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 Neopestalotiopsis foedans causing pestalotia spot in leaf on coconut in Brazil.

The coconut (Cocos nucifera L., Arecaceae) is one of the most important tropical species used by humans. In Brazil, its cultivation has been expanding in the recent years (Souza et al. 2020) and many diseases have emerged. The pestalotia spot, caused by Pestalotiopsis guepinii (Desm.), is a leaf disease of the coconut characterized by elliptic lesions with defined dark borders varying in size from 3 to 5 mm (Cardoso et al. 2003). In January of 2018, leaves with symptoms of pestalotia spot were obtained from ten year old coconut plants "dwarf variety" in a commercial planting in the city of Neópolis (10°20'S/36°42'W), Sergipe, Brazil. Disease incidence was 80% on 60 plants observed. Twenty samples of symptomatic tissues were collected and disinfested for 2 min in 1% sodium hypochlorite, washed in sterile water, placed on PDA (potato dextrose agar), and incubated at 25 ± 1°C with a 12-h photoperiod for 4 days. Five isolates were obtained, and pure cultures deposited in Phytopathogen Collection of the Federal University of Alagoas, accession numbers: COUFAL0240 to COUFAL0244. Seven day old colonies grown on PDA at 25°C, were whitish with aerial mycelium on the surface and abundant production of black conidiomata. Conidia were fusiform, straight to slightly curved with five cells, three median cells with brown coloring being the second and third being darker and the apical and basal cells, hyaline. Fifty conidia were measured and varied in size from 20.02-24.26 x 5.37-7.50 µm. The conidia presented two to four apical appendages and one basal appendage (Fig. S1). The morphological characteristics coincide with the Neopestalotiopsis foedans (Sacc. & Ellis), Maharachchikumbura et al. (2014). Molecular identification was conducted using partial nucleotide sequences from the ITS (ITS1/ITS4) region (GenBank no. MT605375 to MT605379) and from the genes TUB2 (Bt2a/Bt2B) (no. MT634202 to MT634206) and TEF-1α (526F/1567R) (no. MT634197 to MT634201). Besides that, the isolates grouped with the ex-type N. foedans species (CGMCC 3.9123) in a phylogenetic tree of Bayesian inference using concatenated sequences (Fig. S2). The pathogenicity was confirmed on seedling from coconut plants "dwarf variety" maintained in a greenhouse. Four plants were used, being one as a control. Spore suspensions of 106 conidia mL-1 was prepared from a 7 days old culture (cultivated at 25ºC). Inoculations were performed by spraying the conidial suspension on two whole leaves per plant (wounded and unwounded). In the control, sterilized distilled water was used. Plants were incubated at 25 ± 1°C and 100% relative humidity. Ten days after inoculation, depressed and necrotic lesions were observed in 100% on the inoculated leaves with wound. No symptoms observed on unwounded leaves, nor in the control treatment. To complete Koch's postulates, the N. foedans fungus was successfully re-isolated from the symptomatic leaves and identified phenotypically in optical microscope. Neopestalotiopsis foedans has already been reported in Calliandra haematocephala (Hassk), Neodypsis decaryi (Jum.), Rhizophora mangle (L.), Thuja occidentalis and Psidium guajava (L.) (Saccardo, 1882; Maharachchikumbura et al. 2014, Solarte et al. 2018). However, this is first report of N. foedans causing leaf spot in coconut in the world. The pestalotia spot is commonly observed in Brazil in C. nucifera and should be considered an important disease for this culture, as this can significantly reduce its photosynthetic area.

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.