Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related. Accepted: 15 October 2000     

Glomales species associated with surface and deep rhizosphere of Faidherbia albida in Senegal

 Five arbuscular mycorrhizal (AM) fungal species were isolated and propagated from surface and deep rhizospheres of Faidherbia albida trees growing in two ecoclimatic zones of West Africa: the semi-arid Sahelian and the more humid Sudano-Guinean areas. Of these species, Glomus aggregatum, Glomus caledonium, and Glomus mosseae were trapped by F. albida roots when cultivated with either surface or deep soils. Glomus fasciculatum was found exclusively at the semi-arid Sahelian sites of Louga and Diokoul and Gigaspora margarita was isolated only from 16.5-m and 34-m-deep samples. Comparable glomalean fungal species richness was identified in deep (1.5–34 m) and surface (0.15 m) samples. The isolation and the propagation of glomalean fungi from F. albida rhizospheres confirmed the presence of viable AM fungal propagules, down to the water table, as deep as 34 m. Accepted: 27 August 2000     

Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization

 Fluorescence in situ hybridization (FISH) was applied to interphasic nuclei isolated from spores of four species of AM fungi : Scutellospora castanea, Glomus mosseae, Glomus intraradices and Gigaspora rosea. Ribosomal DNA loci were visualized using digoxigenin-labeled 25 S rDNA probes obtained by nested PCR. Several hybridization sites were detected per nucleus and an internuclear variability was observed in the number of loci. This is the first report of successful application of FISH to analyse the genomes of glomalean fungi. Accepted: 16 September 1998     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism)

PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum, and did not recognise sequences from Glomus claroideum. Sequence differences between isolates were detected by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non-denaturing conditions. Isolates of G. mosseae, G. caledonium and G. coronatum could be separated by their SSCP patterns, while three isolates of G. geosporumshowed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased Growth and Yield of Hydroponically Grown Greenhouse Tomato Plants Inoculated with Arbuscular Mycorrhizal Fungi and Fusarium oxysporum f. sp. radicis-lycopersici

The arbuscular mycorrhizal fungi Glomus monosporum, G. vesiculiferum, G. deserticola, G. intraradices, G. mosseae, and two unidentified species were tested to determine their effect on plant growth and fruit production of tomato (Lycopersicon esculentum Mill.) cv. Trust inoculated with Fusarium oxysporum f. sp. radicis-lycopersici (FORL) under near-commercial greenhouse conditions. Inoculation with G. monosporum and G. mosseae significantly increased fruit yield and fruit number of tomato plants grown hydroponically in sawdust. Plant height and plant dry weight increased significantly when inoculated with G. monosporum and G. mosseae. Further, plants inoculated with G. monosporum and G. mosseae showed significantly lower FORL root infection than the untreated control plants.     

Cellular localization of a plant protein PSAM 1 in arbuscular mycorrhizas of Pisum sativum

Psam 1 is a single-copy gene which is activated during early plant-fungal interaction in wild-type pea inoculated with Glomus mosseae and which codes for PSAM 1, a putative protein of 108 amino acids. A synthetic peptide was designed in an antigenic region of this protein to produce a polyclonal antibody against PSAM 1 and to investigate its cellular localization. Western blot analysis revealed that a polypeptide of about 14.5 kDa accumulated more in mycorrhizal than non-mycorrhizal pea roots. The PSAM 1 antigen was immunolocated in planta in arbuscule-containing cells of mycorrhizal roots and especially in the cytoplasm surrounding young arbuscules in cortical cells, which suggests that its accumulation is somehow related to the symbiotic state of these cells. Received: 27 June 1998 / Accepted: 27 July 1998     

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

 Strips of horticultural film (16–32 cm²) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm² of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season. Accepted: 11 October 1998     

A procedure for the establishment of Glomus mosseae in dual culture with Ri T-DNA-transformed carrot roots

 A stepwise procedure was investigated to determine the optimal conditions for the establishment of Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe in dual in vitro culture with Ri T-DNA-transformed roots of Daucus carota L. Glomus mosseae spores germinated best in 10 mm Tris or MES-buffered medium at pH values just above neutral. Growth of hyphae from germinated spores was much greater in the presence of Tris than MES, eg. 8 mm versus 4 mm per spore for Tris and MES, respectively, at pH 7.2. Roots exhibited a broad pH optimum for growth of 6.0–7.0 in both MES and Tris, but did not grow well above pH 7.5. In addition, purified gelling agent, gellan gum, was utilized to lower the P concentration of media. With these factors combined, mycorrhizas were successfully established in 14% of dual cultures. Accepted: 5 March 1997     

The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi

A unique oligonucleotide pair, GOCC56:GOCC427, was designed that correctly primed specific amplification of a approximately 370-bp sequence spanning the ITS and 5.8S rDNA regions of Glomus occultum and Glomus brasilianum. In addition, this primer pair successfully detected G. occultum and G. brasilianum DNA in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn (Zea mays) roots using modified ITS1:ITS4 primers. A second primer pair, GBRAS86:GBRAS388, primed specific amplification of a approximately 200-bp sequence spanning the ITS and 5.8S rDNA regions present only in G. brasilianum and Glomus strain GR582. Combined use of both primer pairs provides the means to detect and differentiate two ancient endomycorrhizal species, G. occultum and G. brasilianum, undetectable by standard root staining procedures. Sequence analysis showed that the purported G. occultum strain GR582 is likely a strain of G. brasilianum.     

Rapid detection of arbuscular mycorrhizae in roots and soil of an intensively managed turfgrass system by PCR amplification of small subunit rDNA

 Arbuscular mycorrhizal fungi (AMF) were detected in uninoculated soil and roots of turfgrass (Agrostis palustris) by direct extraction and PCR amplification of the small subunit rRNA gene. Sequence analysis of the cloned PCR product confirmed the identity of the amplified DNA as an AMF sequence having 95% identity to Glomus intraradices. The sensitivity of the method was gauged by comparison with the most probable number analysis of infective propagules in an intensively managed turfgrass system having 56 propagules per 100 g soil. In contrast to the heavily managed system, infective propagule numbers were high in systems under moderate and limited management. The method described may be useful for rapid investigations of genetic diversity and community structure of AMF. Accepted: 8 January 1999     

Phylogenetic position of an arbuscular mycorrhizal fungus,Acaulospora gerdemannii, and its synanamorphGlomus leptotichum, based upon 18S rRNA gene sequence

We examined the phylogenetic position of an arbuscular mycorrhizal fungus which produces two types of spore,Acaulospora gerdemannii andGlomus leptotichum, based upon the DNA sequence of the 18S rRNA gene. DNA was extracted separately from bothGlomus-like orAcaulospora-like spores and partial 5′-terminus segments of 18S rRNA gene were amplified by the PCR method. Several clones derived from each spore type were sequenced and compared. The sequences from both spore types agreed well, confirming that these morphologically different spores were formed by the same fungus. Nucleotide substitutions were found among several clones, suggesting polymorphism of the rRNA gene in glomalean fungi. Further phylogenetic analysis based upon the whole sequence of the 18S rRNA gene showed thatA. gerdemannii may be within the order Glomales but is far from the fungi that have been analyzed and probably should be in a new family.     

In vitro regeneration of loblolly pine and random amplified polymorphic DNA analyses of regenerated plantlets

 Adventitious buds were induced from organogenic callus derived from mature zygotic embryos of three lines (E-311, E-440, and E-822) of loblolly pine (Pinus taeda L.) within 27 weeks of culture. The influence of cytokinins, silver nitrate, and low-temperature treatment on the differentiation of adventitious buds was analyzed. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). After adventitious shoots had rooted on TE medium supplemented with 0.5 mg/l IBA, 2 mg/l BA, and 0.5 mg/l gibberellic acid, 498 regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1 :>: 1) soil mixture; 351 of these survived in the field. Total DNA was extracted from 21 regenerated plantlets randomly chosen from the 151 regenerated plantlets of line E-822. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed that 21 primers gave 107 clear reproducible bands, with the amplification products being monomorphic for all of the plantlets of line E-822 tested. A total of 2,247 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested plantlets. These results suggest that somatic organogenesis can be used for clonal micropropagation of some lines of loblolly pine without the fear of the appearance of unwanted somaclonal variants. Received: 5 August 2000 / Revision received: 5 September 2000 / Accepted: 10 October 2000     

Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency. Received: 14 May 1998 / Accepted: 3 September 1998     

Detection of the 3-phosphoglycerate kinase protein of Glomus mosseae

 The Glomus mosseae 3-phosphoglycerate kinase (PGK) gene encodes a polypeptide of 416 amino acids. A synthetic peptide was designed to the C-terminus of the polypeptide for the production of a polyclonal antibody. The antibody was tested against the synthetic peptide in an immuno-dot blot and was then used to investigate the asymbiotic and symbiotic accumulation of the PGK protein. Western blot analysis revealed that a polypeptide of approximately 45 kDa accumulated in G. mosseae-colonised tomato roots; this is similar to the theoretical molecular weight of 44.764 kDa. The protein was not detected in non-mycorrhizal roots. Quantitative immuno-dot blotting revealed that the polypeptide accumulated in germinating spores and hyphae of G. mosseae and also in tomato roots colonised by G. mosseae. The amount detected in the mycorrhizal root system was significantly higher than that found in germinating sporocarps. The variation in the levels of glycolytic activity in the symbiotic and asymbiotic developmental stages of G. mosseae is discussed. Accepted: 20 April 2000     

A molecular marker that segregates with sorghum leaf blight resistance in one cross is maternally inherited in another

Leaf blight-resistant sorghum accession SC326-6 was crossed to the susceptible cultivar BTx623 to analyze the genetic basis for resistance. Field scoring of inoculated F₂ progeny revealed that resistance was transmitted as a dominant single-gene trait. By combining the random amplified polymorphic DNA (RAPD) technique with bulked-segregant analysis, it was possible to identify PCR amplification products that␣segregated with disease response. Primer OPD12 amplified a 323-bp band (D12R) that segregated with resistance. Creation of longer primers, or SCARs (sequence characterized amplified regions) for D12R resulted in the amplification of a single major band of the predicted size from all the resistant F₂ progeny and the resistant parent SC326-6, but not from BTx623 or 24 of 29 susceptible F₂ progeny. The SCAR primers also amplified a single band with DNA from IS3620C, the female parent in a cross with BTx623 that has been used to produce a recombinant inbred population for RFLP mapping. An equivalent band was amplified from all 137 recombinant inbred progeny, indicating that organelle DNA is the amplification target in this cross. Received: 31 July 1998 / Accepted: 23 November 1998     

Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This “autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang. Received: 11 August 1998 / Accepted: 2 October 1998     

Use of random amplified polymorphic DNA markers for the detection of Azospirillum strains in soil microcosms

Probes for the detection of Azospirillum strains were obtained from DNA fragments generated by random amplification of polymorphic DNA (RAPD) and tested to assess their specificity towards DNA extracted from pure cultures. The most specific probe, referred to as α4, produced a hybridization signal only with amplified DNA of A. lipoferum ATCC29731. This strain was inoculated, together with two other Azospirillum strains, in soil microcosms of different complexity and its presence tested with the probe α4. This probe confirmed its high specificity with amplified DNA extracted from the soil microcosm and in the presence of other A. lipoferum strains, indicating that the strategy for bacterial detection, based on RAPD markers, is useful for monitoring the presence of a particular strain under environment-like conditions. Other RAPD-derived probes, when tested on soil samples, did not show the same level of specificity as that shown on DNA from pure cultures. This result suggests that some precautions are necessary in the choice of a really specific RAPD marker. In a further development of this strategy, the α4 probe was sequenced and two pairs of “nested” primers were designed, which enabled a diagnostic polymerase chain reaction from soil samples that was specific for the A. lipoferum species. Received: 7 July 1997 / Accepted: 14 October 1997     

Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)_n, (CATTA)_n, (CATATCATTT)_n and (TATTT)_n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus- specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contributed to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F₁ progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)₅₃ to (TATTT)₆₅], while the remainder of the sequence was unchanged. Received: 11 March 1997 / Accepted: 21 June 1997