Sunflower (Helianthus annuus L.) is a major oilseed crop in the world, but recently it has become a popular ornamental flower in China (Kaya ET AL., 2012). Notably, stem rot was frequently observed on 30-day-old sunflower plants in Sichuan Province, P. R. China. In May 2021, 207 plants with stem rot and premature death were found among the investigated 1732 plants, with 11.95% incidence rate. Stem rot were mainly observed on the base stem of plants. Infected stems developed brown lesions that became slightly sunken and covered with white mycelial mats. With disease progress, the affected plants eventually wilted at early stage (Fig. 1A). For pathogen isolation, mycelia were harvested directly from diseased tissue and cultured on potato dextrose agar (PDA) at 25±2℃ in incubator without light. Eighteen fungal isolates with similar colony morphology were consistently isolated by purifying from different sampling areas. These isolates would form radial colonies with white aerial mycelia possessing branches and septa, and some could form clamp connection structure on PDA. Their average growth rate was 27.6 ± 0.09 mm per day (Fig. 1B & C). White granular sclerotia were abundantly produced on 6-day-old colonies. The sclerotia with a diameter of 1.18 ± 0.26 mm (n=348) gradually changed from white to light yellow and finally to brown with age (Fig. 1B). A representative isolate NCSF5.20-8 was identified accurately by multi-locus approach, and its genomic DNA was extracted for amplifying and sequencing the fragments of ITS, 18 S and TEF1-α according to previous description (Zhong et al., 2021). The nucleotide BLAST search of these sequences was nearly 100% identical to Athelia rolfsii (the teleomorph of Sclerotium rolfsii Sacc.) with accession number MN258360.1 (499/499 bp, 100%), AY665774.1 (1428/1428 bp, 100%), and JF267794.1 (489/490, 99%), respectively. The obtained sequences were deposited in GenBank with accession number OK635580.1, OM319631.1 and OK665849.1. Phylogenetic analysis based on simultaneously available ITS, 18S and TEF1-α sequences of Athelia genus indicated that NCSF5.20-8 could be clustered into the A. rolfsii clade with a support value of 100% (Fig. 2). Combining the above morphological characteristics and molecular identification, the isolate NCSF5.20-8 was finally confirmed as the identity of A. rolfsii (Mordue 1974; Zhong et al., 2021). In order to fulfil Koch's postulates and prove the pathogenicity of NCSF5.20-8 to sunflower, mycelial plugs were leaned on the base stem of healthy sunflower plants (cv. Guangwu muntain) with 45-day-old, while plants with same growth treated by PDA plug free of mycelium were used as control. All the inoculated plants were kept in a greenhouse at 25±2°C with 14 h photoperiod and 80% relative humidity. The experiments were conducted for triple biological repeats. Two days after inoculation, all the plants inoculated with NCSF5.20-8 had typical symptoms of stem rot, whereas, the control plants remained asymptomatic (Fig. 1D). Using above mentioned protocol, A. rolfsii was re-isolated only from the symptomatic stem of inoculated plants. To our knowledge, stem rot caused by A. rolfsii on sunflower has been reported in Italy and Turkey (Infantino et al., 1997; Cer & Morca, 2020), but it is the first report in China. For this pathogen has a wide host range with serious destructiveness (Punja, 1985), our research is beneficial to develop strategies to mitigate future losses of sunflower in China.
Keywords: Athelia rolfsii; stem rot; sunflower (Helianthus annuus).