Multiple Modes of Nematode Control by Volatiles of Pseudomonas putida 1A00316 from Antarctic Soil against Meloidogyne incognita

Pseudomonas putida 1A00316 isolated from Antarctic soil showed nematicidal potential for biological control of Meloidogyne incognita; however, little was known about whether strain 1A00316 could produce volatile organic compounds (VOCs), and if they had potential for use in biological control against M. incognita. In this study, VOCs produced by a culture filtrate of P. putida 1A00316 were evaluated by in vitro experiments in three-compartment Petri dishes and 96-well culture plates. Our results showed that M. incognita juveniles gradually reduced their movement within 24–48 h of incubation with mortality ranging from 6.49 to 86.19%, and mostly stopped action after 72 h. Moreover, egg hatching in culture filtrates of strain 1A00316 was much reduced compared to that in sterile distilled water or culture medium. Volatiles from P. putida 1A00316 analysis carried out by solid-phase micro-extraction gas chromatography–mass spectrometry (SPME-GC/MS) included dimethyl-disulfide, 1-undecene, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy)-octadecane. Of these, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, and 2-undecanone had strong nematicidal activity against M. incognita J2 larvae by direct-contact in 96-well culture plates, and only 2-undecanone acted as a fumigant. In addition, the seven VOCs inhibited egg hatching of M. incognita both by direct-contact and by fumigation. All of the seven VOCs repelled M. incognita J2 juveniles in 2% water agar Petri plates. These results show that VOCs from strain 1A00316 act on different stages in the development of M. incognita via nematicidal, fumigant, and repellent activities and have potential for development as agents with multiple modes of control of root-knot nematodes.


INTRODUCTION
Plant parasitic nematodes (PPNs) cause serious damage to a wide range of crops worldwide . To date, more than 4100 species of PPNs have been described (Kyndt et al., 2014). Among these, the root-knot nematode (Meloidogyne spp.), found all over the world in tropical, subtropical, and temperate regions, is the most economically important plant nematode (Jones et al., 2013). The destruction caused by PPNs has been assessed to be more than 100-150 billion US dollars per year, of which more than half are due to Meloidogyne spp. (Nicol et al., 2011;Li et al., 2015;Kim et al., 2016). Among the Meloidogyne spp., Meloidogyne incognita is the most destructive because of its wide host range, including most flowering plants, as well as its short generation time, high reproduction rate, and ability to form complex diseases with other soil-borne pathogens such as fungi (Vos et al., 2013). M. incognita can infect the roots of over 2000 plant species and interferes with normal plant uptake of nutrients and water. Moreover, it causes physiological plant disorders (Jang et al., 2014), making it perhaps the most damaging of all crop pathogens (Trudgill and Blok, 2001). At present, the main method of controlling plant nematodes involves using chemical nematicides. However, these chemicals are toxic, have side effects against other organisms, and can adversely affect human health and the environment (Riga, 2011). The rotation and resistance of crop varieties are complementary strategies to control PPNs, but their effectiveness is limited (Jones et al., 2013). Therefore, new economical, effective, and environmentally friendly PPN controls are urgently needed.
Biological control is one such way to reduce pest losses, but more investigation on novel microorganisms in the environment is needed to speed the development of new agents for controlling root-knot nematodes . These pathogens inhabit the soil and typically are infected with indigenous bacteria and fungi, suggesting the possibility of using microorganisms to control PPNs (Zhang et al., 2015). In fact, there are specific microorganisms in soil, such as nematophagous bacteria and fungi, which have complex strategies for capturing, killing, and digesting PPNs, and they often target a specific stage of the nematode life cycle . Many secondary metabolites of microorganisms have nematicidal activity (NA) and therefore could become substitutes for highly toxic chemical nematicides. Through comparative genomic studies, isolation and purification, the nematicidal compounds alkaline metalloproteinase AprA and two metabolites, hydrogen cyanide and cyclo(L-Pro-L-Ile), were previously identified from Pseudomonas putida strain 1A00316 and shown to have good NA against M. incognita (Guo et al., 2016). Bacillus cereus strain S2 produced sphingosine and showed high NA against M. incognita (Gao et al., 2016). Moreover, Purpureocillium lilacinum, with the adjuvant avermectin, has been used effectively to control PPNs (Fisher, 1990;Kiewnick and Sikora, 2006).
Pseudomonas species are ubiquitous in nature and produce many secondary metabolites active against important plant pathogens (Giles et al., 2014). Members of the genus are physiologically and metabolically multifunctional, readily colonizing terrestrial and aquatic habitats such as soil, plants, and water (Troxler et al., 2012). Among the species of this genus, P. putida has been isolated from many niches and survives in soil containing organic pollutants and heavy metals. For example, P. putida JMQS1 isolated from detergent-contaminated soil exhibited quorum sensing along with its ability to degrade phenol (Antony and Jayachandran, 2016). Some strains of P. putida inhabit the rhizosphere or are endophytes that promote plant growth, making them ideal for biocontrol. The nematicidal effects of P. putida against M. incognita were noted previously; strain 1A00316 isolated from Antarctic soil showed good inhibition of M. incognita in vitro and in pot experiments, with biocontrol efficiency of nematodes as high as 71.67%. In addition, strain 1A00316 itself could induce systematic resistance in tomato by increasing the activity of three defense enzymes: phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase in tomato plants (Tang et al., 2014). Hydrogen cyanide and cyclo(L-Pro-L-Ile) also were identified from strain 1A00316 and exhibited NA against M. incognita (Guo et al., 2016), but little is known about whether volatile organic compounds (VOCs) are produced by the strain, or if they have potential for use in biological control against M. incognita.
Compared to solid nematicides, the greatest advantages of volatile nematicidal compounds are their good dispersibility and penetration in the soil. In the past, fumigants such as methyl bromide, which was used as nematicide, have been identified as contributing to the reduction of the ozone layer and overall poor air quality, so it is important to search for new green soil fumigation agents. Accordingly, this investigation focused on the identification and evaluation of VOCs from strain 1A00316. In the present study, we report (i) the identification the volatile compounds in the fermentation broth of P. putida 1A00316; and (ii) evaluation of their nematicidal, fumigation, and chemotaxis activities against M. incognita.

Collection of M. incognita Eggs and Second-Stage Juveniles and Propagation of Caenorhabditis elegans
Meloidogyne incognita eggs were collected from the roots of infested tomato plants (Solanum lycopersicum L.), which were previously infected with the nematodes in the greenhouse at 23-26 • C, and relative humidity 40-60%. The tomato plants were watered manually once a day. After 45 days, the plants were uprooted, the roots were rinsed free of soil with tap water, and the egg masses were picked into a bottle with a dissecting needle (Lee et al., 2014). After shaking the egg mass with 1% NaOCl (sodium hypochlorite) solution by hand in the bottle for 3 min, the solution was passed in turn through a series of filters with pore sizes of 74, 45, and 25 µm, and the sterilized eggs were collected from the 25-µm filter by spraying with sterile distilled water (SDW; Seo et al., 2013). Second-stage juveniles of M. incognita were obtained by using a modified Baermann funnel method under sterile conditions (Barker et al., 1985;Southey, 1986). Caenorhabditis elegans N2 (Bristol, wild type), was purchased from the Caenorhabditis Genetics Center (CGC). C. elegans was maintained at 20 • C on nematode growth medium plates seeded with Escherichia coli OP50.

Nematicidal Activity of Volatiles
A three-compartment Petri plate ( Figure 1A; 85 mm diameter; Fernando et al., 2005) was used to study the NA of bacterial VOCs. Three milliliters of the original culture filtrate was added into one compartment and 200 nematodes of either M. incognita or C. elegans were introduced onto the surface of layers of 2% water agar (WA) in the other two compartments. Control plates contained uninoculated 2216E medium in place of the culture filtrate. Plate lids were immediately sealed with Parafilm (Bemis) to avoid escape of the volatiles, and the plates were incubated at 28 • C in the dark. There were three replicates for each treatment and the experiments were repeated twice. After 24, 48, and 72 h, the numbers of mobile and immobile nematodes were counted under a dissecting microscope (Jiang Nan JS25B).
In order to confirm the NA of bacterial VOCs produced by strain 1A00316, activated charcoal was introduced into one of the three compartments and culture supernatant and nematodes were added to each of the other two compartments of the Petri plate ( Figure 1B; Gu et al., 2007). The activated charcoal can adsorb volatiles, blocking their activity and resulting in no loss of nematode viability. At the same time, 100 eggs  of M. incognita were immersed in 100 µL of SDW in wells of 96-well tissue culture plates surrounded by four adjacent wells containing culture filtrate of strain 1A00316. SDW and 2216E media in the surrounding wells served as controls. The experiment was repeated three times. The numbers of eggs hatching were counted after 2, 4, 6, 8, and 10 days of exposure with an inverted microscope (XDS-1B COIC, Chongqing Mike Photoelectric Instrument Limited Company, China).

Identification of Volatiles from Strain 1A00316
Strain 1A00316 was cultured for 48 h as described above, and volatiles were collected and analyzed by using SPME-GC/MS (Azenha and Vasconcelos, 2002;Dìaz et al., 2004). Fiber (65 µm PDMS/DVB fiber, Supelco, Bellefonte, PA, United States) used for SPME was first preconditioned with helium at 250 • C for 20 min. The extractions were performed in 20-mL headspace vials (22.5 mm × 75.5 mm) filled with 9 mL fermentation broth and a magnetic stirring bar. The vials were fixed inside a thermostatic water bath and samples were equilibrated at 60 • C for 1 h. The VOCs from 9 mL 2216E medium were used as controls. After the extraction, the fiber was inserted into the injection port of a gas chromatograph [Hewlett-Packard (HP) 7890A] coupled with a mass spectrometer (HP 5975C, Agilent Technologies,  United States) and desorbed for 5 min at 250 • C (Gu et al., 2007). The chromatographic separation was performed on a HP-5MS (30 m × 0.25 mm) × 0.25 µL column and helium was used as the carrier gas at a constant flow of 1 mL/min. The column was held at 40 • C for 2 min, then increased to 180 • C at a rate of 4 • C/min, held for 0 min, then increased to 240 • C at a rate of 5 • C/min, and held for 6 min. The MS detector was programmed as follows: EI ion source operating at 70 eV, acquisition range between m/z 35 and 550. The temperature of the transfer line and iron trap were 250 and 300 • C, respectively. The identification of a volatile compound was based on a comparison of the substance with GC/MS system data banks (NIST 08 Library). Each sample was tested twice.

Nematicidal Activity of Commercial VOCs
The NA of commercial VOCs was tested against M. incognita J2 larvae at a dose range of 10-1000 mg/L, and the 50% lethal concentration (LC 50 ) values were calculated. Stock solutions of pure compounds were prepared in methanol to overcome insolubility, whereas aqueous Tween-20 (0.3% v/v) was used for further dilution. Test solutions (200 µL) at various concentrations were added to detachable 96-well tissue culture plates and a suspension of 40-50 J2 juveniles was added to each well. SDW and a mixture of methanol and Tween-20 served as controls (Aissani et al., 2013). Plate lids were sealed with Parafilm to avoid evaporation and plates were kept at 20 • C in the dark. Juveniles in solutions of VOCS were observed with the aid of an inverted microscope after 24, 48, and 72 h and were categorized as either motile or immotile/paralyzed. The experiments were performed three times, and every treatment was replicated three times.

Fumigant Activity of VOCs against J2
The various commercial VOCs were introduced into one well in 96-well tissue culture plates surrounded by four wells containing 50 nematode J2 juveniles suspended in distilled water. SDW and a mixture of methanol and Tween-20 served as controls. Percentages of nematode death were recorded in response to the fumigant activity of the VOCs in the adjacent wells. Assessments were made at 24, 48, and 72 h (Ntalli et al., 2011). The experiments were performed three times, and every treatment was replicated three times.

Effect of VOCs on Egg Hatching
Whether VOCs could inhibit egg hatching of M. incognita was tested over a dose range of 20-1000 mg/L. One hundred eggs of M. incognita suspended in 10 µL SDW were introduced into detachable 96-well tissue culture plates and combined with 200 µL of a range of concentrations of commercial VOCs. SDW as well as a mixture of methanol and Tween-20 served as controls. Each treatment had three replicates and the experiments were repeated twice.
At the same time, 100 eggs of M. incognita in 100 µL SDW were introduced into wells of 96-well tissue culture plates and surrounded by four wells containing 200 µL of one of the VOCS at 1000 µg/mL. SDW and 2216E media served as controls. Plate lids were sealed with Parafilm and plates were incubated in the dark at 20 • C. The numbers of J2 hatchings were counted after 2, 4, 6, 8, and 10 days of exposure under the inverted microscope and hatch rate was measured. Each treatment had four replicates and the experiments were repeated twice.

Chemotaxis of J2 Nematodes to Culture Filtrate and VOCs
Chemotaxis was assessed on Petri plates containing 2% WA (Tajima et al., 2001). A 5 mm filter paper disc immersed in various concentrations of culture filtrate or solutions of volatile substances was placed on the test area (A) of 35 mm Petri dishes, while a filter paper immersed in 2216E medium or a mixture of methanol and Tween-20 was added to the opposite side of the plate (area B) as a control (Figure 2). Subsequently, 150 J2 juveniles of M. incognita were added to the center (area D) of the Petri dish, and the dish was incubated in a dark cabinet at 20 • C for 8 h (Hu et al., 2012). The numbers of J2s in areas A and B were then counted under a dissecting microscope to calculate the chemotaxis index (C.I.; Saeki et al., 2001), calculated after 8 h as. C.I. = (the number of nematodes in test area − the number of nematodes in control area)/(the number of nematodes in test area + the number of nematodes in control area).
For 0 < C.I. < 1, M. incognita was attracted to the tested sample; if −1 < C.I. < 0, the tested sample repelled M. incognita; and if C.I. = 0, the sample had no effect on the nematode. Experiments were performed in triplicate, and treatments were replicated three times.

Data Analysis and Statistics
Mortality values for in vitro bioassays against M. incognita were corrected by Abbott's formula (Abbott, 1925). The LC 50 was calculated by Probit analysis. Data from the chemotaxis assay were analyzed using a homogeneity test of variance. If the variance was homogeneous (P ≤ 0.05), a paired Student's t-test was chosen; otherwise, the Wilcoxon rank sum test was used. Data from all assays except the chemotaxis assay were analyzed by one-way variance with SPSS 20. Means among treatments were compared by Fisher's least significant difference (LSD) test at the P = 0.05 level.

Nematicidal Effects of Strain 1A00316 VOCs
We evaluated effects of VOCs produced by culture filtrate on M. incognita J2s by the three-compartment Petri plate method. Juveniles of M. incognita gradually reduced movement within 24-48 h and mostly were immobile after 72 h of incubation, with mortality ranging from 6 to 100% (Figure 3). We also found that strain 1A00316 had strong NA (≥80%) against M. incognita J2 juveniles, but not to C. elegans (Supplementary Table S1), indicating that C. elegans was not sensitive to these VOCs. Moreover, most of the VOCs were adsorbed in the plates containing activated charcoal, and NA values decreased from 86% to less than 10%. In addition, neither culture filtrate nor 2216E medium affected egg hatching after 2 and 4 days, although hatching slowly declined after 6 days, and reached 11.24% at 10 days, a value significantly less than control values in SDW or 2216E medium (Figure 4). These results suggest that the culture filtrate of strain 1A00316 contains VOCs that kill nematodes and inhibit egg hatching, and they are consistent with the hypothesis that VOCs from strain 1A00316 are responsible for the NA.
With the aim of exploring the potency of seven of these VOCs [(Z)-3-decen-1-ol acetate was not commercially available], their NA was tested on juveniles of M. incognita in vitro.  Table S2).

Effect of Commercial VOCs on Egg Hatching of M. incognita
The effect of the seven commercial organic compounds was measured in vitro by direct-contact. The results of the tested concentrations suggested that all seven VOCs had adverse effects on egg hatching of M. incognita (see Supplementary  Tables S3-S9). (Z)-hexen-1-ol acetate at concentrations from 1-(Ethenyloxy)-octadecane >1000 >1000 (Z)-3-decen-1-ol acetate ND ND FL, fiduciary limits; ND, not determined.

Chemotaxis of Culture Filtrates and VOCs by J2 Juveniles of M. incognita
Chemotaxis of culture filtrates from strain 1A00316 is presented in Figure 8. The results show that the higher concentrations of culture filtrates (original concentration and 1/3 original concentration) immobilized J2 nematodes (−1 < C.I. < 0) and acted as an attractant (0 < C.I. < 1) at from 1/5 to 1/20 original concentration. Interestingly, many nematodes were found in the test area (A), and the seven VOCs at concentrations of 1-10,000 mg/L showed C.I. ranging from −1 to 0 ( Table 3). These results indicated that the seven VOCs repelled M. incognita J2 juveniles. Among them, the C.I. of different concentrations of (Z)-hexen-1-ol acetate, dimethyl-disulfide, and 2-nonanone showed no significant difference from the control. However, with the increase of concentration of 2-octanone, 1-undecene, 1-(ethenyloxy)-octadecane, and 2-undecanone, the absolute value of the C.I. gradually increased and the four VOCs had greater ability to repel nematodes. Comparing the C.I. of all seven VOCs at different concentrations, we found that 2-undecanone showed the greatest effect, with a C.I. value of −0.725 at 10,000 µg/mL.

DISCUSSION
In a preliminary experiment, different concentrations of culture filtrate from strain 1A00316 were added to 96or 24-well tissue culture plates to test NA against eggs or J2 juveniles of M. incognita. This work showed effects  greater than 90% killing of M. incognita J2 juveniles in the control group situated near culture filtrates, leading us to test SDW and culture filtrates in separate plates.
The egg hatching in different 24-well plates containing 150 eggs, with SDW coexisting with different concentrations of culture filtrate in other wells, had significantly different inhibition effects on egg hatching after 10 days (Supplementary Figure S1). These results suggested that VOCs could contribute to the decrease in egg hatching in culture filtrates of strain 1A00316. Based on the above experiment, we hypothesized that strain 1A00316 may produce volatiles to kill M. incognita J2 juveniles and inhibit egg hatching, and that there may be multiple modes of nematode control of M. incognita.
Subsequently, we identified eight VOCs with SPME-GC/MS analysis: dimethyl-disulfide, 2-nonanone, 2-undecanone, 2-octanone, 1-undecene, 1-(ethenyloxy)-octadecane, and (Z)-hexen-1-ol acetate. Dimethyl-disulfide, 2-nonanone, and 2-undecanone exhibited strong NA (NAs > 80%) against both juveniles and eggs at a concentration of 0.5 mmol by fumigation after 7 days of exposure . In another study, dimethyl-disulfide had the strongest NA (LC 90 = 0.162 mmol/L) against Bursaphelenchus xylophilus exposed for 24 h in directcontact (Yu et al., 2015). 2-Nonanone and 2-undecanone were reported to induce paralysis in M. incognita and M. javanica by direct-contact (Ntalli et al., 2011). However, to our knowledge, no NA has been reported for 2-octanone, 1-undecene, 1-(ethenyloxy)-octadecane, and (Z)-hexen-1-ol acetate against M. incognita. In our study, we found the strongest direct contact NA by dimethyl-disulfide, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, and 2-undecanone, but only 2-undecanone had fumigant activity against J2 juveniles. In addition, the seven VOCs had the ability to inhibit egg hatching of M. incognita both by direct-contact and as a fumigant. Our further analysis focused on chemotaxis by J2 juveniles of M. incognita, and we found that they were repelled by higher concentrations of culture filtrate and attracted to lower concentrations. In recent years, there has been a proliferation of research on the chemotaxis of nematicidal VOCs. Hu et al. (2012) reported that Chaetomium globosum NK102 repelled M. incognita chemotaxis, but in that study the whole colony was regarded as a research object and the authors did not explore which factors acted as repellants of the nematode. In our study, all of the identified VOCs had a phobotactic effect on the nematodes, which explains the repellant activity of higher concentrations of the culture filtrates against M. incognita. In nature, VOCs can also exhibit an attractant effect: for example, the bacterium Bacillus nematocida B16 lures the nematode by emitting six potent VOCs, of which benzyl benzoate, benzaldehyde, 2-heptanone, and acetophenone were potent attractants, with the bacteria then entering the nematode intestine and causing death (Niu et al., 2010). These examples demonstrate the diversity and complexity of the VOCs against nematodes.
In summary, the results of our experiments using threecompartment Petri dishes and identification by GC/MS were consistent with the hypothesis that strain 1A00316 may produce VOCs with multiple modes of nematode control. We identified eight VOCs from strain 1A00316. Among them, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z)-hexen-1ol acetate, and 2-undecanone had strong NA in direct contact with M. incognita J2 juveniles, but only 2-undecanone had fumigant activity. In addition, the seven VOCs inhibited egg hatching both by direct-contact and as a fumigant. All of the seven VOCs repelled M. incognita. Our results showed that the VOCs from strain 1A00316 have at least three modes by which to control M. incognita: NA, fumigant activity, and repellent activity. The VOCs also acted on different stages in the nematode life cycle including J2 juveniles and eggs. The multiple modes of action of the VOCs produced from P. putida 1A00316 are consistent with the potential of the strain to be an effective biocontrol agent against M. incognita in the greenhouse. Further investigation is needed to understand the molecular mechanisms responsible for the NA of the VOC compounds produced by strain 1A00316.

AUTHOR CONTRIBUTIONS
YZ conceived and designed the work that led to the submission, acquired data, and played an important role in interpreting the results. ZS and GL provided the strain Pseudomonas putida 1A00316. MC and LZ provided the suggestions and helped to perform the analysis with constructive discussions. DH and WC helped to perform the analysis with constructive discussions. LT and DW drafted and revised the manuscript. ZY provided a platform for the experiments. JZ drafted and revised the manuscript and approved the final version.