Evaluation and identification of metabolites produced by Cytobacillus firmus in the interaction with Arabidopsis thaliana plants and their effect on Solanum lycopersicum

Abstract

Currently, the use of bio-inputs is increasing due to the need to reduce the use of agrochemicals. However, one of the limitations is to preserve the viability of the living microorganisms, so it is important to find an alternative that allows us to obtain different metabolites to produce it. We evaluated three different interactions (contact, diffusible and volatile compounds) in vitro in Arabidopsis thaliana (At) seedlings with the strain Cytobacillus firmus M10 and its filtered secondary metabolites (M10F). The results showed that the seedlings inoculated by contact with the filtrate (AtM10F) presented increases in root length (30 %) and leaf area (33 %), as well as in the volatile interaction (At/M10F) with respect to the uninoculated treatment. For both interactions, the seedlings inoculated with the bacteria by contact (AtM10) and volatile (At/M10) obtained greater biomass (48 and 57 %). Subsequently, an evaluation at the end of the A. thaliana cycle showed that the treatments obtained by contact and distance when reinoculated with the bacteria and the filtrate (AtM10, At-M10 and AtM10F) obtained 50 % more seed yield than the control treatment, while AtM10F presented 72 %, while At/M10F presented the highest no. of siliques and seeds, which increased the yield by 65 %. In the Solanum lycopersicum (Sl) experiment, the filtrate (SlM10F) showed significant differences in seedling height, leaf length and width (23, 24 and 36 %, respectively). It also promoted an increase in fresh and dry weight, producing a greater root area and larger leaves compared to the control (Sl) and the bacteria (SlM10). We performed a qualitative characterization of the secondary metabolites present in the filtrate, where we found 2,4-DTBP, sylvopinol, isophthaladehyde, and eicosane of interest with possible growth-promoting effects on A. thaliana and tomato. We identified volatile compounds present in plant-microorganism and plant-filtrate interactions as possible precursors in the induction of plant growth, among which phenols, alcohols, aldehydes, alkanes, and alkenes stand out. Most of the analyzed compounds have not been found in the literature with reports of growth promoters, is important to mention that due to their characteristic functional groups they can derive and trigger the synthesis of new molecules with agronomic application.

Keywords: Diffusible compounds; Filtered metabolites; PGPRs; Plant-bacteria interaction; Plant-filtrate interaction; VOCs.

Graphical abstract

Fig. 1

Morphological analysis of Arabidopsis thaliana seedlings. Comparison of the means of root length (A) and shoot area (B) in A. thaliana seedlings at 14 and 21 days in direct and indirect interactions. Control treatments for contact and diffusion inoculation: At, uninoculated seedlings, AtLB and At-LB inoculated with LB broth. Contact interaction inoculated by C. firmus (AtM10) and its filtered (AtM10F). Interaction of bacteria and filtered by distance (At-M10 and At-M10F). Control treatments of volatiles (At/ and At/LB) and interaction of bacteria (At/M10) and filtrate (At/M10 F). (C) Evaluation of the fresh weight of A. thaliana seedlings at 21 days, interactions by contact and diffusion in the medium (n = 27), and interactions by volatile compounds (n = 25). The bars represent the mean ± standard deviation. Different letters indicate statistically significant differences using the Tuckey test (p ≤ 0.05).

Fig. 2

Evaluation of the effect on the development of A. thaliana Col-0 seedlings inoculated in direct and indirect interactions at 14 or 21 days. (A), contact interaction. (B), diffusion interaction. (C), volatile interaction. At, uninoculated seedlings; AtLB, At-LB and At/LB, seedlings inoculated with the LB culture medium; AtM10, At-M10 and At/M10, seedlings inoculated with the bacteria M10; AtM10F, At-M10F and At/M10F, seedlings inoculated with the M10 filtrate.

Fig. 3

Morphological analysis of variables measured in S. lycopersicum seedlings at 15, 30, and 45 days. (A) seedling height (mm), (B) stem diameter (mm), (C) blade length (mm), and (D) blade width (mm). Sl, uninoculated seedlings; SlM10, seedlings inoculated with the M10 strain; SlM10F, seedlings inoculated with M10 filtrate. The bars represent the mean ± standard deviation. Different letters indicate significant statistical differences using the Tuckey test (n = 15; p ≤ 0.05).

Fig. 4

Comparison of the growth and development of S. lycopersicum seedlings after 45 days. Sl, uninoculated seedlings; SlM10, seedlings inoculated with the M10 strain; SlM10F, seedlings inoculated with M10 filtrate.

Fig. 5

Chromatogram of the M10 Filtered extract with Dichloromethane (Dcm) with method No. 1. The numbers in each peak represent a different identified compound. 1) Phenol, 2,4-bis(1,1-dimethylethyl); 2) Isophthalaldehyde; 3) Eicosane; 4) Benzenemethanol, 3-hydroxy-5-methoxy-; 5) Nonacosane; 6) Benzene, 1-methoxy-3-(methylthio)-; 7) 3-Demethyl-3-ethylthiocolchicine; 8) Propanedinitrile, (phenylmethylene)-; 9) Cholestane, 3-thiocyanato-, (3.alpha.,5.alpha.)-; 10) 6-Norlysergic acid diethylamide; 11) Nonane, 2,2,4,4,6,8,8-heptamethyl-; 12) 2,4(1H,3H)-Quinolinedione, 3-benzo; 13) N-Deacetyl-N-ethoxycarbonylcolchicine; 14) 3,6-Dibutyl-1,2-dihydro-1,2,4,5-tetrazine; 15) Benzeneacetic acid, .alpha.,3,4-tris[(trimethylsilyl)oxy]-, trimethy ester; 16) Pentadecane, 2-methyl-; and 17) .alpha.-D-Glucofuranose, 3-O-(2,3,5-O-acetyl-.beta.-D-lyxofuranosyl)-1,2:5,6-DI-O-isopropylidene-. Some of the peaks that are not selected belong to the sample blanks used. Abundance; time (min).

Fig. 6

Comparison of the VOCs obtained in the interaction of plants with the M10F filtrate at 14 days. The numbers in each peak represent a different identified compound. Black chromatogram indicate the control of uninoculated plants; 1) Dihydro-O,N-dimethyldehydrococcinine methine; 2) 1-Ethyl(dimethyl)silyloxyoctadecane; 3) 1,2-Benzisothiazole-3-propanoic acid; 4) 1-Oxa-3-aza-2-silacyclopentan-5-one, 2,2-dimethyl-3-phenyl-; 5) Phosphoric acid, 2-(methoxyimino)ethyl bis(trimethylsilyl)ester; 6) Tricyclo[5.3.0.0(3,9)]dec-4-ene; 7) 1H-Purine, 8-methyl-; 8) 2-Methyl-6-(5-methyl-2-thiazolin-2-ylamino)pyridine; 9) 3-Cyclopentylcyclopentene; 10) Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, [1S-(1.alpha.,2.beta.,4.beta.)]-; and 11) Benzonitrile, m-phenethyl-. Some of the peaks that are not selected belong to the sample blanks used. Purple chromatogram show the VOCs of the M10F filtrate blank; 1) 1-Propanone, 1-[4-[(trimethylsilyl)oxy]phenyl]-; 2) (E)-4-Chloro-3-methyl-3-hexen-2-one; 3) 3,6-Bis(N,N-dimethylamino)-9-methylcarbazole; 4) Nonanal; 5) Anthranilic acid, N-methyl-, butyl ester; 6) Isobornyl acetate; 7) Quinazoline, 2-(p-anisyl)-4-methylthio-6-nitro-; 8) Phosphine oxide, bis(pentamethylphenyl); 9) Octadecane, 3-ethyl-5-(2-ethylbutyl)-; 10) 1,2-Indandione, 3,3-dimethyl-; 11) N,N'-di-sec-Butyl-p-phenylenediamine; 12) Hexadecane; 13) 4H-1,4-Epoxy-4a,7-methanonaphthalene, 1,5,6,7,8,8a-hexahydro-, (1.alpha.,4.alpha.,4a.alpha.,7.alpha.,7a.beta.)-; 14) 2,5-Cyclohexadien-1-one, 2,5-dimethyl-4-[(2,4,5-trimethylphenyl)imino]-; and 15) Mercaptoacetic acid, bis(trimethylsilyl)-. Green chromatogram represents the VOCs of the interaction with M10F filtrate; 1) 5-Acetamido-4,7-dioxo-4,7-dihydrobenzofurazan; 2) 13H-Dibenzo[a,i]carbazole; 3) Morphinan-6-ol, 7,8-didehydro-4,5-epoxy-3-methoxy-17-methyl-, acetate (ester), (5.alpha.,6.alpha.)-; 4) 1-Dimethyl(chloromethyl)silyloxyoctadecane; 5) 2,4-Cyclohexadien-1-one, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-; 6) 1-Pentene, 1,3-diphenyl-1-(trimethylsilyloxy)-; 7) Dicyclopropylmethanimine, N-cyano-; 8) Ethane, 1-(4,4,4-trifluoro-1,3-dithiobutyl)-2-(3,3,3-trifluoro-1,2-dithiopropyl)-; and 9) Indole-2-one, 2,3-dihydro-N-hydroxy-4-methoxy-3,3-dimethyl-. Some of the peaks that are not selected belong to the sample blanks used. Abundance; time (min).

Fig. 7

Comparison of the VOCs obtained in the interaction of plants with M10 bacterial at 14 days. The numbers in each peak represent a different identified compound. Black chromatogram represents the control of uninoculated plants; 1) Dihydro-O,N-dimethyldehydrococcinine methine; 2) 1-Ethyl(dimethyl)silyloxyoctadecane; 3) 1,2-Benzisothiazole-3-propanoic acid; 4) 1-Oxa-3-aza-2-silacyclopentan-5-one, 2,2-dimethyl-3-phenyl-; 5) Phosphoric acid, 2-(methoxyimino)ethyl bis(trimethylsilyl)ester; 6) Tricyclo[5.3.0.0(3,9)]dec-4-ene; 7) 1H-Purine, 8-methyl-; 8) 2-Methyl-6-(5-methyl-2-thiazolin-2-ylamino)pyridine; 9) 3-Cyclopentylcyclopentene; 10) Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, [1S-(1.alpha.,2.beta.,4.beta.)]-; and 11) Benzonitrile, m-phenethyl-. Some of the peaks that are not selected belong to the sample blanks used. Purple chromatogram represents the VOCs of the M10 bacteria blank; 1) 1,3-Bis(trimethylsilyl)benzene; 2) Benzene, 1,2-dichloro-; 3) Anthranilic acid, N-methyl-, butyl ester; 4) Isobornyl propionate; 5) 2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-ethyl-5-(1-methylethyl)-1,3-bis(trimethylsilyl)-; 6) Tridecane; 7) Octadecane, 1,1′-[1,3-propanediylblsilyl)-; 8) Phenol, 4,6-di(1,1-dimethylethyl)-2-methyl-; 9) 11H-Dibenzo[b,e][1,4]diazepin-11-one, 5,10-dihydro-5-[3-(methylamino)propyl]-; 10) Dithioerythritol, tetrakis(trimethylsilyl)-; 11) 3,4-Dihydrocoumarin, 6-amino-4,4-dimethyl-; 12) 1-(3,6,6-Trimethyl-1,6,7,7a-tetrahydrocyclopenta[c]pyran-1-yl)ethanone; and 13) Oxacyclohexadecan-2-one. Green chromatogram represents the VOCs of the interaction with M10 bacteria; 1) Limonene; 2) Acetonitrile, 1-(6-chloro-2-pyridyl)-1-(4-cyanomethylphenyl)-; 3) Dibenz[b,d]cycloheptane, 2,4,7-trimethoxy-11-acetamino-; 4) 1H-Indole, 1,3-dimethyl-5,6-dimethoxy-2-(3,5-dimethoxyphenyl)-; 5) 2H-1,4-Benzodiazepin-2-one, 7-chloro-1,3-dihydro-5-phenyl-1-(trimethylsilyl)-; 6) E-6-Octadecen-1-ol acetate; 7) Benzoic acid, 5-methyl-2-trimethylsilyloxy-, trimethylsilyl ester; 8) Silanamine, N-[2,6-dimethyl-4-[(trimethylsilyl)oxy]phenyl]-1,1,1-trimethyl-; 9) 4,7-Methano-1H-indene, octahydro-2-(1-methylethylidene)-; 10) Tricyclo[4.2.1.1(2,5)]decan-3-ol; 11) Eicosane; 12) 3-Methoxyphenyllactic acid, di-TMS; 13) Malonic acid, bis(2-trimethylsilylethyl ester; 14) Indole-2-one, 2,3-dihydro-N-hydroxy-4-methoxy-3,3-dimethyl; 15) N-Methyl-1-adamantaneacetamide; and 16) Bicyclo[4.3.0]nonane, 7-methylene-2,4,4-trimethyl-2-vinyl-. Some of the peaks that are not selected belong to the sample blanks used. Abundance; time (min).