L.J.C. Autrey and S. Saumtally Sugar Industry Research Institute, Reduit, Mauritius

Transcription

1 Pathology CULTURAL CHARACTERSTCS OF MYCOVELLOSELLA KOEPKE (KRUGER) DEGHTON ON VAROUS MEDA AND THER RELATONSHP WTH VARETAL REACTON ON CANE LEAF AGAR L.J.C. Autrey and S. Saumtally Sugar ndustry Research nstitute, Reduit, Mauritius ABSTRACT Six methods were investigated to is,olate the fungus Mycovellosiella koepkei the causal agent of yellow spot disease. The most successful one consisted in transferring spores from field collected material or from leaves left to sporulate in a humid chamber to agar plates, using a sterile fine needle, and then streaking them by means of a platinum wire loop. Little contamination was occasioned by this method in comparison with the other ones used. Among five media, Czapek yeast extract was the best for isolation but cane leaf agar (CLA) was more appropriate for maintenance of cultures as growth was slightly faster and sporulation much better on CLA than on the other media. Colony morphology was the same on all media but the length of conidia varied, being greatest on CLA and least on carrot decoction agar. n pure culture the conidia were shorter than on infected leaves. The fungus grew faster and sporulated better when CLA was prepared from susceptible sugarcane varieties rather than from resistant ones. n vitro culture on CLA could provide an interesting method of screening for varietal resistance to yellow spot disease. NTRODUCTON Yellow spot is considered to be the most important leaf spot disease of sugarcane in the tropics having a marked depressing effect on sucrose content and cane yield (Autrey3, Ricaud9, Ricaud and Autreyl0 and Ricaud"). n Mauritius, the disease was first observed in 1964 (Antoine2), but it assumed greater importance in 1977 on variety Saipan 17 which was resistant for several years but was found to be highly susceptible (Anon.') probably due to variation in the pathogen. The causal organism is the fungus Mycovellosiella koepkei (Kruger) Deighton, previol~sly known as Cercospora koepkei Kruger. The fungus has recently been described by Deighton4 and its perfect form in not known. Keywords: Yellow spot disease, in vitro screening 336

2 L.J.C. AUTREY AND S. SAUMTALLY 337 The fungus is difficult to isolate (Hughes and Ocfemia6) but aseptic cultures have been successfully attempted by Matsumoto and Yainamoto7, Praksain and Satyanarayana8 and more recently by Ricaud (unpublished) and Autrey (unpublislied). Praksam and Satyanarayana8 found the conidia were longer in culture on cane leaf extracts than those found naturally on infected leaves. The present paper describes methods of isolation and the cultural characteristics of the fungus on five media. The use of cane leaf agar as a possible method for assessing the reaction of sugarcane clones is discussed. MATERALS AND METHODS Methodsof isolation and media used solations were made from the lower epidermis of infected leaves from five sugarcane varieties. The following methods were used: (a) Leaf sections placed 011 media Leaf sections (0.5 x 0.5 cm) were surface sterilised in 70% alcohol for 30 sec. followed by 2 min. in 1:1000 mercuric chloride and again in 70% alcohol for 30 sec. After drying between sterile blotting papers the leaf sections were placed on the media. (b) Leaf strip stuck to the lid of a petri dish (Matsurnoto and Yaniarnoto7) An infected leaf strip (6 x 2 cm) collected from the field was stuck with cellotape to the lid of a Petri dish containing a specific growth medium. The strip was removed after three days to prevent heavy contamination. (c) Dlrect plating from field collected rnaterial With a fine needle, lesions were~scraped lightly and the spores inoculated on agar slants. (d) solation fro171 leaf after sporulation in a rnoist chamber nfected leaves were placed in a moist chamber for 24 h and the fungus isolated as in (c). (e) Streaking rnethod Field collected material or leaves left to sporulate in a moist chamber were used and the organism isolated as described in (c) except that inoculations were made in Petri dishes and the inoculated spores were then spread all ovei the medium by means of a platinum wire loop. (f) solation by a centrifugation-dilution method A suspension was made in sterile distilled water and centrifuged at 5000 G in a MSE Superminor bench centrifuge. The pellet was washed, resuspended and centrifuged again. The pellet was finally resuspended in a small amount of a sterile water which was then serially diluted up to 1: 1000; 0.1 ml of each dilutioli \\,as then plated on five growth media. The following media were tested: (a) Potato dextrose agar (PDA), prepared from 200 g of scrubbed potato tubers which had been sliced, steamed for 1 h, filtered through muslin and the filtrate sup-

3 338 PATHOLOGY plemented with 20 g of glucose and 12 g agar. The broth was made up to 1 L with distilled water. The ph of this medium was 6.1. (b) Czapek yeast extract (CYE) of ph 7.7 and consisting of the following ingredients per litre: sodium nitrate 2 g, di-potassium hydrogen phosphate 1 g, magnesium sulphate 7H g, glucose 20 g, yeast extract 1 g and agar 12 g. (c) Cane leaf agar (CLA), ph 5.4 and carrot decoction agar (CDA), ph 5.9. These media were prepared from cane and carrot leaves respectively. The leaves were cut into small pieces, blended, steamed for 1 h, filtered through muslin and the filtrate incorporated with 20 g glucose and 12 g agar. Distilled water was added to make up the volume to 1 L. Sterilisation was at 105 OC for 15 min. (d) Malt extract agar (MEA), ph 5.4 The medium was purchased from Oxoid Ltd, U.K. Frequency distribution of spore length of M. koepkei on various media and from infected leaves The length of conidia of M. koepkei from field collected leaves was compared to those obtained from cultures on the fungus on CLA, CDA and CYE. Correlation between growth and sporulation M. koepkei and varietal resistance Cane leaf agar was prepared from leaves of variety S 17 for routine cultivation of the fungus. n order to study the cultural characteristics of the organism, the medium was also prepared from four other varieties, M 124/59, M 356/53, M 1227/62 and B 3337 which have differential reaction to M, koepkei in the field. Preparation of CLA was carried out using separately young (second) and old (seventh) leaves from stalks of each variety. After pouring in Petri dishes, the media were inoculated with a sterile needle carrying spores of the fungus and incubation was at room temperature (25-30 "C) for 15 days. Colonies were measured and their surface was scraped to give a spore suspension in 1 ml distilled water, 0.01 ml of which was placed in a cavity slide and the spores counted under the low power objective of a Leitz Ortholux 1 microscope. Four replicate samples were measured from each suspension. The experiment was repeated twice with old leaves. RESULTS Methods of isolation and culture media used Methods (a), (b), (c), (d), (e) and (f) brought about heavy contamination by fast growing fungi which tended to swamp the slow growing M. koepkei. The fungus could not be isolated at all using methods (a) and (f) but it was isolated with difficulty by the four other methods. The contaminants were checked to a great extent when using methods (b), (c) and (d) by circumscribing a colony as soon as it appeared and removing it from the plate. Method (e) was however the most successful and contamination was due mostly to yeasts which formed small distinct colonies and did not interfere with isolation of M. koepkei to a great extent. CYE was the best medium for isolation as fewer contaminants grew on it, but CLA was more appropriate for the maintenance of the fungus. Growth on that medium was slightly faster and sporulation much better than on other media

4 L J.C. AUTREY AND S. SAUMTALLY 339 (Figure 1). Subsequent transfers, even on CLA, brought a gradual deterioration in the structure of the mycelium and sporulation became sparse. 6 FGURE 1. Colonies of Mycovellosiella koepkei on Czapek yeast extract (top) and cane leaf agar showing marked sporulation on the latter medium.

5 Colony morphology was similar on all media. After 15 days the colony was about 7 mm in diameter, with a flat elevation but a slightly raised centre. The surface was silvery grey and sporulation was more abundant at the centre. Frequency distribution of spore length in M. koepkei Conidia obtained from sporulating colonies on CLA, CDA and CYE were shorter than those from infected leaves of sugarcane variety S 17. Their size varied on the three media as well, being longest on CLA and shortest on CDA as illustrated Number 35 - sugar cane leaves (var. S 17) extract agar FGURE 2. Frequency distribution of conidial length in Mycovellosiella koepkei from infected sugar cane leaves and from cultures on three synthetic media.

6 L.J.C. AUTREY AND S. SAUMTALLY 341 in Figure 2 from data obtained from 100 spores. No measurement of conidia was attempted from colonies growing on PDA and MEA owing to poor sporulation of the fungus. Correlation between growth and sporulation of M. koepkei and vbrieta~ resistance Data on colony diameter and spore counts taken after 15 days on CLA media prepared from five sugarcane varieties indicated that for both parameters highest values were obtained for M 1227/62, S 17 and B 3337, susceptible to the disease and least for M 124/59 known to be resistant 'while with the slightly susceptible clone M 356/53, they were of intermediate value. Typical data obtained in two tests are given in Table. There was a slight trend for better growth and sporulation on CLA from older rather than young leaves. TABLE. Growth and sporulation of Mycovellosiella koepkei after 15 days on cane leaf agar from young and old leaves of five sugar cane varieties Colony diameter No. of conidialcolony (X1000) Variety Reaction Young leaves Old leaves Young leaves Old leaves Trial 1 Trial 2 Trial 1 Trial 2 M Resistant M Slightly susceptible M Susceptible B 3337 Highly susceptible S 17 Highly susceptible DSCUSSON Pricking yellow spot lesions with a very fine needle was found to be the simplest and most effective way to isolate M koepkei solation was better carried out in 1 Petri dishes than on slants since the inoculum could be dispersed over a wider surface i with a wire loop. Consequently this method induced the development of widely separated single colonies. Contaminants such as yeasts and saprophytic fungi could then be easily detected after h and removed by circumscribing the colonies with a sterile needle. The incorporation of antibiotic in the isolation medium used by Autrey (unpublished), Praksam and Satyanarayana8, Ricaud (unpublished) was found to be unnecessary since bacterial contamination was negligible. CYE was the most appropriate medium for isolation probably because its high ph was unfavourable to the growth of many fungal contaminants which prefer acidic conditions (Harringan and McCances). However CLA, as already reported by

7 PATHOLOGY medium for maintaining M. koepkei, despite its low ph, probably because the nutritional requirements of the fungus were satisfied by this complex medium. Sterilisation of CLA and CDA had to be limited to 105 "C for 15 min., since it had been found in preliminary investigations that at 121 "C for 15 min. growth was much affected. t is believed that this was due to the destruction of heat labile substances in the leaf extracts. Prior to this project, it was difficult to isolate and maintain M. koepkei in pure culture. The use of the streaking method for plating spores on CYE and the maintenance of the colonies thus obtained on CLA, have now been adopted as standard procedures since they can be reproduced to order. n sharp contrast to the observations of Praksam and Satyanarayana9, conidia of M. koepkei growing on artificial media were found to be shorter than those from infected leaves. This could be attributed to genetic differences in the M. koepkei isolates involved. The variations observed in spore length on various media, with highest values on CLA, could be due to nutritional factors. Praksam and Satyanarayanas also found that conidia were longer on CLA than on oatmeal agar. A positive correlation between growth and sporulation of M. koepkei and varietal resistance was apparent on CLA. The fungus was found with a larger colony diameter and heavier sporulation on CLA prepared from susceptible varieties than from resistant ones. No such behaviour has been reported for yellow spot disease or for any other fungal disease of sugarcane. Many factors are known to be involved in the reaction of plants to disease as reviewed by Tarr12 and Woods and Jellis3. The correlation obtained in vitro possibly indicates the presence of a thermolabile growth factor favourable to the fungus in susceptible clones; the more so as sterilisation at 121 "C for 15 min. of CLA from S 17 was found to be unfavourable to growth and sporulation. There appears to be some correlation between the finding of better growth and sporulation of the fungus on CLA from old leaves rather than young ones and field observations which indicate that the pathogen does not infect the very first leaves even under conditions favourable to infection in highly susceptible varieties such as B 3337 and S 17 (Ricaud9 and Ricaud"). Further work must be carried out with a larger number of varieties of known reaction to the disease to determine whether growth characteristics on leaf extracts could be correlated with varietal reaction; if so, this could lead to a new approach for screening for resistance to the yellow spot pathogen. n vitro culture on CLA would be rapid and would be quite useful for screening a large number of varieties very early in a selection program. ACKNOWLEDGEMENTS. The authors are indebted to Drs. C. Ricaud and R. Julien, Director and Assistant Director respectively of the Mauritius Sugar ndustry Research nstitute, for their help and suggestions in the preparation of the manuscript as well as permission for its publication.

8 L.J.C. AUTREY AND S. SAUMTALLY 343 REFERENCES 1. Anon. (1977). Rep. Maurit. Sug. nd. Res. nst. 24: Antoine, R. (1965). Cane diseases: 6. Yellow spot. Rep. Maurit. Sug. nd. Res. nst. 12: Autrey, L.J.C., Ricaud, C., Sullivan, S, and Dhayan, S. (1983). Control of yellow spot disease of sugar cane by aerial applicat~on of fungicide. Sugar Y Azucar 78: Deighton, F.C. (1979). Studies on Cercospora and allied genera. V. New specles and redispositions. Commw. Mycol. nst. Mycol. Pap. 144; 56 p. 5. Harringan, W.F. and McCance, M.E. (1966). Moulds and yeasts. n Laboratory Methods in Miclobiology. Academic Press, London and New York 1966, p Hughes, C.G. and Ocfemia, (3.0.(1961). Yellow spot disease. Sugar Cane Diseases of the World. Vol. 1. Elsevier Pub. Co., Amsterdam, p Matsumoto, T. and Yamarnoto, W. (1934). Three important diseases of sugar cane in Taiwan (Formosa). J. Soc. T op. Agric. 6: Plaksam, P. and Satyanarayana, V. (1969). Studies on yellow spot disease of sugal cane. Proc. SSCT 13: Ricaud, C. (1974). Factols affecting yellow spot development, its control and effect on cane and sugar yields. Proc. SSCT 15: Ricaud, C. and Autrey, L.J.C. (1986). Yellow spot disease. Sugar Cane Diseases. Elsevier Pub. Co. Amsterdam (in press). 11. Ricaud, C., Autrey, L.J.C. and Sullivan, S. (1980). Losses from the recurrence of yellow spot eplphytotics in Mauritius. Suga~ Y Azucar 75 (7): Tarr, S.A.J. (1972). The principles of plant pathology. Macrnillan Press Ltd. London and Basingstoke. 632 p. 13. Wood, R.K.S. and Jellis, G.J. (1984). Plant diseases. nfection, damage and loss. Blackwell Scientific Publications. Oxford. 327 p.