PHYTOCHEMICAL AND ANTIMICROBIAL PROPERTIES OF LEAF EXTRACTS OF Calliandra calothyrsus, Leucaena diversifolia AND Sesbania sesban

Phytochemical compounds are secondary metabolites of plants useful as antimicrobial agents. Botanicals are being explored for bioactive compounds with antimicrobial properties against phytopathogens. Little information is available on the phytochemical and antimicrobial activity of Calliandra calothyrsus, Leucaena diversifolia and Sesbania sesban against Cercospora zeae-maydis and Xanthomonas campestris pv. musacearum. The aim of the study was to determine the phytochemical and antimicrobial properties of leaf extracts of C. calothyrsus, L. diversifolia and S. sesban against C. zeae-maydis and Xc. pv. musacearum. Dried leaves were extracted in methanol and aqueous solvents and screened for phytochemical and antimicrobial activity using Kirby-Bauer’s disk diffusion and poisoned food technique methods. Sesbania sesban extracts contained all the phytochemical tested; tannins, terpenoids, steroids, saponins, flavonoids, and alkaloids, Leucaena diversifolia lacked alkaloids while Calliandra calothyrsus lacked steroids and alkaloids. The extracts were active against Cercospora zeae-maydis and Xc.pv. musacearum with Sesbania sesban having greater radial inhibition activity. There was no significant difference in the antimicrobial activity between the lowest concentrations (25% and 25mg/ml) and highest concentrations (75% and 75mg/ml) in all the three plant extracts. Growth inhibition observed could be as a result of the different chemical compound observed in the extracts. Presence of alkaloids in Sesbania sesban could explain the greater growth inhibition of the pathogens under study. The results form the basis for further research that could lead to isolation and development of antimicrobial agents. Therefore, these plants can be used as an alternative to synthetic chemicals to control Cercospora zeae-maydis and Xanthomonas campestris pv. Musacearum.


INTRODUCTION
. Sesbania sesban (L.) Merrill is a multipurpose tree that is widely distributed in tropics and subtropics of Africa and Asia and usually planted by smallholder farmers mostly for its fodder and soil improvement values (Nigussie and Alemayehu, 2014; Mythili and Ravindhran, 2012). It is used also as a source of green manure, anti-inflammatory activities, reproduction and milk production enhancement, nitrogen fixation, bioenergy source, antibacterial and anti-parasitic  (Gomase et al., 2012;Kathiresh et al., 2012). While plants are being considered reliable sources of antimicrobial compounds, the antimicrobial activity of C. calothyrsus, L. diversifolia and S. sesban against Cercospora zeae-maydis and Xanthomona scampestris pv. musacearum has not been documented. Similarly, because of the effect of environment on phytochemical constituents, it is necessary to study the phytochemical composition and distribution among the three plant species. This will go along with identifying alternative measures of disease control using botanicals and understanding the variation in phytochemical components of plant in varying ecological zones. This study therefore aimed at identifying the phytochemical constituents and evaluating the antimicrobial properties of C. calothyrsus, L. diversifolia and S. sesban leaf extracts.

Collection and processing of plant materials
Leaves of C. calothyrsus, L. diversifolia and S. sesban were collected in nonwoven bags from demonstration plots of Maseno University located 0° 10' 0" South, 34° 36' 0" East along Kisumu Busia road in western Kenya. They were transported to the botany laboratory for extraction. They were washed in tap water, air dried under shade for 14 days with periodic turning of two days then crushed into fine powder using electrical motor. Fine powder was used for extraction with both methanol and aqueous solvents according to Dent et al. (2013) where 50 grams of each powdered plant leaf materials were separately kept in 500 ml conical flask and 500 ml methanol and aqueous added and thoroughly mixed respectively. The mixtures were left to stand overnight on a shaker for complete extraction, filtered using muslin cloth followed by Whatman no 1 filter paper. Methanol was evaporated using rotary vacuum evaporator with the water bath temperature of 45 o C. The filtrate was used to test for the presence of phytochemicals and antimicrobial activity of the extracts.

Phytochemical screening of the extracts
The presence of steroids, alkaloids, flavonoids, saponins, tannis and terpenoids in leaves of C. calothyrsus, L. diversifolia and S. sesban were determined as indicated below. (50)  Flavonoids: 2 ml of 2.0% NaOH mixture was mixed with aqueous plant crude extract. A concentrated yellow color was produced, which became colorless when 2 drops of dilute H2SO4 acid was added. Colorless appearance indicated presence of flavonoids (Gul et al., 2017).

Steroids: Fifty
Tannins: A small quantity of the extract was boiled with 5 ml of 45% solution of ethanol for 5 minutes, cooled and filtered. 1ml of filtrate was diluted with distilled water and two drops of ferric chloride added. A transient greenish to black color indicated the presence of Tannins (Sheel et al., 2014).

Isolation of fungal pathogen Cercospora zeae-maydis.
Maize leaves showing characteristic symptoms were collected from the fields, cut into pieces of approximately 5cm, placed on sterile moist blotter in a sterile petridish and incubated at 25 o C for 5 days for the pathogen to sporulate. Conidia were picked with an isolation needle under dissecting microscope and plated on PDA. Plates were incubated at 25 o C for 5-7 days and hyphal tips from the advancing colony margins with typical morphological characteristics were transferred onto PDA with isolating needle as pure culture and kept at 5 o C (Nega  et al., 2016).

Determination of antimicrobial activity
Methanol extracts were reconstituted by Dimethyl sulfoxide (DMSO) to make concentrations of 12.5mg/ml, 25mg/ml/ 50mg/ml and 75mg/ml while aqueous extracts were reconstituted into concentrations 12.5%, 25%, 50% and 75% and used for antimicrobial studies. Disc diffusion method was used to assess the sensitivity of the bacterial pathogen to plant extracts (Bauer et al., 1966). Colonies from pure culture were lawn spread on MHA plates and discs impregnated with 10µl of each test extract placed on the surface aseptically while discs impregnated with pure water and DMSO served as negative control. Every treatment was replicated thrice, plates arranged in completely randomized design and incubated at 30 o C for 48 hours and zone of inhibition measured in millimeters. Antifungal activity of the extracts was determined using poisoned food technique according to Durgeshlal et al. (2019), by dispensing 4 ml of each extract in petri plates and adding 16 ml of PDA then mixing and allowing them to set. A 5mm mycelia plug from 7 day old mycelia was inoculated at the center of the plates and plates without extracts served as control in triplicates. Plates were incubated for 7 days at 28 o C where fungi mycelia radial growth was measured and inhibition percentage determined using the formula of

Phytochemical screening
Phytochemical screening revealed that terpenoids, flavonoids and saponins were present in the leaf extracts of the three plant species (Table 1). Tannins were present in C. calothyrsus and S. sesban but absent in L. diversifolia. Steroids were present in L. diversifolia and S. sesban but absent in C. calothyrsus. Alkaloids were present only in the leaf extracts of S. sesban. Calliandra calothyrsus had higher concentrations of tannins, terpenoids, saponins and flavonoids while S. sesban contained higher amounts of steroids and alkaloids.

Antimicrobial activity of leaf extract of S. sesban, C. calothyrsus and L. diversifolia against Xc.pv. musacearum and Cercospora zeae-maydis
The antimicrobial activity of leaf methanol and aqueous extracts of S. sesban , C. calothyrsus and L. diversifolia against Xc. pv musacearum and Cercospora zeaemaydis were significantly different with the P value of (P=0.0014 and P=0.0001) in methanol extract and (P=0.0016 and <.0001) in aqueous extract for Xc. pv musacearum and Cercospora zeae-maydis respectively. Sesbania sesban produced largest mean zone of inhibition of 13.9 mm for bacteria and inhibition percentage of 72.2% for fungi in methanol extracts and 13mm for bacteria and 78.3 % for fungi in aqueous extracts compared to C. calothyrsus and L. diversifolia (Tables 2 and 3). There was no significant (p≤ 0.05) difference in the mean zone of inhibition between different concentrations for both methanol and aqueous extracts against Xc. pv musacearum except for L. diversifolia whose concentrations exhibited significant difference against Cercospora zeae-maydis with concentration 75% having greatest mean inhibition percentage in both methanol and aqueous extracts (Tables 2 and 3). Means followed by the same letters down the column are not significantly different at P = 0.05. Means followed by the same letter down the column are not significantly different at P = 0.05.

DISCUSSION
Plant species used in traditional medicines continue to be reliable sources for discovery of useful compounds (Musyimi et al., 2008; Emitaro et al., 2018). There was a significant inhibition of radial growth of Xc. pv musacearum and Cercospora zeae-maydis by the leaf extracts from S. sesban, C. calothyrsus and L. diversifolia. Sesbania sesban extract was more effective against Xc. pv musacearum and Cercospora zeae-maydis pathogen as it produced large zones of inhibition compared to C. calothyrsus and L. diversifolia. The difference in performance could be attributed to high concentration of saponins, steroids and alkaloids in the leaf extract of S. sesban (Table 1). These compounds are known to have antibacterial and antifungal activity when they work synergistically with flavonoids. It may also be because active compounds were polar which dissolved in methanol and aqueous solvents readily than those in C. calothyrsus and L.

CONCLUSION
This study aimed at isolating, identifying and evaluating the antimicrobial properties of the compounds from the leaf extracts of S. sesban, C. calothyrsus and L. diversifolia. Phytochemical screening revealed that terpenoids, flavonoids and saponins were present in the leaf extracts of the three plant species. Tannins were only present in C. calothyrsus and S. sesban. Steroids were present in L. diversifolia and S. sesban while alkaloids were present only in the leaf extracts of S. sesban. The study found a variation in the concentration of phytochemical compounds in the leaf extracts of three plant species even though the plants were from the same ecological zone. Antimicrobial activity of plant extracts is depended on the secondary metabolites that the plant synthesise. Sesbania sesban, Callindra calothyrsus and Leucaena diversifolia extracts showed antimicrobial activities against Xc. pv musacearum and Cercospora zeae-maydis which could form a basis of developing botanical pesticides to avoid the adverse effects of synthetic chemicals. The results in this study supports the use of plant extracts in controlling plant pathogens as they are readily available, cheap and ecofriendly. Future studies should focus on identifying the active ingredients in the extracts of S. sesban, C. calothyrsus and L. diversifolia for development of chemicals to optimize their use by smallholder farmers in disease control to reduce dependence on synthetic pesticides.