【作者】 赵妍；

【导师】 屠康；

【作者基本信息】 南京农业大学 ， 食品科学， 2010， 博士

【摘要】 樱桃番茄是我国广泛种植的经济作物之一,在我国经济发展和食物供给中占有重要地位。生物防治(Biological control)作为一种可有效控制果蔬类产品采后病害的方法引起了研究人员的广泛关注,本论文将重点研究拮抗酵母对樱桃番茄采后病害的防治效果及其作用机理。此外,采后热处理(Post-harvest heat treatment)是一种无毒无残留的物理处理方法,合适的热处理方法能较好的维持果蔬贮藏品质、减少病虫害损失。因此,本论文以樱桃番茄果实为研究材料,以酵母菌Pichia guilliermondii为生物防治拮抗菌,并结合得到国内外广泛认可的热处理条件(38℃热空气处理),研究生物防治结合热空气处理对樱桃番茄采后病害的防治效果及其作用机理。具体研究内容和结果如下：1.拮抗酵母对樱桃番茄采后病害防治效果的研究对于采摘于绿熟期的樱桃番茄果实而言,拮抗酵母P. guilliermondii的使用可以有效地防治由病原菌Botrytis cinerea、Alternaria solani和Rhizopus stolonifer所引发的番茄果实贮藏期间灰霉病、果腐病和根霉霉烂病的发生和发展。在本试验条件下,对于病害的防治效果和拮抗酵母的使用浓度正相关,酵母菌的浓度越大,对于病害侵染的防治效果越好；当酵母菌菌悬液浓度为108 CFU/mL时,对樱桃番茄采后三种主要病原菌的防治效果均最好。在樱桃番茄采后的三种主要病原真菌中,拮抗酵母Pguilliermondii对于R. stolonifer的抑制效果最为显著。对于三种不同成熟度的樱桃番茄果实而言(红熟期、粉红期、绿熟期),采用浓度为108CFU/mL的拮抗酵母菌悬液均可以显著地减少其采后自然病害的发生和发展；其中对于绿熟期的樱桃番茄果实则可以完全地防治贮藏期间自然病害的发生。与此同时,拮抗酵母P. guilliermondii的使用不会影响樱桃番茄果实的主要贮藏品质指标,包括：果实失重率；总可溶性固形物、可滴定酸、维生素C含量以及果实颜色。2.拮抗酵母生物防治作用机理的初步研究本章首先研究了拮抗酵母P. guilliermondii的菌悬液、活菌液、高压灭菌液以及过滤液对樱桃番茄采后人工接种病害的防治效果,结果表明：该拮抗酵母不产生体外抗菌素；其生物防治作用效果和其与病原菌之间对于营养物质的竞争有关。其次研究了拮抗酵母的接种时间对于防治果实根霉霉烂病的影响,结果表明：相对于病原微生物,拮抗酵母接种于伤口处的时间越早,对樱桃番茄采后病害的防治效果越好；其生物防治作用效果和其与病原菌之间对于生存空间的竞争有关。最后研究了拮抗酵母对于樱桃番茄果实抗病性防御酶的诱导作用,结果表明：酵母菌P. guilliermondii诱导了樱桃番茄果实细胞中过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)、几丁质酶(CHI)以及p-1,3葡聚糖酶等防御酶活性的提高,增强了其在贮藏期间对于病害的防御能力。3.采后热空气处理对樱桃番茄贮藏品质的影响及贮期主要病害的控制由于在生产实践当中,单一使用生物防治的方法其防病保鲜效果可能难以与化学杀菌剂相抗衡,因此,我们尝试将生物防治与采后热处理这一安全、有效、无残留的处理方式相结合,以期增强对果蔬采后病害的防治效果。在将此两种方式结合使用之前,有必要研究一下热处理对樱桃番茄贮藏品质的影响及贮期主要病害的控制,以便于选择最适宜的热处理条件。本章试验所选用的采后热处理方式为热空气处理(Hot Air Treatment, HAT),选择国际上惯常使用的应用于果蔬采后防病保鲜的热空气处理温度(38℃),得出以下试验结论：(1)38℃热空气处理可以有效地防治樱桃番茄果实在室温下贮藏期间的三种主要病害；在本试验条件下,热处理时间越长,对于病害的防治效果越好；但即使是处理时间最短的24 h,也可以显著地降低三种病害的发病率并减小果实的病斑直径,且该防治效果可以维持到贮藏期结束；(2)对于不同处理组果实的品质指标而言,38℃热空气处理24h不会影响樱桃番茄果实的贮藏品质；而48 h、72 h的热空气处理会使番茄果实总可溶性固形物、可滴定酸含量以及维生素C含量不同程度地下降；此外,72 h的热空气处理还会加速采后番茄果实颜色的转变,加速果实的衰老。所以,综合考虑本章的试验结果,本着节约能源与社会资本的原则,选择38℃热空气处理24 h作为最佳采后热空气处理条件,它将在下几章的研究中与生物防治相结合,以期增强对果蔬采后病害的防治效果。4.拮抗酵母结合热空气处理对樱桃番茄采后病害的防治效果本部分研究首先通过拮抗酵母P. guilliermondii在不同温度下生长曲线的测定,得出拮抗酵母P. guilliermondii不耐热,因此,生物防治与热空气处理的结合顺序为先热处理,再拮抗酵母处理。接下来,研究了单独热空气处理、拮抗酵母处理以及二者结合对樱桃番茄采后三种主要病原微生物B. cinerea、A. solani和R. stolonifer的防治效果,结果表明：对于樱桃番茄果实贮期灰霉病而言,单独热空气处理防治效果要显著好于拮抗酵母处理；对于果腐病而言,单独拮抗酵母处理防治效果要显著好于热空气处理；而对于根霉霉烂病而言,当贮期结束时,两种处理方法的防治效果相差无几。因此,不同的病原菌对热处理或者生物防治的敏感度不同,单一使用一种采后处理方式可能无法防治多种病原菌的侵染,考虑到果实采后其贮藏环境中微生态的复杂多样性,若将热空气处理与拮抗酵母处理结合应用,则可以同时增强对樱桃番茄果实贮期多种病害侵染的防治效果,极大地降低果实采后贮藏期间的病害发生率。扫描电镜观察结果显示：热空气处理通过抑制病原菌菌丝生长与孢子萌发来防治病原真菌对果实的侵染,而拮抗酵母则通过营养物质和生存空间的竞争抑制病原菌,此外,酵母细胞可以紧密地附着于病原菌菌丝体上抑制其生长。本章的最后,使用电子鼻来检测樱桃番茄果实贮藏期间挥发性气味的浓度,并将检测结果与不同处理组果实病害发生情况建立联系,结果显示：经过热空气结合拮抗酵母处理的樱桃番茄果实伤口处腐烂程度最低、溢出的挥发性气味最少。5.拮抗酵母结合热空气处理对樱桃番茄果实抗病性防御反应的诱导由于目前的研究对于热处理对果蔬表皮和伤口处潜在病原菌的直接杀死作用和拮抗酵母通过营养及生存空间的竞争来抑制病原菌的生长已经有了较为明确的认识,因此本章将重点研究热空气处理结合拮抗酵母P. guilliermondii对樱桃番茄果实采后主动防御反应(active defense responses)的诱导。试验结果表明：(1)单独热空气处理或者单独拮抗酵母处理均可以有效地防治樱桃番茄果实贮藏期间灰霉病、果腐病以及根霉霉烂病的发生和发展,但若将两种采后处理方法相结合,则可以显著地提高对于三种病害的防治效果；(2)热空气处理和拮抗酵母均可以诱导樱桃番茄果实的抗病性防御反应,热空气处理可以调节番茄果实细胞中H202的代谢,而拮抗酵母则可以诱导番茄果实细胞中木质素的生物合成以及β-1,3葡聚糖酶的积累；(3)结合处理对于樱桃番茄果实抗病性防御反应的诱导要显著地好于二者单独应用,包括对于H202代谢的调节、提高木质素生物合成、诱导β-1,3葡聚糖酶这几个方面。6.拮抗酵母采前喷施对樱桃番茄贮藏品质的影响及贮期自然病害的防治由于许多病原微生物在果实生长期间就已侵入果实并长期潜伏,而不表现症状,直到果实成熟采收和环境条件适合的时候才发病,此类病害的防治主要应加强采前的田间管理,以清除病原、减少果实采后病害的发生。因此,本章的研究重点是拮抗酵母P. guilliermondii采前喷施对樱桃番茄果实贮藏品质的影响以及贮期自然病害的防治,通过对于试验数据的分析,可以得出以下几点结论：(1)拮抗酵母P. guilliermondii采前喷施以及其喷施次数均不会影响樱桃番茄果实采前生长时期以及采后贮藏阶段的果实品质；(2)拮抗酵母采前喷施可以显著地降低樱桃番茄果实贮藏期间自然病害的发生率,在本试验条件下,其采前喷施次数越多,对自然病害的防治效果越好；(3)P. guilliermondii采前喷施可以诱导樱桃番茄果实抗病性防御酶活性的升高,在本试验条件下,其采前喷施次数越多,对于酶活性的诱导能力越强；(4)在本试验条件下,拮抗酵母P. guilliermondii采前喷施的次数越多,其在番茄果实表皮的定殖生长能力就越强。更多还原

【Abstract】 Cherry tomato is an important agricultural commodity and widely planted in China. Because many postharvest pathogens infect through wounds caused during harvest and are readily accessible to treatment with antagonists, biological control is considered a desirable alternative to chemical control. Furthermore, heat treatment is a non-damaging physical treatment that can be used to alleviate physiological diseases of various commodities. Therefore, in this paper, we will select cherry tomato fruit as experimental material, and our purpose is to research the effect and mechanism of antagonistic yeast (Pichia guilliermondii), alone or in combination with heat treatment (Hot air treatment,38℃) on the reduction of postharvest disease on cherry tomato fruit.The contents and results are as follows: 1. Biological control of the postharvest pathogens Botrytis cinerea, Alternaria solani and Rhizopus stolonifer on cherry tomato fruit by P. guilliermondiiIn mature green cherry tomato fruit, P. guilliermondii controlled three postharvest pathogens of tomato (B. cinerea, A. solani and R. stolonifer). Moreover, there was a close link between the concentration of the antagonistic yeast and the control effect. Cell suspensions at 108 CFU mL-1 were most effective in controlling all three tomato pathogens. P. guilliermondii showed efficacy against all three pathogens, but it appeared to be most effective against R. stolonifer.In tomato fruit at three different stages of maturity (red, pink, or mature green), a cell suspension of P. guilliermondii at 108 CFU mL-1 significantly decreased the rate of natural infection. Based on the results of an inoculation experiment, lower yeast concentrations (107 CFU mL-1) could effectively inhibit infections caused by all three pathogens, and it may be possible to use lower numbers of cells of the antagonistic yeast to control natural diseases. In this study, P. guilliermondii did not significantly affect fruit quality parameters such as FW loss, total soluble solids content, titratable acidity content, ascorbic acid content, and fruit colour of cherry tomato fruit during postharvest storage. 2. Mechanism of the yeast P. guilliermondii against postharvest pathogens on cherry tomato fruitThe yeast P. guilliermondii, was examined for its effects to control R. stolonifer on cherry tomato fruit during storage time, and in order to highlight the reason for biocontrol, possible mode of action was discussed. Results showed that the yeast autoclaved culture and culture filtrate had no effect in controlling postharvest disease caused by R. stolonifer, and inoculating P. guilliermondii prior to R. stolonifer had better biocontrol efficacy. Moreover, rapid colonization of the yeast on wound sites was observed during initial three days at 20℃, and then the population stabilized for the remaining four days. This phenomenon indicated that at room temperature, P. guilliermondii could acclimatize itself to the environment of tomato fruit wounds and occupy the living space quickly. All of the above results indicated that P. guilliermondii did not produce an antifungal substance, however, competition for nutrients and space on wounds appeared to play a role in the activity of biocontrol and it might be one of the mechanisms. In addition, the fruit inoculated with P. guilliermondii changed the expression of activities of peroxidase (POD), polyphenoloxidase (PPO), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), chitinase (CHI) andβ-1,3-glucanase, all of which were correlated with the onset of induced resistance. This result suggested that cherry tomato fruit was capable of responding to the yeast P. guilliermondii, which could activate defensive enzymes, thereby induced host disease resistance. This might be also one of the mechanisms.3. Effects of postharvest hot air treatment on quality parameters and decay development of cherry tomato fruitCompared to synthetic fungicides, biocontrol agent such as antagonistic yeast lacks eradicative ability, its spectrum of activity is narrower, and the effect of environmental factors is generally greater. It is therefore necessary to use an integrated strategy rather than a single approach. Heat treatment is a non-damaging physical treatment that can be used to alleviate physiological diseases of various commodities. The current study investigated the effects of heat treatment and antagonistic yeast, either alone or in combination, on the quality parameters and incidence of fungal infection in cherry tomato fruit.Hot air treatment (38℃) was used in this experiment and the results are as follows:(1) hot air treatment controlled three postharvest pathogens of tomato (B. cinerea, A. solani and R. stolonifer) effectively. Moreover, longer heat treatment gave a lower incidence of disease. Although cherry tomato fruit received hot air treatment (38℃) for only 24 h, the postharvest gray mold decay, black spot decay and Rhizopus decay were all significantly reduced. (2) hot air treatment (38℃for 24 h) did not significantly affect fruit quality parameters. However, hot air treatment (38℃for 48 h or 72 h) impaired the contents of total soluble solids, titratable acidity and ascorbic acid of cherry tomato fruit. Furthermore, hot air treatment (38℃for 72 h) made fruit color change fast. Therefore, considering all of the above results and the rule for saving energy and social capital, hot air treatment (38℃for 24 h) was selected in the following experiments.4. A combination of P. guilliermondii and hot air treatment prevents cherry tomato spoilage by fungiResults showed that antagonistic yeast P. guilliermondii’s ability to survive was inhibited gravely by the 38℃hot air treatment and this yeast was not heat-resistant, thus controlling postharvest diseases by applying P. guilliermondii after postharvest hot air treatment is recommended. In addition, for controlling various diseases, heat treatment or antagonistic yeast alone is not satisfactory; the best method, according to this research, is the combination of hot air treatment and P. guilliermondii, which has significant efficacy in controlling postharvest diseases of cherry tomato fruit caused by B. cinerea, A. solani and R. stolonifer.Scanning electron microscopy (SEM) observation indicated that hot air treatment at 38℃for 24 hours can inhibit hyphae growth and spore germination of R. stolonifer. Furthermore, P. guilliermondii multiplied rapidly on fruit wounds, and its cells had a strong capability of competing for nutrient substance and living space with pathogen. Although the combination of hot air treatment and P. guilliermondii not to eradicate pathogens existing on fruit wounds, the cells of yeast P. guilliermondii reproduced on fruit wounds and firmly attached to fungal hyphae, inhibiting their ability to infect cherry tomato fruit and competing for nutrients and living spaces to provide persistent protection. Furthermore, the result of electronic nose also showed that a combination of hot air treatment and P. guilliermondii is one of the most effective techniques at controlling postharvest fungal spoilage in cherry tomato fruit.5. Antagonistic yeast in combination with hot air treatment reduces diseases and elicits the active defense responses in harvested cherry tomato fruitIn recent years, considerable amount of research has focused on the active resistance in harvested fruits and vegetables, which acts as an important manageable form of crop protection. Results showed that hot air treatment at 38℃for 24 h in combination with P. guilliermondii at 108 CFU mL-1 was the most effective approach to reduce various infections on cherry tomato fruit’s wounds. Moreover, the combined hot air and P. guilliermondii treatment stimulated a rapid increase of H2O2 and higher lignin deposition in cherry tomato fruit showing that the oxidative burst and biological synthesis of lignin might play important roles in the fruit’s active defense responses. In addition, the reduction of the fruit’s susceptibility to pathogens by the combined treatment was positively correlated with higher activities of phenylalanine ammonia-lyase (PAL) andβ-1,3-glucanase in cherry tomato fruits, both of which are associated with plant defense responses.6. Effects of preharvest P. guilliermondii treatment on quality parameters and decay development of cherry tomato fruitMany pathogenic microorganisms have penetrated the fruit during the period of fruit growth, but the symptom of diseases usually be shown until fruit being stored with appropriate environmental conditions. In order to reduce the incidence of postharvest diseases, the primary prevention for such diseases should be taken to strengthen the field management before fruit harvest. Therefore, the purpose of this study was to evaluate the effects of preharvest P. guilliermondii treatment on quality parameters and decay development of cherry tomato fruit. Based on the results of this experiment, we suggested that:(1) both preharvest P. guilliermondii treatment and the spraying frequency did not significantly affect fruit quality parameters regardless cherry tomato fruit growing or stored after harvest. (2) preharvest P. guilliermondii treatment significantly reduced the incidence of postharvest natural decay, and the efficacy of biological control was positively correlated with the spraying frequency of P. guilliermondii. (3) preharvest P. guilliermondii treatment induced the defensive enzymes of cherry tomato fruit, such as POD, PAL andβ-1, 3-glucanase, which was related to host disease resistance. Furthermore, the efficacy of induction was positively correlated with the spraying frequency of P. guilliermondii. (4) the ability of P. guilliermondii to colonize on the surface of fruit was also positively correlated with the spraying frequency of antagonistic yeast.更多还原