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.

First report of Cotton leafroll dwarf virus infecting cacao (Theobroma cacao L.) trees in Brazil.

Cotton leafroll dwarf virus (genus Polerovirus, family Solemoviridae) has been commonly reported affecting cotton plants (Gossypium spp., family Malvaceae) and several weed species (Ramos-Sobrinho et al., 2021; Sedhain et al., 2021). During a recent survey, cacao (Theobroma cacao L.) trees exhibiting virus-like symptoms such as leaf mosaic, vein clearing, and yellow spot were observed in the south part of the state of Bahia, northeastern Brazil, in 2022. Leaf samples were randomly collected from symptomatic cacao plants (n=30) growing in an affected area of approximately 30 ha. Total RNA obtained from pooled cacao samples were subjected to Illumina HiSeq 2500 sequencing as previously described (Keith et al., 2021), and partial sequences of cotton leafroll dwarf virus (CLRDV), and other virus-specific sequence contigs, were de novo assembled according to Ramos-Sobrinho et al. (2021). To further investigate the presence of CLRDV in cacao leaves, total RNA was individually extracted using a modified silica protocol (Rott and Jelkmann, 2001) and used as template for cDNA synthesis with random hexamers using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, CA, USA) following the manufacturer´s protocol. Detection of CLRDV was carried out by reverse transcription-polymerase chain reaction (RT-PCR) with the primers PL4F and o3-R, which amplify the open reading frame 3 (ORF3) encoding the capsid protein (Corrêa et al., 2005). Expected size amplicons (~0.6 kb) were observed from 16 out of 30 symptomatic plants, indicating ~53% of the cacao trees were infected by CLRDV. Considering 14 symptomatic plants tested negative for CLRDV, the symptoms observed here could also be caused by other viral groups or abiotic stress. To confirm the detection of CLRDV, the first half (~3.5kb) of the viral genome was amplified from two representative cacao samples using the primers P20F and P22R (Avelar et al., 2020). The RT-PCR products were gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) and Sanger sequenced. The RNA Illumina sequencing from pooled cacao samples (n=30) yielded 34,610,572 million trimmed reads. Two contigs of 868 and 839 nucleotides (nt) in length, and sharing high nt identity with CLRDV isolates, were assembled from 6,903 and 10,271 reads, at a coverage depth of 795 and 1,224x, respectively. Together, these contigs represent ~29% of the complete viral genome and included part of the 5´-untraslated region, ORF0 and the second half of ORF1-ORF2. Additional CLRDV-like contigs were observed across the viral genome, but they were not considered for further analyses due to the poor sequence quality. The Illumina- and Sanger-derived ORF0 and partial ORF1-ORF2 sequences shared >97% nt identity, suggesting they were congruent. Pairwise sequence comparisons for ORF0, encoding the gene silencing suppressor P0, indicated the cacao-associated isolates shared 99.7 and 99.2% nt and amino acid (aa) identity one with another, respectively. The ORF0 nt sequences showed 91.9-93.8 and 90.7-93.6% identity, while the aa sequences shared 85.8-88.5 and 86.2-90.0% similarity, with CLRDV isolates previously reported in South America and the USA, respectively. Finally, the ~3.5kb nt sequences of cacao-infecting CLRDV isolates shared 92.9-95.8% identity with CLRDV genomes deposited in NCBI-GenBank. The Bayesian phylogenetic tree reconstructed based on ORF0 nt sequences showed the new sequences were more closely related to CLRDV-atypical isolates (GenBank accession nos. KF359946, KF359947, KF906260, and KF906261). Together, these results suggest the new ORF0 sequences belong to CLRDV and were deposited in GenBank under accession nos. ON954058-ON954059. To our knowledge, this is the first report of CLRDV infecting cacao plants, expanding the range of malvaceous hosts of this polerovirus. CLRDV is largely known for causing yield losses in cotton crops, but additional studies are needed to determine if CLRDV infection is detrimental to cacao production.

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.

First report of Colletotrichum tropicale causing anthracnose on Passiflora edulis in Brazil.

Brazil is the world's largest producer and consumer of yellow passion fruit (Passiflora edulis f. flavicarpa), mainly for the manufacture of concentrate and frozen juice as well as for fresh consumption (Faleiro et al. 2005). Between June and July 2018, passion fruit plants with symptoms of anthracnose were observed in commercial planting in the municipality of Coruripe (20 ha), northeastern state of Alagoas, Brazil. Approximately 70% of the plants showed leaves with relatively large, watery, circular spots that affected 30% of the leaf surface. Small fragments taken from the transition region of symptomatic tissue were superficially disinfected in 70% ethanol for 30 s and in 1% NaClO for 1 min, rinsed in sterile distilled water (SDW), dried on filter paper, plated on potato dextrose agar (PDA-Kasvi) incubated at 25°C under white light and 12 h photoperiod, for 3 days. Two isolates were obtained and deposited in the Collection of Phytopathogens at the Universidade Federal de Alagoas (COUFAL0281 and COUFAL0282). To identify the isolates, partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin (TUB2) genes and of the rDNA-ITS (ITS) region were amplified by PCR. The partial sequences were deposited at GenBank (MT299339, MT334694, MT310553, MT299340, MT334695 and MT310554). Based on the BLASTn analysis, sequences of the PCR products showed high nucleotide similarity with sequences of the species C. tropicale (CBS 124949/ex-type and ICMP 18672), for GAPDH (98.94% and 100%), TUB2 (99 and 100%) genes and ITS (100%). This result was also confirmed in the phylogenetic tree of Bayesian Inference assembled with concatenated data (GAPDH, TUB2 and ITS). The colonies of the isolates were white with a white reverse, with dense mycelium, and mean growth rate of 7.54 mm/day, after 7 days on PDA medium at 25° C. Conidia were subcylindrical with rounded ends, hyaline, smooth walls and measured 13.63-20.59µm (= 17.54µm; n= 50) in length and 4.40-7.93 µm (= 5.88 µm; n= 50) in width. Appressoria were melanized, subglobose, irregular and measured 7.44 - 18.57 µm (= 10.04 µm; n= 50) in length and 5.49-10.16 µm (= 7.66 µm; n= 50) in width. These morphological characteristics were consistent with those described for Colletotrichum tropicale E.I. Rojas, S.A. Rehner & Samuels (Rojas et al. 2010). To confirm pathogenicity, 30 µL of a 106 conidia/mL sterile distilled water (SDW) conidia suspension, together with a drop of 20% Tween were deposited on the adaxial surface of passion fruit leaves wounded with a sterile needle, with four repetitions. The control consisted of leaves inoculated only with SDW. The leaves were placed in a plastic Gerbox box with sterilized filter paper moistened with SDW and maintained in a Biochemistry Oxygen Demand (BOD) incubator stove at 25 ºC and photoperiod of 12 h. After 7 days, typical anthracnose symptoms were observed on inoculated leaves. The pathogen was re-isolated and confirmed by morphological characterization, according to Koch's postulates. No symptoms were observed in the negative control. The occurrence of this species has been frequently reported in several other crops grown in northeastern Brazil (Silva et al. 2017; Veloso et al. 2018; Vieira et al. 2018; Costa et al. 2019). Additionally, many of these crops are grown in close proximity to the passion fruit orchards, thus favoring pathogen movement between hosts, probably, due to the anthropic influence, circulation of animals and insects, as well as wind driven rain splashes. However, this is first report of C. tropicale in Passiflora edulis in the world.

Contrasting genetic structure between two begomoviruses infecting the same leguminous hosts.

Begomoviruses are whitefly-transmitted, ssDNA plant viruses and are among the most damaging pathogens causing epidemics in economically important crops worldwide. Wild/non-cultivated plants play a crucial epidemiological role, acting as begomovirus reservoirs and as 'mixing vessels' where recombination can occur. Previous work suggests a higher degree of genetic variability in begomovirus populations from non-cultivated hosts compared with cultivated hosts. To assess this supposed host effect on the genetic variability of begomovirus populations, cultivated (common bean, Phaseolus vulgaris, and lima bean, Phaseolus lunatus) and non-cultivated (Macroptilium lathyroides) legume hosts were sampled from two regions of Brazil. A total of 212 full-length DNA-A genome segments were sequenced from samples collected between 2005 and 2012, and populations of the begomoviruses Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) were obtained. We found, for each begomovirus species, similar genetic variation between populations infecting cultivated and non-cultivated hosts, indicating that the presumed genetic variability of the host did not a priori affect viral variability. We observed a higher degree of genetic variation in isolates from MaYSV populations than BGMV populations, which was explained by numerous recombination events in MaYSV. MaYSV and BGMV showed distinct distributions of genetic variation, with the BGMV population (but not MaYSV) being structured by both host and geography.

Circular DNA genomics (circomics) exemplified for geminiviruses in bean crops and weeds of northeastern Brazil.

Circomics was coined to describe the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) and pyro-sequencing to investigate the genome structures of small circular DNAs. A batch procedure is described using 61 plant samples from Asia, South America and Central America which revealed 83 contig sequences of geminiviral DNA components and 4 contig sequences of DNA satellites. The usefulness of this approach is validated for the Brazilian begomoviruses, and the sequence fidelity is determined by comparing the results with those of conventional cloning and sequencing of Bolivian begomoviruses reported recently. Therefore, circomics has been proven to be a major step forward to economize costs and labor and to characterize reliably geminiviral genomes in their population structure of the quasispecies.

Caracterização molecular de cultivares de cana-de-açúcar utilizando marcadores ISSR / Molecular characterization of the sugarcane cultivars obtained by issr markers

A diversidade genética de catorze cultivares de cana-de-açúcar (Saccharum oficinarum) foi acessada por meio de marcadores moleculares ISSR. Objetivou-se caracterizar molecularmente as cultivares estudadas. Foram utilizados trinta e sete primers de ISSR, dos quais, oito foram eficientes na amplificação do DNA das amostras analisadas, sendo sete primers suficientes para distinguir todas as cultivares de cana-de-açúcar envolvidas nas análises. A faixa de amplicons variou de 300 a 2000 pb. As cultivares RB 92579 e RB 863129 apresentaram maiores coeficientes de similaridade (77 por cento) enquanto as cultivares RB 961 e RB 931611 formaram o grupo com menor similaridade (22 por cento). Os resultados indicam que os marcadores ISSR foram úteis na análise da diversidade genética e geração de padrões genéticos (fingerprint), em germoplasma de cana-de-açúcar. Marcadores ISSR cultivar-específico foram obtidos com o primer UBC 817 para as 14 cultivares testadas. Num próximo trabalho, mais primers ISSR serão utilizados para buscar mais polimorfismos dessas e de outras cultivares de cana-de-açúcar.

Genetic diversity of fourteen sugarcane cultivars was accessed by ISSR molecular markers. With the aim to characterizing and validating the efficiency of these markers in the fingerprint of studied cultivars, thirty seven ISSR primers were used, from which, eight were efficient for the DNA amplification. Seven primers were efficient to discriminate the fourteen studied sugarcane cultivars. The amplicons varied from 300 to 2000 bp. The cultivars RB 92579 and RB 863129 presented higher similarity coefficient (77 percent) while the cultivars RB 961 and RB 93611 formed the group with lower similarity (22 percent). The results suggested that ISSR markers were useful in the analysis of the genetic diversity and in the fingerprint in sugarcane germosplasm. In the next step more ISSR primers will be used in order to obtain more polymorphism from these varieties and to analyze more sugarcane cultivars.