Mechanisms Of Antagonists And Chemical Compounds Against Postharvest Diseases And Cloning Of Disease Resistant Genes From Fruit

Postharvest decay caused by fungal pathogens is the major losses in fresh fruits. Application of fruit with biological antagonists has demonstrated an efficient way for controlling postharvest diseases of fruit, reducing fungicidal dosage, thus increasing the food safety and reducing latent environmental danger. However, application of antagonist alone is not usually effective in controlling postharvest diseases as comparison with using chemical fungicides. Therefore, in order to control postharvest diseases effectively, this study was mainly to investigate mechanisms of fruit disease control using biological antagonists and/or chemical compounds, and cloning and analysis of disease-resistant genes. The results were presented as following: 1. Antagonists Cryptococcus laurentii and Bacillus subtilis could effectively control postharvest diseases in jujube fruit caused by Penicillium expansum and Alternaria alternata. C. laurentii showed the better effect against postharvest diseases than B. subtilis at the same concentration. As to the same antagonist, the higher concentration of antagonist was, the better of controlling efficacy was. Inoculation of postharvest pathogen in the yeast wounds obviously enhanced the populations of the antagonistic yeast. However, B. subtilis did not show the same results. 2. There were significant differences in sensitivity to the fungicides (Deccozil, Sportak, Iprodine and Stroby) among the different yeasts. R. glutinis was more sensitive to Deccozi, Iprodione and Stroby as compared to other yeasts. Combination yeasts with fungicide could more significantly enhance biocontrol ability of yeasts against the pathogenic fungi in vitro. C. laurentii was the most effective in controlling spore germination of P. expansum and A. alternata when combined with stroby at the concentration of 100 μl/L. The addition of 2% (w/v) sodium bicarbonate (SBC) in the suspensions of antagonistic yeast C. laurentii or T. pullulans significantly limited spore germination and germ tube elongation of P. expansum and A. alternata. Biocontrol activity of C. laurentii or T. pullulans was significantly increased when yeast combined with SBC. Effects of C. laurentii with and without SBC on controlling P. expansum and A. alternata were better than those of T. pullulans. 3. The abilities of C. laurentii or B. subtilis to induce disease resistance in jujube fruit against diseases by P. expansum or A. alternata were investigated. Induced resistance by C. laurentii or B. subtilis was distance-and time-dependent, and limited to the fruit tissue closely surrounding the antagonists' application sites. C. laurentii could induce stronger defense response in jujube fruit and reduce lower disease incidence and lesion diameter than B. subtilis. This increased resistance was related with temporal differential induction of chitinase, β-1, 3-glucanase, phenylalanine ammonia lyase (PAL), peroxidase (POD) and Polyphenol oxidase (PPO). 4. Preharvest treatments with 2 mM salicylic acid (SA) and 0.2 mM methyl jasmonate (MeJA) significantly reduced lesion diameters on sweet cherry fruit caused by Monilinia fructicola compared with control postharvest treatments. Preharvest treatment of sweet cherry with SA or MeJA induced β-1, 3-glucanase, PAL and POD activities and enhanced the content of ethylene during the early storage time. The efficacy of inducing resistance in sweet cherry fruit preharvest-treated with SA or MeJA to M. fructicola was better than that for fruit with postharvest treatments, especially, at 25°C. Activities of β-1, 3-glucanase and PAL in SA-or MeJA-treated cherry fruit stored at 25°C for both pre-and postharvest treatments were significantly higher than those in fruit stored at 0°C. SA with a concentration of 2 mM showed direct fungitoxicity on M. fructicola and significantly inhibited mycelial growth and spore germination of the pathogen. MeJA at 0.2 mM had little inhibitory effect on mycelial growth and spore germination of M. fructicola. The fruit preharvest-treated with MeJA expressed higher activity of β-1, 3-glucanase and PAL than fruit treated with SA and the control during the early storage time. 5. Treatment of peach fruit with C. laurentii at 1 × 108CFU/ml alone, or combining C. laurentii at 5 × 107CFU/ml with MeJA at a concentration of 0.2 mM all resulted in a lower lesion diameter of brown rot and blue mold caused by M. fructicola and P. expansum compared with the controls in peach fruit. MeJA at 0.2 mM inhibited mycelial growth of P. expansum. However, it had a little effect on M. fructicola. MeJA and C. laurentii alone or in combination induced higher activities ofchitinase, β-1, 3-glucanase, PAL and POD than applying the yeast alone at both 25 and 0°C. 0.2 mM MeJA increased population of C. laurentii, which induced stronger disease resistance in fruit than MeJA or yeast alone, and resulted in a lower lesion diameter of brown rot and blue mold caused by M. fructicola and P. expansum. 6. A pair of degenerate primer was designed according to the sequences of some β-1, 3-glucanase genes in different plants. And one fragment was amplified from jujube fruit cDNA. After that, β-1, 3-glucanase genes (Glu-1 and Glu-2 full-length cDNAs) were cloned according to the strategy of RACE. RT-PCR results showed that transcription level of Glu-1 could be induced by yeast antagonist C. laurentii, and this was consistent with the enzyme expression of β-1, 3-glucanase; however, Glu-2 did not showed the same results as Glu-1.