苹果轮纹病拮抗真菌Pa2的分离与鉴定

doi:10.16420/j.issn.0513-353x.2023-0292

园艺学报 ›› 2024, Vol. 51 ›› Issue (5): 1113-1125.doi: 10.16420/j.issn.0513-353x.2023-0292

苹果轮纹病拮抗真菌Pa2的分离与鉴定

史冰柯¹, 王卓妮¹, 覃艮红¹, 黄天祥¹, 王丽¹, 涂洪涛¹^,³, 袁洪波¹^,²^,^*(), 侯珲¹^,³^,^*()

¹ 中国农业科学院郑州果树研究所，郑州 450009
² 中国农业科学院西部农业研究中心，新疆昌吉 831100
³ 中国农业科学院中原研究中心，河南新乡 453004

收稿日期:2023-12-11 修回日期:2024-03-28 出版日期:2024-05-25 发布日期:2024-05-29
通讯作者: * E-mail：yuanhongbo@caas.cn，houhui@caas.cn
基金资助:
新疆维吾尔自治区青年自然科学基金项目(2022D01B226); 中国农业科学院科技创新工程项目(CAAS-ASTIP-2016-RIP); 河南省重点研发与推广专项(232102110052); 中国农业科学院郑州果树研究所科研业务费专项(ZGS202205); 中国农业科学院郑州果树研究所科研业务费专项(1610192023311)

Isolation and Identification of Endophytic Fungus Pa2 Antagonistic Against Apple Ring Rot Caused by Botryosphaeria dothidea

SHI Bingke¹, WANG Zhuoni¹, QIN Genhong¹, HUANG Tianxiang¹, WANG Li¹, TU Hongtao¹^,³, YUAN Hongbo¹^,²^,^*(), HOU Hui¹^,³^,^*()

¹ Zhengzhou Fruit Research Institute，Chinese Academy of Agricultural Sciences，Zhengzhou 450009，China
² Western Agricultural Research Center，Chinese Academy of Agricultural Sciences，Changji，XinJiang 831100，China
³ Zhongyuan Research Center，Chinese Academy of Agricultural Sciences，Xinxiang，Henan 453004，China

Received:2023-12-11 Revised:2024-03-28 Published:2024-05-25 Online:2024-05-29
Contact: * E-mail：yuanhongbo@caas.cn，houhui@caas.cn

摘要/Abstract

摘要：

为了分离和筛选对苹果轮纹病菌（Botryosphaeria dothidea）有抑制效果的拮抗真菌，通过组织分离法从苹果轮纹病抗性品种的枝条上分离出内生真菌。结果从40个分离菌株中得到5个拮抗真菌菌株，其中鉴定为砖红镰刀菌（Fusarium lateritium）的Pa2菌株对苹果轮纹病的抑制效果最好，抑制率为77.49%。菌株Pa2上清液不仅可以破坏B. dothidea菌丝细胞膜，还可以诱导果实中致病相关基因MdPR1的表达。在苹果接种B. dothidea后5 d，菌株Pa2对苹果轮纹病的控制效果为63.63%。

关键词: 苹果轮纹病, 拮抗真菌, 砖红镰刀菌, 防效

Abstract:

In order to isolate and screen antagonistic fungi against apple ring rot caused by Botryosphaeria dothidea，the present study isolated endophytic fungi from the branches of apple ring rot resistant cultivar by tissue isolation methods. The results showed that five antagonistic fungal strains were obtained from 40 isolated strains，among which strain Pa2，identified as Fusarium lateritium，had the best inhibitory effect on B. dothidea with 77.49% inhibition rate. Strain Pa2 supernatant treatment can not only destroy the hyphae cell membrane，but also induce pathogenesis-related gene（MdPR1）expression in apple fruit. The result showed that the control effect of strain Pa2 on apple ring rot was 63.63% at 5 dpi.

Key words: apple ring rot, antagonistic fungus, Fusarium lateritium, control effect

史冰柯, 王卓妮, 覃艮红, 黄天祥, 王丽, 涂洪涛, 袁洪波, 侯珲. 苹果轮纹病拮抗真菌Pa2的分离与鉴定[J]. 园艺学报, 2024, 51(5): 1113-1125.

SHI Bingke, WANG Zhuoni, QIN Genhong, HUANG Tianxiang, WANG Li, TU Hongtao, YUAN Hongbo, HOU Hui. Isolation and Identification of Endophytic Fungus Pa2 Antagonistic Against Apple Ring Rot Caused by Botryosphaeria dothidea[J]. Acta Horticulturae Sinica, 2024, 51(5): 1113-1125.

图/表 14

表1 本研究中所用的引物信息

Table 1 Primer information used in this study

基因 Gene	引物 Primer	引物序列（5′-3′） Primer sequence
ITS	ITS1	TCCGTAGGTGAACCTGCGG
	ITS4	TCCTCCGCTTATTGATATGC
Tubulin	Bt2a	GGTAACCAAATCGGTGCTGCTTTC
	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC
MdEF1α	MdEF1α-q_F	ACATTGCCCTGTGGAAGTT
	MdEF1α-q_R	GTCTGACCATCCTTGGAAA
MdPR1	MdPR1-q_F	GCAGCAGTAGGCGTTGGTCCCT
MdPR1	MdPR1-q_R	CCAGTGCTCATGGCAAGGTTTT

图1 苹果树枝干韧皮部内生真菌菌株Pa2与苹果轮纹病菌的对峙试验（A）及对照病菌菌丝（B）与处理菌丝（C）

Fig. 1 Experiment（A）on the confrontation between endophytic fungal strain Pa2 in the phloem of apple tree branches and Botryosphaeria dothidea，and the effects of control pathogen hyphae（B）and growth on treated hyphae（C）

表2 菌株Pa2对不同病原菌的抑制效果

Table 2 Effect of strain Pa2 on different pathogenic fungi

病原真菌 Pathogenic fungi	抑制率/% Inhibition
苹果轮纹病菌 Botryosphaeria dothidea	77.49 ± 0.92
苹果腐烂病菌 Valsa mali	75.34 ± 1.14
苹果炭疽病菌 Colletotrichum gloeosporioides	74.50 ± 0.39
梨树腐烂病菌 Valsa pyri	74.65 ± 2.11

图2 菌株Pa2在PDA培养基上的菌落正背面（A、B）及其菌丝（C）和分生孢子（D）形态

Fig. 2 Morphology of colony front（A）and back（B），hyphae（C），and conidia（D）of strain Pa2 on PDA medium

图3 基于ITS（A）和Tubulin（B）基因序列构建的系统发育树数字代表置信度。

Fig. 3 The phylogenetic tree based on sequences of ITS（A）and Tubulin（B）gene Numbers represent confidence.

图4 菌株Pa2上清液对苹果轮纹病菌的抑制作用

Fig. 4 Suppression of strain Pa2 supernatant on Botryosphaeria dothidea

图5 菌株Pa2上清液对苹果轮纹病菌细胞质膜的影响

Fig. 5 Effect of strain Pa2 supernatant on the hyphal membrane of Botryosphaeria dothidea

图6 不同浓度菌株Pa2上清液对苹果轮纹病菌细胞膜通透性的影响不同字母表示经邓肯氏新复极差法检验在P < 0.05水平差异显著。

Fig. 6 Effect of strain Pa2 on cell membrane permeability of Botryosphaeria dothidea Different letters indicate significant difference at P < 0.05 level by Duncan’s multiple range test.

图7 菌株Pa2在苹果上的定殖能力（5 d）

Fig. 7 Colonization ability of strain Pa2 on apple（5 d）

图8 菌株Pa2对苹果轮纹病的抑制作用

Fig. 8 Suppression of strain Pa2 cell suspension on apple ring rot

表3 菌株Pa2对苹果轮纹病的防治效果

Table 3 Effectiveness of strain Pa2 in the control of apple ring rot

处理 Treatment	平均病斑长度/cm Lesion length		发病率/% Disease incidence		防治效果/% Control effect
处理 Treatment	3 d	5 d	3 d	5 d	3 d	5 d
对照Control	2.19 ± 0.04 a	3.58 ± 0.06 a	100.00 ± 0.00 a	100.00 ± 0.00 a	—	—
Pa2孢子悬浮液Pa2 spore suspension	0.68 ± 0.00 b	1.30 ± 0.06 b	76.67 ± 4.71 b	100.00 ± 0.00 a	68.95 ± 0.66 b	63.69 ± 1.90 b
多菌灵Carbendazim	0 ± 0.00 c	0 ± 0.00 c	0 ± 0.00 c	0 ± 0.00 b	100.00 ± 0.00 a	100.00 ± 0.00 a

图9 菌株Pa2对苹果轮纹病菌多菌灵抗性菌株Bd7的抑制作用（A）及防治效果（B）

Fig. 9 Inhibition（A）and control（B）effect of strain Pa2 on carbendazim-resistant strains of Botryosphaeria dothidea Bd7

表4 菌株Pa2对苹果轮纹病菌多菌灵抗性菌株Bd7引起的苹果轮纹病的防治效果

Table 4 Effectiveness of strain Pa2 in the control of apple ring rot caused by carbendazim-resistant Botryosphaeria dothidea Bd7

处理 Treatment	平均病斑长度/cm Lesion length		发病率/% Disease incidence		防治效果/% Control effect
处理 Treatment	3 d	5 d	3 d	5 d	3 d	5 d
对照Control	2.10 ± 0.02 a	3.32 ± 0.14 a	100.00 ± 0.00 a	100.00 ± 0.00 a	—	—
Pa2孢子悬浮液 Pa2 spore suspension	0.78 ± 0.03 b	0.97 ± 0.05 b	86.67 ± 4.00 b	90.00 ± 0.00 b	62.90 ± 1.92 b	70.78 ± 3.00 b
多菌灵Carbendazim	2.12 ± 0.02 a	3.29 ± 0.06 a	100.00 ± 0.00 a	100.00 ± 0.00 a	0 ± 0.00 c	0 ± 0.00 c
戊唑醇 Tebuconzole	0 ± 0.00 c	0 ± 0.00 c	0 ± 0.00 c	0 ± 0.00 c	100.00 ± 0.00 a	100.00 ± 0.00 a

图10 qR T-PCR检测菌株Pa2对病程相关基因MdPR1表达的影响 * 代表经t检验在P < 0.05水平差异显著。

Fig. 10 qR T-PCR assay for MdPR1 gene expression induced by strain Pa2 Asterisks indicate a significant difference at the P < 0.05 level based on Tukey’s tests.

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苹果轮纹病拮抗真菌Pa2的分离与鉴定

Isolation and Identification of Endophytic Fungus Pa2 Antagonistic Against Apple Ring Rot Caused by Botryosphaeria dothidea

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苹果轮纹病拮抗真菌Pa2的分离与鉴定

Isolation and Identification of Endophytic Fungus Pa2 Antagonistic Against Apple Ring Rot Caused by Botryosphaeria dothidea

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