First Report of 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 ddHO. 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, fusiform conidia and round conidiomata, and agreed with those previously reported for the morphology of strains (Phillips et al. 2013). During recent months, was reported to cause stem canker and leave wilt on a number of plant species in China (Huang et al. 2020; Ju et al. 2020; Wang et al. 2020a, 2020b, 2020c), confirming the expansion and host promiscuity of this pathogen. Stem canker on soybean has been thoroughly associated to strains; however, this is the first report of causing this disease. We believe that our results will help to better understand the pathogens affecting soybean production in China.