| China is a large agricultural country. Development of agriculture is the basis for economic growth. Agricultural harmful microorganisms can attack crops and contaminate agricultural products in various aspects of agricultural production. The harmful microorganisms will cause severe crop failures, quality decline of agricultural production and huge economic losses. They may also produce toxins and do harm to human and animals. Furthermore, Agricultural harmful microorganisms will contaminate the environment causing potential long-term risk to human health, even affecting sustained and stable development of economy.There are many physical or chemical methods for controlling of agricultural harmful microorganisms; however, there are different defects of the methods in application. Sometimes, physical methods could not entirely kill microbes, especially bacteria producing spores. Chemical methods usually could cause environmental pollution.In this study, we propose to apply environment-friendly technologies to control agricultural harmful microorganisms. We can utilize advanced oxidation technology (AOT) to sterilize the raw materials, wastes and excreta in agricultural production, or sterilize the progress rooms or storage room for agricultural products. We can also apply resistance gene for crop breeding, which contribute to controlling of plant pathogens and decreasing or avoiding environmental pollution and health risk causing by mass farm chemicals.This paper includes the two following sections:control of harmful microorganisms using exogenous hydroxyl radicals and control of plant pathogens by disease resistance genes.Section Ⅰ Control of Harmful Microorganisms Using Exogenous Hydroxyl RadicalsAdvanced oxidation technology can produce hydroxyl radicals by reactions. Hydroxyl radical, a strong oxidant, can oxidate all kinds of organics and degraded them in to water, carbon dioxide and other harmless substances. In this paper, hydroxyl radicals were generated by generated by strong ionization discharge. As the models, Escherichia coli (Gram-negative bacteria), Bacillus subtilis (Gram-positive bacteria) and its spores were used to reveal the biological effects and mechanisms of exogenous hydroxyl radicals on agricultural harmful microorganisms. The major works are as the follows:1. Biological effects and mechanisms of exogenous hydroxyl radicals on Gram-negative bacteriaE. coli were treated by exogenous hydroxyl radical solutions with different concentrantion. The results showed that0.4mg/L hydroxyl radical solution could entirely kill bacteria. It indicated that hydroxyl radical could efficiently kill Gram-negative bacteria. The assays on biomolecules (including DNA, RNA and protein) revealed that hydroxyl radical could enter the cell and destroy these biomolecules. The assays of cellular electrolytes leakage and MDA contents indicated that hydroxyl radical could lead to membranes damage. Cellular structures were also observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that cells were deformed and a great space appeared between the bacterial cell wall and membrane in the sample treated by0.2mg/L hydroxyl radical solution. After treated by1.0mg/L hydroxyl radical solution, bacteria kept rod-shaped; however, irregular wrinkles appeared on the surface of cells. Furthermore, the nucleoid changed vague, even disappeared in cells treated by hydroxyl radicals. The above results revealed that, exogenous hydroxyl radicals kill Gram-negative bacteria by damaging intracellular biomolecules and extracellular structures including membrane and cell wall.2. Biological effects and mechanisms of exogenous hydroxyl radicals on Gram-positive bacteriaThe study on lethal effects showed that exogenous hydroxyl radicals could also efficiently kill B. subtilis and its spores; however, the lethal concentrations were0.8mg/L and1.0mg/L, respectively, which were more higher than that (0.4mg/L) on E. coli. Under the SEM, treated B. subtilis cells were still rod-shaped. Like E. coli, wrinkles appeared on the surface of cells treated by0.2mg/L hydroxyl radical solution. After treated by1.0mg/L hydroxyl radical solution, wrinkles were more clear and regular. Under the TEM, the nucleoid become vague after treated by hydroxyl radicals. The SEM results showed that0.4mg/L hydroxyl radical solution could not lead to obvious change on the surface of spores. After treated by1.0mg/L hydroxyl radical solution, violent change was observed on the spores. Spores become small, about one ninth of the controls, and had many granules on the surface. The TEM results revealed that the surface structure of damaged spore were in disintegration and the inside of spore was showed in deep color, which indicated that the inside of spore with high-density. We proposed that the cell wall of Gram-positive bacteria and spore wall were also the major target attacked by hydroxyl radicals.3. Tracing the lethal effect of hydroxyl radicals on E. coli by green fluorescence from EGFP.Using genetic engineering method, recombinant enhanced green fluorescent protein (EGFP) was prepared and the engineered bacteria E. coli-EGFP strain was constructed in this study. In vitro, hydroxyl radical solution could efficiently decrease the green fluorescence intensity of purified recombinant EGFP. When treated by0.2mg/L hydroxyl radical solution, the fluorescence intensity was48.25%. When the concentration of hydroxyl radical reached to0.4mg/L, the fluorescence signal disappeared. In vivo, the fluorescence intensity deceased to33.22%after treated by0.3mg/L hydroxyl radical solution. When the concentration of hydroxyl radical reached to0.8mg/L, the fluorescence signal disappeared. Combined with the lethal effect on engineered bacteria, relative fluorescence intensity was related to mortality of engineered bacteria when the concentration range of hydroxyl radical reached was0.2to1.0mg/L. The data met the curve equation:y=0.0009x2-0.0276x+0.0525(R2=0.9923). This result indicated that the lethal effect of hydroxyl radical solution on engineered bacteria could be evaluated by fluorescence signal of EGFP.Section Ⅱ Control of crop pathogens by disease resistance genesBreeding and application of resistant cultivars is the most economical, safe and effective way to control crop disease. The key of this way is to discover and use new resistance resources. Wheat powdery mildew, one of the most severe diseases, is a great threat to wheat production. Haynaldia villosa, a wild relative of wheat, carres Pm21gene conferring the highest resistance to wheat powdery mildew. However, there is little known about Pm21gene. This paper attempts to develop CISP markers around6VS bin FL0.45-0.58carring Pm21gene based on comparative genomics and construct a high-density comparative genomic map. According to the map, we try to clone the Pm21candidate gene using the RGA method. The major works are as the follows:1. Fine-localization of Pm21gene in H. villosaIn this paper, four reported6VS-specific markers were utilized to perform chromosome localization analysis by using the deletion materials. It indicated that chromosome bin FL0.45-0.58carring Pm21gene was between the marker Xcinau188 and CINAU91. The126VS markers reported could be localized to Brachypodium distachyon chromosomes. And a low-density comparative genomics map was constructed. In the target region of the short arm of B. distachyon chromosome3(3BdS), two hundred genes was selected to analyzed the colinearity between B. distachyon and rice. Among them,69genes with good colinearity were used to designed94CISP primers, and226VS-specific markers were founded. Four CISP markers were further localized on the6VS bin FL0.58-1.0, ten on FL0.45-0.58and8on FLO-0.45. So the bin FL0.45-0.58carring Pm21gene was between the CISP marker6VS-CISP027and6VS-CISP145. A high-density comaparative genomics map were further constructed.2. Cloning of Pm21candidate gene using the RGA method.As seed sequences, the conserved NBS domains of plant resistance (R) genes were used to perform BLAST analysis. In B. distachyon genome,299R genes were found. Among them,30R genes were on the3BdS chromosome. According to the high-density comaparative genomics map, only R5-R8gene locus corresponded to the region of6VS FLO.45-0.58in H. villosa. The conserved sequences of R5-R7were used to design degenerate primers, and three RGA fragments were PCR-amplified from H. villosa. Among them, only Hv-R1was localized on the bin FL0.45-0.58. Transcriptional analysis showed that Hv-R1constitutively expressed and the expression level was enhanced after inoculation by Blumeria graminis f. sp. tritici (Bgt). The full length sequence was obtained by TAIL-PCR. The result showed that Hv-R1gene was5309bp in length wih a250-bp intron and the coding region was2568bp in length. The predicted protein,855aa in length, had high homology (72%-77%) with R5-R7genes of B. distachyon. Protein domain analysis revealed that Hv-R1was with the classic CC-NBS-LRR domains. Promoter motif analysis indicated that CAAT-box, TATA-box, W-box, MYB and MYC transcription factor motif existed in the upstream of Hv-R1gene. These motifs might play critical roles in the expression regulation of Hv-R1gene. This study will contribute to revealing the function of Hv-R1gene and providing a new strategy for cloning of target gene in orphan crops.These researches revealed the lethal effects and mechanisms of exogenous hydroxyl radicals on E. coli, B. subtilis and its spores, and constructed engineered bacteria with green fluorescence for fast tracing lethal effect of exogenous hydroxyl radicals. These results provided a basis for green controlling of agricultural harmful microorganism by using exogenous hydroxyl radicals. In this paper, a Pm21candidate gene, Hv-R1was obtained from H. villosa using a RGA cloning method combined with comparative genomics. This work contributed to discoving the function of the candidate R gene and made a basis for breeding applification of this gene in controlling wheat powdery mildew. |
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