Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
METHOD FOR CULTURING PHYTOPLASMA
Document Type and Number:
WIPO Patent Application WO/2013/186591
Kind Code:
A1
Abstract:
The present invention relates to a method for culturing a phytoplasma colony, preferably under axenic conditions, and a kit for culturing a phytoplasma colony.

Inventors:
BERTACCINI ASSUNTA (IT)
CONTALDO NICOLETTA (IT)
WINDSOR GEORGE DAVID (GB)
Application Number:
PCT/IB2012/052965
Publication Date:
December 19, 2013
Filing Date:
June 12, 2012
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV BOLOGNA ALMA MATER (IT)
PHYTOPLASMAS IN VITRO LTD (GB)
BERTACCINI ASSUNTA (IT)
CONTALDO NICOLETTA (IT)
WINDSOR GEORGE DAVID (GB)
International Classes:
C12N1/20; C12Q1/02; C12Q1/24; C12Q1/68
Other References:
BERTACCINI, A, CONTALDO N, CALARI A, PALTRINIERI S. WINSOR H, WINDSOR D: "Preliminary results of axenic growth of phytoplasmas from micropropagated infected periwinkle shoots", 18TH CONGRESS OF THE IOM (INTERNATIONAL ORGANISATION FOR MYCOPLASMOLOGY), 11-16 JULY 2010, CHIANCIANO TERME, ITALY, 16 July 2010 (2010-07-16) - 16 July 2010 (2010-07-16), pages Abstr. 147, 153, XP009165186
A BERTACCINI ET AL: "In vitro micropropagation for maintenance of mycoplasma-like organisms in infected plant tissues", HORTSCIENCE 27(9), vol. 27, no. 9, 1992, pages 1041 - 1043, XP055043941, Retrieved from the Internet [retrieved on 20121113]
I M LEE: "Prospects for in vitro culture of plant-pathogenic mycoplasmalike organisms", ANNUAL REVIEWS OF PHYTOPATHOLOGY, 1 January 1986 (1986-01-01), pages 339 - 354, XP055043697, Retrieved from the Internet [retrieved on 20121109]
S K GHOSH ET AL: "Isolation, cultivation and characterization of mycoplasma-like organisms from plants", PROCEEDINGS OF THE INDIAN NATIONAL SCIENCE ACADEMY, vol. 41, no. 4, 1975, pages 362 - 366, XP055043860, Retrieved from the Internet [retrieved on 20121112]
JANA FRÁNOVÁ: "Difficulties with conventional phytoplasma diagnostic using PCR/RFLP analyses", BULLETIN OF INSECTOLOGY, vol. 64, no. Supplement, 22 October 2011 (2011-10-22), pages S287 - S288, XP055043943, Retrieved from the Internet [retrieved on 20121113]
B. SCHNEIDER ET AL: "Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas", MICROBIOLOGY, vol. 143, no. 10, 1 October 1997 (1997-10-01), pages 3381 - 3389, XP055043944, ISSN: 1350-0872, DOI: 10.1099/00221287-143-10-3381
SASKIA A HOGENHOUT ET AL: "Phytoplasmas: bacteria that manipulate plants and insects", MOLECULAR PLANT PATHOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 9, no. 4, 1 July 2008 (2008-07-01), pages 403 - 423, XP002660927, ISSN: 1464-6722, [retrieved on 20080628], DOI: 10.1111/J.1364-3703.2008.00472.X
GAIL E. GASPARICH: "Spiroplasmas and phytoplasmas: Microbes associated with plant hosts", BIOLOGICALS, vol. 38, no. 2, 1 March 2010 (2010-03-01), pages 193 - 203, XP055043942, ISSN: 1045-1056, DOI: 10.1016/j.biologicals.2009.11.007
PADOVAN ET AL.: "Molecular detection of the Australian grapevine yellows phytoplasmas and comparison with grapevine yellows phytoplasmas from Italy", AUSTRALIAN JOURNAL OF GRAPE WINE RESEARCH, vol. 1, 1995, pages 25 - 31
LEE ET AL.: "Use of mycoplasma like organism (MLO) group specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant", PHYTOPATHOLOGY, vol. 84, 1994, pages 559 - 566
"Laboratory guide for identification of plant pathogenic bacteria", 2001, APS PRESS
MICROBIOLOGY, vol. 143, pages 3381 - 3389
Attorney, Agent or Firm:
PREDAZZI, Valentina et al. (Piazza di Pietra 39, Roma, IT)
Download PDF:
Claims:
CLAIMS

1 . A method for the culture of phytoplasma comprising the following steps: a) placing at least one micropropagated phytoplasma infected plant sample in Mycoplasma Experience Liquid medium that does not comprise thallium acetate (MEL/-) and incubating said sample at a temperature of about 0 to about 35°C for a period of time sufficient to bring the pH of the medium lower than 7.0, or to reduce the the pH of the medium;

b) sampling and diluting at least one aliquot of the culture broth obtained in the preceding step and plating said aliquot on at least one plate with Mycoplasma Experience Solid medium that does not comprise thallium acetate (MES/-); and c) incubating said plate under an atmosphere of about 90 to about 99% inert gas and about 1 to about 10% carbon dioxide at a temperature of about 0 to about 35°.

2. The method according to claim 1 , wherein said temperature in steps a) and/or c) is from about 15°C to about 30°C, optionally wherein the temperature is about 25± 1 °C.

3. Th e method according to any one of claims 1 or 2, wherein said atmosphere in step c) comprises from about 3% to about 7% carbon dioxide and from about 93% to about 97% nitrogen , optionally wherein the atmosphere comprises about 95% nitrogen and about 5% carbon dioxide.

4. The method according to any one of claims 1 to 3, wherein said step b) is carried out by using a MEL/- comprising a pH indicator.

5. The method according to claim 4, wherein said pH indicator is phenol red.

6. The method according to any one of claims 1 to 5, wherein the dilution in step b) is a dilution of at least 1 :9.

7. The method according to any one of claims 1 to 6, further comprising a step

d) of recovering at least one cultured phytoplasma colony from the plate of step c).

8. The method according to any one of claims 1 to 7, further comprising a step a') prior to step b) wherein an aliquot of the culture broth obtained in a) is sampled and diluted in fresh MEL/- and subsequently cultured according to the conditions set out in a).

9. The method according to any one of claims 1 to 8, wherein said plant sample is pre-treated by moistening it in said MEL/- medium, and optionally cut or sliced.

10. The method according to any one of claims 1 to 9, further comprising a step of phytoplasma characterisation and/or identification.

1 1 . The method according to claim 10, wherein said identification is carried out by PCR using 16S and/or tuf as target genes.

12. The method accordi ng to claim 1 1 , further comprising a Restriction Fragment Length Polymorphism analysis of the PCR obtained amplicons.

13. The method according any one of clai m s 1 to 12, wherein said micropropagated plant sample is infected with at least one phytoplasma strain belonging to stolbur and/or 'Candidatus Phytoplasma ' genera and taxa.

14. The method according to any one of claims 1 to 13, wherein said at least one plant sample comprises shoots, leaves or phloematic tissues.

15. The method according to any one of claims 1 to 14, wherein said at least one plant sample comprises plant materials from Catharanthus genus or other plant species, cultivars or varieties.

16. The method according to claim 15, wherein said plant materials are shoots of Catharanthus roseus sp.

17. A Phytoplasma colony obtainable by the method according to any one of claims 1 to 16.

18. A culture sample obtainable from step a) of the method according to any one of claims 1 to 17.

19. A kit for culturing at least one phytoplasma colony comprising at least one aliquot of Mycoplasma Experience Liquid without thallium acetate (MEL/-); at least one aliquot of Mycoplasma Experience Solid Media without thallium acetate (MES/-); control mea ns selected from mean s for determ i n i ng the p H and instructions for use.

20. The kit according to claim 19, further comprising at least one primer pair for amplifying the 1 6S phytoplasma gene and/or at least one primer pair for amplifying the tuf phytoplasma gene, and/or a combination thereof.

21 . The kit of claim 20, further comprising at least one culturing plate with or without MES/-; and/or at least one sterile tube; and/or an aliquot of sterile water; and/or at least one sterile tip; and/or at least one aliquot of restriction enzyme Tru\ and/or 7sp509; and/or at least one aliquot of Taq DNA polymerase; and/or at least one aliquot of dNTP mix.

Description:
METHOD FOR CULTURING PHYTOPLASMA

DESCRIPTION

The present invention relates to a method for cu lturing at least one phytoplasma colony, preferably under axenic conditions. The present invention also provides a kit for culturing at least one phytoplasma colony.

PRIOR ART

The evidence that several plant diseases were associated with phloem colonization by prokaryotes morphologically similar to mycoplasmas was first shown in 1 967. Since then , several hundreds of plant syndromes have been reported to be associated with the so-called mycoplasma-like organisms. Due to the lack of in vitro growth of these microorganisms, they were poorly characterized until the last ten years, when ribosomal rDNA sequencing provided evidence that these wall-less prokaryotes colonizing plant phloem and insects constitute a large monophyletic group within the class Mollicutes. This group has been identified as Phytoplasma genus.

It was demonstrated and confirmed that these organisms are associated with many plant d iseases worldwide and that genetically indistinguishable phytoplasmas can be associated with diseases and induce different symptoms and/or affect different plant species, and also that different phytoplasmas can be associated with similar symptoms in the same or in different plant host(s).

Plants infected by phytoplasmas exhibit an array of symptoms that suggests profound disturbances in the normal balance of growth regulators. Symptoms include virescence/phyllody (development of green leaf like structures instead of flowers), sterility of flowers, proliferation of auxiliary buds resulting in a witches' broom behaviour, abnormal internodal elongation, and generalized stunting.

Lots of cultivated plants are affected by phytoplasma infection, not only in countries where agriculture is still not very well advanced, but also in the so called more adva nced cou ntries where these pathogens severely damage both herbaceous and woody plants. The major diseases known to be associated with phytoplasmas are, in tropical areas, coconut lethal yellowing, sandal spike disease, pawlownia witches' broom, corn stunt, and rice yellow dwarf diseases. Forest trees are very often severely destroyed by phytoplasma epidemics in countries such as India and Central Africa, but also in US and in Europe. Elm yellows or witches' broom is a disease that almost eliminated historical as well as new elm plantations in Europe and in North America. Among fruit plants, grapevine, apple, pear, plum, apricot, cherry, citrus and the majority of small fruit are more or less severely affected by phytoplasma associated diseases described as yellows, decline, proliferation, and witches' broom. It has also been described that the phytoplasma infection of poi nsettia with branching leads to an i mproved branching than poinsettia without infection, hence, in some cases these prokaryotes have also a potential beneficial effect in some hosts.

The pathogen identification has re l i ed fo r m o re th a n 20 yea rs on microscopic observations (DAPI stain ing) or electron microscopy detection; however, in the last 20 years the applications of DNA-based technology allowed the differentiation of molecular clusters within these prokaryotes.

Phytoplasmas are wall-less prokaryotes with sizes varying from 200 to 800 nm, they are pleomorphic, and survive and multiply only in isotonic habitats, such as plant phloem or insect haemolymph; therefore they are strictly host-dependent, but they can multiply in insect vectors and also infect their eggs. The phytoplasma chromosome is very small (680-1 ,600 kb) and phylogenetic studies propose that the common ancestor for phytoplasmas is Acholeplasma laidlawii in which the triplet coding for tryptophan (trp) is UGG, while in other prokaryotes, including mycoplasmas and spi roplasmas, trp is coded by U GA. Phytoplasmas are genetically distinguishable from mycoplasmas infecting humans and animals by the presence of a spacer region (about 300 bp) between 1 6S and 23S ribosomal regions, which codes isoleucine tRNA (tRNAIIe) and part of the sequences for alanine tRNA (tRNAAIa). Sequencing of complete rRNA genes for two phytoplasma strains shows that tRNA coding for valine and asparagine are located downstream from the 5S rRNA gene, and th is is a unique feature of phytoplasmas. Early phytoplasma identification and classification systems proposed were based on specificity of vector transmission, on range of host plants and, more recently, on symptom expression of a common host such as periwinkle.

Because of the inability to isolate phytoplasmas in pu re cu lture, their identification has relied to date on serological methods or by the use of specific cloned DNA probes. From these studies, it appeared possible that several phytoplasma groups could be clearly distinguished by their chromosomal and extra chromosomal DNA sequences. The detection approach based, for example, on 1 6S gene of phytoplasma, provides rapid and reliable means for preliminary classification for epidemiological studies on diseases associated with phytoplasma presence. Polymerase chain reaction with primers from sequencing of randomly clon ed phytoplasm a D NA, from 1 6S rRNA, from ri bosomal protei n gene sequences, from SecY and Tuf genes, and from membrane associated protein genes open ed new paths for research on phytoplasma identification and classification. RFLP analysis, together with the sequencing of 16Sr phytoplasma genes, was the first step on the way to allowing the construction of phylogenetic trees of many microorganisms, especially those in the Mollicutes taxon.

The infection of plants by phytoplasmas is mainly performed by insect vectors belonging to a few species such as leafhoppers, plant hopper, cixiid, and psyllids. The main characteristic of insect vectoring is the ability to pass from infected to healthy plants due to the insect's phloem-feeding ability. In particular, the insect can acquire the pathogens by sucking the plant liquid; in this way, the alimentary canal, the haemolymph, as well as the salivary glands of the insect, are infected and the insect can transmit pathogens to healthy plants. There is usually a latent period in which the insect is infected but it is not able to transmit the pathogen to healthy plants; this period can last from a few hours to a few weeks.

Phytoplasmas are also transmitted by the majority of the dodder species, as well as by means of agricultural practices such as grafting, cutting, or other ways to propagate plant germoplasm avoiding sexual reproduction . Very recently, the possibility of phytoplasma transmission by seed has also been reported for some combinations of phytoplasma/plant species.

Control of epidemic outbreaks can be carried out theoretically either by controlling the vector or by eliminating the pathogen from the infected plants by antibiotics, mainly tetracyclines, or by other chemicals. Both protection measures are ineffective under field conditions: the first because it is impossible to eliminate all vectors from environments, and the second because the use of antibiotics is very expensive, not allowed in several cou ntries, and does not necessarily eliminate the infection completely. Therefore, the only real way to control phytoplasma infection is to prevent the outbreaks by finding phytoplasma-resistant varieties. Research into this important area is still very limited even though some basic knowledge about epidemiology, and physiopathology of phytoplasma associated with diseases is available.

The phytoplasma-related searches are still in their infancy and it would be crucial, for a better understanding and fighting of these organisms, to analyse them in detail. In order to do so, some researchers have been trying without success for a long period of time (about 20 years or more) to culture phytoplasmas in order to gain more consistent knowledge about these pathogens and diseases correlated to them, and thus define possible strategies to prevent and/or treat some plant diseases.

Hence, a method of culturing said organisms in an axenic way, would be essential for a better understanding of these organisms and to develop strategies for protecting plants from the same. SUMMARY OF THE INVENTION

In the present application, it is reported for the first time a method for culturing a phytoplasma colony from a plant sample infected by such a pathogen. The cu ltu ring method may optionally include a further step of recovering a phytoplasma colony. In particular, the inventors of the present specification have fou n d cond ition s th at a l low substantially pure culturing of phytoplasma microorganisms present in the plant sample, minimising at the same time the culture of the other possible organisms that may be present in the starting plant material. The present invention also provides a method of producing an axenic culture of phytoplasma. Axenic means pure cultures of phytoplasmas that are completely free of the presence of other organisms.

The conditions that the inventors found to allow the growth of phytoplasmas in vitro comprise the use of a micropropagated phytoplasma infected plant material as starting material, two specific media, namely Mycoplasma Experience Liquid (MEL/-) and Mycoplasma Experience Solid Media (MES/-), and specific conditions, defined in terms of the temperature and gas percentage.

In addition, the method hereafter described is characterized by a step of transferring from a liquid culture in MEL/- to a solid one in MES/- that is preferably carried out at a well defined moment identified by the reaching of an acid pH of the liquid culture (pH of less than about 7.0). A convenient method for ascertaining when the liquid culture medium has become acidic involves the use of phenol red indicator which displays a colour change at a pH of less than or equal to about 6.8. Accordingly, the transfer step may be carried out once the liquid culture medium is at a pH of less than or equal to about 6.8.

Alternatively, the transfer step may be carried out once a noticeable reduction in the pH of the liquid culture medium is displayed, e.g. a reduction in pH of about 0.20, about 0.30, about 0.40, about 0.50, or about 1 .0 pH units. The original pH of the liquid culture medium will be around physiological pH, e.g. in the range of about pH 7.0 to about pH 8.0, preferably from about pH 7.2 to about pH 7.6 , more prefera bly from a bout pH 7.3 to about pH 7.5. Since growth of phytoplasma will lead to an increase in acidic by-products, a noticeable reduction in pH of the liquid culture medium is indicative of growth . Therefore, where the original pH is about 8.0, a noticeable reduction might be a reduction to a pH of about 7.8, 7.7, 7.6, 7.5 or 7.0, and similarly for other starting pH values.

The transfer step may alternatively be carried out following a set amount of time without any monitoring of the pH of the liquid medium.

It is the presence of the technical features/steps above in the method that make possible culturing and, optionally recovering, of colonies belonging to the Phytoplasma genus.

The invention also provides a kit for culturing at least one phytoplasma colony, the kit comprising at least one aliquot of each suitable med iu m and optionally comprising control elements as set out in the detailed description of the invention.

The main advantage of the method or kit herein described is therefore the possibility of isolating phytoplasmas free from the other plant pathogens and thus having a reliable method for obtaining a source of phytoplasma colonies from different phytoplasma strains on which any further analysis can be carried out in order to improve the scientific knowledge about this parasite and its pathogenetic mechanisms. As a con seq u en ce, strategies a im i ng to treat and/or prevent phytoplasma related plant diseases can be better defined. In other words, the setup of the method here described will allow the validation of phytoplasmas as real etiological agents of many plant diseases (satisfaction of Koch's postulates) and the study of cycles of diseases to develop the correct strategies to fight and red uce the socio-economic i mpact that phytoplasma d iseases have in all agricultural areas of the world . In addition, a faster production of diagnostic reagents can be achieved, as well as a more detailed knowledge about basic mechanisms that regulate the survival of such pathogens, which are among the smallest known living organisms that can be obtained.

Aspects of the present invention have a variety of commercial applications. For example, the present invention allows for:

(i) detection of phytoplasma infection by culture;

(ii) the improvement of diagnostic techniques by allowing the preparation of more specific antisera because multiple antigens can be prepared from cultures in the same time for very effective and broad spectrum antisera preparation;

(iii) the rapid assessment in vitro of compounds/chemical agents that show potential to inhibit the growth of phytoplasmas causing diseases to plants or crops of economic importance;

(iv) the production of high titre broth cultures of phytoplasma leading to the development of novel methods of plant infection with the consequences of (a) acceleration of selection and/or screen ing of plants resistant to phytoplasma infection, (b) infection of plants causing horticultural benefits including, for example dwarf or shrubby plants (such as poinsettia), small flowers, unusually coloured flowers or leaves, and/or production of small, ornamental fruits;

(v) the study of the pathogenesis of phytoplasma in plant infection with in vitro procedures leading to the possible amelioration of deleterious effects in the absence of elimination, or the possible modification and/or increase of such effects where they confer a horticultural benefit;

(vi) the genetic modification of phytoplasma in vitro in order to confer some benefit, for exam ple to (a) eliminate or attenuate pathogenesis, (b) secrete substances having fungicidal and/or bactericidal properties, (c) secrete compounds of benefit to the plant, (d ) provide a horticultural modification, such as those described in (iv)(b) above; and

(vii) isolation from insect vectors infected with phytoplasmas using media developed for culture of plant phytoplasmas which would allow the study of the passage of phytoplasmas from the gut to the salivary glands, such study could allow for the development of compounds which disrupt the passage through the insect without the elimination of the vector or closely related insects i.e. targeting phytoplasma transmission rather than the insect.

A first aspect of the present invention is therefore a method for the culture of phytoplasma comprising the following steps:

a) placing at least one micropropagated phytoplasma infected plant sample in Mycoplasma Experience Liquid medium that does not comprise thallium acetate (MEL/-) and incubating said sample at a temperature of about 0 to about 35°C for a period of time sufficient to bring the pH of the medium lower than about 7.0, or to reduce the pH of the medium;

b) sampling and diluting at least one aliquot of the culture broth obtained in a) and plating said aliquot on Mycoplasma Experience Solid medium that does not comprise thallium acetate (MES/-); and

c) incubating said plated aliquot under an atmosphere of about 90 to about

99% inert gas and about 1 to about 10% carbon dioxide at a temperature of about 0 to about 35°C.

A second aspect of the present invention is a phytoplasma colony obtainable by the above method.

A third aspect of the present invention is the acid liquid broth obtainable in step d) of the above method.

A fourth aspect of the present invention is a kit for culturing at least one phytoplasma colony comprising at least one aliquot of Mycoplasma Experience Liquid without thallium acetate (MEL/-); and at least one aliquot of Mycoplasma Experience Solid Media without thallium acetate (MES/-).

The teachings of the present description also enable a person skilled in the art to carry out a method for the screening in vitro of compounds inhibiting phytoplasma's growth, comprising the following steps

a') placing at least one micropropagated phytoplasma infected plant sample in Mycoplasma Experience Liquid medium that does not comprise thallium acetate (MEL/-) and incubating said sample at a temperature of about 0 to about 35°C for a period of time sufficient to bring the pH of the medium lower than about 7.0, or to reduce the pH of the medium or

a") placing at least one phytoplasma colony in Mycoplasma Experience Liquid medium that does not comprise thallium acetate (MEL/-) and incubating said sample at a temperature of about 0 to about 35°C for a period of time sufficient to bring the pH of the medium lower than about 7.0, or to reduce the pH of the medium.

b') sampling and diluting at least one aliquot of the culture broth obtained in a) and plating said aliquot on at least two different plates or in at least two different liquid aliquots with Mycoplasma Experience Solid medium that does not comprise thallium acetate (MES/-); and

c') incubating each of said aliquots under an atmosphere of about 90 to about 99% inert gas and about 1 to about 10% carbon dioxide and at least one test compound or a mixture of test compounds at a temperature of about 0 to about 35°C wherein at least one aliquot is plated as control without test compounds; d') comparing the at least one control plate with the plates treated with at least one test compound or a mixture of test compounds.

A sixth aspect of the present invention is a kit for the screening in vitro of compounds inhibiting phytoplasma's growth comprising at least one aliquot of Mycoplasma Experience Liquid without thallium acetate (MEL/-); and at least one aliquot of Mycoplasma Experience Solid Media without thallium acetate (MES/-) distributed in at leas two ready-to-use plates or suitable for the preparation of at least two plates thus allowing the comparison of at least one test compound on at least one plate of said solid medium with respect to a control plate.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 is a photograph showing plating from a MEL/- broth culture of

'Candidates Phytoplasma asteris' strain CY (CY-TO 94).

Figure 2 is a h igh power photograph showing a colony of the same organism ['Ca. P asteris' strain CY (CY-TO 94)] after two sub-cultures.

Figure 3 is a photograph showing reticulated colonies and individual colonies of the same organism ['Ca. P. asteris' strain CY (CY-TO 94)].

Figure 4 is a photograph showing separated colonies of Mycoplasma arginini. Figure 5 is a photograph of PCR results in agarose gel 1 % stained with ethidium bromide and photographed under UV light. The results were obtained with primers M1/M2 on the broth growth of the for tubes of CH-1 (1 -4) n. 5 is water negative PCR control. M, 1 kb DNA ladder.

Figure 6 is a photograph PCR results as before obtained with primers

R16(I)F1/R1 on the broth growth of the for tubes of CH-1 (1 -4) W is water negative PCR control. M, 1 kb DNA ladder.

Figure 7 is a photograph of RFLP results obtained after Tru1\ digestion of amplicons in figure 5. P, PhiX174 Haelll digested marker.

Figure 8 is a photograph of RFLP results obtained after Tru1\ digestion of amplicons in figure 6. P, PhiX174 Haelll digested marker.

Figure 9 is a photograph showing reticulated colonies and individual colonies of PD44A.

Figure 10 is a photograph of PCR results obtained with primers M1/M2 on broth growth (2-4) and colonies (5-7) of PD44A n. 1 is water negative PCR control. M, 1 kb DNA ladder.

Figure 1 1 is a photograph of RFLP results obtained after Tru1\ digestion of amplicons in figure 10. P, PhiX174 Haelll digested marker.

Figure 12. Acid colour change of PD44A growing in MEL/- medium (right) on the left the negative control.

Figure 13 is a photograph of isolation in plate from a MEL/- broth culture of STOL59.

Figure 14 is a photograph of separate colonies of STOL59.

Figure 15 is a photograph of RFLP results obtained after Tru1\ digestion of amplicons M1/M2 of samples from P1 and P2 in experiment 2. P, PhiX174 Haelll digested marker. 1 , STOL59 from P1 ; 2, STOL59 from P2; 3, TBB from P1 ; 4, TBB from P2; 5, PD44A from P1 ; 6, PD44A from P2; 7, CY-T094 from P1 ; 8, CY-T094 from P2; 9, PD44 from P1 ; 10, PD44 from P2; 1 1 , PD44 * from P2; STOL59 from P1 ; PD44 * from P1 .

DETAILED DESCRIPTION OF THE INVENTION

Hence, the present invention relates to a method for the substantially pure culturing of phytoplasma, i.e. a method for the growth of phytoplasmas, while minimising growth of living organisms or living cells of any other species. For the first time, it is also provided a method that allows culturing of phytoplasmas in an axenic form and that allows also the isolation of single phytoplasma colonies from solid medium.

The present invention will be described below by means of individual embodiments and features which are described separately. For the avoidance of doubt, the present invention also encompasses subject-matter resulting from any such individual embodiment or feature being combined with one or more additional embodiments or features.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology etc).

In the present specification, the term "about" means plus or minus 20%; more preferably plus or minus 10%; even more preferably plus or minus 5%; most preferably plus or minus 2%.

In the present specification, the term "substantially pure" means at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, most preferably at least 99% pure.

I n the present specification, the term "comprises" means "includes" or "contains", i.e. other integers or features may be present, whereas the term "consists of" means "consists exclusively of".

The terms "comprises-comprising" can be substituted by the term "consists of-consisting of" in any embodiment herein described.

I n the present specification th e term "colony" refers to a g rou p of microorganisms grown from a single parent cell or a small cluster of parent cells; thus, the expression "phytoplasma colony" indicates a circumscribed number of phytoplasma cells, normally derived from a single cell or small cluster of cells growing on a solid or semisolid medium.

For the first time it is hence possible to have single colony cultures of organisms belonging to the same phytoplasma strain.

In particular, the method for culturing of the present invention comprises different steps of which the first, namely step a), consists in recovering at least one micropropagated plant sample that has been i nfected by phytoplasma. The expression "micropropagated phytoplasma infected plant sample" refers to a plant or portion or tissue thereof which is infected by one or more strains belonging to 'Candidatus Phytoplasma' genus, but not by other plant pathogens, that can e.g. be obtained from a collection of micropropagated phytoplasma infected plant samples. Shoots are normally only infected by phytoplasmas, however time to time in some cases other organisms (endophytes) could be detected on shoot growing medium, such shoots should be discarded and not used for the cultivation. The use of micropropagated phytoplasma infected shoots is not crucial for cultivation and the medium for micropropagation is described (Bertaccini et al., 1992; Murashige and Skoog, 1 962) O ne of these collections is the "Phytoplasma collection in micropropagated shoots" of the Phytoplasmology Laboratory, Plant Pathology, DiSTA - Alma Mater Studiorum - University of Bologna, Italy (http://www.IPWGnet.org)., other collections available are in greenhouse maintained plants and are located in Udine (Italy) and Bordeaux (France). The use of such plant materials avoids external contamination that arises when samples from plants infected not only by phytoplasmas but also by other microorganisms are used. In fact, there are problems in culturing phytoplasmas when other microorganisms, such as endophytes, are present in the same sample, which usually happens, for example, in field-collected plant materials.

By way of example, the infected plant sample used as starting material can be infected with strains from stolbur and/or 'Candidatus Phytoplasma' genus or phytoplasmas. Phytoplasma strains for use in the method of the present invention include 'Candidatus Phytoplasma asteris' strain CY (CY-TO 94), 'Candidatus Phytoplasma aurantifolia' (TBB61 ), 'Candidatus Phytoplasma pyri' (PD 44) pear decline, stolbur phytoplasma strain PD 44A(10PD), stolbur phytoplasma strain CH- 1 (C H-1 99), and stolbur phytoplasma strain STOL (STO L 59). It is to be understood that such phytoplasma taxon are merely possible embodiments of the invention in suit and that any other 'Candidatus phytoplasma' genera as well as particular phytoplasma strains can be used for infecting plant materials in order to obtain the micropropagated plant sample infected by phytoplasma for use in the method herein described.

The plant sample may comprise or consist of one or more plant portions or tissues infected by phytoplasma. Such plant materials can be, for example, shoots, leaves, lymph or any part containing phloematic tissues where phytoplasmas are located. In one exemplified embodiment of the invention, the plant materials are from Catharanthus genus; however, it is evident that in order to carry out the method of the invention any plant species, genus, and/or variety infected by phytoplasma can be used as a source for the infected material suitable for use in the method as herein descri bed . Accord ing to a non-limiting example, the micropropagated plant material comprises or consists of shoots of Catharanthus roseus sp. infected by a stolbur phytoplasma strain.

The plant sample as above described is placed, according to step a) of the method of the invention, in a suitable liquid growth medium that shall not comprise thallium acetate. Thallium acetate has been shown to be inhibitory to the growth of the cultured phytoplasmas for each of the strains tested as listed above and in Table 1 , so that the absence of thallium acetate is essential at least for these particular phytoplasma strains. A suitable medium for the method of the invention is the medium Mycoplasma Experience Liquid (MEL/-), available from Mycoplasma Experience (http://www.mycoplasma-exp.com/hompag.htm), which is obtainable from Phytoplasmas in vitro under product codes pIV L1 , L5 or L1 0 (1 1, 51 or 1 01 batches of the liquid medium). MEL/- is a liquid culture medium that comprises a broth base, heat inactivated pig serum, yeast extract and pure chemical nutrients. MEL additionally comprises thallium acetate, whereas MEL/- is free from thallium acetate. In particular, the MEL media comprise

Broth base/Agar - 50 to 80%

Pig serum - 5 to 25%

Yeast extract - 2.5 to 15%

Pure chemical nutrients - 0.25 to 2.5 %

The skilled person can hence either commercially obtain the MEL media from Mycoplasma Experience or develop suitable growth media for carrying out the invention without use of inventive skill.

MEL/- is a culture liquid that has proven, differently from several other media, to be suitable for culturing phytoplasma in substantially pure, or more preferably axenic, conditions. The inventors have found that, in order to avoid the inhibition of the phytoplasma growth, is essential that the MEL used does not comprise thallium acetate (i.e. MEL/-) since it has been demonstrated to inhibit the growth of some phytoplasma strains.

The starting plant material can be used as such, namely as recovered, or, for example, it can also be pre-treated by moistening it in said MEL/- medium or in a sterile isotonic solution (such as brine, pbs, or other commonly used sterile isotonic solutions) and optionally cut, sliced, grinded, partially homogenised, or partially disrupted for a period of time sufficient to disintegrate the plant material, for example, for a period of time of from about ten seconds up to about two minutes, preferably from about thirty seconds up to about one minute. Preferably, following moistening of the plant material, it is cut or sliced, which provides a gentle manipulation of the plant material to enable release of the phytoplasma from sieve tubes, while avoiding damage to the phytoplasma that might occur with more vigorous grinding or homogenising.

Since the method herein described is intended to produce a substantially pure, preferably an axenic, culture of phytoplasmas, it is clear that the preparation of the plant material (e.g. by slicing, grinding, partial homogenisation or disruption) has to be carried out in sterile conditions.

The preparation of the material as described above is not essential, but the preparation step has the advantage of facilitating the transfer of the phytoplasmas from the plant material into the culturing medium.

The plant material placed in the MEL/- is then incubated, according to step a) of the method herein described, at a temperature of about 0 to about 35°C until the pH of the medium reaches a value that is lower than 7.0, or until there is a noticeable reduction in the pH of the medium, thus obtaining a culture broth. By "culture broth" is meant the M EL/- comprising the sample treated as described which contains, at the desired pH, at least one viable phytoplasma cell.

The temperatu re su itable for achieving the phytoplasma growth is a temperature near to the approximate ambient temperature at which growth of the relevant phytoplasma strain occurs within a suitable host plant in the natural environment. The majority of phytoplasma strains are found in host plants that are native to tropical climates, whereas certain phytoplasma strains are found in host plants that grow in temperate climates, e.g. stone fruits. Accordingly, suitable temperatures may be within the range of about 0°C to about 35°C, preferably within the range of about 15°C to about 30°C, and more preferably within the range of about 20°C to about 28°C. In certain embodiments, the temperature under which the phytoplasma is cultured is selected from about 20, 20.5, 21 , 21 .5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27,5 and 28°C. Once an appropriate temperature for the phytoplasma strain in question has been determined , the temperature is preferably regulated, for example by culturing in an incubator which can maintain a predetermined temperature with an accuracy of ±1 °C. In a particular embodiment of the method, a highly efficient temperature for allowing phytoplasma growth, is a temperature of about 25± 1 °C.

During the incubation as described hence, the pH of the culture broth may advantageously be monitored in order to asses the phytoplasma rate of growth. This can be achieved by any suitable means known by a skilled person in the art for such purpose such as, for example, a pH paper indicator or a pH meter. Merely by way of example, the pH can be monitored by taking at least one aliquot of the liquid broth at regular time intervals and testing it using a pH meter. Step a) of the method herein described could include a pH measurement before placement of the sample in the MEL/- medium. I n one embodiment, a pH indicator can be also comprised in the culturing medium so that an easy monitoring and early detection of pH changes can be achieved . Preferably, the pH indicator is an indicator capable of showing, by a colour change, when a basic medium becomes acidic. Any suitable indicator known in the art can be used. In an embodiment, the pH indicator present in the MEL/- medium can be phenol red that changes from a red to yellow colour when a pH of less than of equal to about 6.8 is reached.

The ti m in g of colou r changes of the liquid broth is different among phytoplasma strains since it is linked to the growth rate of the strain as well as its concentration in the starting plant material. As shown in table 1 of example 1 below, this timing may range from about 13 days to more than 100 days starting from the incubation according to step a) above described.

When the pH of the liquid broth reaches a pH value of less than about 7.0, namely it becomes acidic, at least one aliquot of said acidic broth can be sampled and diluted. The dilution of the acidic broth can be carried out at any pH acid value reached in a range lower than 7.0 such as, by way of example, at a pH value of less than 6.9, 6.8, 6.7, 6.5, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, or 5.0.

Alternatively, when there has been a noticeable decrease in the pH of the liquid broth, at least one aliquot of said broth can be sampled and diluted. For example, the dilution may be carried out following a reduction in the pH of the liquid broth of about 0.20, about 0.30, about 0.40, about 0.50, or about 1 .0 pH units. In this case, the dilution of the broth can be carried out at any pH acid value reached, e.g. at a pH value of about 7.5, 7.4, 7.3, 7.2, 7.1 , 7.0 6.9, 6.8, 6.6, 6.7, 6.5, 6.4, 6.3, 6.2, 6.1 or 6.0.

By way of example, the dilution is of at least about 1 :10 or 1 :9 in fresh medium hence, for any about 100μΙ of culture broth, at least about 900μΙ or at least about 1 ml of fresh medium can be used for the dilution step. Subsequently, a portion of said diluted aliquot, for example about 50 μΙ, is plated on a plate containing Mycoplasma Experience Solid Media (MES/-). The volume in microlitres of the diluted aliquot to be plated can be readily determined by the skilled person taking into account the necessity of allowing the complete absorption of said aliquot onto the solid medium. In addition, or alternatively, a diluted aliquot can be transferred into a fresh MEL/- for carrying out a more extensively liquid culture or for other purposes such as, for example, carrying out identification tests on it (see below for more details).

In fact, the method of the invention, can comprise a further step a') prior to step b) wherein an aliquot of the culture broth obtained in a) is sampled and diluted in fresh MEL/- and subsequently cultured according to the conditions set out in a).

The diluted sample as described is plated , according to step b) of the method , on a plate comprising solid medium. The solid medium to be used according to the method of the invention, is as above indicated the MES/- medium which is also available from Mycoplasma Experience (http://www.mycoplasma- exp.com/hompag.htm), and which is also obtainable from Phytoplasmas in vitro under product codes pIV S1 , S5, or S10 (11, 51 or 10I batches of the solid medium). It is essential that this medium does not comprise thallium acetate (MES/-) since this compound inhibits the growth of some phytoplasma strains, as explained above. MES/- is a solid culture medium that comprises a broth base, heat inactivated pig serum, yeast extract and pure chemical nutrients, the medium being solidified with agarose. MES additionally comprises thallium acetate, whereas MES/- is free from thallium acetate.

In particular, the MES media comprise

Broth base/Agar - 50 to 80%

Pig serum - 5 to 25%

Yeast extract - 2.5 to 15%

Pure chemical nutrients - 0.25 to 2.5 %

The skilled person can hence either commercially obtain the MES media from Mycoplasma Experience or develop suitable growth media for carrying out the invention without use of inventive skill.

The plate on which the diluted aliquot has been absorbed is then incubated i n step c) under conditions suitable for substantially pure, preferably axenic, culturing of phytoplasma colonies. In particular, the axenic conditions found by the inventors of the present method, that allow specifically the growth of at least of one colony of cells belonging to Phytoplasma genus are: an atmosphere of about 90 to about 99% inert gas and about 1 to about 10% carbon dioxide at a temperature of about 0 to about 35°C. The incubation of the plate according to step c) as above described under said conditions is a key point of the method for culturing and subsequently recovering, if desired, single phytoplasma colonies. I n fact, when different conditions were tested such as aerobic growth, no phytoplasma colony was observed. Accordingly, step c) should be carried out in an atmosphere that does not comprise oxygen. Since carbon dioxide is necessary to achieve growth of the phytoplasma, the atmosphere should include at least about 1 % carbon dioxide. Preferably, the atmosphere should comprise from about 1 % to about 10% carbon dioxide, more preferably the atmosphere should comprise from about 2% to about 8% carbon dioxide, even more preferably from about 3% to about 7% carbon dioxide, most preferably from about 4% to about 6% carbon dioxide. In a preferred aspect, the atmosphere should comprise about 5% carbon dioxide. The remainder of the atmosphere should predominantly be an inert gas or a mixture of such gases. Accordingly, the atmosphere should comprise from about 90% to about 99% inert gas, more preferably from about 92% to about 98% inert gas, even more preferably from about 93% to about 97% inert gas, most preferably from about 94% to about 96% inert gas. In a preferred aspect, the atmosphere should comprise about 95% inert gas. Preferred inert gases include argon and nitrogen, particularly nitrogen. Accordingly, the atmosphere may preferably comprise about 5% carbon dioxide and about 95% nitrogen. In particular, optimum growth conditions occur in an atmosphere of about 95% N 2 and about 5% C0 2 at a temperature of about 25 ± 1 °C.

The growth of phytoplasma colonies on the plate as described normally occurs in 2-5 days from the incubation according to step c).

Once phytoplasma colonies are grown, they can be recovered from plates by standard procedures of colony picking and can be grown in MEL/- medium under the conditions described for step a).

I n order to confirm the presence of phytoplasma cells, the method can further comprise a step of phytoplasma characterisation and/or identification. This can be carried out according to any procedure known by the skilled person at different stages of the method above; for example, identification tests can be performed either on the culture broth according to step a), on the sample according to step b) and/or on phytoplasma colonies obtainable according to step c). The identification tests can be based on morphological or genetic differences between phytoplasma cells with respect to close related microorganisms inspected, for example, by routine laboratory tests and analysis. Thus, for example, phytoplasma colonies can be observed under an optical bifocal microscope and compared in shape and dimension with reference photographs of phytoplasma, such as those provided in Figures 1 -3. Phytoplasma colonies may also be compared to those of mycoplasmas (Figure 4), the most closely related organisms. Phytoplasma colonies are generally reticulated, unlike mycoplasma colonies, but can also be present in colonial form similar to mycoplasma colonies, as shown in Figure 3.

In addition, or alternatively, the identification can be based on molecular tests, and a preferred molecular methodology that can be used for such purpose is a PCR analysis based on the amplification of genes or portions of genes known to be specific for phytoplasma detection. Different genes have been described in the art as reliable genes for phytoplasma identification and among these the 16S and tuf genes can be used to asses the presence of phytoplasma in the sample under examination. Hence, the identification step can be carried out by PCR using 16S and/or tuf as target genes. Merely by way of example, direct PCR can be performed using universal primers for phytoplasma detection such as those designed by Gundersen and Lee, 1 996, and commonly indicated R16F2/R2 (Gundersen and Lee, 1996.- Ultrasensitive detection of phytoplasma by nested PCR assays using two universal primer pairs.- Phytopathologia mediterranea, 35: 144-151 ), followed by nested-PCR with both, universal (16R758f/16R1232r=M1/M2 described in Padovan et al., 1995.- Molecular detection of the Australian grapevine yellows phytoplasmas and comparison with grapevine yellows phytoplasmas from Italy.- Australian Journal of Grape Wine Research , 1 : 25-31 ) and group specific primers for the strain cultivated up to now, in particular 16Sr(l)F1 /R1 (Lee et al., 1994.- Use of mycoplasma like organism (M LO) group specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant.- Phytopathology, 84: 559-566). It is also possible to use in the identification step different molecular methodologies since, as known by the state of the art, phytoplasmas are microorganisms morphologically and genetically similar to mycoplasmas. Hence, in one embodiment, the method of the present invention may further comprise, after the P C R step, a Restriction Fragment Length Polymorphism (RFLP) analysis. RFLP analyses with specific restriction enzymes such as Tru1\, Tsp509\ can confirm the presence of phytoplasma. Alternatively, or in addition, direct sequencing of PCR products can give a further final confirmation of phytoplasma identity.

The skilled person will be readily able to identify techniques suitable for the characterisation step above according to routine laboratory guides and manuals (see "Laboratory guide for identification of plant pathogenic bacteria" APS Press, 2001 ).

Depending on the needs of the user, the method can simply comprise an identification step in order to verify the presence of phytoplasmas in the sample, or it could comprise more complex characterisation step/s as described above.

Colonies and culture broths found to be positive to phytoplasmas by any suitable genetic and/or morphological analysis can be stored, preferably at -80°C, for future investigations and scientific purposes. Therefore, a further aspect of the present invention is a phytoplasma colony, e.g. picked, re-suspended and/or cultured in MEL/- as described above and/or a sample of culture broth obtainable at the end of step a) of the method above described.

Following the teachings above, the present description hence enables a skilled person to carry out a method for the screening in vitro of compounds inhibiting phytoplasma growth without use of inventive skill.

An example of such method is a method comprising the following steps a') placing at least one micropropagated phytoplasma infected plant sample in Mycoplasma Experience Liquid medium that does not comprise thallium acetate (MEL/-) and incubating said sample at a temperature of about 0 to about 35°C for a period of time sufficient to bring the pH of the medium lower than about 7.0, or to reduce the pH of the medium;

b') sampling and diluting at least one aliquot of the culture broth obtained in a) and plating said aliquot on at least two different plates or in at least two different liquid aliquots with Mycoplasma Experience Solid medium that does not comprise thallium acetate (MES/-); and

c') incubating each of said aliquots under an atmosphere of about 90 to about 99% inert gas and about 1 to about 10% carbon dioxide and at least one test compound or a mixture of test compounds at a temperature of about 0 to about 35°C, wherein at least one aliquot is plated as control without test compounds; d') comparing the at least one control plate with the plates treated with at least one test compound or a mixture of test compounds.

All the steps can be carried out as in the culturing method described above, and all the possible embodiments applicable to the culturing method described above can be applied to the screening method herein described; however, step c') will be carried out in parallel on various plates, at least one plate will be prepared as in step c) of the culturing method described above, i.e. incubating the aliquot plated thereon under an atmosphere of about 90 to about 99% inert gas and about 1 to about 10% carbon dioxide at a temperature of about 0 to about 35°C; and one or more plates will be used for the screening of test compounds by incubating each plated aliquot under an atmosphere of about 90 to about 99% inert gas and about 1 to about 10% carbon dioxide and at least one test compound or a mixture of test compounds at a temperature of about 0 to about 35°C.

The method may further comprise a step d) of comparing the at least one control plate with the plates treated with at least one test compound or a mixture of test compounds.

The method hence allows the parallel testing of several test compounds on the same phytoplasma broth culture.

The method can be carried out even on single colonies prepared with the culturing method of the invention, hence according to steps a), b) and c) of the culturing method, wherein step a') of the screening method is substituted by step a") of

- placing at least one phytoplasma colony in Mycoplasma Experience Liquid medium that does not comprise thallium acetate (MEL/-) and incubating said sample at a temperature of about 0 to about 35°C for a period of time sufficient to bring the pH of the medium lower than about 7.0, or to reduce the pH of the medium. It is understood that the phytoplasma colony can be readily obtained according to the culturing medium herein described.

Another aspect of the present invention is a kit for culturing at least one phytoplasma colony. Said kit for cultu ring at least one phytoplasma colony comprises at least one aliquot of Mycoplasma Experience Liquid without thallium acetate (MEL/-); and at least one aliquot of Mycoplasma Experience Solid Media without thallium acetate (M ES/-) ; control mean s selected from mea ns for determining the pH and, optionally at least one primer pair for amplifying the 16S phytoplasma gene and/or at least on e pri m er pa i r for amplifying the tuf phytoplasma gene, and/or a combination thereof, reagents suitable for amplification (e.g. amplification buffer, MgCI2, dNTPs, Taq Polymerase); and instructions for use.

A phytoplasma infected plant sample, preferably a micropropagated plant sample, can be used with the kit of the invention in order to carry out the method of for the culture of phytoplasma as described herein. Alternatively, colonies or broths obtainable by step a) or c) of the culturing method herein described can be used.

The kit may comprise the culturing media described above in ready to use aliquots, i.e. tubes or flasks of Mycoplasma Experience Liquid without thallium acetate (MEL/-) and plates of Mycoplasma Experience Solid Media without thallium acetate (MES/-). As pH variation (noticeable pH decrease of the liquid medium) is essential in order to establish the successful growth of phytoplasmas in the liquid medium, the kit will be supplied with means for monitoring the pH variation during the liquid culturing step (i.e. step a) of the culturing method herein described or step a') of the screening method herein described.

I n one embodiment of the invention the Mycoplasma Experience Liquid without thallium acetate (MEL/-) aliquots of the kit will comprise phenol red.

The kit may further comprise one or more of the following means suitable for carrying out the method herein described, such as, e.g. at least one culturing plate with or without MES/-; and/or at least one sterile tube; and/or an aliquot of sterile water; and/or at least one sterile tip; and/or at least one aliquot of restriction enzyme Tru\ and/or 7sp509; and/or at least one aliquot of Taq DNA polymerase; and/or at least one aliquot of dNTP mix.

The kit of the present invention as described above is also suitable for the screening in vitro of compounds inhibiting phytoplasma's growth, in this embodiment the kit will advantageously comprise at least one aliquot of Mycoplasma Experience Liquid without thallium acetate (MEL/-); the at least one aliquot of Mycoplasma Experience Solid Media without thallium acetate (MES/-) will be conveniently provided in at least two plates of Mycoplasma Experience Solid Media without thallium acetate (MES/-), thus allowing the comparison of at least one test compound on at least one plate of said solid medium with respect to a control plate.

Alternatively, any embodiment of the kit herein described may comprise a liquid or agar diagnostic medium on a cellulose pad. A membrane filter on such a cellulose pad allows developments of red colonies of phytoplasma.

The next section aims to illustrate the method for culturing phytoplasma colonies according to the present invention and therefore aims to clarify the invention without of course being limitative of the same.

EXAMPLES

Example 1 : first isolation and culturing phytoplasma colonies

Two micropropagated phytoplasma strains were employed CH-1 a n d PD44A for phytoplasma isolation in the laboratory of the Bologna University. Two lengths of stem per shoot of approximately 0.5-1 cm in length were moistened with 1 .0 ml of M E L/- and sliced along their axes by cutting the stems with sterile scalpels whilst held with sterile forceps. After slicing, the liquid plus plant pieces were a l l tra n sferred to a 4 ml vacuette tube (Greiner bio-one, ref 454001 ) containing 2.5 ml of Mycoplasma Experience Liquid Medium (MEL/-). This was repeated for each plant giving two broth cultures with stem pieces from all shoots used. The experiment was carried out in two different times according with the different strain of phytoplasma.

a) Strain CH-1. Isolations were carried out from shoots labelled CH 1 a and CH 1 b for a total of 4 tubes. The tubes were incubated at a temperature of 25 ± 1 °C for 4 days, after which 300 μΙ of broth culture from each of the 4 tubes was transferred into 4 new 8 ml vacuette tubes (Greiner bio-one, ref 454001 ) containing the same medium. These 4 tubes were incubated under the same conditions until colour change from orange-red (pH above 7.0) to yellow (pH below 6.8), which could indicate growth of phytoplasmas, appeared (nine months). Aliquots of 100 μΙ from the 4 tubes were transferred to 4 tubes of fresh medium which all gave acid colour changes after incubation for 3 days; after 10 days incubation, PCR testing was positive for the presence of phytoplasma DNA. PCR amplification was carried out using as template 1 μΙ of the pellets obtained from full speed centrifugation of 100 μΙ medium, resuspended in 10 μΙ of sterile distilled water. Primers employed for these latter amplifications were M1 /M2 generic for phytoplasma 16Sr gene, and R16(I)F1/R1 , specific for phytoplasma groups 16Srl, II and XII (PCR results shown in Figures 5 and 6). RFLP analyses confirmed that 16SrXI I phytoplasmas were present in the broth media of the 4 tubes after transfers (as shown in Figures 7 and 8). Using primers M1/M2, amplicons of about 550 and 1 ,100 bp were obtained from these tubes and direct sequencing confirmed 16SrXII phytoplasma presence. Further PCR amplifications were performed using primers Tufu f/r (Schneider et al. 1997). Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas (Microbiology, 143: 3381 -3389) resulted in two out of the 4 tubes providing expected length bands (1 ,000 bp). Direct sequencing confirmed the presence of stolbur phytoplasmas.

b) Strain PD44A. Isolations were carried out from shoots labelled PD44Aa and PD44Ab for a total of 4 tu bes. The tubes were incubated at a temperature of 25 ± 1 °C and inspected for signs of a colour change from orange- red (pH above 7.0) to yellow (pH below 6.8) wh ich could indicate growth of phytoplasmas. After about two months, colour changes was present and 100 μΙ of broth culture from each of the 4 tubes was transferred into 4 new 8 ml vacuette tubes (Greiner bio-one, ref 454001 ) containing the same medium and incubated under the same conditions. Moreover, 50 μΙ of acid broth cultures were inoculated onto 6 cm diameter plastic plates containing 8 ml of Mycoplasma Experience Solid Medium (MES/-) (MES without thallium acetate) and incubated upside down in an atmosphere of 95% N 2 / 5% C0 2 at 25 ± 1 °C. Colonial growth of phytoplasmas occurred in two to five days (colonies shown in the photograph of Figure 9). 100 μΙ of liquid medium were transferred to a 2 ml eppendorf tube, under sterile conditions. After a centrifugation at 1 1 ,000 rpm for 20-30 min, the supernatant was transferred to a new 2 ml tube and stored in a -20°C, the pellet was resuspended in 10 μΙ sterile double-distilled water for the subsequent PCR analyses. Colonies were picked with sterile tips and resuspended in a 2 ml tubes with 10 μΙ sterile double- distilled water. One μΙ samples from both Eppendorf tubes (from liquid and solid media) were diluted 1 :30 for the subsequent PCR analyses. PCR was performed using universal primers for phytoplasma detection (R16F2n/R2, Gundersen and Lee, 1996), followed by nested-PCR with universal primers (16R758f/16R1232r=M1/M2, Padovan et al., 1995), using 1 μΙ of 1 :30 dilution as template from direct PCR (PCR results shown in Figure 10). RFLP analyses with specific restriction enzymes (Tru\, Tsp509\) confirm the presence of phytoplasma in the colonies tested (as shown in Figure 1 1 ), as well as in the tubes with acid colour change (tubes shown in Figure 12). Direct sequencing of PCR products gave further confirmation of phytoplasma identity.

Colonies and liquid broth positive to molecular tests were stored at -80°C for up to 1 year. To check their viability after some months, samples were thawed on ice and transferred to a 4 ml vacuette tube (Greiner bio-one, ref 454001 ) containing 2.5 ml of fresh MEL medium. The tubes were incubated at a temperature of 25 ± 1 °C and inspected for signs of a colour change. This was observed after 5-10 days, confirming the viability of the frozen material and the subsequent microbial growth. PCR/RFLP and sequencing confirmed phytoplasma identity also for these colonies.

Example 2: method of culturing phytoplasma colonies

Eleven micropropagated phytoplasma infected shoots (see table 1 below) were prepared in the laboratory of the Bologna University and transferred to the laboratories of Mycoplasma Experience as below described. Some shoots were transported in sterile water [listed in Table 1 (a) below] and some shoots were rooted in agar [listed in Table 1 (b) below].

Two lengths of stem of approximately 0.5-1 cm in length were moistened with 0.5 ml of MEL/- and sliced along their axes by cutting the stems with sterile scalpels whilst held with sterile forceps. After slicing, the liquid plus plant pieces were al l transferred to a 4 m l vacuette tu be (Greiner bio-one, ref 454001 ) containing 1 .5 ml of Mycoplasma Experience Liquid Medium (MEL/-). This was repeated for each plant giving two broth cultures with stem pieces from all plants, in all a total of 22 cultures.

The tubes were incubated at a temperature of 25 ± 1 °C and inspected for signs of a colour change from orange-red (pH above 7.0) to yellow (pH below 6.8) which could indicate growth of phytoplasmas. The timing of colour changes was different among different phytoplasma strains, as shown in the table below. When acid colour changes occurred, 50 μΙ of acid broth cultures were inoculated onto 6 cm diameter plastic plates containing 8 ml of Mycoplasma Experience Solid Medium (MES/-) (MES without thallium acetate) and incubated up -side down in an atmosphere of 95% N 2 / 5% C0 2 at 25 ± 1 °C. Colonial growth of phytoplasmas occurred in two to five days (indicated by "+" in Table 1 below). The acid colour changes indicating phytoplasma growth (confirmed by plating) cou ld also be inoculated (1 :10) in l iq u id M E L/- (MEL without thallium acetate). This was performed with 'Candidatus Phytoplasma aurantifolia' (TBB61 ) (agar shoot culture No 2) and stoibur phytoplasma strain CH-1 (CH-1 99). Table 1.

a)

Infected shoots in sterile Culture No Day of acid change Growth on water plating

'Ca. P.asteris' 1 63 +

Strain CY (CY-TO 94) 2 50 +

'Ca. P. aurantifolia' 1 No change by day 140

(TBB61 ) 2 69 +

'Ca. P. pyri' 1 48 +

(PD 44) Pear Decline 2 62 ?+

PD 44A(10PD) 1 69 +

2 96 +

Stolbur phytoplasma 1 78 +

Strain CH-1 (CH-1 99) 2 70 +

Stolbur phytoplasma 1 86 +

Strain STOL, (STOL 59) 2 67 + b)

Infected shoots in Agar Culture No Day of acid change Growth on plating

'Ca P. asteris' Strain CY 1 13 +

(CY-TO 94) 2 20 +

'Ca P. aurantifolia' 1 No change by day 138

(TBB 61 ) 2 44 +

Ca P. pyri 1 26 +

(PD 44) Pear decline 2 46 +

Stolbur phytoplasma Strain 1 86 +

CH-1 2 52 + (CH-1 99)

Stolbur phytoplasma 1 33 +

Strain STOL (STOL 59) 2 21 + Example 3: Identification of phytoplasma colonies

To confirm the presence of phytoplasma in both , liquid and solid media, molecular tests based on PCR were performed at different stages of the method above described on selected strains. After five subcultures in solid medium, photographs of the growth were taken (photographs shown in Figures 13 and 14). Two kinds of preparation were then tested:

P1 (Colony). One colony was picked and placed in 10 μΙ Synergy (Millipore) 18 M ohm water (autoclaved in borosilicate glass) in a 0.5 ml Sarstedt PP tube. Overnight drying at 36°C and storage at -70°C was then carried out.

P2 (Wash). The surface growth was washed with 0.5 ml water (as above), placed in a 2.0 ml Sarstedt PP tube and stored at -70°C.

Samples processed from P1 and P2 (1 μΙ of each) were diluted to a ratio of about 1 :30 for the subsequent PCR analyses (results shown in Table 2). PCR was performed using un iversal primers for phytoplasma detection (R16F2n/R2, Gundersen and Lee, 1996), followed by nested-PCR with universal primers (16R758f/16R1232r=M1 /M2, Padovan et al., 1995), using 1 μΙ of 1 :30 dilution as template from direct PCR. RFLP analyses with the specific restriction enzyme Tru\ confirmed phytoplasma identity (the results of which are shown in Figure 15). Table 2

Infected shoots in sterile water * Preparation PCR/RFLP results

'Ca. P. pyri' colony Pear decline (PD 44 * ) Pear Decline wash 'Ca. P asteris'

Infected shoots in Agar Preparation PCR/RFLP results

'Ca P. asteris' Strain CY colony negative

(CY-TO 94) wash 'Ca. P asteris'(very weak)

Ca P. pyri colony stolbur (PD 44) Pear decline wash negative

'Ca P. aurantifolia' colony negative

(TBB 61 ) wash negative

Stolbur phytoplasma colony Stolbur

Strain STOL (STOL 59) wash Stolbur