CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.

Biofilm formation by strains of Burkholderia cenocepacia lineages IIIA and IIIB and B. gladioli pv. alliicola associated with onion bacterial scale rot.

The Burkholderia genus has high ecological and nutritional versatility, having species capable of causing diseases in animals, humans, and plants. During chronic infections in humans, biofilm formation is a characteristic often associated with strains from different species of this genus. However, there is still no information on the formation of biofilms by plant pathogenic strains of B. cenocepacia (Bce) lineages IIIA and IIIB and B. gladioli pv. alliicola (Bga), which are associated with onion bacterial scale rot in the semi-arid region of northeast Brazil. In this study, we performed an in vitro characterization of biofilm formation ability in different culture media by the phytopathogenic strains of Bce and Bga and investigated its relationship with swarming motility. Our results indicated the existence of an intraspecific variation in biofilm formation capacity in vitro by these bacteria and the existence of a negative correlation between swarming motility and biofilm formation for strains of Bce lineage IIIB. In addition, histopathological analyses performed using optical microscopy and scanning electron microscopy revealed the formation of biofilm in vivo by Bce strains in onion tissues.

Association of "green spot" and defoliation to Tenuipalpus heveae incidence and chemical control screening of Hevea brasiliensis diseases / Associação da presença de "pinta verde" e desfolha com presença do ácaro Tenuipalpus heveae e avaliação de fungicidas para controle de doenças em painéis de seringueira Hevea brasiliensis

According to IBGE data, in 2016, Brazil produced closer to 56 thousand hectares of rubber tree generating a total latex production of 315.62 tons in commercial areas and 1.6 thousand tons in native forests. But this growth in crop production is related to the raise of economic losses generated by foliar and bark (panel) diseases caused by Phytophthora spp, Colletotrichum sp., Lasiodiplodia spp., Colletotrichum sp., Ceratocystis fimbriata, Microcyclus ulei (leaf blight) and the leaf anomaly that we call here as "Green Spot". Also, due to the tapping method and the incisions or "injuries" made in this process, opportunistic pathogens can usually infect and end up growing in the bark. This type of infection that is also difficult to control with the adoption of chemical fungicides, can create additional damage to the rubber panels. Pathogens such as Oidium heveae, C. gloeosporioides (anthracnosis), Phomopsis sp. and Phytophthora spp. were identified in 42% of the areas of growers or farms. Treatments T3- (thiophanate-methyl (350 g.ha-1 active ingredient) and T4- (metiram (1.75 kg.ha-1 active ingredient)) were superior against rot diseases in the bark of rubber trees. It was described for the first time the anomaly of the green spot associated to the Tenuipalpus heveae mite in 65% of the samples and had its etiology confirmed by artificial inoculation in rubber tree seedlings. In the USP-ESALq, Dr. Kitajima, E., did not confirm the presence of rhabdovirus-like particles common to tenuipalpids that transmit the virus in coffee and citrus (nuclear and cytoplasmic viruses).

Segundo dados do IBGE, em 2016, o Brasil registrou uma área cultivada de 156,06 mil hectares, dos quais 146,4 mil hectares foram colhidos, promovendo uma produção total de 315,62 toneladas em lavouras comerciais. Já a exploração em áreas de vegetação natural, resultou em 1,6 mil toneladas de látex e coágulo. Microcyclus ulei também chamado de Mal-de-Folhas é conhecida mundialmente como a doença mais grave da seringueira, mas a intensificação do cultivo permitiu prejuízos econômicos promovidos por doenças foliares como Phytophthora spp, Colletotrichum sp., Alternaria spp., Oidium sp. Entre outros, além da anomalia de Pinta Verde. No tocante às doenças de sapé, são citadas as patologias fúngicas nos painéis Lasiodiplodia spp., Colletotrichum sp., Ceratocystis fimbriata, Erythricium salmonicolor e Phytophthora spp.,. Muitos patógenos permitem a entrada nas lesões de outros fungos oportunistas. Devido ao modo de exploração (corte) causar lesões no tronco que facilitam a inoculação de patógenos principalmente através de instrumentos de sangramento, causando danos nos painéis de seringueiras e são patógenos de difícil controle e poucos resultados significativos com o uso de fungicidas ou produtos químicos. Os patógenos, como Oidium heveae, C. gloeosporioides (antracnose), Phomopsis sp. e Phytophthora spp. foram identificados em 42 % das áreas de produtores ou fazendas. Os tratamentos 3 e 4 foram os melhores do controle dos fungos e esta podridão de painel nas seringueiras (T3- (tiofanato-metílico(350 g / ha ingrediente ativo), T4- (metiram (1,75 kg / ha ingrediente ativo). Foi descrita pela primeira vez a anomalia da pinta verde associada ao ácaro Tenuipalpus heveae¨em 65% das amostars e teve a sua etiologia confirmada por inoculação artificial em mudas de seringueira. Análises em microscopia eletrônica realizadas na USP-S-ESALq pelo Dr. Kitajima, E., não confirmaram a presença de partículas tipo rabdovírus comum aos tenuipalpídeos que transmitem a virose em cafeeiro e citros (vírus tipo nuclear e citoplasmático).