Essential Oils as Biocontrol Agents of Early and Late Blight Diseases of Tomato under Greenhouse Conditions

Tomato production worldwide is usually restrained by various infections, among them mainly the late and early blight caused by Phytophthora infestans and Alternaria solani, respectively. Lately, there has been a growing concern over the use of synthetic fungicides on environmental and food safety, hence the need to explore other alternatives that are friendly to the user, the consumer, and the general environment. &is research sought to test the potency of ginger, garlic, and Mexican marigold essential oils against the early and late blight diseases of tomato under greenhouse conditions. A synthetic fungicide (Ridomil Gold®) was used as a positive control while distilled water acted as a negative control. &e extraction of essential oils was done by dry steam distillation and then mixed with tween twenty before being topped up with sterile distilled water. &ey were then used to spray tomato plants that had been inoculated with A. solani and P. infestans isolates under greenhouse conditions.&e tomato plants were evaluated for growth, yield, and disease severity. &e data obtained was subjected to ANOVA and separation of means was conducted using Student–Newman–Keul (SNK) test at 95% level of confidence. &e three essential oils had a significant potency against the two diseases which is comparable to the synthetic fungicide.Marigold essential oil was also found to have a significant impact on the general growth of sprayed tomato plants. Essential oils of the three plants can be further explored as alternative products management of the two diseases.


Introduction
Tomato cultivation in Kenya has intensified in recent years for both processing and fresh market [1] and for commercial and home consumption [2]. It constitutes 7% of the total horticultural produce and 14% of the total vegetables grown in Kenya [1]. e fruit is always in high demand since it is consumed by nearly all the households. It is rich in lycopene which is an antioxidant that is gaining popularity because of its role in mitigating heart disease, cancer, and muscular degradation [3]. It is also rich in minerals such as iron and vitamins A, C, and E [4]. Tomato is hence a common ingredient in many recipes including stews, soups, and salads [3]. Kenya is among the leading tomato producing countries in sub-Saharan Africa with a total production of about 410,033 tons of tomato fruits [1]. e largest percentage of tomatoes produced in Kenya is under open field where they are vulnerable to diseases and pests [5]. Diseases such as late blight (Phytophthora infestans) and early blight (Alternaria solani) affect the quality, quantity, and profitability of tomato [2]. Early blight is more aggressive under heavy rainfall, high humidity, and temperatures ranging between 24 and 29 o C [6]. e disease can affect all the above ground parts of all stages of growth and development of the tomato plant [7]. Irregular black/brown spots appear on aging leaves and then enlarge creating lesions that result in complete leaf fall. e symptoms begin from older lower leaves and then progress to the upper leaves [8]. Late blight is also known to cause extensive losses on susceptible tomato plants when environmental conditions are conducive [9]. e symptoms appear as irregular watersoaked lesions which extend fast into pale green to brown and then cover the stem and the surface of large leaves [10]. Under high humidity, a grey to white moldy growth covers the lesions consisting of sporangiophores that produce sporangia where the zoospores are formed [10]. e two diseases can cause tomato losses of close to 100% [11].
For disease control in the field, farmers rely largely on synthetic fungicides. However, there is a blooming concern on the use of synthetic fungicides due to their toxicity and residue retention in the food products [12]. Consequently, the consumer markets have come up with strict quality requirement in view of maximum residue levels of pesticides in fresh fruits and vegetables [2]. e fresh fruit and vegetable products that are not able to meet the market requirements have been denied access to the lucrative markets resulting in losses. e rejected products are usually redirected to the local markets where they are sold at low prices to unsuspecting consumers who find them appealing to the eyes and end up consuming the chemical residues. In addition, failure of small-scale farmers to use protective gear during chemical application and to adhere to dilution instructions given by the manufacturers poses a great health risk to them [2]. Some synthetic chemicals are also reportedly toxic to beneficial and nontarget organisms such as natural enemies and pollinators [13][14][15]. e worldwide trend to explore organic/biological pesticides as alternative options to synthetic ones is gaining popularity because of the negative effects resulting from excessive use of the latter [16]. Most of organic pesticides are easily biodegradable, nonpoisonous, and safe to nontarget organisms and antagonists and leaves no harmful residues on the food products [17].
Several plant compounds have proved to have fungicidal activity against fungal pathogens under both in vitro and in vivo domain [18]. Essential oils are aromatic and volatile liquids which have abundant bioactive compounds [19,20] that have demonstrated antifungal activity but are highly degradable under natural conditions [21]. ey contain different compounds [22] which have fungicidal, nematicidal, insecticidal, acaricidal, and bactericidal properties [23]. Essential oils are becoming popular because they are safe to use and more acceptable by consumers and they potentially have a multipurpose functional use [24]. e current study is aimed at investigating the antimicrobial activity of ginger (Zingiber officinale), Mexican marigold (Tagetes erecta), and garlic (Allium sativum) essential oils against tomato late and early blight diseases under greenhouse conditions.

Collection of Diseased Tomato Plant Materials.
Sampling was done in Mwea, Kirinyaga County, because it is an area renown for tomato cultivation and blight diseases are rampant. e area lies within 0.6897°S, 37.3400°E. It is characterized by annual rainfall ranging between 800 mm and 1250 mm usually in two seasons. Annual temperature ranges between 19.6°C and 26.3°C. It has gentle rolling slopes with black cotton soils [25]. Tomato leaves with early and late blight symptoms were randomly collected from the farms by physical examination. ey were taken to the laboratory in cool boxes and refrigerated at 4 o C for processing and further analysis.

Isolation of the Pathogens.
e pathogen isolation followed the modified procedures of Naik et al. [26]. V8 and Potato Dextrose Agar (PDA) were the standard media used for isolation of P. infestans and A. solani, respectively, from the diseased tomato samples. e tomato leaves bearing blight symptoms were first washed under clean running tap water and then surface sterilized in 1% sodium hypochlorite for three minutes. ey were rinsed in three changes of sterilized distilled water and blotted dry using sterilized blotting paper. A sterilized scalpel was used to cut infected leaf tissues of 3 mm × 3 mm size towards the healthy tissues where the blight pathogens were suspected to be more active. e surface sterilized tissues were directly plated on the sterilized PDA and V8 agar for early and late blight separately and then incubated in the laboratory at room temperature (25 o C) for three days. e colonies were then subjected to single spore isolation and subcultured separately on their specific media to obtain pure strains for identification. e isolates were identified using morphological microscopic and macroscopic features [11] and comparing with established identification keys [27]. e isolates were then maintained on plates awaiting their inoculation on tomato plants in the greenhouse.

Extraction of Essential Oils from the Test Plants.
Mexican marigold leaves were collected from the field while garlic cloves and ginger rhizomes were bought from the open air market. Five (5) kg each of the raw plant products were prepared for essential oil extraction following the procedure described by Mugao et al. [11,22]. e essential oils were extracted following the vertical steam distillation procedure described by Adams [28] as modified by Mugao et al. [11,22]. e essential oils were put in air tight bottles and stored in the refrigerator at 4°C awaiting the greenhouse experiment.

Experimental Design and Layout.
e greenhouse experiment was a split plot laid out in a Randomized Complete Block Design (RCBD) with five treatments replicated four times. ere were two main plots representing the plants inoculated by the two pathogens (P. infestans and A. solani).
e five treatments comprised of essential oils of Mexican marigold, ginger, and garlic, the positive control (Ridomil Gold ® synthetic fungicide), and the negative control (distilled water). Ridomil Gold ® is a curative fungicide manufactured by Syngenta (https://www.syngenta.co.ke/ product/crop-protection/ridomil-gold-mz-68-wg) and comprises Metalaxyl-M 40 g/Kg and Mancozeb 640 g/Kg formulated as wettable granules. e test tomato variety was Kilele F1 also developed by Syngenta and not characterized as being resistant to both early and late blight of tomato (https://www.syngenta.co.ke/product/seed/tomato/kilele-f1). One-month-old pregerminated tomato seedlings were planted in a greenhouse in plastic pots filled with sterilized potting mixture of soil, sand, and well decomposed manure in a ratio of 3 : 2 : 1. e greenhouse temperatures were 21-29 0 C and the humidity was 67%-73%.

Preparation of Inoculum.
Fourteen-day-old cultures of A. solani and P. infestans were used as source of the spores. Spore suspension was prepared by adding 5 ml of sterile distilled water to a pure 14-day-old culture in a Petri dish. Dislodging of spores was done with a bent glass rod and the content sieved using a three-layer cheese cloth to remove the mycelia.
e hemocytometer slide was used under a microscope to ascertain the spore suspension concentration and then standardized to 1 × 10 6 spores per millimeter with sterile water. e inoculum was stored in the laboratory at 4 o C awaiting inoculation.

Inoculation and Application of Treatments.
Inoculation was done on actively growing tomato plants three weeks after transplanting by spraying every plant with the 20 ml of the inoculum using a hand sprayer. Symptoms of disease development began to appear on the 6 th day after inoculation. Application of treatments was done at fourteen days after inoculation through foliar sprays conducted using a hand sprayer. In the preparation for spraying, 5 ml of each essential oil was first mixed with 5 ml of tween 20 and then topped with sterile distilled water to up to 500 ml and mixed thoroughly. Ridomil Gold ® solution was prepared following the manufacturer's instructions of 2.5 g per liter of water. Subsequent applications were done after every fourteen days up to 60 days after planting.

Data Collection and Analysis.
Disease severity was scored per treatment after every two weeks using a 0-5 scale [29] based on the size and number of lesions on the infected leaves as follows: (i) 0 � healthy (no visible lesions on the leaf ) (ii) 1 � up to 10% of the infected leaf area (iii) 2 � 11%-25% of the infected leaf area (iv) 3 � 26-50% of the infected leaf area (v) 4 � 51-75% of the infected leaf area (vi) 5 � more than 75% of the infected leaf area e disease scales were converted into percentage for each plant using the formula described by Chaerani et al. [30] as provided in the equation.

Disease severity �
Sum of all ratings × 100 No. of leaves sampled × maximum disease scale .
(1) e disease severity on each plant was determined according to the average of all leaves of a plant. counted based on visual assessment [31].
Data was also collected on growth and yield parameters including plant height, branch number, leaf number, days to 50% flowering, and number and weight of marketable fruits. All the data was subjected to Analysis of Variance (ANOVA) using XLSTAT version 2019 and separation of means was conducted using Student-Newman-Keul (SNK) test at 95% level of confidence.

Influence of the Test Pathogens on Disease Severity.
Severity of early blight disease on tomato plants inoculated with A. solani increased from week 2 to week 4 before decreasing in the subsequent weeks. On the other hand, severity of late blight disease on tomato plants inoculated with P. infestans kept increasing throughout the experimental period (Table 1). In week 2, the severity of the disease did not vary significantly (p > 0.05) between the two pathogens. Within weeks 4-10, the average disease severity differed significantly between the two pathogens with P. infestans recording a higher disease severity than A. solani (Table 1).

Efficacy of Essential Oils on Control of Early and Late
Blight of Tomato. Severity of early and late blight diseases was significantly reduced by application of essential oils from the selected plants. e essential oils from the three plants gave similar results to the synthetic fungicide used as positive control but varied significantly with the negative control (distilled water). For both pathogens, disease severity in the negative control treatment continued to increase as it decreased in the other treatments (Table 2).

Effects of the Essential Oils on the Leaf Number of Tomato.
e number of leaves on the tomato plants inoculated with A. solani isolate varied between the treatments but the leaves increased as the plants continued to grow (Table 3). Among the tomato plants inoculated with A. solani, the number of leaves was the same (p > 0.05) in all the treatments in the second week. In the fourth week, tomato plants treated with Mexican marigold essential oil had lower number of leaves but did not differ significantly from the control while in week six the number of leaves did not vary significantly (p > 0.05) between treatments. However, week eight had significantly (p < 0.05) lower number of leaves in the control but did not differ significantly in the International Journal of Agronomy other treatments. In week ten, the number of leaves on tomato plants treated with ginger, garlic, and Mexican marigold essential oil did not vary significantly but it was significantly higher than that of plants treated with Ridomil Gold ® and the control.
In tomato plants inoculated with P. infestans, the number of leaves increased in all the treatments as the plants continued to grow (Table 3).
ere was no significant (p > 0.05) difference in leaf number between all treatments in weeks two and four. In weeks six and eight, there was

Effect of the Test Pathogens on Leaf Number of Tomato.
e number of leaves increased in all the inoculated tomato plants as the weeks progressed (Table 4). However, the number of leaves varied significantly (p < 0.0001) between tomato plants inoculated with different test pathogens in the first eight weeks. More leaves were recorded in tomato plants inoculated with A. solani as compared to P. infestans. e number of tomato leaves did not differ significantly (p > 0.05) between the two pathogens in week 10 (Table 4).

Effects of the Essential Oils on Height of Inoculated
Tomato Plant. As expected, the plant height increased with the age of the tomato plants (Table 5). For tomato plants inoculated with A. solani, there was no significant (p > 0.05) difference in plant height between treatments in weeks two, four, and six. In week eight, there was no significant difference between all the essential oils and synthetic fungicide treatments but these treatments recorded significantly (p < 0.05) higher plant height value than the control. In week ten, tomato plants sprayed with Mexican marigold essential oils recorded the highest plant height value (70.10 cm) while the control recorded the lowest (55.77 cm). e plant height of tomato plants sprayed with garlic and ginger essential oil did not vary significantly with those sprayed with Ridomil Gold ® (Table 5).
Tomato plants inoculated with P. infestans also did not differ significantly in height in all treatments during the second week. In week four, the tomato plants in the control treatment showed significantly (p < 0.05) lower plant height value than all the other treatments which did not show significant variation among them in plant height. In week six, the plant height varied significantly (p < 0.05) between the treatments with plants in the control treatment being shorter than the plants in other treatments. Tomato plants sprayed with Mexican marigold, ginger, and garlic essential oils did not differ significantly in plant height but Mexican marigold treatment was significantly different from both the positive and negative control treatments. Results of weeks eight and ten showed a similar trend. Mexican marigold essential oil maintained the highest number of leaves in the two weeks. e plant height of tomato plants in the garlic, ginger, and positive control (Ridomil Gold) treatments did not have significant difference. e tomato plants in the negative control treatment were the shortest (Table 5).

Effect of the Test Pathogens on Tomato Plant Height.
e plant height increased in all the tomato plants inoculated with the test pathogens (Table 6). However, the plant height varied significantly between the tomato plants inoculated with A. solani and P. infestans in week 2 and week 10. Tomato plants inoculated with P. infestans were taller than those inoculated with A. solani (Table 6). In weeks 4, 6, and 8, the plant height increased in all the tomato plants but did not differ significantly between the test pathogens ( Table 6).

Effects of Essential Oils on Branch Number.
e branch numbers obtained across the treatments varied significantly (p < 0.05) with tomato plants sprayed with Mexican marigold essential oil recording the highest number of branches among the plants inoculated with A. solani. However, the branch number in the Mexican marigold treatment did not significantly (p > 0.05) differ from the ones recorded from tomato plants sprayed with ginger (Table 7). e rest of the treatments did not differ significantly in tomato branch numbers. Among tomato plants inoculated with P. infestans, the number of branches differed significantly (p < 0.05) between treatments with the negative control recording the lowest branch number while tomato plants sprayed with Mexican marigold essential oil showed more branching but it did not differ significantly from the branching in garlic and ginger treatments. However, tomato plants sprayed with Mexican marigold essential oil had significantly higher number of branches compared to the synthetic fungicide (positive control) treatment (Table 7).

Effects of Essential Oils on Days to Flowering.
e treatments significantly (p < 0.05) influenced the number of days to flowering on the tomato plants inoculated with A. solani (Table 7). Plants sprayed with Mexican marigold essential oil took significantly longer time to flower followed by the positive and the negative control treatments. Tomato plants sprayed with ginger and garlic essential oils took shorter time to flower as compared to the other treatments. Days to flowering did not differ significantly (p > 0.05) among tomato plants inoculated with P. infestans (Table 7).

Effects of Essential Oils on Fruit Number.
e number of tomato fruits varied significantly (p < 0.05) between the treatments and the control (Table 7). Among tomato plants inoculated with A. solani, the control recorded the lowest  (Table 7). Among tomato plants inoculated with P. infestans, all the treatments recorded significantly higher fruit weight than the negative control but only Mexican marigold treatment was significantly different from the positive control (Ridomil Gold) in terms of fruit number (Table 7).

Effects of Essential Oils on Fruit
Weight. e average fruit weight also varied significantly (p < 0.05) between the treatments. For the plants inoculated with A. solani, the negative control treatment recorded the lowest fruit weight compared to the other treatments which did not differ significantly in fruit weight. For the plants inoculated with P. infestans, ginger treatment recorded the highest fruit weight which was not significantly different from fruit weight recorded in Mexican marigold treatment. Tomato plants sprayed with essential oils from garlic and Mexican marigold did not differ significantly from the ones sprayed with Ridomil Gold ® . e control recorded significantly lower fruit weight than all the other treatments (Table 7).

Effects of the Test Pathogens on Growth and Yield of Tomato.
e branch number, days to flowering and fruit number did not differ significantly between tomato plants inoculated with the different test pathogens (Table 8). However, the tomato fruit weight differed significantly (p < 0.0001) between the test pathogens with tomato plants inoculated with P. infestans recording a lower fruit weight (Table 8).

Correlation between Disease Severity, Growth, and Yield of Tomato Plants.
In the plants inoculated with A. solani, the plant height had a significant (p < 0.05) positive correlation with the number of leaves, fruit number, and fruit weight but was negatively correlated with disease severity. ere was no significant (p > 0.05) correlation between plant height and branch number and days to flowering. e leaf number was also positively correlated with the fruit number and fruit weight and negatively correlated with disease severity but had no significant (p > 0.05) correlation with branch number and days to flowering. Disease severity was also found to negatively influence the fruit number and fruit weight but did not significantly (p > 0.05) affect the branch number and days to flowering. e branch number had no significant (p > 0.05) effect on days to flowering. Likewise, the days to flowering had no significant (p > 0.05) effect on fruit number and fruit weight (Table 9). In the plants inoculated with P. infestans, all the growth and yield parameters were significantly (p < 0.05) and positively correlated with each other and negatively correlated with disease severity (Table 9).

Discussion
e essential oils from the three plants were found to have significant efficacy against the early and late blight diseases of tomato that was comparable to that of Ridomil Gold synthetic fungicide. is is in consonance with the findings of Lengai et al. [2] and Nashwa and Abo-Elyousr [32] who reported that ginger, garlic, and turmeric extracts had a similar effect to Ridomil fungicide in their fungicidal properties against tomato early blight. According to Nashwa [18], Ridomil and the plant extracts efficacy were reflected in   International Journal of Agronomy the fruit yield of tomato when compared with the untreated control. Jambhulkar et al. [33] reported that the mechanism of disease suppression may be either by active antimicrobial compounds acting on the pathogen directly by destroying their membranes or by the compounds inducing systemic resistance in host plants thus lowering disease development [34]. e antifungal and antibacterial effects of such compounds are a result of many compounds acting synergistically and there would be negligible chance of pathogens developing resistant races after application of essential oils [24]. Essential oils contain secondary metabolites such as terpenes, organosulfur, phenols, and alkaloids which are antimicrobial [35]. Several other studies have shown that essential oils have remarkable antimicrobial activity against pathogens causing plant diseases. Ngadze [36] reported higher antimicrobial effects of marigold extracts on tomato late blight than garlic extracts. Mexican marigold essential oil reportedly contains antibacterial properties against Staphylococcus aureus and Bacillus subtilis [37] as influenced by the presence of dihydrotagetone and α-ocimene compounds found in the essential oil. Marigold essential oil has also been reported to have antifungal properties expressed by compounds such as limonene, 1, 8-cineole, and α-pinene found in the essential oil [37].
is shows that essential oils have some broadspectrum antimicrobial properties. Reports of Mahmoud et al. [38] showed that garlic essential oil portrayed high antimicrobial activity against damping off disease in peanuts. Diallyl disulfide in garlic essential oil portrayed an antifungal activity against Phytophthora nicotianae that cause tobacco black shank disease [39]. According to Singh et al. [40], ginger essential oils contain phenolic compounds   such as shogaols, zingerone, and gingerols that portray antimicrobial potency. Silvia et al. [41] reported that fungi are more susceptible to ginger components than bacteria. e antimicrobial potential of the essential oils may be determined by the chemical composition, method of extraction, and the conditions to which the plant material was subjected in the preparation for essential oil extraction [41]. e essential oils used in this study were extracted from fresh material which reportedly contains higher concentrations of oxygenated compounds (such as borneol, neral, 1,8-cineole, α-terpineol, and geranial) than dried ginger essential oils and therefore low antifungal and antibacterial properties [41].
is could have been the reason behind the higher antifungal activity revealed by the essential oils that significantly reduced the disease severity in the treated plants as compared to the untreated ones.
e disease severity may also be influenced by the susceptibility of the cultivar used and the aggressiveness of the test pathogens. P. infestans caused a higher disease severity on the tomato plants in the negative control. ese results are similar to Quintanilla et al. [42] who reported that P. infestans caused damage above 65% in potato varieties. is shows that pathogen species respond differently to the different essential oils and synthetic fungicides. P. infestans seemed to be more susceptible to essential oils and the Ridomil fungicide and this corroborated the earlier findings by Lengai et al. [2]. e essential oils used in this study appeared to further influence the growth and yields of inoculated tomato plants as compared to the control. ere was a positive correlation between plant height with number of leaves and fruit number. e leaf number was also positively correlated with the fruit number and fruit weight. All these parameters were negatively correlated with disease severity.
is corroborated the theory of Naing et al. [13] that some organic pesticides induce disease resistance systems of the plant which lead to healthy growth of the plants and thus better production. Ahmad et al. [43] also reported that tomato plants sprayed with extracts of garlic and ginger were taller and yielded more than the untreated control. Nashwa and Abo-Elyousr [32] also showed that the Eucalyptus camaldulensis (eucalyptus), Ocimum basilicum (sweat basil), Allium sativum (garlic), Nerium oleander (oleander), Datura stramonium (jimsonweed), and Azadirachta indica (neem) plant extracts boosted the yield of tomato as compared to the control. However, Stangarlin et al. [12] cited a contrasting finding that there were no fruit yield differences in plants sprayed with several plant extracts.
In this study, the plants that were sprayed with Mexican marigold essential oil performed better than those in the other treatments. Apparently, Mexican marigold repelled some greenhouse pests such as whiteflies more than ginger and garlic thus improving the growth and consequently the yields of the treated plants. Similar observation was made by Rizvi and Jaffar, [44] who reported that increase in yield may also be due to reduced pests during growth and fruit development due to repellence property of the essential oils. However, some plant extracts and essential oils have growth boosting effects which improve the yield of the plants [13,45,46]. Garlic contains allicin compound which is said to promote the plant growth and fruit yield in tomato plants [43] but also contains diallyl disulfide compound which reportedly restricted the growth of tobacco plants [39].

Conclusion
e study showed that the essential oils from Mexican marigold, ginger, and garlic were as effective as the Ridomil Gold ® synthetic fungicide in managing the early and late blight diseases of tomato. Consequently, the treatments significantly boosted the growth and yields of tomato plants.
ese essential oils can therefore be incorporated in the early and late blight management programs as eco-friendly option to synthetic pesticides. is will lower the chemical residue levels in fruits and vegetables thus improving the fruit and vegetable quality and reducing the risks and hazards of toxic fungicides. is will in turn ensure access to the best markets of the fresh produce resulting in increased income for the producers and the country at large.
is study used the vertical steam distillation method to extract the essential oils from fresh samples of the selected test plants. However, there is a need to compare the extraction efficiency of this method with other methods.

Data Availability
Most of the data used to support the findings of this study are included in 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 study.