安纳托(BixaorellanaL.)在中国广泛种植。它的种子被用作药物和收敛解热剂。自2019年以来,在湛江的田间(约30公顷)上观察到炭疽病型病变(21º18\'12\'\'N,110º17\'22\'\'E),广东省,中国。病害发生率约为70%(n=100个来自约3公顷的调查植物)。早期症状是叶子边缘或尖端的黄色斑点。斑点逐渐扩大,变成深棕色,最终合并成大型不规则或圆形病变(补充图1-A)。对来自10个植物的10个有症状的叶子进行取样。将病变的边缘切成2×2mm的碎片,并用75%乙醇消毒表面30秒,用2%次氯酸钠消毒60秒。之后,碎片在无菌水中冲洗三次,置于马铃薯葡萄糖琼脂(PDA)培养基上,并在28℃下孵育3天。通过将菌丝尖端转移到新的PDA平板上获得纯培养物。获得20个分离株。根据形态特征和ITS分析,确认了来自20个分离株的三个代表性单孢子分离株(BOC-1,BOC-2和BOC-3)相同,并用于进一步研究。此外,这三个分离物保存在广东海洋大学水生生物博物馆的真菌收藏中。在28℃黑暗中孵育6天后,PDA上的菌落为白色至灰色,有棉质菌丝体。分生孢子是单细胞的,透明玻璃,圆柱形,棒酸盐,两端呈钝角;它们的尺寸为9.6至18.5µm×3.5至5.5µm(n=50)。Pressoria呈椭圆形至不规则形状,呈深棕色,他们测量了6至9µm×4.5至8µm(n=30)(补充图1-D,E和F)。这些形态特征与siamenseColletotrichum的描述相符(Prihastuti等人。2009;Sharma等人。2013).对于分子鉴定,内部转录间隔区(ITS),甘油醛-3-磷酸脱氢酶(GAPDH),几丁质合成酶(CHS-1),使用引物对ITS1/ITS4,GDF1/GDR1,CHS-79F/CHS-354R扩增分离物的肌动蛋白(ACT)基因座,和ACT-512F/ACT-783R,分别(Weir等人。2012).序列以编号保存在GenBank中。MZ047377-MZ047379(ITS),MZ126934-MZ1269346(GAPDH),MZ126904-MZ1269046(CHS-1),和MZ126844-MZ1268446(ACT)。根据ITS的串联数据生成了系统发育树,GAPDH,CHS-1和ACT序列将三个分离株与C.siamense(类型菌株MFLU090230)聚集在一起,(补充图2)。分别在25~29℃、相对湿度80%的温室中对3株分离株的致病性进行了检测。用孢子溶液(1×105/mL)接种Annatto种子(n=5,2个月大),直至流失。而对照植物用无菌蒸馏水喷雾。.实验重复了三次。10天后,在接种的叶子上观察到炭疽病病变,而对照植物保持健康(补充图1-G,和H)。基于形态学和ITS分析,从所有接种的叶中重新分离相同的病原体。据报道,C.siamense在广泛的宿主中引起炭疽病(Weir等人。2012;Wang等人。2017;Liu等人。2017;卓等人。2017),但不是在B.orellana.据我们所知,这是C.siamense在中国的B.orellana上引起炭疽病的第一份报告。本研究为本病的控制提供了重要的参考信息。
Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18\'12\'\'N, 110˚17\'22\'\'E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.