CHAPTER 3 Greenhouse and laboratory assessment of rotational crops for allelopathic 32

CHAPTER 3 Greenhouse and laboratory assessment of rotational crops for allelopathic 32

32

CHAPTER 3

Greenhouse and laboratory assessment of rotational crops for allelopathic potential that affects both crops and weeds

MI Ferreira 1 and CF Reinhardt 2

2*

1 Institute for Plant Production, Department of Agriculture Western Cape, Private Bag X1, Elsenburg,

7607, South Africa

Department of Plant Production and Soil Science, University of Pretoria, Pretoria, 0002, South Africa

[email protected]

*Current address: South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe,

4300

INTRODUCTION

Chemical interference was described by Hoffman et al. (1996) as a significant coevolutionary force in plant communities, but it may be much more important as a mechanism in recipient than in origin communities (Hierro & Callaway, 2003).

Alterations in the environment by various plant interference mechanisms can differentially affect neighbouring plant species. Allelopathy is defined as any direct or indirect, inhibitory or stimulative, effect by one plant (including micro-organisms) on another through the production of a chemical compound(s) (Rice, 1984). The phenomenon encompasses both detrimental and beneficial interactions between plants through chemicals released by the donor (Xuan & Tsuzuki, 2002).

According to Kato-Noguchi (2000), chemicals with allelopathic activity are present in many plants and in many organs, including leaves, flowers, fruits and buds. They are of varied chemical nature, e.g., phenolics, terpenes, alkaloids, flavonoids, etc.

(Gupta, 2005). In agricultural ecosystems it is one of the important mechanisms of interference, affecting crop performance (Batish et al., 2002). Allelochemicals appear to affect all aspects of crop development including germination, radicle and plumule

(coleoptile in monocots) growth, seedling growth, metabolism, plant growth, flowering and fructification. Belz (2004) suggested that crop allelopathy can be exploited for weed management through the release of allelochemicals from intact roots of living plants or decomposition of plant residues and that in annual crops, root exudation of the phytotoxins by the crop is the preferred mechanism.

33

Kumar et al. (2009) suggested that one approach to understanding the allelopathic effects of crop residues is to separate soil effects occurring during the growth of crops from their residue effects. Another approach is to determine which parts of the cover crop-root, shoot, or root plus shoot-has the most suppressive effects on emergence and growth. Nevertheless, Olofsdotter et al. (1995) and Wu et al. (2000;

2001) cautioned that an essential need in studying crop allelopathy is simulation of the natural release of allelochemicals so that chemical interference from living donor plants on living receiver plants can be measured.

The complicated nature of interference among plants makes it difficult to separate its components in natural environments (Oasem & Hill, 1989). Therefore, the relative importance of competition and allelopathy as mechanisms of plant interference is generally unknown (Hoffman et al., 1996). Furthermore, the interaction of allelochemicals with soil components upon release from the plant is important in determining whether inhibition of the target plant is likely to occur in the field (Blum,

1996).

Separation of allelopathic effects from those of competition is a major experimental challenge (Oasem & Hill, 1989), but many research reports proved its feasibility. In a study carried out by Caussanel et al. (1977) it was shown that root exudates of C.

album (white goosefoot) retarded the radicle growth of Zea mays (maize) in culture solution. An aqueous extract of the weed also inhibited the growth of maize roots.

Further studies carried out by Caussanel (1979), showed that white goosefoot exerted an inhibitory influence on maize growth. He demonstrated that the effect could not be attributed to competition alone. Bhatia et al. (1984) also reported an inhibitory effect of white goosefoot on Triticum aestivum L. (wheat) seedlings.

Chemical effects of white goosefoot seeds on germination were reported by

Stefureac and Fratilescue-Sesan (1979) who found that seeds of white goosefoot placed in Petri-dishes with seeds of meadow fescue, wheat (cv. Dacia) or Medicago

sativa (lucerne) inhibited the germination of all three species.

Quasem and Hill (1989) successfully segregated competitive and allelopathic effects of white goosefoot on tomato. Reinhardt et al. (1997) reported that white goosefoot

34 caused inhibition of maize and soybean root growth. The presence of white goosefoot residual material in soil caused growth reduction of wheat, Lactuca sativa

L. (lettuce), lucerne, and various other crop species (Reinhardt et al., 1994).

Furthermore, white goosefoot residues in the soil have been found to be phytotoxic and to affect the nutrient uptake process in maize and soybean (Reinhardt et al.,

1994). A better understanding of toxic weed root exudates that inhibit crop growth will lead to more effective decision-making in crop rotation systems (Rice, 1984).

Kumar et al. (2009) noted that for most plant species, shoot extracts were more effective than root extracts in inhibiting seed germination and growth of downy broom. Kumar et al. (2009) reported that shoot extracts of two goldenrod species

(Euthamia graminifolia L. Nutt. and Solidago canadensis L.) had inhibitory effects on both germination and growth of radish (Raphanus sativus L.) and lettuce. In contrast, root extracts had no inhibitory effects on germination of these two species, but suppressed root growth. On the other hand, rye (Secale cereale L.) root residues were found to be more suppressive than shoot tissues on growth and emergence of barnyardgrass (Echinochloa crus-galli L. Beauv.) and growth of sicklepod (Senna

obtusifolia L. Irwin and Barneby) (Brecke & Shilling 1996; Hoffman et al., 1996).

Aqueous shoot extracts of buckwheat stimulated Powell amaranth (Amaranthus

powellii S. Wats.) germination slightly, but inhibited radicle growth (Kumar et al.,

2009). Aqueous soil extracts from buckwheat-amended soil inhibited germination of

Powell amaranth whilst extracts from unamended soil showed no effect.

According to Hoffman et al. (1996) competitive hierarchies often form during early stages of plant growth, and therefore interference should be measured between germinating seeds and between seedlings. Typical field studies cannot separate the effects of competition from allelopathy since they happen simultaneously between roots and shoots. In view of this, artificial environments must be devised that remove any possibility of competition while allowing chemical exchange to take place (Smith

et al., 2001). Therefore, the primary objectives of this research were to evaluate the possible role of allelopathy from seeds, seedlings, roots and above-ground plant material, under controlled conditions.

35

MATERIALS AND METHODS

The plant series used in the laboratory and green house, consisted of the rotational crops barley (Hordeum vulgare L. v. Clipper), canola (Brassica napus L. v. ATR

Hyden), wheat (T. aestivum v. SST 88), lupine (Lupinus angustifolius L. v. Tanjil), lucerne (M. sativa L. v. SA standard), medic (M. truncatula Gaertn. v. Parabinga) and rye grass (Lolium multiflorum Lam. v. Energa) in a lay-out for Experiments 1-4 as represented in Table 1 (Appendix A, Figure A3, A4 & A5).

Table 1 Schematic representation of experimental design for Experiments 1-4

Treatment number

1 Barley

2 Canola

Barley

1

Barley

Canola

Canola

2

Barley

Canola

Wheat

3

Barley

Canola

Plant donors

Lupine

4

Barley

Canola

Lucerne

5

Barley

Canola

Medic

6

Barley

Canola

Rye grass

7

Barley

Canola

Control

8

Barley

Canola

3 Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat

4 Lupine

5 Lucerne

6 Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

Lupine

Lucerne

Medic

7 Rye grass Rye grass Rye grass Rye grass Rye grass Rye grass Rye grass Rye grass Rye grass

The research approach for Experiment 1 and 2, although similar in concept to that followed by Hoffman et al. (1996) and Kato-Noguchi (2000) for assessing whether crop seeds and seedlings release phytotoxins that affect the germination and development of radicles of rotational crops, was different in terms of both experimental method and plant series investigated.

For Experiment 3 and 4, research methods were similar to those followed by

Reinhardt et al. (1994), Hoffman et al. (1996) and Smith et al. (2001), for assessing whether crop root exudates and above-ground plant material release phytotoxins that affect the growth and yield of rotational crops. The nature and extent of experiments conducted for this study which was done under controlled conditions, had a similar lay out (Table 1) to Exp 1 in Chapter 2, and therefore a dilution series was not considered, as it replicated treatments from the field experiment in order to compare and explain field data.

36

Experiment 1: The first experiment was set up in the laboratory to observe the mutual effect of seed leachates from the plant series. Ten seeds of each plant type were placed in Petri-dishes in combinations with ten seeds of each of the other species in the series. Seeds were placed on filter paper in 9.5 cm diameter Petri-dishes and moistened with 5 ml distilled water. The lay-out was done according to a Randomised

Block design with ten replicates, equalling 100 seeds per species. Control Petridishes contained only one seed type (not in combinations). Petri-dishes were sealed with Parafilm® and placed in an incubator set at 12h/12h day/night cycle and a temperature range of 25/15 °C. Germination was determined after 7 and 14 days of incubation, by counting the number of germinated seeds and measuring the length of the radicle. A seed was regarded as germinated when the radicle was at least 2 mm long, and was then removed from the Petri-dish.

Experiment 2: The second experiment was conducted in the laboratory to study the effect of seedling leachates from all the plants in the series on germination and early development of all the other species. One hundred seeds of each plant type in the series were germinated in Petri-dishes. The seedlings were allowed to develop until they reached a length of roughly 50 mm, after which ten seedlings from each species were placed in a 4 cm porcelain Buchner funnel and washed with 5 ml distilled water to yield a leachate. This leachate was funnelled into 9.5 cm diameter Petri-dishes into which 10 seeds from each plant type had been placed on Whatman 9 cm filter paper according to a Randomised Block design with ten replicates, equalling 100 seeds per species. Control treatments were treated with distilled water only. Petri-dishes were sealed with Parafilm® and incubated at a 12h/12h day/night cycle with a temperature range of 25/15 °C. Germination was determined after 7 and 14 days of incubation, by counting the number of germinated seeds and measuring the length of the radicle.

Experiment 3: This experiment was conducted in the greenhouse to determine the effects of root exudates from each plant in the series on the growth of themselves and all other species. Ten crop seeds of each plant type were planted in separate donor pots filled with 6 kg of leached river sand, and thinned to five plants of similar size one week after emergence. Treatments in the greenhouse were replicated three times in a Randomised Block design. Pots were over-irrigated twice a week, from the first week after planting with 100 ml water to provide for sufficient drainage per pot. At

37 the time of planting this was 150 ml water (100 ml drainage), reaching 900 ml per pot twice a week (300 ml drainage), as plants matured. All water leached from the same plant type was collected in the same container and used as root leachate on acceptor pots in which five plants were grown in the same growth medium. No planting was done in control pots, but the leachate was collected for use as control treatment.

Of the leachate collected, 100 ml was used twice a week at planting and increasing to 300 ml at maturity, to irrigate the acceptor (same as donor) species as well as each of the other plant types. The first irrigation occurred at the time of planting, and thereafter twice a week for five weeks after emergence. Once a week, Multifeed was applied as balanced plant nutrition at a concentration of 1g ℓ -1 , to each pot by using a volume of 50 ml at the time of planting and reaching 200 ml at five weeks.

Experiment 4: The fourth experiment was conducted in the greenhouse to study the effects of above-ground plant residue leachates from the plant series on the growth of the plant series itself. Plant material from each plant species was collected in the field and air-dried, after which it was ground to a coarse powder. This substratum was mixed shallowly into pots filled with 6 kg of leached river sand, at a rate of 15 g per pot (equivalent to 5 t plant residues per hectare), in which the donor plant itself, as well as all the other plant types, were planted separately (five plants per pot).

Treatments in the greenhouse were replicated three times in a Randomised Block design. Since chemical products of the decomposition process are soluble in a weak carbonic acid solution, the surface irrigation would have leached allelochemicals into the soil, resulting in their absorption by the plant. This leachate from five donor plants was used to treat five acceptor plants planted in the same growth medium, but without residues mixed into pots. At the time of planting this was 50 ml leachate, reaching 600 ml per pot per week, as plants matured. Once a week, Multifeed was applied as balanced plant nutrition at a concentration of 1g ℓ -1 , to each pot by using a volume of 50 ml at the time of planting and reaching 200 ml at five weeks.

In the greenhouse, plant height was determined for all plants on a weekly basis, starting at one week after emergence. After five weeks all plants were cut off at ground level. Thereafter, all the above-ground plant parts were dried at 60°C for 72 hours and dry mass recorded.

38

All data were analysed statistically (ANOVA) with the statistical program SAS. Least significant differences were used to identify significant differences between means at the 5% level of probability.

RESULTS

Experiment 1

Seed leachate: laboratory

Barley

No significant differences between seed leachate treatments were recorded in barley radicle length (Table 2). At 14 days, leachates from wheat and medic seeds had reduced barley cumulative germination significantly from that attained in the control treatment.

Table 2 Effects of seed leachates on barley radicle length and germination

Plant type

Seed leachate

Barley radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

26.4a

25.2a

23.5a

13a

12.2a

13a

25.7a

21.6a

77ab

97a

67 b

73ab

90ab

70 b

80ab

97a

LSD (P≤0.05) 18.1

25

*Means followed by the same letter are not significantly different at the 0.05 probability level

Canola

Canola radicle length was significantly reduced by leachates from barley, lupine and lucerne seeds (Table 3). At 14 days, leachates from lupine seeds had reduced canola cumulative germination significantly from that attained at the control treatment.

Table 3 Effects of seed leachates on canola radicle length and germination

Seed leachate

39

Plant type

Canola radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

10.5 c

22.4a

12.4abc

5.8 c

10.8 bc

22.3ab

23.4a

23.7a

73ab

97a

70ab

60 b

100a

93a

90ab

93a

LSD (P≤0.05) 11.5

33

*Means followed by the same letter are not significantly different at the 0.05 probability level

Wheat

The radicle length of wheat was significantly reduced by leachates from barley, wheat and lupine (Table 4). Lupine seed leachates also significantly reduced wheat cumulative germination.

Table 4 Effects of seed leachates on wheat radicle length and germination

Plant type

Seed leachate

Wheat radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

8.5 bc

19.5ab

9.5 bc

5 c

20.2ab

15.6abc

24.8a

27.9a

53 bc

70ab

93a

27 c

83ab

83ab

93a

87ab

LSD (P≤0.05) 12.5

38

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lupine

No significant differences between seed leachate treatments were observed in lupine radicle length (Table 5). Barley seed leachate had reduced lupine cumulative germination significantly from that attained at the control treatment.

Table 5 Effects of seed leachates on lupine radicle length and germination

Plant type

Seed leachate

Lupine radicle length (mm)

Cumulative germination

% at 14 days

40

8.9ab

11.9a

9.6ab

13.5a

9.1ab

8.4ab

40ab

70a

43ab

63a

47ab

53a

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

2.8 b

6.8ab

13 b

53a

LSD (P≤0.05) 9 40

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lucerne

The radicle length of lucerne was significantly inhibited by seed leachates from barley and lupine (Table 6). Lupine seed leachate had reduced lucern cumulative germination significantly from that attained at the control treatment.

Table 6 Effects of seed leachates on lucerne radicle length and germination

Plant type

Seed leachate

Lucerne radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

3.7 bc

20.8ab

11.9abc

0 c

17.1abc

20.0ab

20.6ab

25.2a

33 c

87a

47 bc

0 d

73ab

57abc

83a

43 bc

LSD (P≤0.05) 17.4

31

*Means followed by the same letter are not significantly different at the 0.05 probability level

Medic

Significant differences in medic radicle length were observed when seeds were treated with barley and lupine seed leachate (Table 7). No differences in cumulative germination were noted.

Table 7 Effects of seed leachates on medic radicle length and dry mass

Plant type

Barley

Seed leachate

Medic radicle length (mm)

Cumulative germination

% at 14 days

13.4 b 57a

41

31.7a

19ab

12.6 b

17ab

19.7ab

31.8a

31.6a

73a

77a

50a

60a

93a

70a

77a

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

LSD (P≤0.05) 16.1

44

*Means followed by the same letter are not significantly different at the 0.05 probability level

Rye grass

The radicle length of rye grass was significantly inhibited by seed leachates from barley, wheat and lupine (Table 8). This growth-inhibiting effect from barley and lupine seed leachates, was also evident in rye grass cumulative germination percentage.

Table 8 Effects of seed leachates on rye grass radicle length and dry mass

Plant type

Seed leachate

Rye grass radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

12.4 cd

33.8ab

15.8 bcd

1.5 d

25.1abc

24.0abc

50 bc

87a

73ab

17 c

90a

97a

Rye grass

Control

28.2abc

36.8a

90a

97a

LSD (P≤0.05) 19.5

34

*Means followed by the same letter are not significantly different at the 0.05 probability level

Experiment 2

Seedling leachate: laboratory

Barley

No significant differences between seedling leachate treatments were recorded in barley radicle length or cumulative germination at 14 days (Table 9).

Table 9 Effects of seedling leachates on barley radicle length and germination

Plant type

Barley

Seedling leachate

Barley radicle length (mm)

Cumulative germination

% at 14 days

35a 73a

42

30.5a

27.3a

36.7a

29.9a

32.7a

33a

40.4a

90a

77a

97a

83a

73a

90a

100a

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

LSD (P≤0.05) 23.4

28

*Means followed by the same letter are not significantly different at the 0.05 probability level

Canola

No significant differences between seedling leachate treatments were recorded in canola radicle length or cumulative germination (Table 10).

Table 10 Effects seedling leachates on canola radicle length and germination

Plant type

Seedling leachate

Canola radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

26a

22a

26a

22.8a

19.2a

21.7a

22.3a

87a

83a

90a

87a

80a

93a

87a

Control

LSD (P≤0.05)

19.5a

15.3

73a

22

*Means followed by the same letter are not significantly different at the 0.05 probability level

Wheat

No significant differences between seedling leachate treatments were observed in wheat cumulative germination (Table 11). After treatment with canola seedling leachates, wheat radicle length was significantly shorter than the control.

Table 11 Effects of seedling leachates on wheat radicle length and germination

Plant type

Barley

Canola

Wheat

Lupine

Seedling leachate

Wheat radicle length (mm)

Cumulative germination

% at 14 days

33abc

25.7 c

44.6a

31 bc

80a

83a

77a

73a

43

40.6ab

35.8abc

41.2ab

41.4ab

83a

87a

70a

87a

Lucerne

Medic

Rye grass

Control

LSD (P≤0.05) 13.5

19

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lupine

No significant differences between seedling leachate treatments were recorded in lupine radicle length (Table 12). The cumulative germination of lupine, treated with lucerne seedling leachates, was significantly less than the control.

Table 12 Effects of seedling leachates on lupine radicle length and germination

Plant type

Seedling leachate

Lupine radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

21.9a

15.2a

26.3a

23a

12.9a

16.5a

24a

27.7a

80ab

90a

87a

90a

57 b

77ab

77ab

93a

LSD (P≤0.05) 14.8

25

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lucerne

No significant differences between seedling leachate treatments were observed in percentage lucerne cumulative germination (Table 13). Rye grass seedling leachate stimulated the growth of lucerne seedlings significantly, as compared to the control, with regard to radicle length.

Table 13 Effects of seedling leachates on lucerne radicle length and germination

Plant type

Barley

Canola

Wheat

Lupine

Seedling leachate

Lucerne radicle length (mm)

Cumulative germination

% at 14 days

18.3abc

21.4ab

20.1abc

14.7 bc

63a

80a

70a

63a

44

12.3 c

22.8ab

26.4a

14.7 bc

63a

70a

57a

73a

Lucerne

Medic

Rye grass

Control

LSD (P≤0.05) 8.7

38

*Means followed by the same letter are not significantly different at the 0.05 probability level

Medic

No significant differences in medic radicle length were observed when treated with seedling leachates (Table 14). The cumulative germination of medic, treated with lupine seedling leachates, was significantly less than the control.

Table 14 Effects of seedling leachates on medic radicle length and dry mass

Plant type

Seedling leachate

Medic radicle length (mm)

Cumulative germination

% at 14 days

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

17.0ab

27.8a

25.8ab

15.6 b

18.5ab

19.2ab

26.8a

24.5ab

73ab

70ab

73ab

63 b

73ab

77a

70ab

77a

LSD (P≤0.05) 10.9

13

*Means followed by the same letter are not significantly different at the 0.05 probability level

Rye grass

No significant differences between seedling leachate treatments were observed in rye grass cumulative germination percentage (Table 15). Seedling leachate from lupine, had significantly inhibited rye grass radicle length.

Table 15 Effects of seedling leachates on rye grass radicle length and dry mass

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Seedling leachate

Rye grass radicle length (mm)

Cumulative germination

% at 14 days

35.6ab

34.7ab

47.4a

31.6 b

36.2ab

39.1ab

46.4a

83a

83a

87a

87a

83a

80a

93a

45

Control 46.1a

90a

LSD (P≤0.05) 13.9

17

*Means followed by the same letter are not significantly different at the 0.05 probability level

Experiment 3

Root exudates: greenhouse

Barley

At three weeks after planting, leachate from the root systems of lucerne and medic had reduced barley height significantly from that attained at the control treatment

(Table 16). This significant growth-inhibiting effect from lucerne and medic on barley, along with lupine, was also evident at five weeks after planting. The dry mass of barley, treated with wheat, lupine and lucerne root leachates, was significantly less than barley treated with control leachate.

Table 16 Effects of root exudates on barley plant height and dry mass

Plant type

Barley plant height at 3 wks (cm)

Root leachate

Barley plant height at 5 wks (cm)

Barley dry mass (g)

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

38.6a

37.5ab

37.6ab

35.7 bc

31.2 d

34.3 c

37.3ab

38.1ab

46.9ab

47.2a

43.9 bc

42.6 c

38.6 d

42.3 c

45.3abc

46.6ab

0.75a

0.61 bcd

0.58 cd

0.56 d

0.54 d

0.63 bcd

0.67abc

0.69ab

LSD (P≤0.05) 2.4

3.2

0.11

*Means followed by the same letter are not significantly different at the 0.05 probability level

Canola

After treatment with barley, canola, lucerne, medic and rye grass root leachates, canola plant height was significantly greater at five weeks after planting (Table 17).

No significant differences between root leachate treatments were recorded in canola dry mass.

Table 17 Effects of root exudates on canola plant height and dry mass

Root leachate

46

Plant type

Canola plant height at 3 wks (cm)

Canola plant height at 5 wks (cm)

Canola dry mass (g)

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

12.5a

13.7a

12.3a

12.9a

12.4a

13.1a

13.1a

12.7a

21.9a

20.5ab

18.8 bc

19.1 bc

20.3ab

21.7a

21.7a

18.1 c

0.67ab

0.70ab

0.60 b

0.63 b

0.65 b

0.71ab

0.77a

0.67ab

LSD (P≤0.05) 1.7

2.1

0.12

*Means followed by the same letter are not significantly different at the 0.05 probability level

Wheat

No significant differences between root leachate treatments were recorded in wheat dry mass (Table 18). Rye grass root leachates increased wheat plant height significantly at three and five weeks after planting.

Table 18 Effects of root exudates on wheat plant height and dry mass

Plant type

Wheat plant height at 3 wks (cm)

Root leachate

Wheat plant height at 5 wks (cm)

Wheat dry mass (g)

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

34.6ab

33.2 bc

34.1 bc

32.1 c

32.5 bc

34.7ab

36.4a

33.5 bc

45.0ab

45.7ab

44.3ab

42.8 b

43.5ab

44.9ab

45.8a

43.1ab

0.96ab

0.87ab

0.93ab

0.77 b

0.87ab

1.00a

0.97a

0.89ab

LSD (P≤0.05) 2.3

2.9

0.19

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lupine

No significant differences between root leachate treatments were recorded in lupine dry mass (Table 19). Root leachates from barley increased lupine plant height significantly at three weeks after planting from that attained at the control. At five weeks after planting, a growth-stimulating effect from barley, medic and rye grass root leachates was evident in lupine plant height.

Table 19 Effects of root exudates on lupine plant height and dry mass

Root leachate

47

Plant type

Lupine plant height at 3 wks (cm)

Lupine plant height at 5 wks (cm)

Lupine dry mass (g)

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

18.6a

17.3ab

17.7ab

16.2 bc

14.1 c

16.3abc

17.1ab

15.5 bc

29.9ab

28.3abc

28.1 bc

27.1 bc

25.7 c

31.3a

29.7ab

25.9 c

0.8ab

0.82ab

0.86a

0.87a

0.73 b

0.87a

0.84a

0.82ab

LSD (P≤0.05) 5.5

3.2

0.1

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lucerne

No significant differences between root leachate treatments were observed at five weeks after planting or in lucerne dry mass (Table 20). Barley root leachate significantly increased lucerne shoot length at three weeks after planting.

Table 20 Effects of root exudates on lucerne shoot length and dry mass

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

Lucerne shoot length at 3 wks

(cm)

11.7a

9.5 b

8.6 b

9.8ab

7.9 b

9.5 b

9.8ab

8.5 b

Root leachate

Lucerne shoot length at 5 wks

(cm)

26.3a

20.7abc

18.1 bc

19.5abc

17.5 c

24.9ab

23.7abc

21.1abc

Lucerne dry mass (g)

0.31a

0.24a

0.20a

0.30a

0.32a

0.32a

0.25a

0.30a

LSD (P≤0.05) 2.1

7.1

0.18

*Means followed by the same letter are not significantly different at the 0.05 probability level

Medic

Treatment with lupine root leachate significantly inhibited both shoot length of medic at three weeks and cumulative germination percentage (Table 21). At five weeks after planting, wheat and lupine root leachates inhibited medic shoot length significantly from that attained at the control. The dry mass of medic treated with lupine root leachates was significantly lower than the control, but in contrast to this, it was significantly increased by lucerne root leachates.

48

Table 21 Effects of root exudates on medic shoot length and dry mass

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic shoot length at 3 wks

(cm)

7.5ab

7.3ab

6.1 bc

3.7 d

6.5abc

Root leachate

Medic shoot length at 5 wks

(cm)

15.5ab

12.5 bc

10.6 cd

7.9 d

15.2ab

Medic dry mass (g)

0.40 b

0.42ab

0.41 b

0.20 c

0.59a

Medic

Rye grass

Control

5.5 c

8.0a

6.4abc

14.6abc

17.5a

15.2ab

0.35 bc

0.46ab

0.41 b

LSD (P≤0.05) 1.7

4 0.17

*Means followed by the same letter are not significantly different at the 0.05 probability level

Rye grass

Lucerne root leachates significantly inhibited rye grass plant height at three weeks after planting (Table 22). No significant differences between root leachate treatments were recorded in rye grass plant height at five weeks. The dry mass of rye grass treated with wheat and lupine root leachates was significantly higher than the control.

In contrast to this, rye grass root leachate, significantly reduced rye grass dry mass.

Table 22 Effects of root exudates on rye grass plant height and dry mass

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

LSD (P≤0.05)

Rye grass plant height at 3 wks

(cm)

30.8a

30.3a

29.7a

29.6ab

26.1 b

27.4ab

29.8a

30.9a

3.5

Root leachate

Rye grass plant height at 5 wks

(cm)

39.2a

38.8a

37.8a

38.2a

36.5a

38.7a

36.4a

40.5a

4.4

Rye grass dry mass (g)

0.72 b

0.78 b

0.97a

0.97a

0.86ab

0.71 b

0.56 c

0.78 b

0.15

49

*Means followed by the same letter are not significantly different at the 0.05 probability level

Experiment 4

Above-ground plant residue leachate: greenhouse

Barley

Leachates from medic plant residues increased barley plant height significantly at three weeks after planting (Table 23). At five weeks after planting, leachate from lucerne had stimulated barley height significantly from that attained at the control treatment. The dry mass of barley treated with wheat plant residue leachate was significantly greater than the control. In contrast to this, the dry mass of barley treated with medic residues, were significantly reduced.

Table 23 Effects of above-ground leachates on barley plant height and dry mass

Plant type

Above-ground leachate

Barley plant height at 3 wks (cm)

Barley plant height at 5 wks (cm)

Barley dry mass (g)

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

25 c

27.7abc

29.3abc

30.3abc

33.7ab

34.4a

32.8ab

26.4 bc

36.9 b

38ab

41.6ab

42.2ab

46.9a

44.8ab

41.1ab

36.7 b

2.09 bc

1.56 bc

3.97a

2.35 bc

1.44 bc

1.36 c

1.69 bc

2.42 b

LSD (P≤0.05) 7.7

9.4

1.04

*Means followed by the same letter are not significantly different at the 0.05 probability level

Canola

Above-ground leachates from lucerne, medic and rye grass increased canola plant height significantly from that attained with the control at three and five weeks after planting (Table 24). The dry mass of canola treated with wheat above-ground leachates was significantly higher than the control.

Table 24 Effects of above-ground leachates on canola plant height and dry mass

Above-ground leachate

50

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

LSD (P≤0.05)

Canola plant height at 3 wks (cm)

5.5 d

7.0 bcd

7.1 bcd

5.7 cd

9.3a

8.1ab

7.7abc

5.5 d

2.1

Canola plant height at 5 wks (cm)

12.6 c

14.2 bc

14.8abc

15.2abc

17.2ab

17.4a

15.7ab

12.3 c

3.1

Canola dry mass (g)

2.52 b

2.23 bc

4.34a

2.19 bc

1.35 c

1.41 bc

1.58 bc

1.77 bc

2.82

Wheat

Lucerne, medic and rye grass above-ground leachates increased wheat plant height significantly more than that attained at the control at three weeks after planting (Table

25). At five weeks after planting, leachate from barley, canola, wheat, lupine and lucerne had inhibited wheat height significantly from that attained at the control treatment. The dry mass of wheat treated with barley, canola, lucerne, medic and rye grass above-ground leachates, was significantly less than the control.

Table 25 Effects of above-ground leachates on wheat plant height and dry mass

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Above-ground leachate

Wheat plant height at 3 wks (cm)

Wheat plant height at 5 wks (cm)

Wheat dry mass (g)

29.1 bc

26.9 bc

28.9 bc

28.8 bc

30.3 b

37.0a

34.0a

33.4 c

37.6 c

37.6 c

36.3 c

36.5 c

46.5a

38.7 bc

2.67 cd

1.89 de

4.67a

3.46 bc

0.93 e

1.01 e

1.8 de

51

Control 26.5 c 44.6ab

3.94ab

LSD (P≤0.05) 3.4

6.4

1.06

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lupine

No significant differences between treatments were recorded in both lupine plant heights at three and five weeks, or dry mass (Table 26).

Table 26 Effects of above-ground plant residue leachates on lupine plant height and dry mass

Plant type

Above-ground leachate

Lupine plant height at 3 wks (cm)

Lupine plant height at 5 wks (cm)

Lupine dry mass (g)

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

17.8ab

9.0 b

19.1a

9.8 b

20.6a

14.7ab

14.9ab

12.7ab

24.1ab

16.2 b

31.8a

16.0 b

29.4ab

27.4ab

25.8ab

22.1ab

2.61ab

2.31abc

3.48a

1.77 bc

0.87 c

1.07 bc

2.06abc

1.90abc

LSD (P≤0.05) 9 14.3

1.64

*Means followed by the same letter are not significantly different at the 0.05 probability level

Lucerne

At three weeks after planting, above-ground leachates from barley, lucerne, medic and rye grass had increased lucerne shoot length significantly from that attained at the control treatment (Table 27). Only medic leachates increased lucerne shoot length significantly from that attained at the control treatment, at five weeks after planting. This growth-stimulating effect was also evident in lucerne dry mass after treatment with barley, canola, wheat, lupine, medic and rye grass leachates.

Table 27 Effects of above-ground leachates on lucerne shoot length and dry mass

Plant type

Barley

Canola

Wheat

Above-ground leachate

Lucerne shoot length at 3 wks

(cm)

9.0a

8.2ab

Lucerne shoot length at 5 wks

(cm)

25.7ab

23.3ab

Lucerne dry mass (g)

3.33 bc

2.62 cd

8.4ab

22.4ab

4.81a

52

9.2a

10a

5.5 bc

28.7a

22.5ab

17.3 b

2.12 d

3.11 c

0.74 e

Lupine

Lucerne

Medic

Rye grass

Control

4.7 c

10.3a

16.9 b

22.8ab

4.21ab

1.67 de

LSD (P≤0.05) 3.4

10 0.96

*Means followed by the same letter are not significantly different at the 0.05 probability level

Medic

At three weeks after planting, leachates from lucerne and medic had stimulated medic shoot length significantly from that attained at the control treatment (Table 28).

This growth stimulating effect from lucerne leachates, was also evident at five weeks after planting. The dry mass of medic, treated with barley, canola, wheat, lupine and rye grass leachates, was significantly greater than the control.

Table 28 Effects of above-ground leachates on medic shoot length and dry mass

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Above-ground leachate shoot length at 3 wks

(cm)

5.8ab

5.8ab

Medic shoot length at 5 wks

(cm)

11.9ab

10.4 b

Medic dry mass (g)

3.52 b

3.09 bc

5.4ab

5.1ab

7.1a

11.5ab

10.4 b

15.1a

6.10a

3.08 bc

1.69 cd

Medic

Rye grass

Control

7.2a

6.1ab

3.7 b

13.5ab

11.7ab

10.7 b

2.23 bcd

2.66 bc

0.73 d

LSD (P≤0.05) 2.6

4.1

1.59

*Means followed by the same letter are not significantly different at the 0.05 probability level

Rye grass

Above-ground leachate from lucerne increased rye grass plant height significantly from that attained at the control at three weeks after planting (Table 29). No significant differences between above ground leachates treatments were observed in rye grass plant height at five weeks. The dry mass of rye grass treated with wheat above ground leachates was significantly higher than the control.

Table 29 Effects of above-ground leachates on rye grass plant height and dry mass

53

24.7ab

23.1ab

20.7 b

33.8ab

33.0ab

35.5ab

1.47 d

1.99 bcd

1.98 bcd

Plant type

Barley

Canola

Wheat

Lupine

Lucerne

Medic

Rye grass

Control

Above-ground leachate

Rye grass plant height at 3 wks

(cm)

22.5ab

21.1 b

Rye grass plant height at 5 wks

(cm)

33.3ab

30.1 b

Rye grass dry mass (g)

2.7 b

2.29 bc

24.9ab

21.7ab

28.3a

34.3ab

35.2ab

38.3a

4.27a

2.43 b

1.57 cd

LSD (P≤0.05) 2.8

5.6

0.78

*Means followed by the same letter are not significantly different at the 0.05 probability level

The methodology followed in Experiment 2, is being suggested as a bioassay to study the effects of seedling leachates on the germination process of crop seeds.

Compared to existing procedure that screen for potential seedling allelopathy under laboratory conditions, the advantages of this method are: a) it can be applied to most grain and leguminous crops; b) the possibility of measuring several response parameters on roots or shoots; c) it is suitable for testing early stages of plant development within a short time of less than a week for donor and receiver germination, totalling roughly two weeks for a data set, and d) the possibility of testing various donor densities, easy handling and low costs of material. In addition, testing of the dose-response method as part of the protocol gives it a wider applicability. However, the dose-response design requires high rates of germination of donor plants especially for the higher densities, which can be a problem for poorly germinating cultivars and/or small quantities of available seeds (Belz, 2004). The assay is, however, reliable, simple, and fast, and facilitates high-throughput screening to screen and select for allelopathic traits in several grain crops.

DISCUSSION

Although results from seed and seedling leachates do not have obvious practical relevance, it was suggested by Hoffman et al. (1996) that competitive hierarchies often form during early stages of plant growth, including between germinating seeds and between seedlings. For this reason and to obtain comprehensive data from all plant parts, results from seeds and seedling leachates indicated allelopathic activity for crop species.

54

Barley

Cumulative germination of barley was inhibited 31% and 28% by wheat and medic seed leachates, respectively. Plant height of barley at 5 weeks after planting was inhibited by root leachates from lupine (9%), lucerne (17%) and medic (9%). The dry mass of barley was reduced after treatment with root leachates from wheat (16%), lupine (19%) and lucerne (22%). This finding is in accordance with those of Xuan et

al. (2005), who also reported plant inhibition by lucerne.

Canola

Canola radicle length was reduced by lupine (76%) and lucerne (54%) seed leachate, respectively. After treatment with lucerne (12%) and medic (20%) root leachates, canola plant height was greater at five weeks after planting. Ground lucerne (40%) and medic (41%) residues stimulated canola with regard to plant height at both three and five weeks after planting.

The effects of lupine, lucerne and medic on barley, canola and wheat are generally similar to those reported by Xuan and Tsuzuki (2002). Many reports have indicated that lucerne (M. sativa L.) plants contain water-soluble allelochemicals that are released into the soil environment from fresh leaf, stem and crown tissues, as well as from dry hay, old roots and seeds.

Wheat

The radicle length of wheat was reduced by seed leachates from barley, wheat and lupine. Lupine seed leachates also reduced wheat cumulative germination by 77%.

Ben-Hammouda et al., (2001) reported that the allelopathic potential of barley increased near physiological maturity. Leaves and roots were the most phytotoxic barley plant parts for durum and bread wheats, respectively. Laboratory experiments

(Qasem, 1994) showed that aqueous extracts of many weed species inhibited germination, coleoptile length, root length, and shoot and root dry weight of wheat and barley seedlings grown in Petri-dishes. Extracts of the fresh materials were inhibitory to cereal seedlings compared to extracts from the dried materials.

55

Rye grass root leachates increased wheat plant height by 9% at three weeks after planting. This growth stimulating effect by rye grass root leachates on wheat plant height was also evident at five weeks after planting. At five weeks after planting, leachate from barley, canola, wheat, lupine and lucerne had inhibited wheat height.

The dry mass of wheat treated with lucerne (76%), medic (74%) and rye grass (54%) above-ground leachates, was less than the control. A transition from stimulatory to inhibitory effects over time was observed for rye grass root leachates and aboveground residues. According to Kruidhof (2008) there are two possible explanations for this. Firstly, it is widely recognised that low concentrations of allelochemicals can be stimulating to weed germination and early growth (Lovett et al., 1989; Belz, 2004).

Secondly, the observed stimulation could be a response to increased nutrient and especially nitrate levels in the residue-amended soil, because nitrate stimulates weed seed germination (Bouwmeester & Karssen, 1993).

Results indicating inhibition of wheat growth by leachates from wheat seeds correspond with those by McCalla and Norstadt (1974), who also showed that the water soluble substances in wheat residues reduced germination and growth of wheat seedlings. Furthermore, in pot experiments, Sozeri and Ayhan (1998) found that mixing wheat straw with soil decreased germination of wheat seeds, and increased seedling mortality.

Lupine

Barley seed leachate reduced lupine cumulative germination (75%) from that attained at the control treatment. In contrast, root leachates from barley increased lupine plant height (15%) at five weeks.

Lucerne

Lupine seed leachate had reduced lucerne cumulative germination by 100%. In contrast, canola (102%) and rye grass (93%) seedling leachate stimulated the growth of lucerne seedlings with regard to radicle length. A growth-stimulating effect was evident in lucerne dry mass after treatment with barley, lupine and rye grass residue leachates.

56

The influence of rye grass on wheat and lucerne contrasted with findings of Breland

(1996), who investigated phytotoxicity after spring grain on a loam soil was undersown with Italian ryegrass (L. multiflorum), following on clover (Trifolium

repens) or no cover crop in the previous year. The ryegrass incorporated by spring rotary tillage reduced radish germination up to 45%. Germination values, in response to leachates from fresh ryegrass, were 64%. At double the amount of crop residues, the corresponding value was 27%.

Lucerne produces allelopathic saponins which might be the major cause of yield reduction in subsequent crops (Hall & Henderlong, 1989). Hall and Henderlong

(1989) indicated that the water soluble fraction from lucerne shoots have the characteristics of phenolic compounds. Among several phenolic compounds assayed for their phytotoxicity on root and shoot growth of lucerne, coumarin and t-cinnamic were most inhibitory. Most parts of lucerne plants contain autotoxic substances that inhibit seed germination and early seedling growth. Chung et al. (2000) reported that chlorogenic acid occurs in relatively large amounts (0.39 mg g -1 ) in lucerne aqueous extracts as compared to salicylic acid (0.03 mg g

-1

), and bioassays suggest that chlorogenic acid is involved in lucerne autotoxicity.

Medic

The radicle length of medic was inhibited by lupine seed (60%) and seedling (18%) leachates as was cumulative germination. Treatment with lupine root leachate inhibited radicle length (51%) of medic at five weeks, cumulative germination percentage and reduced medic dry mass. In contrast, medic dry mass was increased

(44%) by lucerne root leachates. At both three and five weeks after planting, aboveground leachates from lucerne had stimulated medic shoot length. The dry mass of medic, treated with lupine above-ground leachates, was greater than the control.

Rye grass

The radicle length of rye grass was inhibited by seed leachates from barley (66%) wheat (57%) and lupine (96%). This growth-inhibiting effect from lupine seed and seedling leachates, was also evident in rye grass cumulative germination percentage. These findings on wheat are in accordance with those by Wu et al.

57

(2000a), who evaluated 92 wheat cultivars for their allelopathic activity on the inhibition of root growth of annual ryegrass. They found significant differences between wheat cultivars in their allelopathic potential at the seedling stage on the inhibition of root elongation of annual ryegrass, with percentage inhibition ranging from 24 to 91 percent.

However, the dry mass of rye grass treated with wheat (24%) and lupine (24%) root leachates was higher than the control, as was dry mass yield of rye grass treated with wheat above-ground leachates. Although the pasture type of rye grass (L.

multiflorum Lam. v. Energa) was used under controlled conditions in order to ensure one seed source and consistent germination, results from the field experiment suggest similar responses for this species and the weed type hybrid (L. multiflorum x

perenne).

Results from the dry mass of rye grass, which was reduced by medic, correspond with those of Fourie (2005) who reported that ‘Paraggio’ medic as a cover crop had a significant negative impact on weed growth during winter. It was speculated that effectively suppressing the winter growing weeds may result in a reduction in the dosage of herbicide applied, and it may minimise the negative effects caused by weeds, such as the harbouring of nematodes and insects during winter (Fourie et al.,

2005). However, such a practice is likely to be exposed to the vagaries of environmental factors, as well as likely being crop and weed-specific.

In contrast, Hoffman et al. (1996) found that rye root residues had more suppressive effects on both emergence and growth of barnyardgrass than did shoot tissues.

Inhibitory effects of both root and shoot extracts of buckwheat on germination of downy brome, although low, (17 to 22%) were similar (Machado, 2007).

Vanillic and o-coumaric acids along with scopoletin may be responsible for the allelopathic effects of barley and wheat (Baghestani et al., 1999). Baghestani et al.

(1999) recommended that an increase in these three allelochemicals may be considered in any cereal breeding programme.

CONCLUSION

58

The allelopathic activity observed for lupine and medic under controlled conditions, corresponds to results obtained in the field and confirms that these leguminous crops should be used prominently, although medic is already planted extensively as rotational crop in the Swartland region. In the long rotation systems of the Overberg region, lupine should be used more frequently in the crop rotation systems used between lucerne plantings. Further studies on the use of crop mulches that do not affect the crop they are used in, yet inhibit or suppress weeds, appear to be warranted. Crop mulches that can provide weed control could reduce dependency on herbicides, in particular those products which are associated with the development of weed resistance. In the case of the mulch being a leguminous plant, the better known attribute of nitrogen fixation will also be achieved.

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