Cultural, Morphological and Biochemical Variability Studies among the Isolates of Alternaria solani, the Causal Agent of Early Blight Disease of Tomato

Nagesh, S. K. Mushrif, C. G. Sangeetha, T. B. Manjunatha Reddy and J. S. Aravinda Kumar

  • Page No:  584 - 593
  • Published online: 31 Oct 2021
  • DOI : HTTPS://DOI.ORG/10.23910/1.2021.2491

  • Abstract

Present experiment was conducted at College of Horticulture, Bengaluru (KA) during year 2017–18 to study the cultural, morphological and biochemical variations among the isolates of the pathogen Alternaria solani, the causal agent of early blight disease in tomato. The results revealed variation among the isolates collected from different regions of Karnataka state, India with regard to the colony characteristics viz., colony colour, mycelial growth pattern, margin of the colony and zonations. Maximum mycelial growth in terms of diameter (90 mm) was observed in the isolates Bagalkot (BaBG) and Chikkamagaluru (CMH) on Czapek’s (Dox) agar medium while the least growth (36.33) was noticed in Bidar (BiHH) isolate. The isolate could grow better on Czapek’s (Dox) agar medium as among the 3 media tested Czapek’s (Dox) agar medium produced maximum growth of 80.70 mm and the least growth (63.70 mm) was noticed in V-8 juice agar. The morphological studies revealed that all the conidia of various isolates varied in length (25.07–42.90 µm), breadth (10.53–21.52 µm) and number of horizontal septa (2–7), longitudinal septa (0–4). Biochemical studies among the isolates revealed significant variation in their enzyme activities. The peroxidase activity was more in Chikamagaluru (CMH) isolate (81.80 Unit g-1 FW) least activity was found in Bidar (BiHH) isolate 11.78 Unit g-1 FW whereas the esterase activity was more Bengaluru (BYC) isolate (69.01 Unit g-1 FW) least activity was found in Bagalkot (BaBG) isolate 11.78 Unit g-1 FW. Existence of variation among the isolates of Alternaria solani isevident from the results obtained.

Keywords :   Alternaria solani, biochemical, cultural, isolates, morphological, variability 

  • Introduction

    Tomato (Solanum lycopersicum L., 2n=24) belongs to the family Solanaceae. It ranks next to potato in world acreage and ranks first among the processing crops. Tomato is being exported in the form of whole fruits, paste and in canned form. Tomato is called as “Poor man’s orange”. The cultivated tomato has a narrow genetic diversity that resulted from its intense selection and inbreeding during period of evolution and domestication thus, these species are more prone to disease epidemics. Among the fungal diseases early blight (EB) is one of the most catastrophic disease which is caused by Alternaria solani(Ellis and Martin) Jones and Grout. It may cause damping-off in the seedbed and a stem canker or collar rot that is destructive to transplants in the field (Sherf and MacNab, 1986; Walker, 1952)

    The fungus is cosmopolitan in nature. It can attack to all parts of the plant. The disease in severe cases can lead to complete defoliation (Peralta et al., 2005). This disease is prevalent and found to be destructive causing the yield losses to the tune of 48–80% (Datar and Mayee, 1984; Mathur and Shekhawat, 1986; Pandey and Pandey, 2002). The disease prevails in the areas of higher humidity, rainfall, and temperature (Sahu et al., 2013). The optimum temperature for fungal growth was 23–28°C and pH 6–8 was found to be optimum (Tong et al., 1994). Penetration can occur at temperatures between 10 to 25°C. This disease epidemics initially progress slowly but accelerate when plants attain maturity. The disease is capable of causing damage to all the aerial parts of tomato, such as stem, leaf and fruit and at all growth stages (Blancard et al., 2012).  It is very difficult to manage, due to its broad host range, extreme variability in pathogenic isolates and prolonged active phase of the disease cycle.

    This fungus produces unique club-shaped conidia, often beaked with horizontal and often vertical septa that may be produced either individually or in a chain, depending on the species. Hyphal cells are darkly pigmented with melanin, which protect hyphae and spores against environmental stress and allows spores to survive in soil for long periods of time.  The fungus overwinters in soil, plant debris, seed and alternate hosts in the form of either conidia or mycelia, which may serve as primary sources of inoculum.

    The variability studies are important tools to document the changes occurring in populations and individuals as variability indicate the existence of different pathotypes. A. solani reproduces asexually; a sexual stage of this fungus is unknown. Despite having asexual reproduction, the isolates of the pathogen A. solani causing early blight disease in tomato exhibit high level of variability within the population in morphological, cultural, biochemical criteria and genetic composition which indicates the possible existence of different patho-types (Van der waals et al., 2004). Such variation may arise out of heterokaryosis or mutation. Because of high variation, A. solani can easily adapt to the changing environment and develop resistance to fungicides. Besides, high variation also affects the rate of disease development and induces infection in promising host lines that have implication for stability of cultivar resistance resulting in higher risk of overcoming existing genetic resistance of the host challenging the control of early blight disease using a completely resistant cultivar (Adhikari et al., 2017). The existence of the high level of variability has been reported in many countries (Pryor and Gilbertson, 2002a; Pryor and Michailides, 2002b; Leiminger et al., 2016; Odilbekov et al., 2016; Mohammadi and Bahramikia, 2019; Chaerani et al., 2017). Morphological characterization is the classical approaches to distinguish fungal species that is one of the main requisites of fungal taxonomy. The present investigation was carried out to find a comprehensive understanding of this causal organism with reference to cultural, morphological and biochemical variation among the isolates of A. solani, from the major tomato growing areas of Karnataka.

  • Materials and Methods

    2.1.  Collection and isolation of the isolates

    The isolates of A. solani were collected from Kolar, Chikkaballapur, Ramanagara, Tumakuru, Bidar, Bagalkot, Chikkamagaluru, Mysuru and Bengaluru districts of Karnataka state during 2017–18. The tomato leaves infected with early blight disease from the above said places were used for the isolation of the fungus that was carried out as per the tissue segment methodology of Rangaswami (1958). The pathogen was purified using single spore isolation method (Riker and Riker, 1936). The identification was done through colony colour, morphology and spore characters. The pure culture of the pathogen was maintained on PDA slants at 27±1oC.

    2.2.  Designation of collected isolates

    The isolates of Alternaria solani collected from various sources from Karnataka were designated based on their locations and sources (Table 1). For example, an isolate designated by BiHH means this isolate was collected from Bidar district (Bi), Humanabad Taluk (H), Hallikhed village (H). 

    2.3.  Cultural variability

    Cultural variability studies were carried out on Potato dextrose agar, Czapek’s (Dox) agar, and V-8 juice agar. The media were prepared as mentioned in the “Ainsworth and Bisby’s Dictionary of Fungi” by Ainsworth (1961). The cultural variability among the isolates viz., colour of the colony, mycelial growth, type of margin, zonations and growth in terms of colony diameter were studied after 14 days of incubation.

    2.4.  Morphological variability

    For the purpose of studying variation in spore morphology of isolates of the pathogen, each isolate was grown on V-8 juice agar medium and incubated at 27°C for 14 days. The slides of the selected fungal cultures or colony were prepared in order to study the fungal morphology such as conidial length, breadth, number of septations and length of the beak. The prepared slides were observed under microscope. Ocular micrometer was calibrated by use of micrometry (Pramila et al., 2014). Conidia were harvested after 14 days from the   V-8 juice agar plates. Conidia were mounted in lacto phenol and measured at 40× magnification with the aid of ocular and stage micrometre in compound microscope (Tuite, 1969).

    2.5. Biochemical variations among the isolates of Alterneria solani

    2.5.1.  Preparations of enzymes extract  Culture condition

    Isolates were grown in 250 ml conical flask containing 100 ml potato dextrose broth. Each flask was inoculated with 2 mycelial discs, each of 5 mm diameter cut from the advancing margin of five-day old culture grown on potato dextrose agar at 25±1°C. The inoculated flask was incubated at 25±1°C for seven days. Then mycelia mat was harvested by filtering through Whatman No 1 filter paper, washed with Phosphate buffer (pH 7.0) and damp dried and frozen overnight at -20°C.  Enzyme preparations           

    The enzyme was extracted by grinding 1 g of freeze-dried mycelium by using pestle and mortar in liquid nitrogen then powder was dispensed and vortexed in 2 ml of 0.1M phosphate buffer (pH 7.0) (Chowdappa and Chandramohanan, 1995). Further centrifuged for 45 min at 10,000 rpm and a supernatant was collected. This supernatant was used for enzyme analysis.  Peroxidase (POD) assay

    Peroxidase activity was determined using the guaiacol oxidation method by Lin and Kao (2001). The reaction mixture contained 0.3 ml of 20 mM guaiacol, 2 ml of 100 mM potassium phosphate buffer (pH 7.0) and 0.2 ml enzyme extract. The reaction was initiated by the addition of 0.5 ml of H2O2 (1%). Increase in absorbance at 470 nm was recorded in 30 sec interval and upto three minute using UV-visible spectrophotometer. The POD activity was calculated by using an absorption coefficient (26.6 mM-cm) at 470 nm for the tetra guaiacol. Enzyme activity was expressed as μmoles of guaiacol oxidized/min /gram fresh wait (Unit g-1 FW).  Esterase assay

    For esterase activity measurement, naphthyl acetate were used as broad spectrum substrate for esterase according to Burlina and Galzigna (1972). The esterase activity was determined spectrophotometrically at room temperature (23 °C) by measuring the increase in Absorbance at 420 nm. The reaction solution contained 1.5 ml 0.1 M Tris/HCl pH 7.4 and 30 µL 100 mM naphthyl acetate dissolved in absolute methanol. For each measurement 200 µL of crude extract was used. Measurements were performed in 1.0 cm cuvettes every 15 seconds over a three-min-period. The esterase activities were corrected for spontaneous hydrolysis of naphthyl acetate. The activities were calculated using the extinction coefficient of naphthol (420 nm)=2,222 M-1 cm-1 (Rudnicka and Kochman, 1984). The blank contained the buffer and corresponding naphthyl acetate. The activity was expressed as µmol of hydrolysed substrate per minute and per gram of fresh weight (µmol min-1 g-1 FW).

    2.6.  Data analysis

    The collected data were compiled and analyzed statistically using the analysis of variance (ANOVA) technique with the help of a statistical software MSTAT-C (Freed and Scott, 1986).

  • Results and Discussion

    3.1.  Cultural variability

    3.1.1.  Colony characters on potato dextrose agar

    The colonies of the BiHH and TGJ isolates were whitish and pinkish white with cottony mycelial growth without any zonations (Table 2). The colonies of BaBG, KSC, RMT and CMH isolates appeared greyish with former three isolates showing cottony mycelial growth with zonation while the latter also had cottony growth but without zonations. The ChCN, BYC and MNY isolates produced light grey coloured colonies with the former two isolates showing cottony types of mycelial growth without zonations whereas the later had compact growth with slight zonations (Figure 1).

    Colony characters on Czapek’s (Dox) agar:  Except the BaBG isolate all other isolates did not show zonations (Table 3). The BiHH isolate produced grey coloured colonies with cottony mycelial growth whereas the CMH isolate colonies appeared dark grey in colour colonies with cottony mycelial growth. The colonies of the ChCN isolate appeared dull grey while it was whitish grey in RMT isolate with the former had cottony type of mycelial growth whereas the later has slightly compact growth (Figure 2).

    Colony characters on V-8 juice agar: The studies showed that all the isolates had no zones except BaBG and CMH isolates (Table 4). The BiHH isolate produced pink coloured colonies with cottony mycelial growth whereas the colonies of BaBG and CMH appeared pinkish white with zonations. The KSC, TGJ and Bangalore isolates produced grey coloured colonies without any zonations. The colonies of ChCN and MNY isolates appeared dull white in colour with no zonations while the RMT isolate had bright white colonies without any zonations (Figure 3).

    In the present studies, variability in cultural characteristics of different isolates of A. solani was noticed on three different solid media. Devi et al. (2017) found variability among the 16 isolates of A. alternata and four isolates of A. solani. Similar results obtained by Anil et al. (2017); Yadav et al. (2016); Waghunde et al. (2018) and Banne et al. (2021) which support our present findings. 

    3.2.  Variability in radial growth of the isolates of A. solani on different media

    The results on the variability in radial growth of different isolates of the pathogen are presented in Table 5 and the results on individual medium are explained hereunder.

    Among different isolates, maximum growth of 87.67 mm was noticed in CMH isolate on potato dextrose agar which was statistically significant over the growth of other isolates followed by BaBG (83.67 mm), BYC (78.33 mm), MNY (75.67 mm), RMT (70.00 mm) and TGJ (68.33 mm) isolates. The least growth was noticed in BiHH isolate (36.33 mm) followed by ChCN (54.00 mm) and KSC (55.67 mm) isolates. 

    On Czapek’s (Dox) agar medium, maximum growth of 90.00 mm was recorded in the isolates CMH and BaBG which were statistically superior over the growth of other isolates followed by BiHH (87.67 mm), RMT (86.67 mm), KSC (87.00 mm), ChCN (80.33 mm) and BYC (77.33 mm). Minimum growth (62.67 mm) was observed in TGJ followed by MNY (64.67 mm) isolates.

    In V-8 juice agar medium, maximum growth (75.67 mm) was recorded in BaBG isolate which was statistically significant over the growth of other isolates followed by TGJ (74.00 mm), BiHH (70.00 mm), RMT (69.33 mm), KSC and MNY isolates (62.67 mm). The least growth (46.67 mm) was recorded in CMH isolate followed by Bangalore isolate (52.00 mm).

    From the mean data of different media, it was observed that the medium Czapek’s (Dox) agar was superior as maximum growth (80.70 mm) of the pathogen irrespective of the isolates was noticed in this medium followed by Potato dextrose agar (67.74 mm). The least growth of the pathogen was noticed in V-8 juice agar (63.70 mm).

    In the present study, a lot of variability was found with respect to the radial growth of the different isolates of the pathogen A. solani. Mohsin et al. (2016) worked on variation studies in twenty-seven isolates of Alternaria porri and revealed that the isolates varied in colony diameter, colony and substrate colour, margin, topography, zonation, pigmentation and sporulation on different culture media. Similarly, Loganathan et al. (2016) observed variability in seventeen isolates of Alternaria spp based onculture colour and conidial dimensions. Results obtained by Banne et al. (2021), Mohsin et al. (2016), Aung et al. (2020); Luo et al. (2018); singh et al., 2014 and Pandey et al. (2021) are confirmatory to our present findings.

    3.3.  Morphological variability

    Nine isolates of Alternaria solani showed morphological variability in respect of conidial length, conidial width, beak length and number of septa (Table 6).

    Average conidial length varied from 27.55 to 39.32 μm (range: 25.07–42.90 µm). Maximum conidial length (42.90 μm) was noticed in ChCN isolate whereas minimum was observed in TGJ isolate (25.07 μm). The average conidial width varied from 15.21 to 19.93 μm (range: 10.53–21.52 μm). Maximum conidial width (21.52 µm) was recorded in ChCN isolate while the minimum was observed in BiHH isolate (10.53 μm). Average beak length varied from 7.64 to 12.73 μm (range: 2.4 to 20.36 μm). Maximum beak length (20.36 μm) was observed in CMH isolate and minimum was observed KSC isolate (2.4 μm). The average number of transverse septa varied from 2.8 to 5.8 (range: 2–7). Maximum number of transverse septa (7) was noticed in BYC isolate and minimum (2) was noticed in RMT and KSC isolates. The average number of longitudinal septa varied from 1.8 to 3.2 (range: 0–4). Maximum number of longitudinal septa (4) was noticed in RMT and MNY isolates whereas longitudinal septum was not noticed in CMH isolate. Thus, the studies on spore morphology revealed variation in morphology of the pathogen. The smallest size of conidia was observed in TGJ isolate whereas, that of largest size was noticed in ChCN isolate.

    In the present investigations, nine single-spore cultures of Alternaria solani showed morphological variability in respect of conidial length, conidial width, beak length and number of septa. The studies on spore morphology revealed that the smallest size of conidia was observed in TGJ isolate whereas, the largest size of conidia was noticed in ChCN isolate. Tymon et al. (2016) observed variability in conidial dimension and septa of Alternaria species associated with potato in the U.S. Northwest. Similar morphological variations among A. solani isolates had also been studied enormously by many researchers (Alhussaen, 2012; Singh et al., 2014; Verma et al., 2007; Kumar et al., 2008; Roopa et al., 2016; Parvin et al., 2021 and Pandey et al., 2021)

    3.4.  Biochemical variability

    The enzymes peroxidase and esterase assist in overcoming the stress conditions in pathogens created by climatic factors or chemical which are used in the management practices. Peroxidase detoxifies the H2O2 and esterase detoxifies chemicals. The primary defence response in the host plant against a pathogen involves the rapid generation of reactive oxygen species (ROS), also known as oxidative burst. ROS include reduced and chemically reactive molecules, such as superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), and hydroperoxyl radical (HO2) (Lehmann et al., 2015). ROS play an important role during the early stages of pathogen infection, which involves direct antimicrobial action, lignin formation, phytoalexin production, and SAR onset (Lamb and Dixon, 1997). The balance between ROS production and anti-oxidation is important for maintaining a healthy biological system (Davies, 2000). To mitigate the cell damage caused by ROS, plants express enzymes that scavenge excess ROS produced in cells under stressed conditions. These enzymes include superoxide dismutase, peroxidase, ascorbate peroxidase and catalase. Esterase helps in modification of cell wall during pathogen attack.

    In present studies, diversity of pathogen was assessed by estimating the peroxidase and esterase activity using spectrophotometer. Significant difference was found in the activity of these two enzymes in the isolates collected from different parts of Karnataka (Table 7; Figure 1). Peroxidase activity was higher in CMH isolate i.e., 81.80 Unit g-1 FW and least activity was found in BiHH isolate 11.78 Unit g-1 FW. Esterase activity was higher in BYC isolate 69.01 Unit g-1 FW and least activity was found in BaBG isolate 6.80 Unit g-1 FW. Similar results obtained by Shahbazi et al. (2010) on biochemical characterization of Alternaria solani isolates which showed variation with respect to peroxidase isoenzymes in different isolates collected from Iran. Similarly, Upadhyay et al. (2018) assessed the diversity among Alternaria solani isolates in tomato from India. Petrunak and Christ (1992) also noticed isozyme variability in isolates of Alternaria solani and Alternaria alternate.

  • Conclusion

    Nine isolates of Alternaria solani infecting tomato exhibited variation with respect to colour of the colony, mycelial growth, margin and zonations on diffent media. Among the media Czapek’s (Dox) agar supported maximum mycelial growth. All the nine isolates differed morphologically with respect to conidial length, conidial width, beak length and number of septa. In addition, isolates exhibited varying capacity to produce esterase and peroxidase. The Esterase activity was more in Bengaluru isolate whereas peroxidase activity was more in Chikkamagaluru isolate.

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