African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 4, Num. 2, 1996, pp. 223-232

E. K. Gichuru, A. W. Mwang'ombe and V. K. Gupta

Department of Crop Science, University of Nairobi, P.O. Box 30197, Nairobi, Kenya

(Received 10 November, 1994; accepted 26 April, 1995)

Code Number: CS96060
Sizes of Files:
    Text:
    Graphics: Tables (gif) - 57.9K

Coffea arabica L. cultivars were inoculated with Colletotrichum coffeanum and thereafter assayed for peroxidase activity. Peroxidase activity of extracts obtained from non-inoculated hypocotyls, detached and attached berries did not reveal any difference among the resistant and susceptible cultivars. Peroxidase activity was found to increase in all C. arabica cultivars studied after inoculation with C. coffeanum. The highest increases in peroxidase activity were in the inoculated susceptible cultivars. In inoculated hypocotyls, there was no clear difference in peroxidase activity between the medium resistant and resistant cultivars. In inoculated detached and attached berries the medium resistant cultivar K7 was comparable to that in the susceptible cultivars, SL 28 and SL 34, at specific sampling periods. Inoculated hypocotyls and detached berries had the highest range of increase in mean peroxidase activity (56-800%) while attached berries had a range of only 12-76%. This study suggests that high peroxidase activity in cultivars of C. arabica is associated with susceptibility rather than with resistance to C. coffeanum.

Key Words: Colletotrichum coffeanum, hypocotyl, coffee berries

RESUME

Des cultivars de Coffea arabica L. ont ete inocules de Colletotrichum coffeanum et ensuite leur activite au peroxidase a ete determinee. Cette activite, determinee dans des extraits des hypocotyles non-inocules, des cerises de cafeier attachees et detachees, n' etait pas differente pour les cultivars resistants et susceptibles. Apres inoculation de Colletotrichum coffeanum, l' activite au peroxidase augmentait dans tous les cultivars. La plus haute augmentation etait observee dans les cultivars susceptibles. Dans des hypocotyles inocules, il n' y avait pas une difference significative d'activite de peroxidase parmi les cultivars de resistance moyenne et les cultivars resistants. Dans les cerises de cafeier detachees et attachees, le cultivar K7 de moyenne resistance etait comparable aux cultivars susceptibles SL 28 et SL 34 e des periodes specifiques d' echantillonage. L' augmentation moyenne d'activite de peroxidase etait la plus haute (56-800%) dans les hypocotyles inocules et les cerises de cafeier detachees tandis que dans les cerises attachees, l' augmentation etait de 12-76%. Cette analyse montre qu' une activite elevee de peroxidase dans les cultivars de C. arabica est associee avec la susceptibilite plut™t qu'avec la resistance contre C. coffeanum.

Mots Cles: Colletotrichum coffeanum, hypocotyle, cerises de cafeier

INTRODUCTION

Peroxidase as a phenol oxidative enzyme together with polyphenol oxidase are considered to play a role in resistance to fungal diseases. These enzymes are thought to oxidize plant compounds to fungitoxic compounds that may inhibit the spread of the infecting pathogen in the plant tissues (Lovrekovich et al., 1968; Macko et al., 1968). However, increase in peroxidase activity in plant tissues after infection may not be responsible for limiting fungal development and appearance of host cell necrosis (Yamamoto et al., 1978) and its association with resistance could be a consequence of, not a determinant in, the resistance (Seevers et al., 1971).

There are contrasting results as to whether high peroxidase activity is associated with resistance or susceptibility in different crops. In wheat (Seevers and Daly, 1970; Southerton and Deverall, 1990) and oat (Yamamoto et al., 1978), high increases in peroxidase activity after infection was associated with resistance reactions. Jennings et al. (1969) attributed high peroxidase activity in maize with susceptibility to Helminthosporium carbonum while Okiror et al. (1982) found similar increases in peroxidase activity in bean lines that were either resistant or susceptible to anthracnose. Rautela and Payne (1970) found higher initial increase of peroxidase activity in infected sugar beets resistant to Cercospora leaf spot but at later stages higher increases were recorded in susceptible varieties. This study was initiated to test the role, if any, of peroxidase in resistance or susceptibility of Coffea arabica to Colletotrichum coffeanum.

MATERIALS AND METHODS

Source of C. arabica Cultivars. Six cultivars of C. arabica viz. SL 28, SL 34 (susceptible); K7, Catimor (moderately resistant); and Rume Sudan and Ruiru 11 (resistant) were used in this study. The seeds were supplied by the Coffee Research Foundation, Ruiru, Kenya.

Growing of Seedlings. The parchment of the coffee seeds was carefully removed by hand before they were sown in moist sterilised sand in boxes with fitting transluscent lids. The sand was kept moist by watering at least once a week. The boxes were kept at room temperature (20-24 C).

Inoculum preparation and inoculation of hypocotyls. A ten-day old culture of C. coffeanum (of an isolate from a plot which has never received fungicides) growing on malt extract agar (MEA) was washed with sterile distilled water (SDW) and the resultant conidial suspension was standardized to 2x106 conidia ml-1 using a haemocytometer. This was used to inoculate seedling hypocotyls using the method of van der Vossen et al. (1976). Hypocotyls of 6 weeks old seedlings were inoculated twice by spraying with conidial suspension at a 48 hr interval using a hand atomiser. Two days after the second inoculation the seedlings were uncovered and incubated in an illuminated incubator set at 18-20¡C for two weeks. Control seedlings were sprayed with SDW and the rest of the procedure was as in the inoculated seedlings. A similar set of non-inoculated hypocotyls of six cultivars was maintained at same conditions to assess normal peroxidase activity at different seedling ages.

Sampling of hypocotyls was conducted on a weekly basis on healthy seedlings, beginning with the fourth week after sowing and continued up to the tenth week after sowing (fourth week after inoculation).

Source and inoculation of detached berries. Berries for use in these experiments were picked 10 weeks after flowering from trees of the six cultivars, SL 28, SL 34, K7, Catimor, Rume Sudan and Ruiru 11 grown in a coffee germplasm collection plot at the Coffee Research Station, Ruiru. This plot does not receive any pest control measures, and the cultural practices include hand weeding and pruning. The berries were surface sterilised using 5% bleach (sodium hypochlorite) for 5 minutes and thoroughly washed in five changes of SDW. The berries were placed in a moist chamber and each berry was inoculated by carefully placing a drop of the inoculum on the surface while avoiding runoff. Control berries were inoculated with drops of SDW. The moist chambers were incubated at room temperature (20-24 C) for 10 days.

Sampling of detached berries for enzyme extraction began from the day of inoculation and continued up to the tenth day at 2-day intervals.

Inoculation of attached berries. The berries that developed from the flowering initiated by the rainfall in April 1992 were used. Inoculation was done in June 1992 when the berries were 10 weeks old and the weather was cool and humid. Ten branches/tree each with approximately 80-105 berries on 4-6 nodes were selected. Two trees per cultivar were used. Berries were inoculated by the method of van der Vossen et al. (1976). The berries were sprayed with standard inoculum to almost runoff using a hand atomiser, and thereafter covered with transparent polythene bag, and the procedure repeated after 48 hours. The berries were then uncovered after four days from the first inoculation. Control berries were sprayed with SDW. Sampling of the attached berries was done weekly up to three weeks from the time of inoculation.

Studies on the variation of peroxidase activity with the age of the coffee berries was carried out at pinhead (2 weeks after flowering), expanding (6 and 10 weeks after flowering), fully expanded (16 weeks after flowering), hard mature (7 months after flowering), and ripe (8 months after flowering) stages.

Enzyme extraction. Enzymes from control hypocotyls were extracted from the top half of the hypocotyls while those from inoculated seedlings were from the visible lesions. Two grammes of the hypocotyl tissue was crushed in 1 ml of 0.05M tris-hydroxymethyl amino methane-HCl buffer (pH 7.5), using prechilled mortars and pestles, and the crushed material was centrifuged at 18000xg for 30 minutes at 4 C. All the peroxidases were assumed to have been extracted into the supernatant. The extracts were stored at -20 C for 4 weeks until peroxidase determinations were carried out.

Enzymes from control berries were extracted from the pericarp tissues. Pinheads were crushed whole. Samples from the susceptible cultivars SL 34 and SL 28 and the moderately resistant K7 showing large lesions were taken only from the areas with the lesions. In the other cultivars without large lesions but showing scabs, samples were taken from the whole pericarp. In each case, 3g of the tissue was thoroughly crushed in 3 ml of tris-HCl buffer (0.05M, pH 7.5) using prechilled mortars and pestles. The crushed material was centrifuged at 18000 x g for 30 minutes at 4 C. The extracts were stored at -20 C for four weeks until analyses were made.

Determination of peroxidase activity. Peroxidase activity of the extracts was determined by the method of Jennings et al. (1969) by placing 0.5 ml of 1:100 dilution of the extracts into a spectrophotometer cuvette into which 0.5 ml of 1% guaiacol solution and 1.5ml tris-HCl buffer (0.05M, pH 7.5) was added. The reaction was initiated by adding 0.5ml of 1% hydrogen peroxide and optical density readings were taken at 485 nm. A blank consisting of 0.5ml of diluted extract, 0.5ml of 1% guaiacol and 2.0ml tris-HCl buffer was used to set the spectrophotometer at 100% transmittance. Changes in the optical density of the reaction mixture were taken at 15 sec. intervals upto 4 min. after mixing all the ingredients. This procedure was repeated three times for each diluted extract and the mean readings calculated.

A graphical representation was made of mean readings versus time. Jennings et al. (1969) defined peroxidase activity as the change in optical density per minute per ml of extract at 485 nm. The change in optical density was calculated from the straight part of the graph and the total peroxidase activity calculated as follows:-

Change in        x       1             x     1       x   100
optical density         ---                 ---
                     time (min)            0.5ml

In the case of berries where the extraction ratio was 1:1 (buffer for tissue), peroxidase activity of 1 ml of the extract was equal to peroxidase activity of 1g of the tissue, but in the case of hypocotyls the extraction ratio was 2:1 (tissue to buffer) meaning that the activity per ml of the extract had to be multiplied by half to get the activity per gramme of the tissue, i.e., change in optical density per gramme of fresh weight of tissue per minute.

RESULTS

Inoculated hypocotyls were scored for CBD during the third week after inoculation using a 1-12 infection grade system. The mean infection grade scores of the cultivars are presented in Table 1. Also presented in Table 1 are the percent infection on attached berries in the third week after inoculation and on detached berries on the tenth day after inoculation. Before the peroxidase activity data was analysed, it was transformed (Ãx+1) and the LSD calculated thereafter.

Peroxidase activity in non-inoculated seedlings. The extracts from different cultivars showed significant differences in their peroxidase activities (P<0.01), but the cultivars could not be grouped into resistant and susceptible categories. The cultivars changed ranks in their peroxidase activities at different times of sampling (Table 2), as indicated by the highly significant cultivar and time interaction (P<0.01). On the eighth week after sowing, the resistant cultivars Rume Sudan and Ruiru 11 had higher peroxidase activities in their extracts when compared to the other cultivars and these differences were significant (P<0.01). The differences between the times of sampling were significant (P<0.01) and in some cases the younger seedlings had higher peroxidase activities than the older ones.

Peroxidase activity in inoculated seedlings. There was an increase in peroxidase activity in the extracts of all the cultivars one week after inoculation (Table 2). The extracts from the susceptible cultivars, SL 28 and SL 34, had a continuous increase in peroxidase activity throughout the study as the symptom development progressed. Extracts from cv Catimor showed an increase in the peroxidase activity from the first week after inoculation but it decreased in the fourth week after inoculation (Table 2). Extracts from cvs K7, Rume Sudan and Ruiru 11 showed a decrease in peroxidase activity in the second week after inoculation when compared to the first week. Later extracts from cv Rume Sudan showed an increase in the peroxidase activity. The cultivar and time interaction was significant (P<0.01), and the cultivars changed ranks with time. The susceptible cultivars, SL 28 and SL 34, were significantly different from all the others in the second week after inoculation (P<0.01), and the mean peroxidase activities of the various cultivars in the study were significantly different (P<0.01). The susceptible cultivars, SL 28 and SL 34, had the highest increase in mean peroxidase activity of 932% and 699%, respectively. There were significant differences in peroxidase activity among the times of sampling (P<0.01) and the highest mean activity was recorded in the third week after inoculation.

Peroxidase activity in non-inoculated detached berries. The peroxidase activity of the extracts from non-inoculated detached berries varied both with the cultivars and the time of recording. There was an initial decrease in the peroxidase activities of the extracts from detached berries after incubation. An increase in the activities was observed starting on the sixth and eighth days after incubation (Table 3). The cultivars changed ranks and the time and cultivar interaction was significant (P=0.01). Cultivar Catimor had the lowest activity in most of its extracts except on the eighth day. There were significant differences among the cultivar means and times of sampling (P<0.01). The extracts from the freshly detached berries had the highest peroxidase activities.

Table 1

Table 2

Peroxidase activity in non-inoculated attached berries. The peroxidase activity of non-inoculated 10-12 weeks old coffee berries served as control for the inoculated attached berries. The cultivar and time of recording interaction was not significant (P<0.05). The cultivars kept rather constant ranks in their peroxidase activities at different weeks of sampling (Table 4). There were, however, significant differences among the cultivar means (P<0.01), but the order of the peroxidase activity in cultivars did not concur with the reaction to CBD.

Peroxidase activity in inoculated attached berries. One week after inoculation, there was an increase in peroxidase activity in the extracts from all the cultivars used except cv K7 (Table 4). The extracts from the susceptible cultivars, SL 28 and SL 34, had the highest increase upto the third week after inoculation when there was a slight decrease and the berries dried up. Peroxidase activity in the extracts from cv K7 started to show an increase in the second week. The cultivar and time interaction was significant (P<0.01) and, in the third week after inoculation, the peroxidase activity was comparably high for cvs K7, SL 28 and SL 34. In the third week after inoculation, the peroxidase activity of the cultivars could be divided into two groups viz. group 1: cvs SL 28, SL 34 and K7 and group 2: cvs Catimor, Rume Sudan and Ruiru 11. There were significant differences among the cultivar means and the times of sampling (P<0.01). Group 1 cultivars had the highest mean peroxidase activities and they were the same ones with active lesions.

Table 3

Table 4

Table 5

Non-inoculated cultivars. The peroxidase activities of non-inoculated C. arabica cultivars did not coincide with susceptibility and resistance to CBD. However, in attached berries six weeks after flowering, the highly resistant cultivars, Rume Sudan and Ruiru 11, contained significantly more peroxidase activity than the other cultivars. The mean peroxidase activities over the time sampled could not differentiate C. arabica cultivars on the basis of their susceptibility or resistance to CBD. For example, in non-inoculated hypocotyls, the resistant cv Ruiru 11 ranked first but the resistant cv Rume Sudan ranked fifth. These results, where no obvious differences were observed between healthy resistant and susceptible cultivars, conform to results obtained by Seevers and Daly (1970) in wheat, Okiror et al. (1982) in beans, and Yamamoto et al. (1978) in oats. However, Fehrmann and Dimond (1967) found that in potato tissues, resistance to Phytophthora infestans was positively correlated to peroxidase activity. Kimenju (1991) also observed that at certain developmental stages of tomato cultivars, peroxidase activity was positively correlated to susceptibility to Clavibacter michiganense subsp. michiganense.

The slight increase in peroxidase activity in non-inoculated detached berries could be due to the response of the berries to injury, rotting and/or secondary infections that may have taken place through the wounds caused by detaching the berries.

Inoculated cultivars. Inoculation of the C. arabica cultivars with C. coffeanum increased the mean peroxidase activity in all the cultivars, with the highest increases being observed in the susceptible cultivars. However, in inoculated attached berries, the medium resistant cv K7 had the highest mean peroxidase activity. Inoculated attached berries of cv K7 had active lesions of CBD two weeks after inoculation and these active lesions were similar to those observed on inoculated berries of the susceptible cvs SL 28 and SL 34. Cultivar K7 had the highest peroxidase activity before inoculation and its percent increase of 11.9% was the smallest after inoculation. In all the cultivars, the least increase in peroxidase activity was in attached berries compared to detached berries and hypocotyls inoculated with the CBD fungus.

The results of this study confirm those of other workers who found greater increase in peroxidase activity in inoculated susceptible maize plants than in resistant ones (Jennings et al. 1969; Kimenju, 1991). Rautela and Payne (1970) reported that in sugar beet varieties infection by Cercospora leaf spot pathogen caused initially higher increases in peroxidase activity in resistant plants but later higher increases were observed in susceptible plants. However, in crops such as wheat increase in peroxidase activity was associated with resistant reactions (Seevers and Daly,1970; Southerton and Deverall, 1990). In oats (Yamamoto et al., 1978) and beans (Okiror et al., 1982), peroxidase activity after inoculation did not show any relationship to either resistance or susceptibility to the pathogens studied.

Rautela and Payne (1970) suggested that failure of peroxidase to arrest infection in susceptible plants was probably because the increase occurred too late or in insufficient amounts. These suggestions do not seem to apply directly to the C. arabica - C. coffeanum association. Therefore, peroxidase activity may not be responsible for resistance reactions seen in this study. In coffee, formation of cork barriers are reported to be essential (Masaba and van der Vossen, 1982) and give resistant coffee cultivars an advantage over susceptible ones throughout the infection process starting from pre-penetration stages. The higher peroxidase activity in susceptible plants could be due to the presence of more infected cells in the susceptible than in the resistant plants.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the generosity of the Coffee Research Station in supplying the plant materials, pathogen and allowing the use of their seedling inoculation facilities for this study.

REFERENCES

Fehrmann, H. and Dimond A.E. 1967. Peroxidase activity and Phytophthora resistance in different organs of the potato plant. Phytopathology 57:69-72.

Firman, I.D. 1964. Screening of coffee for resistance to coffee berry disease. East Africa Agricultural and Forestry Journal 29:192-194.

Jennings, P.H., Brannaman B.L. and Zoheille F.P., Jr. 1969. Peroxidase and polyphenoloxidase activity associated with Helminthosporium leaf spot of maize. Phytopathology 59:963-967.

Kimenju, J.W. 1991. Identification of Resistance to Clavibacter michiganense subs. michiganense in Tomato Using Isozyme Patterns, Restriction of Vascular Discolouration and Bacterial Cell Count. M.Sc. Thesis, University of Nairobi.

Lampard, J.F. and Carter G.A. 1973. Chemical investigations on resistance to coffee berry disease in Coffea arabica. An antifungal compound in coffee cuticular wax. Annals of Applied Biology 73:31-37.

Lovrekovich L., Lovrekovich H. and Stahman M.A. 1968. The importance of peroxidase in the wildfire disease. Phytopathology 58: 193-198.

Macko, V., Woodbury, W. and Stahman, M.A. 1968. The importance of peroxidase in the germination and growth of mycelium of Puccinia graminis f.sp. tritici. Phytopathology 58:1250-1254.

Masaba, D.M. and van der Vossen, H.A.M. 1982. Evidence of cork barrier formation as a resistance mechanism to berry disease (Colletotrichum coffeanum) in arabica coffee. Netherlands Journal of Plant Pathology 88: 19-32.

Nutman, F.J. and Roberts, F.M. 1960. Investigations on a disease of Coffea arabica caused by a form of Colletotrichum coffeanum. 1. Some factors affecting infection by the pathogen. Transactions of British Mycological Society 43:489-505.

Okiror, M.A., Gupta, V.K. and van Breukelen E.W.M. 1982. Genetic and biochemical differences between susceptible and resistant lines of common beans (Phaseolus vulgaris L.) to anthracnose (Colletotrichum lindemuthianum). Theoretical and Applied Genetics 62:355-359.

Rautela, G.S. and Payne, M.G. 1970. The relation of peroxidase and orthodiphenol oxidase to resistance of sugarbeet to Cercospora leaf spot. Phytopathology 60:238-245.

Seevers, P.M. and Daly, J.M. 1970. Studies on the stem rust resistance controlled at the Srb locus. II Peroxidase activities. Phytopathology 60: 1642-1647.

Seevers, F.M., Daly, J.M. and Catedral, F.F. 1971. The role of peroxidase isozymes in resistance to wheat stem rust. Plant Physiology 48: 353-360.

Sempio, C., Della Torre, G., Ferranti, F., Barberini, B. and Dravoli, R. 1975. Defence in bean resistance to rust. Phytopathology 283: 244-266.

Southern, S.G. and Deverall, B.J. 1990. Changes in phenylalanine ammonia-lyase and peroxidase activities in wheat cultivars expressing resistance to the leaf rust fungus (Puccinia recondita f.sp. tritici). Plant Pathology 39:223-230.

Van der Vossen, H.A.M., Cook, R.T.A. and Murakaru, G.N.W. 1976. Breeding for resistance to coffee berry disease caused by Collectotrichum coffeanum Noack (sensu Hindorf) in Coffea arabica L. 1. Methods of preselection for resistance. Euphytica 25:733-756.

Veech, J.A. 1969. Localization of peroxidase in infected tobaccos susceptible and resistant to Black Shank. Phytopathology 59:566-571.

Yamamoto, H., Hokin, H. and Tani, T. 1978. Peroxidase and polyphenoloxidase in relation to the crown rust resistance of oat leaves. Phytopathology Z. 91:193-202.

Copyright 1996 The African Crop Science Society

The following images related to this document are available:

Line drawing images