草业学报 ›› 2022, Vol. 31 ›› Issue (7): 64-75.DOI: 10.11686/cyxb2021197
戈建珍(), 傅文慧, 张露, 蔺宝珺, 赵帅, 白玛噶翁, 寇建村()
收稿日期:
2021-05-11
修回日期:
2021-08-20
出版日期:
2022-07-20
发布日期:
2022-06-01
通讯作者:
寇建村
作者简介:
E-mail: jiancun02@163.com基金资助:
Jian-zhen GE(), Wen-hui FU, Lu ZHANG, Bao-jun LIN, Shuai ZHAO, Ma-ga-weng BAI, Jian-cun KOU()
Received:
2021-05-11
Revised:
2021-08-20
Online:
2022-07-20
Published:
2022-06-01
Contact:
Jian-cun KOU
摘要:
为了解白三叶青贮后不同浓度多菌灵农药的降解情况和果园喷施多菌灵对白三叶青贮过程中细菌多样性的影响,扩大具有农药残留的果园覆盖植物利用途径,在喷施不同浓度[2.0 (RU-),2.5 (RU),3.0 g·L-1 (RU+)]的多菌灵后进行白三叶青贮,以喷水为对照,发酵60 d。采用Miseq高通量测序技术,分别在青贮开始前、青贮第3和60天取样,对白三叶中细菌群落多样性进行研究。结果表明,青贮后多菌灵降解率达到59.6%以上,喷施量越高,降解率越高;多菌灵处理显著(P<0.05)增加了白三叶青贮发酵的乳酸、乙酸、丙酸含量,乳酸在多菌灵RU-、RU、RU+浓度下分别增加了47.55%、63.24%、71.08%,乙酸分别增加了24.49%、44.90%、46.94%,丙酸分别增加了66.67%、187.50%、250.00%,有利于白三叶青贮饲料营养成分的保留;多菌灵喷施显著(P<0.05)改变了白三叶青贮菌群群落构成,增加了青贮的菌群丰度、多样性;多菌灵处理中乳球菌属、魏斯氏菌属、泛菌属、Rosenbergiella、假单胞菌属、寡养单胞菌属、肠杆菌属、芽孢杆菌属菌群丰度增加,不利于青贮发酵,但随着青贮时间的推移,寡养单胞菌属、肠杆菌属、芽孢杆菌属等菌群丰度减少。喷施多菌灵后有利于青贮发酵微生物菌群的丰度增加,对青贮发酵有害菌、多菌灵降解菌群丰度及青贮微生物多样性产生影响,青贮后多菌灵残留高于欧洲食品安全局规定的作物类动物饲料中农药最大残留量,达不到饲用标准。研究结果可为具有农药残留的白三叶青贮饲料的饲用安全性和果园覆盖植物资源的开发利用提供理论依据。
戈建珍, 傅文慧, 张露, 蔺宝珺, 赵帅, 白玛噶翁, 寇建村. 多菌灵在果园白三叶青贮中的降解及其对微生物群落的影响[J]. 草业学报, 2022, 31(7): 64-75.
Jian-zhen GE, Wen-hui FU, Lu ZHANG, Bao-jun LIN, Shuai ZHAO, Ma-ga-weng BAI, Jian-cun KOU. Degradation of carbendazim in orchard white clover silage and its effect on the microbial fermentative community[J]. Acta Prataculturae Sinica, 2022, 31(7): 64-75.
项目Item | 空白对照 CON | RU- | RU | RU+ | 标准误SEM | P-value |
---|---|---|---|---|---|---|
乳酸Lactic acid (mg·mL-1) | 2.04±0.09c | 3.01±0.20b | 3.33±0.11a | 3.49±0.10a | 0.172 | 0.017 |
乙酸Acetic acid (mg·mL-1) | 0.49±0.05c | 0.61±0.04b | 0.71±0.06a | 0.72±0.04a | 0.738 | 0.030 |
丙酸Propionic acid (mg·mL-1) | 0.24±0.04c | 0.40±0.06b | 0.69±0.49b | 0.84±0.45a | 0.101 | 0.049 |
pH | 4.92±0.06a | 4.65±0.04b | 4.53±0.03c | 4.62±0.04b | 0.706 | 0.044 |
氨态氮NH3-N (g·kg-1) | 2.86±0.13a | 2.23±0.13c | 2.60±0.23ab | 2.42±0.19bc | 0.082 | 0.031 |
干物质DM (g·kg-1) | 45.84±0.21a | 45.73±0.15a | 43.45±0.11c | 44.22±0.21b | 0.309 | 0.032 |
粗蛋白CP (g DM·kg-1) | 18.36±0.21c | 19.22±0.19a | 18.79±0.16b | 19.27±0.15a | 0.119 | 0.032 |
粗纤维CF (g DM·kg-1) | 17.00±0.20b | 16.97±0.15b | 17.23±0.15ab | 17.37±0.15a | 0.065 | 0.027 |
酸性洗涤纤维ADF (g DM·kg-1) | 24.63±0.12c | 25.07±0.12b | 26.10±0.20a | 24.77±0.21bc | 0.162 | 0.028 |
中性洗涤纤维NDF (g DM·kg-1) | 24.43±0.15c | 25.93±0.21a | 25.00±0.17b | 24.53±0.15c | 0.157 | 0.030 |
可溶性碳水化合物WSC (g DM·kg-1) | 2.48±0.09b | 3.80±0.01a | 3.83±0.02a | 3.88±0.02a | 0.178 | 0.002 |
粗脂肪EE (g DM·kg-1) | 3.51±0.08a | 3.56±1.00a | 3.69±0.61a | 3.46±0.18a | 0.055 | 0.067 |
表1 多菌灵对果园白三叶青贮饲料品质及营养价值的影响
Table 1 Effect of carbendazim on the quality and nutritional value of white clover silage in orchard
项目Item | 空白对照 CON | RU- | RU | RU+ | 标准误SEM | P-value |
---|---|---|---|---|---|---|
乳酸Lactic acid (mg·mL-1) | 2.04±0.09c | 3.01±0.20b | 3.33±0.11a | 3.49±0.10a | 0.172 | 0.017 |
乙酸Acetic acid (mg·mL-1) | 0.49±0.05c | 0.61±0.04b | 0.71±0.06a | 0.72±0.04a | 0.738 | 0.030 |
丙酸Propionic acid (mg·mL-1) | 0.24±0.04c | 0.40±0.06b | 0.69±0.49b | 0.84±0.45a | 0.101 | 0.049 |
pH | 4.92±0.06a | 4.65±0.04b | 4.53±0.03c | 4.62±0.04b | 0.706 | 0.044 |
氨态氮NH3-N (g·kg-1) | 2.86±0.13a | 2.23±0.13c | 2.60±0.23ab | 2.42±0.19bc | 0.082 | 0.031 |
干物质DM (g·kg-1) | 45.84±0.21a | 45.73±0.15a | 43.45±0.11c | 44.22±0.21b | 0.309 | 0.032 |
粗蛋白CP (g DM·kg-1) | 18.36±0.21c | 19.22±0.19a | 18.79±0.16b | 19.27±0.15a | 0.119 | 0.032 |
粗纤维CF (g DM·kg-1) | 17.00±0.20b | 16.97±0.15b | 17.23±0.15ab | 17.37±0.15a | 0.065 | 0.027 |
酸性洗涤纤维ADF (g DM·kg-1) | 24.63±0.12c | 25.07±0.12b | 26.10±0.20a | 24.77±0.21bc | 0.162 | 0.028 |
中性洗涤纤维NDF (g DM·kg-1) | 24.43±0.15c | 25.93±0.21a | 25.00±0.17b | 24.53±0.15c | 0.157 | 0.030 |
可溶性碳水化合物WSC (g DM·kg-1) | 2.48±0.09b | 3.80±0.01a | 3.83±0.02a | 3.88±0.02a | 0.178 | 0.002 |
粗脂肪EE (g DM·kg-1) | 3.51±0.08a | 3.56±1.00a | 3.69±0.61a | 3.46±0.18a | 0.055 | 0.067 |
图1 果园生草白三叶青贮发酵过程对不同浓度多菌灵降解率的影响不同字母表示差异显著(P<0.05)。Different letters indicate significant differences at P<0.05.
Fig.1 The effect of the fermentation process of orchard grass white clover silage on the degradation rate of different concentrations of carbendazim
测序样本名称 Sequencing sample name | 原始PE reads数目 PE_reads | 去除嵌合体后有效序列数目 Nochimera | 有效序列平均长度 AvgLen (bp) | 各样品OTU总数OTUs |
---|---|---|---|---|
不喷施多菌灵(0 d,A) | 70933 | 66907 | 462 | 82 |
不喷施多菌灵(3 d,B) | 63698 | 54955 | 463 | 83 |
喷施多菌灵(3 d,C) | 59416 | 53199 | 463 | 87 |
喷施多菌灵(60 d,D) | 79630 | 68540 | 463 | 90 |
不喷施多菌灵(60 d,E) | 63874 | 58218 | 462 | 91 |
表2 优化后数据质量统计
Table 2 Data quality statistics after optimization
测序样本名称 Sequencing sample name | 原始PE reads数目 PE_reads | 去除嵌合体后有效序列数目 Nochimera | 有效序列平均长度 AvgLen (bp) | 各样品OTU总数OTUs |
---|---|---|---|---|
不喷施多菌灵(0 d,A) | 70933 | 66907 | 462 | 82 |
不喷施多菌灵(3 d,B) | 63698 | 54955 | 463 | 83 |
喷施多菌灵(3 d,C) | 59416 | 53199 | 463 | 87 |
喷施多菌灵(60 d,D) | 79630 | 68540 | 463 | 90 |
不喷施多菌灵(60 d,E) | 63874 | 58218 | 462 | 91 |
图2 OTU韦恩图韦恩图中不同颜色的圈表示不同的分组,图中的数字分别代表了每个分组特有或共有的OTU数目。A:不喷施多菌灵处理白三叶青贮第0天;B:不喷施多菌灵处理白三叶青贮第3天;C:喷施多菌灵处理青贮白三叶第3天;D:喷施多菌灵处理白三叶青贮第60天;E:不喷施多菌灵处理白三叶青贮第60天。下同。Different colored circles in the Wenn diagram represent different groups, and the numbers in the diagram represent the number of unique or common OTUs for each group. A: White clover silage treatment day 0 without carbendazim; B: White clover silage treatment day 3 without carbendazim; C: White clover silage treatment day 3 with carbendazim spray; D: The 60th day of white clover silage treatment with carbendazim; E: The 60th day of white clover silage treatment without carbendazim. The same below.
Fig.2 OTU Venn diagram
图3 DNA序列数据和微生物多样性指数分析a) Chao 1 指数,Chao 1指数越大,OTU数目越多,说明该样本物种数比较多;b)香农指数,Shannon指数越大,说明群落多样性越高。a) Chao 1 index, the greater the Chao 1 index, the greater the number of OTUs, indicating that there are more species in the sample; b) The Shannon index, the greater the Shannon index, the higher the community diversity.
Fig.3 DNA sequence data and microbial diversity index analysis
图4 基于Unifrac距离的PCoA分析同一个组的样本使用相同颜色和形状表示。x轴与y轴分别代表第一和第二主坐标。主坐标后的百分比代表此坐标对样本差异的贡献率,度量了此主坐标对原始信息量提取的多少。样本点距离的远近代表了样本中微生物群落的相似性,距离越近,相似度越高;聚集在一起的样本由相似的微生物群落构成。The samples in the same group are represented by the same color and shape. The x-axis and y-axis represent the first and second principal coordinates, respectively. The percentage after the principal coordinate represents the contribution rate of this coordinate to the sample difference, and measures how much the principal coordinate extracts the amount of original information. The distance between the sample points represents the similarity of the microbial communities in the sample. The closer the distance, the higher the similarity; The clustered samples are composed of similar microbial communities.
Fig.4 PCoA analysis based on Unifrac distance
图5 细菌群落分布使用RDP classifier贝叶斯算法对97%相似水平的OTU代表序列进行分类学分析,并在各个水平统计每个样本的群落组成,各分组在不同分类水平(门、科、属)下前30的菌群分布绘制为柱状图。Use RDP classifier Bayesian algorithm to perform taxonomic analysis on 97% similar level OTU representative sequences, and count the community composition of each sample at each level, and each group is in the top 30 under different classification levels (phyla, family, genus), the bacterial community distribution is plotted as a bar graph.
Fig.5 Bacterial community distribution
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