Identification Of Alfalfa(Medicago Sativa L.) Fusarium Root Rot-Resistant Cultivars And Proteomic Analysis Of Alfalfa Response To Fusarium Proliferatum

Alfalfa(Medicago sativa L.)root rot caused by Fusarium species is a potential and chronic devastating disease in alfalfa-growing areas worldwide,have occurred in most of the alfalfa production field.Root rot injury is characterized by blackish-brown lesions appearing on root surfaces,usually expanding from where a lateral root emerges from a taproot or a main root;The number of new shoot was decreased in severely affected crowns;Nitrogen fixation declined;Alfalfa stands are capable of surviving for 10 years or more,but because of this disease,the majority of the fields show a progressive deterioration after the second year;Yield and quality also effected greatly.In China,reported pathogens for alfalfa root rot are mainly F.solani,F.oxysporum and F.acuminatum.In order to check if there are new Fusarium pathogens caused alfalfa root rot in China or in the world and explore the mechanism of alafalfa response to Fusarium spp.,we collected root rot disease samples of alfalfa from Langfang of Heibei,Linhe of Inner Mongolia and Yanggao of Shanxi in 2012-2013,to determine the dominant pathogens of alfalfa in these places.Pathogens were isolated from the samples and purified in PSA and MGA plate.Representative strains were selected based on biological characteristics,and then ITS and TEF-1? sequences of the fungi were amplified and sequenced.Morphological and molecular biological methods were used for identification of the representative fungi,and then determined pathogenicity by back infestation test.Then,according to the results of experiment 1,L1 strain was a strong pathogen causing alfalfa root rot.L1 strain used to evaluate 14 alfalfa cultivars by improved hydroponics screening and soil culture test.Moreover,cell morphology and infection mechanism were observed by transmission electron microscope(TEM).Last,for investigate the response mechanism of alfalfa after inoculated Fusarium sp.,we used a proteomic approach to identify inducible proteins in alfalfa during a compatible interaction with F.proliferatum strain L1.Samples used for the two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry were from roots and leaves of alfalfa cultivar AmeriGraze 401+Z and WL656 HQ plants grown under a hydroponic condition and at 4 days post inoculation with L1.Analysis of differentially expressed proteins found in the 2-DE gels followed by protein characterizations using the MASCOT MS/MS ion search software and associated databases identified multiple proteins that might involve in resistance to F.proliferatum infection.The main results were listed as follows:We isolate 6 representative strains,named C1,C2,C3,C4,C5 and L1.The results show that: C1 and C2 were identified to be F.tricinctum,C3,C4 and C5 were F.solani,L1 was F.proliferatum.The result of inoculation showed that L1(DI 80.88%,DSI 61.35%)and C4(DI 82.22%,DSI 59.6%)strains had higher pathogenicity than others,and C2 showed weakest pathogenicity(DI 54.44%,DSI 29.63%).There were no significant difference at disease incidence and disease severity index in C1,C3 and C5 strains,the disease incidence were 66.66%,62.22% and 65.55% respectively;Disease severity index were 42.59%,40.37% and 38.89%,respectively.For the important,this was the first reported that F.tricinctum and F.proliferatum can cause root rot on alfalfa in China.Inoculation method with traditional method of soil culture method and improved water inoculation method,there was no one immune variety among 14 alfalfa varieties.For improved hydroponics screening,disease development was fast,5-7 days after inoculation,plant showed symptoms.14 days after inoculation,the vaccinated root show severely necrosis,the above part of plant turned to yellow.Disease severity index between 41.5% and 63.7%,AmeriGraze 401+ Z and Affinity showed strong disease resistance and the disease severity index were 41.5% and 43.0% respectively.Followed were Beaver(44.4%),Zhongmu.No2(47.4%),Affinity(50.4%)and Alfking(50.4%).Zhaodong(56.7%)and WL656HQ(63.7%)were more sensitive than others.For the soil culture test,the early stage after inoculation,the disease symptoms develop slowly.After 80 days inoculation,evaluated the disease severity index,disease incidence and growth index,inoculated plants showed roots necrotic.Above-ground,symptoms of the disease are manifested in the form of chlorosis of leaves and lower plants.Disease severity index between 38.9% and 60.0%,AmeriGraze 401+Z and Affinity showed strong disease resistance,and the disease severity index were 38.9% and 40.4% respectively.Based on the comprehensive evaluation of the two methods,we found that AmeriGraze 401+Z was the most strongest disease resistance veriety and WL656 HQ was the most susceptible.A total of 66 and 27 differentially expressed proteins were found in the roots and leaves of the plants inoculated with L1,respectively.These proteins included s-adenosine methionine synthetase,glutathione transferase and HSP70,etc.The proteins were invoved in defense and stress response,metabolism,photosynthesis and protein synthesis.Thirteen identified proteins were validated for their expressions by quantitative reverse transcription polymerase chain reaction(qRT-PCR).Our results suggested that some of the identified proteins might play important roles in alfalfa resistance against Fusarium spp.This finding should facilitate further dissections of molecular mechanisms controlling alfalfa root rot disease resistance and potentially for other legume crops.