Efficacy evaluation of a neem (Azadirachta indica A

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Efficacy evaluation of a neem (Azadirachta indica A. Juss) formulation against root-knot nematodes Meloidogyne incognita Article in Crop Protection · June 2009 DOI: 10.1016/j.cropro.2009.01.011

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Crop Protection 28 (2009) 489–494

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Efficacy evaluation of a neem (Azadirachta indica A. Juss) formulation against root-knot nematodes Meloidogyne incognita N.G. Ntalli a, U. Menkissoglu-Spiroudi a, *, I.O. Giannakou b, D.A. Prophetou-Athanasiadou c a

Pesticide Science Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece c Laboratory of Applied Zoology and Parasitology, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 November 2008 Received in revised form 20 January 2009 Accepted 22 January 2009

The nematicidal–nematostatic activity of AzaNema, 1% azadirachtin, against Meloidogyne incognita was evaluated both with motility and pot bioassays at different rates equal or greater than that recommended for nematode control (6 L ha1). To avoid issues that could have interfered with efficacy, such as formulation inconsistency and storage instability, technical grade azadirachtin 11.8% was used to verify the test product’s active ingredient (azadirachtin A) and to study the direct motility effect of azadirachtin on second stage juveniles (J2). Finally, the stability of both azadirachtin formulation types was studied under experimental conditions. Azadirachtin did not provide adequate control of nematodes, at the recommended or higher doses. In pot bioassays, a compensatory effect was achieved with 30.72 mg a.i g1 of soil but, in general, one application per biological cycle failed to protect the plants from Meloidogyne incognita. In motility experiments, only exposure of J2 to doses higher than 12.8 mg a.i ml1 resulted in a significant increase of immotile J2 for both AzaNema and azadirachtin technical grade. Nevertheless, the test product was found, after HPLC analysis, consistent with active ingredient content. Moreover, azadirachtin A concentration remained stable during the experiments, regardless of the formulation type tested. In conclusion, further research will be necessary to improve the efficacy of azadirachtin against Meloidogyne incognita. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Root-knot nematodes Motility Azadirachtin AzaNema HPLC

1. Introduction Tomato culture is one of the most economically important crops in Greece with a production of 270,000 tons (Olympios, 2008). Root-knot nematodes (Meloidogyne spp.) cause severe annual yield reduction or even total crop loss, due to complexes involving plant parasitic nematodes and soil borne pathogens (Back et al., 2002) and the use of nematicides is an important control measure. According to EU legislation existing and new active substances are evaluated under Council Directive 91/414/EEC and those approved are included in Annex I in order to get further authorization by each member state. The non-inclusion in the Annex I of 91/414/EEC of many chemical nematicides, coupled with the inconsistent efficacy of others mainly due to biodegradation (Giannakou et al., 2005), creates an urgent need for alternative nematode control methods.

* Corresponding author. Aristotle University of Thessaloniki, Faculty of Agriculture, Pesticide Science Laboratory, P.O. Box 251, 54124 Thessaloniki, Greece. Tel./fax: þ32 31 099 8835. E-mail address: [email protected] (U. Menkissoglu-Spiroudi). 0261-2194/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2009.01.011

Azadirachtin is a limonoid secondary metabolite (C35H44016, a tetranotriterpenoid) of the Indian Neem tree (Azadirachta indica A. Juss). It is extracted from the soft pressed neem kernels (neem cake) after appropriate solvent extraction, and is available as a yellowish brown powder (technical grade azadirachtin) at a concentration of 12–26% (Immaraju, 1998). This technical grade material is used for the production of a wide range of commercial formulations exhibiting good efficacy against more than 400 insect species (Immaraju, 1998; Mordue and Blackwell, 1993; Isman, 2006; Schmutterer, 1988; Nisbet et al., 1996; Seljasena and Meadow, 2006; Thoeming et al., 2006; Lababidi, 2002; MartinezVillar et al., 2005; Deota and Upadhyay, 2005; Zabel et al., 2002; Cowles, 2004; Greenberg et al., 2005; Nathan et al., 2006; Liang et al., 2003; Elling et al., 2002; Sharma et al., 2006; Ishaaya et al., 2007). In the search for eco-friendly insecticides that can be integrated in organic pest control programs, azadirachtin has been probably best investigated and exempted from residue tolerance requirements by the US-Environmental Protection Agency, fact that implies its friendly environmental profile (Immaraju, 1998). Azadirachtin was first isolated from the seeds of Azadirachta indica by Butterworth and Morgan in 1968. Margosan O was the

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first formulated neem product in the United States registered in July 1985 (Immaraju, 1998), while the total synthesis of azadirachtin, was first reported in 2007 (Veitch et al., 2007). Rapidly, azadirachtin became the modern paradigm for development of botanical insecticides. However, it was frequently characterized by poor efficacy results under field conditions. This has been mainly attributed to its relatively slow action on pest insects (Isman, 2006; Schmutterer, 1988), its storage instability (Immaraju, 1998), the formulation inconsistency (Isman et al., 1990) mainly attributed to the variability in azadirachtin content (Sidhu et al., 2003), and the degradation of light, pH (Sundaram, 1996; Sundaram et al., 1997; Caboni et al., 2002, 2006), temperature (Sundaram, 1996; Jarvis et al., 1998; Barrek et al., 2004; Stark and Walter, 1995), and microbial activity (Stark and Walter, 1995). Azadirachtin is used throughout the European Community for insect control in organic farming and up to now the decision for azadirachtin inclusion in the Annex I of 91/414/EEC is pending. In Italy AzaNema 1% EC (Sipcam S.p.A., Pero, Milan, Italy), is registered for nematode control at the dose of 4–8 L ha1 applied at monthly intervals (3–4 weeks) on different crops with a maximum residue level (MRL) of 0.5 mg kg1, and a preharvest interval (PHI) of 3 days. Several reports suggest neem products, such as seed powder, seed kernel powder, seed cake powder, dry leaf powder and aqueous neem extracts, exhibit good efficacy against root-knot nematodes (Akhtar, 2000; Akhtar and Malik, 2000; Chitwood, 2002; Akhtar et al., 2005; Adegbite and Adesiyan, 2005; Agbenin et al., 2005; Bharadwaj and Satyawanti, 2006; Oka et al., 2007; Javed et al., 2007a,b; Bharadwaj and Sharma, 2007; Javed et al., 2008a,b; Ambrogioni et al., 2003). Relatively few studies have examined registered neem emulsifiable concentrate formulations showing efficacy at high concentration levels (Javed et al., 2007a,b, 2008b), even though this type of formulation is considered to be one of the most promising due to its high storage stability (Isman, 2006). Additionally, no studies address in detail the effects of such neem formulated products on behavior of juveniles’, invasion and subsequent development in host roots when used at their registered doses and application intervals. Therefore, the objectives of this study were to: (1) evaluate the effect of a commercial azadirachtin formulation (AzaNema 1% EC, SipCam S.p.A, hereafter referred as test product) on motility of second stage juveniles (J2) in 24 well-plate bioassays and on nematode development, using pot bioassays; (2) study motility of J2 in response to technical grade product (azadirachtin 11.8% Sipcam S.p.A); and (3) study the stability/degradation of azadirachtin test solutions under experimental conditions and confirm the test product’s active ingredient content using the technical grade product as a reference standard. 2. Materials and methods 2.1. Nematode cultures A population of Meloidogyne incognita, originally obtained from tomato roots collected from a greenhouse in Vasilika, Thessaloniki, Northern Greece, was reared on tomato (Solanum lycopersicum L.) cv. Belladonna, a cultivar which is very susceptible to root-knot nematodes. All plants were maintained in a growth chamber at 25– 28 C, 60% RH and 16 h photoperiod, in plastic pots (18 cm diameter) containing a 10:1 (v:v) mixture of peat and perlite. Plants were inoculated after 7 weeks at the five-leaf stage. After 30 days, the plants were uprooted. Roots were washed free of soil and cut into 2 cm pieces. Eggs were extracted with sodium hypochlorite following Hussey and Barker (1973). Second-stage juveniles (J2) were allowed to hatch in modified Baermann funnels at 28 C. All J2 hatching in the first 3 days were discarded and J2 collected during the subsequent 24 h were used in the experiments.

2.2. Azadirachtin effect on J2 motility 2.2.1. Experiment 1 In the first experiment, solutions of AzaNema (a.i. azadirachtin 1%, SipCam S.p.A) in water were prepared at the following concentrations: 0.2, 0.8, 3.2, 12.8 and 51.2 mg a.i ml1. NemathorinÒ 150 EC (a.i. fosthiazate 15%, Hellafarm Co) at 13.3 mg a.i ml1, served as the chemical control tested, while plain water served as the control. The lowest azadirachtin concentration as well as the fosthiazate test concentration, represent the recommended doses of the respective commercial products for nematode control (6 L ha1 and 3 kg ha1 respectively) assuming that: (1) all of the nematicide is dissolved in the aqueous phase; (2) the water content is at field capacity (about 20% v/v for a sandy loam soil); and (3) pesticides remain within the top 15 cm of the surface soil as indicated by Weber et al. (2000). The commercial products were used to obtain working solutions containing double of the test concentration. Afterwards working solutions were mixed with nematode suspension, at a ratio of 1:1 (v/v), to obtain the final test concentrations. Approximately 50 J2 were added to each well to assay for effects on motility. The treatments were replicated six times in a completely randomized design; the assay was performed twice. The hard plastic lids provided with the multiwell plates were used to reduce evaporation. Multiwell plates were maintained in the dark at 28 C. Juveniles were observed with the aid of an inverted microscope (Euromex, Holland) at 40 after 24, 48 and 96 h and were ranked into two distinct categories: motile or immotile. 2.2.2. Experiment 2 In the second experiment, technical grade azadirachtin 11.8% was used to prepare a stock solution in methanol (2000 mg ml1) which was then dissolved in water containing Tween-20 (2% v/v), to overcome insolubility and achieve emulsion. Final concentrations of methanol and Tween-20 in each well never exceeded 1.5%. Preliminary trials showed that nematodes exposed to these products at these concentrations were not affected. Plain water as well as a mixture of water, Tween-20 and methanol (1.5% v/v) (carrier control) served as controls. The experimental procedure, design and time replication were identical to those described previously. 2.3. Effect of azadirachtin on nematode life cycle Freshly collected clay loam soil with 1.3% organic matter and pH 7.8 was obtained from a field of the Agricultural University Farm, Thessaloniki that had no history of azadirachtin application. It was poured through a 3 mm sieve to separate soil from debris and it was partially air dried overnight. After confirmation of the absence of nematodes using the modified Baermann funnel method, the soil was mixed with sand at a ratio 2:1 to obtain the mixture hereafter referred as soil. Afterwards, it was distributed into eight plastic bags (1 kg fresh weight each) representing the experimental treatments. Sub samples were removed for the determination of soil moisture (by oven drying at 110 C for 24 h) and maximum water holding capacity (gravimetrical measurement following saturation of the soil with water and allowing to drain for 24 h) (Pantelelis et al., 2006). The soil in each plastic bag was inoculated with a nematode suspension of 2500 J2, mixed thoroughly by shaking and allowed to equilibrate in dark at 10 C for 24 h. Subsequently, soil was sieved again through a 3 mm sieve, returned into the plastic bags and treated with the respective test solution. After the combined application of nematode suspension and test solution, the moisture content of the soil was adjusted at 24% of maximum water holding capacity. Preliminary studies showed that this moisture level, for this soil type, is necessary for the uniform distribution of nematodes and chemical, and does not allow leaching.


The azadirachtin test product was used at the following concentrations: 0.03, 0.12, 0.48, 1.92, 7.68 and 30.72 mg a.i g1, while fosthiazate was used at 2 mg a.i g1 to serve as chemical control. The lowest azadirachtin concentration used, and the fosthiazate test concentration, represent the recommended doses of the corresponding commercial products (Weber et al., 2000). One bag of soil received plain water together with the nematode suspension to serve as control. After an additional 24 h equilibration period at 10 C the soil from plastic bags was sieved for the last time, to ensure uniform distribution, and then it was placed in 200 g plastic pots and transplanted with tomato plants, cv. Belladonna. According to preliminary experiments using the Bearmman method, the number of J2 that survived in water control after sieving as before mentioned was found more than 95% of the initial inoculum. Plants used for the experiment were 7 week old, at the six-leaf stage. Plants were maintained at 27 C, 60% RH at 16 h photoperiod, and each pot received 30 ml of water every 3 days. Thirty days afterwards, plants were uprooted and gently washed in running tap water to remove adhering soil particles. Roots were cut at the soil line, blotted dry and weighted. Then they were stained with acid fuchsin solution according to Byrd et al. (1983), and the following variables were assessed: fresh root weight, fresh shoot weight, and total number of female nematodes per gram of root. The treatments were arranged in a completely randomized design with five replicates and the experiment was conducted twice. 2.4. Chemical analysis 2.4.1. Determination of azadirachtin A Azadirachtin reference standard was obtained from Sipcam S.p.A. All solvents used were of HPLC grade, purchased from Merck (Germany). The content of the test product in azadirachtin A, was determined by HPLC analysis according to Kaushik (2002). Azadirachtin A was used as a lead or marker compound for the purpose of quantification, since ‘‘azadirachtin, sensu lato’’ is neither completely defined nor quantifiable (FAO). The reference standard was diluted in methanol to obtain a stock solution of 1000 mg a.i ml1 and working standards were further prepared by serial dilution of the stock solution in water containing Tween-20 (1% v/v). A five point calibration curve in the range of 3–300 mg a.i ml1 was constructed and used for quantification. 2.4.2. Instrumentation and accuracy Analysis was carried out on an Agilent 1100 HPLC system equipped with a reversed-phase column, Kromasil 100 C18 (5 mm, 250 mm 4 mm) (MZ), attached to a guard column packed with the same stationary phase. A 20 ml loop was used for sample injection. The mobile phase was acetonitrile:water (40:60) while wash was performed with methanol:water (80:90). The elution was isocratic with a flow rate of 1.0 ml min1. The detector was a VWD Agilent 1100 operating at 215 nm. The software supporting the analysis was Agilent Chemstation for LC Revision A.10.02. The detection system precision (standard deviation) was determined under conditions of repeatability (intra day repeatability) and intermediate precisions (inter day repeatability), by performing ten injections of the same

491

calibration standard either in the same day or on different days according to Quality Control criteria of SANCO/2007/3131 Document. 2.4.3. Stability assays Azadirachtin test solutions at two concentrations levels (12.8 and 51.2 mg a.i ml1) were prepared from the test and the technical grade products. All the solutions were produced and maintained at 28 C in the dark. At prefixed intervals of 24, 48, 72 and 96 h aliquots of test solutions were removed for HPLC analysis. The stability tests were replicated twice. 2.5. Statistical analysis The motility experiments were performed twice, as a split plot design, with six replications. The main factor was exposure time and the sub-factor was application rate. Since paralysis in carrier control was not significantly different from that in plain water, paralysis data for both experiments were expressed as the percentage immotile J2 increase over water control (Puntener,1981) and they were analyzed (ANOVA) using a five by three factorial approach (eight application doses by three exposure times) combined over time. Since normality and homogeneity of variance test indicated no problems no data transformation took place. Azadirachtin activity on the biological cycle of nematodes was studied in a complete randomized design with five replications and was performed twice. Data were transformed to Oc þ 1 to overcome normality and homogeneity problems. In all cases concerning both biological and stability assays, since ANOVAs indicated no significant treatment by time interaction (between runs of experiment), means were averaged over experiments. Treatments means were compared using Tukey’s test at P 0.05. 3. Results 3.1. Azadirachtin effect on J2 motility Analysis of variance indicated significant influence of exposure dose, time as well as their interaction on J2 motility (P 0.01) (Table 1). The relative percentage of immotile J2 after immersion in different test product doses, for various intervals is presented in Fig. 1. Azadirachtin had no significant effect on J2 motility at the recommended dose (0.2 mg a.i ml1) or at most of the higher test doses, regardless of the exposure time. In general, at concentrations of 0.2, 0.8 and 3.2 mg a.i ml1, motility of J2 was similar to the water control, unlike fosthiazate treatment in which all J2 were immotile. Only exposure of J2 to the test product dosages of 12.8 and 51.2 mg a.i ml1, for 24–96 h, resulted in a significant increase of immotile J2 by 21–35% respectively. Similar results were obtained when J2 were exposed to azadirachtin solutions of the same concentrations prepared from technical grade azadirachtin 11.8% (Fig. 2). Significantly (P 0.05) more immotile J2 were counted after exposure to a technical grade azadirachtin dose of 51.2 mg a.i ml1. Only this test concentration significantly increased immotile J2 by 11–20% after exposure of 48–96 h, respectively. No significant motility effect was

Table 1 Analysis of variance of the effect of time (24, 48 and 96 h), dose (0.2, 0.8, 3.2, 12.8 and 51.2 mg a.i. ml1) and their interaction on motility of second stage juveniles of Meloidogyne incognita. Factor

df

Time Dose Time dose

2 5 10

MS

F

P

AzaNema

Azadirachtin

AzaNema

Azadirachtin

AzaNema

Azadirachtin

60.5 42625.7 22.9

146.3 27627.6 53.3

7.59 5129.86 2.76

14.47 3231.86 6.24

0.02 0.01 0.03

0.01 0.01 0.01

492


fosthiazate

a

a

100

3.2 75

12.8 51.2

50

b

c

fg fg

ef

fg g

24 h

b

e

g g

48 h

3.3. Chemical analysis 3.3.1. Determination of azadirachtin A The content of the test product was confirmed by HPLC analysis and complied with its manufacturing specifications, since its average measurement did not differ from that declared. Chromatograms shown in Fig. 4 representing test solutions, prepared with the same azadirachtin concentration (30 mg a.i ml1), from formulated and technical grade product, are identical considering retention time and attenuation areas. This confirms the formulated fosthiazate a

a

0.2 0.8

100

3.2 12.8

75

51.2 50 b c

cd d d d

0 48 h

b 0.12

0.48

1.92

7.68

30.72

Dose µg azadirachtin g-1 soil

Increased dosage of test product did not result in a significant reduction (P > 0.05) of female nematodes in the roots, compared with the control treatment (Fig. 3). No nematodes were observed in roots in the fosthiazate treatment. Significantly fewer nematodes were observed only at the highest azadirachtin dose (30.72 mg a.i g1). Shoot and root fresh weights were not significantly different amongst treatments (P > 0.05) and no phytotoxicity was observed in any treatment (data not shown).

24 h

10

control fostiazate 0.03

3.2. Effect of azadirachtin on nematode life cycle

d d d d

20

96 h

observed on J2 after exposure to a mixture of water, Tween-20 and methanol (data not shown). For this reason the percentage immotile J2 increase was calculated over water control.

d d d d d

a

0

Fig. 1. Percentage (%) increase of immotile Meloidogyne incognita J2 over water control, after exposure to different doses of AzaNema EC (azadirachtin concentrations of 0.2–51.2 mg ml1). Fosthiazate (NemathorinÒ 150EC) was tested at 13.3 mg ml1 as the chemical control. Means followed by the same letter are not significantly different according to Tukey’s test (P 0.05, df ¼ 10) Error bars represent standard deviations.

25

a

b

e

Exposure time (h)

a

a

cd

d

d

25

a

a

0.8

0

immotile J2s increase over water control

30

0.2

a

Females gr-1

immotile J2s increase over water control

a

96 h

Exposure time (h) Fig. 2. Percentage (%) increase of immotile Meloidogyne incognita J2 over water control, after exposure to different doses of technical grade azadirachtin 11.8% (azadirachtin concentrations of 0.2–51.2 mg ml1). Fosthiazate (NemathorinÒ 150EC) was tested at 13.3 mg ml1 as the chemical control. Means followed by the same letter are not significantly different according to Tukey’s test (P 0.05, df ¼ 10). Error bars represent standard deviations.

Fig. 3. Effect of AzaNema EC, applied at the concentration range of 0.03–30.72 mg g1 in pot bioassays, on the number of M. incognita females per g of root. Fosthiazate (NemathorinÒ 150EC) was used as chemical control at 2 mg g1. Means followed by the same letter are not significantly different according to Tukey’s test (P 0.05, df ¼ 6). Error bars represent standard deviations.

product’s concentration in azadirachtin A, which is the main active ingredient against nematodes Meloidogyne incognita. The retention time for azadirachtin A was 11.357 0.06 min. The standard deviation under conditions of repeatability (intra day repeatability) was 1.46% and intermediate precisions (inter day repeatability) was 2.15% coinciding with the relative SANCO/2007/3131 requirements. 3.3.2. Stability assays Azadirachtin A residue in 51.2 mg a.i ml1 test solutions of formulated and technical grade azadirachtin after incubation for prefixed intervals at 28 C, are presented in Fig. 5. The fate of azadirachtin A residues measured in solutions of 12.8 mg ml1 was similar (data not shown). Total azadirachtin concentrations observed in test solutions were stable under experiment conditions for 72 h. On the fourth day after experiment establishment, total azadirachtin residues averaged 88 and 84% of nominal levels based on application doses and estimated enclosure volumes for test product and technical grade azadirachtin, respectively (Fig. 5). 4. Discussion Application of azadirachtin at the recommended dose used either as formulated product or as technical grade (azadirachtin 11.8%), did not affect motility. Specifically, in 24 well-plates experiments, increased numbers of immotile J2 compared to the control treatment were found only when the test product was used at doses of 12.8–51.2 mg a.i ml1. These results are in agreement with those reported by Javed et al. (2008b), who observed that the refined product ‘‘AzaÓ’’ (1% azadirachtin) at 5–10 mg a.i ml1 was neither nematostatic nor nematicidal to J2. On the contrary, our results do not coincide with the findings of Ambrogioni et al. (2003) who observed paralysis of J2 after 4 days of exposure to 1.92 mg a.i ml1 of ‘‘OikosÓ’’ (0.32% azadirachtin). When technical grade azadirachtin 11.8% was used, increased numbers of immotile J2 compared to the control treatment were obtained only after exposure at the dose of 51.2 mg a.i ml1. The difference on J2 motility effect in the above mentioned high concentration treatments between the test product and technical grade azadirachtin 11.8%, confirms the lack of efficacy of lower concentrations and could possibly be attributed to the different concentration and type of adjuvants in test solutions. Indeed, it is noted by Mordue and Blackwell (1993) that the selection of particular solvents during the formulation process is essential for successful control. In addition, it is reported that pH affects the efficacy of azadirachtin (Sundaram, 1996; Sundaram et al., 1997; Caboni et al., 2002, 2006). However, in all cases the poor efficacy observed in multiwell plates experiments


493

Fig. 4. HPLC chromatograms of (a) technical grade azadirachtin 11.8%, and (b) AzaNema EC solutions at concentration 30 mg ml1.

azadirachtin residues (µg ml-1)

cannot be attributed to the carrier’s pH (7.8), since the total azadirachtin residues remained stable throughout the experiment duration, according to the results by the stability tests (Fig. 5). In pot experiments azadirachtin applied at the recommended dose rate (0.03 mg a.i g1) once per nematode biological cycle (30 days at 28 C) failed to protect plants from infection because J2 invaded the roots, moulted and eventually became adults. Only the highest test concentration (30.72 mg a.i g1) achieved adequate control similar to that given by the organophosphate nematicide fosthiazate. Javed et al. (2007a,b, 2008b), reported good nematode control working with a commercial product (AzaÓ) containing 1% of azadirachtin at doses of 50–100 mg a.i g1, which are similar to the effective dose found in this study. The poor efficacy of the test product against nematodes cannot be attributed to reasons like poor product stability and inconsistency in azadirachtin formulation since its azadirachtin content was confirmed by HPLC analysis. The before mentioned reasons though, have provoked ineffective insect control (Immaraju, 1998; Isman et al., 1990). On the contrary, the limited efficacy of the test product must strongly be associated with the incapacity of its active ingredient (azadirachtin) to control root-knot nematodes at the recommended rate. This is also supported by lack of efficacy against J2 when the technical grade was used at equal concentrations to the test product. Similarly was reported that the nematostatic–nematicidal effect of neem

80

Technical grade azadirachtin (11.8%) Neemazal® EC (1%)

60

a a

40

a

a

a

a

48

72

b b

20

0 24

96

Hours of exposure to 28oC Fig. 5. Stability study of AzaNema EC and azadirachtin technical grade 11.8% test solutions at a concentration level of 51.2 mg ml1 stored at 28 C. Means followed by the same letter are not significantly different according to Tukey’s test (P 0.05, df ¼ 3).

formulations is not due to the azadirachtin content (Javed et al., 2008b). Based on our results we could conclude that the test product applied at the recommended dose (6 L ha1) at monthly intervals, is unlikely to control root-knot nematodes since it cannot reduce J2 motility, root penetration and development. To have practical value there need to be studied possible ways to enhance its activity, such as tank mixtures with other nematicides, both in terms of efficacy, residues and cost effectiveness. To the best of our knowledge this is the first efficacy evaluation data available for a commercial neem product tested at the recommended dose and application intervals against root-knot nematodes Acknowledgements The authors are grateful to Sipcam S.p.A., and Hellafarm Co for supplying the test substances. Special thanks are due to Mr. T. Koufakis and AGRIS SA for providing seeds and seedlings. The project is part of a PhD thesis funded by the Greek State Scholarships Foundation of Greece. References Adegbite, A.A., Adesiyan, S.O., 2005. Root extracts of plants to control root-knot nematode on edible soybean. World J. Agric. Sci. 1, 18–21. Agbenin, N.O., Emechebe, A.M., Marley, P.S., Akpa, A.D., 2005. Evaluation of nematicidal action of some botanicals on Meloidogyne incognita in vivo and in vitro. J. Agric. Rural Dev. Trop. Subtrop 106, 29–39. Akhtar, M., 2000. Nematicidal potential of the neem tree Azadirachta indica (A. Juss). Integ. Pest Manag. Rev. 5, 57–66. Akhtar, M., Malik, A., 2000. Roles of organic soil amendments and soil organisms in the biological control of plant-parasitic nematodes: a review. Bioresource Technol 74, 35–47. Akhtar, H., Anita, S., Prabhat Kumar, S., 2005. Studies on the management of rootknot nematode, Meloidogyne incognita-wilt fungus, Fusarium oxysporum disease complex of green gram, Vigna radiata cv ML-1108. J. Zhejiang Univ. Sci. 6B, 736–742. Ambrogioni, L., Caroppo, S., Capella, A., 2003. Attivita’ e modalita’ di azione del formulato Oikos nei confronti dei nematode galligeni. Inftore. Fitopatol 5, 35–38. Back, M.A., Haydock, P.P.J., Jenkinson, P., 2002. Disease complexes involving plant parasitic nematodes and soilborne pathogens. Plant Pathol 51, 683–697. Barrek, S., Paisse, O., Grenier-Loustalot, M.-F., 2004. Analysis of neem oils by LC-MS and degradation kinetics of azadirachtin-A in a controlled environment. Characterization of degradation products by HPLC-MS-MS. Anal. Bioanal. Chem. 378, 753–763. Bharadwaj, A., Satyawanti, S., 2006. Biocontrol of Meloidogyne incognita in Lycopersicon esculentum with AM fungi and oil cakes. J. Plant Pathol 5, 166–172. Bharadwaj, A., Sharma, S., 2007. Effect of some plant extracts on the hatch of Meloidogyne incognita eggs. Int. J. Botany 3, 312–316.

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