Morphological, cultural, pathogenic and molecular variability amongst Indian mustard isolates of Alternaria brassicae in Uttarakhand

Alternaria blight (Alternaria brassicae) causes severe foliar damage to Indian mustard in Uttarakhand. Ten (10) isolates of A. brassicae were collected from different hosts and characterized for cultural, morphological, pathogenic and molecular variations. A. brassicae colonies varied in their cultural behaviour ranging from cottony, flurry to feathery, with smooth to rough margins. Colour of colonies ranged between white, off white to light brown. Colony growth varied from slow, medium to fast with fast growth in isolate KM and least in JD on the potato dextrose agar (PDA) medium. Significant morphological variations in conidia length, conidia width, and number of horizontal septa were observed in the isolates. Average conidial size ranged from 105 to 135 × 10 to 20 μm. Isolates exhibited variations in disease index, number and size of the lesions. The dendrogram analysis, based on molecular (random amplification of polymorphic DNA, RAPD) basis revealed two groups at 15% similarity coefficient. Group I was composed of seven isolates namely, VR, DV, P7, LM. P10, KR and ND with 18% similarity (82% dissimilarity) while group II was composed of only three isolates namely, JD, KA and AS with only 24% similarity (76% dissimilarity).


INTRODUCTION
Alternaria brassicae (Berk.) Sacc., is an important necrotrophic pathogen causing Alternaria blight disease in Indian mustard [Brassica juncea (L.) Czern and Coss.]. It is very difficult to manage the disease, due to no proven source of resistance reported till date in any of the hosts (Meena et al., 2010b). The yield loss due to this pathogen is up to 47% in the entire mustard growing area (Meena et al., 2010a). One of the significant aspects of biology of an organism is the morphological and physiological characters of an individual within a species, which are not fixed. This holds true with fungi also, although it is not frequent in asexually produced individuals of the progeny. Variability studies are important to document the changes occurring in populations and individuals as variability in morphological and physiological traits indicate the existence of different pathotypes. Alternaria blight severity on oilseed Brassicas differ season to season, region to region and also individual crop to crop in India . This might be due to the existence of variability among geographically similar isolates of A. brassicae. The variability is a well known phenomenon in genus Alternaria and may be noticed as changes in spore shape and size, growth and sporulation, pathogenicity, etc. Diversity appears even in single spore isolates.
Many reports on the existence of variability among different Alternaria species from different hosts have been reported by earlier workers (Pryor and Gilbertson, *Corresponding author. E-mail: gohartajkhan@rediffmail.com. 2002; Pryor and Michailides, 2002;Quayyum et al., 2005;Kumar et al., 2008) as also within A. brassicae species (Kaur et al., 2007). Recently, Meena et al. (2012) studied the aggressiveness, diversity and distribution of A. brassicae isolates infecting oilseed Brassica in India. Variation in pathogen populations can generally be detected with methods like morphological, cultural, pathogenic and molecular specificity. DNA markers have become a powerful tool to study taxonomy and molecular genetics of a variety of organisms. The Random Amplified Polymorphic DNA (RAPD) allows quick assessment of genetic variability, and has been used to study inter-and intra-specific variability amongst the isolates of several fungal species. Reports are available who studied the genetic variation within and between Alternaria species by random amplified polymorphic DNA (RAPD) molecular marker Tewari, 1995, 1998;Pryor and Michailides, 2002;Kumar et al., 2008). Since the crop and disease are of paramount importance to the Uttarakhand state and no studies on pathogenic and genetic variability have been conducted. Keeping this in mind, the present investigation focused on morphological, cultural, pathogenic and molecular variability of ten mustard isolates of A. brassicae in Uttarakhand.

Collection of A. brassicae isolates
Plant material infected with A. brassicae was sampled randomly from different cultivars of B. juncea grown in the field of Crop Research Centre of G. B. Pant University of Agriculture and Technology, Pantnagar Uttarakhand, India. The isolates of A. brassicae were collected and designated as BJABI stands for Brassica juncea Alternaria brassicae isolates (Table 1). These selected infected spots were washed 3 to 4 times in sterilized distilled water and then surface sterilized by dipping in 4% NaOCl solution for 1 min, followed by washing with sterilized water 3 to 4 times. Surface sterilized leaf spot pieces were then aseptically transferred into 9 cm Petri dishes containing potato dextrose agar (PDA) and incubated at 25±2°C for seven days. Thereafter, growing mycelia from margin of apparently distinct colonies of the leaf spot pieces on the medium were aseptically transferred into another Petri plate containing PDA medium, where it was grown for 15 days at 23±2°C in the BOD incubator. On the basis of their conidiophore and conidial morphology as described by Simmons (2007), the pathogen was identified as A. brassicae (Berk.) Sacc. and purified by single spore isolation method. The isolated fungal pathogen cultures were maintained on PDA slants at 4°C.

Morphological variability of different isolates of A. brassicae
Ocular micrometer was calibrated and by use of micrometry , morphological variability among the 10 isolates of A. brassicae was studied in 2010 to 2011. Total of thirty conidia from each slide were examined at 40X magnification of light microscope and measured using ocular and stage micrometer. The average was used to calculate the conidial length, width and number of horizontal septa.

Cultural characteristics of different isolates of A. brassicae
The culture character was recorded on day 10 of inoculation of all isolates of A. brassicae. Characters like colony color, appearance, growth, shape, margin, sporulation and zonation were recorded by direct observation of culture-grown Petri plate on PDA which was incubated in B.O.D. incubator at 25°C temperature and 100% relative humidity.

Pathogenic variability of different isolates of A. brassicae
In order to confirm the identification of the disease and its causal agent, the pathogenicity test was conducted under polyhouse conditions in pot experiments using B. juncea cultivar Divya. Seedlings were raised in pots filled with sterilized soil. Spores from the colony were scraped in autoclaved distilled water and spore suspension of 2 × 10 3 spores/ml concentration was prepared (Giri et al., 2013). Such spore suspension of pathogenic inoculum of the isolates (AS, KA, LM, ND, P7, P10, and VR) was sprayed on 3rd/4th true leaves of 30 days old plant of B. juncea cultivar Divya by drop plus agarose artificial inoculation method (Giri et al., 2013). Three quantitative characters namely, disease index, average number of spot/10 cm 2 and average spot size (cm) were recorded on leaves at different time intervals after pathogen inoculation.

Molecular variability of different isolates of A. brassicae
Molecular variability among ten single spore cultures of A. brassicae was analysed by RAPD molecular marker. Genomic DNA of ten single spore cultures of A. brassicae was isolated separately by using standard cetyl trimethyl ammonium bromide (CTAB) extraction method of Doyle and Doyle (1990). Molecular variability among A. brassicae isolates were studied by using twenty six RAPD primers from Life Tech Company (Table 2). The polymerase chain reaction (PCR) master mix was prepared with 1X Taq polymerase buffer, 1.8 mM MgCl 2 , 0.4 mM dNTPs, 0.4 pM primers and 1.5 U of Taq polymerase. Thereafter, 20 µl of master mix was added with 5 µl (50 ng) DNA in PCR tubes. Forty PCR amplification cycles were carried out in PCR machine [Eppendorf, Germany; model: Mastercycler (R) family] by denaturation at 94°C for 1 min, annealing at 37°C for 1 min and extension at 72°C for 1 min. Each PCR amplification reaction was preceded by an initial denaturation at 94°C for 4 min followed by final extension at 72°C for 10 min. The amplified products were separated by electrophoresis in 1.5% (w/v) agarose (Genei, Bangalore) gel with 1X TBE buffer, stained with ethidium bromide (0.5 μg/ml) at 90 V for 3.0 to 3.5 h and photographed using gel documentation system (Alpha Innotech, USA Alpha Innotech, USA; model: AlphaImager TM 3400). The sizes of the amplification product were estimated using 100 bp to 3.0 kb ladder (Ф × 174 DNA/ BsuRI (Hae III), Fermentas. All the reactions were repeated in at least two independent experiments. All the amplified bands were scored as present or absent for each DNA sample and further, the RAPD reaction results were analyzed using software Gene Profiler. In order to analyze the relatedness among the species, a dendrogram based on unweighted pair group method with arithmetic average (UPGMA) and Nei and Li genetic distance matrix (Nei and Li, 1979) value was obtained.

Cultural characteristic of A. brassicae isolates
Isolates of A. brassicae showed variable cultural characteristics like colony color varied from white, off white to light brown, appearance of the colony from cottony, flurry to feathery, colony growth varied from slow, medium to fast, colony margin from wavy, smooth to rough ( Figure  2). Based on these characteristics, all A. brassicae isolates could be grouped into three colony types. Group 1 isolates (DV and P7) produced white colonies with a fluffy appearance.
The colony was circular in shape with smooth margins. Group 2 isolates (KA, LM, ND, P10 and VR) produced off white colonies with a cottony and feathery appearance. The colony was circular in shape with all types of margins. Group 3 isolates (AS, JD and LR) produced light brown colonies with cottony appearance. The colony was circular in shape with wavy and rough margins (Table 4). Such kind of variability among the different A. brassicae isolates were also reported by Vishwanath (1999) and Meena et al. (2012).

Pathogen aggressiveness of A. brassicae isolates
Different isolates of A. brassicae showed variable response on host B. juncea cultivar Divya. Variation in the disease index (Figure 4a), average number of spots/10 cm 2 (Figure 4b) and average size of spot (cm) (Figure 4c) on same host depending on aggressiveness of isolates revealed that the variability exist among A. brassicae isolates. ND was found to be the most aggressive whereas AS was found to be the least aggressive isolate. A similar study was conducted by Michereff et al. (2003) who studied 38 isolates of Alternaria brassicicola and estimates variability based on disease development and pathogen physiology and found that A. brassicicola isolates were highly variable. In another study, Kaur et al. (2007) reported the pathogenic variability among A. brassicae isolates considering only percent disease severity.

Molecular variability of A. brassicae isolates
Analysis by 26 RAPD primers revealed a high level of genetic variability among ten isolates of A. brassicae of different cultivars of B. juncea. Amplification of DNA of all the A. brassicae isolates produced 1014 scorable and reproducible RAPD markers. On an average, 39 bands were produced. The dendrogram prepared by using the similarity coefficients ( Figure 5) clustered the ten representative isolates into two major groups that is, Groups I and II at only 15% similarity coefficient (85% dissimilarity). Group I was composed of seven isolates namely, VR, DV, P7, LM. P10, KR and ND with 18% similarity (82% dissimilarity) while group II was composed of only three isolates namely, JD, KA and AS with only 24% similarity (76% dissimilarity).
Seven isolates of the group I were sub-clustered into two minor clusters, of which one was composed of six isolates namely, DV, P7, LM. P10, KR and ND with 24% similarity (76% dissimilarity) while another was composed   of remaining one isolates VR 18% similarity (82% dissimilarity). Likewise, group II was sub-clustered into two minor clusters, of which one was composed of two isolates namely, KA and AS with 27% similarity (73% dissimilarity) while another was composed of remaining one isolates JD with 23% similarity (87% dissimilarity). The present results indicated high genetic divergence among the 10 isolates of A. brassicae. Polymorphism within an Alternaria species by RAPD molecular marker has been described by many workers Tewari, 1995, 1998;Kumar et al., 2008). Sharma and Tewari (1995) observed polymorphism among A. brassicae isolates from different geographical regions of the world. However, in 1998 they found low intra-regional variation among Indian and Canadian isolates of A. brassicae with 75% similarity among them. Although, the genus Alternaria is known as an imperfect fungus, it shows genetic variability within a species and this variability might be due to the existence of mutation, somatic hybridization, heterokaryosis, uniform host selection, extensive dispersal or of a cryptic sexual stage. High degree of genetic variability was observed among only ten isolates of A. brassicae from different B. juncea cultivars growing in Pantnagar region of Uttarakhand. This could be the probable possible reason behind extreme and different disease reaction of germplasm at Pantnagar from observations at most of other locations. In order to provide a better picture of the pathogenic as well as genetic divergence among A. brassicae populations of India, there is need to conduct similar holistic investigation among higher number of A. brassicae isolates which could be helpful to generate resistant material against Alternaria blight in oilseed Brassicas.

Conclusion
The variation in cultural, morphological, pathogenic and molecular characters of isolates observed indicated the existence of different strains of pathogen. Similar characters have formed the basis for defining the existence of different strains among the species of fungi imperfection.