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a Figures in parenthesis are original values before arc-sine transformation. b No allelopathic effect.

a Figures in parenthesis are original values before arc-sine transformation. b No allelopathic effect.

and to detect the sensitivity of the target weed to minute quantities of allelopathic substances, incised plant parts of aquatic weeds would serve better than whole plants. For screening of different allelopathic substances for inhibition of water hyacinth, a specific bio-assay method was designed with cut leaves of water hyacinth (Kathiresan, 2000; Kannan, 2002). This bioassay involved exposure of cut leaves of water hyacinth to graded doses of plant materials to be tested (dissolved in water). The leaves of water hyacinth plants (with healthy leaves submerged in water) were detached by cutting the petiole with a razor blade with care to retain the incision point below water level. The detached portions of leaf with a part of petiole intact was kept submerged in water for 90 seconds to ensure that no air was trapped internally. Then these leaves were transferred to scintillation vials with water where in different plant products were dissolved and individually compared with an untreated control. The percent fresh weight reduction of the cut leaves was calculated using the formula

Initial weight of the cut leaves - weight after 24 hr of treatment

Initial weight of cut leaves

5.2. Dose Response Studies

Even if a plant product proves appreciably allelopathic on aquatic weeds in bio-assay, tracing the pattern of allelopathic inhibition with graded doses of the plant product becomes vital as aquatic systems requires enormous quantities of plant product due to the quantum of water body (dilution), which in many cases is not practically feasible. Furthermore, this dose response has to be plotted for differing morpho-physiological states of the weed that occurs in common, as the quantity of allelochemical required for a knock down effect is less with a small statured weed compared to that of larger sized weed. For this purpose, before screening of allelopathic plant products on water hyacinth, the latter was classified in to different morphological stages (large, medium and small) prevalent in the state of Tamilnadu, India using discriminant analysis (Kannan and Kathiresan, 1998). Both whole plant and cut leaf bio-assays were done on each of the three stages of water hyacinth. Higher (10 to 30 g L-1) doses were used in whole plants to impart a near lethal effect on the whole plant and lower doses were used in cut leaf bio-assays to cause some allelopathic injury, if not lethal. The doses used for water hyacinth cut leaf bio-assay were 30, 25, 20, 15 10, 5 2.5, 1, 0.5, 0.25, and 0.1 g L-1. The dose response data revealed that cut leaf-bioassay was superior to whole plant assay. For example, C. amboinicus at as low as 0.1 g L-1 dose caused 24% fresh weight reduction in water hyacinth within a week of exposure (Kathiresan, 2000). Therefore, cut leaf bio-assay could be useful to detect allelopathic potential of plant products which otherwise may have been missed if whole plant assay was used. In contrast, the dose response study data revealed that lethal doses for large, medium, and small plants of water hyacinth were relatively closer. C. amboinicus at low (10 g L-1) dose caused death of the water hyacinth after 20 days whereas at high (25 g L-1) dose caused death within one week.

5.3. Field Testing of Allelopathic Substances

In many instances, the materials or plant substances that prove to be allelopathic in laboratory or pot culture experiments may not elucidate similar magnitude of allelopathic response on aquatic weeds in aquatic environments, watersheds, or wetlands. Hence, it is imperative to confirm plant products for their allelopathic potential on weeds in their own natural habitat. A search was made on allelopathic plant products for use in water hyacinth control programs at Department of Agronomy, Annamalai University. Ten of 55 different plant products that showed allelopathic suppression of water hyacinth within 48 h of treatment were selected and tested for their efficacy in natural habitats. The field testing was done in a two tier model. First, the ten plant products were tested in microponds (simulated natural habitat). Second, the plant products that confirmed to be allelopathic in microponds were further evaluated in natural watersheds.

5.4. Preparation of Microponds

Small (microponds) ponds of 1m x 1m x 1m dimension were dug in the field and canals with running water on both sides of the pond were maintained to minimize seepage loss of water from the ponds. Initially, the microponds were conditioned by flooding and maintaining water as needed for 20 days. After 20 days, healthy water hyacinth plants were introduced and allowed to grow for 2-3 days. The plant products to be tested were dissolved in the water of microponds, separately. Allelopathic potential of the plant products was evaluated in comparison with untreated control using data on fresh weight, number of healthy leaves, and chlorophyll content of the water hyacinth.

5.5. Evaluation in Natural Habitats

Top three best performing plant products in the micropond study were selected for realistic confirmation of their allelopathic potential in natural habitats. To this end, three watersheds of 168, 123, and 492 m2 area were selected at three different locations within a radius of 50 km from Annamalai University. Water hyacinth was allowed to establish in watersheds and the surface area of water was completely covered by weed. Each watershed was divided in to three equal strips using polyethylene sheets stretched between bamboo poles running down the entire depth of water, with the poles anchored to the bottom of the watershed. The plant products were applied to water individually to one of the strips in each watershed. The data were collected on fresh weight, number of healthy leaves, and chlorophyll content at five days interval. Results proved that the plant products showing higher magnitude of allelopathic inhibition on water hyacinth in initial bio-assays like C. amboinicus continued to retain and exhibit the same magnitude of inhibition on water hyacinth with out any dissipation in natural watersheds or aquatic environments.

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