Journal of Chemical Ecology. Vol. 22. No. 10, 1996

EXPERIMENTS ON BIOASSAY SENSITIVITY IN THE STUDY OF ALLELOPATHY

ESPEN HAUGLAND*

and L A R S O L A V

BRANDSAETER

The Norwegian Crop Research Institute. Plant Protection Center Department of Herbology. N-1432 Aas, Norway (Received August 16, 1995: accepted May 16, 1996)

Abstract--The purpose of these experiments was to study the effects of various experimental procedures and conditions on bioassay sensitivity in alleIopathic studies. The following factors were considered: bioassay methods, choice of test species, the effect of osmotic potential on germination and growth of the test species, growth in dark or in light and the effect of concentrating the extracts in vacuo. Extracts from rape and rye were used in the studies to act as allelopathic agents. Ryegrass appeared as the most sensitive test species when grown horizontally on quartz sand, while radish was the most sensitive test species when grown on filter paper in transparent boxes at a 45 ° angle. Root length was the most sensitive growth variable measured. Percent germination appeared a more sensitive measure than the speed of germination index, " S " , when germinated seeds were counted after one and two days for radish and ryegmss, respectively. A rise in the osmotic potential affected germination more than root length, and radish appeared more sensitive to a rise in osmotic potential than ryegrass. A confounding of germination and root length inhibition may therefore, give misleading results. Key Words--Allelopathy, bioassay, evaporator, germination, growth conditions, growth parameters, osmotic potential, radicle elongation, sensitivity, test species.

INTRODUCTION B i o a s s a y s are u s e f u l and n e c e s s a r y t o o l s t b r s t u d y i n g the allelopathic potential o f plant o r soil e x t r a c t s and for e v a l u a t i n g the activity o f the e x t r a c t s d u r i n g purification and identification o f allelopathic c o m p o u n d s . N e a r l y all r e p o r t s o n

*To whom correspondence should be addressed. 1845 (NFIB-O3~I/96/I(XK)-IB45~

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HAUGLAND AND BRANDSAETER

allelopathy describe some type of bioassay method used to demonstrate allelopathic activity (Leather and Einhellig, 1986). Generally there are two types of measurements for testing biological activity of allelopathic compounds: the measurements of specific biological activity (e.g., inhibition of photosynthesis) or measurements of some aspects of growth (e.g., germination, root dry weight). Putnam and Duke (1978) and Leather and Einhellig (1986) reviewed various bioassays for the study of allelopathy. The most widely used bioassay to test allelopathic activity in an extract is the technique of seed germination in petri dishes on filter paper, sand, soil or agar. Numerous studies also include plant growth bioassays, but the effect on germination is often not separated from the effect on growth (e.g., Liu and Lovett, 1993; Kato-Noguchi et al., 1994). Various factors influence the sensitivity of a bioassay. Different test species may have varying sensitivity to specific allelopathic compounds (e.g. Krogmeier and Bremner, 1989) or to aqueous extracts (e.g., White et al., 1989). In some studies the main goal is to rank different species in their sensitivity to the active compounds (e.g., Smith and Martin, 1994), but in other studies it is important to select the most sensitive test species for detecting low limits of the allelopathic compounds. In both types of studies it is important to realize the possibility of an interaction between the test species and the bioassay method. Recording germination is a commonly used measurement, but several investigations have revealed that this is not the most sensitive parameter (Leather and Einhellig, 1986). Root length has often shown to be a more sensitive parameter, but roots are not as easy to measure as germination, and the design of the bioassay method may influence the growth of the roots. Water potential is important in the germination process (Bradbeer, 1988) but will also influence the growth of the seedlings. The osmotic potential will influence the results of the bioassay test, and variation in sensitivity to osmotic potential between test species may occur. Although Anderson and Loucks (1966) demonstrated the importance of solution osmotic potential when testing plant extracts, very few studies on allelopathic effects include osmotic effect (Leather and Einhellig, 1986). Wardle et al. (1992) found that the allelopathic effects could be substantially overestimated when the osmotic effects were not taken into account. Interactions between experimental conditions can also affect the results, as found between osmotic potential and water uptake in light or in dark for lettuce (Lactuca sativa L.) (Nabors and Lang, 1971 ). Clearly a variety of bioassay methods and experimental factors influence the results of bioassays in the study of allelopathy. The lack of standardized bioassays makes comparisons between different studies very difficult, and even reports originating from the same laboratory differ in the conduct of the assay (Leather and Einhellig, 1986). Therefore, the aim of the present investigation was to study if, and in case to what extent, bioassay method, test species,

EXPERIMENTS ON BIOASSAY SENSITIVITY

1847

osmotic potential, light conditions, and evaporator treatments influence the results and the sensitivity of growth parameters in a bioassay.

METHODS AND MATERIALS

General Winter-rope (Brassica napur L. ssp. oteifera DC. cv. "'Bambu") and winter-rye (Secale cereale L. cv. " D a n k o " ) were greenhouse-grown in pots under day/night regime of 16/8 hr (High-pressure sodium lamps/15000 lux) and a temperature of 20°C. Above ground plant tissue from 40 days old plants of rape and rye were collected and cut separately into pieces of 2 to 3 cm length. Half of the plant matter was oven dried at 50°C for 72 hr befbre extraction. In the dry matter extraction a concentration of 1 g dry matter per 20 ml distilled water was prepared. As the fresh plant material was too voluminous, it was not possible to obtain the same concentration in fresh matter extraction. In this procedure, concentrations of 0.52 g dry matter of rye per 20 ml and 0.68 g dry matter of rape per 20 ml were made. The mixture of plant matter and distilled water was agitated for 5 hr on an orbital shaker at room temperature (24°C). Extracts were filtered through two metal-filters, 0.630 mm and 0.212 mm, before vacuum-filtration through filter paper (Whatman #1). To obtain the same concentration in the fresh matter extracts as in the dry matter extracts (1 g/20 ml on a dry matter basis), the fresh-matter extracts were reduced about 50% in vacuo (40°C), and then diluted to give concentrations of 1 g dry matter/20 ml. The extracts were stored at - 2 0 ° C until used. Radish (Raphanus sativus L., cv "Cherry, Belle orig.") and Italian ryegrass (Lolium muhiforum Lain. var. italicum cv "Mondora") were used as test species in the germination and growth bioassays. A 2 mm radicle length was considered as fulfilled germination. When used in growth assay, the seedlings were pregerminated for 48 hr. Two types of bioassay methods were used in the experiments. Method 1 included plastic dishes (18 cm in diameter, 4 cm deep) containing 150 g of quartz-sand (mean particle size -- 0.28 ram) covered with a glass plate. The quartz sand was moistened with 45 mt extract or distilled water (control). The seeds or seedlings were placed on the surface of the sand. As the accuracy of measurements of radicle elongation in petri dishes is questionable (Leather and Einhellig, 1988), a second bioassay method (Method 2) was developed to obtain easy and fast measurements of root length. For this method transparent square boxes (24 cm long, 11 cm wide and 7 cm high) were used with two layers of filter paper (Munktell 1701, art. no. 261 024) in the bottom of the box. The filter papers were moistened with 30 ml extract or distilled water (control). The

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HAUGLAND AND BRANDSAETER

seedlings of the test species were placed on the filter paper along a horizontal line in the upper part of the box with the root pointed downwards. The boxes were arranged at a 45 ° angle which gave straight roots and root length was easy to measure. The transparent box-system was also used in the germination assay. It was then placed horizontally with two layers of filter paper in the bottom supplied with 20 ml extract or distilled water. All experiments were conducted under the same conditions, in a constantly illuminated (Luma Standard LE 58W Vit-S) growth chamber at 22°C.

Screening Experiment with Test Species and Plant Extracts Using Bioassay Method 1 The experiment was established as a screening experiment with the experimental factors of test species (radish and ryegrass), plant extracts (rape and rye), extracts from fresh or dried material, and two concentrations (1 g/20 ml and l g/60 ml). The dry and fresh matter extracts of rape and rye (1 g/20 ml) were diluted to give the concentration 1 g/60 ml. Bioassay Method 1 was used in this experiment. Fifty seeds of Italian ryegrass or radish were placed on the surface of the sand. Germinated seeds were counted after five days for radish and six days for ryegrass. Root length, shoot length, root dry weight, and shoot dry weight were measured on the seeds that germinated. A 2 k factorial design was used with k = 4 (Montgomery, 1984).

Experiment with Bioassay Methods and Test Species This experiment was set up to separate the effects of germination and growth inhibition, to compare the two bioassay methods, and to study the interaction between bioassay methods and test species. The experiment covered the following concentrations: 0 (distilled water), 1 g/120 ml and 1 g/60 ml. Only dry matter extract of rape was used. To study the effect on germination, the extracts (20 ml each) were added to the transparent square boxes with filter paper. One hundred seeds of radish or ryegrass were placed in each box. Germinated seeds were counted and removed daily for five days. Both bioassay methods were used in the study of seedling growth with 20 seedlings of radish or ryegrass in each box/dish~ Root length, shoot length, root dry weight, and shoot dry weight were measured after four days for both test species.

Experiment on the Effect of Osmotic Potential on Germination and Growth The experiment was conducted to evaluate the effects of osmotic potential on germination and growth of radish and ryegrass. Five concentrations of polyethylene glycol 6000 MW (PEG) were evaluated: 0%, 5%, 10%, 15%, and 20% on a weight basis. Effects on germination were evaluated using horizontally

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EXPERIMENTS ON BIOASSAY SENSITIVITY

placed boxes and germinated seeds were counted and removed daily for five days. Bioassay Method 2 was used to evaluate the effects on root and shoot growth. Root and shoot length were measured after four days. Osmotic potentials for dry matter extracts of rape and rye and PEG solutions were determined using a vapor-pressure osmometer. The potentials were the same for the rye and rape extracts and were for the 1/120, 1/60, and 1/20concentrations 24, 54, and 157 mOsm, respectively. The 5, 10, and 15% PEGsolutions were selected to fit these figures and showed potentials of 17, 65, and 133 mOsm, respectively, while the 20% PEG concentration showed an osmotic potential of 252 mOsm.

E~periment on the Effect of Light on Root Length Inhibition To study whether light conditions could affect radish root length sensitivity to allelopathic compounds, radish seedlings were grown in light or dark with distilled water or dry matter extract of rape (1 g/120 ml). Dark growth conditions were obtained by covering the transparent boxes with aluminum foil. Root length was measured after four days.

Eweriment on the Effect of Evaporator Treatment This experiment was conducted to evaluate whether an evaporator could have any deviating effect beyond the effect of concentrating the extracts. The evaporation procedure was as follows: the volume of the sample was reduced 50% in vacuo (40°C) followed by diluting the concentrated extracts with distilled water to obtain the starting concentrations of 1 g/120 ml and 1 g/60 ml, respectively. Dry matter extracts of rape and rye were used with radish seedlings as test species in bioassay Method 2. Root length was measured after fbur days.

Statistical Considerations Speed of germination index " S " was calculated as described by Bradbeer (1988) and Wardle et al. (1991) as

s=

T+2+T+T+

x 100

Where Ni, N 2. . . . . N s are the proportion of seeds that germinated on day 1, 2 . . . 5. Except for percent germination and speed of germination, all variables were transformed to logarithms to homogenize variances and to allow proportional comparisons to be made. To evaluate the sensitivity of test species by using the actual values of root and shoot measurements (and not relative values), the interactions including the test species factor and the concentration factor can be used. When plotting logarithmic transformed values, the lines which can be

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HAUGLAND AND BRANDSAETER

drawn for each species between concentrations would be parallel if there is no difference in sensitivity between the test species. If there are differences in sensitivity between test species, these lines would not be parallel, which would be shown by a significant interaction term of test species × concentrations. The advantage of using this technique for testing sensitivity in comparison with relative values (percent of control), is the avoidance of the less statistically appropriate relative values. The experiments were established as complete factorial designs and analysis of variance was performed. Interactions including up to three factors were used in the statistical models, except for interactions with the replicate effect, which were all included in the error term. Logarithmic transformed values are presented as geometric means. All experiments were repeated three times, each time representing one replicate in the statistical analysis.

RESULTS

Screening Experiment with Test Species and Plant Extracts Using Bioassay Method 1 Radish was the more sensitive test species for all parameters observed (Table 1). Root length was the most sensitive parameter. Percent germination was about as sensitive as the measurements on shoots, but much less sensitive than root length. There were significant differences between effects of extracts from rape and rye on root length, root dry matter, and shoot dry matter, with the extract from rye giving the greatest reduction in growth of the test species. Although the effect was significant, the difference in root length between extracts from rape and rye was only 1 ram. Extract from a dried material was much more effective in reducing plant growth than those made of fresh material. As expected, phytotoxicity was more severe at the higher concentration than the lower one.

Experiment with Bioassay Methods and Test Species Significant differences in cumulative germination between days, extract concentrations, and test species were recorded (Figure I). The results for test species are presented in separate figures for convenience. There were greater differences between the concentrations during the first two days than later on, especially with ryegrass as test species. Sensitivity of the germination parameter was highest for radish at day one and at day two for ryegrass. The figures indicate a delay in germination for both species for both extract concentrations, which was also shown significantly by the speed of germination index " S " (Table 2). Radish germinated faster than ryegrass, while ryegrass germination seemed to be less affected by increasing extract concentration.

EXPERIMENTS ON BIOASSAY SENSITIVITY

185 1

TABLE 1. MAiN EFFECTS OF TEST SPECIES, TYPE OF EXTRACT, AND CONCENTRATIONS ON GERMINATION, ROOT LENGTH AND DRY MATTER (DM) AND SHOOT LENGTH AND DM. VALUES WITHIN MAIN EFFECTS AND MEASUREMENT ARE CONSIDERED SIGNIFICANTLY DIFFERENT AT THE 5% LEVEL WHEN FOLLOWED BY DIFFERENT LETTERS (PERCENT OF CONTROL 1N PARENTHESES)

Root

Test species: ryegrass radish Ettract from: rape rye Material: fresh dried Concentration: I g/60 ml I g/20 ml

Shoot

Germination % (% of control)

length mm (%)

DM mg (%)

length mm (%)

DM mg (%)

69a (79) 43b (47)

8a (41) 2b (17)

16a {58) 19a (31)

21a (76) 2b (42)

21a (76) 91b (48)

57a (64) 55a (62)

5a (32) 4b (26)

19a (49) 15b (42)

7a (62) 6a 156)

49a (65) 39b (59)

74a (83] 38b (43)

14a (49) lb (10)

37a (72) 7b ~17)

14a (90) 3b (29)

83a (93) 22b (32)

69a (78) 43b (48)

9a (41) 2b (18)

25a (59) l i b (31)

10a (76) 4b (43)

64a (76) 29b (49)

Root and shoot length and shoot DM were more sensitive parameters in bioassay Method 2 than in Method 1 (Table 3). In this experiment, ryegrass was the more sensitive test species, while root length was the most sensitive parameter. The variance analysis showed, however, significant interaction in root length between test species, extract concentration and bioassay method (P = 0.0001), indicating that length of radish roots was most sensitive when using bioassay Method 2, while length of ryegrass roots was most sensitive when grown in Method 1. The Effect o f Osmotic Potential on Germination and Growth The effects of PEG on germination are shown in Figure 2. All main factors and interactions appeared significant in the statistical analysis. Increasing proportion of PEG reduced the germination of ryegrass, and a considerable decrease in germination was recorded when PEG-concentration exceeded 15 %. In radish this "threshold value" appeared already above 10% PEG. In the speed of germination index "S" (Table 4), radish had the fastest germination at 0 and 5% PEG, while ryegrass was the faster one at 10% PEG and above. In root length ryegrass was the least sensitive at 5, 10, and 15% PEG, while radish appeared less sensitive to changed osmotic potential than ryegrass at 20% (Table

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HAUGLAND AND BRANDSAETER

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FIG. 1. The interaction between day of recording, plant extract concentration and the test species ryegmss (a) and radish (b) in cumulative germination (Pdays × tes~species.......... walion = &01L

5). Germination was more affected by increased osmotic potential than root length,

The Effect of Light on Root Length Inhibition Radish root length was significantly longer when grown in dark than in light (P = 0.0001). There was also a significant interaction between the light

EXPERIMENTS ON BIOASSAYSENSITIVITY

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TABLE 2. THE INTERACTIONBETWEEN TEST SPECIES AND EXTRACT CONCENTRATION IN SPEED OF GERMINATIONINDEX 'S' (PERCENT OF CONTROL IN PARENTHESES)a

Concentration: 0 1 g/120mt I g/60ml

Ryegrass

Radish

40(100) 36 (90) 33 (83)

70 (1(30) 53 (76) 44(63)

"P,~,,,~............. ,n,,,,,,, = 0,0003

factor and extract concentration (P = 0.006), showing that radish root length was a more sensitive parameter when grown in dark than in light.

The Effect of Evaporator Treatment The phytotoxicity of the extracts was not changed during evaporation. Neither could a significant interaction between the evaporation factor and the extracts be shown.

TABLE 3. MAIN EFFECTS OF TEST SPECIES, TYPE OF B1OASSAY METHOD, AND CONCENTRATIONS ON ROOT LENGTH AND DRY MATTER (DM) AND SHOOT LENGTH AND DM. VALUES WITHIN MAIN EFFECTS AND MEASUREMENT ARE CONSIDERED SIGNIFICANTLYDIFFERENT AT THE 5 %-LEVEL WHEN FOLLOWED BY DIFFERENT LETTERS (PERCENT OF CONTROL IN PARENTHESES) Root

Test ~Tqecies: ryegrass radish Bioassay: Method I Method 2 Concentration: 0 1 g/120 ml 1 g/60 ml

Shoot

length mm (%)

DM mg (%)

length mm (%)

DM mg (%)

36a (62) 29b (69)

20a (89) 40b (109)

76a (114) 15b (14l)

32a (114) 130b (149)

27a (68) 38b (63)

26a (85) 31a (112)

38a (141) 29b (I 15)

62a (150) 67a (113)

56a (100) 28b (53) 21c (42)

31a (100) 30a (107) 24a (89)

27a (100) 37b (141) 37b (142)

54a (100) 70b (149) 71b (146)

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HAUGLAND AND BRANDSAETER

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100 9o 80

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FIG. 2. The interaction between day of recording, polyethylene glycol 6000 MW (PEG)concentration and the test species ryegrass (a) and radish (b) in cumulative germination. PEG concentrations are 0% (-$-) 5% (-II-). 10% (-O-), 15% (-x-) and 20% (-*-) on a weight basis (pj~y~ PEC-~,,,~,'~.,,-.,-.~~p~i~ = 0.0001).

DISCUSSION

Percent germination has commonly been used to measure the effects of atlelopathic compounds. This is a rapid method for a large number of samples. However, Leather and Einhellig (1985) indicated that percent germination may not be the most sensitive parameter. Speed of germination may be a more sensitive indicator of allelopathic effects (Wardle et al., 1991) as allelochemicals may delay germination rather than affect the final germination percentage. In the present investigation percent germination was a more sensitive indicator than

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TABLE 4. THE INTERACTION BETWEEN TEST SPECIES AND POLYETHYLENE GLYCOL 6000

MW (PEG)-CoNCENTRATION IN SPEED OF GERMINATION INDEX " S ' ' S (% of control)

ryegrass PEG-concentration: 0 5 10 15 20

41 38 33 22 5

(1001 (93) (80) (54) (121

radish

71 50 30 9 2

(11301 (70) (42) (131 (3}

speed of germination, one and two days after initiating the germination bioassay for radish and ryegrass, respectively. In addition to being more sensitive, recording data one or two days after sowing is less time consuming than daily recordings for several days required for computing the speed of germination index. In the present investigation, root length was the most sensitive parameter for detecting growth retarding substances. These results are consistent with previous studies (e.g., Leather and Einhellig, 1986; Wardle et al., 1991; Wardle et al., 1992). The screening experiment, in which the effect on germination and growth was confounded, indicated that radish was a more sensitive test species than ryegrass. The experiment which included both bioassay methods on the other hand showed that test species sensitivity depends on bioassay method, and ryeTABLE 5. THE INTERACTION BETWEEN TEST SPECIES AND POLYEFHYLENE GLYCOL 6000 M W (PEG)-CoNcENTRA-I-I