Nemato-toxic analysis of several chopped plant leaves against Meloidogyne incognita affecting tomato In vitro and In pots

Tomato plant is affected by several pathogens, including root-knot nematodes (RKNs), belonging to the genus Meloidogyne. Meloidogyne incognita is among the most potent pests infecting tomato roots. Therefore, it is of interest to discuss the management of Meloidogyne incognita using selected botanicals such as Cammelina benghalensis, Evolvulus nummularius, Gomphrena celosioides, Lindenbergia indica, Scoparia dulcis and Vernonia cinerea. The second-stage juveniles (J2s) of M. incognita were directly treated with the aqueous extracts of the botanicals at varied concentration ranging from 10-100%. 100% concentration of Lindenbergia indica was found to be the most toxic against the survival of J2s of M. incognita as compared to other concentrations. In vitro tests also showed the maximum inhibition in egg hatching at 100% concentration after seven days in the extract of Lindenbergia indica. Moreover, botanicals significantly reduced the infestations in relation to number of root galls, eggmasses/root and nematode population/250 g soil in pots. The plant treated with Scoparia dulcis leaves showed the highest nematicidal efficacy with maximum reductions in all the pathological parameters as compared to the untreated control. All treatments resulted in increased growth, physiological parameters and decreased pathological parameters of tomato.

25%, and 10% were made by dissolving the required amount of distilled water.

Eggs hatching test:
To the hatching experiment, five healthy uniform size eggmasses of M. incognita were picked up from the infected brinjal plant root and placed in Petri dishes, each containing 10 ml of different concentrations of six plant extract. Petri dishes containing five egg masses in distilled water were employed as a control. For seven days, all of the Petri plates in the laboratory were left at room temperature to allow the eggs to hatch. Each treatment was carried out five times. A binocular microscope was used to count the number of hatching juveniles with the help of a counting dish.

Juveniles' mortality test:
For the in-vitro mortality test, 120 freshly hatched second-stage juveniles of M. incognita present in 0.2 ml water are transferred to 9.8 ml different concentrations of each Plant extract in Petri plates. Double Distilled Water in Petri dishes served as control. Each treatment was replicated five times. The number of living and dead juveniles was counted using a binocular microscope following incubation periods 24, 48, and 72 hours. The nematodes that showed any motion were regarded as alive, but the worms that did not show any mobility and had a straight body shape were considered dead. The total number of dead and alive nematodes was counted. The data on concentrations and mortality rates were analysed and the LC-50 values for all treatments were calculated.
The formula was used to quantify the percent inhibition in egg hatching or mortality.

Pots experiment:
The pot study was carried out in a greenhouse at Department of Botany, Aligarh Muslim University, Aligarh. Six-inch clay pots were filled with 1 kg autoclaved soil in a 3: 1 ratio (sandy loam: farmyard manure). The soil was amended with 30 gm of freshly cut leaves from the tested plant. The soil of each pot, also amended with 5 gm dry powder of leaves of the Lindenbergia indica. The required amount of water was given regularly into the pots for proper decomposition of freshly chopped leaves. The seeds of the tomato cultivar Pusa-Ruby were purchased from IARI, New Delhi. The seeds were surface-sterilized in 0.01 percent HgCl2 for two minutes before being washed three times with Double Distilled Water (DDW). Surface sterilized seeds sown in pots for prepared nursery. Single healthy seedlings were transferred from nursery to each treated pot, including control and properly maintained the pots. Each pot was inoculated with 1500 freshly hatched secondstage juveniles of M. incognita by making holes in the rhizosphere. A fully randomized design (CRD) was used in the trial, with five replications of each treatment and control. Untreated uninoculated and inoculated plants were taken as control. Throughout the experiment, pots were supplied with the needed amount of water on a regular basis.

Observation and data collection:
Ninety days after inoculation, tomato plants were uprooted from their pots and roots were cut from the shoot. To avoid eggmasses being damaged, the roots of each tested plant were carefully cleaned in a bucket of water. The data were collected as growth, yields, physiological and pathological parameters such as shoot and root lengths, fresh weights, dry weights, chlorophyll, carotenoid contents, nitrate reductase activity (NRA), Proline, number of galls, eggmasses/root, and nematode population/250 gm soil. The population of the root-knot nematodes was assessed by Cobb's sieving and decanting technique [15].

Estimation of chlorophyll and carotenoids content:
The chlorophyll and carotenoid content of the fresh leaves was determined by Mackinney's method. One gm of fresh leaves detached from the plant and thoroughly ground with a mortar and pestle. After that, add 20 mL of 80% acetone to the pulp. After centrifuging the mixture at 5000 rpm for 5 minutes, the supernatant was collected in a volumetric flask. The Residues were washed three times with 80 percent acetone, each time using the same volumetric tube and the final volume was labelled with 80 percent acetone. A spectrophotometer (Shimadzu UV-1700, Tokyo, Japan) was employed to analyse absorbance at 645 and 663 nm for chlorophyll and 480 and 510 nm for carotenoid beside the blank (80% acetone). The chlorophyll and carotenoid content of the extract (mg g−1 tissue) was estimated using the equation below.

Estimation of Proline content:
Proline content in leaf tissues was determined using a ninhydrin reaction [16]. For this purpose, 0.25gm leaf sample was grinded in 5 ml of 3% sulfosalicylic acid with the help of a mortar pistil. This sample was centrifuged at 10000 rpm for 10 minutes. 1ml supernatant, ninhydrin acid and glacial acetic acid (1:1:1) was incubated at 90℃ for 1 hour to colorimetric measurements. The reaction accrued then cooled in an ice bath. After this, 2ml of toluene was added and vigorously shake the sample. A chromophore was extracted, and its absorbance was measured at 520 nm using a spectrophotometer (UV 1700, Shimadzu, Japan).  Jaworski (1971) technique was used to calculate nitrate reductase activity in fresh leaves. Each sample received 200 mg of chopped leaves, which were put to plastic vials. 2.5 ml of phosphate buffer pH 7.5 and 0.5 ml of potassium nitrate solution were added to each vial, followed by 2.5 ml of 5 percent isopropanol. These vials were incubated for 2 hours at 28 ±2°C in the dark in a BOD incubator. For the colour development, 0.3 ml of sulphanilamide solution and NED HCl were added to 0.4 ml of the incubated mixture in a test tube and left for 20 minutes. 5 ml. Distilled water was used to dilute the mixture. A spectrophotometer was used to measure absorbance at 540 nm (UV 1700, Shimadzu, Japan). A blank was run simultaneously with each sample. Using known graded concentrations of NaNO2 (sodium nitrite) solution, a standard curve was plotted. The absorbance of each sample was compared with that of the calibration curve and NRA activity (μ mole NO2 (FW) g −1 h −1 ) was calculated.

Statistical analysis:
Data of hatching and mortality presented are mean values. Experimental data of pot experiment was analysed by one-way analysis of variance (ANOVA) using SPSS-17.0 statistical software (SPSS Inc., Chicago, IL, USA). The differences between treatments were determined by Duncan's Multiple Range Test. Means values were considered significant at P ≤ 0.05.

Results: Effect of extract on juveniles' mortality:
The results shown in Table 1        Effect on number of galls, eggmasses, Eggs /eggmass and nematode population: Table 5 revealed that soil amendment of the chopped leaves of Scoparia dulcis, was most effective to the suppress effect of nematodes among the all-selected botanicals. The amendment of Scoparia dulcis indicate least number of galls in the roots was recorded i.e. (78) followed by Vernonia cinerea (85), Cammelina benghalensis (89), Gomphrena celosioides (95), while Evolvulus nummularius @ 30gm/pot was found minimum effective against nematode with maximum number of galls (98) as compared to untreated inoculated control (136). The minimum number of eggmasses (92) was observed in Scoparia dulcis, followed by Vernonia cinerea (97), Cammelina benghalensis (106), Gomphrena celosioides (115) while Evolvulus nummularius (124) was found maximum number of eggmasses on the roots of plant as compared to control (160). In the same context, the use of Scoparia dulcis leaves was shown to be the most significant. (P≤0.05) reduction in nematode population (1012), followed by Vernonia cinerea (1107), Cammelina benghalensis (1165), and Gomphrena celosioides (1218) while least reduction was observed in Evolvulus nummularius (1329) compared to untreated inoculated control (1680) Table 5. Results from Table 5 shows that a significant (P ≤ 0.05) reduction in eggmasses, eggs/eggmass nematode population and number of galls was found in all the treated pots. The Table 5 indicated that Scoparia dulcis show a maximum reduction (@ 92 @139 @1012 @78) whereas Evolvulus nummularius show minimum reduction (@124 @186 @1329 @102) in these parameters.

Discussion:
According to the findings, the nematicidal ability of selected plant extracts is due to certain phytochemicals. Existing research shows that many plants or their derived secondary metabolites or phytochemicals have nematicidal potential against a wide variety of plant-parasitic nematodes, including root-knot nematodes, M. incognita [20,21,22]. This in-vitro study found that the aqueous extracts of Lindenbergia indica, Scoparia dulcis, Veronia cinerea, Cammelina benghalensis Gomphrena celosioides and Evolvulus nummularius showed significant nematicidal efficacy against juvenile's mortality and egg hatching of M. incognita. Among all botanical extracts evaluated, the leaf extracts of Lindenbergia indica and Scoparia dulcis were shown to be the most efficient in lowering egg hatching and juvenile mortality. The result of in vitro suggested that Lindenbergia indica was most effective against nematode. So, to enhance the suppression effect of other botanical against nematodes, the powder of the Lindenbergia indica (5 gm) was amended in the soil of each pot, in starting the experiment excluding control. The nemato-toxic potential was found to be directly related to the extract concentration, i.e., the higher the concentration, the larger the nemato-toxic potential, and vice versa. In all treatments, juvenile mortality increases as the concentration increases from 10% to 100% and the exposure period increases from 24 to 72 hours. In all treatments, juvenile mortality increases as the concentration increases from 10% to 100% and the exposure period increases from 24 to 72 hours. As result, it can be concluded that nematode toxicity is dependent on the duration of exposure and the concentration of extracts [30,31]. Olabiyi [32] also reported that aqueous marigold root extracts were treated to root-knot nematode-infested tomato seedlings, enhanced plant height, leaf and fruit yield and plant leaf and fruit production compared to the control treatment. Elbadri et al. [33] reported that neem leaves extract has chemicals as, aldehydes, phenols, amino acids and fatty acids, terpenes, which are antagonistic to root-knot nematodes. Different strategies were used to inhibit the egg hatching and increased larval mortality of juveniles for nematodes management [34,35]. This has been reported that glycoside (asparagusic acid) obtained from Asparagus officinalis, suppressed Meloidogyne spp. [36]. Recently, two nematicidal chemicals nonacosane-10 ol and 23a-homostigmast-5-en-3b-ol were isolated from the roots of assessed. This significant drop in nematode infestation characteristics might be attributed to compounds found in degraded leaves with ovicidal or larvicidal activities, inhibiting nematode reproduction. The inadequate penetration in the second stage of juvenile and subsequent delays in their feeding and reproductive efforts might ascribe decreased root-knot proliferation. The decline in the number of nematodes might be to account for the plant's increased growth. Limited distractions to the plants result in a well and healthy growth [41]. Oka (2010) [42] observed that the use of plant parts can alter the physical structure and fertility of the soil, resulting in greater plant tolerance to nematode infection in terms of plant growth. The use of botanicals increases plant length (shoot and root) compared to their controls (UIC). There was also significant improvement in the fresh and dry weight of the roots and shoots as a result of the influence of botanical amendment. The above results confirm with khan et al.  2021), in which the nematicidal efficacy of botanicals was tested. This was found increase in tomato growth in treated soils compared to untreated soils might be due to an increase in soil nitrogen availability caused by the addition of botanicals. Adding botanical substances to the soil creates a healthy environment for root development. This increases soil nutrients and releases such toxic compounds, which might significantly minimise the nematode infestation [46,47]. Nitrate reductase (NR) is an important enzyme that functions as a key enzymatic source of nitric oxide in the plant cell. It controls plant development as well as tolerance to abiotic and biotic stress [48]. These findings were similar to reported by Berger et al. [49] found that photosynthesis rates drop when plants come into contact with pathogens. Based on safe, cost-effective and environmentally acceptable methods, organic amendments, plant extracts and bio-pesticides are being utilized primarily [50]. Botanicals in the soil benefited the host plants by fighting the nematode penetration or directly triggering the plant's defensive systems. Resistance or defensive responses are reported in host plants against plant diseases by substances from biocontrol agents and chemicals present in antagonistic plants extracts [51]. The tested aqueous leaf extracts and chopped leaves had significant nematicidal potential against M. incognita. More research is needed to isolate and characterize nematotoxic compounds of these botanicals using advanced techniques to be used in plant-parasitic nematode management in the future instead of hazardous chemical nematicides.

Conclusion:
Data shows that the botanical extracts examined delay egg hatching and cause mortality of second-stage juveniles of M. incognita. The inhibitory action of extracts is attributed to chemical compounds contained in extracts with ovicidal or larvicidal activities. Data also suggests that applying selected botanicals to the soil as organic amendments function as nematicides and can be successfully utilized to eliminate root-knot nematodes in place of traditional chemical nematicides. Thus, these selected botanicals can be considered to promote organic farming and sustainable management of nematodes. However, further study is needed to investigate the phytochemicals of the selected botanicals that inhibit the nematodes.