Volatile Organic Compounds Produced by Kosakonia cowanii Cp1 Isolated from the Seeds of Capsicum pubescens R & P Possess Antifungal Activity

Abstract

The Kosakonia cowanii Cp1 strain was isolated from seeds of Capsicum pubescens R. & P. cultivated in Michoacan, Mexico. Genetic and ecological role analyses were conducted for better characterization. The results show that genome has a length of 4.7 Mbp with 56.22% G + C and an IncF plasmid of 128 Kbp with 52.51% G + C. Furthermore, pathogenicity test revealed nonpathogenic traits confirmed by the absence of specific virulence-related genes. Interestingly, when fungal inhibitory essays were carried out, the bacterial synthesis of volatile organic compounds (VOCs) with antifungal activity showed that Sclerotinia sp. and Rhizoctonia solani were inhibited by 87.45% and 77.24%, respectively. Meanwhile, Sclerotium rolfsii, Alternaria alternata, and Colletotrichum gloeosporioides demonstrated a mean radial growth inhibition of 52.79%, 40.82%, and 55.40%, respectively. The lowest inhibition was by Fusarium oxysporum, with 10.64%. The VOCs’ characterization by headspace solid–phase microextraction combined with gas chromatography–mass spectrometry (HS-SPME-GC–MS) revealed 65 potential compounds. Some of the compounds identified with high relative abundance were ketones (22.47%), represented by 2-butanone, 3-hydroxy (13.52%), and alcohols (23.5%), represented by ethanol (5.56%) and 1-butanol-3-methyl (4.83%). Our findings revealed, for the first time, that K. cowanii Cp1 associated with C. pubescens seeds possesses potential traits indicating that it could serve as an effective biocontrol.

Role of Volatile Organic Compounds Produced by Kosakonia cowanii Cp1 during Competitive Colonization Interaction against Pectobacterium aroidearum SM2

“…Bacterial competitive colonization interaction assays were performed on healthy fruits of tomato (Solanum lycopersicum) and jalapeño chili (Capsicum annum) to assess the potential of the strain K. cowanii Cp1 (previously isolated and characterized [26]) against the P. aroidearum SM2 strain and its ability to reduce soft rot symptoms. The fruits were inoculated as previously described above in the infection methodology, with 10 µL of each bacterial suspension added at a concentration of 1 × 10 8 CFU/mL, according to the treatments shown in Table 1.…”

“…The test of the cell-free filtrate's susceptibility towards P. aroidearum SM2 was conducted via the disk diffusion method, where paper filters were impregnated with 10 µL of cell-free filtrate at 2, 4, 6, 8, and 10 h of bacterial growth at 37 • C and 120 rpm and placed on TSA medium (Difco Laboratories; Detroit, MI, USA) inoculated with 100 µL (1 × 10 6 CFU/mL) of P. aroidearum SM2 and incubated at 37 • C for 24 h [39,40]. Third, to determine whether volatile organic compounds (VOCs) could inhibit the growth on the pathogenic P. aroidearum SM2, the double culture method described previously was used [26], as illustrated in the Supplementary Materials (Figure S1). In this experiment, a two-compartment Petri dish plate device was used; on the bottom side of this device, K. cowanii Cp1 was inoculated at 100 µL (10 8 CFU/mL) onto TSA medium (Difco Laboratories; Detroit, MI, USA) and grown for 2, 4, or 6 h at 37 • C to allow time to produce any VOCs.…”

“…The tryptic soy medium without the bacterial strain was used as a control. Once the bacterial cultures were obtained, the VOCs were analyzed using the following methodology [26]: the samples were incubated at 50 • C for one hour, and then the VOCs were collected on a divinylbenzene/carboxen/polydimethylsiloxane fiber (DVB/CAR/PDMS, Supelco, Sigma-Aldrich, Visalia, CA, USA). After this, manual injection was carried out in splitless mode; the injection port and transfer line temperature were set to 250 • C using a 7820A GC with a 5975C MSD (Agilent Technologies, Inc., Santa Clara, CA, USA) and HP-5MS 30 m, 0.25 mm, and 0.25 µm GC Column Capillaries (Agilent Technologies Inc., Santa Clara, CA, USA).…”

“…Therefore, it is of great significance to find beneficial bacterial species with faster colonization responses in plant tissue, with metabolic activities that have an extensive potential to control pathogenic microorganisms in agricultural systems. In this context, we recently isolated and proved the ability of Kosakonia cowanii to colonize Capsicum annuum L. tissue without causing detrimental effects [25,26]. K. cowanii is a member of the family Enterobacteriaceae with a significant metabolic ability, showing plant-promoting activities through auxin (IAA) and siderophore production [27,28], including the production of a high concentration of exopolysaccharides with effects on plant drought tolerance [28], and with the ability to produce active volatile organic compounds against certain fungal pathogens and to colonize the seeds of C. annuum L. [25,26].…”

“…In this context, we recently isolated and proved the ability of Kosakonia cowanii to colonize Capsicum annuum L. tissue without causing detrimental effects [25,26]. K. cowanii is a member of the family Enterobacteriaceae with a significant metabolic ability, showing plant-promoting activities through auxin (IAA) and siderophore production [27,28], including the production of a high concentration of exopolysaccharides with effects on plant drought tolerance [28], and with the ability to produce active volatile organic compounds against certain fungal pathogens and to colonize the seeds of C. annuum L. [25,26]. Therefore, in the present report, we hypothesize that the plant tissue colonization competition of the K. cowanii Cp1 strain can reduce infection with pathogenic bacterial species.…”