澳洲坚果(MacadamiaternifoliaMaiden和Betche)属于Proteaceae家族(Li等人。2022年)。在广西(中国南部)的丘陵地区,澳洲坚果树是重要的收入来源。近年来广西种植面积不断增加,到2022年底超过53333公顷,但这种增加也与紧急情况有关,澳洲坚果病.南宁市某人工林37/241棵澳洲坚果树(发病率15%)出现叶枯病症状,中国广西,六月期间,2022年。受感染树木的疾病严重程度为5%至60%。这种疾病是从叶子的尖端或边缘发展而来的,导致叶子变成棕色,后来逐渐枯萎(图。1A).从五棵澳洲坚果树(每棵树两片叶子。此后,从10个病变的边缘切除的小段(3至4mm²)在75%乙醇中进行表面灭菌30s,在1%次氯酸盐中进行表面灭菌90s,在无菌水中冲洗第1页,共6页2,在接种到马铃薯葡萄糖琼脂(PDA)培养基上之前。平板在白天在照明下孵育,在25℃下,夜间黑暗持续5天。通过传代培养菌丝尖端产生22个纯化的菌落,其中8个表现出相似的形态,并进一步表征。PDA上的菌落是灰色的,具有白色的外环和表面平坦的草坪(图。1B).比尼迪虫是表面的,半浸入PDA上,从孤立到聚集,球状至亚球状,棕色至黑色和渗出的黄色粘液性肿块(图1C)。α-分生孢子是单细胞的,透明椭圆形或梭形,和测量4-8×1.9-4μm(n=30),而β分生孢子是透明的,长,直的或弯曲的,测量20-23×0.9-2μm(n=30)(图1D-E)。形态特征类似于香港DiaportheHongkongensis(Dissanayake等人。2015).使用内部转录间隔区(ITS)区域将八个形态相似的分离株鉴定为D.hongkongensis,但只有一个孤立,选择JG11用于进一步的分子鉴定。五个靶基因,包括ITS地区,平移延伸因子1α(EF1-α),β-微管蛋白基因(TUB2),钙调素(CAL),和组蛋白H3(HIS)扩增和测序使用引物ITS1/ITS4,EF1-728F/EF1-986R,Bt2a/Bt2b,CAL-228F/CAL-737R,和CYLH3F/H3-1b,分别(Carbone和Kohn1999)。EF1-α的序列以登录号OQ932790(ITS)和OR147955-58保存在GenBank中,浴缸,CAL和他的基因,分别。对GenBank的BLAST搜索显示,EF1-α,浴缸,CAL,JG11的HIS序列与D.hongkongensisNR111848的第2页,共6页3页(99.22%的同一性)相似,KY433566(99.72%),MW208603(99.42%),MW221740(99.80%),和MW221661(99.79%),分别。用IQ-TREE软件进行串联序列的系统发育分析。JG11与其他枯草杆菌分离株归入同一进化枝(图。2).在温室中对健康的澳洲坚果树进行了致病性实验。使用三棵澳洲坚果树作为阴性对照,其中每棵树五片未受伤的叶子用无菌蒸馏水喷雾。用浓度为1×106的分离物JG11的分生孢子悬浮液喷洒其他三棵澳洲坚果树的每棵未受伤的五片叶子。每个治疗独立重复3次,每棵树5片叶子(Liu等人。2023年;哈维尔等人。2023年;张等人。2022年)。将塑料袋放置在所有接种的叶子上。温室的日平均温度和相对湿度分别为32℃和65%,分别。两天后,接种孢子悬浮液的叶片出现褐变,并向外扩张。5天后,所有接种真菌孢子的澳洲坚果叶开始枯萎,而对照组仍然无症状(图。1H-I)。D.从接种的叶片中不断重新分离和纯化,并通过形态学鉴定和分子分析确认身份,完成了科赫的假设。D.hongkongensis已经报道了桃子(Zhang等人。2021),葡萄藤树干(Dissanayake等人。2015年)和杉木(廖等人。2022年)。据我们所知,这是中国首例红豆杉致澳洲坚果叶枯病的报道。这些发现为未来研究这种新出现的澳洲坚果病的流行病学和控制提供了基础。
Macadamia (
Macadamia ternifolia Maiden and Betche) belongs to the Proteaceae family (Li et al. 2022). In the hilly areas of Guangxi (southern China),
macadamia trees are an important source of revenue. The planting area in Guangxi has increased in recent years, exceeding 53,333 hectares by the end of 2022, but this increase is also associated with emergency of, macadamia diseases. Leaf blight symptoms were observed in 37/241
macadamia trees (15% incidence) in a plantation in Nanning, Guangxi province in China, during June, 2022. Disease severity on infected trees ranged from 5% to 60%. The disease developed from the tips or margins of leaves, causing the leaves to turn brown, and later gradually withered (Fig. 1 A). Ten leaves with lesions were collected from five
macadamia trees (two leaves per tree. Thereafter, small segments (3 to 4 mm²) excised from the margins of ten lesions were surface sterilized in 75% ethanol for 30 s and 1% hypochlorite for 90 s and Page 1 of 6 2 rinsed in sterile water, before plating onto potato dextrose agar (PDA) medium. Plates were incubated under lighting during the daytime, and darkness at night-time for 5 days at 25℃. Twenty-two purified colonies were generated by subculturing hyphal tips, of which eight exhibited similar morphology and were further characterized. The colonies on PDA were gray with a white outer ring and flat lawn on the surface (Fig. 1 B). The pycnidia were superficial to semi-immersed on PDA, solitary to aggregated, globose to sub-globose, brown to black and oozed yellow mucilaginous masses (Fig.1 C). The α-conidia were unicellular, hyaline elliptical or fusiform, and measuring 4-8 × 1.9-4 μm (n=30) , whereas the β-conidia were hyaline, long, straight or curved, measuring 20-23 × 0.9-2 μm (n=30) (Fig. 1 D-E). The morphological features were similar to Diaporthe hongkongensis (Dissanayake et al. 2015). The eight morphologically similar isolates were identified as D. hongkongensis using the internal transcribed spacer (ITS) region, but only one isolate, JG11, was selected for further molecular identification. Five target genes, including the ITS region, translation elongation factor 1 alpha (EF1-α), beta-tubulin genes (TUB2), calmodulin (CAL), and histone H3 (HIS) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, CAL-228F/CAL-737R, and CYLH3F/H3-1b, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank under accession numbers OQ932790 (ITS) and OR147955-58 for EF1-α, TUB, CAL and HIS genes, respectively. BLAST search of GenBank showed that ITS, EF1-α, TUB, CAL, and HIS sequences of JG11 were similar to Page 2 of 6 3 those of D. hongkongensis NR111848 (99.22% identity), KY433566 (99.72%), MW208603 (99.42%), MW221740 (99.80%), and MW221661 (99.79%), respectively. Phylogenetic analysis of concatenated sequences was performed with IQ-TREE software. JG11 was grouped in the same clade as other Diaporthe hongkongensis isolates (Fig. 2). Pathogenicity experiments were carried out on healthy
macadamia trees in a greenhouse. Three macadamia trees were used as negative controls where five uninjured leaves per tree were sprayed with sterile distilled water. Uninjured five leaves per tree of three other macadamia trees were sprayed with conidia suspension of the isolate JG11 at a concentration of 1×106. Each treatment was repeated 3 times independently, with 5 leaves per tree (Liu et al. 2023; Havill et al. 2023; Zhang et al. 2022). Plastic bags were placed over all inoculated leaves. The average daily temperature and relative humidity in the greenhouse were 32°C and 65%, respectively. Two days later, browning appeared on the leaves inoculated with the spore suspension and expanded outward. After 5 days, all macadamia leaves inoculated with the fungal spores began to wither, while controls remained asymptomatic (Fig. 1 H-I). D. hongkongensis was consistently re-isolated and purified from inoculated leaves and the identity was confirmed by morphological identification and molecular analysis, completed Koch\'s postulates. D. hongkongensis has been reported on peach (Zhang et al. 2021), grapevine trunk (Dissanayake et al. 2015) and Cunninghamia lanceolata (Liao et al. 2022). To our knowledge, this is the first report of D. hongkongensis causing leaf blight on macadamia in China. These findings provide a foundation for future research on the epidemiology and control of this newly emerging disease of macadamia.