Moricca S, Uccello A, Ginetti B, Ragazzi A
Dipartimento di Biotecnologie Agrarie, Sezione di Protezione delle Piante. Piazzale delle Cascine 28, I-50144, Florence, Italy.
Plant Dis. 2012 Nov;96(11):1699. doi: 10.1094/PDIS-06-12-0543-PDN.
Between 2007 and 2011, Acer pseudoplatanus and Quercus robur trees declined in the North Park and the Boscoincittà Park in Milan, Italy (lat. 45° 27' 47″ N, long. 09° 11' 16″ E, elev. 121 m). Symptoms included extensive lengthwise bark cracks, with necrosis of the wood tissue underneath. Isolations were conducted from bark and infected wood after surface sterilization with 10% HO for 10 min, followed by five washings in sterilized water. Tissue was placed on potato dextrose agar (PDA) in petri dishes amended with 0.06 g/l streptomycin at pH 6.5. The dishes were incubated in the dark at 24°C. Colonies reached a diameter of 7 to 8 cm in 7 days. They were whitish grey on the upper surface and blackish at the bottom, and consisted of dense aerial mycelia. The conidia were hyaline, ellipsoid, smooth, non-septate, thin-walled, with their upper end wider than their lower end, and measured 14 to 20 × 4.5 to 7 μm. The causal organism was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (basionym Fusicoccum parvum Pennycook & Samuels; teleomorph Botryosphaeria parva Pennycook & Samuels) based on biometric characteristics, common taxonomic keys, and the sequence information from the rDNA ITS region and the elongation factor 1-alpha (EF1-α) gene (GenBank Accession Nos. HQ893535 and HQ893537, respectively). All morphological traits corresponded to those of the holotype (1), while BLAST searches of the GenBank database revealed for both loci 100% homology with reference sequences. To confirm the involvement of N. parvum in the tree death, and fulfil Koch's postulates, 20 2-year-old seedlings of A. pseudoplatanus and Q. robur (10 each) were artificially infected in May with N. parvum in 2008 and in 2009. A bark portion was removed aseptically and a PDA disc (0.5 cm diameter) of N. parvum mycelium was placed on the wound. Control seedlings (3 per species) received sterile PDA discs. The inoculation site was wrapped in Parafilm for 10 days. After 3 weeks, infected seedlings showed sunken bark lesions associated with necrosis and bark cracks. When the bark was stripped, the wood below these lesions was also necrotic. Control seedlings developed no symptoms. Mycelium of N. parvum was successfully reisolated from the infected tissue. N. parvum is an important member of the Botryosphaeriaceae, a family of cosmopolitan endophytes that have been known for many years (2). The pathogen has been in recent years linked to complex syndromes, in which a prominent symptom is the formation of bark cankers. Although common on fruit crops, it has increasingly been reported on forest trees (3,4). References: (1) P. Crous et al. Stud. Mycol. 55:248, 2006. (2) P. A. Saccardo. Michelia 1:1, 1877. (3) M. E. Sánchez et al. Plant Dis. 87:1515, 2003. (4) B. Slippers and M. J. Wingfield. Fung. Biol. Rev. 21:90, 2007.
2007年至2011年间，意大利米兰的北公园和博斯科因奇塔公园（北纬45°27′47″，东经09°11′16″，海拔121米）内的悬铃木和英国栎树出现衰退现象。症状包括树皮出现大量纵向裂缝，其下方的木质组织坏死。用10%过氧化氢进行10分钟表面消毒后，再用无菌水冲洗五次，然后从树皮和受感染木材上进行分离。将组织置于添加了0.06克/升链霉素、pH值为6.5的马铃薯葡萄糖琼脂（PDA）平板上。平板在24°C黑暗条件下培养。菌落7天内直径达到7至8厘米。其表面呈灰白色，底部为黑色，由密集的气生菌丝组成。分生孢子透明，椭圆形，表面光滑，无隔膜，薄壁，上端比下端宽，大小为14至20×4.5至7微米。根据形态特征、常用分类检索表以及核糖体DNA内转录间隔区（rDNA ITS）和延伸因子1-α（EF1-α）基因的序列信息（GenBank登录号分别为HQ893535和HQ893537），确定致病微生物为细小新壳梭孢（Pennycook & Samuels）Crous、Slippers & A.J.L. Phillips（异名：细小壳梭孢Pennycook & Samuels；有性型：细小葡萄座腔菌Pennycook & Samuels）。所有形态特征均与模式标本相符，而在GenBank数据库中对这两个基因座进行BLAST搜索时发现，与参考序列的同源性均为100%。为证实细小新壳梭孢与树木死亡有关，并满足柯赫氏法则，2008年和2009年5月，分别对20株2年生的悬铃木和英国栎幼苗（各10株）进行人工接种细小新壳梭孢。无菌条件下切除一块树皮，并在伤口处放置一片直径0.5厘米的细小新壳梭孢菌丝体PDA平板。对照幼苗（每个物种3株）接种无菌PDA平板。接种部位用保鲜膜包裹10天。3周后，受感染幼苗的树皮出现凹陷病斑，伴有坏死和树皮裂缝。剥去树皮后，病斑下方的木材也已坏死。对照幼苗未出现症状。从受感染组织中成功重新分离出细小新壳梭孢的菌丝体。细小新壳梭孢是葡萄座腔菌科的重要成员，该科是一类世界性的内生真菌，多年来一直为人们所熟知。近年来，该病原菌与复杂的病害综合征有关，其中一个突出症状是树皮溃疡的形成。虽然它在果树作物上很常见，但在林木上的报道也越来越多。参考文献：（1）P. Crous等人，《Stud. Mycol.》55:248，2006年。（2）P. A. Saccardo，《Michelia》1:1，1877年。（3）M. E. Sánchez等人，《Plant Dis.》87:1515，2003年。（4）B. Slippers和M. J. Wingfield，《Fung. Biol. Rev.》21:90，2007年。
