Analysis on Biochemical Basis of Root knot nematode (Meloidogyne incognita) Resistance in Tomato (Lycopersicon esculentum Mill.)

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1 Research Journal of Agriculture and Biological Sciences, 4(6): , , INSInet Publication Analysis on Biochemical Basis of Root knot nematode (Meloidogyne incognita) Resistance in Tomato (Lycopersicon esculentum Mill.) 2 3 C.Indu Rani, D. Veeraragavathatham and S. Sanjutha Assistant Professor (Horticulture), Directorate of Extension Education, TNAU, Coimbatore, Pin , Tamil Nadu, India. 2 Dean, Horticultural College and Research Institute, TNAU, Coimbatore, Pin , Tamil Nadu, India. Senior Research Fellow (Horticulture), Directorate of Extension Education, TNAU, Coimbatore, Pin , TamilNadu, India. 3 Abstract: Identification of nematode resistant donors and utilization in development of F hybrids by hybridization with high yielding lines will be a boon to tomato growers for maximizing the production of quality tomatoes without much residue of chemicals through the use of nematicides. With this aim the biochemical constituents were estimated in the best performing hybrid among the resistant hybrids CLN 2026C x SL 20, CLN 2026E x SL 20, LE 82 x SL 20 and CLN 464A x SL 20, its parents and susceptible check COTH- with respect to field resistance to root knot nematode. The study on biochemical basis of resistance to root knot nematode showed that among the parents CLN 2026C and SL 20, SL 20 showed the highest level of phenol, peroxidase and polyphenol oxidase activity. Among the hybrids, CLN 2026C x SL 20 had the highest level of peroxidase and polyphenol oxidase enzyme activity and had a high level of total phenols. The susceptible check (COTH ) registered the lowest value for all the biochemical constituents. Native gel electrophoretic separation of crude enzyme extract from tomato roots showed peroxidase and polyphenol oxidase pattern. Strong induction of peroxidase and polyphenol oxidase enzyme activity was observed in resistant male parent (SL 20) and resistant hybrid (CLN 2026C x SL 20) when compared to the susceptible check (COTH ). Similar isozyme banding pattern was observed in all the four genotypes for both enzymes. The evaluation of genotypes based on biochemical constituents and isozyme studies revealed the superiority for resistance in the parents CLN 2026C and SL 20 and in the hybrid CLN 2026C x SL 20. The identified hybrid with resistance to root knot nematodes combined with high yield and processing qualities could be exploited for commercial cultivation. Key words: total phenol, orthodihydroxy phenol, chlorogenic acid and ascorbic acid INTRODUCTION Tomato (Lycopersicon esculentum Mill.) is one of the most economically important vegetables in Asia. Tomato plants are severely affected by several plant parasitic nematodes, especially by the root knot [6] nematodes, Meloidgyne spp. At present, only limited information is available on acceptable processing varieties with root knot nematode resistance. Resistance to root knot nematode in tomato was found sixty years ago in Lycopersicon peruvianum [] accession PI by Bailey. The resistance was transferred and expressed in F hybrid derived from [0] L. peruvianum x L. esculentum crosses. Further works demonstrated that the gene Mi conferred resistance, which is a dominant gene. The use of resistant varieties is the most economical and easy practice. The biochemical constituents like phenols, orthodihydroxy phenol, IAA oxidase, chlorogenic acid and ascorbic acid are available in tomato roots and these biochemical constituents possess [2] nematode resistant properties. This present study was undertaken to study the biochemical basis of resistance to root knot nematode in order to find out the possible correlation between isozymes of peroxidase and polyphenol oxidase with resistance in the selected hybrid by studying isozyme banding pattern. With this aim, the biochemical constituents were estimated in all the hybrids and their parents. Corresponding Author: C. Indu Rani, Assistant Professor (Horticulture), Directorate of Extension Education, TNAU, Coimbatore, Pin , TamilNadu, India. indunathan@gmail.com 866

2 MATERIALS AND METHODS An attempt was made to develop F hybrids for high yield coupled with processing qualities and resistance to root knot nematode (Meloidogyne incognita). With this aim, to asses the biochemical constituents in tomato roots for root knot nematode resistance eighty hybrids and their 4 parents were evaluated for processing qualities and root knot nematode resistance. Fourteen genotypes of diverse origin collected from distinct geographical regions were used for the study. The parents selected were mentioned in Table I. The parents numbered to 0 were used as female parents (Lines) and to 4 were used as male parents (Testers). Ten lines and four testers were crossed in a line x tester fashion, to incorporate processing characters to nematode resistant lines in one set. In another set the parents numbered from to 0 were used as male parents (Testers) and to 4 were used as female parents (Lines). Four lines and ten testers were crossed in a line x tester fashion to incorporate nematode resistance to processing lines. Eighty hybrids were obtained at the end of the season. Based on the per se performance of 80 hybrids and their parents in the first season, twenty three hybrids, were found to be highly resistant to root knot nematode based on the observations like, root gall index, total phenol (mg/g), Ortho dihydroxy phenol (mg/g), IAA oxidase (mg/00mg), Chlorogenic acid (mg/g) and Ascorbic acid in roots (mg/00g). To confirm the potential yield and resistance to root knot nematode, a total number of four hybrids from the resistant twenty three hybrids were selected based on their per se performance and the results are presented in Table II. The results revealed that all the four hybrids showed consistent resistance to root knot nematode as exhibited by their lowest value of root gall index viz., CLN 2026C x SL 20, CLN 2026E x SL 20, LE 82 x SL 20 and CLN 464A x SL 20 even in artificial inoculation. Among these four hybrids, a comparative study was carried out in selected resistant hybrid, CLN 2026C x SL 20 its parents (male and female) and susceptible check COTH- for peroxidase and polyphenol oxidase activity. The selected hybrid, parents and check, which had different disease reaction to root knot nematode, were studied for isozyme banding pattern for peroxidase and polyphenol oxidase. Plants have a wide range of phytochemicals which impart protective action against nematodes. Among these an aromatic ring bearing a hydroxyl substituent called phenolic substituent has antifungal, antibacterial and antiviral activities. Accumulation of phenolic compounds in host parasite reaction is the general phenomenon of resistance and breakdown of these [] compounds determined the degree of resistance. The biochemical constituents viz., total phenol, orthodihydroxy phenol, Chlorogenic acid and ascorbic acid in root were estimated by following standard procedures. Enzymes: For the assay of enzymes and electrophoretic studies, the best performing hybrid with respect to field resistance to root knot nematode, its parents and susceptible check COTH- were used for the study. The root samples were collected after 30 days after inoculation for assaying the biochemical parameters. IAA Oxidase: IAA oxidase in root was estimated as per the procedure suggested by Sadasivam and [7] Manickam. Polyphenol oxidase and Peroxidase: Polyphenoloxidase and peroxidase activity were assayed [9] following the method the method of Srivastava. Assay of Enzymes by Electrophoresis: The root knot nematode resistant F hybrid CLN 2026C x SL 20 exhibiting highest level of resistance was raised along with their corresponding parents for isozyme analysis. The variety COTH was used as check. Polyphenol Oxidase: After native electrophoresis, the gel was equilibrated for 30 min in 0. M phosphate buffer (ph 7.0) containing 0.% p-phenylenediamine followed by 0 mm catechol in the buffer. The addition of catechol was followed by a gentle shaking which resulted in the appearance of dark brown color [3] discrete brown bands. Peroxidase: To study the expression pattern of different isoforms of peroxidases, activity gel electrophoresis was carried out. For native anionic polyacrylamide gel electrophoresis, resolving gel of 8 %t acrylamide concentration and stacking gel of 4 % acrylamide concentration were prepared. After electrophoresis, the gels were incubated in the solution containing % benzidine in Acetate buffer. Then drops of 30 % H2O 2 were added with constant shaking till the bands appear. After shaking, the gel was washed [8] with distilled water and photographed. RESULTS AND DISCUSSION Among the biochemical parameters the total phenol content in roots indicates the degree of resistance to [5] root knot nematodes. Phenol and orthodihydroxy phenol contents were found to be more in the parent SL 20. All the four hybrids produced more phenol than the parents implying more degree of resistance in hybrids than the parents. The increased levels of orthodihydroxy phenols might have resulted as a means 867

3 Table : Fourteen genotypes of diverse origin collected from distinct geographical regions Parents Type Source. CLN 2026C High Yielding and Processing AVRDC, Taiwan 2. CLN 2026E High Yielding and Processing AVRDC, Taiwan 3. CLN 466J High Yielding and Processing AVRDC, Taiwan 4. CLN 466S High Yielding and Processing AVRDC, Taiwan 5. CLN 464A High Yielding and Processing AVRDC, Taiwan 6. PT 467A Processing AVRDC, Taiwan 7. PT 476A Processing AVRDC, Taiwan 8. CO 3 High Yielding HC & RI, TNAU, Coimbatore. (Induced Mutant of CO ) 9. LE 82 High Yielding and Processing HC & RI, TNAU, Coimbatore. (A recurrent selection from EC.8265) 0. Arka Ahuti Processing IIHR, Bangalore. Hisar N Root knot nematode resistant HAU, Hariyana 2. Hisar N2 Root knot nematode resistant HAU, Hariyana 3. Patriot Root knot nematode resistant PAU, Ludhiana 4. SL 20 Root knot nematode resistant IARI, New Delhi Table 2: Per se performance of selected hybrids and parents in Tomato Genotypes Fruit Yield Root Total Ortho IAA Chlorogenic Ascorbic acid weight per plant gall phenol dihydroxy oxidase acid in roots (g) (kg) index (mg/g) phenol (mg/g) (mg/00mg) (mg/g) (mg/00g) Natural Artificial condition inoculation CLN 2026C x SL CLN 2026E x SL LE x SL --- CLN A x SL --- CLN 2026C CLN 2026E CLN 464A LE SL MEAN SE CD (0.05)

4 Table 3: Enzyme activity in CLN 2026C, SL 20, CLN 2026C x SL 20 and susceptible check (changes in OD/min/g. sample) S.No Genotype Peroxidase Polyphenol oxidase. CLN 2026C SL CLN 2026C x SL Susceptible check (COTH ) Fig. : Isozyme banding pattern of Peroxidase Fig. 2. Isozyme banding pattern of Polyphenol Oxidase of defensive reaction following the root knot nematode infestation since orthodihydroxy phenols are known to be reactive and upon oxidation yield quinones which are still more toxic to invading organisms. This finding [2] is in line with the reports of Farkas and Kiraly. The parent SL 20 showed highest value for IAA oxidase, Chlorogenic acid and ascorbic acid in roots. Among the four hybrids CLN 2026C x SL 20 recorded highest value for IAA oxidase, Chlorogenic acid and ascorbic acid followed by CLN 2026E x SL 20. This could have helped in less accumulation of IAA thereby preventing the formation of giant cells. (Table. III). A comparative study was carried out in selected resistant hybrid, CLN 2026C x SL 20 its parents (male and female) and susceptible check COTH- for peroxidase and polyphenol oxidase activity. Gel electrophorosis study was carried out for peroxidase and polyphenol oxidase. The selected hybrid, parents and check, which had different disease reaction to root knot nematode, were studied for isozyme banding pattern for peroxidase and polyphenol oxidase. Quantification of two enzymes viz., peroxidase and polyphenol oxidase activity clearly showed that the resistant hybrid and its parents had more activity than the susceptible check variety COTH-. Isozyme banding patterns were similar in the resistant hybrid, its parents and susceptible check (Figure ). Studies on the changes in the peroxidase and polyphenol oxidase isozymes clearly indicated that there was enhancement of activities of these enzymes in the CLN 2026C x SL 20. Many studies have been reported on the relations between plant infection by pathogen and the accumulation of phenolic compounds in the plants. Many of these compounds, especially oxidized forms, are toxic to certain pathogen and may take part in the plant defence mechanisms. Peroxidase and polyphenol oxidase are capable of oxidizing [4] phenolic compounds. Native gel electrophoretic separation of crude enzyme extract from tomato roots showed peroxidase and polyphenol oxidase pattern. The induction pattern varied with plant type. Generally strong induction of peroxidase and polyphenol oxidase enzyme activity was observed in resistant male parent (SL 20) and resistant hybrid (CLN 2026C x SL 20) than the susceptible check (COTH ). Similar isozyme banding pattern was observed in all the four genotypes compared for both the enzymes (Figure 2). Based on the investigation, among the twenty three resistant hybrids, four hybrid combinations viz., CLN 2026C x SL 20, CLN 2026E x SL 20, LE 82 x SL 20 and CLN 464A x SL 20 have been adjudged as superior in respect of yield per plant, processing qualities and resistance to root knot nematode (Meloidogyne incognita). However, these four hybrid combinations have to be tried in larger area of land so as to assess their potential yield under optimum cultural practices. 869

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