Proc. Indian Acad. Sci., Vol. 88 B, Part 2, Number 1, January 1979, pp. 19-24, ~) printed in India

Histopathologicai studies on ragi (Eleusine coracana (L.) Gaertn.) infected by Sclerophthora macrospora (Sacc.) Thirum. Shaw and Naras.

S R A G H A V E N D R A * and K M SAFEEULLA Downy Mildew Research Laboratory, Mysore University, Manasagangutri, Mysore 570 006 *Department of Microbiology, University of Agricultural Sciences, Dharwad 580 005

MS received 12 December 1977; revised 23 August 1978

Abstract. Downy mildew infected ragi plants exhibits a wide range of symptoms. Histopathological studies of the diseased plant have revealed that the fungus mycelium is present in root, stem, floral parts and seed causing morphological and anatomical changes. The mycelium develops profusely in the sub-stomatal spaces and from this the sporangiophores emerge through the stomata. In the leaf tissue invaded by the fungus, the chloroplasts and leucoplasts are either few or absent. Cells of the mesophyll in the case of the diseased leaves are distorted. Sometimes, the cells of the invaded tissue dissolve and the mycelium or sex organs occupy the space thus created. In the leaf tissue, the intercellular spaces surrounding the vascular sheaths are the primary centres of mycelial development. The sex organs are mostly confined to the vicinity of the vascular bundles. In the infected leaf, very few epidermal hairs are developed.

Keywords. Histopathology; Eleusine coracana ; Sclerophthora ; macrospora.

1. Introduction

Sclerophthora macrospora causes the downy mildew disease o f a number of gramini- cious plants including ragi which is a very important food crop in Karna taka (Ragha- vendra 1974; Safeeulla et al 1976). The affected plants exhibit varied symptoms depending on the host and environmental factors. The plants become stunted with shortened internodes and profuse tillerings. The leaves become chlorotie. The fungus incites partial or complete proliferation of the spikelets into leafy struc- tures often involving the entire earheads and converting them into brush-like struc- tures. The proliferation of the floral parts is common. The lemma, palea, ovary and sometimes the glumes in each flower develop into leaf-like structures. Such an ear is commonly called the ' Green e a r ' or ' crazy t o p ' (Thirumalachar and Nara- simhan 1949).

The present investigations include histopathological study of root, stem, apical meristem, leaf, floral pr imordium and floral parts of infected plants.

2. Materials and methods

The roo t , stem, leaf, terminal buds, young and old inflorescences of healthy as well as the infec tedragi plants were fixed in formalin acetic alcohol. To facilitate

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Proc. (B)--2

20 S Raghavendra and K M Safeeulla

infiltration, roots, young and old leaves, stems and inflorescences were cut into 3 mm to 5 mm bits. After 3 days of fixation, the material was stored in 70 ~o ethyl alcohol. Mierotome sections were prepared following the customary methods of embedding, dehydration and staining with Schiff's reagent with fast green as the eounterstain. Heidenhain's iron alum hematoxylin with orange G as the counter stain was also used in some cases. For detecting mycelium, free hand sections of fresh material were taken and stained with 4 ~ zinc-iodide. Young and oldl eaves, malformed spikelets from infected plants as well as healthy ones were stained with cotton blue or trypan blue after clearing the tissue by boiling for about 10 min. in 95 ~o alcohol and later heating in 2 ~ KOH solution in an oven at 40~ for 12 hr.

Infected ragi plants were uprooted and washed in tap water. The roots were cut into 5 mm pieces. These were treated with chlorine water for 3 hr and later washed for 4 hr in tap water. Subsequently, the material was boiled in 2 ~o KOH for about 10 min and again washed for an hour to remove the KOH. The root bits thus cleared were stained in lactophenol cotton blue for 2-3 hr and mounted in pure lacto-phenol for microscopic examination.

3. Observations

Downy mildew myeelium was observed in all parts of the infected plants and parti- cularly in the vicinity of the vascular bundles (figures 11, 14, 15). The myeelium is aseptate, branched and eoenocytic. The morphology of the myeelium varies con- siderably depending upon its maturity and the plant part it colonises. The mycelium has been detected in root, stem, leaf and floral parts (figures 1-15). Oospores are found in roots (figure 1). When the fungus occupies the stem apex, it branches profusely (figure 3). The hyphase in stem tissues are narrow and intercellular. Haustoria comprise several finger like processes (figure 5).

Leaves of infected plants were generally rough, leathery with warty surface. In the leaf tissue, the inter-cellular spaces surrounding the vascular sheath are the pri- mary centres of mycelial development. The bundle sheath cells, and later the sur- rounding pareuehyma cells collapse as myeelial strands enlarge (figures 10-13). At this stage, the mycelium consists of large globular protoplasmic masses having thin walls. The intercellular eoenocytie mycelium starts in the form of a narrow strip and, while enlarging, pushes aside host cells and fills in the available space (figure 10). In the cell invaded by the fungus, the chloroplasts are either few or totally absent which lead to chlorotie symptoms. The mycelium is thin walled, multinueleate and branched, of extremely variable diameter and with haustorial processes entering the host cells. Sometimes the mycelium is found in the cavities created by the dis- solution of the host cells. The myeelium shows maximum enlargement in the regions which develop oogonia and oospores. In the leaves, the myeelium which gives rise to the sporangiophores occupies the substomatal spaces (figure 12). The mesophyll cells of the diseased tissue are irregular in shape. In the infected leaf, very few epi- dermal hairs are developed, The epidermal cells near the stomatal regions of the diseased leaves are usually bigger compared to the cells in the healthy leaves. At the time of the formation of the sex organs, the hyphae enlarge (figures 11, 13, 14). The myeelium that invades the bundle sheath develops into oogonia and finally oospores (figures 11, 14, 15). With the presence of oospores around the vascular

Histopathologieal studies on E. coracana 21

Figures 1-8. Histopathology of Eleusine coracana infected by Sclerophthora. 1. Root L. S., Oospores x 97. 2, 3. Primary mycelium stem and stem apex L. S., x 230, x 217. 4. L. S. Floral primordium, mycelium x I10.5. Mycellium showing hausto- rium x 514. 6. Whole mount, ovary, mycelium x 70. 7. Basal region of ovary, L.S. , myceUium • 124. 8. Mycellium with pollen grains x 398.

22 S Raghavendra and K M Safeeulla

Figures 9-15. Histopathology of Eleusine coracana infected by Sclerophthora. 9. T. S. Ragi leaf uninfected • 372. 10. T. S. infected leaf, mycelium x 422. 11. T. S. leaf portion, mycelium in the bundle sheath cells • 436. 12. T. S. leaf por- tion, mycelium in the sub. stomatal base x 680. 13. L. S. Leaf mycelium x 265. 14. L. S. leaf, oospores near vascular bundles, x 255. 15. T. S. leaf, oospores near vascular bundles x 410.

Histopathological studies on E. coracana 23

bundles, the xylem elements become very much enlarged and distorted. In the longitudinal sections of the infected leaves, chains of oospores are seen near the vascular bundles (figures 14, 15). After the development of the oospores, the remain- ing mycelium disintegrates.

Downy mildew mycelium occurs in abundance in the glumes. In the flower it is present in the anther tissue and is associated also with the pollen grains (figure 8). In the ovary wall, the ramification of mycelium is seen (figures 6, 7). The mycelium has been detected in floral primordium, ovary, style, stigma, anthers, endosperm and embryo (figures 4, 6-8).

4. Discussion

Ragi plants systemically infected with S. macrospora are stunted with chlorotic leaves. The hypertrophy and proliferation of the floral organs result in a ' crazy top ' symptom. Anatomical study of the systemically infected plants revealed the presence of two types of mycelia, the primary mycelium which is extensively branch- ed, thin walled and located in the root and stem and the secondary mycelium with enlarged hyphae mostly found in the mesophyll. The association of myeelium with the conducting tissue suggests nutritional relationship between the host and parasite. Due to the disintegration of chloroplasts, the infected leaves show chlorotic symptoms. Similar observations have been made by others in corn, sorghum and rice (Ullstrup 1952, Whitehead 1958, Akai et al 1964).

The oospores develop profusely in the vegetative leaves as well as in the proli- ferated ears but rarely in roots. The development of antheridia, oogonia, and oospores is according to the description given by Ullstrup (1952) in corn, Whitehead (1958) in sorghum and Akai et al (1964) in rice. The presence of oospores in the root is of epidemiological significance. Ullstrup (1952), Whitehead (1958) ,were not able to locate the oospores in the root of corn and sorghum.

The effect of the fungus is more marked in the flower. Its development is affected by the intensity of infection. The fungus enters the anther, ovary and ramifies their tissues. The pistil becomes sterile, curled and leaf-like contributing to the brush-like appearance of the inflorescence. The association of the mycelium in the floral parts indicates that the fungus could be seed-borne, particularly in those cases where seeds are formed from infected ears. The changes produced in the inflorescence and the proliferation of the spikelet have been described by Ullstrup (1952) in corn, Whitehead (1958) in sorghum and Akai (1964) in rice.

Singh and Pushpavathy (1965) reported myeelium in floral primordia, flowers, ovary and anther tissue in bajra infected with Sclerospora graminicola (Sate.) Schroet. Jones et al (1972) have observed myeelium in style, ovary wall, and nucellus of the corn flower infected by Sclerospora sorghi Weston and Uppal. The present study confirms the views expressed by these authors for other hosts like corn, rice and sorghum. Singh (1969) has reported the presence of mycelium in leaves but not in roots or seeds of corn infected by Sclerphthora rayssiae Kenneth var zeae.

The presence of the pathogen in all parts of the plant has been observed in infected ragi plants, which indicates that the pathogen is not only systemic but may be seed- borne also.

24 S Raghavendra and K M Safeeulla

Acknowledgements

The au thors wish to record the i r thanks to Agr i cu l tu r a l Research Service, U S A for

the f inancial help u n d e r the PL-480 (FG-IN-414) , AT-CR-352). T h a n k s are due to the au thor i t i es o f the Univers i ty for l a b o r a t o r y facil i t ies. The au tho r s wish to t h a n k

D r M A Rau for cor rec t ing the manuscr ip t .

References

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