PTS and PTSO, two organosulfur compounds from onion by‑products as a novel solution for plant disease and pest management

Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 https://doi.org/10.1186/s40538‑023‑00452‑1 RESEARCH Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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Chemical and Biological Technologies in Agriculture PTS andPTSO, two organosulfur compounds fromonion by‑products asanovel solution forplant disease andpest management Ana Falcón‑Piñeiro1, David García‑López1, Lidia Gil‑Martínez1, José M. de la Torre1, María Dolores Carmona‑Yañez1, Antoine Katalayi‑Muleli1, Enrique Guillamón1, Belén Barrero‑Domínguez2, Silvia López‑Feria2, Dolores Garrido3 and Alberto Baños1,4* Abstract Background Over the past decade, the great impact of agricultural crop diseases has generated considerable economic losses and has compromised the production of edible crops at a time when the world population is only expected to rise, leading to the search for new pest management strategies. Besides that, the environmen‑ tal impact resulting from the continued use of chemical pesticides has led to the search for natural and sustain‑ able alternatives. One of the existing solutions that currently stands out for its effectiveness is the use of bioactive plant extracts. This study aims to evaluate the antimicrobial activity of propyl propane thiosulfinate (PTS) and propyl propane thiosulfonate (PTSO), two organosulfur compounds (OSCs) derived from Allium cepa, against a wide range of target bacteria and fungi. To this end, various in vitro procedures were conducted as well as soil sanitization tests using sterile substrate inoculated with soil‑borne pathogens. In addition, this study also evaluates the pesticidal activ‑ ity of both compounds through in vitro mortality and repellence tests. Results PTS and PTSO revealed inhibition activity on all the pathogens tested, belonging to different taxonomic groups. Moreover, both significatively reduced the population of bacteria and fungi in soil. The quantification of active substances in soil carried out in parallel to the microbial quantification showed that their use reduces the risk of resi‑ due accumulation since they break down quickly when applied. The set of antimicrobial tests performed demon‑ strated that the antifungal effect of both compounds is higher than the bactericidal effect. Lastly, PTS and PTSO showed a concentration‑dependent significant biocidal and repellent effect against aphids. Conclusions The results presented in this work demonstrate that both PTS and PTSO have a significant antimicrobial and pesticidal activity against the great majority of phytopathogens tested, being a promising tool to improve pest management in crops. Keywords Propyl‑propane‑thiosulfinate, Propyl‑propane‑thiosulfonate, Allium cepa, Phytopathogens, Antimicrobial, Soil sanitization, Botanical pesticide, Insecticidal activity, Integrated pest management *Correspondence: Alberto Baños [email protected] Full list of author information is available at the end of the article Page 2 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 Graphical Abstract Background The global human population is predicted to number between 9.4 and 10.1 billion in 2050, an increase by about 1.5 billion compared to 2020 [80]. Around 80% of agricultural production is dedicated to human nutrition [24]. This includes not only the direct use of agricultural products as food, but also the use of crops and other vegetal matter to feed animals, which are in turn intended for human consumption. Keeping in mind the estimated population growth, the production of edible crops might need to increase by up to 119% [20]. This imposes a serious challenge, which involves adopting changes to ensure the transformation of agricultural and food systems toward greater sustainability, and to reduce waste and spoilage [54]. Plant diseases caused by biotic factors are the main responsible for the decrease in crop productivity; in fact, pests and pathogens bring about 40 billion dollars losses a year worldwide [77], which means reductions between 21 and 30% globally in major crops [68]. Plant pathogen control will be even more challenging as climate change conditions progress [11]. Since the environment has a great impact on plant pathogenesis [70], global warming is directly related to disease incidence and severity [81]. Higher temperatures are correlated with soil degradation and less water availability, and foster the emergence of new pathogens and a changeable geographic distribution [67]. Over the last several decades, synthetic agrochemicals have contributed to increase food production worldwide through controlling crop diseases, but with a severe environmental impact [65]. Their application has not only gradually disrupted biological control by natural enemies, but also caused disease outbreaks and the development of resistance [57]. Moreover, synthetic pesticides severely damage non-target organisms, such as pollinators, and human health [73]. In this regard, plant-derived secondary metabolites are receiving increasing attention and gradually replacing synthetic biocides and soil disinfectants from disease management protocols [85]. Many products based on antimicrobial phytochemicals isolated from plants have been developed over the past few years as novel eco-friendly non-synthetic plant protection measures [55]. The extraction of phytochemicals from medicinal and fragrant plants is quite common [35, 58]; however, the extraction of bioactive compounds from by-products from the food industry or second-class plant material, such as grape cane waste [63] and pepper leaves [56], through clean extraction methodologies has become an innovative strategy that contributes to the revaluation of agricultural waste and support circular economy [71]. In recent years, the functional properties of organosulfur compounds (OSCs) obtained from onion (Allium cepa) and garlic (Allium sativum), such as antioxidative, immunomodulatory and antimicrobial activity, have been deeply studied [61]. OSCs are secondary metabolites that are biosynthesized by the plant as a defence mechanism against biotic and abiotic stressors [61]. Garlic bulbs are rich in alliin (S-allyl cysteine sulfoxide) and in a lower degree methiin (S-methyl-l- cysteine sulfoxide), while onion bulbs contain methiin but also isoalliin (S-propenyl-l-cysteine sulfoxide) and propiin (S-propyl-l-cysteine sulfoxide) [61]. Cysteine sulfoxides are natural constituents of fresh bulb tissue, non-volatile and odourless [10, 66]. The disruption of the bulb tissue triggers an enzymatic reaction carried out by alliinase, that catalyses the conversion of these Page 3 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 precursors to thiosulfinates [62], volatile compounds to which the antimicrobial activity of Allium genus plants are mainly attributed and bear the primary responsibility for their organoleptic properties [41, 60]. According to the existing literature, the antimicrobial effect of thiosulfinates is primarily due to their ability to inhibit thiol-containing enzymes by oxidizing protein cysteine or glutathione residues [8]. Enzymes containing thiol include the main enzymes of microbial metabolism as well as bacterial enzymes of the acetyl- CoA-forming system, and RNA polymerase [8]. In onion, propiin turns into propyl-propane thiosulfinate (PTS), a labile compound that changes into dipropyl disulphide and propyl-propane thiosulfonate (PTSO) through dismutation or disproportionation reactions [27]. Whereas bioactive properties of Allicin—that represent about 75% of thiosulfinates in garlic—has been thoroughly investigated in several fields of study, from antimicrobial therapy for human infections to integrated pest management [15, 19], information regarding the antimicrobial activity of PTS and PTSO from onion is limited and focused on the potential of thiosulfinates against human and animal infections. Recent studies have shown broad-spectrum antibacterial activity of PTS and PTSO and its gaseous form against clinical isolates of bacteria and Candida species that are resistant to at least one group of antibiotics [74, 75]. In a previous study we demonstrated the invitro and in planta antifungal activity of volatile organosulfur compounds PTS and PTSO from onion against Verticillium dahliae [23], the most devastating soil-borne pathogenic fungi affecting olive trees [51]. Moreover, the same study showed the potential of both compounds as soil sanitizers, as they reduced V. dahliae population in an artificially infested substrate. As previously mentioned, PTS and PTSO are volatile compounds [42]. Owing to their low molecular weight (< 300 g/mol), they can diffuse through plant cell membranes and soil, playing a key role in the functioning of the whole ecosystem [34]. The study of the active properties of their gaseous phase is thus of interest to back up their use against phytopathogens and inpest managementsystems, especially in the present context in which the search for alternatives to conventional pesticides has become one of the main focuses of modern agriculture research [33]. Within this context, the aim of the present study was to evaluate the bactericidal, fungicidal, pesticidal and repellent activity of PTS and PTSO obtained from low-quality onions not suitable for human consumption, through invitro methodologies and performing soil sanitization trials. Materials andmethods Compounds andreagents Standardized fractions of PTS and PTSO at 20% were supplied by DOMCA SA (Granada, Spain). Both compounds were obtained from onions that had been discarded as they were not suitable for human consumption, following the methodology described by Hu etal. [30] to obtain the allyl derivatives form garlic. Summarizing, onions were chopped and immersed in a solution of Ethanol (70%) in a percentage equivalent to four times their weight. The extraction was carried out for 2weeks at room temperature, then the mixture was filtered, and the solution was concentrated and extracted with Ethyl acetate (EtOAc). The EtOAc extract was concentrated and fractionated by 2 sequential column chromatography’s, taking the trichloromethane (CHCl3) fraction from the first column, and then using EtOAc/hexane as mobile phase in the second column to obtain purified PTS and PTSO. All reagents were purchased from Sigma-Aldrich Química S.L. (Spain), unless otherwise stated. Phytopathogens strains andgrowth media used Bacteria and fungi used in this study were obtained from the Spanish Collection of Type Cultures (CECT), the German Collection of Microorganisms and Cell Cultures (DSMZ), the plant pathogen collection of DMC Research and the culture collection of the Department of Crop Protection, Institute for Sustainable Agriculture, Spanish National Research Council (Córdoba, Spain), which are listed in Table1. Each phytopathogen grew on a specific culture medium and time, indicated by the corresponding culture collection. For the antimicrobial activity tests against pathogenic bacteria, Mueller–Hinton Agar and Mueller–Hinton broth supplied by Scharlau (Barcelona, Spain) were used as culture media [18]; for the invitro antimycotic test, Rose-Bengal agar supplied by Scharlau and RPMI-1640 medium with l-glutamine [17] supplied by Labclinics (Barcelona, Spain) were used. Insects Adult individuals of the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) were supplied by TECNOVA Technological Center (Almería, Spain). The individuals were reared on courgette leaves at the TECNOVA Experimental Center greenhouse for future experiments. Soil The soil used in this study was superficially collected from an olive grove in Linares, Jaen (30U 444908.38 4209274.77 UTM WGS84), owned by the cooperative DCOOP, the world’s largest producer of olive oil. This soil Page 4 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 was chosen because no biocide product had been applied on the farm in the last two years, as it was the control farm of an experimental field trial. The soil was dried in an oven at 50 °C and passed through a 2mm pore sieve to remove plant material, soil macrofauna and stones [9]. Then, it was stored in polyethylene bags for future analysis and characterized according to the procedures previously described. Soil pH, which was determined in a 1:1 water suspension according to the international standard (International Society of Soil Science, ISSS), was 8.71 ± 0.01, that is to say, moderately basic according to the criteria established by the United States Department of Agriculture [48]. Moreover, the soil, classified as sandy loam, contained 6.5 ± 0.22% fine silt, 4.9 ± 0.85% coarse silt, 21.5 ± 0.07% clay, 67.1 ± 0.72% sand (determined through Robinson pipette method [64]), and 1.13% organic matter (soil organic matter fractionation was measured according to [79]). Lastly, this soil presented a maximum Water Holding Capacity (mWHC) of 0.414g H2O per g soil dry matter (determined by the Keen—box method [37]). Based on this parameter, it was determined that the soil had 95.46% dry matter of field-moist soil and a water content of natural moist soil of 0.04g water/g dry matter. In vitro antimicrobial activity againstpathogenic bacteria The antibacterial activity of organosulfur compounds PTS and PTSO was evaluated by performing different testing procedures. The disk diffusion method proposed by Bauer etal. [1] and modified by Calvo and Asensio [14] was used to evaluate the antibacterial activity. Agar plates were inoculated using bacterial suspension adjusted to 106CFU/ml, so that the growth after incubation was confluent. Sterile 6mm cellulose disks (Whatman® antibiotic test discs, Buckinghamshire, UK) impregnated with 20µl of PTS or PTSO at 5, 10 and 25µg/µl were placed in the centre of inoculated agar plates. The inhibition zone of bacterial growth was measured after 48h incubation. Determination of the minimum bactericidal concentration (MBC) was performed by the broth microdilution method, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) collected in the standard Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically [18], to establish the lowest concentration of each antimicrobial agent that reduces the viability of the initial inoculum by 99.9%. 1:2 decreasing dilution were prepared from an initial solution of each compound at 10,000µg/ml so that the following concentrations were obtained: 5000; 2500; 1250; 625; 312.5; 156.25; 78.125; 39.06; 19.53; and 9.76 µg/ml. Each dilution was inoculated with bacterial suspension so that the final concentration in each well was 105CFU/ml, and incubated overnight at room temperature. As positive control, a mix of ampicillin and streptomycin (100,000 and 25,000µg/ml, resp.) was used. As negative control, liquid media without antimicrobial agent was inoculated with bacteria. Bacterial growth was tested by culturing in agar plates, and the lowest concentration of PTS/PTSO in which no growth was observed was established as the MBC. The antibacterial activity of the gaseous phase of PTS and PTSO was assessed through a previously described procedure [23]. Bacterial suspensions adjusted to 106CFU/ml were spread on agar plates. Sterile 6mm Table 1 Bacterial and fungal strains used along with their references and source of isolation Reference Isolation Bacterial strain Erwinia persicina DSM 19328 Tomato plant (Lycopersicon esculentum) Xanthomonas campestris CECT 97 Brussels sprout (Brassica oleracea var. gemmifera) Pseudomonas savastanoi CECT 5023 Olive tree (Olea europaea) Pseudomonas syringae DMC 15 Peach (Prunus persica) Clavibacter michiganensis sp. michiganensis CECT 790 Tomato plant (Lycopersicon esculentum) Agrobacterium tumefaciens CECT 4119 Crown gall of apple seedling (Malus spp) Fungal strain Geotrichum candidum DSM 1240 Tomato plant (Lycopersicon esculentum) Alternaria alternata CECT 2662 Lycopersicon spp Fusarium oxysporum f. sp. cubense DMC 02 Banana tree (Musa paradisiaca) Fusarium graminearum DSM 1095 Maize (Zea mays) Phytophthora cinnamomi CECT 20186 Avocado pear root (Persea americana) Penicillium expansum DMC 01 Apple (Malus domestica) Penicillium digitatum DMC 07 Sweet orange (Citrus sinensis) Phyllosticta spp DMC 10 Olive tree (Olea europaea) Page 5 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 cellulose disks were placed, not in the centre of the plate, but in the centre of the lid of the petri dish, and they were impregnated with 20µl of PTS or PTSO solutions. The same PTS and PTSO concentrations as in the disk diffusion assay were used, i.e., 5, 10 and 25µg/µl. Plates were incubated for 48h and subsequently growth inhibition zones were measured. All invitro assays were performed in duplicate. In vitro antimicrobial activity againstpathogenic fungi The antifungal activity of PTS and PTSO was evaluated following the same methodology described for bacteria, using the appropriate liquid and solid media indicated in section “Phytopathogens strains and growth media used”. The disk diffusion method and the gas phase activity test were carried out with no modifications, with the exception of the incubation time, which was 5days. Regarding the determination of the Minimum Fungicidal Concentration (MFC), the broth microdilution method was also carried out according to the standard reference method for broth dilution antifungal susceptibility testing of filamentous fungi of the CLSI, which does not differ from that described for the determination of MBC [17]. For the positive control, natamycin (50,000µg/ml) was used instead of ampicillin and streptomycin. Moreover, in a fourth trial, the influence of both organosulfur compounds on mycelial growth was determined. Different volumes of PTS and PTSO at 20% were added to Rose-Bengal medium to obtain supplemented agar plates at 25, 50, 100, 250 and 500µg/ml. Each fungal strain was grown on Rose-Bengal agar for 3 days. From these cultures, agar plugs of 5mm diameter were obtained, which were distributed among the supplemented agar plates [47]. In addition, non-supplemented plates with 5mm agar plugs from each strain tested were incubated as control of fungal growth. For 17 days of incubation the diameter of the mycelium over time was measured, compared to the mycelial growth of each fungus when growing on non-supplemented Rose Bengal agar plates. Each experiment was repeated twice. In vitro activity againstaphids In this study, the contact toxicity and repellent activity were evaluated for a liquid blend of PTS and PTSO in proportion 1:1 (w/w) at different concentrations (5000; 2500; 1000; and 500µg/ml). Since the treatments were prepared in water, blank control included only water. The contact toxicity of PTS and PTSO was assessed by the leaf immersion method, as previously reported [69]. Circular cuttings of courgette leaves of 55 mm diameter were immersed in the treatment and control solutions for 5s, air-dried and placed on 60mm diameter petri dishes. Twenty-four adults were transferred to each treated leave cutting in petri dish using a brush. Decis® Protech, a deltamethrin-based pyrethroid insecticide purchased from Bayer CropScience S.L. (Barcelona, Spain) (Ref 84942464) was used as positive control at the dose indicated on the label. The plates were wrapped with Parafilm® purchased from amcor (Valencia, Spain) to prevent the aphids to scape, and maintained in a climate chamber at 25 ± 1°C, 75 ± 5 relative humidity and Light:Dark photoperiod of 14:10 [89]. Mortality was recorded after 24h. An aphid was considered dead if it did not move its legs when touching its abdomen with a brush and if the body turned black [86, 87]. The repellent activity was assessed by a choice assay in 90mm diameter petri dishes [72]. N, N-diethyl-meta- toluamide (DEET), an active ingredient used in many repellent products, was purchased from Sigma–Aldrich at 97% (Ref. D100951) and diluted to 2000µg/ml as positive control [32]. Circular cuttings of courgette leaves of 25mm diameter were immersed in the treatment and control solutions for 5s and dried at room temperature, as in the previous trial. A treated leaf and a negative control leaf were placed in each petri dish on a moist filter paper disk to maintain humidity [88]. Then, 24 adults were introduced into each petri dish using a brush. The parafilm-sealed petri dishes were maintained in the conditions previously indicated. The repellent effect was observed after 24h and expressed as percentage of repellence according to the following formula [59]: where C is the number of aphids on the control leaf, and T is the number of aphids on the treated leaf. Each experiment was performed in triplicate. Soil sanitization The study of the persistence of phytopathogenic microorganisms in soil was carried out by microcosm systems [22]. The ability of a powder blend of PTS and PTSO in proportion 1:1 (w/w) to reduce the population of a pathogenic microorganism artificially inoculated in soil was determined against the bacterium A. tumefaciens and the fungus F. oxysporum, both pathogens that inhabit the soil, where they can survive for long periods [49, 90]. Two different concentrations of active substances, 100µg/g (50µg/g of each one) and 500µg/g (250µg/g of each one), were tested; and the efficacy of a treatment based on a single application was compared with the efficacy of a treatment consisting of 3 applications of the same dose separated in time. In addition, non-inoculated soil was used as sterility control, while untreated inoculated soil was used to follow up microbial growth. Finally, as positive control, the assay included a study group of inoculated soil that was treated with soil fumigant Metam % repellence =[(C−T)/(C+T)]x100 Page 6 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 sodium (C2H4NNaS2) (EPA Reg. No. 45728-16) [76]. Metam sodium 42.1% aqueous solution was purchased from Eastman Chemical Company (Madrid, Spain), diluted in sterile water and set to 60µg/g, in accordance with the recommended application rates [44]. Each study group consisted of four replicates. Table 2 shows the experimental design of the assay and details of the different groups of study. The experimental microcosm unit consisted of a polypropylene box with a drainage system, of 28 cm length × 5cm width × 17cm height and 1.75l capacity. The soil was autoclaved through 4 cycles of 20min at 121°C in a steam sterilizer (Raypa, Terrasa, Spain) for 4 successive days [38], interspersed with incubations at 4°C, to eliminate vegetative forms by heat shock [45]. After drying in an oven at 50°C, 700 g of sterile soil were introduced into each microcosm unit on a bed of sterile gravel to facilitate drainage and prevent soil compaction. Subsequently, soil was inoculated with 140ml of microbial suspension previously adjusted to 109 CFU/ ml, so that the soil moisture was adjusted to 60% of the water holding capacity (WHC) [82]. The negative control group was inoculated with 140ml of distilled water. Microcosms were then placed in a room at 25°C (optimal growth temperature of the two phytopathogens used), where they were kept until the end of the trial. Four days after inoculation, every study group was sampled to establish the starting microbial population. Next, the treatments were applied to the corresponding group at the appropriate dose. Microbial population was quantified 1, 2, 4, 7, 11, 15, 31 and 45days after treatment. The second and third application of the PTS/PTSO powder treatment was added to the corresponding microcosm units 10 and 30days after the first application. At each enumerated date, 25g of soil were diluted in 225ml of buffered peptone water (Scharlau). A lab paddle blender (MASTICATOR, UIL, Barcelona, Spain) was used to homogenize the samples. Serial dilutions were prepared from the supernatant, cultured in the appropriate solid medium, and incubated at 25°C for 3days [21]. In the cases in which no microbial growth was observed on the plate, to confirm the absence of microorganisms a preenrichment step was carried out in a non-selective nutrient medium. Microorganism population was expressed as Log 10CFU/g soil. PTS and PTSO concentrations achieved by each application protocol were assessed by High-performance liquid chromatography using a UV detector (HPLC–UV). Fifty grams of soil were mixed with 100ml of acetone, homogenized with vortex for 1min and extracted in a sonication bath for 10min. Supernatant was separated from the soil by filtration and the process of extraction was repeated adding 20 millilitres of acetone to the solid residue. Then, the supernatant from both extractions was evaporated until dryness in a vacuum rotator and reconstituted with 10ml of methanol (MeOH) vortexing for 30s. Finally, the extract was filtered through a nylon filter of 0.2µm (Sigma–Aldrich, Darmstadt, Germany) and injected into the HPLC system. For the PTS and PTSO determination, an Agilent 1260 Infinity LC (Agilent Technologies, Santa Clara, CA, USA) system was used. The separation of the compounds was accomplished using a Zorbax Eclipse Plus RRHD (50 × 2.1mm, 1.8mm) column at 25°C, and the gradient and mobile phases described by Sorlozano-Puerto etal. [75]. Wavelength of detection was set at 200 nm. A calibration curve using PTS and PTSO standards was made for the quantification. Table 2 Experimental design of soil sanitization assay through microcosm system Group Treatment dose Number of treatment applications Microorganism concentration Negative control – – – Positive control – – 107 CFU/g soil Metam Sodium 60 µg/g 1 application: 4 days after inoculation 107 CFU/g soil PTS/PTSO 100 µg/g 1 application: 4 days after inoculation 107 CFU/g soil PTS/PTSO 100 µg/g 3 applications: 4 days after inoculation 10 days after 1st application 30 days after 1st application 107 CFU/g soil PTS/PTSO 500 µg/g 1 application: 4 days after inoculation 107 CFU/g soil PTS/PTSO 500 µg/g 3 applications: 4 days after inoculation 10 days after 1st application 30 days after 1st application 107 CFU/g soil Page 7 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 Statistical treatment GraphPad prism 8.0 software (GraphPad Software Inc., San Diego, California) was used for statistical analysis. The data obtained in the invitro antimicrobial activity assays were analyzed using descriptive statistics. Shapiro–Wilk normality tests were used to determine normal distribution of all data subjected to ANOVA. A one-way ANOVA test supplemented with Tukey’s post hoc test was used to compare every treatment and control of the invitro assays against aphids with each other. Repeated measures two-way ANOVA test supplemented with Dunnett’s post-hoc test was used for evaluation of statistically significant inhibition of mycelial growth and to establish significant differences between microorganism survival in treated soil and the positive control, considering different treatments and time points. Differences were considered statistically significant when p < 0.05. Results In vitro assessment ofantibacterial activity The antibacterial activity of PTS and PTSO was tested against different bacteria involved in infectious processes of agricultural crops. As shown in Table3, both compounds displayed antimicrobial activity against all the bacterial strains included in the study in various degrees. Moreover, in most cases the bacteriostatic effect rises as the concentration of the product increases, being considerably more modest in the case of P. syringae. Regarding the volatility-linked activity assay, PTS and PTSO inhibited growth of all bacteria tested without coming into direct contact with either the medium or the microorganism, but rather by diffusion of their gas phase, as shown in Fig.1. Xanthomonas campestris, C. m. michiganensis and A. tumefaciens were the most sensitive, showing growth inhibition zones of at least 40mm in all cases (Tables3 and 4). Erwinia persicina and specially P. syringae were, on the other hand, the most resistant strains to PTS and PTSO in both tests, with the smallest inhibition zones among the strains studied. Furthermore, in both agar tests PTSO displayed a greater capacity to inhibit the growth of P. savastanoi, P. syringae, C. m. michiganensis and A. tumefaciens than PTS. Whereas the results of the diffusion and volatility tests in agar appear to suggest that PTSO may have higher antibacterial capacity, the results obtained in the MBC test indicated that it was significantly more active than PTS. As shown in Table5, the lower MBC of PTS is 156.25µg/ml (Me = 312.5 µg/ ml), while MBC data for PTSO ranges from 156.25 to 19.53µg/ml (Me = 78.125µg/ml). In vitro antimycotic activity As for antifungal activity, all phytopathogenic fungi used in this study were sensitive to both organosulfur compounds in a dose dependent manner according to the results of the disk-diffusion method, presented in Table6. Table7 details the results obtained in the MFC determination. In all cases, the MFC of PTSO for each strain was at least one dilution lower than the corresponding concentration of PTS. The highest values obtained, which ranges from 625 to 156.25µg/ml, correspond to the two Penicillium species used. On the other hand, A. alternata, F. oxysporum and F. graminearum were the most sensitive, displaying PTSO MFC values of 39.06, 19.53 and 9.76µg/ml, respectively. The fungicidal activity of the gas phase of the compounds was also demonstrated, since the volatility test generated growth inhibition halos whose diameters were similar to the halos obtained by the agar diffusion test (Table8 and Fig.2). To complete the in vitro assessment of antimycotic activity, the influence of both organosulfur compounds on the mycelial growth was studied. The results of the mycelial growth inhibition test, represented in Figs.3 and 4, also indicates that most fungal species were sensitive to at least the two highest concentrations evaluated (250 and 500µg/ml), showing significant inhibition (p < 0.05). In the case of F. graminearum, which, as in the MFC test, turned out to be the most sensitive, all concentrations of both products completely inhibited the mycelial growth (p < 0.0001), with the exception of PTSO at 25µg/ ml. Moreover, the treatments of 50 and 100µg/ml of PTS Table 3 Antimicrobial activity of PTS and PTSO against phytopathogenic bacteria by disk‑diffusion method, expressed as the average diameter ± standard deviation of inhibition zone (mm) Species PTS (µg/µl) PTSO (µg/µl) 5 10 25 5 10 25 E. persicina 18.0 ± 0.71 23.0 ± 1.87 29.5 ± 1.12 17.5 ± 2.06 20.3 ± 1.79 29.0 ± 0.71 X. campestris 31.5 ± 1.12 36.3 ± 1.09 47.3 ± 1.48 41.0 ± 1.58 48.5 ± 2.06 61.3 ± 1.48 P. savastanoi 12.8 ± 1.30 22.0 ± 1.58 25.3 ± 1.48 25.5 ± 2.06 38.0 ± 1.87 45.5 ± 1.66 P. syringae 11.3 ± 1.09 11.8 ± 1.48 14.5 ± 1.12 19.3 ± 1.09 19.5 ± 1.50 29.8 ± 1.09 C. m. michiganensis 38.0 ± 1.58 46.3 ± 0.83 59.3 ± 1.09 51.5 ± 1.12 56.0 ± 1.58 75.0 ± 1.41 A. tumefaciens 27.5 ± 1.12 36.5 ± 2.18 48.8 ± 2.38 54.3 ± 0.83 64.0 ± 1.58 75.3 ± 2.68 Page 8 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 and PTSO successfully controlled the development of P. cinnamomi, Phyllosticta spp and F. graminearum in agar plates. On the other hand, only the exposure to PTS and PTSO at 500µg/ml achieved a significant reduction of P. digitatum and G. candidum growth (p < 0.05). Penicillium expansum was the most resistant fungal strain, since Microorganisms PTS PTSO (at 5, 10 and 25 µg/µl) (at 5, 10 and 25 µg/µl) E. persicina DSM 19328 X. campestris CECT 97 P. savastanoi CECT 5023 P. syringae DC3000 C. m. michiganensis CECT 790 A. tumefaciens CECT 4119 Fig. 1 Antibacterial activity of the gaseous phase of PTS and PTSO against phytopathogenic bacteria Table 4 In vitro antimicrobial activity of PTS and PTSO against phytopathogenic bacteria via the gas phase, expressed as the average diameter ± standard deviation of inhibition zone (mm) Species PTS (µg/µl) PTSO (µg/µl) 5 10 25 5 10 25 E. persicina 19.0 ± 1.41 25.0 ± 3.16 40.8 ± 2.59 15.8 ± 2.59 22.3 ± 2.17 28.5 ± 2.69 X. campestris 42.8 ± 3.96 50.8 ± 2.59 59.3 ± 3.67 44.3 ± 3.11 54.5 ± 3.84 67.3 ± 3.03 P. savastanoi 16.3 ± 0.83 19.5 ± 1.66 26.8 ± 1.09 26.5 ± 1.12 36.3 ± 1.92 47.0 ± 1.41 P. syringae 0.0 ± 0.00 7.5 ± 1.66 13.3 ± 1.48 20.0 ± 1.41 23.5 ± 1.12 27.8 ± 1.48 C. m. michiganensis 15.0 ± 3.36 25.0 ± 1.41 40.5 ± 1.12 44.8 ± 2.86 55.0 ± 3.39 65.8 ± 1.92 A. tumefaciens 15.5 ± 2.18 26.5 ± 1.12 41.0 ± 2.55 49.5 ± 1.80 57.5 ± 2.69 60.5 ± 2.69 Page 9 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 none of the evaluated concentrations of PTS and PTSO significantly inhibited mycelial development. Although in the case of the other fungal strains no differences were observed between compounds, the effectiveness of PTSO was lower against Phyllosticta spp, F. oxysporum and F. graminearum. In vitro activity againstaphids Insecticidal and repellent capacity of PTS and PTSO were assessed against adult aphids and presented in Fig.5. In the contact toxicity assay, the mortality rate of the aphid population after 24h of exposure to impregnated leaves was investigated. We found that the exposure of A. gossypii to both compounds significantly reduced the population at all concentrations evaluated with respect to the negative control (p < 0.05). The average mortality of aphids treated with 500 and 1000µg/ml of PTS and 500µg/ml of PTSO were 17, 19 and 19% respectively, which is lower than the mortality rate of the population treated with the insecticide used as positive control (28%). On the other hand, the death of individuals treated with PTS ≥ 2500µg/ml and PTSO ≥ 1000µg/ml equalled or exceeded the mortality rate of the positive control. However, none of the treatments showed significant differences compared to the positive control, except for the group of aphids treated with PTSO at 5000µg/ml which registered a significant increase of mortality, reaching 42% after 24h of exposure (p < 0.05). Moreover, whereas the insecticidal capacity of PTSO was found to be higher than that of PTS, the behaviour of both compounds was quite homogeneous in terms of repellent activity. At concentrations of 500 and 1000µg/ml, PTS and PTSO demonstrated a repellent action of 53–56% and 39–47%, respectively. Even though these treatments showed no significant differences, neither between them nor with respect to the positive control (39% repellence), the responses were stronger with higher concentrations, displaying a significant increase of the repellent activity with respect to the control (p < 0.05). Antimicrobial effect andPTS/PTSO quantification insoil When non-treated, A. tumefaciens and F. oxysporum generated growth curves according to what was expected, reaching the concentration of 108CFU/g of soil at the end of the study. Although Metam sodium and the two concentrations of PTS and PTSO tested significantly reduced the population of A. tumefaciens and F. oxysporum when compared to the untreated soil (p < 0.05), the behaviours of the bacterium and the fungus in the presence of the antimicrobials were very unlike. As shown in Fig. 6, within 24 h of exposure A. tumefaciens density were narrowed 3 logarithmic units (from 107 to 104CFU/g) and, surprisingly, thereupon the population remained steady till the end of the assay. In contrast to the bacterial response, F. oxysporum was drastically affected by all the treatments during the first 11days of sampling. As illustrated in Fig.7, from day 15, the progressive recovery of the fungus was observed in soil treated with Metam sodium and a single application of PTS and PTSO at 100 Table 5 Minimum bactericidal concentration (MBC) of PTS and PTSO against phytopathogenic bacteria Species MBC (µg/ml) PTS PTSO E. persicina 312.5 156.25 X. campestris 156.25 19.53 P. savastanoi 312.5 39.06 P. syringae 156.25 78.125 C. m. michiganensis 312.5 78.125 A. tumefaciens 625 78.125 Table 6 Antimicrobial activity of PTS and PTSO against phytopathogenic fungi by disk‑diffusion method, expressed as the average diameter ± standard deviation of inhibition zone (mm) Species PTS (µg/µl) PTSO (µg/µl) 5 10 25 5 10 25 G. candidum 15.5 ± 1.80 26.0 ± 3.16 36.8 ± 3.34 27.3 ± 2.86 34.8 ± 3.11 44.8 ± 2.59 A. alternata 38.5 ± 3.35 44.8 ± 2.17 53.3 ± 2.05 30.8 ± 0.83 42.0 ± 2.12 56.3 ± 1.64 Phyllosticta spp 64.0 ± 1.41 68.8 ± 0.83 75.5 ± 1.12 55.0 ± 1.63 64.5 ± 2.05 79.5 ± 1.25 P. cinnamomi 28.5 ± 1.12 42.5 ± 3.64 54.0 ± 1.41 39.5 ± 2.69 52.0 ± 1.41 66.3 ± 2.59 F. oxysporum 26.5 ± 2.18 46.5 ± 1.66 60.5 ± 2.69 41.8 ± 3.03 51.3 ± 2.59 54.0 ± 1.41 F. graminearum 51.5 ± 2.06 58.0 ± 1.22 65.3 ± 2.17 50.8 ± 2.59 57.8 ± 0.83 69.0 ± 2.12 P. expansum 34.3 ± 3.03 39.8 ± 1.64 48.5 ± 1.50 35.8 ± 3.34 46.3 ± 0.83 54.8 ± 1.48 P. digitatum 15.8 ± 0.83 20.5 ± 0.50 34.0 ± 1.22 12.0 ± 2.55 18.5 ± 1.12 26.0 ± 1.22 Page 16 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 capacity exhibited by 0.1% PTS and PTSO significantly exceed the effect of DEET. While PTSO shows greater biocidal activity against A. gossypii, the greater volatility of PTS compared to PTSO, as demonstrated by HPLC–UV determination, explains why PTS has a greater repellent capacity. Bioactive compounds from plant material with proven antibacterial, antifungal and insecticidal activity are an 0 1 2 3 4 5 6 7 8 9 Samplingtimes (days) F. oxysporum Log10 (CFU/g soil) Positive control MetamSodium PTS/PTSO 100 µ g/g1Application PTS/PTSO 100 µ g/g3Applications PTS/PTSO 500 µ g/g1Application PTS/PTSO 500 µ g/g3Applications 0124 711153 14 5 **** **** **** **** **** Fig. 7 Density of F. oxysporum in soil expressed as Log10 CFU/g of soil over time. Significant reductions in the population of each treated microcosm in comparison with the non‑treated group (control) were established according to the Dunnett test at a 95% confidence level. Values are means with SD in bars. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 respect to control 0113145 0 50 100 150 200 250 Sampling times(days) PTS( µ µ g/gsoil) 100 µ g/g1Application 100 µ g/g3Applications 500 µ g/g1Application 500 µ g/g3Applications 0113145 0 50 100 150 200 250 Sampling times(days) PTSO ( µ µ g/gsoil) 100 µ g/g1Application 100 µ g/g3Applications 500 µ g/g1Application 500 µ g/g3Applications Fig. 8 Concentration of PTS (left) and PTSO (right) reached in soil 1 h (day 0), 11 days, 31 days and 45 days after the first application of the treatment Page 17 of 20 Falcón‑Piñeiroetal. Chem. Biol. Technol. Agric. (2023) 10:76 important natural source for the development of new environmentally safe plant protection products [5, 31]. Onion is one of the most cultivated and consumed vegetables worldwide [78]. Taking into account those that do not reach the consumer due to their low quality and the inedible parts, onion processing creates massive wastes that are a rich source of bioactive compounds [6, 52]. Therefore, to orientate the valorisation of onion solid wastes towards the formulation of novel and sustainable products suitable for agricultural production could not only reduce the environmental impact but also cover important needs for the agrifood sector [36, 40]. Even thought our results suggest that PTS and PTSO from A. cepa display strong activity against pathogenic microorganisms and aphids, and provide useful information that support their use for crop disease control, the present work has not evaluated how these organosulfur compounds influence soil microbial populations, natural enemies or pollinators. Therefore, further studies should focus on the analysis of the effect of PTS and PTSO on soil microbiome and non-target species. Conclusions PTS, and specially PTSO, showed antimicrobial effect against a wide range of bacteria and fungi infecting plants. This study revealed that the degree of efficacy of PTS and PTSO depend on the target species, being more effective against the fungal strains evaluated. Despite their rapid volatilization from soil, the combination of these compounds successfully controlled soilborne microorganism population. Both of them had similar, if not more, biocidal and repellent effect than commercial fumigants. Although PTS and PTSO will be further evaluated in field experiments for potential control of pathogen populations in crops, these results encourage their use for the development of sustainable biopesticides that contribute to environmental health. Moreover, both metabolites are found to be promising candidates for Integrated Pest Management, whose bases include sustainable pest control, reduction of pesticide residues and the use of natural resources. Abbreviations PTS Propyl‑propane thiosulfinate PTSO Propyl‑propane thiosulfonate OSCs Organosulfur compounds EtOAc Ethyl acetate CECT Spanish collection of type cultures DSMZ German collection of microorganisms and cell cultures WHC Water holding capacity MBC Minimum bactericidal concentration CLSI Clinical and Laboratory Standards Institute MFC Minimum fungicidal concentration DEET N, N‑diethyl‑meta‑toluamide HPLC–UV High‑performance liquid chromatography UV detector MeOH Methanol SSCPs Small secreted cysteine‑rich proteins Acknowledgements We would like to acknowledge the work of Jose Manuel García‑Madero in editing the text and TECNOVA Technological Center (Almería, Spain) for providing the aphids. Author contributions AB and DG designed the study. AF‑P, AB and AKM conducted in vitro analy‑ ses. AF‑P and DG‑L conducted experiments with aphids. AF‑P and MDC‑Y performed soil sanitization assays. LG‑M and JMdlT conducted HPLC–UV determination. BB‑D and SL‑P provided soil samples and data. AB supervised the experiments. AF‑P analyzed data and wrote the manuscript. AB, DG and EG reviewed the manuscript. All authors read and approved the manuscript. Funding This research has been carried out within the project GRUPO OPERATIVO SALUD‑ OLIVAR from the Spanish Rural Development Program (2014–2020) funded by the Spanish Ministry of Agriculture, Fisheries and Food and co‑ financed by 80% by the European Agricultural Fund for Rural Development (FEADER) (Total investment 384.830,96 EUR). It has also received funding from European Union’s Horizon 2020 research and innovation program under Grant agreement no. 887281 (BIOVEXO). This study is part of the Industrial Doctorate of the doctoral student Ana Falcón Piñeiro, granted by the State Research Agency of the Spanish Government, with the following reference: DIN2019‑010792. Availability of data and materials All data generated during this study are included in this manuscript. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 DMC Research Center, Camino de Jayena, 82, 18620 Alhendín, Spain. 2 Dcoop Sociedad Cooperativa Andaluza, Carretera Córdoba S/N, 29200 Antequera, Málaga, Spain. 3 Department of Plant Physiology, University of Granada, Fuen‑ tenueva S/N, 18071 Granada, Spain. 4 Department of Microbiology, University of Granada, Fuente Nueva S/N, 19071 Granada, Spain. Received: 5 May 2023 Accepted: 1 August 2023 References 1. Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibil‑ ity testing by a standardized single disk method. Am J Clin Pathol. 1966;45:493–6. 2. Aguinaga‑Casañas MA, Mut‑salud N, Falcon‑Piñeiro A, Alcaraz‑Martinez Á, Guillamón E, Baños A. 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