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- Program for chestnut blight biocontrol based on hypovirulencePublication . Gouveia, Maria Eugénia; Coelho, Valentim; Araújo, Arsénio; Castro, João Paulo; Rodrigues, OrlandoChestnut Blight is an introduced disease in Portugal associated with virulent strains of the Asian fungus Cryphonectria parasitica (Murril) Barr. The disease causes great economic losses and presents a significant risk to the chestnut ecosystem. Eradication efforts and cultural sanitary measures didn’t control the disease and actually more than 10% of chestnut trees are blighted. A new method based on hypovirulence was achieved and a program for biological control of Chestnut Blight was implemented for field extended application. The program is a transfer initiative that encourages the application of Hypovirus CHV-1 strains as a component of the integrated management of the disease. Hypovirus CHV-1 strains were timely approved by national authorities (Ministry of Agriculture-DGAV) and stakeholders and schedules of field applications implemented. General achievements and results of the program will be presented and evaluated.
- Effect of competitive interactions between ectomycorrhizal and saprotrophic fungi on Castanea sativa performancePublication . Pereira, Eric Carvalho; Coelho, Valentim; Tavares, Rui Manuel; Lino-Neto, Teresa; Baptista, PaulaIn Northeast of Portugal, the macrofungal community associated to chestnut tree (Castanea sativa Mill.) is rich and diversified. Among fungal species, the ectomycorrhizal Pisolithus tinctorius and the saprotroph Hypholoma fasciculare are common in this habitat. The aim of the present work was to assess the effect of the interaction between both fungi on growth, nutritional status, and physiology of C. sativa seedlings. In pot experiments, C. sativa seedlings were inoculated with P. tinctorius and H. fasciculare individually or in combination. Inoculation with P. tinctorius stimulated the plant growth and resulted in increased foliar-N, foliar-P, and photosynthetic pigment contents. These effects were suppressed when H. fasciculare was simultaneously applied with P. tinctorius. This result could be related to the inhibition of ectomycorrhizal fungus root colonization as a result of antagonism or to the competition for nutrient sources. If chestnut seedlings have been previously inoculated with P. tinctorius, the subsequent inoculation of H. fasciculare 30 days later did not affect root colonization, and mycorrhization benefits were observed. This work confirms an antagonistic interaction between ectomycorrhizal and saprotrophic fungi with consequences on the ectomycorrhizal host physiology. Although P. tinctorius is effective in promoting growth of host trees by establishing mycorrhizae, in the presence of other fungi, it may not always be able to interact with host roots due to an inability to compete with certain fungi.
- Tinta do castanheiro. diagnóstico e controlo da dispersão no campoPublication . Rosário, Jorge Nuno; Gouveia, Maria Eugénia; Coelho, ValentimA Doença da Tinta do Castanheiro (DTC) é uma doença endémica nos soutos Portugueses apresentando surtos epidémicos cíclicos que causam grande mortalidade das árvores. Os Oomicetas, Phytophthora cinnamomi, Rands e P. cambivora (Petri) Buisman, parasitas associados com a DTC causam podridões radiculares que invariavelmente levam à morte dos castanheiros e perda total de produção e inviabilizam ainda a utilização do solo para a produção do castanheiro europeu. O tratamento do solo é difícil uma vez que não se conhece o comportamento das substâncias orgânicas no solo, é necessário tratar todo o perfil do solo e é difícil antecipar o efeito no conjunto dos microrganismos do solo. Neste trabalho utilizou-se solo de castanheiro onde DTC era evidente e plantaram-se castanheiros para avaliar e identificar as espécies de Phytophthora presentes e comparar num esquema de ensaio completamente casualizado com 10 repetições. Foi avaliada a resposta das plantas quando se utilizaram substâncias fungicidas seletivas, silicato de potássio, e fosfonato de potássio. Os resultados do ensaio em estufa permitirão obter informação para delinear estratégias de proteção para impedir a dispersão da doença quando os castanheiros evidenciam sintomas de DTC no campo.
- 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.
- 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.
- A remoção do inóculo de Cryphonectria parasitica na sustentabilidade dos soutosPublication . Gouveia, Maria Eugénia; Coelho, ValentimCryphonectria parasitica (Murrill) Barr. fungo associado ao cancro do castanheiro foi detectado pela primeira vez em Itália em 1938. Motivou grande preocupação em toda a Europa uma vez que o fungo evidenciou elevada agressividade em Castanea sativa. Em Portugal os prejuízos associados a esta doença foram noticiados apenas a partir de 1989. A partir de então o avanço da doença foi muito rápido, com estimativas de 6 % de árvores doentes na região de Trás-os-Montes. Com o objectivo de avaliar a eficácia da remoção dos cancros por excisão e/ou corte dos ramos, método utilizado no programa de erradicação desta doença, e avaliar o seu efeito na recuperação das árvores, realizaram-se ensaios num souto localizado em Parada–Bragança. Os resultados de 3 anos de observações evidenciam a eficácia da excisão dos cancros como método de remoção de inóculo apresentando no entanto uma acção limitada na recuperação das árvores. A presença de inóculo na área envolvente constitui um factor preponderante no aparecimento de novas infecções. Para que o efeito da remoção do inóculo possa contribuir para a recuperação das árvores e conter a dispersão da doença as acções de erradicação devem ser concretizadas em toda a extensão do foco da doença.
- Caracterização da estrutura populacional de Cryphonectria parasitica para aplicação da luta biológica por hipovirulência: um caso de estudo na Serra da PadrelaPublication . Pereira, Eric Carvalho; Prospero, Simone; Araújo, Arsénio; Coelho, Valentim; Rigling, Daniel; Gouveia, Maria EugéniaO castanheiro europeu (Castanea sativa Mill) é a principal espécie do sistema agroflorestal nas regiões montanhosas de Portugal. O castanheiro é atualmente afetado por vários agentes patogénicos agressivos, destacando-se o fungo Cryphonectria parasitica, que causa o Cancro do Castanheiro, que provoca a morte da árvore. A Hipovirulência, associada à presença de Cryphonectria hypovirus 1 no fungo fitoparasita, é um método específico e eficiente no controlo biológico do Cancro do Castanheiro. Para uma aplicação eficaz deste método no campo é necessário conhecer as características das estirpes agressivas de C. parasitica presentes no campo. Na Serra da Padrela foi selecionado um souto para realizar este estudo. Foi efetuado uma caracterização dos cancros ativos, e posteriormente o seu tratamento por Hipovirulência. Dos isolados obtidos dos cancros, 99% eram agressivos, sendo identificados 3 isolados como hipovirulentos. Todos foram classificados como vc-type EU-11, estando em maior abundância o mating-type 2, numa proporção de 2:1 comparativamente ao mating-type 1. Os cancros agressivos foram tratados com as estirpes hipovirulentas compatíveis tendo-se observado ao fim de 1 ano uma recuperação das árvores afetadas tendo-se confirmado a presença das estirpes hipovirulentas em cancro anteriormente agressivos.
- Potential of local hypovirulent strains of Cryphonectria parasitica for biological control of chestnut blightPublication . Gouveia, Maria Eugénia; Coelho, Valentim; Monteiro, Maria do LoretoCryphonectria parasitica, introduced in the NE of Portugal since 1989, is now an important and widespread pathogen of chestnut (Castanea sativa Mill.) which causes losses in chestnut fruit production and led to decline and death of many chestnut trees. Hypovirulence, a virus mediated attenuation of fungal pathogenesis, has not been frequent until now in Portugal but recently some cases of healing cankers were reported by chestnut growers. We assessed these sites and one of them, where healing cankers were clustered, was intensively surveyed. From all the trees with healing cankers, white and orange phenotypes of C. parasitica were isolated, which are both of the same vc type (EU-11). White isolates of C. parasitica showed reduced pigmentation in colony colour which was not completely stable, suppressed conidiation, reduction of phenol oxidase but no significant differences in lesion length and sporulation on chestnut twigs on dormant chestnut trees. Hypovirulent strains have dsRNA virus in the cytoplasm which is the typical element that reduces virulence in C. parasitica. Bands of dsRNA were identified in all of the white isolates and some variation in band dimension and number of bands was detected. The L-dsRNA (12 kbp), which is the diagnostic fragment of C. parasitica infection, was detected. The presence of white phenotypes of infected C. parasitica (CHV) and the fact that spreads occur in natural conditions is promising for its development as a biological control agent of Chestnut Blight.
- Eficácia do tratamento do Cancro do Castanheiro em ensaios de inoculação com estirpes hipovirulentas de Cryphonectriaparasitica(CHV1) em Trás-os-Montes (Portugal)Publication . Pereira, Eric Carvalho; Araújo, Arsénio; Prospero, Simone; Coelho, Valentim; Rigling, Daniel; Gouveia, Maria EugéniaO castanheiro europeu (Castanea sativa Mill) é a principal espécie do sistema agroflorestal nas regiões montanhosas de Portugal. O castanheiro é atualmente afetado por vários agentes patogénicos agressivos, destacando-se o fungo Cryphonectria parasitica, que causa o Cancro do Castanheiro, que provoca a morte da árvore. A Hipovirulência, associada à presença de Cryphonectria hypovirus 1 no fungo fitoparasita, é um método específico e eficiente no controlo biológico do Cancro do Castanheiro. Para uma aplicação eficaz deste método no campo é necessário conhecer as características das estirpes agressivas de C. parasitica presentes no campo. Na Serra da Padrela foi selecionado um souto para realizar este estudo. Foi efetuado uma caracterização dos cancros ativos, e posteriormente o seu tratamento por Hipovirulência. Dos isolados obtidos dos cancros, 99% eram agressivos, sendo identificados 3 isolados como hipovirulentos. Todos foram classificados como vc-type EU-11, estando em maior abundância o mating-type 2, numa proporção de 2:1 comparativamente ao mating-type 1. Os cancros agressivos foram tratados com as estirpes hipovirulentas compatíveis tendo-se observado ao fim de 1 ano uma recuperação das árvores afetadas tendo-se confirmado a presença das estirpes hipovirulentas em cancro anteriormente agressivos.