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Production of antibacterial compounds by phylloplane yeasts
M. Cristina Romero1, Enso H. Reinoso1, M. Inés Urrutia2 & Alejandro Moreno Kiernan2
1Micología Clínica e Industrial, Microbiología, Fac. Ciencias Veterinarias, 2Fac. Ciencias Agrarias y Forestales - Universidad Nacional de La Plata - Av. 60 e/ 119 y 120 s/nro, La Plata -, ARGENTINA - (Correponding author: • Calle 528 bis nº 1632, 1900 La Plata, Argentina - Fax: 0054-221-422 2904; E-mail: The phylloplane supports an abundant and varied microflora including both normal and accidental inhabitants. Yeasts, filamentous fungi and bacteria dominate at different times during the stages leaf development, but fungi usually outnumber other microorganisms in biomass terms by a 0.50 to 1 ratio. Similar yeasts occured throught the year and on diverse plant species, although their densities were variable and influenced by weather and plant deseases. Cuticle and exudates utilization, morphological adaptation to extreme environments, tolerance to pollutants and plant inhibitors such as phytoalexins had been suggested as possible reasons for the ecological success of yeasts on leaf surface. Although antimicrobial compounds had been reported from a number of yeast species, other studies did not confirm antibacterial or antifungal activities. So, the aims of this study were to screen normal yeast population, not decaying-material nor pathogenic species, from the phylloplane and to detect antibacterial activity. Leaves of Triticum aestivum and Malus domestica were weekly collected during 3 months. For apple, the first leaf from the apex were sampled from several branches chosen at random, and for wheat, only the lower leaves were taken. Six grams of leaf of each plant was cut and placed in 300 ml flask, containig 3 mm ∅ glass beads and 50 ml sterile distilled water, shaked 2 h at 200 rpm and 25ºC. The washings were serially diluted, 101 to 106, and 0.1 ml each sampled types was spread on malt-extract agar (MEA, Oxoid) with 5 g tetracycline hydrochloride / l. The plates were incubated at 25 ºC for 7 days, and then the colonies were transferred to fresh MEA. The isolated yeasts were identified by theirs morphological and physiological characteristics according to Kurtzman & Fell (1998). Yeast metabolites were extracted from isolates grown in 100 ml MEA in shaking incubator, for 7 days at 25 ºC and 200 rpm. Then, the cultures were centrifuged at 1.600 to 2.500 x g to sediment the cells. The supernatants were filtered through 0.45 µm pore size filter and 10 ml ethyl-acetate was added, mixed several times, and then the upper solvent phases were removed into a glass vial. The extracted solvent was pooled and spun at 1.600 x g for 30 s; the clear upper portion was removed and evaporated to dryness. All the procedures were repeated two times. To screen for antibacterial substances, samples were taken from the cultures 10 h after inoculation during the logarithmic phases. The extracts were resuspended in 0.4 ml ethyl-acetate, and 25 µl was loaded onto a thin-layer chromatography (Merck Art. 5735, 60F254). Pseudomonas fluorescens, gram-negative standard, and Staphylococcus aureus, gram-positive one, isolated from apple leaves and contaminated soils, respectively, were used to assay the antibacterial activities of the yeast metabolites. A dish with 20 ml phylloplane-agar (2 g macerated leaves with 50 µg fructose, 15 µg sucrose, 15 µg maltose, 10 µg raffinose and 10 µg yeast-extract / ml, and 2 % agar; PHY-a), then the dish were gently agitated to ensure an even layer of agar. A dried TLC-plate was placed on the agar surface so the plastic back was in contact. Other 150 ml molten PHY-agar with 1 ml culture of the gram-negative or gram-positive bacteria and 0.7 ml of 2 % (wt/vol) 2,3,5-triphenyltetrazolium chloride (Sigma) were then plated on petri-dished and allowed to solidify. Plates were uncubated overnight at 25 ºC, and areas of bacterial growth were indicated by the red pigmentation from the tetrazolium salt. Clear areas due to inhibition of bacterial growth indicated the location of antibacterial compounds. The Rf of each clear spot and the internal antibiotic standard, novobiocin, were recorded to calculate the Rf values between samples, and these data were corrected with the Rf-novobiocin. In total, 42 isolations representing 14 different yeast genera were isolated, identified and screened for antibacterial substances. Aureobasidium spp., Candida spp., Cryptococcus spp., Debaryomyces spp., Filobasidium spp., Hansenula spp., Pichia spp., Rhodotorula spp., Saccharomyces spp., Sporobolomyces spp., Torulaspora spp., Torulopsis spp., Tremella spp. and Trichosporon were isolated from apple and wheat leaves. C. albicans, C. javanicis, C. foliarum, C. bogoriensis, F. unigutulatum, H. wingei, H. trichocarpa, H. alni, H. populi, H. dryadoides, P. opuntiae, P. amethionina, P. heedii, P. antillensis, R. marina, R. graminis, S. cerevisiae, S. roseus, T. beigilii, T. dulcitum, Torulaspora delbruckii, Torulaspora pretoriensis and Tremella foliacea were isolated from leaves. Normal soil and other plant-materials, like stems, woody and nonliving parts did not comprise a significant fraction of leaf-species. On the contrary, a higher variability and yeast numbers were found on fruit skins, seeds, petals and nectaries. The Rf of the antibacterial compounds from the apple and wheat leaf-extracts were significantly different, in relation to the tested yeasts and leaf-plant. Relatively low Rf, between 0.17-0.19, were observed in Citeromyces spp., Cryptococcus spp., Rhodotorula spp. and Sporobolomyces spp. filtrates, with even lower values, 0.13-0.15, from wheat leaf samples. Other genera, like Candida spp., Debaryomyces spp., Hansenula spp., Torulopsis spp. and Saccharomyces spp., had metabolites with close or identical Rf-values no matter the leaf-plant sample was, and ranging from 0.23-0.27. An inhibition zone at Rf 0.28-0.30 was the detected position for Pichia spp., Torulaspora spp., Trichosporon spp. and Aureobasidium spp. cultures. Generelly, the higher Rf-values of each genera were produced by isolates from apple leaves, being the data from wheat samples lower. Considerable variability had been noticed in the biochemical and physiological characteristics of the antibacterial compounds obtained in the cultures of the assayed yeasts. Aureobasidins had been reported as antifungal agents producted by A. pullulans, they are cyclical depsipeptides (molecular weight 1,070-1,148), therefore, this yeast produced both antibacterial and antifungal compounds. Possibly, the antimicrobial production depended from other biological and culture conditions not tested in this study; thus, further work on the identification and activities of the antibiotics must be done, together with the antifungal potencial of these metabolites. Under the bioassay conditions, our results showed that the ability to produce antagonistic activity towards bacteria was an usually feature of yeasts isolated from the phylloplane, more than from soil samples. A succession of yeast populations on the leaf surface occured, with bacteria colonizing initially as the bud breaks and then being mostly replaced by yeasts and filamentous fungi. Competition for available nutrients was an important factor of this succession. Antagonism of filamentous fungi by yeasts had been widely reported, but antagonism of bacteria by yeasts was not often detected. The synthesis of antibiotiocs was shown to occur towards the end of the logarithmic phase of growth, when nutrients become limited. In a natural environments, this situation was common as nutrients were utilized by competing microorganisms. Based on ours findings, these antibiotics may have a role in the inhibition of growth of competing bacteria on the phylloplane. Although, we did not identify the antibactreial substances, we just distinguished between them by the Rf-values. In summary, in the phylloplane these antibiotic substances allowed the yeasts to survival and dominance the habitat, and we confirmed that yeasts are promising source of novel antagonistic activity towards bacteria. These data could be usefull to treat different plant diseases; so further researches must point out to clear the relationship bteween a known bacterial desease and the yeast species capable to attack in nature. Different types of biocontrol had been developed recently, as yeasts and insects, and these kind of treatment are not pollutant one, cheeper and easy to produce.



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