SOME OBSERVATIONS ON THE DOWNY MILDEW DISEASE OF

r Frequent field observations made in the vineyards of Jaffna showed that the disease is confined to the rainy season of the year. The developmeht of symptoms on various parts of grape vine has been observed. Invasion of the host tissue was accompanied by changes in colour and appearance of the affected omon. Sporangia of Plmmopa~a vificola germinated after 18 h of incubation at 2.5' * IOC and 100% r h and their single thick germ tubes penetrated the stomata by about 48 h. Penetration strictly occurred through stomata. Following penetration the fungus formed intercellular hyphae and colonised the spongy and pallisade parenchyma cells of the host. The external appearance of sporulating structures took about five days and this occurred stricdy through the stomata. The lesions remained productive upto the ninth day. during incubation.


Introduction
Grape vine (Vitis vinifera) cultivation in Sri Lanka has been a success and the grapes produced in the vineyards of Jaffna and the eastern regions are suffi.cient to meet the needs of the whole country.In tropical climates the grape vine is evergreen and it has been found in the Jaffna region that by forcing the vine into two growth cycles, one in the wet season and the other in the dry season, it produces profitably.
The most destructive fungal disease of grape vine is the downy mildew caused by Plasrnopara viticola (Berk.& Curt.)Berl.& de Toni.This disease has been observed in almost all the vineyards in the Jaffna region during the rainy season and periods of dew from October to March.The development of the fungus was favoured by the high 5umidity and low temperature of rainy weather and the presence of dew.There was a gradual weakening of the plants due to infection and the yield was reduced because of the severe yearly attacks by the fungus.There have been instances where grape cultivation was completely abandoned as a result.
The present investigation was made in order t o obtain basic information with regard to the occurrence and biology of the fungus in the vineyards in the Jaffna region.This is indeed the first report of such a study in Sri Lanka on the downy mildew disease of grape vine.

Experimental
Frequent field observations were made in the vineyards of Jaffna apd information regarding the occurrence of the disease and the development of symptoms was recorded.
Following the observations on the development of the symptoms on various parts of the grape vine, the different developmental stages of the fungus, namely the germination of sporangia, internal colonization and sporulation, were examined in detail under laboratory conditions.
For the laboratory experiments healthy mature leaves were collected from the field, brought to the laboratory and maintained at a temperature of 2 5 O ~ * 1 in an illuminated incubator (Gallenkarnp Cooled Incubator. . . ..).
These materials were used as the source of host tissue throughout the experiment.All observations were made on the grape vine variety Israel blue which is the commonly cultivated variety in Sri Lanka Inoculum was obtained from mature vine leaves infected with Plasmopara viticola.,These affected leaves collected from the field were placed in pairs, under surface to under surface, in plastic boxes and kept in refrigerators at 4OC.When required these leaves were taken out and shaken in a petri dish of sterile distilled water (SDW).The sporangial suspension thus prepared was used to inoculate fresh sets of leaves or Ieaf discs of 2 cm diameter.The concentration of the sporangial suspension was adjusted to 2.4x107 sporangia ml-I with SDW.
Inoculation was done by spraying the sporangial suspension with a hand sprayer.Leaf discs of 2 cm diameter or-entire leaves were inoculated on the lower surface and kept on moist filter paper discs in Petri dishes.'The Petri dishes were incubated at 25' k 1°c and at 100% rh in an illuminated incubator.
Assessment of the development of the fungus was one as follows:-

Germination of sporangium
Five inoculated leaf discs were removed from the illuminated incubator at two-hour intervals during incubation and sellotape impressions of the inoculated surface were prepared.The preparations were stained with cotton blue in lactophenol and mounted in 50'70 glycerine on a clean glass slide.The impressions were observed under a light microscope (x 400) for germination of sporangia.The numbers of germinated and ungerminated sporangia were determined from counts of 200-300 sporangia in five microscopic fields, randomly selected from each of the five leaf discs.A sporangium was considered to have germinated when the germ tube length exceed its breadth.4From the above 'data the mean percentage germination of sporangia was determined.The length of germ tube of about fifty geminated sporangia was measured after '24 hours of incubation, under the microscope (x 400) with the use of a calibrated eye-piece pticule.

Infection
Observations were made on hand sections and scrapings of the host leaf, taken at six-hour intervals after incubation.The time of appearance of initial symptoms and formation of visible lesions were noted..The amount of infection was determined at 24-hour intervals after inoculation by measuring the diameter of visible lesions.The lesions were assumed to be circular and the diameter of each lesion was measured thrice at right angles to each other, under the microscope (x 40).The amount of infection was expressed as mean perceritage area of infection taken from sets of five leaves.

Sporulation
The inoculated leaf discs were incubated at 25O * 1°C and 100% r h sets of five leaf discs were removed from the illuminated incubator at six-hour intervals commencing 48 hours after inoculation and assessed for sporulation, by the following.twomethods:

Sporangiophore production
The incubated leaf discs were boiled in 1: 1 acetic acid-acetone mixture in a water bath for 15 minutes to decolourise the host tissue.After decolourisation the host materid was placed on a glass slide, stained with cotton blue in lactophenol, mounted in glycerine and covered with a clean cover slip.These preparations were observed under the.rnicroscope and the number of sporangiophores present per microscopic field (x 40) was determined.These values were then converted t o number of sporangiophores produced per mm2 area of the host tissue.

Sporangia production
The incubated leaf discs were shaken with one ml of SDW in Mc Carty bottles for one minute.The numbei. of sporangia present in the sporangial suspension was determined by using a haemocytometer.These values were then converted to number of sporangia produced per cm2 area of the leaf surface.

Perennation of the fungus
The perennation of the fungus during unfavourable seasons has in most cases been ascribed to the production of oospores as reported by Lafon & ~u i l t .~ Attempts were made to look for these by periodical sectioning of infected leaves fallen on the ground and leaf-debris during the disappearance'of the fungus.

Development of symptoms on grape vine
All green parts of the grape vine, namely the leaves, tendrils, stems, inflorescences and berries were found to .beaffected by the fungus.Invasion of the host tissue was accompanied by changes in colour and appearance of the affected portion.The colour changes and symptoms produced on various parts of grape vine are given below:

Leaves
The leaf was found to be the most susceptible tissue to fungal attack.The first evidence of infection was the appearance of light yellow translucent spots or 'oil spots' bn the upper leaf surface resulting from the internal colonization by the fungus within the host tissue.The leaves later became mottled and soon whit; patches of downy growth of the sporulating structures were formed on the lower surface of the leaves.These were due to the sporangiophores and sporangia of the fungus.The sporangiophores were branched and were of determinate growth.These were found to arise sGgly or in tufts from the epidermis strictly through the stomata The branches of sporangiophores were found to arise perpendicularly from the main.axis; the branchlets produced single elliptical sporangia at their terminal ends, on sterigmata, The mature sporangiophores were hyaline and did not take up the stain cotton blue in lactophenol.
When the disease became severe in a vineyard, the lower surface of the leaves was fully covered with the fungus.The lesions covered with the sporulating structures turned brown and finally became necrotic.Badly infected leaves became dry and crumpled and finally dropped from the plant, Vine plants that had been poorly cared for were completely defoliated due to the infection.

Stems
Stems were found to be affected only during growth up to a distance of about 80-100 mm from the apex.The infected portions.ofthe stems deve-loped brown streaks and at later stages of infection they became hooked at the tips.The nodes were found to be more susceptible than the internodes.No sporangiophore production was observed on the surface of the stems.

Tendrils
Young tendrils were infected by the fungus and showed symptoms of infection as brown streaks.The symptoms first appeared at the tips and then gradually spread to other portions of tendrils.Here too the tendrils became hooked at the tips at later stages of infection.No external production of sporangia ,was observed.

Inflorescence
Brown irregular patches or longitudinal streaks appeared on the inflorescence axes due to fungal attack; very often the infected inflorescence curled up, became dry and failed to develop fruits.On the inflorescence axis no external production of sporangia was observed.The affected inflorescences were often shed from the plant.

Berries
Downy mildew attack on the berries was apparent from the initial formation until ripening.After flowering, the developing bunches which were subjected to fungal attack showed yellowish brown patches of infection due t o the penetration of the fungus through stomata of the stalks and the fruits.As the berries matured, the diseased parts became brown but at no stage was external production of sporangia observed.However, white masses of sporulating structures were produced on the stalks of the benies where the stomata present were still functioning.Berries severely affected at an early stage failed to develop further, but when affected at a later stage of development became shrivelled arid were found to be shed easily from the plants.During the period of investigation it was not possible to observe the white cottony growth of the mildew on parts other than leaves in the fields.

Germination 'of Sporangium
Spbrangial suspensions when examined under the microscope showed presence of three stages of sporangia depending on the maturity.Sporangia of the first category were immature, small (20-26 p m in length and 12 -16 p m in breadth), finely granular and were filled with inclusions.
These sporangia readily took up the staincotton blue in lactophenol.
Their hyaline wall was thin and uniform in thickness.Papillae were not distinct in these sporangia.These sporangia were capable of germinating after about 48 hours of incubation.Sporangia of the second category were fairly Niranjani Rarnanathan and A. Sivapalan mature (24 -32 pm in length and 15 -18 pm in breadth), slightly brownish with dense inclusions and oil globules and were found to germinate directly within 24 hours forming single thick germ tubes.Sporangia of this type and their germ tubes readily took up the stain.The germ tubes were found to arise from the papillav ends.Sporangia of the third category were large (30 -37 pm in length and 15 -18 pm in breadth) brownish and coarsely granular.These did not germinate directly by producing single germ tubes; instead, they germinated by producing zoospores.Presence of zoospores was only observed in the sporangia of this category.Counts on the number of zoospores present in these sporangia indicated that about 4 -16 zo,ospores were usually produced from a single sporangium, irrespective of the conditions in which these sporangia were formed.The zoospores were about 2 pm in diameter on an average.The sporangia have been observed to burst open at the papillary end and the zoospores were found to escape through this opening.The liberated zoospores have been observed to reach and germinate on the lower leaf surface of vine leaves.Germination by zoospore formation has been found to be less frequent than direct germination of sporangia In all experiments on gennination,the germination of only the second category of sporangia was assessed.
In the study of gennination of sporangia, inoculated leaf discs incubated at 25O + 1°C and at 100% r h in an illuminated incubator were assessed on the germination after short periods Sporangia started to germinate after about 16 hours producing thick, single germ tubes.However, a considerable number of sporangia was found to have germinated 18 hours after incubation.The germination values increased significantly up to 48 hours (Table 1).
The length of g e q tube of germinated sporangia increased with increae in period of incubation (Table 1) but since the germinated sporangia were found to reach and penetrate through the stomata or perish after 48 hours, it was difficult to make further measurement on the germ tube length after 48 hours of incubation.

Internal colonization
The germ tubes that penetrated the stomata were found to reach the sub-stomata1 cavity on the lower surface of the host leaf.Observations showed that 'on the lower leaf surface, one stoma was penetrated by about three germ tubes on an average but the number varied from 1 to 5. The average number of stomata per microscopic field (x 400) on the lower surface of the host was found to be 6 and about 5m of the stoma were penetrated.It was also observed that the penetration of the host occurs strictly through stomata.Direct penetration through the epidermis or by other means has not been observed.The, germinated sporangia which entered the host tissue fonned intercellular hyphae and colonised the spongy and the palisade parenchyma cells of the host.Globular haustoria with short necks were observed inside these host cells.
Following the internal colonization, the host tissue changed in colour from green to pale yellow depending on the severity of infection.This discolouration or the appearance of oil' spots of infections on the upper leaf surface was observed 60 hours after incubation and was the first visible symptom of infection.
The mean percentage area of infection increased with increase in period of incubation and reached 100% by the twelfth day after incubation (Table 2).  . .
Following internal colonization, the emergence of spor+giophores took place through the stomata on the lower leaf surface.The first emergence of sporangiophore initials was observed around 65 hours after incubation, and clusters of sporangiophores continued to emerge out of the stomata after 72 hours of incubation.About 25% of the total stomata present in a micros-

Period of incubation (days)
Mean % area of infection copic field (x 400) showed emergence of sporangiophore initials.Mature sporangiophores were present only on the fifth day after incubation.The mature sporangiophores were branched or unbranched and varied in axial length from 116 pm -286 I m and in breadth from 16 -26 pm.They produced 3 -7 branches and 3 -7 branchlets on each branch.The mature sporangioph&res emerged out of the stomata at the rate of about 4 per stoma on an average but the number varied from 1 -12 per stoma, on a lesion that was five days old.
In such a lesion, about 90% of the total stomata in a microscope field ( x 400) showed presence of sporangiophores.The number of immature sporangiophores increased up to the sixth day after incubation and gradually decreased thereafter.At the same time the number of mature sporangiophores increased up to the eighth day and then became static (Table 3).By this time the lesion had attained its full growth.
The length measurements of sporangiophores showed that the sporangiophores increased in length with time from the third day after incubation.The increase was rapid up to the eighth day and thereafter the length of the sporangiophore remained more or less constant (Table 4), Sporulation or sporangia production was measured by detenpining the amount of sporangia of the three categories produced per sq.mm area of the lesion (Table 5).The amount of.immaturesporangia produced was high on the fifth day after incubation and thereafter it started decreasing.From the .. . .fourth day onwards the number of fairly mature .sporangia increased in number and reached a maximum value after nine days The amount of fairly mature sporwgia then decreased slowly and became static on the eleventh day.Similarly the amount of mature sporangia present increased from the fifth day and reached its maximum value between the eighth and the ninth days.The value became static on the eleventh day after decreasing slowly.
During these investigations oospores have never been observed though attempts were made to look for them.

Discussion
It has been observed for several years that the downy mildew disease of grape vine is a destructive fungal disease.This is usually severe during periods of high humidity and low temperature or rainy weather conditions.
The fungus responsible for grape vine downy mildew Plasmopara viticola (Berk.& Curt.)Berl.& de Toni. is an obligate parasite which carmot therefore be easily cultivated in vitro or on inert nutritive media.
Obseniations on the occurrence of d o k y mildew in the vineyivds showed that infections begin in September or October when the rainy season starts and last till April with the greatest severity of the disease observed in late February or March.
Almost all green parts of the vine were affected by this fungus as already ob'served -by Ramanathan and ~ivapalan,6 but the most prominent symptoms were on leaves.It was suggested by Rives ~a f o n ' that only the young actively growing organs are susceptible to infection.The infections occurred an the lower leaf surface and undoubtedly through the stomata, as already confirmed by Ravaz and verge?
The first symptom of infeetion was the formation of translucent yellow 'oil spots' on the upper surface which is,followed by appearance of a white downy growth of spo.rulating structures on the under leaf surface corresponding to the oil spots.Apart from leaves, the bemes, tendrils, inflo- rescences and stems too developed infection2 but at no stage was external production of sporangiophores observed on parts other than leaves during this study.Infection on these parts appexred as only brown, irregular or linear patches or streaks zis a result of internal colonization.However there are reports by several authors on the formation of ~orangiophores and sporangia on the surface of bemes and inflorescences.But these workers claim that these organs are susceptible as long as the stomata on them remain functioning for the reason that penetration of host and emergence of sporangiophores occur strictly through stomata Weakening of leaves due to infection led to their premature senescence.The yield and life span of the grape vine was highly reduced by the downy mildew attacks.
The infection process of the fungus was initiated by germination of sporangia during favourable conditions.Sporangia directly germinated by emitting the entire contents and fonnirig thick germ tubes which penetrated stomata on the host leaf and colonised the inner tissues.The external appearance of sporulating structures through stomata of the lower leaf surface took about five days and lesions remained productive up to the ninth day.
The peremation of the fungus during unfa~ourabIe seasons has been

.
attributed by several workers3 to the sexual reproductive structures -known as the oospores.Observations towards the eqd of the season failed to reveal the presence of oospores or the mode of perennation. of the fungus.The disease is completely absent during the season from May to September.In these circunistances the mode of overwintering may be by preservation of mycelia which remain between the bud scales in diseased plants until budburst as suggested by ~a l e t ~ and ~o u b a l s .~

Table 3 .
The production of spormgiophores of Plasmopara mticola on detached leaves of grape vine at 2 5 ' ~ and 100% relative humidity.

Table 4 .
Growth of sporangiophore of Plasmopara mticola on detached leaves of grape vine a t 2 5 ' ~ and 100% relative humidity.

Table 5 . Production of sporangia of Plasmopara viticola on detached leaves of grape vine at 2 5 O ~ and 100% of relative humidity. Period of incubation (days) Mean ,. number of sporangia produced/ mmL area of host leaf.
= Fairly mature sporangia m. = Mature sporangia.