BIOLOGY AND MANAGEMENT OF LEAFHOPPERS AND ASTER YELLOWS PHYTOPLASMA IN MICHIGAN CELERY AND CARROT AGROECOSYSTEMS By Patrick T . Stillson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Entomology Master of Science 20 20 ABSTRACT BIOLOGY AND MANAGEMENT OF ASTER LEAFHOPPERS AND ASTER YELLOWS PHYTOPLASMA IN MICHIGAN CELERY AND CARROT AGROECOSYSTEMS By Patrick T . Stillson Managing aster yellows phytoplasma ( Candidatus Phytoplasma s p.) and its leafhopper vector s is complex and requires better control methods than those currently used and a greater understanding of the phytoplasma - insect relationship . In this thesis, I determine the effectiveness of a decision support tool focused on manag ing phytoplasma infected leafhoppers and determine whether leafhoppers in celery and carrot field edges contribute to phytoplasma transmission within the crop fields . W e informed farmers about phytoplasma infectivity on their farms via a web - based te xt messaging system to shift county - level management of Macrosteles quadrilineatus from using leafhopper abundance to infectivity . We found that infected M . quadrilineatus abundance decreased after farmers were informed about their numbers , suggesting that our decision support tool allowed growers to successfully manage infected M . quadrilineatus . We also identified temporal differences in infected and uninfected leafhopper peak abundance in celery but not in carrot cropping systems , suggesting that farmers should account for these phenological shifts across crops and over time. In the field edge survey s , leafhoppers were collec ted from celery and carrot fields and field edges . I identified leafhoppers through DNA barcoding and conducted real - time PCR to det ermine phytoplasma infection status. The most abundant species were M . quadrilineatus ( 57% ) and Empoasca fabae ( 2 3 %). Our results confirmed that M. quadrilineatus was the primary vector in celery and carrots , although there is evidence that E. fabae may al so vector this pathogen . iii This thesis is dedicated to my husband Chris topher for his constant love and support iv ACKNOWLEDGMENTS I would like to thank my amazing advisor, Zsofia Szendrei, for providing the opportunity to join her l ab and for her encouragement and guidance throughout my M I would also like to thank my graduate committee members: Eric Benbow and Carolyn Malmstrom for challenging me and providing valuable advice and expertise which helped improve my research. I would like to t hank my fellow lab members for their support and friendship over the past two years: Logan Appenfeller, Eli Bloom, Natalie Constancio, Margie Lund, Josh Snook, Thomas W ood, and Jen Zavalnitskaya . Without them, the office would have been qui et and lonely , and a lot less productive or fun . I would also like to thank the undergrad uate students who helped with my research : Joe Burke , Danielle Miner, and Jack Rumery . Their h ard work included collecting leafhoppers , extracting DNA, and running qPC R to identify phytoplasma infected leafhopp ers, which helped me finish my research on time . I would like to thank Mark Crossley and Dr. Benjamin Werling for connecting us with celery and carrot growers and for helping to collect leafhoppers for this resear ch . I would also like to thank the celery a nd carrot growers who let us collect leafhoppers form their farms. Additionally, I would like to thank my husband for always believing in me, and for providing constant love and support . I would also like to thank my brother Tim Stillson for making the phy toplasma cycle figure used in Figure s 1.3 and 1.5. Lastly, I would like to acknowledge my funding sources that made this research possible: t he Michigan Celery Research Commission, the Michigan Carrot Committee, t he Michigan Vegetable Council, MDARD Specialty Crops Block Grant . v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ...................... vii LIST OF FIGURES ................................ ................................ ................................ ................... viii CHAPTER 1: Biology and management of aster leafhoppers and aster yellows phytoplasma in Michigan celery and carrot agroecosystems ................................ ................... 1 1 Economic impact ................................ ................................ ................................ .................... 1 2 Aster leaf hoppers ................................ ................................ ................................ .................... 2 2.1 Biology ................................ ................................ ................................ .............................. 2 2.2 Life history ................................ ................................ ................................ ........................ 3 2.3 Migration ................................ ................................ ................................ ........................... 3 3 Phytoplasma ................................ ................................ ................................ ............................ 4 3.1 Biology ................................ ................................ ................................ .............................. 4 3.2 Identification ................................ ................................ ................................ ...................... 5 3.3 Transmission ................................ ................................ ................................ ...................... 5 4 Aster leafhoppers as vectors of aster yellows phytoplasma ................................ ................ 7 4.1 Management ................................ ................................ ................................ ...................... 7 4.2 Monitoring ................................ ................................ ................................ ......................... 8 5 Leafhoppers of Michigan ................................ ................................ ................................ ....... 9 5.1 Vectors of aster yellows ................................ ................................ ................................ .... 9 5.2 Non - vector leafhoppers ................................ ................................ ................................ ... 10 6 Knowledge gaps ................................ ................................ ................................ .................... 10 7 Thesis objectives ................................ ................................ ................................ ................... 11 LITERATURE CITED ................................ ................................ ................................ .............. 13 CHAPTER 2: A novel plant pathogen management tool for vector management ............... 17 1 I ntroduction ................................ ................................ ................................ .......................... 17 2 Materials and methods ................................ ................................ ................................ ......... 20 2.1 System description ................................ ................................ ................................ ........... 20 2.2 Aster leafhopper diagnostics ................................ ................................ ........................... 22 2.3 Disseminating information ................................ ................................ .............................. 23 2.4 Statistical analysis ................................ ................................ ................................ ............ 24 3 Results ................................ ................................ ................................ ................................ ... 28 3.1 Leafhopper collections ................................ ................................ ................................ .... 28 3.2 Text messaging and infectivity ................................ ................................ ........................ 29 3.3 Leafhopper populations acro ss cropping systems ................................ ........................... 30 3.4 Leafhopper populations within cropping systems ................................ ........................... 32 3.5 Leafhopper populations across sampling points ................................ ............................... 33 4 Discussion ................................ ................................ ................................ .............................. 34 5 Conclusion ................................ ................................ ................................ ............................. 36 APPENDIX ................................ ................................ ................................ ................................ .. 38 LITERATURE CITED ................................ ................................ ................................ .............. 47 vi CHAPTER 3: Identifying leafhopper targets for controlling aster yellows in carrots and celery ................................ ................................ ................................ ................................ ............ 53 1 Introduction ................................ ................................ ................................ .......................... 53 2 Materials and methods ................................ ................................ ................................ ......... 54 2.1 Study system ................................ ................................ ................................ .................... 54 2.2 Leafhopper identification and phytoplasma d etection ................................ ..................... 56 2.3 Detection of phytoplasma ................................ ................................ ................................ 58 2.4 Data analysis ................................ ................................ ................................ .................... 58 3 Res ults ................................ ................................ ................................ ................................ ... 59 3.1 Celery collections ................................ ................................ ................................ ............ 62 3.2 Carrot collections ................................ ................................ ................................ ............. 62 3.3 Phytop lasma infectivity ................................ ................................ ................................ ... 66 4 Discussion ................................ ................................ ................................ .............................. 69 5 Conclusions ................................ ................................ ................................ ........................... 72 Acknowledgements ................................ ................................ ................................ .................. 72 LITERATURE CITED ................................ ................................ ................................ .............. 73 CHAPTER 4: Conclusions and future directions ................................ ................................ .... 81 APPENDIX ................................ ................................ ................................ ................................ .. 84 LITERATURE CITED ................................ ................................ ................................ .............. 90 vii LIST OF TABLES Table S2.1. The number of c ommercial celery farms and fields for aster leafhopper collect ions during the 2 014 2019 growing seasons in Michigan . A 1 indicat es a sampled field in a year . A ................................ ................................ .................. 39 Table S2. 2 . The number of c ommercial carrot farms and fields for aster leafhopper collect ions during the 2014 2019 grow ing seasons. A 1 indicat es a sampled field in a year that the field was not sampled. ................................ ................................ ................................ ....... 40 Table S2. 3 . Weekly mean densities of as ter leafhoppers (leafhoppers per 100 sweeps) collected throughout the 2014 2019 growing seasons in Michigan. Leafhoppers were collected with sweep nets from commercial carrot and celery farms. ................................ ................................ ............. 4 1 Table S2. 4 . Weekly mean abundances of aster yellows phytoplasma infected aster leafhoppers collected throughout the 2014 2019 growing seasons in Michigan. Leafhoppers w ere collected with sweep nets from commercial carrot and celery farms and identified as infected using qPCR based diagnostic methods. ................................ ................................ ................................ ............. 4 2 Ta ble 3.1. Leafhoppers collected from commercial celery and carrot farms in Michigan, USA, from 2018 to 2019. Field ed ges were defined as areas bordering the crop field or between adjacent fields where crops were not growing. Fractions indicate the number of individuals that generated - time PCR with universal phytoplasma primers 24 out o f the total number of individuals collected . ................................ ................................ ................................ .... 6 0 Table 3.2. Known leafhopper vectors of aster yellows phytoplasma or other phytoplasmas for the species collected in this study. Phytoplasma abbreviations are AWB = alfalfa witches broom, AshY = ash yellows, AYp = aster yellows, Cp = clover phyllody, CYE = clover yellow edge, EastX = Eastern X, EAYp = European aster yellows, GFD = Grape flavescence doree, NAGVY = North American grapevine yellows IIIB, Sp = stolbur, SGP = strawberry green petal. Diplocolenus su bg. verdanus , Doratura stylata , Forcipata loca , and Idiocerus raphus were omitted as there is no record of whether they or their congeners vector phytoplasmas. ................................ ............. 6 7 Table 3.3. Known leafhopper vectors of aster yellows phytoplasma or other phytoplasmas for t he genera collected in this study. Commellus sp., Draeculacephala sp . , Erythroneura sp . , and Graphocephala sp . were omitted as there is no record of whether species in these genera vector phytoplasmas . ................................ ................................ ................................ ................................ . 6 8 Table S4.1. Voucher specimens deposited at the Alb ert J. Cook Arthropod Research Collection (Michigan State University) . ................................ ................................ ................................ .......... 8 5 viii LIST OF FIGURES Figure 1.1. (A) Adult aster leafhopper on celery. (B) Aster leafhopper nymph on oats. ............... 2 Figure 1.2. Generalized migration map of aster leafhopper movement from Tex as and Mexico, to the Midwest. Migration begins in mid - May and lasts through early - June. During this time, the lea fhoppers move using seasonal wind currents. This map is based on those provided by Hoy at al. 18 for 1988 1990. ................................ ................................ ................................ ......................... 4 Figure 1.3. Pathway for phyt oplasma acquisition and transmission by hemipteran vectors. ......... 6 Figure 1.4. Summary of the current diagnostics work flow to inform growers about leafhopper infectivity in their fields. Aster leafhoppers are collected from celery or carrot fields, DNA is extr acted from the leafhoppers, qPCR is performed to determine if the leafhoppers are infected with aster yellows, and then the results are provided to farmers via a text message. ...................... 9 Figure 1.5. Current knowledge gaps in our understanding of phytoplasma acquis ition and transmission. These relate to how the vectors feed on the plants, molecular and physiological responses to the phytoplasma, and how the plants interact with the pathogen. Aster yellows phytoplasma = AYp, chrysanthemum yellows phytoplasma = CYp ................................ ............. 11 Figure 2.1. Aster yellows phytoplasma is transmitted by aster leafhoppers to carrots and celery. The economic damage is caused by the phytoplasma; therefore, it is important to assess the proportion of the leafhopper population that is infected . Leafhopp er management that is based on detecting the pathogen in the leafhopper could reduce yield losses. In our system, t he results of disease diagnostics are sent to farmers via a group text messages indicating rates of leafhopper infectivity and the action thr eshold for car rots and celery. If the text message indicates that infected leafhoppers have been detected, then farmers will respond by applying insecticides to their fields (research - mediated management). If the text message indicates that leafhoppers are not infected with the phytoplasma, then management action is not needed and we recommend growers not use insecticide management . Leafhoppers are collected regularly during the growing season and text messages are sent out approximately 24 h after collecti ng leafhoppers from the field, providing an opportunity for quick response, if necessary. ................................ ................................ ................ 1 8 Figure 2.2. Map of Michigan, USA, symbols i ndicate the locations of commercial carrot and celery fields where aster leafhoppers were collected from 2014 - 2019. Lea fhoppers were collected using sweep nets and were transported to the laboratory to determine aster yellows infectivity . 2 1 Figure 2.3. Abundance (mean ± SEM) of aster yellows phytoplasma infected leafhoppers during the 201 6 2019 growing season by the numb er of weeks since farmers received a text message indicating leafhopper infectivity. Text messages were sent to inform stakeholders of the percent of aster yellows phytoplasma infected aster leafhoppers in the population and the action threshol d for carrot and celery. Different letters above bars denote significant differences in abundance of infected leafhoppers across weeks. ................................ ................................ ................................ 29 ix Figure 2.4. (A) Mean ± SEM aster leafhopper density (leafhoppers per 100 sweeps) and (B) mean ± SEM abundance of aster leafhopper s infected with aster yellows phytoplasma found in carrots and celery. Leafhoppers were collected with sweep nets from commercial celery and ca rrot farms in Michigan from 2014 to 2019 and tested for phytoplasma using a qPCR based diagnostic method. Asteris k indicates significant differences between carrot and celery . ................................ ........... 3 0 Figure 2.5. Cross correlation analysis of the abundance of aster yellows phy toplasma infected aster leafhoppers in celery and carrot fields in Michigan from 2014 to 2019. Dotted lines indic ate a 95% confidence interval and each lag represents a week. (A) The cross - correlation value at a lag of - 2 indicates that the pattern of infe cted leafhopper abundance in carrot may be delayed by two weeks when compared to the weekly population pattern ob served in celery . (B) Total number of aster yellows phytoplasma infected aster leafhoppers plotted by week across the season in Michigan celery and carrot fields. The weeks on the x - axis correspond with the weeks of the calendar year. The line for carrots is shifted by two weeks to illustrate the two - week lag that was identified in the cross correlation. ................................ ................................ ................................ .. 3 1 Figure 2.6. Cross corre lation analysis of the density of aster leafhoppers (leafhoppers per 100 sweeps) and the abundance of aster yellows phytoplasma infected aster leafhoppers in celery fields in Michigan from 2014 to 2019. Dotted lines indicate 95% confidence interval and eac h lag represents a week. (A) The cross - correlation value at a lag of - 2 indicates that the weekly patterns of infected leafhoppe r abundance may be delayed by two weeks when compared to the weekly pattern of uninfected individuals . (B) Total density of aste r leafhoppers and the total abundance of aster yellows phytoplasma infected aster leafhoppers in Michigan celery fields , plotted by week. The line for infected leafhoppers was shifted two weeks to illustrate the two - week lag that was identified in the cros s - correlation. ................................ ................................ ................................ . 3 2 Figure 2. 7 . Correlation of leafhopper density and infectivity between sampling sites as a function of distanc e bands split by (A) celery and (B) carrot. (A) Celery sampling sites within 2.5 km were similar in leafhopper density and infec tivity. However, no correlation was found between sites beyond 2.5 km apart for either density or infectivity. (B) Carrot sites within 6 km where dissimilar in leafhopper density. No strong correlation was found for leafhopper density in carrot for sites i n distance bands beyond 6 km, nor was any correlation observed in the infected leafhopper population between carrot sites at any distance. ................................ ................................ ............ 3 3 Figure S2.1 . number of sites within each distance band for (A) celery and (B) carrot sites . ................................ ................................ .. 4 3 Figure S2.2. Temporal relationship between aster leafhopper populations in celery and carrot using c ross correlat ion analysis for aster leafhopper density (leafhoppers per 100 sweeps) in celery and carrot fields in Michigan during the 2014 2019 growing seasons. Dotted lines indicate 95% confidence interval and each lag represents a week. ( A) No correlation was found between leafhopper densities between the two crops. (B) Density of aster leafhoppers plotted by week across the season in Michigan celery and carrot fields. The weeks on the x - axis correspond with the weeks of the calendar year. ................................ ................................ ................................ ...... 4 4 x Figure S 2.3. Cross corr elation analysis for aster leafhopper density (leafhoppers per 100 sweeps) and the number of aster yellows phytoplasma infected leafhoppers in carrot fields in Michigan during the 2014 2019 growing seasons. Dotted lines indicate 95% confidence interval and each lag represents a week. (A) No correlation was found between leafhopper density and infectivity in carrots. (B) Density of aster leafhoppers and total number of aster yellows phytoplasma infected leafhoppers plotted by week across the season in Michig an carrot fields. The weeks on the x - axis correspond with the weeks of the calendar year. ................................ ................................ ........... 4 5 Figure 3.1. Map of collections sites from Michigan, USA. Symbols indicate locations of celery and carrot fields where leafhoppers were collected in 2018 and 2019. Leafhoppers were collected using sweep nets and transported to the laboratory for identification and to determine phytoplasma infectivity. ................................ ................................ ................................ ................................ ...... 5 5 Figure 3.2. Leafhoppers were collected in celery and carrot field edges in Michigan in 2018 and 2019. ( A) A erial view of a celery field with boxed area magnified in B. (B) The surveyed field edge types are indicated by the yellow lines, consisting of vegetation between adjacent crop fields and edges between fields and non - agricultural veg etation, including weed y herbaceous plants growing along roads or paths adjacent to fields, or plants naturally growing along wooded edges. 5 7 Figure 3.3 Leafhopper species collected from Michigan, USA, celery and carrot farms from 2018 to 2019. (A ) Agalli a sp. , (B) Aphrodes bic inctus , (C) Athysanus argentarius , (D) Balclutha sp., (E) Colladonus clitellarius , (F) Commellus sp., (G) Cuerna sp.*, (H) Diplocolenus subg. verdanus , (I) Doratura stylata , (J) Draeculacephala sp . , (K) Elymana inornata , (L) Empoasca fabae , (M) Endria inimica , (N) Erythroneura sp . , (O) Forcipata loca , (P) Graphocephala sp . , (Q) Idiocerus raphus , (R) Idiocerus sp . , (S) Jikradia olitoria , (T) Latalus sp., (U) Macrosteles quadrilineatus , (V) Neokolla hieroglyphica* , (W) Norvellina sp . , ( X) Paraphlep sius sp . , (Y) Psammotettix lividellus , (Z) Scaphytopius sp. Note: * indicates that only nymphs were collected, all other leafhoppers were collected as adults or as both adults and nymphs. See acknowledgements for photo credits . ................................ ................................ ................................ ................................ .. 6 1 Figure 3.4. Mean ± SEM M acrosteles quadrilineatus and Empoasca fabae abundance in commercial carrot and celery fields and field edges in 2018 and 2019 . Numbers above bars indicate the number of individuals collected for each species and location. Asterisks indicate st atistically - value - ................................ ................................ ................................ ............. 6 4 Figure 3.5 . Mean ± SEM of the eight most abundant leafhopper species in celery and carrot fields and fi eld edges ( 50 individuals collected); excluding Macrosteles quadrilineatus and Empoasca fabae . Leafhoppers were collected from commercial carrot (A, B) and celery (C, D) farms in Michigan in 2018 and 2019. Numbers above bars indicate the number of leafho ppers collec ted for each crop and location. ................................ ................................ ................................ .................. 6 5 Figure S4.1. Erythroneura sp . v oucher specimen. Dorsal (A) and ventral (B) view . .................. 8 7 Figure S4.2. Morphotyp e 15 v oucher specimen. Dorsal view. ................................ ..................... 88 xi Figure S4.3. Morphotype 1 6 v oucher specimen. Dorsal (A) and lateral (B) v iew. ...................... 89 1 CHAPTER 1: B iology and management of aster leafhoppers and aster yellows phytoplasma in Michigan celery and carrot agroecosystems 1 E conomic impact Celery ( Apium graveolens L.; Apiales: Apiaceae) and carrot ( Daucus carota subsp. Sativus (Hoffm .) Schübl. & G. Martens; Apiales: Apiaceae) are both economically important crops. In 2018, the USA produced over $1 billion worth of carrots and celery on 110,000 acres , 1 while Michigan produced 4,000 acres of carrots worth approximately $14.5 million and 1,900 acres of celery worth approximately $19.5 million. 1 Aster yellows phytoplasma ( Candidatus Phytoplasma sp.) , a bacter i al plant pathogen, causes considerable damage to vegetables, field crops, and ornamentals in North America and Europe. 2 This disease can infect more than 350 plant species, including important vegetable crops such as carrots, celery, onions, lettuce, and potatoes, which become unma rketable when infected. 3 Aster yellows phytoplasma is vectored by 24 leafhopper species in North America. 4 The primary vector in North America is the aster leafhopper ( Macrosteles quadrilineatus Forbes, formerly considered a part of M. fascifrons Stål; Hem iptera: Cicadellidae) due to its abundance in sensitive crops. 5,6 The primary strategy to control phytopla smas in commercial agriculture is through controlling the vector populations with insecticides. 7,8 Traditionally , insecticides have been applied proph ylactically on a calendar - basis to control leafhopper populations, but due to numerous negative effects of this practice, growers, in collaboration with researchers, have developed better approaches that involve the use of an aster yellows phytoplasma infe ctivity index to guide insecticide applications. 9 Current methods for identifying phytoplasma infected ast er 2 leafhoppers have a relatively quick turnaround of about 24 h ours , 10 but it is unknown whether other leafhopper species contribute to the spread of the pathogen or maintain it in the vegetation around fields, and it is important to identify those that ca n may help minimize the spread of the disease. 2 A ster leafhoppers 2.1 Biology Adult aster leafhoppers (Fig 1.1A) are light yellow - green, with gray - green wings. Sizes range from 3 4 mm with the females generally larger than males. The most distinct ive fea ture s are the markings on the head with 4 6 dark brown - black lines and 2 spots. 1 1 Aster leafhoppers are polyphagous and feed on over 300 species of plants, 12 although adults prefer cereal crops over carrots when presented with a choice. 13 Aster leafhoppers feed on plants using a piercing/sucking type mouth which they insert into leave s and feed on sap within the phloem. While they feed, they secrete saliva to protect and guide their mouth parts. 11 Figure 1.1. (A) Adult aster leafhopper on celery. (B) Aste r leafhopper nymph on oats. 3 2.2 Life history Aster leafhoppers reproduce sexual ly and are hemimetabolous with egg, nymph, and adult stages. 11 Adult females typically remain on the plants where they molted to adult, while males move among plants attempting to find unmated females. 14 Once mated, females will move to younger plants to l ay eggs. 14 A generation is about 27 34 days (average 30 days to get from egg to reproductive adult) . 11 The adult lifespan is about 18 20 days (Beanland et al, 2000). A n a dult aster leafhopper s life span decrease s from 20 to 7 days when temperatures are above 30°C, although all life stages can survive between 0 35°C. 15 Eggs are laid on leaf veins, close to the petiole, and take about 7 8 days to hatch. Aster leafhoppers will la y eggs on cereals and wild plants, suc h as clovers, grasses, and weeds, at the end of the growing season. These eggs will overwinter until spring when the nymphs will move to available host plants (Fig 1.1B) . 11 Typically, females will lay one to five eggs a day, 11 and approximately 30 eggs are laid in a lifetime. 16 Females can lay eggs between 5 35°C. 15 Newly hatched nymphs will feed on the plant from which they emerged and continue to grow and molt, completing 5 nymphal instars before the final molt to an adult. 11 When eggs are laid on less s uitable host plants, the nymphs have a lower survival rate and longer developmental time compared to nymphs laid on preferred host plants. 17 For example, lettuce is one of the few vegetable host plants that is suitable for egg laying. 11 2.3 Migration In th e southern USA, aster leafhoppers have a continuous life cycle and do not undergo diapause as it does not get cold enough to require a quiescent state. 18 During the spring, the winds move in a northerly direction from states like Texas, to the Great Plains states, then into the Midwest (including Michigan), and Canada ; thus, some of these southern populations will migrate 4 to the northern U.S. using wind cu rrents. 18 The wind speed and direction may change every year, w hich contributes to variability in the numbers of leafhoppers that arrive in Michigan each year (Fig 1.2). 18 Figure 1.2. Generalized migration map of aster leafhopper movement from Texas a nd Mexico, to the Midwest. Migration begins in mid - May and lasts through early - June. During this time, the leafhoppers move using seasonal wind currents . This map is based on those provided by Hoy at al. 18 for 1988 1990. 3 P hytoplasma 3.1 Biology Phytoplasmas ( Candidatus Phytoplasma spp., Acholeplasmatales: Acholeplas mataceae) are a genus of globally distributed pathogens that infect 98 plant families, consisting of several hundred plant species, including peanuts, fruit trees, lettuce, and canola. 2 - 3 These small, wall - less, obligate parasitic nano - microbes require pla nt hosts or insect vectors for survival. 19 Their genomes are the smallest of any self - replicating organism ranging from 530 1,350 kb. 19 Officially, there are no named phytoplasma species as they cannot be grown in axenic culture, therefore the group 5 has be en granted the taxonomic classification of Candidatus . 19 On e study has demonstrated that it may be possible to grow phytoplasmas in axenic culture, but this work is still in the e arly stages. 20 Currently, there are 32 sub - groups that are distinguished by t heir 16S rDNA sequences, the species of plants they affect, and their disease symptoms. 19 Common d isease symptoms include yellow or purple leaves, virescence, phyllody, proliferat health, and premature dea th. 19 3.2 Identification Infected plants vary in the severity of their symptoms, ranging from asymptomatic to yellowing and rapid decline. In both cases, these plants could be tested to reveal infections of the same magnitude. 19 To determine which phytopl asma subgroup caused an infection in both plants or insects, 16S rDNA universal primers are used in conventional PCR. 21 To identify phytoplasma subgroups, PCR products can be di gested with restriction enzymes, which cleave the PCR product into phytoplasma subgroup specific segments , and the banding patterns allow sorting into subgroup s . 19 This method is useful in identifying the specific phytoplasma subgroup that may be causing t he infection, but it cannot determine phytoplasma titer in plants. Phytoplasmas are unevenly distributed in plants, thus it may be most effective to test samples using tissue taken from multiple parts of the plant . 19 3.3 Transmission Phytoplasmas are vecto red by herbivorous h emipterans, which secrete phytoplasma in their saliva as th ey feed on the phloem. The main vectors are leafhoppers , treehoppers (Cicadelloidea), psyllids (Psylloidea), and planthoppers (Fulgoroidea). 19 Transmission can also occur in lab oratory settings by grafting infected plant tissue onto healthy plant s from a r elated species or by attaching dodder ( Cuscuta sp p ., Solanales: Convolvulaceae) to both the infected and healthy plants. These 6 methods will connect the phloem of the two specime ns allowing transmission of phytoplasma from the infected plant to the healthy plant. 19 In agriculture, leafhoppers are the most common source of phytoplasma infection. Leafhoppers acquire phytoplasmas by feeding on infected plants. The phytoplasma replicates within the leafhopper during the latency period where it will migrate to the saliva ry glands. Once the infection has reached a sufficiently high titer in the salivary glands, the leafhopper is infectious. The leafhopper can inoculate uninfected plants when feeding for the remainder of its life. 19 This method is similar for all Hemipteran vectors of phytoplasmas (Fig 1.3) , however, the rate of infectivity varies d epending on the specific vector and the phytoplasma acquire d . For efficient vectors, transmission can be near 100%, 22 but for poor vectors , rates can be as low as 40%. 23 Figure 1.3. P athway for phytoplasma acquisition and transmission by hemipteran vector s . 7 4 A ster leafhoppers as vectors of aster yellows phytoplasma Aster leafhoppers are known to vector at least four different phytoplasmas , including clover phyllody, E uropean aster yellows, North American aster yellows, and stolbur. 4 T he most prevalent of the four is North American aster yellows which aster leafhoppers can transmit to at least 191 plant species. 19 The major factor limiting the spread of the phytoplasma s preferences. Male aster leafhoppers are twice as likely to be infected by aster yellows phytoplasma than females, although females have a higher rate of success at inoculating plants. 2 4 This is likely due to the different beha viors of the sexes : m ales jump around from plant to plant looking for mates , while females are less mobile. 2 4 When aster leafhoppers are infected with aster yellows phytoplasma , the infection benefits females by ex tending their lifespan ( ~ 10 days) and do ubling the number of eggs they can lay compared to uninfected aster leafhoppers. 16 4.1 Management Aster leafhopper s often form large populations in carrot and celery fields, but their feeding does little damage to crops . Thus , the primary reason for keepin g aster leafhopper abundance low is not to prevent direct crop damage but rather to limit leafhopper transmission of aster yel lows phytoplasma . O nce infected, aster leafhoppers can transmit phytoplasma for the remainder of their li ves . 2 4 A cultural manag ement method for aster leafhopper control is to avoid planting host plants in adjacent fields, such as celery and alfalfa. Whe n one crop is harvested, the aster leafhoppers will move over to the adjacent field in search of a new food source, (personal obse rvation, Z. Szendrei). Another cultural management method is controlling the host plant reservoirs in the field edge as 8 these may provide shelter for aster leafhoppers when the field is being treated with insecticides or when harvested. 2 5 Foliar insectic ides are the most common chemical control for aster leafhoppers. Plants may need to be sprayed frequently, especially when repeated rain events wash the insecticides off the plants. 2 5 In celery, since aster leafhoppers prefer younger plants, frequent scout ing and management is critical (personal observation, Z. Szendrei). In carrots, it is unknown if aster leafhoppers prefer younger plants. New means of control are being investigated . F or example, several carrot cultivars have been bred to b e resistant/tol erant to aster yellows phytoplasma. 2 5 Selectively breeding crops is currently the only means of preventing aster yellows phytoplasma from infecting a crop, but there are many susceptible cultivars currently is use . 4.2 Monitoring Aster leafh oppers can be m onitored using yellow sticky traps, inverted cage trapping, or sweeping. 14 Sweeping has been identified as the best way to catch an even ratio of males and females, unlike the other two methods. 14 Males are more likely to be caught with yellow sticky traps as they are actively flying between plants looking for unmated females , while f emales are more likely to be caught by inverted cage trapping as they are more sedentary. 14 Sweeping can be combined with laboratory techniques to identify the infectivity lev els of aster leafhoppers in the field. Currently in Michigan, a qPCR protocol 10 is used to determine the infectivity level of field caught aster leafhoppers, then an action threshold is calculated, and sent to growers in a text message ( Fig 1.4). 9 Figure 1.4 . Summary of the current diagnostics workflow to inform growers about leafhopper infectivity in their fields. Aster leafhoppers are collected from celery or carrot fields, DNA is extracted from the leafhoppers, qPCR is performed to de termine if the leaf hoppers are infected with aster yellows, and then the results are provided to farmers via a text messag e . 5 L eafhoppers of Michigan 5.1 Vectors of aster yellows In Michigan, there are several leafhoppers known to vector aster yellows, in addition to the a ster leafhopper, including: Aphrodes bicinctus (Schrank), Athysanus argentarius (Metcalf), Endria inimica (Say), Fieberiella florii (Stål), and Scaphytopius acutu s acutus (Say). 4,2 6 All of xcept F. florii which predominantly feeds on ornamental shrubs and fruit trees. 2 3,26 30 Grasses, clovers, and cereals are all common plants along celery and carro t field edges , and aster leafhoppers can feed on them . If an aster leafhopper infects grasses or cereals in the field edge, other leafhoppers that normally do not interact with infected crops, can acquire aster yellows. This can lead to an overwintering source of aster yellows in the resident leafhopper populations as well as in the plants near cro p fields. 3 1 Besides feeding on plants in the field edge, A. bicinctus can vector aster yellows to celery. 2 8 Thus other leafhopper species are likely present in Michigan that can infect celery with aster yellows, although their populations are likely small er than those of aster leafhoppers. 10 5.2 Non - vector leafhoppers Besides the known vectors, potato leafhoppers ( Empoasca fabae Harris) are abundant in celery and carrot fields and are the second most prominent leafhopper species collected in sweep nets (per sonal observa tion, P. Stillson). Currently, it is unknown if potato leafhoppers vector aster yellows, but due to their abundance in numerous crops , understanding how they interact with this pathogen is important. 6 K nowledge gaps There are many gaps in ou r knowledge of phytoplasma transmission. For example , we need to better understand the factors that contribute to phytoplasma acquisition and infection in both the insect vector as well as in the host plant (Fig 1.5). Does the mann er in which the leafhoppe r feed s (phloem vs. xylem feeders) affect the probability of acquiring phytoplasma from infected plants? Different leafhopper species feed in different ways and on different plant tissues, but in some polyphagous leafhoppers, like the potato leafhopper, th is changes based on the plant they are feeding on. 3 2 After leafhopper s acquire the phytoplasma, the pathogen must migrate to the salivary glands by moving through the midgut and salivary glands. These barriers are likely what preve nts ingested phytoplasma from making leafhoppers into vectors, but what exactly is preventing the migration is unknown. After becoming a vector, some leafhoppers develop detrimental side effects due to the phytoplasma infection, such as shortened lifespans . 3 3 It is unknown why thi s may occur, but it may potentially be due to an immune response in the leafhopper s. In addition to the knowledge gaps associated with phytoplasma acquisition and transmission, there are some gaps associated with the disease ecology. It is unknown how many total plants an infectious leafhopper can infect during its life as this can be affe cted by their sex , 14 11 age when they acquire the phytoplasma, 28 how long it takes to transmit the phytoplasma , and how long latency period s are for different leafhoppers (e. g. for chrysanthemum yellows phytoplasma latency period lasts 18 days for Macrosteles quadripunctulatus and 30 days for Euscelidius variegatus ) . 22 ,34 Additionally, there is not much research on which leafhoppers may contribute to outbreaks in different sus ceptible cropping systems as most work focuses on a single species and pot ential vectors are ignored. Figure 1.5. Current knowledge gaps in our understanding of phytoplasma acquisition and transmission. These relate to how the vectors feed on the plants, molecular and physiological responses to the phytoplasma, and how the plan ts interact with the pathogen. Aster yellows phytoplasma = AYp, chrysanthemum yellows phytoplasma = CYp. 7 T hesis objectives I conducted my research at commercial celery and carrot farms to address knowledge gaps associated with vector identification and management within susceptible cropping systems . These crops are both economically important in Michigan and susceptible to aster yellows phytoplasma. 12 My first goal was to determine w hethe r - based decision support tool was useful to celery and carrot farmers in controlling the populations of aster yellows infected leafhoppers. The objectives of this work were 1) to determine whether o ur de cision support tool informed farmer management and directed insecticide applications at the infected population rather than the overall leafhopper populations, 2) to determine whether there were similarities between leafhopper abundances and infectivi ty be tween celery and carrot systems during the growing season and if these similarities could be used to improve aster yellows management across cropping systems. My second goal was to identify the different leafhopper species associated with commercial c elery and carrot farms and how they might interact with aster yellows phytoplasma. The objectives of this work were 1) to identify the species of leafhoppers that reside within crop fields and the field edges, 2) to determine whether there are differences in le afhopper distributions between the field and the field edge and if any species were found among both cropping systems, 3) to determine whether any of the collected leafhoppers are known vectors of aster yellows or new potential vectors for the phytopl asma. 13 LITERATURE CITED 14 LITERATURE CITED 1 USDA NASS National Agricultural Statistics Service. Available online: https://www .nass.usda.gov/ (accessed on 9 January 2020). 2 Bertaccini A and Duduk B, Phytoplasma and phytoplasma diseases: A r eview of recent research, Phytopathol Mediterr 48 :355 378 (2009). 3 Lee I - M, Davis RE, and Gundersen - Rindal DE, Phytoplasma: phytopathogenic mollicutes, Annu Rev Microbiol 54 :221 255 (2000). 4 Tsai JH, Vector transmission of mycoplasmal agents of plant d iseases, In: The mycoplasmas, vol. Ill, ed. by Whitcomb RF and Tully JG, Academic Press Inc. New York, NY, pp. 265 307 (1979). 5 Lee I - M, Martini M, Bottner KD, Dane RA, Blac k MC, and Troxclair N, Ecological implications from a molecular analysis of phyto plasmas involved in an aster yellows epidemic in various crops in Texas, Phytopathology 93 :1368 1377 (2003). 6 Christensen NM, Axelsen KB, Nicolaisen M, and Schulz A, Phytopl asmas and their interactions with hosts, Trends Plant Sci 10 :526 535 (2005). 7 W eintraub PG and Beanland L, Insect vectors of phytoplasmas, Annu Rev Entomol 51 :91 111 (2006). 8 Frost KE, Esker PD, Van Haren R, Kotolski L, and Groves RL, Factors influenci ng aster leafhopper (Hemiptera: Cicadellidae) abundance and Aster yellows phytopl asma infectivity in Wisconsin carrot fields, Environ Entomol 42 :477 490 (2013). 9 Chapman RK, Control aspects of mycoplasma disease: aster yellows disease, INSERM 33 :251 262 (1974). 10 Demeuse KL, Grode AS, and Szendrei Z, Comparing qPCR and nested PCR d iagnostic methods for aster yellows phytoplasma in aster leafhoppers, Plant Dis 100 :2513 2519 (2016). 11 Capinera JL, editor, Encyclopedia of entomology, Springer Science & B usiness Media, p. 4346 (2008). 12 Gavloski J, Leafhoppers in Manitoba; biology, behaviour and potential for vectoring plant diseases, Univ Manit Ext :5 (2007). 13 Szendrei Z, The impact of plant associations on Macrosteles quadrilineatus management in carrots: plant association effects on leafhoppers, Entomol Exp Appl 143 :191 198 (201 2). 15 14 Beanland L, Madden LV, Hoy CW, Miller SA, and Nault LR, Temporal distribution of aster leafhopper sex ratios and spatial pattern of aster yellows phytoplasma disease in lettuce, Ann Entomol Soc Am 98 :756 762 (2005). 15 Bahar MH, Wist TJ, Bekkaoui D R, Hege dus DD, and Olivier CY, Aster leafhopper survival and reproduction, and aster yellows transmission under static and fluctuating temperatures, using ddPCR for phytoplasma quantification, Sci Rep 8: 227 (2018). 16 Beanland L, Hoy CW, Miller SA, and Na ult LR, Influence of aster yellows phytoplasma on the fitness of aster leafhopper (Homoptera: Cicadellidae), Ann Entomol Soc Am 93 :271 276 (2000). 17 Bosco D, Minucci C, Boccardo G, and Conti M, Differential acquisition of chrysanthemum yellows phytoplasm a by th ree leafhopper species, Entomol Exp Appl 83 :219 224 (1997). 18 Hoy CW, Heady SE, and Koch TA, Species composition, phenology, and possible origins of leafhoppers (Cicadellidae) in Ohio vegetable crops, J Econ Entomol 85 :2336 2343 (1992). 19 Dickin son M a nd Tuffen M, The phytoplasmas: an introduction, in: Phytoplasmas: methods and protocols, ed. by Dickinson M and Hodgetts J, Humana Press, Totowa, NJ, pp. 1 - 14 (2013). 20 Contaldo N, Bertaccini A, Paltrinieri S, Windsor HM, and Windsor GD, Axenic cu lture o f plant pathogenic phytoplasmas, Phytopathol Mediterr 51 :607 617 (2012). 21 Christensen NM, Nicolaisen M, Hansen M, and Schulz A, Distribution of phytoplasmas in infected plants as revealed by real - time PCR and bioimaging, Mol Plant Microbe Interac t 17 :11 75 1184 (2004). 22 Bosco D, Galetto L, Leoncini P, Saracco P, Raccah B, and Marzachì C, Interrelationships Cicadellidae), J Econ Entomol 100 :1504 1511 (2007). 23 Chiyk owski LN, Athysanus argentarius, an introduced European leafhopper, as a vector of aster yellows in North America, Can J Plant Pathol 1 :37 41 (1979). 24 Beanland L, Hoy CW, Miller SA, and Nault LR, Leafhopper (Homoptera: Cicadellidae) transmission of aste r yellows phytoplasma: does gender matter?, Environ Entomol 28 :1101 - 1106 (1999). 2 5 Delahaut KA, Aster leafhopper, University of Wisconsin Extension (1997). 2 6 Chiykowski LN, Scaphytopius acutus (Say), a newly discovered vector of celery - infecting a ster - yellows virus, Can J Bot 40 :799 801 (1962). 16 2 7 Chiykowski LN, Endria inimica (Say), A new leafhopper vector of a celery - infecting strain of aster - yellows virus in barley and wheat, Can J Bot 41 :669 672 (1963). 2 8 Chiykowski LN, Transmission of a celery - i nfecting strain of aster yellows by the leafhopp er Aphrodes bicinctus, Phytopathology 77 :522 524 (1977). 2 9 Westdal PH, Barrett CF, and Richardson HP, The six - spotted leafhopper, Macrosteles fascifrons (Stål.) and aster yellows in Manitoba, Can J Plant Sc i 41 :320 331 (1961). 30 Grant JA, Caprile JL, Coates WW, Van Steenwyk RA, and Daane KM, UC IPM Pest management guid elines: cherry, UC ANR Publication 3440. 3 1 Lee I - M, Gundersen - Rindal DE, and Bertaccini A, Phytoplasma: ecology and genomic diversity, Phy topathology 88 :1359 1366 (1998). 3 2 Chasen EM, Dietrich C, Backus EA, and Cullen EM, Potato l eafhopper (Hemiptera: Cicadellidae) e cology and i ntegrated p est m anagement f ocused on a lfalfa, J Integr Pest Manag 5 :1 8 (2014). 3 3 Madden LV and Nault LR, Diffe rential pathogenicity of corn stunting mollicutes to leafhopper vectors in Dalbulus and Baldulus species, Phytopathology 73 :1608 1614 (1983). 34 Palermo S, Arzone A, and Bosco D, Vector - pathogen - host plant relationships of chrysanthemum yellows (CY) phyt oplasma and the vector leafhoppers Macrosteles quadripunctulatus and Euscelidius variegatus, E ntomol E xp A ppl 99 :347 354 (2001). 17 CHAPTER 2: A novel plant pathogen management tool for vector management 1 I ntroduction Decision support systems have existed for decades to manage insect pests across many cropping systems. 1 4 These tools often provide management thresholds based on scouting data and promote control of pest insects that damage crops while preventing unnecessary insecticide applications. 4,5 While these decision support systems are increasingly adopted in agriculture, few are available specific ally for insect vect or management. 5 M oreover, the impleme ntation of these decision support systems may be complicated by behavioral and biological differences between populations of pests infected and uninfected with plant disease . When data needed for decisions support tools are lacking, a calendar - based spray schedule is sometimes followed where insecticide applications are performed without knowledge of pest abundance. 6,7 However, calendar - based management approaches are not ideal given the use of insecticides is cost p rohibitive, environmentally damaging, and increases insecticide resistance . 8,9 When decision support based on abundance thresholds does exist, these tools are again inadequate because the abundance of infected vectors is frequently a better predictor of pathogen pr e valence in crops than vector abundance alone . 7,10,11 Therefore, c ontemporary pest management is shifting to t h e use of diagnostic s to identify and verify the presence of insect vectored pathogens which can then inform pest management. 12,13 However, decision support systems linking the results of diagnostic laboratories to farmers remain rare, indicating that improving the delivery of diagnostics to farmers could enhance insect vectored plant pathogen management and reduce the use of calendar sprays (Fig 2. 1). 18 Figure 2. 1. Aster yellows phytoplasma is transmitted by aster leafhoppers to carrots and celery. The economic damage is caused by the phytoplasma; therefore, it is impo rtant to assess the proportion of the leafhopper population that is infected . Leafhopper management that is based on detecting the pathogen in the leafhopper could reduce yield losses. In our system, t he results of disease diagnostics are sent to farmers v ia a group text messages indicating rates of leafhopper infectivity and the action threshold for car rots and celery. If the text message indicates that infected leafhoppers have been detected, then farmers will respond by applying insecticides to their fie lds (research - mediated management). If the text message indicates that leafhoppers are not infected with the phytoplasma, then management action is not needed and we recommend growers not use insecticide management . Leafhoppers are collected regularly duri ng the growing season and text messages are sent out approximately 24 h after collecting leafhoppers from the field, providing an opportunity for quick response, if necessary. 19 Time delays between the insect vector acquiring the pathogen and transmi tting it (latency) may contribute to differences between the population abundances of infected and uninfec ted individuals . 14 , 15,16 Infections may occur at various spatial scales, both within a cropping system (as a patchwork of infected and unin fected plants) and across cropping systems due to differences in host suitability and management . This heterogeneity means that some insects are infected and some are not , reflecting differences in the infectivity status of their host plants. 17 19 Identifying patterns between the abundance of infected and uninfected individuals is challeng ing, but it is necessary to shift pest management from abundance to infectivity - based models 17,20 and reduce uncertainty in the appropriate timing of insecticide applications. We used an infectivity - based decision - support system to provi de celery ( Apium graveolens , L. , Apiaceae ) and carrot ( Daucus carota subsp . S ativus , Hof fm., Schübl. & G. Martens , Apiaceae ) farmers with rapid diagnostic s information to manage an economically damaging insect - vectored plant pathogen, aster yellows phytoplasma ( Candidatus Phytoplasma spp.) . This pathogen is transmitted by its primary vector, the a ster leafhopper ( Macrosteles quadrilineatus, Forbes). 21 The main objective of this support tool was to support farmer s in changing their manag ement strategy from one of abundance - based insecticide applications to a directed spray program focusing on the infected population. We identified leafhopper population patterns before and after farmers received diagnostics results , and to understand the r elationship between infected and uninfected vector populations , we examined temporal and spatial patterns . Our results help identify the spatial scale at which decisions support tools can inform management and indicate that temporal shifts in management ba sed on infectivity thresholds may help to reduce the prevalence of an economically important plant pathogen in two high value vegetab le cropping systems. 20 2 Materials and m ethods 2.1 System description 2.1.1 Pathogen - vector system Aster yellows phytoplasma is a cell wall - less bacteria that is transmitted by phloem feeding insects; it is one of the largest and most diverse group of phytop lasmas. 22 This pathogen can infect over 300 plant species , including crops (e.g., carr ots, celery, lettuce) and ornamentals. 22,23 Plants infected with aster yellows phytoplasma are unmarketable due to chlorotic, deformed, a nd stunted growth 23,24 and farmers have reported yield losses of up to 10% due to a ster yellows phytoplasma . 25 Aster leafhoppers are the main vector of aster yellows phyt oplasma ; 21 while aster leafhopp ers cause minimal damage to most crops, leaving small marks where they fed, once infected they transmit the phytoplasma in a persistent manner for the remainder of their lives. 26 Ast er leafhoppers acquire phytoplasmas from the environment while feeding on infected plants 27 and remain latent for two to three weeks before becoming infectious; once infectious they remain so for the rest of their lives. 26,28 Aster leafhoppers annual ly migrat e north from the southern USA in early May, acquir ing aster yellows along the wa y . 21 Little is known about overwintering aster leafhopper populations and sources of aster yellows in the Midwestern USA . 26 However, once in the Midwest, aster leafhoppers move short distances between adjacent crops , field s , and field edges to feed on grasses and weeds 29 which are known disease reservoirs. 30 Currently, insecticides are applied when leafhopper abundance is high , but this practice is unnecessary, as uninfected leafhoppers rarely cause direct damage to plants and the relationship between population abundance and infectivity is unknown . 27 21 2.1.2 Cropping systems We studied leafhopper populations and the incidence of aster leafhoppers infected with aster yellows phyt oplasma in two cr opping systems, carrots and celery. While these crops are taxonomically similar, their production methods differ. Celery is grown in greenhouses for eight weeks before transplanting into fields, and farmers continue to transplant weekly fo r approximately t wo months providing a mixture of plant age classes throughout the growing season. 31 Carrots are direct seeded over a shorter period of time and are more similar in age across fields . 32 All farms in the study were large - scale commercial operations (field sizes from 1.2 36. 2 ha ) and used synesthetic pesti cides for pest management . Fungicides were applied weekly in both celery and carrots; however, insecticide application frequency varied based on scou ting reports . Overall, aster leafhoppers were collected from 10 celery and 12 carrot farms, totaling 40 and 2 0 different fields respectively, between 2014 and 2019 (Fig 2 .2 ; Tables S 2. 1 S 2. 2). Figure 2 .2 . Map of Michigan, USA, symbols i ndicate the locations of commercial carrot and celery fields where aster leafhoppers were collected from 2014 - 2019. Leafhop pers were collected using sweep nets and were transported to the laboratory to determine aster yellows infectivity. 22 2.2 Aster leafhopper diagnostics 2.2.1 Leafhopper collection Leafhoppers were collected weekly from mid - May through early August, 2014 2019 ( n = 365 samples ). Crop consultants performed sampling using a sweep net (38 cm diameter aerial net), with a minimum of 100 sw eeps per field. Fourteen celery and five carrot farms were scouted, on average, each year, with weekly scouting consisting of at l east one field per farm sampled; in larger farms samples were taken from multiple fields (Tables S 2. 1 S 2. 2). The numbers of collected leafhoppers varied depending on leafhopper presence and abundance in fields at a given time. Consultants reported the de nsity of aster leafhoppers found within the field to each farmer for the respective survey as the abundance of leafhoppers col lected per 100 sweeps. After collection, leafhoppers were transferred to plastic bags, placed in a cooler, transported to our labo ratory at Michigan State University, East Lansing, MI, USA , and stored at - 20°C overnight . Since aster leafhoppers are the onl y leafhopper of economic concern in celery and carrot, 21 scouts sorted leafhoppers morphologically into aster leafhoppers and all other leafhoppers . L eafhoppers not identified as aster leafhoppers in were excluded from subsequent analys e s. 2.2.2 Laboratory processing We performed DNA extractions to determine the number of aster leafhoppers infected with aster yellows phytoplasma. One to three adult aste r leafhoppe rs ( three leafhoppers were used when more than 50 leafhoppers were collected from one field) were placed in a 2 ml homogenization tube (Sarstedt, Nümbrecht, Germany), along with high salt extraction buffer 33 (70 µl) and three homog enization beads (2.3 mm diameter, zirconia/silica; BioSpec Products, Inc., Bartlesville, OK). Aster leafhoppers were homog enized for 60 s at 4.0 m/s (FastPrep - 24, MP Biomedicals, Santa Ana, CA). D n easy Blood & Tissue DNA isolation kit (Qiagen, Valencia, CA ) was used to 23 protocol to includ e incubating samples in the proteinase K/Buffer ATL solution for 1 h. DNA was suspended in elution buffer (100 µl for samples with 1 2 leafhoppers and 200 µl for samples with 3 leafhoppers). Varying elution buffer volumes were used to standardize the DNA c oncentration across samples. Final DNA concentrations ranged from 0.50 350 ng/µl. The presence of aster yellows phytoplasma was detect ed using a TaqMan qPCR assay 34 with universal phytopl asma primers and probe 35 (Therm o Fisher Scientific, Waltham , MA) . Leafhopper samples with a cycle threshold < 32 were recorded as positive for aster yellows phytoplasma. 34 2.3 Disseminating information 2.3.1 Infectivity threshold calculations Action thresholds in pest control are designed to decrease pest popul ations before disease transm ission can cause economic damage. 36 When working with vectored pathogens, action thresholds must take into account both pest abundance and the proportion of the infected population, pr oviding a better predictor o f disease incidence. 11,37 We used the following equations in determining an action threshold: 38 Percent of infected leafhoppers = (infected leafhoppers / total leafhoppers) 100, (1) Aster yellows index = percent of infected leafhoppers leafhoppers per 100 sweeps, (2) Celery threshold = (35 / aster yellows index) 100, (3) Carrot threshold = (50 / aster yellows index) 100, (4) where the values of 35 and 50 in eqns. 3 4 represent constants based on resistance to aster yellows phytoplasma in celery and carrot respectively . 38 When the number of infected leafhoppers increase, values found with eqns. 3 4 decrease indicating t hat insecticide applications should take place when leafhoppers are found at or above these threshold values . C onversely, if no infected 24 leafhoppers are detected, then the equation gives an illegal fraction, suggest ing that an infinite number of leafhopper s can be caught and an action remains unnecessary. 2.3.2 Text messages Beginning in 2016, we contacted celery and carrot farmers and encouraged them to e nroll to receive group text messages providing the percent of infected aster leafhoppers and management thresholds determined by each leafhopper survey. The text message (E z texting.com) was sent to those signed up for the group messaging system the day aft er leafhoppers were collected, with a standard turnaround time of 24 h from collection. Text messages were sent out from 201 6 to 2019 in May , June, July, and August (30 in 2016, 31 in 2017, 43 in 2018, and 25 in 2019). Text messages were sent 1 to 8 times per week based on the number of collections performed by crop consultants. Over the course of our study, the number of people receiving our text messages increased approximately 16% from 36 in 2016 to 42 in 2019. Each text message was based on information from leafhoppers collected in a single field but in order to keep the precise location confidential, we identified the county as the sample origin in the message. The messages also included the date , percent of aster leafhoppers testing positiv e for aster yellows phytoplasma, and the threshold adjusted for level of infectivity of aster leafhoppers per 100 sweeps for carrots and celery (Fig 2. 1). 2.4 Statistical analysis 2.4.1 Text messaging and infectivity To determine whether the abundance of i nfected aste r leafhoppers in the fields decreased after farmers received text messages indicating that infectivity was greater than 0% , we calculated one - text messag one - - Wallis rank sum test ) 39 was used to determine differences in the number of infected 25 leafhoppers across the three time points and Dunn 40 was used to identify pairwise differences between weeks. 2.4.2 Leafhopper populations across and within cropping systems Insect abundance is well known to change as ho st plant suitability varies. 19,38 However, whether differences in the abundance of infected and uninfected leafhoppers varies across cropping systems is relatively unknown and likely driven by both host plant su itability and pestici de management practices. 6 To examine these population patterns, we used a Kruskal - Wallis 39 to compare the mean abundance of infected and mean density of leafhoppers across the two crops (carrot and celery). These analyses , however , do not account for variation across the cropping systems between infected and uni nfected leafhopper populations. 10,19,41 Insect populations can temporally vary in abundance across plant resources 19 suggesting that differences in plant management across our study systems may drive temporal differences in leafhopper s over the production season. To examine when population s of leafhoppers in carrot and celery were most similar over time , we performed a cross - ) . 39 We evaluated the correlation of we ekly population patterns at four time lags (two positive and negative) centered on zero, with a correlation at zero indicating that no temporal lag existed across the croppin g systems, a negative lag indicat ing that populations in carrot were temporally de layed when compared to celery, and a positive lag indicat ing the opposite, where populations in celery were temporally delayed when compared to carrot. To prepare our data, l eafhopper densities ( abundance of leafhoppers collected per 100 sweeps ) and infect ed leafhopper abundances were summed by week across years ( n = 365 collections; Tables S 2. 3 S 2. 4) (2014 2019) and by crop (celery and carrot) yielding one time point for each week of the season. There were 15 and 13 time points (weeks of sampling) in cel ery 26 and carrot respectively. For the purpose of analyses comparing carrot and celery, the first two time points were removed from the celery data to align the sampling week s between the two crops, but when comparing timepoints within the celery system, all 15 time points are used (Tables S 2. 3 ). Prior to evaluating our data with the cross - correlation function, we confirmed that our data met the assumptions of the analysis usi ng the Kwiatkowski - Phillips - Schmidt - Shin test for stationarity . 42 We then evaluated the relationship bet ween the populations of infected and uninfected individuals across the cropping systems (13 sampling weeks are used for this analysis) by finding the sample cross - correlati on function, , for the aforementioned lags k : 43 , (5) , (6) where is the sample cross - covariance function and numerator of our desired statistic, . In eqns . 5 6, n is the number of weeks in the sampling season (13 sampling weeks), x t and y t are the total density of leafhopper s per week for celery and carrot, and are the mean density of leafhoppers across all weeks for celery and carrot, and SD x and SD y are the sta ndard deviation of leafhopper densities across all weeks for celery and carrot, respectively. We also determi ned the 95% confidence interval for the cross - correlation function: 39 , ( 7 ) Where n is the number of time points (13 weeks) used in eqns . 5 6. Cross correlation values found at each lag (4 total) and at no lag using eqn . 6 were compared to the 95% confidence interval . We also used the vector of cross correla tion values for each lag to find two - sided p - values which were computed using the pnorm n . 39 Values 27 found with eqn . 6 that were above the 95% confidence interval (eqn. 7) and with p - values below a n - level of 0 .05 indicated a correlatio n between population patterns of infected and uninfected leafhoppers across the cropping systems. We also suspected that temporal differences existed between infected and uninfected leafhopper populations within each cropping system. 15 Therefore, we carried out an additional cross - correlatio n analysis that addressed differences in the population patterns between uninfected and infected individuals within each cropping system. Several mechanisms exist that could explain the tempo ral differences between the infected and uninfected populations. We speculated that the latency period would give rise to a lagged correlation between the uninfected and infected populations, whereby the population of infected individuals would be most sim ilar to the population of uninfected individuals when delayed by up to two weeks which would allow for the mechanisms of disease acquisition and transmission to take place. 44 We also suspected that diseased plants could promot e greater leafhopper abundance. 45 While we could not test this directly, we assumed that patterns of infected individuals could be a proxy of plant infectivity in the field. Therefore, we also investi gated whether populations of uninfected leafhoppers were most sim ilar to the infected population when delayed for up to two weeks temporally. Therefore, this approach accounts for two lags in the positive and negative direction (four lags total) and no lag , where the density of uninfected individuals were treated as the . 5 cropping system with 15 and 13 sampling weeks for celery and carrot respectively (Tables S 2. 4 ). 2.4. 3 Spatial variation in le afhopper populations To determine whether leafhopper samples of similar density and infectivity clustered together, we calculated spatial autocorrelation as a function of distance bands using with 28 the moran.mc function ( n = 2000 simulations) in R , 46 split by cropping system (carrot and celery). P - values below a n - level of 0 .05 indicated a correlation between population s of infected and uninfected leafhoppers across collection points within each distance band . Scouts did not coll e ct spatial data for all samples, therefore we subset our data to those where the collection point was known. In sum, there were 18 and 7 unique collection points (fields) for celery ( n = 191 samples) and carrot ( n = 65 samples), respectively. Distance ban d s were defined based on a priori knowledge of sites and allowed to vary across cropping systems. For example, in celery sites less than 2.5 km apart were known to be fields within a farm, and these coordinates were placed within one distance band (Fig S 2. 1 ). Using a priori knowledge to create distant bands has important practical implications, as strong positive correlations within farms would suggest that sampling need not occur in multiple fields to inform leafhopper management farm - wide. 3 Results 3.1 L eafhopper collections From 2014 2019, a total of 8,343 aster leafhoppers were collected, and 99 infected leafhoppers were detected (Tables S 2. 3 S 2. 4 ). In carrot and celery, there were 1,870 and 6,473 leafhoppers and 39 (2.09%) and 60 (0.93%) infected indi viduals respectively , which was similar to others studies that detected 0.09% 6.25% infectivity. 15 During the growing season, the number of infected leafhoppers peaked at week 26 in celery and week 28 in carrots, while the total number of leafhoppers peaked during week 24 in celery and 31 in carrots. 29 3.2 Text messaging and infectivity There was a 29.17% decrease in the mean number of infected leafhoppers between the week before a text message was sent compared to the week of sending a text message ( 2 = 6.63, df = 2, p - value = 0.06; Fig 2. 3). There was also a 73.33% decrease in the mean number of infected leafhoppers between the week a text message was sent and the following week ( 2 = 6.63, df = 2, p - value = 0.39), and an 81.11% redu ction between the week before a text message was sent and the week after the text message was sent ( 2 = 6.63, df = 2, p - value = 0.02) ( See supplementary Information for follow - up discussion) . Figure 2. 3. Abundance (mean ± SEM) of aster yellows phytoplasma infected l eafhoppers during the 201 6 2019 growing season by the number of weeks since farmers received a text message indicating leafhopper infectivity. Text messages were sent to inform stakeholders of the percent of aster yellows phytoplasma infected aster leafhop pers in the population and the action threshold for carrot and celery. Different letters above bars denote significant differences in abundance of infected leafhoppers across weeks. 30 3.3 Leafhopper populations across cropping system s While we collected m ore leafhoppers in celery, the mean density of uninfected leafhoppers in carrots was 1.84 times higher than the mean density in celery ( 2 = 5.75, df = 1, p - value = 0.02 ; Fig 2. 4A). However, no difference was found between the mean abundance of infected l eafhoppers when comparisons were made between the two crops ( 2 = 0.26, df = 2, p - value = 0.61; Fig 2. 4B). Figure 2.4. (A) Mean ± SEM aster leafhopper density (leafhoppers per 100 sweeps) and (B) mean ± SEM abundance of aster leafhoppers infected with a ster yellows phytoplasma found in carrots and celery. Leafhoppers were collected with sweep nets from commercial celery and carrot farms in Michigan from 2014 to 2019 and tested for phytoplasma using a qPCR based diagnostic meth od. Asterisk indicates signi ficant differences between carrot and celery . 31 When we compared the weekly population patterns of infected and uninfected leafhoppers between the two cropping systems, we found no temporal relationship when comparing the density of uninfected leafhoppers in celery to that in carrot (Fig S 2. 2 A, B). Howe ver, the population of infected leafhoppers in carrot lagged that in celery by two weeks ( r = 0. 79, p - value = 0.004 ; Fig 2. 5A) indicating that the temporal pattern of infected individuals across weeks 24 34 in carrot was similar to the population pattern i n celery across weeks 22 32 (Fig 2. 5B). Figure 2. 5. Cross correlation analysis of the abundance of aster yellows phytoplasma infected aster leafhoppers in celery and carrot fields in Michigan from 2014 to 2019. Dotted lines indicate a 95% confidence interval and each lag represents a week. (A) The cross - correlation value at a lag of - 2 indicates that the pattern of infected leafhopper abundance in carrot may be delayed by two weeks when compared to the week ly population pattern observed in celery . (B) Total number of aster yellows phytoplasma infected aster leafhoppers plotted by week across the season in Michigan celery and carrot fields. The weeks on the x - axis correspond with the weeks of the calendar yea r. The line for carrots is shifted by two weeks to illustrate the two - week lag that was identified in the cross correlation. 32 3.4 Leafhopper populations within cropping systems When we compared the weekly population pattern between uninfected and infecte d leafhoppers within cropping systems, we found support for a temporal relationship between infected and uninfected individuals in celery ( r = 0. 61, p - value = 0.02 ; Fig 2. 6A). Patterns of infected leafhopper abundance across weeks 22 34 were similar to the pattern of uninfected leafhoppers across weeks 20 32 (Fig 2. 6B), indicating that the population pattern of infected individuals was similar to that of uninfected individuals but at a two - week delay. No temporal relationship was found between the density o f uninfected and infected leafhoppers in carrot (Fig S 2. 3 A, B). Figure 2. 6. Cross correlation analysis of the density of aster leafhoppers (leafhoppers per 100 sweeps) and the abundance of aster yellows phytoplasma infected aster leafhoppers in celery fi elds in Michigan from 2014 to 2019. Dotted lines indicate 95% confidence interval and each lag represents a week. (A) The cross - correlation value at a lag of - 2 indicates that the weekly patterns of infected leafhopper abundance may be delayed by two weeks when compared to the weekly pattern of uninfected individuals . (B) Total density of aster leafhoppers and the total abundance of aster yellows phytoplasma infected aster leafhoppers in Michigan celery fields , plotted by week. The line for infecte d leafhop pers was shifted two weeks to illustrate the two - week lag that was identified in the cross - correlation. 33 3.5 Leafhopper populations across sampling points When we compared the abundance of infected and uninfected leafhoppers across sites by cropping syst em, celery fields within 2.5 km were similar in leafhopper density ( Moran s I = 0.56, p - value = 0.03) and infectivity ( Moran s I = 0.70, p - value .001). Celery fields > 2.5 km apart, however, did not correlate strongly in leafhopper density o r infectivity (Fig 2. 7A, S 2. 1A). When evaluated, carrot fields within 6 km where highly dissimilar in leafhopper density ( Moran s I = - 0.65, p - value = 0 .99), while fields in distance bands > 6 km suggested no positive or negative correlation in leafh opper density across fields (Fig 2. 7B, S 2. 1B). No correlation was observed in the infected leafhopper population between carrot fields at any distance (Fig 2. 7B, S 2. 1B). Figure 2.7. Correlation of leafhopper density and infectivity between sampling sites as a function of distance bands split by (A) celery and (B) carrot. (A) Celery sampling sites within 2.5 km were similar in leafhopper density and infectivit y. However, no correlation was found between sites beyond 2.5 km apart for either density or infec tivity. (B) Carrot sites within 6 km where dissimilar in leafhopper density. No strong correlation was found for leafhopper density in carrot for sites in dis tance bands beyond 6 km, nor was any correlation observed in the infected leafhopper population be tween carrot sites at any distance. 34 4 Discussion Few decision support tools exist for insect vector management, and those that do, focus mainly on insect abu ndance rather than pathogen vector prevalence. 5 We addressed this gap by developing a decision support tool which informed farmers of vector infectivity in two cropping systems, carrot and celery . Using our tool, w e found when we sent out text messages reporting infected leafhoppers were pres ent, there was a decrease in infectivity, but this downward trend could have been associated with several factors including a natural decline in leafhoppers over the season as well as changes in weather. Additionally, we were unable to explore this trend f urther as there were no controls to compare to , as not sending farmers the text messages would be ethically questionable. Despite these issues, farmers found the tool useful and with the diagnostics data we gathered, we made some important temporal and spa tial discoveries in this pathosystem. We iden tified that temporal differences and spatial correlations exist between uninfected and infected leafhopper populations and that these depend on the crop context. Specifically, in celery o ur results indicate d a temporal difference between populations of inf ected and uninfected leafhoppers with a 2 - week delay between leafhopper populations which were uninfected compared to those infected with phytoplasma. In practice, this suggests that aster leafhopper management should be delayed to focus control on the dis ease carrying vectors, rather than the inconsequential damage caused by leafhopper feeding. 47 By targeting pesticide applications to align with peak abundance of infe cted leafhoppers, the number of applications r equired to control the disease may decline, which would result in increased profits for small - scale vegetable farmers and a reduction in non - target impacts. 48 Our results also imply that leafhopper diagnostics could begin two weeks after peak leafhopper abundance is detected in celery fields. From a biological viewpoint, the relationship between the abundance of infected and uninfected individuals within a popul ation of 35 aster leafhoppers is not well understood and may depend on the latency of aster yellows phytoplasma within the vector and host plant. 44 The applicabilit y of our decision support tool is likely most useful for pathogens transmitted in a persistent manner and where the transmission from the vector to crop is delayed relative to non - persistently transmitted pathogens. The lag between detection of pathogens i n the vector to transmission to the crop allows management actions to occur before much of plant infection occurs. If pathogen transmission to plants take place in a short period of time (e.g. a single insertion of mouthparts), while diagnostics may reduce overall disease transmission, due to the time between sampling and information del ivery to farmers, there could be significant crop infection occurring. In the spatial analysis we determined that celery fields located within a 2.5 km radius have similar i nfectivity patterns, meaning that our diagnostic efforts can eliminate multiple sam ples originating from celery fields located near each other without losing relevant information. Aster leafhoppers stay in a relatively small geographic area when ideal host s are available at the end of their spring migration. 49 They reproduce and feed until host plant quality declines which signals the need for dispersal. 49 Since they have many host plants, the availability of ideal hosts in a small area is relatively high therefore leafhoppers are likely to travel short dist ances. This may explain why spatial patterns were similar in celery fields that were nearby. In carrots, the lack of spatial correlation may be due to the greater distance a mong fields (1.6 30.6 km between fields). The differences in aster leafhopper tem poral patterns between the two cropping systems is interesting and could be due to variations in the establishment of plants. For instance, celery seedlings are transplanted from greenhouses while carrots are direct seeded. 31,32 Our results i ndicate that celery seedlings likely provide an early season host for aster yellows phytoplasma infected leafhoppers which may later prefer and move to direct seeded carrots. This relationship may be driven by the 36 palatability of the host plants, which is known to mediate insect populations, including leafhoppers. 19,50 For example, a s plants mature they may become less palatable which may influence shifts in insect populations to a more palatable resource. 51,52 In addition, infected insect vectors may demonstrate behavioral differences when compared to uninfected individuals and these behavioral differences may influ ence the presence of infected individuals in certain crops. 53 55 Regardless of the mechanism behind the observed pattern, our results demonstrate the need for crop specific understanding of aster leafhopper management . While fee - based phytoplasma testing is available at many plant diagnostic laboratories, these are focused on testing plant materi a ls and not phytoplasma vectors (Szendrei Z, pers. C omm. ) . Currently our aster leafhopper decision support tool is available to farmers in Michigan and is conducted only by our laboratory. In order to increase its sustainability and availability in a broad e r geographic range, commercial diagnostic laboratories that can process leafhoppers in 24 h will need to become involved. This will also mean a necessary change in funding structure, with a move away from the current grant funded effort to a per - sample pr o cessing fee. Thus far, the large volume of samples processed by our laboratory seemed an impediment for adoption by commercial laboratories (Szendrei, Z. pers. C omm). With our current results reporting on the spatiotemporal patterns in the aster yellows s y stem, we could meaningfully reduce the number of samples needed from the field, which could make the diagnostics more appealing to adoption by commercial laboratories. 5 Conclusion Decision support tools deliver time sensitive information to farmers throu gh the integration of pest monitoring, weather/computer modeling, and alert systems. 1,3 The use of these tools can 37 lead to reductions in pesticide applications on farms, increases in beneficial insects, and increased farmer profits. 1,2 Our decision support tool used a novel combination of scouting by crop consultants, r apid disease diagnostics, and a web - based text messaging system to provide county - level recommendations for pest management. While we cannot identify a causal link between our decision support tool and the reductions of the infected population, the pattern s we observed have important practical outcomes. I f such decision support tools are implemented on a large enough scale , they may have the potential to minimize yield loss and decrease the incidence of and potential for disease over time. These tool s shoul d be implemen ted alongside existing control measures for aster yellows phytoplasma rather than in isolation, given that evidence indicates cultural measures, including weed control, can reduce disease incidence. 47 38 APPENDIX 39 Supplementary Tables for Chapter 2 Table S 2. 1. The number of c ommercial celery farms and fields for aster leafhopper collect ions during the 2014 2019 growing seasons in Michigan . A 1 indicat es a sampled field in a year . A hat the field was not sampled. Farm Field 2014 2015 2016 2017 2018 2019 Farm 1 Field 1 1 0 0 0 0 0 Farm 1 Field 2 1 1 1 1 1 1 Farm 1 Field 3 1 1 1 1 1 1 Farm 1 Field 4 0 0 0 0 1 1 Farm 1 Field 5 0 0 0 0 1 0 Farm 1 Field 6 0 0 0 0 0 1 Farm 1 Field 7 0 0 0 0 0 1 Farm 2 Field 8 1 0 0 0 1 0 Farm 2 Field 9 0 1 1 1 1 1 Farm 2 Field 10 0 0 0 0 1 0 Farm 2 Field 11 0 0 0 0 1 0 Farm 2 Field 12 0 0 0 0 1 0 Farm 2 Field 13 0 0 0 0 0 1 Farm 2 Field 14 0 0 0 0 0 1 Farm 2 Field 15 0 0 0 0 0 1 Farm 2 Field 16 0 0 0 0 0 1 Farm 3 Field 17 1 1 1 0 1 0 Farm 4 Field 18 1 1 0 0 0 0 Farm 5 Field 19 1 1 1 1 1 1 Farm 5 Field 20 0 1 1 1 1 1 Farm 5 Field 21 0 0 0 0 0 1 Farm 6 Field 22 1 0 0 0 0 0 Farm 6 Field 23 1 0 0 0 1 0 Farm 6 Field 24 1 0 0 1 1 0 Farm 6 Field 25 0 1 1 1 1 1 Farm 6 Field 26 0 0 1 0 0 0 Farm 6 Field 27 0 0 0 1 0 0 Farm 6 Field 28 0 0 0 0 1 0 Farm 6 Field 29 0 0 0 0 1 0 Farm 6 Field 30 0 0 0 0 1 1 Farm 6 Field 31 0 0 0 0 1 0 Farm 6 Field 32 0 0 0 0 1 0 Farm 7 Field 33 1 0 0 1 1 0 Fa rm 7 Field 34 0 1 1 1 0 0 Farm 7 Field 35 0 0 0 0 1 0 Farm 8 Field 36 0 0 1 0 0 0 Farm 9 Field 37 0 0 1 0 0 0 Farm 9 Field 38 0 0 0 0 1 0 Farm 10 Field 39 0 1 1 1 1 1 Farm 10 Field 40 0 0 0 0 1 1 Totals 11 10 12 11 25 17 40 Table S 2. 2 . The number of c ommercial carrot farms and fields for aster leafhopper collect ions during the 2014 2019 growing seasons. A 1 indicat es a sampled field in a year that the field was not sampled. Farm Field 2014 2015 2016 2017 2018 2019 Farm 1 Field 1 1 0 0 0 1 0 Farm 2 Field 2 1 1 0 0 0 0 Farm 3 Field 3 1 0 0 0 0 0 Farm 4 Field 4 1 1 0 1 1 1 Farm 5 Field 5 1 0 0 0 1 1 Farm 5 Field 6 0 0 0 0 0 1 Farm 5 Field 7 0 0 0 0 0 1 Farm 5 Field 8 0 0 0 0 0 1 Farm 5 Field 9 1 0 0 0 0 0 Farm 5 Field 10 0 0 0 0 1 0 Farm 6 Field 11 1 1 1 0 0 0 Farm 7 Field 12 0 1 0 0 0 0 Farm 8 Field 13 0 1 0 0 0 0 Farm 9 Field 14 0 0 1 0 0 1 Farm 9 Field 15 0 0 0 0 0 1 Farm 9 Field 16 0 0 0 0 1 0 Farm 9 Field 17 1 0 0 0 0 0 Farm 10 Field 18 0 0 0 1 0 0 Farm 11 Fie ld 19 1 0 0 1 0 0 Farm 12 Field 20 0 0 0 1 0 0 Totals 9 5 2 4 5 7 41 1 : Number of samples t aken across years per sampling week. 2 : T otal number of leafhopper s per 100 sweeps summed across years . 3 : Mean number of leafhopper s per 100 sweeps averaged across years . Table S 2. 3. Weekly mean densities of aster leafhoppers (leafhoppers per 100 sweeps) collected throughout the 2014 2019 growing seasons in Michigan. Leafhoppers were collected with swee p nets from commercial carrot and celery farms. Week Carrot Celery N 1 Total 2 Mean 3 N 1 Total 2 Mean 3 20 NA NA NA 1 2.67 2.67 21 NA NA NA 6 5.56 0.93 ± 0.43 22 3 3.94 1.31 ± 0.68 36 156.43 4.35 ± 0.64 23 4 20.91 5.23 ± 0.75 36 79.92 2.22 ± 0.35 24 3 3 3.29 11.10 ± 10.29 46 197.09 4.28 ± 0.72 25 6 12.22 2.04 ± 0.92 27 42.56 1.58 ± 0.28 26 4 19.84 4.96 ± 2.77 21 42.54 2.03 ± 0.55 27 6 29.18 4.86 ± 2.67 16 23.36 1.46 ± 0.33 28 14 58.06 4.15 ± 1.34 21 53.96 2.57 ± 0.71 29 5 34.33 6.87 ± 5.42 16 35.77 2 .24 ± 0.94 30 9 32.06 3.56 ± 1.54 29 94.83 3.27 ± 0.67 31 16 177.35 11.08 ± 3.03 9 49.78 5.53 ± 1.96 32 4 20.29 5.07 ± 2.57 10 55.19 5.52 ± 2.72 33 5 10.63 2.13 ± 1.18 4 6.54 1.63 ± 0.45 34 4 9.48 2.37 ± 1.40 4 7.34 1.83 ± 0.82 42 1 : Number of samples taken across years per sampling week. 2 : T otal number of infected leafhopper s summed across years . 3 : Mean number of infected leafhopper s averaged across years . Table S 2. 4 . Weekly mean abundances of aster yellows phytoplasma infected aster lea fhoppers collected throughout the 2014 2019 growing seasons in Michigan. Leafhoppers were collected with sweep nets from commercial carrot and celery farms and identified as infected usi ng qPCR based diagnostic methods. Week Carrot Celery N 1 Total 2 Mean 3 N 1 Total 2 Mean 3 20 NA NA NA 1 0.00 0.00 21 NA NA NA 6 1.00 0.17 ± 0.17 22 3 0.00 0.00 36 4.00 0.11 ± 0.07 23 4 0.00 0.00 36 4.00 0.11 ± 0.07 24 3 0.00 0.00 46 11.00 0.24 ± 0.11 25 6 1.00 0.17 ± 0. 17 27 10.00 0.37 ± 0.15 26 4 8.00 2.00 ± 1.22 21 13 .00 0.62 ± 0.30 27 6 10.00 1.67 ± 1.05 16 2.00 0.13 ± 0.09 28 14 15.00 1.07 ± 0.73 21 7.00 0.33 ± 0.20 29 5 0.00 0.00 16 4.00 0.25 ± 0.17 30 9 0.00 0.00 29 2.00 0.07 ± 0.05 31 16 4.00 0.25 ± 0.14 9 0.00 0.00 32 4 0.00 0.00 10 0.00 0.00 33 5 1.00 0.2 0 ± 0.20 4 1.00 0.25 ± 0.25 34 4 0.00 0.00 4 1.00 0.25 ± 0.25 43 Suppleme ntary Figures for Chapter 2 Figure S 2. 1. number of sites within each distance band for (A) celery and (B) carrot sites . 44 Figure S 2. 2 . Temporal relationship between aster leafhopper population s in celery and carrot using c ross correlation analysis for aster leafhopper density (leafhoppers per 100 sweeps) in celery and carrot fields in Michigan during the 2014 2019 growing seasons. Dotted lines indicate 95% confidence interval and each lag repre sents a week. (A) No correlation was found between leafhopper densities between the two crops. (B) Density of aster leafhoppers plotted by week a cross the season in Michigan celery and carrot fields. The weeks on the x - axis correspond with the weeks of the calendar year. 45 Figure S 2.3. Cross correlation analysis for aster leafhopper density (leafhoppers per 100 sweeps) and the number of aster yel lows phytoplasma infected leafhoppers in carrot fields in Michigan during the 2014 2019 growing seasons. Dotted lines indicate 95% confidence interval and each lag represents a week. (A) No correlation was found between leafhopper density and infectivity i n carrots. (B) Density of aster leafhoppers and total number of aster yellows phytoplasma infected leafhoppers plotted by week across the season in Michigan carrot fields. The weeks on the x - axis correspond with the weeks of the calendar year. 46 Supplement ary Information for Chapter 2 Text messaging and infectivity follow up discussion During the growing season in celery , there is a natural increase in abundance peaking in late June early July followed by a decline in abundance within the crop . 29 This also occurs in carrots, but at a slower rate, with more leafhoppers being found with in fields during August (pers onal observation, P. Stillson). After the leafhoppers reach peak abundance in cele ry, they move from the field to the edge for the remainder of the season, likely due to an increase in preferred plants in the edge (see chapter 3, and Jubenville 2015). 29 I n addition , weather may contribute to fluctuations in abundance , but in this chapt er, on abundance or infectivity during the season . Beyond these variables, we were unable to provide controls d ue to the nature of th is research and the ethical implications of not providing infectivity reports to a ll farmers. In order to rectify this in future stud ies that may follow up on this research, I suggest comparing two groups of farmers: those consenting to not receive the tex t messages and those that receive infectivity text m e ssages. This study could then be used to improve our understanding of how the text messaging tool works. Alternatively, all farmers could still receive the text messages, if a comparable control were to be used, such as comparing infectivity between commercial and organic farms . A t th is time, we have only worked with one farm that used organic management , and only one of their fields was organic (two collections in 2019). 47 LITERATURE CITED 48 LITERATURE CITED 1 Jones VP, Brunner JF, Grove GG, Petit B, Tangren GV, and Jones WE, A web - based decision support system to enhance IPM programs in Washington tree fruit. 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Rep. 2 :578 doi:10.1038/srep00578 (2012). 51 Dethier VG, E volution of feeding preferences in phytophagous insects, Evolution 8 :33 54 (1954). 5 2 Claridge MF and Wilson MR, Seasonal changes and alternation of food plant preference in some mesophyll - feeding leafhoppers, Oecologia 37 :247 255 (1978). 5 3 Stafford CA, Walker GP, and Ullman DE, Infection with a plant virus modifies vector feeding behavior, Proc Natl Acad Sci 108 :9350 9355 (2011). 5 4 Bosque - Pérez NA and Eigenbrode SD, The influence of virus - induced changes in plants on aphid vectors: Insights from luteo virus pathosystems, Virus Res 159 :201 205 (2011). 52 5 5 Chuche J, Boudon - Padieu E, and Thiéry D, Host preferences of the leafhopper Scaphoideus Phytopathogenic Mollicutes 6 :38 - 45 (2016). 53 CHAPTER 3 : Identifying leafhopper targets for controlling aster yellows in carrots and celery 1 Introduction Aster yellows phytoplasma ( Candidatus Phytoplasma sp.) is an insect - vectored plant pathogen 1 broom, and ultimately premature death. 2 Even when infected crops reach harvest, th ey are often unmarketable. 1,3 Aster yellows has been reported to reduce yields by 10% 4 and is one of the most widespread phytoplasmas, affecting 14 vegetable crops across various plant families. 5 7 It is vectored by at least 24 leafhoppers, 8 which must acq uire the phytoplasma from the environment by feeding on infected plants, 1 since phytopl asmas are rarely transovarial. 1,9 Not all leafhoppers can transmit aster yellows, which may be associated with a narrow diet breadth where the leafhoppers do not feed on the infected plant or do not feed on the phloem of the infected plant. 7 If a leafhoppe r feeds on an infected plant, the phytoplasma must successfully migrate to the salivary glands before transmission is possible. 10,11 Polyphagous leafhoppers can acquire phytoplasma from crops or weedy host plants and then spread it among susceptible crop f ields or between the field and field edge. 7 Movement of phytoplasmas in agroecosystems is primarily facilitated by polyphagous leafhoppers feeding locally on infected ho st plants, 1 and seasonal migrations of some leafhopper species from overwintering to su mmer habitats. 12 14 In North America, the main vector of aster yellows phytoplasma is the migratory Macrosteles quadrilineatus the aster leafhopper ( Forbes; Hemiptera: Cicadellidae), a polyphagous species with over 300 host plants, 15 and a broad geographic distribution. 12 Macrosteles quadrilineatus may move between different crops, between 54 fields, and into field edges to feed on grasses and weeds. 16 This movement among various host plants can increase the chances of other leafhopper v ectors acquiring aster yellows. 1,17 Currently, M. quadrilineatus is the focus for controlling aster yellows phytoplasma for vegetable farmers in the Midwest, USA. However, agroecosystems can have diverse leafhopper communities. These leafhopper vectors may then create disease reservoirs in the field edge, especially in perennial weeds that can be a source of infection every year. 1,18,19 The identity and vector status of these other leafhopper taxa is understudied and may be important for developing sustaina ble management methods for aster yellows. To investigate if additional leafhopper species are important aster yellows vectors, we collected leafhoppers from commercial celery and carrot farm s in Michigan during the 2018 and 2019 growing seasons. We collect ed leafhoppers from the crops and the field edges using sweep nets, identified the leafhoppers to the lowest taxonomic level possible using DNA barcoding, conducted molecular diagnostics to determine if they contained phytoplasma, and compared leafhopper s pecies abundances in the different crops and locations. 2 Materials and m ethods 2.1 Study system Leafhoppers were collected using sweep nets (38 cm diameter aerial net) from mid - May throug h early August in the 2018 and 2019 growing seasons. All farms sur veyed (Fig 3. 1) were large - scale commercial operations, managed with synthetic pesticides. Sweep net samples were taken between 11:00 and 14:00 on clear days when insecticides had not been r ecently applied. In 2018, leafhoppers were collected three times f rom the field edge (June 26, July 10, and August 1) from one celery farm and weekly from inside seven celery and five carrot fields (n = 36 55 collections). In 2019, collections from both withi n the fields and from the edges were conducted weekly at ten celer y and seven carrot farms (n = 226 collections). A minimum of 100 sweeps from inside the crop fields were taken from randomly chosen sites, approximately >10 m into the field, away from the f ation around crop fields, along driveways, or along wooded edges (Fig 3. 2A, B). In both years, sweeps were taken within randomly selected 5 m sections of the field edge; the total number of sweeps varied by field edge due to the variability in the amount o f vegetation available for sweeping (200 500 sweeps/field). After collection, all leafhoppers were transported in a cooler from the field to the laboratory, where they were stored at - 20°C. Figure 3. 1. Map of collections sites from Michigan, USA. Symbols indicate locations of celery and carrot fields where leafhoppers were collected in 2018 and 2019. Leafhoppers were collected using sweep nets and transported to the laboratory for identific ation and to determine phytoplasma infectivity. 56 Figure 3. 2. Leafhoppers were collected in celery and carrot field edges in Michigan in 2018 and 2019. ( A) Aerial view of a celery field with boxed area magnified in B. (B) The surveyed field edge types are indicated by the yellow lines, consisting of vegetation betwee n adjacent crop fields and edges between fields and non - agricultural vegetation, including weedy herbaceous plants growing along roads or paths adjacent to fields, or plants naturally growing al ong wooded edges. 2.2 Leafhopper identification and phytoplas ma detection In the laboratory, leafhoppers were sorted into groups upon arrival from the field: M. quadrilineatus , Empoasca fabae the potato leafhopper (Harris), and other leafhoppers grouped based on morphological similarities. Macrosteles quadrilineat us and E. fabae were sight identified and were placed into homogenization tubes for DNA extraction. All M. quadrilineatus (n = 2,883) DNA was extracted following Demeuse et al.; 20 modifications to this protocol included individually extracting DNA from eac h leafhopper and eluting DNA in 50 µl EB elution buffer (Qiagen). To identify the other leafhopper species by DNA barcoding, we used a modified Dellaporta DNA extraction to minimize DNA fragment ation. 21 Leafhoppers (n = 2,166) were placed individually in 2 ml homogenization tubes (Sarstedt, Nümbrecht, Germany ), along with 3 homogenization beads (2.3 mm diameter, zirconia/silica; BioSpec Products, Bartlesville, OK, USA), and 400 µl Dellaporta buff er (1 ml of 100 mM Tris, pH 8.0, 1 ml of 500 mM EDTA, 1.25 57 ml - mercaptoethanol and 6.75 ml of Ultrapure water). Leafhoppers were homogenized for 10 s at 4.0 m/s (FastPrep - 24, MP Biomedicals, Irvine, CA, USA). Afterwards, 52.8 µl 10% SDS was added, samples were vortexed then incubated at 65°C for 1 0 min. After incubation, 128 µl 5 M potassium acetate was added. Samples were vortexed then centrifuged for 10 min at 15,000 rcf. Supernatant was removed and placed in a clean 1.7 ml centrifuge tube. Afterwards, 240 µl cold isopropanol was added to the sup ernatant and the samples were incubated at room temperature for 5 min. Samples were mixed by gentle inversion. Samples were placed in a - 20°C freezer for 1 h and then centrifuged in a 4°C refrig erated centrifuge (Centrifuge 5810 R, Eppendorf, Hamburg, Germ any) for 20 min at 15,200 rcf. Supernatant was removed and 800 µl 70% ethanol was added to the pelleted DNA. Samples were again mixed by gentle inversion and then placed back in the refrigerated centrifuge for 10 min. The supernatant was removed, and pelle ts allowed to air dry. Pellets were suspended in 50 µl EB elution buffer (Qiagen). We used PCR to amplify the cytochrome c oxidase subunit I (COI) gene using the Ron and Nancy primer set (Thermo Fisher Scientific, Waltham, MA, USA), 22 cleaned the PCR product with QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA), and submitted the DNA to sequencing. The seq uences were compared to the National Cen ter for Biotechnology Information genomic database (NCBI), and the leafhoppers were identified based on sequence match and were also compared to previously identified, morphologically similar, specimens in the Albert J. Cook Arthropod Research Collection a t Michigan State University and on Bug Guide. 23 After identification, leafhoppers were divided into commonly collected ( ) or rare (< 50 leafhoppers collected) species. For future reference, one adult specimen of each morphological group was pinned, or one nymph was preserved in 70% ethanol. Voucher 58 specimens were stored in the Albert J. Cook Arthro pod Research Collection, Michigan State University (voucher number: 2019 - 09). 2.3 Detection of phytoplasma All leafhoppers were evaluated for the presence of aster yellows phytoplasma with a TaqMan assay, 20 using universal phytoplasma primers and probe (T hermo Fisher Scientific). 24 For M. quadrilineatus , we used a cycle threshold (Ct) value < 32 to determine positives, as establish ed for our regular diagnostic work for farmers. 20 All non - M. quadrilineatus with Ct - values also tested using conventional PCR with P3/P7 universal phytoplasma primers 25 to verify the presence of phytoplasma. The PCR products were run on a 1% aga rose gel precast with GelRed (Biotium, Fremont, CA, USA) for 1 h at 90 V. Bands were visual ized with a UV transilluminator (Bioolympics, Thousand Oaks, CA, USA). In addition, we searched the literature to determine which of the collected leafhoppers are kn own vectors for aster yellows phytoplasma or other phytoplasmas, or if there are other memb ers of the genus that are phytoplasma vectors. Vector status for leafhoppers found through the literature search was determined through transmission studies where le afhoppers inoculated healthy test plants or inoculated sucrose solutions. We then compared the collected leafhopper species to this dataset. 2. 4 Data analysis To identify which leafhopper species may be feeding on the crops, or moving between the crops an d field edge, we determined if there were differences in leafhopper species abundance betwe en species found in both locations and crops. We used a generalized linear model, where crop type and field location were fixed factors. Differences among means of t ested factors were determined with post - 26 The total number of leafhoppers per 100 sweeps was used for each of 59 the most abundant leafhopper species ( genera or specie ). Leafhoppers per 100 sweeps was used to standardize leafhopper densities across collections with different numbers of sweeps. We performed separate statistical analyses for celery and carrot. To determine if there were differe nces in the number of infected M. quadrilineatus between the crop and field edge, we used a generalized linear model, where field location (inside or outside field) was used as a fixed factor. Differences among means of tested factors was again determined with post - crop (carrot or celery) as a fixed factor. All statistical analyses were conducted in R v.3.6.0. 27 3 Results In total, we collected 5,049 leafhoppers from ce lery and carrot fields and their field edges combined during the 2018 and 2019 growing seasons. We identified 25 genera and 14 species, with an additional 16 morphotypes identified to family level (Cicadellidae; Table 3. 1, Fig 3. 3A - Z). Eight genera and fou r species represented 94% of collected leafhoppers (Table 3. 1). The most abundant species were M. quadrilineatus (57%) and E. fabae (23%). 60 Table 3. 1. Leafhoppers collected from commercial celery and carrot farms in Michigan, USA, from 2018 to 2019. Field edges were defined as areas bordering the crop field or between adjacent fields where crops were not growing. Fractions indicate the num ber of individuals that generated cycle th - time PCR with universal phytoplasma primers 24 out of the total number of individuals collected . Genera/Species Celery field Celery edge Carrot field Carrot edge 2018 total 2019 total Ct - value or range Agallia sp. 2 7 10 24 5 38 - Aphrodes bicinctus 0 49 0 29 9 69 - Athysanus argentarius 0 2 0 6 0 8 - Balclutha sp. 1 29 ½ 23 1/21 34 36.97 Colladonus clitellarius 1 0 0 2 1 2 - Commellus sp. 0 0 0 2 0 2 - Cuerna sp. 0 0 1 2 0 3 - Diplocolenus subg. V erdanus 0 0 0 32 0 32 - Doratura stylata 0 0 0 1/191 0 1/191 34.93 Draeculacephala sp . 0 1/23 3 45 1 1/70 39.90 Elymana inornata 0 0 0 2 0 2 - Empoasca fabae 6/235 6/418 409 75 11/304 5/833 25.20 40.00 Endria inimica 0 0 0 6 0 6 - Erythroneura sp . 1 0 0 0 1 0 - Forcipata loca 0 8 0 1 0 9 - Graphocephala sp . 0 1/1 27 1 1/28 1 40.00 Idiocerus rap hus 3 1 0 1 3 2 - Idiocerus sp . 0 1/11 0 0 1/10 1 36.51 Jikradia olitoria 0 6 0 2 0 8 - Latalus sp. 0 2 2 4/135 0 4/139 35.39 37.03 Macrosteles quadrilineatus 1/447 3/582 7/1423 1/431 3/707 9/2176 17.56 31.73 Neokolla hieroglyphica 0 0 41 5 0 46 - Norvellina sp . 1 1 0 0 1 1 - Paraphlepsius sp . 0 11 5 2 2 16 - Psammotettix lividellus 8 1/187 1 18 0 1/214 36.93 Scaphytopius sp . 2 10 ½ 4 ¼ 14 39.30 Unknown Cicadellidae 0 5 6 24 4 31 - Total leafhoppers collected 701 1353 193 2 1063 110 1 3948 61 Figure 3. 3. Leafhopper species collected from Michigan, USA, celery and carrot farms from 2018 to 2019. (A ) Agallia sp. , (B) Aphrodes bicinctus , (C) Athysanus argentarius , (D) Balclutha sp., ® Colladonus clitellarius , (F) Commellus sp., (G) Cuerna sp.*, (H) Diplocolenus subg. V erdanus , (I) Doratura stylata , (J) Draeculacephala sp . , (K) Elymana inornata , (L) Empoasca fabae , (M) Endria inimica , (N) Erythroneura sp . , (O) Forcipata loca , (P) Graphocephala sp . , (Q) Idiocerus raphus , ® Idiocerus sp . , (S) Jikra dia olitoria , (T) Latalus sp., (U) Macrosteles quadrilineatu s , (V) Neokolla hieroglyphica* , (W) Norvellina sp . , (X) Paraphlepsius sp . , (Y) Psammotettix lividellus , (Z) Scaphytopius sp. Note: * indicates that only nymphs were collected, all other leafhopper s were collected as adults or as both adults and nymphs. See acknowledgements for photo credits . 62 3.1 Celery collections We collected 2,054 leafhoppers from 2018 and 2019 from celery farms, with 701 leafhoppers (34% of the total) collected from within the c elery fields and 1,353 (66%) from the field edge. A total of 18 genera and 9 species were identified. Macrosteles quadrilineatus (50%), E. fabae (32%), and Psammotettix lividellus (Zetterstedt; 9%) were the most abundant leafhopper taxa ( ollected of each ). Erythroneura sp. (Fitch) was only found i n celery fields but not in field edges. When comparing the abundances of the eight most abundant leafhopper taxa within and outside celery fields, they were all predominantly fou nd in the field edge. Macrosteles quadrilineatus was 1.65 times more abundant in celery field edges than within the field (p - value E. fabae was 2.23 times more abundant in field edges than within celery fields (p - value = 0.03; Fig 3.4). Psammotettix lividellus and Balclutha sp. (Kirkaldy) were both found primarily outside celery fields with 1.13 (p - value = 0.97) and 1.21 (p - value = 0.97) times greater abundances in the edge respectively than in the celery field. Three other taxa Latalu s sp. (DeLong & Sleesman), Aphrodes bicinctus (Schrank), and Dra eculacephala sp. (Ball) were found only in the field edge ( Fig 3.5C, D) . Doratura stylata (Boheman) was absent from celery fields and edge collections . 3.2 Carrot collections We collected 2, 995 leafhoppers from carrot farms in 2018 and 2019, with 1,932 leafhoppers (65%) collected from within carrot fields and 1,063 (35%) from the field edges. A total of 23 genera and 13 species were identified. The most abundant leafhopper taxa were M. quadri lineatus (62%), E. fabae (16%), D. stylata (6%), and Latalus sp. (5%) ( collected for each ). Leafhoppers found only in carrots included Commellus sp. (Osborn & Ball), Cuerna sp. (Melichar), Diplocolenus subg. V erdanus (Oman), Doratura stylata , Elymana inornata 63 (Van Duzee), Endria inimica (Say), and Neokolla hi eroglyphica (Say). When comparing the abundances of the eight most abundant leafhopper taxa within and around carrot fields, M. quadrilineatus had 1.75 times greater abun dance within the carrot fields than in the field edge (p - E. fabae had 4 .20 times greater abundance within the field (p - value = 0.99; Fig 3.4 ), as did P. lividellus with 1.44 times greater abundance in the field (p - value = 0.99) than in the f ield edge . Conversely, Latalus sp. , Balclutha sp., and Draeculacephala sp . had greater abundances within the field edges than in the carrot fields, with 3.00 (p - value = 0.97), 2.00 (p - value = 0.99), and 1.94 (p - value = 0.78) times more leafhoppers collecte d respectively. Two other taxa A. bicinctus , and D. stylata were only found in the carrot field edge ( Fig 3.5A, B) . 64 Figure 3.4 . Mean ± SEM Macrosteles quadrilineatus and Empoasca fabae abundance in commercial carrot and celery fields and field edges in 2018 and 2019 . Numbers above bars indicate the number of individuals collected fo r each species and location. Asterisks indicate statistically significant differences between field and ed - value - 65 Figure 3.5 . Mean ± SEM of the eight most abundant leafhopper species in celery and carrot fields and field edges ( individuals collected); excluding Macrosteles quadri lineatus and Empoasca fabae . Leafhoppers were collected from commercial carrot (A, B) and celery (C, D) farms in Michigan in 2018 and 2019. Numbers above bars indicate the number of leafhoppers collected for each crop and location. 66 3. 3 Phytoplasma infect ivity Across the two study years, 12 M. quadrilineatus tested positive for aster yellows using the Ct - value threshold of 32 typically used in our detection assay. 20 Twenty - seven individuals from nine other taxa had Ct - values between 25.2 and 40: 16 E. faba e , 4 Latalus sp., 1 Balclutha sp., 1 Draeculacephala sp., 1 D. stylata , 1 Graphocephala sp. (Van Duzee), 1 Idiocerus sp. (Lewis), 1 P. lividellus , and 1 Scaphytopius sp. (Ball; Table 3.1). One E. fabae tested positive for aster yellows p hytoplasma using P3 /P7 primers, while all the other leafhoppers were negative for aster yellows with this primer set. In addition, we found three known aster yellows phytoplasma vectors in our collections including A. bicinctus , Athysanus argentarius (Metc alf), and E. inimic a but none of these produced Ct - Agallia sp. (Curtis), Paraphlepsius sp. (Baker), and Scaphytopius sp. may potentially be vectors since there are aster yellows vectors in thes e genera. Of those we identified to species, Colladonus clitellarius (Say) , E. inornata, and N. hieroglyphica while not known to transmit aster yellows, other species in their genera are aster yellows vectors (Table 3.2, 3.3). There was no difference in the number of infecte d M. quadrilineatus between crops (p - value = 0.61) or between the crop field and the field edge (p - value = 0.67). 67 Table 3. 2 . Known leafhopper vectors of aster yellows phytoplasma or other phytoplasmas for the species collected in this study. Phytoplasma abbreviations are AWB = alfalfa witches broom, AshY = ash yellows, AYp = aster yellows, Cp = clover phyllody, CYE = clover yellow edge, EastX = Eastern X, EAYp = European aster yellows, GFD = Grape flav escence doree, NAGVY = North American grapevine yellow s IIIB, Sp = stolbur, SGP = strawberry green petal. Diplocolenus subg. V erdanus , Doratura stylata , Forcipata loca , and Idiocerus raphus were omitted as there is no record of whether they or their congen ers vector phytoplasmas. Species V ector s AYp V ector s o ther phytoplasmas Congener v ector s AYp Congener v ector s other phytoplasmas References Aphrodes bicinctus Yes EAYp, Sp, SGP, Cp, CYE - A. albifrons 8, 28, 29 Athysanus argentarius Yes - - - 30 Colladonus clitellarius - EastX, AshY C. gemi na tus, C. montanus montanus C. gemi na tus, C. montanus montanus 8, 31 36 Elyma na inor na ta - - E. sulphurella E. virescens 37 39 Empoasca f abae - - - E. decipiens, E. papayae 40 42 Endria inimica Yes - - - 43 Jikradia olitoria - N/A GVY - - 44 Macrosteles quadrilineatus Yes EAYp, Sp, Cp M. sexnotatus M. cirstata, M. laevis, M. quadripunctulatus, M. sexnotatus, M. striifrons, M. viridigriseus 19, 28, 45 59 Neokolla hieroglyphic a - AWB N . severini N. confluens, N. severini 8 , 50, 60 Psammotettix lividellus - GFD - P. cephalotes, P. striatus 8, 18, 61 - about whether the species or members of the genus can vector AYp or other phytoplasmas 68 Table 3. 3 . Known leafhopper vectors of aster yel lows phytoplasma or other phytoplasmas for the genera collected in this study. Commellus sp., Draeculacephala sp . , Erythroneura sp . , and Graphocephala sp . were omitted as there is no record of whether species in these genera vector phytoplasmas . Genus V ect or s AYp V ector s other phytoplasmas References Agallia sp. A. constricta - 62, 63 Balclutha sp. - B. punctata 64 Cuer na sp. - C. septentrio na lis 60 Latalus sp. - Latalus sp. 65 Norvelli na sp . - N. seminuda 45 Paraphlepsius sp . P. aper tinus, P. irroratus P. irroratus 8, 31, 63, 66 Scaphytopius sp . S. acutus acutus, S. acutus delongi S. acutus acutus, S. acutus delongi , S. magdalensis 8, 31, 35, 67 72 - whether the species or members of the genus can vector AYp or other phytoplasmas. 69 4 Discussion Our leafhopper survey confirmed that M. quadrilineatus is the primary leafhopper vector of aster yellows phytoplasma in Michigan celery and carrot agroecosystems , which is consistent with findings from Ohio 12 and Wisconsin 73 carrot fields, and is the first study to confirm this in Midwestern celery fields. While other leafhopper species reside in and near these crops, we did not find strong evidence that they cont ribute to phytoplasma infections within these crops. Additionally, we determined that the leafh opper communities were different between the two cropping systems with the field edges characterized by a greater diversity of species than the crop fields. With 5 it is essential to identify its leafhopper vectors. Our results indicated that across both celery and carrot cropping systems, M. quadrilineatus was the most abundant species , and although carrots overal l had more diversity in leafhopper taxa, the edges of both crops were comparable in leafhopper abundance and composition. The known aster yellows phytoplasma vectors collected were A. bicinctus , A. argentarius , and E. inimica (only in carrot) which were all found in the field edge and are known to feed on grasses, cereals, and clover. 30,43,74 We als o collected Scaphytopius sp. from both cropping systems and while they are likely to be Scaphytopius acutus (Say), a known vector of aster yellows, 75 we did not find strong evidence that this leafhopper is vectoring phytoplasma (Ct = 39.3, n = 1). Unlike s ome of the other leafhopper species in our collections, Scaphytopius sp. was found in both carrot and celery fields and field edges, indicating tha t it is likely to frequently move to new host plants. Doratura stylata and Latalus sp. had the lowest Ct - valu es (Ct = 34.93 , n = 1; Ct = 35.39 37.03 , n = 4), besides M. quadrilineatus and E. fabae . While D. stylata has not been reported in the literature a s a phytoplasma vector, Latalus sp. has been reported as a vector, 70 but we were unable to verify the real - time PCR findings with conventional PCR and sequencing. High Ct - values can potentially result when non - vector leafhoppers f e ed on an infected plant and the phytoplasma is present in the digestive tract. 76 Because of this, the only way to confirm new vectors is through transmission assays which involve having suspected vectors feed on phytoplasma infected plants to acquire the pathogen, followed with them inoculating healthy plants or a sucrose solution. 43,75,77 If the disease is detected in the plant, after a l atency period lasting up to a month, or in the sucrose solution after inoculation, then the leafhopper is a vector for the phytoplasma. 43,75,77 Nev ertheless, real - time PCR is known to be more sensitive for aster yellows detection than conventional PCR ; 20 t hus we cannot exclude the possibility that some of the leafhoppers with high Ct - values are in the early stages of infection. We verified one E. fab ae with conventional PCR and sequencing as containing aster yellows phytoplasma. Empoasca fabae has previousl y been detected with a strain of aster yellows, although the authors did not determine vector status. 78 Two other Empoasca spp. A re known phytoplas ma vectors: Empoasca papayae (Oman) vectors Papaya Bunchy Top associated with Candidatus Phytoplasma aurantif olia , 40,42,79 and Empoasca decipiens (Paoli) vectors chrysanthemum yellows phytoplasma, which is closely related to aster yellows (Table 3. 2). 41 Although we hypothesized that field edges may be disease reservoirs and a source of infection for the crops, ou r findings indicate that the edge may not be the primary source of phytoplasma infection. Since more M. quadrilineatus were positive for aster ye llows in samples collected from within crop fields, compared to field edges, this could indicate that aster yel lows phytoplasma is brought into the field by migrating M. quadrilineatus that later move from the crop to the field edge. 28,29 In the field edge , infected M. quadrilineatus can infect plants which may become disease reservoirs and sources of aster yellows phytoplasma for other leafhoppers. 71 Based on our findings, M. quadrilineatus had higher abundance in carrot fields compared to celery which could be due to differences in the management of the two crops. For example, celery is transplanted in the spring, w hile carrots are direct seeded, thus celery is available earlier in the season for M. quadrilineatus colonization than carrots. Carrots are grown in counties North of the celery producing area (Fig 3. 1), accentuating the difference in developmental stages between the two crops. The management intensity of the two crops is also different with more frequent insecticide applications in celery compared to carrots ( personal observation, Z. Szendrei). This is likely due to the direct damage to celery stems and le aves by annually occurring pests such as caterpillars and aphids and the relatively higher value of celery compared to carrots ($19.5 million and $14.5 million respectively in Michigan in 2018). 80 Areas surrounding crop fields such as field edges can play an important role in the lifecycle of vectored pathogens not only for creating disease reservoirs but by managing vector populations using trap c rops. 81 Difference in host plant preference could be used to attract M. quadrilineatus away from crops to trap crops planted in field edges. For trap crops to work effectively, we need to identify plants that are more attractive to M. quadrilineatus compar ed to the vegetable crops. 82 By planting trap crops we may be able to mostly contain the leafhoppers in the fie ld edge, especially when crops are in their most susceptible developmental stages. It will also be important to screen for aster yellows resistan t hosts, from which phytoplasma cannot be acquired and transmitted to other plants. Since other leafhopper spec ies are likely not as important in aster yellows transmission, focusing on M. quadrilineatus behavioral management could potentially be an effect may implement other control measures, such as mowing weedy field margins, thus reducing potential alternative hosts for M. quadrilineatus . These management strategi es can also be paired 72 with diagnostics - based support tools that inform growers about leafhopper infectivity. 83 By utilizing multiple methods for management, farmers will be able to better control aster yellows phytoplasma in a sustainable way. 5 Conclusio ns Insect - vectored plant pathogens are challenging to manage with sustainable methods, especially when both the vector and pathogen have wide host ranges. Here, we made an important first step by confirming that M. quadrilineatus is an important vector of aster yellows and that E. fabae may potentially be another vector in celery and carrot agroecosystems. The next step will be to conduct transmission tests to determine if E. fabae can vector aster yellows, as they are often abundant in aster yellows susceptible crops, many of which also have M. quadrilineatus . 84,85 Both leafhoppers are found in the field and field edges of celery and carrot fields, and if E. fabae can vector aster yellows, limiting where the leafhoppers can acquire the pathogen by using disease resistant trap crops will minimize phytoplasma prevalence. A cknowledgements Leafhopper photos were used w ith permission from: David Cappaert (Fig 3 .3 C ), Ken Childs ( F ig 3 .3 A, 3 .3 D, 3. 3E , 3.3J, 3.3Z ), Peter Cristofono ( F ig 3 .3 S ), Charley Eiseman ( F ig 3 .3 N), Judy Gallagher ( F ig 3 .3 V), Joyce Gross ( F ig 3 .3 X ), Jon Hart ( F ig 3 .3 P, 3 .3 W), MJ Hatfield ( F ig 3 .3 F), Al ain Hogue ( Fig 3.3G, 3 .3 H, 3 .3 M, 3 .3 T), Kyle Kittelberger ( F ig 3 .3 B, 3 .3 O, 3 .3 R), Ilona Loser ( F ig 3 .3 K, 3 .3 Q), Tom Murray ( F ig 3 .3 U), Claude Pilon ( F ig 3 .3 I, 3 .3 Y), and Joh n Schneider ( F ig 3 .3 L) . 73 LITERATURE CITED 74 LITERATURE CITED 1 Weintr aub PG and Beanland L , Insect vectors of phytoplasmas. 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Am J Plant Sci Biotechnol 141 : 425 - 462 (2011) . 7 9 Pérez KA, Piñol B, Rosete YA, Wilson M, Boa E and Lu cas J, Transmission of the phytoplasma associated with Bunc hy Top Symptom of papaya by Empoasca papayae Oman. J Phytopathol 158 :194 196 (2010). 80 USDA NASS National Agricultural Statistics Service. Available online: https://www .nass.usda.gov/ (accessed on 9 January 2020). 81 Sharon R, Soroker V, Wesley SD, Zahavi T, Harari A and Weintraub PG, Vitex agnus - castus is a preferred host plant for Hyalesthes obsoletus. J Chem Ecol 31 :1051 - 1063 (2005). 82 Szendrei Z, The impact of plant associations on Macrosteles quadrilineatus management in carrots: plant association effects on leafhoppers. Entomol Exp Appl 143 :191 198 (2012). 83 Stillson PT , Bloom EH , Gutierrez Illan J and Szendrei Z , A novel plant pat hogen management tool for vector management . Pest Manag Sci (under review). 8 4 Poos FW and Wheeler NH , Studies on host plants of the leafhoppers of the genus US Dep Agric ( 1943 ) . 8 5 Delahaut KA , Aster leafhopper. University of Wisconsin Extension ( 1997 ) . 81 CHAPTER 4: Conclusions and future directions In this thesis, I hav e discussed potential strategies for controlling M. quadrilineatus and aster yellows phytoplasma through a pathogen - based decision support tool (Chapter 2) . I have also confirmed that M. quadrilin eat us is the main vector of aster yellows in these celery and carrot agroecosystems and identif ied other leafhopper species that may contribute to transmission (Chapter 3). Our knowledge of phytoplasma transmission has greatly increased in recent years, 1 3 but management strategies are lacking. The research presented in this thesis helps outline potential strategies for controlling M. quadrilineatus and helps to identify possible vectors of disease transmission in celery and carrot agroecosystems to aid in managing aster yellows phytoplasma. In Chapter 2, my research focused on determining if the Vegetable Entomology ion support tool, designed to target aster yellows phytoplasma infected M. quadrilineatus , improved management of infected l eafhoppers within celery and carrot farms. Decision support tools have become an important component of IPM (Integrated Pest Managem ent) and in recent years, have become more common with the advancement of internet based programs and mobile devices 4 6 and identifying if these tools work as they are designed to is crucial to proper pest management. Besides verifying the efficacy of the support tool , I identified patterns in M. quadrilineatus abundance and infectivity during the growing season and found tempo ral trends between infectivity across carrot and celery systems. Additionally, a spatial component was analyzed to identify how far from the collection origin are infected leafhopper results useful. The results from this chapter have useful implications f or management of aster yellows in various cropping systems. We determine that our decision support tool worked to control infected 82 M . quadrilineatus populations in celery and carrot systems and similar tools could be applied to other aster yellows suscepti ble cropping systems as well as to other pathosystems where the pathogen is latent for several weeks before it is transmittable . We also determined that in celery systems, a high abundance of M. quadrilineatus proceeds an increase in infectivity by two wee ks, demonstrating that a high abundance of leafhoppers does indicate infected leafhoppers are present in the crop. We also foun d that in our system infectivity in celery proceeds that in carrots, indicating that management in carrots should take place afte r the disease has been detected in celery, not before. Additionally, our spatial findings indicate that if an infected leafhopp er is found at a celery farm, farmers should manage all of their fields, not just the field that the leafhopper was found at. Thi s decreases the amount of sampling farmers need to do and will maintain the same level control. Both the support tool and these temporal and spatial findings provide Michigan farmers with additional guidelines for managing M. quadrilineatus . In Chapter 3, my research focused on identifying vectors of aster yellows phytoplasma in the crop field and the field edges of celery and ca rrot agroecosystems. This research determined that although there are aster yellows vectors in the edges of both cropping systems , these vectors are likely not contributing to the spread of aster yellows within the crops. Instead, M. quadrilineatus is like ly the primary vector and moves the pathogen to the edge, infecting the other leafhoppers. As molecular diagnostics have become l ess expensive over the past few decades, studies have focused on investigating new vectors to improve disease management and li mit transmission . W hile these studies are becoming more common, 7 10 my research is novel as it takes into account both known pest species and non - pest species and compares their populations across two distinct susceptible cropping systems. 83 This study prov ided important foundational research in managing aster yellows in celery and carrots. I identified that other leafhopper species in these crops are likely not contributing to the spread of aster yellows as most non - M. quadrilineatus that had Ct - values 40 were in the edge while infected M. quadrilineatus were found within the fields, thus we need to focus management on M. quadrilin eatus. This may be possible using trap crops 11 that M. quadrilineatus prefer to vegetable crops. Beyond this, the trap crops need to be resistant or immune to aster yellows to prevent creating a disease reservoir that may increase the likelihood of other l eafhopper species becom ing vectors. In addition, E. fabe was identified as a pot ential aster yellows vector. Because of its high abundance in the same susceptible crops as M. quadrilineatus . Empoasca fabae is likely acquiring the pathogen from reservoirs t hat M. quadrilineatus creates and so identifying if it can vector the disease th rough transmission tests is essential to determine if this pest also needs to be managed to control aster yellows outbreaks. Overall, this thesis was the first to explore the u se of a decision support tool to manage M. quadrilineatus that also controlled aster yellows phytoplasma. It was also the first to explore the leafhopper species diversity within Michigan celery and carrot agroecosystems to determine whether other vectors are present and contributing to the spread of aster yello ws in these crops. 84 APPENDIX 85 Record of Deposition of Voucher Specimens The specimens listed below have been deposited in the named museum as samples of those species or other taxa , which were used in this research. Voucher recognition labels bearing the voucher number have been attached or included in fluid preserved specimens. Voucher Number: 2019 - 09 Author: Patrick T . Stillson Title of thesis : B iology and management of leafho ppers and aster yellows phytoplasma in Michigan celery and carrot agroecosystems Museum(s) where deposited: Albert J. Cook Arthropod Research Collection, Michigan State University (MSU) Table S4.1. Voucher specimens deposited at the Albert J. Cook Arthro pod Research Collection (Michigan State University) . Family Genus - Species Life Stage Quantity Preservation Cicadellidae Agallia quadripunctata Adult 1 Pinned Cicadellidae Aphrodes bicinctus Adult 1 Pinned Cicadellidae Aphrodes bicinctus Nymph 1 Ethanol Cicadellidae Athysanus argentarius Adult 1 Pinned Cicadellidae Balclutha sp. Adult 1 Pinned Cicadellidae Colladonus clitellarius Adult 1 Pinned Cicadellidae Commellus sp. Adult 1 Pinned Cicadellidae Cuerna sp. Nymph 1 Ethanol Cicadellidae Diplocolenu s subg. Verdanus Adult 1 Pinned Cicadellidae Doratura stylata Adult 1 Pinned Cicadellidae Doratura stylata Nymph 1 Ethanol Cicadellidae Draeculacephala sp . Adult 1 Pinned Cicadellidae Draeculacephala sp . Nymph 1 Ethanol Cicadellidae Elymana inornata A dult 1 Pinned Cicadellidae Empoasca fabae Adult 1 Pinned Cicadellidae Endria inimica Adult 1 Pinned Cicadellidae Endria inimica Nymph 1 Ethanol Cicadellidae Erythroneura sp . Adult 0 Fig S4.1 Cicadellidae Forcipata loca Adult 1 Pinned Cicadellidae Gra phocephala sp . Adult 1 Pinned Cicadellidae Idiocerus raphus Adult 1 Pinned Cicadellidae Jikradia olitoria Adult 1 Pinned 86 Table S4.1 Family Genus - Species Life Stage Quantity Preservation Cicadellidae Latalus sp. Adult 1 Pinned Cicadellidae Ma crosteles quadrilineatus Adult 20 Pinned Cicadellidae Neokolla hieroglyphica Nymph 1 Ethanol Cicadellidae Norvellina sp . Adult 1 Pinned Cicadellidae Paraphlepsius sp . Adult 1 Pinned Cicadellidae Paraphlepsius sp . Nymph 1 Ethanol Cicadellidae Psammotet tix lividellus Adult 1 Pinned Cicadellidae Scaphytopius sp . Adult 1 Pinned Cicadellidae Morphotype 1 Nymph 1 Ethanol Cicadellidae Morphotype 2 Nymph 1 Ethanol Cicadellidae Morphotype 3 Nymph 1 Ethanol Cicadellidae Morphotype 4 Nymph 1 Ethanol Cicadel lidae Morphotype 5 Adult 1 Pinned Cicadellidae Morphotype 6 Adult 1 Pinned Cicadellidae Morphotype 7 Adult 1 Pinned Cicadellidae Morphotype 8 Adult 1 Pinned Cicadellidae Morphotype 9 Adult 1 Pinned Cicadellidae Morphotype 10 Adult 1 Pinned Cicadellid ae Morphotype 11 Adult 1 Pinned Cicadellidae Morphotype 12 Adult 1 Pinned Cicadellidae Morphotype 13 Adult 1 Pinned Cicadellidae Morphotype 14 Adult 1 Pinned Cicadellidae Morphotype 1 5 Adult 0 Fig S4.2 Cicadellidae Morphotype 1 6 Adult 0 Fig S4. 3 87 F igure S4.1. Erythroneura sp . v oucher specimen. Dorsal (A) and ventral (B) view . 88 Figure S4.2. Morphotyp e 15 v oucher specimen. Dorsal view . 89 Figure S4. 3 . Morphotype 1 6 v oucher specimen. 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