EVALUATING THE EFFECTS OF SOLID VERMICOMPOST APPLICATIONS

ON PLANT GROWTH AND PEST DENSITIES OF NAVEL ORANGE TREES

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In

Plant Science

By

Deborah L. Nardo

2020 SIGNATURE PAGE

THESIS: EVALUATING THE EFFECTS OF SOLID VERMICOMPOST APPLICATIONS ON PLANT GROWTH AND PEST DENSITIES OF NAVEL ORANGE TREES

AUTHOR: Deborah L. Nardo

DATE SUBMITTED: Fall 2020

Department of Plant Science

Dr. Anna L. Soper ______Thesis Committee Chair Plant Sciences

Dr. Jon C. Phillips ______Department Chair Agribusiness & Food Industry Management /Agricultural Science

Dr. Srdjan Lemez ______Assistant Professor Kinesiology

Ben Lehan, M.S. ______Lecturer Plant Sciences

ii ACKNOWLEDGEMENTS

This thesis is possible due to the guidance and support of my colleagues, professors, and mentors at Cal Poly Pomona. Dr. Anna Soper has been an invaluable mentor and advisor since I began my undergraduate research at Cal Poly Pomona. I greatly appreciate her unwavering support. Dr. Jon Phillips afforded me the opportunity to teach in his department under his guidance and to assist him in ancillary projects. His support has been tremendous. Dr. Srdjan Lemez has propelled me to continually push myself in academic endeavors. I would not have set out to master SPSS and JASP if it hadn't been for his perspective on statistics. In addition, he inspires me to continually improve my writing skills. Professor Ben Lehan has been an enormous influence on my research. I am grateful for the support he has given me since my project began. Dr.

Valerie Mellano, Chair of the Plant Sciences Department, continues to be an inspiration to me. Renee Murphy, colleague and fellow M.S. candidate, has assisted me in numerous aspects of my research project. I cannot begin to thank her enough.

Muriel Walker-Waugh, Chemistry Professor at Santa Monica College, has been my ultimate mentor since my Freshman year. I aspire to her level of greatness on a daily basis.

This project was funded by a grant from the Agricultural Research Institute, Grant

Number 15-04-219.

iii ABSTRACT

The citrus industry has been devastated by pests and diseases in the past two decades that creates challenges in successful production. Unique control methods are needed to combat these threats in order to save commercial trees from catastrophic losses and reduce dependency upon pesticides. Vermicompost, a soil medium created from decomposition of organic matter by worms, has been demonstrated to improve plant yield, growth, and quality in a wide range of species. Furthermore, it has shown promise in decreasing pest densities and increasing resistance to pest and disease attacks. To evaluate the effects of solid vermicompost on citrus tree growth and pest densities, 40 field-planted two-year-old citrus trees were subjected to the following treatments: a layer of vermicompost below and surrounding the roots, a layer of vermicompost above the soil around the root zone, a layer both above and below the soil, and a control group with no treatment given. Trunk diameter, fruit yield, weight, and quality, and Asian Citrus

Psyllid, Citrus leafminer, and citrus thrip density levels were measured. Data were analyzed with SPSS statistical software (Version 26) in a two-fold procedure of descriptive i.e. frequencies and variance) and inferential (i.e. ANOVA, MANOVA, and

Repeated Measures ANOVA) statistics with post-hoc Tukey analysis for growth and quality data, and RMANOVA with post-hoc Fisher's LSD for pest density (P<0.05).

Results showed that trees treated with solid vermicompost layered around the root zone at planting had greater trunk diameter growth. Results for pest pressure, fruit production, and fruit quality were statistically insignificant. Further studies should focus on pairing solid vermicompost at the root zone with foliar aqueous vermicompost applications, as well as monitoring these trees in subsequent growing seasons.

iv TABLE OF CONTENTS

SIGNATURE PAGE ...... ii

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF FIGURES ...... viii

LITERATURE REVIEW ...... 1

California Citrus Industry ...... 1

Citrus Growth Characteristics ...... 2

Citrus Pests and Biotic Pressures ...... 3

Current Methods of Pest Control ...... 6

Vermicompost ...... 7

Conventional Composting vs Vermicomposting ...... 8

Vermicompost and Pest Damage ...... 10

Vermicompost and Plant Growth Improvements ...... 11

Vermicompost and Citrus ...... 12

STUDY PURPOSE AND OBJECTIVES ...... 13

METHODOLOGY ...... 15

Planting of Trees ...... 15

Soil Profile ...... 16

Vermicomposting Material ...... 17

v Irrigation ...... 17

Plant Growth Assessments ...... 17

Fruit Assessments ...... 18

Pests...... 18

Asian Citrus Psyllid ...... 18

Citrus Thrips ...... 19

Citrus Leafminer ...... 19

Statistics and Data Analysis ...... 20

RESULTS ...... 21

Plant Growth Results ...... 21

Fruit Production Results ...... 21

Pest Density Results ...... 22

Asian Citrus Psyllid ...... 22

Thrips ...... 22

Leafminer ...... 22

DISCUSSION ...... 24

Limitations and Future Directions ...... 26

CONCLUSION ...... 28

Key Findings ...... 28

Practical Implications and Proposed Impact ...... 28

vi SOURCES ...... 32

FIGURES ...... 38

APPENDIX...... 42

vii LIST OF FIGURES

Figure 1: Trunk Diameter Growth (mm)...... 38

Figure 2: Mean Fruit Pieces per Tree...... 38

Figure 3: Mean Fruit Weight (oz)...... 39

Figure 4: Mean Sugar Content (Brix)...... 39

Figure 5: Mean ACP Densities...... 40

Figure 6: Mean Citrus Thrips Densities...... 40

Figure 6: Mean Citrus Leafminer Densities...... 41

viii LITERATURE REVIEW

California Citrus Industry

Citrus was first introduced into California in the late 1700s by Catholic priests onto Mission property gardens up and down the state. The first official orchard in

California was planted at the San Gabriel Mission in 1804 (Tufts, Allen, & Brooks,

1946). Quickly gaining popularity as a home garden tree, the first commercial orchard was planted in 1841 near downtown Los Angeles. By 1900, the California citrus industry was booming and there were over 4.5 million trees in commercial production (Brown,

1983).

Continuing its expansion through the 1940s, the state experienced its first massive decrease to the citrus industry shortly after World War II (Johnston & McCalla, 2004).

Citrus tristeza, a viral disease, and cottony cushion scale, paired with the urbanization of

Southern California, decreased California citrus acreage from 330,000 to 230,000 acres statewide (Johnston & McCalla, 2004). Over three million orange trees grafted on sour orange rootstock were lost from citrus tristeza over the course of a decade. This led to a massive eradication effort in the 1960s (Bar-Joseph, Roistacher, Garnsey, & Gumpf,

1981). With the loss of available land in Southern California, the industry transitioned to more affordable land in the San Joaquin Valley, where the majority of commercial citrus in the state is still grown today.

Current commercial citrus production in the state continues to center in Fresno,

Kern, and Tulare counties. There are about 270,000 acres of bearing commercial orchards that are mainly in production of fresh fruit orchards (Geisseler & Horwath, 2014).

Because California has 16 distinct climate zones, fresh citrus can be harvested year-

1 round. The value of citrus at the point of shipment for the 2018 season reached a little over $3.389 billion for the state, making citrus one of the top five agricultural commodities in California (Babcock, 2018).

Citrus Growth Characteristics

Navel orange trees take about 10-15 years to reach full maturity, and semi-dwarf varietals like the Thomson Improved will generally grow up to 10 to 15 feet tall

(Mukhopadhyay, 2004). As citrus trees grow, they produce flush. Flush is the new growth on the tree that appears in the area between the bud break area and the shoot expansion. Citrus trees have two significant flushes per year with one taking place in the late Spring, and the other occurring in early Fall in Southern California (Reuther, 1973).

The first three years of fruit production on navel trees are considered the juvenile stage. The majority of orange blossoms that grow on trees during this stage do not fully develop into fruit. These blossoms will bloom and then most will drop once the bloom has completed. During this time, the bulk of the fruit that begins to develop will also drop from the tree long before reaching maturity (Mukhopadhyay, 2004). A growing tree will only cultivate a small load to maturity as a measure of self-preservation. These oranges in the juvenile stage will take anywhere from seven to 12 months to ripen and mature

(Reuther, 1973).

The Thomson Improved navel orange varietal is a bud sport selection that was taken from the Washington navel in 1890 in Duarte, CA. It was considered to be a beneficial varietal to plant because the fruit reaches maturity and is ready to be harvested two weeks before the Washington navel matures (Hodgson, 1967). In addition, the fruit

2 has an attractive appearance as compared to other navels, with a smoother, thinner rind, and an elongated shape as compared to the Washington navel.

Trees of the Thomson Improved varietal are semi-dwarfed and are more compact in size than other navel orange tree types. They are less heat and cold resistant so this varietal performs best in temperate climate zones such as Los Angeles County (Hodgson,

1967).

Citrus Pests and Biotic Pressures

The citrus industry has sustained sizable economic losses due to biotic pressures, especially in the last few decades. There are a number of diseases and insect pests that are threatening commercial citrus tree production and the future of the California citrus fruit industry as a whole.

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a critical agricultural insect pest that vectors the bacteria Candidatus liberibacter liberibacter, the causal agent of

Huanglongbing (HLB), also known as citrus greening disease. The HLB disease was first thought to be a viral infection, but is now known to be a bacterial infection that causes significant damage and then death to citrus trees over a period of about five years (Park &

Louzada 2018). Citrus greening disease conversely influences the ability of the phloem in the tree to transport sugar and nutrients to areas of the tree, causing the tell-tale fruit damage and bitterness. Attempts to inject antibiotics into HLB infected trees have provided only short remission of HLB symptoms (Kumar & Kiran, 2018). Leaves of

HLB-infected trees begin to turn yellow and mottled before dropping prematurely. Fruit begins to grow irregularly, with a forming a hard, green peel at the bottom. Fruit becomes bitter and inedible, and eventually trees will die after a few years of infection.

3 Once the trees are infected, there is no known cure, making HLB one of the most damaging diseases in agriculture today (Monzo & Stansley, 2017).

The insect has the capability to spread citrus greening disease to each tree it feeds off of. ACP feeds on the sap of citrus trees. The insect cannot cause tree death without the HLB bacterial infection being present. Because the Asian Citrus Psyllid mainly feeds on new growth, this causes younger trees to be the most vulnerable, due to the majority of tree mass consisting of tender, fresh shoots (Munir & He, 2018). This is substantially devastating to the trees, by impacting both the systemic and structural components.

Citrus thrips (Scirtothrips citri) are one of the major pests of the California citrus industry, causing huge losses to commercial groves annually due to their damage. The first instar larvae of the insect feed off of fruit and the trees’ tender leaves (Ladaniya,

2008). They concentrate the majority of their damage to the area under the sepals on young citrus fruit. Second instar larvae cause the most damage to the crop, feeding off of the fruit buds and causing scarring and scabs by puncturing the citrus rind cells

(Broughton, 2018). The result of this is a white or silver trail that becomes larger as the fruit grows, forming into damaged tissue rings on mature fruit. Although this damage is cosmetic in nature and does not harm the texture or flavor of the fruit, the California citrus industry is a fresh fruit industry (Ferguson & Grafton-Cardwell, & University of

California, 2014). Damaged exteriors render fruit unpalatable in the eyes of consumers.

The greatest damage caused by citrus thrips in California occurs to San Joaquin

Valley navel oranges and satsuma mandarins. These two citrus types not only receive ring scars from the damage under the sepal of the fruit, but also incur a second type of scarring down at the stylar end of the fruit (Tanigoshi, 1991). Stylar, or bottom-end,

4 scarring is smoother than the damage at the calyx end. When these two forms of damage occur together, it can become severe enough to restrict fruit growth asymmetrically and cause a deformity to the shape of the fruit. Citrus fruit are most vulnerable to thrip damage from the time of petal fall through the time when the fruit reaches about one-and- a-half inches in diameter, and fruit located on the outside canopy receives the most damage (Morse & Brawner, 1986).

A third insect that causes damage to the California citrus industry is the Citrus leafminer, Phyllocnistis citrella, which was first detected in the state in 2000 in Imperial

County, and has spread up through the San Joaquin Valley over the past 20 years. The larvae of this insect feed off of citrus tree growth by creating serpentine mines into leaves from the new flush that is tender (Arshad, Ullah, & Afzal, 2018). As the larvae feed their way through the leaves, they leave behind a dark trail of frass just below the leaf surface.

Damage from the leaf mining results in the flush curling and twisting instead of expanding normally. At the end of the larval stage, the insect will emerge from the leaf mine at the leaf’s edge and roll the edge of the leaf to create a barrier around itself to safely pupate (Hoy, Nguyen, Hall, Pomerinke, Pena, Browning, & Stansly, 1995).

Mature trees are generally safe from extensive leafminer damage because they contain dense canopies of foliage that is much older than flush, so the damage to the flush growth will generally be negligible (Arshad, Ullah, & Afzal, 2018). However, younger trees lack mature foliage and their growth is entirely comprised of young flush growth.

These trees under four years old will suffer much higher damage which can reduce growth needed to reach maturity (Pena, Hunsberger, & Schaffer 2000). Although young trees that are infested with heavy populations of leafminers are not likely to die from the

5 damage, a commercial citrus grower can face huge economic losses from the reduction in growth of these trees.

Current Methods of Pest Control

The standard method of control for citrus pests in nurseries revolves around chemical spraying. For the Asian Citrus Psyllid, the neonicotinoid imidacloprid is a systemic applied at the root zone, while a pyrethroid cyfluthrin is sprayed foliar to combat the insect (CDFA, 2018). These treatments must be reapplied twice yearly to maintain control. Citrus leafminer is controlled with the ryanoid insecticide cyantraniliprole. Citrus thrips build resistance to pesticides quickly, so pesticides such as carbamates, organophosphates, and pyrethroids must be rotated.

The issue of pesticide resistance is a major issue in citrus pest control. A study on citrus pesticides in Florida revealed that a resistance ratio of 35 was found with imidacloprid and a ratio of 6.9 to 17.9 was found with chlorpyriphos for Asian Citrus

Psyllid (Tiwari, Mann, Rogers, & Stelinski, 2009). This means that resistance can become a serious issue in citrus pest control if pesticide resistance management methods are not utilized. Currently in California, these resistance management methods are not in place.

In addition, the public perception of pesticides has continued to be viewed in a negative light. In a public research assessment of modern technology risks, respondents to the survey evaluated the words "pesticides" and "weed killers" as the most dangerous and detrimental food chemicals (Peterson, 2000). Public health concerns contributed to the outlook on pesticides in agriculture crops.

6 These issues of pest resistance and negative public perceptions of pesticides result in a need for other methods of control when combating citrus pests. Alternative methods that are not chemical in nature can reduce pest resistance, and some of these methods that are biological or organic in control also have some other benefits to plant and soil health.

One such method of resistance is vermicompost.

Vermicompost

Vermicompost is a product created from the breaking down of organic material by specific species of beneficial microorganisms and worms. Earthworms have shown to play a critical role in conditioning and processing the organic content in soil and breaking it down into smaller, immediately usable components (Edwards, 2004). This in turn increases the surface area of the matter so that plants and microorganisms can process it more efficiently at a faster rate than other forms of nutrients allow.

The action of the organic material moving through the digestive system of red wiggler earthworm, Eisenia fetida, alters the chemical, biological, and physical characteristics of the organic matter that is processed (Edwards, 2004). The carbon and nitrogen ratio of the matter is lowered. Nutrients such as nitrates, phosphorus, magnesium, calcium, and potassium are compacted and made immediately available for plant uptake. Beneficial populations of microbes that are naturally grown inside the earthworm digestive tract are added to the material and released as earthworm castings, while heavy metals from the material, such as lead, zinc, cadmium, copper, and manganese are removed and stored in the earthworm’s gut (Hussain, Abbasi, & Abbasi,

2016). Moreover, pathogens that are harmful to host crops are destroyed in the process

(Jack & Thies, 2006).

7 The earthworms act as a blender and filter for the material, while adding oxygen for the beneficial microorganisms in the mixture (Lim, Wu, Lim, & Shak, 2015). The result of this is a nutrient dense, porous, stable material with a high capacity for water holding. It provides numerous benefits for plant growth, soil health, and beneficial populations of microbes. Red wiggler worms are capable of eating 50-100% of their weight in a day (Hussain, Abbasi, & Abbasi, 2016). A flow-through structure, 2.5 meters by 1.5 meters, can process up to 100 pounds of organic material each day, producing an average of 75 pounds of casting in return.

Conventional Composting vs Vermicomposting

Vermicompost is one of two major types of compost media. The other is conventional compost. Conventional composts use thermophilic methods by which a favorable environment is introduced to microorganisms in which to process and deconstruct the organic matter efficiently and quickly, altering the material into a pathogen-free, stable soil material easily usable immediately by plants (Fracchia,

Dohrmann, Martinotti, & Tebbe, 2006). This process, however, relies on high rates of temperature to kill harmful organisms within the matter. In this process, it was shown in one study that beneficial bacteria species were also destroyed, which narrowed the species range in the material (Frederickson, Butt, Morris, & Daniel, 1997). Bacteria species that are gram positive such as Actinobacteria and Bacillus were greatly affected.

Alternatively, vermicompost temperatures did not naturally increase in the process of creation and alteration. The thermophilic phases of standard compost practices were not reached. Vermicompost was constantly in a mesophilic state that allowed for a greater diversity to be maintained of microorganisms that were not killed by a high

8 temperature (Tognetti, Laos, Mazzarino, & Hernandez, 2005). Despite lacking an increase in temperature, the vermicompost also increased the quality of nutrients above regular thermophilic composting (Frederickson, Butt, Morris, & Daniel, 1997). This allows for stabilization and elimination of harmful pathogens in a wide range of substrates of organic matter (Lazcano, Gomez-Brandon, & Dominguez, 2008).

In addition, when microbial populations of thermophilic compost were compared to the microbial populations in vermicompost, it was discovered in a study that these populations were vastly different. Genetic analysis performed on the thermophilic compost were primarily found to be from the Firmicutes and Actinobateria phyla

(Fracchia, Dohrmann, Martinotti, & Tebbe, 2006). The analysis on the vermicompost yielded bacteria from uncultured Bacteriodetes, Chloroflexi, Gemmatimonadetes, and

Acidobacteria phyla. The vermicompost contained double the number of distinct phyla which gave it an advantage over the regular thermophilic compost. Variation in feedstock from this study also resulted in variations of species of bacteria in the final composting materials.

Expanding on the concept of variation in feedstock changing the composition of the compost, a research study analyzed the difference in composition of bacteria in thermophilic composts and vermicomposts fed various livestock manures (Aira, Olcina,

Perez-Losada, & Dominguez, 2016). Cattle, horse, and pig manures were used to create both thermophilic and vermicompost amendments. These composts and vermicompost amendments varied widely depending upon the species the manure was derived from, and there were many differences in species flourishing between the composts from worm castings and thermophilic processes from the same animal manure. The result of this

9 research experiment showed that vermicomposts created from the same species of worms can vary depending upon the organic matter it is derived from and it is necessary to keep that in mind when choosing a proper amendment for a crop or species.

Vermicompost and Pest Damage

The effect of adding various amounts of vermicompost into a potting medium that was soilless, was examined for pest population levels and damage from insects such as aphids, cabbage white caterpillars, and peppers in tomato and pepper crops (Arancon,

Galvis, & Edwards, 2005). In soilless potting media, it was discovered that the vermicompost as a substitution for soil correlated with a significant reduction in populations mealybugs and aphids on the crops. The study found that not only were the pest populations reduced, but significant reduction in plant and fruit damage was observed as well. A level of 40% vermicompost yielded results far superior to 20%, showing that using a higher amount of the material did translate to greater pest reduction.

A similar study evaluated vermicompost treatments on tomato and cucumber production (Edwards, Arancon, Vasko, Bennett, Askar, Keeney, & Little, 2010). These treatments greatly reduced the populations of insect pests such as citrus mealybug, green peach aphid, leucaena psyllid, and spotted spider mites on the plants. One of the most relevant portions of this research was the finding that vermicompost applied over time on the plants has a beneficial aspect for the suppression of insect pest Heteropsylla cubana, the leucaena psyllid. This insect is in the same family as the Asian Citrus Psyllid, so this research suggests that there could be benefits in protecting citrus nursery stock and established citrus orchards from the insect that vectors Huanglongbing disease.

10 Pest damage is shown to reduce the efficiency of growth of plants by inhibiting processes that are basic. Research found that citrus trees greatly affected by the citrus leafminer showed a substantial decrease in photosynthetic rates, necessary for plant growth, flowering and fruit production (Schaffer, Pena, Colls, & Hunsberger, 1997).

These species of leafminers are increasingly becoming immune to chemical pesticide treatments, as are other insects and plant diseases. The microecology of defenses in a plant is still not completely known at this point although it is believed that repeated use of heavy pesticide treatments can disrupt the network of organisms that work in conjunction with a plant to fight off predator pests (Pimentel & Edwards, 1982).

Vermicompost and Plant Growth Improvements

A research study on vermicompost evaluated the effects of solid worm casting treatments on petunia plants grown in greenhouses (Arancon, Edwards, Babenko,

Cannon, Galvis, & Metzger, 2008). The result of these vermicompost treatments meant a significant improvement in overall growth and in flowering for these indoor plants. The research study also noted there was an increased efficiency in utilization of fertilizer components and nutrients when mixed with solid vermicompost. By replacing an average synthetic fertilizer in the control group with 50% vermicompost, yields on the flowering plants were higher in overall plant size without compromising soil quality. This is a potential positive implication for citrus cultivation applications.

The increased fertilizer efficiency due to vermicompost as noted in the above study correlates with the importance of speedy growth and pest and disease resilience in nursery stock species. When considering young citrus trees, rapid growth equals shorter growing periods, quicker delivery to customers, and earlier planting into citrus groves for

11 commercial production. Smaller trees and trees with slower growth rates are the trees that are most susceptible to the effects of weather and other environmental factors that can cause abiotic damage that can in turn cause trees to be unable to fight off biotic pests and diseases (Ferguson, Grafton-Cardwell, & University of California, 2014).

Vermicompost and Citrus

There is currently limited research on the effects of vermicompost on citrus tree production. One study dealing with citrus and vermicompost tested the result of adding vermicompost to Azotobacter in the soil media in order to promote lime tree growth from seed germination (Yadav, Jain, & Jakar, 2014). The treatment group with vermicompost added yielded the best results of the various soil amendment groups. The seedlings grew higher, had a greater number of leaves, a larger stem diameter and a greater fresh and dried weight. In addition, the seedlings grown with vermicompost had significantly longer tap roots, larger tap root diameters, larger number of secondary roots, and higher nitrogen and chlorophyll content in the leaves.

The positive effects of vermicompost on citrus seedlings reinforces the potential of these treatments on young navel trees that have been grafted onto rootstock. Reducing pest damage and improving plant growth across many species and varietals, including closely related lime, suggest that further research into solid vermicompost treatments on young navel tree production would be beneficial for the commercial citrus industry in

California, as well as the backyard grower.

12 STUDY PURPOSE AND OBJECTIVES

To date, there is little research reported on vermicompost treatments and the effect of the soil amendment on citrus tree production. One study focused on lemon quality and the availability of nutrients when various pruning and nutrient management methods are used (Ghosh, Dey, Bhowmick, Bandyopadhyay, & Medda, 2017). Another study explored the use of organic media to compare success of air layering acid lime trees

(Kanpure, Barholia, Yadav, Singh, & Gurjar, 2015). Neither of these studies focus on plant growth and pest density as they relate to solid vermicompost treatments. Performing experiments on the effect of vermicompost treatments on citrus tree pest resistance and overall plant health and growth translates into new solutions in combating citrus pests and diseases in a more permanent and proactive basis. There is no known cure for many of the top citrus diseases at this time, so we must determine long term means to contend with diseases, while saving our citrus trees from damage and extermination.

As the literature from past research has shown, vermicompost is an organic and non-pesticide means of reducing pest populations and damage and increasing plant and fruit growth in a wide variety of plant species (Jack & Thies, 2006). Testing this material as an alternative to traditional chemical pesticides in combating citrus diseases can prove to be beneficial in a wide variety of applications. Chemical pesticides destroy natural soil microbial populations in the process of treating against a single pest or small group of pests or pathogens. Vermicompost uses the assumption that there is a natural destruction of pathogens due to the coelomic fluid contained in the guts of earthworms. This has been correlated with the decrease in phytopathogenic fungi in the soil (Plavsin, Velki,

Ecimovic, Vrandecic, & Cosic, 2017). It also decreases pathogenic bacterial populations

13 even in pathogen-dense substrates like human waste (Fracchia, Dohrmann, Martinotti, &

Tebbe, 2006).

The purpose of this research project was to determine if vermicompost would affect tree growth, fruit yield and quality, and pest densities of Thomson improved navel orange trees. The objective of the research project is to analyze a number of the potential effects of solid vermicompost treatments on navel orange trees. Three key areas are analyzed in the scope of growth, yield, and quality. The specific variables that were measured include trunk diameter below the graft, fruit load, fruit weight, fruit sugar content (Brix analysis), average density of Asian Citrus Psyllid, and citrus thrips, and number of leaves affected by citrus leafminers.

14 METHODOLOGY

This research project was conducted with a sample size of 40 Thomson Improved

(TI) navel orange cultivar on Carrizo rootstock that were subjects in a previous aqueous vermicompost study at Cal Poly Pomona (Lasiter, 2019). The trees were approximately two years old at the end of that research trial, and had been previously grown in one- gallon plastic pots with coir-like media and citrus potting soil. The trees had been divided into sample groups for the aqueous experiments and were already tagged with tree numbers. They were also color-coded by the groups they were divided into in the first research trial.

Planting of Trees

The trees were planted on September 23, 2019, in a citrus grove at Spadra Ranch, part of Cal Poly Pomona, located in the City of Pomona. They were placed in two rows in a simple random placement, with the randomization of the trees being chosen by random.org. The numbers chosen were attributed first to the easterly row, moving southward, then to the westerly row, moving southward.

The trees were divided into four groups, with ten trees in each group. The first group was a control group, which received no vermicompost treatment. The second group received one dry gallon of solid vermicompost mix added under the soil in the root area during planting. This dry gallon of material was applied in a ¼ inch layer around the root area. The third group was planted in the soil and then one dry gallon of solid vermicompost mix was placed above the soil around the base of the tree in a ¼ inch layer. This treatment was repeated every 60 days for the duration of the project. The

15 fourth group received both treatments of solid vermicompost below the soil at planting, and above the soil every 60 days for the duration of the project.

The treatment chosen for each tree was not randomized. As the trees were taken from a previous aqueous vermicomposting study, consistency was used so that any interested parties could compare the two studies. The control group stayed the same for this study. The trees that previously received an aqueous root drench in the original study received the beneath soil solid treatment. The group that previously received a foliar drench for the first experiments, received the above soil line solid treatments every 60 days in this trial. The group that previously received both root and foliar drenches, received below and above soil solid vermicompost treatments in this research project.

Additional vermicompost treatments were applied on November 25, 2019,

January 21, 2020, March 23, 2020, May 21, 2020, and July 21, 2020. One dry gallon was placed around the base of each tree in a quarter in layer in the above ground treatment group, and in the combination below and above ground treatment group.

Soil Profile

The soil around these trees was sampled on February 10, 2020 and the profile was evaluated by FGL Environmental in Santa Paula, California, on February 24, 2020

(Appendix A). The soil is a silt loam and was at optimum moisture at the time of sampling, at 16.7% The Nitrate and Phosphorus levels in the soil were both rated very high. The Nitrate level was 209 lbs/AF, and the Phosphorus was 2820 lbs/AF. The

Potassium level was optimal, as were all of the secondary and micronutrients. The pH level of the soil at the time of testing was 7.53, which came in slightly alkaline. Soil salinity and SAR were satisfactory, but limestone was a moderate problem at 5.4%

16 Vermicomposting Material

The solid vermicomposting material used in this study is Wormgold Plus, produced by California Vermiculture (Appendix A). This product was chosen for its uniformity in production and because laboratory assessment already exists on the product.

Wormgold Plus contains worm castings with kelp and rock dust. The pH value is

7.30 and the ECe measure of media salinity is 13.10. The total nitrogen on a dry weight basis is 1.06%. The total carbon on a dry weight basis is 12.01%. The carbon to nitrogen ratio is 11:8. The organic matter on a dry weight basis is 25.03%. The moisture content of the media is 34.8%. The half saturation percentage is 56.5%.

Irrigation

This research project utilized drip irrigation devices to control the output of water on the trees. A multioutlet emission device (Rainbird) with removable port plugs was hooked up to the Spadra Ranch water system, which runs on well water, so chlorine was not an issue with this experiment. Quarter inch tubing was run down the rows and utilized tubing stakes and emitters for each individual tree. Trees were watered once per week. In the winter months, each application was 0.5 gallons per tree, in Spring 1.6 gallons, and in

Summer and Fall through harvest 2.4 gallons per application. These application rates were based on tree canopy size and duration of time after planting.

Plant Growth Assessments

The growth of the plants was measured and analyzed. Since citrus trees do not grow straight and tend to branch out in a non-uniform manner, measurement of the trunk size both below the graft line was taken when the trees were planted on September 23,

17 2019, and again at the end of the study on September 22, 2020. These measurements were taken by digital calipers and measuring tape for maximum accuracy. The difference between the beginning and ending measurements were recorded for analysis.

Fruit Assessments

Fruit production was counted and measured and compared to determine if there was correlation between the groups. Fruit production was determined by counting the number of navel oranges produced per tree, along with the size and weight of each piece of fruit. The results were compared between the treatment groups.

Fruit quality was compared between the four treatment groups. At the end of the study, each piece of fruit was analyzed by Hanna Instruments Electronic Refractometer

H196811 to record the brix levels in order to determine sugar content. These results were compared between the research groups to determine relevance.

Pests

Asian Citrus Psyllid

Pest assessments were analyzed and recorded on a monthly basis from October

2019 through August 2020. Yellow trap cards were set at each tree on 54" tall metal trap stands (Gempler), as the trees were too small to support the weight of the sticky trap cards. The cards were set and collected monthly and then frozen and stored for analysis.

These cards were analyzed with the assistance of Environmental Scientists from the

California Department of Food and Agriculture Pest Detection Emergency Projects

Branch and the Pest Exclusion Branch. Psyllids were counted and recorded from each card. In addition to analyzing the trap cards monthly, scientists observed the trees on site at Spadra Ranch, performing shake tests to analyze pest density of ACP. A ruler was

18 utilized to tap five individual flush points on each tree three times each. A white cardboard box bottom was held underneath to catch insects for identification. A hand lens was utilized to aid in insect identification. All ACP that landed in the box bottom were identified and counted. Eggs, nymphs and adult psyllids were counted and recorded.

Citrus Thrips

Thrips assessments were analyzed and recorded on a monthly basis from October

2019 through August 2020. Blue trap cards were set at each tree on 54" tall metal trap stands (Gempler). The cards were set and collected monthly and then frozen and stored for analysis. These cards were analyzed with the assistance of Environmental Scientists from the California Department of Food and Agriculture Pest Detection Emergency

Projects Branch and the Pest Exclusion Branch. Thrips were counted and recorded from each card. In addition to analyzing the trap cards monthly, scientists observed the trees on site at Spadra Ranch, performing shake tests to analyze pest density of ACP. A ruler was utilized to tap three individual flush points on each tree three times each. A white cardboard box bottom was held underneath to catch insects for identification. A hand lens was utilized to aid in insect identification. All thrips that landed in the box bottom were identified, counted, and recorded.

Citrus Leafminer

Leafminer assessments were analyzed and recorded on a monthly basis from

April 2020 through August 2020. Visual inspections to analyze leafminer damage and the presence of disease were carried out on the trees. Ten random leaves per tree were removed and observed for leafminer damage each month. The number of leaves per tree with damage were recorded.

19 Statistics and Data Analysis

The data were analyzed in SPSS (Statistical Package for Social Sciences by IBM,

Version 26). The trunk diameter was analyzed utilizing descriptive statistics and a one factor ANOVA. The fruit assessments, such as load, fruit size, and Brix levels (sugar content) data were analyzed in a two-fold procedure of descriptive statistics and

MANOVA. Multivariate tests reduce the occurrence of Type I errors that take place when numerous ANOVA's are independently conducted. The MANOVA determined if the independent variables of the vermicompost treatments changed the outcome variables of fruit load, fruit weight, and Brix levels. The pest densities of Asian Citrus Psyllid,

Citrus leafminer, and Citrus thrips testing were analyzed using repeated measures

ANOVA (RMANOVA's) with a randomized design. This analysis was chosen due to the data collection taken on a monthly basis. Post-hoc corrections are utilized when numerous statistical tests are executed simultaneously, which can cause the probability of significant results to be increased. Where applicable, the ANOVA and MANOVA treatment variations utilized a Tukey post-hoc analysis, and the RMANOVA treatment variations utilized a Fisher's Least Significant Difference (LSD) post-hoc analysis. The calculated value (P-Value) level of statistical significance was assessed as statistically significant at percentage of less than 5 with a confidence interval of 95%.

20 RESULTS

Plant Growth Results

Results of this study showed that there was a statically significant difference in trunk diameter based on vermicompost treatments, F (3,39)= 33.150 , p= <0.001, Wilk's

Λ= 0.874, partial η2= 0.886 (Figure 1). The root treatment and the dual treatment both had a larger diameter growth than the control group and the top treatment group. The root treatment had the highest growth, with a mean growth of 19.630 millimeters over the course of the experiment. The dual treatment was close behind in growth at 19.620 millimeters of growth. The two groups that were not treated under the soil at the root zone did experience the same level of growth. The control group had a mean growth of

16.940 millimeters, and the group with the top treatment had a mean growth of 16.930.

Fruit Production Results

This study's results determined there was no statistical significance found between the treatment groups for fruit load, fruit weight, and fruit sugar content based on solid vermicompost treatments, F (3,39)= 0.026 , p= 0.988, Wilk's Λ= 0.926, partial η2= 0.032.

Between the 40 trees in this experiment, there were 32 pieces of fruit successfully grown to harvest. The treatment on top of the soil produced the most fruit, but not statistically significant as compared to the other groups (Figure 2). The control and root treatment groups produced a similar amount of fruit, and the dual treatment produced the least amount.

Fruit weight was also not significant between the treatment groups. Pieces of fruit varied widely in weight, from 2.5 ounces to 6.3 ounces. The top of soil treatment produced the highest mean weight fruit, and the dual treatment had the second highest

21 mean weight (Figure 3). The control group had the lowest mean weight, but was still within a statistically non-significant range with the other treatment groups.

The Brix level between the treatment groups was consistent and no significance was found in the sugar levels between the fruit from these groups. The control group had the highest mean in Brix level at 9.600, but the other groups all fell between 9.412 and

9.456 (Figure 4).

Pest Density Results

Asian Citrus Psyllid

Results of this study showed there was no statistical significance between treatment groups as it pertained to the pest pressure of Asian Citrus Psyllids, F (3,39)=

0.117 , p= 0.975, Wilk's Λ= 0.646, partial η2= 0.044 (Figure 5). There was a low level of these insects found overall, which contributed to the non-significant results. Most trees had no adults or nymphs in the monthly checks, and of those that did, the distribution was fairly evenly distributed between the groups.

Thrips

This study's results determined there was no statistical significance found between the treatment groups for the pest pressure of thrips on the trees F (3,39)= 0.814, p= 0.615,

Wilk's Λ= 0.339, partial η2= 0.022 (Figure 6). No group was consistently higher than another from a month-to-month standpoint and there seemed to be no differentiation between the thrips and the vermicompost treatments.

Leafminer

This study's results determined no statistical significance between the treatment groups for leaves mined by the insects, F (3,39)= 0.105 , p= 0.981, Wilk's Λ= 0.476,

22 partial η2= 0.003 (Figure 7). The control group had consistently more leafminer activity than the other groups, but not significantly so. The root treatment and the top treatment were where the lowest incidence of activity was found, and the dual root and top treatment landed between those groups and the control.

23 DISCUSSION

The objective of this research project was to examine the effects of solid vermicompost treatments on some of the growth characteristics and pest densities of navel orange trees. These effects on growth of the trees include trunk diameter growth, fruit load, fruit weight, and fruit quality. In addition, the densities of three key citrus pests, Asian Citrus Psyllid, Citrus leafminer, and citrus thrips have also been evaluated.

Based on vermicompost treatment studies on annual plants such as petunias, tomatoes, cucumbers, and peppers, as well as the previous aqueous vermicompost study on these same citrus research trees, it has been theorized that trees treated with solid vermicompost treatments will not only experience greater growth, but also have a decrease in pest densities of these citrus pests found on the trees.

The significance found in this research experiment related to the diameter growth between the trees. The two groups that were given a layer of vermicompost treatment under the soil surrounding the roots at the time of planting had a greater increase in diameter size than the groups that were not treated below the soil. The consistency between the groups shows that vermicompost can be used to aid young trees in growing larger, in a shorter period of time. It would not be unreasonable to infer that this could aid in trees reaching maturity sooner than trees not treated with vermicompost. As young citrus trees consist solely of new flush growth, and flush growth is the most attractive to insects such as ACP, thrips, and citrus leafminer, any treatment that aids in fast growth for citrus trees under five years is especially beneficial.

The fruit load on the trees was minimal, as the trees were two to three years old and this was the first year that any of these trees bore fruit. The majority of trees did not

24 bear any fruit, and most of the fruit developing on the trees did not make it to harvest, as expected. This is a natural defense of young citrus trees to prematurely drop fruit that the tree cannot hold. Consequently, this factor greatly affected the outcome of the weight data and the sugar content (Brix) evaluations. Pieces of fruit that were dropped from the trees during the growing season were smaller in size and could not have reached the same level of sweetness as those that remained on the trees to maturity. As these were not included in the data sets, the information for these categories was greatly limited. The pieces of fruit that did grow to harvest had Brix levels that were consistent and expected from commercial T.I. navel crops. Although there were no statistically significant differences in the treatment groups as it pertained to sugar content, the trees performed uniformly, indicating that there were no significantly significant differences in nutrient uptake from any of the treatment groups as it related to fruit development. Past research studies on fruit quality related to vermicompost treatments that found significant differences between treatment groups focused on smaller crops such as peppers and tomatoes that are annual commodities and not fruit trees.

There was no significance related to any of the pest pressure with solid vermicompost treatments. Although two of the treatment groups received an amendment of vermicompost around the base of the tree bimonthly, it made no statistically significant difference in terms of pest pressure above the ground in the canopy. It is possible that the benefits of the vermicompost treatments do not extend past the areas in which they physically cover. A previous vermicompost study on these trees using aqueous vermicompost solution showed a significant difference in pest pressure of leafminers when sprayed in a foliar treatment on the leaves.

25 In addition, there was no variation on the amount of vermicompost applied under the soil in the root zone and above the soil around the base of the tree in this research study. It is possible that the amendments were not sizeable enough or frequent enough for the effects of the solid treatment to affect the leaf canopy several feet above. It is also probable that the amendments placed above the soil volatilized and did not penetrate the soil deep enough to affect the roots below. Vermicompost is available in aqueous form which might be more efficient in penetrating the soil below and reaching the root zone with available nutrients that are readily available for immediate plant uptake.

Limitations and Future Directions

The limitations of this study include the number of different treatment groups and the number of trees available in total. Because this research study is the second part of a single grant utilizing the same trees, there were not enough specimens left from the first trial to use various amounts of vermicompost using the same procedures. One previous research study on vermicompost treatments noted that using 0.25Kg of vermicompost yielded almost the same results as using 0.5 Kg of the material (Ullah, Riaz, & Arshad,

2019). Given that the cost of Wormgold is around $500 for 2000 dry pounds of material, and this study holds beneficial solutions for the citrus industry, further studies on the amount of vermicompost used versus the effect of the treatments could be advantageous.

Further, no study has been previously conducted on using solid vermicompost in the soil and a foliar vermicompost tea spray in conjunction on citrus trees. Vermicompost tea treatments sprayed directly on the leaves could reduce pest populations in a more cost-effective manner, while the solid treatments could boost plant growth by giving nutrients down at the root zone. As these were the two areas found to be significant in the

26 two parts of the citrus vermicompost treatment research on these trees, a continued grant could explore the relationship of these two media, and determine the cost and pest thresholds that are most financially productive in increasing plant health while combating pests and diseases.

Another area for future study is in fruit production. Because these trees were in their first season of fruit bearing, and only a small portion of the trees bore fruit to harvest, following these trees for another few seasons with the continued methods or a combination of methods from both research trials might give a clearer indication of whether vermicompost treatments will increase fruit load, size, and quality (sugar content) as fruit production increases and matures on the trees.

This study utilized trees located on a farm, in real-world working conditions. As a result, it was impossible to control the pest factors on these trees, and the pest pressure was limited to the pests at Spadra Ranch in Pomona, California. In some aspects, this is helpful as it shows how a real citrus orchard will control ACP, thrips, and leafminers in farming conditions. However, a negative of this factor is it was impossible to ascertain the pest load in the surrounding areas. ACP pest pressure is low in this area.

Finally, the location where these trees were planted on Spadra Ranch had previously been dormant. Directly adjacent to this location is a group of citrus trees in conventional production. Although the trees in this research trial were essentially treated organically, as nothing was applied to them other than vermicompost, the same is not true for the conventionally grown trees in the same area. Pesticides are utilized in the area on other crops to abate insect and weed pests. This may have had an overall effect on the pest pressure in the general area, reducing available pest loads overall.

27 CONCLUSION

Key Findings

Results from this research study indicate that solid vermicompost treatments placed under and around the root zone at planting can increase plant growth on young citrus trees, allowing them to reach maturity sooner, and taking them out of their most vulnerable stages of development, when the new growth is most susceptible to pest and disease attacks. These treatments can aid growers in utilizing non-chemical means to help combat these citrus pests and diseases that threaten the citrus industry in California.

The significant diameter growth increase in size on the trees did not translate into greater fruit yield, fruit size or fruit quality (Brix content) for the treatment groups planted with vermicompost below the soil surrounding the root zone. The benefit of growth on trunk size may need further growing seasons observed to note statistically significant effects on these characteristics overall.

In addition, unlike small annual crop commodities that have been studied, the solid vermicompost treatments did not decrease pest pressure within the treatment groups of the two-year-old citrus trees. The navel orange trees are larger than small pepper, tomato, and cucumber crops that have been studied in the past, and the vermicompost did not perform as expected based on previous research trials cited.

Practical Implications and Proposed Impact

The California citrus industry is valued at $3.389 billion at the point of shipment

(Babcock, 2018). There are approximately 270,000 acres in production, with the majority of the production in nine counties (Geisseler & Horwath, 2014). Navel oranges account

28 for 45% of citrus, mandarins and lemons are 20% each, Valencia oranges are 10% and grapefruit makes up about 3% (USDA & CDFA, 2018).

HLB disease is the most immediate threat to the California citrus industry, having already devastated citrus production in Florida over the past fifteen years. It was first introduced in that state in 2005 and since has quickly infected all of the commercial citrus growing regions there. Production has decreased by 60%, with losses hitting just under

$1 billion per year (Nupane, Moss, van Bruggen, 2016). Florida’s citrus production is primarily for juice. California grows fresh fruit citrus, so the effects of HLB on the

California market has the potential to be even more devastating than the damage to

Florida’s citrus industry.

The Asian Citrus Psyllid was first detected in California in 2008. The USDA and

CDFA quickly and aggressively worked together and mobilized to begin a monitoring program in both commercial and residential areas. Sticky traps are placed and checked on a regular basis in areas around the state with citrus trees to determine if ACP is present in the area. In addition to this, a survey program is set up that analyzes leaf and insect samples to test for the presence of ACP and HLB (Arredonado & Delgado, 2016).

California has adopted quarantine areas that restrict citrus tree and fruit movement to contain psyllids to the areas they have been detected. Growers of citrus trees and fruit are in mandatory programs to ensure the agricultural products they sell are free from ACP and HLB. When ACP and HLB are detected, treatment programs are established in order to prevent the spread of HLB in quarantined areas. The current treatment protocol is a systemic spray of Imidacloprid and a foliar spray of beta-cyfluthrin, applied twice a year

(CDFA, 2018). Trees that test positive for HLB are removed and destroyed.

29 In Florida, the citrus industry now relies on resetting trees, which means that unproductive trees in commercial groves are analyzed for pests and diseases, removed and replaced with young trees of the same cultivar (Zekri, 2014). Resetting has several issues. First, there is the cost of the new trees and the labor associated with analysis and replacement. Second, younger trees need far more maintenance than mature trees require.

And since these young trees are replaced randomly in the groves where the unproductive trees have been removed, grove workers must keep detailed records to ensure they are giving every single young tree the extra attention needed for successful growth and production (Zekri, 2014). Lastly, these young trees must compete with the mature, established trees for sunlight, nutrients, and water. They also compete with weeds that increase from the loss of full canopy that the older trees protected against. Weed and pest abatement for these young trees are critical. Therefore, this study is particularly beneficial to growers utilizing resetting as their primary means of combatting HLB, as the statistical significance of greater trunk diameter growth from vermicompost treatments provides these growers a means of boosting tree growth and potentially decreasing the most vulnerable stages of development.

The pesticide solution for treating ACP and HLB is problematic. In the long run, systemic and foliar pesticides kill off beneficial microorganisms and insects that assist in an ecological defense system for the trees. The more these pesticides are applied, the less effective they become. Due to this predicament, it is urgent to devise new solutions for the integrated pest management plan and to address resistance issues that will be the least damaging for beneficial microbial and insect communities.

30 With all that is at stake in the citrus industry in California, this research study has the potential for wide-reaching implications in how the Asian Citrus Psyllid and

Huanglongbing disease are managed and controlled. Studying methods of increasing plant growth can lead to additional research and clues on how to decrease the length of time that young trees are in their most vulnerable stages of growth where they are most susceptible to pests and diseases. The significance of using Wormgold solid vermicompost instead of pyrethroid and neonicotinoid pesticides to increase plant growth in a shorter time could aid in both conventional and organic production, as well as protect home grown citrus across the state. It is only a matter of time before ACP and HLB spread from residential areas in California to the large-scale agricultural valleys. It is essential to find a way for citrus trees to survive with the invasion with the least amount of negative environmental impacts.

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37 FIGURES

Figure 1: Mean change in trunk diameter growth in millimeters with vermicompost treatments on TI navel trees at Spadra Ranch from September 2019-September 2020.

Figure 2: Mean number of pieces of fruit grown with vermicompost treatments on TI navel trees at Spadra Ranch from September 2019-September 2020.

38

Figure 3: Mean weight in ounces of fruit grown with vermicompost treatments on TI navel trees at Spadra Ranch from September 2019-September 2020.

Figure 4: Mean Brix assessment of fruit grown with vermicompost treatments on TI navel trees at Spadra Ranch from September 2019-September 2020.

39

Figure 5: Mean Asian Citrus Psyllid densities with vermicompost treatments on TI navel trees at Spadra Ranch evaluated monthly from October 2019-August 2020.

Figure 6: Mean Citrus thrips densities with vermicompost treatments on TI navel trees at Spadra Ranch evaluated monthly from October 2019-August 2020.

40

Figure 7: Mean citrus leafminer damage of leaves with vermicompost treatments on TI navel trees at Spadra Ranch evaluated monthly from October 2019-August 2020.

41 APPENDIX

Wallace Labs 2019 Soils Report California Vermiculture Wormgold Plus

42

FGL Environmental February 2020 Analysis on TI Navel soil at Spadra Ranch

43