Introduction

Citrus greening, sometimes known as likubin, has seriously affected citrus trees of all cultivars since the 1950s. It causes great damage to the citrus industry by shortening the lifespan of trees. Citrus greening was first reported from South Africa in 1947, although the disease had been known since 1929. Likubin was reported from Taiwan in 1951, while a similar disease known as huang-lungpin (yellow shoot) was reported in 1943 in mainland China (Matsumoto et al. 1961). This virus-like disease is known as leaf mottle yellows in the Philippines, citrus dieback in India, and citrus vein phloem degeneration in Indonesia. It has devastated all citrus-growing areas in Asia, and is also widespread in South Africa. The greening disease is transmitted mainly through vegetative propagation, and is spread by psyllid vectors. The establishment of a pathogen-free nursery system is the only way to control the disease. Precise and rapid indexing techniques are indispensable for the management of a pathogen-free nursery system.

Causal Agent and Symptoms

The fastidious bacteria causing greening (GFB) cannot be cultured. They are submicroscopic walled prokaryotes, existing in a sieve tube. Electron microscopy, using serial sections and stereomicrographing, can confirm the presence of mature forms of the pathogen, generally rigid rods measuring 350-550 x 600-1500 nm, surrounded by a two-layered envelope, 20 _ 25 nm thick ( Fig. 1). However, GFB bodies are pleomorphic during growth.

The Asian greening organism produces symptoms in either a warm climate (27 - 32°C) or a cool one, and is classified as the heat-tolerant form. The South African greening organism belongs to a heat-sensitive form which induces severe symptoms at cool temperatures (22 - 24°C). Apart from pummelo, most citrus cultivars are susceptible to the Asian form of GFB. Even pummelo has become infected by the pathogen in Taiwan since 1970. Pummelo trees growing in the Philippines, Malaysia, Southern China, Palau and Vietnam were also found to be infected with GFB in recent years. The mandarin isolates of Asian GFB induce severe greening symptoms in mandarin and sweet orange trees, but only mild symptoms in pummelo. The pummelo isolate causes severe symptoms in pummelo, and mild symptoms in mandarin and sweet orange. Some isolates of GFB from greening-affected sweet orange and mandarin trees caused severe symptoms in both mandarin and pummelo. A symptomless mild strain of GFB was also isolated from healthy-looking mandarin trees.

It is assumed that Asian GFB might include a number of severe strains such as mandarin strain and pummelo strain, as well as the common strain and mild strain. The Asian form of greening has been spread rapidly by the Asian psyllid ( Diaphorina citri), while the African form of greening is transmitted by the African psyllid ( Trioza erytreae) in a persistent manner.

Although the disease syndrome differs to some extent according to the variety of citrus, common symptoms are yellowing of the veins and adjacent tissues, followed by yellowing or mottling of the entire leaf, occasionally with corking of the veins. This is followed by premature defoliation, dieback of twigs, decay of feeder rootlets and lateral roots, decline in vigor, and ultimately the death of the entire plant. Diseased leaves become hardened and curl outward, while young leaves which develop after premature defoliation are small and slender with symptoms resembling those of zinc deficiency ( Fig. 2). Trees affected with greening become stunted, bear multiple off-season flowers, most of which fall off, and produce small misshapen fruit with thick, pale green peel ( Fig. 3).

Diagnosis and Detection

Greening is tentatively diagnosed in the field by foliage and fruit symptoms. Further diagnosis requires indexing on susceptible mandarin seedlings by graft-inoculation. Because of the low population and uneven distribution of GFB within the plant, bioassay with an indicator plant requires numerous test plants and inoculum buds, and is time-consuming.

DNA probes and PCR with adequate primer pairs were developed through DNA cloning and sequencing of GFB. Dot hybridization (DH) with a selective DNA probe consistently showed a specific and sensitive reaction to DNA extracted from greening-affected citrus plants. DH with a DNA probe has been widely applied to indexing and in ecological studies of the pathogen ( Fig. 3). The detection of GFB in symptomless citrus plants, as well as alternative host plants, can be accomplished by the DH test. For this reason, it is often used to index citrus foundation stocks and pathogen-free seedlings. The sensitivity of GFB detection has recently been enhanced by the development of polymerase chain reaction (PCR) with adequate primer pairs followed by electrophoresis analysis ( Fig. 4).

Detection by Dot Hybridization

Extraction of Dna:

• 1. Collect plant leaves and twigs. Strip the midribs from the leaves and peel the bark from the twigs. Keep the midribs and bark for air-drying.
• 2. Freeze 0.5g of chopped dried tissue in liquid nitrogen and grind to a fine powder in a coffee blender, or with a mortar and pestle.
• 3. Add 3 ml of DNA-extraction buffer* and 0.3 ml Sarkosyl (10%). Stir the tissue-powder suspension.
• 4. Transfer the suspension to a 1.5 ml eppendorf tube and incubate at 55°C/1hr.
• 5. Centrifuge the tube at 6,000 rpm/5 min.
• 6. Save supernatant (0.8 ml), add 100 ul of 5M NaC1 and 100 ul of CTAB/Nac1 (10%CTAB in 0.7 M NaC1), incubate at 65°C/10 min.
• 7. Add equal volume of chloroform/isoamyl alcohol (24:1), mix thoroughly and spin at 11,000rpm/5min. Save the aqueous suspension.
• 8. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly and spin at 11,000 rpm/5 min. Save the aqueous suspension.
• 9. Add 0.6 volume of isopropanol to precipitate the nucleic acid. Spin at 12,000 rpm/30 min.
• 10. Wash pellet with 70% ethanol to remove the residual CTAB. Briefly dry pellet and resuspend in 100 µl of TE* buffer. Store at -20°C.
• * DNA-extraction buffer (pH8.0): 100 mM Tris-HC1, 100 mMDTA, 250 mMNaC1, 100ug/ml proteinase K (may be omitted).
• * TE buffer (pH 8.0): 10mM Tris, 1 mM EDTA.

Dot Hybridization on Nylon or NC Membrane

• 1. Transfer 5 µl purified G-DNA into an eppendorf tube, add 5 µl 6X SSC*, and denature the G-Dna in a boiling water bath for 10 min. Chill quickly on ice.
• 2. Blot 3 µl DNA samples on Nylon of NC membrane.
• (Make a dilution series of DNA samples with 6 X SSC if necessary).
• 3. Treat the membrane in UV linker (120,000µ joules) or vacuum (80-85°C/2 hrs.) for cross-linkage.
• 4. Place membrane in hybridization bottle and add 2X SSC for rinsing. Add prehybridization solution* (10ml/bottle) and incubate at 68°C/2 hrs. in rotary hybridization oven.
• 5. Pour out prehybridization solution and add hybridization solution (10 ml), incubate at 68°C/12-16 hrs. in rotary hybridization oven.
• 6. Pour out Hybridization solution* (save for further use).
• Prehybridization solution: 6X SSC (1X SSC = 0.15M NaC1 and 0.15 M Sodium citrate, pH7.0), 0.1% (w/v) N-lauroylsarcosine, Na-salt (Sigma), -0.02% (w/v) SDS (Lauryl sulfate = Sodim dodecyl sulfate), Add to the freshly prepared solution 1% (w/v) blocking reagent (BM Co.)
• Hybridization solution: Add µ1 l of boiled dig or biotin-labeled DNA probe to 10 ml prehybridization solution.

Detection and Coloring

• 1. Wash filters 2 times, 5 min at room temperataure (RT) with at least 50 ml of 2X SSC, 0.1% (w/v) SDS, per 100 cm ² filter and 2 times, 15 min at 68°C with at least 100 ml of 0.1X SSC, 0.1% (w/v) SDS.
• 2. Wash filters briefly (1 min) in Buffer # 1.
• Buffer #1: 100 mM Tris-HC1, 150mM NaC1; pH 7.5.
• 3. Incubate for 30 min with about 100 ml Buffer #2.
• Buffer # 2: 0.5% (w/v) blocking reagent (BM) in buffer # 1.
• 4. Wash again briefly with buffer # 1.
• 5. Dilute anti-dig-AP solution (BM) for dig-labeled DNA probe or Streptavidin-AP solution (BRL) for biotin-labeled DNA probe to 150 mU/ml (1:50000) in Buffer #1. Incubate filter for 30 min with about 20 ml of diluted AP solution.
• 6. Remove unbound antibody-conjugate by washing 2 times, 15 min with 100 ml of Buffer #1.
• 7. Equilibrate membrane for 2 min with 20 ml of Buffer # 3.
• Buffer #3: 100 mM Tris-HC1, 150 mM NaC1, 50 mM MgC1; pH 9.5.
• 8. Incubate filter in the dark at room temperature with about 10 ml freshly prepared color solution.
• Color solution: 50µl NBT (50 mg/ml), 25µl BCIP (50 mg/ml), in 7 ml Buffer # 3.
• 9. When the desired spots are detected, stop the reaction by washing the membrane for 5 min with 50 ml of stopping solution to terminate reaction.
• Stopping solution: 10mM Tris-HC1, 1mMEDTA; pH 8.0.

Detection by PCR Analysis

GFB-Dna Amplification in PCR Thermocycler

The GFB-DNA is amplified by PCR with primer pairs by using GFB-DNA extract as a template in DNA thermocycler. The components and PCR cycles are as follows: (see Formula 1)

PCR cycles:

Step 1 : 94°C 4 min; 56°C 1 min; 72°C 2 min.

1 cycle

Step 2 : 94°C 1 min; 56°C 1 min; 72°C 2 min.

30 cycles.

Step 3 : 72°C 10 min.

Step 4 : 4°C soaking

Electrophoresis Anslysis of PCR Products

• 1. Prepare 1.4% agarose gel plate in TAE* buffer (0.04 M Tris-acetae, 0.001 M EDTA, pH 8.0).
• 2. Load 3-5 ul of PCR products* into each well, and run the gel in an electrophoresis chamber at 100 volts for about 30 min when the markers reach neara the bottom line. (Running buffer: 0.5 X TAE buffer, pH 8.0)
• 3. Stain gel with Ethidium bromid (0.5 ug/ml) solution for 3-5 min, and soaking in water several min.
• 4. Observe the amplified GFB-DNA band under UV-chamber and take a picture of the electrophoretic plate.
• * 50X TAE buffer (per liter): 242g Tris base, 57.1 ml glacil acetic acid, 100 ml 0.5 M EDTA (pH 8.0).
• * Loading buffer: 0.25% Bromophenyl blue, 30% glycerol (1:4).

Index of Images

Figure 1 Electron Micrograph of the Fastidious Bacteria (GFB) Which Causes Asian Greening Disease (Likubin).

Figure 2 Leaf Symptoms of Greening-Affected Ponkan Mandarin, Showing Yellowing and Mottling, Compared to Healthy Leaves (Left).

Figure 3 Fruits from Greening-Affected Tankan Tangor, Showing Atrophy and Poor Coloring, Compared to Healthy Fruit (Right).

Figure 4 Detection of Greening Fastidious Bacteria (GFB) by Dot Hybridization Tests, with GFB-Dna Extract (a), and by PCR Amplification of GFB-Dna Followed by DH with PCR Products (B). D, Diseased Sample from Go-Infected Ponkan Mandarin for Positive Check; H, Healthy Sample from Shoot-Tip Grafting Ponkan for Negative Check; 6-1 - 6-9, Healthy-Looking Citrus Samples Collected from Field; 1-1, 4-5, 5-1, Citrus Samples with Greening Symptoms.

Figure 5 Detection of Greening Fastidious Bacteria (GFB) by PCR Amplification Followed by Electrophoresis. Lane M, 100BP Dna Ladder As a Marker, Lane 1 - 10, PCR Products from Different Dna Templates of Diseased Citrus Tissues Collected from Different Countries Including Taiwan (Lane 1 - 4), Okinawa, Japan (Lane 5), China (Lane 6), Malaysia (Lane 7), Vietnam (Lane 8), Thailand (Lane 9), and Saudi Arabia (Lane 10); Lane 11, PCR Product of Healthy Sample from a Pathogen-Free Mandarin by Shoot-Tip Grafting. the Arrow Indicates the Position of Go-Specific Dna Fragments (228 BP) Amplified by PCR.

Formula 1 Components of PCR

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