谷物中重要真菌产毒预测微生物学和扩增片段长度多态性分型研究
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摘要
目的：
(1)对安徽、江苏、河北、河南四省部分地区2008年产小麦、玉米的真菌污染状况和菌相分布进行调查,为进行粮食中主要污染真菌产毒预测微生物学研究提供菌株；
(2)对可能影响禾谷镰孢产毒的关键因子进行筛选,为不同自然条件下预测禾谷镰孢的产毒情况提供资料,为粮食防霉提供实验手段；
(3)对从安徽、江苏、河北、河南四省小麦、玉米样品中分离出的50株串珠镰孢进行AFLP分型,通过DNA指纹图谱鉴定来自我国不同地区同一作物、同一地区不同作物中串珠镰孢纯化株的种群亚型和菌株的亲缘关系。
方法：
(1)将小麦、玉米样品消毒后点种于PDA培养基上,28±1℃培养5-7天后进行菌落计数并鉴定菌相；
(2)以禾谷镰孢为研究对象,以玉米和小麦为研究基质,构建L18-Hunter和Plackett-Burman模型,筛选对禾谷镰孢毒素产量有较大影响的重要因素变量(温度、通气量、水分含量、pH值、光照、培养基量、基质成分、培养时间等)。同时进行毒素产量随时间而变化的趋势研究；
(3)对从安徽、江苏、河北和河南四省玉米和小麦样品中分离的50株串珠镰孢进行DNA提取纯化,经PstⅠ和MseⅠ双酶切,对酶切片段进行预扩增和选择性扩增后进行聚类分析。
结果：
(1)安徽、江苏、河北和河南四省小麦样品真菌污染率几乎100%,优势菌是交链孢霉,其次为根霉和镰孢；玉米样品全部被真菌污染,优势菌四省各异,镰孢均是四省玉米污染的重要优势菌,其中串珠镰孢最常见；
(2)L18-Hunter设计的实验结果表明,按a=0.05标准,三株禾谷镰孢中仅菌株3.4522产3-A-DON、15-A-DON以及10种毒素总和的模型拟合结果有统计学意义(P分别为0.0425、0.0118和0.0401),模型R2分别为0.80、0.86和0.81。回归方程系数效应检验结果显示,水分含量(P值分别为0.0293、0.0074和0.0189)和培养时间(P值分别为0.0302、0.0074和0.0189)是影响禾谷镰孢3.4522产3-A-DON、15-A-DON及10种毒素总量的关键因子。
以L18-Hunter设计实验结果为基础进行的Plackett-Burman设计实验结果表明,按a=0.05标准,就DON绝对值、DON对数值、ZEN对数值、DON及其衍生物的对数值、B类单端孢霉烯族化合物对数值、毒素总量绝对值及毒素总量对数值而言,该模型显著(P值分别为0.0345、0.0022、0.0029、0.0068、0.0067、0.0148和0.0006)。模型R2分别为0.81、0.93、0.92、0.89、0.90、0.86和0.95。回归方程系数显著性检验结果显示,在不同的培养条件下,培养时间、温度和初始pH值是影响禾谷镰孢3.4522产毒的关键因子。禾谷镰孢产毒量随培养时间延长而增加,多在14-28天达高峰,此后下降。
(3)50株串珠镰孢的AFLP分型结果显示,8对引物共扩增出1346个条带,其中多态性条带1322个,每对引物产生的多态性位点百分率在95.21%～100%之间,表明串珠镰孢在DNA水平上存在较大变异,其中来自河北玉米样品的串珠镰孢较其它省份多态性高。
50株串珠镰孢菌株间的相似系数在36%～81%,在相似系数为0.63时,50株串珠镰孢菌株可分为5组,其中49株来自玉米,其种群分5个组,一株来自小麦。AFLP分型结果显示,不同作物别、不同区域同一作物来源的串珠镰孢存在较大遗传差异,但该差异与样品种类和来源没有明显相关关系。
结论：
(1)四省2008年产小麦样品真菌污染严重,优势菌是交链孢霉；玉米样品全部被真菌污染,串珠镰孢为重要的优势菌相。
(2)培养时间、一定范围(20～50%)内的水分含量、温度和初始pH值是影响禾谷镰孢产毒的关键因子。禾谷镰孢产毒量随时间延长而增加,产毒高峰多在14-28天。
(3)串珠镰孢在DNA水平上存在较大变异,其中分离自河北玉米样品的串珠镰孢较其它省份多态性高。不同作物别来源(玉米或小麦)、不同区域同一作物来源串珠镰孢存在较大遗传差异,但该差异与样品种类和来源没有明显相关关系。
Objective:
(1) To study fungi invasion of wheat and corn collected from Anhui, Jiangsu, Hebei and Henan provinces harvested in 2008 and provide fungal strains for predictive microbiology studies.
(2) Screening of the key factors which may affect the toxin production by Fusarium graminearum in order to predict the toxin production under different natural conditions. The results obtained from predictive microbiology offer the technical support for controlling the cereal invasion by fungi.
(3) Fifty strains of F. moniliforme isolated from wheat and corn from 4 provinces were employed in amplified fragment length polymorphism (AFLP) molecular typing studies to identify the subtype and analyze the homology similarity among them.
Method:
(1) Wheat and corn kernels were inoculated onto PDA medium after sterilization with 75% ethanol followed by being rinsed with distilled water. The colony forming unit of fungi were counted and identified after incubation at 28±1℃for 5-7 days.
(2) F. graminearum inoculated on wheat kernals and corn flakes was used in predictive microbiological test. The L18-Hunter and Plackett-Burman model were constructed to screen the important factors (temperature, ventilatory capacity, water content, pH value, illumination, the amount and ingredients of the medium, incubation time etc.) which may affect the toxin production by F. graminearum. The time course of toxin production was also studied.
(3) Fifty purified F. moniliforme strains isolated from wheat and corn collected from Anhui, Jiangsu, Hebei and Henan provinces were extracted for DNA. The DNA was cut by both Pst I and Mes I enzymes and the fragments were analyzed after pre-amplification and amplification.
Results:
(1) Fungi were isolated from almost all of wheat samples and the predominant is Alternaria species. While, all corn samples were positive for fungi and the major is Fusarium species. Among which, F. moniliforme was the important.
(2) The L18-Hunter experimental results showed that only the model fitting results of 3-A-DON、15-A-DON and total of 10 kinds of toxins produced by the strain 3.4522 were statistically significant (P=0.0425,0.0118 and 0.0401 respectively) according to a=0.05 criterion and R2 of the model was 0.80,0.86 and 0.81, respectively. The coefficients of the regression equations were had a effect test and it showed that water content (P=0.0293, 0.0074 and 0.0189, respectively) and incubation time (P=0.0302,0.0074 and 0.0189, respectively) were the key factors that affected the level of 3-A-DON,15-A-DON and total of 10 kinds of toxins produced by F. graminearum strain 3.4522.
The results of Plackett-Burman experiment designed on the basis of L18-Hunter showed that the model fitting results of the absolute value of DON, logarithm value of DON, ZEN, DON and its derivatives and type B trichothecenes as well as the absolute and logarithm value of total toxin were statistically significant (P=0.0345,0.0022,0.0029,0.0068,0.0067,0.0148 and 0.0006, respectively) according to a=0.05 criterion and R2 of the model was 0.81,0.93, 0.92,0.89,0.90,0.86 and 0.95, respectively. The coefficients of the regression equations were had a effect test and it showed that incubation time, temperature and initial pH value of the media were the key factors that affected toxin production by F. graminearum strain 3.4522. The level of toxins produced by F. graminearum increased with the incubation time extended and reached the highest at the time of between 14 and 28 days.
(3) Based on AFLP results,50 strains of F. moniliforme generated 1346 bands and 1322 bands were polymorphic. Percentage of polymorphism fragments produced by each pair of AFLP primer varied between 95.21% and 100%. The considerable variation existed among F. moniliforme at DNA level. The F. moniliforme strains from Hebei corns had a higher polymorphism than those from other provinces.
The similarity coefficients of the strains were between 36% and 81%. The 50 F. moniliforme strains could be divided into 5 groups on the basis of the similarity coefficient of 0.63, in which 49 strains from corns and only 1 strain from wheat. The cluster analysis showed obvious differences in homology similarity of F. moniliforme strains isolated from different crops harvested in the same region and that from the same crop harvested in different regions, but these differences showed no obvious relations with the species and source of the samples.
Conclusion:
(1) Wheats collected from four provinces were seriously invaded by fungi and the majority was Alternaria species. While all corn samples were positive for fungi and Fusarium moniliforme was the predominant.
(2) Incubation time, water activity within a certain range (20～50%), temperature and pH value were the key factors that affected toxin production of F. graminearum. The level of toxin production by Fusarium Graminearum increased with the incubation time extended and reached the highest at the time of between 14 and 28 days.
(3) Considerable genetic variation existed in 50 strains of F. moniliforme at DNA level. The F. moniliforme strains from Hebei corns had a higher polymorphism than those from other provinces. There were obvious differences in homology similarity of F. moniliforme strains from different crops harvested in the same region and that from the same crop harvested in different regions, but these differences showed no obvious relations with the species and source of the samples.
(1)对安徽、江苏、河北、河南四省部分地区2008年产小麦、玉米的真菌污染状况和菌相分布进行调查,为进行粮食中主要污染真菌产毒预测微生物学研究提供菌株；
(2)对可能影响禾谷镰孢产毒的关键因子进行筛选,为不同自然条件下预测禾谷镰孢的产毒情况提供资料,为粮食防霉提供实验手段；
(3)对从安徽、江苏、河北、河南四省小麦、玉米样品中分离出的50株串珠镰孢进行AFLP分型,通过DNA指纹图谱鉴定来自我国不同地区同一作物、同一地区不同作物中串珠镰孢纯化株的种群亚型和菌株的亲缘关系。
方法：
(1)将小麦、玉米样品消毒后点种于PDA培养基上,28±1℃培养5-7天后进行菌落计数并鉴定菌相；
(2)以禾谷镰孢为研究对象,以玉米和小麦为研究基质,构建L18-Hunter和Plackett-Burman模型,筛选对禾谷镰孢毒素产量有较大影响的重要因素变量(温度、通气量、水分含量、pH值、光照、培养基量、基质成分、培养时间等)。同时进行毒素产量随时间而变化的趋势研究；
(3)对从安徽、江苏、河北和河南四省玉米和小麦样品中分离的50株串珠镰孢进行DNA提取纯化,经PstⅠ和MseⅠ双酶切,对酶切片段进行预扩增和选择性扩增后进行聚类分析。
结果：
(1)安徽、江苏、河北和河南四省小麦样品真菌污染率几乎100%,优势菌是交链孢霉,其次为根霉和镰孢；玉米样品全部被真菌污染,优势菌四省各异,镰孢均是四省玉米污染的重要优势菌,其中串珠镰孢最常见；
(2)L18-Hunter设计的实验结果表明,按a=0.05标准,三株禾谷镰孢中仅菌株3.4522产3-A-DON、15-A-DON以及10种毒素总和的模型拟合结果有统计学意义(P分别为0.0425、0.0118和0.0401),模型R2分别为0.80、0.86和0.81。回归方程系数效应检验结果显示,水分含量(P值分别为0.0293、0.0074和0.0189)和培养时间(P值分别为0.0302、0.0074和0.0189)是影响禾谷镰孢3.4522产3-A-DON、15-A-DON及10种毒素总量的关键因子。
以L18-Hunter设计实验结果为基础进行的Plackett-Burman设计实验结果表明,按a=0.05标准,就DON绝对值、DON对数值、ZEN对数值、DON及其衍生物的对数值、B类单端孢霉烯族化合物对数值、毒素总量绝对值及毒素总量对数值而言,该模型显著(P值分别为0.0345、0.0022、0.0029、0.0068、0.0067、0.0148和0.0006)。模型R2分别为0.81、0.93、0.92、0.89、0.90、0.86和0.95。回归方程系数显著性检验结果显示,在不同的培养条件下,培养时间、温度和初始pH值是影响禾谷镰孢3.4522产毒的关键因子。禾谷镰孢产毒量随培养时间延长而增加,多在14-28天达高峰,此后下降。
(3)50株串珠镰孢的AFLP分型结果显示,8对引物共扩增出1346个条带,其中多态性条带1322个,每对引物产生的多态性位点百分率在95.21%～100%之间,表明串珠镰孢在DNA水平上存在较大变异,其中来自河北玉米样品的串珠镰孢较其它省份多态性高。
50株串珠镰孢菌株间的相似系数在36%～81%,在相似系数为0.63时,50株串珠镰孢菌株可分为5组,其中49株来自玉米,其种群分5个组,一株来自小麦。AFLP分型结果显示,不同作物别、不同区域同一作物来源的串珠镰孢存在较大遗传差异,但该差异与样品种类和来源没有明显相关关系。
结论：
(1)四省2008年产小麦样品真菌污染严重,优势菌是交链孢霉；玉米样品全部被真菌污染,串珠镰孢为重要的优势菌相。
(2)培养时间、一定范围(20～50%)内的水分含量、温度和初始pH值是影响禾谷镰孢产毒的关键因子。禾谷镰孢产毒量随时间延长而增加,产毒高峰多在14-28天。
(3)串珠镰孢在DNA水平上存在较大变异,其中分离自河北玉米样品的串珠镰孢较其它省份多态性高。不同作物别来源(玉米或小麦)、不同区域同一作物来源串珠镰孢存在较大遗传差异,但该差异与样品种类和来源没有明显相关关系。
Objective:
(1) To study fungi invasion of wheat and corn collected from Anhui, Jiangsu, Hebei and Henan provinces harvested in 2008 and provide fungal strains for predictive microbiology studies.
(2) Screening of the key factors which may affect the toxin production by Fusarium graminearum in order to predict the toxin production under different natural conditions. The results obtained from predictive microbiology offer the technical support for controlling the cereal invasion by fungi.
(3) Fifty strains of F. moniliforme isolated from wheat and corn from 4 provinces were employed in amplified fragment length polymorphism (AFLP) molecular typing studies to identify the subtype and analyze the homology similarity among them.
Method:
(1) Wheat and corn kernels were inoculated onto PDA medium after sterilization with 75% ethanol followed by being rinsed with distilled water. The colony forming unit of fungi were counted and identified after incubation at 28±1℃for 5-7 days.
(2) F. graminearum inoculated on wheat kernals and corn flakes was used in predictive microbiological test. The L18-Hunter and Plackett-Burman model were constructed to screen the important factors (temperature, ventilatory capacity, water content, pH value, illumination, the amount and ingredients of the medium, incubation time etc.) which may affect the toxin production by F. graminearum. The time course of toxin production was also studied.
(3) Fifty purified F. moniliforme strains isolated from wheat and corn collected from Anhui, Jiangsu, Hebei and Henan provinces were extracted for DNA. The DNA was cut by both Pst I and Mes I enzymes and the fragments were analyzed after pre-amplification and amplification.
Results:
(1) Fungi were isolated from almost all of wheat samples and the predominant is Alternaria species. While, all corn samples were positive for fungi and the major is Fusarium species. Among which, F. moniliforme was the important.
(2) The L18-Hunter experimental results showed that only the model fitting results of 3-A-DON、15-A-DON and total of 10 kinds of toxins produced by the strain 3.4522 were statistically significant (P=0.0425,0.0118 and 0.0401 respectively) according to a=0.05 criterion and R2 of the model was 0.80,0.86 and 0.81, respectively. The coefficients of the regression equations were had a effect test and it showed that water content (P=0.0293, 0.0074 and 0.0189, respectively) and incubation time (P=0.0302,0.0074 and 0.0189, respectively) were the key factors that affected the level of 3-A-DON,15-A-DON and total of 10 kinds of toxins produced by F. graminearum strain 3.4522.
The results of Plackett-Burman experiment designed on the basis of L18-Hunter showed that the model fitting results of the absolute value of DON, logarithm value of DON, ZEN, DON and its derivatives and type B trichothecenes as well as the absolute and logarithm value of total toxin were statistically significant (P=0.0345,0.0022,0.0029,0.0068,0.0067,0.0148 and 0.0006, respectively) according to a=0.05 criterion and R2 of the model was 0.81,0.93, 0.92,0.89,0.90,0.86 and 0.95, respectively. The coefficients of the regression equations were had a effect test and it showed that incubation time, temperature and initial pH value of the media were the key factors that affected toxin production by F. graminearum strain 3.4522. The level of toxins produced by F. graminearum increased with the incubation time extended and reached the highest at the time of between 14 and 28 days.
(3) Based on AFLP results,50 strains of F. moniliforme generated 1346 bands and 1322 bands were polymorphic. Percentage of polymorphism fragments produced by each pair of AFLP primer varied between 95.21% and 100%. The considerable variation existed among F. moniliforme at DNA level. The F. moniliforme strains from Hebei corns had a higher polymorphism than those from other provinces.
The similarity coefficients of the strains were between 36% and 81%. The 50 F. moniliforme strains could be divided into 5 groups on the basis of the similarity coefficient of 0.63, in which 49 strains from corns and only 1 strain from wheat. The cluster analysis showed obvious differences in homology similarity of F. moniliforme strains isolated from different crops harvested in the same region and that from the same crop harvested in different regions, but these differences showed no obvious relations with the species and source of the samples.
Conclusion:
(1) Wheats collected from four provinces were seriously invaded by fungi and the majority was Alternaria species. While all corn samples were positive for fungi and Fusarium moniliforme was the predominant.
(2) Incubation time, water activity within a certain range (20～50%), temperature and pH value were the key factors that affected toxin production of F. graminearum. The level of toxin production by Fusarium Graminearum increased with the incubation time extended and reached the highest at the time of between 14 and 28 days.
(3) Considerable genetic variation existed in 50 strains of F. moniliforme at DNA level. The F. moniliforme strains from Hebei corns had a higher polymorphism than those from other provinces. There were obvious differences in homology similarity of F. moniliforme strains from different crops harvested in the same region and that from the same crop harvested in different regions, but these differences showed no obvious relations with the species and source of the samples.
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