First Report of Fusarium commune causing damping off and wilt in Cannabis sativa (L.) in Pennsylvania.

Abstract

Cannabis sativa is a crop prescribed for medical use in Pennsylvania, with an estimated state revenue of $1.5 billion dollars in 2023 (Cannabis Benchmarks, 2024). In August of 2023, mature Cannabis sativa plants at an indoor facility (102m2) in Chambersburg, PA, were observed with wilted yellowing leaves, browning stems/roots, and withering. Of the 490 plants at the facility, 70% displayed the described symptoms, likely exposed to the pathogen through the site's irrigation system. Cultivar "BDC" displayed the most severe symptoms; root samples from 3 soil-grown, mature, symptomatic "BDC" plants were harvested for testing. In a laminar flow hood, roots were surface sterilized with 3% sodium hypochlorite for 5 minutes, rinsed with sterile deionized water, cut into 1cm long with a heat-sterilized scalpel, and embedded into Potato Dextrose Agar (PDA) plates. After 2-3 days of incubation at 27°C, a fast-growing fungus with violet aerial mycelium and burgundy-colored bottom surface grew from all root segments. When plated on SNA, the mycelium was hyaline with some white aerial growth. The culture's morphology was consistent with Fusarium spp. (Mirghasempour SA, et al, 2022 & Yin, et al, 2022). DNA was extracted using Quick DNA Fungi/Bacterial Kit (Zymo Research Irvine, CA, USA). PCR was performed to amplify fungal RNAP II with RPB2 5f2F/7cR primers (Donnell et al., 2015 & O'Donnell et al., 2022) and TEF1 using primers Fa6/Ra7, followed by nested primers Fa/Ra (Karlsson et al., 2015). Amplicons were Sanger sequenced, trimmed, and compared to known sequences using BLAST. Amplicons from the isolates showed 100% identity to Fusarium commune accessions MH822055 (658bp/658bp) and MZ513475 (1778bp/1778bp) (Skovgaard, O'Donnell et Nirenberg). Consensus sequences were deposited in GenBank with accession numbers PP844864 (TEF1) and PP844863 (RPB2). PDA culture isolated from roots was diced and transferred to 500mL of V8 broth. A negative control was prepared using sterile PDA. The broths were placed on a shaker and incubated at 150 rpm and 27°C for 48 hours. Light microscopy revealed mycelial growth, microconidia and macroconidia in the inoculated suspension, as described in Chen, D et al (2023). Growth was absent in the control broth. To test Koch's postulates, each broth was diluted to 1L and 30 rockwool cubes (150 cm3) were soaked in the broths: 15 in the inoculated, 15 in the control. The stems of 30 unrooted "BDC" C. sativa cuttings were dipped in rooting gel (Clonex, Growth Technology, Somerset, UK) and embedded into the cubes. Cuttings were grown indoors, at 27°C for 14 days under LED lights with an 18-hour photoperiod. Inoculated cuttings displayed wilting/yellowing leaves, browning stem bases, and white mold growing on stems. Control cuttings had no disease symptoms and rooted successfully. Stem samples from 2 infected and 2 control cuttings were plated as previously described. After 48 hours at 27C, mycelial growth was observed from stems of infected plants but not from controls; hyphal transfers were re-plated on PDA. DNA extraction and PCR amplification for RPB2 and TEF1 amplicons was performed on the cultures, sequenced and aligned with the previous sequences. All sequences were identical to the original sequences from infected plants. F. commune is a wide-spread pathogen, with global distribution and broad host range. Based on our results, it is likely that there could be future outbreaks of the pathogen in Cannabis grows, causing economic damage to cultivators.