拮抗细菌KRS022的鉴定及对大丽轮枝菌的抑制效果

doi:10.3864/j.issn.0578-1752.2023.04.005

Abstract

Abstract:

【Objective】To clarify the taxonomic status of antagonistic bacterium KRS022 and its inhibitory effect on a variety of pathogenic fungi, and to focus on the control efficacy on Verticillium dahliae, and this study will provide important resources for the research and development of biocontrol preparations against V. dahliae. 【Method】The classification of KRS022 was determined by morphological, physiological and biochemical tests, and 16S rDNA-gyrB gene sequence tandem analysis. The inhibitory effects of KRS022 on various fungi were tested by confrontation and fumigation methods. The effect of KRS022 on the morphology of V. dahliae hyphae and conidia was determined by co-culture of cell-free supernatant and fumigation. The control efficacy of KRS022 on Verticillium wilt of cotton and tobacco was determined by pot experiment and biomass measurement. The RT-qPCR method was used to detect the expression levels of plant resistance-related genes. 【Result】The strain KRS022 was gram-negative bacterium, and it was identified as Pseudomonas alcaligenes. KRS022 has the characteristics of phosphorus and potassium solubilization, nitrogen fixation and siderophore production, and the function of protease, amylase and catalase activities. KRS022 belongs to aerobic alkali-producing bacterium, and 100 μg·mL^-1 ampicillin can be used as the screening condition for natural resistance of this strain. KRS022 showed different degrees of antifungal activity against a variety of pathogenic fungi (such as Verticillium dahliae, Fusarium oxysporum, Fusarium graminearum, Magnaporthe oryzae, Botrytis cinerea, Colletotrichum gloeosprioides, Colletotrichum fructicola). The inhibitory rates of confrontation and fumigation against V. dahliae were 75.19% and 99.78%, respectively. The inhibition rates of PDA plates with fermentation liquid, spreading plates and fumigation against Vd991 were 96.21%, 99.72% and 99.44%, respectively. The co-culture of KRS022 cell-free supernatant with conidia of Vd991 completely inhibited spore germination. The hyphae of Vd991 were enlarged and thickened after fumigation treatment of KRS022. The results of pot experiment showed that KRS022 could significantly inhibit the occurrence of Verticillium wilt of cotton and tobacco. After treatment of KRS022 bacterial solution, the biomass of V. dahliae was significantly reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 1.75 and 2.57 folds higher than that in the treatment group (KRS022+Vd991), respectively. Moreover, KRS022 could also promote plants growth, and the leaf dimension and single leaf area of the tobacco treated with KRS022 fermentation broth increased by 65.7% and 146.4%, respectively. At the same time, KRS022 could stimulate the plants resistance-related genes expression. Cotton treated with KRS022 fermentation broth, the salicylic acid (SA) pathway marker gene GhPR1, jasmonic acid (JA) pathway marker gene GhAOC4 and ethylene (ET) pathway marker gene GhEIN2 were significantly up-regulated, the up-regulation folds were 7.23, 1.69 and 15.05 that of water treatment, respectively. Cotton treated with KRS022 fermentation broth and inoculated with V. dahliae, the expression of GhAOC4 and GhEIN2 was significantly induced, and the up-regulation folds were 68.09 and 11.87 that of cotton plants inoculated with V. dahliae, respectively. The up-regulated expression folds of NbHSR203, NbHIN1, NbPR1, NbPR2, NbPR5, NbRbohA and NbRbohB after KRS022 cell-free supernatant injection were 1.98, 2.79, 2.52, 1.25, 1.70, 3.28 and 3.44 that of LB treatment, respectively. 【Conclusion】The antagonistic bacterium KRS022 was identified as P. alcaligenes. It has the characteristics of siderophore production, phosphorus and potassium solubilization, and nitrogen fixation, which suggested KRS022 may have the function of plant growth promotion. It can inhibit the conidia germination and hyphae growth of V. dahliae. In addition, KRS022 can protect plants from V. dahliae infection and trigger plant immunity response. Together, KRS022 is a potential biocontrol microbial resource with development prospect for the prevention and control of Verticillium wilt and other fungal diseases.

Key words: Pseudomonas alcaligenes, Verticillium wilt, Verticillium dahliae, biocontrol, plant immunity

LUO WanZhen, WANG Dan, QI HongYue, WANG Tong, LIU Zheng, TIAN Li, DAI XiaoFeng, CHEN JinYin, MIJITI Maihemuti. Identification of Antagonistic Bacterium Strain KRS022 and Its Inhibition Effect on Verticillium dahliae[J].Scientia Agricultura Sinica, 2023, 56(4): 649-664.

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基因Gene	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	参考文献Reference
GhUB7	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG	[26]
GhPR1	ACCTCAACGCTCACAACACA	GGTCCACTGGAGTGCACAAG	[26]
GhEIN2	TTTTGATCTGGTAGCCCCC	CAATATGAAACCTGCCGCAT	[26]
GhAOC4	GGCATCACGGCTGGACTCT	GCGATGGTGGCACTGGC	[26]
NbEF-1α	TGAGTTCGAGGCTGGTATCT	CACTTGGTGGTGTCCATCTT	[25]
NbHSR203	GGCAGTGGAGGAGCTTAAAT	GCTATGTCCCACTCCATTGTTA	[25]
NbHIN1	ATCCTCGGAGTGATTGCATTAG	TGTTGTTTGTGGTGGACAAATC	[25]
NbPR1	CCGCCTTCCCTCAACTCAAC	GCACAACCAAGACGTACTGAG	[25]
NbPR2	AGGTGTTTGCTATGGAATGC	TCTGTACCCACCATCTTGC	[25]
NbPR5	GGGCCAATCTTGGAGCATTA	CAGTCTCCAGTCTCACAATTACC	[27]
NbRbohA	CTTGCTCGTCAACATCGTG	GGAGAAATCTTGTTGAGAGC	[28]
NbRbohB	TAATACAAGGAGGGCATATT	GCAGAACGAGCATCACCT	[28]

特性 Characteristics	KRS022	产碱假单胞菌 P. alcaligenes	特性 Characteristics	KRS022	产碱假单胞菌 P. alcaligenes
铁载体Siderophore	+	+	厌氧试验Anaerobic test	-	-
解磷Phosphorus solubilization	+	+	过氧化氢酶活性 Catalase activity	+	+
解钾Potassium solubilization	+	+	葡萄糖酵解试验 Glycolysis test	-	-
固氮Nitrogen-fixing	+	+	卡那霉素^aKanamycin	-	-
明胶水解Gelatin hydrolysis	+	+	氨苄青霉素^aAmpicillin	+	+
蛋白酶活性Protease activity	+	+	头孢霉素^a Cephalosporin	-	-
淀粉酶活性Amylase activity	+	+	氯霉素^aChloramphenicol	-	-
甲基红试验MR test	-	-	利福平^aRifampicin	-	-
柠檬酸盐利用Citrate utilization	+	+	血平板试验CAMP test	+	+
吲哚试验Indole test	-	-

Identification of Antagonistic Bacterium Strain KRS022 and Its Inhibition Effect on Verticillium dahliae

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