黄淮海夏玉米区玉米籽粒带菌检测分析

Detection and Analysis of Fungi Carried by Maize Grain in Huang-Huai-Hai Summer Maize Region

【目的】明确黄淮海夏玉米区玉米籽粒带菌量,为玉米安全生产、储藏加工以及检疫提供参考依据。【方法】分别于玉米乳熟期及完熟期,采集黄淮海夏玉米区4个省(河北、河南、山东、安徽)90个市/县的玉米果穗,每个市/县采集表面未发生病症且果穗饱满的玉米穗4个,共采集720个样本。对所有样本进行籽粒外部及内部带菌量及带菌种类的检测,外部检测采用洗涤检测法,通过统计菌落总数与稀释倍数,计算籽粒表面的孢子负荷量及分离到的各菌属的分离比例;内部检测采用PDA平板法,对籽粒外部检测过的10个籽粒消毒后置于PDA平板上进行培养,统计每个籽粒带菌情况,计算籽粒带菌率及各菌属的分离频率。并且对籽粒内部分离频率较大的菌群进行形态学和分子鉴定。【结果】供试样本带菌量较大,乳熟期籽粒孢子负荷量在0—1 886个/粒,均值为439个/粒,籽粒带菌率在0—65.0%,均值为23.6%;完熟期籽粒孢子负荷量在18—2 658个/粒,均值为942个/粒,籽粒带菌率在10.0%—100.0%,均值为59.6%。完熟期带菌量大于乳熟期,但部分地区乳熟期带菌量仍较大。不同地区玉米籽粒带菌量存在差异,河南省的玉米籽粒带菌量较大,安徽省带菌量最少,河北省与山东省居中且差异不明显。玉米籽粒内部以及外部均携带的真菌类群有镰孢菌(Fusarium spp.)、青霉菌(Penicillium spp.)、曲霉菌(Aspergillus spp.)、链格孢菌(Alternaria spp.)、木霉菌(Trichoderma spp.)、根霉菌(Rhizopus spp.)、蠕孢菌(Hel-minthosporium spp.)、毛霉菌(Mucor spp.)。乳熟期籽粒外部和内部镰孢菌的分离比例分别为59.1%和36.1%,说明玉米在乳熟期时即有大量镰孢菌侵入;籽粒外部青霉菌和曲霉菌的分离比例分别为8.9%与0.7%,籽粒内部的分离频率分别为6.0%与1.9%,说明乳熟期青霉菌与曲霉菌也已经开始侵染玉米果穗。完熟期籽粒外部与内部镰孢菌的分离比例分别为71.9%和58.5%,籽粒外部青霉菌和曲霉菌的分离比例分别为17.0%和0.9%,籽粒内部分离频率分别为9.3%和2.6%,说明镰孢菌、青霉菌、曲霉菌为本研究黄淮海夏玉米区玉米籽粒携带的主要真菌。形态与分子鉴定结果显示,镰孢菌属中轮枝镰孢(F.verticillioides)的分离频率为29.7%,层出镰孢(F.proliferatum)的分离频率为25.9%,禾谷镰孢(F.graminearum)的分离频率为1.3%,表明轮枝镰孢为优势菌;青霉菌主要分离到绳状青霉(P.funiculosum)和草酸青霉(P.oxalicum),分离频率分别为5.0%和3.6%;曲霉菌主要为黄曲霉(A.flavus)和黑曲霉(A.niger),分离频率分别为1.4%和1.2%。【结论】表面无症状的玉米籽粒在乳熟期及完熟期均携带大量病原菌,且完熟期带菌量大于乳熟期;镰孢菌在黄淮海夏玉米区的分离频率最大,轮枝镰孢为当地玉米籽粒携带的优势真菌。

【Objective】The objective of this study is to determine the amount of fungi carried in maize grains in Huang-Huai-Hai summer maize region,and to provide reference for safe production,storage,processing and quarantine of maize.【Method】A total of 720 maize ears were collected at the stages of milk-ripening and full-ripening in 90 cities or counties in 4 provinces(Hebei,Henan,Shandong,Anhui)of the Huang-Huai-Hai summer maize region,4 full maize ears with no symptom on the surface were sampled in each city or county.All samples were tested for the amount and species of fungi carried,including external and internal tests of maize grains.Washing detection method was used to calculate the spores load on the grain surface,the spore load on the grain surface and isolation rate of each genus were calculated by counting the total number of colonies and dilution multiple.The PDA plate method was used for internal detection.Ten grains tested externally were sterilized and cultured on PDA plate.The fungi carrying rate of grains and the isolation frequency of each genus of fungi were calculated by counting the fungi carrying rate of each grain.In addition,the morphological and molecular identification of the higher frequency fungi were carried out.【Result】The samples carried a large number of fungi.The spores load of the tested samples ranged from 0 to 1 886 per grain,with an average of 439 spores per grain at milk-ripening stage,the fungi-carrying rate of grains ranged from 0 to 65.0%,with an average of 23.6%.The spores load ranged from 18 to 2 658 per grain,with an average of 942 spores per grain at full-ripening stage,the fungi-carrying rate of grains ranged from 10.0%to 100.0%,with an average of 59.6%.The amount of fungi carried in full-ripening stage was higher than that in milk-ripening stage,but in some areas,the amount of fungi carried was still large in milk-ripening stage.There were differences in the amount of fungi carried in maize grains in different areas.The amount of fungi carried in maize grains in Henan Province was the largest,and Anhui Province had the least amount of fungi,Hebei Province and Shandong Province were in the middle and the difference was not significant.Fungal communities carried both inside and outside of maize grains included Fusarium spp.,Penicillium spp.,Aspergillus spp.,Alternaria spp.,Trichoderma spp.,Rhizopus spp.,Hel-minthosporium spp.,Mucor spp.The isolation rate of Fusarium spp.in the grain external and internal detection was 59.1%and 36.1%respectively at milk-ripening stage,indicating that a large number of Fusarium spp.occurred at milk-ripening stage of maize.The isolation rate of Penicillium spp.and Aspergillus spp.in external grains was 8.9%and 0.7%respectively,and the isolation frequency in internal grains was 6.0%and 1.9%respectively,indicating that Penicillium spp.and Aspergillus spp.had begun to infect maize ear at milk-ripening stage.The isolation rate of Fusarium spp.in the grain external and internal detection was 71.9%and 58.5%respectively at full-ripening stage,the isolation rate of Penicillium spp.and Aspergillus spp.in external grains was 17.0%and 0.9%respectively,and the isolation frequency in internal grains was 9.3%and 2.6%respectively,indicating that Fusarium spp.,Penicillium spp.and Aspergillus spp.were the main fungi carried by maize grains in Huang-Huai-Hai summer maize region.Morphological and molecular identification results showed that F.verticillioides was the dominant strain in Fusarium spp.,the isolation frequency of F.verticillioides,F.proliferatum,F.graminearum was 29.7%,25.9%,1.3%,respectively.P.funiculosum and P.oxalicum were the dominant strains in Penicillium spp.,and the isolation frequency was 5.0%and 3.6%,respectively.A.flavus and A.niger were the dominant strains in Aspergillus spp.,and the isolation frequency was 1.4%and 1.2%,respectively.【Conclusion】The maize grains with no surface symptom carried a large number of fungi in both milk-ripening and full-ripening stages,and the amount of fungi carried in the full-ripening stage is higher than that in the milk-ripening stage.Fusarium spp.is the most frequently isolated fungi in Huang-Huai-Hai summer maize region and F.verticilliflora is the dominant strain.It is the dominant pathogen carried by local maize grains.