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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 E F F I C A C Y 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 F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R 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  Efficacy o f rosemary essential oil was assessed against t w o - s p o t t e d spider mites (Tetranychus urticae) and greenhouse whiteflies (Trialeurodes vaporariorum) as w e l l as its effects on the tomato host plant and bio-control agents. Laboratory bioassay results indicated that pure rosemary o i l and E c o T r o l ™ (a rosemary oil-based pesticide) caused complete mortality o f spider mites and whiteflies at concentrations that are not phytotoxic to the host plant. T h e predatory mite, Phytoseiuluspersimilis,  is less susceptible to rosemary o i l and E c o T r o l ™ 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 whiteflies. Rosemary o i l repels both spider mites and whiteflies and can affect o v i p o s i t i o n behavior. Rosemary o i l and rosemary oil-based pesticides are non-persistent i n the environment and their lethal and sub-lethal effects fade w i t h i n one or two days. E c o T r o l ™ is safe to tomato foliage, flowers 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 c o u l d reduce a twospotted spider mite population b y 5 2 % . A t that rate, E c o T r o l ™ d i d not cause any mortality among predatory mites Phytoseiulus persimilis nor d i d it affect their eggs. T o x i c i t y o f i n d i v i d u a l and incomplete mixtures o f constituents o f rosemary o i l to spider mites indicated significant synergy among the constituents. Highest mortality was only obtained when all 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 controlling spider mites and whiteflies in greenhouse tomato plants.  Table of contents ABSTRACT  II  TABLE OF CONTENTS  IV  LIST O F T A B L E S  VI  LIST O F FIGURES  VII  ACKNOWLEDGMENTS  VIII  CHAPTER ONE: INTRODUCTION  1  1.1 MAJOR OBJECTIVES  1  1.2 HISTORICAL PERSPECTIVE 1.3 GREENHOUSE TOMATO 1.4 TWO-SPOTTED SPIDER MITE 1.5 GREENHOUSE WHITEFLY 1.6 GREENHOUSE PEST MANAGEMENT 1.7 PLANT ESSENTIAL OILS 1.8 ROSEMARY OIL REFERENCES  2 4 5 8 9 10 12 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  2.1.1 2.1.2 2.1.3 2.1.4 2.1.5  20  Tetranychus urticae (Two-spotted spider mite) Trialeurodes vaporariorum (Greenhouse whitefly) Phytoseiulus persimilis (Predatory mite) Encarsia formosa (Parasitic wasp) Rosmarinus officinalis (Rosemary plant)  2.2 MATERIALS AND METHODS  2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6  20 20 21 22 22 24  Rosmarinus officinalis essential oil and commercial pesticides Spider mites Greenhouse white/lies Phytoseiulus persimilis Athias-Henriot and Encarsia formosa Gahan Plant material General growing conditions for plants, mites and whiteflies  24 24 24 24 25 25  2.2.7 Calculating lethal concentration 50 (LC )  25  50  2.2.8 Residual toxicities 2.2.9 Choice test bioassay for spider mites 2.2.10 Oviposition choice test bioassay for whiteflies 2.2.11 Trans-laminar activity of commercial pesticides 2.2.12 Data Analysis 2.3 RESULTS  29 30 31 32 32 33  2.3.1 Lethal concentration 50 ( L C , )  33  5 0  2.3.2 Residual toxicity 2.3.3 Choice tests 2.3.4 Trans-laminar activity 2.4 DISCUSSION  2.4.1 Efficacy against pests 2.4.2 Effects on bio-controls 2.4.3 Persistence in the environment  35 38 41 42  42 43 44  iv  2.4.4 Repellent effects 2.4.5 Rosemary oil as a pesticide: advantages and limitations REFERENCES  44 45 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 I T S 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 3.1.2 Rosmarinus officinalis essential oil 3.2  54 54  MATERIALS AND METHODS  56  3.2.1 Rosmarinus officinalis essential oil 3.2.2 Spider mites 3.2.3 General growing conditions for plants and mites 3.2.4 Calculating lethal concentration 50 ( L C , ) of the oil  56 56 56 57  5 0  3.2.5 3.2.6 3.3  Comparative toxicities Data Analysis  58 58  RESULTS  59  3.3. J Essential oil constituents 3.3.2 Lethal concentration 50 of the oil 3.3.3 Comparative toxicities of individual constituents and blends thereof. 3.4  DISCUSSION  59 59 ..60 66  3.4.1 Rosemary oil as an acaricide 3.4.2 Synergy among constituents  66 67  REFRENCES  69  CHAPTER FOUR: INTEGRATED C O N T R O L OF TWO-SPOTTED SPIDER MITES ON 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 FIELD E F F I C A C Y O F A R O S E M A R Y OILB 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 AND ASSESSING T H E PHYTOTOXIC EFFECTS OF T H E PESTICIDE ON T O M A T O PLANTS 72 4.1 INTRODUCTIUON  4.1.1 4.1.2 4.1.3 4.1.4  4.2 MATERIALS AND METHODS  4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6  ,  72  72 72 72 72  Two-spotted spider mite Phytoseiulus persimilis Rosemary oil. Phytotoxic effect Commercial pesticide Plant materials Two-spotted spider mites & Phytoseiulus persimilis Efficacy test Phytotoxicity tests Data Analysis  4.3 RESULTS  4.3.1 Efficacy test 4.3.2 Phytotoxicity test 4.4 DISCUSSION  4.4.1 Efficacy of EcoTrol'" 4.4.2 Phytotoxicity trials REFERENCES CHAPTER FIVE: S U M M A R Y AND CONCLUSION REFERENCES APPENDIX  74  74 74 74 75 76 77 78  78 80 83  83 84 86 88 94 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 TABLE 4.4. ANALYSIS OF VARIANCE OF NUMBER OF SPIDER MITE'S EGGS  79 80  vi  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 FIGURE 2.10.  RESIDUAL TOXICITY OF ROSEMARY OIL 1% TO E. FORMOSA  37  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% FIGURE 2.12.  39  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 % FIGURE 2.14.  40  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  vn  Acknowledgments M a n y people contributed significantly to this project. M y research supervisor and three committee members guided this study from its beginning to its final completion, and m y gratitude for their valuable input and support is profound. I take this opportunity to thank Professor M u r r a y B . Isman for his great help and support. Without his support, this study c o u l d not have been accomplished. I also thank D r s . Judith H . M y e r s , D a v e R. G i l l e s p i e and M i c h a e l T . W a n for p r o v i d i n g critical feedback and guidance on m y project. I must express m y warmest gratitude to m y lovely w i f e M r s . M a r y a m A n t i k c h i w h o helped me w i t h designing and b u i l d i n g the electronic micro-sprayer and supported me during m y project. I also have to thank D r . Shahriyar M i r a b b a s i f r o m the Department o f Electrical E n g i n e e r i n g o f U B C and M r . K e y v a n 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. M a n y thanks to M r . R u b e n H o u w e l i n g and H o u w e l i n g ' s Nurseries L t d . for providing plant materials, A p p l i e d B i o n o m i c s for p r o v i d i n g the bio-control agents and M r . R o d B r a d b u r y from Ecosafe Natural Products Inc. for p r o v i d i n g essential oils and analyzing the samples. Special thanks to M r . A m a n d e e p B a l , w h o maintained m y communication link w i t h the growers. M y deepest appreciation also goes to all colleagues and friends at the Centre for Plant Research at U B C , M r s . N a n c y B r a r d , D r . Y a s m i n A k h t a r , M r . Ikkei S h i k a n o , M r . D a v i d N o s h a d , M s . C r i s t i n a M a c h i a l , M s . N y s s a T e m m e l , M r . A l a i n B o u c h a i r and M r . B r i a n K i n g for their help and support. T h i s study was funded in part by a grant from E c o S M A R T Technologies Inc. and a research contract from the B C Greenhouse G r o w e r s A s s o c i a t i o n . F i n a l l y , I w o u l d like to thank m y f a m i l y , m y parents and m y in-laws w h o fully supported me during m y study.  viii  Chapter One: Introduction 1.1 Major objectives T h e 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 t w o - s p o t t e d spider mite and the greenhouse w h i t e f l y ) . T h i s pesticide was introduced to the U S market in 2002. It has been used m a i n l y on berries, grapes, nuts and tree fruits. The product also has been used on vegetables, but its use in the greenhouses has been limited to a few growers. A l t h o u g h this is a natural product and has been used in the U n i t e d States, it requires regulatory a p p r o v a l b y the Pest Management Regulatory A g e n c y ( P M R A ) i n order to be used b y Canadian growers. Similar to all new pesticides, it requires some assessment before entering the market. The following questions need to be addressed: •  Is this product efficacious against pests?  •  C a n 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 limitations for its application?  •  C o u l d pests evolve resistance after repeated applications?  •  W h a t is the mechanism o f toxicity o f this product?  1  •  C a n environmental factors affect its efficacy?  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 conditions concluding w i t h a small-scale greenhouse trial.  1.2 Historical perspective M o s t 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 w i t h the environment. F o r 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 availability o f the desirable species and desired traits o f these species. O v e r long periods o f time and many generations o f human selection, domesticated plant varieties and a n i m a l breeds emerged w i t h traits that w o u l d not survive without human involvement in such tasks as selective breeding, planting, w e e d i n g , harvesting, pest control and storage. These manipulations to the environment resulted in new problems that did not exist before. In cropping systems in particular, monoculture o f plants p r o v i d e d an aggregated food source for pests and suitable growing conditions for their offspring. W i t h increasing human population the necessity o f p r o v i d i n g more f o o d increased. T h e y not only had to develop their agriculture practices to produce more y i e l d , but also had to protect their products f r o m 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 c h e m i c a l 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. M a n y farmers took D D T as a guaranteed pest control tool that c o u l d completely solve the pest p r o b l e m ; however, that feeling o f relief q u i c k l y faded due to a n e w phenomenon; pesticide resistance. Intensive application o f pesticides is the most important factor in the quick build-up o f resistance in most pest populations. Pesticide application removes the susceptible pests f r o m 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 multiply w h i l e pesticides eliminate susceptible ones. E v e n t u a l l y , resistant pests outnumber susceptible ones and the pesticide is no longer effective. In addition to the pest resistance p r o b l e m , extensive application o f pesticides caused damage to the environment, non-target organisms, higher animals in ecosystem and impacted human health. N u m e r o u s studies have been conducted on this subject and many books have been written to describe its detection, magnitude, m e c h a n i s m , side effects and management (1,2). Recently, some scientists have taken a different approach towards pest management by using plants as potential sources o f pesticides. Plants have evolved different defense mechanism against herbivores. Secondary metabolites o f plants including a w i d e array o f chemicals- are good examples o f these mechanisms. O u r ancestors were aware o f the healing and medicinal properties o f some plants and their extracts. T h e y 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 t h o u g h these natural based- products seem to be  3  safer than conventional synthetic products, there are many u n k n o w n aspects that need to be addressed before extensive application. In the present study, a rosemary o i l (Rosmarinus officinalis) based- pesticide has been tested against t w o major pests o f greenhouse tomato (Lycopersicon esculentum), the two-spotted spider mite (Tetranychus urticae), and the greenhouse whitefly (Trialeurodes vaporariorum).  1.3 Greenhouse Tomato T h e greenhouse vegetable industry is an important and g r o w i n g segment o f Canadian agriculture. A c c o r d i n g to Agriculture 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 i n 2000. O f f i c i a l statistics (Statistics Canada P u b l . 20-202 for 2000) value the Canadian greenhouse industry at $1711 M and the greenhouse vegetable portion at $505 M . T h e m a i n greenhouse vegetable crops in Canada are tomatoes (468 ha), cucumbers (190 ha), sweet peppers (144 ha) and lettuce (21 ha) (5). D u r i n g the 1990s, the total area under glass and plastic more than doubled to nearly 1,500 hectares. B y 2 0 0 3 , it had reached nearly 1,900 hectares. In 2003, revenue f r o m greenhouse sales reached a record high o f almost $2.1 b i l l i o n ; nearly double what it had been just six years earlier. F l o w e r s accounted for about 7 0 % o f sales and vegetables the remaining 3 0 % . In the early 1990s, revenues from the comparable greenhouse and field vegetables were roughly the same. H o w e v e r , since 1996, revenues f r o m greenhouse vegetables have increased at a m u c h more rapid pace than field vegetables. F o 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 m i l l i o n . T h i s was more than three times higher than the value o f $171.7 m i l l i o n for the same four vegetable  4  crops produced in the field. Farmers grow more tomatoes than any other vegetable crop, whether it's i n the greenhouse or in the field. Tomatoes alone, account for over one-half o f revenues f r o m the sale o f greenhouse vegetables. T h e y also cross the border in both directions. Canadian greenhouse growers have been shipping hothouse tomatoes to the United States in rising numbers. In recent years, Canada has enjoyed a trade surplus i n tomatoes, shipping far more south o f the border than A m e r i c a n farmers ship north. (6)  1.4 Two-spotted spider mite Spider mites belong to the f a m i l y Tetranychidae o f the order Prostigmata. T h e y are so named because many members o f this f a m i l y produce silk w e b b i n g on host plants. M o s t spider mite species are polyphagous. The Tetranychidae is a large f a m i l y o f w o r l d w i d e distribution. T h e f a m i l y consists o f two subfamilies: B r y o b i n a e and Tetranychinae. M o s t pest species belong to the Tetranychinae. T h e two-spotted spider mite Tetranychus urticae K o c h is the most important species in this subfamily. T h e twospotted spider mite is the most c o m m o n name for this species. It is also k n o w n informally by many other names (e.g. the glasshouse spider mite, the y e l l o w spider mite). N o t very appropriately, it is called the red spider mite or red spider i n some literature presumably because o f the red/orange color or the overwintering f o r m , or in reference to a species complex i n c l u d i n g T. cinnabarinus.  T h e two-spotted spider mite is an important pest o f  greenhouses throughout the w o r l d . It is a cosmopolitan 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 w h i c h over 300 species are grown in greenhouses. Its life c y c l e consists o f eggs, one larval stage, two n y m p h a l stages and an adult stage.  5  T h e eggs are often laid in clusters on the under surface o f leaves. T h e y are spherical in shape and translucent, pale in color. A s they develop, they become more y e l l o w i s h and red eyespots inside the eggshell can be seen. S i x - legged larvae are pale to y e l l o w i s h when first hatched and become y e l l o w i s h green after feeding. Eight-legged nymphs are y e l l o w i s h green w i t h dark spots. T h e i r body is o v o i d in shape w i t h short legs. A d u l t females are about 400-500 urn in length and males are smaller w i t h a pointed hysterosoma. T h e females (summer form) are y e l l o w i s h to greenish in color w i t h two black spots on the dorsolateral idiosoma, but are darker in color, often orange or red in the overwintering f o r m . T h e c o l o r o f mites may vary depending on the host plant and other environmental factors. T w o spotted spider mites often feed on cell chloroplasts on the under surface o f the leaf. T h e upper surface o f the leaf develops characteristic w h i t i s h or y e l l o w i s h stippling, w h i c h may j o i n and become brownish as mite feeding continues. A s mites move around, their w e b b i n g can span leaves and stems. H e a v y damage may cause leaves to dry and drop, and the plant may be covered w i t h w e b b i n g and may die prematurely. Development occurs between 12 and 4 0 ° C . D e v e l o p m e n t a l time f r o m egg to adult decreases w i t h increasing temperature and is less than a week at optimal temperatures for development (30-32°C). U n d e r a diurnal temperature c y c l e o f 15 to 2 8 ° C , development time is about 16 days. M a l e s develop slightly faster than females. M a l e s are attracted to a sex pheromone f r o m dormant female deutonymphs. They guard their territory and fight against any other invading males. M a t i n g occurs as soon as females emerge. Females start to lay eggs w i t h i n a couple o f days o f adulthood. The rate o f oviposition and fecundity varies w i t h food plant and temperature. A n average female  6  can lay over ten eggs per day and produce over 100 eggs during t w o weeks at about 25°C. T h e sex ratio is h i g h l y female biased, w i t h a female to male ratio o f about 3:1. T. urticae disperses b y active w a l k i n g or by passive transport in the w i n d , o n plants, o n tools or on people. Diapause is induced by short day lengths, lack o f f o o d supply and l o w temperature, and is n o r m a l l y terminated by a f i x e d period o f c h i l l i n g . G r a v i d females seek a protected niche at the end o f summer. Diapausing adults are orange/red in color (7). T h e e c o n o m i c threat posed by these mites is constantly increasing because o f the development o f resistance, and resurgence o f mite populations f o l l o w i n g use o f nonselective 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 r o m more than 60 countries (9). Spider mites and especially two-spotted spider mites have been on the priority lists f o r pest management in B C greenhouses f o r several years. T h e current control method f o r spider mites in B C greenhouses is based o n bio-control agents that include s i x different predatory mites (Phytoseiulus persimilis californicus  Athias-Henriot, Metaseiulus occidentalis  Carte, Persimilis longipes Evans, Galendromus  Nesbitt,  occidentalis  Amblyseius Nesbitt and  Amblyseius cucumeris Oudemans), one predatory midge (Feltiella acarisuga V a l l o t ) and two predatory bugs (Deraeocoris brevis U h l e r and Orius sp.). P r o b l e m s w i t h bio-control include limited efficacy against high populations o f spider mites, constraints, and b i o control agents' susceptibility to most pesticides.  7  1.5 Greenhouse Whitefly T h e greenhouse whitefly,  Trialeurodes  vaporariorum  W e s t w o o d , is another  important pest o f greenhouse crops w o r l d w i d e . Its life c y c l e consists o f eggs, a crawler stage, three n y m p h a l stages, a pupa and adults. W h i t e f l y adults are tiny, white moth-like insects. T h e y lay eggs on the underside o f leaves. E g g s hatch i n 10 to 14 days. A f t e r three molts i n about 14 days, they pupate and the adult emerges 6 days later. A d u l t s begin to lay eggs 4 days after emergence. E a c h female is capable o f l a y i n g 400 eggs over a period o f up to 2 months, although usually far fewer eggs are produced. T h e length o f the life cycle is temperature dependent. A d u l t s live 30 to 60 days and feed by s u c k i n g sap f r o m the plant. Two  species  of  whitefly  infest  British  Columbia  greenhouse  crops.  T. vaporariorum W e s t w o o d (the greenhouse w h i t e f l y ) is the most c o m m o n one. H o w e v e r Bemesia tabaci Gennadius (the sweet potato whitefly) was introduced to Canada in recent years o n imported plant materials and has been found in some greenhouses. Greenhouse w h i t e f l y has a w i d e host range and is k n o w n to develop o n more than 250 ornamental and vegetable plants. Poinsettia, hibiscus, nicotiana, aster, calendula, cucumber, lantana, tomato, grape, ageratum, bean, and begonia are a m o n g the more c o m m o n l y infested plants (10). B. tabaci is more difficult to control, o w i n g to its high e g g l a y i n g capacity and wide host range. Sweet potato whitefly is smaller than the greenhouse whitefly and is more off-white o r y e l l o w - w h i t e in color. Sweet potato whitefly is typically found lower in the plant canopy than greenhouse whitefly, w h i c h is often found o n the developing leaves at the g r o w i n g points. B o t h whitefly species can cause severe damage to foliage by reducing v i g o r and by coating the g r o w i n g points, leaves, and fruits w i t h excreta  8  (honeydew). T h e excreta becomes a food source for fungal moulds to develop. T h i s m o u l d coats pepper and tomato fruit requiring extra fruit c l e a n i n g costs prior to sale. Sweet potato w h i t e f l y can also transmit viruses and cause abnormal fruit discoloration. L i k e the two-spotted spider mite, the greenhouse w h i t e f l y is a m o n g the most important pests o f B C greenhouses. C o n t r o l mostly relies on bio-control agents, such as the parasitic wasp Encarsia formosa (11,12). L i k e spider mites, whiteflies 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 ability to control environmental factors that have great impact o n plants. In modern greenhouses, almost all environmental factors are under control, for instance temperature, moisture, light, nutrients and even the c o m p o s i t i o n o f different gases in the air that plants need for growth. In greenhouses, plants constantly g r o w and provide fruit throughout the year regardless o f the outside conditions. These controlled conditions are not only suitable for plants but also for a wide range o f pests, w h i c h normally cannot survive outside the greenhouse. H a v i n g optimal conditions for growth and an aggregated f o o d source allows many pests to achieve e p i d e m i c populations. A s the easiest control measure, pesticides have been extensively used inside greenhouses to control pests d u r i n g the production season. T h i s strong selection pressure resulted in emergence o f highly resistant populations o f pests to almost a l l pesticides that have been applied so far. B i o - c o n t r o l agents were introduced to greenhouses as an alternative solution. Different predators or parasitoids o f insect or mite pests were identified and  9  released inside greenhouses to reduce their populations. T h i s method has been successful for several pests. In Canada, the greenhouse pest management is largely based o n biological control. A survey conducted in 2002 indicated that 9 3 % o f tomato growers (n= 165) use b i o l o g i c a l control f o r insect and mite control (12). A l t h o u g h b i o l o g i c a l control methods were successful f o r most pests, they have limitations and cannot be used in a l l situations. F o r example, it has been shown that activity and survival o f the predatory mite Phytoseiulus persimilis can be affected by different levels o f humidity (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. T h u s , utilization o f bio-control agents for controlling greenhouse pests may o n l y be effective when combined w i t h other strategies (16). A s an example, N i c e t i c et al. (17) found that a combination 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. W h e n 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 oils o f plants might be considered a g o o d option for use i n combination w i t h other pest control methods.  1.7 Plant essential oils Plant essential oils are odorous compounds obtained through steam distillation o f herbs and m e d i c i n a l plants (18). These oils have been used traditionally as healing medicines i n many countries and ancient people were also aware o f their pesticidal properties, however, only in recent years have these oils been c o m m e r c i a l i z e d as pest control products (3). M o s t o f these oils are environmentally non-persistent, and non-toxic  10  to humans (with some exceptions) (19-21), fish (with some exceptions) and w i l d l i f e (2224) Plant essential oils are generally mixtures o f m o n o - and sesquiterpenes (e.g., ctterpineol and pulegone) and phenolics or monophenols (e.g., t h y m o l , carvacrol, and eugenol). T h e y are often quite volatile and are c o m m o n l y used as fragrances and as flavoring agents in f o o d (25). T h e y are sometimes incorporated into natural pest control products. F o r 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). H o u g h - G o l d s t e i n (27) reported antifeedent effects o f essential oils against the Colorado potato beetle  Leptinotarsa  decemlineata L . w h i l e Sharma and Saxena (28) showed their effectiveness as growth inhibitors on houseflies Musca domestica L. M a n y researchers have reported repellent, antifeedent and toxic properties o f selected essential oils 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 citronellic a c i d were found toxic to the c o m m o n housefly, western corn rootworm Diabrotica  vergifera vergifera, and the two-spotted spider mite (30). T h y m e  o i l was also found to be toxic to the tobacco c u t w o r m Spodoptera litura (31). C h o i et al. (32) tested a total o f 53 esential oils against T. urticae and P. persimilis v i a fumigation. Caraway seed, citronellal, l e m o n , eucalyptus, pennyroyal and peppermint oils were found to be highly toxic to both mites. Rosemary o i l w a s also found to be toxic to the predaceous mites Amblyseius  barkeri Hughes, A. zaheri a n d Typhlodromus  athiasae  Porath (33) w h i l e it showed repellent properties against the o n i o n aphid Neotoxoptera formosana Takahashi (34) and the green peach aphid Myzus persicae Sulzer (35). B o t h  11  contact and fumigant toxicities o f eugenol and methyl eugenol were demonstrated to the A m e r i c a n cockroach Periplaneta americana L. (36). The mechanisms o f toxicity o f essential oils have not been f u l l y identified. H o w e v e r a recent investigation using the A m e r i c a n cockroach points to the octopaminergic nervous system as the site-of-action o f some essential oils in insects (37).  1.8 Rosemary oil Rosmarinus officinalis L . is an evergreen perennial w o o d y shrub w i t h aromatic, needle-like leaves and gray, scaly bark. Rosemary bushes can grow up to 6 ft (1.8 m) tall w i t h a spread o f 4-5 ft (1.2-1.5 m). T h i s plant belongs to the f a m i l y L a m i a c e a e formerly k n o w n as the Labiatae. It is used for flavoring 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, inflammatory diseases and hepatotoxicity (38). Anti-cancer (39-41) and anti-viral (42) properties o f rosemary have also been reported. Beside the great m e d i c i n a l 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 oil o f rosemary has repellent and deterrent properties against Thrips tabaci and affect host plant selection and acceptance (44). It also showed ovicidal activity against t w o stored-product insects (45). Essential oil o f rosemary is a c o m p l e x mixture o f different constituents. A recent study detected 33  12  compounds in the oil (46). T h e m a i n components o f the o i l are oc-pinene, 1,8-cineole, camphor, p-pinene, and borneol.  References 1) E b i n g , W . (Ed.). (1997). Molecular mechanisms of resistance to  agrochemicals.  B e r l i n : Springer, pp 21-75. 2) R o u s h , R . T . , & Tabashnik, B . E . (1990). Pesticide resistance in arthropods. N e w Y o r k : C h a p m a n & H a l l . 303p. 3) Isman, M . B . (2000). Plant essential oils for pest and disease management. Crop Protection, 4)  19:603-608.  Introduction to the greenhouse industry. (2003). Agriculture and Agri-Food  Canada.  Retrieved N o v e m b e r 3 , 2 0 0 5 , f r o m http://res2.agr.gc.ca/harrow/publications/ Introduction_e.htm 5) Horticulture and greenhouse products, by province (2001 Census o f Agriculture). Statistics Canada. Retrieved N o v e m b e r 3, 2005, f r o m http://www40.statcan.ca/101/cst01/agrc31 k.htm 6)  Study: H i g h - t e c h vegetables: the b o o m i n g greenhouse vegetable industry. (2005). The Daily. Statistics Canada. Retrieved N o v e m b e r 3, 2005, f r o m http://www.statcan.ca/Daily/English/050322/d050322e.htm  7) Z h a n g , Z . (2003). Mites of greenhouses: identification, biology and control. W a l l i n g f o r d : C A B I publishing, p p 54-61. 8) C r a n h a m , J . E . , & H e l l e , W . (1985). Pesticide resistance in Tetranychidae. I n : World crop pest - spider mites: their natural enemies and control, A m s t e r d a m : Elsevier, pp 405-421. 9) The Database o f Arthropods Resistance to Pesticides. (2004). Michigan State University-Center for Integrated Plant Systems. Retrieved N o v e m b e r 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: Princeton, pp 284-289. 11) Greenhouse vegetable production guide for commercial growers. (1997). Province of British Columbia, Ministry of Agriculture, Food and Fisheries, p p 88-89. 12) M u r p h y , G . D . , Ferguson, G . , F r y , K . , Lambert, L., M a n n , M . , & M a t t e o n i , J . (2002). The use o f biological 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 o f humidity on the activity o f Phytoseiulus persimilis Athias-Henriot and its prey, Tetranychus urticae K o c h . ( A c a r i n a : Phytoseiidae, Tetranychidae). Canadian Journal of Zoology, 4 4 : 863-871. 14) E v e r s o n , P. (1980). The relative activity and functional response o f Phytoseiulus persimilis ( A c a r i n a : Phytoseiidae) and Tetranychus urticae ( A c a r i n a : Tetranychidae): the effect o f temperature. Canadian Entomologist, 121: 17-24. 15) E v e r s o n , P. (1979). The functional response o f the Phytoseiulus persimilis  (Acarina:  Phytoseiidae) to various densities o f Tetranychus urticae ( A c a r i n a : Tetranychidae). Canadian Entomologist,  111: 7-10.  16) Z h a n g , Z . Q . & Sanderson, J . P. (1995). Two-spotted spider mite ( A c a r i : Tetranychidae) and Phytoseiulus persimilis ( A c a r i : Phytoseiidae) o n greenhouse roses: Spatial distribution and predator efficacy. Journal of Economic  Entomology,  88:352-357. 17) N i c e t i c , O . , W a t s o n , D . M . , Beatti, G . A . C , Meats, A . , & Z h e n g , J . (2001). Integrated pest management o f two-spotted spider mite Tetranychus urticae o n  15  greenhouse roses using petroleum spray o i l and the predatory mite Phytoseiulus persimilis. Experimental & Applied Acarology, 2 5 : 3 7 - 5 3 . 18) Y a t a g a i , M . (1997). M i t i c i d a l activities o f tree terpenes. Current Topics of Phytochemistry,  1:87-97.  19) R o e , F . J . C . (1965). C h r o n i c toxicity o f essential oils and certain other products o f natural origin. Food and Cosmetics Toxicology, 33: 311-324. 20) C o c k a y n e , S. E . , & G a w k r o d g e r , D . J . (1997). Occupational contact dermatitis i n an aromatherapist. Contact Dermatitis, 37: 306-309. 21) Hjorther, A . B . , Christophersen, C , Hausen, B . M . & M e n n e , T . (1997). Occupational allergic contact dermatitis f r o m carnosol, a naturally-occuring c o m p o u n d present in rosemary. Contact Dermatitis, 37: 99-100. 22) K u m a r A n u j , D . , F l o r e n c e , V . , B r o u g h t o n , M . J . , & Sriharan, S. (2000). E f f e c t o f root extracts o f m e x i c a n m a r i g o l d , Tagetes minuta (Asterales: Asteraceae), on s i x nontarget aquatic macroinvertebrates. Environmental Entomology, 29: 1 4 0 - 1 4 9 . 23) Wager-Page, S. & M a s o n , J . R. (1997). Ortho-aminoacetophenone, a non-lethal repellent: T h e effect o f volatile cues vs. direct contact on avoidance behavior by rodents and birds. Pesticide Science, 46: 55-60. 24) C l a r k , L . & A r o n o v , E . V . (1999). H u m a n food flavor additives as bird repellents: I. Conjugated aromatic compounds. Pesticide Science, 55: 903-908. 25) Isman, M . B . (1999). Pesticides based on plant essential oils. Pesticide Outlook, 10: 68-72. 26) O b e n g - O f o r i , D., R e i c h m u t h , C . H . , B e k e l e , A . , & Hassanali, A . (1997). B i o l o g i c a l activity o f 1,8 -cineole, 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) H o u g h - G o l d s t e i n , J . A . (1990). Antifeedant effects o f c o m m o n herbs on the C o l o r a d o potato beetle (Coleoptera: Chrysomelidae). Environmental Entomology, 19: 234-238. 28) Sharma, R . N . , & Saxena, K . N . (1974). Orientation and developmental inhibition in the housefly b y certain terpenoids. Journal of Medical Entomology, 11: 6 1 7 - 6 2 1 . 29) G e n g a i h i , E . , A m e r , S . E . & M o h a m e d . S . A . A . (1996). B i o l o g i c a l activity o f thyme o i l and t h y m o l against Tetranychus urticae K o c h . Anzeigerfur  Scchadlingskunde  Pflanzenscutz Umweltsschutz, 6 9 : 157-159. 30) L e e , S., T s a o , R., Peterson, C . & Coats, J . R . (1997). Insecticidal activity o f monoterpenoids to western corn rootworm (Coleoptera: C h r y s o m e l i d a e ) , two-spotted spider mite ( A c a r i : Tetranichidae), and house f l y (Diptera: M u s c i d a e ) . Journal of Economic Entomology, 9 0 : 883-892. 31) Isman, M . B . , W a n , A . J . & Passreiter, C M . (2001). Insecticidal activity o f essential oils to the tobacco c u t w o r m , Spodoptera litura. Fitoterapia, 7 2 : 65-68. 32) C h o i , W . , L e e , S., Park, H . , & A h n , Y . (2004). T o x i c i t y o f plant essential oils to Tetranychus urticae ( A c a r i : Tetranychidae) and Phytoseiulus persimilis ( A c a r 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 o n three predacious mites o f the f a m i l y Phytoseiidae ( A c a r i : Phytoseiidae). Acta Phytopathologica  et Entomologica Hungarica, 34: 3 5 5 - 3 6 1 .  17  34) Masatoshi, H . , & H i r o a k i , K . (1997). Repellency o f rosemary o i l and its components against the o n i o n a p h i d , Neotoxoptera formosana (Takahashi) ( H o m o p t e r a , A p h i d i d a e ) . Applied Entomology & Zoology, 3 2 : 303-310. 35) Masatoshi, H . (1998). R e p e l l e n c y 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., P a n g , F . Y . , H u a n g , Y . , K i n i , M . R . & H o , S . H . (1998). Insecticidal and repellent properties o f nine volatile constituents o f essential oils against the A m e r i c a n cockroach, Periplaneta americana (L.). Pesticide Science, 54: 261-268. 37) E n a n , E . (2001). Insecticidal activity o f essential oils: octopaminergic sites o f action. Comparative Biochemistry and Physiology, 130: 325-337. 38) A b u - A m e r , K . M . , S e n , P., & al-Sereiti, M . R . (1999). P h a r m a c o l o g y o f rosemary (Rosmarinus officinalis L i n n . ) and its therapeutic potentials. Indian Journal of Experimental Biololgy, 37: 124-30. 39) O f f o r d E . A . , M a c e K . , A v a n t i O . , & Pfeifer A . M . A . (1997). M e c h a n i s m s involved in the chemoprotective effects o f rosemary extract studies i n human liver and bronchial cells. Cancer Letters, 114: 275-281. 40) Singletary, K . , M a c D o n a l d , C , & Wallig, M . (1996). Inhibition b y 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: 4 3 48. 41) P l o u z e k , C . A . , C i o l i n o , H . P . , Clarke, R., & Y e h , G . C . (1999). Inhibition o f P glycoprotein activity and reversal o f multidrug resistance i n vitro b y rosemary extract. European Journal of Cancer, 35: 1541-1545.  18  42) A r u o m a , O . I., Spencer, J . P. E . , R o s s 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 . , & H a l l i w e l 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 l s : their antibacterial properties and potential applications in foods- a r e v i e w . International Journal of Food Microbiology, 94: 2 2 3 - 2 5 3 . 44) K o s c h i e r , E . H . , & Sedy, K . A . (2003). Labiatae essential oils affecting host selection and acceptance o f Thrips tabaci lindeman. Crop Protection, 22: 929-934. 45) T u n c , I., Berger, B . M . , Erler, F., & Dagli, F. (2000). O v i c i d a l activity o f essential oils f r o m five plants against t w o stored-product insects. Journal of Stored Products Research, 36: 161-168. 46) Santoyo, S., C a v e r o , S., Jaime, L . , Ibanez, E . , Senorans, F . J . , & R e g l e r o , G . (2005). C h e m i c a l c o m p o s i t i o n and antimicrobial activity o f Rosmarinus officinalis L . essential o i l obtained v i a supercritical f l u i d 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 o n virtually every major food crop and ornamental plant. A b o u t 1200 species o f spider mites are k n o w n i n the w o r l d (1). It is the most polyphagous species o f spider mites and has been reported from over 150 host plants o f e c o n o m i c value (2). T h e greatest p r o b l e m w i t h this mite is its ability to evolve resistance rapidly to pesticides only after f e w applications (3). Spider mites have e v o l v e d resistance to more than 80 acaricides to date and resistance has been reported from more than 60 countries (4). Spider mites and especially two-spotted spider mites have been a priority pest i n B C greenhouses f o r several years. T h e current control method f o r spider mites i n B C greenhouses is mostly r e l y i n g o n bio-control method based on six different species o f predatory mites, one predatory midge and t w o predatory beetles. The p r o b l e m w i t h these bio-control agents is their l i m i t e d efficacy against higher populations o f spider mites and their susceptibility to most pesticides.  2.1.2 Trialeurodes vaporariorum (Greenhouse whitefly) Greenhouse whitefly, Trialeurodes vaporariorum W e s t w o o d , is another important pest o f greenhouse crops w o r l d w i d e . T w o species o f whitefly infest B r i t i s h C o l u m b i a greenhouse crops. T. vaporariorum W e s t w o o d (the greenhouse w h i t e f l y ) is the more  20  c o m m o n one. H o w e v e r Bemesia tabaci Gennadius (the sweet potato w h i t e f l y ) was also introduced to Canada i n recent years on imported plant materials and has been found i n some greenhouses. Greenhouse whitefly has a wide host range and is k n o w n to develop o n more than 250 ornamental and vegetable plants. Poinsettia, hibiscus, nicotiana, aster, calendula, cucumber, lantana, tomato, grape, ageratum, bean, and begonia are a m o n g the more c o m m o n l y infested plants (5). L i k e spider mites, whiteflies have also evolved resistance to many pesticides (6).  2.1.3 Phytoseiulus persimilis (Predatory mite) T h e predatory mite, Phytoseiulus persimilis A t h i a s - H e n r i o t , has been studied extensively w i t h respect to its potential for biological control o f tetranychid mites o n vegetables and ornamentals in greenhouses (7,8) P. persimilis is a selective predator that is able to rapidly suppress spider mites (9,10). Since j u v e n i l e development and reproduction o f P. persimilis depends on the availability o f spider mites as prey (11), it often disappears f r o m 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). T h u s utilization o f the predatory mite for controlling spider mites i n greenhouses m a y only be effective when c o m b i n e d w i t h other strategies (13) . A s an example, N i c e 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 o n greenhouse roses (14) .  21  2.1.4 Encarsia formosa (Parasitic wasp) Encarsict formosa G a h a n is used w o r l d w i d e for c o m m e r c i a l control o f whiteflies in greenhouse crops. C o m m e r c i a l use began in Europe in the 1920s and it shipped to Canada i n the 1930s, but by 1945 interest waned due to the development o f pesticides. A f t e r 1970, use was reinitiated and has expanded f r o m 100 hectares o f greenhouse crops to 4800 hectares in 1993 (8). In Canada, the greenhouse vegetable industry is largely based o n b i o l o g i c a l controls. A survey conducted in 2002 indicated that 9 3 % percent o f the tomato growers (total o f 165 growers) use b i o l o g i c a l 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 o r more pesticides, either in laboratory tests or under conditions o f practical use i n 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. M o s t o f the pesticides were found to be toxic to E. formosa. Selective materials o f interest for possible combination w i t h E. formosa include insecticidal soap, buprofezin, azadirachtin, abamectin, and resmethrin (16). W h e n 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 oils o f plants may be a good option for use in combination w i t h other pest control methods.  2.1.5 Rosmarinus officinalis (Rosemary plant) Plant essential oils are obtained through steam distillation o f herbs and medicinal plants (17) M o s t o f these oils are environmentally  non-persistent, a n d non-toxic to  humans (with some exceptions) (18-20), fish (with some exceptions) and w i l d l i f e ( 2 1 -  22  22). Rosemary o i l has been traditionally used as a medicine f o r c o l i c , 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. T h e aromatic vapor o f rosemary has o v i c i d a l a n d larvicidal effects o n several stored product pests (25,26) and the two-spotted spider mite (27) as a fumigant. T h e o i l can have sub-lethal effects as w e l l , for example acting as a repellent to onion thrips, Thrips tabaci (28). C h o i et al. (29) tested a total o f 53 essential oils against T. urticae and P. persimilis as a fumigant. C a r a w a y seed, citronella, l e m o n , eucalyptus, pennyroyal a n d 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 m y 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 whitefly and their bio-control agents.  23  2 . 2 Materials and methods 2.2.1 R o s m a r i n u s officinalis essential oil a n d c o m m e r c i a l pesticides Pure Rosmarinus officinalis essential o i l (Intarome T O , Lot# 0 2 1 3 1 4 2 M B - 1 0 0 % ) , three c o m m e r c i a l pesticides, Hexacide™(5% rosemary o i l ) , E c o T r o l ™ ( 1 0 % rosemary o i l ) and Sporan™(17.6% rosemary o i l ) and a blank formulation o f E c o T r o l were obtained f r o m E c o S M A R T Technologies Inc (Franklin, T N , U S A ) .  2.2.2 S p i d e r mites Spider mites originated f r o m a research colony maintained o n tomato plants for more than five years without  any pesticide exposure at A g r i c u l t u r e and A g r i - F o o d  Canada ( A g a s s i z , B C , Canada). These mites were reared o n three-week-old vine tomato plants (Lycopersicon  esculentum M i l l c v . Clarance) provided by H o u w e l i n g ' s Nurseries  (Delta, B C , Canada).  2.2.3 G r e e n h o u s e whiteflies Greenhouse whiteflies obtained f r o m A p p l i e d B i o n o m i c s L t d . (Sidney, B C , Canada) originated f r o m a c o m m e r c i a l colony maintained o n tobacco plants for more than 10 years without any pesticide exposure. A d u l t whiteflies were transferred to threew e e k - o l d tomato plants (Lycopersicon  esculentum M i l l c v . Clarance) i n the greenhouse  inside fine mesh cages that a l l o w e d air circulation but prevented insects f r o m escaping.  2.2.4 Phytoseiulus persimilis Athias-Henriot a n d Encarsia Gahan  formosa  Predatory mites and parasitic wasps were purchased f r o m A p p l i e d B i o n o m i c s (Sidney, B C , Canada). Predatory mites were transferred to a spider mite colony maintained o n tomato plants caged i n the greenhouse. Parasitic wasps were introduced to a whitefly c o l o n y maintained o n caged tomato plants inside the greenhouse.  24  2.2.5 Plant material T h r e e - w e e k - o l d tomato plants (Lycopersicon esculentum M i l l c v . Clarance) provided by H o u w e l i n g ' s Nurseries (Delta, B C , Canada) were transferred to plastic pots containing a mixture o f regular peat (50%), fine bark (25%) and p u m i c e (25%) provided by West C r e e k F a r m s ( L a n g l e y , B C , Canada) in greenhouse at the U n i v e r s i t y o f B r i t i s h Columbia.  2.2.6 General g r o w i n g c o n d i t i o n s for plants, mites a n d whiteflies Plants infested w i t h mites o r whiteflies were kept inside isolated cages w i t h i n the greenhouse at 24± 6 ° C , 4 0 - 6 0 % relative humidity ( R H ) and under natural daylight. Plants were irrigated three times per week, t w o times w i t h water and one time w i t h watersoluble fertilizer (Peters E X C E L  15-5-15 C a l - M a g ) (The Scotts C o m p a n y , M a r y s v i l l e ,  O H , U S A ) . A d u l t female mites were transferred to clean plants, a l l o w e d to oviposit for 48 hours, and then removed f r o m the plant. Development o f these eggs resulted in a cohort o f evenly aged mites that were used for a l l bioassays. A d u l t whiteflies were used for all bioassays.  2.2.7 Calculating lethal concentration 50 ( L C  5 0  )  Different bioassay methods were used f o r spider mites and whiteflies. F o r spider mites a leaf disc painting method was used f o r calculating L C  5 0  o f the rosemary o i l and  three pesticides. Tests were conducted in disposable plastic Petri dishes (3 c m diameter). M i t e s were treated w i t h s i x n o m i n a l concentrations (2.5, 5, 10, 2 0 , 4 0 and 80 m l litre "') o f the essential o i l o r the c o m m e r c i a l pesticides and their blank formulation (only for E c o T r o l ) , using a spreader sticker adjuvant (Latron B - 1 9 5 6 , 60 m g litre "') ( R o h m and Haas, P h i l a d e l p h i a , P A , U S A ) diluted in distilled water. L e a f discs (3cm diameter), were  25  cut f r o m leaves o f greenhouse-grown plants using a cork borer. A 2 0 u L aliquot o f each concentration was painted o n the under side o f the leaf disc w i t h a micropipette g i v i n g concentrations o f • (6.25, 12.50, 25.10, 5 0 . 2 1 , 100.43 and 200.87 f i g / c m ) . A f t e r d r y i n g 2  at r o o m temperature f o r 5 minutes, each disc w a s placed i n the bottom o f a Petri dish atop a 3 c m diameter disc o f W h a t m a n N o . 1 filter paper wetted w i t h 5 0 u L distilled water (Figure 2.1). F i v e adult female spider mites w e r e introduced into each Petri dish and the covered dishes w e r e p l a c e d i n a growth chamber at 2 6 ± 2 ° C , 5 5 - 6 0 % R H and a 16/8h L D photoperiod. M o r t a l i t y w a s determined under a dissecting m i c r o s c o p e 2 4 hours after treatment. M i t e s were considered dead i f appendages d i d not m o v e w h e n prodded w i t h a fine paintbrush. C o n t r o l mites were h e l d o n leaf discs painted w i t h the carrier solvent alone. A l l treatments were replicated f i v e times.  Figure 2.1. Leaf disc painting method  A f u m i g a t i o n chamber w a s used to test fumigant toxicity o f rosemary o i l to adult whiteflies. Tests were conducted in disposable plastic containers ( 4 c m diameter x 6.5 c m height). W h i t e f l i e s were treated w i t h same s i x n o m i n a l concentrations (2.5, 5,10,20,40  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 filter paper were placed and pushed o n top o f the cotton pad. A 50 \i\ aliquot o f each concentration was added to the filter paper w i t h a micropipette producing concentrations o f H (1.353, 2.713, 5.432, 10.864, 21.729 and 43.459 fxg/cm ). A n o t h e r 3  filter paper was placed on top o f the previous one w i t h a 1 c m gap to prevent insects f r o m having direct contact w i t h the treated paper. Fifteen adult whiteflies were introduced to each container. C o n s i d e r i n g the fact that rosemary o i l contains compounds that are highly volatile, bioassays were conducted in two different ways. In one group, containers were covered w i t h a plastic l i d to trap a l l the volatiles (closed-container). In the other group, containers were covered w i t h a dense net to prevent whiteflies f r o m escaping but allowed volatiles to evaporate (open-container) (Figure 2.2). A leaf disc painting method was also used to measure the contact toxicity o f rosemary o i l to whiteflies using plastic containers instead o f Petri dishes. Containers were placed in a growth chamber at 26±2 ° C , 5 5 - 6 0 % R H and a 16/8 h L D photoperiod. Mortality was determined under a dissecting microscope 24 hours after treatment. Whiteflies were considered dead i f appendages d i d not m o v e when prodded w i t h a fine paintbrush. C o n t r o l whiteflies were held in containers treated w i t h the carrier solvent alone ( 7 0 % aqueous methanol). A l l treatments were replicated five times.  27  Figure 2.2. Fumigation chamber (open and closed container)  A n electronic micro-sprayer was developed to measure the direct contact toxicity o f the toxicants to test organism m i m i c k i n g spraying practices inside greenhouses ( A p p e n d i x one). T o x i c i t y o f rosemary o i l to the predatory mite P. persimilis and to the parasitic wasp E. formosa w a s measured using a leaf disc painting method, a direct contact method and a fumigation method. F o r predatory mites, ~ 5 0 spider mite eggs w e r e placed onto each leaf disc ( 3 c m diameter) as food source. A d u l t predatory mites w e r e used for bioassays. T o m a t o leaves containing both spider mites and predatory mites were placed inside a Petri dish ( 1 0 c m diameter) on top o f an ice pack inside a S t y r o f o a m b o x i n order to i m m o b i l i z e the predators during bioassay. A d u l t parasitic wasps were collected by an aspirator and transferred i n sealed plastic test tubes. F o r easier h a n d l i n g , they were kept at 5 ° C f o r 2 minutes p r i o r to bioassay to i m m o b i l i z e them. F i v e adult mites and five adult wasps were then transferred to each fumigation chamber. E a c h treatment w a s replicated 5 times. D i r e c t contact toxicity o f the c o m m e r c i a 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 v e adult predatory mites were transferred to a leaf disc (3cm diameter) containing ~ 5 0 spider mite eggs and then sprayed w i t h the sprayer. F i v e adult female spider mites were put on leaf discs and then sprayed w i t h sprayer. Controls were sprayed w i t h carrier solvent alone ( 7 0 % aqueous methanol). A l l treatments were kept inside a growth chamber at the same conditions described above. M o r t a l i t y was measured 24 hours after treatment. M i t e s or wasps were considered dead i f they did not move their appendages w h e n prodded w i t h a paintbrush. M o r t a l i t y in control groups was corrected by A b b o t t ' s f o r m u l a . E a c h treatment was replicated five times.  2.2.8 Residual toxicities Residual toxicity o f rosemary o i l against greenhouse whiteflies and parasitic wasps and o f three rosemary oil-based pesticides against two-spotted spider mites and predatory mites was measured. Three- w e e k - o l d tomato plants were sprayed i n d i v i d u a l l y w i t h rosemary o i l (10 m l 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 - 1 9 5 6 , 60 m g litre "') diluted in distilled water used as the carrier solvent. C o n t r o l plants were sprayed w i t h 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 ( 3 c m diameter) were cut from each i n d i v i d u a l 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 v e 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 C h o i c e test b i o a s s a y f o r s p i d e r 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/cm ) dissolved in a spreader sticker adjuvant (Latron B-1956, 60 mg litre ) 2  -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 O v i p o s i t i o n c h o i c e test b i o a s s a y 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 containerfilledwith 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  A C  B  A  B  A  C  A  A  B  C  B  A C  C  B  B  B  A  A C  C  Figure 2.4. Oviposition cages - A = after 24 hours, B = after 48 hours, C = after 72 hours  31  2.2.11 T r a n s - l a m i n a r activity o f c o m m e r c i a l p e s t i c i d e s F i v e - w e e k - o l d tomato plants were sprayed from above w i t h Hexacide™ (7.5 m l litre " ' ) , E c o T r o l ™ (7.5 m l litre "') o r Sporan™ (7.5 m l litre  - 1  ) using a spreader  sticker adjuvant (Latron B - 1 9 5 6 , 6 0 m g litre " ' ) diluted i n distilled water as the carrier solvent (each plant received ~ 80 ± 10 g o f sprayed material). L e a f discs ( 3 c m ) were cut f r o m tomato plants and were placed o n top o f a wetted filter paper d i s c (as described above) w i t h either the upper surface (sprayed) or undersurface (not sprayed) f a c i n g up (Figure 2.5). F i v e adult female spider mites were introduced into each Petri d i s h and the covered dishes were p l a c e d i n a growth chamber at 2 6 ± 2 ° C , 5 5 - 6 0 % R H and a 16/8h L D photoperiod. M o r t a l i t y w a s determined under a dissecting m i c r o s c o p e 2 4 hours after treatment as described above.  Figure 2.5. Trans-laminar effects of rosemary oil-based pesticides  2.2.12 Data A n a l y s i s Mortality  observations were analyzed using the S P S S p r o g r a m ( C h i c a g o , I L ,  U S A ) , version 11.5 for analysis o f variance ( A N O V A ) . T u k e y ' s test was used to compare means. Probit analysis w a s used to determine L C  5 0  ,  using the E P A probit analysis  program version 1.5. A b b o t t ' s f o r m u l a was used to correct mortality i n controls.  32  2 . 3 Results  2.3.1 Lethal concentration 50  (LC ) 5 0  L C ^ o f pure rosemary o i l was 13.19 m l litre " ' ( s 33.09 n g / c m ) f o r spider mites 2  (Table 2.1). H e x a c i d e (containing 5 % rosemary oil) and E c o T r o l (containing 1 0 % rosemary oil) were found to be two times more active than Sporan (containing 1 8 % rosemary oil). A l t h o u g h Sporan contains more rosemary o i l as active ingredient it showed lower activity against mites. T h i s might be due to difference i n formulation. N o mortality was observed in control mites treated with carrier solvent. In fumigation tests against whiteflies, rosemary o i l i n closed containers was three times more t o x i c than in open containers. T h e results also showed that the rosemary o i l is more toxic to adult whiteflies 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 i a fumigation. T o x i c 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 2  X Value  95%  13.19  2.139  10.05-17.78  33.09  N/A  5  4.01  2.583  2.36-5.46  10.05  N/A  Leaf disc painting  5  5.51  1.358  4.03-7.06  13.79  N/A  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  Toxicant Organism Bioassay method  N  Rosemary  TSM  Leaf disc painting  5  Hexacide™  TSM  Leaf disc painting  EcoTrol ™  TSM  Ec-Blank  L  C  5 0  (ml litre  CI  [ig/cm  2  Lig/cm  Based o n direct contact toxicity, three c o m m e r c i a l pesticides at their recommended label rate produced no mortality among predatory mites indicating that two-spotted spider mites are more susceptible to rosemary o i l . N o mortality was observed i n control mites (Figure 2.6).  34  3  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 R e s i d u a l toxicity Residues o f all three pesticides were found to be moderately toxic to spider mites within the first hour after spraying. H o w e v e r , toxicity decreased significantly after 24 hours. There was a significant interaction between the toxicity o f residues and time [F(3,32)= 7.203, p<0.05]. T h i s result clearly shows that rosemary o i l is not persistent in the environment due to its volatile nature (Figure 2.7). N o mortality was observed among controls. Residues o f three commercial pesticides d i d not show statistically significant toxicity to P. persimilis (Figure 2.8). T i m e had a significant m a i n effect o n the toxicity o f the residues [F (1,32) = 5.538, p< 0.05] while no significant difference was found among  35  pesticides ( F (3,32) = 1.026, p> 0.05 (p= 0.394)). There was n o 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  80  T  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 whiteflies w i t h i n the first hour after spraying but not significantly toxic after 24 hours (Figure 2.9). N o mortality was observed in control whiteflies.  100  -I  80 -  lhr  60 -  co o  24hrs 40 20 n  b ^ rosemary 1%  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)  T h e results indicate that rosemary o i l residue is considerably t o x i c to E. formosa within the first hour but that toxicity decreased almost three folds after 24 hours (Figure 2.10). B o t h t i m e ( F (l,16)=35.588,p<0.05) and treatment ( F (1,16) = 84.390, p<0.05) had significant m a i n effects o n toxicity o f the residues. There was a significant interaction between time and treatment (F (1,16)= 21.844,/?<0.05).  37  100 80 • Rosemary 1% Control  1 hour  24 hours Time  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 C h o i c e tests Rosemary o i l has a significant deterrent effect on mites but this effect declined over time. There was a significant interaction between the location o f mites and time (F (3,72)= 81.203, p< 0:05) and treatment had a significant m a i n effect ( F (1,72)=985.289, p< 0.05). D u r i n g first 12 hours mites aggregated more o n the control disc o r at locations far from the treated disc w i t h i n the test arena. A f t e r 24 hours, they started to spread on both discs and (Figure 2.11), after 48 hours they were almost equally dispersed o n both discs. M i t e s laid greater number o f eggs o n the control discs than o n treated discs. A l t h o u g h they started to oviposit eggs on both treated and control discs after 12 hours, the final numbers o f eggs after t w o days was significantly higher o n control discs (Figure 2.12).  38  ] Treated I Control  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.  Treated  control  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  Results f r o m whitefly o v i p o s i t i o n choice tests indicated that whiteflies l a i d SI gnificantly  more eggs o n control leaves than on treated leaves w i t h i n a l l three intervals.  B o t h rosemary o i l ( F (l,42)=77.931,/><0.05) and time ( F (2,42) =29.366, p<0.05) had main effects o n o v i p o s i t i o n rate. There was a significant interaction between time and pesticide (F (2,42)=6.273,/?<0.05). S i m i l a r to spider mites, rosemary o i l deters whiteflies, h o w e v e r , the effect does not diminish as fast is it d i d for spider mites. Whiteflies laid three times more eggs o n control leaves than o n treated leaves after 48 hours (Figure 2.13).  J Rosemary 1 % I Control  24hrs  48hrs  72hrs  Time 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 T r a n s - l a m i n a r activity N o mortality was observed among mites that were placed o n the un-sprayed surface o f the leaf discs (Figure 2.14) w h i l e significant toxicity w a s observed among mites that were placed o n the sprayed surface, indicating that rosemary o i 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 whitefly, causing complete mortality in the laboratory at concentrations that cause no phytotoxicity to host plants (Chapter three). Rosemary o i l was found to be more toxic to spider mites as a contact toxicant w h i l e it was more effective against whiteflies as a fumigant. C h o i et al. (11) evaluated the toxicity o f 53 essential oils i n c l u d i n g 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, citronella Java, lemon, eucalyptus, pennyroyal and peppermint o i l , w h i c h were h i g h l y toxic (mortality > 90%) to the tested mites. H o w e v e r , Sampson et al. (32) tested 23 different essential oils including rosemary o i l against turnip aphids and found most acted as contact toxicants causing mortality in aphids after 1 hour. W h e n tested against whiteflies, toxicity o f rosemary o i l varied significantly between open and closed containers. This result leads to the c o n c l u s i o n that rosemary o i l activity can be affected by its volatilization and by environmental conditions. T h u s , in greenhouses it might not be as toxic as it is in a laboratory setting. T u n c et al. (13) found that vapors o f some essential oils were toxic to the cotton aphid and the two-spotted spider mite. T h e y found that the quantity o f oil needed for pest control differed in heated and cooled greenhouses compared to ambient greenhouses under plastic. T h e y suggested that essential o i l particles suspended in the air might be lost due to air circulation or adherence to surfaces inside the greenhouse.  42  2.4.2 Effects o n b i o - c o n t r o l s Predatory mites P. persimilis are less susceptible to rosemary o i l than twospotted spider mites (Table 2.1). W h e n both mites were directly sprayed w i t h different pesticides containing rosemary o i l , no mortality was found among predators w h i l e up to 6 0 % mortality was observed in spider mites (Figure 2.6). These results are very p r o m i s i n g in terms o f compatibility o f these pesticides in an I P M program f o r c o n t r o l l i n g spider mites. T h i s difference i n toxicity level between spider mites and predators might be due to differential metabolism o f rosemary o i l - b a s e d pesticides in predatory and phytophagous mites. A n important aspect o f acaricides research is identification o f suitable and novel target sites. Little is k n o w n about the mode and site o f action o f rosemary o i l and other plant essential oils in the mites. T h e octopaminergic nervous system is considered to be the site-of-action o f certain essential oils i n the A m e r i c a n c o c k r o a c h (33) and fruit f l y (34), but this m a y not be the case for two-spotted spider mite and there is a possibility that the essential oils have more than one site o f action since they are c o m p l e x mixtures. U n l i k e predatory mites, E. formosa is more susceptible to rosemary o i l both in fumigation and leaf disc painting bioassays compared to the greenhouse whiteflies. E. formosa is susceptible to more than one hundred crop protection products. H o w e v e r , there are some selective compounds that have fewer side effects on parasitic wasps (16). F r o m m y results, I conclude that rosemary o i l can affect the adult parasitoid i f hit directly by a sprayed pesticide. H o w e v e r , immature parasitoids developing inside whitefly nymphs might not be affected b y pesticide ( A p p e n d i x two). O n the other hand E. formosa is highly mobile and might escape direct spray exposure. In order to reduce side effects o f rosemary o i l - b a s e d pesticides to parasitic wasps, growers can either apply the pesticide 48-72 hours prior to parasitoid release or after whitefly nymphs have been parasitized.  43  2.4.3 P e r s i s t e n c e in the environment A s D e k e y s e r (35) mentioned, new pesticides should be safer towards non-target organisms and have shorter environmental persistence than existing products. M y results clearly indicate that the rosemary oil-based pesticides are not environmentally persistent. In all experiments, toxicity o f residues significantly declined after 24 hours. Essential oils are mixture o f odorous and volatile compounds that can easily break d o w n i n the environment (27). M a n y environmental factors affect the breakdown o f essential oils, most importantly, temperature and light. Essential oils m a y break d o w n faster at higher temperatures and w i t h direct light exposure. L i m i t e d residual toxicity is an important advantage for these pesticides. G r o w e r s can apply them closer to harvest time. It is also important to have a safer environment f o r bio-control agents w i t h fewer pesticide residues. O n the other hand, q u i c k breakdown o f essential oils i n the environment reduces the risk o f pesticide resistance i n the pest population.  2.4.4 Repellent effects In addition to pesticidal properties, sub-lethal effects (repellent, deterrent, antifeedant) o f many plant essential oils have been reported against several pests (27). Trongtokit et al. (36) tested 38 different essential oils on human subjects as repellents to mosquitoes. T h e y found that diluted essential oils couldn't provide a satisfactory level o f repellence w h i l e undiluted o i l c o u l d provide effective repellence f o r up to 2 hours. A m o n g essential oils that they tested, clove o i l provided the longest duration o f repellence (up to 4 hours) against mosquitoes. In another study, T r a b o u l s 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, w h i l e Z h a n g et al. (38) reported repellent effects o f  44  ginger o i l to Bemisia argentifolii on tomato plants. K o s c h i e r et al. (28) reported that essential oils f r o m plants i n the mint f a m i l y could affect host plant selection and acceptance by Thrips tabaci L i n d e m a n . Plant essential oils cannot only repel arthropods, but they also can affect vertebrate behavior. C l a r k et al. (39) reported that some essential oils i n c l u d i n g rosemary, can cause violent undirected locomotory behavior in b r o w n tree snakes, Bioga irregularis, therefore the oils can be used as snake repellents. A c c o r d i n g to m y choice tests results, rosemary o i l is significantly repellent to two-spotted spider mites. It repelled mites for about 6 hours and then mites gradually started to move toward the treated discs. H o w e v e r , both mites and whiteflies preferred untreated leaves f o r o v i p o s i t i o n . Repellent effects o f rosemary o i l cannot be considered as a stand-alone control method but can be c o m b i n e d w i t h other methods to improve pest management strategies. F o r instance, rosemary o i l application might be combined w i t h trap-plants as a " p u s h 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 f o r small-scale pest control in greenhouses. These pesticides meet the major characteristics o f an I P M - c o m p a t i b l e pesticide. A s m y results indicate, under laboratory conditions, 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 d o w n very fast i n the environment. Other advantages o f essential oil-based pesticides include l o w m a m m a l toxicity, safety to terrestrial and aquatic species (with some exception) (21 -22), rapid pest  45  mortality due to their neurotoxic mode o f action and l o w cost, a result o f their extensive w o r l d w i d e use as fragrances and flavoring (40). F i n a l l y , an important characteristic o f rosemary o i l is its c o m p l e x chemical composition. L i k e other essential oils, natural rosemary o i l is a c o m p l e x mixture o f terpenoids. Considering that target site resistance is an important problem f o r mite control, it is less likely 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 w i t h many other constituents present (41). A l t h o u g h rosemary oil-based pesticides meet most requirements o f I P M compatible pesticides, they have some disadvantages that must be considered before extensive application. F o r example, rosemary o i l does not have trans-laminar activity. B o t h spider mites and whiteflies 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 m o d i f y the efficacy o f the rosemary o i l . Ebert et al. (42) reported differences in the efficacy o f spinosad and azadirachtin w h e n applied w i t h different application equipment (carbon dioxide powered h i g h - v o l u m e sprayer, D R A M M c o l d fogger or an electrostatic spraying system). A s described before essential oils might breakdown faster at high temperatures so, some application equipment m i g h t damage the o i l during spraying and reduce its efficacy. In order to be effective, the pesticide must hit the target, so application must be conducted i n such a w a y that it provides complete coverage to the w h o l e plant.  46  In addition to application methods, effects o f environmental factors on the efficacy o f the pesticide must be addressed. For instance, temperature, moisture and light effects o n toxicity and degradation o f pesticides should be studied. T o x i c 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 w i t h the constituents o f the oil (Chapter three).  47  References 1)  Z h a n g , Z . (2003).  Mites  of Greenhouses:  identification,  biology  and  control,  publishing. W a l l i n g f o r d : C A B I International, pp 54-61. 2)  Jeppson, L . R . , K e i f e r , H . H . & B a k e r , T . W . (1975). Mite injurious to economic plants, B e r k e l e y : University o f C a l i f o r n i a press, C A , p p 370-376.  3)  C r a n h a m , J . E . , & H e l l e , W . , (Eds). (1985). Pesticide resistance in Tetranychidae. In: World crop pest - spider mites: their natural enemies and control,  Amsterdam:  E l s e v i e r , pp 4 0 5 - 4 2 1 . 4)  The Database o f Arthropods Resistance to Pesticides. (2004). Michigan State University-Center for integrated plant systems. 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D . , & G i l s t r a p , F. E . (1982). Influence o f prey availability o n reproduction and prey consumption o f Phytoseiulus persimilis, Metaseiulus  occidentalis  (Acarina:  Amblyseius  Phytoseiidae).  californicus , and  International  Journal  of  Acarology, 8: 85-89. 11) Kennet, C . E . , & H a m a i , J . (1980). O v i p o s i t i o n and development i n predaceous mites fed w i t h artificial and natural diets. Entomologica experimentalis et applicata, 28: 116-122. 12) E v e r s o n , P. (1979). T h e functional response o f Phytoseiulus persimilis  (Acarina:  Phytoseiidae) to various densities o f Tetranychus urticae ( A c a r i n a : Tetranychidae). Canadian Entomologist, 111: 7-10. 13) Z h a n g ,  Z.Q. &  Sanderson, J . P . (1995).  Tetranychidae) and Phytoseiulus persimilis  Twospotted  spider  mite  (Acari:  ( A c a r i : Phytoseiidae) o n greenhouse  roses: Spatial distribution and predator efficacy. Journal of Economic  Entomology,  88: 352-357. 14)  N i c e t i c , O . , W a t s o n , D . M . , Beatti, G . A . C . , M e a t s , A . , & Z h e n g , J . (2001). 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, 2 5 : 3 7 - 5 3 . 15) M u r p h y , G . D . , F e r g u s o n , G . , F r y , K . , Lambert, L . , M a n n , M . , & M a t t e o n i , J . (2002). The use o f b i o l o g i c a l control in Canadian greenhouse crops. Integrated Control in Protected Crops, Temperate Climate, IOBC/wprs Bulletin, 25(1): 193-196. 16) H o d d l e , M . S . , V a n D r i e s c h e , R . G . , & Sanderson, J . P . (1998). B i o l o g y and use o f the whitefly parasitoid Encarsia formosa, Annual Review of Entomology, 4 3 : 645-669.  49  17)  Yatagai M . (1997). Phytochemistry,  18)  Miticidal  activities  o f tree  terpenes.  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Ortho-aminoacetophenone, a non-lethal repellent: T h e effect o f volatile cues v s . direct contact o n avoidance behavior by reodents and birds. Pesticide Science, 4 6 : 55-60.  23) Burt, S. (2004). Essential oils: T h e i r antibacterial properties and potential application in food-a review. International Journal of Food Microbiology, 24)  Managena, T., & antimicrobial Rosmarinus Microbiology,  Muyima,  N.Y.O.  (1999).  Comparative  activities o f essential oils o f Artemisia officinalis  94: 223-253. evaluation  afra, Pteronia  o f the  incana and  o n selected bacteria and yeast strain. Letters in Applied  28: 291-296.  50  25) Papachristos, D . P . , & Stampoulos, D . C . (2004). Fumigant toxicity o f three essential oils o n the eggs o f Acanthoscelides  obtectus (Say) (Coleoptera: Bruchidae). Journal  of Stored Products Research, 4 0 : 517-525. 26) T u n c , I., Berger, B . M . , Erler, F., & D a g l i , F . (2000). O v i c i d a l activity o f essential oils f r o m plants against t w o stored-product  insects. Journal  of Stored  Products  Research, 3 6 : 161-168. 27)  Isman, M B . (2000). Plant essential oils f o r pest and disease management. Crop Protection, 19: 603-608.  28)  K o s c h i e r , E . A . , & Sedy, K . A . (2003). Labiate essential oils affecting host plant selection and acceptance o f Thrips tabaci L i n d e m a n . Crop Protection, 2 2 : 929-939.  29)  C h o i , W . , L e e , S., Park, H . , & A h n , Y . (2004). T o x i c i t y o f plant essential oils to Tetranychus urticae  ( A c a r i : Tetranychidae)  and Phytoseiulus  persimilis  (Acari:  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 family Phytopathologica  31)  Phytoseiidae ( A c a r i : Phytoseiidae).  Acta  et Entomologica Hungarica, 34: 3 5 5 - 3 6 1 .  A k h t a r , Y . , & Isman, M B . (2003). L a r v a l exposure to oviposition deterrents alters subsequent oviposition 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 i m e r , N . , D e m i r c i , B . , Baser, K . C , K h a n , I. A . , Spiers, J . M , & W e d g e , D . E . (2005). Insecticidal activity o f 23 essential oils and their major compounds against adult Lipashis pseudobrassicae  Davis (Aphididae:  Homoptera). Pest Management Science, 6 1 : 1122-1128.  51  33)  E n a n , E . (2001). Insecticidal activity o f essential o i l s : octopaminergic site o f action. Comparative Biochemistry and Physiology Part C, 130: 325-327.  34)  E n a n , E . (2005). M o l e c u l a r and pharmacological analysis o f an octopamine receptor f r o m A m e r i c a n c o c k r o a c h and fruit f l y in response to plant essential oils. 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(2003). A e r o s o l i z e d essential oils a n d individual product compounds as b r o w n treesnake repellents. Pest Management  natural  Science 5 8 :  775-783. 40)  Isman, M . B . (2006). B o t a n i c a l insecticides, deterrents, and repellents i n modern agriculture and an increasingly regulated w o r l d . Annual Review of Entomology, 5 1 : 45-66.  52  41)  F e n g , R. & Isman, M . B . (1995). Selection f o r resistance to azadirachtin i n the green peach aphid, Myzus persicae. Experientia, 5 1 : 831-834.  42)  Ebert, T . A . , D e r k s e n , R . , D o w n e r , R. A . , & K r a u s e , C . R . (2003). C o m p a r i n g greenhouse sprayers: the dose-transfer process. Pest Management Science, 60: 507513.  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 plants 1  3.1 INTRODUCTION 3.1.1 T w o - s p o t t e d s p i d e r mite T h e two-spotted spider mite, Tetranychus urticae K o c h , is one the most important pests o f fruit, vegetable and ornamental plants w o r l d w i d e (1). T h e mite has been reported from about 1200 species o f plants (2), o f w h i c h more than 150 are e c o n o m i c a l l y important (3). T h e e c o n o m i c threat posed by these mites is constantly increasing because o f the development o f pesticide resistance, and resurgence o f mite populations f o l l o w i n g 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 r o m more than 6 0 countries (5). In the U . S . A . in 2 0 0 1 , spider mites control programs cost approximately U S D 8 m i l l i o n in cotton alone (National Cotton C o u n c i l o f A m e r i c a ) . Spider mites impose a great expense to greenhouse growers w o r l d w i d e in terms o f damage and control cost and are therefore considered one the most important pests o f greenhouses production.  3.1.2  Rosmarinus officinalis essential oil Plant essential oils are obtained through steam distillation o f herbs and medicinal  plants (6). These oils have been used traditionally as healing medicines i n many countries and ancient people were also aware o f their pesticidal properties, however, o n l y in recent  ' M i r e s m a i l l i , S . , B r a d b u r y , R., and Isman, M . B . (2006). Pest Management  Science: In press ( A c c e p t e d on 27 September 2005)  54  years these oils have been c o m m e r c i a l i z e d as pest control products (7). M o s t o f these oils are environmentally non-persistent, and non-toxic to humans (with some exceptions) (810), fish (with some exceptions) and w i l d l i f e (10-13). R o s e m a r y (Rosmarinus officinalis L.) o i l has been traditionally used as a medicine for c o l i c , nervous disorders and painful menstruation. Recent studies revealed that the rosemary o i l is an effective antibacterial agent, w h i c h 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 f o o d spoilage bacteria and yeast strains (15). Rosemary o i l is relatively effective against insect a n d mite pests. It has been shown that the aromatic vapor o f rosemary has o v i c i d a l and larvicidal effects o n several stored product pests (16-17) and the two-spotted spider mite (7) as a fumigant. T h e o i l can have sub-lethal effects as w e l l , f o r example acting as a repellent to o n i o n thrips, Thrips tabaci L i n d (18). Synthetic acaricides usually contain a single active c o m p o u n d ; however, botanical pesticides such as plant essential oils are c o m p l e x mixture o f several constituents. In the present study, w e characterize the toxicity o f rosemary o i l and its major constituents as residual acaricides against T. urticae.  55  3.2 3.2.1  MA TERIALS AND METHODS R o s m a r i n u s officinalis essential o i l  2  Pure Rosmarinus officinalis essential o i l (Intarome T O , Lot# 0 2 1 3 1 4 2 M B - 1 0 0 % ) was  obtained  from  EcoSMART  Technologies Inc. ( F r a n k l i n , T N , U S A ) . M a j o r  constituents o f the essential o i l were identified by gas chromatography/mass spectroscopy on a V a r i a n 3900 system w i t h a Saturn 2 1 0 0 T i o n trap mass selective detector (Walnut Creek, C A , U S A and using a W C O T fused silica 3 0 m x 0.25 m m I D c o l u m n w i t h a C P S i l 8 C B l o w bleed M S coating, a 1 u.1 injection v o l u m e and pure h e l i u m as the carrier at 1.0 m l / m i n .  T h e temperature program used was 80°C f o r 0.5 m i n , an increase o f  8.0°C/min for 8.0 m i n , f o l l o w e d b y an increase o f 50°C/min f o r 3.2 m i n . C i n n a m i c alcohol ( S i g m a , St. L o u i s , M O , U S A ) was used as an internal standard.  3.2.2  S p i d e r mites T w o colonies o f T. urticae were used in this study. T h e first c o l o n y was collected  from the U B C horticulture greenhouse and reared on three-week-old green bush bean plants (Phaseolus vulgari c v . Speculator #24A Stokes). T h e second c o l o n y originated f r o m a research c o l o n y maintained o n tomato plants f o r more than f i v e years without any pesticide exposure at A g r i c u l t u r e and A g r i - F o o d Canada, A g a s s i z , B C . These mites were reared o n three-week-old  vine tomato  plants (Lycopersicon  esculentum M i l l var.  Clarance) p r o v i d e d b y H o u w e l i n g ' s Nurseries (Delta, B C , Canada).  3.2.3 General g r o w i n g c o n d i t i o n s for plants a n d mites Plants contaminated w i t h mites were kept inside an isolated greenhouse section at 24± 3°C, 4 5 - 6 0 % relative humidity ( R H ) under natural daylight. Plants were irrigated three times per week, t w o times w i t h water and one time w i t h water-soluble fertilizer 2  A n a l y z e d by R o d 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 C o m p a n y , M a r y s v i l l e , O H , U S A ) . A d u l t female mites were transferred to clean plants, allowed to oviposit for 48 hours, and then removed f r o m the plant. Development o f these eggs resulted in a cohort o f evenly aged mites that were used for all bioassays.  3.2.4  C a l c u l a t i n g lethal concentration 50 ( L C  3 0  ) of the oil  A leaf disc painting 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 c m diameter). T h e bean colony o f mites was treated w i t h six n o m i n a l 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 )] o f the essential o i l in 7 0 % 2  aqueous methanol as the carrier solvent. The tomato c o l o n y was treated w i t h the same six concentrations, using water plus a spreader sticker adjuvant (Latron B - 1 9 5 6 , 60 m g litre" ) as the carrier solvent. 1  L e a f discs (3cm diameter), were cut f r o m 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 w i t h a micropipette. A f t e r d r y i n g at room temperature for 5 m i n , each disc w a s placed in the bottom o f a Petri dish atop a 3 c m diameter disc o f W h a t m a n N o 1 filter paper wetted w i t h 50 u L o f distilled water. F i v e adult female spider mites were introduced into each Petri dish and the covered dishes were placed in a growth chamber at 26±2 °C, 5 5 - 6 0 % R H w i t h a 16/8h L D photoperiod. Mortality was determined under a dissecting microscope 24h after treatment. M i t e s were considered dead i f appendages d i d not move when prodded w i t h a fine paintbrush. Control mites were held on leaf discs painted w i t h the carrier solvent alone ( 7 0 % aqueous methanol, or Latron B - 1 9 5 6 60 m g litre" ). A l l treatments were replicated five times. 1  57  3.2.5  C o m p a r a t i v e toxicities Based o n the 1 0 0 % lethal concentration and f o l l o w i n g the natural composition o f  the o i l indicated by G C / M S (Table 3.1), individual constituents were tested at levels equivalent to those f o u n d 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 9 9 % , p-cymene 9 9 % , a-terpineol 9 7 % , b o r n y l acetate 9 7 % , borneol  9 9 % , camphor 9 6 % , d-limonene 9 7 % and camphene 95%) were obtained f r o m S i g m a A l d r i c h (St. L o u i s , M O , U S A ) .  In order to identify the contribution o f each constituent  to the toxicity o f the o i l , we made a blend o f all major constituents as w e l l as blends each lacking one o f the ten major constituents (Figure 3.1). W e compared the toxicity 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, w h i c h contributed to the toxicity  o f the o i l  (active  constituents) and compared them w i t h those w h i c h did not affect the toxicity (inactive constituents). T h e leaf disc painting method was used for a l l bioassays.  3.2.6  Data A n a l y s i s M o r t a l i t y observations were analysed using the S P S S program ( C h i c a g o , I L ,  U S A ) , version 11.5 for analysis o f variance ( A N O V A ) . T u k e y ' 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 GC-MS  analysis indicated that there are ten major constituents i n the o i l ,  comprising 9 2 . 8 % o f the total weight. 1, 8-Cineole w a s the most abundant c o m p o u n d (31.5%), followed b y camphor (20.0%) and a-pinene (17.5%) (Table 3.1). There are more than 4 0 other compounds i n the rosemary o i l , w h i c h are mostly monoterpenes, but their concentration i n the o i l is very l o w (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 c o n c e n t r a t i o n 50 of the oil The L C  5 0  o f rosemary o i l was 1% (10 m l litre  ) ( 9 5 % confidence interval ( C I )  = 6.95 - 13.11) f o r adult female spider mites reared on bean plants and 1.3% (13.0 m l litre "') ( 9 5 % C I = 10.05 - 17.78) for those reared o n tomato plants. C o m p l e t e mortality  59  (100%) o f mites was obtained w i t h a 2 % (20 m l litre "') concentration o f the o i l o n bean plants and 4 % (40 m l litre "') o n tomato plants. N o mortality w a s observed in the controls.  3.3.3 C o m p a r a t i v e toxicities of individual c o n s t i t u e n t s a n d b l e n d s thereof F o r the bean host strain o f mites, bioassay o f single constituents revealed that t w o constituents (1,8-cineole and a-pinene) were significantly toxic at the tested concentration (P< 0.05), one (P-pinene) was slightly but not significantly t o x i c and the remaining seven (p-cymene, borneol, bornyl acetate, camphor, d-limonene, 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  W h e n tested against tomato host strain o f mites, three constituents (camphor, pcymene and camphene) were found non-toxic to mites, five (bornyl acetate, P-pinene, d limonene, a-terpineol and borneol) were moderately toxic and t w o (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 w i t h artificial mixtures showed that the greatest mortality was obtained when a l l ten constituents were present (full mixture). T h e mortality  caused b y the  artificial mixture o f a l l ten constituents d i d not differ significantly f r o m that caused b y 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-elimination assays indicated that the absence o f 1,8-cineole or a-pinene caused the largest decrease in toxicity o f the blend. R e m o v a l o f  61  p-cymene, a-terpineol or b o r n y l acetate also had a significant effect ( P < 0.05) on the toxicity o f the blend but less so than f o r 1, 8-cineole or a-pinene. Excluding five remaining constituents (camphor, camphene, borneol, d-limonene and p-pinene) f r o m 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 b o r n y l acetate were found to contribute to the toxicity o f the o i l whereas P-pinene, p-cymene and borneol had only  a moderate influence on the toxicity.  C a m p h o r , camphene and  d- limonene were found to be inactive when tested individually and their absence d i d not have any effect o n the toxicity o f the mixture (Figure 3.4)  62  100  -T  80 60 de  40  de  20 0 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)  O u r comparison between the toxicity 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 f u l l mixture o f a l l constituents (both actives and inactives). T h e inactive constituent blend d i d not cause any mortality in either strain, but w h e n added to the active constituents b l e n d , toxicity became equivalent to the natural o i l (Figures 3.5,3.6 and 3.7).  63  a  BMl  BM2  BFM  Rosemary oil 2%  Control  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  TM1+2  TM3  T F M  Rosemary oil  Control  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 3.4.1  DISCUSSION R o s e m a r y oil a s a n acaricide O u r results clearly indicate that rosemary o i l can be considered an acaricide  against the two-spotted spider mite, causing complete mortality  i n the laboratory at  concentrations that cause no phytotoxicity to host plants (Chapter four). Rosemary o i l and most o f other plant essential oils are environmentally non-persistent and break d o w n easily in presence o f light. Some essential oils are not t o x i c to non-target organisms and can be used in conjunction w i t h biological control. Furthermore, most plant essential oils including rosemary o i l are safe f o r humans and other m a m m a l s and many o f them are used as flavorings i n 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 toxicity o f rosemary o i l to humans or other mammals has not been reported. L i k e other essential oils, natural rosemary o i l is a c o m p l e x mixture o f terpenoids. Considering that targetsite resistance is an important problem f o r mite control, it is more probable that mites w i l l evolve resistance faster to an acaricide based o n 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). Little is k n o w n about the exact site o f action o f rosemary o i l and other plant essential oils on the two-spotted spider mites. T h e octopaminergic nervous system is considered to be the site-of-action o f essential oils i n the A m e r i c a n cockroach (21), but this may not be the case f o r two-spotted spider mite and there is the possibility that the essential oils have  66  more than one site o f action since they are c o m p l e x mixtures. Further studies need to be done to f i n d the exact mechanism(s) o f action o f the essential oils in spider mites.  3.4.2 S y n e r g y a m o n g constituents W e observed that i n d i v i d u a l constituents differ in their toxicity 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, b o r n y l acetate) that were not toxic to mites feeding on beans were relatively toxic to mites o n tomatoes. S i m i l a r results were reported when major constituents o f two other essential oils were used alone against two post-harvest insect pests (22). T o corroborate the role o f i n d i v i d u a l constituents in the toxicity o f rosemary o i l to spider mites, we eliminated each individual constituent from a synthetic mixture that simulated natural rosemary o i l . W e f o u n d that the absence o f some constituents (1,8-cineol or a-pinene) in the artificial mixture caused a significant decrease i n toxicity ( 8 4 % and 8 0 % respectively), w h i c h tempted us to conclude that these constituents are the major contributors to the o i l ' s toxicity.  H o w e v e r , w h e n we m i x e d  these active constituents together we found that their toxicity level was not as high as w e expected. T h e toxicity o f our artificial mixtures only reached the level o f the natural rosemary o i l when we m i x e d the blends o f active constituents w i t h inactive ones. This indicates that the  'inactive'  constituents  have some  synergistic  effect  on  active  constituents, and although not active individually, their presence is necessary to achieve full toxicity. A c t i v e constituents on the other hand might have an antagonistic effect on each other since their toxicity level is significantly greater w h e n tested i n d i v i d u a l l y and not in a mixture w i t h other active constituents. The highest mortality rates were obtained  67  in both strains w h e n a l l the constituents were present in the mixture ( 9 6 % in tomato mites and 9 2 % in bean mites). K n o w i n g the role o f each constituent in the toxicity o f the o i l gives us the ability to screen different rosemary oils and choose the most effective one for pest control proposes. It might be also possible to artificially 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. 2  nd  ed.,  Ithaca: C o m s t o c k P u b l i s h i n g and C o r n e l l University press, N Y , p p 4 6 8 - 4 7 0 . 2) Z h a n g , Z . (2003).  Mites  of greenhouses:  identification,  biology  and  control,  W a l l i n g f o r d : C A B I p u b l i s h i n g , p p 54-61. 3) Jeppson, L . R . , K e i f e r , H . H . & B a k e r , T . W . (1975). Mite injurious to economic plants, B e r k e l e y : U n i v e r s i t y o f C a l i f o r n i a press, C A , p p 370-376. 4)  C r a n h a m , J . E . , & H e l l e , W . ( E D s ) . (1985). Pesticide resistance i n Tetranychidae. In: World crop pest - spider mites: their natural enemies and control, A m s t e r d a m : Elsevier, pp 4 0 5 - 4 2 1 .  5) The Database o f Arthropods Resistance to Pesticides. M i c h i g a n state university. Center for integrated plant systems. Retrieved A p r i l 28, 2 0 0 5 , f r o m : http://www.pesticideresistance.org / D B / index.html. 6) Y a t a g a i , M . (1997).  Miticidal  activities  o f tree  terpenes.  Current  Topics  of  Phytochemistry, 1: 87-97. 7) Isman, M . B . (2000). Plant essential oils for pest a n d disease management. Crop Protection, 19: 603-608. 8) R o e , F . J . C . (1965). C h r o n i c toxicity o f essential oils and certain other products o f natural origin. Food and Cosmetics Toxicology, 33: 311 - 3 2 4 . 9) C o c k a y n e , S. E . & G a w k r o d g e r , D . J . (1997). Occupational contact dermatitis i n an aromatherapist. Contact Dermatitis, 37: 306-309. 10) Hjorther, A . B . , Christophersen, C , Hausen, B . M . & M e n n e , T . (1997). Occupational allergic contact dermatitis f r o m carnosol, a naturally-occuring c o m p o u n d present in rosemary. Contact Dermatitis, 37: 99-100.  69  11) K u m a r , A . , D u n k e l , F . V . , B r o u g h t o n , M . J . , & Sriharan, S . (2000). Effect o f root extracts  o f mexican  marigold,  Tagetes minuta (Asterales: Asteraceae), on s i x  nontarget aquatic macroinvertebrates. Environmental Entomology, 29: 1 4 0 - 1 4 9 . 12) W a g e r - P a g e , S . , & M a s o n , J . R. (1997). Ortho-aminoacetophenone, a non-lethal repellent: T h e effect o f volatile cues vs. direct contact o n avoidance behavior b y reodents and birds. Pesticide Science, 46: 55-60. 13) C l a r k , L . & A r o n o v , E . V . (1999). H u m a n food flavor additives as bird repellents: I. Conjugated aromatic compounds. Pesticide Science, 5 5 : 903-908. 14) Burt, S. (2004). Essential o i l s : Their antibacterial properties and potential application in food-a review. International Journal of Food Microbiology, 9 4 : 2 2 3 - 2 5 3 . 15) M a n a g e n a , antimicrobial Rosmarinus Microbiology,  T., &  Muyima,  N.Y.O.  (1999).  Comparative  activities o f essential oils o f Artemisia officinalis  evaluation  qfra, Pteronia  o f the  incana and  on selected bacteria and yeast strain. Letters in  Applied  28: 291-296.  16) Papachristos, D . P . , & Stampoulos, D . C . (2004). Fumigant t o x i c i t y o f three essential oils on the eggs o f Acanthoscelides  obtectus (Say) (Coleoptera: Bruchidae). Journal  of Stored Products Research, 4 0 : 517-525. 17) T u n c , I., Berger, B . M . , E r l e r , F., & D a g l i , F. (2000). O v i c i d a l activity o f essential oils f r o m plants against t w o stored-product insects. Journal of Stored Products  Research,  36: 161-168. 18) K o s c h i e r , E . A . , & Sedy, K . A . (2003). Labiate essential o i l s affecting host plant selection and acceptance o f Thrips tabaci L i n d e m a n . Crop Protection, 2 2 : 929-939.  70  19) Pintore, G . , U s a i , M . , B r a d e s i , P., Juliano, C , Boatto, G . , T o m i , F., Chessa, M . , C e r r i , R., & Casanova, J . (2002). C h e m i c a l composition and antimicrobial activity o f Rosmarinus  officinalis  L . o i l from Sardinia and C o r s i c a , Flavour and  Fragrance  Journal, 17: 15-19. 20) F e n g , R. & Isman, M . B . (1995). Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae. Experientia. 5 1 : 831-834. 21) E n a n , E . (2001). Insecticidal activity o f essential oils: octopaminergic site o f action. Comparative Biochemistry and Physiology, 130 ( C ) : 325-327. 22) B e k e l e , J . & Hassanali, A . (2001). B l e n d effects in the toxicity o f the essential o i l constituents o f Ocimum kilimandascharicum  and Ocimum kenyense (Labiateae) on  two post- harvest insect pests. Phytochemistry, 57: 3 8 5 - 3 9 1 .  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 T w o - s p o t t e d s p i d e r mite Two-spotted spider mite, Tetranychus urticae K o c h is one o f the most important pests o f the greenhouse crops. T h e current control method f o r spider mites in B C greenhouses is mostly r e l y i n g on bio-control method.  4.1.2 Phytoseiulus  persimilis  T h e predatory mite Phytoseiulus persimilis A t h i a s - H e n r i o t is a selective predator that is able to rapidly suppress spider mites. H o w e v e r , it might not provide acceptable control for higher populations o f prey particularly on greenhouse tomato (1).  4.1.3 R o s e m a r y oil Rosemary o i l is relatively effective against many insect and mite pests (2-4). In this study, the efficacy o f E c o T r o l ™- a rosemary oil-based pesticide- c o m b i n e d w i t h Phytoseiulus persimilis against two-spotted spider mites on tomato plants was evaluated under the greenhouse condition.  4.1.4 Phytotoxic effect T h e impetus on the use o f plant essential oils for insect pest and pathogen control originates from the need f o r control w i t h reduced environmental and health impacts i n comparison to the highly effective synthetic pesticides. A l t h o u g h essential oil-based  72  pesticides are considered l o w - r i s k pesticides, phytotoxicity to greenhouse crops constitutes one possible obstacle f o r their use i n practice. In a f e w 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 phytotoxicity in limonene treated plants, w h i l e Chiasson et al. (6) did not observe any phytotoxicity among lettuce, roses and tomatoes that were treated w i t h a Chenopodium-based pesticide. The active ingredients i n pesticides do not necessarily cause phytotoxicity. Plant damage can result f r o m the solvents i n a pesticide formulation, impurities i n the spray water, using more pesticide than prescribed on the label, o r poorly m i x i n g spray e m u l s i o n . B l a n k et al. (7) found phytotoxicity in 2 5 % o f kiwifruits treated w i t h mineral o i l m i x e d with diazinon w h i c h resulted in up to 1 7 % reduction i n the y i e l d . A l t h o u g h l o w levels o f phytotoxicity might not be a p h y s i o l o g i c a l threat for plant, cosmetic damages can reduce marketability o f the product. In this study phytotoxic effects o f three rosemary oil-based pesticides to foliage, fruits and f l o w e r s o f greenhouse tomato plants have been investigated.  73  4.2 Materials and methods 4.2.1 C o m m e r c i a l pesticide C o m m e r c i a l pesticides, Hexacide™(5% rosemary o i l ) , E c o T r o l ™ ( 1 0 % rosemary oil) and Sporan™(17.6% rosemary o i l ) were obtained f r o m E c o S M A R T Technologies Inc. ( F r a n k l i n , T N , U S A ) .  4.2.2 Plant materials Three week- o l d tomato plants (Lycopersicon esculentum M i l l c v . Clarance) were provided b y H o u w e l i n g ' s Nurseries (Delta, B C , Canada) (for phytotoxicity tests) and B e v o Farms L t d . ( L a n g l e y , B C , Canada) (for the efficacy test). Plants were transferred to plastic pots containing a mixture o f regular peat (50%), fine bark (25%) and pumice (25%) provided by West C r e e k Farms ( L a n g l e y , B C , Canada). F o r efficacy tests, plants were kept inside isolated cages w i t h i n the greenhouse and f o r phytotoxicity tests plant were kept on a regular bench at the same greenhouse at 2 4 ± 3 ° C , 4 5 - 6 0 % R H under natural daylight. Plants were irrigated three times per week, t w o times w i t h water and one time w i t h water-soluble fertilizer (Peters E X C E L 15-5-15 C a l - M a g ) (The Scott Company, Marysville, O H , U S A ) .  4.2.3 T w o - s p o t t e d s p i d e r mites & Phytoseiulus  persimilis  Spider mites originated from a research colony maintained o n tomato plants f o r more than five years without any pesticide exposure at A g r i c u l t u r e and A g r i - F o o d Canada ( A g a s s i z , B C , Canada). Predatory mites were purchased f r o m A p p l i e d B i o n o m i c s (Sidney, B C , Canada).  74  4.2.4 Efficacy test A 2 x 2 factorial design was used w i t h t w o factors (pesticide or predator) and t w o levels (absence or presence) in each factor. Treatments were randomly assigned inside cells o f cages. D a t a was analyzed by two-way A N O V A ( S P S S , C h i c a g o , I L , U S A ) . T w o tomato plants were placed inside each cell (total o f 40 plants). E i g h t extra plants were randomly placed inside some cells as indicators to estimate initial density o f mites prior to pesticide application 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 AD  D _  n  • C 2  A B  A  © AA 3  D  Figure 4.1. Experiment layout. A= Control, B = Predator, C= Pesticide, D= Predator + Pesticide^ = extra plant,® = HOBO Data Logger.  A p p r o x i m a t e l y 15-20 adult spider mites were placed o n each plant. A f t e r one week, extra plants were removed f r o m the cells and numbers o f spider mites o n their foliage counted under a stereomicroscope. Based on the initial 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 w i t h E c o T r o l ™ at 7.5 m l litre  (Label rate- water was used as carrier solvent as  recommended b y manufacturer). A f t e r 7 days, tomato plants were f u l l y harvested and placed inside paper bags. B a g s then were put inside a c o l d room (4°C) to prevent further development o f mites during data collection. N u m b e r s o f spider mites and predatory mites were counted o n a l l foliage under a stereomicroscope. T h e single apical leaflet at  75  the end o f each leaf w a s selected as a sub-sample for counting the n u m b e r o f spider mite and predatory mite eggs (Figure 4.2).  Figure 4.2. Sub-sampling for counting number of eggs  4.2.5 Phytotoxicity tests Pesticides were tested at three concentrations ( O n e h a 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 f o r each concentration. C o n t r o l plants were sprayed w i t h carrier solvent (water) alone. S p r a y e d plants were randomly p l a c e d o n a greenhouse bench (Figure 4.3). Cuttings made f r o m stems containing flowers or fruits were transferred to n e w pots placed inside a steam r o o m f o r one week. Plants were then transferred to regular benches where they remained for one w e e k prior to the experiment. Fruits and  flowers  were sprayed w i t h the same pesticides at the same concentrations. In order to determine the phytotoxic effect o f pesticides o n f o l i a g e , m e d i u m size leaflets were selected as sub-samples. D a m a g e was defined i n five grades f r o m grade 0  76  (no damage) to grade 5 (completely burned leaflet). F i v e leaflets o f the same size f r o m each plant were selected r a n d o m l y f r o m a l l tested plants f o r damage assessment. D a m a g e to flowers was defined as n o damage (grade 0), m i n o r b u r n i n g sign o n petals (grade 1) and major burning sign or flower abortion due to sever damage (grade 2). D a m a g e to fruits w a s defined as n o damage (grade 0) or any particular b u r n i n g s i g n , w h i c h might cause cosmetic damage to fruit (grade 1). Effects were recorded 24 hours, 4 8 hours and 72 hours after spraying. F i v e flowers and five fruits from each plant w e r e used for damage assessment.  Figure 4.3. Phytotoxicity tests  4.2.6 Data A n a l y s i s Observations were analyzed using the S P S S program, v e r s i o n 13 for analysis o f variance ( A N O V A ) . D a t a were transformed to logarithm values w h e n necessary.  77  4.3 Results 4.3.1 Efficacy test A p p r o x i m a t e l y 100 ± 8 mites were found on each plant (i.e. o n eight extra plants randomly placed inside cages) prior to spraying pesticide o r releasing predators. Temperature w i t h i n cages during the experiment was 24 ± 6 ° C and relative humidity was 60± 1 5 % . N u m b e r s o f mites i n the blocks treated with pesticide o r predator was significantly decreased compared to controls (Table 4.1). Pesticide application showed significant effect on the number o f mites. A significant effect was also found w i t h predator introduction. N o significant interaction between the t w o 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 absent  present  Total  Predator absent  Mean TSM 183.3750  Std. Deviation 65.67264  Mean LogTSM 5.1616  Std. Deviation LogTSM .36771  N 4  present  47.7500  .08998  4  absent  115.5625 87.8750  4.17333 84.32862 29.41194  3.8630  Total  4.5123 4.4365  .73707 .31937  8 4  present  42.5000  3.6480 4.0422  .52862  4  .58405  8  Total  65.1875  21.42429 33.99573  absent  135.6250  69.46158  4.7991  .50191  8  present Total  45.1250  14.56206 67.33981  3.7555 4.2773  .36938  8  .68677  16  90.3750  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  7.075  15  Corrected Total  N u m b e r s o f spider mite eggs were not significantly affected b y pesticide or predators (Table 4.3), although predators alone appeared to suppress spider mite eggs. N o interaction was found between the t w o 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 absent  present  Total  Predator absent present Total absent present Total absent present Total  Mean Egg 159.7500 54.8750 107.3125 73.8750 75.5000 74.6875 116.8125 65.1875 91.0000  =  =  Std. Deviation 90.92625 22.26498 83.05546 35.33972 44.43160 37.17616 78.64792 34.35210 64.40471  =  =  Mean LogEggs 4.9228 3.9092 4.4160 4.2288 4.1056 4.1672 4.5758 4.0074 4.2916  Std. Deviation LogEggs .66619 .56090 .78651 .42463 .87568 .64051 .63646 .68883 .70472  N 4 4 8 4 4 8 8 8 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  7.450  15  Corrected Total  N o significant 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)) o n b l o c k s sprayed w i t h pesticide compared to those not sprayed.  4.3.2 Phytotoxicity test H e x a c i d e and E c o T r o l were not phytotoxic to foliage, flowers or fruits (grad 0). Sporan (containing 1 8 % rosemary o i l ) caused first grade damage (burning signs on less than 1/5 o f leaflet) to 1 2 % o f foliage at the recommended label rate and second grade (burning signs o n between 1/5 to 2/5 o f the leaflet) damage to 3 2 % o f leaflets after 24 hours. N o additional damage was found at day t w o or day three o f data collection (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.  H e x a c i d e and E c o T r o l were not phytotoxic to flowers. Sporan caused second grade damage (some petals o f f l o w e r s demonstrated m i n o r b u r n i n g sings) i n 4 0 % o f tested flowers at its label rate, and second grade damage (complete b u r n i n g or f l o w e r abortion) i n 5 6 % o f f l o w e r s and third grade damage (3/5 o f f l o w e r s demonstrated b u r n i n g signs) i n 3 6 % o f flowers at double the label rate after 2 4 hours (Figure 4.5). N o additional damage w a s f o u n d at day t w o o r day three o f data c o l l e c t i o n . N o n e o f the pesticides w e r e found to be phytotoxic to fruits (Figure 4.6).  81  Figure 4.5. Phytotoxicity toflowers.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 E c o T r o l ™ In this study, the efficacy o f EcoTrol™ ( 1 0 % rosemary oil) at its recommended label rate (7.5 m l litre  f o r spider mites on tomato) was evaluated against two-spotted  spider mites on greenhouse tomato plants, both individually and in c o m b i n a t i o n w i t h predatory mites, Phytoseiulus persimilis under greenhouse conditions. In previous laboratory bioassays, contact toxicity o f rosemary o i l based-pesticides was observed w i t h two-spotted spider mites. H o w e v e r , in the present study, the same degree o f mortality was not observed. There was considerable variation a m o n g observations requiring transformation o f the data to logarithmic f o r m . Based o n the average number o f mites on each b l o c k (Log-transformed), we can conclude that pesticide application, predator release and the combination o f pesticide and predators, suppress the mite population by 52 ± 16%, 74 ± 2 % and 76 ± 1 2 % respectively (Table 4.1). Several factors m a y 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 w i t h i n the canopy o r remain at parts o f the plant that pesticide cannot reach. In addition, environmental conditions such as temperature and light m a y accelerate the degradation o f the o i l . There was a fluctuation o f 12 degrees i n temperature and 3 0 % i n relative humidity w i t h i n the greenhouse during the experiment. T h i s fluctuation might be a reason f o r differences in efficacy i n different blocks and cages. M o r e o v e r , it was shown that this product has repellent effects (Chapter two) so there might be another scenario. M i t e s that were not directly hit by pesticide might move to the non-sprayed parts o f the plants enhancing their s u r v i v a l . H o w e v e r , this should not affect numbers o f eggs o f spider mites and predatory mites.  83  A c c o r d i n g to the final number o f mites we might conclude that the combination o f pesticide and predators was twice as effective as the pesticide alone. H o w e v e r , statistical analysis d i d not show a significant interaction between pesticide and predators (Table 4.2). T h i s difference (15%) was not statistically significant but in this experiment may be b i o l o g i c a l l y significant. There are many factors that affect the statistical significance 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 limitations, I c o u l d only conduct the trial once w i t h four replicates for each treatment ( w h i c h is the m i n i m u m requirement by P M R A ) (8). Further experiments are needed to clarify the efficacy o f this pesticide against spider mites i n the greenhouse. A c c o r d i n g to m y results, I conclude that EcoTrol™ can effectively suppress (not control) the spider mite population on greenhouse tomato and it is safe for predatory mites and their eggs at the tested concentration.  4.4.2 Phytotoxicity trials A c c o r d i n g to P M R A efficacy guidelines for plant protection products (8) three concentrations o f EcoTrol™ were used for phytotoxicity tests. Effects were recorded for three continuous days. Phytotoxicity tests were performed on foliage, flowers and fruits. N o sign o f phytotoxicity was found among tested tomato plants. Certain plant essential oils have recently been used as least-toxic herbicides (910). U n l i k e essential oil-based pesticides, the herbicides contain higher amounts o f essential oils. F o r instance, M a t r a n E C ™, a contact, non-selective, broad spectrum, foliar herbicide developed by E c o S M A R T Technologies Inc. contains 5 0 % c l o v e o i l . It might  84  be possible for E c o T r o l to cause phytotoxicity i f used at higher concentrations, however, m y results show that E c o T r o l is safe to tomato even when applied at double the recommended label rate. T h i s is a very important aspect o f this pesticide w h i c h makes it a favorable option for I P M programs in greenhouse tomato plants.  85  References 1) E v e r s o n , P. (1979). T h e functional response o f Phytoseiulus persimilis  (Acarina:  Phytoseiidae) to various densities o f Tetranychus urticae ( A c a r i n a : Tetranychidae). Canadian Entomologist, 111: 7-10. 2)  Isman, M . B . (2000). Plant essential oils for pest and disease management. Crop Protection, 19: 603-608.  3) K o s c h i e r , E . A . , & S e d y , K . A . (2003). Labiate essential o i l s affecting host plant selection and acceptance o f Thrips tabaci L i n d e m a n . Crop Protection, 2 2 : 929-939. 4)  C h o i , W . , L e e , S . , Park, H . , & A h n , Y . (2004). T o x i c i t y o f plant essential oils to Tetranychus  urticae  ( A c a r i : Tetranychidae)  a n d Phytoseiulus  persimilis  (Acari:  Phytoseiidae). Journal of Economic Entomology, 97: 553-558. 5) Ibrahim, M . A . K a i n u l a i n e n , P . , A f l a t u n i , A . T i i l i k k a l a , K . & . H o l o p a i n e n , J . K . (2001). Insecticidal, repellent, antimicrobial activity and phytotoxicity  o f essential  oils: W i t h special reference to limonene and its suitability f o r control o f insect pests. Agricultural 6)  and Food Science in Finland, 10: 243-259.  Chiasson, H . , Bostanian, N . J . , & V i c e n t , C . (2004). A c a r i c i d a l properties o f a Chenopodium-based botanical. Journal of Economic Entomology, 97: 1373-1377.  7) B l a n k , R . H . , O l s o n , M . H . , T o m k i n s , A . R., Greaves, A . J . , W a l l e r , J . E . , & P u l f o r d , W . M . (1994). Phytotoxicity investigation o f mineral o i l and d i a z i n o n sprays applied to k i w i f r u i t i n winter-spring for armoured scale control. New Zealand Journal of Crop and Horticultural 8) A n o n y m o u s .  (2003).  Science, 22: 195-202. Efficacy  guidelines  for plant  protection  products.  Pest  Management Regulatory Agency, D I R 2 0 0 3 - 2 4 , 49p.  86  9) G h o s h e h , H . Z . (2005). Constrainst in implementing b i o l o g i c a l w e e d control: A review. Weed Biology and Management, 5: 83-92. 10) T w o r k o s k i , T . (2002). H e r b i c i d e effects o f essential oils. Weed Science, 50: 425-431.  87  Chapter five: Summary and Conclusion T h e major objective o f this research was to study the efficacy o f a rosemary oil-based pesticide (EcoTrol™) to be used on greenhouse tomato plants against important pests o f this crop (two-spotted spider mite and greenhouse w h i t e f l y ) . A c c o r d i n g to P M R A efficacy guidelines for plant protection products (1) there are some issues that need to be addressed before m a k i n g a decision about this product. Some o f these questions have been answered based on the results o f m y study.  1) Is this product efficacious against pests? T h e laboratory results showed that rosemary o i l can cause contact and fumigant toxicity in t w o - s p o t t e d spider mites and greenhouse whiteflies. E c o T r o l at its recommended label rate in particular, caused >80% mortality among spider mites when tested under laboratory conditions. H o w e v e r , when tested in a greenhouse, lower mortality was observed but suppression o f the mite p o p u l a t i o n was observed. T h e answer to this question depends on our definition o f efficacy. A c c o r d i n g to the results (considering the limitations o f greenhouse trial) it is clear that E c o T r o l alone might not control a large mite p o p u l a t i o n inside a greenhouse. H o w e v e r , it might effectively restrain the population either below the economic threshold or make it more manageable w i t h other control measures such as predatory mites.  2) Is it hazardous to human health? H u m a n s have used plant essential oils as flavors and food additives f r o m ancient times (2). These oils have been used traditionally as healing medicines in m a n y countries and ancient people were also aware o f their pesticidal properties (3). R o s e m a r y 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 t h o u g h some essential oils in very high concentration and over long periods o f time can cause chronic toxicity or some dermal problems (11-13), in general, essential oils are safe for humans and other m a m m a l s in l o w concentrations.  3) Is it persistent in the environment? T h e short answer to this question is no. M y results indicate that both pure rosemary o i l and E c o T r o 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 w a s detected after 48 hours.  4) What are the effects of this product on the host plant? I did not find any sign o f p h y t o t o x i c i t y on tested plants (foliage, flowers and fruits) w i t h tested concentrations (one-half, recommended and doubled label rate). A c c o r d i n g to m y results E c o T r o l is safe for greenhouse tomato plants (Lycopersicon esculentum M i l l c v . Clarance) at its label rate within the environmental conditions that I used for m y experiment.  5) Is it toxic to bio-control agents? In both laboratory experiments and the greenhouse trial, E c o t r o l was not found to be toxic to predatory mites w h e n directly sprayed o n plants. C o n t a c t toxicity bioassays showed that predatory mites are less susceptible to rosemary o i l than spider mites. H o w e v e r , 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 whiteflies.  89  Residual toxicity assays indicate a significant decrease in toxicity o f residues to bio-control agents after 24 hours. T h u s , the best strategy for protecting bio-control agents from side-effects o f E c o T r o l is to either release them w i t h a short delay (48-72 hours) after the pesticide application or a p p l y i n g the pesticide after the bio-control agents are established on the pest p o p u l a t i o n .  6) Are there any limitations for its application? Rosemary o i l cannot pass through the plant tissues and does not have translaminar 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 i n 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 solid statement about this issue but I have a theory. L i k e other essential oils, natural rosemary o i l is a c o m p l e x mixture o f terpenoids. C o n s i d e r i n g 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 w i t h many other constituents present (14). Little is k n o w n about the exact site o f action o f rosemary o i l and other plant essential oils on the t w o spotted spider mites. T h e octopaminergic nervous system is considered to be the site-of-  90  action o f essential oils in the A m e r i c a n cockroach (15), but this m a y not be the case for two-spotted spider mite and there is the possibility that the essential oils have more than one site o f action since they are c o m p l e x mixtures. A s mentioned before, E c o T r o 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 i n d i v i d u a l constituents differ in their toxicity to the two host strains o f mites, and it seems that they are more toxic to mites that feed o n 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 i m i l a r results were reported w h e n major constituents o f two other essential oils were used alone against two post-harvest insect pests (16). T o corroborate the role o f individual constituents in the toxicity o f rosemary o i l to spider mites, I eliminated each i n d i v i d u a l constituent f r o m 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 ( 8 4 % and 8 0 % respectively), w h i c h tempted me to conclude that these constituents are the major contributors to the o i l ' s toxicity.  However, when I mixed  these active constituents together I found that their toxicity level was not as high as I expected. T h e toxicity o f m y artificial mixtures only reached the level o f the natural rosemary o i l w h e n I m i x e d the blends o f active constituents w i t h inactive ones. T h i s indicates that the  'inactive'  constituents  have some  synergistic  effect  on  active  constituents, and although not active i n d i v i d u a l l y , their presence is necessary to achieve f u l l toxicity. A c t i v e constituents on the other hand might have an antagonistic effect on  91  each other since their toxicity level is significantly greater w h e n tested i n d i v i d u a l l y and not i n a mixture w i t h other active constituents. T h e highest mortality rates were obtained in both strains when a l l the constituents were present in the mixture ( 9 6 % in tomato mites and 9 2 % in bean mites). K n o w i n g the role o f each constituent in the toxicity o f the o i l gives us the ability to screen different rosemary oils and choose the most effective one for pest control proposes. It might be also possible to artificially create a blend o f different constituents, base o n their activity and their effect o n the pest.  9) Can environmental factors affect its efficacy? In this study, I d i d not test the effect o f environmental factors o n the toxicity or degradation level o f rosemary o i l but it is obvious that any factor that can accelerate the volatilization o f the o i l , can affect its toxicity or degradation level. Further studies are needed to find the effect o f factors such as temperature, moisture or light on toxicity level o f the o i l . A b i o 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 toxicity. F o r instance, toxicity o f the rosemary o i l to the same species o f pest m i g h t be different on various host plants. I observed a s m a l l difference in toxicity 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 i n tomato plants). Further studies are needed to indicate the effect o f host plant on the toxicity o f rosemary o i l .  92  10) What are the most effective ways of using this product? A c c o r d i n g to the results o f this study, I can make some suggestions for effective use o f E c o T r o l as part o f an I P M program for mite and w h i t e f l y management i n the tomato greenhouse. A ) A s seen in the greenhouse trial, E c o T r o l cannot provide complete control o f pests and must be c o m b i n e d w i t h other control measure such as bio-control agents. A n effective monitoring system can detect pests before they become a major p r o b l e m . E c o T r o l can then be used for spot spraying and small-scale control o f pests in lower populations f o l l o w i n g bio-control agent release. B ) In order to reduce the side effect o f E c o T r o l on the bio-control agents, predators and parasitoids should be released w i t h a short delay (48-72 hours) after pesticide application. If necessary, additional applications should be made after the bio-controls are established on the plants. C ) E c o T r o l application can also be combined with cultural control methods such as trap plants. C o n s i d e r i n g the repellent effect o f the E c o T r o l , for m o b i l e pests like whiteflies, it can be use to repel the adults f r o m major host plants and push them toward trap plants (i.e. tobacco) where they can be controlled locally (push-pull strategy).  In general, E c o T r o l can be considered a favorable option for a c h e m i c a l control portion o f a greenhouse I P M program.  93  References 1) A n o n y m o u s .  (2003).  Efficacy  guidelines  for plant  protection  products.  Pest  Management Regulatory Agency, D I R 2 0 0 3 - 2 4 , 49p. 2) Isman, M . B . (2006). B o t a n i c a l insecticides, deterrents, and repellents i n modern agriculture and an increasingly regulated w o r l d . Annual Review of Entomology, 5 1 : 45-66. 3) Isman, M . B . (2000). Plant essential oils f o r pest and disease management. Crop Protection, 19:603-608. 4) A b u - A m e r , K . M . , S e n , P., & al-Sereiti, M . R . (1999). P h a r m a c o l o g y o f rosemary (Rosmarinus officinalis L . ) and its therapeutic potentials. Indian Journal of Experimental Biololgy, 3 7 : 124-30. 5) O f f o r d E . A . , M a c e K . , A v a n t i O . , & Pfeifer A . M . A . (1997). M e c h a n i s m s involved in the chemoprotective effects o f rosemary extract studies i n human liver and bronchial cells. Cancer Letters, 114: 275-281. 6)  Singletary, K . , M a c D o n a l d , C , & W a l l i g , M . (1996). Inhibition b y 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: 4 3 48.  7) P l o u z e k , C . A . , C i o l i n o , H . P . , Clarke, R., & Y e h , G . C . (1999). Inhibition o f P glycoprotein activity and reversal o f multidrug resistance i n vitro b y rosemary extract. European Journal of Cancer, 35: 1541-1545.  94  8) A r u o m a , O . I., Spencer, J . P. E . , R o s s i , R., A e s c h b a c h , 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 . , & H a l l i w e 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 l s : their antibacterial properties and potential applications in foods- a r e v i e w . InternationalJournal  of Food Microbiology, 9 4 : 2 2 3 - 2 5 3 .  10) Prichard, A . J . N . (2004). T h e use o f essential oils to treat snoring.  Phytotherapy  Research, 18: 6 9 6 - 6 9 9 . 11) R o e , F . J . C . (1965). C h r o n i c toxicity o f essential oils and certain other products o f natural o r i g i n . Food and Cosmetics Toxicology, 33: 311-324. 12) C o c k a y n e , S. E . , & G a w k r o d g e r , D . J . (1997). Occupational contact dermatitis i n an aromatherapist. Contact Dermatitis, 37: 306-309. 13) Hjorther, A . B . , Christophersen, C , H a u s e n , B . M . & M e n n e , T . (1997). Occupational allergic contact dermatitis f r o m carnosol, a naturally-occuring c o m p o u n d present i n rosemary. Contact Dermatitis, 37: 99-100. 14) F e n g , R. & Isman, M . B . (1995). Selection for resistance to azadirachtin i n the green peach aphid, Myzuspersicae.  Experientia. 5 1 : 831-834.  15) E n a n , E . (2001). Insecticidal activity o f essential o i l s : octopaminergic site o f action. Comparative Biochemistry and Physiology Part C, 130: 325-327. 16) B e k e l e , J . & Hassanali, A . (2001). B l e n d effects i n the toxicity 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-sprayer  1  Electronic micro-sprayer was developed to measure the direct contact toxicity o f the toxicants to test organism m i m i c k i n g spraying practices inside greenhouses. A mechanical switch was installed o n an airbrush (Badger 2 0 0 N H - I L , U S A ) l i n k e d to a dishwasher solenoid controlled electronically b y a digital timer. T h e timer is constructed using a N E 5 5 5 chip. N E 5 5 5 c h i p is a highly stable controller capable o f p r o d u c i n g accurate t i m i n g pulses. T h i s c h i p has 8 input terminals as shown in the diagram b e l l o w (Figure A . l ) . W h e n the l o w signal input is applied to the reset terminal (pin 4 ) , the timer output remains l o w regardless o f the threshold voltage (pin 6) or the trigger voltage (pin 2). O n l y when the high signal is applied to the reset terminal, the timer's output changes according to threshold voltage and trigger voltage. W h e n the threshold voltage exceeds 2/3 o f the supply voltage (pin 8) w h i l e the timer output (pin 3) is h i g h , the timer's internal discharge turns o n , l o w e r i n g the threshold voltage to b e l o w 1/3 o f the supply voltage. D u r i n g this time, the timer output is maintained l o w . Later, i f a l o w 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 d r i v i n g the timer output again at high. T h e start and stop buttons are held at the " + " potential b y the resistors R 4 and R 6 . A t the rest position, C 2 is short circuited with the ground. B y pressing the start button, this short-circuit is annihilated, the output is supplied v i a T l and C 2 starts to be loaded v i a 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 , w h i c h is connected to the reference voltage ( p i n 5). O n c e 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, I C w i l l be reset, and the output goes to zero immediately. T h e timer can turn the p o w e r o n and o f f within the intervals between 0.1 second to 1 minute. W h e n the power is on, the solenoid p u l l the mechanical switch i n and that push the airbrush bottom so it sprays. T h e longer the p o w e r is o n , the longer it sprays (Figure A . 2 ) .  VI 12V +V  o  RV1 - Dl " DIODE  Grid Trg Out Rst  RLY1 5VCOIL  Vcc  Die  ^  Thr Ctl  LED1  R5 lk  j 6 SI o  -  6 o  s : 2  - CI " luE  • C2 " luF  VUy  NPN  Figure A . l . Digital timer circuits  97  Figure A.2. Electronic micro-sprayer. A= mechanical switch, B = Airbrush, C = Solenoid, D = digital timer  T h e timer w a s calibrated i n five stages base o n the amount o f aliquot that spray. In stage one it can d e l i v e r 20p.gr ± 5 and i n f o l l o w i n g 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 timer was calibrated once p r i o r to each experiment.  98  II. Sub-lethal effects of rosemary oil T h e effect o f rosemary o i l o n parasitization b y E. formosa w a s investigated. Leaflets o f tomato containing whitefly n y m p h s were placed inside eppendorf tubes  filled  w i t h M S ( M 5 5 2 4 ) standard tissue culture m e d i a (Figure A . 3 ) . T h i r t y w h i t e f l y n y m p h s were selected o n each leaflet and remaining nymphs were r e m o v e d . T h e leaflet was placed i n Petri dishes ( 1 0 c m diameter) and sprayed w i t h rosemary o i l (10 m l litre " ' ) o r 7 0 % aqueous methanol as carrier solvent using electronic m i c r o sprayer o r not sprayed at a l l . F i v e adult E. formosa were introduced to each Petri d i s h and the sealed dishes were place inside a growth chamber at 2 6 ± 2 ° C , 5 5 - 6 0 % R H and a 16:8 L D photoperiod. A f t e r 72 hours numbers o f parasitized n y m p h s were counted. A l l treatments w e r e replicated five times.  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 parasitized w h i t e f l y nymphs were p i c k e d and prepared as described above inside Petri dishes w i t h 3 0 parasitized nymphs o n each leaflet. L e a f l e t s were sprayed w i t h rosemary o i l ( 1 0 m l litre " ' ) , 7 0 % aqueous methanol b y micro-sprayer or not sprayed at a l l . A l l leaflets were placed inside the growth chamber at the conditions mentioned above. N u m b e r s o f emerged wasps were counted after one week.  99  Rosemary o i l d i d 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 limitations, the experiments had inadequate sample size and lacked statistical power.  Rosemary 1%  Carrier solvent  N o 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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