Semi-selective medium for Fusarium graminearum detection in seed samples

Os fungos do gênero Fusarium causam doenças de difícil controle, em diferentes culturas, inclusive em cereais de inverno e milho. Dentre as espécies do gênero, merece destaque Fusarium graminearum . Desenvolver um meio semi-seletivo para detecção e edificação de F. graminearum em sementes, foi o objetivo do presente trabalho. Em diversos experimentos foram testados diferentes meios para meio semi-seletivo acrescido ou não de agentes microbianos. Os fungicidas testados forma iprodiona, nista tina, tr iadimenol, e os bactericidas sulfa to de estreptomicina e sulfa to de neomicina. Foram testadas cinco amostras de sementes de trigo, cevada, aveia, feijão comum e soja para a detecção de F. graminearum utilizando os meios Nash & Segalin, M. & Reis, E. M. Meio semi-seletivo para detecção de Fusarium graminearum em amostras de sementes. Summa Phytopathologica, v.36, n.4, p.304-307, 2010.

quarter dextrose agar (1/4PDA; potato 50g; dextrose 5g and agar 20g ), either un supplemen ted or supplemented with various concentrations of the antimicrobial agents cited above. The selected components and concentrations (g.L -1 ) of the proposed medium, Segalin & Reis agar (SRA-FG), were: iprodiona 0.05; nystatin 0,025; triadimenol 0.015; neomycin sulfate 0.05; and streptomycin sulfate, 0.3 added of ¼ potato sucrose agar. In the isolation from seeds of cited plant species, the sensitivity of this medium was similar to that of NSA but with de advantage of maintaining the colony morphological aspects similar to those observed in potato-dextrose-agar medium.
The aim of this work was to develop a sensitive and semiselective culture medium for the isolation and identification in situ of F. graminearum from seed samples.
The F. graminearum isolate used in this study was isolated from wheat seeds in 2001/2002 and was deposited at the collection of University of Passo Fundo, Laboratory of Plant Pathology FG 21-06 and preserved in PDA slants at 5°C. During the trials F. graminearum was maintained in potato-dextrose-agar (PDA) plates and serially transferred every month. For each experiment fresh cultures were prepared by inoculating PDA plates and incubating them at 25°C ± 2°C under a 12 h light: 12 h dark photoperiod provided by day light fluorescent tubes (Osram 40 W) for six to seven days until the plates were completely covered with mycelia. A sterilized 5.0mm diameter cork borer was used to cut mycelial disks which were transferred to the center of 9.0cm diameter Petri dishes containing the previously selected media and incubated as described above. Radial growth was measured every day by using a digital vernier caliper (Mitutoyo Digimatic Caliper -Mitutoyo Sul Americana Ltda, SP).The experiments were evaluated during seven days when the PDA control plates were completely covered with mycelia.
To screen possible selective agents radial growth experiments were conducted using F. graminearum PDA mycelial disks placed onto unsupplemented CMA, CZA, NSA, PDA, 1/4PDA, PDA-S and 1/ 4PDA-S or the same media supplemented with antifungal and six antibacterial agents at the following concentrations sulfate (NY). Radial growth was measured as described above and the colony color assessed with the aim of selecting the concentrations of these agents which allowed the highest F. graminearum growth and which did not alter the pigmentation in the medium. The unsupplemented media and PDA acted as control.
For each plant specie tested, 25 seeds were placed in 11 cm x 11 cm x 3.5 cm germination boxes (Gerboxes) containing NSA, SRA-FG or PDA at 25°C :f: 20°C under a 12 h light 12 h dark photoperiod, illumination being provided by 40W daylight-type fluorescent tubes (Osram) placed 35cm to 40 cm above the boxes. Four replicates (100 seeds) were used for each species, totaling 400 seeds evaluated. After seven days, the seeds were placed under a stereomicroscope (50 x) to evaluate the presence of F. graminearum and the percentage incidence of F. graminearum incidence per colony developed from the seeds was calculated.
The experiment was analyzed as a randomized factorial with two factors (a) culture media and plant species using ANOVAR and the Tukey-test (P<0.05.
Most selective media are, in fact, semi-selective since they allow the development of other organisms apart from the target organism which means that the medium developed by us may also be useful for taxa other than F. graminearum.
Previous experiments (data not showed) aimed at selecting fungicides for the control of seed-transmitted pathogenic fungi of winter cereals and maize showed that F. graminearum was not affected by the fungicides guazatine, iprodione and triadimenol thus we chose these fungicides for the basis of our semi-selective media. We also tested the antibacterial agent neomycin, effective against Gram-positive bacteria and the fungi Rhizopus and Mucor, and the broad-spectrum antibiotic streptomycin sulfate along with the broad-spectrum antifungal agent nystatin, all of which had been shown to have little effect on Fusarium species (12).
F. graminearum radial growth experiments showed that 1/4PDA was the best basal medium and the growth was slower on this medium on PDA-S. Slow growth of the contaminants was used as a criterion for the best semi-selective media. The medium ¼ PDA also preserved all morphological characteristics, useful for identification and also allowed abundant sporulation of F. graminearum, which usually does not sporulate on some artificial media (2). The other media were not as satisfactory for F. graminearum identification because colony morphology on these media was altered. We therefore chose 1/4PDA as the basal culture medium to test the antibiotic and antifungal selective agents and compared this medium with the commercial PDA.
Previous work has shown that the fungicide iprodione is relatively non-toxic to F. graminearum; our experiments showed that when iprodione was incorporated into 1/4PDA at 0.06 g. L-1 the resultant medium (1/4 PDA-IP60) allowed the second highest radial growth following the PDA control. However, the most typical F. graminearum colony characteristics occurred on 1/4PDA supplemented with 0.05 g.L -1 iprodione (1/4PDA-IP50), thus this concentration was selected for the final semi-selective medium. All iprodione supplemented media allowed better F. graminearum radial growth than the NSA control.
F. graminearum radial growth on media supplemented with the fungicide guazatine was lower than on the control media and inversely proportional to guazatine concentration, leading us to discard the use of guazatine as a semi-selective agent for F. graminearum.
The effects of nystatin were also inversely proportional to the radial growth of F. graminearum and there was some slight change in colony color. Nevertheless we decided to incorporate 0.025 g.L -1 of nystatin into the final medium because this was the highest concentration which did not produce a noticeable color change in the F. graminearum colonies but allowed higher growth than NSA medium. The 1/4PDA-IP50 control resulted in the lowest F. graminearum radial growth while 1/4PDA supplemented with 0.01 g.L -l iprodione (l/4PDA -IP10) led to the second highest radial growth, which was exceeded only by the 1/4PDA control; however 0.01 g.L -1 iprodione did not control contaminant fungi.
For media containing streptomycin sulfate, F. graminearum radial growth was highest in the presence of 0.2 g.L -1 and on 1/ 4PDA and 1/4PDA supplemented with 0.05 g.L -1 iprodione and 0.025 g.L -l nystatin (1/4PDA-IP50/NY25). Since there was little difference in F. graminearum radial growth between the concentrations 0.2 g.L -l and 0.3 g.L -1 concentrations streptomycin, we decided to select the latter to ensure a more effective selective medium against bacteria. We also found that NSA resulted in the lowest F. graminearum radial growth, compared with the other media tested.
When the antibiotic neomycin sulfate was tested we found that compared with NSA, 1/4PDA and IP50/NY25 the lowest F. graminearum radial growth occurred at 0.1, 0.15 and 0.2 g. L -l neomycin and that at these concentrations the fungal colonies were bleached or whitened. However, when 1/4PDA was supplemented with 0.05 g. L -1 neomycin (l/4PDA-NE50) there was little difference between F. graminearum radial growth on this media and that on NSA, and 1/4PDA-NE50 had the advantage of causing no alteration in the color of F. graminearum colonies.
In the experiment with the fungicide triadimenol F. graminearum radial growth was highest on the unsupplemented 1/4PDA control and 1/4PDA supplemented with 0.3 g L -1 streptomycin sulfate, 0.05 g. L -1 of both neomycin sulfate and iprodione, 0.05 g. L -l nystatin and 0.01 g. L -1 triadimenol but we decided that 0.01 g.L -1 a triadimenol was the best concentration to inhibit contaminant fungi since it allow F. graminearum growth without altering its characteristic coloration.
Based on F graminearum radial growth the most suitable semiselective medium for F. graminearum was that consisting of 1/ 4PDA supplemented with (g. L -1 ): streptomycin sulfate, 0.3; iprodione, 0.05; neomycin sulfate, 0.05; nystatin, 0.025; and triadimenol 0.015. This medium was denominated Segalin & Reis semi-selective for F graminearum (SRA-FG). 1/4PDA can be made in the laboratory or prepared from commercial media (Acumedia, Lansing, MI, USA) by using 1/4 of the recommended quantity with the addition of extra agar to give a final agar concentration of 20 g.L -1 .
Standard culture media such as PDA support the growth of many fungal species and often require specialized pretreatment samples, and errors in such processes can compromise the investigation validity due to the presence of undesirable contaminants (5, 8). In many agronomic situations, especially the examination of seeds for plant pathogenic fungi, semi-selective media can reduce the need for asepsis and thus save time and material.
Reis (12) developed a semi-selective medium that restricts the development of undesirable fungi and bacteria but is selective for the pathogen Bipolaris sorokiniana (Sacc.) Shoem. and other members of the Dematiaceous hyphomycetes such as Alternaria and Drechslera, facilitating detailed studies on the biology and ecology of the Table 1. Sensitivity of potato dextrose agar (PDA), Nash & Snyder agar (NSA) and Segalin-Reis agar (SRA-FG) for the detection of Fusarium graminearum on seeds of different cultivars of the cereals wheat, barley, oat, beans, and soybean. 1 After seven days, the seeds were evaluated for F. graminearum in petry plates containing PDA, NSA and SRA-FG media. Plates were incubated as described above and calculating the percentage incidence of F. graminearum. 2 Typical colony color (+) and different (-). 3 Means followed by the same lower-case letter in the columns or upper-case letter in the rows did not differ statistically (Tukey-test, P= 5%). CV= Coefficient of variation.

pathogens.
The seed tests showed that, compared with NSA, SRA-FG exhibited statistically similar sensitivity and selectivity for the detection of F. graminearum in most of the seeds evaluated (Table 1), and SRA-FG had the advantage of facilitating the identification of F. graminearum based on colony color and morphology.
For some samples, the streptomycin concentration had to be raised to 1 g.L -1 to control bacteria and 0.005 g.L-1 pentachloronitrobenzene was added to suppress yeasts. For the legumes P. vulgaris and G. max, which normally have a low incidence of F graminearum, the percentage of F. graminearum incidence on NSA was statistically higher than on SRA-FG and PDA, whereas for the cereals Triticum aestivum L, Hordeum vulgare L and Avena sativa L there was no statistical difference between NSA and SRA-FG (Table 1). Nash & Snyder (7) medium is considered standard for the isolation of Fusarium species but our results confirm the usefulness of SRA-FG, a medium that facilitates the morphological identification of F. graminearum.
The SRA-FG medium described in this paper is sufficiently sensitive and selective for F. graminearum identification and has the advantage over other media of maintaining the morphology and coloration of F. graminearum colony while inhibiting the growth of other microorganisms. This medium may help in carrying out other detailed biological and epidemiological studies on F. graminearum. Work is currently underway testing SRA-FG medium for its ability to recover F. graminearum from other crops as well as from roots, crop residues and soil.