Alicia G. Luque

Soybean sudden death syndrome species diversity within North and South America revealed by multilocus genotyping.

Sudden death syndrome (SDS) of soybean has become a serious constraint to the production of this crop in North and South America. Phenotypic and multilocus molecular phylogenetic analyses, as well as pathogenicity experiments, have demonstrated that four morphologically and phylogenetically distinct fusaria can induce soybean SDS. Published molecular diagnostic assays for the detection and identification of these pathogens have reported these pathogens as F. solani, F. solani f. sp. glycines, or F. solani f. sp. phaseoli, primarily because the species limits of these four pathogens were only recently resolved. In light of the recent discovery that soybean SDS and Phaseolus and mung bean root rot (BRR) are caused by four and two distinct species, respectively, multilocus DNA sequence analyses were conducted to assess whether any of the published molecular diagnostic assays were species-specific. Comparative DNA sequence analyses of the soybean SDS and BRR pathogens revealed that highly conserved regions of three loci were used in the design of these assays, and therefore none were species-specific based on our current understanding of species limits within the SDS-BRR clade. Prompted by this finding, we developed a high-throughput multilocus genotyping (MLGT) assay which accurately differentiated the soybean SDS and two closely related Phaseolus and mung BRR pathogens based on nucleotide polymorphism within the nuclear ribosomal intergenic spacer region rDNA and two anonymous intergenic regions designated locus 51 and 96. The single-well diagnostic assay, employing flow cytometry and a novel fluorescent microsphere array, was validated by independent multilocus molecular phylogenetic analysis of a 65 isolate design panel. The MLGT assay was used to reproducibly type a total of 262 soybean SDS and 9 BRR pathogens. The validated MLGT array provides a unique molecular diagnostic for the accurate identification and molecular surveillance of these economically important plant pathogens.

High prevalence of oral colonization by Candida dubliniensis in HIV-positive patients in Argentina

Candida dubliniensis is a recently described yeast species, closely related to Candida albicans. This work represents the first general survey of the carriage of C. dubliniensis in the oral cavities of HIV-positive patients in Argentina. We studied 133 strains isolated from 162 HIV-positive patients, using the following identification tests: chlamydospore production on corn meal agar with Tween 80; colony color on CHROMagar Candida media; differential growth at 45 degrees C on potato dextrose agar; D-xylose assimilation; chlamydospore formation on sunflower seed agar (SSA); carbohydrate assimilation profiles using the API 20 C Aux commercial kit and PCR using primers that hybridize to the class IV intron of the ACT1 gene. Out of the 133 strains, 21 were identified as C. dubliniensis, representing approximately 13% of the 162 patients in this study. From these data, we conclude that although the PCR assay is the most reliable method, clamydospore formation on SSA is an easier and less expensive test for the screening of C. dubliniensis in the routine laboratory. Our results show that C. dubliniensis has a high prevalence among HIV-positive patients in Argentina.

Systemic infection caused by Trichosporon asahii in a patient with liver transplant

Trichosporon species are emerging pathogens capable of causing severe infections in immunocompromised patients. In this paper, we report a case of systemic infection in a liver transplant patient caused by Trichosporon asahii to show the etiologic agents aggressiveness and poor therapeutic results with the different antifungals employed.

Metabolic profiles of soybean roots during early stages of Fusarium tucumaniae infection

Soybean germplasm exhibits various levels of resistance to Fusarium tucumaniae, the main causal agent of sudden death syndrome (SDS) of soybean in Argentina. In this study, two soybean genotypes, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae. Disease symptoms were scored at 7, 10, 14, and 25 days post-inoculation (dpi). The greatest difference in the area under the disease progress curve (AUDPC) values among genotypes was observed at 25 dpi. In order to detect early metabolic markers that could potentially discriminate between susceptible and resistant genotypes, gas chromatography-mass spectrometry (GC-MS) analyses of root samples were performed. These analyses show higher levels of several amino acids and the polyamine cadaverine in the inoculated than in the uninoculated susceptible cultivar at 7 dpi. Principal component analysis (PCA) revealed that the metabolic profile of roots harvested at the earliest time points from the inoculated susceptible genotype was clearly differentiated from the rest of the samples. Furthermore, variables associated with the first principal component were mainly amino acids. Taken together, the results indicate that the pathogen induced the susceptible plant to accumulate amino acids in roots at early time points after infection, suggesting that GC-MS-based metabolomics could be used for the rapid characterization of cultivar response to SDS.

Comparison of inoculation methods for characterizing relative aggressiveness of two soybean sudden-death syndrome pathogens, Fusarium virguliforme and F. tucumaniae

Fusarium tucumaniae and F. virguliforme are the primary etiological agents of sudden-death syndrome (SDS) of soybean in Argentina and the United States, respectively. Five isolates of F. tucumaniae and four isolates of F. virguliforme were tested for relative aggressiveness to soybean, using a toothpick inoculation method and two versions of a soil infestation inoculation method. Partially resistant soybean cultivar RA629 and susceptible cultivar A6445RG were inoculated separately with each of the nine isolates. Two experiments for each inoculation method were performed. Analysis of variance identified a significant three-way interaction of soybean cultivar*experiment*SDS pathogen (P = 0.01) using the different methods. When the two soil infestation methods were used, F. virguliforme was more aggressive than F. tucumaniae; however, when using the toothpick method, isolates of F. virguliforme and F. tucumaniae were equally aggressive. Although all three methods discriminated levels of partial resistance of the genotypes to SDS, results of the present study indicated that soil inoculations with sorghum infested grain represent the best method for evaluating soybean cultivar resistance to SDS. The existence of interactions among the host, pathogen and environmental conditions highlights the need for additional studies to improve the reproducibility of tests for screening soybean germplasm for resistance to SDS.

Oxidative stress response involving induction of protective enzymes in Candida dubliniensis

Candida dubliniensis is a yeast species closely related to Candida albicans, but in contrast to C. albicans, limited information is available on the virulence factors of this important fungal pathogen. The objective of the present study was to determine if this species was able to evoke an adaptive response to oxidants. C. dubliniensis, treated with a low concentration of either H(2)O(2) or methyl viologen (a superoxide generating agent), mounts an adaptive response that results in increased survival against lethal doses of both oxidants. This response was characterized by the induction of enzymes with known antioxidant function. C. dubliniensis strains were less resistant to oxidants than C. albicans, displaying higher susceptibility to their toxic effects. The adaptive response described here might be responsible, among other factors, for the ability of this pathogen to cause infections in individuals with impaired immunity.

Adherence, colonization and dissemination of Candida dubliniensis and other Candida species

Adherence to host cells is essential for yeasts to develop their full pathogenic potential since it triggers the process that leads to colonization and enables their persistence in the host. The aim of this work was to study the in vitro adherence of Candida dubliniensis and other Candida species, as well as the relation of adherence with the colonization and dissemination of these yeasts in an experimental mice model. Clinical isolates of Candida dubliniensis, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis were tested for their in vitro ability to adhere to buccal epithelial cells and in vivo to colonize and disseminate in an experimental infant mice model. Although C. dubliniensis isolates showed variable adherence values, their ability to colonize and disseminate in mice tissue was almost null. All C. albicans strains showed high levels of adherence and a prolonged gastrointestinal (GI) tract colonization. Both C. glabrata and C. krusei, showed a minor i...

Design, Characterization, and In Vitro Evaluation of Antifungal Polymeric Films

The objective of the present paper was the development and the full characterization of antifungal films. Econazole nitrate (ECN) was loaded in a polymeric matrix formed by chitosan (CH) and carbopol 971NF (CB). Polyethylene glycol 400 and sorbitol were used as plasticizing agents. The mechanical properties of films were poorer when the drug was loaded, probably because crystals of ENC produces network outages and therefore reduces the polymeric interactions between the polymers. Polymers–ECN and CH–CB interactions were analyzed by Fourier-transform infrared spectroscopy (FTIR), thermal gravimetry analysis, and differential thermal analysis (DTA-TGA). ECN did not show structure alterations when loaded into the films. In scanning electron microphotographs and atomic force microscopy analysis, films prepared with CB showed an evident wrinkle pattern probably due to the strong interactions between the polymers, which were observed by FTIR and DTA-TGA. The in vitro activity of the formulations against Candida krusei and Candida parapsilosis was twice as greater as the commercial cream, probably as a result of the antifungal combination of the drug with the CH activity. All these results suggest that these polymeric films containing ECN are potential candidates in view of alternatives dosages forms for the treatment of the yeast assayed.

Estudio taxonómico de aislamientos clínicos de Trichophyton en Rosario, Argentina

Resumen es: Debido al pleomorfismo y la variabilidad cultural que presentan las especies del genero Trichophyton, los metodos de identificacion basados exclusivament...Due to the pleomorphism and cultural variability displayed by species of the genus Trichophyton, the identification methods based solely on morphological features are usually insufficient for their classification. The goal of the present work was to test a set of phenotypic methods in order to identify fungal isolates that belong to the aforementioned genus. These methods were based on a molecular taxonomic technique used as standard. Clinical isolates (56) were used as samples along with 6 reference strains. Macro and micromorphological studies were performed as well as biochemical and physiological tests such as in vitro hair perforation, nutritional requirements in Trichophyton agar media, urease production and growth on bromocresol purple-milk. solids-glucose (BCP-MS-G) agar. Additionally, PCR fingerprinting using the (GACA)4 primer was employed. As a result of the PCR method, specific profiles were observed for Microsporum canis, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton interdigitale. Identical profiles were obtained for Arthroderma benhamiae y Trichophyton erinacei. Of the total number of clinical isolates, 39 matched the T. rubrum profile while 13 corresponded to A. benhamiae and 4 to T. interdigitale. The most useful phenotypic test to differentiate between T. rubrum and T. mentagrophytes complex strains was alkalinization of the BCP-MS-G medium. Phenotypic tests did not allow differentiation among the T. mentagrophytes complex species. On the other hand, the molecular technique allowed characterization of T. rubrum isolates as well as of those observed in our study and included in the T. mentagrophytes complex: T. interdigitale and Trichophyton sp., the anamorph of A. benhamiae.

Detection and chemical control of Cercospora sojina infecting soybean seed in Argentina

Frogeye leaf spot (FLS) of soybean caused by Cercospora sojina Hara is an important disease in the Argentine Pampas region. The primary inoculum sources of the disease are infected seeds and plant debris. Thus, use of pathogen-free or fungicide-treated seeds is crucial to prevent the introduction and further spread of the disease. The aims of this research were i) to quantify C. sojina incidence in harvested soybean seeds, ii) to compare media-based incubation methods for detection of the pathogen in seeds, and iii) to evaluate the effect of fungicide seed treatments in reducing FLS incidence. Among eight different detection methods evaluated, no difference in pathogen incidence was found, but the water restriction blotter method should be recommended as it is more time and cost-effective. Out of 13 different fungicide seed treatment combinations in three independent experiments, benzimidazole seed treatments resulted in zero FLS incidence. Any other of the tested fungicidal active ingredients either did not control the disease or did so poorly only when initial infection levels were low. Treating seeds with benzimidazole fungicides may help to reduce FLS incidence during initial soybean field stands when using infected seed lots.