| The endophytic fungi are a huge potential to exploit novel and highly active secondary metabolites.So it is necessary to carry out the systematic investigation of endophytic fungi.Chinese dragon’s blood and agarwood can be formed by plant under machine injure,fungi infection,insect bite,etc.Chinese dragon’s blood and agarwood are generated by Dracaena cambodiana and Aquilaria sinensis,respectively,during a long time.But yield is low in natural conditions.With increasing consumption of Chinese dragon’s blood and agarwood in recent years,over-exploitation and over-cutting of original plants in China caused depletion of the natural resources.The objectives of this study were to isolate,identify and screen antimicrobial activity of endophytic fungi from D.cambodiana,A.sinensis and Dalbergia odorifera.The effective strains,which could induce D.cambodiana to produce Chinese dragon’s blood and A.sinensis to produce agarwood,were study in this paper.Endophytic fungi were isolated from 450 surface-sterilized tissues of the root,stem and leaf of three plants.Of the 581 isolates recovered,172 were from Dracaena cambodiana,128 from Aquilaria sinensis,and 281 from Dalbergia odorifera.According to morphological characteristics,581 isolates were complicated to 336,and were identified and belonged to 46 taxa in 32 genera and 8 morphological groups.Colonization rates and isolation rates of endophytic fungi were statistical analysis.There was the highest colonization rate(80.0%) in Dracaena cambodiana.The isolation rates were 0.85 to 1.87.At tissue level,the infection degree of the different organs of different plants was also different.The distribution of endophytic fungi in various plants in this study was not uniform and there was a large degree of difference in the quantity of endophytic fungi.Endophytic fungi of Dracaena cambodiana were belonged to 41 taxa in 23 genera.Fusarium sp.1(6.7%) was the most dominant endophytes in Dracaena cambodiana.Endophytic fungi of A.sinensis were belonged to 28 taxa in 17 genera.Fusarium sp.4(7.1%),Cladosporium edgeworthrae(5.7%),Fusarium sp.1(5.7%) and Glomerularia sp.(5.7%) were most commonly isolated from A. sinensis.Endophytic fungi of Dalbergia odorifera were belonged to 20 taxa in 12 genera.Alternaria alternate(8.3%) was the most dominant endophytes in Dalbergia odorifera and was mostly isolated from the leaf.A total of 336 fungal isolates were used to screen the antimicrobial bioactivities to 6 clinical microbial pathogens according to agar block method.29 strains(21.64%) from Dracaena cambodiana,4 strains(7.14%) from A. sinensis and 3 strains from Dalbergia odorifera could inhibit some of tested human pathogenic bacteria and fungi.The effective isolates were from the stem of Dracaena cambodiana.All effective isolates failed to show antimicobial activity to Ec.The inhibition spectrum was narrow.The effective strains mostly inhibited Bacillus subtilis and Aspergillus fumigatus.Endophytic fungi had the same antimicrobial activity for fungi pathogen as for bacteria pathogen.The inhibition zones ranged from 7 mm to 27 mm.7 isolates exhibited strong antimicrobial activity with more than 20 mm inhibition zone diameter,and mostly inhibited Aspergillus fumigatus.4 isolates exhibited broad-spectrum antimicrobial activity.The effective isolates were identified to 10 taxa.Most effective fungi distributed in the genera of Calcarisporium,Fusarium,Epicoccum,Aspergillus, Penicillium,Trichoderma,mitosporic Eremomycetaceae and unknown fungi.In this study,the antimicrobial spectrum of endophyte had certain differences between different genera and strains,furthermore,the activity of different strains from the same genus or species also had differences,for example,Fusarium isolates.In co-cultivation system of endophytic fungi and seedling of Dracaena cochinchinensis,we failed to find effective fungi with promoting growth and secretion of red substance.Endophytic fungi were inoculated in Dracaena cambodiana and the dead stem of D.cambodiana.No strains could promote the dead stem of D. cambodiana to produce red resin.10 strains could promote D.cambodiana to secrete red resin.Most effective fungi were belonged to the genera of Fusarium, Ovulariopsis,Cephalosporium,Acrostalagmus and unknown fungi.RP-HPLC(DAD) method was used to test 5 samples.The contents of 8 components were higher in the treatments of DC-1-12 and DC-1-22 than in natural Chinese dragon’s blood.The treatment DC-1-22 was particularly prominent.In our experiment,red resin could be observed around fungal inoculation site after 3-5 months later.It indicated that fungi could promote D. cambodiana to produce dragon’s blood and augment the output of effective components of dragon’s blood.Endophytic fungi were isolated again from the xylems with red resin in treatment DC-1-12 and DC-1-22.According to morphological characteristics and molecular characteristics,the new isolate and strain DC-1-22 were the same strain.The xylem with red resin inoculated by DC-1-22 was detected by the microscopic observation.The hyphae penetrated into the plant body from inoculation site.The hyphae grew and spread in intercellular space,and colonized in secondary vascular.Fungi initiated plant’s defense response.Plant produced a red secretion with defensive effect.In the initial stage,red secretion formed in the internal of vascular,then in the external vascular,then in ground tissue.The vascular was damaged at later stage.Endophytic fungi were inoculated in Aquilaria sinensis.7 strains could promote A.sinensis to secrete brown resin.Most effective fungi distributed in the genera of Fusarium,Cephalosporium,Penicillium and unknown fungi.GC-MS method was used to test the volatile oil of sample AS-1-11 and AS-1-17.The contents of some components were higher in the treatments of AS-1-11 and AS-1-17 than in agarwood.In our experiment,brown resin could be observed around fungal inoculation site after 4-6.It indicated that fungi could promote A.sinensis to produce agarwood.And A.sinensis could produced agarwood under the machine injure.Endophytic fungi were isolated again from the xylems with brown resin of treatment AS-1-11 and AS-1-17.According to morphological characteristics and molecular characteristics,the new isolates and strain AS-1-11 and AS-1-17 were the same strains. The xylem with brown resin of treatment AS-1-17 was detected by the microscopic observation.The hyphae penetrated into plant body from inoculation site.The hyphae grew and spread in intercellular space,and colonized in wood ray and vessel.Plant produced a brown secretion with defensive effect.It was the product of plant’s defense response. |
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