First Report of Stagonosporopsis vannaccii Causing Leaf Spot on Crassocephalum crepidioides in China.

Crassocephalum crepidioides (Benth.) S. Moore, native to tropical Africa, is an important invasive weed in many countries, seriously threatening the safety of agricultural ecosystem. During December 2018, 100% of C. crepidioides plants exhibited leaf spots in the Kudzu (Pueraria lobata) garden in Tianlin County, Baise City, Guangxi, China (24°40'20.42″N, 106°11'33.51″E), but Kudzu was not affected by this disease. The leaf spots appeared as small brown spots surrounded by a yellow-green halo initially, enlarged to subrotund or irregular in shape, slightly sunken, then developed as a dark brown to dark spot with grey-white necrotic center (Supplementary Fig. 1 a,b), and exuded an orange droplet under high humidity conditions (Supplementary Fig. 1 c). Symptomatic leaf tissues were cut into small pieces (5 x 5 mm) from the junction of necrotic and healthy tissues, and small pieces were disinfected in 75% ethanol solution for 30 s and 0.1% mercury dichloride for 30 sec, then rinsed with sterile water 3 times. These tissues were plated onto potato dextrose agar (PDA) medium and incubated in a thermostatic incubator at 28°C under natural sunlight conditions. Four isolates with similar morphological features were obtained after purification. Colonies of these isolates exhibited creme-orange margins and aerial mycelium was sparse. The colonies formed concentric circles on the surface that were fusco-black, violet-slate and vinaceus-grey (from centre to edge), fusco-black on the reverse after 7 days (Supplementary Fig. 2 a,b), and then the pycnidia and conidia produced for about 30 days (Supplementary Fig. 2 c). Pycnidia of representative isolate YTH-12 were black, subglobose, and unilocular, 95.60-168.27 μm (average 128.32 µm) (n = 40) in diameter. The ostiole was single and central, slightly papillate to papillate and occasionally rostrate (Supplementary Fig. 2 d). Conidia were hyaline, oval to elliptical, aseptate, 2.30 to 5.83 × 1.42 to 3.50 µm (average, 4.36 × 2.03 µm) (n = 50) (Supplementary Fig. 2 e). These morphological characters are consistent with those described for Stagonosporopsis vannaccii (Crous et al. 2019). To further identify the isolate YTH-12, the rDNA internal transcribed spacer (ITS) region, 28s large subunit ribosomal RNA (LSU), RNA polymerase II second largest subunit (RPB2) gene and β-tubulin (TUB2) gene were amplified by polymerase chain reaction using the primer pairs ITS1/ITS4 (White et al. 1990), LR0R (Rehner and Samuels 1994)/LR7 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/fRPB2-7cR (Liu et al. 1999), respectively. The PCR products were purified and sequenced by Sangon Biotech Co. Ltd. (Shanghai, China). The sequences were deposited in GenBank (accession nos. MN892355, MN893911, MN905510 and MN905511). The ITS (522 bp), LSU (1313 bp), TUB2 (380 bp) and RPB2 (1193 bp) nucleotide sequences showed 100% identity to S. vannaccii strain LFNO148 (accession nos. MK519453, MK519452, MK519454 and MN534891). Phylogenetic analysis based on the multi-locus sequences of ITS, LSU, RPB2 and TUB2 was performed in MEGA version 6.0 (Chen et al. 2015). The relative stability of the branches was evaluated by bootstrapping with 1000 replications. The isolate YTH-12 was placed in the same clade as S. vannaccii with 100% bootstrap support. Based on morphology and molecular analyses, this pathogen was identified as S. vannaccii. To satisfy Koch's postulates, the isolate YTH-12 was inoculated on leaves of C. crepidioides plants. Twenty punctured leaves and twenty unwounded leaves were inoculated with a 5-mm-diameter mycelial disc, respectively. Leaves inoculated with sterile PDA discs were used as blank controls. Plants were maintained in a growth chamber (25°C-28°C and relative humidity 80%-90%). Brown spots were observed on inoculated leaves (both punctured and unwounded) about 30 hours after inoculation and typical symptoms appeared about 55 hours after inoculation (Supplementary Fig. 1 d), and the diseased leaves produced black pycnidia and orange droplet 10 days after inoculation (Supplementary Fig. 1 e). All inoculated leaves developed symptoms similar to those on the naturally infected plants in the garden and the disease incidence reached 100%, whereas the control leaves remained symptomless (Supplementary Fig. 1 f). The same fungus was re-isolated from inoculated leaves. To our knowledge, this is the first report of S. vannaccii causing leaf spot on C. crepidioides in China. So far, Stagonosporopsis vannaccii has been reported as a plant pathogenic fungus only in Brazil, causing anthracnose symptoms on pods of soybean (Crous et al. 2019). Crassocephalum crepidioides is a widely distributed weed. If S. vannaccii has strong host specificity, it is possible to be used as a biocontrol fungus to control the weed. Conversely, if the fungus has a wider host range, C. crepidioides may act as a good bridge to spread the pathogen. This study helps to deepen the understanding of S. vannaccii and its associated plant diseases.