Uncovering the presence of CVPD disease in citrus varieties of South Sulawesi, Indonesia: A molecular approach

Abstract

Background

The citrus vein phloem degeneration (CVPD) disease is one of important diseases that infects citrus plants and threatens global citrus production and quality due to its severe symptoms and rapid spread. In the 2000s, South Sulawesi Province as one of the citrus producers in Indonesia reported CVPD infection. To date, it is still uncertain as to whether the citrus production center has already been rid of the CVPD infection, keeping in mind the low prevalence of certified citrus saplings use and sub-optimal management of plantations by farmers.

Results

Field observation results revealed varied chlorosis symptoms from young to productive cultivation, which certainly makes it appealing to find out the presence of the causative bacterium, as it has yet to be known whether all the leaves with positive chlorosis symptoms carry the bacterium Candidatus Liberibacter asiaticus. Citrus saplings that appear healthy may carry CVPD pathogens as the incubation period of CVPD pathogens in the host plant spans three to five months. Thus, it is necessary to find the right, rapid way to detect the presence of CVPD pathogens in the citrus plant. The most effective method to use is PCR as the bacterium Candidatus L. asiaticus is non-culturable in vitro, but it is detectable using 16S rDNA. Sampling of leaves with CVPD symptoms was conducted purposively from eight varieties in five citrus cultivation locations, i.e., Pangkep, Sidrap, Bantaeng, Luwu Utara, and Kepulauan Selayar Regencies. DNA isolation was carried out following the Genomic DNA Kit (Geneaid) procedure, followed by detection using the specific pathogenic primer pair OI1 (5′ GCG CGT ATG CAA TAC GAG CGG C 3′) and OI2c (5′ GCC TCG CGA CTT CGC AAC CCA T 3′).

Conclusion

The PCR visualization result shows seven positive samples with DNA fragments measuring 1160 bp. The seven samples were samples of the Key lime, tangerine, Mandarin (cv. batu 55), and Mandarin (cv. selayar), each being derived from Sidrap, Luwu Utara, and Bantaeng. The average disease incidence rate was 66.56 %. Based on the field observation results, the insect vector Diaphorina citri was nowhere to be found in the five citrus cultivation locations in South Sulawesi.

1. Background

Citrus vein phloem degeneration (CVPD) is the primary threat to global citrus production and quality.1, 2 It is variably known as citrus greening, yellow shoot, leaf mottle (the Philippines), likubin or decline (Taiwan), citrus dieback (India), and blotchy-mottle or mottling disease (Africa), but its international name is huanglongbing (HLB) (China).³ In Indonesia, CVPD infected citrus crops when there was a report of severe damages to citrus cultivation at production centers.4, 5 HLB was first encountered in Indonesia in 1964 in coincidence with the naming of citrus vein phloem degeneration (CVPD) which caused growth disorder and even death to grafted citrus plants.⁴ The disease is caused by the Gram-negative non-culturable bacterium in the α-proteobacteria group, Candidatus Liberibacter spp.6, 7, 8 It can be transmitted through vegetative propagation material, whose spread is geographically caused by infected sapling transport, or through vector in its spread between plants in the same cultivation area.

Indonesia houses various local citrus species and varieties with nationally acknowledged superiority throughout the archipelago from Sabang to Merauke. The Indonesian Ministry of Agriculture has issued a decree on national agricultural estate locations and established national citrus development estates, one of which is South Sulawesi Province. However, some citrus development centers in South Sulawesi with their respective superior citrus varieties were reported to be CVPD-infected, i.e., Jeneponto, Sidrap, Bantaeng, and Luwu Utara Regencies.9, 10 To date, it is still uncertain as to whether the citrus production center has already been rid of the CVPD infection, keeping in mind the low prevalence of certified citrus saplings use¹¹ and sub-optimal management of plantations by farmers. The symptoms triggered by this disease infection resemble those caused by nutritional deficiencies. Field observation results revealed varied chlorosis symptoms from young to productive cultivation. The symptom variation in pattern from mild to severe chlorosis in citrus cultivation has led to an interest in finding out the presence of the causative bacterium as it has yet to be known whether all the leaves with positive chlorosis symptoms carry the bacterium Candidatus Liberibacter asiaticus. Citrus saplings that appear healthy may carry CVPD pathogens as the incubation period of CVPD pathogens in the host plant spans three to five months.¹² Thus, it is necessary to find the right, rapid way to detect the presence of CVPD pathogens in the citrus plant. To the present day, CVPD has yet to receive any significant resistance from the citrus species. There has been no effective therapy,⁷ and even the formulated methods of CVPD mitigation have yet to be well-established up until now. However, correct detection enables preventive measures.¹³

CVPD disease detection can be performed in numerous ways, including visual observation, chemical testing, shield budding and grafting, and molecular detection. However, the most effective method to use is the PCR one¹⁴ as the bacterium Candidatus L. asiaticus is non-culturable in vitro but can be detected with its 16S rDNA using specific primers.15, 16 The PCR method has high levels of accuracy and capacity, involves a small amount of DNA, and offers the best alternative for effective and efficient performance.17, 18, 19 This research detected the presence of the bacterium Candidatus L. asiaticus in citrus leaves with signs of chlorosis with the PCR method using specific primers to ensure that the signs point to the CVPD disease.

2. Methods

2.1. Plant sampling

Sampling of citrus leaves with CVPD symptoms was conducted purposively at citrus cultivation locations in Pangkep, Sidrap, Bantaeng, Luwu Utara, and Kepulauan Selayar Regencies (Fig. 1). Symptomatic leaves were collected from citrus plants assumed to be CVPD-infected. The symptomatic leaf samples were wrapped in plastic bags and coded by tree point and name of place of origin. The samples extracted were stored in an ice-gel-containing coolbox (Fig. 2).

Fig. 1.

CVPD disease survey locations in South Sulawesi. The red areas represent locations of sampling of leaves with CVPD symptoms. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

The research schematic stages.

2.2. Disease incidence

Disease incidence observation was conducted on citrus cultivation exhibiting CVPD disease symptoms over an expanse in each location. The observation data collected were tabulated and calculated to obtain a disease incidence rate. The disease incidence rate is given by the formula below20, 21:

$$\mathrm{D}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{I}\mathrm{n}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}(\%)=\frac{\mathrm{n}}{\mathrm{N}}\text{x 100}$$

• Where:

• P = Disease incidence

• n = Number of infected plants

• N = Total number of observed plants

2.3. DNA isolation

The DNA extraction of the citrus plants was conducted following the Genomic DNA Kit (Geneaid) protocol. A young leaf sample was weighed at 50–100 mg and added with 400 µl of GP1 buffer. It was then vortexed and incubated in a water bath at 60 °C for 30 min. The mix was turned upside down every 10 min. As much as 100 µl of GP2 buffer was added, followed by vortexing, incubation in ice for 10 min, and centrifugation at 10,000 g for 5 min. A filter column was set in a 2 ml tube. The supernatant was pipetted and transferred to the filter column and then centrifuged at 1,000 g for 1 min. The column was then discarded. The supernatant was added with 700 µl of GP3 buffer and quickly turned upside down. A GD column was set in a 2 ml tube, and all solution was pipetted into it before being centrifuged for 2 min. To the GD column 400 µl of W1 buffer was added, followed by centrifugation at 10,000 g for 1 min. The supernatant was then removed. The GD column was centrifuged for 3 min and then transferred to a 1.5 ml tube, added with 100 µl of pre-heated elution buffer/TE precisely at the center of the column for 5 min, and centrifuged at 10,000 g for 1 min. The GD column was then discarded. The solution derived was a DNA solution, which was later added with 3 µl of RNAse.

2.4. DNA amplification and electrophoresis

The isolated DNA of the symptomatic sample was used in the PCR reaction. The tube with materials for the PCR reaction contained 3 µl of target DNA, 1 µl of OI1 primer, 1 µl of OI2c primer, 6.25 µl of KAPA PCR Mix, and 3 µl of ddH₂O. The tube was inserted into a PCR machine. The 16 s rDNA fragment was amplified with the pair of CVPD pathogen-specific primers OI1 (5′ GCG CGT ATG CAA TAC GAG CGG C 3′) and OI2c (5′ GCC TCG CGA CTT CGC AAC CCA T 3′). The DNA amplified with the primers measured approximately 1160 bp.²² The PCR program utilized was a combined modification of23, 24, 25 method and²⁶ method. The DNA amplification consisted of initial denaturation at 92 °C for 30 s, followed by 40 cycles each at 92 °C for 60 s (denaturation), 55 °C for 30 s (annealing), and 72 °C for 90 s (extension), and a final cycle at 72 °C for 10 min.

Electrophoresis was carried out by first weighing agarose at 3.6 g, added with 180 ml of 1x TE buffer. The solution was heated in a microwave for 5 min and then added with 1.5 µl of red gel. The solution was poured into an agar cast to be coupled with a comb and then left to solidify. After the agar solidifies, the comb was removed. The agar was then inserted into a tank containing 1x TE buffer. The DNA sample that had gone through the PCR stage was inserted into each agar well. On the right side and left side of the well, a marker solution was added. Electrophoresis was performed at 120 v for 70 min. The agar was inserted into gel documentation for visualization. The electrophoresed PCR product was documented and analyzed through a DNA band measuring 1160 bp.

2.5. Data analysis

A descriptive data analysis was conducted with the indicator of the CVPD bacterium presence: if a DNA band (i.e., the 16S rDNA of Candidatus L. asiaticus) measuring 1160 bp was present, then it was declared that the bacterium was present or the sample was positive (+), and if no DNA band in that size was present, then the bacterium was said to be absent or the sample was declared negative (−).

3. Results

3.1. CVPD disease infection symptoms

The visual observation results showed varied chlorosis symptoms across five regencies in South Sulawesi. The morphologies of symptomatic leaves in Selayar Regency were as follows: a leaf was dark green in the lamina, with irregular yellowish green blotchy mottles, and dark green in both the midrib and veins (Fig. 3a); a leaf was dark green in the lamina, with irregular yellowish blotchy mottles, and yellowish green in both the midrib and veins (Fig. 3b); a leaf was dark green in the lamina, with irregular bright yellow spots, and dark green in both the midrib and veins (Fig. 3c); a leaf was yellowish green in the lamina and dark green in both the midrib and veins (Fig. 3d); and the last leaf was pale yellow in the lamina and yellowish green in both the midrib and veins (Fig. 3e).

Fig. 3.

The morphologies of symptomatic leaves in five regencies.

The morphologies of symptomatic leaves in Pangkep Regency were as follows: a leaf was dark green in the lamina, with yellowish green blotchy mottles, and dark green in both the midrib and veins (Fig. 3f); a leaf was dark green in the lamina, with bright yellow blotchy mottles in the area on the right side of the midrib, and dark green in both the midrib and veins (Fig. 3g); a leaf was dark green in the lamina, with irregular bright yellow spots, and dark green in both the midrib and veins (Fig. 3h); a leaf was predominantly with an uneven bright yellow color in the lamina, with a small part of it being in dark green, and the midrib and veins were both dark green (Fig. 3i); and the last leaf was dark yellow in the lamina and dark green in both the midrib and veins (Fig. 3j).

The morphologies of symptomatic leaves in Bantaeng Regency were as follows: a leaf were yellowish green in both the lamina and the midrib and dark green in the veins (Fig. 3k-l); a leaf was yellow in the lamina, yellowish green in the midrib, and dark green in the veins (Fig. 3m); a leaf was irregular yellow in the lamina and yellowish green in the midrib and veins, with some of the veins being identical with the lamina in the yellow color (Fig. 3n); and the last leaf was pale yellow in the lamina and yellowish green in the midrib and veins (Fig. 3o).

The morphologies of symptomatic leaves in Luwu Utara Regency were as follows: a leaf was dark green in the lamina, with irregular dark yellow blotchy mottles, and yellowish green in the midrib and veins (Fig. 3p); a leaf was yellowish green in the lamina and dark green in the midrib and veins (Fig. 3q); a leaf was bright yellow in the lamina, yellowish green in the midrib, and dark green in the veins (Fig. 3r); a leaf was irregular yellowish green in the lamina, yellowish green in the midrib, and pale green in the veins (Fig. 3s); and the last leaf was yellow in the lamina and midrib and pale green in the veins (Fig. 3t).

The morphologies of symptomatic leaves in Sidrap Regency were as follows: a leaf was dark green in the lamina, with yellow blotchy mottles, and dark green just like the lamina in the midrib and veins (Fig. 3u); a leaf was pale green in the lamina and midrib and dark green in the veins (Fig. 3v); a leaf was black-spotted irregular yellowish green in the lamina and yellowish green in the midrib and veins (Fig. 3w); a leaf was irregular yellowish green in the lamina and yellowish green in the midrib and veins (Fig. 3x); and the last leaf was irregular pale yellow in the lamina, midrib, and veins (Fig. 3y).

3.2. Disease incidence

The mean disease incidence in citrus plants that showed CVPD symptoms was 6.56 % (Table 1).

Table 1.

Mean disease incidence percentage.

Cultivation area in south sulawesi	Total number of trees	Number of observed trees	Number of infected trees	Disease incidence (%)
Pangkep Regency	224	72	48	66.67
Sidrap Regency	352	52	35	67.31
Bantaeng Regency	144	40	29	72.50
Luwu Utara Regency	530	107	74	69.16
Selayar Regency	108	35	20	57.14
Average				66.56

3.3. CVPD disease detection

The detection of CVPD disease requires specialized methods as the bacteria are difficult to identify. Table 2 present the methods used to detect CVPD disease.

Table 2.

Summary of CVPD detection methods.

No.	Detection method used	Name of citrus variety	Area	Citation
1.	Histological analysis	Mexican lemon Citrus aurantifolia	Colima, Mexico	27
2.	Loop-mediated isothermal amplification (LAMP)	Sweet orange Citrus sinensis	Sao Paulo, Brazil	28
3.	Specific 16S rDNA primers	Tangerine Citrus nobilis Lime Citrus aurantifolia	Bali, Indonesia	29
		Citrus nobilis, C. amblycarpa, C. reticulata, C. aurantifolia, C. limon and C. maxima	Bali, Indonesia	30
		Jeruk Siam Citrus nobilis var. microcarpa	Pontianak, West Kalimantan, Indonesia	31
		Mandarin Citrus reticulata	Assam, India	32
		Citrus maxima, Citrus jambhiri, Citrus macroptera, Citrus medica, and Citrus reticulata	Manipur, India	33
		Citrus aurantifolia, Citrus sinensis	Uttar Pradesh, India	34
		Citrus reticulata	Bhutan	35
		Mandarin Orange, Acid lime	Tamil Nadu, India	36
		Citrus maxima	Hainan, China	37
4.	Tissue print elution(TPE)-qPCR	Citrus sinensis Citrus reticulata	China	38
5.	HLB rapid detection kit	Citrus nobilis	Koto Tinggi, West Sumatera, Indonesia	39
6.	Multiplex qPCR	Navel orange tree Citrus sinensis	Davis, California	40
7.	Simple alkaline heat DNA lysis followed by loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB)	Citrus reticulata	Chiang Mai, Thailand	41
8.	Real-time recombinase polymerase amplification (RPA)	Citrus sinensis, Citrus hystrix	Brazil	42

The DNA isolated from symptomatic citrus plants was shown to contain DNA fragments in the agarose gel electrophoresis (Fig. 4). Total DNA isolation was performed to obtain a good-quality DNA template for subsequent PCR amplification processes. The DNA quality obtained was fairly good. DNA fragments appeared in all samples with average thickness nearly identical to one another in all columns and with clarity, except in columns 1, 9, and 14 where the fragments appeared thin. No smear was discovered in all sample columns.

Fig. 4.

Total DNA of symptomatic leaves. M = marker; 1–3 = mandarin (cv. selayar); 4–6 = pomelo; 7–9 = mandarin (cv. batu 55); 10–12 = tangerine; 13 = sweet santang orange; 14 = dekopon; 15–16 = Key lime; 17–18 = kaffir lime. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The PCR amplification results from the symptomatic citrus leaf DNA samples showed that 7 of the 70 samples contained DNA in parallel with the positive control, namely in columns 22 (Mandarin (cv. Batu 55)), 29 (Mandarin (cv. Batu 55)), 30 (Mandarin (cv. Batu 55)), 40 (Key lime), 51 (tangerine), 66 (Mandarin (cv. Selayar)), and 69 (Mandarin (cv. Selayar)) (Fig. 5). This marks that the 7 samples contained the pathogenic bacterium Candidatus L. asiaticus causative of the CVPD disease, as detected from the 16S rDNA primers. The positive samples were from four citrus cultivars, namely, Mandarin (cv. Batu 55), Key lime, tangerine, and Mandarin (cv. Selayar).

Fig. 5.

DNA visualization from the CVPD disease detection in symptomatic leaf samples in five citrus cultivation locations. M = marker; 1 = positive control; 2–7 = Mandarin (cv. Selayar); 8–13 = pomelo; 14–19 = dekopon; 20 = positive control; 21–30 = Mandarin (cv. Batu 55); 31–38 = kaffir lime; 39 = positive control; 40–49 = Key lime; 50–57 = positive control; 59–64 = sweet santang orange; 65–70 = Mandarin (cv. Selayar). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

The citrus plants observed were of eight varieties from five regencies, i.e., pomelo from Pangkep Regency, Key lime and kaffir lime from Sidrap Regency, Mandarin (cv. Batu 55) from Bantaeng Regency, tangerine, sweet santang orange, and dekopon from Luwu Utara Regency, and Mandarin (cv. Selayar) from Selayar Regency. In each regency, a district whose majority of residents engaged in citrus agribusiness and in which a citrus development center with the largest potential area and the highest number of citrus trees in the regency was selected. The citrus plant age in the five regencies ranged between 5 and 15 years. The farmers in the locations conducted citrus propagation on generative and vegetative bases. Seed-based generative propagation is typically designated for the sowing of rootstocks, which constituted an important factor in the provision of citrus rootstocks given that in Indonesia it still relies on sowing. Vegetative propagation through bud shielding and grafting requires scions and rootstocks.

The CVPD disease may vary in terms of symptoms, types, and modes of transmission, and the symptoms may resemble the symptoms of other diseases. Based on the observation in the five research locations, the symptoms varied from mild to severe across the citrus varieties and cultivation locations. The diagnoses of the patterns of CVPD symptom expressions may also vary for each variety. According to,⁹ there are four types of CVPD symptoms in citrus foliage, i.e., type I (mottling), type II (mild chlorosis with green veins), type III (severe chlorosis with green veins), and type IV (yellowing in the veins). However, there are other characteristics of infected citrus trees in cultivation: one yellow shoot or more, infected leaves generating unclearly bordered blotchy mottles, asymmetrical blotchy mottles, thickened leaves with enlarged veins, and possible Zn-deficiency-like symptom followed by leaf-fall and twig dieback.43, 44 As pointed out by,⁴⁴ CVPD symptoms include blotchy mottle leaf (BML), little leaf chlorosis (LLC), and nutritional deficiency, which typically occur simultaneously. The interaction between pathogenic invasion and plant defensive response facilitates typical leaf symptoms.⁴⁴

Yellow shoot and spots on the leaf are specific symptoms in CVPD-infected citrus plants45, 46 and manifestations of various nutritional disorders.⁴⁷ However, it is still unclear as to how nutritional contents and distribution change in response to CLas invasion as the development of the symptoms is yet to be characterized comprehensively. The observation of CVPD disease symptoms in the citrus cultivation areas in five regencies in South Sulawesi generally unveiled symptoms such as yellow shoot, yellow-green blotchy mottle leaf, and asymmetrical blotchy mottle patterns. The differences in symptoms might be attributed to differences in plant age, attack intensity, climatic condition, and the Candidatus L. asiaticum strain attacking the plants.⁴⁸ They might also be caused by influences from the environment, sapling origins, and variation in disorderly bacterial distribution in the plants.49, 50 Chlorosis symptoms occur due to reduced chlorophyll production, which causes the plant photosynthesis activity to be decreased and infected plants to exhibit necrosis and leaf-fall symptoms.51, 52, 53 There are a variety of interactions between nutrients and CVPD pathogens in citrus plants. The pathogens usually trigger nutritional disorders from starch accumulation, physically block the transport tissue for nutrient mobility, and transfer redistributed nutrients in the phloem⁵⁴. The plant nutrient balance becomes indirectly influenced after the CVPD pathogenic infection.⁴⁴

The research findings of⁴⁴ showed that the levels and distribution of various nutrients such as P, Mn, and B vary with the occurrence of symptoms and the subsequent development of the symptoms. The contents and distribution of other nutrients, such as K, Ca, Mg, and Zn, are affected by the development of symptoms and the presence of CVPD pathogens. Carbon metabolism disorder and Zn source imbalance in citrus plants are important determining factors in CVPD pathogenesis.55, 56 Previous studies have reported carbon compound accumulation and starch synthesis that substantially cause physiological disorders such as phloem dysfunction and chloroplast disintegration in CVPD-infected leaves.⁵⁷ The observation of disease incidence found that 57–72 % of the citrus plants exhibited CVPD disease symptoms (Table 1), with the average exceeding 50 % (i.e., 66 %). Up to 10 % of the citrus plants were CVPD-positive, as shown by the PCR detection results; 7 of 70 samples were positive, with the presence of 1160-bp-long DNA fragments.

Based on the method to detect CVPD disease, Table 2 shows the detection technique applied to each citrus variety sampled for identifying the presence of Candidatus bacteria. The detection of bacterial presence in the symptomatic citrus leaf samples in this study was carried out with the polymerase chain reaction (PCR) technique to amplify the 16S rDNA of Candidatus L. asiaticus. PCR is a sensitive, rapid, accurate detection technique. It uses the specific primer pair of 011 forward primer and 012c reverse primer to amplify the 16S rDNA of Candidatus L. asiaticus that measures approximately 1160 bp.17, 18, 19 The results of the PCR detection with the specific primer pair showed 1160-bp-long DNA fragments in 7 citrus leaf samples, which means that the samples were positive for Candidatus L. asiaticus infection. The samples were of the Key lime, tangerine, Mandarin (cv. Batu 55), and Mandarin (cv. Selayar) varieties from Sidrap, Malangke Barat, and Bantaeng. The other two regencies, Pangkep and Selayar, were negative as no specific 1160-bp-long DNA fragments were found. Previously, the CVPD disease was reported in several citrus production centers in South Sulawesi, including Sidrap, Bantaeng, and Luwu Utara Regencies (Asaad, 2001, 2006). The three regencies were with their respective mainstay citrus commodities. Sidrap Regency, particularly Pitu Riawa and Pitu Riase Districts, is a center for Key lime production. Bantaeng is a new production center; in 2011, it was appointed to be a basis for Mandarin (cv. Batu 55) production. Malangke and Malangke Barat Districts in Luwu Utara Regency had the tangerine as their signature commodity until the 1990s when they were still declared CVPD-free. However, in 1999 farmers discovered greening symptoms, which were assumed to be signs of CVPD. As of 2004, 75–100 % of citrus plants in a number of cultivation locations died. Finally, in 2002–2005 the Malangke tangerine was declared extinct. At the end of the year 2009, citrus planting at the demplot (a plot of land for fruit-planting) in Waeto Village, Malangke Barat District, used saplings certified by the Research Institute for Citrus and Sub-tropical Fruits (Balitjestro) of Batu, and the plants started to bear fruits in mid-2013. However, in 2015, 700 citrus plants in the demplot died from a flood that inundated the plants for over one month. The farmers in Luwu Utara Regency remained highly enthusiastic about the tangerine commodity as proven by the tangerine cultivation that started in 2017 by farmers in Pembuniang Village, Malangke Barat District, being able to yield 10 tons of citrus in a harvest, which happened twice a year.

Based on the PCR detection results, CVPD was identified in three locations, Sidrap, Malangke Barat, and Bantaeng. Most of the citrus leaf samples which visually exhibited typical CVPD symptoms turned out to show no signs of the presence of DNA fragments after PCR visualization result amplification, which means that the samples did not contain the pathogenic bacterium Candidatus L. asiaticum. This suggests that the presence of CVPD symptoms in citrus plants does not necessarily indicate that the plants are CVPD-infected. Transmission of the CVPD disease may occur through the insect vector Diaphorina citri Kuwayama (Homoptera: Psyllidae)⁵⁸ or through infected samplings that are propagated by grafting or bud shielding. Grafting and bud shielding are highly effective in transmitting the bacterium Candidatus L. asiaticus. It is challenging to monitor the mobility or traffic of planting materials from one area to another area, opening up the chance of CVPD infections. The samples from three locations were found to be positive for CVPD infection, which probably was caused by disease transmission during propagation by bud shielding/grafting, in which case the shoots were probably extracted from infected parent plants and carried from infected places,59, 60, 61 given that no vector insects were found in the field. This is supported by the finding of CVPD-positive Mandarin (cv. Selayar) in Bantaeng Regency based on PCR detection. The priority citrus plants developed in the regency, i.e., Mandarin (cv. Batu 55), were also detected as CVPD-positive based on PCR detection. Thus, there was a high likelihood that infection occurred during propagation by bud shielding/grafting, or it was feared that the saplings distributed by local farmers were already CVPD-infected. Similarly,³⁰ discovered that five of six citrus varieties in Taro Village, Gianyar, Bali, Indonesia were CVPD-infected. This location is a new citrus cultivation area which previously was reported to be CVPD-free. It is assumed that the CVPD infection of the citrus plants in this location originated from propagation in another region that had already been infected. In addition, in the five research locations, a number of drawbacks that might threaten the sustainability of the citrus agribusiness were found: persistent bad-quality sapling traffics took place between production centers, the distribution of propagation materials from disease-free parent trees all the way to farmers did not follow the national standard flow, there was a difficulty in accessing rootstocks and scions which led to procurement from other areas and overdependence on sapling producers, and the saplings procured were at times short of the standard. The CVPD control program has grown in complexity as it requires integrated implementation due to the complex interaction between host plants, pathogenic bacteria, insect vector behavior, and farmer habit or behavior in citrus cultivation.⁶²

5. Conclusion

The citrus plants in the five regencies under study exhibited varied chlorosis symptoms with a mean incidence rate of 66.56 %. However, the insect vector Diaphorina citri was nowhere to be found in the five citrus cultivation areas in South Sulawesi. PCR amplification detected 7 of 70 samples as positive for CVPD as marked by 1160-bp-long DNA bands. The 7 samples were of the varieties of Key lime, tangerine, Mandarin (cv. Batu 55), and Mandarin (cv. Selayar) from Sidrap, Luwu Utara, and Bantaeng.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The author declares no competing interests.

Funding

Not applicable.

Authors contribution

MT designed the research, conducted the experiments, analyzed the data, and writing-original draft. TK conceptualization, supervised experiments and interpreted the data, AN supervised, review and editing, ET supervised and analyzed the data.