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Tomato colonization by wt R. solanacearum and a hrp mutant

ABSTRACT: Tomato seeds (Solanum lycopersicum Mill., cultivar Marion) were germinated in vermiculite and 10-day old seedlings were transplanted into 4-inch plastic pots containing a mixture of composted pine bark and vermiculite (3:1) amended with lime and fertilizer. Plants were incubated in a growth chamber (constant 30oC, 14-h photoperiod; 180 ?mol s-1 m-2 light intensity) for 18-21 days before inoculation. Seedlings received a complete liquid fertilizer approximately 7 and 14 days after transplanting. Unwounded roots were inoculated with R. solanacearum by drenching the soil in each pot with 40 ml of water containing 1´107 CFU ml-1 of the wild type or 1´108 CFU ml-1 of the hrp mutant. Non inoculated plants received 40 ml of water. Four days after inoculation, a piece of each stem from about 1-cm above the soil line to just below the cotyledon node was excised, immediately plunged into liquid nitrogen, and stored at -80oC. A separate 0.5-cm stem section from the cotyledon node was removed aseptically from each plant and the number of bacteria in the section determined by dilution plating. Stem pieces from plants systemically colonized by R. solanacearum were pulverized in a mortar under liquid nitrogen, and total RNA was extracted with the Tri-Reagent (Molecular Research Center) and then treated with RNase-free recombinant DNase (Ambion) according to the recommended protocols from TIGR and stored at -70oC. Keywords: Direct comparison

ORGANISM(S): Solanum tuberosum Solanum lycopersicum

PROVIDER: GSE8212 | GEO | 2007/09/01

SECONDARY ACCESSION(S): PRJNA101129

REPOSITORIES: GEO

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Project description:Aspergillus flavus is one of the major fungal molds that colonize peanut in the field and during storage. The impact to human and animal health and to economy in agriculture and commerce are significant since this mold produces the most potent natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect to inhibit aflatoxin formation through down-regulating aflatoxin pathway gene expression in A. flavus as demonstrated by genechip analysis in liquid medium and peanuts. The results showed that aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98%) inhibited by B. megaterium. The expression of many of the aflatoxin biosynthetic genes in the fungus was confirmed to be turned down. Some of the target genes down-regulated by B. megaterium within the whole genome and within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX) were identified. These target genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was found to be significantly down-regulated. The effect of B. megaterium on aflatoxin biosynthesis and genes expression of pathogen was firstly tested in potato dextrose broth (PDB) and glucose minimal salts medium (MM). The cell suspension of B. megaterium (concentration in PDB and MM was finally adjusted to 108 CFU/ml) or sterile distilled water as a control was added into the 100 ml beaker flask containing 15 ml PDB or MM, respectively. Then 100 M-NM-<l of spore suspension (5 M-CM-^W 106 spores/ml) of A. flavus were added into each beaker flask. After 48 h of incubation at 28M-BM-0C at 200 rpm, mycelia were collected, fresh frozen with liquid nitrogen, ground to a fine powder in liquid nitrogen, and stored at -80M-BM-0C for further analysis. The effect of B. megaterium on aflatoxin biosynthesis and genes expression in the A. flavus fungal pathogen was also tested in two types of peanut kernels, UF 715133-1 and Jinhua 1012, respectively. Peanut kernels were wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer and then 20 M-NM-<l of 1 M-CM-^W 108 CFU/ml cell suspension of B. megaterium was inoculated on wounded peanut kernels respectively. Sterile distilled water was also used for inoculation as control. Two hours after bacterial inoculation, 10 M-NM-<l A. flavus spore suspension was inoculated into each wound at a concentration of 106 spores/ml. The kernels were placed in artificial weather chamber to maintain high humidity (85%) and incubated at 28M-BM-0C for 7 days. Each treatment was replicated three times with 20 peanut kernels in each test. The mycelia on kernels were harvested at day 7 and fresh frozen immediately in liquid nitrogen, ground into powder, and stored at -80M-BM-0C for further analysis.

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Tomato colonization by wt R. solanacearum and a hrp mutant

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