Liu Jintao, Wang Rufeng, Qian Ning, Li Jialing, Zhao Wensheng, Xing Junjie, Yang Jun
China Agricultural University, 34752, College of Plant Protection, China Agricultural University, 2 West Yuanmingyuan Road, Beijing, Beijing, China, 100193;
China Agricultural University College of Plant Protection, 539443, Department of Plant Biosecurity, Beijing, China;
Plant Dis. 2022 Jul 11. doi: 10.1094/PDIS-05-22-1077-PDN.
Palm grass (Setaria palmifolia) has been used as an ornamental plant and vegetable crop (Wu, 2009; Plarre, 1995). In June 2019, 2-10 mm severe leaf lesions with gray centers and brown-yellow edges were observed on the leaves of palm grass in Liuyang city (28°43'N, 114°12'E), Hunan province, China (Fig. 1A). Disease incidence on leaves was 20 - 40%. The infected leaves were collected and disinfected with 75% alcohol for 30 sec and 1% sodium hypochlorite for 1 min, followed by three rinses in sterilized ddH2O, dried on sterilized filter paper, and incubated on water agar for 48 h under continuous fluorescent light at 26℃. Then, typical pyriform and 2-septate conidia (23.97 - 30.37 × 7.42 - 9.98 μm, N = 30) appeared at the lesions (Fig. 1B). Four single-spore were captured, and then grew on oatmeal tomato agar for seven days under continuous fluorescent light at 26℃ to obtain four isolates (LY-ZY-7a, -7b, -9b and -9c) and produce conidia for inoculation tests. The colony morphology of LY-ZY-7b on OTA was gray and floccose, and the growth rate was 6.15 - 6.31 mm/d at 26 °C (Fig. 1C). Spores of LY-ZY-7b were washed off with sterilized ddH2O plus 0.025% Tween-20 to make spore suspensions. For scratch inoculation, 10 μL spore suspension (1 × 105 spores/mL) was inoculated on the wound scratched with a sterilized pin along the vein (3 mm × 3 mm) on palm grass middle leaf of 4-week-old seedlings. The inoculated leaves were sealed in a 15-cm Petri dish. For spray inoculation, 20 mL spore suspension (5 × 104 spores/mL) was made and sprayed on ten healthy palm grasses of 4-week-old seedlings. Plants used as negative controls were sprayed with sterilized ddH2O plus 0.025% Tween-20 (Liu et al. 2022; Zhang et al. 2014). After inoculation, all plants were put into transparent boxes to maintain > 95% humidity and covered with black plastic bags for one day. Then, the boxes containing the plants were placed in a growth chamber at 26°C (12 h light / 12 h darkness photoperiod). After six days, typical blast-type lesions with brown-yellow edges were visible on the leaves. Control plants did not show symptoms (Fig. 1D, 1E). Microscopical examination showed that the conidia and conidiophore recovered from the lesion of the inoculated plants have the same morphology as those recovered from natural infected tissues (Fig. 1F, 1G). The colony morphology of the pathogen isolated from the artificially inoculated tissue was consistent with that of isolate LY-ZY-7b (Fig. 1C). The spore suspension (5 × 104 spores/mL) of isolate LY-ZY-7b and one rice-infecting strain P131 (Yang et al., 2010) was made and sprayed onto 4-week-old seedlings of three rice cultivars. But unfortunately, isolate LY-ZY-7b could not cause any disease lesions on the tested rice cultivars, whereas strain P131 produced many typical blast lesions on rice leaves (Fig. 1H). Then, the fungal genetic identity of four isolates (LY-ZY-7a, -7b, -9b, and -9c) was confirmed by comparison of the sequence obtained from partial DNA of Actin (ACT), ITS, and RPB1 loci from our isolates and those previously published by Klaubauf et al. 2014. The nucleotide sequences of ACT, ITS, and RPB1 were submitted to GenBank ON228695-ON228697 (ACT), ON210978-ON210980 (ITS), ON228698-ON228701 (RPB1). A phylogenetic tree deduced from a maximum likelihood analysis based on combined ACT-ITS-RPB1 sequence data of Pyricularia showed that these four isolates (LY-ZY-7a, -7b, -9b, and -9c) clustered together on Pyricularia oryzae, with a high bootstrap support value (Fig. 2). Based on morphological characteristics and molecular phylogeny, these four isolates were identified as P. oryzae (Klaubauf et al. 2014; Qi et al. 2019). To our knowledge, this is the first report of blast disease on palm grass caused by P. oryzae in China, which will help develop disease management strategies against palm grass blast. Moreover, as a host of P. oryzae, palm grass might contribute as an inoculum source for blast diseases on cereal crops (such as rice, wheat, and barley) caused by P. oryzae in the field.
棕叶狗尾草（Setaria palmifolia）一直被用作观赏植物和蔬菜作物（Wu，2009；Plarre，1995）。2019年6月，在中国湖南省浏阳市（北纬28°43′，东经114°12′）的棕叶狗尾草叶片上观察到严重的叶斑，病斑长2 - 10毫米，中央灰色，边缘棕黄色（图1A）。叶片发病率为20% - 40%。采集感染的叶片，先用75%酒精消毒30秒，再用1%次氯酸钠消毒1分钟，然后在无菌双蒸水中冲洗三次，在无菌滤纸上干燥，并在水琼脂上于26℃连续荧光灯下培养48小时。之后，在病斑处出现典型的梨形和具2个隔膜的分生孢子（23.97 - 30.37 × 7.42 - 9.98μm，N = 30）（图1B）。捕获4个单孢子，然后在燕麦番茄琼脂上于26℃连续荧光灯下培养7天，获得4个分离株（LY-ZY-7a、-7b、-9b和-9c），并产生分生孢子用于接种试验。LY-ZY-7b在燕麦番茄琼脂上的菌落形态为灰色、絮状，在26℃下生长速率为6.15 - 6.31毫米/天（图1C）。用无菌双蒸水加0.025%吐温-20冲洗LY-ZY-7b的孢子，制成孢子悬浮液。对于划痕接种，将10μL孢子悬浮液（1×10⁵个孢子/毫升）接种在4周龄幼苗的棕叶狗尾草中叶上，用无菌针沿叶脉划痕处（3毫米×3毫米）。接种的叶片密封在15厘米的培养皿中。对于喷雾接种，制备20毫升孢子悬浮液（5×10⁴个孢子/毫升），并喷洒在10株4周龄的健康棕叶狗尾草幼苗上。用作阴性对照的植株喷洒无菌双蒸水加0.025%吐温-20（Liu等，2022；Zhang等，2014）。接种后，将所有植株放入透明盒中以保持湿度>95%，并用黑色塑料袋覆盖一天。然后，将装有植株的盒子置于26℃的生长室中（12小时光照/12小时黑暗光周期）。6天后，叶片上可见边缘棕黄色的典型稻瘟病型病斑。对照植株未出现症状（图1D、1E）。显微镜检查表明，从接种植株病斑处回收的分生孢子和分生孢子梗与从自然感染组织中回收的形态相同（图1F、1G）。从人工接种组织中分离的病原菌的菌落形态与分离株LY-ZY-7b一致（图1C）。制备分离株LY-ZY-7b的孢子悬浮液（5×10⁴个孢子/毫升）和一个侵染水稻的菌株P131（Yang等，2010），并喷洒在三个水稻品种的4周龄幼苗上。但遗憾的是，分离株LY-ZY-7b在测试的水稻品种上未引起任何病害病斑，而菌株P131在水稻叶片上产生了许多典型的稻瘟病斑（图1H）。然后，通过比较我们分离株以及Klaubauf等人2014年发表的Actin（ACT）、ITS和RPB1基因座部分DNA序列，确认了四个分离株（LY-ZY-7a、-7b、-9b和-9c）的真菌遗传同一性。ACT、ITS和RPB1的核苷酸序列已提交至GenBank，登录号分别为ON228695 - ON228697（ACT）、ON210978 - ON210980（ITS）、ON228698 - ON228701（RPB1）。基于稻瘟病菌ACT-ITS-RPB1联合序列数据的最大似然分析推导的系统发育树表明，这四个分离株（LY-ZY-7a、-7b、-9b和-9c）在稻瘟病菌上聚在一起，具有较高的自展支持值（图2）。基于形态特征和分子系统发育，这四个分离株被鉴定为稻瘟病菌（Klaubauf等，2014；Qi等，2019）。据我们所知，这是中国首次报道由稻瘟病菌引起的棕叶狗尾草稻瘟病，这将有助于制定针对棕叶狗尾草稻瘟病的病害管理策略。此外，作为稻瘟病菌的寄主，棕叶狗尾草可能会成为田间由稻瘟病菌引起的谷物作物（如水稻、小麦和大麦）稻瘟病的接种源。
