Genetic transformation of 'Hamlin' and 'Valencia' sweet orange plants expressing the cry11A gene of Bacillus thuringiensis as an additional tool for the management of Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Candidatus Liberibacter spp., the bacteria associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. HLB management has heavily relied on insecticide applications to control the ACP, although there are efforts towards more sustainable alternatives. In previous work, our group assessed the potential bioactivity of different strains of Bacillus thuringiensis (Eubacteriales: Bacillaceae) (Bt) containing cry/cyt genes as feasible tools to control ACP nymphs. Here, we report an attempt to use the cry11A gene from Bt to produce transgenic sweet orange plants using two promoters. For the genetic transformation, 'Hamlin' and 'Valencia' sweet orange seedlings were used as sources of explants. Transgenic plants were detected by polymerase chain reaction (PCR) with specific primers, and the transgene copy number was confirmed by Southern blot analyses. Transcript expression levels were determined by qPCR. Mortality assays of D. citri nymphs were carried out in a greenhouse, and the effect of the events tested ranged from 22% to 43% at the end of the five-day exposure period. To our knowledge, this is the first manuscript reporting the production of citrus plants expressing the Bt cry11A gene for the management of D. citri nymphs.

Genetic Structure and Molecular Variability Analysis of Citrus sudden death-associated virus Isolates from Infected Plants Grown in Brazil.

Citrus sudden death-associated virus (CSDaV) is a monopartite positive-sense single-stranded RNA virus that was suggested to be associated with citrus sudden death (CSD) disease in Brazil. Here, we report the first study of the genetic structure and molecular variability among 31 CSDaV isolates collected from both symptomatic and asymptomatic trees in CSD-affected areas. Analyses of partial nucleotide sequences of five domains of the CSDaV genomic RNA, including those encoding for the methyltransferase, the multi-domain region (MDR), the helicase, the RNA-dependent RNA polymerase and the coat protein, showed that the MDR coding region was the most diverse region assessed here, and a possible association between this region and virus adaption to different host or plant tissues is considered. Overall, the nucleotide diversity (π) was low for CSDaV isolates, but the phylogenetic analyses revealed the predominance of two main groups, one of which showed a higher association with CSD-symptomatic plants. Isolates obtained from CSD-symptomatic plants, compared to those obtained from asymptomatic plants, showed higher nucleotide diversity, nonsynonymous and synonymous substitution rates and number of amino acid changes on the coding regions located closer to the 5' end region of the genomic RNA. This work provides new insights into the genetic diversity of the CSDaV, giving support for further epidemiological studies.