Integration of pre- and postharvest treatments for the control of black spot caused by Alternaria alternata in stored persimmon fruit

In Israel, black spot caused by Alternaria alternata is the main postharvest factor that impairs the quality and reduces the storability of persimmon fruit (Diospyros kaki cv. Triumph). The fungus infects the fruit in the orchard and remains quiescent until harvest. After harvest, the pathogen slowly colonizes the fruit during storage at 0 °C, which elicits black spot symptom development 2–3 months after storage entry. A commercial postharvest dip treatment in chlorine at 500 mg L^?1, released from sodium troclosene tablets, effectively controlled black spot in fruit stored for up to 2 months. However, decay incidence increased as the length of storage was extended beyond 2.5 months. The long incubation period that precedes black spot symptom development after harvest enabled the development of a series of integrative approaches for application at the pre- and postharvest stages, in combination with the commercial chlorine dip treatment, to improve the control of black spot disease. Preharvest treatments included treatment with the cytokinin-like N₁-(2-chloro-4-pyridyl)-N₃-phenylurea (CPPU) 30 d after fruit set, or a single spray with the curative fungicide polyoxin B 14 d before harvest, and when one of these was applied in combination with the postharvest chlorine dip treatment, the black spot infected area was reduced by 3 and 60%, respectively, compared with the chlorine dip alone. At the postharvest stage, fogging during storage, or post-storage on-line spraying with sodium troclosene, when applied in combination with the postharvest chlorine dip, improved the percentage of marketable fruit by 2 or 10%, respectively, compared with the chlorine dip alone. The results indicate that postharvest pathogens that show a slow colonization pattern might enable the integration of pre- and postharvest disease control methods to improve quality and reduce postharvest disease development.     

p-香豆酸通过加速伤口处聚酚软木脂和木质素的沉积促进马铃薯块茎愈伤

【目的】研究p-香豆酸（p-Coumaric acid,p-CA）处理对马铃薯块茎伤口处聚酚软木脂和木质素积累的影响及相关机理。【方法】用0.5 mmol∙L^-1 p-CA浸泡切半‘大西洋’马铃薯块茎10 min,以蒸馏水处理作为对照,于室温条件下避光贮存进行愈伤。测定损伤块茎在愈伤期间的失重率以及损伤接种硫色镰刀菌（Fusarium sulphureum）块茎的病情指数,观察伤口处聚酚软木脂和木质素的积累,测定伤口处苯丙烷代谢关键酶和过氧化物酶活性,以及苯丙烷代谢产物和H₂O₂含量。【结果】p-CA处理显著降低了损伤块茎的失重率和损伤接种块茎的病情指数,14 d时,处理块茎的失重率和病情指数分别比对照低38.46%和43.18%。p-CA处理加速了块茎伤口处聚酚软木脂和木质素的积累,14 d时,处理块茎的聚酚软木脂和木质素的细胞层厚度分别比对照高26.43%和30.26%。p-CA处理还明显提高了块茎伤口处苯丙氨酸解氨酶、肉桂酸-4-羟化酶、4-香豆酰辅酶A连接酶和肉桂醇脱氢酶的活性,7 d时,处理块茎的酶活性分别比对照高32.17%、23.51%、25.09%和23.08%。14 d时,p-CA处理块茎的肉桂酸、p-香豆酸、咖啡酸和总酚含量分别比对照高34.26、38.29%、19.31%和41.04%;21 d时,处理块茎的阿魏酸、芥子酸含量分别比对照高38.33%和20.47%。此外,p-CA处理促进了块茎伤口处肉桂醇、松柏醇、芥子醇和木质素的积累,14 d时,处理块茎的肉桂醇和木质素含量分别高于对照36.93%和31.66%;21 d时,处理块茎的松柏醇和芥子醇含量分别高于对照41.43%和34.05%。p-CA处理还提高了块茎伤口处H₂O₂含量和过氧化物酶活性,7 d时,处理块茎的H₂O₂含量和过氧化物酶活性分别高于对照40.25%和27.01%。【结论】p-CA处理可通过激活苯丙烷代谢,提高H₂O₂含量和过氧化物酶活性,促进马铃薯块茎伤口处聚酚软木脂和木质素的积累,从而加速块茎愈伤。     

采前与采后处理结合控制柿子贮藏过程中链格孢菌(Alternaria alternata)引起的黑斑病的研究

由链格孢菌引起的黑斑病是导致以色列柿子(Diospyros kaki cv.Triumph)贮藏过程中果实品质下降、贮期缩短的主要因素。其病原真菌在采收前的果园中即开始侵染果实,但一直处于休眠状态。采收后,在0℃贮藏环境中病原菌开始繁殖,当贮藏2～3个月后,柿果表面出现黑斑症状。商业上采用二氯异氰尿酸钠片剂溶解后生成500 mg.L-1的氯水浸果,可在2个月的贮期内有效控制黑斑病的发生。然而,当贮期超过2个半月后,柿子果实的腐烂率则不断增加。在柿子采后出现黑斑症状之前病原真菌较长的潜伏期内,采前与采后阶段可采取一系列的综合技术措施,并与商业氯水浸果处理相结合来提高对贮藏柿果黑斑病的防控效果。采前处理包括柿子坐果30 d后,以1-(2-氯-4-吡啶)-3-苯基脲(CPPU)处理或采收前14 d以治疗性杀菌剂多抗霉素(polyoxin B)进行喷果处理。试验结果表明,与采后单独使用氯水浸果处理相比,将CPPU或polyoxin B处理与采后商业处理相结合,可使柿果黑斑浸染面积分别减小3%和60%;采收后贮藏期间二氯异氰尿酸钠喷雾处理或采后立即用二氯异氰尿酸钠喷果处理与采后商业氯水处理相结合比单独使用氯水处理可分别提高柿子好果率2%和10%。研究结果表明,对于具有较长潜伏侵染方式的采后病原菌来说,可应用采前和采后处理相结合的方法来提高果实的贮藏品质及减轻采后病害的发生。     

Expression of an Antifungal Peptide in Saccharomyces: A New Approach for Biological Control of the Postharvest Disease Caused by Colletotrichum coccodes

ABSTRACT A cecropin A-based peptide inhibited germination of Colletotrichum coccodes at 50 muM. The DNA sequence encoding the peptide was cloned in pRS413, using the Saccharomyces cerevisiae invertase leader sequence for secretion of the peptide, and expressed in yeast. Yeast transformants inhibited the growth of germinated C. coccodes spores and inhibited decay development caused by C. coccodes in tomato fruits. Expression of the antifungal peptide in yeast therefore represents a new approach for the biological control of postharvest diseases.     

The Analysis of Fruit Protection Mechanisms Provided by Reduced-Pathogenicity Mutants of Colletotrichum gloeosporioides Obtained by Restriction Enzyme Mediated Integration

ABSTRACT Colletotrichum gloeosporioides is an important postharvest pathogen that attacks ripe avocado fruit. Two reduced-pathogenicity mutants, Cg-M-142 and Cg-M-1150, previously obtained by restriction enzyme mediated integration, were used for the sequential analysis of the induction of biocontrol in avocado fruit. Plant biochemical indicators, such as H(+)-ATPase activity and levels of reactive oxygen species, phenylalanine ammonia lyase, epicatechin, and an antifungal diene, were investigated. The main difference between Cg-M-142 and Cg-M-1150 was the lack of appressorium formation by the latter. Preinoculation of avocado fruit with Cg-M-142 enhanced H(+)-ATPase activity and the production of reactive oxygen species. These early signaling events were followed by higher phenylalanine ammonia lyase activity and higher levels of epicatechin and the antifungal diene, and decay was delayed. Unlike Cg-M-142, Cg-M-1150 did not activate early signaling events related to fruit resistance. We suggest that the initiation of early signaling events affecting fruit resistance is determined by the capability of the pathogen to interact with the fruit during appressorium formation. Furthermore, the intensity of the fruit defense response determines the level of resistance during fruit storage.     

Effect of Modified Atmosphere for Control of Black Spot, Caused by Alternaria alternata, on Stored Persimmon Fruits

ABSTRACT Modified atmosphere packaging (MAP) of persimmon fruit resulted in the accumulation of acetaldehyde to a level of 80 mug/ml; ethanol to a level of 900 mug/ml; and CO(2) up to 30%. When fruits were stored at -1 degrees C for 4 months in such atmospheres, the incidence of black spot disease, caused by Alternaria alternata, was reduced. The effects of each of these gases were examined to determine their individual involvement in the inhibition of Alternaria development during storage. When A. alternata, grown at 20 degrees C on potato dextrose agar or inoculated in persimmon fruit, was exposed for 24 h to different levels of each volatile, acetaldehyde was the most fungistatic but only at concentrations higher than those that accumulated under MAP; CO(2) was moderately inhibitory at concentrations from 10 to 60%, whereas ethanol had no effect. Similar inhibitory effects were obtained with acetaldehyde at 620 mug/ml or 30% CO(2) when in vitro cultures of A. alternata and infected fruits were exposed for up to 2 weeks at 20 degrees C, but 1,000 mug of ethanol per ml had only a transitory inhibitory effect under these conditions. Based on analysis of the effect of concentration versus time for each gas accumulating in MAP, we suggest that the increasing concentration of CO(2) during storage is the principal factor in the inhibition of black spot disease development.     

Development of Colletotrichum gloeosporioides Restriction Enzyme-Mediated Integration Mutants as Biocontrol Agents Against Anthracnose Disease in Avocado Fruits

ABSTRACT Reduced-pathogenicity mutants of the avocado fruit pathogen Colletotrichum gloeosporioides isolate Cg-14 (teleomorph: Glomerella cingulata) were generated by insertional mutagenesis by restriction enzyme-mediated integration (REMI) transformation. Following seven transformations, 3,500 hygromycin-resistant isolates were subjected to a virulence assay by inoculation on mesocarp and pericarp of cv. Fuerte avocado fruits. Fourteen isolates showed a reduced degree of virulence relative compared with wild-type Cg-14. Two isolates, Cg-M-142 and Cg-M-1150, were further characterized. Cg-M-142 produced appressoria on avocado pericarp similar to Cg-14, but caused reduced symptom development on the fruit's pericarp and mesocarp. Isolate Cg-M-1150 did not produce appressoria; it caused much reduced maceration on the mesocarp and no symptoms on the pericarp. Southern blot analysis of Cg-M-142 and Cg-M-1150 showed REMI at different XbaI sites of the fungal genome. Pre-inoculation of avocado fruit with Cg-M-142 delayed symptom development by the wild-type isolate. Induced resistance was accompanied by an increase in the levels of preformed antifungal diene, from 760 to 1,200 mug/g fresh weight 9 days after inoculation, whereas pre-inoculation with Cg-M-1150 did not affect the level of antifungal diene, nor did it delay the appearance of decay symptoms. The results presented here show that reduced-pathogenicity isolates can be used for the biological control of anthracnose caused by C. gloeosporioides attack.