Morphological , cultural , pathogenic and molecular studies of Alternaria brassicae infecting cauliflower and mustard in India

Dark leaf spot (Alternaria brassicae) is one of the important diseases in crucifers causing serious yield and quality loss in production due to the seed borne nature of the pathogen. Variation in morphology and cultural characteristics among 32 representative Indian geographical isolates of Alternaria brassicae, the causal agent of Alternaria blight of cauliflower (Vegetable) rapeseed-mustard (Oil seed), was studied. All the isolates showed high level of variability in vitro in respect of conidial length, width, and number of septa. Conidia of Uttar Pradesh isolate (CaAbU4) were smallest in size with lowest number of septa. Substantial variation was found in mycelial growth, sporulation among these isolates in different nutrient media. All the isolates did not grow and sporulate abundantly on the same nutrient medium. However, Potato Dextrose Agar, Cauliflower (Host) Agar medium and Carrot Potato Agar were good for all the cultures. Variation in mycelial growth, sporulation was also observed. Cluster analysis of data on cultural variability among thirty two A. brassicae isolates found a close relationship among isolates of both origins viz, from Cauliflower and mustard. Isolates from Uttar Pradesh, Delhi, Haryana and West Bengal were found to be more similar to each other whereas the Rajasthan isolates along with Tamil Nadu and Kerala isolate were distantly related to others. All the isolates were pathogenic in nature but not directly related to the cultural and the morphological characteristics. These isolates were further molecularly characterized by using internal transcribed spacer region where all the isolates were found 56% similar to each other and 99% similar to the A. brassicae isolates present in NCBI database.

This species have the ability to survive in seeds for several months at different temperatures and relative humidity (Kumar and Gupta, 1994;Abul-Fazal et al., 1994).Losses up to 30% and 47% were caused by Alternaria brassicae in cauliflower (Brassica oleracea var botrytis) (Tamayo et al., 2001) and Indian mustard (Brassica juncea) (Chattopadhyay, 2008) respectively.Morphological characteristics of conidia and conidiophores and sometimes host plant association, provide the major taxonomic criteria for delimitation of fungal species (David, 1991).However, the classification of small spored species, including host-specific toxin producing fungi, has been particularly confused, because of the simple and convergent morphology of conidia and facultative parasitism, resulting in an ambiguous host range (Simmons, 1999).However, according to the study of Pattanamahakul and Strange (1999), the taxonomy of Alternaria on brassicas has been based principally on morphology and sometimes host plant association of each of the species occurring (A. brassicicola, A. brassicae and A. raphani) has a distinct morphology considering the diversity of conidium shapes and sizes among Alternaria spp.All commercial cultivars of brassicas are susceptible to this pathogen (Tewari, 1991).Till now no resistance are found among the crucifers against Alternaria brassicae.Development of resistant cultivars requires knowledge of pathogen variation present in different regions where these crucifers are grown.Severity of Alternaria blight on Brassicas differs among seasons and regions as also between individual crops within a region.This may be due to existence of variability among isolates of Alternaria species.Many reports on the existence of morphological variability within the isolates of other Alternaria species have been reported by earlier workers (Verma and Saharan, 1994;Varma et al., 2006).Variability in the morphological characteristics in A. brassicae isolates of different regions of India have been reported (Meena et al., 2005;Kaur et al., 2007;Singh et al., 2007;Goyal et al., 2011).
Some researchers have worked on cultural variability in Alternaria species in respect of mycelial growth and sporulation (Ansari et al., 1989), media (Patni et al., 2005).Variability on the basis of morphology, sporulation, growth and other cultural characteristics also have been reported earlier (Kaur et al., 2007).Morphological and cultural variability among the oilseeds Brassica isolates of A. brassicae from different geographical regions of India were reported from across the rapeseed-mustard growing region of India (Goyal et al., 2011).However variability among the A. brassicae isolates from vegetable crop like Cauliflower is still missing.
The molecular approaches have been used increasingly in taxonomy and systematics of filamentous fungi including phytopathogens at the species and subspecies level (Benali et al., 2011).Variation in nuclear ribosomal DNA sequences among Alternaria species pathogenic to crucifers has recently been reported from A. brassicae, A. brassicicola (Schwein) Wiltshire, A. raphani J. W. Groves & Skolko and A. alternata (Fr.)Keissl.(Jasalavich et al., 1995).
The objective of this study was to evaluate isolates of A. brassicae collected from both crucifer crops cauliflower (vegetable) and mustard (oilseed) at morphological and cultural level and further relate at molecular level.

Collection and maintenance of Alternaria brassicae isolates from black spot infected leaf samples
Alternaria brassicae isolates were obtained from black leaf spot infected leaf samples of both cauliflower and mustard (Table 1) collected during winter season in 2009 to 2011 from seven different regions of India namely Delhi, Uttar Pradesh, Rajasthan, Haryana, West Bengal, Tamil Nadu and Kerala.To obtain isolates from infected cauliflower and mustard leaves and other plant tissues, blighted leaf and stem pieces (2mm) were surface sterilized with 0.1% Mercuric Chloride (HgCl2) for one minute, rinsed in sterile water 3x for 10 min and placed on Potato Dextrose Agar (PDA) plates.Fungal growth was observed after 5-7 days of incubation at 25°C. A. brassicae isolates growing on the leaf/stem pieces were transferred onto other PDA plates until purification by single spore technique.These isolates were purified and preserved as PDA slants at 4°C.

Single spore isolation
A total of 32 putative single spore Alternaria colonies were randomly picked from the lesions on infected plant parts under binocular microscope, and seeded onto the surface of water agar using tip of a sharp sterile inoculating needle.Inoculated plates were incubated on laboratory bench at room temperature (20-24°C).Conidial germination on the plates was checked daily and upon germination, agar blocks bearing single germinated conidia were cut out and aseptically seeded on PDA plates.The plates were incubated for 14 days at room temperature and natural lighting conditions (20-24°C and 12-h light).Plates of primary media were centrally inoculated with 2-mm diameter plugs taken from the edge of actively growing 4 day old cultures, and then incubated at 25°C after 72 h, the colony diameters were measured and agar blocks were transferred to the fresh media.Spore yields were determined with a haemocytometer after 24 h of incubation on different media.

Evaluation of morphological characters
All the isolates were microscopically identified based on their morphology on PDA using light microscope (Carl Zeiss, Germany) and available literature (Ellis, 1971).For all isolates, morphological characteristics of the colony and sporulation apparatus were determined from single-spored colonies as described earlier.The nature of mycelia growth, shape of conidia was noted.The size and shape of conidia (length and width) was determined using ocular and stage micrometer.Numbers of septa were also recorded.

Effect of different types of media type on growth and sporulation
The objective of this experiment was to establish the suitable growth conditions for the in vitro growth and sporulation of Alternaria pathogen.Investigation was done on growth rate and conidia formation of the Alternaria pathogen.Seven types of media were tested to determine their effect on growth and sporulation of the pathogen.These were potato dextrose agar (PDA), Cauliflower agar media (CAM), Carrot Potato Agar (CPA), Oat Meal Agar (OMA), Czapex Dox Agar (CDA), V8-Juice Agar (VJA) and Corn Meal Agar (CMA).The different media served the purposes of growing of cultures, cleaning of cultures and sporulation of cultures.Growth media for Alternaria species was i) Potato dextrose agar (PDA) made of Dextrose (20 g), agar (15 g) and Potatoes infusion from 200 g in 1000 ml water, ii) Cauliflower Agar Media (CAM) made of Dextrose (20 g), agar (15 g) and Cauliflower infusion from 200 g in 1000 ml water, iii) Carrot Potato agar (CPA) 24 g in 1000 ml distilled water, iv)Oat Meal Agar (OMA), 38 g in 1000 ml distilled water, v) Czapex Dox Agar (CDA) 49 g in 1000 ml distilled water, vi) V8 Juice Agar (VJA) 44.3 g in 1000 ml distilled water and vii) Corn Meal Agar (CMA) 17 g in 1000 ml distilled water.All synthesized media were of Himedia.The agar plates were autoclaved at 121°C for 15 min and approximately 20 ml were dispensed into sterile 90 mm plastic Petri dishes to form a layer of 2 mm deep.The media was inoculated using a uniform culture plug of 4 mm in diameter obtained from 14-day-old culture plates and placed in the centre of each dish.The plates were incubated at room temperature.The experiment was arranged in a completely randomized design with 3 replicates.Fungal growths (Radial growth in cm) of different isolates were studied.Measurements on radial colony diameter were taken on 7th day after inoculation whereby six plates were sampled each time for each media treatment.

Measurement of fungal sporulation
To determine conidial concentration of each isolates, cultures grown in the seven different media plates were considered.Ten milliliter (10 ml) of sterile distilled water was added to culture plate and using a sterile glass slide, the culture surface was gently scrapped to make a conidial suspension.Conidial concentration was determined using a haemocytometer.

Pathogencity testing of A. brassicae isolates
Twenty-two A. brassicae isolates from Cauliflower were tested for symptom production on a susceptible cultivar of Cauliflower viz., DC-23000.Similarly ten A. brassicae isolates from Mustard were tested on a susceptible variety of mustard viz., Pusa Jagganath.The seeds of cauliflower were sown in nursery beds during October, 2009October, -2010October, and 2010October, -2011 seasons.Cauliflower Seedlings were transplanted after two weeks in fields with a spacing of 30 cm x 40 cm.Similarly mustard seeds were sown on fields.After 60 days leaves from both the cauliflower and mustard plant were taken for the detached leaf inoculation method.Test Leaves were properly washed under running tap water and then surface wiped off with 70% alcohol and 2 microlitres of 4 x10 4 spores ml -1 spores were inoculated with a fine needle (Dispovan, 2.5 ml) while sterile distilled water was applied on control.The leaves were placed inside moist chambers in green house conditions and were observed for appearance of disease symptoms on 7 day after inoculation.Experiments were conducted in completely randomized design (CRD).Each treatment consisted of three replicates.Symptoms observed were ranked as minus (-) for no symptom and plus (+) for black leaf spot with yellow halos on inoculated leaves.Appearance of symptom again divided into three groups viz., black spot with diameter 0.2-0.5cmranked as single plus (+), spot with diameter 0.6-1.0cmranked as double plus (++) and spot diameter more than 1cm were ranked as three plus sign (+++).

DNA extraction and purification
All the 32 A. brassicae (22 from cauliflower and 10 from mustard) isolates were grown on Potato Dextrose Broth (PDB) medium in Sharma et al. 3355 100 ml capacity conical flask for 7 days at 28°C.Mycelial mats were harvested by Whatman No.1 filter papers and lyophilized.DNA was isolated according to a modified Cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1990).Around 300 mg of lyophilized fungal material was ground in liquid nitrogen, dispersed in 800 µl of 2% CTAB extraction buffer at 65°C in water bath for 30 min.An equal volume of Chloroform: isoamyl alcohol (24:1, v/v) was added, mixed well and centrifuged (4000 rpm, 10min).The upper aqueous phase was transferred to a fresh tube.Nucleic acids were precipitated by adding 0.6 volume of ice cold isopropanol, and collected by centrifugation (14000 rpm for 30 min).Pellet was washed twice with 70% ethanol and then air dried by putting the tube face down on a paper towel.

Internal transcribed spacer (ITS) region analysis
Thirty two A. brassicae isolates were analyzed by amplifying the regions of the rDNA repeat from the 3'end of the 18s and the 5' end of the 28s gene using PCR conditions with the two universal primers, ITS1-3' TCC GTA GGT GAA CCT GCG G 5' and ITS4-3' TCC TCC GCT TAT TGA TAT GC 5' which were synthesized on the basis of conserved regions of the eukaryotic rRNA gene (White et al., 1990;Jasalavich et al., 1995).The PCR-amplification reactions were performed in a 25 µl mixture containing 50 mM KCl, 20 mM Tris HCl (pH 8.0), 2.0 mM MgCl2, 20 µM of each of the four deoxynucleotide triphosphates, 20 pmol of each primer, 50 ng/µl of template and 2.5 U of Taq polymerase.These reactions were subjected to an initial denaturation hot start at 94°C for 4 min, followed by 40 cycles of denaturation at 94°C for 35 s, primer annealing at 60°C for 1 min 30 s and primer extension at 72°C for 3 min and a final extension for 10 min at 72°C in a thermal cycler.Aliquots (4 µl) of the amplified products were analysed by electrophoresis in 1.5% (wt/vol) agarose gel in 1X TAE buffer (40 mM Tris, 20mM acetic acid, 1mM EDTA [pH8]), stained with ethidium bromide (1 µg/ml) and electrophoresis was carried out at 70 volts for 2 h in TAE buffer.The molecular marker was 100 bp ladder (Biomatrix Co. Ltd.).The desired bands were cut from the gel with minimum quantity of gel portion and the amplified PCR product was eluted using QIAGEN DNA gel extraction kit.

Nucleotide sequencing and in silico analysis
The sequencing of the PCR product was carried out in automated Sequencer at Xcelris Lab., Bangalore, India.Related sequences were searched for homology using BLAST bioinformatic search tool available at the Gen-Bank database (http://www.ncbi.nlm.nih.gov/blast/)(Altschul et al., 1997).Sequences used for comparison were obtained from NCBI database (http://www.ncbi.nlm.nih.gov) and the details are given in the results.
The multiple sequence alignment and pair wise alignment were performed using the Clustral W algorithm in Bioedit (Hall, 1999) and Phylogenetic analysis was done using MEGA5.0(Tamura et al., 2011) software.To assess the possible phylogenetic relationship neighborhood-joining bootstrap tree was created using CLUSTAL W 1.6 matrix by the CLUSTAL X programme ver.1.81 (Thompson et al., 1997).The ITS sequences were submitted to GenBank.Sequences used for comparison were obtained from NCBI database (http://www.ncbi.nlm.nih.gov) and the details are given in the results.

Statistical analysis
Averages of colony diameter on each media plates and number of conidia/ ml for each isolates were taken for subsequent data analysis.Analysis of variance and separation of means to determine differences in growth rates on media types was analyzed by performing two-way ANOVA using statistical software PRISM version 3.0 at p<0.0001.Similarly the conidial characters were also statistically analyzed at p<0.05 by performing one way ANOVA and least significant difference was calculated by student's t-test.

Morphological characterization
Differences in rates of linear growth and colour of the colony were observed among all 32 isolates of A. brassicae (Table 1; Figure 1) obtained from the infected Cauliflower and Mustard leaves.The color of the colonies and the conidia grown on PDA showed little variation.The color of the A. brassicae isolates varies between light olive gray to olivaceous black.The mycelia colour varies between brown and golden.The conidia characteristics were also similar to each other among the isolates viz., conidia color was golden or brown with mostly smooth surface.Most of the conidia were long obpyriform in shape with long beak.
The 32 single-spore cultures of A. brassicae showed significant (P < 0.05) morphological variability in respect of conidia length, conidia width and number of septa (Table 2; Figure 1).Average conidial length, which varied from 37.88 to 57.65 μm, was highest in West Bengal cauliflower isolate (CaAbW1) that is, 57.65 μm and lowest in Delhi mustard isolate (MAb2) that is, 37.88 μm.Average conidial width, which varied from 6 to 9.5 μm, was highest in West Bengal cauliflower (CaAbW2) isolates that is, 9.5 μm and lowest in Delhi cauliflower isolate (CaAbD1) that is, 6 μm.The average number of transverse septa, which varied from 2.33 to 6, was highest in Delhi cauliflower (CaAbD4) isolates that is, 6 and lowest in Delhi mustard isolate (MAb6) that is, 2.33.

Effect of different media on sporulation
Sporulation of each A. brassicae isolates on the 7 th day on different media was almost similar.The lowest sporulation was observed for the isolates of Uttar Pradesh (CaAbU2, CaAbU3), Delhi (CaAbD3) and Haryana (CaAbH1) which was in a range 2.67-6.33x 10 4 /ml.Moderately sporulating within a range of 7-10 x 10 4 /ml were   The size and shape of conidia (length and width) was determined using ocular and stage micrometer.Numbers of septa were also recorded.
Cluster analysis also revealed cultural variability among thirty two A. brassicae isolates and found a close relationship among Delhi and Uttar Pradesh isolates in respect of  mycelial growth and sporulation on different nutrient media.

Pathogencity
All the A. brassicae isolates both from cauliflower and mustard were found to be pathogenic in nature (Table 3).

ITS analysis
DNA was successfully extracted from the fungal isolates using CTAB method and yielded the PCR products of amplicons of ~550 to 600bp.Preliminary fingerprinting of the 32 isolates using internal transcriber spacer primers ITS1-5'TCC GTA GGT GAA CCT GCG 3' and ITS4-5' TCC TCC GCT TAT TGA TAT GC 3' confirmed that these isolates to be of Alternaria species (Figures 4 and   5).Analysis of the ITS regions revealed 90-100% identity among the thirty two isolates in the A. brassicae species group.All the ITS sequences were submitted to NCBI database (Table 1).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987).The optimal tree with the sum of branch length = 0.20085009 is shown.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches (Felsenstein, 1985).The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site.The analysis involved 37 nucleotide sequences.Codon positions included were 1st+2nd+3rd+Noncoding.All positions containing gaps and missing data were eliminated.There were a total of 513 positions in the final dataset.Evolutionary analyses were conducted in MEGA5 (Tamura The phylogenetic tree (Figure 6) formed three distinct clusters of the A. brassicae isolates but there are not of much difference.First cluster comprises of only one isolate that is CaAbD2, second group was formed constituting only two isolates of Rajasthan origin and cauliflower as source plant of isolation viz., CaAbR3 and CaAbR4 which are 99% similar to each other.The third group containing the rest of all isolates having >56% similarities with each other.When some other accessions of A. brassicae were considered from NCBI it showed a close relationship with the isolates in the present study.

DISCUSSION
In a recent report Goyal et al. (2011) reported variation in conidial morphology, mycelial growth, sporulation of thirteen isolates of A. brassicae collected from different geographical zones were dependant on temperature and geographical origin.Similarly, variability in the morphological characteristics in A. brassicae isolates of different regions of India have been reported (Meena et al., 2005;Kaur et al., 2007;Singh et al., 2007).Some researchers have worked on cultural variability in Alternaria species in respect of mycelial growth and sporulation (Ansari et al., 1989;Patni et al., 2005;Kaur et al., 2007).Cauliflower and mustard are winter crops and are affected by A. brassicae.This present study was to understand the variability among the isolates infecting both the crops grown in different states of India on basis of morphology, cultural, pathogenic and molecular level.Significant variation in growth, sporulation and conidial morphology of A. brassicae isolates were found on different nutrient media irrespective of crop and geographical states.All the isolates were found pathogenic in nature against their respective host.
Due to the systematic and taxonomic usefulness the ITS region has been used in classifying fungi (Chillali et al., 1998) as it is suitable size for PCR amplification, restriction analysis and sequencing procedures, and because ITS regions are variable among species as well (Jung et al., 2002).Molecular relationships amongst Alternaria species based on nuclear ribosomal DNA and hostspecific toxins (Kusaba andTsuge, 1994, 1995) or with other related fungi have been analyzed (Pryor and Gilbertson, 2002;Chou and Wu, 2002).Variation in nuclear ribosomal DNA sequences among Alternaria species pathogenic to crucifers has been reported from one isolate each of A. brassicae, A. brassicicola, A. raphani and A. alternata (Jasalavich et al., 1995).BLAST analysis of the internal transcribed spacer region of all thirty two A. brassicae isolates in this study showed high similarity among the isolates with A. brassicae of the NCBI database.
All the thirty two isolates of Alternaria brassicae though different at cultural and morphological level were found to be pathogenic in nature.Irrespective of the host and geographical origin the A. brassicae isolates are dispersed in the whole country sharing a single gene pool.

Figure 1 .
Figure 1.Spore morphology and cultural characters on PDA plates and microscopic images of Alternaria brassicae isolates.

Figure 1 .
Figure 1.Spore morphology and cultural characters on PDA plates and microscopic images of Alternaria brassicae isolates.

Figure 2 .
Figure 2. Dendrogram showing cultural variability in mycelial growth of 32 Alternaria brassicae isolates at different nutrient media.

Figure 3 .
Figure 3. Dendrogram showing cultural variability in sporulation of 32 Alternaria brassicae isolates at different nutrient media.

Figure 4 .
Figure 4. Amplification of the internal transcribed spacer region of the twenty two A.brassicae isolates collected from the infected cauliflower host from different part of India with the universal primer ITS1 and ITS4.Lane M: 100 bp ladder (GeneDirex RTU, Biochem Life Science, India).

Figure 5 .
Figure 5. Amplification of the internal transcribed spacer region of the ten A.brassicae isolates collected from the infected cauliflower host from different part of India with the universal primer ITS1 and ITS4.Lane M: 100bp ladder (Gene Direx RTU, Biochem Life Science, India).

Figure 6 .
Figure 6.Dendrogram representing Internal Transcribed Spacer region of thirty two Alternaria brassicae isolates.

Table 1 .
Morphological and cultural characteristics of Alternaria brassicae isolates (32) collected from Cauliflower and Mustard grown in different states of India.
Ca: Cauliflower, M: Mustad, Ab: Alternaria brassicae, U: Uttar Pradesh, D: Delhi, R: Rajasthan, W: West Bengal, H: Haryana, K: Kerala, T: Tamil Nadu.All the morphological characters such as culture appearance on PDA plate, Mycelial colour, conidia appearance were observed and data were collected.Accession numbers of ITS sequences of the A. brassicae isolates deposited to NCBI database.

Table 3 .
Pathogenicity testing of A. brassicae isolates on the respective hosts cauliflower and mustard.