Riccioni L, Haegi A, Valvassori M
Consiglio per la Ricerca e la Sperimentazione in Agricoltura-Centro di Ricerca per la Patologia Vegetale (CRA-PAV), Via C.G. Bertero 22, I-00156 Rome, Italy.
Plant Dis. 2008 Jul;92(7):1132. doi: 10.1094/PDIS-92-7-1132C.
Lentil (Lens culinaris Medik.) is a traditional crop in Sicily, Italy. Near Villalba (Caltanissetta), a local lentil landrace, "Lenticchia di Villalba", is commonly grown. From 2002 to 2004, wilt was observed in five lentil fields (≈1 ha each) at rates from 5 to 20%. Affected plants were yellow and stunted with discoloration in the vascular tissue of stems and crowns. Pieces of brown vascular tissue from stems were disinfested in 2% sodium hypochlorite for 2 min, rinsed with sterile distilled water, placed on potato dextrose agar, and incubated at 23°C. Isolates with morphological characteristics of Fusarium oxysporum Schlecht.:Fr. (2) were consistently recovered from affected plants. For molecular identification of five isolates, the rDNA internal transcribed spacer (ITS) region and a portion of the elongation factor EF-1α were sequenced using ITS5/4 and EF1/2 primers, respectively (1). Two sequences of the ITS region were obtained: a 468-bp sequence from isolates ER1259, ER1260, and ER1275 (submitted as GenBank Accession No. EU159118) and a 483-bp sequence from isolates ER1274 and ER1276 (submitted as GenBank Accession No. EU281661). The two sequences shared 93% similarity. A sequence homology search using the NCBI BLAST program revealed that the first sequence had 100% homology with the ITS sequences of more than 50 F. oxysporum isolates of various formae speciales in GenBank and the second shared 100% homology with the ITS sequences of five isolates of F. redolens Wollenw. (e.g., GenBank Accession No. X94169 of the strain CBS 360.87). Amplification of the EF-1α produced a sequence from isolates ER1274 and ER1276 (submitted as GenBank Accession No. EU281660) with 99 to 100% homology to sequences of F. redolens and a sequence from strains ER1259, ER1275, and ER1260 (submitted as GenBank Accession No. EU281659) with 100% homology to that of more than 50 F. oxysporum strains in GenBank. Although F. redolens and F. oxysporum are morphologically similar, recent molecular studies have shown that they are distinct and phylogenetically distant species (3). On the basis of genetic sequences, isolates ER1274 and ER1276 were identified as F. redolens. These isolates were evaluated for pathogenicity on lentil. For each isolate, 10 2-week-old seedlings of "Lenticchia di Villalba" were inoculated by submerging roots in a suspension of 2.5 × 10 conidia/ml for 10 min. Plants were put into separate tubes containing 70 ml of a nutritional liquid medium (7 ml of HydroPlus Olikani per liter; Yara, Nanterre, France) and incubated in a growth chamber at 20°C with 12 h of light per day. Seedlings dipped in sterile water served as the control treatment. The pathogenicity test was repeated twice. Inoculated seedlings started to wilt 1 week after inoculation and developed root rot and vascular discoloration. After 2 weeks, 70% of the inoculated plants were affected by both isolates and 40 and 10% died when inoculated with ER1274 and ER1276 isolates, respectively. F. redolens was consistently reisolated from the stems of wilted plants. Noninoculated plants remained healthy. Currently, only F. oxysporum f. sp. lentis Vasud. and Sriniv. has been reported as the cause of Fusarium wilt of lentil. To our knowledge, this is the first report of F. redolens as a pathogen on lentil. References: (1) R. P. Baayen et al. Phytopathology 91:1037, 2001. (2) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (3) K. O'Donnell et al. Mycologia 90:465, 1998.
小扁豆（Lens culinaris Medik.）是意大利西西里岛的一种传统作物。在卡尔塔尼塞塔省的维拉巴附近，当地一种名为“Villalba小扁豆”的小扁豆地方品种普遍种植。2002年至2004年期间，在五个小扁豆田（每个田约1公顷）中观察到枯萎病，发病率为5%至20%。受影响的植株发黄且生长受阻，茎和冠部的维管组织变色。将茎部褐色维管组织切块在2%次氯酸钠中消毒2分钟，用无菌蒸馏水冲洗，置于马铃薯葡萄糖琼脂上，在23°C下培养。 consistently recovered from affected plants. For molecular identification of five isolates, the rDNA internal transcribed spacer (ITS) region and a portion of the elongation factor EF-1α were sequenced using ITS5/4 and EF1/2 primers, respectively (1). Two sequences of the ITS region were obtained: a 468-bp sequence from isolates ER1259, ER1260, and ER1275 (submitted as GenBank Accession No. EU159118) and a 483-bp sequence from isolates ER1274 and ER1276 (submitted as GenBank Accession No. EU28;661). The two sequences shared 93% similarity. A sequence homology search using the NCBI BLAST program revealed that the first sequence had 100% homology with the ITS sequences of more than 50 F. oxysporum isolates of various formae speciales in GenBank and the second shared 100% homology with the ITS sequences of five isolates of F. redolens Wollenw. (e.g., GenBank Accession No. X94169 of the strain CBS 360.87). Amplification of the EF-1α produced a sequence from isolates ER1274 and ER1276 (submitted as GenBank Accession No. EU281660) with 99 to 100% homology to sequences of F. redolens and a sequence from strains ER1259, ER1275, and ER1260 (submitted as GenBank Accession No. EU281659) with 100% homology to that of more than 50 F. oxysporum strains in GenBank. Although F. redolens and F. oxysporum are morphologically similar, recent molecular studies have shown that they are distinct and phylogenetically distant species (3). On the basis of genetic sequences, isolates ER1274 and ER1276 were identified as F. redolens. These isolates were evaluated for pathogenicity on lentil. For each isolate, 10 2-week-old seedlings of "Lenticchia di Villalba" were inoculated by submerging roots in a suspension of 2.5 × 10 conidia/ml for 10 min. Plants were put into separate tubes containing 70 ml of a nutritional liquid medium (7 ml of HydroPlus Olikani per liter; Yara, Nanterre, France) and incubated in a growth chamber at 20°C with 12 h of light per day Seedlings dipped in sterile water served as the control treatment. The pathogenicity test was repeated twice. Inoculated seedlings started to wilt 1 week after inoculation and developed root rot and vascular discoloration. After 2 weeks, 70% of the inoculated plants were affected by both isolates and 40 and 10% died when inoculated with ER1274 and ER1276 isolates, respectively. F. redolens was consistently reisolated from the stems of wilted plants. Noninoculated plants remained healthy. Currently, only F. oxysporum f. sp. lentis Vasud. and Sriniv. has been reported as the cause of Fusarium wilt of lentil. To our knowledge, this is the first report of F. redolens as a pathogen on lentil. References: (1) R. P. Baayen et al. Phytopathology 91:1037, 2001. (2) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (3) K. O'Donnell et al. Mycologia 90:465, 1998.
从受影响的植株中一直分离到具有尖孢镰刀菌（Fusarium oxysporum Schlecht.:Fr.）形态特征的菌株(2)。为对五个分离菌株进行分子鉴定，分别使用ITS5/4和EF1/2引物对rDNA内转录间隔区（ITS）和延伸因子EF-1α的一部分进行测序(1)。获得了ITS区域的两个序列：分离菌株ER1259、ER1260和ER1275的一个468 bp序列（提交为GenBank登录号EU159118）以及分离菌株ER1274和ER1276的一个483 bp序列（提交为GenBank登录号EU281661）。这两个序列的相似性为93%。使用NCBI BLAST程序进行的序列同源性搜索显示，第一个序列与GenBank中50多个不同专化型尖孢镰刀菌分离株的ITS序列具有100%的同源性，第二个序列与五株变红镰刀菌（F. redolens Wollenw.）分离株的ITS序列具有100%的同源性（例如，菌株CBS 360.87的GenBank登录号X94169）。对EF-1α的扩增产生了分离菌株ER1274和ER1276的一个序列（提交为GenBank登录号EU,81660），与变红镰刀菌的序列具有99%至100%的同源性；以及分离菌株ER1259、ER1275和ER1260的一个序列（提交为GenBank登录号EU281659），与GenBank中50多个尖孢镰刀菌菌株的序列具有100%的同源性。尽管变红镰刀菌和尖孢镰刀菌在形态上相似，但最近的分子研究表明它们是不同的且在系统发育上距离较远的物种(3)。基于基因序列，分离菌株ER1274和ER1276被鉴定为变红镰刀菌。对这些分离菌株进行了对小扁豆的致病性评估。对于每个分离菌株，将10株2周龄的“Villalba小扁豆”幼苗的根部浸入2.5×10个分生孢子/ml的悬浮液中10分钟进行接种。将植株放入装有70 ml营养液培养基（每升7 ml HydroPlus Olikani；雅苒公司，法国楠泰尔）的单独试管中，在生长室中于20°C下培养，每天光照12小时。浸入无菌水中的幼苗作为对照处理。致病性试验重复两次。接种的幼苗在接种后1周开始枯萎，并出现根腐和维管组织变色。2周后，70%的接种植株受到两种分离菌株的影响，接种ER1274和ER1276分离菌株的植株分别有40%和10%死亡。从枯萎植株的茎中一直重新分离到变红镰刀菌。未接种的植株保持健康。目前，仅尖孢镰刀菌扁豆专化型（F. oxysporum f. sp. lentis Vasud. and Sriniv.）被报道为小扁豆枯萎病的病因。据我们所知，这是关于变红镰刀菌作为小扁豆病原菌的首次报道。参考文献：(1) R. P. Baayen等人，《植物病理学》91:1037，2001年。(2) P. E. Nelson等人，《镰刀菌属：鉴定图谱手册》。宾夕法尼亚州立大学出版社，大学园，1983年。(3) K. O'Donnell等人，《真菌学》90:465，1998年。
