PRELIMINARY STUDIES ON THE PREVALENCE AND CONTROLLING EFFICIENCY OF PASTEURIA PENETRANS, A PARASITE OF MELOIDOGYNE SPP. IN SRI LANKA

: Prevalence ofPastezrria penetl-ans in Sri Lanlra was investigated and the effects of microenvironmental factors on its prevalence were determined. Naturally-occurring Paster~riri isolates were then evaluated for the efficiency of controlling Meloiclogyne spp.. Randomly collected 804 soil samples were tested for the presence of Pastcuria using second stage juveniles of Meloidogyrze. Controlling efficiency of Pasteuria was evaluated on greenhouse-grown tomato (Lycoj~ersicon csculentu.nl var. T-146) plants. Out ofthe tested soil samples, 26% reported Paste~cria spores in varying concentrations. Microenvironmental factors such as soil drainage and cropping history showed a significant influence on the prevalence oflUastcuria whereas cropping pattern had no significant effect on its prevalence. However, the interaction between the cropping history and cropping pattern also had a significant effect on the prevalence of hsteuria. Uncler greenhouse conditions, Pasteuria isolates showed 48% control of the nematode in tomato (var. T-146) and the root powder prepared from above tomato plants gave 8 x lo5 sporeslg root (dw). The~.efore,~naturall.y-occurring P. pe7tetrnn.s isolates of the tested areas of Sri Lanlta could be used as a potential biocontrol agent of Meloidogyrle spp..


INTRODUCTION
Root-knot nematodes (Meloidogyne spp.) are serious pests of many agriculturally-important crops grown in Sri Lanka.4.9.10 The pest can be controlled by cultural and chemical-methods but these control measures are associated with practical limitations and environmentally-unfavourable effects. Crop rotation has limited success in the control of root-knot nematodes and fallowing though effective for the control of the pest, would not be a n economical option for the farmer. Use of nematicides is associated with many hazardous effects such as destruction of non-target organisms, contamination of ground water and bioaccumulation. Above all, small scale farmers are unable to afford the cost of chemical control of the nematodes. Therefore, biological control could '"orresponding author.
be considered as an alternative approach in managing root-knot nematodes and it can be effectively incorporated in the integrated manageme~lt of the pest.
Pasteuria penetrans, has been identified as a promising biocontrol agent of Meloidogyne spp. by several workers.'.fl,l2 When the bacterium is added to soil in pots, small plots, container-grown crops and. polytunnels, significant reduction of root-knot nematodes has been attained.j.~.~ Pasteuria is a soil inhabiting, mycelial and endospore-forming bacterium and has two modes of action against the Meloidogyne spp.. It can inhibit the egg production of adult female nematode and the entry of the infective stage of the nematode into host roots. Therefore, a reduction in the nematode population over time can be seen.
Although the root-knot nematodes have been a problem in Sri Lanka for many decades, investigations so far conducted on potential biological control agents are limited. Therefore, the present study was conducted to investigate prevalence of P. penetrans in Sri Lanka, determine the effect of microenvironmental factors on its prevalence and evaluate the controlling efficiency of Meloidogyne spp. by naturally-occurring Pasteuria.

Experimental region
Kandy District of Sri Lanka was selected as the region of investigation.

Additional information on the sampling site
Microenvironmental factors which could have an influence on the growth and development of Meloidogyne and Pasteuria were recorded. The factors recorded were, cropping pattern (i.e. continuous or on and off cultivation), cropping history (i.e. for how long the same crop has been in a particular land) and drainage condition.

Detection of Pasteuria penetrans i n soil
Second stage juveniles (J,s) were used to detect the Pasteurza spores in s 0 l l . 1~~~ J,s were initially isolated from the infected roots of several vegetable crop species collected from the Gelds at Marassana, Thalatuoya and Thannekumbura in Kandy District. After isolatio~l J,s"ere cultured on greenhouse-grown tomato plants (var. T-146).
Detection of Pastez~ria in soil samples was done on plastic cylinders having 7.5x7.5 cm dimensions. One end of the cylinder was sealed by a piece of muslin and a piece of paper towel was laid over it. Ten grams of soil was spread on the paper towel and approximately 15 J,s were added on to the soil. Two-day-old J,s were used and they were free from pre-encumbered P a s t e~~r i a spores. These J,s were extracted after 24 h S and examined under the light lnicroscope (10x10) for spores attached on the cuticle. Soil samples which gave spore encumbered J,s were considered as Paster~ria positive soils. Average number of sporesIJ, were recorded by examining 5 nematodes per extraction. Determining t h e Pasteuria spore concentration Dried root powder was prepared from T, treated plants eight weeks after the inoculation of Leloidogyne.lQried root powder weighing 0.5g was ground with 50ml water and the homogenate was filtered through a 38 micrometer sieve. The filtrate was observed using ahaemocytometer (mag 10x40) to quantify the spore concentration.

Statistical analysis
The effect of Pasteuriu in the reduction of Meloidogylze population was tested by 't' test.  (Figures 1 and 2).

RESULTS
Among the tested microenvironmel~tal factors, cropping history and soil drainage showed a significant influence on the prevalence of'Pustez~/~ia (p=0.05). Out of the total Pastel~l-in positive locations, 87% were obtained f?om welldrained soils. Locations having longer cropping histories showed high % prcvalencc of the bjocontrol agent. Paste~sl-ia were reported from 74% sampling sites which h a d more than 30 year cropping histories. I n contrast, Pastez~l-!;a positive soils were reported only from 9% sampling sites which had a cropping history of less t h a n 15 years. Cropping pattern in the sampling location did not show a significant effect on the prevalence of the biocontrol agent. Hox~cver, the interaction between cropping history and cropping pattern had a significant effect 011 the prevalence of P a s t e~~r i a .
I n the prcselit study, high collcentrations of Pcistcz/~-za were reported from locatioiis under tea or mixed cropping cultivations, which hacl longer croppmg histories (more than 30 years) and well-drained soils. Numbers of clifferent growth stages of MeLozdogy~ze spp fouild In T, and T, treatmcats are shown 111 Figure 3 No male nen~atodes were found In the tested samples Total nematode population of Meloidogyne spp. i n T, treatment was significantly lower t h a n t h a t of T, treatment. Significa~ltly low number of eggs were found in Pastez~riu treated root systems compared to the plants which were not treated with Pastez~ria (p=0.05). P. pe~zetra~ls found from different locations of' Kandy District gave 48% control of the MeloicLogylze spp. in greenhouse-grown tomato (var. T-146) plants. Root powder prepared from T, plants had an average spore concentration of 8 x 10: sporeslg root. Since Pastez~ria spores can survive in the soil for several years without losing their viability's the effect of cropping pattern may not have an effect on its prevalence. Ability to infect a wide range of hosts by Meloidogylze would be the reason for the interaction between the cropping pattern and cropping history on the prevalence of Pastez~ria.

Controlling efficiency
Pastez~ria isolates of different locations of Kandy District, showed. a 48% control of the Meloidogyne spp. in tomato (var. T-146) under greenhouse conditions. Ilowever, the field performances of these isolates of Pastez~ria could vary from the greenhouse condition.

Spore concentration
P.penetrans can reduce fecundity of Meloidogylze at concentrations as low as 1 x 10.1 sporeslg soil and the number of root-knot nematodes invading roots is substantially reduced as the spore concentration approaches 1 x 10"pores/ g soil.5,*7 Since the spore concentration ofthe root powder obtained from the present study was lxlOj sporeslg root (dry weight) an effective control of the nematode could be obtained.

Potential use in an Integrated Management Programme
Present study revealed the presence of P. pe~zetrans, in many locations of the Kandy District. Further indepth studies are essential to investigate the field performance of the biological control agent under local environmental conditions. P.penetrans can be introduced and once introduced, the biocontrol agent will get augmented in the fields provided the availability of the host plants.14 Therefore, microenvironmental factors which favour the gradual increase of the biocontrol agent can be promoted to enhance the naturally-inhabiting populations of Pasteuria. Future investigations have to be focused on the determination of ecological factors which are conducive for the biological control. by Pastez~ria. It will be helpful to develop a n integrated management programme especially for small-scale vegetable cultivations which are being heavily infested with Meloidogyne spp. a t present.