绣球花(绣球花),通常被称为大叶绣球,是绣球花科的一个物种,以其观赏价值而著称。其特点是色彩鲜艳的萼片和郁郁葱葱,引人注目的花序,这个物种在全球范围内被认为是盆栽和景观植物。值得注意的是,2022年,在位于南京市的16个温室内,年龄在6至12个月之间的大约40%的大叶树植物中观察到了令人震惊的茎腐病发生率(N31°14',E118°22),江苏省,中国。该病的最初症状表现为幼苗和茎基部的湿灰黑色斑点,在茎中进展为坏死的灰白色变色,并伴随着受影响部位的灰霉病的生长。这种感染最终导致叶子枯萎和幼苗死亡。对于病原体鉴定,切除病变和健康切片界面处的茎组织,用75%乙醇表面灭菌30秒,然后用3%次氯酸钠处理2-3分钟,随后在空气干燥之前用无菌水冲洗三次。然后在马铃薯葡萄糖琼脂(PDA)培养基上培养2-3mm的切片,补充50mg/mL利福平(RFP),并在25℃下孵育3-5d(Zhou等人。2022年)。在孵育2-3天后,观察到真菌菌落的显着生长。随后将来自这些菌落外围的菌丝体簇转移到新鲜的PDA平板上,并在25℃下再孵育5-7天。命名为JSNJ2022-2,现在保存在江苏省农业科学院,被选中进行详细检查。这个菌落表现出絮凝的质地,颜色从灰白色到浅棕色。它的特点是不规则形成的存在,菌丝内的硬核。观察到分生孢子细长而直立,在其四肢有树枝状的分支。分生孢子像葡萄一样聚集在分生孢子上。这些分生孢子通常是无色或灰色的,椭圆形,光滑透明,测量值在6.4-12.2×7.3-18.2μm(n=50)之间。对于遗传分析,基因组DNA(gDNA)使用DNA安全植物试剂盒(天根生物技术,北京,中国)。使用ITS1/ITS4的一组通用引物进行聚合酶链反应(PCR)扩增(White等人。1990),对应于甘油醛-3-磷酸脱氢酶(G3PDH)的特定序列的引物,热休克蛋白60(HSP60),和DNA依赖性RNA聚合酶亚基II(RPB2)(Yang等人。2020)。对得到的PCR产物进行测序,得到的序列被提交到GenBank数据库,登录号分别为OP131597、OP142320、OP142321和OP142322。从JSNJ2022-2分离株获得的序列的BLAST分析显示了高度的遗传相似性,从99%到100%,具有已知的灰葡萄孢菌序列(材料MK051124.1、MH796662.1、MH479931.1和KU760986.1)。为了阐明分离物的系统发育位置,使用最大似然法构建了一个系统发育树,由1,000个引导复制支持,在Mega7软件中(Kumar等人。2016)。该分析的结果证实,所研究的菌株聚集在与灰霉病菌相同的分支内。建立该分离株的致病性,科赫的假设(Falkow1988)被采用。健康的盆栽H.macrophilla幼苗,大约三个月大,用在PDA上培养的6毫米直径的JSNJ2002-2菌丝体塞在幼苗基部接种3天,随后用湿润的脱脂棉覆盖。对照植物用湿润的脱脂布处理,减去病原体。接种后,这些植物被放置在25℃的生长室中,相对湿度范围为60-80%。经过3天的潜伏期,接种的植物表现出与最初在温室中观察到的症状相同的症状。从这些接种的植物中成功地重新分离出病原体,并在形态上重新确认为灰霉病菌,从而满足科赫的假设标准。据我们所知,该报告代表了中国首例引起巨大叶枯病的灰霉病的发病率。
Hydrangea (Hydrangea macrophylla), commonly referred to as big leaf hydrangea, is a species within the Hydrangeaceae family notable for its ornamental value. Characterized by its vividly colored sepals and lush, striking inflorescences, this species is globally esteemed as both a potted and landscape plant. Notably, in 2022, an alarming incidence of stem rot was observed in approximately 40% of H. macrophylla plants aged between six and twelve months within 16 greenhouses situated in Nanjing City (N 31°14\', E 118°22\'), Jiangsu Province, China. Initial symptoms of the disease manifested as wet gray-black spots at the base of the seedlings and stems, progressing to a necrotic gray-white discoloration in the stems and accompanied by the growth of gray mold on the affected parts. This infection ultimately led to the wilting of the leaves and the death of the seedlings. For pathogen identification, stem tissues at the interface of diseased and healthy sections were excised, surface-sterilized with 75 % ethanol for 30 s, followed by a 2 - 3 min treatment with 3% sodium hypochlorite, and subsequently rinsed three times with sterile water before air drying. Sections measuring 2 - 3 mm were then cultured on potato dextrose agar (PDA) medium, supplemented with 50 mg/mL rifampicin (RFP), and incubated at 25 ℃ for 3 - 5 d (Zhou et al. 2022). Upon 2 - 3 days of incubation, notable growth of fungal colonies was observed. Mycelial clusters from the periphery of these colonies were subsequently transferred to fresh PDA plates and incubated at 25 ℃ for an additional 5 - 7 d. A particular colony, designated JSNJ2022-2 and now preserved at the Jiangsu Academy of Agricultural Sciences, was selected for detailed examination. This colony exhibited a flocculent texture, with a coloration ranging from grey-white to light brown. It was characterized by the presence of irregularly formed, hard sclerotia within the hyphae. The conidiophores were observed to be slender and erect, featuring dendritic branches at their extremities. The conidia were clustered on the conidiophore like grapes. These conidia were generally colorless or grey, oval in shape, smooth and transparent, and measured between 6.4 - 12.2 × 7.3 - 18.2 μm (n = 50). For genetic analysis, genomic DNA (gDNA) was extracted using the DNA secure Plant Kit (Tiangen Biotech, Beijing, China). Polymerase chain reaction (PCR) amplification was performed using a set of universal primers of ITS1/ITS4 (White et al. 1990), primers corresponding to the specific sequences of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (Yang et al. 2020). The resultant PCR products were sequenced, and the resulting sequences were submitted to the GenBank database, under the accession numbers OP131597, OP142320, OP142321, and OP142322, respectively. BLAST analysis of the sequences obtained from the isolate JSNJ2022-2 revealed a high degree of genetic similarity, ranging from 99 to 100%, with known sequences of Botrytis cinerea (accessions MK051124.1, MH796662.1, MH479931.1, and KU760986.1). To elucidate the phylogenetic position of the isolate, a phylogenetic tree was constructed using the maximum likelihood method, supported by 1,000 bootstrap replications, in the Mega7 software (Kumar et al. 2016). The results of this analysis confirmed that the strains under study clustered within the same branch as B. cinerea. To establish the pathogenicity of the isolate, Koch\'s postulates (Falkow 1988) were employed. Healthy potted H. macrophylla seedlings, approximately three months old, were wound inoculated at the base of the seedlings with a 6 mm diameter mycelium plug of JSNJ2002-2 cultivated on PDA for 3 days, which was subsequently covered with moistened degreasing cotton. Control plants were treated with moistened degreasing cloths minus the pathogen. Post-inoculation, these plants were placed in a growth chamber maintained at 25 ℃ with a relative humidity range of 60 - 80%. After a 3-d incubation period, the inoculated plants displayed symptoms identical to those initially observed in the greenhouse. The pathogen was successfully re-isolated from these inoculated plants and was morphologically re-confirmed as B. cinerea, thus satisfying the criteria of Koch\'s postulates. To our knowledge, this report represents the first documented incidence of B. cinerea causing stem rot in H. macrophylla in China.