Control of the root-knot nematode, Meloidogyne incognita, using plant bioproducts

Escola de Ciências Manuel Maria Gomes Rodrigues Control of the root-knot nematode, Meloidogyne incognita , using plant bioproducts janeiro de 2022 Universidade do Minho janeiro de 2022 Manuel Maria Gomes Rodrigues Control of the root-knot nematode, Meloidogyne incognita , using plant bioproducts Master's dissertation Plant Molecular Biology, Biotechnology and Bioentrepreneurship Work supervised by Professora Doutora Maria Teresa da Silva Craveiro Martins de Almeida Doutora Maria Clara Almeida Vieira dos Santos ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii Acknowledgements This work is included in the EOIS-CropProt - Essential oils, infusions, and silicon in crop protection. A study using tomato plants, as a model, to disclose the biopesticides induced defense mechanisms of plants, through an omics approach funded by the European Regional Development Fund (ERDF), through the “COMPETE 2020” and “PORTUGAL 2020” programs, and by national funds from the Foundation for Science and Technology (FCT), under the funding program 02/SAICT/2017 (POCI-01-0145-FEDER-031131). It was carried out at the Biodiversity Laboratory, Center for Molecular and Environmental Biology, University of Minho. First of all, I would like to express my heartfelt gratitude to my supervisors Prof. Maria Teresa Martins de Almeida and Dr. Maria Clara A. Vieira dos Santos for their continuous support and valuable comments throughout this work. For always being available whenever I needed any guidance, and for making sure I conducted my work in the best and most efficient way possible. Their patience, guidance and support, helped me become a better person and to complete my dissertation to the best of my abilities. To Dr. Sofia Costa, I would like to thank for her insight and dedication, as her feedback, and help with the statistical analysis of this work was indispensable. To Dr. Ana Martins I would like to thank for all the support and encouragement given throughout my time in the laboratory. Lastly, I would like to thank Dr. Artur J. Ribeiro for his availability, supply of samples, and the good disposition with which he always received me. iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v Controlo de nemátodes-das-galhas-radiculares, Meloidogyne incognita , por bioprodutos vegetais Resumo Os nemátodes-das-galhas-radiculares (NGR), Meloidogyne spp., são fitoparasitas responsáveis pela redução do rendimento e qualidade de culturas. Diferentes abordagens têm sido implementadas para controlar as suas populações. Vários extratos botânicos e óleos essenciais (OEs) foram referidos pela atividade nematodicida contra NGR e são cada vez mais considerados para uso em programas integrados de gestão de pragas e doenças (IPM). Neste estudo, foram selecionadas diversas espécies vegetais pela disponibilidade e potencial uso como pesticidas botânicos. A atividade nematodicida de extratos hidroetanólicos (EHEs) e OEs de Cymbopogon citratus , Mentha aquatica , M. piperita , M. pulegium , Satureja montana e Thymus vulgaris foi avaliada sobre a mortalidade e infetividade de jovens do 2º estádio (J2) de Meloidogyne incognita . Os EHEs mais eficazes foram os de C. citratus , Mentha. aquatica e M. pulegium , com valores de LC50 de 8,63, 5,98 e 5,32 mg/mL (p<0,05). Os OEs mais eficazes foram os de C. citratus , S. montana e T. vulgaris , os quais induziram cerca de 100% de mortalidade de J2 após 24h, à concentração de 2000 ppm. Com base nos resultados de mortalidade obtidos com EHEs, foram selecionados os EHEs e OEs de C. citratus , M. aquatica e M. pulegium para testar o seu efeito na infetividade de J2, em ensaios de micro-vaso. Após exposição de J2 a concentrações selecionadas durante 48h, estes foram inoculados em plântulas de tomateiro cv. Tiny Tim com 10 dias. Sete dias após a inoculação, os J2 no interior das raízes foram corados com fucsina ácida, e contabilizados. Após exposição a LC50 de EHEs de C. citratus , M. aquatica e M. pulegium , houve uma redução de 66,7, 60,9 e 69% de J2. Após exposição a concentrações selecionadas de OEs de C. citratus (250 ppm), M. aquatica e M. pulegium (2000 ppm), houve uma redução na infetividade de 87,5, 77,8 e 84,4%, respetivamente. O efeito do OE de C. citratus encapsulado em microcápsulas de queratina na mortalidade de J2 foi investigado, após 48h. Verificou-se que o OE encapsulado afetou um número superior de J2 do que na sua formulação livre (p>0.05). Foram obtidas imagens por microscopia de fluorescência que indicaram a internalização do OE pelos J2; além disso, os J2 que morreram após incubação com o OE encapsulado apresentavam alterações morfológicas do intestino. Os resultados obtidos parecem indicar que os EHEs de C. citratus , M. aquatica e M. pulegium e OEs de C. citratus , M. pulegium , S. montana e T. vulgaris têm potencial como agentes nematodicidas e como novas formulações que possam ser incorporadas em programas IPM. Palavras-chave: ação anti-nemátode; encapsulação; extratos botânicos; gestão; óleos essenciais vi Control of the root-knot nematode, Meloidogyne incognita , using plant bioproducts Abstract Root-knot nematodes (RKN), Meloidogyne spp. are plant pathogens responsible for yield reduction and quality loss in crops. Several approaches have been implemented in order to control its populations. Various botanical extracts, and essential oils (EOs), have been reported to have nematicidal activity against RKN and are increasingly being considered for use in integrated pest management (IPM) programs. In this work, various plant species were selected based on their availability and potential use as botanical pesticides. The nematicidal activity of hydroethanolic extracts (HEEs) and EOs of Cymbopogon citratus , Mentha aquatica , M. piperita , M. pulegium , Satureja montana and Thymus vulgaris was evaluated on their effect on mortality and infectivity on second-stage juveniles (J2) of Meloidogyne incognita. The most effective HEEs were of C. citratus , Mentha aquatica and M. pulegium , with LC50 values of 8.63, 5.98 and 5.32 mg/mL (p<0.05). The most effective EOs were of C. citratus , S. montana and T. vulgaris , which induced the mortality of approximately 100% of J2 after 24h of exposure to the concentration of 2000 ppm. Based on the mortality results obtained with HEEs, the C. citratus , M. aquatica and M. pulegium HEEs and EOs were selected to test their effect on J2 infectivity, in micro-pot assays. After J2 exposure to selected concentrations for 48h, they were inoculated on 10-day old tomato cv. Tiny Tim seedlings. Seven days after inoculation, J2 inside the roots were stained with acid fuchsin, and counted. After exposure to LC50 of C. citratus , M. aquatica and M. pulegium HEEs, there was a reduction of 66.7, 60.9 and 69% of J2. After exposure to concentrations of C. citratus (250 ppm), M. aquatica and M. pulegium (2000 ppm) EOs there was a reduction in infectivity of 87.5, 77.8 and 84.4%, respectively. The effect of C. citratus EO encapsulated in keratin microcapsules on the mortality of J2 was investigated, after 48h. The encapsulated EO affected a superior number of J2, than in its free formulation (p>0.05). Images were obtained by fluorescence microscopy, which indicated the internalization of the EO by the J2, and the J2 that died after incubation with the encapsulated EO presented morphological changes in the intestine The results obtained seem to indicate that the HEEs of C. citratus , M. aquatica and M. pulegium and EOs of C. citratus , M. pulegium , S. montana and T. vulgaris have potential as nematicidal agents and as new efficient formulations that can be incorporated in IPM programs. Keywords: anti-nematode action; botanical extracts; encapsulation; essential oils; management. vii Index Resumo ..................................................................................................................................... v Abstract ................................................................................................................................... .vi List of figures………………………………………………………………………………………………………...... ix List of tables………………………………………………………………………………………………………....... xi I. Introduction ......................................................................................................................... 17 1. Root-knot nematodes ( Meloidogyne spp.) .......................................................................... 2 2. Root-knot nematode control .............................................................................................. 3 3. Botanical extracts and essential oils with nematicidal activity ............................................. 5 3.1 Cymbopogon citratus ............................................................................................ 7 3.2 Mentha species. ..................................................................................................... 8 3.3 Satureja montana ............................................................................................. 100 3.4 Thymus vulgaris ................................................................................................ 111 4. Encapsulated botanical extracts and essential oils (EOs) with nematicidal activity ........... 133 5. Objectives ..................................................................................................................... 144 II. Materials and methods ..................................................................................................... 305 1. Meloidogyne incognita isolate ........................................................................................ 166 2. Effect of plant bioproducts on M. incognita second-stage juveniles (J2) mortality ............ 166 2.1 Hydroethanolic extracts (HEEs) .......................................................................... 177 2.2 Essential oils (EOs) .............................................................................................. 18 3. Effect of plant bioproducts on M. incognita second-stage juveniles (J2) infectivity ............. 18 4. Effect of encapsulated Cymbopogon citratus essential oil, on Meloidogyne incognita secondstage juveniles (J2) mortality ............................................................................................... 19 5. Data analysis ................................................................................................................ 200 III. Results ............................................................................................................................ 372 1. Effect on Meloidogyne incognita second-stage juveniles (J2) mortality ............................ 233 1.1 Hydroethanolic extracts (HEEs) .......................................................................... 233 1.1.1 Cymbopogon citratus .......................................................................... 233 1.1.2 Mentha aquatica ................................................................................ 244 1.1.3 Mentha piperita .................................................................................. 244 1.1.4 Mentha pulegium ............................................................................... 255 1.1.5 Satureja montana ............................................................................... 266 1.1.6 Thymus vulgaris ................................................................................... 27 3 epidermis of the root where it will deposit the eggs within a protective gelatinous matrix outside her body (Perry et al ., 2009). More than 100 species of Meloidogyne genus have been described (Bernard et al ., 2017). Around the world, M. arenaria, M. hapla, M. incognita and M. javanica are serious ubiquitous pests being considered as “major” species (Rusinque et al ., 2017). Meloidogyne incognita has been considered as one of the most prevalent species in warmer conditions of southern Europe, and also in glasshouses in northern Europe and is able to infect almost all cultivated plants, and exponentially increase population levels due to rapid parthenogenetic multiplication, meaning that the females reproduce without the presence of a male (Wesemael et al ., 2011). Its genome was sequenced in 2008 in order to provide insights into the adaptations required by metazoans to successfully parasitize and counter defenses of immunocompetent plants (Abad et al ., 2008). 2. Root-knot nematode control The main objective of RKN control is to keep nematode populations below threshold levels, and several approaches have been implemented, such as: chemical control, cultural control and biological control. However, management of RKN under intensive crop cultivation systems is hard due to their resilience and ubiquity, and no single management strategy has proven successful at eliminating RKN, so it relies mainly on the use of integrated pest management (IPM) programs (Rusinque et al ., 2017). The major control method of RKN is based on the use of chemical nematicides, since these are effective and fast acting (Taimoor and Shahina, 2018). Fumigant nematicides are considered more effective in controlling RKN and in increasing crop yield than non-fumigant nematicides, due to their broad-spectrum activity (Netscher and Sikora, 1990). The downside is that they are expensive, can accumulate in plant tissues, and are environmentally hazardous and toxic causing environmental pollution, and endangering the life of animals and humans. As a result, many of the chemical nematicides have been banned (91/414/EEC) or are under evaluation (2009/1107/EU) by strict legislations in European Union countries, thus reducing control options. Hence, there is a global interest in developing less toxic and more environment-friendly alternatives. Cultural practices are the most economical means of managing insect-pests and diseases, including nematode problems (Ghule et al ., 2014). Cultural control includes strategies such as: crop rotation with, cover crops, soil solarization, trap cropping, biofumigation, and use of resistant 4 cultivars. Crop rotation is one of the oldest and most important methods for managing nematodes in annual crops. However, this strategy does not allow intensive agricultural practices and resistant or tolerant crops may not be an economically attractive option. Additionally, crop rotation may further increase selection pressure for virulent populations. Furthermore, the success of this control strategy is limited because of the wide host ranges of most RKN species and the frequent occurrence of infestations induced by more than one species (Wesemael et al ., 2011). Cover cropping has been used in IPM protocols to reduce PPN incidence and replenish soil nutrient levels. Cover crops help improve soil fertility, soil structure, decrease soil erosion, and suppress weeds, insects, nematodes, and other plant pathogens (Gill and Mcsorley, 2011). The major limitation is the wide host range of RKN (Van der Putten et al ., 2006). Solarization is usually performed in midsummer to maximize soil-heating effects. The soil is covered with plastic film for at least 2 weeks, with the intent to destroy egg masses, reducing the population density of RKN, but it is only viable in regions where sufficient solar energy is available for long periods of time (Mediterranean countries). This method is also only effective up to a depth of 20 cm in the soil, and as J2 are able to migrate deep into the soil escaping from high temperatures (Bello et al ., 2000). Trap crops are plants used to suppress or directly control nematode populations. These must be good hosts to allow the easy infection of the nematode species. After RKN invade the roots and establish a feeding site, the females become sedentary and before the nematodes complete their life cycle, the crop is destroyed, in order to destroy the root systems and all the nematodes trapped within. This way a new soil infestation is avoided, reducing the nematode population. Some examples of trap crops are tomatoes and carrots. This technique implies the destruction of plants with no economic return (Sacchi et al ., 2021). Biofumigation consists in the action of volatile substances produced in the bio-decomposition of organic matter. Some examples of biofumigants successfully used in crops are cow manure, and crop remains from rice and ornamental gardens (Bello et al ., 2000). When biofumigation is used in combination with other techniques, such as solarization, it is a very useful and efficient method to control RKN in IPM programs (Medina-Míguez, 2002). However, biofumigants are slow acting upon target organisms, which requires the application of organic amendments at high rates in order to have an effect on nematode populations (Van der Putten et al ., 2006). Plant resistance is probably the most environmentally safe method to control RKN (Ibrahim et al ., 2016). It can be, either naturally occurring and transferred to crop cultivars through conventional breeding methods with wild relatives, or engineered through molecular techniques. The most widely 5 used and investigated approach is the integration of Mi gene, which was introgressed from the wild tomato species Lycopersicon peruvianum (L.) Mill, however, resistance mediated by Mi gene is lost at soil temperatures above 28 °C, and races breaking resistance have been found in M. incognita (Dropkin, 1969; Starr et al ., 2002). Resistant cultivars are also only available for a limited number of crops and PPN, and it has been stated that virulence against Mi gene can develop in some cases after just as few as five plantings (Noling, 2000). Biological control is based on the use of biopesticides, such as antagonistic agents and botanical extracts, to control or reduce population densities of pathogens and pests. Parasitism and predation are the main mechanisms of action of these antagonists, but antibiosis, induced resistance of the host plant, and direct competition for space and limited resources, are some other factors that may lead to the reduction of the infection level (Wiratno et al ., 2020). However, biological control agents will generally provide too little control to be effective alone, as they are required in large amounts, and tend to be difficult to establish in the field (Paul et al ., 2015). 3. Botanical extracts and essential oils with nematicidal activity Plants can be used as pesticides themselves, or they can serve as model compounds for the development of chemically synthesized, easily biodegradable derivatives, with low plant and human toxicity (Chitwood, 2002). The presence of bioactive compounds with nematicidal properties in a wide range of plants, acting as repellents, inducing paralysis, reducing hatch or even causing death, presents a potential alternative to the use of synthetic nematicides (Stamp, 2003; Renčo et al ., 2014). Several secondary metabolites from plant extracts and essential oils are among these compounds and a substantial number in several major classes have been reported to show nematicidal activity, with the most important ones being alkaloids, monoterpenoids, flavonoids, diterpenoids, and polyphenols (Andrés et al ., 2012, Renčo et al ., 2014). The recognition of the importance of plants secondary metabolites in terms of resistance to pests has increased, and so, plant extracts and essential oils (EOs) are increasingly being considered for use in IPM programs (Wiratno et al ., 2020) Several botanical extracts, and EOs have been reported to have nematicidal activity against Meloidogyne spp. Some examples of commercial biological nematicides derived from plant material, with the ability to suppress RKN in an ecologically sustainable manner, are Dazitol® 6 (capasaicin and essential oil of mustard) and Nemakill® (cinnamon oil, clove oil, and thyme oil mixture) (Watson and Desaeger., 2019). Different plant extracts from aromatic plants, in the solid state or as aqueous solutions have been tested against M. incognita and had significant effects on the hatching, and mortality of J2 after 24h of exposure to low concentrations (1-100 mg/L). The extract of garlic ( Allium sativum L.) significantly reduced hatching activity to below 8% at the concentration of 1 mg/L, and the extract of Foeniculum vulgare Mill. was toxic to J2, with an obtained LC50 of 43 mg/L (Ibrahim et al ., 2006; Caboni et al ., 2013). There are also examples, where powder and extracts of different plant parts have been used for the management of RKN. Neem ( Azadirachta indica A. Juss.) is the bestknown example that acts by releasing pre-formed nematicidal constituents into soil (Prakash et al ., 2014).The effects of methanolic or ethanolic extracts of various plants (mainly the aerial parts) against M. incognita have also been investigated, and some ethanolic extracts, like the extract of tobacco leaves ( Nicotiana tabacum L.) and clove buds ( Syzygium aromaticum L.) showed high nematicidal activity against J2 after 24h of exposure, with LC50 values that were 5-10 times lower than the LC50 of the synthetic pesticides chlorpyrifos, carbosulfan and deltamethrin. (Wiratno et al. , 2009; Pavaraj et al. , 2012). Essential oils from various plants have nematicidal properties due to the presence of secondary metabolites, such as carvacrol and thymol (Oka et al ., 2000), which have been shown to completely inhibit hatching of M. incognita eggs at low concentrations (2, 4 mg/L) (Ibrahim et al ., 2006). Among the several plants tested in direct contact assays, the EOs of aromatic plants of the Poaceae and Lamiaceae families, from the Cymbopogon and Mentha genus, for example, have been widely studied in regards to nematicidal activity towards Meloidogyne incognita and M. javanica (Oka et al ., 2000; Ntalli et al ., 2010; Gupta et al ., 2011; Andrés et al ., 2012; Caboni et al ., 2013). Essential oils are generally more complex and have more concentrated active compounds than tinctures or extracts, and investigations are focused mainly on the nematicidal properties of EOs (Amora et al ., 2017). This study is part of a project (POCI-01-0145-FEDER-031131) in which different plant species were selected based on their availability and potential use as botanical pesticide against different organisms. 7 3.1 Cymbopogon citratus Figure 1. Wild lemongrass, Cymbopogon citratus. (Source: Zebua, 2020) Plants of the genus Cymbopogon are widely distributed across all continents and Cymbopgon. citratus (DC.) Stapf (Fig. 1), commonly known as wild lemongrass, is one the most important in terms of EO production (Avoseh et al ., 2015). This plant is used in traditional and modern medicine in many countries for a wide range of applications, based on its antibacterial, antifungal, antiprotozoal, antiinflammatory, antioxidant, cardioprotective, antitussive and antiseptic activities. Its leaves are used to make tea as a homemade remedy for cough, flu, gingivitis, digestive problems and stomachache. In addition to its therapeutic uses, aqueous extract and essential oils from this plant are also used in the food, perfume and cosmetics industries, due to their typical lemon and rose-like aroma. The EO of C. citratus contains various monoterpenes, with citral being the most abundant (65%−85%) (Ekpenyong et al ., 2015). Citral is a mixture of terpenoids neral and geranial, and is the main component responsible for the typical aroma (Ekpenyong et al ., 2014). The nematicidal activity of C. citratus EO is also mainly attributed to the content of citral (Barros et al ., 2020). It has been shown by different authors that the use of leaf extracts of lemongrass can be of economic benefit in the management of nematode pests, since it has been proven to be effective in controlling M. incognita , by reducing the number of formed galls (Corbani and Mazzonetto., 2013; Izuogu et al ., 2015). The nematostatic effect of C. citratus EO has been demonstrated, after C. citratus EO (1000 ppm, dissolved in ethanol – 10% vol/vol and Tween 20 – 0.3% vol/vol) affected the mobility of 69.3% of J2 of M. javanica , after 48h (Oka et al ., 2000). Different studies have also described the mortality that EOs of Cymbopogon grasses caused to J2 of M. incognita. 8 The mortality of M. incognita J2 was reported after C. citratus EO (1:2 in Texapon N-40) caused the mortality of 63% of J2 at the concentration of 625 ppm, and 100% at the concentration of 1250 ppm, after 16 days of exposure (Moreira et al ., 2009). In a different study, the EO (1000 ppm, dissolved in Tween 80 – 0.5% vol/vol) caused the mortality of 100% of J2 and the mortality of approximately 70% of J2 at the concentration of 120 ppm, after 48h of exposure (Gupta et al ., 2011). Similarly, C. citratus EO (dissolved in 1% Tween 80), was tested on M. incognita J2 at different concentrations, and a LC50 value of 166 ppm was obtained after 48h (Barros et al ., 2020). 3.2 Mentha species Figure 2. Mentha species: water mint, Mentha aquatica (A), peppermint, Mentha piperita (B) and pennyroyal mint, Mentha pulegium (C). (Source: Newman, 2020; Bolshakov, 2019; Barros, 2012) The genus Mentha (Lamiaceae) comprises several aromatic species, which are cultivated around the world due to their distinct aroma and commercial value. Fresh and dried plant material, raw extracts and essential oils of mint plants are used for preparation of herbal teas and as supplements to various commercial products including cosmetics, foods and drugs (Mahendran and Rahman, 2020). In addition to the traditional flavoring uses, Mentha spp. extracts and EOs are well recognized for their folk medicinal uses due to their anti-inflammatory, antiemetic, diaphoretic, antispasmodic, anti-catharrhal, analgesic, antibacterial and antioxidant properties. The traditional biological and pharmacological attributes can be linked to the high content of secondary metabolites such as terpenoids, alcohols, rosmarinic acid, and antioxidant phenolics among others (Anwar et al ., 2019). Mentha aquatica L. (Fig. 2A) is used in beverages, salads or cooked foods. Besides the food applications, water mint is also been consumed as tea, and used in traditional medicine for the A B C 9 treatment of external inflammation (mouth or throat problems) and in inflammation-related diseases, such as rheumatism (Anwar et al ., 2019). Among the diversity of plants, Mentha piperita L. (Fig. 2B) is one of the herbs most widely used worldwide, with a long history of safe use in medicinal preparations. Peppermint is among the most popular single ingredient herbal teas and its oil is one of the most popular widely used essential oils. (Mahendran and Rahman., 2020). Mentha pulegium L. (Fig. 2C) is a traditional culinary herb, folk remedy, and abortificient. Pennyroyal mint leaves, both fresh and dried, are especially noted for repelling insects. Its oil is poisonous, yet it is used to make herbal teas, which, although not proven to be dangerous to healthy adults in small doses, is not recommended, due to its known toxicity to the liver (Miraj et al ., 2016). The nematostatic activity of water extracts, methanolic extracts and EOs of M. piperita , M. pulegium , and M. spicata , has been previously evaluated. The methanolic extracts were not biologically active, and the water extracts exhibited nematostatic activity against Meloidogyne incognita J2 after 72h of exposure (EC50 values of: 1.00, 0.75, and 0.30 mg/mL, respectively) (Caboni et al ., 2013). The nematostatic and nematicidal activity of Mentha piperita , M. pulegium and M. spicata EOs on RKN have also been evaluated by various authors (Table 1). In a study with Meloidogyne javanica, Mentha piperita and M. spicata EOs affected the mobility of 71.8 and 100% of J2, after 48h (Oka et al ., 2000). The nematostatic effect of M. spicata EO against Meloidogyne incognita was reported, with an EC50 of 358 ppm, after 72h (Caboni et al ., 2013). The effect of Mentha pulegium EO against Meloidogyne javanica J2 was also reported after the EO exhibited nematostatic activity, with an EC50 value of 3890, after 48h (Ntalli et al ., 2010). In a work with Meloidogyne incognita , Mentha piperita and M. spicata EOs induced the mortality of 35 and 50% of J2 , after 24h (Pandey et al ., 2000). In a similar study with Meloidogyne javanica, Mentha spicata EO induced 100% mortality, after 12h (Andrés et al ., 2012). Furthermore, it has also been reported that M. piperita EO induced 99.1% mortality of Meloidogyne javanica J2, after 24h (Amora et., al 2017). 10 Table 1. Reported effects of Mentha spp. essential oils on the mobility and mortality of second-stage juveniles (J2) of Meloidogyne incognita and of M. javanica. Plant species Meloidogyne spp. Time (h) Concentration tested Assay EC50/ LC50 (ppm) Effect (%) Solvents Reference Mentha piperita M. javanica 48 1000 mobility - 71.8 Ethanol (10% vol/vol); Tween 20 (0.3% vol/vol) Oka et al ., 2000 M. spicata M. javanica 48 1000 mobility - 100 M.piperita M. incognita 48 1000 mortality - 35 DMSO (10% vol/vol); Tween 80 (4.5% vol/vol) Pandey et al ., 2000 M. spicata M. incognita 48 1000 mortality - 50 M pulegium M. javanica 24 - mobility 4520 - Tween 20 (0.3% vol/vol) Ntalli et al ., 2010 96 - 3150 - M. piperita M. javanica 12 1000 mortality - a DMSO; Tween 20 (0.5% vol/vol) Andrés et al ., 2012 M. spicata M. javanica 12 1000 mortality 293 100 M. piperita M. incognita 72 1500 mobility a - Tween 20 (0.3% vol/vol) Caboni et al ., 2013 M. pulegium M. incognita 72 1500 mobility a - M. spicata M. incognita 72 1500 mobility 358 - M. piperita M. javanica 24 2500 mortality - 99.1 DMSO (2% vol/vol) Amora et al ., 2017 a - Essential oil was not biologically active - Missing data indicate lack of information 3.3 Satureja montana Figure 3. Wintor savory, Satureja montana. (Source: Wirth, 2021) The genus Satureja (Lamiaceae) contains around 200 species of aromatic herbs and shrubs. Satureja montana L. (Fig. 3) is a perennial small herb native to arid, sunny, and rocky regions in the Mediterranean area, but it is cultivated throughout Europe. It is an aromatic plant widely used as a spice and as a traditional medicinal herb (Hudz et al ., 2020). Ethanolic extracts and EOs of 11 S. montana revealed antioxidant and antimicrobial properties. The leaves, flowers, and stems are used for herbal tea and, in traditional medicine, to treat various ailments, due to several activities such as digestive, expectorant and laxative, among others. Furthermore, it is often used in Mediterranean cooking preparations and even as natural antibacterial agent in food packaging (Maccelli et al ., 2019). The nematicidal activity of S. montana EO can be attributed to its content in carvacrol and thymol (Andrés et al ., 2012). Satureja montana EO obtained from flowers and leaves (1000 ppm, dissolved in water with DMSO and Tween 20 – 0.5% vol/vol) has been shown to possess nematicidal activity against M. javanica J2, after 12h, with a LC50 of 41 ppm (Andrés et al ., 2012). In a study with M. chitwoodi, two EOs of S. montana displayed hatching inhibitions of over 90%, after 72h of exposure to the concentration of 2000 ppm, and were further tested at the concentration 125 ppm, presenting EC50 values of 61 and 33 ppm (Faria et al ., 2016). 3.4 Thymus vulgaris Figure 4. Thyme, Thymus vulgaris. (Source: Gort, 2008) The genus Thymus (Lamiaceae), thyme, comprises over 350 aromatic plant species with quite different botanical characteristics and broad chemical heterogeneity. Thymus spp. are used as drug because they possess a wide range of biological activities including antimicrobial, antibacterial, antispasmodic, antifungal and antioxidant properties due to the presence of flavonoids, polyphenol and phenolic acids (Hosseinzadeh et al ., 2015). Its nematicidal activity can be explained by its content in carvacrol and thymol (Andrés et al ., 2012). Among the different species, Thymus vulgaris L. (Fig. 4) is a flowering plant native to Southern Europe with a worldwide 12 distribution (Hosseinzadeh et al ., 2015). It is an important medicinal plant that has been used for centuries as spice, homemade remedy, drug, perfume and insecticide (Salehi et al ., 2018). The undiluted aqueous extract of T. vulgaris shoots has been shown to affect 100% of M. incognita J2 in regards to mobility, after 72h (Korayem et al ., 1993). Several studies have also reported the effects of the EO on the mobility and mortality of M. incognita and M. javanica (Table 2). The nematostatic activity of T. vulgaris EO was reported in a study with M. javanica , after the EO affected the mobility of 67.5% of J2, after 48h of exposure (Oka et al ., 2000). The effects of T. vulgaris EO on M. incognita J2 was noted, after the EO induced the mortality of approximately 70% of J2, after 48h (Barros et al ., 2019). In a work with M. javanica , T. vulgaris EO induced 100% mortality of J2, after 12h, and presented a LC50 value of 224 ppm (Andrés et al ., 2012). Furthermore, it was also demonstrated in a different study, that T. vulgaris EO induced 99.4% mortality of M. javanica J2, after 24h of exposure (Amora et., al 2017). Table 2. Reported effects of Thymus vulgaris essential oil on the mobility and mortality of second-stage juveniles (J2) of Meloidogyne incognita and of M. javanica. Plant species Meloidogyne spp. Time (h) Concentration tested Assay EC50/ LC50 (ppm) Effect (%) Solvents Reference Thymus vulgaris M. javanica 48 1000 mobility - 67.5 Ethanol (10% vol/vol); Tween 20 (0.3%) Oka et al ., 2000 T. vulgaris M. javanica 12 1000 mortality 224 100 DMSO; tween 20 (0.5% vol/vol) Andrés et al ., 2012 T. vulgaris M. javanica 24 2500 mortality - 99.4 DMSO (2% vol/vol) Amora et al ., 2017 T. vulgaris M. incognita 48 1000 mortality - 70 Tween 20 (1% vol/vol) Barros et al ., 2019 - Missing values indicate lack of information Botanical or plant-derived compounds show great promise as an alternative to chemical control because they have a specific mode of action, are eco-friendly, do not leave toxic residues in the edible plant parts, and can be prepared locally (Taye et al ., 2012). However, they may present limited persistence in field conditions, and the chemical composition of plant secondary metabolites varies considerably between plant species and varieties, level of maturity, extraction process, and even within the same variety from different geographic areas (Andrés et al ., 2012). 19 described. This procedure was repeated 3 times to ensure no residues of HEEs or EOs were left in the J2 suspensions. The suspensions were transferred to glass beakers and the volume was adjusted to 10 mL. Tomato seeds cv. Tiny Tim placed in plastic pots (40 cm3) containing 25 cm3 of autoclaved vermiculite moistened with nutrient solution (N:P:K, 6:3:6) were transferred to a growth chamber at 25 °C with a 12-hour photoperiod. After 10 days, the plants were uprooted and, after cleaning the roots to eliminate the vermiculite, transferred to pots containing 25 cm3 of autoclaved soil mixture (turf:vermiculite:sand; 1:1:1). Then the plants were inoculated with 100 J2/pot obtained as described. Controls consisted of plants inoculated with J2 exposed to autoclaved sterile water and each treatment was replicated five times. The plants were kept in an air-conditioned room in the conditions described in 2.1. Seven days after inoculation, the plants were uprooted and the roots were washed, weighed and stained with acid fuchsin (Byrd et al ., 1983). The numbers of nematodes inside the roots were counted using a stereomicroscope. 4. Effect of encapsulated Cymbopogon citratus essential oil, on Meloidogyne incognita secondstage juveniles (J2) mortality The Cymbopogon citratus EO was selected for an assay against M. incognita J2, under an encapsulated formulation (ECCEO) in keratin microcapsules, kindly provided by Dr. Artur J. Ribeiro, of the Center for Biological Engineering (CEB) of the University of Minho, and kept in the dark, at 4 °C. The nematicidal activity of ECCEO on M. incognita J2 was tested in vitro , using the methodology described in 2.2.2. The bioassay consisted of ECCEO at the concentration of 250 ppm and C. citratus EO at the same concentration (prepared as described). For each treatment, 5 repetitions were prepared, and autoclaved deionized water and keratin as free protein, at the concentration present in the bioassay (100 ppm) were used as controls. The plates were sealed with parafilm, and incubated in the dark with agitation (≈ 90 rpm), at room temperature, for 48h. The number of mobile and immobile J2 was recorded as described in 2.2.1. In order to check the intake of the encapsulated EO by M. incognita J2, a new bioassay was performed with keratin microcapsules, and keratin, labelled with fluorescein isothiocyanate (FITC) (also provided by Dr. Artur J. Ribeiro). The bioassay was prepared using the same methodology previously referred in 2.4. After 48h, J2 exposed to ECCEO, and also free keratin stained with FITC, used as a control, 20 were mounted on glass slides in water, and photographed using a Olympus BX63 fluorescence microscope equipped with a DP74 camera. Imaging was processed with the green channel fluorescence, DIC and brightfield, using the Olympus cellSens software. Observations were made in at least three J2 specimens in each experimental condition. 5. Data analysis Statistical analyses were performed using IBM SPSS Statistics for Windows, version 27.0 (Armonk, NY: IBM Corp). Data on the effect of HEEs and EOs on the mortality of J2 of Meloidogyne incognita was converted into percentages of inhibitory effect, and corrected using the Abbott's formula (Abbott, 1925): cm = (mt – mc) / (100 – mc) x 100 where cm= corrected mortality (%), mt = mortality in treatment (%), mc = mortality in control (%). Datasets results of the biological replicates on number of dead J2 were normalized and compared using Generalized Linear Models (GLZM) assuming a normal distribution and identity link function (linear function), and the replicates were subjected to pairwise comparison, using the Least Significant Difference test (LSD) at probability levels of 5% (p<0.05). When a culture plate spilled, presented severe fungal contaminations, or the solution tested caused no effect in J2 as opposed to its biological replicates, as was the case with some EOs bioassays (due to improper dissolving), a new replicate was prepared. The experimental data was subjected to GLZM analysis, and when applicable, biological replicates were discarded. Significant differences among the effect of different HEEs and EOs in J2 mortality (untransformed data) were investigated for each exposure time, through the use of Generalized Linear Mixed Models (GLMM), with a normal distribution, using the LSD test (p<0.05). In these models the plant species and the concentrations tested were used as fixed effects, and the bioassay replicates were used as random effects. Model outputs were corrected by the Satterthwaite approximation and using Robust Estimation. Significant differences among HEEs or EOs of different plant species were investigated with pairwise LSD test (p<0.05). Data of the in vitro nematicidal activity of the HEEs and EOs (untransformed data) were subjected to Probit analysis for each exposure time, in order to estimate the LC50 values for a maximum of 20 iterations at the different times of exposure. 21 Datasets results of the biological replicates on number of J2 per root gram, in infected roots were normalized and compared by GLZM analysis, using the same settings as described in J2 mortality analysis. Significant differences among the effect of HEEs and EOs in J2 infectivity (untransformed data) were investigated after 7 days, through the use of GLMM analysis, considering the plant species as fixed effects, and the bioassay replicates as random effects. Model outputs were corrected using the same settings as described in J2 mortality analysis. Significant differences among the effect of ECCEO, C. citratus EO and keratin in J2 mortality were investigated after 48h, through the use of GLMM analysis, considering each sample as a fixed effect. Model outputs were corrected as described above. III. Results 23 1. Effect on Meloidogyne incognita second-stage juveniles (J2) mortality The effect of different HEEs and EOs on the mortality of M. incognita J2 was evaluated in vitro. 1.1 Hydroethanolic extracts (HEEs) 1.1.1 Cymbopogon citratus The C. citratus HEE caused no mortality of J2 in the first 24h, at any of the concentrations tested (Fig. 5). A clear increase in mortality was observed with the increase of exposure time. No significant differences were detected for the concentrations of 7.5 and 10.0 mg/ml at any time of exposure (p>0.05). After 96h, the C. citratus HEE affected 75.42, 86.25 and 88.75% of J2, at the concentration of 5.0, 7.5, and 10.0 mg/mL, respectively. No significant differences were observed among the different bioassays, after 72 and 96h of exposure (p=0.19, p=0.57, respectively). Figure 5. Corrected percentage cumulative mortality of Meloidogyne incognita second-stage juveniles exposed to different concentrations of Cymbopogon citratus hydroethanolic extract at 5.0, 7.5 and 10.0 mg/mL. Values are means of combined data from three biological replicates with four repetitions for each treatment. Error bars represent the standard error. 0 20 40 60 80 100 024 48 72 96 Corrected cumulative mortality (%) Time (hours) 5.0 mg/mL 7.5 mg/mL 10.0 mg/mL 24 1.1.2 Mentha aquatica The M. aquatica HEE caused no mortality of J2 after 24h, at any of the concentrations tested (Fig. 6). After 48h, the highest nematicidal activity was observed at the concentration of 7.5 mg/mL, having affected 77.50% of J2 in comparison with 68.33% for the concentration of 10.0 mg/mL. After 72h, the HEE reached its maximum effect for the concentration of 5.0 mg/mL (64.58%). No significant differences were detected for the concentrations of 7.5 and 10.0 mg/mL, at any time of exposure (p>0.05), nor among the different bioassays. Figure 6. Corrected percentage cumulative mortality of Meloidogyne incognita second-stage juveniles, exposed to different concentrations (5.0; 7.5; 10.0 mg/mL) of Mentha aquatica hydroethanolic extract , after 24, 48, 72 and 96h. Values are means of combined data from three biological replicates with four repetitions for each treatment. Error bars represent the standard error. 1.1.3 Mentha piperita The M. piperita HEE caused no mortality of J2 after 24h, at any of the concentrations tested (Fig. 7). A clear dose-response effect was observed afterwards. After 48h, the HEE affected 27.08 and 42.92% of J2 at the concentrations of 7.5 and 10.0 mg/mL, respectively. A clear increase in effect was observed over time, and after 96h of exposure, the HEE affected 29.17, 72.08 and 80.83% of J2, at the concentration of 5.0, 7.5 and 10.0 mg/mL, respectively. No significant differences were observed among the different bioassays, after 72h of exposure (p=0.11). 0 20 40 60 80 100 024 48 72 96 Corrected cumulative mortality (%) Time (hours) 5.0 mg/mL 7.5 mg/mL 10.0 mg/mL 25 Figure 7. Corrected percentage cumulative mortality of Meloidogyne incognita second-stage juveniles, exposed to different concentrations (5.0; 7.5; 10.0 mg/mL) of Mentha piperita hydroethanolic extract , after 24, 48, 72 and 96h. Values are means of combined data from three biological replicates with four repetitions for each treatment. Error bars represent the standard error. 1.1.4 Mentha pulegium The M. pulegium HEE presented nematicidal activity after 24h (Fig. 8). A clear dose-response effect was not observed, since no significant differences were detected for the concentrations of 5.0 and 10.0 mg/mL, at any time of exposure (p>0.05). The HEE affected 9.17% of J2 at the concentration of 2.5 mg/mL, and 72.08 and 71.25% at the concentrations of 5.0 and 10.0 mg/mL, respectively. The mortality of J2 increased over time with 15.83% of J2 affected at the concentration of 2.5 mg/mL, and 85.00 and 80.42% affected at the concentrations of 5.0 and 10.0 mg/mL, after 96h (p<0.05). No significant differences were observed among the different bioassays (p>0.05). 0 20 40 60 80 100 024 48 72 96 Corrected cumulative mortality (%) Time (hours) 5.0 mg/mL 7.5 mg/mL 10.0 mg/mL 26 Figure 8. Corrected percentage cumulative mortality of Meloidogyne incognita second-stage juveniles, exposed to different concentrations (2.5; 5.0; 10.0 mg/mL) of Mentha pulegium hydroethanolic extract , after 24, 48, 72 and 96h. Values are means of combined data from three biological replicates with four repetitions for each treatment. Error bars represent the standard error. 1.1.5 Satureja montana The S. montana HEE caused no mortality of J2 after 24h, at any of the concentrations tested (Fig. 9). A clear dose-response effect was observed afterwards. After 48h, the S. montana HEE affected 5.65 and 13.13% of J2 at the concentration of 7.5 and 10.0 mg/mL, respectively. A clear increase in mortality was observed with the increase of exposure time, for the concentration of 10.0 mg/mL, having affected 38.75% of J2, after 96h. The HEE caused no mortality of J2 at the concentration of 5.0 mg/mL, when compared with the control. No significant differences were observed among the different bioassays, after 72 and 96h (p=0.07, p=0.88, respectively). 0 20 40 60 80 100 024 48 72 96 Corrected cumulative mortality (%) Time (hours) 2.5 mg/mL 5.0 mg/mL 10.0 mg/mL 27 Figure 9. Corrected percentage cumulative mortality of Meloidogyne incognita second-stage juveniles, exposed to different concentrations (5.0; 7,5; 10.0 mg/mL) of Satureja montana hydroethanolic extract , after 24, 48, 72 and 96h. Values are means of combined data from three biological replicates with four repetitions for each treatment. Error bars represent the standard error. 1.1.6 Thymus vulgaris The T. vulgaris HEE presented nematicidal activity after 24h of exposure (Fig. 10) causing 5.31, 6.56 and 9.06% mortality of J2 at the concentration of 5.0, 7.5 and 10.0 mg/mL, respectively. After 96h, the T. vulgaris HEE affected 86.88% of J2 at the concentration of 10.0 mg/mL. However, the results obtained in the prepared replicates were significantly different at every observation time (p<0.05), most likely due to contaminations, as this HEE was more prone to fungal contaminations. 0 20 40 60 80 100 024 48 72 96 Corrected cumulative mortality (%) Time (hours) 5.0 mg/mL 7.5 mg/mL 10.0 mg/mL 28 Figure 10. Corrected percentage cumulative mortality of Meloidogyne incognita second-stage juveniles, exposed to different concentrations (5.0; 7,5; 10.0 mg/mL) of Thymus vulgaris hydroethanolic extract , after 24, 48, 72 and 96h. Values are means of combined data from three biological replicates with four repetitions for each treatment. Error bars represent the standard error. 1.1.7 Effect of hydroethanolic extracts (HEEs) on M. incognita second-stage juveniles (J2) After 24h, only the Mentha pulegium and T. vulgaris HEEs affected J2 of Meloidogyne incognita (Table 4). After 48h, the Mentha aquatica and M. pulegium HEEs presented the highest nematicidal activity, having affected an estimated mean of 44.55 and 46.75% of J2 (p=0.51). After 96h, the highest nematicidal activity was reported for C. citratus , M. aquatica and M. pulegium (p>0.05). The HEE of S. montana presented significantly lower nematicidal activity when compared to the other HEEs tested. 0 20 40 60 80 100 024 48 72 96 Corrected cumulative mortality (%) Time (hours) 5.0 mg/mL 7.5 mg/mL 10.0 mg/mL 35 Figure 15. Average number of second-stage juveniles (J2) of Meloidogyne incognita per root gram, after 7 days of infection. J2 were previously exposed to hydroethanolic extracts of Cymbopogon citratus (8.5 mg/mL), Mentha aquatica (6.0 mg/mL) or M. pulegium (5.0 mg/mL) for 48h. Error bars represent standard error. Columns with the same letter are not significantly different (p> 0.05) according to LSD test. 2.2 Essential oils (EOs) Data regarding the effect of C. citratus , M. aquatica and M. pulegium EOs in the infectivity of J2 of Meloidogyne incognita is available on Fig. 16. On average, after 7 days of infection there were 24, 43 and 30 J2 per root gram, in the roots of plants infected with J2, exposed to selected concentrations of C. citratus (250 ppm), Mentha aquatica (2000 ppm) and M. pulegium (2000 ppm), which were significantly less than the average number of 192 J2 per root gram observed in the control . At the selected concentrations these EOs reduced the infectivity of J2 by 87.5, 77.8 and 84.4%, respectively. No significant differences were observed among the effect of the tested EOs, at the selected concentrations (p>0.05) 0 20 40 60 80 100 120 140 160 180 200 220 J2 per root gram Control C. citratus M. aquatica M. pulegium a b 36 Figure 16. Average number of second-stage juveniles (J2) of Meloidogyne incognita per root gram, after 7 days of infection. J2 were previously exposed to selected concentrations of essential oils of C. citratus , (250 ppm), Mentha aquatica (2000 ppm) or M. pulegium (2000 ppm) for 48h. Error bars represent standard error. Columns with the same letter are not significantly different (p>0.05) according to LSD test. 3. Effect of encapsulated Cymbopogon citratus essential oil on Meloidogyne incognita secondstage juveniles (J2) mortality The effect of encapsulated Cymbopogon citratus EO (ECCEO) on M. incognita J2 was tested. At the concentration of 250 ppm, the ECCEO caused an average mortality of 31% of J2, which was significantly higher than the 24% caused by C. citratus EO, at the same concentration (Table 6). No significant differences were observed among the results obtained with J2 exposed to keratin, when compared with the results obtained with J2 exposed to deionized water (p>0,05). Table 6. Effect of Cymbopogon citratus essential oil (EO) and of encapsulated C. citratus EO (both at 250 ppm), and keratin (100 ppm) on the mortality of second-stage juveniles (J2) of Meloidogyne incognita , after 48h. Data are average % estimated by GLMM analysis. Sample Dead J2i) (%) Deionized water 6 (±1,90)c Keratin 11 (±5.53)c Cymbopogon citratus 24 (±3.55)b Encapsulated C. citratus 31 (±3.55)a i) Percentages are average of five repetitions (± standard error) for each treatment. Values followed by the same letter are not significantly different according to LSD test (p>0.05) 0 20 40 60 80 100 120 140 160 180 200 220 J2 per root gram Control C. citratus M. aquatica M. pulegium a b 37 The intake of ECCEO by M. incognita J2 was observed by fluorescence microscopy. The J2 of M. incognita incubated with ECCEO showed fluorescence in the digestive system (Fig. 17A, B). A noticeable fluorescence could be observed in the intestine of J2 that died after incubation with ECCEO for 48h (Fig. 17A). Figure 17. Fluorescence imaging of Meloidogyne incognita second-stage juveniles (J2), incubated for 48h, with Cymbopogon citratus essential oil encapsulated in keratin FITC labeled (A-D) and exposed to keratin labeled with FITC (E-H). Left images (A, C, E, G), dead J2; Right images (B, D, F, H), alive J2. C - imaged with DIC; D, G, H – brightfield images. Arrows indicate the median intestine region. a b a b c d A B E F C D G H Dead Alive Keratin ECCEO 38 The M. incognita J2 that died after incubation also showed some morphological changes in the intestine region (Fig. 17A, C). In the J2 that were alive after incubation, these kind of morphological changes were not observed (Fig. 17D), or in J2 exposed just to keratin (Fig. 17H). Labeled keratin was used as a control, and the fluorescence of exposed J2 could also be observed along their digestive system (Fig. 17E, F). No morphological changes were observed in the intestine of J2 that were dead after incubation with this protein, for 48h (Fig. 17G, H). IV. Discussion 40 The search for green pesticides from natural sources is important for the development of new botanical pesticides, and control of RKN through the use of plant extracts and essential oil (EO) based formulations can be an economically rewarding for agricultural systems, due to the lack of syntethic nematicides options, and since plant extracts and EOs can be a source of cheap and effective nematicides (Corbani et al ., 2013; Watson and Desaeger, 2019). In this study the hydroethanolic extracts (HEEs) and EOs of Cymbopogon citratus , Mentha aquatica , M. piperita , M. pulegium , Satureja montana and Thymus vulgaris effect on the mortality of J2 of Meloidogyne incognita was evaluated. Only the Mentha pulegium and T. vulgaris HEEs affected J2 after 24h. The M. pulegium and M. aquatica HEEs were the most effective (p>0.05), followed by the C. citratus HEE, with LC50 values of 5.32, 5.98 and 8.63 mg/mL, respectively (Table 4). None of the tested HEE presented mortality values of over 90% after 96h, at the maximum concentration tested of 10.0 mg/mL. The HEEs of C. citratus and M. aquatica seem to have reached their maximum effect at the concentration of 7.5 mg/mL, and M. pulegium at the concentration of 5.0 mg/mL, since no differences in results were observed at higher concentrations (p>0.05). Every HEE presented some level of fungal contaminations over time, and it seemed to be proportional with the increase in concentration, with T. vulgaris HEE being the most prone to contamination throughout the bioassays. The C. citratus, M. aquatica, and M. pulegium HEEs were tested in regards to their effect in the infectivity of J2. After 7 days of infection, the number of J2 per root gram was reduced by 66.7, 60.9 and 69% (Fig. 15). There is little information regarding the nematicidal activity of HEEs of the plant species used in this study, as only the methanolic extracts of M. piperita and M. pulegium have been previously tested in regards to their effect on the mobility of J2 of Meloidogyne incognita , and the maximum concentration tested of 1000 ppm was not enough to affect 50% of exposed J2. (Caboni et al ., 2013). Of the tested EOs, Mentha aquatica and M. piperita EOs had no effect on the mortality of J2. The remaining EOs had an effect on J2 after 24h, and the most effective EOs were of C. citratus , followed by S. montana and T. vulgaris (Table 5). These three EOs affected approximately 100% of J2 after 24 of exposure to the maximum concentration tested of 2000 ppm (Figs. 11, 13 and 14). After 48h, these EOs also affected more than 90% of J2 at the concentration of 1000 ppm, although only the C. citratus EO had effect on J2 at concentrations lower than 500 ppm (Fig. 11). As opposed 41 to what was observed in HEEs bioassays, no sort of contamination was observed throughout the EOs bioassays, most likely due to the antimicrobial nature of EOs (Ibrahim et al ., 2006). The equivalent EOs of the HEEs tested on the infectivity of J2 were also tested in regards to their effect on infectivity. Since no dose-response effect was observed for the EOs after 48h of exposure, concentrations were selected based on the mortality results (Figs. 11 and 12). Therefore, of the EOs tested on the effect on the infectivity of J2, C. citratus EO was the most effective, as it required a lower concentration, followed by the M. pulegium and M. aquatica EOs. Despite not having an effect in the mortality of J2 , M. aquatica EO was able to affect the infectivity of J2, at the concentration of 2000 ppm. No significant differences in effect was observed among the results obtained with the selected concentrations of EOs (p>0.05). Significant differences were observed however, among the results obtained in biological replicates (p<0.05), which may have been caused by improper dissolution of EOs. Cymbopogon citratus EO has been tested by several authors in regards to its effect on RKN J2, and in reports with similar conditions, such as exposure time and concentrations, the EOs presented superior results to the obtained in this study (Gupta et al ., 2011; Barros et al ., 2020). This may be due to the use of surfactants such as Tween 80, which may have an influence in J2 health. Results obtained by different authors with M. piperita EO are variable. Some works report some noticeable effects in regards to mobility of Meloidogyne javanica (Oka et al ., 2000; Ntalli et al ., 2010) and mortality of J2 of M. incognita and M. javanica (Pandey et al ., 2000; Amora et al ., 2017), while other studies report no nematicidal effect in J2 of these two species (Caboni et al ., 2013; Andrés et al ., 2012). Mentha pulegium EO has been previously tested and caused no effect on J2 of Meloidogyne incognita , after 72h to exposure (1500 ppm) (Caboni et al ., 2013), which differs with the results obtained with the provided EO, as it affected 22.9 and 86.7% of J2, after 72h of exposure to the concentrations of 1000, and 2000 ppm. In this work, S. montana EO was effective against J2 of M. incognita , since 100% mortality of J2 was induced, at the concentration of 1000 ppm, after 24h, and the nematicidal activity of this EO has also been previously reported in another work, after inducing the mortality of 100% of M. javanica J2, at the same concentration, after 12h (Andrés et al ., 2012). In regards to T. vulgaris EO, in a similar study, it proved effective as it induced 99.4% mortality at the concentration of 2500 ppm, after 24h (Amora et al ., 2017), which was also observed in the results obtained in this study, since the EO caused the mortality of 96.3% of J2 after 24h, at the concentration of 2000 ppm. A lower effect than the observed in this work has also been reported, after the EO caused the mortality of approximately 70% of J2, after 42 48h of exposure to the concentration of 1000 ppm (Barros et al ., 2019), and a higher effect was reported (100% mortality of J2 at the concentration of 1000 ppm, after 12h exposure (Andrés et al ., 2012). The inconsistency in results observed among the EOs bioassays, is most likely due to improper dissolution of EOs, since, with the small solution volumes prepared, a slight fraction of undiluted EO is enough to highly influence the final concentration of the work solutions, and since no surfactants were used, when preparing the EO solutions. Overall, the tested EOs, were more biologically active against M. incognita J2, since some were able to affect 100% of J2 at certain concentrations after 24h, as oppose to the tested HEEs, that were not able to affect over 90% of J2, at the maximum concentration tested of 10.0 mg/mL, even after 96h. The S. montana HEE presented the lowest nematicidal activity of the tested HEEs, while the EO was one of the most effective, despite not having any effect at concentrations lower than 500 ppm. C. citratus was the plant that presented the highest nematicidal potential of the tested species, and was selected for a bioassay under an encapsulated formulation. The effect of keratin encapsulated Cymbopogon citratus EO (ECCEO) on the mortality of J2 was investigated. After 48h, the ECCEO affected a superior number of J2, than the C. citratus EO in its free formulation (p>0.05). Free keratin was used as a control, and had no significant effect in the mortality of J2, when compared with the results obtained with J2 exposed to deionized water (p<0.05) (Table 6). Images obtained by fluorescence microscopy show fluorescence throughout the digestive system of J2 incubated with ECCEO and with keratin (Fig. 17). Some fluorescence was found in the anterior region of the J2 in both types of exposure, either to the ECCEO, or to the protein, which may indicate that some fluorescein could have diffuse in this region, since no fluorescence was observed in J2 that were exposed to deionized water. However, a noticeable fluorescence was observed in J2 that died after incubation with ECCEO (Fig. 17A), when compared with J2 that died after exposure to only keratin (Fig. 17E), which may indicate that C. citratus EO (inside the keratin nanoparticles) was ingested. The M. incognita J2 that died after incubation with ECCEO showed some morphological changes in the intestine middle region (Fig. 17C). The ECCEO had no apparent effect in J2 that were alive after incubation, since no differences in fluorescence or morphology could be observed (Fig. 17B, D), when compared with J2 incubated with only keratin (Fig. 17F, H). These results seem promising, since the encapsulation is a potential solution to the persistence problems of botanical extracts and EOs, and to the solubility problems of EOs (Devi et. al 2019). More biological replicates are required in order to properly evaluate and confirm the effect of ECCEO on M. incognita J2. 43 The present study on plant extracts and EOs supports their biopesticidal nature. The results obtained seem to indicate that the HEEs of C. citratus , Mentha aquatica and M. pulegium , and EOs of C. citratus , M. pulegium , Satureja montana and Thymus vulgaris show potential for the development of new nematicidal formulations, that are sustainable to the environment and human health, and can be incorporated in IPM programs. However, considering that the chemical composition of plant extracts and EOs varies upon various factors such as agronomical factors, plant age, and technical procedures, such as plant material drying and EO extraction, a standardization of these parameters is needed in order to obtain safe and effective commercial formulations (Andrés et al ., 2012; Ashoka et al ., 2017). Additional trials are needed to further validate their potential application in the management of RKN. V. References