Fusarium solani Causing Root Rot Disease on Gastrodia elata in Shaxi, China

Gastrodia elata, a traditional and important medicinal plant in China, it is used to numerous medical reasons. It is widely planted in Shaxi, Guizhou Province, China. G. elata grown in Guizhou is of high quality and an important source of income for the region. However, a root rot disease has been reported on G. elata in Guizhou in recent years, with an incidence rate of approximately 25%; this disease has markedly affected the plant growth and development. It causes what is referred to as a “rotten nest” and “empty nest”, significantly reducing the yield and medicinal value of G. elata. Eighty diseased G. elata samples were collected from August to December 2020 in Shaxi. Tissue dissection was used to isolate the pathogen on an ultra-clean workbench. In short, thew surface of G. elata was wiped with 75% alcohol for 30 s and then rinsed three to four times with sterile water. After the surface had dried, the skin from an infected area of the plant was cut into a net shape using a sterile scalpel. Eighty diseased tissue samples were placed on PDA (potato dextrose agar) medium using a sterile medical syringe needle and placed in an incubator at 25 °C for 7 days, and 61 fungal isolates with the same morphological characteristics were obtained from the diseased samples. Pure cultures of a putative fungal pathogen designated SX13 were obtained using the single-spore isolation and cultured on PDA medioum for identification and analysis. The colony grew in a circular shape, and the early hyphae were compact and white. A light-yellow ring appeared in the outer circle of the hyphae, and could be seen on both sides of the plate. The upper side of the colony turned white subsequently, and the lower side was light yellow. Identification of SX13 as Fusarium solani was primarily done based on morphological characteristics (Chitrampalam et al., 2018). Colonies produced macroconidia, which were sickle-shaped with two to five septa; most of them had three septa (length by width: 17.28 to 36.23 μm by 4.33 to 6.43 μm). Smaller conidia were fusiform, renal, or oblong, with no or one septum (length by width: 5.56 to 14.35 μm by 2.93 to 5.76 μm). Chlamydospore were also observed with diameters of ranging from 3.43 to 13.12 μm. Identification of SX13 was verified through DNA sequencing. Genomic DNA was extracted using the Biomiga Fungal gDNA Kit. The internal transcribed spacer (ITS) region (primers ITS5/ITS4) (Schoch et al., 2012), β-tubulin (primers T1/T2) (O’Donnell and Cigelnik, 1997), and actin gene (ACT) region (primers ACT-512F/ACT-783R) (Carbone and Kohn, 1999) were PCR amplified, sequenced, and subjected to NCBI BLASTn homology matching analyses (GenBank Accession Nos. MW888340, MW892976 and MZ440809). High levels of sequence homology were observed with a F. solani reference sequence (Accession Nos. MT560378, ITS=100%; KU938955, β-tubulin=100%; KM231197, ACT=99%). To complete Koch's postulates, a conidial suspension (106 spores/mlcollected from isolate SX13 was inoculated onto nine G. elata root samples. Sterile water was used as a negative control, and the pathogenicity assay was repeated three times. Following inoculation, plants were kept under high relative humidity in the dark at 25 °C for 7 days. Symptoms similar to the original outbreak were observed on all inoculated plants. In contrast, the negative control plants were healthy and unaffected. The SX13 was re-isolated successfully from the diseased tissues and verified based on morphology and sequencing as described above. To the best of our knowledge, this is the first report of F. solani causing root rot disease on G. elata in China. These findings provide a basis for further research on the management of this disease.