Phytoplasma mali
(apple proliferation)

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

See also CABI/EPPO (1998, No. 249).

Apples are the main host, and most cultivars are susceptible (EPPO/CABI, 1997).

Apple cultivars known to be affected by AP phytoplasma include: susceptible, Belle de Booskop, Gravestein, Golden Delicious and Winter Banana (Anon., 1997); highly susceptible, Florina, Prima and Priscilla (Loi et al., 1995); of medium susceptibility, Idared, McIntosh, Starking and Starkrimson (Németh, 1986); and tolerant, Roja de Benejama (Anon., 1997), Antonokova, Cortland, Spartan, Yellow transparent, Wealthy (Németh, 1986). In northern Italy serious epidemics have been reported to occur on the cultivars Golden Delicious, Florina, Canadian Renette and Granny Smith, grafted on different rootstocks (Osler et al., 2001).

The cultivars Prima, Florina and Priscilla, which are known to be resistant to scab (Venturia inaequalis), were derived from cultivars susceptible to apple proliferation such as: Golden Delicious, Starking Delicious, McIntosh, Jonathan, Rome Beauty and Malus floribunda 821 (Kartte and Seemüller, 1988). These authors stated that 'care should be taken that the high susceptibility of some Malus spp. is not combined in the breeding products'.

The phytoplasma can also be artificially inoculated to Malus baccata, M. coronaria, M. domestica, M. floribunda, M. fusca, M. gloriosa, M. ionensis, M. platicarpa, M. purpurea, M. robusta (Németh, 1986).

Magnolia species and cultivars have been found to be hosts for phytoplasmas of both the apple proliferation and aster yellows phytoplasma groups, although the specific phytoplasma within the AP group/clade was not identified. Infected plants have distorted and stunted growth (Kaminska, 2003).

In the field, the disease seems to spread naturally by root fusion and by insect vectors. In terms of the vectors, AP was experimentally transmitted from apple to apple and to Catharanthus roseus by the leafhopper vector Philaenus spumarius; from apple to C. roseus by Aphrophora alni and Lepyronia coleoptrata; and from infected celery plants to apple seedlings by Arhianus interstitialis (Hegab and El-Zohairy, 1986). These experiments showed that nymphs can acquire the pathogen and transmit it in the adult stage. Adults were found to maintain the ability to transmit the phytoplasma up to the end of their life. The incubation period in apple lasts 1-2 years (Krczal et al., 1988; Krczal and Bliefernich, 1992).

More recently, the leafhopper Fiebierella florii was considered as a putative vector of apple proliferation phytoplasma. AP-DNA was revealed in the total DNA extracted from F. florii trapped in orchards with proliferation-diseased trees (Vega et al., 1993; Bliefernich and Krczal, 1995). Also, it has recently been demonstrated that the psyllids Cacopsylla costalis, C. mali, and C. melanoneura are vectors (Grando et al., 1998; Jarausch et al., 2003; Tedeschi et al., 2003; Tedeschi and Alma, 2004) and, according to Tedeschi and Alma (2004), psyllids seems to be the most important vectors for AP.

Schmid (1975) reported that, in terms of spread, 73% of trees in an apple orchard became infected over a 12-year period; an average increase of 18% per year was noted.

As indicated above, the distribution of the phytoplasma within the infected tree is restricted to the functional phloem elements, with colonization of above-ground parts following a seasonal pattern. The phytoplasma disappears in the above ground parts during winter, when the complete inactivation of sieve-tubes in aerial parts of pome fruit trees occurs. During this period the phytoplasma still survives in the roots and in spring begins to re-colonize the stem and shoots after the development of a new phloem circle. The pathogen can then be acquired by sap-sucking insects, such as those listed above, within which it can multiply, circulate to the salivary glands, and be expelled during feeding probes to infect other plants. Once a vector is infected, it retains the ability to transmit the phytoplasma for the duration of its life span, and overwintering adult psyllid vectors have been shown to still carry the phytoplasma the next spring (Tedeschi et al., 2003; Tedeschi and Alma, 2004).

Temperature seems to have a significant impact on disease expression and therefore impact. An AP survey conducted in Germany (Seemüller et al., 1998) showed that the phytoplasma was widely distributed outside of the area in southwest Germany where the disease is of greatest economic importance. In the southwest region it seems that the warmer temperatures enhance the growth of the phytoplasma, leading to high populations in the tree tops and more obvious symptoms. However, in the warmer regions of southern Europe, such as in the Emilia-Romagna region of Italy, temperatures may be too high for good symptom development, whereas in the more northerly areas of Europe the temperatures may be too cool for good symptom development. This suggestion corresponds at least in part with results of an earlier study by Ducroquet et al. (1986) who found that symptoms of AP developed at temperatures of 21-24°C, but not between 29 and 32°C.

Biological Assay

Positive identification requires transmission to a woody indicator species (see Detection and Inspection Methods). Malus dawsoniana, a very sensitive indicator, when grafted in June on the scion, develops symptoms the following autumn. Using the double budding technique, the reaction appears after budbreak. (Anon., 1997). The use of DAPI reagent (1,6 diamidino 2-phenylindole) can help to detect the fluorescence of phytoplasmas in the sieve tubes of phloem tissue under the bark of infected apple trees

Serological Assay

Apple proliferation can be detected in infected apple trees using monoclonal antibodies (MAbs) to AP phytoplasmas obtained from AP phytoplasma-infected Catharanthus roseus as source of antigen. These MAbs react specifically in ELISA and immunofluorescence (IF) (Loi et al., 1998). Their patent is pendent. ADGEN Ltd. have also produced an AP phytoplasma specific antisera, which works with apple extracts if there is sufficient antigen present. Usually the concentration of phytoplasma is a limiting factor.

Molecular Assay

DNA extraction

Midrib and main vein tissue (1 g) should be used from leaves collected in August-September, avoiding any large necrotic areas. Use the following enriched phytoplasmas procedure (Ahrens and Seemüller (1992) to obtain DNA 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 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 fraction 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

Polymerase Chain Reaction (PCR)

Several primers intended for diagnostic procedures for the diagnosis of phytoplasma diseases have been identified, among them are those specific to the apple proliferation group (see Lee et al., 1993, 1995; Firrao et al., 1994; Jarausch et al., 1994; Lorenz et al., 1995; Smart et al., 1996).


Molecular diagnosis of apple proliferation 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 (AP, PD, ESFY, PYLR)

Reference: Lorenz et al. (1995)

Primer pair: fPD/r01

Sequence:
5' - GAC CCG TAA GGT ATG CTG -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: fAT/rAS

Sequence:
5' - CAT CAT TTA GTT GGG CAC TT - 3'
5' - GGC CCC GGA CCA TTA TTT ATT - 3'

Amplified product size: ca 500 bp

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

Reference: Smart et al. (1996)

Primer pair: AP5-AP4

Sequence:
5' - TCT TTT AAT CTT CAA CCA TGG C - 3'
5' - CCA ATG TGT GAA ATC TGT AG - 3'

Amplified product size: ca 483 bp

Phytoplasma: specific for apple proloferation phytoplasma

Reference: Jarausch et al. (1994)

Primer pair: AP3-AP5

Sequence:
5' - CTA AAA CTC ACG CTT CAG CTA CTC - 3'
5' - TGA GAT TTG CTA AAA CTC ACG CTT - 3'

Amplified product size: ca 670 bp

Phytoplasma: specific for apple proliferation phytoplasma

Reference: Firra et al. (1994)


The pair fO1/rO1, AP3-AP5 and fPD/rO1 prime in the 16S rDNA sequence and are, respectively, the first AP group specific, the second AP specific, and the third pome fruit subgroup specific. The pair fAT/rAS primes in the 16S/23S rDNA spacer region and is specific for pome fruit subgroup phytoplasmas.

The pair AP5-AP4 was designed from a randomly cloned nucleotide sequence (Gene Bank accession number L22217) of the German isolate AT (1812 bp) of the previously cloned Hind III fragment IH196 (3,7 Kb) sequenced from both ends. This pair is specific for AP phytoplasma.

Molecular diagnosis of apple proliferation phytoplasma by nested PCR

Primer pair: R16F2/R2

Sequence:
5' - ACG ACT GCT AAG GAC TGG - 3'
5' - TGA GGG GCG GTG TGT ACA AAC CCC G - 3'

Amplified product size: ca 1200 bp

Phytoplasma: all phytoplasmas

Reference: Lee et al. (1993)

Primer pair: R16(X)F1/R1

Sequence:
5' - GAC CCG CAA GTA TGC TGA GAG ATG - 3'
5' - CAA TCC GAA CTG AGA CTG T - 3'

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 This pair primes on the 16S rDNA sequence. (Lee et al., 1995). All these primer pairs work at different reaction mixtures and conditions.

The following are reaction mixtures and conditions for PCR with different primer pairs designed for diagnosis of AP group and AP phytoplasma:

Primer pairs: fO1/rO1; fPD/rO1

Reaction mixture: final volume: 40 µl
template, 100-200 ng
Primers: 0.5 µM
Buffer: 1x; MgCl2 1.5 mM
dNTPs: 100 µM
Enzyme: 0.20-1 Unit/reaction.
Reaction conditions: 35 cycles
95°C for 30 s denaturation
55°C for 75 s annealing
72°C for 90 s extension

Primer pair: fAT/rAS

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 pair: AP5/AP4

Reaction mixture: final volume: 40 µl
template: 10-100 ng
Primers: 0.5 µM
Buffer: 1x
dNTPs: 125 µM
Enzyme: 0.5 Unit/reaction
Reaction conditions:
95°C for 60 s predenaturation step
40 cycles
95°C for 10 s denaturation
58°C for 15 s annealing
72°C for 45 s extension
72°C for 240 s elongation

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

Primer pairs: AP3-AP5:

Reaction mixture: final volume: 50 µl
template: 2 µl
Primers: 300 ng
Buffer: 1x
dNTPs: 100 mM
Enzyme: 1 Unit/reaction

Reaction conditions: 25 cycles
94°C for 30 s denaturation
63°C for 30 s annealing
72°C for 30 s extension
72°C for 600 s 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 AP phytoplasma with RFLP analysis of amplified products obtained with different primers

Primer pair: f01/r01; fPD/r01
Endonuclease: Ssp I, Sfe I
Phytoplasma: AP
Reference: Lorenz et al. (1995)

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

Primer pair: AP5-AP4
Endonuclease: Ssp I, Spe I, Hinf I
Reference: Jarausch et al. (1994)


Dot-Blot Hybridization

Differential diagnosis of apple proliferation and European stone fruit yellow phytoplasmas can be made by oligonucleotide hybridization in the presence of tetramethylammonium chloride (Malisano et al., 1996). This diagnostic approach may be more practical than PCR plus RFLP in differentiating the two phytoplasmas that cause apple proliferation and plum leptonecrosis, which are economically significant diseases in the main European fruit tree-growing areas. It is also suitable for automatic evaluation of a large number of samples.

DNA Amplification

The primer pair P1-P4 should be used:

P1 5' - CAG CAG GYC CGC GTA ATA CAT A - 3'
P4 5' - RMC CCG AGA AGC TAT TCA CCG - 3'

Y = C or T; R = A or G; m = A or C

This pair primes on 16S rDNA sequence and amplify a segment of ca 865 bp (Firrao et al., 1993).

- Add 2 x SSC to 5-45 µl of PCR mixture (1 x SSC: 150 mM NaCl and 15 mM Na citrate, pH 7.0).
- vacuum-filter on nylon membranes with a 96-well dot-blot manifold apparatus
- place dried membrane sequentially on 3 mm paper sheets saturated with: 1.5 M NaCl and 0.5 NaOH for 5 min; 0.5 M Tris pH 7.4; 0.5 M Tris pH 7.4 and 1.5 M NaCl for 5 min.
- dry and bake for 30 min at 120°C
- pre-hybridize in 5 x SSC; -0.1% laurosylsarcosine; 0.02% SDS; 1% blocking reagent; 42°C for 2 h
- hybridize in solution as above containing 20 pmol oligonucleotide probe at 42°C for 6 h
- washings :2 x 30 min in 5x SSC at 4°C; 2 x 20 min at 57°C in tetramethylammonium chloride wash solution (wash solution: 3 M TMACl, 50 mM Tris-HCl; 2 mM EDTA; 0.1% sodium dodecyl sulfate (SDS), pH 8)
- digoxigenin labelling and detection can be performed according to manufacturer's instructions.

Probe sequences:

LN 11 5' -GTG CGT AGG CGG TTA AA- 3'
AP 11 5' -GTG TGT AGG CGG TTA AA- 3'

(Malisano et al., 1996)

Requirements for positive molecular diagnosis:

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

- In case of negative result, the use of universal rRNA primers is recommended to check the presence of phytoplasmas other than apple proliferation phytoplasma. Suggested universal primers: 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.

Apple proliferation is routinely detected by visual observation (see Symptoms) and by field testing on Malus domestica Golden Delicious (side interstock grafting) using five repetitions for 2 years (OEPP/EPPO, 1991/1992).

For laboratory diagnosis at least five samples per plant should be randomly collected in order to avoid false negative results due to the low titre and erratic distribution of these pathogens in the phloem of the plant. Samples from leaves, petioles, shoots and canes should be collected at the end of August. The use of fresh material is recommended in diagnostic assays. However, if this is not possible, the test material should be stored at -20°C. (See Diagnosis.)

The criteria used to differentiate apple proliferation from the other phytoplasmas and the related Mollicutes are: full-length of 16S rDNA; 16S/23S spacer region sequence; RFLP of PCR-amplified ribosomal DNA products; and 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 pests in the orchards is also essential. Removal of root suckers is a control measure to reduce the diffusion of leafhoppers living on weeds. Phytoplasmas are sensitive to tetracycline, but this antibiotic is bacteriostatic rather than bactericidal. After antibiotic treatment, symptoms can noticeably diminish for 2 years (Casanova et al., 1980), but often this is only observed as a temporary effect (Schmid, 1983). The use of tetracycline can unintentionally select for resistant bacterial populations. At the present time, resistant rootstock is the most efficient way to control the disease (Seemüller, 1990).