Proc. Indian Aead. Set., Vol. 85 B, No. 5, 1977, pp. 319-326.

Phyllody disease of Raphanus sativus L. and variation in the epidermal structure

S. MISRA AND M. G. GUPTA

Department of , University of Rajasthan, Jaipur 202004

MS received 6 July 1976

ABSTRACT

A phyllody disease of suspected MLO etiology was noticed on a plant of Raphanus sativus in the fields of Sanganer, Jaipur, nearly 3 years ago; and later in other localities in and around Jaipur. A study of the various features of epidermal structure in the normal as well as diseased material has shown that apart from tee stomatal types, which usually remain unchanged, the epidermal structure undergoes considerable change from normal to the diseased condition. The , and carpel of of diseased show phyllody and the becomes sepaloid. And that the degree of phyllody in these organs increases in the direction of carpels.

1. INTRODUCTION

ON a botanical trip to Sanganer, Jaipur, nearly 3 years back we came across a plant of radish which was severely affected by a disease usually referred to as phyllody. In the following years the disease was observed again but in some other localities within and round about Jaipur.

As a teratological phenomenon, however, the phyllody in case of radish was recorded about a century ago l-a and was described by the authors under anomalies like phyllody of the corolla, phyllody of the entire , and phyllody of the . Recently Hulbary et al. 4 reported prolification in the flowers of radish and considered it as an anomaly of unknown causal mechanism.

During the past 8 or 9 years diseases like Sesamum phyllody,5 phyllody of pea, e leucerne witches'-broom, 7 etc., in which floral anomalies of the above types as well as witches'-brooms are produced as characteristic symptoms have been ascribed to infections by certain resembling those 319 B7--May 77 320 S. MISRA AND M. G. GUPTA of the class mollicutes.8,9 This interested us in taking up the material of radish for morpho- and histopathological studies. The present contri- bution concerns a part of this study and describes our observations on the epide~-mal structures in the normal as well as diseased material and also explores the possibility of using it for the purpose of assessing the real nature of the various transformations in the so called phyllodic flowers.

As far as known to us, studies of this type have not been done so far. However, in case of the phyllody disease of Sesamum mulayanum, recently, Kavathekar et al. 1° have attempted to bring out certain features of epidermal structure of the transformed in comparison to that of the normal floral organs. 2. MATERIALS AND METHODS

The materials, both normal as well as diseased, were collected during the third week of January 1973 and were fixed in acetic-alcohol. They were washed and then preserved in 70~ alcohol. A portion of the diseased plant was prepared into a type herbarium sheet and deposited in the Rajasthan University Botanic Herbarium under No. RUBL 11839.

The preserved materials were used for making preparations of epidermal peels from various parts of the entirely phyUodic and normal flowers as well as normal and diseased . A peel was usually taken off with the help of forceps after putting the plant part under water. It ~as stained in aqueous safranin, washed thoroughly with water, mounted in dilute solu- tion of glycerine and thereafter the coverglass was sealed with cutex nail- polish. Since peeling was not possible in normal petal and stamen whole mounts were prepared for the study of these organs.

The stomatal counts were done under high power of the microscope ~45 × objective and 15 × eye piece) and by taking the microscopic field as the unit area. Later the readings were converted to the number per mm ~. For the comparison of total epidermal pattern camera-lucida drawings were made at uniform magnification.

3. OBSERVATIONS

Observations of diseased plants in the field have made it clear that symp- toms like colour breaking in the corolla, virescence, phyllody and proliti- cation in flowers, are characteristic of the phyllody disease of radish (Plato I, figures A, B, C, D). And that their occurrence usually depends on the stage at which the plant has been infected and also on the severity of the infection.

GROSS MORPHOLOGY OF THE EPIDERMIS.--The , in both the normal as well as diseased material, is amphistomatic. Its epidermis on either side PHYLLODY DISEASE OF Raphanus sativus L. 321

(Explanations given in p. 326) 322 S. Mtsag ANO M. G. GUPTA is composed of cells with irregular orientation and sinuate outline in the normal, and with mostly longitudinal orientation and less sinuate to almost smooth outline in the diseased material (figures 1, 2 and 7, 8).

The sepal also is amphistomatic in both normal as well as diseased conditions. Its epidermis on either side is usually made up of cells longi- tudinally elongated and with smooth outline, except in case of the abaxial surface of the diseased sepal where the cells are slightly sinuate in outline (figures 3, 4 and 9, 10).

The petal in the normal condition is astomatic and its epidermis on either surface is made up of highly characteristic polygonal ceils. In the diseased condition the petal is amphistomatic and its epidermis on either side is composed of cells which are mostly elongated longitudinally and with a smooth outline (figures 5 and 11, 12).

The stamen in the normal condition has a typical anther-filament type structure and is astomatic. In the diseased condition it is laminar and amphistomatic and with the epidermis on either surface consisting of cells elongated and with shghtly sinuate outline which is more conspicuous in the case of abaxial epidermis (figures 13, 14).

The carpel (one lateral half of the ) in the normal condition is hypostomatic and its epidermis on the abaxial side is composed of cells small and mostly elongated. In the diseased condition it is amphistomatic and with the epidermis on either side composed of ceils vertically elongated, and with slightly sinuate outline at least in case of the abaxial epidermis (figures 6 and 15, 16).

STOMATAL TYPES.~With regard to the occurrence of the various stomatal types there appears to be hardly any difference between the normal and diseased materials. In general the stomata of the anisocytic type are the most common (Plate II, figures 17, 21, 22). Next in abundance are those of the anomocytic type. Besides, there are stomata of several unusual types, common among these are: (i) a combination of 2-3 stomata with one subsi- diary cell common (figure 2); (ii) contiguous or paired stomata (figures 4, 12, 15); ('tii) stomata with one or both the guard ceils degenerating (Plate II, figures 18, I9); and (iv) abortive stomata, with the guard cells undergoing arrest of development at an early stage (figure 4).

While stomata of all these types are found to occur on almost all the stomatic surfaces, those of the contiguous type being abundant especially S. Misra and M. G. Gupta Proc. Indian Acad. SeL, Plate I Vol. 85 B, No. 5, 1977, pp. 319-326

Figures A, B, C, D. Habit photographs of normal and diseased twigs. A--Diseased twig with less affected flowers; B--Diseased twig with flowers showing increasing phyllody in acropetal direction. C and D--Nermal twigs with flowers and fruits respectively. (.facing page 322) S. Misra and M. G. Gupta Proc. Indian Acad. Sci., Plate H Vol. 85 B, No. 5, 1977, pp. 319-326

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Figures 17 to 22. Comparison of stomatal types; 17. Stomata healthy, on abaxial epidermis of normal leaf; 18. Stomata with degeneration of one guard cell, on abaxial epidermis of diseased leaf; 19. Same, later stage of degeneration, on adaxialepidermis of diseased leaf; 20. Stomata with distended guard cells and deformed openings, on abaxial epidermis of diseased sepal; 21. Stomata, on abaxial epidermis of maturing normal carpel; 22. Single stornatal apparatus of 21 magnified. (facing page 323) PHYLLODY DISEASE OF Raphanus sativus L. 323 in the case of abaxial epidermis of the normal sepal (figure 4) and adaxial epidermis of the diseased petal (figure 12) as well as carpel (figure 15). The stomata of the third and fourth types which together come under the category of degenerating stomata are more frequent on the diseased in comparison to the normal material. Of the category of unusual types of stomata there is one more type which is uncommon. This type (5) is recognized by an excessive distention in the guard cells and characteristic deformity of the stomatal opening (Plate II, figure 20). It is found to occur on the abaxial surface of the diseased sepal only and is probably confined to it. The stomata of the 3, 4 and 5 types invariably degenerate, hence may be found at different stages of degeneration. The dimensions of the well developed stomata range between 14.6/~ x 14.6/z and 21 "9/~ x 17.0/z in the normal, and between 18-3/z x 14.6/z and 25.5/z x 29.0/~ in the diseased material. Based on these figures the length/width ratio of the stomata comes to 1 or more than 1 and never tess than 1 in the normal, but ranges between more than 1 and less than 1 in the case of diseased material. FREQUENCY OF STOMATA.--The frequency of stomata calculated in res- pect of stomatic surfaces of the various organs under normal as well as diseased conditions is recorded in table 1. Each figure is an average of fifteen readings taken with respect to each surface.

Table 1.

Normal Diseased Organs Adaxial Abaxial Adaxial Abaxial

1. Leaf 248 297 160 215 2. Sepal 17 198 230 253

3. Petal .... 154 209

4. Stamen .... 132 259

5. Gynoeeium (carpels) .. 44 4-149" = 193 248 275

* developing stomata. A comparison between frequencies pertaining to individual organs under normal and diseased conditions shows that: In leaf there is considerable decrease in frequency from the normal -to the diseased condition. 324 S. MISRA AND M. G. GUPTA

While in the case of petal, stamen and adaxial surface of the carpel, the increase is from zero in the normal to the respective number in the diseased condition; in the case of adaxial surface of the sepal, it is from a very low number to a considerably high number in the same sequence. In case of the abaxial surface of sepal as well as carpel the increase from the normal to the diseased condition is, however, moderate.

A comparison between frequencies pertaining to the various organs under normal and diseased conditions yields the following information"

The diseased carpel is the only organ in which the frequency with respect to the two surfaces equals or reaches very near that of the normal leaf.

The diseased petal is the only organ in which the frequency with respect to the two surfaces reaches very near that of the diseased leaf.

The diseased sepal is an organ in which the frequency with respect to at least one surface reaches quite near that of the corresponding surface of the normal leaf. But with respect to the other surface, it comes very near that of the corresponding surface of the diseased stamen.

In the case of diseased stamen, while the frequency with respect to one surface comes quite near that of the diseased petal, with respect to the other surface it lies very near that of the sepal.

TOTAL EPIDERMAL PATTERN.--A comparison between the total epidermaI pattern on the various stomatie surfaces has revealed some close similarities which would be taken up under discussion. 4. DISCUSSION

In a recent review on flower abnormalities Meyer ix has classified the various reports made on the subject from the year 1900 onwards. Under phyllcdy, entire flower, corolla, and ovules are the sub-heads under which Raphan.us has been included. Our observations on the phyllcdy of Raphanus have made it clear that all these reports on the flower abnormality of Raphanus including that of prolification of flower by Hulbary 4 could be regarded as descriptions of individual symptoms of the phyllcdy disease given at different occasions.

A comparison between various features of the epidermal shucture in normal and diseased conditions reveals that while the stomatal types usually remain unchanged, features like outline of epidermal cells, fcequency and lcngth/width ratio of stomata, and distribution of stomata of the degene- PHYLLODY DISEASE OF Raphanus sativus L. 325 rating types, undergo considerable change from normal to the diseased condi- tion. The information gained from the study of va~'ious features of the epidermal structure is analysed below.

Out of all floral organs the sepal undergoes minimum change though in respect of frequency of stomata the change undergone by its adaxial surface is of considerable magnitude and is indicative of a leaning towards the condition of a normal leaf. However, in regard to the total epidermal patte~'n the diseased sepal appears quite close to the diseased leaf.

The carpel undergoes change which in comparison to that of sepal is of a higher order because one of its surfaces changes from astomatic to ~tomatic. Considering the frequency of stomata the diseased carpel lies nearest to the normal leaf, and in the matter of total epidermal pattern also it lies almost in a similar situation.

The Petal has undergone change higher than that of the carpel because in it the situation changes with respect to both the surfaces. In matters of frequency of stomata as well as epidermal pattern the petal comes close to the diseased leaf, although with respect to its adaxial surface it is closer to the maturing normal carpel.

The stamen seems to have undergone change of the highest order because of its total transformation. In terms of frequency of stomata the diseased stamen on one side comes close to the diseased petal and in turn to the diseased leaf; but on the other side, it comes closer to the diseased sepal. In respect of epidermal pattern also it comes very close to the diseased sepal. From the analysis presented above it could possibly be inferred that while the sepal, petal and carpel have undergone phyllody the stamen seems to have taken the course towards sepalody. Further, the phyllody of sepal, petal and carpel is of the order of increasing magnitude and this order seems to increase along sequence in which the organs have been arranged.

ACKNOWLEDGEMENT

We are grateful to Professor B. Tiagi, Life Sciences, for the encouragement.

REFERENCES

1. *Masters, M.T., Vegetable Teratology, Robert Hardwicks, London (1869). 2. *Penzig, O., Pflanzen-Teratologie, A. Ciminago, Genna (1890). 3. *Worsdell, W. C., The Principlesof Plant-teratology, Vol. II, RoyalSoc., London(1916). 326 S. MISRA AND M. G. GUPTA

4. Hulbary, R. L., Rao, A. N. and Michel, B.E., Proc. Iowa Acad. Sci. 64 127 (1957). 5. *Choopanya, D. In: Second Int. Symp. Plant Pathol. IARI, New Delhi, p. 116(1971). 6. *Hampton, R. O., Stevens, J. O. and Allen, T. C., Plant Dis. Rep. 53 499 (1969). 7. Bowyer, J. W., Antherston, J. G., Teakle, D. S. and Athern, G. A., Aust. J. Biol. Sci. 22 271 (1969). 8° Bos, L., Symptoms of Diseases in Plants, Oxford and IBH (1970). 9. Maramorosch, K., Rev. MicrobioL 28 301 (1974). 10. Kavathekar, A. K., Gaaapathi, P. S. and Paliwal, G. S., Phyton 15 153 (1973). 11. Meyer, Vesta, G., Bot. Rev. 32 165 (1966). * Not seen in original.

EXPLANATION OF FIGURES

Figares I to 16. Comparison between epidermal patterns of the normal and diseased materials~ 1, 2. Normal leaf--abaxial and adaxial epidermis, epidermal cells with sinuate outline; 3, 4. Normal sepal--adaxial and abaxial epidermis, former with walls of adjoining cells separativg. intermittently; 5. Normal Petal--epidermis; 6. Normal carpel--abaxial epidermis, many stomata still developing; 7, 8. Diseased leaf--adaxial and abaxial epidermis, outline of cells less sinuate; 9, 10. Diseased sepal--adaxial and abaxial epidermis, outline of epidermal ceils more sinuate; 11, 12. Diseased petal--abaxial and adaxial epidermis; 13, 14. Diseased stamen-- adaxial and abaxial epidermis, outline of cells as in 9, 10; 15, 16. Diseased carpel--adaxiaL and abaxial epidermis (all, × 210).