Activity of Vetiver Extracts and Essential Oil against Meloidogyne incognita

In the U.S., vetiver cv. Sierra was purchased from Agriflora Tropicals, Caguas, Puerto Rico and grown in the greenhouses of the Mycology and Nematology Genetic Diversity and Biology Laboratory (2-mon-old plants) and the Invasive Insect Biocontrol and Behavior Laboratory (4-yr-old plants) at USDA ARS, Beltsville, Maryland. Vetiver plants that were harvested after 2 mon of growth had been planted in Promix PGX (Premier Tech Horticulture, Quakertown, PA) in one-gallon (3.8 L) pots and maintained at 24°C to 29°C, with natural and supplemental lighting combined for a 16-hr daylength. Vetiver plants that were harvested after 4 yr of growth had been planted in Promix BX in 19 gallon pots (72 L) and maintained at 18°C to 24°C, with natural and supplemental lighting combined for a 16- to 18-hr daylength. Roots were chopped into 1 cm pieces and dried at room temperature (23-25°C) for 5 to 7 d. The dried material was ground by a milling machine (Thomas-Wiley, Laboratory Mill Model 4, Swedesboro, NJ) and passed through a sieve with a pore size of 2 mm. The powders were stored at 4°C until use.

In Thailand, 1-yr-old roots of field-grown vetiver cv. Songkha 3 were collected from plants grown at the Land Development Department, Nakhon Ratchasima province, Thailand. The vetiver plants were cultivated in Pak Chong series, red brown earth loam soil. In addition, because French marigold (Tagetes patula L.) has been widely studied for nematode management, plants of cv. Durango Mix (AGA Agro Co., Ltd.) were purchased from an orchard (Phu Ruea District, Loei Province, Thailand) to compare their activity with vetiver. Roots of vetiver and French marigold were chopped into 1 cm pieces and dried at room temperature (28-30°C) for 5 to 7 d. The dried material was ground by a milling machine (Hammer Mill, Department of Farm Machinery, Kasetsart University, Thailand) and passed through a sieve with a pore size of 2 mm. The powders were stored at room temperature until use.

Shoot and root extracts from 2-mon-old and 4-yr-old vetiver plants were prepared from cv. Sierra in the U.S. Since vetiver shoots can be harvested and used as soil mulch (Lim, 2016), studies with shoots focused on water-soluble compounds that might more readily leach into the soil. Research on root extracts was conducted with aqueous extracts, and with ethanol extracts that would provide material for GC-MS investigation of secondary metabolites. Procedures for making extracts were similar to those described in Meyer et al. (2006). To summarize, water-soluble compounds were extracted from dried, powdered shoots and roots (10% dry weight plant material/volume water) on a mechanical rotary shaker (VWR, Advanced Digital Shaker, Radnor, PA) at 100 rpm for 24 hr at room temperature (25°C). The mixture was filtered through eight layers of cheesecloth, centrifuged for 10 min at 3,000 g, and the supernatants were then sequentially filtered through syringe filters: 1.0 µm, 0.45 µm (Whatman, Clifton, NJ) and 0.2 µm (Nalgene, Rochester, NY), and stored at 4°C until use.

To prepare ethanol extracts, dried root powder was immersed in 95% ethanol (10% dry weight plant material/volume ethanol) and placed on a shaker as described above. The solution was then vacuum filtered through Whatman No. 1 filter paper, and the filtered solution was evaporated in a rotary evaporator at 45°C, 172 bar (Heidolph 2 Rotavac, Schwabach, Germany) to nearly dry. The small amount of remaining ethanol was air dried and the extracts were stored at -20°C.

Haitian vetiver oil (VO), also from V. zizanioides, was purchased as a commercial product (Texarome, Inc. TX, USA). The vetiver oil had been extracted by water distillation, with 99% purity.

Ethanol root extracts from French marigold and from 1-yr-old vetiver plants (cv. Songkha 3) were prepared in Thailand. Dried powder from vetiver roots or French marigold roots was immersed in 95% ethanol (20% dry weight plant material/volume ethanol) for 7 d in the dark at room temperature (28-30°C) without shaking. The solution was then vacuum filtered through a Whatman No. 1 filter paper and concentrated in a rotary evaporator at 50°C, 200 bar (Heidolph Hei-VAP Value, Schwabach, Germany). The concentrated extract was then transferred to a separatory funnel and partitioned with dichloromethane (Sac Science-Eng, Ltd, Thailand) at a ratio of 1:3. The dichloromethane phase was kept and re-evaporated at 50°C, dried with nitrogen, and finally stored at −20°C (procedure modified from Laksanaphisut, 2010).

Meloidogyne incognita (Kofoid & White) Chitwood Race 1 (originally isolated in Maryland) was maintained on pepper (Capsicum annuum L.) cv. PA-136 in a greenhouse with temperatures and lighting as described above for vetiver plants. Procedures for J2 collection were like those in Meyer et al. (2016). Pepper roots from 2-mon-old plants infected with M. incognita were collected from the greenhouse; egg masses were handpicked from roots and surface sterilized in 0.6% sodium hypochlorite for 3.5 min, rinsed in sterile distilled water (SDW), and transferred to a hatching chamber (25-µm-diam. Spectra/Mesh Nylon Filter, Spectrum Laboratories Inc., Rancho Dominguez, CA) in a storage dish. The hatching chamber was incubated at 27°C and 40 rpm in a refrigerated incubator shaker (Innova 4230, New Brunswick Scientific, Edison, NJ) for 3 d, and the J2 were then collected and used for assays. J2 were adjusted to a final concentration of 20 per 10 µl.

Laboratory assays with aqueous and ethanol extracts were conducted in 96-well polystyrene plates, following the procedures in Meyer et al. (2006). Approximately 20 J2 were added to each well in 10 µl SDW, and then 190 µl of extract, or of vetiver oil, was added to each well. The microwell plates were covered by a plastic adhesive sealing film (Excel Scientific, Inc., Victorville CA) and the lids were sealed with Parafilm (Bemis, Neenah, WI). The plates were incubated at 26°C.

For microwell assays of vetiver shoot aqueous (VSA) extracts and vetiver root aqueous (VRA) extracts from 2-mon-old plants, 100 µl of a 50.0 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO) stock solution was added to 9.9 ml of 100% vetiver aqueous extract (100% was the undiluted extract, and was filtered through a 0.2 µm syringe filter prior to addition of the antibiotic). The streptomycin sulfate was used to eliminate the growth of microbes that sometimes occurred in the aqueous extract assays despite sterile filtering. The extract solutions were then diluted to 75%, 50%, and 25% with SDW, and 190 µl of each extract was added to 10 µl J2 in SDW in each well. There were eight aqueous extract treatments: VSA and VRA at final concentrations in the wells of 94%, 71%, 47%, and 24%. Control treatments were SDW and the highest concentration of the antibiotic, equivalent to that in the 94% treatment: 0.5 mg/ml streptomycin sulfate, also diluted with 10 µl J2 in SDW in each well for a final streptomycin sulfate concentration of 0.48 mg/ml. Treatments were replicated in eight wells in each of the two trials, for a total of 16 wells. Active J2 (those exhibiting any movement within 5 seconds) and inactive J2 (no movement after 5 sec) were counted after 1 and 2 d (Days 1 and 2) incubation in the treatments. Following the Day 2 count, the J2 were rinsed two times with SDW and incubated in the second SDW rinse. Active vs. inactive J2 (those exhibiting body movements and those that were not) were counted on Day 3. J2 not active after the water rinse were considered dead.

Vetiver root ethanol (VRE) extracts and French marigold root ethanol (FMRE) extracts were prepared at 0.1 and 0.01 mg/ml concentrations in a solvent (referred to as CTD) of equal parts castor oil (BASF, Ludwigshafen, Germany), Tween 80 (Sigma, St. Louis, MO), and dimethyl sulfoxide (DMSO: Fisher, Fairlawn, NJ). To dissolve the extracts, a stock solution was prepared of 10 mg of crude ethanol extract in 1.0 ml of 100% CTD. To prepare the extract concentration of 0.1 mg/ml, 100 µl of this solution was then added to 9.9 ml SDW and was sequentially filtered through 1.0 µm, 0.45 µm, and 0.2 µm sterile filters. To prepare the 0.01 mg/ml extract, 1 ml of the 0.1 mg/ml filtered solution was added to 9.0 ml SDW. After addition of 190 µl extract to 10 µl J2 in SDW, the final extract concentrations in the wells were 0.095 mg/ml and 0.0095 mg/ml, respectively. The treatments at each of the two concentrations were (1) 2-mon VRE, (2) 1-yr VRE, (3) 4-yr VRE, (4) VO (vetiver oil), and (5) FMRE; these comprised treatments 1 through 10. Control treatments were: (11) SDW; (12) 1% CTD (which was 0.95% after dilution in the wells); and 13) 0.1% CTD (0.095% after dilution in the wells). Treatments were each replicated in eight wells for each of the two trials, for a total of 16 wells per treatment. Counts were performed as described above.

Chemotaxis assays were conducted with methods modified from Laznik and Trdan (2013). For our studies, 1.4% water agar (Noble agar, Difco, Leeuwarden, The Netherlands) was poured into plastic plates that were sold as lid sizes of 100- and 60-mm diam. The corresponding plate bases were 85-mm diam. (used for ethanol extracts; 25 ml agar added per plate), and 53-mm diam. (used for aqueous extracts, 10 ml agar per plate). Dried aqueous extracts were dissolved in SDW to obtain 0.1 g/ml. The treatments tested with aqueous extracts were: (1) 2-mon VRA and (2) 2-mon VSA. SDW was the control. Dried ethanol extracts were dissolved in 95% ethanol to obtain 0.1 g/ml. The treatments tested with ethanol extracts were: (1) 2-mon VRE, (2) 1-yr VRE, (3) 4-yr VRE, (4) VO, (5) FMRE, and (6) 95% ethanol. SDW was the control. Each plate was divided into three areas (Figure 1): the starting area, the treatment area, and the control area. Extract treatments (10 µl) were gently pipetted onto the agar at the treatment point, 10 µl of live J2 (ca. 20 J2) onto the starting point, and 10 µl of water onto the control point. Ethanol-dissolved treatments and the ethanol control were air dried for 10 min to allow ethanol to dissipate. The plates were incubated at 26°C for 24 hr. Each treatment was replicated with 5 plates in each of the two trials, for a total of 10 plates. The J2 were counted in each area and the chemotaxis index was calculated using the formula: CI = (number of nematodes in the treatment area – number of nematodes in the control area)/total number of nematodes in the assay. The interpretations of the CI values for the treatments are summarized as follows (from Laznik and Trdan, 2013): ≥0.2 indicated an attractant; between 0.2 and 0.1, a weak attractant; from 0.1 to -0.1, without effect; between -0.1 and -0.2, a weak repellent; and ≤-0.2, a repellent.

Figure 1

GC-MS analysis of the volatile constituents of VRE and VO was carried out using an Agilent 6890 N GC instrument coupled with a 5973 mass selective detector. The instrument was equipped with a DB-5 capillary column of length 30 m, internal diam. 0.25 mm, and film thickness 0.25 µm. The carrier gas was helium at a constant flow rate of 2.0 ml/min. The oven temperature was maintained at 50°C for 5 min and ramped to 280°C at 10°C/min and held there for 3 min. Diluted samples (in methanol) of 2 µl were injected in the split mode with a split ratio of 25:1 and the inlet temperature was 280°C. The mass detector scanned from 4.5 min to 30 min at a mass range from 40 to 400 (EI, 70 eV). The MS ion source temperature was 230°C and the quadrupole temperature was 150°C. The components were identified by comparing mass spectra with the NIST mass spectra library in the GC/MS data system.

Data were analyzed with the statistical package JMP Version 12.1.0 (SAS Institute, Inc., 2015). Differences among treatments were determined by analysis of variance (ANOVA) and means were compared using Tukey–Kramer’s adjustment for multiple comparisons (P ≤ 0.05).

An initial screening of 2-mon VSA, 2-mon VRA, and 4-yr VRA indicated that aqueous extracts from the younger plants showed greater activity against M. incognita J2 than extracts from the older plants (unpubl. data), so aqueous extract studies focused on the 2-mon-old plants. On Day 1, J2 inactivity in all 47%, 71%, and 94% treatments was significantly higher than in the streptomycin sulfate or water controls (Table 1). The 94% and 71% VRA had a greater effect than any of the other treatments, with ca. 5 times more inactive J2 than in the streptomycin sulfate control. On Day 2, 94% VRA again caused the highest J2 inactivity (4½ times greater than in the streptomycin sulfate control), with results similar to 94% VSA and 71% VRA. All treatments except 24% VRA resulted in higher J2 inactivity than in the streptomycin sulfate control. On Day 3, following a 1-d rinse in water, every treatment had significantly higher mortality than in the controls (Table 1). Compared with the streptomycin sulfate control, mortality ranged from 2 times greater (in 24% VRA) to more than 3 times greater (47% VSA and all 71% and 94% treatments). For all but one treatment, the J2 mortality in the water rinse was no different from the percentage inactive J2 on Day 2 (Table 1). Only 47% VSA resulted in significantly more nonviable J2 on Day 3 than inactive J2 on Day 2. No treatment showed a significant increase in J2 activity following the water rinse.

The aqueous extracts used for the microwell assays were not dried to determine weights. However, weights were estimated based on the weights of the dried aqueous extracts used for the chemotaxis assays. For 30 g of dry, powdered plant material, the dry weight of the crude aqueous shoot extract (384 mg) was almost twice the weight of the crude aqueous root extract (207 mg). An estimate of the extract weights used in the microwell assays would be: 94% VSA (9.02 mg/ml), 94% VRA (4.87 mg/ml), 71% VSA (6.82 mg/ml), 71% VRA (3.68 mg/ml), 47% VSA (4.51 mg/ml), 47% VRA (2.43 mg/ml), 24% VSA (2.30 mg/ml), and 24% VRA (1.24 mg/ml). The 24% VRA was likely about half the concentration of the 24% VSA, which might have resulted in this root extract causing significantly lower mortality than the highest root and shoot concentrations.

On Day 1 in the ethanol extracts, J2 activity was similar among the 0.95 mg/ml FMRE and VRE (crude extract) treatments, with more than twice as many inactive J2 than in SDW or the 0.95% CTD control (Table 2). In contrast, 0.95 mg/ml VO treatment resulted in only 20% inactive J2, which was not significantly different from the controls. Unlike the higher concentration of vetiver extracts and FMRE, no treatment at the lower concentration (0.095 mg/ml) was active against J2. Results on Day 2 were similar to those on Day 1, with the 0.95 mg/ml FMRE and VRE all resulting in more than 3 times as many inactive J2 as in 0.95% CTD. The 0.95 mg/ml VO again had no effect on J2 activity. Most of the 0.095 mg/ml treatments, including FMRE, were not effective against J2. At 0.095 mg/ml, only 2-mon VRE was antagonistic to J2, with 2½ times more inactive J2 than in 0.095% CTD. On Day 3, after incubation in a water rinse, the 0.95 mg/ml FMRE and vetiver extract treatments all resulted in greater mortality than the VO or the SDW and 0.95% CTD controls. The 0.95 mg/ml FMRE treatment was the most nematotoxic (Table 2), increasing J2 mortality by more than 6 times compared with mortality in 0.95% CTD. The 0.95 mg/ml VRE treatments increased J2 mortality by more than three times compared with 0.95% CTD. Mortality in FMRE was significantly affected by extract concentration, and was twice as great in the higher concentration of FMRE. However, mortality in 0.095 mg/ml VRE and VO treatments was not significantly lower than in the comparable 0.95 mg/ml VRE and VO treatments. Among the 0.095 mg/ml treatments, all but vetiver oil and extract from 2-mon-old cv. Sierra plants increased mortality compared with the comparable CTD control.

J2 activity and mortality were also compared among days to determine if the treatments were nematostatic or nematotoxic. No difference in J2 activity was found between Day 1 and Day 2 for any treatment. However, on Day 3, J2 mortality increased compared with inactive J2 on Day 2 in almost all extracts (Table 2). The one exception was 0.095 mg/ml VRE from 2 mon-old plants, in which J2 inactivity on Day 2 was the same as J2 mortality after the rinse on Day 3. There were also no significant differences in percent inactive or dead J2 among Days 1, 2, and 3 in vetiver oil, in CTD, or in the SDW control.

The chemotaxis assay used to evaluate the repellent/attractant effects of crude aqueous extracts from vetiver shoots and roots demonstrated that J2 were repelled by 0.1 g/ml VRA and VSA (Table 3). The two aqueous extracts had similar chemotaxis indices. In chemotaxis assays with VO and crude ethanol extracts from French marigold and vetiver roots, the ethanol control did not attract or repel J2 (Table 4). French marigold and vetiver root extract treatments were repellent, although the 1-yr VRE was not significantly different from the water control (Table 4). The root extract from 2-mon VRE (cv. Sierra, greenhouse, U.S.) was twice as repellent as extracts from 1-yr VRE (cv. Songkha 3, field, Thailand) and 4-yr VRE (cv. Sierra, greenhouse, U.S.), but was only significantly greater than the effect of 1-yr VRE. Repellency by French marigold root extracts was similar to repellency with all of the vetiver root extract treatments. VO was not a repellent or an attractant, with a CI that was not significantly different from 1-yr VRE, 4-yr VRE, water, or ethanol in its effects on J2.

The major component peaks of the three VRE extracts and the vetiver oil (VO) were detected and identified by GC/MS analysis including MS library matching (Table 5, Figs. 2 and 3). The VRE extracts, which represented different vetiver cultivars, growing conditions, and ages, contained similar constituents that differed mainly in their concentrations. Two of the major components in all three extracts and the VO were the sesquiterpene acid 3,3,8,8-tetramethyltricyclo[5.1.0.0(2,4)]oct-5-ene-5-propanoic acid and the sesquiterpene alcohol 6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-ol (Fig. 2A-D; 3 A,B and Table 5). Among the extracts, the percentage area of 3,3,8,8-tetramethyltricyclo[5.1.0.0(2,4)]oct-5-ene-5-propanoic acid was lowest in the extract from 4-yr, greenhouse-grown vetiver. However, it was even lower in the VO, with the percentage areas in the extracts being 20 to 60 times greater than in the VO. Unlike the extracts from the greenhouse-grown cv. Sierra, the extract from 1-yr, field-grown vetiver cv. Songkha 3 had two other major components, which are esters of fatty acids: ethyl hexadecanoate (ethyl palmitate) and ethyl 9,12-octadecadienoate (ethyl linoleate) (Figs. 2B; 3C,D and Table 5). Vetiver oil had a major component that differed from those found in the extracts: the sesquiterpene alcohol γ-costol (2-(4a,8-dimethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-2-yl)-prop-2-en-1-ol) (Fig. 3E, Table 5).

Figure 2

Figure 3