Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M_r) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M_r of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50 °C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.     

Study on the role of patulin on pathogenicity and virulence of Penicillium expansum

Although the antibacterial activity and toxicity to humans and animals of the mycotoxin patulin are well known, its role in the postharvest decay of apples by Penicillium expansum has never been investigated. In the present study the gene disruption technique was used to alter the sequence of 6-methyl-salicylic acid synthase, an enzyme involved in the first committed step of patulin biosynthesis. Thirty-nine mutants were obtained, however only two of them (M5 and M21) passed the sub-cultural and molecular confirmation tests. They proved to produce 33-41% less patulin than their wild-type (WT) strain, although no difference in the growth and morphology of the colony was observed. Moreover, the mutants showed a significantly reduced pathogenicity and virulence on artificially inoculated apples. In particular, a 33-34% and 47-54% reduction of disease incidence and severity were recorded for M5 and M21, respectively. As confirmation, when the biomass of the mutants was quantified in vivo by Real-time PCR, a significant difference was recorded as compared to the WT and even between mutants. Moreover, when patulin production potential of mutants was restored by exogenous application of the mycotoxin, their ability to cause the disease was not significantly different from that of WT. Finally, mutants showed an increased susceptibility to the application of the antioxidant quercetin, their pathogenicity and virulence being significantly reduced at only 1/100 of the concentration needed for the WT. Based on these findings, patulin seems to have a role in the development of blue mold decay on apples.     

Artificial aging of Uva di Troia and Primitivo wines using oak chips inoculated with Penicillium purpurogenum

BACKGROUND: Two red wines (Primitivo and Uva di Troia) treated with oak chips inoculated with Penicillium purpurogenum were analysed in order to assess their contents of furfural, cis‐β‐methyl‐γ‐octalactone, syringol, eugenol, vanillin and 4‐vinylguaiacol. Two different sizes of oak chips (small and big, of length 2 and 8 mm respectively) and two different degrees of toasting (low and high) were used in the study. Aroma compounds were analysed by gas chromatography/mass spectrometry to determine differences among samples after 15 days of chip contact time. RESULTS: Big oak chips inoculated with P. purpurogenum increased the level of 4‐vinylguaiacol, while small oak chips inoculated with P. purpurogenum, in some conditions, increased the level of eugenol. Chip size and degree of toasting also played an important role in the content of eugenol. CONCLUSION: The use of oak chips inoculated with mould might be a promising alternative to barrel aging. Moreover, different fungal inocula could contribute to the enrichment of wine with specific compounds (e.g. 4‐vinylguaiacol and eugenol). Copyright © 2011 Society of Chemical Industry     

Penicillium sp.X-1液态发酵生产生淀粉酶的优化

通过摇瓶发酵,研究了培养基成分对Penicillium sp.X-1液态发酵产生淀粉酶的影响。结果表明：碳源、氮源及MgCl2对产酶有较大的影响,经响应面优化得到的培养基组成为：玉米粉42 g/L,豆饼粉30 g/L,MgCl216 mmol/L,在最优条件下酶活达到239 U/mL,与采用基本培养基的相比,酶活提高了7.5倍.     

Phosphate-solubilizing Microbe from Saprists Peat Soil and their Potency to Enhance Oil Palm Growth and P Uptake

Constraint of peat land utilization is low nutrent content. This research was conducted to determine the potency of phosphate solubilizing microbes (PSM) in solubilizing P yto increase the growth and P uptake of plant in peat soil. Based on their ribosomal DNA the best PSM were identified as Burkholderia gladioli nd Penicillium aculeatum that yield the highesr growth aphosphate uptake of oil palm seedling.     

Activity of Electrolyzed Oxidizing Water Against Penicilium expansum in Suspension and on Wounded Apples

ABSTRACT: Spores of Penicillium expansum , the primary organism responsible for the occurrence of patulin in apple juice, were exposed to electrolyzed oxidizing (EO) water in an aqueous suspension and on wounded apples. Full-strength and 50% EO water decreased viable spore populations by greater than 4 and 2 log units, respectively. Although EO water did not prevent lesion formation on fruit previously inoculated with P. expansum , cross-contamination of wounded apples from decayed fruit or by direct addition of spores to a simulated dump tank was substantially reduced. EO water, therefore, has potential as an alternative to chlorine disinfectants for controlling infection of apples by P. expansum during handling and processing operations.     

高产纤维素酶青霉菌的筛选和及产酶条件的研究

本文从稻田土壤中分离到—株产纤维酶活力高的X-5菌株，经形态特征及生理生化特征初步鉴定为半知菌亚门，丝孢纲，丝孢目，丛梗孢科，青霉属（Penicillium）。对青霉菌X-5菌株进行发酵培养条件的研究结果表明，以5．0％稻草粉为碳源，3．0％豆饼粉为氮源，装液量为60ml，接种量为5．0％，培养温度为26～28℃，初始pH4．5-6．0，培养120h，产酶活力最高，CMC酶活为75．72IU／ml，FP酶活为6．68IU／ml.     

α-松油醇对指状青霉抑霉唑抗性菌株Pdw03的抑制作用

本研究探讨了α-松油醇对指状青霉抑霉唑抗性菌株Pdw03的抑制作用及其抑菌机理。体外实验结果表明α-松油醇抑制Pdw03生长的最小抑菌浓度（minimal inhibitory concentration,MIC）和最小杀菌浓度（minimal fungicidal concentration,MFC）均为4.00 μL/mL;体内实验研究发现MFC和5 MFC α-松油醇可明显降低接种Pdw03椪柑果实的绿霉病发病率和发病直径;碘化丙啶（propidium iodide,PI）染色实验发现,经1/2 MIC α-松油醇处理后,Pdw03的细胞膜完整性降低,麦角固醇含量显著降低（P＜0.05）,麦角固醇合成相关基因ERG9表达量显著下调（P＜0.05）。综上,α-松油醇通过抑制ERG9基因的表达影响菌株Pdw03的生长,可作为采后柑橘病害防治的有效替代品。     

Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites

Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis activity. The present research work aimed to study the biotransformation processes of IMP and evaluated the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new derivatives. Ten transformed products were isolated and purified, and their structures were identified accurately based on spectroscopic data. Eight metabolites (2–8 and 10) were novel and previously unreported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of all products (1–10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8 had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future therapeutic regimens for treating osteoporosis.     

Inhibition of Penicillium expansum by an oxidative treatment

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H₂O₂) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO₄, against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L⁻¹ and 400 mmol L⁻¹ for NaClO and H₂O₂, respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L⁻¹ NaClO and 200 mmol L⁻¹ H₂O₂, in the presence of 6 mmol L⁻¹ CuSO₄, inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H₂O₂ in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.