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The prevalence of tomato root galls was assessed by counting galled plants compared to the 30 plants selected on each diagonal row. Thus, the prevalence of root galls per tomato variety was calculated according to the following formula.

Assessment of the severity of root galls
The severity of tomato root galls was assessed basing on the Zeck scale (1971). This scale, which corresponds to a rating ranging from 0 to 10, galls observed on the plant root systems, was summarized as follows: 0 (root system completely free from galls), 1 (very few small galls), 2 (numerous small galls), 3 (numerous small galls of which some are grown together), 4 (numerous small and some big galls), 5 (25% of the roots severely galled), 6 (50% of the roots severely galled), 7 (75% of the root severely galled), 8 (no healthy roots but plant is still green), 9 (roots rotting and plant dying) and 10 (plant and roots dead).

         

The severity scores recorded on tomato plant root systems made it possible to calculate gall severity according to the Mckinney formula (1923).

         

Collecting soil and root samples
Soil and root samples from the uprooted plants were taken, making sure to remove the top soil layer. Soil samples, approximately 500 g each, were taken at a depth of approximately 20 cm. Soil and root samples from each plant were put in different labeled plastic bags. All those samples were placed in a polyethylene bag and sent to the laboratory.
Extraction and identification of root-knot nematodes
Soil samples collected per tomato variety in the plot were homogenized so as to constitute a composite sample. Plant parasitic nematodes were extracted from 100 ml of soil samples by the Whitehead tray method (Coyne et al., 2010). Five replicates were made per composite sample.
            The extraction of nematodes, from the roots, was done per tomato variety. The roots were washed with running water and cut into approximately 5mm-explants with scissors. The root explants were homogenized in order to constitute a composite sample. Plant parasitic nematodes were extracted from 5 g of tomato root explants by the Baermann maceration method (Coyne et al., 2010). Five replicates were made per composite sample.
            Three 5 ml-aliquots of nematode suspensions after extraction were placed on a counting plate and mounted under an optical microscope. Root-knot nematodes were described, identified basing on the Perry et al. key (2009) and quantified.
Quantification of nematodes

To quantify the extracted nematodes, three 5 ml-aliquot were taken per nematode suspension. The number of individuals per 5 ml-aliquot was noted and the average numbers of individuals were calculated according to the following formula 1.

Contribution to root-knot nematode control by using garlic and onion extracts
Assessment of the in vitro effect of garlic and onion aqueous extracts on root-knot

• Preparation of root-knot nematode aliquots

The nematode suspensions from tomato root galls were concentrated to an average number of 100 ± 3 individuals of second larval stage per 100 µl of nematode suspension.

• Preparation of garlic and onion aqueous extracts

Garlic and onion cloves were stripped of their husks. Then, 100 g of garlic and onion cloves were weighed separately, diced and crushed in 100 ml of distilled water in a household blender. The ground material was collected and filtered twice in a row using hydrophilic cotton. The first filtration was done using hydrophilic cotton in a funnel and the second one with a syringe containing densified hydrophilic cotton. Each aqueous extract was used to test its effectiveness on root-knot nematodes, particularly individuals of the 2nd larval stage.

• Exposure of individuals to garlic and onion extracts

The effectiveness of garlic and onion aqueous extracts on root-knot nematodes second larval stage individuals was assessed with three concentrations of aqueous extracts (S, S/2 and S/4). Five replicates were made per concentration. A volume of 100 µl of nematode aliquot containing on average 100 ± 3 individuals was taken with a micropipette and transferred into each Eppendorf tube containing 2 ml of extracts of different concentrations. Control individuals were exposed to 2 ml of sterile distilled water. The Eppendorf tubes were slightly shaken and incubated in the container in the laboratory at room temperature (27 ± 2 °C).

• Determination of nematode immobility and mortality rates

After 24-hour incubation, the individuals were observed under an optical microscope in order to count immobile nematodes. After counting, the nematodes were transferred to sterile distilled water and incubated for 24 hours under the same conditions. Another observation was made to verify whether immobile individuals were dead or recovered by stimulation with a needle. The individual was considered dead in the absence of movement following the injection (Cayrol et al., 1989). The number of dead and recovered individuals were made per concentration. The experiment was repeated three times and the immobility and mortality rates of nematodes concentration were calculated as follows.

Assessment of the in vivo effect of garlic and onion aqueous extracts on root-knot nematodes

• Acquisition of tomato plants

Tomato variety F1 seeds were acquired on the market and germinated on moistened blotting paper in the tanks for 7 days. The seedlings were transferred into cavities containing humus-rich soil previously sterilized two times in an autoclave at 121 °C under 1 bar for 30 minutes. Tomato young plants, after 21 days, were transplanted into 30 plastic pots perforated at the base and containing 500 g of the same sterilized soil. The tomato plants were left in culture for nine days under an insect-proof shelter and were watered at a frequency of 2 days.

• Preparation of garlic and onion extracts

Garlic and onion extracts were prepared as previously described with 1 kg of cloves. Concentration of extract with the most important nematicidal activity was selected for the test.

• Preparation of nematode inoculum

Root-knot nematodes extracted from tomato roots were used for aqueous extract effectiveness test. They were concentrated in a suspension to an average number of 100 ± 3 second larval stage individuals per 4 ml of aliquot.

• Inoculation of tomato plants

Five treatments were performed so as to assess the in vivo effectiveness of garlic and onion extracts on root-knot nematodes. These included T0 (inoculated and untreated plants), T1 (uninoculated and untreated plants), NA (plants inoculated and treated with garlic extract), NO (plants inoculated and treated with onion extract) and NAO (plants inoculated and treated with both garlic and onion extracts).
Each plant was inoculated with 4 ml-aliquot containing 100 ± 3 individuals of second larval stage of root-knot nematodes. This aliquot was deposited using a micropipette into four holes of approximately 5 cm deep made around the tomato plant and near the roots. Each treatment was replicated seven times.

• Assessment of treatment effectiveness

Treatment effectiveness was assessed, 30 days after, using disease and plant development parameters. Disease development parameters included the number of root-knot nematode individuals in the tomato roots and soil, the number of galls and the nematode reproductive factor. Plant development parameters were size and number of leaves per plant.
Statistical analyses
The data were analyzed using Statistica 7.1 software. Root-knot nematode numbers in soils and roots were transformed log function before being subjected to one-way analysis of variance, including tomato variety or treatment. Likewise, immobility and mortality rates were transformed by angular function before subjecting them to one-way analysis of variance (extract concentrations or type of extract). Homogeneous groups were determined using Fisher’s LSD test at 5% level for each analysis.

RESULTS

Phytosanitary status of tomato fields

Symptoms on tomato plants

Three types of symptoms, namely root deformation, aerial organ discoloration and root system reduction were observed on tomato plants. Root deformation, manifested by the presence of rounded excrescence of varying diameter, was characteristic of tomato root galls (Fig. 1A). Aerial organ discoloration is reflected by plant leaf chlorosis (Fig. 1B). Root system reduction was also observed on tomato plants (Fig. 1C).

Root knot prevalence and severity

Tomato plants belonging to varieties PADMA and COBRA 26, showed root galls. Root gall prevalence and severity, however, varied depending on tomato varieties and exceeded 50% (Table 1). Root gall prevalence was 88.33% in variety PADMA, against 81.67% in variety COBRA 26. Root gall severity was 52% for variety PADMA, while it was 62.19% regarding variety COBRA 26.

Table 1: Average root gall prevalence and severity on tomato roots

 

Nematodes extracted from tomato soil and root samples

Individuals of various development stages belonging to Meloidogyne genus were identified from tomato soil and root samples. These included eggs, 1st and 2nd larval stage. Adult males and females were extracted, in addition to these different larval stages (Fig. 2).

Numbers of Meloidogyne sp. individuals in tomato soil and root samples
Average numbers of root-knot nematodes varied in soils and roots depending on tomato varieties (Table 2). The average numbers of root-knot nematodes were statistically different in soils and roots depending on tomato varieties. The average numbers of root-knot nematodes were higher in the tomato variety COBRA 26 cultivation soils and roots with 31 and 798 individuals, respectively. the average numbers of root-knot nematodes, whatever the tomato variety, were higher in tomato roots compared to those in soils.

Table2. Average numbers of root-knot nematodes in soil and roots per tomato variety

 

In vitro effectiveness of plant extracts on 2nd larval-stage root-knot nematodes
Immobility and mortality rate depending on extract concentrations

Average immobility rates of the 2nd larval stage remained relatively constant regardless of garlic and onion aqueous extract concentrations. The average mortality rates of these individuals, in contrast, went up with increasing concentrations of garlic and onion aqueous extracts (Table 3). Average mortality rates varied from 43.57 to 72.99% at S/4 and S concentrations of onion extract, respectively. As for garlic extract, the average mortality rates switched from 17.69 to 53.56% respectively for the S/4 and S concentrations. Statistical analyses revealed a concentration effect on the average mortality rate of root-knot nematodes (P ≤ 0.05). Average mortality rates were higher in crude extracts with 72.99% for onion and 53.56% for garlic (Table 3).

Table 3: Average immobility and mortality rates of 2nd larval-stage Meloidogyne sp exposed to garlic and onion extracts depending on their concentrations

 

Average immobility and mortality rates depending on the types of extracts

Garlic and onion aqueous extracts caused average immobility rates of 2nd larval stage root-knot nematodes to be higher than 76% (Table 4). No significant difference, however, was noted between the aqueous extracts of both plants in terms of average immobility rates. In contrast, the average mortality rates varied significantly depending on plant extracts. Statistical analyses showed that onion aqueous extract induced an average mortality rate of 55.52% higher than the one caused by garlic extract (29.04%).

Table 4: Average immobility and mortality rates of 2nd larval stage root-knot nematodes depending on garlic and onion extracts

IR: average immobility rates; MR: average mortality rates

 

In vivo effectiveness of garlic and onion extracts on root gall development
Effect of treatments on disease development
Number and average reproductive factor of 2nd larval stage root-knot nematodes and the number of root galls varied depending on the treatments (Table 5). Statistical analyses revealed a significant difference between the treatments in terms of numbers, reproductive factor and number of root galls. The analyses showed that the average numbers of root-knot nematodes in soils and roots were the highest with 245 individuals in the inoculated and untreated plants. The numbers of nematodes, in contrast, were lower (i.e. two individuals) in the soils and roots of the plants inoculated and treated with both garlic and onion extracts than those of plants inoculated and treated separately with garlic or onion extract.
The reproductive factor of Meloidogyne sp. was higher, that is, 2.45 in the inoculated and untreated tomato plants than in the plants inoculated and treated with extracts with a reproductive factor lower than 1. No significant difference was observed in nematode reproduction between tomato plants treated with a single extract and those treated with a combination of both extracts for nematode reproduction.

The number of root galls was lower in plants treated with plant extracts compared to inoculated and untreated plants. However, the roots of tomato plants treated with garlic extract did not exhibit root galls. Tomato plants inoculated only with nematodes were more favorable to the development of nematodes and root galls.

Table 5: Parameters of root gall development on tomato plants per treatment

Treatments	Average numbers of nematodes	Average reproductive factor	Average numbers of root galls
T0	0c	0c	0c
T1	246 ± 1a	2.45 ± 0.25a	11 ± 9.01a
NA	5 ± 0.04b	0.02 ± 0.00b	0c
NO	5 ± 0.04b	0.5 ± 0.01b	3 ± 0.93b
NAO	2 ± 0.06c	0.4 ± 0.01b	1 ± 0.69b
P	˂0.05	˂0.05	˂0.05

T0: uninoculated and untreated plants; T1: inoculated and untreated plants; NA: plants inoculated and treated with garlic extract; NO: plants inoculated and treated with onion extract; NAO: plants inoculated and treated
with both garlic and onion extracts.

 

Effect of treatments on tomato plant development
Tomato plants reacted differently in their development (Fig. 3). Indeed, the size and average number of tomato leaves per plant varied depending on the treatments (Table 6). Statistical analyses showed a treatment effect on the tomato plant development. Tomato plants inoculated and treated with both garlic and onion extracts showed a higher average height (22.63 cm) and a higher average leaf count per plant (36 leaves) than the plants that were subjected to other treatments.

Table 6: Plant sizes and leaves numbers of tomato plants per treatment

T0: uninoculated and untreated plants; T1: inoculated and untreated plants; NA: plants inoculated and treated with garlic extract; NO: plants inoculated and treated with onion extract; NAO: plants inoculated and treated
with both garlic and onion extracts.
Averages with the same letter, in each column, are statistically similar according to Fisher’s

 

DISCUSSION

The characteristic symptoms of root-knot nematode infection were observed on tomato plants during phytosanitary surveys carried out in tomato fields. These included root galls, root system reduction and plant leaf chlorosis among others. Root-knot nematodes, particularly individuals of the 2nd larval stage, once in the central cylinder of the root thanks to their salivary secretions, induce the formation of permanent nourishing sites made up of hypertrophied cells (Djian-Caporalino et al., 2018). Giant cells therefore divert nutrients in favor of nematodes in order to complete their development cycle. The increase in the volume of corrupt plant cells therefore leads to the formation of gall, characteristic of root-knot nematode infection. The permanent diversion of nutrients in favor of nematodes leads to leaf discoloration resulting in chlorosis, followed by plant wilting. Root galls, chlorosis, tomato plant root system reduction have previously been observed by Mokrini et al. (2016) in a survey of tomato farms in Morocco.
Furthermore, the phytosanitary surveys carried out in the tomato fields showed that the plants of all tomato varieties PADMA and COBRA 26 showed the characteristic symptoms of root-knot nematode infection. The prevalence and severity of these symptoms was over 50% regardless of the variety of tomato. This strong presence of the characteristic symptoms of root-knot nematode infection on tomato plants could be explained by the fact that these tomato varieties are sensitive to this category of nematodes. These varieties are best known for their resistance and tolerance to fungal, bacterial and viral infections. But it is clear that they are sensitive to root-knot nematode infection, even though they are one of the main varieties of tomato grown in the area of Yamoussoukro. Mokrini et al. (2016) showed that root-knot nematodes have a strong preference for Solanaceae and legumes. Thus, tomato yield in the area, already subject to numerous phytosanitary constraints, is strongly and negatively impacted by root-knot nematodes.
Baermann’s maceration method, used for nematode extraction, revealed the presence of individuals of several developmental stages of root knot nematode belonging to the genus Meloidogyne. Their presence in the roots might be due to their plant-parasitic way of life. Indeed, plant parasitic nematodes are obligate biotrophic organisms that only live dependent on a host plant (Dinh et al., 2014). Part of their life cycle takes place in the roots of host plants. Thus, water and mineral salts extracted and sugars synthesized by plants are diverted by plant parasitic nematodes for their development (Bleve-Zacheo et al., 2007).
The number of nematodes, in addition, was found to be higher in the roots than in the soil. This might be due to the fact that nutrients (minerals absorbed) are found in roots for nematode development and reproduction. Jia et al. (2012) showed that root exudates (sugar, salts, enzymes) from plants sensitive to nematodes favor nematode egg hatching, which would explain their high proportion in roots.
On the basis of the foregoing, the in vitro effectiveness tests carried out with crude aqueous extracts of garlic (Allium sativum) and onion (Allium cepa) showed that they have nematicidal activity. This nematicidal activity might be due to the presence of sulfur compounds such as dimethyl-dipropyl and dimethyl-disulfide in Alliaceae extracts (Djian-Caporalino et al., 2018). Indeed, these compounds could act on nematode development and reproduction so as to modify their physiology, by affecting ion absorption, by modifying membrane permeability, enzymatic activity, cell division and electron transportation, thus leading to their paralysis and then their death (Anaya et al., 2006). Studies carried out by Youssef et al. (2016), showed the nematicidal effectiveness of garlic and acetyl salicylic acid on root-knot nematodes. These authors have shown, through their study, that the extracts significantly reduced, either by watering or by foliar spraying, the development parameters of diseases caused by nematodes. This reduction therefore improved the growth and development of the plants. Bakr (2018) also demonstrated that the highest concentrations of garlic and onion aqueous extracts were more effective in reducing the numbers of Meloidogyne sp. infecting tomatoes. Onion aqueous extract, in this study, has a stronger nematicidal activity than that of garlic extract. Bakr (2018), in the same perspective, showed the larvicidal potential of onion extract, through the reduction in the number of individuals of the 2nd larval stage of Meloidogyne incognita. Joshi et al. (2013), in addition, reported the ovicidal and larvicidal potential of onion extract on 2nd larval stage Meloidogyne incognita. In view of the significant in vitro nematicidal activity of onion and garlic aqueous extracts, their nematicidal potential has been verified in vivo.
Under semi-controlled conditions, all treatments carried out with garlic and onion aqueous extracts reduced nematode reproductive factor and root gall development in tomatoes compared to other treatments. However, garlic extract reduced root gall development more than that of onion. El-Saedy et al. (2014), showed that garlic oil helped control effectively Meloidogyne incognita in tomatoes. Likewise, the work carried out by Abd Elgawad et al. (2009) showed that treating the soil with a product containing garlic extract reduced root gall index. In contrast, the combination of garlic and onion extracts in root-knot nematode control has helped improve the development of tomato plants. This improvement might be due to a synergy of activity of phytochemicals with nematicidal activity present in plant extracts. According to El-Nagdi et al. (2010), turmeric and ginger-based aqueous extract treatment improves the availability of nutrients, which could be explained by a high absorption of these nutrients by the roots.

CONCLUSION

Root galls, leaf chlorosis and root system reduction, characteristic of root-knot nematode infection, are found on the plants of different varieties of tomato grown in the area of the Autonomous District of Yamoussoukro. These symptoms are not only preponderant, but also severe on the plants regardless of the variety of tomato. Meloidogyne sp., only, found at different development stages was associated with tomato root galls, leaf chlorosis and root system reduction with an important presence in soils and roots of variety COBRA 26. The crude garlic and onion aqueous extracts used against root-knot nematode developed an important nematicidal activity under controlled and semi-controlled conditions. Treatment carried out with both garlic and onion extracts reduces root gall development and improves tomato plant development. There is therefore an interest in combining garlic and onion aqueous extracts in an integrated tomato pests and disease management program. An inclusion of Alliaceae crops in an association or rotation with host crops, particularly tomato, could be made in order to study the impact on nematode development and tomato yield.

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