INFLUENCE OF NODULE BACTERIA STRAINS OF SINORHIZOBIUM MELILOTI ON THE AMINO ACID CONTENT IN THE TOPS OF ALFALFA UNDER DIFFERENT WATER SUPPLY

Objective. To study the amino acid composition in the tops of alfalfa under different water supply (60 % MVC (maximum water capacity) and 30 % MVC) depending on the inoculum strain (Sinorhizobium meliloti 425a, T17, AC08). Methods. Microbiological (bacterial cultivation), physiological (vegetation experiment), biochemical (determination of amino acid content in the tops of plants). Results. It was established that the qualitative amino acid composition in the tops of ALFALFA was identical in all variants. However, quantitative content of the studied samples differed depending on the inoculum strain and water supply. It was found that aspartic and glutamic acids, leucine and lysine are dominant in the tops of alfalfa under different water supply. In the absence of moisture, an increase in the content of individual amino acids was reported at the background of Sinorhizobium meliloti AC08 and T17, while inoculation with Sinorhizobium meliloti 425a resulted in a decrease in the content of all amino acids except tyrosine. In plants exposed to water stress, a tendency towards decrease in the content of glutamic acid at the background of bacterization with Sinorhizobium meliloti 425a and increase — under inoculation with Sinorhizobium meliloti T17 and AC08 was reported, which may support the use of the latter to increase the ability of symbiotic systems to recover during a post-stress period. Conclusion. Pre-sowing inoculation of alfalfa of the variety Laska with new active strains of Sinorhizobium meliloti AC08 and T17 helps to increase the content of amino acids, in particular essential, in the tops of plants, both under optimal conditions of moisture and insufficient water supply. The prospects of using new strains of rhizobia in the cultivation of alfalfa for green mass under the conditions of optimal water supply and drought has been verified.

Draft Genome Sequence of Pseudomonas sp. LAB-08 Isolated from Trichloroethene-Contaminated Aquifer Soil

Pseudomonas sp. LAB-08 was isolated from a phenol-fed bioreactor constructed with contaminated aquifer soil as the inoculum. Strain LAB-08 utilized phenol as a sole carbon and energy source. Here, we report the genome sequence and annotation of Pseudomonas sp. LAB-08.

Evaluation of a Selective Enrichment Most Probable Number Enumeration Method for Viable Listeria spp. in Dairy Products

A most probable number (MPN) method for enumerating low numbers of Listeria spp. in dairy foods was developed by adapting the U.S. Food and Drug Administration (FDA) Listeria isolation methodology. Milk, cheese, and other milk products were diluted and homogenized in enrichment broth (1 g/10 ml). Homogenates were inoculated with L. monocytogenes Lm82, a streptomycin-resistant variant of strain Scott A, at <1 to 320 CFU/g and further diluted in FDA enrichment broth to give 0.1, 0.01, and 0.001 g of food sample per 10 ml. Dilution aliquots (10 ml) in triplicate or quintuplicate were incubated at 30°C for 48 h before being subcultured on Oxford agar at 35°C. Esculin-hydrolyzing colonies on Oxford agar were confirmed as the inoculum strain by their ability to grow on Trypticase soy agar containing streptomycin. Differences between inoculum and MPN values were evaluated by using tabulated 95% confidence limits. The calculated MPNs agreed with the inoculum levels in 91% (58 of 64) of noncheese dairy foods and in 49% (56 of 112) of 15 varieties of ripened cheeses. Competitive microflora affected by cheese age and the kind of milk used may account for the suboptimal performance of the MPN method with the cheeses.

Phage coating of soybean seed reduces nodulation by indigenous soil bradyrhizobia

Inoculation of soybean with Bradyrhizobium japonicum is often unsuccessful owing to the failure of inoculum strains to nodulate soybeans (Glycine max (L.) Merr.) in the presence of indigenous strains of rhizobia in soil. Previous studies have shown that it is possible to reduce nodulation with indigenous strains of rhizobia by amending the soil with a bacteriophage specific for the indigenous strain. The objective of the current study was to determine whether the coating of seed with phage affected nodule occupancy and soybean growth. A phage specific for B. japonicum USDA 469 and a symbiotically superior strain of rhizobium (B. japonicum USDA 110) were coated together onto soybean seed and planted into both greenhouse and field soil previously inoculated with B. japonicum USDA 469. The phage coated onto seed reduced nodulation by B. japonicum USDA 469 to 48% occupancy, compared with 64% for the untreated control value. Nodulation by the superior inoculum strain was increased from 48 to 82% occupancy by coating seed with the homologous phage and B. japonicum USDA 110. The rate of nitrogenase activity (on a per plant basis) was increased by coating seed with the phage and B. japonicum USDA 110. No other plant or symbiotic parameters were affected by phage coating of seed. These results indicate that the nodulation of soybeans can be significantly affected by the coating of seed with phage specific for undesirable strains of rhizobia in soil and the concurrent coating of seed with desirable strains of rhizobia. Key words: competition, rhizobiophage, rhizobia, soybeans.

ELISA evaluation of the competitive abilities of two Rhizobium meliloti strains

The double antibody sandwich enzyme-linked immunosorbent assay (DAS-EL1SA) was used to determine the competitive abilities of Rhizobium meliloti strains BALSAC and NRG-185 which are used in commercial alfalfa inoculants for eastern and western Canada, respectively. The ratio of NRG-185:BALSAC in the inoculum applied to aseptically grown alfalfa (Medicago sativa L.) was adjusted to 0:100, 1:99, 50:50, 90:10, 99:1, or 100:0. The ratios between nodules containing only the BALSAC strain and nodules containing only strain NRG-185 were similar to their respective inoculum strain ratios. Nodules containing both strains occurred only with the inoculum strain ratio of 50:50. Total plant weights were highest with the two single strain inocula and were decreased 5–19% with dual strain inoculation. The decrease in total plant weight was significantly correlated with decreases in nodule number and nodule weight. The results suggest that R. meliloti strains BALSAC and NRG-185 have equal competitive abilities, but are somewhat antagonistic, resulting in decreased nodulation and plant growth when both are present in an inoculum.

Rhizobium trifolii Mutant Interactions During the Establishment of Nodulation in White Clover

A more refined bacteroid isolation procedure was developed and used to investigate the establishment of nodulation of white clovers by Rhizobium tri/olii strain T1. Combinations of a variety of bacterial strains were mixed together for inoculation of clovers. The results showed that in nodules containing two different strains of Rhizobium only one type of inoculum strain could be reisolated from any one plant cell. Furthermore, it was found that nodules which resulted from inoculation with cell mixtures containing the non-invasive Agrobacterium tume/aciens strain B6S3 and a nodulating Rhizobium strain at various cell ratios did not contain strain B6S3. However, a number of invasive but non-nodulating mutants of Rhizobium could be readily reisolated from nodules resulting from inoculation with cell mixtures containing both the mutant and its parent strain. Moreover, some combinations of symbiotic mutants showed a cooperative interaction giving rise to nitrogen-fixing nodules. From an investigation of the genetic characteristics of the bacterial cells isolated from nodules, it was concluded that this interaction between different symbiotically defective mutants of Rhizobium is a form of cross-feeding and not genetic complementation. Furthermore, there are four possible steps in the nodulation process where the intra-nodule interactions can occur between different strains of Rhizobium.

Studies in the nodulation of Kenya white clover (Trifolium semipilosum) under field conditions in south-east Queensland

The nodulation of Kenya white clover (Trifolium semipilosum) was examined under field conditions to evaluate reported establishment problems attributed to poor nodulation. Two isolates of Rhizobium identified as strain CB782 were obtained from nodules of T. semipilosum located in separate field sowings each of which was considered an establishment failure but which had recovered to healthy stands at the time of sampling. The two isolates were compared with a laboratory maintained culture of CB782 for nodulation and nitrogen fixation. Both isolates and the original strain were equally effective in N-fixation and all three increased plant dry weight, per cent plants with effective (pink) nodules and amount of nitrogen fixed compared with an uninoculated control. Increasing the level of seed application of inoculum up to the normal rate increased effective nodulation, but excess inoculum levels resulted only in greater numbers of smaller effective (pink) nodules without any increase in nodule dry weight. High inoculum levels decreased the number of white and green nodules compared with the control. Over 50 per cent effective nodulation was obtained with less than 50 Rhizobium seed-1 at sowing. Serological identification and effectiveness tests of strains in nodules confirmed that most white and green nodules on T. semipilosum resulted from infection by naturally occurring white clover rhizobia. These were ineffective in nitrogen fixation with T. semipilosum but fully effective on T. repens. Serological typing of nodules also indicated that normal levels of inoculation resulted in more than 80 per cent of nodules being formed by the specific inoculum strain. We conclude that field sowings of T. semipilosum can be effectively nodulated by the specific inoculum strain CB782 which is both effective and persistent under south-east Queensland conditions. We suggest that most reported nitrogen deficiency symptoms of establishing T. semipilosum are due to infection by Rugose Leaf Curl Virus.

Correction - Studies in the nodulation of Kenya white clover (Trifolium semipilosum) under field conditions in south-east Queensland

The nodulation of Kenya white clover (Trifolium semipilosum) was examined under field conditions to evaluate reported establishment problems attributed to poor nodulation. Two isolates of Rhizobium identified as strain CB782 were obtained from nodules of T. semipilosum located in separate field sowings each of which was considered an establishment failure but which had recovered to healthy stands at the time of sampling. The two isolates were compared with a laboratory maintained culture of CB782 for nodulation and nitrogen fixation. Both isolates and the original strain were equally effective in N-fixation and all three increased plant dry weight, per cent plants with effective (pink) nodules and amount of nitrogen fixed compared with an uninoculated control. Increasing the level of seed application of inoculum up to the normal rate increased effective nodulation, but excess inoculum levels resulted only in greater numbers of smaller effective (pink) nodules without any increase in nodule dry weight. High inoculum levels decreased the number of white and green nodules compared with the control. Over 50 per cent effective nodulation was obtained with less than 50 Rhizobium seed-1 at sowing. Serological identification and effectiveness tests of strains in nodules confirmed that most white and green nodules on T. semipilosum resulted from infection by naturally occurring white clover rhizobia. These were ineffective in nitrogen fixation with T. semipilosum but fully effective on T. repens. Serological typing of nodules also indicated that normal levels of inoculation resulted in more than 80 per cent of nodules being formed by the specific inoculum strain. We conclude that field sowings of T. semipilosum can be effectively nodulated by the specific inoculum strain CB782 which is both effective and persistent under south-east Queensland conditions. We suggest that most reported nitrogen deficiency symptoms of establishing T. semipilosum are due to infection by Rugose Leaf Curl Virus.