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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

RESISTANCE TO ALS-INHIBITORS IN TWO POPULATIONS OF PAPAVER RHOEAS L.

VAROTTO S.*, CAMPANI A.**, FORLANI G.**, GASPARETTO M.A.***, SCARABEL L.***, SATTIN M.***

* Dipartimento di Agronomia Ambientale e Produzioni Vegetali Università degli Studi di Padova, Via Romea, 16, 35020 Legnaro (PD)

serena.varotto@unipd.it

** Dipartimento di Biologia Università di Ferrara, Via Borsari 46, 44100 Ferrara

flg@unife.it

*** Centro Studio Biologia e Controllo Piante infestanti CNR, Via Romea 16, 35020 Legnaro (PD) sattin@pdadr1.pd.cnr.it

ALS, target-site, sulfonylureas, Papaver rhoeas, herbicides

Herbicide resistance is the ability of a plant to survive herbicide treatment at a dose that would normally control that species. This phenomenon first appeared in the late 1960s with the selection of weeds resistant to s-triazine herbicides as a consequence of the repeated use of these compounds. To date, resistance has developed to almost all major groups of chemical herbicides. Among the different mechanisms which can confer herbicide resistance, target site mutations involve amino acid substitutions in the functional protein that make it less sensitive to inhibition by herbicides.

Acetolactate synthase (ALS) is the first enzyme in the synthesis of branched-chain amino acids and is the target site of four herbicide families, the sulfonylureas (SU), imidazolinones (IM) triazolpyrimidines (TP) and pyrimidinyloxybenzoates (POB). In plants ALS is encoded in the nucleus but localised in the chloroplast. The amino acid sequence of the mature ALS protein appears to be highly conserved across plant species. The resistance worldwide reported in several biotypes is always due to the possession of an ALS enzyme with decreased sensitivity to ALS inhibitors. Sequencing to date has revealed that particular mutations in specific regions of the ALS gene can confer resistance to one or more of these ALS inhibitors in resistant weeds.

We have characterized two Italian populations of Papaver rhoeas L. in which resistant biotypes to ALS were selected by the repeated use of two different herbicides: population 9805 from Puglia was selected by tribenuron (SU), while the selecting agent of population 0010 was chlorsulfuron (SU). A greenhouse screening revealed that population 9805 is highly resistant to SUs tested and a much lower resistance to imazethapyr (IM) and florasulam (TP). The resistance was not dose-dependent, suggesting that the resistance mechanisms involved is a target site. Population 0010 presented a very similar pattern of resistance. This type of cross-resistance is typical of a target-site mutation in the ALS sequence. To elucidate the genetic and molecular basis of resistance in field-isolate plants of P. rhoeas, the ALS nucleotide sequence was amplified and cloned. Five particularly conserved regions of the DNA sequence, in which mutations conferring resistance are usually located, were sequenced. The nucleotide sequence of the region 1 of the resistant P. rhoeas biotype (9805R) differed from that of the two susceptible biotypes (9807S and 9808S) by a single nucleotide substitution at the variable Pro codon at position 257, predicting a Pro to His. Only one other nucleotide difference was detected at position 300 determining a silent mutation in the susceptible biotype 9807S. Region 2 from the three P. rhoeas biotypes examined didn’t show any nucleotide mutation. The nucleotide sequence of the region 1 of the resistant P. rhoeas biotypes (0010) differed from that of some susceptible biotypes (0010) by a nucleotide substitution at the variable Pro codon at position 257, predicting a Pro to Thr.

Biochemical characterisation of ALS in extracts from sensitive and resistant biotypes failed due to very low enzyme levels. Cell suspension cultures were thus established from in vitro grown seedlings, which retained differential tolerance. The evaluation of specific activities in cultured cells ruled out the possibility that resistance may be due to an overexpression of the target enzyme. When ALS activity in partially purified preparations was assayed in the presence of increasing doses of chlorsulfuron and tribenuron, a remarkable variation in enzyme sensitivity was found, with a 600-fold difference in concentrations causing 50% inhibition. ALS tolerance to IM and TP was increased only slightly, a result that is consistent to the data obtained in vivo. Interestingly, the Pro to Thr transition in biotype 0010 conferred significantly higher tolerance to the IM imazethapyr than that (Pro to His) in biotype 9805R.