Morphological and molecular characterization of Oomycetes associated with root and crown rot of cucurbits in Kermanshah province, Iran

INTRODUCTION

Oomycetes such as Pythium and Phytophthora are among the most well-known plant pathogens around the world that cause rotting of seeds, root, and crown, damping and decay of the lower parts of the stem, tubers, and corms, and soft rot of fruits in contact with soil (Erwin & Ribeiro 1996, Kucharek & Mitchell 2000).

The genus Pythium and Phytophthora are taxonomically classified in the Kingdom Stramenopila, phylum Oomycota, class Oomycetes (Ainsworth 2008, Dick 1990). The traditional classification of genus Phytophthora is mainly based on the morphological characteristics of sporangia, gametangia, and oospores (Newhook et al. 1978, Stampset al. 1990, Tucker 1931, Waterhouse 1963). Waterhouse (1963) divided the genus into six distinct groups based on morphological characteristics. She published the key for identifying isolates based on the characteristics of sporangium, antheridium shape, and homothallic or heterothallic tendency. Pythium spp. are traditionally classified according to sexual and non-sexual structures, in which the forms of sporangium and oogonium ornamentations are the main traits (Schroederet al. 2013). The main constraints for the identification and classification of these species are: the lack of clear and distinct morphological characteristics, the high number of species, low number of traits, difficulty and inefficiency in culturing isolates and, comparison of their morphological characteristics with each other by microscope (Balaet al. 2010, Robideauet al. 2011, Wanget al. 2003). If there is an adequate database of reference strains, DNA-based identification can be done quickly and easily by a non-specialist and precise results can be achieved in the shortest time (Robideauet al. 2011).

Cooke et al. (2000) published the first datasets of ITS region sequences that included all known and available Phytophthora species. They introduced sequences in this region as a barcode for identification of species of this genus. In the following, Levesque & de Cock (2004) provided similar comprehensive datasets for the identification of Pythium species. They subdivided the genus into 11 clades (A to K), using the ITS sequences and the large subunit ribosomal DNA (28S rDNA). Villa et al. (2006) analyzed ITS I and II regions and 5.8S rDNA gene, cytochrome oxidase II gene (cox II), and the β-tubulin gene. The β-tubulin gene was analyzed in 58 isolates representing 39 species of Pythium and 17 isolates representing nine species of Phytophthora to examine the phylogenetic relationships between the isolates and these two genera. The results of the parsimony analysis of these three regions were four monophyletic groups. Those were completely inconsistent with the classification of isolates based on the morphology of sporangium. Further research revealed that the species belonging to the clade K were correctly intermediate between Pythium and Phytophthora, in terms of morphological and phylogenetic properties. Therefore the new genus Phytopythium was proposed for members of this clade (Balaet al. 2010, de Cocket al. 2015).

Iran is one of the top four countries in the world in cucurbits production and has a long history in cucurbit cultivation (Pitratet al. 1997). Thereby, we aimed the current study to evaluate the diversity and distribution of plant-associated oomycetes. It was found that cucurbit fields in Kermanshah Province were the habitat of diverse species of oomycetes phytopathogens.

MATERIALS AND METHODS

Sampling, isolation and maintaining of isolates

Diseased samples were collected randomly from different cucurbits fields (including cucumber, watermelon, melon, and squash) in Kermanshah province, western Iran. During late May to late September 2014, cucurbit fields were visited. Crown and roots of plants showing symptoms of foliar blight were examined carefully. Samples with characteristic symptoms of oomycetes blight or seedling damping-off were collected, kept in paper bags, and transferred to the laboratory. To isolate oomycetes, 2-5mm pieces were prepared from the border of healthy and infected tissues of crown, root or stem, surface sterilized with 70% ethanol for 10 seconds, air dried on sterile filter paper, and transferred to cornmeal agar-PARP (CMA-PARP) (Jeffers & Martin 1986). The Petri dishes were kept at 25°C and the purification was carried out using the hyphal-tip method (Tuite 1969). The purified isolates were transferred to tubes containing CMA medium and kept at 15°C.

Identification of isolates

Preliminary identification of the oomycetes isolates was based on morphological and physiological examination and compared with available pieces of literature (Dick 1990, Van der Plaats-Niterink 1981). The morphological and physiological characteristics that were examined and recorded are as follows: morphology of sporangium (elliptical, egg-shaped, inverted pear-shaped, lime-shaped, spheroid, filamentous), oogonium surface decorations (flat or decorated), the amount of space that has been captured by oospore in oogonium (plerotic or aplerotic), the origin (diclinous and monoclinous), the connection type of antheridium to oogonium (paragynous or hypogynous), the diameter of the mycelium, formation of hyphal swelling, physiological characteristics including colony morphology on a variety of media such as Corn Meal Agar (CMA), Malt Extract Agar (MEA), Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) and Hemp Seed Agar (HSA), growth rate on different culture media, and growth temperatures. To ensure long-term preservation of isolates, pure cultures of all identified species were deposited at Iranian Fungal Culture Collection (IRAN …C) at the Iranian Research Institute of Plant Protection, Tehran, Iran.

DNA extraction and PCR amplification

Genomic DNA of selected isolates grown in PDB medium and extracted using the DNG^TM-PLUS kit (CinnaGen, Iran). ITS-rDNA region and mitochondrial cytochrome oxidase gene of sub-unit II (cox II) were amplified using the primer pairs ITS6/ ITS4 (White et al., 1990) and FM66/ FM58 (Martin, 2000), respectively. The PCR mixture was prepared by mixing the following: 50 ng of template DNA, one micromole of each primer, 100μM dNTPs, 0.4 μmol Taq DNA polymerase (Sinagen, Iran), 1.5 μmol of MgCl₂, 2.5 μl polymerase chain reaction buffer (200 μm Tris-HCl with pH 8 and 500 mM KCl), and 100 μM BSA for 25 μl reactions. Cycling conditions consisted of an initial denaturation at 95 °C for 2 minutes, 30 PCR cycles of denaturation at 95 °C for 20 seconds, annealing at 55 °C for 25 seconds, and extension at 72 °C for 50 seconds. These were followed by a final extension at 72 °C for 10 minutes using a Biometra thermo-cycler (Tpersonal, Germany). The PCR products were purified and sequenced from both direct and reverse directions by Macrogen, Inc. (South Korea). The sequences were manually edited using the Bioedit software (Hall, 1999). Edited sequences were submitted to the GenBank (http: //www.ncbi.nlm. nih.gov/genbank) (Tables 1 and 2).

Multiple sequence alignments of the newly generated sequences and sequences of the valid species, derived from the GenBank (Tables 2 and 3), were performed with Clustal X software version 2.0.11 (Thompsonet al. 1997), checked and improved manually where necessary. The neighbor-joining algorithm was used to generate the initial tree with bootstrap analysis with 500 replicates, using MEGA5 software (Tamura et al. 2011).

RESULTS AND DISCUSSION

Identification of oomycetes isolates

During the field surveys, a total of 313 samples of diseased plants were collected and 347 isolates of oomycetes were isolated. As many as nine species of Pythium (including P. aphanidermatum, P. dissotocum, P. catenulatum, P. kashmirense, P. middletonii, P. nodosum, P. oligandrum, P. torulosum, and P. ultimum), two Phytopythium species (Pp. mercuriale and Pp. litorale), and three phytophthora species (including Ph. melonis, Ph. nicotianae, and Ph. parasitica) were identified. Those were identified on the basis of the morphological and physiological characteristics and sequence data obtained from ITS–rDNA region and cox II locus. Based on the available literature, Pp. mercuriale (among the species identified in this study) is a new record for the Iranian mycobiota. Moreover, Pp. mercuriale, P. torulosum, P. kashmirense, and P. nodosum are reported for the first time as oomycetes associated with root and crown rot of cucurbits. Furthermore, P. dissotocum, Pp. litorale, and P. catenulatum are reported for the first time from diseased cucurbits in Iran. Morphological description of this seven newly-recorded species in this study is given in alphabetical order as follows:

Pythium catenulatum,V.D. Matthews (1931) 

The colonies had a rose-shaped pattern on CMA, PDA, and MEA, chrysanthemum colony pattern on HSA, and intermediate growth pattern on PCA. Hypha were up to 4μm wide. Hyphal swelling, 10 to 20μm in diameter and usually found in chains of three to eight (Fig. 1, a1), each producing one to three germination tubes. No chlamydospore and appressorium were observed. Sporangia were composed of jagged and flaccid mycelia, 17 to 20μm in diameter, with either regular or irregular splitting (Fig. 1, a2). They produced zoospore at 20 to 25 °C. The cysts were about 8 to 9 μm in diameter. The oogonia were spherical in shape, 19 to 25 μm in diameter, with smooth walls without decorations, formed terminally or intercalary. Antheridia were commonly seen in diclinous and paragynous forms and there were more than one (often five) antheridium per oogonium (Fig. 1, a3). The oospores were spherical in shape, smooth, often aplerotic, rarely plerotic, with a wall thickness of 1.5μm on an average. The minimum, optimum, and maximum growth temperatures were 7, 30 and 37 °C respectively. The average daily growth rate was 15 mm at 25 °C on CMA. The species was placed in clade B of ITS and cytochrome oxidase II phylogenetic trees (Fig. 2 and Fig. 3).

Pythium dissotocum Drechsler (1930)

The colonies were submerged on CMA and had no colony pattern. However, radiate growth pattern was observed on PDA and an intermediate state of chrysanthemum, rose-shape, and radiate colony patterns were observed on MEA, PCA, and HSA. The hypha were up to 7μm wide, the sporangia were filamentous, slightly swollen, branched, and tree-like (Fig. 1, b1), and the discharge tube was long (up to 11μm) (Fig. 1, b3). The encysted zoospores were 8–9μm in diameter. The oogonia were approximately spherical 20 to 24μm formed terminally, intercalary or laterally (Fig. 1, b2).

The antheridia was commonly monoclinous (Fig. 1, b2) with a stalk accurately below oogonium (paragynous) or without a stalk (hypogynous) or diclinous. For every oogonium, there were more than one to three antheridia. The oospore were spherical, ranging from 17 to 21μm (avg. 19μm) in diameter, smooth, aplerotic (Fig. 1, b2) or nearly plerotic. The minimum, optimum, and maximum growth temperatures were 5, 20-28 and 36°C, respectively. The average daily growth rate was 18mm at 25°C on CMA. This species was placed in clade B and subclade B2 of ITS and cytochrome oxidase II phylogenetic trees.

Pythium kashmirense B. Paul (2008)

No colony pattern on CMA, chrysanthemum colony pattern on MEA, and Rose-shaped colony pattern with large sections were observed on HSA, PDA, and PCA. The mycelium was highly branched, up to 6μm wide. There was no chlamydospore, hyphal swelling, and appressorium in this species. The sporangium was filamentous, tumescent, with complex and contiguous tumescence (Fig. 1, c1). Vesicle and zoospores formed after 24 hours incubation at room temperature (20 to 25 °C). The oogonia were spherical, often intercalary, 11 to 22 μm in diameter (avg. 16.4 μm). The oospores were spherical and plerotic, 10 to 21 μm in diameter (avg. 16.1 μm), with a wall thickness of 1-2μm. The antheridia were diclinous, wrapped around oogonia and formed a ring (Fig. 1, c2). The minimum, optimum, and maximum growth temperatures were 5, 25-30 and 38 °C respectively. The average daily growth rate was 15mm at 25 °C on CMA. This species was placed in clade B of ITS phylogenetic tree.

Pythium nodosumB. Paul, D. Galland, T. Bhatn & Dulieu (1998)

The colonies had radiate growth pattern on CMA, PDA, and PCA. However, there was no pattern on HSA and MEA. The hypha were 5-7 μm wide and the sporangia were varying in shape spherical, subglobose, pear-shaped or egg-like, mostly intercalary and sometimes terminally (Fig. 1, d2), 10-25 μm in diameter. The oogonia were smooth-walled, spherical, 12 to 27 μm. Antheridia, one or more, surrounding oogonium and forming node around it (Fig. 1, d1). After fertilization, the node disappeared and only one antheridium remained, which had the appearance of a bell-like cell (Fig. 1, d3). The oospores were spherical and smooth-walled, single, aplerotic (Fig. 1, d3), 10 to 22 μm in diameter, and a wall thickness of about 1 μm. The minimum, optimum, and maximum growth temperatures were 10, 20-25 and 35 °C, respectively. The average daily growth rate was 17 mm at 25 °C on CMA. This species was placed in clade J of ITS and cytochrome oxidase II phylogenetic tree.

Pythium torulosum Coker & P. Patt (1927)

The colonies had subsurface growth on CMA, rose-shaped colony pattern on PCA, and uniform colony pattern on MEA, PDA, and HSA. The hypha were 5μm wide and there was no chlamydospore, hyphal swelling or appressorium. The sporangia were tumescent branches, which ran out of the main mycelium and made up the various bead-like elements in different sizes (Fig. 1, e1). The encysted zoospores were 7-8 μm in diameter. The oogonia were smooth, 15 to 23 μm (avg. 20.5) spherical, produced laterally, and intercalary or on short lateral appendages (Fig. 1, e2, e3). The antheridia were sausage-shaped and curved to club-shaped, mostly monoclinous, 5-10 3-6 μm and attached to the oogonium from their tip. One, two or sometimes three antheridia are attached to each oogonium. The stalk of oogonium or the main mycelium was the origin of monoclinous antheridium (Fig. 1, e4). The oospores were plerotic, 13 to 19 μm in diameter and the wall thickness was up to 2μm. The minimum, optimum, and maximum growth temperatures were 5, 25-30 and 35 °C, respectively. The average daily growth rate was 14mm at 25 °C on CMA. This species was placed in clade B of ITS and cytochrome oxidase II phylogenetic tree.

Phytopythium litorale (Nechw.) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque (2014)

 The colonies had satellite growth pattern on CMA, rose-shape on PDA and PCA, and radiate on HSA and EMA. The hypha were 5 μm wide and the sporangium was spherical or egg-like, 20-31 17-28 μm (avg. 25.5 22.5), with the papilla up to 70 μm (Fig. 1, f1). This papilla could form a discharge tuber or germinate directly and become branched. Sporangia were proliferating (Fig. 1, f2 and f3). The encysted zoospores were about 8- 10μm. The minimum, optimum, and maximum of growth temperatures were 5, 30 and 35 °C, respectively. The average daily growth rate was 10mm at 25 °C on CMA. The oogonium and oospore did not produce, and therefore, it was a heterothallic organism. This species was placed in clade K of cytochrome oxidase II phylogenetic tree.

Phytopythium mercuriale (Belbahri, B. Paul & Lefort) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque (2014) 

Colonies had subsurface growth on CMA, with a slight satellite colony pattern. The colony growth pattern was chrysanthemum, with aerial mycelia and bulk cotton form in the center on PDA and HSA. However, it was rose-shaped on MEA and cottony colony pattern on PCA. The main hyphae was up to 5μm wide. The sporangia, rarely produced in water, were mostly spherical, with papilla measuring up to 23-27 μm (Fig. 1, g1). The zoospores were produced at 17-27 °C and the discharge tube was short and about 4μm. Old sporangia often germinate from their papilla. The oogonia were spherical, measuring up to 22-28 μm, smooth-walled mostly produced terminally or laterally on the short branches. The antheridia were often diclinous, numerous, wrapped around oogonium and created a node (Fig. 1, g2). However, the oospores were not observed. The chlamydospores were mainly spherical, measuring up to 25-44 μm, thin-walled, terminally or intercalary (Fig. 1, g3). The minimum, optimum, and maximum growth temperatures were 8, 25-30 and 35 °C, respectively. The average daily growth rate was 8 mm at 25 °C on CMA. This species was placed in clade K of ITS phylogenetic tree. This species was reported for the first time in Iran.

Table 1. Isolates of Pythium, Phytopythium and Phytophthora were used for phylogenetic analyses based on ITS-rDNA sequence in this study. Newly generated sequences are in bold. 

Table 2. The list of species and isolates of Pythium and Phytopythium were used for phylogenetic analyses based on cox II sequence. Newly generated sequences are in bold.

Fig 1. Morphological features of Pythium and Phytopythium species. a: Pythium catenulatum isolate Pc36-1C. a1: Catenulate globose hyphal swelling. a2: Irregular inflated sporangia. a3: Diclinous antheridia and oogonium. b: Pythium dissotocum isolate Pd32-1C. b1: Filamentous dendroid sporangia. b2: Oogonium, monoclinous antheridium, and aplerotic oospore. b3: Zoospores and vesicle. c: Pythium kashmirense isolate Pk83-1C. c1: Filamentous-inflated and continuous type of sporangia. c2: Diclinous antheridia wrapping around the oogonium. d: Pythium nodosum isolate Pn86-1C. D1: Oogonium surrounded by antheridia forming nodes. d2: Inercalary sporangium. d3: Oogonium with a bell-like antheridial cell. e: Pythium torulosum isolate Pt35-1W. e1: Flamentous inflated sporangia. e2, e3, and e4: Oogonium and monoclinous antheridium. f: Phytopythium litorale isolate Phl11-1W. f1: Sporangium with papilla. f2: Internal extended proliferation. f3: Internally nested proliferation. g: Phytopythium mercuriale isolate Pm23-1C. g1: Papillate sporangium. g2: Oogonium surrounded by diclinous antheridia forming nodes. g3: Chlamydospores. (Bars = 20μm).

ACKNOWLEDGMENTS

The authors would like to acknowledge Razi University for financial support of this project.