First Report of Dieback of Quercus suber Trees Associated with Phytophthora quercina in Morocco.

Cork oak (Quercus suber L.) is an evergreen tree native to SW Europe and NW Africa. It covers 2·106 ha in the western Mediterranean basin, forms heterogeneous forest ecosystems and represents an important source of income derived from cork production. While in Iberia, Italy, Tunisia and Algeria, drought and several endemic pathogens have been associated with cork oak decline (Moricca et al. 2016; Smahi et al. 2017), in Morocco there is no evidence, apart from overgrazing and human intervention (Fennane and Rejdali 2015), of a pathogen associated with oak decline. In December 2019, extensive dieback and mortality of 60-year-old cork oak trees were observed in a natural stand of ca 150 ha located 5 km east from Touazithe, in Maâmora forest, Morocco (34°13'38''N, 6°14'51''W - 87 m a.s.l.). Two years before, Q. suber seedlings from a local nursery were planted to increase tree density. Symptoms in trees and planted seedlings included chlorosis, reddish-brown discoloration of the whole crown and dieback starting in the upper crown. Root rot and lack of fine roots were observed. Tree mortality was estimated at ca 30%, and disease incidences of trees and seedlings were 45 and 70%, respectively. A Phytophthora species was consistently isolated from the rhizosphere of 3 symptomatic trees randomly selected at the site using leaves as bait (Jung et al. 1996). On carrot agar Phytophthora colonies were uniform and cottonwool-like. Sporangia were typically terminal, with ovoid, and obpyriform shape, mostly papillate, measuring 30.7 ± 4.7 µm length and 22.7 ± 4.1 µm wide. Oogonia were produced in single culture, and they were globose to subglobose, elongated to ellipsoid, 32.1 ± 2.9 µm in diameter and 46.1 ± 4.8 µm in length. Oospores were usually spherical, thick-walled, and measured 28.1 ± 2.4 µm. Antheridia were paragynous, mostly spherical, measuring 12.2 ± 1.4 µm. Isolates had minimum and maximum temperatures of 5 °C and 30 °C, respectively, and a growth optimum at 20 °C. Apart from the small size of sporangia, features were typical of Phytophthora quercina Jung. The identity of a representative strain (TJ1500) was corroborated by sequencing the ITS and mitochondrial cox1 gene regions, and BLAST search in GenBank showed 100% homology with sequences of the ex-type culture of P. quercina (KF358229 and KF358241 accessions, respectively). Both sequences of the representative isolate were submitted to GenBank (accessions OP086243 and OP290549). The strain TJ1500 is currently stored within the culture collections of the Mendel University in Brno and the University of Sassari. Its pathogenicity was verified and compared with a P. cinnamomi strain in a soil infestation test with one-year-old cork oak seedlings (Corcobado et al. 2017). Five months after inoculation, the symptoms described were observed in the seedlings, and fine root weight of plants inoculated with the TJ1500 strain and P. cinnamomi was reduced by 19 and 42%, respectively, in relation to non-inoculated controls. The pathogen was re-isolated from the necrotic roots, thus fulfilling Koch's postulates. So far, P. quercina has been reported associated with chronic mortality of cork oak in new plantations in Spain (Martín-García et al. 2015; Jung et al. 2016) and natural forests in Italy (Seddaiu et al. 2020). To our knowledge this is the first report of P. quercina in Morocco. Givenat Morocco is an important cork producing country, our finding warns about the risk this pathogen poses to Q. suber and other North African oaks.

Phylogeography of the wide-host range panglobal plant pathogen Phytophthora cinnamomi.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen.

Endemic and Emerging Pathogens Threatening Cork Oak Trees: Management Options for Conserving a Unique Forest Ecosystem.

Cork oak (Quercus suber) forests are economically and culturally intertwined with the inhabitants of the Mediterranean basin and characterize its rural landscape. These forests cover over two million hectares in the western Mediterranean basin and sustain a rich biodiversity of endemisms as well as representing an important source of income derived from cork production. Currently cork oak forests are threatened by several factors including human-mediated disturbances such as poor or inappropriate management practices, adverse environmental conditions (irregular water regime with prolonged drought periods), and attacks of pathogens and pests. All these adverse factors can interact, causing a complex disease commonly known as "oak decline." Despite the numerous investigations carried out so far, decline continues to be the main pathological problem of cork oak forests because of its complex etiology and the resulting difficulties in defining suitable control strategies. An overview of the literature indicates that several pathogenic fungi and oomycota can play a primary role in the etiology of this syndrome. Therefore, the aim of this review is to analyze the recent advances achieved regarding the bio-ecology of the endemic and emerging pathogens that threaten cork oak trees with particular emphasis on the species more directly involved in oak decline. Moreover, the effect of climate change on the host-pathogen interactions, a task fundamental for making useful decisions and managing cork oak forests properly, is considered.

Diplopimarane, a 20-nor-ent-pimarane produced by the oak pathogen Diplodia quercivora.

In this study a new 20-nor-ent-pimarane, named diplopimarane, was isolated together with sphaeropsidins A (9) and C (10), and (+)-epiepoformin (11) from organic crude extracts of Diplodia quercivora, a recently described oak pathogen originally found on declining Quercus canariensis trees in Tunisia. Diplopimarane was characterized as (1S,2R)-2,8,8-trimethyl-2-vinyl-1,2,3,4,5,6,7,8-octahydrophenanthrene-1,9,10-triol by spectroscopic, X-ray, optical, and chemical methods. It exhibited a wide range of activities including remarkable phytotoxicity on nonhost plants such as tomato cuttings, moderate antifungal activity against important plant pathogens, and moderate zootoxicity against Artemia salina. Its derivatives (2-4 and 6) were also tested for their phytotoxic and zootoxic activities. All these derivatives proved to be active against A. salina at 200 µg/mL, while 2 and 6 were also active on tomato cuttings. The other secondary metabolites (9, 10, and 11) herein reported for D. quercivora exhibited phytotoxic, antifungal, and zootoxic activity. This is the first report on the secondary metabolites secreted in vitro by this oak pathogen that could be key components of its adaptative strategies.

Phylogeography, origin and population structure of the self-fertile emerging plant pathogen Phytophthora pseudosyringae.

Phytophthora pseudosyringae is a self-fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping-by-sequencing. Coalescent-based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide.

Potassium Phosphonate Induces Resistance in Sweet Chestnut against Ink Disease Caused by Phytophthora Species.

Ink disease, caused by Phytophthora spp., represents a serious threat to sweet chestnuts throughout their distribution area. Among the control strategies, new perspectives have been offered by using potassium phosphonate, which indirectly controls Phytophthora diseases by acting on both host physiology and host-pathogen interactions. In this study, we tested in planta the effectiveness of trunk injection with K-phosphonate against seven different Phytophthora species associated with ink disease. For the two most aggressive species, P. cinnamomi and P. ×cambivora, the treatments were repeated at two different environmental conditions (a mean temperature of 14.5 °C vs. 25 °C) and tree phenology stages. The results obtained in this study demonstrated that K-phosphonate could contain the development of Phytophthora infection in phloem tissues. However, its effectiveness varied based on the concentration applied and the Phytophthora species tested. A concentration of 280 g/L of K-phosphonate was the most effective, and in some cases, callus formation around the necrotic lesion was detected. Overall, this study broadens the knowledge of endotherapic treatments with K-phosphonate as an effective measure for managing chestnut ink disease. Interestingly, the increase in mean temperature had a positive impact on the development of P. cinnamomi lesions on chestnut phloem tissues.

Phytophthora: an ancient, historic, biologically and structurally cohesive and evolutionarily successful generic concept in need of preservation.

The considerable economic and social impact of the oomycete genus Phytophthora is well known. In response to evidence that all downy mildews (DMs) reside phylogenetically within Phytophthora, rendering Phytophthora paraphyletic, a proposal has been made to split the genus into multiple new genera. We have reviewed the status of the genus and its relationship to the DMs. Despite a substantial increase in the number of described species and improvements in molecular phylogeny the Phytophthora clade structure has remained stable since first demonstrated in 2000. Currently some 200 species are distributed across twelve major clades in a relatively tight monophyletic cluster. In our assessment of 196 species for twenty morphological and behavioural criteria the clades show good biological cohesion. Saprotrophy, necrotrophy and hemi-biotrophy of woody and non-woody roots, stems and foliage occurs across the clades. Phylogenetically less related clades often show strong phenotypic and behavioural similarities and no one clade or group of clades shows the synapomorphies that might justify a unique generic status. We propose the clades arose from the migration and worldwide radiation ~ 140 Mya (million years ago) of an ancestral Gondwanan Phytophthora population, resulting in geographic isolation and clade divergence through drift on the diverging continents combined with adaptation to local hosts, climatic zones and habitats. The extraordinary flexibility of the genus may account for its global 'success'. The 20 genera of the obligately biotrophic, angiosperm-foliage specialised DMs evolved from Phytophthora at least twice via convergent evolution, making the DMs as a group polyphyletic and Phytophthora paraphyletic in cladistic terms. The long phylogenetic branches of the DMs indicate this occurred rather rapidly, via paraphyletic evolutionary 'jumps'. Such paraphyly is common in successful organisms. The proposal to divide Phytophthora appears more a device to address the issue of the convergent evolution of the DMs than the structure of Phytophthora per se. We consider it non-Darwinian, putting the emphasis on the emergent groups (the DMs) rather than the progenitor (Phytophthora) and ignoring the evolutionary processes that gave rise to the divergence. Further, the generic concept currently applied to the DMs is narrower than that between some closely related Phytophthora species. Considering the biological and structural cohesion of Phytophthora, its historic and social impacts and its importance in scientific communication and biosecurity protocol, we recommend that the current broad generic concept is retained by the scientific community.

Sphaeropsidones, phytotoxic dimedone methyl ethers produced by Diplodia cupressi: a structure-activity relationship study.

Sphaeropsidone and episphaeropsidone are two phytotoxic dimedone methyl ethers produced by Diplodia cupressi, the causal agent of a canker disease of cypress in the Mediterranean area. In this study, eight derivatives obtained by chemical modifications and two natural analogues were assayed for phytotoxic and antifungal activities, and a structure-activity relationship was examined. Each compound was tested on nonhost plants and on five fungal pathogenic species belonging to the genus Phytophthora. The results provide insights into structure-activity relationships within these compounds. It was found that the hydroxy group at C-5, the absolute C-5 configuration, the epoxy group, and the C-2 carbonyl group appear to be structural features important in conferring biological activity. The conversion of sphaeropsidone into the corresponding 1,4-dione derivative led to a compound showing greater antifungal activity than its precursor. This finding could be useful in devising new natural fungicides for practical application in agriculture.

Phytophthora heterospora sp. nov., a New Pseudoconidia-Producing Sister Species of P. palmivora.

Since 1999, an unusual Phytophthora species has repeatedly been found associated with stem lesions and root and collar rot on young olive trees in Southern Italy. In all cases, this species was obtained from recently established commercial plantations or from nursery plants. Morphologically, the Phytophthora isolates were characterized by the abundant production of caducous non-papillate conidia-like sporangia (pseudoconidia) and caducous papillate sporangia with a short pedicel, resembling P. palmivora var. heterocystica. Additional isolates with similar features were obtained from nursery plants of Ziziphus spina-christi in Iran, Juniperus oxycedrus and Capparis spinosa in Italy, and mature trees in commercial farms of Durio zibethinus in Vietnam. In this study, morphology, breeding system and growth characteristics of these Phytophthora isolates with peculiar features were examined, and combined mitochondrial and nuclear multigene phylogenetic analyses were performed. The proportion between pseudoconidia and sporangia varied amongst isolates and depended on the availability of free water. Oogonia with amphigynous antheridia and aplerotic oospores were produced in dual cultures with an A2 mating type strain of P. palmivora, indicating all isolates were A1 mating type. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora; thus, they constitute a separate taxon. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in stem inoculation tests.

The Destructive Tree Pathogen Phytophthora ramorum Originates from the Laurosilva Forests of East Asia.

As global plant trade expands, tree disease epidemics caused by pathogen introductions are increasing. Since ca 2000, the introduced oomycete Phytophthora ramorum has caused devastating epidemics in Europe and North America, spreading as four ancient clonal lineages, each of a single mating type, suggesting different geographical origins. We surveyed laurosilva forests for P. ramorum around Fansipan mountain on the Vietnam-China border and on Shikoku and Kyushu islands, southwest Japan. The surveys yielded 71 P. ramorum isolates which we assigned to eight new lineages, IC1 to IC5 from Vietnam and NP1 to NP3 from Japan, based on differences in colony characteristics, gene x environment responses and multigene phylogeny. Molecular phylogenetic trees and networks revealed the eight Asian lineages were dispersed across the topology of the introduced European and North American lineages. The deepest node within P. ramorum, the divergence of lineages NP1 and NP2, was estimated at 0.5 to 1.6 Myr. The Asian lineages were each of a single mating type, and at some locations, lineages of "opposite" mating type were present, suggesting opportunities for inter-lineage recombination. Based on the high level of phenotypic and phylogenetic diversity in the sample populations, the coalescence results and the absence of overt host symptoms, we conclude that P. ramorum comprises many anciently divergent lineages native to the laurosilva forests between eastern Indochina and Japan.

Green pesticides based on cinnamate anion incorporated in layered double hydroxides and dispersed in pectin matrix.

This paper reports the preparation of green pesticides based on nano-hybrids composed of a Layered Double Hydroxide (LDH) with cinnamate anion. The dispersion into a pectin matrix was obtained using high energy ball milling in wet conditions. Structure and physical properties of the fillers and the composites films were evaluated. Controlled release of cinnamate was followed using UV spectrophotometry and the release kinetics were found to be dependent on the filler loading. The experimental results were analyzed by the Gallagher-Corrigan model. Antimicrobial activity was evaluated on different bacterial strains, as well as plant pathogens belonging to the genus Phytophthora using modified agar diffusion, broth microdilution and dual culture methods, respectively. Experimental results suggested the possibility to use the analyzed composites as green protective coatings for crops' protection.

Bioactive Metabolites from Pathogenic and Endophytic Fungi of Forest Trees.

BACKGROUND: Fungi play an important role in terrestrial ecosystems interacting positively or negatively with plants. These interactions are complex and the outcomes are different depending on the fungal lifestyles, saprotrophic, mutualistic or pathogenic. Furthermore, fungi are well known for producing secondary metabolites, originating from different biosynthetic pathways, which possess biological properties of considerable biotechnological interest. Among the terrestrial ecosystems, temperate forests represent an enormous reservoir of fungal diversity. This review will highlight the goldmine of secondary metabolites produced by pathogenic and endophytic fungi of forest trees with focus on their biological activities. METHODS: A structured search of bibliographic databases for peer-reviewed research literature was undertaken using a research discovery application providing access to a large and authoritative source of references. The papers selected were examined and the main results were reported and discussed. RESULTS: Two hundred forthy-one papers were included in the review, outlined a large number of secondary metabolites produced by pathogenic and endophiltic fungi and their biological activities, including phytotoxic, antifungal, antioomycetes, antibacterial, brine shrimp lethality, mosquito biting deterrence and larvicidal, cytotoxic, antiproliferative and many other bioactivities. CONCLUSION: The findings of this review confirm the importance of secondary metabolites produced by pathogenic and endophytic fungi from forest plants growing in temperate regions as an excellent prospects to discover compounds with new bioactivities and mode of actions. In addition, the potential of some metabolites as a source of new drugs and biopesticides is underlined.

Multiple new cryptic pathogenic Phytophthora species from Fagaceae forests in Austria, Italy and Portugal.

During surveys of Phytophthora diversity in natural and semi-natural Fagaceae forests in Austria, Italy and Portugal, four new cryptic species were isolated from rhizosphere soil samples. Multigene phylogeny based on nuclear ITS, ß-tubulin and HSP90 and mitochondrial cox1 and NADH1 gene sequences demonstrated that two species, P. tyrrhenica and P. vulcanica spp. nov., belong to phylogenetic Clade 7a, while the other two species, P. castanetorum and P. tubulina spp. nov., clustered together with P. quercina forming a new clade, named here as Clade 12. All four new species are homothallic and have low optimum and maximum temperatures for growth and very slow growth rates at their respective optimum temperature. They differed from each other and from related species by a unique combination of morphological characters, cardinal temperatures, and growth rates. Pathogenicity of all Phytophthora species to the root system of their respective host species was demonstrated in soil infestation trials.

Bioactive Secondary Metabolites Produced by the Oak Pathogen Diplodia corticola.

Three new lactones and a new fatty acid ester, named sapinofuranones C and D, diplopyrone B, and diplobifuranylone C, respectively, were isolated from Diplodia corticola, together with sphaeropsidins A and C, diplopyrone, diplobifuranylones A and B, diplofuranone A, and the (S,S)-enantiomer of sapinofuranone B. Sapinofuranones C and D, diplopyrone B, and diplobifuranylone C were characterized as (5S)-5-((1,S-1,6-dihydroxyhexa-2,4-dienyl)-dihydrofuran-2-one, 4,5-dihydroxy-deca-6,8-dienoic acid methyl ester, (5S)-5-hydroxy-6-(penta-1,3-dienyl)-5,6-dihydro-pyran-2-one, and 5'-((1R)-1-hydroxyethyl)-2',5'-dihydro-2H-[2,2']bifuranyl-5-one by spectroscopic and chemical methods, respectively. The relative configuration of sapinofuranone C was assigned by X-ray diffraction analysis, whereas its absolute configuration was determined by applying the advanced Mosher's method to its 11-O-p-bromobenzoyl derivative. The same method was used to assign the absolute configuration to C-5 of diplopyrone B and to that of the hydroxyethyl of the side chain of diplobifuranylone C, respectively. The metabolites isolated were tested at 1 mg/mL on leaves of cork oak, grapevine cv. 'Cannonau', and tomato using the leaf puncture assay. They were also tested on tomato cuttings at 0.2, 0.1, and 0.05 mg/mL. Each compound was tested for zootoxic activity on Artemia salina L. larvae. The efficacy of sapinofuranone C and diplopyrone B on three plant pathogens, namely, Athelia rolfsii, Fusarium avenaceum, and Phytophthora nicotianae was also evaluated. In all phytotoxic assays only diplopyrone B was found to be active. It also showed strong inhibition on the vegetative growth of A. rolfsii and P. nicotianae. All metabolites were inactive in the assay performed for the zootoxic activity (A. salina) even at the highest concentration used (200 µg/mL). Diplopyrone B showed a promising antioomycete activity for the control of Phytophthora spp. also taking into account the absence of zootoxic activity.

Diversity of Phytophthora Species from Declining Mediterranean Maquis Vegetation, including Two New Species, Phytophthora crassamura and P. ornamentata sp. nov.

The Mediterranean basin is recognized as a global biodiversity hotspot accounting for more than 25,000 plant species that represent almost 10% of the world's vascular flora. In particular, the maquis vegetation on Mediterranean islands and archipelagos constitutes an important resource of the Mediterranean plant diversity due to its high rate of endemism. Since 2009, a severe and widespread dieback and mortality of Quercus ilex trees and several other plant species of the Mediterranean maquis has been observed in the National Park of La Maddalena archipelago (northeast Sardinia, Italy). Infected plants showed severe decline symptoms and a significant reduction of natural regeneration. First studies revealed the involvement of the highly invasive wide-host range pathogen Phytophthora cinnamomi and several fungal pathogens. Subsequent detailed research led to a better understanding of these epidemics showing that multiple Phytophthora spp. were involved, some of them unknown to science. In total, nine Phytophthora species were isolated from rhizosphere soil samples collected from around symptomatic trees and shrubs including Asparagus albus, Cistus sp., Juniperus phoenicea, J. oxycedrus, Pistacia lentiscus and Rhamnus alaternus. Based on morphological characters, growth-temperature relations and sequence analysis of the ITS and cox1 gene regions, the isolates were identified as Phytophthora asparagi, P. bilorbang, P. cinnamomi, P. cryptogea, P. gonapodyides, P. melonis, P. syringae and two new Clade 6 taxa which are here described as P. crassamura sp. nov. and P. ornamentata sp. nov. Pathogenicity tests supported their possible involvement in the severe decline that is currently threatening the Mediterranean maquis vegetation in the La Maddalena archipelago.

Afritoxinones A and B, dihydrofuropyran-2-ones produced by Diplodia africana the causal agent of branch dieback on Juniperus phoenicea.

Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Phoenicean juniper in Italy. Additionally, six others known metabolites were isolated and characterized: oxysporone, sphaeropsidin A, epi-sphaeropsidone, R-(-)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS(*),6R(*),7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR(*),6R(*),7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host (Phoenicean juniper) and non-host plant (holm oak, cork oak and tomato) by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. This study represents the first report of secondary metabolites produced by D. africana. In addition, the taxonomic implications of secondary metabolites in Botryosphaeriaceae family studies are discussed.