Climate change mitigation and seasonal infestation patterns of citrus psyllid Diaphorina citri: implications for managing huanglongbing (HLB) disease in tangerine citrus

The citrus psyllid, Diaphorina citri, is a recognized vector of the devastating Huanglongbing (HLB) disease in citrus plants, posing a significant threat to global citrus production. As climate change continues to influence agroecological conditions, comprehending the population dynamics of this vector is imperative for effective disease management. This study delved into the relationship between climate variations and D. citri infestation patterns in tangerine citrus orchards from January to December 2018. Leveraging a dataset of 12 monthly observation sets, we comprehensively analyzed the abundance of the citrus psyllid population. Our investigation unveiled a clear association between climate variability and the incidence of citrus psyllid infestations and HLB occurrences. Importantly, our findings revealed that D. citri infestations were confined to the budding phase. Contrary to expectations, rainfall and temperature did not correlate positively with the development of the D. citri population or the prevalence of HLB disease in tangerine citrus. These results suggest other factors significantly affect HLB spread, impacting disease management. Further research is needed to identify and understand these factors, providing valuable insights for mitigating climate change’s impact on citrus cultivation.


Introduction
The infection of Huanglongbing (HLB) disease in citrus plants is heavily influenced by climatic conditions.Factors such as temperature, humidity, rainfall, and seasons play a crucial role in the spread and development of this disease.Optimal temperatures for the bacteria causing HLB, Candidatus Liberibacter asiaticus, and high humidity create ideal conditions for vector insects like psyllids.Unusual or extreme rainfall patterns can impact the movement of vector insects, while seasonal changes play a role in the insect's life cycle [1] The use of healthy citrus seeds is pivotal for successful citrus cultivation.The adoption of pure and healthy citrus seeds has been reported to enhance productivity and profitability for farmers in the tangerine cultivation regions of West Kalimantan [2].Conversely, using suboptimal and unhealthy seeds renders citrus plants vulnerable to disease infections and climatic stress, resulting in dwindling productivity necessitating eradication measures [3].Healthy citrus seeds must conform to specific standard criteria, including freedom from five systemic diseases, i.e., Huanglongbing (HLB), Citrus Tristeza Virus (CTV), Citrus Vein Enation Virus (CVEV), Citrus Exocortis Viroid (CEV), and Citrus Psorosis Virus (CPsV) [4].Currently, HLB is a widespread disease in Indonesia with global implications, transmitted via bacterial transfer (Candidatus Liberibacter asiaticus) by the citrus psyllid Diaphorina citri (Hemiptera: Psylidae) [4,5].The surrounding climate conditions profoundly influence insect biology.The phenomenon of climate change, characterized by fluctuations in temperature and rainfall patterns across various regions, impacts the population dynamics of insects, including D. citri, which is closely associated with citrus [6,7].The impact of climate change on D. citri includes alterations in its geographic distribution and infestation intensity on citrus plants.Changes in geographic distribution are attributed to temperature shifts, with rising air temperatures making previously unsuitable regions conducive for psyllid population growth [7,8].The heightened intensity of psyllid infestations on citrus is linked to shifts in vulnerable plant growth periods that coincide with psyllid infestation periods [9].Thus, climate change can affect various aspects of the relationship between the psyllid D. citri and HLB, including insect population development, geographic distribution, and disease dynamics.It presents an additional challenge in controlling HLB, necessitating adaptations in plant protection strategies against ongoing climate changes.
The development and distribution of citrus HLB disease also change concurrent with the dynamics of D. citri populations in tangerine orchards in Indonesia.This study aimed to analyze the relationship between rainfall levels and the infestation rate of psyllids and the spread of HLB disease.The results of this analysis are expected to serve as a foundation for developing HLB disease control techniques as a mitigation response to global climate change.

Location
This research was conducted on tangerine orange trees in Sambas Regency (latitude 1° 21' 45.0684" N, longitude 109° 16' 59.3508" E) from January to December 2018.The trees were two years old from planting or at the onset of fruiting.This study involved a survey and observations to evaluate the relationship between the population of D. citri, the occurrence of HLB disease, and temperature and rainfall conditions.Climatic data were obtained from the Mempawah climatology station, West Kalimantan, for 2018.

Population of D. citri on tangerine orchards
The D. citri population was observed on purposively selected citrus trees, specifically citrus orchards, where the presence of D. citri populations had been identified.Observations were conducted on ten selected sample trees characterized by active budding.The sample trees were selected diagonally and represented tangerine orange orchards.Population observations and distribution of the citrus psyllid were conducted by directly counting the number of adults on shoots in the four cardinal directions of the trees.Observations were performed every two weeks for 12 months, resulting in 24 data points.

Symptoms and incidence of HLB disease on the tangerine orchards
The HLB disease symptoms were observed by visually assessing plants displaying symptoms such as yellowing shoots and reduced somewhat pointed leaf size.The observation involved counting the number of symptomatic plants and the number of plants in the orchards.Disease severity on each symptomatic plant was assessed based on symptom scoring on leaves and canopy [5].The severity score for HLB disease was calculated using the formula described in [9].
For laboratory validation of HLB disease, 30 leaf samples showing HLB symptoms were collected from 10 different orchards.Laboratory validation was conducted using the PCR method at the Indonesian Citrus and Subtropical Crops Research Institute in Tlekung, Batu City.

Data Analysis
The data analysis involved the examination of climatological variables (rainfall and temperature) in conjunction with the population of D. citri, its distribution, and disease occurrence in the field, as well as validation.Descriptive statistical analysis was conducted according to the methodology outlined in [10].Linear regression analysis investigated the associations between temperature and rainfall variables and the D. citri population.These analyses were conducted using Microsoft 365 Excel.The results of linear regression analysis revealed a negative relationship between temperature and rainfall in the D. citri population.It indicates that both factors have a limited impact on the D. citri population, with a low R² value of 0.047 for temperature fluctuations, which is below 5%, and an even lower R² value of 0.0009 for rainfall, indicating an influence of less than 1% (Figure 2).This model suggests a weak correlation, as the R-value is far from 1, indicating that temperature and rainfall fluctuations do not positively correlate with the development of the D. citri population.It suggests the presence of other, more influential factors.

HLB Infection Symptoms Based on Visual Observations in 2-Year-Old Tangerine Citrus Trees
The distribution of visual disease symptoms corresponded with characteristic signs of uneven yellowing and reduced leaf size (Fig. 3).Symptoms on fruits included asymmetric appearances upon splitting and brown-colored abortive seeds.The distribution of visual symptoms of plant diseases appears closely related to distinctive features such as uneven yellowing and reduction in leaf size.This correlation indicates a potential connection between specific disease infestations and identifiable characteristics arising from uneven yellowing and leaf size reduction.Understanding these facts can provide profound insights into the nature and development of plant diseases, enabling the implementation of more targeted approaches in diagnosis and control strategies.

PCR Validation of visual symptoms of HLB
The validation of visually observed HLB symptoms was conducted through a molecular examination using a method developed by Himawan al. [10], Liu Y et al. [11], Sankaran et al. [12]Martinelli et al. [13].Laboratory analysis using PCR yielded results indicating that of the 30 samples collected from 10 orchards displaying HLB symptoms (3 samples per orchard), 4 of the sample trees, or 13.3% indicated positive for HLB infection (

Discussion
The population fluctuation of D. citri in tangerine citrus orchards correlates with citrus shoots (Figure 1).The highest population occurs in March, coinciding with the peak shoot formation period in citrus trees or during budding.In March 2018, the peak budding phase reached 46% per tree, with shoots aged 1-15 days.This shoot age provided an ideal medium for D. citri to oviposit.Female adults lay eggs on young shoots starting at 1-35 days; after 36 days, no adults were found laying eggs, and the highest nymph development was found in shoots aged 1-12 days [14].The use of insecticides by farmers is done once a week and depends on the availability of pest attack conditions.Biotic and abiotic factors are essential in influencing the development of this insect population in the natural environment.Biotic factors, such as the budding phase of the plants and the presence of natural enemies, are closely linked to the population of this insect.A sustained budding pattern in citrus trees supports the development of D. citri from the egg stage to adulthood because this insect exclusively lays eggs on young shoots [15,16].Furthermore, with an ample supply of shoots, D. citri can produce up to 9 generations within a year [17] Abiotic factors, such as rainfall, significantly impact the biological activities of insects, such as pests and natural enemies in citrus farming ecosystems [9,18,19].Low rainfall, around 35 mm per month, did not significantly affect the D. citri population, particularly in the egg stage, which is deposited on plant shoots.However, when rainfall exceeds 100 mm per month, it is observed that the psyllids population decreases (Figure 1).It suggests that although D. citri could lay many eggs if accompanied by high rainfall, the eggs laid on shoots will fall, reducing the percentage of successfully developing nymphs.
With an average daily temperature of approximately 29.6°C at that time, these conditions were generally considered to support the development of the D. citri population.Studies by Aidoo et al. [9], Murugan [18] and Guessab et al. [20]showed a positive correlation between weather parameters and D. citri population fluctuations.It affirms that weather factors played a crucial role in the population fluctuation of D. citri.In this study, temperature and humidity did not strongly influence the development of D. citri population in tangerine citrus.Instead, shoot availability was suspected to have a strong connection with psyllid population fluctuations.Therefore, further research is needed to evaluate other factors to understand this insect's ecology and population dynamics comprehensively.Other influential factors in the fluctuation of the D. citri population that need consideration include biotic factors related to the role of natural enemies, such as parasitoids and predators of the psyllids.
The HLB disease incidence validation results showed that only 13% of plants displaying HLB symptoms were confirmed molecularly.From this data, it can be concluded that the observed visual symptoms, such as HLB disease symptoms, are predominantly caused by factors other than the HLB pathogen (87%).HLB-infected citrus trees exhibit abnormal growth, characterized by uneven yellowing of leaves, similar to nutrient deficiency symptoms, with shoots growing upward and smaller leaf sizes [21].Previous research has confirmed that citrus trees in the field experience growth and developmental hindrances due to HLB infection [15,22].This study revealed that D. citri associated with tangerine plants did not conclusively identify as a vector of HLB based on the evaluation of the relationship between the presence of D. citri, HLB incidence in the field, and HLB disease validation by its causal pathogen on a molecular level.However, further research is needed to validate the role of D. citri in the spread of HLB.
The impact of Huanglongbing (HLB) on the leaf size of citrus plants has been the subject of extensive research.HLB, caused by the bacterium Candidatus Liberibacter asiaticus (CLas), has been associated with various physiological and biochemical changes in citrus plants.Studies have shown that HLB-affected citrus trees exhibit symptoms such as root loss, branch die-back, yellow shoots, and blotchy mottle chlorotic patterns on leaves [23].These symptoms are indicative of the detrimental effects of HLB on citrus plants.Additionally, the asymmetrical pattern of blotchy yellowing or mottling on leaves is a typical symptom of HLB in citrus [24].

Conclusion
In summary, our study revealed that neither rainfall nor temperature showed a straightforward or positive correlation with the development of the D. citri population or the incidence of HLB disease in tangerine citrus.It suggests that these environmental variables alone do not drive the dynamics of this insect or the disease.Furthermore, our findings indicate that D. citri was not the exclusive agent responsible for the spread of HLB, implying the involvement of other contributing factors.Further investigations are warranted to identify and understand these additional factors, which could play a more substantial role in disseminating the disease to address the challenges posed by climate change and its potential impact on the proliferation of HLB.These insights are crucial for developing effective strategies to mitigate the consequences of climate change on tangerine citrus cultivation.

3 .
IOP Publishing doi:10.1088/1755-1315/1346/1/0120033 Results and Discussion 3.1 Result3.1.1Population of Diaphorina citri on Tangerine Citrus TreesThe population of D. citri exhibited fluctuations throughout the 12-month observation period.Population fluctuations were closely associated with the age and presence of shoots.The highest population was observed in March, with numbers reaching up to 17 individuals per shoot, but gradually decreased to only one individual per shoot in December.The decline in the D. citri population was attributed to the aging of shoots, which became less suitable for psyllid development (Figure1).

Figure 3 .
Figure 3. HLB was infected on Tangerine orange trees, showing minor and deficiency-like symptomsThe intensity of HLB incidence based on visual symptoms ranged from 0% to 44% in each sample orchard.The highest intensity was observed in the Tekarang sub-district, while the lowest was in the Galing and Teluk Keramat sub-districts.

Table 1 .
Climate variables in Sambas district in 2018

Table 2 .
Results of PCR tests on 30 shoot samples showed HLB disease symptoms on tangerine citrus trees aged two years in Sambas Regency in 2018.