Screening,Purific Ation Of Antagonistic Chemicals And Molecular Marker Of Penicillium Striatisporum Pst10, A Fungal Biocontrol Agent For Chilli Pepper Blight

Phytophthora root rot (PRR), caused by Phytophthora capsici Leonian,is a common and destructive disease of greenhouse and field-grown chilli peppers(Capsicum frutescens L.) in China and the worldwide. The disease caused serious economic losses of chilli peppers in more than20Provinces of China. It has been difficult to control PRR with chemical methods since the special biologial characteristics of pathogen of Phtophthora sp. and complicated condition of soil. The biocontrol efficacy of disease suppression of PRR could not meet the requirement of agricultural production although there are a few reports about application of various of biocontrol agents that were used for disease control.In order to exploit new microorganism for disease control of crops and obtain natural and microbial-soured chemicals against plant pathogens, we carried out some research works as following:Two hundred and five antagonistic microbes against P.capsici were selected from soil which grew chilli pepper continuously in past decades, composting which was made with different raw material and composting technique and plant of varieties of vegetables crops, among them there were32antagonistic fungi,78antagonistic actinomyces and95antagonistic bacteria. Strain A576,A329,B105,BS211,Tpc and-Pst10showed strongly and lasting antagonism against P.capsici on plates in vivtro and was cultured in liquid medium respectively.The antagonistic activity of SLCF of each culture was examined and the effects on disease controlof PRR in pot experiment were compared. The results showed PstlO was better than other microbes and could be a potential candidate for PRR control. PstlO was identified as Pencillium striatisporum, a rare species, by Agricultural Culture Collection of China (ACCC) and named as PstlO in this research.This is the first study that reported Penicillium striatisporum was isolated in China and applied as biocontrol agent for disease control of plants.PstlO showed very high antagonistic effects on mycelium growth of Phytophthora spp., Cladosporium cucumerium, and Sclerotinia sclerotiorum, and the inhibition zone was30mm and lasted more than30days in in vitro assays. The toxicity of sterilized liquid culture filtrates (SLCF) of Pst10grown in potato dextrose broth (PDB) was tested against Phytophthora capsici mycelium growth and sporangia/spore formation or germination. The SLCF completely inhibited mycelium growth and even at a100-fold dilution led to abnormal mycelium. The disease incidence of PRR was zero after6days transplantation in pot experiment with substrate treated with SLCF and still45%plants were healthy after13days transplantation. Composted pig manure slightly increased the colonization of Pst10in the chilli rhizosphere. In pot tests, the number of Pst10in chilli rhizosphere with treatment of Pst10and organic fertilizer was three folds of that in treatment of Pst10singly after10days of transplantation and the number increased to17folds after20days of transplantation. Strain PstlO showed strong ability of colonization and maintainment of dominatnt group since the number of Pst10in chilli pepper rhizosphere remained2.55×104cfu/g after40days. The lowest incidence of PRR was in the treatment consisting of SLCF, conidial suspension of Pst10and organic fertilizer with10%plants were infected by P.capsici after14days of transplantation, while the disease incidence of control was100%.There were obvious difference about growth and secretes of mycelium and conidia production of Pst10when cultured on5different solid medium respectively, and OAT was optimum medium for conidia production. Also, the diffence of biomass of mycelium, sphere appearance of mycelium and antibiotic activity of SLCF was significant when Pst10was cultired in9kinds of different liquid media. PEDB was optimum medium when considering biomass of mycelium and bioactivity of SLCF. Pst10grew well in9types of media. The SLCF of Pst10cultured in Vogel or Jackson medium had no antagonistic activity against P.capsici although Pst10displayed favourable growth in this two media. There was no corelation between mycelium growth and antibiotics production. The studies on culture conditon for antibiotics production showed that:inoculum had no impact on antibiotics prodcution, The antibiotics could be produced by Pst10at20-37℃,and28℃was the best temperature.Gas flux could influence production of antibiotics of Pst10,butthe difference was not significant when the flask of250mL was filled with20-100mL medium.The antibiotics was produced in a large amount by Pst10after4days incubation and the antagonistic activity was highest on the8th days of incubation and this activity lasted for7-8days.The methods for activity examination of SLCF were studied. The antagonistic activity of SLCF prepared by centrifugation with high speed was higher than that prepared by filtration through0.22μm Millipore membrane when the inoculum of Pst10was1-4disks in100mL medium contained in250mL flask, and the following experiment showed part of chemicals were adhered to Millipore membrane But the activity disparity between the two methods reduced with inoculum increasement. This result maybe suggest that the total activity of SLCF was not impacted by inoculum, while the varities of chemicals produced by Pst10were influenced by inoculum,that means Pst10produced much more antagonistic chemicals which were adhered to Millipore membrane with small inoculum,and more chemicals which were easily passed through Millipore membrane were produced by Pst10when with a large amount of inoculum. So, varieties of antifungal chemicals were produed by Pst10. The method of direct dropping of SLCF was the most sensitive among three methods of plate-hole-inhibiton zone, plate containing antibiotics-growth inhibition of mycelium and dropping SLCF directly. We also find antibiotic activity was affected by medium composition.Compared with plate method used for activity examination, the TLC method suggested more information about antibiotics since chemicals could be seperated primarily at the same time of activity examination. We could know how many types of chemicals and the polarity of antibiotic chemicals exsisted in SLCF by numbers, size and position of inhibition zone on TLC plate.Liquid cultures of Pst10grown in PEDB were filtered through two-layers of cheesecloth in a funnel and centrifuged,and the supernatants were collected and concentrated at45℃on a rotary evaporator to a final volume with1%of SLCF. The concentrated supernatants were extracted with ethyl acetate and consequently the antagonistic fraction was seperated by TLC. Under254UV,6bands were observed and the chemicals contained in each band were recovered.The bioassay results of chemicals recovered from each band showed the5th band contained antagonistic chemicals. The chemicals coming from the5th band was seperated by Silica column and elute was collected in tube. The elutes in the5-8th tubes had antagonism against P.capsici. The elutes in the four tubes was combined and concentrated and then fractioned by HPLC. Four fractions with antibiotic activity were obtained. Two purified fractions, named fractionl and fraction5, were collected in a large amount when HPLC condition was changed. The two fractions were analysized by1HNMR,13CNMR, dimension NMR, UV spectrum and finally were identifed as Calbistrin A and Calbistrin B respectively,which were isomer with formula of C31H40O8and molecular weight of540.26. Calbistrin A and Calbistrin B had activity against Bacillus sp.,but had no activity against Pseudomonas sp. and Actinomyces sp.tested. The activity of Calbistrin B is higher than that of Calbistrin A. The MIC of Calbistrin A and Calbistrin B was7.5μg/mL and1.25μg/mL seperately. Calbistrin B had no toxicity to seed germination and root growth when the concentration applied was lower than10μg/mL which was8folds of MIC to P.capsici.Forty-one transformants with Hygromycin B resistance marker were obtained with Agrobacterium tumefaciens-mediated transformation (ATMT), and the transformation rate was0.82%. There was no difference between wild type and all the transformants of Pst10about growth and antagonism against P. capsici. Twelve transformants with GFP-Geneticin resistance dual marker were obtained with ATMT with0.24%of transformation rate. Fourteen transformants with YFP-Geneticin resistance dual marker were obtained with ATMT with0.28%of transformation rate. There was no obvious difference about growth and antagonism against P. capsici between wild types and several transformants with GFP/YFP-Gen dual markers and the green or yellow fluorescent protein was expressed normally, which resulted in a molecular marker for patent protection of Pst10.