A New Species of Ultratenuipalpus (Acari: Tenuipalpidae) from Brazil and Re-Description of Ultratenuipalpus meekeri (De Leon), the Type Species of the Genus, with DNA Barcodes.

Species of the genus Ultratenuipalpus bear a broad subquadrate propodosoma with many large, flattened, lanceolate to ovate dorsal setae. They also bear some plesiomorphic character states, such as the presence of three pairs of ventral ps setae. Here, we describe Ultratenuipalpus parameekeri Castro, Ochoa & Feres sp. nov. based on adult females, males, and immatures, collected on ferns from Brazil. We also re-describe Ultratenuipalpus meekeri (De Leon), the type species of the genus, based on types and newly collected material from Mexico, and include additional novel data (e.g., dorsal and ventral ornamentation, leg chaetotaxy, and setal measurements) in a standardized form. We include highly detailed images obtained using LT-SEM, accompanied by DNA barcodes, for both species. The ontogenetic additions of leg chaetotaxy are presented and discussed.

High susceptibility of Tetranychus merganser (Acari: Tetranychidae), an emergent pest of the tropical crop Carica papaya, towards Metarhizium anisopliae s.l. and Beauveria bassiana strains.

Background: The mite Tetranychus merganser is considered to be an emerging pest of various crops in tropical countries. It is one of the most detrimental pests in the papaya orchards of some regions of México. The current field control of Tetranychus spp. involves the extensive use of chemicals that have some degree of toxicity to humans or the environment and may cause selective resistance. The use of biological alternatives such as parasitoids and mite predators have limited effectiveness. In order to find effective but non-toxic alternatives for mite pest management, bio-products that are able to be mass produced and applied to large production areas have been sought, including the entomopathogen fungi. B. bassiana and M. anisopliae s.l. are the fungi most extensively used for the biological control of insect pests. Although they do not cause natural epizootic diseases in mites, there are reports that show that they infect T. urticae, and should be evaluated for use in the biological control of papaya's mite pests. Methods: A T. merganser colony was established and the susceptibility of adult females to 30 entomopathogenic fungi strains was evaluated under laboratory conditions with an in vitro mass screening bioassay. Ten strains of Metarhizium anisopliae sensu lato (s.l.), eleven of Beauveria bassiana, nine of Lecanicillium sp. and one of Hirsutella thompsonii var. sinematosa were tested. The infectivity of adult females was evaluated calculating the percentage of mortality. To calculate the LC50 and LT50 of the most virulent strains, a bioassay was performed using serial concentrations (1×104-1×108 conidia/mL) for each strain. Strains showing ability to infect eggs laid were evaluated with a novel egg-infectivity bioassay. The internal transcribed spacer (ITS) region of the more lethal strains were sequenced. Results: T. merganser and T. urticae were found in orchards of Carica papaya (Maradol variety and Tainung hybrid) in Campeche, México. All tested strains of M. anisopliae s.l. and B. bassiana were infectious to the adult female of T. merganser at a concentration of 1×108 conidia/mL. Six strains of M. anisopliae (Ma002, Ma003, Ma004, Ma005, Ma014 and Ma034) caused 100% mortality, and one of B. bassiana (Bb016) caused 95% mortality. The most virulent was Ma034, with an LC50 of 1.73×106 conidia/mL followed by Ma005 and Ma003. Ma005 and Ma034 were the fastest strains to reach LT50,achieving this in less than 3.7 days. Additionally, Ma034 and Ma014 strains were infectious to more than 70% of the eggs. Conclusions: T. merganser and T. urticae are present in the papaya orchards of Campeche, México. The high susceptibility of T. merganser adult females and eggs toward several M. anisopliae s.l. or B. bassiana strains suggests that these fungi are a viable alternative to control this emergent pest. The most virulent strain, Ma034, was also infective to eggs, and is the most promising to be tested in the field.

Brevipalpus mites associated with coffee plants (Coffea arabica and C. canephora) in Chiapas, Mexico.

Tenuipalpid mites of the genus Brevipalpus are of significant economic and quarantine importance in agriculture. They can damage and vector phytopathogenic viruses in coffee plantations and other crops. In this study, we focused on: identification of the Brevipalpus species, assessment of the spread of Brevipalpus-associated viruses (CoRSV, CiLV-N, CiLVC and CiLVC2), and mite population fluctuations over the course of 1 year. The study was conducted in coffee plantations in Soconusco, a coffee-producing region in Chiapas, Mexico. The collected mites of the Brevipalpus phoenicis sensu lato species complex (635) were identified as Brevipalpus papayensis (80.2%) and B. yothersi (19.8%) based on morphological and molecular characteristics. Their population abundance was low and there were no indications for virosis. The highest mite abundance was recorded in August-September and the lowest in February-March. An interaction was observed between mite abundance and coffee species in open-growth and shaded cultivation at various altitudes. Brevipalpus papayensis was most abundant in Coffea arabica var. Bourbon, in shaded (80%) growing conditions at an altitude of 1300 m above sea level. In C. canephora (in open-growth cultivation conditions at low altitude), B. yothersi was more abundant than in C. arabica, and as abundant as B. papayensis. We are of the opinion that, at this moment, B. papayensis and B. yothersi do not present risks to the production of coffee for the studied plantations. However, as the coffee-producing regions of Mexico are ecologically diverse, it will be important to continue examining the status of Brevipalpus mite populations in other regions in Mexico.

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.

First detection of orchid fleck virus in orchids in Mexico.

For the first time, an isolate of the dichorhavirus orchid fleck virus (OFV, family Rhabdoviridae) was found infecting an orchid plant in Mexico. The infected sample of Epidendrum veroscriptum was collected in a nursery in Lagunillas, municipality of Zihuateutla, Edo. Puebla. Mites gathered on this plant were analyzed by light and scanning electron microscopy, which consistently indicated the presence of adults of the species Brevipalpus californicus, the common vector of OFV. Viral identification was based on symptoms, cytopathology, and reverse transcriptase-PCR/sequencing of genome fragments of the RNA1 and 2 molecules. Since isolates of OFV causing citrus leprosis have been previously detected in the Mexican states of Chiapas, Querétaro, and Jalisco, we promote a pertinent discussion and thought-provoking questions regarding the epidemiology and putative evolution of OFV.

Dispersal and resistance to starvation in Raoiella indica (Acari: Tenuipalpidae).

Raoiella indica Hirst (Tenuipalpidae) is an obligate phytophagous mite that has rapidly colonized wide areas of tropical America and causes severe injuries, mainly to plants in the families Arecaceae, Heliconiaceae, Zingiberaceae and Musaceae. This study evaluates biological attributes of the species that could explain its potential as invasive pest, its capacity to survive when deprived of food and its dispersal mechanisms, including passive displacement by wind and possible phoretic association with insects. This mite has a higher resistance to starvation than other phytophagous mites, with a maximum 12 days for adult females. Its main dispersal is by wind, even at low velocities. Phoresy on insects was not observed.

Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit-specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease.

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

Genome assembly of citrus leprosis virus nuclear type reveals a close association with orchid fleck virus.

The complete genome of citrus leprosis virus nuclear type (CiLV-N) was identified by small RNA sequencing utilizing leprosis-affected citrus samples collected from the state of Querétaro, Mexico. The nucleotide identity and phylogenetic analysis indicate that CiLV-N is very closely related to orchid fleck virus, which typically infects Cymbidium species.

Análisis de la variación intraespecífica de Conidiobolus coronatus usando RAPD y secuencias ITS / Intraspecific variation analysis of Conidiobolus coronatus using RAPD and ITS sequencing

Introducción El hongo Conidiobolus coronatus (C. coronatus) tiene una extensa distribución de hábitats y organismos; se encuentra en forma saprófita, ataca insectos y mamíferos, incluido el humano, aunque de este los reportes son pocos y están restringidos a zonas tropicales. El objetivo de este trabajo fue determinar si existe o no variación genética entre aislamientos de C. coronatus provenientes de lesiones en humanos y otras fuentes. Métodos Once aislamientos de C. coronatus obtenidos de suelo, insectos y humanos se analizaron con las técnicas de polimorfismo derivado de la amplificación aleatoria de ADN (RAPD) y secuenciación del espaciador interno transcrito (ITS1-5.8S rADN-ITS2); para el análisis de las regiones ITS se usaron los métodos de máxima parsimonia, neighbour-joining (NJ) y mínima evolución. Resultados El análisis de los polimorfismos de C. coronatus con las técnicas RAPD e ITS evidenció altos niveles de variación intraespecífica en los aislamientos evaluados. Los aislamientos obtenidos de lesiones en humanos presentan la mayor divergencia genética respecto de los obtenidos de otras fuentes; la mayor distancia genética entre grupos de aislamientos se da entre los provenientes de humanos y los tomados del insecto Lycoriella ingenua. Conclusiones Este trabajo es el primero en evaluar y demostrar que existe variación intraespecífica a nivel molecular de C. coronatus que, a su vez, está relacionada con la fuente de la que se obtuvieron (AU)

Introduction The fungus Conidiobolus coronatus (C. coronatus) has an extensive distribution of habitats and hosts. It is found saprophytically, and attacks insects and mammals, including humans. Although there are few reports on humans, and they are restricted to tropical areas. The aim of this work was to determine whether genetic variation exists between C. coronatus isolates coming from human lesions and other sources. Methods A total of 11 C. coronatus isolates obtained from soil, insects and humans were analyzed with the random amplification of polymorphic ADN (RAPD) and internal transcribed spacer (ITS) techniques; the maximum parsimony, neighbour-joining and minimum evolution methods were used for the ITS analysis. Results The analysis of the C. coronatus polymorphisms showed high intra-species variation levels between the evaluated isolates. The isolates coming from human lesions showed the greatest genetic divergence compared with the remaining isolates. The greatest genetic distance between isolate groups was found between those coming from humans and those taken from the insect Lycoriella ingenua. Conclusions This is the very first work evaluating and demonstrating that within species variation exists at molecular level in C. coronatus, and is related to the source where the isolates were taken from (AU)

[Intraspecific variation analysis of Conidiobolus coronatus using RAPD and ITS sequencing]. / Análisis de la variación intraespecífica de Conidiobolus coronatus usando RAPD y secuencias ITS.

INTRODUCTION: The fungus Conidiobolus coronatus (C. coronatus) has an extensive distribution of habitats and hosts. It is found saprophytically, and attacks insects and mammals, including humans. Although there are few reports on humans, and they are restricted to tropical areas. The aim of this work was to determine whether genetic variation exists between C. coronatus isolates coming from human lesions and other sources. METHODS: A total of 11C. coronatus isolates obtained from soil, insects and humans were analyzed with the random amplification of polymorphic ADN (RAPD) and internal transcribed spacer (ITS) techniques; the maximum parsimony, neighbour-joining and minimum evolution methods were used for the ITS analysis. RESULTS: The analysis of the C. coronatus polymorphisms showed high intra-species variation levels between the evaluated isolates. The isolates coming from human lesions showed the greatest genetic divergence compared with the remaining isolates. The greatest genetic distance between isolate groups was found between those coming from humans and those taken from the insect Lycoriella ingenua. CONCLUSIONS: This is the very first work evaluating and demonstrating that within species variation exists at molecular level in C. coronatus, and is related to the source where the isolates were taken from.

Repellency of the oily extract of neem seeds (Azadirachta indica) against Varroa destructor (Acari: Varroidae).

A crude oil extract of neem seed (Azadirachta indica, Sapindales: Meliaceae) was evaluated for repellency on Varroa destructor Anderson and Trueman. Burgerjon's tower was used to spray worker bee pupae with 0.0, 0.3, 0.7, 1.3, 2.6, 5.3, 10.6 and 21.1% neem extract concentrations. Sprayed pupae were attached to observation arenas and incubated at 32 ± 2°C and 70 ± 10% RH. The ability of V. destructor to locate and feed on treated and untreated pupae was monitored from 30 min to 72 h after spray. Higher and more stable repellency was achieved with 2.6, 5.3, 10.6 and 21.1% neem extract. At the highest concentration, 98% of V. destructor were prevented to settle on bee pupae, resulting in 100% V. destructor mortality at 72 h.

Identification and characterization of a novel etiological agent of mango malformation disease in Mexico, Fusarium mexicanum sp. nov.

The primary objective of this study was to characterize Fusarium spp. associated with the economically devastating mango malformation disease (MMD) in Mexico. In all, 142 Fusarium strains were isolated from symptomatic mango inflorescences and vegetative tissues in eight geographically diverse Mexican states from 2002 through 2007. Initially, all the Mexican isolates were screened for genetic diversity using appolymerase chain reaction and random amplified polymorphic DNA markers and were grouped into seven distinct genotypes. Based on results of these analyses, evolutionary relationships and species limits of the genetically diverse MMD-associated Fusarium spp. were investigated using multilocus DNA sequence data and phylogenetic species recognition. Maximum parsimony analyses of a five-locus data set comprising 5.8 kb of aligned DNA sequence data indicated that at least nine phylogenetically distinct Fusarium spp. within the Gibberella fujikuroi species complex are associated with MMD, including one species within the African clade (Fusarium pseudocircinatum), two species within the Asian clade (F. mangiferae and F. proliferatum), and at least six species within the American clade (F. sterilihyphosum and five undescribed Fusarium spp.). Molecular phylogenetic analyses indicate that a novel genealogically exclusive lineage within the American clade was the predominant MMD associate in Mexico. This new Fusarium sp. caused MMD and could be distinguished from all other known species morphologically by the production of mostly sterile, coiled hyphae which are typically associated with sporodochial conidiophores together with unbranched or sparsely branched aerial conidiophores. Koch's postulates were completed for isolates of the new species on nucellar seedlings of mango cv. Ataulfo. This pathogen is formally described herein as F. mexicanum.

The population level of Eotetranychus lewisi and the concentration of carbohydrates in peach trees.

Lewis spider mite Eotetranychus lewisi (McGregor) is the most important pest in peach orchards, Prunus persica (L.) (Batsch), in North-Central Mexico. In autumn 2003 and spring 2004, two glasshouse experiments were carried out to assess the influence of that mite on the concentration of total soluble sugars and starch in leaves, bark and roots of 'diamante mejorado' peach trees. Apical leaves of peach trees were inoculated with three mite densities per leaf: (A) 10-20, (B) 21-40, (C) 41-80; a mite-free control was added. In 2003, at 81 days after infestation (DAI), cumulative mite-days per leaf (CMD) were 153, 1313, 2844 and 4771 in control and treatments (A), (B) and (C), respectively. In the same order, these CMD caused reductions in total soluble sugars (TSS): in leaves, 45, 50 and 61%; in bark, 9, 20 and 33%; in roots, 8, 20 and 26%. Reductions of starch concentration in leaves were 17, 43 and 56%; in bark, 25, 55 and 32%; in roots, 17, 22 and 32%. In 2004, at 77 DAI and 57, 1043, 2426 and 3996 CMD for control and treatments (A), (B) and (C), respectively, resulting reductions of TSS were: in leaves, 3, 7 and 15%; in bark, 0.8, 3 and 5%; in roots, 57, 60 and 78%, whereas reductions in starch concentration were: in leaves, 30, 34 and 44%; in bark, 18, 24 and 41%; in roots, 17, 47 and 48%. The higher reductions in roots found in 2004 are attributed to cumulative injury affecting food reserves.