Phytophthora estuarina Marano, A.L. Jesus & Pires – Zottar., sp. nov.
Index Fungorum number: IF 551608, Facesoffungi number: FoF 01275, Fig. 2
Etymology — “estuarina” refers to the estuarine habitat in where this species was isolated.
Holotype — SP 466380
Mycelium well-developed on PYGs, aerial mycelium scanty, hyaline, branched, aseptate, hyphae 3.75 – 5 μm thick (av. 4.85 μm); hyphal swellings sphaerical, globose, tubular to irregular. Zoosporangiophores undifferentiated of the vegetative hyphae, long, simple or sympodially branched, bearing one terminal zoosporangium. Zoosporangia produced abundantly in water cultures, non-caducous, semipapillate or apapillate, ovoid to obpyriform, 55 – 83 × 43 – 63 μm (av. 77 × 54 μm), internally proliferating in both a nested and extended way; secondary lateral zoosporangia regularly formed; transparent lens-shaped plug material prior to zoospore differentiation; wall rough after zoospore discharge; basal-plug present. Zoospores formed inside the zoosporangium and discharged by an elongate, vase-shaped, and semi-persistent vesicle, 33 – 80 μm long when expanded, through which zoospores swim away; encysted zoospores 7.5 – 12.5 μm diam. (av. 10.1μm). The vesicle shrinks completely in length and width in up to 1 h after zoospore release. Chlamydospores and sexual structures not observed. Gametangia not produced in single culture or when paired with tester strains of P. capsici A1 (CBS 111334) and A2 (CBS 370.72). Auto-sterile when the isolates were paired with each other. Radial growth rates on PYGs (photoperiod: 12 h) at near the optimum temperature (30 °C)= 12 ± 1 mm/d (n = 10); at near the maximum temperature (35 °C) = 2 ± 1 mm/d (n = 10); no growth during five days at 40 °C and even after subsequent incubation at room temperature (~20 °C).
Culture characteristics — colonies cottony, with scanty aerial mycelium and no defined growth pattern on PYGs.
Material examined — BRAZIL, São Paulo, Cananéia, BParque Estadual da Ilha do Cardoso^ (PEIC), 25°03′05″– 25°18′18″S; 47°53′48″–48°05′42″W, Perequê river (salinity 1.3 %), from leaves of Laguncularia racemosa onto PYGs medium, 27 Feb 2013, A.L. Jesus, C.L.A. Pires-Zottarelli & A.V. Marano (SP 466380, holotype), ex-types CCIBt 4157, MMBF 14/15; Ibid., permanent shallow lagoon (salinity 2.8 %), from leaves of Rhizophora mangle, on Sorghum sp. seeds, 30 Aug 2012 A.L. Jesus, C.L.A. Pires-Zottarelli&A.V. Marano (SP 466372, paratype), ex-paratypes CCIBt 4116, MMBF 06/15.
Notes — The isolates of P. rhizophorae and P. estuarina were recovered from mangrove swamps, which exhibited salinity concentrations between 0.8 and 2.8 % and, therefore, the habitat of the ITS Clade 9 members is expanded to include estuaries. Both P. rhizophorae and P. estuarina appear as welldelimited species and along with ten other species, they consistently form a high temperature-tolerant subclade within Clade 9, supported by strong bootstrap (100 %) in our ITS phylogeny (Fig. 1). Phytophthora rhizophorae is phylogenetically related with P. virginiana and P. parsiana, while P. estuarina appear as closely related to P. macilentosa and P. irrigata in our ITS phylogeny. The two new species share the presence of ovoid to obpyriform, apapillate and non-caducous zoosporangia, which are terminal and internally proliferating in both a nested and extended way (Figs. 2 and 3). These characteristics appear to be common to most members of Clade 9. Phytophthora estuarina has additionally semipapillate zoosporangia, a characteristic that is present in a few species from this clade, such as P. constricta (Rea et al. 2011). During zoospore discharge, it develops an elongate and semi-persistent vesicle, which completely retracts in length and width in up to 1 h after zoospore release and acquires a characteristic morphology after shrinkage (Fig. 3). The zoosporangia have wrinkled walls after zoospore release and the shrunken vesicle remains constricted at the apex of the zoosporangium. This process of vesicle development is peculiar and has not been previously reported for Phytophthora species.

Fig. 1 Phylogramgenerated fromMaximum likelihood (ML) analysis (PhyML 3.1, Guindon and Gascuel 2003) based on entire ITS rDNA sequences showing the phylogenetic placement of Phytophthora rhizophorae and P. estuarina within Phytophthora Clade 9.ML bootstrap support values < 50 % are marked with (-). Clades that do not appear in the Bayesian analysis are indicated with a zero. Bayesian posterior probability values (MrBayes 3.2, Ronquist et al. 2012) > 0.50 are labelled numerically. Scale bar indicates the average number of substitutions per site. New taxa are in blue and species for which obtained sequences are based on type material have names in bold.

Fig. 2 Phytophthora estuarina (holotype) a, b Zoospore differentiation inside the zoosporangium and discharge of zoospores through an elongate semipersistent vesicle After shrinkage, the vesicle acquires a characteristic morphology (arrows) c Secondary lateral zoosporangium and empty zoosporangium with characteristic rough walls after zoospore discharge d Internal proliferation of the zoosporangium e Hyphal swellings f Colony with scanty aerial mycelium and no defined growth pattern onto PYGs culture medium (CCIBt 4116). Scale bars: a – e = 10 μm.

Fig. 3 Phytophthora rhizophorae (holotype) a Apapillate zoosporangia during different stages of zoospore differentiation b, c Empty zoosporangium with internal proliferation c General aspect of the zoosporangiophore with both nested and extended internal proliferation d, e Nested proliferation of the zoosporangium f – h Differentmorphologies of hyphal swellings i Petaloid colony pattern onto PYGs culture medium (CCIBt 4121). Scale bars: a – i = 10 μm.