杧果采后炭疽病生防菌株筛选及其培养特性研究

doi:10.16420/j.issn.0513-353x.2020-0760

摘要/Abstract

摘要：

用组织分离法从红花龙胆（Gentiana rhodantha Franch. Ex. Hemsl）茎秆内分离芽孢杆菌菌株,通过与杧果炭疽病病原菌（Colletotrachum gloeosporioides）进行对峙培养,筛选出拮抗效果显著的菌株。采用平板试验研究拮抗菌株发酵液对杧果炭疽病病原菌菌丝生长和孢子萌发的影响,采用添加拮抗菌悬浮液或无菌发酵液的涂膜剂验证对杧果炭疽病的防治效果,用高效液相色谱—质谱联用技术对菌株发酵液中的抑菌成分进行检测,通过单因素试验研究菌株最佳发酵条件。经形态学、生理生化特性及16S rDNA和gyrB序列分析确定拮抗菌株SB023为贝莱斯芽孢杆菌（Bacillus velezensis）。该菌株的发酵液可显著抑制杧果炭疽病菌菌丝生长及孢子萌发;加入10% 的SB023无菌发酵滤液的涂膜剂可完全抑制病原菌从伤口侵入杧果果实,且对杧果无致病性。SB023菌株抑菌成分为表面活性素C、杆菌抗霉素D、杆菌抗霉素L和伊枯草菌素Iturin A。研究表明,以KMB为基础培养基、初始pH 7、培养温度为28 ~ 30 ℃以及以碳源为麦芽糖、氮源为酵母粉和无机盐为硫酸镁时菌株SB023生长最快,发酵液抑菌活性最强。

关键词: 杧果, 炭疽病, 贝莱斯芽孢杆菌, 拮抗, 抑菌成分, 发酵条件

Abstract:

Bacillus strains were isolated from Gentiana rhodantha stems with highly antifungal activity resist mango anthracnose was screened by confrontation culture with Colletotrachum gloeosporioides. The fungistatic effect of mycelial growth and conidia germination was measured by in vivo experiment. The control efficiency of antagonistic strain against anthracnose was tested by surface coating method using wounded mango fruit. The structure of the antifungal substances in fermentation broth of antagonistic bacteria was detected by high performance liquid chromatography-mass spectrometry（HPLC-MS）,and single factor experiment was used to study on the optimal growth and fermentation conditions,respectively. Based on morphological,physiological and 16S rDNA and gyrB sequences analysis,the strain SB023 which significant antifungal activity against mango anthracnose was identified as Bacillus velezensis. The mycelial growth and spore germination of pathogen were significantly inhibited by the fermentation broth of strain SB023. The control effect of SB023 to against anthracnose of mango reached 100% then coated with 0.1% Tween-80,2% sodium alginate and 10% fermentation broth. The main antifungal substances were Surfactin C,Bacillomycin D,Bacillomycin L and Iturin A in broth. The fermentation study showed that the optimal culture medium for KMB with yeast extract,maltose,magnesium sulfate,initial pH 7.0 and incubated at 28-30 ℃.

Key words: mango, anthracnose, Bacillus velezensis, antagonistic bacteria, antifungal substance, fermentative conditions

中图分类号:

S667.7

张晓勇, 李树江, 严凯, 翁贵英, 张韵霞, 龙巧芳, 梁文, 蒋芹娜, 杨友联. 杧果采后炭疽病生防菌株筛选及其培养特性研究[J]. 园艺学报, 2021, 48(11): 2171-2184.

ZHANG Xiaoyong, LI Shujiang, YAN Kai, WENG Guiying, ZHANG Yunxia, LONG Qiaofang, LIANG Wen, JIANG Qinna, YANG Youlian. Screening and Culture Characteristics of Biocontrol Strains of Postharvest Anthracnose of Mango[J]. Acta Horticulturae Sinica, 2021, 48(11): 2171-2184.

图/表 10

表1 菌株SB023发酵液对杧果炭疽病菌菌落及孢子萌发的抑制效果

Table 1 Inhibited effect of cell-free fermentation on mycelial growth and spore germination of Colletotrachum gloeosporioides

发酵液添加量/% Contents of fermentation broth	菌落直径/mm Colony diam	菌丝生长抑制率/% Mycelial growth rate	孢子萌发率/% Spore germination	孢子萌发抑制率/% Inhibition rate to spore germination	孢子芽管生长率/ （μm · d^-1） Growth rate of germ tube
0（对照Control）	90.00 ± 0.00 a	—	97.23 ± 3.24 a	—	257.83 ± 18.04 a
2.5	55.10 ± 1.41 b	38.89 ± 1.57 e	78.84 ± 4.33 b	18.91 ± 2.20 c	241.33 ± 6.03 a
5.0	41.30 ± 1.41 c	54.44 ± 1.57 d	34.47 ± 5.56 c	64.55 ± 5.45 b	183.33 ± 23.15 b
10	24.50 ± 0.71 d	72.78 ± 0.79 c	10.23 ± 2.32 d	100.00 ± 0.00 a	0 c
15	11.50 ± 0.71 e	87.22 ± 0.79 b	0 e	100.00 ± 0.00 a	0 c
20	7.10 ± 0.14 f	92.11 ± 0.16 a	0 e	100.00 ± 0.00 a	0 c

表2 菌株SB023对杧果炭疽病的防治效果

Table 2 The control effect of strain SB023 against mango anthracnose

处理 Treatment	病斑面积/ mm²Lessons area	抑制率/% Inhibition rate	病情指数 Disease index	防治效果/% Control efficiency
涂膜剂 + 菌悬液 + 接病菌 Coating agents + suspension + inoculation	0 c	100.00 ± 0.00 a	0 b	100.00 ± 0.00 a
涂膜剂 + 发酵液 + 接病菌 Coating agents + fermentation broth + inoculation	0 c	100.00 ± 0.00 a	0 b	100.00 ± 0.00 a
涂膜剂 + 菌悬液Coating agents + suspension	0 c	—	0 b	—
涂膜剂 + 接病菌Coating agents + inoculation	189.50 ± 45.05 a	5.50 ± 1.45 c	75.35 ± 3.25 a	4.17 ± 1.23 c
接病菌 + 涂膜剂 + 发酵液 Inoculation + coating agents + fermentation broth	135.25 ± 21.21 b	32.37 ± 7.24 b	71.33 ± 4.54 a	9.28 ± 1.67 b
接病菌（对照）Inoculation（Control）	201.55 ± 53.85 a	—	78.63 ± 6.25 a	—

图1 菌株SB023对胶孢炭疽菌的拮抗效果（A）与空白对照（B）

Fig. 1 The antagonism of strain SB023 against Colletotrachum gloeosporioides （A）and the control（B）

图2 菌株SB023在NA培养基上的菌落（A）及形态特征（B）

Fig. 2 Colonies on NA medium（A）and morphological characteristics（B）of strain SB023

图3 基于16S rDNA 和gyrB序列构建的NJ系统发育树

Fig. 3 Neighbor-joining phylograms inferred from combined 16S rDNA and gyrB dates

图4 菌株SB023发酵液对胶孢炭疽菌菌丝生长（A ~ F）及孢子萌发（G ~ L）的影响箭头所示为孢子形成的囊泡状结构。

Fig. 4 Antifungal activity of cell-free fermentation broth on mycelial growth（A-F）and conidia germination （G-L）of Colletotrachum gloeosporioides Arrows indicate vesicle-like structures of condidium.

图5 菌株SB023对杧果炭疽病的防治效果

Fig. 5 The control effect of strain SB023 against mango anthracnose

图6 菌株SB023发酵液高效液相色谱图

Fig. 6 HPLC chromatogram of SB023 fermentation broth

图7 保留时间为11.100（A）、13.566（B）、13.965（C）和15.415 min（D）产物的质谱图

Fig. 7 Mass spectrum at the acquisition time of 11.1（A）,13.566（B),13.965（C）and 15.415 min（D）

图8 菌株SB023在不同培养基（A）、pH（B）、温度（C）、碳源（D）、氮源（E）及无机盐（F）条件下生长及其发酵液抑菌活性

Fig. 8 Cell expansion and fermentation broth antifungal activity of strain SB023 in different mediums（A）,pH values（B）,temperatures（C）,carbon sources（D）,nitrogen（E）and mineral salts（F）

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