First Report of Basal Stem Rot of Achnatherum inebrians Caused by Fusarium pseudograminearum in China.

Abstract

Drunken horse grass (Achnatherum inebrians) is a perennial bunchgrass that is widely distributed in arid and semi-arid grasslands in northwest China (Zhang et al., 2021). In July 2023, Basal stem rot was found in artificially grown drunken horse grass plots in Yuzhong County (35.85° N, 104.12° E), Gansu Province, China, with an average incidence of 5.2%. Diseased plants showed crown and basal stem rot with chocolate brown discoloration at the base of the stem and slight constriction of some basal stems. Five field's foci were surveyed and at least 6 basal stems per focus were collected. Infected basal stems were surface-sterilized (75% ethanol for 30 s and 1% NaClO for 90 s), rinsed three times with sterilized water, placed on potato dextrose agar (PDA), and incubated at 22°C in the dark for 3 days. Isolates were purified by single spore cultures (Leslie and Summerell, 2006). The average mycelial growth rate was 4.8 to 7.5 mm/day at 25°C on PDA, and the colonies produced aerial mycelium varying from rose to yellow white, and rose to burgundy pigment diffused into the agar. Macroconidia of the isolates were produced on carnation leaf agar (CLA) incubated under black light and observed to be abundant, but no microconidia were found. Macroconidia were relatively slender, curved to almost straight, commonly 3-6 septate, averaging 30.1 × 3.8 μm (n=50). The morphological characteristics of this fungus fully fit the description of F. pseudograminearum (Aoki and O'Donnell, 1999). To obtain the phylogenetic support, DNA of three representative isolates YZ-Y-1, YZ-Y-2 and YZ-Y-3 was extracted by using an HP Fungal DNA Kit (D3195), and a portion of the RNA polymerase II second largest subunit (RPB2) gene and elongation factor 1 alpha (EF1-α) gene were amplified using primers RPB2-5f2 and RPB2-7cr (O'Donnell et al. 2010) and EF1 and EF2 (O'Donnell et al. 1998), respectively. Results of sequences were deposited in GenBank (accession nos. PP179044 to PP179049). A nucleotide BLAST search revealed RPB2 and EF1-α sequences to be 99.8 and 100% similar to the corresponding sequences of the ex-type strain NRRL 28062 F. pseudograminearum accessions numbers MW233433 and MW233090, respectively. For pathogenicity tests, 15 μl of conidia suspension (1×106 conidia/ml) was inoculated into the stem bases of 10 healthy drunken horse grass seedlings (around 3 weeks old) using a sterile syringe, then wrapped with moistened sterile gauze, while the other 10 drunken horse grass seedlings were injected with sterile water as a control. All seedlings were placed in a greenhouse with a plastic cover at 15-22°C and 90-100% relative humidity. All inoculated drunken horse grass seedlings showed symptoms similar to those of natural infection with stem basal rot, whereas uninoculated drunken horse grass seedlings remained healthy after 15 days. Fungi re-isolated from the basal stems of inoculated plants were confirmed phenotypically and molecularly as F. pseudograminearum. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of drunken horse grass in China. The disease has become a potential threat to the growth of drunken horse grass in China.