First report of black root rot (Berkeleyomyces rouxiae) on greenhouse hemp (Cannabis sativa) in Kentucky

Hemp, Cannabis sativa, was reintroduced to the US in 2014 after 50 years of prohibition. Since then, growers have focused primarily on cannabinoid production using female plants. Thus, most modern hemp has been asexually propagated in greenhouses and transplanted into fields. In December 2019, a commercial transplant producer in Fayette County, Kentucky reported 30% dieback on ‘Seagull’ hemp. Plants were potted into artificial media (unknown origin) immediately upon removal from the mist bench, and symptoms developed approximately two weeks afterward. Scattered plants developed stunting, yellowing, and wilting symptoms and then died within one week of symptom development. Roots had brown to black lesions distributed along roots that progressed into necrosis. Aleuriospores (chlamydospores) were visible under the dissecting microscope. Root pieces were surface sterilized with 10% household bleach for 2 min and then plated onto cut centers of ~1 cm thick sterilized carrot cross section (Williamson 2014). Resulting colonies were single-spored and maintained on PDA plates on laboratory benchtops (23°C, fluorescent lights). Three isolates were selected for pathogenicity testing and identification. Resulting colonies initially appeared white but darkened to black within two days following the development of aleuriospores (chlamydospores). Aleuriospores were dark brown, formed in chains of 4 to 5, and measured 10.00 to 14.27 × 5.62 to 11.23 μm (n=50). Endoconidia were hyaline, cylindrical, and measured 11.63 to 34.10 × 3.95 to 5.58 μm (n=50). To confirm pathogenicity, soil was washed from three 4-week-old hemp plants (proprietary cultivar) and roots were soaked in a 1×106 spore/mL conidial suspension for 5 minutes. In addition, the roots of three control plants were mock inoculated using sterile water. Inoculated and control plants were transplanted into sterile potting media and maintained in separate trays. Inoculated plants developed leaf yellowing and wilting symptoms 14 dai. Roots examined 21 dai had scattered brown lesions throughout and were colonized by the fungus. Aleuriospores and endoconidia isolated from the symptomatic roots were identical to the inoculum. Control plants did not develop symptoms after 21 dai. For molecular analyses, DNA was extracted from an actively growing (5 d) representative isolate (19FY005) on PDA using Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA). Fragments of commonly used diagnostic loci ITS, ACT, RPBII and Mcm7 were amplified as described by de Beer et al (2014), Fourie et al (2015) and Duong et al (2012). Amplicons were sequenced by Sanger sequencing and the consensus sequences were compared with the NCBI GenBank database by BLASTn. ACT amplicon (OK135163) top hit was B. rouxiae (MF967149.1) with 100% similarity, Mcm7 (OK135165) top hit was B. rouxiae (MF967103.1) with 100% similarity, RPBII (OK135166) top hit was B. rouxiae (MF967194.1) with 99.80% similarity, and ITS (OK135164) top hit was B. rouxiae (MF952402.1) with 99.82% similarity. Berkeleyomyces spp (syn Thielaviopsis sp.) is the causal agent of black root rot in field crops such as cotton and tobacco and in ornamentals such as holly and pansy; it is also a common disease in greenhouse production systems. Introduction of a cryptic sister species in 2017 may suggest that reports of black root rot in many crops may be either B. basicola or B. rouxiae, both species are indistinguishable by morphology (Nel et al. 2017). Although both species have wide host ranges with no host specificity, speciation may be important in terms of pathogenicity, host susceptibility, and other cultural factors. As hemp acreage increases across the US, black root rot may become a more prominent disease in greenhouse transplant production systems and in fields.