Antagonistic Activity of Volatile Organic Compounds Produced by Acid-Tolerant Pseudomonas protegens CLP-6 as Biological Fumigants To Control Tobacco Bacterial Wilt Caused by Ralstonia solanacearum

Abstract

Tobacco bacterial wilt, which is caused by Ralstonia solanacearum, is a devastating soilborne disease of tobacco worldwide and is widespread in the continuously acidic fields of southern China. Here, the fumigation activity under different pH conditions, component identification, and bioactivity of the volatile organic compounds (VOCs) produced by an acid-tolerant strain, Pseudomonas protegens CLP-6, were investigated. There was a wide antimicrobial spectrum of the VOCs against phytopathogens, including four bacteria, eight fungi, and two oomycetes. The antagonistic activity of the VOCs against R. solanacearum was proportionally correlated with the concentration of the inoculum, amount, culture time, and culture pH for CLP-6. The number of gene copies of R. solanacearum was significantly inhibited by VOCs produced at pH 5.5 in vivo. The control effect of VOCs emitted at pH 5.5 was 78.91% for tobacco bacterial wilt, which was >3-fold greater than that at pH 7.0. Finally, the main volatile compounds were identified by solid-phase microextraction (SPME)-gas chromatography-mass spectroscopy (GC-MS) as S-methyl thioacetate, methyl thiocyanate, methyl disulfide, 1-decene, 2-ethylhexanol, 1,4-undecadiene, 1-undecene, 1,3-benzothiazole, and 2,5-dimethylpyrazine, and the inhibition rates of 1,3-benzothiazole, 2-ethylhexanolmethyl thiocyanate, dimethyl disulfide, and S-methyl thioacetate were 100%, 100%, 88.91%, 67.64%, and 53.29%, respectively. S-Methyl thioacetate was detected only at pH 5.5. In summary, VOCs produced by P. protegens CLP-6 had strong antagonistic activities against phytopathogens, especially R. solanacearum, under acidic conditions and could be used to develop a safe and additive fumigant against R. solanacearum on tobacco and even other Solanaceae crop bacterial wilt diseases in acidic fields. IMPORTANCE VOCs produced by beneficial bacteria penetrate the rhizosphere to inhibit the growth of plant-pathogenic microorganisms; thus, they have the potential to be used as biological agents in controlling plant diseases. Tobacco bacterial wilt, which is caused by the acidophilic pathogen R. solanacearum, is a major bacterial disease in southern China and is prevalent in acidic soil. In this study, we discovered that the VOCs produced by P. protegens CLP-6 had excellent inhibitory effects on important plant pathogens. Moreover, two of the VOCs, namely, 1,3-benzothiazole and 2-ethylhexanol, had excellent inhibitory effect on R. solanacearum, and another VOC substance, methyl thiocyanate, was produced only at pH 5.5. The VOCs produced by the acid-tolerant strain P. protegens CLP-6 may have potential as environment-friendly microbial fumigant agents for bacterial wilt of tobacco or even other Solanaceae crops in acidic soils in China.

Keywords: Pseudomonas spp.; Ralstonia solanacearum; microbial fumigation; tobacco bacterial wilt; volatile organic compounds.

FIG 1

Activity of VOCs adsorbed by activated carbon in two sealed base plates. The top plate contained CLP-6/blank, while the bottom plate contained R. solanacearum and activated carbon. (a) R. solanacearum and activated carbon. (b) CLP-6 and R. solanacearum. (c) CLP-6, R. solanacearum, and activated carbon.

FIG 2

Antagonistic activity of VOCs against pathogenic fungi evaluated in two sealed base plates. One plate contained CLP-6/blank, while the other contained a culture of pathogenic fungi. (a to j) The species of pathogenic fungi used were Fusarium graminearum (a), F. oxysporum f. sp. cucumerinum (b), F. oxysporum (c), F. solani (d), Alternaria alternata (e), Colletotrichum destructivum (f), C. gloeosporioides (g), and C. chlorophyti (h), with two oomycetes, i.e., Phytophthora capsica (i) and P. infestans (j). (k and l) The experimental method is shown.

FIG 3

Antagonistic activity of VOCs against pathogenic bacteria evaluated using two inverse face-to-face petri dishes. The top plate contained CLP-6/blank, and the bottom plate contained a culture of pathogenic bacteria. The species of pathogenic bacteria used were Ralstonia solanacearum (a), Dickeya chrysanthemi (b), Pseudomonas syringae pv. tabaci tox⁺ (c), and P. syringae pv. tabaci tox⁻ (d).

FIG 4

Effect of the concentration of the inoculum on the antagonistic activity of VOCs produced by CLP-6 against R. solanacearum (concentration of 10⁸ CFU/mL). One drop equals 10 μL. Means with different letters for each inoculation concentration (10⁵ CFU/mL, 10⁶ CFU/mL, 10⁷ CFU/mL, and 10⁸ CFU/mL) denote significant differences (P < 0.05).

FIG 5

Relationships among different volumes of CLP-6 inocula, fumigation time, and antagonistic activity. The top plate contained CLP-6, while R. solanacearum was cultured on the bottom plate. One drop equals 10 μL. (A) Qualitative analysis. (a-d) treated with 10 μL, 40 μL, 80 μL, 160 μL CLP-6 suspensions; (e) Untreated bacteria without CLP-6. (B) Quantitative analysis. Means with different letters of fumigation time (24 h, 48 h, 72 h, 96 h, and 120 h) denote significant differences (P < 0.05).

FIG 6

Volatile compounds produced by P. protegens CLP-6 reduced the virulence of R. solanacearum in tobacco. Disease incidence (A), disease index (B), disease control effect (C), and the degree to which tobacco plants grew (D) in soil mixed with R. solanacearum exposed to VOCs produced by P. protegens CLP-6 cultured at pH 5.5 or pH 7.0 were measured. Panel D shows four treatments, i.e., R. solanacearum exposed to VOCs produced by CLP-6 cultured at pH 5.5 (a), R. solanacearum exposed to blank pH 5.5 NA medium (b), R. solanacearum exposed to VOCs produced by CLP-6 cultured at pH 7.0 (c), and R. solanacearum exposed to blank pH 7.0 NA medium (d).

FIG 7

GC-MS spectra of volatile compounds produced by strain CLP-6 grown on NA plates at pH 5.5 (a) and pH 7.0 (b) for 72 h.

FIG 8

Antagonistic activity of component standards of VOCs against R. solanacearum (concentration of 10⁸ CFU/mL). One drop equals 10 μL. (a) S-Methyl thioacetate. (b) Methyl thiocyanate. (c) Methyl disulfide. (d) 1-Decene. (e) 2-Ethyhexanol. (f) (4E)-Undeca-1,4-diene. (g) 1-Undecene. (h) 1,3-Benzothiazole. (i) 2,5-Dimethylpyrazine. (j) CK.