荧光标记解淀粉芽孢杆菌WK1在山核桃树体和土壤中的定殖规律

doi:10.3969/j.issn.1004-1524.2021.01.10

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 77-86.DOI: 10.3969/j.issn.1004-1524.2021.01.10

荧光标记解淀粉芽孢杆菌WK1在山核桃树体和土壤中的定殖规律

高竞(), 方伟, 顾佳悦, 严淑娴, 邵帅, 梁辰飞, 秦华, 陈俊辉, 徐秋芳^*()

浙江农林大学环境与资源学院,浙江省森林生态系统碳循环与固碳减排重点实验室,浙江杭州 311300

收稿日期:2020-07-06 出版日期:2021-01-25 发布日期:2021-01-25
通讯作者: 徐秋芳
作者简介:*徐秋芳,E-mail:xuqiufang@zafu.edu.cn
高竞(1996—),男,云南宁洱人,硕士研究生,主要从事土壤微生物方面的研究。E-mail:1564020143@qq.com
基金资助:
浙江省省院合作林业科技计划(2017SY01);浙江省重点研发计划(2015C02013)

Colonization of fluorescently labeled Bacillus amylolyticus WK1 in Carya cathayensis Sarg. and soil

GAO Jing(), FANG Wei, GU Jiayue, YAN Shuxian, SHAO Shuai, LIANG Chenfei, QIN Hua, CHEN Junhui, XU Qiufang^*()

Zhejiang Provincial Key Laboratory of Carbon Cycling in Forest Ecosystems and Carbon Sequestration, College of Environmental and Resource Sciences, Zhejiang A&F University, Hangzhou 311300, China

Received:2020-07-06 Online:2021-01-25 Published:2021-01-25
Contact: XU Qiufang

摘要/Abstract

摘要：

为探明解淀粉芽孢杆菌WK1在山核桃树和林地土壤的定殖动态及其最佳接种方式,采用电击转化方法导入绿色荧光蛋白(GFP)质粒,构建荧光标记菌株(GFP-WK1)。通过叶面喷施(喷叶法)、根系浇灌(灌根法)和树干滴注(挂液法)3种方式接种GFP-WK1菌液,定期测定GFP-WK1在山核桃树体和土壤中的定殖数量,以及不同土壤pH条件下GFP-WK1在山核桃幼苗根、茎、叶中的定殖量,分析GFP-WK1的定殖能力和移动规律。结果表明:GFP-WK1能够通过喷叶法、灌根法和挂液法的方式在土壤和山核桃树体定殖,GFP-WK1在山核桃树体和土壤之间有良好的移动性,定殖量可保持在10⁴~10⁶ CFU·g^-1。相较于灌根法和喷叶法,挂液法处理下GFP-WK1定殖稳定。不同土壤pH条件下,GFP-WK1在山核桃幼苗中的定殖量均表现为根>茎>叶,在pH值为6.8的土壤条件下定殖效果最好,植物体内的定殖量也相对较高。从短期定殖效果和施用方便的角度,田间实际应用推荐灌根法,并将土壤pH值调到6.8;长期使用的话,建议选择挂液法,以保证树体的菌株定殖量稳定。

关键词: 山核桃, 解淀粉芽孢杆菌, 干腐病, 绿色荧光蛋白, 定殖

Abstract:

In order to determine the colonization dynamics and the best way of Bacillus amylolyticus WK1 inoculation in hickory (Carya cathayensis Sarg.) and soil, electric shock transformation method was used to introduce green fluorescent protein (GFP) particles to construct the fluorescent labeled strain (GFP-WK1). The liquid GFP-WK1 was inoculated by leaf spraying, root irrigation and trunk injection, and the colonization number of GFP-WK1 strain was determined in soil, roots and leaves of hickory at regular intervals during 60 days. The liquid GFP-WK1 was added to soil with pH of 5.6, 6.8 and 7.9, respectively, in order to find the suitable soil pH for WK1 colonization. The results showed that GFP-WK1 could be colonized in both soil and inner tissues of hickory trees by leaf spraying, root irrigation and trunk injection, which indicated its good mobility between soil and hickory tree as well as inner tissues. The GFP-WK1 was maintained in the range of 10⁴-10⁶ CFU·g^-1 during 60 days. Compared with root irrigation and leaf spraying, GFP-WK1 strain was more stable in hickory tree and soil by trunk injection. The GFP-WK1 was more abundant in soil with pH of 6.8. The colonization number in hickory seedlings decreased in the order of roots>stems>leaves. It was recommended to inoculate GFP-WK1 by root irrigation in respect to colonization effect in tissue and the convenience of application in short-term, combined with adjusting soil pH to 6.8. For the long-term consideration, trunk injection was a better choice.

Key words: Carya cathayensis Sarg., Bacillus amylolyticus, dry rot, green fluorescent protein, colonization

中图分类号:

S476.1

高竞, 方伟, 顾佳悦, 严淑娴, 邵帅, 梁辰飞, 秦华, 陈俊辉, 徐秋芳. 荧光标记解淀粉芽孢杆菌WK1在山核桃树体和土壤中的定殖规律[J]. 浙江农业学报, 2021, 33(1): 77-86.

GAO Jing, FANG Wei, GU Jiayue, YAN Shuxian, SHAO Shuai, LIANG Chenfei, QIN Hua, CHEN Junhui, XU Qiufang. Colonization of fluorescently labeled Bacillus amylolyticus WK1 in Carya cathayensis Sarg. and soil[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 77-86.

图/表 7

图1 GFP-WK1在荧光显微镜下的影像(A)和GFP质粒PCR验证结果(B) M为DNA相对分子质量标准,1和2为GFP-WK1,3为WK1。

Fig.1 Fluorescence microscope image of GFP-WK1 (A) and PCR inspection result of GPF plasmid (B) M was DNA molecular marker, 1 and 2 were GFP-WK1, and 3 was WK1.

图2 WK1和GFP-WK1菌株的生长曲线

Fig.2 Growth curve of WK1 and GFP-WK1

图3 未标记菌株(WK1)和GFP-WK1菌株的抑菌效果

Fig.3 Bacteriostatic effect of GFP-WK1 and WKI

图4 不同方法接种的GFP-WK1在山核桃林土壤和植物体内的定殖示意图图中箭头所指亮点为GFP-WK1菌体。a,不接种菌株的空白对照在绿色荧光下的土壤样品;b,接种WK1菌株在绿色荧光下的山核桃树皮样品;c,灌根法接种GFP-WK1菌株在绿色荧光下的土壤样品;d,灌根法接种GFP-WK1在白光下的树皮样品;e,灌根法接种GFP-WK1在绿色荧光下的树皮样品;f,灌根法接种GFP-WK1在绿色荧光下的树叶样品;g,挂液法接种GFP-WK1在白光下的树皮样品;h,挂液法接种GFP-WK1在绿色荧光下的树皮样品;i,挂液法接种GFP-WK1在绿色荧光下的树叶样品。

Fig.4 Colonization of GFP-WK1 inoculated with different methods in soil and Carya cathayensis The bright spot indicated by the arrow in the figure is GFP-WK1. a, Soil of blank control under green fluorescence without strain inoculation; b, Carya cathayensis bark inoculated with WK1 under green fluorescence; c, Soil inoculated with GFP-WK1 by root irrigation under green fluorescence; d, Carya cathayensis bark inoculated with GFP-WK1 by root irrigation under white light ; e, Carya cathayensis bark inoculated with GFP-WK1 by root irrigation under green fluorescence; f, Carya cathayensis leaf inoculated with GFP-WK1 by root irrigation under green fluorescence; g, Carya cathayensis bark inoculated with GFP-WK1 by trunk injection under white light; h, Carya cathayensis bark inoculated with GFP-WK1 by trunk injection under green fluorescence; i, Carya cathayensis leaf inoculated with GFP-WK1 by trunk injection under green fluorescence.

表1 不同接种方法GFP-WK1的定殖动态

Table 1 Colonization dynamics of GFP-WK1 inoculated by different methods

接种方法 Inoculation method	t/d	定殖数量Colonization number/(10⁶ CFU·g^-1)
接种方法 Inoculation method	t/d	土壤Soil	根Root	叶Leaf
喷叶法Leaf spraying	3	0.15±0.05 b	0.46±0.21 b	17.36±4.62 a
	7	0.32±0.12 b	0.91±0.37 b	8.21±2.25 a
	15	0.12±0.04 b	0.33±0.12 b	3.12±0.20 a
	30	0.04±0.01 c	0.19±0.03 b	0.78±0.02 a
	60	0.03±0.01 b	0.12±0.04 b	0.34±0.14 a
挂液法Trunk injection	3	0.09±0.03 a	0.13±0.04 a	0.24±0.11 a
	7	0.41±0.15 a	0.22±0.08 b	0.61±0.20 a
	15	0.46±0.13 a	0.57±0.21 a	0.54±0.20 a
	30	0.34±0.10 b	0.68±0.20 a	0.56±0.18 a
	60	0.20±0.08 b	0.50±0.13 a	0.51±0.11 a
灌根法Root irrigation	3	21.10±7.56 a	18.67±5.45 a	9.20±3.22 b
	7	13.63±3.52 a	7.37±2.02 b	5.21±1.85 b
	15	4.87±1.03 a	0.42±0.10 b	0.99±0.24 b
	30	1.23±0.41 a	0.11±0.03 c	0.66±0.26 b
	60	0.11±0.02 a	0.04±0.01 b	0.04±0.01 b

图5 当土壤pH值分别为5.6(A)、6.8(B)、7.9(C)时GFP-WK1在山核桃苗上的定殖动态同一时间柱上无相同字母的表示不同部位间差异显著(P<0.05)。

Fig.5 Colonization dynamics of GFP-WK1 in pecan seedlings under soil pH of 5.6 (A), 6.8 (B), and 7.9 (C) Bars marked without the same letters indicated significant difference within different parts at P<0.05 in the same time.

图6 GFP-WK1在不同pH值土壤上的定殖动态同一时间柱上无相同字母的表示不同处理间差异显著(P<0.05)。

Fig.6 Colonization of GFP-WK1 in soil under different pH value Bars marked without the same letters indicated significant difference within treatments at P<0.05 in the same time.

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荧光标记解淀粉芽孢杆菌WK1在山核桃树体和土壤中的定殖规律

Colonization of fluorescently labeled Bacillus amylolyticus WK1 in Carya cathayensis Sarg. and soil

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