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

doi:10.3969/j.issn.1004-1524.2021.01.10

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (1): 77-86.DOI: 10.3969/j.issn.1004-1524.2021.01.10

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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

Abstract

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

CLC Number:

S476.1

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.

Figures/Tables 7

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.

Fig.2 Growth curve of WK1 and GFP-WK1

Fig.3 Bacteriostatic effect of GFP-WK1 and WKI

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.

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

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.

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.

References 35

[1]	刘锦, 于炜. 山核桃干腐病研究进展[J]. 浙江林业科技, 2017,37(6):99-102.
	LIU J, YU W. Researches on canker of Carya cathayensis[J]. Journal of Zhejiang Forestry Science and Technology, 2017,37(6):99-102.(in Chinese with English abstract)
[2]	吴继来, 潘伟华, 王方, 等. 9种药剂防治山核桃干腐病试验研究[J]. 江苏林业科技, 2018,45(4):25-28.
	WU J L, PAN W H, WANG F, et al. Control efficacy of 9 kinds of pesticides to Chinese hickory stem canker in field[J]. Journal of Jiangsu Forestry Science & Technology, 2018,45(4):25-28.(in Chinese with English abstract)
[3]	周国英, 宋光桃, 李河. 油茶病虫害防治现状及应对措施[J]. 中南林业科技大学学报, 2007,27(6):179-182.
	ZHOU G Y, SONG G T, LI H. Present situation and countermeasures to control Camellia oliefera pest and disease[J]. Journal of Central South University of Forestry & Technology, 2007,27(6):179-182.(in Chinese with English abstract)
[4]	刘凤英. 筛选小麦内生细菌防治小麦纹枯病[D]. 开封: 河南大学, 2008.
	LIU F Y. Selection of wheat endophytic bacteria for biological control of wheat sharp eyespot[D]. Kaifeng: Henan University, 2008.(in Chinese with English abstract)
[5]	宋光桃, 周国英, 罗秋良, 等. 油茶林土壤放线菌的分离及其拮抗油茶炭疽病菌的筛选[J]. 福建林学院学报, 2009,29(4):297-300.
	SONG G T, ZHOU G Y, LUO Q L, et al. Isolation of actinomycetes and screening of their antagonistic strains to Colletotrichum gloeosporioides in soil of Camellia oleifera forest[J]. Journal of Fujian College of Forestry, 2009,29(4):297-300.(in Chinese with English abstract)
[6]	司美茹. 青海高原(西部)土壤放线菌资源及应用研究[D]. 杨凌: 西北农林科技大学, 2003.
	SI M R. A study resources and application of soil actinomycetes from the western part of Qinghai Plateau[D]. Yangling: Northwest A & F University, 2003.(in Chinese with English abstract)
[7]	郭润芳, 刘晓光, 高克祥, 等. 拮抗木霉菌在生物防治中的应用与研究进展[J]. 中国生物防治, 2002,18(4):180-184.
	GUO R F, LIU X G, GAO K X, et al. Progress in biocontrol research with Trichoderma[J]. Chinese Journal of Biological Control, 2002,18(4):180-184.(in Chinese)
[8]	BAKER C J. Inhibitory effect of Bacillus subtilis on Uromyces phaseoli and on development of rust pustules on bean leaves[J]. Phytopathology, 1983,73(8):1148.
[9]	谢栋, 彭憬, 王津红, 等. 枯草芽孢杆菌抗菌蛋白X98Ⅲ的纯化与性质[J]. 微生物学报, 1998,38(1):13-19.
	XIE D, PENG J, WANG J H, et al. Purification and properties of antifungal protein X98Ⅲ from Bacillus subtilis[J]. Acta Microbiologica Sinica, 1998,38(1):13-19.(in Chinese with English abstract)
[10]	权春善, 王军华, 徐洪涛, 等. 一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究[J]. 微生物学报, 2006,46(1):7-12.
	QUAN C S, WANG J H, XU H T, et al. Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity[J]. Acta Microbiologica Sinica, 2006,46(1):7-12.(in Chinese with English abstract)
[11]	邓建良, 刘红彦, 刘玉霞, 等. 解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质鉴定[J]. 植物病理学报, 2010,40(2):202-209.
	DENG J L, LIU H Y, LIU Y X, et al. Identification of the antifungal substances from Bacillus amyloliquefaciens strain YN-1[J]. Acta Phytopathologica Sinica, 2010,40(2):202-209.(in Chinese with English abstract)
[12]	陈志谊, 刘永峰, 刘邮洲, 等. 植物病害生防芽孢杆菌研究进展[J]. 江苏农业学报, 2012,28(5):999-1006.
	CHEN Z Y, LIU Y F, LIU Y Z, et al. Research progress in biocontrol of Bacillus spp. against plant diseases[J]. Jiangsu Journal of Agricultural Sciences, 2012,28(5):999-1006.(in Chinese with English abstract)
[13]	康贻军, 程洁, 梅丽娟, 等. 植物根际促生菌的筛选及鉴定[J]. 微生物学报, 2010,50(7):853-861.
	KANG Y J, CHENG J, MEI L J, et al. Screening and identification of plant growth-promoting rhizobacteria[J]. Acta Microbiologica Sinica, 2010,50(7):853-861.(in Chinese with English abstract)
[14]	陈中义, 张杰, 黄大昉. 植物病害生防芽孢杆菌抗菌机制与遗传改良研究[J]. 植物病理学报, 2003,33(2):97-103.
	CHEN Z Y, ZHANG J, HUANG D F. Research progress on antimicrobial mechanism and genetic engineering of Bacillus for plant diseases biocontrol[J]. Acta Phytopathologica Sinica, 2003,33(2):97-103.(in Chinese with English abstract)
[15]	黎志坤, 朱红惠. 一株番茄青枯病生防菌的鉴定与防病、定殖能力[J]. 微生物学报, 2010,50(3):342-349.
	LI Z K, ZHU H H. Identification, colonization and disease prevention capacity of an antagonistic bacterium against Ralstonia solanacearum[J]. Acta Microbiologica Sinica, 2010,50(3):342-349.(in Chinese with English abstract)
[16]	常文智, 马鸣超, 陈慧君, 等. 胶质类芽胞杆菌对花生生长和土壤微生物学性状的影响[J]. 应用与环境生物学报, 2014,20(2):185-191.
	CHANG W Z, MA M C, CHEN H J, et al. Effects of Paenibacillus mucilaginosus on peanut growth and soil microbiological characteristics[J]. Chinese Journal of Applied and Environmental Biology, 2014,20(2):185-191.(in Chinese with English abstract)
[17]	PANDIN C, LE COQ D, CANETTE A, et al. Should the biofilm mode of life be taken into consideration for microbial biocontrol agents?[J]. Microbial Biotechnology, 2017,10(4):719-734. URL PMID
[18]	ONGENA M, JACQUES P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008,16(3):115-125. URL PMID
[19]	程敏. 山核桃干腐病抑制菌的筛选鉴定及抑制机理的探究[D]. 杭州: 浙江农林大学, 2016.
	CHENG M. Screening and identification of antifungal strains against Carya cathayensis canker and exploration of antagonistic mechanism[D]. Hangzhou: Zhejiang A&F University, 2016.(in Chinese with English abstract)
[20]	魏春燕, 邢永秀, 莫遥, 等. 绿色荧光蛋白基因标记的固氮菌DX120E在甘蔗植株内的定殖[J]. 作物学报, 2014,40(6):1132-1139.
	WEI C Y, XING Y X, MO Y, et al. Colonization of nitrogen fixing bacterial strain Klebsiella sp. DX120E labeled with green fluorescent protein (GFP) gene within sugarcane plants[J]. Acta Agronomica Sinica, 2014,40(6):1132-1139.(in Chinese with English abstract)
[21]	ERRAMPALLI D, LEUNG K, CASSIDY M B, et al. Applications of the green fluorescent protein as a molecular marker in environmental microorganisms[J]. Journal of Microbiological Methods, 1999,35(3):187-199. URL PMID
[22]	MONTEIRO R A, SCHMIDT M A, DE BAURA V A, et al. Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae(Burkholderiales, Oxalobacteraceae)[J]. Genetics and Molecular Biology, 2008,31(4):932-937.
[23]	SARROCCO S, FALASCHI N, VERGARA M, et al. Use of Fusarium oxysporum f. sp. dianthi transformed with marker genes to follow colonization of carnation roots[J]. Journal of Plant Pathology, 2007,89(1):47-54.
[24]	亓雪晨, 齐俊生, 王琦, 等. 枯草芽孢杆菌启动子片段的克隆及其在绿色荧光蛋白标记中的应用[C]// 中国植物病理学会. 中国植物病理学会2006年学术年会论文集, 2006: 557.
	YIN J F, ZHOU X Y, LI J Q, et al. A study on the survival dynamics of Bacillus subtilis in water using green fluorescent protein labeling[J]. Chinese Journal of Analytical Chemistry, 2007,35(10):1405-1409.
[26]	杜芳, 何鹏飞, 吴毅歆, 等. GFP标记内生枯草芽孢杆菌Y10及其在白菜体内的定殖[J]. 生态学杂志, 2015,34(7):2064-2070.
	DU F, HE P F, WU Y X, et al. Colonization of GFP-tagged endophytic Bacillus subtilis Y10 in Chinese cabbage[J]. Chinese Journal of Ecology, 2015,34(7):2064-2070.(in Chinese with English abstract)
[27]	金勤, 朱丹雪, 周国英, 等. 绿色荧光蛋白标记枯草芽孢杆菌Y13^UV在油茶体内的定殖[J]. 林业科学, 2017,53(7):111-117.
	JIN Q, ZHU D X, ZHOU G Y, et al. Colonization of GFP-tagged Bacillus subtilis Y13 ^UV in Camellia oleifera [J]. Scientia Silvae Sinicae, 2017,53(7):111-117.(in Chinese with English abstract)
[28]	TIMMUSK S, GRANTCHAROVA N, WAGNER E G H. Paenibacillus polymyxa invades plant roots and forms biofilms[J]. Applied and Environmental Microbiology, 2005,71(11):7292-7300. URL PMID
[29]	王珍, 曹翠玲, 库永丽, 等. 巨大芽胞杆菌WY4的GFP标记及其在小白菜上的定殖[J]. 农业生物技术学报, 2016,24(12):1925-1934.
	WANG Z, CAO C L, KU Y L, et al. Bacillus megatherium WY₄ labeled by GFP and its colonization in Chinese cabbage (Brassica chinensis)[J]. Journal of Agricultural Biotechnology, 2016,24(12):1925-1934.(in Chinese with English abstract)
[30]	蒋晓玲, 何鹏飞, 王娅玲, 等. 玉米内生细菌Y19的荧光标记及其在玉米体内的定殖应用效果[J]. 玉米科学, 2015,23(3):50-56.
	JIANG X L, HE P F, WANG Y L, et al. GFP-tagging and colonization effect of an endophityic bacterial strain Y19 in maize[J]. Journal of Maize Sciences, 2015,23(3):50-56.(in Chinese with English abstract)
[31]	CAO Y, ZHANG Z H, LING N, et al. Bacillus subtilis SQR 9 can control Fusarium wilt in cucumber by colonizing plant roots[J]. Biology and Fertility of Soils, 2011,47(5):495-506.
[32]	ZHANG N, WU K, HE X, et al. A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N₁₁[J]. Plant and Soil, 2011,344(1/2):87-97.
[33]	ANDREOGLOU F I, VAGELAS I K, WOOD M, et al. Influence of temperature on the motility of Pseudomonas oryzihabitans and control of Globodera rostochiensis[J]. Soil Biology and Biochemistry, 2003,35(8):1095-1101.
[34]	田涛, 亓雪晨, 王琦, 等. 芽孢杆菌绿色荧光蛋白标记及其在小麦体表定殖的初探[J]. 植物病理学报, 2004,34(4):346-351.
	TIAN T, QI X C, WANG Q, et al. Colonization study of GFP-tagged Bacillus strains on wheat surface[J]. Acta Phytopathologica Sinica, 2004,34(4):346-351.(in Chinese with English abstract)
[35]	洪鹏, 安国栋, 胡美英, 等. 解淀粉芽孢杆菌HF-01发酵条件优化[J]. 中国生物防治学报, 2013,29(4):569-578.
	HONG P, AN G D, HU M Y, et al. Optimizing fermentation condition for Bacillus amyloliquefaciens HF-01[J]. Chinese Journal of Biological Control, 2013,29(4):569-578.(in Chinese with English abstract)

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

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