Identification Of Stem Base Rot Pathogens Of Zingiber Officinale Roscoe In Sichuan And Chongqing And Virus Elimination ＆ Micro-propagation Via In Vitro Ginger Nodal Segments

The purpose of this study is to isolate and identify the pathogen of ginger stem-based rot in Sichuan and Chongqing,and to establish a new type of seedling degerming and virus-free technology based on adventitious buds induced by in vitro ginger nodal segment and a micro-propagation technology of virus-free seedlings with high efficiency and wide adaptability.The results are as follows:（1）Isolation and identification of root rot pathogen of ginger in Sichuan＆ChongqingFungi Z5 isolated from the stem base of diseased ginger and fungi S8 isolated from the rhizosphere soil of diseased ginger were pathogenic to healthy ginger.The results of BLAST online comparison analysis showed that the r DNA-ITS sequences of fungi S8 and Z5 are 90%＆80%similar to those of Fusarium oxysporum and Gongronella butleri,respectively.According to the morphological characteristics of the pathogen,strain S8 was identified as Fusarium oxysporum.The strain Z5 is highly similar to Gongronella butleri and needs further analysis and identification.（2）’Qianwei Maliujiang’secondary shoot tip detoxification and rejuvenationThe adventitious buds were induced from the nodal segment of’Qianwei Maliujiang’in vitro plantlets.Histological study showed that the adventitious buds directly occurred in the intermediate meristem of ginger nodal segment.Through secondary shoot tip culture,the regenerated plants did not carry the pathogen of ginger stalk rot,and 100%TMV（Tobacco mosaic virus）was removed.Combined with rejuvenation and transplanting,the virus removal technology of’Qianwei Maliujiang’seedlings was successfully established.The protocol is as follows:0.2～0.3 cm nodal segment were cut and placed on MS+0.5 mg·L^-1NAA+1.0 mg·L^-1 TDZ medium for 12 days,the shoot tips（0.8～1.2 mm）were stripped on MS medium for 15 days,the second shoot tips were stripped（0.8～1.2 mm）,and the shoot tips were rejuvenated on MS+0.25 mol L^-1 sucrose medium for 60 days.（3）Micropropagation technology system of virus-free in vitro ginger seedlingsVirus-frees In vitro’Qianwei Maliujiang’was used as material to establish the seedling rapid propagation protocol with nodal segment induction of adventitious buds as the core.The results showed that when the length of nodal segment was 2～3 mm,the bud induction rate was high,and the average bud induction rate was 83.4%.There was no significant difference in the number of adventitious buds from single nodal segment.The optimal medium was MS+3.0 mg·L^-1 6-BA+0.2 mg·L^-1 NAA,and the proliferation coefficient reached 6.3.It was found that the genetic stability of ginger plants treated by this process was relatively stable.The transplant matrix containing peat and vermiculite with the volume ratio of 1:1 gave best result in survival rate of virus-free plantlets（100%）.We tested the genetic stability of regenerated in vitro ginger plants after transplanting by RAPD-PCR and ISSR-PCR,founding that the genetic stability of regenerated ginger plants treated by this process was relatively stable.We used four Z.officinale Roscoe varieties and one Curcuma variety to verify the broad spectrum of the protocol.This study successfully isolated and identified the pathogen of ginger stalk rot in Sichuan＆Chongqing,which can provide a theoretical basis for targeted prevention and control of ginger stalk rot in Sichuan＆Chongqing.The efficient and broad-spectrum virus-free micropropagation technology system of ginger seedlings is successfully established with adventitious buds induced by nodal segment as the core,which provided a new protocol for the breeding of ginger varieties.