Viable but nonculturable bacteria in drinking water

Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.     

微生物在水环境中的存活和生长

在灭菌自来水模拟水体中,研究了7种细菌的存活和生长规律。Klebsiella pneumo-niae,Enterobacter aerogenes,Agrobacterium tumefatciens,在7天内平板计数降至0,而水体中镜检细菌总数(AODC)和活菌直接计数(DVC)结果无大变化,说明细菌已变成活的非可培养状态。Micrococcus,flavus 和 Streptococcus faecalis 的可培养菌数也可降至0。Pseudomonas sp.在48小时内由10~5降至10~2cfu/ml,随即升至10~6 cfu/ml 并持续到实验终了(41天)。Bacillus subtilis 在48小时平板计数降至10~2cfu/ml 并维持在该水平至实验结束(38天)。研究结果表明仅用涂布平板法检测多种细菌在水环境中的生存和分布是不合适的。     

Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni.

Seven strains of Campylobacter jejuni, isolated from various sources [human (n = 2), chicken (n = 3), water (n = 2)], were studied under starvation conditions in filter-sterilized and pasteurized surface water by acridine orange direct count (AODC), viable count (DVC) and culture methods. Plate counts showed a rapid decline (2 log-units/day) for all strains under these conditions. Only one of the seven strains (14%) showed a (prolonged) viable, non-culturable 'state'. The ability of these viable, non-culturable cells to colonize the intestine was tested on day-old chicks. The infectious oral dose of freshly cultured cells of this model was 26-260 cfu; 1.8 x 10(5) viable, non-culturable C. jejuni were introduced to day-old chicks orally. Campylobacter jejuni was not isolated from the caeca of the chicks after incubation for 7 d. Also, passage through the allantoic fluid of embryonated eggs did not recover viable, non-culturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni

G.J. MEDEMA, F.M. SCHETS, A.W. VAN DE GIESSEN AND A.H. HAVELAAR. 1992. Seven strains of Campylobacter jejuni , isolated from various sources [human ( n = 2), chicken ( n = 3), water ( n = 2)], were studied under starvation conditions in filter-sterilized and pasteurized surface water by acridine orange direct count (AODC), viable count (DVC) and culture methods. Plate counts showed a rapid decline (2 log-units/day) for all strains under these conditions. Only one of the seven strains (14%) showed a (prolonged) viable, non-culturable 'state'. The ability of these viable, non-culturable cells to colonize the intestine was tested on day-old chicks. The infectious oral dose of freshly cultured cells of this model was 26–260 cfu; 1.8×10⁵ viable, non-culturable C. jejuni were introduced to day-old chicks orally. Campylobacter jejuni was not isolated from the caeca of the chicks after incubation for 7 d. Also, passage through the allantoic fluid of embryonated eggs did not recover viable, nonculturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

A tentative direct microscopic method for counting living marine bacteria.

Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.     

Effects of Pharmaceutical Wastes on Microbial Populations in Surface Waters at the Puerto Rico Dump Site in the Atlantic Ocean

A series of cruises during 1979 and 1980 to the pharmaceutical dump site located 64 km north of Arecibo, Puerto Rico, in the Atlantic Ocean, was carried out to evaluate effects of wastes on the ecology of the microflora of surface waters of the dump site. In addition to bacteriological monitoring of the waste plume created by the release of wastes from the disposal barge, stations along a series of transects, extending north from coastal waters through and beyond the dump site, were sampled. Largest numbers of culturable bacteria on marine agar were found at stations closest to shore and in the vicinity of the dump site. Bacteria recovered on marine agar were predominantly Vibrio and Aeromonas spp., with the relative abundance of these organisms decreasing as gram-positive organisms (staphylococci, micrococci, and bacilli) became dominant in areas immediately affected by waste dumping. Total numbers of bacteria (determined by acridine orange direct counts [AODC]), which were relatively stable throughout the region, and a direct estimate of viable cells (DVC), i.e., those cells responsive to additions of yeast extract and nalidixic acid, were determined by acridine orange staining and epifluorescence microscopy. Heterotrophic bacterial activity, measured by the uptake (V_max) of ¹⁴C-labeled amino acids, declined relative to distance from land. Increases in specific activity indices (DVC/AODC and V_max/AODC) were observed near the dump site. The composite results of this study, i.e., increased specific activities (determined by two methods), increased numbers of culturable marine bacteria, and marked alteration of the taxonomic composition of the culturable bacterial community in waters within and surrounding the Puerto Rico dump site, indicate demonstrable changes in the marine microbial community in the region used for waste disposal.     

Characterization of Bacillus and Pseudomonas strains with suppressive traits isolated from tomato hydroponic-slow filtration unit

Bacillus and Pseudomonas spp. are known to be involved in plant pathogenic fungi elimination during the slow filtration process used in tomato soilless cultures. We isolated 6-8 strains of both Bacillus and Pseudomonas from the top, middle, and bottom sections of filters and identified them after 16S rDNA sequencing. Four Pseudomonas strains were identified as Pseudomonas fulva, 5 as Pseudomonas plecoglossicida, and 12 as Pseudomonas putida. The use of specific oligonucleotide polymerase chain reaction primer sets designed from gyrB gene sequences additionally permitted the identification of 17 Bacillus cereus and 3 Bacillus thuringiensis strains. Ribotyping with EcoRI pointed out an important polymorphism within Bacillus and Pseudomonas strains. Molecular characterization did not reveal a correlation between the location of isolates within the filter (top, middle, or bottom) and bacterial identification or riboclusters. Functional aspects assessed by community-level physiological profiling showed marked phenotypic differences between Pseudomonas communities isolated from the top and bottom filter layers; differences were lower between Bacillus communities of different layers and far less noticeable between mixed communities of Bacillus and Pseudomonas. These strains were tested for several suppressive activities. Conversely to most Bacillus, the majority of Pseudomonas strains were auxin producers and promoted the growth of tomato plantlet roots. On the other hand, only Bacillus strains displayed antagonistic activities by inhibiting the growth of pathogenic fungi frequently detected in soilless cultures. Siderophores were produced by nearly all bacteria, but at higher amounts by Pseudomonas than Bacillus strains. The biocontrol agent potentiality of certain strains to optimize the slow filtration process and to promote the suppressive potential of nutrient solution is discussed.     

Development of a direct viable count procedure for some Gram-positive bacteria

The direct viable count (DVC) is a procedure for enumerating viable-nonculturable cells. It should be noted, however, that bacteria demonstrating the viable but nonculturable phase have to date included only Gram-negative species, mainly because the DVC procedure does not lend itself to the analysis of Gram-positive bacteria since the DVC procedure is dependent on the bacterium being sensitive to nalidixic acid. The authors report here concerning studies on an analogous procedure for the direct enumeration of viable-nonculturable Gram-positive bacteria.
To facilitate a differential DVC for Gram-positive bacteria, ciprofloxacin, enoxacin, norfloxacin or isopropyl cinodine were substituted for nalidixic acid. These antibiotics were chosen because, like nalidixic acid, they are DNA gyrase inhibitors. The concentrations used for each antibiotic were 1000 μg ml^-1, 100 μg ml^-1 and 10 mg ml^-1. Pure cultures of Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Listeria monocytogenes and Bacillus subtilis were obtained from the culture collection at the University of Wyoming and a faecal streptococcus was isolated from the Laramie wastewater treatment plant. An antibiotic and optimal concentration thereof was found which gave enlarged cells for all the organisms except the faecal streptococcus isolated from the wastewater plant for which no enlarged cells were ever seen. The antibiotic and concentration thereof which gave the optimal percent enlarged cells in the DVC procedure varied between organisms.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

The use of Alcalase 2·5L in the acridine orange direct count technique for the rapid enumeration of bacteria in beef mince

The acridine orange direct count (AODC) technique was used to count bacterial numbers in beef mince. The ability of Alcalase 2·5L to degrade beef mince was compared with the previously used Alcalase 0·6L. Both methods were evaluated against the standard plate count. The results showed that Alcalase 2·5L can be used in the AODC technique for the rapid counting of bacterial numbers in beef mince. Prediction equations obtained for the relationship between the AODC and standard plate counts were validated with commercial beef mince samples.     

Characterization and resuscitation of viable but nonculturable <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> VIB283

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4°C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10¹⁰ to 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.     

Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities

Plant growth promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. In search of efficient PGPR strains with multiple activities, a total of 72 bacterial isolates belonging to Azotobacter, fluorescent Pseudomonas, Mesorhizobium and Bacillus were isolated from different rhizospheric soil and plant root nodules in the vicinity of Aligarh. These test isolates were biochemically characterized. These isolates were screened in vitro for their plant growth promoting traits like production of indoleacetic acid (IAA), ammonia (NH(3)), hydrogen cyanide (HCN), siderophore, phosphate solubilization and antifungal activity. More than 80% of the isolates of Azotobacter, fluorescent Pseudomonas and Mesorhizobium ciceri produced IAA, whereas only 20% of Bacillus isolates was IAA producer. Solubilization of phosphate was commonly detected in the isolates of Bacillus (80%) followed by Azotobacter (74.47%), Pseudomonas (55.56%) and Mesorhizobium (16.67%). All test isolates could produce ammonia but none of the isolates hydrolyzed chitin. Siderophore production and antifungal activity of these isolates except Mesorhizobium were exhibited by 10-12.77% isolates. HCN production was more common trait of Pseudomonas (88.89%) and Bacillus (50%). On the basis of multiple plant growth promoting activities, eleven bacterial isolates (seven Azotobacter, three Pseudomonas and one Bacillus) were evaluated for their quantitative IAA production, and broad-spectrum (active against three test fungi) antifungal activity. Almost at all concentration of tryptophan (50-500 microg/ml), IAA production was highest in the Pseudomonas followed by Azotobacter and Bacillus isolates. Azotobacter isolates (AZT(3), AZT(13), AZT(23)), Pseudomonas (Ps(5)) and Bacillus (B(1)) showed broad-spectrum antifungal activity on Muller-Hinton medium against Aspergillus, one or more species of Fusarium and Rhizoctonia bataticola. Further evaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits on soil-plant system is needed to uncover their efficacy as effective PGPR.     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

Survival ofLegionella pneumophila in the aquatic environment

Survival ofLegionella pneumophila SG 1 in seawater and river water was assessed using plate counts on buffered charcoal yeast extract agar amended with α-ketoglutarate (BCYEα) and [³H]thymidine-labeling. The [³H]thymidine-labeling method for assessing survival ofL. pneumophila in aquatic environments was compared with viable counts, direct fluorescent microscopy (DFA), and acridine orange direct counts (AODC). Protozoa were isolated from the samples employed in the study and identified by characteristic trophozite and cyst morphology. Selective filtration employing 2.0 μm Nucleopore filters was used to determine the effect of grazing on survival ofL. pneumophila in seawater and river water.Legionella viability as measured by plate counts (CFU/ml), declined to a greater extent than cell lysis, assessed by thymidine, DFA, and AODC counts, suggesting thatL. pneumophila survives in aquatic habitats to a greater extent than revealed through culturable counts.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages. Received 16 December 1997/ Accepted in revised form 9 March 1998     

Effect of fusaric acid and phytoanticipins on growth of rhizobacteria and Fusarium oxysporum

Suppression of soilborne diseases by biocontrol agents involves complex interactions among biocontrol agents and the pathogen and between these microorganisms and the plant. In general, these interactions are not well characterized. In this work, we studied (i) the diversity among strains of fluorescent Pseudomonas spp., Bacillus spp., and Paenibacillus sp. for their sensitivity to fusaric acid (FAc) and phytoanticipins from different host plants, (ii) the diversity of pathogenic and nonpathogenic Fusarium oxysporum isolates for their sensitivity to phytoanticipins, and (iii) the influence of FAc on the production of pyoverdine by fluorescent Pseudomonas spp. tolerant to this compound. There was a great diversity in the response of the bacterial strains to FAc; however, as a group, Bacillus spp. and Paenibacillus macerans were much more sensitive to FAc than Pseudomonas spp. FAc also affected production of pyoverdine by FAc-tolerant Pseudomonas spp. strains. Phytoanticipins differed in their effects on microbial growth, and sensitivity to a phytoanticipin varied among bacterial and fungal strains. Biochanin A did not affect growth of bacteria, but coumarin inhibited growth of Pseudomonas spp. strains and had no effect on Bacillus circulans and P. macerans. Conversely, tomatine inhibited growth of B. circulans and P. macerans. Biochanin A and tomatine inhibited growth of three pathogenic isolates of F. oxysporum but increased growth of three nonpathogenic F. oxysporum isolates. Coumarin inhibited growth of all pathogenic and nonpathogenic F. oxysporum isolates. These results are indicative of the complex interactions that can occur among plants, pathogens, and biological control agents in the rhizosphere and on the root surface. Also, these results may help to explain the low efficacy of some combinations of biocontrol agents, as well as the inconsistency in achieving disease suppression under field conditions.     

Phytase activity in some groups of bacteria. Search for and cloning of genes for bacterial phytases

A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.     

Influence of tomato genotype on growth of inoculated and indigenous bacteria in the spermosphere

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent, Bacillus cereus UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786-4790, 1999). Here we report results of studies examining the host effect on the support of growth of Bacillus and Pseudomonas strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of B. cereus UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24 B. cereus strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of B. cereus UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains Pseudomonas fluorescens 2-79 or Pseudomonas aureofaciens AB254. Thus, while the host effect on growth extended to all strains of B. cereus examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of B. cereus UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous Bacillus and fluorescent Pseudomonas bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous Bacillus soil bacteria, while the line that supported growth of inoculated B. cereus UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of B. cereus UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains-both Bacillus and Pseudomonas spp.-did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.