Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens

Two Aureobasidium pullulans strains (L1 and L8), effective against some fruit postharvest pathogens were evaluated for VOCs production as a part of their modes of action towards five pathogens (Botrytis cinerea, Colletotrichum acutatum, Penicillium expansum, Penicillium digitatum and Penicillium italicum). The VOCs were assayed with a double petri dish assay against conidia germination of target pathogens. Results obtained showed that the VOCs generated by the antagonists inhibited significantly the conidia germination of all pathogens compared to the control. In particular, the conidia germination of all Penicillium was completely inhibited by VOCs produced by L1 and L8. In in vivo tests, apples and oranges were artificially inoculated with pathogen conidia and then biofumigated with VOCs emitted by both antagonists. The antagonistic treatment controlled significantly pathogen infection, confirming the results obtained in vitro tests. The best L1 and L8 VOCs activity was observed on apple inoculated with B. cinerea where the lesion diameter reduction observed was greater than the 88%. The compounds emitted by L1 and L8 strains were identified with the solid-phase microextraction (SPME)–gas chromatographic technique. Compounds as 2-phenyl, 1-butanol-3-methyl, 1-butanol-2-methyl and 1-propanol-2-methyl belonging to the group of alcohols were mainly produced for both strains, in the first 96 h of growth. These compounds were confirmed by comparison with standards. The pure compounds of VOCs cited above were used to determine the EC₅₀ values for conidia germination of pathogens. The 1-propanol-2-methyl was the VOC least active against all tested fungi, with the EC₅₀ values over 0.8 μl ml⁻¹, while the 2-phenethyl alcohol was the most active with EC₅₀ values lower than 0.8 μl ml⁻¹, except for the C. acutatum (1.97 μl ml⁻¹). The present study demonstrated, for the first time, that the production of VOCs could play an essential role in the antagonistic activity of two A. pullulans strains against five fruit postharvest pathogens.     

Aureobasidium pullulans as a biocontrol agent of postharvest pathogens of apples in Uruguay

Aureobasidium pullulans was the microorganism most frequently recovered from the surface of apple fruit (cv. Red Delicious) stored in commercial cold chambers for 6 months. In the present work, 10 isolates of Aureobasidium pullulans were assayed to determine if they could control blue and grey mold disease of apple during cold storage. Although nine of 10 isolates, significantly reduced the percentage of decayed wounds when compared to the control, one of them, designated isolate ApB, showed the highest levels of protection. ApB was able to grow in a wide range of temperatures lower than 35°C, which is an important human health safety factor. ApB was resistant to thiabendazole, iprodione and imazalil, the most commonly commercially applied fungicides in postharvest treatment of apples in Uruguay. Regarding the mechanisms of action of the selected biocontrol agent, lytic enzymes did not seem to play a central role. ApB depleted iron from nutrient media, which may be an important aspect of its ability to inhibit Botrytis cinerea. Further experiments are needed, however, to determine if the depletion of iron is caused by the production of siderophores, by the immobilization of iron in an insoluble pigment, or a combination of both.     

Single-leaf resolution of the temporal population dynamics of Aureobasidium pullulans on apple leaves

The abundance of phylloplane microorganisms typically varies over several orders of magnitude among leaves sampled concurrently. Because the methods traditionally used to sample leaves are destructive, it has remained unclear whether this high variability is due to fixed differences in habitat quality among leaves or to asynchronous temporal variation in the microbial population density on individual leaves. We developed a novel semidestructive assay to repeatedly sample the same apple leaves from orchard trees over time by removing progressively more proximal approximately 1-cm-wide transverse segments. Aureobasidium pullulans densities were determined by standard leaf homogenization and plating procedures and were expressed as CFU per square centimeter of segment. The A. pullulans population densities among leaves were lognormally distributed. The variability in A. pullulans population densities among subsections of a given leaf was one-third to one-ninth the variability among whole leaves harvested concurrently. Sequential harvesting of leaf segments did not result in detectable changes in A. pullulans density on residual leaf surfaces. These findings implied that we could infer whole-leaf A. pullulans densities over time by using partial leaves. When this successive sampling regimen was applied over the course of multiple 7- to 8-day experiments, the among-leaf effects were virtually always the predominant source of variance in A. pullulans density estimates. Changes in A. pullulans density tended to be synchronous among leaves, such that the rank order of leaves arrayed with respect to A. pullulans density was largely maintained through time. Occasional periods of asynchrony were observed, but idiosyncratic changes in A. pullulans density did not contribute appreciably to variation in the distribution of populations among leaves. This suggests that persistent differences in habitat (leaf) quality are primarily responsible for the variation in A. pullulans density among leaves in nature.     

Role of microbial immigration in the colonization of apple leaves by Aureobasidium pullulans

The role of microbial immigration in the veinal colonization pattern of Aureobasidium pullulans on the adaxial surface of apple leaves was investigated in two experiments at two periods (early and late seasons) in 2004 by applying green fluorescent protein (GFP)-tagged blastospores to the foliage of orchard trees. Individual leaves were resampled by a semidestructive method immediately after inoculation (t(0)) and about 1 (t(1)), 2 (t(2)), and 3 (t(3)) weeks later. At t(0), there were no significant (P < or = 0.05) differences in densities (cells/mm(2)) on veinal (excluding midvein) sites and those on interveinal sites, but at all points thereafter, densities were significantly higher on veins. GFP-tagged A. pullulans cells remained primarily as singletons on interveinal regions (> or =90% at all points), while > or =20% of cells over veins at t(3) were in colonies of > or =4 cells. The colonies that developed from single cells placed on midveins and other veins were significantly larger than those that developed on interveinal regions of detached field and seedling leaves incubated under controlled conditions. Colonies primarily developed linearly along veins, reaching average colony sizes (72 h) of 24.4 +/- 12.7 (mean +/- standard deviation) cells. In contrast, colonies on interveinal regions tended to average only 2.9 +/- 1.3 cells, with less linearity. To examine the potential role of A. pullulans growth-inhibiting factors associated with interveinal features, single GFP-tagged A. pullulans cells in droplets previously incubated on interveinal sites were placed on midveins and compared to midvein colonies derived from cells in a water-only suspension. No differences in colony size resulted. Our results indicate that immigration limitation and growth-inhibiting factors are not the primary factors responsible for A. pullulans veinal colonization patterns in the field. Rather, indirect evidence suggests that growth-promoting substances occur locally in the veinal areas.     

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.     

Siderophore production by the marine-derived Aureobasidium pullulans and its antimicrobial activity

Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, only the yeast strain HN6.2 which was identified to be Aureobasidium pullulans was found to produce high level of the siderophore. Under the optimal conditions, this yeast strain could produce 1.1 mg/ml of the siderophore. The crude siderophore produced by the yeast strain HN6.2 was able to inhibit cell growth of Vibrio anguillarum and Vibrio parahaemolyticus, isolated from the diseased marine animals.     

出芽短梗霉A5产胞外多糖发酵条件的优化及产物结构鉴定

基于筛选获得能够生产分子量较高且无色素的普鲁兰多糖酵母菌株,对其进行菌株鉴定、产多糖发酵条件优化和多糖产物鉴定,旨在为工业上普鲁兰多糖发酵提供新的菌株来源。以YPD固体培养基为筛选培养基,氯霉素为筛选压力,曲利苯蓝为筛选指示剂;通过形态学,ITS间隔序列分析对筛选出的A5菌株进行鉴定。采用单因子优化A5菌株的最佳发酵条件;利用普鲁兰酶酶解并结合薄层层析法、红外光谱以及凝胶渗透色谱进行结构鉴定和分子量的测定。A5菌株鉴定为出芽短梗霉属,并被命名为出芽短梗霉A5。最优的发酵条件8%(w/v)麦芽糖,1%(w/v)酵母粉,2%(w/v)蛋白胨,0.5%(w/v)K_2HPO_4,0.06%(w/v)(NH_4)_2SO_4,0.03%(w/v)CaCl_2,pH6,7%(v/v)接种量;经过结构鉴定得知:该菌株的胞外产物是普鲁兰多糖,分子量为63.84 kDa。由此获得了一株生产普鲁兰多糖的出芽短梗霉菌株A5,产物无色素且分子量较高。经过初步的发酵条件优化,在最佳发酵条件下发酵培养后,获得普鲁兰多糖的产量为22.9 g/L。综合上述结果可知,菌株A5能够作为工业上生产普鲁兰多糖的重要候选菌株。     

The life style of Aureobasidium pullulans and the multiple forms of its polygalacturonase

The life style of Aureobasidium pullulans on pectin medium and its production of extracellular polygalacturonases are closely related. Polygalacturonases with random action pattern (EC 3.2.1.15) were formed in the first phases of cultivation, whereas exopolygalacturonases (EC 3.2.1.67) with terminal action pattern on pectin were produced during the whole growth of this yeast-like fungus. The production and inactivation of individual enzyme forms during cultivation were strongly dependent on the pH value of the pectin medium. Various kinds of stress can support the prolongation of the phase of endo-acting enzyme production, as well as the increase of their activity.     

短梗霉黑色素的分离纯化及结构的初步分析

采用热碱提取、水煮酸沉法从短梗霉发酵液中提取得到黑色素粗品,再经DMSO萃取、酸性甲醇(pH=2)沉淀得到不含多糖和蛋白的短梗霉黑色素。此黑色素不溶于水及常规有机溶剂,可溶于碱性溶液和DMSO;离子交换色谱分析表明黑色素组分均一,出峰时间26±0.5 m in;紫外光谱谱图最大吸收峰为215 nm左右,未见蛋白(280 nm)与核酸(260 nm)的特征吸收峰;红外光谱谱图具有黑色素3μm和6μm的特征吸收峰,并含大量的羟基、氨基,与核磁共振和液质联机谱图结合分析推出短梗霉黑色素可能含有酚羟基、羧基和吲哚等官能团,主要结构骨架为5,6-二羟基吲哚-羧酸和多巴醌,推断该黑色素为酪氨酸酶控制合成的真黑素。     

Olfactory responses of Grapholita molesta to different developmental stages of immature fruit of peach,plum, and apple

The Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is known to shift its host use from stone fruits (the primary hosts) to pome fruits (secondary hosts) as the season progresses. Grapholita molesta recognizes potential hosts by semiochemicals from fruits, but few studies have addressed the olfactory responses of G. molesta to host fruits during the seasonal transition of the moth from stone to pome fruits. So, we investigated the olfactory responses of larvae and gravid females of G. molesta to different fruit stages (collected on May 31, June 11, and June 25) of immature peach, plum, and apple fruits, using both multiple and two choice tests. In the multiple choice tests, for fruits collected after June 11, larvae showed a significant preference for peach as their first choice, compared to plum or apple. The final choice rate (when larvae had stayed on the fruit for more than 1 h) of larvae was also highest for peach fruits collected on June 11 and June 25. In two choice tests (Y-tube test), the response rate and overall choice rate of larvae and mated ovipositing adult females were also highest on peaches collected on May 31 (larvae) and June 25 (adults), respectively. In conclusion, we found that both the adults and larvae of G. molesta were highly attracted to immature peach fruits in laboratory choice tests.     

Induction and Inducers of the Pectolytic System in Aureobasidium pullulans

The strain of Aureobasidium pullulans NRRL Y-2311 (CCY 27-1-98), known as a hyperproducer of endo-1,4-β-xylanase, exhibited good growth on pectin or pectate. Growth on these carbon sources is associated with an inducible production of significant amounts of pectolytic enzymes, of which exopolygalacturonase (EC 3.2.1.67) and endopolygalacturonase (EC 3.2.1.15) were identified. The two enzymes are not produced on D-glucose or under carbon starvation conditions. The enzymes can be induced in glucose-grown cells by D-galacturonic acid and its oligomers. Thus, D-galacturonic acid, the monomer derived from the polysaccharide, appears to be the natural inducer or a precursor of an inducer of pectolytic enzymes in the studied yeast. Received: 4 November 1995 / Accepted: 11 December 1995     

Monitoring plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae), populations in apple and peach orchards in the mid-Atlantic

We evaluated responses of plum curculio, Conotrachelus nenuphar (Herbst), to four trap types in commercial and unsprayed apple and peach orchards. Trap types included black pyramid and clear Plexiglas panel traps deployed outside the orchard 2 m from the border row, and branch-mimicking cylinder and trunk-mounted screen traps attached to trees in the border row. Bait treatments evaluated in conjunction with each trap type included the synthetic fruit volatile benzaldehyde, the aggregation pheromone grandisoic acid (GA), benzaldehyde in combination with GA, and an unbaited control treatment. In commercial apple orchards, significantly more plum curculio were captured in traps baited with benzaldehyde + GA compared with traps baited with other treatments. Furthermore, significantly more plum curculio were captured by screen traps baited with benzaldehyde + GA compared with unbaited control traps. Significantly more plum curculio were captured by screen traps compared with other trap types in an unsprayed apple orchard. Very few captures were recorded in commercial peach orchards. Dissections of trapped females indicate that bivoltine populations are present in the mid-Atlantic. In general, correlations between timing and amount of trap captures and timing and amount of fruit injury inflicted concurrently or 1 wk after trap captures were very weak for all trap types and bait combinations. Our results agree with previous studies in the northeastern United States in which trap captures are increased by presence of semiochemical baits but fail to serve as reliable tools to determine need for and timing of insecticide application against plum curculio.     

Strain and medium-related variability in the yeast-mycelial transition of Aureobasidium pullulans

Abstract Morphogenetic observations on the yeast-mycelial (Y-M) transition of Aureobasidium pullulans were strain-specific and considerable differences occurred between solid and liquid medium. Strains that showed maximum Y-M response in both liquid and solid media would be the most useful for kinetic and biochemical studies.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae.

A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L. Li and L. G. Ljungdahl, Appl. Environ. Microbiol. 60:3160-3166, 1994). Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium. S. cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction. Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4. These proteins were recognized by anti-APX-II antibody. The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S. cerevisiae. Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media. No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region. A comparison of the A. pullulans and S. cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S. cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S. cerevisiae cells.     

Chemical and biological characterization of siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 and its antibacterial activity

After analysis using HPLC and electronic ion spray mass spectroscopy, the purified siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 was found to be fusigen. The purified desferric fusigen still had strong inhibition of growth of the pathogenic Vibrio anguillarum while the fusigen chelated by Fe³⁺ lost the ability to inhibit the growth of the pathogenic bacterium. The added iron in the medium repressed expression of the hydroxylase gene encoding ornithine N⁵-oxygenase that catalyzes the N⁵-hydroxylation of ornithine for the first step of siderophore biosynthesis in the yeast cells while expression of the hydroxylase gene in the yeast cells grown in the medium plus ornithine was enhanced.     

Isolation and structural elucidation of a dihydroubiquinone-9 from the fungus Aureobasidium pullulans

Abstract A naturally occurring member of ubiquinone (Q) group, a dihydroubiquinone-9 (Q-9 (H₂)), has been isolated as a minor ubiquinone component from the fungus Aureobasidium pullulans . By ultraviolet absorption, mass and nuclear magnetic resonance spectrometric studies, the structure of Q-9 (H₂) was found to be 2,3-dimethoxy-5-methyl-6-IX-dihydromultiprenyl⁹-1,4-benzoquinone (I).