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Assessing the efficacy and persistence of rosemary oil as a miticide/insecticide for use on greenhouse… Miresmailli, Saber 2006

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ASSESSING THE EFFICACY A N D PERSISTENCE OF R O S E M A R Y OIL AS A MITICIDE / INSECTICIDE FOR USE O N GREENHOUSE TOMATO by SABER MIRESMAILLI B.Sc, The University of Tehran, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES (Plant Science) THE UNIVERSITY OF BRITISH C O L U M B I A January 2006 © Saber Miresmailli, 2006 Abstract Eff icacy o f rosemary essential oi l was assessed against two-spot ted spider mites (Tetranychus urticae) and greenhouse whitefl ies (Trialeurodes vaporariorum) as wel l as its effects on the tomato host plant and bio-control agents. Laboratory bioassay results indicated that pure rosemary o i l and EcoT ro l ™ (a rosemary oi l-based pesticide) caused complete mortality o f spider mites and whitef l ies at concentrations that are not phytotoxic to the host plant. The predatory mite, Phytoseiuluspersimilis, is less susceptible to rosemary o i l and EcoTrol™ than twospotted spider mites both in the laboratory and the greenhouse, whereas the parasitic wasp, Encarsia formosa, is more susceptible to rosemary o i l than whitef l ies. Rosemary o i l repels both spider mites and whitef l ies and can affect oviposi t ion behavior. Rosemary o i l and rosemary oi l -based pesticides are non-persistent in the environment and their lethal and sub-lethal effects fade within one or two days. EcoTro l ™ is safe to tomato fol iage, f lowers and fruits even at double the recommended label rate. A greenhouse trial indicated that a single application o f E c o T r o l ™ at its recommended label rate could reduce a twospotted spider mite populat ion by 52%. A t that rate, EcoTrol™ did not cause any mortality among predatory mites Phytoseiulus persimilis nor d id it affect their eggs. Tox ic i ty o f indiv idual and incomplete mixtures o f constituents o f rosemary o i l to spider mites indicated signif icant synergy among the constituents. Highest mortality was only obtained when al l constituents were present in the mixture. In general, E c o T r o l ™ was found to be a suitable option for small-scale I P M programs for control l ing spider mites and whitefl ies in greenhouse tomato plants. Table of contents A B S T R A C T II T A B L E O F C O N T E N T S I V L I S T O F T A B L E S V I L I S T O F F I G U R E S V I I A C K N O W L E D G M E N T S VI I I C H A P T E R O N E : I N T R O D U C T I O N 1 1.1 MAJOR OBJECTIVES 1 1.2 HISTORICAL PERSPECTIVE 2 1.3 GREENHOUSE TOMATO 4 1.4 TWO-SPOTTED SPIDER MITE 5 1.5 GREENHOUSE WHITEFLY 8 1.6 GREENHOUSE PEST MANAGEMENT 9 1.7 PLANT ESSENTIAL OILS 10 1.8 ROSEMARY OIL 12 REFERENCES 14 C H A P T E R T W O : L E T H A L A N D S U B - L E T H A L E F F E C T S O F ROSMARINUS OFFICINALIS E S S E N T I A L O I L A N D T H R E E R O S E M A R Y O I L - B A S E D P E S T I C I D E S T O TETRANYCHUS URTICAE K O C H , TRIALEURODES VAPORARIORUMXVESTWOOD, PHYTOSEIULUS PERSIMILIS A T H I A S - H E N R I O T , A N D ENCARSIA FORMOSA G A H A N , O N LYCOPERSICON ESCULENTUM. 20 2.1 INTRODUCTION 20 2.1.1 Tetranychus urticae (Two-spotted spider mite) 20 2.1.2 Trialeurodes vaporariorum (Greenhouse whitefly) 20 2.1.3 Phytoseiulus persimilis (Predatory mite) 21 2.1.4 Encarsia formosa (Parasitic wasp) 22 2.1.5 Rosmarinus officinalis (Rosemary plant) 22 2.2 MATERIALS AND METHODS 24 2.2.1 Rosmarinus officinalis essential oil and commercial pesticides 24 2.2.2 Spider mites 24 2.2.3 Greenhouse white/lies 24 2.2.4 Phytoseiulus persimilis Athias-Henriot and Encarsia formosa Gahan 24 2.2.5 Plant material 25 2.2.6 General growing conditions for plants, mites and whiteflies 25 2.2.7 Calculating lethal concentration 50 (LC50) 25 2.2.8 Residual toxicities 29 2.2.9 Choice test bioassay for spider mites 30 2.2.10 Oviposition choice test bioassay for whiteflies 31 2.2.11 Trans-laminar activity of commercial pesticides 32 2.2.12 Data Analysis 32 2.3 RESULTS 33 2.3.1 Lethal concentration 50 ( L C 5 0 , ) 33 2.3.2 Residual toxicity 35 2.3.3 Choice tests 38 2.3.4 Trans-laminar activity 41 2.4 DISCUSSION 42 2.4.1 Efficacy against pests 42 2.4.2 Effects on bio-controls 43 2.4.3 Persistence in the environment 44 iv 2.4.4 Repellent effects 44 2.4.5 Rosemary oil as a pesticide: advantages and limitations 45 REFERENCES 48 C H A P T E R T H R E E : C O M P A R A T I V E T O X I C I T Y O F ROSMARINUS OFFICINALIS L . E S S E N T I A L O I L A N D B L E N D S O F ITS M A J O R C O N S T I T U E N T S A G A I N S T TETRANYCHUS URTICAE K O C H ( A C A R I : T E T R A N Y C H I D A E ) O N T W O D I F F E R E N T H O S T P L A N T S 54 3.1 I N T R O D U C T I O N 54 3.1.1 Two-spotted spider mite 54 3.1.2 Rosmarinus officinalis essential oil 54 3.2 M A T E R I A L S A N D M E T H O D S 56 3.2.1 Rosmarinus officinalis essential oil 56 3.2.2 Spider mites 56 3.2.3 General growing conditions for plants and mites 56 3.2.4 Calculating lethal concentration 50 ( L C 5 0 , ) of the oil 57 3.2.5 Comparative toxicities 58 3.2.6 Data Analysis 58 3.3 R E S U L T S 59 3.3. J Essential oil constituents 59 3.3.2 Lethal concentration 50 of the oil 59 3.3.3 Comparative toxicities of individual constituents and blends thereof. ..60 3.4 DISCUSSION 66 3.4.1 Rosemary oil as an acaricide 66 3.4.2 Synergy among constituents 67 R E F R E N C E S 69 C H A P T E R F O U R : I N T E G R A T E D C O N T R O L O F T W O - S P O T T E D S P I D E R M I T E S O N G R E E N H O U S E T O M A T O : S T U D Y I N G T H E F I E L D E F F I C A C Y O F A R O S E M A R Y O I L -B A S E D A C A R I C I D E S C O M B I N E W I T H P R E D A T O R Y M I T E PHYTOSEIULUS PERSIMILIS A N D A S S E S S I N G T H E P H Y T O T O X I C E F F E C T S O F T H E P E S T I C I D E O N T O M A T O P L A N T S 72 4.1 INTRODUCTIUON , 72 4.1.1 Two-spotted spider mite 72 4.1.2 Phytoseiulus persimilis 72 4.1.3 Rosemary oil. 72 4.1.4 Phytotoxic effect 72 4.2 MATERIALS AND METHODS 74 4.2.1 Commercial pesticide 74 4.2.2 Plant materials 74 4.2.3 Two-spotted spider mites & Phytoseiulus persimilis 74 4.2.4 Efficacy test 75 4.2.5 Phytotoxicity tests 76 4.2.6 Data Analysis 77 4.3 RESULTS 78 4.3.1 Efficacy test 78 4.3.2 Phytotoxicity test 80 4.4 DISCUSSION 83 4.4.1 Efficacy of EcoTrol'" 83 4.4.2 Phytotoxicity trials 84 REFERENCES 86 C H A P T E R F I V E : S U M M A R Y A N D C O N C L U S I O N 88 REFERENCES 94 A P P E N D I X 96 V List of tables TABLE 2.1. TOXICITY OF ROSEMARY OIL AND THREE COMMERCIAL PESTICIDES TO TWO-SPOTTED SPIDER MITE AND GREENHOUSE WHITEFLY ; 34 TABLE 3.1. MAJOR CONSTITUENTS OF ROSEMARY OIL 59 TABLE 4.1. MEAN OF TWO-SPOTTED SPIDER MITES ON TREATED AND NON-TREATED TOMATO PLANTS 78 TABLE 4.2. ANALYSIS OF VARIANCE OF NUMBER OF SPIDER MITES 79 TABLE 4.3. MEAN OF TWO-SPOTTED SPIDER MITE'S EGGS ON TREATED AND NON-TREATED TOMATO PLANTS 79 TABLE 4.4. ANALYSIS OF VARIANCE OF NUMBER OF SPIDER MITE'S EGGS 80 v i List of figures FIGURE 2.1. LEAF DISC PAINTING METHOD 26 FIGURE 2.2. FUMIGATION CHAMBER (OPEN AND CLOSE CONTAINER) 28 FIGURE 2.3. CHOICE TEST BIOASSAY 30 FIGURE 2.4. OVIPOSITION CAGES 31 FIGURE 2.5. TRANS-LAMINAR EFFECTS OF ROSEMARY OIL-BASED PESTICIDES 32 FIGURE 2.6. DIRECT CONTACT TOXICITY OF THREE COMMERCIAL PESTICIDES TO P. PERSIMILIS AND T. URTICAE ON TOMATO PLANTS 35 FIGURE 2.7. RESIDUAL TOXICITY OF THREE ROSEMARY OIL-BASED PESTICIDE TO TWO-SPOTTED SPIDER MITE ON TOMATO PLANTS 36 FIGURE 2.8. RESIDUAL TOXICITY OF THREE PESTICIDES AT THEIR RECOMMENDED LABEL RATE TO P. PERSIMILIS ON TOMATO PLANTS 36 FIGURE 2.9. RESIDUAL TOXICITY ROSEMARY OIL 1 % TO GREENHOUSE WHITEFLY ON TOMATO PLANTS 37 FIGURE 2.10. RESIDUAL TOXICITY OF ROSEMARY OIL 1% TO E. FORMOSA ON TOMATO PLANTS 38 FIGURE 2.11. NUMBER OF TWO-SPOTTED SPIDER MITES STAYING ON LEAF DISCS WHEN GIVEN A CHOICE BETWEEN A TREATED AND NON-TREATED LEAF DISC WITH ROSEMARY OIL 1% 39 FIGURE 2.12. NUMBER OF TWO-SPOTTED SPIDER MITES EGGS ON LEAF DISCS WHEN GIVEN A CHOICE BETWEEN A TREATED AND NON-TREATED LEAF DISC WITH ROSEMARY OIL 1 % 39 FIGURE 2.13. NUMBER OF GREENHOUSE WHITEFLY EGGS ON TOMATO LEAVES WHEN GIVEN A CHOICE BETWEEN A TREATED AND NON-TREATED LEAF WITH ROSEMARY OIL 1 % 40 FIGURE 2.14. TRANS-LAMINAR ACTIVITY OF ROSEMARY OIL 41 FIGURE 3.1. TOXICITY OF PURE CONSTITUENTS OF ROSEMARY OIL TO TWOSPOTTED SPIDER MITE ON BEAN PLANTS 60 FIGURE 3.2. TOXICITY OF PURE CONSTITUENTS OF ROSEMARY OIL TO TWOSPOTTED SPIDER MITE ON TOMATO PLANTS 61 FIGURE 3.3. TOXICITY OF DIFFERENT BLENDS OF PURE CONSTITUENTS OF ROSEMARY OIL TO TWO-SPOTTED SPIDER MITE ON BEAN PLANTS 62 FIGURE 3.4. TOXICITY OF DIFFERENT BLENDS OF CONSTITUENTS OF ROSEMARY OIL TO TWO-SPOTTED SPIDER MITES ON TOMATO PLANTS 63 FIGURE 3.5. TOXICITY OF DIFFERENT BLENDS OF ROSEMARY OIL CONSTITUENTS TO TWO-SPOTTED SPIDER MITES ON BEAN PLANTS 64 FIGURE 3.6. TOXICITY OF DIFFERENT BLENDS OF ROSEMARY OIL CONSTITUENTS TO TWO-SPOTTED SPIDER MITES ON TOMATO PLANTS 64 FIGURE 3.7. TOXICITY OF DIFFERENT BLENDS OF ROSEMARY OIL CONSTITUENTS TO TWO-SPOTTED SPIDER MITES ON TOMATO PLANTS IL CONSTITUENTS TO TWO-SPOTTED SPIDER MITES ON TOMATO PLANTS ....65 FIGURE 4.1. EXPERIMENT LAYOUT 75 FIGURE 4.2. SUB-SAMPLING FOR COUNTING NUMBER OF EGGS 76 FIGURE 4.3. PHYTOTOXICITY TESTS 77 FIGURE 4.4. PHYTOTOXICITY TO FOLIAGE 81 FIGURE 4.5. PHYTOTOXICITY TO FLOWERS 82 FIGURE 4.6. PHYTOTOXICITY TO FRUITS 82 FIGURE A. l . DIGITAL TIMER CIRCUITS 97 FIGURE A.2. ELECTRONIC MICRO-SPRAYER 98 FIGURE A.3. SUB-LETHAL EFFECTS OF ROSEMARY ON E.FORMOSA 99 FIGURE A.4. EFFECT OF ROSEMARY OIL ON PARASITIZING PATTERN OF E. FORMOSA 100 FIGURE A.5. EFFECT OF ROSEMARY OIL ON HATCHING PATTERN OF E. FORMOSA 100 v n Acknowledgments M a n y people contributed signif icantly to this project. M y research supervisor and three committee members guided this study from its beginning to its final complet ion, and my gratitude for their valuable input and support is profound. I take this opportunity to thank Professor Murray B. Isman for his great help and support. Without his support, this study could not have been accompl ished. I also thank Drs. Judith H . Myers , Dave R. Gi l lesp ie and Michae l T. Wan for prov id ing cr i t ical feedback and guidance on my project. I must express my warmest gratitude to my lovely wi fe M r s . M a r y a m An t i kch i who helped me wi th designing and bui ld ing the electronic micro-sprayer and supported me during my project. I also have to thank Dr . Shahriyar Mi rabbas i f rom the Department o f Electr ical Engineer ing o f U B C and M r . Keyvan T i v and M r . A l i M o f i d i for great help and technical support for electronic micro-sprayer. Many thanks to M r . Ruben Houwel ing and Houwe l ing 's Nurser ies L td . for providing plant materials, App l i ed B ionomics for provid ing the bio-control agents and M r . Rod Bradbury from Ecosafe Natural Products Inc. for prov id ing essential o i ls and analyzing the samples. Special thanks to M r . Amandeep B a l , who maintained my communicat ion l ink with the growers. M y deepest appreciation also goes to al l colleagues and friends at the Centre for Plant Research at U B C , Mrs . Nancy Brard, Dr . Yasmin Akhtar , M r . Ikkei Shikano, M r . Dav id Noshad, M s . Cr is t ina Mach ia l , M s . Nyssa Temmel , M r . A l a i n Bouchai r and M r . Br ian K i n g for their help and support. Th is study was funded in part by a grant f rom E c o S M A R T Technologies Inc. and a research contract f rom the B C Greenhouse Growers Associat ion. F ina l l y , I wou ld l ike to thank my fami ly, my parents and my in- laws who ful ly supported me during my study. v i i i Chapter One: Introduction 1.1 Major objectives The major objective o f this research was to study the efficacy o f a rosemary oil-based pesticide (EcoTrol™) for use on greenhouse tomato plants against the important pests o f greenhouse crops (the two-spot ted spider mite and the greenhouse whi tef ly) . Th is pesticide was introduced to the U S market in 2002. It has been used mainly on berries, grapes, nuts and tree fruits. The product also has been used on vegetables, but its use in the greenhouses has been l imited to a few growers. A l though this is a natural product and has been used in the Uni ted States, it requires regulatory approva l by the Pest Management Regulatory Agency ( P M R A ) in order to be used by Canadian growers. Similar to all new pesticides, it requires some assessment before entering the market. The fol lowing questions need to be addressed: • Is this product efficacious against pests? • Can it be hazardous to human health? • What environmental risks does it pose? • Is it persistent in the environment? • What effects might it have on non-target terrestrial and aquatic species? • What are the effects o f this product on the host plant? • Is it toxic to bio-control agents? • A r e there any l imitations for its appl icat ion? • Cou ld pests evolve resistance after repeated appl icat ions? • What is the mechanism o f toxicity o f this product? 1 • Can environmental factors affect its eff icacy? In this research, I tried to answer some o f these questions. I focused mainly on the efficacy o f this pesticide against the pests and its effects on their bio-control agents and host plants under laboratory condit ions concluding with a small-scale greenhouse trial. 1.2 Historical perspective Most present agricultural practices involve growing highly domesticated plants and animals. A n i m a l and plant domestication involved a gradual shift in the pattern o f human interaction with the environment. For thousands o f years prior to domestication, humans subsisted by scavenging, gathering plants and hunting animals as they were found in the environment. In the domestication process, humans manipulated animals, plants and the environment in various ways to increase the avai labi l i ty o f the desirable species and desired traits o f these species. Over long periods o f t ime and many generations o f human selection, domesticated plant varieties and animal breeds emerged with traits that wou ld not survive without human involvement in such tasks as selective breeding, planting, weeding, harvesting, pest control and storage. These manipulat ions to the environment resulted in new problems that did not exist before. In cropping systems in particular, monoculture o f plants provided an aggregated food source for pests and suitable growing condit ions for their offspring. Wi th increasing human population the necessity o f prov id ing more food increased. They not only had to develop their agriculture practices to produce more y ie ld , but also had to protect their products f rom damaging factors, most importantly pests. A s a consequence, many pest control methods have been developed and this remains an important subject o f research. Introduction o f synthetic chemical pesticides and their extensive use in agriculture was a new chapter in the history o f pest control . Development o f insecticides, such as D D T , brought hope to the agricultural industry. Many farmers took D D T as a guaranteed pest control tool that could completely solve the pest problem; however, that feel ing o f rel ief quickly faded due to a new phenomenon; pesticide resistance. Intensive application o f pesticides is the most important factor in the quick bui ld-up o f resistance in most pest populations. Pesticide appl icat ion removes the susceptible pests f rom the population and only those having resistance genes w i l l survive, passing the resistance trait on to their offspring. The percentage o f resistant pests in a population continues to mult ip ly whi le pesticides eliminate susceptible ones. Eventual ly , resistant pests outnumber susceptible ones and the pesticide is no longer effective. In addition to the pest resistance problem, extensive application o f pesticides caused damage to the environment, non-target organisms, higher animals in ecosystem and impacted human health. Numerous studies have been conducted on this subject and many books have been written to describe its detection, magnitude, mechanism, side effects and management (1,2). Recent ly , some scientists have taken a different approach towards pest management by using plants as potential sources of pesticides. Plants have evolved different defense mechanism against herbivores. Secondary metabolites o f plants -including a wide array o f chemicals- are good examples o f these mechanisms. Our ancestors were aware o f the healing and medicinal properties o f some plants and their extracts. They also used some plants to repel insects and other pests. In recent decades scientists started to investigate these ancient traditions in search o f possible substitutes for synthetic pesticides and drugs (3). A l though these natural based- products seem to be 3 safer than conventional synthetic products, there are many unknown aspects that need to be addressed before extensive appl icat ion. In the present study, a rosemary o i l (Rosmarinus officinalis) based- pesticide has been tested against two major pests o f greenhouse tomato (Lycopersicon esculentum), the two-spotted spider mite (Tetranychus urticae), and the greenhouse whitef ly (Trialeurodes vaporariorum). 1.3 Greenhouse Tomato The greenhouse vegetable industry is an important and growing segment o f Canadian agriculture. Acco rd ing to Agr icul ture and A g r i - F o o d Canada (4), the estimated value o f the greenhouse industry was $80 M in 1988, reaching $600 M in 2000. Of f ic ia l statistics (Statistics Canada Pub l . 20-202 for 2000) value the Canadian greenhouse industry at $1711 M and the greenhouse vegetable port ion at $505 M . The main greenhouse vegetable crops in Canada are tomatoes (468 ha), cucumbers (190 ha), sweet peppers (144 ha) and lettuce (21 ha) (5). Dur ing the 1990s, the total area under glass and plastic more than doubled to nearly 1,500 hectares. B y 2003, it had reached nearly 1,900 hectares. In 2003, revenue from greenhouse sales reached a record high o f almost $2.1 b i l l ion ; nearly double what it had been just six years earlier. F lowers accounted for about 7 0 % o f sales and vegetables the remaining 30%. In the early 1990s, revenues from the comparable greenhouse and field vegetables were roughly the same. However , since 1996, revenues f rom greenhouse vegetables have increased at a much more rapid pace than field vegetables. Fo r example, in 2003, the farm gate value o f the four main vegetable crops produced under glass or plastic (tomatoes, cucumbers, lettuce and peppers) amounted to $605.8 mi l l i on . Th is was more than three times higher than the value o f $171.7 mi l l i on for the same four vegetable 4 crops produced in the field. Farmers grow more tomatoes than any other vegetable crop, whether it's in the greenhouse or in the field. Tomatoes alone, account for over one-half o f revenues f rom the sale o f greenhouse vegetables. They also cross the border in both directions. Canadian greenhouse growers have been shipping hothouse tomatoes to the Uni ted States in r ising numbers. In recent years, Canada has enjoyed a trade surplus in tomatoes, shipping far more south o f the border than Amer ican farmers ship north. (6) 1.4 Two-spotted spider mite Spider mites belong to the fami ly Tetranychidae o f the order Prostigmata. They are so named because many members o f this fami ly produce si lk webbing on host plants. Most spider mite species are polyphagous. The Tetranychidae is a large fami ly o f wor ldwide distribution. The fami ly consists o f two subfamil ies: Bryobinae and Tetranychinae. Mos t pest species belong to the Tetranychinae. The two-spotted spider mite Tetranychus urticae K o c h is the most important species in this subfamily. The two-spotted spider mite is the most common name for this species. It is also known informal ly by many other names (e.g. the glasshouse spider mite, the ye l low spider mite). No t very appropriately, it is cal led the red spider mite or red spider in some literature presumably because o f the red/orange color or the overwintering form, or in reference to a species complex including T. cinnabarinus. The two-spotted spider mite is an important pest o f greenhouses throughout the wor ld . It is a cosmopoli tan species and also is the most polyphagous species o f spider mite. It has been reported on about 1200 host plant species in 70 genera o f wh ich over 300 species are grown in greenhouses. Its l i fe cycle consists o f eggs, one larval stage, two nymphal stages and an adult stage. 5 The eggs are often laid in clusters on the under surface o f leaves. They are spherical in shape and translucent, pale in color. A s they develop, they become more yel lowish and red eyespots inside the eggshell can be seen. S ix - legged larvae are pale to yel lowish when first hatched and become yel lowish green after feeding. Eight- legged nymphs are ye l lowish green with dark spots. Their body is ovo id in shape wi th short legs. Adu l t females are about 400-500 urn in length and males are smaller wi th a pointed hysterosoma. The females (summer form) are ye l lowish to greenish in color wi th two black spots on the dorsolateral id iosoma, but are darker in color, often orange or red in the overwintering form. The color o f mites may vary depending on the host plant and other environmental factors. T w o spotted spider mites often feed on cel l chloroplasts on the under surface o f the leaf. The upper surface o f the leaf develops characteristic whi t ish or ye l lowish st ippl ing, wh ich may jo in and become brownish as mite feeding continues. A s mites move around, their webbing can span leaves and stems. Heavy damage may cause leaves to dry and drop, and the plant may be covered with webbing and may die prematurely. Development occurs between 12 and 40°C. Developmental t ime f rom egg to adult decreases with increasing temperature and is less than a week at opt imal temperatures for development (30-32°C). Under a diurnal temperature cycle o f 15 to 28°C, development time is about 16 days. Ma les develop slightly faster than females. Males are attracted to a sex pheromone from dormant female deutonymphs. They guard their territory and fight against any other invading males. Ma t ing occurs as soon as females emerge. Females start to lay eggs within a couple o f days o f adulthood. The rate o f oviposit ion and fecundity varies with food plant and temperature. A n average female 6 can lay over ten eggs per day and produce over 100 eggs during two weeks at about 25°C. The sex ratio is h ighly female biased, wi th a female to male ratio o f about 3:1. T. urticae disperses by active wa lk ing or by passive transport in the w ind , on plants, on tools or on people. Diapause is induced by short day lengths, lack o f food supply and low temperature, and is normal ly terminated by a f ixed period o f ch i l l ing . Grav id females seek a protected niche at the end o f summer. Diapausing adults are orange/red in color (7). The economic threat posed by these mites is constantly increasing because o f the development o f resistance, and resurgence o f mite populations fo l low ing use o f non-selective synthetic pesticides that eliminate natural enemies such as predaceous mites and spiders (8). Spider mites have evolved resistance to more than 80 acaricides to date and resistance has been reported f rom more than 60 countries (9). Spider mites and especial ly two-spotted spider mites have been on the priority lists for pest management in B C greenhouses for several years. The current control method for spider mites in B C greenhouses is based on bio-control agents that include six different predatory mites (Phytoseiulus persimilis Athias-Henriot , Metaseiulus occidentalis Nesbitt, Amblyseius californicus Carte, Persimilis longipes Evans, Galendromus occidentalis Nesbitt and Amblyseius cucumeris Oudemans), one predatory midge (Feltiella acarisuga Val lo t ) and two predatory bugs (Deraeocoris brevis Uhler and Orius sp.). Problems wi th bio-control include l imited eff icacy against high populations o f spider mites, constraints, and bio-control agents' susceptibi l i ty to most pesticides. 7 1.5 Greenhouse Whitefly The greenhouse whi tef ly, Trialeurodes vaporariorum Westwood, is another important pest o f greenhouse crops wor ldwide. Its l ife cycle consists o f eggs, a crawler stage, three nymphal stages, a pupa and adults. Whi te f ly adults are t iny, white moth-l ike insects. They lay eggs on the underside o f leaves. Eggs hatch in 10 to 14 days. Af ter three molts in about 14 days, they pupate and the adult emerges 6 days later. Adu l ts begin to lay eggs 4 days after emergence. Each female is capable o f lay ing 400 eggs over a period o f up to 2 months, although usually far fewer eggs are produced. The length o f the life cycle is temperature dependent. Adul ts l ive 30 to 60 days and feed by sucking sap f rom the plant. T w o species o f whitef ly infest Br i t ish Co lumb ia greenhouse crops. T. vaporariorum Westwood (the greenhouse whitef ly) is the most common one. However Bemesia tabaci Gennadius (the sweet potato whitef ly) was introduced to Canada in recent years on imported plant materials and has been found in some greenhouses. Greenhouse whi tef ly has a wide host range and is known to develop on more than 250 ornamental and vegetable plants. Poinsettia, hibiscus, nicot iana, aster, calendula, cucumber, lantana, tomato, grape, ageratum, bean, and begonia are among the more commonly infested plants (10). B. tabaci is more di f f icul t to control, owing to its high egg laying capacity and wide host range. Sweet potato whitef ly is smaller than the greenhouse whitef ly and is more off-white or yel low-whi te in color. Sweet potato whitef ly is typical ly found lower in the plant canopy than greenhouse whitef ly, which is often found on the developing leaves at the growing points. Both whitef ly species can cause severe damage to fol iage by reducing v igor and by coating the growing points, leaves, and fruits wi th excreta 8 (honeydew). The excreta becomes a food source for fungal moulds to develop. This mould coats pepper and tomato fruit requir ing extra fruit c leaning costs pr ior to sale. Sweet potato whi tef ly can also transmit viruses and cause abnormal fruit discoloration. L i k e the two-spotted spider mite, the greenhouse whitef ly is among the most important pests o f B C greenhouses. Contro l mostly relies on bio-control agents, such as the parasitic wasp Encarsia formosa (11,12). L i k e spider mites, whitef l ies have also been evolved resistance to many pesticides (1). 1.6 Greenhouse pest management A s explained before, most domesticated plants cannot survive without human involvement. Greenhouse plants are among the most human-dependent ones. Greenhouses provide the abi l i ty to control environmental factors that have great impact on plants. In modern greenhouses, almost al l environmental factors are under control , for instance temperature, moisture, light, nutrients and even the composi t ion o f different gases in the air that plants need for growth. In greenhouses, plants constantly grow and provide fruit throughout the year regardless o f the outside condit ions. These control led conditions are not only suitable for plants but also for a wide range o f pests, wh ich normally cannot survive outside the greenhouse. Hav ing opt imal condit ions for growth and an aggregated food source al lows many pests to achieve epidemic populations. A s the easiest control measure, pesticides have been extensively used inside greenhouses to control pests dur ing the product ion season. This strong selection pressure resulted in emergence o f h ighly resistant populations o f pests to almost a l l pesticides that have been applied so far. B io-cont ro l agents were introduced to greenhouses as an alternative solution. Different predators or parasitoids o f insect or mite pests were identif ied and 9 released inside greenhouses to reduce their populations. Th is method has been successful for several pests. In Canada, the greenhouse pest management is largely based on biological control . A survey conducted in 2002 indicated that 9 3 % o f tomato growers (n= 165) use b io logical control for insect and mite control (12). A l though bio logical control methods were successful for most pests, they have l imitations and cannot be used in al l situations. For example, it has been shown that activity and survival o f the predatory mite Phytoseiulus persimilis can be affected by different levels o f humidi ty (13), temperature (14) and pest density (15). Because o f these limitations, bio-control agents might not be able to reduce pest populations to an acceptable level by themselves. Thus, ut i l izat ion o f bio-control agents for control l ing greenhouse pests may only be effective when combined with other strategies (16). A s an example, N ice t ic et al. (17) found that a combinat ion o f petroleum spray o i l and the predatory mite Phytoseiulus persimilis can be used to control two-spotted spider mites on greenhouse roses. When the use o f a pesticide is necessary, materials should be selected that are least harmful to the predators and parasites released into the greenhouse. Essential o i ls o f plants might be considered a good option for use in combination with other pest control methods. 1.7 Plant essential oils Plant essential oi ls are odorous compounds obtained through steam disti l lation o f herbs and medic inal plants (18). These oi ls have been used tradit ionally as heal ing medicines in many countries and ancient people were also aware o f their pesticidal properties, however, only in recent years have these oils been commerc ia l ized as pest control products (3). Mos t o f these oi ls are environmental ly non-persistent, and non-toxic 10 to humans (with some exceptions) (19-21), f ish (with some exceptions) and wi ld l i fe (22-24) Plant essential o i ls are generally mixtures o f mono- and sesquiterpenes (e.g., ct-terpineol and pulegone) and phenolics or monophenols (e.g., thymol , carvacrol , and eugenol). They are often quite volat i le and are commonly used as fragrances and as f lavoring agents in food (25). They are sometimes incorporated into natural pest control products. For example, 1,8-cineole, a major component o f o i l o f eucalyptus and rosemary is active against four stored products beetles (26). Hough-Go lds te in (27) reported antifeedent effects o f essential oi ls against the Colorado potato beetle Leptinotarsa decemlineata L . whi le Sharma and Saxena (28) showed their effectiveness as growth inhibitors on housefl ies Musca domestica L. Many researchers have reported repellent, antifeedent and toxic properties o f selected essential oi ls against many agriculturally important pests. T h y m o l showed both repellent and toxic effects against the two-spotted spider mite (29). In another study, thymol and citronel l ic ac id were found toxic to the common housefly, western corn rootworm Diabrotica vergifera vergifera, and the two-spotted spider mite (30). Thyme o i l was also found to be toxic to the tobacco cutworm Spodoptera litura (31). Cho i et al. (32) tested a total o f 53 esential oi ls against T. urticae and P. persimilis v i a fumigation. Caraway seed, ci t ronel lal , lemon, eucalyptus, pennyroyal and peppermint oi ls were found to be highly toxic to both mites. Rosemary o i l was also found to be toxic to the predaceous mites Amblyseius barkeri Hughes, A. zaheri and Typhlodromus athiasae Porath (33) whi le it showed repellent properties against the onion aphid Neotoxoptera formosana Takahashi (34) and the green peach aphid Myzus persicae Sulzer (35). Both 11 contact and fumigant toxici t ies o f eugenol and methyl eugenol were demonstrated to the Amer ican cockroach Periplaneta americana L. (36). The mechanisms o f toxic i ty o f essential oi ls have not been fu l ly identi f ied. However a recent investigation using the Amer ican cockroach points to the octopaminergic nervous system as the site-of-action o f some essential oi ls in insects (37). 1.8 Rosemary oil Rosmarinus officinalis L . is an evergreen perennial woody shrub with aromatic, needle-l ike leaves and gray, scaly bark. Rosemary bushes can grow up to 6 ft (1.8 m) tall wi th a spread o f 4-5 ft (1.2-1.5 m). Th is plant belongs to the fami ly Lamiaceae formerly known as the Labiatae. It is used for f lavoring food and beverages and it also used in cosmetics and aromatherapy. Rosemary and its constituents, have a therapeutic potential in treatment or prevention o f bronchial asthma, spasmogenic disorders, inf lammatory diseases and hepatotoxicity (38). Ant i -cancer (39-41) and anti-viral (42) properties o f rosemary have also been reported. Beside the great medic inal value o f rosemary for humans, the essential o i l o f rosemary has strong antibacterial activity against microorganisms such as Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O 157:H7, Shigella dysenteria, Bacillus cereus and Staphylococcus aureus (43). It has been reported that the essential oi l o f rosemary has repellent and deterrent properties against Thrips tabaci and affect host plant selection and acceptance (44). It also showed ovicidal act ivi ty against two stored-product insects (45). Essential oi l o f rosemary is a complex mixture o f different constituents. A recent study detected 33 12 compounds in the oi l (46). The main components of the o i l are oc-pinene, 1,8-cineole, camphor, p-pinene, and borneol. References 1) Eb ing , W . (Ed.) . (1997). Molecular mechanisms of resistance to agrochemicals. Ber l in : Springer, pp 21-75. 2) Roush, R.T. , & Tabashnik, B . E . (1990). Pesticide resistance in arthropods. N e w York : Chapman & Ha l l . 303p. 3) Isman, M . B. (2000). Plant essential oi ls for pest and disease management. Crop Protection, 19:603-608. 4) Introduction to the greenhouse industry. (2003). Agriculture and Agri-Food Canada. Retrieved November 3, 2005, f rom http://res2.agr.gc.ca/harrow/publications/ Introduction_e.htm 5) Hort iculture and greenhouse products, by province (2001 Census o f Agr icul ture). Statistics Canada. Retr ieved November 3, 2005, f rom http://www40.statcan.ca/101/cst01/agrc31 k.htm 6) Study: High- tech vegetables: the booming greenhouse vegetable industry. (2005). The Daily. Statistics Canada. Retr ieved November 3, 2005, f rom http:/ /www.statcan.ca/Dai ly/Engl ish/050322/d050322e.htm 7) Zhang, Z . (2003). Mites of greenhouses: identification, biology and control. Wal l ing fo rd : C A B I publ ishing, pp 54-61. 8) Cranham, J . E . , & He l le , W . (1985). Pesticide resistance in Tetranychidae. In: World crop pest - spider mites: their natural enemies and control, Amsterdam: Elsevier , pp 405-421. 9) The Database o f Arthropods Resistance to Pesticides. (2004). Michigan State University-Center for Integrated Plant Systems. Retrieved November 3, 2005, from http://www.pesticideresistance.org / D B / index.html 14 10) Cranshaw, W . (2004). Garden insects of North America. N e w Jersey: Pr inceton, pp 284-289. 11) Greenhouse vegetable production guide for commercial growers. (1997). Province of British Columbia, Ministry of Agriculture, Food and Fisheries, pp 88-89. 12) Murphy , G . D . , Ferguson, G . , Fry , K. , Lambert, L., M a n n , M . , & Mat teoni , J . (2002). The use o f b io logical control in Canadian greenhouse crops. Integrated Control in Protected Crops, Temperate Climate, IOBC/wprs Bulletin, 25(1): 193-196. 13) M o r i , H . , & Chant, D. A . (1966). The influence of humidi ty on the activity o f Phytoseiulus persimilis Ath ias-Henr iot and its prey, Tetranychus urticae K o c h . (Acar ina: Phytosei idae, Tetranychidae). Canadian Journal of Zoology, 44: 863-871. 14) Everson, P. (1980). The relative activity and functional response o f Phytoseiulus persimilis (Acar ina : Phytoseiidae) and Tetranychus urticae (Acar ina : Tetranychidae): the effect o f temperature. Canadian Entomologist, 121: 17-24. 15) Everson, P. (1979). The functional response o f the Phytoseiulus persimilis (Acar ina: Phytoseiidae) to various densities o f Tetranychus urticae (Acar ina : Tetranychidae). Canadian Entomologist, 111: 7-10. 16) Zhang, Z . Q . & Sanderson, J . P. (1995). Two-spotted spider mite (Aca r i : Tetranychidae) and Phytoseiulus persimilis (Acar i : Phytoseiidae) on greenhouse roses: Spatial distribution and predator eff icacy. Journal of Economic Entomology, 88 :352-357 . 17) N ice t ic , O. , Watson, D. M . , Beatt i , G . A . C , Meats, A . , & Zheng , J . (2001). Integrated pest management o f two-spotted spider mite Tetranychus urticae on 15 greenhouse roses using petroleum spray o i l and the predatory mite Phytoseiulus persimilis. Experimental & Applied Acarology, 25: 37-53. 18) Yatagai , M . (1997). M i t i c ida l activities o f tree terpenes. Current Topics of Phytochemistry, 1:87-97. 19) Roe , F. J . C . (1965). Chron ic toxici ty o f essential oi ls and certain other products o f natural or ig in. Food and Cosmetics Toxicology, 33: 311-324. 20) Cockayne, S. E. , & Gawkrodger, D. J . (1997). Occupational contact dermatitis in an aromatherapist. Contact Dermatitis, 37: 306-309. 21) Hjorther, A . B. , Christophersen, C , Hausen, B . M . & Menne, T . (1997). Occupat ional al lergic contact dermatitis f rom carnosol, a natural ly-occuring compound present in rosemary. Contact Dermatitis, 37: 99-100. 22) K u m a r A n u j , D. , F lorence, V . , Broughton, M . J . , & Sriharan, S. (2000). Ef fect o f root extracts o f mexican mar igold, Tagetes minuta (Asterales: Asteraceae), on six nontarget aquatic macroinvertebrates. Environmental Entomology, 29: 140-149. 23) Wager-Page, S. & Mason , J . R. (1997). Ortho-aminoacetophenone, a non-lethal repellent: The effect o f volat i le cues vs. direct contact on avoidance behavior by rodents and birds. Pesticide Science, 46: 55-60. 24) Clark , L . & A ronov , E . V . (1999). Human food f lavor additives as bird repellents: I. Conjugated aromatic compounds. Pesticide Science, 55: 903-908. 25) Isman, M . B . (1999). Pesticides based on plant essential o i ls. Pesticide Outlook, 10: 68-72. 26) Obeng-Ofor i , D. , Reichmuth, C . H . , Bekele , A . , & Hassanal i , A . (1997). B io log ica l activity o f 1,8 -c ineole, a major component o f essential o i l o f Ocimum kenyense 16 (Ayobangira) against stored product beetles. Journal of Applied Entomology, 121: 237-245. 27) Hough-Goldste in , J . A . (1990). Antifeedant effects o f common herbs on the Colorado potato beetle (Coleoptera: Chrysomel idae). Environmental Entomology, 19: 234-238. 28) Sharma, R . N . , & Saxena, K . N . (1974). Orientation and developmental inhibit ion in the housefly by certain terpenoids. Journal of Medical Entomology, 11: 617-621. 29) Gengaih i , E . , Amer , S .E . & Mohamed. S . A . A . (1996). B io log ica l activity o f thyme oi l and thymol against Tetranychus urticae K o c h . Anzeigerfur Scchadlingskunde Pflanzenscutz Umweltsschutz, 69: 157-159. 30) Lee , S., Tsao, R., Peterson, C . & Coats, J .R. (1997). Insecticidal activity o f monoterpenoids to western corn rootworm (Coleoptera: Chrysomel idae) , two-spotted spider mite (Aca r i : Tetranichidae), and house f ly (Diptera: Musc idae) . Journal of Economic Entomology, 90: 883-892. 31) Isman, M . B . , W a n , A . J . & Passreiter, C M . (2001). Insecticidal activity o f essential oi ls to the tobacco cutworm, Spodoptera litura. Fitoterapia, 72: 65-68. 32) C h o i , W . , Lee , S., Park, H . , & A h n , Y . (2004). Tox ic i ty o f plant essential oi ls to Tetranychus urticae (Acar i : Tetranychidae) and Phytoseiulus persimilis (Acar i : Phytoseiidae). Journal of Economic Entomology, 97: 553-558. 33) M o m e n , F . M . , & A m e r , S . A . A . (1999). Effect o f rosemary and sweet marjoram on three predacious mites o f the fami ly Phytoseiidae (Acar i : Phytosei idae). Acta Phytopathologica et Entomologica Hungarica, 34: 355-361. 17 34) Masatoshi , H . , & H i roak i , K . (1997). Repel lency o f rosemary o i l and its components against the onion aphid, Neotoxoptera formosana (Takahashi) (Homoptera, Aphid idae) . Applied Entomology & Zoology, 32: 303-310. 35) Masatoshi , H . (1998). Repel lency o f rosemary o i l against Myzus persicae in a laboratory and in a screenhouse. Journal of Chemical Ecology, 24:1425-1432. 36) N g o h , S.P. , Pang, F . Y . , Huang, Y . , K i n i , M . R . & H o , S . H . (1998). Insecticidal and repellent properties o f nine volat i le constituents o f essential o i ls against the Amer ican cockroach, Periplaneta americana (L.). Pesticide Science, 54: 261-268. 37) Enan, E . (2001). Insecticidal activity o f essential o i ls: octopaminergic sites o f action. Comparative Biochemistry and Physiology, 130: 325-337. 38) A b u - A m e r , K . M . , Sen, P., & al-Sereit i , M . R . (1999). Pharmacology o f rosemary (Rosmarinus officinalis L inn . ) and its therapeutic potentials. Indian Journal of Experimental Biololgy, 37: 124-30. 39) Of ford E . A . , Mace K. , Avan t i O . , & Pfeifer A . M . A . (1997). Mechanisms involved in the chemoprotective effects o f rosemary extract studies in human liver and bronchial cells. Cancer Letters, 114: 275-281. 40) Singletary, K. , M a c D o n a l d , C , & Wal l ig, M . (1996). Inhibit ion by rosemary and carnosol o f 7, 12- dimethylbenz[a]anthracene ( D M B A ) - i n d u c e d rat mammary tumorigenesis and in vivo D M B A - D N A adduct formation. Cancer Letters, 104: 43-48. 41) Plouzek, C . A . , C io l ino , H . P. , Clarke, R., & Y e h , G . C . (1999). Inhibit ion o f P-glycoprotein activi ty and reversal o f mult idrug resistance in vitro by rosemary extract. European Journal of Cancer, 35: 1541-1545. 18 42) A ruoma, O. I., Spencer, J . P. E . , Ross i , R., Aeschbach, R., K h a n , A . , M a h m o o d , N . , M u n o z , A . , M u r c i a , A . , Butler, J . , & Hal l iwel l B . (1996). A n evaluation o f the antioxidant and antiviral action o f extracts o f rosemary and provincial herbs. Food and Chemical Toxicology, 34: 449-456. 43) Burt , S. (2004). Essential o i ls : their antibacterial properties and potential applications in foods- a review. International Journal of Food Microbiology, 94: 223-253. 44) Koschier , E . H . , & Sedy, K . A . (2003). Labiatae essential oi ls affecting host selection and acceptance o f Thrips tabaci l indeman. Crop Protection, 22: 929-934. 45) Tunc , I., Berger, B . M . , Er ler, F., & Dagl i , F. (2000). Ovic idal act iv i ty o f essential oils f rom five plants against two stored-product insects. Journal of Stored Products Research, 36: 161-168. 46) Santoyo, S., Cavero, S., Jaime, L., Ibanez, E. , Senorans, F .J . , & Reglero, G . (2005). Chemica l composi t ion and antimicrobial activity o f Rosmarinus officinalis L . essential o i l obtained v ia supercrit ical f lu id extraction. Journal of Food Protection, 68: 790-795. 19 Chapter Two: Lethal and sub-lethal effects of Rosmarinus officinalis essential oil and three rosemary oil-based pesticides to Tetranychus urticae Koch, Trialeurodes vaporariorum Westwood, Phytoseiulus persimilis Athias-Henriot, and Encarsia formosa Gahan, on Lycopersicon esculentum 2.1 Introduction 2.1.1 Tetranychus urticae (Two-spotted spider mite) Two-spotted spider mite, Tetranychus urticae K o c h , occurs on vir tual ly every major food crop and ornamental plant. About 1200 species o f spider mites are known in the wor ld (1). It is the most polyphagous species o f spider mites and has been reported from over 150 host plants o f economic value (2). The greatest problem wi th this mite is its abil i ty to evolve resistance rapidly to pesticides only after few applications (3). Spider mites have evolved resistance to more than 80 acaricides to date and resistance has been reported from more than 60 countries (4). Spider mites and especial ly two-spotted spider mites have been a priori ty pest in B C greenhouses for several years. The current control method for spider mites in B C greenhouses is mostly re ly ing on bio-control method based on six different species o f predatory mites, one predatory midge and two predatory beetles. The problem wi th these bio-control agents is their l imi ted eff icacy against higher populations o f spider mites and their susceptibil ity to most pesticides. 2.1.2 Trialeurodes vaporariorum (Greenhouse whitefly) Greenhouse whi tef ly, Trialeurodes vaporariorum Westwood, is another important pest o f greenhouse crops wor ldwide. T w o species o f whitef ly infest Br i t ish Co lumb ia greenhouse crops. T. vaporariorum Westwood (the greenhouse whi tef ly) is the more 20 common one. However Bemesia tabaci Gennadius (the sweet potato whitef ly) was also introduced to Canada in recent years on imported plant materials and has been found in some greenhouses. Greenhouse whitef ly has a wide host range and is known to develop on more than 250 ornamental and vegetable plants. Poinsettia, hibiscus, nicot iana, aster, calendula, cucumber, lantana, tomato, grape, ageratum, bean, and begonia are among the more commonly infested plants (5). L i k e spider mites, whitefl ies have also evolved resistance to many pesticides (6). 2.1.3 Phytoseiulus persimilis (Predatory mite) The predatory mite, Phytoseiulus persimilis Athias-Henr iot , has been studied extensively wi th respect to its potential for biological control o f tetranychid mites on vegetables and ornamentals in greenhouses (7,8) P. persimilis is a selective predator that is able to rapidly suppress spider mites (9,10). Since juveni le development and reproduction o f P. persimilis depends on the availabil i ty o f spider mites as prey (11), it often disappears f rom the greenhouse after reducing pest mite populations and thus provides short-term control . Studies o f functional responses o f P. persimilis to different densities o f spider mites suggested that they might not provide acceptable control for higher populations o f prey (12). Thus uti l ization o f the predatory mite for control l ing spider mites in greenhouses may only be effective when combined wi th other strategies (13) . A s an example, N ice t i c et al. found that a combination o f petroleum spray o i l and the P. persimilis can be used to control two-spotted spider mites on greenhouse roses (14) . 21 2.1.4 Encarsia formosa (Parasitic wasp) Encarsict formosa Gahan is used wor ldwide for commercia l control o f whitef l ies in greenhouse crops. Commerc ia l use began in Europe in the 1920s and it shipped to Canada in the 1930s, but by 1945 interest waned due to the development o f pesticides. Af ter 1970, use was reinitiated and has expanded from 100 hectares o f greenhouse crops to 4800 hectares in 1993 (8). In Canada, the greenhouse vegetable industry is largely based on b io log ica l controls. A survey conducted in 2002 indicated that 9 3 % percent o f the tomato growers (total o f 165 growers) use b io logical control agents for insect and mite control (15). M o r e than seventy papers have been published that examine interactions between E. formosa and one or more pesticides, either in laboratory tests or under condit ions o f practical use in greenhouses. Standardized methods for determining the effects o f pesticides on E. formosa have been developed and the effects o f more than one hundred different compounds on E. formosa have been determined. Mos t o f the pesticides were found to be toxic to E. formosa. Selective materials o f interest for possible combinat ion with E. formosa include insecticidal soap, buprofezin, azadirachtin, abamectin, and resmethrin (16). When the use o f a pesticide is necessary, materials should be selected that are least harmful to the predators and parasites released into the greenhouse. Essential oi ls o f plants may be a good option for use in combinat ion wi th other pest control methods. 2.1.5 Rosmarinus officinalis (Rosemary plant) Plant essential oi ls are obtained through steam dist i l lat ion o f herbs and medicinal plants (17) Mos t o f these oi ls are environmental ly non-persistent, and non-toxic to humans (with some exceptions) (18-20), fish (with some exceptions) and wi ld l i fe (21-22 22). Rosemary o i l has been tradit ionally used as a medicine for co l ic , nervous disorders and painful menstruation. Recent studies revealed that the rosemary o i l is also an effective antibacterial agent (23-24). Rosemary o i l is relatively effective against insect and mite pests. The aromatic vapor o f rosemary has ovic idal and larvic idal effects on several stored product pests (25,26) and the two-spotted spider mite (27) as a fumigant. The o i l can have sub-lethal effects as we l l , for example acting as a repellent to onion thrips, Thrips tabaci (28). Cho i et al. (29) tested a total o f 53 essential o i ls against T. urticae and P. persimilis as a fumigant. Caraway seed, ci tronel la, lemon, eucalyptus, pennyroyal and peppermint o i l were found to be highly toxic to both mite species. Rosemary o i l was also found to be toxic to the predaceous mites Amblyseius barkeri Hughes, A. zaheri and Typhlodromus athiasae Porath (30). In my study, lethal and sub-lethal effects o f rosemary o i l and three rosemary o i l -based pesticides were evaluated against the two-spotted spider mite and the greenhouse whitef ly and their bio-control agents. 23 2 .2 Materials and methods 2.2.1 Rosmarinus officinalis essential oil and commercial pesticides Pure Rosmarinus officinalis essential o i l (Intarome T O , Lot# 0213142MB-100%) , three commercia l pesticides, Hexacide™(5% rosemary oi l ) , EcoTro l™(10% rosemary oi l ) and Sporan™(17.6% rosemary oi l) and a blank formulation o f E c o T r o l were obtained f rom E c o S M A R T Technologies Inc (Frankl in, T N , U S A ) . 2.2.2 Spider mites Spider mites originated f rom a research colony maintained on tomato plants for more than five years without any pesticide exposure at Agr icu l ture and Ag r i -Food Canada (Agass iz , B C , Canada). These mites were reared on three-week-old v ine tomato plants (Lycopersicon esculentum M i l l cv. Clarance) provided by Houwe l ing ' s Nurseries (Delta, B C , Canada). 2.2.3 Greenhouse whiteflies Greenhouse whitef l ies obtained f rom App l ied B ionomics L t d . (Sidney, B C , Canada) originated f rom a commercia l colony maintained on tobacco plants for more than 10 years without any pesticide exposure. Adu l t whitef l ies were transferred to three-week-old tomato plants (Lycopersicon esculentum M i l l cv. Clarance) in the greenhouse inside fine mesh cages that a l lowed air circulat ion but prevented insects f rom escaping. 2.2.4 Phytoseiulus persimilis Athias-Henriot and Encarsia formosa Gahan Predatory mites and parasitic wasps were purchased f rom A p p l i e d B ionomics (Sidney, B C , Canada). Predatory mites were transferred to a spider mite colony maintained on tomato plants caged in the greenhouse. Parasit ic wasps were introduced to a whitef ly colony maintained on caged tomato plants inside the greenhouse. 24 2.2.5 Plant material Three-week-old tomato plants (Lycopersicon esculentum M i l l cv. Clarance) provided by Houwe l ing ' s Nurser ies (Delta, B C , Canada) were transferred to plastic pots containing a mixture o f regular peat (50%), fine bark (25%) and pumice (25%) provided by West Creek Farms (Langley, B C , Canada) in greenhouse at the Univers i ty o f Br i t ish Co lumbia . 2.2.6 General growing condit ions for plants, mites and whiteflies Plants infested wi th mites or whitefl ies were kept inside isolated cages wi th in the greenhouse at 24± 6°C, 40 -60% relative humidity (RH) and under natural daylight. Plants were irrigated three times per week, two times wi th water and one t ime wi th water-soluble fert i l izer (Peters E X C E L 15-5-15 Ca l -Mag) (The Scotts Company , Marysv i l l e , O H , U S A ) . Adu l t female mites were transferred to clean plants, a l lowed to oviposit for 48 hours, and then removed f rom the plant. Development o f these eggs resulted in a cohort o f evenly aged mites that were used for al l bioassays. Adu l t whitef l ies were used for al l bioassays. 2.2.7 Calculating lethal concentration 50 ( L C 5 0 ) Different bioassay methods were used for spider mites and whitef l ies. Fo r spider mites a leaf disc paint ing method was used for calculat ing L C 5 0 o f the rosemary o i l and three pesticides. Tests were conducted in disposable plastic Petri dishes (3 cm diameter). Mi tes were treated wi th six nominal concentrations (2.5, 5, 10, 20, 40 and 80 ml litre "') o f the essential o i l or the commercia l pesticides and their blank formulat ion (only for EcoTro l ) , using a spreader sticker adjuvant (Latron B-1956, 60 mg litre "') (Rohm and Haas, Phi ladelphia, P A , U S A ) diluted in dist i l led water. L e a f discs (3cm diameter), were 25 cut f rom leaves o f greenhouse-grown plants using a cork borer. A 20 u L al iquot o f each concentration was painted on the under side o f the leaf d isc wi th a micropipette g iv ing concentrations o f • (6.25, 12.50, 25.10, 50.21, 100.43 and 200.87 f i g / cm 2 ) . A f te r dry ing at room temperature for 5 minutes, each disc was placed in the bottom o f a Petri d ish atop a 3 c m diameter disc o f Whatman N o . 1 fi lter paper wetted wi th 50 u L dist i l led water (Figure 2.1). F i ve adult female spider mites were introduced into each Petr i d ish and the covered dishes were p laced in a growth chamber at 26±2 °C, 55 -60% R H and a 16/8h L D photoperiod. Morta l i ty was determined under a dissecting microscope 24 hours after treatment. M i tes were considered dead i f appendages d id not move when prodded wi th a fine paintbrush. Cont ro l mites were he ld on leaf discs painted w i th the carrier solvent alone. A l l treatments were repl icated f ive t imes. A fumigat ion chamber was used to test fumigant toxic i ty o f rosemary o i l to adult whitef l ies. Tests were conducted in disposable plastic containers (4cm diameter x 6.5 c m height). Whi tef l ies were treated w i th same s ix nominal concentrations (2.5, 5,10,20,40 Figure 2.1. Leaf disc painting method 26 and 80 m l litre "') o f the essential o i l , using 7 0 % aqueous methanol as the carrier solvent. A cotton pad (5cm diameter) was placed in the bottom o f the plastic container and a Whatman N o . l f i l ter paper were placed and pushed on top o f the cotton pad. A 50 \i\ aliquot o f each concentration was added to the filter paper with a micropipette producing concentrations o f H (1.353, 2.713, 5.432, 10.864, 21.729 and 43.459 fxg/cm 3 ) . Another filter paper was placed on top o f the previous one with a 1 cm gap to prevent insects f rom having direct contact wi th the treated paper. Fifteen adult whitef l ies were introduced to each container. Consider ing the fact that rosemary o i l contains compounds that are highly volati le, bioassays were conducted in two different ways. In one group, containers were covered wi th a plastic l id to trap a l l the volati les (closed-container). In the other group, containers were covered wi th a dense net to prevent whitef l ies f rom escaping but al lowed volati les to evaporate (open-container) (Figure 2.2). A leaf disc paint ing method was also used to measure the contact toxici ty o f rosemary o i l to whitef l ies using plastic containers instead o f Petri dishes. Containers were placed in a growth chamber at 26±2 °C, 55-60% R H and a 16/8 h L D photoperiod. Mortal i ty was determined under a dissecting microscope 24 hours after treatment. Whitef l ies were considered dead i f appendages d id not move when prodded wi th a fine paintbrush. Contro l whitef l ies were held in containers treated wi th the carrier solvent alone (70% aqueous methanol). A l l treatments were replicated f ive t imes. 27 Figure 2.2. Fumigation chamber (open and closed container) A n electronic micro-sprayer was developed to measure the direct contact toxici ty o f the toxicants to test organism mimick ing spraying practices inside greenhouses (Append ix one). Tox ic i t y o f rosemary o i l to the predatory mite P. persimilis and to the parasitic wasp E. formosa was measured using a leaf disc painting method, a direct contact method and a fumigat ion method. For predatory mites, ~50 spider mite eggs were placed onto each leaf disc (3cm diameter) as food source. Adu l t predatory mites were used for bioassays. Tomato leaves containing both spider mites and predatory mites were placed inside a Petri dish (10cm diameter) on top o f an ice pack inside a Styrofoam box in order to immobi l i ze the predators during bioassay. Adu l t parasit ic wasps were col lected by an aspirator and transferred in sealed plastic test tubes. Fo r easier handl ing, they were kept at 5°C for 2 minutes pr ior to bioassay to immobi l ize them. F i ve adult mites and five adult wasps were then transferred to each fumigat ion chamber. E a c h treatment was replicated 5 times. Direct contact toxic i ty o f the commerc ia l pesticides Hexacide™ (7.5 m l litre " ' ) , 28 EcoTrol™ (7.5 m l litre ) or Sporan™ (7.5 m l litre " ' ) against predatory mites and spider mites were measured using the electronic micro-sprayer. F i ve adult predatory mites were transferred to a leaf disc (3cm diameter) containing ~50 spider mite eggs and then sprayed wi th the sprayer. F ive adult female spider mites were put on leaf discs and then sprayed wi th sprayer. Controls were sprayed wi th carrier solvent alone (70% aqueous methanol). A l l treatments were kept inside a growth chamber at the same conditions described above. Morta l i ty was measured 24 hours after treatment. Mi tes or wasps were considered dead i f they did not move their appendages when prodded with a paintbrush. Morta l i ty in control groups was corrected by Abbot t ' s formula. Each treatment was replicated f ive times. 2.2.8 Residual toxicities Residual toxici ty o f rosemary o i l against greenhouse whitef l ies and parasitic wasps and o f three rosemary oi l-based pesticides against two-spotted spider mites and predatory mites was measured. Three- week-old tomato plants were sprayed indiv idual ly wi th rosemary o i l (10 ml litre " ' ) , Hexacide™ (7.5 m l litre " ' ) , EcoTrol™ (7.5 m l litre "') or Sporan™ (7.5 m l litre ~')[each plant received ~ 80 ± 10 g o f sprayed material]. A spreader sticker adjuvant (Latron B-1956, 60 mg litre "') di luted in dist i l led water used as the carrier solvent. Contro l plants were sprayed wi th carrier solvent alone (n= five plants for each treatment). Treated and un-treated plants then placed randomly on a greenhouse table. L e a f discs (3cm diameter) were cut f rom each indiv idual plants 1 and 24 hours after spraying. L e a f discs were placed inside a Petri dish or a plastic container as described before. F i ve adult female spider mites or five adult predatory mite plus ~50 spider mite eggs (as a food source for predators) were placed on the sprayed surface o f 29 the leaf disc. Fifteen adult whiteflies or fifteen adult parasitic wasps were placed inside the container. Covered Petri dishes and containers were held inside a growth chamber for 24 hours at 26±2 °C, 55-60% RH and a 16/8h LD photoperiod. Mortality was determined under a dissecting microscope 24 hours after treatment. Whiteflies, wasps, predators or mites were considered dead if appendages did not move when prodded with a fine paintbrush. All treatments were replicated five times. 2.2.9 Cho ice test b ioassay for spider mites Choice test bioassays were conducted for two-spotted spider mites using rosemary oil. Two leaf discs (3cm diameter) were cut from tomato plants and placed on top of a wetted Whatman No. 7 filter paper placed inside a disposable plastic Petri dish (10cm diameter). One leaf disc was painted with a 20 uL aliquot of rosemary oil (10 ml litre "'; « 25 |xg/cm2) dissolved in a spreader sticker adjuvant (Latron B-1956, 60 mg litre - 1) diluted in distilled water as the carrier solvent and the other one was treated with the carrier solvent alone. Thirty female adult mites were placed in the middle of the Petri dish between the two leaf discs (Figure 2.3). The number of mites found standing on each leaf disc was counted under a dissecting microscope after 1,12,24 and 48 hours. Numbers of eggs on treated and non-treated leaf discs were counted at the end of experiment at 48hours. All treatments were replicated 10 times. Figure 2.3.Choice test bioassay 30 2.2.10 Oviposit ion choice test bioassay for whiteflies An oviposition choice test was conducted for whiteflies using rosemary oil (10 ml litre "'). Styrofoam containers used for packing 4 * 1 solvent bottles, were used as oviposition cages for whiteflies (31). Each Styrofoam container provided four cages (cells) (24-cm length, 16.5-cm diameter). A single plastic mesh and a dense net were used to cover the top of the container and were secured by thumbtacks to prevent whiteflies from escaping. Thirty adult whiteflies were introduced to each cage containing a control and treated tomato leaf. Each leaf (approximately containing seven leaflets) was sprayed with rosemary oil (10 ml litre or carrier solvent (70% aqueous methanol) alone as control on both sides of the leaf. After drying for 30 min, leaves were placed inside a container filled with agar solution (3 gr litre "') (Figure 2.4). Boxes were placed inside a growth chamber at 26±2 °C, 55-60% RH and a 16/8h LD photoperiod. Eggs were counted on each leaf at 24,48 and 72 hours after introduction of whiteflies. Treatments were randomized among cages. All treatments were replicated eight times (total of 6 boxes). C B B A A C C A B A A B C B A C A C B C C B B A Figure 2.4. Oviposition cages - A = after 24 hours, B = after 48 hours, C = after 72 hours 31 2.2.11 Trans-laminar activity of commercial pesticides Five-week-o ld tomato plants were sprayed from above wi th Hexacide™ (7.5 m l litre " ' ) , EcoTrol™ (7.5 m l litre "') or Sporan™ (7.5 m l litre - 1 ) using a spreader sticker adjuvant (Latron B-1956, 60 m g litre " ') di luted in dist i l led water as the carrier solvent (each plant received ~ 80 ± 10 g o f sprayed material). L e a f discs (3cm) were cut f rom tomato plants and were placed on top o f a wetted f i l ter paper d isc (as described above) wi th either the upper surface (sprayed) or undersurface (not sprayed) fac ing up (Figure 2.5). F ive adult female spider mites were introduced into each Petri d ish and the covered dishes were p laced in a growth chamber at 26±2 °C , 5 5 - 6 0 % R H and a 16/8h L D photoperiod. Morta l i ty was determined under a dissecting microscope 24 hours after treatment as described above. Figure 2.5. Trans-laminar effects of rosemary oil-based pesticides 2.2.12 Data Ana lys is Morta l i ty observations were analyzed using the S P S S program (Chicago, I L , U S A ) , version 11.5 for analysis o f variance ( A N O V A ) . Tukey ' s test was used to compare means. Probit analysis was used to determine L C 5 0 , using the E P A probit analysis program version 1.5. Abbo t t ' s formula was used to correct mortal i ty in controls. 32 2 .3 Results 2.3.1 Lethal concentration 50 ( L C 5 0 ) L C ^ o f pure rosemary o i l was 13.19 m l litre " ' ( s 33.09 ng/cm 2 ) for spider mites (Table 2.1). Hexac ide (containing 5 % rosemary oil) and E c o T r o l (containing 10% rosemary oi l) were found to be two times more active than Sporan (containing 18% rosemary oi l ) . A l though Sporan contains more rosemary o i l as active ingredient it showed lower activity against mites. This might be due to difference in formulat ion. N o mortality was observed in control mites treated with carrier solvent. In fumigat ion tests against whitef l ies, rosemary o i l in closed containers was three times more toxic than in open containers. The results also showed that the rosemary oi l is more toxic to adult whitef l ies as a fumigant than as a contact toxicant. Rosemary o i l was less toxic to P.persimilis than to spider mites. Rosemary was more toxic to E. formosa through direct contact than v ia fumigat ion. Tox ic i t y to parasitic wasps was two times greater in closed containers than in open containers (Table 2.1). 33 Table 2.1 .Toxicity of rosemary oil and three rosemary oil-based pesticides to two-spotted spider mite, greenhouse whitefly, predatory mite and parasitic wasp. Ec-Blank= EcoTrol blank fomulation without rosemary oil, TSM= Two-spotted spider mite, WF= Whitefly, PP= Phytoseiulus persimilis, EF= Encarsia formosa, N= Number of replicates for each tested concentration (total of 6 concentrations), CI= confidence interval, N/A= not applicable Toxicant Organism Bioassay method N L C 5 0 (ml litre 2 X Value 9 5 % CI [ig/cm2 Lig/cm 3 Rosemary TSM Leaf disc painting 5 13.19 2.139 10.05-17.78 33.09 N/A Hexacide™ TSM Leaf disc painting 5 4.01 2.583 2.36-5.46 10.05 N/A EcoTrol ™ TSM Leaf disc painting 5 5.51 1.358 4.03-7.06 13.79 N/A Ec-Blank TSM Leaf disc painting 5 82.14 0.905 57.52-165.5 220.38 N/A Sporan ™ TSM Leaf disc painting 5 11.44 1.623 7.56-15.91 28.70 N/A Rosemary WF Fumigation-Close 5 4.93 0.571 3.27-6.39 N/A 2.68 Rosemary WF Fumigation-Open 5 18.26 0.565 10.66-31.78 N/A 9.92 Rosemary WF Leaf disc painting 5 27.03 4.107 22.26-33.09 N/A 14.68 Rosemary PP Leaf disc painting 5 16.62 3.163 13.53-20.71 41.73 N/A Rosemary EF Leaf disc painting 5 9.51 1.419 5.84-12.91 N/A 5.16 Rosemary EF Direct spray 5 5.49 0.644 3.15-7.39 N/A N/A Rosemary EF Fumigation-Close 5 5.81 1.288 4.00-7.62 N/A 3.15 Rosemary EF Fumigation-Open 5 10.21 1.394 7.80-13.19 N/A 5.54 Based on direct contact toxic i ty, three commercia l pesticides at their recommended label rate produced no mortality among predatory mites indicat ing that two-spotted spider mites are more susceptible to rosemary o i l . N o mortality was observed in control mites (Figure 2.6). 34 Figure 2.6. Efficacy (% mortality) of three commercial rosemary oil-based pesticides directly sprayed on P. persimilis (PP) and T. urticae (TSM) on tomato plants. TSM= two-spotted spider mite, PP= P. persimilis. Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (FO.05, F(7,32)= 36.196) 2.3.2 Residual toxicity Residues o f al l three pesticides were found to be moderately toxic to spider mites within the first hour after spraying. However , toxicity decreased signif icant ly after 24 hours. There was a signif icant interaction between the toxici ty o f residues and time [F(3,32)= 7.203, p<0.05]. Th is result clearly shows that rosemary o i l is not persistent in the environment due to its volati le nature (Figure 2.7). N o mortality was observed among controls. Residues o f three commercia l pesticides did not show statistically signif icant toxicity to P. persimilis (Figure 2.8). T ime had a signif icant main effect on the toxicity o f the residues [F (1,32) = 5.538, p< 0.05] whi le no significant difference was found among 35 pesticides (F (3,32) = 1.026, p> 0.05 (p= 0.394)). There was no interaction between time and pesticides (F (3,32) = 1.026,/?> 0.05 (p= 0.394)). 24hrs Hexacide EcoTrol Sporan Control Figure 2.7. Efficacy (% mortality) via residual toxicity of three rosemary oil-based pesticide to two-spotted spider mite on tomato plants. EC=EcoTrol, HE= Hexacide, SP= Sporan. Bars representing means (± SE), n=5 replicates with 5 adult female mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (/><0.05, F(7,32)= 18.836) 100 T 80 4 • 1 hr 24hrs Hexacid Ecotrol Sporan Control Figure 2.8. Efficacy (% mortality) via residual toxicity of three rosemary oil-based pesticides at their recommended label rate to P. persimilis on tomato plants. Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. 36 Rosemary o i l residue was slightly toxic to whitefl ies wi th in the first hour after spraying but not signif icant ly toxic after 24 hours (Figure 2.9). N o mortality was observed in control whitef l ies. 100 -I 80 -60 -co o 40 -20 - b n ^ rosemary 1% lhr 24hrs Control Figure 2.9. Efficacy (% mortality) via residual toxicity of rosemary oil 1% to greenhouse whitefly on tomato plants. Bars representing means (± SE), n=5 replicates with 15 adult whiteflies per replicate. Bars marked with the same letter do not differ significantly, Tukey (/><0.05, F (3,16)= 18.113) The results indicate that rosemary o i l residue is considerably toxic to E. formosa within the first hour but that toxici ty decreased almost three folds after 24 hours (Figure 2.10). Bo th t ime (F ( l ,16)=35.588,p<0.05) and treatment (F (1,16) = 84.390, p<0.05) had signif icant main effects on toxici ty o f the residues. There was a signif icant interaction between time and treatment (F (1,16)= 21.844,/?<0.05). 37 100 80 • Rosemary 1% Control 1 hour 24 hours T i m e Figure 2.10. Efficacy (% mortality) via residual toxicity of rosemary oil 1% to E. formosa on tomato plants. Bars representing means (± SE), n=5 replicates with 15 adult mites per replicate. 2.3.3 Choice tests Rosemary o i l has a signif icant deterrent effect on mites but this effect decl ined over t ime. There was a signif icant interaction between the location o f mites and time (F (3,72)= 81.203, p< 0:05) and treatment had a significant main effect (F (1,72)=985.289, p< 0.05). Dur ing first 12 hours mites aggregated more on the control disc or at locations far f rom the treated disc wi th in the test arena. Af ter 24 hours, they started to spread on both discs and (Figure 2.11), after 48 hours they were almost equally dispersed on both discs. Mi tes la id greater number o f eggs on the control discs than on treated discs. A l though they started to oviposit eggs on both treated and control discs after 12 hours, the final numbers o f eggs after two days was signif icantly higher on control discs (Figure 2.12). 38 ] T rea ted I C o n t r o l 1 hr 12hrs 24hrs 48hrs Figure 2.11. Number of two-spotted spider mites staying on leaf discs when given a choice between a treated and non-treated leaf disc with rosemary oil 1%, Bars representing means (± SE), n=10 replicates with 30 adult spider mites per replicate. T r e a t e d con t ro l Figure 2.12. Number of two-spotted spider mites eggs on leaf discs when given a choice between a treated and non-treated leaf disc with rosemary oil 1%. (F (1,18)=34.503,p<0.05). Bars representing means (± SE), «=10 replicates with 30 adult spider mites per replicate. 39 SI Results f rom whitef ly oviposi t ion choice tests indicated that whitef l ies la id gnif icantly more eggs on control leaves than on treated leaves wi th in al l three intervals. Both rosemary o i l (F (l,42)=77.931,/><0.05) and time (F (2,42) =29.366, p<0.05) had main effects on oviposi t ion rate. There was a significant interaction between time and pesticide (F (2,42)=6.273,/?<0.05). S imi la r to spider mites, rosemary oi l deters whitef l ies, however, the effect does not diminish as fast is it d id for spider mites. Whitef l ies laid three times more eggs on control leaves than on treated leaves after 48 hours (Figure 2.13). 24hrs J Rosemary 1% I Control 48hrs Time 72hrs Figure 2.13. Number of greenhouse whitefly eggs on tomato leaves when given a choice between a treated and non-treated leaf with rosemary oil 1%. Bars representing means (± SE), n=8 replicates with 30 adult whiteflies per replicate. 40 2.3.4 Trans-laminar activity N o mortality was observed among mites that were placed on the un-sprayed surface o f the leaf discs (Figure 2.14) whi le signif icant toxici ty was observed among mites that were placed on the sprayed surface, indicating that rosemary oi l does not have trans-laminar activity. Figure 2.14. Efficacy (% mortality) of three rosemary oil-based pesticides applied at their label rate on tomato leaves when exposed to two-spotted spider mites that were placed either on upper surface or under surface of the leaf discs. Bars representing means (± SE), n=5 replicates with 5 adult female mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (P<0.05, F(3,16)= 36.196). 41 2 .4 Discussion 2.4.1 Efficacy against pests M y results clearly indicate that rosemary o i l can be considered an acaricide /insecticide against the two-spotted spider mite and the greenhouse whi tef ly, causing complete mortality in the laboratory at concentrations that cause no phytotoxici ty to host plants (Chapter three). Rosemary o i l was found to be more toxic to spider mites as a contact toxicant whi le it was more effective against whitefl ies as a fumigant. C h o i et al. (11) evaluated the toxici ty o f 53 essential o i ls including rosemary against eggs and adults o f two-spotted spider mites as fumigants. Rosemary o i l was not very toxic (mortality <60%) comparing to caraway seed, ci tronel la Java, lemon, eucalyptus, pennyroyal and peppermint o i l , which were h ighly toxic (mortality > 90%) to the tested mites. However , Sampson et al. (32) tested 23 different essential oi ls including rosemary o i l against turnip aphids and found most acted as contact toxicants causing mortality in aphids after 1 hour. When tested against whitef l ies, toxici ty o f rosemary o i l varied signif icant ly between open and closed containers. This result leads to the conclusion that rosemary o i l activity can be affected by its volat i l izat ion and by environmental condit ions. Thus, in greenhouses it might not be as toxic as it is in a laboratory setting. Tunc et al. (13) found that vapors o f some essential o i ls were toxic to the cotton aphid and the two-spotted spider mite. They found that the quantity o f oi l needed for pest control differed in heated and cooled greenhouses compared to ambient greenhouses under plastic. They suggested that essential o i l particles suspended in the air might be lost due to air circulat ion or adherence to surfaces inside the greenhouse. 42 2.4.2 Effects on bio-controls Predatory mites P. persimilis are less susceptible to rosemary o i l than twospotted spider mites (Table 2.1). When both mites were directly sprayed w i th different pesticides containing rosemary o i l , no mortality was found among predators whi le up to 6 0 % mortality was observed in spider mites (Figure 2.6). These results are very promis ing in terms o f compatibi l i ty o f these pesticides in an I P M program for control l ing spider mites. This difference in toxic i ty level between spider mites and predators might be due to differential metabol ism o f rosemary o i l -based pesticides in predatory and phytophagous mites. A n important aspect o f acaricides research is identif ication o f suitable and novel target sites. Li t t le is known about the mode and site o f action o f rosemary o i l and other plant essential oi ls in the mites. The octopaminergic nervous system is considered to be the site-of-action o f certain essential o i ls in the Amer ican cockroach (33) and fruit f ly (34), but this may not be the case for two-spotted spider mite and there is a possibi l i ty that the essential o i ls have more than one site o f action since they are complex mixtures. Un l i ke predatory mites, E. formosa is more susceptible to rosemary o i l both in fumigation and leaf disc paint ing bioassays compared to the greenhouse whitef l ies. E. formosa is susceptible to more than one hundred crop protection products. However , there are some selective compounds that have fewer side effects on parasitic wasps (16). F rom my results, I conclude that rosemary o i l can affect the adult parasitoid i f hit directly by a sprayed pesticide. However , immature parasitoids developing inside whitef ly nymphs might not be affected by pesticide (Appendix two). O n the other hand E. formosa is highly mobi le and might escape direct spray exposure. In order to reduce side effects o f rosemary o i l -based pesticides to parasitic wasps, growers can either apply the pesticide 48-72 hours prior to parasitoid release or after whitef ly nymphs have been parasit ized. 43 2.4.3 Persistence in the environment A s Dekeyser (35) mentioned, new pesticides should be safer towards non-target organisms and have shorter environmental persistence than exist ing products. M y results clearly indicate that the rosemary oil-based pesticides are not environmental ly persistent. In al l experiments, toxici ty o f residues signif icantly decl ined after 24 hours. Essential o i ls are mixture o f odorous and volati le compounds that can easily break down in the environment (27). M a n y environmental factors affect the breakdown o f essential o i ls, most importantly, temperature and light. Essential o i ls may break down faster at higher temperatures and wi th direct light exposure. L im i ted residual toxici ty is an important advantage for these pesticides. Growers can apply them closer to harvest t ime. It is also important to have a safer environment for bio-control agents wi th fewer pesticide residues. O n the other hand, quick breakdown o f essential oi ls in the environment reduces the risk o f pesticide resistance in the pest population. 2.4.4 Repellent effects In addit ion to pesticidal properties, sub-lethal effects (repellent, deterrent, antifeedant) o f many plant essential oi ls have been reported against several pests (27). Trongtokit et al. (36) tested 38 different essential oils on human subjects as repellents to mosquitoes. They found that diluted essential oi ls couldn' t provide a satisfactory level o f repellence whi le undiluted o i l could provide effective repellence for up to 2 hours. A m o n g essential o i ls that they tested, c love o i l provided the longest duration o f repellence (up to 4 hours) against mosquitoes. In another study, Trabouls i et al. (37) found terpineol and 1,8-cineole were very effective against Culex pipiens molestus bites for 1.6 and 2 hours respectively, whi le Zhang et al. (38) reported repellent effects o f 44 ginger o i l to Bemisia argentifolii on tomato plants. Kosch ier et al. (28) reported that essential oi ls f rom plants in the mint fami ly could affect host plant selection and acceptance by Thrips tabaci L indeman. Plant essential oi ls cannot only repel arthropods, but they also can affect vertebrate behavior. C la rk et al. (39) reported that some essential o i ls inc luding rosemary, can cause violent undirected locomotory behavior in brown tree snakes, Bioga irregularis, therefore the oi ls can be used as snake repellents. Acco rd ing to my choice tests results, rosemary oi l is signif icant ly repellent to two-spotted spider mites. It repelled mites for about 6 hours and then mites gradually started to move toward the treated discs. However, both mites and whitef l ies preferred untreated leaves for oviposi t ion. Repel lent effects o f rosemary o i l cannot be considered as a stand-alone control method but can be combined wi th other methods to improve pest management strategies. For instance, rosemary o i l application might be combined wi th trap-plants as a "push-p u l l " tactic to repel pests from major host plants and attract them to trap-plants. 2.4.5 Rosemary oil as a pesticide: advantages and limitations Rosemary o i l can be considered a good option for small-scale pest control in greenhouses. These pesticides meet the major characteristics o f an IPM-compat ib le pesticide. A s my results indicate, under laboratory condit ions, rosemary o i l can provide complete mortality in pest populations at concentrations that are not harmful to host plants (Chapter four). It is less toxic to predatory mites and breaks down very fast in the environment. Other advantages o f essential oil-based pesticides include low mammal toxici ty, safety to terrestrial and aquatic species (with some exception) (21 -22), rapid pest 45 mortality due to their neurotoxic mode of action and low cost, a result o f their extensive wor ldwide use as fragrances and f lavor ing (40). F ina l ly , an important characteristic o f rosemary o i l is its complex chemical composit ion. L i k e other essential o i ls, natural rosemary o i l is a complex mixture o f terpenoids. Consider ing that target site resistance is an important problem for mite control , it is less l ikely that mites w i l l evolve resistance to a mixture o f different active compounds than to an acaricide based on a single active ingredient. It has been reported that green peach aphids Myzuspersicae Sulzer developed resistance to pure azadirachtin (the major ingredient o f neem insecticide) but not to a refined neem seed extract containing the same absolute amount o f azadirachtin but wi th many other constituents present (41). A l though rosemary oi l-based pesticides meet most requirements o f I P M -compatible pesticides, they have some disadvantages that must be considered before extensive appl icat ion. Fo r example, rosemary o i l does not have trans-laminar activity. Both spider mites and whitef l ies aggregate on the under-surface o f leaves so they might not be affected by rosemary o i l sprays to the upper-surface o f the plants. O n the other hand, the sprayers themselves might modi fy the eff icacy o f the rosemary o i l . Ebert et al. (42) reported differences in the eff icacy o f spinosad and azadirachtin when appl ied wi th different application equipment (carbon dioxide powered h igh-volume sprayer, D R A M M cold fogger or an electrostatic spraying system). A s described before essential oi ls might breakdown faster at high temperatures so, some application equipment might damage the o i l during spraying and reduce its eff icacy. In order to be effective, the pesticide must hit the target, so appl icat ion must be conducted in such a way that it provides complete coverage to the whole plant. 46 In addit ion to application methods, effects o f environmental factors on the eff icacy o f the pesticide must be addressed. For instance, temperature, moisture and light effects on toxic i ty and degradation o f pesticides should be studied. Tox ic i t y o f the o i l might also vary on different host plants due to synergistic or antagonistic effects o f plant secondary metabolite wi th the constituents o f the oi l (Chapter three). 47 References 1) Zhang, Z . (2003). Mites of Greenhouses: identification, biology and control, publishing. Wal l ing ford : C A B I International, pp 54-61. 2) Jeppson, L . R . , Ke i fe r , H . H . & Baker, T . W . (1975). Mite injurious to economic plants, Berke ley : Univers i ty o f Cal i forn ia press, C A , pp 370-376. 3) Cranham, J . E . , & He l le , W. , (Eds). (1985). Pesticide resistance in Tetranychidae. In: World crop pest - spider mites: their natural enemies and control, Amsterdam: Elsevier , pp 405-421. 4) The Database o f Arthropods Resistance to Pesticides. (2004). Michigan State University-Center for integrated plant systems. 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Integrated pest management o f twospotted spider mite Tetranychus urticae on greenhouse roses using petroleum spray o i l and the predatory mite Phytoseulus persimilis. Experimental & Applied Acarology, 25: 37-53. 15) Murphy , G . D . , Ferguson, G . , F ry , K., Lambert, L. , M a n n , M . , & Matteoni , J . (2002). The use o f b io logical control in Canadian greenhouse crops. Integrated Control in Protected Crops, Temperate Climate, IOBC/wprs Bulletin, 25(1): 193-196. 16) Hodd le , M . S . , VanDr iesche, R . G . , & Sanderson, J .P. (1998). B io logy and use o f the whitef ly parasitoid Encarsia formosa, Annual Review of Entomology, 43 : 645-669. 49 17) Yatagai M . (1997). M i t i c ida l activities o f tree terpenes. Current Topics of Phytochemistry, 1: 87-97. 18) Roe , F. J . C . (1965). Chron ic toxici ty o f essential oi ls and certain other products o f natural or ig in. Food and Cosmetics Toxicology, 33: 311-324. 19) Cockayne, S. E . , & Gawkrodger, D. J . (1997). Occupat ional contact dermatitis in an aromatherapist. Contact Dermatitis, 37: 306-309. 20) Hjorther, A . B . , Christophersen, C , Hausen, B . M . , & Menne , T. (1997). Occupat ional al lergic contact dermatitis f rom carnosol, a natural ly-occuring compound present in rosemary. Contact Dermatitis, 37: 99-100. 21) Kumar , A . , Dunke l , F. V . , Broughton, M . J . , & Sriharan, S. (2000). Effect o f root extracts o f mexican mar igold, Tagetes minuta (Asterales: Asteraceae), on six nontarget aquatic macroinvertebrates. Environmental Entomology, 29: 140-149. 22) Wager-Page, S. , & Mason , J . R. (1997). Ortho-aminoacetophenone, a non-lethal repellent: The effect o f volat i le cues vs. direct contact on avoidance behavior by reodents and birds. Pesticide Science, 46: 55-60. 23) Burt, S. (2004). Essential o i ls: Their antibacterial properties and potential application in food-a review. International Journal of Food Microbiology, 94: 223-253. 24) Managena, T., & M u y i m a , N . Y . O . (1999). Comparat ive evaluation o f the antimicrobial activit ies o f essential oi ls o f Artemisia afra, Pteronia incana and Rosmarinus officinalis on selected bacteria and yeast strain. Letters in Applied Microbiology, 28 : 291-296. 50 25) Papachristos, D .P . , & Stampoulos, D . C . (2004). Fumigant toxic i ty o f three essential oi ls on the eggs o f Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). Journal of Stored Products Research, 40: 517-525. 26) Tunc, I., Berger, B . M . , Er ler , F., & Dag l i , F. (2000). Ov ic ida l activity o f essential oi ls f rom plants against two stored-product insects. Journal of Stored Products Research, 36: 161-168. 27) Isman, M B . (2000). Plant essential oi ls for pest and disease management. Crop Protection, 19: 603-608. 28) Kosch ier , E . A . , & Sedy, K . A . (2003). Labiate essential o i ls affecting host plant selection and acceptance o f Thrips tabaci L indeman. Crop Protection, 22: 929-939. 29) C h o i , W . , Lee , S., Park, H . , & A h n , Y . (2004). Tox ic i ty o f plant essential oi ls to Tetranychus urticae (Aca r i : Tetranychidae) and Phytoseiulus persimilis (Acar i : Phytoseiidae). Journal of Economic Entomology, 97: 553-558. 30) M o m e n , F . M . , & A m e r , S . A . A . (1999). Effect o f rosemary and sweet marjoram on three predacious mites o f the fami ly Phytoseiidae (Acar i : Phytoseiidae). Acta Phytopathologica et Entomologica Hungarica, 34: 355-361. 31) Akhtar , Y . , & Isman, M B . (2003). Larva l exposure to oviposi t ion deterrents alters subsequent oviposi t ion behavior in generalist, Trichoplusia ni and specialist, Plutella xylostella moths. Journal of Chemical Ecology, 29: 1853-1870. 32) Sampson, B . J . , Tabanca, N . , K i r imer , N . , Demi rc i , B . , Baser, K . C , Khan , I. A . , Spiers, J . M , & Wedge, D. E . (2005). Insecticidal activity o f 23 essential oi ls and their major compounds against adult Lipashis pseudobrassicae Dav is (Aphid idae: Homoptera). Pest Management Science, 61: 1122-1128. 51 33) Enan, E . (2001). Insecticidal activity o f essential o i ls : octopaminergic site o f action. Comparative Biochemistry and Physiology Part C, 130: 325-327. 34) Enan, E . (2005). Mo lecu la r and pharmacological analysis o f an octopamine receptor f rom Amer i can cockroach and fruit f ly in response to plant essential o i ls. Archives of Insect Biochemistry and Physiology, 59: 161-171. 35) Dekeyser, M . A . (2005). Acar ic ide mode o f action. Pest Management Science, 61: 103-110. 36) Trongtokit , Y . , Rnogsr iyam, Y . , Komalamisra , N . , & Apiwathnasorn, C . (2005). Comparat ive repellency o f 38 essential oi ls against mosquito bites. Phytotherapy Research, 19: 303-309. 37) Trabouls i , A . F., E l - H a j , S. , Tuen i , M . , Taoub i , K . , A b i Nader , N . , & M r a d , A . (2005). Repel lency and toxici ty o f aromatic plant extracts against the mosquito Culex pipiens molestus (Diptera: Cul ic idae) . Pest Management Science, 61 : 597-604. 38) Zhang, W. , M c A u s l a n e , H.J . , & Schuster, D. J . (2004). Repel lency o f ginger o i l to Bemisia argentifolii (Homoptera: Aleyrodidae) on tomato. Journal of Economic Entomology, 97: 1810-1318. 39) Clark , L., & Shiv ik , J . (2003). Aerosol ized essential o i ls and indiv idual natural product compounds as brown treesnake repellents. Pest Management Science 58: 775-783. 40) Isman, M . B . (2006). Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated wor ld . Annual Review of Entomology, 51: 45-66. 52 41) Feng, R. & Isman, M . B . (1995). Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae. Experientia, 51: 831-834. 42) Ebert, T . A . , Derksen, R., Downer, R. A . , & Krause, C . R. (2003). Compar ing greenhouse sprayers: the dose-transfer process. Pest Management Science, 60: 507-513. 53 Chapter three: Comparative toxicity of Rosmarinus officinalis L. essential oil and blends of its major constituents against Tetranychus urticae Koch (Acari: Tetranychidae) on two different host plants1 3.1 INTRODUCTION 3.1.1 Two-spotted spider mite The two-spotted spider mite, Tetranychus urticae K o c h , is one the most important pests o f fruit, vegetable and ornamental plants wor ldwide (1). The mite has been reported from about 1200 species o f plants (2), o f wh ich more than 150 are economical ly important (3). The economic threat posed by these mites is constantly increasing because o f the development o f pesticide resistance, and resurgence o f mite populations fo l lowing use o f non-selective synthetic pesticides that eliminate natural enemies such as predaceous mites and spiders (4). Spider mites have evolved resistance to more than 80 acaricides to date and resistance has been reported f rom more than 60 countries (5). In the U . S . A . in 2001, spider mites control programs cost approximately U S D 8 mi l l ion in cotton alone (National Cotton Counc i l o f Amer ica) . Spider mites impose a great expense to greenhouse growers wor ldwide in terms o f damage and control cost and are therefore considered one the most important pests o f greenhouses product ion. 3.1.2 Rosmarinus officinalis essential oil Plant essential oi ls are obtained through steam dist i l lat ion o f herbs and medicinal plants (6). These oi ls have been used traditionally as heal ing medicines in many countries and ancient people were also aware o f their pesticidal properties, however, only in recent ' M i resmai l l i , S. , Bradbury, R., and Isman, M . B . (2006). Pest Management Science: In press (Accepted on 27 September 2005) 54 years these oi ls have been commerc ia l ized as pest control products (7). Mos t o f these oi ls are environmental ly non-persistent, and non-toxic to humans (with some exceptions) (8-10), fish (with some exceptions) and wi ld l i fe (10-13). Rosemary (Rosmarinus officinalis L.) o i l has been tradit ionally used as a medicine for co l ic , nervous disorders and painful menstruation. Recent studies revealed that the rosemary o i l is an effective antibacterial agent, wh ich can control many food micro-organisms such as Listeria monocytogenes, Salmonella typhimurium, Escherichia coli 0 1 5 7 : H 7 , Shigella dysenteria, Bacillus cereus and Staphylococcus aureus (14). It also can inhibit the activity o f food spoilage bacteria and yeast strains (15). Rosemary o i l is relatively effective against insect and mite pests. It has been shown that the aromatic vapor o f rosemary has ov ic ida l and larv ic idal effects on several stored product pests (16-17) and the two-spotted spider mite (7) as a fumigant. The o i l can have sub-lethal effects as we l l , for example acting as a repellent to onion thrips, Thrips tabaci L i n d (18). Synthetic acaricides usually contain a single active compound; however, botanical pesticides such as plant essential oi ls are complex mixture o f several constituents. In the present study, we characterize the toxici ty o f rosemary o i l and its major constituents as residual acaricides against T. urticae. 55 3.2 MA TERIALS AND METHODS 3.2.1 Rosmarinus officinalis essential o i l 2 Pure Rosmarinus officinalis essential o i l (Intarome T O , Lot# 0213142MB-100%) was obtained f rom E c o S M A R T Technologies Inc. (Frankl in , T N , U S A ) . Ma jo r constituents o f the essential o i l were identif ied by gas chromatography/mass spectroscopy on a Var ian 3900 system with a Saturn 2100T ion trap mass selective detector (Walnut Creek, C A , U S A and using a W C O T fused si l ica 30m x 0.25 m m ID co lumn wi th a C P -Si l 8 C B low bleed M S coating, a 1 u.1 injection volume and pure hel ium as the carrier at 1.0 ml /min . The temperature program used was 80°C for 0.5 min , an increase o f 8.0°C/min for 8.0 m in , fo l lowed by an increase o f 50°C/min for 3.2 min . C innamic alcohol (S igma, St. Lou i s , M O , U S A ) was used as an internal standard. 3.2.2 Spider mites T w o colonies o f T. urticae were used in this study. The first colony was collected from the U B C horticulture greenhouse and reared on three-week-old green bush bean plants (Phaseolus vulgari cv . Speculator #24A Stokes). The second colony originated f rom a research colony maintained on tomato plants for more than f ive years without any pesticide exposure at Agr icu l ture and Ag r i -Food Canada, Agass iz , B C . These mites were reared on three-week-old vine tomato plants (Lycopersicon esculentum M i l l var. Clarance) provided by Houwe l ing 's Nurseries (Delta, B C , Canada). 3.2.3 General growing condit ions for plants and mites Plants contaminated wi th mites were kept inside an isolated greenhouse section at 24± 3°C, 4 5 - 6 0 % relative humidi ty (RH) under natural dayl ight. Plants were irrigated three times per week, two times wi th water and one time wi th water-soluble ferti l izer 2 Ana lyzed by Rod Bradbury at Ecosafe Natural Products Inc. (Saanichton, B C , Canada) 56 (Peters E X C E L 15-5-15 C a l - M a g ) (The Scotts Company, Marysv i l l e , O H , U S A ) . Adu l t female mites were transferred to clean plants, a l lowed to oviposit for 48 hours, and then removed f rom the plant. Development o f these eggs resulted in a cohort o f evenly aged mites that were used for al l bioassays. 3.2.4 Calculating lethal concentration 50 ( L C 3 0 ) of the oil A leaf disc paint ing method was used to determine L C 5 0 o f the rosemary o i l . Tests were conducted in disposable plastic Petri dishes (3 cm diameter). The bean colony o f mites was treated wi th six nominal concentrations [(2.5, 5,10,20,40 and 80 m l litre "') • (6.25, 12.50, 25.10, 50.21, 100.43 and 200.87 fxg/cm 2)] o f the essential o i l in 7 0 % aqueous methanol as the carrier solvent. The tomato colony was treated wi th the same six concentrations, using water plus a spreader sticker adjuvant (Latron B-1956, 60 mg l i tre" 1) as the carrier solvent. L e a f discs (3cm diameter), were cut f rom leaves o f greenhouse-grown plants using a cork borer. A 20 u L aliquot o f each concentration was painted on the under side o f the leaf disc with a micropipette. Af ter dry ing at room temperature for 5 m in , each disc was placed in the bottom o f a Petr i d ish atop a 3 cm diameter disc o f Whatman N o 1 filter paper wetted with 50 u L o f dist i l led water. F ive adult female spider mites were introduced into each Petri dish and the covered dishes were placed in a growth chamber at 26±2 °C, 55-60% R H wi th a 16/8h L D photoperiod. Mortal i ty was determined under a dissecting microscope 24h after treatment. Mi tes were considered dead i f appendages d id not move when prodded wi th a fine paintbrush. Control mites were held on leaf discs painted wi th the carrier solvent alone (70% aqueous methanol, or Latron B-1956 60 mg l i tre" 1). A l l treatments were replicated five times. 57 3.2.5 Comparative toxicities Based on the 100% lethal concentration and fo l lowing the natural composit ion of the o i l indicated by G C / M S (Table 3.1), individual constituents were tested at levels equivalent to those found in the L C 1 0 0 o f the o i l (20 m l litre " ' for beans and 40 m l litre for tomatoes) (Figures 3.2 and 3.3). Individual constituents (a-pinene 9 8 % , P-pinene 99%, 1,8-cineole 99%, p-cymene 99%, a-terpineol 97%, borny l acetate 9 7 % , borneol 99%, camphor 96%, d-l imonene 9 7 % and camphene 95%) were obtained f rom Sigma-A ld r i ch (St. Lou i s , M O , U S A ) . In order to identify the contribution o f each constituent to the toxici ty o f the o i l , we made a blend o f al l major constituents as we l l as blends each lacking one o f the ten major constituents (Figure 3.1). We compared the toxici ty o f the complete and incomplete blends to that o f pure rosemary o i l . In the next step, we made blends o f those constituents, wh ich contributed to the toxic i ty o f the o i l (active constituents) and compared them wi th those wh ich did not affect the toxic i ty (inactive constituents). The leaf disc paint ing method was used for a l l bioassays. 3.2.6 Data Analys is Mortal i ty observations were analysed using the S P S S program (Chicago, IL , U S A ) , version 11.5 for analysis o f variance ( A N O V A ) . Tukey ' s test was used to compare means. Probit analysis was used to calculate L C 5 0 , using the E P A probit analysis program version 1.5. 58 3.3 RESULTS 3.3.1 Essential oil constituents G C - M S analysis indicated that there are ten major constituents in the o i l , compris ing 92 .8% o f the total weight. 1, 8-Cineole was the most abundant compound (31.5%), fol lowed by camphor (20.0%) and a-pinene (17.5%) (Table 3.1). There are more than 40 other compounds in the rosemary o i l , wh ich are mostly monoterpenes, but their concentration in the o i l is very low (19). Table 3.1. Major constituents of rosemary oil (Intarome TO, Lot# 0213142MB-100%) Constituent % v/v Camphene 8.0 1, 8-Cineole 31.5 P-Pinene 6.8 Camphor 20.0 p-Cymene 0.9 Borneol 1.2 d-Limonene 3.7 a-Terpineol 1.1 Bornyl acetate 2.2 a-Pinene 17.5 Other compounds 7.2 3.3.2 Lethal concentration 50 of the oil The L C 5 0 o f rosemary o i l was 1% (10 ml litre ) (95% confidence interval (CI) = 6.95 - 13.11) for adult female spider mites reared on bean plants and 1.3% (13.0 m l litre "') (95% CI = 10.05 - 17.78) for those reared on tomato plants. Complete mortality 59 (100%) o f mites was obtained wi th a 2 % (20 ml litre "') concentration o f the o i l on bean plants and 4 % (40 ml litre "') on tomato plants. N o mortal i ty was observed in the controls. 3.3.3 Comparative toxicities of individual constituents and blends thereof For the bean host strain o f mites, bioassay o f single constituents revealed that two constituents (1,8-cineole and a-pinene) were significantly toxic at the tested concentration (P< 0.05), one (P-pinene) was slightly but not signif icantly toxic and the remaining seven (p-cymene, borneol, bornyl acetate, camphor, d-l imonene, camphene and a-terpineol) were slightly or non-toxic to mites (Figure 3.1) a a Figure 3.1. Efficacy (% mortality) of pure constituents of rosemary oil to two-spotted spider mite on bean plants at concentration equivalent to their proportion at 100% lethal concentration of the oil (2%). Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (PO.05, F(l 1,48)= 82.364) 60 When tested against tomato host strain o f mites, three constituents (camphor, p-cymene and camphene) were found non-toxic to mites, f ive (bornyl acetate, P-pinene, d-limonene, a-terpineol and borneol) were moderately toxic and two (a-pinene, 1, 8-cineole) were highly toxic (Figure 3.2). a Figure 3.2. Toxicity of pure constituents of rosemary oil to two-spotted spider mite on tomato plants at concentration equivalent to their proportion at 100% lethal concentration of the oil (4%). Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (PO.05, F(l 1,48)= 23.196) Bioassays wi th art i f ic ial mixtures showed that the greatest mortal i ty was obtained when al l ten constituents were present (full mixture). The mortality caused by the arti f icial mixture o f al l ten constituents d id not differ signif icant ly f rom that caused by pure rosemary o i l for either strain o f mites (P>0.05 (p= 0.989, Figures 3.3 and 3.4). In the bean host strain, component-el imination assays indicated that the absence o f 1,8-cineole or a-pinene caused the largest decrease in toxicity o f the blend. Removal o f 61 p-cymene, a-terpineol or bornyl acetate also had a significant effect (P< 0.05) on the toxicity o f the blend but less so than for 1, 8-cineole or a-pinene. Excluding five remaining constituents (camphor, camphene, borneol, d-limonene and p-pinene) f rom the mixtures did not significantly affect the toxicity o f the blends (Figure 3.3). Eliminated constituent Figure 3.3. Efficacy (% mortality) of different blends of pure constituents of rosemary oil to two-spotted spider mite on bean plants at concentration equivalent to their proportion at 100% lethal concentration (2%)- Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (PO.05, F(12,52)= 57.787) In the tomato host strain, a-pinene, 1, 8-cineole, a-terpineol and borny l acetate were found to contribute to the toxicity o f the oi l whereas P-pinene, p-cymene and borneol had only a moderate influence on the toxicity. Camphor , camphene and d- limonene were found to be inactive when tested individual ly and their absence did not have any effect on the toxici ty o f the mixture (Figure 3.4) 62 100 -T 80 -60 40 20 0 de de M° <& * .j? ^ <?• <& & ^ A J> 6' ^ 1 Eliminated constituent Figure 3.4. Efficacy (% mortality) of different blends of constituents of rosemary oil to two-spotted spider mites on tomato plants at concentration equivalent to their proportion at 100% lethal concentration (4%)- Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (PO.05, F(12,52)= 46.312) Our comparison between the toxici ty o f a mixture o f effective constituents versus a mixture o f non-effective constituents showed that the effective constituents alone are not as toxic as the ful l mixture o f al l constituents (both actives and inactives). The inactive constituent blend d id not cause any mortality in either strain, but when added to the active constituents blend, toxici ty became equivalent to the natural o i l (Figures 3.5,3.6 and 3.7). 63 a B M l BM2 BFM Rosemary Control oil 2% Figure 3.5. Efficacy (% mortality) of different blends of rosemary oil constituents to two-spotted spider mites on bean plants at concentration equivalent to their proportion in the 100% lethal concentration (2%)- BMl (Active) = a-Pinene + 1,8 Cineole + a- Terpineol + Bornyl acetate + p-Cymene , BM2 (Inactive) = b-Pinene + Borneol + Camphor + Camphene+ d- Limonen, BFM = Full mixture of all constituents, Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (P<0.05, F(4,20)= 43.636 Figure 3.6. Efficacy (% mortality) of different blends of rosemary oil constituents to two-spotted spider mites on tomato plants at concentration equivalent to their proportion in the 100% lethal concentration (4%)- TM1 (Active) = a-Pinene + 1,8 Cineole + a- Terpineol + Bornyl acetate, TM2 (Moderately active) = b-Pinene + Borneol + p-Cymene, TM3 (Inactive) = Camphor + Camphene+ d- Limonen, TFM = Full mixture of all constituents, Bars representing means (± SE), n=5 replicates with 5 adult mites per ) replicate. Bars marked with the same letter do not differ significantly, Tukey (/><() .05, F(5,24)= 83.867) 64 T M 1 + 2 T M 3 T F M R o s e m a r y o i l C o n t r o l 4% Figure 3.7. Efficayc (% mortality) of different blends of rosemary oil constituents to two-spotted spider mites on tomato plants at concentration equivalent to their proportion in the 100% lethal concentration (4%)- T M 1 (Active) = a-Pinene + 1,8 Cineole + a- Terpineol + Bornyl acetate, T M 2 (Moderately active) = b-Pinene + Borneol + p-Cymene, T M 3 (Inactive) = Camphor + Camphene+ d- Limonen, T F M = Full mixture of all constituents, Bars representing means (± SE), n=5 replicates with 5 adult mites per replicate. Bars marked with the same letter do not differ significantly, Tukey (/><0.05, F(4,20)= 54.706) 65 3.4 DISCUSSION 3.4.1 Rosemary oil as an acaricide Our results clearly indicate that rosemary o i l can be considered an acaricide against the two-spotted spider mite, causing complete mortality in the laboratory at concentrations that cause no phytotoxicity to host plants (Chapter four). Rosemary o i l and most o f other plant essential o i ls are environmentally non-persistent and break down easily in presence o f light. Some essential oi ls are not toxic to non-target organisms and can be used in conjunct ion with biological control. Furthermore, most plant essential oi ls including rosemary o i l are safe for humans and other mammals and many o f them are used as f lavorings in foods, beverages and medicines. In some rare cases, chronic exposure to rosemary o i l in high concentration has caused contact dermatitis (8-10) but acute toxici ty o f rosemary o i l to humans or other mammals has not been reported. L i k e other essential o i ls, natural rosemary o i l is a complex mixture o f terpenoids. Consider ing that targetsite resistance is an important problem for mite control , it is more probable that mites w i l l evolve resistance faster to an acaricide based on a single active ingredient than to a mixture o f different active compounds. It has been reported that green peach aphids Myzus persicae Sulzer developed resistance to pure azadirachtin (the major ingredient o f neem insecticide) but not to a refined neem seed extract containing the same absolute amount o f azadirachtin but with many other constituents present (20). Li t t le is known about the exact site o f action o f rosemary o i l and other plant essential o i ls on the two-spotted spider mites. The octopaminergic nervous system is considered to be the site-of-action o f essential oi ls in the Amer ican cockroach (21), but this may not be the case for two-spotted spider mite and there is the possibi l i ty that the essential oi ls have 66 more than one site o f action since they are complex mixtures. Further studies need to be done to f ind the exact mechanism(s) o f action o f the essential oi ls in spider mites. 3.4.2 Synergy among constituents W e observed that indiv idual constituents differ in their toxic i ty to the two host strains o f mites, and it seems that they are more toxic to mites that feed on tomato than on bean plants. W e found that some constituents (viz. borneol, bornyl acetate) that were not toxic to mites feeding on beans were relatively toxic to mites on tomatoes. S imi la r results were reported when major constituents o f two other essential o i ls were used alone against two post-harvest insect pests (22). To corroborate the role o f indiv idual constituents in the toxicity o f rosemary o i l to spider mites, we el iminated each indiv idual constituent from a synthetic mixture that simulated natural rosemary o i l . We found that the absence o f some constituents (1,8-cineol or a-pinene) in the artificial mixture caused a significant decrease in toxicity (84% and 8 0 % respectively), wh ich tempted us to conclude that these constituents are the major contributors to the o i l ' s toxici ty. However , when we mixed these active constituents together we found that their toxici ty level was not as high as we expected. The toxic i ty o f our art i f icial mixtures only reached the level o f the natural rosemary o i l when we mixed the blends o f active constituents w i th inactive ones. This indicates that the ' inact ive ' constituents have some synergistic effect on active constituents, and although not active indiv idual ly, their presence is necessary to achieve ful l toxicity. Ac t i ve constituents on the other hand might have an antagonistic effect on each other since their toxic i ty level is signif icantly greater when tested indiv idual ly and not in a mixture with other active constituents. The highest mortality rates were obtained 67 in both strains when al l the constituents were present in the mixture (96% in tomato mites and 9 2 % in bean mites). K n o w i n g the role o f each constituent in the toxici ty o f the o i l gives us the abil i ty to screen different rosemary oi ls and choose the most effective one for pest control proposes. It might be also possible to art i f ic ial ly create a blend o f different constituents, base on their activity and their effect on the pest. 68 References 1) Johnson, W . T . , & L y o n , H . H . (1991). Insects that feed on trees and shrubs. 2nd ed., Ithaca: Comstock Publ ish ing and Cornel l Universi ty press, N Y , pp 468-470. 2) Zhang, Z . (2003). Mites of greenhouses: identification, biology and control, Wal l ing ford : C A B I publ ishing, pp 54-61. 3) Jeppson, L . R . , Ke i fe r , H . H . & Baker, T . W . (1975). Mite injurious to economic plants, Berkeley: Univers i ty o f Cal i fo rn ia press, C A , pp 370-376. 4) Cranham, J . E . , & Hel le , W . (EDs) . (1985). Pesticide resistance in Tetranychidae. In: World crop pest - spider mites: their natural enemies and control, Amsterdam: Elsevier , pp 405-421. 5) The Database o f Arthropods Resistance to Pesticides. M ich igan state university. Center for integrated plant systems. Retr ieved A p r i l 28, 2005, f rom: http://www.pesticideresistance.org / D B / index.html. 6) Yatagai , M . (1997). M i t i c ida l activities o f tree terpenes. Current Topics of Phytochemistry, 1: 87-97. 7) Isman, M . B . (2000). Plant essential oi ls for pest and disease management. Crop Protection, 19: 603-608. 8) Roe , F .J .C . (1965). Chron ic toxici ty o f essential oi ls and certain other products o f natural or ig in. Food and Cosmetics Toxicology, 33: 311 -324. 9) Cockayne, S. E . & Gawkrodger, D. J . (1997). Occupat ional contact dermatitis in an aromatherapist. Contact Dermatitis, 37: 306-309. 10) Hjorther, A . B . , Christophersen, C , Hausen, B . M . & Menne , T . (1997). Occupat ional al lergic contact dermatitis f rom carnosol, a natural ly-occuring compound present in rosemary. Contact Dermatitis, 37: 99-100. 69 11) Kumar , A . , Dunke l , F .V . , Broughton, M . J . , & Sriharan, S. (2000). Ef fect o f root extracts o f mexican mar igold, Tagetes minuta (Asterales: Asteraceae), on six nontarget aquatic macroinvertebrates. Environmental Entomology, 29: 140-149. 12) Wager-Page, S. , & M a s o n , J . R. (1997). Ortho-aminoacetophenone, a non-lethal repellent: The effect o f volat i le cues vs. direct contact on avoidance behavior by reodents and birds. Pesticide Science, 46: 55-60. 13) Clark , L . & Aronov , E . V . (1999). Human food f lavor additives as bird repellents: I. Conjugated aromatic compounds. Pesticide Science, 55: 903-908. 14) Burt , S. (2004). Essential o i ls : Their antibacterial properties and potential appl ication in food-a review. International Journal of Food Microbiology, 94: 223-253. 15) Managena, T., & M u y i m a , N . Y . O . (1999). Comparat ive evaluation o f the antimicrobial activit ies o f essential oi ls o f Artemisia qfra, Pteronia incana and Rosmarinus officinalis on selected bacteria and yeast strain. Letters in Applied Microbiology, 28: 291-296. 16) Papachristos, D .P . , & Stampoulos, D . C . (2004). Fumigant toxic i ty o f three essential oi ls on the eggs o f Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). Journal of Stored Products Research, 40: 517-525. 17) Tunc, I., Berger, B . M . , Er ler , F., & Dag l i , F. (2000). Ov ic ida l activity o f essential o i ls f rom plants against two stored-product insects. Journal of Stored Products Research, 36: 161-168. 18) Koschier , E . A . , & Sedy, K . A . (2003). Labiate essential o i ls affecting host plant selection and acceptance o f Thrips tabaci L indeman. Crop Protection, 22: 929-939. 70 19) Pintore, G . , Usa i , M . , Bradesi , P., Jul iano, C , Boatto, G . , T o m i , F., Chessa, M . , Cerr i , R., & Casanova, J . (2002). Chemica l composit ion and ant imicrobial activity o f Rosmarinus officinalis L . o i l from Sardinia and Cors ica , Flavour and Fragrance Journal, 17: 15-19. 20) Feng, R. & Isman, M . B . (1995). Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae. Experientia. 51: 831-834. 21) Enan, E . (2001). Insecticidal activity o f essential o i ls: octopaminergic site o f action. Comparative Biochemistry and Physiology, 130 (C) : 325-327. 22) Bekele , J . & Hassanal i , A . (2001). B lend effects in the toxic i ty o f the essential o i l constituents o f Ocimum kilimandascharicum and Ocimum kenyense (Labiateae) on two post- harvest insect pests. Phytochemistry, 57: 385-391. 71 Chapter four: Integrated control of two-spotted spider mites on greenhouse tomato: studying the field efficacy of a rosemary oil-based acaricides combine with predatory mite Phytoseiulus persimilis and assessing the phytotoxic effects of the pesticide on tomato plants 4.1 Introductiuon 4.1.1 Two-spotted spider mite Two-spotted spider mite, Tetranychus urticae K o c h is one o f the most important pests o f the greenhouse crops. The current control method for spider mites in B C greenhouses is mostly re ly ing on bio-control method. 4.1.2 Phytoseiulus persimilis The predatory mite Phytoseiulus persimilis Ath ias-Henr iot is a selective predator that is able to rapidly suppress spider mites. However , it might not provide acceptable control for higher populations o f prey particularly on greenhouse tomato (1). 4.1.3 Rosemary oil Rosemary o i l is relatively effective against many insect and mite pests (2-4). In this study, the eff icacy o f EcoTro l ™- a rosemary oi l-based pesticide- combined with Phytoseiulus persimilis against two-spotted spider mites on tomato plants was evaluated under the greenhouse condit ion. 4.1.4 Phytotoxic effect The impetus on the use o f plant essential oi ls for insect pest and pathogen control originates from the need for control wi th reduced environmental and health impacts in comparison to the highly effective synthetic pesticides. A l though essential oi l-based 72 pesticides are considered low-r isk pesticides, phytotoxicity to greenhouse crops constitutes one possible obstacle for their use in practice. In a few cases essential o i l -treated plants have become attractive to plant damaging insects and phytotoxic effects on cultivated plants have been observed. Ibrahim et al. (5) reported phytotoxici ty in l imonene treated plants, whi le Chiasson et al. (6) did not observe any phytotoxici ty among lettuce, roses and tomatoes that were treated with a Chenopodium-based pesticide. The active ingredients in pesticides do not necessarily cause phytotoxici ty. Plant damage can result f rom the solvents in a pesticide formulat ion, impurit ies in the spray water, using more pesticide than prescribed on the label, or poorly m ix ing spray emulsion. B lank et al. (7) found phytotoxici ty in 2 5 % o f kiwifrui ts treated wi th mineral o i l mixed with diazinon wh ich resulted in up to 17% reduction in the y ie ld . A l though low levels o f phytotoxicity might not be a physio logical threat for plant, cosmetic damages can reduce marketabil i ty o f the product. In this study phytotoxic effects o f three rosemary oi l-based pesticides to fol iage, fruits and f lowers o f greenhouse tomato plants have been investigated. 73 4.2 Materials and methods 4.2.1 Commercial pesticide Commerc ia l pesticides, Hexacide™(5% rosemary o i l ) , EcoTro l™(10% rosemary oi l) and Sporan™(17.6% rosemary oi l) were obtained f rom E c o S M A R T Technologies Inc. (F rank l in ,TN, U S A ) . 4.2.2 Plant materials Three week- o ld tomato plants (Lycopersicon esculentum M i l l cv. Clarance) were provided by Houwe l ing ' s Nurseries (Delta, B C , Canada) (for phytotoxici ty tests) and Bevo Farms L td . (Langley, B C , Canada) (for the eff icacy test). Plants were transferred to plastic pots containing a mixture o f regular peat (50%), f ine bark (25%) and pumice (25%) provided by West Creek Farms (Langley, B C , Canada). Fo r eff icacy tests, plants were kept inside isolated cages wi th in the greenhouse and for phytotoxici ty tests plant were kept on a regular bench at the same greenhouse at 24± 3°C, 45 -60% R H under natural daylight. Plants were irrigated three times per week, two times wi th water and one time wi th water-soluble ferti l izer (Peters E X C E L 15-5-15 C a l - M a g ) (The Scott Company, Marysv i l l e , O H , U S A ) . 4.2.3 Two-spotted spider mites & Phytoseiulus persimilis Spider mites originated from a research colony maintained on tomato plants for more than f ive years without any pesticide exposure at Agr icu l ture and A g r i - F o o d Canada (Agassiz , B C , Canada). Predatory mites were purchased f rom App l i ed B ionomics (Sidney, B C , Canada). 74 4.2.4 Efficacy test A 2 x 2 factorial design was used with two factors (pesticide or predator) and two levels (absence or presence) in each factor. Treatments were randomly assigned inside cells o f cages. Data was analyzed by two-way A N O V A ( S P S S , Ch icago, IL , U S A ) . T w o tomato plants were placed inside each cel l (total o f 40 plants). Eight extra plants were randomly placed inside some cells as indicators to estimate init ial density o f mites prior to pesticide appl icat ion or predator release. Three H O B O data-loggers (Contoocook, N H , U S A ) were randomly installed inside cells to collect temperature and moisture data at 10 minutes intervals (Figure 4.1) B #i | A D D A B ©3 A A A D _ n • 2 C A D Figure 4.1. Experiment layout. A= Control, B = Predator, C= Pesticide, D= Predator + Pesticide^ = extra plant,® = HOBO Data Logger. Approx imate ly 15-20 adult spider mites were placed on each plant. Af ter one week, extra plants were removed f rom the cells and numbers o f spider mites on their foliage counted under a stereomicroscope. Based on the init ial density o f the spider mites, Phytoseiulus persimilis was introduced to treatments D and C at ratio o f 1:20 predators to prey. T w o hours after releasing the predators, treatments B and D were sprayed wi th EcoTro l ™ at 7.5 m l litre (Label rate- water was used as carrier solvent as recommended by manufacturer). Af ter 7 days, tomato plants were fu l ly harvested and placed inside paper bags. Bags then were put inside a co ld room (4°C) to prevent further development o f mites during data col lect ion. Numbers o f spider mites and predatory mites were counted on al l fol iage under a stereomicroscope. The single apical leaflet at 75 the end o f each leaf was selected as a sub-sample for count ing the number o f spider mite and predatory mite eggs (Figure 4.2). 4.2.5 Phytotoxicity tests Pesticides were tested at three concentrations (One ha l f o f label rate [3.75 m l litre " ' ] , label rate [7.5 m l litre "'] and doubled label rate [15 m l litre "']). F i v e plants o f equal age were used for each concentration. Contro l plants were sprayed w i th carrier solvent (water) alone. Sprayed plants were randomly p laced on a greenhouse bench (Figure 4.3). Cutt ings made f rom stems containing flowers or fruits were transferred to new pots placed inside a steam room for one week. Plants were then transferred to regular benches where they remained for one week prior to the experiment. Frui ts and flowers were sprayed wi th the same pesticides at the same concentrations. In order to determine the phytotoxic effect o f pesticides on fo l iage, medium size leaflets were selected as sub-samples. Damage was def ined in five grades f rom grade 0 Figure 4.2. Sub-sampling for counting number of eggs 76 (no damage) to grade 5 (completely burned leaflet). F i v e leaflets o f the same size f rom each plant were selected randomly f rom al l tested plants for damage assessment. Damage to flowers was def ined as no damage (grade 0), minor burning sign on petals (grade 1) and major burning sign or flower abortion due to sever damage (grade 2). Damage to fruits was defined as no damage (grade 0) or any particular burning s ign, wh i ch might cause cosmetic damage to fruit (grade 1). Effects were recorded 24 hours, 48 hours and 72 hours after spraying. F i v e flowers and f ive fruits from each plant were used for damage assessment. Figure 4.3. Phytotoxicity tests 4.2.6 Data Ana lys is Observations were analyzed using the S P S S program, version 13 for analysis o f variance ( A N O V A ) . Da ta were transformed to logarithm values when necessary. 77 4.3 Results 4.3.1 Efficacy test Approx imate ly 100 ± 8 mites were found on each plant (i.e. on eight extra plants randomly placed inside cages) prior to spraying pesticide or releasing predators. Temperature wi th in cages during the experiment was 24 ± 6°C and relative humidi ty was 60± 15%. Numbers o f mites in the blocks treated with pesticide or predator was signif icantly decreased compared to controls (Table 4.1). Pesticide appl icat ion showed significant effect on the number o f mites. A significant effect was also found wi th predator introduction. N o signif icant interaction between the two factors was observed (Table 4.2). Table 4.1. Average number of spider mites on each block, TSM= two-spotted spider mite, LogTSM= Log transferred number of spider mites. Pesticide Predator Mean TSM Std. Deviation Mean LogTSM Std. Deviation LogTSM N absent absent 183.3750 65.67264 5.1616 .36771 4 present 47.7500 4.17333 3.8630 .08998 4 Total 115.5625 84.32862 4.5123 .73707 8 present absent 87.8750 29.41194 4.4365 .31937 4 present 42.5000 21.42429 3.6480 .52862 4 Total 65.1875 33.99573 4.0422 .58405 8 Total absent 135.6250 69.46158 4.7991 .50191 8 present 45.1250 14.56206 3.7555 .36938 8 Total 90.3750 67.33981 4.2773 .68677 16 78 Table 4.2. Results from analyze of variance of number of spider mites (Log transferred) following pesticide and predator treatment, (a) R Squared = 0.777 (Adjusted R Squared = 0.777) Source Sum of Squares df Mean Square F Sig. Corrected Model 5.500 (a) 3 1.833 13.976 .000 Intercept 292.721 1 292.721 2231.313 .000 Pesticide 0.884 1 0.884 6.738 .023 Predator 4.356 1 4.356 33.206 .000 Pesticide * Predator 0.260 1 0.260 1.984 .184 Error 1.574 12 .131 Total 299.795 16 Corrected Total 7.075 15 Numbers o f spider mite eggs were not signif icantly affected by pesticide or predators (Table 4.3), although predators alone appeared to suppress spider mite eggs. N o interaction was found between the two factors (Table 4.4) Table 4.3. Average number of spider mite's eggs on each block, Log Eggs= Log transferred number of spider mite's eggs. ^ = = = _ _ _ = ^ = ^ _ _ _ Pesticide Predator Mean Egg Std. Deviation Mean LogEggs Std. Deviation LogEggs N absent absent 159.7500 90.92625 4.9228 .66619 4 present 54.8750 22.26498 3.9092 .56090 4 Total 107.3125 83.05546 4.4160 .78651 8 present absent 73.8750 35.33972 4.2288 .42463 4 present 75.5000 44.43160 4.1056 .87568 4 Total 74.6875 37.17616 4.1672 .64051 8 Total absent 116.8125 78.64792 4.5758 .63646 8 present 65.1875 34.35210 4.0074 .68883 8 Total 91.0000 64.40471 4.2916 .70472 16 79 Table 4.4. Results from analyze of variance of number of spider mites eggs (Log transferred) following pesticide and predator treatment, (a) R Squared = 0.313 (Adjusted R Squared = 0.141) Source Sum of Squares df Mean Square F Sig. Corrected Model 2.33 (a) 3 0.778 1.824 .196 Intercept 294.688 1 294.688 691.127 .000 Pesticide 0.248 1 0.248 0.581 .461 Predator 1.292 1 1.292 3.031 .107 Pesticide * Predator 0.793 1 0.793 1.859 .198 Error 5.117 12 0.426 Total 302.137 16 Corrected Total 7.450 15 N o signif icant difference was found among the number o f predators (F (1,6) = 0.00, P> 0.05 (1.00)) or their eggs (F (1,6) = 1.720, P>0.05 (0.238)) on blocks sprayed with pesticide compared to those not sprayed. 4.3.2 Phytotoxicity test Hexacide and E c o T r o l were not phytotoxic to fol iage, f lowers or fruits (grad 0). Sporan (containing 18% rosemary oi l) caused first grade damage (burning signs on less than 1/5 o f leaflet) to 12% o f foliage at the recommended label rate and second grade (burning signs on between 1/5 to 2/5 of the leaflet) damage to 3 2 % o f leaflets after 24 hours. N o addit ional damage was found at day two or day three o f data col lect ion (Figure 4.4). 80 Figure 4.4. Phytotoxicity to foliage. EC = EcoTrol, HE= Hexacide, SP= Sporan, a=one half of label rate, b= label rate, c = doubled label rate. Hexacide and E c o T r o l were not phytotoxic to f lowers. Sporan caused second grade damage (some petals o f f lowers demonstrated minor burning sings) in 4 0 % o f tested f lowers at its label rate, and second grade damage (complete burning or f lower abortion) in 5 6 % o f f lowers and third grade damage (3/5 o f f lowers demonstrated burning signs) in 3 6 % o f f lowers at double the label rate after 24 hours (Figure 4.5). N o addit ional damage was found at day two or day three o f data co l lect ion. N o n e o f the pesticides were found to be phytotoxic to fruits (Figure 4.6). 81 Figure 4.5. Phytotoxicity to flowers. EC = EcoTrol, HE= Hexacide, SP= Sporan, a=one half of label rate, b= label rate, c = doubled label rate. Figure 4.6 Phytotoxicity to fruits. EC = EcoTrol, HE= Hexacide, SP= Sporan, a=one half of label rate, b= label rate, c = doubled label rate. 4.4 Discussion 4.4.1 Efficacy of EcoTrol™ In this study, the eff icacy o f EcoTrol™ (10% rosemary oi l ) at its recommended label rate (7.5 m l litre for spider mites on tomato) was evaluated against two-spotted spider mites on greenhouse tomato plants, both indiv idual ly and in combinat ion with predatory mites, Phytoseiulus persimilis under greenhouse condit ions. In previous laboratory bioassays, contact toxici ty o f rosemary o i l based-pesticides was observed wi th two-spotted spider mites. However, in the present study, the same degree o f mortality was not observed. There was considerable variat ion among observations requir ing transformation o f the data to logarithmic form. Based on the average number o f mites on each b lock (Log-transformed), we can conclude that pesticide appl icat ion, predator release and the combination o f pesticide and predators, suppress the mite populat ion by 52 ± 16%, 74 ± 2 % and 76 ± 12% respectively (Table 4.1). Several factors may have lead to this difference. A s mentioned before (Chapter two), this product does not have trans-laminar activity and cannot affect mites that move deep wi th in the canopy or remain at parts o f the plant that pesticide cannot reach. In addit ion, environmental condit ions such as temperature and light may accelerate the degradation o f the o i l . There was a fluctuation o f 12 degrees in temperature and 3 0 % in relative humidi ty wi th in the greenhouse during the experiment. Th is f luctuation might be a reason for differences in eff icacy in different blocks and cages. Moreover , it was shown that this product has repellent effects (Chapter two) so there might be another scenario. Mi tes that were not directly hit by pesticide might move to the non-sprayed parts o f the plants enhancing their surv ival . However , this should not affect numbers o f eggs o f spider mites and predatory mites. 83 Accord ing to the final number o f mites we might conclude that the combinat ion o f pesticide and predators was twice as effective as the pesticide alone. However , statistical analysis d id not show a signif icant interaction between pesticide and predators (Table 4.2). Th is difference (15%) was not statistically signif icant but in this experiment may be b io logical ly signif icant. There are many factors that affect the statistical signif icance o f the results such as sample size or number o f replicates or number o f repeated trials. In this study, because o f time and space l imitat ions, I could only conduct the trial once wi th four replicates for each treatment (which is the m in imum requirement by P M R A ) (8). Further experiments are needed to clar i fy the eff icacy o f this pesticide against spider mites in the greenhouse. Accord ing to my results, I conclude that EcoTrol™ can effectively suppress (not control) the spider mite populat ion on greenhouse tomato and it is safe for predatory mites and their eggs at the tested concentration. 4.4.2 Phytotoxicity trials Acco rd ing to P M R A eff icacy guidelines for plant protection products (8) three concentrations o f EcoTrol™ were used for phytotoxicity tests. Effects were recorded for three continuous days. Phytotoxici ty tests were performed on fol iage, f lowers and fruits. N o sign o f phytotoxici ty was found among tested tomato plants. Certain plant essential oi ls have recently been used as least-toxic herbicides (9-10). Un l i ke essential oi l -based pesticides, the herbicides contain higher amounts o f essential o i ls. Fo r instance, Matran E C ™, a contact, non-selective, broad spectrum, fol iar herbicide developed by E c o S M A R T Technologies Inc. contains 5 0 % clove o i l . It might 84 be possible for EcoT ro l to cause phytotoxicity i f used at higher concentrations, however, my results show that E c o T r o l is safe to tomato even when appl ied at double the recommended label rate. Th is is a very important aspect o f this pesticide wh ich makes it a favorable option for I P M programs in greenhouse tomato plants. 85 References 1) Everson, P. (1979). The functional response o f Phytoseiulus persimilis (Acar ina : Phytoseiidae) to various densities o f Tetranychus urticae (Acar ina : Tetranychidae). Canadian Entomologist, 111: 7-10. 2) Isman, M . B . (2000). Plant essential oi ls for pest and disease management. Crop Protection, 19: 603-608. 3) Kosch ier , E . A . , & Sedy, K . A . (2003). Labiate essential o i ls affect ing host plant selection and acceptance o f Thrips tabaci L indeman. Crop Protection, 22: 929-939. 4) C h o i , W . , Lee , S. , Park, H. , & A h n , Y . (2004). Tox ic i ty o f plant essential o i ls to Tetranychus urticae (Acar i : Tetranychidae) and Phytoseiulus persimilis (Acar i : Phytoseiidae). Journal of Economic Entomology, 97: 553-558. 5) Ibrahim, M . A . Ka inu la inen, P., Af la tun i , A . T i i l i kka la , K . &. Holopainen, J .K . (2001). Insecticidal, repellent, ant imicrobial activity and phytotoxici ty o f essential o i ls : W i th special reference to l imonene and its suitabil i ty for control o f insect pests. Agricultural and Food Science in Finland, 10: 243-259. 6) Chiasson, H . , Bostanian, N . J . , & Vicent , C . (2004). Acar i c ida l properties o f a Chenopodium-based botanical. Journal of Economic Entomology, 97: 1373-1377. 7) B lank, R . H . , O lson , M . H . , Tomkins , A . R., Greaves, A . J . , Wal le r , J . E . , & Pul ford , W . M . (1994). Phytotoxic i ty investigation o f mineral o i l and d iaz inon sprays appl ied to k iwi f ru i t in winter-spring for armoured scale control . New Zealand Journal of Crop and Horticultural Science, 22: 195-202. 8) Anonymous. (2003). E f f i cacy guidelines for plant protection products. Pest Management Regulatory Agency, D IR2003-24, 49p. 86 9) Ghosheh, H . Z . (2005). Constrainst in implementing b io log ica l weed control : A review. Weed Biology and Management, 5: 83-92. 10) Tworkosk i , T. (2002). Herbic ide effects o f essential o i ls. Weed Science, 50: 425-431. 87 Chapter five: Summary and Conclusion The major objective o f this research was to study the eff icacy o f a rosemary oi l-based pesticide (EcoTrol™) to be used on greenhouse tomato plants against important pests o f this crop ( two-spotted spider mite and greenhouse whi tef ly) . Accord ing to P M R A eff icacy guidelines for plant protection products (1) there are some issues that need to be addressed before mak ing a decision about this product. Some o f these questions have been answered based on the results o f my study. 1) Is this product efficacious against pests? The laboratory results showed that rosemary o i l can cause contact and fumigant toxicity in two-spot ted spider mites and greenhouse whitefl ies. EcoTro l at its recommended label rate in particular, caused >80% mortal i ty among spider mites when tested under laboratory condit ions. However , when tested in a greenhouse, lower mortality was observed but suppression o f the mite populat ion was observed. The answer to this question depends on our definit ion o f eff icacy. Accord ing to the results (considering the l imitations o f greenhouse trial) it is clear that E c o T r o l alone might not control a large mite populat ion inside a greenhouse. However , it might effectively restrain the populat ion either below the economic threshold or make it more manageable wi th other control measures such as predatory mites. 2) Is it hazardous to human health? Humans have used plant essential oils as flavors and food additives f rom ancient times (2). These oi ls have been used traditionally as heal ing medicines in many countries and ancient people were also aware o f their pesticidal properties (3). Rosemary in 88 particular plays a great role in modern medicine as anticancer (4-7), antiviral (8) and antibacterial (9) agent. It has even been used recently as a treatment for snoring (10). A l though some essential o i ls in very high concentration and over long periods o f time can cause chronic toxic i ty or some dermal problems (11-13), in general, essential oi ls are safe for humans and other mammals in low concentrations. 3) Is it persistent in the environment? The short answer to this question is no. M y results indicate that both pure rosemary o i l and EcoT ro l disappear quickly in the environment and there is a significant decrease in the toxicity o f their residues after 24 hours. In most cases, no toxicity was detected after 48 hours. 4) What are the effects of this product on the host plant? I did not find any sign o f phytotoxic i ty on tested plants (foliage, f lowers and fruits) wi th tested concentrations (one-half, recommended and doubled label rate). Accord ing to m y results EcoT ro l is safe for greenhouse tomato plants (Lycopersicon esculentum M i l l cv. Clarance) at its label rate within the environmental condit ions that I used for my experiment. 5) Is it toxic to bio-control agents? In both laboratory experiments and the greenhouse trial, Ecotro l was not found to be toxic to predatory mites when directly sprayed on plants. Contact toxici ty bioassays showed that predatory mites are less susceptible to rosemary o i l than spider mites. However , unlike predatory mites, parasitic wasps were found to be more susceptible to rosemary o i l both as a contact and fumigant toxicant compare to greenhouse whitef l ies. 89 Residual toxicity assays indicate a significant decrease in toxicity o f residues to bio-control agents after 24 hours. Thus, the best strategy for protect ing bio-control agents from side-effects o f E c o T r o l is to either release them wi th a short delay (48-72 hours) after the pesticide appl icat ion or app ly ing the pesticide after the bio-control agents are established on the pest populat ion. 6) Are there any limitations for its application? Rosemary o i l cannot pass through the plant tissues and does not have trans-laminar activity. It cannot affect the pests that aggregate on the under-side o f the leaves. T o be effective, it should directly hit the target and this is an obstacle for its use in tomato greenhouses where the plant canopy get dense during the season and pesticides can not penetrate deep enough to reach the pests. 7) Is there potential for pests to evolve resistance to this product? It is hard to make a sol id statement about this issue but I have a theory. L i k e other essential o i ls, natural rosemary o i l is a complex mixture o f terpenoids. Consider ing that targetsite resistance is an important problem for mite control , it is more probable that mites w i l l evolve resistance faster to an acaricide based on a single active ingredient than to a mixture o f different active compounds. It has been reported that green peach aphids Myzuspersicae Sulzer developed resistance to pure azadirachtin (the major ingredient o f neem insecticide) but not to a refined neem seed extract containing the same absolute amount o f azadirachtin but wi th many other constituents present (14). Li t t le is known about the exact site o f action o f rosemary o i l and other plant essential oi ls on the two-spotted spider mites. The octopaminergic nervous system is considered to be the site-of-90 action o f essential o i ls in the Amer ican cockroach (15), but this may not be the case for two-spotted spider mite and there is the possibi l i ty that the essential o i ls have more than one site o f action since they are complex mixtures. A s mentioned before, EcoT ro l is not persistent in the environment so fewer pests w i l l encounter the pesticide and selection pressure w i l l be lessened. 8) What is the mechanism of toxicity of this product? I observed that indiv idual constituents differ in their toxic i ty to the two host strains o f mites, and it seems that they are more toxic to mites that feed on tomato than on bean plants. I found that some constituents (viz. borneol, bornyl acetate) that were not toxic to mites feeding on beans were relatively toxic to mites on tomatoes. S imi lar results were reported when major constituents o f two other essential o i ls were used alone against two post-harvest insect pests (16). To corroborate the role o f indiv idual constituents in the toxicity o f rosemary o i l to spider mites, I el iminated each indiv idual constituent f rom a synthetic mixture that simulated natural rosemary o i l . I found that the absence o f some constituents (1,8-cineol or a-pinene) in the artificial mixture caused a significant decrease in toxicity (84% and 8 0 % respectively), wh ich tempted me to conclude that these constituents are the major contributors to the o i l ' s toxici ty. However , when I mixed these active constituents together I found that their toxici ty level was not as high as I expected. The toxici ty o f my art i f icial mixtures only reached the level o f the natural rosemary o i l when I mixed the blends o f active constituents wi th inactive ones. This indicates that the ' inact ive ' constituents have some synergistic effect on active constituents, and although not active indiv idual ly , their presence is necessary to achieve ful l toxicity. Ac t i ve constituents on the other hand might have an antagonistic effect on 91 each other since their toxic i ty level is signif icantly greater when tested indiv idual ly and not in a mixture wi th other active constituents. The highest mortal i ty rates were obtained in both strains when al l the constituents were present in the mixture (96% in tomato mites and 9 2 % in bean mites). K n o w i n g the role o f each constituent in the toxici ty o f the o i l gives us the abi l i ty to screen different rosemary oils and choose the most effective one for pest control proposes. It might be also possible to art i f ic ial ly create a blend o f different constituents, base on their act ivi ty and their effect on the pest. 9) Can environmental factors affect its efficacy? In this study, I d id not test the effect o f environmental factors on the toxici ty or degradation level o f rosemary o i l but it is obvious that any factor that can accelerate the volat i l izat ion o f the o i l , can affect its toxicity or degradation level . Further studies are needed to f ind the effect o f factors such as temperature, moisture or l ight on toxici ty level o f the o i l . Ab io t i c environmental factors can change the toxicity o f the o i l , but biotic factors might also play a role in their toxici ty. For instance, toxic i ty o f the rosemary o i l to the same species o f pest might be different on various host plants. I observed a smal l difference in toxic i ty o f rosemary o i l to spider mites on bean plants and tomato plants. Host plant chemicals can affect (either as a synergist o f antagonist) the essential o i l (i.e. phenolics in tomato plants). Further studies are needed to indicate the effect o f host plant on the toxici ty o f rosemary o i l . 92 10) What are the most effective ways of using this product? Acco rd ing to the results o f this study, I can make some suggestions for effective use o f EcoTro l as part o f an I P M program for mite and whitef ly management in the tomato greenhouse. A ) A s seen in the greenhouse tr ial, EcoT ro l cannot provide complete control o f pests and must be combined wi th other control measure such as bio-control agents. A n effective monitor ing system can detect pests before they become a major problem. E c o T r o l can then be used for spot spraying and small-scale control o f pests in lower populations fo l lowing bio-control agent release. B ) In order to reduce the side effect o f EcoTro l on the bio-control agents, predators and parasitoids should be released wi th a short delay (48-72 hours) after pesticide appl icat ion. If necessary, addit ional applications should be made after the bio-controls are established on the plants. C ) EcoTro l appl icat ion can also be combined with cultural control methods such as trap plants. Consider ing the repellent effect o f the Ec o T ro l , for mobi le pests l ike whitef l ies, it can be use to repel the adults f rom major host plants and push them toward trap plants (i.e. tobacco) where they can be control led local ly (push-pull strategy). In general, E c o T r o l can be considered a favorable option for a chemical control portion o f a greenhouse I P M program. 93 References 1) Anonymous. (2003). E f f i cacy guidelines for plant protection products. Pest Management Regulatory Agency, D IR2003-24, 49p. 2) Isman, M . B . (2006). Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated wor ld . Annual Review of Entomology, 51: 45-66. 3) Isman, M . B . (2000). Plant essential oi ls for pest and disease management. Crop Protection, 19:603-608. 4) A b u - A m e r , K . M . , Sen, P., & al-Sereit i , M . R . (1999). Pharmacology o f rosemary (Rosmarinus officinalis L.) and its therapeutic potentials. Indian Journal of Experimental Biololgy, 37: 124-30. 5) Of ford E . A . , M a c e K. , Avan t i O . , & Pfeifer A . M . A . (1997). Mechanisms involved in the chemoprotective effects o f rosemary extract studies in human liver and bronchial cells. Cancer Letters, 114: 275-281. 6) Singletary, K . , M a c D o n a l d , C , & Wal l ig, M . (1996). Inhibit ion by rosemary and carnosol o f 7, 12- dimethylbenz[a]anthracene ( D M B A ) - i n d u c e d rat mammary tumorigenesis and in vivo D M B A - D N A adduct formation. Cancer Letters, 104: 43-48. 7) P louzek, C . A . , C io l ino , H . P. , Clarke, R., & Y e h , G . C . (1999). Inhibit ion o f P-glycoprotein activity and reversal o f mult idrug resistance in vitro by rosemary extract. European Journal of Cancer, 35: 1541-1545. 94 8) A ruoma, O. I., Spencer, J . P. E . , Ross i , R., Aeschbach, R., K h a n , A . , M a h m o o d , N . , M u n o z , A . , M u r c i a , A . , But ler, J . , & Hal l iwe l l B . (1996). A n evaluation o f the antioxidant and antiviral action o f extracts o f rosemary and provencial herbs. Food and Chemical Toxicology, 34: 449-456. 9) Burt , S. (2004). Essential o i ls : their antibacterial properties and potential applications in foods- a review. InternationalJournal of Food Microbiology, 94: 223-253. 10) Pr ichard, A . J . N . (2004). The use o f essential o i ls to treat snoring. Phytotherapy Research, 18: 696-699. 11) Roe , F. J . C . (1965). Chron ic toxici ty o f essential oi ls and certain other products o f natural or ig in. Food and Cosmetics Toxicology, 33: 311-324. 12) Cockayne, S. E. , & Gawkrodger, D . J . (1997). Occupat ional contact dermatitis in an aromatherapist. Contact Dermatitis, 37: 306-309. 13) Hjorther, A . B . , Christophersen, C , Hausen, B . M . & Menne, T . (1997). Occupational al lergic contact dermatitis f rom carnosol, a natural ly-occuring compound present in rosemary. Contact Dermatitis, 37: 99-100. 14) Feng, R. & Isman, M . B . (1995). Selection for resistance to azadirachtin in the green peach aphid, Myzuspersicae. Experientia. 51: 831-834. 15) Enan, E . (2001). Insecticidal activity o f essential o i ls : octopaminergic site o f action. Comparative Biochemistry and Physiology Part C, 130: 325-327. 16) Bekele , J . & Hassanal i , A . (2001). B lend effects in the toxic i ty o f the essential o i l constituents o f Ocimum kilimandascharicum and Ocimum kenyense (Labiateae) on two post- harvest insect pests. Phytochemistry, 57: 385-39. 95 Appendix I. Electronic micro-sprayer1 Electronic micro-sprayer was developed to measure the direct contact toxici ty o f the toxicants to test organism mimick ing spraying practices inside greenhouses. A mechanical switch was installed on an airbrush (Badger 2 0 0 N H - I L , U S A ) l inked to a dishwasher solenoid control led electronically by a digital timer. The t imer is constructed using a N E 5 5 5 chip. N E 5 5 5 chip is a highly stable controller capable o f producing accurate t iming pulses. Th is chip has 8 input terminals as shown in the diagram bel low (Figure A . l ) . When the low signal input is appl ied to the reset terminal (pin 4), the timer output remains low regardless o f the threshold voltage (pin 6) or the trigger voltage (pin 2). On ly when the high signal is appl ied to the reset terminal, the timer's output changes according to threshold voltage and trigger voltage. When the threshold voltage exceeds 2/3 of the supply voltage (pin 8) whi le the timer output (pin 3) is h igh, the timer's internal discharge turns on , lower ing the threshold voltage to below 1/3 o f the supply voltage. Dur ing this t ime, the timer output is maintained low. Later, i f a low signal is applied to the trigger voltage so that it becomes 1/3 o f the supply voltage, the timer's internal discharge turns off, increasing the threshold voltage and dr iv ing the timer output again at high. The start and stop buttons are held at the "+ " potential by the resistors R 4 and R 6 . A t the rest posit ion, C 2 is short circuited with the ground. B y pressing the start button, this short-circuit is annihi lated, the output is supplied v ia T l and C 2 starts to be loaded v ia R l and R V 1 . R V 1 is a variable resistor. A s value o f R V 1 increases, it takes longer for ' Digital timer designed and developed by Maryam Antikchi, Department of Engineering Physics, U B C 96 C 2 to reach the same value as the cross point between R 2 and R 3 , wh ich is connected to the reference voltage (pin 5). Once C 2 is loaded, the output goes to zero and C 2 w i l l discharge. I f the Stop button is pressed during this process, IC w i l l be reset, and the output goes to zero immediately. The timer can turn the power on and of f wi thin the intervals between 0.1 second to 1 minute. When the power is on, the solenoid pul l the mechanical switch in and that push the airbrush bottom so it sprays. The longer the power is on, the longer it sprays (Figure A .2 ) . j 6 o SI 6 - o s 2 : Grid Vcc Trg Die Out Thr Rst Ctl - CI " luE VI 12V +V o RV1 • C2 " luF - Dl " DIODE RLY1 5VCOIL R5 lk ^ LED1 VUy N P N Figure A. l . Digital timer circuits 97 Figure A.2. Electronic micro-sprayer. A= mechanical switch, B = Airbrush, C = Solenoid, D = digital timer The timer was calibrated in five stages base on the amount o f al iquot that spray. In stage one it can del iver 20p.gr ± 5 and in fo l lowing stages this amount w i l l be doubled up to 1 OOpgr. In order to reduce errors and increase the accuracy o f the tests, the t imer was calibrated once pr ior to each experiment. 98 II. Sub-lethal effects of rosemary oil The effect o f rosemary o i l on parasitization by E. formosa was investigated. Leaf lets o f tomato containing whitef ly nymphs were placed inside eppendorf tubes filled wi th M S (M5524) standard tissue culture media (Figure A . 3 ) . Thir ty whi tef ly nymphs were selected on each leaflet and remaining nymphs were removed. The leaflet was placed in Petri dishes (10cm diameter) and sprayed wi th rosemary o i l (10 m l litre " ') or 7 0 % aqueous methanol as carrier solvent using electronic m ic ro sprayer or not sprayed at a l l . F i ve adult E. formosa were introduced to each Petri d ish and the sealed dishes were place inside a growth chamber at 26±2 °C, 55-60% R H and a 16:8 L D photoperiod. Af ter 72 hours numbers o f parasit ized nymphs were counted. A l l treatments were repl icated five t imes. Figure A .3. Sub-lethal effect of rosemary on parazitation by of K formosa In order to find the effect o f rosemary on emergence o f E. formosa adults, tomato leaflets containing parasit ized whitef ly nymphs were p icked and prepared as described above inside Petr i dishes w i th 30 parasit ized nymphs on each leaflet. Leaf lets were sprayed w i th rosemary o i l (10 m l litre " ' ) , 7 0 % aqueous methanol by micro-sprayer or not sprayed at a l l . A l l leaflets were placed inside the growth chamber at the condit ions mentioned above. Numbers o f emerged wasps were counted after one week. 99 Rosemary o i l d id affect parasitization (Figure A .4) or emergence (Figure A .5 ) o f E. formosa. Because o f fungal infection o f many leaflets and resource l imitat ions, the experiments had inadequate sample size and lacked statistical power. Rosemary 1% Carrier solvent No spray Figure A.4. Effect of rosemary oil 1% on parasitizing pattern of E. formosa. Bars representing means (± SE), n=5 replicates with 30 whitefly larva and 5 parasitic wasp per replicate. Bars marked with the same letter do not differ significantly, Tukey (P>0.05 (0.305), F(2,12)= 1.314) Rosemary 1% Carrier solvent No spray Figure A.5. Effect of rosemary oil 1% on emergence of E. formosa. Bars representing means (± SE), n=5 replicates with 30 parasitized whitefly larva per replicate. Bars marked with the same letter do not differ significantly, Tukey (/>>0.05 (0.464), F(2,12)= 0.820) 100 

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