A reappraisal of the Pyriculariaceae in Iran

INTRODUCTION

The family of Magnaporthaceae was proposed by Cannon (1994) to accommodate Magnaporthe R. A. Krause & R.K. Webster and its related genera such as Gaeumannomyces Arx & D.L. Olivier. This family was originally considered as a single-family placed in the order Magnaporthales and it is closely associated with the Diaporthales and Ophiostomatales based on the phylogenetic studies performed previously (Zhang et al. 2011, Besi et al. 2009, Thongkantha et al. 2009). The family is characterized by nonstromatic black perithecia, usually with long hairy necks, persistent asci, and elongate fusiform or filiform ascospores. Typically, it includes necrotrophic pathogens that are known to infect grasses. The Magnaporthaceae members are hyphomycetes and are diverse but they can be categorized as two morphotypes named as Pyricularia-like or Phialophora-like. Recently, the phylogenetic study performed by Klaubauf et al. (2014) using a multigene approach (LSU, ITS, RPB1, actin and calmodulin) resulted in the establishment of a new family termed as Pyriculariaceae Klaubauf, Lebrun & Crous.

The Pyriculariaceae was introduced as new family containing new genera, new species, new combinations, and neotypification (basionym). Pyricularia oryzae Cavara belonging to the Pyriculariaceae is the causal agent of the rice blast disease, a serious threat for rice (Oryza sativa L.) cultivation worldwide. This fungus annually causes yield losses averaging upwards of 30% (Yaegashi 1977, Landschoot & Jackson 1989). A close relative species of P. oryzae is Pyricularia grisea, which is indistinguishable in terms of conidium, perithecium, and ascopore morphology. Pyricularia grisea isolates collected from Digitaria were shown to form a distinct clade by the phylogenetic analysis (Couch & Kohn 2002, Hirata et al. 2007). Currently, the Magnaporthaceae sensu stricto contains 11 genera including Buergenerula, Bussabanomyces, Endopyricularia, Gaeumannomyces, Harpophora, Kohlmeyeriopsis, Magnaporthiopsis, Nakataea, Omnidemptus, Pyriculariopsis and Slopeiomyces.

To re-evaluate the systematic relationships among Magnaporthaceae sensu lato species and revision of the Pyriculariaceae in Iran, we phylogenetically analyzed relationships among isolates representing a wide range of host plants by using partial DNA sequences of multiple genes such as ITS, MCM7, and calmodulin. Additionally, morphological features of some identified species were described in this study.

MATERIALS AND METHODS

Fungal isolates

During spring, summer and fall of 2012 and 2013, a comprehensive sampling of various host plants including rice, corn, sorghum, barley, wheat, tea, citrus orchards and forests in the south of Caspian Sea (Astara to Gonbad-e Qabus) were conducted (Fig. 1). Furthermore, numerous samples were collected from other provinces such as Khorasan Razavi, Arak, and Qazvin. Subsequently, the collected samples were transferred to the Mycological laboratory of the University of Tehran. For morphological studies, a single spore generated on the surface-sterilized leaf under at 25°C temperatures was transferred to plates filled with water agar and autoclaved rice leaves (WA+ rice leaves). The pictures were taken via the Olympus microscope model BH2.

DNA extraction and sequencing

DNA extraction was done according to the protocol developed by Zhong and Stephenson (2001). PCR amplification of the internal transcribed spacer (ITS) and DNA replication licensing factor (MCM7) gene was performed based on the protocols described by Zhang et. al. (2011) and for calmodulin (CAL) gene, the protocols established by Hirata et al. (2007) was employed. Sequence alignments were conducted with Clustal X 1.8 (Thompson et al. 1997). The distance matrix was calculated using Kimura’s two-parameter method (Kimura 1980) and analyzed with the Maximum Likelihood (ML) using the software called MEGA 5.10 (Tamura et al. 2011). The statistical accuracy of the tree was tested by bootstrap analysis (1000 repetitions) and the generated sequences have been deposited in the GenBank.

 Fig. 1. Schematic representation of the sampling locations in south of Caspian coast

Fig. 2. Pyricularia oryzae. a. conidiophore; b-c. conidia. Pyricularia grisea d-e. conidiophores; f. conidia. Pseudopyricularia higginsii g-h. conidiophores; i. conidia. Pseudopyricularia sp. ck1 j-k. conidiophore; l-m. conidia. Pseudopyricularia sp. 16d. n-o. conidiophore; p-r. conidia. Nakataea oryzae s-t.conidiophore; u-v. conidia. — Scale bars = 10 µm.

Phylogeny

A total of 31 strains belonging to 6 species were studied. The isolate numbers, sources, hosts and GenBank accession numbers for the ITS region are included in Table 1.

The results of alignment indicated a total of 512, 410 and 618 nucleotide characters including gaps in ITS, CAL, and MCM7, respectively. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. Initial tree(s) for the heuristic search were automatically obtained by applying Neighbor-Join and Bio NJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated.

There were three main clades in the phylogenetic tree constructed based on the ITS and MCM7 regions. The Clade A containing ninety-nine species were supported with high bootstrap (99%) whereas that of the Clade B including two species was 100%. In addition, the bootstrap of the clade C possessing five species was 99%. The aforementioned trees were constructed based on the sequencing of the ITS region. The clade A contained 15 species with high support while both clade B and C had four species supported with 94% and 99% bootstrapping, respectively, in the phylogenetic tree generated based on the analysis of the MCM7. In clade A, species P. oryzae and P. grisea were placed within subclade Ι. These species are different in phylogeny, morphology, and host plants. Conidiophores and conidial size are diverse in two species. Species of the Pseudopyricularia higginsii, Pseudopyricularia sp.1, and Pseudopyricularia sp.2wereclustered within subclade ΙΙ. These four species are variable in terms of morphological features.

Magnaporthiopsis poae and M. rhizophila were grouped in the clade B in the constructed tree based on the ITS region and the clade C, which was made based on the MCM7, respectively. Species positioned into these groups generate Phialophora-like anamorph and these species are necrotrophic pathogens on grass root. Nakataea oryzae isolates placed in the basal clade of the phylogenetic tree generated based on the ITS region and were placed in the clade B based on the MCM7. This species is different from the Pyricularia concerning to the anamorphic and phylogenetic characteristics (Fig. 3, 4).

Table 1. Overall characterizations of fungal species used in this study.

Fig. 3. The maximum likelihood tree inferred from the ITS sequences for 37 species. The numbers above the branches show the bootstrap values in 1000 replicates. The length of branches is proportional to the number of base changes, indicated by the scale bar. Gaeumannomyces graminis.

Fig. 4. The maximum likelihood tree inferred from the MCM7 sequences datasets for 24 species. The numbers above the branches show the bootstrap values in 1000 replicates. Gaeumannomyces graminis.

Species belonging to the Pyriculariaceae were grouped in the two distinct clades based on the sequencing of the CAL gene. Species of both P. oryzae and P. grisea were grouped in the clade A, which was supportedby the high bootstrap value. These species are morphologically and phylogenet-ically different and have different host plants. The species of Pseudopyriculariahigginsii, Pseudopyriculariasp.1 and Pseudopyricularia sp.2 weresupported with 95% bootstrap in the clade B. All five species were collected from sedge, but these species were different based on morphological characteristics (Fig. 5). It is required to morphologically and phylogenetically investigate this species in further details.

Fig. 5. The maximum likelihood tree inferred from the calmodulin sequences datasets for 24 species. The numbers above the branches show the bootstrap values in 1000 replicates. Barretomyces calatheae.