Characterization of Rhizoctonia solani Isolates from Tobacco Fields Related to Anastomosis Groups 2-1 and BI (AG 2-1 and AG BI)

Tobacco has been reported to be infected by Rhizoctonia solani isolates belonging to anastomosis groups 1 through to 5. Ten pathogenic isolates of the fungus were collected from tobacco fields in Italy and France that anastomosed in high frequencies with AG BI tester isolates and in low frequencies with tester isolates of all described subgroups of AG2, although morphology and thiamine requirement of the isolates were similar to AG 2-1. Biomolecular evaluations by means of electrophoresis of polygalacturonase isozymes and RFLPs of ribosomal DNA internal transcribed spacers were carried out. The isolates shared a common pectic zymogram, distinct from those of AG BI and AG 2-subgroups, while RFLPs of rDNA-ITS evidenced a limited genetic variation within the homogeneous group and a closer similarity to AG 2-1. As far as priority is due to the anastomosis behaviour, the isolates should be ascribed to AG BI. However, tobacco isolates differ from tester strains of the known AG BI in their morphology, thiamine requirement, pathogenicity and biomolecular features. In addition they do not anastomose with both AG 3 and AG 6. Therefore they may represent a new subgroup.     

Prolific production of sclerotia in soil by Rhizoctonia solani anastomosis group (AG) 11 pathogenic on lupin

Rhizoctonia solani anastomosis group (AG) 11 causes serious damping‐off and hypocotyl rot of narrow‐leafed lupins (Lupinus angustifolius) in the northern grain‐belt of Western Australia. R. solani AG‐11 produced abundant sclerotia in sand overlaid on potato dextrose agar. Sclerotia were produced in larger numbers in natural Lancelin sand than in Geraldton loamy sand collected from the northern grain‐belt of Western Australia. The majority of the sclerotia produced were in >250 to <500 μam size range. The germination levels of sclerotia in the first two cycles of drying and germination were not significantly different. Sclerotia still retained 50% germination after four such cycles, indicating that they may have the ability to withstand the climatic cycles of the Mediterranean environment of southwestern Western Australia. The radial growth of the mycelium from sclerotia, however, declined with each drying and germination cycle. Inoculum potential of the pathogen increased with the size of sclerotia resulting in more severe lupin hypocotyl rot with larger sclerotia. The number of sclerotia produced in soil increased with increasing density of lupin seedlings. The results also indicate that R. solani AG‐11 can produce sclerotia on infected plant tissues as well as in soil. This is the first report of the prolific production of sclerotia by AG‐11 and their significant role in infection of lupins in soil in Western Australia.     

Saprophytic and pathogenic behaviour of R. solani AG2-1 (ZG-5) in a soil amended with Diplotaxis tenuifolia or Brassica nigra manures and incubated at different temperatures and soil water content

The Brassicaceae species Diplotaxis tenuifolia and Brassica nigra contain high concentrations of glucosinolates, the precursors of isothiocyanates (ITCs) that can have biofumigation effects in amended soils. In a laboratory experiment, incorporation of these plants as green manures into soil was expected to suppress Rhizoctonia solani AG2-1 (ZG5), the causal agent of damping-off in canola (Brassica napus). The manures were incorporated at 1 (1% w/w) or 5 (5% w/w) g fresh material per 100 g dry soil and incubated for 6 months at 10, 20, or 30°C and at soil water contents of 10%, 40%, or 70% of water holding capacity. R. solani survived for up to 6 months as a saprophyte in un-amended soil at all soil water contents and at 10 and 20°C. A temperature of 30°C suppressed R. solani below the level of detection in all treatments after one week. At 1% concentration, the green manures increased the colonisation of the soil by R. solani, which caused severe damping-off of canola subsequently sown in this soil treatment. Soil amendments at 1% temporarily increased soil microbial activity. The addition of B. nigra or D. tenuifolia green manure at 5% concentration suppressed the saprophytic growth of R. solani incubated at 10 or 20°C over all soil water contents and significantly increased the microbial activity at all soil temperatures and water contents. Canola sown into these pots did not succumb to damping-off. The efficiency of hydrolysis of glucosinolates in the 5% treatment in the first week of incubation ranged from 1.6% for 2-propenyl ITC, extracted from soil containing tissues of B. nigra, to 3.4% for 3-butenyl ITC extracted from soil containing tissues of D. tenuifolia. 2-propenyl ITC could not be detected after 7 d of incubation. In the longer term (weeks to months), the increase of microbial activity, caused by adding green manures at 5%, or volatiles from the green manures, most likely played a dominant role in suppressing R. solani. The impact of ITCs, if any, appears to be short-term (days). Responsible Editor: Peter A. H. Bakker     

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.     

Tolerance to the fungal pathogen Rhizoctonia solani AG4 of transgenic tobacco expressing the maize ribosome-inactivating protein b-32

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might functi on as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wun1 gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn     

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.     

广东省水稻纹枯病菌遗传多样性与致病力分化的研究

利用随机引物扩增多态性DNA（PAPD）分子标记技术分析了来自广东省7个县市48个水稻纹枯病菌菌株的遗传多样性,以筛选出的10个随机引物对菌株进行PAPD－PCR扩增,共产生了98个PAPD分子标记,其中89．9％的片段具有多态性。48菌株间的遗传相似性系数（以Nei基因一致度表示）在0．56－0．949之间,用UPGMA和类分析可将它们分为5个RAPD遗传聚类群（A、B、C、D、E）,相同地区来源的菌株基本上聚类在同一组群内。在温室中对供试菌株进行致病性测定,结果表明所有菌株对水稻品种Tetep都有致病性,菌株间致病力差异显著（α＝0．05）,病情指数范围为0．73－18．7,平均感指病指数为5．24。试验分析结果表明水稻纹枯病菌具有丰富的遗传多样性,不同县市的菌株存在很大的遗传分化现象（FST＝0．579）,RAPD遗传聚类群的划分与菌株的地理来源有明显的相关性,但菌株的致病力差异与菌株的来源、遗传聚类组群的划分没有明显的相关性。     

Induced Resistance in Cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2–2 by Infection of the Cotyledons

Localized infection in cucumber cotyledons with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with Rhizoctonia solani AG2–2 and Fusarium oxysporum f. sp. cucumerinum in the roots. The plants were unprotected in soil that was infested heavily with R. solani or in contact with the mycelium, and induced resistance was not observed. Wounding of the root also negated the effect of induced resistance to F. oxysporum .     

Application of endophytic bacteria for the biocontrol of Rhizoctonia solani (Cantharellales: ceratobasidiaceae) damping-off disease in cotton seedlings

The antifungal potentialities of three endophytic bacterial strains, Stenotrophomonas maltophila H8 (Xanthomonadales: Xanthomonadaceae), Pseudomonas aeruginosa H40 (Pseudomonadales: Pseudomonadaceae) and Bacillus subtilis H18 (Bacillales: Bacillaceae) were evaluated against the phytopathogenic fungus Rhizoctonia solani in cotton seedlings under greenhouse conditions. The bacterial strains were applied as a soil drench or talc-based bioformulation in R. solani-infested soil and non-infested soil. Results indicated that the soil drench treatment was more efficient than talc-based bioformulation. A significant increase of seed emergence and seedling survival with a clear reduction of disease severity was achieved with the endophytic bacterial treatments. At the same time, the fresh weight, dry weight, shoot length and root length of the treated plants were markedly enhanced. Moreover, there was an apparent induction of the antioxidant enzymes (peroxidase, polyphenol oxidase and catalase) of the treated seedlings. Gas chromatography–mass spectrometry revealed the presence of various bioactive compounds in the bacterial supernatant. The antagonistic activity of the bacterial strains against R. solani was attributed to their capability to produce a broad spectrum of antifungal compounds in addition to bioactive molecules that can trigger the systemic resistance in the infected seedlings.     

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

 A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T₀, T₁, and T₂ transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants. Received: 11 August 1998 / Accepted: 26 August 1998     

Pathogenic and Genetic Variation among the Isolates of Rhizoctonia solani (AG 1‐IA), the Rice Sheath Blight Pathogen

Sheath blight disease of rice caused by Rhizoctonia solani is one of the most dreaded plant diseases faced by the rice farmers all over the world. None of the commercially cultivated rice varieties have sufficient level of field resistance, and the disease is presently being managed by chemical pesticides. In this study, 40 isolates of rice sheath blight pathogen, collected from diverse rice ecosystems from 12 different states of India, were characterized for their morphological, pathological and genetic variation. The isolates showed wide morphological variation in terms of size of sclerotia and abundance of sclerotia production. The virulence of each pathogen isolate was studied on four rice varieties, that is TN1, IR 64, Tetep and Swarnadhan in glasshouse, and observations were taken by measuring the relative lesion height. The relative lesion heights produced by these isolates on four different rice varieties varied widely. Genetic variation of the isolates was analysed using ISSR markers. The primers based on AG, GA, AC and CA repeats were informative and revealed polymorphism among the isolates. The polymorphism information content (PIC) of the primers ranged from 0.80 to 0.96, while the resolving power (Rp) ranged from 3.7 to 15.35. Largely, grouping of the isolates happened based on their geographical origin. One isolate from Titabar, Assam, and another from Adialabad, Telangana, were quite distinct from rest of the isolates.     

High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG-1 IA (Thanatephorus cucumeris) from Texas

To date, much of the genetics of the basidiomycete Thanatephorus cucumeris (anamorph = Rhizoctonia solani) remains unknown. Here, we present a population genetics study using codominant markers to augment laboratory analyses. Seven single-copy nuclear RFLP markers were used to examine 182 isolates of Rhizoctonia solani AG-1 IA collected from six commercial rice fields in Texas. Thirty-six multilocus RFLP genotypes were identified. Population subdivision analyses indicated a high degree of gene flow/migration between the six geographic populations. Tests for Hardy-Weinberg equilibrium (HWE) among the 36 multilocus RFLP genotypes revealed that four of the seven loci did not significantly differ from HWE. Subsequent analysis demonstrated that departures from HWE at the three remaining loci were due to an excess of heterozygotes. Data presented here suggest that R. solani AG-1 IA is actively outbreeding (heterothallic). Possible explanations for heterozygote excess, which was observed at all seven RFLP loci, are discussed.     

5-(pyrrolidin-2-yl)-1H-tetrazole and 5-[(pyrrolidin-2-yl)methyl]-1H-tetrazole: proline surrogates with increased potential in asymmetric catalysis

Catalytic enantioselective methodology has dramatically been enriched by the re-discovery of the simple amino acid proline as a chiral catalyst in the year 2000. Although no catalyst offers such a simple and broad access to quite complex reaction products, as does proline, its synthetic potential is not unrestricted, what is especially connected to its poor solubility in organic media. Exchange of the carboxylic moiety by a tetrazole unit leads to proline surrogates, that by far outperform proline with respect to yield, enantioselectivity, reaction time, substrate and solvent scope, catalyst loading, and stoichiometry of the compounds used in excess. These factors are discussed and critically compared with selected representative proline-catalyzed reactions.     

Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

Response of the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae), to conophthorin and other angiosperm bark volatiles in the avoidance of non-hosts

1 Seventeen non‐host angiosperm bark volatiles, seven of which are antennally active to Ips pini (Say), the pine engraver (PE), were tested for their ability to disrupt the response of the PE to pheromone‐baited traps. 2 Four green leaf volatiles (GLVs) were tested [1‐hexanol (Z)‐3‐hexen‐1‐ol, hexanal, and (E)‐2‐hexenal]. None had any disruptive effect singly, as a group or in all possible blends based on functional groups, despite the fact that the two aldehydes were antennally active. These compounds may have, in some instances, actually masked the disruptive effect of other compounds. The PE thus differs in its response from other Scolytidae, including other Ips spp. 3 Eight non‐host volatiles that were antennally active to other bark beetles, but not to PEs, had no disruptive effect, validating the use of coupled gas chromatographic‐electroantennographic detection analyses to detect compounds with potential behavioural activity. 4 The bicyclic spiroacetal conophthorin, (E)‐7‐methyl‐1,6‐dioxaspiro[4.5]decane, was disruptive when tested alone. When blends of two aldehydes [salicylaldehyde and nonanal] plus an alcohol and a phenol [benzyl alcohol and guaiacol] were combined with conophthorin, an enhanced disruptive effect was revealed. No single compound, other than conophthorin, disrupted the pheromone‐positive response and no blend that did not contain conophthorin was consistently disruptive to both sexes. Conophthorin seems to be a critical component in the non‐host angiosperm message for I. pini during its host selection phase. 5 Combination of the repellent synomones, verbenone and ipsenol, with the five disruptive non‐host volatiles may provide a potent treatment to protect trees, logs or stands from attack by the PE.     

Analysis of Phaseolus vulgaris Response to Its Association with Trichoderma harzianum (ALL-42) in the Presence or Absence of the Phytopathogenic Fungi Rhizoctonia solani and Fusarium solani

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.     

Human cytomegalovirus-induced DNA polymerase and its interaction with the triphosphates of 1-(2''-deoxy-2''-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, -5-iodocytosine, and -5-methylcytosine.

Human cytomegalovirus-induced DNA polymerase and cellular DNA polymerase alpha were purified by successive chromatography on DEAE-cellulose, phosphocellulose, heparin agarose, and single-stranded DNA agarose columns. The purified virus-induced DNA polymerase was resolved to consist of two polypeptides corresponding to molecular weights of 140,000 and 58,000, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-induced DNA polymerase and cellular alpha polymerase were examined for their sensitivities to the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAUTP), -5-iodocytosine (FIACTP), and -5-methylcytosine (FMACTP). The inhibitive effects of these triphosphates on the DNA polymerases were competitive with regard to the natural substrates; thus FMAUTP competes with dTTP, and FIACTP and FMACTP compete with dCTP. The inhibition constants (Ki) for FMAUTP, FIACTP, and FMACTP of virus-induced DNA polymerase are 0.06, 0.30, and 0.47 microM, respectively. Cellular DNA polymerase alpha is much less sensitive to these inhibitors, and its Ki values for FMAUTP, FIACTP, and FMACTP are 0.45, 3.10, and 2.90 microM, respectively. In addition, human cytomegalovirus-induced DNA polymerase, but not cellular DNA polymerase alpha, can utilize these analog triphosphates as alternate substrates for their corresponding natural deoxyribonucleoside triphosphates in in vitro DNA synthesis.     

Liquid chromatography-tandem mass spectrometry identification of metabolites of three phenylcarboxyl derivatives of the 5-HT(1A) antagonist,N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) trans-4-fluorocyclohexanecarboxamide (FCWAY), produced by human and rat hepatocytes

We have previously described fluorine-18 radiolabeled FCWAY [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) trans-4-fluorocyclohexanecarboxamide] as a high affinity ligand for imaging the 5-HT(1A) receptor in vivo. In a search for radiopharmaceuticals with unique imaging applications using positron emission tomography (PET), we have also developed three new phenylcarboxamide analogues of FCWAY. Two of these analogues were generated by replacing the fluorocyclohexane carboxylic acid with fluorobenzoic acid (FBWAY) or with 3-methyl-4-fluorobenzoic acid (MeFBWAY). The final analogue was generated by replacing the pyridyl group with a pyrimidyl group and the fluorocyclohexane carboxylate with fluorobenzoic acid (FPWAY). We evaluated the metabolic profile of these compounds using either human or rat hepatocytes to produce metabolites and LC-MS/MS to identify these metabolites. We also compared the metabolic rate of these compounds in human or rat hepatocytes. These in vitro metabolism studies indicate that hydrolysis of the amide linkage was the major metabolic pathway for FPWAY and FBWAY in human hepatocytes, whereas aromatic oxidation is the major metabolic pathway for MeFBWAY. The comparative metabolic rate in human hepatocytes was FPWAY>FBWAY>MeFBWAY. In rat hepatocytes, aromatic oxidation was the major metabolic pathway for all three analogs and the rate of this process was similar for all of the analogues. These in vitro metabolic studies demonstrated species differences prior to the acquisition and interpretation of in vivo results.