Document Type : Original Article
Authors
1 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
2 Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran
Abstract
Keywords
Main Subjects
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
RESULTS
Morphology
One hundred and thirty isolates were collected from leaves having symptoms of the blast and leaf spot. Morphological characterization of the tested isolates, including colony color, shape, color and the size of conidia and conidiophores demonstrated that all the examined isolates were belonged to the six species comprising Pyricularia oryzae (80 isolates), P. grisea (30 isolates), Nakataea oryzae (5 isolates), Pseudopyricularia higginsii (8 isolates), Pseudopyricularia sp.1 (10 isolates) and Pseudopyriculariasp.2 (4 isolates). Species of the P. oryzae and P. grisea aredifferent in terms of morphological features as well as host plants. Conidiophores sizes are 100-250 µm in P. oryzae and 70-175 µm in P. grisea; and the conidial size are 16-25×7-10 µm in P. oryzae; and 26-31× 6-8 µm in P. grisea (Fig. 2. a, b, c, d, e, f).
The genus Nakataea has gray to a dark gray colony; some conidiophores are branched, 130-460 µm long; conidia falcate, sigmoid, curved, 45-76 × 10-14 µm, septate and two middle cells brown (Fig. 2. s, v). The genus Nakataea has some similarity to Pyricularia in overall morphological characters, but it differs in falcate to sigmoid conidia with darker median cells (Luo & Zhang 2013).
Pseudopyriculariahigginsiihas 3-septate conidiophores with 45-125 µm length; 2-septate conidia with the size of 22–29 × 6–7 μm (Fig. 2. g, i). Pseudopyricularia sp.2, conidiophores without septa or rarely 2-septa, the size of 87–140 (–170) × 5–6 µm. Conidia solitary, dry, fusiform or cylindrical, hyaline, 23–29 × 5–8 µm, smooth, 2-septate, hilum often protuberant (Fig. 2. n-r). Pseudopyricularia sp.1, conidiophores branch, straight, 1–2(–4)-septate, 65–125(–140) × 4–5 µm. Conidia solitary, dry, obpyriform to obclavate, hyaline, (17–) 22–27(–29) × (5–) 6–8 µm, smooth, 1-septate, hilum often protuberant (Fig. 2. j, m). The Pseudopyricularia sp.1 species is similar to Pyricularia bothriochloaein having 1-septate conidia. However, the conidia ofPseudopyricularia sp.1are larger than that of Pyricularia bothriochloae. Additionally, it differs in conidiophores septa and size.
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.
|
Species |
Strain code |
Source |
Host |
ITS |
|
Pyricularia oryzae |
UTFC-PO1 |
Mazandaran, Iran |
Echinochloa crus-galli |
KP144439 |
|
P. oryzae |
UTFC-PO2 |
Mazandaran, Iran |
Paspalum distichum |
KP144440 |
|
P. oryzae |
UTFC-PO3 |
Guilan, Iran |
Oryza sativa |
KP144441 |
|
P. oryzae |
UTFC-PO4 |
Mazandaran, Iran |
Zea mays |
KP144442 |
|
P. oryzae |
UTFC-PO5 |
Golestan, Iran |
Setaria viridis |
KP144443 |
|
P. grisea |
UTFC-PO6 |
Mazandaran, Iran |
Digitaria sp. |
KP144438 |
|
P. grisea |
UTFC-PO7 |
Mazandaran, Iran |
Digitaria sp. |
|
|
Pseudopyricularia higginsii |
UTFC-PO8 |
Mazandaran, Iran |
Cyperus sp. |
KP144446 |
|
P. higginsii |
UTFC-PO9 |
Mazandaran, Iran |
Cyperus sp. |
|
|
Pseudopyricularia sp.1 |
UTFC-PO10 |
Golestan, Iran |
Cyperus alternifolius |
KP144447 |
|
Pseudopyricularia sp.1 |
UTFC-PO11 |
Golestan, Iran |
Cyperus alternifolius |
KP144448 |
|
Pseudopyricularia sp.1 |
UTFC-PO12 |
Golestan, Iran |
Cyperus alternifolius |
KM207211 |
|
Pseudopyricularia sp.2 |
UTFC-PO13 |
Golestan, Iran |
Juncus sp. |
KM207210 |
|
Pseudopyricularia sp.2 |
UTFC-PO14 |
Golestan, Iran |
Juncus sp. |
---- |
|
Nakataea oryzae |
UTFC-MO1 |
Guilan, Iran |
Oryza sativa |
KP144444 |
|
N. oryzae |
UTFC-MO2 |
Guilan, Iran |
Oryza sativa |
KP144445 |
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.
DISCUSSION
Three distinct families are distinguished in order Magnaporthales, including Magnaporthaceae (based on Nakataea), Pyriculariaceae (based on Pyricularia), and Ophioceraceae (based on Ophioceras) according to the phylogenetic analysis conducted previously (Zhang et al. 2011, Klaubauf et al. 2014). The Magnaporthaceae and Pyriculariaceae are mainly included fungal plant pathogens such as Gaeumannomyces, Nakataea and Pyricularia causing destructive diseases on cereals and grasses. The Magnaporthaceae is clearly distinguished from the Pyriculariaceae by their asexual morphs, which are phialophora- or harpophora-like, or with falcate versicolored conidia formed on brown and erect conidiophores but the Pyriculariaceae is evidently characterized by pyriform 2-septate conidia and rhexolytic secession (Klaubauf et al. 2014).
Based on the results presented here, we phylogenetically identified three main clades in Magnaporthaceae sensu lato. The first one is the Pyriculariaceae, the early diverging lineage that contains several members such as Pyricularia oryzae, P. grisea, Pseudopyricularia higginsii, Pseudopyricularia sp.1, and Pseudopyricularia sp.2. Species of P. oryzae and P. grisea placed in the subclade I. Based on the phylogenetic analysis these species were clustered in two distinct subclades that could be separated based on both host plants and morphological characteristics. Couch and Kohn (2002) described Magnaporthe oryzae as a new species, which is distinct from M. grisea. In M. grisea s. l., two distinct clades were observed, which they are different from each other in terms of host plants. M. grisea is able to infect Digitaria, a grass grown in tropical and warm temperate regionswhile M. oryzae is cable of infecting Oryza sativa and other cultivated grasses. Subclade II included species of the Pseudopyricularia higginsii, Pseudopyricularia sp.1 and Pseudopyricularia sp.2 that they were morphologically different. These abovementioned species werephylogenetically distinct but they were monophyletic and sister groups. The Phylogenetic analysis demonstrated that the Pseudopyricularia species were far away from Pyricularia s. str. and they placed in a clade containing the three other known species of Pseudopyricularia. This genus has conidia that are solitary, obclavate, pale to medium brown, finely roughened, guttulate, 2-septate; hilum truncate, slightly protruding, unthickened, not darkened and conidiophores are solitary, erect, straight or curved, branched or not, medium brown, finely roughened and septate (Klaubauf et al. 2014).
Previously, several isolates were characterized as the representative of P. higginsii belonging to a complex of three related species, P. cyperi, P. kyllingae and P. higginsii, which were classified as Pseudopyricularia. Species in this complex are morphologically similar to each other in terms of conidial size and it would be required to resolve the phylogeny of P. higginsii (Fig. 3, 4, 5).
Analysis of the ITS and MCM7 indicated interrelationships of Magnaporthaceae senu lato. Both the second clade constructed based on the sequencing of the ITS region and the third one made based on that of the MCM7 were mainly contained the Magnaporthaceae sensu stricto such as Magnapothiopsis poae and M. rhizophila. The species of these groups have Phialophora-like anamorph and these species are necrotrophic pathogens on grass root. Nakataea oryzae isolates made the basal clade of the phylogeny tree based on the sequencing of the ITS region and the second clade based on that of the MCM7. These species are different from the Pyricularia genus in boththe anamorphic and phylogenetic characteristics. It is different from Pyricularia in conidial shape. Based on Melbourne Code (Article 59.1), the name for the rice blast fungus should be Pyricularia oryzae while that of the stem rot is Nakataea oryzae (Luo et al 2013).
Evntually, this is a preliminary morphological and phylogenetic study on some genus and species of Magnaporthaceae sensu lato in Iran. Pervious phylogenetic analyses (Zhang et al. 2011, Luo & Zhang 2013, Klaubauf et al. 2014) and also this study shed light on the evolution of Magnaporthaceae sensu lato. The taxonomic reappraisal proposed here is a step toward defining monophyletic genera in Pyriculariacea. More species such as Pyricularia, Pseudopyricularia and Nakataea species should be investigated in the further phylogenetic analysis to gain a better understanding of their evolution and to re-evaluate the current taxonomy.
ACKNOWLEDGMENTS
We gratefully acknowledge the High Council for Research of the University of Tehran for financially supporting and providing materials under grant number 7110022/6/29.
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