Mezzalama Monica, Guarnaccia Vladimiro, Martino Ilaria, Tabome Giulia, Gullino Maria Lodovica
University of Turin, 9314, DISAFA, Largo Paolo Braccini 2, Grugliasco, TO, Italy, 10095;
DISAFA, 165514, University of Torino, largo braccini 2, Torino, Italy, 10095;
Plant Dis. 2021 Jun 15. doi: 10.1094/PDIS-01-21-0075-PDN.
Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 62,587,469 t, with an area that covers 628,801 ha, concentrated in northern Italy (ISTAT 2020). Fusarium species are associated with root and crown rot causing failures in crop establishment under high soil moisture. In 2019 maize seedlings collected in a farm located in San Zenone degli Ezzelini (VI, Italy) showed root and crown rot symptoms with browning of the stem tissues, wilting of the seedling, and collapsing due to the rotting tissues at the base of the stem. The incidence of diseased plants was approximately 15%. Seedlings were cleaned thoroughly from soil residues under tap water. Portions (about 3-5 mm) of tissue from roots and crowns of the diseased plants were cut and surface disinfected with a water solution of NaClO at 0.5% for 2 minutes and rinsed in sterile H20. The tissue fragments were plated on Potato Dextrose Agar (PDA) amended with 50 mg/l of streptomycin sulfate and incubated for 48-72 hours at 25oC. Over the 80 tissue fragments plated, 5% were identified as Fusarium verticillioides, 60% as Fusarium spp., 35% developed saprophytes. Fusarium spp. isolates that showed morphological characteristics not belonging to known pathogenic species on maize were selected and used for further investigation while species belonging to F. oxysporum were discarded. Single conidia of the Fusarium spp. colonies were cultured on PDA and Carnation Leaf Agar (CLA) for pathogenicity tests, morphological and molecular identification. The colonies showed white to pink, abundant, densely floccose to fluffy aerial mycelium. Colony reverse showed light violet pigmentation, in rings on PDA. On CLA the isolates produced slightly curved macronidia with 3 septa 28.1 - 65.5 µm long and 2.8-6.3 µm wide (n=50). Microconidia were cylindrical, aseptate, 4.5 -14.0 µm long and 1.5-3.9 µm wide (n=50). Spherical clamydospores were 8.8 ± 2.5 µm size (n=30), produced singly or in pairs on the mycelium, according to the description by Skovgaard et al. (2003) for F. commune. The identity of two single-conidia strains was confirmed by sequence comparison of the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (O'Donnell et al. 2010). BLASTn searches of GenBank, and Fusarium-ID database, using the partial tef-1α (MW419921, MW419922) and rpb2 (MW419923, MW419924) sequences of representative isolate DB19lug07 and DB19lug20, revealed 99% identity for tef-1α and 100% identity to F. commune NRRL 28387(AF246832, AF250560). Pathogenicity tests were carried out by suspending conidia from a 10-days old culture on PDA in sterile H2O to 5×104 CFU/ml. Fifty seeds were immersed in 50 ml of the conidial suspension of each isolate for 24 hours and in sterile water (Koch et al. 2020). The seeds were drained, dried at room temperature, and sown in trays filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 25°C and RH of 80-85% for 14 days with 12 hours photoperiod. Seedlings were extracted from the substrate, washed under tap water, and observed for the presence of root and crown rots like the symptoms observed on the seedlings collected in the field. Control seedlings were healthy and F. commune was reisolated from the symptomatic ones and identified by resequencing of tef-1α gene. F. commune has been already reported on maize (Xi et al. 2019) and other plant species, like soybean (Ellis et al. 2013), sugarcane (Wang et al. 2018), potato (Osawa et al. 2020), indicating that some attention must be paid in crop rotation and residue management strategies. To our knowledge this is the first report of F. commune as a pathogen of maize in Italy. References Ellis M L et al. 2013. Plant Disease, 97, doi: 10.1094/PDIS-07-12-0644-PDN. ISTAT. 2020. http://dati.istat.it/Index.aspx?QueryId=33702. Accessed December 28, 2020. Koch, E. et al. 2020. Journal of Plant Diseases and Protection. 127, 883-893 doi: 10.1007/s41348-020-00350-w O'Donnell K et al. 2010. J. Clin. Microbiol. 48:3708. https://doi.org/10.1128/JCM.00989-10 Osawa H et al. 2020. Journal of General Plant Pathology, doi.org/10.1007/s10327-020-00969-5. Skovgaard K 2003. Mycologia, 95:4, 630-636, DOI: 10.1080/15572536.2004.11833067. Wang J et al. 2018. Plant Disease, 102, doi/10.1094/PDIS-07-17-1011-PDN Xi K et al. 2019. Plant Disease, 103, doi/10.1094/PDIS-09-18-1674-PDN.
玉米（Zea mays L.）是意大利一种具有重要经济意义的谷类作物；目前产量为62587469吨，种植面积达628801公顷，集中在意大利北部（意大利国家统计局，2020年）。镰刀菌属与根腐病和冠腐病有关，在土壤湿度较高的情况下会导致作物定植失败。2019年，在意大利维琴察省圣泽诺内-德格列埃泽利尼的一个农场采集的玉米幼苗出现了根腐和冠腐症状，茎组织褐变、幼苗萎蔫，并因茎基部组织腐烂而倒伏。病株发生率约为15%。在自来水中将幼苗上的土壤残渣彻底清理干净。从患病植株的根和冠上切取约3 - 5毫米的组织部分，用0.5%的NaClO水溶液进行表面消毒2分钟，然后在无菌水中冲洗。将组织碎片接种在添加了50毫克/升硫酸链霉素的马铃薯葡萄糖琼脂（PDA）上，在25℃下培养48 - 72小时。在接种的80个组织碎片中，5%被鉴定为轮枝镰孢菌，60%为镰刀菌属，35%生长出腐生菌。选择表现出不属于已知玉米致病物种形态特征的镰刀菌属分离株用于进一步研究，而尖孢镰刀菌属的物种则被丢弃。将镰刀菌属菌落的单个分生孢子在PDA和香石竹叶琼脂（CLA）上培养，用于致病性测试、形态学和分子鉴定。菌落呈现白色至粉红色，丰富，气生菌丝浓密絮状至蓬松。菌落背面在PDA上呈浅紫色色素沉着，呈环状。在CLA上，分离株产生稍弯曲的大型分生孢子，具3个隔膜，长28.1 - 65.5微米，宽2.8 - 6.3微米（n = 50）。小型分生孢子圆柱形，无隔膜，长4.5 - 14.0微米，宽1.5 - 3.9微米（n = 50）。球形厚垣孢子大小为8.8 ± 2.5微米（n = 30），根据斯科夫加德等人（2003年）对腐皮镰刀菌的描述，单个或成对地在菌丝体上产生。通过对翻译延伸因子-1α（tef - 1α）和RNA聚合酶II亚基（rpb2）基因片段进行序列比较，确认了两个单分生孢子菌株的身份（奥唐奈等人，2010年）。使用代表性分离株DB19lug07和DB19lug20的部分tef - 1α（MW419921，MW419922）和rpb2（MW419923，MW419924）序列在GenBank和镰刀菌鉴定数据库中进行BLASTn搜索，结果显示tef - 1α的同源性为99%，与腐皮镰刀菌NRRL 28387（AF246832，AF250560）的同源性为100%。通过将来自PDA上10日龄培养物的分生孢子悬浮在无菌水中至5×104 CFU/ml进行致病性测试。将50粒种子浸入每种分离株的50毫升分生孢子悬浮液中24小时，然后浸入无菌水中（科赫等人，2020年）。沥干种子，在室温下干燥，然后播种在装有80:20（v/v）的白色泥炭和珍珠岩蒸汽混合物的托盘中，在25°C和80 - 85%的相对湿度下保持14天，光周期为12小时。将幼苗从基质中取出，在自来水中冲洗，并观察是否存在根腐和冠腐症状，如同在田间采集的幼苗上观察到的症状一样。对照幼苗健康，从有症状的幼苗中重新分离出腐皮镰刀菌，并通过对tef - 1α基因重新测序进行鉴定。腐皮镰刀菌已在玉米上报道过（Xi等人，2019年）以及其他植物物种上，如大豆（埃利斯等人，2013年）、甘蔗（Wang等人，2018年）、马铃薯（Osawa等人，2020年），这表明在作物轮作和残茬管理策略中必须予以一定关注。据我们所知，这是腐皮镰刀菌作为意大利玉米病原菌的首次报道。参考文献：埃利斯M L等人，2013年。《植物病害》，97，doi: 10.1094/PDIS - 07 - 12 - 0644 - PDN。意大利国家统计局，2020年。http://dati.istat.it/Index.aspx?QueryId = 33702。2020年12月28日访问。科赫，E.等人，2020年。《植物病害与保护杂志》。127，883 - 893 doi: 10.1007/s41348 - 020 - 00350 - w。奥唐奈K等人，2010年。《临床微生物学杂志》。48:3708。https://doi.org/10.1128/JCM.00989 - 10。Osawa H等人，2020年。《普通植物病理学杂志》，doi.org/10.1007/s10327 - 020 - 00969 - 5。斯科夫加德K，2003年。《真菌学》，95:4，630 - 636，DOI: 10.1080/15572536.2004.11833067。Wang J等人，2018年。《植物病害》，102，doi/10.1094/PDIS - 07 - 17 - 1011 - PDN。Xi K等人，2019年。《植物病害》，103，doi/10.1094/PDIS - 09 - 18 - 1674 - PDN。
