Morphological and molecular characterization of Fusarium spp pathogenic to pecan tree in Brazil.

The occurrence of Fusarium spp associated with pecan tree (Carya illinoinensis) diseases in Brazil has been observed in recent laboratory analyses in Rio Grande do Sul State. Thus, in this study, we i) obtained Fusarium isolates from plants with disease symptoms; ii) tested the pathogenicity of these Fusarium isolates to pecan; iii) characterized and grouped Fusarium isolates that were pathogenic to the pecan tree based on morphological characteristics; iv) identified Fusarium spp to the species complex level through TEF-1α sequencing; and v) compared the identification methods used in the study. Fifteen isolates collected from the inflorescences, roots, and seeds of symptomatic plants (leaf necrosis or root rot) were used for pathogenicity tests. Morphological characterization was conducted using only pathogenic isolates, for a total of 11 isolates, based on the mycelial growth rate, sporulation, colony pigmentation, and conidial length and width variables. Pathogenic isolates were grouped based on morphological characteristics, and molecular characterization was performed by sequencing TEF-1α genes. Pathogenic isolates belonging to the Fusarium chlamydosporum species complex, Fusarium graminearum species complex, Fusarium proliferatum, and Fusarium oxysporum were identified based on the TEF-1α region. Morphological characteristics were used to effectively differentiate isolates and group the isolates according to genetic similarity, particularly conidial width, which emerged as a key morphological descriptor in this study.

First Report of Fusarium equiseti Associated on Pecan (Carya illinoinensis) Seeds in Brazil.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an important producing nut tree that has been intensively cultivated in the state of Rio Grande do Sul (Brazil) in recent decades. This species is commonly grown in association with other crops and more often with cattle or sheep. An elevated incidence of the fungal genus Fusarium was observed during a quality control seed assay of pecan seeds obtained from orchards in the city of Anta Gorda (28°53'54.7″ S, 52°01'59.9″ W). Concomitantly, seedlings of this species, cultivated in a nursery, showed foliar necrosis, wilt, and root rot. The fungus was thereafter isolated from the seeds (from original seeds lots) and subcultured from single spores. Cultures were purified in order to perform pathogenicity tests. The isolated Fusarium sp. was increased on autoclaved wet corn kernels that were incubated for 14 days (1), and then were mixed with commercial substrate (sphagnum turf, expanded vermiculite, dolomitic limestone, gypsum, and NPK fertilizer) in plastic trays (capacity 7 L), with drainage holes. Twenty seeds were sowed and 90 days later, evaluations were undertaken. Forty percent of the seedlings presented symptoms, i.e., foliar necrosis and wilt owing to root rot. Fusarium sp. was re-isolated from the affected roots by transferring hyphal tips to potato dextrose agar (PDA) and carnation leaf agar (CLA) medium in petri dishes in order to identify the species morphologically. On PDA, the colony pigmentation was yellowish brown and the aerial mycelium was whitish to peach; macroconidia were relatively long and narrow (31.75 × 4.02 µm), with 5 septa on average, and whip-like bent apical cells (2). Chlamydospores were not observed on PDA or CLA. Primer pairs ITS1 and ITS4 (3) and EF1-T and EF1-1567R (4) were employed to amplify the internal transcribed spacer (ITS) and elongation factor-1α (TEF 1-α) regions, respectively. The resulting DNA sequences showed 99% for ITS and 98% for TEF 1-α similarity with Fusarium equiseti (Corda) Sacc. and phylogenetic analysis grouped it with sequences of this species. The consensus sequence was submitted to GenBank and received the accession numbers KC810063 (ITS) and KF601580 (TEF 1-α). The pathogen was re-isolated on PDA and CLA substrate in order to complete Koch's postulates. The pathogenicity test was repeated with the same conditions described before and the results were confirmed. No symptoms were observed on the control seedlings. This species is considered a weak parasite (2); however, it has been reported causing wilt in Coffea arabica in Brazil (5). This pathogen could cause serious damage and high losses to seedling in commercial nurseries. Besides that, it could also carry the disease to the field causing further damage on established plants. To our knowledge, this is the first to report of F. equiseti causing foliar necrosis and wilt on C. illinoinensis in Brazil. References: (1) L. H. Klingelfuss et al. Fitopatol. Brasil. 32:1, 2007. (2) W. Gerlach and H. Nirenberg. The Genus Fusarium - a Pictorial Atlas. Biologische Bundesanstalt für Land- und Forstwirtschaft, Braunschweig, Germany, 1982. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA, 1990. (4) S. A. Rehner and E. A. Buckley. Mycologia 97:84, 2005. (5) L. H. Pfenning and M. F. Martins. Page 283 in: Simpósio de Pesquisa dos Cafés do Brasil, 2000.

First Report of Ilyonectria robusta Associated with Black Foot Disease of Grapevine in Southern Brazil.

In August 2012, symptoms of black foot disease were observed on 21-year-old grapevines (Vitis labrusca cv. Bordô; own-rooted cultivar) at Nova Pádua city, Rio Grande do Sul state, Brazil. Symptomatic plants showed reduced vigor, vascular lesions, decline and death of vines, and necrotic lesions on roots. Isolation of fungi associated with necrotic root tissue was made on potato dextrose agar (PDA) medium containing 0.5 g L-1 streptomycin sulfate. Cultures were incubated at 25°C for 7 days in darkness, and single-spore cultures were obtained from the colonies emerging from the diseased tissue. For morphological characterization, cultures were transferred to PDA and spezieller nährstoffarmer agar (SNA) medium with addition of two pieces of 1 cm2 filter paper. One representative isolate (Cy9UFSM) was used for morphological and molecular characterization and pathogenicity confirmation. After 10 days growth on PDA at 20°C in the dark, colonies were umber to chestnut in color (3), appeared cottony to felty in texture, and sporulated profusely. After 5 weeks on SNA and under dark conditions at 20°C, cultures formed macroconidia predominantly on simple conidiophores, 1 to 3 septate, with both ends slightly rounded. Macroconidia varied in size depending on the number of cells as follows: one-septate (23-) 27.7 (-31) × (4.5-) 5.8 (-7) µm; two-septate (26-) 30.1 (-34) × (5-) 5.6 (-6) µm; and three-septate (24-) 31.2 (-35) × (5-) 5.8 (-6.5) µm. Microconidia were observed and did not have a visible hilum (6-) 11.2 (-17) × (3.5-) 4.2 (-5) µm (n = 30 observations per structure). Brown, thick-walled globose to subglobose chlamydospores were produced abundantly on PDA, (8.5-) 13.8 (-17) µm. To confirm the species, primer pairs H3-1a and H3-1b (2) were used to amplify a portion the histone H3 gene. Sequence of this region showed 98% similarity with a reference sequence for Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous (GenBank Accession No. JF735530). Thus, both morphological and molecular criteria supported identification of the strain as I. robusta. This isolate was deposited in GenBank as accession KF633172. To confirm pathogenicity, 4-month-old rooted cuttings of Vitis labrusca cv. Bordô were inoculated by immersing roots in a conidial suspension (106 ml-1) for 60 min. After inoculation, the cuttings were planted in 1-L bags containing commercial substrate (MecPlant). Thirty days later, each plant was re-inoculated by applying 40 ml of a conidial suspension (106 ml-1) to the commercial substrate. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. After 4 months, the inoculated plants showed a 22.5% reduction of root mass, with root and crown necrosis, browning of vessels, and 20% mortality. Control plants treated with water remained symptomless. The fungus was re-isolated from blackened tissue of wood from the basal end of rooted cuttings, thereby satisfying Koch's postulates. I. robusta was first associated with black foot disease of grapevine in Portugal in 2012 (1). To our knowledge, this is the first report in southern Brazil of I. robusta associated with black foot disease of grapevine. References: (1) A. Cabral et al. Mycol. Prog. 11:655, 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.

First Report of Fusarium sambucinum Associated on Pinus elliottii Seeds in Brazil.

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.

First Report of Pestalotiopsis clavispora Causing Leaf Spot of Carya illinoensis in Brazil.

Conspicuous leaf spots in combination with fruit spots were observed for the first time in April and May 2010 on a 30-ha pecan [Carya illinoensis (Wangenh.) K. Koch] orchard in the state of Rio Grande do Sul, Brazil. Initially, tiny grey spots were observed on leaves and, over time, the spots expanded to become gray to light brown circles surrounded by a dark brown border, followed by leaves falling. Eventually, fruits were also attacked, with typical symptoms beginning with tiny water soaked spots which then became necrotic. The disease was also observed in pecan nursery and field seedlings. Isolation of the pathogen from symptomatic leaves and morphological identification by conidia characters (number of cells, color, hyaline terminal cells, number of appendages) revealed Pestalotiopsis sp. (2) as the causal agent of the disease. Conidia constituted of transverse septa with four dark intermediate sections and two hyaline terminal sections. One of the terminal sections presented two or three apical appendages. Conidia averaged 6.88 µm wide × 31.00 µm long, not considering the apical appendages. Primers ITS 1 and ITS 4 were used to amplify the internal transcribes spacer ITS 1-5.8S-ITS 2 region. Nucleotide sequences were 99% similar to Pestalotiopsis clavispora (G.F. Atk.) Steyaert. Conidia produced on potato dextrose agar medium were used to inoculate 8 plants with a spore suspension of 2.0 × 106 conidia/ml. Eight additional plants were used as control (non-inoculated). The inoculation was performed by spraying the suspension onto the leaves of Pecan seedlings and the plants were incubated for 72 h in a humid chamber (1). All inoculated plants showed symptoms 25 days after inoculation and the fungus was reisolated. The pathogenicity test was repeated once. Ten more isolates collected from four different cities in the same state were identified as Pestalotiopsis spp. by morphological characterization and pathogenicity was confirmed. Because this disease causes losses on production of nuts indirectly by reducing photosynthetically active area when the pathogen attacks leaves and directly when attacking fruits, it may restrict the production where the pathogen occurs. On some orchards in the state of Rio Grande do Sul, the attack rate reached 80% of the plants. P. clavispora has been reported causing stem end-rot of avocado in Chile (3), but this note constitutes the first report, to our knowledge, of P. clavispora causing leaf spot on C. illinoensis in Brazil. References: (1) A. C. Alfenas and F. A. Ferreira. Page 117 in: Métodos em Fitopatologia. A. C Alfenas and R. G. Mafia (eds.). Editora: UFV, Viçosa, 2007. (2) S. S. N. Maharachchikumbura et al. Fungal Diversity 50:167, 2011. (3) A. L. Valencia et al. Plant Dis. 95:492, 2011.