| In this paper,the pathogenic bacteria of walnut root was identified,the effects on its leaves' physiological and biochemical characteristics,and the element content of roots and their correlation with diseased degree were also studied.On the basis of screening biocontrol bacterium,the colonization results on rhizosphere soil and roots were analyzed and explored.The key findings as follows:1.Use the microscope,the morphological identification resutls showed that the pathogenic bacteria was Fusarium solani.Sequencing aligment of ITS and TEF-la,molecular analysis identified the pathogenic bacteria was F.solani.Combined the results of morphological and molecular identifications,we finalized that the walnut root rot pathogen was F.solani.After artificial inoculation pathogenic bacteria,we found that the incidence of Juglans sigllata Dode was susceptible cultivars for F.solani,with more serious and earlier onset.The morbidity of Mianyang walnut and Xinjiang walnut was mild,which was all in 1 and 2-class level.2.F.solani had different effects on walnut leaf photosynthesis,the content of chlorophyll,soluble sugar and MDA.The photosynthetic rate and the transpiration rate of J.sigllata were significantly reduced,while Mianyang walnut and Xinjiang walnut were less affected.After vaccination of F.solani,there was no obvious difference of chlorophyll content between Mianyang walnut and Xinjiang walnut,but the chlorophyll content of 1-3 years of J.sigllata were reduced compared with the control group,which were significantly lower than Mianyang and Xinjaing walnut.After F.solani inoculation,J.sigllata leaf soluble sugar and MDA content decreased rapidly,and reahced their minimum at 50 d of inoculation;Mianyang walnut's and Xinjiang walnut's soluble sugar and MDA content were decline slowly,and were obvious different from Juglans sigllataDode's.3.F.solani affected four of walnuts' defense enzymes.After the pathogenic bacteriainoculation,the SOD activity of J.sigllata,Mianyang and Xinjiang walnut increased,then decreased rapidly,only the SOD activity of Mianyang walnut remained in a highter level,and the activity of Xinjiang walnut SOD reach the second peak at 40d of inoculation.POD activity change of each group was roughly the same,maintaining an upward trend,and the up trend of Mianyang and Xinjiang walnut was particularly prominent.The same trend of PPO activity of each group was showing a decline after the first rise,and J.sigllata appeared relatively late peak compared with Mianyang and Xinjiang walnut.There was no significant difference among each group of walnut leaves of CAT activity performance in varieties.4.F.solani affected the content of root element.After inoculation,the organic matter content of Mianyang and Xinjiang walnut increased,but the organic matter content of J.sigllata was reduce rapidly after the first increased phenomenon;after inoculation of pathogenic bacteria,the total N,P,K content decreased,and J.sigllata's total N,P,K content decreased more significantly;after inoculation pathogenic bacteria,the total Ca and Mg content of the root of Mianyang and Xinjiang walnut were slightly increased,while the Ca content of J.sigllata were significantly increased.5.The results showed that soluble sugar,MDA,organic matter,Ca,Mg conten were highly significant correlation with infected time,resistance,and onset index.There was significant positive correlation between the four kinds of defense enzyme activity and resistance,while a very significant negative correlation with onset index;only the POD activity was significant positive correlation with infected time;and there was no correlation among the CAT activity,variety resistance and onset index.At the root indicators,the organic matter,and N,P,K content were associated with plant resistance,which were significantly positively correlated with the onset of the index.In addtion,there was a significant correlation between onset index and infected time,which showed a significant negative correlation variety resistance,while the Ca and Mg content were opposite with infected time and plant resistance.6.The element content of walnut rhizosphere soil and root rot correlation studiesshowed that the organic matter,N,P,K,Mn and Zn content of rhizosphere soil were significant correlated with root rot susceptible classification,while no significant correlation with the content of Ca and Mg.Through field investigation we found that the occurrence of walnuts root rot had no significant with the altitude,slope direction,slope of the location and soil pH.7.Adoption of the primary and secondary screening of antagonistic experiments,theresults showed that the inhibition rate of fungus(ZJ-1),bacteria(XJ-1)and reddish purple Streptomyces YSSPG3 for F.solani reached about 80%.After the combination of morphological and molecular identification,fungi(ZJ-1)was identified as Trametes versicolor while bacteria(XJ-1)as Bacillus amyloliquefaciens.Bio-control experiments results indicated that all of these three bio-control microbe had effective control effect,and early prevention was better.8.The usage of real-time PCR technology to detect the colonization of bio-control microbe in Walnut rhizosphere showed that after 60d of application of the B.amyloliquefaciens,S.Purpeofuscus and T.versicolor,the relative content of F.solani in J.sigllata rhizosphere soil decreased significantly compared with control group.The decrease of the relative content of F.Solani in B.Amyloliquefaciens treatment was greater than the others.Bio-control effect was:B.amyloliquefaciens>S.purpeofuscus>T.versicolor.When the colonization was stable,the relative content of B.Amyloliquefaciens and S.purpeofuscus had no significant difference with the control group(P>0.05),while the relative content of T.versicolor in healthy plants was 2.6 times than that in infected plants.Colonization effect was:B.amyloliquefaciens>S.purpeofuscus>T.versicolor.The colonization of B.amyloliquefaciens in J.sigllata roots was successfully by using the real-time PCR,with the increasing of walnut root diameter size,along with the migration. |
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