Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues

A "universal non-metazoan" polymerase chain reaction (UNonMet-PCR) that selectively amplifies a segment of nonmetazoan Small Subunit (SSU) rDNA gene was validated. The primers used were: 18S-EUK581-F (5'-GTGCCAGCAGCCGCG-3') and 18S-EUK1134-R (5'-TTTAAGTTTCAGCCTTGCG-3') with specificity provided by the 19-base reverse primer. Its target site is highly conserved across the Archaea, Bacteria, and eukaryotes (including fungi), but not most Metazoa (except Porifera, Ctenophora, and Myxozoa) which have mismatches at bases 14 and 19 resulting in poor or failed amplification. During validation, UNonMet-PCR amplified SSU rDNA gene fragments from all assayed protists (n = 16 from 7 higher taxa, including two species of marine phytoplankton) and Fungi (n = 3) but amplified very poorly or not at all most assayed Metazoa (n = 13 from 8 higher taxa). When a nonmetazoan parasite was present in a metazoan host, the parasite DNA was preferentially amplified. For example, DNA from the parasite Trypanosoma danilewskyi was preferentially amplified in mixtures containing up to 1,000 x more goldfish Carassius auratus (host) DNA. Also, the weak amplification of uninfected host (Chionoecetes tanneri) SSU rDNA did not occur in the presence of a natural infection with a parasite (Hematodinium sp.). Only Hematodinium sp. SSU rDNA was amplified in samples from infected C. tanneri. This UNonMet-PCR is a powerful tool for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.     

Effect of forest fire on number, viability and post-fire re-establishment of arbuscular mycorrhizae

 Forest fire can affect arbuscular mycorrhizal (AM) fungi by changing the soil conditions and by directly altering AM proliferation. We studied the effects of a severe forest fire at Margalla Hills near Islamabad on the number and viability of AM fungal propagules in the burnt soil and their role in the re-establishment of post-fire infection in colonized plants. Compared with a nearby control area, the burnt site had a similar number of total spores but a lower number of viable AM fungal propagules. The roots of the two most frequent species at the burnt site, Dodonaea viscosa and Aristida adscensionis, showed a gradual increase in percentage root length colonized by AM fungi in general and hyphal infection in particular. Our results indicate resumption of mycorrhizal activity following the fire, probably from AM hyphae in the roots of these dominant shrubs. Accepted: 18 July 1997     

Effects of inoculation of phosphate-solubilizing microorganisms and an arbuscular mycorrhizal fungus on mungbean grown under natural soil conditions

 The effect of inoculation of the phosphate-solubilizing microorganisms (PSM) Bacillus circulans and Cladosporium herbarum and the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum with or without Mussoorie rockphosphate (MRP) was studied in a P-deficient natural non-disinfected sandy soil on mungbean (Vigna radiata). The AM levels increased following the addition of MRP or inoculation with PSM or G. fasciculatum. Both grain and straw yield of mungbean increased following inoculation with PSM or the AM fungus. In general, the increase in yield was higher in the presence of MRP and inoculation with a combination of PSM and AM fungus. Highest N and P uptake by mungbean was recorded after treatment with a combination of B. circulans, C. herbarum and G. fasciculatum in the presence of MRP. Generally the PSM population increased after AM fungus inoculation. Accepted: 13 October 1997     

Seasonality of mycorrhizae in coastal sand dunes of Baja California

 Populations of arbuscular mycorrhizal fungi were estimated from spores associated with seven plant species in coastal dunes at El Socorro, near Ensenada, Baja California, during six months in 1992. The seasonal patterns of percent root colonization were also described in the same species during the wet season (January–March) and the dry season (April–July). Comparisons were made between the pioneer species (Abronia maritima) in the mobile dunes and six species (Abronia umbellata, Atriplex julacea, Camissonia californica, Haplopappus venetus, Helianthus niveus and Lotus spp.) in the fixed dunes. Mycorrhizal colonization in Abronia maritima was slight (<1%) and we observed few spores (<1/g soil). All of the species in the fixed dune formed mycorrhizae with up to 80% colonization in early summer, and no more than 4 spores/g soil by late summer. The highest percentages of total colonization and abundance of spores did not coincide temporally for any of the seven species, but the percentages were higher in summer than in spring. Arbuscules were more abundant when the soil was moist, and vesicles more abundant when it was dry. Accepted: 16 August 1995     

A method for direct soil extraction and PCR amplification of endomycorrhizal fungal DNA

 DNA from endomycorrhizal fungi was extracted directly from a weathered soil (alfisol) mixed with sand. Mycorrhizae were established in a greenhouse culture of Glomus clarum with Sudan grass (Sorghum vulgare var. sudanense) host plants. The extraction procedure included enzymatic digestion of cell walls, sodium dodecyl sulfate lysis of cells, polyvinylpolypyrrolidone absorption of organic compounds, and ethanol precipitation of the DNA. DNA in the extracts was amplified by the polymerase chain reaction using primers from the nuclear 17S rRNA sequence that were general to fungi or were specific to endomycorrhizae. Accepted: 17 July 1996     

The use of arbuscular mycorrhizae to control onion white rot (Sclerotium cepivorum Berk.) under field conditions

 A field experiment was carried out to determine the effects of the inoculation of onion (Allium cepa L.) with Glomus sp. Zac-19 on the development of onion white rot (Sclerotium cepivorum Berk.) and on onion production. Mycorrhization delayed onion white rot epidemics by 2 weeks and provided a significant protection against the disease for 11 weeks after onion transplanting, as compared with nonmycorrhizal controls. Mycorrhizal plants showed an increase of 22% in yield, regardless of the presence of the white rot pathogen. Accepted: 8 January 1996     

Rapid typing of truffle mycorrhizal roots by PCR amplification of the ribosomal DNA spacers

DNA analyses were developed to type mycorrhizas of two Tuber species of commercial value (T. melanosporum, T. borchii) and a competitive fungus (Sphaerosporella brunnea) which forms ectomycorrhizas with plants usually considered hosts for truffles. Polymerase chain reaction (PCR) amplification of DNA isolated from fruitbodies, mycelia, mycorrhizas and leaves of host plants, was performed with a primer pair for an internal transcribed spacer ITS1-4. ITS amplification followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products clearly distinguished the two Tuber species at the fruitbody, mycorrhiza and mycelium levels. Accepted: 6 September 1996     

Abundance of arbuscular mycorrhizal fungi spores in different environments in a tropical rain forest, Veracruz, Mexico

 The aim of this study was to compare mycorrhizal abundance and diversity in sites with different regimes of disturbance in a tropical rain forest at Los Tuxtlas, Veracruz, Mexico. Arbuscular mycorrhizal spores were quantified at two sites: closed canopy and gaps in the forest. Data were recorded during dry, rainy, and windy ("nortes") seasons. Spores of eight Glomus species, sporocarps of three Sclerocystis species, three species of Acaulospora and two of Gigaspora were found. Significant differences in the number of species and spores were found among seasons. The highest numbers of species and spores were observed during the dry season, with a marked decrease during the rainy season. Our results show that disturbance does not but seasonality does affect abundance and richness of mycorrhizal spores in this tropical wet forest. Accepted: 11 October 1998     

Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of <Emphasis Type="Italic">Viola calaminaria</Emphasis> and effect of these fungi on heavy metal uptake by clover

 The ability of arbuscular mycorrhizal (AM) fungi from a metal-tolerant plant (Viola calaminaria, violet) to colonise and reduce metal uptake by a non-tolerant plant (Trifolium subterraneum, subterranean clover) in comparison to a metal-tolerant AM fungus isolated from a non-tolerant plant was studied. AM spores from the violet rhizosphere and from violet roots were characterised by polymerase chain reaction (PCR) amplification of the SSU rDNA, and sequencing. Subterranean clover was grown in pots containing a soil supplemented with Cd and Zn salts and inoculated either with a mixture of spores extracted from the violet rhizosphere or with spores of a Cd-tolerant Glomus mosseae P2 (BEG 69), or non-inoculated. The diversity of fungi, including AM fungi, colonising clover roots was assessed and analysed using terminal-restriction fragment length polymorphism. At least four different Glomus species were found in the violet rhizosphere. After 8 weeks in a growth chamber, colonisation of clover roots with spores from the violet rhizosphere increased Cd and Zn concentrations in clover roots without significantly affecting the concentrations of metals in the shoot and plant growth. G. mosseae P2 reduced plant growth and slightly increased the Cd concentration. Only one AM fungus (Glomus b) from the violet rhizosphere colonised clover roots, but other fungi were present. AM fungi from heavy metal-contaminated soils and associated with metal-tolerant plants may be effective in accumulating heavy metals in roots in a non-toxic form. Accepted: 7 July 2000     

Arbuscular mycorrhizae in a tropical sand dune ecosystem on the Gulf of Mexico

 Root samples of 37 species distributed on the beach and along a successional gradient (from mobile to stabilized areas) in a tropical sand dune system on the Gulf of Mexico showed that 97% of the species were mycorrhizal. The mycorrhizal inoculum potential of the sand from several dune areas was compared using two different bioassays. Firstly, the field rate of colonization by arbuscular mycorrhizal fungi of Chamaecrista chamaecristoides seedlings transplanted to random plots in the foredunes and in the mobile area was measured. The seedlings were harvested at intervals during 3 weeks to record mycorrhizal structures. In the mobile area, no mycorrhizal colonization was observed during the experiment. In the foredunes, hyphae and external mycelium were present in 40% of the seedlings as early as 8 days after transplanting. After 15 days, arbuscules and vesicles were observed in 60 and 20% of the seedlings, respectively, and after 21 days, 100, 46 and 20% of the seedlings showed hyphae, arbuscules and vesicles, respectively. Secondly, maize seedlings were transplanted to pots previously filled with sand from the foredunes, mobile dunes, grassland and a Dyphisa robinoides shrub area. After 1 month, the lowest mycorrhizal inoculum potential was recorded for the mobile dunes and the highest for the shrub area. As expected, mycorrhizal inoculum potential increased with dune stabilization. Accepted: 17 July 1996     

Studies of iron transport by arbuscular mycorrhizal hyphae from soil to peanut and sorghum plants

 The influence of an arbuscular mycorrhizal (AM) fungus on phosphorus (P) and iron (Fe) uptake of peanut (Arachis hypogea L.) and sorghum (Sorghum bicolor L.) plants was studied in a pot experiment under controlled environmental conditions. The plants were grown for 10 weeks in pots containing sterilised calcareous soil with two levels of Fe supply. The soil was inoculated with rhizosphere microorganisms only or with rhizosphere microorganisms together with an AM fungus (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe). An additional small soil compartment accessible to hyphae but not roots was added to each pot after 6 weeks of plant growth. Radiolabelled P and Fe were supplied to the hyphae compartment 2 weeks after addition of this compartment. After a further 2 weeks, plants were harvested and shoots were analysed for radiolabelled elements. In both plant species, P uptake from the labelled soil increased significantly more in shoots of mycorrhizal plants than non-mycorrhizal plants, thus confirming the well-known activity of the fungus in P uptake. Mycorrhizal inoculation had no significant influence on the concentration of labelled Fe in shoots of peanut plants. In contrast, ⁵⁹Fe increased in shoots of mycorrhizal sorghum plants. The uptake of Fe from labelled soil by sorghum was particularly high under conditions producing a low Fe nutritional status of the plants. These results are preliminary evidence that hyphae of an arbuscular mycorrhizal fungus can mobilise and/or take up Fe from soil and translocate it to the plant. Accepted: 6 March 1998     

Overwinter survival of arbuscular mycorrhizal hyphae is favored by attachment to roots but diminished by disturbance

 We investigated the overwinter survival in the field of indigenous arbuscular mycorrhizal (AM) hyphae either connected to corn roots or detached from them, and either intact or disrupted. We buried soil-filled pouches which either allowed root entry or excluded roots in the root zone of a field-grown corn (Zea mays) crop in eastern Canada. Following crop harvest in the fall, pouches either remained undisturbed, were disturbed outside the pouch, or were disturbed both inside and outside the pouch. Total and metabolically active AM hyphae in undisturbed pouches declined 20% and 33% (average of coarse- and fine-mesh treatments), respectively, from fall to spring, presumably because of death overwinter. In the spring, living hyphae were more abundant in the presence of roots than in their absence, suggesting that attachment or proximity to roots favored overwinter survival. Total hyphal density, metabolically active hyphal density, and the proportion of total living hyphae progressively diminished with increased disturbance. Accepted: 9 August 1997     

Determination of the nitrogen source for arbuscular mycorrhizal fungi by 15N application to soil and plants

The source of nitrogen in the spores of arbuscular mycorrhizal (AM) fungi was quantified by a ¹⁵N-labeling technique. N was applied as coated urea to the soil and in solution to plant shoots. Soil-applied fertilizer had a significant effect on spore % ¹⁵N (P<0.01), with a 24–75% contribution to spore N. Fertilizer applied to either alfalfa shoots or bahia grass shoots had little effect on spore % ¹⁵N, accounting for 0–14% or 1–9% of spore N, respectively. These results indicate that AM fungi obtain spore N mostly from the soil. The small amount of spore N originating from shoot-applied N may have been obtained via root exudation. Accepted: 6 November 2000     

Rapid and effective detection of anthrax spores in soil by PCR

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.     

The presence of the arbuscular mycorrhizal fungus Glomus intraradices influences enzymatic activities of the root pathogen Aphanomyces euteiches in pea roots

 Fungal enzyme activities were quantified in an interaction study between the fungus Glomus intraradices and the pea pathogen Aphanomyces euteiches. Fungal and host enzymes were separated by polyacrylamide gel electrophoresis and the activity of A. euteiches–specific glucose-6-phosphate dehydrogenase (Gd), phosphoglucomutase and peptidase (PEP) enzymes were quantified by densitometry. The activity of A. euteiches–specific enzymes increased until 14 days after inoculation with A. euteiches, and then decreased. The plants preinoculated with G. intraradices showed no symptoms of severe root rot even though the pathogen was present and active in these plants. Thus, plants preinoculated with G. intraradices were more tolerant of infection with A. euteiches than non-mycorrhizal plants. This effect was evident even though the A. euteiches infection levels of mycorrhizal and non-mycorrhizal plants were the same. A. euteiches enzyme activities in the mycorrhizal plants were different to those in non-mycorrhizal plants. The peaks of PEP and Gd enzyme activity of A. euteiches were lower and the development of A. euteiches PEP activity was later in the mycorrhizal plants than in the non-mycorrhizal plants. Accepted: 14 November 1996     

Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria

 Colonization of Hordeum vulgare L. cv. Salome (barley)and Triticum aestivum L. cv. Caprimus (wheat) roots by the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith leads to de novo synthesis of isoprenoid cyclohexenone derivatives with blumenin [9-O-(2′-O-β-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] as the major constituent and to transient accumulation of hydroxycinnamate amides (4-coumaroylagmatine and -putrescine). Accumulation of these compounds in mycorrhizal wheat roots started 2 weeks after sowing together with the onset of arbuscule formation and proceeded with mycorrhizal progression. Highest levels were reached in 3- to 4-week-old secondary roots (root branches of first and higher order) characterized by the formation of vesicles. In the final developmental stages, the fungus produced massive amounts of spores, enclosing the stele of older root parts (older than 5 weeks) characterized by cortical death. In these root parts, the secondary compounds were detected in trace amounts only, indicating that they were located in the cortical tissues. Some rhizosphere bacteria tested, i.e. Agrobacterium rhizogenes, Pseudomonas fluorescens, and Rhizobium leguminosarum, markedly stimulated both fungal root colonization and blumenin accumulation, thus, acting as mycorrhiza-helper bacteria (MHB). Application of blumenin itself strongly inhibited fungal colonization and arbuscule formation at early stages of mycorrhiza development. This was associated with a markedly reduced accumulation of the hydroxycinnamate amides 4-coumaroylputrescine and -agmatine. The results suggest that both the isoprenoid and the phenylpropanoid metabolism are closely linked to the developmental stage and the extent of fungal colonization. Their possible involvement in the regulation of mycorrhiza development is discussed. Accepted: 18 September 1998     

Mycorrhizae in Adenostoma fasciculatum Hook. & Arn.: a combination of unusual ecto- and endo-forms

 The mycorrhizal status of Adenostoma fasciculatum, the dominant shrub in California chaparral, has been unclear. In two typical, nearly monospecificstands, A. fasciculatum was found to have arbuscules and intercellular hyphae. Antisera detected hyphae of the arbuscular mycorrhizal (AM) fungal genera Acaulospora, Glomus, and Scutellospora, although we found only spores of Glomus. Some roots had partial sheaths and inter- and intracellular septate fungi without indications of root necrosis. Ectomycorrhizal root tips were also found, including Cenococcum and other unknown taxa. Sporocarps of EM fungi including species of Rhizopogon, Pisolithus, Balsamia, Laccaria, Hygrophorus, and Cortinarius were found in the stand, with no other EM or arbutoid mycorrhizal plants nearby. These observations indicate that A. fasciculatum forms mycorrhizae with AM, septate, and EM fungi, but often fails to form easily recognizable mycorrhizal structures. Accepted: 5 September 1998     

Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland

 Analysis of the community of arbuscular mycorrhizal (AM) fungi in roots of Fragaria vesca growing in a heavy metal contaminated site was carried out on a Zn waste site near Chrzanow (southern Poland). The waste substratum was characterized by high contents of Pb, Zn, Cd, Cu and As, and by low levels of N, P and organic matter. Spores of Glomales were isolated by wet sieving and DNA was isolated from individual spores. Nested polymerase chain reaction (PCR) with taxon-specific primers was used to identify the species Glomus mosseae, Glomus intraradices and Glomus claroideum. Spores of other fungi were morphologically characterized and new taxon-discriminating molecular probes were developed for two of them (Glomus sp. HM-CL4 and HM-CL5) based on variations in the large ribosomal subunit (25S rDNA). High sequence similarities were found between Glomus sp. HM-CL4 and Glomus gerdemanii, and between Glomus sp. HM-CL5 and Glomus occultum. The designed primers were used to characterize the population of AM fungi colonizing the roots of F. vesca collected from the Zn waste site. The analysis, carried out on roots stained with trypan blue, showed that the most effective colonizer was closely related to G. gerdemannii. G. claroideum and the G. occultum-like fungus were slightly less common whilst frequencies of G. intraradices and G. mosseae in roots were much lower. The analysis of mycorrhiza stained with rhodizoniate to localize heavy metal accumulation showed that the stain does not influence the PCR reaction. Seventy percent of the root samples containing positively stained fungal hyphae were found to be colonized by G. mosseae. The data obtained demonstrate the usefulness of nested PCR for studies carried out in polluted areas. It will enable selection of AM fungi which are able to colonize plant roots under heavy metal stress conditions, as well as the identification of fungi showing high in situ accumulation of potentially toxic elements. Accepted: 7 July 2000     

Interactions between a mycophagous Collembola,dry yeast and the external mycelium of an arbuscular mycorrhizal fungus

 A plant growth system with root-free hyphal compartments was used to examine the interactions between a mycophagous Collembola (Folsomia candida Willem), dry yeast and an arbuscular mycorrhizal (AM) fungus [Glomus caledonium (Nicol. & Gerd.) Trappe and Gerdemann] in terms of Collembola reproduction, AM-hyphal length and AM-hyphal P transport. Collembola reproduction was unaffected by AM mycelium, but a supplement of dry yeast increased the Collembola population size. The addition of dry yeast increased AM-hyphal P transport by increasing hyphal length. Collembola without yeast affected neither AM-hyphal growth nor AM-hyphal P transport, whereas Collembola with yeast decreased AM-hyphal P transport by 75% after 8 weeks. The hyphal density of G. caledonium remained unaffected by Collembola except after 4 weeks in combination with yeast, when a 33% reduction was observed. The results of this experiment show that the interaction between F. candida and the external mycelium of G. caledonium is limited under the conditions imposed. Accepted: 27 February 1996     

Localized alteration in lateral root development in roots colonized by an arbuscular mycorrhizal fungus

 The morphological responses of root systems to localized colonization by endophytes is not well understood. We examined the responses of lateral roots to the arbuscular mycorrhizal (AM) fungus Gigaspora margarita Becker & Hall inoculated locally into the soil. Peanut (Arachis hypogaea L.) and pigeon pea (Cajanus cajan (L.) Millsp.) were examined. Root boxes filled with nutrient-poor soil in were inoculated in one half with the fungus and in the other half with a sterilized inoculum. Responses were apparent after 30 days but not after 20 days. Overall, lateral root development was more advanced in inoculated soil. This was clearly observed for 2nd- and 3rd-order lateral roots, but less clear for 1st-order lateral roots in both species, although percentage of colonized root length was higher in 1st-order lateral roots. Whilst in peanut the responses were clearly evident at the level of lateral roots initiated on more proximal parts of the tap root axis, they occurred on more distal parts in pigeon pea. We conclude that plants under nutrient-poor conditions give priority to mycorrhizal roots when partitioning assimilation products within the root system. Thus, AM formation may induce local morphological alteration of root systems. Accepted: 29 August 1996