Cacao swollen shoot virus detection and DNA barcoding of its vectors and putative vectors in Theobroma cacao L. by using polymerase chain reaction

Abstract

Cacao swollen shoot virus (CSSV) is an endemic pathogen causing significant economic losses to cacao (Theobroma cacao L.) production in West Africa. There is limited updated report on the occurrence, spread, genetic diversity and species of CSSV and its mealybug vectors, especially in Nigeria. Nigeria is presently lagging behind in the search for resistance to CSSV and its vectors in T. cacao L. The present study aimed to map and screen for the presence of CSSV and its natural vectors - female mealybugs (Pseudococcidae: Hemiptera) in cacao plantations in Nigeria. Symptomatic and asymptomatic cacao leaves and whole female mealybug samples were collected from major cacao-growing areas in Nigeria - Abia, Akwa Ibom, Cross River, Edo, Ondo and Oyo States. A total of 2568 cacao leaves from 1052 cacao trees were screened with polymerase chain reaction (PCR) using an open reading frame 1 (ORF 1) CSSV-specific primer pair. PCR screening of the mealybug species was performed using the cytochrome c oxidase subunit I (COI) gene. A combination of scanning electron microscopy (SEM) and histology for morphological identification and DNA barcoding enabled to characterise the female mealybug species. The results revealed that CSSV and its mealybug vectors are present in the major cacaogrowing areas in Nigeria. Although CSSV and its vectors have been previously reported in Cross River, Ondo and Oyo States, our results present the first documented evidence of CSSV emergence and its mealybug vectors in Abia, Akwa Ibom and Edo States. We also present the first report of Pseudococcus jackbeardsleyi (Gimpel and Miller) mealybug species on cacao in Nigeria. In conclusion, it is pertinent to re-establish coordinated routine survey and monitoring of CSSV and its mealybug vector presence in T. cacao L. in Nigeria.

Keywords: COI – cytochrome coxidase subunit I; Cacao swollen shoot virus; DNA barcoding; Jack Beardsley mealybug; PCR – polymerase chain reaction; Theobroma cacao.

Fig. 1

An archetypical disease triangle of cacao-CSSV-mealybug interactions (red arrows show potential mealybug movements and CSSV transmission, and the green arrow indicates potential loss of mealybug infectivity potential and subsequently, CSSV non-transmission)

Fig. 2

A) Adaxial surface of symptomatic cacao leaf infected with CSSV; B and C) mealybug infestation on stems and D–G) pods

Fig. 3

DNA fragments of CSSV PCR products with expected size (410 bp); L – HyperLadder^TM 100 bp (Bioline, UK), (+/–) – PCR positive/negative

Fig. 4

Phylogenetic comparison of ORF1 DNA sequences (323 bp) of CSSV isolates from Nigeria, Ghana and Togo

Fig. 5

Cacao-growing areas surveyed for CSSV and mealybug vector presence in Nigeria

Fig. 6

Low vacuum ESEM of whole adult female mealybug sample showing A) body segments; B) lateral filaments (lf), cerarii, three pairs of legs, circulus (cr) (3^rd/4^th abdominal segments); C–D) head and eye (ey) with preocular (po) and ocular (o) cerarii and antenna (an); E) coxa (cx), femur (fm) tibia (ti) and tarsus (tr); F) wax (w) from trilocular pores

Fig. 7

High vacuum gold-coated SEM images of adult female mealybug showing A) claw (cl), claw digitules (cd) and tarsal digitules (td); B) conical setae (cs); C) trilocular pores (tp) and conical setae (cs) on the anal lobe

Fig. 8

A) Whole-body slide with 18–19 pairs of cerarii showing (1) 8–9 segmented antennae, (2) a claw with no denticle and (3) setae with three coni; B) whole-body slide with 17 pairs of cerarii (1) 8 segmented antenna, (2) setae with three coni and (3) claws with no denticles; C) whole-body slide with 18 pairs of cerarii showing (1) presence of 8 segmented antennae, (2) setae with three coni and (3) claw with no denticles; D) whole-body slide with 17 pairs of cerarii showing (1) 8–9 segmented antenna, (2) claw with no denticles and (3) coni with 3–4 setae

Fig. 9

Nigerian mealybug post-SEM COI PCR images showing the expected 398-bp DNA fragments (L – HyperLadder^TM 100 bp (Bioline, UK), (+) – PCR positive, (‒) – PCR negative (non-mealybug), O – negative control (water, no DNA) and j – positive control (DNA of a known mealybug)

Fig. 10

Cytochrome c oxidase phylogeny (neighbour-joining method) of mealybug samples (with M prefixes) collected from Nigerian cacao; the Planococcus citri and Planococcus minor clades are magnified and highlighted in blue in comparison with reference samples (AB, AF, EU, GQ and JN prefixes)