In Vitro Control of Phytophthora infestans and Alternaria solani Using Crude Extracts and Essential Oils from Selected Plants

Tomato production is constrained by fungal diseases especially the early and late blight caused by Alternaria solani and Phytophthora infestans , respectively. Control of the two diseases is usually by use of synthetic fungicides which have a long residue eﬀect and also contribute to environmental pollution. Innovative use of biocontrols may oﬀer an eco-friendly and more sustainable solution. This study tested the in vitro eﬃcacy of crude extracts and essential oils of ginger, garlic, tick berry, and Mexican marigold in inhibition of radial growth of A. solani and P. infestans . Extraction of the crude extracts was done using distilled water, ethanol, and methanol solvents, while essential oils were extracted using the dry steam distillation method. The extracts and essential oils were used to amend the growth media of the test pathogens before introducing the precultured pathogens. Sterile distilled water and synthetic fungicide, Ridomil Gold ® , were used as positive and negative controls, respectively. Fungal growth inhibition was determined by measuring the radial growth of the test pathogens. Both the crude extracts and the essential oils portrayed some eﬃcacy against the test pathogens. Garlic crude extracts were found to be the most eﬀective, while ethanol was the most suitable extraction solvent. Essential oils were more eﬀective in restricting the pathogen growth than crude extracts. Ginger and garlic oil was found to be as eﬀective as the synthetic fungicide, and thus it was concluded that the two plants have strong antifungal properties with high potential of being utilized as biofungicides. However, eﬀective utilization of these products in farmers’ ﬁelds may require industrial formulation to improve their eﬃciency.


Introduction
Tomato (Lycopersicon esculentum Mill.) is the second most widely grown vegetable crop in East Africa and in the whole world after potato [1]. e crop is grown for its fruits which have high nutritive value rich in antioxidants such as lycopene, minerals such as potassium, magnesium, calcium, iron, and zinc as well as vitamins such as vitamins A, B group, C, and E [2]. In Kenya, the crop is one of the most important vegetable crops in the horticultural industry [3,4]. Kenya is among Africa's leading producers of tomatoes and is ranked 6 th in Africa with a total production of 410,033 tonnes [5]. Tomato production is a major source of income for small-scale holders and medium-scale commercial farmers [6]. Tomatoes enjoy a ready market in Kenya with considerably high demand throughout the year [7].
In Kenya, tomato production is mainly conducted in the open field or in the green houses [4]. However, production in open fields is highly constrained by pests and diseases [8] especially the early and late blight diseases caused by the fungus Alternaria solani and Phytophthora infestans, respectively [9]. e two diseases affect the leaves, stems, and fruits of tomato causing severe damage and high production losses of about 95.8% [10]. e symptoms of early blight on tomatoes include irregular black to brown spots that begins on older leaves where they enlarge to form lesions and may eventually lead to leaf fall [11]. e symptoms of late blight disease are black or brown lesions on leaves and stems. e lesions are water-soaked in appearance and are usually small at the beginning and then expand to become necrotic [12]. e fungus attacks the fruit at the fruit stalk and cause large sunken areas with concentric rings and a black velvety appearance [11]. Infected stems and petioles collapse while the fruits become greasy and decay and fall off the plant [13].
In Kenya, late blight disease of tomato ranks among the top constraints of tomato production, and together with early blight, they cause 95.8% of all the preharvest tomato losses [14]. e pathogens are spread by wind, water, and weeds of the solanaceae family, volunteer and susceptible tomato plants [8]. Tomato plants can be infected by the early blight fungus at any stage of growth but becomes more severe at the fruit set [15]. e disease is more severe in areas with high humidity, low temperatures, and high rainfall [16]. e development of late blight disease on susceptible tomato plants is favoured by cool moist whether with high relative humidity and temperature ranging between 18-22°C [8].
e causal pathogen, P. infestans, has a sophisticated morphology which makes it difficult to manage in the field [17].
Tomato farmers have for a long time relied on the use of synthetic fungicides to control the early and late blight diseases in an effort to meet the high demand of tomato which is faced by seasonal shortages [18]. In Kenya, control of the two diseases use up to forty pesticide applications per crop season [14]. However, the chemicals are expensive and increases the production cost by 20% [19]. In addition, synthetic fungicides have many other detrimental effects. For example, they pollute the environment by contaminating the soil, water bodies, and food subsequently causing harm to the living things including humans and nontarget beneficial organisms such as micro-organisms and pollinators [20]. Some of the active ingredients in the agrochemicals are hormone disruptors and may cause infertility, carcinogenesis, and mutations that result in the resistance of the pathogens to the agrochemicals [21,22]. Synthetic pesticides also pose problems within the markets due to their high chemical residue levels which makes some of the products rejected in the market resulting in losses [20,23].
Current crop protection efforts focus on developing biopesticides that are environmental friendly and effective in managing the plant pests and diseases. Natural plant products have been shown to be good sources of agrochemicals since they are easily biodegradable and do not pollute the environment [24]. Various natural products of plant origin have been proven to be effective in controlling various foliar pests and pathogens in a similar way as the synthetic pesticides [24,25]. e antimicrobial activity of several plant products against fungal pathogens have been studied under both in vitro and in vivo conditions [25]. Cold extracts of Azadirachtin indica were shown to inhibit the growth of Fusarium oxysporum which causes root and stem rot of cucumber [26]. is study aimed at evaluating the in vitro efficacy of crude extracts and essential oils of selected plants in inhibiting the growth of A. solani and P. infestans.
e study also tested whether the extraction solvent had a significant influence on the efficacy of the crude extracts on the test pathogen.

Collection and Isolation of Fungal Pathogens.
Tomato farms within Mwea Subcounty of Kirinyaga county in central Kenya were randomly sampled for collection of diseased plant samples. e area was selected because of its long history in tomato production and the high prevalence of early and late blight diseases. Diseased tomato leaves bearing symptoms of blights were identified by physical examination and collected randomly from identified farms. e diseased leaf samples were placed in cool boxes and taken to the Microbiology Laboratory at the University of Embu and stored at 4°C in a refrigerator awaiting further processing and analysis. e isolation of A. solani and P. infestans from the infected plant samples was conducted following the modified procedures of [27] using standard media of potato dextrose agar (PDA) and V8 juice agar, respectively. e infected tomato plant leaves were first washed under running water and then dipped into 1% sodium hypochlorite to surface sterilize them for three minutes and rinsed in three changes of distilled water. e cleaned samples were blotted dry using a sterilized blotting paper.
A sterilized scalpel was used to cut sections of infected leaf tissues measuring 3 mm × 3 mm towards the healthy portions where the pathogens were likely to be more active. e dried early and late blight infected tissues were directly plated on sterile PDA and V8 agar, respectively, and then incubated in the laboratory at room temperature (25°C) for 3 days. Single spore isolation was done to obtain pure cultures by isolating a section of hypha from the three-day old culture and introducing it into the growth medium and then incubating at room temperature. Fungal identification was done after 8 days using morphological characteristics and comparing with established keys [28] to confirm the identity of the target pathogens. Morphological identification was based on growth patterns, colour of mycelia, and microscopic examinations of vegetative and reproductive structures [28].

Preparation of the Test Plant Materials.
e crude plant extracts and essential oils were obtained from garlic (Allium sativum) cloves, ginger (Zingiber officinale) rhizomes, tick berry (Lantana camara) leaves, and Mexican marigold (Tagetes erecta) leaves. Fresh tick berry and Mexican marigold leaves were collected from the field, while ginger rhizomes and garlic bulbs were obtained from the local open air market. For extraction of crude extracts, 1 kg each of tick berry and Mexican marigold leaves were washed under tap water, rinsed in three changes of distilled water, and dried using sterilized blotting paper. ey were then separately oven-dried at 40°C for two days before being crushed to powder using a kitchen blender. Five-hundred grams each of garlic cloves and ginger rhizomes were peeled, washed under tap water, and rinsed in three changes of distilled water then dried using sterilized blotting papers. ey were then cut into small pieces using a sterilized scalpel and oven-dried at 40°C for five days after which they were crushed to powder using a kitchen blender. e dry powders were put in air tight dull bottles and stored for later use in the laboratory under room temperature. For extraction of essential oils, 1 kg each of tick berry and Mexican marigold leaves were washed under tap water, rinsed in three changes of distilled water, and then air-dried in the laboratory to evaporate the moisture content. One kilogram each of garlic cloves and ginger rhizomes were also washed with tap water and then rinsed with three changes of distilled water. ey were also air-dried in the laboratory and later peeled. All the fresh materials were stored in the laboratory under room temperature awaiting the extraction of essential oils.

Extraction of Test Plant Crude Extracts.
e extraction of test plant crude extracts was done following a modified procedure described by Handa et al. [29]. Two-hundred (200) grams dry powder of each of the four plant products were soaked separately in 500 ml of sterile distilled water, analytical grade ethanol (95%), and methanol (95%) for 72 hrs.
e content was then filtered using Whitman's (No.2) filter paper. For water extraction, the filtrate formed the stock solution and was refrigerated at 4°C for further use in testing the efficacy on the fungal pathogens. Ethanol and methanol filtrates were evaporated using a rotary evaporator, and the remaining small amounts of the alcoholic solvents were evaporated in the oven at 40°C. e remaining contents were stored in the refrigerator at 4°C and later used to test their efficacy on the growth inhibition of the fungal pathogens.

Extraction of Essential Oils from the Test Plants.
Extraction of essential oils was done following a modified procedure described by Adams [30]. e vertical steam distillation unit, consisting of a hot plate, boiling flask, biomass flask, still head, condenser, and receiving flask (separating funnel), was used for the dry steam distillation of plant material. e fresh materials of each plant were put separately in the biomass flask, and the distillation unit was switched on. Steam was produced in the boiling flask by heating distilled water with the hot plate. e steam moved upward into the biomass flask where essential oils and other water-soluble plant compounds were moved as vapour through the still head, condensed in the water-cooled condenser, and collected in the receiving flask (separating funnel). e receiving flask separated the heavier-than-water oils from the lighter-than-water oils while allowing excess water-soluble compounds to be drained out and collected separately.

Experimental Design and Layout.
e experiment was conducted at the University of Embu Microbiology Laboratory. ere were two sets of factorial in vitro experiments that were laid out in a completely randomized design replicated four times. e first experiment consisted of crude extracts from the four test plants extracted using the three different solvents (sterile distilled water, ethanol, and methanol). e second set of experiment consisted of four essential oils extracted from the four test plants. All the biocontrol products were tested against A. solani and P. infestans.
e experiment was conducted following the modified procedure of Al-samarrai et al. [31,32]. e methanol and ethanol crude plant extracts were first diluted with 2 ml dimethyl sulfoxide (DMSO) before diluting with water in the ratio of 1 : 1. Five-hundred (500) ml each of PDA and V8 agar were amended separately with 5 ml each of the four different crude extracts and 1 ml each of the four essential oils separately and dispensed into separate Petri dishes. Five ml of distilled water and 1.5 g of Ridomil Gold ® (metalaxyl-M and S-isomer, Mancozeb) dissolved in 5 ml of distilled water were mixed with the media as the negative and positive controls, respectively. In the second experiment, 1 ml of distilled water and 1.5 g of Ridomil Gold ® dissolved in 5 ml of distilled water were mixed with the media as the negative and positive controls, respectively. Fungal culture discs of 5 mm were cut from one week old cultures of A. solani and P. infestans using a sterilized cork-borer and cultured at the centre of each Petri dish per replicate and incubated at room temperature (25°C ± 2) for seven days in order to monitor the growth of the test pathogens.

Data Collection and Analysis.
Fungal colony diameter of each treatment was measured using a pair of dividers and a ruler after every two days up to the seventh day. Data obtained were subjected to analysis of variance (ANOVA) using XLSTAT version 2019, and separation of means was conducted using Student Newman's Keul (SNK) test at a 95% level of confidence.

Effect of Water-Extracted Crude Extracts on the Fungal Pathogens.
e results obtained from the study showed that all the water-extracted plant extracts were effective in inhibiting the growth of the test pathogens as compared with the negative control (Table 1). However, the rate of inhibition varied significantly (p < 0.0001) among the different test plant extracts as compared with the positive and negative controls within a period of 7 days. Both pathogens recorded incremental growth from day one to day seven in all the treatments except the positive control with synthetic fungicide where no pathogen growth was recorded. Varied effects of the different extracts were recorded in all the sampling days, but a clear separation was observed on the 5 th and 7 th days where all the treatments were significantly different from each other for both pathogens. Garlic extracts were consistently the most effective in inhibiting the growth of both pathogens followed by ginger, tick berry, and Mexican marigold in that order (Table 1).

Effect of Methanol-Extracted Crude Extracts on the Fungal Pathogens.
e results showed that all the methanolextracted plant extracts were effective in inhibiting the growth of the test pathogens as compared with the negative control, but their efficacy against both pathogens varied International Journal of Agronomy 3 significantly (p < 0.0001) between the treatments ( Table 2). e positive control with Ridomil Gold ® synthetic fungicide recorded 100% growth inhibition of both pathogens throughout the 7 days' testing period. On the other hand, the negative control exhibited the highest pathogen growth which was significantly (p < 0.05) different from the other treatments throughout the testing period. e general trend exhibited near the end of the 7 days testing period showed that garlic extracts were the most effective in inhibiting the growth of both pathogens followed by ginger, tick berry, and Mexican marigold in that order (Table 2). e plant extracts portrayed varied effects on specific pathogens over the testing period. Ginger extracts performed better than the garlic extracts in controlling the growth of A. Solani on the 1 st and 3 rd days of testing but the two switched positions on the 5 th and 7 th day. e efficacy of tick berry and Mexican marigold against A. solani were similar on day 1 and day 5, but tick berry performed significantly better than Mexican marigold on the 3 rd and 7 th day. On the other hand, garlic extracts were consistently the most effective in inhibiting the growth of P. infestans followed by ginger extracts. Mexican marigold performed significantly better than tick berry against P. infestans at the initial testing stages (days 1-3) but the two switched positions in the later stages (days 5-7).

Effect of Ethanol-Extracted Crude Extracts on the Fungal
Pathogens. All ethanol-extracted plant extracts were effective in inhibiting the growth of the test pathogens, but their efficacy varied significantly (p < 0.0001) among them and from the positive and negative controls (Table 3). e positive control maintained 100% inhibition of the pathogen growth, while the negative control recorded the highest pathogen growth. e four crude extracts demonstrated varied reactions to the pathogens at the initial testing stages. Unlike the trend observed with water and methanolextracted products, tick berry appeared to be more effective than ginger in controlling the growth of A. solani from day 1 to day 5. However, the latter became more effective than the former on day 7. e response of P. infestans to ethanolextracted plant products assumed a similar trend to that observed with water and methanol solvents. By the end of the testing period, it was evident that garlic was still the most effective biocontrol against both pathogens followed by ginger and tick berry. Mexican marigold was the least effective in the control of both pathogens.

Comparative Effect of Extraction Solvents on the Fungal
Pathogens.
e type of solvent used in extraction of the crude plant extracts was found to have a significant influence on the efficacy of the extracts (Figure 1). e results showed that ethanol-extracted products recorded the best (p < 0.05) efficacy against both pathogens by the end of the testing period (day 7), while water-extracted products were the least effective. On the other hand, the growth of the test pathogens did not vary significantly (p > 0.05) within the same extraction solvent.

Comparative Response of the Two Fungal Pathogens to the Crude Extracts.
e average growth of the two pathogens varied significantly under the control treatment and when subjected to ginger and garlic extracts but did not vary significantly (p > 0.05) when treated with both tick berry and Mexican marigold extracts (Figure 2). e growth of A. solani was significantly lower than that of P. infestans when treated with ginger extracts. However, garlic extracts had a significantly higher growth inhibition on P. infestans as compared with that in A. solani.

Antimicrobial Activity of the Essential Oils from the Test
Plants on the Test Pathogens. Essential oils extracted from the four test plants varied significantly (p < 0.05) in their antimicrobial activity against the growth of the test pathogens (Table 4). Ginger and garlic oils produced similar results to the positive control treatment with synthetic fungicide where no growth of the test pathogens was observed throughout the testing period. e antimicrobial activity of essential oils obtained from Mexican marigold and tick berry were also significantly (p < 0.05) different from the negative control in inhibiting the growth of both pathogens. Comparative microbial activity of Mexican marigold and tick berry essential oils against the growth of P. infestans was not significantly different except on day 1 International Journal of Agronomy where tick berry essential oils appeared to be more effective than Mexican marigold. However, the two plants varied significantly in controlling the growth of A. solani where essential oils from Mexican marigold were found to be more effective than those from tick berry (Table 4).

Comparative Response of the Fungal Pathogens to Essential
Oils and Crude Extracts. From the results of the study, it was evident that the test plants had varying biocontrol potential against the two test pathogens as compared with the negative and positive controls. However, the essential oils were found to be more effective than the crude extracts in inhibiting the growth of the test pathogens ( Figure 3). Essential oils from ginger and garlic caused 100% growth restriction of the two pathogens just like the synthetic fungicide. Likewise, the efficacy of Mexican marigold and tick berry was found to improve when essential oils were used as compared with raw extracts.

Effect of the Extraction Method of the Plant Product on the Test Pathogens.
e growth inhibition on the test pathogens was found to be significantly (p < 0.0001) influenced by the method used to extract the biocontrol products (Table 5).
e essential oils were more effective in pathogen growth inhibition than crude extracts extracted using extraction solvents. e three extraction solvents were also found to significantly influence the efficacy of the extracts in pathogen growth inhibition. For A. solani, water extraction produced relatively better results than ethanol and methanol on the 1 st and 3 rd day, respectively. However, towards the end of the testing period, ethanol extraction produced the best results   infestans, a clearer trend was exhibited where ethanol extraction consistently recorded the lowest pathogen growth followed by methanol. However, methanol and water extraction methods recorded similar results on the 1 st and 7 th days of testing (Table 5).

Discussion
From the study, it was evident that the crude extracts from all the plants (ginger, garlic, tick berry, and Mexican marigold) that were tested were effective in inhibiting the growth of A. solani and P. infestans as compared with the control. However, the inhibition varied with the type of the plant with garlic showing the highest rate of growth inhibition on the two test pathogens. is was an indication that the tested plants contain certain antifungal compounds. is observation supports other previous reports from different authors on the antifungal properties of various plants. Reports of Daniel et al. [33] showed that garlic effectively inhibited Botrytis cinerea in vitro. Tick berry extracts were reported to effectively inhibit the growth of P. infestans [34,35] and Colletotrichum falcatum that causes red rot disease of sugar cane [36]. Mexican marigold extracts were also reported to significantly suppress the root-knot nematodes that infect tomato [37] and to have antibacterial    effects against Escherichia coli and Staphylococcus aureus [38]. Extracts from ginger were found to have fungicidal properties against Fusarium oxysporum f.sp. lycopersici [39]. Sumira et al. [40] reported that crude extracts of Origanum vulgare had significant antimicrobial activity against 39 bacteria, 2 yeast, and 16 fungi species. Different scientists have been applying different concentrations of crude extracts in efficacy studies. In this study, 5 ml of each plant extracts were used to amend 500 ml each of PDA and V8 agar. Sifat and Monjil [41] used a 10% concentration of crude garlic extract which caused 95.50% growth inhibition of Rhizoctonia spp, while 20% concentration showed excellent mycelial inhibition of Rhizoctonia solani causing sheath blight of rice. Bhuiyan et al. [42] found a 20% concentration of garlic extracts to be effective in controlling the growth of Colletotrichum dematium. Garlic and ginger extracts inhibited growth of Penicillium spp and Aspergillus spp in in vitro [43]. Ginger essential oil was found effective in inhibiting the growth of Fusarium oxysporum, Rhizoctonia solani, and Botryodiplodia theobromae causing decline in the disease of guavas [44].
From the study, it was evident that the two test pathogen species responded differently to different crude extracts. Ginger extracts were more effective against A. solani than P. infestans and vice versa for the garlic extracts. is observation can, therefore, be assumed to indicate that the efficacy of the crude extracts may be determined by the sensitivity of the target pathogens to the extracts.
is study did not analyse the actual compounds that were responsible for antifungal growth inhibition in the selected test plants. However, analysis of the biochemical compounds in the test plants showed that the chief constituent compounds in ginger, garlic, Mexican marigold, and tick berry were gingerols, allicin, phenols, tagetone, and lantacin, respectively (data not presented). e active antifungal compounds in these plants have been reported in some previous studies. Vidyasagar and Tabassum [45] reported that Mexican marigold plant species contain compounds such as piperitone and piperitenone which inhibit fungal mycelial growth by modifying the structure of the mycelia. Sarfraz et al. [46] reported that allicin from garlic effectively controlled Alternaria brasicicola, Verticillium dahlia, Verticillium longisporum, and B. cinerea. Reports of Mahadyet al. [47] showed that ginger has shogaols that contain both antifungal and antibacterial properties. Tick berry reportedly contains flavonoids, alkaloids, and tannins [48] that have been shown to inhibit the growth of Alternaria alternata [49].
e results of this study showed that the extraction solvent had a significant influence on the efficacy of the crude extracts. is corraborates the findings of other scientists who reported that the activity of crude extracts varies depending on the plant material, the method of extraction, and the solvent used [50][51][52]. In this study, the most effective crude extracts were those extracted with ethanol followed by methanol extracts and then water extracts. Similar results were reported by Singh and Srivastava [49] where ethanolic extracts of Lantana camara were the most effective in inhibiting the growth of Alternaria alternata followed by methanolic and water extracts. In vitro antibacterial activity of ethanolic extracts of tick berry was also reported to be more effective than of water extracts in inhibiting the growth of both Gram-negative and Gram-positive bacteria [53]. e higher level of effectiveness in ethanol as a solvent is attributed to the fact that ethanol is a polar solvent and therefore it produces a higher concentration of phenolics [54]. e low efficacy of aqueous plant extracts is as a result of water not extracting nonpolar active compounds in plant materials [55]. e use of water in extraction also increases the amount of impurities that lower the quality of the extracts [50]. However, hot water extracts from ginger were found to inhibit the growth of Fusarium oxysporum, Aspergillus Niger, and A. Flavus and reduced rotting of yam tubers [56]. Dabur et al. [57] obtained better results with tick berry water extracts than with organic solvents.
From the results of this study, essential oils from the four test plants were also effective in inhibiting the growth of the test pathogens as compared with the negative and positive controls. Similar results have been reported in other previous studies. Essential oils of tick berry were reported to exhibit significant antibacterial activities against different strains of Gram-positive and Gram-negative bacteria [58][59][60]. Tick berry essential oils also exhibit insecticidal, antifeedants, and antihelminthic properties [61]. Garlic essential oils are reported to exhibit insecticidal properties [62,63] and nematicidal properties [64]. Mahmoud et al. [65] reported extensive inhibitory effect of garlic essential oil on F. solani, M. phaseolina, R. solani, and S. Rolfsii. Essential oils of ginger were reported to have a substantial fungicidal effect on the Penicillium expansum, A. flavus, A. alternata, Fusarium oxysporum [66], A. parasiticus, and A. flavus [67]. According to Saha et al. [68], essential oils of Mexican Mean values followed by the same letter within the same column are not significantly different at p � 0.05.
International Journal of Agronomy marigold portrayed antifungal properties against Pyricularia grisea, Sclerotium rolfsii, A.solani, and F. oxysporum. e essential oils were found to have a higher efficacy against the test pathogens than the crude extracts. Garlic and ginger essential oils caused total growth restriction in both pathogens just like the synthetic fungicide, Ridomil. ese findings were in agreement with those of Sumira et al. [40] who reported that essential oils of Origanum vulgare species had a significantly higher antimicrobial activity against 39 bacteria, 2 yeast, and 16 fungi species as compared with crude extracts.

Conclusion and Recommendations
From the findings of this study, it was evident that garlic, ginger, tick berry, and Mexican marigold have antifungal properties against A. solani and P. infestans. e four plants are therefore recommended for industrial utilization for production of less cumbersome fungicidal products that are efficient for use under field conditions. e antimicrobial scope of these test plants can also be expanded by testing their efficacy on a wide range of disease causing microorganisms. e efficacy of tick berry and Mexican marigold which was found to be lower than that of ginger and garlic can be improved through integrated utilization. Since ginger extracts were found to be more effective against A. solani than P. infestans and vice versa for the garlic extracts, this study recommends further analysis between these two plants to ascertain this observation before issuing a specific recommendation. In addition, since this study was conducted in vitro, there is also need to conduct a confirmatory in vivo experiment in a greenhouse. is study also recommends ethanol as the most effective extraction solvent as compared with methanol and water.

Data Availability
Most of the data used to support the findings of this study are included within the article. Additional data are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.