Brazil is the second-largest producer of beef in the world, and the Brazilian beef production system is extensive, characterized by free-range animal grazing in large pasture areas during a significant portion of the animal's fattening period. Tropical forages, particularly grasses of the Urochloa genus (syn. Brachiaria), are commonly planted for this purpose. Despite limited literature on viral infections in forages, it is known that these plants are currently affected by such pathogens. These infections contribute to a decline in pasture quality, and the identification of these pathogens is not always straightforward, making it challenging to establish specific measures for diagnosis and proper management. High-throughput sequencing (HTS) has been crucial in identifying new viral species due to the substantial amount of data generated. Therefore, the aim of this study was to detect a new viral species infecting forage plants using high-throughput sequencing. A novel emaravirus was discovered through HTS in a commercial hybrid cultivar derived from Urochloa ruziziensis, U. decumbens, and U. brizantha. A genome sequence was confirmed through Sanger sequencing and comprises five segments of negative-sense RNA. RNA1 consists of 6,867 nucleotides, encoding a RNA-dependent RNA polymerase (RdRp) protein of 2,289 amino acids. RNA2 consists of 2,043 nt, encoding a glycoprotein (G) of 681 aa. RNA3 consists of 981 nt, encoding a nucleocapsid protein (NC) of 327 aa. RNA4 consists of 1,041 nt, encoding a movement protein of 374 aa. RNA5 consists of 1,008 nt, encoding a gene silencing suppressor protein. Phylogenetic analysis based on predicted amino acid sequences of RdRp and G showed a 33.9% closeness to Emaravirus tritici, while analysis based on amino acid sequences of NC revealed a distant relationship to fifteen different species, namely E. vitis, E. sorbi, E. actinidiae, E. syringae, E. cercidis, E. cajani, E. aceris, E. populi, E. pistaciae, E. rubi, E. rosae, E. fraxini, E. kiwii, E. toordali, and E. fici. These data suggest that the discovered virus is a member of a new species in the genus Emaravirus, for which the binomial name Emaravirus brachiariae is proposed.

The tomato crop (Solanum lycopersicum L.) is of great importance for national and international agriculture. Several viruses can affect this vegetable crop causing considerable impact. Among the viruses that infect tomato, isolates from species classified within the genus Begomovirus (family Geminiviridae) are of particular impotance. Begomoviruses have single-stranded circular DNA genomes that can be mono- or bipartite, which are separated into two large groups: begomoviruses from the Old World and the New World. Transmission of begomoviruses occurs via a complex of cryptic species of Bemisia tabaci (Aleyrodidae, Hemiptera) predominantly B. tabaci Middle East Asia Minor 1 – MEAM 1 (former biotype B) and B. tabaci Mediterranean MED (former biotype Q). The range of host plants for begomoviruses is wide, including cultivated and uncultivated dicots. There is considerable diversity in the begomovirus species described to date; and several new species have been characterized in the last decade. This increasing begomovirus variability occurs via three distinct mechanisms: mutation, recombination and reassortment. One of the promising strategies to control begomoviruses is the use of cultivars with resistance/tolerance that are obtained by cultivars that carry the Ty-1 and Ty-3 genes. With the increasing diversity of species, the use of tools such as High-Throughput Sequencing (HTS) has enabled allowed largescale study of viral diversity. Such studies have already shown that tomato plants that carry the Ty-1 gene reduce the number of begomoviruses that infect tomato plants and impact the frequency and prevalence of these viruses, acting as a ‘filter’ on these in tolerant cultivars. In addition, new and/or recombinant species can overcome tolerance factors present in the host plant. Therefore, the objective of this study was to monitor and characterize the viral diversity of the Geminiviridae family and subviral agents associated with the Begomovirus genus associated with tomato crops from the five geographic regions of Brazil via sequencing with HTS technology. To this end, 154 samples from symptomatic tomato plants with and without the Ty-1/Ty-3 factor were collected in five Brazilian regions: North (13 samples), Northeast (36), South (24), Southeast (39) and Central. Oeste (42), enriched through Rolling Circle Amplification (RCA). The enriched samples were grouped into two pools: BP1 (samples from the North, Northeast and South regions) and BP2 (Southeast and Central-West) and submitted to HTS sequencing using the Illumina NovaSeq-6000 platform. The sequences were assembled into contigs using the CLC genomics Workbench 7.5 software, analyzed in Geneious R11.1, compared with the sequence bank deposited in GenBank using the BLASTn algorithm. From the sequencing of the BP1 pool, 17 viral species were recovered, 16 corresponding to the genus Begomovirus and one to the genus Topilevirus, in addition to four new species of begmovirus. 14 viral species were recovered from the sequencing of the BP2 pool, one of them corresponding to a new probable species of begomovirus and also alphasatellite sequences associated with begomoviruses. Species-specific primers were used to recover the genomes by PCR in each sample individually (Chapter 2). The Ty-1/Ty-3 tolerance factors impacted viral diversity and the number of infected samples, with samples lacking Ty-1/Ty-3 having the highest number of detected species and infections. The number of mixed infections was also higher in tomato samples that did not have Ty-1/Ty-3. In chapter 3, recovery by PCR assays with species-specific primers allowed the detection of a new bipartite begomovirus, until and not absent in Brazil, (tomato chlorotic mottle Guyane virus – ToCMoGV) in sample AM-035, collected in Iranduva, state from Amazonas, northern Brazil. Molecular analyzes and phylogenetic reconstructions demonstrated that ToCMoGV detected in Brazil consists of a strain with high divergence from the isolate from French Guiana, with which it shares 92% nucleotide identity. In chapter 4, through molecular characterization, phylogenetic construction and identity analysis using the Sequence Demarcation Tool (SDT), it was demonstrated that viral isolates deposited in GenBank identified as tomato yellow spot virus (ToYSV) and Leonurus mosaic virus (LeMV) together ToYSV isolates recovered by HTS in this study were closely related. Some shared the same iterons and shared identities equal to and/or greater than 91%, leading to the conclusion that they were wrongly named and that they are a single virus, thus proposing that only a single name be proposed. Chapter 5 portrays the detection of a new begomovirus isolate infecting tomato plants in the northern region of Brazil. The new begomovirus isolate wasdetected in sample TO-083, originating from Araguaína, state of Tocantins. The new isolate is bipartite, with DNA-A and DNA-B segments, from which infection clones were produced and inoculated in tomato plants. The molecular characterization of the new isolate identified the GGTGT/ACACC iteron and the Rep domain related to the iteron (Rep– IRD): MPRQPTTFRL. Isolate TO-083 was then named tomato golden leaf spot virus (ToGLSV). In chapter 6, two new species of monopartite begomoviruses were recovered by PCRs with specific primers. The new species #1 was detected in northeastern Brazil, in samples: CE-001, originating from the state of Ceará, PE-011 and PE-012, both from Pernambuco. The new species #2 was detected in samples PR-173 and PR-174, both collected in the state of Paraná, southern Brazil. Recombination analyzes demonstrated that the two new species are recombinants of other Brazilian begomoviruses that infect tomato. New species #1 is phylogenetically closer to tomato interveinal chlorosis virus (ToICV) while new species #2 is closer to tomato mottle leaf curl virus (ToMoLCV). Further studies will be used to biologically characterize these new species of begomoviruses affecting tomato. Chapter 7 portrays the detection and molecular characterization of a new species of bipartite begomovirus infecting tomato plants in the central-western region of Brazil. The new bipartite species was detected in tomato samples from the state of Goiás (GO) and the Federal District (DF), it has the DNA-A segment of 2561 nucleotides and the DNA-B of 2527. It is more phylogenetically related to the golden tomato vein virus (TGVV) with which it shares 89% nucleotide identity. Recombination analyzes detected a recombination event with tomato leaf curl purple vein virus (ToLCPVV) and tomato chlorotic mottle virus (ToCMoV). Studies for the biological characterization of this new bipartite species will be used.

The cocoa tree (Theobroma cacao L.), is the most important species in the Theobroma genus. This is due to its global commercialization of chocolate, which is produced from the almonds of the fruit. Ecuador is the seventh largest cocoa-producing country in the world, and the crop is grown in 16 of the 24 provinces. Although the coastal region is the primary region for cocoa production, the cocoa tree also thrives in other parts of the country. However, it is vulnerable to diseases like brown rot and descending death, which are caused by Phytophthora and Lasiodiplodia species, respectively. To accurately identify these pathogens, morphological characteristics are compared with molecular data from various genomic regions. This approach has led to the discovery of new species and/or new reports of pathogens in several crops. The information available suggests that black pod and dieback of cacao trees in Ecuador are caused by different species of Phytophthora and Lasiodiplodia. The objectives of this work are: (i) to identify the Phytophthora species responsible for black pod and canker, (ii) to identify the Lasiodiplodia species responsible for dieback, (iii) to determine the pathogenicity and aggressiveness of the Phytophthora and Lasiodiplodia isolates, and (iv) update the list of pathogens associated with cocoa in Ecuador. To achieve these objectives, pods with brown rot symptoms and plant tissues with dieback symptoms were collected from various provinces such as Esmeraldas, Guayas, Los Ríos, Manabí, Morona Santiago, Pichincha, and Santo Domingo de los Tsáchilas for indirect isolation. To identify the species, the partial region of the β-tubulin (Phytophthora spp.) or elongation factor (Lasiodiplodia spp.) gene was amplified and sequenced. Representative isolates were then selected for additional sequencing of the ITS and COX2 genomic regions for Phytophthora spp. and ITS, β-TUB, and RPB2 for Lasiodiplodia spp. Nucleotide sequences were compared and added to the GenBank database for Bayesian Inference (BI) and Maximum Likelihood (ML) analyses. Out of the 181 isolates from Phytophthora collected on pod and dieback of cacao trees, only P. palmivora was identified. Phylogenetic analyses confirm that the 166 isolates of Lasiodiplodia belong to L. theobromae, L. pseudotheobromae, L. brasiliensis and L. laeliocattleyae and the first report of L. brasiliensis and L. laeliocattleyae on Theobroma cacao. All species found were pathogenic on Theobroma cacao. The correct etiology of the black pod and dieback is essential to guide improvement programs and efficient control of cocoa diseases in Ecuador.

Meloidogyne incognita is a well-known root-knot nematode species that infects cotton globally, with the recent release of the resistant cultivar IMA 5801B2RF in Brazil for its control. Meloydogine enterolobii, though not historically considered a major threat to cotton production, has gained attention due to recent reports in Brazil and United States of severe damage in cotton resistant cultivars to M. incognita, highlighting its potential epidemic importance. In 2019, the first infection by M. enterolobii on resistant cotton was reported in Minas Gerais state, Brazil. Subsequently, in 2021 M. enterolobii was detected again in the municipality of São Desidério, western Bahia state, on the same resistant cotton cultivar. A continuous study surveyed cotton fields cultivated with resistant ‘IMA 5801B2RF’ from six municipalities in Bahia state, where all six populations from three different geographical origins were identified by Esterase phenotypes (EST) and SCAR markers as M. incognita, but M. enterolobii was not found again, confirming its restricted occurrence in western Bahia state. A bioassay with the resistant cotton cultivar in greenhouse conditions showed high reproduction of M. enterolobii (RF=12.8) but not of the field populations of M. incognita (FR<1.0), indicating the lack of virulence of these populations to the resistant cotton. Subsequently, the genetic variability of seven populations of M. enterolobii from different geographical origins and races, was evaluated using 44 RAPD and 7 AFLP primers. The analysis grouped two guava (race 1) and two Brazilian cotton populations (race 2) with high bootstrap support. However, pepper populations did not cluster with other M. enterolobii race 1 populations, and the sweet potato population showed the highest divergence. Mitochondrial (COII), ribosomal DNA (ITS, D2-D3), and HSP90 gene studies revealed limited interactions related to geographical origin or races of M. enterolobii. The North Carolina Differential Hosts Test (NCDHT) identified two physiological races: race 1 and race 2, with distinct pathogenic profiles. We evaluated the efficacy of current Brazilian cultivars as alternatives for Meloidogyne spp. race tests, and tomato ‘Santa Clara’, pepper ‘Magali R’, watermelon ‘Crinsom sweet’, peanut ‘IAC Tatu’, tobacco ‘NC4’, and cotton ‘FM966’ can be recommended as a substitute for old cultivars suggested in NCDHT. Genetic resistance is seen as a promising approach for managing root-knot nematodes (RKN). We tested twenty-four cotton accessions, aimed to identify sources of resistance to M. enterolobii in Embrapa’s cotton germplasm including different Gossypium species and hybrids under greenhouse conditions. Artificial inoculations were performed, and after 120 days, various variables including gall index, egg mass index, total number of eggs per gram of root, and reproduction factor were assessed. While some genotypes showed susceptibility, others, particularly Upland genotypes and hybrids, exhibited varying levels of resistance. Notably, genotype CNPA GO 2002-2043/5 consistently demonstrated high resistance. Despite the aggressiveness of M. enterolobii, certain cotton genotypes with known resistance QTLs showed significant reductions in nematode populations after inoculation, highlighting the potential of selecting resistant cotton genotypes as a viable strategy to mitigate nematode impact on cotton crops.

Begomovirus (family Geminiviridae) corresponds to the largest genus of plant viruses, encompassing viral species that have one or two single-stranded circular DNA molecules, separately encapsidated in 18-30 nanometer particles. Isolates with a bipartite genome (with DNA–A and DNA–B components) predominate in tomato in the New World. Begomoviruses are transmitted with great efficiency by the Bemisia tabaci Middle East Asian Minor 1 (MEAM-1 = biotype B) vector, which is widely distributed, with a polyphagous feeding habit and high adaptability to different environmental conditions. The introduction of B. tabaci MEAM 1 in the early 1990s was the triggering factor for begomovirus epidemics in tomato in Brazil, with a subsequent increase in the number of new viral species. Furthermore, distinct patterns of diversity and dynamics of viral subpopulations were observed in different environments. In this scenario, weeds play an important role as they function as natural reservoirs of begomovirus. New viral species have been reported in weeds, including areas in and around commercial tomato fields. Some weed begomoviruses have been identified and biologically characterized, however it is believed that these studies have not been extensive enough to estimate the actual diversity of new species in weeds. In the country, four begomoviruses were reported concomitantly infecting tomato and weeds. Globally, more than 40 begomoviruses have been described in Malvaceae species, and some of these have been detected originally infecting Solanaceae species. The small and bipartite genome, together with the transmission efficiency and polyphagy of the vector, provide favorable conditions for the occurrence of mixed infections and recombination and pseudo-recombination events between begomoviruses, thus contributing to the frequent alteration of the genetic structure of viral populations in our conditions. Different strategies have been employed to analyze the evolutionary processes capable of shaping the genetic-molecular structure of begomoviruses. The main strategy is to obtain the complete viral genome (DNA–A and DNA–B) for further analysis using different programs and evolutionary models. The metagenomics combined with High Throughput Sequencing (HTS) has allowed to determine a great viral diversity present in Brazil. A high frequency of mixed infections has been detected with many of them involving potential new species capable of inducing severe symptoms in plants. In the present work, four contigs obtained from HTS of Malvaceae leaf samples were selected for study and characterization, in according to the methodology carried out by the LVV-Fito team and summarized below. Initially foliar samples of weeds showing typical symptoms of begomovirus (generalized chlorosis, mosaics, and golden spots) were collected in tomato production areas and/or areas close to tomato cultivation, in the five regions of the country. Samplings were carried out from 2001 to 2020, with a total of 78 leaf samples from plants of the Malvaceae family analyzed (selected using the year/place of collection as criteria). Total DNA was extracted via CTAB and organic solvents and stored at -20 °C. The initial confirmation of the presence of begomovirus infection in the samples was made through PCR (polymerase chain reaction) assays using degenerate primers ‘PAR1c496’ and ‘PAL1v1978’. Viral circular DNAs were enriched in positive samples via rolling circle amplification (RCA). High-throughput sequencing (HTS) was performed on an Illumina HiSeq2500 platform. 5 The viral contigs were annotated and the reads were mapped back to the annotated genome using the ‘Map to reference’ tool available in the Geneious 11.1.5 program. About 7,391,728 million readings were obtained from the pool of 78 samples. After assembly, using the CLC Genomics Workbench 11 program, 10,679 contigs were obtained. Four contigs were selected. Three of these contigs corresponded to complete DNA–A and exhibited identity levels below 91%, consistent with the current taxonomic criteria for defining new species within the genus Begomovirus. The DNA component– Potential new species #1 showed 79% identity with Sidastrum golden leaf spot virus (HM357458), new species #2 showed 81% identity with Oxalis yellow vein virus (KM887907), while new #3 showed 78% identity with Sida yellow mosaic Alagoas virus (JX871383), confirming the occurrence of three new species of begomovirus in weeds. Sequences of DNA – B components were obtained and analyzes performed, including confirmation of cognates species. The fourth contig showed 98,24% identity with Sida micranta mosaic virus (SiMMV) (KC706535). After using specific primers, already available, a result of 41 positive samples for SiMMV was obtained. The characterization of these three species, as well as the occurrence and distribution of Sida micranta mosaic virus in five Brazilian regions will be presented in this dissertation.

Viruses are widespread, yet little is understood about these biological entities. The evaluation is less than 1% of the total eukaryotic virosphere known. Common knowledge is constrained to viruses that cause diseases and economic losses. Other ecological relationships and rising factors of viruses are unexplored. The segment of plant viruses is not distinct. The understanding of diversity, evolutionary forces, and their impact is unknown. However, with the High throughput sequencing (HTS) advent, the study of the virome got tangible and fast-growing. Likewise, the bioinformatic tools improved the comprehension of viral diversity and evolution patterns. The SARS-CoV 2 pandemics, and the possible rising of new viral diseases in humans and in other economically important eukaryotes have demonstrated that it is necessary to explore the virosphere. The Orchidaceae family is the second-largest family of flowering plants. They grow in tropical areas as Bromeliaceae family specimens. The viral diversity of both families is not entirely investigated, except for the economic interest plants. In this work, the virome bioprospection research was conducted with specimens of Bromeliaceae and Orchidaceae family found in the Brazilian central plateau ornamental market. DNA and RNA HTS from these plants were performed using the Illumina NovaSeq 6000 and HiSeq 2000 platforms, respectively. Bioinformatics programs, CLC Genomic Workbench v. 20.0 and Geneious R11.1, were used to study the results found in the HTS. Two new DNA and seven RNA viruses were described infecting orchids and bromeliads. In the first chapter, a bibliographic review of the ornamental plant market and of the incidence of viruses on them was developed with the aim of exploring the status quo of research in the area. In the second chapter, the exploration of the RNA HTS results of 54,088,166 reads and 16,560 contigs was carried out with the goal of characterizing the possible RNA viruses present. For this, the contigs obtained were BLASTn confronted against a viral sequence 5 database. 15 hitherto uncharacterized viral RNA sequences were identified. A total of seven new viral species were characterized. One sequence belonging to the genus Alphaendornavirus (family Endornaviridae), two belonging to the genus Totivirus (family Totiviridae), three belonging to the genus Polerovirus (family Solemoviridae), two RNA 1 and two RNA 2 belonging to the genus Dichorhavirus (family Rhabdoviridae) and one RNA 1 and an RNA 2 belonging to the genus Nepovirus (family Secoviridae). Three RNA 1 sequences related to the genus Sadwavirus, family Secoviridae, were identified. However, no RNA 2 was found. According to the genus classification criteria adopted by the International Committee on Taxonomy of Viruses (ICTV), the information found on these previous sequences is insufficient to establish new species. The seven new species were named Alphaendornavirus monocotyledonae (OQ866133); Dichorhavirus monocotyledonae (OQ866129, OQ866130, OQ866131, OQ866132); Nepovirus monocotyledonae (OQ866134, OQ866135); Polerovirus monocotyledonae 1 (OQ866138); Polerovirus monocotyledonae 2 (OQ866139, OQ866140); Totivirus monocotyledonae 1 (OQ866136); Totivirus monocotyledonae 2 (OQ866137). The third chapter is a preface containing information about the collection sites, sample cataloging and methodologies used in subsequent chapters. The fourth and fifth chapters contains the result of the bioinformatic treatment of the 11,060,510 reads and 163,808 contigs generated by HTS. The fourth chapter is a disease note of a mixed infection of two known begomoviruses, tomato severe rugose virus (ToSRV) and tomato chlorotic mottle virus (ToCMoV), in orchids of the genus Dendrobium. The fifth chapter is a characterization of two high divergently new begomovirus-like pathogens infecting orchids of genus Spathoglottis. The two new viral sequences were named Spathoglottis-associated mottle virus 1 (OQ791967) and Spathoglottisassociated mottle virus 2 (OQ791968) and should be considered new species. Primers were designed for both sequences and confirmed the infection in orchids sample of the genus Spathoglottis.

Root-knot nematodes (Meloidogyne spp.) represent one of the most important groups of root pathogens due to the damage induced in a wide range of hosts. Parasitism by Meloidogyne spp. is a major obstacle in the commercial production of carrots (Daucus carota) in subtropical regions, causing yield and quality losses. In Brazil, the most important species in carrot crop are M. javanica and M. incognita. The cultivar ‘Brasília’ is the main source of resistance in carrot against these two species. However, molecular marker systems linked to resistance factors for Brazilian carrot cultivars are still not available, which would facilitate the selection process. Tomato (Solanum lycopersicum) is also severely affected by Meloidogyne spp. with special emphasis on M. enterolobii due to its recent geographic expansion and the ability to “break-down” the resistance conferred by the Mi-1.2 gene. However, alternative sources of resistance in tomato to M. enterolobii are not yet available. Furthermore, new methodologies for nematological evaluation are necessary for the selection of resistance plants in both vegetable crops. In this context, the present work aimed to study different aspects of carrot and tomato interactions with Meloidogyne species. In carrots, a study was carried out with an F2 population of 108 plants of the cultivar ‘Brasília #83147’ (which segregates for resistance against M. javanica). Plants were inoculated with 8000 eggs + J2 of M. javanica 30 days after sowing. Total DNA was extracted from foliar samples and evaluated (via PCR) with codominant SCAR/STS markers (SQ1850R / SQ1700S and SQ6650R / SQ6590S) previously identified in close linkage with a resistance locus (Mj-1) to M. javanica in ‘Brasilia’. As for the nematological analyses, the root systems of individual carrot plants were evaluated at 120 days after inoculation (DAI) for the reproduction factor (FR = pf/pi) and the individual quantification of galls and egg mass. Roots of individual contrasting plants (resistant and susceptible) were vernalized and subjected to selfing (S1 generation). Inoculation bioassay in conjunction with marker analyses confirmed that resistance is not fixed in this population, with many susceptible individuals being detected. However, it was possible to identify individuals with extreme resistance (immunity-like response) and with the presence of codominant SCAR/STS markers. These individuals can now be used as parents to facilitate genetic studies as well as the development of new Brazilian carrot cultivars. In addition, the scale proposed in this work was presented as an extremely useful tool, being easy and quick to use for germplasm assessment. In tomato, the reaction of 24 germplasm accessions was verified under different inoculum levels of M. enterolobii. The potential residual effect of the Mi-1.2 gene was also studied in comparative assays using isogenic lines (isolines) contrasting for this resistance factor: ‘Rio Grande’ (mi-1.2/mi-1.2) versus ‘Nemadoro’ (Mi-1.2/ Mi-1.2). The allelic status of each individual plant was confirmed via the use of molecular markers linked to the Mi1.2 locus. This molecular marker was also employed to evaluate the use of the breeding use of this gene in the Brazilian varietal panorama. Accessions were inoculated with four levels of M. enterolobii inoculum (1000, 2000, 4000, and 8000 eggs + J2s) 15 days after transplanting. For the nematological analyses, the tomato roots (at 45 days after inoculation) were evaluated according to the gall index (GI), egg mass index (EMI), number of eggs per gram of root (NOGR), and the reproduction factor (RF). All evaluated tomato accessions showed susceptible responses, demanding the search of novel sources of resistance against this emerging pathogen. In turn, the Mi-1.2 gene did not confer a significant residual effect against M. enterolobii. Although extremely effective against at least 13 Meloidogyne species, this gene was not able to interfere in the infection process of this nematode. In the M. enterolobii inoculation process, the most appropriate inoculum levels ranged from 1000 to 2000 eggs + J2 to obtain the maximum manifestation of nematological traits. Given the importance of tomato and carrot in Brazil, the results of this study contribute substantially to breeding programs aimed at developing cultivars resistant to different Meloidogyne species.

In Brazil, the tomato (Solanum lycopersicum L.) is one of the most important vegetable crops both economically and socially. The production costs and yield levels of this crop suffer recurrent fluctuations due to several factors, among which diseases such as begomoviruses (caused by a complex of Begomovirus species) stand out. These pathogens have one or two single-stranded circular DNA molecules, encapsidated in 18- 30 nanometer particles. Begomoviruses with a bipartite genome (presenting DNA–A and DNA–B components) predominate in tomato in the New World, being transmitted by different cryptic species of the Bemisia tabaci complex. These vectors have polyphagous feeding habits, being widely distributed, with an extensive circle of hosts, including several weeds. The introduction of B. tabaci Middle East Asia Minor 1 – MEAM-1 (= biotype B) took place in the early 1990s, triggering epidemics of begomoviruses in tomato, with an exponential increase in the number of Begomovirus species. The presence of weeds, mainly from the Solanaceae family, plays a very important role as reservoirs of tomato-infecting begomoviruses. New viral species have been reported in weeds, and such interactions increase the occurrence of natural mechanisms that generate genetic variability, which can lead to the surfacing of new viral variants. The advent of High Throughput Sequencing (HTS) technology has led to the discovery of new viral species associated with tomato cultivation. In this context, one of the objectives of this work is to study viral diversity in tomato and weeds from the Solanaceae family. One hundred and thirty-five (135) tomato and weed leaf samples exhibiting typical begomovirus-like symptoms (apical chlorosis, golden mosaic, and chlorotic spots) were collected in the North (15), Northeast (43), South (11), Center West (45), Southeast Brazil (18) and Uruguay (3). Collections were carried out from 2003 to 2022, and leaf samples were analyzed from S. lycopersicum (92), S. aethiopicum (4), S. paniculatum (3), S. sessiliflorum (1), S. melongena (4), S. mammosum (1), Capsicum species (12), Nicandra physalodes (8), Physalis species (6), S. betaceum (2), S. viarum (1), and S. americanum (1). These samples were selected using year/place of collection as criteria. The extracted total DNA was used as template for rolling circle amplification (RCA). Initially, the RCA of each sample was used to confirm the presence of begomovirus via PCR assays using degenerate primers for DNA–A (PAR1c496 and PAL1v1978) and for DNA–B (PBL and CRC). After this step, the RCA products of the 135 samples were used to form a pool and sequenced on an Ilumina NovaSeq 6000 platform. A total of 19,735,294 million reads was obtained. These reads were used to assemble contigs in CLC Genomics Workbench 11. 41,659 contigs were obtained, 173 of which showed (after analysis with RefSeqViral) identities with begomoviruses (170), topilevirus (1), gemycircularvirus (1) and one alphasatellite. Sixteen out of the 170 begomovirus-like contigs showed nucleotide identity below 91%, being consistent with the current taxonomic criteria for defining new species within the genus Begomovirus. Contigs C195, C18367 and C2046 showed identities of 99.1%, 99.67% and 99.92% with tomato apical leaf curl virus (ToALCV; MH539677), plant associated genomovirus 12 (MT214094), and Alphasatellitidae species (MT214093), respectively. With the exception of ToALCV, the other viruses will be fully characterized in the future. Three contigs were considered as potential novel species within the Begomovirus genus. These contigs (C16, C21 and C230) showed 80%, 94% and 80% identity with melochia mosaic virus (KT201151), tomato bright yellow mottle virus (KC791691) and ToBYMV (KC791691), respectively. The alignment of these three contigs showed a typical genomic organization of bipartite begomoviruses. The common regions of C16 and C230 were determined and analyzes were carried out with their iterons and other motifs verifying them as cognate bipartite genomes. On the other hand, contig C21 displayed a typical genomic organization of a monopartite begomoviruses, also representing a potential new viral species. In conclusion, with the employment of HTS it was possible to observe the presence of high levels of genetic variability and diversity of begomoviruses in members of the Solanaceae family, indicating their relevant role as viral reservoirs. Research actions are being conducted aimed at producing infectious clones to uncover the range of hosts as well as sources of resistance against these new viruses.

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Unconventional fruits in Brazilian agriculture, such as Syzygium jambos (Myrtaceae), are widely studied for medicinal use, but the diseases that deteriorate these fruits are neglected. Fungal genera from the families Nectriaceae, Bionectriaceae, and Botryosphaeriaceae cause significant diseases in agriculturally and commercially important plants, but they are rarely reported in native species and/or causing fruit rot. The identification of these fungi is challenging, especially the Calonectria-like group (Nectriaceae) that includes phylogenetically and morphologically related fungi to the genus Calonectria. Since 1995, the submission of sequences from different genomic regions has been consolidated in GenBank for the recognition of phylogenetic species. This rule has facilitated species identification, but the lack of uniformity and the increasing volume of data make it difficult or even impossible to study host range and geographical distribution comprehensively. Therefore, this study aimed to conduct a meta-analysis of the information available in GenBank for Calonectria-like fungal complex and to morpho-molecularly identify the isolates from the Calonectria-like, Clonostachys, Cylindrocladiella, Gliocephalotrichum, Gliocladiopsis and Neofusicoccum obtained from fruits collected in Distrito Federal, Goiás, Minas Gerais, Paraná, Pernambuco, Santa Catarina and São Paulo. The meta-analysis revealed discrepancies among the genomic regions deposited for each isolate, with the sequences from the elongation factor region (n=5,125) and beta-tubulin region (n=5,287) being more representative compared to calmodulin, histone, RNA Polymerase 2, internal transcribed spacer, and the large ribosomal subunit (28S rDNA). The exchange of samples between research groups led to the "Matryoshka Effect," evidenced by the over-representation of isolates. The analysis reveals that 78.3% of the analyzed sequences have information about the collection location, while only 12% have information about the substrate/host. While most genera have a broad geographical distribution, the genera Curvicladiella and Xenocylindrocladium are restricted to South America and Asia, respectively, with the majority of species found in Brazil (90%) and China (100%), respectively. The 86 morphomolecularly identified isolates belong to the following genera: Calonectria (n=15), Clonostachys (n=8), Cylindrocladiella (n=28), Gliocladiopsis (n=18), Gliocephalotrichum (n=2), and Neofusicoccum (n=15). Among these, seven known species have been documented, while one, two, and three new species will be proposed for Clonostachys, Calonectria and Cylindrocladiella genera, respectively. The species Cylindrocladiella vitis, Gliocladiopsis hennebertii, Neofusicoccum occulatum, and N. umdonicola are reported for the first time in Brazil while new host combinations were recorded for C. pseudocholeuca, C. rogersoniana, Cy. infestans, Cy. lageniformis, Cy. peruviana, Gliocephalotrichum simplex, Gliocladiopsis tenuis, N. batangarum, and N. parvum on fruits of the Dypsis madagascariensis, Eugenia aggregata, Eugenia involucrata, Euterpe edulis, Spondias mombin, Syzygium jambos and Terminalia catappa. This study demonstrates the need for comprehensive surveys on the diversity of fungal species in native plants and highlights the risk of transmission to economically important plants.

Photosynthesis, stomatal conductance, and transpiration are plant physiological activities that influence the productive potential of crops. The interference of root-knot nematodes in plant physiology is still poorly studied, as well as the capacity of rhizobacteria to mitigate physiological damage caused by nematodes. The lack of alternative methods of control to the nematode Meloidogyne enterolobii hinders efficient and sustainable management friendly to the environment. The objectives of this study were to evaluate in vitro the effect of a bionematicide, with the active ingredient Bacillus methylotrophicus, on the eggs and second stage juveniles (J2s) of M. enterolobii; and to monitor changes in the physiology of guava plants inoculated with M. enterolobii and treated with the bionematicide over time. In vitro experiments were conducted on M. enterolobii eggs and J2s. The treatments included concentrations of 1%, 10%, 25%, 50%, and 70% of the bionematicide, with water as control treatment. The experimental design was completely randomized with six replicates and two repetitions over time. The treatments with eggs were evaluated at 2, 4, 6, 8, and 10 days after incubation by counting the number of damaged eggs. The treatments with juveniles were evaluated at intervals of 24 to 96 hours by counting the number of dead juveniles. The monitoring of guava tree physiological activity was assessed in greenhouse and field conditions. The treatments included: 1) non-inoculated and non-treated guava cv. Pedro Sato (control); 2) non-inoculated and non-treated guava cv. Paluma guava tree (control); 3) guava 'Pedro Sato' treated with B. met and noninoculated; 4) 'Paluma' treated with B. met and non-inoculated; 5) Paluma' inoculated and non-treated with B. met; 6) 'Pedro Sato' inoculated and non-treated with B. met; 7) 'Pedro Sato' inoculated and treated with B. met, and 8) 'Paluma' inoculated and treated with B. met. The treatments were arranged in a completely randomized design (DIC) and randomized block design (DBC) in the greenhouse and field, respectively. Four applications of the bionematicide were made, at doses of 3 mL/L of water per plant in the greenhouse and 1 mL/L of water per plant in the field experiment. Between the second and third application, the plants were inoculated with 5000 eggs and eventual juveniles of M. enterolobii in the greenhouse, and 6000 eggs and eventual juveniles of the nematodes in the field. In the greenhouse, seven evaluations of physiological parameters of the guava trees (stomatal conductance, water use efficiency, photosynthesis, and transpiration) and six evaluations of total chlorophyll content were performed throughout cycle of the guava plant development. At 132 days after inoculation (DAI), the roots were weighed, and the gall index, egg mass index, and reproduction factor were determined. In the field, ten evaluations of physiological parameters of the guava trees (stomatal conductance, water use efficiency, carboxylation efficiency, photosynthesis, and transpiration) were performed, and productivity was estimated through harvest and subsequent fruit weighing. In vitro, damage to the eggs increased for all treatments, with no difference between doses after six days of exposure. J2 mortality increased up to 48 hours in the lower doses (1%, 10%, and 25%), remaining close to 100% in the higher doses (50% and 70%), with no difference between doses after 48 hours of exposure. In the greenhouse, changes in physiological parameters were concentrated in the first 44 DAI. Meloidogyne enterolobii and B. met combined reduced stomatal conductance and transpiration at 26 and 44 DAI, respectively. Photosynthesis was lower at 26 DAI in treatments that received both nematode and bacterium in combination. Meloidogyne enterolobii and B. met, either isolated or combined, showed reduced water use efficiency at 26 and 44 DAI. Gall index, egg mass index, and reproduction factor were higher in treatments that received the nematode. In the field, there was no difference over time for physiological variables for all treatments, and there was no pattern in the changes caused by isolated or combined M. enterolobii and B. met. Meloidogyne enterolobii reduced stomatal conductance in eight evaluations, water use efficiency and photosynthesis in three evaluations, and transpiration in seven evaluations. The only increase conditioned by the nematode was observed in water use efficiency at 369 DAI. Bacillus methylotrophicus reduced photosynthesis at 421 DAI but increased it at 527 DAI; at 318 and 369 DAI, there was an increase in water use efficiency. Meloidogyne enterolobii and B. met combined reduced stomatal conductance in eight evaluations and transpiration in four evaluations. Reduction in water use efficiency and photosynthesis was observed at 421 DAI. Meloidogyne enterolobii and B. met combined increased water use efficiency at 369 DAI and carboxylation efficiency at 224 and 465 DAI. The application of B. met did not mitigate the negative effects of M. enterolobii on the physiological activities of the guava trees, and isolated or combined M. enterolobii and B. met did not alter guava tree productivity. Bacillus methylotrophicus caused damage to the eggs and mortality of M. enterolobii J2. Under controlled conditions, M. enterolobii and B. met combined reduced stomatal conductance, water use efficiency, photosynthesis, and transpiration of guava trees during the initial phase of fruit development. Isolated M. enterolobii and B. met reduced water use efficiency at 26 and 44 DAI. The nematological parameters were not influenced by the bacterium. In the field, the absence of alterations may be due to the influence of environmental factors, inoculum density, bacterium application method, and combinations of nematode and bacterium application over time. Reduction in physiological parameters by the nematode was dominant. The application of B. met did not mitigate the negative effects of the nematode on the physiological activity of the guava trees. Isolated or combined M. enterolobii and B. met did not alter guava productivity.

Onion (Allium cepa L.) is an important vegetable cultivated since ancient times and distributed worldwide. It is the third most cultivated vegetable, after potatoes and tomatoes. An outbreak of onion bacterial blight occurred in 2018, and since then, has been threatening onion quality and productivity in the Brazilian Cerrado within the states of Goiás, Minas Gerais and Distrito Federal. Samples were collected isolates identified as being Xanthomonas euvesicatoria pv. allii (Xea). To increase knowledge about the pathogen, the objective of this work was to structure a new collection of isolates, identify, characterize, and develop a new PCR detection protocol for Xea. The 2021 isolate collection had its genus confirmed by the XgumD F7/R7 primers and the variability by the BOX-PCR molecular marker technique. Haplotypes were selected for sequencing four genomic regions for multilocus sequence analysis. For the development of a detection protocol, six genomes available in the GenBank of Xea were reannotated and then, by comparative genomics, we searched for genes present among all isolates and absent for other species and pathovars. For the design of the primers the sequences of the exclusive genes were used in the software NEB LAMP Primer Design Tool. The synthesized primers were evaluated for the amplification capacity of Xea isolates, sensitivity, and specificity with onion leaf microbiota. All isolates were identified as belonging to the genus Xanthomonas and by BOX-PCR it was possible to identify the presence of four haplotypes in addition to the six haplotypes from the original collection. By multilocus sequence analysis, the clustering of the new isolates with the originals of 2018/2019 with high values of posterior probability was observed, while only the isolate A-2021-01 clustered in a more distant clade with isolates of the pathovar alfalfae. Only the 5038_ID9 primers were able to amplify all haplotypes only with the expected 200 pb fragment and amplification sensitivity of concentrations greater than 50 pg. None of the microbiota isolates were amplified with these primers.

Meloidogyne enterolobii is an emerging nematode species in Brazil. This species has been considered one of the most aggressive among root-knot nematodes and has been causing damage to vegetables and other crops of economic importance, including under the presence of genetic resistance to other species of this genus. Genetic resistance in plants, whenever available, is the most efficient and economical way to control root-knot nematodes. In this sense, the search for sources of resistance is of great importance for the control of this nematode. Crops of economic importance, including pulses, as peas (Pisum sativum L.), chickpeas (Cicer arietinum L.) and lentils (Lens culinaris Medik.), as well as vegetables such as pepper (Capsicum spp.) and sweet potato (Ipomoea batatas (L.) Lam.), are drastically affected by Meloidogyne spp. The reaction to M. enterolobii was evaluated in 14 genotypes of pea, 06 of chickpeas, 01 of lentil, 27 of pepper (Capsicum chinense), 07 of bell pepper (C. annuum) and 08 of sweet potato. All experiments were carried out in a greenhouse, in a completely randomized design with 06 replications and each plant was inoculated with 5000 eggs and eventual second-stage juveniles (J2). For the reaction of pulses and sweet potato genotypes to M. enterolobii, the experiments were repeated twice in time. As for the reaction of peppers, two experiments were installed, one for C. chinense and another for C. annuum. Sixty-five days after inoculation (DAI) for pea, chickpea, lentil and pepper crops and at 90 DAI for sweet potato. The following nematological variables were evaluated: gall index (GI), egg mass index (IMO), number of eggs per gram of root (NOGR) and the reproduction factor (FR). None of the pea, chickpea, lentil and pepper genotypes evaluated showed resistance to M. enterolobii. However, 03 sweet potato genotypes (BGBD 1399, MD 1609024 and MD 1610036) were classified as resistant. Given the importance and prospects of expansion of these crops in Brazil, the results of this study significantly contribute to the knowledge of their reaction of genotypes of these crops to this species of nematode, as well as to identify potential sources of resistance for breeding programs aimed at the development of cultivars, and meeting the continuous need for research to select genotypes with resistance to M. enterolobii.

Sugarcane (Saccharum spp.), a species belonging to the Poaceae family, is the main source of raw material to produce sugar and fuel ethanol in Brazil, which is why it occupies a prominent role in the country's economy. The crop is affected by the pineapple rot disease, caused by Thielaviopsis sp., a fungus that mainly damages bud sprouting. The objective of this work was to evaluate in vitro and in vivo the efficiency of 15 strains of Trichoderma in the control of this disease, in addition to a commercial strain included as a reference in the tests. Different laboratory methods were adopted, such as pairing of cultures, exposure to volatile organic compounds (VOCs) in shared atmosphere and exposure to autoclaved crude extracts of the antagonist. All Trichoderma strains were able to inhibit the mycelial growth of Thielaviopsis sp. The other tests carried out continued with the strains that presented the best performances, namely CEN281 (T. afroharzianum), CEN1277 (T. asperelloides), CEN1513 (T. koningiopsis), CEN1546 (T. afroharzianum) and CEN219 (T. harzianum) the commercial strain. The inhibition of mycelial growth after exposure to VOCs in chronology was also addressed, as this is one of the main mechanisms of action of antagonistic fungi. The in vivo experiments consisted of the inoculation of the pathogen and antagonists in “CTC20” cane stalks. There was a reduction in the severity of the disease by the Trichoderma strains both in laboratory tests and in greenhouse tests. In the latter, plant height, root and shoot dry mass, stem diameter, and disease severity were evaluated. The main time of pathogen inhibition occurred after 72 hours of antagonist growth for most strains in the VOC assay in chronology. Disease severity was reduced by approximately 70% by one of the selected strains. The tests conducted in a greenhouse complemented the previous ones, conducted in vitro and in vivo, proving the effectiveness of the studied strains in controlling pineapple rot in sugarcane. It is inferred, based on the tests carried out, that the biocontrol agent can be used, in the treatment of stalks prior to planting, as a prophylactic measure for the control of pineapple rot and, also, as a rooting promoter for the crop, including in the production of pre-sprouted seedlings (PSS). In all tests carried out, a strain from the Embrapa Genetic Resources and Biotechnology Collection, identified as belonging to the species T. asperelloides (CEN1277), stood out in the tests carried out, with results similar to those verified with the commercial strain already registered for disease control. Therefore, this strain can be made available for the development of a new product, as demanded by the biological fungicide market.