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- Alternaria alternata an emerging pathogen with great impact on olive grovesPublication . Carvalho, Teresa; Materatski, Patrick; Osa, Nicolás; Patanita, Mariana; Varanda, Carla; Ribeiro, Joana A.; Albuquerque, André; Campos, Maria Doroteia; Félix, M.R.Downy mildew, powdery mildew, grey mould, black rot, and grapevine trunk diseases are among the most important fungal or oomycete diseases affecting grapevine, with a high economic impact in all growing areas. In recent decades, extensive efforts have been made to reduce the use of agrochemicals in viticulture, turning the management of these diseases a major challenge. Given the urgent need to identify and develop new sustainable and effective protection strategies, a better understanding of plant defence mechanisms is essential to develop more tolerant plants and provide valuable insights for disease management. Although recent advances in molecular technologies have allowed the identification of several resistance genes, little is known about the complexity of the molecular mechanisms involved in grapevine-pathogen interactions. This chapter aims to bring together the most up-to-date findings on the regulation of grapevine defence mechanisms against major fungal and oomycete diseases by enumerating promising candidate genes that offer broad possibilities for inducing resistance to specific pathogens. We also focus on the use of functional genomics as a tool to study grapevine immunity to different pathogens, with particular emphasis on new emerging molecular technologies that will undoubtedly contribute to a sustainable plant breeding.
- qPCR as a sensitive tool for detecting Fusarium spp. in tomato plantsPublication . Campos, Maria Doroteia; Varanda, Carla; Patanita, Mariana; Ribeiro, Joana A.; Campos, Catarina; Materatski, Patrick; Albuquerque, André; Osa, Nicolás; Félix, M.R.The need to increase food production together with the demand to reduce the application of synthetic chemicals that have consequences on increasing the carbon footprint and negative impacts on the environment and human health, led to the search for alternative methods to protect plants against pathogens. The use of the highly sensitive real-time quantitative PCR (qPCR) arises as an extremely useful tool for studying various agents of infection in plants, such as fungi, viruses, or bacteria leading to a better control of diseases and limiting the use of chemical defence strategies. Given the high incidence of diseases in tomato plants caused by Fusarium spp., their consequent negative economic impacts, and the fact that most phytosanitary treatments are based on the application of synthetic fungicides, the establishment of a molecular-based tool that enables their early and accurate detection is of great interest. Furthermore, it will provide an additional tool for the screening of resistant plants. In the presented study, a TaqMan®-based qPCR method targeting the Fusarium spp.- specific internal transcribed spacer (ITS) region was developed for the simultaneous detection and quantification of a panoply of Fusarium species that affect tomato. As a proof of principle, the new qPCR assay was used to assess Fusarium spp. contamination of tomato field plants and of plants grown under controlled conditions. qPCR combined with the chemistry of TaqMan® MGB probes represents a highly specific and sensitive detection system, even when low amounts of target DNA are present, as in the case of early plant–fungi interactions.
- Identification of candidate genes involved in olive response to anthracnose for a sustainable disease managementPublication . Inácio, Diogo; Félix, M.R.; Campos, Catarina; Patanita, Mariana; Ribeiro, Joana A.; Varanda, Carla; Materatski, Patrick; Albuquerque, André; Osa, Nicolás; Peixe, A.; Campos, Maria DoroteiaAnthracnose is a disease that affect the olive tree caused by fungi of the genus Colletotrichum. These fungi are responsible to premature fruit drop and a consequent decrease in the oil quality, and also cause defoliation of trees compromising the production of the following years. Currently the best control strategy is based on application of synthetic fungicides, with a regulatory pressure in agriculture worldwide to limit its use. Looking for a sustainable disease management, the present study reports the transcriptional changes of olive genes that encode enzymes directly associated with the maintenance of the balance of oxidative oxygen species (ROS), in response to Colletotrichum sp. infection. The cultivar selected for the studies was ‘Galega vulgar’, known to be extremely susceptible to this disease. Plants used in the experiments were from in vitro culture (to warranty their healthy status), transplanted to pots and maintained under controlled conditions. Leaves of the olive plants were inoculated with a spore suspension of Colletotrichum sp., and leaf samples were collected before fungi inoculation (T0) and at 10 days (T1) and 35 days (T2) after inoculation. Confirmation of the presence of the fungi in inoculated plants was performed following a real-time qPCR approach. The selected target genes for expression analysis were Superoxide dismutase (SOD), Endochitinase_EP3-like (CHI2), Glutathione S-transferase L3-like (TransFL3), Glutathione peroxidase 2 (PEROX2), Glutathione S-transferase THETA 1 (THETA), Glutathione S-transferase DHAR2-like (TransfDHAR), Glutathione peroxidase 5 (PEROX5) and Glutathione peroxidase 8 (PEROX8). Following a qPCR approach, a general up-regulation of the target genes was detected, but only CHI2 revealed a significantly up-regulation (p<0.005) between T0 and T2 in response to Colletotrichum sp. infection, being a promising candidate to be later used in functional analysis. We emphasize the importance of this study for the identification of candidate genes to incorporate new sources of resistance of olive trees to anthracnose with the promotion of the development of sustainable management strategies.
- Papel da proteína de planta Dicer-like 4 nas infeções mistas dos vírus OMMV e OLV-1Publication . Varanda, Carla; Castanho, Beatriz; Albuquerque, André; Ribeiro, Joana A.; Patanita, Mariana; Osa, Nicolás; Campos, Maria Doroteia; Félix, M.R.; Materatski, PatrickOs vírus Olive mild mosaic virus (OMMV) e Olive latent virus 1 (OLV-1) estão largamente disseminados nos olivais portugueses, aparecendo frequentemente em infeções mistas. Estudos anteriores realizados em plantas de Nicotiana benthamiana, revelaram que quando os vírus estão presentes em infeções mistas, observa-se uma intensificação dos sintomas, sugerindo um efeito sinérgico. Neste trabalho, pretendeu-se testar se o mecanismo de defesa das plantas baseado no silenciamento genético de RNA está envolvido na exacerbação dos sintomas verificada nas infeções mistas de OLV-1 e OMMV. Para tal, avaliou-se a expressão do gene que codifica a proteína de planta Dicer-like 4 (DCL4), componente primário da defesa antiviral contra vírus de RNA. Plantas de N. benthamiana foram inoculadas por transmissão mecânica, com OMMV, com OLV-1 e com uma mistura de OMMV + OLV-1. A expressão dos genes da cápside de cada um dos vírus foi determinada por qPCR e usada para estimar a presença dos mesmos, assim como do gene que codifica a DCL4, aos 5 e 12 dias após inoculação. A análise dos resultados permitiu observar que a exacerbação dos sintomas verificada em infeções mistas, não é traduzida numa maior acumulação viral. De facto, ambos os vírus apresentaram uma menor acumulação quando na presença do outro vírus. Quanto à DCL4, verificou-se que esta se expressa mais na planta na presença dos dois vírus em simultâneo, especialmente aos 5 dias após a inoculação. Na presença apenas do vírus OLV-1, a DCL4 expressa-se menos quando há mais expressão do vírus (aos 12 dias). No caso do OMMV, a DCL4 expressa-se mais quando há maior expressão do vírus. Este trabalho parece assim sugerir que a DCL4 pode estar associada ao silenciamento do vírus OMMV ao passo que o OLV-1 parece conseguir ‘iludir’ o mecanismo de defesa da planta, mas tal não acontece quando na presença de OMMV. Este estudo permite incrementar o conhecimento das interações vírus-planta, essencial para o desenvolvimento de plantas resistentes a vírus, o que atualmente se reveste de extrema importância no âmbito de uma agricultura sustentável.
- Insights into grapevine defence response against fungal diseases towards a sustainable plant breedingPublication . Patanita, Mariana; Félix, M.R.; A. Ribeiro, Joana; Varanda, Carla; Albuquerque, André; Materatski, Patrick; Osa, Nicolás; Campos, Maria DoroteiaDowny mildew, powdery mildew, grey mould, black rot, and grapevine trunk diseases are among the most important fungal or oomycete diseases affecting grapevine, with a high economic impact in all growing areas. In recent decades, extensive efforts have been made to reduce the use of agrochemicals in viticulture, turning the management of these diseases a major challenge. Given the urgent need to identify and develop new sustainable and effective protection strategies, a better understanding of plant defence mechanisms is essential to develop more tolerant plants and provide valuable insights for disease management. Although recent advances in molecular technologies have allowed the identification of several resistance genes, little is known about the complexity of the molecular mechanisms involved in grapevine-pathogen interactions. This chapter aims to bring together the most up-to-date findings on the regulation of grapevine defence mechanisms against major fungal and oomycete diseases by enumerating promising candidate genes that offer broad possibilities for inducing resistance to specific pathogens. We also focus on the use of functional genomics as a tool to study grapevine immunity to different pathogens, with particular emphasis on new emerging molecular technologies that will undoubtedly contribute to a sustainable plant breeding.
- CRISPR/Cas13 system: a technology for the successful control of plant virusesPublication . Ribeiro, Joana A.; Varanda, Carla; Materatski, Patrick; Campos, Maria Doroteia; Albuquerque, André; Patanita, Mariana; Osa, Nicolás; Félix, M.R.Viruses are among the most important causal agents of infectious diseases, having relatively small genomes that comprise RNA or DNA. They have the ability to rapidly replicate and spread throughout a crop, being very difficult to monitor and causing devastating diseases in many agricultural systems. These can lead to significant losses in crop quality and yield, resulting in extreme economic impacts worldwide and threatening the provision of adequate nourishment for a continuous growing population. There are no efficient chemical products that can eliminate an infecting plant virus without perturbing host cells. Therefore, preventive sanitary measures, such as the use of viral resistant or tolerant plants, are usually the only options. Conventionally, these resistant/tolerant plants were generated through a very time- consuming classical breeding process. However, nowadays, molecular plant breeding plays a key role to prevent and control plant viruses. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems allowed the development of a new technology that paves the way towards a new horizon for crop improvement. The first CRISPR/Cas systems studied were very useful for DNA-targeting, however, more recently identified types, such as CRISPR /Cas13, can specifically cleave single- stranded RNA in eukaryotic cells. Therefore, CRISPR/Cas13 is a promising tool for engineering plant immunity against a broad range of RNA viruses, which are the most abundant class of viruses in plants. The present work aims to bring together the most up-to-date information on CRISPR/Cas13 system to control plant viruses, discussing the limitations and future challenges for its application to produce virus resistant plants towards a more sustainable agriculture.
- Olive viruses: plant pathogens or plant protect agents?Publication . Félix, M.R.; Campos, Maria Doroteia; Patanita, Mariana; Ribeiro, Joana A.; Materatski, Patrick; Albuquerque, André; Osa, Nicolás; Varanda, CarlaHarmful symptoms in olive trees such as chlorotic lesions, defoliation, fruit and leaf deformation, stem and tree death, and poor olive oil quality are currently associated with viruses. In Portugal, virus affecting olive trees have been studied since the 90’s, and to date eleven virus have been detected in Portuguese olive orchards, out of a total of 17 detected worldwide in this host. Viral diseases are not treatable in an agronomic context and knowledge of these agents, and their epidemiology, is mandatory to establish and improve sanitary regulations and early detection tools. In dozens of olive orchards sampled in the north and south of Portugal, infection rates reach up to 100%. In these surveys, virus belonging to the Alpha- and Beta-necrovirus genera were the most prevalent. Viral genomes were molecular characterized, and their replication strategies have been studied, allowing the design of molecular tools for accurate diagnosis of olive viruses. Despite their harmful effects, viruses have the ability to move inside the plants and can be useful vectors to be developed for general biotechnology. The well-known viral genomes of necroviruses, mainly in the context of their pathogenic interactions with host plants, led us to design vectors for plant protection use i) based on virus-induced gene silencing (VIGS), using the natural plant gene silencing strategy to protect plants against viruses; ii) based on the expression of antimicrobial peptides (AMPs), using the ability of virus genome replication to produce antibiotics and antifungal molecules to prevent diseases caused by bacteria and/ or fungi; and iii) based on CRISPR technology for gene editing. The presence of these viral vectors in plants confers them protection against specific untreatable or harmful diseases, leading us to reduce chemical treatments and to increase production.