南瓜(南瓜),属于葫芦科(葫芦科),在世界各地广泛种植。2023年6月,许多南瓜植物(简历。Miben)在随州(31.99°N,113.02°E),湖北省,中国。9个地区的发病率从30%到40%不等,总计6.3公顷。症状是不规则形状的病变,沿着中脉扩展,直到叶片变成棕色和枯萎。如前所述进行真菌分离(Liu等人。2023年)。收集20个具有典型症状的南瓜叶样品,并切成1cm×1cm的碎片。患病组织在75%乙醇中表面灭菌30秒,置于马铃薯葡萄糖琼脂(PDA)培养基上,25℃孵育3天。然后,将新出现的单个真菌菌丝尖端转移到PDA平板上以获得纯化的分离株.PDA平板上总共18个分离株最初为白色,然后发展为深灰色。在绿豆培养基上生长的5日龄培养物产生的分生孢子为黑色,单细胞,光滑,球形或扁圆形,尺寸范围为14.5至20.8μm×13.3至20.5μm(n=50)。因此,分离株在形态学上被鉴定为Nigrosporasphaerica。此外,提取分离物(HB-P1,HB-P2和HB-P3)的基因组DNA,以扩增和测序内部转录间隔区(ITS)的区域(White等人。1990),核大亚基rRNA(nLSU)(O\'Donnell1992;RehnerandSamuels1994),和β-微管蛋白(TUB2)(Glass和Donaldson1995),引物ITS1/ITS4,LROR/LR3和Bt2a/Bt2b,分别。序列提交给GenBank,登录号为PP348112、PP348113、PP348114(ITS),PP411414、PP411415、PP411416(nLSU)、和PP357438、PP357439、PP357440(TUB2)。BLASTn显示序列ITS,nLSU,HB-P1,HB-P2和HB-P3的TUB2具有>99%的核苷酸同一性((ITS:100%,508/508bp,MF996488.1;99.8%,506/507,ON326588.1;100%,500/500,MK748317.1),(nLSU:99.83%,573/574,KT462720.1;99.83%,574/575bp,KT462720.1;99.65%,575/577,KT462720.1),和(TUB2:100%,388/388,MN719407.1;99.74%,387/388,MN719407.1;100%,387/387,MN719407.1))与Nigrosporasphaerica,分别。多位点(ITS,nLSU和TUB2)系统发育分析表明,分离株为黑质孢子菌。在南瓜植物上测试了三个分离株的致病性(cv。Miben).通过喷洒叶子(1×106孢子/ml)接种15株南瓜植物,分别,用无菌水处理10株南瓜植株作为阴性对照。所有植物都在人工气候箱中孵育(龙越,上海)25℃12天。该实验重复三次。十二天后,接种的南瓜植物出现了叶枯病的症状,而对照植物保持健康。然后,从接种南瓜植物的每片叶子中重新分离病原体,而不是从对照植物中分离。以前曾有报道称,在马来西亚,Nigrosporasphaerica会在西瓜上引起叶斑病(Ismail和AbdRazak2021)。据我们所知,这是中国南瓜叶枯病的首次报道。这种新的疾病会导致叶枯病,这可能会影响南瓜的生产力。
Pumpkin (Cucurbita moschata), which belongs to the gourd family (Cucurbitaceae), is widely planted throughout the world. In June 2023, many pumpkin plants (cv. Miben) displayed leaf blight and chlorosis in fields located in Suizhou (31.99°N, 113.02°E), Hubei Province, China. The disease incidence ranged from 30 to 40% in nine fields, 6.3 ha in total. The symptoms were irregularly shaped lesions that expanded along the mid-vein until the leaf turned brown and wilted. Fungal isolations were performed as described previously (Liu et al. 2023). Twenty pumpkin leaf samples with typical symptoms were collected and cut into 1 cm×1 cm pieces. The diseased tissue was surface-sterilized in 75% ethanol for 30 sec, plated on potato dextrose agar (PDA) medium and incubated at 25℃ for 3 days. Then, the emerging single fungal hyphal tip was transferred onto PDA plates to obtain purified isolates. A total of eighteen isolates on PDA plates were initially white and then developed to dark gray. The 5-day-old cultures growing on mung bean medium produced conidia that were black, single-celled, smooth, spherical or oblate, and ranged in size from 14.5 to 20.8 μm×13.3 to 20.5 μm (n=50). Therefore, the isolates were morphologically identified as Nigrospora sphaerica. Moreover, the genomic DNA of the isolates (HB-P1,HB-P2, and HB-P3) was extracted for amplification and sequencing of the regions of internal transcribed spacer (ITS) (White et al. 1990), nuclear large subunit rRNA (nLSU) (O\'Donnell 1992; Rehner and Samuels 1994), and β-tubulin (TUB2) (Glass and Donaldson 1995), with primers ITS1/ITS4, LROR/LR3, and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank under accession numbers PP348112, PP348113, PP348114 (ITS), PP411414, PP411415, PP411416 (nLSU), and PP357438, PP357439, PP357440 (TUB2). BLASTn showed that the sequences ITS, nLSU, and TUB2 of HB-P1, HB-P2, and HB-P3 had >99% nucleotide identities ((ITS: 100%, 508/508 bp, MF996488.1; 99.8%, 506/507, ON326588.1; 100%, 500/500 ,MK748317.1), (nLSU: 99.83%, 573/574, KT462720.1; 99.83% , 574/575 bp, KT462720.1; 99.65%, 575/577, KT462720.1), and (TUB2: 100%, 388/388, MN719407.1; 99.74%, 387/388, MN719407.1; 100%, 387/387, MN719407.1)) with Nigrospora sphaerica, respectively. A multilocus (ITS, nLSU and TUB2) phylogenetic analysis indicated that the isolates were Nigrospora sphaerica. Pathogenicity of three isolates were tested on pumpkin plants (cv. Miben). Fifteen pumpkin plants were inoculated by spraying the leaves (1×106 spores/ml), respectively, and 10 pumpkin plants were treated with sterile water as a negative control. All plants were incubated in an artificial climate box (LongYue, ShangHai) at 25℃ for 12 days. The experiment was repeated three times. Twelve days later, the inoculated pumpkin plants developed symptoms of leaf blight, while the control plants remained healthy. Then, pathogens were re-isolated from the each leaf of inoculated pumpkin plants and not from the control plants. Nigrospora sphaerica has been previously reported to cause leaf spot on watermelon in Malaysia (Ismail and Abd Razak 2021). To our knowledge, this is the first report of N. sphaerica causing leaf blight on pumpkin in China. This new disease can cause leaf blight, which may affect pumpkin productivity.