THE BACTERIOLOGY OF THE ROOT REGION OF THE OAT PLANT GROWN UNDER CONTROLLED POT CULTURE CONDITIONS

SUMMARY: The bacteriology of the root region of oat plants grown under controlled conditions has been studied by means of improved techniques for separate estimation of the microfloras of the rhizosphere soil and of the root surface. The plate count of bacteria in the root region increased during the growing period of the plants; superphosphate produced a greater increase, which was probably due to increased plant growth, as no such effect was observed in uncropped soil.
The numbers of acid producing and dicalcium phosphate dissolving bacteria were increased in the root region, but the latter were not preferentially stimulated. Dressings of superphosphate and dicalcium phosphate also did not preferentially stimulate either group. No evidence was obtained, by the plate method used, of the presence of organisms capable of dissolving variscite, strengite, or gafsa rock phosphate, although the plants showed appreciable response to gafsa rock phosphate.     

Microbial Ecology of Activated Sludge: I. Dominant Bacteria

Over 300 bacterial strains were isolated from seven samples of activated sludge by plating on sewage agar. Gram-negative bacteria of the genera Zoogloea and Comamonas predominated. Many isolates (51%) showed sudanophilic inclusions of poly-β-hydroxybutyric acid, whereas 34% accumulated iodophilic material on media containing starch. A large number required either vitamins or amino acids, or both, for growth. None of the isolates tested for their ability to bring about changes in autoclaved sewage produced an effluent comparable in quality to the activated sludge control, although the Zoogloea did produce activated sludgelike flocs. A study of 150 bacterial strains isolated from raw sewage revealed that they differed from the sludge isolates in several respects. Coliforms, which constitute nearly a quarter of the sewage isolates, were rarely encountered in sludge.     

Inability to demonstrate calcite precipitation by bacterial isolates from travertine

Bacteria were isolated from 10 actively depositing travertine sites in Europe and North America. Forty‐four isolates were characterized and 33 (75%) were identified as Pseudomonas. Thirty isolates produced base on glucose‐peptone media and 32 isolates produced ammonia from amino acids. Because of the low bacterial biomass in the travertines, the formation of bases, including ammonia was not considered to be significant in carbonate precipitation. None of the bacteria was observed to precipitate calcite from supersaturated calcium carbonate solutions in vitro.     

Asaia lannaensis sp. nov., a new acetic acid bacterium in the Alphaproteobacteria

Asaia lannaensis sp. nov. was described for two strains isolated from flowers of the spider lily collected in Chiang Mai, Thailand. The isolates produced acetic acid from ethanol on ethanol/calcium carbonate agar, differing from the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis, but did not grow in the presence of 0.35% acetic acid (v/v). The new species is the fourth of the genus Asaia, the family Acetobacteraceae.     

Nutritional Requirements of the Nematophagous Fungus Hirsutella rhossiliensis

Six natural media were examined for growth and sporulation of six isolates of the nematophagous fungus Hirsutella rhossiliensis , using solid and/or liquid culture. Twenty carbohydrates, 19 nitrogen (N) compounds, and nine vitamins were also tested for their effects on growth, sporulation, and spore germination of a further three isolates (ATCC46487, OWVT-1 and JA16-1). Variations in nutritional requirements existed among the fungal isolates. In general, V-8 juice agar (VA), cornmeal agar and potato dextrose agar were good media for growth, and malt extract agar, VA and yeast dextrose agar were good for sporulation of all six isolates. Glycogen was the best and sucrose, inulin, D- ( + ) - trehalose and soluble starch were also good carbon (C) sources for growth and spore germination of the three isolates ATCC46487, OWVT-1 and JA16-1 in both liquid and solid culture. None of the isolates utilized D- ( + )xylose as a C source. L- sorbose, D- ribose, citric acid and D- fructose were poor for growth of all isolates. The best C source for sporulation was D- ( + )-trehalose for ATCC46487, D- sorbitol for OWVT-1 and D- ( + )-cellobiose for JA16-1. Casein was the best N source for growth of ATCC46487 and OWVT-1, while peptone was best for JA16-1. L- asparagine, L- proline, and peptone were also good for growth of all three isolates. L - cystine was not utilized by H. rhossiliensis and DL- methionine inhibited growth of all isolates. Spore germination of all isolates was well supported by most N compounds examined but was inhibited by L- cystine. No significant difference in sporulation of ATCC46487 was observed among the N sources. DL- threonine was the best N source for spore production by OWVT-1 and L- phenylalanine was best for JA16-1. Vitamins generally enhanced fungal growth and sporulation, with thiamine having the greatest influence. Excluding some vitamins individually from the medium containing all other test vitamins sometimes increased growth and/or sporulation of certain isolates.     

Mechanical properties of hydrocolloid gels filled with internally produced CO2 gas bubbles.

Agar (2%), alginate (1% algin), and kappa-carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0-2.5% sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0-2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas-filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2-5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distinction of biovar 2 strains of Agrobacterium from other chromosomal groups by differential acid production

Biovar (bv) 2 strains of Agrobacterium produced more acid from glucose than strains from the other two biovars This property caused the development of a clear zone around bv-2 colonies growing on potato glucose agar supplemented with 0.8% calcium carbonate. This simple cultural test produced results after 2 d at 28°C, and distinguished all 22 bv-2 strains tested from strains of other chromosomal groups of Agrobacterium ; none of the 42 bv-1 and 16 bv-3 strains tested produced a clear zone around their colonies.     

Oxidase testing from Kligler's iron agar and triple sugar iron agar slants

Many members of the familyVibrionaceae have been implicated as causative agents of diarrhea. Most of these organisms are non-lactose fermenters, and all are oxidase-positive. If the oxidase test could be reliably performed on growth from the surface of Kligler's iron agar and/or triple sugar iron agar slants, it would aid in the screening of potential stool pathogens. Forty-six isolates from the generaAeromonas, Plesiomonas, andVibrio were inoculated onto Kligler's iron agar and triple sugar iron agar slants, incubated overnight, and tested for oxidase activity. All 46 isolates produced alkaline over acid, with or without gas, Kligler's iron agar slants and were oxidase-positive. On triple sugar iron agar slants, 13 isolates produced these same patterns, and all were oxidase-positive. Acid over acid, with gas, triple sugar iron agar slants were produced by 18 isolates, and all were oxidase-positive. Acid over acid, without gas, triple sugar iron agar slants were produced by 15 isolates, and all were oxidase-negative. These negative oxidase tests were due to low pH. Oxidase tests performed from the surface of Kligler's iron agar and triple sugar iron agar slants used to screen stool isolates were reliable, provided the slants were acid over acid with gas, or alkaline over acid with or without gas. Kligler's iron agar is recommended with this procedure, since most potential stool pathogens of both theEnterobacteriaceae and theVibrionaceae will produce an alkaline over acid, with or without gas, slant, and false negative oxidase tests will be minimized.     

一些细菌和真菌的解磷能力及其机理初探

4株细菌和8株真菌培养6天后,发现培养液中有机酸含量大幅度增加,PH大幅度地下降,磷的含量大幅度增加,真菌比细菌表现出更强的溶解磷矿粉的能力,不同的微生物分泌有机酸的数量和种类差别很大,菌分泌的有机酸种类比细菌要多。但是,培养液中有机酸总量与解磷量之间并不存在显的相关性。     

Nitrogen utilization in bacterial isolates from the equine cecum.

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Method to recover a lipophilic drug from hydroxypropyl methylcellulose matrix tablets

A reverse-phase high-performance liquid chromatographic (HPLC) method for recovery of the lipophilic drug, alprazolam, from matrix tablets containing the hydrophilic polymer hydroxypropyl methylcellulose (HPMC) was developed. Lipophilic drugs, such as alprazolam, are difficult to completely extract and quantitate from tablets containing HPMC polymer. The percentage of recoveries of alprazolam from placebo powder spiked with alprazolam stock solution and from placebo powder mixed with alprazolam powder were about 100% and 85% to 95%, respectively. The validated method using water to completely dissolve HPMC before the addition of a strong solvent to dissolve and extract the drug from the HPMC solution was shown to be the most reproducible method. Different molecular weight distributions of the HPMC polymer, such as HPMC-K4M and HPMC-K100LV, did not influence the dissolution results of alprazolam using this validated method. Similarly, the excipients composing the matrix tablet formulations, such as dicalcium phosphate dihydrate, dicalcium phosphate anhydrous, calcium sulfate dihydrate, sucrose, dextrose, and lactose monohydrate, did not influence the percent recovery of alprazolam. The recovery method reported herein was shown to be the most efficient to achieve complete recovery of alprazolam from powder blends and tablets containing a variety of excipients and different grades of HPMC.     

Genetic variability of aflatoxin B<Subscript>1</Subscript> producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains isolated from discolored rice grains

Twenty-two aflatoxin B₁ (AFB1) producing Aspergillus flavus strains were isolated from 1,200 discolored rice grain samples collected from 20 states across India and tested their potential to produce AFB1 on different agar media. Further these isolates were characterized through randomly amplified polymorphic DNA method. All the strains of A. flavus were produced AFB1 on yeast extract sucrose agar media and none of the strains on A. flavus and A. parasiticus agar. Among the 22 strains, two strains from Tamil Nadu (DRAf 009) and Maharashtra (DRAf 015) produced high amount of AFB1 in all the media tested. To assess the genetic variability in A. flavus, the isolates were analyzed by using random amplified polymorphic DNA markers. Isolates showed 17–80% similarity with standard culture of A. flavus (MTCC 2799).     

Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.     

The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa

Valdensinia heterodoxa (Sclerotiniacae) is a potential fungal bioherbicide for control of salal (Gaultheria shallon). The effect of culture media, substrates and relative humidity (RH) on growth, sporulation and conidial discharge of V. heterodoxa was determined for two isolates PFC2761 and PFC3027 in vitro. Culture media significantly affected the growth, sporulation, and conidial discharge of V. heterodoxa. Of eight agar media used, colony radial growth was optimal on salal oatmeal agar and salal potato dextrose agar for isolates PFC2761 and PFC3027, respectively; whereas sporulation was at an optimum on salal oatmeal agar for both isolates. Of the eight liquid media tested, mycelial production was highest on wheat bran–salal–potato dextrose broth. Growth on solid substrates greatly stimulated sporulation and conidial discharge of V. heterodoxa. Of the 12 solid substrates used, the greatest numbers of discharged conidia were observed from wheat bran and wheat bran–salal within 14 d of sporulation. Sporulation on solid substrates continued for 42 d. RH significantly affected the sporulation and conidial discharge for both isolates across all solid substrates tested. No conidia were produced or discharged below 93 % RH on wheat bran–salal and millet. With an increase of the RH from 93 to 97 %, sporulation and the number of discharged conidia increased significantly for both isolates on wheat bran–salal, but not on millet.