Study On The Biological Characteristics Of T-DNA Marker Insertional Mutants ZS-1T20440 Of Coniothyrium Minitans And Cloning The Gene Involving In The Production Of Anti-fungal Substances

Sclerotinia sclerotiorum is an important pathogen with worldwide distribution,and Coniothyrium minitans has great potential for biological control of plant diseasescaused by S. sclerotiorum and other ScIerotinia spp. C. minitans could produceantifungal substances (AFS) in certain medium, but usually could not produce AFS inPDA. In this paper, C. minitans mutants which could produce AFS against S.sclerotiorum in PDA medium were screened out from Conithyrium minitans ZS-1 ofT-DNA inserts library, and mutant ZS-1T20440, one of screened producingabundant-AFS mutants was systematically studied.The growth rate, conidial germination, bacteriostatic ability againstXanthomonas oryzae pv. oryzae, parasitic ability on sclerotia of S. sclerotiorum ofZS-1T20440 were tested. The growth rate of ZS-1T20440 on PDA plate was 1.23mm/d at 20℃, it is significantly lower than that of original strain ZS-1, whose growthrate was 2.77 mm/d. Conidial germination of ZS-1T20440 on PDA was much fasterthan that of original strain ZS-1, at the same condition, there were up to 65.5 % and97.4 % conidia of ZS-1T20440 germinated after 24 h and 36 h incubation, but only45.3 %and 84.2 % conidia of ZS-1 were germinated. ZS-1T20440 could produceantibacterial substance as ZS-1 did. ZS-1T20440 could parasitize the sclerotia of S.sclerotiorum, but the virulence on S. sclerotiorum was significantly weaker thanoriginal strain ZS-1, at the same conidiation, 97 % sclerotia of S. sclerotiorum wereparasitized by ZS-1, but only 50 % were parasitized by ZS-1T20440. The AFSactivity against S. sclerotiorum in cultured liquid could increase with the extension ofincubation time, but the AFS production could not increase at later stage of incubation.The pH value in filtrate of ZS-1T20440 would decrease to 4.67 at later stage, but thepH value in filtrate of ZS-1 would increase to 6.29.Southern blot showed that only one copy of T-DNA was inserted in ZS-1T20440,and a DNA fragment of 738 bp from genomic DNA was amplified with reverse-PCR.Sequencing analysis showed that 245 amino acids were encoded. The deduced aminoacid sequences shared high identity to the GPI-anchored protein of AspergiUusfumigatus Af293. Therefore the gene cloned here was named CMGPI-like 1. Southernblot showed that there was only one copy of CMGPI-like 1 in ZS-1 genomic DNA,and CMGPI-like 1 gene could not express in ZS-1T20440, but expressed in ZS-1.CMGPI-like 1 was firstly identified to involve in the production of AFS in C. minitans,and it is possible that AFS production pathway negatively.