Effect of phytotoxicity of allelopathic extracts on growth of various plant species

How does the effect of the extracts of allelopathic plant on the growth of leguminous plants, measured in terms of percentage germination depends on the type of the allelopathic plant chosen- Ocimum tenuiflorum (Tulsi) and Azadirachta indica (Neem) and the type of plant taken-Vigna radiata, Vigna unguiculate, Cicer arietinum, Cicer arietinum L, Pisum sativum ?

On a tour of a plant nursery the manager was talking about weed removal from plants. He mentioned the concept of plant allelopathy in passing and it happened to catch my attention. When I went home I decided to do some further research on the topic and found out that a lot of common plants are allelopathic in nature. What i didn’t understand is why this is not a developed technique. To understand the real world relevance of the allelopathic properties of plants i decided to experiment with the resources readily available to me. In environmental sciences in the 9th grade we studied the economic structure of india briefly wherein i found out that most indians stick to a diet of basic legumes. Seeing that my house had multiple variations of leguminous seeds it was evident that this study would be relevant if it had a positive outcome.

Legumes, or pulses, are flowering plants in the Leguminosae family, (Fabaceae, Leguminosae, or Papilionaceae, commonly known as legume, pea, or bean Family, in the Order of Fabales, is a Family, the third largest land plant Family in terms of number of Species). Legumes have often been associated with poverty throughout history. In cultures where a portion of the population can obtain protein from animal sources, beans are seen as food only fit for peasants; the “poor man’s meat”. Eating beans is a cheap way of maintaining important nutritional requirements, but is also accompanied with a negative stigma associated with the lower class; those that could not afford meat had to depend on beans. Food security, lowering the risk of climate change and meeting the increasing demand for energy will increasingly be critical challenges in the years to come. Producing sustainably is therefore becoming central in agriculture and food systems. Legume crops could play an important role in this context by delivering multiple services in line with sustainability principles. In addition to serving as fundamental, worldwide source of high-quality food and feed, legumes contribute to reduce the emission of greenhouse gases, as they release 5–7 times less GHG per unit area (Wong and Ng)compared with other crops; allow the sequestration of carbon in soils with values estimated from 7.21 g kg⁻¹ DM, 23.6 versus 21.8 g C kg⁻¹ year (Genomics in Leguminous Plant-Nutrition Research | Frontiers Research Topic); and induce a saving of fossil energy inputs in the system thanks to N fertilizer reduction, corresponding to 277 kg ha⁻¹ of CO₂ per year (Farkas and Mohácsi-Farkas)

Allelopathy is a common biological phenomenon by which one organism produces biochemicals that influence the growth, survival, development, and reproduction of other organisms. These biochemicals are known as allelochemicals and have beneficial or detrimental effects on target organisms (Latif et al.) Plant allelopathy is one of the modes of interaction between receptor and donor plants and may exert either positive effects (e.g., for agricultural management, such as weed control, crop protection, or crop re-establishment) or negative effects (e.g., autotoxicity, soil sickness, or biological invasion). To ensure sustainable agricultural development, it is important to exploit cultivation systems that take advantage of the stimulatory/inhibitory influence of allelopathic plants to regulate plant growth and development and to avoid allelopathic autotoxicity. Allelochemicals can potentially be used as growth regulators, herbicides, insecticides, and antimicrobial crop protection products (Genomics in Leguminous Plant-Nutrition Research | Frontiers Research Topic).

This section refers to a study- “Phytotoxic Activity of Ocimum tenuiflorumExtracts on Germination and Seedling Growth of Different Plant Species” (Islam and Kato-Noguchi) by A. K. M. Mominul Islam1 and Hisashi Kato-Noguchi published in “The Scientific World Journal”. An investigation was carried out to study the effect of methanolic extract of O.tenuiflorum on various seeds – cress, alfalfa, lettuce, Italian ryegrass and barnyard grass. A bioassay of germination percentage was measured as an index to understand the effect on germination. It was observed that increasing the concentration of the extract taken has reduced the germination percentage especially in terms of seedling growth. Though the result in case of barnyard grass was different. A positive impact of the extract on germination percentage was observed in case of barnyard grass unlike the other species taken.

Type of seed that is grown: V.unguiculate (cowpea) , C. arietinum L (black chickpea) , V.radiata (mung  beans) , P. sativum (pea) and C. arietinum (white chickpea) . The commonly seen legumes available at home  were picked for the experiment so that the research is relevant to daily life.

Type of solution that the seed is grown in: O.tenuiflorum (Tulsi), A.indica (Neem) and Water (control)

Percentage germination – the % of seeds that have germinated after 48 hours.

Formula used: Percentage germination = \(\frac{Number\ of\ seed\ germinated}{total\ number\ of\ seeds\ taken}\) × 100

• The seeds grown in either the Ocimum tenuiflorum solution or the Azadirachta indica solution will show slower growth than the seeds grown in water.

• The experiment uses 50 plastic containers which are a hazard to the environment thus making this experiment environmentally unsustainable. If the plastic containers are cleaned and upcycled for home usage instead, this issue can be taken care of.
• Seeds that are used are used up in compost so that their usage is not a waste.

• Heat resistant gloves: worn during the boiling of water in order to prevent any injuries or burns from the heat.
• Lab coat: worn while boiling water in order to prevent any scope of spillage which might cause skin burns.

• Pick out O. tenuiflorum leaves from a plant
• Clean out 1 container
• Place 1 container on a top pan balance
• Tare the weight of the container
• Add whole leaves of approximately the same sizes 1 by 1
• Keep adding leaves till the sum of their weight is 10g
• Grind the leaves into fine pieces using a blender along with 100ml of water
• Transfer the pasty outcome to a heat safe container with a lid
• Boil 500ml of water
• Add the water to the container with the leaves
• Cover the container with a lid and leave it out for 24 hours
• Using a fine thread, soft, muslin cloth as a filter drain out all the liquid from the mixture
• Leave the cloth with the leaves in it hovering over the container for 2 hours to make sure maximum liquid is drained out.
• Set the acquired extract aside till the final set up.
• Do steps 1-to-14 for the A.indica leaf

• Get a fresh roll of clean white cotton sheet
• Count 55 containers of the identical dimensions and kind
• Place 1 container on a top pan balance
• Tare the weight of the container
• Add thin cotton sheets of approximately the same dimensions 1 by 1
• Keep adding cotton until the weight is 5g
• Repeat steps 1-to-6 for the remaining 54 containers.

• Bring freshly bought packets of each kind of seed
• Pick out 5 large containers
• Count 165 seeds of each kind
• Keep all 5 sets of 165 counted seeds in each of the 5 containers
• Fill all containers with enough water to submerge the seeds fully
• Leave the seeds in the water for 2 hours
• Using a sieve drain out the water from all containers and leave the seeds to dry under the fan for 5 hours

• Layout the containers in two -5×5 grids in two sets as indicated in the diagram below.

• Count 15 seeds of one kind and scatter in one container.
• Repeat for all the containers in the coloumn.
• Each coloumn will be a different seed.
• Add 30 cc of Ocimum tenuiflorum extract to 5/5 rows of the first grid containers.
• Add 30 cc of Azadirachta indica solution to 5/5 rows of the second grid of containers.
• Place a separate row of 5 unique seed containers in the same order.
• Add 30 cc of water to these 5 containers.
• Leave the containers at room temperature by an open window, in direct sunlight.
• After 2 days (48.00 hours), count the number of seeds that has germinated.

Mean number of seeds = \(\frac{Trial-1\ +\ Trial-2\ +\ Trial-3\ +\ Trial-4\ +\ Trial\ 5}{5}\)

The mean has been expressed up to two decimal places and not rounded off to get a more accurate result for the percentage germination.

Standard deviation (SD) = \(\frac{\sum^{i=5}_{i=1}(trial\ value-mean\ value)^2}{5}\)

Formula:\(\frac{mean\ number\ of\ seeds\ germinated}{15}\) × 100 = percenatge germination

The graph above is a bar graph showing the percentage germination of various types of seeds for all the three extract types taken – O.tenuiflorum, A.indica and control (water).

• It is clearly observed that from the graph that the percentage germination is maximum for water (control) in all types of seeds. This claims that both the extracts has a negative impact on the growth of plant or precisely the rate of germination of the seeds.
• In all cases except that for V.radiata, the percentage germination is more for A.indica than that in O.tenuiflorum. This allows us to claim that A.indica is more beneficial for the growth of the seeds or precisely the germination of the seeds in comparison to O.tenuiflorum.
• For all the three types of the solutions, the maximum effect is observed in case of V.radiata in comparison to all other seeds. Thus, it can be claimed that among all the type of seeds taken, the maximum growth rate or rate of germination is obersved for V.radiata.
• The maximum difference between O.tenuiflorum and A.indica is found in case of V.unguiculate. This means that A.indica is more effective in comparison to O.tenuiflorum in case of V.unguiculate in comparison to other seeds.
• The maximum difference between the values of percentage germination for the extracts and control is found in case of P.sativum and C.arientum L in comparison to other seeds. This indicates that the allelopathic extracts taken has the most negative impact on the growth of plant in case of P.sativum.

Formula used:

Total percentage germination against type of seeds = \(\frac{Sum\ of\ \%\ germination\ for\ the\ three\ extracts\ -\ O.tenuiflorum,A.indica\ and\ control\ (water)}{3}\)

Figure - 9  compares the total percentage germination against the type of seeds. This graph shows that the value is maximum for V.radiata. Thus, it can be claimed that among all the type of seeds taken, the rate of germination is maximum for V.radiata. More generally, it can be said that V.radiata germinates much faster than all the other type of seeds chosen. The least value of percentage germination is observed for P.sativum which means that among all other type of seeds chosen, the rate of germination is least for P.sativum.

Formula used:

Total percentage germination = sum of value of percentage germination f or each type of seeds

This graph compares the total percentage germination against the type of plant extract taken- O.tenuiflorum, A.indica and control (water). For both A.indica and O.tenuiflorum, the value is lesser than that for control (water). This confirms that both of these allelopathic extracts have a negative impact on the growth of plant. Comparing the values for O.tenuiflorum and A.indica, it can be said that the negative impact on the growth of plant is more with O.tenuiflorum in comparison to A.indica as O.tenuiflorum shows a lower value of percentage germination than that for A.indica.

Figure - 7 and Figure - 11 clearly confirms that both the extracts – O.tenuiflorum and A.indica has a negative impact on the growth of the plants in case of germination percentages. Thus, the null hypothesis has been rejected and the alternate hypothesis has been accepted.

As the data is collected into three individual groups- (O.tenuiflorum, A.indica and control) for five categories ( five seeds) and the groups are independent of each other, the most suitable test would be ANOVA.

Null hypothesis (H₀) - The effect of allelopathic extract on the percentage germination of the seeds does not depend on the type of allelopathic extract used.

Alternate hypothesis (H₁) - The effect of allelopathic extract on the percentage germination of the seeds does not depend on the type of allelopathic extract used.

significance level (α) = 0.05

Total number of values (N) = 15

Number of values in each group (n) = 5

Number of categories or groups (a) = 3

Degrees of freedom in between (\((df_{between})\) = a − 1 = 3 − 1 = 2

Degrees of freedom in within \((df_{within})\) = N − a = 15 − 3 = 12

Total Degrees of freedom \((df_{total})\) = N − 1 = 15 − 1 = 14

Using (2,12), F = 3.7389 ; ∴ if F is greater than 3.7389, reject the null hypothesis

Sum of square between \((SS_{between})\) = \(\frac{138.67^2\ +\ 142.59^2\ +\ 233.34^2}{5}\ -\ \frac{514.6^2}{15}\) = 1147.555

Sum of square within \((SS_{within})\) = 10587.85

Sum of square total \((SS_{total})\) = 11735.41

As the value of F is lower than 3.7839, the null hypothesis is accepted and the alternate hypothesis is rejected.

How does the effect of the extracts of allelopathic plant on the growth of leguminous plants, measured in terms of percentage germination depends on the type of the allelopathic plant chosen- Ocimum tenuiflorum (Tulsi) and Azadirachta indica (Neem) and the type of plant taken-Vigna radiata, Vigna unguiculate, Cicer arietinum, Cicer arietinum L, Pisum sativum ?

• The rate of germination is maximum for V.radiata. More generally, it can be said that V.radiata germinates much faster than all the other type of seeds chosen. The least value of percentage germination is observed for P.sativum which means that among all other type of seeds chosen, the rate of germination is least for P.sativum.
• Both of these allelopathic extracts have a negative impact on the growth of plant. Comparing the values for O.tenuiflorum and A.indica, it can be said that the negative impact on the growth of plant is more with O.tenuiflorum in comparison to A.indica as O.tenuiflorum shows a lower value of percentage germination than that for A.indica.
• A.indica is more beneficial for the growth of the seeds or precisely the germination of the seeds in comparison to O.tenuiflorum. Among all the type of seeds taken, the maximum growth rate or rate of germination is obersved for V.radiata. A.indica is more effective in comparison to O.tenuiflorum in case of V.unguiculate in comparison to other seeds. The allelopathic extracts taken has the most negative impact on the growth of plant in case of P.sativum.
• The null hypothesis has been rejected and the alternate hypothesis has been accepted.
• Both the allelopathic extracts have been found to show phytotoxic effect on the growth of plants which means that both the extracts have an inhibitory effect on the growth of plant especially in terms of germination of seeds. This fact is also in agreement with the literature reference drawn in the background information.
• The statistical test was done – an ANOVA test was conducted. The result concluded is that the way allelopathic solutions affect the growth of plant species does not depend on the type of the allelopathic solutions used.

• All the factors that may affect the accuracy of the data has been controlled. For example, the volume of the solution, the time period, the part of the plant used to make the extract were kept same.
• The data has been processed in a multi-dimensional way to get a better understanding of the trends observed. For example, instead of deducing the trends from Figure-7, the total percentage germination has been calculated against both the type of seeds and also against the type of the extract has been displayed in Figure-9 and Figure-11. This makes the result more reliable as it takes into account of all the cases instead of relying on one of them.
• Moreover, the features in the Graph are also in coherence. For example-both Figure-7 and Figure-9 shows that the growth or percentage germination is maximum in case of V.radiata irrespective of the type of the extract used.
• Considering various types of seeds allows to have a more comprehensive idea about the effect of the solution on the growth of plants. Had the investigation been done only with one kind of seeds, the result would have been less generic and thus less reliable.

• There are various factors that impacts the germination of seeds. Intensity of sunlight exposed to the seeds is an important factor among them. Variation in the intensity of sunlight received by the seeds may cause inaccurate results and unfair comparison. To optimize this, all the seeds were kept near the same window.
• Environmental factors like temperature and humidity also affects the germination of seeds. Thus, any variations in these conditions will introduce an unfair comparison. To optimize this, the trial with all the seeds were conducted within the same time period so that the environment in which the seeds grow remains the same for all the seeds.
• The rate of germination also depends on the age of the seeds. Though the seeds were bought from the same store and were claimed to be of the same age yet that cannot be ensured. To ensure this, genetically engineered seeds may be used.
• Moreover, the biological composition of the seeds also plays a pivotal role here as germination involves a lot of biochemical reactions and expression of genes followed by release of some hormones or phytochemicals are required for that process. Thus, use of genetically engineered seeds can solve this issue as well.
• The number of seeds germinated was counted manually. Any fatigue of the experimenter will introduce a human error in this process. Thus, to avoid this, the data has been collected in five trials and mean value has been calculated arithmetically.

The investigation can be more quantitatively refined if the length of the growing radicles and plumules are measured and compared at regular time intervals. A line graph can be made to analyse a trend in the growth of the germinated seed over the period of a week using the mean values of the lengths of the radicals and plumules at the end of each day. Using the line graph, a trend line can be obtained to show clearly the overall change in the length of the seed radicals and seed plumules. Another way to analyse the effectiveness of individual allelopathic plants is to test different concentrations of each plant extract. 1%, 2%, 3%, 4% and 5% solutions (with respect to mass of leaves added to 100 cc of water) of the same plant extract can be made by altering the mass:volume ratio of the leaves. Each of the 5 different concentrations of the extracts on one type of seed can be tested. 5 trials for each concentration and find either the % germination or the growth of the seeds over a regular time interval are done. A line graph can be made to analyse trends and find the effectiveness of one unique plant extract.

Farkas, J., and Cs Mohácsi-Farkas. “Safety of Food and Beverages: Spices and Seasonings.” Encyclopedia of Food Safety, edited by Yasmine Motarjemi, Academic Press, 2014, pp. 324–30. ScienceDirect, doi:10.1016/B978-0-12-378612-8.00290-0.

Fengjie, Lei. “Advances in Research on Allelopathy of Ginseng and American Ginseng.” China Journal of Chinese Materia Medica, Sept. 2010. DOI.org (Crossref), doi:10.4268/cjcmm20101701.

Genomics in Leguminous Plant-Nutrition Research | Frontiers Research Topic.https://www.frontiersin.org/research-topics/3625/genomics-in-leguminous-plant-nutrition-research.%20Accessed%206%20Mar.%202021.

Islam, A. K. M. Mominul, and Hisashi Kato-Noguchi. “Phytotoxic Activity of Ocimum Tenuiflorum Extracts on Germination and Seedling Growth of Different Plant Species.” The Scientific World Journal, 17 June 2014, doi:https://doi.org/10.1155/2014/676242.

Latif, S., et al. “Chapter Two - Allelopathy and the Role of Allelochemicals in Plant Defence.” Advances in Botanical Research, edited by Guillaume Becard, vol. 82, Academic Press, 2017, pp. 19– 54. ScienceDirect, doi:10.1016/bs.abr.2016.12.001.

Wong, J. H., and T. B. Ng. “4.60 - Plant Biochemistry: Antifungal Proteins Protecting Plants from Fungal Pathogens.” Comprehensive Biotechnology (Second Edition), edited by Murray Moo-Young, Academic Press, 2011, pp. 745–56. ScienceDirect, doi:10.1016/B978-0-08-088504-9.00013-1.