team members - agricultural innovation marketplace€¢ wagner lucena (co-leader), liziane lima ......

Identification of novel Bacillus thuringiensis effectors active against thecoffee berry borer, leaf-cutter ants and burrowing nematodes through

prospection of tropical agroecosystems and pangenomics

Team members

Institutions

• César Rodríguez (Co-leader), Rebeca Mora, Adrián Pinto • Wagner Lucena (Co-leader), Liziane Lima • Isabela Tristão, Leonardo Pepino, María Grossi

• Universidad de Costa Rica• Embrapa Cotton• Embrapa Cenargen (Genetic Resources and Biotechnology)

Expected vs. Achieved Results

• At least 400 new Bt strains from Costa Rican and Brazilian agroecosystems, incorporated along with their metadata into databases and collections

• Draft sequences of at least 40 Btplasmids

• Annotated supercontigs

• A database of Cry/Cyt proteins and other virulence factors

• A database of replication and maintenance modules of the plasmids sequenced

• Sequence comparisons, similarity matrixes, and dendrograms to highlight novel effectors

• Rearings for each pests



• 60 new Bt strains from leaf-cutter ants (Costa Rica) + whole Costa Rican Btcollection recovered from coffee plantations and protected areas revived (n=120)

• Illumina sequences for five plasmid DNA pools representing 82 isolates

• Annotated contigs (two assembly strategies: 18000/17500, n50 ca. 3kb)

• Two databases with BLAST and HMM hits to curated Cry/Cyt/VIP protein sequences and Bt plasmids

• Dendrograms, heatmaps, and sequence comparisons to highlight diversity and novel alleles

• Rearings for CBB and leaf-cutter ants

Achieved Results



Achieved Results



Cry1Ac32 6F HMM(2)

Cry1Ac32 6F HMM(3)

Cry1Ac32 6F HMM

Cry1Ac32 6F HMM(5)

Cry1Ac32 4D HMM(2)

Cry1Ac32 4D HMM

Cry1Ac12 4D HMM(2)

Cry1Ac32 4D BLA

Cry1Ac12 4D HMM(7)

Cry1Bd3 REF

Cry1Ac12 4D HMM(8)

Cry1Ac12 6F HMM(6)

Cry1Ac12 REF

Cry1Ac32 REF

Cry1Ac12 6F HMM(7)

Cry1Ac12 6F HMM(3)

Cry1Ac12 6F HMM(5)

Cry1Ac12 6F HMM

Cry1Aa22 6F BLA

Cry1Ac12 6F HMM(2)

Cry1Ac12 4D HMM(3)

Cry1Ac12 4D HMM(4)

Cry1Ac12 4D HMM

Cry1Ac12 4D HMM(6)

Cry1Ac12 4D HMM(9)

Cry1Ac12 4D HMM(9)

Cry1Ab34 REF

Cry1Aa21 REF

Cry1Aa8 REF

Cry1Aa22 REF

Cry1Aa21 4D HMM(4)

Cry1Aa21 6F HMM(4)

Cry1Aa21 6F HMM(3)

Cry1Aa22 6F HMM

Cry1Aa21 6F HMM(2)

Cry1Aa21 6F HMM

Cry1Aa21 4D HMM(3)

Cry1Aa21 4D BLA

Cry1Aa21 4D HMM

Cry1Aa21 4D HMM(2)

Cry1Aa22 4D HMM

Achieved Results



Impacts

• Infrastructure improvements• Sustainability for current insect rearings• Curated databases of Bt effectors• Training in bioinformatics• Potential for novel allele discovery



Lessons Learned and Bottlenecks

• It is impractical to isolate pure pDNA in screening campaigns• Do not trust public databases, always curate• Always double check outputs from bioinformatic pipelines• It is hard to establish new rearings

• Apply for collect permits and clarify IP issues beforehand• Most sequences will be truncated



Next Steps

• Obtain sequences for two more pools (CR), and a similar # from Brazil• Continue attempts to establish nematode colony• Perform objective 3• Write scientific paper on allele diversity• Reconstruct full sequences (visual check of contigs with hits)



team members - agricultural innovation marketplace€¢ wagner lucena (co-leader), liziane lima ......

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