Fábio N. Silva

Brazilian Begomovirus Populations Are Highly Recombinant, Rapidly Evolving, and Segregated Based on Geographical Location

ABSTRACT The incidence of begomovirus infections in crop plants sharply increased in Brazil during the 1990s following the introduction of the invasive B biotype of the whitefly vector, Bemisia tabaci. It is believed that this biotype transmitted begomoviruses from noncultivated plants to crop species with greater efficiency than indigenous B. tabaci biotypes. Either through rapid host adaptation or selection pressure in genetically diverse populations of noncultivated hosts, over the past 20 years various previously unknown begomovirus species have became progressively more prevalent in cultivated species such as tomato. Here we assess the genetic structure of begomovirus populations infecting tomatoes and noncultivated hosts in southeastern Brazil. Between 2005 and 2010, we sampled and sequenced 126 DNA-A and 58 DNA-B full-length begomovirus components. We detected nine begomovirus species in tomatoes and eight in the noncultivated host samples, with four species common to both tomatoes and noncultivated hosts. Like many begomoviruses, most species are obvious interspecies recombinants. Furthermore, species identified in tomato have probable parental viruses from noncultivated hosts. While the population structures of five well-sampled viral species all displayed geographical subdivision, a noncultivated host-infecting virus was more genetically variable than the four predominantly tomato-infecting viruses.

Synonymous site variation due to recombination explains higher genetic variability in begomovirus populations infecting non-cultivated hosts.

Begomoviruses are ssDNA plant viruses that cause serious epidemics in economically important crops worldwide. Non-cultivated plants also harbour many begomoviruses, and it is believed that these hosts may act as reservoirs and as mixing vessels where recombination may occur. Begomoviruses are notoriously recombination-prone, and also display nucleotide substitution rates equivalent to those of RNA viruses. In Brazil, several indigenous begomoviruses have been described infecting tomatoes following the introduction of a novel biotype of the whitefly vector in the mid-1990s. More recently, a number of viruses from non-cultivated hosts have also been described. Previous work has suggested that viruses infecting non-cultivated hosts have a higher degree of genetic variability compared with crop-infecting viruses. We intensively sampled cultivated and non-cultivated plants in similarly sized geographical areas known to harbour either the weed-infecting Macroptilium yellow spot virus (MaYSV) or the crop-infecting Tomato severe rugose virus (ToSRV), and compared the molecular evolution and population genetics of these two distantly related begomoviruses. The results reinforce the assertion that infection of non-cultivated plant species leads to higher levels of standing genetic variability, and indicate that recombination, not adaptive selection, explains the higher begomovirus variability in non-cultivated hosts.

Synergism and negative interference during co-infection of tomato and Nicotiana benthamiana with two bipartite begomoviruses

In Brazil, at least eight begomoviruses including Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) infect tomatoes. ToYSV symptoms in tomato and Nicotiana benthamiana appear earlier and are more severe compared to those of ToRMV. We investigated the role of several factors in this differential adaptation. To analyze infection kinetics, a single leaf was inoculated and subsequently detached after different periods of time. Viral DNA accumulation was quantified in plants, viral replication was analyzed in protoplasts, and tissue tropism was determined by in situ hybridization. Results indicate that ToYSV establishes a systemic infection and reaches a higher concentration earlier than ToRMV in both hosts. ToRMV negatively interferes with ToYSV during the initial stages of infection, but once systemic infection is established this interference ceases. In N. benthamiana, ToYSV invades the mesophyll, while ToRMV is phloem-restricted. During dual infection in this host, ToYSV releases ToRMV from the phloem.

Molecular variability of cowpea mild mottle virus infecting soybean in Brazil

Molecular variability was assessed for 18 isolates of cowpea mild mottle virus (CPMMV, genus Carlavirus, family Betaflexiviridae) found infecting soybean in various Brazilian states (Bahia, Goiás, Maranhão, Mato Grosso, Minas Gerais, Pará) in 2001 and 2010. A variety of symptoms was expressed in soybean cv. CD206, ranging from mild (crinkle/blistering leaves, mosaic and vein clearing) to severe (bud blight, dwarfing, leaf and stem necrosis). Recombination analysis revealed only one CPMMV isolate to be recombinant. Pairwise comparisons and phylogenetic analysis were performed for partial genomes (ORF 2 to the 3’ terminus) and for each ORF individually (ORFs 2 to 6), showing the isolates to be distinct. The topology of the phylogenetic tree could be related to symptoms, but not to the year of collection or geographical origin. Additionally, the phylogenetic analysis supported the existence of two distinct strains of the virus, designated CPMMV-BR1 and CPMMV-BR2, with molecular variations between them.

The diversification of begomovirus populations is predominantly driven by mutational dynamics

Abstract Begomoviruses (single-stranded DNA plant viruses) are responsible for serious agricultural threats. Begomovirus populations exhibit a high degree of within-host genetic variation and evolve as quickly as RNA viruses. Although the recombination-prone nature of begomoviruses has been extensively demonstrated, the relative contribution of recombination and mutation to the genetic variation of begomovirus populations has not been assessed. We estimated the genetic variability of begomovirus datasets from around the world. An uneven distribution of genetic variation across the length of the cp and rep genes due to recombination was evident from our analyses. To estimate the relative contributions of recombination and mutation to the genetic variability of begomoviruses, we mapped all substitutions over maximum likelihood trees and counted the number of substitutions on branches which were associated with recombination (ηr) and mutation (ημ). In addition, we also estimated the per generation relative rates of both evolutionary mechanisms (r/μ) to express how frequently begomovirus genomes are affected by recombination relative to mutation. We observed that the composition of genetic variation in all begomovirus datasets was dominated by mutation. Additionally, the low correlation between the estimates indicated that the relative contributions of recombination and mutation are not necessarily a function of their relative rates. Our results show that, although a considerable fraction of the genetic variation levels could be assigned to recombination, it was always lower than that due to mutation, indicating that the diversification of begomovirus populations is predominantly driven by mutational dynamics.

A Phytoplasma Belonging to a 16SrIII-A Subgroup and dsRNA Virus Associated with Cassava Frogskin Disease in Brazil

Cassava frogskin disease (CFSD) is a particular threat in cassava because symptoms remain hidden until harvest and losses can be total. The information related to the etiological agent of this disease is contradictory, because some authors believe it is caused by phytoplasmas while others believe that it is caused by a virus. In order to refine detection protocols and to characterize organisms associated with CFSD in Brazil, 32 symptomatic and 20 asymptomatic cassava plants were collected in Minas Gerais state. Total DNA was extracted and used for nested polymerase chain reaction (PCR) to detect phytoplasmas. Because endophytic Bacillus spp. led to false positives, primers were designed to facilitate the detection of phytoplasma in the presence of bacteria. In addition, double-stranded (ds)RNA was extracted from tubers and used in reverse-transcription PCR for the detection of the RNA-dependent RNA polymerase gene from Cassava frogskin virus segment 4. The detected phytoplasma was identified as belonging to the group 16SrIII-A by restriction fragment length polymorphism (RFLP), sequencing, and RFLP in silico. This is the first report of a phytoplasma belonging to the 16SrIII-A group associated with cassava plants, the first molecular characterization of a phytoplasma associated with CFSD in Brazil, and a first report of phytoplasma and a dsRNA virus (possible reovirus) co-infecting cassava plants with CFSD symptoms.

Production of polyclonal antiserum against Cowpea mild mottle virus coat protein and its application in virus detection

Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to yield losses in the main producing regions of Brazil. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein gene of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of the purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/ToF mass spectrometer analyses. A rabbit was immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antiserum was demonstrated by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection.

A new disease in Citrus aurantifolia in Oman, “sudden decline”, is associated with a pathogen complex including a 16SrII group phytoplasma

Witches’ broom disease of lime is a devastating disease in the Sultanate of Oman. In recent years a new disease has been observed, which promotes a sudden decline in Citrus aurantifolia (acid lime). The etiological agent of this new disease has not yet been determined. In this study the presence of phytoplasmas and RNAs was evaluated in plants showing the sudden decline symptomatology. The results reveal the presence of a complex of at least three different pathogens: 16SrII phytoplasmas, Citrus tristeza virus and Citrus exocortis viroid.

First report of phytoplasma associated with Eucalyptus urophylla showing witches’ broom in Brazil

Eucalyptus production is of great importance to Brazils economy. Diseases affecting the growth and the architecture of these trees lead to large losses in yield and quality of the wood. The occurrence of eucalyptus trees showing witches’ broom and little leaf symptoms in different areas of Brazil is affecting seedling production. In this work, a phytoplasma was found associated with these symptomatic plants and was identified as belonging to the 16SrII group. This is the first report of a phytoplasma associated with eucalyptus in Brazil.

Differential expression and phytohormone unbalance in Citrus aurantifolia plants during “sudden decline of lime”, a new phytoplasma disease of citrus

For decades, the production of acid lime (Citrus aurantifolia) in Oman has been affected by diseases caused by phytoplasmas, notably witches’ broom disease of lime (WBDL). In recent years, a new phytoplasma strain in Oman has been observed that promotes the sudden decline of lime (SDL). The molecular mechanisms behind its pathogenicity and mode of interaction with citrus host plants is still completely unknown. In this study, we evaluated the differential expression of genes in symptomatic and asymptomatic lime trees in Oman using a real-time quantitative PCR assay. Among 27 regulatory and biosynthesis-related genes tested in Citrus aurantifolia plants during phytoplasma infection, we verified the presence of 14 responsive genes in plants showing SDL symptoms, revealing a specific set of SDL-responsive genes. Quantification data of endogenous 3-indoleacetic acid and jasmonic acid show an unbalanced hormonal content in symptomatic lime trees, corroborating the gene expression data. The identification of regulatory genes differentially expressed in plant-phytoplasma interactions during SDL will help to elucidate the mechanisms possibly involved in defense responses, development and death-triggered signals in infected citrus plants.