iotype Distribution of Vascular Wilt Pathogen Pseudomonas solanacearum in Sri Lanka

$he:bacterial wilt caused by Pseudomonas solanacearum E. F. Smith is one of'the @aj.or diseases of solanaceous crops, The disease occurs mostly in warm climates. It is Briown,to infect various other hosts such as ~anand'~eanuts:~aste/t~in~ed bean'. 'has An erratic distribution.in soil.%lso variation within the species based on nk~rphological, physiological pioperties' has been well d o c ~ t n e n t e d . 2 . ~ ~ ~ ~ ' ~ ~ ~ ~


Introduction
$he:bacterial wilt caused by Pseudomonas solanacearum E. F. Smith is one of'the @aj.or diseases of solanaceous crops, The disease occurs mostly in warm climates.It is Briown,to infect various other hosts such as ~a n a n d ' ~e a n u t s : ~a s t e / t ~i n ~e d bean'.'has An erratic distribution.insoil.%lsovariation within the species based on nk~rphological, physiological pioperties' has been well d o c ~t n e n t e d . 2 .~~~~' ~~~~ Haywards grouped P. solanacearum into four biotypes.This c~assificationwas $aed on their ability in oxydizing disaccharides-lactose, maltose, cellobiose and hxose alcohols -n a n n ~t o l , sorbitol and dulcitol.Seneviratne1o reported an occurrence ~f b i o t ~~e s 2, 3, and 4 in the hill country of Sri Lanka.Further studies were done to eqablish the distribution of biotypes throughout major agricultural areas of the island.An attempt was made to relate it to environmental factors.

2,l Field survey
So!anaceous crops infected with Pseudomonas solanacearum Smith were collected fro@ various agroecological regions of Sri Lanka.Whole plant samples were brought td the laboratory and vascular tissues were used for the isolation of pathogen from infected tomato, potato, capsicum and brinjal plants.Potato tubers were also used for $he isolation of bacterium.
Selection of sampling locations were based on the previous cropping histo*, soil type, altitude, rainfall and temperature of the region.
The popular climatic division of the island is classified into two major divisionr "wet" and "dry" zone.This expresses the regional hygro-climatic differentiation based on climatic reality which also reflects a land use and crop cultivation pattern, Although the wet and dry zones ( In the present study agro-ecological regions classified according to 75% expectancy values of annual rainfall and elevation (Land and Water in 1979) was u s 4 for the selection of sampling locations.This apparently coincides with the wet and dry zone classification proposed by Wickramatilaka (1963) on effective dry period basis (Figure 1 ).
The land area under wet and dry zones are 1.6 million and 4.9 million ha respectively.The average annual rainfall in wet zone is about 32 million acre feet (1 Ac.ft.= 760 nimlha )and 57 million acre feet in the entire island, that is rainfall in the wet zone is 72% higher over dry zone.3

Dry Zone
In the dry zone, wet and dry periods alternate once a year.The rainy period is only 3 -4.months (Oct./Nov.to January) but the rains are disproportionately high.The dry period, maximum of 8 months (Feb.to Sept./Oct.) is extremely dry.The 75% expectancy value of annual rainfall is around 508 -762 mm.Soils in the dry zone are mainly reddish brown earths.But red yellow latosols and regosols, low hurnic gley soils and solodized solonitz are also found in some regions (DL,).
The annual average temperature however does not relate to dry and wet conditions.The average annual records reveals homogenous temperature in the! lowlands and the rapidly decreasing temperature ~n the h~ghlands.In the lowland dry zone up to 150 m the temperature varies from 26.5Oto 28'~, the spatial variations of temperature in the region are slight.
These climatic conditions have produced a suitable niche to many crops and a wide variety of vegetation in the dry zone.Many grain crops, pulses, vegetables of various families, tubers and industrial crops are the major economic crops grown in, the area.Among the common annual crops, rice is by far the most important crop during Maha.Although the soils are flooded for 3 -4 months during paddy crops, solanaceous vegetables mainly capsicum, tomato and brinjal are grown undel: irrigation in these fields during the Yala season.However, in Jaffna potato crop is given high priority.

Wet Zone
The Wet zone -Dry zone boundary that rims from Negombo via Kurunegala, and then to Matale, along the top ridge of Knuckles through the Corbet's gap to Pidurutalagala massif to go south of Nuwara Eliya across the high plains.Then it goes round Balangoda in an arc along the Sabaragamuwa hill country to reach Matara.The South West quarter of the island thus forms the Wet zone.However, the boundary is not rigid and merge with dry zone through the intermediate zone.
The 75% expectancy values of annual rainfall range from 889-3 175 mm.Soils are mainly red yellow podzolic type.Variation on the horizons are found.From a climatic standpo~nt, the wet zone is thus characterized by greater a i cultivation advantages in terms of more intensive diversified land use.In the Pidurutalagala massif, the central highlands of Sri Lanka rise to a maximum altltude -Biotype Distribution o f Vascular Wrlt Pathogerl hf.2,524 m, which produces a considerable vertical thermal contrast between highlands and lowlands.Temperature variations are very prominent, it falls quickly as the altitude increases (Table 1) Because of the thermal requirements, the optimum condition for wetland rice dc'eurs in the hill country only up to about 1,200 rn (4,500 ft).However, other crops such as potato, tomato, brinjal and capsicum aregrown.New areas have been cleared for,potato cultivation in the hill country.
< Samples were collected from potato fields elther grown m traditional potato

Pathogenicity tests
Pathogenicity of bacterial isolations were confirmed by reinoculating tomato plants.
Five weeks old tomato cv.Marglobe plants raised in sterile soil in plastic pots (15.5 cm diameter) were stem inoculated with bacterial suspension containing 106 cells/ml.Pacterial suspension was prepared with 48 h cultures grown in calc~um carbonate agar.Threelop Idrops of the suspension was placed on the axil of the third leaf from the top, and the stem was then p r l c k d with a syringe needle (gauge 25) through the inoculum drop.Inoculated plants were placed in the green house ( 2 4 ' ~ night, 284 3 3 ' ~ day).Plants were observed for the wilt and yellowing after 3 days from inoculation.

Biotype separation
~l l isolations were s u b j e c t d to biochemical tests for thejr ability to oxidize disaccharides and hexose alcohol proposed by Hayward!Bacterial cultures were inoculated to culture tubes (1 x 12.5 cm) containing the following medium.
Medium was adjusted to pH 7.1 with IN NSOH before adding 1.5 g agar.Inoculated slopes were incubated at 2 7 ' ~ for 14 days.Slopes were observed fo; acid production on the 3rd, 7th and 14th day before discarding.Although this confirms to the results previously reported by Senevuatnelo from the samples collected at Gorandiyatenna, it was decided to subject the bacterium ' ; ' for further biochemical and taxonomical study.

. Results and Discussion
The biochemical tests used were Oxidase test with a platinum loop6, Arginine -dihydrolase test14 Nitrate reduction and Carbon source utilization in Ayers et al.
mineral salts medium in addition to Acid production tes-is.'Results are summarized in table 2. .,

Acrd produalon tests
,-.Although the carbon source utilization and acid production tests indicated a presence of biotype 4, it is evident from table 2 that the results are not conclusive 0 enough.Therefore all isolates were then grown at 27 C in King's medium B agar (oxidase positive), composed of proteose peptone (Difco) 20.0 g; KJ-lPOI.3H,02.5 g; MgS0,.7H,O6.0 g; agar 15.0 g; glycerol 15.0 ml i n 1 L of distilled water and examined with a long save(375 n l n ) -ultra violet lamp Gelman, universal UV unit for fluorescence.Green fluorescence was detected.
Fluorescent and the non-fluoresxnt were later separated out carefully for pathogenicity tests.Non-fluo;escent bacteria was then i d e n t ~f ~e d as pathogenic Pseudomonas solanocenrum biotype 3 whereas fluorescent bacteria was identified as Pseudomonas marginalis.When artificially inoculated tubers showed soft rot, and sljght wilting of plant with browning of leaf margins.However when bothstrains were inoculated to potato plants wilting was seveie and prominent, with conspicuous.vascular wilt symptoms.I t is evident from these results that in the hill country wet and intermediatezone (IV, & IV,) Pseudomonos marginalis prevails in association with Pseudomomzs solanaceorurn.Probably it has a synergistic effect in wilt production.
Although Seneviratne (1964) reported the presence of biotype 4 in the hill country, 1 luve found only 2 biotypes namely 2and 3 amongthesamples collected.The distribution of biotype 2 was restricted to potato and within ihe 1 6 ' ~ isotherm (Figure 2).Absence of biotype 2 in potato crops in the low land dry zone holds evidence for the effect of temperature on the survival.Figure 3   The characteristic feature of the distr~bution of biotype 2 is that it 1s restr~cted to a small area in the hill country delimited by 1 6 ' ~ isotherm.Thls indicated its ' temperature dependency rather than rainfall or soil types.
In view of the fact that both biotypes 2 and 3 were found in new clearings and ~t s .occurrence in the hill country where the distribution through irrigation water is ;unlikely it is suggested that these biotypes are indigenous to S n Lanka.The broad *, spectrum host range of biotype 3 and its ability to surviveeven at higher temperatures .such as 31°c has made the occurrence of biotype 3 islandwide.
Figure I ) are not internatronally valid terms defined by climatic indices, they are realistic to climatic situation in the island.Of course viewed from the other dry regions of the eafih the "Driest" in the dry zone -Mahalewayasaltern in Hambantota still records a n average rainfall of 929 mm per year.

2 : 4 ,
, .fields, newly opened areas or paddy fields.Potato tubers and plants were collected from Hawaeliya -1981.Nuwara Eliya -1981, Sita Eliya -1981, Rahangala -1981 sad Yalapatwela -1981.Infected tomato plants were obtained from Kandy, University fdrm -1980.Diseased tomato plants were also collected from Rahangala -1981, and Bandarawela -1981.Bacterium was also isolated from the tomato samples collected from Mirahawatte and Welimada -1981.Several samples from each location were used for the isolation of the pathogen.The tomato samples collected from low country wet zone Weboda (1979), Mawanella 1979 , and Mapalana 1980 were also used for this study.Isolation of the Pathogen Isolations were made from potato (Solanum tuberosum L.), tomato (Lycopersicon esculentum Mill), Egg plant (Solanum melongenae L) and capsicum (Capsicum afinum var.grossum ) plants showing typical symptoms of bacterial wilt in the field.Bacterium was isolated from the vascular tissues of infected stem and was used to streak plants.Potato tubers from infected plants were also used for the isolation of the pathogen.Bacterial ooze from clean freshly cut surface of tubers were taken in sterile Water as inoculum.1 Axenic cultures were prepared by streaking agar plates with the bacterial silmples.The medium used for culturing and routme maintenance was as follows.(calcium carbonate agar) peptone 5.0 g; yeast extract 0 5 g; glucose 5.0 g; K2HP0~0.2 g; MgSOd.7H20,0.2g;c ~c o ~, ~. o g; agar 18.0 g; distilled water 1 L. Nutrient medlum was sterilized at 15 psi for 20 min.Inoculated plates were maintained at 21 0.10C in precision low temperature ,in&bators.
parts of sterile, cooled basal medium added 1 part of membrane filter sterilized 105" (w/v) of each carbon source.'-Medium was then dispensed aseptically in a horizontal Laminar ~i o o ~ Cabinet (Environmental Control 1NC.) to sterile plugged tubes to a depth of about 4 mm.

I
t was found that in the Yala season (N: E. monsoon, December -February) Solanurn melongenoe L. (egg plant) was the dominant solanaceous crop grown in the Dry Zone farms visited.Pseudomowas solanacearum isolated from diseased plant from almost all the sites except Kuchchavely in Trincomalee were pathogenic strains.The wilting : of pdtato samples.collected at ~u c h c h a v e l ~ was duc to Fusarium oxysportlm and !j other Fuscrium spp.! Jn he biochemical tests, the development of a yellow colour in t h e medium j indicated production of acid from she oxjdation of carbon sources.I n most cases jwhere t h e r e is positive acid production slight yellow colour appeared around " j inocalum by the 3rd day and .itwas yellow throughout the medium after I week of growth a t 2 7 ' ~.

I'igore 3
Map o f the ;mnuaJ avcrige temperatures i n Sri Lutka, ~sotherrns in "c.:Seneviratnelo suggested a correlation between cropping history, soil type and lbiotype occurrence.The samples collected from the farms L n new clearings of the > Kalawewa and Madatugama (Capsicum) showed biotype 3 only.Only biotype 3 was .isolatedfrom intermediate zone lV,, IM, and lL, agroecological regions.Even in the new clearings and in potato crops in paddy fields at Yalapatwela only biotype 3 was %detected.

:
Acknowledgements ' I am indebted to Messers P. Thirukumaran and S. Ravindran for the technical assistance over a period of 3 years.My thanks arc also due to Dr. S. N. de S. Seneviratne for his encouragements prior t o the commencement of this study.I greatly appreciate the financial assistance and other help rendered by the -Natural Resources, Energy and Science Authority of Sri Lanka and rts staff without which the project would have been impossible.