Evaluation of different formulations of potential biocontrol yeast isolates efficacy on apple blue mold at storage condition

Post-harvest pathogens cause major losses in apple production. Biological control by using epiphytic yeasts against Penicillium expansum has been considered as an alternative method for controlling the post-harvest decays. The yeast isolates Rhodotorula mucilaginosa, Pichia guilliermondii, which showed high biocontrol efficacy against P. expansum, were selected for formulation tests. Formulation is an important step in developing a biocontrol product. The successful delivery of biocontrol agents, shelf life, stability and effectiveness in commercial conditions depend on the formulation. In the formulation, the carrier is the primary material used to allow a bioproduct to be dispersed effectively. Yeast isolates were grown in a cane molasses-based medium. Harvested yeast cells were combined with inorganic (talk, kaolin) and organic (Rice bran, wheat bran) carriers. Viability of the yeast cells in formulations stored at 4°C and 24°C was determined each month during 6 months storage. After 6 months storage to evaluate efficacy of formulations, all formulations were tested on apple to control blue mold in storage condition. High stability of antagonistic yeasts was achieved by using organic and inorganic carriers. Rice bran and wheat bran stimulated the germination of the yeasts cells during storage period. Both of the yeasts had significantly highest viable yeast cell content over 6 months in formulation containing wheat bran as a carrier. P.guillermondii in all formulations had significantly higher shelf life and was effective than R. mucilaginosa.     

Effects of Preharvest Applications of CaCl2, 2,4-D and Benomyl and Postharvest Hot Water, Yeast and Fungicide Treatments on Development of Decay on Satsuma Mandarins

In this study, an integrated approach was evaluated for the control of postharvest decays of mandarin including some pre‐ and post‐harvest treatments under storage conditions. The efficacy of the treatments both as alone and in combination was evaluated during 3 years. Preharvest application of benomyl resulted in significantly less decay of mandarin fruit after storage in 3‐year tests. Calcium chloride (CaCl₂), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and gibberellic acid (GA₃) as stand‐alone treatments or combinations were not effective in controlling Penicillium and total decay infections on inoculated mandarin. Postharvest application of imazalil (200 μg/ml) in solution heated to 54°C for controlling postharvest green and total decay of mandarin was significantly effective for 3 months under storage conditions. The biocontrol activity of yeast (Metschnikowia pulcherrima) was improved when yeast treatment was combined with imazalil (200 μg/ml) at postharvest. The data suggest that preharvest application of benomyl and postharvest treatments of imazalil, hot water and yeast may reduce postharvest green mould and total decay of mandarin under storage conditions.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

Biological Control of Postharvest Diseases of Apple and Pear under Semi-commercial and Commercial Conditions Using Three Saprophytic Yeasts

The yeastsCryptococcus laurentii(strain HRA5),Cryptococcus infirmominiatus(strain YY6), andRhodotorula glutinis(strain HRB6) were tested as biocontrol agents of postharvest diseases of apple and pear in semi-commercial and commercial trials. The yeasts effectively controlled decay when applied in a drench or line spray. The yeasts were not adversely affected when treated fruits were stored in a controlled atmosphere consisting of 1% oxygen and 99% nitrogen. In a commercial trial, the most effective treatments for control of blue mold of pear were a combination ofC. laurentiiandC. infirmo-miniatus(91% control) and the commercially recommended high rate (528 μg/ml) of thiabendazole (88% control). In the commercial apple trial, the most effective treatments for blue mold wereC. infirmo-miniatuscombined with 264 μg/ml thiabendazole (91% control),C. infirmo-miniatuscombined withC. laurentii(84% control), and thiabendazole alone at 528 μg/ml (79% control). The combination ofC. laurentiiwith 264 μg/ml of thiabendazole was significantly more effective for control of blue mold on pear than thiabendazole at 528 μg/ml whenever any thiabendazole-resistant spores were present in the inoculum.     

Control of green mold and sour rot of stored lemon by biofumigation with Muscodor albus

Control of postharvest lemon diseases by biofumigation with the volatile-producing fungus Muscodor albus was investigated. In vitro exposure to M. albus volatile compounds for 3 days killed Penicillium digitatum and Geotrichum citri-aurantii, causes of green mold and sour rot of lemons, respectively. Lemons were wound-inoculated with P. digitatum and placed in closed 11-L plastic boxes with rye grain cultures of M. albus at ambient temperature. There was no contact between the fungus and the fruit. Biofumigation for 24–72 h controlled green mold significantly, even when treatment began 24 h after inoculation. Effectiveness was related to the amount of M. albus present. In tests conducted inside a 11.7-m³ degreening room with 5 ppm ethylene at 20 °C, green mold incidence on lemons was reduced on average from 89.8 to 26.2% after exposure to M. albus for 48 h. Ethylene accelerates color development in harvested citrus fruit. M. albus had no effect on color development. Biofumigation in small boxes immediately after inoculation controlled sour rot, but was ineffective if applied 24 h later. G. citri-aurantii may be less sensitive to the volatile compounds than P. digitatum or escapes exposure within the fruit rind. Biofumigation with M. albus could control decay effectively in storage rooms or shipping packages.     

Biocontrol of tomato wilt by Penicillium oxalicum formulations in different crop conditions

Eight formulations of Penicillium oxalicum (FOR1 to FOR8) were obtained by the addition of various ingredients, in two separate steps of the production and drying of P. oxalicum conidia. These formulations were then evaluated against tomato wilt in three glasshouse (G1 to G3) and two field (F1 and F2) experiments. All formulations were applied to seedlings in seedbeds 7 days before transplanting at a rate of 10⁷ spores g⁻¹ seedbed substrate. The conidial viability of each formulation was estimated by measuring germination just after fluid bed-drying, before seedbed application and after 1 and 2 years of storage at 4 °C under vacuum. The densities of P. oxalicum were estimated in the seedbed substrate and in the rhizosphere of three plants per treatment just before transplanting. Initial conidial viability of formulations just after fluid bed-drying was approx. 80%, except for FOR1, FOR4, and FOR7 which were 60%. The initial viability was maintained up to 40–50% for 2 years of storage at 4 °C under vacuum, except for FOR1. All formulations had 50% viability at application time. Populations of P. oxalicum in the seedbed substrate just before transplanting were >10⁶ cfu g⁻¹ soil in G3 and F2; populations in rhizosphere were also >10⁶ cfu g⁻¹ fresh root, except for FOR3, FOR5, and FOR6 in G2. A range of 22–64% of disease reduction was observed with all formulations, although these reductions were not significant (p = 0.05) for FOR1, FOR4, and FOR5 in any experiment. Contrast analysis showed significant differences between biological treatments and untreated control (p = 0.05) in all experiments, but no significant differences between biological and chemical treatments. Initial conidial viability of P. oxalicum in formulations and populations of P. oxalicum in the seedbed substrate explained 78.26% of the variability in P. oxalicum populations in tomato rhizosphere before transplanting. Disease incidence in untreated plants was negatively correlated (r = −0.54) with the percentage of disease control. The relationship between the viability of formulations, the populations of P. oxalicum in seedbed and rhizosphere, and the control of tomato wilt is discussed.     

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.     

Antagonistic yeasts and thermotherapy as seed treatments to control Fusarium fujikuroi on rice

Bakanae disease, caused by Fusarium fujikuroi, is the most important seedborne disease of rice. Biological control and physical treatments can be effective tools to control seedborne diseases. Sixty-two isolates of yeasts and yeast-like fungi were obtained from different rice seeds. Four yeast isolates were selected in dual culture assays for mycelial growth inhibition, and in seed tests for reduction of infection rate. The isolates R23 and R26 were identified as Metschnikowia pulcherrima, the isolate R9 as Pichia guilliermondii, and the isolate SB1 as Sporidiobolus pararoseus. Rice seeds treated with P. guilliermondii R9, M. pulcherrima R23 and R26 significantly reduced the infection rate of F. fujikuroi, compared to some commercial biofungicides. The four selected yeasts reduced the bakanae disease severity in rice plants grown in greenhouse trials. Antagonist seed dressing resulted in reduction of the disease index from 93.0% in the untreated control to 20.0% in P. guilliermondii R9 treated seeds, and to 28.5% in M. pulcherrima R23 treated seeds. Selected antagonists were also used in combination with thermotherapy, which contributed to increase their efficacy. Thus, P. guilliermondii R9 and M. pulcherrima R23 combined with thermotherapy at 60 °C for 10 min decreased the bakanae disease index below 5%, and improved the seed germination rate compared to the single treatments, showing a seed priming effect. This is the first report about the use of antagonistic yeasts for seed dressing of rice to control F. fujikuroi on rice seeds, and biological treatment may be improved through combination with thermotherapy.     

Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici)

Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4%, respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis, and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature. The application of P. guilliermondii is more effective for preserving chilli fruits than conventional preservation with chlorinated water.     

嗜线虫致病杆菌抑制灰葡萄孢的效应

[背景] 灰葡萄孢（Botrytis cinerea）是引起葡萄采后病害的主要病原菌之一,严重影响葡萄的贮期和品质,给葡萄产业带来极大损失。利用拮抗微生物抑制采后病原菌生长已逐渐成为防治葡萄采后灰霉病的重要手段。[目的] 利用昆虫病原线虫共生细菌广谱高效的抑菌特性,从现有共生细菌资源中筛选对灰葡萄孢具有高拮抗作用的菌株,为葡萄采后灰霉病的抑制提供新的材料和研究方向。[方法] 通过平板对峙培养法和菌丝生长速率法分离筛选拮抗共生细菌,并对优选的高效拮抗共生细菌进行16S rRNA基因序列进化分析,采用扫描电镜观察其对灰葡萄孢菌丝生长的影响,利用损伤接种法对红地球葡萄防治效果进行验证。[结果] 初步分离筛选共获得9株拮抗菌,复筛与复测得到一株抑菌效果显著的共生细菌（命名为ALL）,经进化分析其为嗜线虫致病杆菌（Xenorhabdus nematophila）,其16S rRNA基因序列的Genbank登录号为MW488402,与菌株Xenorhabdus nematophi la NC116聚于同一分支,相似性达99.79%。扫描电镜观察该菌株导致灰葡萄孢菌丝扭曲变形、表面皱缩、失水塌陷,该菌株发酵（36 h）上清液浓度为1%时对灰葡萄孢菌丝抑制率达44.5%。在葡萄常温防效实验中,与对照组比较,ALL菌株发酵上清液对灰霉菌防治效果较好,3 d后防效为63.50%。[结论] 本研究应用昆虫病原线虫共生细菌生物防治葡萄贮期灰霉病,筛选出一株高效拮抗灰葡萄孢的昆虫病原线虫共生细菌,而且其上清液对灰葡萄孢具有良好的抑制效果,为生物防治贮期葡萄灰霉病提供了新的生物材料和相关研究基础。     

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.     

Microbiology of pozol,a Mexican fermented maize dough

Mexican fermented maize dough, pozol, including traditional banana leaf-wrapped samples and material in plastic bags, was purchased. All samples were pH 4.7 to 5.7 approx. 12 h after preparation, pH declining to 3.6 to 3.9 after 6 to 9 days storage at ambient temperature. These latter samples had dry matter contents of 31% to 48% (w/w), 0.35% to 0.75% titratable acidity as lactic acid and lactic acid bacteria as predominant microbial flora at about 10⁸ c.f.u./ml. The lactic acid bacteria included strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus confusus, Lactococcus lactis and Lactococcus raffinolactis. Fungi were not found in the samples stored in plastic bags. The samples wrapped in banana leaf, however, developed a large surface mycoflora within 2 days. This included Geotrichum candidum, yeasts and moulds. The majority of the lactic acid bacteria and approx. 50% of yeasts hydrolysed starch to some extent. No Geotrichum isolate hydrolysed starch. Lactate was assimilated by all the Geotrichum isolates and by 17 of 39 yeast strains.     

Intraspecific variation ofDrosophila buzzatii larval breeding success onOpuntia: A yeast-plant-insect relationship

The cactophilic species,Drosophila buzzatii, normally breeds in decaying pockets ofOpuntia cladodes, in which there is a complex interaction with the microbial flora, especially yeast species. Isofemale lines were used to estimate genetic variation among larvae reared on their natural feeding substrate. Four naturally occurring cactophilic yeast species isolated from the same Tunisian oasis as theDrosophila population were used. Two fitness components were studied for each line, viability and developmental time. Genetic variations amongD. buzzatii lines were observed for both traits. A significant yeast species x isofemale line interaction for viability was also evidenced, suggesting the occurrence of specialized genotypes for the utilization of breeding substrates. This genetic heterogeneity in the natural population may favor a better adaptation to the patchily distribution of yeasts.     

Formulation of a Streptomyces Biocontrol Agent for the Suppression of Rhizoctonia Damping-off in Tomato Transplants

Formulations of a Streptomyces biological control agent for Rhizoctonia damping-off in tomato seedlings were developed for the first time from vegetative propagules obtained from actively growing, nonsporulating liquid cultures. Alginate beads, durum flour (starch) granules, and talcum powder formulation of this new actinomycetous antagonist (Streptomyces sp. Di-944) isolated from the rhizosphere of field-grown tomato (Lycopersicon esculentum) suppressed damping-off caused by Rhizoctonia solani in tomato plug transplants (cv. Bonny Best) in a peat-based, soilless potting mix under greenhouse conditions. For formulations, vegetative biomass of Streptomyces sp. Di-944 from 3-day-old liquid fermentation in yeast extract–malt extract–glucose broth was lyophilized and pulverized to obtain fragments of viable vegetative filaments. The pulverized biomass had an initial viable count of 2 × 10⁷colony forming units/g and retained 100% viability for 2 weeks when stored at 4°C. Formulations stored at 4°C had a longer shelf life than those stored at 24°C based on viability at 2-week intervals over a 6-month storage period. In addition, dual culture tests showed declining efficacy for surviving Streptomyces propagules in formulations during this storage period. At 4°C, the powder and granular formulations were found to be the most stable and were shown to be 100% viable after 14 and 10 weeks of storage, respectively. However, at the end of 24 weeks, the number of viable propagules in the powder and granular formulations declined to 1.2 × 10⁵ and 7 × 10³ colony forming units/g, respectively. Alginate beads were the least stable in storage. Even at 4°C, 6.9 × 10⁴ and 7.3 × 10² viable propagules/g formulation were detected at the end of 12 and 24 weeks, respectively. The talcum powder formulation delivered to tomato seeds as a seed-coating was the most effective biocontrol treatment. It suppressed damping-off in 10-day-old tomato transplants by almost 90% compared to 30 and 22% damping-off reduction when alginate beads or starch granules were delivered concomitantly with tomato seeds. Seed-coating with powder formulation of the biocontrol agent was as effective as drench application of the fungicide, oxine benzoate (No-Damp), in controlling Rhizoctonia damping-off and superior to the commercial biocontrol agent, Streptomyces griseoviridis (Mycostop), applied to tomato seeds as seed-coating.     

Effect of different isolates of Beauveria bassiana on field populations of Lygus hesperus

Lygus hesperus (Knight) (Hemiptera: Miridae) is a particularly damaging pest of many crops in the Western United States. Current control tactics are chemically based and there is some concern over resistance building up in populations. Based on previous laboratory studies conducted in California and Mississippi, USA, two new isolates of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) were selected for field-testing against L. hesperus in California. Alfalfa plots were treated with one of three isolates of B. bassiana (a commercial isolate, an isolate from CA (WTPB2) or an isolate from MS (TPB3)) or the chemical pesticide Warrior T. More than 75% of the adults collected from plots 3 days after application with B. bassiana were infected but no differences in percentage infection occurred among fungal treatments. In addition, approximately 30% of the insects collected from control plots or plots treated with Warrior T were also infected. PCR analysis using SSR markers revealed that the isolate causing most of the infections in fungus treated plots was the isolate applied. A mix of infections was found in control plots and plots treated with Warrior T. Despite high levels of infection, no significant reductions of adult populations occurred until 10–14 days after application when plots treated with Warrior T or B. bassiana had about half the numbers of adult L. hesperus as the control plots.     

Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum

Fungal isolates, with known activity against Sclerotinia spp. in laboratory assays, were tested for their ability to control Sclerotinia minor in four field experiments (1998–2000). In the first experiment, eight fungal isolates (Trichoderma hamatum LU595, LU593, LU592, Trichoderma virens LU555 and LU556, Coniothyrium minitans LU112, Clonostachys rosea LU115 and Trichoderma rossicum LU596) were evaluated by incorporating spore suspensions into transplant potting mix and planting lettuce seedlings into a S. minor infested field site. At harvest, Trichoderma hamatum LU595, LU593, T. virens LU555 and C. minitans LU112 reduced disease by 30–50% compared with the untreated control under very high disease pressure (100%). In further field experiments C. minitans LU112 and T. hamatum LU593, applied as maizemeal–perlite soil amendments or incorporated into the potting mix, reduced S. minor disease over a range of disease pressures (29–91%). Disease control was equivalent or greater than that achieved with the standard carbendazim fungicide treatment. Both isolates were shown to effectively colonize the lettuce rhizosphere and surrounding soil and this colonization may have protected the roots from infection by S. minor. Multiple applications of C. minitans LU112 or T. hamatum LU593 formulations gave no added disease control compared with a single application at planting. Commercial formulations of both C. minitans LU112 and T. hamatum LU593 applied as transplant treatments, solid substrate soil amendments or as a spore drench gave consistent disease control and are currently being developed further.     

Morphological and ecological similarities: wood-boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov.

Ascomycete yeasts that both ferment and assimilate xylose were reported previously as associates of insects living in woody substrates. Most notable have been reports of Pichia stipitis-like yeasts that are widely associated with the wood-boring beetle, Odontotaenius disjunctus (Coleoptera: Passalidae), in the eastern United States. Our continuing investigation of insect gut yeasts has lead to the discovery of two new xylose-fermenting yeasts that phylogenetic analysis places as sister taxa. The beetle hosts, O. disjunctus and Phrenapates bennetti (Coleoptera: Tenebrionidae), are similar in habitat and appearance, and the presence of similar gut yeasts is an additional common feature between them. Here we describe the new yeast genus Spathaspora, the type species S. passalidarum, and its sister taxon Candida jeffriesii and discuss their natural history, including a comparison with Pichia stipitis, another member of a guild of xylose-fermenting yeasts with similar metabolic traits. In addition a morphologically distinct yeast ascospore type is described for Spathaspora.     

An unusual Xanthophyllomyces strain from leaves of Eucalyptus globulus in Chile

Xanthophyllomyces sp. was isolated as an epiphytic red yeast from leaves of Eucalyptus glo-bulus in Concepción, Chile. Sexual reproduction was by basidiospores produced from one or rarely two metabasidia arising from a yeast cell without preceding paedogamy. The main carotenoid pigment was astaxanthin. This isolate did not cluster with the X. dendrorhous complex (including Phaffia rhodozyma) in ITS and 26S rDNA-based phylogenetic analyses. The phylloplane may be a further habitat for Xanthophyllomyces, in addition to the well-known spring sap-flows of deciduous trees and the recently-characterised ascostromata of Cyttaria hariotii.     

Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase

The inulinase gene cloned from the marine-derived yeast Pichia guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for overexpression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase, 286.8 ± 5.4 U/ml and 8873 ± 55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-l fermentor were attained at 120 h of the fermentation and fermentation efficiency was 13.04 μg ± 0.4 of protein/ml/d. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60 °C, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine-derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industies.