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Grey mould caused by Botrytis cinerea leads to severe economic loss on commercial tomato production. Application of beneficial microorganism offers an eco-friendly alternative for mitigation of tomato fungal disease damage, considering negative influences of fungicides. In the present study, an antagonistic Trichoderma afroharzianum isolate TM24 was evaluated for its biocontrol potential on tomato grey mould. The isolate TM24 showed obviously antagonistic effect on B. cinerea mycelium growth and production of glucanase and chitinase. Leaf spraying with spore suspension of isolate TM24 showed a biocontrol efficiency of over 54% against tomato grey mould in greenhouse pot experiment. The activities of plant defense-related enzymes including polyphenol oxidase, phenylalanine ammonialyase, superoxide dismutase, and peroxidase were all increased to varying degrees in tomato leaves after isolate TM24 treatment. Transcriptome analysis showed that, a total of 1941, 1753 and 38 differentially expressed genes (DEGs) were obtained at 24, 48 and 72 hpi, respectively, in tomato leaves pretreated with T. afroharzianum TM24, and then challenged with B. cinerea inoculation. The DEGs were mainly enriched in MAPK signaling pathway and plant hormones signal transduction pathway. Multiple genes that regulated crucial nodes of defense-related pathways, like flavonoid, phenylpropanoid, jasmonic acid and ethylene metabolisms were also identified, which may have positive correlations with the biocontrol potential of isolate TM24 in tomato plants. These promising results provided valuable information on using T. afroharzianum TM24 as a beneficial biocontrol agent in tomato grey mould management.

Camellia chrysantha is cultivated as rare genetic resource of the family Theaceae, which mainly distributed in the South of Guangxi Zhuang Autonomous Region in China. Rhizosphere microbiota may play important roles in C. chrysantha growth and homeostasis through interaction of the plant and soil ecosystem. In order to investigate the bacterial community composition in the rhizosphere soil of three C. chrysantha cultivars under different growing conditions, Illumina MiSeq high-throughput sequencing and real-time quantitative PCR methods were used in this study. Results indicated that Proteobacteria, Acidobacteria, Chloroflexi, Actinobacteria, Bacteroidetes, and Gemmatimonadetes were the top six phyla across all the six rhizosphere soil samples. The richness and diversity of bacterial community in the rhizosphere soil from cultivar “Fangpu” grown understorey (LF) in retail planting area were significantly different from the other samples. Principal coordinate analysis revealed that bacterial community composition of the rhizosphere soil from cultivar “Fangpu” grown understorey (LF) and perennial grown undershed (PFC) was obviously separated from each other as well as the other four rhizosphere soil samples. The growing conditions displayed more influences on the bacterial community composition than plant cultivars, and close correlations were found between the soil physicochemical characters of the geographical origins and the bacterial communities. This work improves understanding of the rhizosphere bacterial community composition and the influencing soil physicochemical factors, and provides basic information for cultivation and protection of the C. chrysantha plants in China.

Plant growth promoting fungi are receiving increased attention as valuable beneficial microorganisms in crop cultivation due to their capacity to produce bioactive substances, promote plant growth and enhance immune defense functions. In this study, a novel Trichoderma isolate, designated as TM2-4, was screened from healthy tomato rhizosphere soil and identified as Trichoderma afroharzianum. Culture filtrate of the isolate TM2-4 displayed obvious bioactive substance production and an evident effect in promoting tomato seed germination, with hypocotyl length, radical length and vigor index increased by 28.7, 19.4 and 62.1%, respectively, after a 100-fold dilution treatment. To assess the promotion effect and related mechanism of isolate TM2-4, the plant biological indexes and gene expression profiles of tomato plants treated with or without T. afroharzianum TM2-4 microbial agent were investigated by greenhouse pot experiment and RNA sequencing. The results demonstrated that T. afroharzianum TM2-4 significantly promoted tomato plant growth in terms of plant height, dry weight, number of leaves per plant and root activity, through efficient colonization in the rhizosphere and root system of the plants. Transcriptome analyses identified a total of 984 differentially expressed genes in T. afroharzianum microbial agent inoculated tomato roots, which were mainly engaged in the biological process of phytohormone homeostasis, antioxidant activity, as well as metabolic pathways including phenylpropanoid biosynthesis and glutathione metabolism. These findings provide useful information for understanding the mechanism of isolate TM2-4 for tomato plant growth promotion, which would facilitate further development of T. afroharzianum TM2-4 microbial agent for use in vegetable crop production.

Peach brown rot, caused by Monilinia fructicola, is one of the most serious peach diseases. A strain belonging to the Actinomycetales, named Streptomyces blastmyceticus JZB130180, was found to have a strong inhibitory effect on M. fructicola in confrontation culture. Following the inoculation of peaches in vitro, it was revealed that the fermentation broth of S. blastmyceticus JZB130180 had a significant inhibitory effect on disease development by M. fructicola. The fermentation broth of S. blastmyceticus JZB130180 had an EC50 (concentration for 50% of maximal effect) of 38.3 µg/mL against M. fructicola, as determined in an indoor toxicity test. Analysis of the physicochemical properties of the fermentation broth revealed that it was tolerant of acid and alkaline conditions, temperature, and ultraviolet radiation. In addition, chitinase, cellulase, and protease were also found to be secreted by the strain. The results of this study suggest that S. blastmyceticus JZB130180 may be used for the biocontrol of peach brown rot.

Camellia chrysantha (Hu) Tuyama, belonging to the Theaceae family, is famous for its large size and golden yellow flowers, which has high ornamental and health care functions (Mo et al. 2013). Anthracnose is one of the most important fungal diseases worldwide, causing serious economic losses to many plants. In October 2019, severe anthracnose symptoms were observed on the leaves of C. chrysantha in a 0.6 hectare field with 15-20% disease incidence in Fangchenggang city, Guangxi Zhuang Autonomous Region of China. Diseased leaves initially appeared irregular chlorotic spots, which afterwards enlarged and coalesced. Finally, the spots became dark brown or black, sunken lesions (8-22 mm in diameter), and covered with plenty of acervuli. For pathogen isolation, the leaf lesions were cut into small tissue pieces (5 mm×5 mm), disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated at 28°C on potato dextrose agar (PDA) plates. A total of 7 fungal isolates with whitish to light grey, dense colonies were recovered at 5 days. These isolates were tentatively identified as belonging to Colletotrichum gloeosporioides species complex through morphological and cultural characters (Weir et al. 2012). The conidia were nonseptate, cylindrical with obtuse to rounded ends, 13.9 to 18.3 (average 16.1) μm × 4.5 to 6.2 (average 5.4) μm (n = 50). For further precise identification, the 7 Colletotrichum isolates were analyzed using partial sequences of genomic loci including the internal transcribed spacer (ITS), β-tubulin (TUB), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and the mating type locus MAT1-2 (ApMat) genes (Liu et al. 2015). The amplification sequences were compared with the sequences registered in the GenBank database based on nucleotide similarity. The above sequences of 4 isolates (JZB-PF4232, JZB-PF2231, JZB-PF42 and JZB-PF22) had 99-100% identity to the sequences of Colletotrichum siamense strains retrieved from GenBank, while the sequences of the other 3 isolates (JZB-PF3231, JZB-PF32 and JZB-PF41) showed over 99% identity with those of the C. fructicola strains. All the sequences were deposited in GenBank with accession number MT708987 to MT709007, MW149430 to MW149433, and MW142259 to MW142282. A multi-loci phylogenetic analysis of the concatenated sequences of ITS, TUB, CAL, ACT, GAPDH, GS and ApMat genes placed the 4 isolates described above in the C. siamense clade, while the other 3 isolates was attribute to the C. fructicola clade. Pathogenicity tests were conducted on 7 healthy 2-year-old C. chrysantha seedlings (cv. Fangpu), consisted of 21 wounded leaves made by a sterile needle, with 3 leaves per seedling. Artificial inoculations were performed by treating each seedling with 20 µl of spore suspension (106 conidia/ml) of each isolate. Leaves of seedlings treated with sterilized water under the same conditions served as controls. The experiment was repeated three times. All the seedlings were covered with plastic bags to maintain high humidity (90% RH) and placed in a greenhouse kept at 25°C with a 16 h light / 8 h dark photoperiod. After 8 days, the inoculated leaves of C. chrysantha plants developed typical dark brown or black lesions, similar to the symptoms in the field, whereas controls remained symptomless. Koch's postulates were fulfilled by re-isolation of the same fungi from symptomatic inoculated leaves, identification confirmed by morphological and molecular characteristics, respectively. C. siamense and C. fructicola have been found to cause anthracnose on Camellia sinensis (Wang et al. 2016; Shi et al. 2018). C. fructicola has also been reported to cause anthracnose on Citrus sinensis in China (Hu et al. 2019). To our knowledge, this is the first report of C. siamense and C. fructicola causing anthracnose on C. chrysantha in China.

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.

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.

Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461 ) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl β- d -N,N′,N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases.

Brown rot caused by Monilinia spp. is among the most important postharvest diseases of commercially grown stone fruits, and application of antagonistic yeasts to control brown rot is one promising strategy alternative to chemical fungicides. In this research, new yeast strains were isolated and tested for their activity against peach brown rot caused by Monilinia fructicola. Three yeast strains were originally isolated from the surface of plums (cv Chinese Angelino) collected in the north of China. In artificially wounded inoculation tests, the yeast reduced the brown rot incidence to 20 %. The population of the yeast within inoculated wounds on peaches significantly increased at 25 °C from an initial level of 5.0×10(6) to 4.45×10(7) CFU per wound after 1 day. The antagonistic strains were belonging to a new species of the genus Candida by sequence comparisons of 26 S rDNA D1/D2 domain and internal transcribed spacer region. The strains are most closely related to C. asparagi, C. musae and C. fructus on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA. However, the strains are notably different from C. asparagi, C. musae and C. fructus, in morphological and physiological characteristics. Therefore, the name Candida pruni is proposed for the novel species, with sp-Quan (=CBS12814T=KCTC 27526T=GCMC 6582T) as the type strain. Our study showed that Candida pruni is a novel yeast species with potential biocontrol against brown rot caused by M. fructicola on peaches.

An antagonistic bacterium, denoted as strain Z01, was isolated from suppressive soils and identified using fatty acid profile analysis and molecular tools. Its efficacy to control Fusarium oxysporum f. sp. conglutinans on rockets (cv. Eruca sativa) was investigated in the presence of the pathogen, and its effects on the growth and pigment contents of rockets, including the plant biomass and chlorophyll and carotenoid contents, were evaluated in the absence of the pathogen. Z01 was identified as Pantoea agglomerans (GenBank accession number is JX257179). Rockets were grown under greenhouse conditions for 21 days at 25 °C and 95 % relative humidity. When the antagonist suspension was applied at 108, 107 and 106 cells/ml by root-dipping for 20 min before transplanting, the disease incidence was 23.5 %, 28.5 % and 60.0 %, respectively. The disease incidence of rockets treated with carbendazim was 22.5 %, and in the inoculated control, the disease incidence was 82.0 %. In the absence of the pathogen, compared with the average plant biomass (10.7 g/4 plants) of the uninoculated control, application of the antagonist at 108 or 107 cells/ml significantly increased the biomass of the rockets by 40.9 % and 39.1 %, respectively, suggesting that Z01 had an additional ability to promote plant growth beyond its role in Fusarium wilt control. This study indicated that P. agglomerans Z01 has a potential to control Fusarium wilt in rockets. This report is the first to describe the application of P. agglomerans to control Fusarium wilt.

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.

The strain M8 of Pichia guillermondii isolated from the carbosphere of apples (cv. Golden Delicious) showed a high efficacy in controlling grey mold, caused by Botrytis cinerea, on apples under semi-commercial conditions. Moreover, Pichia guilliermondii M8 produced high amounts of active exo-1,3-β-glucanase in Lilly-Barnett minimal salt medium with different carbon sources, which greatly inhibited B. cinerea in vitro and in vivo tests. Therefore, an exo-1,3-β-glucanase gene, named as PgExg1 (GenBank accession number HQ113463 ) was cloned from the genomic DNA of the strain M8 by genome walking. The sequencing and the nucleotide BLAST analysis indicates that no introns are present inside the gene, which was confirmed by amplifying the full gene from complementary DNA (cDNA) of the yeast. An open reading frame of 1224 bp encoding a 408-amino acid (aa) protein with a calculated molecular weight (Mr) of 46.9 kDa and an isoelectric point (pI) of 4.5 was characterized. Protein BLAST and phylogenetic tree analysis of the deduced amino acid sequences from the PgExg1 gene suggested that the glucanase produced by PgExg1 gene belongs to the Glycoside Hydrolase Family 5. Expression of PgExg1 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis with enzyme assay, yielded homogeneous recombinant PgExg1. At its optimal pH of 5.0 and its optimal temperature of 40 °C, the recombinant enzyme protein showed the highest activity towards laminarin, while the highest stability was obtained when the enzyme was stored at pH of 7.0 and temperature of 4 °C.

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 109 and 108 cells ml−1 inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH4)2SO4, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 108 cells ml−1 followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.

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.

Few strains of Metschnikowia pulcherrima (Pitt) M.W. Miller are under development for control of postharvest pathogens on fruit. A substrate was developed to optimize the biomass production of M. pulcherrima strain BIO126. Different complex nutrient sources, with or without pH control, were tested. Growth in yeast extract provided at concentrations ≥30 g·L–1yielded the highest biomass. The addition of two carbon sources, d-mannitol and l-sorbose, at 5 g·L–1each, significantly improved yeast growth. The greatest amount of yeast growth occurred when pH values of the medium ranged from 5.0 to 7.5. A combination of yeast extract, d-mannitol, and l-sorbose (YEMS), probably with diauxic utilization, showed a synergistic effect, widening the exponential phase (maximum specific growth rate of 0.45 h–1) and increasing the final cell number (1.5 × 109cells·mL–1) and dry biomass (6.0 g·L–1) in well-controlled batch fermentation. In efficacy trials on ‘Golden Delicious’ apples, M. pulcherrima grown in YEMS effectively reduced incidence and severity of Botrytis cinerea (51.1% and 70.8%, respectively) and Penicillium expansum (41.7% and 14.0%, respectively). Also on ‘Gala’ apples, the best reduction of grey and blue mould incidence was obtained with cells grown in YEMS (58.1% and 50.5%, respectively).

The permafrost soil of Mo-he in Northeast China presents a typical cold environment colonized by psychrophilic microorganisms. This study is aimed at assessing the bacterial communities of permafrost soil of Mo-he in China by sequencing the 16S rRNA genes and Mothur analysis. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species was performed by comparative analysis with the GenBank/EMBL/DDBJ database. A total of 266 clones were obtained with the average length of 1,050 bp. Mothur analysis showed that the coverage value of clone library was 53.78 %, Shannon diversity (H) was 4.03, Simpson diversity value was 0.018, and 74 operational taxonomic units were generated. Through phylogenetic assignment using BLASTN by more than 97 % similarity, a total of 87 tentative taxa were identified. The majority of bacterial sequences recovered in this study belonged to the Acidobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Chloroflexi and Chlorobi. Among them, Acidobacteria are dominant community, accounting for 30.1 % of total bacteria, followed by Proteobacteria which accounted for 22.2 %. This result reflected the acidic characteristics of the permafrost soil of which pH value was 6.0. Our study indicated that the permafrost soil of Mo-he in China has a high diversity of bacteria and represents a vast potential resource of novel bacteria. As far as we knew, this is the first report on bacterial diversity of permafrost soil of Mo-he in China.

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.

Natamycin is an important polyene macrolide antifungal agent produced by several Streptomyces strains and is widely used as a food preservative and fungicide in food, medicinal and veterinary products. In order to increase the yield of natamycin, this study aimed at cloning and overexpressing a natamycin-positive regulator, slnM2, with different promoters in the newly isolated strain Streptomyces lydicus A02, which is capable of producing natamycin. The slnM gene in S. lydicus is highly similar to gene pimM (scnRII), the pathway-specific positive regulator of natamycin biosynthesis in S. natalensis and S. chattanoogensis, which are PAS-LuxR regulators. Three engineered strains of S. lydicus, AM01, AM02 and AM03, were generated by inserting an additional copy of slnM2 with an ermEp* promoter, inserting an additional copy of slnM2 with dual promoters, ermEp* and its own promoter, and inserting an additional copy of slnM2 with its own promoter, respectively. No obvious changes in growth were observed between the engineered and wild-type strains. However, natamycin production in the engineered strains was significantly enhanced, by 2.4-fold in strain AM01, 3.0-fold in strain AM02 and 1.9-fold in strain AM03 when compared to the strain A02 in YEME medium without sucrose. These results indicated that the ermEp* promoter was more active than the native promoter of slnM2. Overall, dual promoters displayed the highest transcription of biosynthetic genes and yield of natamycin.

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.