The species Carissa grandiflora A. DC., commonly called Natal plum, is a shrub native to the coastal region of Natal, South Africa. In southern Spain, Natal plum is used as an ornamental plant due to its beautiful flowers and red ripen fruits. In March 2019 and 2020, we surveyed nine public gardens in the cities of Cadiz and Sanlucar de Barrameda (Andalusia, Spain); and Natal plum fruit showing anthracnose symptoms were observed in six (55% prevalence) of them. Affected fruits showed necrotic and circular lesions with acervuli in the center (Fig. 1a) causing the complete mummification of the fruit (Fig. 1b). Affected fruits were collected from four gardens and disinfested according to Moral et al. (2010). Six fungal isolates were recovered from small (3-4 × 1-2 mm) pieces of the affected fruits in Potato Dextrose Agar (PDA), and hyphal tips from them were transferred to fresh PDA to obtain pure cultures. The six isolates were initially identified as Colletotrichum karstii according to their morphology and the sequences of the ITS1-5.8S-ITS2 (ITS) region (Damm et al. 2012). The six Colletotrichum isolates showed similar colony morphology and their ITS sequences were identical. Overall, C. karstii isolates showed cylindrical and straight conidia that were 12.1 to 14.2 μm long and 4.9 to 5.6 μm wide (n = 50). The aerial mycelia of the fungus varied from grayish-white to dark gray. A multilocus approach was conducted for more precise identification of the Colletotrichum species. For that, ITS, beta-tubulin (TUB2), actin (ACT), partial sequences of the chitin synthase 1 (CHS-1), histone 3 (HIS3), and a 200-bp intron fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of a representative isolate (FITP19001) were amplified and sequenced according to Damm et al. (2012). GenBank Accession Nos. for ITS, TUB2, ACT, CHS-1, HIS3 and GADPH: MT757643, MT759805, MT759806, MT759807, MT759808 and MT759809, respectively. Sequences showed 100% identity with homologous sequences belonging to C. karstii (GenBank taxid:1095194). To test Koch's postulates, 10 unripen and 10 ripen C. grandiflora fruits, harvested from asymptomatic plants, were inoculated. For each group, five fruits were inoculated using a drop of 10 µl of 5 × 104 conidia per ml suspension of C. karstii (FITP19001) and another five fruits were inoculated using a mycelial plug of the same isolate. Inoculated fruits were incubated in a humid chamber at room temperature (19-24ºC) under light for two weeks. Non-inoculated control fruits were treated with sterile water or a PDA plug and incubated under the same conditions. The pathogenicity test was conducted twice. After 10 days, typical anthracnose symptoms developed on both unripen and ripen inoculated fruits, but not on non-inoculated controls. Overall, the severity of anthracnose lesions was higher on ripen fruits than in the unripen fruits. Likewise, the severity of symptoms was higher on the fruits inoculated using a mycelial plug than on those fruits inoculated with a spore suspension. The species C. karstii was reisolated from lesions of all inoculated fruits as described above but not from non-inoculated fruits. The species C. karstii has been described affecting numerous species worldwide (Damm et al., 2012). Previously, C. gloeosporioides was reported causing fruit anthracnose of Natal plum in Florida (Alfieri et al., 1984). To our knowledge, this is the first report of C. karstii causing anthracnose on the fruit of Natal plum in Spain and worldwide.

中文翻译：

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导致Carissa grandiflora果实炭疽病的Colletotrichum karstii首次报告在西班牙。

Carissa grandiflora A. DC。种，通常称为纳塔尔李，是一种灌木，原产于南非纳塔尔沿海地区。在西班牙南部，纳塔尔李子因其美丽的花朵和红色成熟的果实而被用作观赏植物。在2019年3月和2020年，我们对加的斯市和Sanlucar de Barrameda市（西班牙安达卢西亚）的9个公共花园进行了调查; 其中有六个（患病率55％）的纳塔尔李子水果显示炭疽病症状。受影响的果实显示出坏死的和圆形的病变，中心有小宫颈（图1a），导致果实完全木乃伊化（图1b）。根据Moral等人的论文，从四个花园中收集了受影响的果实并进行了除虫。（2010）。在马铃薯右旋糖琼脂（PDA）中从受影响的小块（3-4×1-2 mm）水果中回收了六种真菌分离物，然后将它们的菌丝尖端转移到新鲜的PDA中以获得纯培养物。根据其形态和ITS1-5.8S-ITS2（ITS）区域的序列，最初将这6个分离株鉴定为Colletotrichum karstii（Damm et al。2012）。六个炭疽菌分离株显示相似的菌落形态，其ITS序列相同。总体而言，卡氏梭菌的分生孢子呈圆柱形和直生分生孢子，长为12.1至14.2μm，宽为4.9至5.6μm（n = 50）。真菌的气生菌丝体从灰白色到深灰色不等。进行了多位点方法，以更精确地鉴定炭疽菌物种。为此，ITS，β-微管蛋白（TUB2），肌动蛋白（ACT），几丁质合酶1（CHS-1），组蛋白3（HIS3）的部分序列，根据Damm等人的方法扩增并测序了代表性分离物（FITP19001）的3-磷酸甘油醛脱氢酶（GAPDH）的200bp内含子片段。（2012）。ITS，TUB2，ACT，CHS-1，HIS3和GADPH的GenBank登录号分别为MT757643，MT759805，MT759806，MT759807，MT759808和MT759809。序列显示与属于卡氏梭菌的同源序列具有100％的同一性（GenBank出租车号：1095194）。为了测试科赫的假设，接种了从无症状植物中收获的10个未成熟和10个成熟的桔梗果实。对于每组，每滴卡氏梭菌悬浮液（FITP19001）每滴10微升5×104分生孢子，接种5个水果，并使用同一分离菌丝体的菌丝接种另外5个水果。将已接种的果实在室温（19-24ºC）的潮湿箱中于光照下孵育两周。用无菌水或PDA塞处理未接种的对照水果，并在相同条件下孵育。进行了两次致病性测试。10天后，未成熟和成熟的接种水果都会出现典型的炭疽病症状，而未接种的对照组则没有。总体而言，成熟果实的炭疽病损害严重程度高于未成熟果实的炭疽病损害。同样，用菌丝体塞接种的水果的症状严重程度高于用孢子悬浮液接种的水果。如上所述，从所有接种水果的病灶中重新分离了卡氏梭菌，但未接种水果中没有。物种C. karstii已被描述影响全世界的许多物种（Damm等，2012）。以前，据报道C. gloeosporioides导致佛罗里达州纳塔尔李的果实炭疽病（Alfieri等，1984）。据我们所知，这是C. karstii在西班牙乃至世界范围内的纳塔尔李子果实中引起炭疽病的首次报道。