小麦(Triticumaestivum)是世界范围内的重要作物,约占全球热量摄入的三分之一。2021年6月,有细菌性疫病症状的叶子,包括平行于静脉的黄色和坏死病变,在科罗拉多州东部五个县的几个田地中发现(Weld,摩根,塞奇威克,巴卡,和基特·卡森)。表现出这些症状的植物散布在田野中,但各县的症状似乎一致。为了确定因果代理并完成科赫的假设,从四个田间样品中切除1cm2有症状的叶面积并在0.5mL无菌水中浸渍。将裂解物铺在酵母提取物葡萄糖碳酸钙培养基(YDC琼脂,1%酵母提取物,2%葡萄糖,2%碳酸钙,1.5%琼脂)分离细菌。黄色的单菌落,选择粘液形态并在新的YDC板上划线。提取分离基因组DNA(ZymoResearchQuick-DNA真菌/细菌Miniprep试剂盒,#D6005),并使用约30ng的gDNA来扩增16SrRNA,gyrB,和所有四个分离株的rpoB基因(Barret等人。,2015年;Deletoile等人。,2009;Krawczyk等人。,2020年;奥吉尔等人。,2019)。清洗扩增的PCR产物(ZymoDNAClean&Concentrator试剂盒,#D4033)和桑格测序,和所有序列已保藏在NCBI(16SrRNA:OR707336,OR707337,OR707338,OR707339),(gyrB:PP407951,PP407952,PP407953,PP407954),(rpoB:PP407955,PP407956,PP407957,PP407958)。对整个基因组的BLAST搜索确定了来自KitCarson县(CO314)的一个分离株和来自Baca县的两个分离株(CO316和CO317)作为泛菌团聚体,对16SrRNA具有100%的同一性,gyrB,和rpoB基因,Weld县的一个分离株(CO315)与Pantoeaallii的所有三个基因均100%相同。完成科赫的假设并确认泛果。作为致病因素,分离物在DifcoTM营养琼脂(NA)培养基上生长为草坪(48h,28℃),悬浮在10mMMgCl2中,最终光密度为0.1(每毫升〜109个菌落形成单位(CFU/mL)),并注射器渗入10天龄小麦幼苗叶片的整个叶面积(var。Hatcher)。10mMMgCl2和不会引起症状的现场分离物的治疗,通过16SrRNA和gyrB测序鉴定为假单胞菌,是阴性对照。将接种的小麦植株转移到生长室(22℃,90%相对湿度)。接种后14天出现症状(dpi),最严重的出现21dpi。通过在具有两个金属珠的组织LyserII(Qiagen)中研磨它们并用0.4mL无菌水稀释,从有症状的叶片中重新分离出四个泛菌分离物中的每一个。将每个分离物的20微升样品镀在NA上(24h,28℃)。菌落的表型与原始分离株相同,和Sanger测序证实了分离株的身份。据我们所知,这是美国小麦成群疟原虫引起疾病的第一份报告,以及作为小麦致病因子的艾丽假丝酵母的第一份报告。该报告与以前的通讯一致,该通讯显示成群假单胞菌在中国引起小麦病害(Gao等人。,2023),和波兰的P.ananatis(Krawczyk等人。,2020)。泛菌属的报告越来越多。作为病原体,近年来表明越来越多的新疾病出现在全球范围内的谷物。
Wheat (Triticum aestivum) is an important crop worldwide, contributing to about one third of the global caloric intake. In June 2021, leaves with bacterial blight symptoms, including yellow and necrotic lesions running parallel to veins, were found in several fields across five counties in eastern Colorado (Weld, Morgan, Sedgwick, Baca, and Kit Carson). Plants exhibiting these symptoms were scattered throughout fields, but symptoms appeared consistent across counties. To determine the causal agent and complete Koch\'s postulates, a 1 cm2 symptomatic leaf area was excised and macerated in 0.5 mL of sterilized water from four field samples. The lysate was spread on yeast extract dextrose calcium carbonate medium (YDC agar, 1% yeast extract, 2% dextrose, 2% calcium carbonate, 1.5% agar) to isolate bacteria. Single colonies of yellow, mucoid morphology were selected and streaked on new YDC plates. Isolate genomic DNA was extracted (Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit, #D6005), and ~30 ng of gDNA was used to amplify the 16S rRNA, gyrB, and rpoB genes of all four isolates (Barret et al., 2015; Delétoile et al., 2009; Krawczyk et al., 2020; Ogier et al., 2019). Amplified PCR products were cleaned (Zymo DNA Clean & Concentrator kit, #D4033) and Sanger sequenced, and all sequences have been deposited in NCBI (16S rRNA: OR707336, OR707337, OR707338, OR707339), (gyrB: PP407951, PP407952, PP407953, PP407954), (rpoB: PP407955, PP407956, PP407957, PP407958). A BLAST search against whole genomes identified one isolate from Kit Carson county (CO314) and two isolates from Baca county (CO316 and CO317) as
Pantoea agglomerans with 100% identity for the 16S rRNA, gyrB, and rpoB genes, and one isolate from Weld county (CO315) was 100% identical to
Pantoea allii for all three genes. To complete Koch\'s postulates and confirm
Pantoea sp. as the causal disease agents, isolates were grown as lawns on DifcoTM Nutrient Agar (NA) medium (48h, 28℃), suspended in 10 mM MgCl2 using a final optical density of 0.1 (~109 colony forming units per milliliter (CFU/mL)), and syringe-infiltrated into the entire leaf area of 10-day-old wheat seedling leaves (var. Hatcher). Treatments of 10mM MgCl2 and a field isolate that does not cause symptoms, identified as Pseudomonas synxantha by 16S rRNA and gyrB sequencing, were negative controls. Inoculated wheat plants were transferred to a growth chamber (22℃, 90% relative humidity). Symptoms developed 14 days post inoculation (dpi), with the most severe appearing 21 dpi. Each of the four
Pantoea isolates were re-isolated from symptomatic leaves by grinding them in a Tissue Lyser II (Qiagen) with two metal beads and diluting with 0.4 mL of sterile water. A 20 µL sample of each isolate was plated on NA (24h, 28℃). The colonies appeared phenotypically identical to the original isolates, and Sanger sequencing confirmed the identities of the isolates. To our knowledge, this is the first report of P. agglomerans causing disease in wheat in the United States, and the first report of P. allii as a wheat disease-causing agent. This report is consistent with previous communications showing P. agglomerans causing wheat disease in China (Gao et al., 2023), and P. ananatis in Poland (Krawczyk et al., 2020). The growing numbers of reports of
Pantoea spp. as pathogens in recent years suggests increasing novel disease emergence on cereals worldwide.