库尔勒香梨黑斑病菌拮抗菌筛选及其抑菌机理

Screening and possible mechanism of strains against black spot of Korla pear

摘要:

【背景】由互隔交链孢(Alternaria alternata)引起的黑斑病是库尔勒香梨采后贮期的主要病害之一，严重影响果实品质，造成巨大的经济损失。【目的】筛选对梨黑斑病菌具有高拮抗活性的菌株并探究其可能的抑菌机制，为香梨采后保鲜提供理论依据。【方法】从库尔勒香梨园健康植株根际采集土壤样品，采用稀释涂布法分离细菌并进行纯化，通过平板对峙法和琼脂打孔法筛选对梨黑斑病菌具有显著拮抗作用的菌株，结合形态学观察和16S rRNA基因序列分析进行初步鉴定；通过平板对峙法对拮抗菌抑菌谱进行测定；采用特异性平板定性检测拮抗菌产酶活性，采用3,5-二硝基水杨酸比色法法及福林-酚法定量酶活；通过孢子萌发抑制试验和扫描电镜观察，测定拮抗菌对梨黑斑病菌的拮抗作用。【结果】筛选得到2株对梨黑斑病菌有较强抑制活性的菌株JE53和JE56，经分析鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa)及枯草芽孢杆菌(Bacillus subtilis)，其发酵上清液对梨黑斑病菌的抑菌圈直径分别为22.49 mm和22.40 mm。抑菌谱测定结果表明，菌株JE53和JE56对马铃薯黑痣病菌等9种植物病原菌均有不同程度的抑制作用。胞外酶测定结果显示这2株菌可产生4种胞外酶；其发酵上清液能够显著抑制病原菌孢子萌发，抑制率分别为70.30%和88.87%；扫描电镜发现这2株菌可导致病原菌菌丝表面粗糙、扭曲变形。【结论】JE53和JE56对梨黑斑病菌有较好的抑制效果，具有进一步开发和应用的潜力。

Abstract:

[Background] Alternaria alternate-induced black spot, among the major diseases of Korla pear during storage, lowers the quality of the fruit, thus causing huge economic loss. [Objective] To screen strains with strong inhibitory effect on A. alternata, explore the underlying mechanism, and thus to lay a theoretical basis for postharvest preservation of Korla pear.[Methods] The rhizosphere soil samples of healthy Korla pear plants were collected and bacteria were isolated and purified by serial dilution and plating. The antagonistic strains were screened by plate confrontation assay and cylinder plate method. The preliminary identification of antagonistic bacteria was based on morphological characteristics and 16S rRNA gene sequence analysis. The antifungal spectrum of antagonistic bacteria was determined by plate confrontation assay. The extracellular enzyme species of antagonistic bacteria were detected by specific plate. The enzyme activity was quantified with 3,5-dinitrosalicylic acid colorimetric method and Folin-phenol method. Spore germination test and scanning electron microscopy (SEM) were employed to detect the antagonistic effect of the bacteria on A. alternaria. [Results] JE53 and JE56 with strong activity against A. alternaria were screened out from 152 isolates, with inhibition zone diameters of 22.49 mm and 22.40 mm, respectively. According to phylogeny analysis, they were identified as Paenibacillus polymyxa (JE53, GenBank accession No. OL966428) and Bacillus subtilis (JE56, GenBank accession No. OL966429), separately. The results of antifungal spectrum showed that JE53 and JE56 had different degree of inhibition on 9 plant pathogens such as Rhizoctonia solani. They produced four kinds of extracellular enzymes. The supernatant of the fermentation broth could significantly suppress the spore germination of A. alternaria, with inhibition rates of 70.30% and 88.87%, respectively. SEM showed that the suppressed hyphae were rough and distorted.[Conclusion] JE53 and JE56 inhibit A. alternaria, which have the potential of further development and application.