Symbiotic properties of 5-methyltryptophan-resistant mutants of Bradyrhizobium japonicum

The effect that resistance to 5-methyltryptophan (MT) has on the symbiotic properties of B. japonicum was examined in a survey of fourteen clones. Resistance to MT often involves a mutational alteration in the regulation of tryptophan biosynthesis.Resistant clones (MT^R) were isolated from agar plates containing MT. In the selection process care was taken to avoid pigmented clones that are likely to accumulate large amounts of indole compounds or show increased tryptophan catabolism. Wild-type control clones (WT_c) were isolated from plates containing no selective agent. In greenhouse studies. Tracy-M soybean plants were inoculated with the two types of clones. After six weeks, plants which were inoculated with the MT resistant clones showed a much greater range of symbiotic effectiveness than did plants that received the control clones.While most MT-resistant clones were poor symbionts or unchanged in their symbiotic performance, one clone was obtained that had significantly improved symbiotic properties. The procedure may offer a way of selecting for clones with improved symbiotic performance. These results also indicate a link between tryptophan biosynthesis and symbiotic effectiveness.     

Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

Effect of selective media on loss of congo red binding inShigella flexneri

Summary The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

The effect of carbohydrate on the development of a Cattleya hybrid in association with its mycorrhizal fungus

To investigate beneficial effects of mycorrhizal fungi to advanced leafy orchids, growth studies on the development of symbiotic seedlings of the orchid Cattleya (aclandiae x schoeffeldiana) x aclandiae were conducted in vitro over a period of 18 months using split plates with minerals and carbohydrates on one side and water agar on the other. Mycorrhizal infection and shoot and root growth of seedlings on the nutrient side were compared to growth on the water agar side with nutrient uptake by the orchid only possible via external mycorrhizal hyphae. Seed germination was followed by mycorrhizal infection and rapid development of protocorms on both nutrient and non-nutrient sides of the plates. With 0.5% starch, development of protocorms was sustained for a least 12 weeks, compared to only 6 weeks with 0.1% starch. Advanced protocorms with two small leaves and a smoll root were transferred at week 22 to new fungal plates. When harvested at week 43, plantlets on 0.5% starch (both nutrient and water agar sides) had 2.7 times the dry weight of plantlets on 0.1% starch. Shoot-root ratios were higher on the lower level of carbon. In all plantlets, mycorrhizal infection involved less than 5% of the root length. With zero, 0.1% or 0.5% starch, the roots were re-infected on transfer to fresh fungal plates but young roots that developed following the transfer stayed free of infection, Plantlets on 0.5% starch (nutrient and water agar side) after 18 months had longer roots than plantlets grown in the absence of starch or on 0.1% starch. Shoots were small but significantly larger on the nutrient side than on the water agar side, independent of the carbohydrate level. The shoot-root ratio was highest on the nutrient side with no starch present. In this latter case, plantlet development was steady but plantlets on the non-nutrient side developed slowly; thus there was little evidence of nutrient translocation by the mycorrhizal fungus from the nutrient to the non-nutrient side in the absence of carbohydrates. Mycorrhizal infection is discussed as a mechanism for heterotrophic carbon assimilation. In advanced leafy orchids of Cattleya, external carbon resulted in increased root growth, decreased shoot/root ratio and sometimes yellowish-green plantlets.     

Effect of presensitization of Nocardia asteroides with specific antibody on the viability of the organism

Nocardia asteroides from various growth phases was treated in vitro with normal rabbit sera, immune rabbit sera containing nocardial polyclonal antibodies and a monoclonal antibody. At intervals, samples were grown in broth or on blood agar plates to determine their viability. Log and stationary phase cells were injected intra-peritoneally into female BALB/c mice and their survival rates in the liver and spleen were determined. Presensitization with antibodies reduced the viability of the log phase cells by 48% and that of the late stationary phase by 4%. The antibody-treated log phase organisms were less viable on the blood agar medium and in the spleen and liver than the control organisms. This indicates that pretreatment with antibody has a lethal effect on N. asteroides and affects its survival in vivo.     

Characteristics of nutrient dynamics in Lake Sagate,Japan, a shallow sand dune lake

We examined differences in digestibility and viability following gut passage through water penny larvae (Psephenus herricki) of Synedra ulna and Achnanthidium lanceoloatum, two common diatom taxa that differ in growth habit and autecological characteristics. Prior to the experiment, diatoms were cultured in Chu-10 media in petri plates to establish a monospecific biofilm to offer grazers. After collection, insects were left to clear their guts over night, allowed to graze for 3 hours on diatom biofilms, and then placed in vials over 1-mm mesh to defecate. Samples from source material and from insect feces were mounted in syrup media and the ratio of chloroplast-containing to empty diatom frustules was microscopically assessed. In addition, subsamples from source material and feces were sprayed onto agar plates prepared with Chu-10 and individual cells were mapped and tracked for 5 days to quantify reproduction. Cells of both S. ulna and A. lanceolatum taken from source material formed colonies on agar. Achnanthidium lanceolatum cells from insect feces also formed colonies, but with lower densities than those from source material. In contrast, none of the S. ulna cells tracked from fecal cultures formed colonies, and the percentage of S. ulna cells that were dead was significantly greater in feces relative to source material. Dead cell percentages of A. lanceolatum were also higher in feces relative to source material, but to a lesser degree than observed for S. ulna. These findings have potential implications for linking patterns of energy transfer in stream ecosystems and the structure and dynamics of benthic microalgal communities.     

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Δxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.     

Construction of a new food-grade expression system for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> based on theta replication plasmids and auxotrophic complementation

A new food-grade expression system was constructed for Bacillus subtilis based on replicative food-grade expression plasmids and auxotrophic complementation. The food-grade B. subtilis host FG01 was created by knockout of the dal locus from the chromosome of B. subtilis 168. Two food-grade expression plasmids pXFGT03 and pXFGT05 were constructed by combining a novel theta-type Bacillus replicon with the B. subtilis endogenous gene dal and P43 promoter; while pXFGT05 was derived from pXFGT03 by deletion of two open reading frames (ORFs) from the original replicon. Upon transformation of FG01 with pXFGT03 or pXFGT05, the host phenotype was complemented on Luria–Bertani agar plates by the plasmid-coded dal gene, which served as a food-grade selection marker for recombinants. Results showed that deletion of the two ORFs had no impact on plasmid replication. A reporter gene bgaB was cloned into pXFGT03 and pXFGT05, respectively, under control of the P43 promoter, and it was successfully expressed in this food-grade expression system. Segregational stabilities of two recombinant plasmids were investigated, and they were fully stable.