Preliminary investigation on the HLB natural infection in citrus and citrus relative species in Mekong Delta, Vietnam

Le Thi Thu Hong, Nguyen Thi Ngoc Truc, Nguyen Thanh Nhan

Southern Fruit Research Institute (SOFRI)

Toïm tàõt

Bãûnh Huanglong bin laì bãûnh dëch nghiãm troüng trãn cáy coï muïi åí Viãût Nam. Nhàòm tçm hiãøu tênh chäúng chëu/ khaïng cuía mäüt säú loaìi thæûc váût hoü haìng cáy coï muïi, âäúi våïi vi khuáøn gáy bãûnh, chuïng täi âaî khaío saït trong tæû nhiãn 83 máùu cáy vaì giaïm âënh laûi bàòng PCR. Kãút quaí bæåïc âáöu cho tháúy táút caí caïc máùu nguyãût quåïi, kim quyït, cáön thàng duì coï triãûu chæïng hay khäng âãöu cho kãút quaí ám tênh qua PCR. Mäüt säú doìng vä tênh cáy coï muïi khaïc nhæ saính chua, saính ngoüt, bæåíi âoí, cam sen, hanh, tàõc daìi, chanh taìu cuîng coï kãút quaí tæång tæû. Viãûc âaïnh giaï âang âæåüc tiãúp tuûc tiãún haình trong nhaì læåïi bàòng láy nhiãùm nhán taûo vaì âaïnh giaï sinh hoüc vaì taûi phoìng thê nghiãûm Viãûn Nghiãn Cæïu Cáy Coï Muïi åí Nháût (NIFTS) bàòng phæång phaïp phán tæí.

1. Introduction Citrus HLB disease causes severe damage to citrus production in Vietnam. All citrus species were infested, Vietnam native such as, Citrus sinensis, Citrus reticulata, Citrus nobilis, Citrus microcarpa, Citrus maxima and Japan native such as Citrus satuma, Citrus unshu, Citrus tankan, Citrus kabuchii, Citrus depressa (Shinji Kawano, 2000) by natural observation. The pathogen has many strains and carried by the vectors Trioza erytreae or Diaphorina citri. Many citrus and citrus relatives were recorded as host such as Murraya paniculata, Citrus aurantifolia, Citrus lemon, Citrus sinensis, Citrus media, Citrus nobilis, Citrus reticulata, Citrus deliciosa, Microcitrus australisiaca, Citrus paradis. The others such as Citrus hystrix, Citrus grandis, Triphasia trifoliata, Fortunella sp., Poncirus trifoliata, Murraya koenigii, Toddalia asiatica, Vepros lanceolata. Oviposition and larvae development of the psylid could be seen on the above species (B.Aubert, 1987). This investigation on the HLB natural infection of some plant species were done with field observation and PCR test in order to preliminary evaluate some plant source of tolerance/ resistance for further study.

2. Materials and Methods Samples of citrus and relative citrus species were collected from Tien giang (TG), Ben Tre (BT), Dong Thap (DT), Vinh Long (VL) and Can Tho (CT) province. PCR was used for HLB detection with some details as: - Total nucleic acid from midrib vein in citrus leaves was extracted using CTAB method according to Nakashima et al, 1996.

129 - Primer: A2J5 (from INRA, French) or OI1, OI2C (From Jircas Japan), originally designed by Jagoueix et al, 1994 for amplification of 16 S rDNA region of HLB pathogen were used. - A thermocycler with the following programs was used for DNA amplification: 35 cycles each at 92 0C for 30s, 540C for 30s, and 720C for 60s for primer OI1, OI2C; 35 cycles each at 94 0C for 30s, 540C for 30s, and 720C for 60s for primer OI1, OI2C; - Eight microlitres of amplified DNA were electroforesed in a 0,8- 1% agarose gel in Tris acetate EDTA buffer. DNA bands were visualized with ultraviolet light after staining in an ethidiumbromide solution.

3. Results and Discussion

Table 1: List of plant species collected for HLB observation Sr./ No Scientific name Vietnamese name Location (province name) 1 Citrus sinensis Cam mat Can Tho, Ben Tre, Tien Giang, Vinh Long 2 Citrus sinensis. Cam sanh Can Tho, Ben Tre, Tien Giang, Vinh Long 3 Citrus sinensis Cam sen Can Tho 4 Citrus aurantium Cam dang Can Tho 5 Citrus sinensis Cam navel Can Tho 6 Citrus sinensis Cam california Can Tho 7 Citrus sinensis Cam soan Can Tho, Ben Tre 8 Citrus maxima Buoi 5 roi Can Tho, Ben Tre, Tien Giang, Vinh Long 9 Citrus maxima Buoi da xanh Can Tho, Ben Tre, Tien Giang, Vinh Long 10 Citrus maxima Buoi do Can Tho, Ben Tre 11 Citrus maxima Buoi banh xe Can Tho, Vinh Long, Ben Tre 12 Citrus Chanh vo ngot Can Tho microacrumen 13 Citrus limon Chanh tau Can Tre, Ben Tre, Tien Giang, Vinh Long 14 Citrus limon Chanh tuong Can Tho, Ben Tre, Tien Giang, Vinh Long 15 Citrus sicilian Chanh khong hat Can Tho, Ben Tre, Tien Giang, Vinh Long 16 Citrus limon Chanh giay Can Tho, Ben Tre, Tien Giang, Vinh Long 17 Citrus microcarpa Hanh Can Tho, Ben Tre, Tien Giang, Vinh Long 18 Wild species Tac dai Can Tho 19 Hybrid Sanh ngot Can Tho 20 Hybrid Sanh chua Can Tho 21 Citrus reticulata Quit tieu Can Tho, Ben Tre, Tien Giang, Vinh Long 22 Citrus reticulata Quit duong Can Tho, Ben Tre, Tien Giang, Vinh Long 23 Citrus reticulata Quit ta Can Tho, Ben Tre, Tien Giang 24 Feronia Can thang Can Tho, Ben Tre, Tien Giang, Vinh Long elephantumcorrea 25 Triphasia trifoliata. Kim quit Can Tho, Ben Tre, Tien Giang, Vinh Long 26 Murraya paniculata Nguyet quoi Can Tho, Ben Tho, Tien Giang, Vinh Long

130 Total of 26 plant species were collected from Can Tho, Ben Tre, Tien Giang provinces with 83 samples. The collection was done on the HLB severe infection areas. Samples of symptom less as well as visible symptoms plants were collected.

Table 2: Evaluation of natural infection of HLB of collected samples by PCR Sr.No Code name Visible symptom PCR reaction 1 Mp-CT 1 Mottling - 2 Fe- CT 2 No symptom - 3 Or-CT 3 No symptom - 4 Mp-CT 4 Zn deficiency - 5 Or-CT 5 No symptom - 6 Sch-CT 6 Mottling + 7 Mp-CT7 Yellow - 8 Mp-CT 8 Yellow - 9 Or-CT 9 Mottling - 10 Mand-CT 10 Nosymptom - 11 Mp-CT 11 Yellow - 12 Sng-CT12 No symptom - 13 Mp-CT13 Yellow - 14 Mp-CT14 Yellow - 15 Sch-CT 15 Symptom + 16 Ltau-CT16 Yellow - 17 Sch-CT17 No symptom + 18 Mp-CT18 Yellow - 19 Mp-CT19 Yellow - 20 Mp-CT 20 Yellow - 21 Mp-CT 21 Yellow - 22 Mp-CT 22 Yellow - 23 Mp-CT 23 Yellow - 24 Ru-CT 24 - 25 Orsen-CT 25 No symptom - 26 Pum-CT 26 Mottling - 27 Pum-CT27 Nosymptom + 28 Ltau-CT 28 Yellow - 29 Pum-CT 29 No symptom - 30 Mp-CT 30 Zn deficiency - 31 Mp-CT 31 Zn deficiency - 32 Mp-CT 32 Zn deficiency - 33 Mp-CT 33 Zn deficiency - 34 Mp-CT 34 Yellow - 35 Mp-CT 35 Yellow -

131 36 Mp-CT 36 Yellow - 37 Mp-CT37 Yellow - 38 Mp-CT 38 Yellow - 39 Mp-CT39 Yellow - 40 Mp-CT 40 Yellow - 41 Mp-CT 41 Yellow - 42 Mp-CT 42 Yellow - 43 Fe-CT43 Yellow - 44 Fe-CT 44 Yellow - 45 Fe-CT 45 Yellow - 46 Fe-CT 46 Yellow - 47 Na-CT47 Yellow - 48 Lvn-CT48 No symptom - 49 OrCali-CT49 Symptom - 50 Ordang-CT 50 No symptom - 51 Pum5R-Ordang-CT 51 No symptom - 52 Na-Orsweet –CT 52 No symptom - 53 Mp-CT 53 NS - 54 Man-CT 54 - 55 Man-CT 55 Yellow - 56 Ha-CT 56 - 57 Ha-CT 57 - 58 Rur-CT 58 - 59 Rur-CT59 - 60 Na- Volka-CT60 No symptom - 61 Sng-CT61 No symptom - 62 ManT-CT62 No symptom - 63 Orsen-CT63 - 64 PumDo-CT64 - 65 Ha-CT65 No symptom - 66 Orsoan-CT66 No symptom - 67 ManTa-CT67 No symptom - 68 Pum-TG1 Symptom, + 69 Orsanh-TG2 Symptom + 70 Chtau-BT1 Symptom - 71 Orsoan-BT2 Symptom - 72 Orsanh-BT3 Symptom - 73 PumLong-BT4 Symptom - 74 Pum- Ruta-BT5 Symptom - 75 Ruta-BT6 - 76 Volka-BT7 Very serve +

132 77 Ruta-BT8 - 78 Ha-CT68 Symptom - 79 Ha-CT69 Symptom - 80 Mp-CT70 Symptom - 81 PumDo-BT9 No symptom - 82 Orsweet –TG3 Mottling + 83 Man-DT1 Mottling +

Among 83 samples detected by PCR, only 7 samples were PCR positive. Of them, the clone Sch- CT6 with 20 years old and still survives well, other Sanh chua such as Sch-CT15, Sch-CT17 were also in the same condition. Further findings for potential HLB tolerance genetic resource should be worked out from these clones. Some clones/ plants of commercial varieties were without symptom. All Can thang (Feronia elephantumcorrea),, Kim quyt (Triphasia trifoliata) and Nguyet quoi (Murraya paniculata) were PCR negative reaction of HLB detection from natural infection. It is known that these citrus relatives are the favorite hosts of the psylla Diaphorina citri. Mechanism for not transferring HLB pathogen or improved PCR protocol detection on these species needs to be further studied. Therefore, these three species, i.e., Can thang (Feronia elephantumcorrea), Kim quyt (Triphasia trifoliata), Nguyet quoi (Murraya paniculata) may be a potential HLB resistance source. We have repeated the PCR detection for 12 samples of 3 citrus related species above but all gave negative reaction (Table 3). While sampling the presence of psylla on these plants were recorded.

Table 3: Some evaluated HLB resistant related-citrus species in MD Sr.No English name Province PCR result 1 Feronia elephantumcorrea Can Tho, Ben Tre, Tien Giang, - Vinh Long 2 Triphasia trifolia Can Tho, Ben Tre, Tien Giang, - Vinh Long 3 Murraya paniculata Can Tho, Ben Tre, Tien Giang, - Vinh Long

Observation on the graft compatility of some combinations Nam roi pummelo/ Can thang, Da xanh pummelo/ Can thang, King mandarin/ Can thang, sweet orange/ Can thang and Nam roi pummelo/ Kim quyt in the nursery/ orchards of farmers as shown in table 4. Pummelo, sweet orange and King mandarin could be grown well and gave fruits through some bud-union ring and abnormal symptoms induced. There was completely incompatibility between Nam roi pummelo and Kim quyt.

4. Conclusion Preliminary observation on the HLB natural infection in citrus and citrus relative species in the M.D. Vietnam has indicated that some citrus clones of local commercial varieties of Sanh ngot, Sanh chua, Cam sen, Buoi do, Buoi dang, Hanh, Tac dai, Chanh tau and the other three citrus relative species , i.e., Can thang, Nguyet quoi, Kim quyt should be of some of potential HLB tolerance/ resistance resources.

133 Further and more molecular studies on the above problem are necessary to identify these genetic characteristics for obtaining a gene of HLB resistant strait.

Table 4: Graft compatibility of some citrus combinations of commercial varieties and ornamental species Sr./ No Rootstock species Scion varieties Evaluation Location 1 Can Thang (Feronia Nam roi (Citrus Bud union ring & An Binh, Vinh elephantumcorrea) maxima) abnormal symptoms Long on leaves 2 Can Thang (Feronia Nam roi (Citrus Bud union ring & Ben Tranh, Tien elephantumcorrea) maxima) abnormal symptoms giang on leaves 3 Can Thang (Feronia Da xanh (Citrus Bud union ring & Long an Tien elephantumcorrea) maxima) abnormal symptoms Giang on leaves 4 Can Thang (Feronia King mandarin Bud union ring Dao thang Tien elephantumcorrea) (Citrus sinensis) giang 5 Can Thang (Feronia Sweet orange Bud union ring Dao thanh- Tien elephantumcorrea) (Citrus sinensis) giang 6 Kim quit (Triphasia Nam roi (Citrus Incompatibility Chau Thanh Tien trifolia) maxima) giang

Figure 1: Some photo related to potential possible tolerance genetic resource of HLB disease

References Aubert B., 1987. Le Greening, une maladie infectieuse des agrumes, d’origine bacterienne, transmise par des Homopteres psyllides. IRFA/ CIRAD-B.P 180-97455 Saint Pierre Cedex. pp 185:118. Shinji Kawano, 2000. Citrus Greening control project in Okinawa, Japan. Managing Banana and Citrus diseases. International Plant Genetic Resource, 2000. pp164: 67-72.

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