Heliconiasubulata是一种常见的观赏植物,它已在中国南方广泛种植,用于绿化公园,道路,和住宅区。在东海岸湿地公园(18°16\'53.37″N,109°30'19.36“E),三亚市,海南省,8月中国2023年31日。叶子的症状是不规则的灰白色,斑点,发展成棕色和黑色,疾病-健康交界处有黄色光环。经过现场调查,发现该疾病的发病率为40%至50%。叶片用70%乙醇消毒1分钟,用无菌水冲洗3次,用0.1%HgCl2消毒1分钟,用无菌水冲洗3次,干,置于马铃薯葡萄糖琼脂(PDA)上,在28℃下孵育7天。从培养物中选择红色分生孢子桩,分散在无菌水中并稀释至含有1至2个分生孢子的20μL。多次吸收20μL孢子悬浮液后,接种在新的PDA平板上,五种纯的单孢子培养物,获得J-1-1至J-1-5。生长7天后,菌落的正面是灰色的气生菌丝体,反面是浅橙色的分生孢子。白色的空中菌丝体,分生孢子,Acervulus,并观察到附睾(补充图。S1).形态特征表明,该分离株具有与先前描述的炭疽菌属相同的特征。(Wang等人。2021)。分离株J-1-1和J-1-5的基因组DNA通过真菌DNA试剂盒(OMEGAbio-tek,广州,中国)。内部转录间隔区(ITS),甘油醛-3-磷酸脱氢酶(GADPH),用引物ITS1/ITS4、GDF/GDR扩增β-微管蛋白2基因(TUB2),和Bt2a/Bt2b,分别(Weir等人。2012).基于测序和基因序列比对分析,发现分离株J-1-1和J-1-5的ITS序列之间的一致性为99.82%。GADPH和TUB2序列之间的一致性为100%。分离株J-1-1和J-1-5的基因序列提交给GenBank,登录号为PP455510/PP455511(ITS),PP510210/PP510211(GADPH)和PP510212/PP510213(TUB2)分别。基于BLAST分析,这三个序列与C.热带菌株FC1(ITS:MT192648,GAPDH:MT155819,TUB2:MT199874;Duan等人。2022年)。MEGA11基于ITS构建了系统进化树,GADPH,和TUB2基因序列通过最大似然法。结果表明,分离株J-1-1和J-1-5与C.tropicaleCBS:124949成簇(补充图。S2).基于形态学和分子生物学分析,两个分离株被鉴定为热带梭菌。为了进一步测试分离株J-1-1和J-1-5的致病性,制备了孢子悬浮液(1×106分生孢子/mL),并将20μL孢子悬浮液接种在健康的H.subulata盆栽植物的叶片上。无菌牙签。在每种处理中接种三片叶子,接种无菌水作为对照。将处理过的植物置于温度为28℃的培养箱中,相对湿度90%,和亮/暗(12h/12h)。15天后,孢子悬浮处理显示出与田间自然患病的H.subulata植物相同的症状,但用无菌水处理的叶子没有被感染(补充图。S1).从病叶获得的分离株的形态与PDA平板上的分离株J-1-1和J-1-5的形态相同。据我们所知,这是H.Subulata的第一份报告,在中国引起炭疽病的新宿主。
Heliconia subulata is a common ornamental plant, it has been widely planted in southern China for greening parks, roads, and residential areas. H. subulata plants with spots on their leaves were observed in East Coast Wetland Park (18°16\'53.37″N, 109°30\'19.36″E), Sanya City, Hainan Province, China on Aug. 31, 2023. The symptoms of the leaves are irregular gray-white, spots, that develop into brown and black, with yellow halos at the disease-health junction. Following an on-the-spot investigation, it was found that the incidence of the disease was 40 to 50%. The leaves were disinfected with 70% ethanol for 1 min, rinsed with sterile water 3 times, disinfected for 1 min with 0.1% HgCl2, rinsed with sterile water 3 times, dried, put on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. The red conidia pile was selected from the culture, dispersed in sterile water and diluted to 20 μL containing 1 to 2 conidia. After absorbing 20 μL spore suspension for many times and inoculating it on the new PDA plate, five pure cultures of single spore, J-1-1 to J-1-5, were obtained. After 7 days of growth, the colonies were grayish aerial mycelium on the front and light orange conidia on the reverse. The white aerial mycelia, conidia, acervulus, and appressorium were observed (Supplementary Fig. S1). The morphological characteristics showed that the isolate had the same characteristics as the previously described Colletotrichum spp. (Wang et al. 2021). The genomic DNA of isolates J-1-1 and J-1-5 were extracted by Fungal DNA Kit (OMEGA bio-tek, Guangzhou, China). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and β-tubulin 2 genes (TUB2) were amplified by primers ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b, respectively (Weir et al. 2012). Based on sequencing and gene sequence alignment analysis, it was found that the consistency between the ITS sequences of isolates J-1-1 and J-1-5 was 99.82%. The consistency between GADPH and TUB2 sequences was 100%. The gene sequences of isolates J-1-1 and J-1-5 were submitted to GenBank with accession numbers PP455510/PP455511 (ITS), PP510210/PP510211 (GADPH) and PP510212/PP510213 (TUB2) respectively. Based on the BLAST analysis, the three sequences were more than 99% identical to those of the C. tropicale strain FC1 (ITS: MT192648, GAPDH: MT155819, TUB2: MT199874; Duan et al. 2022). A phylogenetic tree was constructed by MEGA 11 based on the ITS, GADPH, and TUB2 gene sequence by the maximum-likelihood method. The results showed that the isolates J-1-1 and J-1-5 were clustered with C. tropicale CBS:124949 (Supplementary Fig. S2). Based on morphological and molecular biological analysis, two isolates were identified as C. tropicale. To further test the pathogenicity of isolates J-1-1 and J-1-5, spore suspensions (1×106 conidia/mL) were prepared and 20 μL spore suspensions were inoculated on the leaves of healthy H. subulata potted plants stabbed with sterile toothpicks. Three leaves were inoculated in each treatment, and sterile water was inoculated as a control. The treated plants were placed in an incubator with a temperature of 28℃, relative humidity of 90%, and light/dark (12h/12h). After 15 days, the spore suspension treatment showed the same symptoms as the naturally diseased H. subulata plants in the field, but the leaves treated with sterile water were not infected (Supplementary Fig. S1). The morphology of the isolates obtained from diseased leaves was the same as that of isolates J-1-1 and J-1-5 on the PDA plate. To our knowledge, this is the first report of H. subulata, a new host of C. tropicale causing anthracnose in China.