Histochemical Analysis of Chickpea Seeds Infected with Fusarium Oxysporum

Disease wilt is caused by Fusarium oxysporum f. Sp. Wilt disease appears in the field in patches at both seedling and adult stages. Seedling wilt is characterized by sudden drooping followed by yellowing and drying of leaves and the whole seedling and apparently healthy roots with reduced proliferation.

Histochemical methods were used to study the localization of various metabolites and food reserves in seeds. Seeds carrying natural infection of, F. oxysporum and healthy seeds were used to analysis proteins, starch, cellulose, phenols and tannins. Methods employed for each are dealt separately. Total proteins: Total protein was localized by the mercuric bromophenol blue method (Hulse JH, 1991; Jukanti A.K. and Gaur PM, 2012; Murti Krishna,1975). Preparation of stain: 10 gm of mercuric chloride was dissolved in 100ml of 95% ethanol. To this 100 mg bromophenol blue was100ml of 95% ethanol. To this 100 mg bromophenol blue was added. Staining procedure: Fresh hand cut sections were stained in mercuric bromophenol blue for 15 min in 0.5% acetic acid to remove the excess dye. The sections were washed in water for 15 minutes and mounted in glycerine. Protein stains blue. Starch: Starch was localized by iodine (IKI) method of Johansen (1940). Preparation of stain: 2gm potassium iodide was dissolved in 100ml distilled water and then 0.2 gm iodine was added to it. Staining Procedure: Fresh hand cut sections were placed in iodine potassium iodine solution for a few minutes and then mounted in the Same solution and observed. Starch grains appear blue to black in colour. Total phenols: Phenol was localized by nitroso reaction (Reeve, 1951). Preparation of stain: Mixture of following reagent was used for staining. (i) 10% Sodium nitrite (ii) 10% to 20% urea (iii) 10% acetic acid. Staining Procedure: Fresh hand cut sections were placed in stains for 3-4 min. and then added 2N sodium hydroxide solution. The section was mounted in glycerine. Phenols give cherry red colour. Tannins: Tannins were localized by Lugol‘s Iodine Method

Staining Procedure: Fresh hand cut sections were treated in Lug a drop of dilute NH4OH solution was added. The sections were mounted in glycerine. Tannins appear brown in colour. Cellulose: Potassium iodide-iodine-sulphuric acid method as described by Johansen (1940) and Purvis et al. (1964) was followed. Preparation of stain: The I-KI solution was prepared and H2SO4 added later. Staining Procedure: Fresh hand cut sections were stained in iodine potassium iodide solution for 15 min and mounted in the same solution. 65% sulphuric acid was then added through the sides of the cover slips. Cellulose cell wall swells and takes a bright blue colour.

Total Protein Normal seed The localization of proteins was maximum in outer layers of the cotyledon and embryonal axis. The sections of normal seed showed that the cells of cotyledon and embryonal axis contained numerous protein bodies arranged densely and invariable stained blue with bromophenol blue. The cells showed a uniform distribution of stain. The vascular strands of cotyledons took a little weaker stain than other cells. Palisade, hourglass and parenchyma layers of seed coat and hilar region remained unstained showing absence of protein bodies in these cells. Seed tissues rich in protein showed high colour intensity. Infected seeds (F. oxysporum) The cells of cotyledons and embryonal axis showed discrete light and dark coloured patches of blue colour. The light stained cells of cotyledons and embryonal axis were deficient in protein bodies and appeared vacuolated. The cells were infected with mycelium of F. oxysporum. The cotyledonary cells towards seed coat stained little darker than other cells of cotyledons. Different layers of seed coat remained unstained. In general, the intensity of blue colour was than the healthy seeds. A positive result was observed by IKI reaction revealing the presence of starch in the cotyledons. Starch grains showed their maximum localization in the cells of cotyledons 4-12 starch grains per cell were compactly arranged and distributed uniformly. Seed coat (palisade, hourglass and parenchyma layer), hilum region and vascular strands of the cotyledons revealed absence of starch grains. Infected Seed (F. oxysporum) The IKI reaction gave a positive result and showed the presence of starch in cotyledons. At places starch grains appeared small and loosely arranged. The number of starch grains varied from 2-6 per cell, was much lesser than the cells of healthy seeds. The cells which were heavily colonized by the fungus gave a very faint to negative reaction with the stain.

CELLULOSE

Normal Seed The IKI-H2SO4 reaction gave positive response. The swollen cell walls of the cells of cotyledon and embryonal axis showed bright blue colour. Other parts of the seed showed a negative reaction. Infected Seed (F. oxysporum)

TOTAL PHENOLS

Normal Seed The nitroso gave positive stain reaction for phenolics, which was characterized by cherry red colour in the tissues. The cherry red colour was seen in the different layers of seed coat, funiculus and counter palisade region of hilum. The hourglass and parenchyma cell layers stained little weaker than palisade cells, indicating maximum localization of phenols in palisade cells. Cotyledon and embryonal axis showed negative reactions. Infected Seed (F. oxysporum) The tissues of infected seed showed maximum intensity of cherry red colour because of increased amount of total phenols due to infection. The palisade and hourglass cells stained little darker than seed coat parenchyma. The cell of cotyledon and embryonal axis also showed weak staining

TANNINS

Normal Seed Staining with Lugol‘s iodine solution indicated outer few layers of cotyledons. Infected Seed (F. oxysporum) The intensity of brown colour was higher in cell layers of seed coat infected with F. oxysporum as compared to healthy seed, indicating an increase in tannins. The formation of brown colour was more towards hilar region. The cells of cotyledon were weakly stained showing its low level as compared to seed coat.

DISCUSSION

An attempt was made to study the changes in important primary metabolites of Chickpea seed naturally infected with F. oxysporum . Proteins A weak reaction of protein was shown by cells infected with F. oxysporum revealing a decrease in total protein compared to healthy seeds. Maheshwari, Chaturvedi and Yadav (1984) observed stronger protein reaction in Protomyces macrosporus infected cells of Coriandrum sativum. But, less densely stained proteinoplasts in groundnut leaves infected by Cercospora arachidicola was observed by Vijaya Kumar (1990). Unlike present studies Ibraheem et.al. (1987) observed an increase in protein content in soybean seeds when inoculated with cultural filtrates of Alternaria alternata, Ulocladium spp., F. oxysporum and F. solani. Biochemical estimations of soybean seeds infected by F. oxysporum and R. Bataticola conducted by sharma (1992) and Mathur (1992) showed a continuous decline in protein content. Decrease in protein contents in pigeon pea seeds infected by different Aspergilli was reported by Shukla et al. (1988). These observations suggest that the level of protein content may vary with host-parasite interaction. Maheshwari et al. (1984) observed stronger protein reduction in Protomyces macrosporus infected cells of Coriandrum sativum. But, less densely stained proteinoplasts in groundnut leaves infected by Cercospora arachidicola was observed by Vijaya Kumar (1990) may vary with host-parasite interaction. Das and Mitra (1998) found that little leaf infected brinjal leaf tissue contained less amount of soluble protein than in healthy ones. Starch Cell in the infected seeds with F. oxysporum showed weak reaction for starch as compared with healthy ones. The number of starch grains was also reduced. Vijaya Kumar (1990) observed decrease in number and size of starch grains in C. arachidicola infected groundnut leaves. Starch is intracellular and occur as membrane bound granules in cell cytoplasm of cotyledons. However, in diseased seeds, starch was comparatively less, cell arrangement distorted and granules deformed. Similar results were also observed by Santra (1983). He found that the polysaccharides are less in the infected plants parts and their surrounding as compared to healthy plants of potato. This may be attributed to degradation of polysaccharides by extra-cellular fungal enzymes and/or absorption of the metabolites by the fungus (Hahn et al.1980). Maheshwari et al. (1985) observed high intensity of carbohydrates in the hypertrophied inflorescence axis in Brassica juncea caused by Aalbugo candida. Infection by Curvularia lunata, Fusarium moniliforme and Phoma sorghina in sorghum seeds caused significant reduction in size of starch granules as compared with those in healthy seeds. Cellulose The presence of cellulose was demonstrated in cell walls of cotyledons and embryonal axis of both the infected and uninfected seeds. The cells of infected seeds showed slightly less amount of cellulose. Similar results also observed by Sharma (1999) in mungbean.

Parasitic interaction of F. oxysporum resulted increase in phenol content. Similarly tannin was also high in F. oxysporum infected seeds. Many authors have also reported high amount of phenol and tannin in seeds infected with fungus. Both the phenol and tannin are regarded as part of host defense mechanism (Bhatia et al., 1972; Chopra et al., 1974; Farkas and Kiraly, 1962). Kamble and Gangawane (1987) reported that the total phenol contents of ground nut seeds was increased due to infection of Curvularia lunata, Aspergillus flavus, Penicllium funiculosum, P. various Fusarium oxysporum. Khirbat and Jalali

was significantly higher in susceptible cultivar after 10 days of inoculation as compared to resistant cultivar. Bhargava, Sharma and Dashaora (2007) observed that resistant cultivar of resistant cultivar of cowpea possed higher concentration of phenols as compared to susceptible, after infection with Meloidogine incognita. The histochemical studies clearly indicate that natural infection of F. oxysporum caused a decrease in total protein and starch, increase in total phenols and tannins while no change in cellulose.

Total proteins A considerable loss in protein content was observed. The protein bodies in cells of cotyledons were disintegration as the cells appeared vacuolated and gave a weak reaction with mercuric bromo phenol in comparison to the healthy seeds that showed prominent protein bodies giving a dark blue colour. Starch The number of starch grains per cell as compared to the cells of healthy seeds was much lesser. Cellulose The cell walls of seeds infected with F. oxysporum and that of healthy seeds gave a similar bright blue colouration showing no effect of pathogen on cellulose.

The tissues of seed infected with F. oxysporum showed deep cherry red colour in seed coat because of increased amount of total phenols and weak staining in the cells of cotyledons and embryonal axis.

The intensity of brown colour was higher in cells of seed coat infected with F. oxysporum as compared to healthy seeds, indicating an increase in Tannins. Whereas weakly stained cells of cotyledon showed its low level. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or

ACKNOWLEDGEMENT:

Authors are grateful to the Director, Mr. Om Maheshwari, Career point university, Kota (Rajasthan) for providing library and laboratory facilities.

1. Jukanti A. K., Gaur PM, Gowda CLL, and Chibbar RN. Nutritional quality and health benefits of chickpea ( Cicer arietinum L.): a review. Br J Nutr., 2012 Aug; 108 suppl 1: pp. S11-26. 2. Bhargava, S.; Sharma, M.K. and Dashora, P.K. 2007. Histopathological and biochemical changes by root-knot nematode, Meloidogyne incognita on resistant and susceptible cultivars of cowpea .J. Mycol. Pl. Pathol. 37(1): pp. 112-116. 3. Chapman, D.M. 1975. Dichromatism of bromophenol blue with an improvement in the mercuric bromophenol blue technique for protein. Stain Technol., 50: pp. 25-30. 4. Chopra, B.L., Jhooty, J.S. and Bajaj, K.L. 1974. Biochemical differences between two varities of watermelon resistant and susceptible to Alternaria cucumerina. Phytopath. Z., 79: pp. 47-52. 5. Das, A.K. and Mitra D.K. 1998. Some physiological changes associated with little leaf disease of brinjal. J. Mycol. Pl. Pathol. 28(3): pp. 355-357. 6. Bulti Merga & Jema Haji, 2019. Economic importance of chickpea: production, value and world trade, Cogent food & Agriculture,5:1, cngent food 7. Farkas, G.L. and Kiraly, Z. 1962. Role of phenolic compounds in the physiology of plant diseases and disease resistance. Phytopathol. Z., 44: pp. 105-150. 8. Hahn, M.G.; Devil, A.G. and Albersteim, P. 1980. Polysaccharides fragments from the wall of soybean cells elicit phytoalexin (antibiotic) accumulation in soybean Cells. Pl. Physiol. Suppl. 65: pp. 136. 9. Haridass, E.T. and Suresh Kumar, N. 1985. Some techniques in the study of Insect-Host Plant Interactions, 118-137. In: Dynamics of Insect Plant Interactions. 10. Ibraheem, S.A.; Okesha, A.M. and Mhathem, K.T. 1987. Interrelationship between protein and oil content of soyabean seed with some associated fungi. J. Agriculture of Water Research, Plant Production, 6: pp. 53-66. 11. Johansen, D.A. 1940. Plant Microtechnique. McGraw-Hill Book Company. Inc. New York. 12. Kamble, B.R. and Gangawane, L.V. (1987). Biochemical changes in groundnut as influenced by fungi. Seed. Res. 15: pp. 106-108. 13. Khirbat, S.K. and Jalali, B.L. 2003. Influence of Ascochyta Rabiei infection on total phenol and tannin content in chickpea (Cicer arientinum L.) leaves. Legume Res. 26(3): pp. 221-223. 14. Maheshwari, D.K., Chaturvedi, S.N and Yadav, B.S. 1985. Histochemical studies on hypertrophied inflorescence axis of B. juncea due to A. candida. Indian Phytopath., 38 : pp. 263-266. 15. Maheshwari, D.K., Chaturvedi, S.N and Yadav, B.S. 1984. Qualitative changes in starch and protein contents induced by Protomyces macrosporum in coriander stem. Indian phytopath., 37 : pp. 170-173. 16. Mazia, D., Brewer, P.A. and Alfert, M. 1953. The cytochemical staining and measurement of protein with mercuric bromophenol blue. Biol. Bull., 104: pp. 57-67. 17. Reeve, R.M. 1951. Histochemical tests for polyphenols in plant tissues. Stain Technol., 26: pp. 91-96. 18. Roy, F., Boye, J. I., and Simpson, B. K. (2010). Bioactive proteins and peptides in pulse crops: pea, chickpea and lentil. Food Res. Int. 43, pp. 432–442. 19. Santra, S.C 1983. Biochemical localization of starch bodies around infected region in wart infected potato plants. Indian phytopath., 36 : pp. 335-336. 20. Sharma, J. 1992. Mycoflora of soyabean seeds and their pathological effects. Ph.D. Thesis, Univ. Raj. Jaipur. 21. Shukla, H.S., Dube, K.S. and Tripathi, S.c. 1988. Change in protein contents of Arhar (Cajanus cajan) due to fungal association. Legume Research., 11: pp. 85-88. Indian Phytopath., 43: pp. 453- 454. 23. Xu, B., Chang, S.K.C., (2009). Phytochemical profiles and health-promoting effects of cool-season food legumes as influenced by thermal processing. J. Agric. Food Chem. 57(22), pp. 10718-10731.

Department of Botany, School of Basic & Applied Science, Career Point University, Kota Rajasthan, India