Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IB using SCAR primers.

AIMS: The aim of this study was to develop a specific and sensitive identification method for Rhizoctonia solani AG 1-IB isolates based on phylogenetic relationships of R. solani AG-1 subgroups using rDNA-internal transcribed spacer (rDNA-ITS) sequence analysis. METHODS AND RESULTS: A neighbour-joining tree analysis of 40 rDNA-ITS sequences demonstrated that R. solani AG-1 isolates cluster separately in six subgroups IA, IB, IC, ID, IE and IF. A molecular marker was generated from a random amplified polymorphic DNA fragment (RAPD). After conversion into a sequence-characterized amplified region (SCAR), a specific primer set for identification of subgroup AG 1-IB was designed for use in a polymerase chain reaction (PCR). The primer pair amplified a single DNA product of 324 bp. CONCLUSIONS: R. solani AG-1 subgroups were discriminated by sequence analysis of the ITS region. The designed SCAR primer pair allowed an unequivocal and rapid detection of R. solani AG 1-IB in plant and soil samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence analysis of the rDNA-ITS region can be used for differentiation of subgroups within AG-1. The use of the developed SCAR primer set allowed a reliable and fast identification of R. solani AG 1-IB and provides a powerful tool for disease diagnosis.

Expression regulation of the endochitinase-encoding gene sechi44 from the mycoparasite Stachybotrys elegans.

The regulation of the gene encoding the extracellular chitinase sechi44 produced by the mycoparasite Stachybotrys elegans was studied using real-time quantitative reverse-transcription polymerase chain reaction. Alteration of sechi44 expression was observed when S. elegans was in interaction with its host, Rhizoctonia solani, and also when the mycoparasite was grown on minimal media amended with different carbon and nitrogen sources. Direct contact with R. solani leading to mycoparasitism significantly up-regulated the expression of sechi44, although the analysis showed that sechi44 was constitutively expressed but at substantially lower levels. In addition, the study of sechi44 over 12 days showed that its expression followed a cyclical pattern with peaks every 2 days, which suggests that this gene has a role not only in mycoparasitism but also in growth. The addition of external carbon sources, such as N-acetylglucosamine, chitin, and R. solani cell wall (simulated mycoparasitism), triggered an increase in the expression of sechi44, which varied with time and carbon source. Among the carbon sources examined, N-acetylglucosamine induced the highest increase in sechi44 transcript levels. The addition of high concentrations of glucose and ammonium triggered a decrease of sechi44 expression, suggesting that sechi44 is subject to glucose and ammonium repression.

Molecular Monitoring of Wild-Type and Genetically Engineered Colletotrichum coccodes Biocontrol Strains In Planta.

Two strains of Colletotrichum coccodes, the wild type (DAOM 183088) and T-20a, engineered with the necrosis- and ethylene-inducing peptide (NEP1) gene for hypervirulence on velvetleaf (Abutilon theophrasti, Medik.), were monitored in planta for the first 2 weeks after infection. Real-time quantitative polymerase chain reaction (QPCR) was used to assess the extent of colonization of both strains on velvetleaf using SYBR Green chemistry. Quantification of both strains was successful as soon as the conidia were sprayed on the leaves and up to 14 days after infection. The increase in fungal DNA amounts corroborated with the appearance of necrotic lesions on velvetleaf leaves infected with the wild-type strain. The wild-type C. coccodes was more efficient at infecting velvetleaf than the transgenic T-20a strain. In addition, detection of host DNA allowed us to quantitatively monitor the decrease in plant DNA amounts in response to wild-type strain infection. Expression of the NEP1 transgene by conventional retro-transcription (RT)-PCR was absent from T-20a growing on either V8 agar or in planta, suggesting that the gene may be silenced. The application of QPCR to monitor fungal growth was proven to detect the target organisms in planta prior to the appearance of symptoms.

Detection of the biocontrol agent Colletotrichum coccodes (183088) from the target weed velvetleaf and from soil by strain-specific PCR markers.

Diagnostic molecular markers, generated from random amplified polymorphic DNA (RAPD) and used in polymerase chain reaction (PCR), were developed to selectively recognize and detect the presence of a single strain of the biocontrol fungus Colletotrichum coccodes (183088) on the target weed species Abutilon theophrasti and from soil samples. Several isolates of C. coccodes, 15 species of Colletotrichum, a variety of heterogeneous organisms and various plant species were first screened by RAPD-PCR, and a strain specific marker was identified for C. coccodes (183088). No significant sequence similarity was found between this marker and any other sequences in the databases. The marker was converted into a sequence-characterised amplified region (SCAR), and specific primer sets (N5F/N5R, N5Fi/N5Ri) were designed for use in PCR detection assays. The primer sets N5F/N5R and N5Fi/N5Ri each amplified a single product of 617 and 380 bp, respectively, with DNA isolated from strain 183088. The specificity of the primers was confirmed by the absence of amplified products with DNA from other C. coccodes isolates, other species representing 15 phylogenetic groups of the genus Colletotrichum and 11 other organisms. The SCAR primers (N5F/N5R) were successfully used to detect strain 183088 from infected velvetleaf plants but not from seeded greenhouse soil substrate or from soil samples originating from deliberate-released field experiments. The sensitivity of the assay was substantially increased 1000-fold when nested primers (N5Fi/N5Ri) were used in a second PCR run. N5Fi/N5Ri selectively detected strain 183088 from seeded greenhouse soils as well as from deliberate-released field soil samples without any cross-amplification with other soil microorganisms. This rapid PCR assay allows an accurate detection of C. coccodes strain 183088 among a background of soil microorganisms and will be useful for monitoring the biocontrol when released into natural field soils.

Quantification of Fusarium solani f. sp. phaseoli in Mycorrhizal Bean Plants and Surrounding Mycorrhizosphere Soil Using Real-Time Polymerase Chain Reaction and Direct Isolations on Selective Media.

ABSTRACT The capacity of the arbuscular mycorrhizal fungus Glomus intraradices in reducing the presence of Fusarium solani f. sp. phaseoli in bean plants and the surrounding mycorrhizosphere soil was evaluated in a compartmentalized experimental system. Quantification of the pathogen and the symbiont in plant tissues, the soil regions of the mycorrhizosphere (rhizosphere and mycosphere), and the bulk soil was accomplished using specific polymerase chain reaction (PCR) primers in real-time PCR assays, culture-dependant methods, and microscopic determination techniques. Nonmycorrhizal bean plants infected with the pathogen had distinctive Fusarium root rot symptoms, while infected plants previously colonized by G. intraradices remained healthy. The amount of F. solani f. sp. phaseoli genomic DNA was significantly reduced in mycorrhizal bean plants and in each mycorrhizosphere soil compartment. The presence of G. intraradices in the mycorrhizosphere was not significantly modified, although the mycorrhizal colonization of roots was slightly increased in the presence of the pathogen. The results suggest that the reduced presence of Fusarium as well as root rot symptoms are caused by biotic and/or abiotic modifications of the mycorrhizosphere as a result of colonization with G. intraradices.

Systemic Induction of Peroxidases, 1,3-beta-Glucanases, Chitinases, and Resistance in Bean Plants by Binucleate Rhizoctonia Species.

ABSTRACT Inoculation of bean hypocotyls with a nonpathogenic binucleate Rhizoctonia (BNR) species induced systemic resistance and protection of the roots and cotyledons to later challenge with the root rot pathogen Rhizoctonia solani or the anthracnose pathogen Colletotrichum lindemuthianum. Bean seedlings that were treated with BNR 48 h prior to their challenge with R. solani or C. lindemuthianum had few necrotic lesions and reduced disease severity as compared with seedlings not treated with BNR. Treatment with BNR 48 h prior to their challenge also elicited a significant and systemic increase in all cellular fractions of peroxidases, 1,3-beta-glucanases, and chitinases compared with the diseased and control plants. Compared with control plants, total peroxidases and glucanases increased twofold and eightfold, respectively, in all protected bean tissues. BNR 232-CG could not be recovered from the challenged hypocotyls or cotyledons, indicating that there was no contact between the inducer and the pathogen. Both the 1,3-beta-glucanases and the peroxidases were positively correlated with induced resistance.

Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani.

Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1-5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300-1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and -5, indicating that the plasmids do not play any role in pathogenicity.

Production of Chitinases and beta-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani.

The in vitro production of chitinases and beta-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO(3). The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for beta-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and beta-1,3-glucanases were detected. S. elegans culture filtrates, possessing beta-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.

High resolution cytochemical study of the vesicular-arbuscular mycorrhizal association, Glomus clarum×Allium porrum.

Roots of leek, colonized or not by Glomus clarum Nicolson & Schenck, were ultrastructurally examined for the presence of different sugars using lectin-gold complexes. N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-galactosamine (GalNAc), l-fucose (l-FUC), d-mannose (d-Man) and sialic acid sugar residues were localized in various cellular components of the mycorrhizal fungus. GlcNAc, GalNAc, d-Man and sialic acid residues were localized in the colonized host, particularly in the plasmalemma and cytoplasm surrounding the arbuscular hyphae. The same sugars were not present in the non-colonized host. These results indicated that the production of these sugars had been triggered during the plant-fungus association. In the interfacial matrix, GalNAc and d-Galactose (d-Gal) residues were present in moderate amounts. In cell walls and protoplasm of non-colonized and colonized hosts, mainly d-Gal was detected. Ribonucleic acid (RNA) was cytochemically localized in the fungus and host by means of a RNAse-gold complex. Of interest, was the specific localization of RNA in the granular wall layer of vesicles. The ultracytochemical results are discussed in relation to host-symbiont interaction, along with speculations on the function and significance of these macromolecules.