Inoculant Maturity Influences Survival of Rhizobia on Seed

Survival of Rhizobium trifolii on seeds of arrowleaf clover (Trifolium versiculosum Savi) and subclover (Trifolium subterraneum L.) was affected by the maturity of peat-, vermiculite-, and charcoal-based inoculants. Ten times more rhizobia survived on seed 4 days after inoculation when inoculants were stored (cured) before being utilized as compared with uncured inoculants. Increasing the curing time of inoculants beyond 4 weeks had little effect on increasing survival of seed-applied rhizobia.     

Identification of sources of microbial pathogens on cantaloupe rinds from pre-harvest to post-harvest operations

Foodborne illness outbreaks involving cantaloupes have increased dramatically in the past 15 years in the United States and other countries. The need for the identification of the microbial sources that contaminate cantaloupe rinds has been raised by various investigators. This study was undertaken to identify the agricultural, industrial and human sources of microbial contamination from the pre- to post-harvest operations of cantaloupes grown at ten different farms in southern Texas. Results indicate that irrigation water contained a wide range of microorganisms that could cause human illness and were able to survive on the rind of cantaloupes before, at, and after harvesting. Fungi, total aerobic bacteria and total coliform bacteria were not completely eliminated by chlorinated water in the disinfection tanks of the six packinghouses under investigation. There were significant (P < 0.05) reductions on rind populations of fungi and total aerobic bacteria as well as drastic reductions in total coliform bacteria on the rinds after the disinfection and rinsing steps in all packing facilities. There was no evidence of the presence of Escherichia coli O157:H7 on packed cantaloupes across packinghouses. Less than a geometric mean of 1 c.f.u. cm⁻² of salmonellae were detected on the surface of packed cantaloupes in two of the packinghouses, and approximately ten times more salmonellae were found on the packed fruit processed in the remaining packinghouses. A similar trend was observed with listeriae. Results suggested that microbial loads originating from river water may survive on the rind or may re-infest cantaloupes after the disinfection and rinsing process at the packinghouses. Disinfection techniques and aseptic handling of cantaloupes at the packing facilities need a closer evaluation to ensure a safe product.     

Survival ofRhizobium trifolii in soil following inoculation of arrowleaf clover

Summary Arrowleaf clover (Trifolium vesiculosum Savi) is an annual forage legume that is sown in south-eastern USA when temperature and moisture conditions may not be suitable for survival of inoculant rhizobia. Survival of two antibiotic-resistant strains of rhizobia on seed or quartz particles was evaluated under controlled conditions in the laboratory. At 30°C and 75% r.h., lime coating of inoculated seed, soil pH (4.2 and 6.7), and surface or subsurface sowing of inoculated seed did not significantly (P = 0.05) affect the survival of either rhizobial strain. The use of gum arabic as an inoculant adhesive resulted in better rhizobial survival than sucrose or water, but even with gum arabic treatment the population of strain 162Y10 declined fromc. 10,000/seed to less than ten/seed in six days. Survival of strain 162Y15 appeared better andc. 200 rhizobia/seed survived for six days. When inoculated seed were incubated at 45°C, soil pH, method of sowing, lime coating of the seed, and r.h. (75% and 100%) did not influence rhizobial survival. The gum arabic adhesive enhanced survival over the use of sucrose but survival was still poor. No viable rhizobia were detected after two days of incubation. High r.h. (100%) and 30°C favoured survival and growth of both rhizobial strains when inoculated onto quartz particles placed on, or mixed into, soil at pH 4.2. Coating inoculated quartz particles with lime increased the growth of rhizobia under these conditions, but sucrose and gum arabic were equally effective as inoculant adhesives.

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Resumen Supervivencia de Rhizobium trifoliien el suelo después de la inoculación de trébol El trébol en punta de flecha arrowleaf (Trifolium vesiculosum Savi) es una leguminosa de forraje anual que se siembra en el S.E. de EUA en un periodo en el cual las condiciones de humedad y temperatura no suelen ser las adecuadas para la supervivencia de inóculo deRhizobium. La supervivencia en semillas o partículas de cuarzo de dos cepas deRhizobium resistentes a antibiôticos se evaluó bajo condiciones controladas en laboratorio. A 30°C y 75% de humedad relativa, ni el recubrimiento con cal de la semilla inoculada, ni la profundidad de la siembra (en superficie o no) ni el pH del suelo (4.2 y 6.7) alteraron significativamente la supervivencia de las dos cepas estudiadas. El uso de goma arábiga como adhesivo para el inóculo comparado con el uso de sucrosa o agua mejoró la supervivencia delRhizobium. Sin embargo incluso con el tratamiento con goma arábiga la población de la cepa 162Y10 declinó desde 10000/semilla hasta valores inferiores a 10/semilla en seis días. La cepa 162Y15 pareció tener mejor supervivencia y 200Rhizobium por semilla sobrevivieron a los seis días. Cuando las semillas inoculadas se incubaron a 45°C, el pH del suelo, el encalado de la semilla y la humedad relativa (75 y 100%) no influenciaron la supervivencia deRhizobium. El uso de goma arábiga mejoró relativamente la supervivencia aunque esta fue pobre de todos modos. No se detectaronRhizobium viables después de un periodo de incubación de dos días. Una elevada humedad relativa (100%) y una temperatura de 30°C favorecieron la supervivencia de ambas cepas cuando estas se inocularon en particulas de cuarzo, las cuales se situaron o, alternativamente, se mezclaron con suelo a pH 4.2. El recubrimiento con cal de las particulas de cuarzo inoculadas favoreció el crecimiento deRhizobium annque tanto sucrosa como goma arábiga fueron igualmente efectivos como adhesivos para el inóculo.

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Résumé Survie dans le sol de Rhizobium trifoliiaprès inoculation de trèfle sagittaire Le trèfle sagittaire (Trifolium vesiculosum Savi) est une légumineuse annuelle fourragère cultivée dans le Sud-Est des USA à une période de l'année où les conditions de température et d'humidité peuvent ne pas convenir à la survie de rhizobiums inoculés. La survie de deux souches de rhizobiums résistants aux antibiotiques sur des graines ou sur des particules de quartz a été evaluóe au laboratoire, dans des conditions contrôlées. A 30°C et r.h. 75%, l'enrobage dans la chaux des graines inoculées, le pH du sol (4, 2 et 6, 7) et le semis, soit en surface, soit recouvert, ne modifient pas significativement (P = 0,05) la survie des deux souches de rhizobiums. L'emploi de gomme arabique comme adhésif de l'inoculum assure une meilleure survie des rhizobiums que le saccharose ou l'eau. Mais, même avec la gomme arabique, la population de la souche 162Y10 décroit en six jours de 10,000 à moins de dix cellules par graine. La survie de la souche 162Y15 semble meilleure: 200 cellules survivantes par graine au bout de six jours. Lorsque les graines inoculées sont incubées à 45°C, le pH du sol, la méthode de semis, l'enrobage de la graine dans la chaux et le r.h. (75 et 100%) n'ont pas d'influence sur la survie des rhizobiums. La gomme arabique améliore la survie mieux que le saccharose, mais trés faiblement. Après deux jours d'incubation, on ne trouve plus de rhizobiums viables. Un r.h. élevé (100%) et une température de 30°C améliorent la survie et la croissance des deux souches de rhizobiums lorsque celles-ci sont inoculées sur des particules de quartz ou mélangées au sol à pH 4, 2. L'enrobage par la chaux des particules de quartz inoculées accroit la survie des rhizobiums dans ces conditions, mais le saccharose et la gomme arabique sont aussi efficaces somme agents d'adhésion de l'inoculum.

     

Survival of Escherichia coli O157:H7 applied to cantaloupes and the effectiveness of chlorinated water and lactic acid as disinfectants

Initial survey of four packinghouses indicated that bacterial and fungal populations on the surface of cantaloupes were significantly reduced by sanitizing procedures particularly on faecal coliforms. In this study, several combinations of disinfectants were tested in an attempt to obtain a more effective antimicrobial activity on aerobic bacteria, fungi and total coliforms. The efficacy of aqueous chlorine (200 mg l^–1) and lactic acid (1.5%) on inactivation of inoculated Escherichia coli O157:H7 on cantaloupe surfaces was investigated using different temperatures (25 and 35 °C) and immersion times (1 and 10 min). Maximal log reductions were achieved with both sanitizing agents when the initial bacterial population of E. coli was 7.42 log c.f.u. cm^–2. A highly significant 7.2 log reduction (P < 0.01) was obtained with a solution of lactic acid with and without Tergitol (0.3%) surfactant when cantaloupes were immersed for 10 min regardless of the temperature of the solution. Although the sanitizers caused substantial mortality, some bacterial cells remained attached at relatively low numbers on the fruit surface. The development of sanitizers more efficacious than chlorine for total elimination of this pathogen from the surface of cantaloupes is needed.     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the context dependent sequence requirements of active site residues in the metallo-beta-lactamase IMP-1

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.     

Identification of residues critical for metallo-β-lactamase function by codon randomization and selection

IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes beta-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the bla(IMP-1) gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitiveness of Rhizobium trifolii Strains Associated with Red Clover (Trifolium pratense L.) in Mississippi Soils

Five strains of Rhizobium trifolii were evaluated in competition with indigenous populations in nodulating red clover (Trifolium pratense L.) cv. Kenland in two different soils in Mississippi. Double antibiotic resistance acquisition was used to measure the proportion of nodules occupied by the introduced mutant strains. In vertisol soil, strains RP113-7, 162BB1, LM1, and 162P17 were recovered in at least 94% of the assayed nodules, whereas TA1 was found in 83.8% of the nodules. At an ultisol location, significant differences were detected within the introduced rhizobia. Strain RP113-7 was recovered at very high rates (99.2% of the assayed nodules), whereas strains 162BB1, LM1, 162P17, and TA1 were all found in 84.9 to 96.0% of the nodules sampled. Forage yield and percent crude protein levels were lower with the less effective but competitive strain (TA1) at both locations. Results indicated that more effective strains of R. trifolii can increase red clover production and symbiotic nitrogen fixation under different environmental conditions in Mississippi.