Phytophthora debattistii Bregant, Montecchio & Linaldeddu, sp. nov.

Index Fungorum number: IF 900191; MycoBank number: MB 900191; Facesoffungi number: FoF13941; Fig. 1

Etymology – in honour and memory of Dr. Renzo De Battisti for his outstanding work on Alpine ecosystems.

Pathogenic on stems and roots of Alnus incana (L.) Moench forming cankers. Sexual morph: Homothallic. Gametangia produced abundantly on carrot agar (CA) after 7–10 days at 20 °C. Oogonia 40–55 μm diam. ( x = 48.8 ± 3.7 μm, n = 50) smooth-walled, terminal. Antheridia 16.5–30×15–28 μm (x=24.4±4.2×20.9±3. 9 μm, n=50), mostly amphigynous (around 70%), sometimes paragynous (around 30%), hyaline, rounded, clubshaped, attached near the oogonial stalk, one per oogonium. Oospores 37.8±2.6 µm diam., spherical, aplerotic. Asexual morph: Sporangia 47–112×27–60 μm (x=78.3 ±14.9×43.4±7.4 µm, n=50), with a length/breadth ratio of 1.8±0.1 (n=50) persistent, non-papillate, mostly ovoid (around 60%) to ellipsoid (around 30%), sometimes elongate (around 8%) rarely distorted (around 2%) abundantly produced on CA plugs flooded in unsterile pond water after 24–36 h of incubation at 20 °C on simple or rarely compound sympodial sporangiophores. Zoospores abundantly produced in liquid cultures after 48 h at 20 °C. Sporangial proliferation mostly external emerging just below the mature sporangium and less frequently internal, both nested and extended. Hyphal swellings globose and catenulate, rarely produced in unsterile pond water and absent in solid culture. Chlamydospores not observed.

Culture characteristics – colony growth pattern cottony on potato dextrose agar (PDA) with an irregular border, stellate on malt extract agar (MEA) and rosaceous to stellate on carrot agar (CA). On PDA and MEA colony growth was slow, whereas on CA colony reached 62±3 mm (n=5) diameter in 7 days at 23 °C.

Cardinal temperature for growth – Minimum < 2 °C, maximum 33 °C and optimum 23 °C. Isolates failed to grow at 35 °C and mycelium did not resume growth when plates were moved to 23 °C or below.

Material examined – Italy, Vigo di Cadore, the centre of the Dolomites, from aerial bark lesions and bleeding cankers on Alnus incana (Betulaceae), 17 December 2019, Carlo Bregant, (CBS H-24767 holotype, a dried culture on CA), ex-holotype culture CB83=CBS 147721.

GenBank numbers – ITS: OP999674, β-tubulin: OQ067250, cox1: OQ067254.

Notes – Our phytophthora-like isolates phylogenetically close to the species in the sub-clade 6b (Fig. 2) and they grouped with Phytophthora megasperma forming a sister clade with ML=100 statistical support. However, nucleotide differences in ITS, β-tubulin and cox1 loci of our isolates and P. megasperma revealed 0.12%, 0.61%, and 0.18% respectively. Our collection morphologically differs from P. megasperma by sporangia shape and sizes, colony growth and formation, a lower minimum temperature for growth, aplerotic oospores and antheridia mostly amphigynous. Further, our collection is phylogenetically closed to P. crassamura that have 0.49%, 0.73%, and 2.97% nucleotide differences in ITS, β-tubulin and cox1 loci respectively. Phytophthora crassamura produces sporangia similar in shape to our collection, but significantly smaller in size (x=60.3±6.0×37. 4±3.6 µm, with a length/breadth ratio of 1.6).

Figure 1 – Phytophthora debattistii (CBS 147721, ex-holotype). a–c Colony morphology after 7 days growth at 20 °C on a potato dextrose agar (PDA), b malt extract agar (MEA), c carrot agar (CA). d–f Non-papillate persistent sporangia on CA after 24–36 h flooding in nonsterile pond water: ovoid, ellipsoid and distorted respectively. g, h Releasing zoospores. i External proliferation. j Internal nested proliferation. k Intercalary globose catenulated hyphal swellings. l Mature oogonia with amphigynous. m Paragynous antheridia and slight aplerotic oospores. Scale bars: d–m=20 μm

Figure 2 – Phylogram generated from maximum likelihood analysis based on combined ITS, β-tubulin and cox1 sequence dataset. Seventy-eight Phytophthora strains were included in the combined sequence analysis, which comprise 2261 characters. Nothophytophthora sp. (BD268) was used as the outgroup taxon. Sequences were aligned with ClustalX v. 1.83, using the default parameters. The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model (Nei and Kumar 2000). The tree with the highest log likelihood (− 21,877.77) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter=0.2597)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 30.10% sites). Bootstrap support equal or greater than 50% are given at the nodes. Ex-type strains are in black bold and newly generated sequences are indicated in blue
bold. Alignments and trees are available in TreeBase (TB2:S29999)