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.

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.

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.

First Report of Brown Rot and Wilt of Fennel Caused by Phytophthora megasperma in Italy.

Fennel (Foeniculum vulgare Mill. var. azoricum (Mill.) Thell.) in the Apiaceae family is native to southern Europe and southwestern Asia. It is an economically important crop in Italy that produces approximately 85% of all fennel worldwide. The main producing regions are Apulia, Campania, Latium, and Calabria. During the late winter of 2004 in the Crotone Province of the Calabria Region, following heavy rains, patches of fennel plants with symptoms of brown, soft rot of the bulb-like structure formed by the thickened leaf bases, development of yellow leaves, stunting, and wilting of the entire plant were observed in fields. A homothallic Phytophthora sp. was isolated consistently from the brownish tissues of the stout stems and leaf bases of symptomatic plants using a selective medium (3). Pure cultures were obtained by single hyphal tip transfers. On potato dextrose agar (PDA), the diameter of oospores varied from 28 to 42 µm (mean = 36.3 ± 0.4). Antheridia were primarily paragynous. Sporangia were not produced on solid media but were formed in sterile soil extract solution. They were nonpapillate, noncaducous, ovoid and obpyriform (25 to 45 × 35 to 60 µm), and internally proliferating. Optimum and maximum temperatures for radial growth of the colonies on PDA were 25 and 30°C, respectively. At 25°C, radial growth rate was approximately 6 mm per day. On the basis of morphological and cultural characteristics, the isolates were identified as Phytophthora megasperma Drechsler. Electrophoretic patterns of mycelial proteins and four isozymes (acid and alkaline phosphatase, esterase, and malate dehydrogenase) on polyacrylamide gels of the fennel isolates were identical to those of reference isolates of P. megasperma of the BHR (broad host range) group included in P. gonapodyides-P. megasperma Clade 6 (1,3), but distinct from those of the isolates of other nonpapillate species included in Waterhouse's taxonomic group VI. Internal transcribed spacer (ITS) regions of rDNA sequences (2) confirmed that fennel isolates belonged to P. megasperma BHR group. Pathogenicity of a fennel isolate from Calabria (IMI 391711) was confirmed by pouring a zoospore suspension at 2 × 104 zoospores per ml on the soil of 10 3-month-old potted fennel plants. The soil of the inoculated and 10 control seedlings was flooded for 24 h. After 10 days, stems and leaf bases of the seedlings showed a brown rot. Chlorosis and wilting of all seedlings developed after 20 days. Controls inoculated with water did not develop any symptoms. The pathogen was reisolated from typical brown rot and tests were repeated with similar results. To our knowledge, this is the first report of P. megasperma causing disease on fennel. References: (1) S. O. Cacciola et al. For. Snow. Landsc. Res. 76:387, 2001. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) H. Masago et al. Phytopathology, 67:425, 1977.

Race 1,2y of Fusarium oxysporum f. sp. melonis on Muskmelon in Sicily.

Muskmelon (Cucumis melo L.) is very important economically to agriculture in Italy. The Sicily area accounts for ≈40% of the total muskmelon production. Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Leach & Currence) W.C. Snyder & H.N. Hans. is the most prevalent and damaging disease of muskmelon in Sicily. Use of cultivars with major resistance genes, Fom 1 and Fom 2, is the most effective control measure for combating the disease. During March 1999, severe infections of Fusarium wilt were noted in a commercial muskmelon crop, cv. Firmo F1, grown in plastic tunnels in Syracuse Province (eastern Sicily). The muskmelon seedlings had been transplanted into the tunnels during January 20 days after soil fumigation with methyl bromide. Firmo F1 possesses both Fom 1 and Fom 2 genes. Of 18,000 Firmo F1 plants, ≈6,500 showed symptoms consisting of stunting, vein clearing; leaf yellowing, wilting, and dying; brown necrotic streak; and gummy exudates on the basal portion of vines. A pinkish white mold developed on dead tissues when infected plants were kept at high relative humidity. The pathogenicity of both a single-conidium isolate of F. oxysporum f. sp. melonis from a symptomatic Firmo F1 plant and two isolates of races 0 and 1, recovered previously from other cultivars in Sicily and used as references, was tested with three differential muskmelon cultivars, Charentais T, Doublon, and CM 17187 (1), as well as three commercial cultivars, Ramon, Cassella, and Geamar (possessing Fom 1, Fom 2, and both Fom 1 and Fom 2 resistance genes, respectively). Muskmelon seedlings were inoculated by the root-dip method (3), using a suspension of 5 × 105 conidia per ml. Inoculated seedlings were transplanted to plastic pots filled with sterilized soil and placed in a greenhouse (25 to 30°C). Symptoms were scored 7 to 10 days after inoculation. The isolate from Firmo F1 was pathogenic to all cultivars tested, the race 0 isolate was pathogenic only to cv. Charentais T, and the race 1 isolate was pathogenic only to cvs. Charentais T, Doublon, and Ramon. F. oxysporum was reisolated from symptomatic plants. Based on its pathogenicity and symptomology, the isolate from Firmo F1 was classified as race 1,2y (yellows), according to the nomenclature proposed by Risser et al. (1). Race 1,2 poses a serious threat to muskmelon production in Sicily, because all currently used cultivars are susceptible to the race, and other control measures, such as preplant soil fumigation with methyl bromide and solarization, are not as effective as use of resistant cultivars. Further study is needed to establish which is the prevalent race of F. oxysporum f. sp. melonis in Sicily. This report confirms that race 1,2 occurs in all major muskmelon-production areas in Italy (2). References: (1) G. Risser et al. Phytopathology 66:1105, 1976. (2) G. Tamietti et al. Petria 4:103, 1994. (3) F. L. Wellman. Phytopathology 29:945, 1939.