cñ[Ptx¡ s$9 % The toxicity of Vicía speci zftrU $95 their utilisation as grain le es oÇ Dissertation for the degree I)octor of Philosophy in Agricultural Science by Dirk Enneking B.Ag.Sc. (Adelaide) Universify of Adelaide Waite Agricultural Research Institute South Australia June, 1994 uplA zIpIA'luaA I)eclaration This work contains no m¿terial which has been accepted for the award of any other degree or diploma in any universþ or other tertiary institution and, to the best of my knowledge and beliet contains no material previousþ published or written by another person, except where due reference has been made in the text. I girre consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopytng SrG* o ?. :. .t..è... 1. .? ^rB:..7. 11 Acknowledgements I would like to tha¡k the many people who have contributed their time, thought and resorrçes to the successfirl completion of this study. The m¡ny discussions with friends and colleagues, and the written correspondence with overseas researchers have heþed to formulate and test many ideas. Scientific collaboration with the late Dr. R L. Davies, Dr. Phil Glatz, Llmne Giles and Dr. Nigel Maxted has been one of the key features of the study. Individual contributions are acknowledged in the relevant sections of this thesis. I would also like to thank the staffat the South Austraïan Department of Agriculture's Northfiekl Piggery and Parafield Poultry Research Centre, particularþ J¡nine Baker and Andrew Cecil, for carrying out the animal experiments. This study would not have been possible without the continuous help of Dr. Max E. Tate, whose initiative, stimulating ideas, encouragement, patience, experience, wisdom, guidance and friendship þ¿yg mede it a tnrly rewarding and unique experience. The unique combination of skills and resources of the Waite Agricultural Research Institute, its staff and students have also contributed significantly to the outcome of this work. I am particularþ indebted to the library staff for their heþ with the obtention of literature, to tle services of the supply unit and to the administrative and technical zupport afforded by the former Department ofAgricultural Biochemistry and the Department of Plant Science. The financial support for this study tlrough a postgraduate scholarship from the Australian Grain Research Development Corporation (GRDC) is gratefirlly aoknowledged. In addition, I would like to thank my parents for first introducing me to vetches; and their understanding and financial support for my oversees travels, which were also funded by contributions fromthe Alf Hannaford Bequest Fund and the Department of Plant Science. The generous help of my oversees hosts with their time, research collections, contacts and hospitality has also been invaluable. Finally I would like to thank Dr. Clive M. Francis for making valuable suggestions to successive drafts of chapters I and 8 and tle Centre for Legumes in Mediterranean Agriculnue (CLIMA) for support during the final stages ofwriting. Summary The worldwide utilisation of Vicia qecies as forage and green nranure crops is well establiúed. Exoept for V. fabaL., the utilisation of Vicia ryecies as grain legrrmes is of minor global economic importance and mainly restricted to the Mediterranean region and South-West Asia, where the grain is used primarily as seed and in ruminant diets. In Australia, the comparatively high seed yields and low production costs for genotypes from qpecies such as V. narbonensis L. (narbon bean or moor's pea) and V. sativa L. (common vetch) have provided an arttraLctwe altemative grain legume option for drylantl farming and have thus stimulated an interest in markets for the gain' Monogastric animals ( incl' humans) are the major global end-users for grain legume products. Because of the well known toxicity of Vicia qpp. seeds to mono gastric animals in particular, this thesis has focussed on those major toxic chemical seed components which are perceivsd ¿s major constraints to the wider utfüsation of these promising crops as grain legumes. A thorough examination of crurent and past practices ofvetch cultivation and utilisation was undertaken to complement this approach. The two major aims were, first to elucidate the nature of the factors reqponsible for the low palatability of narbon beans to pigs, and second to review the available information about the toxicity of Vicia species and their utilisation as grain legumes. The potent feed inhibitory activity of Namoi vetch (V. villosa Roth cv. Namoi) provided a usefirl model for the initial stage of tl.is study. Its antifeedant activity was shown to be due to tl.e toxic amino acid ca¡lavanine. Inclusion of canavanine in pig diets at a concentration equivalent to that found in Namoi vetch seed accounted firlly for the feed inhibitory activity of this legume. The novel effect of this well knor¡m arginine analogue may well be explicable in terms of the inhibition of the arginine pathway leading to nitric oxide which is now knor¡m to be involved in the control of peristalsis. The e4perience gained with Namoi vetch in the feed-intake bioassay proved to be invaluable for the isolation of the much less potent y-glutamyl-S-ethenyl-cysteine feed inhibitor from the narbon bean. A quantitative assessment of this factor's feetl inhibitory activity was not permitted due to the untimely death of our veterinary colleague, Dr. Richard Davies. There is, however, a clear correlation between the total S-ethenyl cysteine content of tl.e tested diets and the negative porcine feed intake teq)onses. TV An important difference between V. villosa and V. narbonensis was noted, as demonstrated by the rate at which the pigs reduced their feed intake. It is remarkable, that the effect o-f canavanine-ssn1¿ining diets becomes evide,nt only after the second meal whereas the pigs immediateþ restrict their feed intake when presented with diets containing S-ethenyl- cysteine. Such a clear delineation of feed-intake reqponses provides a siryle and general classification for feed-intake inhibitors, and may be worthy of finther detailed physiological studies. The antifeedant effects of these compounds suggest that tley have evolved as part of the plants' anti-predator defence strategy. Particular attention, including a detailed review ofits economic botany, has been given to V. nørbonensis, a relatively r¡nhown but promising grain crop for Australia. With the chemical identþ of the unpalatability established, the selection of more palatable genotypes is likely to provide access for the grain to monogastric feed markets. The historical evidence suggests that V. narbonens¡s is a niche crop of particular value for specific agricultrual applications, its conversion into a broad acre crop is a challenge for the funue. V. sativa was investigated as a direct consequence of a request to chemically examine the toxin content of the cultivar Blanche Fleur. By the time that investigation commenced, Blanche Fleur, which was originally introduced to Australia as ahay, forage and green Íumrre crop, had already been prematurely promoted and exported as a cheap replacement for red lentils (Lens culinøris Med.) in ignorance of this qpecies' well documented content of y- glutamyl-B-cyanoalenine and the favism toxin, vicine. A 1992 commentary article to Nature on our observations led to a ban on its importation by India and Eg¡rpt. Subsequent poultry bioassays established that the cyano-alanine content was substantially altered by cooking to produce some as yet un-identified nitrile component, but the feed inhibitory activity of the cooked gtain was undiminisþed. Acid hydrolysis of Blanche Fleur, however, removed both, the readily detectable nitrile absorbance as well as the poultry feed-intake inhibition. This observation could potentially formthe basis for a simple post-harvest detoxification process for V. sativa and other feed stuffs containing acid labile antinutritive factors. Unforn¡nately, cases of poisoningby Vicia qpecies continue to be reported. These can be grouped into those caused by V. sativa and its related qpecies (cyanogenic glycoside Vicianme: HCN poisoning; and anti-nutritional effects of B-cyanoalanine) and those caused by v canavânine containing species (V. villosa, V. benghalensis, V. ervilia etc.). Farmers need to be mede aware of the well documented biochemical distinctions between Vicia cultivars to prevenfthe accidental intoxication of their livestock with seeds containing high concentrations of canavanine or vicienine. Finally, au overview of the voltrminous and widely dispersed vetch literature, coupled with the observations in this thesis, suggest that the utilify and value of each of the thtee Vicia model speoies examined in this thesis can be markedly enhanced by tle following strategies: 1. Provision of zufficient altemative feed sources to allow feed intake to be regulated by p alatability, thus min imi sing toxin ingestion. 2. Adaptation to Vicia toxns a) through selection of a digestive flora capable of detoxification (in the case of ruminants) and b) through selection ot of animal ^sdification genoq¡pes with improved biochemical tolerance or even resistance to toxicþ. 3. Detoxification prior to ingestion (Post-harvest detoxification) 4. Plant selection or genetic msdification of specific toxin bioqmtletic pathways to provide cultivars with optimum toxin concentration and distribution in strategic tissues (minimis¿1ion oftoxins in the end product). The inevitable conclusion from this thesis is that by incrementing our current flndamental knowledge of the biological chemistry of their naturally occurring anti-predator metabolites, we will promote the intelligent usage of Vicia species as higbly nutritious grains for a zustainable agriculture. This thesis has resulted:m2 publications in refereed journals VI Publications Tate, M. E.;Enneking,D. (1992). A Mess Of Red Pottage. Nature 359, 357-8 Enneking, D.; Giles, L.C.; Tate, M. E.; the late Davies, R L.(1993). Canavanine: A Natural Feed-Intake Inhibitor For Pigs, (Isolation, Identification And Siguificance) J. Sci.