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- A Reliable Molecular Diagnostic Tool for CA90 (Castanea sativa × Castanea crenata) Hybrid Identification Through SSRPublication . Yussif, Toufiq Soale; Cruz, Nadine Evora da; Coelho, Valentim; Gouveia, Maria Eugénia; Choupina, AltinoChestnut trees are an essential source of both food and timber. However, the severe threats from invasive pests and diseases compromise their existence and productivity. In Europe, chestnut hybridization programs have been initiated to produce resilient rootstocks in response to ink disease. However, the gap in the identification of these hybrid plants is typically based on field observations and Morphological features and remains a challenge. Our study presents a marker set for distinguishing between chestnut hybrid CA90 (Castanea sativa × Castanea crenata), a hybrid with demonstrated resistance to Phytophthora cinnamomi, and other varieties using microsatellite (SSR) markers and bioinformatics tools. We used 35 chestnut samples, including three CA90 controls, hybrids sampled within Portugal, with an aim to define the profiles of the chestnut hybrids and varieties in this study based on band patterns and SSR motifs. We selected and modified nine distinct SSR primers with null allelic features from 43 already developed simple sequence repeat (SSR) markers. PCR amplification and agarose gel electrophoresis were used to amplify and visualize the DNA bands. To confirm genetic variations, 27 amplified bands were sequenced by Sanger sequencing. This analysis identified 31 SSRs across 22 SSR-containing sequences, with trinucleotide (67.74%) repeats being the most common, followed by repeats of dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%). A total of 18 alleles were observed for the nine loci. The alleles ranged from one to three per locus for the 35 samples. The novel locus CP4 could only be found in CA90 hybrids. This tool can aid in identifying and selecting disease-resistant hybrids, thereby contributing to chestnut production and management strategies.
- A Reliable Molecular Diagnostic Tool for CA90 (Castanea sativa × Castanea crenata) Hybrid Identification Through SSRPublication . Yussif, Toufiq Soale; Cruz, Nadine Evora da; Coelho, Valentim; Gouveia, Maria Eugénia; Choupina, Altino BrancoChestnut trees are an essential source of both food and timber. However, the severe threats from invasive pests and diseases compromise their existence and productivity. In Europe, chestnut hybridization programs have been initiated to produce resilient rootstocks in response to ink disease. However, the gap in the identification of these hybrid plants is typically based on field observations and morphological features and remains a challenge. Our study presents a marker set for distinguishing between chestnut hybrid CA90 (Castanea sativa × Castanea crenata), a hybrid with demonstrated resistance to Phytophthora cinnamomi, and other varieties using microsatellite (SSR) markers and bioinformatics tools. We used 35 chestnut samples, including three CA90 controls, hybrids sampled within Portugal, with an aim to define the profiles of the chestnut hybrids and varieties in this study based on band patterns and SSR motifs. We selected and modified nine distinct SSR primers with null allelic features from 43 already developed simple sequence repeat (SSR) markers. PCR amplification and agarose gel electrophoresis were used to amplify and visualize the DNA bands. To confirm genetic variations, 27 amplified bands were sequenced by Sanger sequencing. This analysis identified 31 SSRs across 22 SSR-containing sequences, with trinucleotide (67.74%) repeats being the most common, followed by repeats of dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%). A total of 18 alleles were observed for the nine loci. The alleles ranged from one to three per locus for the 35 samples. The novel locus CP4 could only be found in CA90 hybrids. This tool can aid in identifying and selecting disease-resistant hybrids, thereby contributing to chestnut production and management strategies.
- Comunidade de microfungos presente nos cancros tratados no tronco e ramos do castanheiro com a estirpe hipovirulenta (CHV-1)Publication . Coelho, Valentim; Guerra, Inês; Gouveia, Maria EugéniaMuitas espécies de fungos têm sido isoladas conjuntamente com Cryphonectria parasitica (Murr.) Barr dos tecidos de castanheiro com sintomas de Cancro do Castanheiro. Diferentes interações e comportamentos ecológicos foram identificados entre os diversos microfungos e C. parasitica. Foi objetivo deste trabalho (1) avaliar a composição de espécies de microfungos que co-ocorrem com C. parasitica após a introdução da estirpe hipovirulenta (2) estudar as interações dos diferentes microfungos com Cryphonectria Hypovirus I. Os fungos foram isolados de tecido vegetal de cancros curados retirados a 3 cm para o exterior e 3 cm para o interior e ainda no local da aplicação da estirpe CHV1. A identificação das diferentes espécies baseou-se em características morfológicas e métodos moleculares por amplificação e sequenciação da região ITS com a utilização dos iniciadores universais ITS1 e ITS4. Foram obtidos 75 isolados e identificadas 15 espécies. Os fungos presentes em maior número foram Biscogniauxia mediterranea, Penicillium glabrum e Cytospora diatrypelloidea. No exterior dos cancros curados as espécies presentes em maior número foram B. mediterranea e P. glabrum. C. diatrypelloidea foi a espécie mais representada no interior dos cancros curados
- Um bioensaio rápido e sensível na deteção de Phytophthora a partir do solo de castanheiros com sintomas da doença da tintaPublication . Machado, Júlia Melo; Coelho, Valentim; Gouveia, Maria EugéniaA doença da tinta do castanheiro (DTC) é uma doença endémica nos soutos portugueses apresentando surtos epidémicos cíclicos que causam grande mortalidade. Os Oomicetas, Phytophthora cinnamomi e Phytophthora cambivora são os parasitas mais frequentemente associados com a DTC embora outras espécies de Phytophthora possam também estar presentes. Os parasitas atacam as raízes e todo o seu ciclo de vida ocorre na rizosfera dependendo diretamente das condições de humidade e temperatura do solo. Causam podridões radiculares que consequentemente levam à perda total da produção e à morte dos castanheiros, inviabilizando ainda a utilização do solo para futura produção do castanheiro europeu. Neste trabalho foram pesquisadas as espécies de Phytophthora presentes no solo das árvores que apresentam sintomas de DTC. Utilizou-se o método de armadilha biológica que confere ao método elevada especificidade e eficácia de deteção. A utilização de discos de folha de castanheiro foi muito eficaz na deteção de Phytophthora quando o parasita se encontra ativo no solo. O bioensaio baseia-se na maximização das condições de formação e libertação dos zoósporos, o inóculo primário no processo de infeção, que permite o posterior crescimento em meio de cultura das diferentes espécies de Phytophthora. O método é um teste de deteção positivo e proporciona maior sensibilidade de deteção permitindo obter resultados confiáveis e rápidos.
- Perfil de utilização de fontes de carbono de isolados Virulentos e Hipovirulentos de Chryphonecria parasiticaPublication . Moura, Luísa; Santos, Fernando; Martins, Rita; Coelho, Valentim; Gouveia, Maria EugéniaO fungo Cryphonectria parasitica responsável pela doença do cancro do castanheiro é muito agressivo, levando à morte das árvores hospedeiras. Cryphonectria hypovirus 1 (CHV1) infeta C. parasitica e reduz a virulência do fungo (hipovirulência) alterando a sua morfologia em meio de cultura (pigmentação e capacidade de esporulação). O controlo biológico por hipovirulência tem sido usado na Europa, desde os anos 80 e tem demonstrando bons resultados no tratamento da doença, assim como acontece também em Portugal. Variações genéticas e fenotípicas de isolados hipovirulentos podem afetar o seu desempenho no controlo do cancro do castanheiro, pelo que a seleção de isolados passa pela caraterização biológica, incluindo a sua capacidade de esporulação. A avaliação do perfil de utilização de fontes de carbono dos isolados hipovirulentos poderá contribuir para uma melhor compreensão do seu metabolismo, complementando assim a sua caracterização biológica. O perfil metabólico de isolados portugueses virulentos e hipovirulentos de C. parasitica, foi avaliado em microplacas Biolog FF. Culturas puras de cada isolado foram cultivadas em meio PDA a 25 °C na ausência de luz durante 7 dias. Os mesmos isolados foram expostos à luz do laboratório para indução da formação de esporos durante 5 dias. As estirpes hipovirulentas não formam esporos em condições de luz natural, pelo que foi induzida a sua produção. Os isolados hipovirulentos inoculados em placas de PDA (7 dias a 25º) foram colocadas numa câmara de cultura durante 15 dias a 25ºC, com fotoperíodo de 18 horas e 10.000 lux. Suspensões de micélio e de conídios induzidos pela luz a 10.000 lux foram utilizadas para inoculação de Microplacas Biolog FF e incubadas a 25ºC durante 7 dias na ausência de luz. A densidade óptica a 490 nm (atividade mitocondrial) foi determinada usando um leitor de microplacas Biolog e o equipamento ASYS UVM 340 (Hitech GmbH) para cada placa, em intervalos de 24 h durante 7 dias. Verificou-se que os hidratos de carbono, aminoácidos, aminas/amidos, polímeros e outros compostos foram mais consumidos pelos isolados hipovirulentos quando se utilizou suspensões de micélio obtido na ausência de luz, o que sugere uma adaptação ecológica e estabelecimento destes isolados (CHV1) após a sua introdução em castanheiros doentes no campo. Contrariamente, o perfil metabólico obtido a partir de esporos revelou maior atividade dos isolados virulentos na utilização dos compostos testados. Este estudo permitirá relacionar os perfis metabólicos obtidos a partir de micélio e de esporos das estirpes hipovirulentas com as suas características biológicas, em avaliação no âmbito do Projeto BioChestnut.
- Almond disease Management. Epidemiology, sampling and control guidelines for bloom, leaves and annual first year twigs diseasesPublication . Gouveia, Maria Eugénia; Pinto, Beatriz; Coelho, ValentimAlmond tree diseases have been poorly researched in Portugal and there is no experimental data that prioritizes sanitary situations. The renewed interest for almond cultivation and introduction of new varieties with greater cultural intensification requires an in-depth knowledge of the pathogens that reduce production, the longevity of the trees and the profitability of the crop. Our studies are focused on orchards of recently planted almonds trees of Spanish varieties located in the north region of Portugal (Alfândega da Fé and Vila Nova de Foz Côa). Initially, the work was focused on the study of cortical cankers associated with Diaphorthe amygdali, an important disease in some of the new varieties. Field observations revealed the presence of a wide range of sanitary situations ranging from grafting incompatibility to epidemics of Monilinia laxa and Polystigma amygdalinum, major diseases of flowers and leaves. Dormant spurs and first year twigs with symptoms of dead buds and cankers were sampled in each studied orchards and the isolation and purification of pathogens was performed. The identification of pathogens by molecular methods showed the prevalence of Monilinia laxa with minor laboratorial detection of Diaphorthe amygdali. Monilinia laxa is associated with extensive rotting of the flowers, which is particularly important when blooming occurs in wet weather. Treatments with fungicides and no pesticides alternatives, treatment thresholds and fungicides efficacy need to be studied to find guidelines for disease management and avoid losses in almond production.
- Dectetion of Verticillium dahliae kleb from olive trees with chronic decline and dieback of branches and shootsPublication . Gouveia, Maria Eugénia; Coelho, ValentimVerticillium wilt of olive tree, associated with the soil borne fungus Verticillium dahliae Kleb., is considered an important disease in all countries of the Mediterranean region. This disease has been increasing in newly established olive orchards which have a negative economic impact in all regions of olive culture. In Portugal Verticillium wilt has not been considered an important disease and few studies were carried out about this disease. Recently, a great number of cases of chronic decline, slow growth and dieback of shoots and branches of olive trees have been reported in all regions where extensive plantations had occurred. These symptoms are characteristic of Verticillium wilt in olive tree, but very often are also associated with adverse environmental or agronomic conditions or inclusively with mechanical damages which hampered diagnosis of biological causes. In some of these reported situations and for diagnostic purposes we isolated Verticillum dahliae from plant tissues of trees with characteristic symptoms of decline and slow growth. The sample of plant tissues were tap water cleaned and surface disinfected with sodium hypochloride and incubated on PDA (Potato Dextrose Agar) in Petri dishes at 22-23 ºC in the dark. Positive isolation of the suspected pathogen has not been always obtained even though the same laboratory procedure was adopted.
- Gnomoniopsis smithogilvyi, the causal agent of chestnut brown rot reported from PortugalPublication . Coelho, Valentim; Gouveia, Maria EugéniaTo ensure fruit quality and promote good storage practices, it is important to identify the fungi associated with chestnut (Castanea sativa) nut rots. In 2018, in the Trás-os-Montes region of Portugal, five orchards were selected and 300 fruits were collected from the ground by hand during the harvest period. In the laboratory, the fruits were observed visually and the number of decayed nuts recorded. Morphological symptoms were evaluated in diseased nuts and with the help of a scalpel, five sections (2 × 2 mm) of an infected cotyledon were cut for fungal isolation. Sections of the nuts were dipped in 70% (v/v) ethanol for 2 minutes and placed in 90 mm diameter Petri dishes containing potato dextrose agar (PDA; 39 g/l) to allow mycelial growth. Plates were incubated at 25 ±2ºC for seven days in the dark. Single colonies were transferred to new PDA plates to obtain purified isolates for subsequent identification. Identification of purified isolates was initially based on morphological characteristics and species identification obtained by molecular methods. DNA extraction was performed using the REDExtract-N-Amp™ Plant PCR Kit (Sigma-Aldrich, USA), and the ITS region amplified using the universal primers ITS1 and ITS4 (White et al., 1990). The amplified ITS region was sequencing by Stabvida Laboratories (Caparica, Portugal). Sequences were compared with published sequences in the GenBank databases using BLAST. Gnomoniopsis smithogilvyi was isolated from chestnut nuts which had cotyledons with a whitish-brown colour (Fig. 1). On PDA, the mycelium of G. smithogilvyi was woolly with a greyish colour and colonies developed in concentric circles (Fig. 2). This fungal species was found in 0 to 4.3% of fruits in the studied orchards (Table 1). Isolates Samil4.1, Samil6.1 and Brg-16 were preserved on agar slopes and kept at 4 ºC in the fungal collection of Instituto Politécnico de Bragança. The GenBank Accession Nos. of these sequences are MN956833, MN960397 and MN960408. G. smithogilvyi was identified in an industrial processing plant in 2017, 2018 and 2019 and the proportion of infected nuts were 8.0, 5.3 and 5.0% respectively. These values are in line with the study conducted by Driss (2019) on industrial samples from Portugal where it was observed 6.4% of the nuts were affected by G. smithogilvyi. This is the first detection of G. smithogilvyi on chestnut nuts in orchards in Portugal. This species has been reported as the main or among the major endophytes of chestnut tree (Visentin et al., 2012) and was isolated from rotten nuts from several countries in Europe and Asia, and in Australia (Lione et al., 2018). The disease symptoms in nuts are a colour alteration and texture degradation and sometimes the kernels are dehydrated (Lione et al., 2018). Several attacks can affect fruit production (Vannini et al., 2017).
- Effect of mycorrhyzal inoculation on tolerance of Castanea sativa Mill plants to Phytophthora cinnamomi infectionPublication . Martins, Anabela; Gouveia, Maria Eugénia; Coelho, Valentim; Estevinho, Isabel; Pais, Maria SaloméTwo different trials (1) and (2) were made in order to test mycorrhizal inoculation effect against Phytophthora cinnamomi in vivo. Castanea sativa Mill seedlings were inoculated with Pisolithus tinctorius at sowing (M1). Two month later, half of the M1 plants were reinoculated (M2) with P. tinctorius. 1) at the same time, M1, M2 and nonmycorrhizal (NM) plants were inoculated with 0.1%, 0.5% and 2% of P. cinnamomi; 2) One month after reinoculation, M2 and nonmycorrhizal (NM) plants were inoculated with 0.1% and 0.5% P. cinnamomi. The results obtained for the two trials were very different in terms of mycorrhizal protection against P. cinnamomi. Trial 1) shows that mycorrhizal inoculation simultaneous to reinoculation could not induce protection against P. cinnamomi. Results of trial 2) showed mycorrhizal protection against P. cinnamomi for both percentages of inoculation. From the results obtained we can consider that mycorrhizal protection against P. cinnamomi depends on the time of mycorrhizal association. Trial 2) shows that 3 month after mycorrhizal fungus inoculation and 1 month after reinoculation plants present some degree of protection against P. cinnamomi, suggesting that after an adequate period, mycorrhization induces plant protection against this pathogenic fungus.
- Eficácia, manutenção e persistência de Cryphonectria hypovirus I (CHV1) como agente de controlo biológico do cancro do castanheiroPublication . Coelho, Valentim; Victor, Carina; Costa, Eduarda; Gouveia, Maria EugéniaO Cancro do Castanheiro (CC), causado por Cryphonectria parasitica (Murr.), é uma das doenças mais importantes em Castanea sativa Mill. no continente europeu. A aplicação de estirpes hipovirulentas compatíveis, como agente de controlo biológico (ACB) é considerado um método eficaz no controlo da doença e promove a recuperação dos castanheiros. O objetivo deste trabalho foi avaliar, em dois soutos na região de Trás-os-Montes: (1) a eficácia do tratamento; (2) a manutenção e persistência de CHV1 nos cancros tratados. O tratamento foi eficaz em praticamente todos os cancros tratados tendo ocorrido cicatrização e regeneração de tecidos corticais. A avaliação da manutenção e persistência de CHV1 nos cancros curados foi realizada por amostragem em 7 cancros curados no local da aplicação e a 3 cm para o exterior e 3 cm para o interior com a utilização de um punção T Lok™ biopsy needle (Jorgensen Laboratories, inc.). Os isolados com hipovirus CHV1 foram identificados pelas caraterísticas morfológicas das colónias depois de 7 dias de crescimento às escuras seguidas de 5 dias a luz difusa do laboratório. Os isolados CHV1 foram detetados em maior número tanto no local de aplicação como também no exterior e interior do cancro tratado.