal transcribed spacer sequence was submitted to GenBank (MG544880) and matched 100% on identity and coverage with multiple Colletotrichum coccodes accessions, including KY364640. ACT and GPDH sequences were used in phylogenetic trees, which further supported identification as C. coccodes. The original isolate was grown on quarter-strength PDA at 23°C. Microsclerotia developed after 7 days within sparse, white mycelium. Conidia were dislodged from 4-week-old plates flooded with sterile deionized water using a sterile micropestle and then filtered through two layers of cheesecloth. Conidia were 18 to 22 μm long and 3 to 5 μm wide and were cylindrical, aseptate, and hyaline, with cylindrical conidiophores. Acervuli on tomato roots were circular with approximately 100-μm-long setae. Two-week-old ‘Sunstart’ tomatoes were used for Koch’s postulates. Eight plants were each drenched with 25 ml of 5 × 10⁵ conidia/ml (Vrisman et al. 2017). Eight plants drenched with 25 ml of sterile deionized water were noninoculated controls. After 11 weeks, root discoloration was observed, with black acervuli with setae visible on 50× magnified root surfaces; control plants were asymptomatic and lacked fungal growth. Symptomatic root cuttings were submerged in 0.6% NaOCl for 30 s and then plated on quarter-strength PDA. Mycelium was macroscopically visible after 1 week at ambient temperature. Microscopic examination indicated the fungus was morphologically indistinguishable from mycelium, conidiophores, and conidia of the original isolate, confirming Koch’s postulates. This experiment was repeated with the same results. Although C. coccodes and Pyrenochaeta lycopersici were recently reported to cause brown root rot complex in high tunnel tomato (Vrisman et al. 2017), P. lycopersici was not found here. This first report of black dot root rot in intensive high tunnel tomato production in KY reiterates the need for aggressive sanitation prior to disease development, because severe pathogen pressure in soil may necessitate relocation of the high tunnel.