Phytopythium leanoi sp . nov . and Phytopythium dogmae sp . nov . , Phytopythium species associated with mangrove leaf litter from the Philippines

The genus Phytopythium is a monophyletic taxon of the Peronosporaceae with characteristics intermediate between Phytophthora and Pythium. In the Philippines, reports of Phytopythium are scarce, with the mangrove-swamp-inhabiting species Phytopythium kandeliae being the only species recorded to date. It was the aim of the current study to investigate the diversity of Phytopythium in mangrove habitats in more detail. Based on culture characteristics, morphology, and molecular phylogenetic position, two new species of Phytopythium are described from Philippine mangroves, P. leanoi USTCMS 4102 and P. dogmae USTCMS 4101. Phytopythium leanoi is a species morphologically similar to P. kandeliae, but with the ability to develop gametangia in a homothallic fashion. The other new species, P. dogmae, is characterized by having a short discharge tube, semipapillate to papillate sporangia and frequently exhibiting a clustering of two sporangia per sporangiogenic hypha. With the addition of the two species described in this study, the genus Phytopythium has grown from around 10 to beyond 20 recognized species over the past decade, and it seems likely that several more species of this genus await discovery.


Introduction
Oomycetes are fungal-like organisms of the eukaryotic kingdom Straminipila.To date, approximately 2000 species striving in different habitats ranging from the arctic to tropics have been described [1].The discovery of new lineages that branch outside the major genera of Peronosporales [2][3][4][5][6][7] has fueled research into the evolution and diversity of cultivable oomycetes of the Peronosporales [8].Of the recently-described oomycete lineages, the former K-clade Pythium species [5,9] have attracted intense research and were segregated taxon from Pythium in 2010 [2] as members of the new genus Phytopythium on the basis of phylogenetic position and morphology.Uzuhashi et al. [7] as well inferred the polyphyly of Pythium based on large ribosomal subunit region (LSU) and cytochrome oxidase II (cox2) gene sequences and proposed four segregate genera, Elongisporangium, Pilasporangium, Globisporangium, and Ovatisporangium.
The genus Ovatisporangium was described specifically for the clade K of Pythium, however, this genus is a synonym of Phytopythium, as the print version of the latter genus description appeared earlier than the former.The characteristic features of Phytopythium as described by Bala et al. [2] are: globose to ovoid shaped sporangia that are often papillate, internal proliferation of sporangia, zoospore discharge similar to Pythium, smooth and large oogonia, thick-walled oospores and elongate to lobate lateral antheridia.Phytopythium sindhum is the type species of this group, isolated from a banana (Musa paradisiaca L.) field in Pakistan.
In the Philippines, mangrove oomycetes were reported by Leaño [10], including a single Phytopythium species, P. kandeliae [1] (basionym Halophytophthora kandeliae), which was isolated alongside Halophytophthora vesicula, H. bahamensis, H. epistomium, and Salispina lobata (homotypic synonym H. spinosa var.lobata) [10].Considering that during the past decade several new species of Phytopythium have been discovered and that mangroves have been found to harbor a variety of oomycete species [10][11][12], it was the aim of this study to evaluate if additional species of the genus Phytopythium might be present in Philippine mangroves.

Material and methods
Isolation, growth on solid media, and Phytopythium strains Fallen senescent mangrove leaves from various areas in the Philippines were collected and placed in sealed plastic bags.Leaves were blot-dried, cut into strips of approximately 1-2 mm width, and transferred onto clarified-vegetable juice agar (VJ) [medium No. 15, NBRC (http://www.nite.go.jp/en/nbrc/cultures/media/culture-list-e.html), using V8 Juice (Campbell, USA) or Gemüsesaft (Alnatura, Germany), with or without addition of seawater] with nystatin (500 mg/mL) and rifampicin (30 mg/mL) or streptomycin (0.5 mg/mL).Coenocytic hyphae growing from the edge of the leaf strips were cut and placed on new VJ media with antibiotics until axenic.Cultures were maintained in VJ with or without antibiotics.Incubation was done at room temperature in the dark for 7 days.Additional strains of Phytopythium kandeliae NBRC 32620, Phytopythium sp.CBS 113.91, Phytopythium sp.CBS 111.91, P. chamaehyphon CBS 259.30, and P. helicoides CBS 286.31 were either purchased from NITE Bioresource Centre (NBRC, Japan) or the Westerdijk Fungal Biodiversity Centre Culture Collection (KNAW, The Netherlands).
The mean radial colony growth of the Philippine strains at 20, 25, 30, and 35°C was assessed on VJ agar [medium No. 15, NBRC; using Gemüsesaft (Alnatura, Germany)] and three media as formulated by CBS [13]: potato carrot agar [PCA; based on Demeter Karotten mit Kartoffeln purée (Alnatura, Germany)], potato dextrose agar (PDA), and peptone yeast-extract glucose agar (PYGA).Colony radial growth was measured for 5 days and values were expressed as mm/day following the method outlined by Solis et al. [14].For an initial testing at room temperature, isolates were also tested for growth on oatmeal and corn meal agar as formulated by CBS [13].

Induction of sporangia and gametangia
Sporangia and gametangia were induced using 6 mL saline (sea salt) solution at 0, 10, 20, and 30 g/L poured onto 3-7-day-old mycelia in VJ agar plugs in 60-mm Petri dishes.Plates were incubated at room temperature until sporangia and gametangia were formed.Alternatively, sporulation was induced in 90-mm culture plates using 6 mL unsterile soil extract (500 g soil in 500 mL distilled water, settled for 1-2 days and filtered with double-layered cheesecloth) and 6 mL saline solution as above.The set-up was incubated in a climate chamber (CMP 6010; Conviron, Germany) with continuous light and alternating constant temperature at 20°C for 6 h and 23°C for 18 h.Structures were observed and photos were taken using a Canon Digital Camera EOS 500D (Canon, Japan) attached to a Motic AE31 trinocular inverted microscope (Motic, Germany).

DNA extraction, PCR, and phylogenetics
DNA extraction was performed on a BioSprint 96 Kingfisher flex robot (Thermo Fisher Scientific, USA) using a Qiagen plant tissue DNA extraction kit (Qiagen GmbH, Germany).Primer pairs for PCR amplification of the internal transcribed spacers (ITS), large nuclear ribosomal subunit (nrLSU), cytochrome oxidase I (cox1), and cytochrome oxidase II (cox2) are listed in Tab. 1.
The PCR reaction mix contained 1× PCR buffer, 0.2 mM dNTPs, 2.0 mM MgCl 2 , 0.8 µg BSA, 0.4 µM of each primer, 0.5 U Taq polymerase and 10-50 ng DNA.Cycling conditions for ITS were as follows: initial denaturation at 94°C for 4 min, followed by 36 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 20 s, and elongation at 72°C for 60 s.Subsequently, a final elongation at 72°C for 4 min was carried out.For LSU, initial denaturation was at 95°C for 2 min followed by 35 cycles of denaturation at 95°C for 20 s, annealing at 53°C for 20 s, and elongation at 72°C for 120 s.Subsequently, a final extension was carried out at 72°C for 7 min.Cycling conditions for the cox2 region were: initial denaturation at 94°C for 4 min, followed by 36 cycles of denaturation at 94°C for 40 s, annealing at 51°C for 40 s, and elongation at 72°C for 40 s.Subsequently, a final elongation was carried out at 72°C for 4 min.The cox1 locus was amplified with the following cycling conditions: initial denaturation at 95°C for 4 min, followed by 36 cycles of denaturation at 95°C for 40 s, annealing at 51°C for 40 s, and elongation at 72°C for 60 s.Subsequently, a final extension was carried out at 72°C for 5 min.PCR reactions were carried out on an Eppendorf Mastercycler Pro (Eppendorf AG, Germany).PCR products were sequenced at SBiK-F sequencing laboratory with the primers used for PCR.Sequences were analyzed, assembled into contigs, and edited using Geneious version 5.0.4 (Biomatters Ltd., USA).Edited contigs in FASTA format and other sequences obtained from the NCBI webserver (https://www.ncbi.nlm.nih.gov/)(Tab.S1) were uploaded to the TrEase webserver (http://www.thines-lab.senckenberg.de/trease/)for the alignment of sequences and phylogenetic tree construction.The sequence alignment was generated using MAFFT with the G-INS-i algorithm [15].Phylogenetic trees were constructed for all loci using the following programs: FastTree for minimum evolution (ME) [16] with 1000 bootstrap replicates using the generalized time-reversible (GTR) model; RAxML for maximum likelihood (ML) with 1000 bootstrap replicates using the GTR-GAMMA model [17]; and MrBayes [18] for Bayesian inference and the calculation of Bayesian posterior probabilities (BPP) using the 6 GTR model and four incrementally heated chains run for 1 000 000 generations, sampling every 10 000th generation, with the first 30% of the trees discarded for ensuring sampling from the stationary phase.After confirming that no strongly supported alternate topologies were present, the individual alignments were concatenated into a single FASTA file and the phylogenetic inference was done on the combined dataset as described above.Phylogenetic trees were viewed and taxon labels were edited using MEGA, version 6 or 7 [19].

Phytopythium cultures
The two Phytopythium isolates, USTCMS 4102 (Fig. 1) and USTCMS 4101 (Fig. 2), were isolated from mangrove leaf litter in the Philippines.Both isolates were obtained from yellow to brown leaves that were in direct contact with the water surface of the estuarine system.Mean colony radial growth (Fig. 3) of Phytopythium sp.USTCMS 4101 and Phytopythium sp.USTCMS 4102 varied in relation to the agar medium used.Specifically, mean colony radial growth on vegetable juice agar was observed to be 11.25 and 6.75 mm/day at 25 and 30°C, respectively, for the former strain, 9.25 and 10.25 mm/day at 25 and 30°C, respectively, for the latter strain.However, USTCMS 4101 was also capable of growing at 35°C with a radial growth similar to the growth observed at 30°C.In contrast, Phytopythium sp.USTCMS 4102 showed limited growth on all culture media when incubated at 35°C.

Morphology
Phytopythium sp.USTCMS 4102 (Fig. 1) was initially identified as Phytopythium aff.kandeliae based on the mode of zoospore release and shape of sporangia.Phytopythium sp.USTCMS 4102 developed non-to semipapillate, obovoid to pyriform sporangia, with a size of 22-34 × 27-43 µm (average 28 × 35 µm; n = 100) within the standard deviation.Proliferation was both internal and external and branching sympodially.Zoospores of this strain developed both within vesicles and sporangia, and, upon  maturity, biflagellate cells moved rapidly inside the vesicle before rupture and release.An operculum was visible once sporangia were empty, which curled distally after some time.Interestingly, USTCMS 4102 is a homothallic species.Oogonia were formed either terminally or intercalary, with a size of 36-46 µm (average 41 µm; n = 100) within the standard deviation; oospores were mostly plerotic with a size of 31-41 µm (average 36 µm; n = 100) within the standard deviation.The antheridia were either lobate with constrictions, elongate, or bilobed and were attached at places all over the oogonial surface.USTCMS 4102 is distinct from P. kandeliae NBRC 32620 (Tab.2) on the basis of development of intercalary or terminal oogonia with an elongate to cylindrical antheridium and on the process by which sporangia proliferate -internal and external for the former and external for the latter.
Sporangia of USTCMS 4101 were obovoid, ovoid to pyrifom and limoniform, with a size of 25-27 × 29-37 µm (average 26 × 33 µm; n = 100) within the standard deviation.A papilla was formed at maturity, which further developed into a short dehiscence tube (mostly 2-10 µm long).Before zoospore release, the protoplasmic component of the sporangia gradually passed through the apical discharge tube until it came to a rest at Tab. 2 Morphological differences between P. kandeliae and P. leanoi.

Discussion
According to de Cock et al. [20], key characteristics for Phytopythium are the following: predominantly papillate sporangia and internal proliferation in most species as well as often constricted and elongate to cylindrical antheridia.However, there is a large degree of variation in sporangium characteristics.Phylogenetically, the Calycofera-Phytopythium lineage [25] is one of the early-diverging branches in the sister clade to Pythium that also harbors Phytophthora [2].This is reflected by some morphological features shared with the one or the other of these genera, i.e., a sporangial shape similar to the majority of Phytophthora species and a process of zoospore release similar to most Pythium species.The strains USTCMS 4102 and USTCMS 4101 constitute two new records of Phytopythium species from the Philippines, adding to P. kandeliae [10].Both strains were isolated from mangrove leaf litter, where they acted as saprotrophs.The strain USTCMS 4102 was inferred to be a member of the Clade 1 [23,24] group of Phytopythium (Fig. 4) and is almost sequence-identical to CBS 113.91 isolated from Taiwan.The ex-type strain of P. kandeliae NBRC 32620 is phylogenetically distinct from the aforementioned strains but sequence-identical to CBS 111.91 in all loci included in this study.Sporangia of P. kandeliae NBRC 32620 and CBS 111.91, as well as those of USTCMS 4102 and CBS 113.91, are operculate and non-to semipapillate [21] (also, this study).However, sporangia for the former two strains were turbinate, obovate to pyriform, and formed an apical translucent crescent-shaped matrix [22] (also, this study), while the sporangia of the latter two strains were obovoid to pyriform and without an apical crescent-shaped translucent matrix.The proliferation of the sporangia was external for P. kandeliae NBRC 32620 and CBS 111.91, while it was both internal and external for USTCMS 4102.With respect to colony morphology, there are also some differences between the groups, as USTCMS 4102 and CBS 113.91 formed rosette-petaloid-like colonies with rounded edges, whereas the colony edges of P. kandeliae NBRC 32620 and CBS 111.91 were acute (Tab.4).Even if these differences might seem minor, they were consistently observed and thus seem stable enough to allow for differentiation.
USTCMS 4101 is a member of the Clade 2 of Phytopythium [23,24], and differs from all other members of the clade by featuring both internal and external proliferation, while other members of this clade are featuring extended or nested-internal proliferation [20].USTCMS 4101 is similar to P. chamaehyphon, P. fagopyri, P. helicoides, and P. palingenes.However, it has a different combination of key morphological characters (e.g., proliferation of the sporangia and the number of sporangia per sporangiogenic hypha; see also Tab. 3).The sister taxon to USTCMS 4101 is P. palingenes, from which it differs in terms of the proliferation of sporangia, the length of discharge tubes (which is longer in P. palingenes), and the number of sporangia per sporangiogenic hypha, which are often two in USTCMS 4101 but usually single in P. palingenes.
Based on phylogenetic distinctiveness and combination of morphological characters, neither USTCMS 4101 nor USTCMS 4102 can be assigned to any known species of Phytopythium and are thus introduced as new species in the "Taxonomy" section of this paper.Considering the recent discovery of some new species of Phytopythium from a variety of habitats and that only mangroves were sampled in the current study, it seems likely that additional species of Phytopythium await discovery in the Philippines and beyond.

Colony characteristics.
Grows on corn meal agar (CMA), oatmeal agar (OA), peptone yeast glucose agar (PYGA), potato carrot agar (PCA), and vegetable juice agar (VJ).Growth on VJ with abundant aerial mycelium.Colonies stellate to rosette-like with rounded edges on all culture media used; except OA and CMA, and when old in VJ agar.Etymology.Dedicated to Irineo J. Dogma Jr., for his pioneering research on "zoosporic fungi" in the Philippines.

Colony characteristics.
Petaloid or rosette-like on VJ agar and PCA media with or without marine water.Grows on CMA, OA, PYGA, and PCA.Mycelia on VJ, PDA, and PYGA are typically with abundant aerial mycelium.

Fig. 2
Fig. 2 Morphology of Phytopythium dogmae USTCMS 4101.Colony patterns on (a) vegetable juice agar, (b) potato carrot agar, (c) peptone yeast glucose agar, and (d) potato dextrose agar.e Immature sporangium.f Developing sporangium with a developing papilla.g Papillate sporangium (arrow).h,i Papilla develops into a discharge tube that guides the discharging protoplasma (arrow) forming an external vesicle that will nest at the apex of the sporangia.i Convex basal plug (arrow).j Development of zoospores in the vesicle.k Zoospores can similarly mature and develop inside the sporangia.l Empty sporangium; note the presence of the short discharge tube (arrow).Scale bars: 20 µm.

Fig. 4
Fig. 4 Minimum evolution phylogenetic reconstruction generated from concatenated sequences of ITS, cox1, cox2, and LSU.Support values from minimum evolution, and maximum likelihood bootstrapping, as well as Bayesian posterior probabilities are given on the branches in the respective order."-" denotes support values below 50 or alternate, unsupported topologies.The scale bar indicates the number of nucleotide substitutions per site.

P. leanoi (USTCMS 4102) Phytopythium sp. (CBS 113.91) P. kandeliae (NBRC 32620 ex-type) Phytopythium sp. (CBS 111.91)
Zoospores developed inside the vesicle and often also inside the sporangium.After some time, zoospores gradually began to move within the vesicle until the rupture of the vesicle and the subsequent release of zoospores took place.No gametangia were observed for USTCMS 4101.In Tab. 3, the morphological differences of species closely related to USTCMS 4101 are summarized.
* No data available.