Botryosphaeria dothidea (Moug.:Fr.) Ces. De Not. causes perennial cankers on apple trees and causes white rot on apple fruit in the field and during storage (1). Prolonged periods of warm wet weather favor rapid disease outbreaks that result in severe losses, which range from 25 to 50% for the southeastern United States (3). A B. dothidea isolate was obtained from decayed 'Fuji' apple fruit exhibiting white rot symptoms from a local farm market in Beltsville, MD, in May 2010. The fruit had characteristic large dark brown lesions with irregular margins and decay expanded unevenly toward the core and the tissue was soft. The pathogen was isolated from symptomatic tissue by spraying the lesion surface with 70% ethanol. The skin with aseptically removed with a scalpel and small pieces of tissue were placed on potato dextrose agar (PDA) and incubated at 20°C. Once fungal growth was evident, the cultures were hyphal-tip transferred to individual PDA plates and incubated at 20°C. The B. dothidea isolate produced black aerial mycelium with a white margin on PDA and had a black reverse. Conidiomata were evident after 10 to 14 days at 20°C only on oatmeal agar. Conidia were hyaline, smooth and straight, fusiform with an subobtuse apex and a truncate base 20 to 26 (24.33) × 4 to 7 (5) μm (n = 50). Genomic DNA was isolated from the fungus and amplified with gene specific primers (ITS 4 and 5) for the ribosomal DNA internal transcribed spacer region ITSI-5.8S-ITS2 as described by White et al. (4). Both forward and reverse strands of the 542-bp amplicon were sequenced and assembled into a contig. The nucleotide sequence (GenBank Accession No. KC473852) indicated 99% identity to B. dothidea isolate CMM3938 (JX513645.1) and to voucher specimens CMW 25686, 25696, and 25222 (FM955381.1, FM955379.1, and FM955377,1). Koch's postulates were conducted using three 'Golden Delicious' apple fruit that were wound-inoculated with 50 μl of a mycelial suspension of the fungus, obtained from aseptically scraping a 7-day-old PDA culture, and was also repeated using 'Fuji' apple fruit. Large, brown, slightly sunken, soft lesions with undefined edges developed 5 days after inoculation at 20°C and water-only inoculated fruit were symptomless. The fungus was reisolated from infected tissue and was morphologically identical to the original isolate from decayed apple fruit. To determine if the B. dothidea isolate was resistant to postharvest fungicides, the minimum inhibitory concentration (MIC) was conducted using the 96 well plate method with a mycelial suspension of the fungus as described by Pianzzola et al. (2). The MIC for the isolate was >1 ppm for Mertect and Scholar and 50 ppm for Penbotec, which are well below the labeled rates for these postharvest fungicides and the experiment was repeated. To our knowledge, this is the first report of B. dothidea causing white rot on apple fruit in Maryland. References: (1) A. R. Biggs and S. S. Miller. HortScience 38:400, 2003. (2) M. J. Pianzzola et al. Plant Dis. 88:23, 2004. (3) T. B. Sutton. White rot and black rot. Pages 16-20 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinckle, eds. The American Phytopathological Society, St Paul, MN, 1991. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Application. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

译文

:Botryosphaeria dothidea(Moug.:Fr。)Ces。德不。在田间和贮藏期间会在苹果树上引起多年生溃疡,并在苹果果实上引起白腐(1)。长时间的温暖潮湿天气有利于疾病的快速爆发,从而导致严重的损失,在美国东南部占25%至50%(3)。 2010年5月,从马里兰州贝尔茨维尔的一家当地农产品市场的腐烂的“富士”苹果果实中获得了表现出白色腐烂症状的芽孢杆菌分离株。该果实具有特征性的深黑褐色病灶,边缘不规则,腐烂向核心扩展不均匀并且组织柔软。通过用70%乙醇喷洒病灶表面,从有症状的组织中分离出病原体。用手术刀无菌去除的皮肤和小块组织被放在马铃薯葡萄糖琼脂(PDA)上并在20°C下孵育。一旦明显的真菌生长,将培养物菌丝尖端转移到单独的PDA板中,并在20°C下孵育。 B. dothidea分离株在PDA上产生黑色气生菌丝,白色边缘,黑色反向。在20°C下10-14天后,仅在燕麦琼脂上可见形瘤。分生孢子是透明的,光滑的和笔直的,梭形的,具有近钝的先端和截形的基部20至26(24.33)×4至7(5)μm(n = 50)。从真菌中分离出基因组DNA,并如White等人所述用核糖体DNA内部转录的间隔区ITSI-5.8S-ITS2的基因特异性引物(ITS 4和5)扩增。 (4)。对542bp扩增子的正向和反向链都进行测序并组装成重叠群。核苷酸序列(GenBank登录号KC473852)表明与双歧双歧杆菌分离物CMM3938(JX513645.1)和凭证样本CMW 25686、25696和25222(FM955381.1,FM955379.1和FM955377,1)有99%的同一性。 Koch的假设是使用三种“黄金美味”苹果果实进行的,这些果实用50μl真菌菌丝体悬浮液伤口接种,这种真菌悬浮液是通过无菌刮擦7天大的PDA培养物获得的,并且也使用“富士”苹果进行了重复水果。在20°C接种5天后,出现了大的,褐色的,略微下陷的,柔软的病灶,边缘模糊不清,接种纯水的果实无症状。该真菌是从受感染的组织中重新分离出来的,其形态与从腐烂的苹果果实中分离出来的原始菌株完全相同。为了确定双歧双歧杆菌分离株是否对收获后的杀真菌剂具有抗性,如Pianzzola等人所述,使用96孔板法对真菌的菌丝体悬浮液进行最小抑制浓度(MIC)。 (2)。对于Mertect和Scholar,分离株的MIC大于1 ppm,对于Penbotec,MIC大于50 ppm,远低于这些收获后杀菌剂的标记率,并重复了实验。据我们所知,这是马里兰州B. dothidea在苹果果实上引起白腐的首次报道。参考文献:(1)A. R. Biggs和S. S. Miller。 HortScience 38:400,2003。(2)M.J.Pianzzola等。植物病。 88:23,2004.(3)萨顿(T. B. Sutton)。白腐和黑腐。第16-20页,在《苹果与梨病简编》中,A。L. Jones和H. S. Aldwinckle编辑。美国植物病理学会,明尼苏达州圣保罗,1991年。(4)T. J. White等。第315页中的“ PCR协议:方法和应用指南”。 M.A.Innis等,编辑。学术出版社,加利福尼亚州圣地亚哥,1990年。

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