球形Adenia,作为一种优秀的室内观赏植物,种植在热带植物博物馆,南京中山植物园,江苏省,中国。2022年9月,在A.globosa幼苗上观察到一种新的茎基腐病,被种植在这里。在大约80%的A.globosa幼苗上观察到茎基腐病。切割幼苗的基茎出现腐烂,和茎尖最终由于失水而变干(图S1A)。为了分离病原体,从热带植物博物馆不同花盆中种植的三个插条中收集了三个患病的茎。从健康和患病组织之间的边缘切除茎切片(3至4毫米),表面在75%乙醇中灭菌30秒,在1.5%NaClO中灭菌90秒,在无菌蒸馏水中冲洗三次,置于马铃薯葡萄糖琼脂(PDA)上,在25℃黑暗中孵育。通过单孢子分离获得纯培养物。获得了8个分离株,并全部鉴定为Lasiodiplodiasp。.培养物的菌落形态,在PDA上生长的像棉花一样,7天后,初级菌丝体呈黑灰色,PDA板的背面与正面的颜色相似(图S1B)。代表性的分离株,选择QXM1-2进行进一步研究。QXM1-2分生孢子呈卵形或椭圆形,平均尺寸为11.6µm×6.6µm(n=35)。分生孢子在早期是无色透明的,并在后期变成深棕色,带有一个隔膜(图S1C)。在PDA平板上培养近四周后,分生孢子产生分生孢子(图S1D)。分生孢子是透明的圆柱形结构,尺寸为(6.4-18.2)µm×(2.3-4.5)µm(n=35)。这些特征与Lasiodiplodiasp的描述一致。(阿尔维斯等人。2008).内部转录间隔区(ITS),翻译延伸因子1-α(TEF1α)和β-微管蛋白(TUB)基因(GenBank登录号OP905639,编号:OP921005和No.分别扩增OP921006)并用引物对ITS1/ITS4(White等人。1990),EF1-728F/EF1-986R(Alves等人2008)和Bt2a/Bt2b(GlassandDonaldson1995),分别。它们与Lasiodiplodiatheobromae菌株NH-1(MK696029)的ITS(504/505bp)具有99.8-100%的同源性,菌株PaP-3(MN840491)的TEF1α(316/316bp),和分离物J4-1(MN172230)的TUB(459/459bp)。通过组合MEGA7中的所有测序基因座来生成邻居连接的系统发育树。分离株QXM1-2聚集在具有100%引导支持的Theobromae进化枝(图S2)。为了测试致病性,在茎基部上用20μL分生孢子悬浮液(1×106分生孢子/mL)接种了三个先前用无菌针打伤的球茎切割幼苗。用20μL无菌水接种的幼苗用作对照。所有植物都用透明的聚乙烯袋覆盖,以保持温室中的水分(25℃,80%相对湿度)。该实验重复三次。接种后7天,在处理过的切割幼苗上发现了典型的茎腐病,对照幼苗没有任何症状(图S1E-F)。同样的真菌,通过形态特征和使用ITS测序鉴定,TEF1α和TUB基因,从接种的茎的患病组织中分离出来,以完成科赫的假设。据报道,这种病原体感染了蓖麻子的分支(Tang等人。2021)和柑橘根(Al-Sadi等人。2014).对于我们的知识,这是在中国首次报道可氏乳杆菌感染球藻。本研究为生物,嗜血杆菌的流行病学。
Adenia globosa, as an excellent indoor ornamental plant, is planted in Tropical Botanical Museum, Nanjing Zhongshan Botanical Garden, Jiangsu Province, China. In September 2022, a new stem basal rot disease was observed on A. globosa seedlings, being planted here. Stem basal rot were observed on approximately 80% of A. globosa seedlings. The basal stem of cutting seedlings appeared decayed, and stem tip eventually turned dry due to water loss (Figure S1A). To isolate the pathogen, three diseased stems were collected from three cuttings planted in different pots of the Tropical Botanical Museum. The stem sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the dark. Pure cultures were obtained by monosporic isolation. Eight isolates were obtained, and all identified as Lasiodiplodia sp.. The colonies morphology of cultures, growing on PDA were cotton-like, the primary mycelia were black gray after 7 days, and the reverse sides of PDA plates were similar to front sides in color (Figure S1B). A representative isolate, QXM1-2 was selected for the further study. Conidia of QXM1-2 were oval or elliptic, with a mean size of 11.6 µm×6.6 µm (n=35). The conidia are colorless and transparent in the early stage, and become dark brown with one-septum in the later stage (Figure S1C). The conidiophores produced conidia after nearly four weeks of cultivation on PDA plate (Figure S1D). The conidiophore was a transparent cylindrical structure, with a size of (6.4-18.2) µm × (2.3-4.5) µm ( n = 35). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1α) and β-tubulin (TUB) genes (GenBank Accession No.OP905639, No.OP921005, and No.OP921006, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. They had 99.8-100% homology to the ITS (504/505 bp) of Lasiodiplodia theobromae strain NH-1 (MK696029), TEF1α (316/316 bp) of strain PaP-3 (MN840491), and TUB (459/459 bp) of isolate J4-1 (MN172230). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate QXM1-2 clustered in the L. theobromae clade with 100% bootstrap support (Figure S2). To test pathogenicity, three A. globosa cutting seedlings that previously had been wounded with a sterile needle were inoculated with 20 μL conidia suspension (1×106 conidia/mL) on the stem base. The seedlings inoculated with 20 µL sterile water was used as the control. All plants were covered with clear polyethylene bags to keep moisture in a greenhouse (25℃, 80% relative humidity). The experiment was repeated three times. After 7 days post-inoculation, typical stem rot were found on the treated cutting seedlings and the control seedlings did not have any symptoms (Figure S1E-F). The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased tissues of the inoculated stems to complete Koch\'s postulates. This pathogen has been reported infecting the branch of castor bean (Tang et al. 2021) and root of Citrus (Al-Sadi et al. 2014). For our knowledge, this is the first report of L. theobromae infecting A. globosa in China. This study provides an important reference for the biology, epidemiology of L. theobromae.