Isolation and Identification of a Bacillus safensis Strain GX-H6 and Its Biocontrol Effect on Bacterial Leaf Streak of Rice

doi:10.13560/j.cnki.biotech.bull.1985.2022-1153

Abstract

Abstract:

Bacterial leaf streak（BLS）, caused by Xanthomonas oryzae pv. oryzicola（Xoc）, is emerging as a serious disease in rice fields of southern China. In order to screen biocontrol bacteria for controlling BLS, we had Xoc wild-type strain GX01 as indicator, a bacterium with antagonistic effect to Xoc was isolated from the samples of peanut rhizosphere soil by using bacterial plate dilution and Oxford cup methods, and named as GX-H6. Based on the morphological, physiological and biochemical data, 16S rDNA and evolutionary tree analysis, this strain was identified to be Bacillus safensis. Antagonistic experiments revealed that the B. safensis GX-H6 strain effectively repressed the growths of several xanthomonads and plant pathogenic fungi, particularly the bacterium Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight（BB）of rice. Further greenhouse and field rice plant experiments showed that this strain effectively controlled BLS and BB of rice. Genome analysis indicated that the GX-H6 strain contained a variety of genes related to antifungal, environmental competition, and the production of lichenysin, plantazolicin and bacilysin. Overall, the B. safensis strain GX-H6 is a potential novel biocontrol agent for developing pesticides and exploring novel bacteriostatic mechanism in future.

Key words: Xanthomonas oryzae pv. oryzicola, xanthomonads, Bacillus safensis, antagonistic ability, biocontrol

REN Pei-dong, PENG Jian-ling, LIU Sheng-hang, YAO Zi-ting, ZHU Gui-ning, LU Guang-tao, LI Rui-fang. Isolation and Identification of a Bacillus safensis Strain GX-H6 and Its Biocontrol Effect on Bacterial Leaf Streak of Rice[J]. Biotechnology Bulletin, 2023, 39(5): 243-253.

Figures/Tables 10

Fig. 1 Morphological characteristics and antagonistic effect of GX-H6 A: Antibacterial effect of GX-H6 on Xoc GX01. B: Colonies morphology of strain GX-H6. C: Microscopic observation of strain GX-H6

Fig. 2 Phylogenetic tree of tested strains and other Bacillus based on 16S rDNA gene and gyrB gene sequence The scale indicates 0.1% sequence difference

Fig. 3 Antagonistic spectrum of GX-H6 against bacteria A: Antagonistic spectrum of GX-H6 against different Xoc strains（1: 3-1 isolate; 2: 4-1 isolate; 3: 5-1 isolate; 4: 6 isolate; 5: 6-2 isolate; 6: 6-3 isolate; 7: 7-1 isolate; 8: 8-1 isolate; 9: 8-2 isolate; 10: 2015 isolate）. B: Antagonistic spectrum of GX-H6 against different Xoo strains and other plant pathogens（1: Xoo PXO99A strain; 2: Xoo K74 strain; 3: Xoo 1-1 isolate; 4: Xoo 1-2 isolate; 5: Xoo 1-3 isolate; 6: Xcc 8004 strain; 7: Xac 9 isolate; 8: Xac 12 isolate; 9: A.avenae subsp. avenae Pas-1 strain; 10: P.syringae pv. tomato DC3000 strain; 11：P.hibiscicola Y2 isolate; 12：P.hibiscicola Y3 isolate）. The data is average diameter± standard deviation in the Figure, and different letters indicate significant differences at the P < 0.05

Fig. 4 Antagonistic spectrum of GX-H6 against six plant pathogenic fungi strains A: Qualitative analysis via plate confrontation method. B: Quantitative analysis via bacterial plate method. The data in the Figure are average diameter± standard deviation, and different letters indicate significant differences at P < 0.05. 1: N. novaehollandiae. 2: C. plurivorum. 3: B. cinere. 4: F. oxysporum f. sp. cubense, Foc. 5: C. gloeosporioides. 6: M. oryzae

Fig. 5 Biocontrol effect of GX-H6 on Xoc in greenhouse plants * indicates significant（P<0.05）

Fig. 6 Biocontrol effect of GX-H6 strain on Xoo in green-house plants * indicates significant（P<0.05）, and ** indicates significant（P<0.01）

Table 1 Experimental results of GX-H6 biocontrol agent against bacterial leaf streak of rice

药剂处理 Treatment	用量 Dose/（L·hm^-2）	药前病情指数 Pre-treament disease index	第3次药后10 d病情指数 Post-treatment disease index	防效 Relative control effect/%	差异显著性 Significance
GX-H6生防菌剂	7.5	0.6	6.8	65.0	bC
	11.25	0.6	6.1	68.6	bBC
	22.5	0.7	5.2	72.8	aAB
20%噻菌铜悬浮剂	1.875	0.7	4.6	76.0	aA
清水对照（CK）		0.7	19.2	─	─

Table 2 Genomic characteristics of B. safensis GX-H6

特征 Characteristics	值 Value
Toal length	3 763 758 bp
GC content	41.51%
ORF number	3 754
ORF total length	3 301 671 bp
Longest ORF length	10 707 bp
Shortest ORF length	90 bp
Average ORF length	879 bp
ORF/Genome（coding percentage）	87.72%

Fig. 7 Genomic characteristics of B. safensis GX-H6 A：The genomic circle map of B. safensis GX-H6. B：GOSlim illustration of B. safensis GX-H6；C：KEGG chart of B. safensis GX-H6

Table 3 Prediction of secondary metabolites in B. safensis GX-H6 genome

区域Region	起始Start	终止End	最似产物Most similar	类型Type	相似度Similarity
1	693 587	724 627	N/A	Betalactone	N/A
2	1 190 336	1 231 436	N/A	T3PKS	N/A
3	1 271 177	1 291 720	N/A	Terpene	N/A
4	1 357 616	1 386 025	Fengycin	Betalactone	53%
5	1 575 524	1 597 704	Plantazolicin	LAP,RRE-containing	91%
6	2 112 286	2 140 932	Carotenoid	Terpene, siderophore	50%
7	2 287 110	2 308 015	N/A	RRE-containing	N/A
8	2 781 557	2 863 632	Lichenysin	NRPS	92%
9	3 002 623	3 012 922	N/A	RiPP-like	N/A
10	3 231 420	3 278 568	Bacillibactin	NRPS	53%
11	3 545 166	3 586 587	Bacilysin	Other	85%

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