In recent years, citrus production has rapidly increased within the state of Georgia (USA), and there are now citrus plantings within at least 32 counties in residential, production, and nursery settings. Among the pathogens capable of infecting citrus are viroids, the smallest plant pathogens. Viroids are comprised of circular, single-stranded RNA ranging from 246-463 nucleotides in length (Ito et al., 2002). Hop stunt viroid (HSVd) is one of several viroids known to infect citrus. This viroid has been previously reported within Arizona, California, Florida, Texas, and Washington in the United States and in other locations throughout the world (Hadidi, 2017). HSVd is often spread mechanically on contaminated tools or through grafting. With a wide host range that includes the families Moraceae, Rosaceae, and Rutaceae (citrus), this viroid can easily move throughout a nursery and spread to other plants (Hadidi, 2017). Symptoms of HSVd include a discoloration and gumming of phloem tissues, stem pitting, bark splitting, and chlorotic and stunted growth in susceptible citrus varieties including tangerines and their hybrids (Hadidi, 2017). There are not typically symptoms on leaves or fruits; however, lime plants have shown some yellowing on leaves (Hadidi, 2017). In May and June of 2020, leaf samples were collected from 12 different citrus plants in nursery settings in Berrien and Mitchell counties in Georgia. The cultivars sampled from Citrus reticulata 'Dekopon'. The sampled trees looked relatively healthy with little or no signs of damage, but were selected for testing to ensure that they were viroid free. Reverse transcription-polymerase chain reaction (RT-PCR) was initially used to verify infection with HSVd. Genomic RNA was extracted from the leaf tissue of twelve plants using the TRIzol reagent (Thermofisher, Waltham, MA). Following cDNA synthesis, samples were tested for the presence of HSVd using the primer pair HSVd-F (5'-GGCAACTCTTCTCAGAATCCAGC-3') and HSVd-R (5'-CCGGGGCTCCTTTCTCAGGTAAGT-3') which produces a 302 bp amplicon (Sano et al., 1988). The PCR reactions for nine of the tested samples did not result in the production of any bands, however the other three samples, all Citrus reticulata 'Dekopon', produced the expected amplicon for HSVd. The amplified products were sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and the identity of the fragment sequences was confirmed using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial sequences from these amplicons (deposited as accession number MT632007) shared 99% identity to corresponding HSVd sequences in Genbank (accession number MG779542). In addition to RT-PCR and sequencing, the recombinase-polymerase-amplification (RPA) technology based AmplifyRP® Acceler8™ end-point detection assay (Agdia® Inc., Elkhart, IN) was performed on previously confirmed tissue according to the manufacturer's instructions. This assay also confirmed the presence of HSVd viroid in the three samples that had been previously confirmed via RT-PCR. To the best of our knowledge, this is the first report of HSVd infecting Citrus reticulata 'Dekopon' in Georgia. If this viroid were to spread within the growing Georgia citrus industry, it could pose a significant threat to citrus plantings that contain susceptible varieties. Nursery stock infected with this viroid should be destroyed, and Georgia nursery producers and citrus growers should take appropriate precautions to prevent the spread of this viroid disease, including properly sanitizing tools used for citrus grafting and pruning. Further research is needed to determine the distribution of HSVd and its potential to impact commercial citrus production in Georgia.

中文翻译：

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美国佐治亚州的Hop特技类病毒感染柑橘树的首次报道。

近年来，在美国佐治亚州内，柑橘的产量迅速增加，现在在居住，生产和苗圃环境中的至少32个县中有柑橘种植。在能够感染柑橘的病原体中，类病毒是最小的植物病原体。类病毒由长度为246-463个核苷酸的环状单链RNA组成（Ito等，2002）。蛇麻草特技类病毒（HSVd）是已知感染柑橘的几种类病毒之一。先前曾在美国的亚利桑那州，加利福尼亚州，佛罗里达州，德克萨斯州和华盛顿州以及世界其他地方报道过这种类病毒（Hadidi，2017年）。HSVd通常在受污染的工具上或通过嫁接机械传播。寄主包括桑科，蔷薇科和芸香科（柑橘），这种类病毒很容易在整个苗圃中移动并传播到其他植物（Hadidi，2017年）。HSVd的症状包括韧皮部组织变色和发粘，茎点蚀，树皮裂开以及易感柑橘品种（包括橘子及其杂种）的褪绿和发育不良（Hadidi，2017）。叶子或果实通常没有症状。然而，石灰植物叶片上会出现黄色（Hadidi，2017）。2020年5月和6月，在佐治亚州Berrien和Mitchell县的苗圃环境中，从12种不同的柑橘植物中采集了叶片样品。从柑桔'Dekopon'中取样的品种。采样的树木看起来相对健康，几乎没有或没有损坏的迹象，但选择进行测试以确保它们不含类病毒。逆转录聚合酶链反应（RT-PCR）最初用于验证HSVd的感染。使用TRIzol试剂（Thermofisher，沃尔瑟姆，MA）从十二种植物的叶子组织中提取基因组RNA。cDNA合成后，使用产生302 bp扩增子的引物对HSVd-F（5'-GGCAACTCTTCTCAGAGTCCAGC-3'）和HSVd-R（5'-CCGGGGCTCCTTTCTCAGAGAAGT-3'）测试样品中HSVd的存在（Sano等。等，1988）。9个测试样品的PCR反应未产生任何条带，但是其他3个样品（均为柑桔'Dekopon'）均产生了预期的HSVd扩增子。使用Sanger测序（Retrogen Inc，San Diego，CA，美国），并使用BLAST分析（https://blast.ncbi.nlm.nih.gov/Blast.cgi）确认了片段序列的同一性。这些扩增子的部分序列（保藏号为MT632007）与Genbank中相应的HSVd序列（保藏号为MG779542）具有99％的同一性。除RT-PCR和测序外，还根据制造商的说明对先前确认的组织进行了基于重组酶-聚合酶-扩增（RPA）技术的AmplifyRP®Acceler8™终点检测法（Agdia®Inc.，Elkhart，IN） 。该测定还证实了先前已通过RT-PCR确认的三个样品中HSVd类病毒的存在。据我们所知，这是HSVd感染佐治亚州网纹柑桔'Dekopon'的第一份报告。如果这种类病毒在不断发展的佐治亚州柑桔产业中传播，可能会对含有易感品种的柑桔种植构成重大威胁。应当销毁感染了这种类病毒的苗圃，佐治亚州的苗圃生产者和柑橘种植者应采取适当的预防措施，以防止这种类病毒疾病的蔓延，包括对用于柑橘嫁接和修剪的工具进行适当的消毒。需要进一步研究以确定HSVd的分布及其对佐治亚州商业柑橘生产的影响。佐治亚州的苗圃生产者和柑橘种植者应采取适当的预防措施，以预防这种类病毒疾病的蔓延，包括对用于柑橘嫁接和修剪的工具进行适当的消毒。需要进一步研究以确定HSVd的分布及其对佐治亚州商业柑橘生产的影响。佐治亚州的苗圃生产者和柑橘种植者应采取适当的预防措施，以预防这种类病毒疾病的蔓延，包括对用于柑橘嫁接和修剪的工具进行适当的消毒。需要进一步研究以确定HSVd的分布及其对佐治亚州商业柑橘生产的影响。