RESUMEN

The objective of this study was to characterize species of the Cladosporium cladosporioides complex isolated from pecan trees (Carya illinoinensis) with symptoms of leaf spot, based on morphological and molecular approaches. Morphological attributes were assessed using monosporic cultures on potato dextrose agar medium, which were examined for mycelial growth, sporulation, color, and conidia and ramoconidia size. Molecular characterization comprised isolation of DNA and subsequent amplification of the translation elongation factor 1α (TEF-1α) region. Three species of the C. cladosporioides complex were identified: C. cladosporioides, Cladosporium pseudocladosporioides, and Cladosporium subuliforme. Sporulation was the most important characteristic differentiating species of this genus. However, morphological features must be considered together with molecular analysis, as certain characters are indistinguishable between species. TEF-1αcan be effectively used to identify and group isolates belonging to the C. cladosporioides complex. The present study provides an important example of a methodology to ascertain similarity between isolates of this complex causing leaf spot in pecan trees, which should facilitate future pathogenicity studies.

Asunto(s)

Carya/crecimiento & desarrollo , Factor 1 de Elongación Peptídica/genética , Enfermedades de las Plantas/genética , Hojas de la Planta/genética , Carya/genética , Carya/microbiología , Cladosporium/genética , Cladosporium/patogenicidad , Filogenia , Enfermedades de las Plantas/microbiología , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/microbiología , Esporas Fúngicas/genética , Esporas Fúngicas/patogenicidad

RESUMEN

Cultivated grapevine (Vitis labrusca and V. vinifera) is of considerable economic importance to the Brazilian fruit industry for both fresh market consumption and for the production of wines, sparkling beverages, and juices. Black foot disease is caused by fungi of the genera Ilyonectria P. Chaverri & C. Salgado (anamorph: Cylindrocarpon Wollew.), Campylocarpon Halleen, Schroers & Crous, and Cylindrocladiella Boesew. In 2012, 4- to 40-year-old grapevines (Vitis spp.) showing reduced vigor, vascular lesions, necrotic root lesions, delayed budding, vine decline, and death were collected from seven locations at Rio Grande do Sul state, Brazil. Fungal isolations were made from root fragments and crown lesions (at least 2 cm above the bottom) on potato dextrose agar (PDA) medium added with 0.5 g L-1 streptomycin sulfate. Eight isolates were obtained and identified on the basis of morphological features and multi-gene analysis (rDNA-ITS, ß-tubulin, and histone H3) as Ilyonectria macrodidyma (Halleen, Schroers & Crous) P. Chaverri & C. Salgado. One representative isolate (Cy5UFSM) was used for more detailed morphological and molecular characterization, and pathogenicity confirmation. When incubated in the dark at 20°C for 7 to 10 days, colonies of felty straw-colored mycelium (3) 4.79 cm diameter on average were observed. No sporodochia or other fruiting bodies were produced on carnation leaf agar (CLA) medium after 30 days. Microconidia that were produced after 5 weeks on spezieller nährstoffarmer agar (SNA) medium with addition of two pieces of 1 cm2 filter paper showed ovoid and ellipsoid shape (6.4 × 3.6 µm) and one-septate macroconidia (17.3 × 4.1 µm). To confirm the species, primer pairs ITS1 and ITS4 (4); Bt2a and Bt2b; and H3-1a and H3-1b (2) were used to amplify the ITS1-5.8S rRNA-ITS2, part of the ß-tubulin and histone H3 genes, respectively. Sequences of these three regions showed 99, 100, and 100% of homology with I. macrodidyma, respectively. To confirm pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated by immersing them in a conidial suspension of the isolate (106 conidia ml-1) for 60 min (1). Thirty days later, inoculation was performed again by drenching the crown with 40 ml of 106 conidia ml-1 suspension to ensure infection of the roots. In the control treatment, plants were inoculated with sterile distilled water. Plants inoculated with I. macrodidyma showed necrosis of the leaf ribs, reduction in root mass, root and crown necrosis, browning of vessels, drying of shoots, and death. I. macrodidyma was re-isolated from the crown necrosis and vascular lesions, confirming Koch's postulates. To our knowledge, this is the first report of I. macrodidyma associated with black foot disease of grapevine in Brazil, which poses considerable threat to the industry unless management options are realized. References: (1) A. Cabral et al. Phytopathol. Mediterr. 51:340, 2012. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) R. W. Rayner. A Mycological Colour Chart. Commonwealth Mycological Institute and British Mycological Society, 1970. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

RESUMEN

An elevated incidence of the fungal genus Fusarium was ascertained during a health quality analysis of a batch of Pinus elliottii Englm. seeds obtained from the Florestas Institute for Agricultural and Forest Research (Fundação Estadual de Pesquisa Agropecuária [FEPAGRO] Florestas) in Santa Maria (29° 39' 55″ S and 53° 54' 45″ W), state of Rio Grande do Sul, Brazil. This genus comprised about 75% of all fungal genera observed in a blotter test. The fungus was then isolated and purified to perform pathogenicity tests. Healthy seeds of P. elliottii were inoculated by contact with fungal mycelium for 48 h (3). Forty-two days after inoculation, a reduction was observed in the germination potential of the seeds; however, those seeds that germinated developed normally until, as seedlings, they suffered damping-off. Fusarium was isolated from the affected vegetal material by transferring mycelium tips to potato dextrose agar (PDA) medium in petri dishes in order to morphologically identify the species. After 72 h, a tan mycelial pad 5.5 cm in diameter had formed. After transfer to carnation leaf agar (CLA), pale orange sporodochia that formed macroconidia could be observed. The macronidia were relatively short and narrow (40.2 × 4.7 µm), each containing a mean of 5 septa; the apical cell was pointed, while the basal one was foot-shaped (2,4). The chlamydospores formed in clusters, while the conidiogenous cells could be seen on top of monophialides. Primer pairs ITS1 and ITS4, EF1-T and EF1-567R, and ßtub-F and ßtub were employed to amplify the three regions ITS1.8S ITS2, elongation factor - 1α (TEF 1-α), and ß-tubulin, respectively. The sequences of these three regions showed 97, 95, and 99% of similarity with Fusarium sambucinum Fückel, respectively. The pathogen was reinoculated on P. elliottii seeds in order to complete Koch's postulates. The pathogenicity test was repeated with the same conditions described before and the results were confirmed. No occurrence of damping-off was observed in the control seedlings. The inoculated seedlings showed, besides damping-off, a visible reduction in root system expansion as well as reductions in fresh and dry tissue weight. F. sambucinum has already been reported on P. radiata D. Don in New Zealand, causing root rot and dieback (1); however, in Brazil, the present study is, to the best of our knowledge, the first to report the association of this pathogen with P. elliottii. References: (1) M. A. Dick and K. Dobbie. N. Z. Plant Prot. 55:58, 2002. (2) W. Gerlach and H. Nirenberg. The Genus Fusarium - A Pictorial Atlas. Biologische Bundesanstalt für Land - und. Forstwirtschaft, Berlin, 1982. (3) M. Lazarotto et al. Summa Phytopathol. 36:134, 2010. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 1st ed. Wiley-Blackwell, Hoboken, NJ, 2006.