Research Note Utilizing cattleianum as a pre-emergent bio- herbicide: A study on its allelopathic effects on the in vitro germination of Lactuca sativa seeds

LOUIS ANTONELLI, TWYLAH M. MORELLI, KATELYN YOCKEY, BRADON MIYAKE, MALIA TALIA, TEUILA SINCLAIR AND SHARAD P. MARAHATTA*

College of Agriculture, Forestry and Natural Resource Management, University of Hawaii at Hilo, Hilo, HI, USA

Abstract: Chemical herbicides have negative impacts on non-targeted organisms, groundwater and human health. Strawberry guava (Psidium cattleianum) foliage contains allelopathic properties, which can be potentially utilized as a substitute for chemical herbicides. The purpose of this experiment was to evaluate the ability of strawberry guava extract to suppress the growth rate of lettuce (Lactuca sativa) through strawberry guava's bio-herbicidal allelochemicals. The experiment involved two trials (Trial I and Trial II) with four treatments of strawberry guava leaf extract solutions. Strawberry guava leaves were harvested from the University of Hawaiʻi at Hilo’s farm, air dried and blended into leaf concentrations of 0%, 5%, 10%, and 20% strawberry guava. Filter papers were dipped in each treatment separately and placed in separate petri dishes. Ten lettuce seeds per petri dish were plated. Each treatment was replicated seven times and both trials were arranged in a randomized complete block design. In Trial I, compared to the 0% treatment, 20% strawberry guava leaf concentration significantly reduced lettuce germination at 24 hours of experiment setting (P ˂ 0.05), while the 5% and 10% did not show significant difference (P ˃ 0.05). At 24 hours in Trial II, compared to 0% strawberry guava leaf concentration, all other treatments reduced lettuce germination (P ˂ 0.05). In both trials, the effects of strawberry guava were not found at 48 hours of experiment setting (P ˃ 0.05). In both trials, compared to 0% strawberry guava leaf concentration all other treatments consistently reduced lettuce root length (P ˂ 0.05). The results from both trials indicated that with a higher percentage of strawberry guava leaf concentration there was a correlation with a decrease in the lettuce seed’s germination rate and root length. Current experiment supports the evidence that strawberry guava’s allelopathic compounds can inhibit a ’s initial growth. Strawberry guava could be potentially developed into a bio-herbicide or as a method of regulating unfavorable plant growth.

Key words: allelopathy, bio-pesticide, herbicide, invasive species, lettuce, strawberry guava.

Introduction 2000; Lau and Marahatta, 2013; Ooka and Owens, 2018). Chemical herbicides have negative impacts on non- targeted organisms, groundwater and human health Strawberry guava is a small , native to the (Kolpin et al., 2000). Strawberry guava (Psidium southeastern regions of Brazil and is under the plant cattleianum) foliage has a known allelopathic family . Strawberry guava plant was property, which can be potentially utilized as a introduced to the Hawaiian Islands in 1825 (Wikler substitute for chemical herbicides (Wikler et al., et al., 2000; Ooka and Owens, 2018) for its edible fruit and ornamental characteristics. Strawberry

*Corresponding author, Email: [email protected]

1 Pac. Agric. Nat. Resour. 2020 Vol. 10: 1-4 2 Research Note: Psidium cattleianum leaves as a pre-emergent bio-herbicide guava multiplies rapidly and thus it is invasive on all strawberry guava leaves were placed in a commercial the main Hawaiian islands; Hawaiiʻi, Oahu, Maui, blender (Winsted Conn, Waring Products Co., CT) Molokai, Kauai, Lanai, and Kahoʻolawe (Ellshoff et and mixed for ~ three minutes. The resulting puree al., 1995). This plant typically grows at a range of 1 was filtered through filter paper to remove pulp, and to 5 meters in height. It spreads by both shoots and other solid plant materials. seeds and it grows quickly; increasing the cover size and forming dense forests. Researchers have To prepare in vitro germination, four different hypothesized that strawberry guava could ultimately treatments (20%, 10% and 5% strawberry guava leaf invade nearly 12,000 ha of conservation land on the concentrations and water control (0% strawberry island of Hawaiʻi alone (Pacific Southwest Research guava leaf concentration) were prepared. Ninety Station, 2016). Potential contributing factors to the mm diameter, 15 mm deep, disposable petri dishes outbreak of this plant in Hawaiʻi include the (Polystyrene Disposable Sterile, VWR International, absence of strawberry guava predators and diseases Radnor, PA, USA) were used and 90 mm diameter (Lau and Marahatta, 2013). filter paper (Whatman #1; W&R Balston Ltd., England, UK) was used as the growing medium as Strawberry guava is one of the major threats to well as a retainer of the treatment solution. Filter native Hawaiian ecosystems and an invasive papers were then soaked in each concentration of nuisance in Hawaii’s rainforest (Smith, 1984; Wikler strawberry guava leaf extract and the control then et al., 2000). There has been previous research placed on the petri dish. Ten lettuce seeds variety showing allelopathic qualities of strawberry guava ‘Anuenue’ were placed onto the saturated surface of on multiple broad-leaf and grass weeds (Lau and the filter paper. Seeds were randomly taken and Marahatta, 2013), and the forest floors of areas evenly placed on the paper surface. Dishes were invaded by strawberry guava contain little to no covered and sealed with parafilm (Bemis Parafilm M additional plant life (Wikler et al., 2000; Lau and Laboratory Wrapping Film; Fisher Scientific, Marahatta, 2013). Moreover, strawberry guava leaf Lenexa, KS) and kept on lab counter in an area with extract has demonstrated positive results for surface adequate light. Each treatment was replicated seven disinfection (Dacoreggio et al., 2019). Given these times and repeated two times (Trial I and Trial II). evidences, the allelopathic chemicals of strawberry Both trials, Trial I and Trial II, were arranged in guava can be potentially utilized to suppress pre- randomized complete block designs. The entire emergent weed seeds. Therefore, the purpose of this procedure of Trial I was repeated in Trial II. experiment was to evaluate strawberry guava’s ability to suppress the growth rate of lettuce (Lactuca The number of germinated seeds were counted at sativa). Lettuce had been used as a standard plant for 24 and 48 hours after setting each trial. After 5-days testing allelopathic effects of strawberry guava of starting the experiment, each plate was (Ferreira and Aquila, 2000). uncovered, and root lengths of five randomly selected seedlings were measured. Methodology To conduct a test of the allelopathic qualities of Statistical analysis strawberry guava, a simulated growth was Data for number of germinated lettuce seeds and conducted using lettuce seeds, variety ‘Anuenue’. root length were pooled and analyzed to one-way The trial utilized four different concentrations of a analysis of variance (ANOVA) using the PROC strawberry guava leaf solution, 0%, 5%, 10% and MIXED procedure in Statistical Analysis System 20%, to test the allelopathic properties of strawberry version 9.4 (SAS Institute, Cary, NC). Moreover, guava on lettuce germination and root growth. One data were checked for normal distribution using hundred grams of freshly harvested, air dried Proc Univariate and homogeneity of variance using strawberry guava leaves and 500 ml of water were the HOVTEST option in PROC GLM before mixed and blended to make the 20% strawberry performing one-way ANOVA. Based on the results guava leaf solution concentration, this was diluted of normality test (p =0.05), untransformed data further to make the 10% and 5% concentrations, were analyzed. Means were separated by Least respectively. First, strawberry guava branches were Significance Difference (LSD) (the t-critical value collected from the Waiakea district within Hilo, for α=0.05) wherever appropriate. Hawai`i, outside the University of Hawai`i at Hilo’s farm. Leaves were then stripped from the collected branches, rinsed under water and air dried. Air dried

Pac. Agric. Nat. Resour. 2020 Vol. 10: 1-4 Louis Antonelli, Twylah M. Morelli, Katelyn Yockey, Bradon Miyake, Malia Talia, Teuila Sinclair and Sharad P. Marahatta 3

Results and Discussion Effects on lettuce root length Compared to 0% strawberry guava leaf extract all Effects on lettuce germination other treatments consistently reduced lettuce root Compared to the 0% strawberry guava leaf length (P ˂ 0.05) (Fig. 2). Results of this experiment concentration, 10% and 20% strawberry guava leaf is consistent to the findings of Chapla and Campos concentrations significantly reduced lettuce (2010), where 5, 10 and 20% guava leaf extract germination at 24 hours of experiment setting (P ˂ reduced lettuce root length at 24, 48 and 72-hrs- 0.05), while the 5% strawberry guava leaf after starting a laboratory experiment. concentration did not show significant difference (P ˃ 0.05) (Fig. 1). The effects of strawberry guava leaf Previous studies on guava and strawberry guava concentration were not found at 48 hours of have identified several chemicals such as terpenoids, experiment setting (data not presented) (P ˃ 0.05). flavonoides, coumarins, cyanogenic acids (Begum et The highest concentration used in this experiment al., 2002; Gutiérrez et al., 2008), ascorbic acid and (20% strawberry guava leaf extract) inversely related carotenoid (Dantas et al., 2013) on these Psidium to a decreased rate in germination by approximately spp. Recently, Dacoreggio et al. (2019) determined 50% (Fig. 1). Overall, an increase in the phenolic compounds content (TPC) and the concentration of strawberry guava leaf extract was antioxidant activity on strawberry guava leaves. found to decrease the number of seeds germinated. These chemicals could have shown the allelopathic Thus, strawberry guava leaf extract may have properties in strawberry guava. herbicidal properties that had negatively affected the germination and root development in lettuce seeds. This result is consistent to the findings of Chapla and Campos (2010) who found an allelopathic effect of 5, 10 and 20% guava (P. guajava) leaf extract on lettuce germination.

Figure 2. Effects of strawberry guava, P. cattleianum, (SG) leaf extract on lettuce, L. sativa, root length at 5 days-after starting the laboratory experiment. Means ± standard error of the means are pooled average of two trials, 7 replications and 5 samples per replication. Means followed by same letter(s) do not differ according to Least Significant Difference (LSD)-test (P Figure 1. Effects of strawberry guava, Psidium < 0.05) (LSD = 3.43 mm). cattleianum, (SG) leaf extract on germinated lettuce, Lactuca sativa, seeds at 24 hours-after starting the laboratory experiment. Means ± standard error of the Ooka and Owens (2018) found an inhibition in means (SEMs) are pooled average of two trials, 7 growth and germination of green onions and lettuce replications per trial and 5 samples per replication. in a higher concentration of strawberry guava leaf Means followed by same letter(s) do not differ extract and thus concluded that strawberry guava according to Least Significant Difference (LSD)-test (P leaves have water soluble biochemicals. Through < 0.05) (LSD = 1.25). our two trials and those of Chapla and Campos (2010) and Ooka and Owens (2018), there is a trend of smaller root length of seeds that have been exposed to high concentrations of strawberry guava or guava. This may prove to be a possible herbicide or weed suppressant that could be used in

Pac. Agric. Nat. Resour. 2020 Vol. 10: 1-4 4 Research Note: Psidium cattleianum leaves as a pre-emergent bio-herbicide agriculture or allow a place for using strawberry Ooka, J. K., and D. K. Owens. 2018. Allelopathy in tropical guava. and subtropical species. Phytochemistry Reviews 17: 1225–1237. doi: 10.1007/s11101-018-9596-7. Results of the present study support the evidence Pacific Southwest Research Station. 2016. Strawberry guava: that strawberry guava’s allelopathic compounds can not all green is good. The USDA Forest Service Research inhibit a plant’s initial growth. Strawberry guava leaf and Development, Pacific Southwest Research Station , extract could be potentially developed into a bio- 800 Buchanan Street, Albany, CA, USA. herbicide or used as a method of regulating https://www.fs.fed.us/psw/topics/biocontrol/strawbe unfavorable plant growth. rryguava/strawberry_guava.shtml (accessed May 14, 2020). Further research is needed to identify the Smith, C.W. 1984. Impact of alien on Hawaii's native allelopathic chemicals in strawberry guava leaf and biota. In: Hawaii’s terrestrial ecosystems: preservation test its bio-herbicidal properties to a wide range of and management: proceedings of a symposium held June crops and weeds. The positive results of such 5-6, 1984 at Hawaii Volcanoes National Park (eds Stone, experiments will determine the potentiality of this C.P. and J.M. Scott) pp 180-250. Honolulu: Cooperative extract as a bioherbicide. National Park Resources Studies Unit, University of Hawaiʻi at Manoa, University of Hawaii Press. References Wikler, C., J.H. Pedrosa-Macedo, M.D. Vitorino, M.G. Caxambu, and C.W. Smith. 2000. Strawberry guava Begum, S., S. I. Hassan, B. S. Siddiqui, F. Shaheen, M. N. (Psidium cattleianum) prospects for biological control. In: Ghayur, and A. H. Gilani. 2002. Triterpenoids from Proceedings of the X International Symposium on leaves of Psidium guajava. Phytochemistry 61: 399-403. Biological Control of Weeds, July 4-14, 1999 at Montana Chapla, T. E., and J. B. Campos. 2010. Allelopathic evidence State University (ed Spencer, N.R.) pp 659-665. in exotic guava (Psidium guajava L.). Brazilian Archives of Biology and Technology 53: 1359–1362. doi: 10.1590/s1516-89132010000600012. Dacoreggio, M. V., L. S. Moroni, and A. P. Kempka. 2019. Antioxidant, antimicrobial and allelopathic activities and surface disinfection of the extract of Psidium cattleianum sabine leaves. Biocatalysis and Agricultural Biotechnology 21:101295. Dantas, A. L., S. de Melo Silva, M. A. C. de Lima, R. L. Dantas, R. M. N. Mendonça. 2013. Bioactive compounds and antioxidant activity during maturation of strawberry guava fruit. Revista Ciencia Agronomica 44:805-814. Ellshoff, Z. E., D. E. Gardner, C. Wikler, and C. W. Smith. 1995. Annotated bibliography of the genus Psidium, with emphasis on P. cattleianum (strawberry guava) and P. guajava (common guava), forest weeds in Hawaiʻi. Honolulu (HI): Cooperative National Park Resources Studies Unit, University of Hawaii at Manoa, Department of Botany. PCSU Technical Report, 95. Ferreira, A., and M. E. A. Aquila. 2000. Alelopatia: uma área emergente da ecofisiologia. Revista Brasileira de Fisiologia Vegetal12:175-204. Gutiérrez, R. M. P., S. Mitchell, and R. V. Solis. 2008. Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology 117: 1-27. Kolpin, D.W., E. M. Thurman, and S. M. Linhart. 2000. Finding minimal herbicide concentrations in ground water? Try looking for their degradates. Science of the Total Environment 248: 115-122. Lau, J.-H., and Marahatta, S.P. 2013. Can strawberry guava, Psidium cattleianum, leaves be used for weed management in Hawaiʻi? NACTA Journal 57:S78 (Abstr).

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