Occurrence and distribution of citrus tatter leaf disease in Zhejiang and genetic diversity analysis of its pathogen

【Objective】Citrus tatter leaf disease is a viral disease that harms citrus trees. Currently, it is showing an increasing trend of spreading and causing plant weakness, death, and even large-scale destruction of orchards. It has become an important obstacle to the sustainable development of the citrus industry. The aim of this study was to clarify the occurrence and distribution of citrus tatter leaf disease in Zhejiang Province, as well as the diversity and genetic differentiation of its pathogen, and to explore the origin and evolution of citrus tatter leaf virus (CTLV).【Methods】From 2019 to 2022, we continued to investigate the occurrence of citrus tatter leaf disease through observing and recording the symptoms of the disease in various citrus production areas in Zhejiang Province. The 46 citrus varieties included Ehime Kashi No. 28, Haruka, Mantouhong, Himekoharu etc.. For the detection of citrus tatter leaf virus, total RNA of the samples was extracted, and RT- PCR amplification was performed with specific primers. The amplification results were detected by agarose gel followed by observation and photography. The target band on the agarose gel was cut off and then the PCR products were recovered using a DNA gel recovery kit followed by a bidirectional sequencing. After aligning the obtained sequences, the similarity of the nucleotide and amino acid sequences of the coat protein genes from different samples were analyzed. All the sequence information were submitted into GenBank website and were also used to construct a phylogenetic tree using the neighbor-joining method of MEGA 5.05 software.【Results】 The results of field investigations showed that the symptoms of citrus tatter leaf disease were generally similar among different citrus varieties. The infected trees usually grew slower and the tree vigor was weaker than the healthy trees. The leaves of the diseased tree showed a systematic slight yellowing at the initial stage and gradually worsened. In the later stages, the leaves became severely yellowed and gradually fell off from the tree, resulting in few or even no fruits. Finally, the entire tree died. When the diseased trees were subjected to strong winds or external forces, the graft union of diseased trees was prone to break, and the cracking surface was smooth. Out of the 951 collected citrus samples, 311 samples were infected with the disease, with a positive detection rate of 32.70%. Apart from Huzhou city, the disease was found to be widely distributed in various citrus production areas in Zhejiang Province. There were also significant differences in the positive detection rates among different counties or districts within the same region. From a variety perspective, out of the tested 43 citrus varieties, 27 showed positive results. The positivity rate of Tangor type was 48.54%, significantly higher than the Tangelo type (12.33% ), Pummelo type (16.54% ), Tangerine type (16.22% ), and Mandatory type (13.85% ), while the positivity rate of Grapefruit type was relatively low. Within the same type, there were also significant differences in the positivity rates of different cultivars. The constructed phylogenetic tree based on the coat protein gene sequence of CTLV showed that all the 144 isolates were clearly divided into two major branches. The first major branch contained 98 isolated samples, and the second contained 46 isolated samples. From the perspective of townships, the isolates from 31 townships distributed in the first branches, while 21 townships samples distributed in the second branches, and the isolates from 16 townships distributed in both branches. From the perspective of counties, 15 and 10 counties-isolates distributed in the first and second branches, respectively, and the isolates from 8 counties distributed in both branches. From the perspective of cities, the first major branch included the isolates from all of the 8 cities in Zhejiang Province, while the second included the isolates from 6 cities. The two major branches were further divided into 12 secondary branches, and the collected samples from Zhejiang Province were distributed in every branch. The samples collected from Taizhou City were also widely distributed in multiple secondary branches. From the perspective of citrus cultivars, among the isolates from 46 citrus cultivars, the isolates from 36 and 22 cultivars had clustered in the first and second major branch, respectively. The isolates from 12 cultivars had distributed in both branches. Among them, 36 and 21 isolates of the most abundant variety, Ehime Kashi No. 28, had clustered in the two major branches, respectively. From the perspective of the citrus types, 10 citrus types were distributed in the first major branch, while the second included 9 types. Among them, the distribution number of the isolates of Tangor type which had the highest quantity were 55 and 26 in the two major branches, respectively.【Conclusion】The occurrence of citrus tatter leaf disease was common in citrus production areas in Zhejiang Province, and it widely harms different citrus types and cultivars. The pathogen population showed rich genetic diversity, but there was no obvious correlation between its differentiation and geographical isolation or host varieties. The results of this study would provide a reference for further exploration of the origin, systematic evolution and pathogenic differentiation of the CTLV. At the same time, it seems to be possible that the proper procedure of variety introduction and nursery tree transportation would be the key to prevent the occurrence of the disease.