Comparison of Eco-Friendly Control Strategies against Root-Knot Nematode in Relation to Growth and Fruit Yield of Pomegranate Orchards, with Special Reference to Structure of Plant-Parasitic Nematodes Community

Eman Alsayed Hammad1* and Atef Mohamed El-Sagheer2

1Nematode Diseases Research Dept., Plant Pathology Research institute, Agricultural Research Center (ARC), Giza, Egypt; 2Agricultural Zoology and Nematology Department, Faculty of Agriculture, Al-Azhar University, Assiut, Egypt.

Received | February 15, 2022; Accepted | May 08, 2022; Published | May 21, 2022

*Correspondence | Eman A. Hammad, Nematode Diseases Research Dept., Plant Pathology Research institute, Agricultural Research Center (ARC), Giza, Egypt; Email: [email protected]

Citation | Hammad, E.A. and El-Sagheer, A.M., 2022. Comparison of eco-friendly control strategies against root-knot nematode in relation to growth and fruit yield of pomegranate orchards, with special reference to structure of plant-parasitic nematodes community. Pakistan Journal of Nematology, 40(1): 22-34.

DOI | https://dx.doi.org/10.17582/journal.pjn/2022/40.1.22.34

Keywords | Root-knot nematode, Eco-friendly control, Pomegranate, Plant-parasitic nematodes community

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

Pomegranate (Punica granatum) is a fruit-bearing woody plant belonging to the family, Lythraceae, subfamily Punicoideae. Pomegranate is widely cultivated under different climatic and soil conditions (da Silva et al., 2013). Pomegranate is able to thrive in arid environments, so it is considered an economical commercial horticultural crop especially in the Middle East region, North and Tropical Africa, Central Asia, and Mediterranean basin (Chandra et al., 2010). In Egypt as a subtropical region with a climate typified by lengthy hot summers and low air humidity, the cultivated area reached 79.4 thousand acres in the 2021 season (CAPMAS, 2021). Several previous investigations have found that phytonematodes infest most Pomegranate orchards (Katooli et al., 2021; Poornima, 2020; Singh et al., 2019) where many parasitic genera were found, such as Meloidogyne sp., Pratylenchus sp., Paratrichodorus sp., Helicotylenchus sp., Rotylenchulus sp., Tylenchorhynchus sp., Longidorus sp., Xiphinema sp., and Criconemella sp. (Pradhan et al., 2020; El-Qurashi et al., 2019; Ibrahim et al., 2010). The root-knot nematode, Meloidogyne spp. management is more challenging than that of other pests due to a diverse group of hosts, short life span, and adaptability to a variety of environmental contexts (Mandal et al., 2021; Mitiku, 2018). For more than 50 years, the chemical nematicides have been effective as practices to control plant-parasitic nematodes (PPNs) diseases (Chen et al., 2020). Due to the detrimental effects of chemical pesticides on humans and the environment, clean agriculture is becoming more popular as an alternative strategy (Rani et al., 2021; Tudi et al., 2021). Therefore, it was necessary to create new safety and inexpensive economic strategies to reduce nematode damage, whether through direct influence or by increasing the plant’s ability to compensate for the loss resulting from the damage (Desaeger et al., 2020; Oka et al., 2000; Stirling, 2018). The use of compounds such as Vermicompost, which depend on increasing plant health by raising the levels of some enzymes responsible for plant resistance to biological stresses resulting from plant pathogens such as amylase, lipase, and protease, is a promising approach in the integration of sustainable control strategies (Mandal et al., 2021; Chanu et al., 2018; Rasmann et al., 2012). On the other hand, bio-agents have proven their effectiveness as a tool for combating root-knot nematode (Tariq et al., 2021; Temitope et al., 2020). Several species of Trichoderma (Deuteromycetes, Dematiaceae) are commonly utilized as commercial formulations of biological control and plant growth promoters (Pocurull et al., 2020). The common saprophytic fungi, Trichoderma asperellum as a microbial inoculant is an effective eco-friendly alternative method of controlling plant pathogens (Temitope et al., 2020; dos Santos et al., 2021). In this aspect, essential oils such as Marjoram emulsion oil (MEO), Majorana hortensis, are emerging as an effective type of eco-friendly product with nematicidal properties (D’Addabbo and Avato, 2021; El-Ashry et al., 2021; Klein et al., 2020; Saroj et al., 2020). Especially if it’s in a more stable form and transmittance, like emulsions (Boyko and Brygadyrenko, 2021; Sharar et al., 2017). Waste of bio-origin as by-products are currently gaining more attention due to their suppression of several plant pathogens, such as root-knot nematodes (Khan et al., 2021). Chitosan as a by-products of the shell of crabs through crustacean farming has been noted as a product to impact the plant’s systemic resistance against root-knot nematodes (Giannakou et al., 2020; Radwan et al., 2012).

Therefore, the current study aims to compare some eco-friendly strategies in the control of root-knot nematode, Meloidogyne incognita, infecting Pomegranate orchards, to chemical control, under greenhouse and open field conditions.

Materials and Methods

The nematicidal effects of some eco-friendly control agents: Marjoram emulsion oil, three commercial bio-agents, Vermicompost with Trichoderma asperellum, and Chitosan oligosaccharide, were evaluated compared to the chemical nematicide Nemaphos against the root-knot nematode and the subsequent impact on plant growth parameters of Pomegranate plants in greenhouse and field experiments. The chemical composition was used to determine each treatment’s induced resistance (IR).

Root-knot nematode preparation

Root-knot nematode, Meloidogyne incognita eggs were isolated from egg-masses on the roots of the tomato plant according to Hussey and Barker (1976). The eggs were transferred to Petri dishes (100mm x15mm) and incubated at room temperature (28±2°C) for three days. The hatched second-stage juveniles (J2) were acquired by using the Baermann plate technique (Seinhorst, 1962). A calculated suspension containing 1000 active juveniles was used as an inoculum.

Preparation of tested treatments

Vermicompost®: A native commercial product of Vermicompost contains some enzymes (such as Amylase, Lipase, Glycolytic digesting enzymes, and Protease enzymes), in addition to earthworms; tiger worms (Eisenia fetida Savigny,1826), African night crawler (Eudrilus eugeniae) and loaded on some organic matter (animal and plant wastes). The product provided by Miegos Company, Egypt and applied at the recommended rate (14 kg /tree).

Agre-Viva®: A commercial bio-product composition of Trichoderma asperellum contained 10% nitrogen, 10% phosphorus, and 10% potassium provided by View Green Company, Egypt, and used at the recommended rate (1L / Feddan).

BioHope®: A commercial bio-product named chitosan (extracted from the shell of crabs) was obtained from Tagrow Company, Egypt, and used at the recommended rate (400g / Feddan).

Marjoram emulsion oil (MEO): Marjoram oil was obtained from Harraz Company for the food industry and natural products, Egypt. The emulsion essential oil was created by combining one volume of MEO (2.5%) with two volumes of surfactant [polyethylene glycol dioleate (non-ionic surfactant) + Toximol (ionic surfactant)] and water. At room temperature, the mixture was vortexed numerous times and subjected to stability testing and specifications. After two days the Stability was tested according to Muschiolik’s protocol (Muschiolik et al., 2006). The transparency of formulation was determined by Date and Nagarsenker’s protocol (Date and Nagarsenker, 2008).

Chemical nematicide

A commercial product of organophosphate systemic nematicide; Nemaphos 40 % EC, common name: Thionazin, chemical composition: Ethyl- 3methyl -4-(methyl thiophenyl)-methylethyl was applied at the rate of 3L / Fadden.

In vitro experiment

Using five rates, the inhibitory effect of Marjoram emulsion oil (MEO) on M. incognita second-stage juveniles (J2) were examined (1000, 2000, 3000, 4000, and 5000ppm). Second-stage juveniles were extracted, numbered, and concentrated in suspension until they reached 100 J2 in 1 ml of distilled water, according to Demeure and Freckman’s (1981) methods. One ml of juvenile suspension was deposited in screw-capped test tubes containing 5 ml of various rates of tested materials and incubated at 25±2°C for 3 days, then, the number of active and inactive juveniles were counted using a nematode counting slide at 24, 48, and 72 hours (Hussey, 1973). Each treatment was repeated five times, with distilled water serving as the control. The revised mortality percentages were calculated using the Schneider-Orelli formula (Schneider and Orelli, 1947):

Pots experiment

The nematicidal characteristics of MEO at 5000 ppm and Vermicompost, Trichoderma asperellum, and Chitosan, were compared against the root-knot nematode, M. incognita, and the influence on Pomegranate growth parameters.

One seedling of Pomegranate cv. Manfaluty (6 month-old) was planted in 20 cm diam Thirty pots containing a sterilized mixture of clay and sandy soil (1:1 w/w), each pot contained 3k, in addition, five pots were filled with 750 g of Vermicompost and 2250 g of soil per pot, which were combined in a 1:1 ratio (sterile soil with sand), and irrigated immediately.

The Pomegranate seedlings were infected with 1000 newly hatched juveniles of M. incognita for each plant after one week. Each application was repeated five times, with the application MEO at 5000 ppm rates being applied three times after inoculation (first, third, and fifth weeks). Treatment by T. asperellum a rate 3.3 ml /pot and chitosan applied at 1.2g /pot as the same applications. Majorana emulsion oil was applied three times, with nematicide Nemaphos 40% EC (6 ml/100 ml water). All treatments were applied as a soil drench. Five inoculated pots were left without adding any materials as a negative control, in addition to another five replicated healthy seedlings without inoculation with nematodes as a positive control. All pots were arranged in the greenhouse at 27±5˚C in a randomized block design. After fifty days of inoculation, the plants were harvested. Juveniles of M. incognita were extracted from soil by sieving and modified Baermann technique (Seinhorst, 1962). Roots were stained by acid fuchsin (Bybd et al., 1983) and examined under a stereomicroscope for counting developmental stages, females, galls, and egg-masses. Root galling or egg masses numbers were rated on a scale of 0-5 where 0= no galls or egg masses, 1= 1-2 galls or egg masses, 2= 3-10 galls or egg masses, 3= 11-30 galls or egg masses, 4= 31-100 galls or egg masses, 5= more than 100 galls or egg masses per root system (Taylor and Sasser, 1978).

Open field experiment

Before the beginning of the experiment, the nematode samples were taken from the infected soil of pomegranate orchards and used as a zero-day. The open-field experiment was carried out in three locations: South Tahrer, Wadi Al-Natroun, and Nubariya of El-Beheira Governorate, with three associated villages as follows:

1. South Tahrer: Umm saber, Omar Shaheen, and Omar Makram.
2. Wadi Al-Natroun: Banaa Salama, Alhamra, and Alwadaa Alfarigh.
3. Nubariya: Elshaaraw, Naguib Mahfouz, and Abu Bakr Alsidiyq.

The investigation was conducted during the season of 2021, from the beginning of the natural growing season to an assessment of the nematodes community, (as shown in Table 3). Ten samples (250g) were monthly taken from the start of the Pomegranate tree’s activity (February) until the fruit yield was harvested (July), from each of the above-mentioned areas.

The investigated trees were 5 years old and spaced (4x3m) apart, and irrigated with a drip irrigation system. On soil naturally infested with M. incognita, the same treatments employed in the pot experiment were repeated. In February and March of 2021. Each treatment is made up of five duplicates, each with ten trees. All applications were made to the soil as a soil drench. Except for Nemaphos, all therapies were applied three times monthly, and samples were obtained two, four, and six months later.

The root-knot nematode was extracted from soil by Cobb’s sieving method (Cobb, 1918) and modified Baermann funnel method (Baermann, 1917). According to Byrd et al. (1983) roots were colored with acid fuchsine in lactic acid and microscopically analyzed for counting root-knot nematode developmental stages and females. The reproduction factor (Rf) is calculated for each treatment by dividing the final population (Pf) by the initial population (Pi).

Chemical analysis of juice

For the study, fifteen mature Pomegranate fruits from each treatment were split into three repetitions. There are 5 fruits in each refined. An electric juicer was used to remove the Pomegranate seeds from the juice solution and store the juice so that, the following properties could be analyzed:

pH: A pH meter was used to determine the hydrogen ion concentration in the juice solution (Mousavi et.al., 2011).

Total soluble solids (TSS) of pomegranate juice: The technique defended by Preece and Moersfelder (2016) was used to determine total soluble solids (TSS) in the juice.

Vitamin C: Vitamin was estimated in the prepared Pomegranate juice according to El-Sharaa and Mussa (2019).

Crude protein and total carbohydrate: To estimate the crude proteins, Bradford’s method (Bradford, 1976) was used. And Hodge and Hofreiter’s methods (Hodge and Hofreiter, 1962) were used to determine the total carbohydrate content.

Fatty acid isolation and extraction: Using processes for synthesizing fatty acid methyl esters (FAME) from lipids and Pomegranate juice, the fatty acids in the extracted Pomegranate juice were identified using gas chromatography technique (ISO 12966, 2015).

Data analysis

All of the experiments were examined separately. Duncan’s Multiple Range Test (DMRT) was used to compare the treatment means (Gomez and Gomez, 1984). WASP1 was used for the analysis, with the crucial difference set at P= 0.05 and the results interpreted.

Results and Discussion

In vitro: Evaluation of Marjoram emulsion oil viability of root-knot nematode, M. incognita

As shown in Figure 1, the inhibitory effect of five concentrations (1000, 2000, 3000, 4000, and 5000 ppm) of Marjoram emulsion oil (MEO) on the mortality of Meloidogyne incognita juveniles was concentration-dependent, i.e. the nematode’s mortality increased as the concentration and exposure time of the MEO increased. After 24 hours of exposure, the highest proportion of juvenile mortality has occurred at 4000 and 5000 ppm concentrations (84.89 and 100 percent, respectively). After 72 hours of exposure, however, both 3000 and 4000 ppm. MEO exhibited a significant mortality rate, with percentages of 95.9 and 100.0 %, respectively. While the activity of M. incognita juveniles were reduced by 35.07 % after 24 hours and 69.96 % after 72 hours when exposed to MEO at 1000 ppm.

 

Greenhouse experiments

Comparative efficacy of Marjoram emulsion oil, bio-agnate, Chitosan, Vermicompost and chemical nematicide on M. incognita infecting Pomegranate seedlings under greenhouse conditions: The effects of some control strategies; MEO, bio-agnate, Chitosan and Vermicompost as an alternative to chemical nematicide for control the root-knot nematode, M. incognita infected pomegranate seedlings were evaluated under greenhouse conditions.

The results indicated that all treatments had a significant effect on the reduction of M. incognita criteria. Data in Table 1 show that the criteria of M. incognita such as numbers of galls, egg-masses, females/root system of Pomegranate seedling, and rate of build-up were suppressed by application of Vermicompost, Trichoderma asperellum, chitosan, and MEO. Whereas, Nemaphos showed a more suppressive effect on nematode reproduction (RF=0.28), being 93.0% reduction when compared to nematode treatment alone. All treatments dramatically reduced nematode final populations when compared to nematode alone, with average values of decrease ranging from 69.6% for T.asperellum treatment to 82.5% with MEO treatment. Nemaphos treatment resulted in the lowest number of galls (29.2 galls/root), while the treatment with Chitosan (89.3 galls/root).

On the other side, the results presented in Table 2 revealed that all the treatments improved plant growth parameters as indicated by the length and fresh weight of root and shoot as well as shoot dry weight when compared to nematode treatment alone. Nemaphos systemic nematicide exceeded the other treatments, with a percentage increase in length of shoots and roots as well as total plant fresh weight and dry shoot weight of 79.9, 201.0, 437.5, and 424.3 %, respectively when compared to nematode treatment alone.

However, in terms of percentage increase of plant fresh weight and shoot dry weight, after nematicide (437.5%, 424.3%), the MEO treatment occupied the second effective treatment (305.2%, 208.0%), followed by Chitosan (56.25%, 105.8%), and Vermicompost (29.1%, 93.4%), and in the last impact was treated by and T. asperellum (17.7%, 44.3%).

 

Table 1: Efficacy of Marjoram emulsion oil and bio-agents on the developmental and reproduction of M. incognita infected Pomegranate plant under greenhouse conditions (27 ± 3 °C).

Treatment	Nematode population			Final population (Pf)	RF*	Red. %	No. of galls	RGI	No. of Egg masses	EI
	J2/ 250g soil	Root
		Develop. stages	Females
Vermicompost	958.4b	72.3b	65.2b	1095.9b	1.1	72.1	124.3b	5.0	49.3b	4.3
T. asperellum	1032.6	87.2b	75.4b	1195.2b	1.19	69.6	132.1b	5.0	60.4b	4.7
Chitosan	848.2c	65.3c	54.8c	968.3c	0.97	75.3	89.3c	5.0	35.1c	4.1
Marjoram emulsion oil	565.3d	81.2d	41.5d	688.0d	0.70	82.5	87.5d	5.0	27.2d	3.8
Nemaphos	230.6e	16.4e	26.2e	273.2e	0.28	93.0	29.2e	3.2	18.3e	3.1
Nematode	3635.3a	203.2a	89.4a	3927.9	3.9	--	259.7a	5.0	76.5a	5.0
LSD 5 %	50.10	17.17	11.51	56.14	--	--	27.75	-	9.77	-

 

Each value presented the mean of five replicates. M. incognita (1000 larva/ plant). Means in each column followed by the same letter(s) did not differ at P≤ 0.05 according to Duncan`s multiple range test. * Reproduction factor (RF) = final population/initial population.

 

Table 2: Efficacy of emulsion oil, bio-agnates and chemical nematicide on growth parameters of Pomegranate plant infested with M. incognita infesting under greenhouse conditions (27±3°C).

Treatments	Plant growth response								Shoot dry wt. (g)	increase %
	Length (cm)				Plant fresh wt.(g)
	Shoot	Inc.%	Root	Inc.%	Shoot	Inc. %	Root	Inc. %
Vermicompost	70.0de	31.6	13.5e	39.2	124.0cd	29.1	13.5d	28.6	79.5de	93.4
T. asperellum	73.3cd	37.8	16.8d	73.2	113.0d	17.7	15.8cd	50.5	59.3f	44.3
Chitosan	76.0c	42.9	15.5d	59.8	150.0c	56.25	16.8c	60.0	84.6d	105.8
Marjoram emulsion oil	74.3cd	39.7	21.2c	118.6	389.0b	305.2	22.9b	118.1	126.6c	208.0
Nemaphos 40%	95.7 a	79.9	29.2a	201.0	516.0a	437.5	26.6a	153.3	215.5a	424.3
Control	90.0 b	69.2	24.0b	147.7	402.0b	318.8	23.1b	120.0	147.0b	257.7
Nematode only	53.2e	0.0	9.7f	0.0	96.0e	0.0	10.5e	0.0	41.1g	0.0
LSD	3.67	-	2.10	-	34.53	-	1.75	-	14.6	-

 

Each value presented the mean of five replicates. Means in each column followed by the same letter(s) did not differ at P≤ 0.05 according to Duncans multiple range test.

 

 

Table 4: Efficacy of emulsion oil and commercial bio-products on M. incognita infected pomegranate after two, forth and six month of application under field conditions.

Treatments	Two month			Four month			Six month
	Final population	*RF	Red. %	Final population	*RF	Red. %	Final population	*RF	Red. %
Vermicompost	1384.7b	0.47	69.4	1587.1b	0.54	67.1	1831.7b	0.62	63.8
T.asperellum	1272.0c	0.43	71.9	1440.6c	0.49	70.2	1618.0c	0.55	68.0
Chitosan	618.8e	0.21	86.3	713.3e	0.24	85.1	900.4e	0.30	82.2
Marjoram emulsion oil	1090.8d	0.37	75.9	1331.4d	0.45	72.4	1460.5d	0.49	71.2
Nemaphos	442.2f	0.15	90.2	592.5f	0.2	87.6	829.7f	0.28	83.5
N.alone	4528.7a	1.5	--	4824.7a	1.6	--	5063.9a	1.70	--
LSD 5%	64.34	--	--	72.24	--	--	80.0		--

 

Number of initial population = 2970 / 250g. soil and root. * Reproudction factor (RF) = final population/initial population. Means in each column followed by the same letter (s) didn’t differ at P≤ according Duncans multiple range test.

 

Table 5: Effect of tested bio-agents against M. incognita on the fruit yield of pomegranate trees.

Treatment	Vermicompost	T. asperellum	Chitosan	Marjoram emulsion oil	Nemaphos	Control
Fruit weight (kg/tree)	18.53d	16.42e	21.45c	23.91b	28.36a	9.12f
Increase %	103.18	80.04	135.20	162.17	210.96	-

 

Means in each column followed by the same letter(s) did not differ at P ≤ 0.05 according to Duncan`s multiple range test.

 

Open field experiment

Structure of plant-parasitic nematodes community inhabiting the rhizosphere of pomegranate fields in different localities of El-Beheira Governorate: The structures of plant parasitic nematodes community inhabiting the rhizosphere of Pomegranate fields in different localities at Beheira Governorate were classified into three regions South Tahrer, Wadi Natrun, and Nubaria through three distracts for each. The data shown in Table 3 indicated that the Pomegranate parasitic nematode community consists of ten genera belonging to seven families, these genera were, Aphelenchus (Aphelenchoididae), Criconemella (Criconematidae), Helicotylenchus (Hoplolaimidae), Longidorus (Longidoridae), Meloidogyne (Meloidogynidae), Pratylenchus (Pratylenchidae), aratrichodorus (Trichodoridae), Rotylenchulus (Hoplolaimidae), Tylenchorhynchus (Tylenchorhynchidae) and Xiphinema (Longidoridae).

Generally, the study noted a marked variation in the community that was associated with tested cultivars of Pomegranate and the nature of the soil in the study locality. Where some genera were absent and associated with cv. Altaayifaa in some localities such as absence Paratrichodorus spp. in Umm Saber distract, and Xiphinema spp in Omar Shaheen distract. Also in cv. Manfaluty, absence of Aphelenchus spp. and Paratrichodorus spp. in Alhamra distract and Criconemella spp. and Longidorus spp. in Banaa Salama distract. Based on population density and frequency of occurrence as the index of prevalence, the genus of Meloidogyne ranked in first-order (1769, 98.0 %) as the dominant genus, followed by Tylenchorhynchus (149.89, 90.22%). In the middle-ranked were Pratylenchus, Rotylenchulus, Criconemella, and Helicotylenchus genera. While in the last ranked with a significant difference came both of Xiphinema spp. (25.0, 38.11%), Longidorus spp (18.11, 32.0%), Aphelenchus spp. (20.56, 28.89%) and Paratrichodorus spp. (6.22, 10.89%), respectively.

Comparative efficacy of emulsion oil and commercial bio-products on M. incognita infected pomegranate trees after two, forth and six months of application under open field conditions: In the natural growing season of 2021, at the newly reclaimed land in Nubariya, Naguib Mahfouz village, Beheira Governorate, an open-field experiment was done on 5-year-old Manfaluty Pomegranate trees are naturally infested with M. incognita. Data are shown in Table 4 indicated that the treated with Vermicompost, T. asperellum, Chitosan, and MEO all have a nematicidal activity against root-knot nematode. Where, Chitosan treatment achieved the best result in the suppression of root-knot nematode population after six months of use, with an 82.2 % reduction, when compared to nematode treatment alone. Meanwhile, T. asperellum and Vermicompost were the least ranked (68.0 and 63.8 % reduction, respectively), while MEO was the most popular (68.0 and 63.8 % reduction, respectively) (71.2 %). Nonetheless, when compared to nematode therapy alone, Nemaphos injection significantly reduced the final population, with a reproduction percentage of 83.5%.

Effect of tested strategies on fruit yield and pomegranate juice quality

Results presented in Table 5 show that all tested treatments resulted in a significant increase in total Pomegranate fruit weights, compared to control. Nemaphos had the highest percentage of fruit weight increase (210.96 %), while Majoram, Chitosan, and Vermicompost treatments gave a relatively lower percentage of yield increase (162.17, 135.20, and 103.18 %, respectively) when compared to untreated plants (9.12kg/tree), T.asperellum caused the lowest increase in overall fruit weight (16.84 kg fruit/tree) with a percentage increase (80.04%).

From each treatment, fifteen ripe Pomegranate fruits were divided into three replicates for this study. Each repetition contains 5 fruits. The juice was extracted using an electric juicer. Pomegranate juices are used to conduct chemical analyses on them, such as determining acidity pH, which revealed that Pomegranate juice is acidic, ranging from 3.88 Nemaphos in the case of treated fruits to 3.41 in the case of untreated fruits (control) Table 6. As for TSS percentages in juice, Nemaphos treatments had the highest proportion of TSS (18.57%) followed by MEO (16.97%), and the lowest was T.asperellum treatment with 14.17 % (Figure 2).

 

The highest content of vitamin C in Pomegranate juice, measured in mg/100ml, was found in the juice produced from Nemaphos treatment, followed by Chitosan (11.66 and 11.23 mg/100ml) As for percentages of total protein, it was 3.92% in the treatment of MEO and 3.32% and 3.01% in the Chitosan and Vermicompost treatments, respectively. Regarding the percentage of total carbohydrates in Pomegranate juice, it was found that Nemaphos treatment had the highest percentage of total carbohydrates at 52.73%, while the fruits of the untreated plants (untreated) had the lowest percentage at 30.52% (Table 6). Results in Table 7 and Figure 2 show that the treatment of Nemaphos recorded the highest total fatty acid content (0.56 %), whereas T. asperellum occupied the lowest (0.36 %). Chitosan gave 0.320 % of saturated fatty acid in Pomegranate juices, while Vermicompost gave 0.264 percent saturated fatty acid percentages in juices and fruits. Pomegranate juice fruits of Nemaphos treatment recorded 0.301 % unsaturated fatty acid percentage, compared to 0.124 % for untreated Pomegranate juice fruits (control).

According to the structure of plant-parasitic nematodes community inhabiting the rhizosphere of Pomegranate fields, the Meloidogyne genus was the most common and harmful, although the variation of genera prevalence. Similar results were reported by some previous studies (Day and Wilkins, 2011; Shreeshail et al., 2015; Tariq-Khan et a l., 2017; El-Qurashi et al., 2019). Where Khan et al. (2005) revealed that infection of pomegranate roots by many populations of plant parasitic nematodes, especially root-knot nematodes, adversely affect the growth and Pomegranate productivity. The variation of genera prevalence may be due to the type of soil, agricultural processes, cultivated variety, and climatic conditions (Korayem et al., 2014; Singh et al., 2019).

 

Table 6: Evaluation of Marjoram emulsion oil and three commercial bio-products as control agents against root-knot nematode on the juice quality of Pomegranate, under field conditions.

Treatments	pH	T.S.S. mg / 100ml	Vitamin C mg/ 100ml	Total protein mg /100ml	Total carbohydrate mg /100ml
Vermicompost	3.70	15.7	10.50	3.01	42.87
T. asperellum	3.60	14.17	10.00	2.52	36.82
Chitosan	3.81	15.77	11.23	3.32	40.25
Marjoram emulsion oil	3.74	16.97	10.33	3.92	45.35
Nemaphos	3.88	18.57	11.66	4.71	52.73
Nematod alone	3.41	13.63	6.95	1.98	30.52

 

Table 7: Effect of Marjoram emulsion oil and three commercial bio-products as control agents against root-knot nematode on the fatty acids of Pomegranate juice of treated and untreated trees under field conditions.

Treatments	Saturated fatty acid %	Unsaturated fatty acid %	Total fatty acid %
Vermicompost	0.264	0.220	0.43
T.asperellum	0.202	0.126	0.35
Chitosan	0.320	0.243	0.46
Marjoram emulsion oil	0.325	0.295	0.54
Nemaphos	0.375	0.301	0.56
Nematod alone	0.190	0.124	0.16

 

The efficacy of emulsion oil, bio-agnates as alternative eco-friendly control strategies for root-knot nematode M. incognita infested pomegranate were evaluated under conditions of greenhouse and commercial open fields. The current study indicated the ability of all strategies to reduce M. incognita criteria, with different reduction percentages from chemical nematicide, and compared to untreated control. Where in protected conditions the use of Marjoram emulsion oil (MEO) and Trichoderma asperellum as separate treatments achieved the highest rate of nematode suppression after chemical nematicide. However, their ranking differs in terms of their effect on the plant vegetative growth, where Chitosan resulted in increasing percentages better than T. asperellum after MEO treatment. The obtained results were similar to the findings of Radwan et al. (2012) and Luong et al. (2020) where reported that the nematicidal activity of Chitosan improved the plants’ health. While, contrary findings were reported by Mota and Santos (2016), where reported that chitosan application alone had not improved the shoot growth of plants. While Kumari et al. (2020) reported that the soil application of T. asperellum improved the plant growth and suppressed root-knot nematode significantly. Moreover, the findings of Arancon et al. (2002), Giannakou et al. (2020), and Temitope et al. (2020) reported the ability of each of the tested strategies to suppress plant-parasitic nematodes as parasites on different varieties of plants. In agreement with the present findings, Arancon et al. (2002), Xiao et al. (2016), Giannakou et al. (2020), and Temitope et al. (2020) reported the ability of each of the tested strategies to suppress plant-parasitic nematodes as parasites on different varieties of plants. Generally, the variations of tested strategies for suppression of root-knot nematode due to the difference in the mechanism of action and the effect of the active ingredient in each strategy (Roberts, 1993; Hildalgo and Kerry, 2008; Collange et al., 2011; Tariq at al., 2020; Singh and Sinha, 2022).

Conclusions and Recommendations

The current study indicates the possibility of using eco-friendly control strategies such as Marjoram emulsion oil, Vermicompost and products of bio-agents, as an alternative strategy for chemical control to suppression of the root-knot nematodes.

Acknowledgements

The authors would like to thank Dr. Mohammed Hefni, Technology of Food Industry Department, Faculty of Agriculture, Al-Azhar University, Egypt for his contribution to the preparation of the emulsion oil.

Novelty Statement

The current study proves the possibility of applying eco-friendly strategies to control root-knot nematodes in open agricultural fields, especially Marjoram emulsion oil and Vermicompost.

Author’s Contribution

EAH conceived and designed the experiment, performed the experiments, wrote and reviewed the manuscript. AME collection and/or assembled and analyze the data, wrote, reviewed the manuscript and designed the experiment.

Funding

Self-funding

Data availability

Availability of data and materials not applicable in this study.

Ethics approval of human data or animal tissues

Not applicable in this section.

Consent for publication

Not applicable in this section.

Conflict of interest

The authors have declared no conflict of interest.

References

Arancon, N.Q., Edwards, C.A., Lee, S.S., and Yardim, E., 2002. Management of plant parasitic nematode populations by use of vermicomposts. In Brighton crop protection conference pests and diseases, V2, pp. 705-710.

Baermann, G., 1917. A simple method for detecting Anclostomum (Nematode) larvae in soil samples. Tijdschr Diergeneeskd., 57: 131–137.

Barker, K.R., and Hussey, R.S., 1976. Histopathology of nodular tissues of legumes infected with certain nematodes. Phytopathology, 66(7): 851-855. https://doi.org/10.1094/Phyto-66-851

Boyko, O., and Brygadyrenko, V., 2021. Nematicidal activity of essential oils of medicinal plants. Folia Oecol., 48(1); 42-48. https://doi.org/10.2478/foecol-2021-0005

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 7(72): 248-254. https://doi.org/10.1016/0003-2697(76)90527-3

Bybd, Jr, D.W., Kirkpatrick, T. and Barker, K.R., 1983. An improved technique for clearing and staining plant tissues for detection of nematodes. J. Nematol., 15(1): 142–143.

Central Agency for Public Mobilization and Statistics (CAPMAS), 2021. Statistical year book, pp. 19.

Chandra, R., Babu, K.D., Jadhav, V.T., Jaime, A., and Silva, T.D., 2010. Origin, history and domestication of pomegranate. Fruit, Vegetable, Cereal Sci. Biotechnol., 2: 1-6.

Chanu, L.J., Hazarika, S., Choudhury, B.U., Ramesh, T., Balusamy, A., Moirangthem, P. and Sinha, P.K., 2018. A guide to vermicomposting-production process and socio economic aspects. Ext. Bull., 81: 30.

Chen, J., Li, Q.X., and Song, B., 2020. Chemical nematicides: Recent research progress and outlook. J. Agric. Food Chem., 68(44): 12175-12188. https://doi.org/10.1021/acs.jafc.0c02871

Cobb, N.A., 1918. Estimating the nema population of the soil; Agric. Teeh. Cire. Bur. PI. Ind. U.S. Dep. Agric., No.1, pp. 48.

Collange, B., Navarrete, M., Peyre, G., Mateille, T., and Tchamitchian, M., 2011. Root-knot nematode (Meloidogyne) management in vegetable crop production: The challenge of an agronomic system analysis. Crop Prot., 30(10): 1251-1262. https://doi.org/10.1016/j.cropro.2011.04.016

D’Addabbo, T., and Avato, P., 2021. Chemical composition and nematicidal properties of sixteen essential oils. A review. Plants, 10(7): 1368. https://doi.org/10.3390/plants10071368

da Silva, J.A.T., Rana, T.S., Narzary, D., Verma, N., Meshram, D.T., and Ranade, S.A., 2013. Pomegranate biology and biotechnology: A review. Sci. Hortic., 160: 85-107. https://doi.org/10.1016/j.scienta.2013.05.017

Date, A.A., and Nagarsenker, M.S., 2008. Parenteral microemulsions: An overview. Int. J. Pharm., 355(1-2): 19-30. https://doi.org/10.1016/j.ijpharm.2008.01.004

Day, K.R., and Wilkins, E.D., 2011. Commercial pomegranate (Punica granatum L.) production in California. Acta Horticulturae, 890, 275-286. https://doi.org/10.17660/ActaHortic.2011.890.39

Demeure, Y.V.E.S., and Freckman, D.W., 1981. Recent advances in the study of anhydrobiotic nematodes. Plant Parasitic Nematodes, 3: 205-226. https://doi.org/10.1016/B978-0-12-782203-7.50014-5

Desaeger, J., Wram, C., and Zasada, I., 2020. New reduced-risk agricultural nematicides-rationale and review. J. Nematol., 52. https://doi.org/10.21307/jofnem-2020-091

dos Santos, P.T., Monteiro de Paula, A., Ferrari, L.H., da Silva, J., Borges, P.J., Navas C.S.M. and Busato, J.G., 2021. Trichoderma-enriched vermicompost extracts reduces nematode biotic stress in tomato and bell pepper crops. Agronomy, 11(8): 1655-1661. https://doi.org/10.3390/agronomy11081655

El-Ashry, R.M., Elsobki, A.E.A.M., and Helaly, S.M., 2021. Effect of different control agents on Meloidogyne incognita kofoid infesting cucumber plants. J. Plant Prot. Pathol., 12(3): 259-268. https://doi.org/10.21608/jppp.2021.66215.1019

El-Qurashi, M.A., El-Zawahry, A., Abd-El-Moneem, K.M.H., and Hassan, M.I., 2019. Occurrence, population density and biological control of root-knot nematode, Meloidogyne javanica infecting pomegranate orchards in Assiut governorate, Egypt. Assiut. J. Agric. Sci., 50(2): 176-189. https://doi.org/10.21608/ajas.2019.41818

El-Sharaa, I., and Mussa, S.A.B., 2019. Determination of vitamin c (ascorbic acid) contents in vegetable samples by UV-spectrophotometry and redox titration methods and estimation the effect of time, cooking and frozen on ascorbic acid contents. Int. J. Prog. Sci. Technol., 15(2): 281-293.

Giannakou, I.O., Tasoula, V., Tsafara, P., Varimpopi, M., and Antoniou, P., 2020. Efficacy of Purpureocillium lilacinum in combination with chitosan for the control of Meloidogyne javanica. Biocontr. Sci. Technol., 30(7): 671-684. https://doi.org/10.1080/09583157.2020.1756227

Gomez, K.A., and Gomez, A.A., 1984. Statistical procedures for agricultural research. John Wiley and Sons.

Hildalgo-Diaz, L., and Kerry, B.R., 2008. Integration of biological control with other methods of nematode management. In Integrated management and biocontrol of vegetable and grain crops nematodes.  Springer, Dordrecht. pp. 29-49. https://doi.org/10.1007/978-1-4020-6063-2_2

Hodge, J.E. and Hofreiter, B.T., 1962. Determination of reducing sugars and carbohydrates, In: Methods in carbohydrate chemistry, (eds. Whistler, R.L. and Wolfron, M.L.). Academic Press, New York, pp. 380–394.

Hussey, R.S., 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Dis. Rep., 57: 1025-1028.

Hussey, R.S., and Barker, K.R., 1976. Influence of nematodes and light sources on growth and nodulation of soybean. J. Nematol., 8(1): 48.

Ibrahim, I.K.A., Mokbel, A.A., and Handoo, Z.A., 2010. Current status of phytoparasitic nematodes and their host plants in Egypt. Nematropica, pp. 239-262.

ISO 12966, 2015. Animal and vegetable fats and oils analysis by gas chromatography of methyl esters of fatty acids.

Katooli, N., Moghadam, E.M., and Aghnum, R., 2021. Identification and distribution of root-knot nematode species in pomegranate orchards of eastern Iran. Pak. J. Phytopathol., 33(2): 293-297. https://doi.org/10.33866/phytopathol.033.02.0691

Khan, A., Shaukat, S.S., and Siddiqui, I.A., 2005. A survey of nematodes of pomegranate orchards in Balochistan province, Pakistan. Nematol. Mediterr., 33: 25-28.

Khan, A., Tariq, M., Ahmad, F., Mennan, S., Khan, F., Asif, M. and Siddiqui, M.A., 2021. Assessment of nematicidal efficacy of chitosan in combination with botanicals against Meloidogyne incognita on carrot. Acta Agric. Scand. B-Soil Plant Sci., 71(4): 225-236. https://doi.org/10.1080/09064710.2021.1880620

Klein, M.L., Chastain, T.G., Garbacik, C.J., Qian, Y.P.L., and Mc Donnell, R.J., 2020. Acute toxicity of essential oils to the pest slug Deroceras reticulatum in laboratory and greenhouse bioassays. J. Pest Sci., 93(1): 415-425. https://doi.org/10.1007/s10340-019-01154-0

Korayem, A.M., Youssef, M.M.A., Mohamed, M.M.M., and Lashein, A.M.S., 2014. A survey of plant parasitic nematodes associated with different plants in North Sinai. Middle East J. Agric. Res., 3(3): 522-529.

Kumari, M., Sharma, M.K., and Baheti, B.L., 2020. Biological control of root-knot nematode, Meloidogyne incognita infecting tomato (Solanum lycopersicum). J. Entomol. Zool. Stud., 8(1): 35-39.

Luong, T.H., Nguyen, T.K., and Jung, W.J., 2020. Nematicidal activity of cinnamon bark extracts and chitosan against Meloidogyne incognita and Pratylenchus coffeae. Nematology, 23(6): 655-666. https://doi.org/10.1163/15685411-bja10067

Mandal, H.R., Katel, S., Subedi, S., and Shrestha, J., 2021. Plant parasitic nematodes and their management in crop production: A review. J. Agric. Natl. Resour., 4(2): 327-338. https://doi.org/10.3126/janr.v4i2.33950

Mitiku, M., 2018. Plant-parasitic nematodes and their management: A review. Agric. Res. Technol., 8: 30-38. https://doi.org/10.19080/ARTOAJ.2018.16.55580

Mota, L.C., and dos Santos, M.A., 2016. Chitin and chitosan on Meloidogyne javanica management and on chitinase activity in tomato plants. Trop. Plant Pathol., 41(2): 84-90. https://doi.org/10.1007/s40858-016-0072-x

Mousavi, Z.E., Mousavi, S.M., Razavi, S.H., Emam-Djomeh, Z., and Kiani, H., 2011. Fermentation of pomegranate juice by probiotic lactic acid bacteria. World J. Microbiol. Biotechnol., 27(1): 123-128. https://doi.org/10.1007/s11274-010-0436-1

Muschiolik, G., Scherze, I., Preissler, P., Weiss, J., Knoth, A., and Fechner, A., 2006. Multiple emulsions-preparation and stability. 13th World Cong. Food Sci. Technol., pp. 43-43. https://doi.org/10.1051/IUFoST:20060043

Oka, Y., Koltai, H., Bar‐Eyal, M., Mor, M., Sharon, E., Chet, I., and Spiegel, Y., 2000. New strategies for the control of plant‐parasitic nematodes. Pest Manag. Sci. Formerly Pestic. Sci., 56(11): 983-988. https://doi.org/10.1002/1526-4998(200011)56:11<983::AID-PS233>3.0.CO;2-X

Pocurull, M., Fullana, A.M., Ferro, M., Valero, P., Escudero, N., Saus, E. and Sorribas, F.J., 2020. Commercial formulates of Trichoderma induce systemic plant resistance to Meloidogyne incognita in tomato and the effect is additive to that of the Mi-1.2 resistance gene. Front. Microbiol., 10: 3042. https://doi.org/10.3389/fmicb.2019.03042

Poornima, K., 2020. Studies on incidence of root knot nematode and its bio management in pomegranate (Punica Granatum L.). Pest Manag. Hortic. Ecosyst., 26(2): 246-253.

Pradhan, P., Patra, M.K., and Sahoo, N.K., 2020. Association of nematodes with fruit crops in Bhubaneswar, India. Int. J. Curr. Microbiol. Appl. Sci., 9(5): 1918-1923. https://doi.org/10.20546/ijcmas.2020.905.219

Preece, J.E., and Moersfelder, J., 2016. Pomegranate: The grainy apple. J. Am. Pomol. Soc., 70(4): 187-193.

Radwan, M.A., Farrag, S.A.A., Abu-Elamayem, M.M., and Ahmed, N.S., 2012. Biological control of the root-knot nematode, Meloidogyne incognita on tomato using bioproducts of microbial origin. Appl. Soil Ecol., 56: 58-62. https://doi.org/10.1016/j.apsoil.2012.02.008

Rani, L., Thapa, K., Kanojia, N., Sharma, N., Singh, S., Grewal, A.S., and Kaushal, J., 2021. An extensive review on the consequences of chemical pesticides on human health and environment. J. Cleaner Prod., 283: 124657. https://doi.org/10.1016/j.jclepro.2020.124657

Rasmann, S., Ali, J.G., Helder, J., and van der Putten, W.H., 2012. Ecology and evolution of soil nematode chemotaxis. J. Chem. Ecol., 38(6): 615-628. https://doi.org/10.1007/s10886-012-0118-6

Roberts, P.A., 1993. The future of nematology: Integration of new and improved management strategies. J. Nematol., 25(3): 383.

Saroj, A., Oriyomi, O.V., Nayak, A.K., and Haider, S.Z., 2020. Phytochemicals of plant-derived essential oils: A novel green approach against pests. In Natural remedies for pest, disease and weed control. Academic Press. pp. 65-79. https://doi.org/10.1016/B978-0-12-819304-4.00006-3

Schneider, P. and Orelli, O., 1947. Entomologisches praktikum [Entomological internship]. Verlag. H. R. Sauerländer Co., Aarau, Switzerland, pp. 237.

Seinhorst, J.W., 1962. Extraction methods for nematodes inhabiting soil. Prog. Soil Zool., pp. 243-256.

Sharar, M., Bozeya, A., Al-Banna, L., and Al-Bawab, A., 2017. Fungicidal and nematicidal activities for essential oils formulated in Janus emulsion. Green Chem. Lett. Rev., 10(3): 121-128. https://doi.org/10.1080/17518253.2017.1306613

Shreeshail, S., Arun, S., Nargund, V.B., Benagi, V.I., Giri, M.S., Anil, P., and Somaning, S., 2015. Survey for different plant parasitic nematodes association with pomegranate wilt complex. J. Pure App. Microbiol., 9(2): 1429-1433.

Singh, S., and Sinha, R.K., 2022. Vermicomposting of organic wastes by earthworms: Making wealth from waste by converting ‘garbage into gold for farmers. Adv. Org. Waste Manag., Elsevier. pp. 93-120. https://doi.org/10.1016/B978-0-323-85792-5.00004-6

Singh, T., Prajapati, A., Maru, A.K., Chaudhary, R., and Patel, D.J., 2019. Root-knot nematodes (Meloidogyne spp.) infecting pomegranate: A review. Agric. Rev., 40(4): 309-313. https://doi.org/10.18805/ag.R-1945

Stirling, G.R., 2018. Biological control of plant-parasitic nematodes. CRC Press. pp. 103-150. https://doi.org/10.1201/9781351071468-9

Tariq, M., Khan, A., Asif, M., Khan, F., Ansari, T., Shariq, M., and Siddiqui, M.A., 2020. Biological control: A sustainable and practical approach for plant disease management. Acta Agric. Scand. B-Soil Plant Sci., 70(6): 507-524. https://doi.org/10.1080/09064710.2020.1784262

Tariq, J.M., Farooq, T., Al-Hazmi, A.S., Hussain, M.D., and Rehman, A.U., 2021. Role of Trichoderma as a biocontrol agent (BCA) of phytoparasitic nematodes and plant growth inducer. J. Invertebr. Pathol., 183: 107626. https://doi.org/10.1016/j.jip.2021.107626

Tariq-Khan, M., Munir, A., Mukhtar, T., Hallmann, J., and Heuer, H., 2017. Distribution of root-knot nematode species and their virulence on vegetables in northern temperate agro-ecosystems of the Pakistani-administered territories of Azad Jammu and Kashmir. J. Plant Dis. Prot., 124(3): 201-212. https://doi.org/10.1007/s41348-016-0045-9

Taylor, A.L., and Sasser, J.N., 1978. Biology, identification and control of root-knot nematodes. North Carolina State University Graphics, pp. 111.

Temitope, A.E., Patrick, A.A., Abiodun, J., Olasekan, A.A., Onye, A.C., Vincent, A.O.T. and Elliseus, R.J., 2020. Trichoderma asperellum affects Meloidogyne incognita infestation and development in Celosia argentea. Open Agric., 5(1): 778-784. https://doi.org/10.1515/opag-2020-0075

Tudi, M., Daniel, R.H., Wang, L., Lyu, J., Sadler, R., Connell, D. and Phung, D.T., 2021. Agriculture development, pesticide application and its impact on the environment. Int. J. Environ. Res. Publ. Health, 18(3): 1112. https://doi.org/10.3390/ijerph18031112

Xiao, Z., Liu, M., Jiang, L., Chen, X., Griffiths, B.S., Li, H., and Hu, F., 2016. Vermicompost increases defense against root-knot nematode (Meloidogyne incognita) in tomato plants. Appl. Soil Ecol., 105: 177-186. https://doi.org/10.1016/j.apsoil.2016.04.003