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[土壤链霉菌100株的鉴定及其拮抗特性]

[Identification and Antagonistic Properties of the Soil Streptomycete Streptomyces sp. 100].

作者信息

Biliavska L A, Efimenko T A, Efremenkova O V, Koziritska V Ye, Iutynska G A

出版信息

Mikrobiol Z. 2016 Mar-Apr;78(2):61-73.

Abstract

AIM

Determination of the taxonomic status of the soil streptomycete Streptomyces sp. 100 and study of its antagonistic properties against phytopathogenic and opportunistic human microorganisms.

METHODS

For the identification of the strain a set of conventional methods morphological and cultural, physiological and biochemical characteristics of the producer, as well as molecular genetic analysis of 16S rRNA gene were used. Streptomycete was cultivated on agar nutrient and liquid soy medium until the stationary phase of growth. The antagonistic activity of the strain was studied by agar diffusion method.

RESULTS

The study of morphological and cultural properties showed that Streptomyces sp. 100 formed the colonies with irregular edges protruding from the depressed center, straight sporophores were short, gathered in whorls; spores were oval, smooth shell dispute. Growing on agar medium (pH 6.8-7.4, temperature 28 °C, microaerophilic conditions) this strain formed mycelium of various colors: the air white, white-yellow, white-brown or substrate tan, cream and yellow, creamy carmine, yellow-brown. A soluble pigment was yellow and yellow-brown, while melanoid pigment was not detected. The morphological, cultural, physiological, biochemical and molecular genetic characteristics of the soil streptomycete let to identify it as Streptomyces netropsis (Finlay et al., 1951) IMV Ac-5025 (UCM Ac-2186) that is an active antagonist IMV Ac-5025 against plant pathogens. Growing on a surface of agaric nutrient media it inhibits phytopathogenic bacteria (Xanthomonas axonopodis pv. glycines 8609, Pseudomonas savastanoi pv. glycinea 8571, P. syringae pv. coronafaciens 9030) and fungi (Alternaria alternata 16814, Fusarium оxysporum 54201) zone of growth inhibition were 20 - 32 mm and 16 - 30 mm respectively. The supernatant of culture medium and the ethanol extract of biomass inhibited the growth of pathogenic bacteria and fungi. The most sensitive to action of a supernatant of cultural liquid were P. syringae pv. atrofaciens 7886 and Clavibacter michiganensis ssp. michiganensis 102, growth inhibition zones - 42 and 30 mm respectively. It should be noted that in the majority of cases the supernatant of cultural liquid suppressed growth of phytopathogenic bacteria in comparison with biomass extract more actively. At the same time only biomass extract inhibited the growth of P. syringae pv. coronafaciens 9060, P. corrugatа 9070, X. anoxopodis pv. glycines 9075, X. anoxopodis pv. glycines 8609 and Pantoea agglomerans 8490. Tolerant to metabolites of S. netropsis IMV Ac-5025 were P. syringae pv. atrofaciens 8291 and X. visicatoriae 7790. The extract of biomass S. netropsis IMV Ac-5025 inhibited growth of all studied strains of phytopathogenic fungi (A. alternata 16814, A. culmorum 00790, F. оxysporum 54201, F. tricinetum 00795, F. oxysporum n.33, Cladosporium herbarum 16863, Cochliobolus spicifas 16860, Nigrospora oryzae 16864). The supernatant of the cultural liquid also showed the oppressing action on fungi, except for Cladosporium herbarum 16863 and Cochliobolus spicifas 16860. The strain was almost ineffective against opportunistic human microorganisms (Escherichia coli ATCC 25922, Bacillus pumilus NCTC 8241, Staphylococcus aureus FDA 209P et al.).

CONCLUSIONS

The lack of action of Streptomyces netropsis IMV Ac-5025 on the opportunistic human microorganisms and the active antagonism of phytopathogens, both, define potential its application for plant protection.

摘要

目的

确定土壤链霉菌Streptomyces sp. 100的分类地位,并研究其对植物病原微生物和人类机会致病菌的拮抗特性。

方法

采用一系列常规方法对该菌株进行鉴定,包括形态学和培养特征、产生菌的生理生化特性,以及对16S rRNA基因进行分子遗传学分析。链霉菌在琼脂营养培养基和液体大豆培养基上培养至生长稳定期。采用琼脂扩散法研究该菌株的拮抗活性。

结果

形态学和培养特性研究表明,Streptomyces sp. 100形成的菌落边缘不规则,从凹陷的中心突出,直孢子丝短,呈轮生状;孢子椭圆形,表面光滑。在琼脂培养基(pH 6.8 - 7.4,温度28℃,微需氧条件)上生长时,该菌株形成各种颜色的菌丝体:气生菌丝白色、白黄色、白棕色,或基内菌丝棕褐色、奶油色、黄色、奶油洋红色、黄棕色。产生的可溶性色素为黄色和黄棕色,未检测到类黑色素。根据土壤链霉菌的形态、培养、生理、生化及分子遗传学特征,将其鉴定为Streptomyces netropsis(Finlay等人,1951年)IMV Ac - 5025(UCM Ac - 2186),它是对植物病原菌有活性的拮抗剂IMV Ac - 5025。在琼脂营养培养基表面生长时,它能抑制植物病原细菌(大豆细菌性斑点病菌Xanthomonas axonopodis pv. glycines 8609、大豆细菌性叶斑病菌Pseudomonas savastanoi pv. glycinea 8571、燕麦晕斑病菌P. syringae pv. coronafaciens 9030)和真菌(链格孢Alternaria alternata 16814、尖孢镰刀菌Fusarium оxysporum 54201),生长抑制圈分别为20 - 32 mm和16 - 30 mm。培养基上清液和生物量乙醇提取物能抑制病原菌和真菌的生长。对培养液上清液作用最敏感的是燕麦萎蔫病菌P. syringae pv. atrofaciens 7886和密执安棒形杆菌密执安亚种Clavibacter michiganensis ssp. michiganensis 102,生长抑制圈分别为42 mm和30 mm。需要注意的是,在大多数情况下,培养液上清液比生物量提取物更能有效抑制植物病原细菌的生长。同时,只有生物量提取物能抑制燕麦晕斑病菌P. syringae pv. coronafaciens 9060、皱缩假单胞菌P. corrugatа 9070、大豆细菌性斑点病菌X. anoxopodis pv. glycines 9075、大豆细菌性斑点病菌X. anoxopodis pv. glycines 8609和成团泛菌Pantoea agglomerans 8490的生长。对Streptomyces netropsis IMV Ac - 5025代谢产物耐受的是燕麦萎蔫病菌P. syringae pv. atrofaciens 8291和番茄疮痂病菌X. visicatoriae 7790。Streptomyces netropsis IMV Ac - 5025生物量提取物能抑制所有供试植物病原真菌菌株(链格孢Alternaria alternata 16814、禾谷链格孢A. culmorum 00790、尖孢镰刀菌Fusarium оxysporum 54201、三线镰刀菌F. tricinetum 00795、尖孢镰刀菌n.33、草本枝孢Cladosporium herbarum 16863、禾本科布氏白粉菌Cochliobolus spicifas 16860、稻黑孢Nigrospora oryzae 16864)的生长。培养液上清液除对草本枝孢Cladosporium herbarum 16863和禾本科布氏白粉菌Cochliobolus spicifas 16860外,对其他真菌也有抑制作用。该菌株对人类机会致病菌(大肠埃希菌Escherichia coli ATCC 25922、短小芽孢杆菌Bacillus pumilus NCTC 8241、金黄色葡萄球菌Staphylococcus aureus FDA 209P等)几乎无作用。

结论

Streptomyces netropsis IMV Ac - 5025对人类机会致病菌无作用,而对植物病原菌有活性拮抗作用,这决定了其在植物保护方面具有潜在应用价值。

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