Fluorescence detection of aluminum in arbuscular mycorrhizal fungal structures and glomalin using confocal laser scanning microscopy

Arbuscular mycorrhizal spores and glomalin-related soil protein (GRSP) isolated from acid soils were analyzed using confocal laser scanning microscopy (CLSM) for Al detection. Mycorrhizal structures of Glomus intraradices produced under in vitro conditions as well as spores and GRSP from neutral and Cu-polluted soils were used as contrasting criteria. Spores and GRSP from soils with 7 and 70% Al saturation showed autofluorescence which increased especially at the highest soil Al level and when Al³⁺ solution was added. G. intraradices spores showed fluorescence only when exogenous Al³⁺ was added. On the contrary, spores and GRSP from neutral and Cu-polluted soils showed little or no significant fluorescence. This fluorescence shown by fungal structures and GRSP when subjected to high Al (of endogenous or exogenous origin) suggest a high capacity for Al immobilization, which could be an effective way to reduce Al activity and phytotoxicity in acid soils.     

Quantitative evaluation of the microbial nutrient sink in soil in relation to a model system for soil fungistasis

A quantitative approach was devised to evaluate the influence of soil microbial activity as a sink for nutrients exuded by fungal spores as a factor in soil fungistasis. The approach was based on measuring the CO₂ evolved from microbial respiration of ¹⁴C-labelled exudates from conidia of Cochliobolus victoriae incubated on soil. The amount of exudate lost by spores on soil was greater than the amount lost by spores incubated on a bed of sand undergoing leaching at a flow rate of 110 ml h^?1. where restriction of germination was similar to that on soil. Increasing flow rates in the leaching system increased spore exudation and reduced germination. Germination of C. victoriae conidia on membrane filters floated on distilled water decreased as the volume of water increased. The results indicate that the microbial nutrient sink of soil is sufficient to impose soil fungistasis.     

Assessment of Bacterial and Fungal Aerosol in Different Residential Settings

The concentration and size distribution of bacterial and fungal aerosol was studied in 15 houses. The houses were categorized into three types, based on occupant density and number of rooms: single room in shared accommodation (type I), single bedroom flat in three storey buildings (type II) and two or three bedroomed houses (type III). Sampling was undertaken with an Anderson six-stage impactor during the summer of 2007 in the living rooms of all the residential settings. The maximum mean geometric concentration of bacterial (5,036 CFU/m³, ± 2.5, n?=?5) and fungal (2,124 CFU/m³, ± 1.38, n?=?5) aerosol were in housing type III. The minimum levels of indoor culturable bacteria (1,557 CFU/m³, ±1.5, n?=?5) and fungal (925 CFU/m³, ±2.9, n?=?5) spores were observed in housing type I. The differences in terms of total bacterial and fungal concentration were less obvious between housing types I and II as compared to type III. With reference to size distribution, the dominant stages for culturable bacteria in housing types I, II and III were stage 3 (3.3–4.7 μm), stage 1 (7 μm and above) and stage 5 (1.1–2.1 μm), respectively. Whereas the maximum numbers of culturable fungal spores were recovered from stage 2 (4.7–7 µm), in housing type I, and from stage 4 (2.1–3.3 μm) in both type II and III houses. The average geometric mean diameter of bacterial aerosol was largest in type I (4.7 μm), followed by type II (3.89 μm) and III (1.96 μm). Similarly, for fungal spores, type I houses had the highest average mean geometric diameter (4.5 μm), while in types II and III the mean geometric diameter was 3.57 and 3.92 μm, respectively. The results indicate a wide variation in total concentration and size of bioaerosols among different residential settings. The observed differences in the size distributions and concentrations reflect their variable airborne behaviour and, as a result, different risks of respiratory exposure of the occupants to bioaerosols in various residential settings.     

Inoculation of seeds and soil with basidiospores of mycorrhizal fungi

It was demonstrated that basidiospores of the fungus Rhizopoyon luteolus, mycorrhizal for Pinus radiata, could be used successfully as seed inoculum after freeze-drying and storage for 3 months at 22°C, provided the inoculum level was increased 100-fold. Spore inoculum applied to seed could be held dry for at least 2 days before planting provided inoculum was increased 10-fold. On sowing freshly inoculated seed to sterile soil, 3 × 10³ basidiospores/seed were adequate for infection but maximum mycorrhizal infection occurred with 3 × 10⁴ spores/seed.A dose-response curve was obtained for mycorrhizal infection when basidiospores were applied to soil. As few as 100 spores/290 cm³ pot were sufficient for mycorrhizal infection although infection increased with greater spore dose to a maximum of 10⁵ spores/pot. Plant growth response was related to intensity of infection. It is suggested that the percentage germination of basidiospores in the rhizosphere may be considerably greater than those reported in studies with synthetic medium. A rhizosphere effect on germination of basidiospores was demonstrated and a method developed to facilitate studies of spore germination in the rhizosphere.     

Arbuscular mycorrhizal abundance in contaminated soils around a zinc and lead deposit

In order to study the variations in spore abundance and root colonization parameters of arbuscular mycorrhizal (AM) fungi in a naturally heavy metals polluted site and their relationships with soil properties, 35 plots in the Anguran Zn and Pb mining region were selected along a transect from the mine to 4500 m away. Within each plot, a composite sample of root and rhizospheric soil from a dominant indigenous plant was collected. The soil samples were analyzed for their physico-chemical characteristics. Spores were extracted, counted and identified at genus level. The roots were examined for colonization, arbuscular abundance, mycorrhizal frequency and intensity. Along the transect, the total and available (DTPA-extractable) concentration of Zn decreased from 6472 to 45 mg kg⁻¹ and 75 to 5 mg kg⁻¹, respectively. For Pb the values varied from 5203 to 0 mg kg⁻¹ and 32 to 0 mg kg⁻¹, respectively. In parallel, root colonization rate in the dominant native plants (except Alyssum sp.) varied from 35% to 85% and the spore numbers from 80 to 1306 per 200 g dry soil along the transect. Spores of Glomus were abundantly found in all plots as dominant, while Acaulospora spores were observed only in some moderately polluted and in control plots. AM fungal propagules never disappeared completely even in soils with the highest rates of both heavy metals. Spore numbers were more affected by Zn and Pb concentrations than root colonization. The variations of AM fungi propagules were better related to available than to total concentration of both metals. Spore numbers were positively correlated with mycorrhizal colonization parameters, particularly with arbuscular abundance.     

Assessing daily egestion rates in earthworms: using fungal spores as a natural soil marker to estimate gut transit time

Earthworms have an important role in ‘bioturbation’—the mixing of soil due to biological processes. Quantification of earthworm bioturbation relies on estimating earthworm egestion rates which in turn depend on two parameters: the gut content of the worms and the gut transit time (GTT). Gut content can be determined relatively easily, but determining GTT is problematic. The present study aimed at estimating daily soil egestion rates of Aporrectodea caliginosa and Lumbricus terrestris, refining the most common approach for estimating GTT by using fungal spores as natural markers in ingested soil. This approach avoids the use of artificial markers that may adversely affect the earthworms. Gut transit time was estimated by tracking the passage of marked soil through the gut by the appearance of the spores in the egested faeces. Gut transit time was estimated to be 9.6?±?0.3 h for A. caliginosa and 11.6?±?0.5 h for L. terrestris. Gut content averaged 465?±?40(± standard error (SE))?mg dw g^?1 dw worm for A. caliginosa and 265?±?80 mg dw g^?1 dw worm for L. terrestris. From these values, daily egestion rates of 1.16 and 0.66 g dw faeces g^?1 dw worm d^?1 were calculated for A. caliginosa and L. terrestris, respectively. Both values compare well to literature values for each species. The presented method for GTT estimation is inexpensive, rapid and easy to evaluate, with spores being a good alternative to existing markers.     

Hyphal growth from spores of the mycorrhizal fungus Glomus caledonius: Effect of amino acids

Hyphal growth from spores of Glomus caledonius (Nicol. and Gerd.) Trappe and Gerdemann was stimulated by cystine, glycine and lysine at optimum concentrations of 4.6, 556 and 825 mg l^?1 respectively. When all three amino acids were supplied together in water agar, five times more growth was obtained than in the controls.     

Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Soil texture and irrigation influence the transport and the development of Pasteuria penetrans, a bacterial parasite of root-knot nematodes

The transport of the spores of Pasteuria penetrans was studied in three contrasted textured soils (a sandy, a sandy-clay and a clay soils), cultivated with tomato, inoculated with juveniles of Meloidogyne javanica and watered with 25 or 150 mm day⁻¹. One month after inoculation of the nematodes, 53% of the spores inoculated were leached by water flow in the sandy soil but only 14% in the sandy-clay soil and 0.1% in the clay soil. No nematodes survived in the clay soil, while the population was multiplied both in the sandy and in the sandy-clay soils. But juveniles of M. javanica were more infected by P. penetrans in the sandy-clay soil than in the sandy soil. Comparing different combinations of bare soils containing 1.1-57% of clay showed that the best spore percolation and retention balance occurred in soils amended with 10-30% clay. However, the spore recoveries decreased when the soil was enriched with more than 30% clay. The role of clay particles on the extractability of spores and on their availability to attach to the nematode cuticle in the soil is discussed.     

Bacteria associated with Glomus clarum spores influence mycorrhizal activity

The effects of bacteria associated with arbuscular mycorrhizal fungal (AMF) spores on spore germination, growth in vitro and on the pea-AMF symbiosis were evaluated. Bacterial colonies were recovered from untreated Glomus clarum NT4 spores and NT4 spores decontaminated with 5% chloramine-T for 30, 45 and 60 min on five different media. Both G+ and G− bacteria were recovered from untreated NT4 spores, whereas only G+ bacteria were isolated from decontaminated spores. An in vitro assessment of the effect of spore-associated bacteria on clean, decontaminated NT4 spores revealed that (i) most of the bacteria isolated from untreated spores generally did not significantly alter spore function, (ii) some bacteria isolated from clean, decontaminated spores inhibited or stimulated NT4 spore germination, (iii) stimulation of spore germination occurred only when bacteria were in contact with spores, and (iv) inhibition of spore germination was the result of volatile bacterial metabolites. A stimulatory bacterial isolate, Bacillus pabuli LA3, significantly (P<0.05) enhanced the shoot growth, AMF-colonization, shoot N content and P use efficiency of NT4-inoculated 6 week-old pea plants over that of plants co-inoculated with an inhibitory bacterial isolate, Bacillus chitinosporus LA6a and NT4.     

Hyphal translocation and uptake of sulfur by vesicular-arbuscular mycorrhizae of onion

Translocation of S by external hyphae of Glomus fascieulatus, a vesicular-arbuscular (VA) mycorrhizal fungus, was demonstrated. When tracers were injected 8 cm from onion roots in soil chambers, both ³⁵S and ³²P appeared in roots of mycorrhizal plants. Neither radionuclide was present in non-mycorrhizal plants.In a second soil-chamber experiment, mycorrhizal onions took up more ³⁵S per unit dry weight than non-mycorrhizal controls when ³⁵S was injected into soil chambers in a region 3–6 cm from roots. Severing of external hyphae between the application area and the roots reduced the concentration of ³⁵S in tops of mycorrhizal plants but not in roots. Volatile ³⁵S per unit dry weight collected from all plants in each treatment was highest in the mycorrhizal-hyphae intact treatment, and higher in the mycorrhizal-hyphae severed treatment than the non-mycorrhizal treatment. Movement of ³⁵S in soil from the area of application to roots was similar for all treatments.Increased uptake of S from soil by VA mycorrhizal plants can result from hyphal translocation of S to infected roots by external mycorrhizal hyphae.     

Development of actinomycetes in brown semidesert soil under low water pressure

Under laboratory conditions, the spores of a xerotolerant Streptomyces odorifera strain germinated in brown semidesert soil even at extremely low soil water pressure (P = ?96.4 MPa, ?964 atm, a _w 0.50); the plantlets increased in length and formed mycelium, on which a new generation of spores was produced (a complete development cycle of the actinomycetes??from a spore to the formation of new spores??passed). The duration of the first cycles of the actinomycetes?? development varied from 13 days at P = ?27 atm to 57 days at P = ?964 atm and was directly proportional to the absolute value of the soil water pressure (P). In the first cycles of the actinomycetes?? development, the rate of increase of the concentration of the germinated spores and mycelium, as well as the logarithms of the mycelium-to-germinated spore concentration ratios, was inversely proportional to the logarithm of P. These relationships indicated that the energy state of the water determined its availability to soil biota and, hence, the activity of its physiological and biochemical processes.     

Fungal air-spora inside the central library of Gorakhpur University

The study of the air-spora in the Central Library of Gorakhpur University indicated the presence of 19 different types of fungal spores. Amongst these Aspergillus niger, A. favus, Cladosporium spp., Curvularia spp., Paecilomyces sp., Penicillium spp., and periconia sp. were more than 80.5% followed by species of Absidia, Alternaria, Chaetomium, Mucor, Rhizopus, Trichophyton, and Cercospora. Fungal spores showed time trends during the study period. The fungal spore concentrations gradually increased from July to September and attained their maximum in September to October and thereafter decreased. Besides fungal spores, small pieces of paper, dust particles and insect derivatives loaded with fungal mycelium and bacterium were also observed throughout the investigation period.     

Species-dependent partitioning of C and N stable isotopes between arbuscular mycorrhizal fungi and their C3 and C4 hosts

Natural ¹³C and ¹⁵N abundances of mycorrhizal fungi are increasingly used in ecology but reference data on arbuscular mycorrhizal fungi (AMF) are scarce. In experiments with nine phylogenetically dispersed AMF strains inoculated on leek (C3 plant) and sorghum (C4) in pot cultures, we measured the ¹³C/¹²C and ¹⁵N/¹⁴N ratios in shoots, roots, AMF spores as well as carbon isotope signature of the C16:1ω5 fatty acid (FA), which is diagnostic for AMF. Spore δ¹³C values varied among AMF strains on any given host. They were significantly lower than shoot δ¹³C for sorghum (−2.5‰ on average) while for leek, no clear C isotope partitioning between spores and host shoots was observed. The FA C16:1ω5 fatty acids were more ¹³C-depleted than spores, without correlation with spore δ¹³C values. For both, sorghum and leek, spore δ¹⁵N was higher (+1–2‰ on average) than for shoots. We found no evidence that isotopic partitioning between the partners drives ¹³C and ¹⁵N abundances in plant–AMF symbiosis. Mycorrhizal roots displayed relatively high δ¹³C typical for heterotrophic organs, and not a mix between AMF and plant signatures. Interestingly, inoculation slightly decreased shoot δ¹³C on leek but not on sorghum, as compared with non-mycorrhizal plants, suggesting that AMF improved the plant's water status, a parameter affecting the δ¹³C of C3 but not C4 plants. Phylogenetically closer AMF displayed more similar spore δ¹³C and induced similar ¹³C and ¹⁵N abundances on leek shoots, but this observation was not confirmed for sorghum. Plant and AMF isotopic abundances hardly correlated with other parameters related to plant development, mineral nutrition or root mycorrhizal colonisation, and these correlations were never consistent between sorghum and leek. Thus, isotopic abundances in plant–AMF symbiosis were rather constrained by each AMF/plant interaction. Nevertheless, our data provide a valuable reference for future investigations of AMF communities and AM symbiosis in situ.     

Late Quaternary environmental change inferred from phytoliths and other soil-related proxies: Case studies from the central and southern Great Plains,USA

This study investigates stable carbon isotopes (δ¹³C), opal phytolith assemblages, burnt phytoliths, microscopic charcoal and Sporormiella spores from modern soils and paleosols in Kansas and Oklahoma. Grass and dicot phytoliths in combination with δ¹³C are used as proxies for reconstructing the structure of grasslands and woodlands. Burnt grass phytoliths and microscopic charcoal are evaluated as proxies for reconstructing paleofire incidence. Concentrations of the fungal spore Sporormiella are used as a proxy for assessing large herbivore activity. These proxies were tested on various modern grassland communities of the central and southern Great Plains, including areas with bison, cattle, and small herbivores, and areas under different fire frequencies.     

Blue-green algae or Azolla additions on the nitrogen and phosphorus availability and redox potential of a flooded rice soil

Freshly-harvested blue-green algae (20 t ha^?1) or Azolla (20 t ha^?1) were incorporated into a waterlogged rice soil and changes of redox-potential and the availability of nitrogen and phosphorus were measured periodically. The soil progressively became reduced where blue-green algae or Azolla had been incorporated. The rate of N release from Azolla was more rapid than from blue-green algae and the amounts released after 7–35 days of flooding were between 41 and 76% and 12 and 35% respectively. Increased P availability was also recorded in soil following the incorporation of blue-green algae or Azolla and reached highest values after 21 days incubation.     

Fluorescence detection of aluminum in arbuscular mycorrhizal fungal structures and glomalin using confocal laser scanning microscopy

Arbuscular mycorrhizal spores and glomalin-related soil protein (GRSP) isolated from acid soils were analyzed using confocal laser scanning microscopy (CLSM) for Al detection. Mycorrhizal structures of Glomus intraradices produced under in vitro conditions as well as spores and GRSP from neutral and Cu-polluted soils were used as contrasting criteria. Spores and GRSP from soils with 7 and 70% Al saturation showed autofluorescence which increased especially at the highest soil Al level and when Al³⁺ solution was added. G.intraradices spores showed fluorescence only when exogenous Al³⁺ was added. On the contrary, spores and GRSP from neutral and Cu-polluted soils showed little or no significant fluorescence. This fluorescence shown by fungal structures and GRSP when subjected to high Al (of endogenous or exogenous origin) suggest a high capacity for Al immobilization, which could be an effective way to reduce Al activity and phytotoxicity in acid soils.     

Nature of the transient activation of soil microorganisms by ethanol or acetaldehyde

Low molecular weight alcohols and aldehydes emanating from undecomposed plant residues cause a rapid activation of microbial respiration and growth in soil. To determine the relative utilization of added volatile or endogenous microbial reserves during a transient period of activation, ethanol-1,2-¹⁴C or acetaldehyde was added to soil. With concentrations of ethanol giving near maximal respiration, 80–90 per cent of the CO₂ evolved in excess of the control came from labelled ethanol. For comparison, similar experiments were performed with spores of Fusarium solani under conditions where ethanol was a required substrate for germination. About 70–90 per cent of the CO₂ evolved in excess of the control came from added ethanol. In both the soil and spore systems enhanced respiration continued for less than 5 hr and ceased when the volatile substrate was depleted. Few endogenous reserves were utilized, and substantial amounts of cellular materials apparently were accumulated during this process. No changes in numbers of soil microorganisms were observed either during the transient activation or during the subsequent 2 weeks.     

影响丛枝菌根真菌孢子萌发的几种因素研究

对丛枝菌根真菌孢子萌发的几种影响因素进行了研究。结果表明,土壤是丛枝菌根真菌孢子萌发最适宜的培养基;寄主植物根的分泌物对孢子萌发有显著的促进作用。重金属Cd和Pb含量过高时(50mg kg)抑制真菌孢子的萌发。培养基中有效P含量较低时(KH2PO4添加量为0～80mg L),对孢子萌发影响较小,高浓度的有效P(KH2PO4添加量大于100mg L)对孢子萌发有一定的抑制作用。培养基的pH值过高(pH8.0以上)或过低(pH5.5以下)抑制孢子萌发。生长激素对孢子萌发率没有显著性影响。对于有休眠现象的丛枝菌根真菌,4℃低温处理4～6周,可打破休眠孢子的休眠,显著提高休眠孢子的萌发率。     

Relative contributions of sedimentation and impaction to deposition of particles in a crop canopy

Lycopodium spores were released steadily into the air during 20–30 min from a line source positioned within a wheat crop. The spores were trapped on sticky strips held at angles, π, of 0, 30, 60 and 90° with respect to the horizontal and oriented to face the mean wind direction and on sticky, vertical glass rods. The aerial spore concentration, C, was measured by small suction traps. Deposits of the spores on wheat leaves were obtained from sections of leaves whose posture in the canopy was nearly horizontal, nearly vertical, or at angles between 30 and 60°. Number of spores per m² for all trapping surfaces were obtained by counting under a microscope. Experiments were conducted on seven different days, encompassing friction velocities, u^*, of 0.27–0.50 m s⁻¹. The rate of deposition on angled surfaces, D(π), was given approximately by D(θ) = D(0) cos (θ) + D(90) sin (θ), where D(0) and D(90) were the observed rate of deposit on horizontal and on vertical surfaces, respectively. Below mid-canopy height, inertial impaction of spores was negligible, so that D(90) = 0 for all the trap surfaces. There, D(0) was mainly due to sedimentation and was very nearly equal to v_s·C, where v_s is the settling speed of the spore in still air. Near the top of the canopy, deposition on sticky surfaces was enhanced by inertial impaction and turbulent deposition, so that D(0) was about twice that expected from sedimentation and D(90) was about five times larger than expected from inertial impaction at the mean wind speed. Nevertheless, considering the vertical distribution of leaf area and the angles of leaves in a wheat canopy, the rate of deposition of spores for the entire depth of a wheat canopy can be calculated with a probable underestimation of only 20% by simply assuming sedimentation on horizontally projected area and impaction on vertically projected area.