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American Foulbrood (AFB), caused by the spore-forming Gram-positive bacterium Paenibacillus larvae, is the most severe bacterial disease affecting honeybees worldwide. Two bacterial isolates showing specific inhibitory activity against P. larvae were identified as Bacillus cereus by 16S rDNA sequencing. Antagonistic compounds were obtained from cell-free supernatants of strains m6c and m387 growing on Trypticase Soy Broth and concentrated by NH4 SO4 precipitation, ultrafiltration and butanol extraction. Both compounds were characterized as bacteriocin-like inhibitory substances (BLIS). BLISm6c and BLISm387 were stable at 70°C for 30 min and active in the pH range from 3 to 7. The antibacterial activity was completely lost at pH values higher than 8 or temperatures >80°C. Both BLIS have a narrow activity range and highly inhibit the growth of P. larvae. BLISm6c and BLISm387 differ from each other and other BLIS reportedly produced by B. cereus with regard to their molecular weights, antibacterial activity, minimal inhibitory concentration values and sensitivity to degradative enzymes. The findings of this study suggest that BLISm6c and BLISm387 can potentially be used to control AFB. SIGNIFICANT AND IMPACT OF THE STUDY: An Integrated Pest Management (IPM) approach is needed to ensure the sustainability of the beekeeping industry due to the increasing demand for organic honey and the reduction of dependence on antibiotics. Biocontrol agents produced by bacteria isolated from apiarian sources seem promising and able to combine with an IPM strategy. The most significant findings of this study are the characterization of bacteriocin-like compounds (BLIS) obtained from two strains of Bacillus cereus isolated from honey. Both BLIS have a narrow activity range and highly inhibit the growth of Paenibacillus larvae, the causal agent of American Foulbrood disease of honey bees.

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The phytosanitary importance of the corn leafhopper, Dalbulus maidis (De Long and Wolcott) (Hemiptera: Cicadellidae) and the planthopper, Delphacodes kuscheli Fennah (Hemiptera: Delphacidae) lies in their ability to transmit phloem-associated plant pathogens, mainly viruses and mollicutes, and to cause considerable mechanical damage to corn plants during feeding and oviposition. Fungi, particularly some members of the Ascomycota, are likely candidates for biocontrol agents against these insect pests, but several studies revealed their failure to invade the insect cuticle possibly because of the presence of inhibitory compounds such as phenols, quinones, and lipids and also by the antibiosis effect of the microbiota living on the cuticular surface of the host. The present work aims to understand interactions between the entomopathogenic fungus Beauveria bassiana (Balsamao-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) and bacterial antagonists isolated from the cuticular surface of D. maidis and D. kuscheli. A total of 155 bacterial isolates were recovered from the insect's cuticle and tested against B. bassiana. Ninety-one out of 155 strains inhibited the growth of B. bassiana. Bacterial strains isolated from D. maidis were significantly more antagonistic against B. bassiana than those isolates from D. kuscheli. Among the most effective antagonistic strains, six isolates of Bacillus thuringiensis Berliner (Bacillales: Bacillaeae (after B. subtilis)), one isolate of B. mycoides Flügge, eight isolates of B. megaterium de Bary, five isolates of B.pumilus Meyer and Gottheil, one isolate of B. licheniformis (Weigmann) Chester, and four isolates of B. subtilis (Ehrenberg) Cohn were identified.

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One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA). PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex) and bceT (enterotoxin-T), but no correlation with the clumping factor (bound coagulase) and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free) and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.

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Las abejas melíferas son afectadas por gran cantidad de enfermedades infecciosas principalmente producidas por bacterias, hongos, virus y parásitos eucariotas. Dentro de las ocasionadas por procariotas, la loque americana es una enfermedad extremadamente grave que afecta a larvas y pupas de abejas; su agente causal es la bacteria esporulada Paenibacillus larvae. La administración de antibióticos es la principal alternativa para el control de esta enfermedad en colmenares con altos niveles de infección. El objetivo del presente trabajo fue determinar, mediante un método biológico, la unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a las proteínas presentes en abejas adultas, larvas menores de 72 horas, larvas mayores de 72 horas, jalea de obreras, miel y polen, con la finalidad de diseñar un modelo de ruta cinética de los antibióticos. Los límites de sensibilidad de la técnica de valoración de estos antibióticos fueron 0,05 μg/ml para tilosina y tilmicosina, y 0,01 μg/ml para oxitetraciclina. Los coeficientes de correlación fueron superiores a 0,90 y los coeficientes de variación intra e inter-ensayo inferiores al 5%. Tanto tilosina como oxitetraciclina presentaron un porcentaje de unión a proteínas de un 15% en promedio en tejidos y subproductos de la colmena, lo cual resultó inferior a lo observado con tilmicosina (29% en promedio). En conclusión, por sus características químicas, su actividad antimicrobiana y su baja tasa de unión a las abejas, larvas y subproductos de la colmena, la tilosina presenta propiedades farmacocinéticas que podrían representar una ventaja terapéutica para el tratamiento de la loque americana en colmenas.

American Foulbrood (AFB) caused by the spore-forming bacterium Paenibacillus larvae is the most serious disease of bacterial origin affecting larvae and pupae of honeybees. Antibiotics are used in many countries for the control of AFB in high incidence areas, but their misuse may lead to antibiotic resistance of bacterial strains and honey contamination. The objective of the present work was to determine, through a biological method, the protein binding of tylosin, tilmicosin and oxytetracycline to worker jelly; honey; pollen; adult bees and larvae in order to propose their kinetic routes. The sensitivity limit of the technique used was 0.05 μg/ml for tylosin and tilmicosin and 0.01 μg/ml for oxytetracycline, respectively. The method had intra and inter-assay correlation coefficients over 0.90, respectively and a coefficient variation of intra-and inter-assay for all antibiotics and processed samples under 5%. Tylosin and oxytetracycline presented lower percentages of protein binding in tissues and hive products (average 15%) in relation to those observed for tilmicosin (29%). In conclusion, tylosin is useful for AFB control in honey bee colonies due to its chemical characteristics, antimicrobial activity and levels of protein binding in bees, larvae, and beehive products.

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One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA). PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex) and bceT (enterotoxin-T), but no correlation with the clumping factor (bound coagulase) and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free) and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.

Se evaluaron 132 aislamientos de Bacillus cereus y 52 de Bacillus megaterium provenientes de mieles de distintos orígenes geográficos para investigar la presencia de secuencias de ADN relacionadas con genes de virulencia y su posible correlación con la actividad hemolítica y coagulasa. Con respecto a los genes de virulencia, se analizaron por PCR secuencias de ADN de los genes nhe (A, B y C), HBL (A, B, C, D), cytK y bceT. La relación entre las variables fue evaluada mediante un análisis de componentes principales, donde se encontró que los aislamientos de B. cereus mostraron una correlación positiva entre actividad de coagulasa (coagulasa libre) y presencia de los genes del complejo HBL y bceT, mientras que en B. megaterium se halló una alta correlación positiva entre actividad de coagulasa (libre y fija) y actividad hemolítica, pero no se observó correlación significativa entre la presencia de genes de virulencia y dichas actividades. Este estudio constituye el primer registro de la presencia de los genes cyt K y NHE en cepas de B. megaterium y el primer trabajo que analiza la relación entre la actividad de coagulasa y la presencia de genes de virulencia en B. cereus y B. megaterium. La presencia del gen cytK en ambas especies resultó totalmente independiente del resto de los factores de virulencia analizados. Nuestros hallazgos sugieren que la miel podría vehiculizar enfermedades transmisibles por alimentos debido a la presencia de cepas de B. cereus y B. megaterium potencialmente tóxicas.

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American Foulbrood (AFB) caused by the spore-forming bacterium Paenibacillus larvae is the most serious disease of bacterial origin affecting larvae and pupae of honeybees. Antibiotics are used in many countries for the control of AFB in high incidence areas, but their misuse may lead to antibiotic resistance of bacterial strains and honey contamination. The objective of the present work was to determine, through a biological method, the protein binding of tylosin, tilmicosin and oxytetracycline to worker jelly; honey; pollen; adult bees and larvae in order to propose their kinetic routes. The sensitivity limit of the technique used was 0.05 µg/ml for tylosin and tilmicosin and 0.01 µg/ml for oxytetracycline, respectively. The method had intra and inter-assay correlation coefficients over 0.90, respectively and a coefficient variation of intra-and inter-assay for all antibiotics and processed samples under 5%. Tylosin and oxytetracycline presented lower percentages of protein binding in tissues and hive products (average 15%) in relation to those observed for tilmicosin (29%). In conclusion, tylosin is useful for AFB control in honey bee colonies due to its chemical characteristics, antimicrobial activity and levels of protein binding in bees, larvae, and beehive products.

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From 2006 to 2009, crown gall and hairy root symptoms were observed on blueberry (Vaccinium corymbosum cvs. O'Neil, Millennia, and Misty) plants from six nurseries in Tucumán, Concordia, Pilar, Morón, and Baradero, Argentina. Bacteria were isolated from galls of all three cultivars and from hairy roots of Millenia and O'Neil onto D1 and D1M agar media at 27°C. Typical Agrobacterium colonies developed in 5 days (2). Seven bacterial strains (five from galls and two from hairy roots) were studied further. All were gram negative, aerobic, and catalase positive with rod-shaped cells that synthesized ß-galactosidase and metabolized D-glucose, D-arabinose, n-acetyl-glucosamine, maltose, mannitol, and malonate. Strains were negative for lysine decarboxylase, H2S production, indole, and 3-ketolactose production. While gall strains were urease positive and citrate variable (mostly positive), hairy root strains were urease negative, citrate positive, had poly-ß-hydroxybutyrate inclusion granules, and clarified acid on potato dextrose agar containing 0.5% CaCO3 (2). Agrobacterium tumefaciens ATCC 15955 and LBA 958 were included as controls. PCR with virA/C primers amplified a 338-bp product corresponding to the virD2 operon and confirmed that the strains harbored a pathogenic plasmid (1). Bacterial strains were assigned to biovars with a multiplex PCR assay targeting 23S rRNA sequences (3). Two strains produced PCR amplicons typical of A. rhizogenes bv. 2. The other five strains produced PCR amplicons typical of A. rubi, which were insensitive to agrocin in a bioassay with A. radiobacter strain K1026. Identity was confirmed by sequencing the 16S rDNA of strains F 266 (GenBank No. GU580894) and F 289 (No. GU580895), which had 99% homology to 16sRNA sequences of A. rubi ICMP 11833 (AY626395.1) and A. rhizogenes ATCC 11325 (AY945955.1), respectively. Pathogenicity of all seven strains was tested on V. corymbosum cv. Misty, Bryophyllum daigremontiana, tobacco cv. Xanthi, tomato cv. Presto, and pepper cv. California Wonder. Plants were inoculated by a needle stabbed into the stems with the appropriate cell suspension (108 CFU/ml) of each strain or with sterile distilled water (control treatment). Two plants of each species were tested per strain. Plants were grown for at least 45 days at 23 ± 3°C and symptoms were recorded. Inoculations with the five strains isolated from galls caused development of spherical, white to flesh-colored, rough, spongy wart-like galls at the inoculation sites. Root strains induced root proliferation on all inoculated plants as well as in a carrot disk bioassay (4). On blueberry plants, galls were dark brown to black, rough, and woody 6 months after inoculation. No lesions were observed on control plants. Bacteria were reisolated from symptomatic tissues of inoculated plants. Enterobacterial repetitive intergeneric consensus-PCR confirmed that the DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of A. rubi and A. rhizogenes causing hairy root and crown gall on blueberry in Argentina. References: (1) J. H. Haas et. al. Appl. Environ. Microbiol. 61:2879,1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) M. H. Ryder et al. Plant Physiol. 77:215, 1985.

RESUMO

From 2007 to 2008, an uncharacterized disease of maize (Zea mays L.) was observed in commercial fields of Laguna Blanca, Formosa, Argentina and from different fields of Santa Fe and Catamarca provinces of Argentina. Symptoms included light-colored necrotic streaks on leaves and tan or white irregular blotches that sometimes were surrounded by reddish purple-to-dark brown margins. Severity of symptoms varied greatly from one field to another. Abundant bacterial streaming was observed from lesions when examined at ×150. Gram-negative, facultatively anaerobic bacteria were consistently isolated from lesions. These formed light yellow-to-orange, glistening, convex colonies on yeast dextrose calcium carbonate agar incubated at 30°C. Ten isolates from ten different symptomatic plants were selected for further study. All isolates were motile, induced a hypersensitive response in tobacco plants, and were oxidase negative. Colonies developed at 37°C. Physiological and biochemical characterization with the API 20E test strips and database (bioMerieux, Buenos Aires, Argentina) showed that the strains belonged to the genus Pantoea. All strains were positive for ß-galactosidase, utilized citrate and tartrate, and produced acid from d-glucose, d-mannitol, d-melibiose, l-arabinose, sucrose, meso-inositol, glycerol, d-sorbitol, and amygdalin. All were negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophane deaminase, H2S production, urease, and reduction of nitrate to nitrite. Variable results were obtained for indole, gelatinase, and l-rhamnose. Their identity was confirmed by sequencing the 16S rRNA gene strain F327 (GenBank Accession No. GU068363). A BlastN search of GenBank revealed 99% nt identity with strains LMG 20103 (AF364847.1), LMG 20105 (AF364845.1), and LMG 2665 (FJ611815.1) of Pantoea ananatis. Pathogenicity was verified on Z. mays (EM 6079 HX, Dow Morgan) by injection-infiltration of bacterial suspensions at 105 CFU/ml. Controls were infiltrated with sterile distilled water. Plants were kept at 26 ± 3°C in a greenhouse. Symptoms were first detected 15 to 17 days after inoculation and then lesions expanded to resemble natural infections within 30 days. Bacteria were reisolated and the original and reisolated strains were compared by using repetitive sequence-based (rep)-PCR with ERIC primers (1) and fingerprints of the reisolated strains were identical to those of the original strains, thereby fulfilling Koch's postulates. No lesions were observed on controls. Known strains of P. stewartii from the United States (SW2, DC400, DC441, and DC283) were also tested for comparison. On the basis of sequencing data, pathogenicity, and physiological tests, the pathogen was identified as P. ananatis (4). To our knowledge, this is the first report of P. ananatis causing a disease of maize in Argentina, although a similar disease has been reported in Brazil (2) and Mexico (3). References: (1) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (2) L. D. Paccola-Meirelles et al. J. Phytopathol. 149:275, 2001. (3) R. Pérez-y-Terrón et al. Australas. Plant Dis. Notes 4:96, 2009. (4) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

RESUMO

During May of 2008 (austral autumn), an uncharacterized disease was observed on Dieffenbachia picta (Lodd.) Schott and Aglaonema commutatum Schott in commercial greenhouses in Pontevedra (34°45'6″S, 58°42'42″W), Argentina. Affected plants showed irregular, brown lesions on leaves, approximately 15 to 20 mm in diameter, surrounded by water-soaked haloes that progressed inward from the margins. Water-soaked rotting symptoms were also observed in petioles. Disease incidence approached 80%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria consistently isolated from lesions formed cream-colored, glistening, convex colonies on sucrose peptone agar and produced a yellowish green, diffusible, nonfluorescent pigment on King's medium B. Four isolates from different symptomatic plants were selected for further study. All were aerobic, gram-negative rods that accumulated poly-ß-hydroxybutyrate inclusions. In LOPAT tests, all induced a hypersensitive response in tobacco plants, caused soft rot of potato tubers, and were positive for levan, negative for arginine dihydrolase, and variable for oxidase. All isolates oxidized glucose, did not hydrolyze starch and were able to rot onion slices. Colonies developed at 41°C but not at 4°C. With the API 20NE test strips and database (bioMerieux, Buenos Aires, Argentina), all isolates matched (99% identity) Burkholderia cepacia, but their inability to metabolize cellobiose and sucrose further identified them as B. gladioli. For molecular identification, 23S rDNA was amplified by PCR using B. gladioli-specific primers LP1 and LP4, which yielded a 700-bp product (3), and PCR-restriction fragment length polymorphism of 16S rDNA using AluI (2). PCR products were identical to those from the type strain for B. gladioli, ICMP 3950, isolated from Gladiolus spp. that had been included in all tests for comparison. Pathogenicity was verified on D. picta and A. commutatum by spraying the plants with bacterial suspensions in sterile distilled water at 108 CFU/ml with and without wounding the leaves with a sterile needle and also by injection-infiltration of bacterial suspensions at 105 CFU/ml. In addition, another host plant, Gladiolus communis L., was inoculated in the same manner. Controls were sprayed or infiltrated with sterile distilled water. After 48 h in a humidity chamber, plants were kept at 25 ± 3°C in a greenhouse. In all hosts, symptoms were first detected 3 days after inoculation and lesions expanded to resemble natural infections within 4 to 7 days. All strains caused necrosis around the inoculation sites and lesions were identical to those induced by the ICMP reference strain. Bacteria were reisolated from each host tested and then the original and reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (1); DNA fingerprints of the reisolated strains were identical to those of the original strains, thereby fulfilling Koch's postulates. No lesions were observed on controls or on plants inoculated by spraying without wounding, suggesting that bacteria gain entry through wounds. On the basis of PCR and physiological tests the pathogen was identified as B. gladioli (2-4). To our knowledge, this is the first report of B. gladioli on Dieffenbachia and Aglaonema spp. References: (1) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (2) C. Van Pelt et al. J. Clin. Microbiol. 37:2158, 1999. (3) P. W. Whitby et al. J. Clin. Microbiol. 38:282, 2000. (4) E. Yabuuchi et al. Microbiol. Immunol. 36:1251, 1992.

RESUMO

Peace lily (Spathiphyllum wallisii Regel) is a popular ornamental potted plant in Argentina. During May of 2008 (austral autumn), necrotic lesions of unknown etiology were observed on S. wallisii in a nursery in Pontevedra (34°45'6″S, 58°42'42″W). Plants first showed water-soaked areas starting from the leaf tips. Infected tissue became irregular, brown, dark-to-black lesions on leaves ~12 to 14 mm in diameter surrounded by yellowish haloes. Disease incidence approached 30%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria isolated from lesions formed white-to-cream, glistening, convex colonies on yeast dextrose calcium carbonate agar. Three bacterial strains isolated from different symptomatic plants were selected for comparative analysis with Pectobacterium carotovorum subsp. carotovorum type strain ATCC 15713. All were facultatively, anaerobic, gram-negative rods, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All strains were oxidase and arginine dihydrolase negative, fermented glucose, did not hydrolyze starch, did not produce lecithinase, indole or the blue pigment indigoidine, reduced nitrates, hydrolyzed gelatin and esculin, able to rot onion slices, caused soft rot of potato tubers, resistant to erythromycin, and grew at 37°C. Acid was produced from cellobiose, d-glucose, d-melibiose, d-mannitol, d-mannose, l-rhamnose, d-sucrose, and l-arabinose but not from inositol and d-sorbitol. Bacteria utilized N-acetyl-glucosamine and citrate but not tartrate, benzoate, or propionate. Their identity was confirmed by 16S rRNA gene sequencing of strain F402Pcc (GenBank Accession No. FJ717337) showing a 99% homology with that of strain ATCC 3326 (FJ 5958691). Pathogenicity was verified on S. wallisii, Dieffenbachia picta, Aglaonema commutatum, and Anthurium andraeanum within the Araceae family by spraying two plants per strain tested with bacterial suspensions (108 CFU/ml) in sterile distilled water with and without wounding the leaves with sterile needles. Controls were sprayed with sterile distilled water. After 48 h in a humidity chamber, inoculated plants and controls were maintained at 25 ± 3°C in a greenhouse. Water-soaked areas developed from 24 to 48 h after inoculation and became necrotic within 4 to 5 days. Lesions expanded to resemble natural infection in S. wallisii within 20 days, while in the rest of the hosts tested, lesions were smaller and remained brown surrounded by yellowish haloes. All strains were reisolated from each host tested. The original and all reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (4) confirming that DNA fingerprints of the reisolated strains were identical to those of the original strains. No lesions were observed on controls. The pathogen was identified as P. carotovorum subsp. carotovorum based on biochemical, physiological, pathogenicity tests, and 16S rRNA sequencing (1-3).To our knowledge, this is the first report of this pathogen on S. wallisii in Argentina although it has been reported as causing tomato pith necrosis (1) and soft rot of vegetables after harvest (3). References: (1) A. M. Alippi et al. Plant Dis. 81:230, 1997. (2) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:381, 2003. (3) L. Halperin and L. S. Spaini. Rev. Argent. Agron. 6:261, 1939. (4) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994.