Brevipalpus yothersi Baker (Tenuipalpidae) development in sweet orange plants is influenced by previous mite infestation and the presence of shelters.

Citrus leprosis is the most important viral disease affecting citrus. The disease is caused predominantly by CiLV-C and is transmitted by Brevipalpus yothersi Baker mites. This study brings some insight into the colonization of B. yothersi in citrus [(Citrus × sinensis (L.) Osbeck (Rutaceae)] previously infested by viruliferous or non-viruliferous B. yothersi. It also assesses the putative role of shelters on the behavior of B. yothersi. Expression of PR1 and PR4 genes, markers of plant defense mechanisms, were evaluated by RT-qPCR to correlate the role of the plant hormonal changes during the tri-trophic virus-mite-plant interplay. A previous infestation with either non-viruliferous and viruliferous mites positively influenced oviposition and the number of adult individuals in the resulting populations. Mite populations were higher on branches that had received a previous mite infestation than branches that did not. There was an increase in the expression of PR4, a marker gene in the jasmonic acid (JA) pathway, in the treatment with non-viruliferous mites, indicating a response from the plant to their feeding. Conversely, an induced expression of PR1, a marker gene in the salicylic acid (SA) pathway, was observed mainly in the treatment with viruliferous mites, which suggests the activation of a plant response against the pathogen. The earlier mite infestation, as well as the presence of leprosis lesions and a gypsum mixture as artificial shelters, all fostered the growth of the B. yothersi populations after the second infestation, regardless of the presence or absence of CiLV-C. Furthermore, it is suggested that B. yothersi feeding actually induces the JA pathway in plants. At the same time, the CiLV-C represses the JA pathway and induces the SA pathway, which benefits the mite vector.

A Breakthrough in Kitavirids: Genetic Variability, Reverse Genetics, Koch's Postulates, and Transmission of Hibiscus Green Spot Virus 2.

Hibiscus green spot virus 2 (HGSV-2), a member of the genus Higrevirus (family Kitaviridae), is a positive-stranded RNA virus associated with leprosis-like symptoms in citrus and green spots on leaves in hibiscus. HGSV-2 has only been reported in Hawaii, and while it is speculated that mites in the genus Brevipalpus might be responsible for its transmission, proper transmission assays have yet to be conducted. This study characterizes additional citrus and hibiscus isolates of HGSV-2 collected from two Hawaiian Islands. We constructed an infectious cDNA clone from a hibiscus isolate of HGSV-2 collected on Oahu and demonstrated its ability to infect several experimental hosts, including Phaseolus vulgaris, Nicotiana tabacum, and N. benthamiana, as well as natural hosts, Citrus reticulata and Hibiscus arnottianus. Bacilliform virions with varied sizes of 33 to 120 nm (length) and 14 to 70 nm (diameter) were observed in partially purified preparations obtained from agroinoculated leaves. Virus progeny from the infectious cDNA clone was found to be infectious after mechanical transmission to N. benthamiana and to cause local lesions. Finally, an isoline colony of the mite Brevipalpus azores had vector competence to transmit a citrus isolate of HGSV-2 collected from Maui to citrus and hibiscus plants, demonstrating the mite-borne nature of HGSV-2. The infectious cDNA clone developed in this study is the first reverse-genetics system for a kitavirid and will be fundamental to better characterize basic biology of HGSV-2 and its interactions with host plants and mite vectors.

Rescue of a Cilevirus from infectious cDNA clones.

Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agroinfiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.

Kitaviruses: A Window to Atypical Plant Viruses Causing Nonsystemic Diseases.

Kitaviridae is a family of plant-infecting viruses that have multiple positive-sense, single-stranded RNA genomic segments. Kitaviruses are assigned into the genera Cilevirus, Higrevirus, and Blunervirus, mainly on the basis of the diversity of their genomic organization. Cell-to-cell movement of most kitaviruses is provided by the 30K family of proteins or the binary movement block, considered an alternative movement module among plant viruses. Kitaviruses stand out for producing conspicuously unusual locally restricted infections and showing deficient or nonsystemic movement likely resulting from incompatible or suboptimal interactions with their hosts. Transmission of kitaviruses is mediated by mites of many species of the genus Brevipalpus and at least one species of eriophyids. Kitavirus genomes encode numerous orphan open reading frames but RNA-dependent RNA polymerase and the transmembrane helix-containing protein, generically called SP24, typify a close phylogenetic link with arthropod viruses. Kitaviruses infect a large range of host plants and cause diseases of economic concern in crops such as citrus, tomato, passion fruit, tea, and blueberry.

A method for reducing the concentrations of Fusarium graminearum trichothecenes in durum wheat grain with the use of Debaryomyces hansenii.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is one of the most dangerous diseases of durum wheat. This hemibiotrophic pathogen transitions from the biotrophic phase, during which it penetrates host tissues and secretes trichothecenes, to the necrotrophic phase which leads to the destruction of host tissues. Yeasts applied to spikes often reduce mycotoxin concentrations, but the underlying mechanisms have not been fully elucidated. Therefore, the aim of this study was to analyze the concentrations trichothecenes in durum wheat grain and changes in the F. graminearum transcriptome under the influence the Debaryomyces hansenii antagonistic yeast strain. Debaryomyces hansenii cells adhered to and formed cell aggregates/biofilm on the surface of spikes and pathogenic hyphae. Biological control suppressed the spread of F. graminearum by 90 % and decreased the content of deoxynivalenol (DON) in spikes by 31.2 %. Yeasts significantly reduced the expression of pathogen's genes encoding the rpaI subunit of RNA polymerase I and the activator of Hsp90 ATPase, but they had no effect on mRNA transcript levels of genes encoding the enzymes involved in the biosynthesis of trichothecenes. The yeast treatment reduced the number of F. graminearum operational taxonomic units (OTUs) nearly five-fold and increased the number of D. hansenii OTUs more than six-fold in the spike mycobiome. The mechanisms that suppress infections should be explored to develop effective biological methods for reducing the concentrations mycotoxins in wheat grain.

Generation of Fusarium oxysporum-suppressive soil with non-soil carriers using a multiple-parallel-mineralization technique.

Disease-suppressive soils exist worldwide. However, the disease-suppression mechanism is unknown, and it's unclear how to produce such soils. The microbiota that develop in a multiple-parallel-mineralization system (MPM) can increase nutrient production efficiency and decrease root disease in hydroponic systems. Artificial media inoculated with MPM microorganisms can degrade organic matter to produce inorganic nutrients similarly to natural soil, but it's unknown whether they can also suppress pathogen growth. Here, we produced an artificial medium that inhibited root disease similarly to disease-suppressive soils. Microbial MPM culture solution was inoculated into non-soil carriers (rockwool, rice husk charcoal, and vermiculite) to test whether it could suppress growth of Fusarium oxysporum f. sp. lactucae J. C. Hubb. & Gerik. We inoculated F. oxysporum f. sp. conglutinans (Wollenweber) Snyder et Hansen strain Cong:11 and F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik into artificial media sown each with Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. var. capitata supplemented with MPM culture microbes. The MPM microorganisms suppressed F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik growth and prevented plant disease. Thus, MPM-inoculated non-soil carriers that can generate inorganic nutrients from organic matter may also suppress disease in the absence of natural soil. Our study shows novel creation of a disease-suppressive effect in non-soil media using the microbial community from MPM culture solution.

Impact of diseases and pests on premature fruit drop in sweet orange orchards in São Paulo state citrus belt, Brazil.

BACKGROUND: Despite technical improvements in the citrus chain and leadership in orange production achieved in the past decades, premature fruit drop remains a major component of crop loss in São Paulo state citrus belt, the largest sweet orange production area in the world. The present study aimed to determine, during five consecutive seasons, the impact of the diseases and pests on premature fruit drop in the orange belt. RESULTS: Fruit drop due to the main diseases and pests averaged approximately 11.0%, which corresponded to approximately 63% of the annual fruit drop. The average fruit drop rate due to fruit borer and fruit flies combined was 4.0%, Huanglongbing (HLB) 3.3%, black spot 2.6%, leprosis 1.0% and citrus canker 0.3%. The average amount of fruit drop (million 40.8 kg boxes) and value of crop losses (million US$ dollars), in five seasons, were 12.7 and 66.2 for fruit borer/fruit flies, 11.0 and 57.9 for HLB, 8.1 and 42.2 for black spot, 3.1 and 15.6 for leprosis, and 0.9 and 4.9 for citrus canker, respectively. CONCLUSION: Fruit borer and fruit flies (combined), HLB, black spot, leprosis and citrus canker are, in this order, the main diseases and pests in the orange belt of São Paulo state. All of these causes significantly increased the overall fruit drop rate in the evaluated seasons. The results will contribute to the development of the Brazilian citrus industry, while showing to other citrus-growing regions the potential that diseases and pests have to jeopardize production. © 2022 Society of Chemical Industry.

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds.

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers' health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A.flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.

Transcriptional responses to Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder & Hansen infection in three Colombian tomato cultivars.

BACKGROUND: Fusarium oxysporum f. sp. lycopersici (Fol) is a compendium of pathogenic and non-pathogenic fungal strains. Pathogenic strains may cause vascular wilt disease and produce considerable losses in commercial tomato plots. To gain insight into the molecular mechanisms mediating resistance to Fol in tomato, the aim of our study was to characterize the transcriptional response of three cultivars (CT1, CT2 and IAC391) to a pathogenic (Fol-pt) and a non-pathogenic (Fo-npt) strain of Fo. RESULTS: All cultivars exhibited differentially expressed genes in response to each strain of the fungus at 36 h post-inoculation. For the pathogenic strain, CT1 deployed an apparent active defense response that included upregulation of WRKY transcription factors, an extracellular chitinase, and terpenoid-related genes, among others. In IAC391, differentially expressed genes included upregulated but mostly downregulated genes. Upregulated genes mapped to ethylene regulation, pathogenesis regulation and transcription regulation, while downregulated genes potentially impacted defense responses, lipid transport and metal ion binding. Finally, CT2 exhibited mostly downregulated genes upon Fol-pt infection. This included genes involved in transcription regulation, defense responses, and metal ion binding. CONCLUSIONS: Results suggest that CT1 mounts a defense response against Fol-pt. IAC391 exhibits an intermediate phenotype whereby some defense response genes are activated, and others are suppressed. Finally, the transcriptional profile in the CT2 hints towards lower levels of resistance. Fo-npt also induced transcriptional changes in all cultivars, but to a lesser extent. Results of this study will support genetic breeding programs currently underway in the zone.

Potential areas for the establishment of citrus leprosis virus vectors, Brevipalpus spp., in Mexico.

Citrus leprosis is a viral disease vectored by the mites Brevipalpus californicus and Brevipalpus yothersi. This work aimed to determine the potential areas for establishment of both mites and viruses in Mexico, based on the geographical distribution of the hosts and the climatic suitability for the vectors. Life tables of both mites were constructed to determine their thermal requirements-base temperature and degree-days required to complete life cycle-and population growth parameters-net reproduction rate, generation time, and intrinsic growth rate. For this, the mites were confined in Citrus aurantium fruits at 20, 22.5, 25 or 30 °C, 60 ± 5% RH and L14:D10 h photoperiod. Maps were generated where the climatic suitability for establishment of the mites and the citrus leprosis viruses was estimated in citrus-producing municipalities. The climatic suitability was determined through historical temperature records to calculate the potential number of generations per year, and ecological niche modeling based on collecting localities and bioclimatic variables using the algorithm Maxent. The base temperature was 9.5 °C for B. californicus and 10.2 °C for B. yothersi; degree-days required to reach adulthood were 372.1 and 331.7 °C, respectively. Potential sites for establishment of B. yothersi are mostly lowlands, whereas for B. californicus they are both lowlands and highlands. Temperature data indicate that B. californicus has fewer sites where it can develop > 16 generations per year than B. yothersi. According to our results, the sites where citrus leprosis is most likely to present high incidence are the sweet orange cultivars bordering the Gulf of Mexico.

Effect of Debaryomyces hansenii and the antifungal PgAFP protein on Alternaria spp. growth, toxin production, and RHO1 gene expression in a tomato-based medium.

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 µg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 µg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

Unravelling the involvement of cilevirus p32 protein in the viral transport.

Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.

Survey of the citrus leprosis vector (Brevipalpus yothersi) and phytoseiids in spontaneous plants of an organic citrus orchard.

Citrus leprosis (CL) is one of the most important viral diseases in sweet orange orchards in Latin America. It is caused by members of at least five species of the so-called Brevipalpus-transmitted viruses (BTV), and the prevalent is Citrus leprosis virus C (CiLV-C). This virus has the broadest host range amongst all CL-associated viruses and is transmitted by Brevipalpus yothersi, a polyphagous mite that can colonize a large variety of host plants, including some spontaneous ground cover plants. But if, on one hand, spontaneous plants can host CL virus and vector, on the other hand, they can offer alternative food for predators, equally common in organic citrus orchards. Brevipalpus yothersi and predator mites were surveyed in 33 spontaneous plants of a Westin sweet orange orchard conducted under organic production system in Brazil, from June 2010 to April 2011. Predatory mites were identified as phytoseiids, and Iphiseiodes zuluagai was the prevalent species, representing 58% of all predators. Other phytoseiids were considered accidental species in the area. Ageratum conyzoides and Alternanthera tenella were the most represented plant host species to predators, comprising 28 and 10% of the total surveyed plants, respectively. Brevipalpus yothersi specimens were detected on various spontaneous species: A. conyzoides, A. tenella, Amaranthus deflexus, Bidens pilosa, Ipomoea quamoclit, I. cairica, Merremia cissoides, Solanum americanum, Panicum maximum, and, predominantly, Commelina benghalensis. The latter has been previously reported as host of CiLV-C as well and, therefore, it is recommended to eliminate this species from citrus orchards.

Selection and application of antifungal VOCs-producing yeasts as biocontrol agents of grey mould in fruits.

Rotting caused by grey mould (Botrytis cinerea) is a concerning disease for numerous crops both pre- and postharvest stages. Application of antagonistic yeasts is a promising strategy for controlling grey mould incidence which could mitigate undesirable consequences of using synthetic fungicides. In this work, a screening for detection of yeasts isolated from figs producers of antifungal volatile organic compounds (VOCs) were performed by confrontation in double dishes systems. Eleven out of 34 yeasts confronted reduced B. cinerea growth parameter in vitro. This reduction was correlated (p ≤ 0.050) with the production of 10 volatile compounds: two acids (acetic acid and octanoic acid), 7 esters (Ethyl propionate, n-Propyl acetate, Isobutyl acetate, 2-methylbutyl acetate, furfuryl acetate, phenylmethyl acetate, 2-phenylethyl acetate) and one ketone (Heptan-2-one). In bases on in vitro assay, Hanseniaspora uvarum 793 was applied to in vivo assays with strawberries and cherries. The reduction of incidence of B. cinerea in strawberries at 7 °C and 25 °C was 54.9 and 72.1% after 6 and 3 days, respectively. The reduction of incidence of B. cinerea in cherries at 7 °C and 25 °C was 48.9 and 45.6% after 5 and 4 days, respectively. These results showed that VOCs produced by Hanseniaspora uvarum 793 are effective in the control of incidence of Botrytis cinerea in fruits, being a potential alternative to chemical fungicide.

Brevipalpus Species Vectoring Citrus Leprosis Virus (Cilevirus and Dichorhavirus).

Citrus leprosis (CL) is one of the most devastating viral diseases of orchards, and industries correspondingly invest highly in the management and control of the virus vector. In Brazil, the disease is caused most predominantly by the citrus leprosis virus C (CiLV-C, Kitaviridae: Cilevirus), and also by citrus leprosis virus N (CiLV-N, Rhabdoviridae: Dichorhavirus). Both viruses are transmitted by false spider mites and at least three different species, Brevipalpus yothersi Baker, B. papayensis Baker, and B. phoenicis (Geijskes) sensu stricto, have been reported in citrus orchards. The main goal of this study was to evaluate the capacity of three Brevipalpus species to transmit citrus leprosis virus (cytoplasmic and nuclear types). The capacity of false spider mites to acquire the virus was accomplished using RT-PCR and the ability to inoculation the virus to host plants (common bean and sweet orange) was assessed via viral transmission assays. Common beans infested with B. yothersi and B. papayensis showed symptoms of CiLV-C in 87.5 and 17% of the plants assessed, respectively. In sweet orange, B. yothersi was exclusively able to inoculate CiLV-C, and around 83% of samples were symptomatic. Host plants infected with CiLV-N showed symptoms only when infested with B. phoenicis sensu stricto (s.s.). All the Brevipalpus species (Acari: Tenuipalpidae) were able to acquire both viruses (CiLV-C and CiLV-N), but not infect plants. These results suggest the existence of virus-vector specificity in the leprosis pathosystem, and this information will be critical for enhancing our further understanding of epidemiological features and disease management.

Yeast communities of secondary peat swamp forests in Thailand and their antagonistic activities against fungal pathogens cause of plant and postharvest fruit diseases.

Secondary peat swamp forest (PSF) arise by degradation of primary PSF as a result of fire and human activities. Yeasts diversity of Kuan Kreng (KK) and Rayong Botanical Garden (RBG) PSF, which are two secondary PSF in southern and in eastern Thailand, respectively, were investigated. Yeasts were isolated from soil and peat soil by the dilution plate and enrichment techniques. From six samples collected from KK PSF, 35 strains were obtained, and they were identified based on the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene 13 species in 12 genera, and one potential new species of the genus Galactomyces were detected. Thirty-two strains were obtained from six samples collected from RBG PSF and 26 strains were identified as 13 known yeast species in 11 genera, whereas six strains were found to represent two potential new species of the genera Papiliotrema and Moesziomyces. Among yeast strains isolated from KK PSF, the number of strains in the phylum Ascomycota and Basidiomycota were equal, whereas there were slightly fewer strains in Ascomycota than in Basidiomycota among the strains obtained from RBG PSF. The yeast strains were evaluated for their antagonistic activities against fungal pathogens which cause rice diseases (Fusarium moniliforme, Helminthosporium oryzae, Rhizoctonia solani, Curvularia lunata and Pyricularia grisea) and postharvest disease of fruits (Phytophthora palmivora, Lasiodiplodia theobromae and Colletotrichum gloeosporioides). Twelve strains of seven species were found to be antagonistic yeast strains. Starmerella kuoi DMKU-SPS13-6, Hanseniaspora lindneri DMKU ESS10-9 and Piskurozyma taiwanensis DMKU-SPS12-2 capable to inhibit R. solani by 70.1-76.2%, Wickerhamomyces anomalus DMKU SPS6-1 and three Rhodotorula taiwanensis strains (DMKU SPS8-1, DMKU ESS9-3, DMKU SPS9-2) inhibited C. lunata by 69.8-71.9%, Hanseniaspora lindneri DMKU ESS10-9 and Scheffersomyces spartinae DMKU SPS9-3 inhibited P. grisea by 81.9-84.4% and four Papiliotrema laurentii strains (DMKU-SPS15-1, DMKU-ESS11-2, DMKU-ESS8-2, DMKU-ESS6-4) inhibited P. palmivora by 53.2-59.5%.

Incidence of Citrus leprosis virus C and Orchid fleck dichorhavirus Citrus Strain in Mites of the Genus Brevipalpus in Mexico.

The incidences of Citrus leprosis virus C (CiLV-C) and Orchid fleck dichorhavirus Citrus strain (OFV-citrus) were determined in field populations of Brevipalpus mites from 15 citrus-producing states in Mexico. Mites were collected from orange, grapefruit, mandarin, lime, and sweet lime orchards. Brevipalpus yothersi (Baker) (Trombidiformes: Tenuipalpidae) was the most abundant species followed by Brevipalpus californicus (Banks) (Trombidiformes: Tenuipalpidae), which confirmed previous reports. The viruses CiLV-C and OFV-citrus were found in both mite species. The incidence of CiLV-C, OFV-citrus and both viruses simultaneously (CiLV-C and OFV-citrus) was 17.2, 10.3, and 3.4% (n = 116) for B. yothersi, and 12.5, 20.8, and 4.1% (n = 24) for B. californicus, respectively. No significant difference was found when the incidence of these viruses was compared between both mite species. The importance of our results in relation to the epidemiology of leprosis is discussed.

Spatiotemporal association between the mite Brevipalpus yothersi and Citrus leprosis virus C in orange orchards.

Citrus leprosis virus C (CiLV-C) is an economically important pathogen and the main causative agent of leprosis disease in citrus orchards. The main vector of this disease, the mite Brevipalpus yothersi, is widely distributed in Mexican orchards on a wide range of citrus species. Despite the importance of both the virus and the mite, field studies recording their occurrence and co-occurrence are practically non-existent. We systematically sampled orange orchards for both CiLV-C and B. yothersi throughout the year. The distribution of the CiLV-C and B. yothersi was evaluated on each sampling occasion and their spatiotemporal associations were determined. Specifically, 100-112 orange trees, distributed in 18 rows (five or six trees per row), were sampled monthly between March 2017 and February 2018 (11 sampling dates). Twenty leaves per tree were sampled on each occasion. The number of mites per tree and the percentage of leaves per tree with disease symptoms were recorded. On each sampling occasion, spatiotemporal associations between mites and disease were determined using the Spatial Analysis by Distance Indices (SADIE) method. CiLV-C and B. yothersi were identified using molecular methods. Throughout the study, the distribution of CiLV-C was aggregated and the distribution of B. yothersi was random. No association was found between the virus and the mite on any of the sampling dates. In total, 173 mites were collected, but only 43 mites were found to be carrying CiLV-C. The reason for this lack of association between the virus and the mite, as well as the impact of our findings on the epidemiology of the disease in orange orchards, are discussed.

Transmission of Citrus leprosis virus C by the Mite, Brevipalpus yothersi (Acari: Tenuipalpidae), on Four Species of Citrus.

Transmission of the virus, Citrus leprosis virus C (CiLV-C) (Cilevirus) by Brevipalpus yothersi Baker, on different citrus species was evaluated under greenhouse conditions. First, the relationship between acquisition access periods (AAPs; 1, 12, 24, 36, and 48 h) and virus concentration in mites was determined. Second, the ability of B. yothersi to transmit CiLV-C to orange, mandarin, grapefruit, and lime trees was measured. We then assessed the establishment of mites on the different citrus species as measured by their population increase on each species. We found no relationship between AAPs and virus load in mites. The virus was found in all mites tested but there was no difference in virus quantities among the treatments. We selected an AAP of 24 h for the transmission experiment. Brevipalpus yothersi transmitted the virus to all citrus species evaluated, but susceptibility was different. The number of infected leaves was greater on orange and mandarin compared with grapefruit and lime. Furthermore, populations of B. yothersi successfully established on orange and mandarin, but not on grapefruit and lime trees. The implications of our results in the virus-mite-citrus plant relationship are discussed.

Reference genes for gene expression studies by RT-qPCR in Brevipalpus yothersi (Acari: Tenuipalpidae), the mite vector of citrus leprosis virus.

Quantitative reverse transcription PCR (RT-qPCR) is a high-throughput method to analyze the transcriptional expression of genes. Currently, no reference genes have been described for evaluating gene expression in Brevipalpus yothersi, the false spider mite, a polyphagous that act as vector of the citrus leprosis virus C (CiLV-C), an important citrus disease. This study aimed to identify the most stable reference genes in B. yothersi. The RT-qPCR expression data for selected genes were evaluated from three conditions: different developmental stages, plant hosts and acquisition of CiLV-C. To analyze the stability of the candidate reference genes we used ΔCq method, GeNorm, NormFinder, BestKeeper and RefFinder. Ubiq and GAPDH are best suited for normalizing gene expression data in viruliferous and non-viruliferous mites. Ubiq, EF1α and GAPDH are the most stable for different developmental stages. RPL13 and RPL32 are the best reference genes for approaches to B. yothersi in different host plants. Considering all the experimental conditions, Ubiq, EF1α, and GAPDH were the most stable genes. Here we developed an accurate and comprehensive RT-qPCR strategy for use in B. yothersi gene expression analysis. These results will improve the understanding of the biology of the false spider mites and their role as virus vectors.