E. N. AYESU-OFFEI and C. ANTWI-BOASIAKO Department of Biological Sciences, Uni ersity of Science and Technology, Kumasi, Ghana

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1 Annals of Botany 78: , 1996 Production of Microconidia by Cercospora henningsii Allesch, Cause of Brown Leaf Spot of Cassava (Manihot esculenta Crantz) and Tree Cassava (Manihot glaziovii Muell.-Arg.) E. N. AYESU-OFFEI and C. ANTWI-BOASIAKO Dertment of Biological Sciences, Uni ersity of Science and Technology, Kumasi, Ghana Received: 28 June 1995 Accepted: 31 May 1996 Sporulation of Cercospora henningsii Allesch has been examined under various relative humidities, and in the presence of free water in lesions on leaves of cassava and tree cassava. Mature conidia of the fungus on both cassava and tree cassava do not germinate in lesions but accumulate, and under the optimum conditions of C and in the presence of free water, they bud and fragment into numerous microconidia. Microconidia are cylindrical, mostly onecelled, and measure µm. Production of microconidia significantly decreases as relative humidity decreases. Microconidia readily germinate by means of a germ tube at 100% relative humidity on both surfaces of host leaf and on glass slides. Some germ tubes form appressoria and symptoms appear on cassava leaves inoculated with microconidia. The results are discussed in relation to possible modes of dispersal of the spores and control of the disease Annals of Botany Comny Key words: Free water, sporulation, budding, microconidia. INTRODUCTION Cassava (Manihot esculenta Crantz) is cultivated in many tropical areas of the world, notably in tropical Africa, Latin America and Asia. An estimated million people in the areas mentioned above, rely on cassava as a major source of carbohydrate (Bellotti and Van Schoonhoven, 1978). Both the tubers and leaves are widely consumed. It has been reported that brown leaf spot of cassava is found wherever the crop is grown (Jameson, 1970; Lozano and Booth, 1974). It is the most important fungal disease of cassava and, in Ghana, it seems almost all cassava plants are affected by the disease. Severe incidences of the disease have been noted, rticularly under wet conditions, in a number of African countries. The disease can cause great loss in leaf and tuber yield (Terry and Oyekan, 1976). The causal fungus has been identified by a number of workers including Leather in Ghana (Leather, 1959) as Cercospora henningsii Allesch, an obligate rasite in the field. Leather (1959) also reported the occurrence of the disease on tree cassava (Manihot glazio ii Muell.-Arg.) in Ghana. The fully formed lesions, on both cassava and tree cassava, are large, necrotic spots, circular (5 12 mm diameter) or irregular in shape and dark brown on both surfaces of the leaf, sometimes with a distinct dark border on the abaxial surface. The conidia of the fungus are described in the literature as filiform, 3 10 septate and µm in size. Observations by the senior author on sporulation of the fungus on naturally infected leaves of cassava and tree cassava, revealed that in addition to conidia of the fungus described in the literature, there was always another type of $ spore present in sporulating lesions. Since no published work is available concerning these spores, investigations were initiated to provide some information on their origin and possible role in the infection process. MATERIALS AND METHODS The varieties of cassava (Ankra) and tree cassava used in the experiments were planted on an experimental plot at the Dertment of Biological Sciences, University of Science and Technology, Kumasi, Ghana. They were naturally infected and were the source of lesions for the investigations. For each experiment, except where otherwise stated, lesions on infected leaves of both cassava and tree cassava were thoroughly washed with water using a soft wad of cotton wool. This treatment removed spores from the surfaces of the lesions. The lesions were cut out from the leaves and immersed in a solution of equal rts of 95% alcohol and 0 001% HgCl solution for 2 min in order to sterilize their surfaces. Next they were thoroughly washed in two changes of sterile distilled water. The wet lesions were then placed in sterile Petri dishes lined with wet filter pers and incubated in the dark at room temperature (25 32 C) to sporulate. Studies on leaf-borne spores To study the types of spores formed during sporulation, the wet sterilized lesions were incubated for 48 h. Some wet unsterilized lesions were similarly incubated. At the end of the incubation period, clean slides were brought into contact with sporulating lesions so that some spores (spore print) adhered to them. The spores were stained with cotton blue 1996 Annals of Botany Comny

2 654 Ayesu-Offei and Antwi-Boasiako Production of Microconidia by Cercospora henningsii Allesch in lactophenol. Camera lucida drawings were made of the spore types observed. Lengths and widths of at least 200 spores of each kind were measured. A notable observation in the present work on the conidia of the fungus already described in the literature, was their ability to bud and also to fragment into smaller portions. Camera lucida drawings were therefore made to illustrate these processes. Effect of incubation period on frequency of types of spores formed in lesions Five wet surface-sterilized sporulating lesions (from cassava) were randomly taken from Petri dishes in an incubator at 24 h intervals beginning from 24 h after incubation. Each group of five lesions was shaken in a test tube containing 1 ml distilled water to make a spore suspension. 10 µl of spore suspension was placed on a slide. After the water had evaporated, 20 µl of cotton blue in lactophenol was dropped onto the spore print. A cover slip (16 mm diameter) was placed on the stained spores and the number of each type of spore in ten microscope fields ( 100) were counted. The experiment was repeated four times. In each experiment, unsterilized but thoroughly washed lesions were used for a second test. Effect of ambient humidity on sporulation and frequency of each spore type Wet surface-sterilized lesions were dried under a fan. Sulphuric acid solutions were prered and placed in plastic chambers ( cm) with tightly fitting lids to maintain relative humidities of 40, 60, 80, 85, 90 and 95%, respectively (Solomon, 1952). Each chamber held 10 ml of the appropriate solution to give the desired humidity. Ten millilitres of sterile distilled water was used where a relative humidity of 100% was required. Ten dry lesions were put into each of a number of watch glasses. A watch glass and its contents was supported at the bottom of one of the humidity chambers by a solid watch glass. Each was incubated at C for 48 h. At the end of the incubation period in each experiment, 1 ml of distilled water was added to five lesions at each humidity level and spore suspensions were prered as previously described. Slides were made for each humidity level and spores were counted as already described. The experiment was repeated four times. Germination of microconidia and infection tests In studies on glass slides, spores from sporulating lesions were deposited on slides. A drop of sterile distilled water was put on each spore print and the slides were placed in humidity chambers at 100% relative humidity. They were incubated in the dark (25 32 C) for 48 h. The incubated spores were air dried and mounted in cotton blue in lactophenol for microscopic examination. In studies on host leaf surfaces, microconidia were deposited on marked areas on both abaxial and adaxial surfaces of leaves of cassava plants growing in pots. Lesions incubated for approx. 6 d generally contained mainly microconidia. Hence, to inoculate with microconidia, spores were transferred from lesions which had been incubated for 6 d into drops of distilled water on slides. Each spore suspension was thoroughly examined under the microscope and those which contained only microconidia were used for the inoculation. Inoculated plants were incubated in humid chambers in a glass house (25 32 C). After 48 h, some inoculated areas were cut out and cleared in a mixture of equal rts (v v) of glacial acetic acid and absolute alcohol, stained with modified acid schiff reagent (Ayesu-Offei and Clare, 1970) and mounted in dilute glycerine for observation under the microscope. The cassava plants were removed from the humid chambers after 48 h incubation and the remaining inoculated spots were observed for a period of 30 d for symptoms. RESULTS Studies on leaf-borne spores Surface sterilized and unsterilized lesions from both cassava and tree cassava sporulated abundantly. Each lesion from both cassava and tree cassava contained two spore-forms. The larger of the two forms, termed macroconidia in this per, has been described for C. henningsii obtained from cassava. The results obtained in the present study are presented in Fig. 1 (A, B, C) and Table 1 and are in general agreement with those already published. Of much interest in the present work were smaller-sized spores which were previously unknown. They are referred to as microconidia in this per. They were cylindrical and A B FIG. 1. Camera lucida drawings of spores of C. henningsii. A, B, C, Macroconidia; D, E, F, G, microconidia;, pilla. C F D G E

3 Ayesu-Offei and Antwi-Boasiako Production of Microconidia by Cercospora henningsii Allesch 655 TABLE 1. Frequency distribution of lengths and widths of spores of C. henningsii formed in lesions on cassa a Macroconidia Microconidia Number of Number of Number of Number of Length of spores Width of spores Length of spores Width of spores spore (µm) counted spore (µm) counted spore (µm) counted spore (µm) counted * * * * * Mean standard error. serates from the rest of the spores by abstriction (Fig. 2C). The protoplast is then set free to develop into an independent microconidium. The remaining cells of the macroconidium then moved out of the disintegrating cell wall to develop into microconidia. A similar constriction may divide any cell of the macroconidium into microconidia followed by the release of the remaining cells. Any of the cells of the macroconidium may also put out a lateral bud, which is then liberated to develop into a microconidium. The cell wall of any macroconidium may also disintegrate to release the cells to develop into microconidia (Fig. 2A, B). Effect of incubation period on the frequency of spores formed in lesions A D FIG. 2. Camera lucida drawings illustrating budding and fragmentation of macroconidia of C. henningsii derived from cassava. D, Macroconidium with a globular bud formed from one of the terminal cells; C, macroconidium after the bud had serated by abstriction; A, B, disintegration of cell walls to release cells of the macroconidium. mostly one-celled. A few were medianly septate. They measured µm (Fig. 1D G, Table 1). Unlike the macroconidia, they had no pillae and had varying numbers of prominent vacuoles in their cytoplasm. Formation of microconidia Our observations indicated that the microconidia were formed by means of budding and fragmentation of the macroconidia. Commonly, a constriction occurs at one end of the macroconidium. The terminal cell of that end of the spore then becomes completely surrounded by the cell wall to form a small globular bud (Fig. 2D). The bud then B C In all four experiments, there were more macroconidia than microconidia in the wet sporulating lesions at the end of 24 h of incubation. But the proportion of microconidia increased with increase in duration of the incubation period. The increase was already aprent where lesions had been incubated for 48 h. After 144 h of incubation, almost all the spores in the lesions were microconidia. The mean number of spores and standard error of each group of four experiments are given in Table 2. Effect of ambient humidity on sporulation The total number of microconidia produced after 48 h of incubation at 100% relative humidity was significantly higher than the total number of macroconidia produced under the same conditions (P 0 001). The mean of the four experiments and the standard error at each humidity level are presented in Table 3. Production of both types of spores decreased as the humidity decreased, but the greater magnitude of effect of reduced humidity was on the microconidia whose production had almost ceased at 40% relative humidity (Table 3). Germination of spores and infection tests The microconidia germinated on both surfaces of the host leaf and on glass slides at 100% relative humidity (Fig. 3). Appressoria were formed at the apices of germ tubes.

4 656 Ayesu-Offei and Antwi-Boasiako Production of Microconidia by Cercospora henningsii Allesch TABLE 2. The effect of incubation period on sporulation and frequency of spore-types of C. henningsii in sterilized and unsterilized lesions from cassa a incubated at C Mean number of spores s.e. Length of Sterilized lesions Unsterilized lesions incubation period (h) Macroconidia Microconidia Macroconidia Microconidia TABLE 3. Effect of ambient humidity on sporulation and frequency of spores of C. henningsii in sterilized lesions from cassa a, 48 h after incubation at C gt Mean number of spores s.e. % Relative humidity Macroconidia Microconidia ap FIG. 3. Camera lucida drawings of germinating microconidia of C. henningsii. gt, Germ tube; ap, appressorium. Water-soaked areas developed at about 30 40% of the points of inoculation with microconidia on the abaxial surfaces of the leaves within 9 18 d after inoculation. Fully formed lesions appeared d after inoculation. Infection rarely occurred on the adaxial surface of leaves. DISCUSSION The work reported in this per has shown that Cercospora henningsii Allesch produces numerous microconidia. The microconidia germinated by means of germ tubes, some of which formed appressoria. Appressoria are formed by germinating spores of some types of rasitic fungi and are often sites at which infection hyphae are formed. Inoculation of cassava, by the authors, with spores from lesions on cassava, readily infected cassava plants. Macroconidia and microconidia generally occurred together in lesions but by incubating lesions for six or more days, it was possible to select spore prints which contained only microconidia and to demonstrate that they alone could cause infection. The authors are unaware of any previous knowledge of the production of microconidia by C. henningsii. These investigations have therefore demonstrated that the fungus produces a larger number of inocula than was previously thought. The observation from the present study is that many of the microconidia budded off from cells of the macroconidia and are therefore blastospores (cf. Talbot, 1971). But a significant number of them were formed by fragmentation of the macroconidia and therefore these could not be described as blastospores. The studies showed that the longer the incubation period of wet lesions, the greater the number of ungerminated spores formed in the lesions, most of them microconidia. This shows that mature spores failed to germinate while still in lesions. It has been suggested that in some fungi autoinhibitors produced by spores prevent germination in sporulating lesions (Pritchard and Bell, 1967; Ayres and Owen, 1970). In the presence of free water, the macroconidia of C. henningsii in lesions, therefore, bud and fragment into numerous microconidia which could then be dispersed by rain drops. Unpublished observations by the authors strongly suggest that rain-splash dispersal of the spores is more important in dispersing secondary inoculum within a farm or plantation than other modes of dissemination. Ayesu-Offei and Carter (1971) made similar observations on leaf scald of barley. The biological advantage of the above observations with regard to water is that numerous microconidia are produced and efficiently dispersed under wet conditions which predispose cassava plants to infection. This probably explains why the disease could be very severe under wet conditions.

5 Ayesu-Offei and Antwi-Boasiako Production of Microconidia by Cercospora henningsii Allesch 657 Our results have suggested that production of microconidia occurred most abundantly when free water was available. Since observations indicate that wet conditions promote severe development of brown leaf spot of cassava, integrated management strategies for the control of the disease should exploit the drought-resistant properties of cassava and encourage large-scale cultivation of the crop in areas of low and moderate rainfall. Some spores of the fungus may be dislodged from lesions in strong wind which causes plants to sway, vibrate or rub against one another. Inter-cropping will increase distances between cassava plants and therefore reduce the incidence of dispersal of the spores from plant to plant through water splash and contact between plants. LITERATURE CITED Ayesu-Offei EN, Carter MV Epidemiology of leaf scald of barley. Australian Journal of Agricultural Research 22: Ayesu-Offei EN, Clare BG Processes in the infection of barley leaves by Rhynchosporium secalis. Australian Journal of Biological Sciences 23: Ayres PG, Owen H Factors influencing spore germination in Rhynchosporium secalis. Transactions of the British Mycological Society 54: Bellotti AC, Van Schoonhoven A Mite and insect pests of cassava. Annual Re iew of Entomology 23: Jameson JD Agriculture in Uganda. 2nd edn. Oxford: Oxford University Press, Leather RI Diseases of economic plants in Ghana other than cacao. Ghana Ministry of Food and Agriculture Bulletin 1. Lozano JC, Booth RH Diseases of cassava (Manihot esculenta Crantz). Pans 20: Pritchard NJ, Bell AA Relative activity of germination inhibitors from spores of smut and rust fungi. Phytothology 57: Solomon ME Control of humidity with potassium hydroxide, sulphuric acid or other solutions. Bulletin of Entomological Research 42: Talbot PHB Principles of fungal taxonomy. London: Macmillan Press. Terry ER, Oyekan JO Cassava diseases of Africa reviewed. SPAN 19: