Screening, Identification And Fermentation Conditions Optimization Of Antagonistic Strains On Tomato Postharvest Diseases Caused By Alternaria Solani

Tomato is an important vegetable crop in our country. The rotting is main problem in storage of tomatoes. Now, the low-temperature storage combine with chemical preservative is one of the most effective measures for controlling tomatoes rotting, but the low-temperature storage can cause chilling injury problem, and chemical preservative can lead to fungicide-resistant,health problem and environment pollution. Biological control as tomato postharvest diseases of effective measures attracted much attention.In this study,15 strains of endogenetic bacteria were isolated from tomato. By plate screening, endophytic bacterium strains 004 was found to have a strong inhibitory effect, which can obviously inhibit the growth of Alternaria solani. Meanwhile, endophytic bacterium strains 004 was identified by series of physiological and biochemical tests and 16S rDNA sequence analyses. In this paper, it was focused on that endophytic bacterium strains 004 was used to study the antisepsis effect in tomato postharvest storage, the effect of different protective agents and rehydration media on the viabilities of the endophytic bacterium strains 004 subjected to freeze-drying, and preliminary analyses of the crude antagonistic substance.1. By plate screening, there were 10 endophytic bacterium strains showed the inhibition effects on the growth of Alternaria solani. The inhibition zone radium of endophytic bacterium strains 004 was 4.0mm, the effect of endophytic bacterium strains 004 is better than other strains. By storage test, it was found that endophytic bacterium strains 004 is highly inhibition effects on Alternaria solani of two varieties tomato, which can delay disease occurs, the rotten rate and the disease index lower than blank group and control group (use iprodione).2. Based on the characteristics of morphology, physiology, biochemistry and the 16S rDNA sequence analysis, endophytic bacterium strains 004 was identified as Bacillus amyloliquefaciens, which had 99% sequence similarity with Bacillus amyloliquefaciens (HM107808). It was measured that endophytic bacterium strains 004 has the 16S rDNA length of 1453bp via PCR. The accession of it in GenBank was HQ840415. By soxhlet extraction method, the crude antagonist substance was extracted with methanol from the precipitate which was obtained by adding 6mol/L HCl to fermentation liquor. The crude extract was run on HPLC, which turned out that there were seven substances. By the test of inhibiting mechanism, it was demonstrated that the mycelium growth of fungi was inhibited seriously by the antagonist substance, and microscope observation of mycelium revealed that the fungi hypha was dissolved, and had an inflated tip, mycelium was deformed and fractured to small segments.3. The optimum compositions of culture medium and fermentation conditions for the antagonistic production of endophytic strains 004 was presented in this study. The optimal compositions of culture medium were as followed:soluble starch 10g, yeast extracting solution 12g, CaCl2 7g, water 1000mL; The optimal fermentation conditions were as followed:the medium initial pH 7.0, the fermentation temperature 30℃, the quantity of medium 100mL in a 250mL flask, the inoculation volume of 2%, the fermentation cycle of 48h, and the rotation speed of 210r/min.4. By studying on the effect of different protective agents and rehydration media, it was indicated that the 10% lactobiose is the best protective agent, the survival rate of endophytic bacterium strains 004 reach up to 80%, and by screening of rehydration agent, the experimental results indicated that 5% concentration proteins is the beatest rehydration agent, the survival rate of endophytic bacterium strains 004 reach up to 86.7%.