Rice is one of the main staple foodstuffs for the world’s population; however, several diseases caused by phytopathogens cause yield losses in rice crops. Rice stripe necrosis virus (RSNV) belonging to the Benyviridae family and Benyvirus oryzae species is the causal agent of rice crinkle, an emerging disease that threatens this important crop. RSNV is transmitted by the plasmodiophoromycete, Polymyxa graminis, which can remain in the soil through resistance structures for decades. The symptoms caused by the virus are often confused with other causes, making a correct diagnosis and the adoption of effective management and control measures difficult. Current RSNV detection methods are mainly based on enzyme-linked immunosorbent assay (PTA-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) assays. In this study, a new detection method based on a TaqMan real-time RT-PCR combined with a direct sample preparation method using crude extracts has been developed. The new protocol has resulted in 100 times more sensitivity than conventional RT-PCR when using plant material and 1000 times more sensitivity for soil solutions. In addition, the high sensitivity exhibited by our method has also been shown when testing direct samples, thus avoiding nucleic acid purification steps.

Brazil is the world’s largest producer and consumer of passion fruit; however, productivity is restricted by phytosanitary factors, mainly viruses. Passion fruit woodiness disease (PWD) is the major viral disease of the crop, caused in Brazil by the cowpea aphid-borne mosaic virus (CABMV), and non-persistently transmitted by aphids. In this study, a protocol was developed and validated using real-time RT-PCR with a specific TaqMan probe combined with a direct sample preparation method for the detection of CABMV. The new protocol is 100 times more sensitive than the PTA-ELISA in detecting CABMV in passion fruit. The nucleotide sequence of the CABMV isolates from Santa Catarina (SC), had high identity (above 99

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 108 to 10 PFU/mL for the phages and from 108 to 102 CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 102 targets/mL in water and plant extracts to 103 targets/g in soil, and the target bacterium with a limit ranging from 103 targets/mL in water and plant extracts to 104 targets/g in soil, using direct methods of sample preparation.

Four pathogenic bacterial species of the genus ‘ Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘ Ca . Liberibacter asiaticus’ (CaLas), ‘ Ca . Liberibacter americanus’ (CaLam) and ‘ Ca . Liberibacter africanus’ (CaLaf), while ‘ Ca . Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter . The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘ Ca. Liberibacter’ in a one-step assay.

Guabiju tree (Myrcianthes pungens) belongs to the Myrtaceae family, with wide occurrence in Rio Grande do Sul (RS), southern Brazil, demonstrates great commercial potential regarding the consumption of its fresh fruit, which has a sweet taste and can be used in drinks, jellies and ice creams, in addition to its nutraceutical properties. As their main characteristic, rusts present the formation of orange pus-tules containing urediniospores of the pathogen on affected organs. The action of the pathogen causes deformation of stems, leaves, flowers and fruits, thus interfer-ing with the physiological processes of the plant. Thus, the present work aimed at presenting information on the occurrence and confirmation of the causal agent of guabiju rust, in addition to reporting the accessions most susceptible and resistant to Austropuccinia in the guabiju working collection of Eldorado do Sul, RS, Brazil. The diagnosis of the disease was based on symptoms, observation of fungal struc-tures by light microscopy and molecular analyses. From microscopy, ellipsoid to ovoid and slightly equinulate urediniospores were observed, characteristic of Aus-tropuccinia sp. The sequence of the internal transcribed spacer (ITS) region of the isolate showed 99.06% similarity with sequences from the same region of A. psidii deposited on the nucleotide database -GenBank (NCBI). This is the first report of rust associated with guabiju in the state of Rio Grande do Sul, Brazil. From the dia-grammatic scale developed, it was possible to identify different levels of susceptibil-ity to A. psidii in guabiju accessions under study.

O endurecimento dos frutos do maracujazeiro (EFM), causado pelo Cowpea aphid-borne mosaic virus (CABMV), é uma das doenças mais importantes da cultura. Trata-se de uma doença endêmica nas principais regiões produtoras do Brasil. A principal forma de controle do EFM é a utilização de diversas estratégias dentro do manejo integrado de doenças e exige uso de mudas sadias, com pelo menos 80cm de altura, produzidas em ambiente protegido sob telado antiafideo, cuidados nas operações de poda e desbaste, eliminação sistemática de plantas com sintomas até o início do florescimento, utilização dequebra-ventos, a realização do planto em períodos de menor incidência de afídeos no campo e a realização de cultivo anual associado à adoção de vazio sanitário.

O setor agrícola e agroindustrial surge como impulsionadores da economia do Brasil e de muitos países em desenvolvimento. Porém suas atividades causam preocupações acerca dos impactos ambientais que têm gerado ao ambiente. Dessa forma, várias pesquisas surgem em busca de alternativas para diminuir os efeitos adversos. As microalgas têm sido utilizadas para minimizar o impacto ambiental causado por algumas atividades do setor agroindustrial, em função de seus vários fatores benéficos. Podem ser aplicadas em diversas vertentes do ramo agrícola e tecnológico. No entanto, apesar dos atributos serem favoráveis ao solo, plantas e ambiente, ainda não é amplamente difundido em território brasileiro. O objetivo do trabalho foi discorrer sobre os benefícios das microalgas em várias atribuições do setor agrícola e agroindustrial. A metodologia utilizada neste trabalho foi a revisão bibliográfica. Foram abordados aspectos sobre os efeitos das microalgas como biofertilizantes na agricultura, o uso na limpeza de efluentes oriundos na suinocultura, como fonte de cultivo em vinhaça e, por fim, os impactos socioeconômicos e ambientais na produção de biocombustíveis. Concluiu-se que a utilização desse micro-organismo nos setores agrícola e agroindustrial é promissor. Seu principal papel se desempenha na busca pela atenuação dos impactos ambientais para agricultura e pecuária, além de auxiliar na redução de custos com produção de biocombustíveis, já que as microalgas não interferem negativamente na produção dos produtos agrícolas e energéticos. Palavras-chave: Biofertilizantes. Efluentes da Suinocultura. Biocombustíveis. Impacto Ambiental. Abstract The agricultural and agro-industrial sectors are the economy driving forces in Brazil and in many developing countries, but their activities cause concerns about the environmental impacts it has generated on the environment, so several studies have emerged in search of alternatives to decrease these adverse effects. Microalgae have been used to minimize the environmental impact caused by some activities in the agro-industrial sector due to its many beneficial factors and can be applied in different aspects of the agricultural and technological fields. However, although the attributes are favorable to the soil, plants and the environment, it is still not widespread in Brazilian territory. The objective of this study was to discuss the benefits of microalgae in various attributions of the agricultural and agro-industrial sector. The methodology used in this work was the literature review. It addressed their effects as biofertilizers in agriculture, their use in cleaning effluents from pig farming and as a source of cultivation in vinasse, and finally, the socioeconomic and environmental impacts on the biofuels production. It is concluded that the use of this microorganism in the agricultural and agro-industrial sectors is promising, with its main performance being the search for the environmental impacts mitigation for agriculture and livestock, as well as helping to reduce costs with the biofuels production, since microalgae do not interfere negatively in the production of agricultural and energy products. Keywords: Biofertilizers. Swine Effluents. Biofuels. Environmental Impact.

Currently, the main arthropod vectored pathogens associated with carrot and celery crop diseases are ˋCandidatus Liberibacter solanacearum´, Spiroplasma citri and different phytoplasma species. Mitigation strategies require elucidating whether these pathogens survive in the weeds of these Apiaceae crops, which can act as reservoirs. Weed surveys were conducted in a vegetative cycle (April to October 2012) in the spontaneous vegetation that surrounded crops affected by ˋCa. L. solanacearum´, S. citri and/or phytoplasmas. Sixty-three species of 53 genera that belong to 23 botanical families were collected in the main carrot and celery Spanish production area. Species were identified, estimating coverage and abundance, and conserved in herbarium. Samples were analysed by nested-PCR with universal primers for phytoplasmas detection, and were sequenced for identification purposes; by conventional PCR for S. citri and real-time PCR for ˋCa. L. solanacearum´. The only detected pathogens were ˋCa. Phytoplasma trifolii´ (clover proliferation group 16Sr VI-A) in Amaranthus blitoides and Setaria adhaerens and ˋCa. P. solani´ (stolbur group 16Sr XII-A) in Convolvulus arvensis. These pathogens were also sporadically detected in celery or carrot crops. Unexpectedly, neither ˋCa. L. solanacearum´ nor S. citri was detected in the weed samples, despite the relatively high prevalence of these pathogens (less than 66 % and 25 %, respectively) in the surveyed plots. This suggests that weeds do not play an epidemiological role as reservoirs in the spread of such organisms in the studied region. The use of pathogen-free seed lots and the control of vectors are crucial for preventing the introduction and spread of these economical important pathogens to new areas.

Passion fruit woodiness is caused by the cowpea aphid-borne mosaic virus (CABMV; genus Potyvirus ) and is the most common passion fruit viral disease in Brazil . The main route of viral transmission is inoculation by aphid vectors through intracellular stylet puncture. The use of oils can affect the acquisition and inoculation of the virus throughout the process of aphid stylet penetration in the cell. This study aimed to determine vegetable oil doses that do not cause phytotoxicity in passion fruit plants and if the use of oil affects the efficiency of acquisition and transmission of CABMV by Aphis gossypii in the plant. The results showed that doses of up to 1.0% of vegetable oil did not cause phytotoxicity symptoms in passion fruit seedlings in the greenhouse. The species A. gossypii was 80% efficient in transmitting CABMV, and the use of vegetable oil reduced the acquisition and transmission of the virus by A. gossypii in passion fruit seedlings by 98.67% and 60%, respectively. The use of vegetable oil may be a strategy to control the transmission of CABMV and thus reduce the economic losses caused by the viral disease.

Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘Ca. Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms.

Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plants were isolated from river water in Europe but not fully analysed, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum.

As mudanças climáticas tornaram-se um dos maiores desafios para ser superado na esfera rural em busca do desenvolvimento sustentável. As consequências dessas mudanças podem afetar não apenas a produtividade no campo, mas também o bem-estar da humanidade que depende dos ecossistemas. Analisar as causas dos impactos proporciona uma visão sistêmica dos fatores e isso ajuda a entender e modificar as ações prejudiciais e criar possíveis soluções de reparação ambiental que mantenha altos índices de produção. Mesmo com tanto impacto gerado pelas mudanças climáticas à agropecuária, existem maneiras que reduzem danos e auxiliam na adaptação às condições climáticas desfavoráveis. O objetivo geral deste trabalho foi identificar como as mudanças climáticas podem modificar o desenvolvimento agropecuário brasileiro. Este trabalho foi elaborado a partir de uma pesquisa bibliográfica, com base de análise em artigos de diversos autores sobre os estudos das condições climáticas, os impactos ambientais e como influenciaram a mudança da vida no campo. Deste modo, é essencial que toda a sociedade, de maneira geral, o que inclui fazendas e cooperativas, realizem a incorporação de uma gestão de riscos consequentes das mudanças climáticas em seus processos de planejamento, onde grande parte disto se associa por identificar a vulnerabilidade atual aos impactos das mudanças climáticas.

Um dos principais métodos de controle da mancha branca do milho, o controle químico com estrobilurinas e fungicidas protetores demonstraram efeito no controle da doença, no entanto, condicionada ao momento de sua realização em estádios fenológicos da cultura. O objetivo deste trabalho foi avaliar o controle e efeito no progresso temporal da mancha branca do milho com a associação dos fungicidas mancozebe e azoxistrobina em diferentes épocas de aplicação, dois cultivares, duas localidades de plantio e o efeito na produtividade da cultura. Os experimentos foram conduzidos nos municípios de Marechal Cândido Rondon e Sertanópolis, PR. Foram utilizados dois híbridos suscetíveis, DKB230PRO3 e DKB290PRO3. O delineamento experimental utilizado foi de blocos casualizados com 4 repetições. As avaliações foram efetuadas 10 dias após cada aplicação, além de uma avaliação no estádio de maturidade fisiológica. A severidade da doença foi avaliada através da quantificação da área foliar lesionada em 10 plantas por parcela e utilizadas no cálculo da Área Abaixo da Curva de Progresso da Doença, submetidos a análise de variância e o teste de médias. As curvas de progresso da doença foram ajustadas aos modelos logístico e exponencial. A produtividade foi estimada após a colheita manual, com o peso de mil grãos e produtividade (kg ha-1). Aplicações dos fungicidas em estádios V8 e V4+V8+VT proporcionaram melhor controle da mancha branca. Aplicações no estádio V4 não proporcionaram severidades diferentes da testemunha sem aplicação. Não foram observados ganhos significativos em produtividade em função da aplicação dos fungicidas.

Genome organization and phylogenetic relationships of olive leaf yellowing-associated virus (OLYaV) with other members of the Closteroviridae family were determined. The complete coding sequence of OLYaV was obtained by high throughput sequencing of total RNA from a 35-year-old olive tree (cv. Zarzaleña) from Brazil, showing olive leaf yellowing disease and deformations in the wood. This represents the first report of OLYaV in this country. A genomic sequence of 16,700 nt containing 11 open reading frames (ORFs) was recovered, representing the complete virus coding capacity. The knowledge of the nucleotide sequence of the genome including the gene that codes the coat protein will facilitate the development of diagnostic tests, which are limited so far to PCR-based methods targeting the HSP70h gene. Interestingly, a thaumatin-like protein (ORF2), previously reported in other unassigned viruses in the Closteroviridae family, persimmon virus B and actidinia virus 1, was identified in the OLYaV genome. Phylogenetic analysis of shared proteins (ORF1a, ORF1b, HSP70h, HSP90h and CP) with all members of the Closteroviridae family provides new insight into the taxonomic position of these three closteroviruses and suggests they could represent a new genus in the family.

Bacteria belonging to ‘Candidatus Liberibacter spp.’ are associated with various severe diseases in the five continents. The African citrus psyllid Trioza erytreae (Hemiptera: Triozidae) is an efficient vector of citrus huanglongbing-HLB disease, absent in the Mediterranean basin. This psyllid is currently present in the islands and mainland Portugal and Spain, where the prevalence of ‘Ca. Liberibacter solanacearum’ (CaLsol) associated to a carrot disease is high. Trioza erytreae normally feeds on citrus plants but has also been observed on other crops. It would be a great concern to the Mediterranean citrus industry if T. erytreae could transmit this bacterium from carrots to citrus and cause disease; therefore, the transmission of CaLsol from carrot plants to citrus plants was experimentally assessed. Although CaLsol was initially detected on receptor citrus plants in transmission assays by dodder and budding, the infection was not established. The feeding behavior by electrical penetration graphs and oviposition of T. erytreae on carrot plants versus citrus plants was evaluated. Trioza erytreae only reached the phloem in citrus plants. However, it was able to acquire CaLsol from infected carrots but unable to transmit it to citrus plants. CaLsol was detected in some carrot plants immediately after 7 and 14 days (inoculation access period), but it was not detected after one month. Trioza erytreae was unable to complete its life cycle on carrot plants. In conclusion, the efficient vector of bacteria associated to huanglongbing was unable to transmit CaLsol from carrot to citrus plants, but it acquired and transmitted the bacterium from carrot to carrot plants with low efficiency.

Trabajo presentado en el Congress of Microbiology and Biotechnology (Microbiotec 19), celebrado en Coimbra del 5 al 7 de diciembre de 2019.

Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD.

Direct systems to process samples allow high-throughput testing or identification of Citrus tristeza virus (CTV) by the sensitive real-time reverse transcription coupled to polymerase chain reaction (RT-PCR) neither with extract preparation nor nucleic acid purification. Immobilized CTV targets are amplified from fresh sections of plant tissues or squashes of fresh or already caught vectors onto paper, nitrocellulose, or positively charged nylon membranes. The printed or squashed support can be stored or mailed at room temperature. These validated user-friendly methods are recommended by IPPC-FAO standard for CTV diagnosis, detection, and identification. The methods are safe, not under current quarantine regulations because they do not involve any risk of introduction of exotic CTV isolates or vectors and are discrete and useful for epidemiological studies or screening for large-scale analyses. In this chapter, tissue-printing and squashing capture methods for direct sample preparation without extract preparation or nucleic acid extraction and purification were coupled with validated real-time RT-PCR detection protocols based on TaqMan chemistry for CTV detection.