The following article was published as:
Santaniello, C. M. & Koning, R. E. (1996).
Are Radishes Really Allelopathic to Lettuce?
The American Biology Teacher 58(2), 102-103.

I interpret the NABT permission: "Authors are granted unlimited noncommercial use" to mean that I am permitted to reproduce this article here. Since this article was published without benefit of a galley proof, some corrections of the markup editor's work are made here. The authors thank the manuscript reviewers for their efforts. The authors especially thank David Hershey for comments about significant digits. Some of those suggestions have been incorporated here, demonstrating the way electronic publishing can be dynamic in improving science communication. The authors welcome further comment to koning@ecsu.ctstateu.edu.


How-To-Do-It

Are Radishes Really Allelopathic to Lettuce?

Catherine M. Santaniello - - Ross E. Koning

Marcus and Burz (1994) published a demonstration of allelopathy in The American Biology Teacher. The thrust of their work was that sprouting radish seedlings release chemicals into the environment that inhibit germination of lettuce seeds. We were interested in duplicating the demonstration because of its potential use in inquiry-based laboratory exercises. Since the published methods were not explicit, we are not sure we used precisely the same procedures. In any case, our results showed no inhibition of lettuce germination or growth by radish in spite of several different approaches.

A simple replication?

A piece of 9-cm diam Whatman #1 filter paper was placed in the bottom of each of three 10 cm diam Petri dishes. Twenty-six radish (Raphanus sativus cv 'Scarlet Globe') seeds were placed on top of the filter paper in the first dish. Twenty-six lettuce (Lactuca sativa cv 'Salad Bowl') seeds were placed on top of the filter paper in the second dish. In the third dish, 13 lettuce and 13 radish seeds were placed on top of the filter paper. Five mL of distilled water was then added to each dish. Over the next several days, we maintained a volume of 3 mL of "free water" in each of the three dishes by tilting the dish and measuring the pooled volume with a 3 mL syringe body and adding water as needed. The dishes were covered and kept at 23° C under a continuous light source for a period of four days. On the fourth day, the germinated lettuce seeds were counted. In all three repetitions of this attempt, the lettuce seeds germinated 100% with or without radishes present:

Figure 1. Photograph showing lettuce seeds sprouted alone in one dish (left) and with radish seeds (right). The smaller seedlings are lettuce, the larger seedlings are radish. In the left dish, 26 lettuce seeds have germinated. In the right dish, 13 lettuce seeds and 13 radish seeds have germinated. The seed coats, cotyledons and species are easier to distinguish in color (here than in the black-and-white photo published in the journal). In this trial we attempted to follow the procedure of Marcus and Burz (1994).
Hoping to see some kind of allelopathic inhibition, we measured each lettuce seedling from the root tip to the end of the cotyledons. This length was 37.4 ± 11.9 (Mean ± SD) mm with radishes present and 42.4 ± 8.58 mm without radishes present. Student t-testing verified that there was no statistical difference.

Do we need more radishes?

We wondered if our particular radish cultivar produced less of any allelopathic chemical than whatever was used by Marcus and Burz. The same basic procedure was followed but with 50 lettuce seeds and varying the number of radish seeds from 0 to 200 among six petri dishes. The maintenance of the free water volume was critical in the first few hours of this project as 200 radish seeds imbibe a considerable volume. There was no strong demonstration of allelopathy in the results.
Table 1. The effect of increasing the number of radish seeds incubated with 50 lettuce seeds in 5 mL of distilled water in a petri dish. The percent germination of lettuce seeds and mean length of lettuce seedlings were observed after 4 days incubation.
Number of Radish Seeds
0122550100200
% Germination96.0%92.0%98.0%82.0%94.0%88.0%
Mean Length (mm)41.9±12.536.5±13.938.7±13.534.3±14.833.9±11.624.8±7.5

Moreover regression analysis gave us the formula:

Axis Length = -0.073 · (# radish seeds) + 39.8
This shows that the length is about 40 mm regardless of radish seeds and that the added radish seeds decrease length in only a small way (0.073 mm). This inhibition could have been due to shading (light competition) as well.

Do the radishes need more time to produce the chemicals?

Thinking that our radishes might need a few days in the petri dish to make the chemicals before the lettuce seeds were exposed, we revised our project. We varied the number of radish seeds as before, but we allowed the radishes to sprout for four days in the light at 23° C. Then we removed the radish seedlings and put 50 lettuce seeds in the remaining 3 mL of free volume. The lettuce were allowed to grow for a period of four days. On the fourth day, the germinated lettuce seeds were counted and the axis length of each of the lettuce seedlings was measured and recorded. Again there was no obvious demonstration of allelopathy.
Table 2. The effect of increasing the number of radish seeds incubaed in 5 mL of distilled water in a petri dish for 4 days before removing the radish seedlings and adding 50 lettuce seeds. The percent germination of lettuce seeds and mean length of lettuce sedlings were observed 4 days after addition of the lettuce seeds.
Number of Radish Seeds
122550100200
% Germination94.0%86.0%92.0%86.0%86.0%
Mean Length (mm)49.2±27.052.6±20.047.0±20.037.6±22.251.5±17.2
A regression analysis gave us the formula:
Axis length = -0.005 · (#Radish seeds) + 47.9
Again the effect of the number of radish seeds in the dish was minimal (0.005mm) and ten times less than we observed in the previous trial. This reduction in effect is probably due to removal of the shading variable.

Maybe we have the wrong species?

Over the range of number of seeds and time of incubation tested, we could not demonstrate allelopathy by radish over lettuce. Although Marcus and Burz (1994) were apparently able to do this, we believe that we can see the smaller lettuce seedlings among the radish seedlings in their Figure 2 even though the caption states that the lettuce "fail to germinate." Furthermore Choesin and Boerner (1991) reported that Brassica napus (turnip), a radish relative, did not appear to have an allelopathic effect upon Medicago sativa (alfalfa). We used Brassica rapa (birds' rape; Wisconsin Fast Plant), another radish relative, in a parallel experiment and found no statistically significant effect upon germination (88 vs 85%) of lettuce seeds or the length the seedlings (33 vs 27 mm). Choesin and Boerner (1991) suggested that the inhibition of growth of nearby species is caused instead by extreme competitiveness (rapid growth, stress tolerant, nutrient acquisition) of Brassica. Perhaps then there is no chemical allelopathy caused by radish either as it grows with lettuce; any reduced vigor among lettuce is instead due to aggressive competition by radish.

Where to go from here?

It seems that many further studies are needed to screen the potential allelopathy of radish. A battery of target species beyond lettuce needs to be tested. Perhaps students in inquiry-laboratories will find a species sensitive to chemicals produced by radish seedlings. Alternatively, a battery of potential allelopathic species could be tested for inhibiting lettuce. There are many other members of the Brassicaceae; some might be allelopathic to lettuce.

In conclusion we found no simple allelopathic demonstration using 'Scarlet Globe' radish and 'Salad Bowl' lettuce, but we did find a whole series of questions to answer and fertile ground for further experimentation in inquiry-based laboratory experiences.

References

Choesin, D. N. & Boerner, R. E. (1991). Allyl Isothiocyanate release and the allelopathic potential of Brassica napus (Brassicaceae). American Journal of Botany 78 (8), 1083-1090.

Marcus, B. A. & Burz, J. (1994). A simple demonstration of allelopathy. The American Biology Teacher 56 (3), 180-181.

Catherine M. Santaniello is a graduate of Eastern Connecticut State University and can be reached at 22 Pautipaug Lane, North Franklin, CT 06254. Ross E. Koning is Professor of Biology at Eastern Connecticut State University in Willimantic, CT 06226.


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