First Report of Polygonatum cyrtonema Hua Root Rot Caused by Trichoderma virens in China.

Abstract

Polygonatum cyrtonema Hua is a traditional Chinese medicine, which has anti-oxidant, anti-inflammatory, immunomodulatory, and other pharmacological effects (Lu et al. 2023). In June 2022, A disease of root rot was observed on P. cyrtonema plants in Tonggu County (28°63'89″N, 114°48'07″E), Jiangxi Province, China. The disease incidence was approximately 30% in a surveyed area of 3 hectares, which contained approximately 20,000 plants. Initially, the above-ground parts of the plants did not show any obvious symptoms. However, the underground roots exhibited red-brown spots that gradually expanded and sunken areas appeared, and the diseased plants presented leaf chlorosis and red-brown discoloration of the tubers, eventually leading to plant death. To identify the pathogen, symptomatic root tissues (0.5×0.5×0.5cm) from the lesion margin surface were sterilized with 75% ethanol for 30s, 3% NaClO for 3 min followed by rinsing three times with sterile water. The sterile root pieces were placed on potato dextrose agar (PDA) and incubated at 25℃. Thirteen pure fungal isolates with the same morphological characteristics were obtained by monosporic isolation from 20 pieces of roots, and the representative isolates, HJGF1-1, HJGF1-2 were used for morphological studies and phylogenetic analyses. Initially, the two colonies on PDA appeared white with cotton-shaped aerial hyphae, which later turned light green to green and formed concentric rings. At the end of the conidiophores, 3 to 6 pear-shaped branches are irregularly gathered, and the angles between the branches are acute. The conidia were mostly solitary ellipsoid or obovate with the size of (3.7-5.9) × (3.6-4.5) μm (n=100). These morphological characteristics are consistent with the description of a Trichoderma spp. (Kamala et al. 2015). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone et al. 1999), and RPB2-5F2/RPB2-7cR (O'Donnell et al. 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates HJGF1-1 (GenBank accession nos. OR229621, OR290791, OR334600) and HJGF1-2 (GenBank accession nos. OR229622, OR290792, OR334600) showed 99%-100% identity with multiple GenBank sequences of Trichoderma virens. A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analyses revealed that the two isolates HJGF1-1 and HJGF1-2 were in the same clade with T. virens strains. The two isolates were identified as T. virens based on the morphological characteristics and molecular phylogeny. To test pathogenicity, ten healthy P. cyrtonema plants (one tuber each, 5 tubers per isolate, n=10) in the field were pin-pricked with a sterile needle and pour-inoculated with 5 mL spore suspension per tuber (1× 107 conidia/ mL) with a temperature of about 28℃. Another five tubers were were pin-pricked with a sterile needle, inoculated with sterile water and served as controls. The resulting symptoms were similar to those on the original infected plants in the field, and control tubers remained symptomless fourteen days after inoculation. T. virens was reisolated from the diseased tubers, nevertheless no pathogenic fungus was isolated from the control tubers. T. virens has been reported causing disease on tulip bulb (Lou et al. 2003) but has not previously been reported causing disease on P. cyrtonema. Although several species of Trichoderma are known to be beneficial microorganisms, the beneficial fungus may have had an evolutionary period of interaction with plants in which it behaved as a plant pathogen (Poveda et al. 2022). To our knowledge, this is the first report of T. virens infecting P. cyrtonema in China. This result may expanded the etiological study of T. virens and the control strategy of P. cyrtonema root rot.