Identification And Mechanism Of Apple Rootstock Superior Line 12-2 Against Apple Replant Disease

Apple replant disease(ARD)is a common occurrence in major apple-growing regions worldwide and limited the sustainable development of apple production significantly.The imbalance of microbial community structure is considered to be the main cause of ARD.Fusarium is one of the main pathogenic fungi causing ARD in China.Among the many methods for improving ARD,the development of improved ARD-resistant rootstocks is a long-term effective measure to prevent and control ARD.There have been many studies of resistant rootstocks in other countries and have been promoted in production.Nonetheless,the large-scale propagation of rootstocks resistant to ARD has not been achieved in China.And the high affinity of rootstocks is the basis for their application.Rootstocks susceptible to ARD such as T337 and M26 are still the main apple rootstocks used for production in China.While Malus hupehensis Rehd.(PYTC),with high grafting affinity,is a rootstock resistant to ARD,but due to its susceptible to salt and drought,it is not the main rootstock in apple production in China.Therefore,it is important to specifically select and use ARD resistant rootstocks in the main apple producing areas of China.Our research group selected a new elite apple rootstock line named 12-2 that is tolerant to ARD.On this basis,this study firstly planted the three rootstocks T337,M26 and 12-2 in replanted soil and sterilized soil respectively.By means of intraspecific comparison,the changes in intraspecific biomass differences for five consecutive months were detected,and a preliminary judgment was made on the ability of 12-2 to withstand ARD.And the effects of specialized ARD-associated Fusarium proliferatum f.sp.malus domestica MR5 on T337,M26 and 12-2 were carried,to further explore the tolerance of 12-2 against ARD harmful Fusarium.At the same time,in order to test whether12-2 has application value,the PYTC with high grafting compatibility was used as the control,and the grafting compatibility tests were carried out with 13 different apple varieties respectively.The previous research of the research group also showed that the lower to the ARD resistance,the more serious to the influence of ARD on the absorption of K⁺and Ca²⁺by rootstock roots.And the K⁺channel genes increase the stress resistance of plants by regulating the uptake of K⁺by rootstock roots.Therefore,this study took this as an entry point,the effects of ARD soil extracts on the net K⁺and Ca²⁺flux in different root zones of T337,M26 and 12-2,the functional analysis of the K⁺channel genes in the roots of 12-2,in order to disclose the ARD response mechanism of 12-2.Our work provides useful theoretical basis and test materials for the breeding of resistant apple rootstocks.The main results are as follows:1.The effects of ARD on the physiological indices of T337,M26 and 12-2:Compared with the sterilized controls,ARD significantly decreased the plant height and stem thickness,the relative chlorophyll contents,leaf antioxidant enzymes activities in some months of T337and M26.And significantly affected leaf photosynthetic and fluorescence parameters of T337and M26,and significantly increased the leaf MDA contents of T337 and M26 in some months.ARD significantly reduced some root growth indices and the dry and fresh weights of T337 and M26 compared with controls on sterilized soil.ARD also significantly reduced root metabolic activity,root antioxidant enzymes activities of T337 and M26,and increased root MDA content of T337 and M26.The above physiological indices were not affected by ARD in 12-2.Preliminary judgment that 12-2 had a certain tolerance to ARD.2.The effects of MR5 infection on the physiological indices of T337,M26 and 12-2:Infection test showed that after inoculation with MR5 spore,the incidence rate of T337,M26and 12-2 was 68.00%,100.00%and 24.00%,respectively.Compared with controls,the leaf lesion area of M26 was the largest,followed by T337,and the smallest in 12-2.MR5significantly reduced the leaf chlorophyll contents,partly leaf photosynthetic and fluorescence parameters of T337 and M26,and significantly reduced the leaf SOD activity of T337 and leaf antioxidant enzymes activities of M26.Compared with the respective controls,M26showed the most severe root damage,T337 showed moderate levels of root damage,and 12-2showed no significant root damage under MR5.MR5 had no significant effect on the root structure,root dry and fresh weight of T337,M26 and 12-2.MR5 significantly reduced the root metabolic activity of T337 and M26.It also reduced their root antioxidant enzymes activities(except CAT for T337)and significantly increased the leaf and root ROS levels,MDA and permeable substance contents of T337 and M26.MR5 had no such effects on 12-2.Therefore,12-2 could alleviate the damage caused by MR5 infection to a certain extent.3.The grafting compatibility test of 12-2 and PYTC under ARD conditions:The graft survival rate and graft interface healing of 12-2 did not differ significantly from those of PYTC.Mechanical strength tests of the grafted interfaces showed that some mechanical strength indices of SH,QNJ,HX,JAS,and YN were significantly higher when they were grafted onto 12-2 rather than the PYTC control.The height and diameter of shoots and the relative chlorophyll content,photosynthetic and fluorescence parameters,peroxidase activity,and malondialdehyde content of leaves were measured on the 13 apple rootstocks grafted onto12-2 and PYTC.Compared with their respective controls,the 13 scion–rootstock combinations showed different levels of performance.Principal component analysis of the above physiological indicators showed that 2001,TMYH,SH,GL,MB,and SF1H grew similarly on the two rootstocks,but TH2H,LG,QNJ,HX,JAS,YN,and LL grew better when grafted onto 12-2 than onto the PYTC control.12-2 therefore showed good grafting affinity,and its tolerance to ARD was better than that of PYTC.4.The effects of ARD soil extracts on the net K⁺and Ca²⁺flux in different root zones of T337,M26 and 12-2:Compared with before immersion in ARD soil extract(Before-ARD),immersion in ARD soil extract for 30 min(ARD-30)had a significant effect on K⁺and Ca²⁺currents at the different zones of the T337 rhizoplane.And K⁺currents at the different zones,Ca²⁺currents in the meristem and elongation zones of M26 rhizoplane.Immersion in ARD soil extract for 5 min(ARD-5)had a significant effect on K⁺currents in the meristem,elongation,and mature zones of 12-2 and on the Ca²⁺currents in the elongation and mature zones.Compared with ARD-5,ARD-30 had a less pronounced effect on K⁺and Ca²⁺currents in the12-2 rhizoplane.The measurement of the changes of net K⁺and Ca²⁺flux in different zones of rootstocks showed that the meristem and elongation zones of T337 root system had the largest changes in net K⁺and Ca²⁺flux.The meristem,elongation zones and meristem,mature zones of M26.And both elongation zones in 12-2.Therefore,12-2 could quickly adjust the net K⁺and Ca²⁺flux to maintain its steady state in response to ARD.5.The K⁺channel gene in 12-2 root was cloned functional verification with the homologous cloning experiment:We used homologous molecular cloning to obtain eight K⁺channel genes from the superior apple rootstock line 12-2(self-named):Ms AKT1-1,Ms KAT3-2,Ms KAT1-3,Ms K2P3-4,Ms K2P3-5,Ms K2P5-6,Ms K2P3-7,and Ms K2P3-8.Their lengths varied from 942 bp(Ms K2P5-6)to 2625 bp(Ms AKT1-1),and the number of encoded amino acids varied from 314(Ms K2P5-6)to 874(Ms AKT1-1).The prediction of subcellular localizations showed that Ms AKT1-1,Ms KAT3-2,and Ms KAT1-3 were localized on the plasma membrane,and the other five K⁺channel genes were localized on the vacuole and plasma membrane.The eight K⁺channel proteins contained?helices,extended strands,?turns,and random coils,four(Ms KAT1-3)to six(Ms KAT3-2)transmembrane structures,two(five K2P)to nine(Ms AKT1-1)protein domains.Semi-quantitative RT-PCR detection of the K⁺channel genes showed that their expression levels were high in roots.The q RT?PCR analysis showed that the relative expression of the eight genes were different under MR5stress.Subcellular localization test of three different family genes with high expression levels,Ms AKT1-1,Ms KAT3-2 and Ms K2P3-7 was carried out,and the results showed that all three genes were localized on the cell membrane.The verification test of transgenic Arabidopsis found that compared with CK,the root length and fresh weight,chlorophyll a content,chlorophyll b content and antioxidant enzymes activities of the experimental group were significantly increased,and the ROS levels,MDA and permeable substance contents were significantly decreased under MR5 stress.It indicated that 12-2 might improve the resistance of 12-2 to MR5 by regulating the expression of K⁺channel genes in roots.