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

Authors: Fulcher, Michael; Owen Smith, Paul

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2023
Publication Date: 11/25/2023
Citation: Fulcher, M.R., Owen Smith, P.C. 2023. First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States. Plant Disease. https://doi.org/10.1094/PDIS-09-22-2121-PDN.
DOI: https://doi.org/10.1094/PDIS-09-22-2121-PDN

Interpretive Summary: First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States: Perilla mint is a foreign invasive weed present in thirty states. This weed produces toxins that cause respiratory illness in livestock and often grows in sensitive environments where chemical herbicide use is not desirable and physical removal of plants is not practical. Developing biological control products that use naturally occurring plant pests or pathogens would make it easier to manage invasive perilla. A fungal plant pathogen was discovered on this weed in Frederick County, Maryland with the cooperation of the National Park Service. The pathogen was identified using DNA sequences and tested for its ability to cause disease on perilla. This fungus will be used by researchers developing new tools to control invasive perilla.

Technical Abstract: 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). Perilla outcompetes desirable plant species and produces toxins that cause interstitial pneumonia in ruminants (Kerr et al. 1986). In August 2021, an anthracnose disease was observed on P. frutescens leaves in Frederick County, Maryland, United States. Symptoms included necrotic lesions with chlorotic halos, were found at a single location, and were concentrated in the lower canopy (Figure S1). Leaves 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 exuded from individual lesions were streaked onto acidified potato dextrose agar. Two single-conidium isolates were collected from each of four leaves, and a single isolate (21-067) was selected at random for morphological characterization and completion of Koch’s postulates. Colony and spore morphology were recorded after 13 days of growth on synthetic low-nutrient agar incubated at 20°C under 24 hr fluorescent lighting. Average radial growth was 1.50 mm day-1 (n = 3). Conidia were single celled, hyaline, glabrous, approximately round at both ends, and measured 19.0 ± 1.28 x 5.86 ± 0.33 µm (n = 50). These characteristics were consistent with species in the Colletotrichum destructivum complex (Damm et al. 2014). Isolate 21-067 was identified to species based on five partial gene sequences: internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), chitin synthase 1 (CHS-1), actin (ACT), and beta-tubulin (TUB2) (Damm et al. 2014). Sequences were deposited in NCBI Genbank under accession numbers OM865278, OM885084, OM885076, OM885060, and OM885068. Pairwise sequence comparisons to references using the Blastn algorithm found a high similarity between isolate 21-067 and 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). A phylogenetic analysis including all species in the C. destructivum complex further supported this identification (Hoang et al. 2018; Kalyaanamoorthy et al. 2017; Katoh et al. 2019; Minh et al. 2019) (Figure S2). Koch’s postulates were performed using P. frutescens 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 H2O and applied with an aspirator to three replicate plants. 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 Colletotrichum shisoi was reisolated from symptomatic tissue. The experiment was completed twice and included a sterile water control that produced no symptoms. 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 P. frutescens.