Lignocellulose Decomposition and Production of Ligninolytic Enzymes During Interaction of White Rot Fungi with Soil Microorganisms

Abstract Four strains of white rot fungi, including two strains of Pleurotus sp., one Dichomitus squalens, and one Ganoderma applanatum, were grown on milled straw. After colonization of the straw by the fungi, sterile or nonsterile plugs of soil were added to the fungal substrates. The influence of the sterile soil and the indigenous soil microbiota on fungal growth, overall respiration, and production of ligninolytic exoenzymes was assessed. A method for extraction of laccase from soil samples was developed. Lignocellulose decomposition, and enzyme production of D. squalens were enhanced by the presence of sterile soil. The availability of inorganic compounds such as manganese may be a trigger for this stimulation. Neither growth nor the production of laccase and manganese peroxidase (MnP) of the Pleurotus strains was markedly affected by the soil microbiota. These fungi were highly competitive with the soil microbiota. It was demonstrated for the first time that the exoenzymes of such fungi are active in nonsterile soil. Enzyme activity in the aqueous phase of soil was high as in the aqueous phase of the straw substrate. D. squalens and G. applanatum did not withstand the competition with the soil microbiota, but the mycelia associated with straw were overgrown by soil microorganisms. Correspondingly, the fungi did not penetrate the soil, decomposition of lignocellulose was impeded, and the activities of laccase and MnP decreased dramatically. Received: 2 April 1996; Accepted: 7 June 1996     

Bacterial conjugation betweenEscherichia coli andPseudomonas spp. donor and recipient cells in soil

Summary Experiments conducted in microcosms containing loam soil samples inoculated with eitherE. coli orPseudomonas spp. donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period.E. coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples. In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation.Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil. The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10^–3 to 10^–4). Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.     

The effects of micro-organisms upon plant growth

Summary Phytotoxins developed during heat-sterilization of soil were removed by 40 per cent of the fungi and ten per cent of the bacteria isolated from inoculated heat-sterilized soil. Detoxication by the fungi was not necessarily associated with marked proliferation in sterile soil,Trichoderma viride was an active colonizer of sterile soil but quite ineffective as a detoxicating agent.The phytotoxins could be readily leached out and their effects transmitted to sterile sand and irradiated soil. Results indicate that the toxicity is not due to changes in pH, soluble manganese or ammonia but is probably due to the formation of toxic organic materials.Primary root growth of subterranean clover is markedly stunted by toxins formed during heat-sterilization of soil and could provide an extremely sensitive bioassay for toxicity.Detoxication by micro-organisms may have been misinterpreted in the past as a direct stimulation of plant growth and caution is required in studies of the effects of micro-organisms upon plant growth.     

The mutual growth ofArthrobacter globiformis andPseudomonas fluorescens in gamma-sterilized soil

Summary Arthrobacter globiformis andPseudomonas fluorescens were grown separately and together in flasks containing soil sterilized by gamma-irradiation. If the soil was held at 60% of its waterholding capacity (W.H.C.) and 25°C the viable cell yield of the 2 organisms in pure culture was approximately equal in 4 days while in mixed culture neither organism predominated and the total yield was about the same as in either pure culture. The pseudomonad dominated the mixed cultures when the incubation temperature was 10°C, when the soil was saturated and when glucose was added to the soil. The arthrobacter dominated the mixed culture when soil moisture was decreased to 40% W.H.C. The 10-day oxygen uptake by the 2 organisms grown together in sterile soil was much less than that shown by an inoculum prepared from a soil dilution.A. globiformis took up less oxygen when grown in the sterile soil than didP. fluorescens. In these short-term experimentsA. globiformis did not demonstrate competitive capabilities which would explain its more frequent occurrence in unamended soil thanP. fluorescens. This work was supported by grant A-1702 from the National Research Council of Canada, and by assistance from Ontario Department of Agriculture and Food.     

Survival in Sterile Soil and Atrazine Degradation of Pseudomonas sp. Strain ADP Immobilized on Zeolite

In laboratory settings, the ability of bacteria and fungi to degrade many environmental contaminants is well proven. However, the potential of microbial inoculants in soil remediation has not often been realized because catabolically competent strains rarely survive and proliferate in soil, and even if they do, they usually fail to express their desired catabolic potential. One method to address the survival problem is formulating the microorganisms with physical and chemical support systems. This study investigates the survival of Pseudomonas sp. strain ADP in sterile soil and its retention of atrazine-degrading functionality. Assessment was conducted with free and zeolite-immobilized bacteria incorporated into the soil. Pseudomonas sp. strain ADP remained viable for at least 10 weeks when stored at 15°C in sterile soil. Cell numbers increased for both free and zeolite-immobilized bacteria during this period, except for free cells when grown in Miller's Luria-Bertani medium, which exhibited constant cell numbers over the 10 weeks. Only the zeolite-immobilized cell retained full functionality to degrade atrazine after 10 weeks in sterile soil regardless of the medium used to culture Pseudomonas sp. strain ADP. Functionality was diminished in free-cell inoculations except when using an improved culture medium. Survival of zeolite-immobilized Pseudomonas sp. strain ADP separated from the soil matrix after 10 weeks’ incubation was significantly (p < .05) greater than in soil inoculated with free cells or in the soil fraction inoculated by release from zeolite-immobilized Pseudomonas sp. strain ADP.     

Survival of Botrytis cinerea in Soil in South-Eastern Spain

The survival of Botrytis cinerea in sterile and unsterile soil at different temperatures and relative air humidities was investigated in south‐eastern Spain. Conidia survived only 7 days at 40^°C but, depending on relative humidity, for 30–90 days at 22^°C. High air humidity (95%) was needed to maintain soil humidity (8%) at a level that favoured conidial survival. Conidia survived better in sterile soil than in unsterile soil, probably because of the presence in the latter of soil microorganisms antagonistic to B. cinerea. Survival of conidia in environmental conditions simulating those in a greenhouse was less than 28 days. Results showed that B. cinerea conidia cannot survive over summer in south‐eastern Spain, and other primary sources of inocula are discussed.     

Effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

A study was conducted to determine the effect of inoculation method and plant growth medium on colonization of sorghum by an endophytic Beauveria bassiana. Colonization of leaves, stems, and roots by B. bassiana was assessed 20-days after application of the fungus. Although B. bassiana established as an endophyte in sorghum leaves, stems, and roots regardless of inoculation method (leaf, seed, or soil inoculation), plant growth medium (sterile soil, non-sterile soil, or vermiculite) apparently influenced colonization rates. Seed inoculation with conidia caused no stem or leaf colonization by the fungus in non-sterile soil but did result in substantial endophytic colonization in vermiculite and sterile soil. Leaf inoculation did not result in root colonization, regardless of plant growth medium. Endophytic colonization was greater in leaves and stems than roots. Endophytic colonization by B. bassiana had no adverse effects on the growth of sorghum plants. Leaf inoculation with a conidial suspension proved to be the best method to introduce B. bassiana into sorghum leaves for plants growing in either sterile or non-sterile soil. Further research should focus on the virulence of endophytic B. bassiana against sorghum stem borers.     

Influence of the herbicide phosphinothricin on growth and nodulation capacity of Rhizobium meliloti

  Rhizobium meliloti proved to be sensitive to low concentrations of the herbicide phosphinothricintripeptide (PTT) and its active ingredient phosphinothricin (PT), which was formerly assumed to be non-toxic for most of the bacteria analysed. Growth was more strongly reduced in sterile synthetic media and less reduced in sterile soil; in unsterile soil only a transient growth reduction was detectable. Sensitivity was also observed in five out of eight other species analysed. In all sensitive species tested, spontaneous resistances to PT occurred. Under sterile conditions, PTT and PT reduced rhizobial nodulation rates of PT-resistant alfalfa plants drastically; however, nitrogen fixation in the few nodules that arose was unaffected. Because of the small number of nodules, the overall fixation rate was strongly diminished. In unsterile soil, nodulation and nitrogen fixation rates were not changed, possibly because of the rapid degradation of PTT and PT in the soil. Using a herbicide as model substance it could be demonstrated that the sensitivity of R. meliloti to chemical additives in the soil can be detected by analysing its growth rate, and that even a weak impact can influence its nodulation capacity. R. meliloti has proven to be a fast, easy and sensitive detection system for bacteriostatic components present in the soil. Received: 12 April 1996 / Received revision: 15 July 1996 / Accepted: 18 July 1996     

Salinity effects on germination,growth, and seed production of the halophyte Cakile maritima

The growth ofEucalyptus regnans seedlings in forest soil is enhanced when it has been air-dried. In undried forest soil seedlings grow poorly and develop purple coloration in the foliage, indicating P deficiency. This paper reports the results of pot experiments designed to investigate the relationship between growth and P acquisition, ectomycorrhizal infection and age of seedlings grown in air-dried and undried soil. The effect on seedling growth of their inoculation with air-dried or undried soil or with ectomycorrhizal roots from plants growing in air-dried or undried soil was also investigated. Ectomycorrhizal root tips were detected in 3-week-oldE. regnans seedlings in both air-dried and undried soil, and from then on the frequency of ectomycorrhizal root tips increased rapidly. In air-dried soil, seedlings were fully ectomycorrhizal at 9 weeks, and the occurrence of maximum ectomycorrhizal infection coincided with enhanced P acquisition and the initiation of rapid seedling growth. In undried forest soil, seedling growth remained poor, even though the seedlings had well-developed ectomycorrhizae and the incidence of ectomycorrhizal root tips was the same as in air-dried soil. The dominant ectomycorrhizae in airdried soil were associated with an ascomycete fungus, whereas in undried, undisturbed soil they were commonly associated with basidiomycete fungi. Inoculation of sterile soil/sand mix with washed ectomycorrhizal roots from air-dried soil increased the P acquisition and growth of the seedlings significantly compared with controls, whereas ectomycorrhizal inocula from undried soil had no effect on seedling growth, although both inocula resulted in a similar incidence of ectomycorrhizal root tips. Similarly, addition of a small amount of air-dried soil into sterile soil/sand mix resulted in a significantly greater increase in the P content and dry weight of the seedlings, compared with the control, than addition of undried soil. In both treatments, the incidence of ectomycorrhial root tips was similar. As (i) the differentiation in seedling growth between air-dried and undried soil occurred after seedlings became ectomycorrhizal, (ii) the dominant ectomycorrhizae in air-dried soil were different from those in undried soil, and (iii) inocula from air-dried soil, but not from undried soil, stimulated seedling growth in sterile soil/sand mix, it is concluded that development of particular ectomycorrhizae may be involved in seedling growth stimulation and enhanced P acquisition associated with air drying of forest soil.     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.     

Growth and nutrition of mycorrhizal guayule plants*

Synthesis of mycorrhiza in guayule plants was achieved by inoculation of 8-day-old seedlings with hyphae and chlamydospores of an undescribed Glomus species. There was a five-fold increase in total dry weight of 30-day-old mycorrhizal- compared to nonmycorrhizal-guayule grown in sterile loamy-sand without additional fertiliser. Thirty-day-old, inoculated- and uninoculated-seedlings were transplanted to sterile or unsterile soil and grown an additional 60 days. The greatest total dry weight of guayule was attained by inoculated transplants grown in sterile soil. Inoculated transplants increased two- to three-fold in total dry weight compared to uninoculated transplants, both grown in unsterile soil. After 90 days, uninoculated plants grown in unsterile soil had formed mycorrhizae with resident vesicular-arbuscular mycorrhizal fungi to the same extent as inoculated-plants grown in unsterile soil. Total mineral uptake increased in inoculated guayule, irrespective of soil treatment or the presence of resident VA mycorrhizal fungi.     

Distribution ofFrankia in soils from forest and afforestation sites in northern Sweden

Summary The presence in soil ofFrankia, capable of forming nitrogen-fixing root nodules onAlnus incana (L.) Moench, was investigated. Intact soil cores from forested as well as disturbed sites were sampled and both alder-rich and alder-free sites were included in the study. Surface-sterilized alder seeds were sown in the soil cores which were kept in sterile culture tubes in a growth chamber. Root nodules with nitrogenase activity developed in soil cores from all sites studied. Thus, infective and effectiveFrankia was present in all of the soils sampled, even from sites free from actinorhizal plants and irrespective of pH and nitrogen content of the soils.     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

Degradation of bromoxynil byStreptomyces felleus in soil

The completein vivo degradation of the herbicide bromoxynil byStreptomyces felleus and soil microorganisms was investigated. Little breakdown occurred in sterile soil. TLC techniques were used to detect two degradation products in non-sterile soil. Authors are obliged to Mrs. E. Chrastinová for technical assistance.     

Influence of fungal isolates on germination and growth of perennial ryegrass

Summary The influence of fungi isolated from perennial ryegrass roots on the germination and development of seedlings of perennial ryegrass was investigated. The basic procedure employed was to sterilise the seed surface and then inoculate with fungi and plant in non-sterile soil. It was realised that the fungal isolate inoculated on to the sterile seed surface would not remain dominant in the root region of the host and would have an influence on the host which would decline with time from when the seed germinated. This was because it would have to face antagonism from the normal components of the root microflora present in the non-sterile soil.Trichoderma viride delayed the emergence of the seedlings and reduced the production of herbage, an observation consistent with results of some other investigators. A sterile hyaline fungus stimulated the emergence of the seedlings, but subsequent tests showed that the presence of the microflora of the seed coat, or the soil microflora, or the sterile hyaline fungus was effective in promoting the rate of germination of seed that had been surface sterilised. Leaching seed in water brought about an increase in the rate of seed germination, and it is suggested that there might be a germination inhibitor soluble in water present in the seed coat, which might be inactivated by saprophytic micro-organisms.     

Intraspecific competition as a regulating factor limiting the number of transconjugants in soil

A laboratory study was carried out to determine survival of transconjugant cells ofPseudomonas fluorescens intro duced into sterile soil. The transconjugant survived significantly better when it was the only strain inoculated into the soil; when introduced into soil pre-colonized by the recipient strain, the transconjugant was undetectable. These results indicate that intraspecific competition is a regulating factor limiting the number of transconjugants in soil.     

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

Studies of Rhizobium meliloti isolated from salt-affected soils

Rhizobium meliloti strains have been isolated from salt-affected, non-cultivated soils of Greece. The effectiveness of the isolates was investigated in test-tubes using nitrogen-free salts agar or soil. In the latter case a new simple and rapid technique was used during which plants of Medicago saliva were grown in large test-tubes containing salt-affected soil under controlled sterile conditions. Field trial measurements in salt-affected soils indicate a good response to inoculation.     

Evidence for species‐specific plant responses to soil microbial communities from remnant and degraded land provides promise for restoration

Below‐ground interactions between soil microbial communities and plants play important roles in shaping plant community structure, but are currently poorly understood. Understanding these processes has important practical implications, including for restoration. In this study, we investigated whether soil microbes from remnant areas can aid the restoration of old‐fields, and whether soil microbes from an old‐field encourages further invasive establishment. In a glasshouse experiment, we measured growth and survival of two native grasses (Austrostipa nodosa and Rytidosperma auriculatum) and an invasive grass (Lolium rigidum) grown in sterile soil inoculated with whole soil from three locations: an old‐field, a remnant grassland, and a seed orchard planted with native grasses 7 years ago. Plants grown in sterile, non‐inoculated soil acted as controls. The orchard inoculant was included to test whether soil microbes from an area cultivated with native grasses induced plant responses similar to remnant areas. The remnant treatment resulted in the highest biomass and no mortality for R. auriculatum. All inoculant types increased the biomass of the invasive species equally. The native grass, A. nodosa, was the most sensitive to the addition of inoculum, whereas the invasive L. rigidum suffered very low mortality across all treatments. Overall, mortality was highest in the old‐field treatment at 42.9%. These results give insights into how soil microbes can affect community structure and dynamics, e.g. the high mortality of natives with old‐field inoculant may be one mechanism that allows invasive species to dominate. Poorer performance of native species with the orchard inoculant suggests it would not make a suitable replacement for remnant soil; therefore, more work is needed to understand the requirements of target species and their interactions before this technique can be exploited to maximum benefit.