Crop Protection 35 (2012) 85e90

Contents lists available at SciVerse ScienceDirect

Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Melia azedarach controls Meloidogyne incognita and triggers plant

defense mechanisms on cucumber
I. Cavoski a, Z.Al. Chami a, F. Bouzebboudja a, N. Sasanelli b, V. Simeone a, D. Mondelli c,
T. Miano c, G. Sarais d, N.G. Ntalli d, P. Caboni d, *
a
International Centre for Advanced Mediterranean Agronomic Studies, Valenzano (Ba), Italy
b
Institute for Plant Protection, C.N.R., Bari, Italy
c
Department of Environmental and Agro-Forestry Biology and Chemistry, Faculty of Agriculture, University of Bari, Bari, Italy
d
Department of Pharmaceutical Chemistry and Technology, University of Cagliari, Cagliari, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Melia azedarach fruit extracts have recently raised a substantial interest for their use in crop protection
Received 25 March 2011 against phytoparasitic nematodes. The effect of M. azedarach on the root-knot nematode Meloidogyne
Received in revised form incognita on cucumber, as well as the effect on the plant deference mechanism, is reported herein.
5 January 2012
Crushed fruits of M. azedarach, tested in the soil at the rates of 30 and 60 g kg 1, exhibited nematicidal
Accepted 17 January 2012
activity similar to the one of fenamiphos (0.02 g a.i. kg 1) in terms of nematode population in roots and
soil as well as reproduction rate. M. azedarach water extracts, rich in aldehydes, alcohols and carboxylic
Keywords:
acids, showed nematicidal activity against M. incognita. Moreover, all M. azedarach treatments decreased
Melia azedarach fruits
Meloidogyne incognita
the activities of catalase (CAT) and peroxidase (POX) involved in host H2O2 detoxification. Soil application
Nematicidal activity of M. azedarach fruits could be favourably considered in the control of M. incognita on cucumber in
Enzymatic activity a sustainable agriculture, since they act directly as nematicidals. Furthermore, M. azedarach elicits plant
defence and helps the host to fight the nematodes infestation in an indirect way.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction activities (Ntalli et al., 2010b). Recently, carboxylic acids and

Environmental and health concerns impose the reduction
of conventional nematicides and the development of safer but
comparatively effective biorational nematode control measures
(Noling and Becker, 1994). Plants may represent a source of natu-
ral pesticides (Sasanelli and D’Addabbo, 1992, 1993; Isman,
2000; Chitwood, 2002; Perez et al., 2003; Matthiessen and
Kirkegaard, 2006).
Melia azedarach L., commonly known as chinaberry, is a plant
species of the Meliaceae family exhibiting a wide range of bio-
logical activities of practical agricultural and pharmaceutical use.
Extracts from various parts of M. azedarach are reported to exhibit
insecticidal (Bohnenstengela et al., 1999; Li, 1999; Céspedes et al.,
2000; Carpinella et al., 2002; Abou-Fakhr Hammad et al., 2005;
Charleston et al., 2005; Isman, 2006; Akhtar et al., 2008;
Rachokarn et al., 2008), antifungal (Carpinella et al., 2003, 2005),
nematicidal (Hasabo and Noweer, 2005; Ntalli et al., 2010a),
antihelmintic (Maciel et al., 2006), cytotoxic and antiproliferative
* Corresponding author.
E-mail address: caboni@unica.it (P. Caboni).
aldehydes from M. azedarach fruits, and in particular furfural,
5-hydroxymethylfurfural and furfurol have proven strong nema-
ticides (Ntalli et al., 2010c). On the other hand, amending the soil
with chinaberry products has been proven to enhance the soil
fertility. Toselli et al. (2010) concluded that M. azedarach leaves
and fruits in combination with inorganic fertilizers and composts
stimulated mineral N release, root N uptake and peach (Prunus
persica L.) growth. Noble et al. (1996) reported that the leaf litter of
M. azedarach can significantly increase the soil ash alkalinity
(an estimation of organic anion content) which results in an
overall pH increase. Furthermore, M. azedarach derivatives have
been reported to stimulate soil microbial biomass (Marino et al.,
2009; Spyrou et al., 2009).
Catalases (CAT) and peroxidases (APX) are H2O2 detoxification
enzymes in plants involved in the enzymatic scavenging of reactive
oxygen species (ROS) (Kim et al., 2005), occurring after plants’
exposure to biotic or abiotic factors (pollutants, pathogen attacks,
chilling and heating). Low activities of such enzymes inside the host
imply the enhanced defence system of the plant.
In the current study, we report on: a) the direct suppressive
effect of M. azedarach crushed fruits (CF) and their aqueous extracts
(AEF) on the root-knot nematode Meloidogyne incognita (Kofoid

0261-2194/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cropro.2012.01.011
86 I. Cavoski et al. / Crop Protection 35 (2012) 85e90
et White) Chitwood infecting cucumber, b) the GC/MS analysis of
the chemical composition of AEF and c) the triggering of plant
defense system induced by the reduction of catalase (CAT) and
ascorbate peroxidases (APX) activities in host roots.
2. Materials and methods
2.1. Chemicals
Ultrapure water was obtained from the Millipore (Billerica, MA)
Milli-Q system. The neem extract derived from Azadirachta indica
A. Juss contained 11% azadirachtin A (w w 1). Fenamiphos CS 240
was obtained from Makhteshim Agan Group.
2.2. Extracts preparation
Ripen fruits of M. azedarach were collected in Cagliari, Italy in
January 2010. A voucher specimen was deposited in the Depart-
ment of Life and Environmental Sciences (Botany and Botanical
Garden Division, Herbarium CAG, Sardinian Section, University of
Cagliari, Italy) for species identification. Mature fruits were initially
grinded in a blender and were successively used to prepare
aqueous extracts (AEF) at two different ratios, 1:5 and 1:10 (w v 1).
Samples were soaked by sonication for 15 min at 40  C and filtered
through a Whatman No. 1 filter paper (Whatman International Ltd.,
Maidstone, England) before their use. In order to calculate the
extraction yields, extracts were vacuum dried by rotary evaporator
(Ika Werke, Germany) at 60  C to constant weight and yields were
calculated as dry weights. Three replicates were carried out for each
extraction type.
2.3. GCeMS analysis
A Trace GC Ultra gas chromatograph (Thermo Finnigan), coupled
with a Trace DSQ mass spectrometry detector, a splitesplitless
injector, and an Xcalibur MS platform, was used to analyse the
M. azedarach aqueous extract (AEF). The column was a fused silica
capillary Varian CP-WAX 57CB (60 m  0.25 mm; film
thickness ¼ 0.25 mm) (Varian Inc.). The injector and the transfer
line temperatures were set at 200  C. The oven temperature was
programmed as follows: 50  C (hold for 1 min), raised to 220  C
(3  C min 1), and isothermally held for 30 min. Helium was used as
carrier gas at 1 mL min 1; 1 mL of AEF extract at a concentration of
2500 mg mL 1 was injected in the splitless mode. MS conditions
were as follows: ionization mode EI positive from 40 to 300 amu.
The components of AEF were identified by (a) comparison of their
relative retention times and mass fragmentation with those of
authentic standards and (b) computer matching against the NIST98
library. Quantitative analysis of each component was carried out
with the external standard method.
2.4. Pot experiments
The Italian population of M. incognita host race 1 (Taylor and
Sasser, 1978), was reared on tomato plants (Solanum lycopersicum
L.) cv. Rutgers in a glasshouse at 25  2  C. Two months later the
number of eggs and juveniles (J2) were quantified in roots (Hussey
and Barker, 1973). The chopped infected roots were then thor-
oughly mixed with 3 kg of steam sterilised sandy soil (pH 7.2;
sand > 99%; silt < 1%; clay < 1% and organic matter ¼ 0.75%) and
were used as inoculum. Appropriate amounts of this inoculum
were added and mixed with steam sterilised soil to give an initial
nematode population density of 5 eggs and juveniles mL 1 of soil
(Pi). Seven treatments were established in the pot experiment:
a and b) crushed M. azedarach fruits incorporated into the
M. incognita infested soil at rates of 30 and 60 g kg 1 soil (CF1 and
CF2); c and d) aqueous extracts (1:5 and 1:10; w v 1) prepared from
the same amount of fruits (AEF1 and AEF2); e) a commercial
formulation of azadirachtin (AZA) derived from neem plants
(A. indica A. Juss) applied at the dose of 0.03 g kg 1 soil (Ntalli et al.,
2009); f) a fenamiphos treatment (FEN) at the rate of 0.02 g kg 1
soil (62.5 L of a commercial formulation at 24% a.i. ha 1) and g) an
infested untreated soil control (CON). All treatments were applied
at transplant. To evaluate the effect of the nematode suppression on
root enzymatic activity M. azedarach treatments were replicated in
a nematode-infested and non-infested soil. Treatments of pots
were performed at 26.8% of soil water holding capacity. Each
treatment was replicated five times in a randomised block design at
25  2  C. In each pot a one-month-old cucumber (Cucumis sativus
L.) seedling, hybrids Sakamari F1, was transplanted. During the
experiment plants were irrigated as needed but no fertilizers were
applied.
Two months later, plants were uprooted and different
assessments were made. Root gall index (RGI) on each root
system was estimated according to a 0e5 scale, where 0 ¼ no
galls; 1 ¼ 1e2 galls; 2 ¼ 3e10 galls; 3 ¼ 11e30 galls; 4 ¼ 31e100
galls and 5 > 100 galls with a root system completely deformed
by the presence of numerous and large galls (Hussey and Barker,
1973). Final nematode soil density in each pot was determined by
processing 500 mL soil by the Coolen’s method (Coolen, 1979).
M. incognita density in roots was assessed according to Hussey
and Barker (1973). Final nematode population density (Pf) in
each pot was determined by summing nematodes recovered from
soil and roots. The reproduction rate of the nematode (r) was also
calculated as ratio between final and initial population density
(Pf Pi 1).
2.5. Enzymes extraction and assays
Fresh cucumber root samples (5 g) were homogenized in an
Osterizer Sunbeam, Designer homogenizer in 50 mL 100 mM
potassium phosphate buffer (pH 7.6) containing 1 mM EDTA-Na2
and 0.5 mM ascorbate. The homogenized samples were centrifuged
at 10,000 rpm for 5 min. The supernatant was used as a crude
enzyme extract in catalase (CAT) and ascorbate peroxidase (APX)
enzyme analyses. All measurements were made at 20  C in a UNI-
CAM BS DISC PD 2000-1 spectrophotometer. APX activity (EC
1.11.1.11) was determined by following the decrease of ascorbate
and measuring the change in absorbance at 290 nm for 1 min in
2 mL of a reaction mixture containing 50 mM potassium phosphate
buffer (pH 7.0), 1 mM EDTA-Na2, 0.5 mM ascorbic acid, 0.1 mM H2O2
and 50 mL of crude enzyme extract at 25  C (Nakano and Asada,
1981). The activity was calculated from the extinction coefficient
(2.8 mM 1 cm 1) for the ascorbate. CAT activity (EC 1.11.1.6) was
determined as a decrease in absorbance at 240 nm for 1 min
following the decomposition of H2O2 (Cakmak et al., 1993).
The reaction mixture (3 mL) contained 50 mM phosphate buffer
(pH 7.0), 15 mM H2O2 and 50 mL of crude enzyme extract at 25  C.
The activity was calculated from the extinction coefficient
(40 mM 1 cm 1) for H2O2.
2.6. Statistical analysis
Enzymatic and nematological data were subjected to statistical
analysis of variance (ANOVA) and means were compared by
Student’s t test and least significant difference (LSD) test at 0.05 and
0.01 levels of significance. Statistical analysis was performed using
the PlotIT program.
 I. Cavoski et al. / Crop Protection 35 (2012) 85e90 87

3. Results Adult nematodes in all M. azedarach treatments were similar to

those counted in FEN and AZA, and significantly less than CON
3.1. AEF extraction yields and chemical composition (Table 2). M. incognita eggs and J2 extracted from soil and roots
were strongly and significantly reduced (P ¼ 0.05) by all soil
M. azedarach fruits aqueous extraction yields at 1:5 and treatments (CF, AEF, AZA and FEN) in comparison to untreated CON.
1:10 (v/v) were 323 and 590 g kg 1, respectively. According to The highest reduction in nematode population density was
the GCeMS analysis (Fig. 1), AEF was found to afford mainly observed in FEN and CF2 treatment. AEF1 and AEF2 achieved
aldehydes, alcohols and carboxylic acids. Specifically, the slightly less nematode suppression, similar to the one exhibited
chemical composition of the aqueous extract was: furfural, acetic by AZA.
acid, 2-furancarboxyaldehyde-5-(methyl), furfurol, hexanoic acid, All treatments significant affected M. incognita reproduction
4-H-pyran-4-one, 2-3-dihydro-2,5-dihydroxy-6-methyl and 2-fur- rate but the treatments exhibiting the highest effect were once
ancarboxyaldehyde-5-(hydroxymethyl) (Table 1). again CF2 and FEN. The growth of cucumber was not influenced by
treatments with CF (data not shown).

3.2. Effect of CF and AEF against M. incognita

3.3. Root enzymatic activities
M. azedarach aqueous extracts and crushed fruits applied to the
soil are effective against M. incognita (Table 2) (Fig. 2) according to The activities of CAT and APX enzymes of cucumber roots,
the assessments outlined hereafter. infected or non-infected with M. incognita, in the different
M. azedarach treatments (CF1, CF2, AEF1 and AZD) significantly soil treatments are presented in Table 3. In general, infested
reduced RGI if compared with control. In general, treatments of roots exhibited significantly higher (P ¼ 0.01) CAT and APX
M. azedarach were infested at the same level to those treated with activities in comparison to non-infested cucumber roots
AZA, with the exception of AEF2 that was less infested. FEN (CAT: 132 and 43 mmol mg 1 min 1; APX: 23918 and 3060 mmol
exhibited the highest activity in terms of RGI. In general, mg 1 min 1). CAT and APX activities in infested roots treated
M. azedarach treatments achieved similar activity to that of the with CF and AEF were reduced with respect to control and
commercial nematicide FEN in terms of nematodes counted in host the lowest values were assessed in CF2, while in AZA treatment
roots. The treatment of AEF1 was the only exception and it was CAT and APX (mmol mg 1 min 1) were much higher. In non-
found similar to AZA. The number of nematodes eggs and juveniles infected plants, CAT and APX activities were similar to those
in all treatments was less than those recorded in CON. recorded in CON.

RT: 0,00 - 87,67

5,73
100
NL:
2,70E7
95
5 TIC MS
meliaacq
54,82
90

85

80

75

70

65

60
Relative Abundance
55

50

45

40
6
61,98

35

30

25
5,90
4,50

20

15
2 72,62

26,67
6,27
10
6,50
34,08
3 52,44

5
26,30
1 28,63 4 49,52 55,82
82,49
29,24 40,60 64,49 73,92
22,92 56,79 59,31
38,14 46,57 47,17 65,83 71,00 76,18 78,53
0,31 7,01 8,39 10,62 33,25 85,01
16,46 19,14 22,52
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Time (min)

1
Fig. 1. GCeMS chromatogram of the aqueous extract of Melia azedarach fruits (AEF) at 1:10 (w v ). Peaks: (1) furfural; (2) acetic acid; (3) furfurol; (4) hexanoic acid; (5) 4-H-pyran-
4-one, 2-3-dihydro-2,5-dihydroxy-6-methyl; (6) 2-furancarboxyaldehyde-5-(hydroxymethyl).
88 I. Cavoski et al. / Crop Protection 35 (2012) 85e90

The reduction of RGI in CF1, CF2, AEF1 and AZD is in accor-

dance to the findings of Akhtar and Mahmood (1994a,b)
who also witnessed a similar effect caused by neem based
products. They attributed this finding to the chemical minerali-
zation of the amendment and the release of ammonia, and
consequently the increase of nitrogen and carbon dioxide levels
in soil (Akhtar, 1993). In fact, ammonia plays an important role in
the suppression of planteparasitic nematodes and fungi
(Rodriguez-Kabana, 1986; Rodriguez-Kabana et al., 1987). More-
over, our report on population reduction in M. azedarach treated
roots is in accordance with the findings of Hasabo and Noweer
(2005) who also found that treating eggplant cv. Baladi with
aqueous extracts of basil, marigold, chinaberry and neem affect
the survival and reduce the population of M. incognita J2. Ntalli
and coauthors (Ntalli et al., 2010a) have reported a strong
nematicidal activity of M. azedarach against RKN and have
attributed it to the paralysis activity of aldehydes, alcohols and
carboxylic acids on J2 (Ntalli et al., 2010b). We also report on the
decrease of the nematode reproduction rate in the M. azeadarch
treated cucumber roots. This can be attributed to the effect
of Meliaceae plant extracts on nematode egg hatch and J2
mortality that has been investigated on the root-knot nematode
M. incognita and Rotylenchulus reniformis Linford & Oliveira, by
Siddiqui and Alam (1987). The enhanced activity of CF with
respect to AEF must be related to the complexity of chemical
composition and to the biodegradation products of CF in soil
like ammonia, nitrates, hydrogen sulfide and other volatile
substances and organic acids that can produce a direct nemati-
cidal effect (Oka, 2010). Also A. indica, Chromoleana odorata L.,
Ricinus communis L. and Jatropha curcas L. root extracts inhibit
root nematode invasion as reported by Adegbite and Adesiyan
(2005). This inhibition of invasion in host as well as the
reduced activity of juveniles in host’s roots, which blocks the
biological cycle completion in host, could be due to repellence
and disorientation properties M. azedarach products. Such
Fig. 2. Roots of cucumber plants with root knots nematode infected and non-infected activity was previously reported concerning tannins against
(right) treated with crushed fresh M. azedarach fruits (CF) or their aqueous extracts
(AEF) at doses reported in the text, azadirachtin A (AZA) and fenamiphos (FEN).
nematodes (Maistrello et al., 2010) while also other chemical
groups of compounds from plant tissues, like terpenoids
and phenolics, seem to be of similar activities. (Akhtar and
Mahmood, 1994a,b; Chitwood, 2002; Adegbite and Adesiyan,
4. Discussion
2005). Moreover, the nematicidal activity of the aqueous
extract of crushed fruits of M. azedarach could be mainly attrib-
According to our results the nematicidal Italian M. azedarach
uted to the soluble furfural which exhibits nematicidal activity
fruits were found to contain aldehydes, carboxylic acids and alco-
against Meloidogyne spp. (Ntalli et al., 2010b).
hols. In a previous study, the methanolic extract of Greek M. aze-
The activities of CAT and APX enzymes of cucumber roots,
darach fruits afforded slightly lower contents in respective
infected with M. incognita were significantly higher with regards
constituents with the exception of furfurol that resulted about five
to non-infested plants (Table 3). CAT and APX are considered
times lower (811 vs 4225 mg kg 1) (Ntalli et al., 2010b).
fundamental antioxidant enzymes in plants (Orvar and Ellis, 1997).
Plants subjected to stress change their enzymatic activities to
overcome the insult (Shigeoka et al., 2002; Simonetti et al., 2010).
Table 1
This is why plantepathogen interactions induce ROS scavengers
Aldehydes, alcohols and carboxylic acids contents in aqueous extract of Melia aze-
darach (AEF at 1:10 w v 1) activities increase (Cavalcanti et al., 2006). CAT and APX activities
1a
in infested roots treated with CF and AEF were reduced with
Retention time Compound Content mg kg
respect to control. The lowest values were assessed in CF2, while
(Rt) (min) (w w 1)
in AZA treatment CAT and APX (mmol mg 1 min 1) were much
26.30 furfural 1140
26.67 acetic acid 2100
higher. Interestingly, the lowest ROS scavengers activity was
30.61 2-furancarboxyaldehyde- 3.5 recorded on CF2 where the highest nematicidal activity was
5- (methyl) present (Table 2). Additionally, the decrease in ROS scavengers
34.08 furfurol 4225 activity could be attributed to the high total polyphenols levels in
40.60 hexanoic acid 1680
CF and AEF (data not shown) that reduce oxygen free radicals. In
54.82 4-H-pyran-4-one,2-3-dihydro- N.C.b
2,5-dihydroxy-6-methyl non-infected plants, CAT and APX activities were similar to those
61.98 2-furancarboxyaldehyde-5- 8870 recorded in CON.
(hydroxymethyl) It seems that M. azedarach treatments control M. incognita on
a
Expressed as dry fruits weight. cucumber by acting directly as nematicidals, reduce the activity of
b
Not calculated. the antioxidant enzymes and trigger the host defense.
 I. Cavoski et al. / Crop Protection 35 (2012) 85e90 89

Table 2
Effect of different soil treatments with crushed fresh Melia azedarach fruits (CF) or aqueous extracts (AEF) on M. incognita population density infesting cucumber plants.
1 1
Treatments Root gall Index (0e5) Eggs þ J2 g root \g root Nematode population Reproduction rate r ¼ Pf/Pi
Density/pot (Eggs þ J2/pot)
CF1 3.6a cb 766 abc 6.9 a 16564 bc 2.6 bc
CF2 3.2 c 543 ab 5.9 a 11331 ab 1.7 ab
AEF1 3.2 c 1290 bc 12.3 a 31867 e 4.9 e
AEF2 2.4 b 695 abc 9.9 a 19570 cd 3.0 cd
AZA 3.4 c 1772 c 15.3 a 25300 de 3.9 de
FEN 1.2 a 78 a 1.3 a 4438 a 0.7 a
CON 5.0 d 5763 d 77.5 b 59179 f 9.1 f
a
Each value is an average of five replications.
b
Data followed in each column by the same letters are not statistically different according to Least significant difference’s test (P ¼ 0.05).

Table 3
Catalase (CAT) and ascorbate peroxidase (APX) enzymes in cucumber roots infested with M. incognita in the different treatments.
1 1 1 1
Treatments CAT (mmol mg min , dw) APX (mmol mg min , dw)

Infested plants Non-Infested plants Student’s Infested plants Non-Infested plants Student’s
t test t Test
CF1 78a cb 42 a **c 23741 e 4284 b **c
CF2 65 ab 53 b ** 17536 c 4250 b **
AEF1 86 c 55 bc ** 13466 b 5141 b **
AEF2 72 b 47 ab ** 9837 a 4345 b **
AZA 119 d e e e 19089 d e e e
FEN 59 a e e e 8998 a e e e
CON 132 e 43 a ** 23918 e 3060 a **
a
Each value is an average of five replications.
b
Data followed in each column by the same letters are not statistically different according to least significant difference test (P ¼ 0.05).
c
** for P ¼ 0.01.

Acknowledgement
Special thanks for helpful suggestions to Antonio Murgia and
Simona Vargiu. This study was supported by a grant from the Italian
Ministero dell’Istruzione, dell’Università e della Ricerca: Research
Program PRIN 2008 “Discovery and evaluation of new microbial
and vegetable biopesticides for the natural insect pests control”.
This work has been a part of Master thesis in Mediterranean
Organic Agriculture course at CIHEAM-IAMB.
References
Abou-Fakhr Hammad, E.M., Nemer, N.M., Hawi, Z.K., Hanna, L.T., 2005. Responses of
the sweetpotato whitefly, Bemisia tabaci, to the chinaberry tree (Melia azedar-
ach L.) and its extracts. Ann. Appl. Biol. 137, 79e88.
Adegbite, A.A., Adesiyan, S.O., 2005. Root extracts of plants to control root-knot
nematode on edible soybean. World J. Agric. Sci. 1, 18e21.
Akhtar, M., Mahmood, I., 1994a. Nematode population and short-term tomato
growth in response to neem-based products and other soil amendments.
Nematropica 24, 169e173.
Akhtar, M., Mahmood, I., 1994b. Potentiality of phytochemicals in nematode
Control: a review. Bioresour. Technol. 48, 189e201.
Akhtar, Y., Yeoung, Y.R., Isman, M.B., 2008. Comparative bioactivity of selected
extracts from Meliaceae and some commercial botanical insecticides against
two noctuid caterpillars, Trichoplusiani and Pseudaletia unipuncta. Phytochem.
Rev. 7, 77e88.
Akhtar, M., 1993. Utilization of waste materials in nematode control: a review.
Bioresour. Technol. 45, 1e7.
Bohnenstengela, F.I., Wrayb, V., Wittec, L., Srivastavad, R.P., Prokscha, P., 1999.
Insecticidal meliacarpins (C-seco limonoids) from Melia azedarach. Phyto-
chemistry 50, 977e982.
Cakmak, I., Strbac, D., Marschner, H., 1993. Activities of hydrogen peroxide-
scavenging enzymes in germinated wheat seeds. J. Exp. Bot. 44, 127e132.
Carpinella, C., Ferrayoli, C., Valladares, G., Defago, M., Palacios, S., 2002. Potent
limonoid insect antifeedant from Melia azedarach. Biosci. Biotechnol. Biochem.
66, 1731e1736.
Carpinella, M.C., Giorda, L.M., Ferrayoli, C.G., Palacios, S.M., 2003. Antifungal effects
of different organic extracts from Melia azedarach L. on phytopathogenic fungi
and their isolated active components. J. Agric. Food Chem. 51, 2506e2511.
Carpinella, M.C., Ferrayoli, C.G., Palacios, S.M., 2005. Antifungal synergistic effect of
scopoletin, a hydroxycoumarin isolated from Melia azedarach L. fruits. J. Agric.
Food Chem. 53, 2922e2927.
Cavalcanti, F.R., Resende, M.L.V., Lima, J.P.M.S., Silviera, J.A.G.S., Oliviera, J.T.A., 2006.
Activities of antioxidant enzymes and photosynthetic responses in tomato pre-
treated by plant activators and inoculated by Xanthomonas vesicatoria. Physiol.
Mol. Plant Pathol. 68, 198e208.
Céspedes, C.L., Calderón, J.S., Lina, L., Aranda, E., 2000. Growth inhibitory effects on
fall armyworm Spodoptera frugiperda of some limonoids isolated from Cedrela
spp. (Meliaceae). J. Agric. Food Chem. 48, 1903e1908.
Charleston, D.S., Kfir, R., Vet, L.E., Dicke, M., 2005. Behavioural responses of
diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) to extracts
derived from Melia azedarach and Azadirachta indica. Bull. Entomol. Res. 95,
457e465.
Chitwood, D.J., 2002. Phytochemical based strageties for nematode control. Ann.
Rev. Phytopathol 40, 221e249.
Coolen, W.A., 1979. Methods for the extraction of Meloidogyne spp. and other
nematodes from roots and soil. In: Lamberti, F., Taylor, C.E. (Eds.), Root-knot
Nematodes (Meloidogyne species), Systematics, Biology and Control. Academic
Press, London, UK, pp. 317e329.
Hasabo, S.A., Noweer, E.M.A., 2005. Management of root-knot nematode Meloido-
gyne incognita on eggplant with some plant extracts. Egypt. J. Phytopathol 33,
65e72.
Hussey, R.S., Barker, K.S., 1973. A comparison of methods of collecting inocula of
Meloidogyne spp. including a new technique. Plant Dis. Rep. 57, 1025e1028.
Isman, M.B., 2000. Plant essential oils for pest and disease management. Crop Prot.
19, 603e608.
Isman, M.B., 2006. Botanical insecticides, deterrents, and repellents in modern
agriculture and an increasingly regulated world. Ann. Rev. Entomol. 51, 45e66.
Kim, S.Y., Lim, J.-H., Park, M.R., Kim, Y.J., Park, T.I., Seo, Y.W., Choi, K.G., Yun, S.J.,
2005. Enhanced antioxidant enzymes are associated with reduced hydrogen
peroxide in barley roots under saline stress. J. Biochem. Mol. Biol. 38, 218e224.
Li, X., 1999. Recent studies on insecticidal activities of limonoids from meliaceous
plants. Entomol. Sin. 6, 283e288.
Maciel, M.V., Morais, S.M., Bevilaqua, C.M.L., Camurka-Vaconcelos, A.L.F.,
Costa, C.T.C., Castro, C.M.C., 2006. Ovicidal and larviocidal activity of Melia
azedarach extracts on Haemonchus contortus. Veter. Parasitol. 140, 98e104.
Maistrello, L., Vaccari, G., Sasanelli, N., 2010. Effect of chestnut tannins on the root-
knot nematode Meloidogyne javanica. Helminthologia 47, 48e57.
Marino, G., Gaggia, F., Saiano, F., Biavati, B., Marangoni, B., 2009. Elimination of
in vitro bacterial contaminants in shoot cultures of ‘MRS 2/5’ plum hybrid by the
use of Melia azedarach extracts. Eur. J. Plant Pathol. 123, 195e205.
Matthiessen, J.N., Kirkegaard, J.A., 2006. Biofumigation and enhanced biodegrada-
tion: opportunity and challenge in soilborne pest and disease management.
Crit. Rev. Plant Sci. 25, 235e265.
Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific
peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867e880.
Noble, D., Zenneck, I., Randall, P.J., 1996. Leaf litter ash alkalinity and neutralisation
of soil acidity. Plant Soil 179, 2293e2302.
90 I. Cavoski et al. / Crop Protection 35 (2012) 85e90
Noling, J.W., Becker, J.O., 1994. The challenge of research and extension to define
and implement alternatives to methyl bromide. J. Nematol. 26, 573e586.
Ntalli, N.G., Menkissoglu-Spiroudi, U., Giannakou, I.O., Prophetou-Athanasiadou, D.A.,
2009. Efficacy evaluation of a neem (Azadirachta indica A. Juss) formulation against
root-knot nematodes Meloidogyne incognita. Crop Prot. 28, 489e494.
Ntalli, N.G., Menkisoglu-Spiroudi, U., Giannakou, I., 2010a. Nematicidal activity of
powder and extracts of Melia azedarach fruits against Meloidogyne incognita.
Ann. Appl. Biol. 156, 309e317.
Ntalli, N.G., Cottiglia, F., Bueno, C.A., Alché, L.E., Leonti, M., Vargiu, S., Bifulco, E.,
Menkissoglu-Spiroudi, U., Caboni, P., 2010b. Cytotoxic tirucallane triterpenoids
from Melia azedarach fruits. Molecules 15, 5866e5877.
Ntalli, N.G., Vargiu, S., Menkisoglu-Spiroudi, U., Caboni, P., 2010c. Nematicidal
Carboxylic acids and aldehydes from Melia azedarach fruits. J. Agric. Food Chem.
58, 11390e11394.
Oka, Y., 2010. Mechanisms of nematode suppression by organic soil amendments.
Appl. Soil Ecol. 44, 101e115.
Orvar, B.L., Ellis, B.E., 1997. Transgenic tobacco plants expressing antisense RNA for
cytosolic ascorbate peroxidase show increased susceptibility to ozone injury.
Plant J. 11, 1297e1305.
Perez, M.P., Navas-Cortes, J.A., Pascual-Villalobos, M.J., Castillo, P., 2003. Nematicidal
activity of essential oils and organic amendments from Asteraceae against root-
knot nematodes. Phytopathology 52, 395e401.
Rachokarn, S., Piyasaengthong, N., Bullangpoti, V., 2008. Impact of botanical
extracts derived from leaf extracts Melia azedarach L. (Meliaceae) and Amar-
anthus viridis L. (Amaranthaceae) on populations of Spodoptera exigua (Hübner)
(Lepidoptera: Noctuidae) and detoxification enzyme activities. Commun. Agric.
Appl. Biol. Sci. 73, 451e457.
Rodriguez-Kabana, R., Morgan-Jones, G., Chet, I., 1987. Biological control of nema-
todes: soil amendments and microbial antagonists. Plant Soil 100, 237e247.
Rodriguez-Kabana, R., 1986. Organic and inorganic nitrogen amendments to soil as
nematode suppressants. J. Nematol. 18, 129e135.
Sasanelli, N., D’Addabbo, T., 1992. The effect of Cineraria maritima, Ruta graveolens
and Tagetes erecta extracts on the hatching of Heterodera schachtii. Nematol.
Medit. 20, 49e51.
Sasanelli, N., D’Addabbo, T., 1993. Effect of Cineraria maritima, Ruta graveolens and
Tagetes erecta leaf and root extracts on Italian populations of Meloidogyne
species. Nematol. Medit. 21, 21e25.
Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y., Yoshimura, K.,
2002. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot.
372, 1305e1319.
Siddiqui, M.A., Alam, M.M., 1987. Efficacy of seed dressing with extracts of neem and
Persia lilak against Meloidogyne incognita and Rotylenchulus reniformis. Nematol
Medit. 15, 399e403.
Simonetti, E., Alba, E., Montes, M.J., Delibes, A., Lopez-Branca, I., 2010. Analysis of ascor-
bate peroxidase genes expressed in resistant and susceptible wheat lines infected
by the cereal cyst nematode, Heterodera avenae. Plant Cell Rep. 29, 1169e1178.
Spyrou, I.M., Karpouzas, D.G., Menkissoglu-Spiroudi, U., 2009. Do botanical pesticides
alter the structure of the soil microbial community? Microb. Ecol. 58, 715e727.
Taylor, A.L., Sasser, J.N., 1978. Biology, Identification and Control of Root-knot
Nematodes (Meloidogyne spp.). North Carolina State University Graphic,
Raleigh, N.C. (U.S.A.), 111 pp.
Toselli, M., Baldi, E., Sorrenti, G., Quartieri, M., Marangon, B., 2010. Evaluation of the
effectiveness of soil-applied plant derivatives of Meliaceae species on nitrogen
availability to peach trees. Sci. Hortic.-Amsterdam 124, 183e188.