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Keywords:

  • Meloidogyne javanica;
  • Myrtus communis;
  • nematicidal plant;
  • nematode control;
  • plant extract;
  • root-knot nematode

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Nematicidal activity of the leaf powder, leaf extracts and formulated leaf extracts of Myrtus communis, an evergreen shrub that is widely distributed in Israel and other Mediterranean countries, was evaluated using the root-knot nematode Meloidogyne javanica in in vitro and pot experiments. Leaf powder added to sand at 0·1% (w/w) reduced the number of juveniles recovered from the sand by more than 50%. Reduction in galling index and number of nematode eggs on tomato roots was also observed by incorporating the leaf powder at 0·1–0·4% (w/w) in the soil in pot experiments. Leaf powder extracts with methanol or ethanol showed the highest nematicidal activity among all extracts tested. Emulsifiable concentrates of leaf-paste extract at a concentration as low as 0·005% (a.i., w/w) reduced the number of juveniles recovered from treated sand and the gall index of cucumber seedlings. The extract paste at 26 g m−2 was also effective in reducing the gall index of tomato plants in field-plot experiments. The leaf powder at 0·2% and the formulated leaf-paste extract at 0·02% were also nematicidal to Tylenchulus semipenetrans and Ditylenchus dipsaci, but not to Pratylenchus penetrans or Steinernema feltiae. At least three nematicidal compounds were found in the leaf extract upon fractionation by thin-layer chromatography. The results suggest that the leaf powder and paste extract of M. communis are potential nematicides against root-knot nematodes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Management of plant-parasitic nematodes has become more difficult because of the restricted use or ban of effective soil fumigants and nematicides, particularly methyl bromide; alternative nematode control methods or compounds with the control efficacy and spectrum of methyl bromide have yet to be established. In addition, demand for environmentally friendly nematicides and those that can be used in organic farming systems is on the rise. The use of organic soil amendments or antagonistic plants constitutes one such environmentally friendly alternative, alone or as part of an integrated control programme (Oka, 2010). Preparations from several parts of the neem tree (Azadirachta indica) are well known as nematicidal amendments of plant origin (Mojumdar, 1995). Tagetes spp. are some of the most investigated plants as inter- or cover crops for nematode control (Gommers & Bakker, 1988). α-Terthienyl was isolated from Tagetes roots and found to be highly nematicidal (Uhlenbroek & Bijloo, 1958), and these plants are effective at controlling the genera Meloidogyne and Pratylenchus (Hackney & Dickerson, 1975; Ploeg, 1999). Essential oils of herb and medicinal plants or their constituents may constitute another source of environmentally friendly nematode-control agents (Oka et al., 2000; Oka, 2001). In previous studies, the leaves of Inula viscosa (Asteraceae), a common weed in Israel and other Mediterranean countries, were shown to have fungicidal and nematicidal activity (Oka et al., 2001, 2003; Wang et al., 2004; Cohen et al., 2006). Nematicidal sesquiterpenic acids (costic and isocostic acid) were isolated from the plant leaf extract, and showed nematicidal activity against the root-knot nematode Meloidogyne javanica at concentrations as low as 50 mg kg−1 sand (Oka et al., 2001). Formulated leaf extracts of I. viscosa showed potential as a nematicide of plant origin (Oka et al., 2003, 2006). Chrysanthemum coronarium, another member of the Asteraceae widely distributed in Israel, was reported to have nematicidal activity against M. javanica and M. artiellia (Pérez et al., 2003; Bar-Eyal et al., 2006).

Myrtus communis (Myrtaceae) is an evergreen shrub that is widely distributed in Israel and other Mediterranean countries. This aromatic plant is known in medicinal folklore and is cultivated in some countries, mainly for extraction of its essential oils. Leaf extract of M. communis has been shown to possess antibacterial activity, and highly effective antibacterial acyrphloroglucinols were isolated from plant extracts (Kashman et al., 1974; Rotstein et al., 1974; Mansouri et al., 2001; Appendino et al., 2002). Antifungal, insecticidal and molluscicidal activities of the plant have also been reported (Garg & Dengre, 1988; Deruaz et al., 1993; Traboulsi et al., 2002). In in vitro tests, the essential oil of M. communis showed very weak nematicidal activity against second-stage juveniles (J2s) and eggs of M. javanica (Oka et al., 2000), and against mixed stages of Bursaphelenchus xylophilus (Barbosa et al., 2010). However, nematicidal activity of the leaf powder or its extracts appears not to have been reported. In the present study, the nematicidal activities of the leaf powder, leaf extracts and formulated leaf-paste extract of M. communis were tested against M. javanica in in vitro and pot experiments. Fractionation of nematicidal compounds in the leaf extract is also described.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Nematodes

Eggs of M. javanica were extracted from tomato roots infected with the nematode (Hussey & Barker, 1973) and spread on a nylon sieve (30 μm) in a Petri dish filled with water. Emerged J2s were collected daily and stored at 15°C. J2s up to 4 days old were used in experiments. Adults and juveniles of Ditylenchus dipsaci and Pratylenchus penetrans were extracted from infected garlic plants and wheat roots, respectively, by the Baermann funnel method (Barker, 1985). Infective juveniles of Steinernema feltiae were obtained from an infected cadaver of the greater wax moth, Galleria mellonella, using a White trap (White, 1927). Tylenchulus semipenetrans J2s were obtained from roots of orange trees by the Baermann funnel method.

Plant material

Fresh leaves of M. communis were collected from Kibbutz Kramim, Israel, and air-dried at 60°C for 48 h. Dried leaves were pulverized in a Cyclone sample mill before use.

Extraction

Myrtus communis leaf powder was extracted with acetone, ethyl acetate, n-hexane, undiluted or diluted (60%) ethanol or methanol, by stirring 0·4 g powder into 10 mL of the selected solvent for 2 h, followed by centrifugation at 2000 g for 10 min. Aqueous extract of the powder was prepared by stirring the powder in water overnight at 4°C. The powder residue after extraction was air-dried, and nematicidal activity was checked by the J2 recovery test described further on.

Formulations of leaf-paste extract

Leaf powder was extracted by mixing 10 g powder in 100 mL methanol or acetone for 2 h. The extracts were filtered, and the solvents were evaporated under reduced pressure at 35°C to obtain paste-like extracts. Emulsifiable concentrates containing 24·4% (w/w) of the methanol or acetone leaf-paste extract as the active ingredient (a.i.) were obtained by mixing 0·4 g paste extract with a mixture of 1·0 g d-limonene (Fluka) and 0·25 g Tween-80 (Sigma). The emulsifiable concentrates of the paste extract obtained by extraction with acetone and methanol were termed AC-EC and ME-EC, respectively. A blank formulation was prepared by replacing the pastes with water in the formulation to check possible nematicidal activity of d-limonene, which was reported to have insecticidal activity (Hink & Fee, 1986).

Nematode recovery test

Effect of leaf powder on M. javanica

The leaf powder was mixed with 10 g dry, untreated, nematode-free dune sand in 25-mL glass vials at concentrations of 0·1, 0·2 and 0·4% (w/w). Dune sand without powder served as a control. Approximately 200 M. javanica J2s in 1·3 mL water were introduced into each bottle. The vials were partly capped to prevent water evaporation, and nematodes were incubated in the sand for 7 days at 27°C in the dark. Live nematodes were recovered from the sand by the Baermann tray method with a 30-μm sieve (Barker, 1985) and counted. This bioassay was used in previous studies, and found to be useful for detecting nematicidal activity of plant tissues or extracts (Oka et al., 2001, 2006). The experiment was performed twice, with four replicates per treatment.

Effect of leaf powder and methanolic extract on other plant-parasitic and entomopathogenic nematodes

Second-stage juveniles of M. javanica (300 nematodes per vial), adults and juveniles of D. dipsaci (300 nematodes per vial) and P. penetrans (200 nematodes per vial), and infective juveniles of S. feltiae (200 nematodes per vial) in 1·3 mL water were introduced separately into glass vials each containing either 10 g sand amended with leaf powder (0·2%, w/w), or 10 g sand mixed with 0·5 mL methanolic extract of leaf powder. Dune sand without powder or extract served as a control. The sand treated with methanolic extract was dried before introducing nematodes. Trials with all the nematode species were performed at the same time. The nematodes were incubated for 7 days at 27°C, and live nematodes were counted after recovery by the aforementioned sieve method. Each treatment consisted of four replicates.

Effect of leaf powder extracts on M. javanica

The leaf powder extracts (1·0 mL) extracted with the various solvents were mixed with dry sand (10 g) in 25-mL glass vials, and the solvents were evaporated to dryness. Only methanol was applied to untreated sand as a control after evaporating to dryness because in a preliminary experiment, none of the solvents in the sand, following evaporation, were found to affect nematode recovery. Approximately 300 M. javanica J2s in 1·3 mL water were introduced into the sand and incubated for 7 days. Live J2s were recovered and counted as described above. Nematicidal activities of the leaf powder residues after extraction were also tested by the same method at a concentration of 0·4% (w/w). The experiment was performed twice, with four replicates per treatment.

Effect of emulsifiable concentrates of leaf powder extracts on M. javanica

AC-EC and ME-EC were added to the sand at concentrations of 0·005, 0·01 and 0·02% (a.i., w/w). Approximately 300 M. javanica J2s in 1·3 mL water were introduced into the sand and incubated for 7 days at 27°C. Untreated sand served as a control. Following incubation, live nematodes were counted by the aforementioned sieve method. The experiment was performed twice, with four replicates per treatment.

Bioassay with cucumber

The leaf powder was mixed into the dune sand (250 g) in 180-cm3 plastic pots at concentrations of 0·05, 0·1 and 0·2% (w/w), and 1000 M. javanica J2s were introduced into the sand via five 2-cm-deep holes, and incubated for 7 days at 27°C in a growth chamber. Untreated sand served as a control. Two germinated cucumber (Cucumis sativus cv. Delila) seeds with roots about 1 cm long were planted in each pot after the incubation period. Pots were irrigated as needed. Seedlings were carefully uprooted 10 days later, and gall index (GI) was assessed on a scale of 0 to 5 (0 = no infection; 1 = 1–20% of roots galled; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; and 5 = 81–100%). The experiment was performed twice, with four replicates per treatment.

AC-EC, ME-EC and the blank formulation were diluted in 25 mL water and added to 250 g dry sand in 180-cm3 pots at concentrations of 0·0025, 0·005 and 0·01% (a.i., w/w). Untreated sand served as a control. Treated and untreated pots were inoculated with 1000 M. javanica J2s by the method already described, and incubated for 7 days at 27°C in a growth chamber. Germinated cucumber seeds were planted, and GI was recorded as described above. The experiment was performed twice, with four replicates per treatment.

Bioassay with tomato

The leaf powder was mixed into 1000 g dry sandy soil (pH 8·8, clay:silt:sand, 15:5:80; organic matter < 0·1%) in 700-cm3 plastic pots at concentrations of 0·1, 0·2 and 0·4% (w/w). Untreated sand served as a control. The sand was inoculated with 3000 M. javanica J2s in 100 mL water and incubated at 27°C. One-month-old tomato (cv. Daniela) seedlings were transplanted into the pots 7 days after treatment, maintained at 27 ± 2°C in a growth chamber, and received 50 mL 0·1% solution of 20-20-20 (N-P-K) fertilizer every week. Fresh shoot weight, GI (0–5) and number of nematode eggs per plant extracted by the sodium hypochlorite method (Hussey & Barker, 1973) were recorded 5 weeks after planting. The number of nematode eggs was transformed to log10(+ 1) before statistical analysis. The experiment was performed twice, with five replicates per experiment.

AC-EC and ME-EC were added to the sandy soil at concentrations of 0·01, 0·02 and 0·04% (a.i., w/w). Untreated sand served as a control. The soil was inoculated with 3000 M. javanica J2s in 100 mL water and incubated for 7 days at 27°C. Tomato seedlings were transplanted and fresh shoot weight, GI (0–5) and number of nematode eggs per plant were recorded 5 weeks after planting. The experiment was performed twice, with four replicates per treatment.

Field-plot experiment

Plots (1 m2 × 1·1-m-deep plastic tubes buried in the field) filled with sandy soil naturally infested with M. javanica were treated with water-diluted ME-EC by manual incorporation into the soil (down to 30 cm) at a dose of 26 g a.i. per plot. The nematode population before application was 2·5 J2s per 50 g soil taken at a depth between 15 and 30 cm. Ten days after treatments, four 1-month-old tomato seedlings (cv. Daniela) were planted in each plot. The plants were irrigated daily with 1 L water per plant, and fertilized once a week with 200 mL per plant of fertilizer solution (0·1% solution of 20-20-20). The plants were uprooted 60 days after planting, and fresh shoot weight, GI and number of nematode eggs per plant root were recorded. GI was assessed on a scale of 0–10 (Bridge & Page, 1980). Untreated plots served as controls. The experiment was performed twice, with five replicates per treatment.

Fractionation of leaf extract

Leaf extract was prepared by mixing 10 g leaf powder in 100 mL methanol for 2 h, and filtering and concentrating the solution by evaporation of the solvent under reduced pressure at 35°C to obtain an extract paste. The paste, diluted (10%) in methanol, was run on a silica-gel thin-layer chromatography (TLC) plate (Merck 5721) with chloroform and methanol (6:4, v/v) as the mobile phase. After the run, some of the TLC plates were cut and stained with iodine vapour to determine the Rf values of the major spots. Silica gel was scraped off the plates from 12 regions with the main spots, extracted with 50 mL methanol and dried. These fractions were diluted in 100 μL ethanol and added to 5 g sand in a 25-mL glass vial. Nematicidal activity of each fraction was tested using 200 M. javanica J2s by the nematode recovery test. The same fractions were also re-run on the TLC plate and stained with iodine vapour to confirm their Rf values.

Data analysis

Data were subjected to anova, and means were separated according to the Tukey–Kramer HSD test (α = 0·05) or Student’s t-test. Data from the two trials were not combined because no significant interactions were found between trials or treatments in a preliminary factorial analysis of variance, and the variance homogeneity was violated according to Levene’s test of anova procedure. All calculations were performed with jmp software (SAS Institute).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Nematode recovery test

Effect of leaf powder on M. javanica

The number of recovered M. javanica J2s was reduced (F = 30·2, < 0·0001 in the first trial, F = 152·3, < 0·0001 in the second trial) by mixing the leaf powder into the sand at 0·1–0·4% in both trials (Fig. 1). Reductions of 86 and 99% were obtained with 0·4% leaf powder in the first and second trials, respectively.

image

Figure 1.  Number of Meloidogyne javanica second-stage juveniles recovered from sand amended with Myrtus communis leaf powder at concentrations of 0·1–0·4% after 7 days’ incubation in the first (a) and second (b) trial. The sand was inoculated with 200 nematode juveniles. Values are means ± SD of four replicates and means were separated using the Tukey–Kramer HSD test (α = 0·05). Cont = control.

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Effect of leaf powder and methanolic extract on other plant-parasitic and entomopathogenic nematodes

Leaf powder and methanolic extract of M. communis significantly reduced the number of recovered M. javanica (Table 1). The number of recovered D. dipsaci was also reduced following sand amendment with the leaf powder at a concentration of 0·4% (w/w) and methanolic extract. The leaf powder and the methanolic extract did not affect the recovery of adults or juveniles of P.  penetrans, or of S. feltiae infective juveniles.

Table 1.   Numbers of nematodes recovered from sand amended with powder or methanolic (ME) extracts of Myrtus communis leaves at 0·4% (w/w) and 0·5 mL per 10 g sand, respectively, after 7 days’ incubation
NematodeControlaLeaf powdera F value P value
  1. J2: second-stage juveniles; JA: juveniles and adults; IJ: infective juveniles.

  2. aValues are means ± SD of four replicates.

  3. *Significantly different from control, according to Student’s t-test (≤ 0·05).

Meloidogyne javanica (J2)160·5 ± 31·384·5 ± 14·3*30·340·0015
Ditylenchus dipsaci (JA)140·5 ± 28·368·0 ± 21·9*16·40·0067
Pratylenchus penetrans (JA)79·3 ± 4·270·7 ± 23·2 0·760·41
Steinernema feltiae (IJ)76·5 ± 16·694·0 ± 15·3 3·920·095
 ControlME extract F value P value
Meloidogyne javanica (J2)114·0 ± 25·152·5 ± 12·3*19·3<0·0001
Ditylenchus dipsaci (JA)80·5 ± 15·846·5 ± 22·0* 6·30·0458
Pratylenchus penetrans (JA)82·0 ± 6·989·0 ± 12·9 0·550·4867
Steinernema feltiae (IJ)57·0 ± 15·147·5 ± 10·4 1·070·3397
Effect of leaf powder extracts on M. javanica

Extracts of M. communis leaves extracted with either water or organic solvents all reduced (F = 21·7, < 0·0001 in the first trial, F = 24·7, < 0·0001 in the second trial) the number of recovered M. javanica J2s in both trials (Fig. 2a,c). The diluted and undiluted methanolic and ethanolic extracts showed the highest nematicidal activity. The powder residues after extraction still showed nematicidal activity (F = 62·0, < 0·0001 in the first trial, F = 45·5, < 0·0001 in the second trial) (Fig. 2b,d), with those after extraction with water or methanol showing the lowest nematicidal activity of all residues.

image

Figure 2.  Number of Meloidogyne javanica second-stage juveniles recovered from sand amended with Myrtus communis leaf-powder extracts (a, c) and powder residues after extraction (b, d) after 7 days’ incubation in the first (a, b) and second (c, d) trials. Leaf powder was extracted with water (Aqueous), diluted (60%) or undiluted methanol (MeOH), diluted (60%) or undiluted ethanol (EtOH), acetone, ethyl acetate (E. acetate) or n-hexane. The sand was inoculated with 300 nematode juveniles. Values are means ± SD of three replicates, and means were separated using the Tukey–Kramer HSD test (α = 0·05). Cont = control.

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Effect of emulsifiable concentrates of leaf powder extracts on M. javanica

Increasing concentrations of AC-EC and ME-EC reduced (F = 54·0, < 0·0001 in the first trial, F = 33·5, < 0·0001 in the second trial) the number of M. javanica J2s recovered from the sand in both trials (Fig. 3). Reduction in J2 recovery did not differ between AC-EC and ME-EC at any concentration except 0·005% in the first trial (Fig. 3a).

image

Figure 3.  Number of Meloidogyne javanica second-stage juveniles recovered from sand treated with formulated 0·005–0·02% acetone (AC) or methanol (ME) extracts of Myrtus communis leaf paste after 7 days’ incubation in the first (a) and second (b) trials. The sand was inoculated with 300 nematode juveniles. Values are means ± SD of five replicates, and means were separated using the Tukey–Kramer HSD test (α = 0·05). Cont = control.

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Bioassay with cucumber

Increasing concentrations of M. communis leaf powder in the sand reduced the GI of cucumber seedling roots in both trials (Fig. 4a,b). No galls were found on the roots of seedlings grown in the sand amended at 0·2% (w/w). AC-EC reduced the GI of cucumber seedlings at 0·005–0·01% (a.i., w/w) in the first trial (Fig. 4c) and at 0·0025–0·01% in the second trial (Fig. 4d). The blank formulation did not affect the GI at any concentration in the first trial (Fig. 4b), but a slight reduction was found at 0·01% in the second trial (Fig. 4d).

image

Figure 4.  Effect of Myrtus communis leaf powder at concentrations of 0·05–0·2% (a, b) and formulated leaf-paste extract obtained by extraction with acetone (AC) and its blank formulation at concentrations of 0·0025–0·01% (c, d) on root gall index of cucumber seedlings grown in Meloidogyne javanica-inoculated sand in the first (a, c) and second (b, d) trials. Germinated cucumber seedlings were transplanted 7 days after treatment, and root gall index was recorded 10 days later. Values are means ± SD of eight seedlings in four replicates, and means were separated using the Tukey–Kramer HSD test (α = 0·05). Cont = control.

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Bioassay with tomato

Amending the sand with M. communis leaf powder at 0·1–0·4% caused reductions of up to 42% and 27% in tomato shoot fresh weight in the first (F = 8·25, = 0·0015) and second trials (F = 5·52, = 0·0085), respectively (data not shown). All concentrations of leaf powder reduced the root GI of tomato in both trials (Fig. 5a,c). A few galls were observed on roots of plants grown in sand amended with the leaf powder at 0·4% in the first trial (Fig. 5a), but there were no galls at all in the second trial (Fig. 5c). The number of nematode eggs per plant was also reduced by the powder (Fig. 5b,d): in particular, no eggs were recovered from plants grown in sand amended with the powder at 0·4% in either trial.

image

Figure 5.  Effect of Myrtus communis leaf powder at concentrations of 0·1–0·4% on root gall index (a, c) and number of nematode eggs (b, d) on tomato plants grown in Meloidogyne javanica-inoculated soil in the first (a, b) and second (c, d) trials. Tomato seedlings were transplanted 7 days after treatment, and root gall index and number of nematode eggs were recorded 5 weeks later. Values are means ± SD of five replicates, and means were separated using the Tukey–Kramer HSD test (α = 0·05). Cont = control.

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At concentrations higher than 0·02% (a.i. w/w), ME-EC reduced tomato shoot fresh weight in the first trial and AC-EC reduced it in both trials (data not shown). Tomato GI was reduced by application of ME-EC (Fig. 6a,c) and AC-EC (Fig. 6e,g) at a concentration of 0·01%, except in the first trial with ME-EC (Fig. 6a). The number of nematode eggs per plant was also reduced by the formulated paste extracts at the concentrations that reduced GI (Fig. 6b,d,f,h). No eggs were recovered from plants grown in sand treated with AC-EC at 0·04% (Fig. 6f,h).

image

Figure 6.  Effect of formulated Myrtus communis leaf-paste extracts obtained by extraction with methanol (ME) (a–d) or acetone (AC) (e–h) at concentrations of 0·01–0·04% on root gall index (a, c, e, g) and number of nematode eggs (b, d, f, h) on tomato plants grown in Meloidogyne javanica-inoculated soil in the first (a, b, e, f) and second (c, d, g, h) trials. Tomato seedlings were transplanted 7 days after treatment, and root gall index and number of nematode eggs were recorded 5 weeks later. Values are means ± SD of five replicates, and means were separated using the Tukey–Kramer HSD test (α = 0·05). Cont = control.

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Field-plot experiment

There was no difference (F = 0·52, = 0·492 in the first trial, F = 4·3, = 0·072 in the second trial) in tomato shoot fresh weight between control and treated plots in either trial (Table 2). GI of tomato roots was reduced by the treatment with ME-EC in both trials, although the reduction rates were relatively low. The number of nematode eggs per plant was also reduced, by more than 50%, by ME-EC treatment.

Table 2.   Effects of an emulsifiable concentrate formulation of Myrtus communis leaf paste extracted in methanol (ME-EC) of leaf powder applied at 26 g m−2 on shoot fresh weight (SFW), root gall index (GI) and number of Meloidogyne javanica eggs per 10 g roots of tomato plants grown in nematode-infested plots
 SFW (g)aGI (0–10)aEggs per 10 g rootsa,b
  1. aValues are means ± SD of five replicates with four plants each.

  2. bNumber of nematode eggs was transformed to log10(+ 1) before statistical analysis.

  3. *Significantly different from control, according to Student’s t-test (≤ 0·05).

Trial 1
 Control38·4 ± 19·27·7 ± 0·2805 162 ± 243 906
 ME-EC46·8 ± 17·75·5 ± 0·7*372 903 ± 143 946*
Trial 2
 Control48·9 ± 11·18·8 ± 0·6187 200 ± 35 545
 ME-EC60·4 ± 6·95·6 ± 1·2*63 675 ± 24 245*

Isolation of nematicidal compounds

The methanol extract of the leaf showed 12 major spots (Rf: 0·15, 0·21, 0·37, 0·41, 0·45, 0·56, 0·62, 0·68, 0·75, 0·81, 0·88 and 0·91) on a TLC plate. Three fractions, with Rf values of 0·62, 0·75 and 0·88, showed nematicidal activity. The number of J2s recovered from the sand treated with these three fractions was 22, 26 and 16, respectively, whereas the mean number of recovered juveniles from sand treated with the other fractions, including the control, was 72·4 ± 9·3 (range 61–92).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In the present study, the nematicidal activity of M. communis was evaluated in juvenile recovery tests, plant galling bioassays in pots with cucumber and tomato, and field plots. Exposure of M. javanica J2s or eggs to an aqueous extract in vitro is the most common test for evaluating its effects on mobility or hatching. This method was not employed here because of the weak nematicidal activity of the aqueous extracts. The recovery test used here evaluated effects on J2 mobility caused by death or paralysis, while the plant bioassays revealed effects on nematode infectivity. Both tests were required as a nematicide may inhibit infection without affecting J2 mobility (Wright, 1981). Extracts prepared with organic solvents can be applied to soil and tested after evaporation. Recovery tests require smaller amounts of the test materials than plant bioassays, and results are obtained in a shorter time. The bioassay with cucumber requires less time, with a smaller amount of test materials, than tomato. The tomato bioassay, however, enabled the phytotoxicity of the test materials and their effect on nematode reproduction rate to be evaluated.

It was shown here that M. communis leaf powder added to sand at concentrations higher than 0·1% reduces the number of recovered M. javanica J2s and the GI of cucumber seedlings. Plants with high nematicidal activity, I. viscosa and C. coronarium, were shown to be effective at reducing galling caused by M. javanica by incorporation of dry and fresh plant materials in sandy soil at concentrations higher than 0·1% and 0·5%, respectively (Oka et al., 2001; Bar-Eyal et al., 2006). In general, heavier soil containing higher clay contents may require higher application rates than sandy soil. Although the M. communis leaf powder showed high nematicidal activity in all assays, that of I. viscosa leaf powder appears to be even higher (Oka et al., 2001). An undesirable feature of plant-derived nematicidal compounds is allelopathy or phytotoxicity to plants. As with I. viscosa, leaf powder and extracts of M. communis were somewhat phytotoxic, reducing the shoot fresh weight of tomato plants at higher application rates. This phytotoxic effect can probably be avoided by delaying planting after incorporation of the nematicidal compound into the soil.

Sensitivity of plant-parasitic nematodes to nematicides varies among species. Generally, J2s of Meloidogyne spp. are very sensitive to plant-derived nematicides (Oka et al., 2001). Myrtus communis powder was also nematicidal to D. dipsaci, but not to P. penetrans. The leaf powder of I. viscosa, at a lower concentration, showed nematicidal activity against Pratylenchus mediterraneus, although this activity was much lower than that against M. javanica J2s (Oka et al., 2001). Similar tolerance of Pratylenchus to plant-derived nematicidal compounds was also observed with thiarubrine C (Sánchez de Viala et al., 1998). The entomopathogenic nematode S. feltiae was not at all affected by the M. communis powder or extract. Bacterial- and fungal-feeding nematodes appear to be much more tolerant to plant-derived nematicides (Oka et al., 2001; Bar-Eyal et al., 2006). Such selectivity or specific activity of plant-derived nematicides to plant-parasitic nematodes provides an advantage over chemical nematicides and fumigants in the protection of non-target and nematode-antagonistic microorganisms, including predatory nematodes.

Extraction method is very important in obtaining the maximum quantity of nematicidal compounds from plant tissue. Various organic solvents with different degrees of polarity were used in this study to extract M. communis leaf powder and to characterize nematicidal compounds in the extract. Diluted and undiluted ethanol and methanol were more efficient at extracting nematicidal compounds from the leaf powder than solvents with lower polarity, suggesting that nematicidal compounds possess an intermediate degree of polarity or that the leaf contains several nematicidal compounds with different degrees of polarity. Indeed, three fractions on the TLC plate showed nematicidal activity. In further experiments, leaf paste was extracted in methanol or acetone following formulation as EC with d-limonene and Tween-80. Because d-limonene has been reported to have insecticidal activity (Hink & Fee, 1986), nematicidal activity of the blank formulation was tested in the cucumber bioassay. No nematicidal activity was found at up to 0·01% in sand, whereas the formulated leaf paste at corresponding doses showed a high level of nematicidal activity.

AC-EC showed a higher level of nematicidal activity than ME-EC in the tomato bioassays. Although these two formulated pastes were examined in separate experiments, the differences in their nematicidal activities suggest that different nematicidal compounds, or yields, were extracted. No attempt was made to characterize the nematicidal compounds in M. communis. The essential oil of M. communis was reported to have weak nematicidal activity against M. javanica eggs and J2s at the high concentration of 1000 μL L−1 (Oka et al., 2000), and against mixed stages of B. xylophilus (Barbosa et al., 2010). The main constituents of the M. communis essential oil were 1,8-cineole, linalool, myrtenyl acetate and myrtenol (Ozek et al., 2000). These constituents may vary with growth conditions and plant chemotypes. Because of the low nematicidal activity of the essential oil, these constituents are not likely to be the main nematicidal compounds in the leaf extracts. In the present study, silica-gel TLC was performed for preliminary isolation of nematicidal compounds. Incubation of nematodes in sand amended with extracts from the TLC silica gel might prove useful for detecting and isolating non-volatile nematicidal compounds. Here, three different fractions with nematicidal activity were found in the leaf extract using this method. Further experiments are required to identify these compounds.

Antibacterial activity of M. communis has been reported by several researchers (Garg & Dengre, 1988; Deruaz et al., 1993; Mansouri et al., 2001; Traboulsi et al., 2002; Amensour et al., 2010), and the antibacterial compound myrtucommulone-A, an acylphloroglucinol, has been isolated and reported to have strong antibacterial activity against Gram-positive but not Gram-negative bacteria (Rotstein et al., 1974). Methanolic leaf extract of M. communis showed antibacterial activity against Listeria monocytogenes and Pseudomonas aeruginosa (Amensour et al., 2010). It is not known whether the antibacterial compound also has nematicidal activity. The family Myrtaceae includes species with ‘biological’ activities, such as Eucalyptus spp., Eugenia caryophyllata and Melaleuca alternifolia. Oils extracted from these plants are used for medicinal purposes. Of particular interest is the formulated product of M. alternifolia (Australian tea-tree) essential oil, which possesses antifungal activity and is available commercially for control of fungal plant diseases (Reuveni et al., 2009).

In summary, the results of the present study indicate that leaf powder and extracts of M. communis can potentially be developed into a commercial nematicide. Although plant materials and extracts can be used in sustainable and organic farming systems, isolation and identification of the nematicidal compounds is essential for further development of commercial products. Synthesis of active compounds or their derivatives with higher nematicidal activity are likely to be a more promising means of developing a nematicide based on M. communis-derived or related compounds. Such ingredients have been well accepted by the insecticide industry, as seen with the development of neonicotinoids and pyrethroids from natural insecticidal compounds.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • Amensour M, Bouhdid S, Fernández-López J, Idaomar M, Senhaji NS, Abrini J, 2010. Antibacterial activity of extracts of Myrtus communis against food-borne pathogenic and spoilage bacteria. International Journal of Food Properties 13, 121524.
  • Appendino G, Bianchi F, Minassi A, Sterner O, Ballero M, Gibbons S, 2002. Oligomeric acylphloroglucinols from myrtle (Myrtus communis). Journal of Natural Products 65, 3348.
  • Barbosa P, Lima AS, Vieria P et al. , 2010. Nematicidal activity of essential oils and volatiles derived from Portuguese aromatic flora against the pinewood nematode, Bursaphelenchus xylophilus. Journal of Nematology 42, 816.
  • Bar-Eyal M, Sharon E, Spiegel Y, 2006. Nematicidal activity of Chrysanthemum coronarium. European Journal of Plant Pathology 114, 42733.
  • Barker KR, 1985. Nematode extraction and bioassays. In: Barker KR, Carter CC, Sasser JN, eds. An Advanced Treatise on Meloidogyne, Vol. 2, Methodology. Raleigh, NC, USA: North Carolina University Graphics, 1935.
  • Bridge J, Page SLJ, 1980. Estimation of root-knot nematode infestation levels on roots using a rating chart. Tropical Pest Management 26, 2968.
  • Cohen Y, Wang WQ, Ben-Daniel BH, Ben-Daniel Y, 2006. Extracts of Inula viscosa control downy mildew of grapes caused by Plasmopara viticola. Phytopathology 96, 41724.
  • Deruaz D, Reynaud J, Raynaud J, 1993. Evaluation of the molluscicidal properties of Myrtus communis L. (Myrtaceae). Phytotherapy Research 7, 42830.
  • Garg SC, Dengre SI, 1988. Antifungal efficacy of some essential oils. Pharmazie 43, 1412.
  • Gommers FJ, Bakker J, 1988. Physiological diseases induced by plant responses or products. In: Poinar GO, Jansson H-B, eds. Diseases of Nematodes, Vol. 1. Boca Raton, FL, USA: CRC Press Inc., 322.
  • Hackney RW, Dickerson OJ, 1975. Marigold, castor bean, and Chrysanthemum, as controls of Meloidogyne incognita and Pratylenchus alleni. Journal of Nematology 7, 8490.
  • Hink WF, Fee BJ, 1986. Toxicity of d-limonene, the major component of citrus peel oil, to all life stages of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae). Journal of Medical Entomology 23, 4004.
  • Hussey RS, Barker KR, 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reporter 57, 10258.
  • Kashman Y, Rotstein A, Lifshitz A, 1974. The structure determination of two new acylphloroglucinols from Myrtus communis L. Tetrahedron 30, 9917.
  • Mansouri S, Foroumadi A, Ghaneie T, Najar AG, 2001. Antibacterial activity of the crude extracts and fractionated constituents of Myrtus communis. Pharmaceutical Biology 39, 399401.
  • Mojumdar V, 1995. Effects on nematodes. In: Schmutterer H, ed. The Neem Tree, Azadirachta indica A. Juss. and Other Meliaceous Plants: Source of Unique Natural Products for Integrated Pest Management, Industry, and other Purposes. Weinheim, Germany: VCH, 12950.
  • Oka Y, 2001. Nematicidal activity of essential oil components against the root-knot nematode Meloidogyne javanica. Nematology 3, 15964.
  • Oka Y, 2010. Mechanisms of nematode suppression by organic soil amendments – a review. Applied Soil Ecology 44, 10115.
  • Oka Y, Nacar S, Putievsky E, Ravid U, Yaniv Z, Spiegel Y, 2000. Nematicidal activity of essential oils and their components against the root-knot nematode. Phytopathology 90, 7105.
  • Oka Y, Ben-Daniel B, Cohen Y, 2001. Nematicidal activity of powder and extracts of Inula viscosa. Nematology 3, 73542.
  • Oka Y, Ben-Daniel B, Cohen Y, 2003. Nematicidal and fungicidal activity in soil of formulations of Inula viscosa (L.) Aiton. Abstracts of 8th International Congress of Plant Pathology, Christchurch, New Zealand, Vol. 2. Sydney, Australia: Horticulture Australia, 275.
  • Oka Y, Ben-Daniel B, Cohen Y, 2006. Control of Meloidogyne javanica by formulations of Inula viscosa leaf extracts. Journal of Nematology 38, 4651.
  • Ozek T, Demirci B, Baser KHC, 2000. Chemical composition of Turkish myrtle oil. Journal of Essential Oil Research 12, 5414.
  • Pérez MP, Navas-Cortés JA, Pascual-Villalobos MJ, Castillo P, 2003. Nematicidal activity of essential oils and organic amendments from Asteraceae against root-knot nematodes. Plant Pathology 52, 395401.
  • Ploeg A, 1999. Greenhouse studies on the effect of marigolds (Tagetes spp.) on four Meloidogyne species. Journal of Nematology 31, 629.
  • Reuveni M, Neifeld D, Dayan D, Kotzer Y, 2009. BM-608 –a novel organic product based on essential tea tree oil for the control of fungal diseases in tomato. Acta Horticulturae 808, 12932.
  • Rotstein A, Lifshitz A, Kashman Y, 1974. Isolation and antibacterial activity of acylphloroglucinols from Myrtus communis. Antimicrobial Agents and Chemotherapy 6, 53942.
  • Sánchez de Viala S, Brodie BB, Rodríguez E, Gibson DM, 1998. The potential of thiarubrine C as a nematicidal agent against plant-parasitic nematodes. Journal of Nematology 30, 192200.
  • Traboulsi AF, Taoubi K, El-Haj S, Bessiere JM, Rammal S, 2002. Insecticidal properties of essential plant oils against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Management Science 58, 4915.
  • Uhlenbroek JH, Bijloo JD, 1958. Investigations on nematicides. I. Isolation and structure of a nematicidal principle occurring in Tagetes roots. Recueil des Travaux Chimiques des Pays-Bas 77, 10048.
  • Wang WQ, Ben-Daniel BH, Cohen Y, 2004. Control of plant diseases by extracts of Inula viscosa. Phytopathology 94, 10427.
  • White GF, 1927. A method for obtaining infective nematode larvae from cultures. Science 66, 3023.
  • Wright DJ, 1981. Nematicide: mode of action and new approaches to chemical control. In: Zuckerman BM, Rhode RA, eds. Plant Parasitic Nematodes, Vol. 3. New York, USA: Academic Press, 4219.