| In China citrus production area,most of the soils are acidic.When the soil pH is lower than 4.5,the citrus is easy to suffer excess manganese toxicity.On the other hand,the frequent application of copper and manganese-containing pesticides resulted in high copper and manganese contents in orchard soil and led to tree toxicity.Citrus tree,a grafted fruit crop,is a combination of rootstock variety and scion variety.Due to the abundance of citrus rootstock varieties and their great differences of genetic background,the absorption and tolerance of copper and manganese should be different for different rootstocks.Therefore,this study was designed to evaluate excess copper and manganese on growth and physiological response of citrus rootstocks,and thus to select citrus rootstocks against excess copper and manganese stresse,which will be helpful for citrus production practice.In this study,trifoliate orange(Poncirus trifoliate(L.)Raf),Ziyang sour orange(Citrus junos(Sieb.)Tanaka),red tangerine(Citrus tangerine Hort.ex Tanaka)and Shatian pomelo(Citrus grandis(L.)Osb.‘Shatianyou')were treated by excess copper and manganese stresses.Copper treatment concentrations were set as 1.5?mol/L,7.5?mol/L,37.5?mol/L,187.5?mol/L,and 937.5?mol/L,which marked as Cu1.5,Cu7.5,Cu37.5,Cu187.5,Cu937.5,respectively.Manganese treatment concentrations were set as 0.01mmol/L,0.05 mmol/L,0.25 mmol/L,1.25 mmol/L,6.25mmol/L,which marked as Mn0.01,Mn0.05,Mn0.25,Mn1.25,Mn6.25,respectively.During the experiment,we observed the toxic symptoms,determined the physiological and biochemical indexes of chlorophyll contents,photosynthetic index,antioxidant enzyme activity and mineral element contents.The principal component analysis was performed to evaluate the tolerance of the four citrus rootstocks to copper or manganese stress.The results are presented as follows.1.Effects of excessive Cu stress on citrus rootstock seedlings1.1 Copper poisoning symptomsUnder excessive copper stress,the leaves of citrus rootstock seedling showed an iron deficiency symptom and plant growth was significantly inhibited.As the stress time increased,the leaves were gradually yellowed,and then abscission.The trifoliate orange seedling was the most sensitive to excessive copper stress,and its leaves become yellowish in 20 days under Cu937.5 stress.And under Cu937.5 stress,the Shatian pomelo,red tangerine,and Ziyang sour orange seedlings also appeared toxic symptoms gradually.Cu stress significantly affected root growth,reduced the root amount,and resulted in short and brown roots.1.2 The biomass changesWith the increase of excess copper stress concentration,the biomass of the 4 rootstock seedlings decreased gradually.The plant height increment of the plant was significantly decreased,while the growth rate of the plant was smaller under the stress of Cu7.5 or Cu37.5.Under the stresse of Cu187.5 and Cu937.5,the plant height increment ratio of the 4 rootstock seedlings was greatly reduced.Compared with the control,under the stress of Cu937.5,the plant height increment of the 4 rootstock seedlings decreased by 69.50%,68.80%,70.02% and 57.20% respectively.With the increase of the copper stress concentration,the biomass dry weight and fresh weight of the upper and underground parts of the rootstock seedlings were decreased.1.3 The leaf chlorophyll content changesWhen Cu concentration was ?37.5 ?mol/L,it had no significant effect on the relative chlorophyll content of the 4 rootstock leaves.The relative leaf chlorophyll content of trifoliate orange decreased significantly after 45 days under Cu37.5 stress,and decreased rapidly under the stress of Cu187.5.After 20 days of stress treatment in Cu187.5,relative chlorophyll content of Shatian pomelo was significantly reduced.All Cu stress treatments had no significant effect on the relative chlorophyll content of red tangerine and Ziyang sour orange leaves.Absolute leaf chlorophyll content declined significantly under excessive copper stress,both the chlorophyll a and chlorophyll b decreased.The changes of chlorophyll content of rootstocks and their copper stress tolerence were positively correlated.The leaf photosynthetic rate decreased with the increase of Cu stress concentration.1.4 Changes in physiological and biochemical indexesUnder the excessive copper stress,leaf cells were severely damaged,and the relative conductivity and MDA value increased significantly,and these changes were negatively correlated with the toxicity tolerance of rootstocks.Under the stress of Cu937.5,the relative electric conductivity of the 4 rootstock seedlings increased by 2.39 times,1.31 times,2.39 times and 1.06 times respectively,as compared with the control.And the leaf MDA value from high to low were Shatian pomolo,trifoliate orange,red tangerine and Ziyang sour orange.The SOD enzyme,POD enzyme and CAT enzyme were increased under the low concentration Cu stress,but with the increase of Cu stress concentration,the overall change was not obvious.1.5 Changes in nutrient element contentsWith the increase of excess copper stress concentration,the contents of leaf and root copper increased.Under the stress of Cu937.5,the leaf Cu contents of trifoliate orange,Ziyang sour orange,red tangerine and Shatian pomolo were 64.31 mg/kg,57.28 mg/kg,76.97 mg/kg and 53.93 mg/kg,respectively.Copper contents in roots from high to low were red tangerine,trifoliate orange,Ziyang sour orange and pomelo.With the increase of copper stress concentration,leaf K contents in trifoliate orange and Ziyang sour orange were decreased significantly,but the content changes of Ca and Mg in leaves were small.The contents of Ca and Mg in roots of the 4 rootstocks dropped significantly as copper stress concentrations increased.The contents of P element in leaves and roots increased first and then decreased with the increase of Cu stress concentration.The content of leaf Fe decreased significantly under the stress of Cu7.5,and the content of Fe in root decreased significantly under the stress of Cu37.5.The Zn content of leaves decreased slowly with the increase of Cu stress concentration,but the root Zn content showed an upward trend.1.6 Rootstocks' excess copper tolerance comprehensive evaluation and its copper poisoning concentration.(1)Comprehensive evaluation of excess copper toleranceIntegrated the physiological and biochemical indicators,using principal component analysis and subordinate function analysis method to evaluate the 4 rootstocks' excess copper stress tolerence,and the results showed that Ziyang sour orange >red tangerine >Shatian pomolo >trifoliate orange.(2)Copper poisoning concentrationAccording to the analysis of the plant growth,biomass,and the physiological and biochemical indicators of the 4 rootstockseedlings,the initial concentration of copper poisoning for trifoliate orange was Cu7.5 ?mol/L,for both Ziyang sour orange and red tangerine was Cu37.5 ?mol/L,for Shatian pomelo was Cu 7.5 ?mol/L.2 Effects of excessive Mn stress on citrus rootstock seedlings2.1 Manganese poisoning symptomUnder the stress of excessive manganese,the 4 citrus rootstock seedlings showed the symptom of iron deficiency yellow,and brown spots occurred on the leaves with the extension of stress time.Trifoliate orange was the most sensitive to excessive manganese stress,and its leaves become yellowish in 20 days under Mn6.25 stress,and with the time extension of this stress,the Shatian pomelo,red tangerine and Ziyang sour orange showed symptoms in turn.Excess manganese stress significantly affected root growth,reduced the root amount,and resulted in short and brown roots.2.2 Biomass changesWith the increase of excess manganese stress concentration,the biomass of the 4 rootstock plants gradually decreased.Under Mn0.5 and Mn0.25 stress,the increment of plant height was reduced by a small amplitude.Under the stress of Mn1.25,the plant height increment of the all rootstock seedlings decreased significantly.Compared with the control,under the stress of Mn6.25,the plant height increment ratio of the 4 rootstock seedling decreased by 73.50%,68.78%,70.17% and 71.17% respectively.With the increase of the manganese stress concentration,the dry weight and fresh weight of the upper and lower parts of the four rootstock seedlings were decreased.2.3 Chlorophyll content changesWhen Mn concentration was ?0.05mmol/L,it had no significant effect on the relative chlorophyll content of the 4 rootstock leaves.The relative chlorophyll content in the leaves of trifoliate orange decreased significantly after 20 days under Mn0.25 or Mn1.25 stress,and decreased rapidly after 10 days in Mn6.25 stress.The relative chlorophyll content of Shatian pomelo dropped significantly after 10 days of stress at Mn6.25;Excessive Mn stress treatment had no significant effect on the relative chlorophyll content of red tangerine and Ziyang sour orange.Leaf absolute chlorophyll content and leaf net photosynthetic rate decreased with the increase of manganese stress concentration.The changes of chlorophyll a and b contents were consistent with leaf net photosynthetic rate.Chlorophyll content changes and manganese poisoning tolerance were positively correlated.2.4 Changes of physiological and biochemical indexesWith the increase of manganese stress concentration,the relative conductivity and MDA value of each rootstock were increased.Under the stress of Mn6.25,the leaf relative electrical conductivity of red tangerine was the highest,as high as 61.26%.The second was trifoliate orange,which was 54.10%.The relative electrical conductivity of Shatian pomelo and Zhiyang sour orange was 38.48% and 32.32% respectively.The MDA value of the leaves of trifolia orange increased first and then decreased with the increase of the manganese stress concentration,however the other 3 rootstocks increased stably.The activity of SOD rised with the increase of manganese stress concentration for all the 4 rootstocks.The changes of POD enzyme and CAT enzyme activity showed a tendency to decrease with the increase of manganese stress.2.5 Changes in nutrient element contentsWith the increase of manganese stress concentration,the manganese contents in leaf and root increased.Under the stress of Mn6.25,the leaf manganese contents of trifoliate orange,Ziyang sour orange,red tangerine and Shatian pomelo were 2217.16 mg/kg,2725.26 mg/kg,3302.93mg/kg and 3836.57mg/kg respectively,and their root manganese contents were 7487.50 mg/kg,12159.50 mg/kg,11814.30 mg/kg and 8675.10mg/kg respectively.Leaf K,Ca and Mg contents of the 4 rootstock seedlings decreased significantly with the increase of manganese stress concentration,and the content of P element in some rootstocks decreased with the increase of Mn stress concentration.The contents of root K and P increased,however Ca and Mg decreased,and the contents of Fe and Zn in leaves decreased with the increase of Mn stress concentration,but the contents of Fe and Zn in roots increased.2.6 Rootstocks' manganese tolerance comprehensive evaluation and manganese poisoning concentration(1)Comprehensive evaluation of manganese toleranceIntegrated the physiological and biochemical indicators,using principal component analysis and subordinate function analysis method to evaluate the 4 rootstocks' manganeser stress tolerence,and the results showed that Ziyang sour orange >Shatian pomolo >red tangerine >trifoliate orange.(2)Manganese poisoning concentrationAccording to the analysis of the plant growth,biomass,and the physiological and biochemical indicators of the 4 rootstock seedlings,the initial concentration of manganese poisoning for trifoliate orange was Mn1.25 mmol/L,for both Ziyang sour orange and Shatian pomelo was Mn0.25 mmol/L,for red tangerine was Mn0.05 mmol/L.3 Conclusions3.1 The four citrus rootstocks' tolerance to excess copper stress was Ziyang sour orange >red tangerine> Shatian pomolo>trifoliate orange.And their tolerance to excess manganese stress was Ziyang sour orange >Shatian pomolo>red tangerine>trifoliate orange.3.2 The initial concentration of copper poisoning for trifoliate orange was Cu7.5 ?mol/L,for both Ziyang sour orange and red tangerine was Cu37.5 ?mol/L,for Shatian pomelo was Cu 7.5 ?mol/.The initial concentration of manganese poisoning for trifoliate orange was Mn1.25 mmol/L,for both Ziyang sour orange and Shatian pomelo was Mn0.25 mmol/L,and for red tangerine was Mn0.05 mmol/L.3.3 In general,the most sensitive rootstock to excessive stress of copper and manganese was trifoliate orange,and the least sensitive rootstock was Ziyang sour orange.The sensitiveness of red tangerine and Shatian pomelo was between trifoliate orange and Ziyang sour orange.Therefore,in acidic soil areas,citrus grafted on trifoliate orange rootstock should be paid more attention to copper and manganese nutrition management to avoid or reduce the harm of excess copper and manganese. |
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