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Banana bract mosaic virus (BBrMV) causes one of the major viral diseases of the fiber crop abaca at a great economic loss in the Philippines. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect BBrMV. A set of six primer pairs was designed based on the conserved coat protein gene sequences of BBrMV. The RT-LAMP can be completed in 30 min and was 100,000-fold more sensitive than RT-PCR. The RT-LAMP assay is suitable for field detection of BBrMV using crude RNA extracts and fluorescent DNA intercalating dyes that produce easily visualized results.

Polyclonal antibodies against abaca bunchy top virus (ABTV) and banana bunchy top virus (BBTV) proteins are necessary for immuno-based detection of these two viruses in abaca (Musa textilis Nee). In this study, recombinant bunchy top viral coat proteins fused with a 6xHis tag at the N-terminus were expressed in E. coli BL21StarTM(DE3)pLysS strain and purified under denaturing conditions. Purified recombinant ABTV and BBTV coat proteins were used as antigens for the production of rabbit polyclonal antibodies. IgG was purified and evaluated by Direct Antigen Coating (DAC)-ELISA and further optimized by testing primary to secondary antibody dilution combinations. Analysis of ABTV and BBTV-infected abaca samples using the optimized DAC-ELISA assay showed that the anti-ABTV CP IgG can react to BBTV and that anti-BBTV CP IgG can react to ABTV, hence, a cross-reaction. The study demonstrates the advantage of using recombinant DNA technology for mass production of antigens for antibody production. Although specificity of the polyclonal antibodies may have been compromised when renatured recombinant proteins were used as immunogens, the ability of the purified IgGs to detect positive abaca samples reveals that the DAC-ELISA can be routinely used for screening disease-free abaca planting materials.

Abaca (Musa textilis Née), an indigenous crop to the Philippines, is known to be the source of the strongest natural fiber. Despite its huge economic contributions, research on crop improvement is limited due to the lack of genomic data. In this study, the whole genome of the abaca var. Abuab was sequenced using Illumina Novaseq 6000 and Pacific Biosciences Single-Molecule Real-Time Sequel. The genome size of Abuab was estimated to be 616 Mbp based on total k-mer number and volume peak. Its genome was assembled at 65× depth, mapping 95.28% of the estimated genome size. BUSCO analysis recovered 78.2% complete BUSCO genes. A total of 33,277 gene structures were predicted which is comparable to the number of predicted genes from recently assembled Musa spp. genomes. A total of 330 Mbp repetitive elements were also mined, accounting to 53.6% of the genome length. Here we report the sequencing and genome assembly of the abaca var. Abuab that will facilitate gene discovery for crop improvement and an indispensable source for genetic diversity studies in Musa.

Deoxyribonucleic acid (DNA) extraction in abaca (Musa textilis Nee), the source of the strongest natural fiber (Manila hemp), is difficult due to its fibrous nature, high cellulose content, and polyphenol compounds. Thus an optimized DNA extraction method is required for extracting high quality abaca DNA for next-generation sequencing applications. This study, hence, aimed to test and compare five different methods for the extraction of high molecular weight DNA from abaca leaves. The methods are the traditional cetyl trimethylammonium bromide (CTAB) method (Protocol 1), the CTAB with polyvinylpyrrolidone (PVP) method (Protocol 2), the CTAB with 0.3 % β-mercaptoethanol method (Protocol 3), sodium dodecyl sulfate method (Protocol 4), and CTAB with Triton X-100 and PVP method (Protocol 5). The use of a high throughput homogenizer (TissueLyserII) was also tested in tandem with select extraction protocols for applications in high-throughput DNA extraction. DNA from two abaca varieties were extracted via Protocol 3 and were sent for sequencing based on the Illumina Novaseq platform. After passing the quality control parameters for library preparation, Protocol 3 was found to be the simplest and most consistent method for extracting average yield DNA with high quality for next-generation sequencing applications, while Protocol 4 was determined to have the shortest processing time. Together with TissueLyserII-facilitated homogenization, Protocol 4 is the most appropriate combination for high-throughput extraction of abaca samples which will be useful for genotyping-by-sequencing (GBS) strategies as a molecular tool for plant breeding.

Abaca bunchy top virus (ABTV) and banana bunchy top virus (BBTV) are pathogens that infect abaca in the Philippines causing significant reduction in abaca fiber yield and quality. In this study, a sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting ABTV and BBTV in abaca, Musa textilis Nee. Five primer sets were designed and tested to detect ABTV while three primer sets were designed and tested to detect BBTV. Out of which only one primer set for each virus was specific to their respective targets that is DNA-N for ABTV and DNA-S for BBTV for successful LAMP based detection applications. The concentrations of Bst DNA polymerase, dNTPs, incubation time and temperature were optimized for both viruses using either GelRed™ or SYBR™ Green I as detection dyes. The ABTV and BBTV LAMP assays were more sensitive than conventional PCR and were able to detect ABTV or BBTV in asymptomatic abaca samples. The developed LAMP protocols provide a simple, fast and accurate detection method that will be indispensable for the production of ABTV- and BBTV-free abaca tissue cultured plantlets. This is the first report of a LAMP-based assay for detection of ABTV and BBTV in abaca.