棉花内生细菌YUPP-10及其分泌蛋白CGTase对棉花枯萎病的防治作用及机理

doi:10.3864/j.issn.0578-1752.2021.17.011

Abstract

Abstract:

【Objective】 Fusarium wilt is one of the most important soil-borne diseases in cotton planting. Chemical methods are mainly used to control this disease, however, it has certain impact on the environment, human and animal safety. Biological control has become an important way to control cotton Fusarium wilt because of its high specificity and safety. The objective of this study is to screen an efficient antagonistic bacteria, and characterize the biocontrol mechanism of bacteria against Fusarium wilt, thus providing a technical basis for cotton Fusarium wilt control with biocontrol bacteria. 【Method】 In a previous study, an endophytic bacterium Bacillus cereus YUPP-10 was isolated from vascular of cotton, which can hydrolyze polysaccharides with β-1,4 linkage. The effects of YUPP-10 on hyphal growth, sporulation, and spore germination of F. oxysporum were tested using the confront culture method, enclosed chamber test and hanging drop method, respectively. The seeds were soaked by YUPP-10, and then, the seeding germination and the biomass of cotton were detected. The cotton were cultivated in substrate with F. oxysporum, and after a week of growth, YUPP-10 cultured with LB liquid medium was treated at different concentrations (1×10 ⁸, 1×10⁷and 1×10⁶ cfu/mL, respectively), and the control efficacy against Fusarium wilt was studied in the greenhouse. The key antibacterial substances of YUPP-10 were obtained by Fosmid library, and the direct effects of recombinant cyclodextrin glycosyltransferase (CGTase) on F. oxysporum hyphal growth, sporulation, and spore germination were studied. The overexpression vector was transformed into Arabidopsis thaliana Col-0 via the floral dip method. The resistance of CGTase-overexpressing transgenic plants against Fusarium wilt was assessed with an in vitro technique. The transcriptional levels of some defense genes were analyzed under pathogen challenge. 【Result】 YUPP-10 significantly inhibited the hyphal growth, sporulation, and spore germination of F. oxysporum, the most inhibition rate of spore yield and germination was 98.41% and 51.65%, respectively. Low concentration of YUPP-10 could promote the germination rate, emergence rate and stem length of cotton seeds. After the treatment of YUPP-10, the diseased plant rate and disease index were significantly lower than those of the control group. The control efficacy was 45.11% at the concentration of 1×10 ⁷ cfu/mL. CGTase was the key antimicrobial substance of YUPP-10, the effects of added CGTase on the transparent circles of carboxymethyl cellulose and glucomannan were measured, the results showed that CGTase could hydrolyze polysaccharides with β-1,4 linkage. CGTase also had significant inhibitory effects on the growth, sporulation and spore germination of the pathogen, the most inhibition rate of spore yield and germination was 62.63% and 30.83%, respectively. The CGTase-overexpressing A. thaliana enhanced disease resistance by enhancing the expression of defense genes. 【Conclusion】 YUPP-10 is an efficient biocontrol agent that inhibits the F. oxysporum growth, promots germination rate, emergence rate and stem length of cotton seeds, and protects cotton plant from F. oxysporum infection. CGTase can inhibit the growth of F. oxysporum, and its transgenic A. thaliana enhances the resistance to Fusarium wilt.

Key words: Bacillus cereus, cotton Fusarium wilt, Fusarium oxysporum f. sp. vasinfectum, cyclodextrin glycosyltransferase (CGTase), control efficacy

ZHOU JingLong,FENG ZiLi,WEI Feng,ZHAO LiHong,ZHANG YaLin,ZHOU Yi,FENG HongJie,ZHU HeQin. Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium YUPP-10 and Its Secretory Protein CGTase Against Fusarium Wilt in Cotton[J].Scientia Agricultura Sinica, 2021, 54(17): 3691-3701.

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References 36

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处理 Treatment	产孢 Sporulation		孢子萌发Spore germination
处理 Treatment	产孢量Spore yield (cfu/mL)	抑制率Inhibition rate (%)	萌发率Germination rate (%)	抑制率Inhibition rate (%)
CK	33.40±1.61a	-	52.87±4.33a	-
1×YUPP-10	0.53±0.19c	98.41	25.56±4.45d	51.65
1/2×YUPP-10	15.20±1.79b	54.49	34.20±3.49c	35.30
1/4×YUPP-10	14.53±0.80b	56.49	39.78±3.42b	27.75

处理 Treatment	发芽率 Germination rate (%)	芽长 Shoot length (cm)
1×YUPP-10	39.08±0.34d	0.23±0.01b
1/2×YUPP-10	81.91±0.88a	0.84±0.32a
1/4×YUPP-10	75.84±0.98b	0.89±0.25a
CK	72.60±1.01c	0.49±0.10ab

处理 Treatment	检测指标 Detection index
处理 Treatment	出苗率Emergence rate (%)	根长Root length (cm)	茎长Stem length (cm)	总鲜重Fresh weight (g)
CK	84.36±0.04b	8.76±0.71a	13.09±0.38b	0.66±0.46a
YUPP-10	94.87±0.04a	10.26±0.66a	14.26±0.39a	0.76±0.49a

处理Treatment	病情指数Disease index	病株率Diseased plant rate (%)	防治效果Control efficacy (%)
1×10⁸	28.23±4.95b	43.04±14.80b	42.79
1×10⁷	27.08±9.54b	42.92±13.54b	45.11
1×10⁶	32.53±3.65ab	56.25±6.05ab	34.06
CK	49.34±9.08a	71.14±13.98a

蛋白浓度 Protein concentration (mg·mL^-1)	产孢 Sporulation		孢子萌发Spore germination
蛋白浓度 Protein concentration (mg·mL^-1)	产孢量 Spore yield (cfu/mL)	抑制率 Inhibition rate (%)	萌发率 Germination rate (%)	抑制率 Inhibition rate (%)
0	35.40±1.51a	-	42.62±2.13a	-
0.12	22.30±0.17b	37.01	38.06±3.35b	10.70
0.24	14.32±1.09c	59.55	32.25±2.19c	24.33
0.48	13.23±0.83c	62.63	29.48±1.62d	30.83

Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium YUPP-10 and Its Secretory Protein CGTase Against Fusarium Wilt in Cotton

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