Lathyrus Lathyrism Newsletter 4 (2005)

Contents

Page Editor's Comment 1 Colin Hanbury -Australia Articles 2 Fatty acid composition of grass pea (Lathyrus sativus L.) Gurusamy Chinnasamy, Arya Kumar Bal, seeds and David Bruce McKenzie- Canada 5 Performance of grass pea (Lathyrus sativus L.) somaclones D. Tsegaye, W. Tadesse and M. Bayable- at Adet, northwest Ethiopia Ethiopia 7 Search for resistance to crenate broomrape (Orobanche J.C. Sillero, J.I. Cubero, M. Fernández- crenata) in Lathyrus Aparicio and D. Rubiales- Spain 10 Characterization of grass pea (Lathyrus sativus L.) entries G. B. Polignano, P. Uggenti, G. Olita, V. by means of agronomically useful traits Bisignano, V. Alba and P. Perrino- Italy 15 Model plant type in Khesari (Lathyrus sativus L.) suitable Vedna Kumari and Rajendra Prasad- India for hill farming 18 Resilience of South Asian disabling conditions: a glimpse M. Miles- UK of lathyrism among comparative histories 22 Considerations on the reintroduction of grass pea in China Hui-Min Yang and Xiao-Yan Zhang- China 27 Effects of drought on stomatal character, photosynthetic Hui-Min Yang, Xiao-Yan Zhang and Gen- character and seed chemical composition in grass pea, and Xuan Wang- China their relationships 28 Scope of growing lathyrus and lentil in relay cropping S. Gupta and M.K. Bhowmick- India systems after rice in West Bengal, India 34 The same goal, a different approach: a new Belgian- Fernand Lambein and Seid Ahmed- Ethiopian project Belgium/Ethiopia THIS VOLUME IS STILL BEING COMPILED- THERE WILL BE MORE ARTICLES TO FOLLOW

ISSN 1832-8431 (Print) and ISSN 1832-844X (Online)

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Lathyrus Lathyrism Newsletter 4 (2005)

Jointly supported by: Third World Medical Research Foundation (TWMRF), PO Box 9171, Portland, Oregon 97207, USA http://www.twmrf.org and Centre for Legumes in Mediterranean Agriculture (CLIMA), University of Western Australia, 35 Stirling Highway Crawley 6009, Australia http://www.clima.uwa.edu.au

Lathyrus Lathyrism Newsletter 4 (2005)

Editor’s comment

Dear Readers

Welcome to the Lathyrus Lathyrism Newsletter Vol. 4 and thank you all for your support of the newsletter. You will see from the variety of articles that there is enthusiasm for Lathyrus related research in many countries, this bodes well for the increased knowledge and cultivation of these useful legumes.

Recently, there has been a change in the editing process. In order to give authors an advantage of on-line publication from now onward articles will be placed on the web page as soon as the editing process is finalised. This will enable the maximum quick exposure of information. Then at the end of the year the volume will be closed off and compiled, for those who require the printed version this is when it will be completed. Hence as you revisit the web page through the year you will notice more articles appearing. Any suggestions for further improvements are welcome.

The Lathyrus Lathyrism Newsletter has been assigned ISSN 1832-8431 (Print) and ISSN 1832-844X (Online) and has been placed on a number of directories of open access journals, ensuring wider exposure and listings in library resources internationally. Citations of articles in the newsletter have been steadily increasing.

Thanks to the Third World Medical Research Foundation (TWMRF) and the Centre for Legumes in Mediterranean Agriculture (CLIMA) for supporting the newsletter from 2000 until now.

Please consider summarising your recent research for the newsletter. Your contribution will be useful not only as science but also toward the greater stability of cropping systems in regions with increasing pressure on natural resources. Most research submissions should be approximately 1500 words and can include a small number of tables or figures, with electronic copies preferred. Introduction, Methods, Results followed by Discussion is the preferred layout for research summaries, although this can be altered as necessary. Abstracts of complete work are also welcome, if they have been published elsewhere then full acknowledgment will be made.

I hope you find the articles contained interesting and useful to your work.

Colin Hanbury

Editor contact details:

Dr Colin Hanbury Department of Agriculture, Western Australia 3 Baron-Hay Court South Perth 6151 Australia

E-mail: [email protected]

1 Lathyrus Lathyrism Newsletter 4 (2005)

Fatty acid composition of grass pea (Lathyrus sativus L.) seeds

Gurusamy Chinnasamy1*, Arya Kumar Bal1, and David Bruce McKenzie2

1. Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada A1B 3X9. 2. Atlantic Cool Climate Crop Research Centre, Agriculture and Agri-Food Canada, 308 Brookfield Road, St. John’s, Newfoundland and Labrador, Canada A1E 5Y7.

*Author for correspondence. Present address: Imaging Program, Lawson Health Research Institute, St. Joseph’s Health Care, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2 Email: [email protected] OR [email protected]

Introduction represent a potential source of several important Grass pea or chickling vetch (Lathyrus sativus L.) is a nutrients for human and animal nutrition. Therefore, it well-established, commercially available, tropical is necessary to analyze grass pea seeds for their semi-arid crop. Pods of this crop are flat, dorsally nutrient composition. The present investigation broad with two ridges, short and 3-5 cm in length. proposes to determine the fatty acid composition of Each pod contains 2-7 seeds. Mature seeds are different lipid classes in mature seeds of Indian grass rhomboid or triangular in shape, dull whitish grey pea. brown and variously mottled (4). Grass pea is a relatively productive crop compared to other pulses in Material and Methods regions characterized by poor soil (7). It is very well Seed materials adapted to adverse climatic conditions and requires Mature seeds of grass pea were procured from a local very little management for crop production. Moreover, market in Kolkatta (Calcutta), West Bengal, India. its deep taproot and nitrogen-fixing ability make this crop an ideal choice for sustainable agriculture. Grass Extraction and estimation of total lipids pea seeds are a major source of protein for large Total lipids were determined by the gravimetric sections of the population in Bangladesh, China, method (2). One gram of dried grass pea seeds in each Ethiopia and India (13, 15). It is also grown to a lesser of three replicates was powdered using a 700S Waring extent in the Middle East, southern Europe and some blender (Waring Products Co., USA) and parts of South America. In India grass pea is homogenized in 10 ml of 50 mM Tris-HCl buffer considered one of the most economical pulses for containing 0.5 M NaCl at pH 7.2. The homogenate fodder and green manure in rice fields during the cool was combined with a mixture of chloroform and winter period (1). methanol in a ratio of 1.25:2.25 (v/v) to extract lipids. The chloroform layer (supernatant) was separated by Lathyrus species contain very high protein, but a centrifugation at 5000 g for 20 min and allowed to neurotoxin, 3-(-N-oxalyl)-L-2,3-diamino propionic stand overnight after combining with a mixture of acid (ODAP), is present in wild and most cultivated chloroform and distilled water in a ratio of 1:1 (v/v). forms that if consumed in sufficient amounts can The chloroform layer was collected in a pre-weighed cause the irreversible crippling disease known as vial for evaporation under nitrogen gas. The vial with lathyrism (10, 17). This toxin to a considerable extent the lipid residue was weighed again to estimate the has been bred out of some cultivars although lathyrism amount of total lipids. in Asia from consuming grass pea is common. Because of its drought tolerance, grass pea has been Separation of lipids and analysis of fatty acid judged to have good potential as a future new pulse composition crop for low rainfall areas of the Canadian prairies (12). Total lipids were fractionated into 5 lipid classes It acts as a ground cover alternative to summer fallow, [phospholipids (PL), monoglycerides (MG), helping to prevent wind and water erosion, as well as diglycerides (DG), free fatty acids (FFA) and adding nitrogen to the soil (11). triglycerides (TG)] by thin layer chromatography and the constituent fatty acids in each lipid class were The nutritional health and well being of humans are separated and estimated using gas chromatography as entirely dependent on plant foods. Plants are critical described in Chinnasamy et al. (6). The measurement components of the dietary food chain in that they of each fatty acid was calculated as a relative weight provide almost all essential minerals and organic percentage to 10 selected fatty acids [C14:0 (myristic nutrients to humans either directly, or indirectly when acid), C14:1 (myristoleic acid), C16:0 (palmitic acid), plants are consumed by animals, which are then C16:1 (palmitoleic acid), C18:0 (stearic acid), C18:1 consumed by humans (9). Grass pea seeds may (oleic acid), C18:2 (linoleic acid), C18:3 (linolenic

2 Lathyrus Lathyrism Newsletter 4 (2005)

acid), C18:4 and C20:4 (arachidonic acid)]. The from C8 to C22 in total lipids of Canadian grass pea double bond index (DBI) was calculated (14) using the seeds. The relative weight percentages of 10 major formula: fatty acids in 5 lipid classes isolated from mature seeds of Indian grass pea are summarized in Table 1. DBI=Σ (% of fatty acid content × no. of double bonds) 100 C18:2, C18:1, C18:0 and C16:0 were the major fatty acids present in PL, MG, FFA and TG separated from Among 10 major fatty acids, sum of all saturated fatty grass pea seeds. DG contained higher quantities of acids, unsaturated fatty acids with one double bond C18:0, C16:0 and C18:2 compared to other fatty acids. and unsaturated fatty acids with more than one double PL and TG showed higher overall DBI than FFA, MG bond gave total saturated fatty acids (TSFA), total and DG. The content of TSFA was higher in DG than monounsaturated fatty acids (TMUFA) and total in other lipid classes. High amounts of TMUFA were polyunsaturated fatty acids (TPUFA) respectively. observed in FFA and TG. PL registered high quantity Unsaturated to saturated ratio (USR) was calculated of TPUFA. TG, PL and FFA showed higher USR by dividing total unsaturated fatty acids by total compared to MG and DG. Although lipids constitute a saturated fatty acids. minor portion of many leguminous seeds, their profiles indicate the desirable nature of fatty acid Statistical analysis constituents present (3). In the present study, grass pea For all sets of data, one way analysis of variance was seeds exhibited a high amount of total unsaturated performed using the SPSS computer package (16). fatty acids (56.37% – 59.98%) and a low amount of Means were compared by Duncan’s multiple total saturated fatty acids (40.01 – 43.65%) in all lipid comparison test at P = 0.05. For the purpose of classes except MG and DG, which contained 31.49 – statistical analysis, data in percentage were 47.29 % total unsaturated fatty acids and 52.72 – transformed to arcsine values (18). 68.53% total saturated fatty acids. Overall, in the present work, Indian grass pea seeds showed higher Results and Discussion total unsaturated fatty acids than total saturated fatty Total lipid content of mature grass pea seeds was acids that are in agreement with fatty acid composition (3-5) 20.93 ± 0.27 mg/g dry weight. The nutritive value of of beach pea and Canadian grass pea seeds . seeds is determined by not only quantity but also by Therefore, grass pea seeds may be important for quality of lipids they contain. Thus, fatty acids present nutritional health and may serve as a valuable in lipids are playing important role in deciding shelf nutritional source. Further in depth study is necessary life, nutrition and flavor of food products (8). Chavan to elucidate the nutritional quality and importance of et al. (3) reported the presence of 20 fatty acids varying grass pea.

Table 1. Composition of major fatty acids (relative weight percentage1) in phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FFA) and triglycerides (TG) isolated from total lipids of mature seeds of Indian grass pea. Values are means (± SE) of three replications.

Fatty acids PL MG DG FFA TG C14:0 0.65 ± 0.82b 4.51 ± 2.78b 2.01 ± 0.24d 2.09 ± 1.61c 1.06 ± 0.85c C14:1 0.66 ± 0.53b 0.77 ± 1.41b 0.91 ± 1.63de 0.49 ± 0.09c 0.82 ± 1.03c C16:0 24.88 ± 1.67a 26.62 ± 2.89a 31.21 ± 0.75a 26.07 ± 3.60ab 24.57 ± 5.25ab C16:1 0.57 ± 0.40b 2.77 ± 2.53b 1.10 ± 0.48de 2.66 ± 2.61c 2.26 ± 1.17c C18:0 16.24 ± 4.90a 21.59 ± 1.82a 35.31 ± 0.92a 15.49 ± 1.15b 14.38 ± 1.96b C18:1 26.20 ± 6.64a 23.44 ± 2.18a 9.30 ± 0.12c 35.99 ± 8.32a 33.91 ± 6.64a C18:2 28.65 ± 11.13a 17.84 ± 8.39a 17.55 ± 1.86b 14.31 ± 2.85b 19.17 ± 3.73ab C18:3 1.16 ± 0.61b 1.57 ± 2.27b 0.75 ± 0.69de 0.81 ± 0.26c 2.55 ± 1.90c C18:4 0.64 ± 0.91b 0.23 ± 0.19b 0.73 ± 0.99e 1.93 ± 1.61c 0.75 ± 1.35c C20:4 0.36 ± 1.32b 0.67 ± 0.70b 1.15 ± 0.89de 0.18 ± 0.04c 0.52 ± 1.03c DBI2 0.92 ± 0.18 0.71 ± 0.20 0.56 ± 0.08 0.79 ± 0.02 0.88 ± 0.04 TSFA3 41.77 52.72 68.53 43.65 40.01 TMUFA4 27.43 26.98 11.31 39.14 36.99 TPUFA5 30.81 20.31 20.18 17.23 22.99 USR6 1.39 0.90 0.46 1.29 1.50

1The value of each fatty acid was calculated as a relative a-e Means in the same column followed by different letters weight percentage to 10 selected fatty acids. are significantly different using Duncan’s multiple 2 Double bond index. comparison test at P = 0.05. 3 Total saturated fatty acids. 4 Total monounsaturated fatty acids. 5 Total polyunsaturated fatty acids. 6 Unsaturated/saturated ratio.

3 Lathyrus Lathyrism Newsletter 4 (2005)

Acknowledgements We wish to thank Dr. P. J. Davis, Rhonda and Dona for the help in fatty acid analysis. This work was supported by the Dean of Science Grant to A. K. Bal.

References 1. Allen ON, Allen EK. 1981. The leguminosae. 10. Hanbury C, White C, Mullan BP, Siddique KHM. University of Wisconsin Press, Madison, WI. 2000. A review of the potential of Lathyrus 2. Bligh EG, Dyer WJ. 1959. A rapid method of sativus L. and L. cicera L. grain for use as animal total lipid extraction and purification. Can J feed. Lathyrus Lathyrism Newsletter 1, 34. Biochem Physiol 37, 911-917. 11. Henkes R. 1995. The remaking of grasspea. The 3. Chavan UD, Shahidi F, Bal AK, McKenzie DB. Furrow 100, 25-26. 1999. Physico-chemical properties and nutrient 12. Kiehn FA, Reimer M. 1992. Alternative crops for composition of beach pea (Lathyrus maritimus the prairies. Agriculture Canada Publication. L.). Food Chem 66, 43-50. 1887/E, Ottawa, Ontario, Canada. 4. Chavan UD. 1998. Chemical and biochemical 13. Kuo YH, Khan JK, Lambein F. 1994. components of beach pea (Lathyrus maritimus Biosynthesis of the neurotoxin β-ODAP in L.). Ph.D. Thesis. Memorial University of developing seeds of Lathyrus sativus. Phytochem Newfoundland, St. John’s, Newfoundland and 35, 911-913. Labrador, Canada. 14. Skoczowski A, Filek M, Dubert F. 1994. The 5. Chinnasamy G, Bal AK, McKenzie DB. 2004. long-term effect of cold on the metabolism of Fatty acid and elemental composition of mature winter wheat seedlings. II. Composition of fatty seeds of beach pea [Lathyrus maritimus (L.) acids of phospholipids. J Therm Biol 19, 171-176. Bigel.]. Can J Plant Sci 84, 65-69. 15. Spencer PS, Roy DN, Ludolph A, Dwivedi MP, 6. Chinnasamy G, Davis PJ, Bal AK. 2003. Seasonal Roy DN, Hugon J, Schaumburg HH. 1986. changes in oleosomic lipids and fatty acids of Lathyrism: evidence for the role of the perennial root nodules of beach pea. J Plant neuroexcitatory amino acid BOAA. Lancet 2, Physiol 160, 355-365. 1066-1067. 7. Duke JA, Reed CF, Weder JKP. 1981. Lathyrus 16. SPSS Inc. 1990. SPSS/PC + StatisticsTM 4.0 for sativus L. In Handbook of legumes of world the IBM PC/XT/AT and PS/2. SPSS Inc., economic importance. Duke JA (Ed.). Plenum Chicago, IL. Press, New York, NY. pp. 107-110 and 345-349. 17. White C, Hanbury C, Siddique KHM. 2001. The 8. Gaydou EM, Rasoarahona J, Bianchini JP. 1983. nutritional value of Lathyrus cicera and Lupinus A micromethod for the estimation of oil content angustifolius grain for sheep. Lathyrus Lathyrism and fatty acid composition in seeds with special Newsletter 2, 49-50. reference to cyclopropenoic acids. J Sci Food 18. Zar JH. 1996. Biostatistical analysis. Third Agric 34, 1130-1136. edition. Prentice Hall, New Jersey, NJ. 9. Grusak MA, DellaPenna D. 1999. Improving the nutrient composition of plants to enhance human nutrition and health. Ann Rev Plant Physiol Plant Mol Biol 50, 133-161.

4 Lathyrus Lathyrism Newsletter 4 (2005)

Performance of grass pea (Lathyrus sativus L.) somaclones at Adet, northwest Ethiopia

D. Tsegaye*, W. Tadesse and M. Bayable

Adet Agricultural Research Center, P.O Box 08, Bahir Dar, Ethiopia.

*Email: [email protected]

Introduction Material and Methods Grass pea (Lathyrus sativus L.) is one of the important Eleven low neurotoxin grass pea somaclones crops of economic significance in Ethiopia. It is the introduced from ICARDA and a local check were fifth most important pulse crop in Ethiopia after faba grown for two consecutive years (2000/01 and bean, field pea, chickpea and haricot bean. It is the 2001/02) at Adet Agricultural Research Center, which cheapest source of protein in the diets of most people. is located at 37029’E and 11016’N latitude, with an Estimates of the total cultivated area and production of altitude of 2240 m above sea level in the Amhara grass pea in Ethiopia was reported to be 83,522 National Regional State, Ethiopia. The soil is vertisol hectares and 92,339 tonnes, respectively. Of this total with pH 6.0. The weather variables during the testing sum, northwest Ethiopia exceeds other regions, having periods were generally conducive for normal growth 39,983 hectares (47.9 %) and 40,840 tonnes of of grass pea. production (44.2 %) (3).

The genotypes were planted in a randomized complete Grass pea is a highly popular food and feed legume in block design with two replications at a seeding rate of the farming system due to tolerance of drought, 40 kg ha-1. A spacing of 60 cm between plots and 20 flooding and disease and its importance in cm between rows were used, with a plot size of 0.8 m ameliorating soil fertility (2). It is commonly grown as x 4 m. The materials were evaluated for stand percent, a double crop after the cereals tef or barley. Despite its days to flowering, days to maturity, plant height (cm), importance, the presence of the neurotoxin β-N-oxalyl number of pods per plant, number of seeds per pod, L-a, β-diaminopropanoic acid (ODAP) is a 100 seed weight (g) and grain yield (kg ha-1). ODAP discouraging factor for grass pea production. content analysis on seed was done at Addis Ababa Irreversible crippling can occur, if the seeds are University, Department of Chemistry, using Rao consumed as a major part of the diet for an extended Method for the 2001/02 cropping season only. period (1). To date no single improved variety has been Analysis of variance was computed for each season developed and released in Ethiopia due to the using an MSTAT computer program (7). inconsistency of ODAP content of the promising grass pea genotypes across environments.

The development of biotechnology and its application Results and Discussion in grass pea has resulted in somaclones with neurotoxin ODAP content of less than 0.1% (100 mg The result of agronomic performance of the tested ODAP\100gm seed) in India (5). As a result the genotypes is presented in Table 1. Genotypes showed government of India released one of the somaclones, significant difference (P<0.05) in days to maturity, Bio L212 (Ratan), for cultivation (8). Low ODAP lines 100 seed weight and grain yield. The grain yield level -1 are also available at the International Center for ranged from 3705 to 5233 kg ha . ILAT-LS-K-290 -1 Agricultural Research in Dry land Areas (ICARDA) was the best yielder (5233 kg ha ) and early in (6). A study was carried out to test the agronomic maturity; whereas the local check was later to mature, performances and the neurotoxin ODAP content of lower in 100 seed weight and grain yield (3705 kg ha- those lines developed by somacloning technique at 1) than the other tested lines. ILAT-LS-K-289 and ICARDA. ILAT-LS-K-444 were promising, both with 0.104 % ODAP (Table 1). ILAT-LS-K-288 and ILAT-LS-K- 33 were also promising with low ODAP contents of 0.119 and 0.125 % seed. The local variety scored the

5 Lathyrus Lathyrism Newsletter 4 (2005)

highest ODAP content, 0.252 %. The results clearly will be included in the regional as well as the national indicate that the somaclone technique is promising in breeding programme for testing under different developing grass pea varieties with ODAP content environmental conditions and for possible release. presumed at a safe level for human consumption (< 0.2 %) (4) in the future. Therefore, the promising lines

Table 1. Agronomic performance of grass pea somaclones at Adet, northwest Ethiopia (2000/01 and 2001/02).

Variety Stand Days to Days to Plant Pods/ Seeds/ 100 seed Seed Seed % flowering maturity height plant pod weight (g) yield ODAP (cm) (kg/ha) (%)* ILAT –LS-K-290 81 64 142 115 71 3 7.3 5233 0.168 ILAT –LS-K-30 81 66 143 111 59 4 8.7 4317 0.216 ILAT –LS-K-104 83 66 145 121 69 4 10.2 5064 0.140 ILAT –LS-K-33 85 64 143 119 63 3 8.9 4261 0.125 ILAT –LS-K-299 84 67 147 112 56 3 7.2 4300 0.206 ILAT –LS-K-289 82 65 143 121 72 4 7.5 4807 0.104 ILAT –LS-K-444 82 64 144 114 64 4 7.6 4464 0.104 ILAT –LS-K-387 78 64 145 122 74 3 7.9 4405 0.211 ILAT –LS-K-190 78 64 142 112 64 4 7.6 4468 0.202 ILAT –LS-K-288 82 66 145 104 56 4 8.5 4209 0.119 ILAT –LS-K-390 85 57 143 104 56 4 7.4 4206 0.168 Local variety 86 54 149 126 67 4 6.5 3705 0.259 Mean 82 64 114 115 64 4 7.9 4462 - S.E. 6.69 1.44 1.18 8.38 18.2 0.32 0.53 504 - LSD (5%) 19.6 4.2 3.4 24.6 53.4 0.95 1.55 1478 - CV (%) 11.5 3.2 1.2 10.3 40.3 12.9 9.4 16.0 - *2001/02 only

References 1. Campbell CG. 1997. Grass pea (Lathyrus sativus 5. Mehta S.L. 1997. Plant biotechnology for L.). Promoting the Conservation and use of removal of ODAP from Lathyrus. In: R. underutilized and neglected crops. 18. Institute of Teklehaimanot and F. Lambein (eds) Lathyrus Plant Genetic and Crop Plant Research, and Lathyrism: A Decade of Progress. University Gatersleben/International Plant Genetic of Ghent, pp 103. Resources Institutes, Rome, Italy. 6. Moneim, AM. Saxena MC, El-Saleh A, Nakkoul 2. Campbell CG, Tiwari KR. 1997. Breeding grass H. 1997. The status of breeding grass pea pea for reduced seed levels of neurotoxin (Lathyrus sativus) for improved yield and quality (ODAP). In: R. Teklehaimanot and F. Lambein at ICARDA. In: R. Teklehaimanot and F. (eds) Lathyrus and Lathyrism: A Decade of Lambein (eds) Lathyrus and Lathyrism: A Decade Progress. University of Ghent, pp. 85-86 of Progress. University of Ghent, pp. 81-82. 3. Dahiya BS. 1976. Seed morphology as an 7. MSTATC. 1989. A micro-computer statistical indicator for low neurotoxin in Lathyrus sativus. program for experimental design, data Qual Plant-Pl Fds Hum Nut 25, 391-394. management and data analysis. Michigan State 4. Central Statistics Authority, Ethiopia (CSA). University. Crop and Soil Science, Agricultural 2001/02. Agricultural Sample Survey. Addis Economics and Institute of International Ababa, pp. 27. Agriculture, Michigan, USA. 8. Santha IM, Mehta SL. 2001. Development of low ODAP somaclones of Lathyrus sativus. Lathyrus Lathyrism Newsletter 2, 42-45.

6 Lathyrus Lathyrism Newsletter 4 (2005)

Search for resistance to crenate broomrape (Orobanche crenata) in Lathyrus

J.C. Sillero1*, J.I. Cubero2, M. Fernández-Aparicio3 and D. Rubiales3

1. CIFA, Dep. Mejora y Agronomía, Apdo. 3092, E-14080 Córdoba, Spain. 2. ETSIAM-UCO, Dep. Genética, Apdo. 3048, E-14080 Córdoba, Spain. 3. CSIC, Instituto de Agricultura Sostenible. Apdo.4084, E-14080 Córdoba, Spain.

* Email: [email protected]

Updated from original publication: Sillero JC, Cubero JI and Rubiales D. 2001. Resistance to broomrape (Orobanche crenata) in Lathyrus. 7th International Parasitic Weed Symposium, (eds. A. Fer et al.), pp. 224-227, Faculté des Sciences,Nantes, France.

Introduction Material and Methods Grass pea has been widely cultivated in South Asia Fourteen accessions belonging to 10 different species of and the Mediterranean area since antiquity for food the genus Lathyrus (Table 1) were screened for and feed uses(4). Its cultivation was in continuous resistance to broomrape under field conditions in recession in last decades, but there is an increasing 1995/96 in at Córdoba (Spain). Accessions were kindly interest in its reintroduction in the cropping systems in provided by IPK (Germany) and USDA (USA). Each marginal areas due to its adaptability to unfavourable accession was sown in a 1m row, surrounded by four environments (4) and its beneficial effects on rows of a faba bean susceptible check (cv. Prothabon). subsequent crops. Recently, plant breeders have been The sowing took place on 26 November 1995, in a field working to improve its utilities (2) as well as to reduce heavily infested with O. crenata seeds. Hand weeding anti-nutritional factors responsible for lathyrism (1). An was done when required, but no herbicides were applied. additional priority for breeding L. sativus and L. The intensity of broomrape attack was evaluated at crop cicera is resistance to crenate broomrape (Orobanche maturity (from early June), by counting the final number crenata) (4). of emerged and non-emerged broomrape shoots per plant. Data were expressed as a percentage of the mean O. crenata is a holoparasitic weed that seriously of its four surrounding rows of Prothabon (=100%). The attacks legume crops, such as faba beans, lentils, peas, most resistant lines were studied in a second field chickpeas, grass peas and vetches (5,6,9). Different season, with field design and evaluation criteria control methods have been proposed to avoid this similar to those described above. serious problem, but none has been completely effective and the development of resistant cultivars is The resistance found in two of the most resistant a major need (8,9). Breeders are actively working on accessions was confirmed in pots and petri dishes this matter and resistance to O. crenata has been experiments. The faba bean cv. Prothabon and a pea found both in cultivated and wild legume species (7,9). variety (cv. Messire) were included as susceptible The genus Lathyrus can be satisfactorily grown in checks. For the pot experiment five day old plants marginal areas, so it is necessary to develop varieties were transplanted into 1 litre plastic pots filled with resistant to broomrape to prevent this parasitic weed vermiculite, previously mixed with 25 mg of problem in infested areas. broomrape seeds (about 8000 seeds). Each genotype was represented by 12 plants, 1 plant/pot. When the The purpose of this study was to search for sources of plants were mature, 90 to 120 days after sowing, the resistance to O. crenata in different species of the plants were extracted, the roots were washed in water genus Lathyrus. and the number of broomrape tubercles was counted. The petri dish experiment was carried out using the procedure described by Sauerborn (10). One month after transplanting the seedlings, 500 seeds per dish were studied and classified to determine the percentages of germination and the number of tubercles per plant.

7 Lathyrus Lathyrism Newsletter 4 (2005)

Table 1. Emerged and non-emerged broomrape attacks in selected accessions of Lathyrus, under field conditions, during the growing seasons 1995/96 and 1996/97 in Córdoba (Spain)1.

1995/96 season 1996/97 season Emerged Non-emerged Emerged Non-emerged Accession Code Species shoots (%) shoots (%) shoots (%) shoots (%) Lat-340 PI 255365 Lathyrus annuus 246 380 Lat-323 LAT 150/88 Lathyrus aphaca 73 91 Lat-341 PI 227529 Lathyrus aphaca 145 727 Lat-321 LAT 201/89 Lathyrus cicera 66 205 Lat-343 PI 208307 Lathyrus cicera 90 141 Lat-322 LAT 165/86 Lathyrus clymenum 0 0 0 0 Lat-344 PI 283488 Lathyrus clymenum 0 0 0 11 Lat-342 PI 229794 Lathyrus choranthus 0 123 16 34 Lat-345 PI 358859 Lathyrus gorgoni 34 129 Lat-348 PI 358864 Lathyrus inconspicuus 74 175 Lat-320 LAT 340/92 Lathyrus ochrus 0 0 0 0 Lat-352 PI 271361 Lathyrus ochrus 0 0 0 0 Lat-354 PI 165528 Lathyrus sativus 130 746 Lat-356 PI 269921 Lathyrus szowitsii 160 139 1 Emerged and non-emerged broomrapes referred to the susceptible faba bean check, cv. Prothabon (=100%).

Table 2. Established broomrape in pot and petri dishes and germination of broomrape seeds in petri dishes in two selected accessions of Lathyrus and two susceptible checks.

Pot experiment Petri dishes experiment No. of established % No. of tubercles/ Line Species broomrapes/ plant germination plant Messire Pisum sativum 9.4 a 53.2 a 26.4 a Protabon Vicia faba 7.6 a 37.6 b 13.3 b Lat-320 Lathyrus ochrus 0.0 b 0.2 c 0.0 c Lat-322 Lathyrus clymenum 0.0 b 0.1 c 0.0 c Data with the same letter per column are not significantly different (P<0.05, Duncan test).

Results

In none of the accessions of L. clymenum and L. Results of in vitro experiments showed that no ochrus studied was there any broomrape emergence broomrape was installed in any of the accessions, in (Table 1), although in one accession of L. clymenum a neither pots nor in petri dishes (Table 2), which few non-emerged tubercles were found in the second suggests a barrier to the broomrape establishment. field season. Low broomrape emergence but high However, when the germination of the broomrape levels of non-emerged tubercles were recorded in both seeds was recorded, almost no germination was found seasons for the L. choranthus accession studied. All in any of the accessions, so the low germination of the the accessions of the species L. annuus, L. aphaca, L. broomrape seeds seems to be the main barrier to the cicera, L. gorgoni, L. inconspicuus, L. sativus and L. broomrape attack. szowitsii were moderately to highly susceptible, all with more than 34% emerged broomrape shoots.

8 Lathyrus Lathyrism Newsletter 4 (2005)

Discussion References

High levels of resistance to O. crenata have been 1. Enneking D (2000) The riddle of lathyrism. found in the genus Lathyrus, especially in the species Lathyrus Lathyrism Newsletter 1, 6. L. ochrus and L. clymenum. In both species the main 2. Lazányi J. 2000. Grass pea and green manure mechanism of resistance seems to be an early barrier efffects in the Great Hungarian Plain. Lathyrus to the establishment of the broomrape, as none or few Lathyrism Newsletter 1, 28-30. broomrape tubercles were recorded under field 3. Linke KH, Abd El-Monein AM and Saxena MC. conditions. This low establishment is due to the low 1993. Variation in resistance of some forage induction of germination of the broomrape seeds. Low legumes species to Orobanche crenata Forsk. levels of induction of germination have been described (7) Field Crops Research 32, 277-285. in other resistant legume species such as chickpea . 4. Robertson LD and El-Moneim AMA. 1997. Status We cannot exclude the presence of additional of Lathyrus germplasm held at ICARDA and its use mechanisms of resistance preventing attachment in breeding programmes. In: Lathyrus Genetic and/or development of tubercles, but the low Resources Network: Proceedigns of a IPGRI- germination precludes its study and quantification. Such a mechanism preventing development of ICARDA-ICAR Regional Working Group Meeting tubercles is suggested in L. chloranthus by the fact (eds. Mathur PN, RAmanatha Rao V and Arora that tubercle formation was allowed, but they did not RK), pp 30-41, IPGRI New Delhi, India emerge. Both mechanisms have been previously 5. Rubiales D. 2001. Parasitic plants: an increasing detected in the genus Cicer (7). threat. Grain Legumes 33, 10-11. 6. Rubiales D. 2003. Parasitic plants, wild relatives Interspecific variation in resistance to broomrape has and the nature of resistance. New Phytologist 160, been described in the genus Lathyrus (3) and has been 459-461. confirmed in this study. All the accessions of the same 7. Rubiales D, Alcántara C and Sillero JC. 2004. species were susceptible (i.e. L. annuus, L. aphaca, L. Variation in resistance to crenate broomrape cicera, L. gorgoni, L. inconspicuus, L. sativus and L. (Orobanche crenata) in species of Cicer. Weed szowitsii) or resistant (i.e. L. clymenum and L. ochrus) Research 44, 27-32. to the broomrape attack. L. clymenum and L. ochrus 8. Rubiales D, Pérez-de-Luque A, Cubero JI and have been used as human food since antiquity (1) and Sillero JC. 2003. Crenate broomrape (Orobanche can also be cultivated for forages. Farmers can grow crenata) infection in field pea cultivars. Crop these two species in heavily infested fields and avoid Protection 22, 865-872. broomrape attack. L. choranthus (at least the line we 9. Rubiales D, Sillero JC, Román MB, Moreno MT, studied) could be used as a ‘trap crop’ to reduce the Fondevilla S, Pérez-de-Luque A, Cubero JI, broomrape seed bank in the soil; as it permitted very Zermane N, Kharrat M and Khalil S. 2002. low emergence of broomrape shoots but allowed a Management of broomrape in Mediterranean relatively high establishment of the parasite, which agriculture. In: Legumed: Grain Legumes in the imply that the stimulation and germination of the Mediterranean Agriculture (ed. European broomrape seeds occur. Several legume species have Association for Grain Legume Research), pp 67- been successfully used with this aim (3,11). 73, Rabat, Morocco. 10. Sauerborn J, Masri H, Saxena MC and Erskine W. 1987. A rapid test to screen lentil under laboratory conditions to susceptibility to Orobanche. Lens Newsletter 14, 15-16. 11. Saxena MC, Linke KH and Sauerborn J. 1994. Integrated control of Orobanche in cool-season food legumes. In: Biology and management of Orobanche (eds AH Pieterse, JAC Verkleij and SJ Ter Borg), pp 419-431. The Netherlands.

9 Lathyrus Lathyrism Newsletter 4 (2005)

Characterization of grass pea (Lathyrus sativus L.) entries by means of agronomically useful traits

G. B. Polignano1 1*, P. Uggenti1, G. Olita1, V. Bisignano1, V. Alba2 and P. Perrino1

1. Istituto di Genetica Vegetale, C.N.R., Via Amendola 165/A, 70126 Bari, Italia 2. Dipartimento di Biologia Difesa e Biotecnologie Agro Forestali, Università della Basilicata, Potenza

*E-mail: [email protected]

Introduction distribution of the residual genetic variation is Grass pea or chickling pea (Lathyrus sativus L.) is a recommended for the benefit of both direct users and diploid (2n=14), self-pollinated annual with branched, crop improvement programmes (12). straggling, or climbing habit, blue (sometimes violet or white) flowers and characteristic smooth seed with In this regard it is important to underline that the pressed sides (16). The center of origin and consumption of L. sativus seeds by humans and diversification of the Lathyrus gene pool is in the animals has been limited by presence of a neurotoxin Mediterranean region (15). The earliest archaeological known as β-N-oxalyl-L-α,β-diaminoproprionic acid remains of Lathyrus appear in the Neolithic in the (β-ODAP) in the seeds, which when taken in large Balkans and Near East of Bulgaria, Cyprus, Iraq, Iran quantity can lead to “lathyrism” a disease causing and Turkey (4). According to Kupicha (8), the genus paralysis of the limbs (3). With that premise, breeding Lathyrus contains about 150 species but only L. programmes evolving genotypes combining high yield sativus is widely cultivated for human consumption, with high protein content and low neurotoxin (ODAP) particularly in Bangladesh, China, Ethiopia, India, are in progress all over the world (7). At the same time Nepal and Pakistan (10). In Italy, this crop has mostly it was felt that it was necessary to evaluate and disappeared and today it is no longer seen as the ‘food describe the genetic diversity available in the grass of the poor’ as it was in the past. Fortunately, grass pea collections (2,6,10,11,12). In other words there is a pea is still used by local populations in marginal areas, need to survey, collect, conserve and characterise the and sold in some marketplaces. valuable resources of the Lathyrus species germplasm for the benefit of both users and crop improvement Emerging global and national strategies on sustainable programmes. farming systems, sustainable development and the preservation of biological diversity reflect concern at The main objective of the present research was to adequate quantification of local biodiversity. study the variation in a collection of grass pea entries Consequently, researchers, farmers and policy makers with respect to yield capacity and other important have focused their attention on the neglected and/or agronomic traits (such as biomass) with the aim of a underutilised crops to improve the food security, direct utilisation of the most promising material and nutrition and economic welfare of humans all around their use in cross combinations for breeding purposes. the world (5). In Italy, among these species the grain legume grass pea has received renewed attention as a Material and Methods local and typical product, it is becoming an exclusive Seventy-six grass pea entries of different geographical and fashionable food for which discerning consumers origin were used. These were subset of the whole are prepared to pay a higher price than for other pulse collection including entries characterized by desirable products. In addition, the most interesting agronomical traits: erect plants, high podded node, early flowering, feature of the species are drought tolerance, resistance high seed yield, big and light seeds, high biomass, low to pests and diseases, adaptability to different types of ODAP content and high protein content. All entries soil as well as to adverse climatic conditions (9). were grown in 2002-2003 winter season on clay-sand Despite these and other advantages, L. sativus is soil at the experimental farm “Pantanello”, belonging inadequately exploited and studied. In fact, it is well to the Basilicata region, at Metaponto (Matera) in known that this crop is grown mainly as landraces; southern Italy. Generally the climate in the Metaponto their genetic diversity is used and maintained largely area (0-300m a.s.l.) is a strong Mediterranean type by a small number of farmers in very limited areas of with an annual rainfall less than 600 mm and an central southern Italy. In other words, valuable genetic annual temperature trend consisting of mild or absent resources of L. sativus are exposed to the threat of winters and hot summers. Sowing was done in mid genetic erosion and disappearance. Therefore November after a deep summer plowing and two collection and storage of germplasm and deeper secondary tillages. During summer tillage 120 kg/ha knowledge of the nature, entity, and geographical

10 Lathyrus Lathyrism Newsletter 4 (2005)

of P2O5 was applied. Harvest occurred at the end of of correlation among traits for these entries. The first June at full maturity stage. principal component accounted for 31% of variation reflected mostly influence on pod and seed traits. The Five randomly chosen plants from each entry from a second component accounts for 17% of the variance single row plot were scored for the 18 quantitative and and thus is comparable in importance to the first. The qualitative descriptors reported in Table 1. Frequency traits with the largest coefficients and which distributions for the qualitative descriptors flower and contribute to it are the length of longest stem, biomass, seed colour were also determined. Multivariate data seed yield and pedicel length. Time to flowering, leaf analysis followed three steps: a) estimation of length, seed thickness, length of internode, leaf width, standardized entry means of 16 quantitative traits; b) time to emergence and height of first podded node derivation of orthogonal, uncorrelated traits for each have some importance in the other components. entry using Principal Component Analysis (PCA); c) Although there is no clear demarcation between clustering entries into similarity groups using important and unimportant principal components, it is uncorrelated traits (PCA coefficients). The SAS interesting to note that yield and some yield procedure PRINCOMP computed the correlation components appear strongly in the first two matrix and determined the principal components (SAS components. Institute, 1987). The sum of the first eight PC axes, representing 88% of total variation were used in Clustering entries based on similarity of the first eight subsequent analyses. Entries were clustered using principal components identified five large groups Ward’s minimum-variance method (SAS Institute, accounting for a 31% share of variance. Cluster 1987). The cluster routine was stopped to form five memberships are reported in Table 3. Cluster I discrete clusters looking for a consensus among the included the highest number of Italian entries; while a four statistics R2 (RSQ), cubic clustering criterion large number of Cyprus entries were in cluster II. (CCC), pseudo-F (PSF) and pseudo-t2 (PST2). Results Entries from the other less represented origins spread of this clustering were combined with results of the over all five groups. PCA analysis as a visual aid in discerning clusters.

Results and Discussion Table 1. Means, minimum and maximum values, Means, minimum and maximum values, coefficients and coefficient of variation (C.V.) for 16 of variation for 16 quantitative traits and the frequency quantitative descriptors and frequency distributions of the flower colour and seed colour are distributions for 2 qualitative descriptors observed reported in Table 1. Entries showed a wide range of in 76 grass pea landraces. variation as evidenced by coefficients of variation. The most variable traits were seed yield, biomass, leaf Descriptora Mean Min Max C.V. width and seeds/pod; and the lowest values of Time to emergence (d) 23.9 21.0 33.0 5.8 Time to flowering (d) 79.5 74.0 90.0 3.1 variation were estimated for time to flowering and Length of longest stem (cm) 74.0 25.0 98.0 18.9 time of emergence; all the other traits showed Height first podded node (cm) 22.2 8.0 48.0 24.1 intermediate values. The variability of means for yield Length of internode (cm) 3.9 2,0 9.0 24.6 components was lower than variability for seed yield. Leaf lenght (cm) 7.9 0.7 9.9 23.9 Leaf width (cm) 0.7 0.2 1.5 34.3 Extreme values for seed yield and biomass were 7.0- Pod lengthb (cm) 3.8 2.7 5.2 12.4 214.0 and 13.0-481.0g respectively. The distribution Pod widthb (cm) 1.3 0.8 2.0 17.7 in frequency classes for flower colour showed that Pedicel lengthb (cm) 5.0 1.7 8.5 23.0 b nearly 51% of entries were characterized by violet Seeds/pod (no.) 2.6 1.0 5.0 33.8 Seed lengthc (cm) 0.8 0.4 1.5 25.0 flowers; while the prevalent seed colour was beige Seed widthc (cm) 0.8 0.4 1.3 23.0 (68.4%). Seed thicknessc (cm) 0.5 0.2 1.0 16.0 Seed yield (g) 82.4 7.0 214.0 48.7 The eigenvalues representing the variance of the Biomass (g) 201.7 13.0 481.0 44.0 principal components, and the cumulative percent of Colour Flower colour White Violet Pink - the eigenvalues indicating percentage contribution to Frequency (%) 37.6 51.1 11.3 - the total variance attributable to each principal component are given in Table 2. Eigenvectors Seed colour White Beige Brown Green indicating the degree of association among original -grey Frequency (%) 3.9 68.4 5.5 7.9 data and each principal components are also reported. aData collected on single plant; b Average of 5 dry pods/plant; The first two PC axes accounted for >48% of the c Average of 5 seeds/ plant. multivariate variation among entries and the first eight axes >88% of variation, indicating a moderate degree

11 Lathyrus Lathyrism Newsletter 4 (2005)

Table 2. Principal component analysis (PCA) of descriptors associated with 76 grass pea landraces showing eigenvalues and proportion of variation associated with the first eight axes and eigenvectors of descriptors.

PC axis 1 2 3 4 5 6 7 8 Eigenvalues 5.01 2.61 1.67 1.38 1.06 0.98 0.71 0.62 Variation (%) com. 31 48 58 67 73 79 84 88 Descriptora Eigenvectors Time to emergence (d) -0.12 0.15 0.40 0.27 0.17 0.44 -0.45 0.35 Time to flowering (d) -0.11 0.04 0.61 0.04 -0.07 0.09 0.31 0.05 Length of longest stem (cm) 0.17 0.45 -0.18 0.00 0.11 0.00 -0.03 -0.40 Height of 1° podded node (cm) 0.25 0.13 0.18 0.36 0.28 0.20 -0.16 -0.49 Length of internode (cm) 0.11 0.21 -0.23 0.23 0.64 -0.14 0.26 0.20 Leaf lenght (cm) 0.08 0.11 0.21 -0.52 0.20 0.42 0.52 -0.13 Leaf width (cm) 0.06 -0.05 -0.41 -0.34 -0.01 0.60 -0.31 0.03 Pod lengthb (cm) 0.38 -0.04 0.13 -0.15 -0.06 -0.10 -0.10 0.14 Pod widthb (cm) 0.42 -0.09 0.07 -0.06 0.00 -0.07 -0.05 0.01 Pedicel lengthb (cm) -0.03 0.42 -0.01 -0.31 0.24 -0.19 -0.11 0.50 Seeds/podb (n.) -0.33 0.31 -0.07 0.02 -0.08 0.08 0.04 -0.08 Seed lengthc (cm) 0.42 -0.11 0.06 -0.01 0.01 -0.01 -0.04 0.11 Seed widthc (cm) 0.41 -0.14 0.07 -0.00 0.04 0.02 0.03 0.13 Seed thicknessc (cm) 0.12 -0.05 -0.31 0.46 -0.21 0.38 0.44 0.32 Seed yield (g) 0.18 0.43 -0.02 0.12 -0.40 -0.01 0.11 0.07 Biomass (g) 0.20 0.44 0.06 -0.05 -0.40 -0.02 -0.08 0.01 aData collected on single plant; b Average of 5 dry pods/plant; c Average of 5 seeds/ plant.

Table 3. Cluster memberships: entry number and Table 4. Cluster means of 16 quantitative geographical origin of 76 grass pea landraces. descriptors observed in 76 grass pea landraces.

Cluster I (n=19) Descriptor Cluster Entry1 Origin2 Entry Origin Entry Origin I II III IV V Time to emergence (days) 24 24 23 24 24 100263 ITA 112411 CYP 115243 ITA Time to flowering (days) 80 99 79 79 79 Length of longest stem (cm) 67 74 75 79 76 100288 ESP 106531 AUS 113090 ITA Height of 1st podded node (cm) 20.7 19.2 21.8 25.0 25.0 115099 ITA 112252 ITA 100290 ITA Length of internode (cm) 3.7 3.9 3.7 3.9 4.5 100291 MAR 103641 ITA 112390 CYP Leaf length (cm) 7.3 8.3 11.0 8.0 7.5 115242 ITA 103203 ITA 113874 ITA Leaf width (cm) 0.7 0.7 0.8 0.8 0.6 115795 BGR 100041 Unk.3 100042 Unk.3 Pod length (cm) 4.0 3.5 3.9 4.0 4.0 100287 AUS Pod width (cm) 1.3 1.0 0.8 1.2 1.2 Cluster II (n=16) Pedicel length (cm) 4.8 5.4 5.1 4.9 5.0 Seeds/pod (n.) 2 3 2 3 2 106529 AUS 111982 ITA 112401 CYP Seed length (cm) 0.9 0.6 0.9 0.9 0.9 112414 CYP 112418 CYP 116171 ALB Seed width (cm) 0.9 0.6 0.8 0.8 0.9 112403 CYP 112399 CYP 112407 CYP Seed thickness (cm) 0.5 0.5 0.4 0.5 0.4 112415 CYP 112417 CYP 112419 CYP Seed yield (g) 83.5 81.9 73.1 94.9 70.0 112408 CYP 112412 CYP 116170 ALB Biomass (g) 195 197 206 228 163 112410 CYP Cluster III (n=15) The mean values of the original traits for each cluster 110434 ITA 110435 ITA 110437 ITA are listed in Table 4. For some traits it was impossible 110492 ITA 115833 HUN 110955 ITA to clearly differentiate the phenotypic diversity among 109680 ESP 110262 ITA 111986 ITA clusters, such as the time to emergence and seed 100289 RUS 111985 ITA 115097 ITA 115834 HUN 100043 Unk.3 103585 ETH thickness; while, by the other traits the clusters were Cluster IV (n=17) better differentiated. In particular, means indicate 112400 CYP 112416 CYP 113873 ITA shorter plants with larger pods and seeds in cluster I. 115653 ITA 103244 ITA 103376 ITA Entries in cluster II with smaller seeds and shorter 103468 ETH 103579 ETH 113949 HUN pods flower nearly three weeks later. Highest yield 115093 ITA 115094 ITA 100293 Unk.3 and biomass means characterize entries in cluster IV, 103212 ITA 110957 ITA 112251 ITA which also showed taller plants and higher first 116250 ALB 113089 ITA podded node. On the contrary, cluster V grouped Cluster V (n=9) entries with longer internode and lower mean values 100044 Unk. 112413 CYP 115096 ITA for yield and biomass. Entries in cluster III had larger 100292 FRA 103237 ITA 106385 AUS 106434 ITA 106530 AUS 115241 ITA leaves; indicated by leaf length and width. 1Mediterranean Germplasm number (MG); 2ISO Country code ; 3Unknown

12 Lathyrus Lathyrism Newsletter 4 (2005)

Plot of PCAs and clusters using the first two axes, PRIN1 and PRIN2

3

2

1

0

-1

-2

-3 -3 -2 -1 01234

Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5

Fig 1. Plot of principal component analysis and clusters using the first two axes PRIN1 and PRIN2. Each cluster is represented by a different symbol.

A combined spatial distribution of entries and clusters production (for example MG 113089, MG 112416) or can be represented in two-dimensional scatter to use them in future selection programs. As reported diagrams as shown in Figure 1. in previous experiences (1,14) grouping germplasm entries into morphologically similar and presumably Conclusions genetically similar groups is useful when little is The phenotypic diversity among grass pea entries was known about the crop history and the population well defined by both Principal Component and Cluster structure; as is the case for the grass pea collection. It analyses. Considering the different morpho-bio- is evident that entries clustered together are more alike agronomic descriptors, it has been possible to observe than entries from other clusters. However, the data a remarkable inter and intra-group diversity. The must be considered with caution because they are an covariation structure in the material studied revealed a expression of linked genetic and environmental different association between traits. The traits with effects. So, it is important to emphasise that the groups dominant roles in the first two components are closely were defined on the basis of results from in a single related to yield and yield components; while, location of southern Italy. The clusters could be vegetative traits like flowering time, height of first different if the examination took place elsewhere. podded node and leaf traits were separately linked to other components. This suggests the possibility of There seems to be no significant differences in obtaining, though selection, suitable genotypes relation to the origin of entries, most of which were combining high yield with desirable traits for direct from Italy and distributed in all groups. However, the release as cultivars in marginal areas of southern Italy. entries from Cyprus with some exception were found to form a distinct group (II), which was characterized Cluster analysis also helped us to differentiate entries by entries showing smaller pods and seeds, longer on the ground of their different levels of similarity. pedicels and later flowering time. A more detailed Five groups were identified with clear-cut differences geographical differentiation was impossible with according to the first principal component, which many origins underrepresented, with the exception of mostly accounted for yield and yield components. Italy and Cyprus. In fact, at the level of five clusters, Smaller differences among groups were seen the proportion of variance accounted for by the according to the second principal component, which clusters is 31%. This is a low percentage for the accounted for vegetative traits. Compared with other variance explained by the identified groups. Thus the groups, group IV show highest mean values for yield, differentiation according to these clusters can only be biomass, seed size and height of first podded node, considered as a preliminary approach until more which are useful agronomic traits to breed new grass detailed analysis and information is available. Finally, pea varieties. With the exception of group V, which the observed wide diversity in the Italian grass pea included the less productive entries, groups I, II and entries distributed in all groups suggests the use of this III were moderately similar. Among the entries tested adapted material to breed new improved grass pea the analysis provides useful information in order to varieties. utilise directly the most promising materials for

13 Lathyrus Lathyrism Newsletter 4 (2005)

References 1. Alba E, Polignano GB, Notarnicola L. 1997. 9. Noto F, Poma I, Gristina L, Venezia G, Ferrotti Diversity analysis in some Amaranthus entries. F. 2001. Bioagronomic and qualitative Agr. Med. 127, 198-204. characteristics in Lathyrus sativus lines. In: 2. Bisignano V, Della Gatta C, Polignano GB. Proceedings 4th European Conference on Grain 2002. Variation for protein content and seed Legumes (eds. AEP), 8-12 July 2001, Cracow, weight in grass pea (Lathyrus spp.) germplasm. Poland. P 183. Plant Genetic Resources Newsletter 132, 30-34. 10. Polignano GB, Uggenti P, Perrino P. 2001. 3. Eyzaguirre PB, Paludosi S, Hodgkin T. 1999. Phenotypic diversity in Bari grass pea (Lathyrus IPGRI’s strategy for neglected and underutilized spp.) collection. In: Proceedings 4th European species and the human dimension of Conference on Grain Legumes (eds. AEP), 8-12 agrobiodiversity. In: Priority-setting for July 2001, Cracow, Poland. Pp 184-185. underutilized and neglected plant species of the 11. Polignano GB, Uggenti P, Bisignano V, Alba E. Mediterranean region (Paludosi S, ed.). Report of 2003. Patterns of variation in Lathyrus sativus and the IPGRI Conference, 9-11 February 1998, some related species. Agr. Med. 133, 1-9 ICARDA, Aleppo, Syria. IPGRI, Rome, Italy. Pp 12. Sarker A, Robertson D, Campbell CG. 2000. 1-20. Lathyrus spp.: Conserved Resources, Priorities 4. Enneking D, M. Wink. 2000. Towards the for collection and future prospects. In: Linking elimination of anti-nutritional factors in grain Research and Marketing Opportunities for Pulses legumes. In: Linking Research and Marketing in the 21st Century (R. Knight, ed.), Kluwer. Pp Opportunities for Pulses in the 21st Century 645-654. (Knight R, ed.), Kluwer. Pp 671-681. 13. SAS/STAT Guide for Personal Computers. 5. Erskine W, Smartt J, Muehlbauer FJ. 1994. Version 6. SAS Institute Inc., Cary, NC USA, Mimicry of lentil and the domestication of 1987. common vetch and grass pea. Economic Botany 14. Souza E, Sorrells ME. 1991. Relationships 48, 326-332. among 70 North American Oat Germplasm: I. 6. Granati E, Bisignano V, Chiaretti D, Polignano Cluster analysis using quantitative characters. GB, Crinò P. 2002. Characterization of Italian and Crop Sci 31, 599-605. exotic Lathyrus germplasm for quality traits. 15. Zeven AC, Zhukovsky PM. 1975. Dictionary of Genetic Resources and Crop Evolution 50, 273- cultivated plants and their centres of diversity. 280. Centre for Agricultural Publication And 7. Kupicha FK, 1983. The infrageneric structure of Documentation, Wageningen, The Netherlands. Lathyrus. Notes from the Royal Botanic Garden 16. Zohary D, Hopf M. 1988. Domestication of Edinburgh 41, 287-326. Plants in the Old World. Clarendon, Oxford. 8. Hanbury CD, White CL, Mullan BP, Siddique KHM. 2000. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Animal Feed Science and Technology 87, 1-27.

14 Lathyrus Lathyrism Newsletter 4 (2005)

Model plant type in Khesari (Lathyrus sativus L.) suitable for hill farming

Vedna Kumari and Rajendra Prasad

CSK, Himachal Pradesh Krishi Vishvavidyalaya, Oilseeds Research Station, Kangra, Himachal Pradesh 176001, India

Introduction

Khesari is grown over large areas in Bangladesh, recorded at appropriate stages; these included plant China, India, Burma, Nepal and Pakistan, as well as height, number of branches, days to flower, days to being cultivated in southern Europe and parts of podding, days to maturity, pods per plant, seeds per pod, Africa and South America. In India, it is widely grown 100 seed weight and seed yield per plot. The seeds were in Madhya Pradesh, West Bengal, Maharastra and biochemically analysed for ODAP content as per parts of eastern Uttar Pradesh despite the Government standard procedure. Mean values of 5 random plants per of India’s ban on its cultivation and sale. The crop has replication and seed yield from each plot were used for adaptive advantages, such as tolerance to moisture computing correlations. Path-coefficient analysis was stress, requirement of minimum management, performed (1). possessing good quality of amino acid composition in its protein and resistance to many biotic stresses. In spite of these attributes, the excessive consumption of Results and Discussion khesari dal causes irreversible paralysis of lower limbs The analysis of variance (not shown) for all characters (neurolathyrism) due to the presence of a neurotoxin except for days to maturity, showed the presence of called (β-N-oxalyl-L-αβ- diaminopropionic acid or substantial genetic variability. Seed yield was ODAP (3). This disorder is not confined to India alone, positively and significantly associated with plant but has been present in other countries including height, days to flowering, days to podding, pods per Germany, Greece, Italy, Algeria, Bangladesh and Iran. plant and seeds per pod (Table 1). Seed weight was The natural germplasm of grasspea in parts of the negatively but non-significantly correlated with seed north-western hills of India has shown large variation yield. Plant height was had a significant positive in seed ODAP content. The landraces grown by correlation with days to flower. Further, days to farmers have poor plant type, low yield potential and flower and days to podding were strongly positively high neurotoxin content (0.2 to >0.7%) that shows correlated. Seed ODAP content was significantly and instability over locations and seasons. The basic cause positively correlated with number of branches, days to of low yields in pulse crops, including grasspea is flowering, days to podding, pods per plant, seeds per attributed to changes in plant type, which mainly pod and seed yield. includes morphological changes, many of which may be related to physiological functions as well. Path-coefficient analysis (Table 2) showed that the seed ODAP content, pods per plant, plant height, days to podding and seeds per pod had direct positive Material and Methods effects on seed yield. The contribution of the The experimental material for the present investigation remaining characters to seed yield was also mainly comprised of 24 diverse and determinate through these characters, their direct effect being landraces/germplasm lines collected from farmers fields negative. Seed ODAP content had the highest from Kangra district as well as from the Division of contribution to seed yield, followed by pods per plant Genetics, Indian Agricultural Research Institute, New and plant height. The effect of number of branches Delhi. All the lines were grown in randomised complete and seed weight was via pods per plant and plant block design with 3 replications at the experimental height respectively. farm area of Himachal Pradesh Krishi Vishvavidyalaya, Palampur (32°6’N, altitude 1290 m). Each plot was 1.5 A comparison of correlations and path analysis x 1.4 m with inter and intra row spacings of 30 and 15 revealed that plant height, days to podding, pods per cm, respectively. Locally recommended practices were plant and seeds per pod were the principal yield used to raise the crop. Observations on various components. The character days to flower also morphological characters and yield components were contributed to seed yield but mainly via pods per

15 Lathyrus Lathyrism Newsletter 4 (2005)

plant. Path coefficient analysis revealed that pods per seed yield components would help to obtain their plant, followed by plant height contributed the most relative importance and consequently model plant toward seed yield, while correlations also showed architecture. This comparison also suggested large similar associations. A strong positive correlation of differences in yield potential due to the differences in plant height, pods per plant and seeds per pod have pods per plant, followed by seeds per pod, days to previously been reported (2,4). podding, days to flowering, number of branches and plant height. Greater seed weight, though desirable in An alternative approach to identify model plant many pulse crops, was found to be a less important characteristics is by comparing highest and lowest yield component in grasspea. yielding lines in all morphological features to identify the character responsible for difference in yield The plant type suggested by correlation, path- potential (Table 3). There were significantly large coefficient analysis and from a comparison of plant differences in pods per plant, days to podding, plant morphology of high and low yielding line, as well as height and seeds per pod which could account for the seed yield pattern of lines with high vis-à-vis low differences in yield potential of these lines. These level of expression of seed yield components agree to characters are incidentally the same ones indicated by a large extent (5). Accordingly, such a model plant was correlations and path analysis. characterised by higher numbers of days to flowering and podding, pods per plant, seed per pod and was Further, a comparison of the pattern of seed yield in definitely a taller plant. lines with high vis-à-vis low level of expression of

Table 1. Correlation coefficients among different pairs of characters in grasspea.

Branches Days to Days to Pods per Seeds per 100 seed Seed yield Seed per plant flower podding plant pod weight per plot ODAP Plant height -0.03 0.44* 0.36 0.32 0.35 0.29 0.47** 0.32 Branches per plant 0.08 0.01 0.36 0.19 0.06 0.22 0.41* Days to flower 0.75** 0.34 0.28 0.10 0.45* 0.44* Days to podding 0.24 0.30 0.12 0.41* 0.40* Pods per plant 0.31 -0.11 0.59** 0.45* Seeds per pod 0.05 0.45* 0.47* 100 seed weight -0.14 -0.23 Seed yield per plot 0.66** * significant P<0.05, ** significant P<0.01

Table 2. Path-coefficients, direct (diagonal in bold) and indirect (off-diagonal) effects of characters on yield.

Plant Branches Days to Days to Pods per Seeds per 100 seed Seed Correlation height per plant flower podding plant pod weight ODAP with yield Plant height 0.212 0.002 0.004 0.033 0.097 0.031 -0.025 0.119 0.47** Branches per plant -0.006 -0.057 0.001 0.001 0.112 0.017 -0.005 0.154 0.22 Days to flower 0.093 -0.004 0.009 0.068 0.106 0.024 -0.009 0.165 0.45* Days to podding 0.076 -0.001 0.007 0.091 0.075 0.027 -0.010 0.149 0.41* Pods per plant 0.067 -0.021 0.003 0.022 0.309 0.027 0.010 0.170 0.59** Seeds per pod 0.075 -0.011 0.002 0.028 0.094 0.087 -0.004 0.176 0.45* 100 seed weight 0.060 -0.004 0.001 0.011 -0.034 0.004 -0.087 -0.087 -0.14 Seed ODAP 0.067 -0.023 0.004 0.036 0.139 0.041 0.020 0.376 0.66** * significant P<0.05, ** significant P<0.01, Residual effect = 0.39

16 Lathyrus Lathyrism Newsletter 4 (2005)

Table 3. Comparison of the highest and lowest Table 4. Pattern of seed yield (g/plot) in lines with yielding lines in grasspea. high and low levels of expression of quantitative traits in grasspea. Character Highest Lowest Difference Seed yield (g/plot) 206.7 81.0 125.7* Character Highest Lowest Difference Plant height 56.3 52.4 3.9** Plant height 123.1 15.1 108.0** Branches per plant 15.4 13.5 1.9 Branches per plant 124.5 15.1 109.4** Days to flower 133.1 131.4 1.7 Days to flower 179.0 31.0 148.0** Days to podding 159.4 145.6 13.8** Days to podding 184.4 35.2 149.2** Pods per plant 50.0 33.8 16.2 Pods per plant 249.0 15.1 233.9** Seeds per pod 3.0 2.6 0.5 Seeds per pod 206.7 15.1 191.6** 100 seed weight 6.2 7.4 -1.2 100 seed weight 60.9 15.1 45.8 * significant P<0.05, ** significant P<0.01 * significant P<0.05, ** significant P<0.01

References

1. Dewey DR, Lu KH. 1959. A correlation and path- 4. Sharma RN, Chitale MW, Ganvir GB, Geda AK, coefficient analysis of components of crested Pandey RL. 2000. Observations on the wheat grass seed production. Agron J 51, 515-518 development of selection criterion for high yield 2. Kumar S, Dubey DK. 2001. Variability, and low neurotoxin in grasspea based on genetic heritability and correlation studies in grasspea resources. Lathyrus Lathyrism Newsletter 1, 15- (Lathyrus sativus L.). Lathyrus Lathyrism 16. Newsletter 2, 79-81. 5. Solanki IS, Singh VP, Waldia RS. 1992. Model 3. Roy DN, Nagarajan V, Gopalan C. 1963. plant type in lentil (Lens culinaris Medik.). Production of neurolathyrism in chicks by Legume Research 15, 1-6. injection of Lathyrus sativus concentrates. Current Sci 32, 116-118.

17 Lathyrus Lathyrism Newsletter 4 (2005)

Resilience of South Asian disabling conditions: a glimpse of lathyrism among comparative histories

M. Miles

West Midlands, UK

E-mail: [email protected]

Some disabling conditions in South Asia, such as foreshadowed there, are reproduced here to contribute lathyrism, iodine deficiency disorders (IDD), cataract, to an appraisal of the broader picture. poliomyelitis, epilepsy and leprosy, have mechanisms and impairment effects that differ substantially. Yet Evidence of Lathyrus sativus (grass pea or chickling they share socio-cultural features that have promoted vetch) cultivation has been found in Indian their strong resilience in face of efforts to eliminate archaeological sites from the second millennium BC them as public health problems. For each condition a (17). A physical impairment attributed to eating khesari 'magic bullet' or much-improved technical fix has dal (the Indian food name of this pulse) was described been applied, often with increasing vigour over in the period 200 BC - 200 CE, in Susruta's decades. Successes are reported in some regions Nidanasthana: "When there is trembling in taking the during some periods (so far as genuine data can be first few steps with limping and when organisation of distinguished from data adjusted to fit externally- the joint gets loose, it is known as Kalayakhanja prescribed 'targets'). Yet the longer view is that [Footnote: Kalaya -- Khesari pulse.]", though some 'vertical' applications of a seemingly effective fix are translators are cautious about identifying lathyrism not enough, without concurrent broad and long-term here (20,23). The condition is mentioned in South Asian measures for poverty alleviation, community health writing in the late 16th century, in the Bhavaprakasa education, and local self-help. The widespread of Bhavamisra, and in Abul Fazal's Ain-i-Akbari (1,23). resurgence of malaria and tuberculosis warns that The latter notes that, "Kisari is the name of a pulse, none of these diseases or conditions is beaten. None resembling peas, eaten by the poor, but is can be relegated to low-profile 'mopping up' work. unwholesome", with footnote on Kisari as Lathyrus The eradication of smallpox may have been seriously sativus. Symptoms were more precisely described by misleading. the physician and surveyor Francis Buchanan reporting on Bihar and Patna in 1811-1812: "It seems Such a hypothesis was examined recently with focus to consist in a weakness and irregular motion of the on leprosy, comparing evidence from the public muscles moving the knees, which are bent and moved careers of other major disabling conditions including with a tremulous irregular motion, somewhat as in the lathyrism (15). The idea was that the stigma and chorea, but not so violent. When the disease has lasted separateness of the 'leprosy world' has been self- some time, and has become confirmed, the legs suffer reinforcing, and obscured some common factors. emaciation. It is not accompanied by fever, but in the People involved with each disability-related condition commencement is often, though not always, attended have something to learn from history and from one with pain" (4). another, though the precise biomedical focus may be dissimilar. There is nothing new in taking a broader The first institutional service for lathyrism sufferers comparative view. In the 1920s, disabling ailments was probably the Mejah Cripples' Asylum (Allahabad, such as goitre, rickets and lathyrism were mapped India), maintained "by the charity of the local rajas together, across South Asia, for epidemiological and land-holders under the supervision of the purposes (13). The historical background of lathyrism Tahsildar" (21). Some blind inmates and some with in South Asia, and socio-cultural features leprosy were also listed, but this asylum arose mainly

18 Lathyrus Lathyrism Newsletter 4 (2005)

to care for sufferers from lathyrism, affecting an grown) has risen sharply. "Evidently, the poor landless estimated 4% of the local population in 1861. Between labourers were being 'saved' from the poisonous seed 1859 and 1868, the Indian Annals of Medical Science not because of the researches and educational carried four detailed papers by James Irving, Civil programme of the last two decades, but solely due to Surgeon, Allahabad, reporting this cumulative the intervention of market forces. The very greed and disaster. He found it "remarkable that thousands of profit motive of the landed gentry, which for centuries people, who know that a particular grain may render was responsible for the perpetuation of neurolathyrism them lame, yet continue to use it for food. Is this among the poor of Rewa, has apparently helped to because they must either eat the poison or starve? Will redeem the poor by putting Lathyrus sativus out of no other grain grow and be productive in the affected their economic reach" (8). [areas]? If not at present - will drainage or other means not render the soil capable of bearing other and During the 1990s, researchers made laudable technical less deleterious crops? Are there no means, in fact, of progress in breeding and testing types of Lathyrus inducing the people to give up the use of the sativus with the neurotoxin significantly reduced, poisonous food?" (10). Forty years later, Irving's while retaining the plant's remarkable capacity to questions were still unanswered. The government flourish in barren conditions. Yet there remain commissioned an enquiry into lathyrism, by Major complex tasks of promoting the new breeds, re- Andrew Buchanan (6). He duly reported, and the report orienting the centuries-long folk awareness of was approved and filed away. A few years later it was lathyrism dangers, organising distribution networks so practically unknown and unobtainable (22). that the new seeds are used by hundreds of thousands of subsistence farmers scattered across South and Sixty years after Irving's questions, the social West Asia and the Horn of Africa, and monitoring the problems were underlined by a senior pathologist, feeding outcomes with humans and livestock. Major Hugh Acton. He went upcountry from Calcutta Technically this process could perhaps succeed in less to examine 204 people with lathyrism, who were than ten years; yet agricultural realities, and the breaking limestone at a kiln. Acton estimated that imminent prospect of water catastrophes in the region, 60,000 people had lathyrism in North Rewah alone, suggest that some decades may pass before the many of whom "migrate to the larger cities, Patna, benefits of current research reach those needing them. Benares, Bombay and Calcutta, and form a large percentage of the beggar population" (2). Acton was a Availability of research funding for technical hard-boiled military scientist, sceptical of anything not advances may depend more on the commercial viewable on a microscope slide; yet he concluded that potential of the grass pea as a strong, high-protein the solution to lathyrism must be "a sociological one", crop, than the protection of subsistence farmers from starting with abolition of the rural debt-slavery that paralysis. Health and nutrition development for rural forced workers to accept risky food in lieu of wages. people tends to be long, slow and of doubtful Sixty more years down the line, Dr Gopalan of the outcome, unless people see a sure and tangible gain Nutrition Foundation of India noted that the Indian for themselves, without drawbacks. The uncertainty government in the 1950s had tried to ban the payment factors can be verified from experiences in the cataract of Lathyrus sativus to agricultural workers in lieu of surgery field. The offer of 'eyesight regained' should cash, and to dissuade rural folk from excessive be overwhelmingly attractive; yet hundreds of consumption of kesari dal. These efforts "had no thousands of South Asians, within reach of low-cost impact whatever" (7). cataract surgery, do not avail themselves of it. There are deep-rooted folk memories of 'development' and Gopalan was publishing on lathyrism as early as 1950. 'new methods' which turned out to have unexpected In a remarkable turn, he noted in 1999 that lathyrism costs and pains. So what could be the drawbacks of had now practically disappeared from some Indian 'new Lathyrus sativus'? One predictable irony of regions where it was long endemic -- not because of history could occur if, in 30 years time, lathyrism has any direct technical advance, but because "Green been eliminated and a million Asian farmers have lost Revolution" investment in wheat and rice has reduced their smallholdings to agrobusinesses that mass- their price, while that of Lathyrus sativus (still widely produce the toxin-free crop with minimal need of

19 Lathyrus Lathyrism Newsletter 4 (2005)

human labour. There is more than one way to cripple a among a village population of 101, and Mackenzie community, even with the most benevolent intentions. reported further cases (11,12).

For further comparison, Gopalan notes "unforeseen The timescale for rural change is usually long, but not factors" introduced by technological intervention, all the inertia and delay can be blamed on villagers. which are moving IDD nearer to the children of urban International action in translating scientific discovery planners. Iodine deficiency, once considered a hill to practical effect, e.g. on iodinisation, smallpox country problem, has been increasing in the densely vaccination, polio immunisation, or detoxifying populated plains of India. This may arise from lathyrus, is also painfully slow-moving. Coindet intensive irrigation and multiple cropping, resulting in published the effective use of iodine against goitre in the depletion of soil micronutrients, plus food 1820, and by 1825 iodine was being applied additives and contaminants that boost goitrogen or successfully by David Scott, District Commissioner reduce body utilisation of iodine (8). Since no microbe and amateur physician, in a remote area of Bengal (18). works in a vacuum, no body lives solely in a Yet prophylactic iodisation of salt took another 160 laboratory, efforts are needed to 'foresee the years to start being taken seriously in South Asia, and unforeseen', without resort to astrology. is still pursued in a half-hearted way. In the specific region where Scott made the first move, goitre Meanwhile, some Afghans still suffer lathyrism, remains endemic. Nearly half the Bangladesh unseen by modern health services. Lathyrus sativus population reportedly had goitres in 1993, though by has been cultivated in many parts of Afghanistan (5), 2000 this dropped below 20% (9,14). and lathyrism seems to have been noticed in 1839, when Indian soldiers on a British military expedition Major Acton in 1922 proposed an evidence-based plan to Kabul (probably via the Bolan Pass, Kandahar and of action against lathyrism, but lamented that "In India Ghazni) suffered physical harm after being reduced to one publishes results and waits patiently for years to minimal food rations. To survive, they had see them carried out into practice." (2). His comment supplemented their diet with khesari dal, though they could draw a grimace of recognition from scientists knew the risks (10). Modern reports of lathyrism in and development agents in any country at any time Afghanistan appeared in 1953 and 1988, at Kabul during the following eighty years. The historical (16,17). In 1999, a medical team visiting Afghanistan's lesson is that technical fixes alone are seldom north-eastern "Wakhan corridor", reported lathyrism sufficient. Concerted and sustained action is needed endemic in several villages (19). The corridor runs on many fronts and at many levels to address issues of north of Chitral and Gilgit in Pakistan, where in 1908 poverty and exploitation, and to enlighten planners as McCarrison found ten cases of lathyrism, all male, well as the masses.

References 1. Abul Fazal. Ain-i-Akbari, transl. HS Jarrett, 1891. 7. Gopalan C. 1983. Recent studies and efforts. In: Calcutta: Asiatic Society of Bengal, vol. II, 151. The Lathyrism Problem: current status and new 2. Acton HW. 1922. An investigation into the dimensions. Nutrition Foundation of India. causation of lathyrism in man. Ind Med Gaz 57, 8. Gopalan C. 1999. The changing epidemiology of 241-247. malnutrition in a developing society - The effect 3. Arya LS, Qureshi MA, Jabor A, Singh M. 1988. of unforeseen factors. Bull. Nutrition Foundation Lathyrism in Afghanistan. Ind J Pediatr 55, 440- of India 20, 1-5. 442. 9. IDD Prevalence and Control Program Data: 4. Buchanan F (n.d.) An Account of the Districts of Bangladesh. At: www.people.virginia.edu/~jtd/ Bihar and Patna in 1811-1812. Patna: Bihar & iccidd/mi/idd_014.htm Orissa Research Society, p. 274. 10. Irving J. 1860. Report on a species of palsy 5. Chrtkova-Zertova A, van der Maesen LJG, prevalent in Pergunnah Khyraghur. Ind Ann Med Rechinger KH. 1979. Papilionaceae I - Vicieae. Sci 7 (No. 13), 127-137. Flora Iranica No. 140. Graz: Akademische Druck, 11. Mackenzie LHL. 1927. Lathyrism in the Gilgit pp. 78-79. Agency. Ind Med Gaz 62, 201-202. 6. Editorial. 1903. The inquiry into lathyrism. Ind 12. McCarrison R. 1926. A note on lathyrism in the Med Gaz 38, 61-62. Gilgit Agency. Ind J Med Res 14, 379-381.

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13. Megaw JWD, Gupta JC. 1927. The geographical 18. Scott D. 1825. Extract from a letter from D. Scott, distribution of some of the diseases of India. Ind Esq. Commissioner, Rungpore district, Med Gaz 62, 299-313. communicated by G. Swinton, etc. Trans Med 14. Miles M. 1998. Goitre, cretinism and iodine in Phys Soc Calcutta, 1, 367. South Asia: historical perspectives on a 19. Simpson RA. 2002. North West Frontier mission continuing scourge. Med Hist 42, 47-67. in Afghanistan. Med J Australia 177 (11), 633- 15. Miles M. 2003. Knowledge and management of 637. disabling conditions in South Asian histories: 20. Singhal GD, Singh LM, Singh KP. 1972. implications for leprosy futures. Diagnostic Considerations in Ancient Indian Ind J Lepr 75, 153-167. Revised version at: Surgery. Allahabad: Singhal, pp. 77-78. www.disabilityworld.org/ 21. Steel CD. 1884. Statistical, Descriptive, and 09-10_03/news/southasia.shtml . Historical Account. Vol VIII, Pt II - Allahabad. 16. Rouault De La Vigne A, Ahmad A. 1953. NW Provinces & Oudh Govt Press, Allahabad, Lathyrisme en Afghanistan. Revue Médicale du pp. 131-132, 203. Moyen-Orient 10, 325-336. 22. Stockman R. 1917. Lathyrism. Edinburgh Med J 17. Saraswat KS. 1980. The ancient remains of the 19 (n.s.), 277-296. crop plants at Atranjikhera (c. 2000 - 1500 B.C.). 23. Wujastyk D. 1998. The Roots of Ayurveda. New J Ind Botan Soc 59, 306-319. Delhi: Penguin, pp. 15, 169.

21 Lathyrus Lathyrism Newsletter 4 (2005)

Considerations on the reintroduction of grass pea in China

Hui-Min Yang* and Xiao-Yan Zhang

College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China

*E-mail: [email protected]

Introduction

Grass pea (Lathyrus sativus L.) is probably the most Undoubtedly a highly efficient crop plant species, drought tolerant legume crop and it is also resistant to which can be used as food and forage, would be an moderate salinity. In drought prone areas the plant is excellent candidate for this effort. considered an insurance crop for subsistence farmers. During drought-triggered famines, grass pea can be Environment protection and ecosystem health the only food available and a lifesaver. In addition, The development of human activities has heavily grass pea is valuable for its abundant nutrients, influenced the environment and the global climate, especially protein and lysine. Historically, grass pea and desertification of the land and the degradation of has been a daily food for millions in Asia and Africa, farmland has been a very serious problem for years in and is now reintroduced into popular use in different the north and west of China. The global changes in parts of the world as a fine green manure, forage, and climate have also affected the environment, especially food. For better and safer use of grass pea, there have that of the rangelands. In the north and west of China, been a number of efforts of different research groups where rangelands are dominant, low precipitation, from different disciplines. salinity, high temperature and high irradiation all lead to more severe drought stress. Water deficit results in As a developing country in which agriculture plays a the degradation of the rangeland and soil infertility. very important role in people’s daily lives, China has The deterioration of the environment has seriously to further develop its food productivity with high affected the food yield and people’s food security, nutritional quality, and meanwhile protect the consequently the protection of environment has environment for coming generations. Grass pea, which become urgent. In order to develop stockbreeding and has been utilized and then banned several decades ago, also protect the environment, forage species with the should be reintroduced into China and utilized again potential to be used as food for the domestic animals in various ways. Below we summarize the history of and as ground cover in these regions should have high grass pea in China and discuss various considerations. priority. The reintroduction of a highly resilient This will contribute to the understanding of grass pea species is a very urgent requirement. The chosen in China by the international community of species should be tolerant to the environmental researchers. stresses and should contribute to the protection of the environment.

The new challenges for China: food, environment, and health The nature and character of grass pea: the scope of its reintroduction Food availability and human health China produced 25% of the global food yield and fed High nutrient value 22% of the world population on only 9% of the world Besides containing high amounts of starch, grass pea farmland. However, yields decreased from 1996 and contains 26.3%-34.3% protein in the seed and 13.8%- people suffered, especially in the areas of the north 20.1% protein in the stem and leaf, which is higher and west of China. For the purpose of national “food than those in Pisum sativum, Vicia faba or Medicago security”, it was necessary to provide sufficient and sativa. The protein contains 17 amino acids in safe food as well as healthy nutrition (7), and efforts sufficient quantities, especially lysine at levels higher have been made to develop better crop varieties to than that in other legumes or in cereal crops (8). improve food quality. In addition, it was also important to modulate food composition, i.e. the ratio High tolerance and resistance to environmental of plant food to meat, in order to provide more stresses nutrients from meat (which contains more proteins, Grass pea plants can grow well under drought, cold lipids, etc.). A resolution to develop stockbreeding in and moderately saline conditions. They fix and use some regions also implied the development of forage. atmospheric nitrogen, require neither fertile soil nor

22 Lathyrus Lathyrism Newsletter 4 (2005)

costly inputs, and can be the main food source when and confirmed that ODAP caused lathyrism and even other crops fail due to drought stress. In addition, the determined the lethal dose of ODAP. However there plant can resist many pests and pathogens, and control was still no effective way to cure lathyrism patients. some weeds (5). Analysis of ODAP Multi-use for daily life Since its discovery in 1964 (16, 20), multidisciplinary Grass pea is a good green manure, forage, and food research in many groups has focused on ODAP. All crop. As a legume plant, grass pea can fix atmospheric these efforts heavily depended on the analytical nitrogen and use it, and so is a good green manure for methods available for quantification of ODAP, over improvement of soil quality (5). It covers the ground time researchers have developed different methods. well, acting to reduce evaporation and conserve soil water. In spite of the neurotoxin ODAP (3-(-N- A paper-based chromatographic method was oxalyl)-L-2,3-diamino propionic acid), the stem, leaf, established to detect the presence of ODAP (6). The and seed of grass pea can be used as fine forage and is research group in Lanzhou University established a suitable for feeding cattle, sheep, etc. In addition, as a paper chromatography-based colorimetric analytical food source it can also serve people in seriously method to quantitatively measure ODAP for the first stressed areas due to high protein content and other time in China (6). This method could quantitatively nutrients, as well as retaining productivity under measure ODAP content, but was not very accurate and stress. could not separate the isomers, a modified method was then developed (3, 11). The method established by Rao (19) proved to be a good and cheap one to measure Studies on grass pea in China: the theoretical and ODAP, it had been used widely by researchers with technological basis for reintroduction some modifications (3, 12, 13), since it could not separate the two isomers, was not reproducible and time- The brief utilization of grass pea in China consuming, its use is therefore limited. The accurate Grass pea-like species have been reported in China in measurement of α- and β-ODAP separately seemed antiquity (10). Although it might not be the same especially necessary, since the former isomer was species as that today, it indicated the existence of proven to be non-toxic. grass pea. There are at least 43 varieties of L. sativus stored in the Institute of Crop Germplasm Resources Zhao et al. (38, 39) developed a capillary zone of China, however most varieties planted at present electrophoresis (CZE) method to measure ODAP, with were introduced from abroad. Before the 1960s, grass the successful separation of the two isomers and pea was widely planted in northwest China, such as another non-protein amino acid. This method still had Shanxi, Sichuan, Yunnan and Gansu provinces. It was the drawback that the high working pH (9.2) easily reported that the planting area exceeded 20,000 ha at caused the hydrolysis of ODAP to DAP. A high that time in Gansu province alone (15), the crop was performance liquid chromatographic (HPLC) method mainly used as green manure and forage. In the 1970s, has been developed and improved, proving efficient a severe drought occurred and caused a terrible famine and based on widely used technology. The HPLC in this area. Since grass pea could cope with the severe method was further developed using off-line pre- drought, poor soil fertility, and cost little, it was column derivatization with 6-aminoquinolyl-N- widely planted and its seed was used as a main food hydroxy-succinimyl carbamate (AQC) (2). Other source. The continuous consumption of grass pea improvements were pre-column derivatization with 1- seeds as almost the only food available caused an fluoro-2, 4-dinitrobenzene(23), or dansyl chloride (25, 26). epidemic of “lathyrism” in this region during the These modified methods could accurately separate the period of famine. two isomers and developed into a classic routine method to measure ODAP. The lathyrism epidemic led to the banning of the utilization and development of grass pea in China, but However, the above methods still have some studies continued. These researches ranged from the problems, such as experimental inconvenience, time- investigation of clinical symptoms, the toxicity consuming derivatization steps, and especially, the mechanism, breeding to produce low-toxicity easy decomposition of the derivatives. Further varieties, and to the development of detoxification optimization of ODAP analysis is expected. methods. Efforts on detoxification Clinical investigations For the safe use of grass pea, much effort on crop The clinical effects of the consumption of grass pea improvement has been addressed, including direct were studied in various kinds of experimental animals seed treatment, mutagenesis and mutant breeding. All to understand how grass pea affects human health (3, 6, these approaches are described below. 14, 36). This contributed to understanding the mechanisms of lathyrism and its clinical symptoms,

23 Lathyrus Lathyrism Newsletter 4 (2005)

It was found that water soaking of grass pea seed (37). In addition, oxalate influenced ODAP content and could lower the ODAP content but not sufficiently for its distribution in the plant(24). continuous safe human consumption (6). Further treatment of the seed into some products could almost Physiological characteristics of grass pea during remove all toxicity and be safe (6), but these methods treatment with Eu3+, Ca2+ and PEG were studied under were too complicated to be widely used and reduced laboratory conditions (21). The results showed effects the nutritional components in the seed, such as water- on the moisture levels, root activity, Na+-K+ ATPase, soluble vitamins and minerals. antioxidases, hydroxyl radicals and MDA content, polyamines, chlorophyll and carotenoid content, Physical and chemical treatments have also been used proline and other free amino acids. This represents a in the detoxification of grass pea. Treatment of the detailed study of this aspect of grass pea and implies seeds with highly charged C6+ ions could induce some that some mechanisms exist in this species to respond mutants (22). Some mutants were also obtained using and cope with environmental stress. the 60Co γ-ray irradiation combined with ethyl methane sulfonate (EMS) treatment (18). These Our group has made some efforts to study the eco- treatments could lower ODAP content to some extent physiology of grass pea under water deficit conditions (34, 35) in the seedlings but not in the F1 seeds. The mutants . Drought may affect stomata, photosynthesis, were not widely used as their characters were unstable and yield of grass pea plant as well as ODAP content or ODAP content was not sufficiently low. Further (35). We found a significant relationship between efforts on this aspect could still give results but have stomata and photosynthesis and yield, and drought not been pursued. Mutation would be a good choice may enhance the correlations (34), more extensive for further improvements if combined with genetic research is proceeding. engineering methods.

Agronomists have also paid much attention to the Concluding remarks breeding selection of low toxicity varieties. Two low Grass pea is a fine green manure, forage, and food toxin varieties were found among 14 local strains (4), species. Its multiple beneficial properties and various further tests were performed by the addition of uses make it suitable for reintroduction into China, phosphorus to the soil in an attempt to select a stable completed studies show good groundwork for these and low toxin variety (3). Yu et al. (36) studied some purposes (33). Reintroduction of grass pea will help local and introduced varieties to investigate the improve food availability and environment protection, relationships between location, species, genus and not only in China, but also in other parts of the world ODAP content. Bao et al. (1) screened 65 varieties with similar environmental conditions. looking for low toxin lines. These comparative studies afforded some data on the development of low toxin The studies on grass pea are being carried out in a varieties. multidisciplinary approach by researchers in China, in some instances ahead of other international efforts. Physiological and eco-physiological studies Development of low toxin varieties could be further Since some low toxin varieties have been obtained, advanced. A lot of work has been carried out in some researchers turned their eyes to the physiology breeding, selection, detoxification (9) and the and eco-physiology of grass pea. metabolic pathway of ODAP (17) in the past. Now attention should be turned to the ecological, Increases in ODAP content were observed under physiological and biochemical properties of grass pea. prolonged water stress or osmotic stress (27-30). In addition, further work should be devoted to Polyamines in the seedlings were closely related to incorporation of molecular and gene engineering ODAP content under water stress induced by PEG technology. (29) (28) (30) treatment . It was found that ABA and H2O2 accumulation affected ODAP content when the plant suffered drought stress. During seed germination, Acknowledgements ODAP content in the radicles increased as water This work was supported by the International deficit extended, while ODAP decreased in the Cooperation Project between China and Australia cotyledons (31, 32). Zhou et al. (40) concluded that ODAP (2005DFA30030-6) and the Cuiying Programme of could scavenge the hydroxyl radical. Extensive Lanzhou University, Lanzhou, China (to H-MY). experimentation showed that ODAP could protect the activity of glycolate oxidase under stress conditions

24 Lathyrus Lathyrism Newsletter 4 (2005)

References 1. Bao XG, Lv FH, Liu SZ, Shu QP. 1995. Sifting by electrophoresis. Journal of Lanzhou and cultivating utilization of low toxin varieties University (Natural Sciences) 28(3), 89-92. of Lathyrus. Pratacultural Science 12, 48-54. 14. Liu XC, Zhang GW, Li YR, Wang JX, Liang 2. Chen X, Wang F, Chen Q, Qin XC, Li ZX. 2000. ZN. 1989. Toxicological study on grass pea vine Analysis of neurotoxin 3-N-oxalyl-L-2,3- (Lathyrus sativus L.) and its toxic-component diaminopropionic acid and its α-isomer in BOAA. Scientia Agricultura Sinica 22(5), 86-93. Lathyrus sativus by high-performance liquid 15. Lv FH, Bao XG, Liu SZ. 1990. A study of chromatography with 6-aninoquinolyl-N- vetchling (Lathyrus L.) germplasm resources. hydroxysuccinimidyl carbamate (AQC) Crop Genetic Resources (China) 33(3), 17-19. derivatization. Journal of Agriculture and Food 16. Murti VVS, Seshadri TR, Venkitasubramanian Chemistry 48, 3383-3386. TA. 1964. Neurotoxic compound of the seeds of 3. Chen YZ, Li ZX, Lv FH, Bao XG, Liu SZ, Liu Lathyrus sativus. Phytochemistry 3, 73-78. XC, Zhang GW, Li YR. 1992. Studies on the 17. Qin XC, Li ZX, Wang YF. 2000. Studies of screening of low toxic species of Lathyrus, Lathyrus sativus and its neurotoxin (ODAP). analysis of toxins and toxicology. Journal of Chinese Bulletin of Life Sciences 12, 52-56. Lanzhou University (Natural Sciences) 28, 93- 18. Qin XC, Wang F, Wang XJ, Zhou GK, Li ZX. 98. 2000. Effect of combined treatment of 60Coγ-ray 4. Chen YZ, Wang XG, Ma ZL, Cai WT. 1980. and EMS on antioxidase activity and ODAP Studies on Lathyrus sativus L. Journal of content in Lathyrus sativus. Chinese Journal of Lanzhou University (Natural Sciences) 16, 114- Applied Ecology 11(6), 957-958. 115. 19. Rao SLN. 1978. A sensitive and specific 5. Das NR. 2000. Lathyrus sativus in rainfed colormetric method for determination of α, β- multiple cropping systems in West Bengal, India- diaminopropionic acid and Lathyrus sativus a review. Lathyrus Lathyrism Newsletter 1, 25- neurotoxin. Analytical Biochemistry 86, 386- 27. 395. 6. Department of Chemistry, Lanzhou University. 20. Rao SLN, Adiga PR, Saima PS. 1964. The 1975. Analysis on the toxin and studies on the isolation and characterization of β-N-oxalyl-α,β- removal of the toxin in Lathyrus sativus. Journal diaminopropionic acid: a neurotoxin from the of Lanzhou University (Natural Sciences) 11(2), seeds of Lathyrus sativus. Biochemistry 3, 432- 45-65. 436. 7. Food and Agriculture Organization of the United 21. Tian H. 2003. Study of Stress Resistance on Nation. 2004. The state of world food security. Different Environment in Lathyrus sativus L. Ph In: Scanes CG, Miranowski JA. Perspectives in D thesis. Lanzhou University, Lanzhou, China. World Food and Agriculture. Iowa, USA: Iowa 22. Wang CY, Yang HM, Wang YF. 1993. Mutation State Press. 3-29. effect of C6+ heavy ion on seed of Lathyrus 8. Hanbury CD, White CL, Mullan BP, Siddique sativus L. Hereditas (Beijing) 15(1), 28-31. KHM. 2000. A review of the potential of 23. Wang F, Chen X, Chen Q, Qin XC, Li ZX. 2000. Lathyrus sativus L. and L. cicera L. grain for use Determination of neurotoxin 3-N-oxalyl-L-2,3- as animal feed. Animal Feed Science and diaminopropionic acid and non-protein amino Technology 87, 1-27. acids in Lathyrus sativus by precolumn 9. Jiao CJ, Wang CY, Li ZX, Wang YF. 2005. derivatization with 1-fluoro-2, 4-dinitrobenzene. Advances in both toxic biochemistry and Journal of Chromatography A 883, 113-118. detoxification of Lathyrus sativus. Acta 24. Xing GM. 2002. Molecular and Physiological Prataculturae Sinica 14(1), 100-105. Responses to Stress in the Wheat Line with 10. Li SZ. 1578. Ben Cao Gang Mu. Vol. 16, issue Resistance Rust and Lathyrus sativus L. Ph D 24 for grain, 160. thesis. Lanzhou University, Lanzhou, China. 11. Li ZX, Cai WT, Chen YZ. 1986. Paper 25. Xing GM, Chen X, Li ZX. 2001. Quantitative chromatography with scanner for determination high-performance liquid chromatography of BOAA of Lathyrus sativus. Journal of determination of neurotoxin β-ODAP and α- Lanzhou University (Natural Sciences) 22(2), isomer in grass pea (Lathyrus sativus). Chemical 76-80. Research in Chinese Universities 17, 224-225. 12. Li ZX, Cai WT, Xu ZG. 1987. The determination 26. Xing GM, Wang F, Cui KR, Li ZX. 2001. Assay of ODAP in Lathyrus sativus using OPT of neurotoxin β-ODAP and non-protein amino colorimetry. Journal of Lanzhou University acids in Lathyrus sativus by high-performance (Natural Sciences) 23(2), 130-132. liquid chromatography with dansylation. 13. Li ZX, Meng YF, Zhang L, Chen YZ. 1992. Analytical Letters 34(15), 2649-2657. Determination of α- and β- 27. Xing GS, Cui KR, Li J, Wang YF, Li ZX. 2001. oxalyldiaminopropionic acid in Lathyrus sativus Water stress and accumulation of β-N-oxalyl-L- α, β-diaminopropionic acid in grass pea

25 Lathyrus Lathyrism Newsletter 4 (2005)

(Lathyrus sativus). Journal of Agriculture and availabilities. Journal of Agricultural Science Food Chemistry 49, 216-220. 142, 675-681. 28. Xing GS, Zhou GK, Li ZX. 2000. Accumulation 35. Yang HM, Zhang XY, Wang GX, Wang YF, of ABA and ODAP in Lathyrus sativus under Qiao LX. 2004. Stomatal characteristics and the water stress. Chinese Journal of Applied contents of seed ODAP, protein and starch in two Ecology 11(5), 693-698. varieties of grass pea under stress condition. 29. Xing GS, Zhou GK, Li ZX, Cui KR. 2000. Journal of Lanzhou University (Natural Studies of polyamine metabolism and β-N- Sciences) 40, 64-67. oxalyl-L-α,β-diaminopropionic acid 36. Yu JZ, Wang HQ, Yu ZC, Chen YZ, Ma ZL, Cai accumulation in grass pea (Lathyrus sativus) WT. 1994. Researches of biological laws of under water stress. Acta Botanica Sinica 42(10), lower noxious Lathyrus sativus and lower poison 1039-1044. varieties selecting. Acta Botanica Boreali- 30. Xing GS, Zhou GK, Li ZX, Cui KR. 2001. Effect Occidentalia Sinica 3, 94-98. of osmotic stress on accumulation of H2O2 and 37. Zhang J, Xing GM, Yan ZY, Li ZX. 2003. β-N- ODAP in grass pea (Lathyrus sativus). Acta oxalyl-L-α,β-diaminopropionic acid protects the Phytophysiologica Sinica 27(1), 5-8. activity of glycolate oxidase in Lathyrus sativus 31. Xue S, Zhang LS, Cao R, Wang MH, Wang PH. seedlings under high light. Russian Journal of 2001. Effect of drought on β-ODAP and amino Plant Physiology 50(5), 618-622. acids in the process of Lathyrus seed 38. Zhao L, Chen XG, Hu ZD, Li Q, Chen Q, Li ZX. germination. Acta Botanica Boreali-Occidentalia 1999a. Analysis of β-N-oxalyl-L-α,β- Sinica 21(4), 620-624. diaminopropionic acid and homoarginine in 32. Xue S, Zhang LS, Cao R, Wang MH, Wang PH. Lathyrus sativus by capillary zone 2001. Effects of water stress on the proteins and electrophoresis. Journal of Chromatography A free amino acid of the Lathyrus sativus seed 857, 295-302. during the germination. Journal of Northwest 39. Zhao L, Li ZX, Li GB, Chen XG, Hu ZD. 1999. Sci-Tech University of Agriculture and Forestry Kinetics studies on thermal isomerization of β-N- (Natural Science Edition) 29(5), 79-83. oxalyl-L-α,β-diaminopropionic acid by capillary 33. Yan ZY, Xing GM, Wang CY, Wang YF, Li ZX. zone electrophoresis. Physical Chemistry 2004. Recent advance in Lathyrus sativus and Chemical Physics 1, 3771-3773. toxin ODAP. Acta Botanica Boreali-Occidentalia 40. Zhou GK, Kong YZ, Cui KR, Li ZX, Wang YF. Sinica 24(5), 911-920. 2001. Hydroxy radical scavenging activity of β- 34. Yang HM, Zhang XY, Wang GX. 2004. N-oxalyl-L-α,β-diaminopropionic acid. Relationships between stomatal character, Phytochemistry 58, 759-762. photosynthetic character and seed chemical composition in grass pea at different water

26 Lathyrus Lathyrism Newsletter 4 (2005)

Effects of drought on stomatal character, photosynthetic character and seed chemical composition in grass pea, and their relationships

Hui-Min Yang1*, Xiao-Yan Zhang1 and Gen-Xuan Wang2

1. College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China. 2. College of Life Sciences, Zhejiang University, Hangzhou 310027, China *Email: [email protected]

Full article published in Journal of Agricultural Science (2004) 142: 675-681.

Summary

The neurotoxin β-N-oxalyl-L-α, β-diaminopropionic observed between stomatal aperture and Pn, E, the acid (β-ODAP) in grass pea (Lathyrus sativus L.) can concentrations of seed ODAP, protein and starch, cause the disease “lathyrism”. Much work has been while a negative correlation appeared between done on lowering toxicity and on selection of low stomatal aperture and WUE. Under drought toxicity varieties, while research on the eco- conditions, R2 values were more comparable with the physiological characteristics of grass pea is very rare. control. Intriguingly, the R2 values of stomatal aperture were higher than of stomatal density, Stomatal character (stomatal density and aperture), especially under drought conditions. These results photosynthetic character (Pn and E) and seed chemical indicate that stomatal aperture may be more closely composition (ODAP, protein and starch) were related to photosynthetic character and seed chemical measured in four varieties of L. sativus at different composition in grass pea, and drought may enhance water availabilities. Under drought conditions, the correlations. stomatal aperture, Pn and E were lower than those under the control, while the other parameters were Grass pea is a potentially valuable feed and food crop higher. A significant positive correlation was observed in semi-arid regions. Our group is continuing research between stomatal density and water use efficiency on the regulation of water and nutrient utilization (WUE), while negative correlations were found efficiency in grass pea. Further researches in grass pea between stomatal density and the remaining are waiting for multidisciplinary efforts. parameters. Obvious positive correlations were also

27 Lathyrus Lathyrism Newsletter 4 (2005)

Scope of growing lathyrus and lentil in relay cropping systems after rice in West Bengal, India

S. Gupta* and M.K. Bhowmick

Pulses and Oilseeds Research Station (PORS) Berhampore-742-101, West Bengal, India.

* Email: [email protected] OR [email protected]

Introduction system; especially in the mono-cropped areas of the The State of West Bengal in the eastern part of India, state situated in the Coastal, Old Alluvial and Terai bordering Bangladesh, is a unique example where rice is (Foot Hills) Zones of West Bengal with total area under cultivated in all the three seasons viz., summer, autumn cultivation comprising nearly 1.36 million hectares. and winter. The State has to feed almost 70 million Recently efforts are also going on to grow black gram people with the support of only 5.8 million hectares of (Vigna mungo) and green gram (Vigna radiata) under cultivable land. Since independence the state has, utera condition in wheat fallows with the objective to therefore, had to resort to more areas under rice than for grow an early duration pulse as a catch crop and to other crops, especially pulses, the productivity of which sustain soil health in wheat-rice rotations. are comparatively low. At present the area under rice occupies about 66 percent of the total gross cropped Experimental Findings and Discussion area, which is about 9.24 million hectares with an average cropping intensity of 171 percent (9). The total The PORS, Berhampore, West Bengal, India has area under pulses has diminished gradually every year engaged in various field experiments since 1970-71 to from 582,000 ha during 1957-58 to 242,000 ha during identify the cultivars suitable for the utera system, to 2002-03 (11). standardize the improved package of practices. As such, a trial was undertaken at the Block Seed Farm, Kandi, It is a general practice of the farmers of this region to Murshidabad, West Bengal in 1970-71 with different sow various winter pulse crops like lentil (Lens culinaris varieties of lentil and lathyrus. Seeds of lentil and L.), lathyrus (Lathyrus sativus L.), chickpea (Cicer lathyrus were broadcast on standing aman (wet season) arietinum L.) and pea (Pisum sativum) in the standing paddy field (variety Bhasamanik) @ 50 kg per hectare rice crop field, just before the harvest to ensure on 13th and 14th November 1970, respectively, 15 days germination using the residual moisture and to avoid before rice harvest. The variety yields are presented in tillage operations during pulse growing. Such a relay Table 1. cropping operation (known by the terms utera or paira) is very popular for growing lathyrus. Subsequently due to high ODAP content of local land races and also with the advent of irrigation facilities, the farmers tended to Table 1. Yield of different varieties of lentil and shift from relay cropping of lathyrus to more lathyrus grown as utera at Block Seed Farm, Kandi, remunerative crops like rapeseed, mustard, potato, other Murshidabad, India in 1970-71. vegetables and winter rice which require more water. Thus the area under lathyrus, in particular, diminished Lentil Yield Lathyrus Yield drastically from 54,000 ha during 1981-82 to 40,000 ha (kg/ha) (kg/ha) during 2000-01 (10). L6-42 578 Kh-3 3878 L6-8 1022 Kh-4 3306 With the backdrop of the aforesaid facts, efforts were L6-85 1056 Kh-18 2817 concentrated to develop high yielding lathyrus varieties LL6-4 1089 Kh-17 2672 with ODAP content less than a critical limit (0.20 %). A L6-77 1111 variety Nirmal (B-1) was developed at the Pulses and B-77 1133 Oilseeds Research Station (PORS). Other varieties L6-21 1478 (BioL 212, BioR 202, LSD-3 and P-24) are also of late L6-36 1500 recommended by the Indian Council of Agricultural L6-20 1556 Research for cultivation in India. Therefore, the PORS L6-23 2256 conducted a series of experiments to establish the Source: Chakraborty et al. (13) suitability for growing pulses in the utera cropping

28 Lathyrus Lathyrism Newsletter 4 (2005)

It is evident from Table 1 that both lentil and lathyrus coastal saline tracts of West Bengal. The trials were laid could be successfully grown under muddy land out in two Government Farms viz., Sub-Divisional conditions in the standing rice crop 15 days before its Adaptive Research Farm, Baruipur and Block Seed maturity, provided that there is no standing water in the Farm, Mathurapur. The yield data obtained are in Table field. The yield ranged from 578 to 2256 kg/ha in case 2. of lentil and 2672 to 3878 kg/ha in case of lathyrus, compared to the respective average yield of 485 kg/ha The variety Nirmal developed at PORS is the most and 511 kg/ha for these two crops in the state (13). adapted (Table 2) in the rice-utera system in the Coastal Zone of the state. The field experiments were conducted During 1999-2000 and 2000-01 different genotypes of under rainfed conditions on a clayey soil where salinity lathyrus were tested for their suitability and develops gradually after cessation of rainfall in the dry competitiveness for growing under utera conditions in winter months, when the crop was grown. muddy land of standing rice fields in mono-cropped

Table 2. Seed yield of lathyrus after rice in utera conditions at 2 sites (Mathurapur and Baruipur) in the coastal zone of West Bengal.

Varieties 1999-2000 2000-2001 Mean yield Mathurapur Baruipur Baruipur (kg/ha) BioL 212 2083 1104 775 1321 BioR 202 1875 1251 650 1259 LSD 3 1782 923 670 1125 P-24 1771 855 570 1065 P-505 1375 1004 670 1016 Nirmal 1729 1529 1295 1494 LSD (P<0.05) 268 162 104 - CV (%) 10.1 9.7 10.2 - Source: Annual Reports (4,5)

Table 3. Performance of lentil genotypes in rice-utera conditions. Likewise, variety suitability and competitiveness of Variety Seed yield (kg/ha) lentil genotypes under rice-utera system were tested for 2001-02 2003-04 two years at PORS. Altogether 8 cultivars were included L 4661 - 1233 in the experimentation during 2003-04, whereas only 3 WBL-75 - 1642 varieties were tested during 2001-02. The yield data of ILL 8006 - 666 individual lentil genotype tested for the two years are B-77 903 1366 presented in Table 3. WBL-58 1158 1708 PL-639 - 908 The duration and other characters of individual variety IPL-304 - 592 recorded during 2003-04 are in Table 4. The rice variety K-75 841 - cultivated in the experiment was MTU 7029, which LSD(P <0.05) 26.8 - matured in 140-145 DAS. Source: Annual Reports (6,8) The maturity period of all lentil varieties (Table 4) were Table 4. Time to flowering and maturity and 1000 delayed for at least 10 days when grown under utera grain weight of 7 varieties of lentil grown in rice- cropping system. Genotypes with characteristics of early utera conditions. vigour and comparatively tall stature could perform well under utera conditions. Similar observations were made Variety Days to 50% Days to 1000 grain of lathyrus cultivars when grown under utera conditions. flowering maturity wt. (g) L-4661 77 122 22.33 Some experiments were conducted to optimise sowing WBL-75 84 127 18.29 times and other agronomic management in the utera ILL 8006 66 114 20.32 system of sowing of pulses in muddy fields of standing B-77 69 120 19.98 rice, utilizing residual moisture and fertilizer. One such WBL-58 68 126 21.48 trial was conducted in 1994-95 with lathyrus under rice- PL-639 70 133 15.62 utera conditions and the results are given in Table 5. IPL-304 73 139 28.76 Source: Annual Report (8)

29 Lathyrus Lathyrism Newsletter 4 (2005)

Table 5. Effect of sowing time and weed management The yield of lathyrus variety Nirmal was maximum on lathyrus yield under rice-utera conditions during when sown 20 days before harvest of rice (Table 5). 1994-95 (rice variety IR-36 and lathyrus variety Keeping the plot weed free after rice harvest was found Nirmal). to be significantly superior to the existing farmers’ practice of not weeding the plot. Treatment Seed yield (kg/ha) Another experiment was conducted during 1999-2000 Sowing time (days before rice harvest) and 2001-2002 to verify the sowing time results of 10 743 1994-95 and to examine whether basal fertilizer by 20 973 means of placement of fertilizer in between the rows of 30 824 standing rice crop had any effect on yield of utera crop LSD (P<0.05) 100 of lathyrus. The results are given in Table 6.

Weed Management No hand weeding 500 The results in the Table 6 clearly corroborated the results One hand weeding at 45 DAS 932 of the previous experiment of 1994-95. Sowing at 15-20 Two hand weedings at 45 and 60 DAS 1100 days before rice harvest gave a better result in respect of LSD (P<0.05) 100 yield, probably due to the fact that earlier sowing might CV (%) 14.04 have more effective utilization of residual moisture in (14) LSD(P<0.05) for sowing time x NS the rice field. Duary et al. were of the same opinion. weeding interaction The utera crop might need some initial nutrition as a Source: Annual Report (3) starter dose of NPK, possibly to enhance symbiotic activities with the Rhizobium bacteria.

Table 6. Effect of sowing time and fertilizer management on seed yield of lathyrus under rice-utera conditions (rice variety MTU 7029 and lathyrus variety Nirmal)

Treatment Seed yield (kg/ha) 1999-2000 2001-2002 Mean Sowing time (days before rice harvest) 0 - 889 889 10 1150 912 1031 15 1225 993 1109 20 1525 - 1525 LSD (P<0.05) 69 - - CV (%) 6 - -

Fertilizer(N:P205:K20 in kg/ha) No fertilizer (Control) 1033 799 916 10 : 25 : 20 (Basal) 1300 890 1095 20 : 50 : 20 (Basal) 1317 1022 1169 10 : 25 : 20 (Basal) + DAP spray (2% 1550 1013 1282 solution) at flowering LSD (P<0.05) 88 - - CV (%) 8 5 - LSD (P<0.05) for sowing time x fertilizer 153 71 - interaction

Source: Annual Report (4,6)

30 Lathyrus Lathyrism Newsletter 4 (2005)

Table 7. Effect of seed soaking and foliar nutrition on lathyrus under rice-utera conditions during 2003-04 (rice variety IR-36 and lathyrus variety BioL 212).

Treatment Mature Branches/ Pods/ Seeds/pod 1000 seed Seed yield plant plant plant weight (g) (kg/ha) height (cm)

Seed priming No soaking 45 3.69 8.94 2.22 63.6 731 Soaking in water for 6 h 50 3.87 9.48 2.54 66.6 802 Soaking in 2% KH2PO4 solution for 6 h 52 3.92 10.70 2.76 70.4 910 Sowing of sprouted seeds 54 4.26 11.55 3.00 71.8 938 LSD (P<0.05) 3.0 0.23 0.90 0.18 1.7 53

Foliar nutrition Control 46 3.68 9.18 2.44 66.4 675 Water spray 48 3.79 9.62 2.54 67.7 798 Urea spray (2% solution) 54 4.29 11.25 2.87 69.5 963 DAP spray (2% solution) 51 4.11 10.84 2.69 68.7 912 KCl spray (2% solution) 51 3.81 9.95 2.63 68.1 878 LSD (P<0.05) 3.4 0.26 1.01 0.20 1.9 58.7 C.V.(%) 8.2 8.1 12.1 9.33 3.5 8.4

Source: Annual Report (8)

Table 8. Effect of sowing time and seed priming on yield and other characters of lentil sown under rice-utera conditions during 2003-04 (lentil variety WBL-58 and rice variety MTU-7029).

Treatment Plant Mature Pods/plant 1000 seed Seed yield population plant height weight (g) (kg/ha) (‘000/ha) (cm)

Sowing time 14 Nov 2003 842 36 64 18.3 1162 21 Nov 2003 940 38 77 19.7 1232 LSD (P<0.05) 85 NS 4.5 NS 41

Seed priming No soaking 773 34 62 17.7 1018 Soaking in water for 6 h 855 35 70 18.5 1174 Soaking in 2% KH2PO4 solution for 6 h 928 36 74 19.0 1266 Sowing of sprouted seeds 1007 42 77 20.7 1330 LSD (P<0.05) 120 3.2 6.4 NS 58 LSD (P<0.05) for sowing time x priming NS NS NS NS NS interaction

C.V. (%) 10.9 7.0 7.3 10.4 3.89 Source: Annual Report (8)

31 Lathyrus Lathyrism Newsletter 4 (2005)

Table 9. Effect of weed management and seed rate on seed yield of lentil under rice-utera conditions.

Treatment Seed yield (kg/ha) 1991-92 1992-93 Mean

Weed management Control 599 741 670 Weed free (Hand weeding at 30, 45 and 815 831 823 60 DAS) One hand weeding (30 DAS) 730 824 777 One hand weeding (45 DAS) - 931 931 Two hand weedings (30 and 50 DAS) 746 918 832 Oxyfluorfen @ 50 g/ha as post- 704 927 816 emergence (30 DAS) LSD (P<0.05) 79 54 - CV (%) 10.2 6.0 -

Seed rate(kg/ha) 60 676 755 716 80 762 969 866 LSD (P<0.05) 57 27 - CV (%) 11.7 5.0 - LSD (P<0.05) for weeding x seed rate NS 65 - interaction

Source: Annual Report (1,2)

As no tillage operation is done for sowing pulses as a Table 8. The sowing of sprouted lentil seeds increased relay (paira) crop, it is difficult to apply fertilizer either the plant stand and yield quite significantly. This was through placement or through top dressing and also true in the case of lathyrus (Table 7). The consequently, no fertilization is made by the farmers to significantly increased yields are possibly due to the succeeding pulse crops (12). Therefore, the scope of increased germination and crop emergence. fertilization becomes confined to foliar spray or top dressing. With this view, an experiment was conducted An experiment was conducted to ascertain the effect of to study the effect of pre-sowing seed soaking and foliar weeding and also to determine the actual seed rate of nutrition on yield improvement in lathyrus. This was lentil. It was felt that seed rate should be higher when done in 2003-04 at PORS Sub-Station at Beldanga and either lentil or lathyrus were sown under utera the results are given in Table 7. conditions in comparison to being sown as a sole crop. The results of two consecutive year- trials (1991-92 and Sowing of sprouted seeds significantly increased yield. 1992-93) are shown in Table 9. Spraying of 2% urea solution at 10 days after rice harvest had the greatest beneficial effect and it was The higher seed rate (80 kg/ha) was effective in followed by 2% solution of di-ammonium phosphate. significantly increasing both the plant stand and yield. Thus, it is recommended that seed rate should be more The effect of seed soaking of lentil genotypes under than double of the normal rate (30-35 kg/ha ) when lentil rice-utera conditions was also tested in the same year at is sown under rice-utera conditions, presumably PORS Sub-Station, Beldanga. The results are given in indicating that lathyrus will respond similarly.

32 Lathyrus Lathyrism Newsletter 4 (2005)

Future strategies References

To achieve higher productivity of the pulses under utera 1. Annual Report (1991-92). Pulses and Oilseeds conditions, the following points should be considered: Research Station (PORS), Berhampore-742101, Govt. of West Bengal, India. 1. Generation and/or selection of efficient genotypes: 2. Annual Report (1992-93). PORS, Berhampore- Location specific high yielding genotypes with early 742101, Govt. of West Bengal, India. vigour, earliness, close canopy, synchronous maturity, 3. Annual Report (1994-95). PORS, Berhampore- resistance to key diseases and insect-pests and tolerance 742101, Govt. of West Bengal, India. to moisture stress need to be developed for various agro- 4. Annual Report (1999-2000). PORS, Berhampore- climatic zones of West Bengal. 742101, Govt. of West Bengal, India. 5. Annual Report (2000-01). PORS, Berhampore- 2. Development of an efficient fertilizer use schedule: 742101, Govt. of West Bengal, India. A. Identification and use of efficient strains of 6. Annual Report (2001-02). PORS, Berhampore- Rhizobium and phosphate solubilizing bacteria 742101, Govt. of West Bengal, India. (PSB) for seed inoculation 7. Annual Report (2002-03). PORS, Berhampore- B. Developing an integrated nutrient management 742101, Govt. of West Bengal, India. schedule for the system as a whole. 8. Annual Report (2003-04). PORS, Berhampore- C. Exploring the possibilities of foliar nutrition. 742101, Govt. of West Bengal, India. D. Maintaining proper plant population: 9. Anonymous (2000). Status of Agriculture 2000. E. Using quality seeds of small-seeded varieties. Dept. of Agriculture, Govt. of West Bengal, India. F. Timely sowing to exploit the residual soil moisture. 10. Anonymous (2002-03). Area, Production and G. Finding the optimum seed rate. Productivity of Some Principal Crops in West H. Seed treatment with effective fungicide to combat Bengal (2002-03). Socio-Economic and seedling mortality and seed borne diseases. Development Branch, Dept. of Agriculture, Govt. I. Following specific technique for pre-sowing seed of West Bengal, India. soaking (seed priming) to enhance drought 11. Anonymous (2003-04). Area, Production and tolerance and seedling vigour. Productivity of Some Principal Crops in West Bengal (2003-04). Socio-Economic and 4. Weed management: Development Branch, Dept. of Agriculture, Govt. A. Choosing genotypes with higher weed suppression of West Bengal, India. ability. 12. Bhowmick MK, Aich A, Aich SS, Shrivastava MP, B. Higher seed rate to increase smothering effect. Gupta S, Man GC. 2005. Crop diversification C. Identification of post-emergence herbicides and through paira (utera) cropping with rabi pulses. efficient application techniques. SATSA Mukhapatra-Annual Technical Issue 9, 43- D. Utilizing residual efficacy of herbicides by applying 60. the same in preceding crops. 13. Chakraborty LN, Sen SN, Mandal SK, Sengupta K E. Timely intercultural operation. and Mukherjee D. 1973. Possibility of utilizing rice fallow in West Bengal. Proc. Sem. on possibility of Epilogue growing a second crop after rice in West Bengal (Das Gupta DK, ed.), Sept. 15, 1973, Calcutta: 71- The above observations indicate some trend in 76. maximizing the productivity of lathyrus and lentil under 14. Duary B, Hazra D, Ghosh AK. 2004. Response of utera conditions in rice. Similar progress can be made lathyrus (Lathyrus sativus L.) to different dates of for other utera crops grown in rotation with wheat, such sowing and fertilizer application under paira as green gram (Vigna radiata) and black gram (Vigna cropping in rice. Indian Agriculturist 48, 157-159. mungo). Further experiments on utera cropping need to be conducted in order to make recommendations to the farmers. However, such research is being continued on this unique system of growing pulses under residual moisture and fertility, paving the way towards increasing cropping intensity in mono-cropped areas. This will practically maintain soil health, productivity and production of high protein legume crops in developing countries, where many people currently suffer from malnutrition. In conclusion, paira (utera) cropping with winter pulses in low land rice-fallows holds great promise, which would not only step up the pulse production but also help in protecting the environment from the risks of high input agriculture.

33 Lathyrus Lathyrism Newsletter 4 (2005)

The same goal, a different approach: a new Belgian-Ethiopian project

Fernand Lambein1* and Seid Ahmed 2

1. Ghent University, Institute for Plant Biotechnology for Developing Countries (IPBO), Belgium http://www.ipbo.ugent.be 2. Ethiopian Institute for Agricultural Research (EIAR) http://www.eiar.gov.et

*Email: [email protected]

Ever since the discovery of the structure and the Improving the commercial value of grass pea can neuro-excitatory activity of β-ODAP (β-N-oxalyl-L- therefore solve one of the problems underlying the α,β-diaminopropionic acid) in grass pea seeds, occurrence of lathyrism. Understanding the research has focused on the reduction/removal of this physiology of drought and salt tolerance and the role secondary metabolite from the plant on the one hand, of β-ODAP can also be important for agronomic and the better understanding of its physiological planning for a better quality product from the best activity in the brain on the other hand. Forty years suitable soil. Ignorance is a socio-political issue that is later, the plant still produces the toxic metabolite out of the reach of science. However, when we find a albeit in a lesser amount, while the understanding of statistically significant link between the incidence of the brain's physiology and the action of β-ODAP on neurolathyrism and illiteracy, we may find out what motoneurons has made great strides. But still no information or habit is protecting literate people from prevention has reached the people at risk, nor has a developing this irreversibly crippling neurolathyrism4. cure reached the victims of neurolathyrism. Only a What condiments are consumed together with grass few years ago an epidemic of neurolathyrism did pea that may protect the consumer from occur in Ethiopia1. Has something been overlooked? neurolathyrism, and what nutrients are present in those Or is there another road? protective condiments?

In many respectable publications, the nutritional An alternative road to improve grass pea and prevent quality of grass pea is praised as being rich in high neurolathyrism may then be to develop varieties of quality protein, a statement that is being repeated grass pea that are richer in those nutrients, together again and again. When calculating the amino acid with the reduction of antinutritional factors such as β- score of grass pea seed, the low score is stunning: only ODAP. A project funded by the Flemish Inter- 20 % of the WHO/FAO proposed standard in which university Council (VLIR) will explore this alternative all essential amino acids are present in well-balanced road. This project: "Improving the Nutritional Quality optimal ratios. This is the lowest amino acid score of of Grass Pea (Lathyrus sativus)" is a collaborative all commercial legumes. This means that even β- effort between the Ethiopian Institute for Agricultural ODAP-free grass pea is not a healthy staple food and Research (EIAR) (formerly the Ethiopian Agricultural needs to be mixed with ingredients richer in those Research Organization, EARO) and Ghent University amino acids that are low in grass pea protein. This has in Belgium. It will be carried out by the Crops been explained before and documented with historical Research Department of EIAR and the Institute for accounts2. The ancient Aztecs of Central America Plant Biotechnology for Developing Countries (IPBO) were smart enough to mix cereals and beans, which in Ghent. The project started on May 1st, 2004 and together form a much better complement of essential will run for four years. amino acids. Essential amino acids that are present at insufficient level in grass pea and are the limiting factor for the low amino acid score are the sulphur containing amino acids cysteine and methionine, Focal points of the project are: needed for our protection against oxidative stress. -Training Ethiopian researchers in plant Oxidative stress is involved in neuronal cell death that biotechnology. occurs in the upper motoneurons in neurolathyrism3. -Dissemination of information to the populations at risk, concerning the prevention of neurolathyrism.

-Selection of both mutants and somaclones for low β- Lathyrism has often been described as a disease of ODAP and improved amino acid composition. poverty, ignorance and drought-triggered famine.

34 Lathyrus Lathyrism Newsletter 4 (2005)

-Study stability of the low-β-ODAP trait under abiotic A mid-term evaluation will be done by an external stress. expert. The project also calls for an open scientific -Examining the potential for applying genetic conference to be organized near the end of the project transformations to grass pea. in April 2008. -Studies on the effect of essential amino acids as food/feed supplements on the nutritional quality of grass pea seed.

References 1. Getahun H, Mekonnen A, Teklehaimanot R, 3. Rao AV, Balachandran B. 2002. Role of oxidative Lambein F. 1999. Epidemic of neurolathyrism in stress and antioxidants in neurodegenerative Ethiopia. The Lancet 354, 306-307. diseases. Nutritional Neurosciences. 5, 291-309. 2. Lambein F, Diasolua Ngudi D, Kuo YH. 2001. 4. Getahun H, Lambein F, Vanhoorne M, Van der Vapniarca revisited: Lessons from an inhuman Stuyft P. 2002. Pattern and associated factors of human experience. Lathyrus Lathyrism the neurolathyrism epidemic in Ethiopia. Tropical Newsletter 2, 5-7. Medicine and International Health 7, 118-124.

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