INVESTIGATING INTERACTIONS BETWEEN ENVIRONMENTAL EFFECTS AND POME FRUIT 
IPM 

By 

Dalton Miner 

A THESIS 

Submitted to 
Michigan State University 
in partial fulfillment of the requirements 
for the degree of 

Entomology-Master of Science 

2023 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ABSTRACT 

Protecting fruit trees from diseases and insect pests is an essential part in maintaining a healthy and 

productive orchard. One of the most impactful diseases apple growers need to manage is the fungal 

disease Apple Scab, Venturia inaequalis. Growers typically use fungicide application as the primary 

method of apple scab management. In addition to managing apple scab, growers need to manage 

environmental conditions affecting their fungicide application. This study evaluated the rainfastness of 

certain fungicides and the addition of a sticker adjuvant and their efficacy after wash off after four levels 

of simulated rainfall (0, 6.3, 12.7, 25.4mm). The rainfast properties were analyzed based on recovered 

residue data, and the efficacy was measured by shoot count infection counts after infield infection 

simulation. This data will contribute data to spray management programs in order to manage apple scab. 

One of the most impactful pests pear growers need to manage is pear psylla, Cacopsylla pyricola Forster 

(Hemiptera: Psyllidae). This study tested two biopesticides as foliar application and trunk injected 

application methods to evaluate the efficacy of trunk injected application. Insect control was measured by 

egg and nymph counts. The results of this study contribute to the practice of trunk injections as a method 

of pesticide application as well as the efficacy of biopesticides and their development into modern pest 

management programs.

 
 
Dedicated to Jeff, Nikki, Abbie and Ron Min

iii 

 
 
 
 
 
 
 
 
 
 
 
 
 
ACKNOWLEDGEMENTS 

There are many people I would like to thank for the completion of my Thesis and Masters Degree. First, I 

would like to thank my advisor, John Wise, for the opportunity to pursue a masters degree and for the 

continued assistance and support. I would like to thank my committee members, George Sundin and 

David Mota-Sanchez for their advice, guidance, facilities, and crew support for completing crucial aspects 

of the research. Thank you to Cory Outwater for all of his help in the maintenance and execution of one 

of the research projects. I would like to thank Chris VanderVoort and Ehab Abdelraheem for their 

chemistry procedure development and for analyzing hundreds of residue samples. I would like to thank 

my lab mate Ignatius Putra for his help in all parts of my research, from picking leaves and counting 

psylla nymphs to writing R code for statistical analysis. Thank you to the staff at Trevor Nichols Research 

Center for their help in their access to the farm and aspects of data collection and analysis.  

iv 

 
 
 
TABLE OF CONTENTS 

CHAPTER 1: INTRODUCTION ................................................................................................................. 1 

CHAPTER 2: RAINFASTNESS OF FUGICIDES AND DISEASE CONTROL IN APPLES ................... 6 

CHAPTER 3: COMPARING FOLIAR APPICATION AND TRUNK INJECTION APPLICATION OF 
A PEPTIDE BASED BIOPESTICIDE FOR PEAR PSYLLA CONTROL ............................................... 29 

CHAPTER 4: CONCLUSION.................................................................................................................... 40 

BIBLIOGRAPHY ....................................................................................................................................... 43 

v 

 
Rainfastness of Fungicides for Disease Control in Apples 

CHAPTER 1: INTODUCTOIN 

The United States apple industry generates roughly $3 Billion, and Michigan is the third most 

valuable state for apple production with a production of approximately $339 Million in 2021. The United 

States has slightly more than 11.4 million acres of apple orchards (Gerlach, 2022). 

All plant diseases are caused by microorganisms (Guest, 2017). The differences in disease 

symptoms result from the nuances of their pathogenicity, fungi are very different from viruses which are 

different from bacteria. Each disease has unique life cycles, with different infection methods or hosts or 

ways of transmission. Apple scab is a fungal disease that threatens the health and productivity of apple 

orchards.  

Apple scab is the most economically important disease of apples in the world and can cause 

significant losses as fruit infection can lower the value of an apple crop or result in total loss of the crop 

(Nu et al., 2019). Apple scab, caused by Venturia inaequalis (Cooke) G. Wint. (Rossouw et al., 2018), is a 

consistent disease problem year-by-year in Michigan orchards. The scab spores overwinter on the ground 

and in leaf litter beneath the tree canopies. Apple scab can be difficult to manage because it has multiple 

infection cycles in one apple growing season with the assistance of asexual conidia reproduction (Apple 

Scab, n.d.). The infection period begins in early spring on young emerging leaves. Apple scab causes 

lesions to appear under specific environmental conditions. The ideal infection conditions for apple scab 

include 17.2-25.6 °C and wet leaves for 9-26 hours (Apple Scab, n.d.). The moisture on the leaves allows 

for apple scab spore germination and penetration into the leaf tissue. These specific weather conditions 

lead to management strategies. Application of fungicides can be timed around infection events which 

would strategize the use of product leading to a more efficient, cost-effective control program. Scab 

infections on the leaves and fruit impair sugar production and fruit sale value, threatening the long-term 

health of the orchard and hurting growers’ livelihood.  

Apple scab can be managed with a multitude of approaches. The most passive method of control 

is to plant scab resistant cultivars of apple trees. This eliminates the opportunity for initial infection to 

1 

 
 
 
take effect. The next cultural method of apple scab management is to manage environmental conditions to 

mitigate spore production and distribution. This would include planting trees further apart, pruning trees 

to increase air flow and sunlight penetration, and to manage leaf litter by removing the leaves or treating 

them to increase decomposition. The most effective and most common method of apple scab management 

is with fungicide applications (Gauthier, 2018). 

Modern fungicide development began after 1940 (Staub et al., 2008). The introduction of modern 

fungicides were organic compounds that were less phytotoxic, easier to use and prepare, and safer for the 

environment because of the volume of product required. Captan was introduced as an active ingredient in 

1952, and mancozeb in 1961. These fungicides, along with many others, would become major 

contributors to plant disease management (Staub et al., 2008). 

Captan, pydiflumetofen and other succinate dehydrogenase inhibitor (SDHI) fungicides are 

registered fungicides for apple scab management. Mancozeb, an ethylene bisdithiocarbamates (EBDCs) 

fungicide, has been used in agriculture for several decades. Growers of apples have used a variety of other 

fungicides to manage apple scab from other groups including demethylation inhibitors (DMI’s) like Indar, 

Topguard, or Rally. Growers may use Quinone outside Inhibitors (QoI) like Flint or Sovran. Farmers and 

growers are still widely using mancozeb because of its broad spectrum of activity against various fungal 

diseases and low production costs (Crnogorac & Schwack, 2009; Hano et al., 2015). Captan, as a key 

alternative to EBDCs, is another widely used broad-spectrum fungicide (Chen et al., 2020), and belongs 

to the thiophthalimide class of fungicides that are used frequently for treating various pre- and post-

harvest plant diseases (Oulkar et al., 2019). Pydiflumetofen is a broad-spectrum SDHI fungicide with 

translaminar mobility in plant tissues and is labeled on apple for control of apple scab (Wise et al., 2022). 

Translaminar mobility properties are defined as the ability of a material to penetrate leaf tissue to 

form a reservoir of active ingredient within the leaf only moving short distances from application rather 

than throughout the whole plant (Contact, Systemic and Translaminar, 2007). This ability is different 

from other products that are contact materials depending on surface contact with the target pest to achieve 

control. This distinction is important when assessing rainfastness and efficacy of products because surface 

2 

 
acting contact products are much more susceptible to wash off as a result of them being on the surface. 

Translaminar products are more protected from wash off as they penetrate into the leaf tissue 

simultaneously improving deposition and rainfastness. The translaminar products would then show 

greater efficacy for a longer period of time as they are less affected by environmental degradation. 

Michigan’s fruit production season regularly receives 25-50cm of rainfall. The impact of 

precipitation on the performance of fungicides sprayed to control crop diseases is a serious factor for 

growers in Michigan. Growers frequently add additional products to their spray mixes to change the 

performance of the fungicides. A common addition is a sticker adjuvant, to improve the sprayed product’s 

ability to remain on the surface of the plant tissue. 

Comparing Foliar Application and Trunk Injection of a Peptide Based Biopesticide for Pear Psylla 

Control 

The United States pear industry generated $373 Million in 2021, and Michigan is one of the 6 

major contributing states to pear production (Pears, n.d.). The United States has roughly 16,875 hectares 

of pear orchard (“Pear Fact Sheet,” n.d.) and it is important to keep pear orchards healthy and free of 

pests. Pests to pear orchards range from bacterial infections to insect pests, or large mammal pests eating 

fruit. 

Pear trees are victims to many insect pests. Pests to pear trees include aphids, codling moths, 

mites, apple maggot, plum curculio, and many more (Apple and Pear Insect Pests | University of 

Maryland Extension, n.d.). One of the most impactful insect pests to the pear tree are psylla (Insect Pests | 

Edible Landscapes, n.d.). Psylla nymphs feed on the sap of trees through piercing stylets through the 

veins of the tree, leading to stunted trees, fewer fruit produced, and declining health of the trees and the 

orchard in highly infested orchards (Howitt, 1993). Psylla have three life stages, egg, nymph, and adult. 

Eggs are laid on both sides of the leaf along the midrib. The egg looks like a grain of rice and is laid white 

but turns yellow as it develops. The nymphs pass through five stages of development. Nymphs begin 

smaller and yellow, and gradually get larger, wider, and darker in color as they age. The adult psylla 

presents two forms, summer, and winter form adults. The adults hold their wings above their body and 

3 

 
 
have reddish-brown bodies, winter form appears almost black. Psylla adults overwinter as winter form 

adults under bark or in leaf litter. Psylla infestations can result in fruit russet, psylla shock, declining tree 

health (Pear Psylla Integrated Pest Management | WSU Tree Fruit | Washington State University, n.d.). 

Crop protection has relied heavily on synthetic pesticides but their use has become challenged 

with new legislations and potential resistance development (Chandler et al., 2011) which opens a niche 

for biopesticides to become an alternative. Biopesticides are pesticides made from living organisms or 

their products (Glare et al., 2016). They can be classified into three types of biopesticides: micro-

organisms, biochemicals, and semiochemicals (Chandler et al., 2011). The rise in use of biopesticides 

have led to the development of an assortment of products, including peptide products such as Spear-T. 

Spear-T uses the GS-Omega/Kappa HXTX-HV1A peptide as the active ingredient that causes 

hyperexcitation of the nervous system of an insect, and remains nontoxic to humans, other mammals, 

birds, fish, and other beneficials (Vestaron, 2023). The GS-Omega/Kappa HXTX-HV1A peptide is 

derived from the venom of the Australian Blue Mountain Funnel Web Spider (Bloomquist et al., 2023). 

Spear-T has been shown to control pear psylla in pear when applied via airblast (Wise et al. 2020). 

Azadirachtin is a product extract of the Neem Tree, Azadirachta indica, and affect insects in multiple 

ways including inhibit food intake, morphogenesis, ovarian development, fecundity, egg viability, and 

molting (Karnavar, 1987). Azadirachtin is approved to be used on any food crop, but is not commonly 

applied because it is quick to degrade in the environment (J. C. Wise, 2016). This is not ideal for a cost-

effective, long-term spray plan because of the frequent reapplication required for adequate control. 

The historical solution to managing pests in orchards is to use broad-spectrum pesticides applied 

by airblast sprayer (J. Wise & Whalon, 2009). Airblast ground sprayer application may be fast and easy, 

but there are significant downsides including off target drift, product use efficiency and efficacy, as well 

as monetary losses (VanWoerkom et al., 2014). Pesticide application is determined necessary at a nymph 

threshold of 0.3 nymphs per leaf. The inefficiencies of airblast sprayer applications arise from the amount 

of wasted product through drift and off target application. There are significant differences in product 

distribution after airblast application with approximately half of the sprayed product reaching the leaves 

4 

 
 
 
and even less contacting any insect pest. (Wu et al., 2020; Pimentel & Levitan, 1986) In fact, growers will 

over apply their product to take into account the loss of product (VanWoerkom et al., 2014). Application 

via trunk injections may be a better option than the traditional airblast sprayer. With more growers 

moving towards safer, and selective insecticides, including biopesticides, optimizing delivery and 

persistence is needed. Trunk injection provides an even and efficacious distribution of the product 

throughout the canopy, when using an adequate number of injection ports (Aćimović et al., 2014). Studies 

have shown that trunk injections are a better method of pesticide delivery in apples to control foliar 

feeding pests including potato leafhopper, rosy apple aphid, spotted tentiform leaf miner, and oriental fruit 

moth (VanWoerkom et al., 2014). This method of pesticide application maximizes the exposure of the 

active ingredient to sucking insects as a result of the product being transported into the leaves after 

injection (J. C. Wise, 2016). This means less product is needed for adequate control because there is no 

wasted pesticide to drift, or off target application saving growers money.  

5 

 
 
 
CHAPTER 2: RAINFASTNESS OF FUNGICIDES FOR DISEASE CONTROL IN APPLES 

Abstract 

Field studies and residue profiling were used to determine the rainfastness and corresponding 

efficacy of popular fungicides after specific simulated rainfall events. Residue profiling was used to 

determine rainfastness and deposition of residue after 0, 6.3, 12.7, and 25.4mm of simulated rainfall. Field 

studies were used to determine efficacy of the remaining residue after the simulated rainfall events. The 

products tested were captan, mancozeb, and pydiflumetofen. A sticker adjuvant was added to evaluate 

efficacy differences. The effect of rainfall had varied effects on the fungicides and the addition of a sticker 

proved useful under certain conditions. This study showed a significant loss of apple scab control for all 3 

products at 25.4mm of rainfall, a significant loss of control for Mancozeb and Pydiflumetofen at 12.7mm 

of rainfall, and loss of control with Pydiflumetofen at 6.3mm of rainfall. The addition of a sticker 

improved performance of Mancozeb and Pydiflumetofen at 0 and 12.7mm for Mancozeb and 6.3mm 

rainfall for Pydiflumetofen. This study will help apple growers make informed decisions on when 

fungicide applications are needed. 

Introduction 

Apple scab is the most economically important disease of apples in the world and can cause 

significant losses as fruit infection can lower the value of an apple crop or result in total loss of the crop 

(Nu et al., 2019). Scab, caused by Venturia inaequalis (Cooke) G. Wint. (Rossouw et al., 2018), is a 

consistent disease problem year-by-year in Michigan orchards. Apple scab causes lesions to appear on the 

leaves and fruit impairing sugar production and fruit sale value. 

Captan, Mancozeb, and Pydiflumetofen are just a few of the fungicides used for apple scab 

management. Considering fungicide resistance is an important factor when establishing management 

programs. There are other fungicides in other fungicide groups with different modes of action that 

manage the fungus pest differently. This varied approach to management prevents resistance development 

because of the constant variety of modes of action. The fungicide groups studied were EBDC’s and an 

6 

 
 
 
SDHI. Other fungicide groups include coppers, DMI’s, QoI’s, and others of which contain products 

including Indar, Rally, or Sovran. Captan and Mancozeb were chosen because they are commonly used 

and frequently used together to control apple scab, and Pydiflumetofen was chosen because it uses a 

different mode of action with a different method of management (Apple Scab, 2017). 

In addition to fungicide selection as an important strategy in management, timing of fungicide 

applications are also important in managing apple scab. The timing of fungicide sprays is dependent on 

the stage of the apple scab lifecycle as well as impending weather events. Apple scab is a polycyclic 

disease, which means after the initial primary infection event, spores will begin to reproduce asexually to 

continue the infection and dispersion process (Apple Scab, 2018). If the bulk of maintenance sprays are 

applied to manage the initial inoculum, that has the potential to lead to lower scab pressure in the orchard 

which helps with management. Timing sprays to manage primary inoculum and impending weather 

events that lead to infection is a strategy used to manage apple scab. 

Michigan’s fruit production season regularly receives 25-50 cm of rainfall, and precipitation 

patterns range widely in the number, duration, and intensity of events. The impact of precipitation on the 

performance of fungicides sprayed to control crop diseases is a serious risk factor for Michigan growers. 

Mancozeb has been shown to wash off leaves easily after 5 millimeters of rainfall (Hunsche et al., 2007). 

It has also been showed that there was 68% wash off at 2mm and 91% wash off at 10mm of rainfall 

(Kudsk et al., 1991). This level of intense wash off following minor rainfall events was not shown by 

Rossouw (2018), where high wash off was shown at 15mm, with little wash off at lower rainfall levels. 

Captan has been found to lose half of its residue with only 1 mm of rainfall (Xu et al., 2008). Little work 

has been done to evaluate rainfast properties of pydiflumetofen. 

To combat environmental effects and optimize product performance, adjuvants can be used to aid 

the application of pesticides, including fungicides. Adjuvants are materials added to a spray mixture to 

affect the distribution, deposition, tissue penetration, rainfastness, and droplet size, with the goal of 

improving control of the target pest (Abbott & Beckerman, 2018). Adjuvants that are generally not water 

soluble aid in resisting product wash off from rain, thus allowing for a longer interval of protection 

7 

 
(Hazen, 2000). Adjuvants can also improve product deposition by reducing the surface tension of spray 

solutions, thus increasing the proportion of leaf surface with active ingredient. Adjuvant products called 

stickers, typically consist of heavy petroleum, water-soluble polymers, acrylic latex, epoxidized seed oils, 

or resins. These materials have a natural tendency to adhere to the leaf surface, increasing its ability to 

resist wash off (Hazen, 2000). This is an important distinction for product efficacy, as an adjuvant that 

increases deposition will increase the total active ingredient deposited on the tree canopy, but does not 

directly improve rainfastness, whereas an adjuvant with sticker directly reduces a compounds’ sensitivity 

to wash-off. 

This study aims to evaluate the impact of rainfall on the performance of mancozeb, captan, 

pydiflumetofen against apple scab, and each with the addition of a sticker adjuvant after three different 

levels of simulated rainfall: 6.35 mm, 12.7 mm, and 25.4 mm of rain. This study will evaluate the impact 

of rainfall through the recovery of residues from each sample combination and compare it to apple scab 

infection and protection rates observed in the field. This study is expected to show a gradual decrease in 

effective residue as the rainfall increases demonstrated by an increase in apple scab infection. 

Field residue trials: 2021 and 2022 

Methods and Materials 

Research was conducted on 12-year-old semi-dwarf apples trees (Malus domestica Borkhausen 

cv. 'Enterprise’), at the Michigan State University Trevor Nichols Research Center (TNRC) (42.5951°N, -

86.1561°W). Plots were randomly assigned to their treatments, each plot had 3 trees with 4 replicates. The 

trees within each plot were assessed for fruit and leaf quality to see if they were able to provide a 

minimum of 40 fruit and 120 leaves as experimental samples. Plots were separated by buffer trees to 

avoid spray drift from treatment plots of the same chemical. 

In 2021, one chemical was sprayed per day, over 3 consecutive days. Mancozeb (Roper 75 DF, 

Loveland Products, Greeley, Colorado) was sprayed first at 3.36 kg/ha (3 lb/acre), followed by captan 

(Captan 80 WDG, Arysta LifeScience, Cary, NC) at 2.8 kg/ha (2.5 lb/acre), followed by pydiflumetofen 

8 

 
 
  
 
 
(Miravis 1.67 SC, Syngenta Crop Protection, Greensboro, North Carolina) at 248.5 ml/ha (3.4 oz/acre). 

Fungicide treatments were applied with an FMC 1029 Airblast sprayer (Jonesboro, AK) calibrated to 

deliver 935 liters/ha (100 gal/acre) of diluent. 

In 2022, in addition to the fungicide-alone treated plots, an adjuvant was added to each fungicide 

treatment solution for comparisons. The adjuvant used was a sticker, Pinene (terpene) Polymers, 

petrolatum, a-(p-Dodecylphenyl)-Omega-hydroxypoly (oxyethylene); (Attach, Loveland Products, 

Greeley, Colorado) at 177 ml/gal for each treatment. The first application included mancozeb (Koverall 

75 DF, FMC Corporation, Philadelphia, PA) at 6.73 kg/ha (6 lb/acre), Mancozeb+Attach at 6.73 kg/ha (6 

lb/acre). The fungicides used the next day were captan at 2.8 kg/ha (2.5 lb/acre), Captan+Attach at 2.8 

kg/ha (2.5 lb/acre), pydiflumetofen at 248.5 ml/ha (3.4 oz/acre), and pydiflumetofen+Attach at 248.5 

ml/ha (3.4 oz/acre). Insecticide treatments were applied with an FMC 1029 Airblast sprayer calibrated to 

deliver 935 liters/ha (100 gal/acre) of diluent. 

The untreated fruit and leaves were picked from the trees the day before the first chemical was 

sprayed to eliminate any chance of contamination of the samples. On the day of spraying, each chemical 

was left to dry for 2 hours before picking. After 2 hours, 40 fruit and 120 leaves were picked from each of 

the 3 tree plots, picking from both sides of the tree, East and West. The fruit and leaves were immediately 

prepared for rain simulation.  

Rain Simulation: 2021 and 2022 

The Generation 3 Research Track Sprayer rainfall simulator (DeVries Manufacturing, Hollandale, 

MN) was set up with the AI 11008VS nozzle (TeeJet Technologies, Wheaton, IL), and ran at 69 kPa (10 

PSI) and 0.8 kilometers/hour (Figure 1). Pre-study test runs provided target times for achieving rainfall 

treatment regimens. Three rain gauges were placed inside the rainfall simulator to accurately assess the 

amount and uniformity of simulated rain events. 

To prepare for rain simulation, oasis foam (Smithers-Oasis Co., Kent, OH) was used to hold all 

the plant material. Rain levels of 0 mm, 6.35 mm (0.25 inch), 12.7 mm (0.5 inch), and 25.4 mm (1 inch) 

9 

 
 
 
 
were tested. Using the oasis foam, all 4 replicates of the given rainfall level were tested at the same time. 

Branches of leaves were trimmed by removing small, underdeveloped leaves and stabbed into the foam 

with the leaves facing upward. Branches with fruit were trimmed by removing all the leaves, leaving only 

the fruit on the branch. The samples were trimmed so that 30 leaves and 10 fruits were remaining in the 

oasis foam. Leaves and fruit were placed in separate bricks of oasis foam but kept together within the 

replicate inside the rain simulation booth. All replicates were put into the rain simulator booth blocking 

each replicate of leaves and fruit together. The samples were treated for 4:30, 9:00, and 18:00 minutes for 

6.35 mm, 12.7 mm, and 25.4 mm rain respectively to properly get the desired rainfall level. The rainfall 

simulator was set to travel at 0.5 mph across the length of 6ft over the tray holding the samples. After the 

rain simulation was complete, the samples were left to dry completely. Shoots not assigned to receive 

rainfall were not placed in the rainfall simulator. 

When the samples were dried completely, they were placed into quart sized Ziploc bags. All 

leaves were removed from the stem and put in the appropriate bag. In 2021, the fruit was removed from 

the stem, then cut into quarters, then put into the appropriate bag. In 2022, the fruit were placed into the 

Ziploc bag whole. The plastic bags of the samples were immediately frozen and left frozen until they 

were ready for residue analysis. 

Residue analysis: 

Captan and Pydiflumetofen 

Lab analysis methods are the same for both chemicals, captan and pydiflumetofen. Samples were 

analyzed using the Quechers method (Anastassiades et al. 2003). Leaves and fruit were both prepared for 

analysis by grinding them as fine possible. This was done with a mortar and pestle, with liquid nitrogen to 

freeze the leaves to allow for the most effective grinding. Ten grams of each were put into 50ml 

centrifuge tubes while the remainder were put into their own centrifuge tubes to be used later if a redo is 

required. The 10-gram samples were used for the analysis.  

10 

 
 
 
 
Acetonitrile (10ml) was added to the 50ml centrifuge tube. The tube was then vortexed for 1 

minute, then 4g of MgSO4 and 1g of NaCl were added to the tube. The tube was then vortexed for 1 then 

centrifuged for 5 minutes at 4000 rpm. The supernatant (1ml) was extracted and put through a 0.22-

micrometer nylon syringe filter into a GC/MS glass vial to be used for UPLC-MS/MS injection. 

The same procedure is used for the leaves with the addition of a cleanup procedure. The 1ml of 

supernatant was put into a small centrifuge tube instead of syringe for filtering and 0.150g of MgSO4 and 

0.05g of PSA was added then vortexed for 1 minute. The tubes were then centrifuged for 5 minutes at 

4000 rpm. The supernatant (1ml) was extracted and filtered through a 0.22-microliter nylon syringe filter 

into a GC/MS glass vial to be used for UPLC-MS/MS injection. 

Mancozeb 

To calibrate the machine, a standard of mancozeb needed to be prepared. Four solutions were 

made of different, known, levels of concentration. The solution standards used were 39.4ppm, 26ppm, 

5.25ppm, and 2.63ppm. These solutions were put through the machine to be analyzed, to establish known 

levels of mancozeb in solution. The different concentrations of solution were then mixed into leaves and 

put through the same machine. This was to confirm the precision of the extraction methods. 

The fruit and leaves were prepared by grinding as fine as possible using a mortar and pestle, and 

liquid nitrogen to freeze the samples to grind easier. Ten grams of fruit and leaves were put into large 

centrifuge vials. 8 milliliters of ultra-pure water and 100 milligrams of L-cysteine were added to the tube 

then vortexed on high for 30 seconds. After, 0.5 grams EDTA-4Na and 10 milliliters 0.05 molar dimethyl 

sulfate solution in acetonitrile were added and then shaken vigorously for 15 minutes using a stir table. 

After 15 minutes, 1 gram NaCl and 4 grams MgSO4 were added then vortexed for 1 minute on high. The 

tubes were then centrifuged for 5 minutes at 4000rpm. 1.5 milliliters of acetonitrile supernatant were 

extracted and placed into a small centrifuge tube along with 150 milligrams anhydrous MgSO4 and 50 

milligrams PSA. The tubes were then centrifuged for 5 minutes at 4000rpm. The supernatant was 

removed from the small centrifuge tube and put through a 0.22-microliter nylon syringe filter into an 

auto-sample vial for UPLC-MS/MS injection. 

11 

 
 
 
 
Potted tree field infection trial: 

2022 Treatments 

Two groups of one-year old potted Gala apple trees, Malus domestica, 52 and 43 trees 

respectively, were selected based on health of the tree to be placed in an apple scab infected apple orchard 

to be exposed to natural infection. Each replicate within the group had equal numbers of trees per 

treatment: captan, mancozeb, pydiflumetofen, and untreated as well as equal numbers of trees per rainfall 

treatment: 0 mm, 6.35 mm, 12.7 mm, and 25.4 mm of rain. The weather was closely monitored for ideal 

infection conditions: temperatures higher than 23.9°C, light wind, and a wetting period of multiple hours 

(3+ hours ideally) after the beginning of a rainfall event. Twenty-four hours prior to the targeted rainfall 

event, the saplings were treated with the selected fungicides at rates equivalent to those described above 

on a concentration basis (1.5 g Captan 80WDG / 500 ml of water) (1.8 g Koverall 75DF / 500 ml of 

water) (0.13 ml Miravis 1.67SC / 500 ml of water), with each tree being sprayed with approximately 62.5 

ml of treatment solution per tree. One drop of the adjuvant R-11 was added to break surface tension, 

prevent beading, and improve coverage of the leaves. Antifoaming agent was added to the Captan 

solutions as Captan is prone to foaming. Applications were made using a handheld 500 ml sprayer bottle. 

After 1 hour of drying, each tree was subjected to its indicated level of simulated rainfall using a hand-

held variable nozzle garden hose using the mist setting and allowed to dry again. 

2022 Field Methods 

Trees were placed in the Botany block of the Michigan State University Plant Pathology Farm on 

42-year-old McIntosh trees on M.7 rootstock (42°41’24.56” N, 84°29’17.93” W). Trees within the Botany 

Block were randomly selected using a random number generator to determine row and position within the 

row. One full replicate from each treatment was randomly placed under the assigned tree after the 

simulated rainfall had dried with every rainfall level within one rep going under the canopy of one tree. 

Trees were placed under the canopy of their assigned trees within the orchard before the rainfall event 

(Figure 2). 

12 

 
 
 
After the rainfall event, the saplings were left in the orchard for the remainder of the wet period, 

until the saplings had dried. After the saplings had dried, they were removed from the orchard to prevent 

further apple scab infection and kept on a cement pad in partial sun. The trees were watered and kept alive 

until symptoms appeared. 

2023 Treatments 

One hundred McIntosh, Malus domestica, tree saplings were selected for this trial. The treatment 

assignments were 4 replicates of 1 untreated control tree and 4 replicates of 4 trees for each of the 4 

rainfall levels for each fungicide treatment. The weather was closely monitored for ideal infection period: 

temperatures higher than 23.9°C, light wind, and a wetting period lasting longer than 3 hours post rainfall 

event. Immediately prior to the targeted rainfall event, the saplings were treated with the selected 

fungicides at rates equivalent to those described above on a concentration basis (1.5 g Captan 80WDG / 

500 ml of water) (1.8 g Koverall 75DF / 500 ml of water) (0.13 ml Miravis 1.67SC / 500 ml of water) 

(0.23ml Attach/500ml water), with each tree being sprayed with approximately 83.3 ml per tree. One drop 

of the adjuvant R-11 was added to break surface tension, prevent beading, and improve coverage of the 

leaves. Antifoaming agent was added to the Captan solutions as Captan is prone to foaming. Applications 

were made using a handheld 500 ml sprayer bottle. After 2 hours of drying, each tree was subjected to its 

indicated level of simulated rainfall using a hand-held variable nozzle garden hose using the mist setting 

and let to dry again. 

2023 Field Methods 

Trees were placed in the Jones block of the Michigan State University Plant Pathology Farm on 

22-year-old McIntosh trees (42°41’20.29” N, 84°29’17.93” W). The Jones block had previously been 

used for Apple Scab trials and had 4 untreated control trees that were expressing high levels of Apple 

Scab. These 4 trees were used as the host trees for the apple tree saplings. There are 4 replicates of each 

treatment and rainfall simulation, one replicate from each treatment was placed under each tree in a 

randomized block design. The trees were placed surrounding the trunk and canopy. Trees were placed 

13 

 
 
 
 
under the canopy of their assigned trees within the orchard approximately 36 hours before the rainfall 

event (Different picture than figure 2). 

After the rainfall event, the saplings were left in the orchard for the remainder of the infection 

period, until the saplings had dried. After the saplings had dried, they were removed from the orchard to 

prevent further apple scab infection and kept on a cement pad in partial sun. The trees were watered and 

kept alive until symptoms appeared. 

Apple Scab Evaluations: 2022 and 2023 

Each potted tree was evaluated for apple scab lesions. Every leaf was counted to determine the 

number of healthy leaves per tree. Each leaf was evaluated for apple scab lesions and a total number of 

lesions was determined for the tree which was used to determine a percent infection for each tree.  

In 2023, 5 random shoots were chosen to be evaluated. Each shoot was counted for the total 

number of leaves on the shoot and the number of leaves with infection. This was to eliminate spur shoots 

from the trunk, or older leaves that were less susceptible to infection or had grown post-infection event.  

Statistical Analysis Methods: 

Multiple statistical analysis was conducted to explore the differences in treatment as it relates to 

rainfastness across all rainfall treatments, comparing deposition differences between the standalone 

product vs the product plus a sticker, as well as the level of rainfastness differences between the 

standalone product vs the product with the sticker. All data were tested for normality and homogeneity 

assumptions using Shapiro-Wilks and Levene’s test. Transformations were done to data when necessary 

to meet normality assumptions, with ranked tests used on data that could not be normalized. In cases 

where an outlier was observed, the data set was windsorized. The extreme outlier was replaced with the 

mean of the remaining data points. All analyses were performed using R Statistical software (v4.1.2; 

RCore Team 2021). 

14 

 
 
 
 
 
 
Residue: 

To evaluate treatment differences in recovered residues across the different levels of rainfall 

within one treatment compound, a Dunnett test was performed on each set of data. The recovered residue 

levels at zero inches were compared to the recovered residue of the other three simulated rainfall levels. 

Significant differences were determined by a p-value of <0.05. 

To evaluate deposition levels of the treatment compounds, t-tests were performed comparing 

treatment differences between the same products with and without the sticker adjuvant. Significant 

differences in residue deposition were defined by a p-value <0.05. 

To evaluate the rainfastness of the treatment compounds, t-tests were performed on the 

proportional differences between the values of zero mm of rain and the other rainfall levels. Proportional 

values were compared between the original product and the product plus the sticker. Significant 

differences in rainfastness were defined by a p-value <0.05. 

In-field Infection: 

To evaluate the differences in apple scab protection within one treatment, a Dunnett test was 

performed on each of the treatment compounds. This compared the difference in percent of leaves 

infected between 0mm of rain and with 6.3, 12.7, 25.4mm. Significant differences were defined by a p-

value<0.05. 

To evaluate the effect of a sticker adjuvant in the protection against apple scab infection, t-tests 

were performed. At the 0mm rainfall level the percentage of infected leaves within one rainfall treatment 

were compared between the standalone product and the product with a sticker adjuvant. Significant 

differences in apple scab infection were defined by a p-value<0.05. 

15 

 
 
 
 
 
 
 
 
2021 Residue 

Results 

All rainfall levels were compared to 0 mm of rainfall to determine significant treatment 

differences (Figure 3). Captan fruit showed no significance in recovered residues between 0 - 6.35mm 

(P=0.0979), and 0 - 12.7mm (P=0.0990). Captan fruit showed a significant difference in residues between 

0 - 25.4mm (P=0.0148). Captan leaves showed no significance in residue levels between any of the 

rainfalls: 0 – 6.35mm (P=0.9977), 0 – 12.5mm (P=0.9999), and 0 – 25.4mm (P=0.8291).  

Mancozeb residues recovered from fruit showed no significant differences between 0 – 6.35mm 

(P=0.9251), and no significance between 0 – 25.4mm (P=0.0990). There was a significant difference in 

residues between 0 – 12.7mm (P=0.0492). Mancozeb residues recovered from leaves showed no 

significance between any of the three rainfall levels: 0 - 6.35mm (P=0.9413), 0 – 12.7 (P=0.9220), and 0 

– 25.4 (P=0.1600). 

Pydiflumetofen residues recovered from fruit showed no significant differences between any of 

the three rainfall levels: 0 – 6.35mm (P=0.3415), 0 – 12.7mm (P=0.2463), and 0 – 25.4mm (P=0.2255). 

Pydiflumetofen residue recovered from leaves showed no significant difference between 0-6.3mm 

(P=0.0778), however there was a significant difference in recobvered residue for 0-12.7mm (P=0.0114) 

and 25.4mm (P=0.0047). 

2022 Residue 

Captan when applied to fruit showed a pattern of gradual wash off as rainfall levels increased, 

with only one significant decrease in recovered residues 0- 25.4mm (p=0.0301). A similar pattern of 

gradual wash off was seen with the addition of a sticker, but with numerically higher residue recoveries. 

Residue recoveries between 0mm and the three rainfall levels were not significantly different. Captan 

when applied to leaves showed strong levels of residue retention with little wash off at every rainfall 

level. The addition of a sticker showed a similar pattern but with slightly higher levels of residue. There 

was no significant increase in deposition with the addition of a sticker. When comparing the true 

16 

 
 
 
 
 
rainfastness of Captan compared to Captan with a sticker, there was no significant difference at any 

rainfall level. 

Mancozeb when applied to fruit showed a pattern of steady residue retention following the lower 

two rainfall levels but showed a highly significant decrease in residues at 25.4mm rainfall (p=0.00076). 

The addition of a sticker to Mancozeb when applied to fruit showed a pattern of strong residue retention 

with no significant decrease in recovered residue. The addition of a sticker resulted in significantly greater 

deposition on fruit (t=-2.6022, df=5.5314, p=0.04372). The addition of a sticker also resulted in 

significantly greater rainfastness at the highest level of rainfall (t=6.0456, df=5.4555, p=0.001311). 

Mancozeb when applied to leaves showed strong residue retention with no significantly different 

recovered residue values. The addition of a sticker to Mancozeb when applied to leaves shows a pattern of 

gradual residual wash off with one significant decrease at 12.7mm of rainfall (p=0.044). There was no 

significant increase in deposition or rainfastness with the addition of a sticker on leaves. While not 

meeting the p=0.050 standard for significance, there was a statistical difference in deposition on leaves 

with a p-value=0.05687. 

Pydiflumetofen when applied to fruit showed no significant loss of residue with an increse in 

rainfall level. There was a conistent amount of residue recovered among all three tested rainfall levels. 

The addition of a sticker although appears to significantly improve deposition did not (p=0.0774). There 

was no significant decrease in recovered residue on the fruit after the addition of a sticker. There was no 

significant improvement in deposition or rainfastness with the addition of a sticker. Pydiflumetofen when 

applied to leaves showed a similar pattern to fruit with a consistent amount of residue recovered from all 

rainfall levels. The addition of a sticker significantly improved deposition on leaves (P=0.00589) and no 

significant improvement of rainfastness.  

2022 In-field infection 

All three rainfall levels were compared to 0mm to determine significant treatment differences 

(Figure 4). Captan leaves showed no significant treatment differences between 0 – 6.35mm (P=0.9929), 

and 0 – 12.7mm (p=0.1950). There was a significant difference between 0 – 25.4mm (P=0.0170). 

17 

 
 
 
 
Mancozeb leaves showed no significant difference between 0 – 6.35mm (P=0.9504), and 0 – 

12.7mm (P=0.5421). There was a significant difference between 0 – 25.4mm (P=0.0376). 

Pydiflumetofen leaves showed no significant difference between 0 – 6.35mm (P=0.5904). There 

was a significant difference between 0 – 12.7mm (P=0.0032), and 0 – 25.4mm (P=0.0042). 

2023 In-field infection: 

Captan with no sticker showed no increase of infection for the two lower rainfall levels, but a 

significantly higher level of infection at 25.4mm of rainfall (p-value=0.0109). The addition of a sticker 

resulted in significantly more infection at all rainfall levels, 6.3mm (t=-17.246, df=3.2092, p=0.00028), 

12.7mm (t=-8.2767, df=3.9601, p=0.0012), and 25.4mm (t=-6.184, df=4.2342, p=0.0029).  

Mancozeb with no sticker showed no increase of infection at the lowest rainfall levels, but with 

significantly higher levels of infection at 12.7mm (p=0.0052) and at 25.4mm (p=0.0007). The addition of 

a sticker to Mancozeb application resulted in significantly higher infection at 6.3mm (p=0.005) and 

25.4mm (p=<0.0001) but not at the 12.7 mm level when compared to 0mm rainfall on mancozeb with a 

sticker.  The addition of the sticker significantly improved protection against infection at 0mm (t=3.6064, 

df=5.8818, p=0.0117) and 12.7 mm (t=4.1532, df=4.4412, p=0.0114) rainfall level, suggesting that the 

increased deposition is responsible for the improved product performance. 

Pydiflumetofen with no sticker showed strong protection at 0mm and significantly higher levels 

of infection at the rest of the rainfall levels, 6.3mm (p=0.0152), 12.7mm (p=0.0176), and 25.4mm 

(p=0.0112). The addition of a sticker to Pydiflumetofen resulted in increased infections only at the 

12.7mm (p=0.0182) and 25.4mm (p=0.0019) rainfall levels when compared to pydiflumetofen plus a 

sticker at 0mm. The addition of a sticker to Pydiflumetofen did not significantly change the infection 

levels when compared to a no sticker application. While not meeting the p=0.050 standard for 

significance, there was a statistical difference in leaf infection between pydiflumetofen alone and with the 

addition of a sticker at 6.3mm of rainfall with a p-value=0.07225, suggesting modest benefit of the 

sticker’s improved rainfastness. 

18 

 
 
 
 
 
 
 
Discussion 

This study demonstrates how different amounts of rainfall affect fungicide residues remaining on 

apple leaves and fruit, and how wash-off affects protection from apple scab infection. Response to 

simulated rainfall varied between Captan, Mancozeb, and Pydiflumetofen. The observed deposition and 

rainfastness with the addition of a sticker adjuvant varied based on the active ingredient and rainfall level. 

Captan showed good rainfastness when applied to leaves in both years of the study. However, 

Captan residues on fruit showed sensitivity to wash-off at one or more rainfall levels. This was also 

shown in Xu et al. (2008) where they showed significant residue wash off after the initial rain with little 

coming off with following rainfall events. Xu et al. (2008) also showed that younger leaves retain residue 

better than mature leaves. That was slightly different than this study where the mature leaves had enough 

remaining residue to protect from infection until the highest rainfall level, 25.4mm (1 inch). Despite there 

being good rainfastness on leaves, the highest level of rainfall resulted in an increased level of infection 

on the leaves. While the numerical values of Captan residues were consistently higher when adding a 

sticker, these patterns were not supported statistically. This was evident in the infection protection where 

Captan alone and Captan plus a sticker remained roughly the same in protection from infection. The 

addition of a sticker resulted in higher levels of infection at all rainfall levels compared to the same 

rainfall levels with no sticker. This suggests that when applying captan, higher rainfall levels will wash 

off sufficient residues to put apple trees at risk of infection. The increased rate of infection of trees at 

0mm rainfall suggests that despite the randomization method, there was still higher pressure in the “with 

sticker” locations. 

Mancozeb also showed good rainfastness on leaves, while sensitivity to wash off was observed 

on fruit when rainfall levels were 12.7mm (0.5 inch) or 25.4mm (1 inch). The amount of residue 

recovered from the leaves gradually decreases as the rainfall increases but remains an insignificant 

difference. This is contradictory to a study done by (Hunsche et al., 2007), who saw a dramatic decrease 

in residue after any amount of rain, followed by not much additional residue loss. Despite there being 

good rainfastness on leaves, the higher levels of rainfall resulted in a level of infection on the leaves. This 

19 

 
 
 
 
is similar to what Rossouw et al (2018) observed while doing wash off studies with Mancozeb. As the 

amount of rain approaches 10mm (0.4 inch) (Rossouw) there was no significant change in remaining 

residue after the first amount of rain. This is similar to the 12.7mm (0.5 inch) results where the amount of 

residue remained insignificant, while the larger rainfall amount decreases significantly to the point of 

protection failure. In this study, there was evidence of improved deposition on leaves and fruit with a 

sticker was added to Mancozeb. This relatively significant increase in in deposition is likely responsible 

for the improved scab control at 0mm rainfall. In addition, the sticker improved rainfastness on fruit at the 

25.4mm rainfall level. The improved deposition and rainfastness translated into improved protection from 

infection. There was significantly less infection at 0mm and 12.7mm of rain with the addition of a sticker.  

This suggests that with mancozeb, lower levels of rainfall may leave “plant protective” levels of residue, 

but higher levels of rainfall leave trees susceptible to infection. 

Pydiflumetofen showed a much more interesting response pattern. Pydiflumetofen showed good 

residue retention on the leaves, while residue patterns on fruit were variable. Despite the residue patterns, 

plant protection from apple scab was lost following most rainfall levels, suggesting changes in 

bioavailability of the compound. When pesticide is absorbed into the cuticle of the leaf, this can reduce 

the biovailability of active product remaining on the leaf surface (Chowdhury 2001). In the case of Zeta-

cypermethrin on cherries, overall residues remained consistent following simulated rainfall, but lethality 

to spotted wing drosophila diminished significantly (Putra, 2019). When the sticker was added to 

pydiflumetofen, there was evidence of improved deposition on leaves and fruit. In this case the residue 

remains extractable from the leaf, but in a form that did not substantially improve the protection against 

apple scab, especially at higher rainfall levels. 

The addition of a sticker aims at improving deposition, rainfastness, and protection from 

infection. This study demonstrated occasions of improved deposition as well as rainfastness, but these 

residue outcomes do not necessarily result in improved protection from apple scab infection.  

Future research should address how the duration and intensity of the rainfall event changes 

residue sensitivity to wash-off. Other research suggests that more intense rain carries larger droplets at a 

20 

 
 
 
 
higher velocity, delivering more force to the leaves and removing more active ingredient more quickly 

(Hunsche et al. 2007).  Our ongoing research will address questions as to the impact of adjuvants on the 

rainfastness of fungicides.  These preliminary data will help apple growers in their disease control 

decision-making. 

Figure 1.1 Field treated apple shoots undergoing simulated rainfall. 

21 

 
 
 
 
 
 
 
 
Figure 1.2 Potted apple trees exposed to infection period in high inoculum orchard. 

22 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3 Recovered Residue values from leaves and fruit from each treatment. Dunnett tests conducted 

compared the significant difference of recovered residue between 0mm and the other three rainfall levels 

(6.3, 12.7, 25.4mm). Significant differences were determined by p-value<0.05, represented by *. 

23 

 
 
Figure 1.4 Percent of leaves with apple scab lesions. Dunnett tests were conducted within each treatment, 

a. Captan, b. Mancozeb, and c. Pydiflumetofen, comparing three different rainfall levels (6.3, 12.7, 

25.4mm) to 0 mm rainfall for all three fungicide treatments. Significant differences of p-value<0.05 are 

represented by * and p-values<0.01 are represented by **. 

24 

 
 
Figure 1.5 Recovered Residue of Captan (2023). Dunnett tests conducted within one treatment compared 

the significant difference of recovered residue between 0mm and the other three rainfall treatments, 

represented by *. T-test comparing significant differences of deposition of active ingredient between 

Captan and Captan with a sticker, based on the recovered residue of the 0mm, represented by †. T-test 

comparing significant differences of rainfastness between the Captan and Captan with a sticker based on 

recovered residue of each rainfall treatment represented by ◊. All significant differences were determined 

by p-value <0.05. 

25 

 
 
Figure 1.6 Recovered Residue of Mancozeb (2023). Dunnett tests conducted within one treatment 

compared the significant difference of recovered residue between 0mm and the other three rainfall 

treatments, represented by *. T-test comparing significant differences of deposition of active ingredient 

between Mancozeb and Mancozeb with a sticker, based on the recovered residue of the 0mm, represented 

by †. T-test comparing significant differences of rainfastness between the Mancozeb and Mancozeb with a 

sticker based on recovered residue of each rainfall treatment represented by ◊. Significant differences of 

p-value<0.05 are represented by *, p-values<0.01 are represented by ◊◊, and p-values<0.001 are 

represented by ***. 

26 

 
 
Figure 1.7 Recovered Residue of Pydiflumetofen (2023). Dunnett tests conducted within one treatment 

compared the significant difference of recovered residue between 0mm and the other three rainfall 

treatments, represented by *. T-test comparing significant differences of deposition of active ingredient 

between Pydiflumetofen and Pydiflumetofen with a sticker, based on the recovered residue of the 0mm, 

represented by †. T-test comparing significant differences of rainfastness between the Pydiflumetofen and 

Pydiflumetofen with a sticker based on recovered residue of each rainfall treatment represented by ◊. 

Significant differences of p-value<0.05 are represented by ◊ and p-values<0.01 are represented by ◊◊. 

27 

 
 
Figure 1.8 In-field infection differences between treatments (2023). Dunnett tests were conducted within 

one treatment compared the significant differences in infection protection between 0mm of rainfall and 

the other three rainfall treatments, represented by *. Treatment differences between regular product and 

the product with the addition of a sticker were compared for significant differences in infection 

protection, represented by †. Significant differences of p-value<0.05 are represented by *, p-values<0.01 

are represented by **, and p-values<0.001 are represented by ***. 

28 

 
 
 
 
CHAPTER 3: COMPARING FOLIAR APPLICATION AND TRUNK INJECTION 

APPLICATION OF A PEPTIDE BASED BIOPESTICIDE FOR PEAR PSYLLA CONTROL 

Abstract 

Laboratory evaluations of in field pear psylla counts were used to determine the effectiveness of 

trunk injections of a peptide based biopesticide as a method of psylla control. Applications of the 

biopesticide were made as a foliar spray and as an injection to compare treatment methods and the 

corresponding control of the pest. The peptide based biopesticide showed little improvement in psylla 

control both as a foliar application and applied via trunk injection.  

Introduction 

Pear Psylla, Cacopsylla pyricola Forster (Hemiptera: Psyllidae), is the number one arthropod pest 

affecting the US pear industry (Horton 1999). Psylla nymphs feed on the sap of trees through piercing 

stylets through the veins of the tree, leading to stunted trees, fewer fruit produced, and declining health of 

the trees and the orchard in highly infested orchards (Howitt, 1993). The historical solution to managing 

pests in orchards is to use broad-spectrum pesticides applied by airblast sprayer (Wise & Whalon, 2009). 

With more growers moving towards safer, and selective insecticides, including biopesticides, optimizing 

delivery and persistence is needed.  

Psylla have three life stages, egg, nymph, and adult. Eggs are laid on both sides of the leaf along 

the midrib. The egg looks like a grain of rice and is laid white but turns yellow as it develops. The 

nymphs pass through five stages of development. Nymphs begin smaller and yellow, and gradually get 

larger, wider, and darker in color as they age. The adult psylla presents two forms, summer, and winter 

form adults. The adults hold their wings above their body and have reddish-brown bodies, winter form 

appears almost black. Psylla adults overwinter as winter form adults under bark or in leaf litter. Psylla 

infestations can result in fruit russet, psylla shock, declining tree health (Pear Psylla Integrated Pest 

Management | WSU Tree Fruit | Washington State University, n.d.) 

29 

 
 
 
Airblast ground sprayer application may be fast and easy, but there are significant downsides 

including off target drift, product use efficiency and efficacy, as well as monetary losses (VanWoerkom et 

al. 2014). Pesticide application is determined necessary at a nymph threshold of 0.3 nymphs per leaf. The 

inefficiencies of airblast sprayer applications arise from the amount of wasted product through drift and 

off target application. There are significant differences in product distribution after airblast application 

with approximately half of the sprayed product reaching the leaves and even less contacting any insect 

pest. (Wu et al., 2020; Pimentel & Levitan, 1986) In fact, growers will over apply their product to take 

into account the loss of product (VanWoerkom et al. 2014). Application via trunk injections may be a 

better option than the traditional airblast sprayer. 

Trunk injection provides an even and efficacious distribution of the product throughout the 

canopy, when using an adequate number of injection ports (Aćimović et al. 2014). Studies have shown 

that trunk injections are a better method of pesticide delivery in apples to control foliar feeding pests 

including potato leafhopper, rosy apple aphid, spotted tentiform leaf miner, and oriental fruit moth 

(VanWoerkom et al., 2014). This method of pesticide application maximizes the exposure of the active 

ingredient to sucking insects as a result of the product being transported into the leaves after injection (J. 

C. Wise, 2016). This means less product is needed for adequate control because there is no wasted 

pesticide to drift, or off target application saving growers money.  

Biopesticides are pesticides made from living organisms or their products (Glare et al. 2016).  

The rise in use of biopesticides have led to the development of an assortment of products, including 

peptides such as Spear-T. Spear-T uses the GS-Omega/Kappa HXTX-HV1A peptide as the active 

ingredient that causes hyperexcitation of the nervous system of an insect, and remains nontoxic to 

humans, other mammals, birds, fish, and other beneficials (Vestaron, 2023). Spear-T has been shown to 

control pear psylla in pear when applied via airblast (Wise et al. 2020). Azadirachtin is a product extract 

of the Neem Tree, Azadirachta indica, and affect insects in multiple ways including inhibit food intake, 

morphogenesis, ovarian development, fecundity, egg viability, and molting (Karnavar, 1987). 

Azadirachtin is approved to be used on any food crop, but is not commonly applied because it is quick to 

30 

 
 
degrade in the environment (J. C. Wise, 2016, ArborJet 2016). This is not ideal for a cost-effective, long-

term spray plan because of the frequent reapplication required for adequate control. 

The objective of this study is to compare the efficacy of two compounds, GS-Omega/Kappa 

HXTX-HV1A and azadirachtin in the control of pear psylla when applied by two different methods, 

airblast sprayer and trunk injection. This study will evaluate the differences between the compounds and 

application methods by evaluating psylla oviposition rates and nymph populations at regular intervals 

post treatment. 

Trunk Injections: 2022 and 2023 

Methods and Materials 

Research was conducted on 38-year-old bartlett pear, Pyrus communis, trees at the Michigan 

State University Trevor Nichols Research Center (TNRC) (42.5951°N, -86.1561°W). Single tree plots 

were randomly assigned, using only healthy trees that had not been previously injected. To prepare trees 

for trunk injections, four holes were drilled into the tree at approximately 25.4cm above the ground on the 

north, east, south, and west sides of the tree. A 6.3mm tree plug (ArborJet, Inc, Woburn, MA) was 

inserted into the drilled holes using the hammer and plug pounder tool. The plugs were inserted until they 

were even with the layer of bark on the tree. 

The injected products were mixed in the Arbor Jet Tree IV canister. Each product was prepared 

with a final volume of 500ml of solution. Spear-T was mixed with 477.3ml and 454.6ml of distilled water 

while the azadirachtin was mixed into 500ml of water. The prepared canister was inserted into the ground 

near the trunk of the tree securing it into the ground with a long thin metal stake. A hand pump was 

attached to the pressurization valve and screwed onto the top of the canister. Four needles attached to long 

tubes were inserted fully into the tree plugs. The canister was pressurized and primed for injection. A full 

injection for one tree took approximately 1-30 minutes. Once all the liquid was out of the canister, the 

tubes connecting the canister to the needles inside the tree were examined for bubbles to indicate all the 

31 

 
 
 
 
solution had been expelled and absorbed by the tree. Once there were bubbles flowing through the tubes, 

the needles were removed from the tree. This process was repeated for all trees. 

GS-omega/kappa-Hxtx-Hv1a (Spear-T L, Vestaron Corporation, Durham, NC) was injected at the 

label rate 28.2L/ha (3gal/acre) converted to 45.4ml/tree from based on the density of trees in the orchard. 

Azadirachtin (Azasol WSP, ArborJet, Inc, Woburn, MA) was injected at the label rate 0.42kg/ha 

(6oz/acre) converted to 4g/tree. Applications were made at petal fall on 31-May 2022 and 24-May 2023. 

Trees were injected one day before foliar application of the same products were used. Spear-T and 

azadirachtin were applied at the recommended rate on the label. Foliar applications were applied with an 

FMC 1029 airblast sprayer at the rate of 934.9L/ha. Pear psylla specimens collected from the population 

used in these experiemts were previously voucherd at the A.J. Cook Arthropod research collection 

(voucher number 2020-07) by Celeste Wheeler.  

Psylla Evaluations: 2022 and 2023 

Forty leaves were collected from each one tree plot. Ten leaves from each side of the tree were 

picked, selecting from high and low branches, as well as interior and exterior leaves. The leaves were 

placed in labeled brown paper bags, then stored in a standard refrigerator at 4.4°C.  

Under a dissecting microscope, the front and back of the leaves were evaluated for psylla eggs, 

and nymphs. A tabletop multiple display clicker counter was used to record the total number of eggs and 

nymphs for all 40 leaves. 

Statistical Analysis 

Multiple statistical analyses were conducted to explore the differences in treatments across all 

evaluation days, as well as comparing each treatment to the other treatments within one evaluation day. 

To evaluate the effects of one treatment across multiple evaluation days, repeated measures analyses were 

conducted for each treatment for both psylla eggs and nymphs. To evaluate treatment differences on each 

day of evaluation, t-tests were conducted comparing each treatment to the other treatments within one 

32 

 
 
 
 
 
 
 
evaluation day for both psylla eggs and nymphs. All data were tested for normality and homogeneity 

assumptions using Shapiro-wilks and Levene’s tests. Transformations were done to data when necessary 

to meet normality assumptions, with ranked tests used on data that could not be normalized. All analyses 

were performed using R Statistical software (v4.1.2; RCore Team 2021). Significant differences were 

determined by a p-value<0.05. 

2022 

Results 

Overall treatment effects were not significant on eggs (f=0.397, df=4, p=0.808) or nymphs 

(f=1.554, df=4, p=0.237). After slicing treatment effects by day of application there were no significant 

differences in treatment effects. 

2023 

Overall treatment effects were not significant in GS-omega/kappa-Hxtx-Hv1a application on eggs 

(f=0.599, df=2, p=0.57) or nymphs (f=1.031, df=2, p=0.395). Application of azadirachtin did not show 

any significant overall treatment effects in eggs (f=1.162, df=2, p=0.356) or nymphs (f=1.646, df=2, 

p=0.246). After slicing treatment effects by day of application comparing compounds results to untreated 

results, there were two instances of improved protection from psylla infestation and one instance of an 

injection performing better than a sprayed application. On day 56 injected azadirachtin significantly 

protected from oviposition compared to untreated trees (t=-7.472, df=3, p=0.03). On day 56 injected spear 

also significantly protected against nymph populations compared to untreated trees (t=-6.248, df=3, 

p=0.05). on day 56 again, injected azadirachtin showed significantly lower nymph populations compared 

to trees sprayed with azadirachtin (t=-6.536, df=3, p=0.044). 

33 

 
 
 
 
 
 
 
 
Discussion 

This study provides new knowledge on the efficacy of biopesticides for control of pear psylla, 

and the influence of delivery system on their performance in pear trees. Psylla population and oviposition 

varied between products and application methods used.  

Azadirachtin was able to improve protection from oviposition from psylla with limited efficacy. 

This protection came only from the injected method of application and on day 56 after injection. Injected 

azadirachtin was also more effective at controlling nymph populations than the foliar sprayed application 

of azadirachtin, suggesting that injection is a superior method of pesticide application. Previous studies 

have shown that a single injection of azadirachtin can provide a whole season of protection (Wheeler et 

al., 2020). Previous studies have also shown that foliar applications of azadirachtin alone, was enough to 

provide significant reduction in egg populations (Nottingham & Sater, 2021). This study may have 

different results than Nottingham because of the date of the study and the environmental conditions of the 

area. Nottingham conducted the study in April and May in Washington with daily temperatures ranging 

from 13-17 degrees Celsius on average while this study was done in June and July in Michigan with daily 

temperatures ranging from 23-27 degrees Celsius. This temperature, climate, and seasonal difference may 

be a cause for behavior or population differences between studies and results. Psylla egg populations from 

wheeler were similar to this study with maximum egg means approximately 400-700 eggs however, 

nymph populations varied. Maximum nymph counts from wheeler were approximately 100 in both 2017 

and 2018 while this study counted a similar number in 2022 but a maximum number of nymphs at 200 in 

2023. 

GS-omega/kappa-Hxtx-Hv1a in Spear-T, was able to provide limited suppression of psylla 

nymph populations when injected. The GS-omega/kappa-Hxtx-Hv1a was unable to show better control 

for the remainder of the trial. Limited efficacy of GS-omega/kappa-Hxtx-Hv1a in pear may have been 

caused by multiple factors. Phytotoxicity was observed in many trees damaging their leaves which may 

be evidence that the peptides were not delivered into the leaf tissues.  

34 

 
 
 
Biopesticides are an emerging contender for safer, more selective chemistries of pesticides to use 

in modern IPM systems. However, as the data show, there may be limited efficacy of control from 

biopesticides in certain cases. These data show varied results compared to others. GS-omega/kappa-Hxtx-

Hv1a (Spear-T) was tested against brown marmorated stink bugs and showed  significantly control but 

not better control than other products (Sutton et al., 2021). GS-omega/kappa-Hxtx-Hv1a (VST-

006340LC) was tested for efficacy against house flies in a greenhouse setting, and was not able to show 

effective, significant control of the fly (Hubbard & Gerry, 2019). More work needs to be done to examine 

the efficacy of GS-omega/kappa-Hxtx-Hv1a in pear controlling psylla as this is one of the first examples 

of injecting GS-omega/kappa-Hxtx-Hv1a to control this pest. Analysis of GS-omega/kappa-Hxtx-Hv1a 

residue and distribution within the canopy should be done to confirm proper delivery and distribution to 

where it remains bioactive and effective. The product Spear-T may not have been suited for injection as it 

was formulated for foliar application. Some of the inactive ingredients may not result in effective 

injection. 

35 

 
 
Figure 3.1 Mean number of psylla eggs(A) and nymphs (B) per 40 leaves for untreated and spear 

treatments. (2022). 

36 

 
 
Figure 3.2 mean number of psylla eggs(A) and nymphs(B) per 40 leaves for untreated and Azasol 

treatments. (2022). 

37 

 
 
Figure 3.3 Mean number of psylla eggs(A) and nymphs (B) per 40 leaves for untreated and spear 

treatments. All significant differences were determined by p-value<0.05. (2023). 

38 

 
 
Figure 3.4 mean number of psylla eggs(A) and nymphs(B) per 40 leaves for untreated and Azasol 

treatments. All significant differences were determined by p-value<0.05. (2023). 

39 

 
 
 
 
Rainfastness of Fungicides for disease Control in Apples 

CHAPTER 4: CONCLUSION 

This study demonstrated how different amounts of rainfall affect fungicide residue remaining on 

apple leaves and fruit, and how the wash-off affects protection from apple scab infection. Moderate 

rainfastness properties were observed for captan and mancozeb while less was observed for 

pydiflumetofen, especially on fruit tissues. There has also been little research in the effects of sticker 

adjuvants on the ability of fungicides to protect against apple scab infection. 

Captan had been observed to be sensitive to wash off as it can lose up to half of its residue after 

only 1mm of rainfall (Xu et al., 2008). This is similar to what this study was able to show on fruit residue, 

while leaf residue showed good rainfastness and good protection from infection. This pattern of residue 

retention generally translated to protection from scab lesions as well. 

Mancozeb has also been shown to be sensitive to wash off as it loses 91% of its residue after 

10mm of rainfall (Hunsche et al., 2007). In contrast, this study showed that there was no significant 

decrease in recovered residues on leaves up to 25.4mm, but a significant loss of residue on fruit following 

25.4mm of simulated rainfall. The addition of a sticker was able to improve rainfastness and deposition in 

fruit residues but not in the leaves. Mancozeb was able to provide adequate protection from infection until 

12.7mm, but the addition of a sticker significantly reduced rates of infection at the 0mm rainfall level. 

This study was able to provide new information on the rainfastness and protective effects of 

pydiflumetofen to control apple scab. This study showed there was a large but statistically insignificant 

decrease in recovered residues on both leaves and fruit at all rainfall levels, but a significant increase in 

rainfastness with the addition of a sticker adjuvant. This interaction between rainfastness and the addition 

of a sticker adjuvant resulted in modest improvement of protection from apple scab, where 

pydiflumetofen lost protection after 6.3mm of rainfall without a sticker and 12.7mm with a sticker. 

Additional research could be done to determine protective levels of residue to control against 

apple scab or using different levels of rainfall to get a more precise model of rainfall and the 

40 

 
 
 
 
 
 
corresponding level of protection. Additional research could be done with the same procedure but with 

different fungicides or stickers to identify rainfast properties of other products. 

Comparing Foliar Application and Trunk Injection of a Peptide Based Biopesticide for Pear Psylla 

Control 

Biopesticides are an emerging contender for safer, more selective chemistries of pesticides to use 

in modern IPM systems. However, there may be limited efficacy in control of psylla, and other common 

insect pests. This study showed the peptide product GS-omega/kappa-Hxtx-Hv1a (Spear-T) had limited 

control of psylla in the field.  

GS-omega/kappa-Hxtx-Hv1a (Spear-Lep) was unable to significantly control Corn Earworm 

(CEW) on hemp in Virginia when compared to untreated hemp plants (Britt et al., 2021). GS-

omega/kappa-Hxtx-Hv1a (Spear-T) was not significantly different from untreated trials in attempts to 

control Western Flower Thrips (WFT) in tomato plants (Bilbo et al., 2020). The same peptide GS-

omega/kappa-Hxtx-Hv1a (VST-006340LC) was unable to show adequate control of house flies in a 

greenhouse setting (Hubbard & Gerry, 2019). 

Despite the peptide being unable to show adequate measures of control as a standalone 

application, the addition of GS-omega/kappa-Hxtx-Hv1a may improve control measures when added to 

other products. To control cabbage worm on collard greens, GS-omega/kappa-Hxtx-Hv1a (Spear-Lep) 

alone was unable to significantly improve control, but when mixed with other products showed moderate 

abilities to control infestation (Dunn et al., 2023). The addition of GS-omega/kappa-Hxtx-Hv1a (Spear-

Lep) was able to slightly improve control of Spotted Fireworm in cranberries (Rodriguez-Saona et al., 

2022). These studies are contradicted however by one study that showed significant improvement in 

brown marmorated stink bug control (Sutton et al., 2021). 

While Sutton et al. was able to show improvement there seems to be a greater number of studies 

unable to show significant improvements in control using GS-omega/kappa-Hxtx-Hv1a as an active 

ingredient in pesticide control. There may be evidence of GS-omega/kappa-Hxtx-Hv1a becoming an 

addition to many spray programs in aiding pest control, but not being the sole method of pest control. 

41 

 
 
 
Additional research could be done to investigate alternative formulations or concentrations of the 

biopesticide to be injected to achieve adequate control of psylla. Residue analysis of the leaves could be 

used to determine the effectiveness of the distribution of the pesticide within the canopy. Bioassays could 

be used to analyze the mortality rate of the product on the psylla nymphs.  

42 

 
 
 
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