Phytoplasma pyri
(pear decline)

Records from Austria, Belgium, Greece, Romania, Switzerland, former USSR (Németh, 1986), India and South Africa (Seemüller, 1990) are based on symptoms and may require further confirmation.

See also CABI/EPPO (1998, No. 269).

Pear is the main host of pear decline phytoplasma. Pear cultivars known to be affected include:

Highly susceptible: Hardy Burré, Magness, Precocious and Williams (Németh, 1986); Max Red Barlett (Anon., 1997); Conference (Davies et al., 1992); Abate Fetel and Kaiser (Giunchedi et al., 1995).

Medium susceptibility: Comice, Concorde (Davies et al., 1992); Montecosa Precoz (Anon., 1997)

The phytoplasma can be graft transmitted to Pyronia veitchii, Pyrus amygdaliformis lobata, P. aromatica, P. betulifolia, P. calleriana, P. ussuriensis (Németh, 1986) and to Catharanthus roseus by dodder bridge and insect vector.

Pear decline can be transmitted by grafting, budding and chip budding. It is not perpetuated by the standard propagation practices when quince rootstocks are used even though several quince rootstocks selections showed a considerable variation in their suitability for overwintering of the pear decline agent (Seemüller et al., 1984a, b; Poggi Pollini et al., 1995a). Furthermore, it is not transmitted during the winter by scions as the phytoplasma remains in the root at that time because of the degeneration of sieve tubes in winter (Seemüller et al., 1986, 1989). Generally pear decline phytoplasma do not persist in quince rootstock during winter due to the resistance of these rootstocks to the phytoplasma.

The pear psyllids Cacopsylla pyricola, C. pyrisuga and C. pyri are the vectors of PD phytoplasma and they are mainly responsible for spreading the disease in the field (Jensen et al., 1964). The epidemiology of the disease is still poorly understood. A limiting factor in epidemiological studies has been the lack of techniques available to detect phytoplasma in the vector. Molecular diagnostic methods have been developed in order to detect phytoplasmas (Davies et al., 1995; Marzachì et al., 1998).

Biological Assay

Positive identification requires transmission on a woody indicator (see Detection and Inspection Methods).

The transmission test performed by grafting root or stem scions onto P. communis cv. Precocious in the glasshouse produces symptoms at the end of the growing season (Seemüller, 1989).

Among several indicators evaluated under controlled conditions, Cydonia oblonga C 7-1 seems the most reliable for screening propagative material. It takes 40 days, after tongue graft transmission, instead of 2 years of field testing to produce symptoms due to the phytoplasma infection They consist of tufts of small leaves (rosette), leaf yellowing and distortion of the leaf blade. The only symptom observed on the other indicators is the presence of elongated petioles (Del Serrone et al., 1998).

The use of DAPI reagent (1,6 diamidino 2-phenylindole) to detect phytoplasmas in the sieve tubes of the phloem tissue under the bark of infected pear trees, can help to eliminate other abiotic causes such rootstocks-scion incompatibility, malnutrition, and winter injury that induce similar symptomatology (Seemüller, 1976).

Molecular Assay

DNA extraction

Midrib and main vein tissue (1 g) should be used from leaves collected in August-September, avoid any large necrotic areas. The enriched phytoplasmas procedure is then used to obtain DNA useful for PCR.

- Incubate the tissue in 6 ml of grinding buffer on ice for 10 min.
- Grind the tissue, then add 8 ml of fresh grinding buffer and grind once more.
- Centrifuge at 1100 g and 4°C for 10 min.
- Decant the supernatant then centrifuge at 14,600 g and 4°C for 25 min.
- Resuspend the pellet in 1.5 ml of warm (60°C) extraction buffer
- Incubate at 60°C for 30 min.
- Add to the lysate an equal volume of chloroform/isoamylalcohol (24:1, v/v).
- Precipitate the aqueous layer with two-third volume of -20°C isopropanol.
- Centrifuge at 15,000 g and 4°C.
- Wash the pellet with 70% ethanol.
- Dry under vacuum.
- Add 50 µg/ml RNAse at 37°C for 30 min.
- Extract with chloroform/isoamylalcohol (24:1, v/v), precipitate with ethanol, wash and dry pellet as described.

Grinding Buffer

potassium phosphate 125 mM
ascorbic acid 30 mM
sucrose 10%
bovine serum albumin (BSA V) 0.15%
polyvinylpyrrolidone (PVP 15) 2%
pH 7.6

Extraction Buffer

Hexadecyltrimethyl-ammonium bromide (CTAB) 2%
NaCl 1.4 M
2-mercaptoethanol 0.2%
Ethylendiaminetetraacetic (EDTA) 20 mM
Tris- hydroxymethylaminomethane hydrochloride (Tris-HCl) 100 mM
pH 8.0

(Ahrens and Seemüller, 1992)

Polymerase Chain Reaction (PCR)

Several primers intended for diagnosis of phytoplasma diseases have been identified.


Molecular diagnosis of pear decline phytoplasma by direct PCR

Primer pair: f01/r01

Sequence:
5' - CGG AAA CTT TTA GTT TCA GT - 3'
5' - AAG TGC CCA ACT AAA TGA T - 3'

Amplified product size: ca 1100 bp

Phytoplasma: all phytoplasma isolates of AP group

Reference: Lorenz et al. (1995)

Primer pair: fPD/r01

Sequence:
5' - GAC CCG TAA GGT ATG CTG A - 3'
5' - AAG TGC CCA ACT AAA TGA T - 3'

Amplified product size: ca 1000 bp

Phytoplasma: all phytoplasma isolates of pome fruit subgroup of AP group (AP, PD)

Reference: Lorenz et al. (1995)

Primer pair: fPD/rPDS

Sequence:
5' - GAC CCG TAA GGT ATG CTG A - 3'
5' - CCC GGC CAT TAT TAA TTT TA - 3'

Amplified product size: ca 1400 bp

Phytoplasma: specific for pear decline phytoplasma

Reference: Lorenz et al. (1995)

Primer pair: fAT/rPRUS

Sequence:
5' - CAT CAT TTA GTT GGG CAC TT - 3'
5' - GCC CCA AGC CAT TAT TGA TT - 3'

Amplified product size: ca 500 bp

Phytoplasma: all phytoplasma isolates of PD and ESFY

Reference: Smart et al. (1996)

The pair fO1/rO1 and fPD/rO1 prime in the 16S rDNA sequence and are, respectively, AP group specific and pome fruit subgroup specific.

The pairs fAT/rPRUS and fPD/rPDS prime in the 16S/23S rDNA spacer region and are specific for PD and ESFY phytoplasmas, and PD phytoplasma only, respectively.

Molecular diagnosis of pear decline phytoplasma by nested PCR

Primer pair: R16F2/R2

Sequence:
5' - ACG ACT GCT AAG GAC TGG - 3' (153-168)
5' - TGA GGG GCG GTG TGT ACA AAC CCC G - 3' (1373-1397)

Amplified product size: ca 1200 bp

Phytoplasma: all phytoplasmas

Reference: Lee et al. (1995)

Primer pair: R16(X)F1/R1

Sequence:
5' - GAC CCG CAA GTA TGC TGA GAG ATG - 3' (197-221)
5' - CAA TCC GAA CTG AGA CTG T - 3' (1198-1297)

Amplified product size: ca 1100 bp

Phytoplasma: all phytoplasmas of AP group (AP, PD, ESFY)

Reference: Lee et al. (1995)

PCR products initially amplified by using the universal primer pair R16F2/R2 are diluted (1/40) with sterile deionized water and used as template DNA for a subsequent PCR with the pair R16(X)F1/R1 These pairs prime on the 16S rDNA sequence (Lee et al., 1995).

All these primer pairs work at different reaction mixtures and conditions.

Primer pairs: fO1/rO1; fPD/rO1; fPD/rPDS

Reaction mixture: final volume: 40 µl
template: 50-100 ng
Primers: 250 nM
Buffer: 1x
dNTPs: 100 µM
Enzyme: 0.20- 1 Unit/reaction
Reaction conditions: 30 cycles
95°C for 30 s denaturation
55°C for 75 s annealing
72°C for 90 s extension

Primer pair: fAT/rPRUS

Reaction mixture: final volume: 30 µl
template: 50 ng
Primers: 0.5 µM
Buffer:1x
dNTPs: 150 µM
Enzyme: 1 Unit/reaction
Reaction conditions: 30 cycles
94°C for 60 s denaturation
55°C for 60 s annealing
72°C for 120 s extension

Primer pairs: R16F2/R2; R16(X)F2/R2

Reaction mixture: final volume: 50 µl
template: 20 ng
Primers: 0.4-1 µM
Buffer: 1x
dNTPs: 200 µM
Enzyme:1 Unit/reaction
Reaction conditions: 94°C for 120 s predenaturation step
35 cycles
94°C for 60 s denaturation
50°C for 120 s annealing
70°C for 180 s extension
72°C for 600s elongation

Restriction Fragment Length Polymorphism analysis (RFLP)

The use of PCR amplification of the 16S rDNA to differentiate phytoplasmas belonging to the same group is difficult due to the high similarity of their sequences so that it is necessary to digest the amplification products by restriction endonucleases.

Molecular characterization of PD phytoplasma with RFLP analysis of amplified products obtained with different primer pairs

Primer pair: f01/r01; fPD/r01
Endonuclease: Sfc I
Phytoplasma: PD
Reference: Lorenz et al. (1995)

Primer pair: R16F2/R2+R16(X)F2/R2
Endonuclease: Rsa I
Phytoplasma: PD
Reference: Lee et al. (1995)

Primer pair: FAT/rPRUS
Endonuclease: Alu I
Phytoplasma: PD
Reference: Poggi Pollini et al. (1995b)


PCR-ELISA

DNA amplification:
use the primer pair: fP1/rP7

P1 5' - AAG AGT TTG ATC CTG GCT CAG GAT T- 3'
P7 5' - CGT CCT TCA TCG GCT CTT-3'

This pair amplify both 16SrRNA gene and the spacer region between 16S rRNA and 23S rRNA

Reaction mixture: final volume: 30 µl
template: 50 ng
Primers: 0.5 µM
Buffer: 1x
dNTPs: 150 µM
Enzyme: 1 Unit/reaction
Reaction conditions: 30 cycles
94°C for 60 s denaturation
56°C for 60 s annealing
72°C for 120 s extension
72°C for 60 s elongation

(Smart et al., 1996).

Products obtained with the fP1/rP7 primers labelled with digoxigenin during the amplification process are hybridized after denaturation with the rPDS probe. This specific capture probe complementary to the inner part of the amplification product was biotinilated in the 5' position. The hybridization product (20 pmoles + 20µl of amplified product made up to 220 µl with hybridization solution) is incubated in an ELISA plate previously coated with streptoavidin. The test is performed according to Boehringer Mannheim manufacturing instructions following the protocol of Poggi Pollini et al. (1997). This technique is highly specific and sensitive.

Requirements for positive molecular diagnosis:

- Water instead of template DNA must be considered and added to reaction mixture for controls in PCR experiments.

- In case of negative result, it is recommended the use of universal primers to check the presence of phytoplasmas other than the pear decline phytoplasma. Suggested universal primers are: fU5/RU3 (Lorenz et al., 1995); F2n/R2 (Lee et al., 1995); and P1/Tint (Smart et al., 1996).

Then use PCR, with other group- or species-specific primers, and RFLP analysis for differentiation (Lorenz et al., 1995; Smart et al., 1996).

- Use DNA of reference strains, belonging to the same and to other groups, in all molecular diagnostic methods.

Pear decline is routinely detected by visual observation (see Symptoms) and by field testing on Pyrus communis Doyenné du Comice; using three replicates for 2 years (OEPP/EPPO, 1991/1992).

For laboratory diagnosis, at least five samples per plant should be randomly collected for phytoplasma diagnosis in order to avoid false negatives caused by the low titre and erratic distribution of phytoplasmas in the phloem of the plant. Samples, consisting of leaves, petioles, shoots and canes, should be collected at the end of August. The use of fresh material is recommended in diagnostic assays. If this is not possible, tissue should be stored at -20°C. (See Diagnostic Methods.)

Criteria that can be used to differentiate pear decline phytoplasma from other phytoplasmas and the related Mollicutes include: full-length sequence of 16S rDNA; 16S/23S spacer region sequence; RFLP of PCR-amplified ribosomal DNA products; RFLP patterns of non-ribosomal DNA (Southern analysis).

Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.

As a preventive measure, the use of healthy propagation material is a prerequisite. Intensive control of weeds and insect vectors in the orchards is also essential. The use of antibiotics (Nyland and Moller, 1973) can select resistant bacterial populations. Resistant rootstocks are the most efficient way to control the disease (Seemüller, 1990).