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Fungi Associated with the Glassy Winged Sharpshooter, Homalodisca coagulata, in Its Native Range


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FUNGI ASSOCIATED WITH THE GLASSY-WINGED SHARPSHOOTER, Homalodisca coagulata IN ITS NATIVE RANGE By S. ELIE BREAUX A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by S. Elie Breaux

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This document is dedicated to Stefanie, always there.

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iv ACKNOWLEDGMENTS I would like to thank the me mbers of my committee for th eir support, perseverance, and knowledge. I consider myself lucky to ha ve found in them the willingness to take a chance on a student. I would like to thank Dr. Linda Young for extensiv e assistance in the statistical analysis po rtion of this study. I would also like to thank my family. My father has always been a student of nature. Raised with his love of the outdoors, the choice to take this path was made without reservation. My mother has always provided every kind of support a son could ask for, free of expectation or judgment. I thank Nicholas and Silas for being so entertaining. They are so differe nt in nature, but time spent with either of them makes one realize what is important. And finally, I woul d like to thank Stefanie. Always generous with encouragement and unwavering in support, there is no way I could have done this without her.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Homalodisca coagulata ................................................................................................1 Feeding Behavior and Nutritional Ecology of Homalodisca coagulata .....................5 Homalodisca coagulata as a Disease Vector................................................................7 Homalodisca coagulata Vector Capacity..............................................................7 Xylella fastidiosa ...................................................................................................8 Potential Natural Control Agents................................................................................10 Parasites and Predators........................................................................................10 Fungal Pathogens of Auchenorrhyncha...............................................................11 Homalodisca coagulata -Associated Fungi.........................................................13 2 INTRODUCTION......................................................................................................20 3 MATERIALS AND METHODS...............................................................................22 Homalodisca coagulata / Hirsutella homalodiscae Interactions in a Crape Myrtle Field Plot................................................................................................................22 Description of Study Site.....................................................................................22 Sampling Program...............................................................................................22 Sticky Trap Grid..................................................................................................24 Diagnostic Bleeds.......................................................................................................25 Sampling Program...............................................................................................25 Bleed Technique..................................................................................................25 Statistical Analysis......................................................................................................26 4 RESULTS...................................................................................................................28

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vi 5 DISCUSSION.............................................................................................................41 6 CONCLUSIONS........................................................................................................47 APPENDIX A SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF LIVE GWSS POPULATIONS AND LEAST MEANS SQUARED ANALYSIS OF SIGNIFICANT EFFECTS...................................................................................48 B SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF CADAVER INCIDENCE AND LEAST MEANS SQUARES ANALYSIS OF SIGNIFICANT EFFECTS..........................................................................................50 C SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF TRAP POPULATION DATA AND LEAST MEANS SQAURES ANALYSIS OF THE EFFECT OF TIME......................................................................................52 D SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF ARCSINE TRANSFORMED LIVE GW SS/CADAVER PROPORTIONS WITH LEAST MEANS SQUARE ANALYSIS OF SIGNIFICANT EFFECTS.................53 E QUINCY NFREC WEATHER DATA FOR 2004 AND 2005..................................55 F RAW DATA FROM 2004 AND 2004 FIELD STUDIES.........................................57 2004 Field Plot Data...................................................................................................57 2005 Field Plot Data...................................................................................................64 LIST OF REFERENCES...................................................................................................91 BIOGRAPHICAL SKETCH.............................................................................................98

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vii LIST OF TABLES Table page 1 Comparison of 2004 and 2005 GWSS populations in field plot for same trees on same week between years........................................................................................28 2 Weeks with significantly higher trap catches of GWSS in 2005.............................38 3 Significant differences in live GWSS numb ers between cultivars with respect to treatment and week in 2005.....................................................................................38 4 Significant differences between treatments with respect to week and cultivar in 2005..........................................................................................................................3 9 5 Significant differences in cadaver inci dence between treatments for a given week and cultivar in 2005........................................................................................39 6 Significant differences in cadaver incide nce between cultivars for a given week and treatment in 2005...............................................................................................40

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viii LIST OF FIGURES Figure page 1 Light micrographs of mycosed glassy-w inged sharpshooters displaying the three major phenotypes.....................................................................................................16 2 Micrographs of the Pseudogibellula dissected from mycosed H. coagulata ...........17 3 Sporothrix isolated from mycosed H. coagulata .....................................................18 4 Light and electron micrographs of the Hirsutella ....................................................19 5 Representative map of crape myrtle field plot.........................................................23 6 Photographs of bleeding procedure..........................................................................26 7 Mean GWSS per yellow stick trap in 2005..............................................................29 8 Mean GWSS per tree for each cultivar in the first irri gated replicate......................32 9 Mean GWSS per tree for each cultivar in the second irrigated replicate.................33 10 Mean GWSS per tree for each cult ivar in the first dry replicate..............................34 11 Mean GWSS per tree for each cult ivar in the second dry replicate.........................35 12 Mean GWSS per tree for entire plot.........................................................................36 13 Total GWSS cadavers observed by week................................................................37

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ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science FUNGI ASSOCIATED WITH THE GLASSY WINGED SHARPSHOOTER, Homalodisca coagulata IN ITS NATIVE RANGE By S. Elie Breaux December, 2005 Chair: Drion G. Boucias Cochair: Russell F. Mizell III Major Department: Entomology and Nematology The glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say), is an efficient vector of the phytopathogenic bacterium Xyllela fastidiosa. Native to the Southeast U.S., it was introduced to California, where its presence has caused a sharp increase in X. fastidiosa -based disease. A complex of three possible fungal pathogens are associated with the glassy-winge d sharpshooter in its native range: Pseudogibellula Sporothrix and Hirsutella In the summer seasons of 2004 and 2005, a variety of cultivars were selected from a crape myrtle field plot and were used to monitor the interaction between Hirsutella and GWSS, as well as the effect of humidity on this interaction. GWSS preference for specific crape myrtle cultivars was also determined in 2005. In both 2004 and 2005, Hirsutella was the predominant mycopathogen detected in sampled GWSS populations. In 2004, there were high numbers of diseased GWSS. In 2005, there was low incidence of the pat hogen within GWSS populations. In 2005, the crape myrtle cultivar, Biloxi, was preferre d over Osage, Miami, and Tonto in

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x one replicate, but differences were insignificant in all ot her cases. The disparity was likely due to the migration of GWSS into the study plot through thes e “Biloxi” trees. For both 2004 and 2005, the incidence of mycosed GWSS cadavers was significantly higher in replicates treated with high humidity. Th is may reflect higher GWSS numbers in the irrigated plots. By the end of the stud y period in 2005, 46% of cadavers killed by Hirsutella were subsequently colonized by Pseudogibellula

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1 CHAPTER 1 LITERATURE REVIEW Homalodisca coagulata The glassy-winged sharpshooter, Homalodisca coagulata (Hemiptera: Auchenorrhyncha: Cicadellidae: Tettigellinae: Proconiini), has a native range that extends from central Florida north to Nort h Carolina and west to eastern Texas and northeastern Mexico (Turner & Pollard 1959). It was intr oduced as egg masses through shipments of nursery stock to California in the late 1980s (Sorenson & Gill 1996) and has become established in several southern Califor nia counties, with most of the state still thought to be at risk for colonization. This introduction is important since the glassywinged sharpshooter (GWSS) vectors a lethal phytopathogenic bacterium, Xylella fastidiosa which infects several economically important host plants. X. fastidiosa is the organism responsible for Pierce’s Disease of grape and the efficiency of GWSS as a vector has made it a serious threat in this crop (Almeida & Purcell 2003, Purcell & Saunders 1999). GWSS is distinctive in morphology when comp ared to other sharpshooters in its native range, including its congeneric, Homalodisca insolita Turner and Pollard’s (1959) description follows: “The adults of coagulata are large, rather slender leafhoppers, measuring 11 to 13 mm. in length. The males are slightly smaller than the females. The head is bluntly angled and the crown nearly flat. The gene ral color is brown. The venter is ivory with conspicuous black markings. At th e base of the abdomen the ivory color extends upward, so that when observed from above, the insects appears to bear an oblique ivory blotch on either side of the abdomen. The face, genital plates, and legs are approximately mandarin orange. In newly transformed adults the veins of

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2 the forewings are bright re d and spots of red occur towards the tips of the wings. Within a few days these areas darken until the dorsal aspect presents a homogenous brown appearance to the naked eye. The nymphs are olive gray. In the first two instars the eyes are reddish; in the later instars they are darker.” Nymphs progress through five instars, w ith lengths of 2.0 mm, 2.5 mm, 4.0 mm, 5.0 mm, and 7.5 8.0 mm for each successive instar (T urner & Pollard 1959). The eyes of the fisrt two instars have a reddish coloration, wh ich darkens in later instars. The ivory colorations on the ventral and pleural regions of the adult abdomen progressively yellow as the insect ages (R. F. Mi zell, personal communication). The GWSS possesses a piercing-sucking t ype mouthpart, which it inserts through the stem into the plant xylem tissue (Turne r & Pollard 1959). Xylem fluid is its only food source, and as xylem fluid is extremely nutrien t poor, the insect spends much of its time feeding and frequently switches hosts to ach ieve optimal nutrient uptake (Mattson 1980, Brodbeck et al. 1993). If approached, the GWSS displays an interesting defensive behavior. When the insect detect s a potential threat, it migrat es to the side of the stem opposite that threat, putting th e stem between itself and danger (Turner & Pollard 1959). After further provocation, it will fly away. GWSS mate in early spring and the firs t egg masses are generally found in April (Turner & Pollard 1959). Females mate more than once, and oviposit on the underside of a leaf, usually inserting a cluster of 3-28 e ggs under the epidermis of the plant. Optimal development of the embryo takes place in temperatures ranging from 16.7 C to 32.9 C (Al-Wahaibi & Morse 2003). As the embryo de velops, eyespots become visible through the leaf tissue, particularly if the leaf is viewed in front of a light source. Hatch rate (embryo survival) is relatively constant throughout this range, although developmental times are greatly reduced at its upper end. High mortality occurs at constant temperatures

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3 above 35 C and incomplete development at temperatures below 11.5 C (Al-Wahaibi & Morse 2003). 113.8 degree-days are requi red from oviposition to hatch. After oviposition, the female coats th e egg mass with brochosomes, ultramicroscopic reticulated bodies produ ced by all of the major subfamilies of Cicadellidae (Rakitov 1995). Brochosomes are fo und in their most conspicuous form in concentrated spots on the forewings of gr avid female leafhoppers. Riley and Howard (1893) first reported the existence of wing spots while studying populations of GWSS, describing them to be made up of a waxy mate rial. Later investigations showed the wing spots to be made up of microscopic prot einaceous particles which were termed “brochosomes” (Tulloch et al. 1952, Tulloch & Shapiro 1953), which are produced in specialized sections of th e Malpighian tubules (Store y & Nichols 1937, Day & Briggs 1958). In addition to the wings, brochosomes are also found on the remainder of the body (Hix 2001). Rakitov (1996) detailed the behavi ors employed by Cicadellids to coat their bodies with brochosomes. These anointing a nd grooming behaviors va ry from nymph to adult, and across interspecific lines. Nym phs of the Cicadellid subfamilies Macropsinae, Iassinae, Idiocerinae, and many Typhlocybinae do not cover themselves in brochosome secretions, but the Ledrinae, Nioniidae, Eurymelinae, Cicadellinae, Coelidiinae, Hyalicinae, and Deltocephalinaes possess brocho somes in all life st ages (Rakitov 1995). Adults of all subfamilies of Cicadellidae cover themselves in brochosomes. To accomplish this, excreta containing brochosomes are transferred to the wings and dorsal integument from the anus with the hind legs, a maneuver that is faci litated by specialized setae located on the distal por tion of the hind leg (Rakitov, http://www.inhs.uiuc.edu/~rakitov/brochosomes.h tml). The legs are us ed to spread the

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4 excreta to cover the entire body except for th e eyes. This occurs both when the excreta are still wet and after they have dried. Nym phs of some species have also been observed bending their abdomens upwards and forwards to deposit excreta along the head and back, or releasing excreta onto the pl ant itself and wallowing in it. Many hypotheses have been fielded as to the function of brochosomes, including water repellence, anti-microbial activity, pr otection from parasites, protection from desiccation, thermoregulation, protection fr om UV light, and a pheromone reservoir (Rakitov 2002). Of these, wate r repellence is the most likel y function of the brochosome coat (Rakitov 1995). The brochosome coat exhibits extreme hydrophobicity, and may have evolved to complement the cuticular li pids found on most insects. Additionally, the presence of brochosomes on egg masses de creases the oviposition efficiency of Gonatocerus ashmeadi a hymenopteran parasitoid of GWSS eggs (Velema et al. 2005). Brochosomes attached to the legs, body and antennae of the wasp, and prompted prolonged grooming sessions and that interrupted parasitization. In the GWSS and many other leafhoppers, the shape of the actual brochosome varies depending on the life stage of the inse ct (Hix 2001). In the GWSS, the majority of brochosomes are essentially spherical in sh ape. The brochosomes that make up the wing spots that are found on gravid females are rod-shaped (Azevedo-Filho & Carvalho 2005), and are transferred to the egg mass after oviposition. The GWSS goes through two or three generations, in a season, depending on climate (Turner & Pollard 1959). GWSS typi cally overwinter as adults, however overwintering nymphs have been found. Re gardless of life stage, hibernation is incomplete. Both nymphs and adults will become active and feed with a sufficient

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5 increase in ambient temperature. Using this strategy, nymphs can eclose into adults and survive to the next season. Attempts to keep the insects in hibernat ion at constant cold temperatures have resulted in death in le ss than two weeks (Turne r & Pollard 1959). This suggests that periodic winter feeding is critical to th e survival of the insect. Feeding Behavior and Nutritional Ecology of Homalodisca coagulata The GWSS is an extremely polyphagous in sect, feeding on the xylem fluid of over 100 known species of plant, in at least 37 di fferent families (Alderz 1980). It feeds only on xylem fluid and possesses a well-defined ci barial pump for this purpose. The enlarged clypeus houses the pump’s attendant muscul ature (Backus 1985). The well-developed cibarial pump allows the GWSS to feed at most commonly found xylem pressures, although feeding will decrease as xylem fluid te nsions rise and eventually cease at ~2.1 MPa (Andersen et al. 1993). Xylophagous in sects such as the GWSS must process nutrients efficiently, as xylem fluid possesse s the lowest nutritiona l value of any plant tissue (Mattson 1980). Xylem flui d contains very dilute quantities of monomeric amino acids, organic acids, and sugars (Andersen et al. 1989). Adult host preference is partially dependent on amino acid profile of the host xyl em fluid, specifically the concentration of the amides (glutamine and asparagine) in re lation to other amino acids (Brodbeck et al. 1990). Amides, representing the main form of nitrogen present in the adult’s preferred plant hosts, can be readily c onverted into other necessary amino acids (Redak et al. 2004). Assimilation efficiency of amides can su rpass the level of 99.9% while that of the other amino acids is normally lower, ranging fr om ~52% to ~98% (Andersen et al. 1989). The ten essential amino acids are not metabo lized with any greater efficiency than nonessential amino acids. Amides are critical and are believed to dictate GWSS’s host switching behavior. Seasonal and diurnal host pr eferences can be correlate to xylem fluid

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6 composition and to the concentration of amid es, with the insects choosing to feed on hosts with higher amide ratios (Brodbeck et al 1990, Andersen et al. 1992). It is believed that carbon may be the limiting nutrient for thes e insects, as opposed to dietary nitrogen (Andersen et al. 1989). Amino acids, although extremely dilute in xylem fluid, are found in abundance relative to the carbon-rich com pounds, such as sugars. In general, the carbon content of xylem fluid is several orders of magnitude below that of phloem or leaf tissue. The primary nitrogenous waste excreted by GWSS is ammonium (Anderson et al. 1989, Brodbeck et al. 1993). At maximal f eeding rate, the GWSS consumes ~10-100 times its dry body weight in xylem fluid per hour. Because it passes an abundance of water through its system, nitrogenous wast es can be eliminated in the form of ammonium, rather than the less toxic ur ea or uric acid more commonly found in terrestrial organisms (Redak et al. 2004). Th is is to the insect’s advantage, as the processing of ammonium into urea or uric acid requires energy. Urea and uric acid contain 636 and 1926 kJ/mol energy, respectivel y, whereas ammonium contains none. (Handbook of Chemistry and Physics, 1971). Despite the obvious nutritional costs a ssociated with xylophagy, it does have advantages over other types of herbivory. Xylem fluid, while nutrient-dilute, contains low concentrations of plant defensive compounds when compared to leaf tissue or phloem (Bernays & Bright 1993). GWSS are able to avoid a deleterious buildup of these plant defensive compounds through host switching. Considerably less research has addressed the nutritional requirements of GWSS nymphs. Many of the plant host prefe rred by adults will not support nymphal

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7 development. Nymphs are less adept at ut ilizing the unbalanced am ino acid profiles that adults seem to prefer, and therefore have a much more limited host range (Brodbeck et al. 1995, 1996). Whereas adults benefit from an am ino acid composition very rich in amides, immature GWSS require a more balanced ratio of amides to the other essential amino acids, even if total concentrations are less. In the case of both nymphal and adult GWSS, total dietary nitrogen has been shown to be of less importance than nitrogen form in the survivorship of both nymphs and a dults (Brodbeck et al. 1999). Homalodisca coagulata as a Disease Vector Homalodisca coagulata Vector Capacity Recent concerns regarding GWSS arose when this vector was introduced to California in 1989 (Sorenson & Gill 1996). As the GWSS became established and its population grew, so did the incidenc e of diseases associated with X. fastisioda (Blua et al. 1999). While California does have a native sh arpshooter complex capable of transmitting X. fastidiosa transmission characteristics have been determined for few of these. For example, the blue green sharpshooter, Graphocephala atropunctata appears to be the most efficient vector of X. fastidiosa (Freitag & Frazier 1954, Hill & Purcell 1995), but typically does not range far into vineyards from its preferred riparian habitat. The majority of known non-GWSS X. fastidiosa vectors are all members of the tribe Cicadellini. The GWSS, and other members of the tribe Proconiini, are larger than the cicadellinines and are able to feed on woody portions of the pl ant (Almeida & Purcell 2003b, Purcell & Saunders 1999). This, in concert with the etiology of X. fastiodosa makes it of serious concern to producers of wine, table and raisin grapes. Because the native sharpshooters have shorter mouthparts, they can only feed on the thinner stems at the exterior of the plant (Almeida & Pu rcell 2003b). By the end of the season, the

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8 pathogen has not spread to th e interior of the plant and it is removed with routine pruning. Alternatively, the GWSS will introduce X. fastidiosa to the central cane of the grape plant when it feeds, a portion of the plant that is not pruned. This allows the bacteria to proliferate in the plant and surviv e the winter season (Purcell & Saunders 1999), resulting in high plant mortality. Two ot her factors that contribute the success of GWSS as a vector are the aforementioned broa d host range and its st rength as a flyer. H. coagulata ’s ability to cover large areas means that it can more effectively carry the bacterium to new sites than native vectors (Blua & Morgan 2003). Perring et al. (2001) showed that GWSS can vector X. fastidiosa at 1000 feet into vi neyards, considerably further than the predominant native vector, Hordnia circellata (Purcell 1974). The ability to travel long distances and the tendency to switch host plants often means that GWSS can infect a greater number of plants over a greater ar ea than native sharpshooters. Xylella fastidiosa Xylella fastidiosa is a gram-negative, xylem-limited bacterium. The different strains of X. fastidiosa are known to be the causative agents in a number of plant diseases, including Pierce’s disease of grape, phony p each disease, citrus variegated chlorosis, oleander leaf scorch, almond scorch, periwi nkle wilt, and alfalfa dwarf (Pooler & Hartung 1995). Pierce’s disease is of greatest economic importance in the U.S., its spread in California vineyards facilitated by the introduction of the gla ssy-winged sharpshooter. Citrus variegated chlorosis (C VC) is not present in the Unit ed States, but has devastated the citrus industries in Argentina and Brazil (Araujo et al. 2002). CVC has the potential to become the most economically important citrus disease throughout the world, and is the focus of a considerable research in Brazil.

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9 Pathogenicity of X. fastidiosa is due to its ability to block xylem vessels, starving the stems and leaves of water and nutrient s transported upward from the roots (Hopkins 1985, Newman et al. 2004). X. fastidiosa ’s ability to colonize bot h the plant’s xylem vessels and the insect vector’s piercing-sucking mouthparts ma y lie in the production of a biofilm composed primarily of complex e xopolysaccharides (da Silva et al. 2001). The biofilm may allow the bacteria to attach and grow on these hydrodynamically turbulent environments. At present, there is no practic al cure for a plant that is systemically infected by X. fastidiosa and the result is generally plant death. Oxytetracycline injections have been shown to provide short-term control of X. fastidiosa populations within the plant, but do not control heavy infections and must be repeated annually. A GWSS feeding on a plant infected with X. fastidiosa can immediately transmit the bacteria to the next plant it f eeds upon (Purcell 1990, Pur cell & Finlay 1979). X. fastidiosa has been observed in the cibarium, the apodemal groove of the diaphragm, and the walls of the precibarial area in H. coagulata (Brlansky et al. 1983). This however, does not account for the insect’s ability to tr ansmit the pathogen dire ctly after feeding on an infected plant (Purcell & Fi nlay 1979). It is believed that this is possible because the vector may harbor X. fastidiosa first within the food groove of its stylets, allowing the bacterium to be immediately transmitted to the next food source as well as to proceed internally to colonize the rest of the insect foregut (Hill & Purcell 1995). While the bacterium can be found in the midgut and hindgut, only those found in the foregut and mouthparts are believed to be capable of transmission (B rlansky et al. 1983). GWSS nymphs that contract the bacterium lose th e ability to transmit it after molting, as the chitinous lining of the foregut is lost in this process, wh ile the midgut remains intact.

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10 Conversely, insects that contract the bacterium as adults rema in infective for the rest of their lives. Potential Natural Control Agents Parasites and Predators The GWSS’s status as an introduced species in California has led to recent attempts to catalogue potential biocontrol agents. (Triapitsyn et al. 1998). Mymarid wasps have emerged as the most promisi ng biocontrol agents to be found thus far (Triapitsyn et al. 1998, Triapits yn & Phillips 2000). Several my marid species within the genus Gonatocerus have been identified as egg parasitoids, however G. ashmeadi G. fasciatus and G. triguttatus are considered to have the most potential for long-term control of GWSS G. ashmeadi is well-established in California and was probably accidentally introduced at the same time as the GWSS (Vickerman et al. 2004). This species is both more aggressive in its para sitism of GWSS eggs a nd lives longer than its congenerics. However, the inability of G. ashmeadi to effectively target early season egg masses results in poor contro l of GWSS (Irvin & Hoddle 2005). G. fasciatus and G. triguttatus were recently released in California to determine if they would be able to better parasitize these early season egg masses (Irvin & Hoddle 2005). GWSS egg masses containing these parasitoids have been found at release sites (CDFA, 2003). In addition, a previously undescribed Zagella species (Trichogrammatidae) has received attention for introduction (Triapitsyn et al. 1998). More recently, two new species of Ufens (Hymenoptera: Trichogrammatidae), U. principalis Owen sp n and U. ceratus Owen sp n ., have been have been found to parasitize GWSS eggs in southern California (AlWahaibi et al. 2005). Attempts to use para sitoid wasps common to other species of

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11 sharpshooters have met with limited success, and extreme male sex biases have been observed (Triapitsyn et al. 2002). Pseneo punctatus (Hymenoptera: Sphecidae: Pe mphredoninae: Psenini) is a predatory wasp found in California that common ly provisions its nests with sharpshooters (Bethke et al. 2001). It is thought that the nati ve smoketree sharpshooter, Homalodisca lacerta is its typical prey, but it has expanded its prey range to include the GWSS as well. Nests examined in the vicinity of th e University of California-Riverside were provisioned almost exclusively with GWSS (Bethke et al. 2001). P. punctatus ’s impact on the GWSS population is unknown. To date, there has been no t horough survey of GWSS predators in either its na tive range or in California, so information regarding this group of organisms is largely unavailable. The remains of GWSS eggs have been found in the guts of the green lacewing, Chrysoperla carnea and the multicolored Asian lady beetle, Harmonia axyridis but only in a laboratory se tting (Fournier et al. 2004). In recent field studies in Quincy, FL, all pr edators observed predating GWSS were generalist in nature and in cluded salticid spiders and predatory Hemiptera (Breaux, personal observation). Fungal Pathogens of Auchenorrhyncha Auchenorrhyncha is the subdivision of th e true bugs (Hemiptera) that includes cicadas (Cicadidae), leafhoppe rs (Cicadellidae), planthoppe rs (Fulgoridae), treehoppers (Membracidae) and spittlebugs (Cercopidae) As with most sucking insects, most pathogens infecting this gr oup are fungi. The majority of research regarding fungal pathogens of this group involves pests of rice and their possible bioc ontrol agents. These fungi fall primarily in the Deut eromycota and the Zygomycota.

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12 Within Zygomycota is Massospora which grows in the abdominal cavities of adult periodical cicadas and is transmitted either sexually or by general contact when in lek mating aggregations (Duke et al 2002, Soper et al. 1976). Also, Sporodiniella (Zygomycota) has been found to infect memb racids in cocoa plantations (Evans & Samson 1977). However, the most common Zygomycetes that infect leafhoppers and planthoppers reside within th e Entomopthorales (Soper 1985). Erynia delphacis Erynia radicans and Neozygites fumosa all attack the brown planthopper, Nilapavarta lugens (Shimazu 1979, Ben-Ze’ev & Kenneth 1981, Samal et al. 1978). However, Erynia radicans previously termed Zoopthora radicans and Entomophthora sphaerosperma possesses a diverse host range, infecting insects from seven orders, including Lepidoptera, Homoptera, Dipter a, and Hymenoptera (Wraight et al. 1990). Its host range includes the potato leafhopper, Empoasca fabae The remaining majority of fungal pat hogens that are associated with the Auchenorrhyncha are in the Deuteromycota. Pathogens that typically possess broad host ranges, such as Beauveria bassiana and Verticillium lecanii have been recorded from a variety of hosts within the Auchenorr hyncha (Srivastava & Nayak 1978, Steenberg & Humber 1999). Three Metarhizium species, M. flavoviride M. anisopliae and M. album represent a significant mortality factor in pl anthoppers and leafhoppers of rice in Asia (Rombach et al. 1986a, Rombach et al. 1986b, Rombach et al. 1987). There is also record of Hirsutella species infecting auchenorrhyncha ns. Hywel-Jones (1997) described Torrubiella pruinosa (teleomorph of Hirsutella versicolor ), Hirsutella nivea and Hirsutella citriformis from cicadellids in Thailand, as we ll as from other closely related families, and Hirsutella guyana was originally described fr om an infected delphacid

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13 (Minter & Brady 1980). H. citriformis is a common pathogen of auchenorrhynchan pests of rice. Paecilomyces farinosus has been isolated from leaf hoppers on rice (Hirashima et al. 1979). Rombach et al. (1986c) found th at field application of Metarhizium flavoviride Metarhizium anisopliae Beauveria bassiana Hirsutella citriformis and Paecilomyces lilacinus all provided up to 100% mort ality in populations of brown planthopper in rice. Homalodisca coagulata -Associated Fungi Until recently, very little a ttention had been directed at the fungal pathogen complex associated with the GWSS. Turner & Pollard (1959) firs t reported mycosed GWSS clinging to trees with their mouthparts s till inserted into the plant, but gave the topic no further consideration. In a later study, cadavers colle cted in a limited survey of GWSS in Poplarville, Mississippi were found to be infected with Pseudogibellula formicarum Trichothecium roseum and Beauveria bassiana with the conclusion drawn that P. formicarum represented the primary pat hogen in the system (Kanga et al. 2004). When insects were kept in inc ubators, bioassays performed with P. formicarum spore suspensions resulted in 75% and 93 % infection at 21 days with applied concentrations of 2 x 108 spores/ml and 2 x 109 spores/ml, respectively. Greenhouse bioassays produced similar mortality at 48% and 81%. In a broader survey in the southeastern U.S. during th e summer months of 2003 and 2004, GWSS were found to harbor species of Hirsutella, Pseudogibellula and Sporothrix (Boucias et al., submitted). In general, the Hirsutella phenotype was most abundant (44%), followed by the Pseudogibellula (10%) and Sporothrix (2%) phenotypes. A fourth group of unidentified, heterogenous phenotypes (44%) were covered with a combination of algae and various fungi. Molecular diagnostics using a multiplex PCR reaction

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14 demonstrated that Hirsutella DNA was present in ~80% of the samples, including examples from the unidentified and Pseudogibellula categories. These results suggest that Hirsutella sp. is the primary fungal pathogen of GWSS, while Pseudogibellula and Sporothrix are secondary mycoparasites (Boucias et al., submitted). The genus Pseudogibellula is characterized by the presence of broad-based synnemata arising mainly from the intersegment al areas of the mycosed insect (Fig. 1A), and only contains one species, P. formicarum (Samson & Evans 1973). These synnemata bear abundant sporulating coni diophores, possessing an arrange ment similar to that of Aspergillus (Fig. 2A,B) Conidia are produced in a sympodial nature. Conidiophores arise from swollen metulae at the apex of long st alks (Fig. 2C). This sympodial type of conidiogenesis differentiates the f ungus from members of the genus Gibellula which are phialidic. While the GWSS-associated Psedugibellula exhibits slight morphological differences in synnemata shape and conidiophor e structure, it most likely is an example of Pseudogibellula formicarum Sporothrix -infected GWSS cadavers possess white, slender synnemata extending up to 2 mm from the body of the insect (Fi g. 1B) (Boucias et al., submitted). These are covered in conidiogenous cells that appear very similar to the vegetative hyphae (Fig. 3A,B). Both liberated conidia and conidiogenous cells bear c onspicuous scars at the point of detachment (Fig. 3C,D). Id entification is hampered by the fact that literature relating the Sporothrix genus is dominated by species that are human pathogens. However, the morphology of this fungus closely matches that of S. isarioides (Hoog 1974). The GWSS Hirsutella forms a tightly appressed myce lial mat over the body of the insect, originating from the intersegment al membranes (Fig. 1C) (Boucias et al.,

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15 submitted). Both hyphae and phialides appear hyaline under magnification, with the entire mycelium appearing white to grey in color. Phialides are so litary and smooth and originate from the hyphae at right angles (Fi g. 4A,B). They possess cylindrical bases and taper to a slender neck bearing a single apic al conidium (Fig. 4C). Growth in liquid media normally includes yeastlike hyphal bodies, while chla mydospores may be present on solid media (Fig. 4D). Morphologically, this fungus shows the most homology to descriptions of H. versicolor (Minter & Brady 1980), but di rect comparisons of cultures show clear differences. This new species of Hirsutella is currently termed Hirsutella homalodiscae sp. nov. (Boucias et al., submitted).

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16 Figure 1. Light micrographs of mycosed gl assy-winged sharpshooters displaying the three major phenotypes: (A) Pseudogibellula ; (B) Sporothrix ; and (C) Hirsutella. Note the well-developed synnemata (denoted with arrows) formed by both the Pseudogibellula and Sporothrix. The Hirsutella phenotype, lacking synnemata, do produce rhizoids (R ) that serve as holdfasts anchoring the mummified cadavers to the plant s ubstrate. Taken from Boucias et al., submitted.

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17 Figure 2. Micrographs of the Pseudogibellula dissected from mycosed H. coagulata. (A and B) Scanning electron micrographs depicting conidi ophores (C) arising from synnemata and (C) light microgr aph depicting the vesicle (V), the metulae layer (M) and the coniodiogenous cells or phi liades (Ph) that gives rise to ellipsoid conidia (Co). Ta ken from Boucias et al., submitted.

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18 Figure 3. Sporothrix isolated from mycosed H. coagulata. (A) Light micrograph of a dissected synnema depicting the coni diophores. Scanning electron microscopy of this structure (B) prov ides details on the abundan ce and arrangement of the conidiophores originating along the length of the st alk-like portion. High magnification of the conidiophores and c onidia (C), note the characteristic pattern of the denticles over the cond iophore surface (D). Taken from Boucias et al., submitted.

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19 Figure 4. Light and electron micrographs of the Hirsutella produced on Riddel mounts (A), on mycosed H. coagulata (B, C), and in liquid broth (D) Solitary phialides are produced at intervals al ong length of the hyphae that extend onto the glass substrate (A). Similarily, scanning electron microscopy detected phialides along the peripheral region s of mummified insects (C, B). High magnification of the conidia suggest th at these possess a mucilaginous coat. The septate hyphal bodies produced in liqui d media (D) are identical to those observed in the hemolymph samples co llected from live infected GWSS. Taken from Boucias et al., submitted.

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20 CHAPTER 2 INTRODUCTION The glassy-winged sharpshooter’s (GWSS) st atus as the primary vector of Pierce’s Disease of grape in California has lead to re search regarding potent ial biological control strategies. Mymarid egg parasitoids have emer ged as the first organisms to be employed in the biological control of GWSS, but recently a complex of potentially pathogenic fungi were discovered to exist in association with GWSS (See Literature Review: Potential Natural Control Agents). Entomopathogenic fungi have been used with some success in the biological control of a number of insect pests (Shah & Pell 2003, Hajek et al. 1996, Milner 1997, Wraight et al. 2000) and these or ganisms could provide another option to be used in the control of the glassy-winged sharpshooter. Within its native range, GWSS is an extremely polyphagous xylophage (Alderz 1980). However, during the summer months la rge numbers can be found to aggregate on different varieties of crape myrtle, Lagerstroemia indica or Lagerstroemia fauriei (Hoddle et al. 2003, Mizell & French 1987). Alth ough not native, crape myrtle is one of the most popular ornamental plants in the s outheastern U.S. (Pettis et al. 2004). These factors, combined with the species’ ease of cultivation, makes it th e ideal candidate for field observation of GWSS. In a mixed crape myrtle planting, the insect’s differential preference for the various cultivar s (Mizell, unpublished data) resu lts in adjacent plants in the same location harboring different insect densities (Breaux, unpublished data). The anatomy of a crape myrtle tree that has been aggressively pruned at the start of the season, with few branchings and widely spac ed leaves, makes visual sampling of both

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21 live and mycosed GWSS simple. The majority of mycosed GWSS that have been found have been attached to the stem of a host plant in feeding positi on (personal observation) The importance of environmental cond itions on the infectivity of entomogenous mycopathogens has been well-documented (Vidal et al. 2003, Tang & Hou 2001, Shipp et al. 2003, Fargues et al. 1997). Optimal c onditions vary among mycopathogens, but high humidity is typically necessary for in fection and sporulation events. This is supported by anecdotal evidence of fungal epizootics occurring in GWSS populations primarily during years that have a cool, wet spring followed by a summer with consistent rainfall (Mizell, personal communication). This thesis describes field experiments th at utilized a crape myrtle field plot to observe the relationship between the glassy -winged sharpshooter and its associated fungal complex in their nativ e environment. The populations of GWSS and fungi were observed and documented over the course of one and a half seasons in 2004 and 2005. A portion of the plot was subjected to high humidity treatment to determine its effect on the activity of the observed fungi It was hypothesized that Hirsutella homalodiscae was the primary pathogen of GWSS in nor th Florida and that its pres ence in the field plot would closely parallel that of GWSS. It was also hypothesized that the artificial high humidity environment created by the misting syst em would increase the incidence of H. homalodiscae -killed GWSS in the irrigate d portions of field plot.

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22 CHAPTER 3 MATERIALS AND METHODS Homalodisca coagulata / Hirsutella homalodiscae Interactions in a Crape Myrtle Field Plot Description of Study Site A crape myrtle field plot was utilized to track a population of GWSS over the course of a portion of the 2004 and the majo rity of the 2005 summer seasons. The plot consisted of four replicates of 14 crape myr tle cultivars, with each cultivar represented by four adjacent trees in each re plicate (Fig. 5). Cultivar groups were arranged in each replicate on a random basis. In the spring of each year, the trees were aggressively pruned to encourage maximal new growth and to re move the majority of cadavers remaining from the previous season. The four replicates were divided into two treatments, misted and ambient. In the misted replicates, a 6’ diameter emitter was staked above each tree in 10 of the 14 cultivars. These 10 cultivars were selected based on th eir attractiveness to GWSS and were: “Biloxi”, “Carolina Beau ty”, “Sioux”, “Apalachee”, “Osage”, “Miami”, “Tuscarora”, “Tuskegee”, “Natchez”, and “Tonto”. The misters were run the first 15 min. of every hour, 24 hours a day, 7 da ys a week. The other half of the two replicates were subjected only to th e prevailing environmental conditions. Sampling Program In 2004, each tree in the 10 chosen cultivar groups was sampled for live GWSS and GWSS cadavers three times between the date s of July 8 and August 28. Because this

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23 Irrigated Dry Irrigated Dry Replicate 1 Replicate 2 : Biloxi : Miami : Osage : Tonto Figure 5. Representative map of crape myrtle field plot with 2005 cultivars indicated. One block equals one tree.

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24 sampling was begun in the middle of the GWSS s eason, an effort was made to remove all previously occurring cadavers from the trees in the week prior to July 8th. Four cultivars were selected for inte nsive sampling and observation of both live and diseased GWSS for the 2005 season. “Osage ”, “Miami”, and “Tonto” cultivars were selected, as they had demonstrated the highest incidence of mycosed GWSS in the sampling performed in the second half of th e previous season. The fourth cultivar, “Biloxi”, was selected because it had s hown high populations but comparatively low cadaver numbers. Each replicate was sampled on a weekly basis for 20 weeks from 5/12/2005 to 9/21/2005. No sampling was performed on weeks 14 and 17. Because the study was begun prior to the immigrati on of GWSS into the plot it was unnecessary to manually remove cadavers from the trees, as the pruni ng had already accomplished this. Individual trees were visually sampled for live GWSS by running a curved tool behind each branch and counting the insects as they displayed evasive behaviors. Sampling was performed between 08:00 and 12:00, a period of lowe r GWSS activity. This minimized sampling error due to fly-off, a concern during hei ghtened GWSS activity in afternoon periods. Immediately following live sampling, each branch of the tree was visually inspected on all sides for the presence of mycosed insects. Those found were marked by tying a piece of surveyor’s tape around the branch 10-15 cm below the cadaver. The tape was then marked with a number. This allowed the resear cher to keep detailed records on individual cadavers and monitor changes in morphology. Sticky Trap Grid As a supplement to the visual popula tion counts, a 229 m grid with 51 locations was set up in the 650 acre (1 mile2) area surrounding the field plot and 27 yellow sticky

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25 traps consisting of 7.5 x 15 cm mailing tubes on 1 m stakes were distributed to half the grid points at random. Trap placement was randomized each week among the 51 locations. The GWSS on the traps were counted weekly from 5/23/2005 to 9/21/2005. They were replaced with new traps after count ing. The sticky trap grid was utilized to provide data on overall GWSS populations to supplement the localized plot sampling. Diagnostic Bleeds Sampling Program Live GWSS were collected in the field plot through sweep ing or gentle beating of the branch. One insect was removed from each of the ten 4-tree cultivar groups. Insects collected from the two misted and dry repli cates were combined, making for one cohort of approximately 20 insects colle cted for both irrigated and dry treatments. As such, each replicate was treated as a monoculture, with cultivar acknowledged only so far that every one was sampled. After collection, insects were transferred to a mesh sleeve cage. When sampling for each treatment was complete, the sleeve cage was placed over a host plant, typically Baccharis halimifolia to ensure leafhopper hea lth until used for bleeding. Bleed Technique Prior to bleeding, insects were removed from the plant and placed in a plastic tube with a foam plug at each end. Carbon dioxide was introduced into the tube through a port at the end until all insects were visibly immob ilized. They were then transferred to a 2 L Erlenmeyer flask, which was filled with CO2 and sealed with a rubber stopper. The leafhoppers were removed from the flas k in groups of three for bleeding. Bleeds were performed under a dissecting microscope, through removal of one antenna at the base with an EtOH-sterilized insect pin (Fig. 6). Applying gentle pressure to the thorax with soft for ceps usually produced a droplet of hemolymph at the site of

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26 antennal insertion. This was done with the in sect placed on its dorsa l side, and using a micropipette, 0.5-1 L of the re sultant droplet of hemolym ph was removed. This sample was then transferred to a previously aliquo tted 9 L droplet of Ri nger’s solution (0.75% NaCl, 0.035% KCl, 0.021% CaCl2) on a glass slide, which was then covered with a glass coverslip. The sample was viewed under phase contrast optics at 100X magnification. Figure 6. Photographs showing removed antenna and hemolymph drop from bleed. A) Antennal insertion point. B) Rem oved antenna. Photographs processed by C. Tipping. Samples displaying characteristic hyphal body morphology were confirmed to be positive at 400X. After bleeding, insects were separated onto caged host plants based on status as positive or negative for hyphal bodies. Statistical Analysis Data collected from the sticky traps and in the field trial were subjected to repeated measures analysis using a mixed model methodology. Week, treatment, position within the plot, and crape myrtle cultivar were used in the analysis to determine which of these variables produced a significant influence on live and mycosed GWSS counts. Factors and combinations of factors pr oducing significant effects were subsequently subjected to least squares means analysis to determine timing and location of these effects. Similarly, cadaver incidence was analyzed with repe ated measures and least squares means

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27 procedures using time, treatment and cult ivar as factors. Cadaver incidence was compared to host GWSS population numbers by first arcsine transf orming the data and then running the same analysis as for the live GWSS numbers with the resultant proportion. T tests were utili zed to compare 2004 and 2005 cadav er incidence at single points in time and to compare between tota l cadaver numbers in irrigated and dry treatments. All analyses were performed using SAS 9.1 for Windows (SAS Institute 2003)

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28 CHAPTER 4 RESULTS In 2004, humidity treatment was a significant factor in cadaver incidence. Average number of cadavers per tree was significantly higher in misted replicates (t = 6.14, p < 0.0001). In a comparison of leafhopper numbers between the same trees for the same weeks in 2004 and 2005, there is no clear trend in which year had higher populations (Table 1). During week 10, there were more in 2004, but weeks 15 and 16 showed more in 2005. The few samples taken in 2004 limits this comparison. Cumulative mean cadaver numbers per tree were significan tly higher in 2004 than 2005 (t = 7.43, p < 0.0001). The last sample time for the plot in 2004 occurred from 8/17/04 to 8/28/04. Table 1. Comparison of 2004 and 2005 GWSS populat ions in field plot for same trees on same week between years. Week T value P < |t| Significantly Greater Year 9 0.7 0.4931 None 10 4.5 <0.00012004 11 0.64 0.5364 None 12 2.88 0.1021 None 15 -4.61 <0.00012005 16 -2.54 0.0291 2005

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29 During this time, mean GWSS cadavers pe r tree was 4.44 3.74 (N = 64). The same trees sampled on 8/24/05 had 0.31 0.48 mean cadavers per tree. The total number of cadavers persisting on these trees at this time point in 2004 was 284 compared to 20 for 2005. In 2005, background GWSS population data from the sticky trap grid indicate two peaks in GWSS numbers over the course of th e study, the highest coming in week 7 and a much smaller peak at week 16 (Fig. 7). Week s 5 through 9 all produced trap catches that were significantly higher than the other week s (Table 2). The second peak at week 16 was not statistically significant. Visual GW SS counts of the “Biloxi” cultivar in the misted portion of the first replicate clos ely mirror this trend with a delay of approximately 1-2 weeks (Fig. 8). Counts in the remainder of replicate 1, as well as all of replicate 2 show little similarity to backgr ound numbers (Figs. 9-11). Population averages 0 10 20 30 40 50 60 70 80 90 34567891011121314151617181920 WeekAvg. Hopper per Trap Figure 7. Mean GWSS per yellow stick trap in 2005. Error bars equa l standard error.

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30 for the entire plot, without respect to cultivar or treatment, show the same first peak at week 5 to week 9, but do not drop as precip itously afterwards, showing instead a slower dropoff and no evident second peak (Fig. 12). Least squares means analysis of significan t effects found in the repeated measures procedures revealed significant differe nces between both cultivar and humidity treatments were primarily due to the action of the crape myrtle cultivar “Biloxi” in the misted portion of the first rep licate (Tables 3 & 4). This cu ltivar group held the highest numbers of leafhoppers of a ny group throughout the entire fi rst population peak, often by a factor of 3 (Fig. 8-11). Mo st significant differences be tween treatment and cultivar were found within this peak (Tables 3 & 4). The exception to this phenomenon is that within the misted portion of the first replicate, leafhopper numbers on the cultivar “Tonto” were significantly lower when compar ed to “Osage” in weeks 16 through 18 and when compared to “Biloxi” in week 19 (Table 3). These differences, however, were not of great enough magnitude to produce a significant difference in GWSS numbers between irrigated and dry treatments (Table 4). The incidence of Hirsutella homalodiscae within the field plot did not follow fluctuations in GWSS populations. Mycosed cad avers appeared with greatest regularity beginning at the tail of the first population pe ak, until week 19 (Fig. 13). This time period was characterized by lower GWSS populations, but also by almost weekly shifts in host cultivar preference. Sticky trap data show that background GWSS populations were much lower after week 11 (Fig. 7). However, mean GWSS populations in the plot rose slightly in week 12 and then began a gr adual decline (Fig. 12). It shou ld be noted that the higher numbers of cadavers found in week 15 and 18 were probably due to the missed sampling

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31 dates from the weeks 14 and 17. Therefore the cadavers numbers found on weeks 15 and 18 reflect a total for two weeks. Significant between treatment differences in cadaver incidence were found in weeks 10-13, 15, 16, 18, and 19 (Table 6). While significant at times, no single cultivar emerged as a definiti ve factor in cadaver incidence (Tables 5 & 6). Irrigation treatment was a moderately significant effect when total cadavers for each treatment were analyzed using a t-test (T = 2.23, p = 0.0295), with more being found in the misted replicates. On 9/14/05, field obser vation indicated that 46% (N = 27) of the remaining cadavers displayed some degree of Pseudogibellula morphology, varying from mounded areas of mycelium at inters egmental areas to fully formed Pseudogibellula -type synnemata (Fig. 1A). A total of 435 GWSS were bled in 2005, 185 from within the treatment plot, and 250 from a secondary plot Four definitive positives were found, all between the dates of 7/12/05 and 7/20/05. Tw o of the positives were collected from within the study plot.

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32 0 10 20 30 40 50 60 70 80 901 3 5 7 9 11 13 15 17 19WeekAvg. Hoppers per Tree Biloxi Osage Miami Tonto Figure 8. Mean GWSS per tree for each cultivar in the first irri gated replicate in 2005. Error bars equal standard error.

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33 0 5 10 15 20 25 301 3 5 7 9 11 13 15 17 19WeekAvg. Hoppers per Tree Biloxi Osage Miami Tonto Figure 9. Mean GWSS per tree for each cultiv ar in the second irrigated replicate in 2005. Error bars equa l standard error.

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34 0 2 4 6 8 10 12 141 3 5 7 9 11 13 15 17 19WeekAvg. Hoppers per Tree Biloxi Osage Miami Tonto Figure 10. Mean GWSS per tree for each cultivar in the firs t dry replicate in 2005. Error ba rs equal standard error.

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35 0 5 10 15 20 25 30 351 3 5 7 9 11 13 15 17 19WeekAvg. Hoppers per Tree Biloxi Osage Miami Tonto Figure 11. Mean GWSS per tree for each cultivar in the sec ond dry replicate in 2005. Error bars equal standard error.

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36 0 2 4 6 8 10 12 14 16 1234567891011121314151617181920 WeekAvg. Hoppers per Tree Figure 12. Mean GWSS per tree for entire plot in 2005. Error bars equal standard error.

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37 New Cadavers by Week0 1 2 3 4 5 6 7 8 12345678910111213141516171819 WeekCadavers Figure 13. Total GWSS cadavers observed by week in 2005.

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38 Table 2. Weeks with significantly higher trap catches of GWSS in 2005. Table 3. Significant differences in live GWSS numbers between cultivars with respect to treatment and week in 2005. Week Estimate T Value Pr > |t| 5 21.3077 4.18 < 0.0001 6 57.0000 11.17 < 0.0001 7 64.6000 12.42 < 0.0001 8 42.0000 8.23 < 0.0001 9 17.4231 3.41 0.0007 Treatment Week Cultivar Cultivar Estimate T Value Pr < |t| Misted 6 Biloxi Tonto -13.3750 -2.81 0.0050 Misted 6 Biloxi Osage 16.1250 3.39 0.0007 Misted 6 Biloxi Miami 17.5000 3.68 0.0002 Misted 7 Biloxi Tonto -26.7500 -5.62 <.0001 Misted 7 Biloxi Osage 28.8750 6.07 <.0001 Misted 7 Biloxi Miami 28.7500 6.04 <.0001 Misted 8 Biloxi Tonto -26.7500 -5.62 <.0001 Misted 8 Biloxi Osage 24.3750 5.12 <.0001 Misted 8 Biloxi Miami 25.8750 5.44 <.0001 Misted 9 Biloxi Tonto -14.8750 -3.13 0.0018 Misted 9 Biloxi Osage 15.5000 3.26 0.0012 Misted 9 Biloxi Miami 17.8750 3.76 0.0002 Misted 10 Biloxi Tonto -14.7500 -3.10 0.0020 Misted 10 Biloxi Osage 16.8750 3.55 0.0004 Misted 10 Biloxi Miami 18.2500 3.84 0.0001 Misted 11 Biloxi Osage 9.5000 2.00 0.0462 Misted 11 Biloxi Miami 10.6250 2.23 0.0258 Misted 16 Tonto Osage -11.6250 -2.44 0.0147 Misted 18 Tonto Osage -9.6250 -2.05 0.0403 Misted 19 Biloxi Tonto -10.7500 -2.29 0.0220

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39 Table 4. Significant differences between treatments with respect to week and cultivar in 2005. Table 5. Significant differences in cadaver incidence between treatments for a given week and cultivar in 2005. Treatment Treatment Week Cultivar Estimate T Value Pr > |t| Irrigated Dry 10 Osage 0.2500 2.34 0.0193 Irrigated Dry 11 Tonto 0.3750 3.51 0.0005 Irrigated Dry 11 Miami 0.2500 2.34 0.0193 Irrigated Dry 12 Miami 0.2500 2.34 0.0193 Irrigated Dry 13 Osage 0.2500 2.34 0.0193 Irrigated Dry 15 Osage 0.3750 3.51 0.0005 Irrigated Dry 16 Tonto 0.2500 2.34 0.0193 Irrigated Dry 18 Tonto -0.3750 -3.51 0.0005 Irrigated Dry 18 Biloxi 0.2500 2.34 0.0193 Irrigated Dry 19 Osage -0.2500 -2.34 0.0193 Treatment Treatment Week Cultivar Estimate T Value Pr > |t| Misted Dry 6 1 22.000 3.51 0.0005 Misted Dry 7 1 35.500 5.66 < .0001 Misted Dry 8 1 33.000 5.26 < .0001 Misted Dry 9 1 21.875 3.49 0.0005 Misted Dry 10 1 21.250 3.39 0.0007 Misted Dry 11 1 14.000 2.23 0.0258

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40 Table 6. Significant differences in cadaver in cidence between cultivars for a given week and treatment in 2005. Treatment Week Cultivar Cultivar Estimate T Value Pr > |t| 1 10 0 5 -0.2500 -2.34 0.0193 1 10 1 5 -0.2500 -2.34 0.0193 1 10 5 6 0.2500 2.34 0.0193 1 11 0 1 0.2500 2.34 0.0193 1 11 0 5 0.2500 2.34 0.0193 1 12 0 6 -0.2500 -2.34 0.0193 1 12 1 6 -0.2500 -2.34 0.0193 1 12 5 6 -0.2500 -2.34 0.0193 1 13 0 5 -0.2500 -2.34 0.0193 1 13 5 6 0.2500 2.34 0.0193 1 15 0 5 -0.2500 -2.34 0.0193 1 15 1 5 -0.2500 -2.34 0.0193 1 15 5 6 0.2500 2.34 0.0193 1 16 0 1 0.2500 2.34 0.0193 1 16 0 6 0.2500 2.34 0.0193 1 18 0 1 -0.2500 -2.34 0.0193 1 18 1 5 0.2500 2.34 0.0193 2 18 0 1 0.3750 3.51 0.0005 2 18 0 5 0.2500 2.34 0.0193 2 18 0 6 0.3750 3.51 0.0005 2 19 0 5 -0.2500 -2.34 0.0193 2 19 1 5 -0.2500 -2.34 0.0193

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41 CHAPTER 5 DISCUSSION The GWSS’s predilection for accessing different hosts is evidenced by the 2005 plot data. Insects in the initi al population peak preferred the cultivar “Biloxi” in the first irrigated plot (Fig. 8) and to a lesser degree in the second (F ig. 9), but those in the dry plots displayed no clear cultivar preference during this period (Figs. 10,11). During the latter portion of the season, there was either no definitive preference or preferences were slight and shifted often (Figs 8-11). With the data available in this study there can be no conclusive explanation for these behaviors, bu t inferences can be drawn from information on GWSS biology. GWSS overwinter as adults or nymphs in a state of incomplete diapause (Turner & Pollard 1959). The considerab le fluctuations in winter temperatures in north Florida (Florida Au tomated Weather Network, http ://fawn.ifas.ufl.edu/) allow both adult and nymphal stages to feed during warmer periods. This allows the nymphs to eclose to adults before spring. As adults become active in the spring, they oviposit on hosts suitable for nymphal development (Ald erz 1980). Crape myrtle is a host preferred primarily by adult GWSS, so the fi rst population peak is likely the F1 generation migrating to the field plot after they eclo se (Ball 1979). The sec ond peak occurred in mid-late August and is probably indicative of the F2 generation. Timing of the beginning of the first population peak (Fig. 7) corres ponds well with Ball’s ( 1979) observations but not with those of Turner and Pollard ( 1959), who found the highest GWSS numbers in peach in the fall. At the end of the season, it is thought that the F2 generation splits survival strategies (Mizell, pers onal communication). A portion of the F2 individuals will

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42 enter reproductive diapause while the rest wi ll continue to mate a nd oviposit. Those in reproductive diapause enter the winter season as adults. Those hatching from egg masses laid by the F2 enter as nymphs. This dichotomy could explain the change in host preference when compared to members of the F1 generation. Studies with Lepidoptera have shown that nutrition play s a role in the induction of diapause (Hunter & McNeil 2004, Mondy & Corio-Costet 2004). Nutrient requirements of those GWSS entering diapause may differ considerably from non-di apausing GWSS, and this could be reflected in the differences in host preference. An alternative explanation is that the shift in host preference is driven by a change in the am ino acid profile of the host xylem fluid (See Literature Review: Nu tritional Ecology of Homalodisca coagulata ). Most crape myrtles in the field plot had completed flowering by the end of the sampling period (personal observation), and this shift in life stage may be reflected in a resultant shift in available nutrients in the xylem fluid. It is unlikely that changes in host preference are related to other effects, such as overcrowding or in creased energy expended while feeding. Host plants the size of the plot crape myrtle s are capable of sustaining extremely high leafhopper densities with little or no change in xylem fluid content (A ndersen et al. 2003) and the feeding apparatus of GW SS allows them to feed at a wide range of fluid pressures (Andersen et al. 1992). The preference for the “Biloxi” cultivar in the first misted replicate during the first population peak is difficult to explain. The fact that this trend is not shown in any of the other “Biloxi” cultivar groups means that simp le host preference is an unlikely cause. It is, however, possible that the increased number s can be attributed to the cultivar group’s position in the plot. The first irri gated replicate is located at the northern edge of the plot,

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43 with the “Biloxi” group at the western corner. The plot is oriented so that this corner is the closest to a natural forest habitat, and therefore may serve as the initial point of GWSS immigration. This is supported by the f act that the “Biloxi” cultivar group in the first misted replicate is the only one that mimics the trap-based background population curve with any consistency. If these four tr ees represent a major immigration point, after which the insects distribute themselves thr oughout the rest of the plot in a relatively homogenous manner, this trend in cultivar pr eference could be accounted for. A similar phenomenon was also observed by Ball (1979), who found that traps bordering woodlands produced the highe st catches of GWSS. The primary focus of the field study was to characterize the action of Hirsutella homalodiscae on a host GWSS population in the na tive range. During 2004, the pathogen was present in epizootic proportions and was probably a major mortality factor, particularly to the F1 and F2 generations. However, sampling in 2004 was not performed with enough frequency to judge this. In 2005, a more typical enzoot ic-type condition was observed, with very few cadavers found and what could only be a negligible effect on host population. For the few sample dates that co incide between the two years, there is no discernable trend regarding which year displa yed greater densities of GWSS within the plot (Table 1). It should be noted that sampling in 2004 began at a later date than in 2005, and GWSS densities for the first population peak were not recorded. There may have been a significant difference between th e two years during this time period. Explanations for this difference in cadaver incidence between 2004 and 2005 are speculative because at this point in time, vi rtually nothing is known about the biology or life cycle of H. homalodiscae and little is known of the overwintering biology of GWSS

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44 (Alderz 1980). Weather data collected from 6/13/04 to 9/21/04 and 6/13/05 to 9/21/05, the time period when cadavers were found in 2005, shows no major deviations in average temperature, rainfall, and relative humidity between the two years (Florida Automated Weather Network, http://fawn.ifas.ufl.edu/ ) (Appendix E). Bioassays indicate that H. homalodiscae like most entompathogenic fungi, em ploys a transcuticular means of ingress into the host insect a nd that sporulating cadavers can act as the source of the infectious inoculum (Blaeske unpublished data). It has also been shown that cadavers from the previous season will develop minor hyphal growth and limited sporulation if put under moist, warm conditions (persona l observation, Blaeske, unpublished data). Therefore, it is assumed that a cadaver affixe d to the host plant could function as both the pathogen’s overwintering reservoi r and the stage that delivers inoculum to the host. Most pathogenic fungi possess an environmentally resistant stage, w ith intrahyphal or compressed hyphae and chlamydospores bei ng most common in the Hyphomycetes (Shah & Pell 2003, Pendland 1982). H. homalodiscae has been shown to produce chlamydospores under in vitro conditions (Boucias et al., su bmitted), but no studies have been performed to search for these structur es in the cadavers. The ability to induce hyphal growth and limited sporulation from ove rwintered cadavers a nd the presence of chlamydospores in culture tend to indicate that the cadavers th at persist on the host plant from one season to the next may represent th e primary source of inoculum for the new season. Some other pathogenic Hyphomycetes are known to possess a soilborne stage (Fuxa & Richter 2004, Sanchez-Murillo et al. 2004, Hu & St. Leger 2002), but it is unlikely that this would be the main reservoir for H. homalodiscae GWSS are thought to

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45 overwinter primarily on their winter host plants (Turner & Pollard 1959, Alderz 1980) and would not contact a soil-based pathogen. Hirsutella homalodiscae produces a single conidium fr om the apical portion of each phialide (Boucias et al., submitted) that does not appear to rely on dissemination away from the cadaver as a route of infec tion, but on contact between sporulating cadaver and nave individual. This is th e case with other species of Hirsutella such as H. thompsonii a mite pathogen (Ignoffo & Mandava 1988). Work has been performed which demonstrates that GWSS display a ggregation behaviors, most likely based on recognition of a conspecific’s silhouette on a branch (Mizel l, unpublished data). Although the color differs, mycosed GWSS cadavers maintain much the same shape on the branch as healthy leafhoppers (personal observation). If the cad aver triggered the same aggregation response as live GWSS, th e need for wind or water based distribution of conidia would be eliminat ed. Nave hosts would be attr acted to infective cadavers. If as above, it is assumed that overwinte ring cadavers represent the primary source of inoculum for H. homalodiscae then the absence of observed infection in the parent generation in the spring could be due to three f actors. First, it may be that environmental conditions are not sufficient to cause new grow th and sporulation in the cadavers from the previous season or to s upport infection in a new host. Secondly, in order to ensure that no cadavers from the prev ious season remained to skew the counts, all host trees in the plot were trimmed aggressively befo re the study. This removed all cadavers remaining from the previous season, and ther efore all possible inoculum within those trees. For the infection cycle to become esta blished in the plot, diseased insects would

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46 have to first become infected elsewhere a nd then migrate into th e study area, and this may explain the absence of infection until after the first population peak. Entomopathogenic fungi are known to alter the activity of thei r insect hosts by altering movement and distribution (Jensen et al. 2001), reducing f eeding rate (Hajek 1989), changing reproductive behavior(Wats on & Peterson 1993), inducing behavioral fevers (Watson et al. 1993) and causing summit behavior (Bidochka et al. 1997). Comparisons between the behaviors of live a nd infected GWSS would have applications in interpreting field population data. Whether infected GWSS are as prone to migration and host switching as healt hy individuals is unknown. For the GWSS/ H. homalodiscae relationship to be fully understood, future research should focus on the transmission biology of th e fungus, in combination with long-term observation of pathogen dynamics in the fiel d. A better understanding of the effect of environmental conditions on the transmissi on potential and infec tion success of the fungus would allow some parallels to be dr awn between laboratory bioassay data and field incidence for the purpose of determini ng whether epizootic c onditions are based on host population effects or simple phenological fluctuations. The locations in California that could benefit from the introduction of native biocontrol agents possess a cool, wet spring but a very dry summer (www.temeculaweather.com, http://www.wunderground.com/US/CA/Napa.html ). Experiments should be conducted to determine the suitability of H. homalodiscae for introduction in this type of environment.

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47 CHAPTER 6 CONCLUSIONS Disease incidence was much higher in 2004 than in 2005 and may have been due to differences in GWSS populations between the two years, but the 2004 data was not collected over a long enough tim e period to determine this conclusively. Humidity treatment was a significant factor with re spect to cadaver inci dence in 2004, but only moderately so in 2005, when there were very few cadavers in any portion of the plot. Cultivar preference was present to an extent in both years, but further investigation would be necessary to separate the effect of cultiv ar from that of immigration of the insect. This study represents an in itial investigation into this system, detailing some methods and techniques that should be of valu e to an individual wishing to further pursue research with these organisms. But while th e methodologies presented here are believed to be sound, one should use caution when dr awing conclusions about any ecological relationship based on one and a half seasons of field data, including this one. For a more full understanding of how GWSS interacts w ith its fungal pathogen complex, longerterm, higher-volume observation and sampling is vital, with the data shown here simply to be used as a guide for experimental desi gn. When combined with these field studies, research into the environmental optima fo r transmission and growth of the pathogen should provide clues as to its suitability for introduction to Californi a for the biological control of GWSS.

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48 APPENDIX A SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF LIVE GWSS POPULATIONS AND LEAST MEANS SQUARED ANALYSIS OF SIGNIFICANT EFFECTS %let path = E:\SAS Output; %macro printRtf(dset); ods rtf file = "&path\test.rtf"; proc print data=&dset; Run; ods rtf close; %mend printRtf; Data; input blk trt week cult count @@; cards; DATA HERE proc mixed; class blk trt week cult; model count= blk trt cult trt*cult week week*trt week*cult week*trt*cult; Random blk*trt blk*trt*cult; lsmeans trt*week*cult / diff; ods output lsmeans = LSMEAN diffs = LSDIFF; run; *proc print data=lsdiff; run; data siglsdiff; set lsdiff; if week=_week and cult=_cult and Probt < 0.05; keep week cult trt _trt estimate tvalue probt; run; title 'significant differences in treatments for a given week and cultivar'; %printRTF(siglsdiff); run; data siglsdiff2; set lsdiff; if week=_week and trt=_trt and Probt < 0.05; keep week trt cult _cult estimate tvalue probt; run; title 'significant differences in cultivars for a given week and

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49 treatment'; %printRTF(siglsdiff2); run;

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50 APPENDIX B SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF CADAVER INCIDENCE AND LEAST MEANS SQUARES ANALYSIS OF SIGNIFICANT EFFECTS %let path = E:\SAS Output; %macro printRtf(dset); ods rtf file = "&path\test.rtf"; proc print data=&dset; Run; ods rtf close; %mend printRtf; Data; input blk trt week cult cadavers @@; cards; DATA HERE proc mixed; class blk trt week cult; model cadaver= blk trt cult trt*cult week week*trt week*cult week*trt*cult; Random blk*trt blk*trt*cult; lsmeans trt*week*cult / diff; ods output lsmeans = LSMEAN diffs = LSDIFF; run; *proc print data=lsdiff; run; data siglsdiff; set lsdiff; if week=_week and cult=_cult and Probt < 0.05; keep week cult trt _trt estimate tvalue probt; run; title 'significant differences in cadavers between treatments for a given week and cultivar'; %printRTF(siglsdiff); run; data siglsdiff2; set lsdiff; if week=_week and trt=_trt and Probt < 0.05; keep week trt cult _cult estimate tvalue probt; run; title 'significant differences in cadavers between cultivars for a

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51 given week and treatment'; %printRTF(siglsdiff2); run;

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52 APPENDIX C SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF TRAP POPULATION DATA AND LEAST MEANS SQAURES ANALYSIS OF THE EFFECT OF TIME Data; input week count @@; cards; DATA HERE proc mixed; class week; model count= week; lsmeans week / diff; run;

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53 APPENDIX D SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF ARCSINE TRANSFORMED LIVE GWSS/C ADAVER PROPORTIONS WITH LEAST MEANS SQUARE ANALYSIS OF SIGNIFICANT EFFECTS %let path = E:\SAS Output; %macro printRtf(dset); ods rtf file = "&path\test.rtf"; proc print data=&dset; Run; ods rtf close; %mend printRtf; Data; input blk trt week cult live mummy @@; y=mummy; n=live+mummy; if n=0 then n=.; if y=0 then p=1/(4*n); if 1<=y<=n-1 then p=y/n; if y=n then p=(n1/4)/n; transp= (360/2*3.14159)*(arsin(SQRT(p)) ); cards; DATA HERE proc mixed; class blk trt week cult; model transp= blk trt cult trt*cult week week*trt week*cult week*trt*cult; Random blk*trt blk*trt*cult; lsmeans week trt*week trt*week*cult / diff; ods output lsmeans = LSMEAN diffs = LSDIFF; run; *proc print data=lsdiff; run; data siglsdiff; set lsdiff; if week=_week and cult=_cult and Probt < 0.05; keep week cult trt _trt estimate tvalue probt; run; title 'significant differences in treatments for a given week and cultivar'; %printRTF(siglsdiff); run;

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54 54 data siglsdiff2; set lsdiff; if week=_week and trt=_trt and Probt < 0.05; keep week trt cult _cult estimate tvalue probt; run; title 'significant differences in cultivars for a given week and treatment'; %printRTF(siglsdiff2); run;

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55 APPENDIX E QUINCY NFREC WEATHER DATA FOR 2004 AND 2005 Average Daily Temperature from 6/13 to 9/210 5 10 15 20 25 30 351 9 17 25 33 41 49 57 65 73 81 89 97DayAverage Daily Temperature 2004 2005 Precipitation from 6/13 to 9/210 0.5 1 1.5 2 2.5 3 3.5 41 9 17 25 33 41 49 57 65 73 81 89 97DayRainfall (cm) 2004 2005

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56 Average Relative Humidity from 6/13 to 9/210 20 40 60 80 100 1201 8 15 22 29 36 43 50 57 64 71 78 85 92 99DayAverage RH (%) 2004 2005

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57 APPENDIX F RAW DATA FROM 2004 AND 2004 FIELD STUDIES 2004 Field Plot Data Tree Code Date Cultivar Live GWSS Cadavers A1 7/8/2004Biloxi 55 1 A2 7/8/2004Biloxi 60 4 A3 7/8/2004Biloxi 54 3 A4 7/8/2004Biloxi 44 3 A5 7/8/2004Carolina Beauty32 5 A6 7/8/2004Carolina Beauty45 4 A7 7/8/2004Carolina Beauty41 6 A8 7/8/2004Carolina Beauty33 1 A13 7/8/2004Sioux 22 0 A14 7/8/2004Sioux 20 3 A15 7/8/2004Sioux 12 1 A16 7/8/2004Sioux 21 0 A17 7/8/2004Appalachee 20 1 A18 7/8/2004Appalachee 21 2 C5 7/12/2004Natchez 25 1 C6 7/12/2004Natchez 25 4 C8 7/12/2004Natchez 1 C9 7/12/2004Osage 14 0 C10 7/12/2004Osage 11 0 C11 7/12/2004Osage 1 C12 7/12/2004Osage 1 C13 7/12/2004Tonto 17 1 C14 7/12/2004Tonto 18 1 C15 7/12/2004Tonto 0 C16 7/12/2004Tonto 2 C17 7/12/2004Biloxi 12 1 C18 7/12/2004Biloxi 8 0 C19 7/12/2004Biloxi 0 C20 7/12/2004Biloxi 0 C21 7/12/2004Carolina Beauty8 1 C22 7/12/2004Carolina Beauty6 0 C23 7/12/2004Carolina Beauty 0 C24 7/12/2004Carolina Beauty 0 D1 7/14/2004Sioux 16 4 D2 7/14/2004Sioux 15 1 D3 7/14/2004Sioux 1

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58 D4 7/14/2004Sioux 0 D5 7/14/2004Appalachee 5 1 D6 7/14/2004Appalachee 4 0 D7 7/14/2004Appalachee 0 D8 7/14/2004Appalachee 0 D9 7/14/2004Miami 13 4 D10 7/14/2004Miami 15 4 D11 7/14/2004Miami 0 D12 7/14/2004Miami 1 D17 7/14/2004Tuscarora 15 1 D18 7/14/2004Tuscarora 11 2 D19 7/14/2004Tuscarora 0 D20 7/14/2004Tuscarora 2 D21 7/14/2004Tuskegee 17 0 D22 7/14/2004Tuskegee 9 2 D23 7/14/2004Tuskegee 0 D24 7/14/2004Tuskegee 0 E1 7/12/2004Tuskegee 44 8 E2 7/12/2004Tuskegee 51 11 E3 7/12/2004Tuskegee 3 E4 7/12/2004Tuskegee 0 E9 7/12/2004Biloxi 33 3 E10 7/12/2004Biloxi 15 2 E11 7/12/2004Biloxi 1 E12 7/12/2004Biloxi 0 E13 7/12/2004Miami 14 1 E14 7/12/2004Miami 13 1 E15 7/12/2004Miami 0 E16 7/12/2004Miami 1 E17 7/12/2004Sioux 11 0 E18 7/12/2004Sioux 13 1 E19 7/12/2004Sioux 0 E20 7/12/2004Sioux 0 E21 7/13/2004Natchez 13 1 E22 7/13/2004Natchez 12 2 E23 7/13/2004Natchez 1 E24 7/13/2004Natchez 1 E25 7/13/2004Appalachee 7 1 E26 7/13/2004Appalachee 12 2 E27 7/13/2004Appalachee 2 E28 7/13/2004Appalachee 0 F5 7/14/2004Tonto 10 1 F6 7/14/2004Tonto 13 1 F7 7/14/2004Tonto 2 F8 7/14/2004Tonto 2 F9 7/14/2004Tuscarora 17 3 F10 7/14/2004Tuscarora 23 2 F11 7/14/2004Tuscarora 1

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59 F12 7/14/2004Tuscarora 2 F17 7/14/2004Carolina Beauty12 1 F18 7/14/2004Carolina Beauty11 1 F19 7/14/2004Carolina Beauty 0 F20 7/14/2004Carolina Beauty 0 F21 7/14/2004Osage 10 4 F22 7/14/2004Osage 10 2 F23 7/14/2004Osage 3 F24 7/14/2004Osage 4 G1 7/9/2004Carolina Beauty14 0 G2 7/9/2004Carolina Beauty14 2 G3 7/9/2004Carolina Beauty 0 G4 7/9/2004Carolina Beauty 1 G9 7/9/2004Tuskegee 13 1 G10 7/9/2004Tuskegee 13 1 G11 7/9/2004Tuskegee 0 G12 7/9/2004Tuskegee 0 G13 7/9/2004Sioux 15 1 G14 7/9/2004Sioux 12 2 G15 7/9/2004Sioux 1 G16 7/9/2004Sioux 1 G21 7/9/2004Tuscarora 17 0 G22 7/9/2004Tuscarora 15 0 G23 7/9/2004Tuscarora 0 G24 7/9/2004Tuscarora 2 H1 7/9/2004Natchez 36 1 H2 7/9/2004Natchez 33 1 H3 7/9/2004Natchez 1 H4 7/9/2004Natchez 0 H9 7/9/2004Miami 8 0 H10 7/9/2004Miami 10 1 H11 7/9/2004Miami 0 H12 7/9/2004Miami 4 H13 7/9/2004Appalachee 18 1 H14 7/9/2004Appalachee 20 1 H15 7/9/2004Appalachee 1 H16 7/9/2004Appalachee 1 H17 7/9/2004Biloxi 6 1 H18 7/9/2004Biloxi 7 1 H19 7/9/2004Biloxi 0 H20 7/9/2004Biloxi 0 H21 7/9/2004Osage 19 1 H22 7/9/2004Osage 13 0 H23 7/9/2004Osage 0 H24 7/9/2004Osage 0 H25 7/9/2004Tonto 18 1 H26 7/9/2004Tonto 12 0 H27 7/9/2004Tonto 2

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60 H28 7/9/2004 Tonto 0 A1 7/20/2004Biloxi 17 4 A2 7/20/2004Biloxi 22 4 A3 7/20/2004Biloxi 4 A4 7/20/2004Biloxi 5 A5 7/20/2004Carolina Beauty12 5 A6 7/20/2004Carolina Beauty7 5 A7 7/20/2004Carolina Beauty 10 A8 7/20/2004Carolina Beauty 2 A13 7/20/2004Sioux 11 5 A14 7/20/2004Sioux 12 6 A15 7/20/2004Sioux 1 A16 7/20/2004Sioux 0 A17 7/20/2004Appalachee 18 4 A18 7/20/2004Appalachee 21 4 A19 7/20/2004Appalachee 2 A20 7/20/2004Appalachee 1 A21 7/20/2004Osage 40 10 A22 7/20/2004Osage 37 7 A23 7/20/2004Osage 5 A24 7/20/2004Osage 4 A25 7/20/2004Miami 22 2 A26 7/20/2004Miami 28 6 A27 7/20/2004Miami 6 A28 7/20/2004Miami 0 B9 7/22/2004Tuscarora 11 6 B10 7/22/2004Tuscarora 14 6 B11 7/22/2004Tuscarora 1 B12 7/22/2004Tuscarora 2 B13 7/22/2004Tuskegee 4 0 B14 7/22/2004Tuskegee 5 1 B15 7/22/2004Tuskegee 0 B16 7/22/2004Tuskegee 2 B17 7/29/2004Natchez 20 4 B18 7/29/2004Natchez 15 0 B19 7/29/2004Natchez 1 B20 7/29/2004Natchez 2 B25 7/29/2004Tonto 76 9 B26 7/29/2004Tonto 40 5 B27 7/29/2004Tonto 12 B28 7/29/2004Tonto 10 C5 8/18/2004Natchez 2 3 C6 8/18/2004Natchez 2 4 C8 8/18/2004Natchez 1 C9 8/18/2004Osage 0 0 C10 8/18/2004Osage 5 0 C11 8/18/2004Osage 2 C12 8/18/2004Osage 3

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61 C13 8/18/2004Tonto 0 1 C14 8/18/2004Tonto 1 1 C15 8/18/2004Tonto 3 C16 8/18/2004Tonto 6 C17 8/18/2004Biloxi 1 2 C18 8/18/2004Biloxi 0 0 C19 8/18/2004Biloxi 0 C20 8/18/2004Biloxi 0 C21 8/18/2004Carolina Beauty1 1 C22 8/18/2004Carolina Beauty2 2 C23 8/18/2004Carolina Beauty 0 C24 8/18/2004Carolina Beauty 1 D6 8/18/2004Appalachee 2 6 D7 8/18/2004Appalachee 0 0 D8 8/18/2004Appalachee 0 D9 8/18/2004Miami 0 5 D10 8/18/2004Miami 1 5 D11 8/18/2004Miami 1 D12 8/18/2004Miami 2 D17 8/18/2004Tuscarora 3 3 D18 8/18/2004Tuscarora 2 3 D19 8/18/2004Tuscarora 0 D20 8/18/2004Tuscarora 3 D21 8/18/2004Tuskegee 0 0 D22 8/18/2004Tuskegee 0 2 D23 8/18/2004Tuskegee 0 D24 8/18/2004Tuskegee 0 E1 8/19/2004Tuskegee 0 9 E2 8/19/2004Tuskegee 2 13 E3 8/19/2004Tuskegee 4 E4 8/19/2004Tuskegee 1 E9 8/19/2004Biloxi 2 3 E10 8/19/2004Biloxi 3 3 E11 8/19/2004Biloxi 1 E12 8/19/2004Biloxi 0 E13 8/19/2004Miami 0 3 E14 8/19/2004Miami 1 3 E15 8/19/2004Miami 5 E16 8/19/2004Miami 2 E17 8/19/2004Sioux 7 1 E18 8/19/2004Sioux 5 2 E19 8/19/2004Sioux 1 E20 8/19/2004Sioux 2 E21 8/19/2004Natchez 7 5 E22 8/19/2004Natchez 5 5 E23 8/19/2004Natchez 2 E24 8/19/2004Natchez 5 E25 8/19/2004Appalachee 1 1

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62 E26 8/19/2004Appalachee 3 2 E27 8/19/2004Appalachee 2 E28 8/19/2004Appalachee 2 F5 8/20/2004Tonto 6 7 F6 8/20/2004Tonto 0 1 F7 8/20/2004Tonto 5 F8 8/20/2004Tonto 4 F9 8/20/2004Tuscarora 2 4 F10 8/20/2004Tuscarora 4 3 F11 8/20/2004Tuscarora 2 F12 8/20/2004Tuscarora 3 F17 8/20/2004Carolina Beauty2 1 F18 8/20/2004Carolina Beauty2 2 F19 8/20/2004Carolina Beauty 1 F20 8/20/2004Carolina Beauty 4 F21 8/20/2004Osage 3 6 F22 8/20/2004Osage 3 3 F23 8/20/2004Osage 4 F24 8/20/2004Osage 6 G1 8/9/2004 Carolina Beauty7 1 G2 8/9/2004 Carolina Beauty9 5 G3 8/9/2004 Carolina Beauty 3 G4 8/9/2004 Carolina Beauty 2 G9 8/9/2004 Tuskegee 0 1 G10 8/9/2004 Tuskegee 2 1 G11 8/9/2004 Tuskegee 0 G12 8/9/2004 Tuskegee 0 G13 8/9/2004 Sioux 12 2 G14 8/9/2004 Sioux 14 3 G15 8/9/2004 Sioux 2 G16 8/9/2004 Sioux 0 G21 8/9/2004 Tuscarora 2 1 G22 8/9/2004 Tuscarora 2 3 G23 8/9/2004 Tuscarora 1 G24 8/9/2004 Tuscarora 2 H1 8/17/2004Natchez 8 1 H2 8/17/2004Natchez 5 3 H3 8/17/2004Natchez 4 H4 8/17/2004Natchez 2 H9 8/17/2004Miami 1 5 H10 8/17/2004Miami 5 8 H11 8/17/2004Miami 3 H12 8/17/2004Miami 5 H13 8/17/2004Appalachee 1 1 H14 8/17/2004Appalachee 0 0 H15 8/17/2004Appalachee 1 H16 8/17/2004Appalachee 0 H17 8/17/2004Biloxi 1 0

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63 H18 8/17/2004Biloxi 0 0 H19 8/17/2004Biloxi 0 H20 8/17/2004Biloxi 0 H21 8/17/2004Osage 0 1 H22 8/17/2004Osage 0 0 H23 8/17/2004Osage 0 H24 8/17/2004Osage 0 H25 8/17/2004Tonto 3 3 H26 8/17/2004Tonto 8 0 H27 8/17/2004Tonto 2 H28 8/17/2004Tonto 4 A1 8/21/2004Tonto 3 6 A2 8/21/2004Biloxi 10 11 A3 8/21/2004Biloxi 5 A4 8/21/2004Biloxi 11 A6 8/21/2004Carolina Beauty0 9 A7 8/21/2004Carolina Beauty0 10 A8 8/21/2004Carolina Beauty 1 A13 8/21/2004Carolina Beauty3 8 A14 8/21/2004Sioux 1 6 A15 8/21/2004Sioux 3 A16 8/21/2004Sioux 0 A17 8/21/2004Sioux 1 4 A18 8/21/2004Appalachee 0 6 A19 8/21/2004Appalachee 4 A20 8/21/2004Appalachee 3 A21 8/21/2004Appalachee 3 18 A22 8/21/2004Osage 4 8 A23 8/21/2004Osage 9 A24 8/21/2004Osage 10 A25 8/21/2004Osage 7 5 A26 8/21/2004Miami 8 11 A27 8/21/2004Miami 13 A28 8/21/2004Miami 9 B9 8/28/2004Miami 1 7 B10 8/28/2004Tuscarora 0 8 B11 8/28/2004Tuscarora 3 B12 8/28/2004Tuscarora 4 B13 8/28/2004Tuscarora 2 2 B14 8/28/2004Tuskegee 3 2 B15 8/28/2004Tuskegee 1 B16 8/28/2004Tuskegee 2 B17 8/28/2004Tuskegee 0 4 B18 8/28/2004Natchez 0 0 B19 8/28/2004Natchez 4 B20 8/28/2004Natchez 3 B25 8/28/2004Natchez 1 18 B26 8/28/2004Tonto 1 8

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64 B27 8/28/2004Tonto 13 B28 8/28/2004Tonto 11 2005 Field Plot Data Date Week Replicate Tree Code Cultivar Tree # Live GWSS Cadavers 5/12/2005 1 Rep. 1-MistedA1 Biloxi 1 0 0 A2 2 0 0 A3 3 0 0 A4 4 0 0 A21 Osage 1 0 0 A22 2 1 0 A23 3 1 0 A24 4 0 0 A25 Miami 1 0 0 A26 2 0 0 A27 3 0 0 A28 4 0 0 B25 Tonto 1 0 0 B26 2 0 0 B27 3 0 0 B28 4 0 0 Rep. 1-Dry C17 Biloxi 1 0 0 C18 2 0 0 C19 3 0 0 C20 4 0 0 C9 Osage 1 0 0 C10 2 0 0 C11 3 0 0 C12 4 0 0 D9 Miami 1 0 0 D10 2 0 0 D11 3 0 0 D12 4 0 0 C13 Tonto 1 0 0 C14 2 0 0 C15 3 0 0 C16 4 0 0 5/13/2005 Rep. 2-MistedE9 Biloxi 1 0 0 E10 2 0 0 E11 3 2 0 E12 4 0 0 F21 Osage 1 0 0 F22 2 0 0 F23 3 0 0 F24 4 0 0 E13 Miami 1 0 0

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65 E14 2 0 0 E15 3 0 0 E16 4 0 0 F5 Tonto 1 0 0 F6 2 0 0 F7 3 0 0 F8 4 0 0 Rep. 2-Dry H17 Biloxi 1 0 0 H18 2 0 0 H19 3 0 0 H20 4 0 0 H21 Osage 1 0 0 H22 2 0 0 H23 3 0 0 H24 4 0 0 H9 Miami 1 0 0 H10 2 0 0 H11 3 0 0 H12 4 0 0 H25 Tonto 1 0 0 H26 2 0 0 H27 3 0 0 H28 4 0 0 5/16/2005 2 Rep. 1-MistedA1 Biloxi 1 0 0 A2 2 2 0 A3 3 0 0 A4 4 0 0 A21 Osage 1 0 0 A22 2 0 0 A23 3 0 0 A24 4 0 0 A25 Miami 1 0 0 A26 2 1 0 A27 3 0 0 A28 4 0 0 B25 Tonto 1 0 0 B26 2 0 0 B27 3 0 0 B28 4 0 0 5/17/2005 Rep. 1-Dry C17 Biloxi 1 0 0 C18 2 0 0 C19 3 1 0 C20 4 0 0 C9 Osage 1 0 0 C10 2 0 0 C11 3 0 0 C12 4 0 0 D9 Miami 1 0 0

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66 D10 2 0 0 D11 3 0 0 D12 4 0 0 C13 Tonto 1 0 0 C14 2 0 0 C15 3 0 0 C16 4 0 0 5/18/2005 Rep. 2-MistedE9 Biloxi 1 1 0 E10 2 0 0 E11 3 0 0 E12 4 0 0 F21 Osage 1 0 0 F22 2 0 0 F23 3 0 0 F24 4 0 0 E13 Miami 1 1 0 E14 2 0 0 E15 3 0 0 E16 4 0 0 F5 Tonto 1 0 0 F6 2 0 0 F7 3 0 0 F8 4 0 0 5/19/2005 Rep. 2-Dry H17 Biloxi 1 0 0 H18 2 0 0 H19 3 0 0 H20 4 0 0 H21 Osage 1 0 0 H22 2 0 0 H23 3 0 0 H24 4 0 0 H9 Miami 1 0 0 H10 2 0 0 H11 3 0 0 H12 4 1 0 H25 Tonto 1 0 0 H26 2 0 0 H27 3 0 0 H28 4 0 0 5/23/2005 3 Rep. 1-MistedA1 Biloxi 1 0 0 A2 2 0 0 A3 3 1 0 A4 4 0 0 A21 Osage 1 0 0 A22 2 1 0 A23 3 0 0 A24 4 0 0 A25 Miami 1 0 0

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67 A26 2 0 0 A27 3 0 0 A28 4 0 0 B25 Tonto 1 0 0 B26 2 0 0 B27 3 0 0 B28 4 0 0 5/24/2005 Rep. 1-Dry C17 Biloxi 1 0 0 C18 2 0 0 C19 3 0 0 C20 4 0 0 C9 Osage 1 0 0 C10 2 0 0 C11 3 0 0 C12 4 0 0 D9 Miami 1 0 0 D10 2 0 0 D11 3 0 0 D12 4 0 0 C13 Tonto 1 0 0 C14 2 0 0 C15 3 1 0 C16 4 1 0 5/25/2005 Rep. 2-MistedE9 Biloxi 1 0 0 E10 2 1 0 E11 3 0 0 E12 4 0 0 F21 Osage 1 1 0 F22 2 0 0 F23 3 0 0 F24 4 0 0 E13 Miami 1 0 0 E14 2 0 0 E15 3 0 0 E16 4 0 0 F5 Tonto 1 2 0 F6 2 1 0 F7 3 0 0 F8 4 0 0 5/26/2005 Rep. 2-Dry H17 Biloxi 1 0 0 H18 2 0 0 H19 3 0 0 H20 4 0 0 H21 Osage 1 0 0 H22 2 0 0 H23 3 0 0 H24 4 0 0 H9 Miami 1 0 0

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68 H10 2 0 0 H11 3 0 0 H12 4 0 0 H25 Tonto 1 0 0 H26 2 0 0 H27 3 0 0 H28 4 0 0 5/30/2005 4 Rep. 1-MistedA1 Biloxi 1 2 0 A2 2 4 0 A3 3 2 0 A4 4 0 0 A21 Osage 1 0 0 A22 2 0 0 A23 3 0 0 A24 4 0 0 A25 Miami 1 0 0 A26 2 2 0 A27 3 0 0 A28 4 0 0 B25 Tonto 1 0 0 B26 2 0 0 B27 3 1 0 B28 4 0 0 5/31/2005 Rep. 1-Dry C17 Biloxi 1 0 0 C18 2 0 0 C19 3 0 0 C20 4 0 0 C9 Osage 1 0 0 C10 2 1 0 C11 3 0 0 C12 4 0 0 D9 Miami 1 0 0 D10 2 0 0 D11 3 0 0 D12 4 0 0 C13 Tonto 1 0 0 C14 2 0 0 C15 3 0 0 C16 4 0 0 6/1/2005 Rep. 2-MistedE9 Biloxi 1 0 0 E10 2 0 0 E11 3 0 0 E12 4 0 0 F21 Osage 1 0 0 F22 2 0 0 F23 3 0 0 F24 4 0 0 E13 Miami 1 0 0

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69 E14 2 0 0 E15 3 0 0 E16 4 0 0 F5 Tonto 1 0 0 F6 2 0 0 F7 3 1 0 F8 4 0 0 6/2/2005 Rep. 2-Dry H17 Biloxi 1 0 0 H18 2 1 0 H19 3 0 0 H20 4 0 0 H21 Osage 1 0 0 H22 2 0 0 H23 3 0 0 H24 4 0 0 H9 Miami 1 0 0 H10 2 0 0 H11 3 0 0 H12 4 0 0 H25 Tonto 1 0 0 H26 2 0 0 H27 3 1 0 H28 4 0 0 6/6/2005 5 Rep. 1-MistedA1 Biloxi 1 9 0 A2 2 11 0 A3 3 7 0 A4 4 6 0 A21 Osage 1 2 0 A22 2 1 0 A23 3 5 0 A24 4 6 0 A25 Miami 1 4 0 A26 2 5 0 A27 3 4 0 A28 4 1 0 B25 Tonto 1 3 0 B26 2 9 0 B27 3 9 0 B28 4 4 0 6/7/2005 Rep. 1-Dry C17 Biloxi 1 4 0 C18 2 1 0 C19 3 4 0 C20 4 6 0 C9 Osage 1 8 0 C10 2 6 0 C11 3 7 0 C12 4 4 0 D9 Miami 1 5 0

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70 D10 2 0 0 D11 3 3 0 D12 4 1 0 C13 Tonto 1 0 0 C14 2 4 0 C15 3 4 0 C16 4 5 0 6/8/2005 Rep. 2-MistedE9 Biloxi 1 3 0 E10 2 6 0 E11 3 8 0 E12 4 1 0 F21 Osage 1 2 0 F22 2 4 0 F23 3 3 0 F24 4 10 0 E13 Miami 1 0 0 E14 2 4 0 E15 3 1 0 E16 4 2 0 F5 Tonto 1 4 0 F6 2 5 0 F7 3 9 0 F8 4 5 0 6/9/2005 Rep. 2-Dry H17 Biloxi 1 2 0 H18 2 2 0 H19 3 2 0 H20 4 5 0 H21 Osage 1 2 0 H22 2 3 0 H23 3 3 0 H24 4 6 0 H9 Miami 1 3 0 H10 2 3 0 H11 3 6 0 H12 4 3 0 H25 Tonto 1 10 0 H26 2 0 0 H27 3 11 0 H28 4 6 0 6/13/2005 6 Rep. 1-MistedA1 Biloxi 1 44 0 A2 2 41 0 A3 3 39 0 A4 4 39 0 A21 Osage 1 13 0 A22 2 16 0 A23 3 11 0 A24 4 12 0 A25 Miami 1 11 0

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71 A26 2 7 0 A27 3 8 0 A28 4 14 0 B25 Tonto 1 10 0 B26 2 10 0 B27 3 13 0 B28 4 16 0 6/14/2005 Rep. 1-Dry C17 Biloxi 1 0 0 C18 2 3 0 C19 3 3 0 C20 4 3 0 C9 Osage 1 1 0 C10 2 7 0 C11 3 3 0 C12 4 1 0 D9 Miami 1 2 0 D10 2 2 0 D11 3 4 0 D12 4 4 0 C13 Tonto 1 0 0 C14 2 3 0 C15 3 4 0 C16 4 4 0 6/15/2005 Rep. 2-MistedE9 Biloxi 1 14 0 E10 2 12 0 E11 3 13 0 E12 4 19 0 F21 Osage 1 10 0 F22 2 9 0 F23 3 6 0 F24 4 15 0 E13 Miami 1 10 0 E14 2 9 0 E15 3 8 0 E16 4 14 0 F5 Tonto 1 17 0 F6 2 18 0 F7 3 17 0 F8 4 13 0 6/16/2005 Rep. 2-Dry H17 Biloxi 1 7 0 H18 2 15 0 H19 3 8 0 H20 4 6 0 H21 Osage 1 10 0 H22 2 10 0 H23 3 9 0 H24 4 9 0 H9 Miami 1 4 0

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72 H10 2 8 0 H11 3 9 0 H12 4 8 0 H25 Tonto 1 9 0 H26 2 11 0 H27 3 10 0 H28 4 20 0 6/20/2005 7 Rep. 1-MistedA1 Biloxi 1 84 0 A2 2 59 0 A3 3 50 0 A4 4 49 0 A21 Osage 1 16 0 A22 2 14 0 A23 3 13 0 A24 4 16 0 A25 Miami 1 12 0 A26 2 8 0 A27 3 11 0 A28 4 14 0 B25 Tonto 1 15 0 B26 2 12 0 B27 3 13 0 B28 4 16 0 6/21/2005 Rep. 1-Dry C17 Biloxi 1 5 0 C18 2 6 0 C19 3 5 0 C20 4 8 0 C9 Osage 1 8 0 C10 2 7 0 C11 3 7 0 C12 4 4 0 D9 Miami 1 4 0 D10 2 5 0 D11 3 4 0 D12 4 3 0 C13 Tonto 1 2 0 C14 2 5 0 C15 3 5 0 C16 4 4 0 6/22/2005 Rep. 2-MistedE9 Biloxi 1 29 0 E10 2 23 0 E11 3 23 0 E12 4 17 0 F21 Osage 1 13 0 F22 2 7 0 F23 3 8 0 F24 4 16 0 E13 Miami 1 12 0

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73 E14 2 18 0 E15 3 13 0 E16 4 16 0 F5 Tonto 1 19 1 F6 2 17 0 F7 3 16 0 F8 4 12 0 6/23/2005 Rep. 2-Dry H17 Biloxi 1 5 0 H18 2 9 0 H19 3 6 0 H20 4 6 0 H21 Osage 1 6 0 H22 2 7 0 H23 3 10 0 H24 4 9 0 H9 Miami 1 8 0 H10 2 4 0 H11 3 5 0 H12 4 8 0 H25 Tonto 1 4 0 H26 2 3 0 H27 3 6 0 H28 4 9 1 6/27/2005 8 Rep. 1-MistedA1 Biloxi 1 82 0 A2 2 89 0 A3 3 65 0 A4 4 40 0 A21 Osage 1 16 0 A22 2 12 0 A23 3 22 0 A24 4 21 0 A25 Miami 1 15 0 A26 2 17 0 A27 3 17 0 A28 4 18 0 B25 Tonto 1 14 0 B26 2 16 0 B27 3 13 0 B28 4 12 0 6/28/2005 Rep. 1-Dry C17 Biloxi 1 2 0 C18 2 3 0 C19 3 3 0 C20 4 3 0 C9 Osage 1 4 0 C10 2 6 0 C11 3 6 0 C12 4 2 0 D9 Miami 1 2 0

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74 D10 2 1 0 D11 3 5 0 D12 4 1 0 C13 Tonto 1 5 0 C14 2 1 0 C15 3 5 0 C16 4 3 0 6/29/2005 Rep. 2-MistedE9 Biloxi 1 7 0 E10 2 2 0 E11 3 5 0 E12 4 7 0 F21 Osage 1 9 0 F22 2 5 0 F23 3 10 0 F24 4 7 1 E13 Miami 1 4 0 E14 2 9 0 E15 3 5 0 E16 4 5 0 F5 Tonto 1 7 1 F6 2 7 0 F7 3 6 0 F8 4 8 0 6/30/2005 Rep. 2-Dry H17 Biloxi 1 6 0 H18 2 8 0 H19 3 4 0 H20 4 4 0 H21 Osage 1 5 0 H22 2 5 0 H23 3 7 0 H24 4 6 0 H9 Miami 1 9 0 H10 2 6 0 H11 3 10 0 H12 4 6 0 H25 Tonto 1 2 0 H26 2 5 0 H27 3 5 1 H28 4 8 1 7/5/2005 9 Rep. 1-MistedA1 Biloxi 1 51 0 A2 2 50 0 A3 3 40 0 A4 4 35 0 A21 Osage 1 15 0 A22 2 14 0 A23 3 17 0 A24 4 13 0 A25 Miami 1 9 0

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75 A26 2 12 0 A27 3 8 0 A28 4 13 0 B25 Tonto 1 11 0 B26 2 13 0 B27 3 11 0 B28 4 17 0 7/5/2005 Rep. 1-Dry C17 Biloxi 1 6 0 C18 2 2 0 C19 3 1 0 C20 4 4 0 C9 Osage 1 6 0 C10 2 7 0 C11 3 5 0 C12 4 4 0 D9 Miami 1 3 0 D10 2 6 0 D11 3 8 0 D12 4 4 0 C13 Tonto 1 2 0 C14 2 1 0 C15 3 1 0 C16 4 2 0 7/6/2005 Rep. 2-MistedE9 Biloxi 1 7 1 E10 2 10 0 E11 3 9 0 E12 4 9 0 F21 Osage 1 8 0 F22 2 7 0 F23 3 5 0 F24 4 8 0 E13 Miami 1 7 0 E14 2 8 0 E15 3 6 0 E16 4 5 0 F5 Tonto 1 12 0 F6 2 7 0 F7 3 13 0 F8 4 8 0 7/7/2005 Rep. 2-Dry H17 Biloxi 1 3 0 H18 2 4 0 H19 3 9 0 H20 4 7 0 H21 Osage 1 8 0 H22 2 7 0 H23 3 7 0 H24 4 7 0 H9 Miami 1 3 0

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76 H10 2 9 0 H11 3 6 0 H12 4 7 0 H25 Tonto 1 5 0 H26 2 5 0 H27 3 4 1 H28 4 10 1 7/12/2005 10 Rep. 1-MistedA1 Biloxi 1 48 0 A2 2 55 0 A3 3 37 0 A4 4 26 0 A21 Osage 1 6 0 A22 2 7 0 A23 3 6 1 A24 4 8 0 A25 Miami 1 3 0 A26 2 8 0 A27 3 8 0 A28 4 10 0 B25 Tonto 1 2 0 B26 2 4 0 B27 3 10 0 B28 4 13 0 7/13/2005 Rep. 1-Dry C17 Biloxi 1 3 0 C18 2 3 0 C19 3 2 0 C20 4 4 0 C9 Osage 1 11 0 C10 2 8 0 C11 3 6 0 C12 4 6 0 D9 Miami 1 3 0 D10 2 2 0 D11 3 3 0 D12 4 2 0 C13 Tonto 1 2 0 C14 2 1 0 C15 3 1 0 C16 4 1 0 7/14/2005 Rep. 2-MistedE9 Biloxi 1 9 0 E10 2 7 0 E11 3 7 0 E12 4 4 0 F21 Osage 1 4 1 F22 2 9 0 F23 3 7 0 F24 4 11 0 E13 Miami 1 4 0

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77 E14 2 7 0 E15 3 3 0 E16 4 4 0 F5 Tonto 1 13 1 F6 2 15 0 F7 3 10 1 F8 4 8 0 7/15/2005 Rep. 2-Dry H17 Biloxi 1 3 0 H18 2 2 0 H19 3 2 0 H20 4 4 0 H21 Osage 1 7 0 H22 2 3 0 H23 3 3 0 H24 4 9 0 H9 Miami 1 0 0 H10 2 4 0 H11 3 6 0 H12 4 2 0 H25 Tonto 1 6 0 H26 2 4 0 H27 3 8 0 H28 4 11 2 7/18/2005 11 Rep. 1-MistedA1 Biloxi 1 48 0 A2 2 48 0 A3 3 17 1 A4 4 15 0 A21 Osage 1 12 0 A22 2 9 0 A23 3 8 1 A24 4 13 0 A25 Miami 1 8 0 A26 2 6 1 A27 3 20 0 A28 4 15 1 B25 Tonto 1 21 0 B26 2 13 0 B27 3 14 0 B28 4 22 0 7/20/2005 Rep. 1-Dry C17 Biloxi 1 2 0 C18 2 1 0 C19 3 3 0 C20 4 4 0 C9 Osage 1 4 0 C10 2 3 0 C11 3 6 0 C12 4 3 0 D9 Miami 1 4 0

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78 D10 2 5 0 D11 3 6 0 D12 4 3 0 C13 Tonto 1 1 0 C14 2 4 0 C15 3 2 0 C16 4 3 0 7/22/2005 Rep. 2-MistedE9 Biloxi 1 3 0 E10 2 3 0 E11 3 3 0 E12 4 4 0 F21 Osage 1 4 1 F22 2 7 0 F23 3 5 1 F24 4 7 0 E13 Miami 1 1 0 E14 2 1 0 E15 3 3 0 E16 4 2 0 F5 Tonto 1 6 0 F6 2 5 1 F7 3 10 2 F8 4 9 0 7/23/2005 Rep. 2-Dry H17 Biloxi 1 3 0 H18 2 3 0 H19 3 2 0 H20 4 11 0 H21 Osage 1 5 0 H22 2 9 0 H23 3 6 0 H24 4 7 0 H9 Miami 1 3 0 H10 2 3 0 H11 3 6 0 H12 4 8 0 H25 Tonto 1 12 0 H26 2 13 0 H27 3 9 0 H28 4 23 2 7/29/2005 12 Rep. 1-MistedA1 Biloxi 1 41 0 A2 2 14 0 A3 3 13 1 A4 4 11 0 A21 Osage 1 11 0 A22 2 11 0 A23 3 11 1 A24 4 4 0 A25 Miami 1 6 0

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79 A26 2 14 0 A27 3 10 1 A28 4 5 2 B25 Tonto 1 13 0 B26 2 5 0 B27 3 7 0 B28 4 11 0 Rep. 1-Dry C17 Biloxi 1 3 0 C18 2 3 0 C19 3 5 0 C20 4 9 0 C9 Osage 1 7 0 C10 2 7 0 C11 3 6 0 C12 4 5 0 D9 Miami 1 6 0 D10 2 8 0 D11 3 10 0 D12 4 5 0 C13 Tonto 1 5 0 C14 2 3 0 C15 3 3 0 C16 4 4 0 Rep. 2-MistedE9 Biloxi 1 3 0 E10 2 2 0 E11 3 7 0 E12 4 3 0 F21 Osage 1 3 1 F22 2 4 0 F23 3 7 1 F24 4 8 0 E13 Miami 1 10 0 E14 2 7 0 E15 3 2 0 E16 4 3 0 F5 Tonto 1 6 0 F6 2 3 1 F7 3 5 2 F8 4 14 0 Rep. 2-Dry H17 Biloxi 1 12 0 H18 2 18 0 H19 3 10 0 H20 4 16 0 H21 Osage 1 17 0 H22 2 16 0 H23 3 14 0 H24 4 12 0 H9 Miami 1 6 0

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80 H10 2 4 0 H11 3 6 0 H12 4 5 0 H25 Tonto 1 26 0 H26 2 24 0 H27 3 18 1 H28 4 40 1 8/2/2005 13 Rep. 1-MistedA1 Biloxi 1 15 0 A2 2 8 0 A3 3 20 1 A4 4 9 1 A21 Osage 1 3 0 A22 2 10 0 A23 3 6 1 A24 4 9 0 A25 Miami 1 14 0 A26 2 15 0 A27 3 9 2 A28 4 13 2 B25 Tonto 1 9 0 B26 2 5 0 B27 3 11 0 B28 4 5 0 Rep. 1-Dry C17 Biloxi 1 5 0 C18 2 4 1 C19 3 4 0 C20 4 11 0 C9 Osage 1 6 0 C10 2 6 0 C11 3 3 0 C12 4 7 0 D9 Miami 1 12 0 D10 2 14 0 D11 3 12 0 D12 4 7 0 C13 Tonto 1 5 0 C14 2 1 0 C15 3 6 0 C16 4 3 0 Rep. 2-MistedE9 Biloxi 1 4 0 E10 2 1 0 E11 3 4 0 E12 4 4 0 F21 Osage 1 5 1 F22 2 6 0 F23 3 6 1 F24 4 2 1 E13 Miami 1 4 0

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81 E14 2 3 0 E15 3 3 0 E16 4 7 0 F5 Tonto 1 8 0 F6 2 3 1 F7 3 9 2 F8 4 4 0 Rep. 2-Dry H17 Biloxi 1 8 0 H18 2 12 0 H19 3 9 0 H20 4 12 0 H21 Osage 1 15 0 H22 2 10 0 H23 3 10 0 H24 4 12 0 H9 Miami 1 5 0 H10 2 12 0 H11 3 13 0 H12 4 5 0 H25 Tonto 1 20 0 H26 2 13 0 H27 3 14 1 H28 4 36 1 8/18/2005 15 Rep. 1-MistedA1 Biloxi 1 11 0 A2 2 10 0 A3 3 12 2 A4 4 14 0 A21 Osage 1 10 0 A22 2 17 0 A23 3 26 2 A24 4 20 0 A25 Miami 1 19 0 A26 2 13 1 A27 3 18 2 A28 4 17 2 B25 Tonto 1 8 0 B26 2 6 0 B27 3 6 0 B28 4 8 0 Rep. 1-Dry C17 Biloxi 1 4 0 C18 2 8 0 C19 3 4 0 C20 4 6 0 C9 Osage 1 9 0 C10 2 6 0 C11 3 4 0 C12 4 11 0 D9 Miami 1 5 0

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82 D10 2 11 0 D11 3 7 0 D12 4 7 0 C13 Tonto 1 3 0 C14 2 1 0 C15 3 2 0 C16 4 3 0 Rep. 2-MistedE9 Biloxi 1 4 0 E10 2 3 0 E11 3 4 0 E12 4 1 0 F21 Osage 1 6 1 F22 2 8 0 F23 3 4 2 F24 4 9 1 E13 Miami 1 6 0 E14 2 11 0 E15 3 6 0 E16 4 9 0 F5 Tonto 1 2 0 F6 2 0 0 F7 3 2 3 F8 4 1 0 Rep. 2-Dry H17 Biloxi 1 9 0 H18 2 11 0 H19 3 6 0 H20 4 9 0 H21 Osage 1 5 0 H22 2 4 0 H23 3 4 0 H24 4 4 0 H9 Miami 1 4 0 H10 2 7 0 H11 3 6 0 H12 4 4 0 H25 Tonto 1 3 0 H26 2 2 0 H27 3 0 1 H28 4 10 0 8/24/2005 16 Rep. 1-MistedA1 Biloxi 1 7 0 A2 2 12 0 A3 3 9 2 A4 4 11 0 A21 Osage 1 16 1 A22 2 15 0 A23 3 27 2 A24 4 24 0 A25 Miami 1 13 0

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83 A26 2 5 1 A27 3 6 2 A28 4 11 2 B25 Tonto 1 2 0 B26 2 2 1 B27 3 1 0 B28 4 2 0 Rep. 1-Dry C17 Biloxi 1 1 0 C18 2 5 0 C19 3 2 0 C20 4 4 0 C9 Osage 1 2 0 C10 2 4 0 C11 3 2 0 C12 4 2 0 D9 Miami 1 7 0 D10 2 3 0 D11 3 3 0 D12 4 3 0 C13 Tonto 1 1 0 C14 2 2 0 C15 3 1 0 C16 4 1 0 Rep. 2-MistedE9 Biloxi 1 1 0 E10 2 0 0 E11 3 1 0 E12 4 1 0 F21 Osage 1 4 1 F22 2 3 0 F23 3 8 2 F24 4 6 1 E13 Miami 1 8 0 E14 2 6 0 E15 3 4 0 E16 4 4 0 F5 Tonto 1 2 0 F6 2 1 0 F7 3 0 3 F8 4 0 1 Rep. 2-Dry H17 Biloxi 1 2 0 H18 2 1 0 H19 3 4 0 H20 4 5 0 H21 Osage 1 4 0 H22 2 1 0 H23 3 2 0 H24 4 6 0 H9 Miami 1 4 0

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84 H10 2 4 0 H11 3 6 0 H12 4 4 0 H25 Tonto 1 2 0 H26 2 1 0 H27 3 0 1 H28 4 7 0 9/7/2005 18 Rep. 1-MistedA1 Biloxi 1 21 0 A2 2 20 2 A3 3 19 2 A4 4 10 0 A21 Osage 1 18 1 A22 2 16 0 A23 3 25 2 A24 4 17 0 A25 Miami 1 13 0 A26 2 9 1 A27 3 0 1 A28 4 5 1 B25 Tonto 1 2 0 B26 2 0 1 B27 3 1 0 B28 4 2 0 Rep. 1-Dry C17 Biloxi 1 5 0 C18 2 3 0 C19 3 0 0 C20 4 4 0 C9 Osage 1 8 0 C10 2 2 0 C11 3 4 0 C12 4 2 0 D9 Miami 1 3 0 D10 2 3 0 D11 3 1 0 D12 4 4 0 C13 Tonto 1 0 0 C14 2 2 0 C15 3 3 0 C16 4 2 0 Rep. 2-MistedE9 Biloxi 1 1 0 E10 2 0 0 E11 3 1 0 E12 4 0 0 F21 Osage 1 2 1 F22 2 2 0 F23 3 2 1 F24 4 2 1 E13 Miami 1 2 0

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85 E14 2 0 0 E15 3 2 0 E16 4 5 0 F5 Tonto 1 1 0 F6 2 0 0 F7 3 1 3 F8 4 0 1 Rep. 2-Dry H17 Biloxi 1 0 0 H18 2 3 0 H19 3 1 0 H20 4 1 0 H21 Osage 1 3 1 H22 2 4 0 H23 3 1 0 H24 4 3 0 H9 Miami 1 6 0 H10 2 4 0 H11 3 8 0 H12 4 4 0 H25 Tonto 1 5 0 H26 2 1 0 H27 3 4 3 H28 4 1 1 9/14/2005 19 Rep. 1-MistedA1 Biloxi 1 27 1 A2 2 25 2 A3 3 24 2 A4 4 13 1 A21 Osage 1 8 1 A22 2 10 0 A23 3 8 2 A24 4 13 0 A25 Miami 1 9 0 A26 2 9 1 A27 3 3 1 A28 4 3 1 B25 Tonto 1 1 0 B26 2 1 1 B27 3 1 0 B28 4 0 0 Rep. 1-Dry C17 Biloxi 1 0 0 C18 2 3 0 C19 3 3 0 C20 4 2 0 C9 Osage 1 0 0 C10 2 7 0 C11 3 3 1 C12 4 1 0 D9 Miami 1 5 0

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86 D10 2 4 0 D11 3 6 0 D12 4 2 1 C13 Tonto 1 0 0 C14 2 1 0 C15 3 2 0 C16 4 2 0 Rep. 2-MistedE9 Biloxi 1 1 0 E10 2 1 0 E11 3 1 0 E12 4 0 0 F21 Osage 1 4 1 F22 2 4 0 F23 3 2 1 F24 4 4 1 E13 Miami 1 9 0 E14 2 1 0 E15 3 2 0 E16 4 5 0 F5 Tonto 1 0 0 F6 2 0 0 F7 3 2 3 F8 4 1 1 Rep. 2-Dry H17 Biloxi 1 3 0 H18 2 3 0 H19 3 2 0 H20 4 2 0 H21 Osage 1 0 2 H22 2 3 0 H23 3 2 0 H24 4 1 0 H9 Miami 1 1 0 H10 2 2 0 H11 3 11 0 H12 4 1 0 H25 Tonto 1 1 0 H26 2 0 0 H27 3 3 3 H28 4 0 0 9/21/2005 20 Rep. 1-MistedA1 Biloxi 1 12 A2 2 8 A3 3 5 A4 4 8 A21 Osage 1 5 A22 2 4 A23 3 3 A24 4 10 A25 Miami 1 4

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87 A26 2 6 A27 3 1 A28 4 4 B25 Tonto 1 1 B26 2 2 B27 3 0 B28 4 1 Rep. 1-Dry C17 Biloxi 1 0 C18 2 1 C19 3 2 C20 4 2 C9 Osage 1 3 C10 2 4 C11 3 5 C12 4 1 D9 Miami 1 2 D10 2 3 D11 3 0 D12 4 2 C13 Tonto 1 2 C14 2 0 C15 3 2 C16 4 3 Rep. 2-MistedE9 Biloxi 1 2 E10 2 1 E11 3 0 E12 4 1 F21 Osage 1 0 F22 2 2 F23 3 2 F24 4 2 E13 Miami 1 2 E14 2 1 E15 3 3 E16 4 4 F5 Tonto 1 5 F6 2 2 F7 3 3 F8 4 3 Rep. 2-Dry H17 Biloxi 1 0 H18 2 3 H19 3 0 H20 4 1 H21 Osage 1 2 H22 2 3 H23 3 4 H24 4 6 H9 Miami 1 4

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88 H10 2 2 H11 3 6 H12 4 1 H25 Tonto 1 0 H26 2 0 H27 3 1 H28 4 1

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89 LIST OF REFERENCES Alderz, W. C., Hopkins, D. L. 1979. Natural in fectivity of two sharpshooter vectors of Pierce’s disease of grape in Florida. Journal of Economic Entomology 72: 19171919. Alderz, W. C. 1980. Ecological observations on two leafhoppers that transmit the Pierce’s disease bacteria. Proceedings of the annual meeting of the Florida State Horticultural Society 93: 115-120. Almeida, R.P.P., Purcell, A.H. 2003. Transmission of Xylella fastidiosa to grapevines by Homalodisca coagulata (Hemiptera: Cicadellidae). Journal of Economic Entomology 96(2): 264-271. Al-Wahaibi, A.K., A.K. Owen and J.G. Morse. 2005. Description and behavioral biology of two Ufens species (Hympenoptera: Trichogr ammatidae), egg parasitoids of Homalodisca species (Hemiptera: Cicadellidae) in Southern California. Bull. Entomol. Res. 95: 275-288. Al-Wahaibi, A.K., Morse, J.G. 2003. Homalodisca coagulata (Hemiptera: Cicadellidae) embryonic development at constant temperat ures. Florida Entomologist 86(4): 477478. Andersen, P.C., Brodbeck, B.V., Mizell, R.F. III. 1989. Metabolism of amino acids, organic acids and sugars extracted from the xylem fluid of four host plants by adult Homalodisca coagulata Entomologia Experimentalis et Applicata 50: 149-159. Andersen, P. C., Brodbeck, B. V., Mizell R. F. III. 1992. Feeding by the leafhopper, Homalodisca coagulata in relation to xylem fluid chemistry and tension. Journal of Insect Physiology 38: 611-622. Andersen, P.C., Brodbeck, B.V., Mizell III, R.F. 2003 Plant and insect characteristics in response to increasing density of Homalodisca coagulata on three host species: a quantification of assimilate extraction. Entomologia Experimentalis et Applicata 107: 57-68 Araujo, W. L., Marcon, J., Maccheroni, W., va n Elsas, J. D., van Vuurde, J. W. L., Azevedo, J. L. 2002. Diversity of endophy tic bacterial populat ions and their interaction with Xylella fastidiosa in citrus plants. A pplied and Environmental Microbiology 68: 4906-4914.

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90 Azevedo-Filho, W. S., Carvalho, G. S. 2005 Brochosomes for eggs of the Proconiini (Hemiptera: Cicadellidae, Cicadellinae) sp ecies associated with orchards of Citrus sinensis (L.) Osbeck in Rio Grande do Sul, Brazil. Neotropical Entomology 34 (3): 387-394. Backus, E. 1985. Anatomical and Sensory Mechanisms of Planthopper and Leafhopper Feeding Behavior. In: Nault, L.R., Rodr iguez, J.G. (Eds.), The Leafhoppers and Planthoppers. John Wiley & Sons, New York, pp. 165. Ball, J. C. 1979. Seasonal patterns of activ ity of adult leafhopper vectors of phony peach disease in north Florida. Envi ronmental Entomology. 8: 686-689. Ben-Ze’ev, I., Kenneth, R.G. 1981. Zoopthora radicans and Zoopthora petchi sp. nov. (Zygomycetes: Entomopthorales), two species of the “ Sphaerosperma group” attacking leafhoppers and froghoppers ( Hom. ) Entomophaga 26 (2): 131-142. Bernays, E. A., Bright, K. L. 1993. Mechan isms of dietary mixing in grasshoppers: a review. Comparative Biochemistry and Physiology 104A: 11-19. Bethke, J. A., Campbell, K. A., Blua, M. J., Redak, R. A., Yanega, D. A. 2001. Range extension of Pseneo punctatus Fox and notes on predation of an introduced sharpshooter, Homalodisca coagulata (Say). Pan-Pacific Entomologist 77: 54-56. Bidochka, M.J., Walsh, S.R.A., Ramos, M.E., St. Leger, R.J., Carruthers, R.I., Silver, J.C., Roberts, D.W. 1997. Cloned DNA probes distinguish endemic and exotic Entomophaga grylli fungal pathotype infections in grasshopper life stages. Molecular Ecology 6: 303-308. Blua, M. J., Phillips, P. A., Redak, R. A. 1999. A new sharpshooter threatens both crops and ornamentals. California Agriculture 53: 22-23. Blua, M. J., and D. J. W. Morgan. 2003b. Dispersion of Homalodisca coagulata (Hemiptera: Cicadellidae), a vector of Xylella fastidiosa into vineyards in southern California. Journal of Ec onomic Entomology 96: 1369-1374. Boucias, D.G., Scharf, D.W., Breaux, S. E., Purcell, D.H., Mizell, R.F. IIIP. Studies on the Fungi Associated with the Glassy-Winged Sharpshooter Homalodisca coagulata with Emphasis on a New Species Hirsutella homalodiscae sp. nov. Biological Control, submitted. Brlansky, R. H., Timmer, L. W., French, W. J., McCoy, R. E. 1983. Colonization of the sharpshooter vectors, Oncometopia nigricans and Homalodisca coagulata by xylem-limited bacteria. Phytopathology 73: 530-535. Brodbeck, B. V., Andersen, P. C., Mizell, R. F. III. 1995. Differenti al utilization of nutrients during development by the xylophagous leafhopper, Homalodisca coagulata. Entomologia Experimentalis et Applicata 75: 279-289.

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98 BIOGRAPHICAL SKETCH Samuel Elie Breaux was born in St. Fr ancisville, LA on July 29, 1979, but moved to Grayton Beach, FL at the age of seven. He graduated from Fort Walton Beach High School in 1997 and enrolled in the University of Florida as an English major in the same year. After one year and the completion of a particularly influentia l class on the biology of fireflies, he chose to pursue a B.S. in entomology. For the majority of time spent in fulfillment of this degree, he also worked in the Fire Ant Biocontrol Laboratory at the USDA-CMAVE under Drs. David Oi and Da vid Williams. Upon graduation in 2001, he spent 2 years teaching conversational English in Japan before being drawn back to the University of Florida and insect pathology. In 2003, he enrolled in an M.S. program in insect pathology to study the fungal pathoge ns associated with the glassy-winged sharpshooter in northern Florid a and southern Georgia.


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Title: Fungi Associated with the Glassy Winged Sharpshooter, Homalodisca coagulata, in Its Native Range
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Permanent Link: http://ufdc.ufl.edu/UFE0013363/00001

Material Information

Title: Fungi Associated with the Glassy Winged Sharpshooter, Homalodisca coagulata, in Its Native Range
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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FUNGI ASSOCIATED WITH THE GLASSY-WINGED SHARPSHOOTER,
Homalodisca coagulata, IN ITS NATIVE RANGE
















By

S. ELIE BREAUX


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

S. Elie Breaux

































This document is dedicated to Stefanie, always there.















ACKNOWLEDGMENTS

I would like to thank the members of my committee for their support, perseverance,

and knowledge. I consider myself lucky to have found in them the willingness to take a

chance on a student. I would like to thank Dr. Linda Young for extensive assistance in the

statistical analysis portion of this study.

I would also like to thank my family. My father has always been a student of

nature. Raised with his love of the outdoors, the choice to take this path was made

without reservation. My mother has always provided every kind of support a son could

ask for, free of expectation or judgment. I thank Nicholas and Silas for being so

entertaining. They are so different in nature, but time spent with either of them makes one

realize what is important. And finally, I would like to thank Stefanie. Always generous

with encouragement and unwavering in support, there is no way I could have done this

without her.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F TA B L E S ......... .. .... ...... ............ ... ............. ... .. .. .. .............. .. vii

LIST OF FIGURES ............. ................... ................. ................ viii

ABSTRACT .............. .................. .......... .............. ix

CHAPTER

1 LITERATURE REVIEW ............................................................ ................ 1

H om alodisca coagulata ........................................... ....... .................... ................
Feeding Behavior and Nutritional Ecology of Homalodisca coagulata .................5
Homalodisca coagulata as a Disease Vector.......................................................
Homalodisca coagulata Vector Capacity ...................... ..................
Xylella fastidiosa ............................. ..... ................. .... ........... .. 8
Potential N natural Control A gents....................................................... ............... 10
Parasites and Predators ....................................................... .. ... .. .. ............ 10
Fungal Pathogens of Auchenorrhyncha........ ........... .....................11
Homalodisca coagulata -Associated Fungi .............................................. 13

2 IN TR O D U C T IO N .............................................................................. ............... 20

3 M ATERIALS AND M ETHODS ........................................ ......................... 22

Homalodisca coagulata/Hirsutella homalodiscae Interactions in a Crape Myrtle
Field Plot............................................................ .. ... ......... 22
D description of Study Site.......................................................... ............... 22
Sam pling Program .............................................. ........ .. ............ 22
Sticky T rap G rid ......... ......................................... ............. ....... ... 24
Diagnostic Bleeds ................. ................. ........... .... ........ .. ............25
Sam pling Program .............................................. ........ .. ............ 25
B leed T ech n iqu e ................................................................. ............ .... 2 5
Statistical A n aly sis............ .................................................................. ........ .. ....... .. 2 6

4 R E S U L T S .............................................................................2 8




v









5 D ISC U S SIO N ............................................................................... 4 1

6 CON CLU SION S ....................................... ... ......... ........ ..... ......47

APPENDIX

A SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF
LIVE GWSS POPULATIONS AND LEAST MEANS SQUARED ANALYSIS
OF SIGNIFICANT EFFECTS .............................................................................48

B SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF
CADAVER INCIDENCE AND LEAST MEANS SQUARES ANALYSIS OF
SIG N IFIC A N T E FFE C T S....................................... .............................................50

C SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF
TRAP POPULATION DATA AND LEAST MEANS SQAURES ANALYSIS
OF THE EFFECT OF TIME .............. ... ........ .......................52

D SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF
ARCSINE TRANSFORMED LIVE GWSS/CADAVER PROPORTIONS WITH
LEAST MEANS SQUARE ANALYSIS OF SIGNIFICANT EFFECTS .................53

E QUINCY NFREC WEATHER DATA FOR 2004 AND 2005..............................55

F RAW DATA FROM 2004 AND 2004 FIELD STUDIES .............................57

2004 F ield P lot D ata ....... .. .. ...... .. ...... ........................ ....................57
2005 Field Plot Data ....... .. .................................. ................................. 64

LIST OF REFEREN CES ..................................................................... ............... 91

B IO G R A PH IC A L SK E TCH ..................................................................... ..................98
















LIST OF TABLES

Table p

1 Comparison of 2004 and 2005 GWSS populations in field plot for same trees on
sam e w eek betw een years. .............................................. ............................. 28

2 Weeks with significantly higher trap catches of GWSS in 2005...........................38

3 Significant differences in live GWSS numbers between cultivars with respect to
treatm ent and w eek in 2005. ............................................ ............................ 38

4 Significant differences between treatments with respect to week and cultivar in
2005....................... ................................... 39

5 Significant differences in cadaver incidence between treatments for a given
w eek and cultivar in 2005. .............................................. ............................. 39

6 Significant differences in cadaver incidence between cultivars for a given week
and treatm ent in 2005. ......................... ............ ............... .... ..... .. 40
















LIST OF FIGURES


Figure page

1 Light micrographs of mycosed glassy-winged sharpshooters displaying the three
m aj or phenotypes. ....................................................................... .. 16

2 Micrographs of the Pseudogibellula dissected from mycosed H. coagulata........... 17

3 Sporothrix isolated from mycosed H. coagulata. .................................................18

4 Light and electron micrographs of the Hirsutella. .............................................19

5 Representative map of crape myrtle field plot. .............................. ......... ...... .23

6 Photographs of bleeding procedure.................................. ..................................... 26

7 Mean GWSS per yellow stick trap in 2005.......... .............................................29

8 Mean GWSS per tree for each cultivar in the first irrigated replicate....................32

9 Mean GWSS per tree for each cultivar in the second irrigated replicate ...............33

10 Mean GWSS per tree for each cultivar in the first dry replicate..............................34

11 Mean GWSS per tree for each cultivar in the second dry replicate.......................35

12 M ean GW SS per tree for entire plot.................................... ......................... 36

13 Total GW SS cadavers observed by week. .................................... .................37















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

FUNGI ASSOCIATED WITH THE GLASSY WINGED SHARPSHOOTER,
Homalodisca coagulata, IN ITS NATIVE RANGE

By

S. Elie Breaux

December, 2005

Chair: Drion G. Boucias
Cochair: Russell F. Mizell III
Major Department: Entomology and Nematology

The glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say), is an

efficient vector of the phytopathogenic bacterium Xyllelafastidiosa. Native to the

Southeast U.S., it was introduced to California, where its presence has caused a sharp

increase in X fastidiosa-based disease. A complex of three possible fungal pathogens are

associated with the glassy-winged sharpshooter in its native range: Pseudogibellula,

Sporothrix, and Hirsutella. In the summer seasons of 2004 and 2005, a variety of

cultivars were selected from a crape myrtle field plot and were used to monitor the

interaction between Hirsutella and GWSS, as well as the effect of humidity on this

interaction. GWSS preference for specific crape myrtle cultivars was also determined in

2005. In both 2004 and 2005, Hirsutella was the predominant mycopathogen detected in

sampled GWSS populations. In 2004, there were high numbers of diseased GWSS. In

2005, there was low incidence of the pathogen within GWSS populations. In 2005, the

crape myrtle cultivar, "Biloxi", was preferred over "Osage", "Miami", and "Tonto" in









one replicate, but differences were insignificant in all other cases. The disparity was

likely due to the migration of GWSS into the study plot through these "Biloxi" trees. For

both 2004 and 2005, the incidence of mycosed GWSS cadavers was significantly higher

in replicates treated with high humidity. This may reflect higher GWSS numbers in the

irrigated plots. By the end of the study period in 2005, 46% of cadavers killed by

Hirsutella were subsequently colonized by Pseudogibellula.














CHAPTER 1
LITERATURE REVIEW

Homalodisca coagulata

The glassy-winged sharpshooter, Homalodisca coagulata (Hemiptera:

Auchenorrhyncha: Cicadellidae: Tettigellinae: Proconiini), has a native range that

extends from central Florida north to North Carolina and west to eastern Texas and

northeastern Mexico (Turner & Pollard 1959). It was introduced as egg masses through

shipments of nursery stock to California in the late 1980s (Sorenson & Gill 1996) and has

become established in several southern California counties, with most of the state still

thought to be at risk for colonization. This introduction is important since the glassy-

winged sharpshooter (GWSS) vectors a lethal phytopathogenic bacterium, Xylella

fastidiosa, which infects several economically important host plants. X fastidiosa is the

organism responsible for Pierce's Disease of grape and the efficiency of GWSS as a

vector has made it a serious threat in this crop (Almeida & Purcell 2003, Purcell &

Saunders 1999).

GWSS is distinctive in morphology when compared to other sharpshooters in its

native range, including its congeneric, Homalodisca insolita. Turner and Pollard's (1959)

description follows:

"The adults of coagulata are large, rather slender leafhoppers, measuring 11 to 13
mm. in length. The males are slightly smaller than the females. The head is bluntly
angled and the crown nearly flat. The general color is brown. The venter is ivory
with conspicuous black markings. At the base of the abdomen the ivory color
extends upward, so that when observed from above, the insects appears to bear an
oblique ivory blotch on either side of the abdomen. The face, genital plates, and
legs are approximately mandarin orange. In newly transformed adults the veins of









the forewings are bright red and spots of red occur towards the tips of the wings.
Within a few days these areas darken until the dorsal aspect presents a homogenous
brown appearance to the naked eye. The nymphs are olive gray. In the first two
instars the eyes are reddish; in the later instars they are darker."

Nymphs progress through five instars, with lengths of 2.0 mm, 2.5 mm, 4.0 mm, 5.0 mm,

and 7.5 8.0 mm for each successive instar (Turner & Pollard 1959). The eyes of the fisrt

two instars have a reddish coloration, which darkens in later instars. The ivory

colorations on the ventral and pleural regions of the adult abdomen progressively yellow

as the insect ages (R. F. Mizell, personal communication).

The GWSS possesses a piercing-sucking type mouthpart, which it inserts through

the stem into the plant xylem tissue (Turner & Pollard 1959). Xylem fluid is its only food

source, and as xylem fluid is extremely nutrient poor, the insect spends much of its time

feeding and frequently switches hosts to achieve optimal nutrient uptake (Mattson 1980,

Brodbeck et al. 1993). If approached, the GWSS displays an interesting defensive

behavior. When the insect detects a potential threat, it migrates to the side of the stem

opposite that threat, putting the stem between itself and danger (Turner & Pollard 1959).

After further provocation, it will fly away.

GWSS mate in early spring and the first egg masses are generally found in April

(Turner & Pollard 1959). Females mate more than once, and oviposit on the underside of

a leaf, usually inserting a cluster of 3-28 eggs under the epidermis of the plant. Optimal

development of the embryo takes place in temperatures ranging from 16.7 C to 32.90 C

(Al-Wahaibi & Morse 2003). As the embryo develops, eyespots become visible through

the leaf tissue, particularly if the leaf is viewed in front of a light source. Hatch rate

(embryo survival) is relatively constant throughout this range, although developmental

times are greatly reduced at its upper end. High mortality occurs at constant temperatures









above 350 C and incomplete development at temperatures below 11.5 C (Al-Wahaibi &

Morse 2003). 113.8 degree-days are required from oviposition to hatch.

After oviposition, the female coats the egg mass with brochosomes,

ultramicroscopic reticulated bodies produced by all of the major subfamilies of

Cicadellidae (Rakitov 1995). Brochosomes are found in their most conspicuous form in

concentrated spots on the forewings of gravid female leafhoppers. Riley and Howard

(1893) first reported the existence of wing spots while studying populations of GWSS,

describing them to be made up of a waxy material. Later investigations showed the wing

spots to be made up of microscopic proteinaceous particles which were termed

"brochosomes" (Tulloch et al. 1952, Tulloch & Shapiro 1953), which are produced in

specialized sections of the Malpighian tubules (Storey & Nichols 1937, Day & Briggs

1958). In addition to the wings, brochosomes are also found on the remainder of the body

(Hix 2001). Rakitov (1996) detailed the behaviors employed by Cicadellids to coat their

bodies with brochosomes. These anointing and grooming behaviors vary from nymph to

adult, and across interspecific lines. Nymphs of the Cicadellid subfamilies Macropsinae,

lassinae, Idiocerinae, and many Typhlocybinae do not cover themselves in brochosome

secretions, but the Ledrinae, Nioniidae, Eurymelinae, Cicadellinae, Coelidiinae,

Hyalicinae, and Deltocephalinaes possess brochosomes in all life stages (Rakitov 1995).

Adults of all subfamilies of Cicadellidae cover themselves in brochosomes. To

accomplish this, excreta containing brochosomes are transferred to the wings and dorsal

integument from the anus with the hind legs, a maneuver that is facilitated by specialized

setae located on the distal portion of the hind leg (Rakitov,

http://www.inhs.uiuc.edu/-rakitov/brochosomes.html). The legs are used to spread the









excreta to cover the entire body except for the eyes. This occurs both when the excreta

are still wet and after they have dried. Nymphs of some species have also been observed

bending their abdomens upwards and forwards to deposit excreta along the head and

back, or releasing excreta onto the plant itself and wallowing in it.

Many hypotheses have been fielded as to the function of brochosomes, including

water repellence, anti-microbial activity, protection from parasites, protection from

desiccation, thermoregulation, protection from UV light, and a pheromone reservoir

(Rakitov 2002). Of these, water repellence is the most likely function of the brochosome

coat (Rakitov 1995). The brochosome coat exhibits extreme hydrophobicity, and may

have evolved to complement the cuticular lipids found on most insects. Additionally, the

presence of brochosomes on egg masses decreases the oviposition efficiency of

Gonatocerus ashmeadi, a hymenopteran parasitoid of GWSS eggs (Velema et al. 2005).

Brochosomes attached to the legs, body and antennae of the wasp, and prompted

prolonged grooming sessions and that interrupted parasitization.

In the GWSS and many other leafhoppers, the shape of the actual brochosome

varies depending on the life stage of the insect (Hix 2001). In the GWSS, the majority of

brochosomes are essentially spherical in shape. The brochosomes that make up the wing

spots that are found on gravid females are rod-shaped (Azevedo-Filho & Carvalho 2005),

and are transferred to the egg mass after oviposition.

The GWSS goes through two or three generations, in a season, depending on

climate (Turner & Pollard 1959). GWSS typically overwinter as adults, however

overwintering nymphs have been found. Regardless of life stage, hibernation is

incomplete. Both nymphs and adults will become active and feed with a sufficient









increase in ambient temperature. Using this strategy, nymphs can eclose into adults and

survive to the next season. Attempts to keep the insects in hibernation at constant cold

temperatures have resulted in death in less than two weeks (Turner & Pollard 1959). This

suggests that periodic winter feeding is critical to the survival of the insect.

Feeding Behavior and Nutritional Ecology of Homalodisca coagulata

The GWSS is an extremely polyphagous insect, feeding on the xylem fluid of over

100 known species of plant, in at least 37 different families (Alderz 1980). It feeds only

on xylem fluid and possesses a well-defined cibarial pump for this purpose. The enlarged

clypeus houses the pump's attendant musculature (Backus 1985). The well-developed

cibarial pump allows the GWSS to feed at most commonly found xylem pressures,

although feeding will decrease as xylem fluid tensions rise and eventually cease at -2.1

MPa (Andersen et al. 1993). Xylophagous insects such as the GWSS must process

nutrients efficiently, as xylem fluid possesses the lowest nutritional value of any plant

tissue (Mattson 1980). Xylem fluid contains very dilute quantities of monomeric amino

acids, organic acids, and sugars (Andersen et al. 1989). Adult host preference is partially

dependent on amino acid profile of the host xylem fluid, specifically the concentration of

the amides (glutamine and asparagine) in relation to other amino acids (Brodbeck et al.

1990). Amides, representing the main form of nitrogen present in the adult's preferred

plant hosts, can be readily converted into other necessary amino acids (Redak et al.

2004). Assimilation efficiency of amides can surpass the level of 99.9%, while that of the

other amino acids is normally lower, ranging from -52% to -98% (Andersen et al. 1989).

The ten essential amino acids are not metabolized with any greater efficiency than

nonessential amino acids. Amides are critical and are believed to dictate GWSS's host

switching behavior. Seasonal and diurnal host preferences can be correlate to xylem fluid









composition and to the concentration of amides, with the insects choosing to feed on

hosts with higher amide ratios (Brodbeck et al. 1990, Andersen et al. 1992). It is believed

that carbon may be the limiting nutrient for these insects, as opposed to dietary nitrogen

(Andersen et al. 1989). Amino acids, although extremely dilute in xylem fluid, are found

in abundance relative to the carbon-rich compounds, such as sugars. In general, the

carbon content of xylem fluid is several orders of magnitude below that of phloem or leaf

tissue.

The primary nitrogenous waste excreted by GWSS is ammonium (Anderson et al.

1989, Brodbeck et al. 1993). At maximal feeding rate, the GWSS consumes -10-100

times its dry body weight in xylem fluid per hour. Because it passes an abundance of

water through its system, nitrogenous wastes can be eliminated in the form of

ammonium, rather than the less toxic urea or uric acid more commonly found in

terrestrial organisms (Redak et al. 2004). This is to the insect's advantage, as the

processing of ammonium into urea or uric acid requires energy. Urea and uric acid

contain 636 and 1926 kJ/mol energy, respectively, whereas ammonium contains none.

(Handbook of Chemistry and Physics, 1971).

Despite the obvious nutritional costs associated with xylophagy, it does have

advantages over other types of herbivory. Xylem fluid, while nutrient-dilute, contains low

concentrations of plant defensive compounds when compared to leaf tissue or phloem

(Bernays & Bright 1993). GWSS are able to avoid a deleterious buildup of these plant

defensive compounds through host switching.

Considerably less research has addressed the nutritional requirements of GWSS

nymphs. Many of the plant host preferred by adults will not support nymphal









development. Nymphs are less adept at utilizing the unbalanced amino acid profiles that

adults seem to prefer, and therefore have a much more limited host range (Brodbeck et al.

1995, 1996). Whereas adults benefit from an amino acid composition very rich in amides,

immature GWSS require a more balanced ratio of amides to the other essential amino

acids, even if total concentrations are less. In the case of both nymphal and adult GWSS,

total dietary nitrogen has been shown to be of less importance than nitrogen form in the

survivorship of both nymphs and adults (Brodbeck et al. 1999).

Homalodisca coagulata as a Disease Vector

Homalodisca coagulata Vector Capacity

Recent concerns regarding GWSS arose when this vector was introduced to

California in 1989 (Sorenson & Gill 1996). As the GWSS became established and its

population grew, so did the incidence of diseases associated with X fastisioda (Blua et al.

1999). While California does have a native sharpshooter complex capable of transmitting

X fastidiosa, transmission characteristics have been determined for few of these. For

example, the blue green sharpshooter, Graphocephala atropunctata, appears to be the

most efficient vector ofX. fastidiosa (Freitag & Frazier 1954, Hill & Purcell 1995), but

typically does not range far into vineyards from its preferred riparian habitat. The

majority of known non-GWSS X fastidiosa vectors are all members of the tribe

Cicadellini. The GWSS, and other members of the tribe Proconiini, are larger than the

cicadellinines and are able to feed on woody portions of the plant (Almeida & Purcell

2003b, Purcell & Saunders 1999). This, in concert with the etiology of X fastiodosa,

makes it of serious concern to producers of wine, table and raisin grapes. Because the

native sharpshooters have shorter mouthparts, they can only feed on the thinner stems at

the exterior of the plant (Almeida & Purcell 2003b). By the end of the season, the









pathogen has not spread to the interior of the plant and it is removed with routine

pruning. Alternatively, the GWSS will introduce X fastidiosa to the central cane of the

grape plant when it feeds, a portion of the plant that is not pruned. This allows the

bacteria to proliferate in the plant and survive the winter season (Purcell & Saunders

1999), resulting in high plant mortality. Two other factors that contribute the success of

GWSS as a vector are the aforementioned broad host range and its strength as a flyer. H.

coagulata's ability to cover large areas means that it can more effectively carry the

bacterium to new sites than native vectors (Blua & Morgan 2003). Perring et al. (2001)

showed that GWSS can vector X fastidiosa at 1000 feet into vineyards, considerably

further than the predominant native vector, Hordnia circellata (Purcell 1974). The ability

to travel long distances and the tendency to switch host plants often means that GWSS

can infect a greater number of plants over a greater area than native sharpshooters.

Xylellafastidiosa

Xylellafastidiosa is a gram-negative, xylem-limited bacterium. The different

strains of X fastidiosa are known to be the causative agents in a number of plant diseases,

including Pierce's disease of grape, phony peach disease, citrus variegated chlorosis,

oleander leaf scorch, almond scorch, periwinkle wilt, and alfalfa dwarf (Pooler &

Hartung 1995). Pierce's disease is of greatest economic importance in the U.S., its spread

in California vineyards facilitated by the introduction of the glassy-winged sharpshooter.

Citrus variegated chlorosis (CVC) is not present in the United States, but has devastated

the citrus industries in Argentina and Brazil (Araujo et al. 2002). CVC has the potential

to become the most economically important citrus disease throughout the world, and is

the focus of a considerable research in Brazil.









Pathogenicity ofX. fastidiosa is due to its ability to block xylem vessels, starving

the stems and leaves of water and nutrients transported upward from the roots (Hopkins

1985, Newman et al. 2004). X. fastidiosa's ability to colonize both the plant's xylem

vessels and the insect vector's piercing-sucking mouthparts may lie in the production of a

biofilm composed primarily of complex exopolysaccharides (da Silva et al. 2001). The

biofilm may allow the bacteria to attach and grow on these hydrodynamically turbulent

environments. At present, there is no practical cure for a plant that is systemically

infected by X fastidiosa, and the result is generally plant death. Oxytetracycline

injections have been shown to provide short-term control of X fastidiosa populations

within the plant, but do not control heavy infections and must be repeated annually.

A GWSS feeding on a plant infected with X fastidiosa can immediately transmit

the bacteria to the next plant it feeds upon (Purcell 1990, Purcell & Finlay 1979). X

fastidiosa has been observed in the cibarium, the apodemal groove of the diaphragm, and

the walls of the precibarial area in H. coagulata (Brlansky et al. 1983). This however,

does not account for the insect's ability to transmit the pathogen directly after feeding on

an infected plant (Purcell & Finlay 1979). It is believed that this is possible because the

vector may harbor X fastidiosa first within the food groove of its stylets, allowing the

bacterium to be immediately transmitted to the next food source as well as to proceed

internally to colonize the rest of the insect foregut (Hill & Purcell 1995). While the

bacterium can be found in the midgut and hindgut, only those found in the foregut and

mouthparts are believed to be capable of transmission (Brlansky et al. 1983). GWSS

nymphs that contract the bacterium lose the ability to transmit it after molting, as the

chitinous lining of the foregut is lost in this process, while the midgut remains intact.









Conversely, insects that contract the bacterium as adults remain infective for the rest of

their lives.

Potential Natural Control Agents

Parasites and Predators

The GWSS's status as an introduced species in California has led to recent

attempts to catalogue potential biocontrol agents. (Triapitsyn et al. 1998). Mymarid

wasps have emerged as the most promising biocontrol agents to be found thus far

(Triapitsyn et al. 1998, Triapitsyn & Phillips 2000). Several mymarid species within the

genus Gonatocerus have been identified as egg parasitoids, however G. ashmeadi, G.

fasciatus, and G. triguttatus are considered to have the most potential for long-term

control of GWSS. G. ashmeadi is well-established in California and was probably

accidentally introduced at the same time as the GWSS (Vickerman et al. 2004). This

species is both more aggressive in its parasitism of GWSS eggs and lives longer than its

congenerics. However, the inability of G. ashmeadi to effectively target early season egg

masses results in poor control of GWSS (Irvin & Hoddle 2005). G. fasciatus and G.

triguttatus were recently released in California to determine if they would be able to

better parasitize these early season egg masses (Irvin & Hoddle 2005). GWSS egg masses

containing these parasitoids have been found at release sites (CDFA, 2003). In addition, a

previously undescribed Zagella species (Trichogrammatidae) has received attention for

introduction (Triapitsyn et al. 1998). More recently, two new species of Ufens

(Hymenoptera: Trichogrammatidae), U. principalis Owen sp. n. and U. ceratus Owen sp.

n., have been have been found to parasitize GWSS eggs in southern California (Al-

Wahaibi et al. 2005). Attempts to use parasitoid wasps common to other species of









sharpshooters have met with limited success, and extreme male sex biases have been

observed (Triapitsyn et al. 2002).

Pseneopunctatus (Hymenoptera: Sphecidae: Pemphredoninae: Psenini) is a

predatory wasp found in California that commonly provisions its nests with sharpshooters

(Bethke et al. 2001). It is thought that the native smoketree sharpshooter, Homalodisca

lacerta, is its typical prey, but it has expanded its prey range to include the GWSS as

well. Nests examined in the vicinity of the University of California-Riverside were

provisioned almost exclusively with GWSS (Bethke et al. 2001). P. punctatus' impact

on the GWSS population is unknown. To date, there has been no thorough survey of

GWSS predators in either its native range or in California, so information regarding this

group of organisms is largely unavailable. The remains of GWSS eggs have been found

in the guts of the green lacewing, Chrysoperla carnea, and the multicolored Asian lady

beetle, Harmonia axyridis, but only in a laboratory setting (Fournier et al. 2004). In

recent field studies in Quincy, FL, all predators observed predating GWSS were

generalist in nature and included salticid spiders and predatory Hemiptera (Breaux,

personal observation).

Fungal Pathogens of Auchenorrhyncha

Auchenorrhyncha is the subdivision of the true bugs (Hemiptera) that includes

cicadas (Cicadidae), leafhoppers (Cicadellidae), planthoppers (Fulgoridae), treehoppers

(Membracidae) and spittlebugs (Cercopidae). As with most sucking insects, most

pathogens infecting this group are fungi. The majority of research regarding fungal

pathogens of this group involves pests of rice and their possible biocontrol agents. These

fungi fall primarily in the Deuteromycota and the Zygomycota.









Within Zygomycota is Massospora, which grows in the abdominal cavities of adult

periodical cicadas and is transmitted either sexually or by general contact when in lek

mating aggregations (Duke et al. 2002, Soper et al. 1976). Also, Sporodiniella

(Zygomycota) has been found to infect membracids in cocoa plantations (Evans &

Samson 1977). However, the most common Zygomycetes that infect leafhoppers and

planthoppers reside within the Entomopthorales (Soper 1985). Erynia delphacis, Erynia

radicans, and Neozygitesfumosa all attack the brown planthopper, Nilapavarta lugens

(Shimazu 1979, Ben-Ze'ev & Kenneth 1981, Samal et al. 1978). However, Erynia

radicans, previously termed Zoopthora radicans and Entomophthora sphaerosperma,

possesses a diverse host range, infecting insects from seven orders, including

Lepidoptera, Homoptera, Diptera, and Hymenoptera (Wraight et al. 1990). Its host range

includes the potato leafhopper, Empoascafabae.

The remaining majority of fungal pathogens that are associated with the

Auchenorrhyncha are in the Deuteromycota. Pathogens that typically possess broad host

ranges, such as Beauveria bassiana and Verticillium lecanii, have been recorded from a

variety of hosts within the Auchenorrhyncha (Srivastava & Nayak 1978, Steenberg &

Humber 1999). Three Metarhizium species, M. flavoviride, M. anisopliae, and M album,

represent a significant mortality factor in planthoppers and leafhoppers of rice in Asia

(Rombach et al. 1986a, Rombach et al. 1986b, Rombach et al. 1987). There is also record

of Hirsutella species infecting auchenorrhynchans. Hywel-Jones (1997) described

Torrubiellapruinosa (teleomorph of Hirsutella versicolor), Hirsutella nivea, and

Hirsutella citriformis from cicadellids in Thailand, as well as from other closely related

families, and Hirsutella guyana was originally described from an infected delphacid









(Minter & Brady 1980). H. citriformis is a common pathogen of auchenorrhynchan pests

of rice. Paecilomycesfarinosus has been isolated from leafhoppers on rice (Hirashima et

al. 1979).

Rombach et al. (1986c) found that field application of Metarhiziumflavoviride,

Metarhizium anisopliae, Beauveria bassiana, Hirsutella citriformis, and Paecilomyces

lilacinus all provided up to 100% mortality in populations of brown planthopper in rice.

Homalodisca coagulata -Associated Fungi

Until recently, very little attention had been directed at the fungal pathogen

complex associated with the GWSS. Turner & Pollard (1959) first reported mycosed

GWSS clinging to trees with their mouthparts still inserted into the plant, but gave the

topic no further consideration. In a later study, cadavers collected in a limited survey of

GWSS in Poplarville, Mississippi were found to be infected with Pseudogibellula

formicarum, Trichothecium roseum, and Beauveria bassiana, with the conclusion drawn

that P. formicarum represented the primary pathogen in the system (Kanga et al. 2004).

When insects were kept in incubators, bioassays performed with P. formicarum spore

suspensions resulted in 75% and 93 % infection at 21 days with applied concentrations of

2 x 108 spores/ml and 2 x 109 spores/ml, respectively. Greenhouse bioassays produced

similar mortality at 48% and 81%.

In a broader survey in the southeastern U.S. during the summer months of 2003 and

2004, GWSS were found to harbor species of Hirsutella, Pseudogibellula, and Sporothrix

(Boucias et al., submitted). In general, the Hirsutella phenotype was most abundant

(44%), followed by the Pseudogibellula (10%) and Sporothrix (2%) phenotypes. A fourth

group of unidentified, heterogenous phenotypes (44%) were covered with a combination

of algae and various fungi. Molecular diagnostics using a multiplex PCR reaction









demonstrated that Hirsutella DNA was present in -80% of the samples, including

examples from the unidentified and Pseudogibellula categories. These results suggest

that Hirsutella sp. is the primary fungal pathogen of GWSS, while Pseudogibellula and

Sporothrix are secondary mycoparasites (Boucias et al., submitted).

The genus Pseudogibellula is characterized by the presence of broad-based

synnemata arising mainly from the intersegmental areas of the mycosed insect (Fig. 1A),

and only contains one species, P. formicarum (Samson & Evans 1973). These synnemata

bear abundant sporulating conidiophores, possessing an arrangement similar to that of

Aspergillus (Fig. 2A,B). Conidia are produced in a sympodial nature. Conidiophores arise

from swollen metulae at the apex of long stalks (Fig. 2C). This sympodial type of

conidiogenesis differentiates the fungus from members of the genus Gibellula, which are

phialidic. While the GWSS-associated Psedugibellula exhibits slight morphological

differences in synnemata shape and conidiophore structure, it most likely is an example

of Pseudogibellula formicarum.

Sporothrix-infected GWSS cadavers possess white, slender synnemata extending

up to 2 mm from the body of the insect (Fig. 1B) (Boucias et al., submitted). These are

covered in conidiogenous cells that appear very similar to the vegetative hyphae (Fig.

3A,B). Both liberated conidia and conidiogenous cells bear conspicuous scars at the point

of detachment (Fig. 3C,D). Identification is hampered by the fact that literature relating

the Sporothrix genus is dominated by species that are human pathogens. However, the

morphology of this fungus closely matches that of S. isarioides (Hoog 1974).

The GWSS Hirsutella forms a tightly appressed mycelial mat over the body of the

insect, originating from the intersegmental membranes (Fig. 1C) (Boucias et al.,









submitted). Both hyphae and phialides appear hyaline under magnification, with the

entire mycelium appearing white to grey in color. Phialides are solitary and smooth and

originate from the hyphae at right angles (Fig. 4A,B). They possess cylindrical bases and

taper to a slender neck bearing a single apical conidium (Fig. 4C). Growth in liquid

media normally includes yeast-like hyphal bodies, while chlamydospores may be present

on solid media (Fig. 4D). Morphologically, this fungus shows the most homology to

descriptions ofH. versicolor (Minter & Brady 1980), but direct comparisons of cultures

show clear differences. This new species of Hirsutella is currently termed Hirsutella

homalodiscae sp. nov. (Boucias et al., submitted).











































Figure 1. Light micrographs of mycosed glassy-winged sharpshooters displaying the
three major phenotypes: (A) Pseudogibellula; (B) Sporothrix; and (C)
Hirsutella. Note the well-developed synnemata (denoted with arrows) formed
by both the Pseudogibellula and Sporothrix. The Hirsutella phenotype,
lacking synnemata, do produce rhizoids (R) that serve as holdfasts anchoring
the mummified cadavers to the plant substrate. Taken from Boucias et al.,
submitted.













































Figure 2. Micrographs of the Pseudogibellula dissected from mycosed H. coagulata. (A
and B) Scanning electron micrographs depicting conidiophores (C) arising
from synnemata and (C) light micrograph depicting the vesicle (V), the
metulae layer (M) and the coniodiogenous cells or philiades (Ph) that gives
rise to ellipsoid conidia (Co). Taken from Boucias et al., submitted.



















































Figure 3. Sporothrix isolated from mycosed H. coagulata. (A) Light micrograph of a
dissected synnema depicting the conidiophores. Scanning electron microscopy
of this structure (B) provides details on the abundance and arrangement of the
conidiophores originating along the length of the stalk-like portion. High
magnification of the conidiophores and conidia (C), note the characteristic
pattern of the denticles over the condiophore surface (D). Taken from Boucias
et al., submitted.

















































Figure 4. Light and electron micrographs of the Hirsutella produced on Riddel mounts
(A), on mycosed H. coagulata (B, C), and in liquid broth (D). Solitary
phialides are produced at intervals along length of the hyphae that extend onto
the glass substrate (A). Similarily, scanning electron microscopy detected
phialides along the peripheral regions of mummified insects (C, B). High
magnification of the conidia suggest that these possess a mucilaginous coat.
The septate hyphal bodies produced in liquid media (D) are identical to those
observed in the hemolymph samples collected from live infected GWSS.
Taken from Boucias et al., submitted.














CHAPTER 2
INTRODUCTION

The glassy-winged sharpshooter's (GWSS) status as the primary vector of Pierce's

Disease of grape in California has lead to research regarding potential biological control

strategies. Mymarid egg parasitoids have emerged as the first organisms to be employed

in the biological control of GWSS, but recently a complex of potentially pathogenic fungi

were discovered to exist in association with GWSS (See Literature Review: Potential

Natural Control Agents). Entomopathogenic fungi have been used with some success in

the biological control of a number of insect pests (Shah & Pell 2003, Hajek et al. 1996,

Milner 1997, Wraight et al. 2000) and these organisms could provide another option to be

used in the control of the glassy-winged sharpshooter.

Within its native range, GWSS is an extremely polyphagous xylophage (Alderz

1980). However, during the summer months large numbers can be found to aggregate on

different varieties of crape myrtle, Lagerstroemia indica or Lagerstroemiafauriei

(Hoddle et al. 2003, Mizell & French 1987). Although not native, crape myrtle is one of

the most popular ornamental plants in the southeastern U.S. (Pettis et al. 2004). These

factors, combined with the species' ease of cultivation, makes it the ideal candidate for

field observation of GWSS. In a mixed crape myrtle planting, the insect's differential

preference for the various cultivars (Mizell, unpublished data) results in adjacent plants in

the same location harboring different insect densities (Breaux, unpublished data). The

anatomy of a crape myrtle tree that has been aggressively pruned at the start of the

season, with few branchings and widely spaced leaves, makes visual sampling of both









live and mycosed GWSS simple. The majority of mycosed GWSS that have been found

have been attached to the stem of a host plant in feeding position (personal observation)

The importance of environmental conditions on the infectivity of entomogenous

mycopathogens has been well-documented (Vidal et al. 2003, Tang & Hou 2001, Shipp

et al. 2003, Fargues et al. 1997). Optimal conditions vary among mycopathogens, but

high humidity is typically necessary for infection and sporulation events. This is

supported by anecdotal evidence of fungal epizootics occurring in GWSS populations

primarily during years that have a cool, wet spring followed by a summer with consistent

rainfall (Mizell, personal communication).

This thesis describes field experiments that utilized a crape myrtle field plot to

observe the relationship between the glassy-winged sharpshooter and its associated

fungal complex in their native environment. The populations of GWSS and fungi were

observed and documented over the course of one and a half seasons in 2004 and 2005. A

portion of the plot was subjected to high humidity treatment to determine its effect on the

activity of the observed fungi. It was hypothesized that Hirsutella homalodiscae was the

primary pathogen of GWSS in north Florida and that its presence in the field plot would

closely parallel that of GWSS. It was also hypothesized that the artificial high humidity

environment created by the misting system would increase the incidence of H.

homalodiscae-killed GWSS in the irrigated portions of field plot.














CHAPTER 3
MATERIALS AND METHODS

Homalodisca coagulata/Hirsutella homalodiscae Interactions in a Crape Myrtle Field
Plot

Description of Study Site

A crape myrtle field plot was utilized to track a population of GWSS over the

course of a portion of the 2004 and the majority of the 2005 summer seasons. The plot

consisted of four replicates of 14 crape myrtle cultivars, with each cultivar represented by

four adjacent trees in each replicate (Fig. 5). Cultivar groups were arranged in each

replicate on a random basis. In the spring of each year, the trees were aggressively pruned

to encourage maximal new growth and to remove the majority of cadavers remaining

from the previous season. The four replicates were divided into two treatments, misted

and ambient. In the misted replicates, a 6' diameter emitter was staked above each tree in

10 of the 14 cultivars. These 10 cultivars were selected based on their attractiveness to

GWSS and were: "Biloxi", "Carolina Beauty", "Sioux", "Apalachee", "Osage",

"Miami", "Tuscarora", "Tuskegee", "Natchez", and "Tonto". The misters were run the

first 15 min. of every hour, 24 hours a day, 7 days a week. The other half of the two

replicates were subjected only to the prevailing environmental conditions.

Sampling Program

In 2004, each tree in the 10 chosen cultivar groups was sampled for live GWSS and

GWSS cadavers three times between the dates of July 8 and August 28. Because this































111
Irrigated] Dry
Y
Replicate 1
: Biloxi
H : Osage


IIrrigated] I Dry
Y
Replicate 2
: Miami
H : Tonto


Figure 5. Representative map of crape myrtle field plot with 2005 cultivars indicated.
One block equals one tree.









sampling was begun in the middle of the GWSS season, an effort was made to remove all

previously occurring cadavers from the trees in the week prior to July 8th

Four cultivars were selected for intensive sampling and observation of both live

and diseased GWSS for the 2005 season. "Osage", "Miami", and "Tonto" cultivars were

selected, as they had demonstrated the highest incidence of mycosed GWSS in the

sampling performed in the second half of the previous season. The fourth cultivar,

"Biloxi", was selected because it had shown high populations but comparatively low

cadaver numbers.

Each replicate was sampled on a weekly basis for 20 weeks from 5/12/2005 to

9/21/2005. No sampling was performed on weeks 14 and 17. Because the study was

begun prior to the immigration of GWSS into the plot it was unnecessary to manually

remove cadavers from the trees, as the pruning had already accomplished this. Individual

trees were visually sampled for live GWSS by running a curved tool behind each branch

and counting the insects as they displayed evasive behaviors. Sampling was performed

between 08:00 and 12:00, a period of lower GWSS activity. This minimized sampling

error due to fly-off, a concern during heightened GWSS activity in afternoon periods.

Immediately following live sampling, each branch of the tree was visually inspected on

all sides for the presence of mycosed insects. Those found were marked by tying a piece

of surveyor's tape around the branch 10-15 cm below the cadaver. The tape was then

marked with a number. This allowed the researcher to keep detailed records on individual

cadavers and monitor changes in morphology.

Sticky Trap Grid

As a supplement to the visual population counts, a 229 m grid with 51 locations

was set up in the 650 acre (1 mile2) area surrounding the field plot and 27 yellow sticky









traps consisting of 7.5 x 15 cm mailing tubes on 1 m stakes were distributed to half the

grid points at random. Trap placement was randomized each week among the 51

locations. The GWSS on the traps were counted weekly from 5/23/2005 to 9/21/2005.

They were replaced with new traps after counting. The sticky trap grid was utilized to

provide data on overall GWSS populations to supplement the localized plot sampling.

Diagnostic Bleeds

Sampling Program

Live GWSS were collected in the field plot through sweeping or gentle beating of the

branch. One insect was removed from each of the ten 4-tree cultivar groups. Insects

collected from the two misted and dry replicates were combined, making for one cohort

of approximately 20 insects collected for both irrigated and dry treatments. As such, each

replicate was treated as a monoculture, with cultivar acknowledged only so far that every

one was sampled. After collection, insects were transferred to a mesh sleeve cage. When

sampling for each treatment was complete, the sleeve cage was placed over a host plant,

typically Baccharis halimifolia, to ensure leafhopper health until used for bleeding.

Bleed Technique

Prior to bleeding, insects were removed from the plant and placed in a plastic tube

with a foam plug at each end. Carbon dioxide was introduced into the tube through a port

at the end until all insects were visibly immobilized. They were then transferred to a 2 L

Erlenmeyer flask, which was filled with CO2 and sealed with a rubber stopper. The

leafhoppers were removed from the flask in groups of three for bleeding.

Bleeds were performed under a dissecting microscope, through removal of one

antenna at the base with an EtOH-sterilized insect pin (Fig. 6). Applying gentle pressure

to the thorax with soft forceps usually produced a droplet of hemolymph at the site of









antennal insertion. This was done with the insect placed on its dorsal side, and using a

micropipette, 0.5-1 iL of the resultant droplet of hemolymph was removed. This sample

was then transferred to a previously aliquotted 9 itL droplet of Ringer's solution (0.75%

NaC1, 0.035% KC1, 0.021% CaC12) on a glass slide, which was then covered with a glass

coverslip. The sample was viewed under phase contrast optics at 100X magnification.














Figure 6. Photographs showing removed antenna and hemolymph drop from bleed.
A) Antennal insertion point. B) Removed antenna. Photographs processed by
C. Tipping.

Samples displaying characteristic hyphal body morphology were confirmed to be positive

at 400X. After bleeding, insects were separated onto caged host plants based on status as

positive or negative for hyphal bodies.

Statistical Analysis

Data collected from the sticky traps and in the field trial were subjected to repeated

measures analysis using a mixed model methodology. Week, treatment, position within

the plot, and crape myrtle cultivar were used in the analysis to determine which of these

variables produced a significant influence on live and mycosed GWSS counts. Factors

and combinations of factors producing significant effects were subsequently subjected to

least squares means analysis to determine timing and location of these effects. Similarly,

cadaver incidence was analyzed with repeated measures and least squares means






27


procedures using time, treatment and cultivar as factors. Cadaver incidence was

compared to host GWSS population numbers by first arcsine transforming the data and

then running the same analysis as for the live GWSS numbers with the resultant

proportion. T tests were utilized to compare 2004 and 2005 cadaver incidence at single

points in time and to compare between total cadaver numbers in irrigated and dry

treatments. All analyses were performed using SAS 9.1 for Windows (SAS Institute

2003)















CHAPTER 4
RESULTS

In 2004, humidity treatment was a significant factor in cadaver incidence. Average

number of cadavers per tree was significantly higher in misted replicates (t = 6.14, p <

0.0001). In a comparison of leafhopper numbers between the same trees for the same

weeks in 2004 and 2005, there is no clear trend in which year had higher populations

(Table 1). During week 10, there were more in 2004, but weeks 15 and 16 showed more

in 2005. The few samples taken in 2004 limits this comparison. Cumulative mean

cadaver numbers per tree were significantly higher in 2004 than 2005 (t = 7.43, p <

0.0001). The last sample time for the plot in 2004 occurred from 8/17/04 to 8/28/04.


Table 1. Comparison of 2004 and 2005 GWSS populations in field plot for same trees on
same week between years.


Week T value P < Itl Significantly
Greater Year
9 0.7 0.4931 None

10 4.5 <0.0001 2004

11 0.64 0.5364 None

12 2.88 0.1021 None

15 -4.61 <0.0001 2005

16 -2.54 0.0291 2005








During this time, mean GWSS cadavers per tree was 4.44 3.74 (N = 64). The same

trees sampled on 8/24/05 had 0.31 0.48 mean cadavers per tree. The total number of

cadavers persisting on these trees at this time point in 2004 was 284 compared to 20 for

2005.

In 2005, background GWSS population data from the sticky trap grid indicate two

peaks in GWSS numbers over the course of the study, the highest coming in week 7 and a

much smaller peak at week 16 (Fig. 7). Weeks 5 through 9 all produced trap catches that

were significantly higher than the other weeks (Table 2). The second peak at week 16

was not statistically significant. Visual GWSS counts of the "Biloxi" cultivar in the

misted portion of the first replicate closely mirror this trend with a delay of

approximately 1-2 weeks (Fig. 8). Counts in the remainder of replicate 1, as well as all of

replicate 2 show little similarity to background numbers (Figs. 9-11). Population averages


90
80
c. 70
S60
CL
. 50
c. 40
0
o
30
< 20
10
0


-



i.iIiIii..,? ',..
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Week


Figure 7. Mean GWSS per yellow stick trap in 2005. Error bars equal standard error.









for the entire plot, without respect to cultivar or treatment, show the same first peak at

week 5 to week 9, but do not drop as precipitously afterwards, showing instead a slower

dropoff and no evident second peak (Fig. 12).

Least squares means analysis of significant effects found in the repeated measures

procedures revealed significant differences between both cultivar and humidity

treatments were primarily due to the action of the crape myrtle cultivar "Biloxi" in the

misted portion of the first replicate (Tables 3 & 4). This cultivar group held the highest

numbers of leafhoppers of any group throughout the entire first population peak, often by

a factor of 3 (Fig. 8-11). Most significant differences between treatment and cultivar

were found within this peak (Tables 3 & 4). The exception to this phenomenon is that

within the misted portion of the first replicate, leafhopper numbers on the cultivar

"Tonto" were significantly lower when compared to "Osage" in weeks 16 through 18 and

when compared to "Biloxi" in week 19 (Table 3). These differences, however, were not

of great enough magnitude to produce a significant difference in GWSS numbers

between irrigated and dry treatments (Table 4).

The incidence ofHirsutella homalodiscae within the field plot did not follow

fluctuations in GWSS populations. Mycosed cadavers appeared with greatest regularity

beginning at the tail of the first population peak, until week 19 (Fig. 13). This time period

was characterized by lower GWSS populations, but also by almost weekly shifts in host

cultivar preference. Sticky trap data show that background GWSS populations were much

lower after week 11 (Fig. 7). However, mean GWSS populations in the plot rose slightly

in week 12 and then began a gradual decline (Fig. 12). It should be noted that the higher

numbers of cadavers found in week 15 and 18 were probably due to the missed sampling









dates from the weeks 14 and 17. Therefore the cadavers numbers found on weeks 15 and

18 reflect a total for two weeks. Significant between treatment differences in cadaver

incidence were found in weeks 10-13, 15, 16, 18, and 19 (Table 6). While significant at

times, no single cultivar emerged as a definitive factor in cadaver incidence (Tables 5 &

6). Irrigation treatment was a moderately significant effect when total cadavers for each

treatment were analyzed using a t-test (T = 2.23, p = 0.0295), with more being found in

the misted replicates. On 9/14/05, field observation indicated that 46% (N = 27) of the

remaining cadavers displayed some degree of Pseudogibellula morphology, varying from

mounded areas of mycelium at intersegmental areas to fully formed Pseudogibellula-type

synnemata (Fig. 1A). A total of 435 GWSS were bled in 2005, 185 from within the

treatment plot, and 250 from a secondary plot. Four definitive positives were found, all

between the dates of 7/12/05 and 7/20/05. Two of the positives were collected from

within the study plot.












90
80
70
60
50
40
30
20
10


* Biloxi
m Osage
E Miami
* Tonto


Figure 8. Mean GWSS per tree for each cultivar in the first irrigated replicate in 2005. Error bars equal standard error.


C)
0)
0)
C)

0

0


-T







SIO I -IIO

Week
















L
C)
C-


0
d)
--
a)


30

25


20


15


10


5


* Biloxi
SOsage
0 Miami
0 Tonto


M LO I*- M) LO *- 0)


Week


Figure 9. Mean GWSS per tree for each cultivar in the second irrigated replicate in 2005. Error bars equal standard error.
















I LTTI IIi


iw


I-
L
C-
C)
0.
r
I-


12
10
8
6
4
2


M- C O I* M) '-


I


CO) LO I'-
r- V- V


Week


Figure 10. Mean GWSS per tree for each cultivar in the first dry replicate in 2005. Error bars equal standard error.


Iii


A'


E Biloxi
SOsage
O Miami
STonto




























- CM LO I *- O)


-- LO I,- O)


Week


Figure 11. Mean GWSS per tree for each cultivar in the second dry replicate in 2005. Error bars equal standard error.


35

S30
I-
25
C.
2 20




> T
S15



5


* Biloxi
H Osage
O Miami
* Tonto
















16


14


12


S10





.
U)

Q.




4


2


0


T
















1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Week


Figure 12. Mean GWSS per tree for entire plot in 2005. Error bars equal standard error.








New Cadavers by Week


1 2 3 4 5 6


7 8 9 10 11 12 13 14 15 16 17 18 19


Week


Figure 13. Total GWSS cadavers observed by week in 2005.


It










Table 2. Weeks with significantly higher trap catches of GWSS in 2005.


Week Estimate T Value Pr > It|
5 21.3077 4.18 < 0.0001
6 57.0000 11.17 < 0.0001
7 64.6000 12.42 < 0.0001
8 42.0000 8.23 < 0.0001
9 17.4231 3.41 0.0007


Table 3. Significant differences in live GWSS numbers between cultivars with respect to
treatment and week in 2005.


Treatment Week Cultivar Cultivar Estimate T Value Pr < |t|
Misted 6 Biloxi Tonto -13.3750 -2.81 0.0050
Misted 6 Biloxi Osage 16.1250 3.39 0.0007
Misted 6 Biloxi Miami 17.5000 3.68 0.0002
Misted 7 Biloxi Tonto -26.7500 -5.62 <.0001
Misted 7 Biloxi Osage 28.8750 6.07 <.0001
Misted 7 Biloxi Miami 28.7500 6.04 <.0001
Misted 8 Biloxi Tonto -26.7500 -5.62 <.0001
Misted 8 Biloxi Osage 24.3750 5.12 <.0001
Misted 8 Biloxi Miami 25.8750 5.44 <.0001
Misted 9 Biloxi Tonto -14.8750 -3.13 0.0018
Misted 9 Biloxi Osage 15.5000 3.26 0.0012
Misted 9 Biloxi Miami 17.8750 3.76 0.0002
Misted 10 Biloxi Tonto -14.7500 -3.10 0.0020
Misted 10 Biloxi Osage 16.8750 3.55 0.0004
Misted 10 Biloxi Miami 18.2500 3.84 0.0001
Misted 11 Biloxi Osage 9.5000 2.00 0.0462
Misted 11 Biloxi Miami 10.6250 2.23 0.0258
Misted 16 Tonto Osage -11.6250 -2.44 0.0147
Misted 18 Tonto Osage -9.6250 -2.05 0.0403
Misted 19 Biloxi Tonto -10.7500 -2.29 0.0220










Table 4. Significant differences between treatments with respect to week and cultivar in
2005.


Treatment Treatment Week Cultivar Estimate T Value Pr > Itl
Misted Dry 6 1 22.000 3.51 0.0005
Misted Dry 7 1 35.500 5.66 < .0001
Misted Dry 8 1 33.000 5.26 < .0001
Misted Dry 9 1 21.875 3.49 0.0005
Misted Dry 10 1 21.250 3.39 0.0007
Misted Dry 11 1 14.000 2.23 0.0258


Table 5. Significant differences in cadaver incidence between treatments for a given
week and cultivar in 2005.

Treatment Treatment Week Cultivar Estimate T Value Pr > Itl
Irrigated Dry 10 Osage 0.2500 2.34 0.0193
Irrigated Dry 11 Tonto 0.3750 3.51 0.0005
Irrigated Dry 11 Miami 0.2500 2.34 0.0193
Irrigated Dry 12 Miami 0.2500 2.34 0.0193
Irrigated Dry 13 Osage 0.2500 2.34 0.0193
Irrigated Dry 15 Osage 0.3750 3.51 0.0005
Irrigated Dry 16 Tonto 0.2500 2.34 0.0193
Irrigated Dry 18 Tonto -0.3750 -3.51 0.0005
Irrigated Dry 18 Biloxi 0.2500 2.34 0.0193
Irrigated Dry 19 Osage -0.2500 -2.34 0.0193











Table 6. Significant differences in cadaver incidence between cultivars for a given week
and treatment in 2005.


Treatment Week Cultivar Cultivar Estimate T Value Pr > Itl
1 10 0 5 -0.2500 -2.34 0.0193
1 10 1 5 -0.2500 -2.34 0.0193
1 10 5 6 0.2500 2.34 0.0193
1 11 0 1 0.2500 2.34 0.0193
1 11 0 5 0.2500 2.34 0.0193
1 12 0 6 -0.2500 -2.34 0.0193
1 12 1 6 -0.2500 -2.34 0.0193
1 12 5 6 -0.2500 -2.34 0.0193
1 13 0 5 -0.2500 -2.34 0.0193
1 13 5 6 0.2500 2.34 0.0193
1 15 0 5 -0.2500 -2.34 0.0193
1 15 1 5 -0.2500 -2.34 0.0193
1 15 5 6 0.2500 2.34 0.0193
1 16 0 1 0.2500 2.34 0.0193
1 16 0 6 0.2500 2.34 0.0193
1 18 0 1 -0.2500 -2.34 0.0193
1 18 1 5 0.2500 2.34 0.0193
2 18 0 1 0.3750 3.51 0.0005
2 18 0 5 0.2500 2.34 0.0193
2 18 0 6 0.3750 3.51 0.0005
2 19 0 5 -0.2500 -2.34 0.0193
2 19 1 5 -0.2500 -2.34 0.0193














CHAPTER 5
DISCUSSION

The GWSS's predilection for accessing different hosts is evidenced by the 2005

plot data. Insects in the initial population peak preferred the cultivar "Biloxi" in the first

irrigated plot (Fig. 8) and to a lesser degree in the second (Fig. 9), but those in the dry

plots displayed no clear cultivar preference during this period (Figs. 10,11). During the

latter portion of the season, there was either no definitive preference or preferences were

slight and shifted often (Figs 8-11). With the data available in this study there can be no

conclusive explanation for these behaviors, but inferences can be drawn from information

on GWSS biology. GWSS overwinter as adults or nymphs in a state of incomplete

diapause (Turner & Pollard 1959). The considerable fluctuations in winter temperatures

in north Florida (Florida Automated Weather Network, http://fawn.ifas.ufl.edu/) allow

both adult and nymphal stages to feed during warmer periods. This allows the nymphs to

eclose to adults before spring. As adults become active in the spring, they oviposit on

hosts suitable for nymphal development (Alderz 1980). Crape myrtle is a host preferred

primarily by adult GWSS, so the first population peak is likely the Fi generation

migrating to the field plot after they eclose (Ball 1979). The second peak occurred in

mid-late August and is probably indicative of the F2 generation. Timing of the beginning

of the first population peak (Fig. 7) corresponds well with Ball's (1979) observations but

not with those of Turner and Pollard (1959), who found the highest GWSS numbers in

peach in the fall. At the end of the season, it is thought that the F2 generation splits

survival strategies (Mizell, personal communication). A portion of the F2 individuals will









enter reproductive diapause while the rest will continue to mate and oviposit. Those in

reproductive diapause enter the winter season as adults. Those hatching from egg masses

laid by the F2 enter as nymphs. This dichotomy could explain the change in host

preference when compared to members of the F1 generation. Studies with Lepidoptera

have shown that nutrition plays a role in the induction of diapause (Hunter & McNeil

2004, Mondy & Corio-Costet 2004). Nutrient requirements of those GWSS entering

diapause may differ considerably from non-diapausing GWSS, and this could be reflected

in the differences in host preference. An alternative explanation is that the shift in host

preference is driven by a change in the amino acid profile of the host xylem fluid (See

Literature Review: Nutritional Ecology of Homalodisca coagulata). Most crape myrtles

in the field plot had completed flowering by the end of the sampling period (personal

observation), and this shift in life stage may be reflected in a resultant shift in available

nutrients in the xylem fluid. It is unlikely that changes in host preference are related to

other effects, such as overcrowding or increased energy expended while feeding. Host

plants the size of the plot crape myrtles are capable of sustaining extremely high

leafhopper densities with little or no change in xylem fluid content (Andersen et al. 2003)

and the feeding apparatus of GWSS allows them to feed at a wide range of fluid pressures

(Andersen et al. 1992).

The preference for the "Biloxi" cultivar in the first misted replicate during the first

population peak is difficult to explain. The fact that this trend is not shown in any of the

other "Biloxi" cultivar groups means that simple host preference is an unlikely cause. It

is, however, possible that the increased numbers can be attributed to the cultivar group's

position in the plot. The first irrigated replicate is located at the northern edge of the plot,









with the "Biloxi" group at the western comer. The plot is oriented so that this comer is

the closest to a natural forest habitat, and therefore may serve as the initial point of

GWSS immigration. This is supported by the fact that the "Biloxi" cultivar group in the

first misted replicate is the only one that mimics the trap-based background population

curve with any consistency. If these four trees represent a major immigration point, after

which the insects distribute themselves throughout the rest of the plot in a relatively

homogenous manner, this trend in cultivar preference could be accounted for. A similar

phenomenon was also observed by Ball (1979), who found that traps bordering

woodlands produced the highest catches of GWSS.

The primary focus of the field study was to characterize the action of Hirsutella

homalodiscae on a host GWSS population in the native range. During 2004, the pathogen

was present in epizootic proportions and was probably a major mortality factor,

particularly to the Fi and F2 generations. However, sampling in 2004 was not performed

with enough frequency to judge this. In 2005, a more typical enzootic-type condition was

observed, with very few cadavers found and what could only be a negligible effect on

host population. For the few sample dates that coincide between the two years, there is no

discernable trend regarding which year displayed greater densities of GWSS within the

plot (Table 1). It should be noted that sampling in 2004 began at a later date than in 2005,

and GWSS densities for the first population peak were not recorded. There may have

been a significant difference between the two years during this time period.

Explanations for this difference in cadaver incidence between 2004 and 2005 are

speculative because at this point in time, virtually nothing is known about the biology or

life cycle of H. homalodiscae and little is known of the overwintering biology of GWSS









(Alderz 1980). Weather data collected from 6/13/04 to 9/21/04 and 6/13/05 to 9/21/05,

the time period when cadavers were found in 2005, shows no major deviations in average

temperature, rainfall, and relative humidity between the two years (Florida Automated

Weather Network, http://fawn.ifas.ufl.edu/) (Appendix E). Bioassays indicate that H.

homalodiscae, like most entompathogenic fungi, employs a transcuticular means of

ingress into the host insect and that sporulating cadavers can act as the source of the

infectious inoculum (Blaeske, unpublished data). It has also been shown that cadavers

from the previous season will develop minor hyphal growth and limited sporulation if put

under moist, warm conditions (personal observation, Blaeske, unpublished data).

Therefore, it is assumed that a cadaver affixed to the host plant could function as both the

pathogen's overwintering reservoir and the stage that delivers inoculum to the host. Most

pathogenic fungi possess an environmentally resistant stage, with intrahyphal or

compressed hyphae and chlamydospores being most common in the Hyphomycetes

(Shah & Pell 2003, Pendland 1982). H. homalodiscae has been shown to produce

chlamydospores under in vitro conditions (Boucias et al., submitted), but no studies have

been performed to search for these structures in the cadavers. The ability to induce

hyphal growth and limited sporulation from overwintered cadavers and the presence of

chlamydospores in culture tend to indicate that the cadavers that persist on the host plant

from one season to the next may represent the primary source of inoculum for the new

season. Some other pathogenic Hyphomycetes are known to possess a soilborne stage

(Fuxa & Richter 2004, Sanchez-Murillo et al. 2004, Hu & St. Leger 2002), but it is

unlikely that this would be the main reservoir for H. homalodiscae. GWSS are thought to









overwinter primarily on their winter host plants (Turner & Pollard 1959, Alderz 1980)

and would not contact a soil-based pathogen.

Hirsutella homalodiscae produces a single conidium from the apical portion of

each phialide (Boucias et al., submitted) that does not appear to rely on dissemination

away from the cadaver as a route of infection, but on contact between sporulating cadaver

and naive individual. This is the case with other species of Hirsutella, such as H.

7th,,,',//ii, a mite pathogen (Ignoffo & Mandava 1988). Work has been performed

which demonstrates that GWSS display aggregation behaviors, most likely based on

recognition of a conspecific's silhouette on a branch (Mizell, unpublished data).

Although the color differs, mycosed GWSS cadavers maintain much the same shape on

the branch as healthy leafhoppers (personal observation). If the cadaver triggered the

same aggregation response as live GWSS, the need for wind or water based distribution

of conidia would be eliminated. Naive hosts would be attracted to infective cadavers.

If as above, it is assumed that overwintering cadavers represent the primary source

of inoculum for H. homalodiscae, then the absence of observed infection in the parent

generation in the spring could be due to three factors. First, it may be that environmental

conditions are not sufficient to cause new growth and sporulation in the cadavers from

the previous season or to support infection in a new host. Secondly, in order to ensure

that no cadavers from the previous season remained to skew the counts, all host trees in

the plot were trimmed aggressively before the study. This removed all cadavers

remaining from the previous season, and therefore all possible inoculum within those

trees. For the infection cycle to become established in the plot, diseased insects would









have to first become infected elsewhere and then migrate into the study area, and this

may explain the absence of infection until after the first population peak.

Entomopathogenic fungi are known to alter the activity of their insect hosts by

altering movement and distribution (Jensen et al. 2001), reducing feeding rate (Hajek

1989), changing reproductive behavior(Watson & Peterson 1993), inducing behavioral

fevers (Watson et al. 1993) and causing summit behavior (Bidochka et al. 1997).

Comparisons between the behaviors of live and infected GWSS would have applications

in interpreting field population data. Whether infected GWSS are as prone to migration

and host switching as healthy individuals is unknown.

For the GWSS/H. homalodiscae relationship to be fully understood, future research

should focus on the transmission biology of the fungus, in combination with long-term

observation of pathogen dynamics in the field. A better understanding of the effect of

environmental conditions on the transmission potential and infection success of the

fungus would allow some parallels to be drawn between laboratory bioassay data and

field incidence for the purpose of determining whether epizootic conditions are based on

host population effects or simple phenological fluctuations. The locations in California

that could benefit from the introduction of native biocontrol agents possess a cool, wet

spring but a very dry summer (www.temeculaweather.com,

http://www.wunderground.com/US/CA/Napa.html). Experiments should be conducted to

determine the suitability ofH. homalodiscae for introduction in this type of environment.














CHAPTER 6
CONCLUSIONS

Disease incidence was much higher in 2004 than in 2005 and may have been due to

differences in GWSS populations between the two years, but the 2004 data was not

collected over a long enough time period to determine this conclusively. Humidity

treatment was a significant factor with respect to cadaver incidence in 2004, but only

moderately so in 2005, when there were very few cadavers in any portion of the plot.

Cultivar preference was present to an extent in both years, but further investigation would

be necessary to separate the effect of cultivar from that of immigration of the insect.

This study represents an initial investigation into this system, detailing some

methods and techniques that should be of value to an individual wishing to further pursue

research with these organisms. But while the methodologies presented here are believed

to be sound, one should use caution when drawing conclusions about any ecological

relationship based on one and a half seasons of field data, including this one. For a more

full understanding of how GWSS interacts with its fungal pathogen complex, longer-

term, higher-volume observation and sampling is vital, with the data shown here simply

to be used as a guide for experimental design. When combined with these field studies,

research into the environmental optima for transmission and growth of the pathogen

should provide clues as to its suitability for introduction to California for the biological

control of GWSS.


















APPENDIX A
SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF LIVE
GWSS POPULATIONS AND LEAST MEANS SQUARED ANALYSIS OF
SIGNIFICANT EFFECTS

%let path = E:\SAS Output;
%macro printRtf(dset);
ods rtf file = "&path\test.rtf";
proc print data=&dset;
Run;
ods rtf close;
%mend printRtf;

Data;
input blk trt week cult count @@;
cards;

DATA HERE

proc mixed;
class blk trt week cult;
model count= blk trt cult trt*cult week week*trt week*cult
week*trt*cult;
Random blk*trt blk*trt*cult;
lsmeans trt*week*cult / diff;
ods output Ismeans = LSMEAN diffs = LSDIFF;
run;

*proc print data=lsdiff;
run;
data siglsdiff;
set Isdiff;
if week= week and cult= cult and Probt < 0.05;
keep week cult trt trt estimate tvalue probt;
run;

title 'significant differences in treatments for a given week and
cultivar';
%printRTF(siglsdiff);
run;

data siglsdiff2;
set Isdiff;
if week= week and trt= trt and Probt < 0.05;
keep week trt cult cult estimate tvalue probt;
run;
title 'significant differences in cultivars for a given week and







49


treatment';
%printRTF(siglsdiff2);
run;



















APPENDIX B
SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF
CADAVER INCIDENCE AND LEAST MEANS SQUARES ANALYSIS OF
SIGNIFICANT EFFECTS

%let path = E:\SAS Output;
%macro printRtf(dset);
ods rtf file = "&path\test.rtf";
proc print data=&dset;
Run;
ods rtf close;
%mend printRtf;

Data;
input blk trt week cult cadavers @@;
cards;

DATA HERE

proc mixed;
class blk trt week cult;
model cadaver= blk trt cult trt*cult week week*trt week*cult
week*trt*cult;
Random blk*trt blk*trt*cult;
lsmeans trt*week*cult / diff;
ods output Ismeans = LSMEAN diffs = LSDIFF;
run;

*proc print data=lsdiff;
run;
data siglsdiff;
set Isdiff;
if week= week and cult= cult and Probt < 0.05;
keep week cult trt trt estimate tvalue probt;
run;

title 'significant differences in cadavers between treatments for a
given week and cultivar';
%printRTF(siglsdiff);
run;

data siglsdiff2;
set Isdiff;
if week= week and trt= trt and Probt < 0.05;
keep week trt cult cult estimate tvalue probt;
run;
title 'significant differences in cadavers between cultivars for a







51


given week and treatment';
%printRTF(siglsdiff2);
run;

















APPENDIX C
SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF TRAP
POPULATION DATA AND LEAST MEANS SQAURES ANALYSIS OF THE
EFFECT OF TIME

Data;
input week count @@;
cards;

DATA HERE

proc mixed;
class week;
model count= week;
lsmeans week / diff;
run;



















APPENDIX D
SAS PROGRAMMING CODE FOR REPEATED MEASURES ANALYSIS OF
ARCSINE TRANSFORMED LIVE GWSS/CADAVER PROPORTIONS WITH LEAST
MEANS SQUARE ANALYSIS OF SIGNIFICANT EFFECTS

%let path = E:\SAS Output;

%macro printRtf(dset);
ods rtf file = "&path\test.rtf";
proc print data=&dset;
Run;
ods rtf close;
%mend printRtf;

Data;
input blk trt week cult live mummy @@;
y=mummy;
n=live+mummy;
if n=0 then n=.;
if y=0 then p=1/(4*n);
if l<=y<=n-l then p=y/n;
if y=n then p=(n- 1/4)/n;
transp= (360/2*3.14159)*(arsin(SQRT(p)) );
cards;

DATA HERE

proc mixed;
class blk trt week cult;
model transp= blk trt cult trt*cult week week*trt week*cult
week*trt*cult;
Random blk*trt blk*trt*cult;
lsmeans week trt*week trt*week*cult / diff;
ods output Ismeans = LSMEAN diffs = LSDIFF;
run;

*proc print data=lsdiff;
run;
data siglsdiff;
set Isdiff;
if week= week and cult= cult and Probt < 0.05;
keep week cult trt trt estimate tvalue probt;
run;

title 'significant differences in treatments for a given week and
cultivar';
%printRTF(siglsdiff);
run;












data siglsdiff2;
set Isdiff;
if week= week and trt= trt and Probt < 0.05;
keep week trt cult cult estimate tvalue probt;
run;
title 'significant differences in cultivars for a given week and
treatment';
%printRTF(siglsdiff2);
run;
















APPENDIX E
QUINCY NFREC WEATHER DATA FOR 2004 AND 2005


Precipitation from 6/13 to 9/21


4
3.5
3
E
S2.5
S2
. 1.5
S1
0.5
0


- L M M IN- cOLO cM M I
NM c1 L ( r- O 0Day

Day


--2004
---2005


Average Daily Temperature from 6/13 to 9/21


35
30
- 25
20 -.-- 2004
S15 -1- 2005


5
0
-- O I-' L0 r -- 0) I- L0 CDO a- 0) I-y
N CO -t L0 (C) I[- CO CO O

Day











Average Relative Humidity from 6/13 to 9/21


W LO (N M 0 CO 0D N- 19t WLO (N M
S N CO 19t LO) LO) 0 M- M- MDC
Day


120

100

80

60
40

20


.- 2004
--2005


6wwOA8~enad~~dAAXOu -
















APPENDIX F
RAW DATA FROM 2004 AND 2004 FIELD STUDIES

2004 Field Plot Data


Tree Code Date Cultivar Live GWSS Cadavers
Al 7/8/2004 Biloxi 55 1
A2 7/8/2004 Biloxi 60 4
A3 7/8/2004 Biloxi 54 3
A4 7/8/2004 Biloxi 44 3
A5 7/8/2004 Carolina Beauty 32 5
A6 7/8/2004 Carolina Beauty 45 4
A7 7/8/2004 Carolina Beauty 41 6
A8 7/8/2004 Carolina Beauty 33 1
A13 7/8/2004 Sioux 22 0
A14 7/8/2004 Sioux 20 3
A15 7/8/2004 Sioux 12 1
A16 7/8/2004 Sioux 21 0
A17 7/8/2004 Appalachee 20 1
A18 7/8/2004 Appalachee 21 2
C5 7/12/2004 Natchez 25 1
C6 7/12/2004 Natchez 25 4
C8 7/12/2004 Natchez 1
C9 7/12/2004 Osage 14 0
C10 7/12/2004 Osage 11 0
C11 7/12/2004 Osage 1
C12 7/12/2004 Osage 1
C13 7/12/2004 Tonto 17 1
C14 7/12/2004 Tonto 18 1
C15 7/12/2004 Tonto 0
C16 7/12/2004 Tonto 2
C17 7/12/2004 Biloxi 12 1
C18 7/12/2004 Biloxi 8 0
C19 7/12/2004 Biloxi 0
C20 7/12/2004 Biloxi 0
C21 7/12/2004 Carolina Beauty 8 1
C22 7/12/2004 Carolina Beauty 6 0
C23 7/12/2004 Carolina Beauty 0
C24 7/12/2004 Carolina Beauty 0
D1 7/14/2004 Sioux 16 4
D2 7/14/2004 Sioux 15 1
D3 7/14/2004 Sioux 1










D4 7/14/2004 Sioux 0
D5 7/14/2004 Appalachee 5 1
D6 7/14/2004 Appalachee 4 0
D7 7/14/2004 Appalachee 0
D8 7/14/2004 Appalachee 0
D9 7/14/2004 Miami 13 4
D10 7/14/2004 Miami 15 4
D11 7/14/2004 Miami 0
D12 7/14/2004 Miami 1
D17 7/14/2004 Tuscarora 15 1
D18 7/14/2004 Tuscarora 11 2
D19 7/14/2004 Tuscarora 0
D20 7/14/2004 Tuscarora 2
D21 7/14/2004 Tuskegee 17 0
D22 7/14/2004 Tuskegee 9 2
D23 7/14/2004 Tuskegee 0
D24 7/14/2004 Tuskegee 0
El 7/12/2004 Tuskegee 44 8
E2 7/12/2004 Tuskegee 51 11
E3 7/12/2004 Tuskegee 3
E4 7/12/2004 Tuskegee 0
E9 7/12/2004 Biloxi 33 3
E10 7/12/2004 Biloxi 15 2
E11 7/12/2004 Biloxi 1
E12 7/12/2004 Biloxi 0
E13 7/12/2004 Miami 14 1
E14 7/12/2004 Miami 13 1
E15 7/12/2004 Miami 0
E16 7/12/2004 Miami 1
E17 7/12/2004 Sioux 11 0
E18 7/12/2004 Sioux 13 1
E19 7/12/2004 Sioux 0
E20 7/12/2004 Sioux 0
E21 7/13/2004 Natchez 13 1
E22 7/13/2004 Natchez 12 2
E23 7/13/2004 Natchez 1
E24 7/13/2004 Natchez 1
E25 7/13/2004 Appalachee 7 1
E26 7/13/2004 Appalachee 12 2
E27 7/13/2004 Appalachee 2
E28 7/13/2004 Appalachee 0
F5 7/14/2004 Tonto 10 1
F6 7/14/2004 Tonto 13 1
F7 7/14/2004 Tonto 2
F8 7/14/2004 Tonto 2
F9 7/14/2004 Tuscarora 17 3
F10 7/14/2004 Tuscarora 23 2
F11 7/14/2004 Tuscarora 1










F12 7/14/2004 Tuscarora 2
F17 7/14/2004 Carolina Beauty 12 1
F18 7/14/2004 Carolina Beauty 11 1
F19 7/14/2004 Carolina Beauty 0
F20 7/14/2004 Carolina Beauty 0
F21 7/14/2004 Osage 10 4
F22 7/14/2004 Osage 10 2
F23 7/14/2004 Osage 3
F24 7/14/2004 Osage 4
G1 7/9/2004 Carolina Beauty 14 0
G2 7/9/2004 Carolina Beauty 14 2
G3 7/9/2004 Carolina Beauty 0
G4 7/9/2004 Carolina Beauty 1
G9 7/9/2004 Tuskegee 13 1
G10 7/9/2004 Tuskegee 13 1
G11 7/9/2004 Tuskegee 0
G12 7/9/2004 Tuskegee 0
G13 7/9/2004 Sioux 15 1
G14 7/9/2004 Sioux 12 2
G15 7/9/2004 Sioux 1
G16 7/9/2004 Sioux 1
G21 7/9/2004 Tuscarora 17 0
G22 7/9/2004 Tuscarora 15 0
G23 7/9/2004 Tuscarora 0
G24 7/9/2004 Tuscarora 2
H1 7/9/2004 Natchez 36 1
H2 7/9/2004 Natchez 33 1
H3 7/9/2004 Natchez 1
H4 7/9/2004 Natchez 0
H9 7/9/2004 Miami 8 0
H10 7/9/2004 Miami 10 1
H11 7/9/2004 Miami 0
H12 7/9/2004 Miami 4
H13 7/9/2004 Appalachee 18 1
H14 7/9/2004 Appalachee 20 1
H15 7/9/2004 Appalachee 1
H16 7/9/2004 Appalachee 1
H17 7/9/2004 Biloxi 6 1
H18 7/9/2004 Biloxi 7 1
H19 7/9/2004 Biloxi 0
H20 7/9/2004 Biloxi 0
H21 7/9/2004 Osage 19 1
H22 7/9/2004 Osage 13 0
H23 7/9/2004 Osage 0
H24 7/9/2004 Osage 0
H25 7/9/2004 Tonto 18 1
H26 7/9/2004 Tonto 12 0
H27 7/9/2004 Tonto 2










H28 7/9/2004 Tonto 0
Al 7/20/2004 Biloxi 17 4
A2 7/20/2004 Biloxi 22 4
A3 7/20/2004 Biloxi 4
A4 7/20/2004 Biloxi 5
A5 7/20/2004 Carolina Beauty 12 5
A6 7/20/2004 Carolina Beauty 7 5
A7 7/20/2004 Carolina Beauty 10
A8 7/20/2004 Carolina Beauty 2
A13 7/20/2004 Sioux 11 5
A14 7/20/2004 Sioux 12 6
A15 7/20/2004 Sioux 1
A16 7/20/2004 Sioux 0
A17 7/20/2004 Appalachee 18 4
A18 7/20/2004 Appalachee 21 4
A19 7/20/2004 Appalachee 2
A20 7/20/2004 Appalachee 1
A21 7/20/2004 Osage 40 10
A22 7/20/2004 Osage 37 7
A23 7/20/2004 Osage 5
A24 7/20/2004 Osage 4
A25 7/20/2004 Miami 22 2
A26 7/20/2004 Miami 28 6
A27 7/20/2004 Miami 6
A28 7/20/2004 Miami 0
B9 7/22/2004 Tuscarora 11 6
B10 7/22/2004 Tuscarora 14 6
B11 7/22/2004 Tuscarora 1
B12 7/22/2004 Tuscarora 2
B13 7/22/2004 Tuskegee 4 0
B14 7/22/2004 Tuskegee 5 1
B15 7/22/2004 Tuskegee 0
B16 7/22/2004 Tuskegee 2
B17 7/29/2004 Natchez 20 4
B18 7/29/2004 Natchez 15 0
B19 7/29/2004 Natchez 1
B20 7/29/2004 Natchez 2
B25 7/29/2004 Tonto 76 9
B26 7/29/2004 Tonto 40 5
B27 7/29/2004 Tonto 12
B28 7/29/2004 Tonto 10
C5 8/18/2004 Natchez 2 3
C6 8/18/2004 Natchez 2 4
C8 8/18/2004 Natchez 1
C9 8/18/2004 Osage 0 0
C10 8/18/2004 Osage 5 0
C11 8/18/2004 Osage 2
C12 8/18/2004 Osage 3










C13 8/18/2004 Tonto 0 1
C14 8/18/2004 Tonto 1 1
C15 8/18/2004 Tonto 3
C16 8/18/2004 Tonto 6
C17 8/18/2004 Biloxi 1 2
C18 8/18/2004 Biloxi 0 0
C19 8/18/2004 Biloxi 0
C20 8/18/2004 Biloxi 0
C21 8/18/2004 Carolina Beauty 1 1
C22 8/18/2004 Carolina Beauty 2 2
C23 8/18/2004 Carolina Beauty 0
C24 8/18/2004 Carolina Beauty 1
D6 8/18/2004 Appalachee 2 6
D7 8/18/2004 Appalachee 0 0
D8 8/18/2004 Appalachee 0
D9 8/18/2004 Miami 0 5
D10 8/18/2004 Miami 1 5
D11 8/18/2004 Miami 1
D12 8/18/2004 Miami 2
D17 8/18/2004 Tuscarora 3 3
D18 8/18/2004 Tuscarora 2 3
D19 8/18/2004 Tuscarora 0
D20 8/18/2004 Tuscarora 3
D21 8/18/2004 Tuskegee 0 0
D22 8/18/2004 Tuskegee 0 2
D23 8/18/2004 Tuskegee 0
D24 8/18/2004 Tuskegee 0
El 8/19/2004 Tuskegee 0 9
E2 8/19/2004 Tuskegee 2 13
E3 8/19/2004 Tuskegee 4
E4 8/19/2004 Tuskegee 1
E9 8/19/2004 Biloxi 2 3
E10 8/19/2004 Biloxi 3 3
E11 8/19/2004 Biloxi 1
E12 8/19/2004 Biloxi 0
E13 8/19/2004 Miami 0 3
E14 8/19/2004 Miami 1 3
E15 8/19/2004 Miami 5
E16 8/19/2004 Miami 2
E17 8/19/2004 Sioux 7 1
E18 8/19/2004 Sioux 5 2
E19 8/19/2004 Sioux 1
E20 8/19/2004 Sioux 2
E21 8/19/2004 Natchez 7 5
E22 8/19/2004 Natchez 5 5
E23 8/19/2004 Natchez 2
E24 8/19/2004 Natchez 5
E25 8/19/2004 Appalachee 1 1










E26 8/19/2004 Appalachee 3 2
E27 8/19/2004 Appalachee 2
E28 8/19/2004 Appalachee 2
F5 8/20/2004 Tonto 6 7
F6 8/20/2004 Tonto 0 1
F7 8/20/2004 Tonto 5
F8 8/20/2004 Tonto 4
F9 8/20/2004 Tuscarora 2 4
F10 8/20/2004 Tuscarora 4 3
F11 8/20/2004 Tuscarora 2
F12 8/20/2004 Tuscarora 3
F17 8/20/2004 Carolina Beauty 2 1
F18 8/20/2004 Carolina Beauty 2 2
F19 8/20/2004 Carolina Beauty 1
F20 8/20/2004 Carolina Beauty 4
F21 8/20/2004 Osage 3 6
F22 8/20/2004 Osage 3 3
F23 8/20/2004 Osage 4
F24 8/20/2004 Osage 6
G1 8/9/2004 Carolina Beauty 7 1
G2 8/9/2004 Carolina Beauty 9 5
G3 8/9/2004 Carolina Beauty 3
G4 8/9/2004 Carolina Beauty 2
G9 8/9/2004 Tuskegee 0 1
G10 8/9/2004 Tuskegee 2 1
G11 8/9/2004 Tuskegee 0
G12 8/9/2004 Tuskegee 0
G13 8/9/2004 Sioux 12 2
G14 8/9/2004 Sioux 14 3
G15 8/9/2004 Sioux 2
G16 8/9/2004 Sioux 0
G21 8/9/2004 Tuscarora 2 1
G22 8/9/2004 Tuscarora 2 3
G23 8/9/2004 Tuscarora 1
G24 8/9/2004 Tuscarora 2
H1 8/17/2004 Natchez 8 1
H2 8/17/2004 Natchez 5 3
H3 8/17/2004 Natchez 4
H4 8/17/2004 Natchez 2
H9 8/17/2004 Miami 1 5
H10 8/17/2004 Miami 5 8
H11 8/17/2004 Miami 3
H12 8/17/2004 Miami 5
H13 8/17/2004 Appalachee 1 1
H14 8/17/2004 Appalachee 0 0
H15 8/17/2004 Appalachee 1
H16 8/17/2004 Appalachee 0
H17 8/17/2004 Biloxi 1 0










H18 8/17/2004 Biloxi 0 0
H19 8/17/2004 Biloxi 0
H20 8/17/2004 Biloxi 0
H21 8/17/2004 Osage 0 1
H22 8/17/2004 Osage 0 0
H23 8/17/2004 Osage 0
H24 8/17/2004 Osage 0
H25 8/17/2004 Tonto 3 3
H26 8/17/2004 Tonto 8 0
H27 8/17/2004 Tonto 2
H28 8/17/2004 Tonto 4
Al 8/21/2004 Tonto 3 6
A2 8/21/2004 Biloxi 10 11
A3 8/21/2004 Biloxi 5
A4 8/21/2004 Biloxi 11
A6 8/21/2004 Carolina Beauty 0 9
A7 8/21/2004 Carolina Beauty 0 10
A8 8/21/2004 Carolina Beauty 1
A13 8/21/2004 Carolina Beauty 3 8
A14 8/21/2004 Sioux 1 6
A15 8/21/2004 Sioux 3
A16 8/21/2004 Sioux 0
A17 8/21/2004 Sioux 1 4
A18 8/21/2004 Appalachee 0 6
A19 8/21/2004 Appalachee 4
A20 8/21/2004 Appalachee 3
A21 8/21/2004 Appalachee 3 18
A22 8/21/2004 Osage 4 8
A23 8/21/2004 Osage 9
A24 8/21/2004 Osage 10
A25 8/21/2004 Osage 7 5
A26 8/21/2004 Miami 8 11
A27 8/21/2004 Miami 13
A28 8/21/2004 Miami 9
B9 8/28/2004 Miami 1 7
B10 8/28/2004 Tuscarora 0 8
B11 8/28/2004 Tuscarora 3
B12 8/28/2004 Tuscarora 4
B13 8/28/2004 Tuscarora 2 2
B14 8/28/2004 Tuskegee 3 2
B15 8/28/2004 Tuskegee 1
B16 8/28/2004 Tuskegee 2
B17 8/28/2004 Tuskegee 0 4
B18 8/28/2004 Natchez 0 0
B19 8/28/2004 Natchez 4
B20 8/28/2004 Natchez 3
B25 8/28/2004 Natchez 1 18
B26 8/28/2004 Tonto 1 8










B27 8/28/2004 Tonto 13
B28 8/28/2004 Tonto 11


2005 Field Plot Data


Date Week Replicate Tree Code Cultivar Tree # Live GWSS Cadavers
5/12/2005 1 Rep. 1-Misted Al Biloxi 1 0 0
A2 2 0 0
A3 3 0 0
A4 4 0 0
A21 Osage 1 0 0
A22 2 1 0
A23 3 1 0
A24 4 0 0
A25 Miami 1 0 0
A26 2 0 0
A27 3 0 0
A28 4 0 0
B25 Tonto 1 0 0
B26 2 0 0
B27 3 0 0
B28 4 0 0
Rep. 1-Dry C17 Biloxi 1 0 0
C18 2 0 0
C19 3 0 0
C20 4 0 0
C9 Osage 1 0 0
C10 2 0 0
C11 3 0 0
C12 4 0 0
D9 Miami 1 0 0
D10 2 0 0
D11 3 0 0
D12 4 0 0
C13 Tonto 1 0 0
C14 2 0 0
C15 3 0 0
C16 4 0 0
5/13/2005 Rep. 2-Misted E9 Biloxi 1 0 0
E10 2 0 0
Ell 3 2 0
E12 4 0 0
F21 Osage 1 0 0
F22 2 0 0
F23 3 0 0
F24 4 0 0
E13 Miami 1 0 0










E14 2 0 0
E15 3 0 0
E16 4 0 0
F5 Tonto 1 0 0
F6 2 0 0
F7 3 0 0
F8 4 0 0
Rep. 2-Dry H17 Biloxi 1 0 0
H18 2 0 0
H19 3 0 0
H20 4 0 0
H21 Osage 1 0 0
H22 2 0 0
H23 3 0 0
H24 4 0 0
H9 Miami 1 0 0
H10 2 0 0
Hll 3 0 0
H12 4 0 0
H25 Tonto 1 0 0
H26 2 0 0
H27 3 0 0
H28 4 0 0
5/16/2005 2 Rep. 1-Misted Al Biloxi 1 0 0
A2 2 2 0
A3 3 0 0
A4 4 0 0
A21 Osage 1 0 0
A22 2 0 0
A23 3 0 0
A24 4 0 0
A25 Miami 1 0 0
A26 2 1 0
A27 3 0 0
A28 4 0 0
B25 Tonto 1 0 0
B26 2 0 0
B27 3 0 0
B28 4 0 0
5/17/2005 Rep. 1-Dry C17 Biloxi 1 0 0
C18 2 0 0
C19 3 1 0
C20 4 0 0
C9 Osage 1 0 0
C10 2 0 0
C11 3 0 0
C12 4 0 0
D9 Miami 1 0 0










D10 2 0 0
D11 3 0 0
D12 4 0 0
C13 Tonto 1 0 0
C14 2 0 0
C15 3 0 0
C16 4 0 0
5/18/2005 Rep. 2-Misted E9 Biloxi 1 1 0
E10 2 0 0
Ell 3 0 0
E12 4 0 0
F21 Osage 1 0 0
F22 2 0 0
F23 3 0 0
F24 4 0 0
E13 Miami 1 1 0
E14 2 0 0
E15 3 0 0
E16 4 0 0
F5 Tonto 1 0 0
F6 2 0 0
F7 3 0 0
F8 4 0 0
5/19/2005 Rep. 2-Dry H17 Biloxi 1 0 0
H18 2 0 0
H19 3 0 0
H20 4 0 0
H21 Osage 1 0 0
H22 2 0 0
H23 3 0 0
H24 4 0 0
H9 Miami 1 0 0
H10 2 0 0
Hll 3 0 0
H12 4 1 0
H25 Tonto 1 0 0
H26 2 0 0
H27 3 0 0
H28 4 0 0
5/23/2005 3 Rep. 1-Misted Al Biloxi 1 0 0
A2 2 0 0
A3 3 1 0
A4 4 0 0
A21 Osage 1 0 0
A22 2 1 0
A23 3 0 0
A24 4 0 0
A25 Miami 1 0 0










A26 2 0 0
A27 3 0 0
A28 4 0 0
B25 Tonto 1 0 0
B26 2 0 0
B27 3 0 0
B28 4 0 0
5/24/2005 Rep. 1-Dry C17 Biloxi 1 0 0
C18 2 0 0
C19 3 0 0
C20 4 0 0
C9 Osage 1 0 0
C10 2 0 0
C11 3 0 0
C12 4 0 0
D9 Miami 1 0 0
D10 2 0 0
D11 3 0 0
D12 4 0 0
C13 Tonto 1 0 0
C14 2 0 0
C15 3 1 0
C16 4 1 0
5/25/2005 Rep. 2-Misted E9 Biloxi 1 0 0
E10 2 1 0
Ell 3 0 0
E12 4 0 0
F21 Osage 1 1 0
F22 2 0 0
F23 3 0 0
F24 4 0 0
E13 Miami 1 0 0
E14 2 0 0
E15 3 0 0
E16 4 0 0
F5 Tonto 1 2 0
F6 2 1 0
F7 3 0 0
F8 4 0 0
5/26/2005 Rep. 2-Dry H17 Biloxi 1 0 0
H18 2 0 0
H19 3 0 0
H20 4 0 0
H21 Osage 1 0 0
H22 2 0 0
H23 3 0 0
H24 4 0 0
H9 Miami 1 0 0










H10 2 0 0
Hll 3 0 0
H12 4 0 0
H25 Tonto 1 0 0
H26 2 0 0
H27 3 0 0
H28 4 0 0
5/30/2005 4 Rep. 1-Misted Al Biloxi 1 2 0
A2 2 4 0
A3 3 2 0
A4 4 0 0
A21 Osage 1 0 0
A22 2 0 0
A23 3 0 0
A24 4 0 0
A25 Miami 1 0 0
A26 2 2 0
A27 3 0 0
A28 4 0 0
B25 Tonto 1 0 0
B26 2 0 0
B27 3 1 0
B28 4 0 0
5/31/2005 Rep. 1-Dry C17 Biloxi 1 0 0
C18 2 0 0
C19 3 0 0
C20 4 0 0
C9 Osage 1 0 0
C10 2 1 0
C11 3 0 0
C12 4 0 0
D9 Miami 1 0 0
D10 2 0 0
D11 3 0 0
D12 4 0 0
C13 Tonto 1 0 0
C14 2 0 0
C15 3 0 0
C16 4 0 0
6/1/2005 Rep. 2-Misted E9 Biloxi 1 0 0
E10 2 0 0
Ell 3 0 0
E12 4 0 0
F21 Osage 1 0 0
F22 2 0 0
F23 3 0 0
F24 4 0 0
E13 Miami 1 0 0










E14 2 0 0
E15 3 0 0
E16 4 0 0
F5 Tonto 1 0 0
F6 2 0 0
F7 3 1 0
F8 4 0 0
6/2/2005 Rep. 2-Dry H17 Biloxi 1 0 0
H18 2 1 0
H19 3 0 0
H20 4 0 0
H21 Osage 1 0 0
H22 2 0 0
H23 3 0 0
H24 4 0 0
H9 Miami 1 0 0
H10 2 0 0
Hll 3 0 0
H12 4 0 0
H25 Tonto 1 0 0
H26 2 0 0
H27 3 1 0
H28 4 0 0
6/6/2005 5 Rep. 1-Misted Al Biloxi 1 9 0
A2 2 11 0
A3 3 7 0
A4 4 6 0
A21 Osage 1 2 0
A22 2 1 0
A23 3 5 0
A24 4 6 0
A25 Miami 1 4 0
A26 2 5 0
A27 3 4 0
A28 4 1 0
B25 Tonto 1 3 0
B26 2 9 0
B27 3 9 0
B28 4 4 0
6/7/2005 Rep. 1-Dry C17 Biloxi 1 4 0
C18 2 1 0
C19 3 4 0
C20 4 6 0
C9 Osage 1 8 0
C10 2 6 0
C11 3 7 0
C12 4 4 0
D9 Miami 1 5 0










D10 2 0 0
D11 3 3 0
D12 4 1 0
C13 Tonto 1 0 0
C14 2 4 0
C15 3 4 0
C16 4 5 0
6/8/2005 Rep. 2-Misted E9 Biloxi 1 3 0
E10 2 6 0
Ell 3 8 0
E12 4 1 0
F21 Osage 1 2 0
F22 2 4 0
F23 3 3 0
F24 4 10 0
E13 Miami 1 0 0
E14 2 4 0
E15 3 1 0
E16 4 2 0
F5 Tonto 1 4 0
F6 2 5 0
F7 3 9 0
F8 4 5 0
6/9/2005 Rep. 2-Dry H17 Biloxi 1 2 0
H18 2 2 0
H19 3 2 0
H20 4 5 0
H21 Osage 1 2 0
H22 2 3 0
H23 3 3 0
H24 4 6 0
H9 Miami 1 3 0
H10 2 3 0
Hll 3 6 0
H12 4 3 0
H25 Tonto 1 10 0
H26 2 0 0
H27 3 11 0
H28 4 6 0
6/13/2005 6 Rep. 1-Misted Al Biloxi 1 44 0
A2 2 41 0
A3 3 39 0
A4 4 39 0
A21 Osage 1 13 0
A22 2 16 0
A23 3 11 0
A24 4 12 0
A25 Miami 1 11 0










A26 2 7 0
A27 3 8 0
A28 4 14 0
B25 Tonto 1 10 0
B26 2 10 0
B27 3 13 0
B28 4 16 0
6/14/2005 Rep. 1-Dry C17 Biloxi 1 0 0
C18 2 3 0
C19 3 3 0
C20 4 3 0
C9 Osage 1 1 0
C10 2 7 0
C11 3 3 0
C12 4 1 0
D9 Miami 1 2 0
D10 2 2 0
D11 3 4 0
D12 4 4 0
C13 Tonto 1 0 0
C14 2 3 0
C15 3 4 0
C16 4 4 0
6/15/2005 Rep. 2-Misted E9 Biloxi 1 14 0
E10 2 12 0
Ell 3 13 0
E12 4 19 0
F21 Osage 1 10 0
F22 2 9 0
F23 3 6 0
F24 4 15 0
E13 Miami 1 10 0
E14 2 9 0
E15 3 8 0
E16 4 14 0
F5 Tonto 1 17 0
F6 2 18 0
F7 3 17 0
F8 4 13 0
6/16/2005 Rep. 2-Dry H17 Biloxi 1 7 0
H18 2 15 0
H19 3 8 0
H20 4 6 0
H21 Osage 1 10 0
H22 2 10 0
H23 3 9 0
H24 4 9 0
H9 Miami 1 4 0










H10 2 8 0
Hll 3 9 0
H12 4 8 0
H25 Tonto 1 9 0
H26 2 11 0
H27 3 10 0
H28 4 20 0
6/20/2005 7 Rep. 1-Misted Al Biloxi 1 84 0
A2 2 59 0
A3 3 50 0
A4 4 49 0
A21 Osage 1 16 0
A22 2 14 0
A23 3 13 0
A24 4 16 0
A25 Miami 1 12 0
A26 2 8 0
A27 3 11 0
A28 4 14 0
B25 Tonto 1 15 0
B26 2 12 0
B27 3 13 0
B28 4 16 0
6/21/2005 Rep. 1-Dry C17 Biloxi 1 5 0
C18 2 6 0
C19 3 5 0
C20 4 8 0
C9 Osage 1 8 0
C10 2 7 0
C11 3 7 0
C12 4 4 0
D9 Miami 1 4 0
D10 2 5 0
D11 3 4 0
D12 4 3 0
C13 Tonto 1 2 0
C14 2 5 0
C15 3 5 0
C16 4 4 0
6/22/2005 Rep. 2-Misted E9 Biloxi 1 29 0
E10 2 23 0
Ell 3 23 0
E12 4 17 0
F21 Osage 1 13 0
F22 2 7 0
F23 3 8 0
F24 4 16 0
E13 Miami 1 12 0










E14 2 18 0
E15 3 13 0
E16 4 16 0
F5 Tonto 1 19 1
F6 2 17 0
F7 3 16 0
F8 4 12 0
6/23/2005 Rep. 2-Dry H17 Biloxi 1 5 0
H18 2 9 0
H19 3 6 0
H20 4 6 0
H21 Osage 1 6 0
H22 2 7 0
H23 3 10 0
H24 4 9 0
H9 Miami 1 8 0
H10 2 4 0
Hll 3 5 0
H12 4 8 0
H25 Tonto 1 4 0
H26 2 3 0
H27 3 6 0
H28 4 9 1
6/27/2005 8 Rep. 1-Misted Al Biloxi 1 82 0
A2 2 89 0
A3 3 65 0
A4 4 40 0
A21 Osage 1 16 0
A22 2 12 0
A23 3 22 0
A24 4 21 0
A25 Miami 1 15 0
A26 2 17 0
A27 3 17 0
A28 4 18 0
B25 Tonto 1 14 0
B26 2 16 0
B27 3 13 0
B28 4 12 0
6/28/2005 Rep. 1-Dry C17 Biloxi 1 2 0
C18 2 3 0
C19 3 3 0
C20 4 3 0
C9 Osage 1 4 0
C10 2 6 0
C11 3 6 0
C12 4 2 0
D9 Miami 1 2 0










D10 2 1 0
D11 3 5 0
D12 4 1 0
C13 Tonto 1 5 0
C14 2 1 0
C15 3 5 0
C16 4 3 0
6/29/2005 Rep. 2-Misted E9 Biloxi 1 7 0
E10 2 2 0
Ell 3 5 0
E12 4 7 0
F21 Osage 1 9 0
F22 2 5 0
F23 3 10 0
F24 4 7 1
E13 Miami 1 4 0
E14 2 9 0
E15 3 5 0
E16 4 5 0
F5 Tonto 1 7 1
F6 2 7 0
F7 3 6 0
F8 4 8 0
6/30/2005 Rep. 2-Dry H17 Biloxi 1 6 0
H18 2 8 0
H19 3 4 0
H20 4 4 0
H21 Osage 1 5 0
H22 2 5 0
H23 3 7 0
H24 4 6 0
H9 Miami 1 9 0
H10 2 6 0
H11 3 10 0
H12 4 6 0
H25 Tonto 1 2 0
H26 2 5 0
H27 3 5 1
H28 4 8 1
7/5/2005 9 Rep. 1-Misted Al Biloxi 1 51 0
A2 2 50 0
A3 3 40 0
A4 4 35 0
A21 Osage 1 15 0
A22 2 14 0
A23 3 17 0
A24 4 13 0
A25 Miami 1 9 0










A26 2 12 0
A27 3 8 0
A28 4 13 0
B25 Tonto 1 11 0
B26 2 13 0
B27 3 11 0
B28 4 17 0
7/5/2005 Rep. 1-Dry C17 Biloxi 1 6 0
C18 2 2 0
C19 3 1 0
C20 4 4 0
C9 Osage 1 6 0
C10 2 7 0
C11 3 5 0
C12 4 4 0
D9 Miami 1 3 0
D10 2 6 0
D11 3 8 0
D12 4 4 0
C13 Tonto 1 2 0
C14 2 1 0
C15 3 1 0
C16 4 2 0
7/6/2005 Rep. 2-Misted E9 Biloxi 1 7 1
E10 2 10 0
Ell 3 9 0
E12 4 9 0
F21 Osage 1 8 0
F22 2 7 0
F23 3 5 0
F24 4 8 0
E13 Miami 1 7 0
E14 2 8 0
E15 3 6 0
E16 4 5 0
F5 Tonto 1 12 0
F6 2 7 0
F7 3 13 0
F8 4 8 0
7/7/2005 Rep. 2-Dry H17 Biloxi 1 3 0
H18 2 4 0
H19 3 9 0
H20 4 7 0
H21 Osage 1 8 0
H22 2 7 0
H23 3 7 0
H24 4 7 0
H9 Miami 1 3 0










H10 2 9 0
Hll 3 6 0
H12 4 7 0
H25 Tonto 1 5 0
H26 2 5 0
H27 3 4 1
H28 4 10 1
7/12/2005 10 Rep. 1-Misted Al Biloxi 1 48 0
A2 2 55 0
A3 3 37 0
A4 4 26 0
A21 Osage 1 6 0
A22 2 7 0
A23 3 6 1
A24 4 8 0
A25 Miami 1 3 0
A26 2 8 0
A27 3 8 0
A28 4 10 0
B25 Tonto 1 2 0
B26 2 4 0
B27 3 10 0
B28 4 13 0
7/13/2005 Rep. 1-Dry C17 Biloxi 1 3 0
C18 2 3 0
C19 3 2 0
C20 4 4 0
C9 Osage 1 11 0
C10 2 8 0
C11 3 6 0
C12 4 6 0
D9 Miami 1 3 0
D10 2 2 0
D11 3 3 0
D12 4 2 0
C13 Tonto 1 2 0
C14 2 1 0
C15 3 1 0
C16 4 1 0
7/14/2005 Rep. 2-Misted E9 Biloxi 1 9 0
E10 2 7 0
Ell 3 7 0
E12 4 4 0
F21 Osage 1 4 1
F22 2 9 0
F23 3 7 0
F24 4 11 0
E13 Miami 1 4 0










E14 2 7 0
E15 3 3 0
E16 4 4 0
F5 Tonto 1 13 1
F6 2 15 0
F7 3 10 1
F8 4 8 0
7/15/2005 Rep. 2-Dry H17 Biloxi 1 3 0
H18 2 2 0
H19 3 2 0
H20 4 4 0
H21 Osage 1 7 0
H22 2 3 0
H23 3 3 0
H24 4 9 0
H9 Miami 1 0 0
H10 2 4 0
Hll 3 6 0
H12 4 2 0
H25 Tonto 1 6 0
H26 2 4 0
H27 3 8 0
H28 4 11 2
7/18/2005 11 Rep. 1-Misted Al Biloxi 1 48 0
A2 2 48 0
A3 3 17 1
A4 4 15 0
A21 Osage 1 12 0
A22 2 9 0
A23 3 8 1
A24 4 13 0
A25 Miami 1 8 0
A26 2 6 1
A27 3 20 0
A28 4 15 1
B25 Tonto 1 21 0
B26 2 13 0
B27 3 14 0
B28 4 22 0
7/20/2005 Rep. 1-Dry C17 Biloxi 1 2 0
C18 2 1 0
C19 3 3 0
C20 4 4 0
C9 Osage 1 4 0
C10 2 3 0
C11 3 6 0
C12 4 3 0
D9 Miami 1 4 0










D10 2 5 0
D11 3 6 0
D12 4 3 0
C13 Tonto 1 1 0
C14 2 4 0
C15 3 2 0
C16 4 3 0
7/22/2005 Rep. 2-Misted E9 Biloxi 1 3 0
E10 2 3 0
Ell 3 3 0
E12 4 4 0
F21 Osage 1 4 1
F22 2 7 0
F23 3 5 1
F24 4 7 0
E13 Miami 1 1 0
E14 2 1 0
E15 3 3 0
E16 4 2 0
F5 Tonto 1 6 0
F6 2 5 1
F7 3 10 2
F8 4 9 0
7/23/2005 Rep. 2-Dry H17 Biloxi 1 3 0
H18 2 3 0
H19 3 2 0
H20 4 11 0
H21 Osage 1 5 0
H22 2 9 0
H23 3 6 0
H24 4 7 0
H9 Miami 1 3 0
H10 2 3 0
Hll 3 6 0
H12 4 8 0
H25 Tonto 1 12 0
H26 2 13 0
H27 3 9 0
H28 4 23 2
7/29/2005 12 Rep. 1-Misted Al Biloxi 1 41 0
A2 2 14 0
A3 3 13 1
A4 4 11 0
A21 Osage 1 11 0
A22 2 11 0
A23 3 11 1
A24 4 4 0
A25 Miami 1 6 0










A26 2 14 0
A27 3 10 1
A28 4 5 2
B25 Tonto 1 13 0
B26 2 5 0
B27 3 7 0
B28 4 11 0
Rep. 1-Dry C17 Biloxi 1 3 0
C18 2 3 0
C19 3 5 0
C20 4 9 0
C9 Osage 1 7 0
C10 2 7 0
C11 3 6 0
C12 4 5 0
D9 Miami 1 6 0
D10 2 8 0
D11 3 10 0
D12 4 5 0
C13 Tonto 1 5 0
C14 2 3 0
C15 3 3 0
C16 4 4 0
Rep. 2-Misted E9 Biloxi 1 3 0
E10 2 2 0
Ell 3 7 0
E12 4 3 0
F21 Osage 1 3 1
F22 2 4 0
F23 3 7 1
F24 4 8 0
E13 Miami 1 10 0
E14 2 7 0
E15 3 2 0
E16 4 3 0
F5 Tonto 1 6 0
F6 2 3 1
F7 3 5 2
F8 4 14 0
Rep. 2-Dry H17 Biloxi 1 12 0
H18 2 18 0
H19 3 10 0
H20 4 16 0
H21 Osage 1 17 0
H22 2 16 0
H23 3 14 0
H24 4 12 0
H9 Miami 1 6 0










H10 2 4 0
Hll 3 6 0
H12 4 5 0
H25 Tonto 1 26 0
H26 2 24 0
H27 3 18 1
H28 4 40 1
8/2/2005 13 Rep. 1-Misted Al Biloxi 1 15 0
A2 2 8 0
A3 3 20 1
A4 4 9 1
A21 Osage 1 3 0
A22 2 10 0
A23 3 6 1
A24 4 9 0
A25 Miami 1 14 0
A26 2 15 0
A27 3 9 2
A28 4 13 2
B25 Tonto 1 9 0
B26 2 5 0
B27 3 11 0
B28 4 5 0
Rep. 1-Dry C17 Biloxi 1 5 0
C18 2 4 1
C19 3 4 0
C20 4 11 0
C9 Osage 1 6 0
C10 2 6 0
C11 3 3 0
C12 4 7 0
D9 Miami 1 12 0
D10 2 14 0
D11 3 12 0
D12 4 7 0
C13 Tonto 1 5 0
C14 2 1 0
C15 3 6 0
C16 4 3 0
Rep. 2-Misted E9 Biloxi 1 4 0
E10 2 1 0
Ell 3 4 0
E12 4 4 0
F21 Osage 1 5 1
F22 2 6 0
F23 3 6 1
F24 4 2 1
E13 Miami 1 4 0










E14 2 3 0
E15 3 3 0
E16 4 7 0
F5 Tonto 1 8 0
F6 2 3 1
F7 3 9 2
F8 4 4 0
Rep. 2-Dry H17 Biloxi 1 8 0
H18 2 12 0
H19 3 9 0
H20 4 12 0
H21 Osage 1 15 0
H22 2 10 0
H23 3 10 0
H24 4 12 0
H9 Miami 1 5 0
H10 2 12 0
H11 3 13 0
H12 4 5 0
H25 Tonto 1 20 0
H26 2 13 0
H27 3 14 1
H28 4 36 1
8/18/2005 15 Rep. 1-Misted Al Biloxi 1 11 0
A2 2 10 0
A3 3 12 2
A4 4 14 0
A21 Osage 1 10 0
A22 2 17 0
A23 3 26 2
A24 4 20 0
A25 Miami 1 19 0
A26 2 13 1
A27 3 18 2
A28 4 17 2
B25 Tonto 1 8 0
B26 2 6 0
B27 3 6 0
B28 4 8 0
Rep. 1-Dry C17 Biloxi 1 4 0
C18 2 8 0
C19 3 4 0
C20 4 6 0
C9 Osage 1 9 0
C10 2 6 0
C11 3 4 0
C12 4 11 0
D9 Miami 1 5 0










D10 2 11 0
D11 3 7 0
D12 4 7 0
C13 Tonto 1 3 0
C14 2 1 0
C15 3 2 0
C16 4 3 0
Rep. 2-Misted E9 Biloxi 1 4 0
E10 2 3 0
Ell 3 4 0
E12 4 1 0
F21 Osage 1 6 1
F22 2 8 0
F23 3 4 2
F24 4 9 1
E13 Miami 1 6 0
E14 2 11 0
E15 3 6 0
E16 4 9 0
F5 Tonto 1 2 0
F6 2 0 0
F7 3 2 3
F8 4 1 0
Rep. 2-Dry H17 Biloxi 1 9 0
H18 2 11 0
H19 3 6 0
H20 4 9 0
H21 Osage 1 5 0
H22 2 4 0
H23 3 4 0
H24 4 4 0
H9 Miami 1 4 0
H10 2 7 0
Hll 3 6 0
H12 4 4 0
H25 Tonto 1 3 0
H26 2 2 0
H27 3 0 1
H28 4 10 0
8/24/2005 16 Rep. 1-Misted Al Biloxi 1 7 0
A2 2 12 0
A3 3 9 2
A4 4 11 0
A21 Osage 1 16 1
A22 2 15 0
A23 3 27 2
A24 4 24 0
A25 Miami 1 13 0










A26 2 5 1
A27 3 6 2
A28 4 11 2
B25 Tonto 1 2 0
B26 2 2 1
B27 3 1 0
B28 4 2 0
Rep. 1-Dry C17 Biloxi 1 1 0
C18 2 5 0
C19 3 2 0
C20 4 4 0
C9 Osage 1 2 0
C10 2 4 0
C11 3 2 0
C12 4 2 0
D9 Miami 1 7 0
D10 2 3 0
D11 3 3 0
D12 4 3 0
C13 Tonto 1 1 0
C14 2 2 0
C15 3 1 0
C16 4 1 0
Rep. 2-Misted E9 Biloxi 1 1 0
E10 2 0 0
Ell 3 1 0
E12 4 1 0
F21 Osage 1 4 1
F22 2 3 0
F23 3 8 2
F24 4 6 1
E13 Miami 1 8 0
E14 2 6 0
E15 3 4 0
E16 4 4 0
F5 Tonto 1 2 0
F6 2 1 0
F7 3 0 3
F8 4 0 1
Rep. 2-Dry H17 Biloxi 1 2 0
H18 2 1 0
H19 3 4 0
H20 4 5 0
H21 Osage 1 4 0
H22 2 1 0
H23 3 2 0
H24 4 6 0
H9 Miami 1 4 0










H10 2 4 0
Hll 3 6 0
H12 4 4 0
H25 Tonto 1 2 0
H26 2 1 0
H27 3 0 1
H28 4 7 0
9/7/2005 18 Rep. 1-Misted Al Biloxi 1 21 0
A2 2 20 2
A3 3 19 2
A4 4 10 0
A21 Osage 1 18 1
A22 2 16 0
A23 3 25 2
A24 4 17 0
A25 Miami 1 13 0
A26 2 9 1
A27 3 0 1
A28 4 5 1
B25 Tonto 1 2 0
B26 2 0 1
B27 3 1 0
B28 4 2 0
Rep. 1-Dry C17 Biloxi 1 5 0
C18 2 3 0
C19 3 0 0
C20 4 4 0
C9 Osage 1 8 0
C10 2 2 0
C11 3 4 0
C12 4 2 0
D9 Miami 1 3 0
D10 2 3 0
D11 3 1 0
D12 4 4 0
C13 Tonto 1 0 0
C14 2 2 0
C15 3 3 0
C16 4 2 0
Rep. 2-Misted E9 Biloxi 1 1 0
E10 2 0 0
Ell 3 1 0
E12 4 0 0
F21 Osage 1 2 1
F22 2 2 0
F23 3 2 1
F24 4 2 1
E13 Miami 1 2 0










E14 2 0 0
E15 3 2 0
E16 4 5 0
F5 Tonto 1 1 0
F6 2 0 0
F7 3 1 3
F8 4 0 1
Rep. 2-Dry H17 Biloxi 1 0 0
H18 2 3 0
H19 3 1 0
H20 4 1 0
H21 Osage 1 3 1
H22 2 4 0
H23 3 1 0
H24 4 3 0
H9 Miami 1 6 0
H10 2 4 0
Hll 3 8 0
H12 4 4 0
H25 Tonto 1 5 0
H26 2 1 0
H27 3 4 3
H28 4 1 1
9/14/2005 19 Rep. 1-Misted Al Biloxi 1 27 1
A2 2 25 2
A3 3 24 2
A4 4 13 1
A21 Osage 1 8 1
A22 2 10 0
A23 3 8 2
A24 4 13 0
A25 Miami 1 9 0
A26 2 9 1
A27 3 3 1
A28 4 3 1
B25 Tonto 1 1 0
B26 2 1 1
B27 3 1 0
B28 4 0 0
Rep. 1-Dry C17 Biloxi 1 0 0
C18 2 3 0
C19 3 3 0
C20 4 2 0
C9 Osage 1 0 0
C10 2 7 0
C11 3 3 1
C12 4 1 0
D9 Miami 1 5 0










D10 2 4 0
D11 3 6 0
D12 4 2 1
C13 Tonto 1 0 0
C14 2 1 0
C15 3 2 0
C16 4 2 0
Rep. 2-Misted E9 Biloxi 1 1 0
E10 2 1 0
Ell 3 1 0
E12 4 0 0
F21 Osage 1 4 1
F22 2 4 0
F23 3 2 1
F24 4 4 1
E13 Miami 1 9 0
E14 2 1 0
E15 3 2 0
E16 4 5 0
F5 Tonto 1 0 0
F6 2 0 0
F7 3 2 3
F8 4 1 1
Rep. 2-Dry H17 Biloxi 1 3 0
H18 2 3 0
H19 3 2 0
H20 4 2 0
H21 Osage 1 0 2
H22 2 3 0
H23 3 2 0
H24 4 1 0
H9 Miami 1 1 0
H10 2 2 0
Hll 3 11 0
H12 4 1 0
H25 Tonto 1 1 0
H26 2 0 0
H27 3 3 3
H28 4 0 0
9/21/2005 20 Rep. 1-Misted Al Biloxi 1 12
A2 2 8
A3 3 5
A4 4 8
A21 Osage 1 5
A22 2 4
A23 3 3
A24 4 10
A25 Miami 1 4










A26 2 6
A27 3 1
A28 4 4
B25 Tonto 1 1
B26 2 2
B27 3 0
B28 4 1
Rep. 1-Dry C17 Biloxi 1 0
C18 2 1
C19 3 2
C20 4 2
C9 Osage 1 3
C10 2 4
C11 3 5
C12 4 1
D9 Miami 1 2
D10 2 3
D11 3 0
D12 4 2
C13 Tonto 1 2
C14 2 0
C15 3 2
C16 4 3
Rep. 2-Misted E9 Biloxi 1 2
E10 2 1
Ell 3 0
E12 4 1
F21 Osage 1 0
F22 2 2
F23 3 2
F24 4 2
E13 Miami 1 2
E14 2 1
E15 3 3
E16 4 4
F5 Tonto 1 5
F6 2 2
F7 3 3
F8 4 3
Rep. 2-Dry H17 Biloxi 1 0
H18 2 3
H19 3 0
H20 4 1
H21 Osage 1 2
H22 2 3
H23 3 4
H24 4 6
H9 Miami 1 4










H10 2 2
Hll 3 6
H12 4 1
H25 Tonto 1 0
H26 2 0
H27 3 1
H28 4 1















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