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Current Issues on Multidisciplinary Microscopy Research and Education 72

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Microscopy Techniques Applied to the Study of Phytoplasma Diseases: Traditional and Innovative Methods Rita Musetti *,1 and Maria Augusta Favali2 1 Dipartimento di Biologia Applicata alla Difesa delle Piante, Universit di Udine, via delle Scienze, 208,

33100 Udine, Italy 2 Dipartimento di Biologia Evolutiva e Funzionale, Universit di Parma, Parco Area delle Scienze 11/A,

43100 Parma, Italy This review describes the main microscopy techniques used for studying phytoplasma diseases, compar-ing classical methods with the most recent progress in this field. The most useful methods both for the di-agnosis and describing the relationship between the pathogen and the host plants, mainly the new ap-proaches and perspectives regarding the application of cytochemical methods for understanding the phy-toplasma pathogenesis process, are considered.

Keywords autoradiography, cryoultramicrotomy, fluorescence microscopy, image analysis, immunogold labelling, light microscopy, phytoplasmas, plant diseases, scanning electron microscopy, transmission electron microscopy, x-ray microanalysis

1. Introduction

Before dealing with the description of the specific microscopy methods, it is necessary to supply some general information on phytoplasma diseases, since there already exists an extensive literature on this subject. Diseases caused by phytoplasmas occur worldwide in many economically important crops: there are more than 300 distinct diseases associated with these pathogens. Phytoplasmas are wall-less prokaryotes, pleomorphic in shape, belonging to the Class Mollicutes; they are bound by a trilamined unit membrane, contain ribosome and DNA and range up 1.2 in diameter. Their shape may be helical, filamentous, beaded or simply spheroid. They are obligate parasites, unculti-vable in vitro therefore grow and reproduce in the phloem of the host plants and in the vector insects. The association of these pathogens with plants exhibiting yellows symptoms was demonstrated by Doi et al., [1] using Transmission Electron Microscope (TEM). Since then, many authors have used light and electron microscopy to reveal phytoplasmas in the phloem tissues and to study cytological interactions between these pathogens and their hosts [2, 3, 4, 5, 6, 7, 8]. But, because of their pleomorphism, it was impossible to distinguish and classify the different phyto-plasmas by means of the traditional morphological techniques [9]. In recent times, immunological tech-niques applied to light and electron microscopy enable the different phytoplasmas to be characterized in situ [10]. The aim of this paper is to indicate the most important microscopy techniques and the new approaches used in the diagnosis and in the studying of the cellular relationships between phytoplasmas and host plants.

2. Light Microscopy

Several staining methods, applied to serial semithin sections of resin-embedded materials, were used to localize and identify phytoplasmas in the infected tissues by means light microscopy. Most of these methods constitute the first steps towards understanding the possible association between phytoplasmas

* Corresponding author: e-mail: [email protected]


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and the disease symptoms in the plants. Moreover, they are fast and less expensive than electron micros-copy techniques. As sieve tubes of phloem have not nucleus, it is possible to use specific stains to evi-dentiate phytoplasma nucleic acid. Giannotti [11] and Karta et al., [12] reported respectively the application of methyl green and Feulgen staining procedure for the detection of phytoplasmas in the tissues of the host plants. Dienesstain was first developed as a specific stain for animal mycoplasma colonies [13]. Deeley et al., [14] applied this method to hand cut or freezing-microtome sections of stem tissues of plants infected by phytoplasmas and other pathogens. Phloem tissues of stems infected by phytoplasmas stained dark blue (Fig. 1), while xylem was turquoise and cortex light blue. The stain was specific for phytoplasma diseases and gave no reaction in healthy tissues and in samples infected by other pathogens. The procedure is quick, of diagnostic value, and can be used as a preliminary method to detect phyto-plasmas in infected plants [15]. Toluidine blue stains of semithin sections [16, 17, 18] or thionin/acridine orange [19] are also reported. The light microscopic detection of phytoplasmas in semithin sections could be a good method only in cells containing high concentrations of the pathogen. Light microscope techniques also contributed to the study of host plant-phytoplasma interactions. Specific staining methods were reported to detect histological changes in plant tissues infected with phytoplasmas [2, 20]. The alterations of cell walls and the localization of several compounds such as suberin, lignin and poly-phenols in plum and apple plants infected with European Stone Fruit Yellows and Apple Proliferation phytoplasmas respectively, were investigated, revealing an increase of these substances in infected plants compared to the healthy ones [8]. Histological root changes observed in Fraxinus americana infected with Ash Yellows Phytoplasma indicated that root damage precedes other symptoms and triggers branch dieback [21].

Fig. 1 Hand cut sections of healthy (a) and phytoplasma-infected (b) Catharanthus roseus L. stems stained with Dienesstain. Arrows show the blue spots indicating phytoplasma presence (bars= 125 ).

3. Fluorescence Microscopy

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74 Rita Musetti et al.: Microscopy Techniques Applied to the Study of Phytoplasma Diseases...

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3.1 DAPI Stain

Fluorescence in plant tissues could be due to natural autofluorescence of the cell walls (i.e. xylem cell walls) or could be caused by the accumulations of substances (i.e. phenols) produced in response to different types of diseases or stress. Therefore, autofluorescence cannot be used as a diagnostic method for phytoplasma diseases. Fluorescence obtained using specific fluorochromes represents a very useful and quick technique for the detection of phytoplasmas in the infected tissues. Simple methods were developed for confirming the presence of phytoplasmas in hand cut or freezing-microtome sections of infected tissues using two DNA-binding fluorochromes, 4-6-diamidino-2-phenylindole (DAPI) or the benzimidole derivate Hoechst 33258 and fluorescence microscope. DAPI is the stain most used for phytoplasma diagnosis [22, 23] both in herbaceous [24] and woody host plants [25, 26, 5, 8]. Following DAPI staining, phloem cells of infected material show a diffuse fluorescence, brighter that the one typical of the nuclei of parenchymal cells. These very bright spots in the phloem tissues due to phytoplasmas are not visible in healthy tissues (Fig. 2). A rapid compression technique developed for unfixed soft tissues, such as leaf midribs and dodder apices has also been reported [27, 28]. DAPI staining techniques permit a rapid and precise localization of phytoplasmas both in fresh and dried samples [28], and not only in leaf or stem tissues, but also in roots and petioles [29].

3.2 Immunofluorescence

The DAPI technique is rapid but not specific for the different phytoplasmas. Serological techniques ap-plied to fluorescence microscopy give a more sensitive and specific diagnosis. The development of probes such as poly- and monoclonal antibodies advanced the art of diagnosing phytoplasma diseases diagnostics

Fig. 2 DAPI staining of hand cut sections of healthy (a) and phytoplasma-infected (b) Catharanthus roseus L. stems. In b, arrow indicates the fluorescent bright spots, visible at phloem level, diagnostic for the presence of phytoplasmas (bars= 192 ).

[30, 31]. Two ways of performing the immunofluorescence test have been used: the direct and the indirect methods [32]. In the first method the antibody produced against the antigen to be detect is conjugated with a fluorochrome, such as fluorescein isothiocyanate (FITC) and stained by exposing test specimens to a solution of the labelled antibody. The indirect method is based on the capability of

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antibodies to serve as immunogens: for example, if a goat is injected with rabbit immunoglobulins, goat antibody against rabbit immunoglobulins can be produced and conjugated with a fluorochrome. The stain is performed in two steps. First, the test specimens are incubated in a solution of unlabeled rabbit antibody produced against the protein to be detect; second, the sites where the rabbit antibodies have attached to the protein are labelled by specific binding with goat fluorescent antibody prepared against rabbit immunoglobulins. The reaction antigen/antibody is specific, and, with the relatively short time required to prepare and observe the specimen, make the immunofluorescence technique an ideal diagnostic tool for plant diseases associated with phytoplasmas. On the other hand, it is very difficult to produce specific antisera against these pathogens, because phytoplasmas are not cultivable in vitro and only partially purified antigens can be obtained from infected host plants [33]..

4. Transmission electron microscopy (TEM)

Because they are small and without a defined shape, to observe phytoplasmas directly the magnification and resolution of an electron microscope is required (Figs. 3 and 4).

4.1 Thin section technique

The most basic procedure is the preparation of thin sections of resin-embedded plant materials and their observation by TEM.

Figs. 3 and 4 Phytoplasmas (arrows) in the phloem cells of Catharanthus roseus L. (Fig. 3) and apple (Fig. 4). Note that phytoplasmas are more numerous in C. roseus (the typical test plant for these patho-gens) than in apple tissues. In fruit and forest trees phytoplasmas are not uniformly distributed in the phloem of the plants. (Fig. 3: bar = 0.5 ; Fig. 4: bar = 0.65 ).

Using this technique, Doi and co-workers [1], were the first to find and describe phytoplasmas associated with the yellows diseases, showing their typical localization in the phloem of the host plants. Since then, the observations of thin sectioned resin-embedded samples enabled, not only the diagnosis, but also the study of plant-phytoplasma interactions [2, 3, 4, 5, 6, 7, 8].

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Many authors described ultrastructural modifications induced by phytoplasmas in the host tissues [4, 5, 6, 8]. These studies demonstrated that the effects caused by the pathogen on the plant tissues can be of different depending on the type of host and the phytoplasma [5, 34]. For example, in the parasitic plant Cuscuta campestris the pathogenic effect of Apple Proliferation phytoplasma results stronger than Clo-ver Phyllody phytoplasma: in fact Apple Proliferation phytoplasmas cause so serious damages to the cell organelles that their ultrastructure is no longer recognizable. In woody infected plants, the cell walls of the phloem results particularly thickened and distorted, and electron-opaque phenolic material is localized in the vacuoles; the presence of phytoplasmas is not easy to demonstrate by TEM because the sieve tubes have often collapsed or are filled with callose [35, 8, 36].

4.2 Immuno-sorbent electron microscopy (ISEM)

Immunological and other works on detection and identification of phytoplasmas have been extensively reviewed [37, 38, 39, 40, 41, 9, 42]. As these prokaryotes are morphologically very variable and feature-less, the recognition on EM grids of phytoplasmas extracted and purified in vitro by negative or positive staining might result very problematical. Derrick and Brlansky [43] developed an ISEM assay for identification of plant viruses and cultivable mycoplasmas. Sinha and Benhamou [44], using the specific antiserum, trapped on the EM grids and decored, partially purified extracts of Aster Yellows phytoplasma from infected aster plants. The method was specific and sensitive, but it was time consuming to obtain purified phytoplasma prepara-tions required to prepare specific antisera. But, once the serum has been produced, the antigens could be detected within few hours in partially purified preparations. The recognition of phytoplasmas by these techniques rested on comparison with control and ELISA results, because individual phytoplasma bodies could not be identified among the back-ground of host derived membrane debris. A difficulty of immunological studies of phytoplasmas is that monoclonal antibodies are not easily produced, and polyclonal antibody preparations initially could contain signifi-cant levels of antibodies directed against the host. Recently, polyclonal and monoclonal antibodies against phytoplasmas become to be available [31, 45, 46, 33] and protocols and new techniques for the diagnosis and characterization of these prokaryotes have been proposed. Vera and Milne [42] proposed a rapid and specific protocol for immunogold lebelling and negatively staining phytoplasmas from crude preparations, that allows phytoplasmas to be easily identified and distinguished from others that are serologically different. The proposed method is useful in diagnostics, in monitoring the steps of phytoplasma purification, and in the study of phytoplasma morphology and multiplication. The authors also suggested that the procedures of extraction and staining did not greatly alter phytoplasma morphology and artifacts did not occur.

4.3 Immuno-electron microscopy (IEM) of thin sections

Immunogold labelling of thin sectioned material is applied successfully in the detection of phytoplasmas both in the phloem of the host plants [47, 40, 9, 10] and in the insect vectors [48]. Uses of pre-embedding and post-embedding techniques were reported [9]. The first better evidentiated outer membrane antigens of microorganisms, the second could identify both internal and external anti-genic sites. Using both techniques, phytoplasmas present specific labelling on the periphery of the cell, showing that phytoplasma antigens are exposed on the surface of these prokaryotes (Figs. 5 and 6). Musetti et al. [10] compared two post-embedding techniques to determine which is more suitable for plant material affected by phytoplasmas. In the first protocol, according to Berryman and Rodewald [49] and modified by Milne et al. [41], a formaldehyde-glutaraldehyde mixture as fixative and LR Gold resin for embedding were used. According to the second one, diluted solution (0.2%) of glutaraldehyde alone was used, and LR White resin was employed. Ultrathin sections were incubated with primary specific


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monoclonal antibody, then with secondary antimouse antibody coated with colloidal gold particles of different size (5, 10, 15 nm) (Figs. 5 and 6). Both fixation and embedding procedures maintained the typical phytoplasma ultrastructure well enough; the choice of gold particle size together with antibody concentrations resulted to be important for the intensity of the labelling. Immunolabelling techniques applied to thin sections of infected plant tissues were essential for distinguishing between phytoplasmas and host organelles, when the former are few and the tissues necrotic. Interesting applications could be the elucidation of phytoplasma biodiversity and the study of infections caused by two or more different phytoplasmas.

Figs. 5 and 6 Phytoplasmas in phloem tissues of Catharanthus roseus L. labelled by immunogold technique. Pri-mary monoclonal antibody was diluted 25 g/ml, the secondary gold conjugated antibody 1:20. Using gold 15 nm in diameter, few particles are visible on phytoplasma membrane (Fig. 6), using 5 nm gold, particles are well distributed over the periphery of the phytoplasmas (Fig. 5). (Fig. 5: bar = 0.25 ; Fig. 6: bar = 0.15 )

4.4 Cryo-ultramicrotomy

Thin sections of specimens prepared by rapid freezing methods (especially high-pressure freezing) give a more accurate picture of the overall structure of the sample, as ultrastructure preservation is superior due to the omission of conventional dehydration and embedding methods. As chemical fixation is avoided, cryosections are very suitable also for immunolabelling applications [50]. DAgostino [51] applied cryo-ultramicrotomy to the study of the structure of Primula Yellows phytoplasmas in Catharanthus roseus L. leaf tissues, comparing the results with those obtained with conventional Epon-Araldite embedding method. According to the author, phytoplasma structural details were better preserved in cryosections: phytoplasmas were referred to two predominant morphotypes, rounded and filamentous, and the high polymorphism of phytoplasmas observed in epoxy sections was probably due to artifacts associated with this technique.

5. Scanning electron microscopy (SEM)

Observations of phytoplasma infected material by SEM were also reported [52, 53]: they are described as short, branched, filamentous forms in sieve elements of infected plants. Marcone and Ragozzino [54] hypothesize that these morphological variations could be various development stages of phytoplasmas.

6. Cytochemical methods

Cytochemical methods were much used in cytology, but their application to plant diseases was not wide-spread, because some were difficult and time consuming. However, cytochemical techniques can result

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very important to detect chemical nature of cellular structures, both normal and pathogen-induced, or to identify enzymatic activities in the infected cell.

6.1 High resolution autoradiography

High resolution autoradiography was very used in virology to localize viral nucleic acids in the cells, by means incorporation of Uridine- 3H or Thymidine -3H. Favali and Lombardo [55] applied this technique to phytoplasmas affecting clover plants, demonstrating that phytoplasmas were capable of active multiplication in the host. The technique consists in the introduction of a soluble radioactively-labeled substance into the sample. The tissue is then washed, fixed, embedded and sectioned and the sections are coated in photographic emulsion and stored in the dark. During this time, some of the electrons emitted by the radioactive decay of the label strike silver crystals in the emulsion and convert them in a latent image, sensitive to reduction to silver by the developer. After appropriate exposure, the emulsion is developed and fixed maintaining the sections in place. When the sections are observed under the electron microscope, silver grains indicate the sites in the sections where the label has been incorporated.

Figs. 7 and 8 Phytoplasmas in phloem cells of white clover (Trifolium repens L.), after 3 hours labelling with thymidine-3H. Note the silver grains on the dividing phytoplasmas (arrows). (Fig. 7: bar = 0.6 ; Fig. 8: bar =0.47 )

6.2 Potassium pyrantimonate precipitation and cerium chloride labelling

For several years, the development of suitable and precise diagnostic techniques was the most urgent problem to be solved. More recently, the study of physiology of phytoplasma diseases became topical [56, 7, 8], because some products of the plant-pathogen interaction could be putative inhibitors of molecular primers used for detecting phytoplasmas in infected material [57]. Potassium pyrantimonate precipitation for detection of bivalent ions as Ca 2+, and cerium chloride label-ling techniques for the detection of H2O2, were recently applied to phytoplasma infected tissues with the aim of studying the movement and accumulation of ions implicated in cell metabolism and in the de-fence-related mechanisms in the diseased plants [58, 36]. Results obtained demonstrated the involvement of these molecules in the infection processes, particularly in the recovery phenomenon.

7. X-ray microanalysis and electron energy loss spectroscopy (EELS)

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X-ray microanalysis and EELS are also used to analyse the elemental composition and distribution of mass within the cells. Both methods were found to be particularly suitable for the detection of deposits of homogeneous composition (i.e. dense cellular inclusions) in infected plant tissues. Regarding phytoplasma diseases, x-ray microanalysis is particularly useful for studying of progression of symptoms in the diseased plant, i.e. correlate leaf decoloration with the presence of specific elements, such as Si, in the infected tissues [59].

8. New approaches and technologies

Recently, the potential of microscopy techniques as investigative tools in plant cell biology has in-creased, because of development of confocal laser scanning and video microscopy, computerized image processing and analysis, and an increasing array of fluorescent probes that can be applied to living cells. In addition, transgenic plants and cells can be generated in which specific components are fluorescently labelled without any invasive experimental manipulation. The application of such techniques to plant-microbe interactions has produced interesting results [60, 61], and likewise in the study of plant-virus relationships [62] but nothing has been reported about phy-toplasmas. Atomic force microscopy was applied, for the first time, to investigate with a molecular resolution scale, a partially purified extract of unfixed phytoplasma cells from C. roseus (Ciancio, personal communica-tion), providing a new perspective in the study of the biology of these prokaryotes under conditions very close to their native cell environment.

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