Eduardo C. Andrade

Asian citrus psyllid RNAi pathway : RNAi evidence

Diaphorina citri, known as the Asian citrus psyllid, is an important pest of citrus because it transmits a phloem-limited bacteria strongly implicated in huanglongbing (citrus greening disease). Emerging biotechnologies, such as RNA interference, could provide a new sustainable and environmentally friendly strategy for the management of this pest. In this study, genome and functional analysis were performed to verify whether the RNAi core genes are present in the Asian psyllid genome and if the RNAi machinery could be exploited to develop a management strategy for this pest. Analyses of RNAi-related genes in the Asian citrus psyllid genome showed an absence of sequences encoding R2D2, a dsRNA-binding protein that functions as a cofactor of Dicer-2 in Drosophila. Nevertheless, bioassays using an in Planta System showed that the Asian citrus psyllid was very sensitive to ingested dsRNA, demonstrating a strong RNAi response. A small dose of dsRNA administered through a citrus flush was enough to trigger the RNAi mechanism, causing significant suppression of the targeted transcript, and increased psyllid mortality. This study provides evidence of a functional RNAi machinery, which could be further exploited to develop RNAi based management strategies for the control of the Asian citrus psyllid.

Genetic diversity studies of Papaya meleira virus

Papaya (Carica papaya) is a fruit crop of great economic and social importance for Brazil and other papaya-producing countries. Brazil is the second largest producer in the world. The papaya sticky disease, caused by Papaya meleira virus (PMeV), has caused great losses in the major Brazilian papaya-producing states. In order to estimate the genetic diversity of PMeV, latex samples were collected from papaya plants in the states of Bahia, Espirito Santo, Pernambuco, Ceara and Rio Grande do Norte, and total RNA was extracted. Specific primer for the replicase region allowed the amplification, by RT-PCR, of a fragment of approximately 560 bp from 31 isolates. The sequence analysis indicated a level of conservation greater than 88% among isolates. Furthermore, comparative analyzes indicated that PMeV has similarity with mycoviruses of the family Totiviridae. This phylogenetic relationship was reinforced by the presence of conserved motifs within in the RdRp regions from mycoviruses.

Genetic variability of papaya lethal yellowing virus isolates from Ceará and Rio Grande do Norte States, Brazil

The papaya (Carica papaya) is a fruit crop of great economic importance throughout the Brazilian northeast, which is responsible for 60% of the national output. Papayas in the states of Ceara and Rio Grande do Norte are affected by lethal yellowing disease, caused by papaya lethal yellowing virus (PLYV). Previous work suggested that PLYV is a putative sobemovirus. To assess the genetic variability of PLYV, foliar samples were collected in October 2008 and October 2009 in commercial fields from Ceara and Rio Grande do Norte states, and total RNA was extracted. Specific primers based on the sequence of a previously characterized PLYV isolate were used for the RT-PCR-based amplification of a 900 bp fragment corresponding to the central region of the viral genome. Fragments from 21 viral isolates were cloned and sequenced. Sequence analyses indicated >97% nucleotide sequence identity among the isolates, 94-100% identity with the previously sequenced PLYV isolate, and a lower but significant identity with sobemoviruses (43-48.5%). These results suggest a low genetic variability among PLYV isolates, and are in agreement with the provisional placement of PLYV in the genus Sobemovirus. Definitive taxonomic conclusions, however, can only be drawn after the determination of the full-length genomic sequence.

Analysis of the full-length genome sequence of papaya lethal yellowing virus (PLYV), determined by deep sequencing, confirms its classification in the genus Sobemovirus

Papaya lethal yellowing virus (PLYV) causes an economically important disease in papayas in northeastern Brazil. Based on biological and molecular properties, PLYV has been tentatively assigned to the genus Sobemovirus. We report the sequence of the full-length genome of a PLYV isolate from Brazil, determined by deep sequencing. The PLYV genome is 4,145 nt long and contains four ORFs, with an arrangement identical to that of sobemoviruses. The polyprotein and CP display significant sequence identity with the corresponding proteins of other sobemoviruses. Pairwise comparisons and phylogenetic analysis based on complete nucleotide sequences confirm the classification of PLYV in the genus Sobemovirus.

RNAi feeding bioassay: development of a non-transgenic approach to control Asian citrus psyllid and other hemipterans

RNA interference (RNAi) involves a natural mechanism of gene regulation and an antiviral defense system in eukaryotic cells, resulting in sequence‐specific degradation of RNAs. Recent studies demonstrated the feasibility of using RNAi‐based strategies to reduce insect pests or increase the health of beneficial insects. This technology permits the development of strategies to control a single insect species, with possibly no negative effects on non‐target species, like honey bees or parasitoids. Focus on the development of topically applied RNAi‐based approaches to manage insect pests and pathogens may provide environmentally sound products for use across all agriculture production systems. Herein presented are methods to develop RNAi‐based control to the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), vector of citrus Huanglongbing (HLB). Described is an RNAi feeding bioassay, called in plant system (iPS), which uses vegetative new‐growth citrus flush to deliver double‐strand RNA (dsRNA) to ACP during natural feeding. The iPS demonstrated delivery of non‐specific dsRNA in ACP 72 h post feeding. Feeding on flushes treated with dsRNA targeting ACP Arginine Kinase (dsAK), caused relative transcript knockdown and psyllid mortality. Increasing the dose of dsAK or use of dsRNA triggers made to different regions of the AK mRNA did not increase psyllid mortality. The iPS bioassay provides an easy system for use across many plants to successfully deliver dsRNA to ACP and other hemipteran insects for research or RNAi development.

RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC)

RNAi technologies are more environmentally friendly, as the technology provides great‐ er specificity in pest targeting, while reducing the potential negative effects on ecosys‐ tems and leaving beneficial insects and other organisms unharmed in crop ecosystems. Consequently, the increase in native fauna improves the efficacy of biological control agents against pests and pathogens. A growing understanding of the ubiquitous nature of RNAi, along with evidence for efficient, non-transgenic, topical applications has al‐ ready begun to garner support among organic and industry producers. Designing solu‐ tions to agricultural problems based upon the same mechanisms used in nature provides newer, safer solutions to pests and pathogens for all agricultural industries.

Biological, serological and molecular comparison between isolates of Cowpea severe mosaic virus.

ABSTRACT Cowpea, Vigna unguiculata, can be affected by several diseases, and those caused by viruses are considered of great importance. Cowpea severe mosaic virus (CPSMV), which belongs to the family Comoviridae, genus Comovirus, stands out for its severity and degree of incidence. Its genetic variability can give rise to different strains around the world, including in Brazil. The objective of the present research was to establish a biological, serological and molecular comparison between five CPSMV isolates obtained from naturally infected plants in different States of the Northeast of Brazil. Isolates were obtained from cowpea: CPSMV-AL (State of Alagoas), CPSMV- CE (Ceara); CPSMV-MC (Piaui), CPSMV-PE (Pernambuco) and an isolate obtained from Crotalaria paulinea, in the State of Maranhao - CPSMV-CROT. A host range study evidenced biological differences among the virus isolates, especially in cowpea genotypes. The isolates were showed to be serologically and molecularly related by polymerase chain reaction �� PCR, using degenerated primers which amplified two conserved regions in the coat protein and in the replicase genes. Cloning and sequence of CPSMV-PE made possible its comparison with other CPSMV isolates and other virus species from the genus Comovirus.

Crop losses in Brazilian cassava varieties induced by the Cassava common mosaic virus

Despite the widespread distribution of the Cassava common mosaic virus (CsCMV) in Brazil, little is known about the losses it causes in yield. The effect of CsCMV on different varieties was evaluated by reference to several agronomic traits. Four field trials were established in 2012/2013 and 2013/2014 using six varieties of cassava. Following mechanical inoculation with CsCMV, the presence of the virus was confirmed using the ELISA assay. The evaluated traits were plant height (PH), dry matter content (DMC), harvest index (HI), aerial part yield (APY), root yield (RoY), and starch yield (StY) in both inoculated and non-inoculated plants. Overall, the presence of the virus contributed little to the reduction in PH, HI, and DMC across the varieties, with PH being significantly reduced by 9.2 and 7.0 % in the BGM0212 and BRS Kiriris varieties, respectively. In contrast, APY, RoY, and StY were reduced by 30.2, 29.3, and 30.0 %, in the virus-infected plants respectively. While the BRS Kiriris and BRS Jari varieties suffered the highest reductions overall and were considered highly susceptible to CsCMV, none of the traits suffered reductions in the inoculated BRS Formosa plants. Although RoY and StY were reduced in inoculated plants of BRS Tapioqueira, crop yield for this variety was the highest. Thus, BRS Formosa and BRS Tapioqueira exhibited tolerance against CsCMV, which warrants further investigation.

Sequence variability in the coat protein gene of Cowpea severe mosaic virus isolates from northeastern Brazil

The comovirus Cowpea severe mosaic virus (CPSMV) causes a disease in cowpea (Vigna unguiculata) but, despite its importance, there are no studies on the genetic diversity of viral populations. We have determined the nucleotide sequences of part of the coat protein (CP) gene of six Brazilian isolates of CPSMV. Genomic fragments (521 nucleotides) were RT-PCR amplified, cloned, and their sequences were compared with each other and with other comoviruses. Sequence comparisons indicated a high degree of conservation for the CP gene, with 92-100% nucleotide and 97-100% amino acid sequence identity among the isolates. There was no correlation between geographical origin and sequence identity or phylogeny among the isolates.

Dinâmica da infestação de Brevipalpus phoenicis (Geijskes, 1939) (Acari:Tenuipalpidae) em pomares cítricos da Bahia, Brasil

The spatio-temporal dynamics of Brevipalpus phoenicis infestation was quantified in two regions of the state of Bahia, Brazil (North Coast-NC and Reconcavo Baiano-RB). In each region, 10 orchards were evaluated monthly (from April 2008 to February 2011), in which 21 plants had inspected three fruits per plant with a magnifying lens (10x) to record the presence or absence of the mite. The average proportion of infested plants varied between 0.38 and 1.0. In fruits, the minimum proportion of infestation was 0.10 and maximum, of 0.73 for NC and 0.66 in the RB. The infestation process had a strong seasonal component with more infested units detected in spring or summer (Nemenyi, p <0.05). The spectral density analysis showed the occurrence of two cycles of infestation: one of 2 to 3 months, possibly related to the mites biology and other larger, annual, arising from the interaction vector-host-environment. In both regions the spatial pattern of mite infestation within and between orchards was random. No symptoms of citrus leprosies were found in RB.