Development of biocontrol agents from food microbial isolates for controlling post-harvest peach brown rot caused by Monilinia fructicola

An unconventional strategy of screening food microbes for biocontrol activity was used to develop biocontrol agents for controlling post-harvest peach brown rot caused by Monilinia fructicola. Forty-four microbial isolates were first screened for their biocontrol activity on apple fruit. Compared with the pathogen-only check, seven of the 44 isolates reduced brown rot incidence by >50%, including four bacteria: Bacillus sp. C06, Lactobacillus sp. C03-b and Bacillus sp. T03-c, Lactobacillus sp. P02 and three yeasts: Saccharomyces delbrueckii A50, S. cerevisiae YE-5 and S. cerevisiae A41. Eight microbial isolates were selected for testing on peaches by wound co-inoculation with mixtures of individual microbial cultures and conidial suspension of M. fructicola. Only two of them showed significant biocontrol activity after five days of incubation at 22 degrees C. Bacillus sp. C06 suppressed brown rot incidence by 92% and reduced lesion diameter by 88% compared to the pathogen-only check. Bacillus sp.T03-c reduced incidence and lesion diameter by 40% and 62%, respectively. The two isolates were compared with Pseudomonas syringae MA-4, a biocontrol agent for post-harvest peach diseases, by immersing peaches in an aliquot containing individual microbial isolates and the pathogen conidia. Treatments with isolates MA-4, C06 and T03-c significantly controlled brown rot by 91, 100, and 100% respectively. However, only isolates MA-4 and C06 significantly reduced brown rot by 80% and 15%, respectively when bacterial cells alone were applied. On naturally infected peaches, both the bacterial culture and its cell-free filtrate of the isolate C06 significantly controlled peach decay resulting in 77 and 90% reduction, respectively, whereas the treatment using only the bacterial cells generally had no effect. Isolate C06 is a single colony isolate obtained from a mesophilic cheese starter, and has been identified belonging to Bacillus amyloliquefaciens. The results have clearly demonstrated that isolate C06 has a great potential for being developed into a biocontrol agent.     

Synergistic effect of oligochitosan and silicon on inhibition of Monilinia fructicola infections

BACKGROUND: Oligochitosan has broad‐spectrum antimicrobial activity and shows an obvious inhibitory effect on phytopathogens. In addition, as an exogenous elicitor, it can induce various defence responses, including affecting the activities of several defence‐related enzymes and substances in some plants. Owing to this dual function of oligochitosan, it can be used to control postharvest diseases of fruits. Silicon, like oligochitosan, also has a dual function. In this study the synergistic effect of oligochitosan and silicon on the decay control of apple fruit was investigated. RESULTS: In vitro, both oligochitosan and silicon significantly inhibited spore germination, germ tube elongation and mycelial growth of Monilinia fructicola, with higher concentrations having a greater effect. The synergistic effect of oligochitosan and silicon at half‐maximal inhibitory concentration on disease control at 25 °C was much better than the effect of oligochitosan or silicon alone, not only in vitro but also in vivo. CONCLUSION: The results showed that a combination of oligochitosan and silicon had a synergistic effect on the control of disease caused by M. fructicola in apple fruit at 25 °C. Copyright © 2010 Society of Chemical Industry     

丁香油、百里酚复合壳聚糖处理对桃采后褐腐病菌的抑制作用

为了进一步提高壳聚糖用于涂膜时的综合抗菌能力,采用滤纸片法及菌丝扩展法测定并对比了丁香油和百里酚对桃采后致病褐腐菌的抗菌能力及用微孔板法测定了它们的最小抑菌浓度,并将丁香油、百里酚复合壳聚糖对桃果进行涂膜,测量果体经过涂膜和接菌后的病斑扩展。结果表明丁香油和百里酚都具有较好的抗褐腐菌能力,百里酚强于丁香油,复合壳聚糖涂膜时二者都可以提高壳聚糖总体抗菌能力且复合百里酚效果较好。二者具有作为壳聚糖涂膜保鲜添加剂的应用潜力。     

Effect of chitosan on incidence of brown rot,quality and physiological attributes of postharvest peach fruit

The effect of chitosan (5.0 and 10.0 mg ml ⁻¹) on the incidence of brown rot (caused by Monilinia fructicola), quality attributes and senescence physiology of peaches was investigated. It was found that both concentrations of chitosan reduced the incidence of brown rot significantly and delayed the development of disease compared with the control, but were less effective than the fungicide prochloraz. Chitosan‐treated peaches were firmer and had higher titratable acidity and vitamin C content than prochloraz‐treated or control peaches. Compared to control (water‐treated) peaches, chitosan‐treated peaches showed lower respiration rate, less ethylene and malondialdehyde (MDA) production, higher superoxide dismutase (SOD) activity and better membrane integrity. Hence it can be concluded that chitosan has the potential to control brown rot, preserve valuable attributes and prolong the shelf life of postharvest peaches, presumably because of its antifungal property and inhibition of the ripening and senescence process of postharvest peaches. © 2000 Society of Chemical Industry     

Influence of additives on adhesion of Penicillium frequentans conidia to peach fruit surfaces and relationship to the biocontrol of brown rot caused by Monilinia laxa

Additives, such as sucrose, d-sorbitol, glycerol, sodium alginate, carboxymethyl cellulose, silica gel, gelatine, non-fat skimmed milk and a commercial adhesive were added to conidia of Penicillium frequentans at two different points in the production process of the formulation of this fungus to improve conidial adhesion. Conidial adhesion was estimated as the number of P. frequentans conidia (no. conidia cm(-2)) and colony-forming units of P. frequentans per unit area (cfu cm(-2)) that adhered to glass slides or to peach surfaces. The P. frequentans conidial concentration had a significant effect on conidial adhesion, while the shelf life of conidia did not have any effect. The highest adhesion of P. frequentans conidia to glass slides was observed when conidial concentrations were greater than 10(6) conidia ml(-1). P. frequentans conidial adhesion was improved when 1.5% sodium alginate or 1.5% carboxymethyl cellulose were added to the conidial mass obtained after production and before drying by the fluid bed drying process. Conidial adhesion was also enhanced when 1.5% sodium alginate, 1.5% carboxymethyl, or 1.5% gelatine were added to conidia after fluid bed drying. P. frequentans formulations with 1.5% sodium alginate or 1.5% carboxymethyl cellulose were more effective in reducing brown rot caused by Monilinia laxa than dried P. frequentans conidia alone. Our results show that additives can improve adhesion of P. frequentans conidia to fruit surfaces, resulting in more effective control of brown rot in peaches.     

Improvement of microwave treatment with immersion of fruit in water to control brown rot in stone fruit

Monilinia spp. are the most important cause of brown rot in stone fruit. Currently, no chemical fungicides are allowed in the European Union to be applied in stone fruit after harvest. Microwave (MW) treatments at 20 kW with fruit immersed in water at 40 °C for 50 or 60 s were selected as effective conditions to control brown rot without affecting the appearance of the fruit. The efficacy of the treatments was analyzed on fruit with different weights and at various infection times and inoculum concentrations. When the MW treatment was applied for 50 s, brown rot control was significantly higher for smaller fruit in comparison with larger fruit and MW efficacy decreased with increasing time between inoculation and treatment from 0 or 24 h to 48 h and inoculum concentration from 10³ to 10⁵ conidia mL^− 1. When the treatment time was increased to 60 s, a better control of brown rot was observed and, in general, none of the studied factors had a significant effect on the efficacy of the treatment. MW treatments were also evaluated on naturally infected fruit; brown rot incidence was significantly reduced to less than 43% when MW treatment was applied for 50 s and to less than 7% when applied for 60 s.Industrial relevanceThis study demonstrated the efficacy of MW treatment to control brown rot in postharvest of stone fruit. A relationship between brown rot reduction and the applied MW energy was also provided, which could be useful for designing specific equipment to process large quantities of fruit in less time without affecting the efficacy of the treatment or the quality of the fruit.     

Effect of Pichia membranaefaciens combined with salicylic acid on controlling brown rot in peach fruit and the mechanisms involved

BACKGROUND: Fruits are usually stored at low temperature to slow physiological metabolism, maintain quality and extend storage life. However, brown rot caused by Monilinia fructicola often occurs in peach fruit at low temperature, resulting in serious losses. As a promising alternative to the use of synthetic fungicides, the antagonistic yeast Pichia membranaefaciens showed a potential effect on controlling post‐harvest brown rot. To improve biocontrol efficacy of the yeast against fungal pathogen, the biocontrol efficacy of P. membranaefaciens combined with salicylic acid (SA) in inhibiting brown rot in peach fruit caused by M. fructicola was investigated. RESULTS: As compared to P. membranaefaciens used alone, the yeast combined with SA significantly reduced lesion diameter caused by M. fructicola, and increased the activities of chitinase, β‐1,3‐glucanase and peroxidase (POD), as well as enhanced the expressions of chitinase and β‐1,3‐glucanase genes in peach fruit. Moreover, SA at 2 mmol concentration did not affect yeast growth, but inhibited pathogen growth. CONCLUSION: The mechanism by which SA enhances the biocontrol efficacy of the antagonistic yeast against brown rot may be related to inducing biochemical defence responses in peach fruit, and a moderate inhibitory effect of SA on growth of M. fructicola. Copyright © 2008 Society of Chemical Industry     

壳寡糖及其磷酸化修饰产物对小鼠脾淋巴细胞增殖及诱生细胞因子的影响

目的:研究壳寡糖及其磷酸化修饰产物对小鼠脾淋巴细胞增殖及诱生细胞因子的影响。方法:采用四甲基偶氮唑盐(MTT)法检测壳寡糖及其磷酸化修饰产物在不同质量浓度条件下对小鼠脾淋巴细胞增殖的影响;用酶联免疫吸附实验(ELISA)法测定白介素2(IL-2)和γ干扰素(IFN-γ)的含量。结果:壳寡糖及其修饰产物对小鼠脾细胞均具有明显的细胞增殖作用,并存在一定的剂量依赖关系,2种实验药物均能提高IFN-γ水平;IL-2测定结果显示,壳寡糖具有明显提高脾细胞分泌IL-2的作用。结论:壳寡糖及其修饰产物具有明显的免疫增强作用,该作用与诱导小鼠脾细胞增殖、提高脾细胞中IFN-γ、IL-2的水平有关。     

桃果实褐腐病拮抗菌的筛选、鉴定及其拮抗活性

以桃褐腐菌(Monilinia fructicola)为靶菌,采用稀释分离法和平板对峙法从桃园土壤中筛选出对病原菌有较强拮抗作用的菌株12a和14b。通过离体和活体实验对拮抗菌抑菌活性进行研究,经菌株形态、生理生化特性及16S rRNA基因序列分析进行菌株鉴定。结果表明:菌株12a和14b分别鉴定为死谷芽孢杆菌(Bacillus vallismortis)和高地芽孢杆菌(Bacillus altitudinis),其无菌发酵滤液和挥发性代谢产物对桃褐腐菌的生长有显著的抑制作用,其中12a挥发性代谢产物对其菌丝生长的抑菌效果更为显著(p<0.05)。两株拮抗菌的菌液浸泡和熏蒸桃果实,延缓了桃褐腐病发病时间,有效地控制了病斑的扩展。      

Effects of potassium sorbate on postharvest brown rot of stone fruit

The effect of potassium sorbate (K-sorb), a low-toxicity chemical, to control Monilinia spp. was investigated. Preliminary in vitro studies found the MIC of K-sorb for conidial germination and mycelial growth was, respectively, 260 and 1,250 mg/ liter. Immersion of naturally infected peach and nectarine fruit in a solution (15 g/liter) of K-sorb for 120 s reduced brown rot by over 80% in four of five trials. Although treated fruits showed a significant reduction in firmness with respect to the control, they did not reach the overripe stage and retained acceptable quality parameters. In an attempt to elucidate the mechanism of action for K-sorb, the inhibition of enzymatic activity by K-sorb was also tested. In a radial diffusion assay, the addition of K-sorb to agarose reduced polygalacturonase (PG) activity across the concentrations considered. The greatest reduction (54.3%, with respect to the control) was obtained at a sorbate concentration of 15 g/liter. PG kinetic activity of Monilinia laxa observed by a spectrophotometric assay peaked after 40 min in all samples tested. PG activity was significantly higher in the control than in the samples with increased K-sorb concentrations. In conclusion, based on these findings, K-sorb can be recommended as a low-toxicity antifungal compound against Monilinia spp. in peaches and nectarines with its mode of action probably depending in part on the inhibition of PG activity in M. laxa.     

Correlation between brown rot decay and Pilodyn measurements

Assessment of brown rot decay is one of the most important issues within remedial treatment of attacked wood constructions. Pilodyn is a well-established tool for the assessment of density of several commercially important plantation species, therefore the authors were interested in its suitability to evaluate rate of decay as well. The Pilodyn measurements performed on Norway spruce wood exposed to several brown rot fungi clearly indicate correlations between needle penetration and mass loss.     

桃褐腐病菌拮抗酵母的筛选、鉴定及其抑菌活性初探

以北京平谷桃果实表皮上分离得到的45株酵母菌为出发菌株,采用平板对峙法,筛选对桃褐腐病菌（Monilinia fructicola）具有较高拮抗作用的生防酵母菌。结果表明,共筛选出具有较好抑制效果的酵母菌6株,其中菌株MLL-9-1拮抗活性最强,对桃褐腐病菌的抑菌率达77.91%;活体筛选试验结果表明,该菌株对桃褐腐病的抑制率达50.30%。生防相关性状检测结果显示,该菌株能够产生蛋白酶,透明圈直径达30.5 mm,说明该菌主要通过分泌蛋白酶达到抑制病原菌的效果。经形态学、生理生化试验及26S rDNA序列分析,鉴定该菌为葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）。     

壳聚糖和壳寡糖对四种青霉病菌生长和病害控制的比较研究

在离体和活体条件下,开展了壳聚糖与壳寡糖对四种同属不同种青霉菌菌丝与孢子抑制作用及病害防治的研究。结果表明:离体条件下,随着壳聚糖与壳寡糖浓度的升高,对四种青霉菌菌丝与孢子的抑制效果逐渐增强,总体上壳聚糖的抑菌效果要好于壳寡糖。其中,产黄青霉菌(Penicillium chrysogenus Thorm)对壳聚(寡)糖的敏感性在四种青霉菌中最强,指状青霉菌(P.digitatum)敏感性最弱。在柑橘果实实验中,壳聚糖或壳寡糖对柑橘指状青霉病害控制效应最弱,对产黄青霉病害控制效应最强。在四种青霉中,壳聚(寡)糖对产黄青霉的抑制作用和病害控制最为明显,且壳聚糖效果好于壳寡糖。     

脱水香菇不同贮藏水分活度下细菌菌落演替规律及关键菌控制

脱水香菇是目前香菇加工产品的主要形式,贮藏环境的湿度和微生物污染是导致脱水香菇品质变化的主要因素。本实验通过高通量测序方法研究了不同水分活度（water activity,aw）（0.43、0.67、0.76、0.84）环境下脱水香菇贮藏50 d,各处理样品细菌群落的变化,并对发现的关键危害菌进行了控制技术研究。结果表明：脱水香菇携带的细菌主要为肠杆菌科,丰度达50%,贮藏50 d后,高aw环境促进了葡萄球菌科和微球菌科微生物生长。对能够导致食品安全问题的金黄色葡萄球菌控制技术研究显示,5 kGy剂量的电子束辐照杀菌几乎可以杀灭脱水香菇中全部的金黄色葡萄球菌。本研究可为脱水香菇的品质控制、延长货架期提供理论基础。     

采后果实褐腐病防治技术研究进展

褐腐病是采后核果果实最重要的侵染性病害之一,会严重危害贮藏期的果实,造成大量损失。低温贮藏、气调贮藏、热处理、辐射处理、微波处理等物理防治技术对采后果实褐腐病的防治有一定的效果。化学杀菌剂能够高效控制采后果实褐腐病,但其有污染环境、易产生耐药性的缺陷,天然杀菌剂的应用、生物防治尤其是拮抗菌防治技术弥补了化学杀菌剂在控制采后褐腐病方面的不足,采前技术如喷钙、使用拮抗菌也能有效控制采后果实褐腐病。采前采后技术综合防治果实褐腐病将是未来的发展方向。本文从物理、化学、生物防治技术及几种技术复合等角度综述了各防治技术对采后果实褐腐病的控制作用,为综合防治褐腐病、减少商业损失提供了一定的科学依据。     

晚熟桃采后褐腐病致病菌的分离鉴定及拮抗菌对其防治效果的研究

为了明确晚熟桃采后褐腐病的主要致病菌以及拮抗菌对其防治效果,以晚熟桃品种\     

壳聚(寡)糖对柑橘酸腐、黑腐病菌的抑制作用及对采后病害的防治

通过体外实验测定不同浓度的壳聚糖和壳寡糖对酸腐和黑腐病菌孢子萌发、芽管长度和菌丝生长的抑制作用;采用刺伤接种方法评价壳聚糖和壳寡糖对柑橘果实两种病害的防治作用。离体实验结果表明,壳聚糖和壳寡糖浓度为10mg/mL时几乎完全抑制两种病原菌的菌丝生长;浓度为0.5mg/mL壳聚糖和壳寡糖能完全抑制柑橘酸腐病菌(Geotrichum candidum)孢子萌发和芽管伸长的效果,而2mg/mL的壳聚糖才能完全抑制柑橘黑腐病菌(Alternaria citri)孢子萌发和芽管伸长,但相同浓度的壳寡糖抑制A.citri的孢子萌发和芽管伸长效果较差。体内实验结果表明,其病害防治效果具有浓度依赖性,高浓度(1%)的壳聚糖和壳寡糖对果实病害的防治效果较低浓度的防治效果好。      

壳聚糖降解制备低聚壳聚糖和壳寡糖的研究进展

壳聚糖降解制备的低聚壳聚糖和壳寡糖具有许多独特的生理活性。介绍和评述了酸降解法、氧化降解法、酶降解法以及复合降解法等各种制备方法的研究进展。     

Yeasts associated with nectarines and their potential for biological control of brown rot

Resident fruit microflora has been the source of biocontrol agents for the control of postharvest decay of fruits and the active ingredient in commercialized biocontrol products. With the exception of grapes and apples, information on the resident microflora of other fruits is only fragmentary, but greater knowledge in this area can be very helpful in developing biocontrol strategies. We characterized the yeast microflora of nectarines (‘Croce del Sud’) from the early stages of fruit development until harvest. The fruit samples were collected from trees in an unmanaged orchard. The resident fruit microflora was separated from the occasionally deposited microorganisms by discarding initial fruit washings before the final wash, followed by sonication and plating on NYDA medium. The isolated yeasts were identified by BIOLOG and by sequencing the D1/D2 domain of a large subunit of the rRNA gene and, where available, the ITS sequence. BIOLOG identified 19 and the genetic analysis 23 species of yeasts. Although the identification by these two systems was not always the same, the predominant yeasts were Rhodotorula spp., Sporodiobolus spp., Cryptococcus spp., Pichia spp., Candida spp. and yeast‐like Aureobasidium pullulans. Several of the taxa appear to represent new species. The preliminary biocontrol tests against brown rot of nectarine fruit caused by Monilinia fructicola indicates significant decay control potential of some of the identified yeast species, namely Cryptococcus magnus, Cryptococcus sp. nov., Sporidiobolus pararoseus, A. pullulans and Rhodotorula sp. nov. Copyright © 2010 John Wiley & Sons, Ltd.