Without skepticism or suspicion, among the diseases that affect the citrus industry in São Paulo and the rest of the world, citrus leprosis (CL) is one of the most disturbing. Chronic in nature, CL causes localized lesions on leaves, branches and fruits (Figure 1). In the absence of adequate management, the main damage results from intense defoliation, fruit fall and progressive death of affected trees. The prevalent form of the disease in Brazil is spread by a tenuipalpid mite, flat, reddish-brown, whose adults measure just 0,3 mm. Once identified as members of the species Brevipalpus phoenicis, were reclassified into the species B. yothersi.
LC was originally described in Florida at the beginning of the 20th century, and from the 1920s onwards in Argentina, Paraguay and Brazil. Currently, it is found in most countries on the American continent, from Argentina to Mexico. Even though it has been undetectable in the USA since the 1960s, its re-emergence in that country is plausible considering its degree of dissemination in Mexico. Although apparently considered restricted to the New World, a 2009 report highlights the presence of LC in South Africa.
CL is a disease that spreads slowly, but is aggressive if inefficiently controlled. The losses are due to the reduction in productivity as a result of the fall of infected leaves and fruits, in addition to the eventual drying out of the branches and the death of the plant. All sweet orange trees are highly susceptible to the disease, with pomelos and tangerines being more tolerant, as lemon trees are apparently immune. CL management is mainly based on the use of specific acaricides against the mite vector, which involves spending approximately 50 million dollars annually. The use of acaricides is determined by the degree of infestation in the orchard, however continuous application of a given product can result in the selection of resistant mite populations. Consequently, the rotation of different active ingredients becomes essential, however, in a complex scenario where an increasing number of active ingredients have been gradually discontinued due to regulatory restrictions. In turn, partial or severe pruning of affected branches contributes to controlling LC. However, the action is economically disadvantageous as it requires abundant labor.
In recent years, highly technological plantations in the state of São Paulo have experienced a high incidence of CL. The concomitance of diseases, such as HLB (huanglongbing ou greening), a bacterial vascular disease (Candidatus Liberibacter asiaticus) spread by the psyllid diaphorina citri, black spot, caused by the fungus Phyllosticta citricarpa (guignrida citricarpa), and citrus canker, caused by the bacteria Xanthomonas citri subsp. citri, has required changes to the phytosanitary management of citrus farming. The use of insecticides and fungicides to control these other diseases can affect the natural enemies of the leprosy mite, favoring its reproduction. Furthermore, the procedure of mixing products to control two or more targets in the same operation in the spray tank, in certain cases, reduces the efficiency of the acaricide. In this context, strategies for controlling CL are being reevaluated.
The challenging severity of CL was discussed in a meeting coordinated by CAL Oliveira (Unesp/Jaboticabal), held in Bebedouro, São Paulo, in 1995. After this event, a multidisciplinary team was organized involving national researchers (Esalq/USP, IAC, IB, Unesp, Fundecitrus, Embrapa, etc.) and foreigners (Univ. Florida, Univ. Queensland, Univ. Okayama, Univ. Greifswald, Univ. Foggia, INRA, etc.), in order to conduct the identification and characterization of the virus that is the causal agent of CL , its epidemiology and virus/vector/host relationships. Research, with the support of Fapesp, has brought significant advances in the pathosystem of citrus leprosis, contributing to the management of the disease. As a corollary, the knowledge generated applies to other viruses transmitted by mites Brevipalpus (VTB), which have a potential negative impact on Brazilian agribusiness, for example on coffee and passion fruit crops.
The causal agent of CL prevalent in the state of São Paulo had its genome revealed in 2006. Being a different specimen from those previously identified, the one commonly recognized as the cytoplasmic leprosis virus was officially designated as citrus leprosis virus C (CiLV-C) by the International Committee on Virus Taxonomy. The unique characteristics of the virus, unprecedented on a taxonomic scale, led to the creation of a new genus, Cilevirus, which in addition to CiLV-C groups other viral species found in Brazil and other countries, and which affect not only citrus crops. Members of the genus Cilevirus, together with those of the genres Higrevirus e Blunervirus, make up the Kitaviridae family, whose name pays homage to Professor Elliot W. Kitajima, due to his contribution to the studies of these viruses.
The discovery of the CiLV-C genomic sequence allowed the establishment of a molecular detection method based on RT-PCR, making viral detection in samples of infected plants and viruliferous mites faster and more accurate. Molecular assays have identified the presence of two strains within the CiLV-C population. One of them, referred to as CiLV-C_CRD (from Cordeirópolis, São Paulo), is present throughout the Brazilian territory, and beyond national borders, from Argentina to Mexico. The second, designated CiLV-C_SJP (from São José do Rio Preto), was identified in this region. Ongoing epidemiological studies demonstrate a high prevalence of the SJP isolate in the citrus belt of the state of São Paulo and in the Triângulo Mineiro, suggesting that viruses of this strain have adaptive advantages.
Symptomological, cytopathological observations and molecular tests proved that CL symptoms can be caused by viruses of different natures. Currently, viruses that are members of two species of the genus are known. Cilevirus (fam. Kitaviridae) and three of the genus Dichorhavirus (fam. Rhabdoviridae).
Cilevirus: the most aggressive and widespread form throughout Brazil and a large part of the American continent is referred to as the cytoplasmic type or citrus leprosis C (LC-C). LC-C has CiLV-C as its main causal agent. Its particles are bacilliform and short, occur in the cytoplasm in cavities of the endoplasmic reticulum and infection results in the appearance of a vacuolated inclusion in the cytoplasm (Figure 2). In Colombia, in addition to CiLV-C, LC-C is also caused by citrus leprosis virus C2 (CiLV-C2). From a molecular point of view, CiLV-C2 is sufficiently distinct from CiLV-C that it is considered a member of a different species to that of CiLV-C. CiLV-C2 isolates were also detected in the states of Hawaii and Florida, in the USA, infecting only hibiscus plants.
Dichorhavirus: another form of leprosis, rare in occurrence and found in higher regions with milder temperatures such as Amparo, Monte Alegre do Sul, São Roque and Ibiúna, in the state of São Paulo, was called nuclear type citrus leprosis (LC -N) (Figure 3). The first virus identified as the causal agent of CL-N was citrus leprosis virus N (CiLV-N). CiLV-N infection results in the emergence of a nuclear inclusion (viroplasm) and the viral particles are in the form of short rods dispersed in the nucleus and cytoplasm (Figure 4). In Mexico, Colombia and South Africa, forms of LC-N were found (Figure 5) caused by isolates of a virus commonly known as the causal agent of orchid fleck virus (OFV). In Mexico, dichorhavirus OFV infects not only sweet orange trees, but a range of citrus plants including sour orange trees, tangerine trees, pomelo trees, and lemon trees. In Brazil, in addition to CiLV-N, the presence of another dichorhavirus was verified, the citrus chlorotic spot virus (CiCSV), which causes chlorotic spots on orange trees in Piauí (Figure 6). On the other hand, analyzes carried out on herbal samples that were collected from trees with LC symptoms in Florida in the 1960s, made it possible to recover part of the genome of what would be a dichorhavirus, tentatively designated as CiLV-N0, but presumably extinct.
The symptoms of CL caused by cile- and dichorhaviruses are, broadly speaking, similar, but have subtle differences. Leaf lesions caused by cileviruses are circular to ellipsoidal spots, light green in color, commonly with concentric rings of gum-containing tissue, reaching 20mm-30mm in diameter (Figure 1). Those caused by dichorhaviruses are generally smaller, with a dense central spot surrounded by a yolk-yellow halo (Figures 3 and 5). CiCSV, on the other hand, produces pale green lesions, forming bands, which can cover almost the entire leaf blade (Figure 6).
Both cile- and dichorhaviruses that cause CL are transmitted by tenuipalpid mites of the genus Brevipalpus. The involvement of these mites in leprosis was only established in 1940, in Argentina. Until recently the species B. phoenicis was recognized as the vector of CiLV-C. After reevaluating the identification criteria in 2015, with the introduction of new morphological parameters and the use of molecular markers, members of B. phoenicis were reevaluated in eight species, including some described and others cryptic. There was therefore a need to reconsider the species that would be vectors of CL. The species B. yothersi (Figure 7) is a vector for CiLV-C and CiLV-C2, while B. phoenicis sensu stricto transmits CiLV-N. OFV strains that infect citrus are transmitted by B. californicus, just as CiLV-N0 probably was. CiCSV infection is associated with infestation by B. ugh. yothersi. Recent surveys show that B. yothersi is the main species of Brevipalpus found in Brazilian citrus groves.
The mites Brevipalpus they are herbivores and suckers (Figure 8). Many species, including those involved in the transmission of CL, reproduce by thelytocal parthenogenesis (females generating females). These females are haploid and would be males if it were not for the presence of the symbiotic bacteria Candidatus Cardinium, which feminizes them. Consequently, the presence of males in natural populations is rare and must be the result of eggs not infected by the bacteria. All phases of development Brevipalpus (larvae, proto- and deutonymph, adults and males) are capable of transmitting CiLV-C, but transovarian transmission has not been detected. The relationship between CiLV-C and the mite is persistent, that is, the virus acquired by the mite circulates throughout its body and is inoculated into the plant along with saliva during the feeding process. Around four hours would be the minimum feeding period for the acquisition of CiLV-C by the mite, and approximately two hours, for its inoculation in the orange tree, requiring around seven hours after acquisition (latency period) for infection to occur. In the mite, CiLV-C was detected in the intercellular spaces. Although confirmation by molecular methods is awaited, this evidence suggests that CiLV-C does not multiply in the mite. Under the electron microscope, the presence of dichorhavirus causes LC-N in viruliferous mites, causes cytological changes similar to those in the cells of leaf lesions (viroplasm in the nucleus and abundant viral particles), clearly indicating that they multiply in the mite.
Although it is known that CL has multiple causal agents, CiLV-C is still, by far, the most important in Brazil. However, under favorable conditions, any of the other identified viruses could spread rapidly and induce losses, perhaps requiring different strategies.
The simultaneous occurrence of CL with other important diseases in citrus production causes significant changes in the phytosanitary management of the orchard infected with CiLV-C. Economic and other crises (Covid-19, for example) contribute negatively to the quality of disease management. New procedures are being studied to improve control of the leprosy mite, such as the development of biological control (with predatory mites and entomopathogenic fungi), analysis of images obtained by satellites and drones, studies on the combination of pesticides and the mite's resistance to available acaricides, evaluation of genetic resistance in citrus and hybrids, transgenics, etc. It is expected that the introduction of these measures in combination with improvements in the definition of critical moments for the application of chemical agents and in the monitoring of the mite vector and the disease through the training of pest pests can keep the causal agents of citrus leprosis under control.
PL Ramos-González, Biological Institute; J. Freitas-Astúa, Instituto Biológico, Embrapa Cassava and Fruit Culture; RB Bassanezi, Fundecitrus, Araraquara, SP; C. Chabi-Jesus, Instituto Biológico, São Paulo, SP, Esalq/USP, Piracicaba, SP; AD Tassi, Instituto Biológico, Esalq/USP, Piracicaba, SP; EW Kitajima Esalq/USP, Piracicaba, SP
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