J. Phytopathology 117, 349—356 (1986)© 1986 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0031-9481

Station de Pathologie Vegetale, Institut National de la RechercheAgronomique (I.N.R.A.), Route de Saint-Cyr, 78000 Versailles, France

Mycoplasmalike Organisms (MLOs) AssociatedWith the Witches' Broom Disease of Poplar

AjAY KUMAR SHARMA and MARIE-TH£RESE COUSIN

Authors' address: Station de Pathologie Vegetale C.N.R.A., Route de Saint-Cyr, 78000 Versailles(France).

With 10 figures

Received January 29, 1986; accepted March 19, 1986

Abstract

The witches' broom disease has been recently observed on poplars in Paris and its suburbs. Theincidence of the disease appeared to be considerably high along main roads. The electron microscopicexamination of 350 nm thick sieve tube sections revealed the presence of wall-less mycoplasmalikeorganisms (MLOs) in diseased samples of Populus alba var. nivea Wesm. They could not be found intheir healthy counterparts.

Zusammenfassung

Mykoplasmaahnliche Organismen bei Pappeln mit Hexenbesenwuchs

In neuerer Zeit wurde in der Pariser Gegend eine hexenbesenahnliche Krankheit auf Pappelnbeobachtet. Diese Krankheit tritt insbesondere entlang der grofien Verkehrswege auf.

Die elektronenmikroskopische Untersuchung von 350 nm dicken Siebrohrenschnitten zeigtedas Vorhandensein von mykoplasmaahnlichen Mirkoorganismen in den kranken Pflanzen vonPopulus alba var. nivea Wesm., nicht aber in gesunden.

The witches' broom disease was observed on Populus nigra L. "Italica" andPopulus canadensis by ATANASOFF (1973) who suggested a combined action ofvirus and mycoplasma with the disease. VAN DERMEER (1980,1981) observed thedisease on Populus canescens. Although mycoplasmas have been mentioned to beresponsible for the symptoms, no supporting evidence has been provided.

Recently many species of Populus planted along polluted highways werefound to suffer from the witches' broom disease. Most frequently the infection

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350 AjAY KUMAR SHARMA and MARIE-THER6SE COUSIN

was severe, resulting in the death of the plant within a short period of time.Taking into account the increasing incidence of the disease, the present workdeals with the electron microscopic localization of the organism associated withthe witches' broom affecting Populus alba var. nivea.

Materials and Methods

Materials

A survey was carried out in and around Paris city to evaluate the incidence of the witches'broom on poplars. Severely infected and healthy white poplars {Populus alba var. nivea) growing onthe campus of the Cite unversitaire in Paris, were selected for the electron microscopic examinationreported in the present study. Twigs from plants affected with the witches' broom and from healthyplants were studied in Versailles laboratory.

Methods

One mm long pieces of healthy and diseased leaf midveins were cut while the samples weresubmerged in the fixative. Fixation in glutaraldehyde, post-fixation in osmium tetroxyde, dehydrationin ethanol and propylene oxide, then embedding in epon resin were carried out following the methodpreviously described (COUSIN and SHARMA 1986). Longitudinal 350 nm thick sections were obtainedusmg the Reichert Ultracut microtome operating with a glass-knife. The sections on the grids werestained with uranyl acetate and lead citrate. Preparations were examined in Philips EM 300 electronmicroscope at 80 Kv.

Observations and Results

Description of symptoms

The first symptom of the witches' broom disease visible on poplars was theinterveinal chlorosis of leaves. As the disease progressed, the yellowing of theleaves increased, while the surface area of leaf-lamina and the petiole length werereduced. The number of leaves increased. The infection, which was partiallylocalized in the beginning, spread over the whole plants within a few months.

Figs. 1—5. Symptoms of the witches' broom in Populus alba var. nivea. Progression of the diseaseduring two successive years

Fig. \. Upper parts of a plant observed in May 1984. Severe infection, general yellowing, some of thebranches without leaves, others bearing small and yellow leaves

Fig. 2. Lower part of the same plant. Excessively sprouted straight and upright lateral shoots onsecondary branches, shortened internodes, witches' broom appearance

Fig. 3. Comparison of diseased (above.) and healthy (below.) leaves. The diseased leaves, increased innumber, showing interveinal chlorosis, reduction in petiole length and lamina size; lamina partially

necrosed in some leaves

Fig. 4. Same plant in May 1985. More severe infection, main trunk and all branches dried, no foliageexcept in very few lateral sprouts on lower branches

Fig. 5. Main trunk close to the ground in May 1985. Almost all lateral shoots with excessive sproutingdried. Only a few of them presented small-sized and yellowing leaves

Mycoplasmalike Organisms of Poplar 351

Excessive sprouting of axial buds could be observed. Newly emerged lateralshoots shovi ed upright growth. This resulted in the witches' broom appearance of

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352 AjAY KUMAR SHARMA and MARIE-THERESE COUSIN

the affected plants or parts of them. In the systemic infection, leaf size wasreduced, internodes shortened and plants remained stunted.

In the last stage, as observed during sampling in May 1984, the symptomsbecame more severe on the whole plant (Fig. 1). The witches' broom wasparticularly visible on lateral shoots (Fig. 2). As compared to their healthycounterparts, these leaves were small and yellow, with lamina sometimes partiallynecrosed (Fig. 3). Periodic observations of the plants used for sampling revealedthat, in the following spring, they produced no flower. The whole plants shedtheir leaves and dried (Fig. 4) except for a few sprouts emerging from the lowerpart of the trunk close to the ground, that were seen to bear small and yellowingleaves (Fig. 5). The witches' broom disease ultimately resulted in the death of theplant.

Other plants of the same species, showing mild symptoms in May 1984,exhibited severe symptoms in the following year.

Electron microscopy

Electron microscopic examination revealed the presence of Mycoplasmalikeorganisms (MLOs) in the longitudinal sections of diseased sieve tube elements(Fig. 6). Such organisms could not be identified in their healthy counterparts.Owing to differences in contrast between sieve lumen and its contents on onehand and cell wall on the other, as observed in semi-thin sections, it has beenfound necessary to print two electron micrographs with different exposure times,using the same negative. Most organisms were seen in the vicinity of sieve plates.Others were scattered among P Protein, fibrils, vesicles and degenerating proplas-tids containing special starch (Fig. 6, detail Fig. 7). In the same preparation, onlya few sieve tubes were infected. But none of them was found completely packedwith MLOs.

With high magnification (Fig. 8), numerous ribosomes and a trilaminar unitmembrane could be detected.

The organisms were polymorphous. A few of them presented spherical, ovalor beaded forms but most of them were filamentous. The various forms observedwere long, branched or unbranched, without any constrictions (Fig. 9) or withconstrictions (Fig. 10).

These studies carried out on 350 nm thick sections indicated the absence ofspiral forms.

Discussion

The present study constitutes the first attempt to examine MLOs in poplarswith the transmission electron microscope. As the pleomorphic wall-less organ-isms could not be detected in healthy poplars, we assume that the MLOsobserved in white poplars {Populus alba var. nivea) play a role in the developmentof the disease.

For the reasons already discussed (COUSIN and SHAKMA 1986, COUSIN et al.1986) semi-thin and longitudinal sectioris were preferred to the usual ultrathinand transverse sections.

Mycoplasmalike Organisms of Poplar 353

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Figs. 6—10. Mycoplasmalike organism^ ;M[.(..)>•; n -> ll:l.•:ied with witches' broom affected Populusalba var. nivea. Studies on longitudinal 350 nm thick sections of mature sieve tubes of diseased

Populus alba var. nivea

Fig. 6. Mycoplasmalike organisms in sieve tube elements of diseased phloem. Lumen of one sieve tubeelements contains MLOs, degenerating proplastids with special starch inside, P-protein and vesicles.

Another sieve tube element with only P-protein (X 19.000) .

J. Phytopathology, Bd. 117, Heft 4 23

354 AjAY KUMAR SHARMA and MARIE-THEUESE COUSIN

Fig. 7. A magnified part of Fig. 6. Polymorphy of the MLOs. Presence of a bleb. In addition to theMLOs, the sieve cell contains P-protein, vesicle and degenerating plastids with special starch inside;

absence of any spiral form (X 58.500)

Fig. 8. A magnified part of Fig. 7. Ultrastructure of the MLOs. Absence of cell wall. Numerousribosomes surrounded by a unit-membrane composed of dark-white-dark layers; width of the space

between the two dark osmiophilic layers being 7 nm (x 93.000)

Mycoplasmahke Organisms of Poplar

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Fig. 9. Long branched filaments dispersed among P-protein (x 76.000)

Fig. 10. Long branched filament terminating into a single protuberance or a chain of protuberancesthat resulted into beaded appearance of the organism; electron dense ribosomes inside MLOs

(X 103.000)

b: bleb, cw: cell-wall of the sieve tube, m: mycoplasmalike organism, p: degenerating proplastid, pm:plasma membrane of the sieve tube, Pp: P-protein, sp: sieve pore, v: vesicle, -» ^ two osmiophilic

layers of the unit-membrane of the MLO

356 AjAY KUMAR SHARMA and MARIE-TH£RESE COUSIN, Mycoplasmalike Organisms of Poplar

Filamentous forms are known to occur in the late stationary growth phase ofthe organisms, as observed during in vitro culture of animal mycoplasma(ROBERTSON et al 1975) or in the ageing plant tissues (HEARON et al. 1976). Theobservation of such forms in the present investigation suggests that the MLOgrowth was in its late stationary phase. These electron microscopic observationsare correlated to the severe symptoms exhibited by the host-plant samples.

Literature

ATANASOFF, D. , 1973: Witches' brooms on stems of Elms and other trees. Archiv. PhytopathologiePflanzenschutz 9 (4), 241—243.

COUSIN, M . T., and A. K. SHARMA, 1986: Association of mycoplasmalike organisms (MLOs) withmild type of Hydrangea virescence: a study with 60—1000 nm thick sections. J. Phy-topathology 115, 274—282.

, , and S. MiSRA, 1986: Correlation between hght and electron microscopic observationsand identification of mycoplasmalike organisms using consecutive 350 nm thick sections.J. Phytopathology 115, 368—374.

HEARON, S. S., R. H . LAWSON, F . F . SMITH, J. T. MCKENSIE, and J. ROSEN, 1976: Morphology offilamentous forms of mycoplasmalike organisms associated with Hydrangea virescence.Phytopathology 66, 608—616.

VAN DER MEER, F. A., 1980: Witches' broom in poplars. Popuher 17 (2), 42—43., 1981: Mosaic, witches' broom and knobbiness of poplar and a virus-like growth reduction of

willow. Popuher 18 (3), 51—59.ROBERTSON, J., M. GOMERSALL, and P. GILL, 1975: Growth, reproduction and isolation of small

viable cells. J. Bacteriol. 124 (2), 1007—1008.