Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.     

Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani

AIM: To identify antimicrobial peptides with high lytic activity against Rhizoctonia solani strain LR172, causal agent of rice sheath blight and aerial blight of soyabeans in the US. METHODS AND RESULTS: Among 12 natural and synthetic antimicrobial peptides tested in vitro, the wheat-seed peptide, purothionin, showed the strongest inhibitory activity that was similar to the antifungal antibiotics, nystatin and nikkomycin Z. Cecropin B, a natural peptide from cecropia moth, and synthetic peptide D4E1 produced the highest inhibitory activity against R. solani among linear peptides. Membrane permeabilization levels strongly correlated with antifungal activity of the peptides. Noticeable changes in membrane integrity were observed at concentrations of >/=0.5 micromol l(-1) for purothionin, 2 micromol l(-1) for cecropin B, D4E1, D2A21, melittin, and phor21, and 8 micromol l(-1) for magainin II and phor14. An increase of nuclear membrane permeabilization was observed in fungal cells treated with cecropin B, but not with purothionin. Diffusion of nuclear content was observed by fluorescent microscopy 10 min after adding a lethal concentration of cecropin B. Evaluation by electron microscopy confirmed severe cytoplasmic degradation and plasma membrane vesiculation. Purothionin and cecropin B were the most stable against proteolytic degradation when added to liquid cultures of R. solani. CONCLUSIONS: Purothionin, cecropin B, D4E1 and phor21 were shown to exhibit high in vitro lytic activity against R. solani strain LR172 for rice and soyabean. These peptides are greater than 16 amino acids long and rapidly increase fungal membrane permeabilization. Resistance to proteolysis is important for sufficient antifungal activity of antimicrobial peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected antimicrobial peptides offer an attractive alternative to traditional chemicals that could be utilized in molecular breeding to develop crops resistant to rice sheath blight and aerial blight of soyabean.     

喀麦隆芋艿根腐病病原菌鉴定

从采自喀麦隆不同地区的芋艿(Xanthosoma mafaffa L.)病根及田土中分离菌株,分别从雅温得(Yaound(?))和巴马约(Mbalmayo)分离到的腐霉菌株XPMY和XPMM1,根据其形态学及生理学特征鉴定为群结腐霉Pythium myriotylum,用上述腐霉菌株的游动孢子悬液或菌丝体片断悬液人工接种,表现叶黄化及根腐等典型症状。用经常伴随群结腐霉的立枯丝核菌Rhizoctonia solani及茄镰孢Fusarium solani人工接种后均未表现症状。研究结果表明:群结腐霉Pythium myriotylum Drechsler是喀麦隆芋艿根腐病的致病菌。     

Molecular characterization of Rhizoctonia solani isolates the incitant of soybean root rot

Rhizoctonia solani isolates used in this investigation were identified as anastomosis-4 (AG-40), collected from different localities from Assiut governorate in Egypt. Pathogenicity test of seven isolates of R. solani was evaluated on soybean Giza 111 cultivar under greenhouse conditions. All tested isolates were able to infect soybean plants causing root rot with different degrees of severities, isolate No. 1, 2 and 3 showed significantly highest root rot severity, while isolate No. 5 gave the lowest percentage of root rot rating. The sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns were used to compare three isolates of R. solani. There are no variations among R. solani isolates except a few exceptions according to their protein patterns. DNA markers obtained from all isolates showed genetic similarity among different isolates obtained from different geographical regions barring few exceptions. Correlation between DNA patterns of R. solani isolates and their virulence was detected, but no correlation with anastomosis groups (AG).     

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     

Interpretation of variety × sowing date × sowing depth interaction for bean–Fusarium–Rhizoctonia pathosystem

Through this study, the sowing date?×?sowing depth?×?variety interaction for Rhizoctonia root rot (RRR) development was investigated in field trials over two growing seasons. Four-way Wald tests indicated that there was a lower RRR incidence in var. Red compared to var. White in the majority of experimental plots. At a 5?cm seeding depth, RRR incidence for plantings on 22 May and 5 June was often lower than that for 5 May date. A hierarchical cluster analysis grouped experimental plots using averages of RRR incidence, Fusarium root rot (FRR) index and the number of seeds per plant. Sowing depth influenced FRR, RRR and productivity more than variety and date factors. Comparison of clusters recognised lower disease and greater production levels for sowing on 22 May and 5 June at 5?cm depth. Thus, shallow seeding of bean varieties in late spring should be incorporated into FRR–RRR-control programmes.     

Interactions between a fluorescent pseudomonad,an arbuscular mycorrhizal fungus and a hypovirulent isolate of Rhizoctonia solani affect plant growth and root architecture of tomato plants

Abstract

Although Rhizoctonia solani is a cosmopolitan soilborne pathogen, the genus includes isolates with different pathogenicity ranging from high virulence to avirulence. The biocontrol strain Pseudomonas fluorescens P190r and the arbuscular mycorrhizal (AM) fungus Glomus mosseae BEG12 were inoculated alone or in combination in tomato plants infested by the mildly virulent pathogen R. solani #235. Plant growth as well as root morphometric and topological parameters were evaluated. The infection of R. solani was significantly reduced by all the combinations of the beneficial microorganisms. Root systems of R. solani‐infected plants were weakly developed but highly branched with a herring‐bone pattern, while those inoculated with the AM fungus, alone or in combination with the bacterial strain, were longer and more developed, and displayed a dichotomous pattern. The interactions among these three microorganisms affected plant growth and root architecture of tomato plants.     

棉立枯丝核菌(Rhizoctonia solani)中的一个dsDNA病毒

自从1962年Hollings在栽培蘑菇(Agaricus bisporus)中发现第一例真菌病毒以来,迄今已在100多种真菌中发现了病毒,多数含双链RNA基因组。1972年Bozarth报道在R.solani中发现病毒,但未报道该病毒的理化性质。1975年Finlker在R.solani的一个强致病力菌株中分离到双链RNA病毒,它含3个组分dsRNA。     

Biocontrol of Rhizoctonia solani damping-off of sugar beet with native Streptomyces strains under field conditions

A 3-year study was conducted to evaluate the effectiveness of two disease-suppressive Streptomyces spp. to control sugar beet Rhizoctonia solani damping off under field conditions. Streptomyces seed treatments reduced seedling damping off in naturally (2005) and artificially (2006 and 2007) infested soils. All biocontrol agents provided better efficacy than Vitavax to control seedling damping-off. There were no significant differences among Streptomyces isolates. Isolate C increased plant stand by 19.5, 50.5 and 53.75% in 2005, 2006 and 2007, respectively. Evaluation of final harvest revealed that the root yield of the biocontrol agents increased compared to untreated control in these years.     

A new formulation system for the application of biocontrol fungi to soil

A new biocontrol formulation system was devised that does not require sterile conditions during preparation. It involves mixing vermiculite and powdered wheat bran with wet or dry fermentor biomass of Trichoderma spp. or Gliocladium virens, moistening with 0.05 N HCl, and drying the mixture. Before application to soil, the preparation (VBA‐FB) is activated by re‐moistening with 0.05 N HCl and incubated at room temperature for 2–3 days to stimulate development of young hyphae of the biocontrol fungus. Populations of biocontrol fungi proliferated to greater than 10⁷ colony‐forming units (cfu) per g of soil when activated VBA‐FB was added to soil. In soil artificially infested with Rhizoctonia solani, seven isolates of the 14 studied added as VBA‐FB reduced survival and 12 reduced saprophytic growth of the pathogen. Of these, two isolates of T. hamatum (TRI‐4, Tm‐23) and one of T. harzianum (Th‐87) were the most effective. Preparations formulated with either wet or dry biomass effectively reduced pathogen survival, but activated VBA‐FB was more effective than non‐activated VBA‐FB. Storage of VBA‐FB at 25°C for 24 weeks before activation reduced viability of isolates considerably more than storage at 5°C for 24 weeks. In addition, VBA‐FB stored at 5°C before activation more effectively reduced survival of R. solani than VBA‐FB stored at 25° C. Survival of R. solani was reduced by activated VBA‐FB applied to several soil types (sandy loam, sandy clay loam, clay). Some nitrogen fertilizers increased the efficacy of VBA‐FB preparations of several isolates.     

Variations in culture morphology and pathogenicity among protoplast-regenerated strains of Rhizoctonia solani

Abstract Protoplast-regenerated cultures derived from mycelia of cereal-infecting field isolates of Rhizoctonia solani exhibited major variations in cultural morphology and in pathogenicity. Each field isplate yielded three of four distinct morphological types of protoplast cultures. The presence of the new morphological phenotypes was attributed to the selection of homokaryons arising from protoplasts with single nuclei. Highly pathogenic field isolates produced protoplast cultures with higher virulence than those from weakly virulent pathogenic isolates, and homokaryotic strains were generally less pathogenic than the parental field isolate.