Studies on transmission of Indian peanut clump virus disease by Polymyxa graminis*

The plasmodiophoromycete fungus, Polymyxa graminis was observed in the roots of Sorghum bicolor, S. sudanense, Pennisetum glaucum, Triticum aestivum, Cyperus rotundus, Eleucine coracana, Zea mays, Tridax procumbens and Arachis hypogaea collected from Indian peanut clump virus (IPCV)-infested fields. Examination of roots of IPCV-infected S. bicolor, S. sudanense, P. glaucum and T. aestivum grown in previously air dried field soil also showed the presence of cystosori of P. graminis. IPCV-infested soil stored at room temperature for 3 years transmitted the virus to A. hypogaea, T. aestivum and S. bicolor. Roots extracted from IPCV-infected P. glaucum and S. bicolor containing cystosori, and dried root fragments incorporated into sterile soil, transmitted the virus to A. hypogaea and T. aestivum. The root extracts contained primary zoospores of the fungus, presumably arising from cystosori. Utilising root fragments of S. sudanense containing cystosori as inoculum P. graminis was shown to infect both monocotyledonous and dicotyledonous plants. Profuse cystosorus production in rootlets only occurred in monocotyledonous plants. In dicotyledonous plants, in general, only few rootlets showed cystosori. Indian isolates of P. graminis appear to differ from isolates from temperate soils in that they can infect dicotyledonous plants and have a much wider host range.     

Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region

A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.     

Antagonism of the yeast‐like phylloplane fungus Sporothrix flocculosa against Erysiphe graminis var tritici

Microscopic studies of the interaction between the yeast‐like fungus Sporothrix flocculosa and the wheat powdery mildew pathogen, Erysiphe graminis var. tritici (Egt) were undertaken in an attempt to determine the potential antagonistic activity of Sp. flocculosa against Egt and to gain a better insight into events underlying this interaction. On detached leaf segments infected with Egt, application of a spore suspension of Sp. flocculosa resulted in a rapid degeneration of Egt mycelium and conidia. Scanning electron microscope observations revealed that the antagonist colonized and developed profusely on Egt within 12 h and induced a general collapse of conidial chains within 24 h. Under transmission electron microscopy, first consistent reactions were observed 12 h following application of the antagonist. These reactions were characterized by a sequence of ultrastructrural changes beginning with retraction of the plasmalemma followed by complete disintegration of the cytoplasm and death of host cells after 24 h. The basis of this antagonistic process appeared to be antibiosis‐dependent since no evidence of cell wall erosion or penetration was observed while death of the pathogen occurred rapidly. This study presents the first report of the potential of Sp. flocculosa as a biocontrol agent of Egt.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

Cloning and Expression Analysis of a Putative ABC Transporter Gene BgABC1 from the Biotrophic Pathogenic Fungus Blumeria graminis f. sp. tritici

A putative ATP‐binding cassette (ABC) transporter gene (BgABC1) was isolated from the biotrophic pathogenic fungus Blumeria graminis f. sp. tritici (Bgt). Analysis of the deduced amino acid sequence of BgABC1 showed that the BgABC1 protein had a conserved nucleotide‐binding fold in the N‐terminus, and six transmembrane domains (TMDs) in the C‐terminus. Analysis of the BgABC1 expression using real‐time polymerase chain reaction showed that expression of the gene was increased significantly when the fungus was growing in wheat seedlings treated with 14α‐demethylase‐inhibiting fungicide, triadimefon. The results indicated that the BgABC1 was involved in the protection of the fungus against fungicide toxicity.     

Mapping of powdery mildew resistance genes in a newly determined accession of Hordeum vulgare ssp. spontaneum

The accession PI466197 of wild barley (Hordeum vulgare ssp. spontaneum) with a newly identified resistance to powdery mildew caused by Blumeria graminis f.sp. hordei was studied with the aim to localise the genes determining resistance on a barley genetic map using DNA markers. Molecular analysis was performed in the F₂ population of the cross between the winter variety ‘Tiffany’ and the resistant accession PI466197, consisting of 113 plants. DNA markers, 17 simple sequence repeats (SSRs), four sequence-tagged sites (STSs) and one cleaved amplified polymorphic sequence (CAPS) marker developed from the Mla locus sequence were used for genetic mapping and a two-locus model of resistance was shown. One of the resistance genes originating from H. vulgare ssp. spontaneum PI466197 was localised between the markers RGH1aE1 and Bmac0213 on the short arm of chromosome 1H, which is the position consistent with the Mla locus. The other gene was proven to be highly significantly linked with GBMS247, Bmac0134 and MWG878 on the short arm of chromosome 2H. The flanking markers were Bmac0134 and MWG878, assigned 4 and 8 cM from the resistance gene, respectively. Until now, no gene conferring powdery mildew resistance originating from H. vulgare has been located on the short arm of barley chromosome 2H.