Isolation Of Endophytic Fungi From Juglans Regia And Castanea Mollissima, Screening Of Potential Biocontrol Fungi Strains And Their Biological Characteristics

In this paper,64endophytic fungi were isolated from the roots, stems, leaves and fruitsof healthy Juglans regia and Castanea mollissima,36isolated from J. regia and28from C.mollissima. Firstly, the isolation mehtod and culture condition of endophytic fungi wereexplored. Finally, the plant materials were sterilized by soaking in alcohol for30s, inmercuric chloride for2-3min and in sterilized water for2-3times. PDA mediumsupplemented with streptomycin was found to be the most suitable medium for the growth ofendophytic fungi. Results showed that9fungal endophytes which average inhibition ratewere more than50%against mycelia growth of Valsa sordida, Botrytis cinerea,Cryphonectria parasitica, Rhizoctonia solani, or Colletotrichum gloeosporioides, werescreened from the64isolated endophytic fungius in dual culture, and3of9which inhibitionrate were more than50%against mycelia growth of all five pathogens. Volatile substancesfrom the9endophytes were tested on the inhibitory effect on mycelia growth of C. parasiticaand C. gloeosporioides, and the test results show that the volatile substances of8endophytescould inhibit the mycelia growth of C. gloeosporioides and C. parasitica, while one couldpromote mycelia growth of the two pathogenic fungius. Fermentation broth of the9endophytes could inhibit mycelia growth and conidia germination of C. gloeosporioides andC. parasitica. What’s more, in the greenhouse and field, the fermentation broth couldsignificantly promote the growth of J. regia seedlings in the greenhouse and effectivelyreduce the occurrence of walnut anthracnose in field condition. The crude extracts wereobtained from fermentation broth with ethyl acetate and rotary evaporated shows a strongerinhibitory effect on mycelia growth of C. gloeosporioides and C. parasitica.In this paper, parts of the endophytic fungi were identificated by morphological andmolecular biology methods.Biological characteristics of the3endophytes HL3R, HSE7and BR3, were tested,which showed the strongest inhibition to the mycelia growth of all the five pathogens. Theresults showed that3fungi grown best on PDA medium compared with other medium; theycould grow in the temperature range from15°C to35°C and grow well in the temperaturefrom20°C to30°C, and25°C was the optimum temperature; sucrose could significantlypromot the mycelia growth of the three kinds of endophytic fungi, and on lactose carbonsource and nitrogen sources of beef extract BR3grow well, HSE7on medium containinginorganic nitrogen (NH4)2SO4and organic nitrogen alanine, and HL3R on mediumcontaining either inorganic nitrogen (NH4)2SO4or NH4NO3;3fungi could grow in a wide range of pH from6to11and the optum pH condition was neutral to alkaline environment.Photoperiod had no significant effect on the3endophytic fungi’s mycelia growth; Vitaminsdid not promote the3fungus’ mycelia growth, but niacin and VB2in the contrary, inhibitedthe growth of BR3; the trace elements could not obviously promote their growth, but to HL3Rthe promotion was a little, not achieve significant level.