Alternaria blight caused by Alternaria brassicae (Berk.) Sacc. is an important fungal problem that can severely affect the foliage and seed germination in crucifers (Kolte 1985; Tewari 1991; Verma and Saharan 1994). The pathogen can survive in seeds for several months at different temperatures and relative humidity (Abul-Fazal et al. 1994; Kumar and Gupta 1994) and spreads during the growing season by wind-blown or rain-splashed spores (MacKinnon et al. 1999). Among the different hosts of A. brassicae in India, mustard [Brassica juncea (L.) Czern] (Kumar 1997) suffered around 20.28 % losses, and cauliflower (Brassica oleracea L. var. botrytis) also suffered severe damage from germination through harvest, storage, and transport in India. Similarly, losses up to 30 % in cauliflower by Alternaria brassicae in Columbia and South America have also been recorded (Tamayo et al. 2001).

Genetic analysis of plant pathogen populations is fundamental to the understanding of epidemiology, host–pathogen coevolution, and resistance management (Milgroom and Fry 1997). PCR-based marker techniques have been used extensively for genotypic identification of phytopathogens at the species and subspecies level. In addition, genetic variation in populations of Alternaria species pathogenic to crucifers has been characterized by random amplified polymorphic DNA (RAPD) (Sharma and Tewari 1995, 1998).

Intraspecific and interstrainal variation among the isolates of Alternaria species, however, is not yet clear. The present study was aimed to reveal genetic variability among the Alternaria brassicae isolates collected from two important infected hosts, cauliflower and mustard, by using molecular markers such as RAPDs, inter-simple sequence repeats (ISSRs) and internal transcribed spacer (ITS). Alternaria brassicae isolates (Table 1) were collected during the winter in 2009 and 2010 from seven states of India viz., Delhi, Uttar Pradesh, Rajasthan, Haryana, West Bengal, Tamil Nadu and Kerala (see online Supplemental Fig. 1), and a single-spore isolate of each was tested for pathogenicity and virulence on detached leaves of their respective and alternate hosts (Sharma et al. 2004). Twenty-two A. brassicae isolates from cauliflower and 10 A. brassicae isolates from mustard were tested for symptom severity on a susceptible cultivar of cauliflower; DC-23000 (Division of Vegetable Sciences, Indian Agricultural Research Institute, New Delhi, India), and of mustard, Pusa Jagganath (Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India). Pathogenic isolates were categorized into two groups based on symptom severity: (1) less virulent (+), causing black spots with a diameter less than 0.5 cm and (2) highly virulent (++), causing spots with a diameter more than 0.5 cm. All isolates from cauliflower and from mustard proved pathogenic and cross inoculable, i.e., they produced symptoms on their respective susceptible host and on the alternate host (Table 1). Cauliflower isolates comprised both less virulent and highly virulent, whereas all mustard isolates were highly virulent.

Table 1 Isolates of Alternaria brassicae collected from different parts of India, NCBI GenBank accession numbers for their ITS sequences and virulence on detached leaves of susceptible cultivars of cauliflower (DC-23000) and mustard (Pusa Jagganath) 7 days after inoculation
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Fig. 1
figure 1

Dendrogram generated by the unweighted pair group method with arithmetic means (UPGMA) of Jaccard’s similarity coefficients based on the amplicons from the 32 isolates of Alternaria brassicae using RAPD primers

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Genomic DNA was isolated according to a modified cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1990). RAPD analysis of all 32 A. brassicae isolates were done using 42 10-mer oligonucleotide primers (Operon Technologies, Alameda, CA, USA) to identify polymorphic amplicons (see online Supplemental Table 1). Similarly, five ISSR primers (SBS Genetech) were used to identify polymorphism. Data were compiled as a binary 0/1 matrix by the presence (1) or absence (0) of a band at a particular position. Only major RAPD and ISSR bands were analyzed statistically. The genetic associations between isolates were evaluated by calculating the Jaccard’s similarity coefficient for pairwise comparisons based on the proportion of shared bands produced by the primers (Kosman and Leonard 2005). Dendrograms were constructed using the nested unweighted pair group method and the arithmetic averages (UPGMA) cluster of NTSYS-PC version 2.02h software (Applied Biostatistics, Setauket, NY, USA). Genetic similarity among the 32 isolates (Table 1) were assessed using 121 polymorphic bands (150–900 bp) amplified by 42 10-mer RAPD primers. The similarity among the A. brassicae isolates ranged from 45 to 100 %. Some isolates were 100 % similar to each other in producing similar polymorphic bands. The mean value of the Jaccard’s similarity coefficient of the RAPD marker was 0.73. RAPD-PCR fingerprint data in the dendrogram (Fig. 1) showed a close genetic relationship among the isolates of A. brassicae. Similarly, of five ISSR primers screened (see online Supplemental Table 1), primers ISSR1, ISSR2 and ISSR3 generated 29 reproducible bands (150–900 bp). The percentage similarity ranged from 60 to 80 %. The mean value of the Jaccard’s similarity coefficient of the ISSR marker was 0.84. Among the A. brassicae isolates, two groups clustered in the dendrogram (Fig. 2) based on the place of collection. The first cluster grouped most of the Delhi isolates including CaAbD1, CaAbD2, CaAbD3, CaAbD4, CaAbD5 and CaAbD6 along with MAbD1 and MAbD2. Two of the Uttar Pradesh A. brassicae isolates CaAbU4 and CaAbU5 with Haryana isolate CaAbH1 and West Bengal isolate CaAbW1 were also present within the cluster. The similarity among the isolates in the first cluster ranged from 70 to 100 %. The second major cluster consisted of the rest of the isolates with a mean similarity of about 80 %.

Fig. 2
figure 2

Dendrogram generated by the unweighted pair group method with arithmetic means (UPGMA) of Jaccard’s similarity coefficients based on the amplicons from the 32 isolates of Alternaria brassicae using ISSR primers

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The rDNA region from the 3′ end of the 18S and the 5′ end of the 28S genes of all isolates was amplified using PCR conditions with two universal primers, ITS1 (5′ TCC GTA GGT GAA CCT GCG G 3′) and ITS4 (5′ TCC TCC GCT TAT TGA TAT GC 3′) (Jasalavich et al. 1995; White et al. 1990), and the amplified PCR product (~600 bp) was eluted using a QIAGEN gel extraction kit (QIAGEN, New Delhi, India). The purified PCR product was sequenced in the automated sequencer at the Xcelris Lab., Bangalore, India, and related sequences were found in a search for homology using BLAST analysis (Altschul et al. 1997). Phylogenetic analysis was done using MEGA5.0 software (Tamura et al. 2011). Fingerprinting of the 32 A. brassicae isolates using the ITS sequence revealed 90–100 % identity among the isolates. All ITS sequences were submitted to NCBI database (Table 1). The phylogenetic tree (Fig. 3) shows three distinct clusters of the A. brassicae isolates, but there are not many differences. The first cluster comprises only isolate CaAbD2, the second group comprises two isolates that originated from Rajasthan on cauliflower, CaAbR3 and CaAbR4 that are 99 % similar to each other. The third group, comprising the 29 remaining isolates, have 56 % similarity with each other. When four other accessions of A. brassicae (AF229463, ABU05253, JF710505 and JF710505) from NCBI were included, they were closely related (99–100 % similarity) to the A. brassicae isolates in our phylogenetic analysis (Fig. 3).

Fig. 3
figure 3

Phylogenetic tree constructed using maximum parsimony based on the ITS region of 32 Alternaria brassicae isolates and reference ITS sequences obtained from GenBank

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The 32 A. brassicae isolates were collected from two crops, cauliflower (22 isolates) and mustard (10 isolates), in various geographical regions, were each pathogenic on susceptible cultivars of both crops and had similar genetic variability. Genetic variability among the A. brassicae isolates in oilseed crops in India has been reported (Meena et al. 2012; Sangwan and Mehta 2007). To understand genetic variability among these isolates, 42 RAPD and five ISSR markers were tested, but little variation was observed. RAPD data revealed no clear grouping of the isolates. Minor variations thus seen may be due to environmental and survival-based adaptations, which vary from habitat to small niche. The ISSR analysis also yielded similar results, but the two major clusters that formed included isolates of mixed geographical origins. Isolates from the states of Delhi and Uttar Pradesh formed a group that also included isolates from Haryana and West Bengal. ITS analysis of the 32 isolates of A. brassicae confirmed 50–100 % similarity among each isolates irrespective of their original host, thus indicating genetic homogeneity among the A. brassicae isolates. On the contrary, in the past, intra-regional variations among A. brassicae isolates were found using RAPD and restriction fragment length polymorphism (RFLP) markers (Sharma and Tewari 1998). In the present study, the low variability among the isolates may be due to Alternaria brassicae causing leaf spot on the two crucifers, mustard and cauliflower, when the two have the same cropping period and environmental conditions are appropriate for fungal growth and survival. With further research using highly sensitive random amplified microsatellites (RAMS) marker (Guo et al. 2004), we should be able to differentiate the Alternaria isolates.