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The insecticidal toxins from Bacillus thuringiensis subspp. kurstaki (antilepidopteran), morrisoni strain tenebrionis (anticoleopteran), and israelensis (antidipteran) did not affect the growth of a variety of bacteria (8 gram-negative, 5 gram-positive, and a cyanobacterium), fungi (2 Zygomycetes, 1 Ascomycete, 2 Deuteromycetes, and 2 yeasts), and algae (primarily green and diatoms) in pure and mixed culture, as determined by dilution, disk-diffusion, and sporulation assays with purified free and clay-bound toxins. The insecticidal crystal proteins from B. thuringiensis subspp. kurstaki and israelensis had no antibiotic effect on various gram-positive bacteria.Key words: insecticidal toxins, Bacillus thuringiensis, microbiostatic, microbicidal.

The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (Mr=132 kDa) and toxins (Mr=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na2CO3, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni.

Publisher Summary This chapter discusses the method for studying bacterial gene transfer in soil by conjugation and transduction. Soil is unique among microbial habitats in that it is a structured environment with a high solid to water ratio. The discreteness of microhabitats in soil, the probability of genetic exchange may be less than the probability in ecosystems wherein water is continuous. Except for periods when soil is saturated with water, individual microhabitats are isolated by the surrounding pore space, movement of bacteria, transducing bacteriophages, and transforming DNA among the microhabitats. However, filamentous fungi appear to be able to bridge pore spaces between microhabitats, even when the pore spaces are not filled with water, as these fungi grow apically from mycelia that have a food and water base in a microhabitat and, therefore, are independent of the nutrient and water conditions surrounding the growing mycelia. Moreover, the extending mycelia probably have a surrounding water film in which bacteria or bacteriophages may be transported from one microhabitat to another.

Studies were conducted to determine whether the differential toxicities of the two valence states of Cr (+3 and +6) occur with fungi as they do with bacteria as noted in previous studies. It was found that mycelial growth rates were inhibited more by Cr/sup 6 +/ than by Cr/sup 3 +/. Hexavalent Cr was also more toxic than equivalent trivalent Cr to spore formation and spore germination of the fungi tested. The greater toxicity of hexavalent Cr than of trivalent Cr to fungi is in agreement with the results observed with the bacterial studies. (JMT)

Volatile compounds evolved from germinating seeds of slash pine, bean, cabbage, corn, cucumber, and pea were evaluated for their ability to support growth of microorganisms in liquid mineral salts media lacking a carbon source. Growth of eight bacteria was measured turbidimetrically and of six fungi as dry weight of mycelium.Volatiles caused increased growth of Pseudomonas fluorescens, Bacillus cereus, Erwinia carotovora, Agrobacterium tumefaciens, A. radiobacter, Rhizobium japonicum, Mucor mucedo, Fusarium oxysporum f. conglutinans, Trichoderma viride, and Penicillium vermiculatum but not of Sarcina lutea, Serratia marcescens, Chaetomium globosum, or Schizophyllum commune. Spores of Trichoderma viride showed higher germination in the presence of volatiles.Effects on growth were apparent only during the first 3 or 4 days after planting the seeds. Killed or dried seeds had no effect. The volatiles did not support microbial growth in the absence of nitrogen nor did they supply growth factors. Passing volatiles through KMnO4 or hydrazone reduced growth of the bacteria, indicating that oxidizable organic compounds, primarily aldehydes, were the active components. The volatiles were not absorbed by sterile soil, clay minerals, or water, but they were absorbed by non-sterile soil and activated charcoal.

The toxicity of a combination of nickel (Ni) and copper (Cu) toward growth of heterotrophic microorganisms was greater than the sum of the toxicity of each metal individually. This synergism between Ni and Cu was potentiated at acidic pH levels, with the potentiation of the toxicity being, for some organisms, an effect of the acidic pH on the toxicity of the Ni, rather than of the Cu, component. For others, however, the potentiation was an effect of acidic pH on both the Ni and Cu components. The potentiation of the Ni-Cu synergistic interaction at acidic pH levels has relevance to the deposition of acid precipitation into environments contaminated with these metals. Furthermore, the occurrence of such synergistic interactions and their mediation by pH should be considered in the methodologies used to establish criteria for tolerable levels of metals in the environment.

The toxicity of lead (Pb) to fungi in pure culture was influenced by several abiotic factors: pH, inorganic anions, clay minerals, and particulate (humic acid) and soluble organic matter. The toxicity of Pb was potentiated under acidic conditions (pH 5 and 6), and phosphate or carbonate anions reduced the toxicity, apparently as a result of the formation of sparingly soluble Pb salts. Clay minerals (montmorillonite greater than attapulgite greater than kaolinite) and particulate humic acid protected against the toxicity of Pb, presumably as the result of sorption, by cation exchange of the Pb to the exchange complexes, which reduced its availability for uptake by the fungi. Soluble organics, such as tryptone, yeast extract, cysteine, succinic acid, and increasing concentrations of neopeptone, also reduced the toxicity of Pb.

Scanning and transmission electron microscopy of Cunninghamella blakesleeana grown in the presence of toxic concentrations of copper and cobalt indicated that copper, but not cobalt, induced both morphological and ultrastructural changes. In contrast to the control or cobalt-grown cultures, the hyphae of copper-grown cultures (called “blue mycelia”) were larger in diameter, had a rough and granular surface, and the cell wall was thicker. The cytoplasm of the blue mycelia was also abnormal and was in a compressed state. X-Ray microprobe analysis indicated a lower content of magnesium and calcium in the blue mycelia and an elevated content of sulphur in both the blue and cobalt-grown mycelia. The protein composition of the cell walls of the blue mycelia, fractionated on a Sepharose-4B column saturated with copper, was different from that of control or cobalt-grown cultures, as shown by their amino acid composition. Hydroxyproline was present only in the cell wall proteins of the blue mycelia, citrulline and cystathionine were present only in the proteins of cobalt-grown cultures, and proline was absent in the cell wall protiens of the control cultures.

Significant vanillin degradation occurred only in soil amended with 9% montmorillonite and not in soil amended with 9% kaolinite or in soil without addition of clay minerals. Progressively decreasing amounts of vanillin were mineralized in the montmorillonite-amended soil with increasing acidification with H/sub 2/SO/sub 4/, and complete inhibition of mineralization occurred at a soil pH of 1.6. 16 references, 1 table.

(1980). Environmental Factors that Influence the Toxicity of Heavy Metal and Gaseous Pollutants to Microorganisms. CRC Critical Reviews in Microbiology: Vol. 8, No. 2, pp. 99-145.

Since Pb has no known biological function, elevated levels of Pb in soils and in other natural environments may adversely affect the indigenous biota, including the microbiota. Elevated levels of Pb in soil may also adversely affect microbemediated ecologic processes. There is, however, relatively little information on the mediating influence of the physicochemical factors of the recipient environment on the toxicity of Pb to microbe-mediated ecologic processes. This present study evaluated the influence of the clay minerals, kaolinite and montmorillonite, particulate humic acids, and compost on the degradation of glucose in soil.

The soil replica plating technique was used to evaluate the influence of clay minerals and pH on antagonistic interactions between fungi and bacteria in soil. In general, the antagonistic activity of bacteria towards filamentous fungi was greater in soil than on agar. The spread of Aspergillus niger through soil was inhibited by Serratia marcescens when the organisms were inoculated into separate sites in soil, and this antagonistic effect was maintained when the soil was amended with 3, 6, 9, or 12% (vol/vol) montmorillonite, whereas the addition of kaolinite at a concentration of 3% reduced the antagonism and at 6, 9, or 12% totally eliminated it. Similar results were obtained with the inhibition of A. niger by Agrobacterium radiobacter and of Penicillium vermiculatum by either S. marcescens or Nocardia paraffinae . When A. niger and S. marcescens were inoculated into the same soil site, A. niger was inhibited in all soils, regardless of clay content, although the extent of inhibition was greater as the concentration of montmorillonite, but not of kaolinite, increased. A. niger was inhibited more when inoculated as spores than as mycelial fragments and when inoculated 96 h after S. marcescens, but a 1% glucose solution reduced the amount of inhibition when the fungus was inoculated 96 h after the bacterium. When the pH of the soil-clay mixtures was altered, the amount of antagonism usually increased as the pH increased. Antagonism appeared to be related to the cation-exchange capacity and the pH of the soil-clay mixtures. Bacillus cereus and another species of Bacillus showed no activity in soil towards A. niger under any of the environmental conditions tested, even though the Bacillus sp. significantly inhibited A. niger and seven other fungi on agar.

Volatile metabolites produced by eight bacterial species greatly inhibited growth and sporulation of all fungi tested, namely Fusarium oxysporum f. conglutinans, Gelasinospora cerealis, Penicillium viridicatum, Trichoderma viride, and Zygorhynchus vuilleminii. Alterations of colony morphology were induced in all test fungi, except G. cerealis. In P. viridicatum, colony contour and color were altered; in F. oxysporum f. conglutinans, T. viride, and Z. vuilleminii, the amount or distribution of aerial growth was altered.

Detached spores of 14 fungi varied in germination after exposure on agar to 10 to 100 parts per hundred million (p.p.hm.) ozone for 1 to 6 h. Large pigmented spores of Chaetomium sp., Stemphylium sarcinaeforme, S. loti, and Alternaria sp. were insensitive to 100 p.p.hm. Spores of Trichoderma viride, Aspergillus terreus, A. niger, Penicillium egyptiacum, Botrytis allii, and Rhizopus stolonifer were reduced in germination primarily by 100 and 50 p.p.hm. for the longer exposures. Small hyaline spores of Fusarium oxysporum, Colletotrichum lagenarium, Verticillium atbo-atrum, and V. dahliae were the most sensitive, as their germination was prevented or reduced by most exposures to 100 and 50 p.p.hm. and occasionally reduced by doses as low as 25 p.p.hm. for 4 and 6 h. Ten parts per hundred million for 6 h had little inhibitory effect, but extended exposures up to 28 h reduced germination of A. terreus, A. niger, and P. egyptiacum spores to below 50% of the controls. The lower doses of ozone sometimes stimulated spore germination. Fungus colonies maintained in an ozone atmosphere had abnormal growth characteristics. Ozone had little inhibitory effect on air-dried spores or spores in a liquid medium.

The respiration of mycelial homogenates of 27 fungal species representing four classes was generally not affected by pH nor by montmorillonite or kaolinite at concentrations below 4%. Respiration was markedly inhibited by montmorillonite at concentrations of 4% and above, but comparable inhibition by kaolinite occurred only at concentrations above 40%. The inhibition was greater as the metabolic activity of the mycelium increased but was not caused by a limitation in carbon substrate. The inhibition was related to the viscosity of the systems, which, in turn, presumably influenced the rate of O2 diffusion. The viscosities of systems containing montmorillonite were vastly greater than of those containing kaolinite. These results were in contrast to those previously obtained with bacteria, which revealed that montmorillonite greatly stimulated respiration.

A replica plating method was developed to study ecology of microorganisms in soil. Precise placement of inocula and amendments at desired loci in sterile soil contained in petri plates were accomplished with a template. Subsequent growth and distribution of individual species, even when part of a mixed population, was measured by periodic transfer with an easily constructed replicator to agar plates of differing nutritional composition or containing selective inhibitors. The method is rapid and reproducible, and permits the study of many variables and interactions in a single soil plate; it can also be used with non-sterile soil and other suitable microbial habitats.

(1972). Activity, Ecology, and Population Dynamics of Microorganisms in Soil. CRC Critical Reviews in Microbiology: Vol. 2, No. 1, pp. 59-137.

Microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores were compared. With the electronic sizing method, early stages of germination (i.e., spore swelling) were detected long before germ tube emergence or significant changes in respiratory rates were observed. This method, which is rapid, easy, sensitive, and reproducible, also permits measuring the germination of spores when similar-size particles are present in concentrations considerably in excess of the number of spores.

A 10 mM concentration of Zn2+ decreased the survival of Escherichia coli; enhanced the survival of Bacillus cereus; did not significantly affect the survival of Pseudomonas aeruginosa, Norcardia corallina, and T1, T7, P1, and phi80 coliphages; completely inhibited mycelial growth of Rhizoctonia solani; and reduced mycelial growth of Fusarium solani, Cunninghamella echinulata, Aspergillus niger, and Trichoderma viride. The toxicity of zinc to the fungi, bacteria, and coliphages was unaffected, lessened, or increased by the addition of high concentrations of NaCl. The increased toxicity of zinc in the presence of high concentrations of NaCl was not a result of a synergistic interaction between Zn2+ and elevated osmotic pressures but of the formation of complex anionic ZnCl species that exerted greater toxicities than did cationic Zn2+. Conversely, the decrease in zinc toxicity with increasing concentrations of NaCl probably reflected the decrease in the levels of Zn2+ due to the formation of Zn-Cl species, which was less inhibitory to these microbes than was Zn2+. A. niger tolerated higher concentrations of zinc in the presence of NaCl at 37 than at 25 degrees C.

Microorganisms are sensitive to heavy metal pollution as are other components of the biota. However, most studies on the interactions between microbes and heavy metals have been conducted in synthetic media or in altered (e.g., sterilized) environmental samples and usually have used only single species. Few studies have evaluated the effects of heavy metals on the activities of natural heterogeneous microbial populations, both autotrophic and heterotrophic, in terrestrial and aquatic environments. These latter studies have shown that heavy metals inhibit primary productivity, nitrogen fixation, the mineralization of carbon, nitrogen, sulfur, and phosphorus, litter decomposition, and enzyme synthesis and activity in soils, sediments, and surface waters. The potential adverse effects of heavy metals on such microbe-mediated ecologic processes need to be incorporated into the methodologies used by regulatory agencies, such as the U.S. Environmental Protection Agency, to prepare environmental risk assessments which, in turn, are used to formulate environmental criteria, such as the Water Quality Criteria, and to evaluate the safety to the environment of exposure to "new chemical substances," as mandated by the U.S. Toxic Substances Control Act of 1976. To provide appropriate data that can be assimilated into regulatory policy, it is essential that microbial ecotoxicity tests be standardized, are neither costly nor difficult to train personnel to conduct, and produce data that can be quantitated.

Rat endothelial cells in culture can be distinguished from fibroblasts and epithelial cells by their reaction with antisera against human factor VIII (AHF) associated proteins.

The toxicity of phenol to filamentous fungi was unaffected by pH or water hardness, but it was potentiated by salinity at levels comparable to those occurring in estuarine and off-shore marine waters. The effect of temperature on the response of the fungi to phenol was highly variable: depending on the fungus, the toxicity of phenol was either not affected, increased, or decreased as the temperature was raised from 22 to 32°C. The toxicity of phenol was not affected by heavy metals. The mediating influences of these abiotic environmental factors on the toxicity of phenol to filamentous fungi were similar to those reported for aquatic macrobiota. This similarity, which has also been noted with the effects of environmental factors on the toxicity of heavy metals, strengthens the concept of using microbial assays to evaluate initially the potential mediating influence of physicochemical factors of natural environments on the toxicity of organic pollutants. An antagonistic interaction in toxicity occurred between phenol and endrin aldehyde orp-chloro-m-cresol, and an additive interaction occurred between phenol and toxaphene, but no interactions were apparent between phenol and other phenolic compounds and pesticides.

A broad spectrum of microorganisms, including eubacteria (nonmarine and marine), actinomycetes, yeasts, and filamentous fungi, were evaluated for their sensitivities to nickel. Wide extremes in sensitivity to Ni were noted among the filamentous fungi, whereas the range of tolerance to Ni of the yeasts, eubacteria, and actinomycetes was narrower. With all microorganisms, the toxicity of Ni was potentiated as the pH was decreased to acidic levels. The mechanism(s) of how pH affects the toxicity of Ni has not been defined, although the formation of hydroxylated Ni species with differing toxicities was not involved. The enhanced toxicity of Ni at acidic levels may have implications for the toxicity of Ni in environments stressed by acid precipitation.

The influence of high pCO2 and low pO2 on the microbiota derived from four tropical soils and on microorganisms in pure culture was investigated. Incubation under atmospheres containing more than 90% CO2 reduced the number of organisms developing on soil dilution plates, but most organisms recovered when the plates were subsequently incubated in air. Inhibition by high CO2 tensions was the result primarily of CO2 concentration and not absence of O2; many organisms developed under 100% N2, and presence of O2 mitigated the inhibitory effects, especially when CO2 concentration was 95% or less. The actinomycete population was most sensitive to high pCO2 and low pO2 and the fungal population least sensitive. Some fungi were capable of developing under 100% N2 and under atmospheres containing as much as 98% CO2; most identified species developed in air after exposure to the various gases. Fusarium oxysporum f. cubense was more tolerant to low O2 and high CO2 tensions than most of the soil mycobiota.