Introduction

Citrus plants are fruit trees that are generally spread in temperate and tropical parts of the world and have been domesticated and farmed for many years1. Citrus is grown in many countries China, Brazil, India and Mexico are the leading citrus producing countries2. Though citrus cultivation is widespread across Pakistan, Punjab province has the most area dedicated to its production3. At present, Pakistan exports 94,806 metric tonnes of citrus fruits, including Kinnow and others, to other countries. These fruits are grown on 183.8 thousand hectares, resulting in a total yield of 1943.7 thousand tonnes4.

Numerous factors, particularly the presence of diseases caused by nematodes, bacteria, viruses, and fungal pathogens, affect citrus fruit production. Citrus gummosis, citrus canker, and citrus greening are common diseases affecting citrus fruits in Pakistan. Citrus canker is the most frequent disease in all citrus-growing regions globally, and it has a severe impact on plant development and fruit quality. Xanthomonas axonopodis pv. citri (Hasse) is the disease's causative agent5. Canker of citrus was initially detected in Pakistan's Punjab area, but the illness has now spread throughout the country. Unfortunately, the disease may harm any cultivar, particularly grapefruit, sweet citrus, limes, and orange rootstocks and their hybrids6.

The gram-negative bacterium Xanthomonas axonopodis pv. citri (Hasse), characterized by a solitary polar flagellum, is the causative agent of citrus canker. It can survive temperatures as high as 35–39 °C and grows aerobically in the 28–30 °C range7. Insect-induced wounds that provide new entry points for the virus and result in merging lesions have been linked to the surge in canker incidence in Brazil and other nations8. Physiological investigations shows that, citrus trees were significantly influenced by the levels of antioxidants and micronutrients, including boron (B), zinc (Zn), manganese (Mn), iron (Fe), copper (Cu), molybdenum (Mo), and chlorine (Cl)9.

Citrus canker can be diagnosed via visual inspection as well as laboratory techniques. Professionals with the necessary expertise may recognize the unique lesions on injured citrus trees. Furthermore, laboratory tests such as the Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) are used to confirm the presence of the Xanthomonas axonopodis pv. citri (Hasse) bacteria, allowing for exact disease diagnosis and appropriate care. Citrus canker causes a wide range of symptoms, including pustules and necrotic lesions with developing corky tissues, oily or moist margins, and a yellow halo. Defoliation, dieback, early fruit drop, and the production of defective fruit are all indicators of how serious the disease is. Fruit lesions have a substantial economic impact since they render the fruit unsuitable for the fresh market and drastically reduce the price of the fruit. Integrated management techniques for citrus canker include creating disease-free stock, using copper sprays, and measures to reduce pathogen spread via leaf miners or wind10.

Citrus is a crucial fruit for Pakistan's economy, but it has been facing challenges due to diseases caused by climate change. A study was conducted to understand the impact of pathogens on Citrus morphology, physiology, and other characteristics. One of the most concerning diseases is citrus canker; a highly contagious bacterial disease that threatens citrus crops in Sargodha, Pakistan, and globally. Understanding how citrus canker affects the morphological and biochemical traits of the fruit is essential for the citrus industry, but there is limited literature on this topic. Effective disease management strategies can be developed by analyzing changes in citrus varieties to create resistance and enhance control measures. This can help reduce the negative effects of the disease on fruit quality and market value.

Materials and methods

Sargodha, a city located in the heart of Punjab, Pakistan, is known for its prime geographical location at 32.0836° N latitude and 72.6711° E longitude. This city has earned the nickname, "California of Pakistan" due to its reputation for producing top quality citrus fruits. Despite facing extreme weather conditions characterized by scorching hot summers and chilling winters, Sargodha's economy thrives on a strong foundation of agriculture and a variety of industries. The city's rich agricultural heritage and diverse industrial sector contribute significantly to its overall growth and development.

Collection of samples

To collect samples, four Tehsils of District Sargodha (Silanwali, Bhalwal, Kotmomin, and Sargodha) were chosen. There were twenty sample locations from these selected tehisls. Five locations (locations A, B, C, D, and E) were selected at each tehsil in a certain manner. At every location, five healthy and five diseased plants (randomly selected) were designated for sample acquisition. The fruits were randomly collected from the Citrus plants. The investigation was carried out on citrus plants aged between 10 and 15 years. A total of twenty leaves (constituting one sample) and twenty citrus fruits (forming one sample) were obtained from each plant. Each individual plant was treated as one replication in the study. Fruits were picked after they had reached commercial ripeness. Leaves were gathered at the same time as fruits. The justification for selecting these samples is their vulnerability to direct influences on crop production and quality as a result of sustained damage.

Assessment of morphological attributes

Twenty fruit samples were collected to analyze the qualitative attributes at the peak of ripeness. A digital calibrated scale was used to determine the weight (g) of the fruits and juice. Fruit peel thickness (mm) was measured using a computerized Vernier calliper. Then rag weights were determined using a computerized calibrated scale. The leaf area was measured using a portable leaf area meter (Model LI 3100, LICOR, USA).

Evaluation of total chlorophyll content

Total chlorophyll content (mg/g)

The methodologies given by Arnon in 1949 were used to determine chlorophyll levels. Approximately 0.5 g of fresh plant leaves were cut and soaked in 80% acetone for 24 h. The extracts were then centrifuged at 10,000g for 5 min, and their absorbance was measured using a UV–visible spectrophotometer at wavelengths of 645, 663, and 480 nm.

Determination of biochemical attributes

Total titratable acidity

The titratable acidity was determined using the standard methods. 5 g of concentrated fruit juice were homogenized in 20 ml of distilled water before being filtered with what man No. 1 filter paper. As an indicator, 20 ml of the filtrate was titrated against 0.05 M NaOH with phenolphthalein addition. The equation for determining titratable acidity was:

$$ {\text{TAA}} = \frac{{{\text{MNaOH}} \times {\text{NaOH}} \times 0.09 \times 100}}{{\text{Juice Sample}}} $$

where: TA is the Titratable acidity, MNaOH is the Molarity of NaOH used, NaOH is the Amount (in) of NaOH used, 0.09 is the Equivalent weight of lactic acid.

Total dissolved solids (TSS)

A digital refractometer was used to quantify total soluble solid at 25 °C (ATAGO, Tokyo, Japan). The reading was taken as Brix after 3 ml of juice was poured onto the prism surface11.

TSS-acid ratio

The TSS-acid ratio was determined by dividing the TSS value by the acidity percentage.

$$ {\text{TSS: Acid Ratio}} = {\text{TSS }}\left( {{\text{Brix}}} \right)/{\text{Acidity}} $$

Determination of ascorbic acid (Vit. C) (mg/100 ml)

Ascorbic acid was tested by titrating the sample against 2–6, dichlorophenol, indophenol, and sodium carbonate, and the sample was prepared by centrifuging 5 ml with 100 ml of 4% oxalic acid. Five ml of the supernatant were collected, combined with ten ml of 4 percent oxalic acid, and then titrated against the dye. Equation (3) was used to determine the amount of vitamin C.

$$ {\text{Amount of ascorbic acid mg/}}100\;{\text{ml sample}} = 0{-}5\;{\text{mg/V}}1\;{\text{ml}} \times {\text{V}}2/5\;{\text{ml}} \times 100\;{\text{ml/V}}.{\text{ of sample}} \times 100 $$

Reducing power ability (RP)

The assessment of the extract's reducing power (RP) was conducted by using 1 ml of distinct extract concentrations ranging from 0.1 to 1.0 mg/ml in water was combined with 2.5 ml of 0.2 M phosphate buffer at pH 6.6 and 2.5 ml of 1% potassium ferricyanide. The resulting mixture was incubated at 50 °C for duration of 20 min. The reaction was then stopped by adding 2.5 ml of 10% trichloro-acetic acid to the mixture. The absorbance was measured at 700 nm after adding 2.5 ml of distilled water and 0.5 ml of 0.1 percent FeCl3. The higher the absorbance value, the greater the reducing power (RP).

Statistical analysis

The data were subjected to statistical analysis. To discover any statistically significant differences between the means and their interactions, an analysis of variance (ANOVA) was performed using a completely randomized design with two components. The Tukey HSD test for all-pairwise comparisons was used at a 5% significance level for variables that were significant, allowing for a thorough comparison of their means. Small letters in tables compare interaction means, whereas capital letters represent the overall mean. Means that share the same letters in a row or column are statistically non-significant (P > 0.05). Furthermore, Spearman’s correlation coefficient was also calculated. All statistical analyses were executed using SPSS version 20 software.

Institutional statement

The Institutional Ethics and guideline Committee of the University of Sargodha (Approval No. 177-Bot-S 127/2021 UOS) has allowed all the protocols used in this experiment. All the experimental methods of this study followed all the appropriate guidance and regulations.

Voucher specimens

Citrus specimens were identified by Dr. Abdul Ghani associate professor of Botany, University of Sargodha, Sargodha, Pakistan and Dr. Mansoor Hameed, Professor at University of Agriculture Faisalabad. Herbarium sheets (number S2023/3421-3467) are deposited at Department of Botany University of Sargodha, Sargodha Pakistan.

Ethics

I Rab Nawaz, Department of Botany, University of Sargodha, Pakistan, declare that most of the sampling sites were my own citrus gardens and some samples were collected from my friends and relatives lands, and the affidavit from my relatives is attached in “Related Files”. I also submit that in case of any issue I Rab Nawaz will be responsible.

Results

Fruit weight, peel thickness, juice weight, rag weight

Among healthy fruits, Bhalwal tehsil had the greatest fresh weight of 149.02, while Silanwali had the lowest at 145.65. Bhalwal had the highest weight of diseased fruits, whereas Silanwali had the lowest weight. The general mean among tehsils showed that healthy fruits weighed the most, whereas ill fruits weighed the least. Furthermore, Bhalwal had the highest mean values for both healthy (125.08) and sick (116.58) fruits, whereas Silanwali had the lowest values (Table 1). Healthy fruits' weight was consistently highest in Bhalwal and lowest in Silanwali, while similar trends were observed for diseased fruits (Fig. 1A). The overall, healthy fruits consistently displayed greater weight than diseased fruits across all tehsils.

Table 1 Morphological characters, Fruit weight and Peel thickness of Citrus reticulata from different Tehsils. Diseased × Tehsil interaction mean ± SE for Fruit weight (g) and Peel thickness (g).
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Figure 1
figure 1

(A–D): Diseased x Tehsil interaction for Fruit weight (g), Peel thickness (g), Juice weight and leaf area from four different tehsils.

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The interaction between healthy and diseased fruits highlighted that peel thickness was the highest among healthy fruits in Sargodha and among diseased fruits in Bhalwal (Table 1). The mean results showed that Bhalwal had the thickest peel, whereas Silanwali had the thinnest. Statistical analysis indicated significant connections for peel thickness between Bhalwal, Sargodha, and Silanwali, but no correlation were found in the other three tehsils. The graph shows that healthy fruits have consistently thicker peels across all tehsils than unhealthy ones. Similarly, Silanwali had somewhat thicker skins in healthy fruits than the other tehsils. In contrast, peel thickness for infected fruits was modest throughout all tehsils, with no significant variations between them. Notably, damaged fruits had more pronounced variability in peel thickness than healthy ones (Fig. 1B).

Variations in juice weight were detected among healthy fruits at the tehsil level, with Bhalwal having the greatest juice weight and Silanwali having the lowest. There were significant disparities in juice weight between healthy and diseased fruits, with healthy fruits weighing about twice as much as diseased fruits. The greatest juice weight for ill fruits was 39.49 g in Kotmomin tehsil, whereas for healthy fruits, it was 56.39 g in Sargodha tehsil (Table 2). Mean values for both healthy and diseased fruits across all tehsils demonstrated significant correlation. Graph visually depicted that juice weight for healthy fruits consistently exceeded that of diseased fruits in all tehsils. The greatest juice weight for healthy fruits was recorded in Kotmomin, while the lowest was in Silanwali. Moderately high juice weight was evident in Sargodha and Silanwali. In contrast, juice weight for diseased fruits displayed a moderate level across all tehsils, with no significant differences (Fig. 1C).

Table 2 Morphological characters, Juice weight and Rag weight of Citrus reticulata at different Tehsils. Diseased × Tehsil interaction mean ± SE for Juice weight (g) and Rag weight (g).
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Rag weight data revealed that among healthy fruits, Bhalwal had the greatest rag weight, which was somewhat less than the maximum value for ill fruits in Bhalwal. In the other three tehsils, healthy fruits had greater rag weights than ill fruits. Finally, the total weight of nutritious fruits at the tehsil level showed minor variability (Table 2). Bhalwal demonstrated the highest value of 39.59, while Silanwali showed the lowest at 33.82. For diseased plants, the highest rag weight was reported in Bhalwal and the lowest in Sargodha. Mean values for both healthy and diseased fruits were the highest in Bhalwal and lowest in Sargodha (Fig. 1D).

Leaf area, total chlorophyll content

The citrus fruit leaf area results in healthy plants showed that the highest measurement recorded in the Kotmomin tehsil and the lowest value found in the Silanwali tehsil (Fig. 2A). Similar marginal differences were seen in diseased plants, with the peak measurement being recorded in the Kotmomin tehsil (19.36 cm2) and the lower value being recorded in the Silanwali tehsil (17.16 cm2) (Table 3). The mean leaf area encompassing all tehsils among healthy plants exhibited a noteworthy correlation with the mean value among diseased plants.

Figure 2
figure 2

(A–D): Showing TSS-acid ratio, vitamin C, acidity (%), and RP (Reducing power) difference respectively of healthy and diseased fruits at studied tehsils.

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Table 3 Analysis of leaf area and total chlorophyll content for Citrus reticulata at different Tehsils. Diseased × Tehsil interaction mean ± SE for Leaf Area (cm2) and Total Chlorophyll content (mg/g).
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In the midst of healthy plants, Bhalwal tehsil had the highest concentration of total chlorophyll (4.81 mg/g), whereas Silanwali tehsil had the lowest (4.23 mg/g). In terms of diseased plants, Silanwali tehsil had the lowest concentration of total chlorophyll (3.15 mg/g), whereas Kotmomin tehsil had the highest concentration (4.21 mg/g) (Table 3). The composite statistics on total chlorophyll content are illustrated a clear distinction between healthy and unhealthy fruits within each tehsil. All tehsils consistently reverberated with the noticeably increased total chlorophyll content of healthy fruits compared to their ill counterparts (Fig. 2B). Vitamin C concentration was noted the highest at Kotmomin site in healthy fruits (Fig. 2C). TSS-Acid ratio was calculated the highest at Sargodha in healthy fruits (Fig. 2D).

Total soluble solids (TSS) and the TSS-acid ratio were highly significant in correlation to disease presence, whereas acidity percentage showed no significant association with disease. In contrast, acidity percentage displayed high significance when examining tehsil variation. In terms of the interaction between disease and tehsil, TSS showed a significant correlation, while the TSS acid ratio and acidity exhibited no significant associations.

Vitamin C, TSS-acid ratio

The highest TSS-acid ratio of 118.76 was detected in healthy fruits from Sargodha tehsil, whereas the minimum ratio of 100.79 was documented in Bhalwal tehsil (Table 4). Similar trends were evident among diseased fruits, where the highest ratio was noted in Sargodha tehsil, and the lowest was in Silanwali tehsil. The average ratio for healthy fruits was significantly higher at 109.41 compared to diseased fruits. Among all tehsils, Sargodha exhibited the highest mean, while Bhalwal displayed the lowest.

Table 4 Analysis of TSS and TSS-Acid ratio of Citrus reticulata from different Tehsils. Diseased × Tehsil interaction mean ± SE for TSS and TSS-Acid ratio.
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The graphical illustration underscores that the TSS-acid ratio in healthy fruits consistently exceeded that in diseased fruits across all tehsils within the Sargodha district (Fig. 2D). The highest vitamin C contents were determined in healthy fruits from Kotmomin tehsil, while the lowest in Silanwali tehsil. Among diseased fruits, the peak vitamin C value was observed in Sargodha tehsil, with the lowest value in Silanwali tehsil. The mean values for both healthy and diseased fruits across the four tehsils were the highest in Kotmomin and the lowest in Silanwali (Table 5).

Table 5 Analysis of TAA, Vitamin C and Reducing power (RP) of Citrus reticulata from different Tehsils. Diseased × Tehsil interaction mean ± SE for TTA, Vitamin C and Reducing power (RP).
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The graph shows that vitamin C levels in fruits consistently outperformed those in sick counterparts across all tehsils in the Sargodha district. Furthermore, in the healthy condition, the box plot showed greater vitamin C concentrations in Kotmomin tehsil, moderately in Bhalwal and Sargodha, and lower levels in Silanwali tehsil. The highest percentage was discovered in Sargodha tehsil, while the lowest was in Silanwali tehsil. Furthermore, changes in ratios were especially noticeable in Kotmomin Tehsil.

TSS, total titratable acidity (TTA), and reducing power ability (RP)

The most substantial concentration of TSS was found in healthy fruits from Silanwali tehsil, while the least concentration was documented in Kotmomin tehsil. Importantly, all interactions exhibited non-significant differences, diverging from the situation observed among diseased fruits. The highest TSS value was recorded in Sargodha tehsil and the lowest value in Kotmomin tehsil. The mean value across all four tehsils exhibited a notable difference with the recorded value for diseased fruits. Moreover, the mean value for healthy and diseased fruits was the highest in Bhalwal tehsil and the lowest in Kotmomin tehsil. The data indicates higher TSS values in healthy fruits compared to diseased fruits in each tehsil. The fluctuations in values were most prominent in Ksotmomin tehsil (Fig. 3A).

Figure 3
figure 3

(A–C): Comparisons of healthy and diseased Citrus fruits at various tehsil sites. HS (healthy samples), DS (disease samples), TSS (Total Dissolved Solids), RP (Reducing Power).

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Among healthy fruits, Bhalwal Tehsil had the highest acidity level, while Sargodha Tehsil had the lowest. Surprisingly, the similar pattern was observed in sick plants, with the highest acidity measured in Bhalwal tehsil and the lowest in Sargodha tehsil (Table 5). The mean value for acidity among healthy fruits across all tehsils demonstrated a non-significant correlation with the mean value among diseased plants. However, the mean acidity value for healthy and diseased plants within each tehsil exhibited a significant correlation, associating Bhalwal tehsil with Kotmomin, Sargodha, and Silanwali tehsils. Overall, acidity was observed to be higher in diseased fruits compared to healthy ones, with marked fluctuations evident in Kotmomin tehsil for both conditions (Fig. 3B).

The results show that healthy fruits have the highest reducing power (RP) capacity in Kotmomin tehsil and the lowest in Silanwali tehsil. In parallel, among diseased fruits, the highest reducing power (RP) ability was documented in Sargodha tehsil, while the lowest was in Silanwali tehsil. The recorded mean value for healthy fruits from all interactions underscored a significant difference compared to the mean value for diseased fruits. Furthermore, when considering the mean value for each tehsil, Sargodha tehsil displayed the highest value among all tehsils. Notably, the highest value was observed in Kotmomin tehsil under healthy conditions, followed by Bhalwal tehsil. Under disease conditions, the maximum value occurred in Sargodha tehsil, whereas the minimum was documented in Silanwali tehsil (Fig. 3C).

The graphical data connected to the different sub-sites at the tehsil level indicated that Chak 126 NB in Silanwali tehsil had the greatest juice weight (Fig. 4A). The sub-site with the highest total fruit weight was Lakhowana (Fig. 4B), whereas Shaheenabad had the highest reduction power (RP). The TTA high value was recorded in healthy fruits at Shaheenabad while the low concentration was noted at Chak 126 NB (Fig. 4C). Results predict that Chak 126 NB has the greatest chlorophyll concentration compared to the other sub-sites (Fig. 4D). Results for different sub-sites at tehsil level, shows that at Bhalwal tehsil the highest juice weight was recorded at Deowal sub-site (Fig. 5A). The overall highest fruit weight was recorded at Phulrwan sub-site (Fig. 5B) while the highest peel thickness was recorded at Phulrwan (Fig. 5C). The highest total titratable acidity (TTA) contents were recorded at Deowal compared to other sub-sites (Fig. 5D).

Figure 4
figure 4

(A–D): Comparisons of different attributes at Silanwali tehsil sites. HS (healthy samples), DS (disease samples), RP (Reducing Power), TSS (Total dissolved solids), Vit.C (Vitamin C), TTA (Total titrated acid).

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Figure 5
figure 5

(A–D): Comparisons of evaluated various attributes at Bhalwal tehsil at different sites. HS (healthy samples), DS (disease samples), RP (Reducing Power), TSS (Total dissolved solids), Vit.C (Vitamin C), TTA (Total titrated acid).

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The data related to the different sub-sites at tehsil level, the results revealed that at Kotmomin tehsil the highest juice weight was recorded at Mateela (Fig. 6A). The overall highest Vitamin C was recorded at Chak 11 SB sub-site (Fig. 6B) while the highest total titratable acidity (TTA) was recorded at Chak 11 SB (Fig. 6C). The highest total chlorophyll contents were recorded at Mareela compared to other sub-sites (Fig. 6D).

Figure 6
figure 6

(A–D): Assessment among Citrus, healthy and diseased fruit attributes at various studied sites of tehsil Kotmomin. HS (healthy samples), DS (disease samples), RP (Reducing Power), TSS (Total dissolved solids), Vit.C (Vitamin C), TTA (Total titrated acid).

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The graphic data related to the different sub-sites at tehsil level, the results revealed that at Sargodha tehsil the highest juice weight was recorded at Kolowal Ningiana (Fig. 7A). The overall highest fruit weight was recorded at Kolowal Ningiana sub-site (Fig. 7B) while the highest total chlorophyll was recorded at Behak Maken (Fig. 7C). The highest total titratable acidity (TTA) was recorded at Khoonan compared to other sub-sites (Fig. 7D).

Figure 7
figure 7

(A–D): Assessment among Citrus, healthy and diseased fruit attributes at various studied sites of tehsil Sargodha. HS (healthy samples), DS (disease samples), RP (Reducing Power), TSS (Total dissolved solids), Vit.C (Vitamin C), TTA (Total titrated acid).

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The graphical data for healthy and diseased citrus fruit and leaves samples represents that the highest concentrations of morphological and physiological parameters (fruit weight, peel thickness, rag weight, leaf area, juice weight, TSS, TAA, TSS acid-ratio, vitamin C (Vit. C), reducing power (RP) and total chlorophyll were recorded the highest at Silanwali tehsil (Fig. 8A,B) compared to other studied tehsils (Fig. 8C). The results predict that total titratable acidity (TTA) was recorded the highest in healthy fruits at all tehsils compared to all other morphological and physiological parameters (Fig. 9).

Figure 8
figure 8

(A–C): Comparisons of healthy and diseased attributes in studied tehsils and at District level respectively. HS (healthy samples), DS (disease samples), RP (Reducing Power), TSS (Total dissolved solids), Vit.C (Vitamin C). TTA (Total titrated acid).

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Figure 9
figure 9

Morphological and Physiological analysis comparison of healthy and diseased samples of Citrus from different tehsils. HS (healthy samples), DS (disease samples), F. weight (Fresh weight), P. thickness (Peel thickness), R. weight (Rag weight), L. area (Leaf area), J. Weight (Juice weight), TSS (Total dissolved solids), TTA (Total titratable acid), Vitamin C (Vit. C), RP (Reducing Power), T.chl.conc. (Total Chlorophyll contents).

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The analysis of variance of data

The data was subjected to variance analysis, which indicated substantial correlation between several parameters and illness state. Fruit weight, juice weight, and total chlorophyll all had significant associations with disease presence. When the interaction between illness and tehsil was taken into account, fruit weight remained very significant, but juice weight showed no significant correlation, and total chlorophyll remained quite significant. Moreover, peel thickness, rag weight, and leaf area demonstrated highly significant correlations with disease status. In the context of tehsil, peel thickness and leaf area maintained significant associations, while rag weight displayed a highly significant correlation. In terms of the interaction between disease and tehsil, peel thickness exhibited a significant connection, rag weight was highly significant, and leaf area showed no significant correlation (Table 6).

Table 6 Analysis of variance of Citrus reticulata from different Tehsils. Analysis of variance (mean squares) for fruit weight, juice weight, total chlorophyll, peel thickness, rag weight and leaf area.
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Vitamin C and reducing power (RP) were significantly associated with both disease status and tehsil variation. When considering the interaction between disease and tehsil, vitamin C displayed a significant correlation, while RP exhibited a highly significant association (Table 7).

Table 7 Analysis of variance of Citrus reticulata from different Tehsils. Analysis of variance (mean squares) for TSS, TSS-Acid ratio, Acidity, Vitamin C and reducing power (RP).
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Discussion

The maximum weight of healthy Citrus reticulata was observed as 164.3 g which aligns with our observations of non-affected fruits. This difference in fruit weight could potentially be linked to environmental factors like agricultural techniques and proper fertilizers application. It is worth mentioning that our findings diverge from the conclusions reached by12, suggesting that the citrus genetic diversity across different genotypes and cultivars may contribute to the observed variation in fruit weight. Results shows that infected fruits weighed less than healthy fruits, possibly due to the impact of infection.

The study revealed significant variations in peel thickness among Citrus reticulate fruits from different areas of Sargodha district, ranging from 3.31 to 4.58 mm. Specifically, fruits from Silanwali, Sargodha and Bhalwal exhibited distinct peel thickness ranges (3.33–3.77 mm, and 3.58–4.58 mm, respectively). These results suggest that agricultural practices, such as nutrient management and pruning, may influence peel thickness. However, a notable difference was observed in diseased affected fruits, which exhibited thinner peels compared to healthy fruits, potentially due to infection-related factors. The juice weight of Citrus reticulata ranged from 52.87 to 56.39 g. This investigation supports13, argument that temperature, together with wind speed, humidity, and rainfall, has a substantial influence on disease spread, particularly in the context of citrus canker disease. Fruits affected by disease exhibited smaller juice content compared to healthy fruits, potentially due to factors like leaf miner infestation and citrus canker attack, leading to reduced photosynthetic rates.

Healthy fruits indicated decreased rag weights, possibly influenced by variables such as time of sampling, environmental factors like humidity, soil composition, temperature, and changes in photoperiod. Strikingly, fiber weights were similar for both infected and uninfected fruits in our analysis. The onset of citrus canker led to a decrease in chlorophyll levels, likely contributing to a reduction in various morphological characteristics. This decrease in chlorophyll disrupts photosynthesis, leading to decreased carbohydrate production, reduced peel weight, rag weight, juice percentage, and leaf area.

The comparative analysis of chlorophyll content in healthy and diseased leaves across various regions, including Silanwali, Sargodha, Kotmomin, and Bhalwal, yielded similar results. However, damaged leaves had lower total chlorophyll concentrations than healthy leaves, which was consistent with14. This variation in chlorophyll content might be attributed to the impact of leaf miner infestation and citrus canker infections. Notably, our results demonstrated higher values, possibly due to genetic diversity and the selection of different leaf ages. In terms of leaf area, differences were noted in correlation to infection levels. Diseased leaves exhibited smaller leaf areas compared to non-diseased leaves. The highest leaf area was observed in Kotmomin, while the lowest was in Silanwali. Leaf miner attack and citrus canker infection likely contributed to the reduction in photosynthetic rates, aligning with Nawaz14.

The Citrus reticulata was examined for ascorbic acid (Vit. C), content results showed greater concentrations of ascorbic acid in healthy fruits. The elevation in acidity in fruits with citrus canker infections might result from increased CO2 concentration due to the disease. As CO2 levels rise, carbonation increases, leading to heightened acidity15, suggesting that chlorophyll destruction reduces CO2 consumption in citrus canker infected leaves, potentially explaining the increase in acidity and pH decrease. Differences were noted in the vitamin C levels when compared to the research conducted by16,17. This study revealed a vitamin C content of 30.60 mg/100 g, surpassing the previous findings. High ascorbic acid content in our samples suggests their potential for combating scurvy, aligning with18.

The findings revealed a notable disparity between healthy fruits and diseased fruits. Changes were seen in sick fruits, which had higher acidity and lower TSS levels. Diseased fruits had a higher TSS/acid ratio than healthy fruits, which might be explained by the influence of infection. Bacterial infection may triggers the breakdown of complex carbohydrates into simpler sugars, consequently leading to increased juice content and decreased total soluble solids in fruits infected with citrus canker19.

The onset of citrus canker infection causes a loss in leaf surface area, interrupting the photosynthetic machinery and resulting in a decrease in the quantity of the examined biochemical contents. This behavior highlights the pathogen's negative influence on the physiological processes of the citrus plant. As a significant challenge in citrus farming, citrus canker adversely affects leaves, fruit, and twigs, thus compromising overall plant health. The findings aligned with20, illustrating the comprehensive impact of citrus canker across various parameters. Evidently, citrus canker impairs tree production and vigor, leading to reduced fruit yields21. Fruit from canker-affected trees is projected to yield up to a 30% reduction in fresh market pack out rates. Reduced chlorophyll levels due to citrus canker disease hinder the process of photosynthesis, limiting carbohydrate production and potentially leading to a decline in biochemical concentrations. Furthermore, a deficiency in potassium triggered by citrus canker is responsible for a reduction in stomatal movement, consequently impacting the efficiency of photosynthesis.

Conclusion

The study highlights the significant impact of citrus canker on the nutritional value and general health of citrus fruits. There were regional differences in fruit quality, with healthy fruits always showing better nutritional qualities than infected ones. The results highlights the significant variations in fruit weight, peel thickness, juice weight, vitamin C content, chlorophyll content and other parameters between healthy and diseased fruits and leaves. Vitamin C concentration is higher in healthy fruits, which emphasizes the potential to help prevent nutritional shortages like scurvy. The study contributes to understanding of the complex relationships between citrus canker, fruit quality, and environmental factors, providing valuable insights for improving citrus production and disease management practices. These findings help to explore additional factors influencing disease susceptibility and fruit quality, ultimately aiming to develop more effective disease management strategies for the citrus industry.

Future strategies

Citrus canker continues to pose a substantial danger to citrus industry due to its historical effect, catastrophic symptoms, and ease of spread. Effective control techniques, including as attentive monitoring, fast discovery, and removal of contaminated plants, are critical to reducing economic losses. Ongoing development on resistant citrus cultivars and sustainable biological control approaches bodes well for a more resilient citrus business. International coordination and stringent biosecurity measures are critical for protecting citrus crops and livelihoods from this tenacious bacterial illness.

Suggestions

To prevent the infection from spreading, dispose of clipped waste away from the orchard. Use copper-based fungicides during the dormant season to prevent canker formation. Reduce mechanical damage to trees, as they create entrance opportunities for the canker infection. Implement tight quarantine procedures to prevent diseased plant material from entering the orchard. To reduce the chance of the disease spreading, train people on correct pruning practices.