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Article

Diversity and Distribution of Phytophthora Species Along an Elevation Gradient in Natural and Semi-Natural Forest Ecosystems in Portugal

by
Carlo Bregant
1,*,
Eduardo Batista
2,
Sandra Hilário
2,3,
Benedetto Teodoro Linaldeddu
1 and
Artur Alves
2
1
Dipartimento Territorio e Sistemi Agro-Forestali, Università degli Studi di Padova, Viale dell’Università, 16, 35020 Legnaro, Italy
2
Centre for Environmental and Marine Studies (CESAM), Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal
3
GreenUPorto—Sustainable Agrifood Production Research Centre/Inov4Agro, Department of Geosciences, Environment and Spatial Plannings (DGAOT), Faculty of Sciences, University of Porto, Campus de Vairão, Rua da Agrária 747, 4485-646 Vila do Conde, Portugal
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(1), 103; https://doi.org/10.3390/pathogens14010103
Submission received: 20 December 2024 / Revised: 9 January 2025 / Accepted: 17 January 2025 / Published: 20 January 2025
(This article belongs to the Special Issue Microbial Pathogenesis and Emerging Infections)
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Abstract

:
Globally, forests are constantly threatened by a plethora of disturbances of natural and anthropogenic origin, such as climate change, forest fires, urbanization, and pollution. Besides the most common stressors, during the last few years, Portuguese forests have been impacted by severe decline phenomena caused by invasive pathogens, many of which belong to the genus Phytophthora. The genus Phytophthora includes a large number of species that are invading forest ecosystems worldwide, chiefly as a consequence of global trade and human activities. This paper reports the results of a survey of Phytophthora diversity in natural and semi-natural forest ecosystems in Portugal along an elevation gradient. Isolations performed from 138 symptomatic plant tissues and rhizosphere samples collected from 26 plant species yielded a total of 19 Phytophthora species belonging to 6 phylogenetic clades, including P. cinnamomi (36 isolates), P. multivora (20), P. plurivora (9), P. cactorum (8), P. lacustris (8), P. pseudocryptogea (8), P. amnicola (6), P. hedraiandra (6), P. pseudosyringae (5), P. thermophila (5), P. bilorbang (4), P. inundata (4), P. asparagi (3), P. citricola (3), P. gonapodyides (3), P. rosacearum (3), P. chlamydospora (2), P. pachypleura (2), and P. syringae (1). Overall, the data obtained highlight the widespread occurrence of P. cinnamomi in natural ecosystems from sea level to mountain habitats. The results of the pathogenicity tests carried out on 2-year-old chestnut plants confirmed the key role of P. cinnamomi in the recrudescence of chestnut ink disease and the additional risk posed by P. pachypleura, P. plurivora, and P. multivora to Portuguese chestnut forests. Finally, three species, P. citricola, P. hedraiandra, and P. pachypleura, are reported for the first time in the natural ecosystems of Portugal.

1. Introduction

In Portugal, forests provide a variety of resources and play a primary role in the national economy [1]. About 36% of the Portuguese mainland is covered by forested areas [2]. According to the last National Forest Inventory, Portuguese forests are mainly composed of Eucalyptus spp. (mostly Eucalyptus globulus) plantations with a surface of over 845,000 hectares. The other key forest species are Quercus suber (720,000 ha) and Pinus pinaster (713,000 ha) [2].
Forest productions, such as timber, cork, pulp, paper, and wooden furniture, are an important source of income and represent approximately 2% of the national gross domestic product and 10% of total Portuguese exports [1].
In Portugal, during the past decades, a change in land management has occurred following the progressive abandonment of agricultural activities and the consequent transfer of land use towards forestry [3]. Both natural and artificial forests in Portugal are constantly threatened by disturbances of natural and anthropogenic origin. Forest fires and drought induced by climate change are currently the most serious abiotic threats to Portuguese forests, inducing phenomena of land degradation and desertification [4].
Although forest fires are natural occurrences in Mediterranean regions, the past few decades have seen a clear increase in the frequency of major forest fire events, especially in Portugal [4,5,6]. Portugal is the European country with the highest percentage of its forestry land lost to wildfires between 2001 and 2021, about 13% [7].
Climate influences the structure and function of forest ecosystems and plays a primary role in forest health. Rapid variation in climatic conditions can directly and indirectly affect the growth and productivity of forests through changes in temperature, humidity, precipitation, and other factors [8,9]. In the last three decades, a significant reduction in summer rainfall characterized the Iberian Peninsula, alternating with extreme events of concentrated rain [10,11]. Severe summer drought events combined with the spread of fire-prone forest species such as E. globulus and P. pinaster create the most suitable conditions for the occurrence of large-scale fires on the Portuguese mainland [12].
Abiotic disturbances and stress conditions can intensify many of the biotic threats to forests, such as the outbreak of invasive pests and pathogens [9,13]. In recent years, a drastic decline in forest ecosystems has characterized large areas of Portugal [14,15,16]. The low diversity of forest species and the dominant occurrence of clonal stands could favour the occurrence of large outbreaks of forest pests and pathogens, at either spatial or temporal scales, whenever disturbances occur [14]. Pine and cork oak forests are the formations most affected by multiple attacks of insects and fungi [14].
Among the most serious pathogens threatening European forests, some species belonging to the family Botryosphaeriaceae (Ascomycota) and the genus Phytophthora (Oomycota) have been assuming a primary role in recent decades [17,18]. These invasive and often polyphagous pathogens can simultaneously affect plants, causing serious decline phenomena [19,20,21].
In Portugal, some recent studies have identified the widespread occurrence, in forest ecosystems, of 22 species of Botryosphaeriaceae [15,22,23,24,25], whereas, until some years ago, little was known about the occurrence and diversity of oomycetes in natural ecosystems in Portugal [26,27]. Although serious outbreaks of ink disease have impacted chestnut forests since the first half of the 19th century and cork oak stands since 1900, scientific interest in Phytophthora disease has grown considerably only in recent years [27,28,29,30]. Recent studies have revealed a widespread presence of P. cinnamomi in chestnut, eucalyptus and cork oak forests, especially in the central and southern areas of the country [29,31,32,33,34,35,36]. In addition, severe Phytophthora outbreaks are devastating riparian and wet habitats populated by Alnus glutinosa in Central Portugal [27,37]. A recent study listed over 30 species and hybrids in forest ecosystems and nurseries in Portugal [38].
Therefore, given the still limited information about the Phytophthora species involved in the severe decline phenomena affecting natural and artificial forest formations in Portugal, a study was conducted to evaluate the diversity and distribution of Phytophthora species in the main habitats along an elevation gradient.

2. Materials and Methods

2.1. Field Surveys and Sampling Procedure

From February to June 2022, a preliminary survey was conducted across Portugal to evaluate the health status of forest formations. Afterwards, twenty-one different sites were randomly selected along the entire elevation gradient from the sea level (0 m a.s.l) to the highest mountains (1900 m a.s.l.). The 21 sites are representative of three different macroclimatic areas, a Mediterranean/coastal zone (A), a temperate zone (B), and a submontane/montane belt (C), according to the climatic zonation of Köppen [39].
At each site, the plants were checked for the presence of typical Phytophthora disease symptoms on the canopy (leaf necrosis, bleeding cankers, epicormic shoots, sudden death, and chlorosis) and root system (exudates, necrosis, and loss of fine roots). At some sites (1, 5, 6, 8, 9, 10, 11, 12, 13, 16, 17, and 21), a linear transect of 50 m was randomly established to evaluate the disease incidence and mortality rate, as reported in Bregant et al. [27] (Table 1).
A total of 138 samples were randomly collected from 26 plant host species, including rhizosphere (106 samples), necrotic leaves, and bark tissues from bleeding cankers (32) (Table 1). The plants sampled were divided into natural, planted, and invasive.

2.2. Isolation of Pathogens

In the laboratory, Phytophthora isolation was attempted as reported in Bregant et al. [27]. Rhizosphere soil samples were placed in plastic cylinders and flooded with distilled water. Young Q. suber, Hedera helix, and Pittosporum sp. leaves were used as bait on the water surface. Cylinders were kept at 20 °C under natural daylight and checked after 12–24 h for 3–5 days. Leaves showing dark spots were divided into small fragments of 5 mm2 and placed on 90 mm Petri dishes containing the selective medium PDA+ reported in Bregant et al. [40].
The isolation of Phytophthora species was also directly attempted from necrotic leaves and bark tissues collected from bleeding cankers. Small fragments taken with a sterile scalpel along the border of the necrosis were placed in Petri dishes containing PDA+.
The plates were incubated in the dark at 20 °C and examined every 12 h. Hyphal tips typical of Phytophthora from the emerging colonies were sub-cultured on potato dextrose agar (PDA) and carrot agar (CA) and incubated at 20 °C in the dark [41].

2.3. Identification of Pathogens

All isolates were initially divided into morphotypes based on colony growth characteristics, including their colony appearance after 7 days of incubation on PDA and CA at 20 °C in darkness, as well as the morpho-biometric data of sporangia and oogonia. All isolates were initially divided into morphotypes. Phytophthora isolates were grouped according to morphological descriptions provided by Erwin and Ribeiro [41]. To enhance sporangia production, CA plugs (5 mm diameter) of each isolate were placed in Petri dishes containing 10 mL of unsterile pond water with 2 mL of carrot broth added. Sporangial production was assessed every 6 h for 4 days by microscopic observation. For all isolates, breeding systems were evaluated on CA Petri dishes after 20 days of incubation at 20 °C. The biometric data of morphological structures were measured with the software Motic Images Plus 3.0 paired with a Moticam 10+ camera connected to a Motic BA410E microscope (MOTIC INSTRUMENTS INC. Viking Way, Richmond, BC, Canada).
Molecular analysis was used to confirm the identity of all isolates at the species level. The genomic DNA was extracted from the mycelium of 5-day-old cultures grown on PDA at 20 °C, according to the protocol reported by Möller et al. [42]. The primers ITS5 and ITS4 were used to amplify and sequence the internal transcribed spacer (ITS) regions, including the complete 5.8S gene [43]. Polymerase chain reaction (PCR) mixtures and amplification conditions were as described by Bregant et al. [27]. PCR amplicons were purified with the DNA NZY Gelpure kit MB01102 (Nzytech, Lisbon, Portugal) following the manufacturer’s instructions. The ITS regions were sequenced by the GATC Biotech (Cologne, Germany). The nucleotide sequences were read and edited with FinchTV 1.4.0 (Geospiza, Inc., http://www.geospiza.com/finchtv, accessed on 1 December 2024) and then compared with reference sequences (ex-type material) retrieved from GenBank using the BLASTn algorithm. ITS sequences from representative isolates obtained in this study were deposited in GenBank www.ncbi.nlm.nih.gov/genbank (accessed on 1 December 2024) (Table 2).

2.4. Phylogenetic Analysis

Molecular phylogeny based on ITS sequences was used to reconstruct evolutionary relationships among the Phytophthora species obtained in this study into the known clades of the genus [44]. Nineteen ITS sequences representative of the Phytophthora species obtained were compiled in a dataset together with thirty-two sequences from ex-type material of Phytophthora species representative of all phylogenetical clades (Table 2). Two isolates of Halophytophthora avicenniae and two of Nothophytophthora caduca, including those obtained in this study, were included as outgroup taxa.
Sequences were aligned with ClustalX v. 1.83 [45] using the parameters reported by Bregant et al. [40].
Phylogenetic reconstructions were performed with MEGA-X 10.1.8, including all gaps in the analyses. The best model of DNA sequence evolution was determined automatically by the software [46]. Maximum likelihood (ML) analysis was performed with a neighbour-joining (NJ) starting tree generated by the software. A bootstrap analysis (1000 replicates) was used to estimate the robustness of nodes.

2.5. Pathogenicity Test

To fulfil Koch’s postulates, the pathogenicity of six representative isolates of Phytophthora obtained from chestnut (P. cactorum CBP168, P. cinnamomi CBP185, P. multivora CBP154, P. pachypleura CBP158, P. plurivora CBP164, and P. pseudocryptogea CBP166) was tested against 2-year-old chestnut plants cultivated in plastic pots (1 L volume). The experimental design consisted of eight seedlings inoculated per isolate. A 5 mm diameter hole was made through the bark of the stem using a cork borer and replaced with an agar plug of the same size taken from the margin of 5-day-old cultures grown on PDA. The inoculation wounds were wrapped with sterile damp cotton wool and covered with aluminium foil. Eight seedlings were inoculated with a sterile plug of PDA as a control. Plants were kept in field conditions ranging from 9 to 29 °C and watered regularly for 30 days.
At the end of the experimental period, symptoms were checked and the extent of the external lesions was measured. Pathogenicity assay data were first checked for normality (Anderson–Darling test) and then subjected to analysis of variance (ANOVA). Significant differences among mean values were determined using Fisher’s least significant differences multiple range test (p = 0.05) after one-way ANOVA using XLSTAT 2008 software (Addinsoft).
Re-isolation was made from small pieces of wood removed from lesion margins onto PDA+. Growing colonies were sub-cultured onto CA and PDA, incubated in the dark at 20 °C, and identified through morphology and ITS sequencing.

2.6. Geographic Distribution of Phytophthora Species

A literature review was conducted, focusing on the terms “Phytophthora” and “Portugal” (source: Scopus, Google Scholar, and GenBank, November 2024). All relevant records containing geographic information were standardized and organized in a single dataset. Records without a clear geographical identification were not included in this analysis.

3. Results

3.1. Field Survey

Symptoms of decline and mortality were recovered in almost all monitored sites in Portugal. More specifically, emerging diseases are affecting all climatic regions in the country, ranging from the Mediterranean vegetation of the Algarve to the montane habitats in Serra da Estrela (>1900 metres a.s.l.).
Affected plants showed mainly typical root and collar rot symptoms, exudates at the lower part of the stem, stunted growth, and, in severe cases, sudden death (Figure 1). In some sites, aerial Phytophthora symptoms were observed on different plant species, involving various plant organs such as leaves and twigs (Figure 1). Moreover, in stems and branches, extensive bleeding cankers were observed and necrosis progressively girdled the circumference of the branch, causing partial or total death of the crown (Figure 1).
Disease incidence ranged from 30 to 100% with an average mortality rate of 11–55% (Table 3). The most affected formation appeared to be the coastal maquis of Pistacia lentiscus and the mountain forests of Betula celtiberica, with a mortality rate of 31 and 55%, respectively.

3.2. Phytophthora Diversity in Portugal

From the 138 samples collected in different habitats across Portugal, 136 Phytophthora isolates were obtained belonging to six different ITS clades (Table 4). Of these, 22 isolates emerged from bleeding cankers and necrotic leaves and 114 from rhizosphere soil samples.
On the bases of morphology, colony appearance, and ITS sequence data, Phytophthora isolates were identified as P. cinnamomi (36 isolates), P. multivora (20), P. plurivora (9), P. cactorum (8), P. lacustris (8), P. pseudocryptogea (8), P. amnicola (6), P. hedraiandra (6), P. pseudosyringae (5), P. thermophila (5), P. bilorbang (4), P. inundata (4), P. asparagi (3), P. citricola (3), P. gonapodyides (3), P. rosacearum (3), P. chlamydospora (2), P. pachypleura (2), and P. syringae (1). In addition to Phytophthora species, two isolates of Halophytophthora avicenniae and two of Nothophytophthora caduca were obtained (Table 4).
The most common and widespread Phytophthora species detected in this study across Portugal was P. cinnamomi. This species was isolated from 12 out of the 26 hosts, in 12 sites distributed across all climatic regions. The other dominant species were P. multivora and P. plurivora, isolated from five and six hosts and four and five sites, respectively.

3.3. Phytophthora Distribution in Portugal

As regards the geographical distribution of the species isolated in this study, a great variability emerged according to the climatic areas. All three zones (coastal/Mediterranean, temperate, and montane regions) showed a wide diversity in Phytophthora assemblages (Figure 2).
Many species are typical of one or two geographical areas; only P. cinnamomi and P. gonapodyides have been isolated in all three areas spanning from sea level to the mountain belt (Figure 2). In addition to P. cinnamomi, P. pseudocryptogea, and P. multivora are the other most frequent species in coastal (23%) and temperate areas (21%), respectively, whereas at higher altitudes, P. pseudosyringae and P. plurivora have been isolated from 24% of the examined samples.
Combining data from the literature review with our present study, a total of 34 different known Phytophthora species and 3 hybrids have been isolated and officially reported in natural and semi-natural ecosystems in Portugal, including 122 host–pathogen interactions (Table 5).
For the 37 Phytophthora species and hybrids for which data on isolation points are available, geographical and altitudinal distribution in Portugal’s mainland were reconstructed (Figure 3 and Figure 4).
Among the different species, P. cinnamomi is the most widespread from north to south and from west to east and has been reported from 29 different hosts (Table 5, Figure 3).
Other species widespread in the country are P. gonapodyides, P. plurivora, P. pseudocryptogea, and P. quercina (Figure 3). The distribution of the remaining species is more localized and fragmented (Figure 3).
Historically, the occurrence of P. cinnamomi appears related chiefly to Q. suber stands in the southern part of Portugal, but our study also revealed a common presence of this species in northern and central Portugal, including mountain areas.
The distribution along altitude shows the potential adaptability of the 37 Phytophthora species and hybrids to different climatic conditions (Figure 4). Most of the species reported in Portugal have been isolated at low altitudes from 0 to 500 metres a.s.l. Some species, such as P. cactorum, P. cinnamomi, and P. gonapodyides, manifest plasticity to all altitudes from sea level to over 1000 m a.s.l. Finally, P. pseudosyringae is the only species isolated exclusively in mountain forests.

3.4. ITS Phylogeny

The phylogenetic relationships among the Phytophthora isolates obtained in this study were elucidated using ITS sequences (Figure 5). In particular, the isolates included in the phylogenetic analysis were distributed in 19 terminal clades with the relative ex-type of 19 formally described species (Figure 5). Isolates of Halophytophthora avicenniae and Nothophytophthora caduca obtained in this study clustered in two basal clades with the relative ex-type strains.
The 19 Phytophthora species belong to 6 of the 12 phylogenetic clades of this genus [44]. Among all, nine species (P. amnicola, P. asparagi, P. bilorbang, P. chlamydospora, P. gonapodyides, P. inundata, P. lacustris, P. rosacearum, and P. thermophila) belong to clade 6, whereas four species reside in the ex-type P. citricola complex in clade 2 (P. citricola, P. multivora, P. pachypleura, P. plurivora). The other clades (1, 3, 7, and 8) are represented by only one or two species.

3.5. Pathogenicity Test

All Phytophthora species proved to be pathogenic to chestnut plants. At the end of the experimental period, inoculated seedlings showed dark brown inner bark lesions that spread up and down from the inoculation point at the collar root (Figure 6). Among the different species assayed, the length of the necrotic lesion differed significantly (Figure 6). The lesions caused by P. cinnamomi were significantly larger than those caused by other species (Figure 6). Also, P. pachypleura, P. plurivora, and P. multivora caused large lesions, while the other species only caused small necrotic lesions. Lesions caused by P. cinnamomi, P. pachypleura, and P. plurivora progressively girdled the twigs, causing shoot blight, browned foliage, and wilting symptoms. Control seedlings remained asymptomatic. Re-isolation was conducted positively for 100% of seedlings inoculated with Phytophthora spp.

4. Discussion

The results obtained in this study have allowed us to clarify both the symptomatology and aetiology related to severe decline phenomena affecting natural and planted forest ecosystems in Portugal from the sea level to the mountain belt. Over the past decades, research concerning the impact of Phytophthora in Portuguese forests has predominantly focused on the central and southern regions of the country, especially on chestnut and cork oak trees [31,32,33,34]. Furthermore, the recent occurrence of new outbreaks has made it possible to associate a large community of Phytophthora spp. with extensive decline phenomena of Eucalyptus globulus plantings and Alnus glutinosa riparian systems [27,29,30,37]. Furthermore, a study conducted from 2010 to 2015 reported a high diversity of oomycetes across Portuguese forests, rivers, and nurseries [38].
Regarding this study, the field surveys conducted in twenty-one Portuguese forest systems highlight and confirm that the severe disease outbreaks and mortality are affecting several woody plant species, from the Mediterranean to the sub-montane and montane forest formations in Portugal. The most impacted formations were the coastal Mediterranean maquis dominated by Pistacia lentiscus and the mountain forests of Betula celtiberica, but Phytophthora-related diseases affect a multitude of plant species including chestnut, oaks, poplars, willows, and eucalyptus.
The results showed a complex of pathogenic Phytophthora species associated with different symptoms, including leaf and shoot blights, bleeding cankers, and root rot on twenty-six different plant species. Overall, nineteen Phytophthora species belonging to six different phylogenetic clades were isolated and identified by means of morphological characters and DNA sequence data. These include Phytophthora amnicola, P. asparagi, P. bilorbang, P. cactorum, P. chlamydospora, P. cinnamomi, P. citricola, P. gonapodyides, P. hedraiandra, P. inundata, P. lacustris, P. multivora, P. pachypleura, P. plurivora, P. pseudocryptogea, P. pseudosyringae, P. rosacearum, P. syringae, and P. thermophila. The Phytophthora diversity found in Portugal includes both cosmopolitan and polyphagous pathogens and rare species known to attack a limited number of plant hosts in a few geographic areas.
The most common species isolated in this study is P. cinnamomi. This invasive pathogen has been isolated from 12 hosts in 12 sites across different climatic regions. This confirms previous studies on the wide occurrence of this pathogen in forest systems of Portugal [29,31,34,38]. It is considered one of the most invasive organisms worldwide [54]. In our study, P. cinnamomi has been isolated from declining trees from the Algarve (Mediterranean maquis) to the undisturbed cold Betula celtiberica forests in the Serra da Estrela (over 1200 m a.s.l.). This finding highlights the strong potential and plasticity of P. cinnamomi to invade, survive, and adapt to different environments, including low-temperature habitats, confirming the recent distribution patterns developed for this species and the results of other recent investigations in Europe and Australia [55,56,57,58,59].
This study has significantly expanded knowledge on the diversity and impact of pathogenic oomycetes in mountainous areas of Europe. Serra da Estrela, located in Central Portugal, is the highest mountain range and largest protected area. It is characterized by heterogeneous ecological conditions based on the various slopes and altitudes from 300 to over 1900 m. a.s.l. [60]. The involvement of both airborne and soilborne Phytophthora species is causing extensive mortality of many natural and planted mountain species such as birch, larch, and common juniper. In addition to P. cinnamomi, some species belonging to clades 1 and 3 and characterized by producing caducous sporangia emerged frequently from tissue samples of different alpine hosts. These included P. pseudosyringae, a typical species of mountain habitats, and the more plastic P. cactorum and P. hedraiandra. As reported in two previous studies conducted in alpine formations, low cardinal temperature for growth, the production of resistant structures, and an aerial lifestyle would seem to favour the affirmation of these species in cold environments [18,40]. Interestingly, the results of this study confirm the wide diffusion of all the species belonging to clade 1a, except P. aleatoria, in the mountainous areas of Europe including the wildest ones not disturbed by humans [18,40,61].
The abundant production of chlamydospores and hyphal swellings by the strains of P. cinnamomi and P. pseudosyringae obtained in Serra da Estrela could explain the adaptation and resistance of these species in a very “hostile” area, characterized by extremely cold winters and very hot and dry summers with frequent forest fires.
The other two most common species obtained in this study were P. multivora and P. plurivora (clade 2). The distribution of P. plurivora in Europe has been documented for a long time in several countries, with population studies indicating a European origin [62]. Its occurrence in the Iberian Peninsula was associated with many hosts in forest and nursery systems [27,38,63,64,65].
However, the introduction of the invasive P. multivora in Europe seems recent and it is currently spreading in Mediterranean regions, thanks to its greater adaptations to heat and dry conditions [27,61,66,67]. In Portugal, P. multivora was recently reported on Acacia dealbata, Acer pseudoplatanus, Alnus glutinosa, Eucalyptus globulus, Fraxinus angustifolia, and Quercus rubra [27,30,38]. The new association of this pathogen with several other native and invasive plant species suggests the good adaptation of this invasive species to Portugal’s climate and confirms the polyphagous nature of this organism reported in Australia and South Africa, posing a serious threat to European forests in the face of climate changes [67,68,69,70].
In addition to P. multivora and P. plurivora, another two clade 2 species of P. citricola sensu latu complex, namely P. citricola and P. pachypleura, have been isolated for the first time from natural forests of sycamore maple and sweet chestnut near Sintra (temperate sites 11 and 12), respectively. Phytophthora citricola s.s. is a pathogen known for a long time to cause severe diseases in wood crops, although many historical reports of this species probably belong to the cryptic species P. multivora and P. plurivora [71]. Phytophthora pachypleura was recently described from ornamental plants in Europe [72]. Before this study, these two species had never been recovered in Portugal and in natural areas of the continent.
Six species were isolated from Castanea sativa in this study. The under-bark inoculation assay confirmed the aggressiveness of P. multivora, P. pachypleura, and P. plurivora on Castanea sativa, with a statistically more extensive necrosis than that of P. pseudocryptogea and P. cactorum and slightly less than that caused by P. cinnamomi. These results confirm that the aetiology of ink disease is rapidly evolving in Europe and now includes P. multivora and P. pachypleura, reported here for the first time as chestnut pathogens.
A conspicuous number of species found in this study belong to the ITS clade 6 sensu [44]. Phytophthora species from clade 6 have an aquatic and saprotrophic lifestyle; however, some species can act as opportunistic or aggressive tree pathogens [61,73]. This result is due to the numerous samples collected along riparian systems or wetlands and confirms the wide diversity of clade 6 species found in previous research in water systems of Portugal and Europe [27,37,38,74].
In particular, P. gonapodyides is widespread in all climatic areas. The other clade 6 species (P. asparagi, P. bilorbang, P. chlamydospora, P. inundata, and P. rosacearum) appear rarer and geographically confined [26,27,38]. Finally, the isolation of P. thermophila from four hosts in north, central, and south Portugal is very important and confirms the common presence of this species in Portugal, recently reported along watercourses [38]. Phytophthora thermophila was previously described from Eucalyptus forests and river systems of Australia and South Africa [75,76]. This species has high optimum and maximum temperatures for growth and a relative capacity for interspecific hybridization [75,77,78]. Some unstable hybrids close to P. amnicola and P. thermophila have also been isolated from riparian systems in this study (Bregant and Alves, unpublished). Future studies are necessary to clarify the pathogenicity of P. thermophila and the role of the related hybrids in order to understand the real risk posed by this pathogen to Portuguese and European forests.
Finally, combining the results of this study with the literature review data, a total of 37 species and hybrids are now officially reported in the forest ecosystems of Portugal. The distribution of Phytophthora covers all climatic areas investigated along the altitudinal gradient. Some species are restricted to one altimetric range whereas others such as P. cactorum, P. cinnamomi, and P. gonapodyides are invasive. A survey is currently in progress to clarify the pathogenicity of the new host pathogens’ association and to understand the susceptibility of native plants to the most invasive pathogens detected in this study.
Three species, P. citricola, P. hedraiandra, and P. pachypleura are here reported for the first time in the natural ecosystems of Portugal, whereas for P. thermophila, this is the first report from declining forests in Europe.

5. Conclusions

Overall, the results obtained have contributed to expanding scientific knowledge about the diversity of Phytophthora in Portuguese forest ecosystems. Portugal is a country characterized by extremely varied climatic and vegetation conditions, with a high human impact on land management. The coasts and surrounding areas are strongly related to the Mediterranean Sea and the Atlantic Ocean, while the internal part has continental influences typical of the Iberian Peninsula. To date, Phytophthora research in Portugal has been conducted chiefly in temperate and Mediterranean areas in the central and southern parts of the country, concentrating on cork oak, chestnut, and eucalyptus.
The results obtained in this study revealed the occurrence of Phytophthora-related diseases on many other tree and shrub species, contributing to expanding the knowledge about the impact of Phytophthora species on natural ecosystems. A total of 34 species and 3 hybrids of Phytophthora are now officially reported in Portugal.

Author Contributions

Conceptualization: C.B., E.B., B.T.L. and A.A.; methodology, C.B., E.B., B.T.L. and A.A.; formal analysis, C.B., E.B. and S.H.; investigation, C.B., E.B. and S.H.; resources, A.A. and B.T.L.; writing—original draft preparation, C.B. and E.B.; writing—review and editing, S.H., B.T.L. and A.A.; supervision, B.T.L. and A.A.; funding acquisition, A.A. and B.T.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been financially supported by the project TESAF1DOR-00414 and the Land Environment Resources and Health (L.E.R.H.) doctoral course (University of Padova). Thanks are due to the Portuguese Foundation for Science and Technology (FCT/MCTES) for the financial support to CESAM (UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Overview of Phytophthora disease symptoms observed in coastal ecosystems (a–e), temperate (f–j), and montane forests (k–o) across Portugal: Acacia longifolia (a,d), Pistacia lentiscus (b), Carpobrotus edulis (c,e), Quercus spp. (f–i), Rhododendron ponticum (j), Betula celtiberica (k,l,n), Castanea sativa (m), and Juniperus communis (o). On the left, starting from the top, colony morphology of Phytophthora amnicola, P. asparagi, P. bilorbang, P. cactorum, P. chlamydospora, P. cinnamomi, P. citricola, P. gonapodyides, P. hedraiandra, P. inundata, P. lacustris, P. multivora, P. pachypleura, P. plurivora, P. pseudocryptogea, P. pseudosyringae, P. rosacearum, P. syringae, and P. thermophila after 7 days of growth at 20 °C on CA in the dark.
Figure 1. Overview of Phytophthora disease symptoms observed in coastal ecosystems (a–e), temperate (f–j), and montane forests (k–o) across Portugal: Acacia longifolia (a,d), Pistacia lentiscus (b), Carpobrotus edulis (c,e), Quercus spp. (f–i), Rhododendron ponticum (j), Betula celtiberica (k,l,n), Castanea sativa (m), and Juniperus communis (o). On the left, starting from the top, colony morphology of Phytophthora amnicola, P. asparagi, P. bilorbang, P. cactorum, P. chlamydospora, P. cinnamomi, P. citricola, P. gonapodyides, P. hedraiandra, P. inundata, P. lacustris, P. multivora, P. pachypleura, P. plurivora, P. pseudocryptogea, P. pseudosyringae, P. rosacearum, P. syringae, and P. thermophila after 7 days of growth at 20 °C on CA in the dark.
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Figure 2. Isolation frequency and distribution of the most common Phytophthora species isolated in this study.
Figure 2. Isolation frequency and distribution of the most common Phytophthora species isolated in this study.
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Figure 3. Distribution of Phytophthora species in Portugal. Red dots are occurrences for this study, black dots are from literature data, and blue dots in the background are for sampling areas.
Figure 3. Distribution of Phytophthora species in Portugal. Red dots are occurrences for this study, black dots are from literature data, and blue dots in the background are for sampling areas.
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Figure 4. Phytophthora diversity along the elevation gradient in Portugal. Data from the study and literature review.
Figure 4. Phytophthora diversity along the elevation gradient in Portugal. Data from the study and literature review.
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Figure 5. Maximum likelihood tree obtained from the internal transcribed spacer (ITS) sequences of Phytophthora species representative of the 12 clades. The tree was rooted to Halophytophthora avicenniae and Nothophytophthora caduca. Data are based on the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap support values in percentage (1000 replicates) are given at the nodes. Ex-type cultures are in bold, and isolates obtained in this study are in red.
Figure 5. Maximum likelihood tree obtained from the internal transcribed spacer (ITS) sequences of Phytophthora species representative of the 12 clades. The tree was rooted to Halophytophthora avicenniae and Nothophytophthora caduca. Data are based on the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap support values in percentage (1000 replicates) are given at the nodes. Ex-type cultures are in bold, and isolates obtained in this study are in red.
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Figure 6. Mean lesion length (±standard deviation) and symptoms on 2-year-old seedlings of Castanea sativa detected after 1 month from the inoculation with Phytophthora spp. Values with the same letter do not differ significantly at p = 0.05, according to the LSD multiple range test.
Figure 6. Mean lesion length (±standard deviation) and symptoms on 2-year-old seedlings of Castanea sativa detected after 1 month from the inoculation with Phytophthora spp. Values with the same letter do not differ significantly at p = 0.05, according to the LSD multiple range test.
Pathogens 14 00103 g006
Table 1. Details of study sites and the samples collected.
Table 1. Details of study sites and the samples collected.
Study SiteClimate ZoneElevation (m a.s.l.)CoordinatesNumber of Samples
LatitudeLongitudeRhizosphereNecrotic Tissues
1A4037.118918−8.567076Pl(5), Pp(2), Qc(2), Eg(1)-
2A5037.162606−8.251300Ces(3), Ph(1)-
3A441.071134−8.657429Ce(1)-
4A040.598795−8.755363Al(6), Ce(2)Ce(2)
5B1240.724190−8.570960Psp(6)-
6B1640.718277−8.569993Qr(3), Ssp(2)Qr(1)
7B640.704444−8.607099Fa(2), Al(1)-
8B540.695340−8.633084Eg(3), Fa(1)-
9B1840.551664−8.575524Eg(3)-
10B43741.554036−8.375251Qr(7), Qs(2)-
11B40038.783018−9.416210Cs(5), Ap(4), Qc(3), Rp(2), Eg(1)Rp(21), Vm(2), Vt(4)
12B30038.781366−9.386651Cs(7), Qr(6), Qs(2), Fm(2)-
13C105640.490891−7.520354Bc(4)-
14C50040.612330−7.519114Qp(1)-
15C109240.442888−7.511881Fe(2), Bc(1), Ps(1)-
16C110740.541458−7.454340Cs(2), Qp(1)-
17C130040.299541−7.537996Ld(2), Sa(1)-
18C145040.328321−7.587890Bc(2), Ld(1)-
19C190040.332200−7.611709Jc(2)Jc(2)
20C90040.327858−7.677459Cs(1)-
21C68040.383422−7.700445Cs(2)-
In brackets the number of plants collected: Acacia longifolia (Al), Acer pseudoplatanus (Ap), Betula celtiberica (Bc), Castanea sativa (Cs), Carpobrotus edulis (Ce), Ceratonia siliqua (Ces), Eucalyptus globulus (Eg), Ficus macrophylla (Fm), Fraxinus angustifolia (Fa), Fraxinus excelsior (Fe), Juniperus communis (Jc), Larix decidua (Ld), Pinus halepensis (Ph), Pistacia lentiscus (Pl), Pinus pinea (Pp), Pinus sylvestris (Ps), Populus sp. (Psp), Quercus coccifera (Qc), Quercus pyrenaica (Qp), Quercus robur (Qr), Quercus suber (Qs), Rhododendron ponticum (Rp), Sorbus aucuparia (Sa), Salix sp. (Ssp), Vinca major (Vm), Viburnum tinus (Vt).
Table 2. Details of isolates included in the phylogenetic analyses. Ex-type cultures are given in bold and newly generated sequences are indicated in italics.
Table 2. Details of isolates included in the phylogenetic analyses. Ex-type cultures are given in bold and newly generated sequences are indicated in italics.
SpeciesITS CladeCollection No.HostITS GenBank Accession No.
Phytophthora cactorum1CBS231.30Syringa vulgarisMG783385
P. cactorum1CBP168Castanea sativaPQ571399
P. hedraiandra1CBS111725Viburnum sp.MG865504
P. hedraiandra1CBP188Betula celtibericaPQ571404
P. citricola2CBS221.88Citrus sinensisMG865475
P. citricola2CBP150Acer pseudoplatanusPQ571402
P. multivora2CBS124094Eucalyptus marginataFJ237521
P. multivora2CBP154C. sativaPQ571407
P. pachypleura2IMI502404Aucuba japonicaKC855330
P. pachypleura2CBP158C. sativaPQ571408
P. plurivora2CBS124093Fagus sylvaticaMG865568
P. plurivora2CBP164C. sativaPQ571409
P. ilicis3P3939Ilex aquifoliumMG865511
P. pseudosyringae3CBS111772Quercus roburMG865574
P. pseudosyringae3CBP195B. celtibericaPQ571411
P. alticola4CBS141718Eucalyptus grandisKX247599
P. palmivora4CBS305.62Areca catechuLC595875
P. cocois5P19948Cocos nuciferaMG865478
P. heveae5CBS296.29Hevea brasiliensisMG865505
P. amnicola6CBS131652waterJQ029956
P. amnicola6CBP134Rhododendron ponticumPQ571396
P. asparagi6CBS132095Lomandra sonderiEU301168
P. asparagi6CBP179Pistacia lentiscusPQ571397
P. bilorbang6CBS161653Rubus anglicandicansJQ256377
P. bilorbang6CBP140R. ponticumPQ571398
P. chlamydospora6P6133Prunus sp.MG865471
P. chlamydospora6CBP148A. pseudoplatanusPQ571400
P. gonapodyides6P7050Alnus sp.MG865501
P. gonapodyides6CBP186B. celtibericaPQ571403
P. inundata6CBS216.85Salix matsudanaMG865516
P. inundata6CBP78Eucalyptus globulusPQ571405
P. lacustris6IMI389725S. matsundanaJQ626605
P. lacustris6CBP162Populus sp.PQ571406
P. rosacearum6CBS124696Malus sp.EU925376
P. rosacearum6CBP116Acacia longifoliaPQ571412
P. thermophila6CBS127954E. marginataEU301155
P. thermophila6CBP90Q. roburPQ571414
P. cinnamomi7CBS144.22Cinnamomum burmanniiMG865473
P. cinnamomi7CBP185C. sativaPQ571401
P. niederhauserii7CBS149824Hedera helixMG865552
P. pseudocryptogea8CBS139749Isopogon buxifoliusKP288376
P. pseudocryptogea8CBP166C. sativaPQ571410
P. syringae8CBS110161S. vulgarisMG865590
P. syringae8CBP220Q. roburPQ571413
P. parsiana9IMI395329Ficus caricaMG865562
P. polonica9CBS119650Alnus glutinosaAB511828
P. boehmeriae10CBS 291.29Boehmeria niveaMG783382
P. kernoviae10IMI393170F. sylvaticaAY940661
P. lilii11CBS135746Lilium longiflorumMG865523
P. castanetorum12CBS142299C. sativaMF036182
P. quercina12CBS784.95Q. roburMG865578
Halophytophthora avicennae-CBS188.85Avicennia marinaHQ643147
H. avicenniae-CBP98E. globulusPQ571415
Nothophytophthora caduca-CBS142350waterKY788401
N. caduca-CBP163Vinca majorPQ571416
Table 3. Symptoms observed on each plant host and disease incidence/mortality rate estimated.
Table 3. Symptoms observed on each plant host and disease incidence/mortality rate estimated.
Plant SpeciesSymptoms ObservedDisease Incidence (%)Mortality Rate (%)
Acacia longifoliaRoot rot, bleeding cankers, canopy decline, sudden death60–8315–28
Acer pseudoplatanusRoot rot, exudates, bleeding cankers, chlorosis, stunted growthndnd
Betula celtibericaRoot rot, bleeding cankers, canopy decline, sudden death9555
Carpobrotus edulisLeaf necrosis, wiltingndnd
Castanea sativaRoot rot, bleeding cankers, chlorosis, canopy decline, sudden death60–10017–26
Ceratonia siliquaRoot rot, chlorosis, canopy declinendnd
Eucalyptus globulusRoot rot, bleeding cankers, chlorosis, canopy decline, sudden death8020
Ficus macrophyllaRoot rot, bleeding cankers, chlorosis, canopy decline, sudden deathndnd
Fraxinus angustifoliaRoot and collar rot, canopy declinendnd
Fraxinus excelsiorRoot rot, canopy declinendnd
Juniperus communisShoot blight, sudden deathndnd
Larix deciduaRoot rot, chlorosis, canopy decline, sudden death5010
Pistacia lentiscusRoot rot, chlorosis, canopy decline, sudden death7631
Pinus halepensisRoot rot, sudden deathndnd
Pinus pineaRoot rot, sudden deathndnd
Pinus sylvestrisRoot rot, canopy declinendnd
Populus sp.Root rot, chlorosis, canopy decline, sudden death7012
Quercus cocciferaRoot rot, canopy decline6017
Quercus pyrenaicaRoot rot, canopy declinendnd
Quercus roburRoot rot, bleeding cankers, chlorosis, canopy decline, sudden death6711
Quercus suberRoot rot, bleeding cankers, chlorosis, canopy decline, sudden deathndnd
Rhododendron ponticumLeaf necrosis, wilting, shoot blight, root rot, sudden death10016
Salix sp.Root rot, chlorosis, canopy decline, sudden death7522
Sorbus aucupariaRoot rot, canopy declinendnd
Vinca majorLeaf necrosis, wiltingndnd
Viburnum tinusLeaf necrosis, wiltingndnd
nd = not determined.
Table 4. Number of isolates obtained from the monitored plants in the investigated sites.
Table 4. Number of isolates obtained from the monitored plants in the investigated sites.
SpeciesITS CladePlant Species *Total Number of IsolatesSites
Halophytophthora
avicenniae		-Eg(2)28
Nothophytophthora
caduca		-Vm(2)211
Phytophthora amnicola6Rp(5), Qr(1)611, 12
P. asparagi6Pl(3)31
P. bilorbang6Rp(4)411
P. cactorum1Qr(5), Qs(1) Cs(1), Jc(1)810, 12, 19
P. chlamydospora6Qs(1), Ap(1)210,11
P. cinnamomi7Eg(7), Qr(6), Qc(5), Bc(4), Qs(2), Al(2), Cs(3), Ce(3), Rp(1), Ces(1), Pl (1), Pp(1)361–4, 6, 10–13, 16, 20, 21
P. citricola2Ap(3)311
P. gonapodyides6Bc(2), Al(1), Qr(1)44, 10, 18
P. hedraiandra1Rp(3), Vm(2), Bc(1)611, 18
P. inundata6Eg(2), Qr(2)46, 8
P. lacustris6Ssp(2), Psp(4), Qr(2)85, 6, 10
P. multivora2Rp(13), Cs(3), Fm(2), Fa(1), Al(1)204, 7, 11, 12
P. pachypleura2Cs(2)212
P. plurivora2Qr(3), Cs(2), Fe(1), Bc(1), Qp(1), Eg(1)910, 12, 14–16
P. pseudocryptogea8Al(2), Pl(3), Ph(1), Cs(2)71, 2, 4, 11, 12
P. pseudosyringae3Jc(2), Bc(1), Sa(1), Ld(1)513, 17, 19
P. rosacearum6Al(3)34, 7
P. syringae8Qr(1)16
P. thermophila6Pp(2), Rp(1), Qr(1), Qs(1)51, 10, 11
* In brackets, the number of Phytophthora isolates on: Acacia longifolia (Al), Acer pseudoplatanus (Ap), Betula celtiberica (Bc), Castanea sativa (Cs), Carpobrotus edulis (Ce), Ceratonia siliqua (Ces), Eucalyptus globulus (Eg), Ficus macrophylla (Fm), Fraxinus angustifolia (Fa), Fraxinus excelsior (Fe), Juniperus communis (Jc), Larix decidua (Ld), Pinus halepensis (Ph), Pistacia lentiscus (Pl), Pinus pinea (Pp), Pinus sylvestris (Ps), Populus sp. (Psp), Quercus coccifera (Qc), Quercus pyrenaica (Qp), Quercus robur (Qr), Quercus suber (Qs), Rhododendron ponticum (Rp), Salix sp. (Ssp), Sorbus aucuparia (Sa), Vinca major (Vm), and Viburnum tinus (Vt).
Table 5. Phytophthora species reported in natural and semi-natural ecosystems in Portugal.
Table 5. Phytophthora species reported in natural and semi-natural ecosystems in Portugal.
SpeciesHostReferences
P. alticolaEucalyptus globulus[29]
P. amnicolaAlnus glutinosa, Castanea sativa, Fagus sylvatica, Rhododendron ponticum, Quercus robur[27,38]; this study
P. asparagiA. glutinosa, Pistacia lentiscus[27]; this study
P. bilorbangR. ponticum, water[27,38]; this study
P. cactorumA. glutinosa, Castanea sativa, Quercus robur, Q. suber, Juniperus communis[27]; this study
P. cambivoraAcer pseudoplatanus, Betula celtiberica, C. sativa, F. sylvatica, Salix caprea, Quercus ilex, Quercus pyrenaica, water[38,47,48,49]
P. castanetorumC. sativa[28,38]
P. chlamydosporaA. pseudoplatanus, A. glutinosa, C. sativa, F. sylvatica, Q. suber[27,38]; this study
P. cinnamomiAbies alba, Acacia longifolia, A. glutinosa, Arbutus unedo, B. celtiberica, Calluna vulgaris, Carpobrotus edulis, C. sativa, Ceratonia siliqua, Cistus crispus, C. ladanifer, C. populifolius, C. salvifolius, E. globulus, F. sylvatica, Genista triacanthos, Phyllirea latifolia, Pinus pinaster, P. pinea, P. lentiscus, Quercus coccifera, Q. ilex, Q. pyrenaica, Q. robur, Quercus rubra, Q. suber, R. ponticum, Ulex spp.[27,29,30,31,32,34,38,50,51]; this study
P. citricolaA. pseudoplatanusThis study
P. citrophthorawater[38,52]
P. condilinawater[26]
P. gonapodyidesA. longifolia, A. glutinosa, B. celtiberica, Q. robur, water[26,27,38]; this study
P. hedraiandraB. celtiberica, R. ponticum, Vinca majorThis study
P. hibernalisE. globulus[30]
P. honggalleglyanawater[38]
P. inundataE. globulus, Q. robur, water[26,38]; this study
P. kelmaniiwater[38]
P. lacustrisA. glutinosa, Populus sp., Q. robur, Salix sp., water[27,37,38]; this study
P. multivoraAcacia dealbata, A. longifolia, A. glutinosa, A. pseudoplatanus, C. sativa, E. globulus, Fraxinus angustifolia, Q. rubra, R. ponticum[27,30,38]; this study
P. niederhauseriiE. globulus[30]
P. pachypleuraC. sativaThis study
P. plurivoraA. glutinosa, A. pseudoplatanus, B. celtiberica, C. sativa, F. sylvatica, Fraxinus excelsior, Prunus lusitanica, Q. pyrenaica, Q. robur[27,38]; this study
P. polonicaA. glutinosa[27]
P. pseudocitrophthorawater[38,52]
P. pseudocryptogeaA. dealbata, A. longifolia, A. glutinosa, C. sativa, P. lentiscus, Pinus halepensis, P. pinea, Q. suber, water[26,27,52]; this study
P. pseudosyringaeA. pseudoplatanus, B. celtiberica, J. communis, Larix decidua, Q. pyrenaica, Prunus avium, Sorbus aucuparia[38,49]; this study
P. psychrophilaQ. ilex[38,49]
P. quercinaA. unedo, C. sativa, Q. ilex, Q. pyrenaica, Q. robur[28,38]
P. ramorumViburnum sp.; water[38,53]
P. rosacearumA. longifolia, A. glutinosa[27]; this study
P. syringaeA. unedo; Q. robur[38]; this study
P. thermophilaP. pinea, Q. robur, Q. suber, R. ponticum; water[38]; this study
P. uliginosaQ. suber[38]
P. × alniA. glutinosa[37,38]
P. × lusitanicawater[38,52]
P. × stagnumwater[38]
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Bregant, C.; Batista, E.; Hilário, S.; Linaldeddu, B.T.; Alves, A. Diversity and Distribution of Phytophthora Species Along an Elevation Gradient in Natural and Semi-Natural Forest Ecosystems in Portugal. Pathogens 2025, 14, 103. https://doi.org/10.3390/pathogens14010103

AMA Style

Bregant C, Batista E, Hilário S, Linaldeddu BT, Alves A. Diversity and Distribution of Phytophthora Species Along an Elevation Gradient in Natural and Semi-Natural Forest Ecosystems in Portugal. Pathogens. 2025; 14(1):103. https://doi.org/10.3390/pathogens14010103

Chicago/Turabian Style

Bregant, Carlo, Eduardo Batista, Sandra Hilário, Benedetto Teodoro Linaldeddu, and Artur Alves. 2025. "Diversity and Distribution of Phytophthora Species Along an Elevation Gradient in Natural and Semi-Natural Forest Ecosystems in Portugal" Pathogens 14, no. 1: 103. https://doi.org/10.3390/pathogens14010103

APA Style

Bregant, C., Batista, E., Hilário, S., Linaldeddu, B. T., & Alves, A. (2025). Diversity and Distribution of Phytophthora Species Along an Elevation Gradient in Natural and Semi-Natural Forest Ecosystems in Portugal. Pathogens, 14(1), 103. https://doi.org/10.3390/pathogens14010103

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