RESEARCH ARTICLE

 

Isolation, identification and pathogenicity of fungi isolated from mouthparts of coconut bug, Pseudotheraptus wayi Brown (Hemiptera: Coreidae) to litchis in South Africa

 

 

M. Netshipise^I; P.S. Schoeman^II; R. Gouws-Meyer^I; S.R. Netshifhefhe^I

^IDepartment of Crop Sciences, Tshwane University of Technology, Pretoria, South Africa
^IISonbesie Consulting, Nelspruit, South Africa

Correspondence

 

 

---

ABSTRACT

The coconut bug, Pseudotheraptus wayi, is a sap-feeding insect known to damage a wide range of host plants. Its feeding mechanism allows it to inject toxic saliva into plant tissue, and potentially, to transmit fungal pathogens. This study aimed to isolate and characterise fungal species associated with the coconut bug mouthparts, and to assess the pathogenicity of the fungal isolates on litchi fruits. A total of 29 fungal species were identified from 16 bugs. Morphological differences in colony colours, patterns and textures further distinguished these isolates. A total of 300 litchi fruits were inoculated with five fungal species, namely: Pestalotiopsis microspora, Xylaria sp., Nigrospora oryzae, Cladosporium cladosporioides, and Purpureocillium lilacinum. Three parameters (weight loss, colour change and disease severity) were assessed on the fruits over 14 days. The results indicated that the weight loss ranged from 21.58% to 18.67% and disease severity from very severe to slight. Pestalotiopsis microspore and Purpureocillium lilacinum were the pathogens with the most and least pathogenicity, respectively. These findings highlight the potential significant threat posed by fungal pathogens and emphasise the need to monitor and control the coconut bug to maintain litchi fruit quality and reduce post-harvest losses. Future research should explore the actual role and transmission dynamics in natural litchi-insect systems (field-based pathogenicity trials) to confirm the findings of this study.

Keywords: fruit quality, fungal species, fungus-insect interactions, Koch's postulates, morphological, characterisation, pathogen

---

 

 

INTRODUCTION

Originating from China and Vietnam, Litchi (Litchi chinensis Sonn.) is cultivated globally as a highly valued tropical fruit, mostly for its unique flavour, nutritional benefits and economic importance (Soni and Agrawal 2017; Zhao et al. 2020). In South Africa, it is an important fruit crop that has shown a slow-rising trend with regard to new planting hectares and production (South Africa Litchi Growers' Association (SALGA) 2025). According to the SALGA census conducted in 2025, the total litchi plantings in South Africa amounted to 1 389 ha, which is 29 ha more than in 2024. The litchi industry faces numerous challenges, such as pests and diseases, which seriously threaten plant vigour and growth, as well as fruit quality (Kumar 2016). Pests such as stink bugs (Hemiptera: Pentatomidae) and coreid bugs (Hemiptera: Coreidae) are considered serious pests of litchi in South Africa (Grové et al. 2015). Among these, the coconut bug, Pseudotheraptus wayi Brown (Hemiptera: Coreidae), is the most dominant sap-sucking bug occurring on litchis (Schoeman 2014).

The coconut bug is a significant pest of numerous commercial crops in eastern and southern Africa, causing direct damage to crops (Egonyu et al. 2014). Coconut bugs feed by injecting toxic saliva with lytic properties that break down fruit cell components. This feeding behaviour causes scarring, fruit deformities, and premature fruit drop (Doh et al. 2014; Schoeman and Morey 2016), ultimately reducing both the quantity of marketable fruit and the income generated from fruit sales. The nymphs and adults are known to inflict 75-100% direct damage to shoots, flowers, and fruits, which may lead to a loss of 80% or more across a range of crops (Maniania and Ekesi 2016). It is a serious pest of avocado (Persea americana: Lauraceae), guava (Psidium guajava: Myrtaceae), litchi (Litchi chinensis: Sapindaceae), loquat (Eriobotrya japónica: Rosaceae), macadamia (Macadamia integrifolia: Proteaceae), mango (Mangifera indica: Anacardiaceae), cashew (Anacardium occidentale: Anacardiaceae), carambola (Averrhoa carambola: Oxalidaceae), coconut (Cocos nucífera: Arecaceae), pecan (Carya illinoensis: Juglandaceae), cinnamon (Cinnamomum verum: Lauraceae), cocoa (Theobroma cacao: Malvaceae) and various leguminous crops (Hill 2008; Nyambo 2009). Mohan et al. (2022) reported yield losses of up to 100% resulting from both natural fall and early fall of coconuts showing symptoms attributed to the coconut bug impacting coconuts in Zanzibar. Yield losses of 76.2% in avocados (Van der Meulen and Schoeman 1994), 11.6% damaged fruit in mangoes (Schoeman 2010), 40% fruit drop in litchis (Schoeman and Mohlala 2013), and 45.65% of aborted immature litchis (Netshipise et al. 2025) have been reported due to coconut bug in South Africa. Van der Meulen (1992) reported that the incidence of coconut bug damaged guavas at harvest could be as high as 52.4% in the Nelspruit region of South Africa.

In addition to attacking crops, insect pests can spread a number of harmful microorganisms, such as bacteria, viruses, and fungi (Moore et al. 2020). They enable the presence of plant diseases by acquiring, transporting, and introducing pathogens into host plants that would otherwise not have been able to proliferate if the pest were absent. Fungi can use a variety of insects as a substrate for growth and reproduction (Biedermann and Vega 2020). Fungal pathogens infiltrate the insect's body through direct penetration of the insect's cuticle and ingestion, then spread internally until reaching the salivary glands and mouthparts. The pathogen is then introduced into new host plants during subsequent feeding (Fereres and Raccah 2015). Some fungi may even persist within the insect throughout its lifespan and be vertically transmitted to its offspring via eggs (Andret-Link and Fuchs 2005). Additionally, fungi also manipulate both the host plant and the insect throughout its life cycle to promote infection and dissemination (Franco et al. 2021). According to Golan and Pringle (2017), pests play a major role in the dispersal of fungi by acquiring fungal propagules while pollinating or simply moving across infected plant surfaces, thereby facilitating the spread of fungal pathogens. During these insect-plant interactions, the fungal spores adhere to the pests' abdomens, legs, or mouthparts, aiding their transmission to new hosts (Sani et al. 2020).

Postharvest fruit diseases caused by fungi are a major challenge to the litchi fruit industry (Dukare et al. 2019), causing fruit rot and spoilage, and ultimately economic losses. According to Jiang et al. (2002), degradation caused by microbial infections accounts for 20 to 30% of litchi loss after harvest and up to 50% before consumption. Some postharvest fungal pathogens include genera such as Alternaria, Aspergillus, Rhizopus, Cladosporium, Neofusicoccum sp., Pestalotiopsis sp., Botrytis, Colletotrichum, Diplodia, Phomopsis, Mucor, Penicillium and Sclerotinia (Nabi et al. 2017; Fourie and Coertzen 2018; Matrose et al. 2021).

Given the economic importance of litchi production in South Africa and globally, it is important to understand the full extent of the damage caused by the coconut bug and associated fungal pathogens. The impact of fungal infections on crops has been studied, but little is known about the potential impact of these fungi linked to coconut bugs on litchi. Thus, this study aimed to isolate fungal species from the mouthparts of coconut bugs and to explore the potential impact of these fungal species on litchi fruit quality. This topic had not been researched in South Africa prior to this study. Understanding the full extent of the damage caused by the coconut bug and associated fungal pathogens on litchi fruit is essential for developing targeted pest management strategies to reduce the risk of fungal transmission.

 

METHODS AND MATERIALS

Collection of coconut bugs

A total of 16 coconut bug adults and nymphs were collected at the ARC-Tropical and Subtropical Crops Division (25°27'06.2" S; 30°58'11.1" E) in Mbombela (Nelspruit), Mpumalanga Province, at the beginning of October in 2023. Collection was carried out by actively searching for coconut bugs early in the morning on randomly selected litchi, macadamia, and mango trees. Coconut bugs (identification confirmed by a specialist entomologist, Dr PS Schoeman) were collected using a 38 cm nylon sweep net, placed in labelled jars for preservation, and taken to the Tshwane University of Technology Crop Protection Laboratory for dissection and fungal isolation.

Preparation of PDA culture

Potato Dextrose Agar (PDA) culture medium was prepared by adding 39 g of PDA powder to a 1 000 ml culture bottle, mixing it well with distilled water, followed by autoclaving and sterilisation for 45 minutes under pressure and temperature of 121 °C. After removal, it was allowed to cool, and 20 ml PDA was poured into individual 90 mm diameter petri dish plates and left in the laminar flow cabinet to solidify before use.

Fungal isolation

The coconut bugs were frozen at 20 °C to cause immobilisation and death. After which they were surface sterilised in a laminar flow chamber by washing them with 70% ethanol and subsequently soaking them for two minutes in a 0.5% sodium hypochlorite solution. The insects were triple-rinsed in sterile distilled water and dried using sterile absorbent paper according to the protocol described by Pestano et al. (2017). Using a sterile razor, the mouthparts and the head of the coconut bugs were dissected to isolate the fungi that could have been ingested. The dissected parts and the intact (thorax/abdomen) of the coconut bugs were plated onto sixteen PDA plates, according to a protocol described by Fourie and Coertzen (2018). The cultured colonies were maintained in an incubator at 25 °C and 80% relative humidity for eight days. The 16 agar plates with fungal colonies were then removed from the incubator, and fungal colonies were sub-cultured onto fresh PDA plates. Each plate was sub-cultured (replicated) three times to obtain pure cultures in a sterile environment (laminar flow), using sterile equipment.

Morphological characterisation

The purified fungal culture isolates were characterised using morphological features, such as colony colour, pattern and texture over eight days according to the protocol described by Tafinta et al. (2013) to distinguish between the fungal species. Fungal isolates from the mouthparts of the coconut bugs were plated onto PDA and each isolate was replicated three times and subsequently incubated at 25 °C for eight days to evaluate their morphological characteristics. The characteristics of the fungal colonies were observed and compared with known reference cultures.

Molecular identification of the fungal isolates

Purified fungal cultures were subjected to DNA extraction. Genomic DNA was extracted from the cultures using the Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, Catalogue No. D6005). The primers used for fungal amplification/ sequencing were ITS1 (5'TCC GTA GGT GAA CCT GCG G 3') and ITS4 (5'TCC TCC GCT TAT TGA TAT GC 3'). The PCR was conducted using Nucleic Acid Detection (NEB) OneTaq 2X MasterMix with Standard Buffer (Catalogue No. M0482S), 10-30 ng/μ! of genomic DNA, 10 μM of both forward and reverse primers, and nuclease-free water (Catalogue No. E476). The thermocycling was conducted at 94 °C for 5 minutes, followed by 35 cycles of 30 seconds at 94 °C, 30 seconds at 50 °C, 1 minute at 68 °C with a final extension of 10 minutes at 68 °C and hold at 4 °C. The integrity of the PCR amplicons was visualised on a 1% agarose gel (CSL-AG500, Cleaver Scientific Ltd) stained with EZ-vision* Bluelight DNA Dye. The NEB Fast Ladder was used on all gels (N3238) as a size standard. Fragments were enzymatically purified using the ExoSAP procedure (NEB M0293L; NEB M0371). The amplicons were purified for sequencing (Zymo Research, ZR-96 DNA Sequencing Clean-up Kit™, Catalogue No. D4050), and sequenced in the forward and reverse direction (Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000) using the ABI 3730%l Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific).

Harvesting of litchis

Ripe litchis of various sizes were harvested from mature litchi trees that were randomly chosen at the ARC-Burgershall premises (25°04'07.5" S; 31°03'08.7" E) in Kiepersol, Mpumalanga Province, in November 2024. All fruits were placed in a cooler box and transported to the Tshwane University of Technology Crop Protection Laboratory, where inoculation of fungal species was done in an aseptic environment. Fruits that had physical damage or injury caused by mechanical factors, insects and disease were discarded, and only those that were considered healthy and of similar size were used.

Fungal inoculum preparation

Five fungal species isolated from the mouthparts of the coconut bugs were used for inoculation. Fresh spore suspensions were prepared from 14-day-old PDA cultures of the selected fungal species, namely: Cladosporium cladosporioides, Nigrospora oryzae, Pestalotiopsis microspora, Purpureocillium lilacinum, and Xylaria sp. These fungi are considered economically significant due to their association with plant diseases. The surface of the cultures was flooded with sterile distilled water after which, the spores were rubbed off with a spreader and gently dislodged with a sterile loop and carefully deposited into 50 ml Schott bottles. The suspension in the Schott bottles was filtered through cheesecloth to remove mycelial fragments and then 200 μ! of the suspension was pipetted into the channels of a mounted haemocytometer on a microscope (Model Stemi SV 11, Zeiss, Thornwood, NY; 40x magnification) for spore counting and concentration adjustment. The suspension concentration was adjusted to 1 x 10⁶ spores/ml for further use in the experiment.

Fungal inoculation

Litchis were washed under running tap water to remove surface dirt and then surface-sterilised by immersion in 70% ethanol for 2 minutes. After the ethanol treatment, the fruits were rinsed three times with sterile distilled water and allowed to air dry in a laminar flow cabinet. Once dried, the fruit was inoculated with the prepared fungal suspensions. A sterilised 23G X 25 mm needle was used to create 0.5 mm holes in litchi fruits. This procedure was designed to simulate the piercing-sucking mouthparts of the coconut bug. The experiment was performed in a completely randomised design and consisted of seven treatments replicated three times. The fungal isolates treatments were: P. microspora, Xylaria sp., N. oryzae, C. cladosporioides, and P. lilacinum, as well as, negative controls 1 and 2. From the total of 420 fruits used for the trial, 300 fruits were inoculated with five different fungal strains, 60 fruits in each treatment, and 120 fruits were used as controls, with 60 as control (CTRL)1 (litchis inoculated with sterile distilled water) and the other 60 as control (CTRL) 2 (litchis not inoculated). Inoculated fruits were inoculated with 0.1 ml of the fungal suspension using a sterilised syringe, and then stored for 14 days in an incubator at 25 °C with 80% relative humidity. Fungi obtained from the inoculated diseased section of the litchis were re-isolated onto PDA and incubated at 25 °C to identify if the morphological characteristics were identical to those of the original isolates, confirming Koch's postulates.

Post-harvest storage to assess the quality of infected litchis

Three parameters (weight loss, fruit colour, and severity of decay) were evaluated for 14 days to assess the damage caused by the selected five fungal species isolated from the mouthparts of coconut bugs. The fruits were stored at 25 °C and 80% relative humidity. To determine the weight loss of the litchi fruits over time, each replicate was weighed every second day, starting on the day of inoculation (Day 0) up to Day 14. The fruit weight was recorded using an electronic weighing scale with an accuracy of 0.01 g (Minh et al. 2019). The following formula was used for the calculation of weight loss:

The number of decayed fruits in each container was determined on Day 14. Based on the disease severity on the pulp of the fruit, a damage severity index was calculated following the methods described by Sivakumar and Korsten (2006). The damage severity was assigned into the scale of the following groups: 0 = 0% (no decay), 1 = 1-10% (slight infection), 2 = 1130% (moderate) 3 = 31-50% (severe), 4 = 51-75% (very severe), and 5 = 76-100% (entire infection).

The following formula was used for the calculation of the severity of decay:

Fruit colour was evaluated visually every second day to assess the colour changes on the pericarp of the inoculated litchis and the control litchis. A high-resolution camera was used to take photographs of the changes in skin colour of the litchi.

Statistical analysis

The weight loss data was statistically analysed using analysis of variance (ANOVA), using Genstat software (VSN International 2022). Means were used to test for differences between the five fungi and two control effects at the 5% level of significance.

 

RESULTS

Fungal isolation

Twenty-nine fungal species were isolated and identified from the coconut bug mouthparts, head, and full bodies (Table 1). The most prevalent genera were Cladosporium and Xylaria, with four species of each observed. The fungal isolates from the mouthparts were classified into nine genera, with a total of thirteen species found on the mouthparts. Four species of the Xylaria genus were identified from the mouthparts only, Cladosporium spp. from the head and mouthparts only, and four fungal genera (Pestalotiopsis, Sordariomycetes, Nigrospora, and Neopestalotiopsis) from the full bodies and mouthparts. Cladosporium sp., Lecythophora sp., and P. lilacinum were isolated from the nymphs.

 

 

Morphological characteristics of fungi

From the fungal species isolated, of particular interest were genera recovered from the insects' mouthparts, as they could be injected into the fruit during feeding. Table 2 shows the morphological characteristics of the fungal isolates from the coconut bug mouthparts. These include Xylaria, Pestalotiopsis, Neopestalotiopsis, Sordariomycetes, Nigrospora, Corynascus, Myceliophthora, Purpureocillium, and Cladosporium. Isolates showed differences in colour colony, texture, and pattern. However, most appeared to be white, cream and beige, with mostly cotton textures and line patterns (Figure 1).

 

 

 

 

Litchi fruit weight loss

Five fungal species found on the mouthparts of coconut bugs are economically important, as they are linked to diseases affecting key crops (Table 3). Litchis infected with fungi exhibited measurable weight loss over the storage period (Table 4). Litchis inoculated with AM2 (P. microspora) recorded the highest percentage of weight loss (21.58%) compared to the other fungal species (Figure 2). NM1 (P. lilacinum) recorded the lowest percentage weight loss (18.67%) (Figure 2). Both controls had weight loss percentages that were the lowest compared to all fungal species, with control 1 (litchis inoculated with sterile distilled water) having 10.03% weight loss and control 2 (litchis not inoculated) having the lowest with 9.24% weight loss. The difference in weight between the initial day and the last day (Day 14) of storage showed that the litchis with fungal infections did not differ statistically significantly from each other, but differed significantly from the controls (Table 5).

 

 

 

 

 

 

 

 

Litchi fruit colour

During the visual observation of the external colour on the fruits, the results showed that all the infected and control fruits had a similar-looking brown-coloured pericarp after 14 days of assessment (Figure 3). There were no visual symptoms on the outside of the infected and controlled fruits, although the fungi had caused internal changes.

 

 

Severity of litchi fruit decay

Litchis exposed to fungal infection showed varying levels of fungal decay internally on the pulp depending on the type of fungus (Figure 4). Each fungus uniquely affected the fruits' pulp with distinct symptoms. Pestalotiopsis microspora infection showed white mycelium on the pulp of the litchi fruit, N. oryzae showed a khaki-black mycelium, P. lilacinum showed mycelium that is white with a black colour at the centre, Xylaria sp. showed pulp with a cocoa colour, and C. cladosporioides showed an olive-grey to dull green mycelia.

 

 

The severity was greater on litchis infected with P. microspora (AM2), having decay damage of 61%, classified as very severely decayed according to the scale used to compare all fungal species (Figure 5). The observations showed that the internal quality of the control groups remained intact with no decay (Figure 4a and b). The infected fruits suggest that the fungal species could alter the fruit's physiological state. Re-isolated fungi from litchi fruits showed identical morphological characteristics (colony colour, pattern, and texture) to those initially isolated from the insect mouthparts, confirming Koch's postulates.

 

 

DISCUSSION

This study presents the first report of fungal species associated with coconut bug in South Africa. The findings contribute to the growing body of knowledge on fungal pathogen transmission by coreid insects, highlighting their potential role as vectors of fungi in agricultural systems. Previous studies have established that insect vectors can play a critical role in the transmission of plant pathogenic fungi, significantly contributing to crop losses and disease outbreaks (e.g., Wielkopolan et al. 2021; Heitmann et al. 2021). The identification of these fungi on the coconut bug underscores its potential role not only as a direct pest but also as a vector of plant pathogens. By linking specific fungal pathogens to economically valuable crops, this study builds upon existing knowledge and provides novel evidence of the coconut bug's capacity to facilitate the spread of fungal diseases.

Morphological characteristics

Morphological characterisation is a fundamental step in the preliminary identification of fungal pathogens at the genus level. The morphological features observed in NM1, P. lilacinum, was reported by Spatafora et al. (2015), who found that colonies of P. lilacinum are typically white with an uncoloured or cream-coloured reverse side. Similarly, the morphological traits of AM2, comprising P. microspora and N. saprophytica, were previously observed by Maharachchikumbura et al. (2014) and De Jesus et al. (2022). These isolates produced white, cottony colonies with aerial mycelia that gradually turned yellowish as the culture aged. Additionally, the morphological features of AM1, identified as X. adscendens, were in agreement with the description by Husbands (2017), who noted that X. adscendens forms colonies with radial strands that begin white and later develop a warty, canary-yellow mycelial layer.

Sharma et al. (2013) and Marin-Felix et al. (2015) described Corynascus verrucosus as producing colonies that are yellowish white with a white margin and a brownish to light brown reverse, features that were also observed in our AM6 isolates (C. verrucosus, Myceliophthora verrucosa, and Corynascus similis). Similarly, isolate AM8, identified as Cladosporium cladosporioides, displayed colony characteristics reported by Bensch et al. (2010), who noted that colonies of C. cladosporioides on potato dextrose agar (PDA) are olive-grey to dull green, with diffuse mycelial mats forming on the surface. In the case of AM3 and AM4, both identified as Xylaria species, the observed morphology closely resembled descriptions provided by Husbands (2017). Colonies were initially white, gradually darkening to a cocoa-brown colour with age, while the area near the point of inoculation remained white. The colonies also exhibited colourless to yellowish exudate in the aerial mycelium and had a reverse that ranged from beige to brownish orange.

Litchi fruit weight loss

The findings from this study demonstrate a significant impact of fungal species associated with the coconut bug on litchi quality in storage. The results indicate that all five fungal species (AM2 = P. microspora, AM3 = Xylaria sp., AM5 = N. oryzae, AM8 = C. cladosporioides, NM1 = P. lilacinum) were capable of inducing damage and weight loss on the litchi fruits, with an average loss of approximately 10 g every two days per replicate. Amongst all these, P. microspora induced the greatest weight loss of 21.58%. This significant reduction aligns with the findings of Suryanarayanan et al. (2012), who reported that P. microspora secrete enzymes like cellulases, pectinases, and ligninases that causes rapid tissue degradation by breaking down the structural integrity of the fruits, leading to softening, wilting, and rotting of the fruit, which ultimately culminates in weight loss. The extent of tissue degradation observed in this study supports the notion that P. microspora has a particularly aggressive mode of infection. Purpureocillium lilacinum and Xylaria sp. induced the least weight loss. This relatively low degradation is consistent with the findings of Chen and Hu (2022), who reported that both Xylaria spp. and P. lilacinum contain enzymes such as serine proteases and chitinases that degrade proteins and chitin which are not the main structural components of fruits cells. As a result, their impact on tissue breakdown is less significant, leading to less weight loss. Both Xylaria spp. and P. lilacinum are reported by Chen et al. (2024) and Corrêa-Moreira et al. (2022) as possible saprophytes, which means they generally feed on dead or decaying organic matter rather than actively attacking healthy living tissue. They may, however, contribute to decay in stressed, weakened or damaged fruits. The reduced weight loss of controls can be attributed to the absence of fungal degradation and minimal or no enzymatic activity occurring to break down the fruit's cellular structure, thus preserving water content and minimising weight loss in the control fruits. Although control 2 had the lowest weight loss percentage of 9.24% compared to control 1, the weight loss in the latter might be because control 1 was inoculated with distilled water, which may have made the fruits more vulnerable to dehydration when the cuticle was compromised, leading to water loss through transpiration or evaporation.

Severity of litchi fruit decay

The varying levels of fungal decay were only observed internally on the pulp of the fruit. These results align with the findings of Ngolong Ngea et al. (2021), who observed that fungal decay often begins inside the fruit, as pathogens typically degrade internal cellular structures, leading to rotting, softening, and internal discoloration. As a result, internal tissue breakdown may occur before any visible external signs of spoilage appear. According to our severity scale, fruits infected with P. microspora had the highest decay severity (61%), classified as very severely decayed. Bhuiyan et al. (2022) describe P. microspora as a species that effortlessly and rapidly spreads throughout the whole infected fruit, leading to compromised shelf life of fruits because of its rapid growth rate, which was also observed in our results. The white mycelium of P. microspora on the litchi aligns with the observations of Lorenzini and Zapparoli (2018) and Valencia et al. (2011), who associated P. microspora decay with a fine white mycelium and necrosis of host tissue in some fruits. The green mycelium of C. cladosporioides aligns with the findings of Briceño and Latorre (2007), who reported that grapes affected by Cladosporium rot are usually associated with a layer of olive-green mould, a hard patch of deterioration, and dehydration. Zhong et al. (2016) and Wang et al. (2021) reported that N. oryzae causes brown streaks and latent infections in rice, which relates to the colour that was observed in our results, but also khaki-black mycelium on the litchi. Purpureocillium lilacinum had the lowest severity decay of 25%, which was classified as moderate decay. This finding supports the study by Castillo Lopez et al. (2014), who reported that P. lilacinum has limited detrimental pathological effects on the fruits. Similarly, Xie et al. (2016) contested that P. lilacinum has a weaker ability to decompose cell walls, meaning it may be less pathogenic, which corresponds to the observed limited decay. Purpureocillium lilacinum is also not likely a fungus associated with post-harvest decay as it is known to be used as a biocontrol agent with antifungal and antibiosis properties (Chaudhary and Kaul 2011).

Litchi fruit colour

In this study, we observed that both the inoculated and control fruits had the same external (pericarp) brown colour, relating to the observation by Bhushan et al. (2015) that the red rind of litchis gradually turns brown during storage. Sivakumar and Korsten (2011) also reported that litchis have a relatively thick, protective rind that can act as a barrier or delayer to an external fungal invasion, preventing visible external changes even if the internal pulp tissue is affected. The lack of change in the external appearance could also be because the fungal spores were inoculated into the fruit, which may have started to proliferate internally on the pulp first, taking advantage of the fruits' sugars and moisture.

Predominant fungal pathogen

Most of these fungus species are predominantly found in fruits. Bateman et al. (2016) described that P. microspora can spread to the host plants through pests. Comparatively, P. microspora recorded the highest weight loss and decay severity. This corresponds with the observations of Maharachchikumbura et al. (2014), Hosking (2023) and Valencia et al. (2011) regarding the ability of the genus Pestalotiopsis to be pathogenic to various fruits such as grapes, avocados, kiwifruit, and blueberries. Inoculated fruit at the culmination of the study had considerable post-harvest infestations, rendering this fruit unmarketable. The internal changes may also make the fruit more prone to secondary infections from other microorganisms, exacerbating the deterioration process.

The fulfilment of Koch's postulates in this study, confirming that the same organism was responsible for the disease, provides clear evidence that fungi associated with the mouthparts of coconut bugs are capable of causing disease in litchi fruits. These findings suggest that the coconut bug may act as a vector, introducing pathogenic fungi during feeding activity and that fungal pathogens introduced by the coconut bugs contribute significantly to post-harvest fruit deterioration. This insect-fungus interaction could play a significant role in pre- or postharvest fruit damage, reducing the market quality of litchis in South Africa. However, it is important to acknowledge that this was an artificial study, and the experimental conditions may not fully represent natural interactions in the field. In natural settings, factors such as pathogen load, environmental conditions, and insect behaviour may influence whether transmission actually occurs. Therefore, while this study demonstrates the potential of these fungi to cause disease, further research is needed to investigate their actual role and transmission dynamics in natural litchi-insect systems.

 

CONCLUSION

The findings of this study indicate that the coconut bug may act as a vector for a range of plant-pathogenic fungi, including genera such as Cladosporium, Corynascus, Myceliophthora, Neopestalotiopsis, Nigrospora, Pestalotiopsis, Purpureocillium, Sordariomycetes, and Xylaria. Several of the fungal isolates recovered from the insects' mouthparts are known to be of significant economic importance due to their pathogenic effects on crops. The statistically significant difference in weight loss between the inoculated and control fruits underscores the critical role these fungal pathogens play in compromising fruit quality. These findings provide valuable insights into the potential post-harvest risks posed by fungal species associated with the coconut bug, highlighting the need for targeted pest and disease management strategies. The evidence suggests that the coconut bug may act as a vector for economically important fungal pathogens, making integrated pest management (IPM) essential for reducing fungal infection rates in litchi orchards. Future research could further explore this interaction through in-orchard artificial inoculation studies to better understand the fungis' effects under field conditions.

 

ACKNOWLEDGMENTS

The authors wish to thank ARC-Tropical and Subtropical Crops for giving us permission to do this work on their premises. We thank the Mpumalanga Department of Agriculture, Rural Development, Land and Environmental Affairs for granting us approval to conduct this research in the province. We also gratefully acknowledge the support from Tintswalo Maud Mathonsi for helping with morphological characterisation. We would like to thank Professor Edna Kunjeku for taking the necessary time and effort to review our work. We thank Marie Smith from Stats4science for assistance with statistical analysis of data. This work was funded by the National Research Foundation (NRF) and Tshwane University of Technology through the seed funds made available to SR Netshifhefhe.

 

AUTHORS' CONTRIBUTIONS

MN: conceptualisation, methodology, investigation, writing -original draft. PSS: conceptualisation, methodology, supervision, writing - review and editing. RGM: conceptualisation, supervision, validation, writing - review & editing. SRN: conceptualisation, methodology, supervision, validation, writing - review & editing.

 

ORCIDS IDS

M. Netshipise: https://orcid.org/0009-0004-3022-191X

S.R. Netshifhefhe: https://orcid.org/0000-0003-2266-4927

P.S. Schoeman: https://orcid.org/0000-0002-8244-4814

R. Gouws-Meyer: https://orcid.org/0000-0001-7928-9422

 

REFERENCES

Andret-Link P, Fuchs M. 2005. Transmission specificity of plant viruses by vectors. Journal of Plant Pathology. 87(3):153-165.         [ Links ]

Bateman C, Sigut M, Skelton J, Smith KE, Hulcr J. 2016. Fungal associates of the Xylosandrus compactus (Coleoptera: Curculionidae, Scolytinae) are spatially segregated on the insect body. Environmental Entomology. 45(4):883-890. https://doi.org/10.1093/ee/nvw070.         [ Links ]

Bensch K, Groenewald JZ, Dijksterhuis J, Starink-Willemse M, Andersen B, Summerell BA, Shin HD, Dugan FM, Schroers HJ, Braun U, Crous, PW. 2010. Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales). Studies in Mycology. 67(1):1-94. https://doi.org/10.3114/sim.2010.67.01.         [ Links ]

Bhuiyan MAB, Islam SMN, Bukhari MAI, Kader MA, Chowdhury MZH, Alam MZ, Abdullah HM, Jenny F. 2022. First report of Pestalotiopsis microspora causing leaf blight ofbanana in Bangladesh. Plant Disease. 106(5):1518. https://doi.org/10.1094/pdis-05-21-1120-pdn.         [ Links ]

Bhushan B, Pal A, Narwal R, Meena VS, Sharma PC, Singh J. 2015. Combinatorial approaches for controlling pericarp browning in Litchi (Litchi chinensis) fruit. Journal ofFood Science and Technology. 52:5418-5426. https://doi.org/10.1007/s13197-015-1712-8.         [ Links ]

Biedermann PH, Vega FE. 2020. Ecology and evolution of insect-fungus mutualisms. Annual Review of Entomology. 65(1):431-455. https://doi.org/10.1146/annurev-ento-011019-024910.         [ Links ]

Bragard C, Baptista P, Chatzivassiliou E, Di Serio F, Gonthier P, Jaques Miret JA, Justesen AF, Macleod A, Magnusson CS, Milonas P, et al. 2023. Pest categorisation of Pestalotiopsis microspora. EFSA Journal. 21(12):e8493. https://doi.org/10.2903/j.efsa.2023.8493.         [ Links ]

Briceño EX, Latorre BA. 2007. Outbreaks of Cladosporium rot associated with delayed harvest wine grapes in Chile. Plant Disease. 91(8):1060. https://doi.org/10.1094/pdis-91-8-1060c.         [ Links ]

Castillo Lopez D, Zhu-Salzman K, Ek-Ramos MJ, Sword GA. 2014. The entomopathogenic fungal endophytes Purpureocillium lilacinum (formerly Paecilomyces lilacinus) and Beauveria bassiana negatively affect cotton aphid reproduction under both greenhouse and field conditions. PloS ONE. 9(8):e103891. https://doi.org/10.1371/journal.pone.0103891.         [ Links ]

Chaudhary KK, Kaul RK. 2011. Compatibility of Pasteuriapenetrans with fungal parasite Paecilomyces lilacinus against root knot nematode on Chilli (Capsicum annuum L.). South Asian Journal of Experimental Biology. 1(1):36-42. https://doi.org/10.38150/sajeb.1(1).p36-42.         [ Links ]

Chen W, Hu Q. 2022. Secondary Metabolites of Purpureocillium lilacinum. Molecules. 27(1):18. https://doi.org/10.3390/molecules27010018.         [ Links ]

Chen W, Yu M, Chen S, Gong T, Xie L, Liu J, Bian C, Huang G, Zheng C. 2024. Structures and biological activities of secondary metabolites from Xylaria spp. Journal of Fungi. 10(3):190. https://doi.org/10.3390/jof10030190.         [ Links ]

Corrêa-Moreira D, De Lima Neto RG, Da Costa GL, De Moraes Borba C, Oliveira MME. 2022. Purpureocillium lilacinum an emergent pathogen: antifungal susceptibility of environmental and clinical strains. Letters in Applied Microbiology. 75(1):45-50. https://doi.org/10.1111/lam.13707.         [ Links ]

De Jesus JMI, Rodrigues ABL, Marques De Paula KL, Gomes Da Cunha M. 2022. Pestalotiopsis microspora causes leaf spot on Adenium obesum. Phytopathology. 170(6):408-413. https://doi.org/10.1111/jph.13091.         [ Links ]

Doh F, Yao MDS, Issali AE, Allou K. 2014. Incidence Pseudotheraptus devastans (Distant) (Heteroptera: Coreidae) on three hybrid varieties of coconut, PB 113+, PB 121+et NVS x GVT in Ivory Coast. International Journal of Biological and Chemical Sciences. 8(6):2650-2658. http://dx.doi.org/10.4314/ijbcs.v8i6.25.         [ Links ]

Dukare AS, Paul S, Nambi VE, Gupta RK, Singh R, Sharma K, Vishwakarma RK. 2019. Exploitation of microbial antagonists for the control of postharvest diseases of fruits: a review. Critical Reviews in Food Science and Nutrition. 59(9):1498-1513. https://doi.org/10.1080/10408398.2017.1417235.         [ Links ]

Egonyu JP, Ekesi S, Kabaru J, Irungu L. 2014. Biology of the coconut bug, Pseudotheraptus wayi, on French beans. Journal of Insect Science. 14(1):58-87. https://doi.org/10.1093/jis/14.1.44.         [ Links ]

Fereres A, Raccah B. 2015. Plant virus transmission by insects. eLS. 14:1-12. https://doi.org/10.1002/9780470015902.a0000760.pub3.         [ Links ]

Franco FP, Túler AC, Gallan ZD, Gonçalves FG, Favaris AP, Peñaflor MFGV, Leal WS, Moura DS, Bento JMS, Silva-Filho MC. 2021. Fungal phytopathogen modulates plant and insect responses to promote its dissemination. The ISME Journal. 15(12):3522-3533. https://doi.org/10.1038/s41396-021-01010-z.         [ Links ]

Fourie DV, Coertzen J. 2018. The dissemination of fungal pathogens on avocado trees in South Africa with reference to vector potential of insect pests. South African Avocado Growers' Association Yearbook. 41:77-85.         [ Links ]

Golan JJ, Pringle A. 2017. Long-distance dispersal of fungi. In: Heitman J, Howlett BJ, Crous PW, Stukenbrock EH, James TY, Gow NAR, editors. The fungal kingdom. Washington, DC, USA: ASM Press; pp. 309-333. https://doi.org/10.1128/9781555819583.ch14.

Grové T, De Villiers EA, Daneel MS. 2015. Insects of cultivated plants and natural pastures in Southern Africa. Pretoria: Entomological Society of Southern Africa. 574-88. http://dx.doi.org/10.1080/0035919X.2015.1088487.

Heitmann N, Glemnitz M, Lentzsch P, Platen R, Müller MEH. 2021. Quantifying the role of ground beetles for the dispersal of Fusarium and Alternaria fungi in agricultural landscapes. Journal of Fungi. 7(10): 863. https://doi.org/10.3390/jof7100863.         [ Links ]

Hill DS. 2008. Pests ofcrops in warmer climates and their control. Skegness Lincs: Springer Dordrecht. https://doi.org/10.1007/978-1-4020-6738-9.         [ Links ]

Hosking JE. 2023. Postharvest decay of blueberry fruit in New Zealand. Master of Science in Horticultural Science thesis, Massey University, Manawatü, New Zealand.         [ Links ]

Husbands DR. 2017. New species and records of carbonaceous Ascomycetes from Guyana, including a new potential pathogen of greenheart. Master's thesis, Purdue University, West Lafayette.         [ Links ]

Jiang YM, Yao LH, Lichter A, Li JR. 2002. Postharvest biology and technology of litchi fruit. Food, Agriculture and Environment. 1:76-81.         [ Links ]

Ju YM, Rogers JD, Hsieh HM. 2018. Xylaria species associated with fallen fruits and seeds. Mycologia. 110(4):726-749. https://doi.org/10.1080/00275514.2018.1469879.         [ Links ]

Kumar R. 2016. Litchi. In: Kumar RM, editor. Abiotic Stress Physiology of Horticultural Crops. Springer India; pp. 235-266. https://doi.org/10.1007/978-81-322-2725-0_14.

Latorre BA, Briceño EX, Torres R. 2011. Increase in Cladosporium spp. populations and rot of wine grapes associated with leaf removal. Crop Protection. 30(1):52-56. https://doi.org/10.1016/j.cropro.2010.08.022.         [ Links ]

Lorenzini M, Zapparoli G. 2018. Identification of Pestalotiopsis bicilita, Diplodia seriata and Diaporthe eres causing fruit rot in withered grapes in Italy. European Journal of Plant Pathology. 151(4):1089-1093. http://dx.doi.org/10.1007/s10658-017-1416-1.         [ Links ]

Ma H, Song Z, Pan X, Qu Z, Yang Z, Li Y, Zhu A. 2022. Four new pale-spored species of Xylaria (Xylariaceae, Xylariales) with a key to worldwide species on fallen fruits and eeds. Biology. 11(6):885. https://doi.org/10.3390/biology11060885.         [ Links ]

Maharachchikumbura SSN, Hyde KD, Groenewald JZ, Xu J, Crous PW. 2014. Pestalotiopsis revisited. Studies in Mycology. 79(1):121-186. https://doi.org/10.1016/j.simyco.2014.09.005.         [ Links ]

Maniania JNK, Ekesi S. 2016. Development and Application of Mycoinsecticides for the Management of Fruit Flies in Africa. In: Ekesi S, Mohamed S, De Meyer M, editors. Fruit Fly Research and Development in Africa - Towards a Sustainable Management Strategy to Improve Horticulture. Springer International Publishing, Cham; pp. 307-324. https://doi.org/10.1007/978-3-319-43226-7_15.

Marin-Felix Y, Stchigel AM, Miller AN, Guarro J, Cano-Lira JF. 2015. A re-evaluation of the genus Myceliophthora (Sordariales, Ascomycota): its segregation into four genera and description of Corynascus fumimontanus sp. Mycologia. 107(3):619-632. https://doi.org/10.3852/14-228.         [ Links ]

Matrose NA, Obikeze K, Belay ZA, Caleb OJ. 2021. Plant extracts and other natural compounds as alternatives for post-harvest management of fruit fungal pathogens: A review. Food Bioscience. 41:100840. http://dx.doi.org/10.1016/jfbio.2020.100840.         [ Links ]

Minh NP, Nhi TTY, Tuyen LK, Phi TH, Khoa DT, Thuan TQ. 2019. Technical factors affecting sea grape (Caulerpa lentillifera) production by cultivation and its stability by post-harvest treatment. Journal of Pharmaceutical Sciences and Research. 11:783-786.         [ Links ]

Mohan C, Josephrajkumar A, Prathibha PS, Sujithra M, Sajan JV, Anes KM. 2022. Pests and their management in coconut. In: Mani M, editor. Trends in Horticultural Entomology. Singapore: Springer; pp. 1411-1439. https://doi.org/10.1007/978-981-19-0343-4_60.

Moore D, Robson GD, Trinci AP. 2020. 21st Century Guidebook to Fungi. Manchester, UK: Cambridge University Press. https://doi.org/10.1017/9781108776387.         [ Links ]

Nabi SU, Raja WH, Kumawat KL, Mir JI, Sharma OC, Singh DB, Sheikh MA. 2017. Post-harvest diseases of temperate fruits and their management strategies - a review. International Journal of Pure and Applied Bioscience. 5(3):885-898. http://dx.doi.org/10.18782/2320-7051.2981.         [ Links ]

Netshipise M, Schoeman PS, Gouws-Meyer R, Netshifhefhe SR. 2025. Assessment of coconut bug, Pseudotheraptus wayi Brown (Hemiptera: Coreidae) damage on litchis in the Limpopo and Mpumalanga provinces of South Africa. African Entomology. 33(1). https://doi.org/10.17159/2254-8854/2025/a20067.         [ Links ]

Ngolong Ngea GL, Qian X, Yang Q, Dhanasekaran S, Ianiri G, Ballester AR, Zhang X, Castoria R, Zhang H. 2021. Securing fruit production: opportunities from the elucidation of the molecular mechanisms of postharvest fungal infections. Comprehensive Reviews in Food Science and Food Safety. 20(3):2508-2533. https://doi.org/10.1111/1541-4337.12729.         [ Links ]

Nyambo B. 2009. Integrated pest management plan (IPMP): The Agricultural Sector Development Program Project. United Republic of Tanzania. http://documents.worldbank.org/curated/en/793561468341086850 [accessed 6 December].

Pestano F, Liverpool E, Gomathinayagam S, Ansari A. 2017. Investigation of entomopathogenic fungi from insects at University of Guyana Turkeyen Campus. Current Trends in Biomedical Engineering & Biosciences. 5(2):555656. https://doi.org/10.19080/ctbeb.2017.05.555656.         [ Links ]

Sani I, Ismail SI, Abdullah S, Jalinas J, Jamian S, Saad N. 2020. A review of the biology and control of whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), with special reference to biological control using entomopathogenic fungi. Journal of Insect Science. 11(9):619. https://doi.org/10.3390/insects11090619.         [ Links ]

Schoeman PS. 2010. Coconut bug. The cultivation of litchi. Nelspruit, South Africa: ARC-Institute for Tropical and Subtropical Crops. 190:1.         [ Links ]

Schoeman PS. 2014. An overview of control strategies of economically important stinkbugs (Heteroptera) and Tortricidae moths occurring on litchis in South Africa. Acta Horticulture. 1029:401-408. https://doi.org/10.17660/ActaHortic.2014.1029.51.         [ Links ]

Schoeman PS, Mohlala R. 2013. The occurrence ofindigenous heteropterans on subtropical fruit in Southern Africa. Acta Horticulturae. 1007:369-376. https://doi.org/10.17660/ActaHortic.2013.1007.41.         [ Links ]

Schoeman PS, Morey L. 2016. Hemiptera (Pentatomidae & Coreidae) occurring on Litchi chinensis Sonn. in the Nelspruit region of the Mpumalanga province of South Africa. African Plant Protection. 19(1):4-7. https://hdl.handle.net/10520/EJC193385.         [ Links ]

Sharma R, Kulkarni G, Shouche YS. 2013. Corynascus verrucosus, second record for science and new for India. Nova Hedwigia. 97(3-4):485-494. https://doi.org/10.1127/0029-5035/2013/0112.         [ Links ]

Sivakumar D, Korsten L. 2006. Influence of modified atmosphere packaging and postharvest treatments on quality retention of litchi cv. Mauritius. Postharvest Biology and Technology. 41:135-142.         [ Links ]

Sivakumar D, Korsten L. 2011. Litchi (Litchi chinensis Sonn.). In: Yahia EM, editor. Postharvest biology and technology of tropical and subtropical fruits. Woodhead Publishing Series in Food Science, Technology and Nutrition. Woodhead Publishing; pp. 361-407. https://doi.org/10.1533/9780857092885.361.

Soni R, Agrawal S. 2017. Litchi chinensis: Taxonomy, botany and its cultivars. Lychee disease management. Springer Singapore, pp.191215. https://doi.org/10.1007/978-981-10-4247-8_12.

South African Litchi Growers' Association (SALGA). 2025. Industry Production Statistics Information. SALGA Industry Census.

Spatafora JW, Quandt CA, Kepler RM, Sung GH, Shrestha B, Hywel-Jones NL, Luangsa-Ard JJ. 2015. New 1F1N species combinations in Ophiocordycipitaceae (Hypocreales). IMA Fungus. 6(2):357-362. https://doi.org/10.5598/imafungus.2015.06.02.07.         [ Links ]

Sudhakara Reddy M, Murali TS, Suryanarayanan TS, Govinda Rajulu MB, Thirunavukkarasu N. 2016. Pestalotiopsis species occur as generalist endophytes in trees of Western Ghats forests of southern India. Fungal Ecology. 24:70-75. https://doi.org/10.1016/j.funeco.2016.09.002.         [ Links ]

Suryanarayanan TS, Thirunavukkarasu N, Govindarajulu MB, Gopalan V. 2012. Fungal endophytes: an untapped source ofbiocatalysts. Fungal Diversity. 54(1):19-30. http://dx.doi.org/10.1007/s13225-012-0168-7.         [ Links ]

Tafinta IY, Shehu K, Abdulganiyyu H, Rabe AM, Usman A. 2013. Isolation and identification of fungi associated with the spoilage of sweet orange (Citrus sinensis) fruits in Sokoto State. Nigerian Journal of Basic and Applied Science. 21(3):193-196. https://doi.org/10.4314/njbas.v21i3.4.         [ Links ]

Valencia A, Torres R, Latorre B. 2011. First report of Pestalotiopsis clavispora and Pestalotiopsis spp. causing postharvest stem end rot of avocado in Chile. Plant Disease. 95(4):492-492. https://doi.org/10.1094/pdis-11-10-0844.         [ Links ]

Van Der Meulen T. 1992. Assessment of damage caused by the coconut bug Pseudotheraptus wayi (Brown) (Hemiptera: Coreidae) on guavas. Fruits. 47:317-320.         [ Links ]

Van Der Meulen T, Schoeman A. 1994. Pest status of the coconut bug Pseudotheraptus wayi Brown (Hemiptera: Coreidae) on avocados in South Africa. Fruits. 49:71-75.         [ Links ]

VSN INTERNATIONAL. 2022. Genstat for Windows 22nd Edition. VSN International, Hemel Hempstead, UK. Genstat.co.uk.

Wang Y, Yao J, Li Z, Huo J, Zhou S, Liu W, Wu H. 2021. Genome sequence resource for Nigrospora oryzae, an important pathogenic fungus threatening crop production. International Society for Molecular Plant-Microbe Interactions. 34(7):835-838. https://doi.org/10.1094/mpmi-11-20-0311-a.         [ Links ]

Wang Z, Zhang R, Xu Y, Zhou C, Jin J, Zhao X, Zhao J, Wang X, Song J, Xiang W. 2024. Postharvest fruit rot on red guava caused by Neopestalotiopsis saprophytica newly reported in Hainan Province China. Journal of Crop Protection. 186:0261-2194. https://doi.org/10.1016/j.cropro.2024.106915.         [ Links ]

Wielkopolan B, Jakubowska M, Obrçpalska-Stçplowska A. 2021. Beetles as plant pathogen vectors. Frontiers in Plant Science. 12:748093. https://doi:10.3389/fpls.2021.748093.         [ Links ]

Xie J, Li S, Mo C, Xiao X, Peng D, Wang G, Xiao Y. 2016. Genome and transcriptome sequences reveal the specific parasitism of the nematophagous Purpureocillium lilacinum 36-1. Frontiers in Microbiology. 7:1084. https://doi.org/10.3389/fmicb.2016.01084.         [ Links ]

Yang Q, Zeng X-Y, Yuan J, Zhang Q, He Y-K, Wang Y. 2021. Two new species of Neopestalotiopsis from southern China. Biodiversity Data Journal. 9:10-26. https://doi.org/10.3897/bdj.9.e70446.         [ Links ]

Zhao L, Wang K, Wang K, Zhu J, Hu Z. 2020. Nutrient components, health benefits, and safety of litchi (Litchi chinensis Sonn.): A review. Comprehensive Reviews in Food Science and Food Safety. 19(4):2139-2163. https://doi.org/10.1111/1541-4337.12590.         [ Links ]

Zhong J, Zhou Q, Hu Y, Zhu HJ, Da Gao B. 2016. Molecular identification of a novel victorivirus from the phytopathogenic fungus Nigrospora oryzae. Virus Genes. 52:156-159. https://doi.org/10.1007/s11262-015-1284-8.         [ Links ]

 

 

Correspondence:
S.R. Netshifhefhe
Email: NetshifhefheSR@tut.ac.za

Received: 17 September 2025
Accepted: 12 November 2025