| Endophytic fungi are rich in species diversity and ubiquity.Eucalyptus is an important fast-growing economical tree cultured in tropical and subtropical,Cylindrocladium leaf blight(CLB)is one serious disease which damage Eucalyptus production and the control of CLB rely on chemical pesticides.In view of this,we isolated and identified,endophytic fungi from leaves of Eucalyptus,and evaluated the diversity of endophytic fungi.The endophyte strains were selected for inhibiting Calonectria pseudoreteaudii.The main results were summarized below:1.Healthy leaves samples were collected from Eucalyptus grown in Fuzhou,Nanping,Ningde,Fujian Zhangzhou and Longyan in Fujian province.A total of 87 endophyte strains which identified as 7 orders and 24species,were isolated form Eucalyptus leaves by tissue isolation method.Most of endophytic fungi belong to Ascomycetes,account for 66.67%.The number of endophytic fungi isoltated form Fuzhou,Nanping,Longyan,Zhangzhou and Ningde is 23,24,12,15 and 13 respectively.The Simpson diversity index of Eucalyptus leaves from the plantation of Fuzhou,Nanping,Longyan,Zhangzhou and Ningde is 0.866,0.808,0.894,0.838 and 0.423 respectively.The Simpson diversity index of all identified endophytic fungi is 0.916,which shows that there is a rich diversity of endophytic fungi in Eucalyptus.Colletotrichum gloeosporioides,Pseudoplagiostoma corymbiae and Pseudoplagiostoma eucalypti is a dominant species while the proportion of this 3 species is 13.8%,13.8%and 19.5%,and even has been isolated in Eucalyptus leaves from three areas.2.By firstly screening and secondary screening assay in the laboratory,one endophytic fungal strain named ND-13,cxhibited antifungal effect againgst C.pseudoreteaudii,and ND-13 was identified Trichoderma harzianum.We also obtain two endophytic bacterial strains named NP-1 and No.7,which can inhibit the growth of C.pseudoreteaudii.The NP-1 has the most significant repress effect on C.pseudoreteaudii.3.The NP-1 was identified by observation of morphological,cultural characters,as well as the determination of physiological and biochemical characteristic.The 16S rDNA sequencewas also to used for identifying NP-1.The results showed that NP-1 is belonged to Pseudomonas bacteria.4.We found that Pseudomonas sp.NP-1 has antagonism to 8 species of plant pathogenic fungi in plate confrontation assay,inhibition rates up to over 60%.The inhibition rate of Sclerotinia sclerotiorum is the highest,which can reach 86%,followed by Magnaporthe oryzae,Calonectria pseudoreteaudii,Nigrospora oryzae,Pestalotiopsis apiculatus,Fusarium graminearum,Fusarium proliferatum and Monilinia fructicola,inhibition rates was 84%,83%,79%,76%,68%,63%,respectively.The plate confrontation assay indicated that,after inoculated with Pseudomonas sp.NP-1 for 0,24,48 and 72,the inhibition rate against C.pseudoreteaudii was 75%,76%,78%and 83%respectively.However,by shaking cultured NP-1 in PDB,there was no suppression on C.pseudoreteaudii of the fermentation broth of NP-1.The phenomenon was different from the other antifungal substance of Pseudomonas,so further research need to be done on the antifungal metabolites of NP-1.Microscopic examination results of Pseudomonas sp.NP-1 against C.pseudoreteaudii cultivated in dual-culture showed that,the morphology of hyphal protoplasm of C.pseudoreteaudii changed clearly with protoplasm not full,the hypha was narrowed and out of shape,even pumped out many hyphae between the diaphragms.Through the microscopic examination of Pseudomonas sp.NP-1 and pathogenic fungi in plate confrontation assay,it shows that compared with control group,the morphology of hyphal protoplasm of the treatment group changed clearly,hyphal protoplasm was not full,the hyphae was narrowed and out of shape,even pumped out many hyphae among the diaphragms.The results of the research provide one potential biocontrol agent for CLB and laid a foundation for the utilization of Eucalyptus endophytic fungi to control C.pseudoreteaudii. |
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