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为了将生防链霉菌ML27和柑橘内生放线菌P3Y2的优良特性整合在一起,对两株菌进行了原生质体融合研究。结果表明,菌株ML27和P3Y2的种子液分别转移到蔗糖浓度为20%和10%以及甘氨酸浓度为1.5%和1.0%的培养液中培养,收获的菌丝体对原生质体形成和再生最有利;分别选用5 mg/m L、90 min,及5 mg/m L、120 min的溶菌酶浓度与酶解时间制备ML27与P3Y2的原生质体能达到较高的形成率和再生率;在60℃条件下,菌株ML27和P3Y2的原生质体热灭活时间分别以10 min和15 min适宜;在20 W紫外灯下15 cm处分别照射7 min和5 min时,菌株ML27和P3Y2原生质体的灭活程度适宜。在此基础上,进行菌株ML27和P3Y2的原生质体融合,根据形态学特征、抑菌活性检测和定殖能力测定初步筛选出1株融合菌株MP45。试验结果可为这两株柑橘病害生防菌株的改良和充分利用奠定基础。
In order to integrate the excellent properties of Streptomyces hygrophila ML27 and citrus endophytic actinomycetes P3Y2, protoplast fusion was performed on the two strains. The results showed that the mycelia of strains ML27 and P3Y2 were cultured in medium with sucrose concentrations of 20% and 10% and glycine concentrations of 1.5% and 1.0%, respectively. The harvested mycelia were most favorable for protoplast formation and regeneration. Protoplasts of ML27 and P3Y2 were prepared by using 5 mg / mL, 90 min, 5 mg / mL and 120 min lysozyme concentration and enzymolysis time, respectively. High rates of formation and regeneration were achieved. Under 60 ℃ , The thermal inactivation time of protoplasts in strains ML27 and P3Y2 was suitable for 10 min and 15 min, respectively. Protoplasts inoculums ML27 and P3Y2 were inactivated at 7 min and 5 min at 15 cm under 20 W UV lamp respectively . On this basis, protoplast fusion of strains ML27 and P3Y2 was carried out. According to morphological characteristics, antibacterial activity detection and colonization ability test, a strain of fusion MP45 was screened out. The test results can lay foundation for the improvement and full utilization of biocontrol strains of two citrus diseases.