Volatile organic compounds from Bacillus spp. reduce egg hatching, motility, infectivity and reproduction of Meloidogyne incognita

Volatile organic compounds from Bacillus spp. reduce egg hatching, motility, infectivity and reproduction of Meloidogyne incognita | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Volatile organic compounds from Bacillus spp. reduce egg hatching, motility, infectivity and reproduction of Meloidogyne incognita Renata Silva Canuto de Pinho, Vicente Paulo Campos, Eduardo Souza Freire, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7543783/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2025 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted 6 You are reading this latest preprint version Abstract Volatile organic compounds (VOCs) produced by microorganisms have several roles, such as communication and antimicrobial defence. Soil and endophytic bacteria employ VOCs in their interactions with plants, other bacteria, fungi and nematodes. In this study, the VOCs produced by 36 endophytic bacterial strains from pepper and tomato plants were tested for their antagonism to the nematode Meloidogyne incognita . The VOCs produced by most bacterial strains (94%) caused a strong decrease in motility, while 69% of the strains caused high mortality rates in second-stage juveniles (J2s) of M. incognita . Among the strains that simultaneously caused a strong decrease in motility and high mortality, Bacillus pumilus strains 51 and 52 and B. sphaericus strain 43 were selected for further studies. These three strains produced VOCs that decreased egg hatching, infectivity and reproduction on tomato plants. Gas chromatography revealed a diverse VOC profile in the three studied strains, with 36% of compounds not previously reported in bacteria. Only decan-2-one and 2-ethylhexanol out of the eight VOCs produced by the bacterial strains significantly reduced motility (3%) and increased mortality (82%) of M. incognita J2s when tested in their purified form. These findings highlight both the potential of VOCs as templates for new nematicides and the bacterial strains as biocontrol agents against M. incognita . Bacillus pumilus Bacillus sphaericus biological control endophytes root-knot nematode Figures Figure 1 Figure 2 Figure 3 Introduction Plant-parasitic nematodes (PPNs) cause diseases in nearly all cultivated plants, resulting in crop losses worldwide (Li et al., 2015 ). Among these parasites, root-knot nematodes ( Meloidogyne spp.) stand out, and the species Meloidogyne incognita , M. javanica , M. hapla , and M. arenaria , are the most damaging due to their distribution and host range (Li et al., 2015 ). To face this problem, control measures that include the use of microorganisms as control agents of PPNs are being rapidly developed and deployed in the field. This strategy has already partially replaced the use of chemical nematicides and is expected to grow even further in the near future (Machado, 2022 ). Microorganisms may have direct or indirect mechanisms of activity against PPNs. The indirect mechanisms involve induction of resistance and stimulation of plant growth, whereas direct mechanisms include production of toxic compounds such as antibiotics, enzymes and volatile organic compounds (VOCs), parasitism and competition (Kohl et al., 2019; Lahlali et al., 2022 ). These mechanisms of activity frequently occur in combination in biocontrol agents and act synergistically against PPNs. Bacterial biocontrol agents are among the most employed in commercial products to control nematodes and the compounds they produce decrease infection by killing the J2s of root-knot nematodes, decreasing their motility or disorienting them (Cruz-Magalhães et al., 2021 ; Terra et al., 2023 ). Several authors have demonstrated that bacteria can inhibit the infection and reproduction of root-knot nematodes (Hu et al., 2017 ; Li et al., 2019 ; Cruz-Magalhães et al., 2021 ; Antil et al., 2022 ). However, most studies focused on water soluble molecules from bacteria, whereas less attention has been given to volatile molecules that are toxic to nematodes (Campos et al., 2010 ). Bacteria in the genus Bacillus and related genera (i.e., Paenibacillus ) are the most studied and used in biocontrol of PPNs (Ye et al., 2022 ; Terra et al., 2023 ; Yin et al., 2023). A great number of studies have also been done with bacteria in other genera, such as Pseudomonas , Actinobacteria and Pasteuria (Ahmad et al., 2021 ; Migunova & Sasanelli, 2021 ). The nematicidal effects of VOCs produced by bacteria has been demonstrated in vitro for some bacterial biocontrol agents (Cheng et al., 2017 ; Chen et al., 2022 ; Ye et al., 2022 ) and in one study (Yin et al., 2021 ) the role of VOCs produced by Bacillus cereus in the reduction of galls in cucumber plants has been established. Nevertheless, due to the diversity of bacterial strains associated with PPNs in nature, the VOCs produced by these bacteria and their antagonistic effects on nematodes still need to be further investigated. Moreover, VOCs are produced in mixtures that are expected to exhibit varying levels of activity depending on their composition. Therefore, studies on this area are certainly warranted. In nature VOCs allow the producing bacterial strain to communicate with plants, fungi, nematodes and other bacteria at both long and short distances (Raza et al., 2020 ; Lammers et al., 2022 ; Grahovac et al., 2023 ). The propagation of VOCs through the air makes them ideal messaging molecules since solvent-dependent compounds would have a limited dispersion (Lammers et al., 2022 ). The most common classes of VOCs produced by bacteria are fatty acids with long carbon chains and sulphur compounds, whereas halogenated compounds are not as common (Schulz & Dickschat, 2007 ). The production of VOCs is influenced by the bacterial strain itself, its species and genus, composition of the substrate, temperature, pH and oxygen levels (Schulz-Bohm et al., 2017 ; Weisskopf et al., 2021 ), among other factors. The VOCs produced by single strains or mixture of strains may be significantly different in terms of composition and concentration (Raza et al., 2020 ). The diversity of VOCs produced by bacteria is so great that a systematic effort was made to organise them in the mVOC database, which contains more than 3500 VOCs from approximately 1350 strains or species (Kemmler et al., 2025 ). The modes of action of bacterial VOCs against PPNs, especially Meloidogyne spp. and Heterodera spp., include interference with chemotaxis by attracting or repelling the motile J2s (Adam et al., 2014 ; Luo et al., 2018 ), predation (Wang et al., 2014 ; Luo et al., 2018 ), fumigation (Yin et al., 2021 ; Diyapoglu et al., 2022 ), induction of host plant resistance and oxidative stress on J2s (Ayaz et al., 2021 ; Poulakis & Tjamos, 2023) and general nematicidal activity on different stages of the nematode (Huang et al., 2010 ; Cao et al., 2019 ; Du et al., 2022 ; Ye et al., 2022 ). Other effects of VOCs on the system, such as modification of rhizosphere microbial communities (Du et al., 2022 ), modification of plant exudates (Cavalcanti et al., 2024 ) and interference with the pathogen through sRNA signalling are less exploited (Terra et al., 2023 ). In this study, we focused on the characterization of a collection of 36 bacterial strains isolated from healthy plant tissues by assaying their antagonistic effects on M. incognita . Three strains were selected and further evaluated for their effects on egg hatching, motility and mortality of J2s, infectivity and reproduction of the nematode in tomato plants. After determining the composition of the VOCs produced by these three strains by gas chromatography coupled to mass spectrometry, some purified compounds were tested for their effects on M. incognita J2s. Materials and Methods Bacterial strains and nematodes Thirty-six endophytic bacterial strains previously obtained from tomato ( Solanum lycopersicum ) and red pepper ( Capsicum annuum ) stems and identified by fatty acid analysis (Silva et al., 2008 ) were used in this study. These bacterial strains were stored at -80ºC in peptone-glycerol medium composed of peptone 20 g, glycerol 10 mL, K 2 HPO 4 1.5 g, and distilled water to 1 L. The strains were routinely grown on tryptic soy agar medium (TSA) (Difco) at 28ºC for 24 h. Bacterial suspensions were prepared with 0.85 g/mL NaCl solution and adjusted to OD 540 = 0.5. Meloidogyne incognita- infected tomato plants were kept in a greenhouse for three months to serve as sources of inoculum. The nematode eggs were obtained from galled roots following the procedure of Hussey and Barker ( 1973 ). Eggs were placed in hatching chambers and J2s no older than 24 h after hatching were collected and used for in vitro hatching, motility, and mortality experiments. They were also used in an infectivity and reproduction experiment with tomato plants. Egg hatching, motility and mortality of J2s exposed to bacterial VOCs The motility and mortality of J2 caused by bacterial VOCs were evaluated according to the method described by Fernando et al. ( 2005 ). Two-compartment Petri plates measuring 9 cm in diameter were used. One of the compartments contained 10 mL of TSA medium and the other the same amount of 2% water-agar. On the TSA medium, 300 µL of each bacterial suspension was deposited and maintained at 28ºC in the dark and 24 h later, a 2-mL suspension containing approximately 200 M. incognita J2s was added onto the water agar surface. After adding the nematodes, plates containing the different bacterial strains were immediately wrapped with a PVC film to prevent volatile escape and organized in a complete randomized design with four replicates in a growth chamber and the whole experiment was done twice. Plates containing TSA medium without bacteria were used as negative controls. After incubating at 28ºC in the dark for 72 h, the J2 suspension was transferred to a polypropylene counting dish to observe the J2s under an inverted microscope, where they were classified as motile and immotile. To confirm the death of the immotile J2s, 20 µL of 1 mol/L NaOH was added to the J2 suspension and the specimens with no movement were considered dead (Chen & Dickson, 2000 ). Values underwent transformation into percentage before statistical analysis. To study the effect of exposure time to volatiles, three bacterial strains, B. sphaericus 43, B. pumilus 51 and 52, were randomly selected and used in these experiments due to the high mortality of the J2s exposed to their VOCs (Table 1 ). The plate setup described above was used. Cell suspensions were added to the TSA compartment and J2s of M. incognita in 2 mL of water were added to the water-agar compartment. For mortality and motility, approximately 100 M. incognita J2s in 2 mL of water were transferred to the water agar compartment and exposed for 24, 48, 72 and 96 h to the bacterial VOCs. The number of motile and dead J2s was determined as described above. Egg hatching experiments were done by adding 1,000 M. incognita eggs in 2 mL of water on the water-agar surface, incubated at 28°C in the dark for 10 days and the number of hatched J2s was determined under a microscope. Controls did not contain any bacterial strain on the TSA medium. The experiments were arranged in a completely randomized design with four replicates and were done twice. Values were transformed into percentages before the statistical analysis. Infectivity and reproduction of J2s exposed to bacterial VOCs Approximately 500 M. incognita J2s were exposed to the VOCs produced by B. sphaericus strain 43 and B. pumilus strains 51 and 52 for 24, 48, 72 or 96 h in two-compartment plates as described above. Controls were J2s exposed to water instead of bacterial suspensions. After exposure, the nematodes were suspended in 2 mL of water and inoculated with a micropipette on tomato through four 1.5-cm deep holes in the substrate around the plant base. The plants used in the inoculations were 25-day old tomato seedlings cv. Santa Cruz grown on 5-kg pots containing 4 kg of a mixture of soil and commercial organic substrate (Carolina Soil, Brazil) (2:1). Seedlings were arranged in a randomized block design, with four replicates, and were placed in a temperature-controlled room at 28°C, with a 12 h photoperiod. Thirty days after J2 inoculation, root systems were harvested, gently washed, weighed and galls were counted. The number of eggs was determined by the method of Hussey and Barker ( 1973 ). The numbers of galls and eggs were divided by the root masses before the statistical analysis. Identification of bacterial VOCs by gas chromatography/mass spectrometry (GC/MS) using headspace-solid-phase microextraction (HS-SPME ) Bacillus sphaericus 43 and B. pumilus 51 and 52 were transferred to vials containing 100 mL of tryptic soy broth (TSB) medium, shaken at 28ºC for 7 days, at 100 rpm. Controls were vials containing TSB medium alone. The supernatants were filtered through durapore PVDF membranes with 0.22 µm pore size. Aliquots of 9 mL were transferred to sterile 40 mL Supelco SPME glass vials sealed with silicone septae and stored at 0–4ºC. A 100-µm fused silica-non-bonded polydimethylsiloxane (PDMS) Supelco Fiber Core was introduced into the headspace of each vial using a Supelco SPME fiber manual holder and the vial was warmed in a 45ºC water bath for 30 min. Then, the SPME fiber was inserted for 3 min into the injector of a Shimadzu Gas Chromatograph (Model GC-2010)-Mass Spectrometer (Model QP2010) running GCMS Solution Release 2.30 Software. The analyses were carried out under the following conditions: injector temperature 220ºC; injection mode: splitless; sampling time: 1.5 min; column: DB-5MS (Agilent JW, 30 m × 0.25 mm × 0.25 µm); carrier gas: helium; column flow: 1.21 mL/min; linear velocity: 39.7 cm/s; split ratio after sampling time: 10:1; oven temperature: 40ºC for 5 min; rate 5ºC/min (48 min) to 280ºC, in which it stayed for 15 min; ion source temperature: 250°C; interface temperature: 280°C; solvent cut time: 1.50 min; detector gain mode: relative; detector gain: 0.0 kV; threshold: 1000; acquisition mode: scan at 0.30 s interval; range: 50–400 m/z. The experimental retention indexes were obtained by injecting a homologous series of alkanes. Mass spectra were compared to Wiley 7.0, NIST 12 and NIST 62 mass spectral libraries. Compounds identified with less than 85% probability to correspond to a record in the mass spectral libraries were considered non identified. Evaluation of motility and mortality caused by synthetic VOCs A total of 23 purified commercial VOCs, eight of them produced by strains 43, 51 and 52 were acquired (Sigma). Each compound (100 µL) was added to a sterilized 3 cm²-filter paper disc that was placed on the surface of TSA medium in one of the compartments of the Petri dish as described above. Solid compounds such as 2,6-bis(1,1-dimethylethyl)-4-methylphenol and 2,6-bis(1,1-dimethylethyl)phenol were heated according to their boiling point to become liquid before pipetting. An aliquot of 2 mL of an aqueous suspension containing approximately 200 M. incognita J2s was added on the other compartment of the plate. Controls were plates containing TSA medium with a paper disk without any chemical compound added. The experimental design was completely randomized with four replicates (plates) for each commercial compound. The plates were sealed with a PVC film to prevent the escape of volatiles and incubated at 28°C for 24 h. Dead and motile J2 were counted as described above. Statistical analyses Values of motile and dead J2 exposed to bacterial VOCs or commercial VOCs as well as values of J2 hatched from eggs exposed to bacterial VOCs, number of galls and eggs underwent analysis of variance (ANOVA) after assuring normal distribution with the Shapiro-Wilk test and homogeneity of variances with the Levene test and the means were grouped according to the Scott-Knott’s (1974) test ( P ≤ 0.05) in the R software (R Core Team, 2019 ). Results Bacterial VOCs decreased J2 motility and increased mortality in vitro The 36 bacterial strains used in this study were distributed in four categories according to their level of VOC activity against the nematode M. incognita (Table 1 ). Most strains (94.4%) produced volatiles that caused a very strong reduction in J2 motility, whereas 30.6 and 38.9% caused very strong and strong J2 mortality, respectively. Bacteria identified as species of the genus Bacillus were the most commonly found. Among the bacteria that produced VOCs causing very strong mortality and low motility were four strains of B. pumilus , three of B. sphaericus and one strain each of B. cereus, B. amyloliquefaciens, Paenibacillus macerans and a non-identified strain. Bacterial VOCs decreased egg hatching and motility and increased mortality of J2s All the three selected bacterial strains tested significantly reduced M. incognita egg hatching after 10 days of exposure across two independent experiments (Fig. 1 ). Hatching of M. incognita eggs exposed to VOCs produced by B. pumilus and B. sphaericus was reduced to approximately 2% of the values observed for the control treatment (Fig. 1 ). Table 1 Classification of bacterial strains into categories of antagonistic activity of their volatile organic compounds (VOCs) on motility and mortality of Meloidogyne incognita J2s. The nematode was exposed to VOCs in two-compartment Petri dishes and motility and mortality were evaluated under a microscope. The antagonistic categories were classified into I- Low (motility: >48.4%; mortality: <47.2%), II- Moderate (motility: 48.3–23.6%; mortality: 47.2–63.6%); III- Strong (motility: 23.5–22%; mortality: 72.6–86.6%); IV- Very strong (motility: 2.7–0%; mortality: 90.5–100%). Each category was composed of combinations of motility and mortality levels that were significantly different according to the Scott-Knott’s test ( P ≤ 0.05). Species Number of strains Codes of strains in each antagonistic category Motility Mortality I II III IV I II III IV Acinetobacter johnsonii 2 1, 10 10 1 Bacillus pumilus subg. B 1 27 27 B. amyloliquefaciens 7 32 21, 22, 28, 36, 41, 50 41,32, 50 21, 22, 28 36 B. subtilis 2 19, 31 31 19 B. sphaericus 5 42, 43, 45, 53, 54 45 53 42, 43, 54 B. pumilus 10 3, 8, 12, 20, 26, 39, 48, 49, 51, 52 3, 8 12, 20, 26, 39 48, 49, 51, 52 B. marinus 1 44 44 Staphylococcus aureus 1 18 18 B. cereus 1 13 13 Paenibacillus gordonae 2 37, 40 37 40 P. macerans 1 47 47 Not identified (P11) 3 2, 4, 11 2, 4 11 Total 36 0 1 1 34 7 4 14 11 The three bacterial strains decreased J2 motility to almost zero and caused mortalities of 100% at 72 and 96 h of exposure (Fig. 2 ). Exposure times lower than 48 h, although significantly decreased motility and caused higher mortality as compared to the control, were not consistent for some treatments across the experiments (Fig. 2 ). Strain 52 was the least effective at 24 h of exposure, while the first significant effects on motility and mortality were observed after 48 h of exposure to the bacterial VOCs. Infectivity and reproduction of M. incognita J2s exposed to bacterial VOCs The number of galls and eggs of M. incognita in tomato roots, also referred to as infectivity and reproduction, respectively, in the control treatment without any exposure to VOCs reduced approximately 60% when the J2s were 72 and 96 h old in comparison with 24 and 48 h old J2s, respectively. The same occurred to the bacterial treatments, where infectivity of M. incognita J2 decreased after exposure to VOCs in a time-dependent manner. The number of galls and eggs was reduced to zero after exposing the J2s for 96 h to VOCs produced by strains 51 and 52 (Fig. 3 ). Exposure times lower than 96 h did not result in the complete suppression of M. incognita , although the number of galls and eggs were lower than the control for strains 51 and 52 at 48 and 72 h. The exposure to VOCs for 24 h was insufficient to significantly reduce the numbers of galls and eggs by all the strains studied (Fig. 3 ). The regression analysis revealed that exposures of 72 h for strains 51 and 52 and of 96 h for strain 43 were sufficient for the numbers of galls and eggs to be completely suppressed (Fig. S1 ). Table 2 Major volatile organic compounds (relative area ≥ 1.0% in the chromatograms) produced by three Bacillus strains identified by gas chromatography-mass spectrometry analyses. The compounds were identified by comparison of the mass spectra to Wiley 7.0, NIST 12 and NIST 62 mass spectral libraries. Compounds B. sphaericus 43 B. pumilus 51 B. pumilus 52 Novel VOCs 1 Known, tested 2 Known, not tested 3 2-methylpropan-1-amine X X 3-methylbutan-1-amine X X 2,5-dimethylpyrazine X X X X 2,6-dimethylpyrazine X X X X 2-ethyl-3-methylpyrazine X X 3,4-dimethylfurazan X X 3-ethyl-2,5-dimethylpyrazine X X 2-ethyl-3,5-dimethylpyrazine X X 4-methylnonane X X 2-butoxyethanol X X 2-methylnonane X X 3-(1,1-dimethylethyl)cyclobut-3-ene-1,2-dione X X 2,6-dimethylheptan-4-one X X 2,2,5-trimethylhexane X X 2,6-diethyl-3,5-dimethylpyrazine X X 1-(2-hydroxyethoxy)tridecane X X 2,3,5,8-tetramethyldecane X X 2(1,1-dimethylethyl)-4-methylphenol X X 1-(1,1-dimethylethyl)-2-methylpropane-1,3-diyl bis(2-methyl propanoate) X X 2,5-dimethylpyrimidine X X Benzene-1,3-diamine X X X Tridecan-1-ol X X Octadecan-1-ol 4 X X Dodecan-6-one X X 2-methylundec-10-enal X X Decan-2-one X X Decane X X 7-methyloct-2-yne X X 2-ethylhexan-1-ol X X 2-propylpentan-1-ol X X 2-ethyl-4-methylpentan-1-ol X X 6-methylundec-3-ene X X Diheptyl ether X X (Z)-5-methyldec-2-ene X X 2,6-bis(1,1-dimethylethyl)-4-methylphenol X X X 2,4-bis(1,1-dimethylethyl)phenol X X 2,5-bis(1,1-dimethylethyl)phenol X X 3,5-bis(1,1-dimethylethyl)phenol 5 X X 2,6-bis(1,1-dimethylethyl)phenol X X 1 VOCs being reported for the first time as produced by Bacillus spp. in this study; 2 VOCs produced by bacteria or other microorganisms and tested against nematodes; 3 VOCs produced by bacteria or other microorganisms, but not tested against nematodes. The searches were done in the mVOC database (Kemmler et al., 2025 ) and in the scientific literature. 4 Tian et al. ( 2020 ) and 5 Mazumdar and Thakur ( 2024 ) reported the indicated VOCs as produced by bacteria. Identification of bacterial VOCs by gas chromatography-mass spectrometry (GC-MS) Analysis by GC-MS of the VOCs produced by B. sphaericus 43 and B. pumilus 51 and 52, revealed the presence of 19, 11 and 14 compounds, respectively (Table 2 ). Only the most abundant compounds, with a relative peak area ≥ 1% in the chromatogram were considered in this analysis. None of the compounds found in the control (TSB medium) presented the same retention time or mass spectrum of the VOCs produced by the bacteria. Although the three bacterial strains were from the genus Bacillus and two of them, strains 51 and 52 were B. pumilus , they secreted relatively different mixtures of volatile compounds. Two compounds, 2,5-dimethylpyrazine and 2,6-dimethylpyrazine were detected in the three strains analysed. Only the two compounds listed above were common to strains 43 and 51, whereas strains 43 and 52 also secreted 2,6-bis(1,1-dimethylethyl)-4-methylphenol, and strains 51 and 52 secreted benzene-1,3-diamine in addition to the two compounds secreted by all three strains (Table 2 ). Activity of purified commercial VOCs A total of 23 purified commercial VOCs were tested, eight were produced by at least one of the selected bacterial strains and the other 15 were used as standards for comparison purposes. Only benzaldehyde, which was not produced by any bacterial strain, caused 100% mortality and completely inhibited the motility of the J2s (Table 3 ). Five other compounds, with only decan-2-one produced by one bacterial strain ( B. pumilus 51), were able to cause high mortality (82–100%) and decrease motility (0- Table 3 In vitro mortality and motility of Meloidogyne incognita second-stage juveniles (J2) exposed to volatile organic compounds from commercial sources. Values between parentheses correspond to the purity according to the manufacturer. Compounds marked with an asterisk were detected in at least one bacterial strain shown in Table 2 . Means followed by the same letter in a column are not significantly different according to the Scott-Knott test ( P ≤ 0.05) Compounds J2 Motility (%) J2 Mortality (%) Control 94.7 h 1.8 g 2,6-bis(1,1-dimethylethyl)-4-methylphenol* (99%) 94.3 h 0.0 h 2,6-dimethylheptan-4-one* (96%) 45.3 e 1.2 g 2,6-bis(1,1-dimethylethyl)phenol* (99%) 90.7 h 1.9 g 2-butoxyethanol* (99%) 16.7 c 2.2 g 2,6-dimethylpyrazine* (98%) 62.0 f 3.6 g 2,5-dimethylpyrazine* (98%) 74.7 g 3.9 g 2-ethylhexan-1-ol* (99%) 34.7 d 31.9 e Decan-2-one* (98%) 3.0 b 82.4 c 2,4,4-trimethylpent-1-ene (96%) 55.7 f 4.2 g Nonane (99%) 94.3 h 1.8 g Terpineol, mixture of isomers (96%) 70.0 g 4.5 g Decanal (98%) 78.7 g 13.6 f Undecan-6-one (97%) 74.3 g 15.8 f 2-phenylethanol (98%) 10.0 b 17.5 f Dodecan-2-one (97%) 45.0 e 25.8 e Octan-1-ol (99%) 4.3 b 63.6 d 3,7-dimethylocta-1,6-dien-3-ol (linalool) (97%) 3.7 b 69.2 d Undecan-2-one (99%) 22.0 c 71.3 d Nonan-2-one (97%) 1.3 b 92.1 b Octan-2-ol (96%) 0.0 a 98.0 b 3,3,5-trimethylcyclohexanone (98%) 0.0 a 98.3 b Octan-3-ol (99%) 0.0 a 98.9 b Benzaldehyde (98%) 0.0 a 100.0 a 3%) of M. incognita J2s (Table 3 ). Among the seven other compounds produced by the bacterial strains, only 2-butoxyethanol had some effect on motility (17%) and 2-ethylhexan-1-ol had a mild effect on mortality (32%) (Table 3 ). Discussion Volatile organic compounds (VOCs) are carbon-based, possess low-molecular weight and high vapor pressure, making them evaporate at room temperatures (Diyapoglu et al., 2022 ). These compounds are produced by all living organisms and some of them are toxic to diverse plant pathogens, including nematodes, fungi, insects and bacteria (Gu et al, 2007 ; Diyapoglu et al., 2022 ). Although there are many publications available in the recent literature on the effects of VOCs on nematodes (Xu et al., 2015 ; Cheng et al., 2017 ; Silva et al., 2018 ; Cruz-Magalhaes et al., 2021; Chen et al., 2021), the diversity of both bacteria and the VOCs they produce, warrant the pursuit of additional studies. In order to expand our knowledge on the diversity and activity of VOCs against M. incognita we studied a collection of endophytic bacterial strains obtained from tomato and pepper. From the 36 bacterial strains used in this study, 34 strains produced VOCs that strongly lowered the motility of M. incognita J2 and 11 strains strongly increased J2 mortality. Bacillus sphaericus 43 and B. pumilus 51 and 52 were randomly selected for their strong effects on J2 motility and mortality also reduced egg hatching and the number of eggs and galls on tomato plants. These bacteria produced different patterns of VOCs that were not related to the species of the strain. The VOC decan-2-one, produced by B. pumilus 51, in its purified form significantly lowered the motility and increased the mortality of M. incognita J2. The highest activities against M. incognita in our study were achieved by VOCs from Bacillus species. This genus is known for its ability to produce volatile compounds active against PPNs (Cruz-Magalhaes et al., 2021; Chen et al., 2022 ; Dai et al., 2023 ; Song et al., 2024 ). Indeed, most (94%) of the bacterial strains included in our study were able to strongly reduce J2 motility, whereas 31% caused mortality rates higher than 90%. All strains that caused high mortality were also included in the category that strongly decreased motility. Other authors have found much lower levels of mortality in their screening experiments, which varied from 3.5 to 12.5% (Gu et al., 2007 ; Lu et al., 2014 ; Costa et al., 2015 ). These results may be explained by the differences in the time of nematode exposure to the bacterial VOCs, which was 72 h in our study and 24 h in the other studies (Gu et al., 2007 ; Lu et al., 2014 ; Costa et al., 2015 ). Further experiments are still needed to estimate the potential of bacterial strains from different isolation sources in their potential to antagonise PPNs through the production of VOCs. While the strains used in our study were obtained from the interior of tomato and pepper plants, other authors collected potential bacterial antagonists from cow dung (Lu et al., 2014 ), tobacco soil (Gu et al., 2007 ) and Meloidogyne exigua egg masses (Costa et al., 2015 ). Bacillus sphaericus strain 43 and B. pumilus strains 51 and 52 were chosen for more detailed analyses on the activity of their VOCs. The VOCs of these strains decreased M. incognita egg hatching, J2 motility and infectivity and reproduction in planta and increased J2 mortality. Our experiments with tomato plants showed a 60% reduction in infectivity and reproduction, which is in the range observed by other authors (Bui et al., 2020 ; Yin et al., 2021 ; Cruz-Magalhaes et al., 2021; Wu et al., 2022; Song et al., 2023). However, the modes of action of the volatiles produced by strains 43, 51 and 52 against M. incognita were not precisely determined. From our study, we know these strains act by reducing egg hatching and motility of J2 and increasing their mortality. The VOCs produced by the bacterial strains employed in this study differed markedly, even though two strains were from the same species, B. pumilus . From a total of 39 compounds produced, only four were shared by two or three strains (Table 2 ), indicating the high diversity of the compounds. A comparison of the 39 VOCs produced by the studied strains with the ones deposited in the mVOC database (Kemmler et al., 2025 ) revealed that only 12.8% (five compounds from this study) were previously tested against nematodes. The majority of the compounds (51%, i.e., 20 compounds) were produced by bacteria or other microorganisms, but not tested against nematodes, whereas the remaining 36% (14 compounds) are being reported for the first time as produced by bacteria (Table 2 ). Although we have been very strict and removed all compounds detected in the uninoculated medium, we cannot completely rule out that some of the VOCs we are reporting for the first time as being produced by bacteria are not artefacts of our experimental set up. These highly diverse compounds are produced in complex mixtures and in order to determine the activity of individual VOCs against nematodes we employed a selected set of purified chemicals commercially available. Out of eight compounds produced by the strains under study, three were previously tested by other authors against nematodes and five were tested for the first time in our study (Table 3 ). Only one of the VOCs, decan-2-one, produced by B. pumilus strain 51, showed a significant reduction in motility and caused high mortality in M. incognita J2s. Decan-2-one was highly active against M. incognita J2s with an activity that did not differ from the fungicides carbofuran and fluensulfone (Pacule et al., 2022 ). Therefore, the toxic activity of strain 51 may be mostly attributed to this volatile, but we still did not determine the major compounds responsible for the toxicity of the other remaining strains studied, 43 and 52. This is frequently complicated when the strains under study produce a great number of compounds with a minor effect that act synergistically for the final activity. This is further complicated when the VOCs are not commercially available in their purified form. Our results revealed some interesting relationships between the activity against M. incognita and the chemical structure of certain compounds. For example, the methyl ketones with 9 and 10 carbon atoms, nonan-2-one and decan-2-one, respectively were more active against M. incognita than the similar molecules with 11 and 12 carbons, undecane-one and dodecan-2-one, respectively (Table 3 ). By the same token, the primary alcohol octan-1ol is significantly less active than the secondary alcohols octan-2-ol and octan-3-ol, all of them with 8-carbon structural isomers (Table 3 ). Similarly, the primary alcohols 2-butoxiethanol and 2-ethylhexan-1-ol showed low activity against M. incognita (Table 3 ). By analysing the results we obtained for the alkane nonane, it is reasonable to expect low activity from 4-methylnonane and 2-methylnonane, both produced by B. sphaericus 43 (Tables 2 and 3 ), but not yet tested against nematodes. The compounds benzaldehyde and 3,3,5-trimethylcyclohexanone were among the most active against M. incognita . The toxicity of 3,3,5-trimethylcyclohexanone seems to be due to the presence of the cyclohexane ring. This hypothesis becomes stronger in light of the results of a screening experiment of 11 VOCs against M. incognita , and only the three compounds with a cyclohexane ring were toxic (Mei et al., 2021 ). The high activity of benzaldehyde may be related to the direct link of the carbonyl group to an aromatic carbon, whereas decanal, which is also an aldehyde, does not have the carbonyl group linked to an aromatic carbon and has shown much lower activity (Table 3 ). This result suggests that methylundec-10-enal produced by B. pumilus 51, also an aldehyde with the carbonyl group linked to a non-aromatic carbon, will probably show low activity against the nematode. The diversity of VOCs detected in the bacterial strains offer a unique opportunity to develop new agricultural products, including nematicides. These VOCs may be employed as sources of lead structures for these developments by chemical modifications in order to improve their stability, spectrum of activity and decrease their toxicity to the environment and human health, as shown for other compounds (Jia et al., 2019 ; Wang et al., 2024 ). In conclusion, the VOCs produced by endophytic bacteria were diverse and showed toxicity against M. incognita when the strains were used in the in vitro and in planta experiments. On the other hand, the individual effects of each compound in the antagonism against M. incognita could not be pointed out for all strains due to the lack of commercially available compounds to be assayed. The majority (25 VOCs) of the compounds produced by the bacterial strains were previously detected in microorganisms, including Bacillus strains, but about 36% of the compounds were not detected in any bacteria (Kemmler et al., 2025 ). This study adds new VOCs produced by Bacillus that could lead to the development of novel nematicides, besides the direct development of the strains as biocontrol agents to be deployed in the field. Declarations Acknowledgements The authors thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and fellowships. 1. Funding: This work was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). 2. Competing interests: The authors declare no competing interests of any kind. 3. 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11:29:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7543783/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7543783/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s41348-025-01201-2","type":"published","date":"2025-12-06T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91420854,"identity":"6e21cfd8-d892-4058-9bec-980f81e09299","added_by":"auto","created_at":"2025-09-16 10:09:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34510,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of \u003cem\u003eM. incognita\u003c/em\u003e second stage juveniles (J2) hatched after ten days of egg exposure to VOCs produced by three selected bacterial strains: \u003cem\u003eBacillus sphaericus \u003c/em\u003estrain 43 and \u003cem\u003eB. pumilus \u003c/em\u003estrains 51 and 52. (a) Experiment I and (b) Experiment II. The number of J2 hatched from eggs exposed to the three bacterial strains was determined after 10 days. Error bars represent the standard error of the means. Means followed by the same letter are not significantly different according to the Scott-Knott test (\u003cem\u003eP\u003c/em\u003e≤ 0.05)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7543783/v1/513b90aa9736338ede5edc7d.png"},{"id":91420853,"identity":"76df0e1e-631f-4e4a-81a1-3986c3fbfb64","added_by":"auto","created_at":"2025-09-16 10:09:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83778,"visible":true,"origin":"","legend":"\u003cp\u003eMotility (a and b) and mortality (c and d) of \u003cem\u003eMeloidogyne incognita \u003c/em\u003esecond-stage juveniles (J2s) exposed to volatile organic compounds (VOCs) produced by \u003cem\u003eBacillus sphaericus \u003c/em\u003estrain 43 and \u003cem\u003eB. pumilus \u003c/em\u003estrains 51 and 52, for 24, 48, 72 and 96 h in two independent experiments. Error bars represent the standard error of the means. In each time of exposure and experiment, means with the same letter do not differ significantly according to the Scott-Knott test (\u003cem\u003eP\u003c/em\u003e≤ 0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7543783/v1/3ef253ef98cd04255821c830.png"},{"id":91421417,"identity":"e5299095-4825-4375-a15c-5bd290632cfc","added_by":"auto","created_at":"2025-09-16 10:17:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55456,"visible":true,"origin":"","legend":"\u003cp\u003eInfectivity and reproduction of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e on tomato after exposure to volatile organic compounds (VOCs) of \u003cem\u003eBacillus sphaericus \u003c/em\u003estrain 43 and \u003cem\u003eB. pumilus \u003c/em\u003estrains 51 and 52 for 24, 48, 72 and 96 h. (a) Number of galls/root mass and (b) Number of eggs/root mass. After exposure to VOCs, J2s of \u003cem\u003eM. incognita\u003c/em\u003e were inoculated on tomato plants and the number of galls and eggs were determined 30 days later. Error bars represent the standard error of the means. In each time of exposure, means with the same letter are not significantly different according to the Scott-Knott test (\u003cem\u003eP\u003c/em\u003e≤ 0.05)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7543783/v1/b2cdd2231a38551aa2edf13b.png"},{"id":97724536,"identity":"782c3140-dd3c-436e-8c55-82fb7faaff21","added_by":"auto","created_at":"2025-12-08 16:12:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1564396,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7543783/v1/0d1d3e76-cc8d-43bd-8202-c22197ac5d99.pdf"},{"id":91420861,"identity":"0f57974c-40e2-4c58-8d8a-8e7d85243f6e","added_by":"auto","created_at":"2025-09-16 10:09:10","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":144352,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialJPDP.docx","url":"https://assets-eu.researchsquare.com/files/rs-7543783/v1/79e6b60db706f3d0e3e5b0b6.docx"}],"financialInterests":"","formattedTitle":"Volatile organic compounds from Bacillus spp. reduce egg hatching, motility, infectivity and reproduction of Meloidogyne incognita","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant-parasitic nematodes (PPNs) cause diseases in nearly all cultivated plants, resulting in crop losses worldwide (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among these parasites, root-knot nematodes (\u003cem\u003eMeloidogyne\u003c/em\u003e spp.) stand out, and the species \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, \u003cem\u003eM. javanica\u003c/em\u003e, \u003cem\u003eM. hapla\u003c/em\u003e, and \u003cem\u003eM. arenaria\u003c/em\u003e, are the most damaging due to their distribution and host range (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To face this problem, control measures that include the use of microorganisms as control agents of PPNs are being rapidly developed and deployed in the field. This strategy has already partially replaced the use of chemical nematicides and is expected to grow even further in the near future (Machado, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroorganisms may have direct or indirect mechanisms of activity against PPNs. The indirect mechanisms involve induction of resistance and stimulation of plant growth, whereas direct mechanisms include production of toxic compounds such as antibiotics, enzymes and volatile organic compounds (VOCs), parasitism and competition (Kohl et al., 2019; Lahlali et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These mechanisms of activity frequently occur in combination in biocontrol agents and act synergistically against PPNs.\u003c/p\u003e\u003cp\u003eBacterial biocontrol agents are among the most employed in commercial products to control nematodes and the compounds they produce decrease infection by killing the J2s of root-knot nematodes, decreasing their motility or disorienting them (Cruz-Magalh\u0026atilde;es et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Terra et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Several authors have demonstrated that bacteria can inhibit the infection and reproduction of root-knot nematodes (Hu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cruz-Magalh\u0026atilde;es et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Antil et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, most studies focused on water soluble molecules from bacteria, whereas less attention has been given to volatile molecules that are toxic to nematodes (Campos et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Bacteria in the genus \u003cem\u003eBacillus\u003c/em\u003e and related genera (i.e., \u003cem\u003ePaenibacillus\u003c/em\u003e) are the most studied and used in biocontrol of PPNs (Ye et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Terra et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yin et al., 2023). A great number of studies have also been done with bacteria in other genera, such as \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003ePasteuria\u003c/em\u003e (Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Migunova \u0026amp; Sasanelli, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe nematicidal effects of VOCs produced by bacteria has been demonstrated \u003cem\u003ein vitro\u003c/em\u003e for some bacterial biocontrol agents (Cheng et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and in one study (Yin et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) the role of VOCs produced by \u003cem\u003eBacillus cereus\u003c/em\u003e in the reduction of galls in cucumber plants has been established. Nevertheless, due to the diversity of bacterial strains associated with PPNs in nature, the VOCs produced by these bacteria and their antagonistic effects on nematodes still need to be further investigated. Moreover, VOCs are produced in mixtures that are expected to exhibit varying levels of activity depending on their composition. Therefore, studies on this area are certainly warranted.\u003c/p\u003e\u003cp\u003eIn nature VOCs allow the producing bacterial strain to communicate with plants, fungi, nematodes and other bacteria at both long and short distances (Raza et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lammers et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Grahovac et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The propagation of VOCs through the air makes them ideal messaging molecules since solvent-dependent compounds would have a limited dispersion (Lammers et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The most common classes of VOCs produced by bacteria are fatty acids with long carbon chains and sulphur compounds, whereas halogenated compounds are not as common (Schulz \u0026amp; Dickschat, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The production of VOCs is influenced by the bacterial strain itself, its species and genus, composition of the substrate, temperature, pH and oxygen levels (Schulz-Bohm et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Weisskopf et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), among other factors. The VOCs produced by single strains or mixture of strains may be significantly different in terms of composition and concentration (Raza et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The diversity of VOCs produced by bacteria is so great that a systematic effort was made to organise them in the mVOC database, which contains more than 3500 VOCs from approximately 1350 strains or species (Kemmler et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe modes of action of bacterial VOCs against PPNs, especially \u003cem\u003eMeloidogyne\u003c/em\u003e spp. and \u003cem\u003eHeterodera\u003c/em\u003e spp., include interference with chemotaxis by attracting or repelling the motile J2s (Adam et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), predation (Wang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), fumigation (Yin et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Diyapoglu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), induction of host plant resistance and oxidative stress on J2s (Ayaz et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Poulakis \u0026amp; Tjamos, 2023) and general nematicidal activity on different stages of the nematode (Huang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Du et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Other effects of VOCs on the system, such as modification of rhizosphere microbial communities (Du et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), modification of plant exudates (Cavalcanti et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and interference with the pathogen through sRNA signalling are less exploited (Terra et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we focused on the characterization of a collection of 36 bacterial strains isolated from healthy plant tissues by assaying their antagonistic effects on \u003cem\u003eM. incognita\u003c/em\u003e. Three strains were selected and further evaluated for their effects on egg hatching, motility and mortality of J2s, infectivity and reproduction of the nematode in tomato plants. After determining the composition of the VOCs produced by these three strains by gas chromatography coupled to mass spectrometry, some purified compounds were tested for their effects on \u003cem\u003eM. incognita\u003c/em\u003e J2s.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains and nematodes\u003c/h2\u003e\u003cp\u003eThirty-six endophytic bacterial strains previously obtained from tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and red pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e) stems and identified by fatty acid analysis (Silva et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) were used in this study. These bacterial strains were stored at -80\u0026ordm;C in peptone-glycerol medium composed of peptone 20 g, glycerol 10 mL, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1.5 g, and distilled water to 1 L. The strains were routinely grown on tryptic soy agar medium (TSA) (Difco) at 28\u0026ordm;C for 24 h. Bacterial suspensions were prepared with 0.85 g/mL NaCl solution and adjusted to OD\u003csub\u003e540\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMeloidogyne incognita-\u003c/em\u003einfected tomato plants were kept in a greenhouse for three months to serve as sources of inoculum. The nematode eggs were obtained from galled roots following the procedure of Hussey and Barker (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Eggs were placed in hatching chambers and J2s no older than 24 h after hatching were collected and used for \u003cem\u003ein vitro\u003c/em\u003e hatching, motility, and mortality experiments. They were also used in an infectivity and reproduction experiment with tomato plants.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEgg hatching, motility and mortality of J2s exposed to bacterial VOCs\u003c/h3\u003e\n\u003cp\u003eThe motility and mortality of J2 caused by bacterial VOCs were evaluated according to the method described by Fernando et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Two-compartment Petri plates measuring 9 cm in diameter were used. One of the compartments contained 10 mL of TSA medium and the other the same amount of 2% water-agar. On the TSA medium, 300 \u0026micro;L of each bacterial suspension was deposited and maintained at 28\u0026ordm;C in the dark and 24 h later, a 2-mL suspension containing approximately 200 \u003cem\u003eM. incognita\u003c/em\u003e J2s was added onto the water agar surface. After adding the nematodes, plates containing the different bacterial strains were immediately wrapped with a PVC film to prevent volatile escape and organized in a complete randomized design with four replicates in a growth chamber and the whole experiment was done twice. Plates containing TSA medium without bacteria were used as negative controls. After incubating at 28\u0026ordm;C in the dark for 72 h, the J2 suspension was transferred to a polypropylene counting dish to observe the J2s under an inverted microscope, where they were classified as motile and immotile. To confirm the death of the immotile J2s, 20 \u0026micro;L of 1 mol/L NaOH was added to the J2 suspension and the specimens with no movement were considered dead (Chen \u0026amp; Dickson, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Values underwent transformation into percentage before statistical analysis.\u003c/p\u003e\u003cp\u003eTo study the effect of exposure time to volatiles, three bacterial strains, \u003cem\u003eB. sphaericus\u003c/em\u003e 43, \u003cem\u003eB. pumilus\u003c/em\u003e 51 and 52, were randomly selected and used in these experiments due to the high mortality of the J2s exposed to their VOCs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The plate setup described above was used. Cell suspensions were added to the TSA compartment and J2s of \u003cem\u003eM. incognita\u003c/em\u003e in 2 mL of water were added to the water-agar compartment. For mortality and motility, approximately 100 \u003cem\u003eM. incognita\u003c/em\u003e J2s in 2 mL of water were transferred to the water agar compartment and exposed for 24, 48, 72 and 96 h to the bacterial VOCs. The number of motile and dead J2s was determined as described above.\u003c/p\u003e\u003cp\u003eEgg hatching experiments were done by adding 1,000 \u003cem\u003eM. incognita\u003c/em\u003e eggs in 2 mL of water on the water-agar surface, incubated at 28\u0026deg;C in the dark for 10 days and the number of hatched J2s was determined under a microscope. Controls did not contain any bacterial strain on the TSA medium. The experiments were arranged in a completely randomized design with four replicates and were done twice. Values were transformed into percentages before the statistical analysis.\u003c/p\u003e\n\u003ch3\u003eInfectivity and reproduction of J2s exposed to bacterial VOCs\u003c/h3\u003e\n\u003cp\u003eApproximately 500 \u003cem\u003eM. incognita\u003c/em\u003e J2s were exposed to the VOCs produced by \u003cem\u003eB. sphaericus\u003c/em\u003e strain 43 and \u003cem\u003eB. pumilus\u003c/em\u003e strains 51 and 52 for 24, 48, 72 or 96 h in two-compartment plates as described above. Controls were J2s exposed to water instead of bacterial suspensions. After exposure, the nematodes were suspended in 2 mL of water and inoculated with a micropipette on tomato through four 1.5-cm deep holes in the substrate around the plant base. The plants used in the inoculations were 25-day old tomato seedlings cv. Santa Cruz grown on 5-kg pots containing 4 kg of a mixture of soil and commercial organic substrate (Carolina Soil, Brazil) (2:1). Seedlings were arranged in a randomized block design, with four replicates, and were placed in a temperature-controlled room at 28\u0026deg;C, with a 12 h photoperiod. Thirty days after J2 inoculation, root systems were harvested, gently washed, weighed and galls were counted. The number of eggs was determined by the method of Hussey and Barker (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). The numbers of galls and eggs were divided by the root masses before the statistical analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of bacterial VOCs by gas chromatography/mass spectrometry (GC/MS) using headspace-solid-phase microextraction (HS-SPME\u003c/b\u003e)\u003c/p\u003e\u003cp\u003e\u003cem\u003eBacillus sphaericus\u003c/em\u003e 43 and \u003cem\u003eB. pumilus\u003c/em\u003e 51 and 52 were transferred to vials containing 100 mL of tryptic soy broth (TSB) medium, shaken at 28\u0026ordm;C for 7 days, at 100 rpm. Controls were vials containing TSB medium alone. The supernatants were filtered through durapore PVDF membranes with 0.22 \u0026micro;m pore size. Aliquots of 9 mL were transferred to sterile 40 mL Supelco SPME glass vials sealed with silicone septae and stored at 0\u0026ndash;4\u0026ordm;C. A 100-\u0026micro;m fused silica-non-bonded polydimethylsiloxane (PDMS) Supelco Fiber Core was introduced into the headspace of each vial using a Supelco SPME fiber manual holder and the vial was warmed in a 45\u0026ordm;C water bath for 30 min. Then, the SPME fiber was inserted for 3 min into the injector of a Shimadzu Gas Chromatograph (Model GC-2010)-Mass Spectrometer (Model QP2010) running GCMS Solution Release 2.30 Software. The analyses were carried out under the following conditions: injector temperature 220\u0026ordm;C; injection mode: splitless; sampling time: 1.5 min; column: DB-5MS (Agilent JW, 30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m); carrier gas: helium; column flow: 1.21 mL/min; linear velocity: 39.7 cm/s; split ratio after sampling time: 10:1; oven temperature: 40\u0026ordm;C for 5 min; rate 5\u0026ordm;C/min (48 min) to 280\u0026ordm;C, in which it stayed for 15 min; ion source temperature: 250\u0026deg;C; interface temperature: 280\u0026deg;C; solvent cut time: 1.50 min; detector gain mode: relative; detector gain: 0.0 kV; threshold: 1000; acquisition mode: scan at 0.30 s interval; range: 50\u0026ndash;400 m/z. The experimental retention indexes were obtained by injecting a homologous series of alkanes. Mass spectra were compared to Wiley 7.0, NIST 12 and NIST 62 mass spectral libraries. Compounds identified with less than 85% probability to correspond to a record in the mass spectral libraries were considered non identified.\u003c/p\u003e\n\u003ch3\u003eEvaluation of motility and mortality caused by synthetic VOCs\u003c/h3\u003e\n\u003cp\u003eA total of 23 purified commercial VOCs, eight of them produced by strains 43, 51 and 52 were acquired (Sigma). Each compound (100 \u0026micro;L) was added to a sterilized 3 cm\u0026sup2;-filter paper disc that was placed on the surface of TSA medium in one of the compartments of the Petri dish as described above. Solid compounds such as 2,6-bis(1,1-dimethylethyl)-4-methylphenol and 2,6-bis(1,1-dimethylethyl)phenol were heated according to their boiling point to become liquid before pipetting. An aliquot of 2 mL of an aqueous suspension containing approximately 200 \u003cem\u003eM. incognita\u003c/em\u003e J2s was added on the other compartment of the plate. Controls were plates containing TSA medium with a paper disk without any chemical compound added. The experimental design was completely randomized with four replicates (plates) for each commercial compound. The plates were sealed with a PVC film to prevent the escape of volatiles and incubated at 28\u0026deg;C for 24 h. Dead and motile J2 were counted as described above.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eValues of motile and dead J2 exposed to bacterial VOCs or commercial VOCs as well as values of J2 hatched from eggs exposed to bacterial VOCs, number of galls and eggs underwent analysis of variance (ANOVA) after assuring normal distribution with the Shapiro-Wilk test and homogeneity of variances with the Levene test and the means were grouped according to the Scott-Knott\u0026rsquo;s (1974) test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) in the R software (R Core Team, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eBacterial VOCs decreased J2 motility and increased mortality\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 36 bacterial strains used in this study were distributed in four categories according to their level of VOC activity against the nematode \u003cem\u003eM. incognita\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Most strains (94.4%) produced volatiles that caused a very strong reduction in J2 motility, whereas 30.6 and 38.9% caused very strong and strong J2 mortality, respectively. Bacteria identified as species of the genus \u003cem\u003eBacillus\u003c/em\u003e were the most commonly found. Among the bacteria that produced VOCs causing very strong mortality and low motility were four strains of \u003cem\u003eB. pumilus\u003c/em\u003e, three of \u003cem\u003eB. sphaericus\u003c/em\u003e and one strain each of \u003cem\u003eB. cereus, B. amyloliquefaciens, Paenibacillus macerans\u003c/em\u003e and a non-identified strain.\u003c/p\u003e\n\u003ch3\u003eBacterial VOCs decreased egg hatching and motility and increased mortality of J2s\u003c/h3\u003e\n\u003cp\u003eAll the three selected bacterial strains tested significantly reduced \u003cem\u003eM. incognita\u003c/em\u003e egg hatching after 10 days of exposure across two independent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Hatching of \u003cem\u003eM. incognita\u003c/em\u003e eggs exposed to VOCs produced by \u003cem\u003eB. pumilus\u003c/em\u003e and \u003cem\u003eB. sphaericus\u003c/em\u003e was reduced to approximately 2% of the values observed for the control treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClassification of bacterial strains into categories of antagonistic activity of their volatile organic compounds (VOCs) on motility and mortality of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e J2s. The nematode was exposed to VOCs in two-compartment Petri dishes and motility and mortality were evaluated under a microscope. The antagonistic categories were classified into I- Low (motility: \u0026gt;48.4%; mortality: \u0026lt;47.2%), II- Moderate (motility: 48.3\u0026ndash;23.6%; mortality: 47.2\u0026ndash;63.6%); III- Strong (motility: 23.5\u0026ndash;22%; mortality: 72.6\u0026ndash;86.6%); IV- Very strong (motility: 2.7\u0026ndash;0%; mortality: 90.5\u0026ndash;100%). Each category was composed of combinations of motility and mortality levels that were significantly different according to the Scott-Knott\u0026rsquo;s test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNumber of strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c10\" namest=\"c3\"\u003e\u003cp\u003eCodes of strains in each antagonistic category\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eMotility\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003eMortality\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAcinetobacter johnsonii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1, 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus pumilus\u003c/em\u003e subg. B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e21, 22, 28, 36, 41, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e41,32, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e21, 22, 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19, 31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. sphaericus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e42, 43, 45, 53, 54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e42, 43, 54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. pumilus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3, 8, 12, 20, 26, 39, 48, 49, 51, 52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3, 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e12, 20, 26, 39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e48, 49, 51, 52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. marinus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePaenibacillus gordonae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e37, 40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP. macerans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNot identified (P11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2, 4, 11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2, 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe three bacterial strains decreased J2 motility to almost zero and caused mortalities of 100% at 72 and 96 h of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Exposure times lower than 48 h, although significantly decreased motility and caused higher mortality as compared to the control, were not consistent for some treatments across the experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Strain 52 was the least effective at 24 h of exposure, while the first significant effects on motility and mortality were observed after 48 h of exposure to the bacterial VOCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eInfectivity and reproduction of\u003c/b\u003e \u003cb\u003eM. incognita\u003c/b\u003e \u003cb\u003eJ2s exposed to bacterial VOCs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe number of galls and eggs of \u003cem\u003eM. incognita\u003c/em\u003e in tomato roots, also referred to as infectivity and reproduction, respectively, in the control treatment without any exposure to VOCs reduced approximately 60% when the J2s were 72 and 96 h old in comparison with 24 and 48 h old J2s, respectively. The same occurred to the bacterial treatments, where infectivity of \u003cem\u003eM. incognita\u003c/em\u003e J2 decreased after exposure to VOCs in a time-dependent manner. The number of galls and eggs was reduced to zero after exposing the J2s for 96 h to VOCs produced by strains 51 and 52 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Exposure times lower than 96 h did not result in the complete suppression of \u003cem\u003eM. incognita\u003c/em\u003e, although the number of galls and eggs were lower than the control for strains 51 and 52 at 48 and 72 h. The exposure to VOCs for 24 h was insufficient to significantly reduce the numbers of galls and eggs by all the strains studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The regression analysis revealed that exposures of 72 h for strains 51 and 52 and of 96 h for strain 43 were sufficient for the numbers of galls and eggs to be completely suppressed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMajor volatile organic compounds (relative area\u0026thinsp;\u0026ge;\u0026thinsp;1.0% in the chromatograms) produced by three \u003cem\u003eBacillus\u003c/em\u003e strains identified by gas chromatography-mass spectrometry analyses. The compounds were identified by comparison of the mass spectra to Wiley 7.0, NIST 12 and NIST 62 mass spectral libraries.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eB. sphaericus\u003c/em\u003e\u003c/p\u003e\u003cp\u003e43\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eB. pumilus\u003c/em\u003e\u003c/p\u003e\u003cp\u003e51\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eB. pumilus\u003c/em\u003e\u003c/p\u003e\u003cp\u003e52\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNovel VOCs\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKnown, tested\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eKnown, not tested\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-methylpropan-1-amine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-methylbutan-1-amine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,5-dimethylpyrazine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-dimethylpyrazine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-ethyl-3-methylpyrazine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3,4-dimethylfurazan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-ethyl-2,5-dimethylpyrazine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-ethyl-3,5-dimethylpyrazine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4-methylnonane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-butoxyethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-methylnonane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-(1,1-dimethylethyl)cyclobut-3-ene-1,2-dione\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-dimethylheptan-4-one\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,2,5-trimethylhexane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-diethyl-3,5-dimethylpyrazine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1-(2-hydroxyethoxy)tridecane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,3,5,8-tetramethyldecane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2(1,1-dimethylethyl)-4-methylphenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" 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colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenzene-1,3-diamine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTridecan-1-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOctadecan-1-ol\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDodecan-6-one\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-methylundec-10-enal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecan-2-one\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7-methyloct-2-yne\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-ethylhexan-1-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-propylpentan-1-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-ethyl-4-methylpentan-1-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" 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align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(Z)-5-methyldec-2-ene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-bis(1,1-dimethylethyl)-4-methylphenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,4-bis(1,1-dimethylethyl)phenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,5-bis(1,1-dimethylethyl)phenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3,5-bis(1,1-dimethylethyl)phenol\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-bis(1,1-dimethylethyl)phenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eVOCs being reported for the first time as produced by \u003cem\u003eBacillus\u003c/em\u003e spp. in this study; \u003csup\u003e2\u003c/sup\u003eVOCs produced by bacteria or other microorganisms and tested against nematodes; \u003csup\u003e3\u003c/sup\u003eVOCs produced by bacteria or other microorganisms, but not tested against nematodes. The searches were done in the mVOC database (Kemmler et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and in the scientific literature. \u003csup\u003e4\u003c/sup\u003eTian et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and \u003csup\u003e5\u003c/sup\u003eMazumdar and Thakur (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported the indicated VOCs as produced by bacteria.\u003c/p\u003e\n\u003ch3\u003eIdentification of bacterial VOCs by gas chromatography-mass spectrometry (GC-MS)\u003c/h3\u003e\n\u003cp\u003eAnalysis by GC-MS of the VOCs produced by \u003cem\u003eB. sphaericus\u003c/em\u003e 43 and \u003cem\u003eB. pumilus\u003c/em\u003e 51 and 52, revealed the presence of 19, 11 and 14 compounds, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Only the most abundant compounds, with a relative peak area\u0026thinsp;\u0026ge;\u0026thinsp;1% in the chromatogram were considered in this analysis. None of the compounds found in the control (TSB medium) presented the same retention time or mass spectrum of the VOCs produced by the bacteria.\u003c/p\u003e\u003cp\u003eAlthough the three bacterial strains were from the genus \u003cem\u003eBacillus\u003c/em\u003e and two of them, strains 51 and 52 were \u003cem\u003eB. pumilus\u003c/em\u003e, they secreted relatively different mixtures of volatile compounds. Two compounds, 2,5-dimethylpyrazine and 2,6-dimethylpyrazine were detected in the three strains analysed. Only the two compounds listed above were common to strains 43 and 51, whereas strains 43 and 52 also secreted 2,6-bis(1,1-dimethylethyl)-4-methylphenol, and strains 51 and 52 secreted benzene-1,3-diamine in addition to the two compounds secreted by all three strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eActivity of purified commercial VOCs\u003c/h2\u003e\u003cp\u003eA total of 23 purified commercial VOCs were tested, eight were produced by at least one of the selected bacterial strains and the other 15 were used as standards for comparison purposes. Only benzaldehyde, which was not produced by any bacterial strain, caused 100% mortality and completely inhibited the motility of the J2s (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Five other compounds, with only decan-2-one produced by one bacterial strain (\u003cem\u003eB. pumilus\u003c/em\u003e 51), were able to cause high mortality (82\u0026ndash;100%) and decrease motility (0-\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e mortality and motility of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e second-stage juveniles (J2) exposed to volatile organic compounds from commercial sources. Values between parentheses correspond to the purity according to the manufacturer. Compounds marked with an asterisk were detected in at least one bacterial strain shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Means followed by the same letter in a column are not significantly different according to the Scott-Knott test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJ2 Motility\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJ2 Mortality\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e94.7 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.8 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-bis(1,1-dimethylethyl)-4-methylphenol* (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e94.3 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0 h\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-dimethylheptan-4-one* (96%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45.3 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.2 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-bis(1,1-dimethylethyl)phenol* (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.7 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.9 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-butoxyethanol* (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.7 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.2 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-dimethylpyrazine* (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.6 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,5-dimethylpyrazine* (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.7 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.9 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-ethylhexan-1-ol* (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34.7 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.9 e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecan-2-one* (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82.4 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,4,4-trimethylpent-1-ene (96%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55.7 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.2 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNonane (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e94.3 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.8 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTerpineol, mixture of isomers (96%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70.0 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.5 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecanal (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e78.7 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.6 f\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUndecan-6-one (97%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.3 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.8 f\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-phenylethanol (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.5 f\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDodecan-2-one (97%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45.0 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.8 e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOctan-1-ol (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.3 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63.6 d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3,7-dimethylocta-1,6-dien-3-ol (linalool) (97%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.7 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e69.2 d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUndecan-2-one (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22.0 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71.3 d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNonan-2-one (97%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.3 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e92.1 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOctan-2-ol (96%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.0 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3,3,5-trimethylcyclohexanone (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.3 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOctan-3-ol (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.9 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenzaldehyde (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100.0 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e3%) of \u003cem\u003eM. incognita\u003c/em\u003e J2s (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the seven other compounds produced by the bacterial strains, only 2-butoxyethanol had some effect on motility (17%) and 2-ethylhexan-1-ol had a mild effect on mortality (32%) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVolatile organic compounds (VOCs) are carbon-based, possess low-molecular weight and high vapor pressure, making them evaporate at room temperatures (Diyapoglu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These compounds are produced by all living organisms and some of them are toxic to diverse plant pathogens, including nematodes, fungi, insects and bacteria (Gu et al, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Diyapoglu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although there are many publications available in the recent literature on the effects of VOCs on nematodes (Xu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cheng et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cruz-Magalhaes et al., 2021; Chen et al., 2021), the diversity of both bacteria and the VOCs they produce, warrant the pursuit of additional studies.\u003c/p\u003e\u003cp\u003eIn order to expand our knowledge on the diversity and activity of VOCs against \u003cem\u003eM. incognita\u003c/em\u003e we studied a collection of endophytic bacterial strains obtained from tomato and pepper. From the 36 bacterial strains used in this study, 34 strains produced VOCs that strongly lowered the motility of \u003cem\u003eM. incognita\u003c/em\u003e J2 and 11 strains strongly increased J2 mortality. \u003cem\u003eBacillus sphaericus\u003c/em\u003e 43 and \u003cem\u003eB. pumilus\u003c/em\u003e 51 and 52 were randomly selected for their strong effects on J2 motility and mortality also reduced egg hatching and the number of eggs and galls on tomato plants. These bacteria produced different patterns of VOCs that were not related to the species of the strain. The VOC decan-2-one, produced by \u003cem\u003eB. pumilus\u003c/em\u003e 51, in its purified form significantly lowered the motility and increased the mortality of \u003cem\u003eM. incognita\u003c/em\u003e J2.\u003c/p\u003e\u003cp\u003eThe highest activities against \u003cem\u003eM. incognita\u003c/em\u003e in our study were achieved by VOCs from \u003cem\u003eBacillus\u003c/em\u003e species. This genus is known for its ability to produce volatile compounds active against PPNs (Cruz-Magalhaes et al., 2021; Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dai et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Indeed, most (94%) of the bacterial strains included in our study were able to strongly reduce J2 motility, whereas 31% caused mortality rates higher than 90%. All strains that caused high mortality were also included in the category that strongly decreased motility. Other authors have found much lower levels of mortality in their screening experiments, which varied from 3.5 to 12.5% (Gu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Costa et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These results may be explained by the differences in the time of nematode exposure to the bacterial VOCs, which was 72 h in our study and 24 h in the other studies (Gu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Costa et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Further experiments are still needed to estimate the potential of bacterial strains from different isolation sources in their potential to antagonise PPNs through the production of VOCs. While the strains used in our study were obtained from the interior of tomato and pepper plants, other authors collected potential bacterial antagonists from cow dung (Lu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), tobacco soil (Gu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and \u003cem\u003eMeloidogyne exigua\u003c/em\u003e egg masses (Costa et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eBacillus sphaericus\u003c/em\u003e strain 43 and \u003cem\u003eB. pumilus\u003c/em\u003e strains 51 and 52 were chosen for more detailed analyses on the activity of their VOCs. The VOCs of these strains decreased \u003cem\u003eM. incognita\u003c/em\u003e egg hatching, J2 motility and infectivity and reproduction \u003cem\u003ein planta\u003c/em\u003e and increased J2 mortality. Our experiments with tomato plants showed a 60% reduction in infectivity and reproduction, which is in the range observed by other authors (Bui et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cruz-Magalhaes et al., 2021; Wu et al., 2022; Song et al., 2023). However, the modes of action of the volatiles produced by strains 43, 51 and 52 against \u003cem\u003eM. incognita\u003c/em\u003e were not precisely determined. From our study, we know these strains act by reducing egg hatching and motility of J2 and increasing their mortality.\u003c/p\u003e\u003cp\u003eThe VOCs produced by the bacterial strains employed in this study differed markedly, even though two strains were from the same species, \u003cem\u003eB. pumilus\u003c/em\u003e. From a total of 39 compounds produced, only four were shared by two or three strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating the high diversity of the compounds. A comparison of the 39 VOCs produced by the studied strains with the ones deposited in the mVOC database (Kemmler et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) revealed that only 12.8% (five compounds from this study) were previously tested against nematodes. The majority of the compounds (51%, i.e., 20 compounds) were produced by bacteria or other microorganisms, but not tested against nematodes, whereas the remaining 36% (14 compounds) are being reported for the first time as produced by bacteria (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although we have been very strict and removed all compounds detected in the uninoculated medium, we cannot completely rule out that some of the VOCs we are reporting for the first time as being produced by bacteria are not artefacts of our experimental set up.\u003c/p\u003e\u003cp\u003eThese highly diverse compounds are produced in complex mixtures and in order to determine the activity of individual VOCs against nematodes we employed a selected set of purified chemicals commercially available. Out of eight compounds produced by the strains under study, three were previously tested by other authors against nematodes and five were tested for the first time in our study (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Only one of the VOCs, decan-2-one, produced by \u003cem\u003eB. pumilus\u003c/em\u003e strain 51, showed a significant reduction in motility and caused high mortality in \u003cem\u003eM. incognita\u003c/em\u003e J2s. Decan-2-one was highly active against \u003cem\u003eM. incognita\u003c/em\u003e J2s with an activity that did not differ from the fungicides carbofuran and fluensulfone (Pacule et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the toxic activity of strain 51 may be mostly attributed to this volatile, but we still did not determine the major compounds responsible for the toxicity of the other remaining strains studied, 43 and 52. This is frequently complicated when the strains under study produce a great number of compounds with a minor effect that act synergistically for the final activity. This is further complicated when the VOCs are not commercially available in their purified form.\u003c/p\u003e\u003cp\u003eOur results revealed some interesting relationships between the activity against \u003cem\u003eM. incognita\u003c/em\u003e and the chemical structure of certain compounds. For example, the methyl ketones with 9 and 10 carbon atoms, nonan-2-one and decan-2-one, respectively were more active against \u003cem\u003eM. incognita\u003c/em\u003e than the similar molecules with 11 and 12 carbons, undecane-one and dodecan-2-one, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). By the same token, the primary alcohol octan-1ol is significantly less active than the secondary alcohols octan-2-ol and octan-3-ol, all of them with 8-carbon structural isomers (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, the primary alcohols 2-butoxiethanol and 2-ethylhexan-1-ol showed low activity against \u003cem\u003eM. incognita\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). By analysing the results we obtained for the alkane nonane, it is reasonable to expect low activity from 4-methylnonane and 2-methylnonane, both produced by \u003cem\u003eB. sphaericus\u003c/em\u003e 43 (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), but not yet tested against nematodes.\u003c/p\u003e\u003cp\u003eThe compounds benzaldehyde and 3,3,5-trimethylcyclohexanone were among the most active against \u003cem\u003eM. incognita\u003c/em\u003e. The toxicity of 3,3,5-trimethylcyclohexanone seems to be due to the presence of the cyclohexane ring. This hypothesis becomes stronger in light of the results of a screening experiment of 11 VOCs against \u003cem\u003eM. incognita\u003c/em\u003e, and only the three compounds with a cyclohexane ring were toxic (Mei et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The high activity of benzaldehyde may be related to the direct link of the carbonyl group to an aromatic carbon, whereas decanal, which is also an aldehyde, does not have the carbonyl group linked to an aromatic carbon and has shown much lower activity (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result suggests that methylundec-10-enal produced by \u003cem\u003eB. pumilus\u003c/em\u003e 51, also an aldehyde with the carbonyl group linked to a non-aromatic carbon, will probably show low activity against the nematode.\u003c/p\u003e\u003cp\u003eThe diversity of VOCs detected in the bacterial strains offer a unique opportunity to develop new agricultural products, including nematicides. These VOCs may be employed as sources of lead structures for these developments by chemical modifications in order to improve their stability, spectrum of activity and decrease their toxicity to the environment and human health, as shown for other compounds (Jia et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusion, the VOCs produced by endophytic bacteria were diverse and showed toxicity against \u003cem\u003eM. incognita\u003c/em\u003e when the strains were used in the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein planta\u003c/em\u003e experiments. On the other hand, the individual effects of each compound in the antagonism against \u003cem\u003eM. incognita\u003c/em\u003e could not be pointed out for all strains due to the lack of commercially available compounds to be assayed. The majority (25 VOCs) of the compounds produced by the bacterial strains were previously detected in microorganisms, including \u003cem\u003eBacillus\u003c/em\u003e strains, but about 36% of the compounds were not detected in any bacteria (Kemmler et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This study adds new VOCs produced by \u003cem\u003eBacillus\u003c/em\u003e that could lead to the development of novel nematicides, besides the direct development of the strains as biocontrol agents to be deployed in the field.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) for financial support and fellowships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. Funding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Competing interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests of any kind.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Authors\u0026rsquo; contribution statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData acquisition\u003c/strong\u003e: RCSP, ESF, AMP, MOS, NPL, RMS; \u003cstrong\u003eData analysis\u003c/strong\u003e: RCSP, DFO, JTS; \u003cstrong\u003eWriting and editing the original manuscript\u003c/strong\u003e: JTS, VPC; \u003cstrong\u003eReview of the final version\u003c/strong\u003e: JTS, DFO, AMP, NPL.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdam M, Heuer H, Hallmann J (2014) Bacterial antagonists of fungal pathogens also control root-knot nematodes by induced systemic resistance of tomato plants. 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Molecules 28(7):3182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28073182\u003c/span\u003e\u003cspan address=\"10.3390/molecules28073182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacillus pumilus, Bacillus sphaericus, biological control, endophytes, root-knot nematode","lastPublishedDoi":"10.21203/rs.3.rs-7543783/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7543783/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVolatile organic compounds (VOCs) produced by microorganisms have several roles, such as communication and antimicrobial defence. Soil and endophytic bacteria employ VOCs in their interactions with plants, other bacteria, fungi and nematodes. In this study, the VOCs produced by 36 endophytic bacterial strains from pepper and tomato plants were tested for their antagonism to the nematode \u003cem\u003eMeloidogyne incognita\u003c/em\u003e. The VOCs produced by most bacterial strains (94%) caused a strong decrease in motility, while 69% of the strains caused high mortality rates in second-stage juveniles (J2s) of \u003cem\u003eM. incognita\u003c/em\u003e. Among the strains that simultaneously caused a strong decrease in motility and high mortality, \u003cem\u003eBacillus pumilus\u003c/em\u003e strains 51 and 52 and \u003cem\u003eB. sphaericus\u003c/em\u003e strain 43 were selected for further studies. These three strains produced VOCs that decreased egg hatching, infectivity and reproduction on tomato plants. Gas chromatography revealed a diverse VOC profile in the three studied strains, with 36% of compounds not previously reported in bacteria. Only decan-2-one and 2-ethylhexanol out of the eight VOCs produced by the bacterial strains significantly reduced motility (3%) and increased mortality (82%) of \u003cem\u003eM. incognita\u003c/em\u003e J2s when tested in their purified form. These findings highlight both the potential of VOCs as templates for new nematicides and the bacterial strains as biocontrol agents against \u003cem\u003eM. incognita\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Volatile organic compounds from Bacillus spp. reduce egg hatching, motility, infectivity and reproduction of Meloidogyne incognita","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 10:09:05","doi":"10.21203/rs.3.rs-7543783/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-10-13T14:06:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-09T09:48:30+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-09T08:54:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2025-09-08T19:01:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-06T17:54:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2025-09-05T07:28:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d997c0e2-01f2-41d0-915d-9234ebe9e8d2","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:08:21+00:00","versionOfRecord":{"articleIdentity":"rs-7543783","link":"https://doi.org/10.1007/s41348-025-01201-2","journal":{"identity":"journal-of-plant-diseases-and-protection","isVorOnly":false,"title":"Journal of Plant Diseases and Protection"},"publishedOn":"2025-12-06 15:57:02","publishedOnDateReadable":"December 6th, 2025"},"versionCreatedAt":"2025-09-16 10:09:05","video":"","vorDoi":"10.1007/s41348-025-01201-2","vorDoiUrl":"https://doi.org/10.1007/s41348-025-01201-2","workflowStages":[]},"version":"v1","identity":"rs-7543783","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7543783","identity":"rs-7543783","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}