金针菇褐化病毒(FvBV)脱毒方法

【目的】评价5种不同脱毒方法对金针菇(Flammulina velutipes)菌株的脱毒效果，筛选出脱毒率高和脱毒后金针菇菌株菌丝生长速度、生物量、漆酶活力等性状改善明显的脱毒方法。【方法】以栽培金针菇菌株F-4889为研究材料，从菌丝体中提取大小约2.0 kb的病毒dsRNA，经RT-PCR鉴定该病毒为金针菇褐化病毒(FvBV)。采用菌丝尖端分离、原基组织分离、原生质体单核化、有性生殖和核迁移5种脱毒方法对金针菇菌株进行脱毒处理，利用dsRNA技术和RT-PCR检测脱毒效果。【结果】菌丝尖端分离脱毒后得到1株脱毒菌株；原基组织分离法未能脱毒；原生质体单核化脱毒法得到3株脱毒单核菌株和2株原单杂交脱毒菌株；有性生殖脱毒法获得脱毒孢子单核菌株23株和单孢杂交脱毒菌株8株；核迁移脱毒后得到5株核迁移脱毒菌株。脱毒率依次为25.0%、0、7.5%、57.5%和100%。脱毒菌株的菌丝生长速度、生物量、漆酶活力等均优于出发菌株、菌丝尖端和原基组织分离菌株。【结论】这5种方法中原生质体单核化、有性生殖和核迁移脱毒法脱毒效果较佳，均能有效脱除FvBV，脱毒率高，脱毒后菌株菌丝生长速度、生物量、漆酶活力等均明显提高。

Elimination of Flammulina velutipes browning virus

Objective] To eliminate virus contamination in Flammulina velutipes, we assessed five detoxification methods. Methods] F. velutipes F4889 was used to isolate 2.0 kb dsRNA, and the virus was identified as F. velutipes browning virus (FvBV) by RT-PCR. Five virus-eliminating methods, i.e. hyphal tips isolation virus elimination (HTIVE), primordium tissue isolation virus elimination (PTIVE), protoplast monokaryotization virus elimination (PMVE), sexual reproduction virus elimination (SRVE) and nuclei migration virus elimination (NMVE) were applied to prepare virus-free strains. The effect of virus-elimination was detected and identified through dsRNA technique and RT-PCR. Results] Only one virus-free strain was obtained by HTIVE method. Strains of PTIVE method remained the virus. PMVE method achieved three monokaryon strains and two protoplast monokaryon hybridization virus-free strains. Twenty-three sporulated monocaryotic strains and 8 single spore hybridization strains were virus-free after using SRVE method. NMVE method acquired 5 virus-free strains. Hyphal growth rate, biomass and laccase activity of virus-free strains by PMVE, SRVE and NMVE methods were obviously superior to the original strain and strains by HTIVE and PTIVE methods. Conclusion] The detoxification effect of PMVE, SRVE and NMVE methods was apparently better than others. These three methods completely eliminated FvBV and had higher virus-free rate. Moreover, mycelia growth rate, biomass and laccase activity of virus-free strains significantly improved.