nematicide effects on non-target soil invertebrate...
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NEMATICIDE EFFECTS ON NON-TARGET SOIL INVERTEBRATE POPULATIONS IN TURFGRASS SYSTEMS
By
BENJAMIN DAVID WALDO
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2018
© 2018 Benjamin Waldo
To Mom, Dad, Sarah, and Frosty
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ACKNOWLEDGMENTS
I would like to thank my parents and sister for their unwavering support. I would
also like to thank the support from the University of Florida community as well as the
experts at the Division of Plant Industry. I would like to specifically thank Dr. Maria
Mendes, Tom Beam, Gideon Alake, Ruhiyyih Dyrdahl-Young, Lesley Schumacher, Dr.
van Santen, Dr. Daniel Carrillo, Dr. Sam Burton, Tina Gu, Brandon Jones, and Meghan
Yokem. I would also like to thank my committee members Drs. Billy Crow, Zane
Grabau, Tesfa Mengistu, Felipe Soto-Adames. I would specifically like to thank Dr.
Crow for his guidance and support as my advisor.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ................................................................................................................... 11
CHAPTER
1 INTRODUCTION TO SOIL INVERTEBRATES ...................................................... 13
Introduction ............................................................................................................. 13
Literature Review .................................................................................................... 14 Functional Groups .................................................................................................. 15
Nematodes ............................................................................................................. 16 Mites ....................................................................................................................... 20 Springtails ............................................................................................................... 21
Oligochaetes ........................................................................................................... 23 Other soil inhabitants .............................................................................................. 24
Pesticides ............................................................................................................... 25 Nematicides ............................................................................................................ 27
Related studies ....................................................................................................... 29
2 MATERIALS AND METHODS ................................................................................ 37
3 NEMATODE RESULTS .......................................................................................... 42
Functional Groups and Ecological Indices .............................................................. 42 Bacterivores ............................................................................................................ 42 Fungivores .............................................................................................................. 43
Omnivores .............................................................................................................. 43 Predators ................................................................................................................ 44 Plant-parasitic ......................................................................................................... 44 Functional Groups Summary .................................................................................. 45
MI ............................................................................................................................ 46 MI25 ........................................................................................................................ 46 ∑MI ......................................................................................................................... 47
PPI .......................................................................................................................... 47 CI ............................................................................................................................ 47 BI ............................................................................................................................ 48 EI ............................................................................................................................ 48 SI ............................................................................................................................ 48
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Faunal Profile .......................................................................................................... 49
Ecological Indices Summary ................................................................................... 51
4 ARTHROPOD AND NON-NEMATODE RESULTS ................................................ 71
Arthropods .............................................................................................................. 71 Mites ....................................................................................................................... 72 Springtails ............................................................................................................... 72 Insects .................................................................................................................... 72
Arthropod Summary ................................................................................................ 73 Non-Arthropods ...................................................................................................... 73 Rotifers ................................................................................................................... 74 Turfgrass Percent Green Cover Results ................................................................. 74 Percent Green Coverage Summary ........................................................................ 76
5 SUMMARY AND CONCLUSIONS .......................................................................... 85
LIST OF REFERENCES ............................................................................................... 96
BIOGRAPHICAL SKETCH .......................................................................................... 107
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LIST OF TABLES
Table page
1-1 Summary of nematode ecological indices. ............................................................. 35
3-1 Nematode families identified from turfgrass plugs and soil samples. ..................... 53
4-1 Non-insect arthropod families identified from Berlese funnel extraction in 2016and 2017 ............................................................................................................ 77
4-2 Insect families identified from Berlese funnel extraction in 2016 and 2017. ........... 78
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LIST OF FIGURES
Figure page
1-1 Faunal Profile visual depiction as described in Ferris et al., 2001. Structure index values on the x-axis are plotted against enrichment index values on the y-axis.. ................................................................................................................ 36
3-1 Population densities of bacterivore nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 54
3-2 Population densities of fungivore nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 55
3-3 Population densities of omnivore nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 56
3-4 Population densities of predatory nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 57
3-5 Population densities of plant-parasitic nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................... 58
3-5 Maturity index (MI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ...................................................................................................... 59
3-6 2-5 (MI25) from mist and soil extraction as affected by different nematicideapplications at all sampling dates. *, **, *** Different from the untreatedaccording to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ............. 60
3-7 Sigma maturity index (∑MI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ...................................................................................................... 61
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3-8 (PPI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ............. 62
3-9 (CI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. Data only shown for 2016. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 63
3-10 (BI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ............. 64
3-11 (EI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ............. 65
3-12 (SI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ............. 66
3-13 Faunal Profile calculated from mist extracted nematodes affected by different nematicide applications in 2016.. ........................................................................ 67
3-14 Faunal Profile calculated from soil extracted nematodes affected by different nematicide applications in 2016.......................................................................... 68
3-15 Faunal Profile calculated from mist extracted nematodes affected by different nematicide applications in 2017. Quadrats are labeled with A, B, C, or D for reference. No significant differences from the untreated control observed. ........ 69
3-16 Faunal Profile calculated from soil extracted nematodes affected by different nematicide applications in 2017. Quadrats are labeled with A, B, C, or D for reference. No significant differences from the untreated control observed. ........ 70
4-1 Population densities of detritivore mites from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ...................................................................................................... 79
4-2 Population densities of predatory mites from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ...................................................................................................... 80
4-3 Population densities of detritivore springtails from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, ***
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Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 81
4-4 Population densities of phytophagous insects from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................................................................... 82
4-5 Population densities of rotifers from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). ...................................................................................................... 83
4-6 Percent green coverage calculated from plot photographs from June 7, 2016 to. March 13, 2018. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively). .................................. 84
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
NEMATICIDE EFFECTS ON NON-TARGET SOIL INVERTEBRATE POPULATIONS IN TURFGRASS SYSTEMS
By
Benjamin David Waldo
August 2018
Chair: William Crow Major: Entomology and Nematology
In turfgrass systems, nematicides are a valuable tool for managing plant-parasitic
nematode populations, but few studies have examined nematicide effects on non-target
invertebrates. Our study evaluated effects of turfgrass nematicide formulations of
abamectin (Divanem SC), fluopyram (Indemnify), furfural (MultiGuard Protect EC), and
fluensulfone (Nimitz Pro G) on non-target nematode populations. A randomized block
design was used with five replications of the four nematicide treatments and an
untreated control. Plots were 6 m2 with 0.6 m untreated borders between adjacent plots.
Data were collected from 1.5 m2 subplots located in the center of the treatment plots.
Nematicides were applied at labeled rates every four weeks as a summer treatment
program from 7 June to 30 August 2016 and 24 April to 18 July 2017 at the University of
Florida Plant Science Research and Education Center in Citra, Florida. Samples were
collected before treatment and two days, two weeks, and eight weeks after the final
treatment for invertebrate community analysis. Data from each nematicide treatment
were compared to the untreated at each sample date using analysis of covariance with
initial population counts serving as the covariate. Abamectin had moderate impacts and
fluopyram had substantial impacts on the soil ecosystem. Furfural and fluensulfone had
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low impacts on the soil ecosystem. However, abamectin and fluopyram plots generally
had the greatest turfgrass percent green cover and furfural and fluensulfone had
relatively small impacts on percent green cover compared to the control. The results of
this study suggest nematicides can impact soil invertebrate densities in bermudagrass.
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CHAPTER 1 INTRODUCTION TO SOIL INVERTEBRATES
Introduction
Turfgrass is an important industry in the state of Florida. Golf courses, athletic
fields, and lawns utilize turfgrass as a playing surface and as ground cover. Turfgrass
cultivation, sales, and maintenance is a billion-dollar industry in Florida (Haydu et al.,
2006). Plant-parasitic nematodes are an important pathogen of turfgrass. Nematodes
like Belonolaimus longicaudatus use hypodermic needle-like stylets to pierce roots and
drain cells of cytoplasm (Huang and Becker, 1997). Plant, water, and nutrient uptake
efficiency declines due to root injury. This feeding behavior can lead to stunted roots
and even death of the plant (Lambert and Bekal, 2002). As an aesthetic crop, turfgrass
has relatively low tolerance of injury. Chemical nematicides are valuable for managing
harmful nematode populations, particularly on golf courses (Crow et al., 2003).
Nematicides have been shown to reduce plant-parasitic nematodes, but few studies
have evaluated the effect on non-parasitic nematodes and other soil invertebrates.
Free-living nematodes, mites, springtails, earthworms, and enchytreids all play roles in
maintaining healthy soil through ecosystem services (Dindal, 1990). Diverse soil
community structure contains members at different trophic levels. Some organisms
affect nutrient cycling by maintaining steady microbial growth and others help
mechanically breakdown organic matter, increasing the exposed surface area for
microbial decomposition (Hopkin, 1997a; Kranz and Walter, 2009). Some members act
as plant pest and pathogen antagonists by competing with or feeding upon detrimental
organisms (Altieri, 1999). Alterations in soil community structure may affect ecosystem
health by altering the ability of soil to function as a living system (Doran and Zeiss,
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2000; Ferris and Bongers, 2006). Studying population changes of functional groups of
soil invertebrate populations can provide insight into potential nematicide effects on soil
ecosystems. To make more informed turfgrass management decisions, more studies
are needed for evaluating impacts of nematicides on soil ecosystems. This study
evaluated nematicide effects on non-target soil invertebrate populations in turfgrass. We
predicted significant changes in community structure with a decrease in organisms
belonging to high trophic level and rapid recovery and increased abundance by low
trophic level organisms after exposure to nematicides.
Literature Review
Soil organisms are important members of nutrient cycles. Organic matter from
dead plants and animals is broken down by soil fauna and flora into nutrient forms that
can be taken up and used by plants (Dindal, 1990). Plants serve as important primary
producers in soil and above ground food webs. Soil macrofauna such as earthworms
provide mechanical breakdown of bulky organic debris and create architectural
modifications in soil through tunneling activity (Lavelle, 1988). The tunnels created by
macrofuana allow air and water to more easily reach plant roots and help make pore
spaces, which can be inhabited by smaller fauna and flora. Mesofauna members such
as mites, nematodes, springtails, enchytreids, and protists, occupy many positions in
the soil food web. They feed on organic matter, microbes, plants, and on other
mesofauna as predators (Orgiazzi et al., 2016). The grazing feeding behavior of
microbial feeders promotes decomposition of organic matter by prolonging the log
growth phase of microbes (Chen and Ferris 1999). Because they occupy a broad range
of ecological roles, some mesofauna members are useful for community analysis of soil
ecosystems (Bongers, 1990). Microflora comprise the microbial and fungal organisms in
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soil. Microbes rapidly breakdown water soluble compounds and fungi breakdown non-
soluble and soluble compounds which play important roles in mineralization and
immobilization of nutrients such as carbon, nitrogen, and phosphorus (Ingham et al.,
1985; Fierer et al., 2007). A balanced soil food web contains macrofauna, mesofauna,
and microfloura at various trophic levels that balance energy flow so that an ecosystem
is stable (De Angelis, 1975). Soil organisms can be more difficult to study than
aboveground species due to their small size, habitat, and biological qualities. In order to
understand ecosystem health in soil, functional groups of biological indicator organisms
are often studied.
Functional Groups
Functional groups are groupings of organisms with similar ecological roles and
are based largely on feeding habits and life history (Ferris et al., 2001). Invertebrate
functional groups have been used to assess soil condition in other studies (Paoletti et
al., 1991). Population shifts of functional groups can provide a snapshot of soil health.
Organisms with r strategist life history are often associated with environmental
disturbance while K strategist organisms are usually associated with environmental
stability (Bongers and Bongers, 1997). Disturbed or stressed environments are
expected to contain high abundances of r strategists and low abundances of K
strategists. Stable food webs are expected to have decreased r strategists and
increased K strategists relative to a disturbed soil ecosystem. Analyzing population
shifts of soil functional groups can reveal successional recovery after an environmental
disturbance such as pesticide exposure.
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Nematodes
Nematodes are extraordinarily abundant in soil and are found in many habitats
(Cobb, 1915). Species diversity has been estimated at one million species with
approximately 23,000 species described (Lambshead, 1993; Blaxter, 2004). Because of
the high abundance, ubiquitous nature, and occupancy of many trophic levels,
nematodes have been used as bioindicators for ecological studies in regards to soil
condition (Zullini and Peretti, 1986; Bernard, 1992; Yeates et al., 1993; Neher and
Cambell, 1994; Bongers and Ferrris, 1999). Groupings are typically at the family level
and reflect life-history strategy in the soil ecosystem. Functional group analysis can be
performed by comparing shifts in community structure of different feeding groups or by
using more advanced metrics.
Terrestrial nematodes feed as microbivores, algivores, fungivores,
phytophagous, omnivores, saprophages, and predators (Yeates et al., 1993). Nematode
stoma morphology is reflective of feeding strategy (Dindal, 1990). Microbivores have a
wide stoma which allows for direct ingestion of unicellular organisms such as bacteria.
This feeding group is often one the most abundant in soil. They have short life cycles
and rapidly colonize disturbed areas (Bongers and Bongers, 1997). The grazing feeding
behavior promotes microbial turnover. Rhabditidae and Cephalobidae are bacterial
feeding nematode families common in soil (Yeates et al., 1993). Algivores, fungivores,
and phytophagous nematodes have a hypodermic-like needle structure called a stylet
that is used for piercing and ingesting fluids from a food source (Huang and Becker,
1997). Nematodes that feed on fungi often have a weaker and smaller stylet than plant
feeders. Algae or fungal hyphae are directly penetrated and the fluid contents are
withdrawn. Like microbivores, fungal feeding nematodes reproduce rapidly and
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stimulate fungal growth from grazing. Common fungal feeding nematodes can be found
in the families Aphelenchoididae and Anguinidae (Yeates et al., 1993). Plant-parasites
have varied feeding habits as endoparasites, ectoparasites, or semi-endoparasites
(Yeates et al., 1993). They may have an odontostylet, stomatostylet, or onchiostylet for
feeding. Plant-parasitic nematodes are considered the most economically important
feeding group, because of the economic losses caused by loss of crop productivity
(Handoo 1998). Heteroderidae and Belonolaimidae are examples of phytophagous
families (Yeates et al., 1993). Omnivores could include many nematode families, but for
ecological analyses, use of the term omnivore is restricted to a few Dorylaimid families
(Yeates et al., 1993). These nematodes have an odontostylet that is used for puncturing
plant or prey cells. Omnivores are large bodied, sensitive to environmental disturbance,
and have relatively slow life cycles. The families Dorylaimidae and Aprocelaimidae
contain omnivorous members (Yeates et al., 1993). Predatory nematodes have a wide
barrel-shaped stoma often accompanied by a mural tooth-like structure or denticles for
piercing prey (Khan and Kim 2007). Others may possess an odontostylet for piercing
prey. Monichidae and Nygolaimidae families have predatory members (Yeates et al.,
1993).
The colonizer-persister value (cp) originally developed by Bongers (1990) is a
system of assigning a value based on r and K strategies of nematodes. Bongers
created a scale from cp-1 to cp-5 values for nematode families. This classification is an
insightful approach to nematode community analysis. The cp-value of a nematode
family more directly reflects the successional state of soil. Lesser cp nematodes are
typically bacterial feeders such as Rhabditids, which exhibit explosive population growth
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in enriched conditions. Enriched conditions are an environmental condition of soil
characterized by an increase in resource availability due to organism mortality, biomass
turnover, or shift toward favorable environment (Odum 1985). Greater cp value
nematodes like large Dorylaimids have long life cycles with low reproduction rates.
Various indices can be calculated using cp values. Maturity index, enrichment index,
plant-parasitic index, channel index, structure index, and faunal profile are examples of
measures of environmental condition based on nematode community structure (Table
1-1) and (Fig 1-1). The absence of a functional group in a given ecosystem can be
meaningful for environmental analysis due to the abundance and redundancy of
nematode fauna (Ferris and Bongers 2009b).
Maturity index (MI) is a measure of the community structure based on the
abundance of colonizers and persisters, but excludes plant-parasitic nematodes. Low
MI values indicate an environment dominated by short-lived colonizers and generally
indicate a disturbed environment. High MI values suggest a stable environment with
long-lived, persisting nematodes. Maturity index cp-2 to 5 (MI25) is similar to MI, except
it excludes cp-1 nematodes. Studies have shown cp-1 nematode abundance can
increase in the presence of a pollutant due to the greater availability of microbial food
sources flourishing on decomposing organisms after pollutant induced mortality
(Bongers and Ferris, 1999). Excluding cp-1 nematodes may provide more resolution to
the MI analysis in the presence of a contaminant such as a nematicide. Sigma maturity
index (∑MI) is the average cp value of all nematodes present including plant-parasitic
members. Including all nematodes provides a broad analysis of soil condition. Low
values are reflective of a disturbed environment, but incorporates plant-parasitic
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nematodes, which typically do not rapidly respond to enriched conditions. High values
are associated with an undisturbed environment. The plant-parasitic index is a maturity
index for plant feeders. It is heavily dependent upon the health of nearby plants. High
values suggest the presence of a diverse and mature plant community while low values
may indicate the absence of mature plant communities, and is likely affected by
nematicide applications. Channel index (CI) is used to infer the primary decomposition
pathway (Ruess and Ferris, 2004). Bacterial dominated decomposition is rapid and
often short lived due to the growth curve of bacteria. Fungal decomposition is slower
and more stable. Bacteria tend to break down water-soluble compounds whereas fungi
can break down both soluble and non-water soluble organic matter. This index
compares bacterivore and fungivore nematode populations. Carbon tends to stimulate
fungal growth and decomposition may influence community structure of nematodes
(Ferris, 2003). High values indicate the fungal decomposition pathway is dominant and
low values indicate dominance of the bacterial decomposition pathway. Basal index (BI)
is a weighted ratio of nematodes characteristic of basal conditions (bacterivores and
fungivores) to all nematodes (bacterivores, fungivores, predators, and omnivores)
(Berkelmans et al., 2003). High values indicate a structured condition and low values
indicate a basal condition that has limited resources and stressed due to adverse
environmental conditions or contamination (Ferris et al., 2001). Low values are
indicative of increasing community complexity with more abundant resources and lower
environmental stress. Enrichment index weighs enrichment nematodes against basal
nematodes that belong to cp-1 and cp-2 groups of bacterivores and fungivores. High
values indicate enriched conditions with abundant food sources and low values suggest
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an unenriched environment with lower food resource availability (Ferris et al., 2001).
Structure index measures the higher cp nematodes to determine the number of trophic
levels and potential for stability. Nematodes in cp 3 to 5 groups are included. High SI
values are associated with greater food web complexity due to the presence of upper
trophic level nematodes. Low values indicate low abundance of upper trophic level
nematodes and potentially less environmental stability. Faunal profile compares the
structure index on the x-axis against the enrichment index on the y-axis to provide a
more comprehensive depiction of faunal relationships. Points falling in the top left
quadrant on the graph indicate enrichment with high disturbance. Points in the top right
corner indicate a low-moderate disturbance with maturing food web. Points in the
bottom right corner indicate a structured food web with no disturbance. Points in the
bottom left corner indicate a stressed and degraded food web.
Mites
Mites (Acari) represent a very diverse and abundant group in soil. They feed as
saprophages, microbivores, algivores, fungivores, predators, and phytophages (Kranz
and Walter, 2009). Mites may inadvertently ingest organisms such as bacteria, algae,
nematodes, protozoans, and plant material while feeding on a preferred food source.
Size of food appears to be an important factor in food selection by mites. Saprophytes
and fungivores are common in suborder Oribata. The oribatid mites possess large
chelicera that are used to shred plant or fungal material. Some mites prefer to feed on
detritus or mycelia while others feed facultative. It is hypothesized that saprophagy
developed as mites feeding on microbes on the surface of plant material began feeding
on dead and dying plant tissue (Norton, 1985). The mechanical breakdown of detritus
through feeding is important for increasing the rate of decomposition of organic material
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(Dindal, 1990). Surface area is increased which provides better exposure for microbial
decomposition. Mites may play a role in the distribution of fungi and microbes by
carrying fungal spores and bacteria on their bodies. As predators, they feed on
nematodes, enchytreids, and microarthropods and their eggs. Chelicerae of predatory
mites are often modified for piercing or grabbing prey with large and well sclerotized
bodies. Mesostigmata, Prostigmata, and Astigmata taxa contain common predatory soil
mites. Reductions in populations of the plant-parasitic nematode Tylenchorhynchus
dubius by predacious mites in laboratory studies has been documented by Sharma
(1971). Astigmatina mites have been documented feeding on plant-parasitic nematodes
and southern corn earworm eggs (Walter et al., 1986; Brust and House, 1988).
Predatory soil mites may play a role in regulating populations of plant pests. Few soil
Acari are phytophagous, but some may feed on ornamental and vegetable crops
(Ascerno et al., 1981). Opportunistic plant-feeding mites such as some Acariformes
mites may feed on living plant tissue as secondary pests and have chelicera designed
for shredding plant material. Obligate plant feeders including taxa within Trombidiformes
typically have a needle-like chelicera for piercing and ingesting plant cell contents. Most
economically important mites inhabit aerial portions of plants.
Springtails
Springtails (Collembola) are hexapods found in many environments, including
the arctic (Block et al., 1994). They are often the most abundant arthropods collected
from leaf litter and soil samples. The name collembola is derived from the Greek
meaning “glue peg” referring to the ventral tube on the first abdominal segment. The
ventral tube was believed to aid the animal in attaching to substrates, but now is
believed to be involved osmoregulation and excretion (Eisenbeis, 1982). The common
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name springtail refers to a forked spring organ called a furcula attached to the ventral
side of the abdomen. The furcula can quickly propel the animal away from predators
(Hopkin, 1997a). Springtails are important contributors to succession by increasing the
rate of decomposition. Body shapes include globular and cylindrical and may contain
showy color patterns. Springtails prefer moist habitats such as leaf litter. Most
springtails feed on decaying vegetation, fungi, or microbes, but some are predators of
arthropods and nematodes (Hopkin, 1997a).
Springtail distribution in soil is often aggregated. This may be due to attraction to
favorable conditions or avoidance of undesirable fluctuations of temperature and
moisture (Nosek, 1981). Stable environments typically contain a relatively constant
species number dominated by a few common species (Curry, 1969). In disturbed
environments, colonizer species are common and species diversity may be low. As the
environment stabilizes, colonizer species may diminish as species diversity increase as
colonizers disappear. Ideally, the study area should be sampled regularly as community
structure can change rapidly. Effects of agriculture on springtail community structure
has been studied for a variety of disturbances including plowing, grazing pressure, crop
rotation, and forestation (Hopkin, 1997b). However, it is often difficult to isolate the
variable of interest from outside environmental factors. Some springtails respond
favorably to agricultural disturbance, especially when fertilizer is used, but diversity
often is reduced. Soil compaction and urbanization can also lead to reduced abundance
and diversity.
Springtails have been used in toxicology laboratory and field studies of pesticides
and pollutants as a non-target organism (Frampton, 1994; Hopkin, 1997a). Folsomia
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candida is often used as a model for laboratory studies as it is cultured relatively easily
on moist plaster of Paris and reproduces parthenogenetically. In field studies, springtails
may be killed by direct exposure to chemical application or from indirect effects such as
reduced food source (Filsner, 1994). Some springtail populations increase after
exposure to pesticides possibly due to reduction of predators or less competition with
other susceptible springtails. The variable responses of springtails to external
environmental conditions can make results of ecological studies challenging to interpret.
Oligochaetes
Oligochaetes include members of the Class Oligochaeta such as the potworms
or Enchytreidae and the common earthworms of Lumbricidae (Dindal, 1990).
Enchytreids are found on every continent, including arctic climates. Moist, acidic and
organic rich soils are ideal conditions for enchytreids, but they may also inhabit littoral
and aquatic areas. A variety of food sources may be utilized by enchytreids, but they
tend to prefer fungi or plant debris. Enchytreids appear to feed on decaying plant matter
processed by microflora and small arthropods such as mites and springtails. The debris
is further processed which provides benefits to the ecosystem by creating more sites for
microflora to continue decomposition, grazing microflora to maintain the growth phase in
populations, and further decomposing plant material. Enchytreids are most commonly
found in the upper 10 cm soil profile (Peachey, 1963).
Lumbricids are common in many habitats. However, moisture is important for
their survival. Ecology of lumbricids is poorly understood, but may be placed into
epigeic, anecic, and endogeic ecological groups (Bouché, 1977). Epigeic earthworms
are found in decomposing organic debris because of poor physical adaptations for
burrowing. The anecic earthworms form vertical burrows and typically feed at the soil
24
surface. Endogeic earthworms create horizontal burrows and feed on decaying plant
roots. The burrowing and feeding habits of these organisms causes architectural
changes in the soil by creating tunnels which air and water may pass through, but also
helps move organic matter through the soil profile. Earthworm castings provide
processed nutrients from organic matter available for the soil ecosystem. Oligochaetes
have been used in pesticide and pollution studies in both terrestrial and aquatic
environments (Chapman et al., 1982; Potter et al., 1990).
Other soil inhabitants
Tardigrades, rotifers, protozoa, and insects may also be encountered, but
usually at lower abundance than the previously discussed organisms. Tardigrades, also
known as “water bears”, are found in aquatic and moist terrestrial habitats (Dindal,
1990). They possess anhydrobiotic capabilities that enable them to survive extended
dry periods. Tardigrades feed on a variety of food sources including algae, bacteria,
fungi, nematodes, and rotifers. Predators of tardigrades include nematodes, protozoans,
and other tardigrades. Patchy distribution is common and abundance decreases at soil
depths below 5 cm (Meyer, 2006). Bioindicator studies have been performed with
tardigrades, but primarily with air pollution evaluation (Hohl et al., 2001; Vargha et al.,
2002).
Rotifers are also associated with moist habitats and are capable of anhydrobiosis
like tardigrades and some nematodes. Asexual species are considered colonizers due
to their ability to be transported in an anhydrobiotic state and to reproduce without a
mate. They inhabit water films around soil particles where they may feed on algae or
bacteria as filter feeders or on ciliates and other rotifers as predators. Rotifer
25
bioindicator use is primarily for freshwater water quality (Marneffe et al., 1998; Duggan
et al., 2001).
Protozoans are a diverse group common in soil. Flagellates, amoebae, and
ciliates are frequently encountered examples (Dindal, 1990). Bacteria are the primary
food source of protozoans and feeding interactions are important for regulating bacterial
community structure and associated nutrient cycles (Clarholm, 1981). Moisture appears
to be the most important abiotic factor by influencing osmotic stress. Temperature
influences cyst formation and vegetation provides additional nutrients utilized by
protozoa. Protozoa have been used as bioindicators in pesticide, oxygen regime, and
forest decline studies (Foissner, 1999).
Some insect and insect-like organisms that have been used as soil bioindicators
include Coleoptera, Diptera, Isopoda, Formicidae, Diplura, Isoptera, and Protura.
(Frouz, 1999; Paoletti et al., 1996; Paoletti and Hassall, 1999; Rainio and Niemelä,
2003). These organisms have been studied in cases of heavy metal contamination and
urban disturbance of soil.
Pesticides
Pesticides are used to manage populations of pests. Previous pesticide use
practices relied on broad spectrum, non-specific control that would kill beneficial
organisms including natural predators and pollinators along with the target pest (Aktar et
al., 2009). With the Food Quality and Protection Act of 1996, the EPA began banning
highly toxic pesticides and requiring future pesticides to have reduced-risk qualities
(U.S. EPA, 1996). The primary goal of the act was to strictly limit the use of pesticides
with high risk of harm to people and the environment. Ideally, conventional pesticides
have low risk to people, non-target organisms, groundwater, low persistence, and high
26
specificity for a particular pest. Golf courses often rely upon pesticides to achieve
pristine turfgrass appearance (Colding et al., 2009). Nematode pathogens, disease,
insect pests, and weeds are managed with chemical products to maintain high quality
playing surface. Cultural, biological, and physical controls can be used with an
integrated pest management plan, but chemical control is heavily relied upon in high-
end turfgrass systems (Cox, 1991). Plant-parasitic nematodes can be managed with
fumigant and non-fumigant compounds. Extensively used fumigant pesticides for
nematode management in agriculture currently in use include 1,3-dichloropropene,
chloropicrin, metam sodium, and dimethyl disulfide. These pesticides are used primarily
in agronomic settings with only 1,3-D labeled for use on established turfgrasses (Crow
et al., 2003).
Several other pesticides that were formerly used for nematode management, but
have had their turfgrass registration cancelled include 1,2–dibromo-3-chloropropane
(DBCP), fenamiphos (Nemacur), and ethoprop (Mocap) (Perry et al., 1970; Crow,
2005). DBCP provided excellent control of nematodes with a long half-life and low use
rate, but is known to cause human male sterility and is no longer used. Both fenamiphos
and ethoprop belong to the organophosphate class and have high acute toxicity.
Because of the health and environmental risks, EPA registrations have been cancelled
for these pesticides. No single product currently registered for turfgrass manages all
plant-parasitic nematodes with high efficacy, but developing chemistries have reduced
risk to non-target organisms and the environment while being more selective on the
target pest (Gentz et al., 2010).
27
Nematicides
Traditionally, broad spectrum soil fumigants like methyl bromide were the first
choice for management of soil inhabiting pests, such as nematodes, before planting
(Noling and Becker, 1994). Carbamates and organophosphates were also used in
control programs (Hough and Thomason 1975; McLeod and Khair, 1975). Pesticides
with low toxicity to mammals and the environment are taking the place of the acutely
toxic and effective pesticides of years past. Chemical companies are developing new
active ingredients and formulations to fill the void vacated by products not surviving EPA
re-registration. Abamectin is known for having insecticidal, acaracidal, and nematicidal
properties (Lasota and Dybas, 1989). In agricultural settings, it is used to manage
insect, mite, and nematode pests of plants. Abamectin is also used for removing
nematode parasites in animals and people. It is composed of macrocyclic lactone
isomers avermectin B1a and B1b. The avermectins were first discovered from the
actinomycete Streptomyces avermitilis in Japan (Crump and Omura, 2011). The
abamectin nematicide product Avid 0.15 EC (Syngenta Crop Protection, Research
Triangle Park, NC) was granted a Section 24(c) label for golf courses in Florida (Foy,
2014). In May 2016, abamectin was approved for nationwide use on turfgrass by the
EPA (U.S. EPA, 2016). Divanem is an abamectin formulation that was released in 2017
by Syngenta Crop Protection (Research Triangle Park, NC) labeled for use on golf
course turfgrass (GreenCast, 2017). This product has demonstrated excellent control of
root-knot nematodes (M. graminis). Abamectin tends to bind to thatch in turfgrass which
is beneficial for reaching root-knot nematodes that are found in thatch, but less effective
against ectoparasites that live deeper in the soil (Crow, 2014).
28
Fluopyram is classified as a succinate dehydrogenase inhibitor in the Fungicide
Resistance Action Committee classification. It is used to manage common fungal
diseases like gray mold, powdery mildew, and Sclerotinia sclerotiorum. It is also being
studied for effects on nematodes such as Meloidogyne incognita and Rotylenchulus
reniformis (Faske and Hurd, 2015). A study by Faske and Hurd (2015) suggests
fluopyram causes nematode paralysis. Nematodes are unable to move and initiate a
feeding site in the plant root. Fluopyram was found to target the succinate
dehydrogenase gene sdh1 in nematodes, the same target site in fungi (Heiken, 2017).
Bayer Cropscience (Research Triangle Park, NC) developed a fluopyram nematicide
under the trade name Indemnify, which has been labeled for use on turfgrass.
Furfural is another active ingredient that can be used for nematode management.
The first large-scale production of furfural occurred in 1921 when Quaker Oats
Company (Chicago, IL) mill in Cedar Rapids developed a method for making furfural
from cereal waste products (Brownlee and Miner, 1948). Oat hulls were combined with
acid in large pressure cookers. Pentosane polysaccharides such as xylose undergo
hydrolysis to form furfural. Today, furfural has several applications. In chemistry, it is
used to remove aromatic compounds to improve viscosity and ignition of petroleum oils
and drying capacity of unsaturated vegetable oils. In biological systems, it has fungicidal
and nematicidal properties (Rodriguez-Kabana et al., 1993). Illovo Sugar has developed
a furfural-based nematicide from sugarcane bagasse in South Africa. In the United
States, the product is labeled as MultiGuard Protect (Agriguard, Cranford, NJ). Furfural
has contact activity on nematodes by acting on the cuticle to cause death (Fourie et al.,
2017). The advantages of furfural include low mammalian toxicity, rapid degradation by
29
microbes, and relatively low cost to produce. However, it is not effective against
nematodes in the top 5 cm of turfgrass soil profile (Crow and Luc, 2014).
Fluensulfone is a new class of chemistry with nematicidal properties. The mode
of action is not fully understood, but it affects movement, feeding, development, and egg
laying in Caenorhabditis elegans (Kearn et al., 2014). Acetylcholinesterase activity is
thought to be inhibited by fluensulfone (Oka et al., 2013). Reduction in M. javanica
infectivity has been documented when juveniles were exposed to fluensulfone (Oka et
al., 2013). Systemic properties of fluensulfone have also been documented (Oka et al.,
2012). A granular formulation of fluensulfone for turfgrass use was launched in 2016
under the trade name Nimitz Pro G (Quali-Pro, Pasadena, TX). The product has low
mammalian and insect toxicity and limited persistence.
Related studies
Other researchers have studied chemical effects on non-target soil organisms in
cropping systems. However, at the time of writing, no other studies have examined
effects of fluopyram, furfural, or fluensulfone on free-living organisms. Avermectins have
been studied in agricultural land treated with avermectin pesticides and feces from
cattle treated with ivermectin for animal-parasitic nematodes (Yeates et al., 2003; Dong
et al., 2008). These studies found avermectins to decrease free-living nematode
abundance and the Dong et al. (2008) study observed a moderate disturbance based
on Maturity Indices, plant parasitic index, fungivore/bacterivore abundance, basal index
and channel index.
Carrascosa et al. (2014) found significant changes in community structure of soil
fumigated with 1,3-dichloropropene and chloropicrin in a strawberry farm. Nematode
abundance increased during the cropping season and tardigrades were nearly absent in
30
fumigated fields. Nematode communities shifted toward a disturbed community
structure. Plant-parasitic index, channel index, basal index, and structure index
decreased after fumigation. Total nematode abundance was variable and higher trophic
nematodes were more abundant in untreated soil. In an experimental insecticide study,
Čerevková et al. (2017) found maize fields treated with granular insecticide treatments
tefluthrin and clothianidin, and seed treatment of clothianidin significantly influenced
nematode abundance, trophic structure, maturity index, and structure index. However,
repeated ANOVA analysis revealed environmental factors between sampling years had
a more profound effect on the population variables than the insecticides. In 2011 the
untreated control plots had greater abundance of nematodes and in 2013 the untreated
plots did not have the greatest abundance. Untreated controls however had the greatest
number of species in 2011 and 2013. Tefluthrin with decomposing non-Bt corn was also
found to reduce abundance of nematodes at higher trophic levels and reduce the soil
food web structure by Neher et al. (2014). The insecticide-treated areas showed a shift
in community structure when compared with decomposing non-Bt corn and Bt corn.
These studies suggest chemical insecticide treatments can have a significant effect on
nematode community structure, but environmental conditions between years plays an
important role as well.
A study by Gan and Wickings (2017) conducted in New York found varying
effects on mites and collembola exposed to fungicides chlorothalonil and propoconazole
and insecticide imidacloprid. Microarthropod community level analyses was performed
on samples taken from golf courses with long-term pesticide management with high,
moderate, or low pesticide rate usage in addition to a short-term rate usage experiment.
31
The researchers found that microarthropod communities were affected differently
depending on the active ingredient, frequency of application, and timing of pesticide
application. High rate and long-term pesticide usage had the greatest impact on
microarthropods. Mites were sensitive to the three pesticides studied and oribatid mites
were the mites most susceptible to pesticides. Large mites (> 2 mm) were present in
low abundance and not greatly affected by pesticides. Small (< 2 mm) decomposer
mites were most susceptible to pesticides. Pesticides generally did not affect springtails,
but rapid recovered occurred after a significant decline. The findings suggest pesticide
management practices can negatively affect microarthropods, but functional groups are
affected differently depending on active ingredient, frequency of application, and timing
of pesticide application.
Cheng et al. (2008) found no significant effects from insecticide, herbicide, and
fungicide treatments on the number of free-living and plant-parasitic nematodes or on
species richness and diversity measures in turfgrass. Nitrogen input encouraged
microbial growth, but nematodes were not significantly affected by an increase in
microbial growth. The high intensity management practices in turfgrass systems were
shown to maintain an enriched and moderately disturbed soil environment. The authors
suggested turfgrass soil ecosystems are relatively resistant to effects of chemical
pesticide treatments due to the high microbial activity, thatch, and organic content of the
turfgrass environment. Microbial activity greatly aids in the breakdown of pesticide
molecules. Thatch and organic matter can serve as a binding site for pesticides and
reduce their ability to contact the target pest (Horst et al., 1996).
32
A study by Yardirn and Edwards (1998) studied the effect of insecticides,
herbicides, and fungicides on nematode populations in tomato agrosystems. Carbaryl,
endosulfan, esfenvalerate, chlorothalonil, trifluralin, and paraquat were used. Generally,
bacterivore nematodes were the most dominant group in the treated plots. In the
fungicide and herbicide treated plots the bacterivore nematodes were significantly
reduced. The authors reported this observation was supported by the work of Ingham et
al. (1986) but in conflict with the observations by Mahn and Kästner (1985) and Schmitt
and Corbin (1981) which saw an increase of bacterivores when exposed to herbicides.
Fungivorous nematodes were decreased in fungicide and herbicide treated plots. Plant-
parasitic nematodes increased in insecticide, herbicide, and fungicide combination plots
and insecticide only treated plots. The authors suggested several reasons for an
increase in plant-parasitic nematode abundance. Some pesticides may stimulate egg
hatching. It is also possible the host plant could be weakened by herbicides and made
more susceptible to nematode parasitism, but this hypothesis can be challenging to
confirm (Abivardi and Altman, 1978). Altered soil pH could also affect nematodes and
their food sources. Reductions of natural predators in the form of entomophagous fungi,
microarthropods, and predatory nematodes could also contribute (Small, 1979; Mankau,
1980; Imbriani and Mankau, 1983; Pullen et al., 1990). Fungicides and insecticides may
directly reduce numbers of predatory organisms that naturally keep plant-parasitic
nematode populations in check.
Neher et al. (2014) studied potential effects of Cry3Bb1 endotoxins on nematode
communities from decomposing Bt corn. Nematodes were not significantly affected by
the presence of decomposing Bt corn. The maturity index and structure index were
33
similar to the non-Bt control. The enrichment index was lower for the Bt treatment than
the non-Bt control. For Bt corn treatments, nematode communities were located in the C
quadrant of the Faunal Profile suggesting an undisturbed and moderately enriched
environment. From previous studies, microbial communities were minimally affected by
Cry3Bb1 endotoxins (Lawhorn et al., 2009). These studies suggest highly specific
pesticides can provide minimal non-target ecological effects even in the presence of
potentially high quantities of Cry proteins.
Stevenson et al. (2002) evaluated the soil invertebrate community structure in a
pesticide-free corn field and a conventional pesticide corn field in Illinois. The pesticide-
free field had significantly greater invertebrate population densities and was dominated
by springtails, whereas the conventional field was dominated by mites. Seasonal
variability in invertebrate densities was observed and was more unstable in the
pesticide-free field. The greatest diversity and abundance of invertebrates occurred in
the pesticide-free field, which broadly suggests a healthier soil ecosystem.
The herbicide acetochlor and the nematicide carbofuran have been shown to
alter nematode community structure in soybean fields when used independently and in
combination (Zhang et al., 2010). Bacterivore numbers were suppressed by the
pesticides, but fungivores, omnivores, and predators were not significantly affected. The
basal index increased while the structure index decreased, indicating a decline in soil
health. Acrobeloides spp. numbers increased in some of the pesticide treated samples.
Some low value cp bacterivorous nematodes such as Acrobeloides spp. appear to be
relatively insensitive to agrochemicals and can actually rebound more quickly due to the
increased availability of decaying organisms.
34
Imidacloprid was found to suppress target pest populations, but also some non-
target hexapod groups over six years in turfgrass (Peck, 2009). Members of Coleoptera,
Hemiptera, Collembola, and Carabidae were suppressed by ≥ 50%. Surface inhabitants
showed no population density reduction, but soil dwelling hexapods may have been
directly affected by imidacloprid toxicity and indirectly affected by reduction in hexapod
prey. Chlorpyrifos was found to alter the structure of nematode and arthropod
communities in turfgrass (Wang et al., 2001). All insect guilds in the study were affected
and some nematode guilds were affected by chlorpyrifos. Species diversity was
relatively consistent across study years, but abundances varied greatly. Environmental
factors may have played a role, as rainfall was much more abundant in the first year of
the study. Abundance of insect herbivores, thatch detritovores, soil predators, soil
herbivores, and free-living nematodes were all significantly reduced by chlorpyrifos.
Numbers of mites were significantly reduced, and springtail family Smithuridae
significantly increased by chlorpyrifos, but the dominant Isotomidae family was not
significantly affected.
35
Table 1-1. Summary of nematode ecological indices.
Calculation Symbol Calculation High value indication
Maturity index MI average cp value excluding plant-feeders and dauerlarvae
stable/enriched environment
Maturity index Cp 2-5
MI25 same as MI, but also excludes cp 1 nematodes
stable environment
∑ Maturity index ∑MI total MI of all nematodes present
stable environment with plant food sources
Enrichment index EI ratio of enrichment opportunists
enriched environment
Structure index SI ratio of non-opportunistic nematodes to all nematodes
high food web complexity
Basal index BI ratio of fungivores and bacterivores to the other major feeding groups
stressed or degraded environment
Channel index CI ratio of fungivores to bacterivores
fungal dominated decomposition channel
Plant parasitic index
PPI sum of weighted proportions of PPN
environment lacking enrichment
36
Figure 1-1. Faunal Profile visual depiction as described in Ferris et al., 2001. Structure index values on the x-axis are plotted against enrichment index values on the y-axis. The quadrat that the intersection point of corresponding SI and EIvalues calculated from an individual sample is the faunal profile assessment.Environmental condition is estimated by location of data points across fourquadrats. Points located in Quadrat A have characteristics of an enrichedenvironment with low-moderate structure. Quadrat B reflects a structured andenriched environment. Quadrat C is a high structure and low-moderateenrichment environment. Quadrat D is a basal environment with low-moderate levels of structure and enrichment.
37
CHAPTER 2 MATERIALS AND METHODS
Study site. Studies were conducted at the Plant Science Research Unit (PSU) in
Citra, FL. The study field was planted with ‘Tifdwarf’ bermudagrass and maintained with
common turfgrass management practices by the staff at PSU. The only chemicals used
for maintenance were fertilizer, plant growth regulator, and herbicides. Plots were
treated with Tribute (thiencarbazone-methyl, foramsulfuron, and halosulfuron-methyl),
Dismiss (sulfentrazone), and Primo (trinexapac-ethyl) for weed control and turf
management at labeled rates. Plots were fertilized with Harrell’s 13-4-13 controlled
release golf course green fertilizer during the growing season. Soil texture was
comprised of 97% sand, 2% clay, and 1% silt. Organic matter was 4% and pH was 7.1.
Treatment applications. The experiment used a randomized-block design with
five treatments and five replicates. In addition to an untreated control, the experimental
treatments used were: Divanem (abamectin), MultiGuard EC (furfural), Indemnify
(fluopyram), and Nimitz Pro G (fluensulfone). Rates were based on the maximum
allowable rate as listed on each label (Table 2-1).
Applications of liquid treatments were made using a CO2-powered backpack
sprayer (Weed Systems, Hawthorne, FL) with TJ-08 nozzles delivering 1,222 liters
solution/ha. Nimitz Pro G was applied using a walk-behind Gandy (Owatonna, MN)
drop-spreader. Plots were 6 m2 with 1.5 m2 data collection plots located in the center of
each larger treatment plot to minimize any cross contamination between plots. All plots
were separated by an untreated 0.6 m border on each side. After each application, all
treated and untreated plots were immediately irrigated with 0.64 cm of water.
38
Treatments were applied every four weeks replicating a summer treatment program
from 7 June to 30 August in 2016 and 24 April to 18 July in 2017.
Sampling. Samples were collected prior to the initial treatment, and 2 days, 14
days, 56 days, and 238 days after the final treatment application (DAFT) each year.
Plugs were collected using a 3.81-cm-diam. ball mark plugger (Turf-Tec International,
Tallahassee, FL) to a depth of 6.35 cm. Eight plugs were collected from the data
collection subplots and combined for analysis. The soil was separated from the thatch
and roots plugs, and nematodes and other invertebrates were extracted from 100 cm3
of this soil by centrifugal-flotation (Jenkins, 1964). Of the eight thatch and root plugs,
four were used for extraction of nematodes and other invertebrates using mist extraction
(Seinhorst, 1950). The misting chamber was a rectangular plexiglass structure that
contains misting nozzles attached to the top of a chamber made. Nozzles were spaced
40 cm lengthwise and 30 cm widthwise along the length of the chamber. Funnels were
placed 68 cm below nozzles in holes cut in a sheet of plexiglass to support each funnel.
A mesh screen was placed on top of the funnel to support the turfgrass plugs. Mesh
holes were 2 mm x 1 mm. Mist was sprayed on the samples for 45 sec every hour
controlled by a solenoid valve (Hunter Industries, San Marcos, California) set on a
recycling timer (Hydrofarm, Petaluma, California). Samples were collected in an
Erlenmyer flask below each funnel. Turfgrass plugs were left in the misting chamber for
72 hr and the collected specimens were preserved in 2% formalin and stored in plastic
centrifuge tubes. The remaining four plugs were used for Berlese funnel extraction of
invertebrates (Berlese 1905). Berlese samples were left for 120 hr in a growth chamber
with 400 watt bulbs. Distance between turfgrass plug and light bulb was 61 cm to
39
expose turfgrass to 35°C from the light source to slow the rate of drying. Specimens
were collected and preserved in 100% ethanol and stored in screw top vials.
Nematodes and rotifers were identified morphologically and counted from soil and mist
extraction samples using an inverted microscope (Olympus Corporation, Shinjuku,
Tokyo, Japan). The primary guides used for nematode identification were Smart and
Nguyen (1985) and Bongers (1988). Rotifers were identified with Shiel (1995) keys.
Mites, springtails, and insects were identified from Berlese extracted samples with a
dissecting microscope (Olympus Corporation, Shinjuku, Tokyo, Japan). Voucher mite
specimens were identified using a compound phase microscope at 100× magnification.
Arthropods were identified using Stehr (1987); Dindal (1990); Christiansen and Bellinger
(1998); and Kranz and Walter (2009) guides.
Data collection. Data plots were photographed every two weeks using a digital
camera mounted on a custom-built photobox throughout the growing period and
continued until grass dormancy in the winter. Digital images were taken in center of data
plots to be analyzed for the number of green pixels (hue 45 to 105, saturation 15 to 100)
present in each image as a measure of turfgrass health using the macro developed by
Karcher and Richardson (2005). Nematodes collected by mist and soil extraction were
counted in gridded counting dishes using inverted microscopes. One-hundred
nematodes were identified from each sample, or the entire sample if the total number
was fewer, to the family level. Due to challenges of mite identification, mites
representative of 1% or greater of all mites counted were identified to family level. All
other arthropods were identified to family level using a dissecting scope at 40×.
40
Statistical analysis. Population counts were analyzed using analysis of
covariance (ANCOVA) using R software version 3.3.2 (R Core Team, 2016). Data were
log transformed to improve normality and homogeneity of variance. Population means
of the different sampling dates were compared to the initial sample means using the
untreated control as a covariate. ANCOVA was chosen to help account for natural
seasonable variation. Nematode families were grouped into the feeding groups
proposed by Yeates et al. (1993). Groups included were bacterivores, fungivores, plant-
parasites, omnivores, and predators. The relative abundance of each feeding group
were evaluated throughout the course of the two-year study. Data generated from both
extraction methods were compared using a t-test at each sampling date to determine if
extraction method had a significant effect on the number of nematodes extracted.
Additional analyses of ecological indices were performed by the Nematode Indicator
Joint Analysis software developed by Sieriebriennikov et al. (2014).The nematode
ecological indices used in this study were: maturity index, maturity index cp-2-5, sigma
maturity index, plant-parasitic index, channel index, basal index, enrichment index,
structure index, and faunal profile based on the works of Bongers (1990); Ferris et al.
(2001); Ferris and Bongers (2009a). Faunal profile was different from the untreated
control if both enrichment index and structure index were determined to be different
from the untreated control using the analysis of covariance.
41
Table 2-1. Nematicide formulations used in the field study and their per-application labeled application rates EC = Emulsifiable Concentrate and G = granular.
Active Ingredient (a.i.)
Trade Name Application rate Formulation
Abamectin Divanem 0.89 liters product/ha (70g a.i./ha) EC
Fluopyram Indemnify 1.25 liters product/ha (500 g a.i./ha)
EC
Furfural MultiGuard Protect
56 liters product/ha (60 kg a.i./ha) 2016*
74 liters product/ha (77 kg a.i./ha) 2017
EC
Fluensulfone Nimitiz Pro G 62.25 kg product/ha (1 kg a.i./ha) G
* Labeled rate changed between 2016 and 2017
42
CHAPTER 3 NEMATODE RESULTS
Four-hundred samples were collected and processed over the two-year study.
Two-hundred samples were processed with sugar flotation and centrifugation extraction
and two-hundred were subjected to mist chamber extraction. Data from three samples
were excluded due to processing error. The combined number of nematodes identified
in all samples was 60,362 out of a total of 199,611 nematodes.
Functional Groups and Ecological Indices
Twenty-four nematode families were identified during the study. Of the families
encountered, seven families were categorized as plant-parasitic and fifteen were
categorized free-living. Nine of the families were bacterial feeding, three were fungal
feeding, one was omnivorous, and five were predatory (Table 3-1). The bacterial
feeding family Cephalobidae was the dominant family representing 30% of all
nematodes across both extraction processes. The dominant fungal feeding family was
Tylenchidae making up 18% of nematodes. Hoplolaimidae was the most abundant
plant-parasitic group at 15% of nematodes and the omnivore family Aporcelaimidae was
fourth most abundant at 13%. Data collected from each sampling method are presented
separately since a significant effect of extraction method on nematode counts was
observed (P < 0.05).
Bacterivores
Bacterivores in abamectin treated plots increased at 238 days after final
treatment (DAFT) relative to the untreated control (P < 0.1) in 2016 from mist extraction
(ME) (Fig. 3-1). Abamectin reduced bacterivore abundance at 2 DAFT from ME and 56
DAFT from soil extraction (SE) relative to the untreated control (P < 0.1) in 2017 (Fig 3-
43
1). Fluopyram decreased bacterivores relative to the untreated control at 2 DAFT and
238 DAFT in 2016 (P < 0.1) (Fig 3-1). Fluopyram reduced bacterivore abundance at 2,
56, and 238 DAFT in 2016 from SE (Fig. 3-1). In 2016, fungivores from SE increased
relative to the untreated control at 238 DAFT (P < 0.1) (Fig. 3-1), but decreased 238
DAFT relative to the untreated control from SE in 2017 (P < 0.05) (Fig. 3-1). Numbers of
bacterivores were reduced (P < 0.1) by fluopyram 2 DAFT, 56 DAFT, and 238 DAFT
relative to the untreated control from SE in 2017 (Fig. 3-1).
Fungivores
Abamectin treated plots had reductions in fungivores (P < 0.1) at 56 DAFT
relative to the untreated control in 2016 from ME and SE (Fig. 3-2). Abamectin reduced
fungivore abundance 238 DAFT from ME in 2017 (Fig. 3-2). Fluopyram reduced
fungivores (P < 0.1) relative to the untreated control at 2, 14 , 56 , and 238 DAFT in
2016 from ME (Fig 3-2). Fungivore abundance decreased in fluopyram treated plots at 2
and 56 DAFT relative to the untreated control (P < 0.1) in 2016 from SE (Fig. 3-2).
Fluopyram reduced fungivore abundance relative to the untreated control 238 DAFT
from SE in 2017 (Fig. 3-2). Fluensulfone treated plots had fewer fungivores relative to
the untreated control (P < 0.1) 56 DAFT from SE in 2016 (Fig. 3-2).
Omnivores
Abamectin decreased omnivores relative to the untreated control (P < 0.1) 56
DAFT from ME in 2016 (Fig. 3-3). Fluopyram reduced omnivore population densities
relative to the untreated control (P < 0.05) at 2, 14, 56, and 238 DAFT in 2016 from ME
and SE (Fig. 3-3). Omnivore abundance was also lower relative to the untreated control
(P < 0.05) at 2, 14, 56, and 238 DAFT from ME and SE in 2017 (Fig. 3-3).
44
Predators
Abamectin reduced predator abundance relative to the untreated control (P <
0.01) at 14 and 238 DAFT from ME in 2017 (Fig. 3-4). Fluopyram treated plots had
greater predator abundance relative to the untreated control at 56 DAFT from ME (P <
0.01) and decreased population densities relative to the untreated control (P < 0.01) at
238 DAFT from ME and SE in 2016 (Fig 3-4). Fluopyram decreased predators relative
to the untreated control (P < 0.05) at 2, 14, and 56 DAFT after final application from ME
in 2017 (Fig. 3-4). Furfural applications decreased predator abundance relative to the
untreated control (P < 0.1) at 238 DAFT after the final treatment from ME in 2016 and
increased abundance relative to the untreated control (P < 0.1) 56 DAFT after final
application from SE in 2017 (Fig 3-4). Fluensulfone increased predator abundance
relative to the untreated control (P < 0.05) 56 DAFT from ME in 2016 and from SE in
2017 (P < 0.05) (Fig 3-4).
Plant-parasitic
Fluopyram lowered plant-parasitic nematode population densities relative to the
untreated control (P < 0.1) at 2, 14, and 238 DAFT from ME in 2016 and 14 DAFT
relative to the untreated control (P < 0.05) from ME in 2017 (Fig. 3-5). An increase in
plant-parasitic nematode abundance was observed in fluopyram treated plots relative to
the untreated control (P < 0.01) from SE at 14 DAFT in 2017 (Fig. 3-5). Furfural
increased plant-parasitic nematode abundance relative to the untreated control 14
DAFT from ME in 2016 (P < 0.05) and from SE in 2017 (P < 0.1) (Fig. 3-5).
Fluensulfone applications decreased plant-parasitic nematode abundance relative to the
untreated control (P < 0.1) at 2 DAFT from SE in 2016 (Fig. 3-5).
45
Functional Groups Summary
Low cp nematodes generally did not increase after nematicide application
disturbance. Furfural and fluensulfone had low impacts on r strategist nematodes.
Abamectin plots had bacterivore increases relative to the untreated control at one day in
each year (P < 0.1) (Figs. 3-1, 3-3, and 3-4) and fluopyram applications at one date in
2016 (P < 0.01) (Fig. 3-1) possibly indicating an opportunistic response to disturbance.
The only reductions of bacterivore nematodes relative to the untreated control were
seen in plots treated by fluopyram both years (P < 0.1) (Fig. 3-1). Fungivores were
susceptible to abamectin, fluopyram, and fluensulfone (P < 0.1) (Fig. 3-2). Increased
abundance relative to the untreated control occurred at the final sampling day in
fluopyram plots in 2016 (P < 0.01) (Fig 3-2). Furfural and fluensulfone had minimal
impacts on low cp nematodes and may be a lower risk to r strategist nematodes. High
cp nematodes were mildly affected by abamectin, furfural, and fluensulfone and greatly
affected by fluopyram. Omnivores were reduced by abamectin and fluopyram
applications (P < 0.1) (Fig. 3-3). Fluopyram reduced omnivore abundance relative to the
untreated control at all sampling dates during both years. Predators were affected by
abamectin, fluopyram, furfural, and fluensulfone (P < 0.1). Abamectin, fluensulfone, and
furfural reduced predator abundance and fluopyram and fluensulfone increased
abundance relative to the untreated control at one sampling date in each year (Fig. 3-4).
Abamectin, furfural, and fluensulfone nematicides had a much lower impact on both K
strategist nematodes than fluopyram. Plant-parasitic nematodes were affected by
fluopyram, furfural, and fluensulfone (P < 0.1). Fluopyram applications increased
abundance relative to the untreated control at one date each year and furfural and
46
fluensulfone applications decreased plant-parasitic nematode abundance relative to the
untreated control both years (Fig. 3-4).
MI
Abamectin lowered MI relative to the untreated control (P < 0.1) at 14 DAFT from
SE and 238 DAFT from ME in 2016 (Fig. 3-5). Fluopyram reduced MI relative to the
untreated control (P < 0.05) at 14, 56, and 238 DAFT in from SE 2016 and at 14 and
238 DAFT from ME in 2016 (Fig. 3-5). Fluopyram reduced MI at 14 and 56 DAFT
relative to the untreated control (P < 0.1) from ME and at 14 DAFT from SE in 2017.
Furfural increased MI relative to the untreated control (P < 0.1) at 14 DAFT and
decreased relative to the untreated control at 238 DAFT from ME in 2016 (Fig. 3-5).
Fluensulfone decreased MI relative to the untreated control (P < 0.1) at 2 DAFT from SE
in 2016 and at 2 and 56 DAFT relative to the untreated control (P < 0.05) from SE in
2017 (Fig. 3-5).
MI25
Abamectin application reduced MI25 relative to the untreated control (P < 0.05)
at 238 DAFT from ME in 2016 (Fig. 3-6). Fluopyram reduced MI25 relative to the
untreated control (P < 0.05) at 14 and 238 DAFT from ME and 14 DAFT and 56 DAFT
from SE in 2016 (Fig. 3-6). Fluopyram reduced MI25 relative to the untreated control (P
< 0.05) at 56 DAFT after the final application from ME in 2017 (Fig 3-5). Fufural
increased MI25 relative to the untreated control (P < 0.1) at 14 DAFT and decreased
relative to the untreated control (P < 0.1) at 238 DAFT from ME in 2016 (Fig. 3-5).
Fluensulfone had greater MI25 relative to the untreated control (P < 0.1) at 2 and 56
DAFT from SE in 2017 (Fig. 3-5).
47
∑MI
Abamectin caused ∑MI reduction relative to the untreated control (P < 0.05) at
238 DAFT from ME in 2016 and increase relative to the untreated control (P < 0.1) at 14
DAFT from SE in 2017 (Fig. 3-7). Fluopyram lowered ∑MI relative to the untreated
control (P < 0.01) at 56 DAFT from SE and increased ∑MI relative to the untreated
control (P < 0.1) at 238 DAFT from SE in 2016 (Fig. 3-7). Fluopyram reduced ∑MI
relative to the untreated control at thirty-four WAT from ME 2016 (Fig. 3-7). In 2017,
∑MI was greater in fluopyram plots relative to the untreated control (P < 0.05) at 14 and
238 DAFT from SE (Fig. 3-7). Furfural increased ∑MI relative to the untreated control (P
< 0.1) at 14 DAFT and reduced ∑MI relative to the untreated control (P < 0.1) at 238
DAFT from ME in 2016 (Fig. 3-7). Fluensulfone increased ∑MI relative to the untreated
control (P < 0.1) at 14 DAFT after the final application from SE in 2017 (Fig. 3-7).
PPI
Abamectin increased PPI relative to the untreated control (P < 0.05) at 238 DAFT
from ME in 2017 (Fig. 3-8). Fluopyram increased PPI relative to the untreated control (P
< 0.1) at 2 DAFT in ME and SE and decreased PPI relative to the untreated control (P <
0.1) at 238 DAFT from ME in 2016 (Fig. 3-8). In 2017, fluopyram caused a PPI increase
relative to the untreated control (P < 0.01) at 238 DAFT from SE (Fig. 3-8).
CI
Abamectin applications reduced CI relative to the untreated control (P < 0.01) at
14 DAFT from SE in 2016 (Fig. 3-9). Significant CI reduction from furfural applications
relative to the untreated control (P < 0.01) was observed at 56 DAFT from ME in 2016
(Fig. 3-9). Fluensulfone reduced CI relative to the untreated control (P < 0.1) at 238
DAFT from SE in 2016 (Fig. 3-9). CI could not accurately be calculated in 2017 due to
48
numerous zero Ba1 bacterial and Fu2 fungal feeding nematode counts that resulted in
non-numeric CI values from by dividing by zero (data not shown).
BI
Abamectin increased BI relative to the untreated control (P < 0.05) at 238 DAFT
from ME in 2016 (Fig. 3-10). Fluopyram increased BI relative to the untreated control (P
< 0.01) at 14 DAFT from ME and SE, but decreased BI relative to the untreated control
(P < 0.01) at 238 DAFT from SE in 2016 (Fig. 3-10). Furfural decreased BI relative to
the untreated control (P < 0.05) at 14 DAFT the final application and increased BI
relative to the untreated control (P < 0.1) 238 DAFT from ME in 2016 (Fig. 3-10).
EI
Abamectin decreased EI relative to the untreated control (P < 0.05) from ME at
14 DAFT in 2016 (Fig. 3-11). EI increased in abamectin plots relative to the untreated
control (P < 0.1) from SE at 14 DAFT and decreased 56 DAFT in 2016 (Fig. 3-11).
Abamectin treated plots had lower EI relative to the untreated control (P < 0.01) at 238
DAFT from ME in 2017 (Fig. 3-11). Fluopyram increased EI relative to the untreated
control (P < 0.1) at 2, 14, 56, and 238 DAFT from ME and SE in 2016 (Fig. 3-11).
Furfural caused an EI increase relative to the untreated control (P < 0.1) at 56 DAFT
and a decrease relative to the untreated control (P < 0.05) 238 DAFT from ME in 2017
(Fig. 3-11). Fluensulfone reduced EI relative to the untreated control (P < 0.05) at 56
DAFT from SE in 2016 (Fig. 3-8) and 238 DAFT from ME in 2017 (Fig. 3-10).
SI
Abamectin decreased SI relative to the untreated control (P < 0.1) at 238 DAFT
from ME in 2016 (Fig. 3-12). Fluopyram lowered SI relative to the untreated control (P <
0.1) at 2, 14, 56 and DAFT from ME and at 2, 14, and 56 DAFT from SE in 2016 (Fig. 3-
49
12). Furfural increased SI relative to the untreated control (P < 0.05) at 14 DAFT from
ME in 2016 (Fig. 3-12). Fluensulfone increased SI relative to the untreated control (P <
0.1) at 14 and 56 DAFT from SE in 2017 (Fig. 3-12).
Faunal Profile
Abamectin. EI and SI were not different from the untreated control at the same
sampling date in abamectin plots during 2016 or 2017 (P > 0.1). Data points in
abamectin plots prior to the first treatment were clustered in quadrat C at the beginning
of the study from ME and SE (Fig 3-13. and Fig. 3-14). ME 2016 data points were
located in quadrats C and D at 2 DAFT, quadrats C and D at 14 DAFT, quadrat C at 56
DAFT, and quadrats C and D at 238 DAFT (Fig. 3-13). SE 2016 data points were
located in quadrats C and D at 2 DAFT, quadrats A, C, and D at 14 DAFT, quadrats C
and D at 56 DAFT, and quadrats C and D at 238 DAFT (Fig. 3-14). ME 2017 data points
were in quadrat C and D at 2 DAFT, quadrats C and D at 14 DAFT, quadrats C and D at
56 DAFT, and quadrats C and D at 238 DAFT (Fig. 3-15). SE 2017 data points were
located in quadrats C and D at 2 DAFT, quadrats C and D at 14 DAFT, quadrats C and
D at 56 DAFT, and quadrats C and D at 238 DAFT (Fig 3-16). There was consistently
low enrichment in abamectin plots with values below 50 in 2016 and 2017 except for
one point at 14 DAFT from SE in 2016.
Fluopyram. EI and SI were different from the untreated control at all four
sampling dates for ME (P < 0.1) and for 2 DAFT, 14 DAFT, and 56 DAFT for SE in 2016
(P < 0.1). No differences from the untreated control occurred in 2017 (P > 0.1). Data
points in fluopyram plots were primarily clustered in quadrat C with one point in quadrat
B from ME prior to the first treatment at the beginning of the study. After fluopyram
applications, data points began shifting toward A and D quadrats in 2016 from ME and
50
SE (Fig. 3-14 and Fig. 3-15). ME 2016 data points were located in all four quadrats at 2
DAFT, quadrat D at 2 DAFT, quadrats C and D at 56 DAFT, and quadrats A and D at
238 DAFT (Fig. 3-14). SE 2016 data points were located in quadrats A and D at 2
DAFT, quadrat D at 14 DAFT, quadrat A at 56 DAFT, and quadrats A, B, and D at 238
DAFT (Fig 3-15). ME 2017 data points were in quadrat D at 2 DAFT, quadrats A and D
at 14 DAFT, quadrats A and D at 56 DAFT, and quadrats C and D at 238 DAFT (Fig.3-
15). SE 2017 data points were located in quadrats A, C, and D at 2 DAFT, quadrats A,
C, and D at 2 DAFT, quadrats A and D at 56 DAFT, and quadrat D at 238 DAFT (Fig 3-
16).
Furfural. EI and SI were not different from the untreated control at the same
sampling date in furfural plots during 2016 or 2017 (P > 0.1). Data points in furfural plots
were primarily clustered in quadrat C prior to the first treatment at the beginning of the
study (Fig. 3-13 and Fig. 3-14). ME 2016 data points were located in quadrats B and C
at 2 DAFT, quadrat C at 14 DAFT, quadrats C and D at 56 DAFT, and quadrats C and
D at 238 DAFT (Fig. 3-13). SE 2016 data points were located in quadrats C and D at 2
DAFT, quadrats C and D at 14 DAFT, quadrats C and D at 56 DAFT, and quadrat D at
238 DAFT (Fig 3-14). ME 2017 data points were in quadrat C and D at 2 DAFT,
quadrats C and D at 14 DAFT, quadrats C and D at 56 DAFT, and quadrats C and D at
238 DAFT (Fig. 3-15). SE 2017 data points were located in quadrats C and D at 2
DAFT, quadrats C and D at 14 DAFT, quadrats C and D at 56 DAFT, and quadrat D at
238 DAFT (Fig 3-16). Enrichment was lower than 50 at all sampling dates except at 2
DAFT after the final treatment from ME in 2016 and 56 DAFT from ME after the final
treatment in 2017.
51
Fluensulfone. EI and SI were not different from the untreated control at the
same sampling date in fluensulfone during 2016 or 2017 (P > 0.1). Data points in
fluensulfone plots were primarily clustered in quadrat C prior to the first treatment at the
beginning of the study (Fig. 3-13 and Fig. 3-14). ME 2016 data points were located in
quadrat C at 2 DAFT, quadrat C at 14 DAFT, quadrats C at 56 DAFT, and quadrats C
and D at 238 DAFT (Fig. 3-13). SE 2016 data points were located in quadrats C and D
at 2 DAFT, quadrats C and D at 14 DAFT, quadrats C and D at 56 DAFT, and quadrat
D at 238 DAFT (Fig 3-14). ME 2017 data points were in quadrat C and D at 2 DAFT,
quadrats C and D at 14 DAFT, quadrats C and D at 56 DAFT, and quadrats C and D at
238 DAFT (Fig. 3-15). SE 2017 data points were located in quadrats C and D at 2
DAFT, quadrats C and D at 14 DAFT, quadrats B, C, and D at 56 DAFT, and quadrat D
at 238 DAFT (Fig 3-16).Enrichment exceeded 50 at only one sampling date (56 DAFT
from SE in 2017) indicating low enrichment.
Ecological Indices Summary
All functional groups analyzed were significantly affected by nematicide
applications. MI was affected by all four nematicides with fluopyram reducing MI at the
more dates than any other single nematicide (P < 0.05). MI25 was affected by all four
nematicides similarly to MI and fluopyram affected MI25 at the most sampling dates.
The four nematicides affected ∑MI with reductions typically occurring in 2016 and
increases in 2017 (P < 0.1). PPI was affected by abamectin and fluopyram (P < 0.1).
Significant increases occurred both years (Figs. 3-7, 3-7, 3-9, and 3-11). Channel index
was affected by abamectin, furfural, and fluensulfone. CI decreases were observed from
these nematicide applications (P < 0.1). BI was affected by abamectin, fluopyram, and
furfural. BI was increased and decreased during the season in 2016 (P < 0.1). EI was
52
significantly affected by all four nematicides. Fluopyram consistently had greater EI
values than the untreated in 2016 (P < 0.1). The other three nematicides had lower EI
than the untreated control at several dates (P < 0.1). SI was significantly affected by the
four nematicides. In 2016, fluopyram had reductions at each sampling date (P < 0.1).
Faunal profile was most affected by fluopyram. Fluopyram plots typically had faster
reductions of structure and higher enrichment as fluopyram was the only nematicide
treatment to have data points falling within quadrat A in 2016 and 2017 (Figs 3-13, 3-14,
3-15, and 3-16).
53
Table 3-1. Nematode families identified from turfgrass plugs and soil samples.
Family Feeding type
Cp value
Proportion of total mist extracted nematodes
Proportion of total soil extracted nematodes
Proportion of total nematodes
Anguinidae fungal 2 <0.01 <0.01 <0.01 Aphelenchidae fungal 2 0.05 0.02 0.03
Aporcelaimidae omnivore 5 0.08 0.17 0.13 Belonolaimidae plant-parasitic 3 <0.01 0.03 0.02 Bathyodontidae bacterial 4 <0.01 <0.01 <0.01 Cephalobidae bacterial 2 0.27 0.33 0.30 Criconematidae plant-parasitic 3 <0.01 0.01 0.01 Diplogasteridae bacterial 1 <0.01 <0.01 <0.01 Diploscapteridae bacterial 1 <0.01 <0.01 <0.01 Diphtherophoridae bacterial 3 <0.01 <0.01 <0.01 Discolaimidae predatory 5 <0.01 <0.01 <0.01 Heteroderidae plant-parasitic 3 0.10 0.05 0.08 Hoplolaimidae plant-parasitic 3 0.08 0.20 0.15 Ironidae predatory 4 <0.01 <0.01 <0.01 Longidoridae plant-parasitic 5 0.02 <0.01 0.01 Monhysteridae bacterial 2 <0.01 <0.01 <0.01 Monochidae predatory 4 0.00 <0.01 <0.01 Plectidae bacterial 2 0.01 <0.01 0.01 Qudsianematidae predatory 4 0.08 0.02 0.04 Rhabditidae bacterial 1 <0.01 0.01 0.01 Trichodoridae plant-parasitic 4 <0.01 <0.01 <0.01 Teratocephalidae bacterial 3 <0.01 0.01 0.01 Thornenematidae predatory 5 <0.01 0.01 0.01 Tylenchidae fungal 2 0.28 0.10 0.18
54
Figure 3-1. Population densities of bacterivore nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
55
Figure 3-2. Population densities of fungivore nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
56
Figure 3-3. Population densities of omnivore nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
57
Figure 3-4. Population densities of predatory nematodes from mist and soil extraction
as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
58
Figure 3-5. Population densities of plant-parasitic nematodes from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
59
Figure 3-5. Maturity index (MI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
60
Figure 3-6. 2-5 (MI25) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
61
Figure 3-7. Sigma maturity index (∑MI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
62
Figure 3-8. (PPI) from mist and soil extraction as affected by different nematicide
applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
63
Figure 3-9. (CI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. Data only shown for 2016. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
64
Figure 3-10. (BI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
65
Figure 3-11. (EI) from mist and soil extraction as affected by different nematicide
applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
66
Figure 3-12. (SI) from mist and soil extraction as affected by different nematicide applications at all sampling dates. *, **, *** Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
67
Figure 3-13. Faunal Profile calculated from mist extracted nematodes affected by
different nematicide applications in 2016. Quadrats labeled with A, B, C, or D for reference. Only fluopyram plots were significantly different from the untreated control. Figures are labeled with P value at a sampling date if structure index and structure index of fluopyram plots were determined significantly different from the untreated control using the analysis of covariance.
68
Figure 3-14. Faunal Profile calculated from soil extracted nematodes affected by different nematicide applications in 2016. Quadrats labeled with A, B, C, or D for reference. Only fluopyram plots were significantly different from the untreated control. Figures are labeled with P value at a sampling date if structure index and structure index of fluopyram plots were determined significantly different from the untreated control using the analysis of covariance.
69
Figure 3-15. Faunal Profile calculated from mist extracted nematodes affected by
different nematicide applications in 2017. Quadrats are labeled with A, B, C, or D for reference. No significant differences from the untreated control observed.
70
Figure 3-16. Faunal Profile calculated from soil extracted nematodes affected by
different nematicide applications in 2017. Quadrats are labeled with A, B, C, or D for reference. No significant differences from the untreated control observed.
71
CHAPTER 4 ARTHROPOD AND NON-NEMATODE RESULTS
Two-hundred arthropod samples were collected and specimens were extracted
using Berlese funnels. Two samples were excluded from data analysis due to storage
contamination. A total of 64,907 arthropods were counted and identified in the study.
Arthropods
Collembola, Insecta, and Acari were the major taxa identified within Arthropoda.
A total of 27,270 mites were counted and made up 42% of arthropods in the study
(Table 4-1). Ten mite families were identified across both years. Of these families,
seven families were categorized as detritivores and three were predatory. Detritivores
were the dominant group comprising 89% of mites identified.
Springtails made up 29% of arthropods in the study with 18,936 individuals
counted. Two springtail families were identified (Table 4-1). One species encountered
was a new record for the state of Florida (Mesaphorura yosii) and one a new record to
Florida and the continental US (Folsomides centralis) (FDACS 2017a and FDACS
2017b). All springtails identified belonged to the detritivore feeding group.
Insects were the third most abundant taxon with 18,699 specimens identified and
belonged to eight orders and sixteen families (Table 4-2). Insecta made up
approximately 28% of arthropods. Mealybugs (Hemiptera: Pseudococcidae) were the
dominant insect group representing 93% of total insects from this study. Three families
were placed in decomposer, eight in phytophagous, and five in predatory/parasitoid
feeding groups. Decomposers were comprised of saprophytes and fungivores.
Phytophages fed on plant parts as their primary source of nutrition. Predators and
parasitoids were grouped together as receiving primary nutrition from other animals.
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Less than 0.1% of arthropods identified belonged to Diplura. These arthropods
were not included in statistical analysis since only a few individuals were encountered.
Most Diplura feed on decaying matter and were considered detritivores in this study.
Mites
Abamectin increased detritivore mite abundance relative to the untreated control
(P < 0.05) at 2 DAFT and 56 DAFT in 2016 (Fig. 4-1). Abamectin treated plots had
greater detritivore mite abundance relative to the untreated control (P < 0.1) at 2, 14,
and 56 DAFT in 2017 (Fig. 4-1). Abamectin plots had greater predatory mite abundance
relative to the untreated control (P < 0.1) at 14 DAFT in 2016 (Fig. 4-2). No significant
differences (P > 0.1) were observed in abamectin treated plots in 2017 (Fig. 4-2).
Fluopyram also caused an increase in predatory mite abundance relative to the
untreated control (P < 0.1) in 2016 at 2, 14, and 56 DAFT (Fig. 4-2). No effects were
observed in fluopyram plots relative to the untreated control (P < 0.1) in 2017 (Fig. 4-2).
Springtails
Abamectin had no significant effects relative to the untreated control in 2016 (P <
0.1), but population numbers in 2017 were reduced relative to the untreated control (P <
0.01) at 2 and 14 DAFT (Fig. 4-3). Fluopyram did not significantly affect springtails
relative to the untreated control in 2016 (P < 0.1). Fluopyram reduced springtail counts
relative to the untreated control (P < 0.1) at 2, 14, 56, and 238 DAFT in 2017 (Fig. 4-3).
Fluensulfone reduced springtails relative to the untreated control (P < 0.1) at 238 DAFT
(Fig. 4-3).
Insects
Abamectin reduced phytophogous insect counts relative to the untreated control
(P < 0.1) at 2 DAFT in 2016 (Fig. 4-4). Fluopyram increased phytophogous insect
73
abundance relative to the untreated control (P < 0.05) at 14 DAFT 2016 (Fig. 4-4) and
increased phytophogous insect abundance relative to the untreated control (P < 0.1) at
2 and 14 DAFT in 2017 (Fig. 4-4). Furfural significantly decreased phytophage
abundance relative to the untreated control (P < 0.05) at 238 DAFT in 2017 (Fig. 4-4).
Detritivore and predatory insect abundances were very low in both years. Total
counts were often below 5 individuals per plot. Due to the low counts, data analysis and
figures were not included for detritivore or predatory insects.
Arthropod Summary
Arthropods were susceptible to effects of nematicide applications. Mites were
significantly affected in both years, however abundance increased after exposure to
abamectin and fluopyram (P < 0.1) (Fig. 4-1 and Fig. 4-2) Springtails were impacted at
more dates than the other arthropod groups. They were reduced relative to the
untreated control (P < 0.1) by abamectin, fluopyram and fluensulfone (Fig. 4-3).
Fluopyram decreased abundance at all sampling dates in 2017 (Fig. 4-3). Springtail and
mite family composition was similar in both years. Abamectin and fluensulfone affected
springtail abundance at fewer sampling dates (Fig. 4-3). Insect abundance was
dominated by phytophages and very few detritivores and predators were present. The
phytophages increased relative to the untreated control (P < 0.1) after applications of
abamectin and fluopyram and decreased after furfural applications relative to the
untreated control (P < 0.1) (Fig. 4-4).
Non-Arthropods
Several invertebrate groups were adversely affected during the extraction and
preservation process. Enchytraeids were present in samples, but their abundance could
not be accurately calculated due to specimen damage during storage. The bodies of
74
many specimens fragmented after storage in the sample refrigerator or in formalin.
Rotifers were also present in many samples. Formalin preservation affected morphology
of rotifers and morphological identification could not be carried out to family level.
Rotifers were identified to the class Bdelloidea. Rotifer extraction was greater from soil
than mist (Fig. 3-19 and Fig. 3-20). Ciliates were also encountered, but not enough
samples contained ciliates to carry out statistical analysis.
Rotifers
Fluopyram plots had an increase in rotifers relative to the untreated control (P <
0.05) at 14 DAFT from in 2016 (Fig. 4-5) and increases relative to the untreated control
(P < 0.1) at 14 and 56 DAFT from ME in 2017 (Fig. 4-5). Fluopyram treated plots had
rotifer decreases relative to the untreated control (P < 0.05) at 2 and 14 DAFT from SE
in 2017. Furfural treated plots had a reduction of rotifers relative to the untreated control
(P < 0.1) at 238 DAFT from SE in 2017 (Fig. 4-5). Rotifer counts in fluensulfone plots
decreased relative to the untreated control (P < 0.1) at 56 DAFT from ME in 2016 and
from SE in 2017 (Fig. 4-5).
Turfgrass Percent Green Cover Results
Photograph data were analyzed for 38 time points across the two-year study. All
four nematicides significantly affected percent green coverage. Abamectin significantly
impacted green coverage relative to the untreated control at 18 dates, fluopyram at 22
dates, furfural at 8 dates, and fluensulfone at 8 dates.
Abamectin. Significant green coverage increases relative to the untreated
control in abamectin plots were observed at 17 dates and 1 decrease relative to the
untreated control at October 10, 2017 (Figs. 4-6). In 2016, greater green coverage
relative to the untreated control (P < 0.1) was present from July 19 until November 22,
75
except for one date (Fig. 4-5 and Fig. 4-6). Positive effects relative to the untreated
control (P < 0.1) in 2017 were observed after March 28 and maintained greater green
coverage until August 29, except for April 11 (Fig. 4-6). One significant reduction
relative to the untreated control (P < 0.1) was observed from September 13 until the end
of the study (Fig. 4-6).
Fluopyram. Fluopyram had significantly greater green coverage relative to the
untreated control at 17 dates and lower green coverage relative to the untreated control
at 5 dates. Positive green coverage responses relative to the untreated control (P < 0.1)
occurred from June 20 until August 2 in 2016 (Fig. 4-5). Significant reductions relative to
the untreated control (P < 0.1) occurred at September 27 and October 11 in 2016 (Fig.
4-5). Increases occurred relative to the untreated control (P < 0.1) in 2017 from
February 28 until July 3 and then at November 7 and December 5 (Fig. 4-5). Three
declines (P < 0.1) relative to the untreated control occurred at August 29, September
13, and October 10 (Fig. 4-5). An (P < 0.01) increase relative to the untreated control
occurred in 2018 on the first photographed date, February 27 (Fig. 4-5).
Furfural. Furfural had significantly greater green coverage relative to the
untreated control at 8 dates across two years. No significant reductions relative to the
untreated control occurred in either year. Greater green coverage relative to the
untreated control (P < 0.1) occurred from August 30 to December 20 in 2016 (Fig. 4-5).
One increase relative to the untreated control (P < 0.01) was observed in 2017, on
November 21 (Fig. 4-5).
Fluensulfone. Fluensulfone significant effects relative to the untreated control on
green coverage were observed at 8 dates across two years. No significant reductions
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relative to the untreated control occurred. Green coverage was greater relative to the
untreated control (P < 0.1) five dates from August 30 to December 20 in 2016 (Fig. 4-5).
Increases relative to the untreated control (P < 0.1) in 2017 occurred at June 6, June
20, and October 24 (Fig. 4-5).
Percent Green Coverage Summary
Abamectin and fluopyram each had increases in green coverage at 17 dates
(Fig. 4-5). Abamectin had lower coverage than the untreated control at one date while
fluopyram had lower coverage at five dates (Fig. 4-5). The overall positive turfgrass
response suggests the greatest benefits to the above ground appearance occurred from
applications of abamectin and fluopyram. However, unexpected decline relative to the
untreated control occurred in fluopyram plots around September in 2016 and 2017 (Fig
4-5). Notable brown appearance of plots was apparent even without statistical analysis.
Despite the early fall decline, the plots had greater green coverage relative to the
untreated control after the turfgrass broke dormancy and began spring growth the
following year (Fig. 4-5). Abamectin plots did not regain green coverage after dormancy
as rapidly as fluopyram plots, but the coverage was greater longer into the season in
abamectin plots (Fig. 4-5). Furfural and Fluopyram treated plots had modest benefits
across the two years. Significant coverage increases occurred around October for both
nematicides, lasting into December in 2016 (Fig. 4-5). Overall, abamectin had the most
favorable impact on percent green coverage, followed by fluopyram. Furfural and
fluensulfone had moderate benefits, but notably no significant reductions relative to the
untreated control.
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Table 4-1. Non-insect arthropod families identified from Berlese funnel extraction in 2016 and 2017
Taxon (class, order, family)
Proportion of total arthropods 2016
Proportion of total arthropods 2017
Proportion of total arthropods combined years
Feeding group
Arachnida Mesostigmata Ascidae 0.03 0.01 0.02 predator Phytoseiidae <0.01 0.01 <0.01 predator Rhodacaridae 0.01 0.02 0.01 predator Prostigmata Iolinidae 0.00 0.01 <0.01 fungivore Sarcoptiformes Acaridae 0.02 0.02 0.02 fungivore Galumnidae 0.22 0.22 0.22 fungivore Liacaridae 0.06 0.05 0.06 fungivore Thyrisomidae <0.01 0.07 0.04 fungivore Tokunocephidae 0.03 0.02 0.03 fungivore Trombidiformes Tarsonemidae 0.01 <0.01 <0.01 fungivore Entognatha Collembola Isotomidae 0.30 0.25 0.29 fungivore Tubergiidae 0.01 <0.01 <0.01 fungivore Diplura Campodeidae <0.01 0.00 <0.01 omnivore
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Table 4-2. Insect families identified from Berlese funnel extraction in 2016 and 2017.
Taxon (class, order, family)
Proportion of total arthropods 2016
Proportion of total arthropods 2017
Proportion of total arthropods combined years
Feeding group
Insecta - Thysanoptera -
Phlaeothripidae <0.01 <0.01 <0.01 fungivore Hemiptera -
Aphididae 0.00 <0.01 <0.01 phytophagous Pseudococcidae 0.26 0.28 0.27 phytophagous
Psocoptera Liposcelididae <0.01 0.02 0.01 fungivore
Hymenoptera - Braconidae 0.00 <0.01 <0.01 parasitoid Formicidae <0.01 0.02 0.01 nectarivore Mymaridae 0.00 <0.01 <0.01 parasitoid Tiphiidae 0.00 <0.01 <0.01 parasitoid Siricidae 0.00 <0.01 <0.01 phytophagous
Coleoptera - Carabidae <0.01 <0.01 <0.01 predator Staphylinidae <0.01 <0.01 <0.01 fungivore Scarabeidae
(adult) <0.01 <0.01 <0.01 phytophagous
Lepidoptera - Arctiidae <0.01 <0.01 <0.01 phytophagous
Diptera Anthomyiidae <0.01 <0.01 <0.01 phytophagous Ceratopogonidae <0.01 <0.01 <0.01 predator Cecidomyiidae <0.01 <0.01 <0.01 phytophagous Muscidae <0.01 <0.01 <0.01 phytophagous
Insecta - Thysanoptera -
Phlaeothripidae <0.01 <0.01 <0.01 fungivore Hemiptera -
Aphididae 0.00 <0.01 <0.01 phytophagous Pseudococcidae 0.26 0.28 0.27 phytophagous
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Figure 4-1. Population densities of detritivore mites from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, ***Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
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Figure 4-2. Population densities of predatory mites from berlese extraction as affected
by different nematicide applications at all sampling dates. *, **, ***Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
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Figure 4-3. Population densities of detritivore springtails from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, ***Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
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Figure 4-4. Population densities of phytophagous insects from berlese extraction as affected by different nematicide applications at all sampling dates. *, **, ***Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
83
Figure 4-5. Population densities of rotifers from mist and soil extraction as affected by
different nematicide applications at all sampling dates. *, **, ***Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
84
Figure 4-6. Percent green coverage calculated from plot photographs from June 7, 2016 to. March 13, 2018. *, **, ***Different from the untreated according to analysis of covariance (P < 0.1, 0.05, 0.01, respectively).
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CHAPTER 5 SUMMARY AND CONCLUSIONS
Abamectin. Abamectin had intermediate effects on nematodes, altering the
community structure of free-living nematodes and causing a shift to a slightly less
mature soil food web. Arthropods were also affected by abamectin, but only springtails
experienced significant population density declines. Abamectin is a valuable nematicide
used to manage a range of nematodes including species of Meloidogyne, Pratylenchus,
and Heterodera (Lumaret et al., 2012). The decrease of fungivore, omnivore, and
predatory nematodes in our studies indicates a long-term negative impact on beneficial
nematodes in the 2016 study year. The 2017 data showed fewer effects on beneficial
nematodes. Effects on nematodes were generally detected later in the season and
could be explained by the characteristic slow movement of this formulation through the
thatch layer of turfgrass. High cp nematodes were mildly affected by abamectin. The
reduction in higher trophic nematodes at the later sampling dates was accompanied by
bacterivore increases at a few dates possibly indicating an opportunistic response to
disturbance. The enrichment index did not indicate an enriched environment; however
this index considers both bacterivore and fungivore abundance and could be offset by
the reduction in Fu2 fungivores. Maturity index, basal index, and structure index values
suggested abamectin plots had a basal environment with reduced abundance of high
trophic nematodes at the end of the study. Abamectin had a positive effect on turfgrass
health as measured by percent green coverage, which is an important consideration.
While the nematicidal properties of abamectin have been known for some time,
few studies have considered the effects of abamectin on free-living nematodes (Brinke
et al., 2010). Ivermectin contained in feces from cattle treated for animal-parasitic
86
nematodes has been shown to affect Tylenchus and Cephalobus free-living nematodes
in pastureland (Yeates et al., 2003). Farmland treated with pesticides including
abamectin, has been shown to undergo moderate disturbance based on maturity
indices, plant parasitic index, fungivore/bacterivore abundance, basal index and channel
index evaluations (Dong et al., 2008). A study on freshwater nematodes found free-
living nematode abundances were impacted by the exposure of ivermectins (Brinke et
al., 2010).
Abamectin caused a reduction in springtails, but an increase in mites and insects
above untreated control densities. Springtail decline occurred in early sampling dates
but, effects were short-lived as populations recovered later in the season. This is
common for r strategists like springtails (Hopkin, 1997b). It is common for bacterial
feeding nematode numbers to increase following disturbance, but the increase in mites
and insects was unexpected due to sensitivity of mites and insects to abamectin.
Acaricides often contain abamectin or related avermectin compounds to control mites in
agricultural settings (Lumaret et al., 2012). Abamectin is recommended in IPM
programs for its reported low toxicity on beneficial arthopods and rapid degradation in
sunlight (Wilson, 1993). However, some non-target arthropods such as beneficial dung
beetles (Coleoptera: Scarabaeidae) and predatory mites (Acari: Phytoseiidae) can be
sensitive to the macrocyclic lactone group (Lumaret and Errouissi, 2002; Lumaret et al.,
2012). It activates glutamate-gated chloride channels in invertebrates causing a
prolonged flow of Cl ions into the post synaptic neuron and results in nerve
hyperpolarization. The binding site for this molecule is only known in invertebrates such
as nematodes, mites, and insects. Minimal activity has been documented against
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fungal, bacterial, or protozoan organisms (Wang and Pong, 1982). However,
avermectins have been documented with selectivity within mite and insect groups
(Förster et al., 2011; Römbke et al., 2010). Förster et al., 2011 found significant
reduction in springtail densities and no significant effect on mite densities in pasture soil
exposed to ivermectin. Soil mites exposed to ivermectins in laboratory and field settings
were not significantly affected except under very high concentrations. Other studies
reported abamectin having low toxicity or sub-lethal effects on non-target mites (Zhang
and Sanderson, 1990). In our study it is possible dead fungal feeding nematodes and
springtails could have served as a food source for microbes that enabled the bacterial
feeding nematodes and fungal feeding mites to flourish with less competition.
Fluopyram. Fluopyram had the most striking results of the nematicides tested. It
reduced beneficial nematode and plant-parasitic nematode densities at intermediate
and long-term sampling intervals. Similar to abamectin, more significant reductions were
observed in 2016 than 2017. The suppression of these nematodes could be attributed
to direct effects on nematodes, since fluopyram has nematicidal and nematistatic effects
on C. elegans (Kearn et al., 2014; Burns et al., 2015). Suppression of free-living
nematodes could also be caused by the decrease in available food from fungicidal
activity of fluopyram, or a combination of nematicidal and fungicidal factors.
Microorganism composition has been shown to affect diversity and abundance of
r strategist nematodes (Hodda and Nicholas, 1985). Based on channel index, the fungal
decomposition pathway was dominant earlier in the season, but transitioned to a more
bacterial dominated pathway. These changes were not significantly different, however.
This could indicate the fungal community in the soil was not severely impacted in early
88
sampling dates. Bacterivores, fungivores, and omnivores were susceptible to fluopyram
at nearly all sampling dates and predators were reduced at the last two sampling dates.
These observations suggest fluopyram has the potential to affect all nematode feeding
groups quickly after application and throughout the season. Low abundance of low
trophic nematodes like bacterivores and fungivores could reduce predator densities
from indirect effects of bottom-up regulation. While fluopyram effects on free-living
nematodes have not been evaluated in other field studies, impacts on non-target
nematodes have been analyzed from other nematicide products. The nematicide
fosthizate reduced the population density of predators, omnivores, and bacterivores in
grassland diversity studies (Eisenhauer et al., 2010), but did not impact fungal feeders.
The ecological indices from our study also demonstrated trophic level diversity
characteristic of degraded basal conditions. The nematicide fosthizate reduced of
predator, omnivore, and bacterivore population densities in grassland diversity studies
(Eisenhauer et al., 2010). Fungal feeders not significantly affected. The ecological
indices from our study also demonstrated trophic level diversity characteristic of
degraded basal conditions. EI values revealed an enriched environment after fluopyram
application. In addition to the reduction of nematode functional group densities, a shift
occurred toward an environment dominated by r strategist nematodes. The soil
condition shifted toward a more basal and degraded environment. In similar studies,
community shift reduction of high trophic nematodes and increase of opportunistic
nematodes also occurred after exposure to bionematicide 1,4-napthoquinone (Chelinho
et al., 2017). The rapid degradation of newly available resources in our study could
have unfavorable consequences on the soil food web. Less carbon is transferred to
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higher trophic animals from the rapid metabolism and respiration of low trophic
nematodes (Ferris and Bongers, 2006). This can result in low predator abundance, as
seen from fluopyram applications in this study. The fungicidal activity of fluopyram could
reduce fungal food sources for fungal feeding soil invertebrates. Fewer trophic linkages
in the nematode food web existed compared to the untreated control based on maturity
index, maturity index 2-5, ∑ maturity index, basal index, and structure index. This
environment is less stable and would likely be more prone to reductions in ecosystem
service benefits. Fuanal profile results were characteristic of disturbed ecosystems
where high trophic nematode abundance is low and opportunistic colonizers are
dominant. Fluopyram significantly increased percent green coverage in treated plots,
but significantly low coverage was also observed in fluopyram plots in both years.
Sampling dates with significantly greater green cover occurred twice when fluopyram
plots had significantly fewer plant-parasitic nematodes than untreated plots, which likely
benefited from lower plant-parasitic nematode pressure. Number of bacterivores,
fungivores, omnivores, and springtails were reduced at dates with reduced green cover,
but these groups were also reduced when green cover was not significantly different or
higher the untreated control. Lower densities of these organisms could contribute to the
decline in turf appearance, but the data presented do not conclusively support this
claim.
The long half-life of fluopyram likely leads to extended activity in soil long after
application as seen in reduction in r strategists at thirty-four weeks in both years.
Springtails were most affected arthropod group. These data suggest fluopyram may
have persistent effects on springtails. The other arthropod groups were largely
90
unaffected, except for increases in predatory mites and phytophogous insects which
increased. The reduction in predatory nematodes could have allowed predatory mites to
increase with reduced competition over shared food resources. Put et al. (2016) found
no reductions of predatory mite Euseius gallicus treated with fluopyram+tebucinazole in
greenhouse assays on vegetables. Low toxicity has been reported for mites, springtails,
and earthworms (EFSA, 2013; EFSA, 2018). Interestingly, rotifers were not adversely
affected by fluopyram in our study, but their numbers increased at multiple sampling
points across both years compared to the untreated. This increase could coincide with
decreases in bacterivore nematode abundance and could be an opportunistic surge in
abundance with reduced competition from nematodes. The advantages of long lasting
plant-parasitic nematode control from fluopyram is accompanied by reduction in
beneficial nematode numbers and shifts toward a less structured environment
dominated by enrichment opportunists.
Furfural. Furfural had a low impact on free-living nematodes. The predatory
nematode functional group was the only feeding group negatively affected by furfural.
The general lack of reduction in functional group densities suggests furfural may have
low risk to free-living nematodes. Abdelnabby et al. (2016) and Abdelnabby et al. (2018)
observed no adverse effects on free-living nematodes. Negative impacts on bacterivore
and fungivore nematodes have been documented in tomato field trials, suggesting
furfural can reduce beneficial nematode numbers (Ntalli et al., 2018). Plant-parasitic
nematodes experienced an increase at two-week sampling dates in each year. These
results may be attributed with temporary Meloidogyne increases occasionally seen with
furfural use (Crow and Luc, 2014). The gelatinous matrix of Meloidogyne egg masses is
91
thought to be dissolved by furfural releasing juveniles into the soil (Steyn and Van
Vuuren, 2006). Soil condition was found to be stable based on maturity indices.
Channel index assessment suggested bacterial decomposition was prominent in furfural
plots. Furfural had a moderate impact on turfgrass green cover. Positive responses in
turf health were observed in both years, but generally after the beginning of September.
Lack of furfural efficacy in the top 5 cm of soil has been observed in other trials (Crow
and Luc, 2014). As a byproduct from sugarcane processing, furfural is readily broken
down by microbes. It has been hypothesized that in field conditions furfural is broken
down before activity on nematodes occurs. This could contribute to the minimal effects
on functional groups observed in our study. Arthropods and rotifers also experienced
low impacts in this study. One reduction occurred in phytophogus insects and
springtails. No published data on furfural toxicity to arthropods are currently available for
comparison. Botanical products like furfural are used in IPM systems and can have
repellant and sublethal effects on non-target arthropods (Desneux et al., 2007).
Botanical product selectivity in other literature suggests selectivity on non-target soil
arthropods is possible (De Souza Tavares et al., 2009; Elias et al., 2013). Compared to
abamectin and fluopyram, furfural may be a lower risk nematicide to free-living
nematodes.
Fluensulfone. Fluensulfone had low impacts on free-living nematodes
comparable to furfural. Fungivores were the only functional group negatively affected at
one sampling date in 2017. Predators increased in abundance at eight weeks in both
years. As a result, structure and food web complexity were greater in fluensulfone plots
than untreated control according to the maturity indices and SI. Turfgrass health
92
moderately improved from fluensulfone treatments. Benefits were observed near the
beginning of September into December of 2016 and in June and July of 2017. The
fluensulfone impact on arthropods was very low in this study. Mite, springtail, and insect
abundances in the fluensulfone treated plots were largely unaffected, suggesting low
ecological risk to soil microarthropods. Rotifers experienced relatively low impacts with
lower abundance detected at one sampling date each year. Laboratory based assays
have found the PPN Meloidogyne javanica to be susceptible to fluensulfone at lower
doses than the bacterivore C. elegans (Kearn et al., 2014). This compound has a mode
of action distinct from the more traditional organophosphate, carbamate, and ivermectin
active ingredients potentially having a lower impact on the soil ecosystem. Fluensulfone
also has systemic activity which could allow for PPN control in plant roots even if the
compound has moved past the rhizosphere. Low toxicity to earthworms has been
documented, but little is known about the impact of fluensulfone on other soil fauna
(Oka et al., 2012; Beknazarova et al., 2016). The low risk perceived to non-target soil
fauna has led to its promotion as an environmental control measure against soilborne
stages of the human parasite Strongyloides (Beknazarova et al., 2016). Another factor
that may have led to limited effects on non-target invertebrates is persistence. The
manufacturer reports a short half-life of 7 to 17 days in soil and the formulation used in
the study has rapid movement through the thatch and soil profile (Oka et al., 2013;
Crow et al., 2017). Short exposure time and reduced toxicity to non-target nematodes
characteristics could contribute to the low impact on free-living nematodes. The results
observed suggest fluensulfone may have a low risk to free-living nematodes.
93
Conclusions. As environmentally conscious management practices continue to
develop, studies evaluating the ecological impact of chemical control measures are
needed. The results of our study indicate nematicides can affect the abundance of
beneficial soil invertebrates. That being said, the impacts of each nematicide varied.
Abamectin had intermediate impacts on free-living nematodes and arthropods while
fluopyram had substantial impacts on nematodes and arthropods. Furfural and
fluensulfone had relatively low impacts on soil invertebrates. These nematicides have
different modes of action and could be rotated to reduce impacts on specific groups of
non-target invertebrates. Nematodes share many basic morphological and physiological
characteristics that would suggest non plant-parasitic nematodes might respond
similarly to nematicidal or nematistatic compounds as their plant-parasitic counterparts.
While evidence from two compounds in this study supports this hypothesis (i.e
abamectin and fluopyram) the other compounds furfural and fluensulfone did not have a
substantial impact on free-living nematode abundance. Furfural can produce short-lived
nematode control. This is likely due to the very short persistence in soil and contact
nature of the nematicide. Rapid microbial decomposition could limit effects on free-living
nematodes. The short persistence may be a major factor in the lower toxicity in our
study. It also had relatively moderate benefit to turfgrass health during the study.
Fluensulfone has been shown to be an effective nematicide in several cropping
systems, including turfgrass. In our field studies, we also found lower risk to beneficial
nematodes and arthropods. Similar to furfural, fluensulfone had relatively moderate
impacts on turfgrass health. While the mode of action is not clearly understood,
elucidation of the target site could strengthen the claim for low environmental toxicity.
94
Of the arthropods encountered, mites and insects experienced the lowest
impacts from the four nematicides. Abamectin and fluensulfone had relatively low
impacts on springtails. Fluopyram had relatively high impacts, similar to its effect on
free-living nematodes. Increases of mite and insect densities were unexpected,
especially from abamectin. It is possible the formulation of these products was not
favorable for arthropod cuticle penetration and the reduction of bacterial and fungal
feeding nematodes could reduce competition for food in abamectin and fluopyram
treated environments.
Severely disrupting the soil ecosystem could result in reduced benefits and
require additional inputs to compensate for lost benefits of the soil ecosystem (De Deyn
et al., 2003). The nematicide impacts on the soil ecosystem could be at least partly
responsible for the temporary, but severe, decline in turf percent green cover observed
in the fluopyram plots at some dates. The perennial monoculture of turfgrass also could
impact functional group diversity. Managed monoculture systems tend to have lower
nematode diversity than unmanaged polycultures such as grasslands or forests
(Quintanilla et al., 2015). The low diversity environment of managed systems makes
responsible management decisions important to maximize benefits from the existing soil
communities. Therefore, nematode management practices in turfgrass should consider
the environmental impacts of nematicides. These results indicate some nematicides can
affect functional group composition over time and potentially alter the stability of soil
ecosystems. Fluopyram could be used sparingly to drastically reduce plant-parasitic
nematode population numbers or even for spot treatments. Abamectin treatments could
be applied following fluopyram during the season to continue green-coverage benefits
95
with reduced impacts on the soil ecosystem. For low soil ecosystem impacts, furfural
and fluensulfone could be used for maintaining areas with low plant-parasitic nematode
population densities as more of a preventative approach.
96
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BIOGRAPHICAL SKETCH
Benjamin was raised in Indiana and where he started a state licensed plant
nursery as part of a high school project. This project propelled him into scientific studies
and he graduated from the University of Evansville with a BS in Applied Biology. Part of
his undergraduate education included a semester studying abroad in Britain where he
studied British history and ornamental gardening practices. He also completed an
internship with Sakata Seed America in Ft. Myers, FL assisting in tomato breeding and
plant pathology departments. In order to further broaden his knowledge of
interdisciplinary plant health, he enrolled at the University of Florida in dual MS
Nematology / Doctor of Plant Medicine programs in 2016.