In vitro and in planta nematicidal activity of Fumaria parviflora (Fumariaceae) against the southern root-knot nematode Meloidogyne incognita

Authors


E-mail: ishratro@hotmail.com

Abstract

Root and stem extracts of Fumaria parviflora showed strong nematicidal activity against Meloidogyne incognita in in vitro and in planta experiments. Phytochemical screening of F. parviflora revealed the presence of seven classes of bioactive compounds (alkaloids, flavonoids, glycosides, tannins, saponins, steroids and phenols). Quantitative determination of the plant extracts showed the highest percentages of alkaloids (0·9 ± 0·04) and saponins (1·3 ± 0·07) in the roots and total phenolic contents in the stem (16·75 ± 0·07 μg dry g−1). The n-hexane, chloroform, ethyl acetate and methanol extracts of roots and stems at concentrations of 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL−1, significantly inhibited hatching and increased mortality of second-stage juveniles (J2s) compared with water controls. Percentage J2 mortality and hatch inhibition were directly related to exposure time. In pot trials with tomato cv. Rio Grande, root and stem extracts at concentrations of 1000, 2000 and 3000 ppm, applied as soil drenches, significantly reduced the number of galls, galling index, eggs masses, eggs and reproduction factor compared with the water control. Regardless of concentration, all the extracts significantly increased the host plant growth parameters studied. The n-hexane extracts from the roots and stem were the most active, followed by the methanol ones, at all concentrations. The in vitro and in planta results suggest that extracts from the roots and stem of F. parviflora may be potential novel nematicides.

Introduction

The southern root-knot nematode (SRKN), Meloidogyne incognita, is the most prevalent and damaging plant-parasitic nematode in Pakistan, infecting about 102 plant species, including tomato (Maqbool & Shahina, 2001). Infected plants show symptoms including root galling, stunting, nutrient deficiency and a reduction in yield. The population of SRKNs in a field can be reduced by several approaches, such as the use of natural antagonists (Tzortzakakis & Petsas, 2003), cultural practices, host-plant resistance and the application of synthetic pesticides (Khattak, 2008). Although soil nematicides are effective and fast-acting, they contaminate ground water, cause atmospheric ozone depletion and are hazardous to human health (Wachira et al., 2009). Numerous plant species, representing 57 families, have been found to contain biologically active phytochemicals which are able to control plant diseases (Sukul, 1992). Various nematicidal compounds of plant origin, including triglycerides, sesquiterpenes, alkaloids, steroids, tannins, diterpenes, flavonoids, phenolics and many essential oils, have been identified in this way (Chitwood, 2002). Many of these phytochemicals are safer to the environment or humans than synthetic nematicides and are generally considered to be non-persistent under field conditions (Chitwood, 2002). Alcoholic and aqueous extracts from as many as 39 plant species have been shown to possess nematicidal activity against M. incognita. Additionally, many of these plant extracts have been reported to possess broad-spectrum activity against diverse insect pests and pathogens (Sarma et al., 1999). Eucalyptus chamadulonsis [camaldulensis], garlic (Allium sativum) and marigold (Tagetes spp.) showed nematicidal activity against M. incognita juveniles under the greenhouse conditions (Kamal et al., 2009).

Fumaria parviflora (Fumariaceae) is a small, scandent, branched annual herb that grows wild in plains and low hills in Europe, Asia and Africa (Mabberley, 1987), and is found in most agricultural fields of Pakistan, particularly wheat fields (Syed et al., 2006). The plant has been reported to possess anthelmintic, hepatoprotective, antipyretic, antidyspeptic, blood purifying, cholagogue, diaphoretic, diuretic and hypoglycaemic properties (Athanasiadou et al., 2001; Hordegen et al., 2003). Phytochemical studies on F. parviflora revealed the presence of a number of alkaloids (adlumidicein, copticine, fumariline, perfumine, protopine, fumaranine, fumaritine, paprafumicin and paprarine), flavonoids, glycosides, tannins, saponins, steroids and triterpenoids (Rahman et al., 1992). Ethanolic extracts of Fumaria indica produced 59% mortality of second-stage juveniles (J2s) of Meloidogyne javanica (Abid et al., 1997). However, there is no reported information on the effect of F. parviflora extracts on plant-parasitic nematodes. Therefore, the present study was undertaken to achieve the following objectives: (i) phytochemical screening of F. parviflora and determination of extractive values in different solvent systems; (ii) in vitro assays of the effects of extracts of F. parviflora on hatching and J2 viability of M. incognita; and (iii) in planta assays of the effects of the extracts on M. incognita in tomato maintained under greenhouse conditions using artificially inoculated tomato plants.

Materials and methods

Nematode inoculum

The nematode population of M. incognita used was obtained from commercial tomato production fields (cv. Rio Grande) at Dargai (Khyber Pakhtunkhwa, Pakistan). Identification was done on the basis of perineal and isozyme patterns. To establish the cultures of M. incognita, a single egg mass was surface-sterilized (1% NaOCl) for 1 min followed by washing with sterile distilled water and inoculating into a pot containing a 2- to 3-week-old tomato plant (cv. Rio Grande) in sterilized soil and kept at 25 ± 5°C with an average relative humidity of 70% and 16-h daylength for 3 months in the Plant Pathology Department of KPK Agricultural University, Peshawar. Multiplication of the pure culture was done by inoculating new tomato seedlings of the same cultivar with at least 15 egg masses in the same growing conditions. Eggs were extracted using 1% NaOCl solution, J2s were hatched from eggs and both (eggs and J2s) were stored in 1% saline at 4°C or immediately used in bioassays (Hussey & Barker, 1973). The eggs were rinsed on a 25-μm-aperture sieve with distilled water (Hussey & Barker, 1973) before use. J2s were obtained using surface-sterilized eggs placed in sterile water in a cavity block, which hatched after 3–4 days.

Plant material and solvent extraction

Fumaria parviflora (mature, flowering plants) was collected in March–April 2009 from the wheat fields of the Agricultural Research Farm, Malakandher, KPK Agricultural University. The plant was authenticated and a voucher specimen (no. ISH-1732) was deposited in the herbarium of the Department of Botany, University of Peshawar, Pakistan. The plant parts (stem and roots), after washing in tap water, were frozen at −80°C for 24 h, lyophilized and crushed in a grinder to a particle size of around 1 mm. The lyophilized powdered plant material of the stem and roots of F. parviflora (350 g each) were separately and successively extracted with n-hexane (200 mL), chloroform (CHCl3; 200 mL), ethyl acetate (EtOAC; 200 mL) and methanol (MeOH; 200 mL) using a Soxhlet extractor (60°C) for about 3–4 h (Al-Shaibani et al., 2009). The extracts were filtered through Whatman no. 1 (11 μm) filter paper (GE Healthcare) and evaporated to dryness at 60°C under reduced pressure using a rotary evaporator (BUCHI, Rotavapor®). The yields of n-hexane, EtOAC, CHCl3 and MeOH extracts from stems (66·0, 52·0, 61·0 and 72·0 mg, respectively) and roots (88·0, 65·0, 57·0 and 71·0 mg, respectively) were recorded. Dried fractions were stored at 4°C in brown bottles for use in the nematicidal bioassays and the in planta study.

Phytochemical screening of Fumaria parviflora

Thin layer chromatography (TLC) of all the extracts (100 mL) from the stems and roots was performed using TLC silica gel plates 20 × 20 cm, 10-μm pore size (Merck). A mixture of n-hexane:EtOAC (3:1, v/v) was used as the mobile phase. The retention factor (Rf) of each spot was determined. All the organic solvents used were of analytical grade (Merck).

Determination of extractive values (percentage in 30 g dried root or stem powder) of F. parviflora was determined following the procedures described by Banso & Adeyemo (2007). Determination of total phenolic contents (in 20 g dried stems or roots) of F. parviflora was performed using the Folin–Ciocalteau spectrophotometric method and pyrocatechol as a standard (Slinkard & Singleton, 1977). Saponins and alkaloids were determined using dried and powdered plant material (root and stem) of F. parviflora according to the procedure of Sofowora (1993).

Qualitative phytochemical tests were performed on the n-hexane, CHCl3, EtOAC and MeOH extracts of the root and stem of F. parviflora. Yield (%) and positive tests on phytochemical screening were performed (Sofowora, 1993) as listed in Table 1.

Table 1. Phytochemical screening, mean percentage extractive values and retention factor (Rf) of various compounds using four different solvent systems from roots and stems of Fumaria parviflora
Plant partExtractChemical componentsaExtractive valuebTotal phenolic contentc
AlkaloidsbSaponinsbSteroidsGlycosidesTanninsFlavonoids Rf values
  1. a+ (present); – (absent).

  2. bResults expressed in percentage per dry plant material (= 10).

  3. cExpressed as μg pyrocatechol g−1 plant tissue.

  4. dNon-alkaloid compound.

Root 0·9 ± 0·041·3 ± 0·07      12·0 ± 0·05
n-Hexane+++0·39, 0·37, 0·4, 0·3811·71 ± 0·02
EtOAC++0·35, 0·376·28 ± 0·04
CHCl3++0·4, 0·36d5·24 ± 0·05
MeOH+0·388·9 ± 0·02
Stem 0·5 ± 0·030·9 ± 0·08      16·75 ± 0·07
n-Hexane+0·38, 0·36, 0·359·42 ± 0·00
EtOAC+0·366·0 ± 0·01
CHCl3++0·35, 0·395·90 ± 0·02
MeOH+0·4, 0·58·0 ± 0·06

In vitro experiments

A microwell bioassay was performed to determine the activity of all the fractions from the stems and roots of F. parviflora against J2s and eggs of M. incognita, using the procedures described by Al-Shaibani et al. (2009). A stock solution of each extract (n-hexane, EtOAC, CHCl3 and MeOH) was prepared by dissolving the respective dried extracts in DMSO and further dilutions were made in distilled water. The final concentration of DMSO never exceeded 1% (v/v). An aliquot of the starting solution (50·0 mg mL−1) was taken for preparation of final concentrations of 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL−1 (Al-Shaibani et al., 2009). Approximately 1000 ± 50 eggs or 200 J2s were deposited in each well of a 24-microwell plate (MultiwellTM, Becton Dickinson) in a final volume of 1 mL at final concentrations of 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL−1 of the plant extract or distilled water, with DMSO (1% v/v) used as a negative control. A total of four replications for each extract concentration, plus the control, were performed and the experiment was performed twice at room temperature (c. 27°C). The microwell plates were incubated under humidified conditions at room temperature for 3 days in the dark. The total number of unhatched eggs or active/inactive J2s in each well was counted after 24, 48 and 72 h incubation. Juveniles were defined as dead if their body was straight and they did not move, even after mechanical prodding (Choi et al., 2007). Percentages of unhatched eggs and J2 mortality were calculated for each well. Eggs and J2 solutions were removed and hatching or mobility were assessed in water for 3 days in each treatment (data not shown).

In planta experiments

Two separate pot experiments were conducted in the greenhouse (adjusted to 24–28°C) of the Plant Pathology Department of KPK Agricultural University during spring and autumn, 2010. Soil (sand:clay-loam, 1:1, v/v) was steam-sterilized, air-dried and placed into 15-cm clay pots (1 kg soil each). Eggs for the experiments were obtained from the roots of 3-month-old tomato (cv. Rio Grande) plants as described before. About 4000 ± 10 eggs + J2s of M. incognita were applied close to the roots of 14-day-old tomato seedlings using a sterilized micropipette. Four extracts (n-hexane, EtOAC, CHCl3 and MeOH) obtained from the roots and stems of the plant as previously described were first dissolved in 5 mL DMSO (1% v/v). The stock solutions (each 50 mg mL−1) were used to prepare three final concentrations, viz. 1000, 2000 and 3000 ppm. Each of the four root extracts (n-hexane, EtOAC, CHCl3 and MeOH) at these three concentrations were applied at a rate of 50 mL to each pot as a root drench application, 2 days after inoculation. The same procedure was used for the corresponding stem extracts at the same concentrations in a simultaneous greenhouse experiment. Plants treated only with distilled water (45 mL + 5 mL 1% DMSO) served as the controls. The treatment combinations were arranged in a completely randomized design and replicated five times, each replicate consisting of a single potted plant. The experiment was repeated twice during spring and autumn, 2010. Every pot was watered three times a week with about 350 mL fresh water and fertilized weekly with 100 mL 0·1%, 20-5-32 + micronutrients hydro-sol fertilizer (Engro Crop Ltd). Both experiments were evaluated after 60 days, when plants were fully developed and showing symptoms (yellowing and stunting). The following variables were assessed: plant height, number of branches, fresh shoot weight, dry shoot weight, fresh root weight, root galling index (GI), egg masses g−1 of root, number of females g−1 of root, eggs g−1 of root and reproduction factor (RF = final population/initial population). The shoot of each plant was cut off at soil level and the roots were carefully washed in tap water, blotted dry, their gall ratings determined and recorded (Taylor & Sasser, 1978). Roots were visually assessed and scored for GI using a 0–5 galling scale, where 0 = no gall on roots, 1 = 1–2, 2 = 3–10, 3 = 11–30, 4 = 31–100 and 5 = >100 galls per root. One gram of roots was stained in phloxine B for 15–20 min. The stained roots were rinsed in water and egg masses were visually counted (Taylor & Sasser, 1978). Eggs were extracted as described above.

Statistical analysis

Data were subjected to analysis of variance (anova) using statistix (NH Analytical Software). Similarity between experiments was tested by preliminary anovas, in order to check the suitability for both repetitions and experiment combinations.

In the hatching and J2 mortality experiments, for each treatment and repetition, the areas under cumulative number of nematodes percentage hatch inhibition (AUCPHI) and mortality (AUCPM) were estimated by trapezoidal integration (Campbell & Madden, 1990). AUCPHI was analysed by anova. Treatment means of AUCPHI and AUCPM were compared using Fisher’s protected least significant difference test (LSD) at = 0·05. In the in planta experiments, GI and RF were transformed into log10 (+ 1) before analysis (Gomez & Gomez, 1984). The data were analysed by anova. Treatment means of the different parameters were compared using Fisher’s protected least significant difference test (LSD) at = 0·05.

Results

Thin layer chromatography (TLC) and qualitative phytochemical screening

TLC plates sprayed with Dragendorrf’s reagent and ceric sulphate (10% H2SO4) (followed by heating) showed black and green spots, indicating the presence of non-alkaloids, while orange-coloured spots appeared when plates were sprayed with Dragendorrf’s reagent alone, indicating the presence of alkaloids. The retention factor of all the tested extracts ranged from 0·35 to 0·40 (Table 1). The TLC examination and Rf values revealed non-alkaloids in n-hexane and EtOAC extracts and alkaloids in MeOH extracts, whereas the CHCl3 extract contained only alkaloids and saponins. Phytochemical screening and quantitative determination of F. parviflora root and stem extracts showed the presence of seven bioactive secondary metabolites (Table 1). The n-hexane root extract contained steroids, tannins and flavonoids, whereas the n-hexane stem extract only had tannins. The EtOAC root extract contained glycosides and tannins, whereas the EtOAC stem extract had glycosides only. The CHCl3 root and stem extracts contained alkaloids and saponins, whereas the MeOH root and stem extracts contained alkaloids only. The fractions were evaluated for their solvent extractive values, with the highest values obtained for all root extracts except CHCl3 (Table 1). The alkaloid and saponin contents (%) of the roots (0·9 ± 0·04 and 1·3 ± 0·07, = 10, respectively) were significantly higher than those of the stem (0·5 ± 0·03 and 0·9 ± 0·08, = 10, respectively). Conversely, total phenolic contents were higher in the stems (16·75 ± 0·07 μg pyrocatechol g−1) than in the roots (12·0 ± 0·05 μg pyrocatechol g−1; Table 1).

In vitro experiments

Influence of root and stem extracts of Fumaria parviflora on hatching

Percentage hatch inhibition of M. incognita eggs was significantly increased (< 0·05) by all concentrations of root and stem extracts throughout the experiment (Fig. 1). Hatching inhibition was significantly increased (< 0·05) in eggs incubated at 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL−1 organic solvent extract concentrations with respect to the control (Fig. 1). However, differences between some concentrations were not found using Fisher’s protected LSD test (Fig. 1). Similar results of hatching inhibition were obtained with the different extracts of stems or roots and for both in vitro experiments. However, although the experiments showed the same patterns of action, they were significantly different. For this reason, they were treated separately for statistical analyses. Hatch inhibition ranged from 0 to 100% with both root and stem extracts and increased as the concentration of the extracts increased (Fig. 1).

Figure 1.

 Cumulative hatch inhibition of Meloidogyne incognita over 72 h incubation at 27°C over a series of concentrations (expressed in mg mL−1) of organic solvent extracts from Fumaria parviflora. Each point represents the average of two experiments with four replicates. Curves on the same graph labelled with the same lower case (first experiment) or upper case letter (second experiment), do not differ (> 0·05) according to Fisher’s protected LSD test.

Nematicidal activity of root and stem extracts of Fumaria parviflora on J2s

Results showed that the viability of J2s of M. incognita was significantly reduced (P < 0·05) over time by all concentrations of root and stem extracts throughout the experiment (Fig. 2). All the organic extracts from the roots and stems at 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL−1 had significant effects on J2 mortality with respect to the control (Fig. 2). However, differences between some concentrations were not found using Fisher’s protected LSD test (Fig. 2). Percentage mortality was similar with all the extracts and was not dependent on the plant tissue (root or stem; Fig. 2). The EtOAC and CHCl3 extracts from the roots and stems significantly increased J2 mortality at all concentrations, with 50·0 mg mL−1 being the most effective. Mortality increased with exposure period and extract concentration. Stem and root extracts had similar effects on J2 mortality, with similar effects in both the in vitro experiments for stem extracts. As in the root extracts, the experiments were significantly different, but with the same pattern of action. For this reason, they were treated separately for statistical analyses.

Figure 2.

 Cumulative mortality of Meloidogyne incognita over 72 h incubation at 27°C in a series of concentrations (expressed in mg mL−1) of organic solvent extracts from Fumaria parviflora. Each point represents the average of two experiments with four replicates. Curves on the same graph labelled with the same lower case (first experiment) or upper case letter (second experiment) do not differ (> 0·05) according to Fisher’s protected LSD test.

In planta experiment

Effects of solvent extracts from roots of Fumaria parviflora

The two experimental repetitions (spring and autumn) were not significantly different and were combined for analysis (Table 2). Root extracts applied at three different concentrations, viz. 1000, 2000 and 3000 ppm, showed significant differences (< 0·05) compared with the control (H2O + DMSO) (Table 2). The four root extracts at all concentrations tested significantly (< 0·05) influenced all the plant and nematode parameters measured, resulting in an increase in plant growth and a reduction of nematode reproduction parameters (Table 2). The n-hexane root extract showed the strongest nematicidal effects at all the tested concentrations, followed by the MeOH, EtOAC and CHCl3 extracts, with reduced numbers of galls, GI, egg masses g−1 root and numbers of females g−1 root. A similar pattern was shown in plant variables, with the greatest increments in plant height, fresh and dry shoot weight in the plants treated with n-hexane extracts, followed by those treated with MeOH extracts. Similar results for these variables were observed in plants treated with EtOAC and CHCl3 extracts, with fresh root weight being highest in plants treated with CHCl3 extracts, followed by the other treatments. No extracts showed any phytotoxic effects at the concentrations tested.

Table 2. Effect of different concentrations of root extracts of Fumaria parviflora on plant growth parameters and nematicidal effect on Meloidogyne incognita in tomato under greenhouse conditionsa
Organic extractppmbNo. ofgallsGalling indexcEgg masses g−1 rootNo. of females g−1 rootPlant height (cm)Fresh shoot weight (g)Dry shoot weight (g)Fresh root weight (g)No. of branches per plantEggs g−1 rootRF (Pf/Pi)d
  1. aData are means of 10 replicate plants per treatment using the combination of two different experiments. Plants were inoculated with 4000 eggs + J2s (Pi) of M. incognita for each extract. Means followed by the same letter do not differ significantly ( 0·05) according to Fisher’s protected LSD test within each extract – lower case letters are for comparisons between different concentrations of the same extract, while upper case letters are for comparisons between the same concentration of the different extracts. Actual data are presented for each organic extract but galling index and RF were transformed into log10 (× + 1) before analysis. NS: non-significant differences after anova.

  2. bThe 0-ppm treatment was a water control mixed with DMSO (1%; v/v).

  3. cGalling index: 0 = no gall on roots; 1 = 1–2; 2 = 3–10; 3 = 11–30; 4 = 31–100; 5 = more than 100 galls per root.

  4. dNematode reproduction factor (RF) = final nematode numbers per plant (Pf)/initial nematode inoculum per plant (Pi).

n-Hexane0104·40 a5·00 a83·50 a92·90 a33·20 c31·80 a9·40 c29·00 a10·70 d12300·0 a3·10 a
100062·40 bD2·40 bB41·10 bB53·10 bC45·00 bA40·50 cA21·60 bA25·30 bB16·90 cA1405·0 bC0·35 bC
200054·50 cD2·20 bC27·30 cB41·60 cC48·70 bA48·50 bA21·70 bA22·10 cC20·0 bA1230·0 bC0·30 bC
300036·50 dC1·30 cC14·70 dC27·40 dC56·80 aA54·00 dA27·30 aA14·90 dB26·70 aA1055·0 bC0·25 bC
EtOAC0104·40 a5·00 a83·50 a92·90 a33·20 c31·80 c9·40 d29·00 a10·70 b12300·0 a3·10 a
100082·60 bB3·50 bA51·40 bA70·80 bA36·40 bcB36·90 bB12·40 cC26·6 bAB12·00 bB2660·0 bA0·65 bA
200073·20 cB3·15 cA46·60 bcA59·00 cB38·70 abB40·70 aB14·00 bC23·50 cBC15·60 aB2290·0 bcA0·56 bcA
300066·00 dA2·50 dA38·50 cA51·40 dA41·10 aC40·90 aC18·40 aBC22·70 cA17·50 aC1970·0 cA0·49 cA
CHCl30104·40 a5·00 a83·50 a92·90 a33·20 c31·80 d9·40 c29·00 a10·70 c12300·0 a3·10 a
100088·70 bA3·50 bA54·70 bA74·60 bA37·80 bB35·70 cB11·60 bC27·70 aA12·70 cB2560·0 bA0·64 bA
200082·30 cA2·75 cB46·90 bA65·30 cA40·20 abB40·20 bB15·60 aBC25·80 bA16·60 bB2170·0 bA0·53 cA
300070·70 dA2·05 dB35·70 cA53·00 dA42·10 aC42·50 aC16·50 aC22·60 cA19·40 aC1980·0 bA0·49 cA
MeOH0104·40 a5·00 a83·50 a92·90 a33·20 c31·80 c9·40 c29·00 a10·70 c12300·0 a3·10 a
100075·90 bC3·00 bA52·20 bA60·70 bB41·90 bA38·50 bAB15·20 bB25·50 bB18·60 bA1900·0 bB0·47 bB
200065·70 cC2·40 cC44·40 cA55·50 bB46·70 aA45·70 aA16·90 bB24·6 bAB20·50 abA1760·0 bB0·43 bB
300053·10 dB1·65 dC25·90 dB39·30 cB47·10 aB47·10 aB19·40 aB20·80 cA22·40 aB1570·0 bB0·39 bB

Effect of solvent extracts from the stem of Fumaria parviflora

The two temporal experiment repetitions were not significantly different and were combined for analysis (Table 3). The results indicated significant effects (< 0·05) of the stem extract treatments on plant parameters and nematicidal effects against M. incognita compared with the control (H2O + DMSO) treatment. Data revealed that all extracts had a nematicidal effect on parameters in nematode parasitism: number of galls and GI, egg masses g−1 root, eggs g−1 root and RF were all reduced by all concentrations of n-hexane, EtOAC, CHCl3 and MeOH extracts (Table 3). All extracts resulted in increases in plant parameters measured (plant height, fresh shoot weight, dry shoot weight, fresh root weight and number of branches per plant). Comparisons between the different extracts at the same concentration showed differences between them in nematode and plant growth parameters at 1000, 2000 and 3000 ppm, with no phytotoxic effects (Table 3). Specifically, the n-hexane and MeOH extracts had the best nematicidal effect with regard to reduction in the number of galls, number of females g−1 root and RF, and the least reduction in plant height, compared with EtOAC and CHCl3 extracts. However, these differences in all nematode and plant parameters studied seemed to vary depending on the concentration of each extract. None of the extracts showed any phytotoxic effects at the concentrations tested.

Table 3. Effects of different concentrations of stem extracts of Fumaria parviflora on plant growth parameters and nematicidal effect on Meloidogyne incognita in tomato under greenhouse conditionsa
ExtractppmbNo. of gallsGalling indexcEgg masses g−1 rootNo. of females g−1 rootPlant height (cm)Fresh shoot weight (g)Dry shoot weight (g)Fresh root weight (g)No. of branches per plantEggs g−1 rootRF (Pf/Pi)d
  1. aData are means of 10 replicated plants per treatment using the combination of two different experiments. Plants were inoculated with 4000 eggs + J2s (Pi) of Meloidogyne incognita for each extract. Means followed by the same letter do not differ significantly ( 0·05) according to Fisher’s protected LSD test within each extract – lower case letters are for comparisons between different concentrations of the same extract, while upper case letters are for comparisons between the same concentration of the different extracts. Actual data are presented for each organic extract but galling index and RF were transformed into log10 (× + 1) before analysis.

  2. bThe 0-ppm treatment was a water control mixed with DMSO (1%; v/v).

  3. cGalling index: 0 = no gall on roots; 1 = 1–2; 2 = 3–10; 3 = 11–30; 4 = 31–100; 5 = more than 100 galls per root.

  4. dNematode reproduction factor (RF) = final nematode numbers per plant (Pf)/initial nematode inoculum per plant (Pi).

n-Hexane0110·50 a5·0 a89·00 a90·40 a32·20 c30·30 c10·00 c29·60 a9·50 d11610·0 a3·10 a
100070·80 bB3·0 bB45·30 bA59·40 bB42·00 bA38·90 bAB16·80 bA23·70 bB15·30 cAB2080·0 bA0·40 bB
200062·30 cC2·75 bBC33·10 cB50·90 cB45·00 bA42·00 bA17·70 bA20·20 cB19·90 bA1595·0 bB0·37 bB
300050·50 dC1·7 cB20·20 dB36·60 dB53·40 aA47·70 aA24·00 aA15·60 dB24·80 aA1560·0 bB0·29 bB
EtOAC0110·50 a5·0 a89·00 a90·40 a32·20 c30·30 c10·00 c29·60 a9·50 c11610·0 a3·10 a
100084·00 bA3·6 bA50·80 bA71·10 bA36·50 bB37·60 bAB12·70 bB25·50 bAB13·90 bB2380·0 bA0·6 aA
200071·30 cAB3·2 bA43·30 bA61·50 cA40·00 bB39·70 bA14·90 bAB22·20 cB16·80 abAB2090·0 bA0·5 bcA
300062·70 cAB2·6 cA34·90 cA49·90 dA45·90 aBC44·40 aA19·00 aB20·70 cA18·30 aB1950·0 bA0·47 cA
CHCl30110·50 a5·0 a89·00 a90·40 a32·20 c30·30 c10·00 c29·60 a9·50 c11610·0 a3·10 a
100085·00 bA3·65 bA53·30 bA71·80 bA36·90 bB36·50 bB14·00 bAB26·50 bA14·00 bB3302·0 bA0·60 bA
200077·90 bA3·05 cAB44·50 cA64·20 cA39·80 bB41·20 aA14·80 abB25·10 bA16·30 bB2428·0 bA0·51 bcA
300068·60 cA2·15 dAB36·50 cA49·50 dA43·80 aC43·40 aA17·00 aB22·60 cA20·60 aB2037·0 bA0·44 cA
MeOH0110·50 a5·0 a89·00 a90·40 a32·20 c30·30 c10·00 c29·60 a9·50 c11610·0 a3·10 a
100079·10 bA3·40 bAB54·30 bA65·60 bAB41·30 bA39·60 bA15·90 bAB26·30 bA17·50 bA2080·0 bA0·50 bA
200068·50 cBC2·60 cC43·20 cA58·00 cAB44·20 bA42·30 abA16·70 abAB22·60 cAB19·70 abAB1815·0 bAB0·43 bcAB
300056·10 dBC1·75 dB28·10 dAB46·30 dA48·60 aB43·80 aA19·20 aB19·70 dA21·00 aB1590·0 bA0·38 cA

Discussion

The objective of the present study was to explore the nematicidal properties of root and stem extracts of F. parviflora against M. incognita. The results of in vitro and in planta experiments showed that the root and stem solvent extracts had nematicidal activity at all concentrations studied. The various organic compounds found in a single plant tissue can confer synergistic nematicidal properties, leading to high nematode mortality (Chitwood, 2002). The solvents used in the present study (n-hexane, EtOAC, CHCl3 and MeOH) have been used by others for the isolation of nematicidal compounds from a number of plants, for example, Impatiens bicolor against M. incognita and M. javanica (Qayum et al., 2011). The present results show that alkaloids, glycosides, phenolics, tannins and saponins are active components of the roots and stem, while the roots have two additional active constituents, viz. flavonoids and steroids. The nematicidal activity could be attributed to individual bioactive compounds of F. parviflora or mixtures. These results agree with those of Rao et al. (2007), who reported the presence of alkaloids, flavonoids, glycosides, tannins, saponins, steroids and triterpenoids in F. parviflora. The main alkaloids of F. parviflora are protopine, fumarizine, papraine, papracine papracinine, paprafumicine and papraraine (Rao et al., 2007). The results of the present in vitro study revealed that the solvent extracts of the roots and stem of the plant had strong nematicidal effects on J2s and eggs of M. incognita and these effects increased with increasing concentration and exposure time. These results are similar to those reported by other researchers, who reported 59% mortality of J2s of M. javanica using crude ethanolic extracts from the leaves and shoots of F. indica (Abid et al., 1997). The present study further revealed that the n-hexane extracts of roots and stems were the most active, leading to 100% J2 mortality and hatch inhibition. Similar results were obtained with the in planta studies, where the application of n-hexane extracts at all concentrations reduced disease parameters and increased plant growth parameters. This could be explained by the presence of active phytochemical constituents, mainly the tannins in the n-hexane extracts of the roots and stem, as demonstrated by phytochemical screening. Although steroids and flavonoids were not detected in the stem extracts in the present study, the combination of all three compounds (viz. steroids, flavonoids and tannins) performed better in an in planta experiment. Tannins in extracts of F. parviflora have been reported to possess anthelmintic activity against gastrointestinal nematodes (Athanasiadou et al., 2001). These authors suggested that tannins have the capacity to bind to proteins that could operate via several mechanisms. Condensed tannins may bind to the cuticle of the larvae, which is high in glycoproteins, and cause their death. The nematostatic effects of tannins from chestnut (Castanea sativa) against the juveniles of M. javanica are well documented (Maistrello et al., 2010), as is the nematicidal potential of flavonoids and steroids (found in the n-hexane fraction here) on Meloidogyne spp. (Chitwood, 2002). In the present case, other components in addition to tannins could have a synergistic effect on J2 mortality and hatch inhibition because the J2s did not move, nor the eggs hatch, after being transferred to water for 3 days. In the in vitro tests, MeOH extracts from the roots and stem were the second most effective in increasing J2 mortality and hatch inhibition at all the tested concentrations, similar to results obtained with methanolic extracts of Melia azedarach fruit against M. incognita by Ntalli et al. (2010). In the present study, the CHCl3 extracts of the roots and stem at all concentrations gave encouraging results. Phytochemical screening of these extracts revealed the presence of alkaloids and saponins, with higher quantities being detected in roots (0·9 ± 0·04 and 1·3 ± 0·07% dry plant material, respectively) than in the stem (0·5 ± 0·03 and 0·9 ± 0·08% dry plant material, respectively). The results are in close agreement with those of other researchers, who reported that the alkaloids of F. parviflora possessed anthelmintic activity, possibly by intercalating with DNA and inhibiting its synthesis (Maqbool et al., 2004). The present results also showed that the EtOAC extracts of the roots and stem had nematicidal activity, which could be attributed to the presence of glycosides, as revealed by the phytochemical screening of the corresponding extracts of F. parviflora. The flavone-C-glycoside, lantanoside, exhibited 90% mortality within 24 h at a concentration of 1% against M. incognita (Chitwood, 2002). Two glycosides (schaftoside and isoschaftoside) isolated from ethanolic extracts of tubers of Arisaema erubescensi possessed strong nematicidal activity against M. incognita (Du et al., 2011). The results of the present in vitro study were further strengthened by complementary data obtained from in planta experiments, where the application of root and stem extracts significantly decreased the parameters of nematode parasitism (number of galls, galling index, egg masses g−1, eggs g−1 and RF) and increased plant growth parameters (plant height, fresh and dry shoot weight and number of branches) relative to the water control treatments.

These studies suggest that different plant extracts from the same plant species vary in their nematicidal effects. These differences could be caused by the presence of different classes and compositions of active compounds in these extracts (Abid et al., 1997). For example, in the present study the superior efficacy of n-hexane extracts could be related to the presence of greater numbers of active compounds in those extracts, as shown by TLC analysis. As was clear from the TLC analysis, the number of active compounds, e.g. non-alkaloids and alkaloids, differed between the stems and roots. Kolapo et al. (2009) also reported that the concentration of phytochemicals (saponins, tannins, alkaloids, phenols and steroids) varied greatly between different plant parts, which supports the present results in relation to the concentrations of total phenolics and saponins. It is hypothesized that these secondary metabolites contribute to the defence of the plant against pests and pathogens, and thus have great potential in environmentally friendly integrated pest management (Abid et al., 1997). In conclusion, the results confirm previous reports of naturally occurring plant-derived compounds possessing nematicidal activity. The beneficial effect of natural phytochemicals is a promising area of nematode management as these phytochemicals are considered inherently less toxic than conventional pesticides and generally affect only the target pests (Chitwood, 2002). Fumaria parviflora has potential as a bionematicide because of the richness and diversity of compounds effective against Meloidogyne spp. More recently, additional phytochemicals have been detected in the roots and stem of F. parviflora and extensive research based on the isolation and purification of bioactive guided fractions is being carried out. However, more specific information is needed on the mode of action, range of activities (structure–activity relationship) and mechanisms involved in nematode suppression by compounds found in F. parviflora.

Acknowledgements

The authors thank the Higher Education Commission (HEC) of Pakistan for providing necessary funds for this research project.

Ancillary