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Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 12 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.812.033 Studies on Cultural and Morphological Variability in Isolates of Exserohilum turcicum, Incitant of Turcicum Leaf Blight of Sorghum M. R. Vinay* and A. R. Sataraddi Department of Plant Pathology, College of Agriculture, Vijayapur, 586101, India University of Agricultural Sciences, Dharwad, 580001, Karnataka, India *Corresponding author ABSTRACT Keywords Turcicum leaf blight, Exserohilum turcicum, Sorghum, Cultural variability Article Info Accepted: 04 November 2019 Available Online: 10 December 2019 Sorghum [Sorghum bicolor (L.) Moench] belongs to family Poaceae. It is termed, as 'poor man's crop' as it performs well even in marginal lands under moisture stress conditions, low availability of fertilizers and other inputs as compared to other crops. The foliar pathogens are potential yield reducers, and may cause substantial yield losses when occur in epidemic form. Leaf blight of sorghum caused by Exserohilum turcicum (Pass.) Leonard and Suggs is a major foliar disease, which substantially damages the foliage. The foliar blights and spots cause direct loss of foliage due to premature drying and early seedlings blights, or loss in other forage components like fodder weight and reduced carbohydrate translocation to other parts of plants due to reduced photosynthesis and transpiration. A total of 20 isolates of E. turcicum were obtained from district of Northern Karnataka. These isolates were grown in Potato Dextrose Agar (PDA). They varied in their cultural and morphological behaviour. All the isolates were found pathogenic to sorghum. The virulent isolate (E04) showed significant variations in colony colour, colony texture, surface and topography, margin, lustre, sporulation and consistency in the media viz., PDA, Malt extract Agar, Saboraud’s Medium, Richard’s Medium, Czapek’s dox medium, Yeast extract mannitol agar and Oat meal agar. The maximum average growth of the isolate (E04) was supported by PDA medium (7.35 cm) whereas minimum growth was recorded in V-8 juice agar medium (3 cm). Conidia were observed in all the isolates except Et07, Et09 and Et17. Among the isolates, conidia size was maximum in isolate Et10 (87.13 × 12.31 µm) with an average of 7-10 septation and minimum in isolate Et14 (33.92 × 12.23 µm) with 3-4 septation. Introduction Sorghum [Sorghum bicolor (L.) Moench] belongs to family Poaceae. It can be easily grown in latitudes ranging from 40 °S to 45 °N. In India, it is mostly grown between 9° and 21° in tropical and sub-tropical climates. Important uses of sorghum include food in the form of unleavened bread or roti, boiled porridge or gruel, fodder, animal feed, building material and fibre, beer, malted beverages and popped grains. About 35-40 per 232 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 cent of grain sorghum produced worldwide is consumed directly as food. Besides, it has several other industrial uses such as production of jaggery, syrup, lactic acid, riboflavin, microbial polysaccharides, antibiotics, ethanol and citric acid (Arun et al., 2009). Yield of sorghum is generally low in tropical countries, where it is mostly cultivated by marginal farmers. Sorghum diseases are considered to be the major constraint in realizing proper yield potential (ICRISAT, 1980). Sorghum crop is attacked by a large number of diseases, infecting, grains, foliage and roots. Leaf blight of sorghum caused by Exserohilum turcicum (Pass.) Leonard and Suggs is a major foliar disease, which substantially damages the foliage. The pathogen exhibits good tolerance to varied ranges of agroclimatic conditions over the globe. The pathogen is worldwide in distribution; though varies in severity and prevalence in different regions, hence in the economic importance (Tarumoto et al., 1977). The typical symptoms of leaf blight are long, 1-2 cm wide, elliptical, necrotic lesions, straw coloured in the centres with dark margin. The colour of the margin ranges from brown, red or purple depending upon host cultivar. Under humid conditions the centres of lesions bear faint to dark grey growth consisting of conidiophores and conidia. Many lesions may develop and coalesce on the leaves, destroying large areas of leaf tissue and giving the crop a distinctly burnt or blighted appearance (Bunker, 2005). Little information is available on the extent of losses caused by E. turcicum. Lee et al., (1986) observed 47 and 38 per cent reduction in fresh and dry matter yield due to leaf blight. E. turcicum known to produce conidiophores which are single or in small groups, straight to flexuous, sometimes geniculate above, septate, smooth, mid to dark brown, up to 300 µm long and 6-9 µm wide. Conidiogenous cells polytretic, integrated, terminal and intercalary, sympodial, cicatrized. Conidia pale to mid olivaceous brown, fusoid, smooth, straight or curved, mostly 4-7 septate, but up to10 distoseptate, measuring 100-135 x 15-25 µm, with a small protruding basal hilum (Sivanesan, 1986). E. turcicum is highly versatile to changing environmental conditions and shows high variability across different agro-climatic regions. Therefore, in order to document the changes occurring in populations and individuals, variability studies are important as it indicates different pathotypes and may open up a new avenue for disease management in the future. Variation in pathogen can be generally detected by their cultural and morphological characters. Materials and Methods Collection of infected isolation of the fungus specimen and Sorghum leaves bearing typical symptoms of the turcicum leaf blight disease were collected at flowering/grain filling stage from farmer’s field in 2017-18 growing seasons in Vijayapur, Dharwad, Bagalkot and Belagavi and used for the study. The infected leaves samples were plucked and kept in polythene bags with labels. They were brought to laboratory for microscopic observation and later kept at 4° C for further studies. Isolation Sorghum leaves showing typical symptoms of turcicum leaf blight were collected from the field. Leaves with symptoms were first washed in tap water and then cut into small 233 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 bits of 2 mm size, containing the discoloured blights. These bits were surface sterilized with 0.1 per cent NaOCl solution for two minutes followed by three changes of sterilized distilled water. These bits were placed on potato dextrose agar under aseptic condition. Inoculated plates were incubated at 27° C and watched for any contamination for seven days. After seven days of incubation, fungal colonies completely covered the plates and become dark greyish in colour indicating the production of spores. A small loop of fungal culture from the colonies was picked and slide was prepared by mixing the culture with a drop of lacto phenol. The slide was observed under low and high power objectives for the presence of conidia. Purification Purification of the fungus was done by single spore isolation techniques. The spore suspension of the fungal isolate was prepared in sterile distilled water. One ml of the suspension from fungal isolate was mixed with 20 ml of molten two per cent water agar. Single spore was marked with ink under stereo binocular microscope. The spore along with water agar was picked and transferred on potato dextrose agar plates and slants. Plates were incubated at room temperature (27 ±1° C) and periodically observed for the germination of spores and pure culture thus obtained were designated as sorghum isolates and were preserved on potato dextrose agar slants in refrigerator for further studies. Identification The pathogen forms grey colonies on the Petri dishes containing PDA. The colonies appeared as whitish grey mycelial growth and turns dark grey with sporulation. Conidiophores were single or in small groups, straight to flexuous, septate, smooth, dark brown. Conidia pale to mid olivaceous brown, fusoid, smooth, straight or curved, mostly 4-7, but up to 11 distoseptate, measuring 100-135 x 15-25 µm, with a small protruding basal hilum. On the basis of these characters the pathogen was identified as Exserohilum turcicum (Sivanesan, 1986). Proving pathogenicity Seeds of Sorghum from disease free plants of the previous season were surface disinfected with 0.1 per cent NaOCl for one minute and sown in pots containing sterilized soil in order to raise healthy seedlings. The pathogen was multiplied by transferring a loopful of the stock culture to 250 ml of potato dextrose broth taken in a 1000 ml flask. The inoculated flask was incubated at 27±1 ° C for seven days. The fungal culture growing on potato dextrose broth was passed through double layered muslin cloth. The spore suspension of the pathogen was collected under aseptic condition in an automizer. The spore suspension was collected separately in an automizer and sprayed on to the foliage at three to four leaf stage to susceptible sorghum variety M 35-1 at 105 spores per ml as suggested by Kadir et al., (2008) and Tosiah et al., (2011). Inoculated seedlings covered with polythene cover to create required moisture content for 24hrs. The seedlings after spray inoculation were kept in green house condition. Periodical observations were made for the development of typical brown spots on the inoculated plants. The pathogen from typical brown spots was re-isolated, compared with the original symptoms as well as published literature for confirmation. 234 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 Cultural and morphological studies of E. turcicum Cultural studies of the pathogen Effect of different media The study was conducted to describe the cultural characters such as colony colour, colony texture, surface topography, consistency, margin and lustre of the leaf blight pathogen on different solid media. 7mm culture discs of pathogen was inoculated separately on different media viz., PDA, Malt extract Agar, Saboraud’s Medium, Richard’s Medium, Czapek’s dox medium, Yeast extract mannitol agar and Oat meal agar, and incubated at 28±2 °C for 12 days. The cultural characters and the colony diameter (mm) on each medium were recorded. Growth characters on PDA was taken as standard while making comparative studies on different media. Morphological Studies Data on radial colony growth of E. turcicum were obtained by multiplying vertical and horizontal growth of hyphea, measured with centimetre calibrated plastic ruler. The values were rated as excellent (≥7.6 cm²), good (6.67.5 cm²), moderate (5.0-6.5 cm²) and poor (< 5.0 cm²) growth as described by Levy (1991) with modification. Conidial suspensions were prepared from 15-day-old cultures on infected sorghum seeds and adjusted to 10 conidia/ml based on counts made with a hemacytometer. Thereafter, 5 ml of the conidial suspension were pipetted into a 9 cm petri dish containing potato dextrose agar. Inoculated plates were incubated for 24 hours under light and darkness at 25 °C and used for percentage germination based on 100 conidia per petridish. Temporary slides of each treatment were viewed under binocular Leica microscope at 40 x and the sporulation were rated excellent for ≥ 20 conidia per microscopic field (++++), good (15-20 conidia per microscopic field +++), fair (10-15 conidia ++), poor ≤ 10 conidia +) and – for no sporulation, as described by Harlapur et al., (2007). To study morphological characters of conidia of all the isolates of Exserohilum turcicum, slides were prepared from twelve days old culture. Temporary slides were prepared in water mount using cotton blue. Data on length, width and septation of conidia were recorded by ocular micrometer by using a recalibrated compound microscope. 10 spores were observed for each isolate to avoid the error while taking observations. Results and Discussion Isolation and maintenance of different isolates of E. turcicum The pathogen was isolated and the fungal cultures on purification showed white colour and fluffy type of mycelium, which gradually turned into greyish, greenish or brownish as the culture started to produce conidia. On repeated isolation, it was found the association of E. turcicum. A total of twenty isolates were obtained and designated with isolate code as Et according to their respective places of collection (Table 1). The pathogen was identified by comparing with relevant literatures and by studying the cultural and morphological characters. The fungal isolate cultures were sub cultured frequently on PDA slants and kept in refrigerator at 4˚ C. Pathogenicity To prove Koch’s postulate, the isolated pathogen were inoculated on healthy fruits and all the isolates tested were pathogenic on sorghum. However, the isolates behaved differently for the ability to produce disease 235 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 symptoms on leaves. Isolate Et04 showed leaf blight symptoms after 6 days of inoculation. Typical symptoms of leaf blight developed on sorghum were similar to those described by Bunker (2006). Cultural and morphological variability The cultural characteristics of isolates are presented in Table 2 (Fig. 1.1 and 1.2). The following observations were made on Incubation period (days) for maximum growth, Colony colour, Pigmentation, Sporulation, Colony texture, Surface texture and Edge of colony. Incubation period (days) for maximum growth All the twenty isolates produced good growth on PDA, but the period taken by different isolates to completely cover the 9 cm petridish were different based on aggressiveness of the isolate. Among isolates Et04 shown lowest Incubation period of 6 days and Et15 shown highest Incubation period. In an average known to take 12 days of Incubation period to completely cover the 9 cm petridish. Colony colour The colony colour of fungus was recorded based on dominant spectral colour from Munsell’s soil colour chart (1954), 12 days after incubation on PDA medium and the results are presented in Table 2. The colony colour varied from gray to black colour. Based on the colony colour all the twenty isolates were grouped in 6 categories i.e., Gray, dull grey, Dark grey, greenish grey, brown, and black. The Et10 (Badami), Et13(Dharwad M), Et18(Khanapur) and Et20(Belagavi) showed Black(2.5Y 2.5/1) colony colour wheres isolate Et04 shown Dark greyish to black (10YR 5/1) colony colour. The isolates Et02 from Vijayapur showed dark Greenish black (5G 2/1) colony colour which was distinctly different from all other isolates. The isolates Et01 (Vijayapur), Et06 (Jamakhandi), Et12 (Dharwad AC), and Et19(Bailhongal) showed similar colony colour i.e Gray (2.5 Y 5/1), while very dark gray colony colour was observed in the isolates Et04, E08 and Et11 from Basavana Bagewadi H, Bilagi and Hunagunda respectively. Et07 (Guledagudda), Et09 (Mudhol), Et16 (Kalaghatagi) and Et17 (Gokak) showed Dull grey colour. Et05 (Indi), Et14 (Navalagunda) and Et15 (Dharwad N) showed brown (10YR 7/2) coloured colony. Pigmentation Based on the pigmentation E. turcicum isolates were grouped into 4 groups i.e., Black, Bluish black, Greenish black and dark grey. The sorghum isolates Et06, Et07, Et11, Et17 and Et20 was in a distinctly different pigmentation i.e., bluish black (2.5/1 10P). With regard to the isolate Et05, Et16 and Et19 showed greenish black (5G 2/1) pigmentation. The isolates like Et02, Et03, Et08, Et09, Et12, Et15 and Et18, showed black colour (16 YR 2/1) pigmentation whereas the isolates Et01, Et04, Et10, Et3 and Et14which were not having similar colony colour but with regard to pigmentation they showed the dark grey (2.5Y5/1) colour pigmentation (Fig. 1.2). Sporulation All the twenty E. turcicum isolates were classified into five groups based on the sporulation as mentioned in Table 2. Two isolates Et08 and Et14 produced excellent sporulation while the isolates Et04, Et13 and Et20 exhibited good sporulation and isolate Et01, Et03, Et12, Et15 and Et18 produced moderate sporulation, Whereas poor sporulation was noticed in the isolate Et02, Et05, Et06, Et10, Et11, Et16 and Et19 and no sporulation was found in Et07, Et09 and Et17 isolates. 236 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 Table.1 Designation of Exserohilum turcicum isolates from different districts of Karnataka Sl. No District Location Isolates identified Isolates designation 1 Vijayapur Hittinahalli E. turcicum Et01 Vijayapur E. turcicum Et02 Basavana Bagevadi E. turcicum Et03 Basavana Bagevadi E. turcicum Et04 Indi E. turcicum Et05 Jamakhandi E. turcicum Et06 Guledagudda E. turcicum Et07 Bilagi E. turcicum Et08 Mudhol E. turcicum Et09 Badami E. turcicum Et10 Hunagunda E. turcicum Et11 Dharwad E. turcicum Et12 Dharwad E. turcicum Et13 Navalagunda E. turcicum Et14 Dharwad E. turcicum Et15 Kalaghatagi E. turcicum Et16 Gokak E. turcicum Et17 Khanapur E. turcicum Et18 Bailhongal E. turcicum Et19 Belagavi E. turcicum Et20 2 3 4 Bagalkot Dharwad Belagavi 237 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 Table.2 Cultural characters of isolates Exserohilum turcicum on potato dextrose agar (PDA) Location Isolate Vijayapur Et01 Incubation period (days) for max. growth 9 Colony colour Pigmentation Sporulation Surface texture Colony texture Edge of colony Dark grey ++ Fluffy Ad pressed Black + Rough Black ++ Rough Moderately wavy Cottony Highly branched Branched Dark grey +++ Smooth Cottony Greenish black + Smooth Wavy Bluish black + Smooth Wavy 9 9 Grey 2.5Y5/1 Greenish black 5G 2/1 Black 2.5Y 2.5/1 Dark greyish to black 10YR 5/1 Brown 10YR 7/2 Grey 2.5Y5/1 Dull grey Dark grey Et02 8 Basavana Bagevadi M Basavana Bagevadi H Et03 14 Et04 6 Indi Et05 9 Jamakhandi Et06 12 Guledagudda Bilagi Et07 Et08 Bluish black Black ++++ Fluffy Rough Et09 8 Dull grey Black - Smooth Ad pressed Moderately wavy Wavy Mudhol Badami Et10 11 Black 2.5Y 2.5/1 Dark grey + Fluffy Wavy 238 Sparsely branched Branched Sparsely branched Branched Branched Sparsely branched Highly branched Highly branched Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 Location Isolate No. of days Colony colour to cover plate Hunagunda Et11 14 Dark grey Dharwad AC Et12 7 Grey 2.5Y5/1 Dharwad M Et13 9 Navalagunda Et14 11 Dharwad N Et15 15 Kalaghatagi Et16 8 Black 2.5Y 2.5/1 Brown 10YR 7/2 Brown 10YR 7/2 Dull grey Gokak Et17 11 Dull grey Khanapur Et18 9 Bailhongal Et19 14 Black 2.5Y 2.5/1 Grey 2.5Y5/1 Belagavi Et20 7 Black 2.5Y 2.5/1 Pigmentati on Sporulati on Colony texture Surface texture Edge of colony Bluish black Black + Smooth Cottony Branched ++ Smooth Ad pressed Dark grey +++ Rough Cottony Sparsely branched Branched Dark grey ++++ Rough Cottony Branched Black ++ Fluffy Cottony Greenish black Bluish black Black + Fluffy Wavy Sparsely branched Branched - Rough Cottony ++ Rough Ad pressed + Smooth Cottony +++ Fluffy Wavy Greenish black Bluish black Where, Score 1. ++++ 2. +++ 3. ++ 4. + 5. Grade Excellent sporulation Good sporulation Moderate sporulation Poor sporulation No sporulation Description ≥ 20 conidia per microscopic field (40X) 15-20 spores/microscopic field (40x) 10-15 spores/ microscopic field (40x) 1-10 spores/ microscopic field (40x) < 1 spores / microscopic field (40x) 239 Highly branched Sparsely branched Highly branched Branched Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 Table.3 Effect of different solid media on the growth of E. turcicum (E004 strain) Sl No. 1. Medium Potato dextrose agar Radial mycelial growth (cm) 7.35 2. 3. 4. Oat meal agar Czapeck’s agar Malt extract agar 6.45 6.50 5.25 5. 6. 7. Sabouraud’s agar Yeast extract agar Richards’ agar 6.75 3.35 5.05 8. V-8 Agar SE(m) CD (P 0.01) CV (%) 3.00 0.057 0.167 2.081 Table.4 Morphological characters of isolates Exserohilum turcicum on potato dextrose agar (PDA) Isolate Size of conidia length x breadth (µm) Et01 Et02 Et03 Et04 Et05 Et06 Et07 Et08 Et09 Et10 Et11 Et12 Et13 Et14 Et15 Et16 Et17 Et18 Et19 Et20 53.11 × 12.34 63.18 × 14.03 59.34 × 14.74 66.41 × 13.05 80.98 × 15.16 81.61 × 17.63 62.13 × 13.2 56.71 × 13.01 38.52 × 11.91 87.13 × 12.31 37.19 × 11.21 53.40 × 13.39 43.45 × 12.65 33.92 × 12.23 61.24 × 11.43 65.38 × 12.47 57.14 × 13.14 68.93 × 11.65 79.27 × 13.11 45.82 ×11.33 240 No. of septa (Range) 6-8 6-9 6-8 6-8 7-9 8-10 4-6 5-7 3-6 7-10 3-5 6-7 3-5 3-4 5-8 4-9 5-9 4-6 3-9 5-8 Chlamydospore presence on 40th DAI + + + + + + Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 232-244 241