Combined use of Aqueous Plant Extracts for Controlling Meloidogyne incognita and Modulating Chemical Constituents in Tomato under Greenhouse Conditions

Doaa Khairy1, Mohamed Ali Osman2 and Fatma Abdel Mohsen Mostafa1*

1Agricultural Zoology Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt; 2Biology Department, Faculty of Science, Imam Abdul Rahman bin Faisal University, Dammam, Saudi Arabia.

Received | December 09, 2021; Accepted | March 19, 2022; Published | April 05, 2022

*Correspondence | Fatma Abdel Mohsen Mostafa, Agricultural Zoology Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt; Email: [email protected]

Citation | Khairy, D., Osman, M.A. and Mostafa, F.A.M., 2022. Combined use of aqueous plant extracts for controlling Meloidogyne incognita and modulating chemical constituents in tomato under greenhouse conditions. Pakistan Journal of Nematology, 40(1): 1-11.

DOI | https://dx.doi.org/10.17582/journal.pjn/2022/40.1.1.11

Keywords | Meloidogyne incognita, Aqueous extracts, Canola, Moringa, Neem

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Root-knot nematodes, Meloidogyne spp. are an ubiquitous pathogens causing major problems to economic vegetable crops globally grown in tropics and subtropics. Meloidogyne incognita (Kofoid and White) Chitwood is among the most widespread pathogen infecting solanaceous plant and considered a main obstacle of tomato (Solanum lycopersicum L.) production. Egypt ranked the fifth worldwide in tomato cultivation producing 6.9 million tons in 2019 (FAOSTAT, 2020). Minimizing the negative influence of root-knot nematodes, Meloidogyne spp., could be accomplished by chemical nematicides. However, the toxic nature of such chemicals to ecosystem, human, plant and animal life has compelled the researchers to use economically and ecofriendly alternatives strategies.

A strong impulse has given to the study of the botanical nematicides because of the presence of different metabolites with an activity against phytoparasitic nematodes (El-Deriny et al., 2020). Nematicidal activities of various botanical extracts have been evaluated by many researchers (Saeed et al., 2015; Kepenekci et al., 2016; Haroon et al., 2018) against Meloidogyne spp. and still increasing since botanical species produce a massive secondary metabolite i.e. alkaloids, terpenoids and phenolic compounds which play a vital role in resistance mechanism against pests and diseases.

Neem trees (Azadirachta indica Juss) are known to have biocide potential against insects, fungi and some plant parasitic nematodes and many neem-based pesticides formulations have been evolved and registered in several countries with commercial names i.e., Bioneem, Neem Azal-T, etc. (Haroon et al., 2018; Metwally et al., 2019). Azadirachtin is the staple compound present in neem with strongest nematicidal activity (Ntalli et al., 2009; Khalil, 2013) and may act as environmentally friendly favorable option in integrated nematodes management programs.

Various parts of moringa (Moringa oleifera Lam) possess several biological activities i.e. antioxidant, antimicrobial and antibacterial, antifungal and anti -inflamatory properties. Phytochemical profiling of Moringa revealed its richness in alkaloids, steroids, tannins, flavonoids, terpenoids, saponins, anthraquinones, reducing sugars along with glucosinolates and isothiocyanates (Izuogu et al., 2013). The toxicity of moringa leaves and seeds against plant parasitic nematodes has been appraised by a (Izuogu et al., 2013; Khairy, 2016; Haroon et al., 2018; Khairy et al., 2021).

Canola plant (Brassica napus) belongs to family cruciferae also received considerable concern as a substitutional approach for controlling plant parasitic nematodes. Glucosinolates found in cruciferous, are naturally secondary metabolites which could be hydrolyzed by myrosinase releasing a number of compounds essentially isothiocyanates (Mojtahedi et al., 1991; Asaduzzaman et al., 2014; Ntalli et al., 2020). The nematicidal properties of root extracts of various plants against parasitic nematodes have been conducted by many investigators (Goswami and Vijayalakshmi, 1986; Onifade and Egunjobi, 1994).

Compatibility of plant extract mixtures against M. incognita had rarely done therefore the current study was carried out to evaluate extracts of some plant parts as potential inducers for controlling Meloidogyne incognita, as well as, improving tomato plant growth and modulating chemical components.

Materials and Methods

Collection of plant parts

Leaves of moringa plant (Moringa oleifera Lam, Fam. Moringaceae) and neem plant (Azadirachta indica A. Juss, Fam. Meliaceae) as well as leaves, stems and roots of canola plant (Brassica napus L., Fam. Cruciferae) were collected from the experimental farm of Faculty of Agriculture, Mansoura University, Mansoura, Egypt and transferred to the Nematological laboratory for plant extracts preparation.

Botanical extracts

Leaves, stems and roots of canola as well as moringa and neem leaves were thoroughly washed and chopped. Twenty-five grams of each plant part were separately ground to fine particles in 100 ml distilled water using an electric blender. Solutions were then centrifuged at 5000 rpm for five minutes, filtered through filter paper Whatman No. 1 and considered as undiluted solutions (Nimbalkar and Rajurkar, 2009). Aqueous leaf extracts of moringa and neem were prepared at 5% concentration. Whereas aqueous extracts of canola parts were prepared at 10% concentration.

Nematode inoculum

Eggs of M. incognita extracted from coleus roots heavily infested with M.incognita (Hussey and Barker, 1973) were used as inoculum.

Experimental design

The experiment was carried out in plastic pots (13 cm-d) filling with 850 g steam sterilized clay loamy soil (Coarse sand 2.88; Fine sand 23.41; Silt 35.09; Clay 38.62%). Seedlings of tomato cv. Kesmat (25 days old) were separately transplanted in each pot and simultaneously soil was drenched with aqueous leaf extracts of moringa and neem singly and in combination with different parts of canola i.e. leaves, stems and roots at the rate of 2 ml/plant/pot. Five days later, 1400 eggs and juveniles of M. incognita were applied to each seedling. Oxamyl (0.3g/ plant/pot), a carbamate systemic nematicide, was introduced two days after nematode inoculation. Uninoculated and untreated seedlings served as negative control, however those received nematode inoculum served as positive control. The experiment was set up in a randomized complete block design (RCBD) system with four replicates for each treatment (25±4ºC). Plants were regularly irrigated. Treatments were as follows: 1- Moringa leaf (Ml); 2-Neem leaf (Nl) 3- Canola leaf (Nl); 4- Canola shoot (Cs); 5- Canola root (Cr); 6- Ml+ Cl; 7- Ml + Cs; 8- Ml+ Cr; 9- Nl + Cl ; 10- Nl + Cs; 11- Nl+Cr; 12- Ml+ Nl+ Cl; 13- Ml+Nl+ Cs; 14- Ml+Nl+ Cr; 15- Oxamyl; 16-Healthy plants and 17- Nematode alone. The experiment was terminated 42 days after inoculation. Plants were harvested and observations on growth criteria were undertaken. Nematode population (J2) was extracted from soil using sieving and modified Baermann-technique (Goodey, 1957). Roots were stained in acid fuchsin lactic acid as described by Byrd et al. (1983) and measured for root galling, number of egg masses, females, and developmental stages. Root gall and egg masses indices (EI) were determined on a scale of 1-5 (Taylor and Sasser, 1978).

Chemical components

Dried weight of tomato leaves (0.2 g) was subjected to chemical analysis for nutrient contents (N, P and K). Total nitrogen (N) was determined according to Kjeldahl method (AOAC, 1980). Total phosphorus(P) was estimated by Fiske and Subbarow (1925), while total potassium (K) was flam photometry estimated (Jackson, 1967). The leaf photosynthesis pigments (chlorophyll A, B) were spectrophotometrically measured using Fadeel‘s method (1962).

Defense related compounds

Total salicylic acid (SA) and total phenol (TP) were estimated in dried leaves of tomato according to the method of Javaheri et al. (2012).

Enzymes activity

Phenylalanine ammonia-lyase (PAL) activity was estimated as described by Chen et al. (2000).

Flavonoides and phenolic active ingredients

The crude aqueous leaf extract of neem was analysed for the estimation of flavonoids and phenolic active ingredients using High Pressure Liquid Chromatography (HPLC). Extraction was carried out at Food Technology Institute, Agric. Research Center, Giza, Egypt.

Statistical analysis

The data were statistically analysed (ANOVA) according to Gomez and Gomez (1984), followed by Duncan’s multiple range test to compare differences between means at P >0.05 (Duncan, 1955).

Results and Discussion

Screened plant extracts of moringa, neem and canola parts combined in mixtures to expand their nematicidal properties against M. incognita as well as to increase tomato growth are shown in Tables (1 and 2). The obtained data indicated that M. incognita infection caused a significant decrement in plant growth parameters with reduction percentage in plant length and total plant fresh weight reached 37.7 and 49.6%, respectively (Table 1). Aqueous leaf extract of neem and canola were the best amongst individual applications and significantly (P<0.05) improved shoot length and weight of tomato infected with M. incognita, respectively. However, dual and triple applications showed synergistic impact on plant growth compared with single application. Hence, triple application of canola (Cl), moringa (Ml) and neem leaf (Nl) extracts significantly (P<0.05) improved tomato shoot length with percentage of increase over control reached 37.5% followed by Ml+Nl+Cr (30.2%) in par with NL+Cs (30.2%) and Nl+Cr (30.2%). All treatments significantly suppressed nematode population whether in soil or root as compared to control (Table 2). The greatest suppression in nematode parameters was recorded with pots received oxamyl (Rf= 0.25). Among dual applications, Nl integrated with Cl extracts were found to have synergistic effect and non-significantly suppressed soil nematode population (Red.= 77.1%, Rf= 0.91) compared to single application of aqueous Cl (Red.=74.07%, Rf= 0.95) or Nl (Red.= 73.46%, Rf= 0.98) extracts.

 

Table 1: Effect of aqueous plant extracts singly and in mixture as potential inducers on growth of tomato infected with Meloidogyne incognita under greenhouse conditions.

Treatments	Plant Growth Response
	Length (cm)		Total Length	% dec. / inc	Fresh weight (g)		Plant F. wt	٪ dec/inc.
	Shoot	Root			Shoot	Root
Ml	18.5 ab	16.3 b-d	34.8	16.8	4.9 ab	1.8a-c	6.7	9.9
Nl	19.3 ab	15.5 b-d	34.8	16.8	5.0 ab	1.8a-c	6.8	11.8
Cl	13.3 c	15.0 b-d	28.3	-5.0	6.2 a	1.0bc	7.2	17.2
Cs	15.0 bc	12.0cd	27.0	-9.3	4.5 ab	1.3bc	5.8	-4.9
Cr	15.0 bc	12 .0cd	27.0	-9.3	5.6 ab	1.8a-c	7.4	21.3
Ml+ Cl	16.3 a-c	21.5bc	37.8	26.8	4.0 ab	3.6a	7.6	24.6
Ml+Cs	15.3 bc	24.3 ab	39.6	32.8	4.2ab	3.9a	8.1	32.8
Ml+Cr	17.3 a-c	21.3 bc	38.5	29.1	5.0 ab	3.2a	8.2	34.4
Nl + Cl	17.1 a-c	19.7 bc	36.8	23.4	5.4 ab	3.4a	8.8	44.3
Nl + Cs	16.5 a-c	22.3 ab	38.8	30.2	5.1ab	4.0a	9.1	49.2
Nl + Cr	16.8 a-c	22.0 ab	38.8	30.2	5.6 ab	3.6a	9.2	50.8
Ml+ Nl + Cl	19.0 ab	22.0 ab	41.0	37.5	4.5ab	3.7a	8.2	34.4
Ml+ Nl + Cs	17.0 a-c	19.3 bc	36.3	21.8	4.5a b	4.0a	8.5	39.3
Ml+ Nl + Cr	15.5 bc	23.3 ab	38.8	30.2	3.8 b	4.4a	8.2	34.4
Oxamyl	20.8 a	27.5 a	48.3	62.0	9.6 a	4.2a	13.8	126.2
Healthy plants	19.3 ab	28.5 a	47.8	60.4	8.0 a	4.1a	12.1	98.4
Nematode only	13.6 c	16.2 b-d	29.8	-	3.3 b	2.8 a	6.1	-

 

Each value is a mean of four replicates. Means marked with the same letter (s) in each column did not significantly differ at P< 0.05 according to Duncan’s multiple- range test. Ml: moringa leaf; Cl: canola leaf; Cs: canola stem; Cr: canola root and Nl: neem leaf.

 

Table 2: Effect of aqueous plant extracts singly and in mixture as potential inducers on Meloidogyne incognita population and reproduction on tomato under greenhouse conditions.

Treatments	Nematode population in		No .eggs/ egg mass	Total final population (Pf)	Rf* (Pf/Pi)	**RGI	**EI
	Root (females and developmental stages)	Soil (J2s)
Ml	10 de	799 bc	192 b	2346	1.68	3.0	2.0
Nl	9 ef	735 b-d	90 b-e	1375	0.98	2.0	2.0
Cl	8 f	718 b-d	100 b-e	1327	0.95	2.0	2.0
Cs	10 de	633 c-e	129 b-e	1547	1.11	2.0	2.0
Cr	10 de	726 b-d	170 bc	1928	1.38	3.0	2.0
Ml+ Cl	9 ef	894 bc	147 b	1346	0.96	2.0	2.0
Ml+Cs	7 f	993 b	172 b	1690	1.21	2.0	2.0
Ml+Cr	12 bc	944 bc	166 b	1787	1.28	2.0	2.0
Nl + Cl	9 ef	635 cd	158 b	1280	0.91	2.0	2.0
Nl + Cs	11 cd	707 b-d	167 b	1722	1.23	2.0	2.0
Nl + Cr	12 bc	809 bc	149 b	1866	1.33	3.0	2.0
Ml+ Nl + Cl	10 de	434 de	151 b	1353	0.97	3.0	2.0
Ml+ Nl + Cs	13 b	611 cd	174 b	2369	1.69	3.0	2.0
Ml+ Nl + Cr	10 de	748 b-d	169 b	1608	1.15	3.0	2.0
Oxamyl	3 g	291 ef	51 c	345	0.25	1.3	0.5
Nematode only	68a	2769 a	364 a	12671	9.05	4.0	3.8

 

Each value is the mean of four replicates. Means marked with the same letter(s) in each column did not significantly differ at P< 0.05 according to Duncan’s mulltiple range test. *Rf: Reproduction factor; Pf: Nematode population in soil+ No. developmental stages +No. females+ (No. eggs/egg mass x No. egg masses), Pi:1400 eggs and juveniles of M. incognita. Ml: moringa leaf; Cl: canola leaf; Cs: canola stem; Cr: canola root and Nl: neem leaf. *Root gall index (RGI) or egg masses index (EI) was determined according to the scale given by Taylor and Sasser (1978) as follows : 0= no galls or egg masses; 1= 1-2; 2= 3-10; 3= 11-30; 4=31-100 and 5= more than 100 galls or egg masses.

However, the highest synergistic action was postulated with the introduction of Nl with both Ml leaf and Cl extracts that significantly (P<0.05) suppressed nematode population in soil (84.33%) with Rf equal 0.97 compared to nematode alone (Rf= 9.05) followed by Ml+Nl+Cs (77.9%) then Ml+Nl+Cr (73.0%). However, aqueous leaf extracts of canola and neem showed more lethal effect on total population (Pf) and number of eggs/ egg mass in comparison to stem and root extracts of canola and moringa leaf.

Root galling and egg mass numbers of tomato plants were significantly (P<0.05) decreased by all treatments of plant extracts (Table 2). Root gall and egg masses indices ranged from 1.3 to 3.0 and 0.5 to 2.0, respectively compared to control plants (RGI=4.0; EI=3.8, respectively). The highest reduction in root galling (95.7%) or egg masses (96.3%) was recorded with oxamyl (RGI=1.3 and EI = 0.5.) Even, significant difference in root galling or number of egg masses in most treatments was not detected (Table 2).

NPK and photosynthesis pigments

Chemical constituents in dried tomato leaves were significantly altered as a result of mixture plant extracts (Table 3). Hence, aqueous leaf extracts of moringa and neem integrated with different botanical part extracts of canola showed better results than did single or dual applications. Meanwhile, Ml + Nl + Cr extract registered the most effective and exhibited the highest percentage of increase in N (29.88%), P (41.37%) and K (40.66%) and total chlorophyll (12.52%).

Resistance related compounds

Herein, phenylalanine ammonia lyase (PAL), total phenol and salicylic acid (SA) activities were significantly raised in tomato plants as a result of root-knot nematode, M. incognita. Plant extracts applied singly or in mixture showed significantly lower activities in such compounds compared to nematode alone. Irrespective to oxamyl, the highest suppression in such criteria was demonstrated with Ml + Nl + Cr extract (Figure 1).

Resistance related compounds

Herein, phenylalanine ammonia lyase (PAL), total phenol and salicylic acid (SA) activities were significantly raised in tomato plants as a result of root-knot nematode, M. incognita. Plant extracts applied singly or in mixture showed significantly lower activities in such compounds compared to nematode alone. Irrespective to oxamyl, the highest suppression in such criteria was demonstrated with Ml + Nl + Cr extract (Figure 1).

 

Estimation of flavonoids and phenolic compounds in aqueous extract of neem leaves

Flavonoids fractioned using HPLC (Table 4) showed the presence of 21 compounds in aqueous leaf extract of neem i.e. Hespiridin, Naringin, Hespirtin and Rosmarinic acid. Also, chemical analysis of aqueous neem leaf extract also revealed the presence of 23 phenolic compounds including e-Vanillic, pyrogallol, benzoic, salicylic acid and gallic acid (Table 4). The chemical structure of selected flavonoids and phenolic compounds is presented in Figures 2 and 3.

 

 

Table 3: Effect of aqueous plant extracts singly and in mixture as potential inducers on chemical constituents in dried leaves of tomato infected with Meloidogyne incognita under greenhouse conditions.

Treatments	Chemical constituents
	N	P	K	Chlorophyll content (m/g)
				Chlo A	Chlo B	Total (A+B)
Ml	2.95fg	0.277 c	2.51 h	0.629 j	0.446 j	1.075
Nl	2.80 hi	0.261 de	2.33 l	0.618 m	0.434 m	1.052
Cl	2.87 gh	0.288 b	2.42 j	0.625 j	0.440 l	1.065
Cs	2.47 m	0.226 hi	1.98 q	0.601 p	0.416 p	1.017
Cr	2.63 k	0.243 fg	2.15no	0.609 o	0.425 o	1.034
Ml+ Cl	2.85 g-i	0.241 gh	2.49 i	0.645 h	0.445 k	1.090
Ml+Cs	2.93 fg	0.252 ef	2.58 g	0.654 g	0.452 g	1.106
Ml+Cr	3.02 ef	0.263 d	2.66 f	0.663 f	0.458 f	1.121
Nl + Cl	2.59 kl	0.212 ij	2.18 n	0.621 l	0.429 n	1.050
Nl + Cs	2.68 jk	0.221 i	2.29 m	0.630 k	0.434 m	1.064
Nl + Cr	2.76 ij	0.232 h	2.37 k	0.638 i	0.440 l	1.078
Ml+ Nl + Cl	3.1 de	0.270 cd	2.79 e	0.671 e	0.464 e	1.135
Ml+ Nl + Cs	3.17 cd	0.279 bc	2.87 d	0.682 d	0.471 d	1.153
Ml+ Nl + Cr	3.26 bc	0.287 b	2.94 c	0.689 c	0.479 c	1.168
Oxamyl	3.34 ab	0.298 a	3.03 b	0.695 b	0.485 b	1.180
Healthy Plant	3.43 a	0.306 a	3.12 a	0.703 a	0.489 a	1.192
Nematode only	2.51 lm	0.203 j	2.09 p	0.613 n	0.425 o	1.038

 

Each value is the mean of four replicates. Means marked with the same letter(s) in each column did not significantly differ at P< 0.05 according to Duncan’s mulltiple range test. Ml: moringa leaf; Cl: canola leaf; Cs: canola stem; Cr: canola root and Nl: neem leaf; N: nitrogen, P: phosphorus; K: potassium.

 

Table 4: Flavonoids and phenolic compounds in aqueous neem leaf extract.

Flavonoids compounds	Contents (μg/100g)	Phenolic compounds	Contents (μg/100g)
Luteo.6-arabinose8-glucose	4250.63	Gallic acid	51.45
Luteo.6-glucose 8- arabinose	1046.68	Pyrogallol	902.16
Apig.6-arabinose8-glucose	1032.97	4-Amino-Benzoic	3.29
Apig.6-rhamnose 8-glucose	2022.61	Protocatechuic	162.29
Apig.6-glucose 8-rhamnose	1764.94	Catechein	39.94
Luteo.7- glucose	390.23	Chlorogenic	100.55
Naringin	796.59	Catechol	76.14
Rutin	93.84	Epicatechin	122.30
Hespiridin	6494.52	Caffien	46.58
Rosmarinic acid	126.88	Caffeic	10.77
Apig.7-0-o-neohespiroside	222.80	Vanillic	111.75
Kamp.3,7-dirhamoside	195.44	p-Coumaric	231.68
Apigenin-7-glucose	203.15	Ferulic	92.36
Quercetrin	45.45	Iso- Ferulic	51.37
Quercitin	26.40	Reversetrol	17.62
Naringenin	43.33	Ellagic	386.71
Hespirtin	302.16	e- Vanillic	4699.53
Kampferol	114.51	α –Coumaric	260.58
Rhamnetin	79.88	Benzoic	387.42
Apigenin	8.80	3,4,5 methoxy Coumaric	59.70
Acacetin	1.24	Coumaric	21.86
		Salicylic acid	236.20
		Cinnamic	28.76

 

Synergistic effects of plant extracts mixture have been widely documented on bacteria and soil borne fungi (Begum and Navaraj, 2012; Al-Terehi et al., 2013; Riveral et al., 2014). However, little attempt has been given to nematodes (Yen et al., 2005; Ibrahim et al., 2007; Khairy, 2016). In the present study, screened plant extracts are combined in mixture to widen the spectrum of nematicidal activity against root-knot nematode, M. incognita thereby ameliorating tomato plant growth criteria. Aqueous leaf extracts of neem and moringa have been previously reported to induce remarkable improvement in plant infected with M. incognita (Nwankwo et al., 2016; Kankam et al., 2018; Khairy et al., 2021) which support the present findings in that aqueous leaf extract of neem and canola were the best amongst individual applications and significantly (P<0.05) improved shoot length and weight of tomato infected with M. incognita, respectively. In all treatments of mixture extracts, dual and triple applications showed synergistic impact on tomato growth compared with single application. Hence, triple application of canola (Cl), moringa (Ml) and neem leaf (Nl) extracts significantly (P<0.05) improved tomato shoot length followed by Ml+Nl+Cr and dual application of Nl+Cs and Nl+Cr. However, the mixture of neem leaf and different parts of canola showed significant augmentation in plant biomass better than did other treatments with a range of 44.3-50.8%. even no significant differences (P<0.05) between treatments were noticed.

In some treatments, the nematicidal efficacy of mixture of aqueous plant parts was considerably increased related to the low activities indicated with singly parts extracts. Individual applications of aqueous leaf extract of canola and neem were found to be more effective in suppressing total nematode population and number of eggs/ egg mass in comparison to stem and root extracts of canola and moringa leaf. These observations are in conformation with the findings of Haroon et al. (2018) who reported. that methanolic neem extract (leaf and root) was the most effective in inhibiting egg hatching of M. incognita, followed by M. oleifera extract. However, the highest synergistic action was accomplished with the combination of leaf extracts of neem, moringa and canola that significantly (P<0.05) suppressed nematode reproduction (Rf =0.97) compared to nematode alone (Rf= 9.05). These findings are consistent with published literature citing the nematicidal activity of Sincocin, a mixture of neem+mustard against M.incognita (Yen et al., 2005; Youssef, 2008). Similarly, root gall and egg masses indices were obviously reduced by all treatments.

Generally, it has been stated that ethanolic extracts of moringa possess growth enhancing compounds that belong to the group of cytokinin which is functional in controlling nematode population density and improving in plant growth and vigor (Guzman, 1984). Also, cultivating rapeseed (canola) cv. Jupiter in M. chitwood infested soil for 2 months and then incorporating shoots into soil resulted in significant (P < 0.05) reduction in final population (Mojtahedi et al., 1991). However, glucosinolates, found in all parts of canola plants, were hydrolyzed by myronase to is thiocyanate which is considered an effective biocide (Ntalli et al., 2020).

Plant extracts were found to induce NPK, chlorophyll and defense related enzymes which lead to systemic resistance in tomato plant infected with M.incognita (Table 3). Meanwhile, Ml + Nl + Cr extract being the most effective and exhibited the highest percentage of increase in N (29.88%), P (41.37%) and K (40.66%) and total chlorophyll (12.52%). A significant increment in NPK and total chlorophyll has been reported in economic plants infected with M. incognita treated with plant extracts and botanical biopesticides (Khairy, 2016; Metwally et al., 2019).

Current study implies that salicylic acid (SA), total phenols and phenylalanine ammonia lyase (PAL) were significantly (P<0.05) fostered in leaves of tomato infected with target nematode related to healthy plants. Phenolic compounds form one of the most significant groups of plant secondary metabolites which render as defense components against plant pathogens (Bahattacharya, 2010). Phenol increment induced by pathogen infestation trigger the RNA transcription; increasing amounts of PAL gives rise to the composition of phenolic compounds (Taiz and Zeiger, 2002).

Phenylalanine (PAL) is an important enzyme for trans-cinnamic acid formation, that is the prospector of phenolic compounds where lignin is the final product (Campbell and Sederoff, 1996). The activity of resistance related compound i.e. phenylalanine ammonia lyase (PAL) was significantly raised in tomato seedlings as a result of M. incognita infection. This finding is consistent with published literature citing that the high PAL activity at control plants treatment may reflect a retard in the host defense, which simplifies any hazards effects induced by the pathogenic organism (Kyndt et al., 2013). Salicylic acid was also found to act as growth promoters and an inducer of resistance to Meloidogyne spp. in tomato (Molinari, 2008; Mukherjee et al., 2012).

In the current investigation, plant extracts applied singly or in mixture showed significantly lower levels of SA, phenols and PAL activity in tested plants infected with the target nematode, M. incognita compared to untreated uninoculated plant. Among plant extracts the greatest suppression in such criteria was accomplished with Ml + Nl + Cr extract. According to Ahmed et al. (2009) the lower levels of phenols in mung bean infected with M. javanica could be attributed to oxidation reaction of phenolic compounds by polyphenoloxidase (PPO), and formation of quinones, which are extremely toxic to nematodes.

The nematicidal properties of moringa leaf (Ml) and neem leaf (Nl) extract against M.incognita could be imputed to the presence biological compounds as tannins, flavonoids, saponins, steroids, triterpenoids alkaloids and reducing sugars. Some of these phytochemicals such as tannins, saponins and flavonoids are reported to cause disruption of membranes in organisms thereby facilitating penetration of toxic principles to the detriment of such organism (Izuogu et al., 2013; Chin et al., 2018; Olajidei, 2018). Flavonoids have been known to exert enormous biological effects including antioxidant and antimicrobial activity against a wide array of pathogens through inhibiting the membrane bound enzymes (Li-Weber, 2009; Dash et al., 2017). Moreover, alkaloids and flavonoids present in botanical extracts showed ovicidal activity against Meloidogyne eggs (Adegbite, 2003).

Also variation in chemical components was observed between canola organs (root extracts, shoot extracts) and between canola genotypes (Asaduzzaman et al., 2014). Root extracts sustained more secondary metabolites compared to shoot extracts. Fourteen secondary metabolites, were identified in such organs including quercitin, rutin, jasmonic acid, vanillc acid, methyl-jasmonate, and 2-phenylethyl glucosinolates. The flavonols i.e. kaempferol, quercetin, and myricetin have been reported to repel and slow second stage juveniles of M. incognita (Wuyts et al., 2006). Whereas, Patuletin, patulitrin, quercetin, and rutin showed lethal impact on Heterodera zeae juveniles (Faizi et al., 2011).

Twenty one flavonoids and twenty three phenolic compounds were evidenced using HPLC in aqueous leaf extract of neem. Hespiridin, Naringin, Hespirtin and Rosmarinic acid have been reported to have nematicidal properties against plant parasitic nematodes (Wuyts et al., 2006; Wang et al., 2012). Also phenolic compounds including e-Vanillic, pyrogallol, benzoic, salicylic acid and gallic acid. Pyrogallol and gallic acid were found to be highly toxic against M.incognita (Ohri and Pannu, 2010).

Conclusions and Recommendations

In conclusion mixtures of plant extracts have shown potentiality to reduce M. incognita reproduction and alter chemical constituents in tomato and could be recommended for sustainable management of Meloidogyne spp. However, isolation and identification of the nematicidal compounds is fundamental for further promotion.

Novelty Statement

The current study indicated the nematicidal properties of combined use of plant extracts against root-knot nematode, M .incognita . Screened plant extracts can act as resistance inducers, plant growth promoters and biocontrol agents within sustainable management.

Author’s Contribution

Doaa Khairy: Conducted the experiment, performed material preparation, data analysis and drafted the manuscript.

Mohamed Ali Osman and Fatma Abdel Mohsen Mostafa: Contributed to the study conception and design, supervised and approved the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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