Hydrilla (Hydrilla verticillata L.f. Royle) was introduced to the surface water of Florida in the 1950s and is today one of the most serious aquatic weed problems in the USA. As a result of concerns associated with the applications of pesticides to aquatic systems, fluridone is the only USEPA-approved chemical that provides systemic control of hydrilla. After a decrease in fluridone's efficacy at controlling hydrilla, 200 Florida water bodies were sampled to determine the extent of the problem and the biological basis for the reduced efficacy. Our studies revealed that hydrilla phenotypes with two- to six-fold higher fluridone resistance were present in 20 water bodies. Since fluridone is an inhibitor of the enzyme phytoene desaturase (PDS), the gene for PDS (pds) was cloned from herbicide-susceptible and -resistant hydrilla plants. We report for the first time in higher plants three independent herbicide-resistant hydrilla biotypes arising from the selection of somatic mutations at the arginine 304 codon of pds. The three PDS variants had specific activities similar to the wild-type enzyme but were two to five times less sensitive to fluridone. In vitro activity levels of the enzymes correlated with in vivo resistance of the corresponding biotypes. As hydrilla spread rapidly to lakes across the southern United States in the past, the expansion of resistant biotypes is likely to pose significant environmental challenges in the future.

Lemna species are widely used in ecotoxicological research to assess the phytotoxicity of environmental compounds. The purpose of the work described in this paper was to further refine the Lemna spp. bioassay, making it more robust and useful in comparing results between laboratories. A nondestructive image analysis method was used to measure growth as affected by herbicides during a time course. We tested 26 herbicides, with as many as 19 different modes of action, on leaf area growth of Lemna paucicostata to establish complete dose-response relationships. By using 3,5-dichlorophenol as a reference compound, different EC50 (concentration that inhibited growth by 50%) values of the herbicides can be compared. The EC50 values ranged from 0.003 microM for sulcotrione to 407 microM for asulam. Complete dose-response parameters were obtained that will better allow comparison of these results to those of other laboratories. These results should give useful information to anyone who works with Lemna species to address questions on herbicide residues in environmental compartments, screening of new herbicidal compounds, or the assessment of phytotoxic side effects of any compound.

SummaryThree natural somatic mutations at codon 304 of the phytoene desaturase gene ( pds ) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone.In in vitro assays, the threonine ( Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of crossresistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R / S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.

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