Antinemic Metabolites of Simarouba glauca against Root Knot Nematode, Meloidogyne incognita Infesting Groundnut, Arachis hypogaea

Groundnut (Peanut) (Arachis hypogaea), an important oil and food crop, is widely grown in tropical and subtropical countries, with a worldwide production estimated at 33·1 million tonnes (Mc Donald et al., 2005). Several species, such as Meloidogyne spp., Pratylenchus brachyurus,  Belonolaimus longicaudatus, Criconemella ornate and Ditylenchus africanus are considered to be pests of great economic importance for the peanut, either worldwide or in specific regions (Dickson and De Waele, 2005). These nematode species can attack the roots, pegs and hulls of the peanut. Adegbite and Adesiyan (2005) and Almohithef et al., (2020) described nematodes as destructive parasites because the nutrients are directly taken by the nematode from the host plant’s cell wall with the aid of a needle-like stylet. Depending on the severity, the losses brought on by root knot nematode might range from minimal to significant (Kavitha et al., 2012). Stunting of plants results from the impeded and altered Root to Shoot (R/S) transfer of nutrients and minerals during root colonisation of nematodes (Gullino et al., 2020). 
       
Botanicals have been in use for a long time for nematode control and these compounds offer many environmental advantages The Simaroubaceae family includes 32 genera and more than 170 species of trees and brushes of pantropical distribution. It is characterized by its content of bitter substances, mostly responsible for its pharmaceutical properties (Fernando and Quinn, 1992; Muhammad et al., 2004 and Babaali et al., 2017). Due to the chemical diversity of the Simaroubaceae family, it is worth noting that it can be characterized as a promising source of bioactive molecules with remarkable research potential. Exploration of quassinoid chemicals is predominant in simaroubaceae family (Curcino Vieira and Braz-Filho, 2006; Kumar et al., 2006; Priyadharshini et al., 2023).
       
In the last few years, gas chromatography mass spectrometry (GC-MS) has become firmly established as a key technological platform for secondary metabolite profiling in both plant and non-plant species (Kell et al., 2005; Robertson, 2005). The occurrence of root knot nematodes in vegetable crops is more predominant in India compared to other cultivated crops. In comparison with other nematode management strategies the phytochemicals derived from botanicals also plays a major role to control plant parasitic nematodes Hitherto, the present study is aimed to investigate the nematicide activity of Simarouba glauca leaf extract and the possible bioactive chemical components against M. incognita by subjecting it to GC-MS analysis.

The leaf and seed of Simarouba glauca were collected from Tamil Nadu Agricultural University, Coimbatore. The plant materials were washed under running tap water and dried for 1 week at room temperature in a shaded area. The dried samples were finely powdered by using an electric blender and stored in a plastic bag container for further use at room temperature.
       
Fifty gram of finely ground leaf and seed samples of Simarouba were packed separately in a porous bag. The solvent (250 ml of methanol) was added to a round bottom flask, which was attached to a Soxhlet extractor and condenser on an isomantle. Extraction was carried out separately for leaf and seed samples. Finally the leaf and seed extracts were evaporated and stored in a refrigerator at 0-4°C in an air tight container for further use. The egg masses of root knot nematode, M. incognita infested Groundnut plants were uprooted from the infested field and used for the collection of egg masses and infective juveniles. The species level confirmation of root knot nematode as Meloidogyne incognita was confirmed based on morphological characters of perineal pattern. Five mature females of Meloidogyne spp. were taken out of the root tissue using forceps, placed in a drop of warm lactophenol on a glass slide and examined under a light microscope to determine perineal patterns (Eisenback et al., 1981).
 
In vitro bioassay
 
Each treatment consisted of 5 replicates of egg mass in 25%, 50%, 100% concentrations of each extract. The experiment was conducted in a 24-well plate maintained in an incubator in the dark at 24°C. Hatched Juveniles were counted after 24, 48 and 72 hours in treatments.  The effect of methanolic extracts on 100 Juveniles of M. incognita at concentrations of 25%, 50%, 100% obtained from the stock solution was evaluated for 24, 48, 72 hours and repeated the each treatment 5 times. Using a double counter, 100 Juveniles between dead and live juveniles were counted after 24, 48 and 72 hours of exposure, considering a young straight or motionless juvenile as dead, which was confirmed by touching them with a fine needle. To compare the results, natural mortality was counted in distilled water which serves as control (Costa et al., 2003).
 
Preliminary phytochemical screening
 
Extraction is the separation of bioactive portions of plant using selective solvents of increasing polarity such asethanol. The purpose of extraction is to separate the soluble plant metabolites, leaving behind the insoluble residue. The initial crude extracts contain complex mixture of many plant metabolites, such as alkaloids, glycosides, phenolics, terpenoids and flavonoids. The methanol extract was tested for alkaloids,  anthroquinones, flavonoids, phenols, steroids, tannins, terpenoids, glycosides, saponins and volatile oils using GC-MS analysis (ee 2).
 
GC-MS (Gas chromatography-mass spectrometry) analysis
 
Volatile compounds like nonpolar components and essesntial oil, fattyacids, lipids and alkaloids that have antimicrobial property are identified by the GC-MS analysis. The methanolic extract of the leaf and seed of Simarouba were used for the GC-MS analysis. 1 μl of the methanolic extract of the leaf and seed of Simarouba were dissolved seperately in HPLC grade methanol and subjected to GC and MS facility available in the Department of Microbiology, TNAU, Coimbatore. The Clarus SQ 8C Gas Chromatography - Mass Spectrometer from Perkin Elmer, were engaged for analysis (Tables 3 and 4).
       
The pot and field experiment were conducted in the Department of Nematology, Centre for Plant Protection studies, TNAU, Coimbatore during 2021-2023 using different botanical formulations of Simarouba glauca. The treatments were applied to 15 days old Groundnut plants at two different time intervals @ 15 days and 45 days after transplantation. The observations includes No. of adult females/5 g root, No. of egg masses/5 g root, Gall index, Root population (5 g of root) and Soil population (250 cc soil) were recorded at 60 days after transplantation.
 
Statistical analysis
 
The data were analysed using (ANOVA) with IBM SPSS version 20.00 software (SPSS Inc., Chicago, IL, USA). Duncan’s Multiple Range Test (DMRT) with 0.05 probability assessed the significant differences between treatments. With the help of probit analysis, the LC₅₀ values of the compound determined.

The crude leaf extract of S. glauca (100%) exposure after 24 hrs on M. incognita observed the least eggs hatched (7.85/egg mass) and juveniles mortality was 60.58/100 juveniles, followed by 50% crude leaf extract on the number of eggs hatched (12.72/egg mass) and juveniles mortality (38.20/100 juveniles). The maximum number of eggs hatched (34.13/egg mass) and the lowest juveniles mortality (30.13/100 juveniles) were recorded in the 25% crude leaf extract compared to control had no juvenile mortality and the number of eggs hatched (75.45/egg mass).
       
The crude leaf extract of S. glauca (100%) exposure after 48 hrs on M. incognita observed the least eggs hatched (12.63/egg mass) and juveniles mortality was 70.23/100 juveniles, followed by 50% crude leaf extract on the number of eggs hatched (29.02/egg mass) and juveniles mortality (48.11/100 juveniles). Recorded the maximum number of eggs hatched (38.22/egg mass) and the lowest juveniles mortality (40.73/100 juveniles) in the 25% crude leaf extract compared to control had no juveniles mortality and the number of eggs hatched (167.21/egg mass) (Table 1).
 

       
After 72 hrs, the exposure of S. glauca crude leaf extract (100%) on M. incognita had the highest juvenile mortality rate and the lowest number of eggs hatched was 92.58/100 juveniles and 16.30/egg mass, followed by 50% crude leaf extract on juveniles mortality (62.44/100 juveniles) and the number of eggs hatched (36.54/egg mass). Recorded the lowest juveniles mortality (46.46/100 juveniles) and the maximum number of eggs hatched (42.39/egg mass) in the 25% crude leaf extract compared to control had juveniles mortality (4.01/100 juveniles) and the number of eggs hatched (286.53/egg mass). They have inhibited the egg hatching by increasing the concentration of the extracts from 25 to 100%. The present study verified the efficacy of methanolic leaf extract for M. incognita egg hatch inhibition and juvenile mortality (Table 1). 
       
The phytochemical tests showed the presence of alkaloids, cardiac glycosides, flavonoids, phenols, phlobatannin, reducing sugars, saponins, steroids, tannins, terpenoids, volatile oils, carbohydrates and protein/amino acids in methanolic extract of S. glauca (Table 2) as described by (Robertson, 2005). The GC-MS analysis has shown the presence of different phytochemical compounds in the methanolic extract of Simarouba glauca. A total of 21 compounds were identified representing 84.49% of total methanolic extract composition. The GC-MS spectrum confirmed the presence of various components with different retention times of Simaruoba leaf and seed as illustrated in Fig 1 and 2, Table 3 and 4. 
 

 

 

 

 

       
Pot and field experiments revealed that Simarouba leaf powder applied @ 10 g/plant in pot and 5 kg/ha under field conditions showed a significant reduction in the incidence of number of female nematode, egg masses, number of galls, total soil population and root population (Table 5 and 6).
 

 

From the results, it is evident that Simarouba glauca contains various phytocomponents that are having antifungal, antimicrobial, nematicidal property and can be effectively formulated and employed for integrated biotic stress management for agricultural crops. The presence of various bio-active compounds with antimicrobial properties detected after GC-MS analysis using the methanolic extract of S. glauca justifies the use of its leaves and seed against agricultural insects, pest and nematodes for their effective control. However, the biological activity of individual compounds against plant pathogens especially nematodes will subjected to understand their biocontrol potential. Evaluation of the nematicidal property of bioactive compounds of Simarouba glauca against root knot nematode, Meloidogyne incognita infesting groundnut revealed that Simarouba leaf powder seed oil cake applied in the soil significantly reduced the soil and root population of root knot nematode in Groundnut. This study provide an insight in development and application of botanical formulations that replace the hazardous chemical compounds in the context of integrated nematode management.

The authors are declaring that there is no conflict of interest in the publication of the paper.