Vrandecic K, Cosic J, Jurkovic D, Duvnjak T, Riccioni L
Faculty of Agriculture in Osijek, 31000 Osijek, Croatia.
Agricultural Institute in Osijek, 31000 Osijek, Croatia.
Plant Dis. 2009 Oct;93(10):1074. doi: 10.1094/PDIS-93-10-1074C.
Sunflower (Helianthus annuus L.) is a crop that is grown worldwide for the production of edible oil. In Croatia, it has considerable economic significance. From 2004 to 2007, sunflower stems showed light-to-dark brown lesions of different sizes and shapes. The lesions were observed for the presence of pycnidia in affected areas. Isolations from infected tissue on potato dextrose agar (PDA) yielded in two fungal species. One, which was isolated in most cases, was the well known sunflower pathogen Diaporthe helianthi Munt. Cvet. Morphological characteristics, stromata pattern, formation of alpha and beta conidia, and ascostromata characteristic of the other isolated fungus matched the description of D. phaseolorum (Cooke & Ellis) Sacc. (2). D. phaseolorum frequency was 5%. On PDA, the fungus formed white, floccose, aerial mycelium that filled a petri dish (9 cm) in 6 days. D. phaseolorum produces conidiomata in black stromatic structures, which consist of pycnidia with alpha and beta conidia. The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform, and 5.6 to 10.0 × 1.9 to 4.8 μm. The beta conidia were hyaline, elongated, filiform, straight, curved at one or both ends, and 11.7 to 27.6 × 0.7 to 2.0 μm. After 50 days, perithecia were formed. Asci were clavate and 27.64 to 40.1 × 5.70 to 8.2 μm. Eight ascospores formed within asci. Ascospores were two-celled, elliptic, hyaline, and slightly constricted at the septa, and 8.93 to 13.5 × 2.1 to 4.0 μm. Amplification and sequencing of the internal transcribed spacer (ITS) rDNA region were performed with ITS4 and ITS5 universal primers (3) on two isolates (Su9 and Su10) and data were deposited in GenBank (Accession Nos. GQ149763 and GQ149764). Comparison of sequences available in GenBank revealed that the ITS sequence was identical to D. phaseolorum found on Stokesia laevis Hill (Greene) (U11323/U11373) and identical to the strain CBS 116020 isolated from Aster exilis Elliot. (AY745018). On the basis of the obtained results of morphological characteristics and molecular approaches, the pathogen was identified as D. phaseolorum. Pathogenicity evaluation consisted of artificial infections on field-grown sunflower plants at the full button stage as described by Bertrand and Tourvielle (1). A leaf test was done by placing a mycelial plug of 5 × 5 mm from a cork borer of two isolates (Su9 and Su10) on the tip of the main vein. The inoculation site was covered with moistened, cotton wool and wrapped in aluminum foil to prevent the inoculum from drying out. Ten plants of each of the four replications were inoculated. Control plants were inoculated with pure PDA plugs. Lesions of 12 to 40 mm long were observed on the sunflower leaf 10 days after inoculation. Control plants did not develop symptoms. The pathogen was reisolated from the infected plants. To our knowledge, this is the first report of the finding of D. phaseolorum on sunflower in Croatia and we have no literature data about the occurrence of this fungus on sunflower in the world. References: (1) F. Bertrand and D. Tourvielle. Inf. Tech. CETIOM 98:12,1972. (2) E. Punithalingma and P. Holliday. No. 336 in: Descriptions of Pathogenic Fungi and Bacteria. CMI/CAB, Kew, Surrey, England, 1972. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.
向日葵（Helianthus annuus L.）是一种在全球范围内种植用于生产食用油的作物。在克罗地亚，它具有相当重要的经济意义。2004年至2007年期间，向日葵茎出现了大小和形状各异的浅褐色至深褐色病斑。在病斑处观察到分生孢子器的存在。从感染组织在马铃薯葡萄糖琼脂（PDA）上进行分离培养，得到了两种真菌。其中一种在大多数情况下被分离出来，是著名的向日葵病原菌向日葵间座壳菌（Diaporthe helianthi Munt. Cvet.）。另一种分离真菌的形态特征、子座模式、α和β分生孢子的形成以及子囊壳特征与菜豆间座壳菌（D. phaseolorum (Cooke & Ellis) Sacc.）（2）的描述相符。菜豆间座壳菌的出现频率为5%。在PDA上，该真菌形成白色、絮状的气生菌丝体，6天内可布满一个9厘米的培养皿。菜豆间座壳菌在黑色子座结构中产生分生孢子盘，其中包含具有α和β分生孢子的分生孢子器。α分生孢子单细胞，透明，椭圆形至梭形，大小为5.6至10.0×1.9至4.8微米。β分生孢子透明，细长，丝状，直形，一端或两端弯曲，大小为11.7至27.6×0.7至2.0微米。50天后形成子囊壳。子囊呈棒状，大小为27.64至40.1×5.70至8.2微米。每个子囊内形成8个子囊孢子。子囊孢子双细胞，椭圆形，透明，在隔膜处略有缢缩，大小为8.93至13.5×2.1至4.0微米。使用ITS4和ITS5通用引物（3）对两个分离株（Su9和Su10）的内部转录间隔区（ITS）rDNA区域进行扩增和测序，并将数据存入GenBank（登录号GQ149763和GQ149764）。与GenBank中可用序列的比较显示，ITS序列与在光滑 Stokesia（Greene）上发现的菜豆间座壳菌（U11323/U11373）相同，也与从细叶紫菀（Aster exilis Elliot）分离的CBS 116020菌株（AY745018）相同。根据获得的形态特征和分子方法的结果，该病原菌被鉴定为菜豆间座壳菌。致病性评估包括按照Bertrand和Tourvielle（1）所述，在田间生长的处于全钮期的向日葵植株上进行人工接种。通过将来自两个分离株（Su9和Su）的5×5毫米菌丝块从打孔器上放置在主脉尖端进行叶片接种试验。接种部位用湿棉球覆盖，并用铝箔包裹以防止接种物干燥。每个重复接种10株植物，共4个重复。对照植株接种纯PDA菌块。接种10天后，在向日葵叶片上观察到12至40毫米长的病斑。对照植株未出现症状。从感染植株上重新分离出病原菌。据我们所知，这是在克罗地亚向日葵上发现菜豆间座壳菌的首次报道，并且我们没有关于该真菌在世界范围内向日葵上发生情况的文献数据。参考文献：（1）F. Bertrand和D. Tourvielle。《信息与技术》CETIOM 98:12，1972年。（2）E. Punithalingma和P. Holliday。《病原真菌和细菌描述》第336号。CMI/CAB，英国萨里郡邱园，1972年。（3）T. J. White等人。《PCR协议：方法与应用指南》第315页。M. A. Innis等人编。学术出版社有限公司，纽约，1990年。
