Studies On Fungal Community In Replanted Soil Around Bohai Gulf And Alleviation Apple Replanted Disease By Mixed Cropping With Allium Fistulosum L.

Apple replanted disease（ARD）has become the limiting factor of the sustainable development of apple industry in China.There were no effective measures to control,due to the complex causes.Based on our previous research,rotation of A.fistulosum L.was useful measure for remitting ARD.However,it always takes a long time to take effect.Under replanted condition,absorption,distribution and utilization of ¹⁵N-urea in Malus hupehensis Rehd.seedlings were measured.The fungal community of ten replanted orchards were investigated by clone libraries in Hebei,Liaoning and Shandong province.In addition,correlations between the relative abundance of fungal genera in soils and the severity of ARD were analyzed.The pathogenicity of four Fusarium were further confirmed by isolation,identification and pathogenicity test.The effects of mixed cropping with A.fistulosum L.on the growth and soil environment were studied.At the same time,the main effective components of allium root exudate were analyzed,to explore the alleviation mechanism of mixed cropping with Allium fistulosum L.on apple replanted disease.These researches could provide the theoretical basis for alleviating ARD.Results were as follows:1.Continuous cropping changed the soil microbial community,increased the number of harmful fungi,reduced the number of bacteria,and reduced the bacteria/fungus ratio.Not only the growth and root architecture of Malus hupehensis Rehd.seedlings were inhibited significantly,but also the root function was influenced,due to Continuous cropping.Root activity and H⁺-ATPase activity were also reduced by 33.41%and 25.69%,respectively,compared to seedlings planted wheat field soils.Further more,Absorption,distribution and utilization of¹⁵N in seedlings were also influenced by continuous cropping.The contribution rates of nitrogen derived from fertilizer（Ndff）in different organs of replanted seedlings were declined,compared to other treatments.What’s more,continuous cropping affected the distribution ratio of ¹⁵N in different organs seriously.Due to more ¹⁵N were consumed in roots,the supply to leaves was reduced.Even worse,the tendency expanded further with the continuous cropping time increases.Moreover,the utilization of nitrogen fertilizer was reduced,due to the continuous cropping.In September,the ratios of¹⁵N utilization was only13.33%,accounting for only 32.81%and 39.61%respectively,compared to seedlings planted wheat field soils and methyl bromide fumigated soils.2.Ascomycota were dominate in the apple replanted soil around Bohai Gulf.Fusarium and Saccharomyces were widely found in all ten replanted orchards and had highly relative abundance.ARD was commonly occurred round Bohai Gulf and the degree of ARD varies greatly among different soils,based on our inhibition experiments.The correlation between soil characteristics and the degree of ARD was analyzed,and the results showed there was no direct correlation between them.The community structure of soil fungi varies greatly in different areas and Laizhou and Pulandian was lower in diversity of fungal community than others,presenting severely ARD.In addition,the relative abundance of main fungi of replanted soil and the degree of ARD were analyzed in the same way.The results showed that Fusarium was correlated positively with the severity of ARD,but Mortierella was negatively correlated.Furthermore,the results of qPCR analysis showed that F.oxysporum was widely found in the replanted soil around Bohai Gulf.Overall,this study demonstrated that ARD was strongly associated with an unbalanced microbial ecosystem with more pathogenic fungi.While,Fusarium was the key factor for ARD around Bohai Gulf.3.The morphological characteristics and sequencing methods were used to identify the isolated Fusarium.The results showed that these Fusarium fungi were mainly divided into four species,just as Fusarium oxysporum,Fusarium proliferatum,Fusarium solani and Fusarium moniliforme.The four Fusarium species showed strong pathogenicity to Malus hupehensis Rehd.seedlings,based on inoculation experiments.Compared with the control,the dry weight of the seedlings decreased 35.60%,51.92%,40.96%and 40.96%after one month of inoculation,respectively.what’s more,the lethal rate of the four Fusarium species was up to 50%two month later.According to the sequencing results of four Fusarium species,specific primers were designed,and we also built the fluorescence quantitative detection method,which was rapid,accurate,sensitive and easy to operate.This detection method provided the methods and basis for the study of dynamic change of the pathogen in the apple replanted soil.4.Evidence indicates that mixed cropping with A.fistulosum and B.juncea significantly improved growth of grafted seedlings and alleviated the ARD,based on our pot and field tests.Compared with replanted soil,the plant height,branch length and dry weight of mixed cropping with A.fistulosum L.were increased significantly,by 6.50%,36.04%and24.28%,respectively.Mixed cropping with A.fistulosumL.improved the soil biological environment by activating antioxidant enzymes,increasing the number of bacteria,decreasing the number of fungi and improving the ratio of bacteria and fungi.In addition,the phlorizin and phloretin were significantly reduced by 78.8%and 81.1%,respectively.The diversity of fungal community was also improved distinctly,such as Margalef index,Pielou index,and Shannon index.The results of clone libraries showed that mixed cropping A.fistulosum L.preferential proliferated antagonistic fungi,such as Mortierella,Trichoderma,Penicillium,and inhibited Fusarium.The number of four Fusarium species were also decreased by qPCR analysis.These results suggested that mixed cropping A.fistulosum L.could improve soil microbial community structure,increase beneficial fungus and inhibit pathogens.As a result,it was an effective method for alleviation ARD.5.Bioactive compounds in root exudates of A.fistulosum L.were identified by Gas Chromatography-Mass Spectrometer（GC-MS）,which indicated that sulfur compounds were abundant.Meanwhile,Dimethyl disulfide and Diallyl disulfide were bioactive compounds in root exudates of A.fistulosum L.,which could inhibite the mycelial growth and spore germination of four Fusarium.Compared with the control,the mycelial growth and spore germination of F.solani were reduced by 34.78%and 23.43%,respectively.In addition,the results showed Dimethyl disulfide showed stronger inhibition than Diallyl disulfide.The pot experiment showed that the root exudates and sulfur compounds significantly increased the plant height and fresh weight of the seedlings and inhibited the number of Fusarium in the replanted soil.Moreover,the root exudates presented more effective effect than others.Therefore,it could be concluded that apple tree mixed cropping with A.fistulosum L.could be as a cultivation pattern conttrolling ARD by exudating effective substances and inhibiting the harmful pathogens.