Genetic characterization of an elite coffee germplasm assessed by gSSR and EST-SSR markers.

Coffee is one of the main agrifood commodities traded worldwide. In 2009, coffee accounted for 6.1% of the value of Brazilian agricultural production, generating a revenue of US$6 billion. Despite the importance of coffee production in Brazil, it is supported by a narrow genetic base, with few accessions. Molecular differentiation and diversity of a coffee breeding program were assessed with gSSR and EST-SSR markers. The study comprised 24 coffee accessions according to their genetic origin: arabica accessions (six traditional genotypes of C. arabica), resistant arabica (six leaf rust-resistant C. arabica genotypes with introgression of Híbrido de Timor), robusta (five C. canephora genotypes), Híbrido de Timor (three C. arabica x C. canephora), triploids (three C. arabica x C. racemosa), and racemosa (one C. racemosa). Allele and polymorphism analysis, AMOVA, the Student t-test, Jaccard's dissimilarity coefficient, cluster analysis, correlation of genetic distances, and discriminant analysis, were performed. EST-SSR markers gave 25 exclusive alleles per genetic group, while gSSR showed 47, which will be useful for differentiating accessions and for fingerprinting varieties. The gSSR markers detected a higher percentage of polymorphism among (35% higher on average) and within (42.9% higher on average) the genetic groups, compared to EST-SSR markers. The highest percentage of polymorphism within the genetic groups was found with gSSR markers for robusta (89.2%) and for resistant arabica (39.5%). It was possible to differentiate all genotypes including the arabica-related accessions. Nevertheless, combined use of gSSR and EST-SSR markers is recommended for coffee molecular characterization, because EST-SSRs can provide complementary information.

First Report of Cercospora apii Leaf Spot on Capsicum chinense in Brazil.

In Brazil, Capsicum chinense Jacq. is the predominant species of commercial hot peppers because of its popular citrus-like aroma and adaptability to different soils and climates (4). In June 2010, 30 samples of C. chinense with severe leaf spot were collected from a field in the city of Viçosa, state of Minas Gerais, Brazil. Symptoms were observed on leaves, calyxes, fruits, and stems on most of the plants found in the area. On leaves, symptoms included amphigenous lesions that were initially circular to ellipsoid, 1 to 5 mm in diameter, whitish to tan in the center, and surrounded by a dark brown or reddish purple border. Lesions coalesce and turned necrotic with age. A fungus isolated from the lesions matched well with the description of Cercospora apii Fresen. It formed erumpent stromata that were dark brown and spherical to irregular; fascicule conidiophores were clear brown or pale, straight or curved, unbranched, geniculate, 22.5 to 80 × 5 to 7.5 µm, 0 to 3 septate, subtruncate apex; and conidia were solitary, hyaline to subhyaline, filiform, base truncate, tip acute, straight to curved, 12.5 to 140 × 3.5 to 5 µm, and 0 to 11 septate (1,2). A sample was deposited in the herbarium of the Universidade Federal de Viçosa, Minas Gerais, Brazil (VIC 31415). Identity was confirmed by amplifying part of the calmodulin gene with species-specific primers CercoCal-apii and CercoCal-R (3) of fungal DNA from a single-spore culture. In amplification reaction, initial denaturation step was done at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C (30 s), annealing at 56°C (30 s), and elongation at 72°C (30 s). Primers CercoCal-apii and CercoCal-R amplified a single DNA product of 176 bp, and coupled with the morphological characteristics, confirmed the identity of the fungus as Cercospora apii. To check pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 21-day-old culture grown on potato dextrose agar (PDA) and placed in contact with the adaxial face of the leaves of 8-week-old C. chinense grown in 2-liter plastic pots with soil substrate. Six plants, one per pot, were inoculated with the isolate and six plants were inoculated with the fungus-free PDA plug. Inoculated plants were maintained in a moist chamber for 24 h and then subsequently kept in a greenhouse at 26°C. Leaf spot was observed in all inoculated plants 15 days after inoculation and symptoms were similar to those expressed in the field. The fungus was reisolated from the inoculated plants and matched well with the description of Cercospora apii. All fungus-free PDA inoculated plants remained healthy. Cercospora apii comprises a complex of 281 morphologically indistinguishable species that can infect an extremely wide host range (2). To our knowledge, this pathogen has the potential to cause significant damage to the hot pepper industry of Brazil. References: (1) C. Chupp. A Monograph of the Fungus Cercospora. Cornell University Press, Ithaca, NY, 1954. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) M. Groenewald et al. Phytopathology 95:951, 2005. (4) S. D. Lannes et al. Sci. Hortic. 112:266, 2007.

First Report of Leaf Blight on Rubus brasiliensis Caused by Colletotrichum acutatum in Brazil.

There are more than 300 blackberry (Rubus) species worldwide. Rubus brasiliensis Mart. is a native Brazilian species found in tropical forests. In January 2009, samples of R. brasiliensis with severe leaf blight were collected from an area of rain forest in the city of São Miguel do Anta, State of Minas Gerais, Brazil. Dark spots began developing in the young leaves and progressed to necrotic spots with occasional twig dieback. From the spots, a fungus was isolated with the following morphology: acervuli that were 20 to 50.0 × 50 to 125.0 µm and hyaline amerospores that were ellipsoid and fusiform and 7.5 to 23.75 × 2.5 to 5.0 µm. On the basis of these morphological characteristics, the fungus was identified as Colletotrichum acutatum. In Brazil, C. acutatum is reported in apple, citrus, strawberry, peach, plum, nectarine, olive, medlar, and yerba-mate, but it was not reported as the causal agent of leaf blight in R. brasiliensis. A sample was deposited in the herbarium at the Universidade Federal de Viçosa, Minas Gerais, Brazil (VIC 31210). One representative isolate, OLP 571, was used for pathogenicity testing and molecular studies. Identity was confirmed by amplifying the internal transcribed spacer (ITS) regions of the ribosomal RNA with primers ITS4 (3), CaInt2 (a specific primer for C. acutatum [2]) and CgInt (a specific primer for C. gloeosporioides [1]). Isolates of C. acutatum (DAR78874 and DAR78876) and C. gloeosporioides (DAR78875) obtained from Australian olive trees were used as positive controls. The primers ITS4 and CaInt2 amplified a single DNA product of 500 bp expected for C. acutatum. OLP 571 was grown for 7 days on potato dextrose agar. Young leaves of R. brasiliensis were inoculated with a conidial suspension (106 conidia/ml) on young leaves. Inoculated plants were maintained in a moist chamber for 2 days and subsequently in a greenhouse at 25°C. Necrotic spots similar to those described were detected on young leaves 3 days after the inoculation. Control leaves, on which only water was sprayed, remained healthy. The same fungus was reisolated from the inoculated symptomatic tissues. To our knowledge, this is the first report of C. acutatum causing leaf blight in the native species of R. brasiliensis in Brazil. References: (1) P. R. Mills et al. FEMS Microbiol. Lett. 98:137, 1999. (2) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

The complete nucleotide sequence and genome organization of bean common mosaic virus (NL3 strain).

The nucleotide sequences of 3 cDNA clones corresponding to entire RNA genome of bean common mosaic virus NL3 strain have been determined. The RNA is 9612 nucleotides long, excluding a 3'-terminal poly(A) tail. A putative start codon located at nucleotide positions 170-172 initiates one large open reading frame that is terminated with a UAA codon at position 9368-9370. The predicted polyprotein has 3066 amino acids and an M(r) of 340.3 kDa. The positions of putative protein cleavage sites have been determined by analogy to consensus sequences in other potyviruses. The nucleotide sequences of the non-translated regions and the predicted amino acid sequences of BCMV NL3, were compared with those of other potyviruses. Comparison of the BCMV NL3 proteins with those of other potyviruses indicated a similar genomic organization, and high percentage of amino acid sequence identity in the cylindrical inclusion protein, nuclear inclusion 'b' protein and coat protein. BCMV NL3 displays the highest amino acid sequence identity with soybean mosaic virus.