A NEW ISOLATE OF SCLEROTIUM ROLFSII SACC . CAUSING BULB ROT IN ONION ( ALLIUM CEPA L . VARIETY POONA RED ) NIRAN

I The causative fungus of a hitherto unreported bulb rot *ease of onion (Rllium cepa L. variety Poona red) was found to be Sclerotium r o w ; Sacc. The vegetative propagative structures of the fungus known as sclerotia, were able to germinate within 24h on a suitable substratum and produce new colonies. The fungus took about ten days to produce fresh sclerotia from germinating sclerotia, under laboratory conditions. The fungal isolate obtained from infected onion plants (Sclerotium rolfsii Isolate 5 ) had a temperature optimum of 35% for germination of sclerotia. It showed luxurious growth and maximum amount of sderodal production at temperatures between 30 and 3s0c. The fungus was able to survive ih a wide range of pH conditions (pH 3 to 9) but had an optimum pH of 5.5 for germination of sclerotia and radial growth.


Introduction
Sclerotium rolfsii Sacc. is a versatile soil borne pathogen attacking several crop plants.7 It is widespread throughout the moist tropics and warmer region of the temperate zone and has an extensive and varied host range.'It has been found to cause a variety of diseases namely damping off of seedlin s, collar or stem rot,' foot rot, crown rot, Sclerotium wilt and B blight.The sequence of events during the maceration or death and decay of host tissue resulting in the formation of symptoms has been found to be complicated as suggested by Punja et al.
The fungus forms sclerotia during unfavourable'conditions and remains in the soil for long periods without being affected by adverse soil and environmental conditions.When favourable conditions intervene and if suitable substrata are available, the sclerotia germinate producing clusters of hyphae around them4 In July, 1986 our attention was drawn t o a disease of Bombay onion (Allium cepa.L.) in the Arali area of Jaffna.The onion plants ofthe variety Poona red were attacked by a fungus.The disease occurred in the seed bed when the plants were about 45 days old.Symptoms were associated with bulb rot.However, the initial symptoms appeared as yellowing of tips of onion scales or leaves.The discolouration was found t o spread down the leaves and the tips became necrotic and brown.The above symptoms were followed by wilting of scathes.Groups or patches of plants showing the above symptoms appeared in the field.On close examination of uprooted diseased plants, the bulbs were found to have withered with the complete loss of their root system.Strands of white fan shaped hyphae were seen on the bulbs.These clusters of hyphae also enveloped the base of the stem near soil level.The rotten plant material and the soil supples taken from around these plants when examined showed presence of numerous, tiny, white or yellow-brown sclerotia at various-developmental stages and in the form of strands.Each of the hyphal strands found on the surface of bulbs consisted of 4 -10 hyphae.From the characteristic growth of hyphae and the size and shape of sclerotia, the fungus was identified as Sclerotium rolfsii Sacc.The identity of the fungus was confirmed by Commonwealth Mycological Institute (CMI Report No: H107/86/YS18).
The fungus was constantly associated with the symptoms produced on onion bulbs in the field.This is in fact the first record of the disease caused by S. rolfsii on onion variety-Poona red in Sri Lanka.The fungus obtained from affected bulbs was referred to as Sclerotium rolfsii -Isolate 5 .

I
Infected onion bulbs (variety Poona red) w,ere coyected from the field and brought to the laboratory.These were washed with tap water, surface sterilized by dipping in 0.5% sodium hypochlorite solution,for 1 min and then washed in 3-4 changes of sterile 'distilled water.Small-pieces from surface sterilized bulbs were transferred on to freshly poured Potato Dextrose Agar (PDA) plates and incubated at roam temperature (30+ 4OC) for seven days.
The morphology of the colony and of the fungus on PDA was noted.Measurements of length and breadth of primary; secondary and tertiary hyphae were made under a light microscope from a 3 day old ,colony of the fungus.H y d sections of mature sclerotia were also made and examined under the microscope.
Following observations on the morphology of the fungus, studies were made on germination of sclerotia, radial growth of mycelium and sclerotial production after different periods of incubation on PDA.For studies on germihation of sclerotia mature brown sclerotia were dislodged from the surface of a ten day old colony with a soft brush and transferred on to fresh PDA plates.Ten sclerotia were transferred an .toeach of 5 PDA plates and incubated at room temperature under .continuousillumination (7 kl).
The percentage sclerotial germination was determined at 24h intervals during incubation: The diameter of colonies formed from the germinating sclerotia were also measurd at 24h intervals along two predetermined diameters at right angles to each other.Radial growth of the fungus was also studied by inoculating 104resh PDA plates at the centre with 0.8cm diameter mycelial discs cut from the advancing region of a 6 day old colony of ~clerotium' rolfsii -Isolate 5 .These plates were also incubated as before for 7 days and the diameter of colonies was measured daily.Sporulation was measured by counting the number of sclerotia produced per plate and then converting the value to number of sclerotia produced per lcm2 of the colony.
With the basic information on the morphology and various developmental phases in the life cycle of S. rolfsii, the effects of some environmental.factors on the above developmental stages were studied.
To study the effect of temperature on sclerotial germination, PDA ' plates inoculated with mature brown sclerotia harvested from a ten day old colony of S. rolfsii were incubated at 10,15,20,25,30,35 and 4 0 ' ~ under continuous illumination for 5 days.The sclerotial germination was determined at 24h intervals at the above temperatures.Five plates were used for each temperature.
Similarly, PDA plates were inoculated with 0.8cm diameter mycelial discs cut from a 6 day old colony and incubated at the above temperatures for 7 days.The diameters of colonies were measured as before on each day during incubation.Ten plates were used at each temperature t o give a total of 20 diameter measurements.
To study the effect of temperature on sclerotial production, PDA plates' were inoculated with mycelial discs as described earlier and incubated at room temperature for 3 days.They were then kept at 10,15,20,25,30,35 and.40'~ for further 4 days.Sclerotial production was estimated at 24h intervals from the third day after initial incubation.Ten plates were used at each temperature.
The effect of pH was studied only on the germination of sclerotia and the growth of the fungus.PH of the medium was adjusted to 3.0,4.0,5.0,5.5, 6.0,7.0,8.0 and 9.0 with a buffer mixture of citric acid/potassium dihydrogen orthophosphate, before being poured into sterile petri dishes.Sclerotia of S. rolfsii -Isolate 5 were transferred t o PDA plates of different pHs and were incubated at room temperature for 3 days.The mean percentage germination of sclerotia was determined each day during incubation.Each set up was replicated 5 times.Similarly, mycelial discs were transferred t o PDA plates at different pHs and were incubated for 7 days at room temperature.The diameters of colonies wdre measured at 24h in'tervals as described earlier.

Results
Colonies of S. rolfsii --Isolate 5 on PDA were pure white with narrow mycelial strands radiating on the colony surface.The ~clerotial initials appeared as tiny white knots of mycelia which later turned pale yellow, pale brown and finally dark brown in colour while they increased in size.Mature brown sclerotia were spherical but slightly flattened below measuring up to about 2mm across when fresh.The outer wall of sclerotia was shiny and smooth.Primary hyphae of the fungus were 5.0 -8.2 m in breadth and grew along the surface of the medium while the secondary hyphae were usually narrow, about 2-4 m in breadth and about 350 m in length and often grew adpressed to the primary hyphae.Tertiary hyphae and other subsequent branches were also narrow and had comparatively short cells.The branching was of tertiary hyphae occurred in a wide angle and was not associated with septation.The tertiary hyphae were usually about 190 m in length and 1.0 -1.8 m in breadth.Clamp connections were only present on the primary hyphae.

PDA (Xg)
Hand sections of the mature sclerotia of S. rolfsii-Isolate 5 showed a sharply differentiated outer rind about 1.2 -2.0/*.m in thickness with strongly pigmented cells.Cortex of the inner region had fairly pigmented small cells and the innermost medulla had large and almost hyaline cells.Reserve materials were found as globules in the cells of cortex and medulla.

Sclerotial germination
Sclerotia germinated within 24h on PDA.The mode of germination was hyphal according to punja4 and the hyphae originated from cells of the medulla in all directions.Mean percentage germination of sclerotia increased significantly at 24h intervals during incubation and reached 100% on the third day after inoculation (Table 1).The germinated sclerotia formed tiny white and circular colonies, consisting of sparse hyphae, which become visible after 24h of incubation.The colony size increased with the period of incubation (Table 1).The above colonies developed sclerotial initials on the aerial hyphae on the fifth day after inoculation. .I Table 2.
Effect of temperature on sclerotial germination of Sclerotium rolfsii -Isolate 5 on PDA.

Radial growth of the fungus
The fungal mycelium showed luxurious growth on PDA.The colony was pwe white and fluffy with dense hyphal strands consisting of 4 -5 individual hyphae per strand.The colonies increased in diameter during incubation and almost covered the entire 9,Scm diameter plate in about.5 days (Table 3).

Period of incubation Mean diameter of colony in cm (houn)
' Temperature (OC)

Production of sclerotia
Sclerotiai initials appeared on the thir'd day after inoculation and turned yellow -brown within two days.The number of immature sclerotia produced increased gradually during the incubation and mature brown sclerotia were seen only on the seventh day after incubation (Table 4).The numbets given within brackets indicate the number of mature brown sclerotia.

Effect of temperature on the germhation of sclerotia
Sclerotia failed to germinate at temperatures below. 2 5 ' ~ (Table 2).Optimum temperature for germination of sclerotia was around 35'~.Mean percentage germination improved with increase in period of incubation.

Effect of temperature on the radial growth of the fungus
Results which appear in Table 3 show that the fungus failed to grow at low temperatures below 1 5 ' ~ while the growth was slow at 20 and 25'~.Best radial growth was observed at temperatures between 30 and 35'C.White knots of sclerotia appeared in the colony on the third day at 30, 35 and 4 0 ' ~ and on the sixth day at 2 5 ' ~.
3.6 Effect of temperature on production of sclerotia 6 The total number of sclerotia and sclerotial initials formed on the colonies was counted on all the plates at different temperatures after different periods of incubation.k o m the above data the number of sclerotia and sclerotial initials formed on lcm2 of the colony was determined.
No sclerotia production was observed at temperatures below 25'~ and maximum amount of sclerotia were produced at temperatures between 30 and 35'~ (Table 4).The mean number of sclerotial initials produced per cm2 area of colony increased with increase in period of incubation.Mature brown sclerotia were observed on the seventh day after initial inoculation at 30,35 or 40'~ and on the eighth day at 2 5 ' .

Effect of pH on sclerotial g e m t i o n
Sclerotia germinated at all pHs tested in this experiment (Table 5).Germination occurred best at pH 5.5.Germination also improved with increase in period of incubation and was maximum on the third day after inoculation at pHs 4 to 7.

Effect of pH on the radial growth of the fungus
The fungus showed luxurious growth a t pHs between 4-7 but growth occurred best at pH 5.5 (Table 6).The fungus was able to survive a wide range of pH conditions.

Discussion
An aerial mycelium composed of both single hyphae and hyphal strands consisting of a few hyphae has been observed on the surface of onion bulbs infected with S. rolfsii which has been described as a plant pathogenic soilborne fungus by cock.^ .
The active spread of the fungus through the soil following the seasonal rain has been observed in the onion fields in the Arali area of the Jaffna region.The infected bulbs became partially or entirely covered with thick wafts of hyphae which radiate upwards from the base of the stem.
The tiny but macroscopic spherical sclerotia produced in large numbers dn and around the rotten bulbs form the outstanding characteristic of the -- organism.The mature sclerotia obtained from diseased bulbs germinated readily on PDA without any period of dormancy.The fungus took about ' " ten days to produce fresh sclaotia from 'germinating sclerotia on artificial medium.The observations that sclerotial initials arise from the aerial strands of a colony tally with those made by Townsend arid ~i l l e t t s .~ Sclerotia of S. rolfsii -Isolate 5 germinated best at 3 5 ' ~ while other stages in the fungal development such as radial growth and sclerotial production required an optimum temperature slightly less than 3 5 ' ~, This is in accordance with the observation that the soil temperature in the Arali area during the period of disease incidence was around 3 5 ' ~.

Photograph 2 :Photograph 3 :Table 1 .
Photograph 2 : The T.S. of a developing sclerotial initial (a) and cluster of hyphae (b) taken from a 3 day old colony of S. rolfsii on PDA,'X40

Photograph 4 :
Photograph 4 : A portion of T.S. through a mature sclerotium obtained from a 7 day old colony of S. rolfsii on PDA.(a) Rind, (b) Cortex, ( c ) Medullary region X400 Although the fungus was found to establish itself & ti wide range ofpH, .. .

Table 5 .
Effect of pH on sclerotial germination of Sclerotiurn rolfsii -Isolate on PDA.

Table 6 .
Effect of pH sn radial growth of Sclerotiurn roifsii -Isolate 5 on PDA. +Mean