绿豆尖镰孢枯萎病抗性鉴定方法

The Method for Evaluation of Mung Bean Resistance to Fusarium wilt

绿豆是我国的主要食用豆类之一。由尖镰孢引起的绿豆枯萎病是一种严重的土传病害,病原菌从根部侵入,引起植株矮化,叶片黄化、枯萎,根茎部维管束变褐,严重时导致植株死亡。防治枯萎病最经济、有效的方法是培育利用抗病品种。本研究在控制条件下以具有不同抗性表型绿豆品种为材料,分别对接种方法、植株生育期、接种体浓度、接种体处理时间及接种后植株培养温度等影响绿豆抗性表型的因素进行比较研究,以期建立一个快速、准确和高效的绿豆枯萎病抗性鉴定方法,为抗病资源的筛选和抗病育种提供技术支持。结果表明,绿豆枯萎病苗期抗性鉴定最适宜的接种方法为剪根浸根法,最适宜接种体浓度为105~106孢子/m L,接种最佳植株生育期为2叶期,最短有效接种体浸根时间为2 min,最适宜发病温度为25℃,接种后14 d调查病情。

Mung bean [Vigna radiata（ L.） R. Wilczek] is one of the most important grain legumes in China,which is often incorporated into various multiple crops and intercropping systems because it is a short-duration legume crop and can improve soil fertility by rhizosphere atmospheric nitrogen-fixing. Fusarium wilt,caused by the fungal pathogen F. oxysporum,is a severe soil-borne disease and seriously limits the yield of mung bean in China. F. oxysporum can infect mung bean at all phenological growth stages. This pathogen firstly infects roots of plants and cause symptoms including leaf chlorosis and necrosis,vascular discoloration of roots and stems. Lastly,the seriously infected plants of mung bean wilt and died. Management of Fusarium wilt is very difficultly due to its soil-borne pathogen and long-term survival in soil. The reproductive and vegetative structures produced by F. oxysporum include microconidia,macroconidia,and chlamydospores. The interaction of F. oxysporum with nematodes adds additional constraints to the management of this disease. Deployment of resistant cultivars is the most economic and effective way to control this disease. In this study,the mung bean cultivars with contrasting resistant phenotype were used as plant materials（ Yulv 2 and Bao 200612-6-1 are resistance cultivars,Jilv 7 and Bailv 6 are susceptible cultivars）,and the factors affecting resistance phenotype including inoculation method（ root cutting inoculation,root dipping inoculation and radicel socking inoculation）,plant development stage（ 2 leaf age,3 leaf age and 4leaf age）,inoculum concentration（ 10^4,10^5,10^6 and 10^7spore/m L）,inoculum treatment time（ 30 s,1 min,2 min,4 min and 6 min） and inoculated plants incubation temperature（ 20 ℃,25 ℃,30 ℃,35 ℃）,were comparatively analyzed. In order to reduce the error,wilt disease severity of mung bean was assessed u-sing a scale of 0-4. A disease index（ DI） was recorded on each plant based on the severity of the leaf disease lesions and differences of replications were analyzed using standard error（ SD） and subjected to one-way ANOVA analyses,and means of different treatments were compared by Duncan＇s new multiple range method. The resistance phenotypic results using different inoculation method showed that significant differences were observed among three inoculation methods,but only root cutting inoculation gave repeatable,accurate and consistent results. Significant differences were also observed between the plant development stages. We obtained accurate and consistent results by inoculating the plants at 2 leaf age,which can shorten inoculation period and accelerate identification process. There were no significant differences between the results by inoculation concentration of 10^5 and 10^6,however,the significant difference were observed between 10^5~ 10^6 and 10^4,10^5~ 10^6 and 10^7,respectively. So,105 and 10^6spore/m L were optimum inoculation concentration,which was consistent with those of other diseases. When the inoculum treatment time was 2-4 min,there was no significant difference between the four cultivars,which could reflect the actual resistance level of four cultivars. As for incubation temperature,there was no significant difference between 25 ℃ and3 0 ℃,even the disease index of four mung bean cultivars at 25 ℃ was higher than at 30 ℃,and the results could reflect the actual resistance level of each cultivar. However,the significant difference existed between25-30 ℃ and both 20 ℃ and 35 ℃. As for surveyed period,the results had no significant difference between 14 d and 21 d although the disease index after inoculation 21 d was higher than those at 14 d. But significant difference was observed between 14-21 d and 7 d. Our results indicated that scoring results between 14-21 d after inoculation could accurately distinguish the resistance level among the tested materials.The optimal survey period should be at 14 days after inoculation under considering the experimental cycle and timeliness. In conclusion,the root cutting technique was the optimal inoculation method. The seedling of mung bean at 2 leaf age was suitable to inoculation with pruned roots dipped in spore suspension of 10^5-10^6spore/m L for 2 min. Inoculated plants should be incubated at 25 ℃. The survey time of disease scoring can effectively shorten experimental period at 14 days after inoculation. This method was rapid and effective for evaluation of mung bean resistance to Fusarium wilt in controlling condition,which would provide technical support for mung bean germplasm resistance screening and breeding program.