Ávalos-Cerdas, Juan Manuel ¹ ; Otero-Colina, Gabriel ² ; Ochoa-Martínez, Daniel L. ³ ; Villegas-Monter, Ángel ⁴ ; Bautista-Martínez;, Néstor ⁵ ; Suárez-Espinosa, Javier ⁶ ; Carrillo-Benítez, María Guadalupe ⁷ ; Tassi, Aline Daniele ⁸ ; Ochoa, Ronald ⁹ and Valdez Carrazco, Jorge Manuel ¹⁰

¹Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.
²Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.
³Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.
⁴Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.
⁵Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.
⁶Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.
⁷Humus y Derivados de Lombriz de México, SPR de RI, Carretera Federal Puebla-Tehuacán Km. 42. C.P. 75214 El Empalme, Tepeaca, Puebla, México.
⁸Laboratório de Bioquímica Fitopatológica, Instituto Biológico, São Paulo, CEP 04014-002, Brazil.
⁹Systematic Entomology Laboratory, USDA-ARS, Beltsville, MD 20705, USA.
¹⁰Colegio de Postgraduados, Campus Montecillo, C.P. 56264 Texcoco, Estado de México, México.

2023 - Volume: 63 Issue: Suppl pages: 45-68

Proceedings of the 9^th Symposium of the EurAAc, Bari, July, 12^th–15^th 2022

Keywords

Abstract

Introduction

Due to their capacity to transmit devastating viruses to plants, mites in the genus Brevipalpus (Acariformes: Tetranychoidea: Tenuipalpidae) are of increasing economic importance across the planet (Mesa et al., 2009; Kitajima et al., 2010). Among the most important Brevipalpus-transmitted viruses or BTVs are those that cause citrus leprosis (CL) disease. CL viruses are divided into two genera – those in genus Cilevirus are cytoplasmic viruses [CiLV-C and citrus leprosis virus C2 (CiLV-C2)], and those in genus Dichorhavirus are nuclear viruses [OFV-citrus, citrus leprosis virus N (CiLV-N), and citrus chlorotic spot virus (CiCSV)] (Cruz-Jaramillo et al., 2014; Roy et al., 2015a, 2015b; Ramos-González et al., 2016, 2017; Chabi-Jesus et al., 2018; Dietzgen et al., 2018).

The capacity of Brevipalpus californicus (Banks) to transmit OFV-citrus and of Brevipalpus yothersi Baker to transmit CiLV-C has previously been investigated and proven (Ramos-González et al., 2016; García-Escamilla et al., 2017). In Mexico, these viruses have allopatric distributions that are associated with different altitudes; CiLV-C is found between 10 and 885 m asl, whereas OFV-citrus is found between 1,239 and 1,710 m asl (Duran-Trujillo, 2017). Likewise, the distributions of these viruses also appear to be associated with climatic conditions, in that CiLV-C is distributed in warmer climates (Izquierdo-Castillo et al., 2011) while OFV-citrus is found in colder, more temperate climates (Roy et al., 2015a).

Indicator plants are used in studies of BTVs to decrease the timeframe for the appearance of symptoms. For example, bean plants (Phaseolus vulgaris L.; Fabaceae) inoculated with B. californicus viruliferous for OFV showed yellow lesions in two–three weeks (Kondo et al., 2003), while bean plants inoculated with B. yothersi infected with CiLV-C showed necrotic spots in five days (Garita et al., 2013, 2014; Tassi et al., 2017). Also, Arabidopsis thaliana L. (Brassicaceae) developed chlorotic symptoms associated with CiLV-N and CiCSV 8–10 days after being inoculated with viruliferous Brevipalpus phoenicis (Geijskes) s.s. or a mixture of B. yothersi and B. aff. yothersi, respectively (Ramos-González et al., 2017; Chabi-Jesus et al., 2018). Arabidopsis thaliana is one of the best host models for the study of plant-pathogen interactions (Nishimura & Dangl, 2010) and has proven to be a useful indicator for several BTVs (Arena et al., 2016, 2017).

García-Escamilla et al. (2017) concluded that the different viruses involved in the citrus leprosis complex are associated with a specific mite vector, indicating that the correct identification of the mites involved is critical. The small size of Brevipalpus mites, combined with observed intraspecific morphological variation (Welbourn et al., 2003; Mesa et al., 2009; Navia et al., 2013), has resulted in numerous misidentifications and suspected and actual synonyms. As an example of the complexities involved in Brevipalpus taxonomy, using detailed analyses of morphological characteristics, Beard et al. (2015) proved that the taxon B. phoenicis alone represented at least eight different species.

Given the intraspecific variation and the existence of species complexes, many species in the genus Brevipalpus are difficult to distinguish when using traditional morphological criteria (Beard et al., 2015). These highly variable morphological features must be confirmed or linked with molecular techniques that allow the detection and separation of cryptic species within groups (Bickford et al., 2007). Several new species have recently been described, based on new morphological criteria and/or molecular markers, for example Brevipalpus incognitus Ferragut & Navia (Navia et al., 2013), Brevipalpus feresi Ochoa & Beard and Brevipalpus ferraguti Ochoa & Beard (Beard et al., 2015).

Brevipalpus lewisi McGregor infests citrus crops in Australia (Smith & Papacek, 1985), China (Hao et al., 2016), Greece (Hatznikolis 1986), South Africa (Saccaggi et al., 2017), and the United States of America (Pritchard and Baker, 1958; Mesa et al., 2009). Currently, no case of leprosis in citrus has been associated with this mite (Ochoa et al., 2016). During field surveys conducted by the authors in the municipality of Tetipac, Guerrero, Mexico, mite specimens were collected and were originally tentatively identified as B. lewisi, based on their morphological characteristics as described by Baker (1949), Baker and Tuttle (1987) and Beard et al. (2012). The main significant characters were the presence of one solenidion on each tarsus II, and the presence of setae f2 in all the examined specimens (n = 20). However, the ornamentations of the cuticle were interpreted as being more like those of B. californicus. Using the above-described specimens, a colony was established and reared on sour orange fruits (Citrus aurantium L.) and re-identified as ''Brevipalpus sp. (Tetipac)'', with the assumption that the species could be B. californicus, B. lewisi, or another related species. The objectives of the present research were to confirm the actual specific identity of these mites and to test their capacity as a vector of leprosis causing viruses (CiLV-C and OFV-citrus).

Material and methods

Establishment of Brevipalpus sp. (Tetipac) colonies

Specimens of ''Brevipalpus sp. (Tetipac)'' were collected in Muyuapan, municipality of Tetipac, Guerrero, Mexico (18°37′32.81″N; 99°41′56.79″W), an area considered free of leprosis (SENASICA, 2016). The host plants were sweet orange (Citrus x sinensis (L.) Osbeck.'Valencia') and Mexican lime [Citrus x aurantifolia (Christm.) Swingle]. Once the mites were obtained, single-parent or isoline colonies were established from a single female, following the method described by García-Escamilla et al. (2017) on sour oranges. A single adult female was placed on each orange and allowed to oviposit. After at least one egg was laid, the female was extracted to be mounted on a slide in Hoyer's solution (Krantz & Walter, 2009) for identification. Only one of the isolines could be established permanently, multiplied on several oranges and became the source of experimental material. About every two months, several specimens were taken from the infested oranges and mounted in temporary slides to confirm they were still morphologically distinguishable as Brevipalpus sp. (Tetipac).

The colony was incubated at 24–27°C and 40–50% relative humidity, with led illumination of 14.76 µmol/m²s and 12:12 h light period. The replacement oranges were obtained from the Campus Montecillo of the Colegio de Postgraduados and other nearby locations in the municipality of Texcoco, Mexico State (19°27′49.59″N, 98°54′19.92″W), which is also a leprosis-free zone (SENASICA, 2016). The oranges were previously cleaned as described by García-Escamilla et al. (2017).

Morphometric characterization

The morphological characterizations were performed for the two mite populations used for the virus transmission experiments: Brevipalpus sp. (Tetipac) and ''B. californicus (Texcoco)'' . The specimens of B. californicus (Texcoco) were obtained from a colony established in 2016 from a female collected on sour orange in Texcoco de Mora, State of Mexico (19°27′46.4″N; 98°54′14.75″W). Twenty adult females taken from each colony were mounted on slides with Hoyer's solution (Krantz & Walter, 2009). The slides were observed using both phase-contrast microscopy (Carl Zeiss GmbH Primo Star mod. 314800509) and differential interference contrast (DIC) microscopy (photomicroscope 3, Carl Zeiss). The observed characteristics were compared against both the original description (Baker, 1949) and redescriptions of B. lewisi (Baker & Tuttle, 1987; Beard et al., 2012).

Thirty-seven measurements were taken for each specimen, using the software ImageJ 1.52a (Table 1), the terminology of Beard et al. (2015) and Tassi (2018) was followed. Each measurement was taken by processing the photomicrographs with a Carl Zeiss photomicroscope 3, DIC optical microscope at magnifications from 640 to 1000X. For lengths, the scale obtained for each photograph was used. ImageJ 1.52 measures the roundness factor as an interval from 0 (completely unrounded) to 1 (perfect roundness); the arithmetical mean was estimated for each specimen. The slides used for this study are in the collection of the Acarology Laboratory of the Colegio de Postgraduados, Montecillo, State of Mexico, Mexico.

The same measurements were taken for 15 specimens from the type series of B. lewisi [hereafter, B. lewisi (type)] and five specimens of the type series of B. californicus, [hereafter B. californicus (type)] from photomicrographs of specimens located in the US National Mite collection held at the Systematic Entomology Laboratory, Beltsville Agricultural Research Station, USDA, United States of America. These photomicrographs were taken using DIC with a Carl Zeiss Axioplan 2 microscope and used for comparisons of the characteristics of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco). All the obtained images were edited using the GIMP 2.8.22 software.

The individual seta length was measured from the base to the apex, while the distance between setae was measured between their bases. The degree of the roundness character was measured using only the cells in the photographs with a defined edge in the ornamentations on the dorsal surface of the propodosoma as valid data. The well-defined cells were mostly confined to the sublateral area of the propodosomal plate. The number of measured cells per specimen were (mean ± standard error), Brevipalpus sp. (Tetipac), 84.57 ± 3.58; B. californicus (Texcoco), 92.67 ± 7.84; B. lewisi (type), 55.3 ± 3.56; B. californicus (type), 89.75 ± 5.51. The values of each variable were compared through analysis of variance (ANOVA) and Tukey multiple comparison (α=0.05). The normality (Shapiro-Wilk test) and variance homogeneity (Bartlett test) assumptions were verified. For the joint analysis of all the variables, we used a multivariate analysis, where principal component analyses (PCA) and a cluster analysis were performed. All the analyses were performed using the SAS v. 9.4 software.

Supplementary observations of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco) were done using SEM to illustrate the chaetotaxy of the idiosoma, the legs, the patterns of reticulation on the cuticle and the microplates. The samples were processed through dehydration in graduated ethanol series, critical point dried and coated in gold (Tassi, 2018) for observation in Jeol JSM IT300 or Quanta FEG650 microscopes, or through freezing in liquid nitrogen and coating in platinum (Bauchan et al., 2019) for observation in a Hitachi S-4700 microscope.

Molecular characterization

DNA extraction

Total DNA was extracted from 10 mites each of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco), both from the respective single-parent colonies, using the QIAamp DNA Micro kit (Cat. No. 56304), according to the protocol outlined by the manufacturer with some modifications (Klimov & OConnor, 2008).

PCR amplification

The D1–D3 (gene 28S, 900 bp) and COI (400 and 650 bp) segments were amplified for both B. californicus (Texcoco) and Brevipalpus sp. (Tetipac). To accomplish this, PCR was performed using GoTag® G2 Green Master Mix by Promega (Cat. No. M7822) with the D23F-D6F primer pair (Park and Foighil, 2000) for 28S, and both the DNF-DNR (Navajas et al., 1996) and LCO1490-HC02198 (Folmer et al., 1994) primer pairs for COI. The reaction mix consisted of 12.5 µL 2X Go Tag® G2 Green Master Mix, 2.5 µL forward primer (10 µM), 2.5 µL reverse primer (10 µM), and 2.5 µL MG water. The volume of the resulting mix was 20 µL, to which 5 µL of template DNA were added. The reaction was done in a Thermo Scientific Type 5020 (model ITCA0096) thermocycler following the indications in Table 2.

The products of the amplification were verified through electrophoresis in agarose gel at 1.2% dyed with ethidium bromide with a molecular weight marker of 100 bp Plus Ladder, for which 6 µL of the PCR product were used. This was run at 90 volts for 60 min in a Thermo Scientific (model 7309 B1) electrophoresis chamber and visualized in a Quantum Vilber Loumat (serial N°13 200819) photodocumenter under UV light.

The PCR product was purified and sequenced by Macrogen Inc, Korea. The obtained sequences were edited with the BioEdit (v. 7.2.5 https://www.mbio.ncsu.edu/bioedit/bioedit.html ) software and compared against other Brevipalpus sequences available in the database of the National Center for Biotechnology Information, NCBI.

Phylogenetic analysis

The sequences were edited in BioEdit v.7.2.5, then aligned with ClustalW from MEGA X v. 10.1.8 and compared with data base of the National Center for Biotechnology Information, (NCBI). Three phylogenetic trees were generated, two for COI fragments amplified with the primers DNF-DNR (Navajas et al., 1996), LCO1490-HC02198 (Folmer et al., 1994), and one for the fragment 28S (D1-D3) amplified with the primers D23F-D6R (Park & Foighil, 2000). The tool Models in MEGA X (v.10.1.8) was used to select nucleotide substitution models. In the construction of the phylogenetic tree generated by the primers DNF-DNR, Cenopalpus pulcher (Canestrini & Fanzago) was used as outgroup with the method of Maximum Likelihood (ML), the model Hasegawa-Kishino-Yano (HKY), discrete Gamma distribution (+G, parameter =0.4220) (Hasegawa et al., 1985). The analysis involved 26 nucleotide sequences with 364 positions, Bootstrap analysis with 1,000 replicates. For the fragment amplified by the primers LCO1490-HC02198 (Folmer et al., 1994), ML, the model Hasegawa-Kishino-Yano (HKY), discrete Gamma distribution (five categories (+G, parameter =0.2048)) were used. The outgroup was Raoiella indica Hirst. This analysis involved 25 nucleotide sequences with 686 positions. For the primers D1-D3, gene 28S, ML, the Tamura-3 parameters model with Gamma distribution (+G=0.2038) of five categories were used. R. indica was included as outgroup. This analysis involved 27 nucleotide sequences, with 1,044 positions, Bootstrap analysis with 1,000 replicates.

CiLV-C and OFV-citrus transmission tests

Once the single-parent Brevipalpus sp. (Tetipac) colonies were established, we confirmed that they were free of CiLV-C and OFV-citrus. To do this, total RNA was extracted from a group of 20 specimens from each colony through CTAB (Locali et al., 2003). Subsequently, RT-PCR was done using the RNeasy Plant Mini Kit Qiagen OneStep RT-PCR kit (Cat. No. 210212) (García-Escamilla et al., 2017) with the primers MPF/MPR (Locali et al., 2003) and CiLV-N-NPF/CiLVN-NPR (Roy et al., 2015b) for the diagnosis of CiLV-C and OFV-citrus, respectively.

Acquisition test

Virus-free specimens of Brevipalpus sp. (Tetipac) from the single-parent colony were used. Moreover, sweet orange leaves with cytoplasmic type leprosis symptoms were collected in Huimanguillo, Tabasco, Mexico (17°59′10.45″N, and 93°35′2.70″W), and sour orange leaves with nuclear type leprosis symptoms in Tolimán, Querétaro, Mexico (20°54′23.20″N and 99°55′41.55″W). These locations have a prevalence of CiLV-C and OFV-citrus, respectively (SENASICA, 2016).

The presence of CiLV-C in the leaves collected in Huimanguillo and OFV-citrus in the leaves collected in Tolimán were confirmed through RT-PCR. In the RT-PCR assays, Nad5f/Nad5mr primers were included as positive controls to avoid false negatives in the analyses, since they amplify a plant DNA segment of 180 bp (Menzel et al., 2002).

For the acquisition tests, observation arenas were made using 12 x 13 cm plastic containers, with the bottom covered with cotton saturated with 100 mL distilled water. Once these arenas were ready, a line of STICK-BUG 50 glue was traced on the edge of positive CiLV-C or OFV-citrus leaves (previously checked to be free of mites or other arthropods) and they were placed in the arenas with their abaxial surface facing up.

Groups of 15 mites were placed on the leaves of sweet orange (for acquisition of CiLV-C) or sour orange (for acquisition of OFV-citrus) in different developmental stages: larvae, nymphs (protonymphs and deutonymphs in unspecified proportions) and adult females, with 10 replicates for each stage for a total of 450 mites. The mites of each group were allowed to feed for 24, 48 or 72 h periods, after which the live specimens were removed and placed in 1.5 mL Eppendorf tubes for storage at -80 °C. Positive controls of B. californicus (Texcoco) and B. yothersi from virus-free colonies were used, two replicates of 15 mites of mixed stages for each species, fed with the leaves infected with OFV-citrus or CiLV-C, respectively. The mites and leaf tissues were analyzed through RT-PCR with the respective methods.

Inoculation tests

Brevipalpus sp. (Tetipac) specimens from the single-parent colony were collected and corroborated to be free of CiLV-C and OFV-citrus through RT-PCR with the methods described above. From the same colonies, 15 nymphs and 15 adult females were transferred to leaves of sour orange positive for OFV-citrus and showing leprosis symptoms for 48 h to acquire the virus (following the procedure previously described). Then, 15 females or 15 nymphs, supposedly carrying OFV-citrus, were placed on each A. thaliana and P. vulgaris indicator plants, with 10 replicates per plant species and mite stage. The plants were observed every other day to register the possible development of lesions. After 22 days, the plants were observed to determine the presence of surviving mites and their descendants. Leaf samples from both plant species were taken and the live mites were recovered to determine the presence of OFV-citrus through RT-PCR (Roy et al., 2015b). The PCR product was sent to Macrogen Inc. for sequencing.

Furthermore, citrus-citrus inoculation tests were done. For this, Brevipalpus sp. (Tetipac) larvae, nymphs and adult females were placed on sweet orange leaves with CiLV-C symptoms or on sour orange leaves positive to OFV-citrus for five days to acquire the respective viruses. Subsequently, a minimum of 15 specimens of each stage (i.e. minimum 45 mites) were transferred to virus-free sweet orange plants, approximately five months old. The CiLV-C inoculation assay was carried out in Cárdenas, Tabasco, Mexico, and included additional plants to which B. yothersi nymphs and adult females subjected to the same CiLV-C acquisition treatment were transferred as positive controls. A positive control was included in the OFV-citrus inoculation assay, consisting of minimum 45 specimens of B. californicus (Texcoco) nymphs and adult females previously fed with sour orange leaves positive to the correspondent virus. In both assays, a completely randomized design was used with eight replicates and negative controls, without mites.

Once the described experiments were established, a monthly photographic record of the plants was done over five months to determine if leprosis symptoms appeared. When similar symptoms developed, samples were taken from the affected tissues and subjected to RT-PCR.

Results

Morphological characterization

SEM and DIC photomicrograph collections of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco) were taken, while only DIC photographs of B. californicus and B. lewisi were available from their respective type series. The former allowed us to observe external structures with great augmentation, while the latter allowed us to observe the spermathecae. Photographs from Beard et al. (2012) were used as reference.

All the mites observed had setae f2 present (Figure 1), palps with four segments and three setae on the palp tarsus. The B. californicus (type) and B. californicus (Texcoco) specimens had two solenidia on each tarsus II, placing them in the californicus group, according to the Baker and Tuttle (1987) classification. In contrast, all the B. lewisi (type) and Brevipalpus sp. (Tetipac) specimens had a single solenidion on each tarsus II, placing them in the cuneatus group, according to the same classification (Figure 2).

The vesicles of all four spermathecae were rounded with a crest or crown on the distal margin and similar in form to each other (Figure 3). Consequently, this character was not useful for separating the four mite taxa. The microplates on Brevipalpus sp. (Tetipac) and B. californicus (Texcoco) were similarly shaped, being round to ovoid with fine longitudinal ridges on the surface, although the ridges were more widely spaced on specimens of the latter species (Figure 4).

The measurements of 37 characteristics for the four taxa are shown in Table 1. A univariate analysis was done to the 17 characteristics that fulfilled the assumptions of the ANOVA (normal distribution and variance homogeneity). Brevipalpus lewisi (type) generally had the greatest dimensions overall. At the other extreme, B. californicus (type) had the lowest dimensions overall, and in 16/17 variables analyzed, it was significantly different from B. lewisi (type). Brevipalpus californicus (Texcoco) and Brevipalpus sp. (Tetipac) were at intermediate positions, forming a block where they were significantly different from each other in only six of the 17 variables analyzed.

The numerical value assigned to the degree of roundness of the reticulations of the propodosomal dorsum placed B. californicus (Texcoco) and Brevipalpus sp. (Tetipac) in the same group, with no significant difference, whereas B. californicus (type) and B. lewisi (type) had significantly lower roundness values (Table 1). The cells on the sublateral propodosoma of B. lewisi (type) were clearly elongate (Figure 5 D), a diagnostic characteristic in its original description (McGregor, 1949).

In the principal component analysis, the first two components (CP1= 75.0%, CP2= 10.78%) explained 86% of the total variability (Figure 6). It is worth highlighting that the analysis separates the specimens 56 [B. californicus (type)] and 51 [B. lewisi (type)]; the rest of the specimens make up a single group, with no separation between the putative species or populations.

The cluster analysis showed the presence of at least seven groups (Figure 7) between the species. Using this number of clusters takes most of the variation of the used data (≈0.85); this is to say, only using the seven clusters, over 85% of the variation is explained.

The data obtained in this study prove that the four ''species'' analyzed make up a single group (based on the 37 parameters of the study) and, according to the information presented, they cannot be separated morphometrically from each other (except for the specimens 51 and 56).

Molecular characterization

Using the jModelTest 2.1.10 software (Darriba et al., 2012), we obtained the best nucleotide substitution model to construct the phylogenetic trees. The best model for the segments COI (DNF-DNR) and 28S (D23F-D6R) was Kamura2-parameter model (TPM1) (Kimura, 1981), while for the other COI segment (LCO1490-HC02198) it was Tamura and Nei model (TrN+G) (Tamura & Nei, 1993). The phylogenetic trees were built with the Maximum likelihood (ML) test with 1,000 replicates (bootstrap) (Figures 8-10). The sequences used for building the phylogeny of the COI fragments (DNF-DNR and LCO1490-HC02198) and 28S (D23F-D6R) are shown in Tables 3–5.

From the sequences obtained in the three phylogenetic trees, we can observe the different lineages of the distinct groups, B. californicus, B. chilensis, B. ferraguti, B. incognitus, B. papayensis, B. phoenicis, B. obovatus and B. yothersi. Brevipalpus sp. (Tetipac) is consistently grouped with B. californicus (Texcoco).

Given the similar results for the segment pair of the mitochondrial region (COI) and the D1-D3 fragment of 28S gene, in association with the consistent data of the morphometry study, we can postulate that Brevipalpus sp. (Tetipac) is a lineage of the species complex B. californicus s.l. aberrant for having a single solenidion on each leg II.

CiLV-C and OFV-citrus acquisition tests

CiLV-C was only detected in adult females of Brevipalpus sp. (Tetipac) at 24 and 48 h after feeding on infected plant tissue (1 out of 10 in both replicates of 15 mites each one). No virus was detected in larvae or nymphs 24, 48 or 72 h after feeding on the infected leaves. In contrast, OFV-citrus was detected in all the development stages of this mite: larvae (28 of 30), nymphs (29 of 30), and adult females (27 of 30) (Table 6). Meanwhile, individual B. californicus (Texcoco) and B. yothersi underwent the same acquisition test protocols and were used as controls for OFV-citrus and CiLV-C, respectively. These mites were positive for these viruses in all developmental stages (data not shown).

CiLV-C and OFV-citrus inoculation tests

There were no symptoms of chlorosis or necrosis similar to those described by Kondo et al. (2003) and Ramos-González et al. (2017) in the A. thaliana and P. vulgaris plants 20 days after they were infested with Brevipalpus sp. (Tetipac) nymphs and adult females previously fed with sour orange leaves infected with OFV-citrus. Specimens of Brevipalpus sp. (Tetipac) were recovered from the test plants, included mixed immature stages on A. thaliana and mixed immatures and some females on P. vulgaris. OFV-citrus was not detected in the plants or the mites.

In the assays carried out in Cárdenas, Tabasco, for the inoculation of CiLV-C, none of the evaluations (20, 42 and 84 days) or treatments detected the presence of typical citrus leprosis symptoms; this included B. yothersi used as a positive control. These results were corroborated by RT-PCR. In every reaction, an amplicon of about 180 bp was visualized in the gel, resulting from a positive amplification of plant DNA by the Nad5f/Nad5mr primers (Menzel et al., 2002). The presence of such amplicons excluded false negatives.

There were symptoms similar to leprosis from the third evaluation (78 days after inoculation, DAI) in the plants located in Tolimán, Querétaro, where the OFV-citrus inoculation was tested. These symptoms occurred in two Brevipalpus sp. (Tetipac) replicates of the nymph treatment (Figure 11A) and in one of the larval observations. Small leprosis-like symptoms appeared in one of eight plants infested by OFV-citrus viruliferous B. californicus (Texcoco) nymphs and adults (Figure 11 C), and infection was confirmed with RT-PCR.

Only very small lesions were caused by Brevipalpus sp. (Tetipac) mites on orange plants at 126 DAI in Tolimán, Querétaro. The lesions were not visually recognized as typical of the nuclear type leprosis (Figure 11 B) and RT-PCR for the presence of OFV-citrus was negative. These samples were looked at using a stereoscopic microscope, but there were no mites of any development stage present on the leaves.

Discussion

In the determination of the identity of the species initially designated Brevipalpus sp. (Tetipac) but later identified as an aberrant morphotype of B. californicus s.l., we mostly considered morphological characteristics presented in taxonomic keys and in both the originals and subsequent descriptions. Using the key in Baker and Tuttle (1987), which uses the number of solenidia on tarsi II among other characteristics to separate species, B. californicus (Tetipac) would be identified as B. lewisi. However, the original description of this species (McGregor, 1949) and several redescriptions (Baker, 1949; Meyer & Ryke, 1959) indicate that ornamentations (cells in the reticulation) in the sub-lateral region of the propodosoma are much longer than wide, a feature that is not present in the Brevipalpus sp. (Tetipac) specimens. The cells appear more like those of B. californicus, being polygonal or rounded (Figure 5 A-C) not elongate, and this was confirmed by the degree of roundness (Table 1).

Despite the long history of using the number of solenidia in taxonomic keys (Baker, 1949; Pritchard & Baker, 1958; Beard et al., 2012) and as the basis for the formation of species groups in addition to other characters (Meyer, 1979; Baker & Tuttle, 1987), De León (1961) and Baker and Tuttle (1964) have observed that this feature is unstable, and some individual specimens can even have one solenidion on one tarsus II and two on the other (Kitajima et al., 2011; Navia et al., 2013; Beard et al., 2015). Therefore, in this study we compared the morphometric data with molecular markers. From this, the mites under study resulted as con-specific with B. californicus s.l., where there was a 100% identity in a COI gene marker with specimens that had two solenidia in tarsus II (GenBank accessions KX100319 and KX100321, Salinas-Vargas et al. 2016). From this, we can point out that there can be erroneous identifications, and the data concerning distribution, range of hosts, etc., assigned to B. lewisi and B. californicus, among other species, should be revised or are simply not valid.

Although the univariate analysis of the B. californicus and B. lewisi type series showed these species as separate entities, in the multivariate analysis, no distinct groups were clearly recognized; specimens of both type series were grouped with B. californicus (Texcoco) and Brevipalpus sp. (Tetipac) (Figures 6 and 7). The specimens 51 [B. lewisi (type)] and 56 [B. californicus (type)] were separated by their dimensions, one for being particularly large and the other for being small (Figure 6). There was variation in the type series of B. californicus and B. lewisi; apparently there is heterogeneity in both, with the presence of morphotypes that merit careful revision. It would be helpful to collect B. californicus and B. lewisi specimens from their respective type locations and type host (both from Citrus in California) for molecular analysis to help define their taxonomic position. Regrettably, there are no DNA samples that could serve to confront morphological and molecular data in the two type series studied.

The presence of different patterns in the microplates that cover the body of many tenuipalpid mites can become a useful feature to identify species (Beard et al., 2015). In the present study the microplates of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco) were highly similar, in line with the postulate that they are a single species. However, this criterion is new and requires equipment with great augmentation for adequate observation, for example standard or low temperature SEM, which might not be available to all researchers.

In pest management and prevention, one must identify the species in question with complete certainty, especially in the case of the genus Brevipalpus, given the presence of different cryptic species and the potential for virus transmission (Childers & Rodrigues, 2011; Navia et al., 2013; Beard et al., 2015). Each mite species can present different adaptations to climatic conditions, different hosts and natural enemies (Navia et al., 2013). Thus, the correct identification of the specimens is vital.

The results of the present study show the importance of contrasting the morphological with the molecular analysis for a correct identification of the species. Navia et al. (2013) refer to the importance of the combination of both identification techniques, in the case of Brevipalpus, to reveal the appearance of cryptic species and avoid incongruences in their identity. As a result of this study, Brevipalpus sp. (Tetipac) was placed within the californicus group; if only the taxonomic keys had been used, it would have been placed in the cuneatus group.

In this research, Brevipalpus sp. (Tetipac) is reported as capable of acquiring CiLV-C and OFV-citrus, much more efficiently in the case of the latter. However, it was unable to inoculate any of these viruses, or its efficiency is so low that inoculation could not be demonstrated in this study. In contrast, García-Escamilla et al. (2017) proved the capacity of B. californicus to inoculate OFV-citrus to several citrus species, and Kondo et al. (2003) also proved transmission of OFV by B. californicus, from Cymbidium sp. (Orchidaceae) to P. vulgaris and Tetragonia expansa Murray (Aizoaceae).

Arabidopsis thaliana has been successfully tested as an indicator plant in BTV transmission tests. For example, Arena et al. (2017) were able to transmit the cilevirus CiLV-C, the tentative cilevirus Solanum violaefolium ringspot virus (SvRSV) and the dichorhaviruses CoRSV and Clerodendrum chlorotic spot virus (CICSV) by B. yothersi mites to this plant. Similarly, Ramos-González et al. (2017) and Chabi-Jesus et al. (2018) proved the capacity of B. phoenicis s.s. and B. yothersi or B. aff. yothersi to transmit, respectively, the dichorhaviruses CiLV-N and CiCSV to the same indicator plant. These results contrast those obtained in the present research, as no visible symptoms were seen in A. thaliana 25 days of being fed on by Brevipalpus sp. (Tetipac) infected with OFV-citrus.

Bean has also proved to be useful as an indicator plant, specifically for the transmission of BTV. Garita et al. (2013, 2014) successfully transmitted CiLV-C to bean with B. yothersi mites, while Kondo et al. (2003) transmitted OFV from orchids to bean with B. californicus. In contrast, when this indicator plant was used to test the transmission of OFV-citrus with Brevipalpus sp. (Tetipac), there were no symptoms nor was this virus detected by RT-PCR in any of the replicates.

In the OFV-citrus inoculation tests into sweet orange, leprosis-like symptoms were observed from 27 to 128 days after inoculation with the viruliferous mites (OFV-citrus), with very small lesions that progressed with time. These lesions had to be observed carefully, as they were very small. Duran-Trujillo (2017) observed that, in the case of sweet orange, nuclear type lesions in their early stages are almost imperceptible, but they maintain edges with a greenish colored halo up to eight months, when the lesions can be clearly observed in Citrus x sinensis'Valencia'. However, 126 DAI with putatively viruliferous Brevipalpus sp. (Tetipac), the samples with symptoms showed no positive results when processed through RT-PCR, excepting the positive control with putatively viruliferous B. californicus.

In the CiLV-C inoculation test carried out in Cárdenas, Tabasco, no leprosis symptoms developed, and all the asymptomatic tissue samples analyzed through RT-PCR turned out negative. This result was expected, as in the CiLV-C acquisition tests through Brevipalpus sp. (Tetipac), there were only two positive cases in 90 replicates. The above data suggest a low, almost null, capacity of Brevipalpus sp. (Tetipac) to acquire the virus, or an enzymatic process of virus degradation, that does not allow its detection through RT-PCR. This result coincides with García-Escamilla et al. (2017), who only found CiLV-C inoculation by B. yothersi. However, plants infested with the positive control (B. yothersi) did not become infected; this could be explained because the efficiency of this species to vector CiLV-C when fed on infected tissues for one to three days is low, as seen by García-Escamilla et al. (2017).

The B. californicus specimens used by García-Escamilla et al. (2017), with which they proved the capacity of this species to acquire and transmit OFV-citrus, were taken from the same single-parent colony used in the present study, herein called B. californicus (Texcoco). Given the taxonomic closeness of these mites with Brevipalpus sp. (Tetipac), we expected this species to have a similar performance. In fact, it did, as it was capable of acquiring OFV-citrus. Ali et al. (2016) proved that Brevipalpus oncidii Baker collected in the field from OFV infected plants carried that virus. However, once the mite was left to incubate without feeding for 48 h, the OFV was no longer detectable through the same RT-PCR test. In the present work, we found a similar result, where Brevipalpus sp. (Tetipac) was able to acquire OFV-citrus, but did not inoculate it to healthy plants, and 22 days after acquiring said virus, it was no longer detectable in the mites using RT-PCR. This suggests that there might be an enzymatic action against the virus, or a barrier in the intestine membranes or salivary glands that does not allow the correct transference of the pathogen into susceptible tissue.

The OFV-citrus inoculation tests with Brevipalpus sp. (Tetipac) were done on A. thaliana, P. vulgaris and Citrus x sinensis'Valencia' with a high number of replicates in each case (8-10 for each plant species). All these plants have shown susceptibility to one or more of the studied viruses, all using several Brevipalpus species as vectors. Therefore, the non-inoculation of CiLV-C and OFV-citrus is postulated to be due to lack of capacity of this mite to act as a vector. Brevipalpus sp. (Tetipac), although morphologically and molecularly close to B. californicus, is a biotype either incapable of transmitting OFV-citrus or whose efficiency is so low that it could not be proven with the methods used. This postulate is supported by the fact that, under the same conditions, the positive control B. californicus carrying OFV-citrus was capable of inoculating citrus plants with this virus.

Acknowledgements

To the National Council of Science and Technology (CONACyT) for the scholarship granted to carry out the graduate studies of the senior author in Mexico. To Dr. Carlos Fredy Ortiz García, and in general Campus Tabasco of the Colegio de Postgraduados. To the Centro Nacional de Referencia Fitosanitaria, DGSV, SADER, where observations under SEM were carried out. To the Comité Estatal de Sanidad Vegetal de Querétaro, DGSV, SADER, Ing. Juan Bautista Castillo Vega and Mr. José Manuel Baltazar for the facilities they gave us to set up the experiment on virus transmission.

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