First Report of Botryosphaeria dothidea Causing Stem Canker on Soybean in China

In September 2020, widespread stem canker on soybean ( ) was detected in southeastern Jiangsu (Nantong municipality; 120.76° E, 32.23° N) in China. Mature plants, 14 weeks of cultivation, exhibited brown necrotic lesions and dried-up stem. The symptoms were observed in eleven soybean fields, 1.6 ha in total, and approximately 80% of the plants were symptomatic. The symptoms were consistent with those previously reported for stem canker on soybean caused by , and (Ghimire et al. 2019; Mena et al. 2020). Small pieces, approximately 0.4 cm in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH O. The pathogen was isolated and cultured on potato dextrose agar (PDA), containing chloramphenicol (50 µg/mL), under darkness at 28 ºC for 7 days. Amplification of internal transcribed spacer ( ), elongation factor 1-α ( ) and β-tubulin ( ) genes was performed using , and primers, respectively (Jia et al. 2019). Sequences were submitted to GenBank under accession numbers MW130133 ( ), MW147481 ( ) and MW147482 ( ). Blast search revealed that the amplified sequences had 99.65% ( ; JZB310202, MN945381), 100% ( ; ZB-77, MH726166) and 99.75% ( ; ZB-1, MN642587) matches to multiple strains, whereas all reported strains showed no nucleotide identity to the amplified sequences. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. , and sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-4291.3981) is shown in Figure 1. strains found causing stem canker on soybean, some strains (which also cause dried-up stem on soybean) (Yang et al. 2019), and strains found in China in other hosts were included in the phylogenetic tree. To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem of 2-week old soybean plants. Mycelial plugs from a 7 day-old culture on PDA were placed on the wounds and covered with Parafilm. Sterilized PDA plugs were used as control. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity. Typical stem canker symptoms were observed 5 days after inoculation (Figure 2). Microscope observations showed the presence of septate mycelium,