Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virus-free garlic was evaluated.

Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Identification of Phaeoisariopsis griseola pathotypes from five States in Brazil

Due to the increased importance of angular leaf spot of common bean (Phaseolus vulgaris) in Brazil, monitoring the pathogenic variability of its causal agent (Phaeoisariopsis griseola) is the best strategy for a breeding program aimed at developing resistant genotypes. Fifty one isolates of P. griseola collected in five Brazilian States were tested on a set of 12 international differential cultivars in the greenhouse. When inoculated plants showed symptoms but no sporulation was observed, they were transferred to a moist chamber for approximately 20-24 h. After this period of time, if no sporulation was observed, the plants were considered resistant; otherwise, they were considered susceptible. From the fifty-one tested isolates, seven different pathotypes were identified. No Andean pathotypes were identified; consequently, all isolates were classified as Middle American pathotypes. Pathotype 63-31 was the most widespread. Pathotype 63-63 overcame resistance genes present in all differential cultivars and also the resistance gene(s) present in the cultivar AND 277. This fact has important implications for breeding angular leaf spot resistance in beans, and suggests that searching for new resistance genes to angular leaf spot must be pursued.

Mosaic in Senna occidentalis in southern Brazil induced by a new strain of Soybean mosaic virus

Plants of Senna occidentalis (sin. Cassia occidentalis) with mosaic symptoms were collected near a soybean (Glycine max) field where some plants exhibited symptoms of mosaic and blistering. A preliminary examination of leaf tissue from diseased S. occidentalis by electron microscopy revealed the presence of pinwheel inclusions as well as long flexuous particles, indicating the presence of a potyvirus. Host range, serology, and amino acid sequence from this potyvirus were similar to those from other Brazilian isolates of Soybean mosaic virus (SMV). The 3'- terminal region of the genomic RNA was cloned and a cDNA sequence of 1.9 kb upstream of the poly (A) tract was determined. The sequence contains a single open reading frame and a 3'- non-translated region (NTR) of 259 bp. The nucleotide sequence of the CP gene of SMV-Soc was 98% identical to that of Brazilian isolates SMV-B, SMV-L, and SMV-FT10. The percentage of nucleotide identity of their 3'-NTR's was 91, 98, and 99% in relation to SMV-L, SMV-B, and SMV-FT10, respectively. In contrast to other Brazilian SMV isolates studied, SMV-Soc was able to infect sunflower (Helianthus annuus). Based on these results, the S. occidentalis isolate was identified as a new strain of SMV belonging to the SMV strain, group G5 and was named SMV-Soc. This is the first report of naturaly occurring SMV infecting plants of S. occidentalis in Brazil, adding this weed as a new source of SMV in the field.

Genetic diversity of begomovirus infecting tomato and associated weeds in Southeastern Brazil

The genetic diversity of begomovirus isolates from tomato (Lycopersicon esculentum) fields in the Southeastern region of Brazil was analyzed by direct sequencing of PCR fragments amplified by using universal oligonucleotides for the begomovirus DNA-A, and subsequent computer-aided phylogenetic analysis. Samples of tomato plants and associated weeds showing typical symptoms of virus infection were collected at seven locations in the states of Minas Gerais, Espírito Santo and Rio de Janeiro. A total of 137 out of 369 samples were infected with a begomovirus based on PCR analysis. Phylogenetic analysis indicated a high degree of genetic diversity among begomoviruses infecting tomatoes in the sampled area. One species (Tomato chlorotic mottle virus, TCMV) occurs predominantly in Minas Gerais, whereas in Rio de Janeiro and Espírito Santo a distinct species, not yet fully characterized, predominates. Phylogenetic analysis further indicates the presence of an additional four possible new species. This high degree of genetic diversity suggests a recent transfer of indigenous begomovirus from wild hosts into tomatoes. The close phylogenetic relationship verified between begomovirus infecting tomato and associated weeds favors this hypothesis.

Progress of soybean charcoal rot under tillage and no-tillage systems in Brazil

The increase in incidence of charcoal rot caused by Macrophomina phaseolina on soybeans (Glycine max) was followed four seasons in conventional and no-till cropping systems. In the 1997/98 and 2000/01 seasons, total precipitation between sowing and harvest reached 876.3 and 846.9 mm, respectively. For these seasons, disease incidence did not differ significantly between the no-till and conventional systems. In 1998/99 and 1999/00 precipitation totaled 689.9 and 478.3 mm, respectively. In 1998/99, in the no-till system, the disease incidence was 43.7% and 53.1% in the conventional system. In 1999/00 the final incidence was 68.7% and 81.2% for the no-till and conventional systems, respectively. For these two seasons, precipitation was lower than that required for soybean crops (840 mm), and the averages of disease incidence were significantly higher in the conventional system. The concentration of microsclerotia in soil samples was higher in samples collected in conventional system at 0 - 10 cm depth. However, analysis of microsclerotia in roots showed that in years with adequate rain no difference was detected. In dry years, however, roots from plants developed under the conventional system had significantly more microsclerotia. Because of the wide host range of M. phaseolina and the long survival times of the microsclerotia, crop rotation would probably have little benefit in reducing charcoal rot. Under these study conditions it may be a better alternative to suppress charcoal rot by using the no-till cropping system to conserve soil moisture and reduce disease progress.

Tomato chlorotic spot virus in hydroponically-grown lettuce in São Paulo State, Brazil

In the regions of Campinas and Sumaré, São Paulo, Brazil, hidroponically grown crops of Lettuce (Lactuca sativa) cv. Verônica, which showed virus-like symptoms were examined by electron microscope, biological, serological and molecular tests. Pleomorphic, enveloped particles (80-100 nm in diameter) were always detected in these samples. Experimentally inoculated host plants, including lettuce, reacted with tospoviruses-induced symptoms. Some differences were observed in Gomphrena globosa, which reacted by showing local lesions and systemic mosaic. Two isolates of Tomato chlorotic spot virus (TCSV) were identified by DAS-ELISA and by RT-PCR. The sequencing and alignment of the RT-PCR coat protein amplified fragments have indicated a high degree of homology with the TCSV sequences stored in the GenBank. This is the first report of losses due to a virus from the genus Tospovirus in commercial hydroponic lettuce crops in Brazil. Further epidemiological studies are needed for better understanding the spread of the virus in hydroponic crops, since Tomato spotted wilt virus (TSWV) is reported to spread through the nutritive solution.

Variability of the 3' terminal of the polymerase gene of Grapevine leafroll-associated virus 3 isolates from Vale do São Francisco, Brazil

Many viral diseases, including leafroll, which is of great economic importance, affect grapevines (Vitis spp.). A complex of eight viruses [Grapevine leafroll-associated virus (GLRaV) -1 to 8] is associated with this disease. The objective of this study was to compare the variability of the 3' terminal region of the polymerase gene of three isolates of GLRaV-3 (Grapevine leafroll-associated virus-3), from Submédio do Vale do Rio São Francisco (Petrolina-PE) with that of other isolates available at the GenBank, including an isolate from North America and another from Southern Brazil. The viral RNA was extracted from three infected ELISA reactive plants and a fragment of 340 bp was amplified, by RT-PCR, using primers that recognize that portion of the polymerase gene found between nucleotides 8267 and 8606. The three isolates from Vale do Rio São Francisco named Pet-1, Pet-2 and Pet-3, showed similarities ranging from 98% and 94%, respectively to the isolates from North America (AF037268) and Southern Brazilian (AF438411). Considering the whole genome, the main variation found was one amino acid change at position 2766 (F2766Y). These preliminary data indicate the existence of a natural variation among GLRaV-3 isolates from grapevines. This could be due to the vegetative propagation and long cycle of the plant, associated with the error-prone nature of RNA-dependent RNA polymerase.

Biological and molecular characterization of an isolate of Tobacco streak virus obtained from soybeans in Brazil

A virus was isolated from soybean (Glycine max) plants with symptoms of dwarfing and bud blight in Wenceslau Braz County, Paraná, Brazil. The host range and properties resembled those of Tobacco streak virus (TSV). The purified virus showed three peaks in a frozen sucrose gradient. Antiserum was produced and the virus was serologically related to TSV. Electron microscopy detected 28 nm spherical particles. Coat protein (CP) had a Mr of 29.880 Da. A fragment of 1028 nt was amplified, cloned and sequenced. One open reading frame with 717 nt was identified and associated to the CP. The CP gene shared 83% identity with the sequence of TSV CP from white clover (Trifolium repens) (GenBank CAA25133). This is the first report of the biological and molecular characterization of TSV isolated from soybeans. It is proposed that this isolate be considered a strain of TSV named TSV-BR.

Detection of Varicosavirus and Ophiovirus in lettuce associated with lettuce big-vein symptoms in Brazil

During surveys undertaken from 1998 to 2003 in the major vegetable growing areas of the city of São Paulo green belt, lettuce (Lactuca sativa) and endive (Cichorium endivia) plants were observed, which showed chlorotic thickening of foliar veins, defective growth and, in some cases, failure to form complete heads. Biological and serological [DAS-Enzyme linked immunosorbent assay (Elisa)] tests together with electron microscope observations, revealed the presence of Lettuce big-vein virus and Mirafiori lettuce virus, in these plants both responsible for the lettuce big-vein syndrome.

First report of Fusarium oxysporum f. sp. lycopersici race 3 on tomato in Brazil

Fusarium wilt, caused by three races of Fusarium oxysporum f. sp. lycopersici, is one of the most important diseases of tomato (Lycopersicon esculentum). Races 1 and 2 are distributed worldwide whereas race 3 has a more limited geographic distribution with no report thus far in Brazil. Seven F. oxysporum isolates were obtained from wilted tomato plants of race 1 and 2-resistant hybrids 'Carmen' and 'Alambra' in Venda Nova do Imigrante (State of Espírito Santo), Brazil. Virulence assays were performed using a set of the race differential cultivars: 'Ponderosa' (susceptible to all races), 'IPA-5' (resistant to race 1), 'Floradade' (resistant to races 1 and 2) and 'BHRS-2,3' (resistant to race 3). All isolates were highly virulent to 'Ponderosa', 'IPA-5' and 'Floradade' and were able to infect only a few plants of 'BHRS-2,3'. An additional virulence test was conducted including the same set of cultivars plus Lycopersicon pennellii 'LA 716'. Identical results were obtained with L. pennellii displaying an extreme (immune-like) resistant response. These results indicated that all seven isolates could be classified as F. oxysporum f. sp. lycopersici race 3. This new Fusarium wilt might became an economically important disease since race 3-resistant cultivars adapted to Brazil are not yet available.

Effect of extracts from citric biomass, rusted coffee leaves and coffee berry husks on Phoma costarricencis of coffee plants

Phoma leaf spot, caused by Phoma costarricensis poses a serious threat to coffee (Coffea arabica) production, especially in the highlands of the state of Minas Gerais, Brazil. Extracts of citric biomass, coffee berry husks and coffee leaves severely affected by rust caused by Hemileia vastatrix, were evaluated against P. costarricensis. In an in vitro assay, aqueous extracts of rusted leaves and berry husks plus the commercial extracts based on citric biomass named Ecolife® and Agromil® were tested at various dilutions on the mycelial growth inhibition of P. costarricensis. In vivo, coffee seedlings maintained in glasshouse, were sprayed with these extracts seven days before inoculation of P. costarricensis. Only extracts from citric biomass had inhibitory effects on the fungus. In vivo, Ecolife® (5 ml/l), Agromil® (5 g/l) and the aqueous extract of rusted coffee leaves (dilution 1:6) reduced Phoma leaf spot. Both, Ecolife® and the extract of rusted coffee leaves were significantly more effective in reducing the area under the lesion progress curve when applied at lower doses, indicating a possible effect on the induction of resistance.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the São Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong Nº2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

Powdery mildew of ornamental species caused by Oidiopsis haplophylli in Brazil

Oidiopsis haplophylli (syn. Oidiopsis sicula) was identified as the causal agent of powdery mildew diseases occurring on five ornamental species in Brazil. This disease was observed in plastic house-grown lisianthus (Eustoma grandiflorum: Gentianaceae), in nasturtium (Tropaeolum majus: Tropaeolaceae) cultivated under open field conditions and in greenhouse-grown calla lily (Zantedeschia aethiopica: Araceae), impatiens (Impatiens balsamina: Balsaminaceae) and balloon plant (Asclepias physocarpa: Asclepiadaceae). Typical disease symptoms consisted of chlorotic areas on the upper leaf surface corresponding to a fungal colony in the abaxial surface. With the disease progression, these chlorotic areas eventually turned to necrotic (brown) lesions. Fungi morphology on all hosts was similar to that described for the imperfect stage of Leveillula taurica (O. haplophylli). The Koch's postulates were fulfilled by inoculating symptom-free plants via leaf-to-leaf contact with fungal colonies. Additional inoculations using an isolate of O. haplophylli from sweet pepper (Capsicum annuum) demonstrated that it is pathogenic to all five species belonging to distinct botanical families, indicating lack of host specialization. This is the first formal report of a powdery mildew disease on lisianthus, calla lilly, impatiens and nasturtium in Brazil. It is, to our knowledge, the first report of O. haplophyllii infecting A. physocarpa, extending the host range of this atypical powdery mildew-inducing fungus. This disease might become important on these ornamental crops especially in protected cultivation and also under field conditions in hot and dry areas of Brazil.

Progress of Asian soybean rust and airborne urediniospores of Phakopsora pachyrhizi in southern Brazil

Asian soybean rust, caused by the fungus Phakopsora pachyrhizi, was reported at epidemic levels in 2003/2004 and is the main soybean disease in Brazil. The aim of this study was to investigate the spread of Asian soybean rust and to quantify airborne urediniospores in the region of Campo Mourão, Paraná State, Brazil. Three experiments were conducted under field conditions during the 2007/08 and 2008/09 crop seasons. Using the disease gradient method, provided by the application of increasing levels of the fungicide tebuconazole, four Asian soybean rust epidemics at different intensities were obtained in each experiment. To quantify the urediniospores, weathercock-type spore collectors were installed during and between the two crop seasons. Disease progress curves were plotted for each epidemic, and maximum severity was estimated. The curves were fit to the logistic model, which provided higher coefficients of determination and more randomly distributed residuals plotted over time. Analyses of the area under the disease progress curve showed that the largest epidemics occurred in the 2007/2008 crop season and that the progress rates were higher for severity, even among plants protected with the fungicide. The number of urediniospores collected in the air was related to the presence of soybean plants in the cultivated crops. The quantity of urediniospores was also positively correlated to the disease severity and incidence, as well as to cumulative rainfall and favorable days for P. Pachyrhizi infection.