Peroxidase activity in extracts of egg-plant (Solanum melongena E.) fruits inoculated with isolates of some fungi

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1 Bot. Bull. Academia Sinica (1988) 29: Peroxidase activity in extracts of egg-plant (Solanum melongena E.) fruits inoculated with isolates of some fungi M. 0. Basalah, A. H. Bahkali and Sher Mohammad Department of Botany, College of Science, King Saud University P. 0. Box 2455, Riyadh 11451, Saudi Arabia (Received May 28, 1988; Accepted Juls 5, 1988) Abstract. The activity of peroxidase was measured in extracts of egg-plant (Solanum melongena L.) fruits, uninoculated and inoculated with isolates of Rhizoctonia solani, Fusarium solani, Alternaria alternata and Rhizopus oryzae, over a period of two weeks. The increase in the activity of peroxidase was apparent about four days after inoculation and increased progressively with increase in incubation time. The rate of increase was the highest in the extracts of fruits inoculated with A. alternata and followed by those inoculated with F. solani, R. solani and Rhizopus oryzae. The host tissue was unable to resist infection by any of the isolates, tested at any stage of infection. Key words: Egg-plant fruit; Fungi; Inoculation; Peroxidase activity; Solanum melongena. Introduction Peroxidase is widespreaded in plants and (Farkas and Lovrekovich, 1965), in bean hypocotyls infected with Rhizoctonia (Maxwell and Bateman, 1967), in wheat leaves infected with has frequently been found in fungi, bacteria Puccinia graminis tritici (Seevers and Daly, and some invertebrate tissues (Saunders et a/., 1970) and in barley infected with Erysiphe 1964). Increased peroxidase activity in plant graminis f. sp. holzlei (Hislop and Stahmann, tissues has been correlated either with disease 1971). resistance as in potato to Phytophthora infestans (Kammermann, 1959; Umaerus, 1959), in sweet potato to Ceratocystis fimbriata (Weber et al.. Recently, it was observed that a-amylase and invertase activities increased in extracts of egg plant (Solanum melongena L.) fruits infected 1967), and in tobacco to tobacco mosaic virus with Rhizoctonia solani and Fusarium solani (Simon and Ross, 1970), or with infection and (Basalah et al., 1986). The objective of present symptoms expression as in cucumber cultivars study was, therefore, to examine if there was infected with cucumber mosaic virus (Wood some possible change in peroxidase activity in and Barbra, 1971; Barbra and Wood, 19721, in extracts of egg-plant fruits inoculated with tobacco leaves injected with wild fire toxin- isolates of Rhizoctonia solani, Fusarium solani, containing filtrates of Pseudomonas tabacci Alternaria alternata and Rhizopus oryzae.

2 Botanical Bulletin of Academia Sinica, Vol. 29, 1988 Results Fig. I. Fungal growth on egg-plant fruit tissue, uninoculated (1) and inoculated with Rhizoctonia solani (2), Fusarium solani (3), Alternaria alternata (4), Rhizopt*s oryzae (5), after 8 days of infection. Egg plant (Solanurn melongena L.) fruit tissue was inoculated with isolates of Rhizoctonia solani, Fusariurn solani, Alternaria alternata and Rhizopus oryzae and incubated for a period of 2 to 14 days. These fungi continued to grow throughout the incubation period. Figure 1 shows the infection of the tissue after 8 days of inoculation and it is indicated that there was increased growth of fungi on the host tissue and the tissue did not show any kind of resistance to any of the isolates. The peroxidase activity in extracts of host tissue, uninoculated and inoculated with isolates of the fungi tested, was determined over a period of 2 to 14 days, and is indicated in Fig. 2. The peroxidase activity in the extracts Materials and Methods Egg-plant (Solanum melongena L. round var.) fruits were secured fresh from the market and stored at 5-10 C. Isolates of Rhizoctonia solani Kuhn, Fusariurn solani (Martius) Appel and Wollenweber, Alternaria alternata (Fr.) Keissler and Rhizopus oryzae Went and Gerlings were obtained from Centralbereau Voor Schimmel Culture, Baarn, Netherland. Stock cultures of the fungi were maintained on test tube slants of potato dextrose agar (PDA) medium. The cultures used as inoculum, were grown in potato dextrose broth. Methods for sterilization of fruits, inoculations, incubation and sampling were similar to those described by Weber et al. (1967). Samples were taken every two days up to 14 days after inoculations. Extraction of peroxidase and its activity was determined according to Dias and Costa (1983). The activity of peroxidase was expressed in mmoles of guaiacol dehydrogenation product (GDHP) formed per gram fresh weight per minute. II Time (days1 after inoculation Fig. 2. Change in peroxidase activity (mmol GDHP g-i fr. wt. min-i) measured in extracts of eggplant fruits inoculated with -0- Rhizoctonia solani, ---A- Fusarium solani, -0- Alternaria alternata, -0- Rlzizopus oryzae and -x- uninoculated. Values are mean (n=3). Standard deviation (calculated for all values) did not exceedk0.1.

3 Basalah et a1.-peroxidase activity in egg-plant fruits inoculated with fungi 277 of healthy fruits decreased gradually with the increase in the incubation period while the extracts from all types of inoculated fruits showed increased peroxidase activity, beginning about 4 days after inoculations and rising consistently in the following days. Though the pattern of increase was similar in all of the inoculated fruits, showing highest activity 8 to 10 days after inoculations, but the rate of increase differed with the fungal isolates and incubation period after inoculation. The extracts of fruits inoculated with A. alternata showed low activity in the first 4 days which increased abruptly after 6 days, being highest after 8 days of inoculations and declined sharply in the following days. Next highest activity was recorded in the extracts of fruits inoculated with F. solani with a sharp increase in the beginning, becoming the highest after 8 days and decreased in the last 6 days, while in the case of R. solani the activity was lower than the extracts of fruits inoculated with F. solani during first 6 days which increased in the next 2 days and declined slowly in the following days. On the other hand, activity, the lowest of all, was recorded in the extracts of fruits inoculated with Rhizopus oryzae, which was slow in the early days of inoculation but increased sharply 10 days after inoculation and declined in the following four days. Figure 3 depicts the peroxidase activity with respect to control (uninoculated), when control is considered as 100% activity. This showed that the activity increased to its maximum, about 8 folds with respect to control, in extracts of fruits inoculated with F. solani, followed by A. alternata, R. solani and Rhizopus oryzae where it was about 7, 6 and 3.5 folds with respect to control, respectively. Discussion ~esuits of peroxidase activity determined in the present study (Fig. 2) reveal that there was marked increase in peroxidase activity in the extracts from the egg-plant fruit tissue inoculated with isolates of Rhizcotonia solani, Fusariurn solani, Alternaria alternata and Rhizopus oryzae as compared to those from uninoculated tissue. These results are supported by some of the previous studies, which showed that in diseases caused by facultative parasites, Tim* (60~1) oftar inoculotie Fig. 3. Peroxidase activity (% of control) measured in extracts of egg-plant fruit tissue inoculated with -0- Rhizoctonia solani, -A- Fusarium solani, -0- Alternaria aiternata and -0- Rhizopus oryzae. Values are mean (n = 3). Standard deviation (calculated for % values) did not exceed k Values for control, mean (n = 3) are 0.15, 0.14, 0.13, 0.11, 0.07 and 0.06 mmol GDHP g-i fr. wt. min-', 2, 4, 6, 8, 10, 12, and 14 days after inoculation, respectively. Standard deviation (calcu~ated for these valaes) did not exceed e0.1.

4 276 Botanical Bulletin of Academia Sinica, Vol. 29, 1988 peroxidase activity is generally higher in diseased tissues than in healthy tissues (Clare et al., 1966; Stahmann et al., 1966; Uritani and Stahmann, 1961). Increased peroxidase activity in the infected plant tissue has also been reported in many other instances (Fehrmann and Diamond, 1971; Wood and Barbra, 1971; Barbra and Wood, 1972; Weber et al., 1967). Kosuge (1969) reviewed numerous instances where phenolic compound and/or peroxidase were apparently involved in disease resistance. However, workers suggested that increased peroxidase activity probably could not be related directly to resistance or susceptability (Seevers and Daly, 1970; Hislop and Stahmann, 1971; Barbra and Wood, 1972). As regards the relationship between increased peroxidase activity and resistance or susceptability to infection in the present study, it is evident from the results (Fig. 2) that there was no significant increase in peroxidase activity in the early phase of infection but it increased during intermediate stage of infection, when there was maximum growth of the fungus on the host tissue (Fig. 1) and when host tissue has started disintegrating which disintegrated almost, completely in the later stage of infection. This shows that host tissue has not shown any retardation in the fungal growth even at higher peroxidase activity. It was also noticed (Fig. 1) that in the uninoculated (control) tissue, there was no tissue disintegration and peroxidase activity de. creased rather than increasing during the incubation period. It is clear from the observations that the host tissue did not show any kind of resistance and fungi continued to grow without any check and finally the tissue became disintegrated. These results may be correlated with some of the previous investigations which indicated that even the susceptable tissue in sweet potato showed increased peroxidase activity following infection by pathogenic isolates of Ceratocystis fimbriata (Weber et al., 1967). It may be suggested, therefore, that increased peroxidase activity in the present study, is probably not involved in resistance mechanism but it may be associated with degeneration of the host tissue. Moreover, there were no chances of increase in peroxidase activity due to fungal autolysis, because according to the method (Weber et al., 1967), adopted for inoculations and sampling, the host tissue containing fungal material was discarded and only fungus free part of the host tissue was used for extractions. Previously, it has been studied that there was also an incease in invertase and a-amylase activity in the egg-plant fruit tissue due to infection by Rhizoctonia solani and Fusarium solani (Basalah et al., 1986). The interpretation of the relationship beteween increased activities of peroxidase and invertase and/or a- amylase in infected tissue becomes somewhat complicated. However, it may be explained to some extent on the findings of Farkas and Lovrekovich (1965) and Tomiyama and Stahmann (1964) who indicated that a treatment of the host tissue with pectic and some other hydrolytic enzymes result in marked stimulation of some enzymes activities. From the above discussion it may be concluded that there seems to be no correlation between resistance to infection and increased peroxidase activity in the extract from eggplant fruit tissue inoculated with Rhizoctonia solani, Fusarium solani, Alternaria alternata and Rhizopus oryzae as it was established in some other instances (Ferhmann and Diamond, 1967). Literature Cited Barbra, D. J. and K.R. Wood Virus multiplication, and ployphenoloxidase activity in leaves of two cucumber (Cucumis satiuus L.) cultivars inoculated with cucumber mosaic virus. Physiol. Plant Pathol. 2:

5 Basalah et al.-peroxidase activity in egg-plant fruits inoculated with fungi Basalah, M.O., A.A. A. Suleiman, and S. Mohammad Invertase and a-amylase activities in eggplant (Solanurn melogena L.) fruits infected with Rhizoctonia solani Kuhn and Fusarium solani (Martius) Appel and Wollenweber. Phyton 46: Clare, B., D. J. Weber, and M. A. Stahmann Peroxidase and resistance to Ceratocystis in sweetpotato increased by volatile materials. Science 153: Dias, M.A. and M. Costa Effect of low salt concentration on nitrate reductase and peroxidase of sugar beet leaves. J Exp. Bot. 34: Farkas, G.L. and L. Lovrekovich Enzyme level in tobacco leaf tissue affected by wild fire toxin. Phytopathol. 55: Fehrmann, H. and A.E. Diamond Peroxidase activity and Phytophthora resistance in different organs of the potato plant. Phytopathol. 57: Hislop, E. C. and M. A. Stahmann Peroxidase and ethylene production by barley leaves infected with Erysiphe graminis f. sp. hordei. Physiol. Plant Pathol. 1: Kammermann, N The peroxidase test as a tool in the selection of potato varieties resistant to leaf blight. Am. Potato J. 36: Kosuge, T The role of phenolics in host response to infection. Ann. Rev. Phytopathol. 7: Maxwell, D. P. and F.D. Bateman Changes in activity of some oxidases in extract of Rhizoctonia infected bean hypocotyls in relation to lesion maturation. Phytopathol. 57: Saunders, B.G., A.G. Holmes-Sidle, and B. P. Stark Peroxidase. Butterworth, Washington. Seevers, P.M. and J. M. Daly Studies on wheat stem rest resistance controlled at the Sr 6 locus. 11. Peroxidase activities. Phytopathol. 60: Simon, T.J. and A. F. Ross Enhanced peroxidase activity associated with induction of resistant to tobacco mosaic virus in hypersensative tobacco. Phytopathol. 60: Stahmann, M. A., B.G. Clare, and W. Woodbury Increased disease resistance and enzyme activities induced by ethylene and ethylene production by black rot infected sweet potato tissue. Plant Physiol. 41: Tomiyama, K. and M. A. Stahmann Alteration of oxidative enzymes in potato tuber tissue by infection with Phytophthora infestans. Plant Physiol. 39: Umaerus, V The relationship between peroxidase activity in potato leaves and resistance to Phytoplzthora infestans. Am. Potato J. 36: Uritani, I. and M.A. Stahmann Changes in nitrogen metabolism in sweet potato with black rot. Plant Physiol. 36: Weber, D. J., B. Clare, and M. A. Stahmann Enzymatic changes associated with induced and natural resistance of sweet-potato to Ceratocystis fimbriata. Phytopathol..57: Wood, K. R. and D. J. Barbra Virus multiplication and peroxidase activity in leavesa of cucumber (Cucumis sativus L.) cultivars systemically infected with W-Strain of cucumber mosaic virus. Physiol. Plant Pathol. 1: M. 0. Basalah, A. H. Bahkali and Sher Mohammad Department of Botany, College of Science, King Saud University P.O. Box 2455, Riyadh 11451, Saudi Arabia Rhizoctonia solani, Fusarium solani, Alternaria alternata, $@ Phizopus oryzae 2j5F!j339S@1232%@?~@ 9?J,!J5?J%$354~5Z,E2@i~1& 0 ~ ~4kz@~+~@&~Xj&~~~1&@@~#$n 9 ~ p ~ ~ 2 t ~ ~ $U 9 $&+$%$% A. alternata 2$ii+F#$~$$37 St;.&% F- solani, R. RRhizofius oryzae 0 R%:Ejjri32& 2i%i&4E%L!3N ~1~.-rnR2EW@FflQ9&% O