What's in my Pot? Six Colletotrichum Species Causing Anthracnose in Brazilian Pecan Orchards.

Pecan (Carya illinoinensis) is one important exotic forest crop cultivated in South America, specifically in Brazil, Uruguay, and Argentina. However, diseases such as anthracnose, favored by high humidity conditions and high summer temperatures, make its cultivation difficult, causing important loss to pecan farmers. This study used morphological and molecular approaches to identify the Colletotrichum species causing anthracnose in pecan plantations in Southern Brazil. The isolates obtained from pecan fruits with anthracnose symptoms were grouped through quantitative morphological characteristics into three distinct morphotypes. Molecular analysis of nuclear genes allowed the identification of six species of Colletotrichum causing anthracnose in pecan: C. nymphaeae, C. fioriniae, C. gloeosporioides, C. siamense, C. kahawae, and C. karsti. Three of these species are reported for the first time as causal agents of anthracnose in pecan. Therefore, these results provide an important basis for the adoption and/or development of anthracnose management strategies in pecan orchards cultivated in southern Brazil and neighboring countries.

First Report of Root-Knot Nematode, Meloidogyne incognita, Infecting Buckwheat, Fagopyrum esculentum, in State of São Paulo, Brazil.

Buckwheat (Fagopyrum esculentum Moench) belongs to the Polygonaceae family and has been widely cultivated due to its high nutritional, nutraceutical, and medicinal properties. Brazil ranks seventh-largest producer, with 66,000 tons produced in 2018. Buckwheat is also valued for its adaptability as a cover crop, in grain fields of soybean (Glycine max (L.) Merr., maize (Zea mays L.), and sorghum (Sorghum bicolor (L.) Moench) (Görgen et al. 2016, Babu et al. 2018) especially in fields highly infested with plant-parasitic nematodes (PPN). PPN cause severe root damage, suppressing plant development and yield production. In October 2018, six samples of roots and soil were collected in symptomatic patches of buckwheat, in Guaíra SP (20° 19' 32"S 48° 13' 15.4"W). Samples were analyzed in the Nematology Laboratory (LabNema), UNESP, Jaboticabal, SP, BR. Plants presented symptoms of yellow leaves and galled and volume-reduced roots. Meloidogyne sp. was found, comprising 6,320 eggs and second-stage juveniles (J2s) from 10 g of root and 1,628 J2s in 100 cm³ of soil. Adult morphological characteristics, isoenzyme phenotype of esterase, and molecular analysis were performed to identify the Meloidogyne species. The perineal patterns presented high and trapezoidal dorsal arch (n=15), and the males showed a trapezoidal labial region, including a high head cap formed by a large round labial disc that is raised above the medial lips and centrally concave (n=15) (Eisenback and Hirscmann 1981). These characteristics are typical in Meloidogyne incognita (Kofoid and White, 1912) Chitwood, 1949 (Nascimento et al., 2020; Eisenback and Hirschmann 1981; Netscher and Taylor 1974). The enzymatic phenotype was performed with females (n=8), and the phenotype I1 was verified, described by Esbenshade and Triantaphyllou (1985) as typical for M. incognita. To confirm the species DNA samples were extracted from individual females (n=6) and PCR with specific primers for M. incognita (Mi-F 5'- GTGAGGATTCAGCTCCCCAG-3' and Mi-R 5'-ACGAGGAA CATACTTCTCCGTCC-3') and M. javanica (Treub) Chitwood 1949 (Fjav 5'-GGTGCGCGATTGAACTGAGC-3' and Rjav 5'-CAG GCCCTTCAGTGGAACTATAC-3') that amplify SCAR markers described by Meng et al. (2004) and Zijlstra et al. (2000), respectively, and specific primers for M. enterolobii Yang & Eisenback 1983 that amplify rDNA-IGS2 region (Me-F 5'-AACTTTTG TGAAAGTGCCGCTG-3' and Me-R 5'-TCAGTTCAGGCAGG ATCAACC-3') described by Long et al. (2006) were tested. A fragment of 955 pb DNA size was amplified in Mi-F/R primer, which confirmed the M. incognita identification (Meng et. al., 2004). The original population was used to execute pathogenicity test. In a greenhouse, single buckwheat seeds (cv. IPR 91 Baili) were sown in six 5L pots filled with autoclaved-soil and inoculated with 3,000 eggs and J2s per pot (n=6) and control (n=6). After 60 days, the nematodes were extracted from roots and the M. incognita was confirmed. An average of 15,738 eggs and J2s were recovered, (reproductive factor = 5.24), which confirmed buckwheat as a host to M. incognita. The inoculated plants showed symptoms as those observed in the field. No symptom or nematode was noted on the control. Meloidogyne incognita has been reported causing high damage to the F. esculentum in California (Gardner and Caswell-Chen 1994) plus several crops in Brazil (Nascimento et al., 2020). However, this is the first report of this nematode infecting buckwheat in Brazil. Given the importance of buckwheat in Brazil, with extensive use as forage, cover crop, and its nutritional properties, this report is essential to specific management measures are adopted to avoid further losses.

Fire ants: What do rural and urban areas show us about occurrence, diversity, and ancestral state reconstruction?

In South America, Solenopsis saevissima and S. invicta are the most common fire ants. Nests are founded in areas under anthropic interference like urban or rural areas, but S. invicta is found preferentially in those with the greatest anthropic interference. However, we do not know the rates at which they exist in anthropized areas next to high density of native vegetation. Areas with 60 to 90% of native Atlantic Forest were selected to verify the occurrence of both species in rural and urban areas. We investigated the molecular diversity and applied the reconstruction of the ancestral state analysis for each species. A total of 186 nests were analyzed and we found that the two species had the same proportion in the urban area. However, S. saevissima had a higher rate of prevalence in the rural area, in addition to having a greater number of haplotypes and ancestry associated with this type of habitat for the region. S. invicta had the same number of haplotypes in both rural and urban regions, and less haplotypic diversity. We conclude that S. saevissima is a species typically associated with rural areas and S. invicta, although present, is not dominant in urban areas.

Analysis of glucose and xylose metabolism in new indigenous Meyerozyma caribbica strains isolated from corn residues.

Aiming to broaden the base of knowledge about wild yeasts, four new indigenous strains were isolated from corn residues, and phylogenetic-tree assemblings on ITS and LSU regions indicated they belong to Meyerozyma caribbica. Yeasts were cultivated under full- and micro-aerobiosis, starting with low or high cell-density inoculum, in synthetic medium or corn hydrolysate containing glucose and/or xylose. Cells were able to assimilate both monosaccharides, albeit by different metabolic routes (fermentative or respiratory). They grew faster in glucose, with lag phases ~ 10 h shorter than in xylose. The hexose exhaustion occurred between 24 and 34 h, while xylose was entirely consumed in the last few hours of cultivation (44-48 h). In batch fermentation in synthetic medium with high cell density, under full-aerobiosis, 18-20 g glucose l-1 were exhausted in 4-6 h, with a production of 6.5-7.0 g ethanol l-1. In the xylose medium, cells needed > 12 h to consume the carbohydrate, and instead of ethanol, cells released 4.4-6.4 g l-1 xylitol. Under micro-aerobiosis, yeasts were unable to assimilate xylose, and glucose was more slowly consumed, although the ethanol yield was the theoretical maximum. When inoculated into the hydrolysate, cells needed 4-6 h to deplete glucose, and xylose had a maximum consumption of 57%. Considering that the hydrolysate contained ~ 3 g l-1 acetic acid, it probably has impaired sugar metabolism. Thus, this study increases the fund of knowledge regarding indigenous yeasts and reveals the biotechnological potential of these strains.

Costus stripe mosaic virus, a tentative new member of the genus Potyvirus.

Morphological, biological, serological, and molecular tests underpin the description of costus stripe mosaic virus (CoSMV) as a new member of the genus Potyvirus, family Potyviridae. Found affecting the native ornamental Costus spiralis in Brazil, the pathogen showed a severely restricted natural and experimental host range. Excluding the poly(A) tail, the CoSMV genome contains a large open reading frame (ORF) of 9,446 nucleotides that encodes a polyprotein with 3,046 amino acids, which is potentially cleaved into ten products, and a small ORF (77 amino acids) knows as PIPO. Genome analysis demonstrated the highest CoSMV nucleotide sequence identity to onion yellow dwarf virus (51.79%). No evidence of recombination was detected in the CoSMV genome, and phylogenetic analysis revealed its basal position in a group formed by members of the genus Potyvirus, along with Cyrtanthus elatus virus A (Vallota speciosa virus) and canna yellow streak virus. CoSMV was not transmitted by aphids of the species Aphis solanella, Myzus persicae or Uroleucon sonchi, which could be due to mutations in the HC-Pro motifs required for aphid transmission. A divergence in the P1 protein cleavage site was found when compared to other members of the family Potyviridae. Based on its unique biological and molecular characteristics and the current species demarcation criteria, we propose CoSMV to be a new tentative member of the genus Potyvirus.

Two epizootic Perkinsus spp. events in commercial oyster farms at Santa Catarina, Brazil.

Perkinsus spp. have been detected in various bivalve species from north-east Brazil. Santa Catarina is a South Brasil state with the highest national oyster production. Considering the pathogenicity of some Perkinsus spp., a study was carried out to survey perkinsosis in two oyster species cultured in this State, the mangrove oyster Crassostrea gasar and the Pacific oyster Crassostrea gigas. Sampling involved eight sites along the state coast, and oyster sampling was collected during the period between January 2013 and December 2014. For the detection of Perkinsus, Ray's fluid thioglycollate medium (RFTM) and histology were used, and for the identification of the species, PCR and DNA sequencing were used. Perkinsus spp. was found by RFTM in C. gigas and C. gasar from São Francisco do Sul. This pathology was also detected in C. gasar from Balneário Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C. gigas and C. gasar from São Francisco do Sul and Perkinsus beihaiensis in C. gasar from Balneário Barra do Sul. This is the first report of P. marinus in C. gigas from South America. Results of this preliminary study suggest that both oyster species tolerate the species of Perkinsus identified, without suffering heavy lesions.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Unveiling the complete genome sequence of clerodendrum chlorotic spot virus, a putative dichorhavirus infecting ornamental plants.

The genus Dichorhavirus includes plant-infecting rhabdoviruses with bisegmented genomes that are horizontally transmitted by false spider mites of the genus Brevipalpus. The complete genome sequences of three isolates of the putative dichorhavirus clerodendrum chlorotic spot virus were determined using next-generation sequencing (Illumina) and traditional RT-PCR. Their genome organization, sequence similarity and phylogenetic relationship to other viruses, and transmissibility by Brevipalpus yothersi mites support the assignment of these viruses to a new species of dichorhavirus, as suggested previously. New data are discussed stressing the reliability of the current rules for species demarcation and taxonomic status criteria within the genus Dichorhavirus.

Citrus leprosis virus N: A New Dichorhavirus Causing Citrus Leprosis Disease.

Citrus leprosis (CL) is a viral disease endemic to the Western Hemisphere that produces local necrotic and chlorotic lesions on leaves, branches, and fruit and causes serious yield reduction in citrus orchards. Samples of sweet orange (Citrus × sinensis) trees showing CL symptoms were collected during a survey in noncommercial citrus areas in the southeast region of Brazil in 2013 to 2016. Transmission electron microscopy analyses of foliar lesions confirmed the presence of rod-like viral particles commonly associated with CL in the nucleus and cytoplasm of infected cells. However, every attempt to identify these particles by reverse-transcription polymerase chain reaction tests failed, even though all described primers for the detection of known CL-causing cileviruses and dichorhaviruses were used. Next-generation sequencing of total RNA extracts from three symptomatic samples revealed the genome of distinct, although highly related (>92% nucleotide sequence identity), viruses whose genetic organization is similar to that of dichorhaviruses. The genome sequence of these viruses showed <62% nucleotide sequence identity with those of orchid fleck virus and coffee ringspot virus. Globally, the deduced amino acid sequences of the open reading frames they encode share 32.7 to 63.8% identity with the proteins of the dichorhavirids. Mites collected from both the naturally infected citrus trees and those used for the transmission of one of the characterized isolates to Arabidopsis plants were anatomically recognized as Brevipalpus phoenicis sensu stricto. Molecular and biological features indicate that the identified viruses belong to a new species of CL-associated dichorhavirus, which we propose to call Citrus leprosis N dichorhavirus. Our results, while emphasizing the increasing diversity of viruses causing CL disease, lead to a reevaluation of the nomenclature of those viruses assigned to the genus Dichorhavirus. In this regard, a comprehensive discussion is presented.

The complete genome sequence of 'Candidatus Liberibacter americanus', associated with Citrus huanglongbing.

Liberibacter spp. form a Rhizobiaceae clade of phloem-limited pathogens of limited host range. Two obligately parasitic species have been sequenced: 'Candidatus Liberibacter asiaticus', which causes citrus huanglongbing (HLB) worldwide, and 'Ca. L. solanacearum', which causes potato "zebra chip" disease. A third (proposed) species, Liberibacter crescens, was isolated from mountain papaya, grown in axenic culture, and sequenced. In an effort to identify common host determinants, the complete genomic DNA sequence of a second HLB species, 'Ca. L. americanus' strain 'São Paulo' was determined. The circular genome of 1,195,201 bp had an average 31.12% GC content and 983 predicted protein encoding genes, 800 (81.4%) of which had a predicted function. There were 658 genes common to all sequenced Liberibacter spp. and only 8 genes common to 'Ca. L. americanus' and 'Ca. L. asiaticus' but not found in 'Ca. L. solanacearum'. Surprisingly, most of the lipopolysaccharide biosynthetic genes were missing from the 'Ca. L. americanus' genome, as well as OmpA and a key regulator of flagellin, all indicating a 'Ca. L. americanus' strategy of avoiding production of major pathogen-associated molecular patterns present in 'Ca. L. asiaticus' and 'Ca. L. solanacearum'. As with 'Ca. L. asiaticus', one of two 'Ca. L. americanus' prophages replicated as an excision plasmid and carried potential lysogenic conversion genes that appeared fragmentary or degenerated in 'Ca. L. solanacearum'.

Assessment of the Entomopathogenic Potential of Fungal and Bacterial Isolates from Fall Armyworm Cadavers Against Spodoptera frugiperda Caterpillars and the Adult Boll Weevil, Anthonomus grandis.

Increased attention is being focused on the biological control of agricultural pests using microorganisms, owing to their potential as a viable substitute for chemical control methods. Insect cadavers constitute a potential source of entomopathogenic microorganisms. We tested whether bacteria and fungi isolated from Spodoptera frugiperda (JE Smith) cadavers could affect its survival, development, egg-laying pattern, and hatchability, as well as induce mortality in Anthonomus grandis Boheman adults. We isolated the bacteria Enterobacter hormaechei and Serratia marcescens and the fungi Scopulariopsis sp. and Aspergillus nomiae from fall armyworm cadavers and the pest insects were subjected to an artificial diet enriched with bacteria cells or fungal spores to be tested, in the case of S. frugiperda, and only fungal spores in the case of A. grandis. Enterobacter hormaechei and A. nomiae were pathogenic to S. frugiperda, affecting the survival of adults and pupae. The fungus Scopulariopsis sp. does not affect the survival of S. frugiperda caterpillars and pupae; however, due to late action, moths and eggs may be affected. Aspergillus nomiae also increased mortality of A. grandis adults, as well as the development of S. frugiperda in the early stages of exposure to the diet, as indicated by the vertical spore transfer to offspring and low hatchability. Enterobacter hormaechei and A. nomiae are potential biocontrol agents for these pests, and warrant further investigation from a toxicological point of view and subsequently in field tests involving formulations that could improve agricultural sustainability practices.

Two Novel Betarhabdovirins Infecting Ornamental Plants and the Peculiar Intracellular Behavior of the Cytorhabdovirus in the Liana Aristolochia gibertii.

Two novel members of the subfamily Betarhabdovirinae, family Rhabdoviridae, were identified in Brazil. Overall, their genomes have the typical organization 3'-N-P-P3-M-G-L-5' observed in mono-segmented plant-infecting rhabdoviruses. In aristolochia-associated cytorhabdovirus (AaCV), found in the liana aristolochia (Aristolochia gibertii Hook), an additional short orphan ORF encoding a transmembrane helix was detected between P3 and M. The AaCV genome and inferred encoded proteins share the highest identity values, consistently < 60%, with their counterparts of the yerba mate chlorosis-associated virus (Cytorhabdovirus flaviyerbamate). The second virus, false jalap virus (FaJV), was detected in the herbaceous plant false jalap (Mirabilis jalapa L.) and represents together with tomato betanucleorhabdovirus 2, originally found in tomato plants in Slovenia, a tentative new species of the genus Betanucleorhabdovirus. FaJV particles accumulate in the perinuclear space, and electron-lucent viroplasms were observed in the nuclei of the infected cells. Notably, distinct from typical rhabdoviruses, most virions of AaCV were observed to be non-enclosed within membrane-bounded cavities. Instead, they were frequently seen in close association with surfaces of mitochondria or peroxisomes. Unlike FaJV, AaCV was successfully graft-transmitted to healthy plants of three species of the genus Aristolochia, while mechanical and seed transmission proved unsuccessful for both viruses. Data suggest that these viruses belong to two new tentative species within the subfamily Betarhabdovirinae.

Species diversity of culturable endophytic fungi from Brazilian mangrove forests.

This study aimed to perform a comparative analysis of the diversity of endophytic fungal communities isolated from the leaves and branches of Rhizophora mangle, Avicennia schaueriana and Laguncularia racemosa trees inhabiting two mangroves in the state of São Paulo, Brazil [Cananeia and Bertioga (oil spill-affected and unaffected)] in the summer and winter. Three hundred and forty-three fungi were identified by sequencing the ITS1-5.8S-ITS2 region of rDNA. Differences were observed in the frequencies of fungi isolated from the leaves and branches of these three different plant species sampled from the Bertioga oil spill-affected and the oil-unaffected mangrove sites in the summer and winter; these differences indicate a potential impact on fungal diversity in the study area due to the oil spill. The molecular identification of the fungi showed that the fungal community associated with these mangroves is composed of at least 34 different genera, the most frequent of which were Diaporthe, Colletotrichum, Fusarium, Trichoderma and Xylaria. The Shannon and the Chao1 indices [H'(95 %) = 4.00, H'(97 %) = 4.22, Chao1(95 %) = 204 and Chao1(97 %) = 603] indicated that the mangrove fungal community possesses a vast diversity and richness of endophytic fungi. The data generated in this study revealed a large reservoir of fungal genetic diversity inhabiting these Brazilian mangrove forests and highlighted substantial differences between the fungal communities associated with distinct plant tissues, plant species, impacted sites and sampling seasons.

Disease caused by Neofusicoccum parvum in pruning wounds of grapevine shoots and its control by Trichoderma spp. and Xenorhabdus szentirmaii.

Neofusicoccum parvum, is a fungal pathogen and one of the etiological agents of dieback disease in grapevines. The fungus causes deterioration of vines due to vascular colonization and/or production of toxins. We report herein the inhibitory effects of Trichoderma spp. isolates and the antifungal effects of cell-free supernatants (CFS) from Xenorhabdus and Photorhabdus bacteria against N. parvum in agar plates. We also evaluated the effects of the most effective fungi and bacteria against the pathogen in pruning wounds of vine shoots. All isolates of Trichoderma exhibited antifungal activity ranging between 82 and 97.5% at 14 days of post-treatment. All Xenorhabdus and Photorhabdus CFS at 10 and 33% concentrations inhibited mycelial growth with X. szentirmaii PAM 11 and PAM 25 causing the highest inhibition (>74%). In the shoot experiments, T. asperellum IB 01/13 and T. asperellum Quality®, X. szentirmaii PAM 11 (undiluted growth culture and CFS) suppressed the fungus by ≥ 93%. Our study highlights the potential of Trichoderma and X. szentirmaii PAM 11 for use as biofungicides in the management of N. parvum in grapevines. Further studies should be conducted to develop formulations of Trichoderma and Xenorhabdus that enhance stability in shelf-life and increase the efficacy of N. parvum control in grapevines under field conditions.

First Report of Aeromonas veronii as an Emerging Bacterial Pathogen of Farmed Nile Tilapia (Oreochromis niloticus) in Brazil.

Brazil is one of the world's leading producers of Nile tilapia, Oreochromis niloticus. However, the industry faces a major challenge in terms of infectious diseases, as at least five new pathogens have been formally described in the last five years. Aeromonas species are Gram-negative anaerobic bacteria that are often described as fish pathogens causing Motile Aeromonas Septicemia (MAS). In late December 2022, an epidemic outbreak was reported in farmed Nile tilapia in the state of São Paulo, Brazil, characterized by clinical signs and gross pathology suggestive of MAS. The objective of this study was to isolate, identify, and characterize in vitro and in vivo the causative agent of this epidemic outbreak. The bacterial isolates were identified as Aeromonas veronii based on the homology of 16S rRNA (99.9%), gyrB (98.9%), and the rpoB gene (99.1%). A. veronii showed susceptibility only to florfenicol, while it was resistant to the other three antimicrobials tested, oxytetracycline, enrofloxacin, and amoxicillin. The lowest florfenicol concentration capable of inhibiting bacterial growth was ≤0.5 µg/mL. The phenotypic resistance of the A. veronii isolate observed for quinolones and tetracycline was genetically confirmed by the presence of the qnrS2 (colE plasmid) and tetA antibiotic-resistant genes, respectively. A. veronii isolate was highly pathogenic in juvenile Nile tilapia tested in vivo, showing a mortality rate ranging from 3 to 100% in the lowest (1.2 × 104) and highest (1.2 × 108) bacterial dose groups, respectively. To our knowledge, this study would constitute the first report of highly pathogenic and multidrug-resistant A. veronii associated with outbreaks and high mortality rates in tilapia farmed in commercial net cages in Brazil.

Hitting the Road: Haplotype Diversity of Fire Ants Nesting on Disturbed Atlantic Forest Habitats.

Ants of the genus Solenopsis are globally distributed, presenting high diversity and many generalist species. In South America, the dominant species is Solenopsis saevissima (Smith, 1855), commonly found nesting in grassy fields surrounding humanized areas. In spite of being so common, there has been no research evaluating the effect of human disturbances on the mitochondrial DNA (mtDNA) haplotype diversity in this species. In this context, we here characterized the mtDNA haplotype diversity in S. saevissima nests by highway roadsides, dust roads, and forest borders of Atlantic Forest, based on partial sequences of cytochrome c oxidase subunit I (COI). Based on the facts that the species is a rapid colonizer of disturbed habitats, we specifically probed how the genetic diversity of native S. saevissima is impacted by highways and roads infrastructure expanding around the rainforest. Species diagnosis was established both by morphological characters and obtained mtDNA COI sequences. Overall, the species exhibited high haplotypes and nucleotide diversity, particularly around forest borders; though all haplotypes seemed closely related across the different habitats. We identified seven mitochondrial haplotypes (H1 to H7), where haplotype H1 was exclusively found in highway roadside nests, and H7 on dust roads; the remaining haplotypes were recorded from all habitats. Haplotype H1 was geographically isolated to the south of the Atlantic Forest, supporting previous suggestions that it acts as a biogeographical barrier. The pattern is suggestive of a recent species expansion, probably resulting from extensive habitat fragmentation. Taken together, our data demonstrates fire ant haplotypes prevailing in some anthropized habitats, characterizing how a native species lining the remnants of the Brazilian Atlantic Forest might be a concern for environmental conservation.

Citrus Bright Spot Virus: A New Dichorhavirus, Transmitted by Brevipalpus azores, Causing Citrus Leprosis Disease in Brazil.

Citrus leprosis (CL) is the main viral disease affecting the Brazilian citriculture. Sweet orange (Citrus sinensis L. Osbeck) trees affected by CL were identified in small orchards in Southern Brazil. Rod-like particles of 40 × 100 nm and electron lucent viroplasm were observed in the nucleus of infected cells in symptomatic tissues. RNA extracts from three plants, which proved negative by RT-PCR for known CL-causing viruses, were analyzed by high throughput sequencing and Sanger sequencing after RT-PCR. The genomes of bi-segmented ss(-)RNA viruses, with ORFs in a typical organization of members of the genus Dichorhavirus, were recovered. These genomes shared 98-99% nt sequence identity among them but <73% with those of known dichorhavirids, a value below the threshold for new species demarcation within that genus. Phylogenetically, the three haplotypes of the new virus called citrus bright spot virus (CiBSV) are clustered with citrus leprosis virus N, which is a dichorhavirus transmitted by Brevipalpus phoenicis sensu stricto. In CiBSV-infected citrus plants, B. papayensis and B. azores were found, but the virus could only be transmitted to Arabidopsis plants by B. azores. The study provides the first evidence of the role of B. azores as a viral vector and supports the assignment of CiBSV to the tentative new species Dichorhavirus australis.

First molecular evidence of Wolbachia occurrence in Amblyomma sculptum (Acari: Ixodidae).

As the main vector for the bacterium Rickettsia rickettsii in Brazil, the tick Amblyomma sculptum is a parasite of great public health importance in this country. Wolbachia is an endosymbiont bacterium highly widespread among invertebrates and because of its impact on its hosts' biology, form a powerful alternative for pests and disease control. The aim of this study was to investigate the occurrence of this bacterium in A. sculptum. For this, 187 adult ticks collected in two municipalities in the interior of the state of São Paulo, Brazil, were analyzed using molecular techniques and bioinformatics tools. A total of 15 ticks were positive for the presence of Wolbachia. Phylogenetic analysis on the 16S rRNA gene indicated that the Wolbachia DNA sequences obtained in this investigation belonged to different clades, probably in supergroups B and F. This was the first study to report the occurrence of Wolbachia in A. sculptum and it enriches knowledge about the susceptibility of ticks to this bacterium. Now that we know that Wolbachia can be found in A. sculptum, the objective for a next study must be to investigate Wolbachia's possible origin in this tick.

Phylogenetic analysis of Xanthomonas based on partial rpoB gene sequences and species differentiation by PCR-RFLP.

The rpoB gene was evaluated as an alternative molecular marker for the differentiation of Xanthomonas species and in order to understand better the phylogenetic relationships within the genus. PCR-RFLP experiments using HaeIII allowed differentiation of Xanthomonas species, particularly those that affect the same plant host such as Xanthomonas albilineans and X. sacchari, pathogenic to sugar cane, Xanthomonas cucurbitae and X. melonis, which cause disease in melon, and Xanthomonas gardneri, X. vesicatoria and X. euvesicatoria/X. perforans, pathogenic to tomato. Phylogenetic relationships within the genus Xanthomonas were also examined by comparing partial rpoB gene sequences (612 nt) and the Xanthomonas species were separated into two main groups. Group I, well supported by bootstrap values of 99 %, comprised X. euvesicatoria, X. perforans, X. alfalfae, X. citri, X. dyei, X. axonopodis, X. oryzae, X. hortorum, X. bromi, X. vasicola, X. cynarae, X. gardneri, X. campestris, X. fragariae, X. arboricola, X. cassavae, X. cucurbitae, X. pisi, X. vesicatoria, X. codiaei and X. melonis. Group II, again well supported by bootstrap values of 99 %, comprised X. albilineans, X. sacchari, X. theicola, X. translucens and X. hyacinthi. The rpoB gene sequence similarity observed among the species in this study ranged from 87.8 to 99.7 %. The results of PCR-RFLP of the rpoB gene indicated that this technique can be used for diagnosis and identification of most Xanthomonas strains, including closely related species within the genus. However, species that showed identical profiles could be differentiated clearly only by sequence analysis. The results obtained in our phylogenetic analysis suggested that the rpoB gene can be used as an alternative molecular marker for genetic relatedness in the genus Xanthomonas. The results of PCR-RFLP of the rpoB gene indicate that this technique can be used for diagnosis and identification of closely related species within the genus, representing a rapid and inexpensive tool that can be easily standardized between laboratories.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters.

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the ß-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.