Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.     

纳他霉素对芒果采后胶孢炭疽菌的抑菌效果及机理

以纳他霉素为抑菌剂, 实验测定了离体条件下不同浓度纳他霉素对胶孢炭疽菌(Colletotrichum gloeosporioides)的孢子萌发及菌丝生长的抑制效果, 以及活体损伤接种炭疽病菌后, 纳他霉素对芒果(Mangifera indica)果实炭疽病的防治效果。通过测定纳他霉素处理后胶孢炭疽菌的细胞膜相对渗透率、可溶性蛋白含量、细胞膜完整性、孢子内活性氧水平和线粒体分布情况, 初步探明其抑菌机理。结果表明, 3 mg∙L ^-1纳他霉素可显著抑制胶孢炭疽菌孢子萌发、芽管伸长和菌落生长, 80 mg∙L ^-1纳他霉素可有效抑制芒果贮存过程中果实炭疽病斑的扩展。纳他霉素处理后胶孢炭疽菌细胞膜相对渗透率和可溶性蛋白含量增加; 2 mg∙L ^-1纳他霉素处理8小时, 处理组胶孢炭疽菌孢子细胞膜损伤染色率为33.6%, 对照组染色率为13.9%; 处理组胞内活性氧产生染色率达46.9%, 比对照组高39.7%; 同时观察到纳他霉素使胞内线粒体分布不均且荧光信号微弱。以上结果表明, 纳他霉素可以破坏胶孢炭疽病菌细胞膜, 诱导活性氧大量积累, 并降低线粒体活性, 从而干扰菌体正常生理活性, 使其代谢活动受影响, 从而达到抑菌目的。     

Bacillus velezensis strain HYEB5-6 as a potential biocontrol agent against anthracnose on Euonymus japonicus

Anthracnose is a foliar disease of the Euonymus shrub caused by Colletotrichum gloeosporioides. In this study, the bacterium HYEB5-6 was isolated from inside one-year-old branches of healthy Euonymus japonicus and showed significant antifungal activities against various phytopathogenic fungi, including C. gloeosporioides s.s. HYCG2-3, in dual culture experiments. The HYEB5-6 isolate significantly decreased lesion diameter and disease index caused by C. gloeosporioides inoculation on detached leaves of E. japonicus. The effects of HYEB5-6 metabolites on the invading structure of the fungus were investigated. Bacterial metabolites inhibited conidial germination, the growth of the germ tube and appressorium formation, possibly through protease and glucanase of HYEB5-6 by managing the mycelial cell wall. The HYEB5-6 isolate also produced a massive biofilm, which might facilitate leaf colonisation. These results indicate that HYEB5-6 has the potential for use as a biological control agent against C. gloeosporioides. The HYEB5-6 isolate was identified as Bacillus velezensis based on its biochemical characteristics and its 16S rRNA gene sequence.     

苹果炭疽菌低毒性菌株生物学特性的研究

研究了温度、pH值及碳源对苹果炭疽病菌(Colletotrichum gloeosporioides)强毒性菌株及低毒性菌株的生长、产孢和孢子萌发的影响。结果表明:菌丝生长温度范围10℃~35℃、最适温度为28℃,低毒菌株生长速率较快,产生分生孢子的温度范围与分生孢子萌发的温度范围为15℃~35℃,低毒菌株孢子萌发率较高,菌丝生长的最适pH值基本相同,pH在3~10的范围内均能生长和产孢,产生分生孢子的最适pH值为4~5,但经离子注入的低毒性菌株C100-2-5的产孢量在不同的pH值和温度下却一直低于经磁场处理的C0.25-1-2和强毒性菌株(对照)。分生孢子的致死温度为47℃、15 min或50℃、5 min。但菌丝的致死温度却不同,对照为60℃、20 min,C0.25-1-2为60℃、25 min,C100-2-5为60℃、30 min,65℃下5 min(及5 min以上)。     

炭疽病菌侵染对荔枝果实生理生化变化的影响

本研究测定了荔枝果实人工接种炭疽病菌后呼吸速率、乙烯释放量的变化和果皮氧化、过氧化作用以及与酚类代谢有关的几种酶活性的变化。结果表明,接种炭疽病菌的荔枝果实呼吸速率和乙烯释放量显著增高,果皮活性氧(O₂^·)产生速率和丙二醛(MDA)含量显著增加,超氧化物歧化酶(SOD)活性显著降低,过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性显著增高。说明炭疽病菌的侵染可导致荔枝果实呼吸速率和乙烯释放量的增高,加速荔枝果皮氧化和过氧化进程,并诱导荔枝果皮PPO、POD、PAL活性增高,是加速采收后荔枝果实衰老、褐变、腐烂的一个重要原因。     

胶孢炭疽菌生防放线菌SD-29的鉴定及抗菌活性评价

【背景】胶孢炭疽菌是引起橡胶炭疽病的一种重要病原菌,可导致橡胶树产胶量下降。【目的】从山东青岛一农田土壤中分离出一株胶孢炭疽菌生防放线菌SD-29,并对其进行鉴定及抗菌活性评价。【方法】采用对峙生长法及菌丝生长速率法对菌株SD-29的拮抗活性进行鉴定;利用乙酸乙酯萃取法提取菌株SD-29发酵液粗提物并进行活性评价;根据菌株SD-29的形态特征、生理生化及16S rRNA基因序列进行鉴定。【结果】菌株SD-29对胶孢炭疽菌具有较强的抑制活性,皿内抑制活性达到82.6%。发酵液粗提物对菌丝生长的EC50为13.6μg/mL,100μg/mL的粗提物对胶孢炭疽菌孢子萌发抑制率达到63.16%,其对感炭疽病橡胶叶片的防治效果达到48.96%。根据该菌的形态特征、生理生化特征及16S rRNA基因序列分析鉴定菌株SD-29为Streptomyces yatensis。【结论】菌株SD-29对胶孢炭疽菌有较强的防治效果,具有潜在的应用价值。     

营养物质对两种炭疽菌潜伏侵染的影响

通过不同浓度的几种糖类对潜伏侵染在青香蕉果实中的colletotrichum musae和芒果果实中的colletotichum gloeosporioides的菌体的影响进行了测定,结果表明,高浓度淀粉可极显著地提高两种炭疽菌的孢子萌发率,并有利于附着胞和分生孢子的形成。单糖和二糖在较低浓度时有利于孢子萌发和产孢,不利于附着胞形成。未成熟果实的坚硬结构和高淀粉含量为病菌提供了以附着胞形式潜伏侵染在寄主中的条件。     

Effect of row spacing on biological control of sicklepod (Senna obtusifolia) with Colletotrichum gloeosporioides

In field experiments conducted over 3 years, the mycoherbicidal fungus Colletotrichum gloeosporioides, formulated either in 20% (v/v) unrefined corn oil and 0.2% Silwet L-77 surfactant or with an invert emulsion, provided season-long control of Senna obtusifolia in narrow (51 cm) rows of soybean. However, in wide (102 cm) rows, one application of either formulation failed to provide season-long control of S. obtusifolia, and two applications were required to achieve season-long weed control. In narrow (51 cm) rows, one application of the fungus either in unrefined corn oil or an invert emulsion controlled S. obtusifolia an average of >90%, and a second application was not required for season long weed control. Soybean yields in wide-row plots treated with two applications of either the fungus/corn oil or fungus/invert emulsion, or with a single application of the fungal treatments in narrow-row soybean plots, were not significantly different from weed-free control plots, or from plots treated with the herbicide chlorimuron. These results suggest that row spacing can affect mycoherbicidal efficacy of this fungus for controlling S. obtusifolia.