枸杞根际根腐病拮抗细菌的筛选及其防效检测

doi:10.16420/j.issn.0513-353x.2023-0298

摘要/Abstract

摘要：

以健康的‘宁杞5号’枸杞根际土壤为材料，通过稀释涂布法分离纯化根际细菌并进行分类鉴定；依据平板对峙试验进行初筛，基于菌丝生长抑制试验进行复筛，并对优选出的菌株进行防效检测。研究结果显示，从健康枸杞根际土壤中共得到18株菌落形态特征存在差异的细菌，属于芽孢杆菌属（Bacillus）和葡萄球菌属（Staphylococcus）；初筛得到对尖孢镰刀菌和立枯丝核菌均具有50%以上抑制活性的细菌11株，均为Bacillus；复筛发现菌株Bacillus sp. JK-B-01的培养液对立枯丝核菌、尖孢镰刀菌、芬芳镰刀菌和腐皮镰刀菌的抑制作用均最强，且培养液内存在可耐高温的抑菌物质；通过盆栽试验证明采用灌根法接种菌株Bacillus sp. JK-B-01能够阻碍尖孢镰刀菌引起的枸杞根腐病发生，说明其对枸杞根腐病有一定的预防效果。本研究中从‘宁杞5号’根际获得的土著芽孢杆菌Bacillus sp. JK-B-01，具有开发为枸杞根腐病生防菌剂的潜力，为宁夏枸杞根腐病的高效防治剂研发拓展了菌种资源。

关键词: 枸杞, 根腐病, 生物防治, 拮抗细菌, 芽孢杆菌

Abstract:

In this study，rhizosphere soil of the healthy plants of Lycium barbarum‘Ningqi 5’was collected for bacteria isolation and screening using dilution coating method based on the colony morphological characteristics，and the purified rhizosphere bacteria were classified based on biochemical and molecular identification. The preliminary and secondary screenings were conducted based on plate confrontation experiments and the growth inhibition of pathogenic mycelium using fermentation broth，respectively. Results indicated that a total of 18 strains of bacteria with different colony morphology characteristics were isolated and screened from the rhizosphere soil of the healthy‘Ningqi 5’，and were identified subsequently，belonging to Bacillus spp. and Staphylococcus sp. There were 11 strains exhibiting notable inhibitory effects on both Fusarium oxysporum and Rhizoctonia solani，with the bacteriostatic rate of more than 50%. All the 11 strains were identified to be Bacillus spp. The culture broth of Bacillus sp. JK-B-01 was found to possess the best inhibitory effect on four pathogens，R. solani，F. oxysporum，F. redolens，and F. solani. There might be high-temperature resistant antibacterial substances present in the extracellular metabolites of Bacillus sp. JK-B-01. In pot experiment，the occurrence of root rot was prevented after inoculation of Bacillus sp. JK-B-01 via root irrigation，suggesting that it had a certain preventive effect on root rot disease of L. barbarum caused by F. oxysporum. In general，an indigenous bacterium Bacillus sp. JK-B-01 was obtained from the rhizosphere of‘Ningqi 5’，and its potential as a candidate strain of BCAs against root rot disease of L. barbarum was proved via in vitro bacteriostatic activity determination and in vivo pot experiment. This study expanded the strain resources for the research and development of efficient BCAs against the root rot disease of L. barbarum in Ningxia.

Key words: Lycium barbarum, root rot, biological control, antagonistic bacteria, Bacillus

韩琴, 高惠惠, 马香, 苏建宇, 徐春燕. 枸杞根际根腐病拮抗细菌的筛选及其防效检测[J]. 园艺学报, 2024, 51(5): 1162-1172.

HAN Qin, GAO Huihui, MA Xiang, SU Jianyu, XU Chunyan. Screening and Identification of Antagonistic Rhizosphere Bacteria and Their Biological Control Effect on Root Rot of Lycium barbarum[J]. Acta Horticulturae Sinica, 2024, 51(5): 1162-1172.

图/表 7

图1 部分细菌的菌落形态

Fig. 1 Colony morphology of some bacteria

图2 基于16S rDNA序列的系统发育学分析

Fig. 2 Phylogenetic tree based on 16S rDNA sequences

表1 枸杞根际分离细菌菌株对供试病原菌生长的抑制作用

Table 1 Inhibitory effect of bacterial strains isolated from Lycium barbarum rhizosphere against pathogenic fungi

菌株编号 Strain No.	立枯丝核菌QX106 Rhizoctonia solani QX106	尖孢镰刀菌QX206 Fusarium oxysporum QX206	尖孢镰刀菌QX306 Fusarium oxysporum QX306
JK-B-01	+++	++	++
JK-B-02	+++	++	++
JK-B-03	+++	++	++
JK-B-04	++	-	-
JK-B-05	-	-	-
JK-B-06	++	++	++
JK-B-07	++	-	-
JK-B-08	-	-	-
JK-B-09	++	-	-
JK-B-10	+++	++	++
JK-B-11	++	-	-
JK-B-12	+++	++	++
JK-B-13	++	++	+
JK-B-14	+++	++	++
JK-B-15	+++	++	++
JK-B-16	-	-	-
JK-B-17	+++	++	++
JK-B-18	+++	++	++

图3 枸杞根际分离细菌菌株JK-B-01、JK-B-02、JK-B-03和JK-B-14对供试病原菌的抑制作用

Fig. 3 Inhibitory effects of bacterial strains JK-B-01，JK-B-02，JK-B-03 and JK-B-14 isolated from Lycium barbarum rhizosphere against pathogenic fungi

图4 培养1和3 d的拮抗菌高温灭菌培养液（Group 1）和过滤除菌培养液（Group 2）对病原菌菌丝生长的抑制作用 Group 1：培养基为2× PDA与高温灭菌培养液按1:1比例混合；Group 2：2× PDA与过滤除菌培养液按1:1比例混合。

Fig. 4 Inhibitory effects of antagonistic bacteria high temperature sterilization medium（Group 1）and filtration sterilization medium（Group 2）cultured for 1 and 3 d on the growth of pathogenic mycelium Group 1：The media were made by mixing 2× PDA and high temperature sterilized culture solution at the ratio of 1:1；Group 2：The media were made by mixing 2× PDA and filter sterilization culture solution at the ratio of 1:1. The same below.

图5 培养3 d的拮抗菌株JK-B-01高温灭菌培养液（Group 1）和过滤除菌培养液（Group 2）对病原菌菌丝生长的抑制作用图中3 d表示打孔接种菌块恒温培养时间为3 d，图5题目中的3 d表示采用拮抗菌株JK-B-01振荡培养3 d的培养液。

Fig. 5 Inhibition of antagonistic strain JK-B-01 cultured for 3-day by high temperature sterilization medium（Group 1）and filtration sterilization medium（Group 2）on the growth of pathogens 3 d in the figure indicates that the constant temperature culture time of the perforated inoculation block is 3 d，and 3 d in the name of Figure 5 indicates the culture solution with antagonistic strain JK-B-01 for 3 d.

图6 拮抗菌菌株JK-B-01对盆栽枸杞根腐病的防效

Fig. 6 Control effect of antagonistic strain JK-B-01 on root rot of Lycium barbarum

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