First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States

Perilla mint (Perilla frutescens (L.) Britt.) is an annual plant native to Asia and considered invasive in North America where it has escaped cultivation as an ornamental (Miller 1947; Swearingen et al. 2010). In August 2021, an anthracnose disease was observed on invasive perilla found along the disturbed margins of a forest in Frederick County, Maryland, United States. Symptoms included necrotic lesions with chlorotic halos, were concentrated in the lower canopy, and caused premature defoliation of lower leaves (Figure S1). Leaves from four plants were surface sterilized by rinsing for 30 s in 70% ethanol, 60 s in 0.8% NaClO, and 60 s in sterile water and then incubated on 2% water agar under ambient laboratory conditions to permit sporulation. After three days, spores that exuded from individual lesions were streaked onto acidified potato dextrose agar. Two single-conidium isolates were recovered from each plant. All eight isolates were identified to species using DNA sequences. A single isolate (21-067) was selected at random for morphological characterization and completion of Koch’s postulates. Morphological features were recorded after seven days of growth on synthetic low-nutrient agar (SNA) and potato dextrose agar (PDA) incubated at 22°C under 12 hr UV-B and white fluorescent lighting. Measurements were based on a minimum of 20 observations per structure. Cultures on SNA were flat, hyaline to pale salmon, lacked sporodochia and grew at a rate of 1.3 mm day-1 (n = 3). Vegetative hyphal width was (minimum-maximum) 1.5-4.0 μm, (average ± standard deviation) 2.7 ± 0.9 μm. Conidiophores arose directly from hyphae, were hyaline, smooth, unbranched and measured 40.0-180.0 x 1.5-4.5 μm, 102.3 ± 33.9 x 2.7 ± 0.8 μm. Conidia were single celled, straight, hyaline, glabrous, rounded at both ends and measured 10.0-20.0 x 3.8-6.3 μm, 15.4 ± 2.5 x 4.9 ± 0.7 μm. Setae, observed only on PDA, were pale brown, 1-2 septate, straight, blunt tipped and measured 42.5-97.5 μm, 70.8 ± 13.4 μm. Appressoria formed on PDA were single-celled, pigmented, smooth, and obovoid with entire margins, measuring 5.2-7.7 x 8.6-13.8 μm, 6.8 ± 0.6 x 10.8 ± 1.4 μm. These characteristics were consistent with members of the Colletotrichum destructivum species complex. Partial DNA sequences from five loci were amplified following the procedures of Damm et al. 2014: internal transcribed spacer region of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and beta-tubulin (TUB2). Pairwise sequence comparisons to references using the Blastn algorithm found 99.8% similarity between isolate 21-067 and ex-type C. shisoi isolate JCM 31818: MH660930 (ITS, 545/546 bp), MH660931 (GAPDH,192/192 bp), MH660929 (CHS-1, 278/280 bp), MH660928 (ACT, 262/262 bp), and MH660932 (TUB2, 511/511 bp) (Altschul et al. 1990; Gan et al. 2019). Subsequently, sequences from all eight isolates were aligned with Clustal Omega (Sievers and Higgins 2018), concatenated, and used in a Bayesian phylogenetic reconstruction (Huelsenbeck and Ronquist 2001) of the C. destructivum species complex (Figure S2), confirming the species identity as C. shisoi. Sequences were deposited in NCBI GenBank under accession numbers OM865277-OM865284 and OM885059-OM885090. Koch’s postulates were fulfilled using perilla grown in a greenhouse until second true leaves emerged. Inoculum washed from two-week-old fungal cultures grown on potato dextrose agar was adjusted to 4 x 104 conidia mL-1 and applied to three replicate plants with an aspirator until runoff. Three plants were sprayed with sterile water as a negative control. Plants were covered with plastic bags for 72 hrs and maintained in a growth chamber at 20°C and 80% RH for 14 days. Inoculated plants displayed disease symptoms similar to those observed under field conditions, and control plants did not develop symptoms. Colletotrichum shisoi was reisolated from symptomatic tissue and reidentified based on morphology. The experiment was completed twice. To the authors’ knowledge, this is the first report of C. shisoi on P. frutescens in the United States. Colletotrichum shisoi has not been reported as a pathogen on other plants in the United States and may have potential use as a biological control agent for invasive perilla.