ANTIBIOSIS AND ANTIXENOSIS IN TOMATO GENOTYPES AGAINST

Anaya significantly harbored the lower number of Helicoverpa armigera adult (0.1, 0.4, 0.4). The lowest number of eggs (0.5) was recorded from adults placed on Anaya variety. There was high and negative significant correlation of r = -0.865 between phenols and adult emergence. Phenols and oviposition were significantly negatively correlated (r = -0.816). Implication. Anaya and Mona hinder the development of Helicoverpa armigera larvae when fed on it. Phenol has negative impact on the development and oviposition of Helicoverpa armigera . Conclusion . It was revealed through this study that availability of promising resistant varieties can effectively combat the damage caused by Helicoverpa armigera and lessen the shortcomings related with the application of conventional chemical insecticides in tomato production.


INTRODUCTION
Tomato, an important vegetable with appreciable quantities of vitamins A, B and C are essential for human growth (Reddy et al., 2023).Among the important nutritional contents in tomato fruit is lycopene which is an important antioxidant that lowers the risk of prostate cancer in men (Dube et al., 2020).Danuta et al. (2020) reported that processed tomato products like pizza sauce, tomato juice, spaghetti sauce, paste and ketchup provide 80% of the lycopene found in food.Tomato production is hampered by insect pests that reduce its yield and increases the cost of production (Mantzoukas and Karnastasi, 2019).Helicoverpa armigera belongs to insect family noctuidae and order lepidoptera.It is a nightmare to tomato farmers and is attracted to tomato plant during the flowering and fruiting stages (Pavunraj et al., 2021).Tomato fruitborer lays eggs on tomato leaves and soon after hatching the first instar larva feeds on tomato foliage.The second and older instars infest and damage tomato fruits, penetrate the fruits through the stem end, feed inside creating a watery hole (Omotoso and Alabi, 2023).Usually, the damaged fruits ripe prematurely or rot due to secondary invasion of fungal diseases hence damage of H. armigera constitutes a serious threat to tomato production (Sharma et al., 2009).
In the quest to control this pest of economic importance and salvage this important crop from H. armigera damage.Although, the use of conventional chemical insecticide provided a succour to the farmers by lessen the damage on tomato caused by H. armigera (Latha et al., 2018).However, the relief is short-lived due to snags associated with the use of synthetic chemical insecticides (Yadav, et al., 2022) such as a threat to the health of the growers and consumers, harmful impact on beneficial and nontarget organism and development of resistance to the chemical insecticides (Ramadan et al., 2020).All these necessitate the application of eco-friendly control option in the management of H. armigera on tomato.
Planting of crops that are resistant to insect pest is among the most promising ways to reduce dependence on synthetic insecticides (Erdogan et al., 2020).Reports from previous researchers have shown that planting of insect-resistant varieties is part of the most effective, cost-effective and environmentally safe management tactics in controlling insect pests (Villegas et al., 2021).However, there is dearth of information in exploring resistant varieties and its mechanism for the management of H. armigera on tomato varieties.This study seeks to elucidate the mechanisms of resistance in different tomato genotypes and determine the phytochemicals that induce their resistance in tomato varieties.

Study location
The experiment was conducted at the Insect Chemical Ecology Laboratory, Department of Crop Protection and Environmental Biology, University of Ibadan, Nigeria.

Helicoverpa armigera culture
Larvae of tomato fruit borer were collected from established insecticide-free tomato field.The collected larvae were reared on fresh leaves of tomato; they were placed singly in a plastic container (7.0 cm diameter x 5.2 cm height) with a lid to avoid cannibalism.The lid was punctured randomly with a pin for proper aeration.Fresh young tomato leaves were placed in the plastic container and changed daily to maintain proper sanitation.When the larvae were fully grown, it pupated.After pupation, the pupae were transferred into another container (19.3 cm diameter x 18.2 cm height) that served as oviposition chamber.A paper towel was positioned at the base of the oviposition chamber and the pupae were placed on the paper towel.A white mesh was placed on the oviposition chamber and held with the lid that was cut with a circular open for proper aeration.Thereafter, those pupae that jiggled were placed on the paper towel using forceps.Immediately after adult emergence, 10% honey was placed in a small tube covered with cotton wool which served as food for the adult H. armigera.The adult mated and laid eggs on the white mesh in the oviposition chamber.The white mesh was removed and replaced with a new one daily.The white mesh with eggs was put in a zip up bag.Oxygen was allowed in the zip up bag for the neonates.

Source of seeds
Tomato seeds NGB00724 and NGB00725 were sourced from National Center for Genetic Resources and Biotechnology.UC82B and Roma VF (Susceptible check) were sourced from National Horticultural Research Institute while Anaya, Mona, Kelvin and Tropimech, were from Agritropic Limited, all in Ibadan, Oyo State, Nigeria.The tomato genotypes were selected based on farmers preferred choice.

Evaluation of antibiosis of tomato varieties
Under no choice test, the experiment was set up using completely randomized design with six replicates.There were eight treatments in this experiment, with each corresponded to a genotype of tomato: NGB00724, NGB00724, Anaya, Kelvin, Mona, Roma VF (susceptible varieties), Tropimech and UC82B.Seven best performed varieties were selected from the previous experiment due to their consistent lower rate of Helicoverpa armigera percentage fruit damage (1.03 -18.00) and the susceptible check with 36.80 percentage fruit damage (Omotoso and Alabi, 2023).
Fourty-eight second instar larvae of Helicoverpa armigera due to the tender nature of first laval instar were transferred from the culture with brush into a plastic container (7.0 cm diameter x 5.2 cm height) each with a pierced lid.The lid was punctured eight times with a pin.Eight tomato genotypes: NGB00724, NGB00724, Anaya, Kelvin, Mona, Roma VF (susceptible varieties), Tropimech and UC82B that were insecticide-free were earlier established on the field and used for this experiment.Eight weeks after transplanting, third tomato leaves from the upper part of each genotype mentioned above were introduced into the experimental set up.The study was conducted under laboratory condition (temperature: 31.8 o C, relative humidity: 67% and photoperiod: Light 12: Dark 12).The experimental set up was observed daily for pupation and mortality, the leaves were changed daily to maintain sanitation till pupation period of the larvae.A tube containing 10% honey was placed in a small tube covered with cotton wool served as food for the adult (Sujana et al., 2012).

Antixenosis of tomato genotypes to Helicoverpa armigera
Antixenosis was evaluated under choice test using tomato plants set up in the Screenhouse of the Department of Crop Protection and Environmental Biology, University of Ibadan, Nigeria.Those eight tomato genotypes earlier used for antibiosis experiment were used for this experiment.The experiment was laid out using a randomized complete block design with six replicates.Sandy-loam soil of 10 kg was filled into a plastic pot (24.5 cm diameter x 21.7 cm height) placed in the screenhouse.
Four-week-old tomato seedlings from the nursery were carefully taken and transplanted at the rate of one seedling per pot in the evening after all pots were filled with sandy-loam soil.All pots were watered with 1.5 L of water between 17: 00 and 18: 00 hrs.One week after transplanting missing stands were supplied with tomato seedlings from the nursery and the potted plants in each replicate were placed in an enclosed fine mesh.The plants were well supported with erect wooden bars to prevent the enclosed mesh from damaging the stem of the tomato plants in the pots; watering was done daily.Five pairs of adult H. armigera from the insect culture (one male: one female) were released into the enclosed mesh for seven days and no fertilizer was applied.A tube containing 10% honey was placed in a small tube covered with cotton wool served as food for the adults (Sujana et al., 2012).

Extraction Procedures
Tomato leaves of: Anaya, NGB00724, NGB00725, Mona, Kelvin, Tropimech, UC82B, Roma VF (Susceptible check) were obtained from the established tomato field eight weeks after transplanting.Third tomato leaf samples from the upper part of plant were collected and put in a well labelled envelop; the samples were stored in freezer until extraction.

Sugar Content Determination
Determination of the total sugar content in tomato leaves was done by the method described by Dubois et al. (1956).The technique described by Saad et al. (2021) was used to determine the reducing sugar content of tomato leaves.

Determination of Phenolic, Flavonoids and Protein contents
Total phenolic content of tomato leaves extracts was measured using the Folin-Ciocalteu reagent method described by Kaur and Kapoor (2002).The aluminum chloride colorimetric method, as reported by Hashemi et al. (2021), was used to determine the total flavonoid content of tomato leaf extracts.The protein content was determined by Kjeldahl method described by Sakar et al. (2020).

Data collection
Data were collected on the following parameters during antibiosis experiment: larval weight, larval period, pupal weight, pupal period, adult longevity and percentage pupation.While the number of eggs on the plants and the number of larvae on each plants were collected from antixenosis study.

Data analysis
Analysis of Variance (ANOVA) was performed using DSAASTAT statistical software (ver.1.101 2011).Significant means were separated using Newman-Keuls Multiple Range Test at p < 0.05.

Antibiosis of tomato genotypes to tomato fruitworm, Helicoverpa armigera
Significant differences (p < 0.05) were shown in the larva weight (g) and larval period (days) as presented in Table 1.Roma VF (Susceptible Check) recorded highest (0.46 ± 0.17 g) larval weight, while the lowest value (0.19 ± 0.04 g) was from Anaya.The same result trend was observed in larval period where Roma VF (SC) had the highest value (14.83 ± 0.17 days) with no significant differences (p > 0.05) from other genotypes except Mona and Anaya.
Adult emergence (days) and longevity (days) of Helicoverpa armigera fed on different tomato genotypes were showed in Table 3.All the genotypes supported Helicoverpa armigera to adult but in varied degree.The lowest percentage of adult emergence (16.7 ± 11.18) was observed on Anaya and Mona and were significantly lower (p = 0.05) than others.Roma VF (Susceptible variety), Tropimech, and NGB00725 recorded the highest adult longevity (6.00 ± 1.21 days, 5.00 ± 1.59 days, 4.00 ± 1.29 days, respectively) with no significant difference (p > 0.05) from Roma VF (susceptible variety).

Antixenosis of tomato genotypes to tomato fruitworm, Helicoverpa armigera
Results on Table 4 revealed the number of Helicoverpa armigera adult and oviposition.The lowest number of Helicoverpa armigera adult (0.1 ± 0.04) was obtained on Mona with a significantly lower value (p = 0.05) than Roma VF, susceptible variety.Roma VF had the highest number of eggs (7.2 ± 1.09).While Anaya had the lowest number of eggs (0.5 ± 0.016) and was significantly lower (p = 0.05) than the susceptible variety.

Percentage sugar and protein contents in genotypes of tomato leaves
The highest percentage of protein content (24.1 ± 0.11 mg/100g) was recorded from UC82B which was significantly different (p < 0.05) from the susceptible check (21.7 mg/100) (Table 6).Protein contents ranged from 22.8 ± 0.21 mg/100g on Mona to (20.0 ± 0.11 mg/100g) on Kelvin and Anaya.The highest similar percentages of 0.5 % of reducing sugars were obtained on Mona, Kelvin and Roma VF (susceptible check).The lowest percentage of reducing sugars (0.3 ± 0.03% each) were observed on NGB00725 and Mona, respectively with a significant difference (p = 0.05) from the susceptible check.Percentages of total sugar content ranged from (2.2 ± 2.39%) on UC82B to (1.8± 0.35%) on Anaya.

Correlation of primary and secondary metabolites in genotypes of tomato leaves with percentage pupation, adult emergence and oviposition
There was negative correlation between phenolics and pupation (r = -0.684)and was not significant (Table 7).The correlation between flavonoids and pupation was negative and not significant (r = 0.742) (p = 0.05).Protein and pupation was positively correlated and non-significant (r = 0.427) (p = 0.05).Pupation and reducing sugar and terpenoids were positively correlated and non-significant.
Phenolics was negatively correlated with adult emergence (r = -0.865)and was significant.There was also negative correlation (r = -0.715) between flavonoids and adult emergence.The correlation between total sugar and adult emergence was positive (r = 0.938) and significant.Terpenoids and adult emergence was positively correlated (r = 0.032) and non-significant.There was a negative correlation between phenolics and oviposition (r = 0.816) and was significant.There was a positive correlation between flavonoid and oviposition (r = 0.646) and was significant.Protein and oviposition was positively correlated (r = 0.282) and non-significant.The correlation of oviposition and reducing sugar and terpenoid was positive and non-significant.Total sugar and oviposition was positively correlated and significant (r = 0.827).

DISCUSSION
Antibiosis mechanism of resistance impairs an insect's metabolic processes.Most times, ingestion of plant metabolites is involved.In this study, reduced larval weight and period were observed on H. armigera fed on Anaya tomato variety.This could be attributed to differences in the nutrition contents of each tomato variety as revealed through varied levels of larval weights and periods (Coelho et al., 2019).This is similar to Kouchi et al. (2014) that reported varied feed conversion efficiency among H. armigera larvae fed with different tomato varieties with Rio grande recorded the lowest feed conversion efficiency of tomato.This is also in consonance with Krisnawati et al. (2017) that reported the evaluation of soyabean genotypes for antibiosis against armyworm through reduced weight of the larvae and duration and stated high antibiosis on genotype G511H/Anj-1-6 with the lowest larval weight.217.9 ± 6.17a 64.5 ± 1.47a 55.3 ± 0.04b Means in a column followed by the same letter(s) are not significantly different at p > 0.05 using Newman-Keuls Multiple Range Test.Values are means ± S. E. of three replicates SC = Susceptible Check Table 6.Percentage sugar and protein contents in tomato leaves of different tomato genotypes.
Treatment Protein (mg/100g) Reducing sugar (%) Total sugar (%) NGB00724 20.5 ± 0.28a 0.4 ± 0.02a 2.0 ± 0.05b NGB00725 20.9 ± 1.02ab 0.3 ± 0.03a 2.1 ± 0.92b Anaya 20.0 ± 0.11a 0.4 ± 0.02a 1.8 ± 0.35a Kelvin 20.0 ± 0.19a 0.5 ± 0.02ab 2.1 ± 0.49b Mona 22.8 ± 0.21c 0.3 ± 0.03a 1.9 ± 0.98a Roma VF (SC) 21.7 ± 0.23ab 0.5 ± 0.02ab 2.1 ± 0.56b Tropimech 22.7 ± 0.11bc 0.5 ±0.01ab 2.2 ± 1.73b UC82B 24.1 ± 0.11c 0.5 ± 0.01ab 2.2 ± 2.39b Means in a column followed by the same letter(s) are not significantly different at p > 0.05 using Newman-Keuls Multiple Range Test.Values are means ± S. E. of three replicates SC = Susceptible Check Anaya tomato variety had the lowest larval period which translates to reduction in the damage caused when compared with others that recorded lengthy days.This could be due to imbalances in the quality and quantity of primary and secondary metabolites require for optimum larval growth and could be an important factor in conferring antibiosis on tomato (Gacemi et al., 2022).Although, the pupal stage is the resting and changing stage where feeding does not take place.However, the amount and quality of food consumed during the larval stage affects pupation and emergence of adult.This was shown in the elongated pupal period obtained on Mona, Anaya and NGB00725.This gave a clue to the effect of antibiosis as it increased the duration of time spent during inactive period, delaying the development and decreases the damage done to the tomato plant.This agrees with Ngugi-Dawit et al. (2020) that indicated that delay in the development of insect pest is a promising indicator of an antibiosis mechanism of defence.Therefore, the low pupation rate observed on larvae that fed on Anaya showed the insect's metabolic processes had been impaired.Also, it is worthy of mention that half of the population of the larvae fed on Anaya and Mona pupated.This indicates a high level of antibiosis to H. armigera.
Lower percentage of adult emergence and longevity observed on larvae fed with Anaya and Mona indicated the apparent antibiosis resistance mechanism operating within these two genotypes.It may be due to the presence of secondary metabolites in optimum amount that have detrimental effect on the percentage of adult emergence.Low level of adult emergence observed on Anaya and Mona affects the population dynamics of the next generation, this retards the pest population.This is similar to de Castro et al. (2015) that reported different responses in the biology of Supputius cincticeps fed on different plants including tomato leaves.The results obtained of this study also show the existence of ample genetic variation among tomato genotypes to provide improved varieties in advancement to insect pest management (Javaid, 2006).
In the antixenosis study, the relatively low number of Helicoverpa armigera adults that settled on Mona, NGB00725 and Anaya indicated low acceptance by H. armigera for shelter.This could be as a result of genetical and morphological variability of the genotypes that deter settling and utilizing those genotypes for oviposition by H. armigera (Kamel and El-Gengaihi, 2009).Therefore, the strongest antixenosis resistance is observed on Mona which attracted lowest number of H. armigera (Kirişik et al., 2020).In the same vein, oviposition diminished on Mona and Anaya due to the inability to suitably utilize it for shelter.This suggests that there are biophysical and biochemical factors that impair oviposition behavior and also elicit different responses from different varieties of tomato plants to Helicoverpa armigera.Smith (2005) also indicated presence of some morphological characters (pubescence, foliage size and shape) in Cucumis species that makes Aphis gossypii look for an alternate host plant.
The studies further examined primary and secondary metabolites of the observed tomato genotypes.Secondary metabolites such as phenolic compounds present in plants confer properties such as antifeedant by repelling phytophagous insects (Talukder et al., 2021).Mrosso et al. (2022) indicated that flavonoids are toxic to whiteflies, thus protecting tomato plants from their infestation and damage.Therefore, it can be inferred from this study that the varieties that showed resistance to H. armigera had some secondary metabolites liable for the resistance in Anaya F1, the most resistant variety, has 75.3 mg/100 flavonoids and 183.9 mg/100 phenol.This aligns with Golan et al. (2017) that observed secondary metabolites preventing oviposition of insect on host plant and disrupt larval growth.This results also corroborates with the findings of Dixit et al. (2017) that indicated that phenolic compounds are toxic to insects and act as feeding deterrents to a wide range of insects including lepidopteran larvae.
It is anticipated that genotypes with higher level of total sugar and reducing sugar would enhance susceptibility of H. armigera damage.In the present study, resistant genotypes, Anaya, Mona and NGB00724 recorded relatively lower level of total sugar and reducing sugar.This agrees with Sun et al. (2021) that reported that sugars and protein are phagostimulants that induce sustained feeding in insect herbivores.However, from the results obtained in this study, those genotypes that showed resistance contained higher levels of protein than the susceptible control, Roma VF.This corroborates with Alabi et al. (2006) that reported higher levels of protein in the floral buds and flowers of resistant cultivars of cowpea to Megalorothrips sjostedti than Vita 7, the susceptible control.
In this study, highly significant negative correlation observed between phenolic content and adult emergence suggested a clue in safe and ecofriendly management of H armigera.Phenolic content was also negatively correlated with oviposition, this suggests the promising impact of phenol content to impair the biology of insect.In this sense, gene coding for phenolic content can be explore to induce resistance to H. armigera.This agrees with Puri et al. (2020) who reported the ability of phenol content to undermine the physiology of insect pest and confirmed phenol stability for use in the management of insect pest.This also suggests that role of phenol in plant resistance is very important in plant defense system preventing crops from the invasion of insects (Ramaroson et al., 2022).

CONCLUSION
The results of this study shows the existence of variation in the quantity of metabolites among tomato genotypes to provide improved varieties in advancement to insect pest resistance in the field of crop protection.Furthermore, it is noteworthy to recognize susceptible genotypes to H. armigera to prevent over-reliance and snags associated with the use of synthetic insecticides in the management of H. armigera.It was observed from the findings of these studies that biochemical compounds can be used in the management of H. armigera.It shows that secondary metabolites, phenol and flavonoids can be explore in the management of H. armigera affecting tomato yield on farmers' field.In addition, biochemicals are the results of primary and secondary metabolic processes which serve as feeding stimulants or deterrents.Some of the secondary metabolites such as phenol function as mechanisms for chemical defense against H. armigera infestation on tomato fruits.This is a good omen for farmers as insect-resistant crop varieties can enhance the livelihoods of farmers, particularly those regions that are heavily dependent in agriculture.Farmers can have more consistent yields, higher incomes, safe food and greater resilience to insect-pest related risks.This can help uplift the farmers out of poverty and contribute to rural development.

Table 1 . Development parameters of Helicoverpa armigera larvae on tomato genotypes.
Means in a column followed by the same letter(s) are not significantly different at p > 0.05 using Newman-Keuls Multiple Range Test.Values are means ± S. E. of three replicates SC = Susceptible Check