| Take-all of wheat, caused by the fungus Gaeumannomyces graminis (Sacc.) Von Arx & Oliver var. tritici Walker (Ggt), is one of the most important root diseases of wheat worldwide. People pay much attention on the biocontrol methods for control of this pathogen currently, because the absence of resistant varieties and effective chemicals, as well as the limitation of crop rotation. Significant advances in control of take-all have been achieved both in research and application by the use of biocontrol microorganisms. However, the effect of many microorganisms was not stable because of the effect of environment. It is necessary to screen some biocontrol factors against take-all disease of wheat which are insensitive to environment. In recent years, bacterial strains which colonize the internal tissue of plants have gained increasing scientific interest. They have more advantages as biocontrol factors because they will hardly be affected by detrimental environmental factors. In this research, five endophytic bacterial strains from our lab were screened against take-all disease of wheat in planta. One of the most effective endophytic bacterial strains, designated EDR4, was studied on antifungal protein from the culture filtrate of endophytic Bacillus subtilis strain EDR4 of wheat, control effect against take-all disease and histiocyte mechanism of strain EDR4 and antifungal protein E2 controlling take-all and inhibiting Ggt. The main results are listed as following:(1) Five of 27 endophytic bacterial strains of wheat in vivo against wheat take-all disease caused by Gaeumannomyces graminis var. tritici were selected in repeat experiments. The results indicated that there were some difference between 5 strains and their ferment filtration in the antagonistic effect on Ggt; In greenhouse experiment, the effects of 5 strains against wheat take-all disease was excellent (up to 57.3%, better 16.8% than the fungicide control). The endophytic bacterial strain EDR4 was studied in more detail.(2) The study provided evidences that mutant strain EDR4 (up to 23.54×103 cfu/g root tissue) can colonize wheat seedlings following root drenched treatment while spraying leave treatment less and soaking seeds treatment the least (1.97×103 cfu/g root tissue), but the number of bacteria decreased after treatment one month in different treatments. It is benefit for colonization of strain EDR4 in root tissue by drenching root and that the colonization exhibiting the trend of increase in on month. However, the conlonization of 2×dilution was less than of the original and spraying leave treatment showed excellent cononization in leaves. So we assumed that the colonization of mutant strain EDR4 could be determined by different treatment and concentration.(3) In sequent field plots of two years, the endophytic Bacillus subtilis strain EDR4 mitigated wheat take-all disease in elongation stage (30~50% control effect), promoted the growth of plants and increased wheat yield.(4) The bacterial strain EDR4, isolated from wheat roots, was identified as Bacillus subtilis on the basis of its morphology and 16S rDNA sequence analysis as well as on physiological and biochemical characteristics. In addition, strain EDR4 showed inhibitory activity on mycelium growth of different familiar plant pathogenic fungi.(5) The proteins are the main antifungal substances produced by B. subtilis strain EDR4 were studied by ammonium sulfate precipitation, and the antifungal proteins were precipitated by 30% and 30%~70% saturation ammonium sulfate separately. The conditions of strain EDR4 growth and secreting antifungal protein were optimized in flasks. The optimal condition for the protein yield was that 3 mL bacterial suspension was inoculated and grew in 50mL LB medium in 250 flask at pH 7.0 and 28℃for 48h, in which the production of crude protein, the growth of bacterial population and the effect of inhibitory activity were the best.(6) An antifungal protein was purified from the culture filtrate of endophytic B. subtilis strain EDR4 of wheat by (NH4)2SO4 precipitation, hydrophobic-interaction chromatography on Phenyl Sepharose 6 Fast Flow, anion-exchange chromatography on DEAE-Sepharose Fast Flow and PAGE. The molecular mass of the protein was about 377.0 kDa determined by GPC using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoeletric focusing PAGE was 6.59. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides which were detected by using a nanoESI-MS/MS (Q-TOF2) System. The antifungal protein demonstrated neitherβ-1, 3-glucanase orβ-1, 4-glucanase or chitinase activities nor protease inhibitory activity. However, it exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. Also,the purified protein exhibited inhibitory activity on mycelium growth of Ggt and other pathogens detected.(7) The mycelia treated with strain EDR4 stopped growing and was swollen, shriveled, ruptured, exuded and died, which were observed by light microscopy and scanning electron microscopy (SEM). Transmission electron microscopic(TEM) studies showed that in Ggt inoculated roots treated with the bacterial strain EDR4 spread hyphal of the take-all fungus in the root tissues was effectively retarded, bacteria cells presented in root cells, caused degeneration of hyphal cytoplasm and morphological hyphal responses were induced in infected and treated host tissues.(8) The effects of E2, a novel antifungal protein from endophytic B. subtilis strain EDR4, on morphology and ultrastructure of mycelial cell of Ggt were investigated under scanning and transmission electronic microscopy. The studies revealed that protein E2 caused a series of pronounced morphology and structural alterations of hyphae including swollen, shriveled and ruptured, cell wall was thickened, new cell wall was formed, the organelles and cytoplasm were degraded.These results provided the foundation for application of strain EDR4 and antifungal protein E2, and are available to explore the mechanisms of inhibiting plant pathogens in more detail.
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