The latest progress in the research of citrus tree virus, interpreted by experts

The latest progress in the research of citrus tree virus, interpreted by experts

Zhou Yan

Citrus recession disease caused by citrus recession virus (CTV) is an important citrus disease that has occurred in all major citrus producing regions of the world since it was first recorded in Australia in 1860, and has dealt a devastating blow to the citrus industry in Brazil, Venezuela, Argentina, the United States, South Africa and other countries. By the 90s of the 20th century, more than 100 million citrus plants had died worldwide due to citrus recession disease. In recent years, the decline of citrus in the main citrus producing areas of Jiangxi, Sichuan, Yunnan, Hunan, Guangxi and other major citrus producing areas in mainland China has occurred frequently, which has caused serious losses to the local citrus industry. This paper reviews the relevant research on citrus recession disease, in order to provide a reference for future research and prevention and control of citrus recession disease.

1 Host range

在自然条件下,CTV 主要为害芸香科 Citrus (柑桔属)、 Poncirus (枳属)和 Fortunella (金柑属)植物。 在实验条件下,CTV 亦可感染芸香科 Aegle (木桔属)、 Aeglopsis (拟硬皮桔属)、Afraegle 、 Atalantia (酒饼勒属)、 Citropsis (樱桃桔属)、 Clausena (黄皮属)、 Eremocitrus (澳沙檬属)、 Hesperthusa 、Merrillia (茉莉果属)、 Microcitrus (澳指檬属)、 Pamburus (灰叶桔属)、Pleiospermium (东方橙属)、 Swinglea (菲律宾木桔属)等植物 ,以及非芸香科的Passifloracaerulea (蓝花西番莲)、 P . gracilis (细柱西番莲)和 Nicotianabenthamiana (本生烟 )等植物 。

2 Types of recessionary diseases and their damages

Depending on the combination of anvils and varieties, there are 3 types of decay disease.

(1) Rapid decay disease: causes the death of sweet oranges, broad-skinned citrus and grapefruit with lime and citron as rootstocks. According to the speed of onset, it can be divided into rapid decay type and general decline type. Rapid decay disease is the most important type of citrus recession disease in South America, the Caribbean, as well as the United States, South Africa, Spain and other countries. The mainland does not use or uses less lime and citron rootstocks, and mainly grows disease-resistant wide-skinned citrus, so there have been sporadic reports of rapid decay diseases only in Jianshui and Binchuan, Yunnan.

(2) Stem trap type recession disease: this type has nothing to do with the rootstock varieties used, and mainly occurs in lime, grapefruit, orange, most pomelos and some sweet oranges (such as golden orange, navel orange, and Pera sweet orange). Prismatic, yellow-brown honeycomb pitfalls of different sizes appeared on the xylem surface of diseased plants, and even glue filling, resulting in easy branch breakage, dwarf plants, weakened tree strength, smaller fruits, and lower quality. Stem punctate recession disease is the main type of citrus recession disease in mainland China, Australia, Japan and other countries.

(3) Seedling yellow recession disease: under the condition of temperature control, it causes severe dwarf and yellowing of lime, Yulik lemon, grapefruit and wild orange seedlings in Daoxian County.

3 Strain differentiation

According to the differences in genome sequences, CTV is divided into multiple major genotypes such as T36, T30, VT, T3, T68, RB, S1 and HA16-5, and with the continuous development and improvement of deep sequencing technology, more and more CTV genotypes have been discovered one after another. Among them, the T36 genotype was associated with tachyaging symptoms, the T3, VT, RB, and T68 genotypes were associated with stem depression point symptoms, the VT genotype was associated with tachyaging and stem depression points, and the S1 and T30 genotypes usually did not cause obvious symptoms. However, recent studies have found that some CTV strains of the same genotype also have different biological traits, and CTV strains in the field often contain multiple genotypes, so it is difficult to accurately judge the biological traits of CTV strains based on genotype alone. In addition, due to the difficulty of CTV full sequence determination, regions such as open reading frame (ORF) 1a, 5' untranslated region (UTR), and RNA-dependent RNA polymerase (RdRp) are often used as important targets for CTV typing, among which ORF1a is the most diverse, which is the most widely used basis for CTV typing.

4 Epidemic law

CTV is mainly transmitted by grafting and a variety of aphids in a non-circulating semi-persistent manner, among which Aphiscitricida (brown orange aphid) has the strongest transmission ability and can efficiently transmit a variety of strains of CTV, especially the strong strains of CTV that are not easy to transmit or have potential harm from other aphids, so brown orange aphid is one of the important reasons for the invasion and rapid epidemic of foreign strong strains of CTV. A gossypii (cotton aphid) and A . Spiraecola (Meadowsweet aphid) is less virulent but is also an important vector of CTV because of its large occurrence in the field. A. craccivora (bean aphid), A . Aurantii (orange binary aphid), Myzuspersicae (peach aphid) and Dactynotusjaceae (finger tube aphid) can also transmit CTV, but their transmissibility is weak. In addition, under experimental conditions, CTV can also be transmitted by knife cutting and two types of dodder (Cuscutasubinclusa and C. americana).

5 Virus origin

The southeastern foothills of the Himalayas are the origin center of citrus, and most of the current citrus cultivars are the hybrid descendants of the earliest three types of citrus, such as citron, broad-skinned citrus and pomelo. Liu et al. (2021) analyzed the CTV strains collected in the natural ecology of wild citrus in the Ailao Mountains of Yunnan Province, China, and the results provided an important theoretical basis for the hypothesis that CTV originated in China and gradually spread to other countries with propagating material.

6 Viral protein function

CTV is a member of the genus Closterovirus, and the sense single-stranded RNA strand that makes up its genome is packed in 2000nm ×11nm helix-symmetrical mitochondria, making it the largest known plant virus with the largest genome. The genome of CTV contains 12 ORFs and is capable of encoding 17 proteins with molecular weights of 6~401kDa. Among them, the L1 and L2 domains composed of papain-like cysteine protease tandem structures in ORF1a are related to viral accumulation, replication, and establishment of primary infection sites, and CTV mutants with L2 domain deletion have a wider host range than wild-type CTV. The non-conserved multifunctional protein p33 encoded by ORF2 is currently a hot topic in the functional research of CTV protein. The transmembrane domain of the C-terminus of p33 protein is associated with the host range of CTV. Because p33 is distributed in plasmodesmata and cytoplasmic vesicles in plants, and can use the cellular secretion pathway and actin network for intracellular transport, and can also oligomerize to form a tubular structure, which affects the movement efficiency of viruses in limes and lemons, it may have atypical mobile protein functions. In addition to being involved in the movement and transport of the virus, p33 is also involved in cross-protection between CTV strains together with L1 and L2. Recent studies have also shown that p33 is necessary for aphids to transmit CTV. In addition, p61, heat shock protein 70 homolog (p65), minor coat protein (CPm), and major coat protein (CP) are involved in the assembly and movement of viruses. p33, p18 and p13 affect the ability of CTV to infect citrus varieties such as lime, lemon, grapefruit and Crimedine. As an RNA-binding protein, p23 regulates the proportion of positive and negative strand RNA during CTV replication, and is related to the production of Miao Huang symptoms and the pathogenicity of the virus. p23, CP, and p20 also have the activity of RNA silencing repressors.

In the process of CTV replication, in addition to the genes at the 5' end that can be directly translated, the synthesis of 10 genes at the 3' end requires the participation of subgenomic RNA (sgRNA) at the same 3' end as mRNA, and the closer to the 3' end, the expression of genes also increases correspondingly, but the expression of CP is higher than that of p 13 and p 18 closer to the 3' end. In addition, the expression of 3' genes is also affected by factors such as the +1 frameshift of the transcription start site, the type of gene upstream regulator, and the presence or absence of NTR in the gene upstream.

7 Virus-plant interactions

Due to the distribution of CTV in the phloem of plants and the slow growth of citrus, it is difficult to study CTV-plant interactions. Ruiz-Ruiz et al. (2018) found that CTV-p23 regulates the replication ratio of positive and negative sense RNA molecules and the movement of CTV between cells through the interaction with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at the cytoplasm and plasmodesmata of Buntophysma, thereby accelerating the pathogenesis of CTV. At the same time, p23 also interacted with the fk506-binding protein (FKBP17-2) to transfer it from Bunsen tobacco chloroplasts to plasmodesmata, thereby down-regulating the expression level of NbFKBP 17-2 mRNA and inhibiting the accumulation of CTV in Bunsen Nicotiana. In addition, p23 directs the CP/NbFKBP17-2 complex along the cell wall. The interaction between CTV and host proteins also attenuates the host's antiviral response. For example, CTV's long-chain non-coding sgRNALMT1 can promote CTV proliferation in plants by inducing the production of AOX-1a, an alternative oxidase, inhibiting salicylic acid (SA)-mediated defense pathways and reactive oxygen species (ROS) accumulation. The CTV-encoded silencing repressors p20 and CP inhibit the host RNA silencing response and promote CTV infection by interacting with AGO1 and AGO4, key components of the RNA silencing mechanism.

p33, as an important multifunctional protein of CTV, also plays an important role in CTV-host interaction. Sun et al. (2019) found that p33 could induce the accumulation of ROS in plants, while activating the expression of plant defense-related genes and the occurrence of programmed death (PCD), thereby inhibiting the invasion of CTV into plant phloem tissues and alleviating the occurrence of symptoms. Further studies have shown that p33-induced a similar miracle protein (MLP2) associated with host defense response inhibits the movement of CTV in the host by recognizing and hijacking p33. At the same time, CmMLP2 has the ability to recombinant the host membrane system, induce host stress response and ROS accumulation, and inhibit CTV replication by merging the endoplasmic reticulum and Golgi apparatus. In addition, p33 attenuated the RNA-silencing antiviral response of plants by interacting with CP, p23, and p20.

8 Mechanism of stem trap formation

The pathogenesis of CTV has always been one of the research focuses, but due to the difficulty in monitoring the movement of CTV in plants and the process of disease development, the progress of related research is relatively slow, and the research on the formation mechanism of stem traps is mainly focused on.

Schneider et al. (1957) hypothesized from anatomical observation of CTV-infected plants that stem pitting may be related to the destruction of the cambium layer and the stunting of normal xylem and phloem cell growth. Brlansky et al. (2002) suggested that the formation and accumulation of gelatinous material in the xylem cavity after CTV invasion of phloem cells is related to the formation of stem pits. Tatineni et al. (2008) speculated that the occurrence of stem pitting symptoms is related to the balance of expression between p33, p13, and p18 genes. Although CTV has long been thought to occur only in the phloem, in recent years it has also been detected in some mature xylem with severe stem trap symptoms. Subsequently, Sun et al. (2019, 2021) confirmed that, in addition to phloem-associated cells, CTV-infected mature xylem tubular cells and xylem parenchyma cells (radiological cells) are also important factors in inducing stem trap symptoms. In the early stage of infection, CTV invaded the protophloem and phloem, and then CTV invaded and colonized the xylem, disrupting the differentiation of xylem cells, resulting in the appearance of colloids and cell deformation. With the division of cambium cells and the radial spread of CTV, it further leads to the enlargement of the cone deformity, which eventually produces stem trap symptoms. The latest studies also showed that with the continuous aggravation of stem trap symptoms, the levels of callose and phloem protein gradually increased, and the expression of β-1, 3-glucanase gene decreased, indicating that the development of stem trap symptoms was related to the increase of phloem occlusion.

9 Mechanism of aphid transmission

Brown orange aphid is the most important vector of CTV, which can be poisoned by feeding on CTV strains for a few seconds to 30 minutes, and maintain the ability to transmit viruses within 24~48h. With the extension of feeding time, the poison transmission ability of brown orange aphid gradually increased, and its poison transmission ability reached the maximum value when feeding for 24 hours. In addition, the ability of brown orange aphid to transmit CTV was also affected by multiple factors, such as the source plant and the type of infected plant, environmental conditions, the genotype of the CTV strain, and the amount of CTV in the aphid.

Satyanarayana et al. (2004) speculated that the proteins encoded by CTV, such as p65, p61 and CP, may be involved in aphid transmission by comparing known aphid-related proteins in other viruses. Herron et al. (2006) found that feeding brown orange aphids with CTVp20 antibodies increased their efficiency in transmitting the CTV strain T66aH-1, suggesting that p20 protein was involved in aphid transmission. However, Zhou et al. (2007) found that there did not seem to be a significant association between the transmissibility of CTV and the p20 gene in P. brown orange aphid, and the CTV strains belonging to group 3 of CP/Hinf I. RFLP had strong transmissibility, suggesting that the CP gene may have played a role in aphid transmission. In recent years, it has been found that the virus-encoded CPm plays an important role in the insect transmission process in the long-line viridae family, such as beetyellowsvirus (BYV) and lettuceinfectiousyellows (LiYV), which are closely related to CTV The aphid transmission process of CTV.

Although various theories have been proposed about the genes that control the ability of CTV to transmit to aphids, it is difficult to construct recombinant viruses or deletions to screen and identify genes related to aphid transmission due to the large size of the CTV genome and the difficulty of obtaining full-length clones. It was not until the end of the 20th century that Satyanarayana et al. (1999) constructed a full-length infectious clone of CTV using the T36 strain as a template for the first time. However, the infected clone could only infect citrus after 4~6 consecutive transgenerations in Bunsen protoplasts, and the success rate was less than 1%. In order to solve this problem, Gowda et al. (2005) placed the whole sequence of CTV downstream of the 35S promoter in the Agrobacterium binary expression vector, and used the method of Agrobacterium inoculation with Bunsen tobacco and then extraction of virus to inoculate citrus, which greatly improved the ability of full-length infectious clones of CTV to infect citrus. On this basis, Harper et al. (2016) and Shilts et al. (2020) found that the p65, p61 and p33 of the high-aphid CTV strain FS577/T68-1 could significantly improve the aphid-transmissibility of the CTVT36 strain by constructing recombinant CTV. Immunofluorescence labeling further showed that CTV specifically bound to glycans on the surface of the foregut (sinus and mouth needle) of A. brown through CPm, and protease treatment did not affect the accumulation of CTV particles in A. brown, but the interaction between p61/p65 and CPm reduced the binding ability of CTV particles in the foregut of Aphids.

10 Prevention and control methods

The use of disease-resistant rootstocks such as citrus aurantium, citrus aurantium, red orange, and sour orange is the most effective way to prevent and control the rapid decay disease. Cross-protection technology can effectively prevent the occurrence of stem pitpoint recession disease, and this technology has been widely used in Brazil, Australia, Peru, South Africa and other countries to prevent and control stem pitpoint decline disease on sweet orange and grapefruit. However, the use of traditional methods to screen for attenuated strains with protective effects is not only time-consuming, but also difficult. Since cross-protection occurs only between CTV strains of the same genotype, the success rate of cross-protection can be improved by clarifying the composition and genotype of local virulent strains and selecting the corresponding attenuated strains through multiplex molecular markers or deep sequencing technology. In addition, the mixed use of attenuated CTV strains of different genotypes can further improve the control effect of cross-protection.

11 Outlook

As an important citrus virus in the world, CTV is still a constant threat to the development of the world's citrus industry. In recent years, most of the stem trap type decline diseases that have broken out in many places in mainland China are related to seedling diseases, so seedling safety is still the cornerstone of ensuring the healthy and sustainable development of the citrus industry. At present, the research on CTV at home and abroad mainly focuses on the monitoring of endemic areas, the identification of new strains, the verification of viral gene function, virus-host-aphid interaction, and the screening and application of protective strains, and the research has been relatively in-depth, but the research on the mechanism of citrus disease resistance is still insufficient. In the future, with the mining and identification of citrus disease resistance/susceptibility genes, as well as the development and maturity of gene editing technologies such as CRISPR-Cas9, it will help to create new varieties resistant to citrus recession disease, so as to provide an important guarantee for the healthy and sustainable development of the citrus industry.