Pleosporaceae is the largest family within the Pleosporales. Its species are parasites or saprobes on wood and dead herbaceous stems or leaves (Sivanesan 1984).
Anamorphic forms of Pleosporaceae had been previously placed in Deutromycota. However, after molecular revolution in fungal taxonomy which commenced in the early 1990s, with analyses of PCR-amplified ribosomal RNA genes (White et al. 1990), anamorphic forms of Ascomycota and Basidiomycota were mostly placed in the orders and families that owned their teleomorphs (Hibbett et al. 2007). Despite efforts to clarify phylogenetic relationships of this group, there is still uncertainty about taxonomic position of some genera. This investigation was performed in order to clarify the issue, and also due to the importance of a natural classification and correct species identification in disease control, plant breeding and establishment of phytosanitary measures (Cai et al. 2009, Hyde et al. 2010).
MATERIALS AND METHODS
We collected abundant anamorphic species of Cochliobolus (Bipolaris and Curvularia) and some Alternaria species from various hosts and different geographical regions of Iran. After morphological and molecular identification, their ITS rDNA regions and RPB2 sequences were compared to each other, and to some other sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/Taxonomy) to verify the placement of these genera and assess which marker is able to determine their taxonomic position.
Fresh specimens were collected from soil and host tissues from different provinces of Iran during 2010 to 2011. Strains were initially identified according to the criteria used by Ellis (1971), and were later verified by molecular identification.
Twenty five isolates from different hosts and geographic origins were selected for molecular analysis. Total genomic DNA was extracted using modified CTAB protocol (Ashktorab et al. 1992). PCR was performed using the primer pairs ITS4 (5′-T CCTCCgCTTATTgATATgC-3′) and ITS5 (5′-ggAA gTAAAAgTCgTAACAAgg-3′) (White et al. 1990) to amplify Internal Transcribed Spacers (ITS) and 5.8S region FRPB2-5f (5′-gAY gAYMgWgATCAYTTYg g-3′) and FRPB2-7CR (5′-CCCATRgCTTgYTTRCC CAT-3′) (Hall lab, fungal specific) to amplify RNA polymerase II, subunit 2. Phylogenetic analysis was performed by using Neighbor Joining and Maximum Likelihood methods. The bootstrap settings were 1000 replicates and retaining groups are those with frequency of more than 50 %. Then, phylogenetic trees were constructed using MEGA 5.
RESULTS AND DISCUSSION
The ITS 4 and ITS 5 primers directed the amplification of a single product. DNA sequencing revealed that these fragments ranged in size from 530 to 569 bp (including primer sequences). Among 558 significant sites, 311 (55.73 %) were conserved and 247 were variable. Phylogram constructed by Neighbor Joining method is shown in Figure 1. On the other hand, applying the primers RPB2-5F and RPB2-7CR resulted in amplification of a single fragment, ranging from 1178 to 1184 bp (including primer sequences). Phylogenetic tree was constructed by using neighbor joining (Fig. 1). Among significant sites, 601 (50.67 %) were conserved and 585 were variable.
Phylogeny of Cochliobolus (clades D1 and D2): RPB2 results were almost similar to ITS. In all the phylograms Curvularia was closely related to Bipolaris. Similar to the findings of a study by Kodsueb et al. (2006) in which Cochliobolus was segregated into two groups as a result of phylogenetic analysis of 28S rDNA region, two groups (D1 and D2) were obtained as a result of analysis of both ITS and RPB2 regions in this study. Clade D1 includes Bipolaris oryzae, B. sorokiniana, Cochliobolus sativus and C. heterostrophus. Conidium morphology is almost similar in members of this clade (morphology of their anamorphs were considered for C. sativus and C. heterostrophus). They have (6-12) pseudosepta and size of their conidim is (30-120 × 14-20) µm. All of them produce a hilum with length of 3-4 µm (Ellis 1971). It should be mentioned that members of this clade have big spore and are highly virulent pathogens (Manamgoda et al. 2011) comparing clade D2. Taxonomic position of Bipolaris oryzae in both ITS and RPB2 analyses was the same (D1). It was placed in a separate branch with C. hetrostrophus and C. sativus in RPB2 analysis, and with B. sorokiniana in ITS analysis.
Bipolaris australiensis, B. spicifera, B. hawaiiensis, Curvularia inaequalis, C. pallescens, C. lunata, C. brachyspora and C. geniculata in clade D2 represent a distinct monophyletic group (similar to Group 2, as defined by Berbee et al. 1999). This clade comprises mild pathogens with both Bipolaris and Curvularia asexual states. The Pseudocochliobolus species were clustered in this group. They have also small spores (18-40 × 6-17 µm) comparing clade D1.
Results indicate that classification derived from both ITS and RPB2 are consistent with morphology of the spore. Although RPB2 analysis had a similar result to ITS, RPB2 segregated small-spore containing species better. As observed in Fig. 2, clade D2 was divided into 2 subclades and Curvularia species were placed in D2-1 with a bootstrap value of 100%. RPB2 seems to be a suitable marker for differentiation of Curvularia and Bipolaris.
Phylogeny of Alternaria and Ulocladium:In this investigation, Embellisia, Alternaria and Ulocladium were placed together in a large clade with a bootstrap value of 100%, which is similar to the results of the study by Pryor and Gilbertson (2000). Within this clade, the following distinct species-clades were revealed: A (A1, A2, A3), B and C. SectionA1: Ulocladium species were composed of a monophyletic group with 99% and 100% bootstrap values in ITS and RPB2 analyses, respectively. Section A2: Alternaria brassicicola was placed in section A2 with Ulocladium botrytis, and lonely in this section in ITS and RPB2 analyses, respectively (Fig. 1). Since the result obtained from RPB2 is consistent with morphological characters described by Lawrence et al. (2013) for section brassicicola of Alternaria species, RPB2 is considered as a suitable marker for segregation of this group.
Section A3: Alternaria panax was placed in A3, which formed a sister group with Embellisia chlamydospora and Embellisia allii in ITS analysis (Fig. 1) and was placed singly in analysis of RPB2. These two genera of Embellisia belong to section Embellisia described by Simmons (1971), whereas A. panax is a type species of section panax characterized by Lawrence et al. (2013), recently. This result also matches with morphology of this genus. Section B: Alternaria tenuis, A. tenuissima, A. citri, A. solani, and A. alternata which have different morphological specifications were placed in a single clade according to ITS results, but RPB2 presented accurate results. As shown in Fig. 1, A. solani and A. dauci were placed in section B1. These two species share some similarities. For example, they both have broadly ovoid, obclavate, ellipsoid, subcylindrical or obovoid (medium) large conidia, disto- and euseptate, solitary or in short to moderately long chains, with a simple or branched, long to filamentous beak (Woudenberg et al. 2013).
Alternaria alternata was placedin section B2. This is a small-spore containing Alternaria species, which was properly isolated from other species by RPB2. The mentioned species had been put in section B, using ITS analysis.
Alternaria longissima and A. padwickii which are big-spore containing Alternaria species were placed in clade C in ITS analysis. So, ITS segregation result was similar to the morphology of these two genera. It seems that RPB2 is a suitable marker for investigating phylogenetic relationships of the selected genera.