Clair Walker

FIRST REPORT OF SPECIES IN THE CLADOSPORIUM CLADOSPORIOIDES COMPLEX CAUSING PECAN LEAF SPOT IN BRAZIL

From January to April 2014, sixteen pecan (Carya illinoinensis) orchards in southern Brazil were surveyed to determine the causal agent of a small black circular leaf spot disease. Forty isolates obtained on potato dextrose agar (PDA) were identified on the basis of morphological features and sequence analysis of the translation elongation factor 1-α gene (Bensch et al., 2012). Morphological features and sequence homology confirmed the isolates to be either Cladosporium cladosporioides, C. pseudocladosporioides or C. subuliforme, all species included in the C. cladosporioides complex. On PDA, C. cladosporioides (2/1PR, GenBank accession No. KT991541) and C. pseudocladosporioides (11RS, KT991560) both produced colonies of greenish-gray pigmentation with velvety and floccose aerial mycelium, whereas C. subuliforme (16/2RS, KT995114) produced colonies with similar pigmentation but more abundant aerial mycelium. Isolates 2/1PR, 11RS and 16/2RS produced on average colonies that were 72.2, 68.1 and 74.6 mm in diameter, respectively; conidia were 5.1 × 2.6 μm, 5.7 × 2.5 μm and 5.3 × 2.9 μm in length; ramoconidia were 23.7 × 4.2 μm, 22.5 × 2.8 μm and 16.0 × 4.3 μm in length. To confirm pathogenicity, young detached leaflets were inoculated with twenty-six isolates. One-tenth ml of conidial suspension (1 × 106 conidia ml-1) divided into six individual drops was applied on each leaflet surface with eight replicates. Leaflets receiving only sterile distilled water served as a non-inoculated control. Black leaf spots were observed after 22 days on all inoculated leaflets. The original Cladosporium species was re-isolated from inoculated leaflets. To our knowledge, this is the first report of C. cladosporioides, C. pseudocladosporioides and C. subuliforme causing pecan leaf spot in Brazil as well as in the world.

CARACTERIZAÇÃO MORFOLÓGICA, MOLECULAR E PATOGENICIDADE DE Fusarium acuminatum E Fusarium verticillioides A Cordia americana

Cordia americana (L.) Gottschling & J. S. Mill. (guajuvira) is a forest species widely distributed in Brazil and represents environmental and economic importance due to the quality of its timber and use in reforestation. In order to evaluate the pathogenicity of Fusarium sp. associated with seeds, characterizing the isolates morphologically and molecularly in order to identify them on the species level, was obtained four fungal isolates that were inoculated in seeds to demonstrate their pathogenicity. The morphological characterization was performed using an identification key for the genus Fusarium and, for the molecular identification, genomic regions Internal Transcribed Spacer (ITS) were sequenced with its elongation factor 1 alpha (TEF1-α). The results of the molecular identification agreed with the morphological characterization and allowed to identify the species Fusarium acuminatum and Fusarium verticillioides , both being pathogenic to Cordia americana , causing damping-off in pre and post emergence, and rotting of roots and necrosis of hypocotyl.

ANTAGONISMO DE Trichoderma SPP. E Bacillus subtilis (UFV3918) A Fusarium sambucinum EM Pinus elliottii ENGELM

Pinus elliottii is an important species in the forest sector, which has restrictions such as seed sanitary quality, especially the association of Fusarium spp., responsible for seedling losses in the nursery. This study evaluated antagonist in vitro and in vivo action of Trichoderma spp. and Bacillus subtilis (UFV3918) in controlling Fusarium sambucinum, responsible for damage in Pinus elliottii seedlings. The in vitro assay considered the inhibition of mycelial growth (paired comparison of cultures) after incubation at 25 ± 2 oC and photoperiod of 12 hours. For in vivo tests (performed in nursery conditions) seeds were initially inoculated with the pathogen and, subsequently, microbiolized with antagonistic agents, before sowing. The techniques of contact with biocontroller on PDA for 48 hours and seed coating, as forms of microbiolization, were used. Both Trichoderma spp. and Bacillus subtilis (UFV3918) were efficient on the in vitro control of F. sambucinum; however, Bacillus subtilis (UFV3918) was more effective against the pathogen on the in vivo biocontrol test, reducing seedlings losses caused by the pathogen and increasing values of length, as well as fresh and dry matter of the seedling.

Susceptibility of Pecan Cultivars to Cladosporium cladosporioides Species Complex

Recent studies have observed symptoms of leaf spot in pecan (Carya illinoinensis) associated with species of the Cladosporium cladosporioides complex (C. cladosporioides, C. pseudocladosporioides, and C. subuliforme). Therefore, the present study aimed to investigate the pathogenicity of these Cladosporium spp. isolated from two pecan cultivars. Twenty-six isolates were used to inoculate leaflets of the cultivars, ?Barton? and ?Shawnee?, with a spore suspension. The suspension was applied through drops on the surface of the leaflets, the material was kept in boxes, ?gerbox?, in the incubation room. After 22 days, leaf spots varied from small circular brown to black stains, which indicated the advancement of the disease. The three Cladosporium sp. were pathogenic to pecan cultivars and there was a difference in susceptibility to C. pseudocladosporioides species between the ?Barton? and ?Shawnee? cultivars, where ?Shawnee? showed greater disease severity.

Fusarium oxysporum and F. verticillioides associated with damping-off in Pinus spp.

Occurrence of Fusarium spp. is one of the problems, most limiting to growth of seedlings, in nurseries. This pathogen can be transmitted via seeds and causes damages to the seedlings during pre- and post-emergence stages. The present study aimed to identify Fusarium spp. at the species level based on morphological and molecular characteristics and to verify the pathogenicity of these isolates in seeds lots of Pinus elliottii and P. taeda. For this, we used two Fusarium isolates and five lots of Pinus spp. seeds. Morphological characterization was performed based on a key, specific to Fusarium spp. identification, whereas, molecular identification was carried out by amplification and sequencing of the regions from internal transcribed spacer (ITS) and the elongation factor 1-α (tef1). The pathogenicity test was conducted through the contact of the seeds with fungal culture for 48 h, followed by sowing them in sand. The variables evaluated were emergency speed index, percentage of emergency, non-emergency seeds, symptomatic seedlings, and seedling damping-off. One isolate, F1UFSM, was identified as F. verticillioides and another isolate, F2UFSM, was identified as F. oxysporum. Both the isolates were pathogenic to the seeds of Pinus spp., causing a reduction in the percentage of emergence and seedling damping-off.

TESTE DE SANIDADE E GERMINAÇÃO EM SEMENTES DE Cabralea canjerana (Vell.) Mart.

The current study aimed to identify pathogens, analyzing different seed treatments and substrates for the germination of Cabraleacanjerana and also to indicate instructions testing for laboratory test of the species germination. First, we assessed the moisture content and determined the thousand seed weight. In order to assess the pathogens associated with the seeds, health testing was performed at different seed treatments: T1 - Control; T2-Captan ® ; T3-Maxim ® ; T4-Sodium hypochlorite; andT5-AgrotrichPlus ® . The germination testwas performed based ona factorial designwithfive seed treatmentsand threesubstrates (on blotting paper, vermiculiteand sand). The seeds showedmoisture contentandthousand seed weight of41.2% and457.3g, respectively. The main fungi genera associated with Cabraleacanjerana seeds were: Penicillium spp., Fusarium spp., Cladosporium spp., Pestalotia spp. and Phomopsis spp..Theseed treatment withCaptan ® , Maxim ® andsodium hypochloritewas effectivein reducing the incidenceof Penicillium spp., but onlythe last twoshowedthe greatest potential forseed germination,regardless theused substrate. Treatment with Agrotrich Plus® had the lowest percentage of germination and increased incidence of Fusarium spp. Asepsis with 1% per 2’ sodium hypochlorite and seeding substrate over vermiculite is indicated for the germination tests of Cabraleacanjerana seeds and the first counting can be performed at the 19 th day and the final one at the 30 th day after the test.