Study On The Effect Of Dark Treatment On The Detection Of Iodine-starch For Citrus Huanglongbing

Citrus Huanglongbing(HLB)is one of the most devastating diseases in citrus production throughout the world.In field,it often misjudged due to complicated symptoms of HLB.How to diagnose accurately will be the most urgent problem to be solved in practice.There are various forms of yellowing of citrus leaf infected by HLB in the field.In China,the chlorosis of leaf base is regarded as a typical symptom,while the mottled leaf is in Florida United States..In order to clarify the leaf with basics of chlorotic,mottled leaves and different symptoms occurred often in field.Red-nose fruits were first selected as research object and standard trees of HLB.Distribution of citrus HLB were compared with leaf with chlorotic base,leaf mottle and different leaf symptoms of trees.It provide a more comprehensive basis for the detection of dark treatment and provide technical support for field diagnosis.Changes in starch content of healthy and HLB affected Olinda orange leaf in different seasons under dark conditions can guide the detection of citrus HLB by iodine-starch method in field.Red-nose fruits were selected as standard trees of HLB.Healthy plants were taken from non-toxic nursery,and Olinda orange lea,ves were used as materials.The dark condition is as follows:Dark treatments began at 8:00 am(T=8:00),12:00 am(T=12:00)and 5:00 pm(T=17:00)respectively with the duration of Oh,24h,48h and 72h.Real-time quantitative PCR and iodine-starch method were used to detect and compare the change of the samples,and the changes in microstructure before and after dark treatment was also carried out.Results are as follows:1.Distribution of Huanglongbing in the fieldChlorotic in leaf base and mottled leaf of trees with red-nose fruits from mandarin cultives 'Nanfeng' and 'Shatang','Newhall' navel orange,and Olinda Valencia orange were first selected to detect respectively through the detection of Huanglongbing distribution base of real-time quantitative PCR.The results show that the average detection rate of chlorotic in leaf base from the four cultivars is 100%,the mottled leaf of'Nanfeng' mandarin,'Newhall' navel orange,'Shatang' mandarin and Olinda orange were 88.89%,88.89%,94.44%and 83.33%,respectively.The detection rate of green leaves with chlorotic in base from all the four cultivars was higher than that of the mottled leaves.Leaves of different symptoms of Olinda orange from red-nose fruits Huanglongbing tree were selected to detect by real-time quantitative PCR.The results also showed that a variety of other symptoms except the normal leaf blade without symptom from Huanglongbing trees showed high detection rate.The detection rate of leaves with uniform yellow symptoms from Huanglongbing tree was highest with 98.51%,followed by the typical symptoms in mottled leaf with 96.13%,zinc shaped blade with the positive rate of 100%,and normal leaf blade with 61.42%.It is also indicated that the symptoms of different yellows are mixed in the field.Therefore,leaves with chlorotic in base should be selected firstly for dark treatment detection of field sampling,followed by mottled leaves and uniform yellowing leaves.2.Detection results of dark treatment in different seasonsSeasonal changes lead to changes in starch accumulation,this paper studied the seasonal variation of dark treatment technology in the detecting effects in order to select the optimal leaf from different season and treatment time in dark condition.Result showed that(1)In spring,the dark treatment time at 5:00pm(T=17:00)has a great influence on the test results,the best time for dark treatment is 72h to avoid sampling at night.(2)In summer,the detection results were relatively stable.The detection of Huanglong disease plants and healthy plants were not affected by dark treatment.(3)In autumn,dark treatment could be carried out in the morning,noon and everning,but the dark treatment time should reach 48h or above.(4)In winter,detection results are similar to those in summer.They are also relatively stable.They are not affected by dark treatment.This is resulted from winter temperature is low and starch accumulates little in leaf.Dark treatment technology has little effect on starch accumulation in citrus leaf.Therefore,4 seasons can be dark treatment.In spring,morning and noon dark treatment 72h can be sampled and detected;In summer,autumn and winter,morning,noon and evening dark treatment 48h can be sampled and detected.3.Comparison of iodine-starch method and dark treatment method and the difference in microstructureThis study will carry out be dark treatment of iodine-starch detection technology and iodine-starch detection method.Uniformity of dark treatment of iodine-starch detection method and qPCR detection method was significantly higher than that of the iodine-starch and qPCR detection method.In addition the detecting effect of leaf under dark treatment by iodine-starch is relatively accurate,showing that dark treatment technology is necessary for detecting HLB in leaf.Furthermore,citrus leaf microstructure changed before and after dark treatment showing that dark treatment is necessary.Starch accumulated in the phloem in Huanglongbing leaves after dark treatment is unable to "transfer",therefore,leaf under dark treatment stain deeply,while healthy leaf is different.Leaf was treated by darkness and its starch consumed and its content decreased significantly.When stained by iodine,it can not change color.It is proved that dark treatment is necessary in iodine-starch detection technology.