Introduction
Eggplant is a popular vegetable in Ghana due to its rich source of vitamins and minerals. The two primary cultivars, Solanum aethiopicum L. (African eggplant) and Solanum melongena L. (aubergine), are predominantly grown for local consumption and export, respectively (European Commission Health and Consumers Directorate-General, 2012; Fening and Billah, Reference Fening and Billah2019a, Reference Fening and Billah2019b; Fening et al., Reference Fening, Billah, Nankinga, Niassy, Ekesi, Migiro and Otieno2020). While eggplants can be cultivated throughout the year, recent yield declines have had a negative impact on export value to Europe. In Ghana, achievable eggplant yields are estimated to reach 15,000 kg ha−1; however, in 2016, the recorded average yield was only 50% of this attainable figure (Ministry of Food and Agriculture (MOFA), 2017). Moreover, the value of S. melongena fruit exports has experienced an annual decline of 11% from 2008 to 2013 (Food and Agriculture Organization – FAO, 2019). Several factors contribute to Ghana's low yields and diminished value of eggplant exports. These include high labour costs, arthropod pests, inadequate water management (Horna and Gruère, Reference Horna and Gruère2006; Horna et al., Reference Horna, Timpo and Gruere2007) and insufficient investment in efficient production technologies (Tsiboe et al., Reference Tsiboe, Asravor and Osei2019).
Among these factors, arthropod pests are a significant concern (Amengor et al., Reference Amengor, Boamah, Akrofi, Gamdoagbao, Egbadzor, Davis and Kotey2017). Both S. aethiopicum and S. melongena are susceptible to various arthropod pests, with the eggplant fruit and shoot borer (EFSB), Leucinodes orbonalis Guenée (Lepidoptera: Crambidae), being the most destructive (EPPO, 2023). The larval stage of L. orbonalis is particularly destructive in its lifecycle. Larvae bore into and feed on the shoots and fruits of eggplants, leading to a reduction in fruit quality and quantity (EPPO, 2023). Infestations associated with L. orbonalis have resulted in significant yield losses, reaching up to 70% in eggplant fields in Ghana's Volta region (Amengor et al., Reference Amengor, Boamah, Akrofi, Gamdoagbao, Egbadzor, Davis and Kotey2017).
Leucinodes orbonalis is classified as an A1 quarantine pest. This classification is based on the regular intercepting of its larvae in eggplant fruits exported from African, Caribbean and Pacific (ACP) countries to Europe, where the European and Mediterranean Plant Protection Organization (EPPO) regions have declared it absent (EPPO, 2023). The presence of A1 quarantine pests can hinder international trade in eggplants, as exemplified by the European Union's (EU) ban on the export of eggplant fruits from Ghana. From October 2015 to December 2017, this ban was imposed due to the frequent interception of L. orbonalis larvae and other quarantine pests at border control points (BCPs) in EU Member States (Fening and Billah, Reference Fening and Billah2019a, Reference Fening and Billah2019b; Fening et al., Reference Fening, Billah, Nankinga, Niassy, Ekesi, Migiro and Otieno2020).
Previously, L. orbonalis was known to be present in sub-Saharan Africa, as reported by Walker (Reference Walker1859), Frempong (Reference Frempong1979) and CABI (2012). However, recent information regarding its distribution suggests that this pest is not as widespread in Africa (EPPO, 2023). Several studies conducted by Hayden et al. (Reference Hayden, Lee, Passoa, Young, Landry, Nazari and Ahlmark2013), Gilligan and Passoa (Reference Gilligan and Passoa2014) and Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) focused on the identification of EFSBs intercepted from African consignments. The findings of Hayden et al. (Reference Hayden, Lee, Passoa, Young, Landry, Nazari and Ahlmark2013) and Gilligan and Passoa (Reference Gilligan and Passoa2014) indicated that the intercepted EFSB specimens from Africa consisted of three distinct species, distinct from the L. orbonalis found in Asia. Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) further identified eight different species of EFSB intercepted from consignments in Africa, namely Leucinodes africensis Mally, Korycinska, Agassiz, Hall, Hodgetts & Nuss, Leucinodes laisalis (Walker), Leucinodes rimavallis Mally et al., Leucinodes ethiopica Mally et al., Leucinodes pseudorbonalis Mally et al., Leucinodes kenyensis Mally et al., Leucinodes ugandensis Mally et al. and Leucinodes malawiensis Mally et al.. However, among the intercepted eggplant fruits from Ghana, only L. africensis and L. laisalis were found.
It is important to acknowledge that the presence of L. africensis and L. laisalis in intercepted eggplant fruits from Ghana does not necessarily indicate that these are the sole Leucinodes species attacking eggplants in farmer's fields. The limited sampling of consignments intended for trade within the EU, which occurs at exit points such as airports, suggests that there may be other Leucinodes species causing damage to eggplants in the country that have yet to be identified (Everett, Reference Everett2000; Surkov et al., Reference Surkov, Oude Lansink, Van Kooten and Van Der Werf2008; Saccaggi and Pieterse, Reference Saccaggi and Pieterse2013; Fening and Billah, Reference Fening and Billah2019a, Reference Fening and Billah2019b; Seidu, Reference Seidu2022).
Furthermore, a significant concern arises regarding the identification of the EFSB species attacking eggplants in Ghanaian farmer's fields. At the BCPs, consignments are usually sent into a containment facility upon arrival; and a visual inspection of consignments is carried out to detect signs or presence of EFSB species infestations by a trained phytosanitary officer. When the presence or signs attributed to EFSB infestations is detected, consignments are bagged, and sent to the laboratory for in-depth examination and taxonomic identification of EFSB species found in consignments (IPPC, 2020). Surprisingly, the pest list of eggplants in Ghana does not include L. africensis and L. laisalis, which have been reported as the species intercepting eggplant consignments (Ministry of Food and Agriculture (MOFA), 2022). Instead, L. orbonalis is listed as the EFSB attacking eggplants on farmers’ fields in Ghana, despite previous reports suggesting its absence in Africa (Mally et al., Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015; EPPO, 2023). This raises the question of whether the EFSB species solely consists of L. orbonalis, as earlier studies suggested (Frempong, Reference Frempong1979; Owusu-Ansah et al., Reference Owusu-Ansah, Afreh-Nuamah, Obeng-Ofori and Ofosu-Budu2001; Mochiah et al., Reference Mochiah, Banful, Fening, Amoabeng, Offei Bonsu, Ekyem, Braimah and Owusu-Akyaw2011; Ofori et al., Reference Ofori, Afful, Quartey, Osae and Amoatey2015; Ministry of Food and Agriculture (MOFA), 2022), or if L. africensis and L. laisalis, as reported by Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) and EPPO (2023), are also present.
Therefore, there is an urgent need to establish the precise identity of the EFSB species attacking eggplants in Ghana to make well-informed decisions. This study aimed to determine the species of EFSB attacking eggplants in eggplant hotspots in southern Ghana, study their phylogenetic relationships and monitor the population of adult males in on-farm conditions.
Materials and methods
Study and sampling sites
In 2022, a survey was conducted to investigate the occurrence of EFSBs in major eggplant production regions of Ghana, specifically the Deciduous Forest and Coastal Savannah agroecological zones. The surveyed regions included Eastern, Greater Accra and Volta (Asenso-Okyere et al., Reference Asenso-Okyere, Twum-Baah, Kasanga and Portner2000; Ministry of Food and Agriculture (MOFA), 2018). The survey spanned from March to November, covering both the major and minor rainy seasons to capture the complete seasonal cycle of the pest.
A total of ten fields were selected for sampling, consisting of six exporter farms and four local eggplant fields across eight study areas: Adeiso, Asuboi, Azagonorkope, Begoro, Legon, Nsawam, Okorase, and Senchi (fig. 1). The aim was to determine the identity of the EFSB. Furthermore, four exporter farms were specifically chosen for monitoring the population of adult EFSB males in on-farm conditions. These farms were Eric and Trosky at Adeiso, Joekopan at Azagonorkope and Tacks at Senchi. The geographical coordinates of the sampling sites (farmer's fields) are provided in table 1.
Figure 1. Map of Ghana showing the study areas and sampling sites. The map of Ghana (A) shows the regional political boundaries in different colours. The inserted purple box demarcates the geographic region where our study was conducted. The sampling sites (B) are shown in the blown-up image. The purple and yellow dots show the sites where fieldwork was conducted in the Eastern, Greater Accra and Volta regions.
Table 1. Study areas and sampling sites of the EFSB (Lepidoptera: Crambidae)
*Exporters farms.
Sampling of EFSB
During the survey, a systematic approach was followed to examine the presence of EFSBs. Seventy-five (75) eggplants were randomly selected using an ‘X’ pattern at each sampling site. The shoots and fruits of these eggplants underwent a thorough examination to identify signs of EFSB infestation. These signs included shoot drooping caused by larval tunnelling inside the shoots, the presence of EFSB larvae within the shoots, and emergence holes created by mature (5th) instar larvae exiting eggplant fruits to pupate in the soil.
Infested eggplant shoots were carefully separated from the plants and opened to extract the EFSB larvae found inside the tunnels. These larvae were preserved in vials containing 95% (v/v) ethanol, appropriately labelled and transported to the laboratory. Upon arrival, they were stored in a refrigerator at 4 °C for identification.
Furthermore, infested eggplant fruits were collected from the sampling sites and placed in containers for transportation to the laboratory. A rearing procedure, adapted from Padfwal and Scrivastava (Reference Padfwal and Scrivastava2018), was employed to rear the EFSB larvae found within the eggplant fruits to the adult stage in a controlled laboratory environment.
EFSB rearing procedure
To facilitate the pupation process of EFSB larvae, the collected infested eggplant fruits were carefully placed in transparent plastic containers. The bottom of each container was covered with muslin cloths, providing a suitable pupation site for the larvae. Another muslin cloth was used to cover the exposed area at the top of the plastic container. This setup ensured a controlled environment for pupation. Once the pupae emerged from the larvae in the rearing cages, they were transferred to glass tubes. These tubes were lined with a muslin cloth at the bottom, providing a comfortable surface for adult emergence. The top of the glass tubes was covered with another muslin cloth. This arrangement allowed for the emergence of adult EFSB specimens while keeping them contained. To provide sustenance for the emerging adults, cotton balls soaked in a 10% sugar solution were placed in the adult cages as a food source. Subsequently, the adult EFSB specimens were humanely euthanised by freezing to preserve them for identification.
Monitoring adult EFSB males’ population in on-farm conditions
During the vegetative stage of eggplant cultivation in Eric, Joekopan, Tacks and Trosky farms, a delta trap was set up to capture adult male L. orbonalis. A delta trap was baited with sex pheromone lures specifically designed for L. orbonalis (P308-Lure manufactured by Chemtica Internacional SA). The active ingredients of the lure were E-11-hexadecenyl acetate and E-11-hexadecenol. The installation of the delta trap occurred at the farms mentioned above, and the trap was monitored every week from the vegetative stage until the maturity stage of the eggplants. Each week, the adult EFSB males captured in the trap were collected. These captured specimens were then identified and counted to determine the population dynamics of the pest.
Morphological identification of EFSB
The dead adults were identified morphologically on a Leica EZ4 D stereomicroscope using the identification keys published by Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) by a curator, H. Davies, at the Insect Museum of the Department of Animal Biology and Conservation Science (DABCS), University of Ghana.
Molecular identification of EFSB
The molecular identification process was performed at the CABI Plantwise Diagnostic and Advisory Service laboratory in the United Kingdom and the National Institute of Agricultural Botany (NIAB) laboratories in the UK. DNA was extracted from adult and larval specimens of the EFSB for samples sent to CABI using the microLYSIS®-PLUS extraction technique. For samples sent to NIAB, DNA was extracted using the Norgen Cells and Tissue DNA kit (Norgen, Thorold, 4Y6, Canada). The extracted DNA was then amplified by PCR using the universal mitochondrial cytochrome oxidase (COI) gene primers LCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ and HCO2198 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994) to amplify a section of the COI gene. The quality of the PCR products was assessed using gel electrophoresis, followed by purification using a commercial kit. Samples were diluted to the required concentrations and submitted for semi-automated Sanger sequencing (Sanger et al., Reference Sanger, Nicklen and Coulson1977; Smith et al., Reference Smith, Sanders, Kaiser, Hughes, Dodd, Connell, Heiner, Kent and Hood1986) on the ABI 3130 Genetic Analyzer. The generated DNA sequences were compared with existing sequences in the Barcode of Life Data System (BOLD) and the National Center for Biotechnology Information (NCBI) to identify the Leucinodes species.
Phylogenetic analysis
The sequences of the EFSB from GenBank that were similar to the DNA sequences of the specimens used for molecular identification were downloaded in FASTA format and were used for phylogenetic analyses. Pairwise comparison was performed to establish similarity of COI sequences of EFSB obtained in this study. The COI sequences of EFSB identified in this study, some reference sequences of Leucinodes species downloaded from GenBank viz. L. orbonalis from Bangladesh, India, Malaysia, Pakistan and Thailand; L. africensis from Bangladesh and Nigeria; L. laisalis from Kenya, Nigeria and South Africa; L. kenyensis and L. rimavallis from Kenya; L. malawiensis from Malawi; and L. pseudorbonalis from Uganda, and a reference sequence of the Mediterranean fruit fly, Ceratitis capitata (included as an outgroup for comparison) were aligned using MUSCLE algorithm (Edgar, Reference Edgar2004), and percentage similarity computed in SDT v 1.2 software (Muhire et al., Reference Muhire, Varsani and Martin2014). Following that, the sequences were aligned using MUSCLE (Edgar, Reference Edgar2004) in Molecular Evolutionary Genetics Analysis Version 11 (MEGA 11) (Tamura et al., Reference Tamura, Stecher and Kumar2021) and used to construct a phylogenetic tree using the Neighbor Joining (NJ) tree algorithm (Saitou and Nei, Reference Saitou and Nei1987) with Tamura-3 parameter (Tamura, Reference Tamura1992). The statistical support for the nodes in the phylogenetic tree was assessed using 1000 bootstrap replicates. All the data used for the phylogenetic analysis can be found in the supplementary file (see Supplementary File 1).
Data analysis of the prevalence of the EFSB male population in eggplant fields
The prevalence of the adult EFSB males was estimated using the fruit fly prevalence estimation indices F/T/W where F = the total number of adult EFSB males captured, T = the number of inspected traps and W = the number of weeks traps exposed in the farmer's field (International Standards for Phytosanitary Measures (ISPM) 30, 2008; Billah and Fening, Reference Billah and Fening2019; Fening and Billah, Reference Fening and Billah2019a, Reference Fening and Billah2019b).
Results
Identification of the EFSB
A total of 834 EFSB (Lepidoptera: Crambidae) were found in the shoot and fruits of the eggplants and pheromone traps mounted at the sampling sites. Following molecular and morphological taxonomic examination, the L. africensis and L. laisalis were identified as the EFSB infesting eggplants in southern Ghana (table 2). The BLAST search for similarity revealed that the generated DNA sequences of EFSB samples 1–83 selected for identification were >99% identical to the mitochondrial COI sequence of the L. africensis identified in eggplant fruits from Nigeria (GenBank Accession number: KM987391.1) (Mally et al., Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015). However, the generated DNA sequences of the EFSB specimens 84 and 85 were both found to be 100% identical to the mitochondrial COI sequence of the L. laisalis identified in eggplant fruits from Nigeria (GenBank Accession number: KM987397.1) (Mally et al., Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015). The DNA sequences generated from the specimens used for identification have been deposited in GenBank and assigned accession numbers (table 2). The complete list of all sequenced specimens and the GenBank accession numbers assigned is included in the supplementary information (see Supplementary table 1).
Table 2. Summary of results of EFSB specimens subjected to molecular identification
The pairwise comparison of the DNA sequences showed a clear species demarcation between the L. orbonalis, L. africensis, L. laisalis, L. rimavallis, L. kenyensis, L. malawiensis and L. pseudorbonalis (fig. 2). Three clusters of closely related sequences having >96% identity were identified. The first cluster comprised the mitochondrial COI sequences of the L. orbonalis identified in eggplant fruits from Bangladesh (accession number: LN624686.1), Thailand (accession number: LN624707.1), India (accession number: LN624690.1), Pakistan (accession number: LN624679.1) and Malaysia (accession number: LN624689.1). Likewise, the second cluster comprised the mitochondrial COI sequences of the L. africensis identified in this study, and those in eggplant fruits from Bangladesh (accession number: OL693251.1), Nigeria (KM987391.1). The third cluster comprised the sequences of the L. laisalis identified in eggplant fruits at Adeiso (Adeiso_1_10) and Legon (Legon_2_9) in southern Ghana, and those from Nigeria (accession number: KM987397.1), Kenya (accession number: KM987403.1) and South Africa (accession number: KM987697.1).
Figure 2. Pairwise sequence similarity of different Leucinodes species. Pairwise sequence comparison of L. africensis and L. laisalis identified in this study and reference sequences downloaded from GenBank: L. orbonalis, L. africensis, L. laisalis, L. rimavallis, L. kenyensis, L. malawiensis and L. pseudorbonalis was performed by aligning the sequences using MUSCLE algorithm and computing percentage similarity in SDT v 1.2 software. The intense crimson colour (as shown in the scale) indicated close similarity in the sequences and was more pronounced within species. Notably, there was a clear species demarcation between sequences from L. africensis and L. laisalis which had been identified in our study.
The neighbour-joining tree grouped all Leucinodes taxa into two major clades (I and II) with the exception of L. malawiensis (fig. 3). In the first clade, all L. orbonalis specimens clustered into one monophyletic clade (subclade A) consisting of two distinct groups. One group comprised a single Malaysian specimen (GenBank accession number: LN624689.1) and the second group formed a polytomy comprising specimens found in eggplant fruits in Bangladesh (GenBank accession number: LN624686.1), Pakistan (GenBank accession number: LN624679.1), India (GenBank accession number: LN624690.1) and Thailand (GenBank accession number: LN624707.1).
Figure 3. Phylogenetic analysis of Leucinodes species. Phylogenetic analyses to show the evolutionary relationship between L. orbonalis, L. africensis, L. laisalis, L. malawiensis, L. pseudorbonalis, L. rimavallis and L. kenyensis were constructed using the Neighbor-Joining method based on the Tamura-3 parameter in MEGA 11. There were two major clades (clade I and clade II). Notably, L. africensis and L. laisalis, which were of significant interest to this study, clustered in separate clades. Generally, there was a clear demarcation between the species whose representative sequences were used in the phylogenetic analyses. The mitochondrial COI sequence of the C. capitata was included as an outgroup.
Likewise, L. pseudorbonalis, L. rimavallis and L. africensis specimens clustered into another monophyletic clade (subclade B). Within this monophyletic clade, three distinct groups were found. One group comprised L. pseudorbonalis specimen identified in fruits from Uganda (GenBank accession number: LN624707.1). The second group comprised L. kenyensis (GenBank accession number: KM987390.1) and L. rimavallis specimens (accession number: LN624678.1) identified in eggplant fruits from Kenya. Similarly, the third group comprised L. africensis specimens found in eggplant fruits in this study and L. africensis identified in eggplant fruits from Nigeria (GenBank accession number: KM987697.1) and Bangladesh (GenBank accession number: OL693251.1) respectively.
Notwithstanding, it is interesting to note that L. africensis specimens clustered into two distinct sub-groups. The first group formed a polytomy comprising all L. africensis specimens identified in this study and the reference specimen imported with fruits from Nigeria to Europe (GenBank accession number: KM987697.1); and the second comprised a single specimen found in eggplant fruits in Bangladesh (GenBank accession number: OL693251.1).
Two groups were identified in the second major clade (clade II) containing all L. laisalis specimens. One group comprised all L. laisalis specimens identified in this study (GenBank accession numbers: OR058652.1 and OR058653.1), and reference specimens identified in fruits from Kenya (GenBank accession number: KM987403.1) and Nigeria (GenBank accession number: KM987397.1) (subclade C). However, the second group comprised a single specimen from South Africa (GenBank accession number: KM987697.1).
The morphological examination of the L. africensis and L. laisalis revealed similar and marked distinguishing features between the two species (fig. 4). Both species were found to possess white-coloured first abdominal segments. However, the remaining abdominal segments of the L. africensis were dark brown, compared to that of L. laisalis, which was light brown. Likewise, the ground colour of the forewings of the L. africensis was white with brown coloured half-moon-shaped patches and black patches at the wing tips, whilst that of the L. laisalis was light brown with brown coloured half-moon-shaped patches and dark brown patches at the wing tips (fig. 4).
Figure 4. Eggplant fruit and shoot borer identified from fruits of eggplants. The adult insects were obtained by following the incubation of larvae-infested eggplant until the larvae pupated, and subsequently, adult forms emerged. The left panel (a) shows female Leucinodes africensis, while the right panel (b) shows female Leucinodes laisalis. Both species can readily be identified by the brown half-moon-shaped patches on their forewings and a white-coloured first abdominal segment. However, the L. africensis possess forewings with a white ground colour and its remaining abdominal segments being dark brown coloured, distinguishing it from the L. laisalis, which possesses forewings with a light brown ground colour, and remaining abdominal segments that are also light brown.
Distribution and abundance of the EFSB species in southern Ghana
Leucinodes africensis was found in the shoot and fruits of eggplants on farmer's fields in all the study areas. However, L. laisalis was found in the shoot and fruits of eggplants on farmer's fields in only five study areas; Adeiso, Nsawam, Okorase and Senchi; and Legon in the Deciduous Forest and Coastal Savannah agroecological zones, respectively (fig. 5). Overall, the abundance of L. africensis was found to be higher than that of L. laisalis (table 3). Similarly, the percentage abundance of L. africensis was also higher than that of L. laisalis in all the study areas where both species occurred. The percentage abundance of the L. africensis identified in the shoot and fruits of eggplants on farmer's fields in southern Ghana was >90% in all the study areas, whereas that of L. laisalis was <10%.
Figure 5. Distribution of the L. africensis and L. laisalis in southern Ghana. This study revealed that both L. africensis (red dots) and L. laisalis (purple dots) were present in southern Ghana. We did not detect any L. laisalis in Begoro, Asuboi and Azagonorkope. The complete map of Ghana on the left shows the regional political boundaries in different colours, and the inserted red box demarcates the geographic region where our study was conducted.
Table 3. Percentage (%) abundance of the L. africensis and L. laisalis in the shoot and fruits of eggplants at different locations in southern Ghana
*Exporter's farms.
Monitoring of EFSB males in on-farm conditions
The L. africensis was the only EFSB identified in the pheromone traps mounted at Eric, Trosky, Tacks and Joekopan farms following a molecular and morphological taxonomic examination of the specimens.
The population of L. africensis males in eggplant fields at the exporter's farms followed an irregular pattern from the vegetative to the maturity stage of the eggplants (fig. 6). Except for Joekopan farms, the number of L. africensis males remained stable at counts of zero from the 3rd to at least the 5th week after transplanting of the eggplants. Likewise, the number of L. africensis males peaked in the 4th, 9th, 11th and 14th weeks after transplanting the eggplants at Joekopan, Trosky, Tacks and Eric farms, respectively. Generally, the relative density of L. africensis males in eggplant fields at all the exporter's farms was low (<2.00) (table 4). The highest relative density of L. africensis males was recorded at Trosky farms, followed by Eric farms, Joekopan farms and Tacks farms.
Figure 6. Weekly trap catches of adult L. africensis males at Eric, Trosky, Tacks and Joekopan farms. This study revealed that adult L. africensis males were attracted to the sex pheromone lure of the L. orbonalis as this EFSB was the only species found in the delta pheromone traps mounted on farmer's fields. The population of adult L. africensis males followed an irregular pattern from the vegetative to the maturity stage of the eggplants in the farmers’ fields, peaking in the 4th, 9th, 11th and 14th weeks after transplanting of the eggplants at Joekopan, Trosky, Tacks and Eric Farms, respectively.
Table 4. Comparison of relative density of adult L. africensis males based on weekly trap catches
*F = the total number of L. africensis males captured, T = the number of pheromone traps inspected and W = the number of weeks pheromone traps were exposed in the eggplant fields.
Discussion
This study aimed to ascertain the identity of EFSB species (Lepidoptera: Crambidae) attacking eggplants on farmer's fields in southern Ghana and monitoring the adult male population in on-farm conditions. Earlier studies by Frempong (Reference Frempong1979), Owusu-Ansah et al. (Reference Owusu-Ansah, Afreh-Nuamah, Obeng-Ofori and Ofosu-Budu2001), Mochiah et al. (Reference Mochiah, Banful, Fening, Amoabeng, Offei Bonsu, Ekyem, Braimah and Owusu-Akyaw2011), Ofori et al. (Reference Ofori, Afful, Quartey, Osae and Amoatey2015) and Ministry of Food and Agriculture (MOFA) (2022) noted the presence of the L. orbonalis on farmer's fields in Ghana. However, this study did not detect any evidence of the L. orbonalis in the shoot and fruits of S. aethiopicum and S. melongena found on farmers’ fields in southern Ghana. Instead, L. africensis and L. laisalis were the only EFSB species identified on farmer's fields in southern Ghana, corroborating the findings of Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015), who identified the L. africensis and L. laisalis in intercepted eggplant fruits from Ghana.
Previous studies by Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) and EPPO (2023) have highlighted that L. africensis infests both S. aethiopicum and S. melongena. Similarly, Boateng et al. (Reference Boateng, Braimah, Glover-Amengor, Osei-Sarfo, Woode, Robertson and Takeuchi2005) documented the presence of Sceliodes laisalis (syn. L. laisalis) in S. melongena fruits in Ghana. However, there is limited literature on the occurrence of L. laisalis in S. aethiopicum fruits. Nevertheless, Mantey (Reference Mantey2021) reported the presence of L. laisalis in S. aethiopicum fruits in eggplant fields at Legon in southern Ghana in an unpublished thesis. The findings of this study support the report by Mantey (Reference Mantey2021) and provide formal confirmation of the presence of L. laisalis in S. aethiopicum fruits in eggplant hotspots in southern Ghana.
The presence of L. africensis and L. laisalis in the shoots and fruits of both S. aethiopicum and S. melongena in southern Ghana has significant implications for the bilateral trade of eggplants between Ghana and European Union (EU) Member States. These implications may also extend to the international trade of other Solanaceae plants, such as Capsicum annum and Solanum lycopersicum, between Ghana and EU Member States, as these crops have been reported as host plants for these pest species (Mally et al., Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015; EPPO, 2023). The implications include but are not limited to interception of produce and, in the absence of robust phytosanitary measures, could negatively impact the export of these Solanaceae crops from Ghana to the EU Member States, a situation that has occurred before in Ghana. The first local ban on the export of eggplants and other crops within the Solanaceae family was issued by the Plant Protection and Regulatory Service Directorate (PPRSD) of the Ministry of Food and Agriculture (MOFA) in September 2011 due to high interceptions of the L. orbonalis (now referring to individuals within the Leucinodes genus native to Africa (EFSA et al., Reference Bragard, Di Serio, Gonthier, Jaques Miret, Justesen, Magnusson, Milonas, Navas-Cortes, Parnell, Potting, Reignault, Thulke, Van der Werf, Vicent Civera, Yuen, Zappala, Gregoire, Malumphy, Czwienczek, Maiorano and MacLeod2021)) and Thrips spp. (European Commission Health and Consumers Directorate-General, 2012); while the EU ban, also due to high interceptions of harmful organisms, including possibly the misidentified L. orbonalis, was issued by the EU in October 2015 and extended to December 2017 (Fening and Billah, Reference Fening and Billah2019a, Reference Fening and Billah2019b).
It is interesting to note that in the neighbour-joining tree, L. orbonalis, L. pseudorbonalis, L. kenyensis, L. rimavallis and L. africensis clustered together into one clade, while L. laisalis clustered into another clade. This suggests that L. orbonalis, L. pseudorbonalis, L. kenyensis, L. rimavallis and L. africensis are more closely related to each other than to L. laisalis as far as mitochondrial COI gene is concerned. The present finding is broadly consistent with the findings of Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015), who also demonstrated that L. orbonalis, L. africensis, L. rimavallis, L. pseudorbonalis and L. kenyensis clustered together in one clade, while the L. laisalis and L. malawiensis clustered together in another clade.
The morphological examination of the L. africensis and L. laisalis showed that both species possessed brown-coloured half-moon-shaped patches in theirs and a white-coloured first abdominal segment. These features have been reported to be characteristic of species found in the Leucinodes genus (Mally et al., Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015). Additionally, differences were observed in the ground colour of the forewings and remaining abdominal segments of the L. africensis and L. laisalis; this is in concurrence with the reports made by Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015). Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) reported that the ground colour of the forewings and remaining abdominal segments of the L. africensis was white and ranged from brown to grey, respectively, whilst that of the L. laisalis had colours ranging from orange-brown to greyish-white, and brown, respectively. Notwithstanding, Mally et al. recommended the use of male genitalia as another diagnostic feature to accurately distinguish between L. africensis and L. laisalis. The male genitalia of adult L. africensis has a long ventrad fibula; an elongated, strong-hooked or straight shaped, sometimes branching distal sacculus process that is projected towards the valva apex; an apically thin juxta; and a posterior phallus with an oval saw blade-shaped sclerotisation. However, the male genitalia of adult L. laisalis has a large and oval sacculus; a ventrad fibula that is broad and strongly sclerotised; well elongated saccus; and a phallus that has a keeled coecum and slim, fingerlike and strongly sclerotised apoderme.
Considering the technical knowledge involved in the use of male genitatlia to distinguish between moth species in general, the differences found in the ground colour of the forewings and abdominal segments between adult L. africensis and L. laisalis could be helpful to farmers in their identification during pest monitoring activities on their farms, which can inform decision-making on the management of the infestations of the L. africensis and L. laisalis. Hence, the extension staff of the PPRSD of MOFA is encouraged to educate farmers on these diagnostic features during focus discussion sessions with farmer groups.
Leucinodes africensis was detected in all the study areas in southern Ghana, and it was found to coexist with L. laisalis in some of these areas. This indicates that the study areas provide favourable conditions for the establishment of these Leucinodes species. Moreover, the presence of both L. africensis and L. laisalis suggests their adaptability to the various environmental conditions in the study areas. The climatic conditions in these areas are predominantly hot and humid throughout the year, with an average annual temperature of 26.1 °C for locations near the coast (Ministry of Food and Agriculture (MOFA), 2018). This observation suggests that L. africensis and L. laisalis are moths that thrive in warm and humid environments.
Leucinodes africensis was found to have a wider distribution and greater dominance in southern Ghana compared to L. laisalis. This aligns with reports by Mally et al. (Reference Mally, Korycinska, Agassiz, Hall, Hodgetts and Nuss2015) highlighting the widespread presence of L. africensis in Africa among other Leucinodes species native to the continent. Additionally, Pace et al. (Reference Pace, Ascolese, Miele, Russo, Griffo, Bernardo and Nugnes2022) reported frequent interceptions of L. africensis in exported eggplant fruits from Ghana. Interestingly, despite using an L. orbonalis sex pheromone lure, there was no evidence of adult L. orbonalis males in the pheromone traps installed on exporter's farms. Instead, only adult L. africensis males were observed through molecular and morphological taxonomic examination. This provides further evidence that L. orbonalis may not be present in Ghana as previously described and suggests the possibility of interspecific pheromone attraction among Leucinodes species. While limited information is available on interspecific pheromone attraction among Leucinodes species, it is plausible that there are similarities in the components of sex pheromones released by adult females of L. africensis and L. orbonalis, considering their close relationship. However, it is essential to exercise caution in interpreting this finding, as factors other than those suggested may influence the behavioural responses of L. africensis to the L. orbonalis sex pheromone lure. Nevertheless, the attraction of L. africensis to the sex pheromone lures of L. orbonalis can be utilised as a population suppression tool for managing L. africensis in eggplant production in Ghana.
There was variation in the number of adult L. africensis males among the exporter's farms and the peak period of the adult L. africensis males. This is attributed to the variation in the growth stage of the eggplant and climatic conditions among the farms (McNeil, Reference McNeil1991; Rhino et al., Reference Rhino, Dorel, Tixier and Risède2010). The EFSB has been reported by many studies to be present on eggplant fields in all the growth stages of eggplant, with their numbers varying throughout the lifecycle of eggplants. For instance, Ofori et al. (Reference Ofori, Afful, Quartey, Osae and Amoatey2015) reported the presence of EFSB previously reported as L. orbonalis in all the growth stages of eggplants in Ghana. Similarly, Taiwo et al. (Reference Taiwo, Olaitan, Abiodun and Abiodun2020) demonstrated the variation in numbers of EFSB previously reported as L. orbonalis at different weeks after transplanting of eggplants and in each growth stage. This has been attributed to the production of secondary metabolites in leaves, shoot, flowers and fruits of eggplants, whose levels vary throughout the lifecycle of eggplants and serve as kairomones for adult EFSB. For instance, Nusra et al. (Reference Nusra, Udukala, Amarasinghe and Paranagama2021) demonstrated that the production of secondary metabolites such as benzyl alcohol, 2,2’-(ethane-1,2-diylbis(oxy))bis(ethane-2-1-diyl) dibenzoate and 3,7-dimethylocta-1,6-dien-3-ol as major constituents of leaves, flowers, fruits and shoots of eggplants, respectively, serves as kairomones for L. orbonalis. During the survey of farmer's fields in this study, a variation in planting dates of eggplants and cultivation period among farmer fields was observed, resulting in variation in onset and duration of growth stages of eggplants among farmer fields. This may explain the difference in the number and peak period of adult L. africensis males among the exporter's farms.
Similarly, climatic conditions such as temperature and rainfall have an effect on the development and survival of moths. Among the climatic conditions that affect the development and survival of moths, temperature and rainfall have a significant relationship with moth abundance in the tropics (Kato et al., Reference Kato, Inoue, Hamid, Nagamitsu, Merdek, Nona, Itino, Yamane and Yumoto1995; Intachat et al., Reference Intachat, Holloway and Staines2001; Brehm et al., Reference Brehm, Colwell and Kluge2007), as revealed by Choi (Reference Choi2008). In this study, the population of adult male moths was monitored in farmer fields in the eastern and volta (found in south-eastern coastal area) regions of southern Ghana. Both regions experience predominantly warm and humid conditions; however, the eastern belt is comparatively warmer (Ministry of Food and Agriculture (MOFA), 2018). Likewise, both regions experience bimodal rainfall every year (Ministry of Food and Agriculture (MOFA), 2018). However, there is variability in rainfall amounts; with the eastern belt experiencing more rainfall than the south-eastern coastal areas (Braimah et al., Reference Braimah, Asante, Ahiataku, Ansah, Otu-Larbi, Yahaya, Ayabilah and Nkrumah2022). The variation in these conditions (especially temperature, humidity and rainfall) among the farms could have influenced the variation in the numbers and peak periods of adult L. africensis males.
The relative density of the adult L. africensis males recorded in all the exporter's farms was low, and this is attributed to the effectiveness of the management practices recommended by the PPRSD of MOFA in its roadmap to manage populations of the L. orbonalis (now referring to Leucinodes spp. native to Africa) (European Commission Health and Consumers Directorate-General, 2012) as the farmers adhered to this management protocol. The management practices recommended by the PPRSD of MOFA in its roadmap to manage populations of the misidentified L. orbonalis included on-farm sanitation, that is, proper disposal of rotten eggplant fruits, use of pheromone traps and application of selective insecticides (Fening et al., Reference Fening, Billah and Kukiriza2017). The extension staff of the PPRSD of MOFA is therefore encouraged to regularly visit exporters’ farms to ensure that farmers adhere to these management practices to increase yield and revenues obtained from exports of eggplants.
In conclusion, this paper presents evidence that challenges the previous description of L. orbonalis presence in Ghana. Through identification efforts, it was determined that L. africensis and L. laisalis are the only species attacking eggplants on farmer's fields in southern Ghana. One notable finding is that L. africensis males were attracted to the sex pheromone lures of L. orbonalis, despite the species being distinct. This suggests the potential use of L. orbonalis sex pheromone lures as a tool to suppress L. africensis populations in eggplant fields. Further investigation and experimentation in this area are strongly recommended. The management protocol implemented by the PPRSD of MOFA in Ghana, aimed at managing L. orbonalis populations (now referred to as African Leucinodes spp.), was found to be effective, resulting in low numbers of L. africensis on exporter's farms. Eggplant farmers are therefore encouraged to adhere to this management protocol.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485324000154.
Acknowledgements
The authors are very grateful for the financial support from GCRF AgriFood Africa Innovation Awards Round 3 grant awarded to Dr Francis Wamonje and Professor Ken Fening. We are also grateful to farmers from the Ghana Association of Vegetable Exporters (GAVEX) who offered their farms for the study.
Competing interests
None.
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