Virus Identification And Analysis Of Yield And Quality Of Virus-free Plantlets At Different Generations On Chrysanthemun Morifolium

Chrysanthemum morifolium belongs to Asteraceae family.It is cultivated in theTongxiang City as major C.morifolium production area in Zhejiang province,China.The vegetative propagation is vulnerable to the accumulation of viruses over subsequent generations and can be spread the viruses.The viral diseases cause significant yield and quality loss on C.morifolium,also even destroy some susceptible varieties.In this study,we investigated the presences of potential viruses on C.morifolium at various growing fields.Furthermore,virus-free plantlets were obtained by repeated shoot tips stripping,and then transplanted in the field.The yield and quality of virus-free plantlets in different generations of C.morifolium were analyzed.The main results are shown as follows:1.The 140 symptomatic samples were collected from Luosibang,Huayuanli,Guihua Village,Yanjing Village,Miaotou Village,Qianlin Village and Agricultural Technology Extension Service Center areas in Tongxiang,Zhejiang province.The results of the survey revealed that the symptoms viral disease of each seven plantation areas were not distinct.C.morifolium plants exhibiting virus-like symptoms,including chlorosis and shrinking,mosaic,vein clearing,wilting,and stunting.It was preliminarily confirmed that C.morifolium in Tongxiang City was infected with plant viruses.2.Viruses Detection.Crude extracts of diseased plants were negatively stained by ammonium molybdate（1%,p H7.0）,then screen with TEM.Slightly curved linear viron particles and banded aggregates were observed in the cytoplasm of C.morifolium leaves.The RNA sequencing revealed that the small RNA sequences obtained from the samples had high nucleotide similar with Chrysanthemum virus B（CVB）and Chrysanthemum virus R（CVR）,ranging from 92.67%to 100%and79.82%to 98.67%,respectively.The specific primers of ten different chrysanthemum viruses were used for RT-PCR assay.The amplified products with expected size of650 bp for CVB and 1641 bp for CVR were purified and sequenced.The obtained sequence shared 91%and 97%nucleotide sequence identity to previously reported strains of CVB and CVR.Overall,we confirmed that all collected C.morifolium samples were only infected by CVB and CVR.3.The establishment of a method for rapid detection of CVB using microfluidic chip.The c DNA of CVB-infected samples were subjected to microfluidic fluorescence reader with five primer combinations were designed in this study such as CVB-12,CVB-13,CVB-14,CVB-15 and CVB-16.Although,all specific primers could quickly detect CVB,the shortest time belongs CVB-13 primer（8 min）;hence it was used as a specific primer combination for the detection of CVB.The sample c DNA was diluted10^-1,10^-2,10^-310^-4and 10^-5with dd H₂O,then.The sensitivity and rapid of the microfluidic chip method and RT-PCR were compared.The results revealed that the rapid detection system allocated the microfluidic chip method had higher detection sensitivity than the RT-PCR detection method.4.Determination of field agronomic traits and yield in different generations of virus-free plantlets.The first-generation virus-free plantlets and the second-generation virus-free plantlets of C.morifolium were investigated to evaluate the agronomic traits on 8^thNovember 2019 and 12^thNovember 2020,respectively.The data demonstrated that the first-generation virus-free plantlets produce plant with 56.11 cm height,the994.65 mm²leaf areas,97 branches,139 petals,7 layers of petals,and 1.36 g a single flower weight;while,the second-generation virus-free plantlets generate plant with47.11 cm height,950.34 mm²leaf areas,83 branches,118 petals,5 layers of petals,0.79 g a single flower weight.The agronomic characteristics of virus-free plantlets in different generations were significantly better than those of common plantlets.The yield per mu of the first and second generation virus-free plantlets was 67.96%and75.27%higher than common plantlets.5.Determination of net photosynthetic rate（Pn）and chlorophyll content of virus-free plantlets in different generations.The Pn of the first-generation virus-free plantlets and common plantlets were measured in September 2019 and November 2019,respectively.The Pn of the second-generation virus-free plantlets and common plantlets were measured in September 2020 and November 2020,respectively.The results showed that the Pn of virus-free plantlets were greater than that of common plantlets during the budding and picking stages in different generations.Regarding the relative chlorophyll content,in the bud stage,the relative chlorophyll content of the first-generation and second-generation virus-free plantlets was significantly higher than that of the common plantlets.The relative chlorophyll contents of the first-generation virus-free plantlets and the common plantlets were 41.78 SPAD and35.62 SPAD,respectively;whereas,relative chlorophyll content of virus-free plantlets and common plantlets were 53.34 SPAD and 46.48 SPAD,respectively.In the harvesting period,the relative chlorophyll content of the first-generation and second-generation virus-free plantlets were slightly greater than the common plantlets,but the difference was not significant.6.Determination of the quality in different generations of virus-free C.morifolium.The results demonstrated that the content of chlorogenic acid(C₁₆H₁₈O₉),luteolin(C₂₁H₂₀O₁₁),and 3,5-O-dicaffeoylquinic acid(C₂₅H₂₄O₁₂)in the first and second generation virus-free plants were congruent with“the Pharmacopoeia of the Peoples Republic of China 2015”regulations.Moreover,the content of chlorogenic acid and3,5-O-dicaffeoylquinic acid in the virus-free plantlets in different generations of C.morifolium were higher than common plants,while,the content of luteolin was lower than ordinary plants.7.The fingerprinting analysis assay indicated showed that of virus-free plantlets and common plantlets has high similarity to each other over 0.98,indicating that virus-free plantlets and common plantlets have the same quality,and the quality of these plantlets were relatively stable.