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CROP ECOLOGY, PRODUCTION, & MANAGEMENT

Winter Annual Legumes for Use as Cover Crops in Row Crops in Northern Regions

II. Frost Resistance Study

^a The Norwegian Crop Research Institute, Plant Protection Centre, Dep. of Herbology, N-1432 Aas, Norway
^b The Norwegian Crop Research Institute, Plant Protection Centre, Dep. of Plant Pathology, N-1432 Aas, Norway

lars.brandsater{at}planteforsk.no

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Several benefits, e.g., weed suppression, are achieved by including a cover crop in a cropping system. A requirement for developing cover crop systems in northern regions is to find species and cultivars which are adapted for the local climate. The basic objective of this study was to investigate the effects of freezing temperatures on subclover (Trifolium subterraneum L.) cultivars; however, because field experiments recently carried out in Norway have shown that subclover only has potential for winter survival near the southern coast, other winter annual legumes were also tested. To make an adequate freezing-test program reflecting field conditions, a factorial experiment including cultivars, age of subclover plants, and daylength treaments was carried out. The study showed that frost resistance of subclover was increased by short-day treatments both before and through the hardening periods. The effect of short-day treatments was more pronounced for the early flowering cultivar Geraldton than for the later flowering cultivar Mount Barker. The freezing program developed for subclover may also be suitable for other winter annual legumes. The results of a winter annual legume experiment showed that hairy vetch (Vicia villosa Roth.) cultivars, especially cv. Hungvillosa, exhibited the best frost resistance, followed by yellow sweetclover [Melilotus officinalis (L.) Pall.], crimson clover (Trifolium incarnatum L.), black medic (Medicago lupulina L.), white clover (Trifolium repens L.), subclover, barrel medic (Medicago trunculata Gaertn.), and snail medic [Medicago scuttelata (L.) Mill.], which exhibited the poorest frost resistance. In terms of relative biomass, the hairy vetch cultivars Welta and Hungvillosa were significantly different at -9°C. In general, hairy vetch exhibited the best frost resistance in this study.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
SPECIES AND CULTIVARS with sufficient winter hardiness for the local climate and latitude are an absolute requirement for developing cover crop systems with winter annual legumes in Norway. Variability in factors such as soil moisture and fertility, juvenile plant growth and hardening, occurrence of pathogens and insect pests, snow and ice cover, and freezing and thawing sequences contribute to a highly complex pattern of winter injury. This complexity makes it difficult to detect small differences among species and cultivars in their ability to cold harden (Enache, 1989).

Subclover sown in the autumn exhibits a winter annual life cycle. This species is thus expected to properly fit the requirements for living mulch cropping systems (Enache, 1989). Experiments carried out with subclover sown in the autumn in New Jersey, USA, have shown excellent weed control, and, for many crops, without adversely affecting the yield (Ilnicki and Enache, 1992). However, field experiments in Norway have clearly shown that subclover only has potential for winter survival near the southern coast. Hairy vetch and other winter annual legumes, have better winter hardiness (Brandsæter and Netland, 1999). Abdul-Baki and Teasdale (1993) obtained promising results in a no-tillage tomato production system using hairy vetch as a cover crop.

Both Enache (1989) and Brandsæter and Netland (1999) have observed differences among cultivars of subclover with respect to winter hardiness in field experiments. The effects of freezing temperatures on subclover have rarely been considered, however (McGuire, 1985). Studies related to the cold hardening process of subclover are not mentioned in the literature reviews by Knight et al. (1974) or McGuire (1985). Subclover is a vernalizable long-day plant in which all strains reveal a marked interaction between vernalization by low temperatures and the need for long days (Evans, 1985).

The objective of this study was to clarify the effects of freezing temperatures on subclover cultivars. To make an adequate freezing-test program designed to mimic field conditions, subclover cultivars, age of the subclover plants, and the influence of day length on the growth and hardening periods in frost resistance were assessed in a factorial experiment. Since other legumes may have better potential as overwintering cover crops, the freezing resistance was also studied in the following species: Crimson clover, black medic, barrel medic, snail medic, yellow sweetclover, and hairy vetch.

	Materials and methods

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Experimental Design, Plant Material and Treatments
For both experiments, about 25 seeds of each species–cultivars were sown in 12-cm (diameter) pots filled with limed peat [L.O.G. `Gartnerjord'. Mixture: 840 g kg^-1 sphagnum peat, 100 g kg^-1 fine sand, 60 g kg^-1 clay, pH 5.5–6.5, 5.5 kg dolomite lime m^-3, 1.2 kg fertilizer (NPK 15-4-12), 0.2 kg F.T.E. no 36 (micro nutrients), and density: 270 kg DM/m^-3 (applied volume)].

The seeds were inoculated with compatible Rhizobium, before sowing. After 1 wk, the seedlings were thinned to eight or five plants per container, for the subclover or winter annual experiments, respectively. The pots were placed randomly on carts in the growth chamber. The carts were rotated twice a week in the chamber to compensate for variations in light levels at different spatial positions.

Experiment 1: Subclover
The trial was designed as a factorial, randomized complete block with two replications. A 2^k factorial design was used with k = 4 (Montgomery, 1984). The following factors were included in the experiment.

1. Two subclover cultivars: (i) the early-flowering cultivar Geraldton, and (ii) the late-flowering cultivar Mount Barker.
   
2. Age of plants: early and late sowing time.
   
3. 3. Short-day (SD) and long-day (LD) treatments during the growth period.
   
4. SD and LD treatments during the cold-hardening period. As shown in Table 1 , all factors were combined in a factorial design and the plants were exposed to freezing at -5°C or 0°C (control). The experiment was replicated in time and each replication consisted of three parallel containers. Containers with early sown plants were fertilized with 100 mL Hoagland solution twice, 3 and 6 wk after germination, and containers with late sown plants were fertilized once, 3 wk after germination. The treatments, including growth periods, hardening, freezing and revival, are presented in Table 1. Visible (without dissection) flowers were recorded prior to the freezing treatment.
   

The pots were fertilized twice during the cultivating period, with nutrient solution (Hydro Superba brun NPK-fertilizer with micro nutrients / Conductivity 1.5; Norsk Hydro, Oslo), 3 and 6 wk after sowing.

The hairy vetches were cut down to 20 cm height from the average height of 35 cm, 30 d after sowing, to prevent them shading and climbing the other species.

The containers with hairy vetches had iron railing (wire support) to keep the plants upright.

The treatments of the plants are presented in Table 1, winter annual legume experiment. All the plants were exposed to equal day length, temperatures, and light intensity prior to the freezing treatment.

Because of lack of seed, hairy vetch AU EarlyCover was only included in one replication. Before the plants were exposed to freezing treatment, visible flowering was recorded (without dissection). Additionally, primary shoot length and number of secondary shoots were recorded in the third replication (see Table 2 for details).

Freezing Test
Controlled freezing of the plant material was conducted in freezing chambers, according to a method described by Larsen (1978). To assure a slow initial freezing, the containers were placed in trays in which the bottom was covered with a 2-cm-thick styrofoam plate. Both experiments included control treatments kept at 0°C for 60 h. In the other freezing chambers, the temperature were decreased by 1°C/h to -2.5°C and kept for 12 h. The temperature was then decreased by 1°C/h until the freezing temperatures were reached.

In the subclover experiment, the second freezing chamber was decreased to -5°C and maintained for 38 h. The choice of freezing temperature was based on results from a pilot experiment (Brandsæter, unpublished).

In the winter annual legume experiment, the temperature in the freezing chambers was allowed to stabilize at 2°C. The temperature was then lowered at a rate of 1°C/h, including 12 h at -2.5°C, to temperatures of -3, -6, and -9°C in the second, third, and fourth chamber, respectively. The minimum temperatures were maintained for 60 h (0°C), 42 h (-3°C), 36 h (-6°C), and 30 h (-9°C), respectively.

For both experiments, the thawing procedure for all units started with an increase in temperature at a rate of 1°C/h until the temperature of +4°C was reached and then maintained for 24 h. The thawed plants were taken out of the freezing chambers and placed in the greenhouse for 3 wk before the freezing resistance, recorded as the parameters shown in Table 3 , were assessed. For the biomass assessments, dead and living tissue was identified by the separating withered and green tissues, and also by testing whether the tissue was connected with a fresh root or not.

In the subclover experiment, interactions including up to two factors were used in the statistical models. The three- and four factor interactions were not included in the analysis because this gave a lack of degrees of freedom in the error term.

	Results

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Subclover Experiment
Parameters for freezing resistance of the cultivars, sowing date, and day length on freezing resistance are shown in Table 4 . Long day during the Growth Period I significantly increased damage and decreased root length of both cultivars. Long day during hardening also reduced root length and biomass compared with short day. There was a tendency for all response variables, particularly for relative root length, that Mount Barker was less damaged by freezing than Geraldton. However, the differences were not significant. Only minor, non-significant differences appeared between dates of sowing.

All two-factor interactions are presented in Table 5 . For visually assessed damage, the interaction between cultivar and day length during the growth period was significant in the statistical analyses. This interaction implied that SD treatment during the growth period, compared with LD treatment, increased frost resistance more in the early-flowering cultivar Geraldton than in the late-flowering cultivar Mount Barker. For relative root length, the interaction between day length during both the growth period and the hardening period was significant. This interaction showed that SD treatment in both the growth and the hardening period gave the most frost hardy plants. All other combinations of day lengths in the growth and hardening period resulted in less frost resistance.

Neither the interaction between cultivar and day length during the hardening period, nor the interaction between sowing date and day length during the hardening period, showed any significant differences or tendencies.

Winter Annual Legume Experiment
The frost resistance of the cultivar–species, in terms of relative biomass (percentage of control) is shown in Fig. 1 . Percentage of surviving plants after freezing is shown in Table 2. Percentage of flowering plants, primary shoot length, and numbers of secondary shoots prior to the freezing treatments are also presented in Table 2.

View larger version (41K): [in this window] [in a new window]   Fig. 1 The biomass given as relative values of control for all treatments. Points at the same temperature associated with different letters are significantly different at P = 0.05. Figures in brackets behind names of cultivars give the mean values for the control (0 C°) biomass, and correspond with 100%, for the respective species–cultivars. indicates that AU EarlyCover was included in only one replication and was therefore not included in the statistical analysis

The differences between the species–cultivars at -3°C were small compared with the results at the other temperatures. Also, they were mostly not consistent with the differences found at the other two freezing temperatures.

At the -6°C, the subclover cultivars, Denmark and Nuba, the barrel medic cultivar, and the snail medic cultivar showed significantly lower frost resistance than the other species–cultivars. Within these four species–cultivars, Denmark was significantly more frost resistant than barrel medic.

The relative biomass of the mentioned species–cultivars were considerably lower than the other species–cultivars and this was also true at the lowest temperature (-9°C). Among the remaining species–cultivars at -6°C, the only significant difference appeared between the yellow sweetclover which showed the highest, and white clover which showed the lowest relative biomass.

At the lowest temperature (-9°C), the hairy vetch Hungvillosa had the highest relative biomass. In this aspect, Hungvillosa was significantly better than hairy vetch Welta at -9°C. White clover and the black medic had the lowest relative biomasses of the remaining species. The differences between hairy vetch Hungvillosa on one hand and white clover, black medic, and crimson clover on the other were significant. Hairy vetch AU EarlyCover appeared as the weakest of the hairy vetches; however, the result was not analyzed statistically.

At -3°C, only the barrel medic showed a significant lower difference in survival percentage than the rest of the species–cultivars. The same pattern as described for the relative biomass, appears for survival at -6°C. The subclovers, Nuba and Denmark as well as the barrel medic and snail medic showed significantly lower survival compared with the remaining species–cultivars. No significant differences appeared within this group. At -9°C, the survival of black medic Virgo Pajberg was significantly lower than the hairy vetches, Hungvillosa and Villana.

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
To develop local suitable cover crop systems (e.g., living mulch), it is essential to find species–cultivars that have suitable life cycles The subclover seems to have an ideal life cycle and characteristics desirable for the potential use as cover crop–living mulch (Ilnicki and Enache, 1992; Brandsæter and Netland, 1999). However, the results of Brandsæter and Netland (1999) show that subclover has poor winter survival and would only have the potential to survive the winter on the southwestern coast of Norway.

Most differences obtained in the subclover experiment seem to be related either to choice of cultivar or to stage in the life cycle. In spite of a lack of significant differences, the late-flowering cultivar Mount Barker seems to be more frost resistant than the early-flowering cultivar Geraldton. The reason for including an early and a late cultivar was to study the interaction with other factors, e.g., day lengths. Short-day treatment in the growth and hardening periods was important for increasing frost resistance. The frost resistance of subclover may decrease under LD treatment because this species is a vernalizable long-day plant (Evans, 1985) and frost resistance of the floral primordia is poor (Morley, 1961). In the growth period, the light in the LD treatment was given as photosynthetically active irradiation in the whole period and this could influence the frost resistance. However, because day length is of vital importance for flower initiation of subclover, it seems reasonable to believe that day length is more important than increased irradiation for flowering initiation.

The results showed that SD during the growth period was more important for improving frost resistance of an early-flowering cultivar than for a late-flowering cultivar. This result is consistent with the fact that only the early cultivar flowered in some of the treatments. This result is consistent with Aitken (1985) who showed that an early variety of subclover had rapid flower initiation when grown under long-day conditions. Furthermore, the result clearly demonstrated that to increase frost resistance SD was needed both during the growth and the hardening period.

On the basis of the results from the first experiment, SD was used in growth and hardening periods of the second experiment. It can be questioned whether the freezing-test program developed for subclover also is suitable for other winter annual legumes. However, it seems reasonable to believe that flower initiation of most winter annual legumes, similar to subclover (Evans, 1985), will reveal an interaction between vernalization by low temperatures and the need for long days. Knight and Hollowell (1958) concluded that crimson clover appears broadly similar to subclover regarding flower initiation. Results of Kurth (1956) indicated that interaction between vernalization and the need for long days also may occur for hairy vetch. Annual Medicago species (black medic, snail medic, and barrel medic) also have their flowering controlled by processes similar to those identified in subclover (Evans, 1985). Black medic is, however, reported to be both an annual, biennial, or perennial species (Lammerink, 1968). If sown in spring, subclover, crimson clover, black medic, and hairy vetch will flower in the summer in Norway (Brandsæter and Netland, 1999; Grønnerød, personal communication). This shows that during the summer time the requirement of long days for initiation of flowering is satisfied for these species under Norwegian conditions. On the basis of the cited literature (Evans, 1985; Knight and Hollowell, 1958; Kurth, 1956) which report that most winter annual legumes respond quite similarly to day length, we presume that the subclover freezing program is adequate also for the other winter annual legumes included in the experiment. Yellow sweetclover is reported to be a biennial species (Lid, 1987). Biennials have an absolute cold requirement, as contrasted to the facultative winter annuals (Salisbury and Ross, 1992). Surprisingly, all yellow sweetclover flowered before freezing (Table 2) and therefore showed no absolute cold requirement in the present study.

The tested species and cultivars in the present study show varying abilities to withstand frost. Percentage survival and relative biomass indicated mostly the same trends, and frost resistance is discussed on the basis of both characteristics.

In earlier experiments by Brandsæter (1996), subclover Nuba showed rather poor frost resistance in a growth chamber experiment but relatively good winter survival in field experiments, compared with other subclover cultivars. Subclover Denmark had not been tested earlier, and in this study it was ranked as good as or better than Nuba; however, the difference was not significant. Similar to the subclovers, the snail medic and barrel medic showed quite poor frost resistance. Overall, the hairy vetch, yellow sweetclover, and crimson clover showed rather good frost resistance. The present results indicate, however, that hairy vetch Hungvillosa is better adapted for cold climates than the more commonly used cultivar Welta. This difference is confirmed in a recent field experiment, where also the cultivar Villana showed better winter survival than Welta (Brandsæter, unpublished). Hairy vetch AU Early-Cover was killed in spring in the same field experiment.

Crimson clover has shown poor winter survival in field experiments by Brandsæter and Netland (1999), but the cultivar used in these experiments was not specified. In the present study, crimson clover showed a relatively good frost resistance, better than black medic, although poorer than the vetches. The difference between crimson clover and black medic was not significant, however. Yellow sweetclover also showed a good frost resistance and was significantly better than white clover.

The barrel medic and snail medic showed poor frost resistance. They had both reached the flowering stage when they were frozen and this could be the reason for their poor survival. Given a shorter period of the long-day treatment before freezing, barrel medic and snail medic might exhibit better frost resistance results, since the flowering would be delayed to after the cool period.

It seems reasonable to believe that the stage of flowering prior to freezing is an important aspect in frost resistance. Winter annual legumes which have reached the flowering stage may exhibit poor frost resistance (Brandsæter, 1996). Consequently, including plants at different growth stages in the experiment might help clarify what is important in conditioning freezing resistance in different winter annual species and cultivars.

Frost resistance is only one factor that determines the species' or cultivar's potential of winter survival in the field. As reported by Brandsæter (1996), subclover Nuba showed relatively good winter survival in field experiments, in spite of the fact that it showed poor frost resistance when tested in freezing chamber. One other example is white clover Milkanova, which is quite persistent in field, but showed only modest frost resistance in the present study. The results from a freezing test can therefore only be used as an indication of which species or cultivars have an apparent potential for winter survival.

In conclusion, the best frost resistance was shown by the hairy vetches, especially Hungvillosa, yellow sweet-clover, crimson clover, and black medic, in that order. Subclover, barrel medic, and snail medic showed the poorest frost resistance.SAS Institute 1988

	NOTES

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Research financed by The Research Council of Norway.

Received for publication June 19, 1998.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 

• Abdul-Baki A.A., Teasdale J.R. A no-tillage tomato production system using hairy vetch and subterranean clover mulches. HortScience 1993;28:106-108.
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• Brandsæter, L.O. 1996. Alternative strategies for weed control: Plant residues and living mulch for weed management in vegetables. Agricultural University of Norway, Doctor Scientiarum Theses 1996:25.
• Brandsæter L.O., Netland J. Winter annual legumes for use as cover crops in row crops in northern regions: I. Field experiments. Crop Sci. 1999;39:1369-1379.[Abstract/Free Full Text]
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• Evans L.T. Trifolium subterraneum. In: Halevy A.H., ed. CRC handbook of flowering. Volume I. Boca Raton, FL: CRC Press, Inc., 1985:636-640.
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• McGuire W.S. Subterranean clover. In: Taylor N.L., ed. Clover science and technology. Agron. Monog. 25. Madison, WI: ASA, CSSA, and SSSA, 1985:515-534.
• Montgomery D.C. Design and analysis of experiments, 2nd ed Toronto: John Wiley and Sons, 1984.
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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Brandsæter, L. O. Articles by Netland, J. Search for Related Content PubMed Articles by Brandsæter, L. O. Articles by Netland, J. Agricola Articles by Brandsæter, L. O. Articles by Netland, J.