RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Production of caffeine alkaloid by Claviceps sorghi

The ergot disease of sorghum (Sorghum bicolor), caused by the fungus Claviceps sorghi, restricted to the Indian sub-continent, is a disease in which the pathogen infects the florets, colonizing the unfertilized ovaries. Losses are higher in hybrid seed production fields due to a higher susceptibility of male sterile lines. The sclerotia of C. sorghi have never been found to contain alkaloids with a tetracyclic ergoline ring system, which is normal in most ergot pathogens. In this work, we show that sclerotia of C. sorghi contain caffeine alkaloid and the ability to produce it in vitro.

Molecular characterization of Brazilian Dicyma pulvinata isolates

Forty-nine Brazilian Dicyma pulvinata isolates were examined by morphological traits and RFLP, RAPD and AFLP analyses. This fungus is a mycoparasite of Microcyclus ulei, the causal agent of the most devastating rubber (Hevea brasiliensis) disease, known as "South American Leaf Blight" (SALB). These isolates were compared with an Indian isolate from Cercosporidium sp., and a French isolate from Cladosporium fulvum. They were also compared with Dicyma ampullifera from Papua New Guinea. The morphological parameters analyzed confirmed the identification of the Brazilian isolates. The graphic representations of the distance matrices of each molecular marker showed similar results. Dicyma pulvinata isolates from M. ulei were closely related, whereas the reference isolates examined were dispersed. Among the D. pulvinata isolates obtained from M. ulei, a significant pairwise distance was obtained, for all the molecular markers, between the isolates from the areas favorable to the occurrence of SALB (North and Northeast of Brazil) and the region of escape for the disease (Mato Grosso State).

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.

Soybean pod blight and root rot caused by lineages of the Fusarium graminearum and the production of mycotoxins

Surveys of soybean (Glycine max) seed grown in South Brazil revealed infection with Fusarium graminearum. To determine if members of this complex were pathogenic to soybean, six strains derived from soybean were added to soil at a rate of 10³ macroconidia/ ml or individual pods were inoculated with 10(4) macroconidia/ml. Seedlings grown in infested soil developed small necrotic lesions in the crown and upper roots. Pods inoculated with conidia developed large (>1 cm), dark brown, necrotic lesions. Younger pods inoculated with the fungus blighted and dropped from the plant. Strains of the F. graminearum complex recovered from lesions on the crown, roots and pods of soybean plants were identified as lineage 1, 2 or 8 by obtaining the DNA sequence from the EF1-alpha gene and comparing it to strains of the known lineage. Two strains of F. graminearum lineage 7 from the U.S. caused similar symptoms of the disease on soybean. Mycotoxin tests on soybean and wheat (Triticum aestivum) indicate that most Brazilian strains produce nivalenol as the major trichothecene mycotoxin rather than deoxynivalenol. In addition, strains from lineages 2 and 8 produce the novel trichothecene, 3-acetylnivalenol.

Ocorrência de Bipolaris maydis causando mancha foliar em Paspalum atratum cv. Pojuca no Brasil

Bipolaris maydis was consistently isolated from infected Paspalum atratum cv. Pojuca plants showing leaf spot symptoms in the Cerrado of Brazil, in 2002. Pathogenicity tests under greenhouse conditions and subsequent reisolations of B. maydis from artificially inoculated Pojuca seedlings confirmed the hypothesis that this fungus was the causal agent of the disease. Symptoms of leaf spot appeared four days after inoculation in 100% of the inoculated Pojuca plants. All seven species of grasses evaluated were susceptible to B. maydis. The occurrence of leaf spot of Pojuca caused by B. maydis is reported for the first time in Brazil.

Distinct features of Pepper yellow mosaic virus isolates from tomato and sweetpepper

Determination of virus diversity in the field is vital to support a sustainable breeding program for virus resistance of horticultural crops. The present study aimed to characterize four field potyvirus isolates found naturally infecting sweet pepper (Capsicum annuum) (Sa66 and Sa115) and tomato (Lycopersicon esculentum) (IAC3 and Sa21) plants. Their biological characteristics revealed differences among the isolates in their ability to infect distinct Capsicum spp. and tomato genotypes, and in the severity of symptoms caused by these isolates compared to the infection caused by an isolate of Pepper yellow mosaic virus (PepYMV). Absence of cross-reaction was found among the studied isolates with antiserum against Potato virus Y (PVY). However, all isolates reacted, at different intensities, with antiserum against PepYMV. All isolates showed high identity percentage (97 to 99%) of the amino acid sequence of the coat protein with PepYMV (accession AF348610) and low (69 to 80%) with other potyvirus species. The comparison of the 3' untranslated region also confirmed this finding with 97 to 98% identity with PepYMV, and of 47 to 71% with other potyviruses. The results showed that PepYMV isolates were easily differentiated from PVY by serology and that the host response of each isolate could be variable. In addition, the nucleotide sequence of the coat protein and 3' untranslated region was highly conserved among the isolates.

Bellulicauda dialii on Dialium guianense (Leguminosae) in Brazil

This is the first report of Bellulicauda dialii on Dialium guianense leaves, and the second record of this fungus, previously known only from Africa (Sierra Leone).

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.

Variability of the wheat powdery mildew pathogen Blumeria graminis f. sp. tritici in the 2003 crop season

Wheat (Triticum aestivum) powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici, is one of the most severe foliar diseases attacking this crop, reducing grain yields by 10% to 62% in Brazil. The disease can be controlled by genetic resistance of the host, but the pathogen has physiological specialization, which enables it to infect wheat cultivars that have remained resistant for years. The objective of this work was to evaluate the variability of pathogenic strains of B. graminis f. sp. tritici collected in Brazil and the effectiveness of wheat resistance genes to powdery mildew in the 2003 crop season. Plants of a differential series were inoculated with each monopustular isolate. Thirty-one combinations of effective and ineffective resistance genes were identified. Only the gene Pm4a+... remained totally effective to all isolates, and gene Pm6 was highly effective (below 10% of susceptibility), whereas genes Pm3a and Pm8 were totally ineffective (susceptible to all isolates). Genes Pm3c, D1, and D2 showed low effectiveness (above 50% of susceptibility), and genes Pm1, 2, 4a, 1+?, and 2+Mld had mean effective results to most strains (susceptibility between 10% and 49%). The virulence formula Pm1, 3c, 4a, 6, 1+?, 2+Mld, 4a+..., D2 (effective genes) / 2, 3a, 8, D1 (ineffective genes) was most frequently found, accounting for 15% of the occurrences. The most frequent number of ineffective genes was seven, ranging from three to ten.

Effect of Fusarium graminearum and infection index on germination and vigor of maize seeds

Pathogens in maize (Zea mays) seeds cause serious problems, such as the loss of their capacity to germinative. The objectives of this study were to identify the optimal period for infection of maize seeds on agar colonized by Fusarium graminearum, when incubated for 4, 8, 16 and 32 h, and to evaluate the effect of the fungus on the germination and vigor of seeds with different infection levels. After the respective incubation periods, the seeds were removed from the culture medium and submitted to the blotter test for 3 min with and without superficial disinfection with 1% solution of sodium hypochlorite. Once the optimal period for seed incubation was identified, seeds from the same sample were again placed on the colonized agar for infection. Germination and vigor tests (accelerated aging and cold test) were performed with a mixture of healthy seeds (placed on PDA medium) and inoculated seeds, resulting in seeds with 0, 20, 40, 60, 80 and 100% rates of infection. The results showed that a period of 32 h was long enough to obtain seeds infected by the pathogen. There were no significant effects of fungal infection on seed germination at any of the infection levels, probably due to the high vigor of the maize seed lot tested. Regarding vigor tests, infection levels differed significantly from the control (0% infection), but there were no significant differences among the infection levels.

Survival of Botrytis cinerea as mycelium in rose crop debris and as sclerotia in soil

Botrytis blight caused by Botrytis cinerea is an important disease of rose (Rosa hybrida) grown in greenhouses in Brazil. As little is known regarding the disease epidemiology under greenhouse conditions, pathogen survival in crop debris and as sclerotia was evaluated. Polyethylene bags with petals, leaves, or stem sections artificially infected with B. cinerea were mixed with crop debris in rose beds, in a commercial plastic greenhouse. High percentage of plant parts with sporulation was detected until 60 days, then sporulation decreased on petals after 120 days, and sharply decreased on stems or leaves after 90 days. Sporulation on petals continued for 360 days, but was not observed on stems after 150 days or leaves after 240 days. Although the fungus survived longer on petals, stems and leaves are also important inoculum sources because high amounts of both are deposited on beds during cultivation. Survival of sclerotia produced on PDA was also quantified. Sclerotia germination was greater than 75% in the initial 210 days and 50% until 360 days. Sclerotia weight gradually declined but they remained viable for 360 days. Sclerotia were produced on the buried petals, mainly after 90 days of burial, but not on leaves or stems. Germination of these sclerotia gradually decreased after 120 days, but lasted until 360 days. Higher weight loss and lower viability were observed on sclerotia produced on petals than on sclerotia produced in vitro

Evaluation of genetic mixtures of sorghum lines for anthracnose resistance management

The main objective of this work was to evaluate the diversification of sorghum (Sorghum bicolor) populations as a way to manage resistance to the sorghum anthracnose fungus Colletotrichum graminicola. A total of 18 three-way hybrids were obtained by crossing six single cross male-sterile F1 hybrids, derived by crossing A (non restorer sterile cytoplasm) and B (non restorer normal cytoplasm) lines, with three fertile R (restorer) lines, previously evaluated for their differential reaction to the pathogen. Variation in the level of resistance was observed, as indicated by the values of the area under the disease progress curve (AUDPC) obtained for each hybrid. Lines contributed differently to the level of resistance of each hybrid. All hybrids in which CMSXS169R was the male progenitor were classified as highly resistant. Some hybrids had a level of resistance superior to the maximum levels of each line component individually.

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.

Virulence pattern of Pyricularia grisea isolates from farmers' fields on newly released upland rice cultivars

The virulence pattern of the isolates of Pyricularia grisea from commercial fields of the upland rice (Oryza sativa) cultivars 'Primavera' and 'BRS Bonança' was analyzed. A hundred and seventy monoconidial isolates of the pathogen virulent to 'Primavera' and 139 to 'BRS Bonança' collected from eight fields, during two years (2001-2003) were tested, under greenhouse conditions, on six newly released rice cultivars. Differences in virulence pattern were observed in pathogenic populations of 'Primavera' and 'BRS Bonança'. Isolates with virulence to improved cultivars were common in samples from farmers' fields in the absence of aloinfection. The virulence frequency of P. grisea isolates collected from 'Primavera'' to cultivars 'BRS Vencedora', 'BRS Colosso', 'BRS Liderança', 'BRS Soberana', 'BRS Curinga' and 'BRS Talento', was high in descending order. On the other hand, in the fungus population of 'BRS BRS Bonança' virulence frequency was high in 'BRS Talento', followed by 'BRS Curinga', 'BRS Vencedora', 'BRS Liderança', 'BRS Colosso' and 'BRS Soberana'. While virulence to 'BRS Talento' was rare among isolates from 'Primavera', it was most frequent in isolates of 'BRS Bonança'. The six improved rice cultivars permitted to differentiating agriculturally important virulences in the pathogen population which can be utilized in selecting breeding lines for specific resistance, in rice blast improvement program.