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Bee pollen is the result of the agglutination of pollen grains collected from flowers and mixed with nectar and salivary secretions by honey bees. Bee pollen is a natural product exposed to environmental conditions and also provides a unique microhabitat for yeasts and bacterial communities. We analyzed 30 fresh bee pollen samples obtained from the main producing areas of Argentina to identify aerobic-spore-forming bacteria. We obtained 73 isolates belonging to 16 different species through isolation on selective and differential media, morphological and biochemical tests, and PCR and RFLP analysis of genes encoding 16S rRNA. Our data revealed that Bacillus cereus sensu stricto was the most predominant species (50%), followed by Bacillus megaterium (40%) and Bacillus subtilis (40%), respectively. In a minor proportion, Paenibacillus polymyxa (20%), Paenibacillus larvae (17%), Bacillus pumilus (13%), Bacillus licheniformis (13%), Bacillus amyloliquefaciens (10%), Lysinibacillus sphaericus (7%), Bacillus coagulans (7%), Rummelliibacillus stabekisii (7%), Bacillus thuringiensis (7%), Bacillus clausii (3%), Paenibacillus alvei (3%), Bacillus simplex (3%), and Paenibacillus amylolyticus (3%) were also found. Our results showed that Argentinean bee pollen could transmit honey bee diseases due to the presence of viable spores of P. larvae and also spores of toxicogenic B. cereus s.s. and B. megaterium strains., Facultad de Ciencias Agrarias y Forestales

This is the fifth and final issue of the 60th volume of the Journal of Apicultural Research (JAR) - an excellent opportunity to reflect on the highlights in honey bee research and revise the latest developments of our journal. In this extended editorial, we briefly review the importance of JAR in a historical context, followed by the latest developments in the field of honey bee research in terms of main contributors and topics. Then, we look into the latest developments of JAR regarding its impact. Finally, we share our view on the future topics in bee research and conclude by thanking all involved parties for the ongoing success of JAR.

Species of Bacillus and Brevibacillus associated with honey bees are interesting sources of bioactive compounds with potential uses beyond the field of apiculture. Most Bacillus species and related genera produce a broad range of antimicrobial compounds, with activity against bacteria and fungi that include peptides, lipopeptides, bacteriocins, and bacteriocin-like inhibitory substances. By using biological tools, we evaluated the antagonistic activity of 34 bacterial strains against Paenibacillus larvae and Ascosphaera apis, the causal agents of American Foulbrood and Chalkbrood diseases of honey bee larvae, respectively. Data reveal that the antagonistic response was strain-specific, species-specific, and also medium-dependent. By using molecular tools, we investigated the distribution of antimicrobial peptide genes in the antagonist strains. The presence of homologous sequences to nine genes encoding for the synthesis of the antimicrobial peptides bacillomycin L (bmyB), fengycin (fenD), bacilysin (bacA), subtilin (spaS), iturin A (ituD, lpa-14; ituC), and surfactin (sfp; srfAA) was assayed by PCR. The distribution and frequency of these genes within the bacterial antagonists were also variable and strain-dependent, being the most common surfactins (srfAA = 44% and lpa-14 = 38%), iturins (ituD = 47%), and bacilysin (bacA = 32%). Moreover, a positive correlation between presence of antimicrobial peptide genes and antagonism was found taking into account that 85% of the antagonists had at least one of the antimicrobial peptide genes. We also identified those antagonists active against different P. larvae genotypes. To our knowledge, this is the first study of the association between the presence of homologous sequences of antimicrobial peptide genes and antagonism against P. larvae and A. apis strains. Cepas de Bacillus and Brevibacillus como antagonistas potenciales de Paenibacillus larvae y Ascosphaera apis. Las especies de Bacillus y Brevibacillus asociadas con abejas melíferas son una fuente interesante de compuestos bioactivos con usos potenciales más allá del campo de la apicultura. La mayoría de las especies de Bacillus producen una amplia gama de compuestos antimicrobianos, con actividad contra bacterias y hongos que incluye péptidos, lipopéptidos, bacteriocinas y sustancias inhibidoras similares a bacteriocinas (BLIS). Con el objeto de buscar alternativas naturales para el control de loque americana y cría yesificada, se emplearon herramientas biológicas para evaluar la actividad antagónica de 34 cepas bacterianas contra cepas de Paenibacillus larvae y de Ascosphaera apis, agentes causales de estas enfermedades. Los resultados obtenidos mostraron que la respuesta antagónica fue medio-dependiente y cepa-dependiente. Se analizó por PCR la presencia de secuencias homólogas a 9 genes relacionados con la síntesis de péptidos antimicrobianos: bacilomicina L (bmyB), fengicina (fenD), bacilicina (bacA), subtilina (spaS), iturina A (ituD, lpa-14; ituC), y surfactina (sfp; srfAA). La distribución y frecuencia de estos genes en las cepas antagonistas también resultó variable y cepa-dependiente, siendo los más comunes surfactinas (srfAA = 44% y lpa-14 = 38%), iturina A (ituD = 47%) y bacilicina (bacA = 32%). Se identificaron antagonistas bacterianos frente a distintos genotipos de P. larvae y se encontró una correlación positiva entre la presencia de genes vinculados con la producción de péptidos antimicrobianos y la inhibición del desarrollo de P. larvae. Este trabajo constituye el primer estudio de asociaciones entre presencia de estos genes y el antagonismo frente a P. larvae y A. apis. Fil: Bartel, Laura Cecilia. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina Fil: Abrahamovich, Eliana. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mori, Consuelo. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: López, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina Fil: Alippi, Adriana Mónica. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina

This study aimed to evaluate the performance of Hicrome Bacillus™ agar for isolation and rapid identification of the aerobic spore-forming bacteria most frequently found in honey samples. A collection of 197 bacterial isolates of Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, and Rummeliibacillus belonging to different species that have been reported in honey were screened for their abilities to grow and for their colony colors and medium appearance in HiCrome Bacillus agar. Also, 21 strains from culture collections were used for comparison and quality controls. A flowchart utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were elaborated for quick presumptive identification. A procedure for direct isolation from honey samples was developed. In conclusion, HiCrome Bacillus agar in combination with simple microbiological tests was highly useful for rapid and reliable identification of most Bacillus, Brevibacillus, Lysinibacillus and Paenibacillus species commonly found in honey samples facilitating isolation from polymicrobial honey., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones en Fitopatología

This study aimed to assess the feasibility of using RFLP of PCR-amplified 16S rRNA gene (s) by using universal primers 27f/1492r and a combination of three restriction enzymes, AluI, CfoI, and TaqI, for a low-cost, rapid screen for a primarily differentiation of isolates of the complex of aerobic spore-forming bacteria commonly found in honey samples. The described method produced unique and distinguishable patterns to differentiate among 80 isolates belonging to 26 different species of Bacillus, Brevibacillus, Lysinibacillus, Rummeliibacillus, and Paenibacillus reported in honey and other apiarian sources., Centro de Investigación y Desarrollo en Fermentaciones Industriales

The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacilluswhen growing in HiCrome Bacillus agar. The species studied are common contaminants of honey. In support to the recent publication entitled “HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples” (2), a collection of 197 bacterial isolates belonging to 24 different species of aerobic spore-forming bacteria have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation. Two simple flowcharts utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were developed for quick presumptive identification., Centro de Investigaciones en Fitopatología

In 1995, fruiting tomato plants (Lycopersicon esculentum Mill. hybrid Tommy) from different commercial greenhouses near La Plata and near Chacabuco (Province of Buenos Aires) had symptoms similar those caused by Erwinia carotovora subsp. carotovora (1,4). Stems of the infected plants were rotted and produced adventitious roots. The cortex on the basal part of the stems turned black and sloughed off easily. The pith disintegrated and stems appeared hollow. Disease incidence of 2% was common, and nearly 10% of the plants in wetter areas of greenhouses were affected. Bacteria consistently isolated from diseased stems formed white-to-cream-colored colonies on yeast dextrose calcium carbonate agar (YDC). Bacteria from purified colonies were gram negative, oxidase negative, arginine dyhidrolase negative, catalase positive, methyl red positive, and facultatively anaerobic. Tests on four strains showed all fermented glucose, reduced nitrates to nitrites, and grew at a maximum temperature of 37 to 40°C. Strains did not hydrolyse starch nor utilize Tween 80. All strains were resistant to erythromycin in an antibiotic disk (15 μg) assay. Acid was produced from D(+)-glucose, D -mannitol, sucrose, D(+)-cellobiose, L(+)-rhamnose, L(+)-arabinose, D(+)-galactose, and D(+)-trehalose, but not from D-arabinose, D-sorbitol, and maltose. Bacteria utilized maleate and citrate but not propionate, benzoate, or malonate. The strains caused soft rot of pepper fruits and carrot slices within 24 h at 25°C. Pathogenicity was confirmed by needle stab inoculation at the primary leaf node on five plants each of 6-week-old greenhouse-grown tomato hybrids Presto and Parador. Inoculum was from 24-h-old cultures on YDC. Control plants were stab inoculated with needles dampened in sterile water. All plants were covered with polyethylene bags for 48 h at 25°C. Within 24 h after inoculation, watersoak and rot were detected; and during the next 48 h, plants wilted. Controls remained healthy. The bacterium was readily isolated from inoculated plants. Tests showed physiological characteristics identical to those of the bacteria used as inoculum. The pathogen was identified as Erwinia carotovora subsp. carotovora based on morphological, biochemical, and physiological characteristics and on pathogenicity. Reactions were identical to those of the type strain ATCC 15713 that had been included in all tests for comparison. Further identity was shown by polymerase chain reaction utilizing ERIC primers to generate DNA profiles (3). Profiles of the pathogen or the type strain were very similar to those from bacteria recovered from inoculated plants. This is the first known occurrence of a disease caused by Erwinia carotovora subsp. carotovora on greenhouse-grown tomato plants in Argentina, although it has been reported as causing soft rot of vegetables after harvest (2). References: (1) B. N. Dhanvantari and V. A. Dirks. Phytopathology 77:1457, 1987. (2) L. Halperin and L. S. Spaini. Rev. Arg. Agron. 6:261, 1939. (3) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (4) D. E. Speights et al. Phytopathology 57: 902, 1967.

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.

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-β-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.

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-β-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.

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 β–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-β-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.

In order to evaluate if the different stages of bee pollen production could lead to changes in the microbiota, counts of filamentous fungi and yeast, culturable heterotrophic mesophilic bacteria, aerobic spore-forming bacteria, sulfite-reducing clostridia, enterobacteria, total and thermotolerant coliforms, and the study of human pathogenic bacteria were performed. Also, the chemical characterization was carried out. We report the analysis of 36 bee pollen samples which were obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in the Southwest of Buenos Aires province, Argentina. In bee pollen samples, Escherichia coli, Salmonella sp., coagulase- positive Staphylococcus, and Clostridium perfringens were not detected. A total of 2.90 x 103 colony forming units of Bacillus cereus group were counted. Culturable heterotrophic mesophilic bacteria as well as yeasts showed the highest values and drying and freezing stages did not reduce either. Although bee pollen samples were manipulated following appropriate practices under good hygienic conditions they presented counts of filamentous fungi and yeasts higher than the Argentine Food Code tolerance while they did fit the European Codes requirements. Thus, this data offers scientific support to suggest a revision of our Code in order to establish an official method for carbohydrate content analysis and to allow higher limits for filamentous fungi and yeasts for bee pollen destined to human consumption., Centro de Investigaciones en Fitopatología, Comisión de Investigaciones Científicas de la provincia de Buenos Aires

We report here the 6,092,003-bp draft genome sequence of Bacillus thuringiensis strain m401, a tetracycline-resistant isolate recovered from honey. The isolate contained three plasmids of 8,307 bp, 9,934 bp, and 69,561 bp and a tetracycline resistance gene with high homology to tet45 in a contig of 236,180 bp.

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 NH4SO4 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. Fil: Minnaard, Jessica. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Alippi, Adriana Mónica. Universidad Nacional de la Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina

The aim of this study was to isolate, identify and analyze the diversity of the causative agents of crown galls and hairy roots from symptomatic plants of Vaccinium corymbossum by means of biological, biochemical and molecular tools. All the bacteria isolated from blueberries (n = 78) were found to be Agrobacterium since they grew on three differential media, provoked cell and/or root proliferation on Kalanchoe, and contained a 730 bp partial sequence that codes for virulence genes within the virC operon found on Ti and/or Ri plasmids. Isolates were highly variable considering the ERIC-PCR patterns as well as biochemical reactions and were all represented by 7 different restriction patterns of the 16SrDNA. While most of the isolates belonged to Agrobacterium bv. 1 (n = 33) or Agrobacterium bv. 2 (n = 31) only fourteen were Agrobacterium rubi. A representative isolate of each of these three groups was further identified by sequencing the approximately 400 bp 16SrDNA. We concluded that Vaccinium plants are particularly susceptible to Agrobacterium bv. 1, Agrobacterium bv. 2, and also to Agrobacterium rubi. To our knowledge this is the first survey of Agrobacterium affecting blueberries in Argentina., Centro de Investigaciones en Fitopatología

La enfermedad más grave de la etapa larval de las abejas (Apis mellifera L.) es la loque americana, causada por la bacteria esporulada Gram (+) Paenibacillus larvae. Es muy contagiosa y posee problemas únicos para su prevención y control debido a que las esporas bacterianas mantienen su capacidad infectiva durante tiempo prolongado y sobreviven bajo condiciones ambientales adversas, no existiendo brotes estacionales ya que se manifiesta en cualquier época del año con la condición que haya cría presente en la colmena. La enfermedad tiene difusión mundial y aparece en la lista de enfermedades de la OIE (Office International des Epizooties - Organización Mundial de la Salud Animal), que incluye enfermedades transmisibles que son consideradas de impacto socio-económico y/o de importancia para la salud pública entre países y que influyen negativamente en el comercio internacional de animales y productos de origen animal. En la Argentina se la detectó por primera vez en 1989 y actualmente está ampliamente diseminada en todas las áreas productoras de miel. Con el objeto de estudiar la diversidad de Paenibacillus larvae se aplicaron procedimientos de microbiología clásica y de biología molecular para caracterizar una colección de 455 cepas del patógeno. El cepario de trabajo se conformó con 106 aislamientos obtenidos de larvas de abejas con síntomas de la enfermedad, con 307 cepas aisladas de mieles contaminadas de distintos orígenes geográficos, 21 cepas provistas por otros laboratorios y 21 cepas de Colecciones Internacionales. Se identificaron 54 aislamientos de restos larvales y/o escamas con síntomas clínicos de la enfermedad y 252 aislamientos de mieles provenientes de distintas localidades de la Argentina. A partir de material de panales con síntomas clínicos remitidos por investigadores de otros países, se obtuvieron 52 aislamientos conformados por 3 aislamientos de Alemania, 5 de Francia, 5 de Italia, 9 de N. Zelanda, 6 de Polonia, 11 de Sudáfrica, 4 de Suecia, 1 de Túnez y 8 de Uruguay. Adicionalmente se aislaron 18 cepas de mieles de Italia, 17 de mieles de EE.UU., 7 de mieles de Francia, 5 de mieles de España, 3 de mieles de Canadá, 2 de mieles de Sudáfrica, 1 de miel de Brasil, 1 de miel de Túnez y 1 de miel de Panamá conformando un total de 55 cepas obtenidas de mieles de otros países productores. Fueron recibidas como cultivo 42 cepas: 2 de Argentina, 2 de Bélgica, 2 de Chile, 2 de Japón, 3 de Inglaterra, 4 de República Checa, 10 de EE.UU. y 13 de origen desconocido. Si bien la colección estuvo integrada por un alto número de cepas provenientes de Argentina (n= 308) el trabajo se realizó también sobre un número considerable de cepas (n=147) de otros orígenes que resultó adecuado para los estudios de diversidad. Adicionalmente, con el fin de incluir controles, se emplearon 5 cepas de Colecciones Internacionales que en la actualidad se clasifican como Paenibacillus larvae pero que pertenecían a la anterior subespecie Paenibacillus larvae subsp. pulvifaciens (1 de EE.UU., 1 de Canadá, 1 de Francia y 2 de origen desconocido). Otras cepas de este grupo proveniente de EE.UU. fueron gentilmente cedidas por el Dr. D. P. Stahly para los estudios con el bacteriófago PPL1c del capítulo III. Se constituyó un segundo cepario con una colección de 282 aislamientos bacterianos de otras especies esporuladas aerobias conformado por 129 aislamientos de Bacillus cereus, 62 de Paenibacillus alvei, 52 de Bacillus megaterium, 8 de Bacillus mycoides, 6 de Bacillus subtilis, 6 de Lysinibacillus sphaericus, 5 de Bacillus thuringiensis, 5 de Brevibacillus laterosporus, 5 de Bacillus circulans, 2 de Bacillus licheniformis, 1 de Bacillus polymyxa y 1 de Bacillus pumilus. Adicionalmente, se emplearon 41 cepas recibidas de Colecciones Internacionales y pertenecientes a distintas especies de los géneros Paenibacillus, Bacillus, Brevibacillus, Lysinibacillus y Virgibacillus. En el Capítulo II se caracterizaron las 455 cepas de Paenibacillus larvae mediante técnicas microbiológicas. El agregado de ácido nalidíxico al medio de cultivo a una concentración final de 9 µg/ml resultó adecuado para la obtención de cultivos puros de Paenibacillus larvae evitando el sobredesarrollo del saprobio Paenibacillus alvei comúnmente presente en las larvas de abejas. Con respecto a los aislamientos provenientes de muestras de mieles, el agregado de la combinación de antibióticos ácido nalidíxico y ácido pipemídico al medio MYPGP permitió obtener colonias de Paenibacillus larvae y mayormente inhibió el desarrollo de otras especies esporuladas presentes en este tipo de muestras, ya que más del 80% de las muestras analizadas contenía más de una especie bacteriana resistente al tratamiento de shock térmico de 80 °C. Independientemente del origen geográfico y/o floral de las mieles analizadas aquí, las especies esporuladas aerobias encontradas con mayor frecuencia han sido Paenibacillus larvae, Paenibacillus alvei, Bacillus cereus, Bacillus megaterium y Bacillus pumilus. Paralelamente y, en menor proporción, se hallaron cepas de Brevibacillus laterosporus, Bacillus mycoides, Bacillus thuringiensis, Bacillus subtilis, Bacillus circulans, Paenibacillus polymyxa, Lysinibacillus sphaericus y Bacillus licheniformis. Mientras que Paenibacillus larvae y Paenibacillus alvei están asociados con enfermedades de las abejas, Bacillus cereus y Bacillus megaterium son especies ubicuas frecuentes en todo tipo de suelos, sus esporas sobreviven la distribución en polvos y aerosoles siendo vehiculizadas desde estos lugares a otros hábitats como las superficies florales. Paralelamente, Bacillus megaterium junto con Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus cereus, Bacillus circulans y Paenibacillus alvei son las especies predominantes en los tractos digestivos de larvas, abejas adultas y heces de las mismas. También en este capítulo, se determinó que la configuración superficial de las esporas bacterianas vistas al MEB resultó coincidente con lo descripto por otros autores para cada una de las especies analizadas. En el capítulo III se demostró que el bacteriófago PPL1c resultó una herramienta útil para la identificación y diferenciación rápida de cepas de Paenibacillus larvae, como complemento de otras características fenotípicas. El fago mostró ser altamente específico a nivel de especie encontrándose un 98 % de cepas de Paenibacillus larvae susceptibles al mismo mientras que el resto de las especies de Paenibacillus, Bacillus, Brevibacillus, Lysinibacillus y Virgibacillus resultaron resistentes. En el capítulo IV se estableció que la técnica de rep-PCR resultó adecuada para determinar la variabilidad de las poblaciones de Paenibacillus larvae dónde se halló una alta homogeneidad genética. El empleo de los cebadores BOX y REP reveló la existencia de 4 perfiles de fingerprints a los cuales denominamos A, B, C y D respectivamente, mientras que las cepas de colección pertenecientes al ex. grupo pulvifaciens mostraron el perfil distintivo E. Cuando se emplearon los cebadores ERIC, con excepción de algunas cepas provenientes de Colecciones Internacionales, todos los aislamientos obtenidos de larvas y mieles presentaron un perfil ERIC-I coincidente con lo descripto también por otros investigadores y sólo se halló el perfil ERIC-II en cepas pigmentadas enviadas de la Colección del SAG. Mientras que el perfil ERIC-I es el de mayor distribución a nivel mundial, el perfil ERIC-II ha sido citado en Alemania, Finlandia, Suecia y Austria y, muy recientemente, en muestras de Canadá y Nueva Zelanda. Las cepas pertenecientes al perfil ERIC I estudiadas en esta tesis a su vez se subdividían en alguno de los 4 perfiles BOX (A, B, C o D) y presentaban colonias "típicas" de Paenibacillus larvae no pigmentadas, de color entre blanquecino y grisáceo y formas ligeramente rugosas en agar MYPGP y en agar Columbia suplementado con sangre ovina (Capítulo II). Por otra parte las cepas de colección del grupo ex pulvifaciens, analizadas aquí (n=5) presentaron un perfil ERIC–IV / BOX-E. Si bien en este trabajo no se encontró ningún representante del genotipo ERIC-II, a excepción de los cultivos enviados por el SAG, el mismo pudo haber pasado inadvertido en el análisis de mieles debido a la alta contaminación de las mismas con otras especies esporuladas Gram (+) que hizo imprescindible someter a las muestras a un shock térmico previo al aislamiento en medios semi-selectivos. En el capítulo IV también se concluyó que el gen 16S rRNA es polimórfico entre las especies esporuladas aerobicas comúnmente presentes en la miel, no obstante, no se detectaron polimorfismos a nivel intra-especie en las cepas de Paenibacillus larvae de distintas regiones geográficas cuando se emplearon las endonucleasas (Alu I, Msp I, Hae III, CfoI, Rsa I y Taq I). En cuanto a las comparaciones entre perfiles de rep-PCR y RFLP se observó que los 4 patrones de BOX-PCR observados (A, B, C y D) y los fingerprints ERIC-I y ERIC-II se correlacionaron con el perfil A obtenido por HaeIII, mientras que las cepas de colección que mostraron un perfil BOX-E y un perfil ERIC-IV se correlacionaron con el perfil B obtenido con la endonucleasa HaeIII. Lo mismo ocurrió con la enzima HinfI dónde las cepas pertenecientes a los perfiles ERIC-III y BOX-E mostraron un patrón B y las cepas con perfiles ERIC-I o ERIC-II y BOX-A, BOX-B, BOX-Cy BOX-D no presentaron un sitio de corte con esta endonucleasa. En el capítulo V se seleccionó un subconjunto de aislamientos de Paenibacillus larvae de distintos orígenes geográficos y perfiles de fingerprints para determinar la sensibilidad/ resistencia a tetraciclina y oxitetraciclina mediante las técnicas de CIM y de difusión en agar. La distribución de la sensibilidad por CIMs de la población bacteriana analizada varió entre 0,062 y 128 µg/ml de Tc sugiriendo una distribución bimodal con dos sub-poblaciones que claramente difirieron en su sensibilidad, con 76% de cepas S (CIM entre 0,062 y 2 µg/ml) y 24 % de cepas dentro del rango de la zona I y R. Las cepas I provenían en su mayoría de Argentina y las S cuyos valores de CIM resultaron los más bajos, de países europeos, lo cual es esperable dado que en casi toda la UE está prohibido el uso de antibióticos en apicultura, mientras que en países como EE.UU., Canadá y Argentina, entre otros, es usual el empleo de OTC para el control de loque americana en las colmenas afectadas. Al comparar las CIM obtenidas por E-test con las obtenidas por dilución en agar, en el caso de agar Iso-Sensi Test hubo concordancia de categoría del 100 %, encontrándose la misma proporción de cepas susceptibles y resistentes que por la técnica de dilución en agar; pero al emplear MYPGP hubo concordancia de categoría del 81,81 %. Se concluye entonces que la combinación de las tiras de E-Test con el agar ISO-Sensi Test representa una alternativa válida para determinar las CIM de tetraciclina en cepas de Paenibacillus larvae. Es importante destacar que todas las cepas de Paenibacillus larvae ensayadas mostraron un desarrollo confluente en Iso-Sensi test, el cual podría usarse también como medio alternativo para el desarrollo de las células vegetativas de este patógeno. Se obtuvieron las secuencias completas de los plásmidos pPL373, pPL374 y pPL395. Dichas secuencias se depositaron en el GenBank con los números de acceso KF 433938 para pPL373, KF 536616 para pPL374 y KF440690 para pPL395, provenientes de las cepas PL373, PL374 y PL395, respectivamente que presentaban resistencia a Tc y OTC. Hemos demostrado experimentalmente la transferencia del plásmido pPL374 en la cepa de Bacillus subtilis m351 y del plásmido pPL373 en la cepa de Bacillus subtilis GSY1104 mediante conjugaciones en medio líquido. Al examinar ambas cepas dadoras de Paenibacillus larvae PL373 y PL374 mediante la técnica de lisis in situ se observaron dos plásmidos de aproximadamente 5.000 bp y 8.000 bp, pero sólo el plásmido más pequeño era el que contenía el gen de resistencia a Tc. Luego de efectuar la secuenciación completa de los plásmidos más pequeños obtenidos de las cepas pPL373 y pPL374, respectivamente confirmamos que el gen de resistencia es el tetL y no el tetK como creímos en un principio y que ambos replican por el mecanismo de círculo rodante encontrado en pequeños plásmidos movilizables de alto número de copias presentes en bacterias Gram (+). Todos los experimentos de movilización por conjugación empleando la cepa PL395 que contiene un único plásmido de 5.000 bp denominado pPL395 resultaron infructuosos, no obstante el ADN plasmídico obtenido de la cepa PL395 si pudo transferirse por electroporación a una cepa de Paenibacillus larvae TcS. Los plásmidos pPL373 y pPL374 también pudieron transferirse por electroporación a la misma cepa de Paenibacillus larvae TcS. En todos los casos los plásmidos se mantuvieron en forma estable en sus respectivos aceptores. Los plásmidos más grandes contenidos en las cepas PL373 y PL374, podrían codificar para funciones conjugativas y, al coexistir en la misma cepa bacteriana aportarían a los plásmidos movilizables pPL373 y pPL374 las funciones conjugativas necesarias para su movilización por conjugación; por lo que se justifica estudiarlos en profundidad y secuenciarlos para clarificar los mecanismos de transmisión plasmídica de Paenibacillus larvae en la naturaleza. La presencia de plásmidos con secuencias muy similares aislados de bacterias Gram (+) provenientes de distintas zonas geográficas y de distintos nichos ecológicos (suelo, hábitats marinos, alimentos) de los géneros Bacillus, Paenibacillus, Sporosarcina, Lactobacillus y Bhargavaea nos sugieren que los genes mob presentes en dichos plásmidos pueden estar involucrados en una exitosa THG. El uso extensivo de Tc y OTC para el control de loque americana en algunos países de América pudo haber contribuido al incremento del número de cepas resistentes de Paenibacillus larvae, favoreciendo la transferencia de estos plásmidos entre cepas del mismo patógeno o desde o hacia otras especies de bacterias Gram (+) de los géneros Lactobacillus, Bacillus y Paenibacillus que pertenecen a la microbiota de la colmena (miel, tractos digestivos de abejas adultas y larvas, superficies florales y polen). El uso prolongado de tetraciclina en las colmenas afectadas por loque americana y otras enfermedades bacterianas estaría favoreciendo la ocurrencia de eventos de transferencia genética horizontal como se demostró en cepas del patógeno provenientes de EE.UU., Facultad de Ciencias Agrarias y Forestales

SUMMARY Paenibacillus larvae larvae, the causative agent of American foulbrood disease of honey bee larvae occurs throughout the world and is found in all beekeeping areas of Argentina. Microbiological analysis of 394 honey samples obtained from bee hives from Buenos Aires province (Argentina) from three years of sampling (1999‐2001) yielded 219 positive cases (55.6%). The incidence of P. l. larvae infected honey samples for 1999 was 68.1% (n = 160), for 2000 47.1% (n = 102 ), and 46.2% for 2001 (n = 132). The mean values of spore contamination for the three-year study showed a continuous reduction, probably due to good practices of disease management by beekeepers by breeding bees for hygienic behaviour and reduction of antibiotic treatments for control of AFB. P. l. larvae populations isolated from honey were characterized on the basis of DNA fingerprints using the repetitive-sequence-based polymerase chain reaction technique (rep-PCR) with BOX- and REP- sequencespecific primers. Four distinctive patterns, named A, B, C, and D, were distinguishable among the isolates. Genotype D was not observed in previous studies; this finding could be correlated with a new introduction of the disease in Argentina since 1997 when only three genotypes (A, B, and C) were confirmed. The rep-PCR fingerprint patterns obtained were compared with the patterns generated by a world-wide collection of P. l. larvae strains. The same 4 genotypes patterns were found within a collection of strains from 18 different countries of the world. It is important to point out that pattern C was only found in Argentina and in one sample from Uruguay located in the border line, suggesting that genotype C could have been derived from genotype A and disseminated to Uruguay from Argentina. These findings support the hypothesis that American foulbrood disease is exposed to a limited selective pressure from climatic and environmental sources.

La loque americaine (AFB), causee par la bacterie sporulante Paenibacillus larvae subsp. larvae, est la maladie la plus grave du couvain de l'Abeille domestique (Apis mellifera L.). Dans les regions fortement touchees, au lieu de bruler le materiel infecte, on utilise des antibiotiques, mais avec le risque de contamination du miel. Il a ete montre par des tests in vitro que les huiles essentielles (h.e.) extraites de plantes aromatiques possedaient une activite antimicrobienne (Alippi et al., 1999b ; Calderone et al., 1994 ; Deans et al., 1992) et certaines d'entre elles ont ete utilisees avec succes pour traiter le couvain plâtre (Colin et al., 1989) et la varroose (Imdorf et al., 1994). Le but de cette etude etait de determiner la toxicite aigue orale des h.e. pures et en melange sur des abeilles adultes par la dose letale 50 (DL 50 ) et d'evaluer la reaction des colonies infectees par l'AFB a differents traitements bases sur les h.e. en mesurant la duree de la prise de nourriture. Les tests ont porte sur les h.e. de (1) sarriette (Satureia hortensis), (2) thym (Thymus vulgaris), (3) citronnelle (cymbopogon citratus) et (4) marjolaine (Origanum vulgare) et sur les melanges suivants : (A) citronnelle + thym, (B) citronnelle + thym + origan + basilic (Ocimum basilicum) et (C) citronnelle + thym + basilic. Les principaux composes de chaque h.e. ont ete determines par chromatographie en phase gazeuse sur colonne de silice methylee et sur une colonne enduite de Carbowax 20 M et par detecteurs a ionisation de flamme (Tab. I). Pour evaluer la toxicite aigue orale, des doses d'h.e. pures et en melange (μg d'h.e. ou de melange par abeille) ont ete choisies conformement aux tests biologiques usuels pour determiner les DL 50 (Cox, 1977). Le dimethoate a ete utilise comme reference toxique et le saccharose comme temoin non toxique. On a fait quatre essais de plein champ, deux avec les h.e. pures, le premier a titre curatif et la second a titre preventif, et deux avec les melanges a titre preventif. Les traitements ont ete administres sous forme de sucre candi dans les trois premiers essais et sous forme de sirop de sucre dans le dernier. On a fait cinq repetitions par traitement et utilise deux temoins: pas de traitement (saccharose) et du tartrate de tylosine comme temoin d'efficacite, a l'exception du premier essai en plein champ qui n'a eu que le temoin non traite. Les resultats montrent que le melange A etait legerement toxique (DL 50 a 24 h de 15,94 μg de melange par abeille et DL 50 a 48 h de 18,75 μg) et que le melange C n'etait pas toxique (DL 50 a 24 h de 122 μg de melange par abeille et DL 50 a 48 h de 356,8 μg). Il n'a pas ete possible de calculer les DL 50 dans le cas du melange B et de toutes le h.e. pures car les donnees de mortalite ont fourni des courbes negatives. Les resultats des essais en plein champ montrent que les h.e. pures ou en melange ne sont pas efficaces pour eliminer les signes cliniques de l'AFB aux doses, formulations ou modes d'administration testes, tandis que le traitement a la tylosine s'est montre tres efficace.

Pseudomonas species causing pith necrosis symptoms on tomato and pepper collected in different areas of Argentina were identified as Pseudomonas corrugata, P. viridiflava and Pseudomonas spp. Their diversity was analysed and compared with reference strains on the basis of their phenotypic characteristics, copper and antibiotic sensitivity tests, serology, pathogenicity, DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis of a 16S rRNA gene fragment. All P. corrugata strains tested were copper-resistant while P. viridiflava strains were more variable. Numerical analysis of phenotypic data showed that all P. corrugata strains formed a single phenon that clustered at a level of about 93%, while all the P. viridiflava strains clustered in a separated phenon at a level of 94%. Genomic analysis by repetitive (rep)-PCR and 16S rRNA-RFLP fingerprinting and serological analysis showed that the two species contained considerable genetic diversity. Inoculations of tomato and pepper plants with strains from both hosts caused similar pith necrosis symptoms. Strains of both P. corrugata and P. viridiflava were grouped according to their geographical origin and not according to the original host. This is the first report of Pseudomonas viridiflava causing pith necrosis on pepper., Facultad de Ciencias Agrarias y Forestales, Instituto de Biotecnologia y Biologia Molecular

SUMMARYAscosphaera apis, the causative agent of chalkbrood disease in honey bee larvae occurs throughout the world and is found in many beekeeping areas of Argentina. Microbiological analysis of 394 honey samples from Buenos Aires province from three years of sampling (1999–2001) yielded 51 positive cases (13% incidence). Eighty-four isolates of A. apis from Argentina and Chile isolated from diseased larvae and/or honey samples were characterized on the basis of DNA fingerprints using the repetitive-sequence-based polymerase chain reaction technique (rep-PCR) with BOX, REP, and ERIC sequence-specific primers. Computer-assisted analysis of combined fingerprints distinguished six groups of patterns, designated A, B, C, D, E and F. Pattern C was the most prevalent and suggests a limited diversity in the populations of A. apis from Argentina and Chile. The results demonstrated the usefulness of rep-PCR genomic fingerprinting to characterize populations of A. apis. In addition, a simple and efficient protocol ...

Se estudió la diversidad de una colección de cepas de Agrobacterium rubi aisladas de arándanos provenientes de distintas regiones de la República Argentina estableciendo su grado de heterogeneidad mediante pruebas microbiológicas clásicas y técnicas de biología molecular. Los resultados obtenidos en las pruebas bioquímicas y fisiológicas, así como mediante rep-PCR y RFLP del gen 23S ADNr, demostraron una alta variabilidad intraespecífica, tanto fenotípica como genotípica. The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplifi ed 23S rDNA showed high phenotypic and genotypic intraspecific variation. Fil: Abrahamovich, Eliana. Universidad Nacional de la Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro En Investigación de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: López, Ana Claudia. Universidad Nacional de la Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro En Investigación de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alippi, Adriana. Universidad Nacional de la Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro En Investigación de Fitopatología; Argentina

La bacteria esporulada gram positiva Paenibacillus larvae es el agente causal de la loque americana de las abejas. El objetivo de este trabajo fue comparar las CIM de tetraciclina obtenidas por el método Etest y las obtenidas por el método de dilución en agar frente a 22 cepas del patógeno, empleando dos medios de cultivo: Iso-Sensitest y MYPGP. Se encontró una concordancia de categoría entre dichos métodos del 100 % usando agar Iso-Sensitest, mientras que con MYPGP la concordancia de categoría fue del 86,36 % (con 3 errores menores). Los resultados de este estudio sugieren que la combinación de las tiras de Etest con agar Iso-Sensitest sería una alternativa rápida y confi able para determinar las CIM de tetraciclina en P. larvae; sin embargo, estos resultados deberán confi rmarse en futuros estudios que contemplen un mayor número de aislamientos., American foulbrood (AFB) is a bacterial disease caused by the spore-forming, grampositive bacterium Paenibacillus larvae, which affects honeybee broods worldwide. The aim of this work was to compare the Epsilometer test (Etest) to the agar dilution method for testing a collection of 22 P. larvae strains to tetracycline by using MYPGP and IsoSensitest agars. Results showed that a categorical agreement of 100% was found when using Iso-Sensitest, while a categorical agreement of 86.36% was found (with 3 minor errors) when MYPGP was tested. In conclusion, the Etest could be a rapid and reliable method for testing MIC values of tetracycline in P. larvae only when used in combination with Iso-Sensitest agar. Nevertheless, these results should be confi rmed with future studies involving a larger number of isolates., Facultad de Ciencias Agrarias y Forestales

SUMMARYThe efficiency of tylosin and erythromycin was evaluated and compared to oxytetracycline hydrochloride and Terramycin for the control of American foulbrood (AFB) (Paenibacillus larvae larvae) in infected honey bee colonies. Oral acute toxicity tests for adult bees of all antibiotics were determined by calculations of LD50 values at 24 h, 48 h and 72 h, using dimethoate as reference compound. All the antibiotics tested showed no toxicity for adult bees, with LD50 values greater than 100 μg per bee. The times of degradation in honey were analysed for tylosin and erythromycin through HPLC and bioassays, respectively. Erythromycin, as ethyl succinate, was not effective for the control of AFB at any of the tested doses and its degradation time in honey was about 35–40 days. On the other hand, tylosin, as tartrate, was highly effective for the control of AFB, and also suppressed disease signs for one year after treatment. Its degradation time in honey was about 60 days. AFB was effectively controlled by ...

SUMMARYPaenibacillus larvae subsp. larvae, the causative agent of American foulbrood, is an economically important pathogen in Argentina. Using the polymerase chain reaction (PCR) with primers that...

The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa prevalence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45±5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4–5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroa jacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface., Facultad de Ciencias Agrarias y Forestales

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 β-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.

During the summers of 2007 and 2008 fruiting tomato plants (Solanum lycopersicum cv. Orco) from commercial greenhouses near La Plata, Argentina (35 ºS 57 ºW) showed abundant adventitious root production, apical chlorosis of leaves and a brown discoloration of the stem pith (Fig. 1). These symptoms were similar to those reported by López et al. (1994) and Catara et al. (2002) on tomatoes affected by Pseudomonas corrugata or Pseudomonas mediterranea. Bacteria consistently isolated from stem lesions formed cream-coloured, glistening, convex colonies on sucrose peptone agar (SPA) and were non-fluorescent on King’s medium B (KMB). Four isolates were selected for further study. All were aerobic, Gram-negative rods with PHB inclusions. In LOPAT tests, all induced a hypersensitive response in tobacco plants, were oxidase positive, did not cause soft rot of potato tubers, and were negative for levan and arginine dihydrolase. Colonies developed at 28ºC and 37ºC but not at 41ºC. Additional characterisation was achieved by API 20 NE tests strips (Biomerieux®, Argentina). Reference strains 536.7 (Spain), 592.4 (Spain) and CFBP 10906 (France) of P. mediterranea and strain NCPPB 2445 of P. corrugata were included in all tests for comparison. Further identity was confirmed by PCR, using species-specific primers PC5/1-PC5/2 for P. mediterranea and primers PC1/1-PC1/2 for P. corrugata (Catara et al., 2002). All the strains were identified by the amplification of a 600 bp DNA fragment characteristic of Pseudomonas mediterranea (Fig. 2; Catara et al., 2002). The isolates of P. mediterranea were also differentiated from those of P. corrugata by PCR/RFLP analysis of 16S rDNA gene by using endonuclease AluI (Fig. 3). Pathogenicity was verified on four-week-old tomato plants (cv. Presto) by injecting bacterial suspensions at 107 cfu/ml or sterile distilled water for controls, after which the plants were kept for 72 h in a humid chamber before incubation at 25°C. After 45 days inoculated plants showed necrotic pith symptoms similar to those observed on field-grown plants, whereas no lesions were observed on controls. Pith necrosis caused by P. corrugata and P. viridiflava has been previously reported in Argentina (Alippi et al., 1993 Alippi et al, 2003). This is the first report of a disease caused by P. mediterranea on greenhouse-grown tomatoes in Argentina and South America.

Se estudió la infección de Xanthomonas campestris pv vesicatoria en hojas de tomate por medio de microscopía óptica y electrónica de barrido. Se determinó que la bacteria penetra en el hospedante por los estomas y se localiza y multiplica en las cámaras subestomáticas que sirven como sitios de supervivencia. Adicionalmente, también penetraría por las bases de los tricomas deteriorados. Luego, las bacterias colonizan los espacios intercelulares del parénquima esponjoso y, al alcanzar las cámaras subestomáticas (no invadidas previamente), se multiplican abundantemente y son expulsadas, aglutinadas en un sustancia de naturaleza mucosa, dando origen a nuevas fuentes de inóculo. Tras la inoculación, se notó una mayor concentración de células bacterianas en la cara inferior de las hojas. Los estomas y las cámaras subestomáticas fueron el nicho ecológico preferido por el patógeno para la posterior colonización del hospedante., Xanthomonas campestris pv vesicatoria infection was followed by light and scanning electron microscopy. Bacteria entered leaf tissue through stomata and multiplied in substomatal chambers which are additional survival sites. The pathogen could also enter through the open base of trichomes. X c pv vesicatoria colonized the intercellular spaces of spongy parenchyma and could reach the uninvaded substomatal chambers and, after extensive multiplication, masses of bacterial cells, agglutinated in a mucous substance, extruded through stomata being the cause of secondary sources of inoculum. Abaxial leaf surfaces contained more bacteria than adaxial tomato leaf surfaces. Stomata and substomatal chambers were the preferred ecological niches of X c pv vesicatoria before tissue colonization., Facultad de Ciencias Agrarias y Forestales

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.

Several laboratory techniques for detecting significant bacteria of diseased honey bees (Apis mellifera L.) were compared. Bacillus larvae, Bacillus alvei and Bacillus laterosporus spores were easi...

American Foulbrood (AFB) of honeybees (Apis melliferaL.), caused by the Gram-positive bacterium Paenibacillus larvae is one of the most serious diseases affecting the larval and pupal stages of honeybees (A. mellifera L.). The aim of the present work was to asses the response of 23 strains of P. larvae from diverse geographical origins to tilmicosin, a macrolide antibiotic developed for exclusive use in veterinary medicine, by means of the minimal inhibitory concentration (MIC) and the agar diffusion test (ADT). All the strains tested were highly susceptible to tilmicosin with MIC values ranging between 0.0625 and 0.5 m gm l 1 , and with MIC50 and MIC90 values of 0.250 m gm l 1 . The ADT tests results for 23 P. larvae strains tested showed that all were susceptible to tilmicosin with inhibition zones around 15 mg tilmicosin disks ranging between 21 and 50 mm in diameter. Oral acute toxicity of tilmicosin was evaluated and the LD50 values obtained demonstrated that it was virtually non-toxic for adult bees and also resulted non-toxic for larvae when compared with the normal brood mortality. Dosage of 1000 mg a.i. of tilmicosin applied in a 55 g candy resulted in a total suppression of AFB clinical signs in honeybee colonies 60 days after initial treatment. To our knowledge, this is the first report of the effectiveness of tilmicosin against P. larvae both in vitro and in vivo. # 2008 Elsevier B.V. All rights reserved.

Of 433 honey samples collected in Argentina, 114 (27%) yielded Bacillus cereus isolates (27%) and 60 (14%) yielded other species of Bacillus. The Argentinian B. cereus isolates were compared with isolates recovered from honey from other countries (n=16) and with strain ATCC 11718. The 133 B. cereus isolates and the ATCC strain were characterized using rep-PCR fingerprinting with primers BOX, REP and ERIC, restriction fragment length polymorphism analysis of a 16S rRNA gene fragment (16S rRNA PCR/RFLP), and morphological and biochemical tests. There was a high degree of diversity, both phenotypic and genotypic among the isolates of B. cereus. Our results demonstrate the usefulness of rep-PCR genomic fingerprinting for characterizing populations of B. cereus. According to our knowledge, this is the first report of phenotypic and genotypic characterization of B. cereus isolates from honey.

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., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones de Fitopatología

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).

The minimal inhibitory concentrations (MICs) of tylosin were determined to 67 strains of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood (AFB) disease, from different geographical origins. MIC values obtained ranged from 0.0078 to 0.5 microg/ml. These very low values imply that no resistance to tylosin was found in any isolate of the Foulbrood pathogen. The measurement of diseased larvae with AFB-clinical symptoms in three different field studies demonstrated that tylosin treatment could be effective in vivo. No negative effects in colonies were noted at any dosage rates or forms of application. These studies demonstrate that tylosin, as tartrate, can be used to treat AFB in honeybee colonies., Facultad de Ingeniería

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.

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones de Fitopatología, Instituto de Biotecnologia y Biologia Molecular, Facultad de Ciencias Exactas

In June 1998, during a cool, humid period, typical bacterial spot symptoms were observed on basil plantlets (Ocimun basilicum L. ‘Royal Louis’ and ‘Zaes’) in a commercial greenhouse in La Plata, Argentina. Affected plants had dark brown to black lesions on cotyledons. Spots on leaves were first water soaked, then became necrotic and progressed inward from the margins. Disease incidence approached 30%. Symptoms were similar to those reported by Little et al. (2) on basil affected by Pseudomonas viridiflava. No pathogenic fungi or viruses were associated with symptomatic plants. Bacterial streaming was observed from lesion margins. Bacteria consistently isolated from leaf lesions formed cream-colored, glistening, convex colonies on sucrose peptone agar and a green fluorescent pigment on King's medium B. Bacterial growth produced a distinctive olive green pigment on glycerol agar medium and a pink pigment on T-5 medium (1). Four isolates selected for further study were aerobic, Gram-negative, non-spore-forming rods. In LOPAT (levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity) tests, all induced a hypersensitive response in tobacco plants, caused soft rot of potato tubers, and were negative for levan, oxidase, and arginine dihydrolase. In addition, strains rotted onion slices and produced a reddish sunken lesion on bean pods. Acid was produced aerobically from D-glucose, mannitol, mesoinositol and sorbitol, but not from D-arabinose, L-rhamnose, melibiose, amygdalin, or sucrose. Bacteria used D-tartrate, pyruvate, and citrate, but not benzoate. The strains did not hydrolyze starch, exhibited an oxidative metabolism of glucose, and did not reduce nitrates to nitrites or accumulate poly-β-hydroxybutyrate inclusions. Negative reactions were obtained with indole, ornithine, and D-tryptophan. Isolates hydrolyzed gelatine, used Tween 80, were positive for catalase, and were unable to grow in the presence of 5% NaCl. Colonies developed at 4°C but not 37°C. Reactions were identical to those of reference strains ICMP 5776 and 12363, which were included in all tests for comparison. Pathogenicity was verified on 35-day-old basil plants by both spraying and infiltration inoculations with bacterial suspensions (108 and 105 cells per ml, respectively). Carborundum was included in the inoculum used for a set of plants inoculated by spraying. Controls were injected or sprayed (with and without Carborundum) with sterile, distilled water. In addition, bean (Phaseolus vulgaris cv. Nag12 INTA) and lettuce (Lactuca sativa cv. criolla), both reported as host plants, were inoculated by spraying with bacterial suspensions of 107 cells per ml plus Carborundum. After 48 h in a humid chamber, inoculated plants and controls were maintained at 23 ± 3°C. Symptoms on basil plants inoculated by injection or spraying with Carborundum were identical to those observed on basil in the field. Symptoms on bean and lettuce were similar to those described for P. viridiflava. The bacterium was reisolated from lesions of all species tested, fulfilling Koch's postulates. No lesions were observed on controls or on plants sprayed without Carborundum, suggesting that bacteria gain entry through wounds. The microorganism was identified by physiological tests and polymerase chain reaction as P. viridiflava. This is the first report of bacterial leaf spot of basil in Argentina. References: (1) R. Gitaitis et al. Plant Dis. 81:897, 1997. (2) E. L. Little et al. Plant Dis. 78:831, 1994.