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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.

One of the biggest threats for the chestnut tree in Portugal and in Europe is the ink disease. The disease is caused by two Phytophthora species (Phytophthora cinnamomi and Phytophthora cambivora). These root parasites have biological and epidemiological characteristics that provide them with rapid adaptation to the environmental conditions and have a high capacity for survival. With this work we intend to develop molecular methods for the detection and identification of Phytophthora in chestnut soils, providing greater sensitivity of detection and greater specificity of identification. In this study, two methods were compared for the detection of Phytophthora from soils infected with chestnut ink disease (natural soil from a stand with diseased chestnut trees and potting soil from greenhouses). In the classical method (baiting method), for the detection of Phytophthora, leaf discs of chestnut (Castanea sativa) were used and subsequent identification of isolates. For molecular methods, soil DNA was extracted from the DNeasy® PowerSoil® Pro Kit (Qiagen) and was amplified from the ITS region of ribosomal DNA (rDNA) using the universal primers ITS4 and ITS6. It was possible to obtain 67% of positive detection from natural soils by baiting method and 75% of positive detection from soil of pots from greenhouse. P. cinnamomi was the species identified from the baiting method and the nucleotide identity of the ITS region of the three identified isolates ranged between 98 and 100%. Trap tissue assays are very useful when the aim is to obtain isolates of different Phytophthora species and DNA-PCR assays are valuable and basic requirements for integrated management programs to evaluate the control measures applied to minimize impacts and control the disease. Uma das maiores ameaças para o castanheiro em Portugal e na Europa é a doença da tinta. A doença é causada por duas espécies de Phytophthora (Phytophthora cinnamomi e Phytophthora cambivora). Estes parasitas radiculares têm características biológicas e epidemiológicas que lhes proporcionam uma rápida adaptação às condições ambientais e têm uma elevada capacidade de sobrevivência. Com este trabalho pretendemos desenvolver métodos moleculares para a deteção e identificação de Phytophthora em solos naturais, proporcionando uma maior sensibilidade de deteção e uma maior especificidade de identificação. Neste estudo, foram comparados dois métodos para a deteção de Phytophthora em solos infetados com a doença da tinta do castanheiro (solo natural de um povoamento com castanheiros doentes e solo de vasos de estufas). No método clássico (tecido de armadilha), para a deteção de Phytophthora, foram utilizados discos de folhas de castanheiro (Castanea sativa) e subsequente identificação dos isolados. Para métodos moleculares, o ADN do solo foi extraído do DNeasy® PowerSoil® Pro Kit (Qiagen) e foi amplificado da região ITS do ADN ribossómico (rDNA) utilizando os primers universais ITS4 e ITS6. Foi possível obter 67% de deteção positiva de solos naturais através do método de tecido armadilha e 75% de deteção positiva de solo de vasos de estufa. P. cinnamomi foi a espécie identificada a partir do método de tecido armadilha e a identidade nucleotídica da região ITS dos três isolados identificados variou entre 98 e 100%. Os ensaios de tecido de armadilha são muito úteis quando o objetivo é obter isolados de diferentes espécies de Phytophthora e os ensaios de DNA-PCR são requisitos valiosos e básicos para programas de gestão integrada para avaliar as medidas de controlo aplicadas para minimizar os impactos e controlar a doença.

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 info:eu-repo/semantics/publishedVersion

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.

Phytophthora cinnamomi is an Oomycete considered one of the most widespread and destructive pathogens on the planet. Its geographic presence is cosmopolitan and the range of hosts is regarded as one of the largest. This pathogen causes enormous economic damages to important cultures worldwide; this has helped to attract the attention of the scientific community. It is the causal agent of chestnut “ink” disease (Castanea sativa Miller). The chestnut tree has different applications, besides chestnut and wood, it plays an important role in soil ecology and biodiversity. Therefore it is important to know the molecular mechanisms (genes and proteins) that are the basis of the Castanea sativa infestation process by Phytophthora cinnamomi and the nature of the pathogen-host interaction in order to establish strategies to combat and control this parasite. With bioinformatics tools, we were able to identify and characterize the Avr3a gene in genomic sequences of P. cinnamomi deposited in the Databases. This gene encodes a recognized 209 aminoacids, protein in the host cytoplasm where it triggers cell death. info:eu-repo/semantics/publishedVersion

Mestrado de dupla diplomação com a Université Libre de Tunis Phytophthora cinnamomi, soil-borne pseudo fungi from the class of oomycete, not only representing a real threat to strategic cultures across the world but also in forestry. This pathogen is able to colonize roots of Castanea sativa causing ink disease. For that many studies are established to well understand the mechanism of infection by exogenous molecules secreted during contact with host cells. Those molecules have been identified thanks to the progress in molecular biology using technics like RNA Seq. The Avr like genes expresses an RXLR protein family (Arginine-X-leucine-Arginine; X could be any amino acid), implicated in the pathogenicity of several pseudo-fungi of the genus Phytophthora, especially in Phytophthora infestans, Phytophthora sojae and Phytophthora palmivora. In this work, we isolate sequence, and characterize the Avr3a and Avr1b homologs in P. cinnamomi using molecular biology techniques such as PCR. In addition, we identified, in genomic sequences deposited in the databases, molecular factors involved in the metabolism and pathogenicity of P. cinnamomi using bioinformatics tools. In order to better understand its role in the pathogenicity mechanism, interference RNA silencing cassettes were designed for the genes encoding AVR proteins. cinnamomi, é um pseudo fungo do solo da classe dos oomicetos, que não só representam uma ameaça real para culturas agrícolas com importância econômica, mas também na silvicultura. Este patógeno é capaz de colonizar as raízes de Castanea sativa causando a doença de tinta. Face ao exposto, muitos estudos têm sido realizados com o objectivo de entender bem o mecanismo de infecção por moléculas exógenas secretadas durante o contato com células hospedeiras. Estas moléculas foram identificadas graças ao progresso da biologia molecular usando técnicas como o RNA Seq. Os genes AVR expressam uma família de proteínas RXLR (Arginina-X-leucina- Arginina; X pode ser qualquer aminoácido), implicada na patogenicidade de vários pseudo- fungos do gênero Phytophthora, especialmente em P. infestans, P. sojae e P. palmivora. Neste trabalho, isolamos, sequenciamos e caracterizamos os homólogos Avr3a e Avr1b em P. cinnamomi utilizando técnicas de biologia molecular como PCR. Além disso, identificamos, em sequências genómicas depositadas nas bases de dados, fatores moleculares envolvidos no metabolismo e patogenicidade de P. cinnamomi usando ferramentas de bioinformática. A fim de compreender melhor o seu papel no mecanismo de patogenicidade, foram desenhadas cassetes de silenciamento por ARN de interferência para os genes que codificam as proteínas AVR.

Appalachian forests are threatened by a number of factors, especially introduced pests and pathogens. Among these is Phytophthora cinnamomi, a soil-borne oomycete pathogen known to cause root rot in American chestnut, shortleaf pine, and other native tree species. This study was initiated to characterize the incidence of P. cinnamomi on surface mined lands in eastern Kentucky, USA, representing a range of time since reclamation (10, 12, 15, and 20 years since reclamation). Incidence of P. cinnamomi was correlated to soil properties including overall soil development, as indicated by a variety of measured soil physical and chemical parameters, especially the accumulation of soil organic carbon. P. cinnamomi was detected in only two of the four sites studied, aged 15 and 20 years since reclamation. These sites were generally characterized by higher organic matter accumulation than the younger sites in which P. cinnamomi was not detected. These results demonstrate that P. cinnamomi is capable of colonizing reclaimed mine sites in Appalachia; additional research is necessary to determine the impact of P. cinnamomi on susceptible tree species at these sites.

Tradução de leaflet do projeto AGFORWARD - Os sistemas agroflorestais com castanheiro (Castanaea sativa Miller) são um sistema tradicional de uso do solo no este da província de Lugo, na Galiza, noroeste de Espanha. Embora os soutos raramente sejam associados a outras culturas (devido à baixa produção sob o coberto arbóreo), ou pastoreados (devido ao receio de danos nas árvores), estes povoamentos criam um mosaico ligeiro de usos da terra que inclui áreas cultivadas e floresta. Contudo, onde os declives acentuados tornam a apanha da castanha não lucrativa, introduz-se o pastoreio de porcos durante o outono e inverno. Os soutos são também uns dos melhores habitats para a produção comercial de cogumelos comestíveis. Existe uma procura crescente dos agricultores para o estabelecimento de soutos com variedades comercialmente atrativas. Além dos benefícios ambientais do castanheiro, pode aumentar-se a rentabilidade das novas plantações através da gestão do andar sob coberto e da pastorícia. O método tradicional de enxertia de variedades em porta-enxertos de castanheiro-bravo (usado em áreas sem doença-da-tinta), assim como em híbridos resistentes àquela doença, é ineficaz e incapaz de responder à procura crescente de plantas. As variedades que se desenvolvem das suas próprias raízes poderiam potencialmente evitar o processo relativamente complexo de enxertia e ajudar ao rápido estabelecimento nas áreas livres da doença-da-tinta.

The most dangerous pathogen affecting the production of chestnuts is Phytophthora cinnamomi a hemibiotrophic that causes root rot, also known as ink disease. Little information has been acquired in chestnut on the molecular defense strategies against this pathogen. The expression of eight candidate genes potentially involved in the defense to P. cinnamomi was quantified by digital PCR in Castanea genotypes showing different susceptibility to the pathogen. Seven of the eight candidate genes displayed differentially expressed levels depending on genotype and time-point after inoculation. Cast_Gnk2-like revealed to be the most expressed gene across all experiments and the one that best discriminates between susceptible and resistant genotypes. Our data suggest that the pre-formed defenses are crucial for the resistance of C. crenata to P. cinnamomi. A lower and delayed expression of the eight studied genes was found in the susceptible Castanea sativa, which may be related with the establishment and spread of the disease in this species. A working model integrating the obtained results is presented info:eu-repo/semantics/publishedVersion

Summary The research was performed in a new and isolated ink disease outbreak. Nine sweet chestnuts of comparable age, growing under same environmental and site conditions, and belonging to three phytosanitary classes (healthy, infected but asymptomatic and symptomatic) were randomly selected. Their ectomycorrhizal community was monitored during two periods, with regard to species abundance, to verify whether the community composition can be useful as an ink disease bioindicator. From the 216 samples, 29 ectomycorrhizal species were recorded, with abundances that changed with the health status of the tree. The results demonstrated that the mycorrhizal community composition was highly related to the ink disease level, allowing the consideration of the use of this parameter as a tool for the quick detection and control of the early stages of the disease.

The two most destructive diseases of chestnut (Castanea sativa Miller) are chestnut blight and ink diseases which are spreading in Italy and through other European countries. These constrain factors play an important role in the evolution of chestnut stands and orchards, affecting their management. Predominance of hypovirulence was observed in the majority of European chestnut stands and orchards. Healing cankers arising from the action of hypovirulent strains of Cryphonectria parasitica were detected in the stands investigated. Factors involved in the natural spread and predominance of the hypovirulent strains are cited. New lines of the parasite spread, but the damages produced are undetectable for the effect of the natural mixed inoculum, which could buffer any variations in the genome lines. Management of chestnut stands and orchards and the spread of hypovirulent strains enhances the natural biological control of the disease. Ink disease attacks produce severe damage in chestnut stands and orchards and influence their evolution Treatments with integrated organic fertilisers have produced the re-growth of symptomatic chestnut trees.

Mestrado com dupla diplomação com o High Institute of Biotechnology of Monastir (ISBM), Tunisia Ink Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is caused by two species: Phytophthora cinnamomi and Phytophthora cambivora, being the first one the foremost pathogen of ink disease in Portugal. P. cinnamomi is one of the most aggressive and widespread plant pathogen with nearly 1,000 host species. This oomycete causes enormous economic losses and it is responsible for the decline of many plant species in Europe and worldwide. Up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels, especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP (glucanase inhibitor protein) and NPP1 (necrosis-inducing Phytophthora protein 1) produced by P. cinnamomi during the infection. The technique of RNA interference was used to knockdown the gip gene of P. cinnamomi. Transformants obtained with the silenced gene have been used to infect C. sativa, in order to determine the effect of gene silencing on the plant phenotype. To know more about the function of GIP and NPP1 involved in the mechanism of infection, the ORF’s of gip and npp1 genes have been cloned to the pTOR-eGFP vector for a future observation of P. cinnamomi transformants with fluorescent microscopy and determination of the subcellular localization. Moreover the prediction by bioinformatics tools indicates that both GIP and NPP1 proteins are secreted. The results allow to predict the secretory destination of both GIP and NPP1 proteins and confirm RNAi as a potential alternative biological tool in the control and management of P. cinnamomi. Keywords: A doença de tinta do castanheiro é considerada uma das mais importantes causas do declínio dos soutos. A quebra da produção de Castanea sativa Mill é causada por duas espécies: Phytophthora cambivora e Phytophthora cinnamomi, sendo o principal causador da doença da tinta em Portugal. P.cinnamomi é um dos patógenos de plantas mais agressivo e generalizado, com cerca de 1.000 espécies de hospedeiros. Este oomiceta provoca enormes perdas económicas e é responsável pelo declínio de muitas espécies de plantas na Europa e no mundo. Até ao momento não se conhecem tratamentos eficazes para combater esses patógenos. Devido à importância do castanheiro a níveis económico e ecológico, especialmente em Portugal, é importante explorar os mecanismos moleculares que determinam a interação entre espécies de Phytophthora e plantas hospedeiras através do estudo das proteínas GIP (proteína inibidora de glucanases) e NPP1 (proteína indutora de necroses em Phytophthora) produzidos por P.cinnamomi durante a infeção. A técnica de RNA de interferência foi usada para o silenciamento do gene gip de P.cinnamomi. Os transformantes obtidos com o gene silenciado foram utilizados para infectar C. sativa, a fim de determinar o efeito de silenciamento de genes no fenótipo da planta. Para saber mais sobre a função de GIP e NPP1 no mecanismo da infeção, estes genes foram clonados no vector pTOR-eGFP para uma posterior observação de transformantes de P.cinnamomi com microscopia de fluorescência a fim de determinar a sua localização subcelular, apesar de, a previsão com ferramentas bioinformática indicarem que tanto GIP como NPP1 são proteínas secretadas para o meio extracelular. Os resultados permitem prever o destino celular de ambas as proteínas e confirmar a técnica de silenciamento de genes por iRNA como uma potencial ferramenta biológica alternativa no controle de P. cinnamomi.

International audience; Genetic variation in 23 populations of European chestnut (Castanea sativa L) to Ink Disease caused by Phytophthora cambivora (Petri) Buisman was studied in three labs. The populations represented three domestication levels (naturalized populations, coppice forests and orchards) and were sampled in 10 locations in five countries (Italy, France, Greece, Spain, and the UK). Adult chestnut susceptibility to Ink Disease was assessed by measuring lesion length following inoculation of excised shoots with P. cambivora. Half-sib families were harvested in most of these populations and the seedlings were root-inoculated. Provenance and family variance components were estimated. Significant variation in the extent of shoot colonization by P. cambivora was observed within and among adult tree populations, suggesting a large amount of genetic variation in resistance. No consistent ranking of the domestication levels for susceptibility was observed. Some results indicate selective pressure exerted by P. cambivora on local populations. Lesion measurements in a set of 48 trees inoculated in 2001 and 2002 were correlated (r = 0.58). One or more resistant trees (lesion length < 10 mm) were identified in 15 populations. Root inoculation of seedlings showed that only three families had a mean value for percentage of infected taproot = 15% or comparable to seedlings of the resistant control 'Marigoule'. Lesion lengths in parent trees and percentage of infection of the taproot of their seedlings were not highly correlated

Chestnut blight and ink disease, the two most destructive diseases of chestnut (Castanea sativa Miller), are spreading through European chestnut orchards and stands, playing an important role in their evolution and affecting management. Predominance of hypovirulence was observed in the majority of European countries. Abnormal cankers arising from the action of hypovirulent strains of Cryphonectria parasitica were detected in the stands investigated. Blight may be now considered as naturalised and endemic in almost all European chestnut stands but, because of the spread of hypovirulence, damage primarily affects weak and stressed trees. Blight can act as a bio-regulator in chestnut ecosystems. Spread of new virulent lines of the parasite, wildfire and other stress factors can change this favourable situation, but in the chestnut stands visited to date this occurs in limited areas and only for a short time. Sylvicultural management and, if necessary, artificial combined inoculation with selected hypovirulent strains enhances natural biological control of the disease. Ink disease attacks produce severe damage in chestnut stands and influence their evolution towards mixed woods. Monitoring of ink disease foci and control of blight evolution are desirable in chestnut management projects., Le chancre de l'écorce et la maladie de l'encre constituent les deux phytopathologies les plus virulentes pour le châtaignier {Castanea sativa Miller). Ces maladies s'étendent dans les vergers et les châtaigneraies d’Europe, jouant un rôle important dans leur évolution et affectant leur gestion. Une prédominance d'hypovirulence a été observée dans la majorité des pays européens. Des chancres anormaux issus de l'action de souches hypovirulentes de Cryphonectria parasitica ont été détectés dans les châtaigneraies inventoriées. Le chancre peut maintenant être considéré comme naturalisé et endémique dans la quasi totalité des châtaigneraies européennes mais, en raison de la diffusion de l'hypovirulence, les dégâts affectent principalement les arbres affaiblis et soumis à des stress écologiques. La maladie peut donc agir comme un biorégulateur dans les écosystèmes dominés par le châtaignier. L’expansion de nouvelles lignes virulentes du parasite, les incendies et d’autres facteurs de stress peuvent modifier cette situation favorable ; cependant, dans les châtaigneraie suivies jusqu’à présent, ceci se produit dans des zones limitées et seulement durant un temps limité. La gestion sylvicole et, si nécessaire, l'inoculation artificielle de souches hypovirulentes sélectionnées augmente le contrôle biologique naturel de la maladie. La maladie de l’encre engendre des dommages sévères dans les châtaigneraies et influence leur évolution vers des boisements mixtes. Le suivi de la maladie de l’encre et le contrôle de l’évolution du chancre de l’écorce doivent être pris en compte dans les projets de gestion durable des châtaigneraies., Turchetti T., Maresi Giorgio. Effects of diseases on chestnut orchards and forest ecosystems. In: Ecologia mediterranea, tome 26,2000. pp. 113-121.

[ES] Por vez primera se ha abordado el estudio de la sobre-expresión de los genes CsTL1 y CsCh3, aislados de castaño y que codifican una proteína tipo taumatina y una quitinasa, respectivamente, con el fin de regenerar plantas de castaño que muestren tolerancia o resistencia a las enfermedades de la tinta y del chancro, causadas por los hongos Phytophthora cinnamomi Rands, y Cryphonectria parasitica (Murr.) Barr utilizando para ello la transformación genética de líneas embriogénicas de castaño europeo. Los genes se clonaron en el vector pK7WG2D que contiene el gen marcador de selección neomicina fosfotransferasa (nptII) y el gen reportero de la proteína verde fluorescente (gfp). La eficiencia de la transformación con el gen CsTL1 fue dependiente del genotipo y varió del 32,5% en la línea CI-9 al 7,1% en la línea CI-3. Se obtuvieron un total de 126 líneas independientes transformadas. También la eficacia de la transformación con el gen CsCh3 varío con el genotipo, alcanzando el 25,0% en la línea CI-9 y el 4,2% en la línea C12- H1, obteniéndose un total de 175 líneas transgénicas independientes. La presencia e integración de ambos genes en el ADN genómico se confirmó mediante PCR y Southern blot. Mediante qPCR se demostró que la expresión de CsTL1 fue 13.5 veces mayor en una línea transgénica comparada con su correspondiente línea no transformada. Se regeneraron plantas transgénicas de castaño después de las etapas de maduración y germinación de los embriones somáticos transformados. Las plantas completan su desarrollo en un invernadero específicamente diseñado para el crecimiento de plantas transgénicas y se han iniciado los ensayos de resistencia a las enfermedades de las plantas regeneradas, [EN] The availability of a system for direct transfer of anti fungal candidate genes into European chestnut (Castanea sativa) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. and blight disease caused by Cryphonectria parasitica (Murr.) Barr. For the first time, a chestnut thaumatin-like protein gene (CsTL1) and a chitinase (CsCh3), both isolated from European chestnut cotyledons, have been overexpressed in three European chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens vector harbouring the nptII selectable and the green fluorescent protein (gfp) reporter genes. The transformation efficiency for CsTL1 was clearly genotype-dependent and ranged from 32.5% in the CI-9 line to the 7.1% in the CI-3 line. A total 126 independent transformed lines were obtained. A similar pattern has been recorded for CsCh3: 25.0% in the line CI-9 and 4.2% in the line C12-H1 and a total number of 175 transgenic lines were recorded. The presence and integration of chestnut genes in genomic DNA was confirmed by PCR and Southern blot analyses. qPCR revealed that CsTL1 expression was up to 13.5 fold higher in a transgenic line compared with its corresponding untransformed line. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos which were acclimatized and grown in greenhouse. Analyses of tolerance/resistance to the fungal diseases on these plants are under way.

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. On the Nordeste Transmontano region (northeast Portugal), the Castanea sativa chestnut culture is extremely important. The biggest productivity and yield break occurs due to the ink disease, caused by Phytophthora cinnamomi which is one of the most widely distributed Phytophthora species, with nearly 1000 host species. The knowledge about molecular mechanisms responsible for pathogenicity is an important tool in order to combat associate diseases of this pathogen. Complete open reading frames (ORFs) of act1, act2 and tub1 genes who participate in cytoskeleton formation in P. cinnamomi were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) polymerase chain reaction (PCR). act1 gene comprises a 1128 bp ORF, encoding a deduced protein of 375 amino acids (aa) and 41,972 kDa. act2 ORF comprises 1083 bp and encodes a deduced protein of 360 aa and 40,237 kDa. tub1 has a total length of 2263 bp and encodes a 453 aa protein with a molecular weight of 49.911 kDa. Bioinformatics analyses shows that actin1 is ortholog to the act1 genes of Phytophthora infestans, Phytophthora megasperma and Phytophthora melonis; actin2 is ortholog to the act2 genes of P. infestans, Phytophthora brassicae, P. melonis and Pythium splendens and tubulin1 shows the highest orthology to P. infestans and P. capsici α-tubulin genes. Analysed 3D structure of the three putative proteins revealed a spatial conformation highly similar to those described for orthologous proteins obtained by X-ray diffraction. COMBATINTA/SP2.P11/02 Interreg IIIA–Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund.

International audience; Soil borne Phytophthora cinnamomi and Phytophthora cambivora are considered the most pathogenic species associated with chestnut (Castanea sativa) decline in Europe. Mapping their incidence and distribution from nursery and plantation soils may offer valuable information for limiting spread. As conventional biological baiting and taxonomic confirmation is generally time consuming, labour, logistically and space intensive, we have focused on the development of a specific touchdown nested multiplex Polymerase Chain Reaction (PCR) approach for the simultaneous detection of both species direct from soil. Pre-existing and novel primers, based on Internal Transcribed Spacer (ITS) sequences, have been evaluated for their specificity and use in a multiplex capacity in various combinations. Coupled to this we have modified a mechanical lysis procedure for DNA extraction from up to 10 g of chestnut under storey soils (ranging from 0.5 to 25 μg DNA g−1 fresh soil). Using serial dilutions and/or polyvinylpolypyrrolidone chromatography purification, both species have been successfully detected, in artificially and naturally infected soils. Levels of assay detection are comparable to other Phytophthora species where PCR based diagnostic systems have been reported. A qualitative evaluation of this approach against conventional baiting is presented.

Tecniche di remote sensing, integrate con i sistemi informativi geografici (GIS), sono state applicate per monitorare la presenza di focolai del mal dell’inchiostro in alcune aree castanicole del centro Italia. Per questo lavoro si è ricorsi all’uso di immagini aeree multispettrali ad alta risoluzione. L’alta definizione delle immagini ha permesso di distinguere i focolai in base alla firma spettrale della chioma. La classificazione a massima verosimiglianza (maximum likelihood) è stata associata ad una metodologia con la quale sono state separate le chiome delle piante morte dalle zone di radura. Il prodotto finale, convalidato da attività di verifica a terra, è stata la produzione di mappe di distribuzione del mal dell’inchiostro di castagno. La qualità del lavoro è stata accertata attraverso la stima dell’accuratezza. La distribuzione ed il gradiente di analisi del mal dell’inchiostro, causato da Phytophthora cambivora sono stati monitorati per 2 anni in un bosco ceduo di castagno del centro Italia. Durante questo periodo, l'incidenza della malattia, la severità e la mortalità delle piante è aumentata sull’intera area. I dati dei parametri di malattia studiati sono stati introdotti in ambiente GIS dovesono stati elaborati come strati tematici. Gli indici di dispersione e i modelli di dispersione empirici sono stati impiegati per studiare la distribuzione spaziale della malattia nell’area oggetto di studio. I semivariogrammi sono stati utilizzati per interpolare i dati spaziali e per mappare il mal dell’inchiostro. Una dipendenza spaziale è stata individuata tra le variabili studiate. Il mal dell’inchiostro ricorre, preferenzialmente, lungo i compluvi dove è presente una grande quantità d’inoculo. Una relazione negativa è stata evidenziata tra l'incidenza della malattia, la severità e la mortalità rispetto alla distanza lineare dai compluvi. Remote sensing techniques were integrated with geographic information systems, and applied to monitoring Ink diseases in central Italy. The multi-spectral high resolution images, acquired with digital sensor mounted on an aircraft, were used to map the chestnut affected areas by Ink disease. The images definition allows to distinguish diseased plants and foci on the base of crown reflectance signature. A novel stratified method of separating dead tree crowns from bare soil, associated with a maximum likelihood classifier is proposed. The delivered product was a distribution map of Ink disease of chestnut that was validated with ground activities. Accuracy assessment is the procedure used to quantify product quality. Distribution and gradient analysis of Ink disease caused by Phytophthora cambivora were studied over a 2 years period in a chestnut forest in central Italy. Ink disease incidence, severity and tree mortality increased during the period over the studied chestnut forest. Disease descriptors and landform datasets were analysed by Geographic Information System software and displayed as a multilayer thematic map. Indices of dispersion and empirical dispersion models were used to study spatial distribution of disease in the investigated area. Semivariograms were used to interpolate spatial data and to map Ink disease. A logical spatial dependence of the studied variables was found. Ink disease occurred preferentially along natural drainages routes where multicyclic inoculum build up was likely to occur. A negative relationship was found between Ink disease incidence, severity and tree mortality with respect to distance from the drainage. Dottorato di ricerca in Protezione delle piante

Actins, as the essential component of cellular microfilament, are ubiquitous and highly conserved proteins that play key roles in several basic functions of organism such as cytoskeleton morphology, cell division, cell motility, cellular signal transduction, cellular interaction and organelle movements, as well as locomotion, phagocytosis, endocytosis and exocytosis . Actins are highly conserved structural proteins, found in all eukaryotes. So, actin gene sequences are used as tools in scientific research, for example, for phylogenetic analysis. Actin in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and filamentous fungi (Candida albicans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Filobasidiella neoformans) where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. Phytophthora cinnamomi is a host-nonspecific, soilborne, pathogen of many plant species. In Portugal it is most important as a pathogen of chestnut trees. The purpose of this study was to clone and determine the phylogenetic retionships evidencided by Phytophthora cinnamomi actins. In order to isolate the actin genes, P. cinnamomi was grown in cellophane-PDA medium and genomic DNA was used as a template in PCR amplification reactions combining degenerate primers Act1, Act2, Act3 and Act4. PCR fragments were purified, cloned into pGEM-T vector and transformants were selected. Complete open reading frames (ORFs) of act1 and act2 genes were achieved by HE-TAIL PCR, and submitted to EMBL databases (Accession numbers AM412175.1 and AM412176.1). Act1 has an 1128bp ORF, encoding a deduced protein of 375aa and 41,972kDa. Act2 ORF has 1083bp and encodes a deduced protein of 360aa and 40,237kDa. Deduced amino acid sequences were analyzed using FASTA programs from EMBL databases. Act1 showed a 98.9% identity with P. melonis actB, 94.4% with P. megasperma actin and 96.0% with P. infestans actin2. Act2 showed a 98.9% identity with Pythium splendens actin and 98.6% with P. brassicae actinA. COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal; Identification, characterization and role of molecular factors associated with the mechanisms of infection of Fagaceae species by Phytophthora cinnamomi” (PTDC/AGRAAM/67628/2006) FCT

Chestnut ink disease represents a great risk for chestnut (Castanea sativa Mill.) in all regions of chestnut production of Europe and North America. Induced by soil borne oomycetes, Phytophthora cinnamomi and P. cambivora, both parasites infect the root system and cause root and collar rot resulting in dieback, decline and finally, the death of infected trees. There are no single control measures for root rot pathogens and control with systemic and selective oomycetes biochemical substances can be important as part of the integrated disease management of these soil borne parasites. In this work we studied the effect of potassium phosphonate to protect roots from P. cinnamomi. Chestnut seedlings were planted in potting mix previously inoculated with P. cinnamomi (Pr 120). One group of five pots, with three seedlings each were submitted to potassium phosphonate foliar spraying (3 ml L -1 Atlante ® ) and another group of five pots were water foliar sprayed. Seedlings had been grown in a nursery for 120 days. At the end of the experiment, visual symptoms of the crown and root system were assessed. Parameters related with roots: root length, root rot length, root number, root rot number, crown diameter, height and biomass were evaluated. Statistic significant differences between treatments were obtained in all root studied variables. Potassium phosphonate by foliar spraying prevented P. cinnamomi infections of roots of chestnut and can be a key factor in the management of ink disease of chestnut.

Aged trees are considered as a valuable source of information on the past ecological conditions and social life where they are found, and their documentation and preservation are vital for the continuity of benefits they provide. Nineteen Castanea sativa Mill. individuals on abandoned agricultural plots near Artvin city center (Turkey) were measured for diameter at breast height (DBH), circumference at breast height (CBH) and height. DBH and CBH ranged between 72 cm and 186 cm, and between 231 cm and 615 cm, respectively. The highest tree is 22.20 m tall and the shortest is 13.65 m. Estimated ages of the trees ranged from 135 to 342 years. With their size and age, these trees can be designated as monumental trees. Protection of these trees will help in conserving C. sativa genetic resources and in passing the information they hold in their annual rings to future generations.