工业大麻(大麻)在意大利种植,直到第二次世界大战结束。从那以后,主要由于法律限制和公众关注,它被废弃并用其他作物代替。2016年通过的公共立法允许生产大麻种子,花和纤维(法律第242/2016号)。在2019年那不勒斯省大麻卫生状况调查中(40°57\'6\“12N,14°22\'37\“56E),在一个私人农场观察到具有根腐病症状的大麻“Kompolty”,并在卡塞塔的CREA植物检疫实验室收集用于进一步分析。死亡一般发生在首次症状出现后2-3周内,发生在CA。10%的植物,由变黄组成,冠层枯萎,树皮下有白色菌丝体和扇形菌丝体覆盖的根迹象。因果代理,从小根段分离,从有症状的植物中切除,表面用2%的次氯酸钠消毒,置于用硫酸链霉素(100mg/L)修正的马铃薯葡萄糖琼脂(PDA)上,并在25°C下在黑暗中孵育5天。将所得菌落边缘处的小片(2-3mm)在PDA上传代培养,并在25°C下在黑暗中孵育一周。来自15个分离株的菌丝体显示出与隔片相邻的梨形肿胀。分生孢子是无价的,透明玻璃,椭圆形到卵圆形,和3-5×2.5-3µm(n=50)。根据形态特征,该真菌被鉴定为念珠菌。前普里尔。(辛格尔顿等人。,1992)一种分类修订为DematophoranecatrixR.Hartig的真菌(Wittstein等人。,2020)。为了确认身份,使用DNeasyPlantMini试剂盒(Qiagen,希尔登,德国)和ITS间隔区用引物ITS1-ITS4进行PCR扩增(White等人。,1990).对536bp的大小预期扩增子进行纯化和测序,将所得序列修剪并以登录号MK937913存入GenBank。BLAST-n分析显示与一些代表性的D.necatrix分离株(MK888684.1;KT343972.1)具有98.83%的核苷酸同一性。为了实现科赫的假设,致病性试验是对15株4周龄盆栽大麻植物“Kompolty”进行的。接种是通过添加3克接种了10个菌丝体塞的小米种子来进行的,取自D.necatrix活跃生长的殖民地的边缘,每升无菌泥炭和珍珠岩基质在单个盆中。此外,用灭菌的小米种子接种十株大麻植物,并作为阴性对照。所有植物均在25°C下孵育。三周后,接种的植物表现出叶面萎黄病,根尖枯萎,两周后死亡,类似于在现场观察到的情况。对照植物没有表现出任何症状。从所有15种接种植物的根中分离出真菌,并根据形态学和分子分析确认为D。用第二对引物EF1-983F/EF1-2218R靶向转录延伸因子1-(Rehner和Buckley。,2005)(MW541068),在BLAST-n分析中显示99.67%nt。据我们所知,这是D.Necatrix在欧洲感染大麻的第一份报告。出现问题的农场有30多年来种植苹果的历史。因此,假设D.necatrix适应新的宿主。需要对大麻疾病的深入了解才能重新启动该地区的大麻种植。
Industrial hemp (Cannabis sativa L.) was cultivated in Italy until the end of the Second World War. Since then, it has been abandoned and substituted with other crops mainly due to legal restrictions and public concerns. Public legislation passed in 2016, has allowed for the production of hemp seeds, flowers and fibers (law n. 242/2016). During a 2019 survey on hemp sanitary status in the province of Naples (40°57\'6\"12 N, 14°22\'37\"56 E), hemp \'Kompolty\' with symptoms of root rot were observed at a private farm and collected for further analysis at the phytosanitary laboratory of CREA in Caserta. Death generally occurred within 2-3 weeks after the appearance of the first symptoms, occurring on ca. 10% of plants, consisting of yellowing, canopy wilt and signs of roots covered with white mycelium and fan-like mycelium under the bark. The causal agent, was isolated from small root segments, excised from symptomatic plants, the surface was disinfected with 2% sodium hypochlorite, placed on potato dextrose agar (PDA) amended with streptomycin sulphate (100mg/L) and incubated in the dark at 25°C for 5 days. Small pieces (2-3 mm) at the edge of the resulting colonies were sub-cultured onto PDA and incubated at 25°C in the dark for one week. The mycelia from 15 isolates showed pear-shaped swellings adjacent to the septa. The conidia were aseptate, hyaline, ellipsoid to ovoid, and 3-5 × 2.5-3 µm (n=50). Based on the morphological characteristics, the fungus was identified as Rosellinia necatrix Berl. ex Prill. (Singleton et al., 1992) a fungus taxonomically revised to Dematophora necatrix R. Hartig (Wittstein et al., 2020). To confirm the identification, total DNA was extracted from five isolates using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the ITS spacer was PCR-amplified with primers ITS1-ITS4 (White et al., 1990). The size-expected amplicons of 536 bp were purified and sequenced, the resulting sequence was trimmed and deposited in GenBank under the accession number MK937913. BLAST-n analysis revealed 98.83% nucleotide identity with some representative isolates of D. necatrix (MK888684.1; KT343972.1). To fulfill Koch\'s postulates, the pathogenicity tests were carried out on fifteen 4-weeks-old potted hemp plants \'Kompolty\'. The inoculation was performed by adding 3 g of millet seeds inoculated with ten mycelial plugs, taken from the margins of a D. necatrix actively growing colony, per liter of sterile peat and perlite substrate in single pots. Moreover, ten hemp plants were inoculated with sterilized millet seed and served as negative controls. All plants were incubated at 25°C. After three weeks, inoculated plants exhibited foliar chlorosis, apical wilting, and death in two weeks, similar to what was observed in the field. Control plants did not show any symptoms. The fungus was isolated from the roots in all fifteen inoculated plants and confirmed to be D. necatrix based on morphological and molecular analysis, carried out with a second primer pair EF1-983F/ EF1-2218R targeting the transcription elongation factor 1- (Rehner and Buckley., 2005) (MW541068) that showed 99.67% nt in BLAST-n analysis. To our knowledge, this is the first report of D. necatrix infecting hemp in Europe. The farm where the problem arose has a history of cultivation for the production of apples for over 30 years. Therefore, an adaptation of D. necatrix to the new host is hypothesized. An in-depth knowledge on the diseases of hemp will be needed to relaunch hemp cultivation in this area.