The role of cuticle and epidermal cell wall in resistance of rapeseed and mustard to Rhizoctonia solani

A study was conducted to determine whether the cuticles in two genera of the family Cruciferae are effective barriers to infection by Rhizoctonia solani, and whether differences in cuticle and epidermal cell wall thickness and morphology of epicuticular wax exist between resistant and susceptible cultivars. As Canola/rapeseed (Brassica napus) and mustard (Sinapis alba) plants develop from 1 to 3 weeks of age, they become increasingly resistant to R. solani AG2-1 seedling root rot. Seven-day-old seedlings of S. alba cultivars are invariably more resistant than B. napus cultivars. Brassica napus cultivars do not show an obvious cuticle layer at 1 week but at 3 weeks the presence of a cuticle is seen through autofluorescence with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular autofluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. Three-week-old plants of S. alba have a much lower percent disease rating and a significantly (p=0.05) thicker cuticle layer than similar-age plants of B. napus. The results suggest that the cuticle plays an important role in the resistance of S. alba and older plants of B. napus to infection by R. solani.     

The in vitro screening of 118 Trichoderma isolates for antagonism to Rhizoctonia solani and an evaluation of different environmental sites of Trichoderma as sources of aggressive strains

Trichoderma isolates were collected from different sources and screened for their in vitro parasitism of Rhizoctonia solani. They were grouped according to the different sources and each group compared statistically. 74% of the total isolates collected were regarded as antagonistic to R. solani in vitro. Isolates associated with pine bark source were very aggressive. The most aggressive strains were isolated from soil samples collected under the Speedling^® trays of a commercial seedling nursery.     

Analysis of whole cellular fatty acids and anastomosis relationships of binucleate Rhizoctonia spp. associated with Ceratobasidium cornigerum

Previous research has demonstrated that whole cellular fatty acids analysis is a useful tool for identifying and establishing taxonomic relationships between anastomosis groups (AGs) and related Rhizoctonia isolates. In this experiment, the composition of fatty acid of 28 isolates of teleomorph genus Ceratobasidium cornigerum, consisting of binucleate Rhizoctonia, AG-A, AG-B(o), AG-C, AG-P, and AG-Q, was evaluated using gas chromatography. Eleven fatty acids identified, i.e., myristic, pentadecanoic, palmitic, 2-hydroxypalmitic, palmitoleic, heptadecanoic, 9-heptadecenoic, stearic, oleic, linoleic, and linolenic acids, were present in isolates of AG-A, AG-B(o), AG-C, AG-P, and AG-Q. The major fatty acids, palmitic, oleic, and linoleic acids, were common in all isolates, constituting 87.1% to 94.7% of the whole cellular fatty acids identified. Isolates within the same AG were closely clustered, whereas isolates from different AGs were clearly and distinctly clustered based on average linkage cluster analysis of whole cellular fatty acids. Principal-component analysis generated from all fatty acids also confirmed the divergent separation of the 5 AGs of binucleate Rhizoctonia.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

Influence of the mycorrhizal fungus,Glomus coronatum,and soil phosphorus on infection and disease caused by binucleate Rhizoctonia and Rhizoctonia solani on mung bean (Vigna radiata)

Root-infecting fungal pathogens and also parasites, which do not cause major disease symptoms cause problems of contamination in pot cultures of arbuscular mycorrhizal (AM) fungi. We investigated the effect of the AM fungus, Glomus coronatum Giovannetti on disease caused by binucleate Rhizoctonia sp. (BNR) and R. solani in mung bean in the absence (P0) and presence (P1) of added soil phosphorus (P). When G. coronatum and BNR or R. solani were inoculated at the same time, G. coronatum improved the growth of the plants and reduced colonization of roots by BNR, but not by R. solani. R. solani reduced the growth of non-mycorrhizal mung bean in P0 soil 6 weeks after inoculation, whereas BNR had no effect on growth. G. coronatum reduced the severity of disease caused by BNR or R. solani on mung bean in both soil P treatments. When G. coronatum was established in the roots 3 weeks before BNR or R. solani was added to the potting mix, there was no significant effect of BNR or R. solani on growth of mung bean. Prior colonization by G. coronatum slightly reduced indices of disease caused by BNR or R. solani. In both experiments, addition of P stimulated plant growth and reduced the colonization of roots by BNR, but had little effect on disease severity. We conclude that the reduction of the effect of BNR or R. solani on mung bean could not be explained by improved P nutrition, but could be attributed to the presence of G. coronatum within and among the roots.     

An alpha-glucan elicitor from the cell wall of a biocontrol binucleate Rhizoctonia isolate

Binucleate Rhizoctonia (BNR) isolate (232-C6) is an effective biocontrol agent for protection of potato from Rhizoctonia canker, a disease caused by Rhizoctonia solani. Production of hydrolytic enzymes is one of the best known inducible defense responses following microbial infection. We isolated and characterized a cell wall alpha-glucan from BNR, which induces beta-1,3 glucanase activities in potato sprouts, the primary site of infection by R. solani. An autoclaving method, previously reported for isolation of oligosaccharide elicitors was used, and the glucan purified by chromatographic techniques. Maximal induction of beta-1,3 glucanase activity in potato sprouts was obtained with 250 microg of the alpha-glucan elicitor after 6 days from inoculation time. Both, BNR mycelium and the alpha-glucan produced a similar kinetic response of beta-1,3 glucanase. However, the alpha-glucan did not induce phytoalexin accumulation, previously correlated with the defense response. Uronic acids (approximately 10% with respect to total neutral sugars) were determined and identified as glucuronic acid by high-pH anion-exchange chromatography. Methylation analysis showed that the glucan consists of (1-->3) and (1-->4)-linked glucose units with preponderance of the first ones. Some of the (1-->4) linkages were branched at position 6. The glucan was partially degraded with amyloglucosidase. This, together with the NMR spectra data and the high optical rotation of the original (+195 degrees ) and degraded glucans (+175 degrees ) proved the alpha configuration. Further methylation of the amyloglucosidase degraded glucans indicated that they consist of (1-->3)-linked glucoses. The present study is the first report on the isolation and characterization of an alpha-glucan from Rhizoctonia, that may be important as a biocontrol factor.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Detoxification of oxalic acid by pseudomonas fluorescens strain pfMDU2: implications for the biological control of rice sheath blight caused by Rhizoctonia solani

Rhizoctonia solani isolates varying in their virulence were tested for their ability to produce oxalic acid (OA) in vitro. The results indicated that the virulent isolates produced more OA than the less virulent isolates. In order to isolate OA-detoxifying strains of Pseudomonas fluorescens, rhizosphere soil of rice was drenched with 100 mM OA and fluorescent pseudomonads were isolated from the OA-amended soil by using King's medium B. These isolates were tested for their antagonistic effect towards growth of R. solani in vitro. Among them P. fluorescens PfMDU2 was the most effective in inhibiting the mycelial growth of R. solani. P. fluorescens PfMDU2 was capable of detoxifying OA and several proteins were detected in the culture filtrate of PfMDU2 when it was grown in medium containing OA. To investigate whether the gene(s) involved in OA-detoxification resides on the plasmids in P. fluorescens PfMDU2, a plasmid-deficient strain of P. fluorescens was generated by plasmid curing. The plasmid-deficient strain (PfMDU2P-) failed to grow in medium containing OA and did not inhibit the growth of R. solani. Both PfMDU2 and PfMDU2P- were tested for their efficacy in controlling sheath blight of rice under greenhouse conditions. Seed treatment followed by soil application of rice with P. fluorescens strain, PfMDU2, reduced the severity of sheath blight by 75% compared with the control, whereas PfMDU2P- failed to control sheath blight disease.     

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Geographic range, impact, and parasitism of lepidopteran species associated with the invasive weed Lantana camara in South Africa

Six isolates of Trichoderma were screened for antagonism to Armillaria in tea stem sections buried in the soil. The inability of Armillaria to invade Trichoderma-colonized stem sections and the reduction of its viability in the plant materials following invasion of these by Trichoderma were used as indicators of antagonism. Four isolates of the species Trichoderma harzianum significantly (P<0.001) reduced the incidence of the pathogen in the plant materials. Isolate T4 completely eliminated the pathogen from plant materials in sterile soil and also antagonized two different isolates of the pathogen in nonsterile soil. Application of this T. harzianum isolate to the soil as a wheat bran culture significantly (P<0.001) reduced viability of Armillaria in woody blocks of inoculum. Soil amendment with coffee pulp also reduced the inoculum viability but did not affect the incidence of Trichoderma in the blocks of inoculum. We conclude that the direct application of wheat bran-formulated T. harzianum into soil surrounding woody Armillaria inoculum sources can suppress the pathogen. Further, no organic amendment is needed to enhance development of the antagonist in the soil as a pre-requisite to suppressing the pathogen.     

The effect of soil compaction on the saprophytic growth of Rhizoctonia solani

The increase in bare patch of cereals associated with minimum tillage practices prompted an investigation of the relationship between soil compaction and saprophytic growth of Rhizoctonia solani. In soils wetter than 10 kPa there was a greater density of hyphae in compacted than in non-compacted soil. In relatively dry soil, however, there was wider exploration by hyphae in non-compacted than in compacted soil. The implications of these findings for disease management are discussed.     

Appearance of wheat-germ agglutinin during maturation of field-grown wheat

Field-grown wheat (Triticum aestivum L.) has been used as a developmental system to study the appearance of wheat-germ agglutinin during grain maturation. The lectin appears at the mid-grain growth period (30–34 days post-anthesis) and continues to be synthesised throughout the late stages of maturation and desiccation. An acidic endopeptidase activity, inhibited by pepstatin-phenanthroline is present in extracts of embryo and endosperm throughout maturation. After in-vivo labelling of immature embryos with [³⁵S]methionine for 3 h and extraction in the presence of proteinase inhibitors, immunoprecipitates with anti-wheat-germ agglutinin were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, and found to contain three ³⁵S-labelled polypeptides of M_r 46000, 18000 and 13000. Comparison of two-dimensional tryptic maps of ¹²⁵I-labelled peptides indicate the three polypeptides are closely related.Abbreviations dpa days post-anthesis - PBS phosphate-buffered saline - RIA radioimmunoassay - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Secondary metabolites from species of the biocontrol agent Trichoderma

Trichoderma species are free-living fungi that are highly interactive in root, soil and foliar environments and have been used successfully in field trials to control many crop pathogens. Structural and biological studies of the metabolites isolated from Trichoderma species are reviewed. This review, encompassing all the literature in this field up to the present and in which 269 references are cited, also includes a detailed study of the biological activity of the metabolites, especially the role of these metabolites in biological control mechanisms. Some aspects of the biosynthesis of these metabolites and related compounds are likewise discussed.     

Control ofRhizoctonia solani in cotton by seed-coating withTrichoderma spp. spores

Summary Coating cotton seeds withTrichoderma spp. reduced the incidence of disease caused byR. solani by up to 83% in the greenhouse.T. hamatum was more efficient in disease control at 20°C, whileT. harzianum was superior at 27°C. Disease severity was reduced by 47–60% in two field experiments, showing no statistical difference from treatment with pentachloronitrobenzene.     

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.Abbreviations PBS phosphate-buffered saline - IgG immunoglobulin - SNA Sambucus nigra agglutinin     

Interrelationships of Rotylenchulus reniformis with Rhizoctonia solani on Cotton

The interrelationships between reniform nematode (Rotylenchulus reniformis) and the cotton (Gossypium hirsutum) seedling blight fungus (Rhizoctonia solani) were studied using three isolates of R. solani, two populations of R. reniformis at multiple inoculum levels, and the cotton cultivars Dehapine 90 (DP 90) and Dehapine 41 (DP 41). Colonization of cotton hypocotyl tissue by R. solani resulted in increases (P ≤ 0.05) in nematode population densities in soil and in eggs recovered from the root systems in both 40- and 90-day-duration experiments. Increases in soil population densities resulted mainly from increases in juveniles. Enhanced reproduction of R. reniformis in the presence of R. solani was consistent across isolates (1, 2, and 3) of R. solani and populations (1 and 2) and inoculum levels (0.5, 2, 4, and 8 individuals/g of soil) of R. reniformis, regardless of cotton cultivar (DP 90 or DP 41). Severity of seedling blight was not influenced by the nematode. Rhizoctonia solani caused reductions (P ≤ 0.05) in cotton growth in 40- and 90-day periods. Rotylenchulus reniformis reduced cotton growth at 90 days. The relationship between nematode inoculum levels and plant growth reductions was linear. At 90 days, the combined effects of these pathogens were antagonistic to plant growth.     

Co-utilization of Bacillus subtilis and Flutolanil in controlling damping-off of tomato caused by Rhizoctonia solani

Damping-off of tomato caused by Rhizoctonia solani was controlled in a pot test using the biological agent, Bacillus subtilis RB14-C, and the chemical pesticide, Flutolanil. The co-utilization of B. subtilis RB14-C, and Flutolanil decreased the amount of Flutolanil used from 375 g/pot when Flutolanil was used alone to 94 g/pot, while exerting the same effect of reducing disease occurrence.     

Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms

A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of¹²⁵I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of¹²⁵I-LFA is not inhibited by 10⁶-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 l of serum were sufficient for the detection of major gangliosides.Abbreviations LFA Limax flavus agglutinin - ELLA Enzyme Linked Lectin Assay - PIM Poly(isobutyl methacrylate) - PVP Polyvinylpyrrolidone mol.wt. 40,000 - PBS Phosphate buffered saline - BSA Bovine serum albumin     

Biosynthesis and properties of the plant cell wall

The characterization of cell wall mutants of Arabidopsis thaliana, combined with biochemical approaches toward the purification and characterization of glycosyltransferases, has led to significant advances in understanding cell wall synthesis and the properties of cell walls. New insights have been gained into the formation of cellulose and the functions of the matrix polysaccharides rhamnogalacturonan-II and xyloglucan.