Control of apple blue mould disease with Torulaspora delbrueckii in combination with Silicon

In this study, Torulaspora delbrueckii alone and in combination with silicon were evaluated for the control of apple blue mould disease caused by Penicillium expansum. In vitro, the antagonistic effects of T. delbrueckii in controlling mycelial growth of P. expansum on potato-dextrose-agar (PDA) in dual cultures, and the growth of P. expansum alone with cell-free metabolites and volatile components of T. delbrueckii were assayed. In vitro, to evaluate the direct effect of silicon on mycelial growth of pathogen, silicon at different concentrations (0.2, 0.4, 0.6, 1 and 2% (wt./vol.)) was added to PDA medium. Silicon at 0.6% (wt./vol.) and above concentrations completely inhibited the mycelial growth of P. expansum. However, it had no significant effect on population dynamics of yeast in vitro and in apple wounds. In vivo, silicon at 0.2 and 1% (wt./vol.) in combination with antagonistic yeast (1 × 10⁸ cell/ml) was a more effective approach to reduce the lesion diameter of blue mould decay of apples than the application of silicon or T. delbrueckii alone at 20 and 4°C, respectively.     

Enhancement of biocontrol activity of Torulaspora delbrueckii with methyl jasmonate against apple blue mould disease

Torulaspora delbrueckii alone and in combination with methyl jasmonate was applied to the control of Penicillium expansum. For evaluation of direct effect of Methyl jasmonate on mycelial growth of pathogen, it was added to potato dextrose agar culture at different concentrations. Effect of methyl jasmonate on population of yeast in nutrient yeast dextrose broth media was determined after 24 and 48 h. Results showed that methyl jasmonate had no significant direct effect on pathogen and yeast. Also, evaluation of methyl jasmonate effect on the population of yeast in apple wounds indicated that methyl jasmonate at different concentrations increased population growth of yeast at 20°C, 8 and 15 days after inoculation in toward the control and it had no significant effect on population dynamics of yeast at 4°C. In vivo, the results indicated that combination of methyl jasmonate with antagonistic yeast reduced the blue mould of apples better than methyl jasmonate and yeast alone.     

Improvement in the biocontrol of postharvest diseases of apples with the use of yeast mixtures

Mixtures of yeasts were tested for theirability to control Penicillium expansum andBotrytis cinerea on Red Delicious apple fruits. The occurrence of synergistic or antagonisticinteractions between yeast strains in differentmixtures was also evaluated. Two strains ofRhodotorula (R. glutinis SL 1 and R. glutinisSL 30) and two strains of Cryptococcus (C. albidus SL 43 and C. laurentii SL 62) were selected fordeveloping yeasts mixtures.The R. glutinis SL 1–R. glutinis SL 30 mixtureexhibited a lower effectiveness than eachstrain alone, against both moulds. Othermixtures (R. glutinis SL 1–C. albidus SL 43 and R. glutinis SL 30–C. albidus SL 43) showedsynergism against P. expansum but not against B. cinerea. The R. glutinis SL 1–C. laurentii SL62 mixture was the only mixture which showedsynergism against gray mould. There was not anymixture, which showed high effectivenessagainst both moulds at the same time. Differentresults could be explained by the dynamics ofthe population of the yeasts.By using yeast mixtures, it was possible toimprove biocontrol without increasing theamount of antagonists applied. The synergismobserved could be useful in enhancingbiological control.     

The infection of apples, cv. Bramley's Seedling, by Nectria galligena Bres

In laborator experiments germinating conidia penetrated leticels and wounds but not the intact surfaces of apples. Date of harvest had no significant effect on the numbers of apples infected with Nectria galligena but the earliest picks rotted first in barn store. Inoculations of unpicked apples resulted in small arrested lesions which only developed into progressive rots after a considerable period in store. Rots developed mosy quickly from inoculations made between mid-August and mid-September. The size of rot increased with spore number and many inoculations with 10–100 conidia remained as arrested lesions. Arrested lesions developed 10–15 days after unripe apples were inoculated and consisted of a zone of fungal colonization surrounded by suberized, necrotic cells in which compounds toxic to both N. galligena and Penicillium expansum were detected. No antifungal compounds were found in progressive rots to mature apples or in healthy apples of any age. Antifungal activity, measured by inhibition of P. expansum, was greatest 15–20 days from inoculation of unripe apples with N. galligena but decreased after a total of 35 days incubation at 20 d? C. Much less antifungal activity was produced in ripe or desert apples.     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

Efficacy of Wickerhamomyces anomalus yeast in the biocontrol of blue mold decay in apples and investigation of the mechanisms involved

Blue mold decay is the one of most important postharvest disease of apples caused by the fungus, Penicillium expansum. This study aimed to investigate the biocontrol efficacy of the yeast, Wickerhamomyces anomalus, on postharvest blue mold decay of apples and the relative defense mechanisms. The results indicated that W. anomalus could significantly reduce the blue mold decay of apples, and the maximum inhibition was obtained when the concentration of W. anomalus was 1?×?10⁸ cells ml^?1. Furthermore, W. anomalus significantly reduced the fruit decay under ambient conditions, without generating any change in fruit quality. In vitro experiments showed that W. anomalus greatly inhibited the spore germination and germ tube elongation of P. expansum. Besides, its ease of adaptation, stable growth and potential colonization of in apple wounds or surfaces indicated that W. anomalus could compete with P. expansum for nutrients and space, leading to considerable inhibition blue mold decay. W. anomalus significantly induced the activities of polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and ascorbate peroxidase (APX) in apples. Moreover, W. anomalus increased the contents of flavonoid and total phenols. All these results suggested that W. anomalus has potential biocontrol efficacy to control the postharvest blue mold decay of apples

     

Control of apple blue mold by <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant strains expressing cecropin A

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.     

Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum

The objectives of this work were to assess the optimum conditions for induction of acid tolerance in the marine yeast Rhodosporidium paludigenum and evaluate the biocontrol activity of non-adapted and acid-adapted yeasts in controlling apple blue mold caused by Penicillium expansum. R. paludigenum grown in malic and lactic acid treatments were stimulated after 12 h incubation. Moreover, medium modified with malic and lactic acid significantly enhanced the acid tolerance of R. paludigenum (p?<?0.05). In acid tolerance response test, the highest viability of R. paludigenum was obtained at initial pH of 5.5 in the NYDB medium modified with malic acid (91.6 %). In addition, all R. paludigenum treatments significantly reduced the disease incidences and lesion diameters of blue mold in apples. Furthermore, there was no significant difference between acid-adapted and unadapted yeasts in the apple wounds after 48 h dynamics. Acid stress improved R. paludigenum viability under acidic conditions. However, there was no significant difference between acid-adapted and unadapted yeasts in controlling P. expansum on apple fruit (p?<?0.05). These results indicate the potential for maintaining the survival level of biocontrol agents by physiological inducement strategy.     

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10⁸ CFU ml^?1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10⁵ spores ml^?1; While the blue mold decay was completely inhibited at concentrations of 5 × 10⁸ CFU ml^?1, at challenged with P. expansum spores suspension of 5 × 10⁴ spores ml^?1_. These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10⁵ CFU per wound to 2.24 × 10⁷ CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.     

Salicylic Acid Enhances Biocontrol Efficacy of the Antagonist Cryptococcus laurentii in Apple Fruit

Biological control and induced resistance are two of the promising approaches to the control of postharvest diseases. This study was conducted to evaluate the efficacy of salicylic acid (SA) alone or in combination with an antagonistic yeast, Cryptococcus laurentii, in controlling the blue mold disease caused by Penicillium expansum on apple fruit wounds. SA alone significantly inhibited the spore germination of P. expansum in vitro when its concentration was increased to 1000 μg ml⁻¹, but it was not effective in controlling the disease in vivo. Simultaneous application of SA and C. laurentii to the wounds on the apple fruit surface showed that SA could improve the efficacy of C. laurentii against P. expansum in a concentration-dependent manner, being most effective at 10 μg ml⁻¹ but less effective at a higher or lower concentrations. Besides reducing the blue mold incidence in the local wound sites, the combination of C. laurentii with SA at 10 μg ml⁻¹ also had a synergistic effect on the induction of fruit resistance to the disease, which might be associated with a rapid increase in peroxidase, phenylalanineamonialyase and lipoxygenase activities. In addition, SA at 100 μg ml⁻¹ or above showed an adverse effect on the growth of C. laurentii in vitro and in vivo, whereas it had no effect when its concentration was decreased to 10 μg ml⁻¹ or lower. This suggested that SA could enhance the biological activity of C. laurentii in apple fruit by inducing resistance to pathogens based on the antagonistic activity of C. laurentii.     

Biocontrol of Botrytis cinerea in apple fruit by Cryptococcus laurentii and indole-3-acetic acid

This study evaluated the effect of a yeast antagonist Cryptococcus laurentii and a plant regulator indole-3-acetic acid (IAA) on inhibition of Botrytis cinerea infection in harvested apple fruit. The results showed that the combined treatment with C. laurentii and IAA at 20 μg/ml was a more effective approach to reduce the gray mold rot in apple wounds than the C. laurentii alone. After 4 days of incubation, gray mold incidence in the combined treatment with C. laurentii and IAA was about 18%, which was a 50% reduction in incidence compared to the treatment with C. laurentii alone. Although IAA had no direct antifungal activity against B. cinerea infection when the time interval between IAA treatment and pathogen inoculation was within 2 h, application of IAA strongly reduced gray mold infection when IAA was applied 24 h prior to inoculation with B. cinerea in apple fruit wounds. Moreover, combination of IAA and C. laurentii stimulated the activities of superoxide dismutase, catalase and peroxidase with above 1.5-fold higher than that treatment with C. laurentii alone at 48 h. Therefore, combination of C. laurentii with IAA, which integrated the dual biological activity from the antagonistic yeast and plant regulator, might be developed to be a useful approach to control gray mold in harvested apple fruit.     

地黄内生放线菌的分离及地黄轮纹病拮抗菌Streptomyces folium leaf-16的新种鉴定

药用植物内生放线菌具有合成天然活性化合物的潜力,放线菌新种是寻找新型抗生素先导化合物的一个重要来源。【目的】挖掘药用植物地黄内生放线菌资源,并对地黄轮纹病拮抗菌株leaf-16进行新种鉴定。【方法】本研究采用五步消毒法分离河南道地药材地黄的内生放线菌,以地黄轮纹病原真菌草茎点霉(Phoma herbarum)为指示菌,采用平板对峙法筛选对该病菌有抑制作用的菌株,16S rRNA基因测序发现一株抗地黄轮纹病的放线菌新种leaf-16。通过形态、生理生化、细胞壁化学组分和分子生物学等特征对菌株leaf-16进行多相分类学鉴定。【结果】经平板对峙实验得到8株抗地黄轮纹病的放线菌,其中菌株leaf-16经16S rRNA基因测序、形态比较、生理生化、化学组分和分子生物学以及DNA-DNA杂交分析,确定菌株leaf-16为1株链霉菌新种,并命名为Streptomyces folium。【结论】菌株leaf-16为1株链霉菌新种,具有抑制地黄轮纹病原真菌的活性,为进一步分离新型抗地黄轮纹病的生物制剂奠定物质基础。     

Types of Rot, Rate of Rotting, and Analysis of Pectic Substances in Apples Rotted by Fungi

The rate at which the fungi grew through apples was determinedin various ways and used to estimate their rate of linear advance.Five fungi were studied;they were Sclerotinia fructigena (firm-browncoloured rot, rapid growth through apples), Botrytis cinerea(soft, light-brown coloured rot, rapid growth through apples),Psyrenochaeta furfuracea (firm to soft rot, variable in colourbut generaly dark, slow growth through apples), Penicilliumexpansion A (soft, white rot, slow growth through apples) andPenicillium expansum B (soft, white rot, medium rate of growththrough apples). S. fructigena which had the highest rate oflinear advance which was about three times that of P. furfuraceawhich had the lowest. Methods for extracting different types of pectic substancesfrom sound and rotted tissues are described, and details aregiven of a rapid and reasonably accurate colorimetric methodof determining the anhydrogalacturonic acid content of theseextracts. The firm-rot fungi reduced the water-insoluble pecticsubstances by 10–20 per cent., but the soft-rot fungicaused much larger changes, up to 70 per cent. being degraded,The firm-rot and soft-rot fungi had different effects on thepectic substances insoluble in dilute acid but soluble in dilutealkali. The soft-rot fungi had little effect on these substances,or reduced their concentration, whereas the firm-rot fungi causedsubstantial increases compared with sound tissue. These resultsare considered in terms of pectic enzyme activity. Analysisof extracts by paper chromatography showed that galacturonicacid, absent from sound tissue, was present in each type ofrotted tissue. Di- and tri-galacturonic acids were present inrots caused by P. expansum, and these rots probably also containedproducts from the break-down of other polysaccharides.     

Screening of antagonistic yeast strains for postharvest control of Penicillium expansum causing blue mold decay in table grape

Blue mold decay caused by Penicillium expansum is one of the most important postharvest diseases of grapes, leading to considerable economic losses. Regarding the increasing demand for pesticide-free foods, this study aimed to find potential yeast strains for biological control of blue mold on table grapes. A total of 50 yeast strains were screened for antagonistic activity against P. expansum using the dual culture method and six strains significantly inhibited the fungal growth. All six yeast strains (Coniochaeta euphorbiae, Auerobasidium mangrovei, Tranzscheliella sp., Geotrichum candidum, Basidioascus persicus, and Cryptococcus podzolicus) reduced the fungal growth (29.6–85.0%) and the decay degree of wounded grape berries inoculated with P. expansum while G. candidum was found to be the most efficient biocontrol agent. On the basis of antagonistic activity, the strains were further characterized by in vitro assays involving inhibition of conidial germination, production of volatile compounds, iron competition, production of hydrolytic enzymes, biofilm-forming capacity, and exhibited three or more putative mechanisms. To our knowledge, the yeasts are reported for the first time as potential biocontrol agents against the blue mold of grapes but more study is required to evaluate their efficiency related to field application.     

Effect of an Antagonistic Yeast in Combination with Microwave Treatment on Postharvest Blue Mould Rot of Jujube Fruit

The potential of using an antagonistic yeast alone or in combination with microwave treatment for controlling blue mould rot of jujube fruit, and its effect on postharvest quality of fruit, was investigated. The results showed that the growth of Penicillium citrinum was completely inhibited by a 2450‐MHz microwave heating for 2 or more minutes in vitro. The population density of P. citrinum in surface wounds of fruit treated with microwave treatment for 2–3 min was significantly lower than that of controls. When tested on jujube fruit, antagonistic yeast or microwave treatment, as stand‐alone treatment, the disease incidence of infected wounds was reduced from 100% to 45.0% and 36.0%, and lesion diameters were reduced from 1.92 cm to 1.50 cm and 1.38 cm, respectively. However, in fruit treated with a combination of Metschnikowia pulcherrima and microwave treatment, the disease incidence of infected wounds and lesion diameters was only 21.0% and 1.00 cm, respectively. The natural decay incidence on jujube fruit treated with the combination of microwave treatment and M. pulcherrima was 6.2% after storage at 2 ± 1°C for 45 days and at 22°C for 7 days. None of the treatments impaired quality parameters of fruits. Thus, the combination of microwave treatment and M. pulcherrima could provide an alternative to synthetic fungicides for controlling postharvest blue mould rot of jujube fruit.     

Identification of siderophores and siderophore-mediated uptake of iron in Stemphylium botryosum

Under iron-deficient conditions Stemphylium botryosum f. sp. lycopersici produces three major siderophores; dimerum acid, coprogen B and an unidentified monohydroxamate siderophore designated as A. The system of siderophores mediating uptake of iron was characterized. It exhibits active transport, saturation kinetics and an optimum at pH 6 and 30°. The rate of iron uptake via dimerum acid and coprogen B was four times higher than siderophore A. S. botryosum was capable of taking up iron from hydroxamate siderophores produced by other fungi, e.g. ferrichrome, fusigen, rhodotorulic acid but not ferrioxamine B. Double labelling experiments suggest that ferric coprogen B accumulates in mycelial cells as an intact chelate.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.