Preliminary Quantification of DNA of Citrus Huanglongbin Pathogen in The Mekong Delta

Transcription

1 Preliminary Quantification of DNA of Citrus Huanglongbin Pathogen in The Mekong Delta Nguyen Thi Ngoc Truc, Le Thi Thu Hong and Nguyen Thanh Nhan Southern Fruit Research Institute, Longdinh, Chauthanh, Tiengiang, Vietnam Abstract We used DNA quatitation marker and spectrophotometric to quantity DNA of Liberibacter in citrus leaf and psyllid in infected citrus of the Mekong Delta. This experiment showed that DNA of Liberibacter asiaticus in psylla were significantly higher than that of leaves of various cultivars. Among the various citrus cultivars, Tieu mandarin had highest quantity of Liberibacter DNA. The older leave gave more DNA quantity, and the longer infested plants gave higher DNA tilter. Introduction Many research on citrus HLB in the Mekong Delta to compare the amount of the Liberibacter present in different samples. This experiment was done symptoms, plant ages studied and presented in this paper. Materials and Methods The leaf samples were collected from total of 14 citrus cultivars with the specific symptom. Nam roi pomelo, King mandarin, Ta mandarin, Soan orange, sweet orange, duong mandarin, tieu mandarin were collected with the like Zn-deficiency symptom. Sanh, Long pomelo were collected with the mottling symptom. Hanh, tau lemon, giay lemon, num lemon were collected with pale yellowing symptom (Hong et al, 2002). Chemicals and reagents. PCR primers used in detection and quantitation were 16 SA2Gorev5 (the specific primers for the plant and for the psyllid) The presence of L.asiaticus were detected in samples of infected Citrus and the psylla vector fed on infected plants. DNA marker: The 1 Kb DNA ladder (U.S Patent No. 4,403,036) is suitable for sizing linear doublestranded DNA fragments from 500 pb to 12 kb.the bands of the ladder each contain from 1 to 12 repeats of a 1018 pb DNA fragment. In addition to these 12 bands, the ladder contains vector DNA fragments that range from 75 to 1636 pb. 1

2 DNA concentration machine Samples of citrus were collected from Tien giang, Ben Tre, Dong Thap, Vinh Long and Can Tho province. PCR was used for HLB detection with some details as: - Total nucleic acid from midrib vein in citrus leaves, and in other parts was extracted using CTAB method according to Nakashima et al, A thermocycler with the following programs was used for DNA amplification: 35 cycles each at 92 0 C for 30s, 54 0 C for 30s, and 72 0 C for 60s. - Eight microlitres of amplified DNA were electroforesed in a 1.4 % agarose gel in Tris acetate EDTA buffer. DNA bands were visualized with ultraviolet light after staining in an ethidiumbromide solution (Hong et al, 2001, Hong et al,2002). DNA quality test method: The extracted DNA from leaves and psyllids was analyzed on a 0.9% agarose gel with TAE as the running buffer. Bands were visualized under UV light after staining with ethidium bromide (Lang, 2002). There are three methods to quantify DNA: using PCR based marker, spectrophotometric quantitation of DNA (Duong HHT, 1998; Lang NT, 2002), the third one is competitors DNA fragment. In this paper, the first and the second method were applied. The known DNA quantitation markers (DNA marker) are taken in PCR based marker method. The marker DNA was used with 3 concentrations: 0.5X, 1X, 2X (5,10, and 20 µl by the order of 0.5 µg/µl. And after staining DNA in agarose gels, DNA quantitation was measured based on the marker bands. Spectrophotometric measurements of nucleic acid solution are typically taken at wavelengths of 260 nm and 280 nm. The A 260 reading is used to determine the concentration of nucleic acid in solution. For a solution with an A 260 = 1.0, the approximation: 1 A 260 unit of dsdna = 50 µg/ ml. The ratio between measurements at 260 nm and 280 nm provides an indication of the purity of a nucleic acid. Dilution factor: 500/1000 µg/ µl DNA concentration (mg/ml)= (ABS 260- ABS 310) X 50 µg DNA/ OD unit X dilution factor Each variety was done 7 samples X 3 replications, then taking the average value Results and Discussion Among 293 samples extracted, there were 13 citrus varieties, 45 leaf samples and 24 psyllid samples. They were used directly to do PCR. After then, 72 samples had to be diluted 20 times, and 97 samples had to be diluted 30 times. 2

3 Table 1: List of the collected Citrus varieties at Mekong delta Seri.No Local name Sientific name Province 1. Buoi nam roi Citrus maxima Can tho, Vinh long 2. Hanh Citrus microcarpa Can tho, Ben tre 3. Cam sanh Citrus sinensis Vinh long, Can tho, Ben tre 4. Chanh tau Citrus limon Can tho, Ben tre 5. Quit ta Citrus reticulata Ben tre, Can tho 6. Cam soan Citrus sinensis Can tho, Ben tre 7. Cam mat Citrus sinensis Can tho, Tien giang 8. Sanh chua/ngot Hybrid Can tho, Ben tre 9. Quyt duong Citrus reticulata Tien giang, Ben tre 10. Quyt tieu Citrus reticulata Dong thap, Ben tre 11. Chanh giay Citrus limon Tien giang, Can tho 12. Chanh num Citrus limon Ben tre, Vinh long 13. Buoi long Citrus maxima Vinh long, Tien giang 14. Buoi duong Citrus maxima Ben tre, Vinh long P C R, M, Figure 1: Result of DNA quality test of 293 collected samples Quantitation of Liberibacter in different HLB infected Citrus varieties at MD. DNA quantitation of Liberibacter in Tieu mandarin was highest ( µg/µl ), while those of Tau lemon, hanh, and nam roi pomelo were lower (Figure 2, Table 2). Figure 2: The PCR result of the different HLB infected Citrus varieties at MD 3

4 Table 2: The DNA concentration of the Liberibacter in 14 collected Citrus varieties Seri. No Variety DNA concentration (µg/µl) 1 Buoi nam roi Hanh Cam sanh Chanh tau Quit ta Cam soan Cam mat Sanh chua/ngot Quyt duong Quyt tieu Chanh giay Chanh num Buoi long Buoi duong The quantitation of Liberibacter in the HLB infected psyllid Figure 3: The PCR result of the HLB infected psyllid at MD There were 24 HLB infected psyllid samples, all of them were PCR positive. And the DNA concentration average of 24 samples was µg/µl, the highest concentration, much more than the DNA concentration in the plants (from to µg/µl). This result was similar to the result of Y. Tian, His result showed that psyllids contain higher amount of Liberibacter per unit of DNA than plant hosts. Within the plant samples tested, periwinkle has higher titer than Citrus plant. That was why the bands of the psyllid samples could be much bigger than that the bands of the plant samples by PCR The quantitation of Liberibacter in the 7 types of HLB infected leaves According to Yoshihiro Ohtsu et al, 1998; Hong et al 2001, there are 7 types of symptom in the HLB infected trees: Mottling: pale, light yellowing with the dark green spots uneven located Yellow spots balanced between the mid vein (Manganese deficiency) Small leaves and green vein (Zinc deficiency) 4

5 Yellow leaf and yellow vein (phytophthora like) Vein swelling and vein corking leaf Light green leaf Complex, oldest leaf The oldest leaf had the highest DNA concentration (Table 3). The HLB infected leaves were collected from the same variety (sweet orange) but different ages (Table 3). The result showed that the quantity of DNA in the older leaves was higher than that of young leaves (Table 4). Table 3: The DNA concentration of the 7 kinds of symptom and in other parts of Citrus tree Seri.No Symptoms DNA concentration (µg/µl) 1 Mottling Yellow spots balanced between the mid vein Small leaves and green vein Yellow leaf and yellow vein Vein swelling and vein corking leaf Light green leaf Complex symptom, oldest leaf Heart of fruit Fruit stalk Trunk Table 4: The quantitation of Liberibacter in the 3 stage of plant age Age (years old) DNA concentration (µg/µl) > Conclusion This preliminary investigation showed that of different citrus cultivars, DNA quantitation of Liberibacter in Tieu mandarin was highest ( µg/µl ), while those of Tau lemon, hanh, and nam roi pomelo were lower Of different sampling parts and symptoms, DNA quantity of fruit heart, and complex -old leaf were highest. The host plant of any citrus cultivars contained lower titer of DNA of Liberibacter as compared to that of the Psylla. Literature Cited Le Thi Thu Hong, Nguyen Thi Ngoc Truc Investigation on some factors affected PCR result in Citrus Huanglongbin detection. Proceedings of the 2001 annual workshop of Jircas Mekong delta Project Nguyen Thi Lang DNA marker. Unpublished document. Nguyen Thi Lang Phuong phap can ban trong cong nghe sinh hoc. NXB Giao duc. 5

6 Nguyen Thi Ngoc Truc, Le Thi Thu Hong Investigation on the present status of Citrus Huanglongbin disease at Tan Phu Thanh village. Proceedings of the 2001 annual workshop of Jircas Mekong delta Project Ho Huynh Thuy Duong, Sinh hoc phan tu. NXB Giao Duc Y. Tian, S. Ke and C. Ke Polymerase Chain Reaction for detection and quantitation of Liberobacter asiaticum, the Bacterium Associated with Huanglongbin (Greening) of Citrus in China. Thirteenth IOCV conference, 1996-Procaryotes and Blight. Ohtsu. Y, Nakashima, K. Promintara, M and Tomiyasu, Y Typical symptoms of Citrus greening on mandarin trees in Nepal, supported by detection and characterization of ribosomal DNA of the causal organisms. Ann. Phytopathol. Soc.Jpn. Tóm lược Để đánh giá hàm lượng DNA của Liberibacter gây bệnh Vàng lá greening trong cây có múi và trong rầy chổng cánh, ở Đồng bằng sông cửu long, chúng tôi đã sử dụng DNA marker và máy định lượng DNA dựa vào sự hấp thu ánh sáng tử ngoại ở bước sóng 260 nm (OD 260 nm- optical Density 260 nm) của các mẫu đo cho phép xác định nồng độ nucleic acid trong mẫu. Kết quả cho thấy, hàm lượng DNA của Liberibacter asiaticus trong rầy cao hơn rất nhiều so với hàm lượng DNA trong cây. Trên các giống khác nhau của ĐBSCL thì quít tiều có hàm lượng DNA của vi khuẩn cao nhất, và triệu chứng lá già nhất thì có hàm lượng DNA cao nhất. Và hàm lượng DNA trên các cây bị nhiễm bệnh càng lâu năm thì càng cao. 6