Phytochemical analysis and antifungal activity of Tithonia diversifolia and Kigelia africana extracts against Fusarium oxysporum in tomato

Fusarium wilt can cause severe losses in many vegetables and flowers, field crops and plantation crops. The main method for controlling Fusarium wilt is through chemical methods which have the disadvantage of polluting the environment and are expensive. Thus, there is a need for more environmentally friendly and cheaper techniques for controlling Fusarium Wilt. This study screened the phytochemical compounds of two plant extracts; Tithonia diversifolia and Kigelia africana and assessed their potency in controlling plant fungal pathogen F. oxysporum, which causes fusarium wilt in tomatoes. The phytochemical analysis revealed that the two plant extracts contained saponins, tannins, terpenoids, flavonoids, glycosides and phenolics except alkaloids and steroids which were only found in T. diversifolia. The mean inhibitory zones ranged from 7.93 to 10.44 mm for T. diversifolia at 25 to 100 g/l. The mean inhibitory zones for K. Africana ranged from 12.07 to 15.56 mm at 25 to 100 g/l. K. Africana extracts had the highest inhibitory (antimicrobial) activity. Combining both extracts was more effective compared to the single extract. The effect of the combined plant extract and positive control was statistically significant (p < 0.05). This study provides scientific evidence that K. africana and T. diversifolia extract possess antifungal activity and can be used as a broad-spectrum in managing microbial diseases.


INTRODUCTION
Soil-borne pathogens escape competition with other microorganisms by penetrating the host plant roots (Haas and Defago, 2005). Plants infected by soil-borne pathogens suffer from various symptoms such as wilt, root rot and damping-off of seedlings. Main agricultural soil-borne fungal diseases include Fusarium wilt (Fusarium oxysporum Schlechtthe), Verticillium wilt (Verticillium dahliae) and damping-off diseases (Ampt et al., 2019). Fusarium wilt can cause severe losses in many vegetables and flowers, field crops and plantation crops (Michielse and Rep, 2009). In tomato, two distinct forms of the pathogen are found and can cause either vascular wilt or a crown and root rot and both occur throughout most tomato growing areas (Larkin and Fravel, 1998). Use of Fusarium resistant tomato cultivars can provide some degree of control of these diseases but the occurrence and development of new pathogenic races is a continuing problem (Jones et al., 1991). Another problem is that F. oxysporum can persist in the soil for a very long time and is spread by insects, water and garden equipment (Agrios, 2005). Therefore, increasing interest in the introduction of bio-control agents for managing soil-borne pathogens mainly due to the nontarget effects of synthetic pesticides (Cook, 1993).
Kenya is one of sub-Saharan Africa's top tomato producers and grows more than 400,000 tonnes of the fruit every year, which corresponds to 14% of the whole vegetable harvest and 7% of its total horticultural production (Government of Kenya (GoK), 2016). Tomatoes are either grown on fields or in greenhouses. Production in the field accounts for 95% while greenhouse production accounts for 5% of the entire tomato production in Kenya (Food and Agriculture Organization [FAO], 2018). Tomatoes are also a source of income to the small-holder farmers hence contributing to food security and economic development (Ongeri, 2014). However, commercial farming of this important crop is under threat from pests and diseases (Berrueta et al., 2012), mainly Fusarium wilt caused by the fungus Fusarium oxysporum (Ajilogba and Babalola, 2013) causing significant yield losses to farmers.
Farmers prefer the use of synthetic fungicides or cultural practices to control Fusarium wilt disease in tomato (Mwangi et al., 2015). However, cultural practices such as crop rotation and shifting cultivation are no longer effective due to insufficient land for cultivation (Bawa, 2016). Furthermore, commercial fungicides are unaffordable to many poor farmers and excessive use is environmentally toxic and can lead to the development of more resistant pathogen strains (Aktar et al., 2009;Njoroge, 2014). Due to these limitations to control Fusarium wilt disease, there is a need to seek alternative control methods of biological origin for the management of the disease. Plant secondary metabolites pose possible alternatives because they are environmentally friendly and have minimal effect on non-target soil microorganisms (Malkhan et al., 2012;Njoroge, 2014). In this study, two plants, Tithonia diversifolia (Hemsl.) A. Gray and Kigelia africana (Lam.) Benth was screened for bioactive compounds and assessed for their potency in controlling plant fungal pathogen F. oxysporum that causes Fusarium wilt in tomatoes. Both plants have antimicrobial substances that have been successfully demonstrated against food and human pathogens Saini et al., 2009;Rejeki and Addy, 2017).

Collection and preparation of plant samples
Plant samples of T. diversifolia (leaves) and K. africana (fruits) were collected from the field. They were washed and dried under shade until all the water molecules evaporated and plants became well dried for grinding. About 100 g of the dried sample was chopped and grounded into a powder. The sample was then mixed with 100 ml of distilled water to make a solution. The water extract was incubated at room temperature for 36 h and then filtered. The extract was then stored in airtight containers in the refrigerator for phytochemical analysis.

Phytochemical analysis of crude extract
All crude extracts were screened for phytochemical contents using standard procedures (Parekh and Chanda, 2008;Yadav and Agarwala, 2011). They were tested for tannins using 5 ml of extract dissolved in 1 ml of distilled water, filtered and then added 2 ml of Iron (III) Oxide. They were also tested for alkaloids (Wagner test), terpenoids (Salkowski test), flavonoids (Sodium hydroxide test), glycosides (Keller-Kilian test) and saponins (Froth test). Phenolics were tested by adding 1ml of ferric chloride solution to 2ml of the plant extract.

Collection and identification of Fusarium oxysporum
Sites with Fusarium wilt symptoms were identified in a tomato plantation. Soil samples were collected at the rhizosphere of the tomato plant. The samples were stored in sterile containers containing sterile distilled water ready for F. oxysporum isolation. They were inoculated into Petri dishes containing Potato Dextrose Agar (PDA) medium and incubated at 28°C for 7 days. After growth, the fungus was sub-cultured to obtain a pure culture. Identification and isolation of F. oxysporum were done according to De Carolis et al. (2012) by determining the colony colour and investigating the presence and shape of the macroconidia, microconidia and chlamydospores. Morphological characteristics were studied using light microscopy.

Antifungal activity test of F. oxysporum
The antifungal activity of the plant extracts was determined using the disc diffusion assay according to Benkeblia (2004). Plates of PDA media were prepared. Sterile filter papers (discs) of 6 mm in diameter were impregnated with 2 ml of the plant extract at the concentration of 25, 50 and 100 g/ml and placed on the culture medium. For controls, discs were saturated with pure water extracts. After 30 min, plates were inverted and incubated at 28°C for seven days to allow growth. Antifungal activity was evaluated by measuring the zone of inhibition against the test organisms and expressed in millimeters. The combined effect of the extract was determined by combining the two plant extracts in the ratio of 0:30, 10:20, 30:30, 20:10 and 30:0 ml of K. africana and T. diversifolia, respectively. A commercial fungicide was used as a control. Preparation of antimicrobial test disc was replicated three times for each test.

Statistical analysis
Statistical Analysis Software (SAS) version 9.4 was used to analyze data on the zone of inhibition. Two -way analysis of variance  (ANOVA) was carried out to determine variation among the zones of inhibition at α=0.05. Least Significance Difference was used to determine the difference among the means.

Phytochemical screening of the plant extracts
Several phytochemical compounds of the plant extracts were identified for both plants (Table 1). These were saponins, tannins, alkaloids, terpenoids, flavonoids, glycosides, phenolics and steroids. However, steroids and alkaloids were only found in T. diversifolia leaf extract and not in K. africana fruit.

Identification of F. oxysporum
F. oxysporum grown on the PDA media produced white mycelia, with a cotton appearance that was pink in colour on the underside of the plate (Figure 1). The microscopic features observed during identification were septations and shapes of conidia and chlamydospores. Masses of conidiophores were produced in the culture. The microconidia were abundant and kidney-shaped. The macroconidia were also abundant, slightly curved (sickle cell shape) with septations.

Antimicrobial activity of the plant extracts
The antifungal activities of T. diversifolia and K. africana aqueous extracts against F.oxysporum were demonstrated by observable zones of inhibition ( Figure  2). The mean inhibitory zones were highest at 100g/l in both plants, although K. africana extract portrayed the highest inhibitory activity (Table 2).
A combined extract of the two plants produced a slightly more antimicrobial activity against F. oxysporum compared to when the extract was used singly. The effect of the plant extract and the positive control was statistically significant (p < 0.05). The mean zone of inhibition of the plant extracts ratios ranged from 17.25 to

Phytochemical analysis of the plant extract
This study revealed the presence of saponins, tannins, alkaloids, terpenoids, flavonoids, glycosides, phenolics and steroids in the aqueous extracts of K. africana and T. diversifolia. These are bioactive compounds with antimicrobial activity and they could have been attributed to the ability in inhibiting growth of F. oxysporum. Alkaloids, tannins, flavonoids and saponins display several biological properties and have been reported to have antimicrobial activity against several pathogenic microorganisms (Avato et al., 2006;Rejeki and Addy, 2017). Terpenoids and phenolics on the other hand are essential oils that inhibit microbial oxygen uptake and oxidative phosphorylation (Griffin et al., 1999). Terpenoids have been isolated and tested against pathogenic bacteria and fungi and showed a high range of inhibition zones (Singh and Singh, 2003). Kigelia africana and T. Diversifolia are widely distributed in nature, easily accessible and have the ability to inhibit Table 3. Zones of inhibition of F. oxysporum when exposed to different ratios of the combined plant extracts of K. africana and T. diversifolia. the growth of pathogens due to their bioactive compounds (Rejeki and Addy, 2017;. They also have medicinal value and have been among many medicinal plants tested for antimicrobial activity (Neela et al., 2014). However, the majority of the studies have been conducted on human bacterial pathogens. For instance, Fasola and Iyamah (2014) found the presence of terpenoids, flavonoids, tannins, alkaloids and saponins in Tithonia diversifolia when used to treat malaria. Similarly, Muyenga et al. (2015) confirmed that K. africana extract contains terpenoids, flavonoids, tannins and saponins that were used to treat various human bacterial diseases. Furthermore, leaf extracts of T. diversifolia have been reported to have phytochemical and antimicrobial activities of pharmaceutical importance (John-Dewole and Oni, 2013; Ogoti et al., 2015). Besides, Odeyemi et al. (2014) revealed the presence of alkaloids, phenol, flavonoids, sesquiterpene, monoterpenes and diterpenes in the leaf extract of T. Diversifolia against common environmental pathogenic bacteria. This study indicates that these two plants K. africana and T. diversifolia possess antifungal activity and can be used as broad-spectrum fungicides against F. oxysporum. As compared to T. diversifolia, extracts of K.africana did not show the presence of alkaloids and steroids. Similar studies indicate that the fruit extract of K. africana contains a high amount of flavonoid and tannin content, with very low content on alkaloid compared to other plants (Rejeki and Addy, 2017;Abdulkadir et al., 2015). The absence of alkaloids in fruits has been reported by Bondjengo et al. (2017) and is attributed to differences in plant species or type of method used in extraction.

Identification of Fusarium oxysporum
Microscopic characteristics observed in this study such as the presence of oval non-septate microconidia and macroconidia with a slight curvature, septate, pointed apical cell and pink in colour confirm that the isolates were of Fusarium oxysporum. This supplements similar studies on the characterization of the fungal pathogen (Shobha and Kumudini, 2012;El Kichaoui, 2016;Bedasa, 2018).

Antimicrobial activities of the plant extract
As the concentration of K. africana extract increased, the mean mycelia diameter decreased (the inhibition zone increased). This supports similar research findings on the anti-fungal activity of K. africana against F. oxysporum (Itonga, 2011;Zofou et al., 2013;Rejeki and Addy, 2017). Kigelia africana extracts are reported to induce the disruption in fungal cell metabolism, increased permeability of fungal plasma membrane and destruction of the conidial wall structure (Al-Mujammaa'e, 2008). Organic extract of the fruit contains unique phytochemical constituents such as naphthoquinones, p-coumaric acid, ferulic acid, phenylpropanoids, kigelinone, dehydro α lapachone and lapachol, β-sitosterol, 3-dimethyl kigelin, ferulic acid and iridoids that toxic to plant fungal pathogen (Chenia, 2013;Bello et al., 2016;Chinsembu, 2019), although they were not identified in this study probably due to the use of aqueous extract alone. Water extract is not very effective on some microorganisms (Itoandon et al., 2012) because only a few phytochemicals can dissolve in water due to its low polarity (Gupta et al., 2010).
The current study revealed that an increase in the concentration of T. diversifolia leaf extracts reduced the mycelia growth of F. oxysporum. Leaf extracts of T. diversifolia are toxic to F. oxysporum and inhibit mycelial growth and spore germination (Enyiukwu et al., 2014;Adekunle, 2005). The plant has a promising broadspectrum antimicrobial activity that has been attributed to various compounds such as aradiol, squalene and a mixture of stigmasterol and sitosterol (Tagne et al., 2018). Combined plant extracts of K. africana fruit and T. diversifolia were more effective to inhibit the growth of F. oxysporum as compared to the single extract. This is because a combination of plant extract ratios increases the antimicrobial activity of the extract compared to when the plant extract is used singly (Akila et al., 2011;Mubarak et al., 2011;Bassolé and Juliani, 2012)

CONCLUSION AND RECOMMENDATION
This study investigated the extracts of K. africana fruit and T. diversifolia leaf ability to inhibit the growth of F. oxysporum which causes Fusarium wilt in Tomatoes. The phytochemical analysis revealed that the two plant extracts contained saponins, tannins, terpenoids, flavonoids, glycosides and phenolics except for alkaloids and steroids which were only found in T. diversifolia. The mean inhibitory zones ranged from 7.93 to 10.44 mm for T. diversifolia at 25 to 100 g/l. The mean inhibitory zones for K. Africana ranged from 12.07 to 15.56 mm at 25 to 100 g/l. K. Africana extracts had the highest inhibitory (antimicrobial) activity. Combining both extracts was more effective as compared to the single extract and this could be attributed to the synergistic effects of combining the two extracts. These plants may provide an effective measure of managing Fusarium wilt disease of tomatoes and could play an integral part in integrated pest management. However, there is a need for more studies on the isolation of the compound responsible for antifungal activity and ex-vitro experiments to test the efficacy of these plant extracts on the field. This study provides scientific evidence that K. africana and T. diversifolia extract possess antifungal activity and can be used as a broad-spectrum in managing microbial diseases.