Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity.

During 25 surveys of global Phytophthora diversity, conducted between 1998 and 2020, 43 new species were detected in natural ecosystems and, occasionally, in nurseries and outplantings in Europe, Southeast and East Asia and the Americas. Based on a multigene phylogeny of nine nuclear and four mitochondrial gene regions they were assigned to five of the six known subclades, 2a-c, e and f, of Phytophthora major Clade 2 and the new subclade 2g. The evolutionary history of the Clade appears to have involved the pre-Gondwanan divergence of three extant subclades, 2c, 2e and 2f, all having disjunct natural distributions on separate continents and comprising species with a soilborne and aquatic lifestyle and, in addition, a few partially aerial species in Clade 2c; and the post-Gondwanan evolution of subclades 2a and 2g in Southeast/East Asia and 2b in South America, respectively, from their common ancestor. Species in Clade 2g are soilborne whereas Clade 2b comprises both soil-inhabiting and aerial species. Clade 2a has evolved further towards an aerial lifestyle comprising only species which are predominantly or partially airborne. Based on high nuclear heterozygosity levels ca. 38 % of the taxa in Clades 2a and 2b could be some form of hybrid, and the hybridity may be favoured by an A1/A2 breeding system and an aerial life style. Circumstantial evidence suggests the now 93 described species and informally designated taxa in Clade 2 result from both allopatric non-adaptive and sympatric adaptive radiations. They represent most morphological and physiological characters, breeding systems, lifestyles and forms of host specialism found across the Phytophthora clades as a whole, demonstrating the strong biological cohesiveness of the genus. The finding of 43 previously unknown species from a single Phytophthora clade highlight a critical lack of information on the scale of the unknown pathogen threats to forests and natural ecosystems, underlining the risk of basing plant biosecurity protocols mainly on lists of named organisms. More surveys in natural ecosystems of yet unsurveyed regions in Africa, Asia, Central and South America are needed to unveil the full diversity of the clade and the factors driving diversity, speciation and adaptation in Phytophthora. Taxonomic novelties: New species: Phytophthora amamensis T. Jung, K. Kageyama, H. Masuya & S. Uematsu, Phytophthora angustata T. Jung, L. Garcia, B. Mendieta-Araica, & Y. Balci, Phytophthora balkanensis I. Milenkovic, Z. Tomic, T. Jung & M. Horta Jung, Phytophthora borneensis T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora calidophila T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora catenulata T. Jung, T.-T. Chang, N.M. Chi & M. Horta Jung, Phytophthora celeris T. Jung, L. Oliveira, M. Tarigan & I. Milenkovic, Phytophthora curvata T. Jung, A. Hieno, H. Masuya & M. Horta Jung, Phytophthora distorta T. Jung, A. Durán, E. Sanfuentes von Stowasser & M. Horta Jung, Phytophthora excentrica T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora falcata T. Jung, K. Kageyama, S. Uematsu & M. Horta Jung, Phytophthora fansipanensis T. Jung, N.M. Chi, T. Corcobado & C.M. Brasier, Phytophthora frigidophila T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora furcata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora inclinata N.M. Chi, T. Jung, M. Horta Jung & I. Milenkovic, Phytophthora indonesiensis T. Jung, M. Tarigan, L. Oliveira & I. Milenkovic, Phytophthora japonensis T. Jung, A. Hieno, H. Masuya & J.F. Webber, Phytophthora limosa T. Corcobado, T. Majek, M. Ferreira & T. Jung, Phytophthora macroglobulosa H.-C. Zeng, H.-H. Ho, F.-C. Zheng & T. Jung, Phytophthora montana T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora multipapillata T. Jung, M. Tarigan, I. Milenkovic & M. Horta Jung, Phytophthora multiplex T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora nimia T. Jung, H. Masuya, A. Hieno & C.M. Brasier, Phytophthora oblonga T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora obovoidea T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora obturata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora penetrans T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora platani T. Jung, A. Pérez-Sierra, S.O. Cacciola & M. Horta Jung, Phytophthora proliferata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora pseudocapensis T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pseudocitrophthora T. Jung, S.O. Cacciola, J. Bakonyi & M. Horta Jung, Phytophthora pseudofrigida T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora pseudoccultans T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pyriformis T. Jung, Y. Balci, K.D. Boders & M. Horta Jung, Phytophthora sumatera T. Jung, M. Tarigan, M. Junaid & A. Durán, Phytophthora transposita T. Jung, K. Kageyama, C.M. Brasier & H. Masuya, Phytophthora vacuola T. Jung, H. Masuya, K. Kageyama & J.F. Webber, Phytophthora valdiviana T. Jung, E. Sanfuentes von Stowasser, A. Durán & M. Horta Jung, Phytophthora variepedicellata T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora vietnamensis T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora ×australasiatica T. Jung, N.M. Chi, M. Tarigan & M. Horta Jung, Phytophthora ×lusitanica T. Jung, M. Horta Jung, C. Maia & I. Milenkovic, Phytophthora ×taiwanensis T. Jung, T.-T. Chang, H.-S. Fu & M. Horta Jung. Citation: Jung T, Milenkovic I, Balci Y, Janousek J, Kudlácek T, Nagy ZÁ, Baharuddin B, Bakonyi J, Broders KD, Cacciola SO, Chang T-T, Chi NM, Corcobado T, Cravador A, Dordevic B, Durán A, Ferreira M, Fu C-H, Garcia L, Hieno A, Ho H-H, Hong C, Junaid M, Kageyama K, Kuswinanti T, Maia C, Májek T, Masuya H, Magnano di San Lio G, Mendieta-Araica B, Nasri N, Oliveira LSS, Pane A, Pérez-Sierra A, Rosmana A, Sanfuentes von Stowasser E, Scanu B, Singh R, Stanivukovic Z, Tarigan M, Thu PQ, Tomic Z, Tomsovský M, Uematsu S, Webber JF, Zeng H-C, Zheng F-C, Brasier CM, Horta Jung M (2024). Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity. Studies in Mycology 107: 251-388. doi: 10.3114/sim.2024.107.04.

Phytophthora : taxonomic and phylogenetic revision of the genus.

Many members of the Oomycota genus Phytophthora cause economic and environmental impact diseases in nurseries, horticulture, forest, and natural ecosystems and many are of regulatory concern around the world. At present, there are 223 described species, including eight unculturable and three lost species. Twenty-eight species need to be redescribed or validated. A lectotype, epitype or neotype was selected for 20 species, and a redescription based on the morphological/molecular characters and phylogenetic placement is provided. In addition, the names of five species are validated: P. cajani, P. honggalleglyana (Synonym: P. hydropathica), P. megakarya, P. pisi and P. pseudopolonica for which morphology and phylogeny are given. Two species, P. ×multiformis and P. uniformis are presented as new combinations. Phytophthora palmivora is treated with a representative strain as both lecto- and epitypification are pending. This manuscript provides the updated multigene phylogeny and molecular toolbox with seven genes (ITS rDNA, ß-tub, COI, EF1α, HSP90, L10, and YPT1) generated from the type specimens of 212 validly published, and culturable species (including nine hybrid taxa). The genome information of 23 types published to date is also included. Several aspects of the taxonomic revision and phylogenetic re-evaluation of the genus including species concepts, concept and position of the phylogenetic clades recognized within Phytophthora are discussed. Some of the contents of this manuscript, including factsheets for the 212 species, are associated with the "IDphy: molecular and morphological identification of Phytophthora based on the types" online resource (https://idtools.org/tools/1056/index.cfm). The first version of the IDphy online resource released to the public in September 2019 contained 161 species. In conjunction with this publication, we are updating the IDphy online resource to version 2 to include the 51 species recently described. The current status of the 223 described species is provided along with information on type specimens with details of the host (substrate), location, year of collection and publications. Additional information is provided regarding the ex-type culture(s) for the 212 valid culturable species and the diagnostic molecular toolbox with seven genes that includes the two metabarcoding genes (ITS and COI) that are important for Sanger sequencing and also very valuable Molecular Operational Taxonomic Units (MOTU) for second and third generation metabarcoding High-throughput sequencing (HTS) technologies. The IDphy online resource will continue to be updated annually to include new descriptions. This manuscript in conjunction with IDphy represents a monographic study and the most updated revision of the taxonomy and phylogeny of Phytophthora, widely considered one of the most important genera of plant pathogens. Taxonomic novelties: New species: Phytophthora cajani K.S. Amin, Baldev & F.J. Williams ex Abad, Phytophthora honggalleglyana Abad, Phytophthora megakarya Brasier & M.J. Griffin ex Abad, Phytophthora pisi Heyman ex Abad, Phytophthora pseudopolonica W.W. Li, W.X. Huai & W.X. Zhao ex Abad & Kasiborski; New combinations: Phytophthora ×multiformis (Brasier & S.A. Kirk) Abad, Phytophthora uniformis (Brasier & S.A. Kirk) Abad; Epitypifications (basionyms): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne, Phytophthora ilicis Buddenh. & Roy A. Young, Phytophthora inundata Brasier et al., Phytophthora megasperma Drechsler, Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Lectotypifications (basionym): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne, Phytophthora ilicis Buddenh. & Roy A. Young, Phytophthora megasperma Drechsler, Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Neotypifications (basionym): Phloeophthora syringae Kleb., Phytophthora meadii McRae Citation: Abad ZG, Burgess TI, Bourret T, Bensch K, Cacciola S, Scanu B, Mathew R, Kasiborski B, Srivastava S, Kageyama K, Bienapfl JC, Verkleij G, Broders K, Schena L, Redford AJ (2023). Phytophthora: taxonomic and phylogenetic revision of the genus. Studies in Mycology 106: 259-348. doi: 10.3114/sim.2023.106.05.

Six new Phytophthora species from ITS Clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan.

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuata, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4-33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

Fungal Planet description sheets: 558-624.

Novel species of fungi described in this study include those from various countries as follows: Australia: Banksiophoma australiensis (incl. Banksiophoma gen. nov.) on Banksia coccinea, Davidiellomycesaustraliensis (incl. Davidiellomyces gen. nov.) on Cyperaceae, Didymocyrtis banksiae on Banksia sessilis var. cygnorum, Disculoides calophyllae on Corymbia calophylla, Harknessia banksiae on Banksia sessilis, Harknessia banksiae-repens on Banksia repens, Harknessia banksiigena on Banksia sessilis var. cygnorum, Harknessia communis on Podocarpus sp., Harknessia platyphyllae on Eucalyptus platyphylla, Myrtacremonium eucalypti (incl. Myrtacremonium gen. nov.) on Eucalyptus globulus, Myrtapenidiella balenae on Eucalyptus sp., Myrtapenidiella eucalyptigena on Eucalyptus sp., Myrtapenidiella pleurocarpae on Eucalyptuspleurocarpa, Paraconiothyrium hakeae on Hakea sp., Paraphaeosphaeria xanthorrhoeae on Xanthorrhoea sp., Parateratosphaeria stirlingiae on Stirlingia sp., Perthomyces podocarpi (incl. Perthomyces gen. nov.) on Podocarpus sp., Readeriella ellipsoidea on Eucalyptus sp., Rosellinia australiensis on Banksia grandis, Tiarosporella corymbiae on Corymbia calophylla, Verrucoconiothyriumeucalyptigenum on Eucalyptus sp., Zasmidium commune on Xanthorrhoea sp., and Zasmidium podocarpi on Podocarpus sp. Brazil: Cyathus aurantogriseocarpus on decaying wood, Perenniporia brasiliensis on decayed wood, Perenniporia paraguyanensis on decayed wood, and Pseudocercospora leandrae-fragilis on Leandrafragilis.Chile: Phialocephala cladophialophoroides on human toe nail. Costa Rica: Psathyrella striatoannulata from soil. Czech Republic: Myotisia cremea (incl. Myotisia gen. nov.) on bat droppings. Ecuador: Humidicutis dictiocephala from soil, Hygrocybe macrosiparia from soil, Hygrocybe sangayensis from soil, and Polycephalomyces onorei on stem of Etlingera sp. France: Westerdykella centenaria from soil. Hungary: Tuber magentipunctatum from soil. India: Ganoderma mizoramense on decaying wood, Hodophilus indicus from soil, Keratinophyton turgidum in soil, and Russula arunii on Pterigota alata.Italy: Rhodocybe matesina from soil. Malaysia: Apoharknessia eucalyptorum, Harknessia malayensis, Harknessia pellitae, and Peyronellaea eucalypti on Eucalyptus pellita, Lectera capsici on Capsicum annuum, and Wallrothiella gmelinae on Gmelina arborea.Morocco: Neocordana musigena on Musa sp. New Zealand: Candida rongomai-pounamu on agaric mushroom surface, Candida vespimorsuum on cup fungus surface, Cylindrocladiella vitis on Vitis vinifera, Foliocryphia eucalyptorum on Eucalyptus sp., Ramularia vacciniicola on Vaccinium sp., and Rhodotorula ngohengohe on bird feather surface. Poland: Tolypocladium fumosum on a caterpillar case of unidentified Lepidoptera.Russia: Pholiotina longistipitata among moss. Spain: Coprinopsis pseudomarcescibilis from soil, Eremiomyces innocentii from soil, Gyroporus pseudocyanescens in humus, Inocybe parvicystis in humus, and Penicillium parvofructum from soil. Unknown origin: Paraphoma rhaphiolepidis on Rhaphiolepsis indica.USA: Acidiella americana from wall of a cooling tower, Neodactylaria obpyriformis (incl. Neodactylaria gen. nov.) from human bronchoalveolar lavage, and Saksenaea loutrophoriformis from human eye. Vietnam: Phytophthora mekongensis from Citrus grandis, and Phytophthora prodigiosa from Citrus grandis. Morphological and culture characteristics along with DNA barcodes are provided.

First Report of Phytophthora taxon niederhauserii Causing Root and Stem Rot of Mimosa in Italy.

Mimosa [Acacia dealbata Link, syn. Acacia decurrens (Wendl. F.) Wild. var. dealbata (Link) F. Muell., Fabaceae] is an evergreen shrub native to southeastern Australia that is cultivated as an ornamental plant in warm temperate regions of the world. In spring 2010, in a commercial nursery in Liguria (northern Italy), 6- to 10-month-old potted plants of A. dealbata showed symptoms of sudden collapse, defoliation, and wilt associated with root and basal stem rot. An abundant gum exudate oozed from the basal stem. A Phytophthora species was consistently isolated from roots and stem on BNPRAH selective medium (4). On V8 agar (V8A), axenic cultures obtained by single hyphal transfers formed stellate to radiate colonies with aerial mycelium whereas on potato dextrose agar (PDA) the colonies grew more slowly than on V8A and showed stoloniform mycelium and irregular margins. Minimum and maximum growth temperatures on PDA were 10 and 35°C, with the optimum at 30°C. In water, all isolates produced catenulate or single fusiform hyphal swellings and ellipsoid, nonpapillate, persistent sporangia. Dimensions of sporangia were 46.1 to 65.4 × 23.1 to 30.8 µm (mean l/b ratio 2.1). All isolates were A1 mating type and produced spherical oogonia with amphyginous antheridia when paired with A2 mating type of P. drechsleri Tucker on V8A plus ß-sytosterol (4). Internal transcribed spacer (ITS) regions of rDNA of the representative Phytophthora isolate IMI 500394 from A. dealbata were amplified and sequenced in both directions with primers ITS6/ITS4. The consensus sequence (GenBank Accession No. JF900371) was 99% similar to sequences of several isolates identified as Phytophthora taxon niederhauserii Z.G. Abad and J.A. Abad (e.g., GQ848201 and EU244850). Pathogenicity tests were performed on 1-year-old potted plants of A. dealbata with isolate IMI 500394. Twenty plants were transplanted into pots (12-cm-diameter) filled with soil infested (4% v/v) with the inoculum of IMI500394 produced on kernel seeds. Plants were kept in a greenhouse with natural light at 25 ± 2°C and watered to field capacity weekly. All inoculated plants showed symptoms of wilt, leaf chlorosis, and basal stem rot within 3 to 4 weeks. Twenty control plants transplanted in autoclaved soil mix remained healthy. P. taxon niederhauserii was reisolated solely from inoculated plants, thus fulfilling Koch's postulates. Since 2003, this pathogen has been found on bottlebrush and rock rose grown in a nursery in Sicily (southern Italy), as well as on Banksia in a nursery in Liguria (2,3). To our knowledge, this is the first report of P. taxon niederhauserii on A. dealbata. P. taxon niederhauserii, recently described as P. niederhauserii sp. nov. (1), is a polyphagous pathogen that was originally reported on arborvitae and ivy in North Carolina in 2001. References: (1) Z. G. Abad et al. Mycologia (in press), 2013. (2) S. O. Cacciola et al. Plant Dis. 93:1075, 2009. (3) S. O. Cacciola et al. Plant Dis. 93:1216, 2009. (4) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

First Report of Phytophthora × pelgrandis Causing Root Rot and Lower Stem Necrosis of Common Box, Lavender and Port-Orford-Cedar in Hungary.

In 2008 and 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown common box (Buxus sempervirens L.), lavender (Lavandula angustifolia Mill. 'Hidcote'), and Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Columnaris') in three ornamental nurseries in western Hungary. Number of affected plants ranged from approximately 100 (Port-Orford-cedar) to 250 (lavender). Isolations from necrotic root collars of each host plant species yielded four Phytophthora isolates developing uniform colonies on carrot agar with a maximum growth temperature of 35 to 36°C. The isolates were homothallic with smooth-walled oogonia (32.2 ± 2.3 to 35.9 ± 3.5 µm), aplerotic oospores (27.5 ± 1.8 to 32.1 ± 3.1 µm) and both amphigynous and paragynous antheridia, and produced chlamydospores (25.8 ± 3.9 to 29.1 ± 5.2 µm) and papillate sporangia (35.2 ± 2.5 to 43.5 ± 5.6 µm long and 27.6 ± 2.2 to 32.0 ± 3.8 µm wide), mostly obpyriform to nearly spherical or rarely distorted with two or three apices. In spring water, sporangia were both caducous with short pedicel and non-caducous. Multiplex ITS-PCR assay of DNA from all isolates, using primers specific for P. nicotianae (NICF1 and NICR2.1) and P. cactorum (CACTF1 and CACTR1) (1), amplified DNA fragments of the expected size for each Phytophthora species. In addition, isoenzyme analysis revealed a characteristic banding pattern of one heterodimer and two homodimer bands at both loci of the dimeric enzyme malate dehydrogenase. These bands comigrated with those of P. × pelgrandis (Gerlach et al.) (CBS 123385) and isolate PD 93/1339 (courtesy of W. A. Man in 't Veld), two natural hybrid strains of P. nicotianae and P. cactorum (2,3), proving that our four isolates can be referred to as this interspecific hybrid. Pathogenicity was tested on 1- or 3-year-old plants of the original host species and cultivars (for common box, cv. Faulkner was used). Cultures were grown for 4 to 6 weeks at 20°C on autoclaved millet grains moistened with V8 broth. Infested and uninfested grains were mixed with autoclaved soil in a ratio of 6% (w/v), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth chamber (20°C, 70% RH, 12-h photoperiod). Pots were flooded for 24 h on the 1st and 21st day after transplanting. All plants in infested potting mix showed symptoms of wilt associated with basal stem and root necrosis, similar to those observed on the plants from the field, within 2 and 3 months on lavender and both common box or Port-Orford-cedar, respectively. Additionally, a reduction of root weight ranging from 35 to 68% compared to the control was recorded. Growth reduction was significant at P ≤ 0.019 according to Mann Whitney test. Control plants remained healthy. The same Phytophthora hybrid was reisolated solely from inoculated plants. In Europe, hybrid isolates of P. nicotianae × P. cactorum have been reported on several ornamental plants, including lavender, in the Netherlands and Germany (2,3). However, to our knowledge, this is the first report of this hybrid in Hungary and as a pathogen of common box and Port-Orford-cedar in the world. References: (1) P. J. M. Bonants et al. Phytopathology 90:867, 2000. (2) W. A. Man in 't Veld et al. Phytopathology 88:922, 1998. (3) H. I. Nirenberg et al. Mycologia 101:220, 2009.

Globisporangium coniferarum sp. nov., associated with conifers and Quercus spp.

During a survey of gardens in Shiraz County, Iran, aimed at identifying oomycetes associated with roots of ornamental trees, a species of Globisporangium with distinctive morphological characters separating it from other known species in this genus was recovered from conifers and occasionally from a Quercus sp. Five isolates of this species were characterised. Phylogenetic analyses of nuclear (ITS and ßtub) and mitochondrial (cox1 and cox2) loci using Bayesian inference and maximum likelihood analyses as well as their distinct morphological and cultural characteristics (e.g., abundant production of chlamydospores; globose, ellipsoid to ovoid sporangia; amorphous oogonia with a smooth wall; paragynous to rarely hypogynous antheridia and 1-5 antheridia per oogonium; mostly plerotic oospores) revealed that these isolates belong to a new Globisporangium species grouping in the phylogenetic clade G of Pythium sensu lato. This paper formally describes Globisporangium coniferarum sp. nov. as a new species and compares it with other phylogenetically related and already known Globisporangium species, including G. nagaii, G. violae, G. paddicum, G. okanoganense, G. iwayamae and G. canariense. Citation: Salmaninezhad F, Aloi F, Pane A, Mostowfizadeh-Ghalamfarsa R, Cacciola SO (2022). Globisporangium coniferarum sp. nov., associated with conifers and Quercus spp. Fungal Systematics and Evolution 10: 127-137. doi: 10.3114/fuse.2022.10.05.

Root and Basal Stem Rot of Rose Caused by Phytophthora citrophthora in Italy.

Approximately 800 ha of cut flower roses are cultivated for commercial production in Italy. During autumn of 2004 in an experimental greenhouse in western Sicily (southern Italy), 60% of 2-year-old plants of rose cv. Red France on Rosa indica cv. Major rootstock grown in soil showed leaf chlorosis and wilt. A dark brown lesion lined by a water-soaked area was noticeable at the stem base near the soil surface. Root rot was found consistently associated with aboveground symptoms and plants collapsed within 4 months after the appearance of the first symptoms. The same symptoms were observed sporadically on rose plants of the same cultivar during the last 6 years in commercial nurseries in western Sicily. In all cases, a Phytophthora species has been consistently isolated from rotted roots and stems on Phytophthora-selective media. Pure cultures were obtained by single-hypha transfers. The species was identified as Phytophthora citrophthora on the basis of morphological characters and electrophoretic analysis of mycelial proteins on polyacrylamide gel (1). On potato dextrose agar, isolates produced petaloid colonies with optimum growth temperature at 25°C. On V8 agar, mono- and occasionally bipapillate, ovoid to limoniform sporangia, measuring 44 to 55 × 27 to 28 µm, with a mean length/breadth ratio of 1.4:1 were produced. All isolates were heterothallic but did not produce gametangia in dual cultures with P. nicotianae isolates of A1 and A2 mating type. Electrophoretic patterns of total mycelial proteins and four isozyme (acid and alkaline phosphatases, esterase, and malato dehydrogenase) of the isolates from rose were identical to those of reference isolates of P. citrophthora, but clearly distinct from isolates of other heterothallic species with papillate sporangia, including P. capsici, P. nicotianae, P. palmivora, and P. tropicalis. All isolates from rose showed the same electrophoretic profiles. Blast search of rDNA-ITS sequence from PCR-amplified ITS4/ITS6 primers (2) of a representative isolate from rose (IMI 392044) showed 98% homology with a reference isolate of P. citrophthora (GenBank No. EU0000631), thus confirming the identification. Pathogenicity of isolate IMI 392044 was tested on 10 12-month-old plants of rose cv. Red France grafted on R. indica cv. Major transplanted in pots containing a mixture of sphagnum peat moss and sandy loam soil (1:1 vol/vol) infested with 80 g of inoculum per liter of mixture. Inoculum was produced by growing the isolate on wheat kernels. Plants transplanted in pots containing noninfested soil served as controls. Plants were kept in a greenhouse at 22 ± 3°C and watered to soil saturation once a week. Inoculated plants developed symptoms of leaf chlorosis and root and crown rot within 15 to 30 days and wilted within 40 to 80 days after inoculation. Control plants remained healthy. P. citrophthora was consistently reisolated from inoculated plants. Root and basal stem rot of rose may be caused by several Phytophthora spp. and has been reported in various countries of Asia, Europe, and North America (3,4). However, to our knowledge, this is the first report in Italy. The occurrence of this disease may be attributed to excessive irrigation practices. References: (1) S. O. Cacciola et al. EPPO Bull. 20:47, 1990. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) Y. Nagai et al. Phytopathology 68:684, 1978. (4) B. W. Schwingle et al. Plant Dis. 91:97, 2007.

First Report of Root and Basal Stem Rot Caused by Phytophthora nicotianae on Tree Aeonium (Aeonium arboreum) in Italy.

The genus Aeonium, family Crassulaceae, comprises approximately 35 species that are native to northern Africa and the Canary Islands. Tree aeonium (Aeonium arboreum (L.) Webb & Berthel.) is a bushy, perennial succulent with rosettes of tender, waxy leaves at the apex of few-branched or occasionally single, naked stems. Mature rosettes bear yellowish inflorescences. Aeoniums are cultivated as ornamentals in gardens and containers. During the summer of 2009, in a garden in eastern Sicily (southern Italy), 3-year-old potted plants of tree aeonium showed stunting, shrivelling, and chlorosis of leaves and drop of external leaves associated with root and basal stem rot. Drops of an amber exudate oozed from the basal stem. Tissues of the basal stem were soft, but no external necrosis was visible. A species of Phytophthora was consistently isolated from symptomatic roots and basal stem tissues on a medium selective for Oomycetes (2). Axenic cultures were obtained by single-hypha transfers. The pathogen was identified by morphological criteria as Phytophthora nicotianae B. de Haan; it formed stoloniferous colonies on potato dextrose agar and grew between 8 and 38°C, with the optimum at 30°C. On V8 juice agar it produced spherical, intercalary chlamydospores (mean diameter of 26 µm) and persistent, mono- and bipapillate, spherical to ovoid, ellipsoid, obpyriform sporangia that measured 29 to 56 × 22 to 45 µm with a mean length/breadth ratio of 1.3:1. All isolates were A2 mating type and formed spherical oogonia (mean diameter 28 ± 2 µm) with smooth walls and amphigynous antheridia in dual cultures with a reference isolate of the A1 mating type of P. nicotianae. BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of a representative isolate from aeonium (IMI 398812, GenBank Accession No. HQ433333) amplified by PCR using the ITS6/ITS4 universal primers (1), revealed 99% similarity with the sequences of a reference isolate of P. nicotianae available in GenBank (Accession No. EU331089.1). Pathogenicity of isolate IMI 398812 was demonstrated by transplanting cuttings of A. arboreum into pots filled with a mixture of steam-sterilized sandy loam soil and inoculum (4% vol/vol) produced by growing the isolate for 20 days on wheat kernels. Ten plants were transplanted into 3-liter pots (two plants per pot) while 10 plants, transplanted into pots filled with a mixture of steam-sterilized soil and noninoculated kernels, were used as controls. Plants were kept in a greenhouse at 25 to 28°C and watered daily to field capacity. Thirty to forty days after the transplanting into infested soil, cuttings developed the same symptoms observed on plants with natural infections. Control plants remained symptomless. P. nicotianae was reisolated from symptomatic plants, thereby completing Koch's postulates. To our knowledge, this is the first report of P. nicotianae on an Aeonium species worldwide. The economic relevance of this disease is minor because aeoniums are not cultivated on a large scale. Moreover, the disease may be easily prevented by avoiding excess irrigation water since aeoniums need a well-drained soil or potting mix and do not tolerate soil waterlogging. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977.

Bud and Root Rot of Windmill Palm (Trachycarpus fortunei) Caused by Simultaneous Infections of Phytophthora palmivora and P. nicotianae in Sicily.

In June 2009 in a commercial nursery in eastern Sicily (Italy), 3-year-old potted windmill palms (Trachycarpus fortunei (Hooker) H. Wendl.) showed a decline in growth, wilt, droop, and basal rot of the youngest leaves. The rot progressed inward and killed the bud. Initially, older leaves remained green but eventually the entire plant collapsed. Root rot was consistently associated with aboveground symptoms. Two Phytophthora species were consistently isolated from the petiole base, heart, roots, and rhizosphere soil of symptomatic plants on a selective medium (2) and occasionally recovered from roots and rhizosphere soil of asymptomatic plants. Pure cultures were obtained by single-hypha transfers and the two species were identified on the basis of morphological and molecular characters as Phytophthora palmivora and P. nicotianae. Both species were recovered from all symptomatic plants. From multiple tissue samples per plant, we recovered either or both species. On potato dextrose agar (PDA), P. palmivora isolates grew between 10 and 35°C, with the optimum at 27°C. On V8 juice agar, they produced elliptical to ovoid, papillate, caducous sporangia (32 to 78 × 23 to 39 µm) with a mean length/breadth (l/b) ratio of 1.8:1 and a short pedicel (mean pedicel length = 5 µm). Isolates of P. nicotianae produced arachnoid colonies on PDA, grew at 37°C but did not grow at 40°C. Sporangia (29 to 55 × 23 to 45 µm) were spherical to ovoid (l/b ratio 1.3:1), papillate and often bipapillate, and noncaducous. Isolates of both species produced amphigynous antheridia and oogonia only when paired with reference isolates of P. nicotianae of the A2 mating type. The internal transcribed spacer (ITS) region of rDNA of two isolates of P. palmivora (IMI 398987 and IMI 398988) and an isolate of P. nicotianae (IMI 398989) from T. fortunei was amplified with primers ITS6/ITS4 and sequenced (1). Blast analysis of the sequences of isolates IMI 398987 and IMI 398988 (GenBank Accession Nos. HQ596556 and HQ596558) showed 99% homology with the sequence of two reference isolates of P. palmivora (GQ398157.1 and GU258862), while the sequence of isolate IMI 398989 (HQ596557) showed 99% homology with a reference isolate of P. nicotianae (EU331089.1). Pathogenicity of isolates IMI 398987 and IMI 398989 was proved by inoculating separately each isolate on 1-year-old potted plants of T. fortunei (10 plants per isolate). A zoospores suspension (2 × 104 zoospores/ml) was pipetted onto the petiole base of the three central leaves (200 µl per leaf) of each plant. Sterile water was used for control plants. All plants were incubated at 25 ± 2°C with 100% humidity for 48 h and then maintained in a greenhouse at 24 to 28°C. Within 3 weeks, all inoculated plants showed symptoms of bud rot. Control plants remained healthy. P. palmivora and P. nicotianae were reisolated only from inoculated plants. Bud rot of palms caused by P. palmivora was reported previously in Italy (3). However, to our knowledge, this is the first report of simultaneous infections of P. palmivora and P. nicotianae as causal agents of this disease. Outbreak of bud rot may have been favored by overhead sprinkler irrigation. The recovery of P. palmivora and P. nicotianae from rhizosphere soil and roots of asymptomatic plants suggests infested soil was the primary inoculum source. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) A. Pane et al. Plant Dis. 91:1059, 2007.

Root and Basal Stem Rot of Mandevillas Caused by Phytophthora spp. in Eastern Sicily.

Approximately 150,000 potted mandevillas (Apocynaceae) are produced each year in the Etna District of eastern Sicily. Since 2004, leaf chlorosis, wilt, and sudden collapse of the entire plant associated with root and basal stem rot of 6- to 12-month-old potted mandevillas, including Mandevilla × amabilis 'Alice du Pont', M. splendens, and M. sanderi 'Alba', 'My Fair Lady', and 'Scarlet Pimpernel', have been observed in six nurseries. Incidence of affected plants varied from 5 to 40%. Four Phytophthora species were consistently isolated from rotted roots and stems on a selective medium (2). Pure cultures of the first species produced colonies with a camellia pattern on potato dextrose agar and grew between 10 and 37°C with an optimum of 27°C. On V8 juice agar they produced ellipsoid to obpyriform (length/breadth [l/b] 1.45:1), nonpapillate sporangia with internal proliferation, coralloid, spherical hyphal swellings and both terminal and intercalary chlamydospores. In dual cultures with A1 and A2 isolates of P. nicotianae, all isolates produced oogonia with amphyginous antheridia only with A2 isolates. Isolates of the second species formed petaloid colonies, had an optimum growth temperature of 25°C, and produced mono- and bipapillate, ovoid to limoniform sporangia (l/b 1.40:1); they did not produce gametangia. Isolates of the third species formed colonies with a slight petaloid pattern and grew between 2 and 30°C with an optimum of 25°C. Sporangia were obpyriform (l/b 1.48:1), nonpapillate, and proliferous. All isolates were A2 mating type. The isolates of the fourth species formed arachnoid colonies, grew between 8 and 38°C with an optimum of 30°C, and produced mono- and bipapillate, ellipsoid, and obpyriform (l/b 1.3:1) sporangia and apical chlamydospores. All isolates were A2 mating type. DNA was extracted from mycelium and amplified by PCR using the ITS 4/ITS 6 primers (1). Blast search of the rDNA-ITS sequence of isolate IMI 397618 (GenBank Accession No. GQ388261) of the first species showed 100% identity with the ITS sequence of an isolate of P. cinnamomi var. parvispora (EU748548). The sequences (GQ463703 and GQ463704) of isolates IMI 397471 and IMI 397472 of the second species showed 99% similarity with the sequences of a P. citrophthora isolate (EU0000631). The sequence of isolate IMI 397473 (GQ463702) of the third species showed 99% similarity with the sequence of a P. cryptogea isolate (AY659443.1), while the sequence of isolate IMI 397474 (GU723474) of the fourth species showed 99% similarity with the sequence of a P. nicotianae isolate (EU331089). The pathogenicity of individual isolates IMI 397618, IMI 397471, IMI 397472, IMI 397473, and IMI 397474 was tested on 3-month-old potted plants (10 plants per isolate) of mandevilla 'Alice du Pont' by applying 10 ml of a suspension (2 × 104 zoospores/ml) to the root crown. Plants were maintained at 25°C and 95 to 100% relative humidity. All inoculated plants wilted after 4 weeks, while noninoculated control plants remained healthy. The four Phytophthora spp. were subsequently reisolated only from symptomatic plants. To our knowledge, this is the first report of P. cinnamomi var. parvispora in Italy and on mandevilla worldwide. In recent years, Phytophthora root and stem rot has become the most serious disease of potted mandevillas in Sicily. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977.

First Report of a Decline and Wilt of Young Olive Trees Caused by Simultaneous Infections of Verticillium dahliae and Phytophthora palmivora in Sicily.

In summer 2008, leaf chlorosis, defoliation, exceptional fruit set, twig dieback, and wilt were observed on 4-year-old olive (Olea europea L.) trees cv. Tonda Iblea in a drip-irrigated orchard in eastern Sicily. Rot of fine roots was associated with these symptoms and on ~15% of symptomatic trees rot extended to the crown and basal stem. Trees declined slowly or collapsed suddenly with withered leaves still attached. Incidence of affected trees was ~10%. A fungus identified as Verticillium dahliae Kleb. was isolated from the xylem of main roots and basal stem. An oomycete identified as Phytophthora palmivora (Butler) Butler was isolated from roots and basal trunk bark. Both pathogens were recovered from symptomatic trees with mean frequency of positive isolations per tree of 80 and 30% for V. dahliae and P. palmivora, respectively. To isolate V. dahliae, wood chips were surface disinfested in 0.5% NaOCl for 1 min and plated onto potato dextrose agar (PDA). The fungus was identified on the basis of microsclerotia, verticillate arrangement of phialides on conidiophores, and hyaline single-celled conidia. Ten monoconidial isolates were characterized by PCR using primer pairs INTND2f/INTND2r and DB19/espdef01 (3). Only 824-bp amplicons, diagnostic of the virulent, nondefoliating V. dahliae pathotype, were obtained. P. palmivora was isolated on selective medium (2) and pure cultures were obtained by single-hypha transfers. Colonies grew on PDA between 10 and 35°C (optimum at 27°C). Chlamydospores and elliptical to ovoid, papillate, caducous (mean pedicel length = 5 µm) sporangia (length/breadth ratio of 1.8) were produced on V8 juice agar. All isolates were paired with reference isolates of P. nicotianae and produced gametangia only with isolates of the A2 mating type. PCR amplicons of a representative isolate generated using primers ITS 6 and ITS 4 (1) were sequenced and found to be identical to those of a reference isolate of P. palmivora (GenBank No. AY208126). Pathogenicity of V. dahliae (IMI 397476) and P. palmivora (IMI 397475) was tested on 6-month-old rooted cuttings of olive cv. Tonda Iblea. Ten cuttings were transplanted into pots with steam-sterilized soil and inoculum of P. palmivora (4% vol/vol) produced on wheat kernels. Ten olive cuttings were inoculated with V. dahliae by injecting the stem with 150 µl of a conidial suspension (107 conidia ml-1) and 10 cuttings were stem inoculated with V. dahliae and transplanted into soil infested with P. palmivora. Controls were 10 noninoculated cuttings transplanted into steam-sterilized soil. Pots were kept in a greenhouse (25 ± 3°C) for 4 months. No aerial symptoms were observed on cuttings transplanted into soil infested with P. palmivora. However, root dry weight was reduced by 40% in comparison with the controls. Cuttings inoculated solely with V. dahliae had a 15% reduction in height compared with the controls but only four cuttings wilted. All cuttings inoculated with P. palmivora and V. dahliae wilted, indicating a synergism between the two pathogens. Controls remained healthy. Each pathogen was reisolated solely from inoculated cuttings and both pathogens were reisolated from cuttings with double inoculations. A similar syndrome 'seca' (drying) was reported in Spain (4). References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) J. Mercado-Blanco et al. Plant Dis. 87:1487, 2003. (4) M. E. Sánchez-Hernández et al. Eur. J. Plant Pathol. 104:34, 1998.

Callistemon citrinus and Cistus salvifolius, Two New Hosts of Phytophthora taxon niederhauserii in Italy.

Bottlebrush (Callistemon citrinus (Curtis.) Skeels., Myrtaceae) and rock rose (Cistus salvifolius L., Cistaceae) are evergreen shrubs native to Australia and the Mediterranean Region, respectively. In the spring of 2003, approximately 2% of a nursery stock of 12-month-old potted plants of C. citrinus and 8% of a nursery stock of 12-month-old potted plants of Cistus salvifolius grown in the same nursery in Sicily, showed symptoms of leaf chlorosis, defoliation, and wilt associated with root and collar rot. A Phytophthora species was consistently isolated from roots and basal stems on BNPRAH selective medium (2). One isolate from rock rose (IMI 391708) and one from bottlebrush (IMI 391712) were characterized. On potato dextrose agar (PDA), the colonies showed stoloniform mycelium and irregular margins; on V8 juice agar (V8A), colonies were stellate to radiate. Minimum and maximum temperatures on PDA were 10 and 35°C, respectively, with the optimum at 30°C. Mean radial growth rate of isolates on this substrate was 9.9 and 11.3 mm/day, respectively. In saline solution (1), both isolates produced catenulate hyphal swellings and ellipsoid, nonpapillate, persistent sporangia with internal proliferations and dimensions of 52 to 70 × 30 to 42 µm and 51 to 85 × 39 to 45 µm. Mean l/b ratio of sporangia for both isolates was 1.8 ± 1. On V8A plus ß-sytosterol, both isolates produced amphyginous antheridia and spherical oogonia in dual cultures with an A2 tester of P. drechsleri Tucker. Conversely, they did not produce gametangia with an A1 tester of P. cryptogea Pethybr., indicating they were A1 mating type. The internal transcribed spacer (ITS)-rDNA sequences of rock rose and bottlebrush isolates showed 100% similarity with those of two reference isolates of P. taxon niederhauserii from GenBank (Accession Nos. FJ648808 and FJ648809). On the basis of the analysis of the DNA, the species isolated from bottlebrush and rock rose were identified as Phytophthora taxon niederhauserii. Pathogenicity tests were carried out on 6-month-old potted plants of C. salvifolius and C. citrinus (10 plants of each plant species for each isolate) transplanted into pots (12 cm in diameter) containing a mixture of 1:1 steam-sterilized, sandy loam soil (vol/vol) with 4% inoculum produced on autoclaved kernel seeds. Plants were maintained at 25 to 28°C and watered to soil saturation once a week. After 2 to 3 weeks, all inoculated plants developed symptoms identical to those observed on plants with natural infections. Ten control plants transplanted into pots containing noninfested soil remained healthy. P. taxon niederhauserii was reisolated solely from inoculated plants. To our knowledge, this is the first report of P. taxon niederhauserii on C. citrinus and C. salvifolius in Italy. This Phytophthora taxon has been reported recently on rock rose in Spain (3). References: (1) D. W. Chen and G. A. Zentmyer. Mycologia 62:397, 1970. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) E. Moralejo et al. Plant Pathol. 58:100, 2009.

Phytophthora taxon niederhauserii, a New Root and Crown Rot Pathogen of Banksia spp. in Italy.

In the last 10 years, various species of Banksia (family Proteaceae) endemic to Australia have been introduced into Italy where cultivation as flower plants is expanding. In the spring of 2003, a decline associated with root and basal stem rot of 2- to 3-year-old plants of Banksia speciosa R. Br., B. baxteri R. Br., and B. prionotes Lindl. grown in the ground was observed in a commercial nursery in Liguria (northern Italy). Aboveground symptoms included leaf chlorosis and wilt. Plants collapsed within 1 to 2 weeks after the appearance of leaf symptoms. A Phytophthora species was consistently isolated from roots and basal stem on BNPRAH selective medium (3). On V8 juice agar (V8A), axenic cultures obtained by single hyphal transfers formed stellate to radiate colonies with aerial mycelium; on potato dextrose agar (PDA). the colonies showed stoloniform mycelium. Minimum and maximum growth temperatures on PDA and V8A were between 5 and 10°C and 38 and 40°C, respectively, with the optimum at 30°C on PDA (mean radial growth rate of 10 isolates ranged between 9.3 and 10.2 mm per day) and 25 to 30°C on V8A (14 mm per day). In saline solution and soil extract, all isolates produced catenulate hyphal swellings and ellipsoid, nonpapillate, persistent sporangia. Sporangia in saline solution varied from 47 to 70 × 30 to 44 µm (mean l/b ratio of 1.5). All isolates were A1 mating type and produced oogonia with amphyginous antheridia when paired with A2 mating type of P. drechsleri Tucker on V8A plus ß-sytosterol (3). The electrophoretic patterns of total mycelial proteins and two isozymes (esterase and malate dehydrogenase) (2) of all isolates from Banksia plants were identical, but distinct from the patterns of isolates of other Phytophthora species, including P. drechsleri, P. megasperma sensu stricto, and P. sojae. Internal transcribed spacer (ITS) regions of rDNA were amplified with primers ITS4/ITS6 and sequences of two isolates, IMI 393960 from B. speciosa and 466/03 from B. baxteri (GenBank Nos. FJ648808 and FJ648809), were 100% identical to sequences of isolates identified as Phytophthora taxon niederhauserii Z. G. Abad and J. A. Abad (GenBank Nos. AY550916, AM942765, and EU244850). Pathogenicity tests were performed on 1-year-old potted plants of B. speciosa with isolates IMI 393960 and 466/03. Twenty plants per each isolate were transplanted into 12-cm-diameter pots containing infested soil prepared by mixing steam-sterilized sandy loam soil with 1% of inoculum produced on autoclaved wheat kernels. Twenty control plants were grown in autoclaved soil mix. Plants were kept in the greenhouse with natural light at 25 ± 2°C and watered to field capacity weekly. All Banksia plants transplanted in infested soil showed symptoms of wilt, leaf chlorosis, and basal stem rot within 2 to 3 weeks. Noninoculated plants remained healthy. P. taxon niederhauserii was reisolated solely from inoculated plants. P. taxon niederhauserii has been reported recently from Banksia spp. in Australia (1), but to our knowledge this is the first report from Italy. P. taxon niederhauserii may represent a threat to the cultivation of many ornamentals including Cystus spp., English ivy, and laurel (4). References: (1) T. I. Burgess et al. Plant Dis. 93:215, 2009. (2) S. O. Cacciola et al. EPPO Bull. 20:47, 1990. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (4) E. Moralejo et al. Plant Pathol, 58:100, 2009.

First Report of Phytophthora tentaculata Causing Root and Stem Rot of Oregano in Italy.

Oregano (Origanum vulgare L.; Lamiaceae) is cultivated for culinary and medicinal purposes and as an ornamental. In October of 2007, 1- to 2-year-old potted plants of oregano showed symptoms of decline associated with root and basal stem rot in a nursery in Liguria (northern Italy) that produces 1 million to 1.5 million potted aromatic plants per year. Aboveground symptoms included leaf russeting and chlorosis, wilt, defoliation and dieback of twigs, browning of the basal stem, and subsequent collapse of the entire plant. Approximately 80% of the plants died within 30 days after the appearance of the first symptoms on the canopy. Approximately 20% of a stock of 30,000 oregano plants was affected. Stocks of other aromatic species, such as mint, lavender, rosemary, and sage, appeared healthy. A Phytophthora species was consistently isolated from symptomatic stems and roots of oregano plants on BNPRAH selective medium (2). Ten pure cultures were obtained by single-hypha transfers, and the species was identified as Phytophthora tentaculata Kröber & Marwitz by morphological criteria and sequencing of the internal transcribed spacer (ITS) region of rDNA using the ITS 4 and ITS 6 universal primers for DNA amplification. Isolates from oregano formed stoloniferous colonies with arachnoid mycelium on potato dextrose agar and had a growth rate of 2 to 3 mm per day at 24°C with optimum, minimum, and maximum temperatures of 24, 8, and 34°C, respectively. Sporangia formed in soil extract solution and were papillate and spherical or ovoid to obpyriform with a length/breadth ratio of 1.3:1. Few sporangia were caducous and all had a short pedicel (<5 µm). Hyphal swellings and chlamydospores were produced in sterile distilled water and corn meal agar, respectively. All isolates were homothallic and produced globose terminal oogonia (mean diameter of 34 µm) with one or occasionally two paragynous, monoclinous, or diclinous antheridia. Amphigynous antheridia were also observed. The sequence of the ITS region of the rDNA (GenBank No. FJ872545) of an isolate from oregano (IMI 395782) showed 99% similarity with sequences of two reference isolates of P. tentaculata (Accession Nos. AF266775 and AY881001). To test for pathogenicity, the exposed root crowns of 10 6-month-old potted plants of oregano were drench inoculated with 10 ml of a suspension of 2 × 104 zoospores/ml of isolate IMI 395782. Sterile water was pipetted onto the roots of 10 control plants. All plants were maintained in 100% humidity at 22 to 24°C in a greenhouse under natural light and watered once a week. Within 3 weeks after inoculation, all inoculated plants developed symptoms identical to those observed in the nursery and died within 30 to 40 days after the appearance of the first symptoms. Control plants remained healthy. P. tentaculata was reisolated solely from symptomatic plants. P. tentaculata has been reported previously on several herbaceous ornamental plants (1,3). However, to our knowledge, this is the first report of this species on O. vulgare. Root and basal stem rot caused by P. tentaculata is the most serious soilborne disease of oregano reported in Italy so far. References: (1) G. Cristinzio et al. Inf. Fitopatol. 2:28, 2006. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) H. Kröber and R. Marwitz. Z. Pflanzenkr. Pflanzenschutz 100:250, 1993.

Four Phytophthora Species Causing Foot and Root Rot of Apricot in Italy.

In the summer of 2006, 1-year-old apricot (Prunus armeniaca L.) trees with leaf chlorosis, wilting, and defoliation associated with root and crown rot were observed in a nursery in Sicily (Italy). Of 3,000 plants, ~2% was affected. Four Phytophthora spp. (45, 25, 20, and 10% of the isolations of the first, second, third, and fourth species, respectively) were isolated from decayed roots and trunk bark on BNPRAH (3). Axenic cultures were obtained by single-hypha transfers. Isolates of the first species formed petaloid colonies on potato dextrose agar (PDA) and had an optimum growth temperature of 25°C. On V8 agar (VA), they produced persistent, papillate (often bipapillate), ovoid to limoniform sporangia (length/breadth ratio 1.4:1). They did not produce gametangia when paired with A1 and A2 isolates of Phytophthora nicotianae. The second species formed arachnoid colonies, had an optimum growth of 30°C, and produced uni- and bipapillate, ellipsoid, ovoid or pyriform sporangia (length/breadth ratio 1.3:1). All isolates were A2. The third species formed rosaceous colonies on PDA, had an optimum temperature of 28 to 30°C, and produced papillate (sometime bipapillate), ellipsoid or limoniform (length/breadth ratio 2:1), caducous sporangia with a tapered base and a long pedicel (as much as 150 µm). All isolates were A1 type. The fourth species formed petaloid-like colonies on PDA and had an optimum growth of 26 to 28°C. On VA, it produced papillate (sometimes bipapillate), ovoid (length/breadth ratio 1.3:1), and decidous sporangia with a short pedicel (<4 µm). The isolates were homothallic and produced oogonia (25 to 31 µm in diameter) with paragynous antheridia and aplerotic oospores. On the basis of morphological and cultural characters, the species were identified as P. citrophthora, P. nicotianae, P. tropicalis and P. cactorum. Identification was confirmed by the electrophoretic analysis of total mycelial proteins and four isozymes (acid and alkaline phosphatases, esterase, and malate dehydrogenase) on polyacrylamide gel (1). Analysis of internal transcribed spacer (ITS) regions of rDNA using the ITS 4 and ITS 6 primers for DNA amplification (2) revealed 99 to 100% similarity between apricot isolates of each species and reference isolates from GenBank (Nos. AF266785, AB367355, DQ118649, and AF266772). The ITS sequence of a P. citrophthora isolate from apricot (IMI 396200) was deposited in GenBank (No. FJ943417). In the summer of 2008, pathogenicity of apricot isolates IMI 396200 (P. citrophthora), IMI 396203 (P. nicotianae), IMI 396201 (P. tropicalis), and IMI 396202 (P. cactorum) was tested on 3-month-old apricot seedlings (10 plants for each isolate) that were transplanted into pots filled with soil prepared by mixing steam-sterilized sandy loam soil (4% vol/vol) with inoculum produced on autoclaved kernel seeds. Ten control seedlings were grown in autoclaved soil. Seedlings were maintained in a screenhouse and watered daily to field capacity. Within 40 days of the transplant, all inoculated seedlings showed leaf chlorosis, wilting, and root rot. Control seedlings remained healthy. All four Phytophthora spp. were reisolated solely from inoculated plants. To our knowledge, this is the first report of Phytophthora root and crown rot of apricot in Italy and of P. tropicalis on this host. References: (1) S. O. Cacciola et al. Plant Dis. 90:680, 2006. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) H. Masago et al. Phytopathology 67:425, 1977.

Blight Caused by Sclerotium rolfsii on Potted Ornamental Citrus in Sicily.

Approximately 140,000 container-grown ornamental citrus plants are produced each year in the province of Catania (eastern Sicily). In the spring of 2006, a severe blight was observed in a commercial nursery in Catania on 2-month-old rooted cuttings of lemon (Citrus limon (L.) Burm.) and calamondin (× Citrofortunella mitis (Blanco) J. W. Ingram & H. E. Moore). Approximately 80% of the nursery stock of 2,000 cuttings was affected. Cuttings were grown in 7.5-cm2 pots made with compressed peat and wood pulp at 28 to 30°C with 95 to 100% relative humidity on benches in a greenhouse, The pot mix was composed of peat, perlite, and soil (2:1:2). Cuttings showed a dark brown necrotic lesion at the base of the stem that extended upward, resulting in chlorosis and wilting of the leaves. An invasive, white, cottony mycelium with a fan-like pattern and numerous, small, brown spherical sclerotia (0.5 to 4.0 mm in diameter) developed on infected tissues, in the potting mix as well as on the pot wall. Herbaceous cuttings collapsed within 2 weeks while woody cuttings gradually died. Symptomatic basal stem sections were disinfected for 1 min in 1% NaOCl, rinsed in sterile water, and plated on acidified (pH 4.5) potato dextrose agar (PDA). Isolations consistently yielded a fungus whose morphological characters corresponded to Sclerotium rolfsii Sacc. On PDA, it produced a septate mycelium with clamp connections and numerous olive brown-to-clove brown sclerotia (1 to 3 mm in diameter). Pathogenicity of two S. rolfsii isolates (IMI 396204 and IMI 396205) from citrus was confirmed on 3-month-old lemon cuttings grown in 10-cm-diameter plastic pots filled with a sterilized mix of peat moss and vermiculite (3:1) (10 cuttings for each isolate). Each pot was inoculated with 15 sclerotia harvested from 6-week-old cultures on PDA and placed on the soil surface around the base of the cutting. Ten noninoculated cuttings served as the control. Cuttings were kept in a growth chamber at 28°C and relative humidity at >95%. All inoculated cuttings showed wilting, blight, and stem rot within 3 weeks after inoculation. White mycelium and sclerotia were produced on the stem base and soil surface. Noninoculated controls remained symptomless. S. rolfsii was reisolated from infected cuttings. The pathogenicity test was repeated once with calamondin cuttings and the results were similar. Blight caused by S. rolfsii is widespread in nurseries of ornamentals in Italy (1). However, to our knowledge, this is the first report of this disease on potted ornamental citrus. Probably high temperature and moisture during rooting were conducive to the disease. References: (1) A. Garibaldi et al. Malattie Delle Piante Ornamentali. Calderini Edagricole, Bologna, Italy, 2000.

First Report of Phytophthora spp. as Pathogens of Pandorea jasminoides in Italy.

In the summer of 2005, approximately 5% of a nursery stock of 12-month-old potted plants of bower vine (Pandorea jasminoides (Lindl.) K. Schum.) in Sicily (Italy) showed wilt, leaf chlorosis, defoliation, root rot, and collapse of the entire plant. Three Phytophthora spp. (20, 50, and 30% of the isolations of the first, second, and third species, respectively) were isolated from rotted roots on BNPRAH selective medium (2). Single-hypha isolates of the first species formed petaloid colonies on potato dextrose agar (PDA) and had an optimum growth temperature of 25°C (9.3 mm/day); on V8 juice agar, they produced uni- and bipapillate, ovoid to limoniform sporangia with mean dimensions of 45 × 30 µm and a mean length/width (l/w) ratio of 1.4:1. They did not produce gametangia when paired with A1 and A2 isolates of Phytophthora nicotianae. The second species formed arachnoides colonies on PDA, had an optimum growth temperature of 30°C (6.9 mm/day) and produced sporangia that were uni- and bipapillate, ellipsoid, ovoid, or pyriform to spherical (dimensions 44 × 34 µm; l/w ratio 1.3:1). All isolates were A2 mating type and produced amphyginous antheridia and spherical oogonia with smooth walls. The third species formed rosaceous colonies on PDA, had an optimum growth temperature of 28 to 30°C (11.9 mm/day), and produced uni- and bipapillate, ellipsoid or limoniform, caducous sporangia (dimensions 52 × 26 µm; l/w ratio 2.1:1) with a tapered base and a long pedicel (as much as 150 µm). All isolates were A1 type and produced amphigynous antheridia and spherical oogonia with smooth walls. The three species were identified as P. citrophthora, P. nicotianae, and P. tropicalis, respectively. The electrophoretic analysis of the mycelial proteins and four isozymes (1) confirmed the identification. Blast analysis of the sequence of the internal transcribed spacer region of the rDNA of a P. tropicalis isolate from bower vine (GenBank Accession No. EU076731) showed 99% similarity with the sequence of a P. tropicalis isolate from Cuphea ignea (GenBank Accession No. DQ118649). The pathogenicity of three isolates from bower vine, IMI 395552 (P. citrophthora), IMI 395553 (P. nicotianae), and IMI 395346 (P. tropicalis), was tested on 3-month-old potted bower vine plants (10 plants for each isolate) by applying 10 ml of a suspension (2 × 104 zoospores/ml) to the root crown. The plants were maintained at 24°C and 95 to 100% relative humidity. All inoculated plants wilted after 4 weeks. Noninoculated control plants remained healthy. The three Phytophthora spp. were reisolated from symptomatic plants. To our knowledge, this is the first report of Phytophthora root rot of bower vine in Italy. References: (1) S. O. Cacciola et al. Plant Dis. 90:680, 2006. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

First Report of Bud Rot of Canary Island Date Palm Caused by Phytophthora palmivora in Italy.

Canary Island date palm (Phoenix canariensis hort. ex Chabaud) is planted as an ornamental in Mediterranean climatic regions of the world. From 2004 to 2006, withering of the spear leaf was observed on screenhouse-grown potted plants of this palm in Sicily (Italy). The first symptom was a dark brown rot that extended from the petiole base of the spear to the adjacent youngest leaves and killed the bud. Dissection of plants revealed a foul-smelling internal rot. After the bud died, external older leaves remained green for months. As much as 10% of plants in a single nursery were affected. A Phytophthora species was consistently isolated from symptomatic plants on BNPRAH selective medium (4). Single zoospore isolates were obtained from the colonies. The species isolated was identified as Phytophthora palmivora (E. J. Butler) E. J. Butler on the basis of morphological and cultural characteristics (3). On V8 juice agar, the isolates produced elliptical to ovoid, papillate sporangia (33 to 77 × 22 to 38 µm) with a mean length/breadth ratio of 1.8. Sporangia were caducous with a short pedicel (mean pedicel length = 5 µm) and had a conspicuous basal plug. All isolates were heterothallic and produced amphigynous antheridia, oogonia, and oospores when paired with reference isolates of P. nicotianae and P. palmivora of the A2 mating type. The oogonium wall was smooth. Identification was confirmed by electrophoresis of mycelial proteins in polyacrylamide slab gels (1). The electrophoretic patterns of total mycelial proteins and four isozymes (alkaline phosphatase, esterase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase of the isolates) from Phoenix canariensis were identical to those of P. palmivora reference isolates, including four Italian ones, two from pittosporum and olive, respectively, and two (IMI 390579 and 390580) from Grevillea spp. Phoenix canariensis isolates were clearly distinct from those of other heterothallic papillate species including P. capsici, P. citrophthora, P. katsurae, P. nicotianae, and P. tropicalis. Pathogenicity of one isolate from Phoenix canariensis (IMI 395345) was tested on 10 2-year-old potted Canary Island date palm plants. An aqueous 105 zoospores per ml suspension (200 µl) was pipetted onto unwounded petiole bases of the three youngest central leaves of each plant. Sterile water was pipetted onto 10 control plants. All plants were incubated in 100% humidity at 24°C for 48 h and maintained in a greenhouse at 20 to 28°C. Within 3 weeks after inoculation, inoculated plants developed symptoms identical to those observed on plants with natural infections. Control plants remained healthy. P. palmivora was reisolated from symptomatic plants. Phytophthora bud rot is a common palm disease worldwide and Phoenix canariensis is reported as a host (2). To our knowledge, this is the first report of Phytophthora bud rot on Phoenix canariensis in Italy. References: (1) S. O. Cacciola et al. EPPO Bull. 20:47, 1990. (2) M. L. Elliot et al., eds. Compendium of Ornamental Palm Diseases and Disorders. The American Phytopathological Society, St. Paul, MN, 2004. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (4) H. Masago et al. Phytopathology, 67:425, 1977.

First Report of Armillaria Butt Rot Caused by Armillaria mellea on Phoenix canariensis in Italy.

During 2006, in a garden in the Mount Etna Piedmont, eastern Sicily (Italy), a 40-year-old specimen of Canary Island date palm (Phoenix canariensis hort. ex Chabaud) with a trunk circumference at breast height of 220 cm showed a rotted lesion with a viscous, brown ooze at the stem base and root initials. The lesion extended to approximately one-third of the trunk circumference. Trunk excavation exposed a wet rot of internal tissues, a cream-colored mycelial mat, and a mushroom-like smell. Although the rot spread inward (approximately 25 cm deep) with decay of nonlignified ground tissues and blackening of wood fibers, the palm did not show symptoms on the canopy. Conversely, ferns, apricot, and cedar trees growing at the same site had died from Armillaria rot over the last 10 years (2). In late autumn, clumps of honey mushroom-like sphorophores with a prominent annulus encircling the stalk formed at the base of the trunk. The spore print of the basidiocarp was light cream. The morphology of 100 basidiospores was determined microscopically. The basidiospores were smooth, elliptical, hyaline, and measured 7 to 9.5 × 5 to 7 µm. The fungus was isolated from diseased tissues on selective benomyl-dichloran medium (3) and was transferred to 2% malt extract agar where it formed ribbon-shaped, fast-growing, and profusely branching rhizomorphs. Armillaria mellea (Vahl.) P. Kumm. was identified on the basis of cultural and morphological characteristics. Identification was confirmed by electrophoresis of mycelial proteins and isozymes in polyacrylamide and starch slab gels (1,2). The electrophoretic patterns of the isolate from P. canariensis were identical to those of reference isolates of A. mellea from grapevine and fern isolated previously at the same site (2). The pathogenicity of the A. mellea isolate from palm (A-palm5) was tested on 20 3-year-old potted seedlings of P. canariensis grown in a greenhouse at 24 ± 4°C. Seedlings were inoculated with wood pieces of holly oak (Quercus ilex L.) colonized by the fungus (two pieces for each seedling) (4). Ten noninoculated plants served as controls. After 12 months, mycelial fans colonizing the root initials, the base of the stem, and the leaf stalks were observed on 14 inoculated seedlings. Although only four infected seedlings showed decline symptoms, the fungus was reisolated from all inoculated plants. No infections were observed in control plants. To our knowledge, this is the first report of Armillaria butt rot on a palm in Europe. References: (1) M. Bragaloni et al. Eur. J. For. Pathol. 27:147, 1997. (2) S. Grasso et al. Plant Dis. 84:592, 2000. (3) T. C. Harrington et al. Armillaria. Page 81 in: Methods for Research on Soilborne Phytopathogenic Fungi. The American Phytopathological Society, St. Paul, MN, 1992. (4) R. Metaliaj et al. Phytopathol. Mediterr. 45:3, 2006.