Journal of Plant Pathology & Microbiology

Biofumigation Potentials of Some Brassica Crops to Control Root-knot Nematodes Meloidogyne spp., on Tomato Under Field Conditions

Shimaa Hassan¹, Amal A Al-Gendy², Sahar H Abdel-Baset^3*, Salah M Abd El-Kareem¹, Samia Massoud⁴ and Mohamed Abdallah^{5 1}Plant Production Department, Faculty of Environmental, Agricultural Sciences, Arish University, Egypt
²Department of Pharmacognosy, Faculty of Pharmacy, Egypt
³Department of Nematology, Plant Pathology Res. Inst., ARC, Giza, Egypt
⁴Department of Agricultural Botany, Faculty of Agriculture, Suez Canal University, Egypt
⁵Faculty of Desert and Environmental Agriculture,, Matrouh University, Egypt
^*Correspondence: Sahar H Abdel-Baset, Department of Nematology, Plant Pathology Res. Inst., ARC, Giza, Egypt, Tel: +0100397094, Email:

Received: 07-Nov-2020 Published: 27-Dec-2020, DOI: 10.35248/2157-7471.20.12.227

Introduction

Tomato (Solanum lycopersicum L.) is the second most important vegetable crops in the world next to potato with approximately 182.3 million tons of tomato fruits produced on 4.85 million ha each year, Europe, America, and Africa produced 13.5, 13.4, and 11.8% of the total tomato yield, respectively [1]. Egypt is the fifth country in the world in terms of the production of tomatoes. It's grown all year round. The cultivated area reached 475.514 thousand feddan produced approximately 7.94 million tons [2].

Among the several pests and diseases that affect tomato plants, the plant-parasitic nematodes pose a major threat, resulting in an estimated annual monitoring loss of USD 80 billion [3]. The most prevalent and destructive among the phytonematodes of tomato are Meloidogyne spp., which causes major economic losses on vegetables, especially in tropical and subtropical areas [4-6]. In Egypt, tomatoes are known to be highly susceptible to root-knot nematodes Meloidogyne spp., caused annual losses of 12% [7].

The continuous use of chemical nematicides to control root-knot nematode had a considerable environmental impact and has resulted in the onset of resistance phenomena within some populations of nematode pests, this situation has led to an increased demand for environment-friendly products in order to reduce the effects of widespread nematicides utilization in crop protection [8].

Several studies reported the ability of certain plants to suppress nematodes through the nematicidal activity of the secondary metabolites [9,10]. Several researches on biofumigation had focused on using brassicaceous crops [11]. The suppressive effect of brassicaceous, biofumigants on Phyto-nematodes were explained in numerous studies under in vitro, in vivo, and field conditions [12-14]. The mechanism responsible for the biocidal effect of decomposing brassica crops was thought to be based on a chain of chemical reactions ultimately resulting in the formation of biologically active products [15]. Brassicaceae plants contain glucosinolate compounds, which are β-D thioglucosides, sulphur containing stable and non-toxic compounds located in the cell vacuoles distinguished from one another by differences in their organic side chains (R groups) and classified as aliphatic, aromatic or indole forms, occur in all parts of the plant and degrade via enzymatic hydrolysis [16]. The fumigant action of these volatile compounds that are released, suppresses plant pathogens soil-borne pathogens [17,18].

The aim of this study was to determine the biofumigation effects of fodder radish, and rocket salade on the root-knot nematodes, Meloidogyne spp., and tomato yield under field conditions in addition to identification of bioactive compounds of the two plants under investigation using Gas liquid chromatography-mass spectrometry (GLC-MS) and correlate between the glucosinolate autolysis products and nematicidal activity.

Materials and Methods

Source of plant seeds

Two brassica plants, i.e., fodder radish (Raphanussativus var. Terranovah-4-169/0300), was obtained from Joordens Zaden Company, Netherlands. Rocket salad(Eruca sativa cv. Baladi) was obtained from Horticultural Research Institute, Agriculture Research Center, Egypt, Giza.

Field experiments

The experiments were conducted at naturally infested with root-knot nematodes Meloidogyne spp., in the experimental farm of Faculty of Environmental Agricultural Sciences, Arish University during two successive seasons of (2017, and 2018) using the same field design plots of applied treatments. The experiments were divided into 16 plots, each plot (2.5 × 3.5 m²), all treatments were added in the two seasons as follows:

1. Biofumigation crops of fodder radish (Raphanussativus var. Terranovah).

2. Biofumigation crops rocket salad (Eruca sativa cv. Baladi ).

3. Nematicide treatment Vydate (oxamyl) 24% L.

4. Control treatment (without any treatment).

Preparation of soil

Sixteen individual plots were arranged 2.5 × 3.5² with the 1m interval between cross plots. Plots were plowed to 30 cm depth by the garden tiller. Seeds of (fodder radish, and rocket salad) were sterilized before using and sowed in arranging plots as a recommended amount (2.4kg/Fed) (15 December 2017). Vydate (oxamyl) 24% L as a nematicide was used at 4L/Fed (recommended dose) was used for comparison. Control treatment kept free without any treatment. The experiments were in a completely randomized block design with four replicates.

Incorporation of brassica plants and evaluation of their biofumigation effects on tomato plants

After 3 months (15 March 2018) at the full blooming stage (3 months after cultivation), 4 plants were randomly selected from each replicate. Shoots and roots were dried at 70˚C till a constant weight to determine both roots, shoot and whole plant dry weights (g/plant). After that, the dried plant materials were subject to the determination of C/N ratio of the whole plant.

Another 4 plants were used to determine nematicidal phytochemical profiles using GlC-MS. The brassica plants were incorporated into the top 25-30 cm of the soil by a garden tractor with a rotary tiller. The plots were irrigated to field capacity and covered with a transparent polyethylene film (50-micron thickness) in order to prevent any evolved gasses from escaping to the atmosphere, as well as, to increase the temperature to accelerate the decomposition process of brassica as biofumigation crops. Irrigation was continued at 3 day-interval during the decomposition period (four weeks). After that, plastic sheets were removed, and the soil was left for two weeks before tomato transplanting. Tomato seedlings were planted, and all soil partitions were planted at the same time with a tomato seedling (Solanum lycopersicum) cv. "Elisa"35 days old with 4-5 true leaves". Seedlings were transplanted in each plot at a distance of 40 cm in the row and 40 cm between rows. Tomato plants were grown by drip irrigation.

Three months after transplanting tomato seedlings, harvest was done and 10 tomato plants from each plot were randomly selected and the growth parameters were: shoot and root length (cm), fresh shoot and root weights (g) as well as dry weight (g). Also, fruit yield weighs (g) were recorded. Increasing percentages fresh weight of the whole plant, and fruit yield (g) % = Treatment - control/control × 100.

The roots of these plants were carefully removed and recorded numbers of galls, gall index, number of egg masses/ root system, as well as, number of juveniles in each plot [19]. Root galling was estimated according to [20], whereas: 0= no galls or egg-mass 1= 1-2 galls or egg-mass 2= 3-10 galls or egg-mass 3= 11-30 galls or egg mass 4= 31-100 galls or egg-mass 5= more than 100 galls or egg-mass. Egg-masses were stained prior to counting by dipping the infected roots in phloxine-B solution (0.015%) for 20 minutes as described by Daykin et al. [21]. Reduction percentages of root knot nematode galls, egg-masses numbers/ root system, and number of the secondstage juveniles (j₂) in 250 g of soil were calculated in comparison with a control treatment.

Preparation of glucosinolates hydrolysis products by natural autolysis

Each plant under investigation (20 g fresh weight) was homogenized with distilled water (200 mL) and left for natural autolysis overnight (15 hours) at room temperature (≈25°C). Each mixture was shaken with dichloromethane (50 mL) for 20 min and centrifuged for 5 min at 3500 rpm. Anhydrous sodium sulfate was used to dry the separated organic layer which then concentrated under nitrogen to about 0.5 mL and kept (in a tightly closed vial) in deep freezer until analysis [22].

Gas Liquid Chromatography-Mass Spectrometry (GLCMS)

1μL of dichloromethane extract of each plant autolysate was injected into an Agilent 6890 gas chromatography (USA) equipped with PAS- 5MS capillary column (30 m × 0.32 mm; 0.25 μm film thickness), attached to an Agilent 5973 quadrupole mass spectrometer. The injector temperature was 250°C and the temperature program started at 45°C isothermal for 3 min and raised to final temperature 280°C at 5°C/min, 10 min isothermal. Helium was the carrier gas (1 ml/min). The mass spectrophotometry detector was operated in an electron impact ionization mode and ionizing energy of 70 eV scanning from m/z 40 to 500. The temperature of ion source was 230°C. The percentage composition was computed from the total ion chromatogram peak areas (Table 1).

Table 1: Chemical analysis of two studied brassica species and its dry weight (g/plant).

Statistical analysis

All experiments were performed twice in a completely randomized design with three replicates in each treatment. Data were subjected to analysis of variance (ANOVA) using MSTAT-C program version 2.10 [23,24]. Means were compared by Duncan’s multiple range test at P ≤ 0.05 probability [25].

Results and Discussion

Effect on nematode population

Results obtained in both seasons 2017 and 2018 were almost identical Table 2 indicated that significant (p≤0.05) reduced nematode parameters on tomato plants, under field conditions. Average percentages of nematode population reduction were calculated compared with control treatment. In general the effect of Raphanus sativus var. Terranovah, as biofumigation crop recorded the average of the percentage reduction in number of galls, egg-masses/root system, and a number of second-stage juvenile (j₂) /250 g of soil (84, 90, and 84%) respectively in season 2017. While, were (90,87, and 88%) in season 2018 respectively. On the other hand, Eruca sativa cv. Baladi as biofumigation crop recorded (75, and 82%) of the percentage reduction in number of galls, (55% and 73%) egg-masses/root system, and a number of second-stage juvenile (j₂) /250 g of soil (77% and 75%) in seasons 2017 & 2018 respectively. The ability of certain plants to suppress nematodes through the nematicidal activity of the secondary metabolites has been reported [26]. Research has furthermore proved that many Brassicaceous species show nematicidal efficacy of plant-parasitic nematodes [27,28]. The plant species that generally are considered for biofumigation are found mostly in family Brassicaceae and include Eruca sativa (rocket salad), and Raphanus sativus (radish), [29,30]. The present results are agreemet with those obtained by Aydınlı et al. [31] reported that used radish (Raphanus sativus) and rocket salad (Eruca sativa) as biofumigation crops decreased gall index and egg masses of root-knot nematode M. arenaria on tomato plants under the greenhouse conditions. The same trend was noticed with El-Nagdi [19], who reported that used two mashed leaves of plants belonging to the genus brassica, cabbage (Brassica oleracea) and kohlrabi (Brassica caulorapa) were used as pre-and at sowing biofumigants for managing root-knot nematode, Meloidogyne incognita on cowpea plants, under greenhouse conditions. They found that the addition of the plant's residue cabbage and kohlrabi 10 days before sowing gave the highest percentages of final nematode reduction (70.4, and 82.1%) respectively. Also, when the C/N ratio of the amendment is less than 20:1, more effect on nematodes is evident [32,33]. On this basis, (R. sativus and E. sativa) residue used in this study, have a C/N ratio (10.1, and 9.20) so more effect of its toxic biofumigants on the nematode population.

Table 2: Effect of Brassica as biofumigation crops (R. sativus, and E. sativa) on root galling and nematode reproduction of Meloidogyne spp., under field conditions during successive seasons 2017 & 2018.

Effect on tomato growth and yield

As shown in Table 3 all plant growth parameters on tomato plants significant increased (p≤0.05) by using the tested treatments. The obtained results indicated that the effect of R. sativus as biofumigation crop recorded the average highest percentages. i.e., the plant growth vigor, increase, and fruit yield per plant compared with control treatment. The increase in fresh weight of whole tomato plant, and fruit yield were (57, and 92%), and (64, and 102%) in seasons 2017 &2018 respectively, this may be attributed to the relatively high biomass accumulation by fodder radish (48.54g/plant) Table 1. The same trend occurred for E. sativa as biofumigation crop increased in fresh weight of whole tomato plant, and fruit yield were (57, and 79%), and (46, and 93%) in seasons 2017 & 2018 respectively. The present results are agreement with those obtained by Anita [6] who, reported that ethanol extracts of cabbage, cauliflower, radish and chinese cabbage leaves reduced population of M. hapla and improved celery plant growth criteria, of which, radish leaf residue increase 41.9% in celery green leaves and stalk yield.

Table 3: Effect of Brassica as biofumigation crops (R. sativus and E. sativa) on the growth of tomato infected with Meloidogyne spp., under field conditions during successive seasons 2017 & 2018.

Gas liquid chromatography-mass spectrometry (GLC-MS) of R. sativus and E. sativa

Analysis of dichloromethane extract of fodder radish and rocket salad indicated the presence of glucosinolates hydrolysis products including isothiocyanate, nitriles and epithioalkane nitriles. Total ion current chromatogram of the GLC analysis of dichloromethane extract of autolysis products of fodder radish, and rocket salade, were shown in Tables 4 and 5, and Figures 1 and 2. The identification was based upon a comparison of mass fragments and their relative intensities with the available literature [23,34-37] and Wiley 9th edition NIST11 (W9N11) USA, database libraries.

Figure 1: Total ion current chromatogram of GLC analysis of fodder radish.

Figure 2: Total ion current chromatogram of GLC analysis of rocket salad.

Table 4: Glucosinolates hydrolysis products obtained by natural autolysis of R. sativus var. Terranovah (Fodder radish).

Table 5: Glucosinolates hydrolysis products obtained by natural autolysis of E. sativa cv. Baladi (Rocket salad).

Bioactive compound names, retention time, relative percentage, and mass spectral data of each compound in each plant extract of glucosinolates hydrolysis products were listed in Tables 4 and 5.

Results in Figure 1, and Table 4, indicated that four glucosinolates were identified in fodder radish through their volatile autolysis products. Gluconapin, the major compound was identified by 3- butenyl isothiocynate while glucoerucin was identified by 4-(methylthio) butyl isothiocynate, which is commonly known as erucin. Sulphoraphane was released from 4-(methylsulfinyl) butyl glucosinolate (glucoraphanin) while 4-(methylsulphony) butyl isothiocyanate commonly known as erysolin was liberated from glucoerysolin.

The results indicated that rocket salad autolysate had five GLS Figure 2, and Table 5. Gluconapin was detected in rocket salad also and its presence was identified through its epithionitrile; 4,5-epithiopentanenitrile. The isomers progoitrin and epiprogoitrin were detected by the two hydrolysis products diastereomers threo and erythro 1-cyano-2-hydroxy-3,4-epithiobutane respectively. The aromatic glucosinolate; gluconasturtiin could be identified through its liberated nitrile named as 1-benzenepropane nitrile. Sativin, the major identified compound is 4-mercaptobutyl isothiocyanate liberated from glucosativin according to several researchers [38-40]. Fechner et al., [41] revealed that sativin was proved to be 1,3-thiazepane-2-thione, a tautomer of 4-mercaptobutyl isothiocyanate with cyclic 7-membered ring structure according to NMR data.

Gluconapin, glucosinolate (glucoraphanin) 4-(methylsulphony) butyl isothiocyanate. An alternative management strategy that is increasingly receiving interest is biofumigation that are a sustainable approach to manage soil-borne pathogens, nematodes, and weeds. This process was defined as a process that occurred, when volatile compounds with pesticidal properties were released during the decomposition of plant materials or animal products [42]. Some isothiocyanates such as benzyl isothiocyanate, methyl isothiocyanate, and phenyl isothiocyanate have been extracted from brassica species and showed nematicidal effects [43]. Other isothiocyanates also showed some nematicide effects, such as ethyl isothiocyanate benzyl thiocyanate, 1-phenylethyl isothiocyanate, and 2-phenylethyl isothiocyanate. However, acryloyl isothiocyanate and allyl isothiocyanate showed the highest effect against nematodes [44]. Glucosinolates and their derivatives, such as isothiocyanates, isolated from various brassica species differ in their toxicity against nematodes. Species containing benzyl or 2-phenylethyl and smaller level allyl isothiocyanate were more effective against Tylenchulus semipenetrans, and M. javanica ,when compared to butyl, ethyl, methyl, phenyl, and 4-methylsulfinyl isothiocyanate [45]. Glucosinolates are one of the most frequently studied groups of defensive secondary metabolites in plants. Upon cellular disruption, e.g. wounding by nematodes, the thioglucoside linkage is hydrolyzed by endogenous enzymes (myrosinases), resulting in the formation of products (e.g. isothiocyanate, thiocyanate, nitrile, epithionitrile, oxazolidine-2-thione) that are active against herbivores and pathogens [46,47]. For instance, glucosinates purified from Brassicaceae (Brassica napus, B. rapa, B. carinata, Lepidium sativum, Raphanus sativus, and Sinapis alba) were not toxic to j₂s of the cyst nematode Heterodera schachtii in their original form, but enzymatic hydrolysis products of glucosinolates (isothiocyanate, sinigrin, gluconapin, glucotropeolin, dehydroerucin) were lethal to the nematode [48]. Similarly, 11 glucosinolates and their degradation products did not affect j₂s of the root knot nematode M. incognita, but myrosinase hydrolysis products (gluconasturtiin, glucotropaeolin, glucoerucin, and sinigrin) were highly toxic [49]. Other studies also report that glucosinolates are only lethal to the cyst nematode Globodera rostochiensis in the presence of myrosinase [50].

Conclusion

From the obtained results, it can be concluded that (fodder radish, and rocked salad) when were utilized as biofumigation crops and compared to nematicide Vydate (oxamyl) 24% L, were effective in reducing population density of root-knot nematode Meloidogyne spp., infecting tomato plants and improved plant growth parameters. The highest percentage of the nematode population reduction occurred when of R. sativus as a biofumigation crop in two successive seasons 2017 & 2018 respectively.

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