Preliminary Study On The Mechanism Of High Temperature Inhibiting The Symptoms Of Lemon Yellow Vein Clearing Disease

Citrus yellow vein clearing disease caused by Citrus yellow vein clearing virus(CYVCV)has become the most threat on the cultivation and production of lemons.Since CYVCV was first discovered in China in 2009,it was widely distributed in major citrus producing provinces of China and has a serious impact on the lemon industry.The symptoms in affected lemon include leaf yellow vein clearing,leaf distortion,tree growth and fruit yield were reduced.The virus has been detected in India,Turkey,Pakistan,China and Iran.Temperature is an important environmental factor that influences plant growth and the development of plant virus infections.The symptoms of CYVCV on Eureka lemons can be reduced at high temperature.But the mechanism is still unknown.In this study,the annual distribution of CYVCV in Eureka lemon seedlings were detected in the field.The expression levels of CYVCV coat protein(CP)gene,CP content,chlorophyll and enzyme activity content,and chloroplast ultrastructure in CYVCVinfected Eureka lemon were studied at 25°C and 37°C,respectively.Furthermore,comparative transcriptome analysis of CYVCV-infected lemons at 25°C and 37°C,was analyzed.The main research results of this paper are as follows:1、 The distribution of CYVCV in Eureka lemon was unevenly.The content of CYVCV in the bark was higher than that in the leaf tissue and varied significantly with temperature.The virus content was the highest at 25°C average temperature in midOctober and the lowest at 37°C average temperature in mid-August.2、 Detected by RT-q PCR and Western blot,the expression of CYVCV CP gene and the content of CP in CYVCV-infected lemons were significantly reduced at 37°C comparing with those at 25°C.The results indicated that the content of CYVCV was positively associated with the severity of symptoms in infected Eureka lemon seedlings.Comparisons of the whole-genome sequences of the CYVCV isolate in Eureka lemon at25°C and 37°C revealed that the sequence identity was 99.72% at nucleotide level and99.48% at amino acid level,indicating that there is a very low level of sequence heterogeneity among CYVCV isolate at different temperatures.3、 The results of immunofluorescence electron microscopy showed that CYVCV was distributed in the phloem,mesophyll tissue and secretory cavity of the lateral veins of the CYVCV-infected plants,as well as in the phloem of the main vein.Compared with37°C,the fluorescence intensity in the lateral veins was higher when treated at 25°C,indicating a high level of virus expression at this time.At 25°C,the chloroplasts of CYVCV-infected plants were swollen and deformed due to the increase in starch grains;the stromal and grana lamellae were loose.No significant changes in chloroplast ultrastructure were observed between CYVCV-infected and mock plants grown at 37°C.Furthermore,CP of CYVCV was detected in chloroplast of CYVCV-infected lemon by Western blot.4、 Chlorophyll content,activity of ascorbate peroxidase(APX),and catalase(CAT)activity were significantly higher in CYVCV-infected lemons at 37°C than those at 25°C.The results of comparative transcriptome analysis showed that resistance-related pathways such as phenylpropanoid biosynthesis,isoflavone biosynthesis,and flavonoid and flavonol biosynthesis.Resistance-related genes AGO4,PAL,starch and sucrose metabolism genes AMF2/3,BAM3,plasma membrane transporter gene GPT2,chlorophyll degradation related gene CHL-1/2,antioxidant enzyme gene APX2 and Transcription Factors(TFs)(MYB1R1 /s3 /12 /14/48/111)may be related to symptom recovery of Citrus yellow vein clearing disease under high temperature.