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Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes ('Santa Clara', susceptible, and its near-isogenic line 'LAM 157', resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

Tropical fruit tree species constitute a yet untapped supply of outstanding diversity of taste and nutritional value, barely developed from the genetics standpoint, with scarce or no genomic resources to tackle the challenges arising in modern breeding practice. We generated a de novo genome assembly of the Psidium guajava , the super fruit “apple of the tropics”, and successfully transferred 14,268 SNP probesets from Eucalyptus to Psidium at the nucleotide level, to detect genomic loci linked to resistance to the root knot nematode (RKN) Meloidogyne enterolobii derived from the wild relative P . guineense . Significantly associated loci with resistance across alternative analytical frameworks, were detected at two SNPs on chromosome 3 in a pseudo-assembly of Psidium guajava genome built using a syntenic path approach with the Eucalyptus grandis genome to determine the order and orientation of the contigs. The P . guineense -derived resistance response to RKN and disease onset is conceivably triggered by mineral nutrients and phytohormone homeostasis or signaling with the involvement of the miRNA pathway. Hotspots of mapped resistance quantitative trait loci and functional annotation in the same genomic region of Eucalyptus provide further indirect support to our results, highlighting the evolutionary conservation of genomes across genera of Myrtaceae in the adaptation to pathogens. Marker assisted introgression of the resistance loci mapped should accelerate the development of improved guava cultivars and hybrid rootstocks.

The gene cluster encodes protein receptors that are potentially able to recognize microbial products and activate signaling pathways that lead to plant cell immunity. Although there are several homologs in the tomato genome, only one of them, named , has been extensively studied due to its functionality against a wide range of (thrips-transmitted) orthotospoviruses. The gene is a dominant resistance gene originally from a wild Peruvian tomato that has been used in tomato breeding programs aiming to develop cultivars with resistance to these viruses. Here, we provide an overview starting from the first reports of Sw-5 resistance, positional cloning and the sequencing of the gene cluster from resistant tomatoes and the validation of Sw-5b as the functional protein that triggers resistance against orthotospoviruses. Moreover, molecular details of this plant-virus interaction are also described, especially concerning the roles of Sw-5b domains in the sensing of orthotospoviruses and in the signaling cascade leading to resistance and hypersensitive response.

Verticillium dahliae is a soil-borne fungal pathogen responsible for vascular wilt diseases in more than 300 dicotyledonous species, including solanaceous vegetable crops. In this study, a collection of 89 Brazilian V. dahliae isolates was characterized by combing molecular information for mating type and physiological race determination, as well as via virulence bioassays employing a set of differentials. Based on the virulence assays, three isolates were classified as race 1, 76 were classified as race 2, whereas ten isolates did not cause any symptom on the tested cultivars.” In race-specific detection, a total of six isolates were identified as race 1, 70 as race 2, and 13 isolates displayed no amplicon with any primer set employed. Therefore, V. dahliae race 2 isolates are currently ubiquitous across major Solanaceae-producing areas in Brazil. Both MAT idiomorphs were detected, but a larger number of the V. dahliae isolates displayed the MAT1–1 (82%) pattern in our molecular analyses. The simultaneous presence of both MAT idiomorphs opens the possibility of sporadic events of sexual reproduction among V. dahliae populations from Brazil, enabling the potential emergence of either new recombinant isolates with broader host ranges or novel physiological races. Our results clearly indicated the need to intensify the search for effective sources of resistance to V. dahliae race 2 in tomato breeding programs under Brazilian conditions.

Reverse transcription-quantitative PCR (RT-qPCR) is an analytical tool for gene expression quantification. Reference genes are not yet available for gene expression analysis during interactions of Ralstonia solanacearum with ‘Hawaii 7996’ (the most stable source of resistance in tomato). Here, we carried out a multi-algorithm stability analysis of eight candidate reference genes during interactions of ‘Hawaii 7996’ with one incompatible/avirulent and two compatible/virulent (= resistance-breaking) bacterial isolates. Samples were taken at 24- and 96-h post-inoculation (HPI). Analyses were performed using the ∆∆Ct method and expression stability was estimated using BestKeeper, NormFinder, and geNorm algorithms. TIP41 and EF1α (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and TIP41 (with BestKeeper), were the best combinations for mRNA normalization in incompatible interactions at 24 HPI and 96 HPI. The most stable genes in global compatible and incompatible interactions at 24 HPI and 96 HPI were PDS and TIP41 (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and PDS / EXP (with BestKeeper). Global analyses on the basis of the three algorithms across 20 R. solanacearum -tomato experimental conditions identified UBI3, TIP41 and ACT as the best choices as reference tomato genes in this important pathosystem.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA–A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA–B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

The discovery rate of new plant viruses has increased due to studies involving high-throughput sequencing (HTS), particularly for single-stranded DNA viruses of the family Genomoviridae. We carried out an HTS-based survey of genomoviruses in a wide range of native and exotic trees grown in the Brazilian Cerrado biome, and the complete genome sequences of two novel members of the family Genomoviridae from two distinct genera were determined. Specific primers were designed to detect these genomoviruses in individual samples. A new gemykolovirus (Tecoma stans associated gemykolovirus) was detected in Tecoma stans, and a new gemykibivirus (Ouratea duparquetiana associated gemykibivirus) was detected in Ouratea duparquetiana. A gemykrogvirus related to Gila monster associated gemykrogvirus (80% pairwise identity) was also detected in foliar samples of Trembleya parviflora. Our pilot study paves the way for a better characterization of this diverse collection of genomoviruses as well as their interactions with the associated tree species.

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5′ ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE–088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE–088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE–088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

Foliar anthracnose is one of the main diseases of onion (Allium cepa L.) under tropical and subtropical conditions. Thus far, only Colletotrichum gloeosporioides has been reported as the causal agent of this disease in Brazil. However, there are no extensive studies characterizing Colletotrichum isolates associated with onion anthracnose in the country. Here, 38 Colletotrichum isolates obtained from onion plants displaying foliar anthracnose across major Brazilian onion-producing regions were characterized using morphometric and molecular information. The Bayesian and Maximum Liklihood methods were used for an initial analysis of the β-tubulin gene (tub2) sequences of all isolates, resulting in the discrimination of nine haplotypes. Three haplotypes grouped with the reference species of the C. acutatum complex and six with the C. gloeosporioides complex. Sequences of either the glyceraldehyde-3-phosphate dehydrogenase (gapdh), actin (act), and calmodulin genes or the intergenic spacer (IGS) region between DNA lyase (apn2) gene and the mating-type mat1–2-1 locus were used to characterize a subset of isolates representing these nine distinct tub2 gene haplotypes. These analyses revealed five anthracnose-inducing Colletotrichum species, including three members of the C. acutatum species complex (C. nymphaeae, C. scovillei, and C. tamarilloi) and two of the C. gloeosporioides species complex (C. fructicola and C. theobromicola). Bioassays confirmed that all these Colletotrichum species are pathogenic to onion, inducing typical anthracnose symptoms on bulbs and leaves. Twenty-six out of 38 isolates were identified as C. theobromicola, indicating this fungus as the prevalent anthracnose pathogen under Brazilian conditions. This Colletotrichum species diversity will affect anthracnose management strategies, including chemical and cultural control as well as the identification and deployment of onion cultivars with species-specific and/or wide-spectrum tolerance/resistance.