Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.22

MOLECULAR BASIS OF RESISTANCE TO ALS-INHIBITORS HERBICIDES IN SCIRPUS MUCRONATUS L. (PALLA)

L. SCARABEL*, N. CARRARO**, M. SATTIN*, S. VAROTTO**

*) Istituto di Biologia Agroambientale e Forestale (IBAF)-CNR-sez. Malerbologia, viale dell’Università, 16 Legnaro (PD)

**) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università degli Studi di Padova, viale dell’Università, 16 Legnaro (PD)

Acetolactate synthase (ALS), herbicide resistance, point mutation, polyploids, rice fields

Acetolactate synthase (ALS) is the target site of five herbicide families known as ALS-inhibitors: sulfonylureas (SU), imidazolinones (IM), triazolopymiridines (TP), pyrimidinylthiobenzoates (PTB) and sulfonylaminocarbonyltriazolinones. During the ‘80s and ‘90s the use of these herbicides increased rapidly worldwide because of their excellent selective control of the main broadleaf weeds by using low dosages. Anyway, only five years after the commercial introduction of the SU (chlorsulfuron) in 1982, the first case of resistance was observed in Lactuca serriola (Mallory-Smith et al. 1990). Since then there has been a steady increase in the number of species affected by ALS resistance: to date at least 25 monocot and 55 dicot species (Heap, 2003).

In rice crops, repeated use of ALS inhibitor herbicides has led to the development of ALS-resistant biotypes. At least three infesting rice species developed populations resistant to ALS-inhibitors in Italy: Alisma plantago-aquatica, Scirpus mucronatus and Cyperus difformis. In most cases, the field-selected ALS-inhibitors resistance is linked to an altered ALS enzyme that is no longer sensitive to the herbicide (target-site resistance). The resistance is due to the possession of particular point mutations in one out of five specific highly conserved regions (Domains) of the ALS gene sequence, that determine one amino acid substitution in the protein sequence. It has become evident that each amino acid substitution shows a peculiar pattern and level of resistance to different ALS-inhibitors.

Three populations of S. mucronatus were screened in our labs: two were selected by the bensulfuron-methyl (SU), while the third one was sensitive to ALS-Inhibitors. One population (9711) presented a high resistance to SU and a low resistance to TP, while the second one (9719) showed a high resistance to both SU and TP. To elucidate the molecular basis of resistance, DNA and RNA were extracted from plants grown in the greenhouse and survived to a treatment with ALS inhibitors at field dose. Degenerated specific primers were constructed and used in PCR and RT-PCR amplifications in order to amplify genomic DNAs and/or cDNAs encompassing the five conserved Domains of the ALS gene. Then, RACE experiments were performed in order to obtain the full-lenght cDNA of the ALS gene of S. mucronatus.

In population 9711 the nucleotide sequence of Domain A differed from that of the susceptible population by a single nucleotide mutation at the variable Pro codon at position 197 (where the amino acid position refers to the Arabidopsis precursor ALS), predicting a Pro to His substitution. Specific primers will be used to amplify the conserved ALS Domains of resistant byotypes in population 9719, that is characterised by a different pattern of resistance.

S. mucronatus is a polyploid plant. Southern analysis using ALS gene as a probe showed tha the species has at least four ALS sequences in its genome. With the aim to characterize the level of ploidy and to understand how the different ALS genes are expressed and regulated in different tissues and during plant development further experiments are in progress.