Jones E E, Brown D S, Bleach C M, Pathrose B, Barclay C, Jaspers M V, Ridgway H J
Department of Ecology, Faculty of Agriculture and Life Sciences, Lincoln University, New Zealand.
Plant Dis. 2012 Jan;96(1):144. doi: 10.1094/PDIS-04-11-0347.
Isolates morphologically identified as Cylindrocladiella parva were isolated from characteristic black foot symptoms on a grapevine (Vitis vinifera) rooted on 101-14 rootstock from Central Otago in 2005 and 101-14 rootstocks from a nursery in the Auckland Region in 2007 and 2008. On potato dextrose agar, the isolates initially produced cottony, white mycelia that turned grayish cream or golden cream within 10 days, the initially tawny colony undersides becoming dark brown with age. Conidia (0 to 1 septate; 16.4 to 17.0 [16.7] × 2.3 to 2.6 [2.5] μm) and abundant chlamydospores were produced. To confirm identity of the isolates, genomic DNA was extracted and the ribosomal DNA (rDNA) and β-tubulin gene were amplified and sequenced (3,4). Sequences of the PCR products were compared with sequences in GenBank. The rDNA (535 bp) and β-tubulin (297 bp) sequences of the four isolates were 100 and 99% identical, respectively, to reported sequences of C. parva in GenBank (AY793454, grapevine isolate (4)/AY793455 for rDNA; AY793486/AY793488, grapevine isolate (4)/AY793489/HM034822 for β-tubulin). Although C. parva was previously isolated from grapevines in New Zealand (2) and rootstocks of mature grapevines, cuttings, and graft unions of grafted young grapevines in South Africa (4), its role as a pathogen of Vitis spp. has not been confirmed (2,4). However, it has been reported as a pathogen of Eucalyptus spp. (1) and was also isolated from Telopea speciosissima and Macadamia integrifolia in New Zealand (2,4). The C. parva isolates were tested as a mixed inoculum (four isolates) for pathogenicity on roots of 10 grapevine rootstock plants each of cvs. 101-14 and Schwarzmann (Sch). The rootstocks were grown in potting mix for 4 months, after which the root systems of all vines were wounded with an asparagus knife with a sharp, square tip, driven vertically down into the soil at four equidistant locations approximately 8 cm from the trunk. Each plant was inoculated with 50 ml of the mixed-isolate conidial suspension (10/ml), or 50 ml water (controls), followed by 50 ml of water. After 7 months of growth, the plants were harvested. For C. parva-inoculated plants, internal blackening of the stem base tissue was observed. Isolations from surface-sterilized trunk bases recovered C. parva from four and nine plants of 101-14 and Sch, respectively, with C. parva infections in 25 and 48%, respectively, of the four wood pieces taken per plant. Plants inoculated with water had no blackening and no C. parva was isolated from their stem bases. Mean shoot dry weights of inoculated plants (17.9 and 15.0 g for 101-14 and Sch, respectively) were significantly lower (P = 0.035) than noninoculated controls (26.5 and 20.0 g for 101-14 and Sch, respectively). Mean root dry weights were reduced by C. parva inoculation, although not significantly (32.7 and 27.0 g for C. parva inoculated 101-14 and Sch, respectively, and 36.2 and 27.4 g for control 101-14 and Sch, respectively). To our knowledge, this is the first report of C. parva as a pathogen of grapevines (2,4) and suggests that along with Cylindrocarpon spp., C. parva is part of the pathogen complex responsible for black foot of grapevines. References: (1) P. W. Crous et al. Plant Pathol. 42:302, 1993. (2) P. D. Gadgil et al. Fungi on Trees and Shrubs in New Zealand. Fungal Diversity Press, Hong Kong, 2005. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) G. J. van Coller et al. Australas. Plant Pathol. 34:489, 2005.
2005年，从奥塔哥中部一株嫁接到101 - 14砧木上的葡萄（欧亚种葡萄）出现的典型黑脚症状中，分离出形态学上鉴定为小柱盘多毛孢菌的菌株；2007年和2008年，又从奥克兰地区一个苗圃的101 - 14砧木上出现的类似症状中分离出该菌株。在马铃薯葡萄糖琼脂培养基上，这些分离菌株最初产生棉絮状白色菌丝体，10天内变为浅灰奶油色或金黄奶油色，最初黄褐色的菌落底面随着时间推移变为深褐色。产生了分生孢子（0至1个隔膜；16.4至17.0 [16.7]×2.3至2.6 [2.5]μm）和大量厚垣孢子。为确认分离菌株的身份，提取了基因组DNA，并对核糖体DNA（rDNA）和β - 微管蛋白基因进行了扩增和测序（3,4）。将PCR产物的序列与GenBank中的序列进行比较。四个分离菌株的rDNA（535 bp）和β - 微管蛋白（297 bp）序列，分别与GenBank中报道的小柱盘多毛孢菌序列（rDNA的AY793454，葡萄分离株(4)/AY793455；β - 微管蛋白的AY793486/AY793488，葡萄分离株(4)/AY793489/HM034822）有100%和99%的同一性。尽管此前在新西兰（2）以及南非的成熟葡萄藤、插条和嫁接幼葡萄藤的嫁接结合处都分离出过小柱盘多毛孢菌（小柱盘多毛孢）（4），但其作为葡萄属植物病原体的作用尚未得到证实（2,4）。然而，它已被报道为桉属植物的病原体（1），并且在新西兰也从特洛皮阿花和澳洲坚果中分离出来（2,4）。将小柱盘多毛孢菌的分离菌株作为混合接种物（四个分离菌株），对10株101 - 14和施瓦茨曼（Sch）葡萄砧木品种的根系进行致病性测试。砧木在盆栽基质中生长4个月，之后用一把带有尖锐方形尖端的芦笋刀在所有葡萄藤根系的四个等距位置垂直向下插入距树干约8厘米处的土壤中造成伤口。每株植物接种50毫升混合分离菌株的分生孢子悬浮液（10个/毫升），或50毫升水（对照），随后再浇50毫升水。生长7个月后收获植株。对于接种小柱盘多毛孢菌的植株，观察到茎基部组织内部变黑。从表面消毒的树干基部进行分离，分别从101 - 14品种的4株和施瓦茨曼品种的9株植株中分离出小柱盘多毛孢菌，每株植物取的四块木材中，小柱盘多毛孢菌感染率分别为25%和48%。接种水的植株没有变黑现象，其茎基部也未分离出小柱盘多毛孢菌（小柱盘多毛孢）。接种植株的平均地上部干重（101 - 14品种为17.9克，施瓦茨曼品种为15.0克）显著低于未接种对照（101 - 14品种为26.5克，施瓦茨曼品种为20.0克）（P = 0.035）。接种小柱盘多毛孢菌使平均根干重降低，尽管不显著（接种小柱盘多毛孢菌的101 - 14品种和施瓦茨曼品种分别为32.7克和27.0克，对照101 - 14品种和施瓦茨曼品种分别为36.2克和27.4克）。据我们所知，这是小柱盘多毛孢菌作为葡萄藤病原体的首次报道（2,4），表明小柱盘多毛孢菌与柱孢属真菌一样，是导致葡萄黑脚病的病原体复合体的一部分。参考文献：(1) P. W. Crous等人，《植物病理学》42:302，1993年。(2) P. D. Gadgil等人，《新西兰树木和灌木上的真菌》，真菌多样性出版社，香港，2005年。(3) N. L. Glass和G. C. Donaldson，《应用与环境微生物学》61:1323，1995年。(4) G. J. van Coller等人，《澳大利亚植物病理学》34:489，2005年。
