YANG Zhen ,ZHANG Lan ,ZHAO Jin-fa ,ZHANG Xing-kai ,WANG Ying ,Ll Tai-sheng ,ZHANG Wei,ZHOU Yan

1 National Citrus Engineering and Technology Research Center,Citrus Research Institute,Southwest University,Chongqing 400712,P.R.China

2 Horticulture Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530000,P.R.China 3 Plant Protection Station of Sichuan Agriculture and Rural Bureau,Chengdu 610041,P.R.China

Abstract In 2009,an emerging citrus viral disease caused by Citrus chlorotic dwarf-associated virus (CCDaV) was discovered in Yunnan Province of China. However,the occurrence and spread of CCDaV in other citrus-growing provinces in China is unknown to date. To better understand the distribution and molecular diversity of CCDaV in China,a total of 1 772 citrus samples were collected from 11 major citrus-growing provinces and were tested for CCDaV by PCR. Among these,134 citrus samples from Guangxi,Yunnan and Guangdong were tested positive for CCDaV,demonstrating that the occurrence and spread of CCDaV are increasing in China. The complete genome sequences of 17 CCDaV isolates from different provinces and hosts were sequenced. Comparisons of the whole-genome sequences of the 17 CCDaV isolates as well as the 15 isolates available in GenBank revealed that the sequence identity was about 99-100%,showing that the CCDaV isolates were highly conserved. Phylogenetic studies showed that the 32 CCDaV isolates belonged to four different groups based on geographical origins and host species,and that CCDaV isolates from China and Turkey were clustered into different groups. The results provide important information for clarifying the distribution and genetic diversity of CCDaV in China.

Keywords:Citrus chlorotic dwarf-associated virus,detection,sequence alignments,phylogenetic tree analysis

1.lntroduction

Citrus (Citrus spp.) is a commercially valuable and nutritionally important fruit crop in the world. In China,citrus has been cultivated for more than 4 000 years. In 2018,China became the greatest citrus producer in the world,with about 41 million tons of production spread over 2.7 million hectares of cultivation area (Zhou 2020).

Worldwide,citrus production has persistently suffered significant yield losses owing to citrus viral diseases,some of which are widely distributed,and the number of viruses infecting citrus is increasing as well. In recent years,some new citrus viruses have been identified,such as Citrus chlorotic dwarf-associated virus (CCDaV),Citrus yellow vein clearing virus (CYVCV),Citrus leaf blotch virus-2 (CLBV-2),and Citrus yellow mottle-associated virus(CiYMaV) (Loconsole et al.2012a,b;Cao et al.2018;Wu et al.2020).

CCDaV is a putative member of the family Geminiviridae,whose viral genome consists of a circular single-stranded DNA of around 3.64 kb with 5 predicted open reading frames (ORFs) (Loconsole et al.2012b). CCDaV is transmitted by grafting and slash-inoculation (Korkmaz and Garnsey 2000) as well as by Parabemisia myricae in a persistent or semi-persistent manner (Korkmaz et al.1995). Field symptoms consist of a V-shaped notch and chlorotic flecking on young leaves,and inverted cupping and variegation on mature leaves (Korkmaz et al.1995).In Turkey,CCDaV has caused over 50% yield loss in grapefruit,wherein affected plants produced fewer and smaller fruits (Korkmaz et al.1995). Currently,this disease is considered to be the most serious viral disease to Turkey citrus industry,only sweet orange showed some resistance(Loconsole et al.2012b). CCDaV was first reported in Turkey’s Eastern Mediterranean Region in the late 1980s(Korkmaz et al.1995). Subsequently,it was detected in China and Thailand (Guo et al.2015;Yang et al.2020).Previous studies reported that the occurrence of CCDaV was restricted to few Eureka lemon (Citrus limon) orchards in Ruili of Yunnan Province in China (Zhou et al.2017b).Recently,however,some citrus viral diseases have spread quickly due to frequent domestic exchange. Thus this study was conducted to determine the new geographic distribution of CCDaV in China. Additionally,the molecular diversity of CCDaV was analyzed by using the complete sequences of 17 CCDaV isolates obtained from different citrus cultivars and citrus-growing provinces.

2.Materials and methods

2.1.The field survey and sampling

The field survey and sampling were conducted between 2017 and 2019. A total of 1 772 citrus samples were collected from 145 orchards in 11 major citrus production regions in China,namely Sichuan,Chongqing,Yunnan,Guizhou,Guangxi,Guangdong,Hubei,Hunan,Fujian,Jiangxi,and Zhejiang.

The samples from 145 orchards were randomly evaluated both for normal appearance and virus-like symptoms of a V-shaped notch and chlorotic flecking on young leaves and inverted cupping and variegation on mature leaves. During sampling,the symptoms on leaves were carefully observed.The relationship between PCR results and symptoms was also studied.

2.2.Nucleic acid extraction and PCR detection

Total DNA was extracted from each plant by using Plant Genomic DNA Extraction Kit (Bioer,China) and amplified by PCR with primer pair Ant2/Sen2 targeting the movement protein gene of CCDaV (Yang et al.2020).

2.3.Cloning and sequencing

For cloning,17 CCDaV-positive samples were randomly selected from different citrus cultivars and geographical origins (Table 1). The complete genome of CCDaV was amplified by using two pairs of previously described primers 1409fw/341rev and 221fw/1542rev (Loconsole et al.2012b).PCR amplicons were purified and cloned into pGEM-T Easy vector (Promega,USA). Three randomly selected positive clones were custom sequenced (Invitrogen,Shanghai,China). The genomic sequences were assembled by using the CLC Sequence Viewer 7 and submitted to GenBank(accession numbers:MT680001 to MT680012,MT683766 to MT683770).

2.4.Genetic diversity and phylogenetic analyses

Multiple nucleotide sequence alignments of the complete genome of the 17 CCDaV isolates from this study,and 15 CCDaV isolates available in GenBank were conducted separately with CLC Genomics Workbench 11.0.1. MEGA 7.0 was used to construct neighbor-joining and maximumlikelihood phylogenetic trees with 10 000 bootstrap replicates. The cut-off value for the condensed tree was 50%.

3.Results

3.1.Detection of CCDaV by PCR

Using PCR,CCDaV was detected in 134 out of 1 772 collected samples. CCDaV-positive samples originated from Yunnan (73/704),Guangxi (60/195),and Guangdong(1/136). CCDaV was not found in Sichuan (0/196),Hunan(0/140),Jiangxi (0/146),Chongqing (0/127),Fujian (0/54),Zhejiang (0/40),Hubei (0/18),and Guizhou (0/16) (Table 2).CCDaV was not detected in Guizhou and Hubei,possibly due to the limited samples.

Table 1 Accession numbers,host,and collection area of Citrus chlorotic dwarf-associated virus isolates used in this study for molecular variability analysis

Table 2 Number of test plants used and number of plants infected in 11 regions of China considered in this study

Among the surveyed citrus cultivars,‘Ruby Green’pomelo (C.grandis) was the most frequently infected cultivar,with an infection rate of 50.8% (61/120). OtherCCDaV-infected cultivars were ‘Thailand Green’ pomelo(C.grandis,24.0%),‘Eureka’ lemon (20.8%),Mexican lime(C.aurantifolia,20.0%),Tahiti lime (C.aurantifolia,18.2%)and ‘Sanhongyou’ pomelo (C.grandis,3.2%). CCDaV was not detected on sweet orange (0/268),tangor (0/251),mandarin (0/65),tangerine (0/138),trifoliate (0/2),grapefruit(0/14) or kumquat (0/19).

3.2.Virus symptoms observed in orchards

Typical leaf symptoms such as V-shaped notch,chlorosis,curling and distortion were observed on CCDaV-infected‘Eureka’ lemon,‘Ruby Green’ pomelo,‘Thailand Green’pomelo and ‘Sanhongyou’ pomelo. Additionally,the three pomelo cultivars ‘Ruby Green’ pomelo,‘Thailand Green’pomelo and ‘Sanhongyou’ pomelo plants developed more severe symptoms than ‘Eureka’ lemon,and young flushes of the pomelo cultivars exhibited vein clearing. Mexican lime and Tahiti lime developed mild leaf curling and distortion,but no chlorosis was observed. These symptoms were not reduced after the leaf matured,and became exacerbated during the summer. In this study,134 out of the 137 symptomatic samples tested positive for CCDaV by using PCR tests. Three ‘Ruby Green’ pomelo samples from Guangxi gave negative results despite showing CCDaV-like symptoms. CCDaV was not detected in any symptomless or healthy control plants.

3.3.Characterization of the CCDaV genome

The genome size of CCDaV isolates collected from ‘Ruby Green’ pomelo (YN-RG-1,GX-RG-1,GX-RG-3) and‘Thailand Green’ pomelo (YN-RG-2) was 3 642 nucleotides(nt). That of CCDaV isolate GD-sh-1 from ‘Shanhongyou’pomelo was 3 640 nt. The CCDaV isolates collected from Mexican lime (YN-ML-66),Tahiti lime (YN-Th-168),and ‘Eureka’ lemon (excepted YN-EL-9,YN-EL-19 and YNEL-72) were 3 641 nt in length. The genome organization of all these isolates was consistent with those published earlier.

Sequence alignments showed that the complete sequence identity of the 32 CCDaV isolates was 98.98 to 99.97% similar. At the amino acid level,the 17 CCDaV isolates also showed high shared identities with the reported 15 CCDaV isolates for ORF1 (97.86 to 100%),ORF2 (98.43 to 100%),ORF3 (96.42 to 100%),ORF4 (97.06 to 100%)and ORF5 (98.52 to 100%).

3.4.Phylogenetic analyses

The phylogenetic tree showed that the 32 CCDaV isolates,including 15 sequences from GenBank,were clustered into four groups (Fig.1). CCDaV isolates from China were phylogenetically distinct from the isolates from Turkey.CCDaV isolates from Turkey and isolate Tha30 from Thailand belonged to group 1. CCDaV isolates from ‘Ruby Green’ pomelo,‘Thailand Green’ pomelo and ‘Sanhongyou’pomelo were grouped together with isolates Tha1-17 and Tha1-19 from Thailand and belonged to group 2. Isolates YN-EL-9 and YN-EL-19 from Ruili,Yunnan Province,belonged to group 3. The remaining CCDaV isolates from Ruili,Yunnan Province,were clustered into group 4.Accordingly,the phylogenetic results indicated that the complete sequence of CCDaV had host specificity,and most of the isolates of the same host were clustered together.

branches. The cut-off value for the condensed tree was 50%. The 17 CCDaV genome sequences from this study are marked by“▲”. Fig.1 Phylogenetic tree generated by the maximum-likelihood method from the alignment of the genome nucleotide sequences of Citrus chlorotic dwarf-associated virus (CCDaV) using MEGA7. Bootstrap values for 10 000 replicates are indicated at the main

4.Discussion

CCDaV,an emerging citrus virus,was first recognized in China in 2009 (Guo et al.2015). Previous studies showed that the occurrence of CCDaV in China was restricted to few ‘Eureka’ lemon trees in orchards in Ruili,Yunnan (Zhou et al.2017b). With the development of the citrus industry in recent years,the incidence areas of some citrus viruses,such as CYVCV,Citrus tatter leaf virus (CTLV),and Citrus psorosis virus (CPsV),have increased rapidly (Xiang et al.2017;Zhou et al.2017a;Li et al.2018). Furthermore,nearly all citrus species and cultivars are susceptible to CCDaV,while only sweet orange has shown some disease resistance (Baker et al.2008). Therefore,it is necessary to understand the new geographic distribution of CCDaV in China to gauge its apparent risks and address its potential impact on citrus industry.

In this study,CCDaV was first detected in ‘Mexican’ lime and ‘Tahiti’ lime from orchards near the CCDaV-infected‘Eureka’ lemon trees in Ruili,with a detection rate of about 20%. Because of the intense and rapid outbreak of CCDaV,vector transmission was suspected. However,P.myricae has never been found in the citrus orchards where CCDaV infection was observed. This observation indicated that low populations of P.myricae can spread CCDaV,or other vector,such as other whitefly species,may have transmitted the citrus disease (Korkmaz et al.1995;Baker et al.2008).

In recent years,‘Ruby Green’ pomelo and ‘Thailand Green’ pomelo from Thailand have been introduced in China because of their production quality and high yield potential.In this study,CCDaV was detected in ‘Ruby Green’ pomelo and ‘Thailand Green’ pomelo from Guangxi,Yunnan and Guangdong provinces with a high infection rate,which suggests that infected planting materials may have been used in citrus plant propagation. It is necessary to use virusfree seedlings or planting material to prevent further spread of CCDaV and reduce the cost of production.

In this study,the CCDaV isolates from China,Turkey,and Thailand shared about 98.98% similarity in identity at the complete genome level. These results indicate that the CCDaV isolates are highly conserved in terms of molecular structure. However,based on phylogenetic analyses on geographical origin and host species,the isolates belonged to four different groups. CCDaV isolates from China were clearly distinct from the isolates from Turkey. While earlier studies showed that CCDaV isolates from China grouped together in phylogenetic trees (Zhou et al.2017b;Karanfil and Korkmaz 2019),the results of this study suggest that CCDaV isolates from China may have different geographical origins and that CCDaV from ‘Ruby Green’ pomelo may have originated from Thailand. Furthermore,based on the phylogenetic analysis of complete genome,isolates YN-EL-9 and YN-EL-19 were included in the same group,indicating that the two isolates may be evolutionarily farther than other CCDaV isolates from Ruili,Yunnan.

5.Conclusion

To the best of our knowledge,this is the first evidence of CCDaV infection outside of Yunnan Province in China.Therefore,the virus is spreading toward neighboring citrus cultivation regions and might be an increasing tendency.The results of this study laid the foundation for further study of the epidemiology and molecular characteristics of CCDaV in China.

Acknowledgements

This research was partially supported by the National Key R&D Program of China (2019YFD1001800),the China Agriculture Research System,Overseas Expertise Introduction Project for Discipline Innovation (B18044),the China Agriculture Research System of MOF and MARA (CARS-26-05B),the Natural Science Foundation of Chongqing,China(cstc2019jcyj-msxmX0557),and the Guangxi Natural Science Foundation,China (2018GXNSFBA050027). We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.