Bioactive Natural Products in Plant Disease Control

Chapter 7

Bioactive Natural Products in Plant Disease Control Kimberly D. Gwinn1 University of Tennessee, Knoxville, TN, United States 1 Corresponding author: e-mail: [email protected]

Chapter Outline Introduction Commercial Microbial Products Commercial Products From Algae and Plants

229 232

References Further Reading

244 246

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INTRODUCTION The agricultural challenge of providing an adequate, safe, and nutritious food supply for all populations is currently unrealized, as globally, approximately one in seven people is chronically undernourished. In addition, the global population is increasing exponentially so this condition will worsen if new methods in all aspects of food production are not developed and implemented [1]. Protection against losses in crop production due to diseases, while reducing negative impacts on consumers, remains a key issue in meeting this global food challenge. As economies and populations expand, greater amounts of conventional pesticides are used to produce needed food, but increased use of conventional pesticides in developing countries often leads to misuse of pesticides [1]. Many of these countries are responding by developing educational outreach and Integrated Pest Management programs. Furthermore, they are investing in the development of pesticides based on natural products such as essential oils and plant extracts [1]. Interestingly, regulation and registration of pesticides and enforcement of their use may be more relaxed and take less time in developing countries and this may spur the rapid development of bioactive natural products (BNPs) as pesticides [2]. In addition, countries such as Brazil and the European Union (EU) are devising registration protocols that result in faster review times for BNPs. The EU countries face a different challenge of reducing reliance on pesticides while maintaining stable crop yields. Although sustainable use of pesticides is allowed in the EU, large numbers of Studies in Natural Products Chemistry, Vol. 56. https://doi.org/10.1016/B978-0-444-64058-1.00007-8 © 2018 Elsevier B.V. All rights reserved.

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consumers have a zero tolerance for pesticides [3]. Consumers in the United States (US) are willing to pay a premium price for organic goods and therefore organic food production and import has increased [4]. As a result, the price premium of many US organic goods has decreased. For example, in 2004, consumers paid 1.5-fold higher price for organic coffee than for conventionally produced coffee but in 2010, this increase decreased to only a 0.51-fold premium. [4]. During the same time, carrots slightly increased in price premium, and milk premiums remained constant. Demands for increased food production and safer food supplies are spurring the development of biopesticides by companies that have historically been pivotal in the development of conventional pesticides [5]. Many of these large multinational companies have partnered with or have acquired niche biopesticide companies, and investment by these firms has resulted in increased grower acceptance and ability to integrate biopesticides into farm management practices [6]. Biopesticides are often developed for crops that are grown commercially as well as in the home garden. For example, biopesticides registered in the US and Canada for control of diseases on tomato are shown in Table 7.1. Sustainability can be incorporated into crop production by expanded use of BNPs for plant protection. In both ancient and modern agriculture, BNPs derived from plants and microorganisms have been used to reduce impact of disease. The use of BNPs for plant protection is often equated with biopesticides and organic agriculture, but strict definitions of biopesticides and organic agriculture do not include all BNPs. Moreover, biopesticides are often used in combination with traditional crop protection products for effective disease control and to reduce development of resistance by the pathogen. To confound the situation further, there is no unified definition of biopesticide [7]. The US Environmental Protection Agency (EPA) defines biopesticides as pesticides derived from natural materials and categorizes them as either biochemical pesticides, containing substances that control pests by nontoxic mechanisms, microbial pesticides, consisting of microorganisms that typically produce BNPs, or plant-incorporated-protectants with activity produced by plants because of added genetic materials [8]. Biopesticides must have a mode of action that is nontoxic to the nontarget pest(s) and a demonstrate minimal toxicity to humans and the environment [9]. Between 1997 and 2010, a majority of pesticide registrations were based on biological systems: natural products (36%), synthetics derived from natural products (6%), and biological control agents (27%) [10]. In this review, discussion will be limited to commercially available microbial- or botanical-derived BNPs that act as antimicrobial agents and/or as natural inducers of the host defense system, and the term, biopesticide, will be used only in reference to BNPs that have met registration requirements of the EPA. Allowances for the use of specific BNPs in organic agriculture systems will be addressed. Genetic modification of plants to produce or overexpress BNP has been recently reviewed [11] and will not be discussed in this review.

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Bioactive Natural Products in Plant Disease Control Chapter

TABLE 7.1 Biopesticide Products Registereda for Control of Diseases of Tomato in the United States Trade Name

Company

Organic

Active Ingredient(s)

Endorse Wettable Powder Fungicide

Arysta LifeScience

No

Polyoxin D zinc salt

FireLine

AgroSource, Inc.

No

Oxytetracycline hydrochloride

FireWall

AgroSource, Inc.

No

Streptomycin sulfate

KeyPlex 350

KeyPlex

No

Brewer’s yeast extract hydrolysate, Saccharomyces cerevisiae

Messenger STS

Plant Health Care, Inc.

No

Harpin protein

OSO 5% SC

Certis USA

No

Polyoxin D zinc salt

Actigard 50 WG

Syngenta

No

Acibenzolar-S-methyl

Agricolle

Biopol Natural

No

Seaweed extract

BacStop

USAgriTech, (Anjon AG)

Yes

Thyme, clove, cinnamon, peppermint, garlic oils

Cinnacure 30%

ProGuard

No

Cinnamaldehyde

Dazitol Concentrate

Natural Forces Avian Control

No

Capsaicin, related capsaicinoids, allyisothiocyanate

Dominus

Isagro

No

Allyl isothiocyanate

Eco E-Rase

IJO Products

Yes

Jojoba oil

EF400

USAgriTech, (Anjon AG)

Yes

Clove, rosemary, and peppermint

FungaStop

Soil Technologies

No

Citric acid, ascorbic acid, mint oil, etc.

Nemitol

Natural Forces Avian Control

No

Capsaicin and related capsaicinoids

Regalia

Marrone BioInnovations

Yes

Reynoutria sachalinensis

Microbial Products

Botanical Products

Continued

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TABLE 7.1 Biopesticide Products Registered for Control of Diseases of Tomato in the United States—Cont’d Trade Name

Company

Organic

Active Ingredient(s)

Sporatec

Brandt

Yes

Rosemary, thyme, and clove oils

Trilogy

Certis USA

Yes

Neem oil, clarified hydrophobic

Fractureb

FMC

No

Banda de Lupinus alba doce (BLAD)

Head’s Up

Bayer

Yes

Saponins of Chenopodium quinoa seed

a

Registration information from https://www.epa.gov and www.hc-sc.gc.ca. Only acibenzolar-S-methyl, seaweed extract, azadirachtin, and clove oil are listed as approved in the EU Pesticides database (http://ec.europa.eu/food/plant/pesticides/eu-pesticides-database-redirect/index_en.htm). Thyme oil and Reynoutria sachalinensis extract have pending approval status. b Fracture and Head’s Up are not listed in the IR4 database but registered for use on tomatoes in the United States and Canada. Data retrieved from the IR-4 Biopesticide Database unless otherwise noted. Products in bold are also registered in Canada. Products in italics are not found in the IR4 database but registered for use on tomato in the United States.

COMMERCIAL MICROBIAL PRODUCTS Actinobacteria, particularly those in the genus Streptomycetes, produce the majority of naturally occurring antibiotics (Fig. 7.1) [12] (chemical structures used in this document were prepared using ChemDraw 15.1). Most of these antibiotics are protein synthesis inhibitors but some disrupt cell membrane integrity or cell wall biosynthesis. Still other antibiotics induce plant host defenses (Table 7.2 and references, therein). Some BNPs have multiple sites of action; for example, ningnanmycin interferes with the assembly of the coat protein of tobacco mosaic virus [25] and can induce resistance of the plant to the pathogen [26]. Because many of these compounds or closely related ones are used in the management of human health, use of these same antibiotics in agriculture can be controversial. Use of antibiotics for plant protection is less than 1% of all US agricultural uses of the compounds [16]. In some countries (e.g., US, EU, and Canada), use of antibiotics for plant disease control in organic agriculture is prohibited. Antibiotic resistance may be slow to develop, as in the case of fire blight where there is little resistance to streptomycin after years of careful application [16,27,28], or it may be rapid, as in the case of Xanthomonas perforans where resistance to kasugamycin developed within one season [29]. Pimaricin is used as a preservative in cheeses and sausages because of its relative lack of toxicity to humans and low probability for development of resistance by spoilage organisms [30].

HN

H2N

H3C

Protein synthesis

NH N

H2N

H 2N

NH2 N

NH2

O

OH

H

H

OH

N H OH

N

H2N

H

O

NH2

N

O

HN

HO

O O

OH

H

(10)

H

N

O

OH

O

H

N H

NH2

N

O O

OH N

O

HNH

NH2 OH

HO

H

H2N NH

HN

HO

O O HO HO

N H

N H OH

OH HN

NH2

HN

HO

O H

O

(2)

HO

(3)

HN

O

HO

O

O

(1)

O

N

NH2

O

N

H

HN

Unknown

NH O

O

HO

O

NH OH

OH

(4) NH2

OH O HO

O

O

OH

O

O

OH OH HN

(5)

(6)

OH

Cell wall biosynthesis

OH

HO OH O

O

OH

OH

Plasma membrane

OH

OH

(9)

OH

O O

O O

O

H

HO

OH OH

H NH2

HO

O OH

HO

OH

H 2N O

O

O

O

NH

O OH

O HO H2N

HN

N

O

(8)

O

H

(7) O

OH

OH

OH

FIG. 7.1 Antibiotics produced by selected species of Streptomyces classified by their mode of action. Numbered compounds are Blasticidin (1); Mildiomycin (2); Gentamicin (3); Kasugamycin (4); Oxytetracycline (5); Streptomycin (6); Pimaricin (natamycin) (7); Polyoxin D (8); Validamycin (9); Ningnanmycin (10).

TABLE 7.2 Selected Biochemical Pesticides Isolated From Actinomycetes Used for Control of Phytopathogens Compound Actinomycete Producer(s) Blasticidin (1) Streptomyces griseochromogenes Mildiomycin (2) Streptoverticillium rimofaciens Gentamicin (3) Micromonospora purpurea Kasugamycin (4) Streptomyces kasugaensis

Oxytetracycline (5) Streptomyces rimosus

Activity (FRAC MOA Code)a

Common Target Organisms

References

Protein synthesis (D2)—inhibits peptide bond formation and peptidyl–tRNA hydrolysis through deformed conformation of tRNA

Bacteria and fungi, particularly rice blast disease caused by Magnaporthe grisea

[12–15]

Protein synthesis (D2)—blocks peptidyltransferase

Fungi. Serine derivative of blasticin active against powdery mildew diseases. Primarily used in Japan

[12, 14, 15]

Protein synthesis—inhibits binding to prokaryotic 16S rRNA and disrupts cell membrane integrity

Bacteria (particularly Gram negative (G
)). Fire blight of apple and pear—Mexico. Various bacteria diseases of vegetable crops (Mexico and Central America)

[12, 14, 16]

Protein synthesis (D3)—inhibits binding of aminoacyl-tRNA to mRNA-30S and mRNA70S

Bacteria and fungi (systemic activity). Fire Blight and several species of Pseudomonas and Xanthomonas, several fungal plant pathogens and for rice blast caused by Magnaporthe grisea

[12, 14, 17, 18]

Protein synthesis—inhibits binding of aminoacyl-tRNA to 30S and 50S ribosomal subunits

Bacteria. Several species of Erwinia, Pseudomonas, and Xanthomonas as well as mycoplasma-like organisms

[12, 14, 16, 18]

Protein synthesis—binds to prokaryotic 16S rRNA and disrupts cell membrane integrity

Bacteria (particularly G
). Fire Blight used primarily in North and South America, Israel, New Zealand. Restricted use in Germany, Austria, and Switzerland. Seed treatment approved by US-EPA

[12, 14, 16, 19]

Pimaricin (natamycin) (7)

Ergosterol-specific inhibition of membrane transport proteins

Fungi. Control various fungal diseases of plants and mushroom, but especially basal rots of ornamental bulbs caused by Fusarium oxysporum and dry bubble disease in mushrooms

[14, 20, 21]

Polyoxins (8)

Cell wall biosynthesis (H4)—blocks chitin synthesis which leads to germ tube and hyphal tip swelling

Fungi. Control of various fungal diseases including those caused by Alternaria, Rhizoctonia, and Magnaporthe grisea

[12, 14]

Cell wall biosynthesis (H4)—degradation of intracellular trehalose; may be host defense inducer

Fungi and Bacteria. Diseases caused by Rhizoctonia, Fusarium. Various bacterial diseases

[12, 22, 23]

Multisite—inhibits the coat protein assembly of virus; may be host defense inducer

Fungi and viruses. Fungal diseases of turfgrasses. Tobacco mosaic and cowpea mosaic viruses. Available in China

[24–26]

Streptomycin (6) Streptomyces griseus Multiple soil actinomycetes

S. cacaoi var. asoensis Validamycin (9) Streptomyces hygroscopicus Ningnanmycin (10) Streptomyces noursei var. xichangensisn

a Fungicide Resistance Action Committee Mode of Action Codes—Different letters are used to denote biochemical mode of action against plant pathogens. D ¼ protein synthesis; H ¼ cell wall synthesis.

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Many microorganisms produce peptides and proteins that are used in defense against other microorganisms. Several of the amino acid-derived antibiotics produced by Streptomyces (e.g., polyoxins, mildiomycin, blasticidin) are classified as pseudopeptides [12,31]). Unlike the antibiotics, few, if any, true antimicrobial peptides (AMPs) have been commercialized as crop protection products. Their limited use may possibly result from their lack of chemical and physical stability (they are easily hydrolyzed and oxidized) [31]. Many AMPs produced by microorganisms are in development for use as infection controls in human health as well as for diabetes and cancer treatments [32]. Development of resistance to AMPs is believed to be unlikely because they target membranes and degradation is difficult without the concomitant degradation of proteins of the pathogen [33]. Some AMPs (e.g., bacteriocins) are used to control foodborne pathogens and spoilage organisms [34]. Plant innate immunity against pathogens is initiated by the recognition of pathogen-associated molecular patterns or chemical elicitors that induce resistance to the pathogen [35]. These compounds can be developed into biopesticides in a purified form [e.g., some antibiotics (Table 7.2); harpin proteins] or as crude extracts (yeast extract hydroxylate) that reduce or prevent disease. These products typically have little or no direct effects on pathogen, the notable exception being the antibiotics. Harpins are glycine-rich and heat-stable proteins produced by pathogens that mostly target the extracellular space of plants [36]. The biopesticide, Harpin, produced by the fire blight pathogen, Erwinia amylovora, induces resistance and is effective against many pathogens on different hosts [14]. Treatment of tomato fruit with Harpin decreased losses due to postharvest fungal pathogens; therefore, Harpin may be an effective strategy for managing postharvest decay of tomato fruit [37]. Furthermore, Harpin can enhance plant growth (e.g., germination, accelerated flowering, or fruit set) [14]. Like Harpin, the biopesticide, Harpin ab stimulates resistant responses, enhances plant growth, and does not act directly on the pathogen [14,38]. Harpin has one active site on the protein, whereas Harpin ab has four active regions—derived from E. amylovora (HrpN and HrpW), Ralstonia solanacearum (PopA), and Pseudomonas syringae (HrpZ) [38]. These additional sites are believed to increase efficacy over a wider range of crops and rates and provide a more stable positive effect on the crop [38]. Harpin ab is registered for use as a postharvest treatment on all food commodities. Yeast extract hydroxylate is a biopesticide derived from Saccharomyces cerevisiae that also induces resistance responses [10]. It is typically combined with chelated micronutrients [39] and is not as well studied as the harpins for disease control. Strobilurin was originally isolated from the wood rotting fungus, Strobilurus tenacellus, but it is also produced by a number of related fungi. Strobilurin and its related derivatives are often referred to as QoI fungicides because in eukaryotic cells, strobilurin and its derivatives bind to the Qo site of cytochrome b, thus blocking electron flow between it and cytochrome c [39]. In

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2002, six fungicides based on strobilurin were commercially available and represented approximately 10% of the global fungicide market [40]. The Fungicide Resistance Action Committee (FRAC) currently lists 17 fungicides that are strobilurin derivatives (Table 7.3) and these remain an important part of plant disease control in many agricultural systems [41]. Pathogen resistance to these compounds develops rapidly, but resistant strains are not maintained when selection pressure is removed (i.e., fungicide is no longer used) [42].

COMMERCIAL PRODUCTS FROM ALGAE AND PLANTS Extracts from many plants, particularly those used in traditional medicine, have activity against pathogens of humans [43] and plants [2,14]. Based on a survey of traditional healers located in the Meru-Central district of Kenya, plants used as traditional biopesticides were given a high species use value leading to speculation that they may have potential for development as biopesticides. Plants identified as potential sources of biopesticides were from five plant families Laminaceae, Fabaceae, Asteraceae, Apocynaceae, and Flacourtiaceae [44]. Diversity of chemical composition varies among species and within cultivars of the same species [45], but some plant families such as the Laminaceae, Fabaceae, Brassicaceae, and the Asteraceae are well known for BNPs that are active against plant pathogens and pests. In order to facilitate development of plant-based pesticides, several factors need to be addressed [2]. First, raw material must be available and grown under conditions to induce standardized production of BNPs. Second, extraction methods must also be standardized. Ideally, the pesticide should degrade rapidly in the environment and not affect nontarget organisms. Finally, the regulatory climate should be designed to facilitate development. If these factors are met, then market demand for plant-based materials will control the development of commercially available products. As demand increases, there will be a need to identify alternative sources of plant-based BNPs. Waste from industries utilizing plant materials have been suggested as sources of BNPs [34]. Inhibition of pathogens by peels from citrus and pomegranate fruits, and pomace from grapes and olives are well documented in the literature (reviewed in [34]), but only a few products from industry wastes (e.g., oriental mustard seed meal [Brassica juncea, Family Brassicaceae [46]; saponins from Chenopodium quinoa [Family Chenopodiaceae] [47]) have been registered as biopesticides. The waste products from BNP harvest may also be used in other industries. For example, harvesting of brown algae for laminarin production is more sustainable if the extracted algal mass is also used for biofuel production. The extracted algae is less efficient for biofuel production, however, because laminarin may constitute up to 35% of the total dry weight and a larger portion of the carbohydrate is needed for biofuel production [48]. Biofuel yield from plant sources is reduced by phenolic-rich, nonstructural extractives that hinder enzyme activity and microorganism growth. Removal of extractives is cost

TABLE 7.3 Strobilurin-Based Fungicides Listed by the Fungicide Action Committee as QoI Inhibitors to Which There Is a High Risk of Pathogen Resistance Chemical Name

Common Name O

Chemical Name

Azoxystrobin Enoxastrobin Flufenoxystrobin

Kresoxim-methyl

O

Coumoxystrobin

O O– Methoxy acrylate

Common Name

HO

Trifloxystrobin

N

O–

Oximino acetate

Picoxystrobin Pyraoxystrobin

O NH2

Mandestrobin

Methoxy acetamide

O– N H Methoxy-carbamates

HO

Fenaminstrobin

N NH2

Oximino acetamide

O O

Dimoxystrobin

O

O

Pyraclostrobin Pyrametostrobin Triclopyricarb

O O N H Dihydro dioxazines

Metominostrobin Orysastrobin

Fluoxastrobin

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prohibitive unless other products are made from the fractions. Preliminary research on the bactericidal and fungicidal activity of extractives supports the development of these as biopesticidal resources ([49], author unpublished). Conifer resins are a renewable resource for BNPs and some resins contain compounds that are antimicrobial ([50], author unpublished). Plant cell cultures may represent cost effective alternatives for BNP production and have the advantage of standardization of the environment that in turn standardizes types and amounts of BNPs [51]. Similar to products derived from microorganisms, some botanical-derived products act as elicitors that induce resistance responses, while others directly influence the pathogen [35]. Salicylic acid and its synthetic analog, acibenzolarS-methyl (ASM), Giant Knotweed (Reynoutria sachalinensis, Family Polygonaceae) extract, and laminarin all induce resistance to pathogens and have little to no toxicity to targeted pathogens. Salicylic acid (Fig. 7.2), a BNP originally described in willow but found in many plants, plays the central role in systemic acquired resistance (SAR) [52]. Because of its phytotoxicity, direct use of salicylic acid in crop protection is limited, but BNP production can be elicited by salicylic acid in cell culture (reviewed in [51]). A methyl esterase converts methyl salicylic acid, proposed to be the phloem-mobile signal, to salicylic acid in distal tissues [52]. The salicylic acid analog, ASM, shown in Fig. 7.2 can be used as a preventative [10], as a seed treatment [53], in trunk injections [54], and can be effective after infection [28]. Treatment with ASM also reduced postharvest food spoilage [55]. Virus-infected plants treated with ASM had reduced virus titers and lesion size [56]. Phytotoxicity can also be caused by ASM treatment [57]. Ethanolic extracts of Giant Knotweed also induce resistance in a wide range of hosts. The extract contains at least three terpene (anthraquinone) compounds, shown in Fig. 7.3 [58,59]. Both the extract and the anthraquinone, physcion, increase defense responses of the plant [58, 60]. Commercial formulations of the extract can be applied as a foliar spray, a seed treatment, or a dip or drench. Synergism between the extract and conventional fungicides or ASM results in enhanced disease control [58]. Another elicitor of induced resistance, laminarin is a common low-molecular weight carbohydrate (oligosaccharide) found in all

S

O

O

OH

S N

OH Salicylic acid

N Acibenzolar-S-methyl

FIG. 7.2 Salicylic acid and its synthetic analog, acibenzolar-S-methyl.

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HO

OH

O

O

OH

O

Emodin

Giant Knotweed OH

O

OH

HO

O

OH

Neem

H

OH

HO O H

Azadirachtin

O HO

O

H

O

O

O H O

O

O O

Resveratrol

Physicon

O

O O

OH

Pink Plume Poppy

Essential oils

O

OH O

HO

Menthol Thymol

Chelerythrine α-terpinene O

O

γ-terpinene

Cinnamaldehyde O

Methyl eugenol

N+ O O

Sanguinarine

Cl–

O O

N+

O OH

Terpinen-4-ol

O

FIG. 7.3 Selected bioactive natural products from Giant Knotweed (Reynoutria sachlinensis); neem (Azadirachta indica); pink plume poppy (Macleaya cordata); and primary components of the essential oils from thyme (Thymus vulgaris), thymol; peppermint (Mentha  piperita), menthol; cinnamon (Cinnamon cassia), cinnamaldehyde; clove (Syzygium aromaticum), methyl eugenol; and tea tree (Melaleuca alternifolia), terpinenes and terpinen-4-ol.

edible plants. Laminarin is typically extracted from brown algae where it is stored for food reserves (up to 35% of dry weight) [48]. The terminal residues of laminarin are either glucose (G chain) or mannitol (M chain) [48, 61]. The “chains” are mixes of b-(1,3)-D-glucans with b(1,6)-branching. The ratio of G and M chains are species and environmentally dependent [48]. Laminarin is well known as a biostimulant for plant growth [48,61] but has also been approved as a biopesticide that induces the SAR response in plants [62]. Pathogen resistance to laminarin is unknown and unlikely to occur. Laminarin use is allowed in organic agriculture as a growth stimulant and is approved as fungicide that can be used in food production. Seeds from plants are versatile sources of BNPs. Seed from C. quinoa must be washed extensively to remove bitter tasting saponins and greater amounts of saponin were extracted at 60°C than 20°C [63]. The mode of action for saponins from C. quinoa in suppressing plant disease is complex and not fully understood. Unlike other botanical-derived biopesticides that induce resistance, extracted saponins have antifungal activity; however, the primary use of the saponins from C. quinoa is as a biopesticide to treat seeds or seed potatoes and induce a systemic resistance response [47]. Seedling treatment as a root or shoot dip is also a registered use of the product [47]. The US EPA considers saponins exempt from tolerance as a result, at least in part, of their low mammalian toxicity and rapid degradation in the environment. The efficacy ratings for treatment of pea root rot and tomato early

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blight were high for the commercial product containing C. quinoa saponins [64]. Oil of Chenopodium (extracted from another member of Chenopodiaceae family, Dysphania ambrosioides, formerly Chenopodium ambrosioides) is a well-known antihelminthic that protected seeds from microbial attack for up to 6 months in storage [65]. Its development as a plant protection product has been limited by its mammalian toxicity, but C. quinoa from which saponin biopesticides are derived does not contain ascaridole or carvacrol, the primary toxic components in Oil of Chenopodium. In addition to the chenopods, extracts and oils from other seed contain compounds that can be used as plant protection products. When oriental mustard seed oil is cold expressed, the remaining seed bran and meal pellet contain both the enzyme, myrosinase, and the glucosolinate, sinigrinallyl isothiocyanate. When hydrated, the enzyme and the BNP react and release allyl isothiocyanate, which can be used as a biofumigant [46]. The biopesticide is typically used to control soil-borne fungi and nematodes. The biopesticide, BLAD (Banda de Lupinus alba doce) is a protein oligomer produced in germinating seed of sweet lupine (Lupinus alba doce, Family Fabaceae) as a product of limited proteolysis of the seed storage protein, b-conglutin [64,65]. The BLAD-oligomer has both lectin binding and chitolytic (b-N-acetyl-D-glucosaminidase and chitosanase) activities and can be used as a preventative and after infection [66,67]. Concerns regarding BLAD as a biopesticide have been aimed at cross reactivity for persons with allergenic responses to legume protein and nontarget impact on honeybees, but BLAD-oligomer was not toxic to honeybees in feeding (LD50 > 109 mg/bee) or contact studies (LD50 > 100 mg/bee) [68]. In addition, in an immunoblot test, sera from persons sensitive to lupins and/or peanuts were positive reaction to lupins and/or peanuts but had no reaction to BLAD. This suggests that BLAD is not a potential allergen [68]. Oil from jojoba (Simmondsia chinensis, Family Simmondsiaceae) is used primarily on powdery mildew, and it can induce systemic resistance [69]. However, the primary mode of action is believed to be the physical blocking of oxygen uptake by the mycelium by the straight chain wax esters that comprise the oil [14]. Azadirachtin and Clarified Hydrophobic Extract of Neem Oil are two biopesticide products obtained from seed of the neem tree (Azadirachta indica, Family Meliaceae). Azadirachtin, the primary BNP associated with neem, deters insect feeding, interferes with the normal life cycle of insects, but was compatible with entomopathogenic fungi [70]. Based on published field studies, Cao et al. [64] classified biopesticides that could be used in organic production in 2013 as: (+) positive evidence for disease control, (0) no evidence for disease control, or () mixed reports with some reports positive and others not. Clarified neem extract was classified as positive (+) for control of dogwood powdery mildew, almond brown rot, and poinsettia powdery mildew. Neem oil received a mixed rating for dogwood Cercospora leaf spot, snapbean gray mold, pumpkin powdery mildew, and sweet cherry powdery mildew but had no effect on crepe myrtle Cecospora leaf spot, dogwood spot anthracnose, tomato early blight,

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grape powdery mildew, or almond scab [64]. Growth of the wood soft fungus, Chaetomium globosum, was slightly inhibited by azadirachtin, and clarified neem oil had greater antifungal activity than did crude neem oil [71] so it is not surprising that the three neem-based biofungicides identified by Cao et al. [64] as positive or mixed positive efficacy for control of plant disease in organic systems have clarified hydrophobic extract instead of the crude oil as the active ingredient. Induction of systemic resistance by oil pressed from the fruit and seeds of the neem tree has also been reported [69], but definitive studies on the relative ability of clarified neem extract, crude oil, and azadirachtin could not be found. Cottonseed oil (Gossypium hirsutum, Family Malvaceae) was rated by Cao et al. [64] as positive for control of tomato early blight, mixed for squash powdery mildew and no effect for tomato late blight or tomato bacterial spot. Antifungal activities of plant essential oils are an active area of research, and much discovery-based research is conducted to determine novel chemical combinations or to discover value-added roles for waste streams in plantbased industries. Antibiotics and essential oils often have additive or synergistic antimicrobial effects that may be used to reduce the amount of antibiotics used in plant protection [72]. Use of essential oils is typically allowed in organic agriculture (Table 7.1), and many are classified as Minimum Risk Pesticide Products by the EPA [73]. Essential oils are particularly important for treatment of postharvest diseases in fruits and vegetables [74]. They can be added directly to the products as a dip in the oil, as either an edible coatings or waxes, or part of a film. Biofumigation with essential oils allows them to be used when the flavor of the essential oil is not compatible with the produce. Essential oils that are common components in biopesticide formulations include thyme and peppermint (Thymus vulgaris and Mentha  piperita, Family Laminaceae), garlic (Allium sativum, Family Amaryllidaceae), clove (Syzygium aromaticum, Family Myrtaceae), and cinnamon (Cinnamomum zeylanicum, and Cinnamon cassia, Family Lauraceae). Usually, these oils are a complex mixture of terpenes and phenols, and chemical composition is dependent upon the genetics, age, and environment of the plant. However, each oil has typical primary compound(s). Selected primary components are shown in Fig. 7.3. The primary component of thyme oils is usually thymol, and peppermint oils typically contain menthol. The primary component in clove oil is methyl eugenol. Cinnamaldehyde is found in most cinnamon oil products and is itself a biopesticide that is generally regarded as safe and can be used in food [75]. Garlic oils contain high amounts of sulfur compounds [76]. In classification of biopesticides allowed in organic agriculture [64], products based on thyme oil had mixed results in reports on all diseases of cucurbits and were ranked as not effective for turnip greens bacterial leaf spot. Although, garlic oil was ranked not effective for tomato early blight [64], the use of garlic bulb extract as both a seed treatment and foliar spray to control Alternaria blight in oriental mustard had a higher benefit-to-cost ratio than use of conventional fungicides [77, 78]. Disease incidence of

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powdery mildew (Podosphaera xanthii) was lower in treatments that alternated clove oil and conventional fungicide than in either treatment alone [79]. Tea tree oil, the volatile essential oil derived mainly from leaves and terminal branches of Melaleuca alternifolia (Family Myrtaceae), contains primarily terpinenic alcohol and terpenes; primary components, as defined by international standard, are shown in Fig. 7.3 [80]. Tea tree oil has a number of antimicrobial properties, and as a biopesticide, it is active against a broad spectrum of plant-pathogenic fungi and viruses [79]. Antifungal and antibacterial activity of the oil are consistent with the loss of membrane integrity and function associated with hydrophobic molecules that constitute the oil. Solutions of tea tree oil inhibited powdery mildew (Blumeria graminis) of barley, and it can be used as a preventative fungicide [81]. Extracts from the vegetative portions of plants are also used as plant protection products. Giant knotweed extract is prepared from leaves of the plant. Saponins and neem extracts can be prepared from the leaves, but seeds tend to be a richer, more standardized source. The leaf extracts of pink plume poppy (Macleaya cordata, Family Papaveraceae) contain several antifungal quaternary benzophenanthridine alkaloids, shown in Fig. 7.3. These alkaloids were effective against powdery mildew of wheat caused by Erysiphe graminis and tomato gray mold caused by Botrytis cinerea [82,83]. Pesticides containing this extract are limited to use on nonfood crops [83]. Biopesticides and other BNP formulations are critical tools in meeting the agricultural challenge of providing an adequate, safe, and nutritious food supply for all populations. The partnership between large multinational companies and niche biopesticide companies has provided resources to research and develop many of these products, and to ensure that biopesticides will continue to increase in their share of the agricultural chemical market.

ABBREVIATIONS AMP BLAD BNP FRAC HrpN HrpW HrpZ popA SAR

antimicrobial peptides Banda de Lupinus alba doce bioactive natural products Fungicide Resistance Action Committee hypersensitive response and pathogenicity from Erwinia amylovora hypersensitive response and pathogenicity from Erwinia amylovora hypersensitive response and pathogenicity from Pseudomonas syringae hypersensitive response and pathogenicity from Ralstonia solanacearum systemic acquired resistance

protein (harpin) isolated protein (harpin) isolated protein (harpin) isolated protein (harpin) isolated

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FURTHER READING [1] N. Asano, T. Yamaguchi, Y. Kameda, K. Matsui, J. Antibiot. 40 (1987) 526–532. [2] S. Monteiro, R. Freitas, B. T. Rajasekhar, A. R. Teixeira, R.B. Ferreira, PLoS One 5 (2010) e8542. https://doi.org/10.1371/journal.pone.0008542 (accessed June 30, 2016).