Can wild species become problem weeds because of herbicide resistance? Brachypodium distachyon: a case study

Comment Can wild species become problem weeds because of herbicide resistance? Brachypodium dJtachyon: a case study Jonathan Gressel* and Yishayahu Kleifeld*

*Department of Plant Genetics, The Weizmann Institute of Science, Rehovot IL 76100, Israel and tDepartment of Weed Science, Neve Yaar Research Center, Haifa IL 31999, Israel

A purported drawback to the use of transgenic herbicide-resistant crops is the fear that the crop or interbreeding wild relatives will become weedy. It has been posited that a change even in a single trait can confer weediness. This hypothesis was tested with Brachypodium distachyon. This innocuous species came into contact with herbicides through the use of crushed rock from its habitat for road foundations. It evolved s-triazine resistance and developed as a monoculture. When true weeds later evolved simazine resistance, B. distachyon was partially competed from the ecosystem and then disappeared upon the use of non-triazine herbicides. Thus, this wild species remained a weed only until true weeds evolved resistance or until other herbicides were used. One gene mutation did not convert it into a weed, which implies that this will be equally improbable in other cases, when the gene codes for an otherwise neutral trait such as herbicide resistance.

Key~vords: transgenic crops; herbicide resistance; Brachypodium distachyon

Much has been said about wild species becoming weedy plants in agriculture. Weeds are a very small sub-set of plant species, having a series of attributes allowing them to compete with each other and with crops in agroecosystems. T h e y have a 'general purpose genotype' (Baker, 1991) coding for a group of properties conferring excellent competitiveness and flexibility. Keeler (1989) has made the point that many crop and wild species have some of these attributes and a change in one or a few properties can pass a species over the threshold to b e c o m e a weed. Conversely, any mutation or added gene must have a fitness advantage for the species to b e c o m e a weed (Levin, 1990). There is continuing evolution that allows wild species to b e c o m e noxious weeds (Baker, 1991). This evolution can be due to changing genetic factors in the species and changing environments. The spectrum of weed flora in crops has changed, owing to changes in cultivation systems, drainage, fertilization, and herbicide use, with some noxious weeds disappearing back to innocuous or wild status and new weeds appearing or gaining in importance (Haas and Str,eibig, 1982). Some weeds that were always naturally resistant to herbicides filled the ecological niche resulting from the elimination of susceptible weeds. It has been theorized that transgenic crops can b e c o m e weeds, or the transfer of genes from transgenic crops to wild relatives may cause the wild relatives to b e c o m e weeds (Keeler, 1989; D a r m e n c y and Gasquez, 1990; Ellstrand and Hoffrnan, 1990; Fitter, Perrins and *To whom correspondence should be addressed

Williamson, 1990; Goldburg et al., 1990; Keeler and Turner, 1991; Warwick, 1991). There are few crops/ wild relatives that are sufficiently genetically compatible to ihterbreed in such a manner (Keeler and Turner, 1991; de Vries, van der Meijden and Brandenburg, 1992; Raybould and Gray, 1993). Even when there was some genetic compatibility, exceedingly high selection pressures had to be used to exclude competition with the weed's own pollen, still resulting in exceedingly infrequent genetic transfers (Kerlan et al., 1991; Lefol et al., 1991; Dale et al., 1992).

Should herbicide-resistant species become weedy? Can a crop b e c o m e a weed if it were to become herbicide resistant? Is herbicide resistance a trait that can have a fitness advantage where the herbicide is not used? These questions were analysed for transgenic crop plants by release of transgenic oilseed rape containing either kanamycin resistance or glufosinate resistance into a variety of ecosystems as representative for all transgenes. The transgenic types were slightly less invasive than the non-transgenic rape (Crawley et al., 1993).

Case study The question remains as to whether the transfer of a herbicide resistance gene from a crop to a wild relative will render it more weedy. This has yet to occur in

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nature, and seems unlikely, as described above. We can try to assess this possibility by looking at wild species that have evolved herbicide resistance. This is not simple, as most wild species live in habitats that are quite remote and very different from agroecosystems, precluding contact with herbicides or pollen from resistant crops. Thus, it was a surprise when a relatively rare, innocuous grass species, Brachypodium distachyon (L.) P. Beauv., appeared as a monoculture along simazine-treated roadsides over ten years ago, having target-site resistance to s-triazinetype herbicides (Gressel et al., 1983). The case history/ interpretation/account of its appearance and almost complete disappearance from this ecosystem can be considered to have general implications. We presume that this phenomenon would occur with most, if not all, wild species, should they evolve resistance to a herbicide. Taxonomists had trouble in classifying this weed as

Other cases An analogous previously reported case was the purported advent of 2,4-D-resistant wild carrot (Daucus carota L.) along a Canadian roadside (Whitehead and Switzer, 1963). A year later there was no longer any viable resistant seed available in their laboratory (C. M. Switzer, personal communication, 1964), nor was a wild resistant population ever reported again. Similarly, Lopochloa phleoides (Vill.)Reichb., another wild species that is not weedy, evolved resistance to simazine along Israeli roadsides (Rubin, Yaacoby and Schonfeid, 1985). It, too, seems to have disappeared from that ecosystem with the changes to other herbicides (Y. Yaacoby, personal communication, 1993). Implications

Brachypodium distachyon = Trachnynia distachya when it first appeared along roadsides, as it was 40-60 cm tall (Gressel et al., 1983). Both texts and herbarium specimens described it as 12-25 cm tall (Bor and Guest, 1968; Feinbrun-Dothan, 1986). It was hard to find the wild type to ascertain if it was naturally resistant; its habitat was described as 'dry stony hills, often calcareous, on sandy gravel and gypsum plain, in rocky limestone desert throughout the Mediterranean and Near-Eastern areas' (Bor and Guest, 1968). It has been reported to occur in and along fields (FeinbrunDothan, 1986) but it is not an agricultural weed in cultivated fields. The wild type was eventually found on soft sandstone/sand cliffs (Gressel et al., 1983). Seed from the puny wild plants grew in the greenhouse as vigorously as the resistant type, but the wild type was sensitive to the triazine herbicides. There is a strong possibility that Brachypodium seed came with the road foundation material of mildly crushed soft sandstone, thus bringing the large quantities of seed needed for selection. Sterilant levels of simazine (10 kg ha -1) were routinely used along the roadsides selecting for the rare resistant individual(s). Seeds of such individual(s) rapidly spread due to vehicular movement, as well as run-off, resulting in hundreds of kilometres of roadsides being covered by this weed. The roads authorities continued to apply simazine; nothing but Brachypodium could grow along the roadsides until seven other grass species and Amaranthus blitoides (covered with Cuscuta) evolved resistance to triazines. These other weedy species were all indigenous to the nearby agroecosystems. Simazineresistant Brachypodium could not compete with the truly weedy species and was displaced, becoming rare. The roads authorities then began using other herbicides (especially diuron), which easily decimated the Brachypodium. One must now go back to the sandstone hills to find Brachypodium; it has reverted back to its niche in the wild. It remains a weed only in some olive orchards and industrial sites where simazine is still used as the sole herbicide. These are all on poor soils, similar to some extent to its wild home, and the other simazine-resistant grasses are taking longer to displace the Brachypodiurn in such habitats.

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We believe that these case histories indicate the following: 1. Wild species can acquire genes for herbicide resistance. In the documented cases the acquisition was by selection; it could occur in other species by crosspollination with crops. 2. Such selections can probably only occur in the rare instances when a large population of the wild species is brought into contact with a herbicide or with a related resistant crop species. Nevertheless, we do not know of instances where crops have conferred any of their natural herbicide resistances on wild r e l a t i v e s . With Brachypodium, selection was possible after moving seed from the wild to a treated area. The same could also occur with other wild species when virgin land is brought into cultivation. It may thus be advisable initially to use mechanical weed control to lower the level of wild species in a new agroecosystem. 3. Genes for herbicide resistance can temporarily elevate wild species to weed status. 4. Resistant wild species will remain weedy until more typical weed species evolve resistance and displace the wild species or until the selective herbicide chemistry is replaced by a different chemistry. Thus, there seems to be little risk of a wild species remaining a weed for long periods.

Caveats Some caveats must be attached to the above: 1. Triazine-resistant individuals are usually inherently less fit than equivalent wild-types (Gressel and Ben Sinai, 1985; Gressel and Segel, 1990; Holt, 1990). This diminished competitive fitness should cause the weed to disappear when the usage of the herbicide that created its niche is discontinued. Not all herbicide resistances result in such large fitness differentials. 2. One type of paraquat resistance is correlated with cross-tolerances to many other tolerances to oxidant stresses (Shaaltiel et al., 1988; Jansen et al., 1990;

Comment G u l l n e r , K o m i v e s a n d K i r a l y , 1991; M a t s u n a k a a n d I t o , 1991; A m s e l l e m el al., 1993) i n c l u d i n g t o l e r a n c e to t r a n s i e n t d r o u g h t stress ( M a l a n , G r e y l i n g a n d G r e s s e l , 1990; P a s t o r i a n d T r i p p i , 1992). T h i s t y p e o f e v o l v e d r e s i s t a n c e m i g h t b e t h o u g h t to h a v e v a l u e , l e a d i n g to c o n t i n u e d w e e d i n e s s e v e n a f t e r t h e selecting herbicides are withdrawn. Such a naturally o c c u r r i n g r e s i s t a n c e g e n e m u s t h a v e less v a l u e in its n a t u r a l h a b i t a t o r it w o u l d h a v e b e e n t h e wild t y p e . The drought/herbicide tolerant biotype may p r e d o m i n a t e in t h e n o n - i r r i g a t e d w i l d , b u t it s h o u l d b e a d v a n t a g e o u s to lose this costly t r a i t u p o n invading farmland. 3. A t l e a s t t w o grass w e e d s p e c i e s h a v e e v o l v e d crossr e s i s t a n c e to a w i d e v a r i e t y o f h e r b i c i d e s , i.e. to all t h e s e l e c t i v e h e r b i c i d e s u s e d in w h e a t ( G r e s s e l , 1988). I t is p r e s u m e d t h a t t h e y h a v e d o n e this b y e v o l v i n g a g e n e r a l m e c h a n i s m o f i m m u n i t y to t h e s e herbicides. Such evolution could result only from c h e m i c a l s e l e c t i o n ; n o such g e n e s a r e k n o w n o r proposed for genetic engineering of crops. Such g e n e t i c m e c h a n i s m s w o u l d n o t b e useful in t r a n s g e n i c c r o p s as t h e r e w o u l d b e n o w a y t o c o n t r o l such a c r o p w h e n it b e c o m e s a v o l u n t e e r w e e d in a subsequent crop. It s e e m s logical to c o n c l u d e f r o m t h e e x a m p l e o f B r a c h y p o d i u m t h a t if a t r a n s g e n i c c r o p t r a n s f e r s h e r b i c i d e r e s i s t a n c e g e n e s to a w i l d r e l a t i v e , t h a t s p e c i e s will m o s t p r o b a b l y b e a w e e d f o r o n l y a s h o r t period.

Acknowledgements The authors acknowledge discussions with various c o l l e a g u e s , b u t t h e view:~ s t a t e d h e r e i n a r e t h e i r o w n responsibilities. J.G. has the Gilbert de Botton Chair of Plant Sciences.

Botanical files: a study of the real chances for spontaneous gene flow from cultivated plants to the wild flora of the Netherlands. Gorteria Suppl. 1, 1-100 EIIstrand, N. C. and Hoffman, C. A. (1990) Hybridization as an avenue of escape for engineered genes. BioScience 40, 438-442 Feinbrnn-Dothan, N. (1986) Brachypodium distachyon. In: Flora Palestina, Vol. 4, pp. 162-163, Israel Academy of Sciences and

Humanities, Jerusalem Fitter, A., Perrins, N. and Williamson, M. (1990) Weed probability challenged. Bio/Technology 8, 473 Goldburg, R., Rissler, J., Shand, H. and Hasselbrook, S. (1990) Biotechnology's Bitter Harvest, Environmental Defense Fund, New

York, 73 pp Gressei, J. (1988) Multiple resistances to wheat selective herbicides. New challenges to molecular biology. Oxford Surv. Plant Mol, Cell Biol. 5, 195-203 Gressel, J. and Ben-Sinai, G. (1985) Low intraspecific competitive fitness in a triazine-resistant nearly nuclear isogenic line of Brassica napus. Plant Sci. 38, 29-32 Gressel, J. and Segei, L. A. (1990) Herbicide rotations and mixtures; effective strategies to delay resistance. In: Managing Resistance to Agrochemicals (Ed. by M. B. Green, H. M. LeBaron and W. K.

Moberg) pp. 430-458 American Chemical Society, Washington, DC Gressel, J., Regev, Y., Malkin, S. and Kleifeid, Y. (1863) Characterization of an s-triazine-resistant biotype of Brachypodium distachyon. Weed Sci. 32, 450-456 Gullner, G., Komives, T. and Kiraly, Z. (1991) Enhanced inducibility of antioxidant systems in Nicotiana tabaeum results in acifluorfen resistance. Z. Naturforsch. 46C, 871-881

Haas, H. and Streibig, J . C . (1982) Changing patterns of weed distribution as a result of herbicide use and other agronomic factors. In: Herbicide Resistance in Plants (Ed. by H. LeBaron and J. Gressel) pp. 57-80, Wiley, New York Holt, J. S. (1990) Fitness and ecological adaptability of herbicide resistant biotypes. In: Managing Resistance to Agrochemicals (Ed. by

M.B. Green, H. M. LeBaron and W. K. Moberg) pp. 419-421, American Chemical Society, Washington, DC Jansen, M. A. K., Malan, C., Shaaltiel, Y. and Gressel, J. (1990)

Mode of photooxidant resistance to herbicides and xenobiotics. Z. Naturforsch. 45C, 563-569 Keeler, K . H . (1989) Can genetically engineeered crops become weeds? Bio/Technology 7, 1134-1139 Keeler, K. H. and Turner, C. E. (1991) Management of transgenic plants in the environment. In: Risk Assessment in Genetic Engineering

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Received 30 September 1993 Revised 11 February 1994 Accepted 11 February 1994