新疆野苹果内生娄彻氏链霉菌A-m1的鉴定和发酵条件优化及抑菌广谱作用

doi:10.11707/j.1001-7488.20200708

摘要/Abstract

摘要：

目的: 研究新疆野苹果内生链霉菌菌株A-m1的分类地位，优化其发酵条件，测定其对多种植物病原物的抑菌活性，明确其广谱抑菌性，为相关病害的生物防治以及该菌株的应用提供参考。方法: 对菌株A-m1菌落培养性状、形态特征、理化性质和16S rDNA序列进行研究；以葡萄座腔菌为靶标菌，确定最高抑菌活性所需的基础培养基，优化碳源和氮源种类，确定最优发酵条件；测定发酵滤液对9种供试植物病原菌的抑菌活性。结果: 菌株A-m1是娄彻氏链霉菌，命名为Streptomyces rochei A-m1。高抑菌活性所需基础培养基为高氏一号，最佳碳源为可溶性淀粉，最佳氮源为硝酸钾；添加培养基体积5%的种子液、发酵温度23~32℃之间、pH 6~8之间、装液量100/500 mL和发酵培养4天为较优的发酵条件。发酵滤液对参试的9株植物病原菌均表现出抑菌活性，具广谱抑菌性；其中对葡萄座腔菌、苹果拟茎点霉的抑菌率可达95%以上，对胶孢炭疽菌、小麦赤霉病菌和小麦纹枯病菌的抑菌率超过80%，表现出强抑菌作用。结论: 链霉菌S. rochei A-m1抑菌谱较广、抑菌活性高，具有防治部分作物真菌性病害的应用潜力。

关键词: 新疆野苹果, 娄彻氏链霉菌, 葡萄座腔菌, 内生菌, 抑菌谱广, 发酵条件

Abstract:

Objective: This paper aims to study the taxonomic status of endophytic actinomycete A-m1, optimize its fermentation conditions, and clarify its antibacterial activity and antibacterial spectrum against pathogens such as apple diseases, and provide biological control for related diseases and further application of the strain. Method: The colony culture characteristics, morphological observation, physicochemical properties and 16S rDNA sequence of strain A-m1 were studied. The basic medium for the highest antibacterial activity was determined by using Botryosphaeria dothidea as the target bacteria, and the types of carbon source and nitrogen sources were optimized. The optimal fermentation conditions were determined; the antibacterial activity of the fermentation filtrate against the nine tested pathogens was determined. Result: The strain A-m1 was identified as Streptomyces rochei by combining morphology, physiological and biochemical characteristics and 16S rDNA molecular identification. The basic medium required for high bacteriostatic activity was Gause 1 cultural medium, the best carbon source was soluble starch, the optimum nitrogen source was potassium nitrate. The optimal fermentation conditions were: adding 5% seed solution of medium volume, the fermentation temperature of 23-32 ℃, pH of 6-8, the liquid volume of 100/500 mL, and fermentation culture for 4 days. The fermentation filtrate showed antibacterial activity against the 9 plant pathogens tested, showing the broad-spectrum of antibacterial activity. Among them, the antibacterial activity of the fermentation filtrate against B.dothidea and Phomopsis mali was more than 95%, and the inhibition rate against Colletotrichum gloeosporioides, Fusarium graminearum and Rizoctonia cerealis was over 80%. Conclusions: S. rochei A-m1 has a broad spectrum of antibacterial activity and high antibacterial activity, and has the potential for controlling fungal diseases of some crops.

Key words: Malus sieversii, Streptomyces rochei, Botryosphaeria dothidea, endophyte, extensive inhibition spectrum, fermentation conditions

中图分类号:

S763.13

李永丽,王亚红,周洲,曲良建. 新疆野苹果内生娄彻氏链霉菌A-m1的鉴定和发酵条件优化及抑菌广谱作用[J]. 林业科学, 2020, 56(7): 70-81.

Yongli Li,Yahong Wang,Zhou Zhou,Liangjian Qu. Identification and Optimization of Fermentation Conditions and Antibacterial Activity of Endophytic Streptomyces rochei A-m1 from the Branch of Malus sieversii[J]. Scientia Silvae Sinicae, 2020, 56(7): 70-81.

图/表 10

表1

菌株A-m1在不同培养基上的形态特征"

培养基 Medium	孢子堆颜色 Spore mass color	气生菌丝颜色 Aerial mycelium	基内菌丝颜色 Substrate mycelium	可溶性色素 Soluble pigment	菌落特征 Colony characteristics
ISP2	棕色 Brown	白色 White	黄色 Yellow	黄色 Yellow	菌落中央凸起，边缘有白色放射状菌丝 The colony is convex in the center and has white radial hyphae on the edge
ISP3	棕色 Brown	白色 White	无色 Colorless	无色 Colorless	菌落中央凸起，边缘有白色放射状菌丝，中间有黑色同心轮纹 The colony is convex in the center and has white radial hyphae on the edge, black concentric wheel pattern in the middle
ISP4	紫色 Purple	白色 White	黄色 Yellow	黄色 Yellow	菌落中央凸起，边缘有不规则放射状菌丝 The colony is convex in the center and has irregular radial hyphae on the edge
ISP5	棕色 Brown	白色 White	黄色 Yellow	无色 Colorless	菌落中央凸起，边缘有不规则放射状菌丝 The colony is convex in the center and has irregular radial hyphae on the edge
LB	无 None	白色 White	黄色 Yellow	黄色 Yellow	菌落圆形，表面有光泽，中央凸起，没有产孢 The colony is round, the surface is shiny, the central bulge, no sporulation
NA	白色 White	白色 White	黄色 Yellow	黄色 Yellow	菌落呈规则圆形，中央凹陷，边缘有白色放射状菌丝 The colonies are regular round, with a central depression and white radial hyphae at the edges
PDA	棕色 Brown	白色 White	黄色 Yellow	黄色 Yellow	菌落中央凸起，边缘有白色放射状菌丝 The colony is convex in the center and has white radial hyphae on the edge
PSA	紫色 Purple	白色 White	黄色 Yellow	黄色 Yellow	菌落中央黑色凸起，外围呈紫色，边缘有白色放射状菌丝 The colony is black in the center, purple in the periphery, and white radial hyphae at the edges
高氏一号 Gause 1	淡紫色 Light purple	白色 White	黄色 Yellow	黄色 Yellow	菌落中央凸起，菌落呈紫色，有黑色同心轮纹，边缘是白色不规则放射状菌丝 The colony is convex in the center, the colony is purple, there are black concentric rings, and the edges are white irregular radial hyphae

表1

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图7

表2

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理化特征 Physiological and biochemical characteristics		菌株 A-m1	娄彻氏链霉菌 S. rocheiD164	哥斯达黎加链霉菌 S. costaricanus	鼠灰链霉菌 S. murinus
碳源利用 Utilization of carbon sources	木糖Xylose	+	+	+	+
	棉子糖Raffinose	+	+	－	－
	L-阿拉伯糖L-Arabinose	+	+	+	－
	L-鼠李糖L-Rhamnose	+	+	－	－
	果糖Fructose	+	+	+	+
	葡萄糖Glucose	+	+	+	+
	肌醇Inositol	－	－	－	－
	蔗糖Sucrose	－	－	+	－
	甘露醇Mannitol	+	+	+	+
	水杨苷Salicin	+	+	+	+
牛奶凝固与胨化 Milk coagulation and peptonization	凝固Coagulation	+	+	+	+
牛奶凝固与胨化 Milk coagulation and peptonization	胨化Peptonization	+	+	+	+
明胶液化Degradation of gelatin		－	NA	+	+
H₂S产生H₂S production		－	NA	－	－
黑色素形成Melanin formation		－	NA	－	－
纤维素分解Cellulose decomposition		+	+	－	+
淀粉水解Starch hydrolysis		+	+	+	+

菌株 Strain	抑菌率Inhibition rate(%)
菌株 Strain	发酵滤液 Fermentation filtrate	70%甲基硫菌灵 70% thiophanate	80%多菌灵 80% carbendazim
葡萄座腔菌 B. dothidea	95.21±0.00	100.00±0.00	91.49±2.80
胶孢炭疽菌 C. gloeosporioides	83.08±1.38	100.00±0.00	100.00±0.00
拟茎点霉 P. mali	91.04±1.55	100.00±0.00	100.00±0.00
番茄灰霉病菌 B. cinerea	77.60±3.67	50.67±1.22	4.27±2.06
南天竹炭疽病菌 C. karstii	66.88±1.10	100.00±0.00	100.00±0.00
君子兰茎基腐病菌 F. oxysporum	64.03±0.82	94.28±0.82	100.00±0.00
小麦赤霉病菌 F. graminearum	82.03±2.28	100.00±0.00	100.00±0.00
小麦纹枯病菌 R.cerealis	80.89±0.96	100.00±0.00	78.66±3.62
小麦全蚀病菌 G. graminis	31.27±2.13	100.00±0.00	100.00±0.00

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