Program Title: Harnessing Abaca Genetic Resources: Integrating Molecular Strategies for Pest Management, Drought Resiliency, and On-site Detection Assays

Program Leader: Dr. Leny C. Galvez

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PROGRAM SUMMARY

 

Objectives of the Program:

 

General:  This program aims to enhance the abaca genetic resources management and utilization through biotic and abiotic stress characterization and development of on-site detection assays for abaca disease and varietal identification

 

Specific:

 

1. To profile the physical and physiological mechanisms and analyze the genetic and functional basis of abiotic (drought) and biotic (ABTV/BBTV) stress;

2. To develop a comprehensive strategy for the identification, on-site detection, and evaluation of abaca responses against the causal pathogen for abaca Fusarium wilt;

3. To develop, package and validate a portable and ultrasensitive genotyping assay for identification of abaca (Musa textilis Née) varieties; and

4. To upgrade and enhance the capability of PhilFIDA-IMBL to effectively and efficiently deliver services to its clientele.

 

Significance/Impact to knowledge advancement and to the society:

 

The Philippine Fiber Industry Development Authority (PhilFIDA), an attached agency of the Department of Agriculture is mandated to promote the growth and development of the Philippine fiber industry in all aspects including research.  PhilFIDA’s R4D program covers all natural fiber crops with abaca being the premier crop, as the focus of the Research and Development, Extension Agenda and Program (RDEAP) which is aligned with the goals of the Agricultural and Fishery Modernization Act (AFMA) of 1997.  Moreover, these R&Ds were also aligned to the Updated Harmonized National Research and Development Agenda (HNRDA-AANR 2022-2028) of the DOST-PCAARRD.

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The abaca (Musa textilis Nee) is classified botanically according to the following taxonomic divisions: Spermatophyta, Angiospermae, Monocotyledonae, Zyngiberales, Musaceae, and Musa (Gutierrez et al., 2023). The anatomy of the plant closely resembles that of the banana plant (Musa sapientum), with around 25 fleshy stalks emerging from its rootstock, creating a "mat" or "hill”.  Each of these stalks develops approximately 12–25 leaves with petioles that overlap, resulting in a compact, shrub-like morphology and the formation of a "false trunk" or "pseudostem" that normally has a diameter of 30–40 cm (Shahri et al., 2014). The height of this perennial herb is normally around 8 meters, and it forms a cluster consisting of short cylindrical corms with developing buds (Gutierrez et al., 2023).

The leaves, which have a length of 1 to 2.5 meters and a width of 20 to 30 centimeters, have a vibrant green hue on their upper surface and a yellowish-green coloration on their underside (Shahri et al., 2014). The stalk of the plant exhibits a height ranging from 2 to 6 centimeters and a diameter ranging from 9 to 30 centimeters. The stems of abaca consist of approximately 93% water and 1.3-5% fiber. The fiber is mostly made up of basic polymers such as hemicelluloses, celluloses, and lignin. The stems have a high crystalline index of over 65% (Gutierrez et al., 2023). These fibers display a diverse range of colors, including white, brown, red, black, or purple. The specific hues of these fibers may vary depending on the plant variety and the position of the petiole. Upon reaching maturity, the plant generates male inflorescence consisting of diminutive dark red bracts organized in spikes. The banana-shaped fruits measure 8 cm in length and 2.5 cm in diameter. These fruits have green to yellow peel and white pulp, which contains sizable, black seeds (Shahri et al., 2014).

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Abaca is one of the country’s major agricultural export commodities. For the period 2010 – 2019, it earned an average of US$119 million annually from the exports of raw fibers and manufactures; of which the pulp exports account for the lion share of 69.3%.  Notably, in 2020 the aggregate export earnings reached USD160.6 million. In addition, the country supplied 86.1% of the world's requirements for raw fiber, fiber craft, cordage, and pulp manufacturing with the rest contributed by Ecuador (13.7%) and Costa Rica (0.2%) (PhilFIDA Report, 2020).  The abaca industry is a major source of livelihood for nearly 1.5 million Filipinos comprising 126,601 farmers cultivating a total abaca area of 162,546.21 hectares (PhilFIDA Report, 2023).

 

The abaca plant is indigenous to the Philippines and has a long history of use.  Our forefathers were already cultivating abaca, extracting the fibers and weaving them into cloth even before the Hispanic times. In the 1820s abaca fibers were exported to the United States (US) as a raw material for the marine cordage of the US Navy (Spencer, 1953).  In the light of the growing global interest for biodegradable products and forest conservation, abaca with its natural and superior material offers enormous potential for industrial use.  It is presently the preferred material in the production of pulp for specialty papers like tea bags, meat/sausage casings, cigarette paper, filter papers, currency notes, stencil papers and other non-woven product applications.  Companies here and abroad are aggressively researching on other applications in highly specialized products.  Glatfelter, the largest producer of abaca pulp in the world, makes tea bags from high quality abaca fiber together with other man-made fiber to achieve rapid release of first-class tea aroma.  It also produces heat-sealable coffee filter paper with abaca fiber as a filtration layer.  These developments in the global market demand a stable supply of high quality abaca fiber. However, local supply is unstable because the abaca production is hampered, among others, by the dreaded viral diseases like bunchy-top, bract mosaic, and mosaic that cause significant reductions in fiber yield.  Moreover, the effect of global climate change is threatening sustainable abaca cultivation.

 

Various cultivars of abaca are cultivated in almost all provinces in the Philippines.  There are as many as 200 cultivars of abaca, mostly landraces, which are attributed to the planting of seeds in the early days of its domestication (Torres and Garido, 1939).  Duplications are also possible because the same cultivar may be given a different name in different regions. There are more than 700 accessions of abaca maintained in field genebanks in the country (Altoveros and Borromeo, 2007), and there are still abaca plants in the wild (Villavicencio et al, 2007).  At PhilFIDA genebanks, there are more than 100 accessions being maintained in three locations.

 

The Philippines currently has a rich abaca genetic diversity, as evidenced by a diversity index of 0.68 (Yllano et al., 2020) and a heterogenous gene pool (Galvez et al., 2021).  The population structure among abaca cultivars has also been previously elucidated by Galvez et al. (2021). Sixty-two abaca accessions were grouped into two types, the first being comprised mostly of M. textilis cultivars, while the second consisted of hybrids such as Daratex, Canton, Mamakaw, Hybrid 1, and Hybrid 2. These listed accessions were also identified to have differentiated from the rest of the accessions as a result of interspecific hybridization.

 

To take full advantage of the rich genetic diversity in the abaca germplasm collections, a high standard of accuracy is essential for genebank management. Each accession must have a true-to-type genetic identity, be accurately labeled, and have intact database records.  The recently published abaca genome sequencing and assembly (Galvez, et al. 2021) will serve as reference to efficiently and accurately genotype the various accessions.

 

The main goal of this program is to enhance the abaca genetic resources management that will ultimately maintain the integrity and sustainability of the Philippine abaca industry.  In order to establish a well-curated germplasm collection, there is a need to discover and validate genetic information of each accession including accurate molecular markers, polymorphism among other Musa species, and differential gene expression in response to biotic and abiotic stresses.  With the recent sightings of Fusarium oxyporum f. sp. cubense Tropical Race 4 (TR4) in abaca, a need for immediate characterization and development of appropriate detection tool is a must.  Additionally, a genotyping assay is a necessary tool to continuously screen for the authenticity of each variety as well as to keep track of the sources of fibers in the market for regulatory purposes.

 

Methodology:

 

This program contains three interconnected projects and the methodologies are discussed specifically in the project methodologies.  Prior to the conduct of the projects, a Permit for the collection and transport of suspected Foc-infected samples will be secured from the BPI Quarantine Office.

 

Project 1.  Profiling, genetic, and functional analysis of abaca (Musa textilis Née) responses to drought and bunchy top disease stresses

Project Leader: Cris Francis C. Barbosa

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Study 1. Morphological and physiological analysis of abaca responses to drought stress

 

1. Plant materials and experimental set-up

2. Identification of drought tolerant variety(ies) based on physical and physiological evaluation after exposure to drought condition simulations

 

Study 2. Morphological and physiological analysis of abaca responses to disease stress

 

1. Plant materials and experimental set-up

2. Virus transmission and disease resistance/tolerance evaluation

 

Study 3. Genetic and functional analysis of abaca responses to drought stress

 

1.  Identification of genetic polymorphisms linked to drought tolerance

2. Identification of differentially-expressed genes associated with drought tolerance

3. Detection and confirmation of candidate genes for resistance to drought

 

Study 4. Genetic and functional analysis of abaca responses to bunchy top disease stress

 

1.  Identification of genetic polymorphisms linked to disease tolerance

2. Identification of differentially-expressed genes associated with disease tolerance

3. Detection and confirmation of candidate genes for resistance to ABTV and BBTV

 

Study 5. Validation of molecular markers for disease and drought resistance based on response-genetic polymorphism-gene expression relationships

 

Project 2. Detection, characterization, and race identification of Fusarium oxysporum f. sp. cubense, and the response of selected abaca accessions to the causal organism of abaca wilt

Project Leader: Jayson C. Asunto

 

Study 1. Polyphasic identification of Fusarium oxysporum f. sp. cubense affecting abaca using molecular and vegetative compatibility group analysis.

 

     a. Sample collection and Isolation

     b. Molecular identification using PCR Analyses

     c.  Vegetative Compatibility Group (VCG) Analysis

 

Study 2.  Evaluation of selected abaca accessions for resistance to Fusarium oxysporum f. sp.  cubense

 

     a. Abaca and Musa sp. Planting Material Collection

     b. Inoculation of Abaca and Musa sp. Planting Material

     c. Physical Observation and Disease Severity Evaluation

     d. RNA Extraction and Sequencing

     e. Identification and Assessment of DEGS

 

Study 3.  Enhancement, packaging and field validation of LAMP detection technique for Fusarium oxysporum f. sp. cubense

 

    a. Development of crude extraction protocol for on-site detection

    b. Optimization of lyophilized LAMP mixes

    c. Field testing and validation

 

Project 3.  Development, packaging and validation of a portable and ultrasensitive genotyping assay for identification of abaca (Musa textilis Née) varieties

Project Leader: Dr. Leny C. Galvez

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This project will have four (4) different studies as follows:

 

Study 1. Identification and mining of highly informative polymorphisms through genotyping-by-sequencing platforms

1. DNA extraction from 30 abaca varieties (each represented by three replicates)

2. Whole genome resequencing/Genotyping-by-sequencing

3. Bioinformatics analysis and polymorphism mining

 

Study 2. Development of DNA extraction protocol(s) for abaca fibers

1. Testing of different approaches and strategies for DNA extraction from abaca dry fibers

2. Statistical comparisons and protocol selection

 

Study 3. Development of a LAMP assay for genotyping of abaca plant- and fiber-derived DNA

         a.  Primer design

         b. Optimization of LAMP conditions

         c. Testing the LAMP assay for varietal identification using abaca leaf and fiber samples

Study 4. Packaging of the developed genotyping assay for on-site variety and fiber identification

         a. Genotyping kit production

         b. Lyophilization of the LAMP-based genotyping kit

 

Study 5. Validation of the packaged genotyping assay in the laboratory and for on-site application

 

         a. Analysis of analytical and diagnostic sensitivity and specificity of the assay

         b. Multivariate and phylogenetic analysis of identified genotypes

         c. Comparison with PCR/HRM-based genotyping through PABAK statistics

 

Project 4. Upgrading of PhilFIDA Immunology and Molecular Biology Laboratory for More Effective and Efficient Delivery of Services

 

1. Acquisition of additional laboratory equipment necessary in the conduct of research projects; and

2. Procurement and acquisition of one (1) unit high capacity and high processing workstation suitable for Big Data analytics.