First Report of Sida micrantha mosaic virus in Phaseolus vulgaris in Brazil.

Snap and common beans (Phaseolus vulgaris L.) are severely affected by Bean golden mosaic virus (BGMV) infection, so far the only begomovirus reported on these crops in Brazil (1). Samples of snap and common beans colonized by the whitefly Bemisia tabaci biotype B and displaying golden mosaic, chlorotic spots, and leaf distortion were collected in three production regions in Goiás State (Goianápolis, Luziânia, and Itaberaí) between 2003 and 2007. Total DNA extracted from leaf samples was used as template in PCR assays using universal primers targeting conserved regions of the DNA-A and DNA-B genomes (3). Begomovirus-specific amplicons were observed only with DNA template from symptomatic plants. Two single amplicons were observed for both genomic segments, indicating the presence of bipartite species in all samples. Sequence analysis of four isolates (named as GO-176, GO-260, GO-354, and GO-368) obtained from common bean samples indicated identity levels of approximately 95% with the DNA-A segment of BGMV (GenBank Accession No. FJ665283). However, the complete DNA-A sequence (GenBank Accession No. HM357459.1) of the GO-060 isolate (from a symptomatic snap bean plant collected in Goianápolis) displayed 76% identity with BGMV (GenBank Accession No. FJ665283) and 95% identity with the DNA-A of a Sida micrantha mosaic virus (SimMV) isolate (GenBank Accession No. EU908733.1) reported to be infecting okra (Abelmoschus esculentus L.) and 94.8% with a SimMV isolate reported to be infecting soybean (GenBank Accession No. FJ686693) in Brazil (2). Koch's postulates were fulfilled for the isolate GO-060 by inoculating a set of soybean and bean accessions via a biolistic approach. The ratio of positive PCR amplicons per total of inoculated plants were 15 of 16 for snap bean cv. Trepador, 9 of 10 for snap bean cv. Fartura, 18 of 24 for common bean cv. Olate Pinto, and 19 of 25 for common bean cv. Carioca. The isolate was also able to infect eight of nine soybean 'Doko' plants. Sequence analysis using symptomatic leaf samples (15 days after inoculation) confirmed SimMV as the causal agent. To our knowledge, this is the first report of a SimMV isolate infecting P. vulgaris. This virus is apparently fast expanding its host range from Malvaceae to Solanaceae species and leguminous hosts after the introduction of B. tabaci biotype B (2). More extensive surveys are necessary to access the current epidemiological importance of SimMV in both snap and common beans in Brazil. References: (1) J.C. Faria and D. P. Maxwell. Phytopathology 89:262, 1999. (2) F. R. Fernandes et al. Arch. Virol. 154:1567, 2009. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Genomic diversity of sweet potato geminiviruses in a Brazilian germplasm bank.

Begomoviruses cause major diseases of sweet potato worldwide impairing considerably the yields of this important food staple. Since sweet potato plants are vegetatively propagated and globally transported, they are prone to accumulate and disseminate geminiviruses. Effective diagnostic tools are, therefore, desirable. We studied the genomic diversity of geminiviruses present in naturally-infected sweet potato accessions belonging to a Brazilian germplasm bank collection. Fifty-five samples from different sweet potato accessions displaying geminivirus-like symptoms were analyzed by combining rolling circle amplification (RCA) with restriction fragment length polymorphism (RFLP) and sequencing. The restriction enzyme MspI (HpaII) revealed diverse band patterns in 55 samples and digestion with BamHI, SstI or PstI resulted in full-length sweet potato geminivirus DNAs of about 3 kb in 46 samples. In addition, smaller fragments were identified as either viral "Defective DNAs" (D-DNAs) or mitochondrial plasmid DNAs. The diversity of sweet potato-associated geminiviruses was found to be very high under Brazilian conditions. Representative viral full-length DNAs have been cloned and sequenced yielding two new tentative species, three strains and several variants of previously described sweet potato geminiviruses. Sequence comparisons identified footprints of recombination in their genomes underscoring the risk of generating new geminiviruses in vegetatively propagated germplasm bank material. The sites of recombination were found in conjunction with predicted hairpin structures. We propose diagnostic routines to screen germplasm bank material for geminiviruses by the rapid and reliable RCA/RFLP as the technique of choice.

Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis.

AIMS: The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method. METHODS AND RESULTS: Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis, using arithmetic means (UPGMA). Isolates grouped according to host, with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus-specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2), and an internal amplification control was developed, to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi. CONCLUSIONS: RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus, with a detection limit of 10 fg. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.

The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein.

Cowpea mosaic virus (CPMV) moves from cell to cell as virus particles which are translocated through a plasmodesmata-penetrating transport tubule made up of viral movement protein (MP) copies. To gain further insight into the roles of the viral MP and capsid proteins (CP) in virus movement, full-length and truncated forms of the MP were expressed in insect cells using the baculovirus expression system. Using ELISA and blot overlay assays, affinity purified MP was shown to bind specifically to intact CPMV virions and to the large CP, but not to the small CP. This binding was not observed with a C-terminal deletion mutant of the MP, although this mutant retained the capacity to bind to other MP molecules and to form tubules. These results suggest that the C-terminal 48 amino acids constitute the virion-binding domain of the MP.

Distribution and genetic diversity of tomato-infecting begomoviruses in Brazil.

Tomato-infecting begomoviruses have been reported throughout Brazil since the introduction of the B biotype of Bemisia tabaci. Here, we report a large scale survey on the distribution and genetic diversity of tomato-infecting begomoviruses. Tomato samples with typical begomovirus symptoms were collected in seven different states, comprising the major tomato growing areas of the country. Viruses were detected by polymerase chain reaction (PCR) using universal primers for the genus Begomovirus. PCR-amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, at least seven previously undescribed species of begomoviruses were found. Four of the new viruses were found exclusively in the Southeastern states, two exclusively in the Northeastern states, and one was found in both regions. Sequence comparisons reveal strong evidence of recombination among the Brazilian begomoviruses. Together, the results indicate the existence of a high degree of pre-existing genetic diversity among tomato-infecting begomoviruses in Brazil and suggest that these viruses have emerged after being transferred from natural hosts to tomatoes, due to the introduction into Brazil of a novel polyfagous biotype of the whitefly vector.

Is TTF1 a good immunohistochemical marker to distinguish primary from metastatic lung adenocarcinomas?

To evaluate the immunohistochemical expression of thyroid transcription factor 1 (TTF1) in primary and metastatic pulmonary adenocarcinomas, and test the diagnostic accuracy of this antibody, two surgical pathologists independently evaluated 34 cases of adenocarcinomas in the lung without clinical data and tried to distinguish between primary and metastatic cases using histological criteria exclusively. Thirteen cases were primary in the lung and 21 were metastases of extrapulmonary adenocarcinomas: 6 from the endometrium, 4 from the ovary, 3 from the colon, 2 from the kidney, 2 from the breast, 2 from the liver and 1 from the prostate. Afterward, the immunoreactivity of TTF1 in these neoplasms was evaluated and correlated with morphological and clinical data. The two pathologists were able to diagnose only 5 out of 13 cases of primary lung adenocarcinomas (sensitivity of 38.46%) and also misdiagnosed two primary malignancies as metastases. After correlation with TTF1 data, the sensitivity increased to 61.53%. The specificity of TTF1 was 100%. In conclusion, TTF1 is a highly specific marker for primary lung adenocarcinomas, and should be included in a panel of antibodies for the differential diagnosis between primary and metastatic adenocarcinomas of the lung.

Widespread Occurrence of Tomato Geminiviruses in Brazil, Associated with the New Biotype of the Whitefly Vector.

Although tomato golden mosaic virus (TGMV) was reported in Brazil more than 20 years ago (3), tomato-infecting geminiviruses have not been of economic significance in the country until recently. However, a sharp increase in the incidence of geminivirus-like symptoms in tomatoes has been reported in several areas of Brazil since 1994. This has coincided with the appearance of the B biotype of Bemisia tabaci, which, as opposed to the A biotype, readily colonizes solanaceous plants (2). We have isolated geminiviruses from symptomatic tomato plants in the Federal District, in two different areas of the state of Minas Gerais, and in the state of Pernambuco. Tomato plants in these areas showed a variety of symptoms, including yellow mosaic, severe leaf distortion, down-cupping, and epinasty. Whitefly infestation was high in all fields sampled, and in some fields, particularly in Pernambuco, incidence of virus-like symptoms was close to 100%, and no tomatoes of commercial value were harvested (1). Using primer pairs PAL1v1978/PAR1c496 and PCRc1/PBL1v2040 (4), DNA-A and -B fragments were polymerase chain reaction (PCR)-amplified from total DNA extracted from diseased plants, cloned, and sequenced. Sequence comparisons of the PCR fragments indicated the existence of at least six different geminiviruses. The nucleotide sequence homologies for DNA-A fragments ranged from 67 to 80% for the 5' end of the cp gene, and from 44 to 80% for the 5' end of the rep gene. Data base comparisons indicated the viruses are most closely related to TGMV, bean golden mosaic virus from Brazil (BGMV-Br), and tomato yellow vein streak virus (ToYVSV), although homologies were less than 80% for the fragments compared. A similar lack of a close relationship with each other and other geminiviruses was obtained with two DNA-B component PCR products compared, corresponding to the 5' end of the BC1 open reading frame. Infectious, full-length genomic clones from the tomato viruses are being generated for biological and molecular characterization. References: (1) I. C. Bezerra et al. Fitopatol. Bras. 22:331, 1997. (2) F. H. França et al., Ann. Soc. Entomol. Bras. 25:369, 1996. (3) J. C. Matyis et al. Summa Phytopathol. 1:267, 1975. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment.

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.