Dos Santos R F, Blume E, da Silva G B P, Lazarotto M, Scheeren L E, Zini P B, Bastos B O, Rego C
Department of Plant Pathology, Federal University of Santa Maria, Santa Maria, Brazil.
CEER Biosystems Engineering, Instituto Superior de Agronomia, Technical University of Lisbon, Lisboa, Portugal.
Plant Dis. 2014 Jun;98(6):845. doi: 10.1094/PDIS-09-13-0935-PDN.
In August 2012, symptoms of black foot disease were observed on 21-year-old grapevines (Vitis labrusca cv. Bordô; own-rooted cultivar) at Nova Pádua city, Rio Grande do Sul state, Brazil. Symptomatic plants showed reduced vigor, vascular lesions, decline and death of vines, and necrotic lesions on roots. Isolation of fungi associated with necrotic root tissue was made on potato dextrose agar (PDA) medium containing 0.5 g L streptomycin sulfate. Cultures were incubated at 25°C for 7 days in darkness, and single-spore cultures were obtained from the colonies emerging from the diseased tissue. For morphological characterization, cultures were transferred to PDA and spezieller nährstoffarmer agar (SNA) medium with addition of two pieces of 1 cm filter paper. One representative isolate (Cy9UFSM) was used for morphological and molecular characterization and pathogenicity confirmation. After 10 days growth on PDA at 20°C in the dark, colonies were umber to chestnut in color (3), appeared cottony to felty in texture, and sporulated profusely. After 5 weeks on SNA and under dark conditions at 20°C, cultures formed macroconidia predominantly on simple conidiophores, 1 to 3 septate, with both ends slightly rounded. Macroconidia varied in size depending on the number of cells as follows: one-septate (23-) 27.7 (-31) × (4.5-) 5.8 (-7) μm; two-septate (26-) 30.1 (-34) × (5-) 5.6 (-6) μm; and three-septate (24-) 31.2 (-35) × (5-) 5.8 (-6.5) μm. Microconidia were observed and did not have a visible hilum (6-) 11.2 (-17) × (3.5-) 4.2 (-5) μm (n = 30 observations per structure). Brown, thick-walled globose to subglobose chlamydospores were produced abundantly on PDA, (8.5-) 13.8 (-17) μm. To confirm the species, primer pairs H3-1a and H3-1b (2) were used to amplify a portion the histone H3 gene. Sequence of this region showed 98% similarity with a reference sequence for Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous (GenBank Accession No. JF735530). Thus, both morphological and molecular criteria supported identification of the strain as I. robusta. This isolate was deposited in GenBank as accession KF633172. To confirm pathogenicity, 4-month-old rooted cuttings of Vitis labrusca cv. Bordô were inoculated by immersing roots in a conidial suspension (10 ml) for 60 min. After inoculation, the cuttings were planted in 1-L bags containing commercial substrate (MecPlant). Thirty days later, each plant was re-inoculated by applying 40 ml of a conidial suspension (10 ml) to the commercial substrate. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. After 4 months, the inoculated plants showed a 22.5% reduction of root mass, with root and crown necrosis, browning of vessels, and 20% mortality. Control plants treated with water remained symptomless. The fungus was re-isolated from blackened tissue of wood from the basal end of rooted cuttings, thereby satisfying Koch's postulates. I. robusta was first associated with black foot disease of grapevine in Portugal in 2012 (1). To our knowledge, this is the first report in southern Brazil of I. robusta associated with black foot disease of grapevine. References: (1) A. Cabral et al. Mycol. Prog. 11:655, 2012. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) R. W. Rayner. A mycological colour chart. Commonwealth Mycological Institute and British Mycological Society, 1970.
2012年8月，在巴西南里奥格兰德州新帕杜阿市，21年生的葡萄树（美洲葡萄品种博尔代；自根品种）上观察到了黑脚病症状。有症状的植株活力下降，出现维管束病变、葡萄藤衰退和死亡，根部有坏死病变。在含有0.5 g/L硫酸链霉素的马铃薯葡萄糖琼脂（PDA）培养基上对与坏死根组织相关的真菌进行分离。将培养物在25°C黑暗条件下培养7天，从患病组织长出的菌落中获得单孢子培养物。为进行形态学特征鉴定，将培养物转移至添加了两片1 cm滤纸的PDA和特殊低营养琼脂（SNA）培养基上。选用一个代表性分离株（Cy9UFSM）进行形态学和分子特征鉴定以及致病性确认。在20°C黑暗条件下于PDA上培养10天后，菌落颜色为棕褐色至栗色（3），质地呈棉絮状至毡状，产孢丰富。在SNA上于20°C黑暗条件下培养5周后，培养物主要在简单分生孢子梗上形成大型分生孢子，具1至3个隔膜，两端稍圆。大型分生孢子大小因细胞数量而异，如下：一个隔膜的（23 -）27.7（-31）×（4.5 -）5.8（-7）μm；两个隔膜的（26 -）30.1（-34）×（5 -）5.6（-6）μm；三个隔膜的（24 -）31.2（-35）×（5 -）5.8（-6.5）μm。观察到小型分生孢子，无明显脐点，（6 -）11.2（-17）×（3.5 -）4.2（-5）μm（每个结构观察30次）。在PDA上大量产生褐色、厚壁的球形至近球形厚垣孢子，（8.5 -）13.8（-17）μm。为确认菌种，使用引物对H3 - 1a和H3 - 1b（2）扩增组蛋白H3基因的一部分。该区域序列与强壮伊氏丝核菌（A.A. Hildebr.）A. Cabral & Crous的参考序列（GenBank登录号JF735530）显示98%的相似性。因此，形态学和分子标准均支持将该菌株鉴定为强壮伊氏丝核菌。该分离株已作为登录号KF633172存入GenBank。为确认致病性，将4个月大的美洲葡萄品种博尔代的生根插条根部浸入分生孢子悬浮液（10 ml）中60分钟进行接种。接种后，将插条种植在装有商业基质（MecPlant）的1升袋中。30天后，通过向商业基质施加40 ml分生孢子悬浮液（10 ml）对每株植物再次接种。每个分离株使用10个单株葡萄重复，包括10株接种水的葡萄藤作为对照。4个月后，接种的植株根质量减少22.5%，出现根和冠坏死、维管束褐变，死亡率为20%。用水处理的对照植株无症状。从生根插条基部变黑的木质组织中再次分离到该真菌，从而满足了科赫法则。强壮伊氏丝核菌于2012年在葡萄牙首次与葡萄黑脚病相关联（1）。据我们所知，这是巴西南部关于强壮伊氏丝核菌与葡萄黑脚病相关联的首次报道。参考文献：（1）A. Cabral等人，《真菌学进展》11：655，2012年。（2）N.L. Glass等人，《应用与环境微生物学》61：1323，1995年。（3）R.W. Rayner，《真菌学颜色图谱》，英联邦真菌研究所和英国真菌学会，1970年。
