research and development of environmentally friendly fungicide derived from botanical materials against phytopathogenic fungi

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY RESEARCH AND DEVELOPMENT OF ENVIRONMENTALLY FRIENDLY FUNGICIDES DERIVED FROM BOTANICAL MATERIALS AGAINST PHYTOPATHOGENIC FUN

INTRODUCTION

Research rationale

Phytopathogenic fungi pose a significant threat to crop productivity, resulting in substantial economic losses and environmental degradation Synthetic fungicides are widely employed to combat these fungi, but their broad usage has generated concerns about their risk of adverse effects on the health of humans and the ecosystem as a whole As a result, there is a growing concern about developing sustainable and environmentally friendly alternatives to synthetic fungicides

Botanical resources, such as the extracts of plants and essential oils, have emerged as an option for naturally produced fungicidal materials These naturally occurring compounds have demonstrated potent antifungal properties and offer several benefits, including their low toxicity, biodegradability, and minimal impact on the environment Furthermore, botanical fungicides can be obtained from a diverse range of plant sources, which makes them a flexible and cost-effective option

Numerous investigations have examined the potential of botanical materials to combat phytopathogenic fungi For example, Ngo MT et al (2019) research discovered six active compounds from Maesa japonica Among them, five acylated triterpenoid saponins were new, and one was a known substance The recently discovered compounds demonstrated notable antifungal properties towards Magnaporthe oryzae in vitro, exhibiting MIC values ranging from 4 to 32 g/mL Furthermore, they greatly suppressed rice blast growth in vivo

Tan et al (2021) demonstrated nine limonoids from Melia dubia, Aphanamixis polystachya, and Swietenia macrophylla exhibit efficacy against phytopathogenic fungi, Fusarium oxysporum, Magnaporthe oryzae, Sclerotium rolfsii, Rhizoctonia solani, Alternaria spp., Botrytis cinerea, and three oomycetes, Phytophthora species Among the tested limonoids, four compunds demonstrated exceptional broad-spectrum antifungal activity against all the phytopathogens It is noteworthy that certain plant extracts and their active components have antifungal properties, leading to some of these extracts' registration as natural agents for managing fungal infections

Vietnam boasts a rich biodiversity, with approximately 20,000 plant species, offering a valuable natural resource that can serve as a foundation for researching and developing herbal medicine Among Vietnam's plant species, vast varieties from the Fabaceae family possess significant medicinal value and have been used in treatments for various ailments One such Fabaceae plant is

Desmodium sequax, a medicinal herb found abundantly in regions including India, the Himalayas, Myanmar, China, Taiwan, Laos, Vietnam, Indonesia, Philippines, and Papua New Guinea (eFloras.org, 2010) In Vietnam, this plant thrives in the wild at elevations ranging from 200 to 1600 meters, commonly found along rivers, forest edges, lawns, and open spaces Specifically, D sequax can be found in northern Vietnam, particularly in Lai Chau, Lao Cai, and Thanh Hoa provinces (Do et al., 2022)

D sequax has been recognized as a medicinal plant and has been employed in traditional medical practices The whole plant is used to heal conjunctivitis and burn wounds, according to traditional knowledge The plant is frequently given in

China to treat placental failure, eye discomfort, internal traumas, and bleeding Traditional Chinese medicine uses D sequax stems to treat pulmonary TB The plant's roots have anti-leprosy properties, help in damage reduction, work as antiseptics and antitussives, prevent asthma, and promote digestion The root is used to cure diarrhea, persistent fever, cough, asthma, snakebites, and scorpion stings in India Internal injuries and bleeding are treated with the seeds (Ma et al., 2011)

Previous research has given solid evidence for D sequax's significant antioxidant potential Chlorogenic acid has been observed as the key active component responsible for these antioxidant effects (Tsai et al., 2011) Siddiqui and Zaman (1998) discovered flavonoids and pterocarpanes in D sequax, including karanjin, lanceolatin-B, pongapin, 5'-methoxypongapin, kanujin, and glabra-II Furthermore, the identification of D sequax phytochemicals was published in the comprehensive research completed by Do et al (2022) and Nguyen Ngoc et al (2022) Naringenin, the phytohormone abscisic acid (ABA), lupeol, methyl p-coumarate, methyl caffeate, 4-hydroxybenzaldehyde, dehydrovomifoliol, and (Z)-2-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid were among the phytochemicals identified Among these substances, naringenin and ABA may be classified as defensive flavanones and phytohormones, respectively

In addition to investigating the antifungal properties of plants, there has been notable progress in the application of nanotechnology for the development of botanical fungicides Nanoparticles offer several advantages when utilized in conjunction with plant extracts Nanoformulations of plant extracts possess numerous benefits that render them well-suited for agricultural purposes These benefits include their environmental friendliness, quick action upon utilization, the ability to degrade, simplicity of application for crops, and controlled discharge capabilities Furthermore, their small size enables them to effectively act as carriers when combined with other formulations, facilitating seamless penetration into various plant tissues Additionally, nanoformulations do not impose detrimental effects on soil microorganisms, and any potential phototoxicity associated with silver-based nanoparticles can be mitigated by employing biocompatible polyvinyl pyrrole compounds for coating purposes (Hazafa et al., 2021)

This discovery suggests the possibility of uncovering additional Vietnamese plant extracts with antifungal properties, which could further contribute to the development of the botanical fungicide industry in Vietnam The utilization of botanical materials as a natural source of compounds with fungicidal properties is a promising appropriate substitute for chemical-based fungicides Several investigations have revealed that plant-based extracts and essential oils have antifungal properties against phytopathogenic fungi However, further research is necessary to determine potent botanical materials and to create environmentally friendly fungicides suitable for sustainable agricultural practices.

Research’s objectives

The main objective of this research was to contribute significantly to the improvement of natural product fungicides by investigating the potential of botanical materials as a viable source of environmentally friendly fungicides for regulating phytopathogenic fungi.

Research questions

1 What are the specific phytochemicals present in Desmodium sequax that exhibit antifungal properties against phytopathogenic fungi?

2 What is the structural composition of phytochemicals originating from the aerial components of the D sequax extract?

3 To what extent is the nanoformulation of D sequax an effective and environmentally friendly fungicide against phytopathogenic fungi?

4 What mechanisms do these phytochemicals employ to inhibit the growth of phytopathogenic fungi?

Significance of the study

This study will be noteworthy for the following:

The findings of this research can serve as an important point of reference for future studies investigating the antifungal potential of plant extracts against phytopathogenic fungi

The purpose of this study is to provide knowledge and raise awareness about botanical fungicides and their application in agriculture practices The results of this research may encourage people to consider the use of these environmentally-friendly fungicides

The outcomes of this study could potentially inform the creation and enforcement of new regulations aimed at promoting a more sustainable environment, particularly in the realm of sustainable agriculture practices The information gathered here can serve as a valuable resource for decision-making and policy implementation.

Scope and Limitations

Scope: Botanical materials for use as environmentally-friendly fungicides Limitations: The experiments for the master study were carried out in duration from June 2022 to June 2023, of which data collection, analysis, and interpretation were performed to meet the research objectives Despite achieving its objectives, the reserach had several undesirable drawbacks First, there is a shortage of time to thoroughly research all active chemicals found in D sequax Secondly, in vivo and field trials of the obtained fungicide need to be conducted in the future Lastly, plant extracts may contain phytotoxic compounds and other metabolites that can harm non-target organisms.

LITERATURE REVIEW

Natural products as a source of pesticides

The global population is projected to reach 9.8 billion by 2050 (United Nations and Department of Economic and Social Affairs, 2017), resulting in complex issues like food security, human health, and environmental sustainability Diseases, insects, and weeds cause major losses in agricultural activities, which are critical for food security and environmental sustainability, reducing worldwide crop output by 31% to 42% yearly (Dang, Q.L., 2012) Many farmers, however, continue to use synthetic pesticides, presenting risks to the well-being of humans and the ecosystem as a whole Studies highlight the need for preventive measures, accurate dosage measurement, and safe and effective pesticide utilization to protect the environment and human health as suggested by studies conducted by Lorenz (2009), Lechenet et al (2017), Vasilieadis (2017), and El-Wakeil (2013)

Chemical pesticides have been widely utilized since the 1940s, however they pose health dangers such as skin irritation, cancer risk, neurological problems, gastrointestinal impacts, and agricultural product contamination They can also harm non-target organisms and disrupt ecosystems and food chains Finding long- term alternatives to synthetic pesticides is difficult because of their high cost, pollution, and health hazards Natural pesticides offer lower toxicity, biodegradability, and target specificity but face challenges such as limited availability, variable efficacy, regulatory hurdles, and the need for research and development (Agrawal and Rathore, 2014; Bhat et al., 2019; Couzigou, 2016)

Natural plant extracts like Timorex Gold™ derived from tea tree plants have shown effectiveness against fungal infestations (APS Education Center, 2016; Gerwick et al., 2014; Sparks et al., 2017) Other products like Biocin-T™, BM608™, and MycoSin™ utilize essential oils and extracts to treat fungal diseases (Dagostin et al., 2011; Hogmann, 2003) Despite the long-standing use of synthetic pesticides and their advanced design, natural products hold promise for human and environmental benefits Regulation and risk reduction strategies are crucial for their safe and sustainable use in agriculture (Nieder et al., 2018; Bhat et al., 2019) Investing in research, development, and effective regulation is essential for promoting sustainable agriculture practices (Arora, 2019).

Phytochemical composition and bioactivity of botanical sources

Phytochemicals are naturally produced compounds found in plants that are biosynthesized by plant metabolisms and provide a variety of health advantages, including antioxidant, anti-inflammatory, and anticancer capabilities (Sultana et al., 2022) In addition, Sultana et al (2022) discovered phytoestrogenic isoflavonoids in clover honey generated from several Trifolium spp., which may have significant advantages for both the pharmaceutical and apiculture sectors Additionally, Hossain et al (2021) reviewed Lasia spinosa (L.), a plant traditionally used to treat various diseases and discovered that it contains several important nutritional and phytochemical components, including alkanes, aldehydes, alkaloids, carotenoids, flavonoids, fatty acids, ketones, lignans, phenolics, terpenoids, steroids, and volatile oil, all of which have excellent bioactivity

Phytochemicals have also been studied for their ability to control phytopathogenic fungi Sobhy et al (2023) examined the antifungal effects of an extract from Cinnamomum camphora on three common phytopathogens, Alternaria alternata, Fusarium solani, and Fusarium oxysporum, and found that the extract was able to inhibit their growth The presence of bioactive plant metabolites, such as phenolic and flavonoid compounds, may be responsible for this effect In a similar study, Ilondu (2013) evaluated the effectiveness of ethanolic leaf extracts from three Vernonia species, Vernonia ambigua, V amygdalina, and V cinerea against Cercosporella apersica and Curvularia lunatus, which cause groundnut leafspot disease, and found that the extracts are more effective than Dithane M45 in inhibiting the growth of the pathogens The extracts contained various compounds, including flavonoids, alkaloids, terpenes, saponins, and tannins, and GC/MS analysis revealed a mixture of compounds that could be responsible for the observed biological activity Additionally, Goudjil et al (2016) investigated the bioactivity of essential oils from Laurus nobilis and Mentha piperita against several phytopathogenic fungi and found that the oils had the potential as natural antimicrobial agents The oils contained various compounds, including 1,8-cineole, bornylene, linalool, sabinene, carvone, limonene, and β-pinene.

Mode of action of plant products against phytopathogens

It is critical to develop effective antifungal agents for the control of plant diseases Several investigations have been conducted to evaluate the antifungal effects of plant extracts against phytopathogenic fungi In one study, plant extracts derived from recently collected weeds were tested against several important phytopathogenic fungi, including Pythium ultimum, Penicillium expansum, and F solani The extracts caused changes in the structure of the fungi's hyphae and spores, resulting in abnormalities (Hashem et al., 2016) Chen et al (2021) focused on the design, synthesis, and evaluation of cryptolepine derivatives as antifungal agents against agriculturally relevant fungi Fungicidal activity was demonstrated against B cinerea, R solani, F graminearum, and S sclerotiorum Compound A3 was the most promising, preventing spore germination, producing the formation of harmful reactive oxygen species, and interfering with the normal functioning of the nucleus

Silva et al (2017) studied the antifungal characteristics and mode of action of CaTI, a trypsin inhibitor isolated from Capsicum annuum seeds, against phytopathogenic fungi CaTI was shown to be capable of inhibiting the development of Colletotrichum gloeosporioides and C lindemuthianum by permeabilizing their membranes and inducing the production of reactive oxygen species (ROS) and nitric oxide (NO), notably in Fusarium species Furthermore, CaTI conjugated with fluorescein isothiocyanate (FITC) was used in the investigation to confirm the presence of the inhibitor within the hyphae of the F oxysporum fungus Furthermore, the study discovered the presence of protease inhibitors in other Capsicum species

According to Lattanzio et al (1994), phenolics' antifungal activities can be related to their lipophilic character and the presence of hydroxyl groups in their molecular structure Because of these properties, they can attach to adhesions and proteins, disrupting membranes, inactivating enzymes, and complexing with metal ions As a result, they have toxic effects on fungi, as shown in Table 2.1 (Cowan, 1999)

Table 2 1 Modes of action of phenolic and O-heterocyclic compunds on fungi

Quinones Bind to adhesion link to cell, enzyme inactivation,

Blind to proteins, bind to adhesions, membrane disruption, enzyme inhibition, substrate deprivation and metal ions complexation

Other research on terpenes has shown that they are efficient against a wide variety of fungus species (Mihai and Popa, 2015) Mendoza et al (1997) discovered that terpenes damage fungal membranes due to their lipophilic characteristics However, these compounds can cause structural changes in hyphae and mycelia, resulting in decreased production of toxins such as aflatoxin (produced by Aspergillus species) and fumonisin (produced by Fusarium species), and thus decreasing the pathogenicity of mycotoxin-producing fungi (Garcia et al., 2012)

Zhou et al (2003) found that verazine and jerveratrum-type alkaloids had effective suppressive activities against the phytopathogenic oomycete

Phytophthora capsici in vitro using plant extracts from Veratrum taliense The intercalation of these alkaloids into the cell wall and/or DNA is thought to be responsible for their antioomycete effect (Houghton et al., 1994).

Phytopathogenic fungi and their effects on plant health

Phytopathogenic fungi are a broad range of microorganisms that may cause plant diseases, resulting in significant agricultural production and quality decreases These fungi may infect a variety of plants and have evolved many techniques to circumvent plant defensive mechanisms

In their natural habitat, plants are regularly exposed to a wide range of diseases such as bacteria, fungus, and viruses Pathogenic elements found in phytopathogenic fungi include enzymes that destroy cell walls, poisons, and growth regulators, all of which aid in their capacity to penetrate and control the host plant Some of these fungi, however, have been researched for their potential to function as biocontrol agents, thus they are not all hazardous They can inhibit the growth and development of other plant diseases through a variety of processes, including resource competition, the release of antimicrobial chemicals, and the induction of resistance in the host plant (Peng et al., 2021; Dean et al., 2012)

Miles Joseph Berkeley originally detected late blight, the cause of the Irish potato famine, and Anton de Bary subsequently termed it Phytophthora infestans

De Bary's research proved unequivocally that white sporulation on diseased potato plants was the root cause of the disease, refuting prior theories (Berkeley, 1846; de Bary, 1876) P infestans is a member of the oomycetes, sometimes known as

"water molds," which are closely related to brown algae but not genuine fungus Peronosporaceae is the family name for its hyaline, coenocytic, and diploid mycelium (Schumann and D'Arcy, 2000; Beagle, 2008)

Between potato seasons, P infestans can live as mycelium in infected tubers or tomato fruit Infected tubers or volunteer sprouts can develop sporangia, which are transferred by air currents to healthy potato leaves Stem lesions or diseased seed potatoes can cause infections Pathogen propagation is aided by movement in infected tuber tissues and asexual reproduction in clonal lineages P infestans germinates indirectly via zoospores in chilly, damp environments, whereas higher temperatures result in direct germination via a germ tube On sporangiophores, new sporangia are formed and can be disseminated by wind or water Sporangia can infect tubers via soil wash, and oospores can be formed by mating and spread through soil or plant tissues (Figrue 2.1) (Schumann and D'Arcy, 2000)

Figure 2 1 Life cycle of Phytophthora infestans (Adapted from Schumann et al., 2018 at Apsnet.org)

Late blight symptoms in potatoes include black or brown lesions on leaves and stems, starting as water-soaked spots and growing rapidly P infestans produces white spores on the underside of leaves in moist environments Without treatment, the disease spreads quickly, killing the plant within days

Potato tubers become infected by sporangia transmitted to the soil from foliar diseases, primarily through fissures, eyes, or lenticels Infected tubers are copper brown, reddish, or purple in hue, with sporulation on their surface Soft rot bacteria attack infected tubers, causing them to be discarded

Tomato late blight symptoms are similar to potato late blight symptoms, with lesions forming on leaves In humid conditions, white sporulation might develop The disease may spread through tomato stems and transplants, as evidenced by a large epidemic in the United States in 2009 (Hu et al., 2011)

Late blight causes dark brown lesions on tomato fruit, which can damage the entire fruit, followed by soft rot and disintegration If sporulation occurs, infected fruit can potentially act as a source of inoculum

Late blight management include choosing resistant types, exercising hygiene, and administering fungicides at the appropriate time Potato farming requires disease-free tubers, the burning or burial of cull heaps and volunteer plants, the promotion of air flow and drainage, and crop rotation Traditional agriculture employs fungicides such as metalaxyl, mancozeb, chlorothalonil, and copper formulations, whereas organic farming depends on copper products (Agrios, 1997; Finckh et al., 2015; Schumann & D'Arcy, 2000)

Rice blast is one of the most devastating grain diseases, posing a significant danger to crop yield, caused by the fungus M oryzae (Ascomycota), impacts rice farming in about 80 nations, resulting in major output losses of 10-30% globally

It appears as little necrotic areas on seedlings that develop with yellowish margins and can target numerous plant elements such as leaves, stems, panicles, and even roots (Boddy, 2016)

According to TeBeest's (2012) review, infections occur when conidia land on rice tissues, germinate, and produce a germ tube and appressorium, the appressorium penetrates the tissue, and the primary infection hypha spreads within susceptible tissues (Figure 2.2) Moderate temperatures (about 24°C) and extended high moisture, as found in flooded rice fields, are ideal for rice blast Infections in early seedlings are initiated by spores from overwintering tissues, and subsequent cycles can occur numerous times during the growth season, possibly creating high disease levels Temperature, rainfall, water depth in the paddy, nitrogen levels in fertilization, and genetic resistance in the cultivar all impact the severity of the disease

Infections in the neck region can severely impact seed set, and the fungus can also infect panicles, branches, and seeds themselves These infections typically manifest as grayish discolorations of the tissues

Figure 2 2 Life cycle of Magnaporthe oryzae (Adapted from Ebbole D J., 2007)

According to TeBeest (2012), the fungus M oryzae presents a number of symptoms that affect various parts of the rice plant Diamond-shaped lesions on the leaves are the most visible sign, with sheath lesions being less common Susceptible cultivars exhibit gray-green lesions with necrotic edges, whereas resistant cultivars have little brown lesions

Infections at the collars of the rice plant result in necrosis and leaf death, extending into the sheath Spore production can occur on these lesions Necks, supporting the panicle, are susceptible to "neck blast," causing panicle failure or collapse Panicle branches may break at lesion sites The fungus can also infect pedicels, leading to reduced seed production and brown spots on the seeds

To manage rice blast, cultural strategies such as crop rotation, proper fertilization, and maintaining appropriate flood levels are recommended High- quality seeds and genetic resistance are important, although the pathogen's genetic variability poses challenges Chemical fungicides can be used as a last resort, with seed treatments and foliar applications However, their effectiveness is limited, and aerial application can be costly

2.4.3 Fusarium oxysporum f sp cubense ( Foc)

Fusarium wilt, often known as Panama wilt, is a plant disease that affects banana plants and is caused by the soil-borne fungus F oxysporum f sp cubense

Overview of the genus Desmodium

The taxonomic framework of the genus Desmodium (GBIF.org, 2023): kingdom: Plantae

Desmodium represents a considerable collection of leguminous plants comprising approximately 350 distinct species These plants find their primary utility as grains and herbal resources However, a mere fraction of approximately

30 species has undergone rigorous examination to elucidate their botanical attributes, chemical composition, and physiological impacts The distribution of these species spans across Vietnam, where certain members hold significance as medicinal ingredients within traditional medical practices (Dang, Q L., 2014; Pham H.,1999)

In China, there is an estimated count of 41 species within the genus

Desmodium (Ma et al., 2011) The official Plant Varieties Resource Information page of the US Department of Agriculture has documented the existence of 211 species (Gao et al., 2015) Similarly, in Vietnam, Desmodium species have been identified and cataloged with a total of 59 species (Pham H.,1999)

The majority of Desmodium genus species are typically encountered as shrubs or occasionally as trees These plants exhibit compound leaves, often featuring an elongated triangular leaflet with a heart-shaped base The leaf stalks possess distinctive wings and are accompanied by additional leaves Inflorescences, consisting of double clusters of flowers, are found in the leaf axils and apex The flowers themselves are typically pink in color As for the fruit, it takes the form of leguminous pods Some species within the genus also display ash-gray gills (Magielse et al., 2013; Zhou et al., 2012)

2.5.1.1 Desmodium sequax Wall (Sinuate-leaf Tickclover)

According to Saisorn and Chantaranothai (2020), D sequax has specific botanical traits It is a shrub that grows to a height of 1 to 2.5 m The stem and twigs are cylindrical, with ascending white hairs with a hooked form covering the surface The leaves have linear stipules that measure 3-5 0.5-1.5 mm and have an acuminate apex The surface of the leaf is heavily coated with whitish-appressed pubescence Petioles range in length from 0.5 to 3 cm and are sparsely coated with ascending pubescence interspersed with uncinate hairs The rachis, which is 5-15 mm in length, also has sparsely ascending pubescence interspersed with uncinate hairs The inflorescences of D sequax are pseudoracemose or panicle-like in structure, featuring one or multiple branches that can grow up to 15 cm in length These inflorescences are terminal or axillary in position Both the rachis and rachilla are covered with spreading pubescence and minutely uncinate hairs The pods of the plant are dark brown, sessile, and indehiscent, and often consist of 3–

11 articulations Resembling moniliform structures, they measure 1.5–3.5 cm in length and 2.5–3 mm in width The pod surface is densely covered with ferrugineous uncinate hairs and does not display a reticulate pattern The sutures on both sides of the pod are equally constricted, each with a depth of less than 0.5 mm The isthmus, located between the articulations, is approximately 3/5 to 2/3 as wide as the pod The articles within the pod are elliptic to quadrangular, measuring 2.5–3.5 mm in length Fruiting pedicels, around 4–5 mm long, are densely covered with ascending pubescence The seeds of D sequax are brown to dark brown in color, reniform in shape, with dimensions of 2–2.3 × 1.3 mm and a thickness of approximately 1 mm This plant species can be found in various regions, including India, the Himalayas, Myanmar, China, Taiwan, Laos, Vietnam, Indonesia, Philippines, and Papua New Guinea It grows best on open ground, grassland, and roadside areas, as well as mixed evergreen and hill evergreen forests at elevations ranging from 500 to 1,680 m (eFloras.org, 2010) The flowering period typically occurs between September and December

2.5.1.2 Desmodium triangulare (Triangular Horse Bush)

Desmodium triangulare is a small perennial plant that features numerous branches Typically, it has a height of 0.5–0.6 m, although some trees may reach up to 1.5 meters The trunk is round and has multiple branches, while the young twigs are generally flattened, edged, and wavy, with soft white hairs

The leaves of the plant are compound and arranged alternately, featuring three leaflets, with the middle leaflet being larger than the other two The entire leaflet is ovate, rhombic, or oval The young leaves located at the top of the plant are often covered in white hairs

The flowers of the plant grow in single clusters in the interstices of the leaves Each cluster has 10–20 white-colored petals with claws The calyx consists of four lobes, with the lower lobe being longer than the other three, and covered in soft hairs The fruit of the plant has a rounded edge and lacks a stalk Each fruit contains nodes in the middle of the seeds, which divide the seeds into 2-3 segments and are covered in soft hairs

D triangulare in Vietnam distributes mainly in lowland, midland and highland provinces such as Lao Cai, Lang Son, Dak Lak, Lai Chau, Hoa Binh, Kon Tum, Gia Lai and Ha Bac

The plant D gangeticum can be classified as both an herb and a shrub, typically reaching a height of 1–1.5 m Its branches are elongated and slender, with young branches displaying slight angles and a hairy texture

The leaves of the plant consist of a single leaflet and are arranged alternately They are oval or ovate, measuring approximately 6–10 cm in length and 3–5 cm in width The base of the leaf is rounded, while the tip is slightly pointed The upper surface of the leaf is smooth and short, whereas the lower surface is covered in fine hairs On closer inspection, two short fibers can be observed at the base of the leaves, which are remnants of two reduced leaflets on the sides The petiole, or leaf stalk, measures 1–2 cm in length and ends in a pointed structure

The inflorescence, or flower cluster, appears as a sparse panicle located at the apex or in the interstices of the plant It is covered in hairs and consists of numerous small flowers arranged in pairs The calyx, the outer part of the flower, has four smooth teeth, while the corolla, the inner part of the flower, is shaped like an inverted oval and possesses flagella and a spatula The lateral wings of the corolla are oblong, and the stamens, the male reproductive organs, are arranged in two bundles The fruit of the plant is curved and contains 7–8 compartments, each housing a single seed

This ancient tropical plant thrives naturally in hill areas, lawns, and alongside the road spanning from the northern to the southern regions

2.5.1.4 Desmodium heterophyllum (L.) DC (Variable-leaf Tick Trefoil)

D heterophyllum is a compact shrub that grows in a crawling manner with abundant branching The branches extend up to 20–40 cm in length and are slender and rigid, spreading over the ground

The leaves of the plant are arranged alternately The lower leaves are typically single, while the upper leaves are compound, consisting of three leaflets The leaflets are oval or elliptical, with an obtuse base and a rounded tip Occasionally, there may be slight notches on the leaf margins The length of the leaflets ranges from 0.7 to 2 cm, while their width spans from 0.5 to 1.2 cm The upper surface of the leaflets has a glossy appearance, while the underside is hairy Among the three leaflets, the terminal leaflet is larger than the lateral leaflets The leaves are accompanied by small stipules

D heterophyllum only grows and grows in areas with hot and humid climates Therefore, the tree grows quite commonly in India, Vietnam, Laos, Cambodia and some provinces in China

2.5.1.5 Desmodium triquetrum (L.) DC (The Trefle Gros)

D triquetrum is characterized as a robust herb, reaching a height of 1–1.5 m Its stem possesses a triangular shape, featuring three distinct sides The leaves exhibit an elongated, heart-shaped triangular leaflet at the base, accompanied by a winged stalk Notably, the leaves also possess pointed triangular scales, measuring 1.5 cm in length and displaying a brown coloration

The inflorescences of the plant occur in double clusters, found both in the axillary regions and at the apex The flowers themselves are pink and are typically arranged in pairs The pods, which vary in color from ash-gray to other shades, can have a different number of nodes, ranging from 4–5 to 8–9 The width of the pods can vary as well, from 2–2.5 to 4–5 mm or even wider Other distinguishing features include the presence or absence of hair on the fruit, as well as variations in the number of nodes, and the width, which can be narrow or wide

The advancements in utilizing natural products as botanical fungicides 37 1 Environmental impact of botanical fungicides and challenges in

Natural products are very valued commodities obtained from plant and microbial biodiversity Despite our limited exploration thus far, there is still much to uncover regarding the potential applications of natural compounds and their combinations in fungal disease control These environmentally friendly and sustainable chemical solutions have become essential not only in agriculture, which includes conventional, low-input farming systems, integrated crop production, and organic agriculture but also in effectively managing fungal disease, which poses environmental and public health risks

Synthetic insecticides have greater durability and efficacy in controlling pests and diseases Phytochemical pesticides, on the other hand, have lesser permanence and efficacy than synthetic counterparts due to their biodegradable nature Despite countless studies on the pest-controlling characteristics of diverse plant-based chemicals, only a small number of these molecules have been successfully used for commercial reasons

Notably, recent advances have resulted in the creation and approval of new classes of natural plant protection agents These items have been effectively incorporated into agricultural methods with the support of organizations authorized to commercialize them, resulting in major commercial accomplishments Jojoba essential oil (marketed as Detur, E-Rasem, Eco E-Rase, Permatrol, Erase TM ), rosemary essential oil (marketed as Ecotrol TM , Sporan TM , Ecosmart), and other examples have emerged as notable examples of these natural plant protection products (Suteu et al., 2020)

2.6.1 Environmental impact of botanical fungicides and challenges in commercialization and application

Fungicides are helpful for treating fungal infections in agriculture, but they have downsides such as safety problems, remains in food, and dangers to the environment and health To find alternatives, researchers have explored botanical fungicides, which offer advantages like environmentally-friendly, effectiveness, selectivity, and biodegradability Studies by Deresa and Diriba (2023) and Yoon et al (2013) highlighted the potential of botanical fungicides in mitigating issues associated with synthetic agents Research has shown the suppressive effects of botanical fungicides on plant diseases For example, extracts from various plants demonstrated growth suppression of S rolfsii, the cause of soybean damping-off disease, in vitro (Fauzi and Sari, 2022) However, environmental impact of botanical fungicides remains insufficiently understood, and more research is needed to assess their risk in soil and water environments

Despite the challenges, botanical fungicides are seen as viable alternatives to synthetic fungicides Researchers have isolated and characterized antifungal plant derivatives, demonstrating the effectiveness of natural products in managing plant diseases However, further research is required to develop commercially viable botanical fungicides and evaluate their environmental and health risks (Tsalidis, 2022)

The commercialization of botanical fungicides is hindered by factors such as low activity levels, low persistence in the environment, and low residual toxicity and shelf life Light, heat, pH, moisture, and other variables in outdoor environments contribute to the fast dissolution of the active ingredient, necessitating repeated applications (Walia et al., 2017) The varying chemical composition of fungicidal plants adds complexity to creating standardized and stable preparations, and stringent biodiversity protection laws complicate the availability of botanical resources Insufficient analytical procedures and reliable standards hinder quality control, while regulatory approval processes slow down the commercialization of botanical fungicides (Bhandari et al., 2021; Regnault- Roger et al., 2008)

2.6.2 Regulatory integration for adoption and utilization of botanical fungicides

According to Isman (2020), farmers require access to extension services that provide complete knowledge on the identification, preparation, and administration of botanical fungicides in order to boost small-scale production

Subsidies can promote their development and usage Raising knowledge about the advantages of natural fungicides over synthetic ones, as well as encouraging sustainable agriculture and organic farming, is critical It is critical to establish a secure market for botanical fungicides and to have the government regulate pricing based on quality Simplifying import and export rules, lowering taxes on botanical fungicides, and offering low-interest loans for large-scale manufacturing will help them gain traction It is required to investigate possible plants with fungicidal qualities, improve extraction and processing procedures, and research climatic conditions for site-specific manufacturing To achieve quality standards, manufacturers of botanical fungicides strive to enhance formulations, stabilize extracts, and maintain consistent chemical compositions It should be explored to market synthetic molecules that look like natural chemicals Collaboration among regulators, industry representatives, academics, and farmers is essential for developing fair regulatory processes and expediting market entry while taking risk assessment into account It is critical to prioritize botanical fungicides in order to increase integrated pest control, organic farming, and sustainable agricultural practices.

MATERIALS AND METHODS

Materials

 Solvents for extraction and isolation:

 Methanol (MeOH), acetone, n -hexane (Hex), dichloromethane

(DCM), ethyl acetate (EtOAc), n -butanol, ethanol and distilled water

 Chemicals and solvents used in the research and analysis are of industry standard and are re-distilled before use

 Reagents in thin layer chromatography: Anisaldehyde-Sulfuric acid (AS) reagent solution;

 Fungal culture medium for activity testing: PDA medium (fresh potato 200 g; glucose 20 g; agar 20 g; pH 6.8–7.0)

 Thin layer chromatography (TLC) was performed on silica pre- coated plates gel 60 Merck F 254 has a thickness of 0.25 mm Observe the plate under ultraviolet light at two wavelengths λ = 254 nm and λ = 365 nm , then spray the reagent and dry at about 100 o C to detect compounds

 Column chromatography (CC) using silica Merck gel particle size ( 40-63 àm and 15-40 àm ) and Sephadex LH - 20

 Instruments in the laboratory: o Extraction funnel 0.5 L, 1 L, and 2 L; o 25 mL glass test tube; o 100 mL, and 250 mL measuring tubes; o Penicillin vials containing the substance; o Eppendorf 1 mL, and 2 mL; o Pipettes 5 mL, 10 mL, and 20 mL; o Globe flask, evaporator 25 mL, 100 mL, 250 mL, 500 mL, and

 Vacuum evaporator of IKA company in Institute for Tropical Technology at VAST

 Ultraviolet lamp with two wavelengths 254 nm and 365 nm in Institute for Tropical Technology at VAST

 Analytical balance AJ-310 (300g/0 001 g) of Japan-origin VIBRA in Institute for Tropical Technology at VAST

 Branson 1510 ultrasonic in Institute for Tropical Technology at VAST

 1D- and 2D-NMR nuclear magnetic resonance spectra were measured on a Bruker Avance 500 FT-NMR Spectrometer in Institute for Tropical Technology at VAST

 ESI -HRMS high-resolution mass spectrometer was measured on the SCEIX X500R-QTOF instrument in Institute for Tropical Technology at VAST.

Methods

3.2.1 Sample plants collection and processing

Desmodium sequax sample was identified by Nghiem Duc Trong,

Department of Botany, Hanoi University of Pharmacy The collected sample was stored in Institute for Tropical Technology at VAST

To ensure accuracy and avoid confusion, it is imperative to use the correct scientific name when collecting and handling the raw material of the plants The raw materials should possess favorable qualities such as good quality, high content of active components, and stability After harvesting, the plant samples were thoroughly washed and then dried in shaded conditions at approximately 40 degrees celsius Subsequently, the dried samples were finely ground and extracted using appropriate solvents to obtain the plant extracts

This extraction technique operates on the principle of partitioning the analyte between two immiscible liquid phases, typically using two solvents, with one potentially containing the analyte The thermodynamic distribution coefficient (Kb) of the extraction equilibrium, which is controlled by parameters such as temperature and the presence of an acidic environment, governs extraction efficiency largely Another important factor is the thermodynamic constant (Kpb) Two extraction methods can be employed: static extraction and continuous flow extraction In analytical applications, the static extraction method is more commonly employed due to its simplicity It is important to follow specific criteria and requirements throughout the extraction process in order to produce the best extraction outcomes.:

 The extraction solvent should be of high purity to prevent further contamination of the sample with analytes

 The solvent should effectively dissolve the analytes while leaving other substances in the sample undissolved

 The extraction system should exhibit a large distribution coefficient to ensure thorough extraction

 Rapid and reversible extraction equilibrium should be achieved to facilitate efficient extraction

 Clear, fast, and easily separable phases should be obtained during the extraction process

 Appropriate selection of an acid environment, including pH and acid type, is crucial

 The extraction process should be conducted at a suitable and constant temperature

 Vigorous shaking or mixing is necessary to facilitate efficient extraction

This extraction method is straightforward and does not require complex equipment; This method is employed in the current study for the comprehensive extraction of D sequax compounds using organic solvents

Chromatography encompasses a range of physicochemical techniques employed for the separation of components within a mixture This separation is accomplished by the differential partitioning of substances between two immiscible phases: stationary and mobile The stationary phase hinders component migration, forcing them to move through the chromatographic system at different rates and become separated over time Each component exhibits a distinct retention time, representing the duration it takes to traverse the system Chromatography serves as a valuable method for the purification and isolation of biological molecules

Column chromatography was conducted under ambient pressure conditions The stationary phase consisted of relatively large particles (50-150 μm) packed into a glass column To keep the surface plate intact, the analyte sample was inserted at the top of the column, above the stationary phase, and covered with a glass cover The solvent used for elution was placed in a container positioned above the column As the solvent flowed through the column, it exited at the bottom and was collected in small vials placed directly at the outlet It is worth noting that this method tends to exhibit slower separation and lower efficiency when compared to high-pressure liquid chromatography (HPLC) Nonetheless, column chromatography offers the advantages of a stationary phase and the availability of inexpensive instruments that can be used with relatively large samples

The procedure for conducting column chromatography involves several steps, including the selection of the solvent, sorbent, loading the dry sorbent onto the column, and introducing the sample The following carriers were used in this investigation to isolate compounds using column chromatography:

 Silica gel 60 (230-400 mesh) as the regular phase sorbent

 Sephadex LH-20 with methanol (MeOH) as the solvent

Thin layer chromatography, also referred to as planar chromatography, primarily relies on the principle of absorption In this technique, a mobile phase composed of one or more solvents travels over a stationary phase, which consists of an inert substance like silica gel or Al2O3 The stationary phase is placed evenly as a thin coating on a flat support material, such as glass, metal, or plastic sheets Thin layer chromatography is the name given to this technology because of the thin covering of the sorbent

The migration coefficient Rf, which defines the degree of analyte migration, may be computed as the ratio of the analyte spot distance to the solvent front distance

𝑅 𝑓 = 𝑎𝑏 a: is the distance from the starting point to the center of the test specimen trace, in cm b: is the distance from the starting point to the measured solvent level along the trace path, in cm

Rf: only has a value from 0 - 1

3.2.3 Organic compound chemical structure determination methods

The process of determining the structure of a substance involves collecting and analyzing data from various sources, each providing specific information about the substance's structure The determined structure must align with all the structural details obtained from these sources Currently, modern techniques for structural analysis are employed, which include absorption spectroscopy methods like ultraviolet/visible (UV/VIS) and infrared (IR) spectroscopy, as well as nuclear magnetic resonance (NMR) and mass spectroscopy (MS) Furthermore, there have been advancements in "coupled" methods that combine high-performance liquid chromatography (HPLC) with other techniques such as HPLC-MS, HPLC-NMR, and more However, among these approaches, spectrometric methods like mass spectrometry (MS) and 1D and 2D nuclear magnetic resonance spectroscopy ( 1 H- NMR, 13 C-NMR, DEPT, HSQC, HMBC, etc.) are the most widely used and popular

3.2.3.1 Nuclear magnetic resonance spectroscopy (NMR)

Nuclear magnetic resonance spectroscopy represents a contemporary and highly effective approach for the structural analysis of compounds By employing both one- and two-dimensional NMR spectroscopy techniques, it becomes feasible to precisely determine the structure of compounds, including their stereochemistry The fundamental principle of NMR (proton spectrum and carbon spectrum) revolves around the distinct resonances exhibited by magnetic nuclei ( 1 H and 13 C) when subjected to an external magnetic field These resonances are manifested as chemical shifts, representing different spectral positions Within the NMR spectrum, two key parameters, namely the chemical shift () and the spin-spin interaction constant (J), are indicative of the chemical structure of a molecule

In 1 H-NMR spectra, the chemical shift (δ) of protons is measured on the TMS (tetramethylsilane) scale, ranging from 0 àg/mL to 14 àg/mL The magnitude of the chemical shift depends on the hybridization of the atoms and the specific chemical structure of the molecule Each type of proton resonates at a distinct magnetic field, resulting in a unique chemical shift value By analyzing the chemical shift, peak area, and spin interactions among the magnetic nuclei, it becomes possible to deduce the chemical structure of the compound The characteristics of chemical shifts and spin interactions (J Hz) provide valuable information that aids in determining the chemical structure of the compound

Every carbon atom exhibits a unique resonance in the NMR field, resulting in a distinct spectral signal The 13 C-NMR spectrum employs a scale measured in àg/mL, similar to the proton spectrum, but with a broader range spanning from 0 àg/mL to 240 àg/mL Furthermore, the 13 C spectrum is obtained using the DEPT (Distortionless Enhancement by Polarization Transfer) technique

The HSQC spectrum provides insights into the correlation between the 1 H and 13 C signals, revealing the direct bonding between protons and carbon atoms

The spectrum reveals the long-range interactions (2 and 3 bonds) between carbon and hydrogen atoms in the molecule, allowing the identification of specific regions and the entire molecule as a whole This spectrum holds particular significance when examining molecules with quaternary carbon atoms, as it highlights the connection between the 1 H proton signal at a specific 13 C atom and the signal of another 13 C atom situated 2 to 3 bonds away, and occasionally even up to four bonds away in certain cases

Mass spectrometry is a complex method that uses specialized equipment called a mass spectrometer to measure the mass-to-charge ratio of ions This method exhibits remarkable sensitivity Initially, the analytes undergo ionization through various methods Subsequently, in a vacuum environment, the resulting ions are separated based on their individual mass-to-charge ratios (m/z ratio), and their intensities are subsequently measured The resulting mass spectra exhibit a pattern of ion occurrence, associating each ion with a specific mass number This aspect greatly aids in quantitative analysis The mass number, directly obtained from mass spectrometry, is unique to each molecule However, challenges arise when multiple analytes are simultaneously introduced, making deconvolution of the spectra exceedingly intricate

This technique finds extensive application in various areas, including:

• Identification of unknown compounds by analyzing the mass of the compound molecule

• Determination of the isotopic composition of the compound's constituents

• Quantification of compounds in a sample using complementary methods (since mass spectrometry alone is not inherently quantitative)

• The HRMS method is employed on the SCEIX X500R-QTOF machine in Institute for Tropical Technology at VAST

3.2.4 Isolation and structural determination of naringenin and abscisic acid from the aerial parts of Desmodium sequax

The identification of D sequax samples was performed by Nghiem Duc

Trong from the Department of Botany at Hanoi University of Pharmacy The dried materials of this particular plant sample, weighing 3.5 kg, underwent extraction using methanol as the solvent The resulting extracts were combined and then evaporated in a vacuum, yielding a total extract weighing 150g This total extract was subsequently dispersed in distilled water and separated into different fractions using ethyl acetate as the solvent

To obtain the ethyl acetate fraction, the solvent layer was evaporated under reduced pressure This process yielded an ethyl acetate fraction weighing 83 g The ethyl acetate fraction was then subjected to separation using a silica gel column

RESULTS

Structural characterization of naringenin and abscisic acid isolated from

from the aerial parts of Desmodium sequax

Compound 1 was obtained as a white powder (refer to Fig 1) The 1 H-NMR spectrum displayed typical signals characteristic of the A2B2 system, specifically two proton signals at δH 7.31 (2H, d, J = 8.4 Hz, H-2′, 6′) and 6.82 (2H, d, J = 8.4

Hz, H-3′, 5′) Additionally, the A-ring proton of the flavonoid scaffold was identified by the pair of double-nose signals at 5.90 (1H, d, J = 1.8 Hz, H-6) and 5.88 (1H, d, J = 1.8 Hz, H-8) The 13 C-NMR spectrum of compound 1 exhibited

13 carbon signals, indicating the presence of a symmetrical benzene ring as evidenced by the two carbon signals at δC 129.0 and 116.3, which are indicative of

2 carbon atoms Consequently, the chemical structure of compound 1 was inferred to be a flavonoid skeleton Additionally, the 1 H-NMR spectrum revealed a triplet signal of a doublet type at δH 5.34 (1H, dd, J = 3.0; 13.2 Hz, H-2); 3.11 (1H, dd, J

= 13.2; 17.4 Hz, H-3a); and 2.70 (1H, dd, J = 3.0; 17.4 Hz, H-3b), which is indicative of the H-2 and H-3 of a flavanone Through NMR analysis and comparison with the spectral data documented in the published literature (Nguyen Ngoc et al., 2022; Cordenonsi et al., 2017), the chemical structure of compound 1 was identified as naringenin.

Figure 4 1 Isolation and structural analysis of naringenin (1) and abscisic acid (2) from D sequax aerial parts Experimental NMR (methanol- d4, 600 MHz) spectra of naringenin (left) and abscisic acid (right) are presented in the form of graphical representations, with the 1 H-NMR spectrum depicted by the red line at the top and the 13 C-NMR spectrum illustrated by the black line at the bottom

Compound 2 is a white powder that exhibits solubility in methanol (Figure 4.1) The 1 H-NMR spectrum reveals the presence of four methyl groups at δH 2.00 (3H, d, J = 1.2 Hz, H-12); 1.03 (3H, s, H-13); 1.06 (3H, brs, H-14); 1.93 (3H, d, J

= 1.2 Hz, H-15) Additionally, the 13 C-NMR spectrum displays 15 carbon signals, suggesting a sesquiterpenoid structure (Figure 4.1) In order to elucidate the structure of compound 2, HSQC and HMBC spectra were employed (Figure S1–S2) The skeleton of this sesquiterpenoid was constructed based on the HMBC correlations of the four methyl groups (Figure 4.1) Notably, significant HMBC correlations of compound 2 were observed, including H-15/ C-5,6, C-1; H-13,14/ C-1,2,3; H-7/ C-1, 6, 7; and H-12/C-8, 9, 10, indicating a megastigmane-type scaffold The positions of the carbonyl group (δC 201.1) and the carboxylic group (δC 171.5) were determined by the HMBC correlations of H-3/C-4 and H-10/C-11 The trans configuration of the double bond between C-7 and C-8 was established through analysis of the coupling constant of 16.2 Hz Likewise, the cis configuration of the double bond between C-9 and C-10 was ascertained by comparing the chemical shifts of H-8 and C-8 with the spectral data of cis, trans- and trans-trans abscisic acid in the reference (Ohkuma et al., 1963; Cornforth et al., 1965; Chen et al., 2020; Nguyen Ngoc et al., 2022) Consequently, the chemical structure of compound 2 was determined to be cis, trans-abscisic acid

4.2 In vitro inhibitory efficacy of naringenin and abscisic acid against

Phytophthora infestans and Magnaporthe oryzae

Figure 4 2 In vitro inhibitory efficacy of naringenin (1) and abscisic acid (2) against

Phytophthora infestans at 24 h after treatment

The antifungal activity of naringenin (compound 1) and abscisic acid

(ABA) (compound 2) was assessed in vitro against P infestans and M oryzae In the preliminary in vitro bioassay targeting P infestans, both compounds 1 and 2 displayed potent activity, exhibiting minimum inhibitory concentration (MIC) in

48 hours, values of 50 and 100 àg/mL, respectively In a dose-dependent bioassay, both naringenin (1) and ABA (2) demonstrated antifungal activity that increased with dosage, effectively inhibiting the growth of P infestans at concentrations exceeding 200 àg/mL (Figure 4.2) Even at a low concentration of 6.4 àg/mL, both naringenin and ABA exhibited inhibitory effects on P infestans growth,

2 suppressing it by 23.8% and 21.9%, respectively Notably, ABA exhibited stronger potency against P infestans when compared to naringenin (1) ABA effectively inhibited the growth of P infestans at 24 h, with an IC50 of 19.7 àg/mL and an IC90 of 72.0 àg/mL, whereas naringenin displayed respective values of 53.7 and 129.2 àg/mL after 24 h treatment (Table 4.1)

Table 4 1 In vitro inhibitory efficacy of flavanone naringenin (1) and phytohormone abscisic acid (2) against Phytophthora infestans and Magnaporthe oryzae

Compound 1= naringenin; 2= abscisic acid (ABA); Fold: This refers to the IC value ratios of compound 1 to compound 2 to P infestans at 24 h after treatment and to M oryzae at

12 h after treatment The numerical value enclosed in parentheses denotes the confidential interval (àg/mL) Different letters in each column indicate statistically significant differences according to Duncan’s multiple-range test (p < 0.05)

Figure 4 3 In vitro inhibitory efficacy of naringenin (1) against Magnaporthe oryzae at

The inhibitory values of ABA were 2.72 and 1.79-fold below compared with those of naringenin at 12 h after treatment (Table 4.1) In contrast, compound

2 was inactive against M oryzae at concentrations below 400 àg/mL Naringenin

(1) completely inhibited the growth of M oryzae at a MIC of 200 àg/mL This flavanone showed moderate antifungal activity against M oryzae in a dose- dependent manner (Figure 4.3) and at 12 h also produced inhibitory values of IC50 and IC90 of 95.0 and 172.7 àg/mL, respectively (Table 4.1)

This is the first study of the antifungal activity of naringenin and ABA against P infestans and M oryzae The study results supported the antimicrobial roles of flavanone (1) and phytohormone (2) in the defense response in the host plants In particular, the remarkable inhibition of ABA against P infestans, for the

Concentration (mg/mL) first time, provided important evidence that ABA, a phytohormone, seems to strongly inhibit the soilborne oomycete such as Phytophthora species.

Analyzing nanoparticle structure of D sequax extract

D sequax extract was prepared using Tween 80 as an emulsifier, as described in Section 3.2.5 This fabrication process resulted in nanodroplets with an average size of 120 nm (Figure 4.4) The nanoparticles containing D sequax extract exhibited stability, as observed in the TEM images, which showed that the particles had a size of 50 nm and were evenly distributed in the dispersion medium (Figure 4.5)

Figure 4 4 The results of the particle size analysis performed on the nano formulation

Figure 4 5 TEM analysis results of the nano formulation (a-b) Bar indicates 50 nm scales (c) a b c

In vitro inhibitory efficacy of nanoformulation of D sequax extract

The in vitro bioassay of nanoformulation of D sequax was performed by poisoned-food technique using PDA medium to observe the mycelial gowth inhibition of various strains of plant pathogenic fungi As shown in Table 4.2, nanoparticles significantly inhibited the mycelial growth of M oryzae and S rolfsii in the concentration ranging from 5000 to 10000 àg/mL The nanoformulation did mycelial growth inhibition from 30.13% to 56.34% over a range of test concentrations and displayed in vitro antifungal activity in a dose-dependent manner (Table 4.2 and Figure 4.6-4.10) However, R solani was not inhibited by this nanoformulation at each concentration after 3 days of incubation (Table 4.2) The positive control, Score EC 250 showed antifungal activity against all the fungi tested The strain R solani less sensitive compared to other fungi tested; it was suppressed 3.05% growth when exposed to D sequax nano formulation after 3 days of inoculation

Table 4 2 Inhibitory potency of nanoformulation D sequax extract against various fungi strains under in vitro condition

Fungi strains Concentration (àg/mL) Inhibitory effect (%)

Figure 4 6 Inhibition effect on mycelium of M oryzae on 3 days of post inoculation

Figure 4 7 Inhibition effect on mycelium of C capsici on 3 days of post inoculation

Figure 4 8 Inhibition effect on mycelium of R solani on 3 days of post inoculation

Control 2500 àg/mL 5000 àg/mL 10000 àg/mL

Control 2500 àg/mL 5000 àg/mL 10000 àg/mL

Control 2500 àg/mL 5000 àg/mL 10000 àg/mL EC 250

Figure 4 9 Inhibition effect on mycelium of F oxysporum on 3 days of post inoculation

Figure 4 10 Inhibition effect on the mycelium of S rolfsii on 3 days of post inoculation

In silico model of defense mechanism of naringenin and abscisic acid

against Phytophthora infestans and Magnaporthe oryzae

Among the compounds investigated, only naringenin (1) exhibited effective control against M oryzae, indicating its potential as a targeted compound against enzymes from this fungus, as suggested through in silico analysis Enzymes involved in the melanin biosynthesis pathway play a crucial role in limiting blast disease caused by M oryzae and are therefore important targets for fungicide development (Gao et al., 2022; Motoyama and Yamaguchi, 2003; Khan et al., 2022) Traditional breeding methods require significant time to develop resistant varieties, hence emphasizing the significance of these catalytic enzymes as targets for blast pathogen control In this study, it focused on two specific enzymes: 1,3,8- trihydroxynaphthalene reductase (ID PDB: 1YBV) and scytalone dehydratase (ID PDB: 1STD), both of which participate in the melanin biosynthetic pathway Control 2500 àg/mL 5000 àg/mL 10000 àg/mL EC 250

Control 2500 àg/mL 5000 àg/mL 10000 àg/mL EC 250

Initially, complex models of the enzymes were downloaded and subjected to re- docking with naringenin (1) The re-docking results yielded RMSD values below 0.5 Å, indicating high reliability of the models The binding analysis revealed favorable interactions between naringenin (1) and both enzymes, with predicted binding energies of -8.7 and -9.2 kcal/mol, respectively Notably, this flavanone exhibited stronger binding energy compared to cycloheximide (-8.4 and -9.1 kcal/mol) when docked with 1YBV (Figure 4.11) and 1STD (Figure 4.12), respectively

Figure 4 11 Molecular docking simulation study on the interaction of naringenin targeting 1,3,8-trihydroxynaphthalene reductase (1YBV) from M oryzae The redocking process is represented by a small image in the top left corner

Figure 4 12 Molecular docking simulation study on the interaction of naringenin targeting scytalone dehydratase (1STD) from M oryzae The redocking process is represented by a small image in the top left corner

In particular, naringenin (1) was enclosed and tightly bound within a hydrophobic pocket formed by specific residues (Val118, Ser164, Ile165, Tyr178, Gly210, Met215, Val219, Cys220, Tyr223, Trp243, and Met283) at the active site of enzyme 1YBV, providing complete protection from the external environment Naringenin (1) established hydrogen bonds with the phenolic hydroxyl group of Tyr178 and the amine group of Ile211 Pi-sulfur interactions were observed between naringenin (1) and Met215 However, no interaction was observed with certain key amino acids (Cys220 and Tyr223), which might explain the difference in potency between compound 1 and the control cycloheximide Notably, these findings indicate a resemblance to the binding pattern of the tricyclazole inhibitor (a co-ligand of 1YBV) (Andersson et al., 1996; Lundqvist et al., 1994) (Figure 4.11) (Table 4.3)

Table 4 3 Molecular docking results of naringenin (1) targeting 1,3,8- trihydroxynaphthalene reductase (1YBV) and scytalone dehydratase (1STD) from

Ile41, Tyr178 (6.51), Ile211 (4.09), Met215 , Tyr216

Lys182 (5.69), Ile211 (2.98), Tyr216, Cys220, Tyr223

Tyr178 (5.65), Met215 , Cys220 , Tyr223, Met

Tyr50 (6.23), Phe158, Asn131 (4.59), Met69, Val75, Phe158

Leu76, Val108, His110, Pro149, Ile151, Arg166, Phe162, Phe53, Val75

LE b 0.46 0.45 0.52 aBinding energy; bLigand efficiency (LE > 0.3 as potential lead compounds); c Control; dMacromolecules in complex with tricyclazole inhibitors (ID PDB: 1YBV); eMacromolecules in complex with salicylamide inhibitors (ID PDB: 1STD) The numerical value enclosed in parentheses denotes the length of the hydrogen bond (Å)

Naringenin (1) was observed to establish a hydrogen bond with Tyr50 and a pi-alkyl bond with Pro149 within the active site of enzyme 1STD These specific amino acids play a crucial role in the mechanism of enzyme inhibition (Lundqvist et al., 1994) Additionally, interactions such as pi-pi T-shaped with Phe53 and pi- alkyl with Val75 were also detected, which align with the interactions of the co- crystallized ligand in 1STD Moreover, the ligand efficiency of naringenin (1) for 1YBV and 1STD was calculated to be 0.43 and 0.46, respectively (Table 4.3), indicating that the structural framework of naringenin (1) holds promise as a potential lead molecule in the search for M oryzae fungicides Based on the observed interactions of naringenin at the active site of M oryzae enzymes, it can be inferred that it functions as a competitive inhibitor for 1YBV and 1STD (Andersson et al., 1996; Lundqvist et al., 1994; Khan et al., 2022; Thuy et al., 2021) (Figure 4.12) (Table 4.3)

Naringenin and ABA were subjected to docking analysis with the enzymes chitin synthase and calmodulin derived from P infestans The synthesis of chitin, a vital process for the survival and reproduction of organisms, is carried out by an integral membrane enzyme known as chitin synthase (Chen et al., 2022) Calmodulin, a calcium-binding protein, plays a crucial role in interacting with and modulating the activity of various proteins (Kumar et al., 2016) Inhibition of calmodulin has been associated with disease resistance in specific contexts Previous studies have indicated that inhibiting calmodulin can diminish the ability of certain bacterial and fungal pathogens to infect plants, making calmodulin inhibitors potential candidates for developing novel strategies to control plant diseases (Steinbach et al., 2007; Li et al., 2022; Ceballos Garzon et al., 2020; Juvvadi et al., 2017) Additionally, recent research has revealed that calmodulin can also interact with DNA and function as a transcription factor However, the three-dimensional structures of chitin synthase and calmodulin in P infestans have not yet been determined To address this limitation, the Swiss-Model tool was utilized to conduct a homology identification study for these two macromolecules Two structures with PDB codes (7JWO and 5A2H) were selected for the study All generated models exhibited a significant similarity (> 90% sequence identity) with chitin synthase (GenBank: KAF4140382.1) and calmodulin (GenBank: AAA21424.1) of P infestans available in the NCBI database (Figure S3-4)

Table 4 4 Molecular docking results of naringenin (1) and abscisic acid (2) targeting chitin synthase and calmodulin from P infestans

Arg497 (4.47), Trp539 (4.06), Lys358, Arg538, Leu493

Leu493 (5.23), Arg497 (3.96), Leu412, Trp539, Tyr433

LE b 0.26 0.23 0.25 aBinding energy; b Ligand efficiency (LE > 0.3 as potential lead compounds); c Control The numerical value enclosed in parentheses denotes the length of the hydrogen bond (Å).

Figure 4 13 Interaction of naringenin (right) and abscisic acid (left) in the chitin synthase and calmodulin binding site suggested by molecular docking studies

The docking analysis of both compounds on chitin synthase revealed their close binding to specific amino acids at the active site (Figure 4.13) The predicted binding energies were -6.9 and -7.6 kcal/mol for compound 2 and compound 1, respectively Both compounds exhibited ligand efficiency values of 0.36 (compound 2) and 0.38 (compound 1), indicating their potential as lead compounds Compound 2 formed interactions with several amino acids, including Arg497, Trp539, Lys358, Arg538, and Leu493 Similarly, compound 1 demonstrated hydrogen bonding with Trp539 and Arg538, as well as Pi-alkyl interactions with Val452 and Leu493 Trp539 is an essential amino acid involved in chitin biosynthesis inhibition, and compounds that inhibit this process have been observed to directly bind to this amino acid (Chen et al., 2022) Furthermore, compound 1 also displayed binding to Val452, an important amino acid, which could account for its lower energy compared to compound 2 In a study of chitin biosynthesis inhibition by the NikZ inhibitor, similar interactions were observed with these residues (Chen et al., 2022)

According to the research, calmodulin has been found to interact with Z- box DNA through Arg127 Consequently, compounds 1 and 2 exhibit strong binding to Arg127, thereby preventing the interaction between calmodulin and Z- box DNA (Kumar et al., 2016) This interaction inhibition has the potential to effectively control certain plant diseases Both compounds demonstrated interactions with Arg127, with predicted binding energies of -4.4 and -5.2 kcal/mol, respectively Additionally, naringenin (1) was observed to interact with Asp123 through a hydrogen bond and with Gly135 through a carbon-hydrogen bond The positive control, cycloheximide, was also docked to calmodulin and displayed binding configurations involving interactions with several amino acids, namely Arg127, Ile126, and Gly135 (Figure S3).

DISCUSSION AND CONCLUSION

Discussion

In Chinese medicinal literature, D sequax has been recognized as a medicinal plant with curative properties for various ailments Therefore, researchers have been exploring the potential agricultural applications of D sequax to offer an alternative to synthetic pesticides Previous studies have revealed the presence of diverse chemical constituents in D sequax, such as karanjin, lanceolatin-B, pongapin, 5′-methoxypongapin, kanujin, and glabra-II, as identified and characterized by Siddiqui and Zaman (1998)

In this particular study, two distinct phytochemicals, namely narignenin (1) and phytoalexin ABA (2), have been successfully identified in the D sequax plant These phytochemicals are recognized for their ability to interact with plants and induce a systemic resistance response when confronted with plant pathogens such as fungi and bacteria Naringenin, for example, has been empirically proven to yield numerous advantageous outcomes in terms of plant-microbe interactions occurring in the root habitat of tomatoes and soybeans Notably, it can stimulate the proliferation of beneficial rhizobacteria, thereby bolstering plant development and augmenting stress tolerance (An et al., 2021; Sun et al., 2022; Ishihara, 2021; Shimizu et al., 2012) Furthermore, naringenin exhibits the ability to enhance the uptake of specific minerals, such as phosphorus, by plant roots In terms of fungal infection prevention, naringenin acts through various mechanisms Firstly, it inhibits the germination of fungal spores, impedes the penetration of fungal mycelia, and indirectly triggers the production of pathogenesis-related proteins and antifungal antibiotics within the plant These responses contribute to the plant's defense against fungal phytopathogens (Ishihara, 2021) Additionally, naringenin directly suppresses the growth and development of fungal phytopathogens by disrupting fungal cell membranes, interfering with cell wall synthesis, and inhibiting fungal enzymes (An et al., 2021; Sun et al., 2022; Ishihara, 2021; Shimizu et al., 2012)

A study conducted by Jiang et al in 2021 examined the induction of the flavonoid biosynthesis pathway during the interaction between Stylosanthes and

C gloeosporioides Within this study, phloretin, naringenin, apigenin, daidzein, quercetin, kaempferol, puerarin, and formononetin were evaluated for their effects on C gloeosporioides' mycelial growth and conidial germination The results indicated that phloretin and naringenin inhibited the mycelial growth of C gloeosporioides by 30% and 20%, respectively, while the other flavonoid compounds had no significant effect (Jiang et al., 2021) Xu et al (2018) reported that naringenin exhibited a moderate inhibitory effect on the mycelial growth of

Botrytis cinerea, with an IC50 value of 502 àg/mL However, when it comes to other plant pathogens such as S sclerotiorum and F graminearum, Sun et al

(2022) observed that naringenin displayed minimal to no inhibitory efficacy Naringenin showed some degree of inhibition against Pythium aphanidermatum and P ultimum (Sun et al., 2022) On the other hand, Li et al (2022) found that naringenin, along with kaempferol, quercetin, and dihydroquercetin, did not inhibit the growth of R solani

Interestingly, in this study, naringenin derived from D sequax exhibited significant inhibitory effects against P infestans and M oryzae These findings align with previous research highlighting the antifungal properties of naringenin (An et al., 2021; Sun et al., 2022; Ishihara, 2021; Xu et al., 2018; Li et al., 2022) Moreover, our study provides novel insights by demonstrating the antifungal efficacy of naringenin against P infestans and M oryzae, presenting IC50 and IC90 values for the first time

The sensitivity of oomycetes, specifically Phytophthora species, to naringenin was apparent Sun et al (2022) previously showed inhibitory effects of naringenin (EC50 = 22.01 mg/L), liquiritigenin (EC50 = 51.43 mg/L), and hesperetin (EC50 = 30.20 mg/L) demonstrated inhibitory effects on the oomycete

P nicotianae (Sun et al., 2022) Similarly, naringenin exhibited suppression against P capsici, with an EC50 value of 50.11 mg/L In the case of soybean, naringenin and genistein had EC50 values of 180 M and 160 M, respectively, against the mycelial development of the oomycete P sojae (Rivera-Vargas et al., 1993) Additionally, Padmavati et al (1997) reported that naringenin effectively inhibited M grisea in rice plants It completely hindered spore germination at a concentration of 70 àg/mL, and when applied using paper discs impregnated with naringenin at 7 g/disc and 14 g/disc, it reduced spore germination by 9.6% and 1.73%, respectively In comparison, kaempferol, quercetin, and dihydroquercetin displayed weaker inhibition of M grisea spore germination than naringenin The antifungal effectiveness of naringenin and kaempferol was attributed to their increased lipophilicity (Padmavati et al., 1997) The present study aligns with the previous findings of Padmavati et al (1997) regarding the antifungal efficacy of naringenin against M oryzae

Siciliano et al (2015) reported a method that was developed to simultaneously determine the levels of certain hormones and phytoalexins in artificially inoculated F fujikuroi on two rice seed cultivars (cv), Selenio and

Dorella The interaction between rice and the pathogen resulted in higher production of the phytohormone ABA in the diseased rice plants of the Dorella cultivar Naringenin levels were in a range from 68 to 61 ng/g in rice cv Selenio and from 90 to 40 ng/g in rice cv Dorella (Siciliano et al., 2015) In plant physiological processes, ABA can inhibit root elongation and promote the growth of lateral roots, aiding in the plant's response to drought stress However, ABA has also been described to have detrimental effects on plant disease resistance (Li et al., 2022; Lievens et al., 2017) For instance, the suppression of ABA has been observed in tobacco infected with Ralstonia solanacearum, barley infected with

M oryzae, and tomato infected with B cinerea, thereby compromising the plants' immune responses (Lievens et al., 2017) On the other hand, ABA has been found to enhance the resistance of Linum usitatissimum to F culmorum and Arabidopsis to Alternaria brassicicola (Boba et al., 2022) It appears that ABA may have diverse functions in plant defense, depending on the infection stage and the type of phytopathogens involved Embrahim et al (2020) demonstrated that ABA isolated from the endophytic fungus F verticillioides inhibited the growth of four endophytic fungal strains, namely Aspergillus austroafricanus, A versicolor, Phoma fungicola, and A tubingensis, as well as the yeast Candida albicans, with

MIC values ranging of 0.6, 0.7, 0.4, 0.7 and 0.4 àg/mL, respectively In a drug repurposing study, Khedr et al (2018) conducted a screening of 11 compounds and identified (+)-(S)-abscisic acid as a potent antifungal inhibitor based on docking models with fungal chorismate mutase The authors also reported in vitro MIC values of (+) (S)-abscisic acid against human pathogens C albicans, C parapsilosis, A niger, and T rubrum, which were 125, 62.5, 125, and 62.5 àg/mL, respectively (Khedr et al., 2018)

Consequently, this study provided evidence of the effective control of the soilborne phytopathogen P infestans by ABA, which is found in the vicinity of the root network and can infiltrate the plant through the surface of the roots It has been shown that ABA can stimulate the production of reactive oxygen species (ROS) in plants, thereby reinforcing cell walls and exerting antibacterial effects (Li et al., 2022) However, the precise mechanism underlying the antimicrobial activity of ABA remains poorly understood The findings of this study, which reveal strong evidence of antifungal potential of ABA against P infestans, present new insights into the role of this phytohormone in the control of soilborne phytopathogenic microorganisms, thereby indicating a promising avenue for further research

Performing in vitro bioassay studies on nanoformulations of D sequax extracts against phytopathogenic fungi can serve as an additional method to evaluate the potential of D sequax as a botanical fungicide The particle size analysis revealed that the nanoformulation of D sequax exhibited an average particle size of 120 nanometers, through dynamic light scattering (DLS) technique TEM analysis demonstrated that the particles had a size of 50 nm and were evenly distributed throughout the dispersion medium, indicating a high level of stability

The presence of compounds 1 and 2 in the extract of D sequax further enhances its capacity to effectively inhibit phytopathogenic fungi

In the review conducted by Worrall et al (2018), it was explained that nanoparticles can be utilized for plant protection through two distinct mechanisms: nanoparticles themselves acting as agents for crop protection, or nanoparticles serving as carriers for existing pesticides or other active substances, such as double-stranded RNA (dsRNA) These nanoparticles can be applied through spray application or by drenching/soaking onto seeds, foliar tissue, or roots When used as carriers, nanoparticles have various benefits, including increased active ingredient shelf life, better solubility of pesticides that are poorly soluble in water, decreased toxicity, and greater targeted absorption into the designated pest and disease species (Hayles et al., 2017) Furthermore, the utilization of nanocarriers can also enhance the efficacy, stability, and endurance of nanopesticides when subjected to environmental factors such as UV radiation and rain This results in a significant reduction in the number of required applications, thereby lowering toxicity levels and reducing overall costs

To address the issues associated with synthetic fungicides, which leave toxic residues in plants and pose risks to humans and the environment, there has been a growing focus on exploring eco-friendly fungicides derived from natural sources Recent research conducted by Bui et al (2021) has investigated the antifungal activity of curcumin-removed turmeric oleoresin (CRTO) nanoemulsion for controlling anthracnose caused by C gloeosporioides in lychee The study encompassed both in vitro and in vivo experiments The results of the study demonstrated that CRTO nanoemulsion effectively inhibited the growth of various Colletotrichum species In the in vitro inhibition test, the nano-formulation of CRTO exhibited an equivalent half-maximal inhibitory concentration of 0.11 mg/mL This concentration was notably lower than the IC50 values observed for CRTO alone (0.33 mg/mL) and a mixture of curcuminoids (0.48 mg/mL), showcasing the enhanced effectiveness of the nano-formulation Moreover, during the field trial, the nano-formulation of CRTO proved to be highly efficient in controlling litchi anthracnose infections These findings highlight the potential of CRTO nanoemulsion as an effective solution for combating fungal diseases, particularly anthracnose, in litchi crops By utilizing eco-friendly formulations derived from natural resources, such as the CRTO nanoemulsion, it is possible to overcome the drawbacks associated with synthetic fungicides This approach offers a promising alternative that not only ensures effective control of plant pathogens but also minimizes the negative impacts on human health and the environment

In our study, the nano-formulation of D sequax demonstrated efficacy against a range of phytopathogenic fungi, with a notable impact observed against

Conclusion

The botanical extract derived from D sequax has been empirically confirmed to contain both naringenin and ABA These constituents have demonstrated substantial antifungal efficacy against phytopathogenic fungi The outcomes of the investigation provide novel substantiation of their fungicidal attributes in relation to P infestans and M oryzae It was worth noting that both naringenin (1) and ABA (2) exhibited considerable inhibitory effects on P infestans in a dose-dependent manner This study held particular significance as it marked the inaugural documentation of ABA's capability to impede the growth of the soilborne oomycete P infestans Moreover, naringenin exhibited moderate inhibition of M oryzae Additionally, a nanoformulation derived from D sequax extract achieved an average droplet size of 120 nm This formulation demonstrated antifungal effectiveness against diverse phytopathogenic fungi, including M oryzae, S rolfsii, F oxysporum, C capsici, and R solani Through the utilization of computational models, we elucidated the interactions between ABA, naringenin, and the binding sites of enzymes found in P infestans and M oryzae Our result of ligand-receptor complex models offers valuable insights into the precise mechanisms of atomic-level interactions This comprehensive analysis provided an insight on the antifungal properties and interaction mechanisms of naringenin (a phytoalexin) and ABA (a phytohormone) within the defense response of host plants The robust antifungal attributes of naringenin and ABA, alongside the nanoformulation derived from D sequax, underscore their potential as environmentally friendly fungicide for the management of fungal diseases in the realm of sustainable agriculture

The present thesis is encompassed within the paper titled:

Insight into the role of phytoalexin naringenin and phytohormone abscisic acid in defense against phytopathogens Phytophthora infestans and Magnaporthe oryzae : in vitro and in silico approaches

Corresponding Author: Assoc Prof Quang Le Dang

Co-Authors: Hieu Nguyen-Ngoc, Dr.; Cuong Quoc Nguyen, B.S.; Kieu Anh Thi

Vo, Master; Thu Trang Thi Nguyen, Dr.; Duc Trong Nghiem, Master; Nguyen Thi

Ha, B.S.; Van Minh Nguyen, Dr.; Gyung Ja Choi, Dr.; Ahmad Ghozali Ardiansyah, B.S.; Cong Thanh Nguyen, Dr.; Hoang Dinh Vu, Dr.; Ngoc Tuan Nguyen, Dr.; Quang De Tran, Dr

The paper has been published in the Journal of Physiological and Molecular Plant Pathology

Date of published: August 17 th , 2023

Agrawal, S., & Rathore, P (2014) Nanotechnology pros and cons to agriculture: a review Int J Curr Microbiol App Sci, 3(3), 43-55 http://dx.doi.org/10.13140/2.1.1648.1926

Ahluwalia, V K., Sachdev, G P., & Seshadri, T R (1966) Chemical Investigation of Leaves of Ougenia Dalbergoides Indian Journal of Chemistry, 4(5), 250-+

An, J., Kim, S H., Bahk, S., Vuong, U T., Nguyen, N T., Do, H L., & Chung, W S (2021) Naringenin induces pathogen resistance against Pseudomonas syringae through the activation of NPR1 in Arabidopsis Frontiers in Plant Science, 12,

Andersson, A., Jordan, D., Schneider, G., & Lindqvist, Y (1996) Crystal structure of the ternary complex of 1, 3, 8-trihydroxynaphthalene reductase from Magnaporthe grisea with NADPH and an active-site inhibitor Structure, 4(10), 1161-1170 https://doi.org/10.1016/S0969-2126(96)00124-4

APS Education Center Common and Trade Fungicides (2016) Retrieved from: https://www.apsnet.org/edcenter/disimpactmngmnt/topc/Documents/CommonAnd TradeFungicides.pdf (accessed on 30/04/2023)

Arora, S (2019) Regulation of pesticides and associated risks In Pesticide risk assessment (pp 290-317) Wallingford UK: CAB International https://doi.org/10.1079/9781780646336.0290

Beagle, R (2008) Pioneering women in plant pathology Pioneering women in plant pathology (http://www.shopapspress.org/piwoinplpa.html)

Bentley S, Pegg KG, Dale JL, 1995 Genetic variation among a world-wide collection of isolates of Fusarium oxysporum f.sp cubense analysed by RAPD-PCR fingerprinting Mycological Research, 99(11):1378-1384; 27 ref https://doi.org/10.1016/S0953-7562(09)81225-2

Berkeley, M J (1948) Front Matter In Observations, Botanical and Physiological, on the Potato Murrain (pp 1-12) The American Phytopathological Society https://doi.org/10.1094/9780890545232.001

Bhandari, S., Yadav, P K., & Sarhan, A (2021) Botanical Fungicides; Current Status, Fungicidal Properties and Challenges for Wide Scale Adoption: A Review Reviews in Food and Agriculture, 2, 63-68 https://doi.org/10.26480/rfna.02.2021.63.68

Bhat, R., Khajuria, M., & Mansotra, D K (2019) A systematic review on global environmental risks associated with pesticide application in agriculture Contaminants in Agriculture and Environment: Health Risks and

Remediation, 1, 96 https://www.aesacademy.org/books/cae-vol-1/08.pdf

Boba, A., Kostyn, K., Kochneva, Y., Wojtasik, W., Mierziak, J., Prescha, A., & Kulma,

A (2022) Abscisic Acid—Defensive Player in Flax Response to Fusarium culmorum Infection Molecules, 27(9), 2833 https://doi.org/10.3390/molecules27092833

Boddy, L (2016) Pathogens of autotrophs In The fungi (pp 245-292) Academic press https://doi.org/10.1016/B978-0-12-382034-1.00008-6

Botta, B., Gacs-Baitz, E., Vinciguerra, V., & Delle Monache, G (2003) Three isoflavanones with cannabinoid-like moieties from Desmodium canum Phytochemistry, 64(2), 599-602 https://doi.org/10.1016/S0031- 9422(03)00201-2

Bruhn, J G., & Bruhn, C (1973) Alkaloids and ethnobotany of Mexican peyote cacti and related species Economic botany, 27(2), 241-251 https://doi.org/10.1007/BF02872994

Bui, V C., Le, T T., Nguyen, T H., Pham, N T., Vu, H D., Nguyen, X C., & Lam,

T D (2021) Curcumin-removed turmeric oleoresin nano-emulsion as a novel botanical fungicide to control anthracnose (Colletotrichum gloeosporioides) in litchi Green Processing and Synthesis, 10(1), 729-741 https://doi.org/10.1515/gps-2021-0071

Cahill, W M., & Jackson, R W (1938) The proof of synthesis and the configurational relationships of abrine Journal of Biological Chemistry, 126(1), 29-36 https://doi.org/10.1016/S0021-9258(18)73890-8

Ceballos Garzon, A., Amado, D., Robert, E., Parra Giraldo, C M., & Le Pape, P (2020) Impact of calmodulin inhibition by fluphenazine on susceptibility, biofilm formation and pathogenicity of caspofungin-resistant Candida glabrata Journal of

Antimicrobial Chemotherapy, 75(5), 1187-1193 https://doi.org/10.1093/jac/dkz565

Chen, K., Li, G J., Bressan, R A., Song, C P., Zhu, J K., & Zhao, Y (2020) Abscisic acid dynamics, signaling, and functions in plants Journal of integrative plant biology, 62(1), 25-54 https://doi.org/10.1111/jipb.12899

Chen, W., Cao, P., Liu, Y., Yu, A., Wang, D., Chen, L., & Yang, Q (2022) Structural basis for directional chitin biosynthesis Nature, 610(7931), 402-408 https://doi.org/10.1038/s41586-022-05244-5

Chen, Y J., Liu, H., Zhang, S Y., Li, H., Ma, K Y., Liu, Y Q., & Tang, C (2021) Design, synthesis, and antifungal evaluation of cryptolepine derivatives against phytopathogenic fungi Journal of Agricultural and Food Chemistry, 69(4), 1259-

1271 https://doi.org/10.1021/acs.jafc.0c06480

Cordenonsi, L M., Sponchiado, R M., Campanharo, S C., Garcia, C V., Raffin, R P.,

& Schapoval, E E S (2017) Study of flavonoids presente in Pomelo (Citrus máxima) by DSC, UV-VIS, IR, 1H and 13C NMR and MS Drug Analytical

Research Porto Alegre Vol 1, n 1 (2017), p 31-37 http://hdl.handle.net/10183/196208

Cornforth, J W., Milborrow, B V., Ryback, G., & Wareing, P F (1965) Chemistry and physiology of ‘dormins’ in sycamore: Identity of sycamore ‘dormin’with abscisin

II Nature, 205, 1269-1270 https://doi.org/10.1038/2051269b0

Couzigou, J M., André, O., Guillotin, B., Alexandre, M., & Combier, J P (2016) Use of microRNA-encoded peptide miPEP172c to stimulate nodulation in soybean New Phytologist, 211(2), 379-381 https://doi.org/10.1111/nph.13991 Cowan, M M (1999) Plant products as antimicrobial agents Clinical microbiology reviews, 12(4), 564-582 https://doi.org/10.1128/cmr.12.4.564

Dagostin, S., Schọrer, H J., Pertot, I., & Tamm, L (2011) Are there alternatives to copper for controlling grapevine downy mildew in organic viticulture? Crop

Protection, 30(7), 776-788 https://doi.org/10.1016/j.cropro.2011.02.031

Daly, A.M and Walduck, G (2006) Fusarium Wilt of Bananas (Panama Disease)

(Fusarium oxysporum f sp cubense) Agnote(151):5 Retrieved from https://www.musalit.org/seeMore.php?id531 accessed in May 10, 2023 Dang, Q L (2014) Thuốc trừ sâu nguồn gốc Thảo mộc, Thông tin kinh tế và công nghệ- công nghiệp hóa chất: Chuyên đề phục vụ lãnh đạo, Số 3

Dang, Q L., Kim, W K., Nguyen, C M., Choi, Y H., Choi, G J., Jang, K S., & Kim,

J C (2011) Nematicidal and antifungal activities of annonaceous acetogenins from

Annona squamosa against various plant pathogens Journal of agricultural and food chemistry, 59(20), 11160-11167 https://doi.org/10.1021/jf203017f

Dang, Q L., Lim, C H., & Kim, J C (2012) Current status of botanical pesticides for crop protection Research in Plant Disease, 18(3), 175-185 https://doi.org/10.5423/RPD.2012.18.3.175

De Bary, A (1876) Researches into the nature of the potato fungus, Phytophthora infestans J Bot Paris, 14, 105-126

Dean, R., Van Kan, J A L., Pretorius, Z A., Hammond-Kosack, K E., Di Pietro, A., Spanu, P D., & Foster, G D (2012) The Top 10 fungal pathogens in molecular plant pathology Molecular plant pathology, 13(4), 414-430 https://doi.org/10.1111/j.1364-3703.2011.00783.x

Deresa, E M., & Diriba, T F (2023) Phytochemicals as alternative fungicides for controlling plant diseases: A comprehensive review of their efficacy, commercial representatives, advantages, challenges for adoption, and possible solutions Heliyon https://doi.org/10.1016%2Fj.heliyon.2023.e13810

Desmodium sequax Wall in GBIF Secretariat (2022) GBIF Backbone Taxonomy

Checklist dataset https://doi.org/10.15468/39omei accessed via GBIF.org on 2023- 07-05

Dita, M., Barquero, M., Heck, D., Mizubuti, E S., & Staver, C P (2018) Fusarium wilt of banana: current knowledge on epidemiology and research needs toward sustainable disease management Frontiers in plant science, 9, 1468 https://doi.org/10.3389/fpls.2018.01468

Do, H T T., Nguyen, T H., Nghiem, T D., Nguyen, H T., Choi, G J., Ho, C T., & Le Dang, Q (2021) Phytochemical constituents and extracts of the roots of Scutellaria baicalensis exhibit in vitro and in vivo control efficacy against various phytopathogenic microorganisms South African Journal of Botany, 142, 1-11 https://doi.org/10.1016/j.sajb.2021.05.034

Do, T H T., Pham, T H., Pham, G V., Vo, K A., Nguyen, T T T., Vu, D H., & Le Dang, Q (2022) Potential use of extracts and active constituent from Desmodium sequax to control fungal plant diseases International Journal of Agricultural

Technology Vol 18(2):489-502 Retrieved from http://www.ijat- aatsea.com/pdf/v18_n2_2022_March/6_IJAT_18(2)_2022_Do,%20T.%20H.%20 T.(144).pdf

Ebbole, D J (2007) Magnaporthe as a model for understanding host-pathogen interactions Annu Rev Phytopathol., 45, 437-456 https://doi.org/10.1146/annurev.phyto.45.062806.094346

EFloras (2010) Flora of China Vol 10 Page 268, 278 Retrieved from http://www.efloras.org/florataxon.aspx?flora_id=2&taxon_id$2317606 (accessed on 05/07/2023)

El-Wakeil, N E (2013) Botanical pesticides and their mode of action Gesunde

Farr, D F., Rossman, A Y., Palm, M E., and McCray, E B (2016) Fungal Databases, Systematic Botany and Mycology Laboratory, ARS, USDA Available online at: http://nt.arsgrin.gov/fungaldatabases/

Fauzi, M T., & Sari, R D (2022) Effectiveness of several plant extracts as botanical fungicides to control damping-off disease caused by Sclerotium rolfsii on soybean

In IOP Conference Series: Earth and Environmental Science (Vol 1107, No 1, p 012035) IOP Publishing https://doi.org/10.1088/1755- 1315%2F1107%2F1%2F012035

Finckh, M R., Tamm, L., & Bruns, C (2015) Chapter 5.1: Organic Potato Disease Management, In: Plant Diseases and Their Management in Organic Agriculture, M.R Finckh, A.H.C Van Bruggen, and L Tamm, Editors 2015, The American Phytopathological Society: St Paul p 239-257 https://doi.org/10.1094/9780890544785.019

Gao, J., Wenderoth, M., Doppler, M., Schuhmacher, R., Marko, D., & Fischer, R (2022) Fungal melanin biosynthesis pathway as source for fungal toxins MBio, 13(3), e00219-22 https://doi.org/10.1128/mbio.00219-22

Gao, X M., Wang, H., Li, Y K., Ji, B K., Xia, C F., Yang, J X., & Hu, Q F (2015) Cytotoxic Chalcones from Desmodium oxyphyllum Journal of the Brazilian Chemical Society, 26, 736-740 https://doi.org/10.5935/0103-5053.20150034

Garcia, D., Ramos, A J., Sanchis, V., & Marín, S (2012) Effect of Equisetum arvense and Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus flavus and Fusarium verticillioides in maize seeds as affected by water activity International Journal of Food Microbiology, 153(1-2), 21-27 https://doi.org/10.1016/j.ijfoodmicro.2011.10.010

Gerwick, B C., & Sparks, T C (2014) Natural products for pest control: an analysis of their role, value and future Pest Management Science, 70(8), 1169-1185 https://doi.org/10.1002/ps.3744

Goudjil, M B., Ladjel, S., Zighmi, S., Hammoya, F., Bensaci, M B., Mehani, M., & Bencheikh, S (2016) Bioactivity of Laurus Nobilis and Mentha Piperita essential oils on some phytopathogenic fungi (in vitro assay) J Mater Environ Sci, 7(12), 4525-4533 http://www.jmaterenvironsci.com/Document/vol7/vol7_N12/478- JMES-2397-Goudjil.pdf

Guo, L., Yang, L., Liang, C., Wang, G., Dai, Q., and Huang, J (2015) Differential colonization patterns of bananas (Musa spp.) by physiological race 1 and race 4 isolates of Fusarium oxysporum f sp cubense J Phytopathol 163, 807–817 doi: 10.1111/jph.12378

Hashem, M., Alamri, S A., Alrumman, S A., & Moustafa, M F (2016) Suppression of phytopathogenic fungi by plant extract of some weeds and the possible mode of action Br Microbiol Res J, 15, 1-13 http://dx.doi.org/10.9734/BMRJ/2016/26629

Hayles, J., Johnson, L., Worthley, C., & Losic, D (2017) Nanopesticides: a review of current research and perspectives New pesticides and soil sensors, 193-225 https://doi.org/10.1016/B978-0-12-804299-1.00006-0

Hazafa, A., Murad, M., Masood, M U., Bilal, S., Khan, M N., Farooq, Q., & Naeem,

M (2021) Nano-biopesticides as an emerging technology for pest management

In Insecticides IntechOpen DOI: 10.5772/intechopen.101285

Hossain, R., Quispe, C., Herrera-Bravo, J., Islam, M S., Sarkar, C., Islam, M T., & Cho, W C (2021) Lasia spinosa chemical composition and therapeutic potential: a literature-based review Oxidative Medicine and Cellular Longevity, 2021 https://doi.org/10.1155%2F2021%2F1602437

Houghton, P J., Woldemariam, T Z., Khan, A I., Burke, A., & Mahmood, N (1994) Antiviral activity of natural and semi-synthetic chromone alkaloids Antiviral research, 25(3-4), 235-244 https://doi.org/10.1016/0166-3542(94)90006-X

Ilondu, E M (2013) Phytochemical composition and efficacy of ethanolic leaf extracts of some Vernonia species against two phytopathogenic fungi Journal of

Biopesticides, 6(2), 165 http://www.jbiopest.com/users/lw8/efiles/vol_6_2_165-

Ishihara, A (2021) Defense mechanisms involving secondary metabolism in the grass family Journal of pesticide science, 46(4), 382-392 https://doi.org/10.1584/jpestics.J21-05

Isman, M B (2020) Commercial development of plant essential oils and their constituents as active ingredients in bioinsecticides Phytochemistry reviews, 19, 235-

Jewsakun, S (1978) Serology, seed transmission on anthracnose disease of pepper and effect of foliar fungicides to the causal pathogens Kasetsart University

Jiang, L., Wu, P., Yang, L., Liu, C., Guo, P., Wang, H., & Luo, L (2021) Transcriptomics and metabolomics reveal the induction of flavonoid biosynthesis pathway in the interaction of Stylosanthes-Colletotrichum gloeosporioides Genomics, 113(4), 2702-2716 https://doi.org/10.1016/j.ygeno.2021.06.004

Juvvadi, P R., Lee, S C., Heitman, J., & Steinbach, W J (2017) Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach Virulence, 8(2), 186-197 https://doi.org/10.1080/21505594.2016.1201250

Kashyap, P L., & Dhiman, J S (2010) Eco-friendly strategies to suppress the development of Alternaria blight and black rot of cauliflower Academic Journal of

Plant Sciences, 3(4), 140-146 Retrieved from https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doie703ef347d a6a47808951d15e98b872f31c5a3

Khan, M A., Al Mamun Khan, M A., Mahfuz, A M U B., Sanjana, J M., Ahsan, A., Gupta, D R., & Islam, T (2022) Highly potent natural fungicides identified in silico against the cereal killer fungus Magnaporthe oryzae Scientific Reports, 12(1), 20232 https://doi.org/10.1038/s41598-022-22217-w

Khedr, M A., Massarotti, A., & Mohamed, M E (2018) Rational discovery of (+)(S) abscisic acid as a potential antifungal agent: a repurposing approach Scientific

Konno, C., Shirasaka, M., & Hikino, H (1979) Cardioactive principle of Aconitum carmichaeli Roots1 Planta medica, 35(02), 150-155 Doi: 10.1055/s-0028-

Kumar, S., Mazumder, M., Gupta, N., Chattopadhyay, S., & Gourinath, S (2016) Crystal structure of Arabidopsis thaliana calmodulin7 and insight into its mode of DNA binding FEBS letters, 590(17), 3029-3039 https://doi.org/10.1002/1873- 3468.12349

Lattanzio, V., Cardinali, A., & Palmieri, S (1994) The role of phenolics in the postharvest physiology of fruits and vegetables: Browning reaction and fungal diseases Italian Journal of food science, 6, 3-3

Le Dang, Q., Do, H T T., Choi, G J., Van Nguyen, M., Vu, H D., Pham, G V., & Nguyen, T T T (2022) In vitro and in vivo antimicrobial activities of extracts and constituents derived from Desmodium styracifolium (Osb.) Merr against various phytopathogenic fungi and bacteria Industrial Crops and Products, 188, 115521 https://doi.org/10.1016/j.indcrop.2022.115521

Lechenet, M., Dessaint, F., Py, G., Makowski, D., & Munier-Jolain, N (2017) Reducing pesticide use while preserving crop productivity and profitability on arable farms Nature plants, 3(3), 1-6 https://doi.org/10.1038/nplants.2017.8

Li, C., Chen, S., Zuo, C., Sun, Q., Ye, Q., Yi, G., & Huang, B (2011) The use of GFP- transformed isolates to study infection of banana with Fusarium oxysporum f sp cubense race 4 European Journal of Plant Pathology, 131, 327-340 https://doi.org/10.1007/s10658-011-9811-5

Li, C., Yang, J., Li, W., Sun, J., & Peng, M (2017) Direct root penetration and rhizome vascular colonization by Fusarium oxysporum f sp cubense are the key steps in the successful infection of Brazil Cavendish Plant Disease, 101(12), 2073-2078 https://doi.org/10.1094/PDIS-04-17-0467-RE

Li, G., Liu, S., Wu, L., Wang, X., Cuan, R., Zheng, Y., & Yuan, Y (2022) Characterization and functional analysis of a new Calcium/Calmodulin-dependent Protein Kinase (CaMK1) in the citrus pathogenic fungus Penicillium italicum Journal of Fungi, 8(7), 667 https://doi.org/10.3390/jof8070667

Li, S., Liu, S., Zhang, Q., Cui, M., Zhao, M., Li, N., & Wang, L (2022) The interaction of ABA and ROS in plant growth and stress resistances Frontiers in Plant

Science, 13 https://doi.org/10.3389%2Ffpls.2022.1050132

Lievens, L., Pollier, J., Goossens, A., Beyaert, R., & Staal, J (2017) Abscisic acid as pathogen effector and immune regulator Frontiers in plant science, 8, 587 https://doi.org/10.3389/fpls.2017.00587

Lợi, Đ T (2006) Những cây thuốc và vị thuốc Việt Nam Retrieved from http://192.168.1.9:8080/dspace/handle/123456789/351553

Lorenz, E S (2009) Potential health effects of pesticides Ag Communications and

Lundqvist, T., Rice, J., Hodge, C N., Basarab, G S., Pierce, J., & Lindqvist, Y (1994) Crystal structure of scytalone dehydratase—a disease determinant of the rice pathogen, Magnaporthe grisea Structure, 2(10), 937-944 https://doi.org/10.1016/s0969-2126(94)00095-6

Ma, X., Zheng, C., Hu, C., Rahman, K., & Qin, L (2011) The genus Desmodium

(Fabaceae)-traditional uses in Chinese medicine, phytochemistry and pharmacology Journal of ethnopharmacology, 138(2), 314-332 https://doi.org/10.1016/j.jep.2011.09.053

Magielse, J., Arcoraci, T., Breynaert, A., van Dooren, I., Kanyanga, C., Fransen, E., & Hermans, N (2013) Antihepatotoxic activity of a quantified Desmodium adscendens decoction and D-pinitol against chemically-induced liver damage in rats Journal of ethnopharmacology, 146(1), 250-256 https://doi.org/10.1016/j.jep.2012.12.039

Manandhar JB, Hartman GL, Wang TC (1995) Anthracnose development on pepper fruits inoculated with Colletotrichum gloeosporioides Plant Dis 79:380–383 https://doi.org/10.1094/PD-79-0380

Mendoza, L., Wilkens, M., & Urzua, A (1997) Antimicrobial study of the resinous exudates and of diterpenoids and flavonoids isolated from some Chilean Pseudognaphalium (Asteraceae) Journal of ethnopharmacology, 58(2), 85-88 https://doi.org/10.1016/S0378-8741(97)00084-6

Mihai, A L., & Popa, M E (2015) In vitro activity of natural antimicrobial compounds against Aspergillus strains Agriculture and Agricultural Science Procedia, 6, 585-

Moriyasu, M., Ichimaru, M., Nishiyama, Y., Kato, A., Wang, J., Zhang, H., & Lu, G B (1997) (R)-(+)-Isotembetarine, a quaternary alkaloid from Zanthoxylum nitidium Journal of Natural Products, 60(3), 299-301 https://doi.org/10.1021/np960420v

Motoyama, T., & Yamaguchi, I (2003) Fungicides, melanin biosynthesis inhibitors Encyclopedia of agrochemicals https://doi.org/10.1002/047126363X.agr102

Ngo, M T., Han, J W., Yoon, S., Bae, S., Kim, S Y., Kim, H., & Choi, G J (2019) Discovery of new triterpenoid saponins isolated from Maesa japonica with antifungal activity against rice blast fungus Magnaporthe oryzae Journal of agricultural and food chemistry, 67(27), 7706-7715 https://doi.org/10.1021/acs.jafc.9b02236

Nguyen Ngoc, H., Nguyen, T T T., Vo, T K A., Nghiem, D T., Nguyen, H T., Tran,

D L., Le., D Q (2022) Study on Chemical Constituents from the Plant

Desmodium sequax Wall, Journal of Medicinal Materials 27, 131–135

Nguyen, M V., Han, J W., Le Dang, Q., Ryu, S M., Lee, D., Kim, H., & Choi, G J (2021) Clerodane diterpenoids identified from Polyalthia longifolia showing antifungal activity against plant pathogens Journal of agricultural and food chemistry, 69(36), 10527-10535 https://doi.org/10.1021/acs.jafc.1c02200

Nguyen-Ngoc, H., Nguyen-Thi-Thu, T., Vo-Thi, K A., Nguyen-Huu, T., Tran-Dai, L.,

Vu, H D., & Le Dang, Q (2023) Chemical constituents of Desmodium triflorum and their antifungal activity against various phytopathogenic fungi Zeitschrift für

Naturforschung C, 78(5-6), 179-187 https://doi.org/10.1515/znc-2022-0048

Nieder, R., Benbi, D K., & Reichl, F X (2018) Health risks associated with pesticides in soils Soil components and human health, 503-573 https://doi.org/10.1007/978- 94-024-1222-2_10

Ohkuma, K., Lyon, J L., Addicott, F T., & Smith, O E (1963) Abscisin II, an abscission-accelerating substance from young cotton fruit Science, 142(3599), 1592-1593 https://doi.org/10.1126/science.142.3599.1592