Partial characterization of bacteriocin-like compounds from two strains of Bacillus cereus with biological activity against Paenibacillus larvae, the causal agent of American Foulbrood disease.

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.

Growth inhibition of Beauveria bassiana by bacteria isolated from the cuticular surface of the corn leafhopper, Dalbulus maidis and the planthopper, Delphacodes kuscheli, two important vectors of maize pathogens.

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.

Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey.

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.

Unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a proteínas presentes en abejas, larvas y productos de la colmena de Apis mellifera L / Binding of tylosin, tilmicosin and oxytetracycline to proteins from honeybees, larvae and beehive products

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.

Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey / Búsqueda de factores de virulencia en cepas de Bacillus cereus y de Bacillus megaterium aisladas de miel

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.

[Binding of tylosin, tilmicosin and oxytetracycline to proteins from honeybees, larvae and beehive products]. / Unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a proteínas presentes en abejas, larvas y productos de la colmena de Apis mellifera L.

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.

First Report of Agrobacterium rubi and A. rhizogenes Causing Crown and Root Gall and Hairy Root on Blueberry in Argentina.

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.

First Report of Leaf Spot Disease of Maize Caused by Pantoea ananatis in Argentina.

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.

First Report of Leaf Spot of Dieffenbachia picta and Aglaonema commutatum Caused by Burkholderia gladioli in Argentina.

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.

First Report of Pectobacterium carotovorum subsp. carotovorum on Spathiphyllum wallisii in Argentina.

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.

Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples / Detección de determinantes de resistencia a tetraciclina y oxitetraciclina en cepas de Bacillus cereus aisladas de muestras de miel

The aim of this study was to investigate the presence of tetracycline and oxytetracycline resistance determinants in Bacillus cereus strains isolated from honey samples. Of a total of 77 isolates analyzed, 30 (39%) exhibited resistance to tetracyclines according to the results of a disk diffusion method. Resistant strains (n=30) were screened by PCR for the presence of the resistant determinants tetK, tetL, tetM, tetO, tetW, otrA and otrB and their MIC values for tetracycline, oxytetracycline and minocycline were assessed. According to the PCR results, 23 isolates (77%) presented at least one tetracycline or oxytetracycline resistance determinant. The tetK genotype was present in 10 isolates while the tetL, tetM, and otrA genotypes were present in 3, 2, and 5 isolates, respectively. In addition, 2 isolates of the tetK plus tetM genotype, 1 of the tetK plus tetL genotype, and 1 of the tetK plus otrA genotype were found. All isolates were tetW, tetO and otrB negatives. On the other hand, 7 isolates (23%) showed a tetracycline-resistant and/or minocyclineresistant phenotype (MIC) but did not carry any of the tet or otr determinants investigated in this study. This research has shown that B. cereus isolates from honey samples contain a variety of tetracycline and oxytetracycline resistance genes, including the tetK and tetL determinants which encode for efflux proteins, and tetM and otrA, which encode for ribosomal protection proteins. These findings indicate that strains isolated from honeys could represent a reservoir for tetracycline resistance genes. To our knowledge, this is the first report of tetracycline-resistant and oxytetracyclineresistant B. cereus strains carrying the tetK determinant, and also the first report of oxytetracycline-resistant and tetracycline- resistant Bacillus species carrying the otrA determinant.

El objetivo del presente estudio ha sido investigar la presencia de diversos determinantes de resistencia a tetraciclina y oxitetraciclina en las poblaciones de Bacillus cereus presentes en la miel. De un total de 77 aislamientos evaluados, 30 (39%) resultaron resistentes a tetraciclina y/o minociclina de acuerdo con los resultados de las pruebas de difusión en disco. Dentro del grupo que presentó un fenotipo resistente, se investigó la presencia de los determinantes tetK, tetL, tetM, tetO, tetW, otrA y otrB por PCR y se determinaron los valores de CIM para tetraciclina, oxitetraciclina y minociclina. De acuerdo con los resultados obtenidos por PCR, 23 aislamientos (77%) presentaron al menos un determinante de resistencia a tetraciclina o a oxitetraciclina; el genotipo tetK se encontró en 10 de esos aislamientos, mientras que los genotipos tetL, tetM y otrA se hallaron en 3, 2 y 5 aislamientos, respectivamente. Ningún aislamiento presentó los genotipos tetW, tetO ni otrB. Adicionalmente, se encontraron los genotipos tetK plus tetM (2 aislamientos); tetK plus tetL (1 aislamiento) y tetK plus otrA (1 aislamiento). Por otra parte, 7 cepas (23%) resultaron resistentes a tetraciclina, oxitetraciclina y/o minociclina por CIM, pero no presentaban ninguno de los determinantes tet u otr estudiados. Estos resultados indican la existencia de un alto porcentaje de cepas de B. cereus aisladas de miel con genes de resistencia a tetraciclina y oxitetraciclina, incluyendo los determinantes tetK, tetL, tetM y otrA. Este estudio constituye el primer registro de la presencia del determinante tetK de resistencia a tetraciclina en B. cereus, como así también la presencia del determinante otrA dentro del género Bacillus.

Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples.

The aim of this study was to investigate the presence of tetracycline and oxytetracycline resistance determinants in Bacillus cereus strains isolated from honey samples. Of a total of 77 isolates analyzed, 30 (39%) exhibited resistance to tetracyclines according to the results of a disk diffusion method. Resistant strains (n=30) were screened by PCR for the presence of the resistant determinants tetK, tetL, tetM, tetO, tetW, otrA and otrB and their MIC values for tetracycline, oxytetracycline and minocycline were assessed. According to the PCR results, 23 isolates (77%) presented at least one tetracycline or oxytetracycline resistance determinant. The tetK genotype was present in 10 isolates while the tetL, tetM, and otrA genotypes were present in 3, 2, and 5 isolates, respectively. In addition, 2 isolates of the tetK plus tetM genotype, 1 of the tetK plus tetL genotype, and 1 of the tetK plus otrA genotype were found. All isolates were tetW, tetO and otrB negatives. On the other hand, 7 isolates (23%) showed a tetracycline-resistant and/or minocycline-resistant phenotype (MIC) but did not carry any of the tet or otr determinants investigated in this study. This research has shown that B. cereus isolates from honey samples contain a variety of tetracycline and oxytetracycline resistance genes, including the tetK and tetL determinants which encode for efflux proteins, and tetM and otrA, which encode for ribosomal protection proteins. These findings indicate that strains isolated from honeys could represent a reservoir for tetracycline resistance genes. To our knowledge, this is the first report of tetracycline-resistant and oxytetracycline-resistant B. cereus strains carrying the tetK determinant, and also the first report of oxytetracycline-resistant and tetracycline-resistant Bacillus species carrying the otrA determinant.

Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols.

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes.

[Optimization of the growth of Paenibacillus larvae in semi-selective media]. / Optimización del desarrollo de Paenibacillus larvae en medios semiselectivos.

The sensitivity of media MYPGP, MYPGP(NALPIA) A (6 microg/ml nalidixic acid and 10 microg/ml pipemidic acid) and MYPGP(NALPIA) B (9 microg/ml nalidixic acid and 20 microg/ml pipemidic acid) for the recovery of viable spores of Paenibacillus larvae from honey, was evaluated by using different incubation times and different spore concentrations. No significant differences between incubation times, spore concentration or culture media were found. In the case of the recovery of vegetative cells from PBS at different incubation times and different dilutions no significant differences were found between the incubation times or the dilutions tested, while significant differences were found in the three media when compared with one another, MYPGP(NALPIA)B providing the lowest recovery of vegetative cells. Considering these results, we propose the use of MYPGP(NALPIA)B to recover spores of P. larvae from honey, specially for honeys with heterogeneous populations of bacterial spores; when culturing vegetative cells, MYPGP or MYPGP(NALPIA)A must be used to obtain good growth.

Geraldton Wax as a New Host for Agrobacterium tumefaciens in Argentina.

During 2004, Geraldton wax plants (Chamaelaucium uncinatum cv. BM Violet) from commercial greenhouses in La Plata, Argentina showed gall-like structures on collars and roots similar to those reported by Carsten et al. (2). No pathogenic fungi were associated with lesions. Bacteria isolated from galls grown on medium 1A and D1M agar yielded colonies typical of Agrobacterium sp. (4). Four isolates were selected for further study. All isolates were aerobic, gram-negative rods and produced 3-ketolactose, but did not produce alkali from l-tartrate or accumulate poly-ß-hydroxybutyrate inclusions. The isolates were able to grow with 2% NaCl, at 35°C, and also on potato dextrose agar medium containing CaCO3 (4) but without acid clarification. Polymerase chain reaction analysis with primers VCF/VCR that amplified the expected 730-bp product of virC operon confirmed that all strains harboured a Ti plasmid (3,4). In addition, strains were screened for extrachromosomal DNA by the in-well lysis and electrophoresis procedure of Eckhardt with minor modifications as reported by Albiach and Lopez (1) and compared with strain Agrobacterium tumefaciens LBA 958, all Argentinean strains tested harbored a plasmid similar in size. Pathogenicity was verified on Geraldton wax and tobacco plants. Bacterial suspensions of each isolate (108 CFU/ml) were pricked into the stems. Control plants were pricked with sterile distilled water. Plants were maintained at 23 ± 3°C and symptoms were recorded after 45 days. Development of almost spherical, white-to-flesh-colored, rough, spongy and wart-like galls at the inoculation sites of the inoculated collars, stems, and trunks were registered. In Geraldton wax, as galls aged, they become dark brown to black, rough, and woody. Bacteria were reisolated from these galls fulfilling Koch's postulates. No lesions were observed on the controls. To our knowledge, this is the first report of A. tumefaciens on C. uncinatum in Argentina. References: (1) M. R. Albiach and M. M. López. Appl. Environ. Microbiol. 58:2683, 1992. (2) E. Carstens et al. Plant Dis. 83:783, 1999. (3) H. Sawada et al. Appl. Environ. Microbiol. 61:828, 1995. (4) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey.

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist/A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level.

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey / Inhibición del crecimiento de Ascosphaera apis mediante cepas de Bacillus y Paenibacillus aisladas de miel

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist / A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

La cría yesificada es una micosis invasiva ocasionada por el hongo heterotálico Ascosphaera apis que afecta exclusivamente a las larvas de las abejas. La enfermedad tiene difusión mundial y en la Argentina se halla diseminada en todas las áreas donde se realiza apicultura. Se estudió la potencialidad de 249 cepas de bacterias esporuladas aeróbicas aisladas de miel como agentes biocontroladores del hongo mediante un ensayo en disco central en condiciones de laboratorio. Se seleccionaron como mejores antagonistas 10 cepas bacterianas identificadas como Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), y Paenibacillus alvei (m321). Para probar la eficiencia de las cepas seleccionadas, se empleó la técnica de cultivo dual con 5 repeticiones de cada combinación antagonista bacteriano / cepa de A. apis, 5 para cada control y 4 medios de cultivo diferentes empleando 10 cepas del hongo de distintos orígenes geográficos. El análisis de la variancia y posterior comparación de medias LSD (Least Square Dfferences) mostró que los mejores antagonistas fueron B. subtilis (m329) y B. megaterium (m435) para el caso del medio YGPSA, mientras que para MYPGP las más eficientes fueron las cepas de B. circulans Fr 231 y m448b.

First Report of Species of Colletotrichum Causing Leaf Blotch of Liriodendron tulipifera in Argentina.

Since 1986, irregular, blotchy lesions of leaves and petioles of tulip tree (Liriodendron tulipifera L.) have been observed each summer in the city of La Plata, Argentina. Leaf spots are initially 2 to 3 mm in diameter and water soaked, and then they become brown or black with faint chlorotic margins that are more noticeable on the adaxial surface. Lesions may attain a diameter of 12 mm. Veins and petioles may also be affected and lesions may coalesce, especially at leaf margins. Surface-sterilized pieces of necrotic tissue collected from eight sites in the La Plata area were plated on potato dextrose agar and incubated at 27°C. Two Colletotrichum species were consistently isolated from the lesions. Single-spore cultures were obtained and identified as C. acutatum (1) and C. gloeosporioides (4). When cultured on V8 medium, mycelial growth of C. acutatum was tufted and pale gray, and the reverse side of colonies was buff to cream or pale gray to tan, but never dark. Hyaline conidia were buff to rosy buff en masse and elliptical or elongated with abruptly tapering ends measuring 8.04 to 13.58 × 2.68 to 3 µm. Micromorphology of conidia were compared with C. acutatum "Herb IMI 232176" (1). C. gloeosporioides cultures were dark gray; their reverse was dark mouse gray to fuscous black. The cirrhus was peach to orange and hyaline conidia were oblong with rounded ends measuring 8.6 to 13.40 × 2.68 to 4.02 µm. Pathogenicity was verified on greenhouse-grown tulip trees and strawberry cv. Pájaro (Fragaria × ananassa) by spray inoculation with conidial suspensions of C. acutatum (107/ml), C. gloeosporioides (106/ml), and a mixture of both species (equal parts of each suspension). Controls were sprayed with sterile distilled water. Irregular, blotchy lesions of tulip tree appeared 30 days after inoculation. Anthracnose symptoms (3) were observed on strawberries 7 days after inoculation. In all cases, lesions produced by the mixture were larger than those obtained with individual species. The inoculated fungi were reisolated from lesions of all species tested, fulfilling Koch's postulates. No lesions were observed on control plants. Voucher specimens were deposited in La Plata Spegazzini Herbarium, Argentina as C. acutatum (LPS 47187) and C. gloeosporioides (LPS 47188); cultures of both fungi were also deposited as LPSC 795 and LPSC 796, respectively. C. acutatum and C. gloeosporioides cause Liriodendron leaf blotch in Argentina. Glomerella cingulata was previously reported affecting L. tulipifera (2), but to our knowledge, this is the first report of tulip tree as a host of C. acutatum. References: (1) B. J. Dyko and J. E. M. Mordue. No. 630 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1979 (2) K. Ito and T. Kobayashi. Bull. For. Exp. Stn. Meguro 146:1, 1962. (3) C. J. Ramallo et al. Plant Dis. 84:706, 2000. (4) J. A. von Arx. Phytopathol. Z. 29:413, 1957.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey.

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist/A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.