Scientia Agriculturae www.pscipub.com/SA E-ISSN: 2310-953X / P-ISSN: 2311-0228 DOI: 10.15192/PSCP.SA.2016.15.2.348355 Sci. Agri. 15 (2), 2016: 348-355 © PSCI Publications Morphological Characterization of Cercospora Zeae-Maydis (Tehon and Daniels) Isolates in Southern and Southwestern Ethiopia Alemu Nega*, Fikre Lemessa, Gezahegn Berecha Department of Horticulture and Plant Sciences Jimma University P.O. Box 307 Jimma, Ethiopia. *Corresponding author email: alemunega531@gmail.com Paper Information ABSTRACT Maize (Zea mays L.) is one of the most important cereal crops cultivated in Received: 13 January, 2016 Ethiopia that ranks second after teff in area coverage and first in total production. Grey leaf spot is one of the major constraints of maize Accepted: 27 April, 2016 production in maize belt area of Ethiopia where warm humid environmental condition is prevailing. In view of the expansion, Published: 1 June, 2016 seriousness, and potential destructiveness of grey leaf spot (GLS), the study was conducted with the objective of characterizing Cercospora zeaemaydis isolates collected from various maize growing areas of Southern and Southwestern Ethiopia using morphological and cultural characteristics. Purified cultures of 10 representative samples were selected from 99 isolates of Cercospora zeae-maydis by categorizing the character of colony appearance on the medium and studied for the variation of colony growth, color, shapes, growth diameter and conidial size, in length and width and the number of septa. Colony growth, color, shapes and growth diameter of every culture were recorded, conidial size (length and width) and shapes were computed from 20 conidia per each selected 10 isolates of Cercospora zeae-maydis. Ninety nine isolates were studied for morphology and most of the conidia shapes were slightly curved (70%) and straight (30%). The mean conidial size was 49.5μm in length and 9μm in width. The number of septa was found to range from 4.7 to 7.0. The study of cultural characteristics showed that variation existed among 10 representative isolates in colony growth, colony color and pigmentation. Among the two culture media tested, the highest mean colony diameter of 57.2mm was recorded on potato dextrose agar (PDA) and the lowest mean mycelial growth of 53.1mm was recorded on malt extract agar (MEA). Colony growth rate (mm/day) of 10 isolates was significantly different after growing the fungus on both PDA and MEA. All isolates had good sporulating capacity on general media (PDA) followed by MEA. Over all, the results of this study indicated wide variations among 10 isolates of Cercospora zeae-maydis based on morphological characteristics. Further investigations are recommended to get full image of the variations of Cercospora zeae-maydis in each maize growing agro-ecological areas of Ethiopia. © 2016 PSCI Publisher All rights reserved. Key words: Cercospora zeae-maydis, cultural characterization, grey leaf spot, maize,morphological characterization. Introduction Maize (Zea mays L.) has a critical nutritional role to play in human as it is the third important cereal crop globally after wheat and rice with regards to cultivation area, total production and consumption (FAO, 2011). Maize is growing productively all over the world, covering the lowland tropical, subtropical and temperate zones wherever rainfall or irrigation is sufficient and grows from sea level to over 3000 meters above sea level (Dowswell et al., 1996). Maize has multiple uses for human and animals as a feed grain, a fodder crop, and also as raw materials for industry. Its grain, stalk, leaves, cobs, tassel and silk all have marketable value in some regions. The principal producers of maize are the U.S., China, Brazil, Argentina, Mexico, Indonesia, India, South Africa, and France (FAO, 2012). In sub-Saharan Africa maize is produced in an estimated Sci. Agri. 15 (2), 2016: 348-355 area of about 26 million hectares with an average yield of 460 million tons. In east Africa, maize occupies about 11 million hectares production area with an average yield of 16 million tons (FAO, 2011). In Ethiopia, about 2.1 million hectares of land was enclosed with maize with an anticipated production of about 7.23 million tons in the 2014/15 cropping season (CSA, 2015). It explains for 16.80% of the 10.14 million hectare (80.78%) of land distributed for all cereals. Maize is one of the most imperative cereal crops grown in the Ethiopia. The total annual production and productivity surpasses all other cereal crops. In terms of area coverage, it is only super passed by tef [Eragrostis tef (Zucc.) Trotter], first in total national production and yield per hectare (CSA, 2015). Maize is widely cultivated in western, southern, and southwestern, and in some northern, northwestern and eastern part of the country and over 90% of the maize produced is used as food (Ferdu et al., 2001). Relatively high productivity, favorable growing conditions, and the technological advances over the last decades have contributed to its increased area of production as compared with other crops. However the national average yield (2.95 tons ha-1) (CSA, 2012) is still very low compared to the global average of 5.21t/ha (FAO, 2011). The low productivity of maize is attributed to many factors like drought stress, declining of soil fertility, plant population, poor agronomic practice, insufficient technology generation, poor seed quality, and disease (CIMMYT, 2004; Tolessa et al., 2002; Tewabech et al., 2002). Previous research indicated that foliar diseases such as Turcicum leaf blight caused by Exserohilum turcicum and common leaf rust stimulated by Puccinia sorghi have been documented as the most imperative maize production problems that occur widely on maize (Assefa, 1999). Currently, grey leaf spot, caused by Cercospora zeae-maydis Tehon and Daniels, is the major maize disease in the country. The disease first became serious in 1997 in the boundary of western Ethiopia. Becuase, there have been extensively disseminated severe outbreaks every year, particularly in the warm and humid areas of the country (Tewabech et al., 2002). Grey leaf spot has become one of the most important yield-limiting diseases of maize in sub-Saharan Africa and it was also reported as a cause of economic losses in South Africa (Ward et al., 1999). The disease is mainly severe and destructive when extensive periods of high relative humidity arise, caused by slow-drying dews and extended long season fogs (Beckman and Payne, 1983). Furthermore, the extensive implementation of reduced tillage practices, maize monocropping and planting of susceptible varieties have contributed in a build-up of inoculums reservoirs and grey leaf spot incidence (Gevers et al., 1994; Pratt et al., 1997). A study performed in Ethiopia by Dagne et al. (2004) designated that gre y leaf spot caused a yield loss of 37%, with predictable higher losses in years of severe epidemics. Losses coupled with grey leaf spot are maximum when photosynthetic tissues are blighted and prematurely killed earlier to grain fill (Donahue et al., 1991). The disease intensity is expected to differ from location to location and also highly variable within regions of the country, zones, districts and areas. Such observable differences in reaction to the disease in the same varieties/cultivar within nearby localities, advocates the existence of strains of the causative organism to grey leaf spot of maize. The aggressiveness of this disease has formed a vital demand from farmers for control measures. For efficient control of grey leaf spot, the variability existent within the pathogen population must be well recognized. Furthermore, despite of the high grey leaf spot incidence in south and southwest Ethiopia, no efforts have been made so far to characterize the variations among isolates in cultural and morphological characteristics. Therefore, the present study was conducted with the objective of characterizing Cercospora zeae-maydis isolates collected from various maize growing areas of Southern and Southwestern Ethiopia using morphological and cultural characteristics. Materials And Methods Study area sample collection A study was carried out in major maize-producing areas/zones of southern (Sidama and Wolaita) and southwestern (Jimma and Illubabore) Ethiopia during the long rainy season (July to September) in 2014 cropping season. The study area is located at 704’N Latitude and 380 31’E Longitude with altitude of 1700m.a.s.l. and 7012’N - 906’N Latitude and 350 27’E - 370 09’E longitude with altitude ranging from 1000 - 2500m.a.s.l. at southern and southwestern Ethiopia, respectively. The infected maize leaf samples with symptoms recognized as that of grey leaf spot (pale brown or grey to tan, long narrow streaks that become dark, greyish-brown rectangular lesions as the disease develops) were collected from 110 farmers fields in eleven districts, and samples were taken to Jimma University College of Agriculture and Veterinary Medicine plant pathology laboratory and kept at 80C in a refrigerator until isolation. The diseased samples were collected when the crops are at the grain filling stage. This is the stages when maize crops are between tasseling and physiological maturity, a period within which grey leaf spot development is considered to be at its peak (Latterell and Rossi, 1983). Morphological and Cultural Studies of C. zea maydis Isolates Isolation and Culture of Pathogens Section of diseased leaf tissue was cut into approximately 5cm2 segments and surface-sterilized in 5.25% of NaOCl solutions for approximately 30 seconds (Asea et al., 2005). After this, the sample was placed on sterile moist blotter in a sterile petridish. Five sections of diseased tissue were placed in each petridish. The petridishes were then left at 25 0C for 5 days of incubation to allow the pathogen to develop and sporulate in growth cabinets under a 12h fluorescent light/dark regime. The 349 Sci. Agri. 15 (2), 2016: 348-355 sporulating diseased section was examined under a binocular microscope for the presence of conidia. Conidia from separate synnemata was picked with an isolation needle and plated on PDA, allowing at least three pickings per leaf sample. The plates were then incubated at 250C for 5-7 days. Hyphal tips from the advancing colony margins were then transferred onto PDA medium as part of the culture purification process. Purified isolates were kept at 5 0C and infrequently sub-cultured onto fresh PDA medium to maintain fungal viability (Kinyua et al., 2010). Morphological and Cultural Identification of Isolates Isolates of Cercospora zeae-maydis obtained from maize leaf samples were evaluated for their morphological and cultural characteristics. Each isolate of Cercospora zeae-maydis were cultured on PDA medium with three replications and randomized on the incubating bench, and investigated for morphological variations such as shape, size (length and width) of conidia, and the number of septa after 5-7 days of incubation. Colony color were observed front side and types of pigments from the reverse side of each Cercospora zeae-maydis isolate were determined by using RGB color chart (Anonymous, 2005). Colony diameter of every culture was recorded daily for 7 days. Growth rate was calculated as the 7-days average of mean daily growth (mm/day). Moreover, colony margin of every culture was recorded. Three cultures of each isolate were investigated. For examination of conidial morphology, all isolates were sub-cultured as indicated above. Cultures were washed with sterile water and drops of the suspension were placed on microscope slides and mixed with lacto phenol blue to stain the conidia. Conidial size (length and width) and septum were computed from 20 conidia per isolate. Length and width of conidia were measured with ocular micrometer (μm) by adjusted stage micrometer, which were fitted into 10x eyepiece and adjusted at 40x objective of the compound microscope (Hanyuma, 2000). Sporulation 15-27 old cultures of each Cercospora zeae-maydis isolate, incubated on PDA media were washed by flooding with 10ml sterilized distilled water, rubbed with sterilized scalpel and transferred to 50ml sterilized beaker and thoroughly stirred for 10-15 minutes with vortex to extract the spores from the interwoven mycelia and then filtered into another sterilized beaker through double layer cheese clothes. The numbers of spore/conidia per milliliter were harvested by haemocytometer. The results were determined for each isolate as the average number of conidia per milliliter after taking haemocytometer counts. The experiment was repeated once. Data Analysis The mean conidial length, width and count of number of septum data were calculated for each isolates. Mean conidial length, width and septum of each isolate were used to make comparison between the isolates obtained from different districts. The data of colony growth diameter and growth rate were subjected to ANOVA to determine the colonial growth diameter and growth rate of each isolate. All the statistical analysis were carried out using SASV.9.2 and the Least Significant Difference (LSD) test was employed for mean comparison of significant variables at the probability level of 5%. Result And Discussion A total of 99 fungal isolates were collected from the infected maize leaf samples during the extensive grey leaf spot biological surveys, covering a wide range of agro-ecological maize belt areas of Southern and Southwestern Ethiopia in 2014. Morphological characteristics of Cercospora zeae-maydis The 99 isolates of Cercospora zeae-maydis collected from the different areas of southern and southwestern Ethiopia varied in terms of morphological characteristics of conidia studied (Table 1 & 2). The results indicated that the shape of conidia was typically slightly curved for some while others were straight shaped. Accordingly, the slightly curved (70%) and straight (30%) shape of the conidia dominated in the populations of the fungus. The conidia are also straight or slightly curved, hyaline and they are wide at the base and get thinner at the tip (Fig. 1). Reportedly, the conidia are hyaline and measure 30180µm in length and 4-9µm in width and have 3 to 10 septa (Nowell, 1997), and also mature conidia are 70-180µm long and 5-6µm wide at the base. They are tapering to 2-3µm at the apex (Latterell and Mosi, 1983). As with most fungi, however, these dimensions as well as those of conidiophores vary considerably with the conditions under which these structure are formed especially with respect to natural or artificial substrate, moisture, light and age of stroma. Conidia are hyaline have 6-10 septa, and germinate readily from any cell, three or four germ tube typically emerging during the infection process on maize leaves (Latterell and Mosi, 1983). Selected 10 purified cultures from the total of 99 isolates of Cercospora zeae-maydis by categorizing the character of colony appearance on the medium and studied for the variation of conidial size, in length and width and the number of septa shown in Table 2. The result revealed that the conidial lengths varied from 30μm - 50μm whereas conidial width (breadth) ranged from 7 - 9μm. The maximum mean number of septa was 7 and the minimum was 4.75. Significant variations were observed with respect to conidial dimensions among the isolates. The maximum length of conidia was observed Bor-S isolates 350 Sci. Agri. 15 (2), 2016: 348-355 (49.5μm) and minimum length of conidia was recorded in Gor-H isolates (30μm). Similarly, the maximum width of conidia was observed in the isolate of Bor-S (9μm) and followed by BS-W, Uru-I, Bed-6 and DS-3 (7.88, 7.63μm, 7.63μm and.7.5μm), respectively. As the length of the conidia/spore size increases, the width and septum also increase simultaneously. Minimum width of conidia was observed in Gor-H isolate (7μm) (Table 2). The mean value of conidial length, width and number of septum designated the presence of variation in morphological characteristic among 10 isolates of Cercospora zeaemaydis (Table 1). Our result also indicated that, there was significant variation in the mean number of length, width and septa of conidia of Cercospora zeae-maydis from location to location. This could be due to the difference in the amount of rainfall, relative humidity as well as the minimum and maximum temperatures received in the assessed regions of south and southwest Ethiopia. This investigation is supported by (Ward and Nowell, 1998), who reported that the ecological conditions have a consequence on the development of grey leaf spot disease on maize. In addition, we obtained that, there was a comparable difference in conidial length, width and septa of the fungus/Cercospora zeae-maydis. Donahue et al. (1991), who reported that conidial length and width can vary among different isolates. Table 1. Percent conidial shape of 10 isolates of Cercospora zeae-maydis collected from different districts of south and southwest Ethiopia in 2014 cropping system Conidial character type Conidial Shape: -Straight -Slightly curved Sub total Number of Isolate Total percent of Isolates 7 3 10 70 30 100 Table 2. Mean conidial length, width and septum of 10 isolates of C. zeae-maydis collected from different districts of South and southwest Ethiopia in 2014. Isolate Ker-J Bor-S BS-W DS-3 SK-J Gor-H Mal-9 Uru-I Gech-7 BD-6 Length (µm) 35.00 49.50 34.50 32.10 38.30 30.00 37.10 31.80 35.10 33.10 Width (µm) 7.50 9.00 7.90 7.50 7.50 7.00 7.10 7.60 7.40 7.60 Septum 6.10 7.00 5.30 4.70 5.80 5.20 5.90 4.80 5.80 5.10 Table 3. Percentage of colonial gross of 10 C. zeae-maydis isolates collected from different locations of South and southwest Ethiopia in 2014. Colony characteristics Colonial gross (morphology): Elevation: -Raised -Growth in medium Sub total Shape: - Round - Curled Sub total Edge: - Smooth - Irregular Sub total Number of Isolates Total percent of Isolates 6 4 10 59.4 39.6 100 8 2 10 79.2 19.8 100 8 2 10 79.2 19.8 100 Cultural characteristics of Cercospora zeae-maydis. The proportion of colonial gross and colonial color of 10 C. zeae-maydis isolates were presented in (Table 3 & 4). Pure cultures of 10 isolates were investigated on cultural characteristics by categorizing the nature of the colony appearance on the medium. The results showed that 59.4% of isolates showed raised colonial growth while 39.6% of the total isolates showed medium in growth. The colonial shape were round (79.2%) and curled (19.8%) with smooth (79.2%) and irregular (19.8%) margin (Table 3). The front (obverse) side of colony color of the isolates ranged from white, gray, pink, to brown. Most isolates had white color (39.6 %) followed by pink (29.7%), grey (19.8%) and brown (9.9%). The reverse side of colony characteristic of most isolates had light pink color (39.6%) followed by corn silk, Indian red/red and light grey (19.8%, 19.8%, and 19.8%), respectively (Table 4). Overall, the study of cultural characteristics showed the existence of variation among the 10 representatives isolates of Cercospora zeae-maydis in colony growth, colony color and pigmentation. This work is 351 Sci. Agri. 15 (2), 2016: 348-355 supported by (Latterell and Rossi, 1983), who reported that the cultural performances of Cercospora zeae-maydis grow on agar media with the dense, sluggish growing colony type characteristics of the genus. Growth character ranges from black, densely sporulating cushin like colonies to white, cottony mycelial growth, one type often ‘mutating’ from the other. Intermediate types include gray, moderately sporulating colonies often with pink, red, or purple pigment, depending on the substrate, due to formation of cercosporin crystals. Table 4. Percentage of colonial color of 10 C. zeae-maydis isolates collected from different locations of South and southwest Ethiopia in 2014. Colonial color: Front (obverse) side: - White - Gray - Pink - Brown Sub total Back (reverse) side: - Corn silk - Light gray - Light pink - Indian red/red Sub total A Number of isolates Total percent of isolates 4 2 3 1 10 39.6 19.8 29.7 9.9 100 2 2 4 2 10 19.8 19.8 39.6 19.6 100 B C D 1 2 3 4 5 6 Figure 1. Infected leaf lesion of maize by GLS (A), Front and back side of colony color (B&C) and Conidia of Cercospora zeae-maydis (D) 352 Sci. Agri. 15 (2), 2016: 348-355 The mean of colony growth diameter (mm/day) and sporulation of Cercospora zeae-maydis isolates were shown in (Table 5 & 6). The results showed that both potato dextrose agar (PDA) and malt extract agar (MEA) media tested maintained the mycelial growth of the isolates of Cercospora zeae-maydis, the causal agent of maize grey leaf spot. The highest average mean colony diameter of 57.2mm was recorded on PDA and the lowest average mean mycelial growth of 53.11mm was recorded on MEA (Fig. 2). The effect of medium also influenced spore production by Cercospora zeae-maydis. A good sporulation was obtained on PDA and sparse sporulation on MEA. Our result indicated that the sporulation of Cercospora zeae-maydis was relatively high on PDA but scarce on MEA. This could be due to faster growth of pathogen on PDA and attaining the average mean of 57.2mm of colony growth followed by slower growth of pathogen on MEA with the average mean of 53.1mm of colony growth in two weeks of incubation. Furthermore, the varied degree of sporulation occurred in both of the media with highest sporulation on PDA followed by MEA could be due to the impression that there is a genetic variation between the isolates of the pathogen. The mean colony growth diameter of isolates Uru-I (62.26mm) was fastest, followed by BD-6 (61.52mm), DS-3 (60.57mm), BS-W (59.67mm) and SK-J (58.12mm), on PDA, respectively (Table 5). While the colony growth diameter of isolates SK-J (56.66mm) was fastest, followed by Gech-7 (54.67mm), Uru-I (53.86mm) and DS-3 (53.83mm), on MEA respectively. Isolates Bor-S (48.5mm) and Mal-9 (48.13mm) were the slowest in the colony growth diameter on both PDA and MEA, respectively. Isolates DS-3, BD-6 and Uru-I have highest colony diameter on both PDA and MEA (60.57mm, 53.83mm), (61.52mm, 53.43mm) and (62.19mm, 53.86mm), respectively (Table 5). There was a statistically significant (p<0.001) difference in mycelial growth among the 10 isolates of Cercospora zeae-maydis on both PDA and MEA of the solid media. Overall, the result revealed that there was significant variation between growth on the two media investigated with the highest vegetative growth on PDA and the lowest growth occurred on MEA and also we observed an effect of medium on colony growth. This investigation is supported by Latterell and Rossi (1974), who reported that various media supported different types or degrees of development of erect or submerged stromata and of sub spherical bodies containing either macro or microspermatia of Cercospora zeae-maydis. Furthermore, there was a statistically significant (P<0.001) difference in growth rates among the 10 isolates. Isolates of Cercospora zeae-maydis isolate SK-J (8.88mm/day) grew significantly faster, followed by Ker-J, Gor-H and Uru-I, 8.65mm/day, 8.52mm/day and 8.49mm/day, respectively on PDA and the lowest growth rate was recorded by isolate BS-W (6.93mm/day) on MEA media (Table 7). 80 Mean of GLS growth (mm) 70 60 50 40 30 20 10 0 PDA MEA Media Standard Error (SE) = 1.26 for PDA, SE = 0.97 for MEA. Fig.2. Mean of colony growth diameter (mm) of GLS isolates after two days incubation on PDA and MEA at 25 0C. 353 Sci. Agri. 15 (2), 2016: 348-355 Table 5. Effect of Potato dextrose agar (PDA) and Malt extract agar (MEA) of solid media on the mycelia growth diameter (mm) of Cercospora zeae-maydis isolate in 2014. Isolate BD-6 Bor-S BS-W DS-3 Gech-7 Gor-H Ker-J Mal-9 SK-J Uru-I CV (%) LSD (0.05) PDA 61.52ab 48.49d 59.67a-c 60.57a-c 55.48a-d 54.21bcd 53.19cd 49.86d 58.12a-c 62.19a 8.16 7.8 MEA 53.43bc 48.13d 52.30c 53.83bc 54.67ab 51.33c 52.10c 48.53d 56.66a 53.86bc 3.43 3.09 Means in a column followed by the same letters are not significantly different according to LSD at 5% probability level. Table 6. Sporulation of Cercospora zeae-maydis identified from infected maize leaf on both solid media of potato dextrose agar (PDA) and malt extract agar (MEA) in 2014. Isolate BD-6 BS-W DS-3 Gor-H Mal-9 Ker-J Bor-S SK-J Gech-7 Uru-I Sporulation/ml in PDA 6.5x107 3.6x107 4.6x107 2.8x107 2.5x106 2.6x107 2.2x106 4.5x107 4.2x107 8.4x107 Sporulation/ml in MEA 2.4x106 1.8x106 2.2x106 1.5x106 1.6x105 4.6x105 1.5x105 1.7x106 1.6x106 1.8x107 Table 7. Mean of Cercospora zeae-maydis growth rate (mm/day) in PDA and MEA solid media Isolate BD-6 Bor-S BS-W DS-3 Gech-7 Gor-H Mal-9 Ker-J SK-J Uru-I CV (%) LSD (0.05) PDA 7.87d 8.30bcd 7.29e 8.00cd 8.22bcd 8.52abc 8.07cd 8.65ab 8.88a 8.49ab 3.71 0.52 MEA 7.98cd 8.79a 6.93e 7.02e 8.69ab 8.34bc 8.02cd 8.58ab 7.96cd 7.63d 3.25 0.44 Means in a column followed by the same letters are not significantly different according to LSD at 5% probability level. Conclusion Our result revealed that there is variability in the cultural characteristics of colony growth, color, shape, growth diameter and morphology of conidial size and shape of Cercospora zeae-maydis. This variation due to differences existing in the climatic conditions in the agro-ecological regions of south and southwest Ethiopia. In general, the results obtained from cultural and morphological characteristics, variation exists among the isolates collected from all the maize growing areas of south and southwest Ethiopia. Therefore, this variation also depended on the difference in environmental condition within and between the regions of the surveyed areas of the country. Over all the contribution of our research project output have been achieved to developmental impact. As a result, we now have a much apparent understanding of the variability of the pathogen population in the country and management strategies which are appropriate for the target beneficiaries’ i.e. small-scale maize farmers. From this study, it is suggested that further investigation of Cercospora zeae-maydis, the causal agent of maize grey leaf spot has been publicized to be more and more important pathogen of maize in Ethiopia. Moreover, further investigations are recommended to complete map of the variations of Cercospora zeae-maydis in each maize belt agro ecological areas of Ethiopia. 354 Sci. 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