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Blackwell Science, LtdOxford, UKWBMWeed Biology and Management1444-61622005 Weed Science Society of JapanSeptember 200553143149Research PaperInhibition by hound’s-tongue seed leachateA. Rashid et al.

Weed Biology and Management 5, 143–149 (2005)

RESEARCH PAPER

Inhibition of seed germination and seedling growth by hound’s-tongue (Cynoglossum officinale L.) seed leachate ABDUR RASHID,† NANCY H. FURNESS, BRIAN E. ELLIS and MAHESH K. UPADHYAYA* Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC, Canada The leaching of phenolics from decoated seeds, seed coats and pericarps of hound’s-tongue (Cynoglossum officinale L.), factors affecting leaching, and seed germination and seedling growth inhibitory activities of the leachate were investigated. Embryonic axes and cotyledons contained a large amount of methanol-soluble phenolic substances. Decoated seeds released phenolics into an aqueous incubation medium and this solution was capable of inhibiting root elongation of several grassy and broadleaf species, but not of hound’s-tongue. The leachate and the phenolic fraction of the leachate inhibited germination of bluebunch wheatgrass (Pseudoroegnaria spicata (Pursh) A. Löve [syn. Agropyron spicatum (Pursh) Scribn. and J. G. Sm.]). The non-phenolic fraction of the leachate did not inhibit seed germination or root and shoot elongation of bluebunch wheatgrass. Low oxygen and an increasing temperature significantly increased the amount of phenolic substances leached from decoated seeds. Increased leaching of phenolic substances related to an increasing temperature was associated with the loss of seed viability. Further investigation of the role of water-extractable hound’s-tongue seed phenolics in the interaction of this species with its biotic environment under field conditions is needed to determine the ecological significance of these findings. Keywords: Cynoglossum officinale L., germination, oxygen, phenolics, seed leachate, seedling growth.

INTRODUCTION Plant phenolics have been implicated in the regulation of seed germination (Rasmussen & Einhellig 1977; Chung et al. 2002), allelopathic interaction between species (Rice 1984; Ridenour & Callaway 2001) and the defense against pathogens (Paul & Sharma 2002; Prats et al. 2003). Mature seeds of hound’s-tongue (Cynoglossum officinale L., family: Boraginaceae), a noxious rangeland weed of

*Correspondence to: Mahesh K. Upadhyaya, Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. Email: [email protected] † Present address: Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada. Received 27 December 2004; accepted 31 May 2005

British Columbia, Canada (Upadhyaya et al. 1988), are innately dormant and contain large amounts of methanol (50% v/v)-extractable phenolic substances (Qi et al. 1993), constituting >90% of the total phenolic pool in the embryo (Stabell et al. 1998). The seed phenolics do not appear to play any direct role in the regulation of germination (Qi et al. 1993), but preliminary laboratory studies showed that the aqueous leachate of decoated hound’s-tongue seeds, which contain phenolic substances, could inhibit root elongation of a number of rangeland grass and weed species. The objective of this study was to characterize the seed germination and root and shoot elongation inhibitory activities of hound’s-tongue seed leachate. Specifically, we were interested in determining: (i) if hound’s-tongue seed leachate inhibits germination and seedling growth of rangeland forage grasses (bluebunch wheatgrass [Pseudoroegnaria spicata (Pursh) A. Löve syn. Agropyron spicatum (Pursh) Scribn. and J. G. Sm.] and orchardgrass [Dactylis

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glomerata L.]) and broadleaf weeds (spotted knapweed [Centaurea maculosa Lam.], lamb’s-quarters [Chenopodium album L.] and hound’s-tongue); and (ii) whether low oxygen (O2), temperature and seed viability influence the leaching of inhibitory substances. MATERIALS AND METHODS Seed source The seeds of bluebunch wheatgrass and orchardgrass were purchased from Richardson Seed Company, Abbotsford, BC, Canada. The seeds of spotted knapweed and lamb’s-quarters and the nutlets of hound’stongue were collected from natural populations in British Columbia. Preparation of hound’s-tongue seed leachate The nutlets of hound’s-tongue were imbibed for 10 min in distilled water, the barbed pericarps were removed using a scalpel and the seeds were allowed to dry overnight. The brittle, black seed coats were then dissected away with a scalpel, taking care to avoid damage to the underlying embryonic tissue. Unless stated otherwise, leachates were prepared by incubating 1 g each of decoated seeds, seed coats or pericarps at 25∞C in 30 mL of distilled water in sealed Erlenmeyer flasks (VWR International, Mississauga, ON, Canada), with no void space, in darkness. The flasks were incubated on a gyratory shaker (75 r.p.m) for 72 h, while the incubation medium was filtered through Miracloth (CalbiochemNovabiochem, La Jolla, CA, USA) and centrifuged at 13 800 g for 5 min, and the supernatant was stored at -20∞C. The effect of low O2 levels on the leaching of phenolic substances was studied by comparing leachates prepared under aerobic and low-O2 conditions.Three replicates of 15 decoated seeds each were shaken for 24, 48 and 72 h in sealed 250 mL Erlenmeyer flasks containing either air (aerobic condition) and 5 mL of distilled water or nitrogen (N) gas (low-O2 condition) and 5 mL of distilled water through which N gas had been bubbled. Bubbling N gas through distilled water lowered the dissolved O2 content of the water by 60% compared to untreated, distilled water.The O2 content was measured using a Clarktype O2 electrode and a YSI Model 53 O2 meter (Yellow Springs Instruments, Yellow Springs, OH, USA). To study the effect of seed viability on the leaching of phenolic substances, decoated seeds were killed by hightemperature incubation in a moist environment. Seeds were placed in sealed Petri dishes (1 g seeds plate-1) with

moist filter paper lining the lids. This created a humid environment without bringing the seeds into contact with free water in order to prevent the leaching of phenolic substances. The Petri dishes were incubated in darkness at 50∞C for 5 h in sealed ‘Frig-O-Seal’ containers (Cascades Inopak, Drummondville, QC, Canada) lined with moist paper towels. The loss of seed viability was verified by determining the germinability of seed samples taken from the treated seed lots. The leaching of phenolic substances from the treated seeds was compared with the untreated seeds (control) at 24, 48 and 72 h. The effects of temperature on leaching and on the subsequent germinability of decoated seeds were studied by incubating seeds (0.33 g) in 10 mL of water at 5, 12, 25, 31, 41 and 55∞C. The phenolic content and inhibition of bluebunch wheatgrass seed germination by the resulting leachates were measured. The leached seeds were transferred to Petri dishes for germination assays. There were three replicates for each temperature treatment. Separation of phenolic substances from hound’s-tongue seed leachate Phenolic substances were separated from hound’stongue leachates using Amberlite XAD-7 (Sigma, St Louis, USA), a non-ionic polymeric resin column. The Amberlite-treated leachate was designated as the ‘nonphenolic fraction’. Substances retained by the Amberlite resin were recovered by washing the resin column with 80% aqueous methanol and evaporation of the methanol in the eluate with a stream of N gas. The volume of the resultant aqueous phase was made up to the original volume of the leachate using distilled water. This fraction was designated as the ‘phenolic fraction’ in this study. The phenolic content (mg mL-1) was assayed using Folin reagent (Ferraris et al. 1987), with chlorogenic acid as a standard. Seed germination assay Twenty bluebunch wheatgrass seeds were incubated in 6.0 cm diameter Petri dishes lined with 5.5 cm filter discs (No. 1; Whatman International, Maidstone, UK) wetted with 0.8 mL aliquots of either distilled water (control), leachate, the phenolic fraction or the nonphenolic fraction. The dishes were incubated at 25∞C in darkness as described above and seed germination (protrusion of radicle ≥5 mm) was monitored at various time intervals. The Petri dishes were arranged in a randomized, complete-block design with three replicates for each treatment.

Inhibition by hound’s-tongue seed leachate Inhibition of shoot and root elongation Hound’s-tongue, orchardgrass, bluebunch wheatgrass, spotted knapweed and lamb’s-quarters seeds were initially germinated in 9 cm Petri dishes lined with two 9 cm Whatman No. 1 filter discs moistened with 5 mL of distilled water. Ten uniform seedlings with ª5 mm radicle lengths were placed in 6.0 cm diameter Petri dishes lined with 5.5 cm Whatman No. 1 filter discs wetted with 0.8 mL aliquots of either distilled water (control) or leachates from decoated seeds, seed-coats or pericarps.The seedlings were incubated at 25∞C in darkness and seedling root lengths were recorded after four days. In separate experiments, following the above procedure, bluebunch wheatgrass seedlings were incubated in distilled water (control), whole leachate, or the phenolic or non-phenolic fraction of the decoated seed leachate. Root and shoot lengths were measured after 48 h in the first experiment and 72 h in the second experiment. A randomized, complete-block design with three replicates for each treatment was used and all experiments were repeated. Where there was no experiment ¥ treatment interaction, data were pooled (Tables 1, 2); otherwise, the results of the two experiments are presented separately. Means were separated

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using Fisher’s protected LSD. Figures 1–4 show the results of one of the two experiments as the results of both experiments were similar. RESULTS The leachate from the decoated hound’s-tongue seeds inhibited the root elongation of rangeland forage grasses (bluebunch wheatgrass and orchardgrass) and broadleaf weeds (spotted knapweed and lamb’s-quarters; Table 1), but had no effect on hound’s-tongue seedling root elongation. The hound’s-tongue seed-coat leachate inhibited orchardgrass, bluebunch wheatgrass and spotted knapweed root elongation, but did not affect lamb’s-quarters or hound’s-tongue seedling root elongation.The pericarp leachate did not affect root elongation in any of these species.The leachates of decoated seeds and seed-coats contained substantially more phenolic substances (per unit weight) than pericarp leachates (Table 3). The phenolic content of hound’s-tongue cotyledons (0.696 ± 0.028 mg) was ª12-fold that of the embryonic axis (0.056 ± 0.002 mg) on a per seed basis (Table 2). However, on a unit fresh weight basis, the embryonic axis (87.0 ± 4.8 mg) had a higher phenolic content than the cotyledons (68.9 ± 3.0 mg).

Table 1. The effect of aqueous leachates of hound’s-tongue decoated seeds, seed coats and pericarps on the root elongation of five species Leachate

Control Decoated seed Seed coat Pericarp Fisher’s protected LSD (0.05)

Root length (mm) Hound’s-tongue

Orchardgrass

Bluebunch wheatgrass

Spotted knapweed

Lamb’s-quarters

20.8 ± 1.7 21.3 ± 2.1 20.7 ± 2.0 22.0 ± 2.0 NS

24.1 ± 2.5 14.2 ± 2.1 22.0 ± 1.7 23.0 ± 2.2 1.7

26.8 ± 2.7 16.4 ± 2.1 24.7 ± 1.9 25.7 ± 1.7 1.8

21.6 ± 2.0 10.0 ± 1.5 15.8 ± 2.0 21.4 ± 2.3 1.7

18.1 ± 1.9 9.5 ± 1.4 18.1 ± 2.2 18.0 ± 1.9 1.5

The values are means ± standard errors of six replicates of 10 seedlings pooled from two experiments. LSD, least significant difference; NS, not significant.

Table 2. The distribution of phenolics in decoated hound’s-tongue seeds Seed part

Embryonic axis Cotyledon

Phenolic content

Level of significance

mg seed-1

mg g-1 fresh weight

0.056 ± 0.002 0.696 ± 0.028

87.0 ± 4.8 68.9 ± 3.0

The values are means ± standard errors of six replicates pooled from two experiments. **Significant at P £ 0.01.

** **

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Bluebunch wheatgrass germination was significantly inhibited by the decoated-seed leachate and the phenolic fraction of the leachate but not by the non-phenolic fraction (Fig. 1). By 17 days, seeds incubated in the leachate and in the phenolic fraction germinated at 12.5% and 7.5%, respectively, compared with 44% of the control. The leachate and phenolic fraction also inhibited bluebunch wheatgrass root elongation in the first experiment by 93% and 61%, respectively, and by 89% and 87%, respectively, in the second experiment com-

pared with aqueous controls (Table 4). In the second experiment, bluebunch wheatgrass shoot elongation was stimulated by the non-phenolic fraction (40%), but the leachate and phenolic fraction had no effect. Decoated hound’s-tongue seeds incubated in a low-O2 environment released more phenolic substances in the medium than those incubated under aerobic conditions and this effect increased with time (2.5-fold at 72 h; Fig. 2).

0.15

Phenolics (mg decoated seed–1)

Germination (%)

75

50

25

0.10

0.05

0 0

5

10

15

20

25

30

0.00 0

Time (days) Fig. 1. The effects of hound’s-tongue decoated-seed leachate, non-phenolic fraction and phenolic fraction on the germination of bluebunch wheatgrass seeds. Values are means ± standard errors of three replicates of 20 seeds each. (), non-phenolic; (), control; (), leachate; (), phenolic.

20

40

Decoated seed Seed coat Pericarp Fisher’s protected LSD (0.05)

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Duration of leaching (h) Fig. 2. The effects of aerobic and low-oxygen conditions on the leaching of phenolic substances from decoated hound’s-tongue seeds.Values are means ± standard errors of three replicates of 15 seeds each. (), low oxygen; (), aerobic.

Table 3. The phenolic content of aqueous leachates from decoated seeds, seed coats and pericarps of hound’s-tongue Leachate

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Phenolic content (mg g-1 fresh weight) Experiment 1

Experiment 2

40.4 ± 3.7 46.6 ± 0.5 8.8 ± 0.8 7.7

51.9 ± 1.3 41.1 ± 2.1 11.2 ± 0.4 4.9

The values are means ± standard errors of three replicates. LSD, least significant difference.

Inhibition by hound’s-tongue seed leachate The incubation temperature during leaching had a significant effect on the level of phenolic substances released from decoated hound’s-tongue seeds (Fig. 3). The phenolic content of the leachate increased as the incubation temperature increased from 5–55∞C. The amount of phenolic substances released at 25∞C (18.8 mg g-1 seed) was twice that at 5∞C (9.3 mg g-1 seed), while the amount released at 55∞C (100.2 mg g-1 seed) was 5.3-fold of that at 25∞C. Germination assays of

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hound’s-tongue seeds incubated at various temperatures showed an abrupt loss of seed germinability at ≥ 25∞C. Seeds did not germinate when incubated in an imbibed state >31∞C. The seeds heat-killed by imbibition at a high temperature (50∞C for 5 h) before leaching released 2.5-fold the quantity of phenolic substances compared with the control seeds (Fig. 4).

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Germination (%)

80 60 60 40 40 20

20

0

Phenolics (mg g–1 decoated seed)

100

80

Phenolics (mg g–1 decoated seed)

120

100

100

80

60

40

20

0 0

10

20

30

40

50

60

0 0

Temperature (°C) Fig. 3. The effect of incubation temperature on the germination and leaching of phenolic substances from decoated hound’s-tongue seeds.Values are means ± standard errors of three replicates of 0.33 g of seeds each. (), phenolics; (), germination.

20

40

60

80

Duration of leaching (h) Fig. 4. The effect of heat-killing on the leaching of phenolic substances from decoated hound’s-tongue seeds. Values are means ± standard errors of three replicates of 1.0 g of seeds each. (), heat-killed; (), control.

Table 4. The effects of decoated hound’s-tongue seed leachate and its phenolic and non-phenolic fractions on shoot and root elongation of bluebunch wheatgrass seedlings Parameter

Length (mm) Experiment 1 (48 h)

Control Non-phenolic fraction Phenolic fraction Leachate Fisher’s protected LSD (0.05)

Experiment 2 (72 h)

Shoot

Root

Shoot

Root

12.5 ± 1.1 13.1 ± 0.7 12.8 ± 0.5 11.0 ± 0.3 NS

8.8 ± 2.2 4.8 ± 1.4 3.4 ± 0.6 0.6 ± 0.2 3.7

13.0 ± 0.4 18.2 ± 0.4 12.1 ± 2.4 16.8 ± 1.3 4.5

3.8 ± 0.8 4.3 ± 0.9 0.5 ± 0.4 0.4 ± 0.3 2.3

The values are means ± standard errors of three replicates of 10 seedlings each. LSD, least significant difference; NS, not significant.

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DISCUSSION Hound’s-tongue seeds have been shown to contain high levels of phenolic substances (Qi et al. 1993; Stabell et al. 1996), but the ecophysiological role(s) of these substances in this species is unknown. In other systems, phenolics have been implicated in allelopathic interactions, defense against soil-borne pathogens, regulation of seed germination and the protection of plants from ultraviolet radiation (Kefeli & Kadyrov 1971; Naqvi & Hanson 1982; Einhellig 1986; Putnam & Tang 1986; Jain et al. 1989; Reigosa et al. 1999; Ohno et al. 2000; Bieza & Lois 2001; Paul & Sharma 2002). Qi et al. (1993) found that the methanol-soluble (50% v/v) phenolic substances in hound’s-tongue seeds are not directly involved in the regulation of seed germination in this species. However, the present work demonstrates that the leachate of decoated hound’s-tongue seeds inhibits root elongation of rangeland forage grasses (orchardgrass and bluebunch wheatgrass) and broadleaf weeds (spotted knapweed and lamb’s-quarters) (Table 1), but not that of hound’stongue seedlings. The seed-coat leachate inhibits root elongation in orchardgrass, bluebunch wheatgrass and spotted knapweed, while pericarp leachate, which has a much lower phenolic content compared to decoated seeds or seed coats on a fresh weight basis (Table 3), has no effect on root elongation in any of the species studied. Both the embryonic axes and cotyledons of hound’stongue seeds contain a high concentration of phenolics. Even though the absolute amount of phenolics found in the embryonic axis was only ª8% of that in the cotyledon, the concentration of phenolics in the embryonic axis was higher than that in the cotyledons (Table 2). Despite this metabolic commitment to phenolic accumulation, the phenolics present in the hound’s-tongue embryo do not appear to be directly involved in the regulation of hound’s-tongue seed germination (Qi et al. 1993; Stabell et al. 1996). Stabell et al. (1996) showed that during seed development, soluble phenolics in the hound’s-tongue embryo polymerize to form an impermeable seed coat that prevents mature seeds from germinating.The present study suggests that phenolics present in mature hound’stongue seed might play a role in the interaction of this species with its biotic environment. In addition to the potential role of seed phenolics in allelopathy (Friedman & Waller 1983), Hendry et al. (1994) demonstrated a correlation between the seed phenolic content and seed bank persistence. They concluded that phenolic compounds might protect seeds against herbivory and microbial decomposition in the soil. The phenolic fraction of the leachate inhibited bluebunch wheatgrass seed germination (Fig. 1) and root

elongation (Table 4). Hound’s-tongue leaves also contain substances, presumably phenolic compounds, with strong seed germination and seedling growth inhibitory activities (Li et al. 2002). Whether the phenolics released by seeds and following decomposition of winter-killed leaves or of dead plants following seed maturation exert an allelopathic influence in nature remains to be investigated. It should be noted that even though the fraction termed ‘phenolic fraction’ in this study contained a high level of phenolic substances, the possible presence of non-phenolic substances, which could influence growth, cannot be ruled out. A number of factors might modulate the allelopathic influence of plants by affecting the release of the active substances. For example, under O2-deficient and waterlogged conditions, the decomposition of rice stubble in the soil increased the release of phytotoxic substances (Chou & Lin 1976). Stressed sunflower plants have been shown to release greater amounts of phenolic substances than non-stressed plants (Koeppe et al. 1976; Hall et al. 1982), which subsequently caused greater inhibition of redroot pigweed (Amaranthus retroflexus L.) germination in the soil (Hall et al. 1982). The results of our study show that low-O2 conditions during leaching increased the release of phenolic substances from decoated hound’s-tongue seeds twofold to fourfold (Fig. 2). The release of phenolic substances from decoated hound’s-tongue seeds increased with the increasing temperatures in this study (Fig. 3). The increased release of phenolics at ≥25∞C was probably partly related to a direct effect on leaching and partly to a loss of seed viability, which could reduce cell integrity. Chou et al. (1981) reported that decomposing rice straw produced maximum phytotoxins at 20–25∞C after 6–9 weeks of incubation under field conditions. Higher temperatures increased the production of phytotoxins, but they were rapidly degraded. Heat-killed decoated hound’s-tongue seeds released significantly greater amounts of phenolic substances than untreated (viable) seeds (Fig. 4). This suggests that even dead seeds might contribute to the competitive success of this species by releasing substances which could discourage the growth of competing species. Friedman and Waller (1983) reported that the rate of release of caffeine from old, dead coffee (Coffea arabica L.) seeds was significantly higher than that from young and viable seeds, highlighting the importance of including non-viable, as well as viable, seeds when evaluating the allelopathic potential of seeds. They suggested that a seed-induced zone of allelopathy might occur close to parent plants where many seeds are released.

Inhibition by hound’s-tongue seed leachate In summary, the results of this study show that: (i) aqueous leachate of decoated hound’s-tongue seeds, which is rich in phenolic substances, inhibits the seed germination of bluebunch wheatgrass and the root elongation of bluebunch wheatgrass, orchardgrass, spotted knapweed and lamb’s-quarters; and (ii) high temperature, O2 deficiency and loss of seed viability increase the release of phenolic substances from the seed. The greater release of phenolics at temperatures >25∞C was likely to be related, in part, to the direct effect of temperature on the leaching process and, in part, to the loss of seed viability. Further investigation of the role of water-extractable hound’s-tongue seed phenolics in the interaction of this species with its biotic environment under field conditions is needed to determine the ecological significance of these findings. ACKNOWLEDGMENTS We thank Natural Sciences and Engineering Research Council of Canada and Jean Bostock Memorial Fund for financial support, and Ms Lisa Yu for technical assistance. REFERENCES Bieza K. and Lois R. 2001. An Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavonoids and other phenolics. Plant Physiol. 126, 1105–1115. Chou C.-H., Chiang Y.-C. and Cheng. H.H. 1981. Autointoxication mechanism of Oryza sativa. III. Effect of temperature on phytotoxin production during rice straw decomposition in soil. J. Chem. Ecol. 7, 741–752. Chou C.-H. and Lin H.-J. 1976. Autointoxication mechanism of Oryza sativa. I. Phytotoxic effects of decomposing rice residues in soil. J. Chem. Ecol. 2, 353–367. Chung I.M., Kim K.H., Ahn J.K., Chun S.C., Kim C.S., Kim J.T. et al. 2002. Screening of allelochemicals on barnyardgrass (Echinochloa crusgalli) and identification of potentially allelopathic compounds from rice (Oryza sativa) variety hull extracts. Crop Prot. 21, 913–920. Einhellig F.A. 1986. Mechanisms and modes of action of allelochemicals. In: The Science of Allelopathy (ed. by Putnam A.R. and Tang C.-S.). John Wiley & Sons, New York, 171–188. Ferraris L., Abbattista-Gentile I. and Matta A. 1987. Variations of phenol concentrations as a consequence of stresses that induce resistance to Fusarium wilt of tomato. Z. Pflanzenk. Pflanzen. 94, 624–629. Friedman J. and Waller G.R. 1983. Seeds as allelopathic agents. J. Chem. Ecol. 9, 1107–1117. Hall A.B., Blum U. and Fites R.C. 1982. Stress modification of allelopathy of Helianthus annuus L. debris on seed germination. Am. J. Bot. 69, 776–783.

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