Phytopathology. 1998 Dec;88(12):1306-14. doi: 10.1094/PHYTO.1998.88.12.1306.
ABSTRACT Diaporthe phaseolorum and Phomopsis longicolla isolates from soybean were examined using traditional mycological characteristics and molecular methods. Cultural characteristics including types of fruiting bodies and conidia were assessed for isolates collected from soybean stems and seeds. Cultures were identified as P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, or D. phaseolorum var. sojae. Molecular markers for these groups were developed and analyzed using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP) and DNA sequencing in the internal transcribed spacer (ITS) and the 5.8S ribosomal DNA. The ITS(4) and ITS(5) primers amplified PCR products for all isolates studied. Gel electrophoresis of undigested PCR products and DNA sequencing produced various fragment lengths including 604 bp for P. longicolla, 602 and 603 bp for D. phaseolorum var. caulivora, 603 bp for D. phaseolorum var. meridionalis, and from 597 to 609 bp for D. phaseolorum var. sojae. Digestion of these PCR products with enzymes AluI, HhaI, MseI, RsaI, and ScrFI resulted in distinct bands for identification of P. longicolla and the varieties of D. phaseolorum I. All P. longicolla, D. phaseolorum var. caulivora, and D. phaseolorum var. meridionalis isolates were distinguished using AluI and HhaI with RsaI or ScrFI. The banding patterns of D. phaseolorum var. sojae isolates were complex and were separated into 11 subgroups after digestion with AluI, HhaI, MseI, RsaI, and ScrFI. Phylogenetic analysis of 20 isolates of D. phaseolorum and P. longicolla based on the DNA sequence of the ITS region resolved six clades termed A, B, C, D, E, and F. Clade A included all sequenced D. phaseolorum var. caulivora isolates, two from Italy and one from the United States. Isolates in clade B were exclusively associated with D. phaseolorum var. meridionalis. Clades A and B formed a well-supported monophyletic group. Isolates in clades C, D, E, and F were morphologically defined as isolates of P. longicolla, D. phaseolorum var. sojae, and Diaporthe spp. The ITS sequences similarity of seven geographically diverse P. longi-colla isolates illustrated that P. longicolla isolates have a similar genetic background, with some affiliations to some D. phaseolorum var. sojae isolates. Morphological characteristics of the isolates along with the terminal clades of the ITS phylogeny suggest that P. longicolla is an individual species, D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis are varieties of D. phaseolorum, and D. phaseolorum var. sojae is either several varieties of D. phaseolorum or possibly several distinct species.
摘要 从大豆中分离出的豌豆壳球腔菌和长蠕孢菌使用传统的真菌学特征和分子方法进行了研究。从大豆茎和种子上采集的分离物评估了包括产孢体类型和分生孢子在内的培养特征。培养物被鉴定为长蠕孢菌、豌豆壳球腔菌 var. caulivora、豌豆壳球腔菌 var. meridionalis 或豌豆壳球腔菌 var. sojae。为这些群体开发了分子标记，并使用聚合酶链反应限制性片段长度多态性 (PCR-RFLP) 和内部转录间隔区 (ITS) 和 5.8S 核糖体 DNA 中的 DNA 测序进行了分析。ITS(4) 和 ITS(5) 引物扩增了所有研究分离物的 PCR 产物。未消化 PCR 产物的凝胶电泳和 DNA 测序产生了不同的片段长度，包括长蠕孢菌的 604 bp、豌豆壳球腔菌 var. caulivora 的 602 和 603 bp、豌豆壳球腔菌 var. meridionalis 的 603 bp 以及豌豆壳球腔菌 var. sojae 的 597 至 609 bp。用酶 AluI、HhaI、MseI、RsaI 和 ScrFI 消化这些 PCR 产物可产生用于鉴定长蠕孢菌和豌豆壳球腔菌 I 变种的独特带型。所有长蠕孢菌、豌豆壳球腔菌 var. caulivora 和豌豆壳球腔菌 var. meridionalis 分离物均使用 AluI 和 HhaI 与 RsaI 或 ScrFI 进行区分。豌豆壳球腔菌 var. sojae 分离物的带型复杂，经 AluI、HhaI、MseI、RsaI 和 ScrFI 消化后分为 11 个亚组。基于 ITS 区域的 DNA 序列对 20 株豌豆壳球腔菌和长蠕孢菌的分析， resolved 六个分支群，称为 A、B、C、D、E 和 F。分支群 A 包括所有测序的豌豆壳球腔菌 var. caulivora 分离物，其中 2 株来自意大利，1 株来自美国。分支群 B 仅与豌豆壳球腔菌 var. meridionalis 有关。分支群 A 和 B 形成了一个支持良好的单系群。分支群 C、D、E 和 F 中的分离物在形态上被定义为长蠕孢菌、豌豆壳球腔菌 var. sojae 和 Diaporthe spp. 的分离物。七个地理上不同的长蠕孢菌分离物的 ITS 序列相似性表明，长蠕孢菌分离物具有相似的遗传背景，与一些豌豆壳球腔菌 var. sojae 分离物有一定的关联。分离物的形态特征以及 ITS 系统发育的末端分支群表明，长蠕孢菌是一个独立的种，豌豆壳球腔菌 var. caulivora 和豌豆壳球腔菌 var. meridionalis 是豌豆壳球腔菌的变种，而豌豆壳球腔菌 var. sojae 可能是几个豌豆壳球腔菌的变种，也可能是几个不同的种。
