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Article

Multiple Botryosphaeriaceae and Phytophthora Species Involved in the Etiology of Holm Oak (Quercus ilex L.) Decline in Southern Italy

by
Carlo Bregant
1,
Francesca Carloni
2,
Gaia Borsetto
1,
Angelo G. Delle Donne
3,
Benedetto T. Linaldeddu
1 and
Sergio Murolo
2,*
1
Dipartimento Territorio e Sistemi Agro-Forestali, Università degli Studi di Padova, Viale dell’Università, 16, 35020 Legnaro, Italy
2
Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, Via Brecce Bianche, 60131 Ancona, Italy
3
Regione Puglia—Sez. Osservatorio Fitosanitario—Sede di Lecce viale Aldo Moro, 73100 Lecce, Italy
*
Author to whom correspondence should be addressed.
Forests 2025, 16(7), 1052; https://doi.org/10.3390/f16071052
Submission received: 6 May 2025 / Revised: 20 June 2025 / Accepted: 22 June 2025 / Published: 24 June 2025
(This article belongs to the Section Forest Health)
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Abstract

In recent years, severe decline and mortality events have been observed in holm oak (Quercus ilex L.) ecosystems in different Italian regions, including Puglia (southern Italy). Given the landscape and ecological relevance of holm oak forests in Apulia, a study was conducted to identify the causal agents related to this complex disease syndrome. The surveys, conducted in winter 2024 in three different woodlands, revealed the widespread occurrence of mature holm oak trees showing sudden death, crown thinning, shoot and branch dieback, sunken cankers, and root rot symptoms. Isolations performed from symptomatic samples collected from both stem and small roots yielded fungal and fungal-like colonies representing two distinct families: Botryosphaeriaceae and Peronosporaceae. Analysis of morphological and DNA sequence data allowed us to identify six distinct species, including Diplodia corticola and D. quercivora (Botryosphaeriaceae), Phytophthora cinnamomi, P. multivora, P. psychrophila, and P. asparagi (Peronosporaceae). For P. asparagi and P. psychrophila, isolated for the first time from declining holm oak trees in Italy, Koch’s postulates were satisfied by inoculating 1-year-old seedlings at the collar in controlled conditions. Thirty days after inoculation, all plants showed the same symptoms observed in the field. Overall, the data obtained highlights the co-occurrence of multiple Botryosphaeriaceae and Phytophthora species on declining holm oak trees and the discovery of a new haplotype of Diplodia quercivora.

1. Introduction

In Italy, oak ecosystems cover a surface of approximately eleven million hectares and represent an important component of landscapes, providing habitat for a myriad of wildlife species and an economic resource of natural and renewable raw materials such as cork [1,2]. Among the ten oak species reported in Italy, two evergreen species, holm oak (Quercus ilex L., Fagaceae Dumort) and cork oak (Quercus suber L.), are the most widespread in southern regions [3,4]. Holm oak is a versatile species common in both forest and urban areas, where it is used for ornamental purposes [5,6]. In Apulia, holm oak ecosystems are widely distributed throughout the regional territory, with larger woodlands in the Gargano plateau and small-sized relict formations in the other areas, including Salento [7]. Over the last two decades, the holm oak formations of Puglia have been severely impacted by decline phenomena, with symptomatic trees showing extensive branch dieback and chiefly sudden death symptoms. Frisullo et al. [8] have established the invasive soil-borne pathogen Phytophthora cinnamomi Rands (Peronosporaceae de Bary) as the causal agent of this decline, while Del Grosso et al. [9] have isolated from symptomatic trees some pathogens belonging to the Botryosphaeriaceae Theiss. & H. Syd., including the primary oak pathogen Diplodia corticola A.J.L. Phillips, A. Alves & J. Luque.
Recent studies have demonstrated that on different woody hosts, including beech, common ash, olive, paulownia, and pomegranate, Botryosphaeriaceae and Phytophthora species can often act synergistically by simultaneously attacking the aerial plant organs and root system and that multiple pathogenic species can be isolated from one site or a single symptomatic tree [10,11,12,13,14,15]. Simultaneous attacks by multiple Botryosphaeriaceae and Phytophthora species, including D. corticola and P. cinnamomi, have also previously been reported on holm oak in Italy by Linaldeddu et al. [16]. Despite Botryosphaeriaceae and Phytophthora species having different pathogenic behavior and being very distant from an evolutionary point of view, they share some key ecological traits, such as high invasiveness, adaptation to new hosts (host jump), and adaptation to ongoing climate change [10,17,18].
Therefore, considering the growing number of pathosystems showing the co-presence of simultaneous attack by multiple pathogenic Botryosphaeriaceae and Phytophthora species and the occurrence of a complex symptomatology in the declining holm oak trees of Salento, the main aim of this work was to ascertain a potential joint attack by multiple species belonging to these two groups of pathogens on holm oak.

2. Materials and Methods

2.1. Field Survey and Sampling Procedure

In January 2024, phytosanitary monitoring was conducted at three different sites in Salento (Lecce province), southern Puglia (Italy) (Table 1). These areas were originally part of much larger forested landscapes but have undergone a drastic reduction over the last centuries. Today, they are considered residual lowland forests, surrounded by intensively cultivated crops, particularly olive orchards.
According to Copernicus Land Monitoring Service and CORINE Land Cover 2018 Level 3 (https://land.copernicus.eu/en/products/corine-land-cover (accessed on 15 June 2025), we found that Site A1 Mazza is classified as CLC 2.4.2 (Complex cultivation patterns), Site A2 Bonata as CLC 2.4.3 (Land principally occupied by agriculture, with significant areas of natural vegetation), and finally Site A3 Gabrieli as 2.2.3 (Olive orchards). The dataset has a Minimum Mapping Unit (MMU) of 25 hectares (ha) for this reason; Q. ilex forest was not detected by this system.
The composition of these study areas has been shaped by the geological substrate, the region’s morphological features, and human activities. The predominant species in the surveyed areas was holm oak, accompanied by other key species of Mediterranean flora, including deciduous oak, Arbutus unedo L., Pistacia lentiscus L., and Phyllirea spp.
All three sites were characterized by a uniform distribution of symptomatic trees showing exudates, epicormic shoots, sunken cankers, and chiefly sudden death symptoms. Given the uniformity of the symptoms detected during the phytosanitary monitoring, a total of eleven still-alive symptomatic holm oak trees were randomly selected for sampling. For each selected tree, after removing the litter layer surface, the root system was assessed, and approximately 300 g of rhizosphere and fine roots were collected around the collar, stored in plastic bags, and labelled. The used instruments were carefully decontaminated by spraying a 90% ethanol solution. The same trees were further examined for the occurrence of bleeding cankers, and by removing the outer bark with a sterile scalpel, a sample of inner bark (5 × 5 cm) was collected from each tree for diagnostic analysis. From ten of the selected trees showing extensive sunken cankers, typical of Botryosphaeriaceae infections, a further bark sample (5 × 5 cm) was collected. All samples were transferred to the laboratory and analyzed within 48 h to isolate the potential causative agents. Phytosanitary parameters such as the disease incidence and mortality rate were estimated in each site along a 25 m-long linear transect and expressed as the number of symptomatic trees out of the total number of trees (DI = n/N × 100) and the number of dead trees out of the total number of trees (M = d/N × 100), respectively [16].

2.2. Pathogen Isolations

For Phytophthora isolation, fine root and rhizosphere samples were placed in glass beakers with approximately 1 L of distilled water. A zoospore trap assay performed with 5–7 young holm oak and cork oak leaves was applied according to Linderman and Zeitoun [19]. After 3–5 days at 20 °C, oak leaves showing initial necrotic lesions were transferred to Petri dishes containing the selective PDA+ media [14]. Necrotic inner bark tissues taken from bleeding cankers were cleaned in sterile water, put to dry in aseptic conditions, cut into small chips (approximately 0.3–0.5 cm2), and placed in Petri dishes containing PDA+.
Samples taken from sunken cankers were initially disinfected with ethanol (70%) for 30 s, and then the outer bark was removed with a sterile scalpel. Longitudinal and cross views were made to observe internal disease symptoms. Isolations were performed from ten small fragments (5 × 5 mm) cut aseptically from the margin of the necrotic lesions. All fragments were placed onto 90 mm Petri dishes containing potato dextrose agar (PDA, Oxoid Ltd., Basingstoke, UK).
After 4–6 days of incubation at 20 °C in the dark, fungal and Phytophthora colonies were sub-cultured onto PDA and carrot agar (CA), respectively, and incubated at 20 °C until the differentiation of reproductive structures was observed.
To enhance sporulation, fungal isolates were cultured on autoclaved holm oak twigs, placed on half-strength PDA, and kept on the laboratory bench, where they received indirect sunlight. Whereas the production of sporangia was stimulated by placing CA plugs (9 mm diameter) of each Phytophthora species into 60 mm Petri dishes containing unsterile pond water and holm oak fine roots [15,16].

2.3. Identification of Pathogens

Morphological identification of the colonies obtained in pure culture was performed based on colony appearance on PDA (fungi) and CA (oomycetes) after 7 days at 20 °C and biometric data of conidia and sporangia observed in water under a light microscope (Motic BA410E microscope) at × 400 magnification.
The identity of all isolates was confirmed by analysis of DNA sequence data. Total DNA, extracted according to protocols reported by Linaldeddu et al. [15], was quantified by Biophotometer (Eppendorf) and amplified with primer pair ITS1/ITS4 [20] using the thermal parameters reported in Linaldeddu et al. [15]. In addition, for the Botryosphaeriaceae species, the primer pairs EF446f/EF1035r were used to amplify and sequence a portion of the translation elongation factor 1 alpha gene (tef1-α) [21].
For Diplodia corticola and Diplodia quercivora Linaldeddu & A.J.L. Phillips, the primers Diplodia_MAT1_391F and Diplodia_MAT1_1325R were used to amplify the MAT1-1-1 gene, whereas the primers Diplodia_MAT2_113F and Diplodia_MAT2_1187R were used for the MAT1-2-1 gene [22]. For PCR conditions, the values reported in Lopes et al. [22] were used.
The amplicons were purified and sequenced with the same primers used for PCR at the Genewiz company (Germany). The raw sequence data were edited with BioEdit software v 7.0.9 and compared using BLASTN analysis in the NCBI database. The species was assigned when the nucleotide identity was more than 99.9% compared with sequences of ex-type culture deposited in GenBank. ITS, tef1-α, and MAT genes sequences of representative isolates of Diplodia corticola (isolate CB1001, accession numbers: PV299150, PV348962, PV348967; isolate CB1004: PV299151, PV348963, PV348968) D. quercivora (isolate CB1006, accession numbers: PV299152, PV348960, PV348965; isolate CB1007: PV299153, PV348961, PV348966), as well as ITS sequences of Phytophthora asparagi Saude & Hausbeck (isolate CB1015: PV299155), P. cinnamomi isolate CB1021 (PV299156), P. multivora P.M. Scott & T. Jung isolate CB1023 (PV299157), and P. psychrophila T. Jung & E.M. Hansen (isolate CB1020: PV299158) were deposited at GenBank.

2.4. Phylogenetic Analysis

Individual phylogenies based on MAT genes and tef1-α sequences for Diplodia corticola and D. quercivora species were performed. Sequences were aligned with ClustalX v. 1.83 [23], using the parameters reported by Lopes et al. [22]. 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 [24]. Maximum likelihood (ML) analysis was performed with a neighbor-joining (NJ) starting tree generated by the software. A bootstrap analysis (1000 replicates) was used to estimate the robustness of nodes.

2.5. Pathogenicity Tests

For two Phytophthora species (P. asparagi and P. psychrophila) first identified and not yet isolated in Italy from holm oak, we set up artificial inoculations to satisfy Koch’s postulates.
During April 2024, seven 1-year-old holm oak seedlings were inoculated at the stem with a representative isolate of each species. A further seven seedlings were inoculated with P. multivora for comparison. Inoculation was performed as reported in Linaldeddu et al. [16], and seedlings were maintained under control conditions at approximately 22 °C for 30 days at the UNIVPM screenhouse (Ancona, Italy). Seven controls were inoculated with a sterile PDA plug. At the end of the experimental period, inner bark necrotic lesions and external (wilting and exudates) disease symptoms were assessed. The size of the necrotic lesion was estimated by peeling off the outer bark. Finally, pathogen re-isolation was conducted by extracting five pieces of symptomatic inner bark tissue and placing them onto PDA+ medium. Pathogenicity assay data were checked for normality, then subjected to analysis of variance (ANOVA). Significant differences among mean values were determined using Fisher’s least significant differences multiple range test (α = 0.05) after one-way ANOVA using the software package XLSTAT® (Addinsoft Pearson Edition 2022, Paris, France).

3. Results

3.1. Symptomatology and Aetiology

Field surveys conducted in three holm oak woodlands allowed us to ascertain a high incidence of symptomatic holm oak trees. The incidence of symptomatic holm oak trees was uniform in all study sites; basically, two different main types of symptoms were detected on declining holm oak trees. Specifically, a group of trees showed leaf yellowing, reduced leaf size, branch dieback, and epicormic shoots (up to 30%), whereas most trees showed sudden death and root rot symptoms (more than 50%). In both groups of trees, aerial bleeding cankers with emission of black exudates induced by Phytophthora spp. and sunken cankers caused by Botryosphaeriaceae were observed (Figure 1).
From the samples collected in the three holm oak stands, eight Phytophthora isolates were successfully obtained from rhizosphere/fine roots, as well as two directly from bleeding cankers. Morphological identification, supported by molecular analysis of the ITS region, revealed the presence of four distinct Phytophthora species: P. cinnamomi (5 isolates), P. multivora (2), P. psychrophila (2), and P. asparagi (1) (Figure 1). In particular, in the site A1, P. cinnamomi was detected from both rhizosphere and tissue samples, while P. multivora and P. psychrophila were only from the rhizosphere. In the site A2, P. multivora was detected in the rhizosphere, whereas in the site A3, P. asparagi, P. cinnamomi, and P. psychrophila were isolated from the rhizosphere of symptomatic trees.
In addition to Phytophthora species, from the inner bark samples taken from the aerial sunken cankers of the same trees, 11 fungal colonies belonging to the Botryosphaeriaceae family were isolated. Based on morphological features and DNA sequence data (ITS and tef1-α), two distinct species were identified, namely Diplodia corticola (9 isolates) and D. quercivora (2). Diplodia corticola has been isolated in all sites, in particular from two trees in site A1 and one tree in site A3, together with P. cinnamomi, whereas from one tree in site A2, together with P. multivora (Table 2).
For each species, BLAST searches against GenBank showed 100% identity to reference ITS sequences of representative strains, including those of ex-type cultures. In the individual phylogenies based on tef1-α sequences, D. corticola isolates clustered within the Lineage A sensu [25,26] (Figure 2). Whereas the phylogeny of tef1-α sequences placed the two D. quercivora strains obtained in this study within a new lineage (haplotype), well supported and separated by 6 bp from the ex-type culture (Figure 2). Only isolates of the idiomorph MAT1-1 were detected for D. corticola, while for D. quercivora, only isolates of the MAT1-2 idiomorph were detected. Phylogenetic analyses based on MAT1-1 and MAT1-2 genes discriminate both species with high bootstrap support values (Figure 3).

3.2. Pathogenicity Test

The three Phytophthora species showed a different degree of virulence on holm oak. The size of necrotic lesions ranges from 8.4 ± 2 mm for P. psychrophila to 18 ± 10 mm for P. multivora. Necrosis spread up and down the inner bark from the inoculation site. All Phytophthora species were successfully re-isolated from the margin of necrotic inner bark lesions of all seedlings, thus fulfilling Koch’s postulates (Figure 4). On control seedlings inoculated with sterile PDA plugs, the wound had healed without any disease symptoms.

4. Discussion

Overall, the data obtained in this study highlight that the severe holm oak decline observed in Salento (southern Apulia, Italy) is due to a simultaneous attack of multiple Botryosphaeriaceae and Phytophthora species capable of infecting both the canopy and the root system. In particular, the co-presence on the same site and tree of Diplodia corticola and Phytophthora cinnamomi attacks confirms what was reported in previous studies on holm oak decline in Italy [16]. These two aggressive pathogens were directly involved in the most severe form of decline observed in the monitored sites, characterized by the sudden death of mature oak trees.
Phytophthora cinnamomi was isolated from declining holm oak in Puglia by Frisullo et al. [8] and proved to be highly aggressive on seedlings of some evergreen Mediterranean oak species, including holm oak, cork oak (Q. suber), and kermes oak (Q. coccifera). Holm oak is confirmed to be extremely susceptible to P. cinnamomi as well, as reported in several studies conducted in the Mediterranean basin [27,28,29,30,31,32,33,34]. In addition to P. cinnamomi, in this study, three other Phytophthora species, namely P. asparagi, P. multivora, and P. psychrophila, were isolated from symptomatic holm oak. When artificially inoculated on holm oak, these species were able to cause both non-specific symptoms, such as chlorosis and wilting, as well as specific symptoms, including inner bark necrosis (Figure 4).
The detection of P. multivora, P. asparagi, and P. psychrophila together with P. cinnamomi in Puglia is of particular concern from an ecological point of view. This pool of Phytophthora species is characterized by a different host range, optimal temperature for growth, ability to differentiate resistance structures, and persistent and caducous sporangia capable of infecting both roots and stems [35,36,37]. Phytophthora multivora is emerging as an aggressive and cosmopolitan species on diverse woody hosts in natural ecosystems, timber plantations, and productive orchards [12,36,38,39,40]. In our investigation, P. multivora was isolated in the same site with P. cinnamomi, highlighting the ability of these two pathogens to coexist in the same environment. Phytophthora asparagi, here reported for the first time on holm oak worldwide, has recently been reported causing root rots of different species of Mediterranean maquis in Italy and Portugal [37,41], suggesting the polyphagous behavior of this species and its ability to survive in Mediterranean environments. The discovery of Phytophthora psychrophila in Puglia confirms the results of a previous study conducted in Mediterranean regions where this pathogen was found to be a species directly associated with holm oak decline [42]. Pathogenicity tests in the present study demonstrated that all Phytophthora tested were able to colonize and necrotize the inner bark of stems on holm oak.
The declining holm oak trees monitored were also positive for members of Botryosphaeriaceae, a large family of plant pathogens [17,43,44]. In particular, Diplodia corticola was the most frequently isolated species, and our field survey clearly revealed that it is the major cause of stem and branch canker on holm oak. The high isolation frequency of D. corticola obtained in this study agrees with earlier studies conducted by Del Grosso et al. (2024) [9] in Apulia. Diplodia corticola is a primary pathogen able to colonize bark and wood tissues, causing extensive necrotic lesions that gradually determine a loss of vitality and eventually the death of the plants [26,45,46,47]. Luque et al. (2000) [48] demonstrated that the virulence of D. corticola is similar in water-stressed and non-stressed cork oak seedlings. In the Mediterranean region, two haplotypes of D. corticola, named A and B, with different geographic distributions were known [25]. The presence of two haplotypes was supported by nine fixed differences in the sequences of the tef1-α region. These differences were confirmed by the results of this study, with all isolates belonging to the lineage A. Diplodia corticola is a heterothallic (self-sterile) species [22]. Both idiomorphs are known to be present in Italy (Bregant and Linaldeddu, unpublished), but in the investigated sites, only isolates belonging to the MAT1-1-1 mating types were obtained.
Diplodia quercivora was originally isolated from Algerian oak (Quercus canariensis Willd.) in Tunisia [25] but was later identified as an aggressive pathogen of live oak (Quercus virginiana Mill.) in Florida [49], cork oak in Portugal and Algeria [26,50], and chestnut oak (Quercus montana Willd.) in West Virginia [47]. Phylogenies based on tef1-α sequences revealed for the first time the existence of two distinct evolutionary lineages (haplotypes) in D. quercivora, for which the acronyms A and B are proposed. The presence of two haplotypes was supported by 6 fixed differences in the sequences of the tef1-α region. Similarly, for D. corticola and D. quercivora, the tef1-α region was shown to be a useful marker to study intraspecies variability, while phylogenies based on concatenated ITS and MAT gene sequences appear to be the best choice for the accurate separation of Diplodia species.

5. Conclusions

The present research allowed us to confirm the combined action of different Botryosphaeriaceae and Phytophthora species in the onset of holm oak decline and mortality in Italy. The recording of four Phytophthora in the monitored holm oak ecosystems can represent a risk for the surrounding crops, including olive orchards dramatically impacted by a severe disease called olive quick decline syndrome (CoDiRO) [51], associated with Xylella fastidiosa [52].
These areas have recently been subjected to a renewal of the olive grove heritage by the introduction of tolerant/resistant varieties. The new olive orchards, cultivated in semi-intensive conditions and supported by irrigation systems, could easily be vulnerable to attacks by Phytophthora, which takes advantage of these conditions.
Therefore, further studies will be necessary to explore the potential host jump of these pathogens from holm oak to olive as well as the susceptibility of the olive varieties recently introduced in Salento towards Botryosphaeriaceae and Phytophthora species, which are emerging as a serious threat to olive production in different countries with a Mediterranean climate, including Italy [15,53,54,55,56,57,58,59,60].

Author Contributions

Conceptualization, B.T.L., C.B. and S.M.; methodology, C.B. and B.T.L.; validation, B.T.L., C.B. and S.M.; formal analysis, C.B., F.C., G.B., A.G.D.D., B.T.L. and S.M.; investigation, C.B., F.C., G.B., A.G.D.D., B.T.L. and S.M.; resources, B.T.L. and S.M.; data curation, B.T.L., C.B. and S.M.; writing—original draft preparation, B.T.L., C.B. and S.M.; writing—review and editing, B.T.L., C.B. and S.M.; supervision, B.T.L., C.B. and S.M.; funding acquisition, B.T.L. and S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

All relevant data are within the paper and its supporting information files.

Acknowledgments

We would like to thank Apulia Phytosanitary Service for involving UNIVPM and UNIPD Phytopathology Research Staff in the research of holm decline. This research was partially funded by the UNIPM p+roject “Innovative detection of plant emergent pathogens” and by the project TESAF1DOR-00414.

Conflicts of Interest

The Authors declare no conflicts of interest.

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Forests 16 01052 g001
Figure 1. Major disease symptoms observed on holm oak trees and related pathogens associated: sudden death of mature trees (a); crown thinning, branch dieback, and abnormal growth of epicormic shoots along stems and branches (b–d); bleeding cankers on the stem (red arrow) (e); large bleeding canker with dark-brown exudation at the collar (f); inner bark and xylem necrotic lesions (g); particular of a sunken canker (h); cross section of a branch showing a characteristic V-shaped necrotic sector in the wood (i). Colony morphology of D. corticola (j), D. quercivora (k), P. asparagi (l), P. cinnamomi (m), P. multivora (n), and P. psychrophila (o) on PDA after seven days of incubation at 20 °C in the dark.
Figure 1. Major disease symptoms observed on holm oak trees and related pathogens associated: sudden death of mature trees (a); crown thinning, branch dieback, and abnormal growth of epicormic shoots along stems and branches (b–d); bleeding cankers on the stem (red arrow) (e); large bleeding canker with dark-brown exudation at the collar (f); inner bark and xylem necrotic lesions (g); particular of a sunken canker (h); cross section of a branch showing a characteristic V-shaped necrotic sector in the wood (i). Colony morphology of D. corticola (j), D. quercivora (k), P. asparagi (l), P. cinnamomi (m), P. multivora (n), and P. psychrophila (o) on PDA after seven days of incubation at 20 °C in the dark.
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Figure 2. Phylogenetic relationships of Diplodia corticola and Diplodia quercivora isolates based on the sequence data from tef1-α region. The phylogeny was inferred using the Maximum Likelihood method based on the General Time Reversible Model. The tree with the highest log likelihood is shown. A uniform distribution was used to model evolutionary rate differences among sites. Bootstrap values are given at the nodes. The tree is drawn to scale, with branch length measured in the number of substitutions per site. Isolates obtained in this study are reported in red. Ex-type culture in bold.
Figure 2. Phylogenetic relationships of Diplodia corticola and Diplodia quercivora isolates based on the sequence data from tef1-α region. The phylogeny was inferred using the Maximum Likelihood method based on the General Time Reversible Model. The tree with the highest log likelihood is shown. A uniform distribution was used to model evolutionary rate differences among sites. Bootstrap values are given at the nodes. The tree is drawn to scale, with branch length measured in the number of substitutions per site. Isolates obtained in this study are reported in red. Ex-type culture in bold.
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Figure 3. Phylogenetic relationships of Diplodia species based on the sequence data from MAT1-1-1 (A) and MAT1-2-1 (B) genes. Phylogeny was inferred using the Maximum Likelihood method based on the General Time Reversible Model. A discrete Gamma distribution was used to model evolutionary rate differences among sites for both phylogenies. The trees with the highest log likelihood are shown. Bootstrap values (>70%) are given at the nodes. The trees are drawn to scale, with branch length measured in the number of substitutions per site. Isolates obtained in this study are reported in red. Ex-type culture in bold.
Figure 3. Phylogenetic relationships of Diplodia species based on the sequence data from MAT1-1-1 (A) and MAT1-2-1 (B) genes. Phylogeny was inferred using the Maximum Likelihood method based on the General Time Reversible Model. A discrete Gamma distribution was used to model evolutionary rate differences among sites for both phylogenies. The trees with the highest log likelihood are shown. Bootstrap values (>70%) are given at the nodes. The trees are drawn to scale, with branch length measured in the number of substitutions per site. Isolates obtained in this study are reported in red. Ex-type culture in bold.
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Figure 4. Mean lesion length (±standard deviation) and symptoms on Q. ilex seedlings detected after one 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 4. Mean lesion length (±standard deviation) and symptoms on Q. ilex seedlings detected after one 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.
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Table 1. Information on study sites and number of sampled trees per site.
Table 1. Information on study sites and number of sampled trees per site.
Study SiteLocalityLatitudeLongitudeArea (ha)Number of Trees Sampled
A1Mazza 40.263960 18.40663010.06
A2Bonata40.253000 18.29566510.03
A3Gabrieli40.086495 18.2216440.42
Table 2. Species of Diplodia and Phytophthora obtained from the sampled trees. Diplodia corticola (Dc), D. quercivora (Dq), Phytophthora cinnamomi (Pc), P. multivora (Pm), P. psychrophila (Pp), and P. asparagi (Pa).
Table 2. Species of Diplodia and Phytophthora obtained from the sampled trees. Diplodia corticola (Dc), D. quercivora (Dq), Phytophthora cinnamomi (Pc), P. multivora (Pm), P. psychrophila (Pp), and P. asparagi (Pa).
Study SiteTreeSunken CankersBleeding CankersRhizosphere/Fine Roots
A1P1DcPcPc
P2DcPcPc
P3Dc, Dq--
P4Dc--
P5ns-Pm, Pp
P6Dc, Dq--
A2P7Dc--
P8Dc--
P9Dc-Pm
A3P10Dc-Pc
P11--Pa, Pp
ns: not sampled.
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Bregant, C.; Carloni, F.; Borsetto, G.; Delle Donne, A.G.; Linaldeddu, B.T.; Murolo, S. Multiple Botryosphaeriaceae and Phytophthora Species Involved in the Etiology of Holm Oak (Quercus ilex L.) Decline in Southern Italy. Forests 2025, 16, 1052. https://doi.org/10.3390/f16071052

AMA Style

Bregant C, Carloni F, Borsetto G, Delle Donne AG, Linaldeddu BT, Murolo S. Multiple Botryosphaeriaceae and Phytophthora Species Involved in the Etiology of Holm Oak (Quercus ilex L.) Decline in Southern Italy. Forests. 2025; 16(7):1052. https://doi.org/10.3390/f16071052

Chicago/Turabian Style

Bregant, Carlo, Francesca Carloni, Gaia Borsetto, Angelo G. Delle Donne, Benedetto T. Linaldeddu, and Sergio Murolo. 2025. "Multiple Botryosphaeriaceae and Phytophthora Species Involved in the Etiology of Holm Oak (Quercus ilex L.) Decline in Southern Italy" Forests 16, no. 7: 1052. https://doi.org/10.3390/f16071052

APA Style

Bregant, C., Carloni, F., Borsetto, G., Delle Donne, A. G., Linaldeddu, B. T., & Murolo, S. (2025). Multiple Botryosphaeriaceae and Phytophthora Species Involved in the Etiology of Holm Oak (Quercus ilex L.) Decline in Southern Italy. Forests, 16(7), 1052. https://doi.org/10.3390/f16071052

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