喷施茉莉酸甲酯对烟草叶片氧化酶活性及叶际微生物多样性的影响

吴加香; 刘涛; 谢永辉; 黄之远; 字淑慧

doi:10.19303/j.issn.1008-0384.2022.06.005

喷施茉莉酸甲酯对烟草叶片氧化酶活性及叶际微生物多样性的影响

doi: 10.19303/j.issn.1008-0384.2022.06.005

吴加香^{1, 2,},
刘涛^{1, 2},
谢永辉³,
黄之远^{1, 2},
字淑慧^{1, 2, ,}

1.
云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心，云南　昆明，650201
2.
云南农业大学农学与生物技术学院，云南　昆明 650201
3.
云南省烟草公司昆明市公司，云南　昆明　650051

基金项目: 云南省科技计划重大科技专项（2017AB002、2018BB015、2018ZF011）；云南省烟草公司科技计划项目（2020530000242026）

详细信息

作者简介:
吴加香（1995-），女，硕士研究生，研究方向：作物栽培理论（E-mail：3252736156@qq.com）

通讯作者:
字淑慧（1971-），女，博士，副教授，研究方向：乌头属植物繁育及规范化种植技术（E-mail：zsh7525@l63.com）

中图分类号: S 572
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- 被引次数: 0
出版历程
- 收稿日期: 2022-02-23
- 修回日期: 2022-06-01
- 刊出日期: 2022-06-28

Effect of Spraying Methyl Jasmonate on Oxidase Activity and Interleaf Microbial Diversity of Tobacco Leaves

WU Jiaxiang^{1, 2
,},
LIU Tao^{1, 2},
XIE Yonghui³,
HUANG Zhiyuan^{1, 2},
ZI Shuhui^{1, 2
, ,}

1.
National-Local Joint Engineering Research Center on Germplasms Innovation & Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, Yunnan　650201, China
2.
College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan　650201, China
3.
Kunming Tobacco Company of Yunnan Province, Kunming, Yunnan　650051, China

摘要

摘要: 目的探索茉莉酸甲酯（MeJA）影响烟草抗氧化酶活性与叶际微生物多样性及其相关性。方法设喷施0.5 mmol·L^-1茉莉酸甲酯（+MeJA）和不喷施（CK）处理，在喷施72 h后分别测定烟草叶片的超氧化物歧化酶（SOD）活性、过氧化物酶（POD）活性、总酚含量和过氧化氢（H₂O₂）积累量，烟草叶际细菌、真菌的丰富度和多样性。结果与−MeJA相比， +MeJA显著提高了烟叶中SOD和POD的活性及H₂O₂的积累，显著降低叶际细菌和真菌的丰富度，极显著提高叶际真菌的多样性；SOD活性和H₂O₂积累量与高氏白粉菌属呈显著负相关，POD活性与青枯菌属和伯克霍尔德氏菌-卡巴拉尼亚-帕拉伯克霍尔德氏菌属呈正相关。结论烟草喷施茉莉酸甲酯不仅能激活叶片中抗氧化酶活性，而且能改变叶际真菌优势菌属占比，烟草叶片中氧化酶活性与真菌菌属占比具有一定的相关关系。
- 茉莉酸甲酯 /
- 烟草 /
- 叶际微生物 /
- 抗氧化酶
Abstract: Objective Effects of spraying methyl jasmonate (MeJA) on the antioxidant enzyme activity and interleaf microbial diversity of tobacco leaves were investigated. Method Superoxide dismutase (SOD) activity, peroxidase (POD) activity, total phenolic content, hydrogen peroxide (H₂O₂) accumulation as well as abundance and diversity of interleaf bacteria and fungi in tobacco leaves were measured 72 h after spraying the plant growth regulator at 0.5 mmol·L^-1 (+MeJA) or without (CK) as control. Result The MeJA spray significantly increased SOD and POD activities and H₂O₂ accumulation but reduced the microbial abundance and increased the fungal diversity in the tobacco leaves. The SOD activity and H₂O₂ accumulation significantly inversely correlated with Golovinomyces, whereas POD positively correlated with Ralstonia and Burkholderia-Caballeronia-Paraburkholderia. Conclusion Tobacco leaves sprayed by MeJA not only activated the antioxidant enzyme activity but also altered the dominant fungi distribution in them, resulting in a correlation between oxidase activity and fungal genus share in tobacco leaves.
- methyl jasmonate /
- tobacco /
- phyllosphere microorganisms /
- antioxidant enzyme

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图 1 茉莉酸甲酯对烟草抗氧化酶活性和总酚含量的影响

POD和SOD活性、H₂O₂含量测定鲜样，TP含量测定干样；图中*表示P＜0.05，**表示P＜0.01；CK—未喷施茉莉酸甲酯，MeJA—喷施茉莉酸甲酯，图2同；A：茉莉酸甲酯处理后烟草叶片中过氧化物酶活性和过氧化氢含量的变化；B：茉莉酸甲酯处理后烟草叶片中超氧化物歧化酶活性和总酚含量的变化。

Figure 1. Effect of methyl jasmonate on antioxidant enzyme activity and total phenol content in tobacco

POD and SOD activities and H₂O₂ content were measured in fresh samples, and TP content in dry samples. * indicates P＜0.05, ** indicates P＜0.01. CK: no spraying, MeJA:spraying with MeJA; Same for Fig.2. Changes in POD activity and H₂O₂ content in tobacco leaves by spraying (A); changes in SOD activity and TP content in tobacco leaves by spraying (B).

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图 2 茉莉酸甲酯对烟草叶际微生物群落丰富度和多样性变化的影响

A：茉莉酸甲酯处理后烟草叶际微生物ACE指数的变化；B：茉莉酸甲酯处理后烟草叶际微生物Shannon指数的变化。

Figure 2. Effects of methyl jasmonate on the richness and diversity of tobacco phyllosphere microbial community

Changes on leaf microbial ACE index of tobacco leaf after spraying (A); changes of microbial Shannon index of tobacco leaf after spraying (B) .

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图 3 茉莉酸甲酯对烟草叶际真菌群落（A）、细菌群落（B）在属水平上组成的影响

Figure 3. Effects of methyl jasmonate on composition of phyllosphere fungal community (A) and bacterial community (B) at genus level in Tobacco

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图 4 不同处理真菌群落（A）、细菌群落（B）PCA分析

R值大小表相关性的大小，P值大小表样本间的相关度。

Figure 4. PCA analysis of fungal community (A) and bacterial community (B) under different treatments

R value size indicates the magnitude of correlation, and P value size indicates the correlation between samples.

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表 1 PCR扩增引物

Table 1. PCR amplification primers

微生物 Microbial	引物 Primers	序列 Sequence
细菌 Bacteria (V3+V4)	Forward	AACMGGATTAGATACCCKG
细菌 Bacteria (V3+V4)	Reverse	ACGGGCGGTGTGTRC
真菌 Fungi (ITS1+ITS2)	Forward	CTTGGTCATTTAGAGGAAGTAA
真菌 Fungi (ITS1+ITS2)	Reverse	GCTGCGTTCATCGATGC

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表 2 PCR扩增反应体系及条件

Table 2. PCR amplification reaction system and conditions

反应体系 Reaction system		反应条件 Reaction conditions
反应成分 Reaction components	添加量 Amount added	反应条件 Reaction conditions
缓冲液5×FastPfu Buffer 5×FastPfu	4 mL	95 ℃预变性3 min Pre-denaturation at 95 ℃ for 3 min ↓ 27个循环（95 ℃变性30 s， 55 ℃退火30 s，72 ℃延伸30 s） 27 cycles (denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 30 s) ↓ 72 ℃稳定延伸10 min Stable extension at 72 ℃ for 10 min ↓ 4 ℃保存 storage at 4 ℃
上下游引物 Upstream and downstream primers	0.8 mL
脱氧核糖核苷三磷酸 dNTPs(2.5 mmol·L⁻¹)	2 mL
快速-pfu 聚合酶 Fast-Pfu Polymerase	0.4 mL
DNA模板 DNA template	10 ng
双蒸水 ddH₂O	至20 mL Up to 20 mL

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表 3 不同处理氧化酶活性与属水平的优势菌群丰度Pearson相关性分析

Table 3. Pearson correlation analysis on oxidase activity and abundance of dominant flora at genus level under varied treatments

属 Genus	超氧化物歧化酶 SOD	过氧化物酶 POD	过氧化氢 H₂O₂	总酚 TP
高氏白粉菌属 Golovinomyces	−0.734*	0.037	−0.435*	−0.426
未分类真菌属 unclassified_k__Fungi	0.237	−0.702	0.534	0.232
毛孢子菌属 Cutaneotrichosporon	−0.601	−0.357	0.979	0.116
青枯菌属 Ralstonia	−0.535	0.867*	−0.406	0.148
红球菌属 Rhodococcus	0.377	−0.793	0.478	0.472
叶杆菌属 Phyllobacterium	0.557	−0.271	−0.111	0.620
伯克霍尔德氏菌-卡巴拉尼亚-帕拉伯克霍尔德氏菌属 Burkholderia-Caballeronia-Paraburkholderia	−0.990	0.963**	−0.600	−0.184
表示在P＜0.05水平上显著相关；表示在P＜0.01水平上显著相关。 Indicates significant correlation at P＜ 0.05 level; ** indicates significant correlation at P＜ 0.01 level.

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