Localisation of phosphomonoesterase activity in ectomycorrhizal fungi grown on different phosphorus sources

Phosphorus (P) is a major limiting nutrient for plants in boreal forest ecosystems where a substantial part of the total P is sequestered in organic compounds. Some ectomycorrhizal (ECM) fungi are known to produce phosphomonoesterases, enzymes that degrade organic P sources. Here, we test 16 ECM species for this enzymatic activity by growing them on media containing orthophosphate, phytic acid or apatite. A method with an overlay gel that determined both phosphomonoesterase activity and its spatial distribution was developed. The phosphomonoesterase activity was not significantly higher when growing on organic P; conversely some isolates only produced measurable enzyme activity when grown on apatite. Species-specific variations with respect to phosphomonoesterase activity as well as growth responses to different substrates were found. The production of phosphomonoesterases was found to be widespread in ECM fungi and the enzyme activity did not need induction by organic P. The enzyme activity was highest in the central parts of the mycelia, potentially reflecting breakdown and recycling of phospholipids from old hyphae or potentially higher mycelial density.     

Acid phosphomonoesterase and phytase activities of wheat (Triticum aestivum L.) roots and utilization of organic phosphorus substrates by seedlings grown in sterile culture

Wheat seedlings exhibited a differential ability to utilize P from a range of organic P substrates when grown in agar culture under sterile conditions. Plants showed limited ability to obtain P from inositol hexaphosphate (IHP), whereas other monoester substrates such as glucose 1‐phosphate (G1P), were equivalent sources of P for plant growth as compared with inorganic phosphate (P_i). Poor utilization of IHP was exemplified by significantly lower rates of dry matter accumulation and reduced P content of tissues, which were generally not significantly different to control plants that were grown in the absence of added P. The inability of wheat seedlings to obtain P from IHP was not associated with poor substrate availability but was due to either insufficient root phytase activity or inappropriate localization of phytase within root tissues. Phytase activities of 4 and 24 mU g ^{? 1} root fresh weight (FW) were determined for crude root extracts prepared from plants that were grown with either adequate P or under deficient conditions, respectively. Similar levels of phytase activity (approximately 12 mU g ^{? 1} FW) were observed in assays using intact roots, although no secreted activity was detected. By comparison, a secreted acid phosphomonoesterase activity was observed, and activities of between 466 and 1029 mU phosphomonoesterase g ^{? 1} root FW were measured for intact roots. On the basis of the differences in enzyme activity, and the observed differences in the ability of wheat seedlings to utilize G1P and IHP, it is evident that low intrinsic levels of phytase activity in wheat roots is a critical factor that limits the ability of wheat to obtain P from phytate when supplied in agar under non‐limiting conditions. This hypothesis was further supported by the observation that the ability of wheat to obtain P from IHP was significantly improved when the seedlings were inoculated with a soil bacterium (Pseudomonas sp. strain CCAR59) that possesses phytase activity.     

Barley genotypes differ in activity of soluble extracellular phosphatase and depletion of organic phosphorus in the rhizosphere soil

In the present investigation we studied the extent of variation among barley genotypes (Hordeum vulgare L. cv. Alexis, Canut, Digger, Etna, Peel) in their ability: i) to induce activity of soluble extracellular phosphatase in rhizosphere soil. ii) to withdraw bicarbonate extractable organic phosphorus (NaHCO₃-P₀). All the genotypes induced 3–4 times higher phosphatase activities in rhizosphere soil as compared to bulk soil. Among the genotypes, there were significant (p>0.01) differences in soluble extracellular and non-soluble phosphatase activities and depletion of NaHCO₃-P₀ in soil near their root mats. Etna induced highest phosphatase activities and depleted most NaHCO₃-P₀ from the rhizosphere soil. A high correlation (r=0.79) was found between the activity of soluble extracellular phosphatase and the quantity of NaHCO₃-P₀ withdrawn from the rhizosphere soil by the barley genotypes.     

Availability of phosphorus and sulfur of insecticide origin by fungi

Thirteen fungal species isolated from soil treated with pesticides were tested for their ability to mineralize and degrade three organophosphate insecticides currently used in Egypt (Cyolan®, Malathion® and Dursban®) in liquid media free from phosporus (P) and sulfur (S). All fungal species grew successfully on the culture media treated with the three used doses of insecticides (10, 50 and 100 ppm active ingredient) but the growth rate varied with the species, the insecticide and the doses. At 10 ppm level, insecticide degradation expressed in term of organic P mineralization (calculated as % of applied P) was the highest with all fungi tested. Organic P mineralization from pesticides was decreased by increasing the dose used to 50 and 100 ppm. The highest amount of P mineralized was observed with Cyolan® followed by Malathion® whilst P mineralization from Dursban® proceeded very slowly. Aspergillus terreus showed the greatest potential to mineralize organic P followed by A. tamarii, A. niger, Trichoderma harzianum and Penicillium brevicompactum whilst the remaining fungi only moderately mineralized the organic P component of the insecticides tested. Organic sulfur mineralization by the used fungal species paralleled, to some extent, organic P mineralization. The extracellular protein content of culture filtrates in the presence of various doses of insecticides was also decreased by increasing insecticide concentrations. The extracellular protein was significantly correlated with P and S mineralization (r = 0.89** and 0.64**, respectively) whilst correlation with cell dry mass was not significant (r = 0.03 and 0.003) suggesting a direct relationship between pesticide degradation and microbial protein production. The addition of P or S to the growth media enhanced extracellular protein excretion, and increased organic P and S mineralization by the most potent species tested (A. niger, A. tamarii, A. terreus and T. harzianum). This increment was significant in most cases, especially at the higher application rates. The relationship between extracellular protein excretion and organic P and S mineralization from insecticides was highly significant with the addition of inorganic phosphorus (r = 0.96** and 0.83**, respectively) or sulfur (r = 0.85** and 0.89**, respectively) to the growth media.     

低分子量有机酸对红壤无机态磷转化及酸度的影响

以鄂南、赣北两红壤样品为材料,加入不同有机酸并经室温培养后,测定不同P组分、pH及活性Al含量的变化。结果表明,供试有机酸均使土壤Ca2－P含量增高,增幅大小依次为柠檬酸＞苹果酸＞琥珀酸＞乙酸;2种土壤的Ca8－P和Ca10－P含量无明显变化规律,Fe-P、Al-P和O－P含量有所下降,除乙酸处理的土壤pH值无显著变化外,其它有机酸的加入使pH下降0．65－1．96;有机酸引起活性Al量增多,除乙酸处理的变化较小外,其它有机酸或混合物的加入使土壤中0．02mol.L^-1CaCl2提取Al增加4．7－50．3倍,1mol.L^-1提取Al增加4．0－67．3倍。可见,有机酸具有双重作用,既增加P的有效性,又增加土壤酸度和Al毒。     

Uptake and transport of phosphorus by Agrostis capillaris seedlings from rapidly hydrolysed organic sources extracted from32 P-labelled bacterial cultures

Cultures of the soil bacterium Serratia liquifaciens grimesii were grown with³² P labelled phosphate, to produce a uniformly³² P labelled source of microbial P. Extracts of the bacteria were prepared by sonication, dialysis and filtration to provide a clear sterile solution which was characterised in terms of dissolved organic and condensed P (DOP and DCP) and molecular weight range. The extract was used as a source of P to Agrostis capillaris L. seedlings in nutrient solution from which orthophosphate was omitted. In a time course experiment, root surface phosphatase activity increased as soon as extract was added to the root medium, DOP was rapidly hydrolysed and orthophosphate concentration increased rapidly. These processes were complete within about 8 h, after which phosphatase activity fell to its original level, and the plants absorbed molybdate reactive P from the nutrient solution so that it reached its original concentration over 48 h. DCP concentrations did not change significantly throughout the experiment. This work clearly demonstrated that DOP but not DCP, as a component of a bacterial extract produced by a relatively straightforward method, was quickly hydrolysed and the P made available for plant uptake.     

Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition

The potential of three arbuscular mycorrhizal fungi (AMF) to enhance the production of antioxidants (rosmarinic and caffeic acids, RA and CA) was investigated in sweet basil (Ocimum basilicum). After adjusting phosphorus (P) nutrition so that P concentrations and yield were matched in AM and non-mycorrhizal (NM) plants we demonstrated that Glomus caledonium increased RA and CA production in the shoots. Glomus mosseae also increased shoot CA concentration in basil under similar conditions. Although higher P amendments to NM plants increased RA and CA concentrations, there was higher production of RA and CA in the shoots of AM plants, which was not solely due to better P nutrition. Therefore, AMF potentially represent an alternative way of promoting growth of this important medicinal herb, as natural ways of growing such crops are currently highly sought after in the herbal industry.     

Response of strawberry to inoculation with arbuscular mycorrhizal fungi under very high soil phosphorus conditions

A field study was done to assess the potential benefit of arbuscular mycorrhizal (AM) inoculation of elite strawberry plants on plant multiplication, under typical strawberry nursery conditions and, in particular, high soil P fertility (Mehlich-3 extractible P=498 mg kg⁻¹). Commercially in vitro propagated elite plants of five cultivars (‘Chambly,’ ‘Glooscap,’ ‘Joliette,’ ‘Kent,’ and ‘Sweet Charlie’) were transplanted in noninoculated growth substrate or in substrate inoculated with Glomus intraradices or with a mixture of species (G. intraradices, Glomus mosseae, and Glomus etunicatum) at the acclimation stage and were grown for 6 weeks before transplantation in the field. We found that AM fungi can impact on plant productivity in a soil classified as excessively rich in P. Inoculated mother plants produced about 25% fewer daughter plants than the control in Chambly (P=0.03), and Glooscap produced about 50% more (P=0.008) daughter plants when inoculated with G. intraradices, while the productivity of other cultivars was not significantly decreased. Daughter plant shoot mass was not affected by treatments, but their roots had lower, higher, or similar mass, depending on the cultivar–inoculum combination. Root mass was unrelated to plant number. The average level of AM colonization of daughter plants produced by noninoculated mother plants did not exceed 2%, whereas plants produced from inoculated mothers had over 10% of their root length colonized 7 weeks after transplantation of mother plants and ∼6% after 14 weeks (harvest), suggesting that the AM fungi brought into the field by inoculated mother plants had established and spread up to the daughter plants. The host or nonhost nature of the crop species preceding strawberry plant production (barley or buckwheat) had no effect on soil mycorrhizal potential, on mother plant productivity, or on daughter plant mycorrhizal development. Thus, in soil excessively rich in P, inoculation may be the only option for management of the symbiosis.     

The biogeochemistry of phosphorus after the first century of soil development on Rakata Island, Krakatau, Indonesia

This study examined the accumulation of organic carbon (C) and fractions ofsoil phosphorus (P) in soils developing in volcanic ash deposited in the1883 eruption of Krakatau. Organic C has accumulated at rates of 45 to 127g/m²/yr during 110 years of soil development, resulting inprofiles with as much as 14 kgC/m². Most soil P is found inthe HCl-extractable forms, representing apatite. A loss of HCl-extractableP from the surface horizons is associated with a marked accumulation ofNaOH-extractable organic P bound to Al. A bioassay with hill rice suggeststhat P is limiting to plant growth in these soils, perhaps as a result ofthe rapid accumulation of P in organic forms.     

Plantago lanceolata L. and Rumex acetosella L. differ in their utilisation of soil phosphorus fractions

To establish relationships between soil phosphorus (P) fractions and leaf P, a mycorrhizal species (Plantago lanceolata L.) was compared with a typically non-mycorrhizal species (Rumex acetosella L.) in a glasshouse experiment. The plants were grown in 40 soils from non-fertilised, abandoned pastures or abandoned arable fields and leaf P concentration were found to be related to various soil P fractions after six weeks of growth. The differences in the P fractions in soil can account for a large share of the variation in leaf P concentration in both species, but the two species differed in their utilisation of P fractions. Leaf P concentration of R. acetosella was more related to extractable soil P than that of P. lanceolata. Rumex acetosella showed a higher maximum P concentration. The P fractions accounting for the largest share of the variation in leaf P concentration was the Bray 1 extractable and the weak oxalate (1 mM) extractable P, and for P. lanceolata also the Na₂SO₄+NaF extractable P fraction. P extracted with these methods accounted for up to 80% of the variation in P concentration in leaves of R. acetosella and 65% of the variation in leaves of P. lanceolata. More P extractable with weak oxalate, Na₂SO₄+NaF and strong oxalate (50 mM) was released from the soil than was taken up by the plants during the experimental period. The Bray 1 extractable P fraction, however, decreased in both unplanted and planted soils. Phosphatase release was not induced in any of the plants during the experimental period, indicating that they were not mobilising soil organic P. However, some of the methods extracted a large share of the organic P and still explained much of the variation in leaf P concentration. Mycorrhizal colonisation of P. lanceolata was inversely related to the extractable soil P. The consistently fast P uptake of R. acetosella indicates that this species have a high demand for P. The differences in P utilisation between R. acetosella and P. lanceolata could be caused by their different mycorrhizal status.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

Studies on the distribution of alkalophilic and alkali-tolerant soil fungi I

Soil fungi were isolated from two different soil types using alkaline and slightly acidic media (alkaline cornmeal agar (AC-MA), pH 9.7; cornmeal agar (CMA), pH 6.0) to study their distribution. Different species were obtained on each isolation medium. The number of species ofAcremonium andFusarium increased on ACMA, though many species growing well in acidic conditions were not detected on ACMA. Most of the fungi isolated on ACMA, especially from the alkaline soils, were alkalophiles or alkali-tolerants that can grow at pH 10.Acremonium alternatum, A. furcatum, Acremonium sp. 6,Gliocladium cibotii (YBLF 575),Phialophora geniculata, Stachylidium bicolor andStilbella annulata were alkalophilic, of whichAcremonium sp. 6 was the most pronounced alkalophile. Ability to grow under alkaline conditions, as well as under acidic conditions, was common in manyAcremonium species. The use of alkaline medium facilitates the isolation of alkalophilic soil fungi.     

Ecological significance of phosphomonoesters and phosphomonoesterase activity in a small Mediterranean river and its estuary

In 1998, the concentration of phosphomonoesters, a biologically available fraction of the dissolved organic phosphorus pool, was measured along Palmones River (a small Mediterranean river in Southern Spain) and its estuary. Due to the extremely low phosphomonoester concentration in the river (usually under detection limits by analytical procedures), a method using chromatographic cartridges to concentrate this compound was used. Phosphomonoester concentration was usually in the nanomolar range, although values up to 6 m were measured in a sampling station near an effluent of domestic sewage and a cellulose pulp plant. Concentrations were always lower than the dissolved reactive phosphorus (mostly orthophosphate). Phosphomonoesters represented a variable percentage of the dissolved organic phosphorus, from negligible percentages up to 56%. Besides, a highly significant correlation between these two phosphorus fractions was found.In addition, phosphomonoesterase (alkaline phosphatase) activity was measured in the water and macrophytobenthos (both algae and aquatic phanerogams) along the river and the estuary. In water, most of the phosphatase activity upstream was regarded as soluble. This was confirmed by the difficulty of performing kinetic studies in the unfiltered samples. On the contrary, the phosphomonoesterase activity in the estuary was attributed to different size fractions, suggesting that phosphomonoesters are used actively as a phosphate source by bacteria and phytoplankton, with relative contributions depending on the sampling period.     

Availability of phosphorus in a calcareous soil treated with rock phosphate or superphosphate as affected by phosphate-dissolving fungi

A study was undertaken to isolate some fungi exhibiting phosphate-dissolution ability, and to test whether these fungi are capable of increasing the amount of available P in a calcareous soil treated with rock phosphate (RP) or with triple superphosphate (TSP) and its subsequent uptake by sorghum (Sorghum bicolor L. Moench).Penicillium sp. and twoAspergillus foetidus (Naka) isolates significantly increased the availability of P in soil treated with RP or TSP during the growing season.Penicillium sp. isolate was more effective in increasing available P in the soil treated with RP or TSP than were Aspergillus isolates. However, the dry matter and P uptake responses to inoculation with these fungi were better in the soil treated with RP than in soil treated with TSP. In the TSP treated soil, the fungi achieved their maximum P releasing capacity two weeks earlier than in soil treated with RP. Positive and significant correlation coefficients among available P, P uptake and dry matter production at different periods of the growing season were observed following inoculation. However, none of these variables were found to be significantly correlated with the fungal populations.     

Biological and geochemical controls on phosphorus fractions in semiarid soils

This study examined the concentration of organic and inorganicphosphorus in surface soils of a Boutelouagracilis-Bouteloua eriopoda grassland, and a Larreatridentata shrubland, in the northern Chihuahuan Desert, NewMexico, U.S.A. In this desert where the grassland vegetation has auniform spatial distribution and individual shrubs have a patchy spatialdistribution across the landscape, vegetation strongly influences thedistribution of soil nutrients. Most studies of soil phosphorusfractions in desert soils have focused primarily on inorganic Pfractions and have demonstrated the importance of geochemical controlson soil P cycling. The research presented here addressed the question ofwhether organic phosphorus, determined by the presence of differentvegetation types, also contributes to soil P cycling. Within soils ofsimilar age, topography, parent material, and climatic regime, sampleswere collected under and between vegetation and analyzed for P fractionsfollowing a modified sequential fractionation scheme. Most soilinorganic P was found in the HCl- and cHCl-extractable forms in both thegrassland and shrubland soils, indicating CaCO₃ control overphosphorus availability. In contrast, most soil organic P was bound toAl and Fe minerals. Labile, plant-available P fractions summed to9.5% of total P in the grassland and 6.1% in theshrubland. Organic P comprised 13.3% of the total phosphorus poolin the grassland and 12.0% in the shrubland. Our results showthat the organic P pool may represent an important, yet oftenoverlooked, source of P in semiarid ecosystems.     

Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.     

Ectomycorrhizal fungi contribute to soil organic matter cycling in sub-boreal forests

Soils of northern temperate and boreal forests represent a large terrestrial carbon (C) sink. The fate of this C under elevated atmospheric CO₂ and climate change is still uncertain. A fundamental knowledge gap is the extent to which ectomycorrhizal fungi (EMF) and saprotrophic fungi contribute to C cycling in the systems by soil organic matter (SOM) decomposition. In this study, we used a novel approach to generate and compare enzymatically active EMF hyphae-dominated and saprotrophic hyphae-enriched communities under field conditions. Fermentation-humus (FH)-filled mesh bags, surrounded by a sand barrier, effectively trapped EMF hyphae with a community structure comparable to that found in the surrounding FH layer, at both trophic and taxonomic levels. In contrast, over half the sequences from mesh bags with no sand barrier were identified as belonging to saprotrophic fungi. The EMF hyphae-dominated systems exhibited levels of hydrolytic and oxidative enzyme activities that were comparable to or higher than saprotroph-enriched systems. The enzymes assayed included those associated with both labile and recalcitrant SOM degradation. Our study shows that EMF hyphae are likely important contributors to current SOM turnover in sub-boreal systems. Our results also suggest that any increased EMF biomass that might result from higher below-ground C allocation by trees would not suppress C fluxes from sub-boreal soils.     

Isolation of fungi from soil using the keratin-baiting technique

Of 236 soil samples baited with wool, some 71.2% (168) produced fungal growth. Gliocladium (25.0%), Paecilomyces (14.8%), Trichophyton (11.9%) species were the most prevalent in soil moistened with sterile distilled water (SDW) containing antibiotics. On the other hand, in soil moistened with SDW only, Trichophyton (32.6%), Paecilomyces (27.5%), Diheterospora (16.5%), Gliocladium (13.6%) and Fusarium (13.1%) species were more common. Of the known potential pathogens, the Paecilomyces and Fusarium species were frequently isolated from soil collected in parks, cleared areas, paddocks, rivers and roadsides. All five sites are areas of human and animal activity. The keratinolytic species were Microsporum cookei, M. gypseum complex, Trichophyton ajelloi and T. terrestre, which are regarded as nonpathogens with the exception of M. cookei and M. gypseum complex.