Isolation and Identification of Pseudomonas koreensis ZF518 and Its Biocontrol Effect on Cucumber Leaf Spot

doi:10.16420/j.issn.0513-353x.2024-0985

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (12): 3346-3362.doi: 10.16420/j.issn.0513-353x.2024-0985

• Plant Protection • Previous Articles Next Articles

Isolation and Identification of Pseudomonas koreensis ZF518 and Its Biocontrol Effect on Cucumber Leaf Spot

DING Changzong¹, LI Lei¹, JIANG Zhenqun¹, SHEN Yuhe¹, GAO Suqin¹, DEJI Yangzong², SHI Yanxia¹, CHAI Ali¹, FAN Tengfei¹, SUN Xianhua¹, LI Baoju¹^,^**(), XIE Xuewen¹^,^**()

¹ State Key Laboratory of Vegetable Biobreeding，Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China
² Institute of Olericulture，Xizang Academy of Agriculture and Animal Husbandry Sciences，Lhasa 850030，China

Received:2025-09-29 Revised:2025-11-04 Online:2025-12-25 Published:2025-12-20
Contact: LI Baoju, XIE Xuewen

Abstract

Abstract:

To obtain biocontrol Pseudomonas strains with high efficacy against cucumber Corynespora leaf spot，a total of 557 fluorescent strains were isolated from 56 cucumber rhizosphere soil samples collected across China. This was achieved through low-temperature enrichment and selective medium screening，with fluorescence detected under 365 nm UV light. Using the plate confrontation assay，strains antagonistic against Corynespora cassiicola were selected. Through in vivo pot experiments，one strain，designated ZF518，was identified as providing stable and effective control of cucumber Corynespora leaf spot，with an inhibition rate of 65.82% in vitro and a control efficacy of 57.08% in planta. Based on a polyphasic taxonomic approach including morphology，physiological-biochemical characteristics，and multi-gene phylogenetic analysis，strain ZF518 was identified as Pseudomonas koreensis. This strain exhibited direct inhibitory effects against various phytopathogenic fungi in vitro and was capable of producing siderophores. These findings indicate that P. koreensis ZF518 is a promising biocontrol resource for managing cucumber Corynespora leaf spot.

Key words: cucumber, Corynespora cassiicola, Pseudomonas, biocontrol agent identification, Biological control, physiological and biochemical characteristics, phylogenetic evolution

DING Changzong, LI Lei, JIANG Zhenqun, SHEN Yuhe, GAO Suqin, DEJI Yangzong, SHI Yanxia, CHAI Ali, FAN Tengfei, SUN Xianhua, LI Baoju, XIE Xuewen. Isolation and Identification of Pseudomonas koreensis ZF518 and Its Biocontrol Effect on Cucumber Leaf Spot[J]. Acta Horticulturae Sinica, 2025, 52(12): 3346-3362.

Figures/Tables 15

References 50

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基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	扩增长度/bp Amplicon length	来源 Source
16S rDNA	27F	GGCGACATGGTCAACGG	1 465	Dutta et al.，2022
	1492R	CGGCTGGCGGCGTATAT	1 465	Dutta et al.，2022
rpoD	rpoD-F	CACGGTTGAGCACATCCTCT	1 105	Dutta et al.，2022
	rpoD-R	GGAGAGTACTTCGCGAGTCG	1 105	Dutta et al.，2022
rpoB	rpoB-F	GTCCGACTTCACTGTGAGCA	1 126	Dutta et al.，2022
	rpoB-R	GTGAAAAAGCCAGCGACGTT	1 126	Dutta et al.，2022
gyrB	gyrB-F	GACAAGCTGGTGTCTTCCGA	1 320	Dutta et al.，2022
	gyrB-R	CATCTCGCCCAGACCTTTGT	1 320	Dutta et al.，2022

基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	扩增长度/bp Amplicon length	来源 Source
16S rDNA	27F	GGCGACATGGTCAACGG	1 465	Dutta et al.，2022
	1492R	CGGCTGGCGGCGTATAT	1 465	Dutta et al.，2022
rpoD	rpoD-F	CACGGTTGAGCACATCCTCT	1 105	Dutta et al.，2022
	rpoD-R	GGAGAGTACTTCGCGAGTCG	1 105	Dutta et al.，2022
rpoB	rpoB-F	GTCCGACTTCACTGTGAGCA	1 126	Dutta et al.，2022
	rpoB-R	GTGAAAAAGCCAGCGACGTT	1 126	Dutta et al.，2022
gyrB	gyrB-F	GACAAGCTGGTGTCTTCCGA	1 320	Dutta et al.，2022
	gyrB-R	CATCTCGCCCAGACCTTTGT	1 320	Dutta et al.，2022

基因 Gene	引物序列（5′-3′） Primer sequence	扩增长度/bp Amplicon length	来源 Source
吩嗪Phenazine（phzCD）	TGCCAAGCCTCGCTCCAAC	1 400	Zhang et al.，2006
吩嗪Phenazine（phzCD）	CCGCGTTGTTCCTCGTTCAT	1 400	Zhang et al.，2006
硝吡咯菌素Pyrrolnitrin（prnC）	CCACAAGCCCGGCCAGGAGC	720	Mavrodi et al.，2001
硝吡咯菌素Pyrrolnitrin（prnC）	GAGAAGAGCGGGTCGATGAAGCC	720	Mavrodi et al.，2001
2-羟基吩嗪 2-hydroxylated（phzO）	AAGTCCAGATGCGAAAGAACG	1 400	Delaney et al.，2001
2-羟基吩嗪 2-hydroxylated（phzO）	GTGAAAAAGCCAGCGACGTT	1 400	Delaney et al.，2001
2,4-二乙酰基间苯三酚 2,4-DAPG（phlD）	GAGGACGTCGAAGACCACCA	745	Mavrodi et al.，2001
2,4-二乙酰基间苯三酚 2,4-DAPG（phlD）	AAGTGGCATGGCTCGAACAAAG	745	Mavrodi et al.，2001
藤黄绿脓菌素 Pyoluteorin（pltB）	CGGAGCATGGACCCCCAGC	900	Mavrodi et al.，2001
藤黄绿脓菌素 Pyoluteorin（pltB）	GTGCCCGATATTGGTCTTGACC	900	Mavrodi et al.，2001

基因 Gene	引物序列（5′-3′） Primer sequence	扩增长度/bp Amplicon length	来源 Source
吩嗪Phenazine（phzCD）	TGCCAAGCCTCGCTCCAAC	1 400	Zhang et al.，2006
吩嗪Phenazine（phzCD）	CCGCGTTGTTCCTCGTTCAT	1 400	Zhang et al.，2006
硝吡咯菌素Pyrrolnitrin（prnC）	CCACAAGCCCGGCCAGGAGC	720	Mavrodi et al.，2001
硝吡咯菌素Pyrrolnitrin（prnC）	GAGAAGAGCGGGTCGATGAAGCC	720	Mavrodi et al.，2001
2-羟基吩嗪 2-hydroxylated（phzO）	AAGTCCAGATGCGAAAGAACG	1 400	Delaney et al.，2001
2-羟基吩嗪 2-hydroxylated（phzO）	GTGAAAAAGCCAGCGACGTT	1 400	Delaney et al.，2001
2,4-二乙酰基间苯三酚 2,4-DAPG（phlD）	GAGGACGTCGAAGACCACCA	745	Mavrodi et al.，2001
2,4-二乙酰基间苯三酚 2,4-DAPG（phlD）	AAGTGGCATGGCTCGAACAAAG	745	Mavrodi et al.，2001
藤黄绿脓菌素 Pyoluteorin（pltB）	CGGAGCATGGACCCCCAGC	900	Mavrodi et al.，2001
藤黄绿脓菌素 Pyoluteorin（pltB）	GTGCCCGATATTGGTCTTGACC	900	Mavrodi et al.，2001

处理 Treatment	抑菌直径/cm Antibacterial diameter	抑菌率/% Inhibition rate	病情指数 Disease index	防治效果/% Control efficiency
ZF518	5.20 ± 0.10 a	65.82 ± 1.27 a	27.92 ± 0.79 f	57.08 ± 1.61 b
ZF549	4.23 ± 0.15 c	53.59 ± 1.93 c	48.92 ± 0.70 e	41.01 ± 0.84 c
ZF557	4.40 ± 0.10 bc	55.70 ± 1.27 bc	58.84 ± 2.35 cd	29.04 ± 2.84 d
ZF561	4.50 ± 0.10 bc	56.96 ± 1.27 bc	62.12 ± 3.71 bc	25.08 ± 4.47 d
ZF577	4.70 ± 0.10 b	59.49 ± 1.27 b	63.15 ± 3.70 bc	23.84 ± 4.46 d
ZF582	4.60 ± 0.10 b	58.23 ± 1.27 b	69.83 ± 2.96 b	15.79 ± 3.57 e
ZF589	4.27 ± 0.20 c	54.01 ± 4.79 c	59.92 ± 3.10 cd	27.73 ± 3.74 d
ZF593	5.07 ± 0.06 a	64.14 ± 0.73 a	8.92 ± 2.11 e	41.01 ± 2.54 c
ZF612	4.50 ± 0.10 bc	56.96 ± 1.27 bc	52.19 ± 2.06 de	37.05 ± 2.49 e
75%百菌清WP 75% Chlorothalonil WP	—	—	27.92 ± 0.79 f	66.32 ± 0.95 a
病原菌对照 Pathogen control	—	—	82.92 ± 7.34 a	—

Isolation and Identification of Pseudomonas koreensis ZF518 and Its Biocontrol Effect on Cucumber Leaf Spot

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生理特性 Characteristic	ZF518	模式菌株 LMG 21318^T	生化特性 Characteristic	ZF518	模式菌株 LMG 21318^T
荧光 Fluorescence	+	+	己二酸 Adipic acid	-	-
氧化酶 Oxidase	-	-	精氨酸	+	+
硝酸盐还原 Nitrate reduction	-	-	D-葡萄糖 Arginine	+	+
精氨酸双水解酶Arginine dihydrolase	+	+	D-甘露醇 D-Glucose	+	+
吐温80水解 Tween 80 hydrolysis	+	+	D-甘露糖 D-Mannitol	+	+
果聚糖Fructan	-	-	L-阿拉伯糖 L-Arabinose	+	+
4 ℃下生长 Growth at 4 ℃			麦芽糖 Maltose	-	-
41 ℃下生长 Growth at 41 ℃	-	-	色氨酸 Tryptophan	-	-
淀粉水解 Starch hydrolysis	+	+	柠檬酸盐 Citrate	+	+

抑菌方式 Antibacterial method	病原真菌 Pathogenic fungi	抑菌直径/cm Antibacterial diameter	抑菌率/% Inhibition rate
平板对峙抑菌谱 Plate standoff antibacterial spectrum	立枯丝核菌 Rhizoctonia solani	6.45 ± 0.24 a	75.88 ± 0.24 a
	辣椒疫霉菌Phytophthora capsici	4.17 ± 0.38 bc	67.72 ± 2.01 b
	多主棒孢菌 Corynespora cassiicola	4.18 ± 0.32 bc	56.23 ± 1.82 c
	大丽轮枝菌Verticillium dahliae	4.38 ± 0.06 b	55.04 ± 0.40 c
	茄病镰孢菌 Fusarium solani	3.68 ± 0.10 cd	48.57 ± 0.70 d
	尖孢镰孢菌 Fusarium oxysporum	3.30 ± 0.45 d	44.01 ± 3.41 e
	茄匍柄霉 Stemphylium solani	2.67 ± 0.21 e	42.18 ± 1.88 e
	灰葡萄孢菌Botrytis cinerea	0.42 ± 0.00 f	4.94 ± 0.00 f
挥发性物质抑菌谱 Antibacterial spectrum of volatile substances	辣椒疫霉菌 Phytophthora capsici	2.47 ± 0.05 a	71.18 ± 2.03 a
	瓜类炭疽菌 Colletrichum orbiculare	0.63 ± 0.05 bc	14.86 ± 1.28 bc
	茄匍柄霉 Stemphylium solani	0.57 ± 0.42 bc	11.85 ± 8.57 bc
	瓜疮痂枝孢霉 Cladosporium cucumerinum	0.33 ± 0.05 c	7.41 ± 1.05 bc
	多主棒孢菌 Corynespora cassiicola	0.27 ± 0.09 c	6.06 ± 2.00 bc
	立枯丝核菌 Rhizoctonia solani	1.20 ± 0.80 b	16.61 ± 12.00 b
	尖孢镰孢菌 Fusarium oxysporum	0.07 ± 0.05 c	1.37 ± 0.99 c
	茄病镰孢菌 Fusarium solani	0.03 ± 0.37 c	0.60 ± 8.34 c

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处理 Treatment	平板抑菌效果 Antibacterial activity		盆栽防病效果 Disease control efficacy in pot experiment
处理 Treatment	抑菌直径/cm Antibacterial diameter	抑菌率/% Inhibition rate	病情指数 Disease index	防治效果/% Control efficiency
菌株发酵液 Fermentation broth	5.65 ± 0.08 a	67.26 ± 0.97 a	36.58 ± 1.44 b	51.65 ± 1.91 b
无菌上清液 Cell-free supernatant	0.07 ± 0.08 b	0.79 ± 1.01 b	73.15 ± 7.42 a	2.81 ± 9.86 a
菌体重悬液 Cell resuspension	5.58 ± 0.12 a	66.47 ± 1.40 a	35.64 ± 1.65 b	52.65 ± 2.20 b
75%百菌清 WP 75% Chlorothalonil WP	—	—	33.85 ± 2.22 b	55.02 ± 2.95 a
清水对照 Water control	—	—	75.27 ± 3.90 a	—
处理 Treatment	抑制孢子萌发效果 Inhibition of spore germination
处理 Treatment	孢子萌发率/% Spore germination rate	抑制率/% Inhibition rate
菌株发酵液 Fermentation broth	3.92 ± 0.80 b	92.50 ± 1.53 b
无菌上清液 Cell-Free supernatant	52.76 ± 0.79 a	-0.91 ± 1.51 a
菌体重悬液 Cell resuspension	4.49 ± 1.97 b	91.41 ± 3.76 b
75%百菌清 WP 75% Chlorothalonil WP	—	—
清水对照 Water control	52.28 ± 3.47 a	—

处理 Treatment	平板抑菌效果 Antibacterial activity		盆栽防病效果 Disease control efficacy in pot experiment
处理 Treatment	抑菌直径/cm Antibacterial diameter	抑菌率/% Inhibition rate	病情指数 Disease index	防治效果/% Control efficiency
菌株发酵液 Fermentation broth	5.65 ± 0.08 a	67.26 ± 0.97 a	36.58 ± 1.44 b	51.65 ± 1.91 b
无菌上清液 Cell-free supernatant	0.07 ± 0.08 b	0.79 ± 1.01 b	73.15 ± 7.42 a	2.81 ± 9.86 a
菌体重悬液 Cell resuspension	5.58 ± 0.12 a	66.47 ± 1.40 a	35.64 ± 1.65 b	52.65 ± 2.20 b
75%百菌清 WP 75% Chlorothalonil WP	—	—	33.85 ± 2.22 b	55.02 ± 2.95 a
清水对照 Water control	—	—	75.27 ± 3.90 a	—
处理 Treatment	抑制孢子萌发效果 Inhibition of spore germination
处理 Treatment	孢子萌发率/% Spore germination rate	抑制率/% Inhibition rate
菌株发酵液 Fermentation broth	3.92 ± 0.80 b	92.50 ± 1.53 b
无菌上清液 Cell-Free supernatant	52.76 ± 0.79 a	-0.91 ± 1.51 a
菌体重悬液 Cell resuspension	4.49 ± 1.97 b	91.41 ± 3.76 b
75%百菌清 WP 75% Chlorothalonil WP	—	—
清水对照 Water control	52.28 ± 3.47 a	—