金线兰茎腐病拮抗木霉菌鉴定及抑菌和促生效果评价

叶炜; 颜沛沛; 王培育; 李尊文; 郭莺; 张剑亮; 林敏水; 江金兰

金线兰茎腐病拮抗木霉菌鉴定及抑菌和促生效果评价

叶炜^{1, 2,},
颜沛沛^{1, 2},
王培育^{1, 2},
李尊文^{1, 2},
郭莺³,
张剑亮⁴,
林敏水⁵,
江金兰^{1, 2, ,}

1.
福建省（山区）作物遗传改良与创新利用重点实验室，福建　三明　365509
2.
三明市农业科学研究院药用植物研究所，福建　三明　365509
3.
福建省亚热带植物生理生化重点实验室，福建　厦门　361006
4.
龙岩草韵堂农业科技发展有限公司，福建　龙岩，364214
5.
永安市林业局，福建　永安　366038

基金项目: 福建省省级财政林业专项资金（闽财资环指〔2020〕10号）；福建省科技计划项目（2023S0018、2023N0047）；三明市科技计划项目（2023-N-6、2023-N-19）

详细信息

作者简介:
叶炜（1980 —），男，博士，副研究员，主要从事园艺植物生物技术研究，E-mail：yewei922@qq.com

通讯作者:
江金兰（1973 —），女，高级农艺师，主要从事园艺植物生物技术研究，E-mail: jjl75@qq.com

中图分类号: S435.672
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- 文章访问数: 13
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- 被引次数: 0
出版历程
- 收稿日期: 2024-04-28
- 修回日期: 2024-05-29
- 网络出版日期: 2024-11-11

Identification and Evaluation of Stem Rot Antagonistic and Growth promotion Effects of Trichoderma on Anoectochilus roxburghii

YE Wei^{1, 2
,},
YAN Peipei^{1, 2},
WAN Peiyu^{1, 2},
LI Zunwen^{1, 2},
GUO Yin³,
ZHANG Jianliang⁴,
LIN Minshui⁵,
JIANG Jinlan^{1, 2
, ,}

1.
Fujian Key Laboratory of Crop Genetic Improvement and Inovative Utilization for Mountain Area, Sanming, Fujian　365509, China
2.
1.Institute of medicinal plant sciences, Sanming Academy of Agricultural Science, Sanming, Fujian　365509, China
3.
Fujian Key Laboratory of Physiology and Biochemistryfor Subtropical Plant, Xiamen, Fujian　361006, China
4.
Longyan Caoyuntang Agricultural Science and Technology Development Co., Ltd, Longyan, Fujian　364214, China
5.
Yongan Forestry Bureau, Sanming, Fujian　366038, China

摘要

摘要: 目的分离具有金线兰茎腐病拮抗作用的木霉菌，为生防菌的开发提供理论基础。方法以金线兰仿野生种植植株为材料，利用组织分离法分离木霉菌，利用形态特征与ITS和rpb2序列同源性分析鉴定其分类，利用平板对峙法鉴定其抗茎腐病能力，并对不同木霉菌的促生长作用进行评价。结果利用组织分离法分离3株木霉菌A21B-1、A21B-2和A21E。经鉴定，3株木霉株分别为哈茨木霉、拟康宁木霉及Trichoderma longifialidicum。对峙生长表明，3种木霉菌株均对茎腐病病原菌尖孢镰刀菌ASP01表现较强的抑制作用，其抑制率分别达75.29%、73.55%和 60.02%。室内防效结果表明，A21B-1菌株对茎腐病有较强的抑制作用，接种15 d后病情抑制率达91.9%，可作为该病的生物防治候选菌株。促进生长试验表明，种植6个月后，施用3个木霉菌的金线兰植株的单株重、株高、茎粗、叶面积及SPAD值较对照均显著提高，其中A21B-2与A21E处理的植株单株重比对照分别提高了58.6%与58.9%，叶面积分别提高66.8%与59.7%，可作为金线兰促进生长的候选菌株。同时，施用木霉菌可有效提高金线兰多糖及金线莲苷的含量，其中A21B-2菌株效果最佳，其多糖及金线莲苷含量均较对照提高89.6%与11.8%，可作为促进金线兰药用成分积累的候选菌株。结论 3种不同类型的木霉菌在金线兰对抗茎腐病、促进生长及提高多糖含量方面有显著作用。
- 木霉菌 /
- 金线兰 /
- 尖孢镰刀菌 /
- 金线莲苷
Abstract: Objective Isolation of stem rot antagonistic Trichoderma from Anoectochilus roxburghii, providing theoretical basis for the development of biocontrol fungi. in A. roxburghii. Method Using wild cultivated A.roxburghiias as materials, Trichoderma strains were isolated using tissue isolation method. Morphological characteristics and homology analysis with ITS and rpb2 sequences were conducted for strains classification. Plate confrontation method were used for evaluating different Trichoderma strains resistance to stem rot ability, and the growth promoting effects of different Trichoderma strains were also processed.[Resulsts]3 Trichodermas trains as A21B-1, A21B-2 and A21E were isolated from the A. roxburghii via tissue isolation method. Combined with morphological characterization and ITS and rpb2 sequences homology identification, 3 Trichoderma strains were identified as T. rugulosum, T. koningiopsis and T. longifialidicum, respectively. Confrontation cultured showed that the 3 Trichoderma strains showed strong inhibitory effects on stem rot pathogen Fusarium oxysporum f. sp. opponiarum ASP01, and their inhibition rates reached 75.29%, 73.55% and 60.02%, respectively. The indoor control results showed that A21B-1 strain had a strong inhibitory effect on stem rot, after 15 d inoculation, the disease inhibition rate reached 91.9%, which could be used as a candidate strain for stem rot biological control fungi. The growth promotion experiments showed that Trichoderma strains significantly increased individual plant weight, height, stem diameter, leaf area, and SPAD value in A. roxburghii after 6 months grown. Among the 3 Trichoderma strains, plants innoculated with A21B-2 and A21E showed significant growth promotion effects with individual plant weight increased by 58.6% and 58.9%, leaf area increased by 66.8% and 59.7% compared to the controls, respectively. They could be used as candidate strains for growth promoting in A. roxburghii. At the same time, the application of Trichoderma strains effectively increased the content of polysaccharides and kinsenoside in A. roxburghii, A21B-2 strain showed the best effects, which content of polysaccharides and kinsenoside increased by 89.6% and 11.8% compared to the controls, could be used a candidate strain for promoting the accumulation of medicinal components in A. roxburghii. Conclusion 3 different strains of Trichoderma have significant effects on inhibiting stem rot disease, promoting growth, and increasing polysaccharide content in A. roxburghii.
- Trichoderma /
- Anoectochilus roxborghii /
- Fusarium oxysporum /
- kinsenoside

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图 1 木霉菌形态特征

a～d，e～h，i～l分别为木霉菌菌株A21B-1, A21B-2与A21E接种于PDA培养7 d后的菌落形态；a，e，i为正面；b，f, j为背面；c，g，k为分生孢子梗与瓶梗形态，分生孢子形态。

Figure 1. Morphological characterization of Trichoderma strains

a-d，e-h，i-l:Colony morphologyof Trichodermastrains A21B-1, A21B-2 and A21E grown on PDA for 7 d; a, e, i are front; b, f, j are back; c，g，k are conidia stem and bottle stem, conidia morphology.

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图 2 基于序列ITS与rpb2多基因进化树分析

标记T的菌株为模式菌，其余为参考菌株。

Figure 2. Phylogentic tree analysis based on ITS and rpb2 sequences

The strains marked with T are type strains, and the rest are reference strains.

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图 3 不同木霉菌与尖孢镰刀菌菌株ASP01对峙培养

a，e：尖孢镰刀菌ASP01；b，f：A21B-1与ASP01对峙培养；c，g：A21B-2与ASP01对峙培养；d，h：A21E与ASP01对峙培养；箭头所指为木霉菌菌丝侵入ASP01菌丝并导致尖孢镰刀菌孢子不能正常发育。

Figure 3. Confrontation culture of different Trichoderma strains against F. oxysporum f. sp. opponiarumASP01

a, e: F. oxysporum f. sp. opponiarum ASP01; b,f: confrontation culture of A21B-1 and ASP01; c, g: confrontation culture of A21B-2 and ASP01; d, h: confrontation culture of A21E and ASP01; the arrow points to the invasion of Trichoderma hyphae into ASP01 hyphae, resulting in the inability of ASP01 spores to develop normally.

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表 1 木霉菌序列登录号及种名

Table 1. Trichoderma sequences accssion number and species

菌株 Strains	登录号 Accession NO.		种名 Species
菌株 Strains	ITS	rpb2	种名 Species
A21B-1	ON209377	OR161371	T. harzianum
A21B-2	ON209378	OR161372	T. konginggipsis
A21E	ON209384	OR161373	T. longifialidiucum

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表 2 不同木霉菌对尖孢镰刀菌株ASP01抑制效果

Table 2. Inhibition effects of different Trichoderma strains against F. oxysporum f. sp. opponiarumASP01

处理 Treatment	病原真菌菌落直径 Colony diameter of pathogens/cm	抑制率/% Inhibition rates
CK	5.41±0.08 a	0±0 c
A21B-1	1.33±0.32 c	75.29±6.45a
A21B-2	1.43±0.40 c	73.55±7.38 a
A21E	2.17±0.16 b	60.02±2.87 b
数据为平均值±标准误，不同小字母表示0.05水平上差异显著。 Dates were presented as mean ± SD, data with different lowercase letters indicated significant difference at 0.05 level.

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表 3 木霉菌对茎腐病室内防效

Table 3. Indoor control effects of Trichoderma strains on stem rot disease

处理 Treatment	7 d		15 d
处理 Treatment	感病面积 Infected areas/mm²	抑制率 Inhibition rates/%	感病面积 Infected areas/mm²	抑制率 Inhibition rates/%
CK	354.0±90.7	0c	995.3±55.6	0 d
A21B-1	5.33±0.94	98.5±0.26a	79.9±14.7	91.9±1.47 a
A21B-2	23.3±5.73	93.4±1.62 b	242.1±38.5	75.6±3.78 b
A21E	11.8±4.09	96.7±1.15 b	334.2±65.8	66.4±6.61c
数据为平均值±标准误，不同字母代表在0.05水平存在显著差异。 Date were presented as mean±SD, Different lowercase letters indicated significant differenceat 0.05 level.

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表 4 木霉菌对金线兰促生效果

Table 4. Growth promotion effects of Trichoderma strains on A.roxburghii

处理 Treatment	株高 Plant highth/ cm	茎粗 Stem diameter/ mm	叶数 Number of leaves	叶长 Leaf Lenth/ mm	叶宽 Leaf Width/ mm	叶面积 Leaf area/ mm²	叶厚 Leaf thickth/ mm	根长 Root lenth/ cm	根粗 Root diameter/ mm	根数 Root number	SPAD	单株重 Single plant weight/g
CK	15.23±1.81 b	2.62±0.30c	6.67±1.37b	29.10±3.02b	26.78±1.84b	784.69±141.95 b	0.33±0.05 b	7.19±1.08b	2.02±0.26b	2.83±0.37 a	46.83±3.84c	3.17±0.44c
A21B-1	19.15±3.26 a	3.05±0.40b	7.67±1.37ab	29.85±2.41 b	28.02±2.99b	984.27±151.95b	0.42±0.07 a	6.68±0.68b	1.95±0.21b	2.83±0.90 a	56.65±4.18 a	3.25±0.45 b
A21B-2	21.02±2.05 a	3.08±0.19 b	8.33±0.47a	39.55±3.08 a	32.95±2.41a	1309.01±177.04a	0.40±0a	8.93±1.20 a	2.53±0.09a	2.33±0.47a	50.53±4.83b	5.03±0.62a
A21E	19.65±2.00a	3.65±0.29 a	6.00±2.45b	38.10±0.73 a	32.85±1.67a	1253.43±87.91a	0.40±0 a	9.14±1.36a	2.10±0.24 b	3.00±0 a	56.95±2.16a	5.04±0.20a
数据为平均值±标准误，不同字母代表在0.05水平存在显著差异。 Date were presented as mean±SD, data with different lowercase letters indicated significant difference at 0.05 level.

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表 5 木霉菌对金线兰促进次生代谢物积累效果

Table 5. Sencondary metabolite accumulated promotion effects of Trichoderma strains on A.roxburghii

处理 Treatment	折干率 Drying rate/%	多糖含量 Content of polysaccharide/(mg·g⁻¹)	黄酮含量 Content of flavone/（mg·g⁻¹）	金线莲苷含量 Content of kinsenoside/（mg·g⁻¹）
CK	11.25±0.71d	6.07±0.14 d	8.15±0.43 a	(14.292±0.24) b
A21B-1	12.88±0.73 cd	7.4±0.68 c	7.2±0.14c	(14.6±0.45) b
A21B-2	13.91±0.59b	11.51±0.35a	7.43±0.24bc	(15.98±0.97) a
A21E	14.58±0.01 a	8.05±0.99 b	7.76±0.78ab	(14.96±1.26) b
数据为平均值±标准误，不同字母代表在0.05水平存在显著差异。 Date were presented as mean±SD, data with different lowercase letters indicated significant difference at 0.05 level.

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