Detection of Citrus Vein Phloem Degeneration (CVPD) disease and the quality of healthy fruits in nutrient deficiency of citrus

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ORIGINAL ARTICLE

Bali Medical Journal (Bali Med J) 2017, Volume 6, Number 3 (IBL Conference 2017 Special Issue): S117-S120 P-ISSN.2089-1180, E-ISSN.2302-2914

S117 Open access: www.balimedicaljournal.org and ojs.unud.ac.id/index.php/bmj

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Published by DiscoverSys

ABSTRACT

Background: Citrus plantation has the main disease called citrus greening disease or Huanglongbing or Citrus Vein Phloem Degeneration (CVPD) which in Indonesia is widely known as CVPD disease. The disease caused by Gram negative bacteria called Liberobacter asiaticus.

The disease symptom is very similar to plant deficiency of nutrients and can be confirmed by using polymerase chain reaction (PCR).

Objective: This study tried to find the nutrients deficiency, quality of the fruits, and to detect the presence of the pathogenic bacteria, L. asiaticus.

Methods: In the study of nutrients deficiency was done by using AAS technique, while the study of the healthy and consumable fruits was

assessed by using AAS and HPLC techniques. Detection of L. asiaticus we used the PCR method.

Results: The results of this study showed that all citrus plants showing the nutrients deficiency were infected by the pathogenic bacteria. Nutrient deficiency of citrus plants was found to be deficient in Fe, Mn, Mg, or Zn and the quality of the fruits, therefore, were physically small and lack of vitamin C and antioxidants.

Conclusion: The results indicated that there is a correlation between the citrus plant’s infected by L. asiaticus caused nutrients deficiency and lead to the quality of the fruits that we consume.

Keywords: Citrus plants, nutrient deficiency, Liberibacter asiaticus, PCR, AAS

Cite This Article: Wirawan, I.G.P., Simanjuntak, S., Sritamin, M., Wijaya, N. 2017. Detection of Citrus Vein Phloem Degeneration (CVPD) disease and the quality of healthy fruits in nutrient deficiency of citrus. Bali Medical Journal 3(3): S117-S120. DOI:10.15562/bmj.v3i3.757

INTRODUCTION

Citrus plant (Citrus spp.) is a horticultural crop with high economic value for farmers. One of the efforts made for the fulfillment of the needs of the citrus fruits carried out increased production of citrus, but still constrained of Citrus Vein Phloem Degeneration (CVPD) disease that caused by a gram negative Liberibacter asiaticus.^1,2 It is transmitted by an insect vector, Diaphorina citric, through the oculation in seedling from CVPD disease-infected citrus plants.^3,4,5 CVPD disease symptoms include chlorosis on some or all of the leaf lamina of bone, dark green leaves. Then, the leaves become thick and look like a particular nutrient deficiency symptoms.^1,2

Several observational studies had carried out in citrus leaves that have symptoms of CVPD in Bangli (Bali) and Wajo Regency (South Sulawesi).

It was found that there were variations in symptoms of chlorosis on each type of citrus plant. In those study, L. asiaticus was detected using Polymerase Chain Reaction (PCR) using a specific primer that amplified the 1160 bp 16S rDNA while nutrient deficiency was determined using Atomic Absorption Spectrophotometer (AAS).^1,2,3 This study tried to determine the unique symptom of the citrus plants affected by CVPD disease and then to elaborate the

relation of nutrient deficiency of the citrus plants.

The model of the molecular mechanism of nutrient deficiency was also formulated.

MATERIALS AND METHOD

This study conducted in several steps which begin with diseased leaves samples collection, isolation of total DNA, DNA amplification by PCR, electrophoresis of amplicon, analysis of nutrient deficiency and content of the fruits with AAS or HPLC. DNA isolation of Genomic DNA is conducted using a KAPA Mini Plant Kit^R with slight modification.

The leaf samples were chopped down and crushed using a mortar into a fine smooth powder. It then be preserved by stored it inside the Eppendorf tube (1.5 ml) and in the liquid nitrogen.

The PCR protocol included lysis suspension of 400 µ l GP1 lysis buffer and 5 µ of RNase. The master mix solution consists of PCR master mix 5 μl, prim- ers OI1 and OI2c 1 μl each, DNA template of 1 μl, and H2O 2 μl. The PCR was conducted in four stages namely pre-treatment at a temperature 92°C for 30 seconds to 1 cycle remedial, denaturation at a temperature 92°C for 60 seconds, annealing at a temperature 60°C for 30 seconds, and elon- gation at a temperature 72°C for 90 seconds. The cycle was repeated 40 times for optimal amplicon Graduate School of Agricultural

Biotechnology, Faculty of Agriculture, Udayana University

*Corresponding author: I Gede Putu Wirawan; Graduate School of Agricultural Biotechnology, Faculty of Agriculture, Udayana University.

PB. Sudirman street. Denpasar, 80232, Bali, Indonesia [email protected]

Received: 2017-07-17 Accepted: 2017-08-20 Published: 2017-08-24

Volume No.: 6 Issue: 3

First page No.: S117 P-ISSN.2089-1180 E-ISSN.2302-2914

Doi: http://dx.doi.org/10.15562/bmj.v6i3.757

ORIGINAL ARTICLE Detection of Citrus Vein Phloem Degeneration

(CVPD) disease and the quality of healthy fruits in nutrient deficiency of citrus

I Gede Putu Wirawan,^* Sheila Simanjuntak, Made Sritamin, Nyoman Wijaya

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S118 Published by DiscoverSys | Bali Med J 2017; 6(3): S117-S120 | doi: 10.15562/bmj.v6i3.757

yield. The amplicon was then visualized using 1%

agarose electrophoresis at 100 volts for 30 minutes to determine whether the gene had been amplified or not.^2,4,6,7

Nutrient analysis was carried out by using AAS with wet ashes method. Initially, HNO₃ and H₂SO₄ were applied to the leaves ashes to break the molecular bonds of complex compounds, where metals form a compound complex with the organic mate- rial. In this method, 1 gr of delicate citrus leaves were used and mixed with 10 ml concentrated HNO₃ and 3 ml 60 % solution of H₂SO₄. The mixture then heated on a hotplate at 100-120°C to acceler- ate the reaction until frothy discharges appear. The product then mixed with 5.0 ml solution nutrients 200 mg/L (standard solution) and solution of 2%

HNO₃, transferred to squash measure, and 2% of HNO₃ was added to provide a 100 ml final solution volume. The solution was homogenized and the absorbance was measured using the flame-AAS instrument at 422.7 nm wavelength.^8,9,10

RESULTS AND DISCUSSION

Various CVPD disease symptoms were observed in the citrus plants from Bangli (Bali) and Wajo (South Sulawesi). The citrus plants from Bangli that we observed was Citrus aurantifolia, Citrus nobilis, and lime, while in Wajo we observed Citrus maxima and Citrus aurantifolia. There are some variety of symptoms of CVPD in leaves as shown in Figure 1. Several citrus varieties showed some symptoms at the same time, starting from mild symptoms to the severe symptom. The chlorosis can be seen from the typi- cal change in lamina color into yellow or, in several cases, the yellowish color of the entire leaf surface.

Some of the leaves were still looked green but they were more rigid and thick (Figure 1 b, c, e and g).

Table 1 Result of analysis nutrients with the AAS methods Symptoms of CVPD

disease in citrus leaf

The Concentration Of Nutrient (%)

Zn Mn Mg Fe

1 3,30* 0,10* 131,1 53,33

2 0* 20,10 88,30 61,83

3 4,38* 0* 78,35 107,94

4 8,16* 0* 155,23 81,32

5 15,33* 0* 80,71 71,73

6 8,81* 0* 94,09 152,93

7 3,88* 0* 85,44 161,41

8 5,96* 0* 75,24 128,77

Description: (*)critical mineral elements, citrus leaf infected by bacteria L. asiaticus, (1) Citrus aurantifolia from Wajo, (2) Slayar from Bangli, (3) Citrus nobilis from Bangli, (4) Lime from Bangli, (5) Citrus maxima from Wajo, (6) Citrus nobilis from Bangli, (7) Citrus nobilis from Bangli, and (8) Citrus nobilis from Bangli

Figure 1 Various Symptoms of CVPD disease in citrus leaf, (a)Citrus aurantifolia from Wajo, (b) Citrus nobilis var. Slayar, (Bangli), (c,f,g,h) Citrus nobilis,var. Siem (Bangli) (d) Lime, (Bangli), and (e) Citrus maxima from Wajo. In this eight types of symptoms, each has a unique symptom due to different nutrients deficiency

Figure 2 Total DNA electrophoresis from leaves samples of citrus having the symptoms of CVPD (1) Citrus aurantifolia from Wajo, (2) Slayar from Bangli, (3) Citrus nobi- lis from Bangli, (4) Lime from Bangli, (5) Citrus maxima from Wajo, (6) Citrus nobilis from Bangli, (7) Citrus nobilis from Bangli, and (8) Citrus nobilis from Bangli

Figure 3 Amplicon visualization of bacteria L.asiaticus, (M) Marker DNA 1kb, (1) Citrus aurantifolia from Wajo, (2) Slayar from Bangli, (3) Citrus nobilis from Bangli, (4) Lime from Bangli, (5) Citrus maxima from Wajo, (6) Citrus nobi- lis from Bangli, (7) Citrus nobilis from Bangli, and (8) Citrus nobilis from Bangli

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Some citrus plants, such as C. nobilis var Siem (Bangli), had mild symptoms namely discrete yellow lamina on most of the leaf surface but the leaves were still had green-colored bones. Furthermore, the leaves become thick and stiff (Figure 1 f and h).

These symptoms were not observed in lemon and orange lemon of Wajo. Bangli cultivars tend to had a mild chlorosis of leaf lamina but still looks green, green leaf’s bones, and thickening and more stiff leaves (Figure 1 a and d). The concentration of the nutrient elements in diseased leaves of CVPD affected plants is presented in Table 1.

Nutrient analysis showed that all plant sample had Zn and Mn deficiency that could also play a role in the symptoms of CVPD. Meanwhile, the level of Mg and Fe were found to be normal in all leaves sample. To further investigate the etiology of the disease, we used DNA analysis to determine whether the plant had CVPD caused by L. asiati- cus or just a nutrient deficiency. Figure 2 shows the result of the electrophoresis of the total DNA samples.

Figure 3 shows the electrophoresis result of the amplicon of the pathogen’s gene from plants with CVPD. The result indicated that the samples from Wajo were infected by L. asiaticus despite their mild symptoms (Figure 3 lines 1 and 5).

Meanwhile, samples from Bangli also showed the same result albeit with severe symptoms (Figure 3 lines 2, 3, 4, 6, and 7). However, Siam lime leaves from Bangli (Figure 3 line 8) was found to be negative for L. asiaticus infection which indicates a merely nutrient deficiency. Combining this finding with the result of the nutrient analysis, it appears that there is a possible interconnection between the severity of CVPD related chlorosis and nutrient deficiency and the importance to differentiate them. The result of this analysis was presented in Table 2.

Based on the result of amplicon visualization, it appeared that not all of the samples were infected by L. asiaticus but all of them experienced a various degree of nutrients deficiency especially Zn and Mn. Sample 1 to 7 experienced both minerals deficiency and CVPD at the same times while sample 8 only had minerals deficiency without any sign of infection. Sample 1 to 7 experienced Zn and Mn deficiency and CVPD at the same time while sample 2 and 6 only experienced Zn deficiency in addition to CVPD.

The interrelation between L. asiaticus infection and mineral deficiency can be explained as follows. First, L. asiaticus entereds the phloem cell and multiply within it by using organic carbon and nitrogen as nutrient resources. The presence of the bacteria inside the cell triggers a reaction Table 2 Analysis of association between L. asiaticus infection and

nutrient deficiency in citrus plant Type of

symptoms Bacteria L.

asiaticus

Concentration of Nutrients in

Citrus leaf (%) Symptoms of CVPD disease in citrus leaf

Zn Mn Mg Fe

1 + * * *** **

2 + * ** *** **

3 + * * *** **

4 + * * *** **

5 + * * *** **

6 + * * *** ***

7 + * * *** ***

8 - * * *** **

Description: (+) positive infected bacteria L. asiaticus

(-) to be negative bacteria L. asiaticus

(*) low nutrient level

(**) medium nutrient level

(***) high nutrient level

Figure. 4 The quality of the fruits of Citrus plant infected by L. asiaticus. The fruit became small with reduced level of vitamin C

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that begins with the production of virulence protein (peroxide) that interfere the metabolism of phloem cell. The plant also has a specific protein that acts as the receptor for the virulentce protein and the complex could interact with membrane domain of channel protein that normally trans- ports inorganic ions into the phloem cell. The interaction decreases the function of the channel protein and interferes the transportation process of several inorganic ions especially Zn and Mn. In the long run, this process would ultimately affect the quality of the fruit. Most notably, the fruit becomes small and have significantly lower level of vitamin C (19.45 ppm per 100 gr compared to the normal level at 49.75 ppm per 100 gr).

Furthermore, the fruit also becomes considerably hard and not as juicy (Figure 4) as the normal fruit which could affect its economic value and would reduce the profit gained by the farmer.

CONCLUSION

In conclusion, L. asiaticus infection could be associated with nutrients deficiency. Zn and Mn contrib- ute to the majority of nutrients deficiency with lesser extent were addressed to Fe and Mg. Finally, the infection and its associated nutrient deficiency affect the quality of the fruits according to its size, consistency and vitamin C contents.

ACKNOWLEDGEMENT

This study is supported by the PNBP research fund of Udayana University, Bali, Indonesia.

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