Mycoplasma-like organisms as the causative agent of witches’ broom of Cedrela (Cedrela toona Roxb.) in Himachal Pradesh, India

S. Nath

Department of Bio-Sciences, M.L.S.M. College, Sunder Nagar,

District Mandi 174 401, India

Cedrela toona is an important timber plant growing extensively in Himachal Pradesh. Unfortunately, a large number of trees exhibit witches’ broom-like disease symptoms in the crown region. The infected plant/branch displays stunted growth, excessive bud sprouting, reduced leaf size and inhibition of flowering. All these symptoms suggest the involvement of mycoplasma-like organisms (MLOs). Histochemical examination of thin stem sections revealed strong positive reaction with Dienes’ stain and fluorescent dye, aniline blue in the phloem elements of infected plants vis-ŕ-vis healthy ones. Moreover, application of tetracycline to diseased plants causes remission of disease symptoms. These experiments establish the association of MLOs with this disease. Tetracycline treatment of infected plants was found to be an effective remedy.

CEDRELA toona Roxb, ‘Tooni’ in vernacular, is an extremely important timber plant growing abundantly throughout Himachal Pradesh at an altitude of 2000–3000 ft. Its aromatic scented wood is used extensively

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Figure 1.  a, A severely affected tree of Cedrela toona exhibiting a number of dense green bunches, i.e. witches’ brooms in the crown region. b, A small healthy plant of C. toona. c, A close view of an early infected plant of C. toona showing the details of witches’ broom-like symptoms, i.e. stunted growth, highly reduced leaves and excessive bud sprouting.

for making furniture, door panels and tea and cigar boxes, etc.1,2. This tree forms a dominant species of forest flora, grows up to a height of 60–70 ft, produces leafy shoots in summer, the leaves wither away during autumn, and it undergoes a period of rest in winter (November to February). In the following spring it resumes active vegetative growth and bears a large number of small white, honey-scented flowers in panicles in April–May.

Most of the trees particularly in Bilaspur–Sunder
Nagar–Mandi belt of Himachal Pradesh have been ruined by a severe witches’ broom disease locally known as ‘Reen’ disease, which is feared to assume an epiphytotic form in the coming years. The disease appears first on the apices of some branches and then affects the whole branch. The affected branches subsequently develop into easily recognizable globular, huge, dense green bunches, showing characteristic witches’ broom-like symptoms in the crown region (Figure 1 a) which persist even during the autumn season. The rest of the tree, however, gives a healthy look.

The severely infected plant/branch exhibits stunted growth, shortened internodes and excessive bud proliferation leading to the formation of thin cylindrical branches which run almost parallel to the main axis of the stem, forming a broom-like structure. The leaves are markedly reduced in size from 1.9 ft to 2–5 inches and even less, i.e. show microphylly in extreme cases (Figure 1 a–c). Induction of flowering and seed setting is completely suppressed (Figure 1 c).

Since such disease symptoms are associated with mycoplasma-like organisms (MLOs) – the pleomorphic, membrane-bound submicroscopic prokaryotic organisms3–7, it was imperative to undertake studies to establish their probable involvement with the disease. Histochemical studies using Dienes’ stain and fluorescent dye, aniline blue, and studies on tetracycline treatment of infected plants were conducted.

Diseased stem material excised from the severely infected branches of the tree was used for histochemical studies. The disease-free material (control) was taken from 2–3-year-old plants grown under insect-proof conditions. The material was fixed immediately either in FAA (formaline:glacial acetic acid:alcohol, 10:5:85 v/v) for Dienes’ staining or in 5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0 for fluorescence microscopy.

Dienes’ stain is highly specific to both the animal and plant mycoplasmas and is, therefore, extensively employed for their detection in infected tissues8–10. Transverse thin sections of healthy and infected stem materials (fixed in FAA) were stained with 0.2% Dienes’ stain (prepared by dissolving 2.5 g of methylene blue, 1.25 g azure II, 10 g of maltose and 0.25 g of sodium carbonate in 100 ml of distilled water) for 10 min (ref. 8), washed 2–3 times with distilled water, mounted in a drop of 1% glycerine and examined under a light microscope.

Distinct deep blue patches, typical of MLOs, were detected throughout the phloem region of all the stem sections of infected plants but not in sections of the

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Figure 2 a, b.  Projection micrographs of T.S. stem of Cedrela toona, showing distinct deep blue patches in the phloem (Ph) region of diseased plants (b) as compared to the healthy ones (a – control) exhibiting no such patches (´  45); c, d. Photomicrographs of T.S. stem of C. toona, exhibiting strong MLO fluorescence in the phloem cells of infected plants (d) in contrast to the healthy ones (c – control) showing no such fluorescence (´  150); Co, cortex’ Pe, pericycle; Ph, phloem; Xy, xylem; Pi, pith.

healthy stem (Figure 2 a, b). This suggests the involvement of MLOs with the disease. Similar observations were recorded in a number of other MLO-infected plants8,9.

Fluorescent dyes, such as aniline blue and DAPI (4,6-diamidino-2 phenyl indole), are also specific in their reactions to MLOs and have also been employed widely for their diagnosis11–14. Healthy and diseased stem material (fixed in 5% glutaraldehyde) was boiled for
5 min in the fixatives and sections were cut as above. The sections were stained with 0.2% aniline blue (standard stain, B.D.H., UK) in 1/15 M K2HPO4, pH 8.0 for 15 min (refs 12, 15), mounted in a drop of 1% glycerine, and examined under Nikon Fluorescence Microscope using exciter filter of wavelength 330–380 nm and an eye piece absorbance filter of 420 nm in visible range.

A strikingly bright fluorescence, characteristic of MLOs, was observed in the phloem cells of all the stem sections of infected plants, but not in sections of healthy stem (Figure 2 cd). This also suggests MLOs to be the etiological agents as was also reported by several investigators in other MLO-infected plants with aniline blue/DAPI or with both the dyes11–14,16,17.

Use of tetracyclines to check MLO-incited diseases in plants, has been in practice for a long time and is also used as a diagnostic aid for determining MLO etiologies of new diseases18–21. Application of tetracyclines either by spraying the foliage or by injecting it into the stem, is considered to be the most effective method. It causes remission of disease symptoms by inhibiting growth and reproduction of MLOs20–24. However, the remission of symptoms occurs for short duration in small plants and for longer duration (2–5 years) in woody trees20,21,25. The remission of disease is characterized by healthy new growth which is free from symptoms but there is no recovery of the distorted tissues20,21.

For tetracycline (oxytetracycline–HCl) treatment, Tooni plants of 6–9 ft height were selected in the field in the beginning of September, 1995. The stem was drilled 1 cm deep and a glass tube with a reservoir of 20 ml capacity was fixed (Figure 3 a). The joint was sealed with ‘M-Seal’ (a putty, Mahindra Engg. and Chemical Products Ltd., Pune) and allowed to dry for an hour. 20 ml of tetracycline solution (1000 µg/l, prepared in distilled water and stored at 5° C) was added to the reservoir, regularly in the morning. The solution diffused into the stem automatically. The reservior was wrapped with thick paper to protect it from photodegradation and its mouth was kept closed with tin foil to avoid evaporation and dust particles.

The treated plants showed an amazing recovery. Disease symptoms started decreasing after 15–20 days and disappeared completely after one month of regular tetracyline treatment. The newly-formed leaves and branches looked normal and completely healthy (Figure 3 b).
The treatment was continued for another month (till 30 October 1995) with a view to studying its effect in the next growing season in which the treated trees exhibited active vegetative growth like the healthy ones. These results also demonstrate the involvement of MLOs with the witches’ broom disease of C. toona like observations on other MLOs-infected plants18–20,22–25.

The experimental plants are healthy till date, even after two years of discontinuing the treatment (Figure 3 c). It appears that application of tetracyclines to MLO-infected plants through the stem, may prove to be an effective control measure of the disease.

The disease is suspected to be transmitted by some insect vectors (leafhoppers) including a moth Magiria robusta, an important pest of C. toona2. Therefore,

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Figure 3. a, A severely infected plant of Cedrela toona showing many witch’s broom-like symptoms. An attached reservoir (R) of 20 ml capacity, on the very first day of its tetracycline treatment (i.e. 1 September 1995), is also seen. b, The same diseased plant of C. toona on the 35th day of its regular tetracycline treatment (1000 µg/l) showing remission of disease symptoms, i.e. new symptomless growth with healthy-looking leaves and branches. c, The same plant of C. toona two years after discontinuing the tetracycline treatment (i.e. in October 1997) looks completely healthy with fully extended leaves.

experiments in this direction have been planned and will be undertaken shortly.


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ACKNOWLEDGEMENTS. I thank Dr G. S. Rawla, Ex-Chairman, Department of Botany, P.U. Chandigarh for providing facilities for Fluorescence Microscopy. I am also indebted to my teacher Dr C. L. Mandahar, Retd. Prof. of Mycology and Plant Pathology from the same University and Dr I. D. Garg, Senior Plant Pathologist, Central Potato Research Institute, Shimla for their encouragement and suggestions.

 

 

Received 30 May 1998; revised accepted 5 September 1998

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