Light and Electron Microscopy Studies on the Infection of a Wild‐type and Metalaxyl‐resistant Isolate of Phytophthora sojae in Soybean Hypocotyls

Resistance of soybean cultivars, depending on single dominant genes to Phytophthora sojae, may easily be overcome by emerging new virulent races. Light microscopy (LM) and electron microscopy (EM) were used to study the infection process of the wild‐type isolate Ps411 and metalaxyl‐resistant mutant Ps411‐M of P. sojae in hypocotyls of soybean seedlings grown from untreated and metalaxyl‐treated seeds. The isolate Ps411‐M of P. sojae exhibited a high degree of resistance to metalaxyl compared to Ps411. The pathogenic fitness of Ps411‐M in hypocotyls of soybean seedlings was lower compared to Ps411. LM observations showed distinct differences in the infection process of both isolates in hypocotyls of treated soybean seedlings. EM studies revealed differences in the prepenetration stage between Ps411 and Ps411‐M on hypocotyls grown from seeds treated with 0.02% metalaxyl until the whole seed surface coated. The number of infection sites was markedly reduced and few hyphae continued to spread. Numerous ultrastructural alterations in hyphae were observed in treated hypocotyls infected with Ps411, including pronounced thickening of hyphal cell walls and encasement of haustorium‐like bodies; electron‐dense material was deposited in host cell walls in contact with hyphal cells. Neither the prepenetration process nor penetration or spread of hyphae in the hypocotyls of the resistant isolate was affected in treated compared to non‐treated tissue. While in treated hypocotyls infected with the wild‐type isolate, host defence reactions were induced, no such reactions were detected in treated hypocotyls infected with the resistant isolate. Hypocotyls from metalaxyl‐treated seeds infected with the wild‐type isolate resembled an incompatible interaction, whereas during infection with the metalaxyl‐resistant mutant, the compatible interaction was not changed.     

Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action

The bacterial strain E1R-j, isolated as an endophyte from wheat roots, exhibited high antifungal activity to Gaeumannomyces graminis var. tritici (Ggt). Strain E1R-j was identified as Bacillus subtilis based on morphological, physiological and biochemical methods as well as on 16S rDNA analysis. This strain inhibited mycelium growth in vitro of numerous plant pathogenic fungi, especially of Ggt, Coniothyrium diplodiella, Phomopsis sp. and Sclerotinia sclerotiorum. In greenhouse experiments, soil drenches with cell densities of 10⁶, 10⁹ and 10¹² CFU ml⁻¹ E1R-j reduced significantly take-all disease, caused by Ggt, in wheat seedling by 62.6%, 68.6% and 70.7%, respectively, compared to the inoculated control, 4 weeks after sowing. Growth parameters such as lengths and fresh weights of roots and shoots of Ggt-inoculated control plants were significantly lower compared to Ggt-inoculated and E1R-j treated plants. Field experiments in the season 2006/2007, heights of wheat plants in the Ggt inoculated plots were significantly reduced compared to the non inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in inoculated control plants were lower compared to the other treatments. In the experimental year 2007/2008, independent treatments with the bacterial strain E1R-j and the fungicide Triadimefon reduced take-all disease in wheat roots by 55.3% and 61.9%, compared to the inoculated control plants. In this season plant height in inoculated control was significantly lower and also the yield parameters seeds per head and especially TKW were drastically reduced compared to the other treatments. E1R-j treatment alleviated the detrimental effects of take-all on grain yield parameters to a similar extent as Triadimefon application. SEM studies revealed that in the presence of E1R-j, hyphae of Ggt showed leakage, appeared ruptured, swollen and shriveled. Following root drench, strain E1R-j was able to colonize endophytically roots and leaves of wheat seedlings. While the population of the bacterial strain in wheat roots steadily increased from the second to the fourth leaf stage, in the leaf tissue the population of the strain rapidly declined. TEM studies also showed that cells of E1R-j were present in roots of wheat seedlings and effectively retarded infection and colonization of Ggt in root tissue; suppression of Ggt by E1R-j was accompanied by disintegration of hyphal cytoplasm. In addition, in the presence of E1R-j cells in Ggt-infected root tissue morphological defense reactions were triggered such as formation of wall appositions and papillae. The results presented indicate that the endophytic strain E1R-j of B. subtilis meets demands required for biocontrol of take-all.     

Purification and characterization of a novel extracellular β-1,3-glucanase complex (Glu<Emphasis Type="Italic">Ggt</Emphasis>) secreted by <Emphasis Type="Italic">Gaeumannomyces graminis</Emphasis> var. <Emphasis Type="Italic">tritici</Emphasis>

β-1,3-Glucanases produced by the take-all disease pathogen Gaeumannomyces graminis var. tritici (Ggt) have been suggested to be implicated in infection and colonization process of the pathogen in wheat. For further studying the role of these enzymes in pathogenesis by cytochemical technique, an extracellular β-1,3-glucanase complex (GluGgt), was purified from culture filtrate of Ggt by (NH₄)₂SO₄ precipitation, hydrophobic-interaction, anion-exchange and size-exclusion chromatography. The complex consisted of two interconvertible isoforms (Ia and Ib), both active proteins Ia and Ib appeared to be glycosylated in native polyacrylamide gel electrophoresis (PAGE). The pI of the active proteins Ia and Ib determined by IEF-PAGE were 6.3 and 6.4, respectively. GluGgt yielded two subunits with molecular masses of 66.2 and 56.0 kDa, respectively, in 15% SDS–PAGE. The complex had a pH optimum of 5.0 and the optimal temperature was 50°C. The enzyme complex proved to be stable at a temperature up to 60°C and retained an optimal high activity in the pH range from 4.0 to 7.0. Enzyme activity of GluGgt was obviously stimulated by Mn²⁺ ions and moderately inhibited in the presence of Hg²⁺ and Co²⁺ ions and KMnO₄. The K _m of GluGgt was estimated to be 1.08 mg ml⁻¹ for laminarin as substrate and the V _max 0.20 μmol min⁻¹.     

Morphoregulatorische Effekte von Xanthen-Derivaten

Summary Of the compounds tested especially xanthene-9-carboxylic acid [I], applied via the roots (10^–4M), strongly affected the morphogenesis of tomato plants. The leaves were deformed, the laminae reduced, the internodes shortened and the growth of axillary buds was stimulated. Geo- and phototropism of wheat and rye seedlings were disturbed. The observed effects are very similar to the influence of the structurally related derivatives of 9-hydroxyfluorenecarboxylic acid-(9) [II], known as morphactins.     

Immunocytochemical Localization of Cell Wall-Bound Thionins and Hydroxyproline-Rich Glycoproteins in Fusarium culmorum-Infected Wheat Spikes

Abstract In the present study, a rabbit polyclonal antiserum against cell wall‐bound thionins from barley leaf and a mouse monoclonal antibody against hydroxyproline‐rich glycoproteins (HRGP) from maize were used to investigate the subcellular localization of thionins and HRGP or extensins in Fusarium culmorum‐infected wheat spikes by means of the immunogold labelling technique. The proteins were localized in cell walls of different tissues including the lemma, ovary and rachis, while the cytoplasm and organelles in these tissues showed almost no labelling. However, accumulation of thionins and HRGP in infected wheat spikes of resistant wheat cultivars differed distinctly from those of susceptible cultivars. Compared with the healthy tissues, labelling densities for the two types of proteins in cell walls of the infected lemma, ovary and rachis increased only slightly in the susceptible cultivar Agent, while in cell walls of infected tissues of the resistant cultivar Arina labelling densities of thionins and HRGP increased markedly. These findings indicated that accumulation of thionins and HRGP in cell walls of infected resistant wheat spikes may be involved in defence responses to infection and in spreading of F. culmorum.     

Induction of Resistance in Tobacco Leaves to Tobacco Mosaic Virus by Dodecylbenzenesulfonate

Application of dodecylbenzenesulfonate (DBS) to half leaves of Nicotiana tabacum cv. Xanthi nc. before TMV inoculation resulted in a marked decrease in lesion number and size as well as in virus content of the lesions in the untreated half leaves. Systemic induction of resistance in untreated leaves of the plants was not detected.     

Microscopic Observation on the Development of Puccinia striiformis in the Spike and Stem of Wheat

The majority of germ tubes of the pathotype CYR32 of Puccinia striiformis f.sp. tritici formed on the surface of spike organs of the susceptible wheat cv. Suwon 11 penetrated through the stomatal pore, only a few germ tubes formed small appressoria over the stomata. In the lemma, palea and glume, the stripe rust fungus spread between the parenchyma cells close to the inner epidermal layer, but the fungus did not develop between the thick‐walled cells near the outer epidermal layer of these organs. In the awn and stem, spread of the stripe rust was confined to the intercellular spaces of the chlorophyll parenchyma, beneath the invaded stomatal pore of the epidermis and the urediniospores to be released disrupted the epidermis. In the caryopsis, the spread of hyphae was restricted to the intercellular spaces of the pericarp cells.     

Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain Reaction

Stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst . Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst . In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants.     

Isolation and identification of N-mercapto-4-formylcarbostyril, an antibiotic produced by Pseudomonas fluorescens.

Pseudomonas fluorescens strain G308 isolated from barley leaves produces a novel antibiotic substance that was purified by preparative TLC and HPLC and identified as N-mercapto-4-formylcarbostyril (Cbs) by LC/DAD, IR, LC-ES(+)/MS, LC-ES(-)/MS, GC-EI/MS, LC-HRES(+)/MS, mass isotope ratios analysis, 1H NMR and 13C NMR analysis. The purified new antibiotic compound is effective against many phytopathogenic fungi in vitro. The compound inhibited at 25 ppm spore germination and germ tube growth of the following fungi; Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Cladosporium cucumerinum and Colletotrichum lagenarium. At concentrations up to 125 ppm, the compound did not interfere with release of zoospores from sporangia and germination of encysted zoospores of Phytophthora infestans.