Phenotypic plasticity of the isolates assigned to Pythium plurisporium

INTRODUCTION

The genus Pythium Pringsh. is a highly diverse, cosmopolitan and heterogeneous group containing more than 230 described species (Hyde et al.2014). Identifying the species of Pythium has often arisen difficulties to researchers due to various reasons such as the absence of certain structures for morphological identification, the lack of identification keys for the species, pleomorphism of the sexual and asexual structures, and the lack of molecular identification data for the species. Molecular phylogenetic studies categorized Pythium spp. into clades with diverse morphological characteristics (de Cock & Lévesque 2004, Villa et al. 2006, Robideau et al.2011, Hyde et al.2014). Uzuhashi et al.(2010) believe that Pythium species belonging to the clades E to J sensu Lévesque & de Cock (2004) should be transferred into two new genera, Globisporangium Uzuhashi, Tojo & Kakish (clades E to J, including a part of clade H) and Elongisporangium Uzuhashi, Tojo & Kakish (another part of clade H) and other species located in the clades A to D (Lévesque & de Cock 2004) should be remained as Pythium sensu stricto.They also described two other genera: Ovatisporangium Uzuhashi, Tojo & Kakish (clade K sensu Lévesque & de Cock (2004), a later synonym to Phytopythium)and Pilasporangium Uzuhashi, Tojo & Kakish (a completely new clade with only one species)among Pythium sensu lato species. However, these genera are still a matter of controversy.

Identification of the morphological features of various species has been a major concern of many researchers (Bala et al. 2010a). Many Pythium species show intraspecific variations for some specific morphological features. Although, phylogenetic analyses using DNA sequences along with other molecular techniques have significantly assisted in the identification of unknown Pythium species, morphological traits comprise fundamental importance to support the identifications defined by molecular techniques.

Pythium plurisporium Abad, Shew, Grand & L.T. Lucas was first isolated from bentgrass (Agrostis palustris Huds.) in North Carolina and formally described as a new species belong to the clade B of ITS phylogenetic tree (Abad et al.1995). This species was characterized by possessing multiple oospores within oogonium. Pythium plurisporium has been reported to be morphologically close to P. multisporum Poitras according to the production of more than one oospore per oogonium; however, the asymmetrical shape of oogonia in P. plurisporium, as well as the existence of swollen elements below oogonial stalk, separate it from P. multisporum. Moreover, P. multisporum belongs to the clade E of Pythium ITS phylogenetic tree (Lèvesque & de Cock 2004), whereas, P. plurisporium is located in the clade B of Pythium ITS phylogenetic tress (Lèvesque & de Cock 2004; Robideau et al. 2011). Pythium plurisporium isolates has been recovered from diseased root and crown of bentgrass. However, it was reported as a secondary colonizer of infected roots. Since its first isolation, no other records have been presented until 2017 (Salmaninezhad & Mostowfizadeh-Ghalamfarsa 2017). During the investigation of rice paddy fields of Fars province, Iran, 1129 Pythium isolates were recovered among which seven isolates were morphologically and phylogenetically similar to P. plurisporium (Salmaninezhad & Mostowfizadeh-Ghalamfarsa 2017). Although, five other isolates were morphologically identified as a new species, phylogenetic analyses revealed that they belong to P. plurisporium. These isolates’ morphological features were completely in the conflict of the original description. These controversial findings led to the conclusion that there could be some intraspecies pleomorphism in the morphological features of the isolates assigned to P. plurisporium,which is discussed in this paper. 

MATERIALS AND METHODS

Sampling and isolation

Sampling was randomly conducted from rhizosphere soil, water ponds and rice seedlings from different rice paddy fields and ornamental trees of Fars province, Iran from 2013 to 2018. Coordinates were recorded for each field by Global Positioning System (GPS) (Table 1). Samples were transported to the Mycology Laboratory of the Department of Plant Protection, Shiraz University. Being washed with distilled water, the rice seedlings were placed on oomycetes semi-selective medium CMA-PARP (Ground corn extract 40 g/L; agar 15 g/L; amended with 10 µg/mL pimaricin, 200 µg/mL ampicillin, 10 µg/mL rifampicin and 25 µg/mL PCNB) (Jeffers & Martin 1986). One hundred grams of each soil sample was placed in a plastic container and flooded with tap water to 1 cm above the soil surface (Tan 1996). Isolates were recovered from either soil or water samples by baiting with 5-mm surface-sterilized bitter orange (Citrus aurantium L.) leaf disks or 5 mm pieces of sterile meadow grass (Poa annua L.) at 25 °C every 8 h for 40 h in total, and plating on CMA-PARP. Isolates were purified by hyphal tip method on water agar (WA, Agar 10 g/L) and stored on CMA (Ground corn extract 40 g/L; agar 15 g/L) slopes at 15 °C.

Morphological characterization

To observe asexual organs (sporangia, vesicle, and zoospores), isolates were transferred to CMA containing sterile hemp (Cannabis sativa L.) seeds or turfgrass (Poa sp.) on agar (Mostowfizadeh-Ghalamfarsa & Banihashemi 2005) for 24 h. Hemp seeds or turfgrass were then transferred to Petri dishes containing distilled water (Ho et al. 2012), sterile soil extract (McLeod et al. 2009) or Schmitthenner solution (Schmitthenner 1973) under fluorescent light for 24 h and were checked every 8 h for six times. Sporangial formation was examined using French bean agar (FBA, French bean extract 30 g/L; agar 15 g/L) (Jeffers & Martin 1968) and sterile soil extract (Mostowfizadeh-Ghalamfarsa et al.2008). Sexual organs were obtained with hemp seed agar (HSA, ground hemp seed extract 60 g/L; agar 15 g/L) and carrot agar (CA, carrot extract 250 g/L; agar 15 g/L) incubated in darkness (Mostowfizadeh-Ghalamfarsa & Banihashemi 2005). To study colony morphology, isolates were grown on CMA, HSA, CA, potato-dextrose agar (PDA, potato extract 300 g/L; dextrose 20 g/L; agar 15 g/L) and malt extract agar (MEA, Malt extract 25 g/L; agar 15 g/L) (Mostowfizadeh-Ghalamfarsa & Banihashemi 2005). Five mm diameter plugs from the edge of a 3 d old culture were placed on Petri dishes containing 20 mm of a particular test media. The plates were incubated at 25 °C for 48 h. Temperature-growth relationships were tested on PDA with three replicate plates per isolate and incubated at 0, 5, 10, 15, 20, 25, 30, 35, and 40 °C. The growth rate was recorded 2–12 d after the onset of linear growth.

Sequencing and phylogenetic analyses

The method described by Mirsoleimani & Mostowfizadeh-Ghalamfarsa (2013) was employed for DNA extraction. Potato extract broth (extract of 300 g/L boiled potato in distilled water) was used for the growth of isolates. Mycelia were harvested, freeze-dried, and DNA extracts were obtained using a DNG^TM-PLUS extraction kit (Sinagene, Iran) according to the manufacture’s instruction. The DNA quality was examined with an MD-1000 Nanodrop machine (NanoDrop Technologies, USA). The primers used for amplification and sequencing of nuclear (Internal transcribed spacers 1, 2 and 5.8S region of rDNA (ITS-rDNA) and β-tubulin gene (Btub)) as well as mitochondrial (cytochrome c oxidase subunit II (cox2)) loci are listed in table 2. The PCR conditions for these loci are listed in table 3. PCR products were purified and sequenced with the primers used for amplification by a dye terminator cycle (Bioneer, South Korea). Sequenced data were deposited into GeneBank and accession numbers were obtained.

Resulting sequences were edited by Bioedit (Hall 1999). The sequence alignment of the amplicons together with data extracted from GenBank (Supplementary Table 1) was conducted by ClustalX (Thompson et al.1997) with subsequent visual adjustment. To reconstruct the phylogenetic trees, Bayesian inference analyses on individual and concatenated ITS, Btub and cox2 loci were carried out with MrBayes v. 3.1 (Rounquist & Huelsenbeck 2003), imposing a general time-reversible (GTR) substitution model with gamma (G) and proportion of invariable site (I) parameters to accommodate variable rates across sites. Bayesian analyses were conducted with the same data set according to Safaeifarahani et al.(2015). The best nucleotide substitution model was determined by MrModelTest v. 2.3 (Nylander 2004). Two independent runs of Markov Chain Monte Carlo (MCMC) using four chains were run over 1,000,000 generations. Trees were saved each 1000 generations, resulting in 10001 trees. Burn-in was set at 5% generations. Pythium nagaii S. Ito & Tokun.was chosen as an outgroup. Phylogenetic trees were edited and displayed with TREEGRAPH (Stöver & Müller 2010). Partition homogeneity tests were conducted on combined nuclear and mitochondrial gene alignments by PAUP* 4.0a136 (Swofford 2002) using 100 replicates and heuristic general search option. Alignments and trees were submitted to TreeBASE (http://www.treebase.org).

Pathogenicity

Resulting isolates were evaluated for their ability to cause stunting, post- and pre-emergence damping-off and seed rot, and their pathogenicity on rice plants. Inoculum preparation was conducted by the method described by Salmaninezhad & Mostowfizadeh-Ghalamfarsa (2019) with mycelium inoculated vermiculite amended with 120 mL/L hempseed extract (extract of 60 g boiled hemp seed).

For pre-emergence damping-off tests, rice seeds were washed and planted in sandy loam (1:1) soil (500 mL) amended with 10 mL inoculum. Post-emergence damping-off was examined on 20 d old seedlings inoculated with 10 mL inoculum per pot (500 mL soil). Control pots contained only hempseed extract amended vermiculite. Symptoms were monitored two weeks after inoculation. To evaluate the ability of various isolates in the colonization of root and crown tissues of rice, after being cut into 0.5 mm pieces, roots and crown were washed, blotted and placed on the CMA-PARP medium at 25 °C. The growth rate was checked every 12 h during a week (Afeck et al. 1990).

RESULTS

Pythium plurisporium isolates from Iran

A total of 12 isolates of Pythium spp. With filamentous sporangia were recovered from rice paddy fields of Fars province. These isolates formed two distinct morphological groups: the first group consists of seven isolates which were morphologically similar to the original description of P. plurisporium (Abad et al. 1996). Morphometric results can be seen in table 4. The representative sequenced isolate of the group was located in the vicinity of P. plurisporium in Clade B of ITS phylogenetic tree (Lévesque & de Cock 2004) (Fig. 1).

Some other recovered isolates from Iran were morphologically different from the others as well as the original description of P. plurisporium.The representative sequenced isolates of this group also appeared as P. plurisporium both in ITS and combined gene trees (Fig. 1 and 2). Variations have been observed in these isolates (table 4). The morphological features of both groups (i.e. Group I and Group II) are described below.

Morphological Group I

Colonies on PDA, MEA, HSA, and CA show rosette pattern, on CMA with uniform pattern (Fig. 3a). Main hyphae up to 6 µm wide. Sporangia are filamentous, slightly inflated with long discharge tube (Fig. 4a). Sporangia were abundantly formed in liquid media after 10 h (Salmaninezhad & Mostowfizadeh-Ghalamfarsa 2017). Vesicles and zoospores are produced on sterile hempseed in water cultures after 12 h at 25 °C. Oogonia are obpyriform, smooth, terminal, aplerotic, consisting of at most two oospores, (Fig. 4b–f), and variable in size (table 4).

Table 1. List of Pythium plurisporium isolates recovered from rice paddy fields of Fars Province of Iran with their GenBank accession numbers.

Supplementary Table 1. GenBank accession numbers of Pythium species used for phylogenetic reconstructions.

Table 2. List of primers used in this study.

^aInternal transcribed spacers 1, 2 and 5.8S gene of rDNA. ^b β-tubulin. ^c cytochrome c oxidase subunit II.

Table 3. PCR conditions for primers used in this study.

^aInternal transcribed spacers 1, 2 and 5.8S region of rDNA. ^b β-tubulin. ^c cytochrome c oxidase subunit II. ^d Temperature ‘ºC’ (time s’)

None of the isolates produced more than two oospores in a single oogonium. Antheridia are 6–12 per oogonium, crook-necked, mostly monoclinous, and sometimes diclinous, with a terminal contact, paragynous (Fig. 4b–f). Antheridium origination in monoclinous oospores is near oogonium stalk. Although no swollen elements were observed in the oogonial stalk, the stalk was swollen itself (Fig. 4). None of these isolates produced any papillae on oogonium. Oospores are mostly globose, aplerotic, up to 30 µm in diam, with a wall which is up to 3 µm thick. Morphometric results are shown in table 4. Colonies on PDA have an average radial growth rate of 5.5 mm/d at 15 °C, 10 mm/d at 20 °C, 25 mm/d at 25 °C, 27 mm/d at 30 °C and 35 °C, 7 mm/d at 40 °C and no growth at 5 °C and 10 °C (Fig. 7). Cardinal temperatures: optimum 35 °C, minimum 15 °C, and maximum 40 °C.

Specimens examined. Iran, Fars Province: Kamfiruz, (30°20.426'N−052°16.460'E), from pond water of Oryzae sativa, 16 Aug. 2014, F. Salmaninezhad Kb440; Kamfiruz, (30°17.131'N−052°19.039'E), from nursery soil of Oryzae sativa, 16 Aug. 2014, F. Salmaninezhad Kh416; Kamfiruz, (30°17. 412'N −052°18.682'E), from nursery soil of Oryzae sativa, 16 Aug. 2014, F. Salmaninezhad Kh423; Kamfiruz, (30°17.396'N−052°18.698'E), from nursery soil of Oryzae sativa, 16 Aug. 2014, F. Salmaninezhad Kh424; Kamfiruz, (30°17.395'N−052°18.696'E), from pond water of Oryzae sativa, 16 Aug. 2014, F. Salmaninezhad Kh425; Kamfiruz, (30°17.392'N−052°18.693'E), from pond water of Oryzae sativa, 16 Aug. 2014, F. Salmaninezhad Kh426; Ramjard, (30°05.671'N−052°35.522'E), from root of Oryzae sativa, 20 May 2014, F. Salmaninezhad KC12. GenBank: ITS = KX228082.

All isolates of the Group I were extremely pathogenic on rice seedlings causing post-emergence damping-off (Fig. 5a; table 5). The representative isolate of this grouped was located in the vicinity of P. plurisporium in the Clade B of ITS phylogenetic tree (Lèvesque & de Cock 2004).

Morphological Group II

Colonies on MEA show no specific pattern, on PDA, HSA and CA show an intermediate pattern between radial to rosette and on CMA show an approximately radial pattern (Fig. 3b). Main hyphae have 2.9–4.3 (av. 3.2) µm width. Sporangia are filamentous, slightly inflated with a rather long discharge tube (Fig. 6a). Vesicles and zoospores are formed plentifully on sterile hempseed in water cultures after 12–24 hours at 20–25 °C. Oogonia are globose, smooth, terminal and intercalary, 25.9–27.2 (av. 26.2) µm. More than 50 % of the oogonia have one or two papillae which are 1.5–6.7 (av. 2.2) µm long (Fig. 6b).

Fig. 2. Phylogenetic relationships of Pythium plurisporium from paddy fields of Fars Province among 36 Pythium species based on the analysis of multigene genealogies of nuclear (ITS and Btub) and mitochondrial (cox2) sequences. Values on branches are posterior probability based on Bayesian analysis greater or equal to 0.5. Pythium nagaii is used as an outgroup taxon.

Fig. 3. Colony morphology of examined groups of Pythium plurisporium in this study: a. Group I; b. Group II after 24 h on various media at 25 °C; top (from left to right): carrot agar, malt extract agar and potato-dextrose agar; bottom (from left to right): cornmeal agar and hempseed agar.

Fig. 4. Morphology of Pythium plurisporium (Group I). a. filamentous, slightly inflated sporangium; b. oospore with monoclinous and diclinous antheridia; c. ellipsoid aplerotic oogonium; d. aplerotic oogonium with two oospores; e. oogonium with swollen stalk; f. Oogonium with 10 to 12 paragynous antheridia. — Scale bars = b-f: 10 µm, a: 20 µm .

Fig. 5. Pathogenicity tests on roots and crown of rice (Oryzae sativa) by representatives of Pythium plurisporium groups. a. Group I (KC12) which causes post-emergence damping-off and root rot (left: control; right: infected crown and roots). b. Group II (045-1) which causes severe root and crown rot; pre- and post-emergence damping-off (left: control; right: infected crown and root).

Fig. 6. Morphological structures of Pythium plurisporium (Group II). A. filamentous slightly inflated sporangium; b. aplerotic oospore with two long papillae; c. aplerotic oospore with a papilla and paragynous antheridium. — Scale bars = a: 20 µm, b-c: 10 µm.

 Fig. 7. Average radial growth rate of Pythium plurisoprium isolates from Iran on potato-dextrose agar at different temperatures; Group I (upper diagram) Group II (lower diagram).

Antheridia are 2–5 per oogonium, crook-necked, mostly diclinous, and sometimes monoclinous, with a terminal contact, paragynous (Fig. 6c). Antheridium origination in monoclinous oospores is near oogonium stalk. Oospores are globose, aplerotic, 22.3–23.2 (av. 23.0) µm with a wall which is 1.3–2.1 (av. 1.5) µm thick. Morphometric results are shown in table 4. Colonies on PDA have an average radial growth rate of 3.5 mm/d at 15 °C, 5 mm/d at 20 °C, 10 mm/d at 25 °C, 11 mm/d at 30 °C and 35 °C, 2 mm/d at 40 °C and no growth at 5 °C and 10 °C (Fig. 7).  Cardinal temperatures: optimum 35 °C, minimum 15 °C, and maximum 40 °C.

Phylogenetic analyses using both nuclear (ITS, Btub) and mitochondrial (cox2) loci revealed that this taxon is located in Clade B in the vicinity of  P. plurisporium in a separate monophyletic group (Fig. 1 and 2).

Specimens examined. IRAN,FARS PROVINCE: Kamfiruz (30°16.545'N−052°19.659'E), from the roots of Oryzae sativa, 16 Aug 2014, F. Salmaninezhad 045-1 (CBS 140940). GenBank: ITS = KX228085; Btub = KX228110; cox2 = KX228123.2. IRAN,FARS PROVINCE: Ramjard (30°06.568'N−052°34.164'E), from Oryzae sativa crown, 9 Nov 2015, F. Salmaninezhad SS. GenBank: ITS = KX228074; Btub = KX228111; cox2 = KX228122. IRAN, FARS PROVINCE: Kamfiruz (30°17.421'N−052°18.692'E), from root of Oryzae sativa, 16 Aug 2014, F. Salmaninezhad PS. GenBank: ITS = KX228086; Btub = KX228112; cox2 = KX228121. IRAN, FARS PROVINCE, Kamfiruz (30°18.199'N−052°17.635'E), from rhizosphere soil of Oryzae sativa paddy fields, Aug 2014, F. Salmaninezhad, HS.

All isolates of the Group II were severe pathogens of rice seedlings, causing pre- and post-emergence damping-off, as well as root, crown, and seed rot (table 5).

Note. The length of papilla was mainly more than 3 µm in the isolate 045-1. Generally, oogonia in this isolate (i.e. 045-1) contained more than one papilla. However, more than 60% of oogonia had only one papilla in other isolates.

DISCUSSION

Among 1129 Pythium isolates recovered from rice paddy fields of Fars Province, Iran, 12 isolates were assigned to P. plurisporium (Salmaninezhad & Mostowfizadeh-Ghalamfarsa 2017). These isolates formed two distinct morphological groups: Group I consists of isolates KC12, Kb440, Kh419, Kh423, Kh424, Kh425, and Kh426. These isolates were recovered from rice root, pond water, and nursery soil (Table 1). All the isolates produced 1 to 2 oospores per oogonium. Colony morphology on all examined media was rosette form, except for CMA, which was uniform.This was in contrast with the original description of P. plurisporium which was reported to be chrysanthemum on CA and CMA (Abad et al. 1996).

Table 4. Comparison of Pythium plurisporium isolates from the original description (North Carolina) and Iran isolates.

Table 5. Pathogenicity results of the Pythium plurisporium isolates examined in this study.