Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

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.     

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.     

Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici

Bacillus subtilis strain Z-14 has biological control activity against the take-all fungus Gaeumannomyces graminis var. tritici (Ggt). In Petri dishes, the crude extract from B. subtilis Z-14 culture filtrate reduced take-all severity in roots of wheat seedlings by 91.3% and potted plants by 69.8% compared to the Ggt-inoculated control. Treatment with the crude extract also significantly (P?<?.05) increased growth of roots’ average length, and fresh weight in comparison with those of the Ggt-inoculated control. B. subtilis Z-14 culture filtrate was relatively thermally stable with 88.2% of the antifungal activity being retained after being heated at 100°C for 30?min. Meanwhile, the antifungal activity remained almost unchanged (>95%) when the culture filtrate was exposed to a pH ranging from 3 to 8, but significantly reduced in basic conditions. This activity was not transferred to the organic solvent phase after treatment with organic extraction agents. B. subtilis Z-14 culture filtrate exhibited a broad spectrum of antifungal activities against various phytopathogenic fungi. Three homologs of iturin A (C14–16) were characterised by liquid chromatography-mass spectroscopy (LC-MS) and electrospray ionisation mass spectrometry/mass spectrometry collision-induced dissociation (ESI-MS/MS CID).     

Susceptibility to take‐all of cereal and grass species,and their effects on pathogen inoculum

Two field trials were conducted to investigate different herbage grasses and cereals for their susceptibility to the disease take‐all, for their impact on concentrations of the pathogen, Gaeumannomyces graminis var. tritici (Ggt), in soil and for their effect on development of take‐all in a subsequent wheat crop. In the herbage grass trial, Bromus willdenowii was highly susceptible to Ggt, produced the greatest post‐senescence Ggt concentrations in soil and highest incidence of take‐all in following wheat crop. Lolium perenne, Lolium multiflorum and Festuca arundinacea supported low Ggt soil concentrations and fallow the least. The relationship between susceptibility to Ggt and post‐senescence concentrations in soil differed between pasture grasses and cereals. In a trial in which Ggt was added to half the plots and where wheat, barley, triticale, rye or fallow were compared, the susceptibility of the cereals to take‐all was not clearly linked to post‐harvest soil Ggt concentrations. In particular, triticale and rye had low and negligible take‐all infection respectively, but greater post‐harvest soil Ggt concentrations than barley or wheat. This indicates that low Ggt concentrations on roots may build up during crop senescence on some cereals. Soil Ggt concentrations were greater following harvest in inoculated plots sown to cereals, but in the second year there was more take‐all in the previously non‐inoculated than inoculated plots. Thus, the grass and cereal species differed in susceptibility to take‐all, in their impact on Ggt multiplication and in associated take‐all severity in following wheat crop.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

Construction of a proteome reference map and response of Gaeumannomyces graminis var. tritici to 2,4-diacetylphloroglucinol

Take-all disease, caused by Gaeumannomyces graminis var. tritici (Ggt), is one of the most serious root diseases in wheat production. In this study, a proteomic platform based on 2-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Tandem Mass Spectrometry (MALDI-TOF/TOF MS) was used to construct the first proteome database reference map of G. graminis var. tritici and to identify the response of the pathogen to 2,4-diacetylphloroglucinol (DAPG), which is a natural antibiotic produced by antagonistic Pseudomonas spp. in take-all suppressive soils. For mapping, a total of 240 spots was identified that represented 209 different proteins. The most abundant biological function categories in the Ggt proteome were related to carbohydrate metabolism (21%), amino acid metabolism (15%), protein folding and degradation (12%), translation (11%), and stress response (10%). In total, 51 Ggt proteins were affected by DAPG treatment. Based on gene ontology, carbohydrate metabolism, amino acid metabolism, stress response, and protein folding and degradation proteins were the ones most modulated by DAPG treatment. This study provides the first extensive proteomic reference map constructed for Ggt and represents the first time that the response of Ggt to DAPG has been characterized at the proteomic level.     

A scanning electron microscope study of interactions between micro-organisms andGaeumannomyces graminis (Syn.Ophiobolus graminis) on wheat roots

Roots of wheat grown in unsterilized sand inoculated withGaeumannomyces graminis (Sacc.) von Arx and Olivier were examined by scanning electron microscopy. Healthy roots had a mucilaginous covering and were sparsely colonized by bacteria, but asG. graminis colonized the roots the mucilage disappeared and the numbers of bacteria on the surface increased. Lysis of the hyphae occurred, apparently caused by bacteria that colonized the hyphae. Inoculation of wheat in axenic culture with a strain ofPseudomonas fluorescens that was antagonistic toG. graminis in agar gave some protection against the pathogen; lysis of hyphae was observed where protection occurred.