Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Cryptogein and capsicein promote defence responses in Quercus suber against Phytophthora cinnamomi infection

The decline of cork oak (Quercus suber) stands in Iberian Peninsula is associated with infection by Phytophthora cinnamomi. Most Phytophthora species secrete elicitins, which can enhance defence reactions against some pathogens. Here cytological and physiological effects of the elicitins cryptogein and capsicein on cork oak root infection by P. cinnamomi were evaluated. The progression of the pathogen in root tissue and its effects on total fatty acid (TFA) composition of roots and leaves were analysed in seedlings. Net photosynthesis (P _n ), stomatal conductance (g _s ), chlorophyll a fluorescence measurements (quantum yield of linear electron transport ? _e , photochemical quenching q _P, non-photochemical quenching NPQ) and carotenoid determinations were carried out in well established (4?months) plants. In elicitin-treated roots, 2?days after inoculation, the pathogen which presented loss of viability and membrane degradation was mainly restricted to the intercellular spaces of the cortical parenchyma, and did not reach the vascular cylinder. Electron dense materials accumulated in the intercellular spaces of the cortex next to disorganized hyphae, suggested to be related with defence reactions. Cryptogein (or its interaction with P. cinnamomi) induced enhanced lipid synthesis in leaves, which may contribute to preserve membrane stability. P. cinnamomi decreased P _n , g _s , ? _e , and q _P, whereas elicitin-treated plants displayed values similar to controls. Overall, the results indicated a resistance response of cork oak against this oomycete, induced by the elicitins.     

Calcium fertilizers induce soil suppressiveness to <Emphasis Type="BoldItalic">Phytophthora cinnamomi</Emphasis> root rot of <Emphasis Type="BoldItalic">Quercus ilex</Emphasis>

Based on the observation that the root disease caused by P. cinnamomi on Q. ilex has a low incidence and severity in soils with medium-high Ca²⁺ content, we studied the ability of Ca²⁺ fertilizers to induce soil suppressiveness to the pathogen. Studies on cultures of P. cinnamomi exposed to different Ca²⁺ fertilizers in vitro showed significant inhibition of sporangial, chlamydospore and zoospore production at millimolar concentrations while mycelial growth was mainly unaffected. Experiments performed with artificially infested soil showed that some Ca²⁺ fertilizers induce a significant decrease on chlamydospore viability. Additionally, greenhouse experiments using artificially infested soils showed a significant reduction of foliar and root symptom severities in Holm oak seedlings growing in soils amended with Ca²⁺ fertilizers. We suggest that limestone amendments in oak rangelands could enhance the suppressiveness of soils to P. cinnamomi, and it is likely that the inhibition of sporangial production was the main mechanism involved.     

Involvement of the β-cinnamomin elicitin in infection and colonisation of cork oak roots by <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis>

The virulence of two wild type (PA45 and PA37) and two genetically modified (13C: hygromycin resistant; FATSS: hygromycin resistant and β-cin knock-down) Phytophthora cinnamomi strains towards cork oak (Quercus suber) was assessed via a quantitative evaluation of disease symptoms arising from a soil infestation assay, and by a histological analysis of root colonization. Comparison of virulence, as expressed by symptom severity, resulted in the following ranking: highly virulent (wild type strains), medium virulence (strain 13C) and weakly virulent (FATSS). Both transgenic strains were compromised in their virulence, as expressed by symptom severity, but strain 13C was much less affected than FATSS. Microscopic observation showed that the FATSS strain was unable to effectively invade the root, while 13C and the two wild type strains were all able to rapidly colonize the whole root, including the vascular tissue. These results strengthen the notion that elicitins are associated, either directly or indirectly, with the infection process of Phytophthora.     

Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain

The evergreen holm oaks (Quercus ilex subsp. ilex and Q. ilex subsp. ballota) are the most representative tree species in the Iberian peninsula and the main tree species in oak‐rangeland ecosystems (dehesas). Oak decline in western, central and southern parts of Spain has been associated with root rot caused by Phytophthora cinnamomi for decades. However, Phytophthora species such as P.  quercina and P. psychrophila have recently been found associated with Quercus decline in eastern Spain where calcareous soils are predominant. Soil and root samples from two Quercus forests presenting decline symptoms in two different geographical areas in eastern Spain (Carrascar de la Font Roja and Vallivana) were analysed by amplicon pyrosequencing. Metabarcoding analysis showed Phytophthora species diversity, and revealed that an uncultured Phytophthora taxon, named provisionally Phytophthora taxon ballota, was the predominant species in both areas. In addition, a real‐time PCR assay, based on the pyrosequencing results, was developed for the detection of this uncultured Phytophthora taxon, and also for the detection of P. quercina. TaqMan assays were tested on soil and root samples, and on Phytophthora pure cultures. The new assays showed high specificity and were consistent with metabarcoding results. A new real‐time PCR protocol is proposed to evaluate the implication of different Phytophthora spp. in oak decline in eastern Spain.     

Early survival of Quercus ilex subspecies from different populations after infections and co‐infections by multiple Phytophthora species

Forests in Europe are threatened by increased diversity of Phytophthora species, but effects on trees of simultaneous infections by Phytophthora and ecological consequences of their coexistence are unknown. This study explored variation in early survival of Quercus ilex to Phytophthora infections and assessed interactions between Phytophthora species when trees were co‐infected. Three Phytophthora species (P. cinnamomi, P. gonapodyides and P. quercina), seeds from 16 populations of Q. ilex (ballota and ilex subspecies) and two infection times were used as sources of variation in two experiments. The influence of Phytophthora species, Q. ilex subspecies and populations on plant germination and survival were analysed using generalized linear mixed models and survival analysis techniques. Germination rates were not influenced by Phytophthora spp. (P = 0.194) but by the subspecies and populations of Q. ilex (P < 0.001). In Phytophthora‐infested soils, Q. ilex subsp. ilex germinated at higher rates than Q. ilex subsp. ballota. Plant survival was strongly influenced by Phytophthora species (P < 0.001), not by the subspecies and populations of Q. ilex. Seedling mortality was reduced and delayed if a less virulent Phytophthora species infected plants prior to infection by a more virulent Phytophthora species. The results help to explain oak decline syndrome and the lack of natural and artificial regeneration of Q. ilex forests. Lack of interspecific variability of early survival to Phytophthora spp. discourages direct sowing for artificial reforestation programmes. Large, thick seeds, giving plants rapid growth, are advantageous traits when soils are infested with Phytophthora spp.     

Evaluating potassium phosphonate injections for the control of <Emphasis Type="Italic">Quercus ilex</Emphasis> decline in SW Spain: implications of low soil contamination by <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis> and low soil water content on the effectiveness of treatments

The Iberian forests are suffering severe disease and mortality as a result of decline, with Quercus ilex the major species at risk. Trunk injections with potassium phosphonate, which have been used successfully to control Phytophthora cinnamomi, were tested against decline. In an area in which P. cinnamomi was isolated, Q. ilex trees showing different degrees of decline were trunk-injected. Soil properties, and measurements of soil water content (θ) and depth to soil water table were assessed at three sites with markedly different decline incidences. Over the 5 years following the initiation of the experiment, mean symptoms among spring-treated trees and autumn-treated trees, or among trees injected twice a year (spring and autumn), once a year, and non-injected, were not significantly different. No effects of the treatments on shoot growth and acorn production were observed. However, θ values under trees which recovered from decline were higher than θ values under trees which did not recover from decline. At the site with the highest incidence of decline and tree mortality, P. cinnamomi was rarely isolated, and the presence of gravel, soil infiltration capacities and water table depth values were significantly higher than at the other sites, water stress being more likely to contribute to decline than P. cinnamomi. In areas in which θ is low, the distribution of phosphonate on the tree would be limited. Since the thresholds for phytotoxicity of potassium phosphonate in Q. ilex trees at the site studied would be higher than the amounts used, rates of the chemical slightly less than those that cause phytotoxicity should be tested.     

Analysis of the genetic diversity and phylogenetic relationships of <Emphasis Type="Italic">Biscogniauxia mediterranea</Emphasis> isolates associated with cork oak

Biscogniauxia mediterranea is one of the most frequent fungal pathogens involved in cork oak decline in the Mediterranean Basin, causing charcoal canker. In Portugal, this disease is widespread on adult declining trees but nowadays it increasingly affects young trees and exhibits atypical symptoms, leading to the hypothesis that some change in the fungus may have occurred. In order to evaluate the genetic diversity and phylogenetic relationship of B. mediterranea associated with cork oak, 102 isolates were obtained from young and adult trees of Quercus suber and other hosts species with different disease expression, from several Mediterranean countries. The collection of isolates was analyzed by individual and multigene phylogenies using Maximum-Likelihood approach based on nucleotide sequences of the internal transcribed spacers of ribosomal DNA, translation elongation factor 1-α and β-tubulin genes, and by microsatellite-primed PCR profiles. Sequence analyses separated the Mediterranean isolates from those from other regions, while MSP-PCR analysis revealed relevant but unstructured diversity among the Mediterranean isolates under study, making this a monophyletic but diverse population. Considering the adaptive capacity of the fungus in the Mediterranean-climate ecosystems and the present climatic change scenario, all conditions are gathered to favor aggravation of the disease in cork oak stands.     

Root infection by Phytophthora cinnamomi in seedlings of three oak species

The combination of soil infestation with Phytophthora cinnamomi and repetitive flooding was studied on 1-year-old plants of Quercus ilex (holm oak) and Q. suber (cork oak). In a second experiment, using 2-year-old plants of the same species and of red oak ( Q. rubra ), the soil infestation was followed by two drought-rewatering cycles. Oak predawn leaf water potential (PLWP) and stomatal conductance (gs) were monitored during both experiments. Root infection, root loss, wilting and mortality were assessed at the end of the experiments. Q. ilex exhibited the highest susceptibility to P. cinnamomi , and Q. rubra the lowest. Root infections caused by P. cinnamomi were more severe in the flooding than in the drought experiment. The most noticeable effect of the infection on plant water relations was a decrease in stomatal conductance. This occurred at different times after inoculation, varying with species susceptibility and experiment. Inoculation with P. cinnamomi induced a decrease of PLWP in Q. ilex plants, and in some Q. suber plants exhibiting a severe root loss. The results further showed that the relationship between PLWP and gs was modified by infection with P. cinnamomi . The combination of flooding and infection with P. cinnamomi acted synergistically on the water relations of Q. ilex . By contrast, there was no significant increase in disease severity due to the postinoculation water stress imposed on the oaks.     

Bacterial community associated with the ambrosia beetle Platypus cylindrus on declining Quercus suber trees in the Alentejo region of Portugal

In Portugal, the oak pinhole borer Platypus cylindrus and its mycobiota have been associated with cork oak (Quercus suber) death, but no knowledge exists regarding the associated bacterial community. However, it is known that some bacteria are important for ambrosia beetle symbiosis and play a role in oak tree health. To explore the bacteria associated with this beetle and its host, with the ultimate goal of highlighting potential roles in oak decline, this study used a culture-dependent approach for strain isolation and phylogenetic identification using 16S rRNA gene sequencing and multilocus sequence analysis (MLSA). The bored galleries of different cork oak trees from a cork stand in Alentejo, together with the body and mycangia of adult beetles, were investigated. The samples revealed a diverse community comprising 500 isolates with 64 distinct types of bacterial colonies. Sixty-eight strains were selected for sequencing and used for phylogenetic analysis, 40 from wood galleries and 28 from beetles. Thirty-two genera of bacteria were identified, 18 of which were described for the first time within oak–beetle interactions. Major taxonomic groups were Actinobacteria in beetles and Enterobacterales in wood galleries. Although specific oak bacterial pathogens were not detected, a group of distinct strains detected in wood galleries, potentially belonging to a new Pectobacteriaceae species, were able to produce mild symptoms on cork oak plantlets. This study reports for the first time the biodiversity of culturable bacteria associated with the Q. suber–P. cylindrus interaction, their relevance to both organisms and the possible contribution to oak decline.     

Interaction between root rot basidiomycetes and Phytophthora species on pedunculate oak

Forest declines are usually complex multifactorial phenomena that involve interactions between different factors. The possible interaction between different types of mycelial pathogens was investigated through artificial inoculation of oak seedlings, involving two root rot basidiomycetes, Collybia fusipes and Armillaria mellea, and two Phytophthora species, P. cinnamomi and P. cambivora. These pathogens were inoculated onto young Quercus robur saplings in greenhouse conditions, either alone or combining a root rot basidiomycete with a Phytophthora species. Three out of the four Phytophthora spp.*root rot basidiomycete combinations tested resulted in significantly greater damage to the oak host than the sum of the damages induced by the individual pathogens. This positive interaction could be significant in oak decline syndrome.     

Susceptibility of common herbaceous crops to Phytophthora cinnamomi and its influence on Quercus root rot in rangelands

The susceptibility to Phytophthora cinnamomi of four crops (wheat, oat, vetch, and yellow lupin) commonly planted in rangeland ecosystems in southern Spain was evaluated. By means of in vitro infection experiments, the presence of the pathogen into the roots of yellow lupin (symptomatic) and vetch (asymptomatic) was observed, but never into wheat and oat roots (asymptomatic). It was also demonstrated that yellow lupin stimulated the production of zoospores of P. cinnamomi. Vetch, wheat and oat did not stimulate zoospore production. Under controlled conditions, only yellow lupin induced an increase in the number of viable chlamydospores in the soil. We concluded that the culture of wheat, oat, and vetch in rangelands did not influence the epidemiology of the Quercus root disease, even when asymptomatically-infected vetch is grown, and these crops can constitute an alternative to the culture of yellow lupin in rangeland ecosystems affected by Quercus root rot.     

Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance

Castanea sativa is susceptible to Phytophthora spp., a serious root pathogen causing ink disease, while C. crenata and C. mollissima show resistance to infection. Interspecific controlled crosses were established for introgression of resistance genes from the resistant species into the susceptible C. sativa, and two mapping populations were created. Phytophthora cinnamomi resistance of each progeny was evaluated by root and excised shoot inoculation tests. The number of days of survival after root inoculation was the best discriminator of resistance to P. cinnamomi while the percentage of shoots with internal lesions was the symptom most associated with survival. The lesion progression rate in the excised shoot inoculation test was strongly and negatively correlated with survival in the root inoculation test. The excised shoot inoculation test appears to be a reliable approach for screening the resistance of chestnut genotypes to P. cinnamomi. Strong genetic correlations were obtained between survival and ink disease symptoms and among symptoms, indicating that common or linked genes might influence resistance to P. cinnamomi. The most resistant genotypes selected from this study will be tested for other commercial variables, such as ease of vegetative propagation and stock–scion compatibility.     

<Emphasis Type="Italic">Lupinus luteus</Emphasis>, a new host of <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis> in Spanish oak-rangeland ecosystems

Phytophthora cinnamomi is an aggressive pathogen on Lupinus luteus (yellow lupin), causing root rot, wilting and death of this crop, common in oak-rangeland ecosystems ('dehesas') in south-western Spain. The oomycete, the main cause of Quercus decline in the region, was isolated from roots of wilted lupins in the field. Artificial inoculations on four cultivars of L. luteus reproduced the symptoms of the disease, both in pre- and post-emergence stages, recovering the pathogen from necrotic roots. These results suggest the potential of yellow lupin as inoculum reservoir for the infection of Quercus roots. This is the first report of P. cinnamomi as root pathogen of L. luteus.     

Elucidation of defence responses and signalling pathways induced in Arabidopsis thaliana following challenge with Phytophthora cinnamomi

Arabidopsis thaliana (Arabidopsis) Col-0 was inoculated with Phytophthora cinnamomi to assess the interaction and defence responses involved. Pathogen ingress and asexual reproduction occurred on root tissue but not leaf tissue. The colonisation of root tissue did not cause disease symptoms or plant death, indicating that Arabidopsis Col-0 was tolerant of the infection. The induction of several plant defence responses including the expression of defence-related genes were found, with differences displayed between inoculated root and leaf tissue. Arabidopsis defence-related gene mutant/over-expressing lines were also inoculated with P. cinnamomi but none of the lines tested exhibited a marked increase in susceptibility to the pathogen.     

Evidence for Phytophthora cinnamomi involvement in Iberian oak decline

Rapid and sometimes extensive mortality and decline of oak, principally Quercus suber and Q. ilex , has occurred in parts of southern Spain and Portugal in recent decades. We report here isolation of the aggressive root pathogen Phytophthora cinnamomi from roots of diseased oaks or from soil at eleven out of thirteen decline foci examined. It is proposed that the introduction and spread of P. cinnamomi may be a major factor in the Iberian oak decline, interacting with drought and other site factors, and leading to stress-related attacks by insects and other fungi. By analogy, it may also be involved in the similar oak declines occurring elsewhere on the Mediterranean.     

European oak declines and global warming: a theoretical assessment with special reference to the activity of Phytophthora cinnamomi

Causes of current severe declines of the deciduous oaks Quercus robur and Q. petraea in northern and central Europe and of the evergreen Q. ilex, Q. suber and other Quercus spp. in the Mediterranean area are reviewed. Factors implicated include drought, pollution, winter cold, flooding, and stress-related attacks by insects and fungi. Additional factors in Mediterranean oak declines include changing land-use patterns and root disease caused by the aggressive, exotic oomycete root pathogen Phytophthora cinnamomi. Under conditions of global warming the survival and degree of root disease caused by this fungus seems likely to be enhanced, while the host range of the organism might also be increased. Application of the CLIMEX climate-matching program suggests that with a mean increase in temperatures of 1.5–3°C the fungus might considerably increase its disease activity in its existing locations, and to some extent spread northwards and eastwards. However, it seems unlikely to become significantly active in areas of Europe with colder winters such as parts of Scandinavia, Russia and the central Danube. The predictive value of research on major environmental problems such as oak declines could be enhanced by more highly coordinated European forestry research programmes.     

Comparison of the Susceptibility of Quercus petraea, Q. robur and Q. rubra to Collybia fusipes

Collybia fusipes is the cause of a root rot of Quercus petraea (sessile oak), Q. robur (pedunculate oak) and Q. rubra (red oak). This parasite is often reported to cause problems in stands of red oaks and field evidence suggests that this North American oak species is more susceptible than the two European oak species. Young saplings of the three oak species and of chestnut, Castanea sativa, in the glasshouse, and also mature sessile and red oaks growing in the same stand were inoculated with C. fusipes to compare their susceptibility. Red oak, both as young seedlings and mature trees, was more susceptible to C. fusipes than sessile oak. Chestnut seedlings were as susceptible as sessile oak. Susceptibility of pedunculate oak seedlings was intermediate between red oak and sessile oak. In one experiment this species was significantly less susceptible than red oak, and in the other it was as susceptible.     

Age‐related susceptibility of Eucalyptus species to Phytophthora boodjera

Phytophthora boodjera is a newly described pathogen causing damping off and mortality of Eucalyptus seedlings in Western Australian nurseries. This study evaluated the age‐related susceptibility of several taxa of mallee Eucalyptus to P. boodjera in sterilized washed river sand‐infestation pot trials. Phytophthora cinnamomi and P. arenaria were included for comparison. Seedlings of Eucalyptus taxa were inoculated at 0, 2, 4, 12 and 88 weeks with individual Phytophthora isolates. Pre‐emergent mortality in the presence of Phytophthora was almost 100%. Post‐emergent mortality was 50–100%, depending on isolate, compared to 0% for the control. Mortality was also high for inoculated 1 month‐old seedlings (46–68%) and root length of surviving seedlings was severely reduced. Death from root infection was not observed for seedlings inoculated at 12 and 88 weeks, but they developed root necrosis and reduced root dry weight compared to non‐inoculated controls. Phytophthora boodjera is a pre‐ and post‐emergent pathogen of mallee eucalypts. These eucalypts are susceptible to P. boodjera at all life stages tested, but the mortality rates declined with plant age. Similar results were obtained for P. cinnamomi and P. arenaria. The events leading to its recent appearance in the nurseries remain unknown and further investigations are underway to determine if this is an introduced or endemic pathogen. The approach used here to understand the impact of a Phytophthora species on multiple hosts at different seedling ages is novel and sets a benchmark for future work.