SJA_42_4_1444-1451

Introduction

Huanglongbing (HLB), also known as citrus greening disease (CGD), is a phloem-limited bacterial disease caused by uncultivable gram-negative bacteria belonging to the genus Candidatus Liberibacter (α-Proteobacteria) (Mubeen et al., 2024). Once a plant is infected with HLB, there is no cure and most trees decline and die within a few years (Braswell et al., 2020; Vashisth and Kadyampakeni, 2020). HLB affects all the citrus varieties (Bové, 2006; Gottwald, 2010). The disease significantly reduces yield and exacerbates the economic value of citrus fruits, ultimately dispatching the entire tree (Bové and Garnier, 2003; Tipu et al., 2020). HLB is a graft-transmissible disease (GTD) in citrus which is systemic in nature (Batool et al., 2007; Bové, 2006). The different means of transmission of HLB are insect and vegetative propagation in nature (Lin and Lin, 1990). Vegetative propagation, including grafting is one of the most efficient ways not only for disease characterization but also transmission from one plant to another (Sajid et al., 2021). All the citrus species and their relatives have been found susceptible to HLB infection. Kinnow from Mandarin and Mosambi from sweet orange recorded as the most susceptible (Knapp et al., 2004). In an experiment, 30 citrus varieties were assessed against CLas to categorize the varieties in sensitive, moderately resistant and tolerant group (Folimonova et al., 2009). Biological characterization is included the host response after the grafting of HLB infected scions (Kamble et al., 2017). Timely detection of pathogen of CGD is effective for the formulation of management strategies (Fareed et al., 2026; Iftikhar et al., 2024). Recently advanced molecular detection methods for HLB have been reviewed by many researchers (Ding et al., 2020; Iftikhar et al., 2021). Molecular detection of HLB through PCR targeting 16S rDNA and β-operon regions is widely used for reliable diagnosis. Primer pairs OI1/OI2C and A2/J5 have been successfully employed to amplify fragments of approximately 1160 bp and 703 bp, respectively (Hocquellet et al., 1999; Razi et al., 2014; Yaqub et al., 2017). Molecular confirmation of HLB in Pakistan has been previously reported from different citrus-growing regions (Chohan et al., 2007; Sajid et al., 2021). In Pakistan, the incidence of CGD or HLB has been reported in several citrus-growing regions (Chohan et al., 2007; Razi et al., 2014; Sajid et al., 2021; Hassan et al., 2025; Shafique et al., 2024), however information on biological characterization through graft transmission is limited. Combining biological indexing with molecular detection helps confirm pathogen transmission and provides reliable diagnosis of infected citrus material.

Layyah district represents an expanding citrus-growing region in southern Punjab where structured biological indexing and molecular confirmation of HLB have not previously been reported. As citrus cultivation is increasing in this region, early detection and pathogen characterization are important for nursery certification and area-wide disease management. Therefore, this study extends a previously conducted survey on the spatial distribution of HLB in Layyah district (Bashir et al., 2023) by integrating biological indexing and molecular characterization.

Materials and Methods

Transmission of CGD through vegetative propagation

Five citrus species viz., Seedless Kinnow (Citrus nobilis x Citrus deliciosa), Mosambi (C. sinensis (L) Osb.), Blood orange (C. sinensis), sweet lime (Citrus limetta) and Salustiana sweet orange (C. sinensis) were maintained in insect-free environment as indictor plants. The infected scions from C. sinensis were used for the grafting on the maintained citrus plants of five varieties (Razi et al., 2014). 10 plants of each variety were grafted and three were controlled without grafting. A total of 50 grafted plants (10 per variety) and 15 control plants were maintained in the experiment. The grafted plants were maintained under insect-free conditions and observed for symptom development for 8–10 months (Figure 1). Transmission percentages among citrus varieties

were compared using analysis of variance (ANOVA) followed by least significant difference (LSD) test at p ≤ 0.05. All statistical analyses were performed using RStudio (version 2026.01.1+403).

Molecular detection of CGD

Leaf samples from symptomatic citrus plants were subjected to polymerase chain reaction (PCR) for molecular detection of HLB following the method described (Sajid et al., 2021). DNA was extracted using the GF-1 plant DNA extraction kit (Vivantis, Malaysia) according to the manufacturer’s protocol. PCR amplification was carried out in a thermal cycler following the protocol described by Sajid et al. (2021) with an initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min (Hocquellet et al., 1999; Jagoueix et al., 1996; Sajid et al., 2021).

Gel electrophoresis

Agarose gel electrophoresis was performed using 0.8% agarose prepared in 0.5× TBE buffer. GelRed stain was added during gel preparation. PCR products were loaded into wells and electrophoresed at 80 V for 45 min.

Results

The bud-woods from branches infected with citrus greening disease were collected and grafted on healthy citrus plants which were maintained and kept in insect free zone. The plants of five citrus varieties viz., Kinnow (Citrus nobilis × Citrus deliciosa), Mosambi (C. sinensis L. Osbeck), Blood orange (C. sinensis), Sweet lime (Citrus limetta) and Salustiana sweet orange (C. sinensis) were grafted by infected bud-woods. The results showed the successful transmission of citrus greening disease regardless of varieties by producing the characteristics symptoms after the 6-8 months of inoculation (Figure 2).

The control plants were symptomless showing the negative transmission of CGD. The means were compared using LSD test and the transmission rate was significantly higher in Mosambi (C. sinensis L. Osbeck) followed by Blood orange with transmission percentages of 51.2% and 46.2%, respectively (p ≤ 0.05). Transmission rates in Kinnow, Salustiana and Sweet lime were not significantly different, with transmission percentages of 30.8%, 20.5% and 23.1%, respectively (Figure 3A). The controls consisted of non-grafted plants while unsuccessful transmission referred to grafted plants that did not develop symptoms or died after grafting. There was no significant difference between the unsuccessful grafting and the control (Figure 3B). The presence of disease was also confirmed through the PCR assay of the transmitted plants.

Molecular detection of CGD

The samples from the symptomatic citrus plants of different varieties viz., Kinnow (mandarin), Mosambi (Sweet orange) and Feutrell’s Early (mandarin) were subjected to PCR to confirm the citrus greening bacterium in leaves samples of above citrus varieties (Figure 4A and Figure 4B). The DNA from samples of citrus varieties infected with CGD bacterium was amplified through PCR by two primer pairs at the size of ~1160 bp and ~703 bp.

Discussion

Our results regarding transmission through vegetative propagation were in accordance with the already published literature discussed here (Bashir et al., 2023, 2026). The HLB mottling symptoms were observed on citrus plants of seven-month post inoculation of infected bud-wood (Lin and Lin, 1990). They also reported the unsuccessful transmission in terms of survival of grafts. From total 58% of grafts were successful and 20% showed the typical greening symptoms. Moreover, about 40% of grafted plants showed the symptoms after the 9 months of post inoculation. Those plants were grafted from the asymptomatic branches of diseased plants. (Van Vuuren and Manicom, 2009) reported that the successful transmission of grafting for the greening bacterium varied from up to 50%. Mosambi showed the maximum disease transmission followed by Kinnow (Sajid et al., 2021). In another experiment it has been found that mosambi (C. sinensis) was highly susceptible against the CGD (Gupta et al., 2017). Our results were in accordance with (Kamble et al., 2017) where they found that Mosambi and Kinnow were the most susceptible followed by lemon and lime. (Kamble et al., 2017) reported the reaction of different indicator plants against the infection of CGD. They reported the successful transmission of CGD through grafting on the indicator plants. Like other literature, C. sinensis was also highly susceptible to HLB. Resistance of root stock against HLB was reported (Alves et al., 2021). They reported the resistance of acid lime rootstock against HLB. Tolerant rootstock can be an economical management practice (Bowman, 2020). The results from our study were confirmed and supported by previous studies (Bové and Garnier, 2003) (Razi et al., 2014). Therefore, the HLB is also transmitted through vegetative propagation among the local germplasms of Pakistan. The uncultivable nature of HLB bacterium in-vitro makes the conditions difficult to study the epidemiology and management of HLB (Ghosh et al., 2021) (Kokane et al., 2020). The size of PCR products was confirmed through already published literature. Our results were in accordance with (Chohan et al., 2007) obtained similar bands using the same primer pairs. Our results were also in accordance with (Sajid et al., 2021). They used the primers to amplify the PCR amplicons at 1160 bp and 703 bp size. The same results were also obtained (Yaqub et al., 2017; Zafarullah and Saleem, 2016).

They used the primer for the amplification of operon regions and 16s rDNA. In different studies HLB bacterium was confirmed through PCR in citrus groves of North-Eastern Indian region (Das et al., 2007), and repeatedly detected (Kokane et al., 2020). HLB bacterium was detected in different citrus species viz., Citrus macroptera, C. maxima, C. medica, C. reticulate and C. jambhiri (Alves et al., 2020). The primer pairs used in this study successfully amplified the expected fragments of the 16S rDNA and β-operon regions. (Zhang et al., 2020) detected the high-level DNA from the infected citrus samples from HLB through qPCR. CLas and CLaf were distinguished through A2/J5 primers. (Hocquellet et al., 1999) reported that CLas was amplified at 703 bp and CLaf at 669 bp. Molecular detection of bacterial pathogens including CGD pathogen is quick and reliable even at low intensity in the symptomatic and asymptomatic samples (Iftikhar et al., 2016). (Diksha et al., 2023) investigated the association between CLas and various citrus cultivars from diverse locations in Western and North-Eastern India by examining the genetic diversity and interrelationships among isolates through the characterization of different genetic markers.

Conclusions and Recommendations

Based on the results from our studies it was concluded that HLB was prevailing in the citrus orchards of newly established areas of southern Punjab (Layyah). The HLB was confirmed through vegetative propagation and a molecular assay (PCR) from the citrus plants of all three varieties during the survey. Early detection through biological indexing and molecular PCR assays is important for monitoring and management of HLB in emerging citrus-growing regions.

Acknowledgements

The first author thanks Department of Plant Pathology, College of Agriculture, University of Sargodha, Sargodha, Pakistan for helping the for the completion of the part of Ph.D. research.

Novelty Statement

This study provides the first integrated biological and molecular assessment of Huanglongbing (HLB) in the citrus-growing region of Layyah, Pakistan, a district for which no comprehensive HLB surveillance has previously been reported. These findings fill a geographic and diagnostic gap in Pakistan’s HLB literature and provide evidence to support region-specific management strategies.

Author’s Contribution

Sonum Bashir: Conducted the research trial.

Yasir Iftikhar: Conceptualized and supervised the trial.

Mustansar Mubeen: Formal analysis, software, writing the original draft, writing-review and editing.

Rida Fatima: Finalization.

Muhammad Asif Shabbir: Helped technical assistance for lab analysis.

Muhammad Ahmad Zeeshan: Co-supervised the trial.

Amjad Ali, Muhammed Tatar and Ahmet Buğra Aykaç: Statistical analysis and visualization.

Umbreen Shahzad: Validation.

Data Availability

Data sharing available on request.

Generative AI or AI assisted technology statement

The author(s) declare that no Gen AI was used in the creation of this manuscript.

Conflict of interest

The authors declare no conflict of interest.

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