| Clonostachys rosea(syn. Gliocladium roseum) is a mycoparasite of various pathogenic fungi, such as Sclerotinia sclerotiorum, Botrytis cinerea, and Fusarium spp., and has shown great potential in biocontrol of plant fungal diseases. However, only a few research on the biocontrol-related genes of C. rosea is available up to now. In this study, quantitative real-time PCR was conductd to verify the differential expression of the unigenes selected from the transcriptome of C. rosea 67-1 associating with sclerotia of S. sclerotiorum. The transaldolase encoding gene Tal67, monooxygenase encoding gene Mon67 and α-galactosidase gene Aga67 were chosen and their sequences were analyzed using bioinformatics tools. Gene deletion and complementation were performed and the functions of Tal67, Mon67 and Aga67 were identified.Seventeen differentially expressed unigenes were selected from the transcriptome and their expression levels were quantified by real-time PCR in which the elongation factor EF1 gene was used as an internal reference. The results were consistent with the transcriptome analysis. Quantitative determination showed that the relative expression levels of Unigene 4498 and Unigene 8262 were upregulated throughout the process of sclerotia induction(0-48 h), peaked at 13.9-fold and 32.3-fold, respectively, higher than the control at 24 h. The relative expression levels of Unigene 13246 decreased at first(8-24 h)and then increased, peaked at 7.4-fold higher than the control at 48 h. The determination of relative expression levels indicated that these genes might involve in the mycoparasitism of C. rosea to sclerotia.DNA sequences of Unigene 4498, Unigene 8262 and Unigene 13246 were obtained from the draft genome sequence of C. rosea 67-1. Bioinformatics analysis showed that Unigene 4498 had a 750 bp open reading frame, encoding a 249 AA protein, and was most closely related to transaldolase from Colletotrichum gloeosporioides with a similarity of 77%. Therefore, termed Unigene 4498 as Tal67. Unigene 8262 had a 1266 bp open reading frame, encoding a 421 AA protein, and was most closely related to monooxygenase from Metarhizium acridum with a similarity of 49%. Therefore, termed Unigene 8262 as Mon67. Unigene 13246 had a 1668 bp open reading frame, encoding a 555 AA protein, and was most closely related to α-galactosidase from Diaporthe ampelina with a similarity of 52%. Therefore, termed Unigene 13246 as Aga67.By the method of PEG mediated protoplast transformation and homologous recombination, we obtained two Tal67 gene-deficient mutant ΔTal67-1 and ΔTal67-8, three Mon67 gene-deficient mutant ΔMon67-22, ΔMon67-26 and ΔMon67-28, two Aga67 gene-deficient mutant ΔAga67-30 and ΔAga67-62, and five ΔTal67-8 complementary strains ?Tal67+.Phenotypes analysis showed that the growth rate of ?Tal67, reduced to 5.3 mm/day, significantly lower than wild type and the complemented strain ?Tal67+(P<0.05). The antagonistic activity of ?Tal67 against Botrytis cinera was 15.8% lower than the wild type and the parasitic rate to S. sclerotiorum decreased by 24.6%. However, the reinsertion of transaldolase gene recovered the fungicidal activity of C. rosea. The efficacy of the mutants against soybean Sclerotinia stem rot and cucumber Fusarium wilt were evaluated in greenhouse, and the the control efficiency of WT reached 65.3% and 75.6%, however, the efficiency of ?Tal67 strain sharply decreased to 17.8% and 23.1%. The complemented strain ?Tal67+ rose back to the ability of WT. There was no difference in growth rate, sporulation and antagonistic activity among the ?Mon67, ?Aga67 and WT. However, the parasitic rate of ?Mon67 and ?Aga67 to S. sclerotiorum decreased by 34.2% and 28.7%, respectively. The efficacy of of ?Mon67 and ?Aga67 against cucumber Fusarium wilt decreased by 51.5% and 41.4%, respectively, compared to WT. The results suggest that Tal67, Mon67 and Aga67 play important roles in the biocontrol activity of C. rosea. |
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