First report of Tomato interveinal chlorosis virus infecting Rhynchosia minima plants in Brazil.

Tomato interveinal chlorosis virus (ToICV; Begomovirus solanumintervenae, genus Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.

Endophytic Fungus of Achyrocline satureioides: Molecular Identification, Chemical Characterization, and Cytotoxic Evaluation of its Metabolites in Human Melanoma cell line.

Endophytic fungi are important sources of anticancer compounds. An endophytic fungus was isolated from the medicinal plant Achyrocline satureioides, and molecularly identified as Biscogniauxia sp. (family Xylariaceae) based on partial nucleotide sequences of the internal transcribed spacer genomic region (GenBank Accession No. ON257911). The chemical characterization and cytotoxic properties of secondary metabolites produced by Biscogniauxia sp. were evaluated in a human melanoma cell line (A375). The fungus was grown in potato-dextrose liquid medium for 25 days, and the extracted compounds were subjected to solid-phase fractionation to obtain the purified FDCM fraction, for which the metabolites were elucidated via ultra-performance chromatography coupled to a mass spectrometer. In the present study, 17 secondary metabolites of Biscogniauxia sp., including nine polyketide derivatives, five terpenoids, and three isocoumarins, were putatively identified. This is the first study to report of the ability of Biscogniauxia sp. in the production of isocoumarin orthosporin; the terpenoids nigriterpene A and 10-xylariterpenoid; the polyketide derivatives daldinin C, 7'dechloro-5'-hydroxygriseofulvin, daldinone D, Sch-642305, curtachalasin A, cytochalasin E, epoxycytochalasins Z8, Z8 isomer, and Z17. Furthermore, this study has reported the biosynthesis of Sch-642305 by a Xylariaceae fungus for the first time. FDCM significantly reduced the viability and proliferation of human melanoma cells at half-maximal inhibitory concentrations ​​of 10.34 and 6.89 µg/mL, respectively, and induced late apoptosis/necrosis and cell cycle arrest in G2/M phase after 72 h of treatment. Given its ability to produce unique metabolites with promising cytotoxic effects, Biscogniauxia sp. of A. satureioides may be a reservoir of compounds with important therapeutic applications.

Cotton Leafroll Dwarf Virus US Genomes Comprise Divergent Subpopulations and Harbor Extensive Variability.

Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9-98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5-99.6% and 81.2-89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (dN/dS < 1) was implicated as the primary selective force acting on viral protein evolution.

High molecular diversity and divergent subpopulations of the begomovirus cnidoscolus mosaic leaf deformation virus associated with Cnidoscolus urens.

Begomoviruses have circular, single-stranded DNA genomes encapsidated into twinned quasi-icosahedral particles and are transmitted by whiteflies of the Bemisia tabaci sibling group. Begomoviruses infect cultivated and non-cultivated plants, causing great losses in economically important crops worldwide. To better understand the genetic diversity of begomoviruses infecting the non-cultivated host Cnidoscolus urens, leaf samples exhibiting virus-like symptoms were collected in different localities in the state of Alagoas, Brazil, during 2015 and 2016. Forty-two complete DNA-A sequences were cloned and sequenced by the Sanger method. Based on nucleotide sequence comparisons, the 42 new isolates were identified as the bipartite begomovirus cnidoscolus mosaic leaf deformation virus (CnMLDV). The CnMLDV isolates were clustered in two phylogenetic groups (clusters I and II) corresponding to their sampling areas, and the high value of Wright's F fixation index observed for the DNA-A sequences suggests population structuring. At least seven independent intraspecies recombination events were predicted among CnMLDV isolates, with recombination breakpoints located in the common region (CR) and in the CP and Rep genes. Also, a high per site nucleotide diversity (π) was observed for CnMLDV isolates, with CP being significantly more variable than Rep. Despite the high genetic variability, strong negative or purifying selection was identified as the main selective force acting upon CP and Rep.

Assessing the diversity of whiteflies infesting cassava in Brazil.

BACKGROUND: The necessity of a competent vector for transmission is a primary ecological factor driving the host range expansion of plant arthropod-borne viruses, with vectors playing an essential role in disease emergence. Cassava begomoviruses severely constrain cassava production in Africa. Curiously, begomoviruses have never been reported in cassava in South America, the center of origin for this crop. It has been hypothesized that the absence of a competent vector in cassava is the reason why begomoviruses have not emerged in South America. METHODS: We performed a country-wide whitefly diversity study in cassava in Brazil. Adults and/or nymphs of whiteflies were collected from sixty-six cassava fields in the main agroecological zones of the country. A total of 1,385 individuals were genotyped based on mitochondrial cytochrome oxidase I sequences. RESULTS: A high species richness was observed, with five previously described species and two putative new ones. The prevalent species were Tetraleurodes acaciae and Bemisia tuberculata, representing over 75% of the analyzed individuals. Although we detected, for the first time, the presence of Bemisia tabaci Middle East-Asia Minor 1 (BtMEAM1) colonizing cassava in Brazil, it was not prevalent. The species composition varied across regions, with fields in the Northeast region showing a higher diversity. These results expand our knowledge of whitefly diversity in cassava and support the hypothesis that begomovirus epidemics have not occurred in cassava in Brazil due to the absence of competent vector populations. However, they indicate an ongoing adaptation process of BtMEAM1 to cassava, increasing the likelihood of begomovirus emergence in this crop.

Evolutionary dynamics of bipartite begomoviruses revealed by complete genome analysis.

Several key evolutionary events marked the evolution of geminiviruses, culminating with the emergence of divided (bipartite) genomes represented by viruses classified in the genus Begomovirus. This genus represents the most abundant group of multipartite viruses, contributing significantly to the observed abundance of multipartite species in the virosphere. Although aspects related to virus-host interactions and evolutionary dynamics have been extensively studied, the bipartite nature of these viruses has been little explored in evolutionary studies. Here, we performed a parallel evolutionary analysis of the DNA-A and DNA-B segments of New World begomoviruses. A total of 239 full-length DNA-B sequences obtained in this study, combined with 292 DNA-A and 76 DNA-B sequences retrieved from GenBank, were analysed. The results indicate that the DNA-A and DNA-B respond differentially to evolutionary processes, with the DNA-B being more permissive to variation and more prone to recombination than the DNA-A. Although a clear geographic segregation was observed for both segments, differences in the genetic structure between DNA-A and DNA-B were also observed, with cognate segments belonging to distinct genetic clusters. DNA-B coding regions evolve under the same selection pressures than DNA-A coding regions. Together, our results indicate an interplay between reassortment and recombination acting at different levels across distinct subpopulations and segments.

Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil.

Black pod disease, caused by Phytophthora species, is among the main limiting factors of cacao (Theobroma cacao L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by Phytophthora capsici, Phytophthora citrophthora, Phytophthora heveae, and Phytophthora palmivora. To assess the diversity of Phytophthora species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as P. citrophthora and P. palmivora were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome c oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting Phytophthora isolates. The morphological characterization and evaluation of the mycelial growth rates for the Phytophthora isolates were performed in vitro. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as P. palmivora (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in Phytophthora clade 2. Therefore, these isolates are considered as a new species herein referred to as Phytophthora theobromicola sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The P. palmivora isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of P. capsici, but no oogonia/antheridia were observed when P. theobromicola was paired with the different tester strains. The P. theobromicola and P. citrophthora isolates showed higher mycelial growth rates, when compared to P. palmivora, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both Phytophthora species, with P. theobromicola inducing higher rates of necrotic lesion expansion, when compared to P. palmivora. Here, two Phytophthora species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed P. theobromicola seems to be prevalent in field conditions. This is the first report of P. theobromicola on T. cacao. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to Phytophthora.

Complete genome sequences of three newly discovered cacao mild mosaic virus isolates from Theobroma cacao L. in Brazil and Puerto Rico and evidence for recombination.

To analyze the DNA virome associated with cacao (Theobroma cacao L.) trees showing virus-like symptoms in Brazil (BR) and Puerto Rico (PR) during 2018-2019, total DNA was isolated from symptomatic leaves and subjected to high-throughput Illumina sequencing. The assembled complete badnaviral genome sequences were verified by PCR amplification, cloning, and DNA sequencing. Based on pairwise distances and phylogenetic analysis, three badnaviral genomes were identified, and these viruses were found to be isolates of the previously described cacao mild mosaic virus (CaMMV). The three genomes were 7,520, 7,524, and 7,514 bp in size for the isolates CaMMV-BR321, CaMMV-BR322, and CaMMV-PR3, respectively. Each genome contained four predicted open reading frames: ORFs 1-3 and ORFY. The CaMMV-PR3 isolate was identified as a probable recombinant, with a CaMMV-BR-like virus as the major parent.

A Complex of Badnavirus Species Infecting Cacao Reveals Mixed Infections, Extensive Genomic Variability, and Interspecific Recombination.

The incidence of cacao swollen shoot disease (CSSD) in cacao (Theobroma cacao L.) has increased in West Africa since ~2000. To investigate the genomic and species diversity of the CSSD-badnaviruses infecting cacao in Côte d'Ivoire and Ghana, symptomatic leaves were subjected to high-throughput sequencing. Among the 30 newly determined genomes, three badnaviruses were identified, Cacao swollen shoot Togo B virus (CSSTBV), Cacao swollen shoot CD virus, and Cacao swollen shoot CE virus (CSSCEV). The phylogenetic trees reconstructed for the reverse transcriptase (RT) and ribonuclease H (RNase H) sequences were incongruent with the complete viral genomes, which had the most robust statistical support. Recombination seems to be involved in the CSSD-badnavirus diversification. The genomic diversity varied among different CSSD-badnaviruses, with CSSTBV showing the lowest nucleotide diversity (π = 0.06236), and CSSCEV exhibiting the greatest variability (π = 0.21911). Evidence of strong purifying selection was found in the coding regions of the CSSTBV isolates.

Characterization of the Complete Genome and P0 Protein for a Previously Unreported Genotype of Cotton Leafroll Dwarf Virus, an Introduced Polerovirus in the United States.

Virus-like disease symptoms consisting of leaf cupping, shortened internodes, and overall stunting were observed in commercial cotton fields in Alabama in 2017 to 2018. To determine the complete genome sequence of the suspected causal polerovirus, symptomatic leaf samples were collected in Macon County, Alabama, and subjected to Illumina RNA sequencing. Based on BLASTn analysis, the Illumina contig of 5,771 nt shared the highest nucleotide identity (approximately 95%) with members of the species Cotton leafroll dwarf virus (CLRDV) (genus Polerovirus; family Luteoviridae) from Argentina and Brazil. The full-length viral genome sequence was verified by reverse transcription (RT)-PCR amplification, cloning, and Sanger sequencing. The complete CLRDV genome of 5,865 nt in length shared 94.8 to 95.2% nucleotide identity with six previously reported CLRDV isolates. The genome of the CLRDV isolate amplified from Alabama samples (CLRDV-AL) has seven predicted open reading frames (ORFs). Viral proteins 1 to 5 (P1 to P5) shared 91.9 to 99.5% amino acid identity with the six CLRDV isolates from Argentina and Brazil. However, P0, the suppressor of host gene silencing, shared 82.4 to 88.5% pairwise amino acid identity with the latter CLRDV isolates. Phylogenetic analysis of the seven full-length CLRDV genomes resolved three sister clades: CLRDV-AL, CLRDV-typical, and CLRDV-atypical, respectively. Three recombination events were detected by the recombination detection program among the seven CLRDV isolates with breakpoints occurring along the genome. Pairwise nucleotide identity comparisons of ORF0 sequences for the three CLRDV-AL field isolates indicated that they were >99% identical, suggesting that this previously unknown CLRDV genotype represents a single introduction to Alabama.

Genetic variability and population structure of the New World begomovirus Euphorbia yellow mosaic virus.

The emergence of begomoviruses (whitefly-transmitted viruses classified in the genus Begomovirus, family Geminiviridae) in Brazil probably occurred by horizontal transfer from non-cultivated plants after the introduction of Bemisia tabaci MEAM1. The centre of diversity of Euphorbia heterophylla (Euphorbiaceae) is located in Brazil and Paraguay, where it is an invasive species in soybean and other crops. Reports of possible begomovirus infection of E. heterophylla in Brazil date back to the 1950s. In 2011, Euphorbia yellow mosaic virus (EuYMV) was described in symptomatic plants collected in the Brazilian state of Goiás. Here we assess the genetic variability and population structure of begomoviruses infecting E. heterophylla in samples collected throughout nine Brazilian states from 2009 to 2014. A total of 158 and 57 haplotypes were compared in DNA-A and DNA-B datasets, respectively. Analysis comparing population structure in a large sampled area enabled us to differentiate two subpopulations. Further, the application of discriminant analysis of principal components allowed the differentiation of six subpopulations according to sampling locations and in agreement with phylogenetic analysis. In general, negative selection was predominant in all six subpopulations. Interestingly, we were able to reconstruct the phylogeny based on the information from the 23 sites that contributed most to the geographical structure proposed, demonstrating that these polymorphisms hold supporting information to discriminate between subpopulations. These sites were mapped in the genome and compared at the level of amino acid changes, providing insights into how genetic drift and selection contribute to maintain the patterns of begomovirus population variability from a geographical structuring point of view.

Cnidoscolus mosaic leaf deformation virus: a novel begomovirus infecting euphorbiaceous plants in Brazil.

Begomoviruses have been detected infecting the weed Cnidoscolus urens (family Euphorbiaceae) since 2004, but the viral species to which these viruses belonged was not known. Here, we report for the first time the complete genome sequence of a bipartite begomovirus obtained from C. urens collected in the state of Alagoas, Brazil. This isolate met the criteria to be classified as a member of a new begomovirus species, and the tentative name cnidoscolus mosaic leaf deformation virus (CnMLDV) is proposed. Pairwise sequence comparisons and phylogenetic analysis showed that the DNA-A genomic component of CnMLDV is most closely related to that of passionfruit severe leaf distortion virus, with 86.3 % nucleotide sequence identity.

Revision of Begomovirus taxonomy based on pairwise sequence comparisons.

Viruses of the genus Begomovirus (family Geminiviridae) are emergent pathogens of crops throughout the tropical and subtropical regions of the world. By virtue of having a small DNA genome that is easily cloned, and due to the recent innovations in cloning and low-cost sequencing, there has been a dramatic increase in the number of available begomovirus genome sequences. Even so, most of the available sequences have been obtained from cultivated plants and are likely a small and phylogenetically unrepresentative sample of begomovirus diversity, a factor constraining taxonomic decisions such as the establishment of operationally useful species demarcation criteria. In addition, problems in assigning new viruses to established species have highlighted shortcomings in the previously recommended mechanism of species demarcation. Based on the analysis of 3,123 full-length begomovirus genome (or DNA-A component) sequences available in public databases as of December 2012, a set of revised guidelines for the classification and nomenclature of begomoviruses are proposed. The guidelines primarily consider a) genus-level biological characteristics and b) results obtained using a standardized classification tool, Sequence Demarcation Tool, which performs pairwise sequence alignments and identity calculations. These guidelines are consistent with the recently published recommendations for the genera Mastrevirus and Curtovirus of the family Geminiviridae. Genome-wide pairwise identities of 91 % and 94 % are proposed as the demarcation threshold for begomoviruses belonging to different species and strains, respectively. Procedures and guidelines are outlined for resolving conflicts that may arise when assigning species and strains to categories wherever the pairwise identity falls on or very near the demarcation threshold value.