INTRODUCTION

Oomycetes form a group of fungus-like microorganisms, which are present in marine, freshwater and terrestrial environments (Dick 2001, Beaks et al. 2012). They belong to the Stramenopiles lineage within the Stramenopiles-Alveolata-Rhizaria (SAR) super group (Kirk et al. 2008, Burki 2014). Of the nine orders within the Oomycetes, Peronosporales, Pythiales, and Saprolegniales are well-studied due to their impact on agriculture and natural ecosystems (Judelson 2012). While Peronosporales are predominantly freshwater saprophytes of plant and animal debris, Pythiales are primarily known as soil-born saprotrophs and necrotrophic pathogens (Cooke et al. 2000; Riethmuller et al. 2002; Levesque & de Cock 2004; Beakes & Sekimoto 2009). The order Saprolegniales is divided into three well-supported families, Saprolegniaceae, Achlyaceae and Verrucalvaceae (Beaks et al. 2014).

Of the 22 genera within the Saprolegniales, Achlya, Saprolegnia, and Aphanomyces seem to be widely distributed throughout the world (Johnson et al. 2002). Since the late 19th century, mycologists have focused on their identification and phylogenetic relationships because some genera can cause severe disease in fish and amphibians (Urban et al. 2015; Rezinciuc et al. 2016; Marano et al. 2017). According to all available identification keys, the zoospore release mechanism and the form and shape of sexual reproductive organs are important features to delineate different Saprolegniales genera (Johnson 1956; Scott 1961; Dick 1969; Seymour 1970; Johnson et al. 2002). However, the lack of type species and sexual structures in vitro and unstable morphological features have forced mycologists to look for alternative tools such as molecular markers (Sandoval-Sierra et al. 2014). Recently, cytochrome c oxidase subunit I (cox1) and internal transcribed spacer (ITS) of rDNA have been accepted as DNA barcodes for identification of oomycetes (Robideau et al. 2011).

The genus Dictyuchus Leitgeb belongs to the family Achlyaceae (Beakes et al. 2014). The distinctive characteristic of the genus Dictyuchus is the dictyucoid discharge mode (Leitgeb 1868; Steciow et al. 2014), meaning releasing of spores individually from cysts in an intact sporangium, leaving a network of cyst walls (Johnson et al. 2002). Currently, there are seventeen records of Dictyuchus species given in the Index Fungorum community resource (Index Fungorum, 2019), but according to the newest literature, D. monosporus and D. pseudodictyon are only valid species (Johnson et al. 2002). The rest are considered as imperfectly known species or excluded from the genus or transferred to other genera. Although most of the difficulties regarding Dictyuchus species delineation stem from its ambiguous sexual behaviour (Humphrey 1890, 1893; Kaufman 1915; Coker 1923), they are still mainly separated based on their dioecious or monoecious behaviour (Johnson et al. 2002), meaning the ability to produce antheridia and oogonia on one single (dioecious) or different (monoecious) hyphae (Johnson et al. 2002). Despite the fact that there are more than 190,000 partial nuclear and mitochondrial sequences assigned to Saprolegniales in the GenBank database (GenBank, NCBI, USA; [Online] http://www.ncbi.nlm.nih.gov/), Dictyuchus is among the least-studied genera. 

During a survey of aquatic oomycetes in twelve different freshwater ecosystems located in northern Iran and north-eastern Germany, only nine out of 150 isolates were identified morphologically as Dictyuchus sp. using their zoospore discharge character. More detailed investigations revealed morphological and morphometric differences among the isolates compared to two other known Dictyuchus species. Phylogenetic analyses of mitochondrial (cytochrome c oxidase I (cox1)) and nuclear rDNA sequences (ITS) were also conducted to investigate the position of this species in the genus Dictyuchus.

MATERIALS AND METHODS

Sampling and isolation

Dictyuchus isolates were isolated by the methods described earlier by Coker (1923), Sparrow (1960) and Seymour (1970) over several months in 2017 (Table 1). Samples of decaying leaves of the dominant local vegetation (Typha spp. L.) collected from Anzali lagoon (37° 28′ 16″ N, 49° 27′ 44″ E) were moved to the mycology laboratory of the University of Guilan, Iran, in separate sterile polyethylene bags. Briefly, the samples were cut into several approximately equal pieces, and after being washed with distilled water, were placed in sterilized plates containing 10 ml sterile distilled water with 10 sterilized hemp (Cannabis sativa L.) seed halves at 20-25°C (Middleton 1943). After three to five days, a piece of mycelia from colonized hemp seed halves was transferred to a fresh CMA-PARP medium (CMA-PARP; 40 g/L ground corn meal, 0.5 g/L ampicillin, 0.01 rifampicin, 0.2 g/L delvocid and 0.1 g/L pentachloronitrobenzene (PCNB), 15 g/L agar) (Kannwischer and Mitchell 1981). This step was repeated three to five times to achieve bacterial free (axenic) colonies. Then, a single hypha was transferred to cornmeal agar (CMA; 40 g/L ground corn meal, 15 g/L agar) (Seymour & Fuller 1987). The hyphal-tip technique was conducted three to five times to obtain a pure culture in CMA. The specimens of this new species were then deposited in the Fungal Herbarium of the Iranian Research Institute of Plant Protection (IRAN).

Characterization of morphological features

For each strain, thirty measurements per replicate were taken of the following characters: breadth of mycelia, length and breadth of sporangia, gemma diamater, and colony and cyst diameter. All measurements and observations were made using an Olympus BH-2 microscope (Olympus Optical, Tokyo, Japan) equipped with AM4023- Digital Microscope 1.3 MPixel 72.5 30 - USB 2.0 (Dino-Lite).

Although temperature and nutrition has been considered effective on initiation and maturation of oomycetes, there was no report on suitable chemically defined medium and temperature for inducing oospore formation by Dictyuchus species. Thus, in order to investigate sexual behavior, several treatments, were used. The nutrition treatments included (1) reciprocal culturing of all isolates with one another and Trichoderma sp. (Brasier et al. 1978) on CMA, (2) hemp seed agar (HSA; 60 g/L ground hemp seeds, 15 g/L agar ) (Hendrix 1964), (3) soybean agar (SA; 100 g/L ground soybean seeds, 15 g/L agar) (Savage et al. 1968), (4) rape seed extract agar (REA; 100g/L ground rape seeds, 15 g/L) (Satour 1967), (5) carrot juice agar (CJA; 250 g/L boiled carrot extract, 20 g/L agar) (Ershad 1971), (6) mPmTG (2, 0.4, 0.4 and 12 g/L glucose, tryptone, peptonized milk and agar, respectively) (Moreau and Moreau 1936b), and (7) immersing colonized CMA in glycerin (4%) (Moreau and Moreau 1936a) and (8) culturing the isolates in Petri dishes containing 10 boiled hemp seeds in distilled lake water and distilled water (50/50). The temperature treatments also included culturing the isolates in 5, 10, 15, 20 and 25°C in Petri dishes containing 10 boiled hemp seeds in distilled lake water and distilled water (50/50).

DNA extraction and PCR

DNA extraction was conducted based on protocol of Montero-Pau et al. (2008). Briefly, 100 µL of alkaline lysis buffer (25 mM NaOH, 0.2 mM disodium EDTA, pH 8.0) was aliquoted into 1.5 mL tubes. Malt extract broth (MEB; 17 g/L malt extract) (Galloway & Burgess 1962) was used for isolates’ growth. Mycelial mass was then transferred to the tube and centrifuged for 30 minutes at 9000 rpm. The tube was incubated at 95°C for 30 minutes and then cooled on ice for five minutes. Finally, 100 µL of neutralizing solution (40 mM Tris-HCl, pH 5.0) was added to the tubes. The final solution was vortexed for 5 minutes and kept at -20°C. Nuclear ITS-rDNA region as well as mitochondrial cox1 was amplified in a Flexible PCR Thermocycler (Analytikjena, Germany) using ITS1/ITS4 (White et al. 1990) and OomCoxI-Levup/OomCoxI-Levlo (Robideau et al. 2011) primers. Thermocycler program for amplification of the ITS region was: 94°C for 2 min of initial denaturation followed by 32 cycles of 94°C for 15 s, 53°C for 15 s, 72°C for 30 s, and a final extension at 72°C for 5 min. Thermocycler program for amplification of the cox1 region was: 95°C for 2 min followed by 35 cycles of 95ºC for 1 min, 55°C for 1 min, 72°C for 1 min. A final extension step was made at 72°C for 10 min. The resulting sequences were edited by using the Bioedit software (Hall 1999) and submitted to GenBank (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov) database (Table 2).

Phylogenetic analysis

The ITS and cox1 sequences of the isolates together with sequences of the representative genera of Saprolegniaceae, retrieved from GenBank, were used to performed phylogenetic analysis. The sequences were aligned using MAFFT (Katoh & Standley 2013), refined manually using BioEdit (Hall et al. 2011), and analyzed with MEGA7 using maximum likelihood method (Kumar et al. 2016).

Table 1. Dictyuchus sp. isolates from Anzali lagoon (Anzali County, Iran) used in this study and their GenBank accession numbers for ITS-rDNA and cox1 regions.

Table 2. Comparison of morphological characters of Dictyuchus sp. isolates used in this study, Dictyuchus monosporus and D. pseudodictyon.

* NA: Not assigned.

RESULTS

In total, nine Dictyuchus sp. isolates were obtained from three different regions in Anzali lagoon, Iran, and studied morphologically, morphometrically, and phylogenetically. The genus Dictyuchus is located within the clade recognized by the presence of eccentric oospores. According to our morphological and morphometric studies, the isolates are separated from their allied species mainly based on its dictyoid and achlyoid spore discharge mode, abundant fusiform and spherical gemma, abundance presence of branched sporangia, and absence of sexual reproduction (Fig. 1, Table 2) (Leclerc et al. 2000; Johnson et al. 2002). The size of sporangia and cyst were also different from their closely related species (Fig. 2, Table 3).

Table 3. Morphometric features (μm) of Dictyuchus sp. isolatesisolated from Anzali lagoon, Iran.

Phylogenetic analyses

The phylogenetic analysis of the ITS sequences (698 bp) of different genera belonging to the family Saprolegniaceae (Saprolegniales, Oomycota), inferred from maximum likelihood method was consistent with the morphology-based taxonomy. According to the ITS derived phylogeny, all the isolates analyzed in this study were associated with Dictyuchus spp. (Fig. 3). The phylogentic analysis of the cox1 sequences (413 bp) also led to robust separation of our isolates from Brevilegnia spp. (Figure 4). ITS and cox1 analyses involved 10 and 6 nucleotide sequences, respectively. However, as it was described before, there were clear different morphological and morphometric characters that raised the question about the novelty of the isolates. The matrix and the resulting tree can be viewed on TreeBASE (accession 23950; http://purl.org/phylo/ treebase/phylows/study/TB2:S23950).

Dictyuchus sp. (Fig. 1) 

Mycelium is dense; main hyphae are slender, sparingly branched, hyaline to dark, with 13.47–43.15 μm (average 29.99 µm) width. The arrangement of mycelia becomes irregular and segmented in old cultures. Sporangia are abundant, mostly branched and fusiform, occasionally curved, renewed sympodially, 80.45–391.84 length×11.78–34.49 width μm (average 210.75 µm), mainly detaching from hyphae regardless of the time the spores become mature, and very long in old cultures. Zoospores rarely form in small sporangia, usually monoplanetic, spores’ discharge behave as dictyoid, in which cysts leaves a network of wall material and sporangium wall may or may not disintegrate in part or entirely, and achlyoid, in which spores clusters (66.25–169.28 μm diam. (average 117.43 μm) do not usually persist at the exit orifice, and may fall away from the sporangium or just collapse. Cysts are 5.94–10.94 μm in diameter. (average 8.82 µm), gemmae is mostly present, spherical and fusiform, when spherical always terminal (77.01–176.11 μm) (average 122.25 μm in diam.), when fusiform mainly intercalary, sometimes catenulate (overabundant in HSA medium) (number of continuous gemma 2–5), and sometimes has irregular shapes in agar media. No specific pattern was observed for any of isolates on CMA. Sexual apparatuses are not produced in any treatments examined. Known distribution – Iran.

Specimen examined – Iran, Guilan Province, Anzali County, Anzali lagoon, on decaying leaves, 37°25'11.0"N 49°26'24.4"E, 20 Jul. 2017, leg. H. Masigol (GenBank Acc. No: ITS – MH253594, MH253589, MH253592, MK400430, MH253595, MH253585, MH253588, MK400431 and MK400432, cox1 – MK396251 and MK396250).

 Fig. 1. Morphological structures of Dictyuchus sp. isolates: a–b. fusiform, intercalary gemma; c–d. spherical, terminal, catenulate gemma; e–f. dark (e) and hyaline (f) slender hyphae; g–h. sympodial renewal of fusiform sporangia, usually with only two successions; i–j. achlyoid discharge mode of zoospores four days after inoculation in water culture; k. immature fusiform sporangia; l. mature sporangia before dictyoid discharge; m–n. immature (m) and mature branched sporangia; o. segmentation of hyphae in one month water culture; p–r, t-v. dictyoid discharge mode of zoospores 10 days after inoculation in water culture in different stage of disintegration; (s) curved and detached immature sporangia. (Scale = 50 μm).

Fig. 2. Boxplots representation of cyst diameter (μm) range (up) and sporangia length (μm) (bottom) of Dictyuchus sp. isolatesinferred from 30 measurements of each character. The measurements have been compared to Dictyuchus monosporus, D. pseudodictyon and D. sterilis (when available).

Fig. 3. Molecular phylogenetic tree of the family Saprolegniaceae (Saprolegniales, Oomycota). The analysis was performed on alignment of the ITS1-5.8S-ITS2 region of nuclea rDNA (610 bp) using the maximum likelihood and Bayesian methods. Numbers next to the branches shows the bootstraps values ≥ 50% and posterior probabilities, respectively.  HQ643117  Aphanomyces euteiches was considered as outgroup.

Fig. 4. Molecular phylogenetic tree of the family Saprolegniaceae (Saprolegniales, Oomycota). The analysis was performed on alignment of the mitochondrial cox1 region (529 bp) using the maximum likelihood and Bayesian methods.Numbers next to the branches shows the bootstraps values ≥ 50% and posterior probabilities, respectively.  HQ708200 Brevilegnia gracilis was considered as outgroup.

Key to Dictyuchus species

1   Antheridia or oogonia or both present…………………………………………………………………...............………….... 2

1* Absence of antheridia and oogonia ……………………………………………………………………..……..................…... 3

2   Dioecious, Anth. or oog. branches, or both, cross-induced……………….………………………….... D. monosporus

2* Monoecious, Antheridia or oogonia self-induced………………………………........…………………. D. pseudodictyon

3 Gemma present, spherical (terminal) or fusiform (intercalary), achlyoid or dictyoid discharge
   mode, mainly branched sporangia, cyst diam. 6-11 μm…………………………………......………….… Dictyuchus sp.

3 Gemma absent, dictyoid discharge mode, cyst diam. 12-17 μm………………………………………… D. sterilis (?)