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Variation in resistance of some forage legumes species to Orobanche crenata Forsk.
Field Crops Research, 32 ( 1993 ) 277-285 Elsevier Science Publishers B.V., Amsterdam
277
Variation in resistance of some forage legumes species to Orobanche crenata Forsk. K.-H. Linkea'b, A.M. A b d E1-Moneim b a n d M.C. S a x e n a b University of Hohenheim, Institute of Plant Production in the Tropics and Subtropics, Stuttgart, Germany blnternational Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria (Accepted 12 March 1992 )
ABSTRACT Linke, K.-H., Abd EI-Moneim, A.M. and Saxena, M.C., 1993. Variation in resistance of some forage legumes species to Orobanche crenata Forsk. Field Crops Res., 32: 277-285. Parasitic weeds of the family Orobanchaceae threaten many forage legumes in the Mediterranean region. Laboratory and field screening of five lines each of six forage legumes ( Vicia narbonensis L., 1~ sativa L., ~: villosa ssp. dasycarpa (Ten.) Cavill., Lathyrus sativus L., L. ochrus (L.) D.C., and L. cicera L.) for resistance to the parasitic weed Orobanche crenata Forsk. revealed high interspecific and intraspecific variation. Lathyrus ochrus lines were free of emerged O. crenata shoots while those of L. sativus and L. cicera were highly susceptible to the parasite. Intraspecific variation in reaction to O. crenata was high within V. narbonensis and V. sativa, with several lines being free of Orobanche parasitism whereas others were highly susceptible. Lines with high resistance to Orobanche can reduce the build-up of a seed bank of the parasite in areas where mainly food legumes are grown and can be of value for developing integrated Orobanche control systems. The laboratory screening, which is faster and cheaper, reliably predicted the field performance and hence retesting in the field can be restricted to those lines found resistant in the laboratory.
INTRODUCTION
Cool-season food and forage legumes are an important component of the crop rotation with cereals in the rainfed farming systems of West Asia and North Africa (WANA). With the increasing demand for livestock in this region, replacement of weedy fallow by fodder crops is one alternative available to farmers. Orobanche crenata is a major root-parasite of many cool-season legume crops in the WANA region. This parasite causes economic losses in such food legumes as faba bean ( Viciafaba L. ), lentil (Lens culinaris Med. ) and field peas (Pisum sativum L. ) and also forage legumes such as Vicia natCorrespondence to: M.C. Saxena, International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria.
0378-4290/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.
278
K.-H LINKE ET AL.
bonensis, V. sativa, V. melanopis, V. ervilia, Lathyrus sativus, L. odoratus, L. cicera, L. inconspicuus and L. angulatus (Winstel, 1972; Abu-Raya et al., 1973; Abu-Irmaileh, 1982; Parker, 1986). At the International Center for Agricultural Research in the Dry Areas (ICARDA), forage legumes are being developed to replace fallow in zones where many food legumes can not thrive, and their low susceptibility to the parasite may reduce the production of new Orobanche shoots and thus decrease the build-up of the seed bank of Orobanche in legume fields. There has been little research on host-plant resistance of forage legumes to O. crenata. Gil et al. ( 1982, 1984) reported that 1I..sativa was not very seriously infested by O. crenata in Spain, and it was possible to identify resistant lines. Work at ICARDA showed large interspecific difference in the susceptibility in the field: V. narbonensis supported 220 O. crenata shoots m -2 as against only two shoots m -2 present in 1I..villosa ssp. dasycarpa (ICARDA, 1989 ). The purpose of this study was to identify lines resistant to O. crenata in six forage legume species, and to compare the screening done in the field with that in the laboratory, to identify a reliable method for screening a large number of breeding lines for resistance to the parasite. MATERIALS AND METHODS
Based on their productivity in advanced yield trials, five pure-lines (accessions/selections) were chosen (Table 1 ) from each of three Vicia spp. and three Lathyrus spp. from ICARDA's breeding material.
Laboratory experiments Laboratory screening was done in Petri dishes (9 cm diameter) under controlled environmental conditions (25 ° C day/20 ° C night; 12-h light period) using the procedure developed earlier for lentil by Sauerborn et al. ( 1987 ). Briefly, the procedure consists of growing forage legume seedlings in plastic TABLE l Pure-lines (accessions/selections) of Vicia and Lathyrus species used in the study Species
Accession / selection a
V. narbonensis V. sativa V. villosa ssp. dasycarpa L. ochrus L. sativus L. cicera
67,577-2391,578-2392,574-2388,573-2378 708-2037,715,1361-1448,1416,2541 569-2404,683,801-2409,956-2410,958-2411 84-539,91-538,94-539,95-540,101-185 188-38,201-455,347,170-439,205-495 111-487,15-486,120-490,143-497,145-498
aSingle number represents a germplasm accession; a second number after hyphen represents a selection within the accession. Numbering of accessions/selections is according to the ICARDA Germplasm Resource Unit designations.
VARIATION IN RESISTANCE OF FORAGE LEGUMES TO OROBANCHE
279
Petri dishes filled with moist quartz sand, topped with a microfibre filter paper carrying 1000 pre-conditioned seeds of Orobanche. The preconditioning of Orobanche seeds is done separately by incubating them in dark for 7 days at 20°C on the same moist microfibre filter paper (9 cm diameter) which is then used in the Petri dishes. The dishes are arranged vertically in a trough, with an opening on the top edge to hold a 5-day-old legume seedling such that the roots grow in between the surface of the filter paper carrying the Orobanche seeds and the cover of the Petri dish. Another opening in the bottom edge carries a polyamide wick for irrigation. This ensures normal growth of the seedling and permits easy examination of attachments of Orobanche to the roots of the host plant without removing the cover of the Petri dish. Six replications were used per entry. Petri dishes were distributed randomly in the incubator. Four weeks after the transplanting of the test seedlings, the number of parasite attachments to the roots were counted and the root length of the test legume per Petri dish determined according to Tennant ( 1975 ). From these data the total number of Orobanche attachments per 100 cm host root length was calculated to make the results comparable among the entries. The experiment was repeated after several weeks and the results of both sets are presented.
Field experiments The test lines were screened in 1988/89 and 1989/90 in a field at the ICARDA research station at Tel Hadya (36°N, 37 ° E, 284 m a.s.1. ) in northern Syria. The Orobanche infestation at this particular site was heavy; the field had been artificially infested eight years ago, and hence the natural infestation developed in the meantime was uniform. A randomized block design with six replications was used. Seed rate was 100 kg h a - 1 and plot size was 0.75 X 2 m (three rows per plot). Sowing dates were 23 November 1988 and 5 December 1989 for the two years. Hand weeding was performed twice to remove all weeds other than Orobanche. The intensity of parasite infestation was evaluated at crop maturity by counting the number of O. crenata shoots emerged per plot and by determining their dry weight. The crop-matter production was also assessed. Results of both laboratory and field experiments were subjected to analysis of variance and tested for the least significant differences (L.S.D.), using a probability level of 0.05 to compare accession means. Correlations between the two sets of laboratory data and two seasons of field data were computed, as was the correlation between field and laboratory results. RESULTS
Laboratory experiments The results of the two screenings (A and B, Table 2 ) were highly correlated (r=0.809, P<0.001 ). Among the six forage legume species, L. ochrus dis-
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TABLE 2 Mean number (each of two experiments) of O. crenata attachments per 100 cm root length in the laboratory, and mean number of O. crenata shoots m -2 in the field, for five accessions within six forage legume species Species
Laboratory A
~Tcia narbonensis 578-2392 0.0 577-2391 6.7 574-2388 3.5 573-2378 12.0 67 11.5 Mean 6.7 L.S.D. 9.6 Vicia sativa 2541 0.0 715 3.5 708-2037 0.0 1361-1448 0.0 1416 6.8 Mean 2. I L.S.D 6.1 Vicia villosa ssp. dasycarpa 956-2401 1.9 958-2411 0.0 569-2404 0.0 683 0.0 801-2409 1.3 Mean 0.6 L.S.D 2.7 Lathyrus ochrus 84-539 0.0 91-538 0.0 94-539 0.0 95-540 0.0 101-185 0.0 Mean 0.0 L.S.D Lathyrus sativus 201-455 14.0 188-38 19.6 205-495 14.3 347 40.1 170-439 18.6 Mean 21.3 L.S.D. 11.8
Field B
Mean
1989
1990
Mean
0.0 24.6 14.9 19.7 18.0 15.4 8.7
0.0 15.6 9.2 15.8 14.7 11.1 5.7
5.9 5.6 8.1 8.5 90.1 23.7 15.9
2.4 2.9 4.8 6.0 10.7 5.3 6.7
4.2 4.3 6.5 7.2 50.4 14.5 11.4
0.0 13.9 0.0 0.0 14.6 5.7 7.3
0.0 8.7 0.0 0.0 10.7 3.9 4.4
0.0 0.0 0.1 1.3 14.3 3.1 3.0
0.0 0.0 0.3 0.2 0.2 0.1 0.3
0.0 0.0 0.2 0.8 7.3 1.6 2.3
3.2 0.0 0.9 1.0 1.8 1.3 1.9
2.5 0.0 0.4 0.5 1.5 1.0 1.6
0.0 0.2 0.3 0.1 0.3 0.2 0.4
0.0 0.0 0.0 0.0 0.0 0.0 -
0.0 0.1 0.1 0.1 0.1 0.1 0.1
0.0 0.0 0.0 0.7 0.0 0.1 0.6
0.0 0.0 0.0 0.3 0.0 0.1 0.2
0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 -
0.0 0.0 0.0 0.0 0.0 0.0 -
25.4 17.8 17.6 26.4 22.9 22.1 16.5
19.7 18.7 16.0 33.2 20.8 21.7 9.8
69.7 80.2 87.4 98.8 97.8 86.7 26.9
1.1 3.3 1.3 6.2 7.8 3.0 4.0
35.4 41.7 44.4 52.5 52.6 44.8 10.0
VARIATION IN RESISTANCE OF FORAGE LEGUMES TO
281
OROBANCHE
TABLE 2 Species
Lathyrus cicera 111-487 15-486 120-490 143-497 145-498 Mean L.S.D Grand mean L.S.D.
Laboratory
Field
A
B
Mean
1989
1990
Mean
11.8 29.2 16.2 16.8 13.0 17.4 11.7
16.5 19.4 15.5 14.7 18.4 16.9 9.0
14.2 24.3 15.9 15.8 15.8 17.2 6.9
68.4 87.8 96.9 62.4 68.1 76.7 20.6
4.4 4.0 5.8 4.0 3.6 4.4 3.9
36.2 45.9 51.4 33.2 35.8 40.5 8.8
8.0 7.4
10.3 7.9
9.2 4.0
31.7 17.9
2.1 3.2
16.9 6.7
played the highest resistance to O. crenata: only one of its lines ('95-540') had Orobanche attachments in two out of four replications in the second screening. Two other species, V. villosa ssp. dasycarpa and K sativa also had low infestation, with the former being not significantly different from L. ochrus. The highest infestation occurred in L. sativus followed by L. cicera and V. narbonensis. Lathyrus sativus and L. cicera can be grouped as susceptible species, and V. villosa ssp. dasycarpa and L. ochrus as resistant ones. In contrast to the large interspecific differences, the intraspecific differences were low in V. villosa ssp. dasycarpa, L. ochrus, L. sativus and L. cicera; the largest differences were observed within V. sativa and V. narbonensis; lines '708-2037', '1361-1448' and '2541' of V. sativa and '578-2392' of V. narbonensis were free of Orobanche attachments and were therefore significantly better than the other accessions within the respective species, which showed high level of Orobanche infestation.
Field experiments The growth and development of Orobanche, like that of the host plant, is affected by the environmental conditions (ter Borg, 1986). Hence, the data on temperature and rainfall were collected and are shown in Fig. 1. The mean March and April temperatures were lower by 2 and 4.8 °C, respectively, in 1990 than in 1989. This should have reduced the germination of Orobanche seeds (ter Borg, 1986 ) in 1990 and may explain why the parasite infestation was lower in 1989/90 than in the 1988/89 season. In both years the total seasonal rainfall was low, with 234 and 233 m m in 1988/89 and 1989/90, respectively, as against a long-term average of 329 mm. However, the 1987/ 88 season was extremely wet (524 m m ) and hence the 1988/89 season started with a higher stored soil moisture content than 1989/90. In addition, the
282
K.-H LINKEETAL. T e m p e r a t u r e (°C)
30
20
P r e c i p i t a t i o n (mm)
!
/
/
fJ
80
//
JJ
jz
J
60
40
[]
10
!iiiii
20
:::::::
Oct
NOV
Jan
Dec
:K
max. temp. 8 8 / 8 9
[]
min. temp. 8 9 / 9 0
[] I
Feb
0 Mar
max. temp. 8 9 / 9 0 I precipit. 8 8 / 8 9
~ ~
Apr
May
min. temp. 8 8 / 8 9 precipit. 8 9 / 9 0
Fig. 1. Monthly average temperature and total precipitation during the 1988/89 and 1989/90 growing seasons at Tel Hadya, Syria.
rainfall in 1989/90 was less than that in 1988/89 in the first three months (Oct., Nov. and Dec. ) and it was only in the subsequent two months (Jan. and Feb. ) that the cumulative total for 1989/90 reached the value of 1988/ 89. This pattern of rainfall distribution in the 1989/90 season accentuated the soil moisture stress for the host plants and should have therefore further affected the development of Orobanche adversely (ter Borg, 1986 ). Frost occurred 52 and 55 times during the growing season in 1988/89 and 1989/90, respectively, damaging the forage legumes, especially in 1989/90 due to its late occurrence. However, the damage was more persistent in the case of L. ochrus than with other species because of its high susceptibility to frost. The infestation of Orobanche in the field was uniform, as revealed by the low standard deviation for replications for the number of Orobanche shoots m-2 in the susceptible species, L. sativus. Because of the special weather conditions described above, the number of Orobanche shoots was considerably lower in 1989/90 than in 1988/89 (Table 2). The relative reaction of any given accession to Orobanche infestation, however, was similar for the two seasons as revealed by a high correlation coefficient (r= 0.674, P < 0.001 ). In conformity with the results of the laboratory study, the field study showed
VARIATION IN RESISTANCE OF FORAGE LEGUMES TO OROBANCHE
283
that L. ochrus was free of Orobanche infestation and L. sativus had the highest level of infestation followed by L. cicera. Vicia villosa spp. dasycarpa was almost free of Orobanche infestation whereas V. narbonensis showed medium and V. sativa low susceptibility. The highest intraspecific variation was in V. narbonensis; its line '67' averaged over two seasons, had seven times as many Orobanche shoots as its second-most susceptible line ('573-2378'). Intraspecific variation in resistance was also obvious in V. sativa, with line' 1416' showing a susceptible reaction, whereas two other lines ('715' and '2541') had no Orbanche shoots, and the remaining two lines displayed only low infestation. Visual observation indicated that Orobanche shoots in V. narbonensis were bigger in size at low infestation than at high infestation. There was, however, a high correlation between the number and dry weight of Orobanche shoots ( r = 0.983, P < 0.001 ) across all the tested lines. The dry-matter production of the forage legume lines tested decreased with increasing Orobanche with a correlation coefficient of - 0.849 ( P < 0.01 ). DISCUSSION
Interspecific variation in resistance to Orobanche as observed with the six forage species tested has also been observed in other crops (Vogt, 1987). However, the research on intraspecific variation has provided limited results because the variation within a species usually is lower than between species. In the past, resistant lines were identified in crops such as faba bean, sunflower (Helianthus annuus L. ) and tomato (Lycopersicon lycopersicum [L. ] Karst ex Farw.) (Cubero, 1986), as well as in V. sativa (Gil et al., 1982). This study has demonstrated that substantial intraspecific variation exists in V. narbonensis and V. sativa in their reaction to O. crenata. The availability of Orobanche-resistant selections of different forage legume species offers options to a farmer wanting to grow forage legumes on a heavily infested field to choose the species most adapted to the given environmental conditions. For example, in an area where frost does not occur, lines ofL. ochrus could be used on Orobanche-infested fields to stop an increase in the seed bank. Growing L. sativus or L. cicera or susceptible lines of V. narbonensis such as line '67' should be avoided. Reduction of the seed bank could be accomplished by such methods as hand weeding, application of preemergence (imazapyr) or postemergence (imazaquin and glyphosate) herbicides and cultivation of non-host crops such as flax (Linum usitatissimum) (Linke and Saxena, 1991 ). Hand weeding is generally expensive and should only be used when there are a few Orobanche plants in the field to prevent seed formation. Growing any of the tested lines of L. ochrus, V. villosa ssp. dasycarpa or lines of V. sativa such as '715' and
284
K.-H LINKE ET AL
'2541' should ensure no parasite shoot emergence. Line '715' of V. sativa, which showed high resistance to O. crenata in this study has been found to be a high- and stable-yielding line across diverse environments in west Asia (Abd et-Moneim et al., 1988). Those lines of V. narbonensis and V. sativa which have intermediate level of resistance to Orobanche, but high yield potential, can be used on parasiteinfested fields, provided they are grown in an integrated Orobanche control system based on manipulation of the date of sowing and/or use of low doses of herbicides. Delaying the sowing on infested fields by 3 to 4 weeks from the date found optimum under parasite-free condition reduces Orobanche attack (Linke and Saxena, 1991 ), probably because of reduced germination of Orobanche seeds (ter Borg, 1986). Under such conditions applications of reduced rates of herbicides ( 15-20 g a.i./ha imazapyr, preemergence; or 2 × 15 g a.i./ha imazaquin or 2 × 40 g a.i./ha of glyphosate as postemergence) can become effective in further reducing the parasite development (Linke and Saxena, 1991 ). None of the L. ochrus lines tested in this study permitted emergence of Orobanche shoots in the field, but they caused stimulation and germination of parasite seeds and in some cases even allowed development of parasitic attachments on roots in the laboratory screening. This was also the case with resistant lines of V. sativa and V. villosa ssp. dasycarpa. These lines thus permit early growth of the parasite in the field but further development is inhibited. Hence, these lines behave like trap crops as has been observed with tomato, kidney beans and flax (Abu-Irmaileh, 1982), resulting in a decrease in the seed bank of Orobanche. In fact, a 30% decrease in the seed bank of Orobanche was demonstrated by Linke (1989) by growing V. villosa ssp. dasycarpa. The inter- and intraspecific differences in susceptibility to Orobanche in the laboratory conformed with those obtained in the field experiments ( r = 0.860, P < 0.001 ). The laboratory screening, which is faster and cheaper, thus reliably predicted the field performance and hence retesting in the field can be restricted to those lines found resistent in the laboratory. ACKNOWLEDGEMENTS This research was partly funded by the Deutsche Gesellschaft ftir Technische Zusammenarbeit (GTZ), Federal Republic of Germany. The technical assistance of Mr. Hasan Masri is greatly appreciated. REFERENCES Abd el-Moneim,A.M.,Coks,P.S. and Swedan,Y., 1988.Yieldstabilityof selectedforagevetches ( Vicia spp.) under rainfed conditions in West Asia. J. Agric. Sci. (Cambridge), 111: 295301.
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Abu-Irmaileh, B.E., 1982. Crop rotation for the control ofbroomrape (Orobanche ramosa L. ). Dirasat, Agricultural Studies, 9:121-126. Abu-Raya, M.A., Radi, A.F. and Darwish Heikal, M.M., 1973. Host parasite relationship of Orobanche species. In: Proceedings of the European Weed Research Council, Symposium on Parasitic Weeds. Malta University Press, Malta, pp. 167-176. Cubero, J.I., 1986. Breeding for resistance to Orobanche and Striga: a review. In: S.J. ter Borg (Editor), Proceedings of a Workshop on Biology and Control ofOrobanche. LH/VPO, Wageningen, Netherlands, pp. 127-139. Gil, J., Martin, L.M. and Cubero, J.I., 1982. Resistencia al jopo (Orobanche crenata Forsk. ) en Vicia satlva L. Anales IN IA, Serie Agricola, 21:175-181. Gil, J., Marlin, L.M. and Cubero, J.I., 1984. Resistance to Orobanche crenata Forsk. in ITcia sativa L. II. Characterization and genetics. In: C. Parker, L.J. Musselman, R.M. Polhill and A.K. Wilson (Editors), Proceedings of the Third International Symposium on Parasitic Weeds, ICARDA/1PSPRG, 7-9 May 1984. ICARDA, Aleppo, Syria, pp. 221-229. ICARDA (International Center for Agricultural Research in the Dry Areas), 1989. 1988 Annual Report, Food Legume Improvement Program. Aleppo, Syria, pp. 214-241. Linke, K.-H., 1989. Sechster Arbeitsbericht zum Orobanche Projekt, August 1989. ICARDA, Food Legume Improvement Program, Aleppo, Syria, 33 pp. (unpubl.). kinke, K.-H. and Saxena, M.C., 1991. Towards an integrated control of Orobanche spp. in some legume crops. In: K. Wegmann and L.J. Musselman (Editors), Progress in Orobanche Research. Eberhard-Karls-Universit~it, TiJbingen, FRG, pp. 248-256. Parker, C., 1986. Scope of agronomic problems caused by Orobanche species. In: S.J. ter Borg (Editor), Proceedings of a Workshop on Biology and Control of Orobanche. LH/VPO, Wageningen, Netherlands, pp. 11-17. Sauerborn, J., Masri, H,, Saxena, M.C. and Erskine, W., 1987. A rapid test to screen lentil under laboratory conditions for susceptibility to Orobanche. LENS Newsletter, 14:15-16. Tennant, D., 1975. A test of a modified line intersect method of estimating root length. J. Ecol., 63: 955-1001. ter Borg, S.J., 1986. Effects of environmental factors on Orobanche host relationships; a review and some recent results. In: S.J. ter Borg (Editor), Proceedings of a Workshop on Biology and Control of Orobanche. LH/VPO, Wageningen, Netherlands, pp. 57-69. Vogt, W., 1987. Untersuchungen zum EinfluB verschiedener Pflanzen auf Keimung und Befall bei Orobanche crenata Forsk., Orobanche ramosa L. und Strlga hermonthica (Del.) Benth. M.Sc. Thesis, University of Hohenheim, Germany, 126 lap. (unpubl.). Winstel, K., 1972. Monographie der Gattung Orobanche. M.Sc. Thesis, University of Hohenheim, Germany, 115 pp. (unpubl.).
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Variation in resistance of some forage legumes species to Orobanche crenata Forsk. K.-H. Linkea'b, A.M. A b d E1-Moneim b a n d M.C. S a x e n a b University of Hohenheim, Institute of Plant Production in the Tropics and Subtropics, Stuttgart, Germany blnternational Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria (Accepted 12 March 1992 )
ABSTRACT Linke, K.-H., Abd EI-Moneim, A.M. and Saxena, M.C., 1993. Variation in resistance of some forage legumes species to Orobanche crenata Forsk. Field Crops Res., 32: 277-285. Parasitic weeds of the family Orobanchaceae threaten many forage legumes in the Mediterranean region. Laboratory and field screening of five lines each of six forage legumes ( Vicia narbonensis L., 1~ sativa L., ~: villosa ssp. dasycarpa (Ten.) Cavill., Lathyrus sativus L., L. ochrus (L.) D.C., and L. cicera L.) for resistance to the parasitic weed Orobanche crenata Forsk. revealed high interspecific and intraspecific variation. Lathyrus ochrus lines were free of emerged O. crenata shoots while those of L. sativus and L. cicera were highly susceptible to the parasite. Intraspecific variation in reaction to O. crenata was high within V. narbonensis and V. sativa, with several lines being free of Orobanche parasitism whereas others were highly susceptible. Lines with high resistance to Orobanche can reduce the build-up of a seed bank of the parasite in areas where mainly food legumes are grown and can be of value for developing integrated Orobanche control systems. The laboratory screening, which is faster and cheaper, reliably predicted the field performance and hence retesting in the field can be restricted to those lines found resistant in the laboratory.
INTRODUCTION
Cool-season food and forage legumes are an important component of the crop rotation with cereals in the rainfed farming systems of West Asia and North Africa (WANA). With the increasing demand for livestock in this region, replacement of weedy fallow by fodder crops is one alternative available to farmers. Orobanche crenata is a major root-parasite of many cool-season legume crops in the WANA region. This parasite causes economic losses in such food legumes as faba bean ( Viciafaba L. ), lentil (Lens culinaris Med. ) and field peas (Pisum sativum L. ) and also forage legumes such as Vicia natCorrespondence to: M.C. Saxena, International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria.
0378-4290/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.
278
K.-H LINKE ET AL.
bonensis, V. sativa, V. melanopis, V. ervilia, Lathyrus sativus, L. odoratus, L. cicera, L. inconspicuus and L. angulatus (Winstel, 1972; Abu-Raya et al., 1973; Abu-Irmaileh, 1982; Parker, 1986). At the International Center for Agricultural Research in the Dry Areas (ICARDA), forage legumes are being developed to replace fallow in zones where many food legumes can not thrive, and their low susceptibility to the parasite may reduce the production of new Orobanche shoots and thus decrease the build-up of the seed bank of Orobanche in legume fields. There has been little research on host-plant resistance of forage legumes to O. crenata. Gil et al. ( 1982, 1984) reported that 1I..sativa was not very seriously infested by O. crenata in Spain, and it was possible to identify resistant lines. Work at ICARDA showed large interspecific difference in the susceptibility in the field: V. narbonensis supported 220 O. crenata shoots m -2 as against only two shoots m -2 present in 1I..villosa ssp. dasycarpa (ICARDA, 1989 ). The purpose of this study was to identify lines resistant to O. crenata in six forage legume species, and to compare the screening done in the field with that in the laboratory, to identify a reliable method for screening a large number of breeding lines for resistance to the parasite. MATERIALS AND METHODS
Based on their productivity in advanced yield trials, five pure-lines (accessions/selections) were chosen (Table 1 ) from each of three Vicia spp. and three Lathyrus spp. from ICARDA's breeding material.
Laboratory experiments Laboratory screening was done in Petri dishes (9 cm diameter) under controlled environmental conditions (25 ° C day/20 ° C night; 12-h light period) using the procedure developed earlier for lentil by Sauerborn et al. ( 1987 ). Briefly, the procedure consists of growing forage legume seedlings in plastic TABLE l Pure-lines (accessions/selections) of Vicia and Lathyrus species used in the study Species
Accession / selection a
V. narbonensis V. sativa V. villosa ssp. dasycarpa L. ochrus L. sativus L. cicera
67,577-2391,578-2392,574-2388,573-2378 708-2037,715,1361-1448,1416,2541 569-2404,683,801-2409,956-2410,958-2411 84-539,91-538,94-539,95-540,101-185 188-38,201-455,347,170-439,205-495 111-487,15-486,120-490,143-497,145-498
aSingle number represents a germplasm accession; a second number after hyphen represents a selection within the accession. Numbering of accessions/selections is according to the ICARDA Germplasm Resource Unit designations.
VARIATION IN RESISTANCE OF FORAGE LEGUMES TO OROBANCHE
279
Petri dishes filled with moist quartz sand, topped with a microfibre filter paper carrying 1000 pre-conditioned seeds of Orobanche. The preconditioning of Orobanche seeds is done separately by incubating them in dark for 7 days at 20°C on the same moist microfibre filter paper (9 cm diameter) which is then used in the Petri dishes. The dishes are arranged vertically in a trough, with an opening on the top edge to hold a 5-day-old legume seedling such that the roots grow in between the surface of the filter paper carrying the Orobanche seeds and the cover of the Petri dish. Another opening in the bottom edge carries a polyamide wick for irrigation. This ensures normal growth of the seedling and permits easy examination of attachments of Orobanche to the roots of the host plant without removing the cover of the Petri dish. Six replications were used per entry. Petri dishes were distributed randomly in the incubator. Four weeks after the transplanting of the test seedlings, the number of parasite attachments to the roots were counted and the root length of the test legume per Petri dish determined according to Tennant ( 1975 ). From these data the total number of Orobanche attachments per 100 cm host root length was calculated to make the results comparable among the entries. The experiment was repeated after several weeks and the results of both sets are presented.
Field experiments The test lines were screened in 1988/89 and 1989/90 in a field at the ICARDA research station at Tel Hadya (36°N, 37 ° E, 284 m a.s.1. ) in northern Syria. The Orobanche infestation at this particular site was heavy; the field had been artificially infested eight years ago, and hence the natural infestation developed in the meantime was uniform. A randomized block design with six replications was used. Seed rate was 100 kg h a - 1 and plot size was 0.75 X 2 m (three rows per plot). Sowing dates were 23 November 1988 and 5 December 1989 for the two years. Hand weeding was performed twice to remove all weeds other than Orobanche. The intensity of parasite infestation was evaluated at crop maturity by counting the number of O. crenata shoots emerged per plot and by determining their dry weight. The crop-matter production was also assessed. Results of both laboratory and field experiments were subjected to analysis of variance and tested for the least significant differences (L.S.D.), using a probability level of 0.05 to compare accession means. Correlations between the two sets of laboratory data and two seasons of field data were computed, as was the correlation between field and laboratory results. RESULTS
Laboratory experiments The results of the two screenings (A and B, Table 2 ) were highly correlated (r=0.809, P<0.001 ). Among the six forage legume species, L. ochrus dis-
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TABLE 2 Mean number (each of two experiments) of O. crenata attachments per 100 cm root length in the laboratory, and mean number of O. crenata shoots m -2 in the field, for five accessions within six forage legume species Species
Laboratory A
~Tcia narbonensis 578-2392 0.0 577-2391 6.7 574-2388 3.5 573-2378 12.0 67 11.5 Mean 6.7 L.S.D. 9.6 Vicia sativa 2541 0.0 715 3.5 708-2037 0.0 1361-1448 0.0 1416 6.8 Mean 2. I L.S.D 6.1 Vicia villosa ssp. dasycarpa 956-2401 1.9 958-2411 0.0 569-2404 0.0 683 0.0 801-2409 1.3 Mean 0.6 L.S.D 2.7 Lathyrus ochrus 84-539 0.0 91-538 0.0 94-539 0.0 95-540 0.0 101-185 0.0 Mean 0.0 L.S.D Lathyrus sativus 201-455 14.0 188-38 19.6 205-495 14.3 347 40.1 170-439 18.6 Mean 21.3 L.S.D. 11.8
Field B
Mean
1989
1990
Mean
0.0 24.6 14.9 19.7 18.0 15.4 8.7
0.0 15.6 9.2 15.8 14.7 11.1 5.7
5.9 5.6 8.1 8.5 90.1 23.7 15.9
2.4 2.9 4.8 6.0 10.7 5.3 6.7
4.2 4.3 6.5 7.2 50.4 14.5 11.4
0.0 13.9 0.0 0.0 14.6 5.7 7.3
0.0 8.7 0.0 0.0 10.7 3.9 4.4
0.0 0.0 0.1 1.3 14.3 3.1 3.0
0.0 0.0 0.3 0.2 0.2 0.1 0.3
0.0 0.0 0.2 0.8 7.3 1.6 2.3
3.2 0.0 0.9 1.0 1.8 1.3 1.9
2.5 0.0 0.4 0.5 1.5 1.0 1.6
0.0 0.2 0.3 0.1 0.3 0.2 0.4
0.0 0.0 0.0 0.0 0.0 0.0 -
0.0 0.1 0.1 0.1 0.1 0.1 0.1
0.0 0.0 0.0 0.7 0.0 0.1 0.6
0.0 0.0 0.0 0.3 0.0 0.1 0.2
0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 -
0.0 0.0 0.0 0.0 0.0 0.0 -
25.4 17.8 17.6 26.4 22.9 22.1 16.5
19.7 18.7 16.0 33.2 20.8 21.7 9.8
69.7 80.2 87.4 98.8 97.8 86.7 26.9
1.1 3.3 1.3 6.2 7.8 3.0 4.0
35.4 41.7 44.4 52.5 52.6 44.8 10.0
VARIATION IN RESISTANCE OF FORAGE LEGUMES TO
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OROBANCHE
TABLE 2 Species
Lathyrus cicera 111-487 15-486 120-490 143-497 145-498 Mean L.S.D Grand mean L.S.D.
Laboratory
Field
A
B
Mean
1989
1990
Mean
11.8 29.2 16.2 16.8 13.0 17.4 11.7
16.5 19.4 15.5 14.7 18.4 16.9 9.0
14.2 24.3 15.9 15.8 15.8 17.2 6.9
68.4 87.8 96.9 62.4 68.1 76.7 20.6
4.4 4.0 5.8 4.0 3.6 4.4 3.9
36.2 45.9 51.4 33.2 35.8 40.5 8.8
8.0 7.4
10.3 7.9
9.2 4.0
31.7 17.9
2.1 3.2
16.9 6.7
played the highest resistance to O. crenata: only one of its lines ('95-540') had Orobanche attachments in two out of four replications in the second screening. Two other species, V. villosa ssp. dasycarpa and K sativa also had low infestation, with the former being not significantly different from L. ochrus. The highest infestation occurred in L. sativus followed by L. cicera and V. narbonensis. Lathyrus sativus and L. cicera can be grouped as susceptible species, and V. villosa ssp. dasycarpa and L. ochrus as resistant ones. In contrast to the large interspecific differences, the intraspecific differences were low in V. villosa ssp. dasycarpa, L. ochrus, L. sativus and L. cicera; the largest differences were observed within V. sativa and V. narbonensis; lines '708-2037', '1361-1448' and '2541' of V. sativa and '578-2392' of V. narbonensis were free of Orobanche attachments and were therefore significantly better than the other accessions within the respective species, which showed high level of Orobanche infestation.
Field experiments The growth and development of Orobanche, like that of the host plant, is affected by the environmental conditions (ter Borg, 1986). Hence, the data on temperature and rainfall were collected and are shown in Fig. 1. The mean March and April temperatures were lower by 2 and 4.8 °C, respectively, in 1990 than in 1989. This should have reduced the germination of Orobanche seeds (ter Borg, 1986 ) in 1990 and may explain why the parasite infestation was lower in 1989/90 than in the 1988/89 season. In both years the total seasonal rainfall was low, with 234 and 233 m m in 1988/89 and 1989/90, respectively, as against a long-term average of 329 mm. However, the 1987/ 88 season was extremely wet (524 m m ) and hence the 1988/89 season started with a higher stored soil moisture content than 1989/90. In addition, the
282
K.-H LINKEETAL. T e m p e r a t u r e (°C)
30
20
P r e c i p i t a t i o n (mm)
!
/
/
fJ
80
//
JJ
jz
J
60
40
[]
10
!iiiii
20
:::::::
Oct
NOV
Jan
Dec
:K
max. temp. 8 8 / 8 9
[]
min. temp. 8 9 / 9 0
[] I
Feb
0 Mar
max. temp. 8 9 / 9 0 I precipit. 8 8 / 8 9
~ ~
Apr
May
min. temp. 8 8 / 8 9 precipit. 8 9 / 9 0
Fig. 1. Monthly average temperature and total precipitation during the 1988/89 and 1989/90 growing seasons at Tel Hadya, Syria.
rainfall in 1989/90 was less than that in 1988/89 in the first three months (Oct., Nov. and Dec. ) and it was only in the subsequent two months (Jan. and Feb. ) that the cumulative total for 1989/90 reached the value of 1988/ 89. This pattern of rainfall distribution in the 1989/90 season accentuated the soil moisture stress for the host plants and should have therefore further affected the development of Orobanche adversely (ter Borg, 1986 ). Frost occurred 52 and 55 times during the growing season in 1988/89 and 1989/90, respectively, damaging the forage legumes, especially in 1989/90 due to its late occurrence. However, the damage was more persistent in the case of L. ochrus than with other species because of its high susceptibility to frost. The infestation of Orobanche in the field was uniform, as revealed by the low standard deviation for replications for the number of Orobanche shoots m-2 in the susceptible species, L. sativus. Because of the special weather conditions described above, the number of Orobanche shoots was considerably lower in 1989/90 than in 1988/89 (Table 2). The relative reaction of any given accession to Orobanche infestation, however, was similar for the two seasons as revealed by a high correlation coefficient (r= 0.674, P < 0.001 ). In conformity with the results of the laboratory study, the field study showed
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283
that L. ochrus was free of Orobanche infestation and L. sativus had the highest level of infestation followed by L. cicera. Vicia villosa spp. dasycarpa was almost free of Orobanche infestation whereas V. narbonensis showed medium and V. sativa low susceptibility. The highest intraspecific variation was in V. narbonensis; its line '67' averaged over two seasons, had seven times as many Orobanche shoots as its second-most susceptible line ('573-2378'). Intraspecific variation in resistance was also obvious in V. sativa, with line' 1416' showing a susceptible reaction, whereas two other lines ('715' and '2541') had no Orbanche shoots, and the remaining two lines displayed only low infestation. Visual observation indicated that Orobanche shoots in V. narbonensis were bigger in size at low infestation than at high infestation. There was, however, a high correlation between the number and dry weight of Orobanche shoots ( r = 0.983, P < 0.001 ) across all the tested lines. The dry-matter production of the forage legume lines tested decreased with increasing Orobanche with a correlation coefficient of - 0.849 ( P < 0.01 ). DISCUSSION
Interspecific variation in resistance to Orobanche as observed with the six forage species tested has also been observed in other crops (Vogt, 1987). However, the research on intraspecific variation has provided limited results because the variation within a species usually is lower than between species. In the past, resistant lines were identified in crops such as faba bean, sunflower (Helianthus annuus L. ) and tomato (Lycopersicon lycopersicum [L. ] Karst ex Farw.) (Cubero, 1986), as well as in V. sativa (Gil et al., 1982). This study has demonstrated that substantial intraspecific variation exists in V. narbonensis and V. sativa in their reaction to O. crenata. The availability of Orobanche-resistant selections of different forage legume species offers options to a farmer wanting to grow forage legumes on a heavily infested field to choose the species most adapted to the given environmental conditions. For example, in an area where frost does not occur, lines ofL. ochrus could be used on Orobanche-infested fields to stop an increase in the seed bank. Growing L. sativus or L. cicera or susceptible lines of V. narbonensis such as line '67' should be avoided. Reduction of the seed bank could be accomplished by such methods as hand weeding, application of preemergence (imazapyr) or postemergence (imazaquin and glyphosate) herbicides and cultivation of non-host crops such as flax (Linum usitatissimum) (Linke and Saxena, 1991 ). Hand weeding is generally expensive and should only be used when there are a few Orobanche plants in the field to prevent seed formation. Growing any of the tested lines of L. ochrus, V. villosa ssp. dasycarpa or lines of V. sativa such as '715' and
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'2541' should ensure no parasite shoot emergence. Line '715' of V. sativa, which showed high resistance to O. crenata in this study has been found to be a high- and stable-yielding line across diverse environments in west Asia (Abd et-Moneim et al., 1988). Those lines of V. narbonensis and V. sativa which have intermediate level of resistance to Orobanche, but high yield potential, can be used on parasiteinfested fields, provided they are grown in an integrated Orobanche control system based on manipulation of the date of sowing and/or use of low doses of herbicides. Delaying the sowing on infested fields by 3 to 4 weeks from the date found optimum under parasite-free condition reduces Orobanche attack (Linke and Saxena, 1991 ), probably because of reduced germination of Orobanche seeds (ter Borg, 1986). Under such conditions applications of reduced rates of herbicides ( 15-20 g a.i./ha imazapyr, preemergence; or 2 × 15 g a.i./ha imazaquin or 2 × 40 g a.i./ha of glyphosate as postemergence) can become effective in further reducing the parasite development (Linke and Saxena, 1991 ). None of the L. ochrus lines tested in this study permitted emergence of Orobanche shoots in the field, but they caused stimulation and germination of parasite seeds and in some cases even allowed development of parasitic attachments on roots in the laboratory screening. This was also the case with resistant lines of V. sativa and V. villosa ssp. dasycarpa. These lines thus permit early growth of the parasite in the field but further development is inhibited. Hence, these lines behave like trap crops as has been observed with tomato, kidney beans and flax (Abu-Irmaileh, 1982), resulting in a decrease in the seed bank of Orobanche. In fact, a 30% decrease in the seed bank of Orobanche was demonstrated by Linke (1989) by growing V. villosa ssp. dasycarpa. The inter- and intraspecific differences in susceptibility to Orobanche in the laboratory conformed with those obtained in the field experiments ( r = 0.860, P < 0.001 ). The laboratory screening, which is faster and cheaper, thus reliably predicted the field performance and hence retesting in the field can be restricted to those lines found resistent in the laboratory. ACKNOWLEDGEMENTS This research was partly funded by the Deutsche Gesellschaft ftir Technische Zusammenarbeit (GTZ), Federal Republic of Germany. The technical assistance of Mr. Hasan Masri is greatly appreciated. REFERENCES Abd el-Moneim,A.M.,Coks,P.S. and Swedan,Y., 1988.Yieldstabilityof selectedforagevetches ( Vicia spp.) under rainfed conditions in West Asia. J. Agric. Sci. (Cambridge), 111: 295301.
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Abu-Irmaileh, B.E., 1982. Crop rotation for the control ofbroomrape (Orobanche ramosa L. ). Dirasat, Agricultural Studies, 9:121-126. Abu-Raya, M.A., Radi, A.F. and Darwish Heikal, M.M., 1973. Host parasite relationship of Orobanche species. In: Proceedings of the European Weed Research Council, Symposium on Parasitic Weeds. Malta University Press, Malta, pp. 167-176. Cubero, J.I., 1986. Breeding for resistance to Orobanche and Striga: a review. In: S.J. ter Borg (Editor), Proceedings of a Workshop on Biology and Control ofOrobanche. LH/VPO, Wageningen, Netherlands, pp. 127-139. Gil, J., Martin, L.M. and Cubero, J.I., 1982. Resistencia al jopo (Orobanche crenata Forsk. ) en Vicia satlva L. Anales IN IA, Serie Agricola, 21:175-181. Gil, J., Marlin, L.M. and Cubero, J.I., 1984. Resistance to Orobanche crenata Forsk. in ITcia sativa L. II. Characterization and genetics. In: C. Parker, L.J. Musselman, R.M. Polhill and A.K. Wilson (Editors), Proceedings of the Third International Symposium on Parasitic Weeds, ICARDA/1PSPRG, 7-9 May 1984. ICARDA, Aleppo, Syria, pp. 221-229. ICARDA (International Center for Agricultural Research in the Dry Areas), 1989. 1988 Annual Report, Food Legume Improvement Program. Aleppo, Syria, pp. 214-241. Linke, K.-H., 1989. Sechster Arbeitsbericht zum Orobanche Projekt, August 1989. ICARDA, Food Legume Improvement Program, Aleppo, Syria, 33 pp. (unpubl.). kinke, K.-H. and Saxena, M.C., 1991. Towards an integrated control of Orobanche spp. in some legume crops. In: K. Wegmann and L.J. Musselman (Editors), Progress in Orobanche Research. Eberhard-Karls-Universit~it, TiJbingen, FRG, pp. 248-256. Parker, C., 1986. Scope of agronomic problems caused by Orobanche species. In: S.J. ter Borg (Editor), Proceedings of a Workshop on Biology and Control of Orobanche. LH/VPO, Wageningen, Netherlands, pp. 11-17. Sauerborn, J., Masri, H,, Saxena, M.C. and Erskine, W., 1987. A rapid test to screen lentil under laboratory conditions for susceptibility to Orobanche. LENS Newsletter, 14:15-16. Tennant, D., 1975. A test of a modified line intersect method of estimating root length. J. Ecol., 63: 955-1001. ter Borg, S.J., 1986. Effects of environmental factors on Orobanche host relationships; a review and some recent results. In: S.J. ter Borg (Editor), Proceedings of a Workshop on Biology and Control of Orobanche. LH/VPO, Wageningen, Netherlands, pp. 57-69. Vogt, W., 1987. Untersuchungen zum EinfluB verschiedener Pflanzen auf Keimung und Befall bei Orobanche crenata Forsk., Orobanche ramosa L. und Strlga hermonthica (Del.) Benth. M.Sc. Thesis, University of Hohenheim, Germany, 126 lap. (unpubl.). Winstel, K., 1972. Monographie der Gattung Orobanche. M.Sc. Thesis, University of Hohenheim, Germany, 115 pp. (unpubl.).