Peanut (Arachis hypogaea L.) is an important oilseed and cash crop cultivated in over 100 countries worldwide. The major producers are China, India and USA (Ding et al. 2022). In September 2022, peanut pods exhibiting black necrotic symptoms on the shell surface were observed in Puyang, Henan Province, China. These black spots often merged to form larger necrotic spots on the shell. Disease incidence was 100% in susceptible varieties. Symptomatic shell pieces were surface sterilized with 75% ethanol for 3 min, rinsed three times with sterile water, and then transferred onto PDA medium supplemented with 25 µg/ml chloramphenicol (Long et al. 2022). Isolation frequency of a fungus with similar-appearing colonies from symptomatic pods was 81.7%. A pure culture of a representative isolate, PYHB, was obtained through single-sporing and maintained on PDA plates at 25℃ in darkness. The colony initially appeared white but turned black within 2 days. The isolate produced dark brown, unicellular chlamydospores, which were arranged in club-shaped chains consisting of two to seven cells. The size of the unicellular chlamydospores varied from 3.34 to 15.27 µm (average:6.81, n = 100) in length and 8.30 to 15.51 µm (average:11.29, n = 100) in width. The endoconidia were hyaline and cylindrical, measuring 7.91-22.94 × 1.69-4.81 µm (average: 12.16 × 3.13, n = 100). Based on morphological characteristics, the isolate was tentatively identified as a Berkeleyomyces sp. (Nel et al. 2018; Long et al. 2022). The ITS region of r-DNA, the ribosomal large subunit (LSU), the minichromosome maintenance complex component 7 (MCM7), and the 60S ribosomal protein RPL10 (60S) genes were amplified using ITS1/ITS4, LR0R/LR5, rouxMCM7-F/rouxMCM7-R and roux60s-F/roux60s-R primers, respectively (White et al. 1990; Vilgalys and Hester 1990; Nakane and Usami 2020). The sequences were deposited in GenBank (ITS: OR053803; LSU: OR053818; MCM7: OR058549; 60S: OR060656). Through BLASTn analysis of the NCBI GenBank database, the generated ITS and LSU sequences showed 100% identity to Berkeleyomyces rouxiae (GenBank MF952418.1 and MF948662.1, respectively) and B. basicola (GenBank MT221585.1 and MH868639.1, respectively). Importantly, the MCM7 and 60S sequences were 100% identical to B. rouxiae (GenBank MF967114.1 and MF967077.1, respectively). Phylogenetic analysis combining ITS, LSU, MCM7, and 60S sequences showed that the isolate PYHB clustered with B. rouxiae. To evaluate pathogenicity, surface-sterilized healthy peanut pods (n = 90) were immersed in a 1×106 spore/ml conidial suspension obtained from isolate PYHB for 5 min and placed in Petri dishes containing moistened cotton at 25°C for 10 days. Pods (n = 90) inoculated with sterile water served as controls. Inoculated pods displayed black necrosis 10 days after inoculation (dai), whereas no symptoms were observed on the control pods at 21 dai. The reisolated pathogen was shown to be identical to the original inoculum through morphological and phylogenetic analysis. Black root rot is a fungal disease caused by Berkeleyomyces spp. (syn. Thielaviopsis spp.) and affects various crops and ornamentals, such as cotton, tobacco, carrot, holly, and pansy (Rahnama et al. 2022). The causal agents B. rouxiae and B. basicola have similar morphological characteristics but can be differentiated through molecular characterization (Nel et al. 2018). To our knowledge, this is the first report of black pod rot in peanut caused by B. rouxiae in China. The finding from this study will contribute to the development of monitoring and management strategies to combat this destructive disease in peanut cultivation.
Keywords: Causal Agent; Crop Type; Field crops; Fungi; Pathogen detection; Subject Areas; oilseeds and legumes.