加纳木霉ACCC30153对山新杨幼苗促生抗病功能分析

黄颖; 苗嘉琪; 遇文婧; 刁桂萍

doi:10.12171/j.1000-1522.20230346

加纳木霉ACCC30153对山新杨幼苗促生抗病功能分析

黄颖^{1, 2,},
苗嘉琪^{1, 3},
遇文婧²,
刁桂萍^1, ,

1.
东北林业大学林学院，黑龙江哈尔滨 150040
2.
黑龙江省森林保护研究所，黑龙江哈尔滨 150040
3.
西安立讯科技技师学院，陕西西安 710000

基金项目: 中央高校基本科研业务费专项（2572022DS07），黑龙江省省属科研院所科研业务费项目（LKSB2024-2、CZKYF2022-1-C048），黑龙江省博士后出站基金项目（2023）。

详细信息

作者简介:
黄颖，博士。主要研究方向：森林保护。Email：729218328@qq.com　地址：150040 黑龙江省哈尔滨市和兴路 26 号东北林业大学林学院

责任作者:
刁桂萍，博士，副教授。主要研究方向：森林保护。Email：dgp2003@126.com　地址：同上。

中图分类号: S792.13
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出版历程
- 收稿日期: 2023-11-28
- 修回日期: 2024-05-29
- 录用日期: 2024-11-25
- 网络出版日期: 2024-11-26
- 刊出日期: 2024-12-24

Growth promotion and disease resistance function of Trichoderma ghanaiana ACCC30153 on Populus davidiana × P. bolleana seedlings

Huang Ying^{1, 2,},
Miao Jiaqi^{1, 3},
Yu Wenjing²,
Diao Guiping^1, ,

1.
School of Forestry, Northeast Forestry University, Harbin 150040, Heilongjiang, China
2.
Heilongjiang Forest Protection Institute, Harbin 150040, Heilongjiang, China
3.
Xi’an Lixun Technology Technician College, Xi’an 710000, Shaanxi, China

摘要

摘要:
目的
探索并分析加纳木霉ACCC30153的促生抗病功能，为该菌株的田间应用提供理论依据和技术支持。
方法
利用加纳木霉 ACCC30153分生孢子悬浮液灌施山新杨幼苗，对山新杨幼苗的生长指标、生理指标和光合作用进行了研究；通过加纳木霉 ACCC30153与病原菌对峙试验及分析相关抗病基因表达差异，明确加纳木霉ACCC30153抗病功能。
结果
（1）灌施不同孢子浓度加纳木霉ACCC30153分生孢子悬浮液能够促进山新杨幼苗的生长。与对照相比，灌施1.5 × 10⁷ cfu/mL孢子浓度60 d后幼苗平均株高增加了63.99%。生理指标测定显示，灌施处理显著提高叶绿素含量、净光合速率、蒸腾速率、气孔导度和胞间CO₂浓度，且显著高于对照组（P < 0.05）。（2）加纳木霉ACCC30153处理显著提高了山新杨叶片内防御酶的活性，包括苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）、过氧化氢酶（CAT）和超氧化物歧化酶（SOD），表明加纳木霉通过提高防御酶活性增强了山新杨的抗病能力。（3）对峙试验结果表明，加纳木霉ACCC30153对多种病原真菌具有显著的拮抗作用。实时荧光定量RT-qPCR结果表明，加纳木霉ACCC30153分泌的溶解酶和蛋白水解酶在其拮抗病原真菌过程中发挥了重要作用。
结论
本研究明确了加纳木霉ACCC30153能够通过提高山新杨叶片内叶绿素含量、增强其光合速率促进山新杨幼苗的生长，并显著提高山新杨幼苗防御酶活性；同时能够分泌溶解酶及蛋白水解酶参与其对病原菌的拮抗作用。
- 加纳木霉 /
- 杨树 /
- 生防作用 /
- 促生作用
Abstract:
Objective
To provide a theoretical basis and technical support for the field application of Trichoderma ghanense ACCC30153, its growth-promoting and disease-resistance functions were analyzed.
Method
The study investigated the effects of irrigating poplar seedlings with a conidial suspension of T. ghanense ACCC30153 on growth indicators, physiological indicators, and photosynthesis. The disease resistance function of T. ghanense ACCC30153 was determined through pathogen confrontation tests and by analyzing the expression patterns of disease resistance genes.
Result
(1) Irrigating with different concentrations of T. ghanense ACCC30153 conidial suspension promoted the growth of poplar seedlings. Compared with the control, the average height of poplar seedlings increased by 63.99% after 60 days of irrigation with a conidial suspension of T. ghanense ACCC30153 at a concentration of 1.5 × 10⁷ cfu/mL. Additionally, physiological measurements demonstrated a significant increase in chlorophyll, net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO₂ concentration in leaves compared to the control group (P < 0.05). (2) Comparison of disease resistance in poplar leaves irrigated with or without T. ghanense ACCC30153 conidial suspension revealed a significant improvement in disease resistance after treatment. Enzymatic activity of defense-related enzymes, including phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), indicated that T. ghanense ACCC30153 enhanced disease resistance by increasing the activity of these defense enzymes in the leaves (P < 0.05). (3)The confrontation experiment showed that T. ghanense ACCC30153 exhibited significant antagonistic effects on against various pathogenic fungi. Furthermore, real-time fluorescence quantitative RT-qPCR analysis revealed that lysozymes and proteolytic enzymes secreted by T. ghanense ACCC30153 played critical roles in its antagonistic effects against pathogenic fungi.
Conclusion
This study demonstrated that T. ghanense ACCC30153 can promote the growth of poplar seedlings by increasing chlorophyll content, enhancing photosynthetic rates, and it significantly increased the activity of defense enzymes. Moreover, the T. ghanense ACCC30153 can secrete lytic enzymes and proteolytic enzymes to participate in its antagonism against pathogenic organisms.
- Trichoderma ghanense /
- poplar /
- biocontrol effect /
- growth promoting effect

HTML全文

图 1 加纳木霉ACCC30153对山新杨幼苗促生作用

CK.对照；T1.孢子浓度1.5 × 10³ cfu/mL；T2.孢子浓度1.5 × 10⁵ cfu/mL；T3.孢子浓度1.5 × 10⁷ cfu/mL。不同小写字母表示同一时间不同处理之间差异显著（P < 0.05）。CK, control; T1, spore concentration of 1.5 × 10³ cfu/mL; T2, spore concentration of 1.5 × 10⁵ cfu/mL; T3, spore concentration of 1.5 × 10⁷cfu/mL. Different lowercase letters indicate significant differences in same time under varied treatments (P < 0.05).

Figure 1. Promotion function of Trichoderma ghanense ACCC30153 on growth of P. davidiana × P. bolleana seedlings

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图 2 不同处理下山新杨移栽苗净光合速率（P_n）日变化特性

Figure 2. Diurnal variation characteristics of net photosynthetic rates (P_n) in P. davidiana × P. bolleana under different treatments

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图 3 不同处理下山新杨移栽苗的净光合速率（P_n）、蒸腾速率（E）、气孔导度（G_s）和胞间CO₂浓度（C_i）

不同小写字母表示同一时间不同处理间的显著性差异（P < 0.05）。Different lowercase letters indicate significant differences between varied processes at the same time (P < 0.05).

Figure 3. Net photosynthetic rate (P_n), transpiration rate (E), stomatal conductance (G_s) and intercellular CO₂ concentration(C_i) of transplanted P. davidiana × P. bolleana seedlings under different treatments

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图 4 山新杨幼苗叶片生理酶活性

Figure 4. Leaf physiological enzyme activity of P. davidiana × P. bolleana seedlings

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图 5 对峙试验

T.加纳木霉ACCC30153；A.叶枯病病原菌；T-A.加纳木霉ACCC30153与叶枯病病原菌对峙；C.烂皮病病原菌；T-C.加纳木霉ACCC30153与烂皮病病原菌对峙。T, T. ghanense ACCC30153; A, A. alternate; T-A, confrontation test of T. ghanense ACCC30153 and A. alternate; C, C. chrysosperma; T-C, confrontation test of T. ghanense ACCC30153 and C. chrysosperma.

Figure 5. Confrontation test

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图 6 加纳木霉ACCC30153不同诱导下溶解酶基因表达量

MM. 微量无机盐基础培养基Mineral inorganic salt medium；CA. 细链格孢菌细胞壁 Cell walls of A. alternata；CC. 金黄壳囊孢菌细胞壁 Cell walls of C. chrysosperma；FA. 细链格孢菌发酵液 Fermentation supernatant of A. alternata；FC. 金黄壳囊孢菌发酵液 Fermentation supernatant of C. chrysosperma

Figure 6. Expression levels of lytic enzyme gene of T. ghanense ACCC30153 under different treatments

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表 1 引物设计

Table 1 Primer design

基因编号 Gene No.	引物序列（5′—3′） Primer sequence (5′—3′)
TghECh	F：GCGACCTGCTCCAGAGATTGAG	R：GCCCGTGTTTGTCACCTCCA
TghChi	F：GGCACTTGGTACGAGCCATTCA	R：CCGCTTGTAGTCCTCGCCATT
TghAsp	F：AGGCGTCCAGGTCATCGTAGA	R：CGGATCGAGCAGCGAGTTGTT
TghAse	F：TGGACTCTGGATGTGCGTCAAC	R：GCCGATCCATGTGCCTTCTTCT
TghXyn	F：TTGTCACTTTCTCCGGCGTCG	R：CCGTGTTCTTGGCCGTAACGA
TghNac	F：CTTTGTCGGCGGCAAGGGAT	R：CGATGATGGACGGCTGGTTGA
TghSep	F：CCGTCGTCTACTGCCTCAACAA	R：GCTCTACGATGCTGCGGTGAA
TghGlu	F：CGGCATCCACAAGCGACACA	R：TCATCGTCTTCGTCCTCGTCCT
TghEgl	F：GAGTTCAACGCCCGCAACCA	R：CGCTCCGGCATTGACCTTCTT
TghGTP	F：CTTTGTCGGCGGCAAGGGAT	R：CGATGATGGACGGCTGGTTGA
actin	F：AAGGAGTCCGTCGTCGAGGTT	R：AGTTGAGGGCGGAGCGGATA
α-tublin	F：CTGGTCACGGCTGGACTACAAG	R：GCATCAAGTCGTCGTCGTCGT
β-tublin	F：GTTACCTGACGTGCTCCACCAT	R：CGGAACATGGCGGTGAACTGT

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表 2 不同处理60 d后山新杨移栽苗的生长状况

Table 2 Growth of P. davidiana × P. bolleana seedlings under different treatments after 60 days

生长指标 Growth indicators	CK	T1	T2	T3
茎粗 Stem diameter/cm	0.21 ± 0.004^a	0.26 ± 0.011^a	0.38 ± 0.005^b	0.55 ± 0.010^b
根长 Root length/cm	2.26 ± 0.261^a	2.29 ± 0.142^a	5.26 ± 0.166^b	6.11 ± 0.211^b
叶片数 Leaf number	13.6 ± 0.050^a	15.2 ± 0.115^a	22.6 ± 0.168^b	23.8 ± 0.008^b
鲜质量 Fresh mass/g	6.05 ± 0.035^a	6.79 ± 0.122^a	9.28 ± 0.135^b	11.55 ± 0.256^b
干质量 Dry mass/g	1.03 ± 0.056^a	2.34 ± 0.045^a	4.54 ± 0.246^b	5.08 ± 0.002^b
注：同行不同小写字母表示不同处理下同一生长指标的差异显著（P < 0.05）。Note: different lowercase letters in the same line indicate significant differences in same growth index under varied treatments (P < 0.05).

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表 3 不同处理下山新杨叶片叶绿素a含量

Table 3 Contents of chlorophyll a in leaves of P. davidiana × P. bolleana under different treatments μg/g

处理 Treatment	0 h	1 h	3 h	6 h	12 h	1 d	5 d	10 d	15 d
CK	0.84^a	1.07^b	2.72^b	2.11^b	3.53^b	3.61^b	3.65^b	3.68^b	3.39^b
T1	0.94^a	2.81^b	2.55^b	3.82^ab	3.42^b	4.16^ab	4.35^b	5.46^a	6.31^a
T2	0.97^a	2.85^b	3.78^ab	5.83^a	5.29^a	7.11^a	7.67^a	6.02^a	7.72^a
T3	0.94^a	5.98^a	4.71^a	4.16^ab	4.53^ab	5.95^ab	5.68^ab	5.80^a	6.74^a
注：同列不同小写字母表示不同处理之间差异显著（P < 0.05）。下同。Notes: different lowercase letters in the same column indicate significant differences under varied treatments (P < 0.05). The same below.

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表 4 不同处理下山新杨叶片叶绿素b含量

Table 4 Contents of chlorophyll b in leaves of P. davidiana × P. bolleana under different treatments μg/g

处理 Treatment	0 h	1 h	3 h	6 h	12 h	1 d	5 d	10 d	15 d
CK	0.81^a	0.24^b	1.85	1.88^b	2.08^b	2.08^b	3.68^b	2.07^b	3.33^b
T1	0.77^a	4.93^a	4.63^a	4.95^a	4.93^a	4.90^a	4.94^ab	5.46^ab	5.32^a
T2	0.91^a	3.73^a	2.65^b	4.23^a	4.48^a	5.04^a	5.71^a	6.66^a	6.16^a
T3	1.21^a	3.67^a	3.92^ab	4.40^a	4.99^a	4.82^a	5.36^a	6.21^a	6.15^a

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表 5 不同处理下山新杨叶片叶绿素总含量

Table 5 Total contents of chlorophyll in leaves of P. davidiana × P. bolleana under different treatments μg/g

处理 Treatment	0 h	1 h	3 h	6 h	12 h	1 d	5 d	10 d	15 d
CK	1.64^a	1.30^c	4.57^b	3.99^b	5.62^b	5.69^b	7.33^b	5.75^b	6.72^b
T1	1.71^a	7.74^b	7.18^a	8.77^ab	8.35^ab	9.06^ab	9.28^ab	10.93^ab	11.63^ab
T2	1.87^a	6.57^b	6.43^ab	10.07^a	9.78^a	12.15^a	13.38^a	12.68^a	13.88^a
T3	2.14^a	9.65^a	8.64^a	8.56^ab	9.51^a	10.76^ab	11.04^ab	12.01^a	12.90^a

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