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作物杂志,2024, 第2期: 221–227 doi: 10.16035/j.issn.1001-7283.2024.02.027

所属专题： 油料作物

• 生理生化·植物营养·栽培耕作 • 上一篇    下一篇

襄阳地区花生茬土壤不产毒黄曲霉菌的筛选及其特性分析

王雪1,2,3(), 李力1, 张姝娟1, 朱梦洁1, 张奇2,3, 李培武2,3, 董菁1, 王盾1,2,3(), 吴垭楠4   

  1. 1襄阳市农业科学院，441057，湖北襄阳
    2中国农业科学院油料作物研究所，430062，湖北武汉
    3湖北洪山实验室，430062，湖北武汉
    4襄阳市公共检验检测中心，441057，湖北襄阳
  • 收稿日期:2022-04-25 修回日期:2023-11-08 出版日期:2024-04-15 发布日期:2024-04-15
  • 通讯作者: 王盾，研究方向为农产品安全，E-mail：wangdunzju@163.com
  • 作者简介:王雪，研究方向为农产品安全，E-mail：mayxuer@qq.com
  • 基金资助:
    国家自然科学基金(32030085);农业农村部生物毒素检测重点实验室开放课题(SWDSJC2018001);湖北洪山实验室(2021hszd015);襄阳市青年英才开发计划(2018);襄阳市农科院青年基金(2020)

Screening and Feature Analysis of Atoxigenic Aspergillus flavus in Reducing Aflatoxin Prodution in Peanuts Soil in Xiangyang

Wang Xue1,2,3(), Li Li1, Zhang Shujuan1, Zhu Mengjie1, Zhang Qi2,3, Li Peiwu2,3, Dong Jing1, Wang Dun1,2,3(), Wu Yanan4   

  1. 1Xiangyang Academy of Agricultural Sciences, Xiangyang 441057, Hubei, China
    2Oil Crops Research Institute of Chinese Academy of Agriculture Sciences, Wuhan 430062, Hubei, China
    3Hubei Hongshan Laboratory, Wuhan 430062, Hubei, China
    4Xiangyang Public Inspection and Testing Center, Xiangyang 441057, Hubei, China
  • Received:2022-04-25 Revised:2023-11-08 Online:2024-04-15 Published:2024-04-15

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摘要：

通过对襄阳花生茬土壤中黄曲霉菌进行初筛，并对筛选出的菌种进行特性分析和测序鉴定，最终确定为黄曲霉菌种，通过液体发酵培养，测定土壤中黄曲霉毒素（AFT）含量，同时筛选不产黄曲霉毒素的黄曲霉菌株，通过平板初筛到几株抑菌效果较好的黄曲霉菌株，将标准产毒菌株与非产毒菌株进行共生培养，测定AFT含量。结果显示，有4株非产毒黄曲霉共生菌株的黄曲霉素毒素含量较对照菌株降低80%以上，其中XZ38抑制产毒率达99.49%，说明非产毒黄曲霉菌株在抑制产毒黄曲霉生长、降低或消除AFT方面有很大作用，可作为田间生物防治黄曲霉污染的备选菌株。

关键词: 黄曲霉菌, 非产毒黄曲霉菌, 特性分析

Abstract:

Several Aspergillus flavus strains were isolated from Xiangyang, Hubei peanut soil medium. Through characteristic analysis and sequencing identification, the trains were identified as Aspergillus flavus strains. Using liquid fermentation culture, aflatoxin content in the soil was detected, and the atoxigenic Aspergillus flavus strains were obtained at the same time. Through the flat screening, the several good Aspergillus flavus strains were screened at the beginning. The standard and non-virulent strains were used in symbiotic culture to determine the AFT content. The results showed that there were four strains of enterotoxigenic symbiotic strains of Aspergillus flavus, toxins aflatoxin content lower than control strains over 80%, including XZ38 inhibitory reached 99.49%. This indiated that non-toxigenic Aspergillus flavus strains had a great role in the inhibition of enterotoxigenic aspergillus flavus growth, reduction or elimination aflatoxin, and could be used as alternative strains for biological control for the field aflatoxin contamination offers a good alternative.

Key words: Aspergillus flavus, Atoxigenic Aspergillus flavus, Characteristic analysis

图1

PCR扩增鉴定电泳图

表1

BLAST软件上菌株分类比对结果

样品
Sample		 同源性
Homology		 AFPA平板菌落颜色
The colony colour
of AFPA plate		 参考物种
Reference
species
GC-01 702/720(98%) 亮橘色 曲霉属Aspergillus
ZY-06 618/628(98%) 亮橘色 曲霉属Aspergillus
ZY-07 605/608(99%) 亮橘色 曲霉属Aspergillus
ZY-08 597/599(99%) 亮橘色 曲霉属Aspergillus
GX-01 625/631(99%) 亮橘色 曲霉属Aspergillus
NZ-01 598/601(99%) 亮橘色 曲霉属Aspergillus
XZ-34 599/600(99%) 亮橘色 曲霉属Aspergillus
XZ-40 662/672(99%) 亮橘色 曲霉属Aspergillus
XZ-63 604/608(99%) 亮橘色 曲霉属Aspergillus
XZ-69 659/669(99%) 亮橘色 曲霉属Aspergillus
YC-01 601/602(99%) 亮橘色 曲霉属Aspergillus
YC-03 616/623(99%) 亮橘色 曲霉属Aspergillus
YC-11 627/630(99%) 亮橘色 曲霉属Aspergillus
YC-14 621/629(99%) 亮橘色 曲霉属Aspergillus
......

图2

不同区域黄曲霉菌产毒力

图3

花生在土壤中产毒株与不产毒株分布比例

图4

产毒株和非产毒株的拮抗试验

图5

非产毒菌对产毒菌的抑菌率

图6

黄曲霉菌的荧光观测图 (a) 产毒株空白对照，(b) 产毒株和非产毒株共同培养。

图7

4种AFT混标的液相标准图谱

表2

AFT标准曲线

AFT标准品
AFT standard		 标准曲线
Standard curve		 R2 检出限
Limit of detection (LOD) (μg/kg)		 定量限
Limit of quantification (μg/kg)
AFG2 Y=392 96.0X-17 563.0 0.999 968 5 0.26 1.3
AFG1 Y=12 069.0X-9167.47 0.999 956 5 0.36 1.3
AFB2 Y=88 965.4X-57 284.3 0.999 972 8 0.18 1.0
AFB1 Y=44 595.7X-35 890.3 0.999 968 7 0.24 1.0

图8

产毒株和非产毒株菌共生菌丝球状态

表3

不产毒黄曲霉菌对产毒菌的抑制效果

菌株
Strain		 产毒量
Toxin
production
(μg/kg)		 抑制产毒量
Inhibition of
toxin production
(μg/kg)		 抑制产毒率
Inhibition rate of
toxin production
rate (%)
SZ3.4408(SZ)(105) 1072.95±1.67 － －
NZ03:SZ(105:105) 197.26±3.05 875.69±3.05 81.61±1.73a
NZ06:SZ(105:105) 676.07±4.56 396.88±4.56 36.99±2.02a
ZY10:SZ(105:105) 1050.52±5.69 22.43±5.69 2.09±3.06b
ZY11:SZ(105:105) 1053.58±5.04 19.37±5.04 1.81±2.33a
XZ21:SZ(105:105) 936.82±4.83 136.13±4.83 12.69±1.85a
XZ38:SZ(105:105) 5.49±0.19 1067.46±0.19 99.49±0.75c
XZ52:SZ(105:105) 869.79±3.66 203.16±3.66 18.93±1.32d
XZ56:SZ(105:105) 869.47±2.92 203.48±2.92 18.96±1.25d
XZ59:SZ(105:105) 192.45±2.04 880.50±2.04 82.06±0.80c
XZ64:SZ(105:105) 604.78±3.06 468.17±3.06 43.63±1.58d
YC10:SZ(105:105) 75.77±1.35 997.19±1.35 92.94±0.67b

图9

YC10（左）和XZ38（右）在显微镜下的孢子形态

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