André Nepomuceno Dusi

Pepper yellow mosaic virus, a new potyvirus in sweetpepper, Capsicum annuum

Summary. A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3′ terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3′-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3′-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested.

Detection of three Allexivirus species infecting garlic in Brazil

Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D). The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR) was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI). By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV), in Brazil.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.

Spread of viruses in garlic fields cultivated under different agricultural production systems in Brazil

Samples from four regions, representing 76% of the garlic growing area in Brazil, were tested by RT-PCR for the presence of Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Garlic virus C (GarV-C), Garlic virus D (GarV-D) and Garlic mite-borne filamentous virus (GarMbFV). The samples (352 bulbs) represented five agricultural systems: traditional common garlic (CG), virus-free common garlic (VFCG), non-vernalized noble garlic (NVNGSG) and vernalized noble garlic cultivated by small growers (VNGSG), and vernalized noble garlic adopted by major growers (VNGMG). Multiple infections were detected in 22% of the samples. Potyvirus species were present in all regions. LYSV prevailed over OYDV while the carlavirus GCLV was less prevalent. GarV-C and GarMbFV were the most prevalent among the allexivirus species. The NVNG production system had a higher prevalence of LYSV and GarV-C. The CG production system, that uses less technology, had the highest prevalence for all species, especially LYSV that prevailed in 94% of the samples. Overall, the regions with higher technological input employing better quality seeds had the lowest viral prevalence for all species. This monitoring provides information to establish a strategy to raise the phytosanitary quality and the national productivity of garlic.

Viral reinfection affecting bulb production in garlic after seven years of cultivation under field conditions

Six sequential field experiments were conducted from 1999 to 2002 to evaluate virus reinfection in garlic cv. Amarante and its effect on bulb production. The treatments in the year 1999 were: T1 – virus-free garlic-seed obtained by thermotherapy and meristem-tip culture and indexed for virus by immuno-sorbent electron microscopy (ISEM) under its first field cycle; T2, T3 and T4 – garlic-seed in the second, third and fourth field cycles, respectively; and C – standard garlic-seed from the grower (with no control of virus infection) as a control. In the years 2000 to 2002, a new plot of virus-free seed was added to the experiment and cloves from the previous treatments were again grown under field conditions. In the fourth year of experiments, the treatments comprised T1 (virus-free garlic seeds under the first field cycle) to T7 (garlic-seed in the sixth field cycle) and C. Two experiments were conducted in the years 1999 and 2000, at two locations and in the years 2001 and 2002 only one experiment per year at one location. The combined analysis of variance for all experiments indicated a significant difference between the treatments for plant height and yield. The bulbs were classified into commercial classes according the Brazilian regulation and the commercial classes 4–7 were 72 %, 60%, 59%, 53% and 35% of the total number of bulbs harvested, from T1 to T5, respectively. Virus reinfection at the end of the second field cycle, determined by serology using antisera against the most common viruses of garlic in Brazil reached 83%. Treatments T1–T7 yielded 118%, 79%, 57%, 51%, 39%, 33% and 31% higher than the control.

Shoot tip culture and thermotherapy for recovering virus-free plants of garlic.

Garlic shoot tip culture associated with dry heat thermotherapy (cloves exposed to 37°C for 35 days) were essential for recovering virus free plants of the cv Amarante. In this condition 70% of the explants developed in vitro and produced plants. A total of 77% of those plants was virus free when indexed by ISEM, which resulted in a final index of 54% of virus free plants from treated cloves. The percentage of regeneration decreased to 20% as the temperature increased up to 40°C. However 90% of those plants were virus free, leading to a final index of 18% virus free plants out of treated cloves.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

BackgroundGarlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors.ResultsIn this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR.ConclusionsThe expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Colonização de plantas de alho por Neotoxoptera formosana no DF

Garlic plants (cv. Amarante) cultivated during the spring in the greenhouse for detection of viruses, were found to be naturally colonized by a dark aphid. For species identification, specimens of the few alatae individuals collected by pan yellow water traps and many apterae individuals were compared to the species descriptions in Blackman & Eastop (1984). The specimens were also compared with Neotoxoptera formosana and N. oliveri from the aphid collection of the Phytopathology Department of the University of Brasilia. The aphid found in the garlic plants belongs to the species Neotoxoptera formosana Takahashi, 1921.

Resistance levels to two strains of Potato virus Y (PVY) in transgenic potatoes cv. Achat

Two transgenic potato clones of cv. Achat, denominated 1P and 63P were challenged with two Potato virus Y strains (PVYO and PVYN), under greenhouse conditions, to be evaluated for resistance to these strains. Optical density values of the Elisa readings of samples from the transgenic plants were compared to readings from samples of the inoculated non-transformed plants. Clone 1P was extremely resistant to both PVY strains, reflected by not being systemically infected. Clone 63P, however, presented partial resistance to both PVY strains as local or systemic infection was delayed in some days. These results confirm the previously reported extreme resistance to PVY of clone 1P.

Resistance of genetically modified potatoes to Potato virus Y under field conditions.

The objective of this work was to evaluate the resistance of genetically modified clones of potato to Potato virus Y (PVY) under field conditions. Genetically modified plants were compared with nontransformed plants of the same cultivar. The plots were flanked with potato plants infected with both PVYo and PVYN strains (spread lines), in order to provide the experimental area with the source of virus, which was naturally spread by the native aphid population. The experiment was weekly monitored by visual inspections and by DAS-Elisa in the plants produced from the harvested tubers, in order to evaluate the resistance of transgenic plants throughout the plant growth cycle. By the end of the third year, no infection symptoms were observed in the 1P clone; clone 63P showed 1% of infection, in contrast to about 90% of nontransformed plants infected. The stable expression of resistance to PVY provided by the coat protein gene was obtained in genetically modified clones of potato plants cultivar Achat under field conditions, during three consecutive years.