Billones R G, Ridgway H J, Jones E E, Jaspers M V
Faculty of Agriculture and Life Sciences, Lincoln University, Canterbury, New Zealand.
Plant Dis. 2010 Dec;94(12):1504. doi: 10.1094/PDIS-07-10-0494.
In a 2008 survey, 120 isolates of the Botryosphaeriaceae were recovered from a representative subsample of Vitis vinifera plants and propagation materials collected in nine New Zealand grapevine nurseries. Isolates were identified by amplified ribosomal DNA restriction analysis (ARDRA) (1) as Neofusicoccum luteum (56%), N. parvum (18%), N. australe (8%), Diplodia mutila (7%), Botryosphaeria dothidea (5%), D. seriata (3%), and N. ribis (2%). One isolate (M353) from 1 cm below the graft union of a nonsymptomatic 1-year-old grafted plant from the Nelson Region was not identified by ARDRA and was morphologically distinct from all others. Mycelium produced by the novel isolate on potato dextrose agar (PDA) was initially moderately dense, flat, and white and turned olivaceous brown within 10 days. The isolate did not produce pycnidia in PDA or prune extract agar, but when grown in water agar with sterile pine needles for 8 weeks at 25°C and a 12-h light/dark regimen, small, black pycnidia covered with mycelium were produced but no conidia were observed. To identify the novel fungus, genomic DNA was extracted and the ribosomal DNA (rDNA), β-tubulin gene, and elongation factor α-1 gene were amplified and sequenced (4). The sequences of the PCR products were compared with sequences present on GenBank. The rDNA (503 bp), β-tubulin (371 bp), and elongation factor α-1 gene (227 bp) sequences of M353 were 100% identical to reported sequences of N. macroclavatum on GenBank (Accession No. DQ093199/198/196 for rDNA, DQ093207/206 for β-tubulin, and DQ093219/217 for elongation factor α-1). These genes differed from the same genes in other Neofusicoccum species by at least 11, 2, and 3 base pairs, respectively. The N. macroclavatum isolate was tested for pathogenicity on wounded grapevine (Sauvignon blanc) green shoots and 1-year-old rooted canes (n = 4 per plant type) using mycelium plugs from a 4-day-old PDA culture. Sterile agar was used for the negative control. Green shoots inoculated with N. macroclavatum developed brown lesions with an average length of 40.5 mm 6 days after inoculation. Bark from inoculated 1-year-old canes was peeled off 28 days after inoculation and brown-to-black lesions on the wood, with an average length of 52 mm, were observed. Control plants produced no lesions. The pathogen was consistently reisolated from the inoculated plants while none were found in negative control plants. To our knowledge, this is the first report of N. macroclavatum as a pathogen of grapevines and the first report of its presence in New Zealand (3). N. macroclavatum was first reported as a pathogen of Eucalyptus globulus in Western Australia in 2005 and has not been reported as a pathogen of grapevines (2). References: (1) A. Alves et al. FEMS Microbiol. Lett. 245:221, 2005. (2) T. T. Burgess et al. Australas. Plant Pathol. 34:557, 2005. (3) J. Sammonds et al. N. Z. Plant Prot. 62:248, 2009. (4) B. Slippers et al. Mycologia 96:83, 2004.
在2008年的一项调查中，从新西兰9个葡萄苗圃采集的葡萄植株和繁殖材料的代表性子样本中分离出120株葡萄座腔菌科菌株。通过扩增核糖体DNA限制性分析（ARDRA）（1）将菌株鉴定为 luteum新壳梭孢（56%）、细小新壳梭孢（18%）、南方新壳梭孢（8%）、残损色二孢（7%）、葡萄座腔菌（5%）、seriata色二孢（3%）和ribis新壳梭孢（2%）。从尼尔森地区一株无症状的1年生嫁接植株嫁接部位以下1厘米处分离得到的一个菌株（M353），经ARDRA鉴定未成功，且在形态上与其他所有菌株不同。该新分离菌株在马铃薯葡萄糖琼脂（PDA）上产生的菌丝体最初中等致密、扁平且呈白色，10天内变为橄榄褐色。该菌株在PDA或李子提取物琼脂中不产生分生孢子器，但在含有无菌松针的水琼脂中于25°C、12小时光照/黑暗条件下培养8周时，会产生覆盖着菌丝体的黑色小分生孢子器，但未观察到分生孢子。为鉴定该新真菌，提取了基因组DNA，并对核糖体DNA（rDNA）、β-微管蛋白基因和延伸因子α-1基因进行了扩增和测序（4）。将PCR产物的序列与GenBank上的序列进行比较。M353的rDNA（503 bp）、β-微管蛋白（371 bp）和延伸因子α-1基因（227 bp）序列与GenBank上报道的大孢新壳梭孢序列100%相同（rDNA登录号为DQ093199/198/196，β-微管蛋白登录号为DQ093207/206，延伸因子α-1登录号为DQ093219/217）。这些基因与其他新壳梭孢属物种的相同基因分别至少相差11、2和3个碱基对。使用4日龄PDA培养物的菌丝体小块，对大孢新壳梭孢菌株在受伤的葡萄（长相思）嫩梢和1年生根蘖上进行致病性测试（每种植株类型n = 4）。无菌琼脂用作阴性对照。接种大孢新壳梭孢的嫩梢在接种6天后出现平均长度为40.5毫米的褐色病斑。接种1年生根蘖的树皮在接种28天后被剥下，观察到木质部上有平均长度为52毫米的褐色至黑色病斑。对照植株未产生病斑。从接种植株中始终能重新分离出病原菌，而在阴性对照植株中未发现病原菌。据我们所知，这是大孢新壳梭孢作为葡萄病原菌的首次报道，也是其在新西兰存在的首次报道（3）。大孢新壳梭孢于2005年在西澳大利亚首次被报道为蓝桉的病原菌，尚未被报道为葡萄的病原菌（2）。参考文献：（1）A. Alves等人，《FEMS微生物学快报》245:221，2005年。（2）T. T. Burgess等人，《澳大利亚植物病理学》34:557，2005年。（3）J. Sammonds等人，《新西兰植物保护》62:248，2009年。（4）B. Slippers等人，《真菌学》96:83，2004年。
