First Report of Boxwood Dieback Caused by Colletotrichum theobromicola in Texas
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In January 2016, a boxwood sample (Buxus sp.) from a Tarrant County landscape was submitted to the Texas Plant Disease Diagnostic Lab exhibiting randomly distributed tan dieback symptoms on approximately 20% of the shrub. Examination beneath the bark tissue revealed discoloration that extended to dead branches. Symptomatic tissue was surface sterilized with 10% bleach for 3 min and then rinsed in sterile distilled water for 1 min. The tissue was blotted dry with sterile paper towels, plated on 1/4-strength potato dextrose agar (QPDA) amended with the antibiotics streptomycin and chlorotetracycline at concentrations of 0.1 mg/ml each, and incubated at 27C in the dark. Following a 10-day incubation at 27C, fungi with acervuli, containing conidia typical of Colletotrichum spp. and black setae, were recovered. The conidia were single-celled, hyaline, and cylindrical, measuring 10 to 20 m long (avg. 15 m) and 5 m wide (n = 10). Based on these morphological characteristics of the fungal colonies and conidia, the fungus was identified as a Colletotrichum spp. DNA was extracted from the isolate (TPDDL2016-14) using the Zymo Research Fungal/Bacteria DNA Kit. Four genes were amplified and sequenced (Weir et al. 2012): ACT (actin), CHS-1 (chitin synthase), ITS (internal transcribed spacer region), and TUB2 (-tubulin 2). BLASTn analysis of these sequences had a 100% homology to C. theobromicola strains. The CHS-1 (GenBank accession no. MF509305) and ITS (MF509302) gene sequences matched with C. theobromicola strain PDC11290 from Louisiana (KP642641.1 and KM505031.1 respectively). The ACT (MF509304) gene matched isolate PDC11305 (KP642639). TUB2 (MF509303) sequence matched with C. theobromicola strain F27 (KC425712.1). To satisfy Kochs postulate, inoculations were conducted during summer 2016 (April to August). Fifteen Japanese boxwoods (Buxus microphylla) were inoculated with plugs of the fungal isolate that had been growing on QPDA plus antibiotics for 7 days. The inoculation was done by cutting the bark off of an area of the stem (1 cm 1 cm) and securing a 0.6 cm agar plug to the wound with Parafilm. Fifteen control plants were inoculated with sterile agar plugs of the same medium. Plants were maintained on an outdoor pad with an average temperature of 32C and were hand irrigated three times a week. Initial symptoms of foliar chlorosis were observed beginning at 3 weeks post-inoculation, progressing to branch. All control plants remained symptomless. C. theobromicola was reisolated on the antibiotic-amended QPDA. The reisolated fungus was morphologically similar to the original isolate and confirmed with sequence analysis of the four previously mentioned genes. Boxwoods are common woody ornamentals in Texas landscapes. Boxwood dieback has been subsequently diagnosed on an additional landscape specimens submitted to the TPDDL from Dallas County in March 2017. A previous report (Singh et al. 2015) has noted the presence of this pathogen in Louisiana, North Carolina, New York, Virginia, Indiana, and South Carolina. To our knowledge, this is the first reported incidence of C. theobromicola causing dieback on boxwood in Texas.
author list (cited authors)
Hawk, T., Rhodes, S. C., McBride, S., & Ong, K. L.
complete list of authors
Hawk, T||Rhodes, SC||McBride, S||Ong, KL