HERBICIDAL POTENTIAL OF LANTANA CAMARA L. ON LUDWIGIA SPP IN PADDY SOIL

This study assessed herbicidal potential of Lantana (Lantana camara L.), on two commonly found paddy field weeds; Ludwigia decurrens Walt. and Ludwigia hyssopifolia (G. Don) Exell. Methodology involved a laboratory bioassay using 1:5 w/v and 1:10 w/v aqueous Lantana dried leaf extracts to assess germination, cotyledon expansion, root and shoot development of Ludwigia spp. Green house experiments included paddy soil amendments using 1g, 2g, 4g dried Lantana leaf residues in 85g soil and 30g, 60g, 120g residues in 2.5 kg paddy soil respectively to test seedling establishment and growth of Ludwigia spp. Toxicity of Lantana on rice (Oryza sativa) was tested by growing rice cv. ‘BG 353’ with L. decurrens in Lantana amended paddy soil. Toxic effects of Lantana on naturally occurring micro-biota were also assessed by diffusion method using 1.5 w/v Lantana dried leaf extracts on pure cultures of fungi and bacteria isolated from paddy soil. Results indicated that 1:5 w/v was the strongest extract to suppress root and shoot development of seedlings of both weeds. An amount of 4g Lantana leaf residue in 85g paddy soil suppressed germination and seedling establishment of L. hyssopifolia while higher quantities such as 120g of dried Lantana leaf residues in 2.5 paddy soil was required to suppress growth of L. decurrens. No potential harmful effects of Lantana were observed on growth of rice seedlings and microbiota of paddy soil. Higher quantities such as 120g in 2.5 paddy soil were required to suppress growth of L. decurrens.

(1:10 boiled extract, 1:10 unboiled extract, 1:5 boiled and 1:5 unboiled extracts) by moistening the filter papers daily with an equal volume of appropriate solutions. Seeds treated with distilled water were used as the control. Seed germination (radicle length of ≥ 2 mm) was recorded until no germination was observed for three consecutive days. Lengths of shoots and roots of the germinated seedlings were measured and the number of seedlings with expanded cotyledons was recorded on the 19 th day after germination.

Germination and/or seedling emergence of Ludwigia spp in paddy soil amended with Lantana leaf residues
Germination of Ludwigia seeds in paddy soil containing different quantities of Lantana leaf residues was monitored in a greenhouse at 29±1 0 C under 12h day length conditions. Dried Lantana leaf residues weighing 1g, 2g and 4g were each mixed with 85 g of pesticide free paddy soil (sandy loam in texture, pH 5.14 at 1:25 water, Organic matter 1.3%, Oslens's Phosphorous 6.5 ppm) in plastic pots (65 mm in diameter and 35 mm in height) to obtain different soil residue combinations. The ratio of residue: soil has been chosen in previous experiments (Madushani andRanwala, 2004, Chaturani andRanwala, 2005). Each pot had 10 weed seeds of either species. Controls contained paddy soil without Lantana residues. Another set of pots containing Lantana residues was kept without seeds of test species to monitor the seedling establishment of the existing seed bank of paddy soil. Pots were arranged in completely randomized design with 10 replicates and kept moist throughout. Emergence of seedlings from introduced seeds of test species and paddy soil seed bank was monitored for 22 days.

Growth of L. decurrens seedlings in leaf residue amended paddy soil (with or without rice plants)
The experiment was designed to monitor the growth of L. decurrens which was able to establish seedlings in soil containing dried Lan-Such usage also provide opportunities to control populations and manage the spread of alien plant invaders at a minimum cost (Saha et al. 2007).
The present work aims to explore further the herbicidal properties of Lantana camara (Lantana) by describing toxic effects produced by aqueous leaf extracts and dried leaf residues on seed germination, cotyledon expansion, seedling establishment and growth of two noxious broad leaved paddy field weeds Ludwigia decurrens Walt. and Ludwigia hyssopifolia (G. Don) Exell., that prevail in wet lowland paddy fields of South East Asia including Sri Lanka (Chandrasena 1988). Investigations also included identification of any potential harmful effects of Lantana on a popular low country grown direct-seeded rice cultivar (Oryza sativa cv. 'BG 353') and naturally occurring bacteria and fungi species found in herbicide free paddy soil obtained from fields of Borelesgamuwa area of the Western Province, Sri Lanka.

Germination and seedling development of Ludwigia spp exposed to Lantana leaf extracts
According to preliminary laboratory trials, weights of 200g and 100g of air dried mature Lantana leaves each were soaked in 1000 ml distilled water at 10ºC for 7 days and 1:5 (w/v) and 1:10 (w/v) aqueous filterates were obtained. Half of each solution was boiled under a Bunsen gas flame for 5 minutes to identify any changes of the filterate. More than two thousand L. decurrens and L. hyssopifilia seeds were hand picked from mature capsules, rinsed under running tap water in muslin bags for twenty minutes and blotted dried. Seeds were placed (10 per Petri plate) on dry filter paper (No. 1 Whatmann International, Maidstone UK) lined on 9 cm diameter glass Petri plates. The experiment was conducted according to a completely randomized design with 20 replicates at 30±1 0 C under 12h day-length conditions. Seeds were subjected to treatments tana leaf residues. Here too, the ratio of residue: soil was maintained as same above but higher quantities of materials were used. Thus, Lantana leaf residues weighing 30 g, 60 g and 120 g were each mixed with 2.5 kg of paddy soil in a total of 50 pots (380 mm in diameter and 120 mm in height). The control contained paddy soil without Lantana residues. To each pot, five 4 week old L. decurrens seedlings (raised in trays in green house conditions up to an approximately 5cm height) were introduced. Each treatment was replicated ten times. Pots were arranged in completely randomized design and plants were kept moist throughout. Plant height and leaves per plant was recorded for L. decurrens seedlings weekly for six weeks (so as to be equivalent with critical period of rice) and the total dry weight was obtained. The same methodology was followed for L. decurrens grown in pots (five 4 week old weed seedlings) with one 2 week old rice plant of the same height (raised in trays in green house conditions) belonging to cultivar 'BG 353', one of the popular cultivars used for directseeded rice cultivations in wet lowlands of Sri Lanka. Additionally, plant height, leaves per plant and biomass of rice seedlings was also measured at the end of six weeks to identify any toxic effects of Lantana on growth of rice.

Effects of Lantana on bacteria and fungi of paddy soil
For isolation of naturally occurring fungi and bacteria, a dilution series of paddy soil suspensions was prepared and 0.1 mL aliquots of 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 dilutions were introduced separately to sterile Potato Dextrose Agar (PDA) and Nutrient agar (NA) media supplemented with Tetracyclin (3µL/mL) and Chloramphenicol (1µL/mL) respectively. The pH was adjusted to 5.14 and incubated at 37 o C for 7 days. The fungi were purified at 37 o C on sterile PDA slants while bacteria were isolated in standard nutrient broths (Carter & Gregorich, 2007) and separately stored at 37 o C. Culture collections of bacte-rial (Streptococcus sp., Staphylococcus sp and Bacillus sp.) and fungal (Aspergillus sp., Trichoderma sp. and Fusarium sp.) obtained from the Department of Plant Sciences, University of Colombo were used as references. Unboiled Lantana (dried) leaf extract (1: 5 w/ v) was introduced to bacterial and fungal cultures containing 5 replicates each. Well diffusion method was used for bacteria cultured on NA as described by Booth (1971). Petri plates were incubated at 37 o C for 20 hrs. Disc diffusion method (Murray et al. 1995) was used to assess toxicity on soil fungi incubated at 30± 1 0 C for 72 h. Appearance of clear inhibition zones on NA and PDA plates in the presence of L. camara extract was considered as a toxic effect produced by Lantana.
All data sets were separately subjected to Analysis of Variance (ANOVA) using software SPSS (version 10) and the significance among treatments was tested using Least Significant Difference (LSD) at 0.05 probability level.

Effect of Lantana leaf extract on germination and seedling development of Ludwigia spp.
Germination, cotyledon expansion, elongation of roots and shoot growth of Ludwigia spp. varied between boiled and unboiled Lantana extracts and of the test species.
The Final Germination Percentage (FGP) of L. decurrens was significantly increased when seeds were in contact with Lantana leaf extract while germination of L. hyssopifolia seeds depended upon the strength of the Lantana extract of the medium. A higher strength (1:5) of Lantana extract significantly (P<0.05) inhibited germination (the boiled extract showing a greater inhibition) and the lower strength (1:10) stimulated germination indicating a higher FGP over the control.
Further contact of germinated Ludwigia seeds with Lantana extract affected cotyledon expansion of young seedlings. The boiled higher strength (1:5) was much effective in inhibiting expansion of cotyledons in both Ludwigia spp while the unboiled extract of the same strength inhibited cotyledon expansion only in L. hyssopifolia. Boiling of the Lantana extract had no effect on elongation of roots and shoots. Nevertheless, higher strength of the Lantana extract produced significant suppressive effects on shoot growth than root growth of young Ludwigia seedlings (Table 1).

Effect of Lantana leaf residues on seedling emergence of Ludwigia spp.
Seedling emergence of L. decurrens was not affected due to the addition of Lantana residues to paddy soil while that of L. hyssopifolia seeds showed a significant suppression when residues were added over and above 2g per 85g to paddy soil. Seedling emergence of monocotyledon and dicotyledon weeds from the existing seed bank of paddy soil showed a decreasing trend with increasing weights of L. camara residues ( Table 2).

Effect of Lantana residues on growth of L. decurrens seedlings grown with or without rice
Although L. decurrens seedlings were able to survive in a paddy soil growing medium containing Lantana leaf resides, their growth, in terms of height, leaf and biomass production was significantly decreased when a higher weight of Lantana residues (60g and 120g ) were mixed with paddy soil. Superscripts x,y,z and p,q,r in a column show significance between concentrations and boiled-unboiled nature of the extract respectively at P ≤ 0.05 significant level However presence of the rice plants in the growing medium favoured growth of L. decurrens rather grown alone. There was no change in leaf production and biomass of rice plants grown with L. decurrens in a medium containing different weights of Lantana resides (Table 3, Figures 1and 2).

Effect of Lantana on bacteria and fungi in paddy soil
Pure cultures of two soil bacteria and three soil fungi were isolated from paddy soil. No clear inhibition zones were observed in any of these species when L. camara leaf extract was in contact with the microbes. The observations were same with known bacteria: Streptococcus sp., Staphylococcus sp. and Bacillus sp. and known fungi: Aspergillus sp., Trichoderma sp. and Fusarium sp. Remarkable antimicrobial activity was recorded only by the antibiotic and antifungal solutions (Table 4).

DISCUSSION
Growth suppressive responses of the two Ludwigia species, L. hyssopifolia and L. decurrens to aqueous Lantana leaf extracts and dried residues varied significantly with regard their identity and growth stage at which the treatments were imposed. Our results indicated that allelochemicals of Lantana could enhance seed germination of Ludwigia spp at 1: 5 and 1:10 w/v concentrations, but able to completely supress the establishment and growth of L. hyssopifolia seedlings via inhibiting elongation of root and shoots. In comparison, the phytotoxic effect of Lantana on L. decurrens was more effective at a later stage during seedling establishment and subsequent growth of seedlings. This indicated that L. hyssopifolia was much susceptible to allelopathic effects of Lantana than L. decurrens. These results may correlate with their differences in germination ecology which in turn depends upon phylogenetic relationships of the genus (Fay andDuke, 1977, Wogu andUgborogho, 2000), physiological and biochemical properties of the species (Kobayashi, 2004).
Phytotoxins often inhibit cell division and expansion (Tomaszewski and Thimann, 1966), often slowdown or stop photosynthesis and change the amount of chlorophyll and respiration of plants leading to death (Batish et al. 2002). Phytotoxic potential of Lantana was more pronounced when Lantana was applied in 'higher amounts' as concentration depended growth suppression has been a characteristic of allelopathic interactions (Ashrafi et al. 2008, Inderjit and Nilsen, 2003, Randhawa et al. 2002. The present study also supports this idea as a higher 'dose' of Lantana (more concentrated extract and/or more heavier weight of residues per unit weight of paddy soil) was found to be more effective for suppression of seedling growth and development of Ludwigia species as well as for the emergence of other associated weed seeds in paddy soil. Further, our results also revealed that boiling of the extract would be more effective in suppressing germination and cotyledon expansion of Ludwigia spp. However, the reasons for such an observation cannot be explained without further investigations. Boiling the extract would have also destroyed microorganisms in the extract enabling a clear vi- Symbols *** and * indicate high and low sensitivity respectively, -indicates insensitive status and NA, not applicable sion of the allelopathic effects.
This work was also be in line with previous findings that crude extracts exhibit greater allelopathic effects than that produced by residues, as the visible changes are a result of reactions of different groups of chemical compounds directly leached out from tissues into the testing medium than the ones that are slowly released by decomposition to the environment (Inderjit and Callaway 2003). Compared to laboratory experiments, soil amendment experiments often interfere with chemical, physical and biological properties of soil and their dynamics in spatial and temporal scales (Bhowmik and Inderjith 2003, Inderjit 2001, Kobayashi, 2004. Therefore, visible effects of laboratory bioassays in allelopathy research may or may not always correspond with observations in either green house studies or in field (Inderjit and Nilsen, 2003). Nevertheless, the results of our laboratory study using crude extracts coincided with that of green house experiment to which crushed residues were directly incorporated, especially when higher amounts were used. However, field trials are recommended for recognizing promising results.

CONCLUSION
The study exhibited potential herbicidal effects of L. camara on Ludwigia spp. in a paddy soil medium containing rice seedlings in their early growth stage equivalent to the critical period. Additionally, it was also shown that L. camara does not possess toxic effect towards micro biota of paddy soil. These findings strengthen evidence for herbicidal property of L. camara and suggest its possible applications towards development of bio-herbicidal mixtures for organic paddy production in Sri Lanka.