Isolation of culturable bacteria from Asian citrus psyllid, Diaphorina citri: Toward potential applications in symbiont-based pest management

Introduction

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), not only causes direct damage by feeding on host plants but also serves as a vector for the bacterium Candidatus Liberibacter asiaticus (CLas), which is a phloem-limited bacterium and the causative agent of Huanglongbing (HLB), a devastating citrus disease (Wang et al., 2017). The spread of HLB poses a significant threat to global citrus production (Gottwald, 2010). Despite extensive efforts to control this vector and mitigate HLB, these measures have failed to prevent the disease’s proliferation. Chemical control methods, though effective in the short term, face increasing challenges due to environmental hazards, disruption of natural enemy populations (Bové, 2006), and the development of pesticide resistance in psyllid populations (Tiwari et al., 2011; Hall et al., 2013; Chen et al., 2017; Pardo et al., 2018). Furthermore, the high cost and non-specificity of chemical treatments have diminished their efficacy. Consequently, the development of alternative pest management strategies is imperative. Among these, biological control methods present a sustainable and environmentally benign approach that could reduce reliance on chemical interventions.

Microbial symbionts in insects play pivotal roles in reproduction, survival, fecundity, oviposition, nutrient provisioning, and pathogen defense (Kikuchi et al., 2012; Gosalbes et al., 2010; Ayoubi et al., 2025; Kashkouli et al., 2019; Karamipour et al., 2016). In recent years, the biological control of insect vectors through the manipulation of their endosymbionts has emerged as a promising strategy for managing both the vectors and their associated pathogens (Nouri et al., 2018; Heck, 2018; Britt et al., 2020; Wu et al., 2025). Recent studies highlight that identifying bacterial endosymbionts in insects, including the D. citri, is critical for understanding host biology, pathogen resistance, and even pathogen transmission dynamics (Ramsey et al., 2015). Endosymbiotic bacteria can significantly influence the growth, reproduction, dispersal, and transmission efficiency of D. citri; thus, characterizing these microbial communities may provide novel avenues for sustainable pest management (Guidolin & Consoli, 2013; Fagen et al., 2012).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), not only causes direct damage by feeding on host plants but also serves as a vector for the bacterium Candidatus Liberibacter asiaticus (CLas), which is a phloem-limited bacterium and the causative agent of Huanglongbing (HLB), a devastating citrus disease (Wang et al., 2017). The spread of HLB poses a significant threat to global citrus production (Gottwald, 2010). Despite extensive efforts to control this vector and mitigate HLB, these measures have failed to prevent the disease’s proliferation. Chemical control methods, though effective in the short term, face increasing challenges due to environmental hazards, disruption of natural enemy populations (Bové, 2006), and the development of pesticide resistance in psyllid populations (Tiwari et al., 2011; Hall et al., 2013; Chen et al., 2017; Pardo et al., 2018). Furthermore, the high cost and non-specificity of chemical treatments have diminished their efficacy. Consequently, the development of alternative pest management strategies is imperative. Among these, biological control methods present a sustainable and environmentally benign approach that could reduce reliance on chemical interventions.

Microbial symbionts in insects play pivotal roles in reproduction, survival, fecundity, oviposition, nutrient provisioning, and pathogen defense (Kikuchi et al., 2012; Gosalbes et al., 2010; Ayoubi et al., 2025; Kashkouli et al., 2019; Karamipour et al., 2016). In recent years, the biological control of insect vectors through the manipulation of their endosymbionts has emerged as a promising strategy for managing both the vectors and their associated pathogens (Nouri et al., 2018; Heck, 2018; Britt et al., 2020; Wu et al., 2025). Recent studies highlight that identifying bacterial endosymbionts in insects, including the D. citri, is critical for understanding host biology, pathogen resistance, and even pathogen transmission dynamics (Ramsey et al., 2015). Endosymbiotic bacteria can significantly influence the growth, reproduction, dispersal, and transmission efficiency of D. citri; thus, characterizing these microbial communities may provide novel avenues for sustainable pest management (Guidolin & Consoli, 2013; Fagen et al., 2012).

Insect endosymbionts have emerged as innovative tools for sustainable pest management strategies (Engel & Moran, 2013; Kashkouli et al., 2019). Among the most promising approaches are heterologous associations, microbial symbiont disruption, the Incompatible Insect Technique (IIT), and paratransgenesis. The IIT method, which leverages Wolbachia-induced cytoplasmic incompatibility (CI), functions by releasing Wolbachia-infected males into target populations. These males mate with wild females, resulting in reproductive failure and population suppression (Zheng et al., 2019; Lim et al., 2024). Paratransgenesis offers another sophisticated approach, involving genetic modification of insect symbionts to alter host traits (Beard et al., 1998). First developed in the 1990s for managing disease vectors and agricultural pests (Beard et al., 1993; Arora & Douglas, 2017), this technique requires symbionts with specific characteristics: laboratory cultivability, genetic transformability, efficient vertical/horizontal transmission to hosts and either high host specificity or non-target safety (Qadri et al., 2020). By exploiting these intimate host-microbe relationships, these methods provide targeted, environmentally sound alternatives to conventional pest control, representing a paradigm shift toward precision agriculture and vector management.

Although paratransgenesis research has primarily targeted medically important insect vectors, its application to insects that transmit phytopathogens remains largely unexplored. A critical first step in developing paratransgenic strategies is the identification and cultivation of suitable vector-associated endosymbionts. In this study, we employed culture-dependent methods to isolate and characterize potential bacterial candidates for such an approach. The ecological significance and biotechnological potential of these isolates are also discussed.

Materials and methods

1. ACP Collection and Rearing

The ACP specimens were collected from field populations in Rudan County (Hormozgan Province) and Jahrom County (Fars Province), Iran, and subsequently transferred to the research greenhouse at the Faculty of Agriculture, Tarbiat Modares University for colony establishment. The insects were maintained on two-year-old key lime (Citrus aurantifolia) seedlings housed in double-layered mesh cages under strict conditions. Environmental parameters were regulated throughout the rearing period, maintaining a temperature of 25±3°C, relative humidity of 60-70%, and a photoperiod of 14:10 hours (light: dark). To optimize host plant quality and promote adult oviposition, regular pruning was conducted biweekly. Plant nutrition was maintained through the application of balanced NPK fertilizer (20-20-20 formulation) supplemented with iron chelate, ensuring adequate micronutrient availability for both plant health and insect development.

2. Insect Dissection and Bacterial Cultivation

 For bacterial isolation, adult ACP specimens were collected from established colonies and immobilized on ice for 1 hour. Following surface sterilization with 70% ethanol and sterile distilled water washes, males and females were separated, and intestinal tissues were aseptically dissected from 20 individuals. The dissected tissues were homogenized in sterile phosphate-buffered saline and transferred to 15 mL conical tubes containing liquid culture medium, Luria-Bertani (LB). These suspensions were incubated at 37°C for 12-13 hours with constant agitation (150 rpm) in an orbital shaker to facilitate bacterial growth. Two distinct culture media were employed for bacterial isolation: LB agar (per liter: 10g tryptone, 5g yeast extract, 10g NaCl; pH 7.0 ± 0.2) and Acetic acid bacteria-specific medium (per liter: 20g D-sorbitol, 5g peptone, 3g yeast extract, 100mg cycloheximide). Inoculation loops were sterilized by immersion in 70% ethanol followed by flaming, with adequate cooling time before use. Quadrant streaking was performed at 45° angles to achieve isolated colonies, with particular attention to maintaining aseptic conditions throughout the process.

3.PCR Amplification

For detection of cultured endosymbiotic bacteria from ACP, single colonies were isolated from solid culture media. 16S rRNA gene amplification was performed using universal primers (F: 5'-AGAGTTTGATCMTGGCTCAG-3' and R: 5'-TACGGYTACCTTGTTACGACTT-3') in 20 μL reaction volumes containing: 10 μL 2× Master Mix Red (Ampliqon), 1 μL of each primer (10 μM), 2 μL template DNA, and 6 μL nuclease-free water. Following brief centrifugation to pellet reaction components, thermal cycling was performed with initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (95°C, 5 sec), annealing (57°C, 45 sec), and extension (72°C, 45 sec), with a final extension at 72°C for 2 min (Applied Biosystems 2720 Thermal Cycler). Amplified products were verified by 1.5% agarose gel electrophoresis before Sanger sequencing (Pishgam Biotech, Iran).

4. Sequencing and Phylogenetic Analysis

The PCR products from the extracted DNA of the cultured endosymbiotic bacteria were submitted for Sanger sequencing. The obtained sequences were analyzed using the NCBI's (National Center for Biotechnology Information) BLAST tool for taxonomic identification. For phylogenetic reconstruction, reference sequences of related bacterial species were retrieved from the NCBI database. Sequence alignment was performed using the PRATT software, followed by phylogenetic tree construction in MEGA X software. When necessary, manual sequence adjustments were made to optimize alignments. The neighbor-joining method was employed for phylogenetic analysis with 1000 bootstrap replicates to assess nodal support, using the Kimura 2-parameter substitution model.

Results

1. Identification and Characterization of Cultured Bacterial Isolates from ACP

Bacterial isolation was successfully performed using both LB agar and acetic acid bacteria-specific media, with numerous distinct colonies observed (Fig. 1). Single colonies were selected based on morphological characteristics (colony color, size, and morphology) and subjected to DNA extraction. A portion of the 16S rRNA gene was amplified by PCR and the products were sequenced. After quality trimming, the obtained sequences were analyzed using the nucleotide BLAST, leading to the identification of four bacterial genera from the psyllid populations. Bacillus sp. and Enterococcus sp. were detected in populations from the colonies collected from Jahrom and Rudan populations, while Staphylococcus sp. was exclusively found in the Jahrom colony and Burkholderia sp. only in the Rudan colony. The 16S rRNA sequences were deposited in the NCBI GenBank under accession numbers PV875931 (Burkholderia), PV875933 (Enterococcus), and PV875935 (Bacillus).

Fig. 1. Culture and isolation of Diaphorina citri gut bacteria. (A) Primary streak plate on nutrient agar for isolation of bacterial colonies from the dissected gut of D. citri. (B) Purified single colonies obtained after subsequent streaking were used for further molecular identification.

The BLAST analysis revealed the following closest matches: Bacillus sp. showed 98% query coverage and 87% identity to Bacillus safensis, Enterococcus sp. showed 100% query coverage and 98% identity to Enterococcus casseliflavus, Staphylococcus sp. showed 100% query coverage and 98% identity to Staphylococcus saprophyticus, Burkholderia sp. showed 92% query coverage and 95% identity to Burkholderia cepacia.

2. Phylogenetic Analysis of Bacterial Isolates

The phylogenetic relationships of the isolated bacteria (Bacillus sp., Enterococcus sp., Staphylococcus sp., and Burkholderia sp.) were analyzed using MEGA10 software with reference sequences from NCBI. For Bacillus species, the phylogenetic tree revealed two sister clades, with isolates from both Jahrom and Rudan counties clustering together in a distinct clade (Fig. 2). Enterococcus sp. isolates formed a single cluster regardless of their geographic origin (Fig. 3). The Staphylococcus sp. isolate from Jahrom showed relationship to the phylogenetic affinity to S. saprophyticus, forming a well-defined branch adjacent to reference strains (Fig. 4). Similarly, the Burkholderia sp. isolate from Rudan demonstrated the highest similarity to B. diffusa (Fig. 5). All phylogenetic analyses were performed using the Neighbor-Joining with 1000 bootstrap replicates. The Tamura-Nei evolutionary model was applied for Bacillus and Burkholderia sp., while all sequences were aligned using the MUSCLE algorithm. Branch support values exceeded 85% bootstrap for all significant nodes, confirming robust phylogenetic relationships. These results demonstrate clear species-level classification for Staphylococcus sp. and Burkholderia sp., while revealing interesting patterns of geographic distribution among the Bacillus populations and consistent evolutionary relationships for Enterococcus isolates across different locations.

Fig. 2. Phylogenetic analysis of bacterial isolate Bacillus sp. from D. citri gut. The evolutionary history was inferred using the Neighbor-Joining method based on the 16S rRNA gene sequence. The bootstrap consensus tree (from 1000 replicates) shows the position of the isolate (highlighted in blue) relative to closely related type strains. The scale bar indicates the number of base substitutions per site.

Discussion

The ACP is recognized as the primary vector of Candidatus Liberibacter asiaticus, the causal agent of citrus greening disease (huanglongbing). In addition to this pathogen, recent studies have reported the presence of other associated bacteria within the insect’s body, which may play a role in host biology, disease transmission, or microbial-host interactions. In this study, as part of efforts to exploit symbiotic bacteria for the biological control of this pest, we aimed to identify culturable bacteria present in the gut of Diaphorinacitri. Four bacterial isolates belonging to the genera Bacillus sp., Burkholderia sp., Enterococcus sp., and Staphylococcus sp. were successfully isolated and identified. Studies indicate that the bacteria associated with D. citri are predominantly citrus-related endophytes (Araújo et al., 2002; Azevedo et al., 2000).

Each genus holds distinct biological significance: certain Bacillus species produce antimicrobial metabolites with demonstrated biocontrol potential against plant pests and pathogens (Fira et al., 2018); Burkholderia comprises both beneficial symbionts and phytopathogens that may influence insect-plant interactions (Compant et al., 2008); while Enterococcus and Staphylococcus, frequently reported as gut symbionts, may contribute to antibiotic resistance or host immune modulation (Dillon & Dillon, 2004). Although previous studies have characterized some D. citri endosymbionts such as Bacillus sp., Staphylococcus sp., Cardinium sp., Hamiltonella sp (Kolora et al., 2015; Hosseinzadeh et al., 2019; Rahimpour et al., 2025; Zanganeh et al., 2025), the full diversity and functional roles of its bacterial associates remain incompletely understood. These findings advance our understanding of the tripartite psyllid-symbiont-pathogen interplay and provide a foundation for developing novel management strategies, including biocontrol approaches or genetic manipulation of endosymbionts. Based on the obtained results, the bacterium Burkholderia sp. was exclusively identified in the Diaphorina citri population from Rudan County. To our knowledge, this bacterium has not been previously reported in the Asian citrus psyllid. Members of the Burkholderia sp. genus (β-proteobacteria) are primarily soil-dwelling bacteria commonly found in the rhizosphere of plants, surrounding environments, and other moist habitats (Woods et al., 2006). Certain Burkholderia species are established as specialized, beneficial symbionts in various hemipteran insects.

Fig. 3. Phylogenetic analysis of bacterial isolate Enterococcus sp. from D. citri gut. The evolutionary history was inferred using the Neighbor-Joining method based on the 16S rRNA gene sequence. The bootstrap consensus tree (from 1000 replicates) shows the position of the isolate (highlighted in blue) relative to closely related type strains. The scale bar indicates the number of base substitutions per site.

Fig. 4. Phylogenetic analysis of bacterial isolate Staphylococcus sp. from D. citri gut. The evolutionary history was inferred using the Neighbor-Joining method based on the 16S rRNA gene sequence. The bootstrap consensus tree (from 1000 replicates) shows the position of the isolate (highlighted in blue) relative to closely related type strains. The scale bar indicates the number of base substitutions per site.

They are typically acquired environmentally by nymphs and colonize the midgut to facilitate host growth (Kikuchi et al., 2005, 2011). Evidence also suggests potential endosymbiotic and vertical transmission, as Burkholderia has been detected in insect bacteriomes and ovaries (Kikuchi et al., 2005, 2011). Its presence across diverse insect hosts, including stinkbugs, leafhoppers, and scale insects, indicates a broader symbiotic role, potentially through nutrient provisioning or metabolic contributions (Takeshita & Kikuchi, 2020). The detection of Burkholderia sp. in D. citri is particularly noteworthy, as this genus encompasses species with diverse ecological roles, ranging from plant pathogens to insect symbionts. Certain Burkholderia sp. strains are known to aid insects in nutrient acquisition or detoxification (Kikuchi et al., 2012).

Bacillus sp. was also isolated from both Jahrom and Rudan Counties psyllid populations. This bacterium has been previously reported from ACP (Kolora et al., 2015). Beyond the ACP, Bacillus sp. has been reported in other insect species, including the kissing bug, Meccus pallidipennis (Jiménez et al., 2021), and the tobacco hornworm Manduca sexta (Van der Hoeven et al., 2008). Members of the Bacillus sp. genus are Gram-positive, rod-shaped, spore-forming bacteria that are widely distributed in soil, plant surfaces, and insect digestive systems, often establishing symbiotic or antagonistic relationships with their hosts. Certain Bacillus species (e.g., B. thuringiensis) are known to function as biological pesticides (Zhao et al., 2016; Guo et al., 2017). Given that some Bacillus strains can produce antimicrobial compounds, their presence in citrus psyllids may influence the insect's susceptibility to Candidatus Liberibacter asiaticus (CLas) or other gut pathogens. Enterococcus sp. was also identified in the psyllid populations collected from both Jahrom and Rudan. Enterococcus species are ubiquitous in nature, commonly found in water, soil, food products, and the gastrointestinal tracts of various hosts (Ran et al., 2015). While these bacteria are typically associated with vertebrate microbiomes, several studies have documented their presence in different insects (Vilanova et al., 2016; Yun et al., 2014; Li et al., 2020). Enterococci may play roles in food digestion and immune regulation (Johnston & Rolff, 2015). Staphylococcus sp. was the other bacterial genus exclusively cultured and isolated from the psyllid population collected from Jahrom. These bacteria are frequently reported as components of the normal surface or gut flora of insects (El Shazely et al., 2019; Oliveira et al., 2014), including Manduca sexta (Van der Hoeven et al., 2008), Brithys crini and Hyles euphorbiae (Vilanova et al., 2016).

Fig 5. Phylogenetic analysis of bacterial isolate Burkholderia sp. from D. citri gut. The evolutionary history was inferred using the Neighbor-Joining method based on the 16S rRNA gene sequence. The bootstrap consensus tree (from 1000 replicates) shows the position of the isolate (highlighted in blue) relative to closely related type strains. The scale bar indicates the number of base substitutions per site.

Staphylococci sp. are Gram-positive, coccoid bacteria, with certain species such as S. aureus being human pathogenic (Oliveira et al., 2014). In D. citri, their presence may result from environmental exposure or contact with plants. Although Staphylococcus sp. is not recognized as a primary insect pathogen, its detection could indicate secondary contamination or alterations in the insect microbiome under stressful conditions. The isolation of Bacillus, Burkholderia sp., Enterococcus sp., and Staphylococcus sp. from ACP carrying the HLB pathogen expands current knowledge about the microbial communities associated with citrus psyllids. The widespread occurrence of these bacteria across diverse insect orders suggests they may not form specialized associations with specific hosts. Instead, they likely confer general beneficial traits when symbiotic, or may represent transient environmental acquisitions. The results raise important questions about potential interactions between these bacteria and psyllid biology, including their possible influence on pathogen transmission dynamics and potential applications in biological control strategies. The presence of these bacterial genera, particularly those with known plant-growth-promoting or antimicrobial properties, warrants deeper exploration of their functional roles in the HLB pathosystem.

While this study successfully isolated and identified several culturable bacterial associates with D. citri, it is important to acknowledge the limitations inherent in the culture-dependent approach employed. Our methodology, while optimal for obtaining live isolates for downstream biotechnological applications, is inherently selective. The use of specific growth media and incubation conditions inevitably favors fast-growing bacteria and may overlook a significant portion of the microbial community, including unculturable, fastidious, or slow-growing symbionts. For instance, well-known primary and secondary symbionts of D. citri, such as CLasand Wolbachia, which are typically detected via molecular methods, were not recovered in our cultures, as reported in previous studies. Consequently, the findings presented here should not be interpreted as a complete census of the D. citri microbiome but rather as a targeted isolation of cultivable components with potential for manipulation. Further studies investigating the microbial composition of field-collected psyllids from different regions and seasons would help elucidate the influence of environmental factors. Finally, functional experiments, including the gnotobiotic rearing of psyllids with the isolated bacteria, are essential to unequivocally determine their roles in host fitness, immune response, and, most critically, their interaction with and impact on CLas acquisition and transmission.