Screening And Cultivation Of The Endophytic Fungi Of Pinus Massoniana And Its Application In Producing Lipid

Endophytic fungi generally refer to the fungus which complete their part or whole lifecycle within plants,without causing obvious disease symptoms.They distribute in subcutaneous tissue of root,stem,leaf and other organs of almost all plants.Endophytic fungi live within the special environment of the plant tissue,and endophytes and the plant benefit each other.Previous studies have documented biological effects and secondary metabolites of the endophytic fungi.Evidences show that endophytic fungi can promote the growth of host,enhance the resistance of the host plant to diseases,pests,biotic and nonbiotic stress,and produce various bioactive metabolites.The majority of isolated endophytic fungi can produce the same or similar active ingredients as which exist in the plant host,with the long-term symbiosis and interaction between endophytes and the plant.Pinus massoniana is an important timber species,and also is a pioneer species for afforestation in barren mountainous areas.They are widely distributed.Pinus massoniana is rich in resin.It is the main species for resin production in China.Up to now,no investigation was performed on the application of endophytic fungi isolated from Pinus massoniana in producing lipid.In this study,endophytic fungi from Pinus massoniana were isolated and identified,and the diversity was analyzed.Resin microbial tapping research was performed by regression analysis of the Pinus massoniana endophytic fungi influence against the resin yield.Endophytic fungi were isolated from Pinus massoniana for producing resin and identified using molecular techniques.This study aims to provide the technological basis for the comprehensive exploitation and utilization of Pinus massoniana,and enhancing resin productivity.The main contents and results are as follows:1)One hundred and two endophytic fungi strains were isolated from the roots,stems and leaves of Pinus massoniana from Wuyi state-owned forest farm of Zhangping,Fujian,with surface sterilization.They belonged to eleven genera,six families,six orders,three classes,three phyla,by morphological identification,which showed that species of endophytic fungi of Pinus massoniana had broad biodiversity.Research showed that the quantity and species of the endophytic fungi had great difference among different organs of Pinus massoniana,with leaves ranking the first,followed by stems and roots.Mucor,Trichoderma,Penicilliu and Aspergillus were isolated from the roots,stems and leaves,the others were isolated from two of the three organs.Aspergillus,Phomopsis,Phoma and Penicillium were the dominant species.The other species,including Fusarium,Paecilomyces,Bionectria,Phyllosticta and Monilia,were not the dominant species.2)Resin microbial tapping was investigated with endophytic fungi isolated from Pinus massoniana for the first time.Ten endophytic fungi isolated from the roots,stems and leaves of Pinus massoniana were selected for the test in Zhuoyang village,Xiaochi town,Longyan city,Fujian province.The resin Microbial tapping was carried out by brushing the fermentation broths of the ten endophytic fungi successively and then mixing them on the slotting instead of chemical stimulant.The results showed that the average single-tree and single-bark resin yield of the Pinus massoniana brushed with the fermentation broths of endophytic fungi were more than that of the Pinus massoniana without brushing.The increasing rate(40.83%)of the resin yield of the Pinus massoniana brushed with the mixed fermentation broths of endophytic fungi was the highest.Strains ZI41155,BS4787 and FH0131,which formed the top three increasing rate of the resin yield of the Pinus massoniana,were selected from the ten endophytic fungi to investigated the influencing factors on resin yield.For this analysis,the method of three factors and three levels orthogonal test was employed,with species,DBH,and infection times as the three factors.The results showed that the effects of the three factors on the resin yield were significantly different.The effect of DBH factor was the greatest,followed by species and infection times.Effect of species on the increasing rate of the resin yield of the Pinus massoniana was significant at ?=0.01,while the effect of infection times was not significant at ?=0.05.Strains ZI41155 formed the highest average increasing rate of the resin yield,followed by strains BS4787 and strains FH0131.The average increasing rate of the resin yield increased with the DBH and infection times of the three classes,respectively.3)Strains ZI41155 was determined as the optimal level in the orthogonal test for resin microbial tapping.The relationship between the increasing rate of resin yield and DBH was investigated,using DBH as the independent variable,and increasing rate of resin yield as the dependent variable.100 Pinus massoniana individuals with DBH>18 cm were ramdomly selected for resin microbial tapping test by brushing the fermentation broths of the strains ZI41155 on the slotting.According to the modeling principle and least square method,regression analysis of the DBH against resin yield was performed with SPSS software,and the regression equation was obtained as Y=-28.608+2.76X.Analysis of variance and significance test of coefficient of regression showed that the regression equation was significant and effective.4)The production of lipid by endophytic fungi isolated from Pinus massoniana was investigated in this study for the first time.The strains ZI41155,BS4787,FH0131 and BS18200,which formed the top four increasing rate of the resin yield of the Pinus massoniana,were selected from the ten endophytic fungi of section two for test.The hyphae of the endophytic fungi were stained with Sudan black B and the lipid bodies could be clearly seen in the cells of the four isolates by optical microscope.Lipids of the four strains were extracted by acid-heating method to determine the lipid content and analyze the lipid components by GC-MS.The results showed that all of the four endophytic fungi could produce lipid.The average lipid contents were up to 20%,and the highest content was 30.56%.The fatty acid contents of the four endophytic fungi accounted for 64%of the lipids,and the unsaturated fatty acid contents were over 46%.The main components of the lipid were palmitate,stearic acid,oleic acid,linoleic acid and elaidic acid etc.Oleic acid accounted for the highest proportion,which was up to 42.08%5)Strains ZI41155,BS4787,FH0131 and BS18200 were identified using molecular techniques.Genomic DNA extraction,PCR amplification and sequencing were done for the four strains.BLAST analysis was carried out by comparing the sequence in the GenBank database.The genetic relationship and phylogeny were determined according to multiple sequence alignment with ClustalX 2.1 software and the phylogeny trees was constructed using Neighbor-Joining method with MEGA 5.0 software.Based on the colony shape feature,strain ZI41155 was preliminarily identified as Paecilomyces,strain BS4787 as Bionectria,and strains FH0131 and BS18200 as Fusarium.