Continuous and long-term monoxenic culture of the arbuscular mycorrhizal fungus Gigaspora decipiens in root organ culture

Establishment of arbuscular mycorrhizal (AM) germplasm collections is complex because of the obligate biotrophic nature of AM fungi. Only a few AM species are routinely maintained in monoxenic culture with Ri T-DNA transformed roots as host. Incorporation of new AM species into this culture system is important for molecular, physiological, and taxonomical studies. Here we report for the first time the successful monoxenic culture of Gigaspora decipiens (JA2 strain) with transformed carrot (Daucus carota) roots. In vitro cultures were established from field-collected spores; sub-culture of newly in vitro formed spores was established over five successive generations for a period of 6 y. Although initial culture of field-collected spores was difficult successive sub-cultures appeared to be adapted to the in vitro growing conditions. The JA2 strain of G. decipiens completed its life cycle while maintaining its morphological characteristics, stability, and propagule viability under the monoxenic conditions over several generations. This stable and homogeneous monoxenic material obtained for G. decipiens is part of the Banco de Glomeromycota In Vitro (BGIV, http://www.bgiv.com.ar), and could facilitate morphological, physiological, and molecular analysis of this AM species.     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Effects of above-ground browsing by mammals on mycorrhizal infection in an early successional taiga ecosystem

Using an exclosure experiment in the willow stage of primary succession on the floodplain of the Tanana River, we tested the hypothesis that browsing can reduce mycorrhizal infection. We measured the effects winter browsing by moose (Alcesalces) and snowshoe hare (Lepusamericanus) had on mycorrhizal infection and fine root biomass of willow (Salix spp.) and balsam poplar (Populusbalsamifera). We found that protection from winter browsing increased ectomycorrhizal infection by 10% in the top 5 cm of the soil profile, by 23% at 5–10 cm, and by 42% at the 10–15 cm depth. Mammal browsing in taiga forests is now recognized as a major cause of the shift from palatable deciduous species such as willow and balsam poplar to less palatable species such as alder and spruce. We suggest that browsing-induced reduction in ectomycorrhizal infection of salicaceous species plays a central role in this shift in plant community composition. Received: 26 March 1996 / Accepted: 26 September 1996     

Effect of an AM fungal consortium and Pseudomonas on the growth and nutrient uptake of Eucalyptus hybrid

 Eucalyptus is an important tree species used for afforestation of large tracts of marginal and wastelands. Eucalyptus-arbuscular mycorrhizal fungal (AMF) interactions in seedling establishment and growth promotion have been inadequately dealt with. Efforts were made to assess the role of AMF-pseudomonad (PRS9, plant growth promotory fluorescent Pseudomonas) interactions in growth promotion and nursery establishment of E. hybrid. Seedlings were subjected to six different treatments: (i) uninoculated control, (ii) 400 AM spores, (iii) 800 AMF spores, (iv) PRS9 (v) 400 AMF spores + PRS9, (vi) 800 AMF spores + PRS9, with the different P regimes of 10, 20 and 30 ppm. Root length, shoot length, root and shoot fresh and dry weights were maximal at 400 AMF spores and 20 ppm soil P. Shoot P content was maximal at 800 AMF spores followed by 400 AMF spores and 400 AMF spores + PRS9. In general, plant growth was greater at 20 ppm P. Root P content increased significantly with 400 AMF spores followed by 800 at 20 ppm P. Independent of soil P levels, the quality index of mycorrhizal treatments without PRS9 was significantly higher than the treatments including PRS9 or PRS9 alone. Mycorrhizal inoculation efficiency was superior at 10 ppm P irrespective of the treatment. AM alone (400 spores) significantly improved the inoculation efficiency. PRS9 in association with AM fungi inhibited growth promotion and nutrient uptake Accepted: 8 September 1999     

Accumulation of sesquiterpenoid cyclohexenone derivatives induced by an arbuscular mycorrhizal fungus in members of the Poaceae

Sixty one members of the Poaceae, including various cereals, were grown in defined nutrient media with and without the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenk & Smith. The roots of all species investigated were colonized by the AM fungus, however, to different degrees and independent of their systematic position. High-performance liquid chromatographic analyses of methanolic extracts from the roots of mycorrhizal and nonmycorrhizal species revealed dramatic changes in the patterns of UV-detectable products along with a widespread occurrence of AM-fungus-induced accumulation of sesquiterpenoid cyclohexenone derivatives. The latter occur most often in the tribes Poeae, Triticeae and Aveneae. Some additional control experiments on plant infection with pathogens (Gaeumannomyces graminis) and Drechslera sp.) or an endophyte (Fusarium sp.), as well as application of abiotic stress, proved that the metabolism of these terpenoids is part of a response pattern of many gramineous roots in their specific reaction to AM fungal colonization. Received: 23 October 1996 / Accepted 11 December 1996     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 July 1996     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19

The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H₂O₂ required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable. Received: 16 September 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Distribution of arbuscular mycorrhizal fungi in stands of the wetland grass Panicum hemitomon along a wide hydrologic gradient

Although wetland plant species usually aggregate into zones that correspond with their water depth/dryness tolerances, it is not known whether associated arbuscular mycorrhizal (AM) fungi show a similar zonation. We assessed the distribution of AM fungi in two similar depressional wetlands dominated by the semi-aquatic grass Panicum hemitomon by sampling soil in plots along dry-to-wet gradients that spanned 80 cm in relative elevation, and identifying/counting viable AM fungal spores. We found that eight of nine AM fungal species were common to both of the wetlands. Within each wetland, there were significant differences in species composition related to relative water depth. The zonation patterns were not identical between wetlands but revealed that certain species were relegated to the drier portions of the gradient in both. No species were relegated to only the wet portions of the wetlands; those that dominated there were also present in the drier areas. Our data show that water depth is an important factor determining the distribution of the AM fungi, even when, as in our study wetlands, the host plant remains constant along a dry-to-wet gradient. This suggests that the fungi are not physiologically equivalent in their tolerance to wetland conditions. Received: 9 October 1998 / Accepted: 24 February 1999     

Influence of edaphic and climatic factors on dynamics of root colonization and spore density of vesicular-arbuscular mycorrhizal fungi in Acacia farnesiana Willd. and A. planifrons W.et.A.

 A study was conducted to assess the dynamics of vesicular-arbuscular mycorrhizal (VAM) fungi associated with Acacia farnesiana and A. planifrons in moderately fertile alkaline soils. The intensity of root colonization by VAM fungi and the distribution of VAM fungal structures varied with host species over a period of time. The occurrence of vesicles with varied morphology in the mycorrhizal roots indicates infection by different VAM fungal species. This was further confirmed from the presence of spores belonging to different VAM fungal species in the rhizosphere soils. Root colonization and spore number ranged from 56% – 72% and 5 – 14 g^{–  1}soil in A. farnesiana and from 60% – 73% and 5 – 15 g^{–  1} soil in A. planifrons. Per cent root colonization and VAM spore number in the rhizosphere soil were inversely related to each other in both the Acacia species. However, patterns of the occurrence of VAM fungal structures were erratic. Spores of Acaulospora foveata, Gigaspora albida, Glomus fasciculatum, G. geosporum and Sclerocystis sinuosa were isolated from the rhizosphere of A. farnesiana whereas A. scrobiculata, G. pustulatum, G. fasciculatum, G. geosporum and G. microcarpum were isolated from that of A. planifrons. The response of VAM status to fluctuating edaphic factors varied with host species. In A. farnesiana though soil nitrogen (N) was positively correlated with root colonization, soil moisture, potassium and air temperature were negatively correlated to both root colonization and spore number. Per cent root colonization and spore number in A. planifrons were negatively related to each other. Further, in A. planifrons as the soil phosphorus and N were negatively correlated with the density of VAM fungal spores, the same edaphic factors along with soil moisture negatively influenced root colonization. Received: 16 May 1995 / Accepted: 7 February 1996     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

Effects of directly soluble and fibrous rapidly acidifying chemical oxygen demand and reactor liquid surface tension on granulation and sludge-bed stability in upflow anaerobic sludge-blanket reactors

 In recent years, it has become clear that the rapidly acidifying chemical oxygen demand (RACOD) content of the waste water and the surface tension of the reactor liquid contribute to the phenomenon of granular growth in upflow anaerobic sludge-blanket reactors (UASB). By adding 20% of directly soluble RACOD, in the form of a sucrose/starch mixture, on top of the original COD load and by adjusting the reactor liquid surface tension below 50 mN m⁻¹ with linear alkylbenzenesulphonate, granular growth and sludge-bed stability could be enhanced significantly within 40 days. Carrot pulp, a waste product having a high short-chain fatty acid precursor potential, was applied as an alternative fibrous RACOD source. Best results were obtained when adding the carrot pulp freshly to the laboratory-scale UASB reactor in an in-recycle liquefying chamber. This concept of adding carrot pulp waste product as a granular growth supplement by means of an in-recycle liquefying chamber therefore merits testing in practice. Received: 30 October 1996 / Received version: 3 February 1997 / Accepted: 10 February 1997     

Growth responses and dependence of Acacia nilotica var. cupriciformis on the indigenous arbuscular mycorrhizal consortium of a marginal wasteland soil

 The responses of Acacia nilotica L. var. cupriciformis to phosphorus application and inoculation with the indigenous consortium of arbuscular mycorrhizal (AM) fungi were evaluated in a nursery experiment using soil from a marginal wasteland. A positive growth response to mycorrhizal inoculation was observed at an Olsen-P level of 20 ppm in the presence of the natural population of AM fungi. There was growth stimulation by either inoculation or additional P at the highest soil P of 40 ppm. Colonization was negatively correlated to soil P but P content of both shoot and root were positively correlated. Inoculation with the indigenous AM consortium significantly increased the uptake of P at all levels of applied P. Acacia is moderately dependent upon the AM symbiosis and exhibited a maximal mycorrhizal dependence (MD) of 18.25% at 20 ppm Olsen-P level under the conditions studied. A sharp and considerable reduction in MD and dry matter yield observed at 40 ppm P suggests that the external P requirement for maximal production of biomass was met at approximately 20 ppm Olsen-P. Accepted: 25 June 1996     

Stabilization and increased production of insecticidal crystal proteins of Bacillus thuringiensis subsp. galleriae in steady- and transient-state continuous cultures

Bacillus thuringiensis subsp. galleriae, grown in continuous cultures, segregated to spontaneous asporogenic variants replacing the wild-type Spo⁺ Cry⁺ strains [Sachidanandham R, Jayaraman K (1993) Appl Microbiol Biotechnol 40:504–507]. Realizing that this was due to specific but unknown nutritional requirements, we undertook further continuous-culture studies to identify growth requirement(s) by pulsing various medium components and growth factors. While carbon, nitrogen and pulses of nutrients exhibited a neutral pulse response, a group of amino acids were shown to improve the stability and volumetric productivity of biomass. The formation of spores and insecticidal crystal proteins was found to be higher with amino acid supplementation. Comparison of carbon-limited steady-state continuous cultures under two different conditions of growth brought forth the stabilizing effects of the amino acid supplementation. Batch experiments carried out with these inputs demonstrated a better carbon utilization, resulting in a higher biomass as well as enhancement of bioinsecticidal activity. Received: 14 May 1996 / Received revision: 9 September 1996 / Accepted: 13 September 1996     

Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10⁻⁵−10⁻⁴. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml). Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996     

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10⁻⁵–10⁻⁷ exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50 % of larvae (LC₅₀) being 2.1 × 10⁵ and 1.3 × 10⁵ cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth. Received: 23 July 1996 / Received revision: 11 November 1996 / Accepted: 15 November 1996     

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol

An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water. The application of an alternating potential of 1.0 V and −0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h. Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately. The bacterial cell density in treated water was always been less than 20 cells/ml. It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling. Received: 19 February 1996 / Received last revision: 23 July 1996 / Accepted: 2 September 1996     

Soil biota responses to long-term atmospheric CO2 enrichment in two California annual grasslands

Root, arbuscular-mycorrhizal (AM), soil faunal (protozoa and microarthropods), and microbial responses to field exposure to CO₂ for six growing seasons were measured in spring 1997 in two adjacent grassland communities. The grasslands showed contrasting root responses to CO₂ enrichment: whereas root length was not affected in the sandstone grassland, it was greater in the serpentine grassland, as was specific root length. AM fungal hyphal lengths were greater in the sandstone, but were unaffected in the serpentine community. This lent support to the hypothesis that there may be a tradeoff in resource allocation to more fine roots or greater mycorrhizal extraradical hyphal length. AM root infection was greater in both communities at elevated CO₂, as was the proportion of roots containing arbuscules. Our data on total hyphal lengths, culturable and active fungi, bacteria, and protozoa supported the hypothesis that the fungal food chain was more strongly stimulated than the bacterial chain. This study is one of the first to test these hypotheses in natural multi-species communities in the field. Received: 23 October 1998 / Accepted: 19 February 1999     

Transformation of tetrachloroethene in an upflow anaerobic sludgeblanket reactor

  Reductive dechlorination of tetrachloroethene was studied in a mesophilic upflow anaerobic sludge blanket reactor. Operating the reactor in batch mode the dynamic transformation of tetrachloroethene, trichloroethene and dichloroethene (DCE) was monitored. Tetrachloroethene was reductively dechlorinated to trichloroethene, which again was dechlorinated at the same rate as DCE was produced. DCE showed a lag period of 40 h before transformation was observed. During normal reactor operation trans-1,2-DCE was the major DCE isomer, followed by cis-1,2-DCE. Small amounts of 1,1-DCE but no vinyl chloride were detected. When the influent tetrachloroethene concentration was increased from 4.6 μM to 27 μM, the transformation rate increased, indicating that the system was not saturated with tetrachloroethene. The main organic component in the effluent was acetate, indicating that the aceticlastic methane-producing bacteria were inhibited by the chlorinated ethenes. Received: 29 July 1996 / Received revision: 13 September 1996 / Accepted: 13 September 1996     

Microbially influenced corrosion of glass

Microbially influenced corrosion of glass and countermeasures were studied by literature research and by an investigation of contaminated glass samples. Such corrosion is often linked to the growth of fungi. It is assumed that glass composition is not the decisive factor in biogenic contamination, but can encourage or retard the microbial activity significantly. It is suggested that a biochemically initiated ion-exchange reaction is most important for corrosion of glass exposed to the atmosphere. Future work will emphasize the biotechnological applications in the recovery of heavy metals from special glasses. Received: 26 September 1996 / Received revision: 17 December 1996 / Accepted: 17 December 1996     

Reversal of the inhibitory effect of surfactants upon germination and growth of a consortium of two strains of Bacillus

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth. Received: 9 July 1998 / Received revision: 26 October 1998 / Accepted: 30 October 1998