Fabio Nascimento da Silva

High-throughput sequencing applied for the identification of viruses infecting grapevines in Brazil and genetic variability analysis

The application of high-throughput sequencing technologies (HTS) enables the recovery of many nucleotide sequence fragments from diseased plants and may help in pathogen identification. This study was designed to identify viruses infecting 15 grapevine (Vitis spp.) samples collected from experimental fields and vine collections and assess the genetic variability of the identified viruses. The virus-enriched dsRNAs were extracted from bark scrapings and sequenced using an Illumina platform. The paired-end reads were analyzed, assembled contigs were generated and identified as related to viruses. Contigs of 14 viruses have been identified, some of them covering large extensions of viral genomes or resulting in assembly of near-complete or complete genomes. Grapevine virus infections are usually mixed and the HTS assays were suitable to identify ten viruses already reported that traditionally infect grapevines in Brazil, one that has been recently identified (Grapevine Syrah virus 1) and others (Grapevine Cabernet Sauvignon reovirus, Grapevine Red Globe virus and Grapevine vein clearing virus) not previously reported in this country. Nucleotide identities among Brazilian isolates identified by HTS and homologous grapevine virus sequences in GenBank were high, ranging from 77% to 99%. Genetic variability analysis of viral sequences obtained by HTS and sequences available in GenBank indicated that the coding regions in the different viral species are under purifying selection, and that recombination events occurred in the majority of the viral species analyzed. The coat protein genes, generally, had lower genetic variability than the replicase and movement protein genes.

Biological multiple indexing and RT-PCR detection of latent viruses in apple plants

This study aimed to evaluate the suitability of a multiple biological indexing method of latent apple viruses. The method consisted of grafting five indicator species and two buds of the plant to be indexed on to one rootstock. Results showed that the sensitivity and efficacy of the MBI is adequate for reliable detection of Apple stem grooving virus, (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) of mother plant stock in certification programs. Sample analysis by RT-PCR confirmed the results of the diagnosis of ASPV and ASGV by multiple biological indexing. However, it did not detect ACLSV diagnosed by LL-S5 in three out of nine analyzed accessions. The coat protein gene of one of the accessions studied was cloned and sequenced. The annealing sites of primers used in unsuccessful amplification attempts were compared with data of the Genbank to explain the non-amplification of certain ACLSV isolates.

Effect of four training systems on the temporal dynamics of downy mildew in two grapevine cultivars in southern Brazil

The aim of this study was to evaluate the temporal dynamics of downy mildew epidemics under four training systems: vertical shoot positioning (VSP), Geneva Double Curtain (GDC), Simple Curtain (SC) and Tendone in two cultivars (‘Cabernet Sauvignon’ and ‘Merlot’). Experiments were conducted at commercial vineyards in southern Brazil, during the 2012–2013 and 2013–2014 growing seasons. Downy mildew incidence and severity were quantified on a weekly basis from the first symptoms appearance until harvest. Training systems and cultivars were compared based on the following variables of downy mildew progress curves: a) beginning of symptoms appearance (BSA); b) maximum disease incidence and severity (Imax, Smax); c) time to reach maximum disease incidence/severity (TRMDI and TRMDS); and d) area under incidence and severity disease progress curve (AUIDPC and AUSDPC). The training systems significantly affected AUSDPC and Smax. AUSDPC was lowest in the VSP system in all cultivars and growing seasons. In addition, ‘Merlot’ showed significantly lower AUIDPC and AUSDPC than ‘Cabernet Sauvignon’ in both growing seasons. The Tendone and GDC systems showed significantly higher AUSDPC in all cultivars and growing seasons. Our results suggested that the use of the VSP system contributes to reduce downy mildew severity on ‘Merlot’ and ‘Cabernet Sauvignon’ in southern Brazil.

Characterization and genetic variability of coat protein genes of Apple chlorotic leaf spot virus isolates from southern Brazil

Apple chlorotic leaf spot virus (ACLSV) infects temperate rosaceous fruit trees worldwide and causes a wide range of diseases that are economically highly damaging. This study was carried out to analyze genetic variability of regional ACLSV isolates and compare it with ACLSV isolates from other parts of the world. Nineteen amplicons of ACLSV, corresponding to the coat protein (CP) gene of isolates from apple, plum, and nectarine, from two states in southern Brazil, have been analyzed for genetic variation and compared phylogenetically among themselves and with other sequences available in GenBank. Sequences identities among complete CP genes of these isolates ranged from 87.5 to 100% and 92.2 to 100% at the nucleotide (nt) and the deduced amino acid (daa) levels, respectively. In the comparison with isolates from Asia, Europe and North America, identities were 68.4 to 100% and 72.5 to 100% at nt and daa levels, respectively. Phylogenetic trees based on nucleotide sequences showed that these isolates grouped into two clusters, cluster 1 containing apple isolates and cluster 2 comprising apple, plum and nectarine isolates. Most Brazilian isolates showed conserved signatures (Ser40, Leu59, Tyr75, Thr130 and Leu184) in their CPs, which place them with type B6 isolates. However, some Brazilian isolates were found to be variants of type B6. These analyzes indicated that Brazilian isolates had lower genetic variability compared to isolates from China, India and Japan and that the CP genes were under negative selection. The greatest diversity of nucleotides was observed in the central portion of the CP gene, represented predominantly by synonymous substitutions. One natural recombinant was detected among ACLSV isolates from Brazil.

Distribution and phylogenetics of whiteflies and their endosymbiont relationships after the Mediterranean species invasion in Brazil

The Bemisia tabaci is a polyphagous insect and a successful vector of plant viruses. B. tabaci is a species complex and in Brazil native species from the New World (NW) group, as well as the invasive species, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) were reported. For better understanding the distribution of the different species four years after the Mediterranean species invasion in Brazil, whiteflies were collected from 237 locations throughout the country between the years of 2013 and 2017, species were identified and the facultative endosymbionts detected. The survey revealed that MEAM1 was the prevalent species found on major crops across Brazil. It is the only species present in North, Northwestern and Central Brazil and was associated with virus-infected plants. MED was found in five States from Southeast to South regions, infesting mainly ornamental plants and was not associated with virus-infected plants. The prevalent endosymbionts identified in MEAM1 were Hamiltonella and Rickettsia; and the mtCOI analysis revealed low genetic diversity for MEAM1. In contrast, several different endosymbionts were identified in MED including Hamiltonella, Rickettsia, Wolbachia and Arsenophonus; and two distinct genetic groups were found based on the mtCOI analysis. Monitoring the distribution of the whiteflies species in Brazil is essential for proper management of this pest.

Relationship among Entomosporium severity, defoliation, and vegetative-reproductive variables in pear in Brazil

The objective of this work was to evaluate the relationship among Entomosporium severity, defoliation, and vegetative-reproductive variables of three European pear cultivars in Southern Brazil. The Packham’s Triumph, Abate Fetel, and Williams pear cultivars were assessed regarding Entomosporium severity, defoliation, and natural leaf fall every two weeks, from the appearance of the first symptoms at the beginning of January until the end of April, during the 2012/2013 and 2013/2014 crop seasons. The vegetative and reproductive variables were evaluated in July, during the dormant period. Data on Entomosporium severity were integrated over time and converted into the area under the disease progress curve (AUDPC). The coefficient of determination (R2) of the linear regression of Entomosporium severity versus defoliation was significant for all studied cultivars and crop seasons. Defoliation in trees affected by Entomosporium was significantly higher than natural leaf fall in the control trees protected by fungicides. A negative correlation was observed among defoliation, branch fertility index, and plant height, indicating that the higher the defoliation, the lower the development of the vegetative and reproductive variables plant height and vigor. Entomosporium severity differed significantly among cultivars when the AUDPC was considered the differentiating epidemiological variable. The Abate Fetel and Williams pear cultivars are the most susceptible and resistant to Entomosporium, respectively.

Characterization of Neofabraea actinidiae and N. brasiliensis as causal agents of apple bull’s-eye rot in southern Brazil

Abstract The causal agents of apple bull’s-eye rot in southern Brazil have recently been described as Neofabraea actinidiae and N. brasiliensis. Isolates of both species were evaluated for response of mycelial growth index (MGI) to different temperatures, enzyme production, mycelial growth inhibition and effective concentrations (EC50 and EC100) of the fungicides triflumizole, pyrimethanil and thiophanate methyl, as well as aggressiveness on fruits of ‘Fuji’ hybrid and ‘Pink Lady’. There was significantly lower mycelium growth in N. brasiliensis compared with N. actinidiae at all temperatures tested. Neither species grew at 3 and 32°C. There were minor differences in production of enzymes in the two species, with all N. brasiliensis isolates showing no production of pectolyase at pH 7. The lowest EC50 and EC100 values were observed with thiophanate methyl. In general, ‘Fuji’ fruits were more susceptible to Neofabraea infection and had larger lesions, while N. brasiliensis isolates showed greater aggressiveness on ‘Fuji’ hybrid and ‘Pink lady’ fruits compared with N. actinidiae.