Abstract：
A survey of Diaporthe/Phomopsis Complex (DPC) species was carried out on 479 asymptomatic soybean (Glycine max (L.) Merrill) seed samples collected from commercial soybean fields in the states of Santa Catarina (20 counties) and Rio Grande do Sul (41 counties), in the 2020/21 (n=186), 2021/22 (n=138) and 2022/23 (n=155) seasons from 120 cultivars. The seeds were provided by seed producers who collected according to the sampling standard of the Ministry of Agriculture, Livestock and Food Supply. From each sample received, 200 symptomless seeds were randomly sorted out. The seeds were surface disinfected by immersion in a sodium hypochlorite solution (1%) for two minutes and placed on Potato Dextrose Agar (PDA). The plates were incubated for 7 days at 23°C with a photoperiod of 12-h. The average prevalence of 73.7% of DPC-infected seeds. Colonies were isolated by transferring mycelial tips to PDA and incubating for 14 days at 25ºC in a 12-h photoperiod. One colony (isolate MEMR0500) had morphological characteristics similar to those reported in Lopez-Cardona (2021). This isolate had a floccose, dense colony ranging from grayish beige to brown with greenish regions and black globose pycnidia (3 to 4 pycnidia/cm²). Alpha-conidia, 5.1 to 7.0 µm x 1.5 to 2.8 µm, were observed after 30 days and were hyaline, aseptate and fusiform (Figure S1). No beta-conidia were observed. Soybean plants of cultivars BMX Cromo IPRO, BMX Zeus IPRO, BRS 5804 RR, FPS 1867 IPRO and NEO 750 IPRO were tested for pathogenicity using the toothpick inoculation method (Siviero and Menten 1995). Non-colonized toothpicks served as a negative control. Plants were incubated for four days at 25°C and 90% relative humidity. Elongated 1.0 to 2.5 cm x 0.5 to 0.9 cm lesions gray-brown/reddish-brown with a depressed center were observed in all inoculated cultivars. The fungus was reisolated and the characteristics of the colonies were identical to those previously isolated. For molecular characterization, DNA was extracted from the mycelia using the CTAB method (Doyle and Doyle 1990). End-point PCR was performed using GoTaq® Flexi DNA Polymerase (Promega, USA) and primer pairs, ITS-4F/ITS-5, T2/Bt2b and EF1-728F/EF1-986R to amplify the internal transcribed spacer (ITS) (Costamilan et al. 2008), β-tubulin (TUB2) (Glass and Donaldson 1995), and translation elongation factor 1-α (TEF1) (Carbone and Kohn 1999) genes, respectively. The amplified fragments were sequenced and submitted to blast search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the sequences available in GenBank. The fragment from ITS (accession number OR912979) showed 99.8% (549/582 bp) identity with Diaporthe ueckeri Udayanga & Castl. [as \'ueckerae\'] [syn. D. miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan] isolate FAU656 (Ac. N. KJ590726). The sequence of TEF (Ac. N. PP372869) showed 99.7% (339/355 bp) identity with D. ueckeri FAU656 (Ac. N. KJ590747), and of TUB (Ac. N. PP372870) showed 98.9% (436/536 bp) identity with D. ueckeri FAU656 (Ac. N. KJ610881). A phylogenetic tree with amplified sequences of each gene and the corresponding representative sequences from the DPC was constructed in MEGA X (Kumar et al. 2018). The MEMR0500 isolate was clustered only with the D. ueckeri clade, confirming the identity of the fungus (Figure S2). In Brazil, this is the first report of the association of this pathogen with soybean seeds. In other countries, this pathogen has been identified as the causal agent of stem canker (Mena et al. 2020; Lopez-Cardona et al. 2021). Further research is needed to analyze the risk of this seed-associated pathogen.
摘要：
对479种无症状大豆（Glycinemax（L.）美林）从圣卡塔琳娜州（20个县）和南里奥格兰德州（41个县）的商业大豆田收集的种子样本，在2020/21年度（n=186），来自120个品种的2021/22（n=138）和2022/23（n=155）季节。种子由按农业部抽样标准采集的种子生产者提供,牲畜和粮食供应。从收到的每个样本中，随机挑选出200个无症状种子。通过浸入次氯酸钠溶液(1%)2分钟对种子进行表面消毒,并置于马铃薯葡萄糖琼脂(PDA)上。将板在23°C下以12小时的光周期孵育7天。DPC感染种子的平均患病率为73.7%。通过将菌丝体尖端转移至PDA并在25ºC下在12小时光周期中孵育14天来分离菌落。一个菌落（分离株MEMR0500）的形态特征与Lopez-Cardona（2021）中报道的形态特征相似。这种分离物具有絮凝剂，密集的菌落范围从浅灰色米色到棕色有绿色的区域和黑色球形的比尼迪亚（3到4比尼迪亚/平方厘米）。阿尔法分生孢子,5.1至7.0µmx1.5至2.8µm,30天后观察到透明，无盐和梭形（图S1）。没有观察到β-分生孢子。大豆品种BMXCromoIPRO，BMXZeusIPRO,BRS5804RR,使用牙签接种方法测试了FPS1867IPRO和NEO750IPRO的致病性（Siviero和Menten1995）。非定殖牙签用作阴性对照。将植物在25°C和90%相对湿度下孵育4天。在所有接种品种中均观察到1.0至2.5cmx0.5至0.9cm的灰褐色/红褐色病变，中心凹陷。重新分离真菌，菌落的特征与先前分离的那些相同。对于分子表征，使用CTAB方法(Doyle和Doyle1990)从菌丝体中提取DNA。使用GoTaq®FlexiDNA聚合酶（Promega，美国)和引物对，ITS-4F/ITS-5，T2/Bt2b和EF1-728F/EF1-986R用于扩增内部转录间隔区（ITS）（Costamilan等人。2008),β-微管蛋白（TUB2）（Glass和Donaldson1995），和翻译延伸因子1-α（TEF1）（Carbone和Kohn1999）基因，分别。对扩增的片段进行测序并提交到blast搜索(https://blast。ncbi.nlm.nih.gov/Blast.cgi）与GenBank中可用的序列。来自ITS的片段（登录号OR912979）与DiaportheueckeriUdayanga和Castl显示99.8％（549/582bp）的同一性。[as\'ueckerae\'][syn。D.奇迹R.G.Shivas,S.M.Thomps.&Y.P.Tan]分离FAU656（Ac。N、KJ590726)。TEF的序列(Ac。N.PP372869）与D.ueckeriFAU656（Ac。N.KJ590747)，和TUB（AC。N.PP372870)与D.ueckeriFAU656(Ac.N、KJ610881）。在MEGAX中构建了具有每个基因的扩增序列和来自DPC的相应代表性序列的系统发育树（Kumar等人。2018)。MEMR0500分离株只与D.ueckeri进化枝聚集，确认真菌的身份（图S2）。在巴西,这是该病原体与大豆种子关联的第一份报告。在其他国家,这种病原体已被确定为茎溃疡的病原体(Mena等人。2020；洛佩兹-卡多纳等人。2021)。需要进一步的研究来分析这种种子相关病原体的风险。
