Dianpeng Zhang

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.

Biocontrol activity of an alkaline serine protease from Aureobasidium pullulans expressed in Pichia pastoris against four postharvest pathogens on apple

The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period.

Heterologous coexpression of Vitreoscilla hemoglobin and Bacillus megaterium glucanase in Streptomyces lydicus A02 enhanced its production of antifungal metabolites.

Streptomyces lydicus A02 is a novel producer of commercially important polyene macrocyclic antibiotic natamycin and a potential biocontrol agent to several plant fungal diseases, including wilt caused by Fusarium oxysporum f. spp. To improve the natamycin production and the antifungal activity of S. lydicus A02, we coexpressed gene vgb encoding Vitreoscilla hemoglobin (VHb) and bglC encoding Bacillus megaterium L103 glucanase, both under the control of the strong constitutive ermE* promoter, in S. lydicus A02. Our results showed that coexpressing VHb and glucanase improved cell growth, and the engineered strain produced 26.90% more biomass than the wild-type strain after 72h fermentation in YSG medium. In addition, coexpressing genes encoding VHb and glucanase led to increased natamycin production, higher endogenous chitinase activity and exogenous glucanase activity, as well as enhanced antifungal activity in the engineered S. lydicus AVG02 and AGV02, regardless of the position of the two genes on the plasmids. Compared with model strains, few reports have successfully coexpressed VHb and other foreign proteins in industrial strains. Our results illustrated an effective approach for improving antifungal activity in an industrial strain by the rational engineering of combined favorable factors.

Efficacy of yeast antagonists used individually or in combination with hot water dipping for control of postharvest brown rot of peaches

The efficacy of the antagonistic yeasts Pseudozyma fusiformata AP6, Metschnikowia fructicola AP47 and Aureobasidium pullulans PL5 were investigated, in combination with hot water dipping (HWD), to control brown rot of peaches caused by Monilinia laxa. The effects on postharvest fruit quality were also evaluated under semi-commercial conditions. In potato dextrose broth, HWD at 50°C for more than 40 s or at 55-60°C for more than 10 s completely suppressed M. laxa spore germination and germ tube elongation. In fruits inoculated by wounding, HWD at 55°C for 50 s significantly reduced brown rot decay and maintained fruit quality. This treatment would be optimal for commercial application. The harvested fruits were dipped in HW at 55°C for 50 s, cooled to 1°C for 10 min, dipped in the selected yeast cell suspension (108 cells ml−1) for 60 s, and then stored at 1°C and 95% relative humidity (RH). After 21 days, the disease incidence was measured and the postharvest quality parameters of the fruits, including firmness, total soluble solids, ascorbic acid and titratable acidity, were measured. When used alone, the antagonists AP6, AP47 and PL5 reduced brown rot incidence on the peaches to 28.3%, 30.0% and 25.8%, respectively, compared with 74.2% for the untreated control. However, when applied together with HWD, AP6, AP47 and PL5 reduced brown rot incidences to 16.7%, 15.8% and 17.5%, respectively, while when HWD was used alone, disease incidence was 30.0%, suggesting that HWD greatly increased the effectiveness of the antagonists. In combination with HWD, the efficacies of AP6, AP47 and PL5 signifi-cantly increased from 61.8%, 59.6% and 65.2% to 77.5%, 78.7% and 76.4%, respectively, and they were similar to the efficacy of tebuconazole (77.5%). Analysis of postharvest quality indicated that none of the treatments damaged fruit quality. Our results showed that the three antagonists combined with HWD at 55°C for 50 s exhibited potential for commercial use, as an effective alternative to fungicides in controlling postharvest brown rot of peaches. This appears to be the first report on combined application of yeast strains M. fructicola or P. fusiformata with HWD to control M. laxa on peaches.ZusammenfassungZur Bekämpfung der Fruchtfäule an Pfirsichen verursacht durch Monilinia laxa wurde die Wirksamkeit der antagonistischen Hefen Pseudozyma fusiformata AP6, Metschnikowia fructicola AP47 and Aureobasidium pullulans PL5 in Kombination mit einer Heißwasserbehandlung (HWD) untersucht. Außerdem wurden die Auswirkungen auf die Früchte nach der Ernte unter semikommerziellen Bedingungen geprüft. Die Sporenkeimung und das Keimschlauchwachstum von M. laxa wurden in Kartoffel-Dextrose-Medium bei 50°C für mehr als 40 s oder bei 55–60°C für mehr als 10 s vollständig unterdrückt. In Früchten, die per Verwundung inokuliert wurden, wird die Fruchtfäule durch HWD bei 55°C für 50 s signifikant reduziert und die Fruchtqualität bleibt erhalten. Diese Behandlung eignet sich optimal zur kommerziellen Anwendung. Die geernteten Früchte werden für 50 s in 55°C heißes Wasser getaucht, anschließend für 10 min auf 1°C abgekühlt, dann für 60 s in die Hefezellsuspension (108 Zellen ml−1) getaucht und bei 1°C und 95% relative Luftfeuchtigkeit (RH) gelagert. Nach 21 Tagen wurde die Krankheitsentwicklung erfasst und die Nacherntequalitätsparameter der Früchte wie Fruchtfleischfestigkeit, Anteil der löslichen Feststoffe, Ascorbinsäuregehalt und Säuregehalt gemessen. In der Einzelbehandlung reduzierten die Antagonisten AP6, AP47 und PL5 das Braunfäulevorkommen an den Pfirsichen auf 28.3%, 30.0% und 25.8%, verglichen mit 74. % bei der unbehandelten Kontrolle. Dagegen wurde in Kombination mit HWD bei AP6, AP47 und PL5 eine Reduktion des Krankheitsauftretens auf 16.7%, 15.8% und 17.5% erreicht, während mit der HWD alleine die Krankheit nur auf 30% reduziert werden konnte. Das zeigt, dass die Wirksamkeit der Antagonisten durch die HWD deutlich gesteigert wird. In Kombination mit HWD wird die Wirksamkeit von AP6, AP47 und PL5 jeweils signifikant von 61.8%, 59.6% und 65.2% auf 77.5%, 78.5% und 76.4% gesteigert und erreichen somit eine ähnlich hohe Wirksamkeit wie Tebuconazol (77.5%). Die Untersuchung der Nacherntequalität hat ergeben, dass keine der Behandlungen die Fruchtqualität gestört haben. Unsere Ergebnisse zeigen, dass die drei Antagonisten jeweils in Kombination mit HWD bei 55°C für 50 s auch im kommerziellen Einsatz eine effektive Alternative zur Fungizidanwendung als Bekämpfung gegen die Braunfäule bei Pfirsichen darstellt. Dies ist nach Meinung der Autoren die erste Arbeit, die eine kombinierte Anwendung der Hefestämme M.fructicola oder P. fusiformata mit einer Heißwasserbehandlung als Bekämpfung gegen M. laxa an Pfirsichen zeigt.

Metschnikowia persici sp nov., A Novel Protease-Producing Yeast Species from China

Three yeast strains, named as FHL-A, FHL-B, and FHL-C, were isolated from peach fruit surfaces collected from different regions in the North of China highly produced protease and were presented as single separate group in the genus Metschnikowia by sequence comparisons of 26S rRNA gene D1/D2 domain and internal transcribed spacer (ITS) region. BLASTn alignments on NCBI showed that the similarity of 26S rRNA gene sequences of the three strains to all sequences of other yeasts accessed into the GenBank/EMBL/DDBJ and other database was very low (≦93%). The phylogenetic tree based on the D1/D2 region of 26S rRNA gene sequences revealed that three strains are most closely related to Metschnikowia koreensis KCTC 7828T (AF257272.1) (sequence similarity: 93.0%) and Metschnikowia reukaufii CBS9709T (AJ716113.1) (sequence similarity: 93.0%). However, the strains are distinguished from M. koreensis by its non-assimilation of galactose, ribitol, and D-xylose, and by its growth at 37 °C or in vitamin-free medium, and are notably different from M. reukaufii by its non-assimilation of galactose, D-xylose, D-arabinose, and D-ribose, and by its growth at 35 °C or in vitamin-free medium. The strain FHL-B formed asci in V8 juice sporulation medium for 3 weeks. Therefore, the name Metschnikowia persici is proposed for the novel species, with FHL-B (= CBS12815T = CFCC 3578T) as the type strain.

Identification and evaluation of Aspergillus tubingensis as a potential biocontrol agent against grey mould on tomato

Grey mould of tomato caused by Botrytis cinerea is a highly damaging disease worldwide. This study was aimed at identifying fungal strain QF05, which was isolated from the sandy soil collected in the Qinghai-Tibet Plateau in China and to evaluate its biocontrol potential on grey mould and growth promotion effect on tomato plants. On the basis of morphological observation and a multigene phylogeny based on the ITS, β-tubulin and calmodulin genes, QF05 was identified as Aspergillus tubingensis. An in vitro dual culture test indicated obvious antagonistic activity by strain QF05 against B. cinerea. Strain QF05 distinctly reduced grey mould in assays on tomato fruits and plants. Treatments with the culture filtrate of strain QF05 and a tenfold dilution of the filtrate gave disease biocontrol efficacy of 34.5 and 31.0% on tomato plants, respectively. QF05 also promoted seed germination and plant growth of tomato. Length and total fresh and dry mass of tomato plants were all significantly increased by 17.8, 25.7 and 56.8%, respectively, after treatment with the tenfold dilution of the culture filtrate. QF05 also had positive biocontrol attributes such as indoleacetic acid and siderophore production, phosphate solubilization, and rhizosphere colonization. All these results indicated the good performance of A. tubingensis QF05 for the biocontrol of tomato grey mould and growth promotion of tomato plants.