Molecular Identification of Cassava Arthropod Pest Complex in the Philippines

doi:10.21203/rs.3.rs-3327078/v1

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Molecular Identification of Cassava Arthropod Pest Complex in the Philippines

https://doi.org/10.21203/rs.3.rs-3327078/v1

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Cassava productivity is severely affected by arthropod pests, which cause damage through feeding and vector transmission. The complex nature of these pests, with morphologically similar species and small sizes, presents challenges in accurately identifying and implementing effective control measures. Accurate identification of arthropod pests infesting cassava in the field is crucial for successful pest management and mitigating the risk of introducing exotic pests through cassava trade and changing climate conditions. Thus, we employed DNA barcoding to generate genetic barcodes of the cassava arthropod pest complex found in major cassava growing areas in the Philippines. Identification to species level was achieved using molecular works with prior morphological identification. Molecular identification offers accurate species resolution of the cassava pest complex even at immature stages, typically hard to identify.

cassava

arthropod pests

DNA barcoding

genetic diversity

molecular identification

Cassava (Manihot esculenta) is a significant food crop in many parts of the world, particularly in Africa, Asia, and Latin America. Southeast Asia's export market of starch, chips, and other cassava-derived products was valued at $US 5 billion (Curran & Cooke, 2008). It is a significant root crop, bioenergy and industrial crop in developing countries. The Cassava pest complex in SE Asia includes native and non-native species and new and old-time invaders, and some species have coevolved with cassava in native South America. In contrast, others are generalist herbivores that have gradually adapted to the crop (Chaya et al., 2021). Mealybugs (Hemiptera: Pseudococcidae) are soft-bodied sap-feeding insects, with eight species relevant to Southeast Asia. One of which is the cassava mealybug Paracoccus manihoti which is part of the cassava arthropod complex that has rapidly spread into Laos, Vietnam, Cambodia, Malaysia and Indonesia, facilitated by highly suitable local climates in the recent years (Chaya et al., 2021). Paracoccus marginatus Williams and Granara de Willink, or papaya mealybug, is another mealybug that is part of the arthropod pest complex pf cassava and was first reported from Asia in 2008, with records from Indonesia and India, and more recently was detected in Cambodia, Thailand and the Philippines. Mites are among the significant pest of cassava in Southeast Asia, with approximately 50 species of mites that feed on cassava worldwide, one member of the green mite complex and 12 species of red mites (Acari: Tetranychidae) occur in the Asia region (Curran & Cooke, 2008). Red mites can cause yield reductions of up to 50% in Indonesia, with the most widespread species, Tetranychus urticae Koch and T. truncatus Ehara. T. cinnabarinus Boisd and the African red mite Eutetranychus africanus (Tucker) are also reported. Whiteflies (Hemiptera: Aleyrodidae) are a global cassava pest that directly and indirectly damages the plant. It transmits cassava mosaic virus (CMV), which causes yield losses of up to 82% and has been reported from Africa, India and Sri Lanka (Chaya et al., 2021).

Recent studies show that cassava production in the Philippines has been increasing (Murayama et al., 2014; Soria & Preciados, 2018). However, invasive insect pests have resulted in severe losses to cassava in Southeast Asia, including the Philippines (Uke et al., 2021; Milenovic et al., 2019). Whiteflies of the Bemisia tabaci species complex are economically important pests of cassava in the Philippines (Milenovic et al., 2019). These pests cause damage to cassava by feeding on the plant sap, leading to stunted growth, reduced yield, and even death of the plant. In addition, cassava phytoplasma disease (CPD) has been identified as a significant threat to cassava production in the Philippines (Plata et al., 2022). The problem of identifying these pests and diseases is that they are small and difficult to detect, especially in the early stages of infestation. These challenges have profound implications for food security and livelihoods in the Philippines. With the implementation of the ASEAN Free Trade Agreement and the World Trade Organization-General Agreement on Tariffs and Trade (WTO-GATT), native pests can potentially be explosive/invasive and reach outbreak levels if not immediately monitored and identified; thus, molecular identification is required, particularly concerning plant quarantine protocols and pest management. Although many cassava pests have been reported in recent reports from the Philippines, the molecular data on these insects is highly scarce and understudied. Here, we address the lack of molecular data through the conduct of molecular techniques, offering reliable identification tool for the cassava pest complex, in addition to classical taxonomic identification. The DNA barcodes of generated, which is already available in GenBank in the study major cassava pests essential for pest identification and management, plant quarantine protocols and sanitary phytosanitary (SPS) operations.

Collection of cassava arthropod pests

Cassava pests were extensively collected from different geographical regions of major cassava growing sites in the Philippines. Selected regions include Region II (Cagayan Valley), Region IV-A (CALABARZON), Region V (Bicol Region), Region VIII (Eastern Visayas), and Region X (Northern Mindanao). Provinces among the selected regions include Isabela and Quirino from Region II; Laguna from Region IV; Camarines Sur and Masbate from Region V; Samar from Region VIII; Bukidnon and Misamis Oriental from Region X. The collection sites were composed of eight provinces (Supp Fig. 1). Three barangays per province were selected per locality. A total of 33 farms served as collection sites. Cassava leaves, and branches infested with arthropod pests were collected and placed in clean plastic bags. Samples for DNA extraction were placed in a vial containing ethanol. All collections were labelled accordingly, and GPS data were recorded (Supp. Table 1). The collected samples were brought to the National Crop Protection Center (NCPC) for processing in the laboratory.

Morphological Identification of cassava arthropod pests

Specimens were viewed in a stereo microscope (ZEISS Stemi 305, Carl Zeiss Microscopy, LLC) and labelled accordingly. Arthropod pests were identified using basic morphological features and classified broadly as mealybugs, whiteflies, red spider mites, scale insects, and beetles. This broad classification was done as a primary identification, prior to molecular works.

Genomic DNA Isolation for Various Arthropod Pests of Cassava

Genomic DNA isolation for various arthropod pests including its immature stages was performed utilizing a solution-based Animal and Fungi DNA Preparation Kit (Jena Bioscience), following the manufacturer's protocol with modification. The DNA extraction process involved the utilization of specific methodologies tailored for each arthropod group, which are comprehensively described in Table I. These protocols were designed to efficiently extract high-quality genomic DNA from various arthropod pests, ensuring optimal yield and purity for downstream genetic analyses and research applications.

Table 1

The ratio of reagents and enzymes used on genomic DNA isolation for various insects in cassava.
Insect (whole or body part for larger insects)	Cell Lysis Solution (uL)	Proteinase K+ (uL)	Protein Precipitation Solution (uL)	Absolute Isopropanol (uL)	Washing Buffer Solution (uL)	DNA Hydration Solution (uL)	RNase (uL)
Scale insects	50	0.3	30	100	100	20	0.3
Whiteflies	50	0.3	30	100	100	20	0.3
Mealybugs	100	0.6	60	200	200	50	1.0
Mites (pooled 3–5 individuals)	30	0.2	15	70	50	20	0.2
	5			3	3
	Time of Centrifugation at 15,000xg (min)

PCR Amplification for Various Arthropod Pests of Cassava

Gene amplification was performed through polymerase chain reaction (PCR) to amplify the target COI gene. The 2xTaq Mastermix (Vivantis) was used with some modifications on the number of reagents and DNA templates used. The volume of reagents used for PCR amplification per insect group is detailed in Table 2. The DNA template used depends on its integrity, preferably at least 50 ng/uL concentration with a 1.5–2.0 purity index. Genomic DNA with high concentrations were diluted with DNA Hydration Solution from the extraction kit to achieve an approximately 50 ng/uL concentration. The PCR conditions and primers used in amplifying a particular insect group, following the literature, were detailed in Table 3.

Table 2

The ratio of reagents (Vivantis) and enzymes used on COI gene amplification for 25 uL PCR volume.
Insect	2xTaq (uL)	MgCl2+ (uL)	Forward Primer [amount and concentration] (uL; pmol/uL)	Reverse Primer [amount and concentration] (uL; pmol/uL)	Nuclease-Free Water (uL)
Scale insects and mealy bugs	12.5	0.5	1; 50	1; 50	5
Whiteflies	12.5	0.5	2.5; 10	2.5; 10	2
Mites	12.5	0.5	1; 10	1; 10	5

Table 3

PCR conditions and primers used on COI gene amplification for various arthropod pests of cassava
Insect Samples	Primer	Gene Region	Sequence	Thermal Profile	Reference
Whitefly	Btab-F	Cytochrome oxidase I	5’GAGGCTGRAAAATTARAAGTATTTGG 3’	94°C for 2mins, 35 cycles of 94°C for 30secs, 46°C for 60secs, 72°C for 1min and 72°C for 5mins	Shatters et al., 2009
Whitefly	Btab-R	Cytochrome oxidase I	5'CTTAAATTTACTGCACTTTCTGCCARATTAG3'		Shatters et al., 2009
Mites	C1-J-1718	Cytochrome oxidase I	5’ GGAGGATTTGGAAATTGATTAGTTCC 3’	94°C for 3mins, 35 cycles of 94°C for 1min, 45–51°C for 1 min, 72°C for 1min 30secs and 72°C for 10mins	Simon et al., 1994
Mites	COI REVA	Cytochrome oxidase I	5’ GATAAAACGTAATGAAAATGAGCTAC 3’		Gotoh et al., 2009
Scale insect and Mealybug	PcoF1	Cytochrome oxidase I	5’CCTTCAACTAATCATAAAAATATYAG3’	95°C for 2mins, initial 5 cycles of 94°C for 40secs, 45°C for 40secs, 72°C for 1min & 10secs, 40 cycles of 94°C for 40secs, 51°C for 40secs, 72°C for 1min & 10secs and 72°C for 5mins	Park et al., 2010
Scale insect and Mealybug	LepR1	Cytochrome oxidase I	5’TAAACTTCTGGATGTCCAAAAA3’		Park et al., 2010

Molecular Detection of Cassava Arthropod Pests Using COI mtDNA Gene Markers

The amplified gene was detected through gel electrophoresis using 1.0% agarose dissolved in TBE and viewed under the Labnet Enduro Touch gel viewer. The amplified COI region of mites samples collected from Bukidnon, showed the expected molecular size of approximately 900 bp. Among the amplified samples in every local population, a minimum of ten amplicons with good and clear bands were chosen for sequencing.

Molecular identification of cassava arthropods based on COI mtDNA sequences

In the present study, we employed molecular techniques to identify various arthropod pests infesting cassava plants based on their COI mtDNA sequences. A total of seven species of hemipteran insects and two species of spider mites were successfully identified using this approach. To ensure the accuracy of our molecular identification, we performed sequence alignment using the BLASTn tool in GenBank, which allowed us to compare our obtained sequences with those deposited in the database (Altschul et al., 1990). The percent identity of the nucleotide sequences ranged from 97–100%, indicating a high level of similarity between our samples and the reference sequences in GenBank.

The arthropod pests that were molecularly identified in this study included Aleurodicus disperses, Bemisia tabaci, Ferrisia virgata, Maconellicoccus hirsutus, Paracoccus marginatus, Parasaissetia nigra, and Pseudaulacaspis pentagona. We also identified two species of spider mites, namely Tetranychus cinnabarinus and Tetranychus kanzawai, which are known to infest cassava plants and cause damage by piercing plant cells and sucking out the contents. Table 4 and Fig. 1 summarise the identified arthropod pests and their corresponding COI mtDNA sequences. Table 5 presents the GenBank Accession number of Philippine cassava sequence barcodes generated by this research.

Table 4

The molecular identity of cassava arthropod pests using partial sequences of COI mtDNA using COI mtDNA through BLASTn alignment in the Genbank database.
Species Identity	Common Name	Query Cover	Percent Homology	Reference Sequence	Collection Site
Aleurodicus dispersus	Whitefly	100	99	KR063274.1	Camarines Sur, Laguna, Bukidnon, Misamis Oriental
Bemisia tabaci	Whitefly	100	98	KY951451.1	Masbate, Quirino, Camirines Sur
Ferrisia virgata	Mealy bug	100	99	LC273485.1	Laguna, Isabela
Maconellicoccus hirsutus	Mealy bug	100	99	KY373158.1	Isabela,Laguna
Paracoccus marginatus	Mealy bug	97	100	KP692579.1	Camarines Sur, Quirino
Pseudaulacaspis pentagona	Armored scale insect	95	100	HM474345.1	Camarines Sur, Misamis Oriental
Parasaissetia nigra	Black soft scale	98	100	KY933367.1	Camarines Sur
Tetranichus cinnabarinus	Mites	100	99	HM753535.1	Isabela; Bukidnon; Quirino; Cagayan De Oro City;
Tetranychus kanzawai	Mites	100	99	KJ729017.1	Camarines Sur; Laguna

Table 5

GenBank accession numbers for deposited COI sequences of identified armoured scale insects in the Philippines.
GenBank Accession Number	Sample Codes	COI phylogenetic ID	Location
MK213555	T_cinnabarinus_AB2_1_contig	Tetranychus cinnabarinus	Brgy. Sto. Niño, Manolo Fortich, Bukidnon
MK213556	T_cinnabarinus_AB2_2_contig	Tetranychus cinnabarinus	Brgy. Sto. Niño, Manolo Fortich, Bukidnon
MK213557	T_cinnabarinus_ABC4_4_contig	Tetranychus cinnabarinus	Brgy. Imbatug, Baungon, Bukidnon
MK213558	T_cinnabarinus_AC3_1_contig	Tetranychus cinnabarinus	Brgy. Balubal, Cagayan de Oro City
MK213559	T_cinnabarinus_AC3_2_contig	Tetranychus cinnabarinus	Brgy. Balubal, Cagayan de Oro City
MK213560	T_cinnabarinus_AI3_2_contig	Tetranychus cinnabarinus	Brgy. Rangayan, Ilagan City, Isabela
MK213561	T_cinnabarinus_AI3_3_contig	Tetranychus cinnabarinus	Brgy. Rangayan, Ilagan City, Isabela
MK213562	T_cinnabarinus_AMQ2_1_contig	Tetranychus cinnabarinus	Brgy. Lusod, Mandela, Quirino
MK213563	T_cinnabarinus_AMQ2_2_contig	Tetranychus cinnabarinus	Brgy. Lusod, Mandela, Quirino
MK213564	T_cinnabarinus_ANVL_20_contig	Tetranychus cinnabarinus	UP Los Banos, Laguna
MK213565	T_cinnabarinus_AS1_1_contig	Tetranychus cinnabarinus	Brgy. Imbatug, Baungon, Bukidnon
MK213566	T_cinnabarinus_AS1_3_contig	Tetranychus cinnabarinus	Brgy. Imbatug, Baungon, Bukidnon
MK213567	T_cinnabarinus_AS3_4_contig	Tetranychus cinnabarinus	Brgy Balubal, Cagayan De Oro City
MK213568	T_cinnabarinus_BS4_4_contig	Tetranychus cinnabarinus	Brgy. Tinangis, Pili, Camarines Sur
MK213569	T_cinnabarinus_HI6_1_contig	Tetranychus cinnabarinus	Brgy. Cabugao, Cauayan, Isabela
MK213570	T_cinnabarinus_HI6_2_contig	Tetranychus cinnabarinus	Brgy. Cabugao, Cauayan, Isabela
MK213572	A_dispersus_BS_3_3_contig	Aleurodicus dispersus	Brgy Bagumbayan, Baao, Camarines Sur
MK213573	A_dispersus_BS_3_4	Aleurodicus dispersus	Brgy Bagumbayan, Baao, Camarines Sur
MK213574	A_dispersus_VL_10	Aleurodicus disperseus	UP Los Banos, Laguna
MK213575	A_dispersus_VS_4	Aleurodicus dispersus	UP Los Banos, Laguna
MK213576	A_dispersus_VS_5	Aleurodicus dispersus	UP Los Banos, Laguna
MK213577	A_dispersus_VM_consensus	Aleurodicus dispersus	UP Los Banos, Laguna
MK213578	M_hirsutus_HI_S4_1	Maconellicoccus hirsutus	Brgy. Cabugao, Cauayan, Isabela
MK213579	M_hirsutus_HI_S4_5	Maconellicoccus hirsutus	Brgy. Cabugao, Cauayan, Isabela
MK213580	M_hirsutus_HI_S4_6	Maconellicoccus hirsutus	Brgy. Cabugao, Cauayan, Isabela
MK213581	P_marginatus_BS_4_2	Paracoccus marginatus	Brgy. Tinangis, Pili, Camarines Sur
MK213582	P_marginatus_BS_4_3	Paracoccus marginatus	Brgy. Tinangis, Pili, Camarines Sur
MK213583	P_marginatus_BS_4_13	Paracoccus marginatus	Brgy. Tinangis, Pili, Camarines Sur

Genetic diversity was conducted on field-collected mites, Tetranychus cinnabarinus, using COI gene. Insect species were not analyzed for genetic diversity due to apparent sequence similarity across various sampling sites. Five distinct haplotypes were identified among this species, indicating the presence of polymorphic sequences (Table 5). The analysis revealed 32 polymorphic sites, consisting of 24 singleton variable sites and eight parsimony informative sites. This variability in nucleotide positions among the haplotypes suggests more genetic heterogeneity within the T. cinnabarinus population. The nucleotide diversity, denoted as Pi, was calculated to be 0.00892 for the T. cinnabarinus mites. This value represents the average proportion of nucleotide differences between the population's randomly selected sequences. The higher Pi value, in this case, suggests a relatively elevated level of genetic variation within the T. cinnabarinus population.

Furthermore, the haplotype diversity (Hd) for the T. cinnabarinus mites was determined to be 0.6364, indicating moderate haplotype diversity within the population. Four distinct haplotypes among the T. cinnabarinus mites contribute to the observed haplotype diversity. Similar to the insects, the mites also exhibited an AT-rich COI region. This observation suggests a common characteristic of an AT-biased composition in the COI region among arthropods.

Table 5

Haplotype diversity of field-collected mites, *Tetranychus cinnabarinus*, using COI gene
	Sequence Haplotypes
	Haplotype 1	Haplotype2	Haplotype3	Haplotype4
Location	Isabela Quirino	Laguna	Camarines Sur, Bukidnon, Cagayan de Oro City, Bukidnon Zamboanga City	Camarines Sur
Number of base differences concerning Haplotype
Hap 1		9	8	29
Hap 2	9		1	27
Hap 3	8	1		26
Hap 4	24	27	26
Nucleotide Composition
A	280	280	280	283
C	105	108	107	103
G	101	100	100	98
T	360	358	359	362
G + C (%)	24.35	24.59	24.47	23.76
A + T (%)	75.65	75.41	75.53	76.24

Phylogeny of cassava arthropod pest

The nucleotide sequences of the cassava pests were aligned, and the consensus sequence was used to construct a phylogenetic tree based on the maximum likelihood algorithm of MEGA 7.0 (Kumar et al., 2016). A bootstrap with 1000 iterations was included to test the strength of the clustering obtained. The bootstrap value of 100% for mealybugs (Pa. marginatus, F. virgata, M. hirsutus) and scale insect P. pentagona, when aligned with GenBank reference sequences, indicates high confidence for the groupings obtained (Fig. 3).

Molecular phylogenetic analysis of red spider mites of cassava using COI mtDNA with evolutionary history inferred by using by Maximum Likelihood method based on the Tamura 3-parameter model (Tamura, 1992) showed the highest log likelihood of -1564.4083. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7, with 740 positions in the final dataset. Mite species show higher evolutionary differences between species of the same genera, with T. kanazawai and T. cinnabarinus as the closely related ones (Fig. 4).

Cassava pests remain a significant challenge for cassava farmers in Asia because of the pest complex nature of these pests. The availability of molecular identification tools such as DNA Barcoding is increasingly essential to support cassava pest management, leading to reduced cassava pest damage (Bellotti, 2001; Chavez et al., 2021; Paul et al., 2022; Bisimwa et al., 2019). The molecular identification of these pests contributes to our understanding of the arthropod community associated with cassava and serves as a valuable resource of molecular data. The results presented in this study underscore the significant role that DNA barcoding plays in the identification of cryptic species and the arthropod pest complex in cassava. DNA barcoding was proven to be a highly effective tool, not only for accurate identification of known pests but also for discovering novel potential pest species. This has implications for the sustainable management of cassava, a vital food, and cash crop for millions of people in tropical and subtropical regions.

One key aspect of our research revealed the limitations of the morphological identification of arthropod pests, especially in immature stages (Ratnasingham and Hebert, 2007). Conventional identification methods have often proven challenging, especially for cryptic species and larval stages (Hebert et al., 2003). Our study showed that integrating DNA barcoding can substantially enhance the resolution and accuracy of pest identification. Accurate species-level identification is still paramount to any pest management strategy, as some treatments, such as biological control, are species-specific. Due to the continuing decline of experts among new insect taxonomists, traditional morphological identification has become challenging (Burger and Ulenberg, 1990; Hardy, 2013).

The arthropod pests that are molecularly identified in this study, Aleurodicus disperses, Bemisia tabaci, Ferrisia virgata, Maconellicoccus hirsutus, Paracoccus marginatus, Parasaissetia nigra, and Pseudaulacaspis pentagona are species known to be economically important pests of cassava, causing significant damage to the crop by feeding on its tissues and transmitting viral diseases. Mealybugs are a leading cassava pest, causing global economic damage to the crop (Jankaew et al., 2019; Rauwane et al., 2018). Ferrisia virgata, M. hirsutus, Pa. marginatus are problematic to differentiate; hence, DNA barcoding of these species is still essential to correctly identify the pests in the field. Due to the minute nature of spider mites and the lack of experts, identifying mite species using molecular tools is necessary. Two species of spider mites, namely Tetranychus cinnabarinus and Tetranychus kanzawai, were identified. Two species of spider mites, Tetranychus cinnabarinus and Tetranychus kanzawai, were identified as pests that could infest cassava plants and cause damage by piercing plant cells and sucking out the contents (Gotoh and Gomi, 2003; Liang et al., 2022). Both spider mites are small in size and can be incidentally consumed by voracious lepidopteran larvae, leading them to avoid such intraguild predation that could affect their survival and development (Kinto et al., 2022). Tetranychus kanzawai, in particular, is an important pest that threatens many crops and ornamental plants in Far Eastern areas (Gotoh and Gomi, 2003).

DNA barcoding has potential implications for pest management and biosecurity. Early detection and identification of invasive species can lead to more effective containment and control strategies (Hajibabaei et al., 2007). On a different note, our study did not find the invasive mealybug, Phenacoccus manihoti which could pose more significant loss in cassava farming. This research presents that this invasive mealybug is not present in the Philippines as of this writing. Despite these promising results, there are still challenges to be addressed. These include the need for extensive reference databases, the complexities of genetic variation within species, haplotype diversity results depicting only the population studied, and potential technical errors (Meyer and Paulay, 2005). Yet, it is clear that the benefits of DNA barcoding in pest identification and management substantially outweigh the limitations.

Implementing DNA barcoding in accurate species identification of cassava arthropod pest complex presents significant information. By enhancing our ability to accurately identify cassava pests in adult and immature stages and identify the pest complex, we can more effectively protect cassava crops and thus safeguard the livelihoods of farmers who depend on them. Our results demonstrate the effectiveness of using COI mtDNA sequences to identify arthropod pests infesting cassava. This approach provides a rapid and accurate means of species identification, contributing to the accurate pest management of this important crop.

mtDNA: Mitochondrial DNA, PCR: Polymerase Chain Reaction,

COI: Cytochrome Oxidase I,

NCPC: National Crop Protection Center

ACKNOWLEDGMENT

We thank the Department of Science and Technology- National Research Council of the Philippines (NRCP) for funding the study. Special thanks to Dr Marieta Bañes Sumagaysay for the financial support to the project; to NRCP staff, especially to Beverly Mae N. de la Cruz, Senior Science Research Specialist, Research Development and Management Division, for accommodating all our inquiries and for the valuable assistance she gave to our team from its proposal until its culmination; To the UPLB Foundation, Inc. headed by Dr Casiano S. Abrigo, Jr. for the administrative services provided to the project; To the personnel of different Regional Crop Protection Centers (RCPCs) and the Department of Agriculture- Regional Field Units (DA-RFOs) for the assistance during the collection trips and capacity building seminars.

AUTHOR CONTRIBUTIONS

MSG and BFC conceptualized the study. MSG and JFL conducted molecular experiments from DNA extraction to sequence analysis. MSG, JFL, BFC, KPA, and MCdLdR, collected the samples. MSG wrote the original draft of the paper. MSG, JFL, CPK and BFC contributed to the manuscript preparation.

FUNDING

This study is funded by the Department of Science and Technology- National Research Council of the Philippines (NRCP).

AVAILABILITY OF DATA AND MATERIALS

This published article and its supplementary information file include all data generated or analyzed during this study.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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Molecular Identification of Cassava Arthropod Pest Complex in the Philippines

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Collection of cassava arthropod pests

Morphological Identification of cassava arthropod pests

Genomic DNA Isolation for Various Arthropod Pests of Cassava

PCR Amplification for Various Arthropod Pests of Cassava

Molecular Detection of Cassava Arthropod Pests Using COI mtDNA Gene Markers

RESULTS

Molecular identification of cassava arthropods based on COI mtDNA sequences

Phylogeny of cassava arthropod pest

DISCUSSION

CONCLUSION

Abbreviations

Declarations

References

Supplementary Files

Status:

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