Fernandez Laura Noemi, Maumary Roxana Lorena, Seimandi Gisela Marisol, Pernuzzi Cristián, Derita Marcos Gabriel, Favaro Maria Alejandra
Facultad de Ciencias Agrarias, Universidad Nacional del Litoral, Kredder 2805, Esperanza, Santa Fe, Argentina, 3080;
Facultad de Ciencias Agrarias, Universidad Nacional del Litoral, Esperanza, Argentina;
Plant Dis. 2023 Oct 11. doi: 10.1094/PDIS-08-23-1543-PDN.
Strawberry ( x ) production in Argentina extends to around 1700 hectares. Coronda city, located in Santa Fe province, is an important strawberry producer due to ideal agroecological conditions for culture and a high specialization for production. In November 2021, anthracnose symptoms were observed on strawberries cvs. 'San Andreas' and 'Splendor' in Coronda (31°58'S, 60°55'W), central Argentina. During these years, the incidence of the disease reached 40% of the production. Symptoms included 2-3 mm circular to irregular dark brown spots which enlarged rapidly and became sunken. Under high humidity conditions, concentric rings of pinhead-size salmon-colored acervuli developed on the lesions. The causal agent was isolated by touching acervuli with a sterile needle and monosporic cultures were obtained on PDA after 10 days at 25°C, with a 12-h light period. Colonies were white to gray on the top and orange on the underside, where concentric rings of salmon acervuli were clearly distinguished. The width and length of one hundred conidia were examined in three isolates (CF1, CF2, and CF3), ranging from 3.27 to 5.53 μm (avg.= 4.3 μm), and from 10.37 to 19.52 μm (avg.= 14.27 μm), respectively. The conidia were hyaline, smooth-walled, aseptate, and cylindric-clavate with one end round and one end acute. These morphological characteristics correspond to species belonging to the C. acutatum complex (Damm et al. 2012; Liu et al. 2022). To accurately identify the species, DNA was extracted from isolates, and ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone (HIS3) genes were partially amplified and sequenced (Vieira et al. 2020). TUB2, GAPDH, and HIS3 sequences presented a 100% of identity with species of . The nucleotide sequences were deposited in GenBank (OR271556-OR271558, TUB2; OR271559-OR271561, GAPDH; and OR271562-OR271564, HIS3). Multilocus phylogenetic analyses performed with reference sequences (Damm et al. 2012) showed that the three isolates clustered with , in accordance with BLAST results. To confirm pathogenicity, each isolate was inoculated in eight detached fruits of the cultivar from which it was originally obtained. Two drops of 10 μl of conidial suspension (1x105 conidia per ml) were deposited in non-wounded areas on fruits previously disinfested with 1% sodium hypochlorite solution for 1 min and rinsed twice with sterile distilled water. Drops of sterile water were deposited in eight fruits as control. Pathogenicity tests were repeated twice. Fruits were kept in moist chamber (80+5% relative humidity) at 25°C for ten days. First symptoms appeared 4 days after inoculation. After that, all of the isolates produced symptoms identical to those previously described, whereas the controls remain symptomless. The pathogen was re-isolated from lesions, and identified as by morphological characteristics and based on the TUB2 sequences, as previously described. Strawberry anthracnose in Argentina was previously associated with and species based on morphological characteristics (Ramallo et al. 2000; Monaco et al. 2000) but molecular identification was not performed until today. To our knowledge, this is the first report of causing anthracnose on strawberry in Argentina. This accurate identification will help to develop more efficient management strategies.
阿根廷的草莓种植面积约为1700公顷。位于圣菲省的科龙达市是重要的草莓产区,因为其具备适宜种植的理想农业生态条件且生产专业化程度高。2021年11月,在阿根廷中部科龙达(南纬31°58′,西经60°55′)的草莓品种“圣安德烈亚斯”和“辉煌”上观察到炭疽病症状。这些年,该病发病率达到产量的40%。症状包括2 - 3毫米的圆形至不规则深褐色斑点,斑点迅速扩大并凹陷。在高湿度条件下,病斑上会形成针头大小的鲑鱼色分生孢子盘同心环。通过用无菌针触碰分生孢子盘分离出病原菌,在25°C、12小时光照周期下培养10天后,在马铃薯葡萄糖琼脂(PDA)培养基上获得单孢培养物。菌落顶部为白色至灰色,底部为橙色,能清晰分辨出鲑鱼色分生孢子盘同心环。对三个分离株(CF1、CF2和CF3)的100个分生孢子的宽度和长度进行了检测,宽度范围为3.27至5.53微米(平均 = 4.3微米),长度范围为10.37至19.52微米(平均 = 14.27微米)。分生孢子无色透明、壁光滑、无隔膜,呈圆柱状 - 棒状,一端圆形,一端尖形。这些形态特征与尖孢炭疽菌复合体的物种相符(达姆等人,2012年;刘等人,2022年)。为准确鉴定物种,从分离株中提取DNA,对β - 微管蛋白(TUB2)、甘油醛 - 3 - 磷酸脱氢酶(GAPDH)和组蛋白(HIS3)基因进行部分扩增和测序(维埃拉等人,2020年)。TUB2、GAPDH和HIS3序列与该物种的序列一致性为100%。核苷酸序列已存入GenBank(OR271556 - OR271558,TUB2;OR271559 - OR271561,GAPDH;OR271562 - OR271564,HIS3)。与参考序列(达姆等人,2012年)进行的多位点系统发育分析表明,这三个分离株与该物种聚类,与BLAST结果一致。为确认致病性,将每个分离株接种到最初分离出它的品种的八个离体果实上。将两滴10微升的分生孢子悬浮液(每毫升含1×10⁵个分生孢子)滴在先前用1%次氯酸钠溶液消毒1分钟并用无菌蒸馏水冲洗两次的果实未受伤部位。在八个果实上滴加无菌水作为对照。致病性试验重复两次。果实置于25°C的保湿箱(相对湿度80±5%)中10天。接种4天后出现最初症状。之后,所有分离株产生的症状与先前描述的相同,而对照果实无症状。从病斑中重新分离出病原菌,并根据形态特征和TUB2序列鉴定为该物种,如先前所述。基于形态特征,阿根廷的草莓炭疽病以前与该物种和其他物种有关(拉马略等人,2000年;莫纳科等人,2000年),但直到现在才进行分子鉴定。据我们所知,这是该物种在阿根廷引起草莓炭疽病的首次报道。这种准确鉴定将有助于制定更有效的管理策略。