Comte Alexia, Gräfenhan Tom, Links Matthew G, Hemmingsen Sean M, Dumonceaux Tim J
Agriculture and Agri-Food Canada Saskatoon Research and Development Centre, Saskatoon, Saskatchewan, Canada.
Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada.
PLoS One. 2017 Mar 3;12(3):e0173495. doi: 10.1371/journal.pone.0173495. eCollection 2017.
We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.
我们使用通用条形码伴侣蛋白60（cpn60）检测了谷物的附生微生物组。对从田间种植的谷物制备的含DNA提取物的种子洗涤液进行微生物群落分析，检测到来自仅鉴定到粪壳菌纲的一种真菌的序列。为了鉴定该真菌序列并完善参考数据库，我们测定了从田间采集的麦角菌（Claviceps purpurea）菌株和参考菌株的cpn60序列。这些数据使我们能够鉴定该真菌序列源自麦角菌，并表明在包括那些未将麦角鉴定为分级因素的农产品上，很容易检测到麦角菌DNA。为了了解谷物中麦角菌DNA的流行程度和水平，我们基于真菌内部转录间隔区（ITS）开发了一种定量PCR检测方法，并将其应用于2014作物年度的137个样品。虽然有证据表明非目标生物导致了基于ITS检测方法的一些假阳性，但定量的麦角菌DNA量与每个谷物批次中鉴定出的麦角菌核的比例密切相关。因此，我们开发了一种靶向cpn60的环介导等温扩增检测方法，并将其应用于相同的谷物洗涤样品。阳性出现时间与通过视觉评级确定的麦角水平呈显著负相关。这些结果表明，基于实验室的和适用于田间的分子诊断检测方法都可用于使用谷物洗涤液检测和定量大宗农产品中的病原体负荷。
