Ink Disease Research Articles

Monitoring ink disease epidemics in chestnut and cork oak forests in central Italy with remote sensing

Forests provide multiple ecosystem services including water and soil protection, biodiversity conservation, carbon sequestration, and recreation, which are crucial in sustaining human health and wellbeing. Global changes represent a serious threat to Mediterranean forests, and among known impacts, there is the spread of invasive pests and pathogens, often boosted by climate change and human pressure. Remote sensing can provide support to forest health monitoring, which is crucial to contrast degradation and adopt mitigation strategies. Here, different multispectral and SAR data are used to detect the incidence of ink disease driven by Phytophthora cinnamomi in forest sites in central Italy, dominated by chestnut and cork oak respectively. Sentinel 1, Sentinel 2, and PlanetScope data, together with ground information, served as input in Random Forests to model healthy and disease classes in the two sites. The results indicate that healthy and symptomatic trees are clearly distinguished, whereas the discrimination among disease classes of different severity (moderate and severe damage) is less accurate. Crown dimension and sampled spectral regions are a critical factors in the selection of the sensor; better results are obtained for the larger chestnut crowns with Sentinel 2 data. In both sites, the red and near infra-red bands from multispectral data resulted well suited to monitor the spread of the ink disease.

The Disease Triangle of Chestnut: A review of host, pathogen, and environmental interactions of chestnuts cultivated in the eastern United States

Chestnuts, the edible seeds of the genus Castanea, are a perennial food crop closely tied to the global migration of humans throughout history and have recently been gaining popularity in agriculture and forest restoration in eastern North America. Cultivation of chestnuts yields nutritionally balanced food while fostering economic development, food security, and environmental health. However, diseases and insect pests threaten successful ecological restoration and food production. In this review we explore conditions affecting chestnuts in the eastern United States through the lens of the disease triangle. A “host” in the disease triangle is not merely a single tree, but a tree including its constituent population of fungal endophytes. Chestnut trees are rich with microbial life, and the sustainability of chestnuts in forest and cultivated settings may lie in understanding and manipulating microbial communities to improve plant health and control destructive diseases. To benefit from the ecological and economic potential of chestnuts on the landscape, it may be necessary to select locally adapted chestnut trees, regardless of pedigree, that are resilient against cosmopolitan pathogens. With transport of plants and pathogens throughout the globe, and with landscape level environmental changes over the last century, chestnut trees in the eastern United States (U.S.) are in a unique disease landscape compared to their ancestors. Diseases of economic concern from fungi and fungal-like organisms include chestnut blight (Cryphonectria parasitica) and ink disease (Phytophthora cinnamomi) on American and European chestnuts, oak wilt (Bretziella fagacearum) on all chestnut species, and the emerging diseases of brown rot (Gnomoniopsis smithogilvyi) and chestnut anthracnose (Colletotrichum henanense). The eastern U.S. has experienced profound environmental changes over the 20th century and into the early 21st century. These changes happen to coincide with the demise of the American chestnut in the eastern forest, efforts to re-establish chestnut as a forest species, and the rise in cultivation of multiple chestnut species and hybrids as a culinary crop. Chestnut trees growing in the early 21st century face different environmental circumstances than the American chestnuts of pre-colonial times, including changes in forest composition, rainfall changes and acidification, industrialized agriculture’s increased chemical inputs, rising global temperatures, and increased levels of carbon dioxide in the atmosphere. We conclude that chestnut tree species for both forestry and agriculture should be considered based on current fitness, adaptability, and economic and ecological value considering continued dynamics in host and pathogens distributions and anthropogenically driven climatic and edaphic conditions.

Phytophthora and Phytopythium species associated with walnut dieback in plantations of northern Patagonia, Argentina

AbstractThe walnut industry in Argentina has grown in recent years, especially in northern Patagonia. However, this expansion has also brought new challenges, including the emergence of diseases threatening the health and productivity of walnut plantations. One of the most important diseases affecting walnut crops worldwide is ‘ink disease’ or ‘walnut dieback'. The aim of this study was to characterize ink disease, identify the aetiological agents, and estimate its incidence in walnut orchards of northern Patagonia, Argentina. Symptomatic trees were observed in the seven surveyed walnut orchards, showing a range of symptoms, including chlorosis and wilting, stem cankers and desiccation. In vivo assay and molecular analyses revealed that not only Phytophthora (P.) but also Phytopythium (Ph.) species are the causal agents of the disease, with P. drechsleri, Ph. mercuriale and Ph. vexans being the most virulent over the commonly used rootstock Juglans hindsii. Notably, Ph. vexans was the most frequently isolated pathogen, suggesting its prevalence and potential impact on the regional walnut industry. Overall, this study provides critical insights into the prevalence and impact of Phytophthora and Phytopythium species on walnut health in northern Patagonia. The presence of multiple pathogenic oomycete species raises concerns about the health of walnut orchards, as well as the potential risk of pathogen transmission to other fruit crops in the region. The findings will help growers to develop better cultural practices and implement measures to guarantee the sustainability of the walnut industry in Patagonia.

85 years counteracting an invasion: chestnut ecosystems and landscapes survival against ink disease

A retrospective examination of early pest and disease outbreaks, such as ink disease, offers new insights into their impact on ecosystems and landscapes. Ink disease, caused by Phytophthora spp., is one of the most destructive diseases affecting Castanea sativa Mill. It first appeared in Europe in the early 18th century, with the initial recorded case in Italy (Piedmont) dating back to 1845. However, its spread instilled significant concerns in several chestnut-growing regions primarily devoted to fruit production. In 1938, a comprehensive assessment of Phytophthora xcambivora outbreaks was conducted in the Bolognese Apennines (Italy), providing a detailed overview of chestnut cultivation status. Thirty-six disease foci were documented, and laboratory tests confirmed the presence of ink disease. To understand the disease’s impact on chestnut ecosystem and landscape over the past 85 years, the 1938 sites were revisited to assess chestnut persistence and phytosanitary status, with Phytophthora species detected using isolation and molecular techniques. Monitoring data revealed that C. sativa still thrives in all but one site, with its wood seemingly able to coexist in dynamic equilibrium with the disease. While P. xcambivora was still detected in several foci, the extent of damage appeared limited. The potential for natural control, likely influenced by the complexity of soil biota, along with the natural spread of hypovirulence in chestnut blight and biological control of the Asian chestnut gall wasp, could explain the continued presence of chestnut in the investigated area and potentially throughout the Italian chestnut range, despite nearly two centuries of ink disease prevalence. Nevertheless, ongoing monitoring is essential to dynamically comprehend the factors at play and their efficacy, particularly in the context of climate change and the possible spread of other Phytophthora species. The survival of chestnut ecosystems amidst ink disease underscores the preservation of the economic, ecological, and landscape values associated with these woodlands.

Post-Transcriptional Gene Silencing of Glucanase Inhibitor Protein in Phytophthora cinnamomi.

Ink disease is considered one of the most significant causes contributing to the decline of chestnut orchards. The reduced yield of Castanea sativa Mill can be attributed to two main species: Phytophthora cinnamomi and Phytophthora cambivora, with the first being the main pathogen responsible for ink disease in Portugal. P. cinnamomi is a highly aggressive and widely distributed plant pathogen, capable of infecting nearly 1000 host species. This oomycete causes substantial economic losses and is accountable for the decline of numerous plant species in Europe and worldwide. To date, no effective treatments are available to combat these pathogens. Given chestnut's economic and ecological significance, particularly in Portugal, it is crucial to investigate the molecular mechanisms underlying the interaction between Phytophthora species and host plants. This can be achieved through the study of the glucanase inhibitor protein (GIP) produced by P. cinnamomi during infection. The technique of RNA interference (RNAi) was employed to suppress the GIP gene of P. cinnamomi. The resulting transformants, carrying the silenced gene, were used to infect C. sativa, allowing for the assessment of the effects of gene silencing on the plant's phenotype. Additionally, bioinformatics tools predicted the secretion of GIP protein. The obtained results validate RNAi as a potential alternative tool for studying molecular factors and for controlling and managing P. cinnamomi.

Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a

Phytophthora cambivora is a major quarantine pathogen that devastates economically important plants across the globe. P. cambivora causes ink disease in chestnut trees and root and stem rot in various fruit trees, resulting in significant yield reductions and plant death. Given the potential dangers of P. cambivora, effective detection methods are needed for both disease management and prevention. In this study, based on the whole-genome screening of specific target genes, a combination of the recombinase polymerase amplification technique (RPA) and CRISPR/Cas12 was established to detect P. cambivora. The RPA-CRISPR/Cas12a assay was able to specifically detect 7 target isolates of P. cambivora but did not detect the following 68 non-target isolates, including 28 isolates of Phytophthora, 3 isolates of Pythium, 3 isolates of Phytopythium, 32 isolates of fungi, and 2 isolates of Bursaphelenchus. The RPA-CRISPR/Cas12a detection method was able to detect 10 pg·μL−1 of P. cambivora genomic DNA at 37 °C within a short time span (60 min). Additionally, this method can identify the presence of P. cambivora in artificially inoculated apple fruits. In summary, compared with conventional detection techniques, the RPA-CRISPR/Cas12a detection method eliminates the need for expensive instruments, long reaction times, and high amounts of raw materials and can detect P. cambivora in imported plants at entry ports, enabling instant prevention and detection.

Portuguese Castanea sativa Genetic Resources: Characterization, Productive Challenges and Breeding Efforts

Chestnuts are multipurpose trees that grow mainly in the Northern Hemisphere due to their aptitude for fruit and wood production. These trees are vastly significant for the economy and wildlife. The widespread distribution of these trees demonstrates their genetic adaptability to many environmental conditions. The main varieties of European chestnut (Castanea sativa Miller) cultivated in Portugal, their productive challenges and breeding and biotechnological efforts developed over the last decades are described. This paper highlights the efforts focused on the improvement of varieties and rootstocks through selection and hybridization of European chestnut with the Asian species Castanea crenata Siebold and Zuccarini and Castanea mollissima Blume, which are resistant to ink disease, which have been the foundation of the Portuguese chestnut breeding programs. Breeding and biotechnological efforts developed over the last decades, focused on ink disease and chestnut blight resistance, are described. The potentialities of this research to stimulate the competitiveness of bioeconomy-based knowledge and innovation in the productive chestnut sector is also discussed.

Distribution, causal agents, and infection dynamic of emerging ink disease of sweet chestnut in Southern Switzerland.

Emerging diseases caused by both native and exotic pathogens represent a main threat to forest ecosystems worldwide. The two invasive soilborne pathogens Phytophthora cinnamomi and Phytophthora × cambivora are the causal agents of ink disease, which has been threatening Castanea sativa in Europe for several centuries and seems to be re-emerging in recent years. Here, we investigated the distribution, causal agents, and infection dynamics of ink disease in southern Switzerland. A total of 25 outbreaks were identified, 19 with only P. cinnamomi, 5 with only P. × cambivora, and 1 with both species. Dendrochronological analyses showed that the disease emerged in the last 20-30 years. Infected trees either died rapidly within 5-15 years post-infection or showed a prolonged state of general decline until death. Based on a generalized linear model, the local risk of occurrence of ink disease was increased by an S-SE aspect of the chestnut stand, the presence of a pure chestnut stand, management activities, the proximity of roads and buildings, and increasing annual mean temperature and precipitation. The genetic structure of the local P. cinnamomi population suggests independent introductions and local spread of the pathogen.

Castanea crenata Ginkbilobin-2-like Recombinant Protein Reveals Potential as an Antimicrobial against Phytophthora cinnamomi, the Causal Agent of Ink Disease in European Chestnut

The European chestnut tree (Castanea sativa Mill.) is widely cultivated throughout the world’s temperate regions. In the Mediterranean region, it has a significant economic role mainly because of the high quality of its edible nuts. The Oomycete Phytophthora cinnamomi is one of the most severe pathogens affecting European chestnuts, causing ink disease and significant losses in production. Ginkgobilobin-2 (Gnk2) in Ginkgo biloba is a secreted protein with a plant-specific cysteine-rich motif that functions as a lectin, and its carbohydrate-binding properties are closely related to its antifungal activity. The binding of lectins to mannose residues of the cell wall of Phytophthora species may disturb and disrup the cell wall structure. This work determined that the amino acid sequence has a signal peptide that directs the final protein peptide to the apoplast. The Cast_Gnk2-like expression was performed and optimized, and different in vitro antagonism tests were done against P. cinnamomi using different purified protein concentrations. As a result of one of these assays, Cast_Gnk2-like significantly reduced the mycelia growth of P. cinnamomi in liquid medium as shown by the mycelia weight (g) in control treatments was 377% higher than in the treatments. These insights reveal the potential of Cast_Gnk2-like for agricultural uses and biotechnological developments for the pathosystem chestnut/P. cinnamomi.

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.

Effect of grafting on phenology, susceptibility to Phytophthora cinnamomi and hormone profile of chestnut

Ink disease caused by the root-rot pathogen P. cinnamomi (Pc) threatens European sweet chestnut (Castanea sativa Mill.) orchards, and growers increasingly graft susceptible C. sativa traditional varieties on Pc-resistant hybrid commercial rootstocks. The influence of the scion, the rootstock, and grafting per se on the vegetative budburst, growth, susceptibility to Pc and defence-related hormone profile of Castanea spp. are unknown. In a greenhouse experiment, these effects were evaluated by reciprocally grafting two Pc resistant C. crenata x C. sativa clones and two Pc susceptible C. sativa clones. Resistance to Pc and the hormone content of leaves and roots were rootstock-dependent, and survival rates of susceptible chestnuts strongly increased when grafted on resistant rootstocks. The scion had no effect on the resistance to Pc and the hormone profile of leaves and roots of grafted trees, but influenced vegetative budburst and primary growth. Grafting per se increased susceptibility to Pc and altered the defence-related phytohormone content of trees, especially in resistant rootstocks, but did not influence budburst and growth of trees. Grafting-induced alteration of the constitutive defense-related hormone profile could explain the increased susceptibility of resistant rootstocks to Pc. Nine days after infection, a dynamic hormonal response consisting of decreased jasmonates (JA and JA-Ile) in leaves and increased ABA and JA-Ile in roots was observed in resistant chestnuts. This is the first study addressing the role of grafting in modulating resistance to the soil-borne pathogen Pc in chestnut trees.

European and American chestnuts: An overview of the main threats and control efforts.

Chestnuts are multipurpose trees significant for the economy and wildlife. These trees are currently found around the globe, demonstrating their genetic adaptation to different environmental conditions. Several biotic and abiotic stresses have challenged these species, contributing to the decline of European chestnut production and the functional extinction of the American chestnut. Several efforts started over the last century to understand the cellular, molecular, and genetic interactions behind all chestnut biotic and abiotic interactions. Most efforts have been toward breeding for the primary diseases, chestnut blight and ink disease caused by the pathogens, Cryphonectria parasitica and Phytophthora cinnamomi, respectively. In Europe and North America, researchers have been using the Asian chestnut species, which co-evolved with the pathogens, to introgress resistance genes into the susceptible species. Breeding woody trees has several limitations which can be mostly related to the long life cycles of these species and the big genome landscapes. Consequently, it takes decades to improve traits of interest, such as resistance to pathogens. Currently, the availability of genome sequences and next-generation sequencing techniques may provide new tools to help overcome most of the problems tree breeding is still facing. This review summarizes European and American chestnut’s main biotic stresses and discusses breeding and biotechnological efforts developed over the last decades, having ink disease and chestnut blight as the main focus. Climate change is a rising concern, and in this context, the adaptation of chestnuts to adverse environmental conditions is of extreme importance for chestnut production. Therefore, we also discuss the abiotic challenges on European chestnuts, where the response to abiotic stress at the genetic and molecular level has been explored.

Resistance, tolerance and competence for a root pathogen in six woody species

The outcome of host–pathogen interactions depends on host defence and pathogen infection strategies. On the host side, two strategies, resistance and tolerance, have long been identified. They differ in their underlying mechanisms and their effects on pathogens. However, the dynamics of disease transmission also rely on host competence. This trait, defined as the ability of the host to allow pathogen transmission, is rarely studied for pathogen-supporting plants. We assessed the competence for Phytophthora cinnamomi of seedlings of five Quercus and one Castanea species and studied how this trait is correlated with resistance and tolerance. P. cinnamomi is responsible for the chestnut ink disease, alongside with Phytophthora × cambivora, which has a much less generalist behaviour. Each seedling was inoculated in minirhizotrons by root–mycelium contact and phenotyped for its resistance, tolerance and competence. The amount of necrosis (lesion length), the effect of infection on plant performance (root growth) and the sporangia number were measured. Moreover, we developed a droplet digital (dd) PCR assay to quantify the pathogen within host tissues near the inoculation point. This measure was used as a proxy of pathogen load. We were thus able to relate individual host competence to the pathogen content measured in the same root fragment. No significant correlation between the phenotyped traits was found at the individual level. However, at the species level, the least competent species (Q. robur, Q. petraea) proved to be the most resistant. These results may have important implications for management of chestnut and oak ecosystems threatened by ink disease.

Biocontrol of Phytophthora xcambivora on Castanea sativa: Selection of Local Trichoderma spp. Isolates for the Management of Ink Disease

Ink disease is a devastating disease of chestnut (Castanea sativa) worldwide, caused by Phytophthora species. The only management measures of this disease are chemical and agronomic interventions. This work focuses on the evaluation of the in vitro antagonistic capacity of 20 isolates of Trichoderma spp. selected in a diseased chestnut orchard in Tuscan Apennines (San Godenzo, Italy) for the biocontrol of Phytophthora xcambivora. Each Trichoderma isolate was tested to investigate pathogen inhibition capability by antagonism in dual cultures and antibiosis by secondary metabolites production (diffusible and Volatile Organic Compounds). The six most performing isolates of Trichoderma spp. were further assessed for their aptitude to synthesize chitinase, glucanase and cellulase, and to act as mycoparasite. All six selected isolates displayed the capability to control the pathogen in vitro by synergistically coupling antibiosis and mycoparasitism at different levels regardless of the species they belong to, but rather, in relation to specific features of the single genotypes. In particular, T. hamatum SG18 and T. koningiopsis SG6 displayed the most promising results in pathogen inhibition, thus further investigations are needed to confirm their in vivo efficacy.

INTEGRATION OF GEOMATIC TECHNIQUES FOR THE 3D REPRESENTATION AND MONITORING OF A VETERAN CHESTNUT TREE

Abstract. This study concerns the application of different geomatic techniques in forestry sector. An accurate 3D model of a veteran chestnut tree (Castanea sativa Mill.) has been realised integrating point clouds by Mobile Laser Scanner (MLS) and photogrammetry techniques. The surveys were conducted in two seasons (summer time: July 22nd 2021; autumn time: December 10th 2021), since chestnut is a deciduous tree and thus taking the chance to detect it also without leaves. Therefore, it was obtained tree’s metric data such as the diameter at breast height (DBH), the total height (TH), the crown basal area, the wood and crown volume. Annual surveys will result in a dataset of virtual trees that will be used to monitor the evolution of these noteworthy plants through time, which are expected to be considered as cultural heritage, given their story and their importance at regional and landscape level. The 3D model was then released for virtualization, making it accessible and available as a data container for different users. Such data will also provide historical memory about the presence of these veteran trees that might be lost in the future if affected by species-specific phytopathologies such as Ink Disease (Phytophthora spp.).

Anthropogenic and landscape features are associated with ink disease impact in Central Italy

AbstractInk disease of sweet chestnut (Castanea sativa) caused by the oomycetes Phytophthora cinnamomi and P. x cambivora is the limiting factor for chestnut cultivation in several European regions. The objective of this study was to explore how the spatial landscape heterogeneity affects the distribution pattern of ink disease over a large chestnut area in Central Italy using an approach that combined remote sensing, ground truthing activities and GIS. A multivariate model was developed that explained a large proportion of the variance of the impact of the disease using the density of roads and drainage networks as predictor variables. The association of these landscape elements, specifically with ink disease foci, also provides practical tool to improve the accuracy of monitoring of this disease and the preparation of risk maps.

Fatores moleculares no metabolismo fundamental de Phytophthora cinnamomi: Molecular factors in the fundamental metabolism of Phytophthora cinnamomic

Phytophthora cinnamomi is a diploid Oomycete that lives in the soil and is the cause of the death of countless plants worldwide. In Portugal the production of European chestnut is found mainly in the northern region where more than 80% is concentrated mainly in the region of Tras-os-Montes, making it a very valuable economic source for the region. European chestnut ink disease is a pathology caused mainly by P. cinnamomi and poses a threat to the preservation of chestnut (Castanea sativa Miller). The main objective of this work is the identification of genes that express proteins related to the fundamental metabolism of P. cinnamomi, through the use of bioinformatics tools. The genome of this microorganism is deposited in the biological database NCBI (National Center for Biotechnology Information) and through the sequences of this genome, ORFs (open reading frames) that code for proteins associated with fundamental metabolism were identified. With bioinformatics tools we can also predict the subcellular location, the three-dimensional structure, the physical-chemical characteristics and the characterization of the domains of these proteins.

Localized reshaping of the fungal community in response to a forest fungal pathogen reveals resilience of Mediterranean mycobiota

Mediterranean forests are facing the impact of pests such as the soilborne Phytophthora cambivora, the causal agent of Ink disease, and this impact is made more severe by global changes. The status and resilience of the soil microbial ecosystem in areas with such a disturbance are little known; however, the assessment of the microbial community is fundamental to preserve the ecosystem functioning under emerging challenges. We profile soil fungal communities in a chestnut stand affected by ink disease in Italy using metabarcoding, and couple high-throughput sequencing with physico-chemical parameters and dendrometric measurements. Since the site also includes an area where the disease symptoms seem to be suppressed, we performed several analyses to search for determinants that may contribute to such difference. We demonstrate that neither pathogen presence nor trees decline associate with the reduction of the residing community diversity and functions, but rather with microbial network reshaping through substitutions and new interactions, despite a conservation of core taxa. We predict interactions between taxa and parameters such as soil pH and C/N ratio, and suggest that disease incidence may also relate with disappearance of pathogen antagonists, including ericoid- and ectomycorrhizal (ECM) fungi. By combining metabarcoding and field studies, we infer the resilient status of the fungal community towards a biotic stressor, and provide a benchmark for the study of other threatened ecosystems.

Identification of Susceptibility Genes in Castanea sativa and Their Transcription Dynamics following Pathogen Infection.

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

A Histopathological Study Reveals New Insights Into Responses of Chestnut (Castanea spp.) to Root Infection by Phytophthora cinnamomi.

The European chestnut (Castanea sativa) is threatened by the hemibiotrophic oomycete Phytophthora cinnamomi, the causal agent of ink disease. Chestnut species have different susceptibility levels to P. cinnamomi, with the Asian species (C. crenata; C. mollissima) exhibiting the highest level of resistance. A histological approach was used to study the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of P. cinnamomi infection and the cellular responses it induces in roots. C. sativa (susceptible) and C. crenata (resistant) plantlets were inoculated with a P. cinnamomi virulent isolate with a zoospore suspension or by direct contact with mycelia agar pieces. Root samples were collected at 0.5, 3.5, 24, 48, and 72 h after inoculation (hai) for microscopic observations. Penetration was observed in both species at 0.5 and 3.5 hai with mycelium and zoospore inoculations, respectively. In both inoculation methods, following penetration into the rhizodermis, P. cinnamomi hyphae grew inter- and intracellularly through the cortex and into the vascular cylinder. C. crenata cells displayed a delay in the pattern of infection by having fewer cell layers colonized compared with C. sativa. At 72 hai, the collapse of the first layers of C. sativa cortical cells was observed, indicating the beginning of necrotrophy. C. crenata was able to respond more efficiently to P. cinnamomi than C. sativa by restricting the pathogen's growth area through the early activation of resistance responses such as callose deposition around some intracellular hyphae, hypersensitive response-like cell death, cell wall thickening, and accumulation of phenolic-like compounds.