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ENTOMOPATHOGENIC FUNGI AND NEMATODES
FOR MICHIGAN TREE FRUIT MANAGEMENT
TARGETING PLUM cuncuuo
(CONOTRA CHEL us NENUPHAR)

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Renee Jean Pereault

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5/08 K'lProj/Acc8Pres/ClRC/DateDue.indd

ENTOMOPATHOGENIC FUNGI AND NEMATODES FOR MICHIGAN TREE
FRUIT MANAGEMENT TARGETING PLUM CURCULIO
(CONOTRA CHEL US NENUPHAR)

By

Renee Jean Pereault

A THESIS
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

MASTERS OF SCIENCE
Entomology

2008

ABSTRACT
ENTOMOPATHOGENIC FUNGI AND NEMATODES FOR MICHIGAN TREE
FRUIT MANAGEMENT TARGETING PLUM CURCULIO (CONOTRACHELUS
NENUPHAR)
By
Renee Jean Pereault
Tree fruit growers are seeking effective alternatives to US EPA-mitigated
insecticides for managing plum curculio, Conotrachelus nenuphar Herbst (Coleoptera:
Curculionidae). Formulations and novel delivery mechanisms of entomopathogenic
nematodes (Steinemema riobrave, Steinemema carpocapsae and Heterorhabditis
bacteriophora) and fungi (Beauveria bassiana and Metarhizium anisopliae) were
investigated in the laboratory and in Michigan orchards. Mortality of fungus-exposed
adults ranged from 38-100% and occurred 17-23 d post treatment in the laboratory. Fall
granular fungus formulation applications failed to reduce overwintering survival of
adults. Larvae were introduced to orchard soils -10, -5, O, 5, 10, 15, or 20 d from
entomopathogen application. Steinemema riobrave consistently reduced adult emergence
most effectively in sandy sites. Beauveria bassiana exhibited limited effectiveness and
sensitivity to environmental and site factors. Both nematodes and fungi were promising

tactics against plum curculio, but more investigation is needed to determine if oviposition

damage and the second generation can be reduced to economic levels.

Dedicated to Michigan Tree Fruit Farmers.

ACKNOWLEDGEMENTS

I thank my major advisor, Mark Whalon for his encouragement and passion.
Thank you to my committee members for reviewing my thesis and in particular Leah
Bauer for guiding me at the Society for Invertebrate Pathology meeting, John Biernbaum
for engaging me in organic horticulture, and David Smitley for additional pest biocontrol
expertise. Special thanks to Diane Alston for technical assistance and review. I thank
Willye Bryan for her dedication to rearing plum curculio in “the cave.” I thank Dan
Nortman, Alex Johnson, Peter Nelson, Soo-hoon Kim, Ki Kim, Zach Koan, Karlyn Page,
Michael Hosking, Jeannette Wilson, Christopher Archangeli, Saunté Sutton, Abbra
Puvalowski, Jennifer Silveri, Andrew Skwiercz, Robert Brown, and Lisa Losievsky for
technical assistance. I thank Richard Humber for entomopathogenic fungus identiﬁcation
and accession and Fred Warner for nematode diagnostics. We thank the Michigan
Agricultural Experiment Station for providing orchard sites and support (Jerry Skeltis,
Gayle “Peach” Byler, and Denise Ruwersma at Clarksville Horticulture Experiment
Station and the staff at Northwest Horticulture Research Station). A special thanks to
farmers for sharing their perceptions, advice, and orchards: Gene Garthe, Bruce Walton,
Sheryl and Alan Kobernik, Jim Laubach, Donald Smeltzer, Merle Brown, Cal Lutz,
Dennis Mackey, Jim Koan, and Steve Tennes. Funding contributions were provided by
Project GREEEN, Michigan Apple Research Committee, Cherry Marketing Institute,

Michigan State Horticultural Society, IR-4, and SARE.

iv

TABLE OF CONTENTS

List of Tables ..................................................................................... vi
List of Figures ................................................................................... viii
Chapter 1
Literature Review ................................................................................... l
Plum Curculio (Conotrachelus nenuphar) ........................................ 1
Geographic Distribution and Hosts .............................. 1
Characteristics and Life History .................................. 1
Pest Status and Management Techniques...........................3
Biological Control Agents ................................................. 5
Natural Enemies ................................................... 5
Entomopathogenic Nematodes .................................. 7
Entomopathogenic Fungi ......................................... 9
Insect Behavioral Responses to Entomopathogens ............ 12
Entomopathogen Experiments Against Plum Curculio ....... 14
Thesis Research ............................................................. 16
Chapter 2
Laboratory Evaluations of Entomopathogenic Fungus Efﬁcacy Against Last Instar Plum
Curculio ............................................................................................ 18
Introduction ................................................................... 18
Materials and Methods ....................................................... 19
Results ......................................................................... 21
Discussion ..................................................................... 22
Chapter 3
Entomopathogenic Nematode and Fungus Field Efﬁcacy Against Last Instar Plum
Curculio ............................................................................................ 24
Introduction ................................................................... 24
Materials and Methods ....................................................... 27
Results ......................................................................... 36
Discussion ..................................................................... 38
Chapter 4
Laboratory and Field Evaluations of Entomopathogenic Fungus Efﬁcacy Against Adult
Plum Curculio .................................................................................... 53
Introduction ................................................................... 53
Materials and Methods ....................................................... 55
Results ......................................................................... 61
Discussion ..................................................................... 67
Appendix ........................................................................................ 71

References ....................................................................................... 73

vi

LIST OF TABLES

Table 2.1. Percent viability of conidia in immersion suspensions on each test

date .............................................................................................. 21
Table 3.1. Soil Analysis for 2006 and 2008 sites ......................................... 51
Table 3.2. Nematode Community Analysis, Michigan State University Diagnostic

Service Laboratory, from pre-application soils, 2008 .................................... 51
Table 3.3. Percent soil moisture in 2008 sites ............................................. 51

Table 3.4. Mean percent reduction from control treatments (Abbott’s Corrected
Mortalities) for plum curculio in S. riobrave high rate treatment. Larvae were applied -10,
-5, 0, 5, 10, 15, or 20 d from S. riobrave application to pots of soil. Percentages with an
asterisk (*) indicate that the corresponding mean adult counts were signiﬁcantly lower

than control within a larva timing (SNK, a=0.05) ........................................ 52
Table 3.5. Spray record for Clarksville orchard blocks in 2007 ........................ 52
Table 4.1. Dish/Wick assay treatments .................................................. 59

Table 4.2. Daily temperatures and precipitation near entomopathogenic fungus
application date (16-Oct) in overwintering trial .......................................... 66

vii

LIST OF FIGURES

Figure 2.1. Mean (:t: SE) percent mortality and percent of larvae with sporulation 14 d
after a 10 s immersion in 1x105 or 1x106 conidia/ml suspensions of M. anisopliae F52, B.
bassiana strain GHA, M. anisopliae 8270, or water control. Bars with different letters
indicate signiﬁcant difference among treatments within mortality or within sporulation
(T ukey’s HSD, a=0.05). ..................................................................... 22

Figure 2.3. Percent mortality of and sporulation on last-instar plum curculio after
exposure to B. bassiana-impregnated fabric as last-instars and subsequent 22 d
containment in sterile soil for pupation .................................................... 24

Figure 3.1. Mean (1 SE) number of adult plum curculio emerged from B. bassiana GHA-
treated soil in pots (10 larvae/pot) over a 50—d period in 2006 at four orchards. Rice
treatments (B. bassiana grown on rice) were not conducted at Koan. Bars with the same
letter within an orchard are not signiﬁcantly different (SNK, alpha=0.05) ............ 46

Figure 3.2. Mean (:1: SE) number of adult plum curculio emerged from entomopathogen-
treated soil in pots over a 50—d period in 2007 at a single orchard. Ten larvae were added
to each pot either immediately, 3 d, or 5 d after entomopathogen application. Bars with
the same letter are not signiﬁcantly different (SNK, alpha=0.05) ..................... 47

Figure 3.3. Mean (:i: SE) number of adult plum curculio emerged from entomopathogen-
treated soil in pots over a 50—d period in 2008. Ten larvae were added to each pot either -
10, -5, 0, 5, 10, 15, or 20 d from entomopathogen application. Note Hb treatments were
not conducted on -10, 10, and 20 (1. Bars with an asterisk are signiﬁcantly different from
the control within the same larva addition timing (SNK, (1:0.05). All orchards combined
and Leelanau Co. Sandy Loam site ......................................................... 48

Figure 3.4. Mean (:I: SE) number of adult plum curculio emerged from entomopathogen-
treated soil in pots over a 50-d period in 2008. Ten larvae were added to each pot either -
10, -5, 0, 5, 10, 15, or 20 d from entomopathogen application. Note Hb treatments were
not conducted on -10, 10, and 20 (1. Bars with an asterisk are signiﬁcantly different from
the control within the same larva addition timing (SNK, o.=0.05). Leelanau Co. Sandy
Loam site and Ionia Co. Loam site. ....................................................... 49

Figure 3.5. Mean (:t: SE) number of adult plum curculio emerged from entomopathogen-
treated soil in pots over a 50—d period in 2008. Ten larvae were added to each pot either -
10, -5, 0, 5, 10, 15, or 20 d from entomopathogen application. Note Hb treatments were
not conducted on -10, 10, and 20 d. Bars with an asterisk are signiﬁcantly different from
the control within the same larva addition timing (SNK, o.=0.05). Eaton Co. Clay Loam
site and Genesee Co. Loam site ............................................................. 50

viii

Figure 4.1. Mortality of adult plum curculio exposed to entomopathogen-treated soil or
peat at a rate of 8 x 1014 conidia/ha after 15 d. Bars with the same letter are not
signiﬁcantly different (SNK, 0.=0.05) ...................................................... 61

Figure 4.2. Mortality of adult plum curculio exposed to entomopathogen-treated soil (low
rate = l x 10'3 conidia! ha; high rate = 5 x 10'3 conidia/ha) after 12 d. Bars with the same
letter are not signiﬁcantly different (Dunnett’s procedure, o.=0.05) ................... 62

Figure 4.3. Mean (:tSE) number of days after treatment exposure until mortality of plum
curculio adults. The exposed adult (sex indicated in the x-axis) was held with an
unexposed mate of the opposite sex ........................................................ 63

Figure 4.4 Mean (18E) percent mortality of plum curculio adults 29 d after treatment.
The exposed adult (sex indicated in the x-axis) was held with an unexposed mate of the
opposite sex .................................................................................... 64

Figure 4.5. Mean (:tSE) percent sporulation of plum curculio adults 37 d after treatment
exposure. The exposed adult (sex indicated in the x-axis) was held with an unexposed
mate of the opposite sex. Bars with same letter indicate that the corresponding means are
not signiﬁcantly different (T ukey, a=0.05) ................................................ 65

Figure 4.6. Mean (:tSE) number of emerged adults in the spring following overwintering
in cages. Groundcover treatments included a granular formulation of B. bassiana or M.
anisopliae, or an untreated control ......................................................... 66

ix

CHAPTER 1
LITERATURE REVIEW
1. Conatrachelus nenuphar
A. Geographic Distribution and Hosts

Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), plum curculio, is
native to North America. Populations are distributed east of the Rocky Mountains
(Chapman 1938). One known exception is a localized infestation in eastern Box Elder
County, Utah, ﬁrst detected in 1980 (Alston et al. 2005).

Hosts are limited to the plant family Rosaceae, and are reviewed and surveyed in
Connecticut by Maier (1990) and in Georgia by Jenkins et al. (20063). Native hosts
include plum, choke cherry, and pin cherry (Prunus spp.), hawthorn (Crataegus spp.),
serviceberry (Amelanchier spp.), and highbush blueberry (Vaccinium spp.) (Antonelli et
al. 1992). Important exotic hosts include apple (Malus spp.), pear (Pyrus spp.), peach,
apricot, and nectarine (Prunus spp.).

B. Characteristics and Life History

The adult is dark gray and brown in color. The rough, deeply ridged elytra and
hunched appearance prompted the nickname “hunchback of the orchard” (Quaintance and
Jenne 1912). The sexes can be separated based on a groove between the 2“‘1 and 3"I pair
of legs on the male’s ventral side, which is absent in females (Thomson 1932).

Univoltine (single brood) populations, such as those in Michigan, undergo an
obligate winter reproductive diapause as adults. Females begin oogenesis and mating
only after spring emergence from overwintering (Smith and Salkeld 1964). Although

geographic strain distributions were illustrated by Chapman (1938), voltinism in the

Midatlantic US remains unclear. Evidence of bivoltine populations has been documented
as far north as West Virginia (Leskey and Wright 2004) and southern Delaware (Stearns
et al. 1935), where historic records assert only univoltine populations occur. Northern and
southern populations have been compared on the basis of molecular markers, fertility of
cross-population matings, and Wolbachia gut symbionts (Zhang 2008).

The majority of adults experimentally labeled with 65 Zn in Quebec overwintered
3-5 m into woodlots with a thick layer of fallen deciduous leaves, adjacent to orchards.
Overwintering mortality is greater for adults in leaves than for adults remaining in such
substrates as orchard turf (Laﬂeur et al. 1987), grass litter (Bobb 1949), and bare soil
(Smith and Flessel 1968). Some adults may penetrate loose soil down to 8 cm
(Armstrong 1958, Smith and Flessel 1968, Laﬂeur et al. 1987). They are not gregarious
in the fall and winter, as are some other species of beetles (Laﬂeur et al. 1987, Smith and
Flessel 1968).

The ﬁrst appearance of adults in spring is associated with temperature and host
plant phenology (Quaintance and Jenne 1929, Whitcomb 1929, Cox 1951, Smith and
Flessel 1968). In spring, adults may congregate in the groundcover at the base of the
perimeter row of fruit trees (Laﬂeur and Hill 1987, Racette et al. 1991, Chouinard et al.
1993, 1994). The aggregations are possibly caused by sounds produced by both sexes
(Mampe and Neunzig 1966). Males may mate with as many as 3 females per day (Yonce
and J acklin 1978), or an average of 10.4 females per 30 days (Johnson and Hays 1969).

Adults feed on leaves and buds before feeding on newly developing fruit (Garman
and Zappe 1929). Adults have depleted fat levels at the time of emergence from

hibernation and require feeding for ovary maturation (Smith and Salkeld 1964).

The female oviposits into fruit. With the proboscis, she cuts a characteristic
crescent-shaped ﬂap in the fruit skin and chews a small cavity underneath, reducing
chances of the egg and developing larva being crushed under the pressure of the growing
fruit (Quaintance and Jenne 1912). A portion of the characteristic oviposition marks
never receive an egg. The pearly white egg hatches within 3-12 (I (Paradis 1956). The
legless larva tunnels and feeds throughout the fruit interior for four instars. Instars are
classiﬁed by head capsule width ranges as quantiﬁedby Garman and Zappe ( 1929) and
Chapman (1938). The last instar exits fruit and enters the ground where it pupates 1-8 cm
below the surface (Quaintance and Jenne 1912).

Garman and Zappe (1929) in Connecticut observed larvae in soil for an average
of 11.6 d after entering soil, the pupal stage lasted 11 d, and the adult remained in the soil
an additional 9.8 d. Intensive fruit feeding by this “summer generation” has been
observed in Michigan cherries (Whalon unpublished) and in apples before adults seek
overwintering habitats (Smith and Salkeld 1964, Racette et al. 1992). The average fall
dispersal rate was 3.3 m/d (Lafleur et al. 1987), with a maximum recorded total distance
of 142 m (Laﬂeur and Hill 1987).

C. Pest Status & Management Techniques

Oviposition scarring and the infestation by feeding larvae diminish the
marketability of fresh produce. Oviposition and feeding can result in mangled, misshapen
fruit if it does not abscise prior to harvest. Fruit abscission caused exclusively by plum
curculio damage is difﬁcult to assess, and is not considered a negative economic
consequence in apples; the abscission may contribute to the process of fruit thinning

(Levine and Hall 1977, Racette et al. 1992).

In the southern US, peaches tend to be the most adversely impacted crop, whereas,
in Michigan damage is most economically important in apples and cherries. Processor
compliance with a national zero-tolerance standard for infested tart cherries (U SDA-
AMS 1941) can result in the rejection of an entire fruit load, resulting in serious
economic consequences for the grower, who must then pay to clean the processing line
and dispose of the rejected load. The zero-tolerance is not likely to change due to
consumer expectations for worm-free fruit, and consumer demand for unblemished fresh
produce necessitates an alternative, lower price use of cosmetically damaged fruit, such
as juice or cider making.

In the upper Midwest, organophosphate insecticides have been used for over 50
years as the primary control for plum curculio; however, US use will be terminated in
2012 in compliance with the Food Quality Protection Act (1996). Newer chemistries,
such as the neonicotinoids, oxadiazines, spinosyns, and insect growth regulators, are
being evaluated as alternatives (Wise et al. 2006a). To reduce the number of synthetic
chemical applications, recent advancements were made in plum curculio monitoring
including traps and lures (Coombs 2001, Leskey and Wright 2004).

Prior to the introduction of synthetic chemical insecticides, cultural and
mechanical controls were practiced by orchardists (Racette et al. 1992). According to
studies of plum curculio movement, overwintering habitat should be managed both
within and up to 300 m from the orchard (Laﬂeur et al. 1987). Habitat management can
consist of a spring burning of overwintering habitat (Stearns et al. 1935) or removal of
neglected or wild hosts (Maier 1990). Alternatively, early-ﬂowering hosts or wild plums

could be kept in borders as refuge or a “trap crop,” (Leskey et al. 2008), and that

concentrated area of plum curculio could be treated with an insecticide (Shapiro-Han et al.
2008, Prokopy et al. 2003, Laﬂeur et al. 1987). Some organic Michigan orchardists use a
“push-pull” strategy, similar to the “trap crop” strategy, wherein lures are placed in

border rows that remain free of kaolin clay, and the concentrated adult plum curculios are
targeted with Pyganic® (Whalon communication). Kaolin clay coverage is widely

adopted by fruit growers for preventing fruit damage from plum curculio and a number of
other pests, but keeping coverage during high precipitation levels in springtime increases
labor costs and fuel consumption for tractors. With no effective control strategies for
plum curculio available for organic producers, organic apple plots may be a source of
infestation into nearby 1PM and conventional plots (Laﬂeur et al. 2007).

Removal of adults from trees can be accomplished by limb jarring, but reduction
of oviposition damage has varied from 1-36% (Chapman 1938). Jarring trees and
collecting adults with hand labor was historically prescribed (Cook 1890), but may now
be prohibitively expensive in large orchards. Removal and destruction of infested fruit
could be accomplished by hand labor, livestock, or with a modiﬁed golf ball collecting
machine (Stedman 1904, Quaintance and Jenne 1912, Chapman 1938). Cultivating the
orchard by disking has been described as another effort to disrupt the soil-dwelling pupal
stage (Garman and Zappe 1929, Steams et al. 1935, Stedman 1904).

II. Biological Control Agents
A. Natural Enemies

Observations of natural enemies killing plum curculio are few, perhaps because 1)

adults are cryptic in appearance and behavior, 2) the egg and instar development takes

place inside the protection of the fruit host, and 3) the 4th instar emerges from fruit and

quickly burrows into soil, another cryptic habitat. A list consisting of several parasitoids
and generalist insect predators is summarized by Racette et al. (1992). The most
important natural enemies of plum curculio in a more recent natural enemy survey were
ants (F ormicidae spp.) carrying away soil-burrowing larvae (Jenkins et al. 2006b).

Vertebrates can remove adult plum curculios or wormy fruit in orchards.
Chouinard et al. (1992) found evidence that toads fed on 65Zn-labeled adults. Free range
foul failed to control plum curculio in a Michigan orchard (Clark and Gage 1996).
Quantitative measures for hogs feeding on infested June drop apples in Michigan showed
some early success (Epstein submitted).

Entomopathogenic fungi and a bacterium have been observed as natural enemies
of the plum curculio. These entomopathogens are known for their inconsistent natural
epizootics and performance as augrnentative applications against pest insect species.
Garman and Zappe (1929) report observations of two diseases of plum curculio: the
green muscardine fungus, Metarhizium anisopliae (Metchnikoff) Sorokin, and an
unidentified white fungus, “Isaria sp.”, perhaps Beauveria bassiana (Balsamo) Vuillimen.
In North Carolina, where plum curculio were collected as larvae in blueberry ﬁelds, 90%
of adults that died in the lab were infected by the entomopathogenic fungus B. bassiana
(McGiffen and Meyer 1986); the contamination source was not identiﬁed. Adults
infected with B. bassiana were collected from spring rearing boxes in Quebec within
mixed deciduous forests (Laﬂeur et a1. 1987). The bacterial pathogen Bacillus
thuringiensis (var. entomocidus or subtoxicus Heimpel) was also isolated from the same

group of adults.

N 0 records of natural infections of entomopathogenic nematodes were found,
although nematodes occurring in plum curculio-infested soil, trapped by waxworms
(Galleria mellonella L.), caused high mortality when applied to plum curculio larvae
(Alston et al. 2005).

B. Entomopathogenic nematodes

Entomopathogenic nematodes of the genera Steinemema and Heterorhabditis are
microbivorous, not directly entomophagous (Kaya and Gaugler 1993). They are
mutualistically associated with the bacteria Xenorhabdus and Photorhabdus, respectively,
which are carried in the nematode’s intestine. There, bacteria reside in a quiescent stage
inside the nematode intestine (Boemare 2002), where the bacteria remain without food.
The bacterium is released when an infective juvenile enters the haemocoel of an insect
host (Forst et al. 1997). The infective juvenile gains entry into the haemocoel by entering
a host spiracle, mouth, or anus. Following multiplication of the bacterium, the host dies
from septicemia within 24-48 h, at which time the nematode feeds on the bacteria and
produce one to several generations before new infective juveniles leave the cadaver.

Sometimes these bacteria are pathogenic to insects when experimentally injected
alone (without the nematode) into insect haemocoel, or ingested by an insect. Reversely,
sometimes nematodes are pathogenic to insects when injected alone into insect
haemocoel; for instance, when axenic (grown without bacteria) Steinemema carpocapsae
are injected into Galleria mellonella, they produce infective juveniles, completing their
life cycle (Forst and Clarke 2002). However, Xenorhabdus poinarii and Steinemema
scapterisci, when injected into a Galleria mellonella larva body cavity without each other,

are not pathogenic; they are only pathogenic when combined (Bonifassi et al. 1999).

While some non-symbiotic bacteria are able to provision some of these nematodes in the
absence of the naturally occurring symbiotic bacteria, the natural bacteria provide
nutrition for the most efﬁcient development.

The complex microbial ecology involved in keeping the natural bacteria
associated with its nematode counterpart complicates the act of mass culturing these
nematodes over many generations in laboratories for subsequent use in augmentative
applications. There is inadequate understanding of the specially required signals,
nutritional substances, and hormonal substances secreted between these symbionts and of
the physiological signiﬁcance of bacterial phase variants which may occur during mass
culture (Grewal et al. 1997).

To eliminate microbial competitors present in the nematode gut or in the insect
haemocoel, these bacteria produce antimicrobial barriers, such as molecules with high
levels of antibiotic activity both in vitro and in viva (Maxwell et al. 1994). The speciﬁc
antibiotics produced by different Xenorhabdus and Photorhabdus spp. vary (Forst and
Clarke 1992). Each nematode thus contains only one bacterial species. These nematodes
are proposed to live in a biologically unique state of “natural monoxeny;” the state occurs
inside of the free-living (soil-dwelling) nematode’s intestine, as well as within the body
cavity of the insect cadaver (Bonifassi et al. 1999).

The insect immune system protects haemolymph with cellular and humoral
responses, and is reviewed with respect to invading nematodes by Dowds and Peters
(2002). The cellular haemocytes can phagocytize small microorganisms (bacteria) or
encapsulate large invaders (nematodes) by nodulation. Haemocytes can trigger deposition

of a layer of melanin around the invader by converting prophenoloxidase to

phenoloxidase. Induced cecropins, constitutive lysozymes, and other antibacterial
peptides may be released as other humoral (cell-free) responses to bacterial invasion, but
these can be overcome by enzymes produced by the bacteria.

Nematodes may resist encapsulation by evasion (where nematode surface lipids
protect against recognition or the nematode molts), tolerance (where nematode numbers
overwhelm the haemocoel), or immune suppression (nematode surface coat proteins).
Either way, nematodes may release bacteria before they are encapsulated, which adhere
to and kill haemocytes, and may cause insect death with or without nematode
reproduction.

C. Entomopathogenic fungi

The deuteromycete, hyphomycete fungi Beauveria bassiana (Balsamo) Vuillemin
and Metarhizium anisopliae (Metschnikoff) Sorokin are facultative pathogens. They are
the causal agents of white and green muscardine disease in insects, respectively.
Although these fungi may enter the host via mouth, anus, or spiracles, direct penetration
of the insect cuticle after attachment of a germinating spore (conidium) is the standard
approach of fungal entry into the insect body. These fungi form an appresorium and
penetration peg, structures also produced by some plant pathogens. Once inside the
haemocoel, the mycelium moves through the body and forms blastospores (hyphal
bodies). Host death is caused by a combination of fungal toxin, physical obstruction of
blood circulation, nutrient depletion, and invasion of organs (Goettel and Inglis 1997).
The hyphae then emerge from the cadaver and produce conidia under appropriate

temperature and humidity conditions, which may then infect new hosts.

In common with entomopathogenic nematodes, host susceptibility to the fungal
pathogen is inﬂuenced by fungal strain, host physiological state, nutrition, defense
mechanisms, cuticular and epicuticular microorganisms, and the environmental
conditions (Goettel and Inglis 1997). Also common to both fungi and nematodes are the
cellular and humoral responses described above.

St. Leger (1993) illustrates the barriers to cuticle penetration by fungi. Preformed
barriers in insect epicuticles include surface structures, a hydrophobic barrier (important
for dry, nonsticky conidia), electrostatic charges, low relative humidity, low nutrient
levels, competing microbial ﬂora, toxic cuticular lipids and phenols, and tanned proteins.
Preformed barriers to the procuticle include protease inhibitors and tanned proteins in
combination with crystalline chitin which results in a stiff and desiccated cuticle. Barriers
in the cuticle that are induced upon recognition of the spore include attachment and
activation of prophenoloxidase, resulting in melanization.

Mucus formed by M. anisopliae may act as both an adhesive and a protectant
from dessication and host polyphenols (Boucias and Penland 1991). The restricted host
range of fungal strains may be a result of speciﬁc requirements for germination,
involving nutrients such as cuticular lipids like hydrocarbons (Charnley 1984). Most
strains within both of these species tend to have a broad host range and little speciﬁcity
for infection site; so appresorium formation occurs immediately upon germination on a
ﬂat cuticular surface, but hyphal growth may be directed away from heavily sclerotized
regions, on the path of least resistance (Pekrul and Grula, 1979). Therefore, sclerotization,

cuticle thickness, and tensile strength of the cuticle formed by chitin lamellae may affect

10

growth behavior of the pathogens during penetration (Zacharuk 1970, Hassan and
Charnley 1989).

Metarhizium anisopliae has low endogenous reserves, and so the host cuticle must
provide nutrients required for penetration, which are freed by the secretion of
extracellular cuticle-degrading enzymes produced by the fungus. Cuticle—degrading
endoproteases contribute more signiﬁcantly to the virulence of pathogens than do other
host speciﬁcity factors. Host recognition mechanisms are keyed to nutrient levels
available on appropriate host cuticles (St. Leger et. al 1992). Only when these
extracellular nutrients are depleted does the pathogen form structures for penetration.
Protein, chitin, and lipids are the main polymers in insect cuticle; a range of cuticle-
degrading enzymes produced by pathogens correspond to these polymers (Charnley and
St. Leger 1991). Using a labeled antibody against a pathogen protease, Goettel et al.
(1989) showed that procuticle penetration involves both enzymatic degradation and
mechanical separation of the lamellae, whereas epicuticle penetration is primarily by
enzymatic degradation only.

When tested, the impact of the cellular host defenses on M. anisopliae var.
acridum appeared to be minimal since the haemocytes remained unattached to fungal
particles and nodules did not incorporate fungal particles (Gillepsie et al. 2000). The
pathogen itself secretes toxins. Destruxins are cytotoxic fungal metabolites produced by
M. anisopliae var. anisopliae that are toxic to haemocytes (Huxham et al. 1989). One
counter to a host resistance mechanism is the detoxiﬁcation of the insect’s

polyphenoloxydase. The destruxins prevent phenoloxydase production by locust

11

haemocytes by destroying the cells that produce it (Cerenius et al. 1990). The metabolites
beauvericin and oosporein produced by Beauveria sp. exhibit antibacterial action.

Inundative releases of M. anisopliae and B. bassiana typically involve application
of aqueous or granular formulations to soils, where these species reside as facultative
saprophytes. A granular formulation of Beauveria brongniartii is commercially available
for European cockchafer biocontrol (Kessler et al. 2004). A simple maize-based B.
bassiana formulation developed for subsistence farmers was effective against banana root
weevil (Nankinga and Moore 2000) and granular M. anisopliae increased yield in ﬁelds
with sugarbeet root maggot (Campbell et al. 2006). A granular formulation of B. bassiana
on rice established in pasture better than a biopolymer when considered for clover root
weevil control (Nelson et al. 2004).
D. Insect responses to entomopathogenic nematodes and fungi

The effectiveness of entomopathogens for insects may be inﬂuenced by
behavioral responses of insects, both by affecting infection rates of insects exposed to
inocula and dispersal of inocula to other insects. Such behaviors may not be limited to
grooming, secretion of antibiotics, nest hygiene in social insects, avoidance, and dispersal.

Horizontal infection has been documented in many insect/entomopathogen
systems. Confoundingly, both insect avoidance of and attraction to entomopathogens has
been described in some systems. In the laboratory, termites were repelled by the
nematode Heterorhabditis indica for up to 17 days (Wang et al. 2002). Colorado Potato
Beetle was not repelled by sporulating cadavers infected with B. bassiana (Klinger et al.
2006). Japanese beetle grubs actively avoided areas where M. anisopliae was added

(V illani et al. 1994), but black vine weevil grubs were attracted to M. anisopliae only

12

when applied in conjunction with plants. This ﬁnding suggests a complex tritrophic
interaction only in the early stages of exploration (Kepler and Bruck 2006). The authors
hypothesize that the fungus may produce volatile compounds attractive to the pest only in
the presence of a plant rhizosphere, but this has yet to be tested.

In the presence of nematodes, white grubs exhibit aggressive grooming behaviors
such as rubbing with an abrasive raster on the abdomen, brushing with legs, or scraping
or chewing with mandibles (Koppenhofer et al. 2000). The spiracles of white grubs are
covered with sieve plates, making them inaccessible to nematodes. In some ants,
grooming, including the use of tibial combs, rubbing of legs, and licking, does not result
in the ingestion of inocula since they are collected in an infrabuccal pocket (Eisner and
Happ 1962). Dissemination of inocula between individuals by grooming was seen in
termites (Kramm et al. 1982). Secretions of venom alkaloids, phenyl acetic acid, or
myrmicacin seemingly reduce pathogenicity of fungi in ants (Oi and Pereira 1993).
Removal of B. bassiana- infected cadavers from ant nests and isolation of infected
nestrnates by burial have been observed (Oi and Pereira 1993).

Since insects are “cold-blooded,” they thermoregulate themselves behaviorally. A
febrile response, or so-called “behavioral fever,” is associated with infection of locusts by
M. anisopliae. Here the individual insect moves to an area of elevated temperature, and
this is thought to suppress pathogen growth and enhance the immune system.

Behavioral modulation of insects by parasites may enhance the parasite’s
transmission potential. For instance, ants and ﬂies infected with the entomopathogenic

fungus Entomopthora have a propensity to disperse to and die at elevated locations, such

13

as clinging (or fastened by fungal rhizoids) to the tops of grass blades, facilitating
dispersal of the pathogen spores (Humber 1981).

In conclusion, the physical, biochemical, and behavioral barriers to
entomopathogen infection of an insect host are abundant, poorly understood, and vary
greatly between entomopathogen strains and insect species. Concepts related to volatile
cues and toxins are only in the early period of scientiﬁc understanding. As conventional
chemical control agents for use in agriculture and forestry are cancelled by the
Environmental Protection Agency, control tactics that are perceived as more natural, such
as entomopathogens, are becoming more signiﬁcant as exhibited by the recent literature.
Forthcoming knowledge of the mechanisms of insect resistance to entomopathogens may
soon prove to increase the efﬁcacy of augmentative applications of entomopathogens as
replacements for conventional insecticides. Further integration of new physiological,
ecological, and behavioral understandings may result in a conservation approach, where
landscapes are manipulated ecologically to enhance crop protection by natural or
naturalized populations of entomopathogens.

E. Entomopathogen experiments against plum curculio

In laboratory assays, entomopathogenic nematodes, particularly strains of
Steinemema riobrave (Cabanillas, Poinar, and Raulston), Steinememafeltiae (Filipjev),
Steinemema carpocapsae (W eiser), and Heterorhabditis bacteriophora (Poinar), have
demonstrated efﬁcacy against last instar plum curculio larvae (Brossard et al. 1989,
Shapiro-ﬂan et al. 2002, Alston et al. 2005). Olthof & Hagley (1993) reported 73-90%
mortality for S. feltiae Biosys27 strain and S. carpocapsae All strain against last instar

plum curculio; however, other authors found low efﬁcacy of both the S. carpocapsae All

14

strain as well as a strain of S. carpocapsae collected in Mississippi (T edders et al. 1982,
Shapiro-Han et al. 2002). Steinemema riobrave 355 strain reduced adult emergence by
77-97% when applied to soil in ﬁeld cages containing plum curculio larvae in Georgia
(Shapiro-ﬂan et al. 2004a). S. feltiae was not effective in the same trial, and demonstrated
lower mortality (22-39%) in another caged trial in a drier climate in Utah (Shapiro-[Ian et
al. 2004a, Alston et al. 2005).

Cold tolerance is generally exhibited by Steinemema spp. while heat tolerance is
generally exhibited by Heterorhabditis spp. (Molyneux 1986, Grewal et al. 1994). A
strain of H. bacteriophora from Utah was overall more virulent than S. feltiae to all plum
curculio life stages, and adults and pupae were more susceptible than larvae (Kim and
Alston 2008). On the surrogate insect species Galleria mellonella, virulence and
reproductive potential of H. bacteriophora was superior to S. feltiae at 30°C, and inferior
at 10°C (Kim and Alston 2008).

Soil applications of S. riobrave 355 strain targeting plum curculio larvae in a wild
plum thicket caused 100% control in two years, while 3-8b strain caused > 87.9% control
(Shapiro-Han et al. 2008). Late-term S. riobrave and S. carpocapsae applications
targeting plum curculio after onset of adult emergence failed to result in signiﬁcant
control (Shapiro-Han et al. 2008). Foliar applications of S. carpocapsae in June did not
reduce plum curculio damage at harvest in apples, but border-row treatments reduced
damage by 75% and 30%, with economically acceptable levels of damage in only one of
the two years (Bélair et al. 1998).

In a laboratory bioassay, a South Carolina strain of the entomopathogenic fungus

B. bassiana caused slower mortality of last instar plum curculio larvae, compared to a

15

South Carolina strain of the fungus M. anisopliae (Tedders et al. 1982). A California
strain of M. anisopliae was not effective in this same study. Strains of M. anisopliae
tested against larvae in small cups of soil in the laboratory showed high variability in
LCso values between strains (Alston et al. 2005), but M. anisopliae was not evaluated in
the ﬁeld. Beauveria bassiana GHA strain did not signiﬁcantly reduce numbers of
emerging adults when applied to soil containing larvae in Georgia (Jenkins et al. 2006b).
While efﬁcacy of nematodes for suppression of plum curculio adults has been evaluated
in the laboratory (Shapiro-[Ian et al. 2002), neither M. anisopliae nor B. bassiana have
been evaluated against plum curculio adults.

Field efﬁcacy of entomopathogens to consistently control plum curculio at levels
greater than 40% has only been demonstrated for S. riobrave 355 strain in the southern
US (Shapiro-Han et al.2004a, 2008) and with H. bacteriophora in residential fruit trees
in Utah (Kim 2007). Kim (2007) demonstrated 56, 69, and 73% control of plum curculio
after one, two, and three years of multiple H. bacteriophora applications. Due to high
costs associated with augmentative applications of entomopathogens and historically
variable ﬁeld results, region-speciﬁc and production system-speciﬁc ﬁeld efﬁcacy
evaluations of candidate strains are necessary. Results could provide growers with the
information needed to weigh these beneﬁts against the higher cost of treatment.

IV. Thesis Research

The goal of the research herein is to demonstrate the efﬁcacy of augmentative
releases of entomopathogenic fungi and nematodes for control of plum curculio in
. Michigan orchards. Adults were targeted with entomopathogenic fungi and larvae were

targeted with both entomopathogenic fungi and nematodes. Field efﬁcacy experiments

16

against larvae were conducted for three years in a total of ten different orchards.
Alternative means of exposing adult plum curculios to fungi were investigated, including
aqueous and granular formulation applications to soils and surfaces that could be adapted
to an autodissemination trap. This research is a stepping stone for farrnscale
implementation of entomopathogens for plum curculio management.

The next phases of this research will be to: 1) develop a phenology model that
predicts larval emergence from fruit to narrow recommended entomopathogen
application timings for tree fruit growers in different regions, 2) improve the
understanding of short-term inundative entomopathogen efﬁcacy persistence in different
formulations and in different soil types under microhabitat moisture manipulation, and 3)
investigate the efﬁcacy of fungus formulations and delivery methods against adults in

orchards.

l7

CHAPTER 2
LABORATORY EVALUATIONS OF ENTOMOPATHOGENIC FUNGUS
EFFICACY AGAINST LAST INSTAR PLUM CURCULIO
1. Introduction

Plum curculio, Conotrachelus nenuphar (Herbst), is a key pest of tree fruit and is
susceptible to disease caused by entomopathogenic fungi. Although initial damage to
fruit would not be prevented within-season, mortality at the last-instar stage would reduce
the emergence of second generation adults that cause damage the following season.
Currently the fungus Beauveria bassiana (Balsamo) Vuillemin strain GHA is registered
with EPA for food crops and labeled for plum curculio. Metarhizium anisopliae
(Metschnikoff) Sorokin strain F52 is registered as a control for greenhouse and nursery
crops, but not currently for food crops. Both have a relatively broad host range.

In a laboratory soil bioassay, a South Carolina strain of B. bassiana caused
mortality of last-instar plum curculio, but caused mortality at a slower rate than a South
Carolina strain of the fungus M. anisopliae (T edders et a1. 1982). Yet both isolates caused
high levels of mortality after 14 d. In a similar laboratory bioassay against last-instars, 17
M. anisopliae isolates resulted in varying LTso (4-22.8 d), indicating differing virulence
between strains (Alston et al. 2005). In Georgia, a ﬁeld application of B. bassiana strain
GHA in Mycotrol-O formulation to orchard soils with plum curculio-infested fruit
overlaid, failed to reduce adult emergence (Jenkins et al. 2006b). The researchers used
naturally plum curculio-infested fruit, with a single B. bassiana application, so the time
between B. bassiana application and larval contact was unknown. Further, there was no

mention of soil type or other edaphic factors that could have inﬂuenced mortality.
18

Another factor that could have contributed to limited ﬁeld efﬁcacy is that B. bassiana
GHA strain may not be as virulent as other strains of entomopathogenic fungi.

Laboratory-based virulence is not the only factor that contributes to successful
ﬁeld efﬁcacy; for instance, a strain collected from an orchard might be expected to persist
better in the ﬁeld following augmentative application if it is locally adapted to both biotic
and abiotic conditions characteristic to that environment. Alternatively, the ‘new
associations’ hypothesis in biological control asserts that the introduction of natural
enemies from the prey species native range may be less effective than novel
combinations of natural enemies and prey (Hokkanen and Pimental 1984). Other factors
such as dose, timing, coverage, pH, and pest age and condition contribute to efﬁcacy
(T anada and Kaya 1993).

The objective of this study was to test the efﬁcacy of the commercial B. bassiana
and M. anisopliae strains, as well as a native strain against pupation-bound last-instar
plum curculio in the laboratory. Bioassays were carried out by standard immersion
exposure methods.

2. Methods
2.1 Larvae and Entomopathogens

Newly emerged (<24 h) last-instar plum curculios were obtained from a colony
maintained by the Pesticide Alternatives Laboratory, Michigan State University, East
Iansing, MI. The colony was reinitiated annually from adults and infested cherries and
apples collected in six orchards in Benzie, Ionia, Leelanau, and Manistee Co., MI. Adults
were collected using circle traps (Mulder et al. 1997) and “Whalon pyramid traps” (1.6 m

x 0.8 m) made from recyclable black corrugated plastic with high-contrast reﬂective tape

19

(Great lakes 1PM, Vestaburg, MI). Both trapping systems were equipped with plum
essence lures and stabilized benzaldehyde vials (Coombs 2001, Leskey and Wright 2004,
Leskey et al. 2005). In order to break diapause and induce mating and oviposition, adults
were exposed to thinning apples that had been dipped in a solution of 750 mg
pyriproxifen (Esteem® 35 WP) per L water (Hoffmann et al. 2007).

We used commercial strains of B. bassiana (GHA strain received as Mycotrol-O®
from Laverlam International, Butte, MT) and M. anisopliae (FSZ strain received as
Taenure® granular formulation, Novozymes Biologicals, Inc., Salem, VA). A strain of
M. anisopliae referred to hereafter as strain 8270 was isolated on a selective medium
from a highly infested organic tart cherry orchard in Leelanau Co., MI in June 2006
(identiﬁed by Richard Humber, accession #8270 at the USDA Agricultural Research
Service Entomopathogenic Fungus Culture Collection (ARSEF), Ithaca, NY). Fungal
inoculum viability was assessed according to Goettel & Inglis (1997) by pouring 50 ul of
each suspension within 30 rrrin of application onto three dishes of potato dextrose agar
and checking 200 spores/dish for the presence of germ tubes after 20 h with an Olympus
BX40 research microscope.

2.2 Immersion Bioassay

Aqueous suspensions of M. anisopliae F52, M. anisopliae 8270, and B. bassiana
GHA were prepared in 10 ml plastic centrifuge tubes. Conidia concentrations were
adjusted to 1 x 105 and 1 x 106 conidia/ml (3270 was tested at 1 x 10‘5 only). Last-instars
were immersed individually for 10 s and placed on sterile Whatman #1 ﬁlter paper to
remove excess liquid. The tubes were vortexed for 15 s between larvae. The larvae were

placed individually in 90 x 10 mm Petri dishes lined with two pieces of sterile ﬁlter paper
20

with 1.5 ml sterile water, sealed with paraﬁlm. Petri dishes were incubated in the dark at
25C for 14 d, after which mortality and signs of sporulation visible under a Nikon
SMZlOOO (Mager Scientiﬁc, Inc., Dexter, MI) stereo dissecting microscope were
recorded.

There were 12 larvae per treatment and the experiment was repeated on ﬁve dates.
The M. anisopliae 8270 treatment was tested on only two of the ﬁve dates. Percent
mortality and percent sporulation were both arcsine square root transformed to meet
normality assumptions and pairwise comparisons were made among all treatments using
Tukey’s HSD, d=0.05 (SAS Institute 2004).
3. Results
3.1 Immersion Bioassay

Spore viability was always greater than 95% (Table 2.1). The F52 106 treatment
resulted in mortality and sporulation (98 and 93%) signiﬁcantly higher than all other
treatments (p<0.05) (Figure 2.2). Mortality and sporulation in the 8207 10‘5 treatment (58
and 50%) was comparable to F52 105 and signiﬁcantly lower than the F52 106, but
signiﬁcantly higher than control. Mortality and sporulation in GHA 105 and GHA 106
were not different from control. Mortality in GHA 10‘5 was not different from F52 105
and 8207 10".

Table 2.1. Percent viability of conidia in immersion suspensions on each test date.

 

Date

 

Isolate 1 2 3 4 5

 

 

Bb GHA 98.83 99 99.33 99.67 98.83
Ma F52 99.83 95.33 99.88 100 99.33
Ma 8270 - - 99.88 - 98.83

 

 

 

 

 

 

 

 

 

21

 

 

1 00% . . .
I I Mortality D Larvae wrth sporulation I

 

80%

60%

40%

 

20%

 

 

 

0%

Percent Mortality or Sporulation

 

 

Treatment
Figure 2.1. Mean (1 SE) percent mortality and percent of larvae with sporulation 14 d after a 10 s

5 6
immersion in 1x10 or 1x10 conidia/ml suspensions of M. anisopliae F52, B. bassiana strain GHA, M.

anisopliae 8270, or water control. Bars with different letters indicate signiﬁcant difference among
treatments within mortality or within sporulation (T ukey’s HSD, a=0.05).
4. Discussion

In the immersion bioassay at optimal conditions, the commercial M. anisopliae
strain F52 resulted in higher mortality and sporulation on plum curculio larvae cadavers
than the commercial B. bassiana strain GHA labeled for plum curculio. More replicates
of the M. anisopliae Michigan soil isolate 8270 may reduce variability and show more
clearly whether 8270 causes higher mortality than GHA.

Laboratory virulence assays, especially those using an immersion method, may
not be indicative of ﬁeld efﬁcacy. For instance, one physical aspect that this immersion
assay does not account for is the difference in hydrophobicity between fungus species
which may interfere with conidia adhesion to the insect cuticle; consequently, dose levels
cannot be measured precisely (Goettel and Inglis 1997). Consecutive immersion of larvae

into a single conidia suspension may have reduced the conidia concentration between
22

irnmersions. Field conditions such as soil texture, temperature, water'activity,
agrochemical exposure, and UV light exposure are common reasons for variation in
entomopathogenic fungus efﬁcacy between laboratory and ﬁeld experiments (McCoy et
al. 1992).

In addition to ﬁeld efﬁcacy trials, registration for food use and a formulation
approved by the Organic Materials Review Institute would be required for organic
grower use of M. anisopliae for plum curculio management. Issues such as difﬁculty
sourcing organic materials and the small organic market size may impede further
investments by the companies that hold rights to the EPA-registered strains and
formulations. The cost of registration processes for entomopathogenic fungi is an
impediment to the registration of other strains (Butt and Coping 2000), so had the 8270
strain been signiﬁcantly more virulent to plum curculio, the time and a high cost to

register the strain would likely inhibit registration.

23

CHAPTER 3
ENTOMOPATHOGENIC NEMATODE AND FUNGUS FIELD EFFICACY
AGAINST LAST INSTAR PLUM CURCULIO
1. Introduction

Plum curculio, Conotrachelus nenuphar (Herbst), has adapted well to cultivated
tree fruits throughout its range in eastern North America (Maier 1990) and is a key pest in
cherry, apple, and peach production. In Michigan, overwintering adults emerge from
obligate diapause in overwintering habitats, migrate into orchards, feed, mate, and
I oviposit in fruits (Smith 1957). Larval feeding occurs exclusively inside fruit throughout
development. Fourth instars exit fruits, burrow into the soil where they pupate at a depth
of 1-8 cm (Quaintance and Jenne 1912), and emerge as summer adults. If early season
control is incomplete, summer adults can contribute signiﬁcantly to late-season damage
(Racette et al. 1992) and become the next year’s ovipositing population.

In the upper Midwest, organophosphate insecticides have been used for over 50
years as the primary control for plum curculio particularly in cherry, but also in apples,
plums, and peaches. However, US use will be terminated in 2012 in compliance with the
Food Quality Protection Act (1996). Newer cherrristries, such as the neonicotinoids,
oxadiazines, spinosyns, and insect growth regulators, are being evaluated as alternatives
to the organophosphates (Wise et al. 2006a). The over-reliance on one class of
insecticides in agricultural systems raises concerns about the potential for insecticide
resistance (Denholrn and Rowland 1992, Nauen and Denholrn 2005, Whalon et al. 2008)
and negative effects on non-target insects and other invertebrates (Croft and Whalon

1982, Theiling and Croft 1988, Desneux et al. 2007). Incorporation of control agents such

24

as entomopathogenic fungi and nematodes into orchard integrated pest management
systems has been proposed as a way to mitigate some of these concerns (Bathon 1996,
Roy and Pell 2000, Lacey and Shapiro-Han 2008).

For producers in the wet and humid Upper Midwest where plum curculio is
problematic, the higher humidity and rainfall may enhance effective augmentative
releases of natural enemies, presenting an opportunity to produce quality fruit that may
approach the cosmetic standards of fruit produced in more arid regions where plum
curculio is not present. Such improvements in pest management will contribute to the
feasibility of increasing fruit produced for local consumption in this region. If production
was maintained and expanded in the Upper Midwest, signiﬁcant reduction in energy
consumption from long-distance western shipping would result.

Historically, only active adult plum curculio were targeted with chemical
insecticide sprays because the organophosphates yielded excellent control, including
“curative” activity on life stages within fruit (Wise et al. 2007). Augmentative
entomopathogen application for control of soil-dwelling life stages offers an opportunity
to suppress the pest at a previously untargeted life stage - a major goal of higher level
IPM programs (Kogan 1988). The plum curculio life cycle includes four duff- and soil-
dwelling stages that may be vulnerable to soil-borne entomopathogens: the soil-
burrowing last-instar larva, the pupa, the emerging adult, and the overwintering adult.

In laboratory assays, entomopathogenic nematodes, particularly strains of
Steinemema riobrave (Cabanillas, Poinar, and Raulston), Steinememafeltiae (Filipjev),
Steinemema carpocapsae (W eiser), and Heterorhabditis bacteriophora (Poinar), have

demonstrated efﬁcacy on last-instar plum curculio, (Shapiro-[Ian et a1. 2002, Alston et al.

25

2005). Olthof & Hagley (1993) reported 73-90% mortality for S. feltiae Biosy827 strain
and S. carpocapsae All strain against last-instar plum curculio; however, other authors
found low efﬁcacy of both the S. carpocapsae All strain as well as a Mississippi-
collected S. carpocapsae strain (T edders et al. 1982, Shapiro-Han et al. 2002).
Steinemema riobrave 355 strain reduced adult emergence by 77-97% when applied to
soil in ﬁeld cages containing plum curculio larvae in Georgia (Shapiro-Han et al. 2004a).
S. feltiae was not effective in the same trial, and demonstrated lower mortality (22—39%)
in another caged trial in a drier climate in Utah (Shapiro-Han et al. 2004a, Alston et al.
2005).

In a laboratory bioassay, a South Carolina strain of the entomopathogenic fungus
Beauveria bassiana (Balsamo) Vuillemin caused slower mortality of last-instar plum
curculio, compared to a South Carolina strain of the fungus Metarhizium anisopliae
(Metschnikoff) Sorokin (Tedders et al. 1982). A California strain of M. anisopliae was
not effective in this same study. Strains of M. anisopliae tested against larvae in small
cups of soil in the laboratory showed high variability in LCso values between strains
(Alston et al. 2005), but M. anisopliae was not evaluated in the ﬁeld. In Georgia, B.
bassiana GHA strain did not signiﬁcantly reduce numbers of emerging adults when
applied to soil containing larvae (Jenkins et al. 2006b). The researchers used naturally
plum curculio-infested fruit, with a single application, so the time between B. bassiana
application and larval contact was unknown. Further, there was no mention of the soil
type or other edaphic factors that could have inﬂuenced mortality.

Field efﬁcacy of entomopathogens to consistently control plum curculio at levels

greater than 40% has only been demonstrated for S. riobrave 355 strain in the southern

26

US (Shapiro-[Ian et al. 2004a, 2008) and with H. bacteriophora in residential fruit trees
in Utah (Kim 2007). Kim (2007) demonstrated 56, 69, and 73% control of plum curculio
after one, two, and three years of multiple H. bacteriophora applications. Due to high
costs associated with augmentative applications of entomopathogens and historically
variable results, region-speciﬁc and production system-speciﬁc ﬁeld efﬁcacy evaluations
of candidate strains are necessary. Efﬁcacy results would provide growers with the
information needed to weigh these beneﬁts against the higher cost of treatment in the
context of reduced environmental and ecosystem services impacts of entomopathogens
(Goettel and Hajek 2000, Barbercheck and Millar 2000).

The overall goal of this study was to evaluate efﬁcacy of commercially available
entomopathogens against plum curculio in Michigan. Our objective was to evaluate ﬁeld
efﬁcacy of ﬁve soil-applied entomopathogens (M. anisopliae, B. bassiana, H.
bacteriophora, S. carpocapsae, S. riobrave, and H. bacteriophora) timed precisely
against emerged last-instars or against last-instars as they emerged from fruit over time.
2. Materials & Methods
2.1 Entomopathogens

We used commercial strains of B. bassiana (GHA strain Mycotrol-O® liquid
formulation, Laverlam International, Butte, MT) and M. anisopliae (F52 strain Taenure®
granular formulation, Novozymes Biologicals, Inc., Salem, VA). The commercial strains
of nematodes included S. carpocapsae (All strain in a vermiculite-based formulation) and
S. riobrave (355 strain in a petroleum-based gel formulation) were supplied by Becker
Underwood (Ames, IA). The H. bacteriophora strain isolated from Utah (Alston et al.

2005) was not formulated.

27

Aqueous suspensions of fungi were prepared by scraping M. anisopliae or B.
bassiana conidia from 2-wk old potato dextrose agar (PDA) plate cultures into sterile
water with 0.01% Tween-80. A granular formulation of B. bassiana was produced in the
laboratory by colonizing broken rice in autoclavable bags following methods of Nelson et
al. (2004). The bags were inoculated with conidia on PDA isolated from a last-instar
plum curculio 8 d after immersion in a Mycotrol-O suspension. The number of conidia
per gram of granular material was approximated by placing a 5 g sample into a ﬂask
containing 100 ml of 0.01% Tween-80. Flasks were placed on a rotating shaker at 180
RPM for 5 min, and the number of spores per ml of solution was determined using a
hemacytometer (4 counts per ﬂask). The mean (:SE) count of 3 ﬂasks was 4.75:0.25 x
108 conidia/g.

Fungal inoculum viability was assessed according to Goettel & Inglis (1997) by
pouring 50 ul of each suspension within 30 min of application onto three dishes of PDA
and checking 200 spores/dish for the presence of germ tubes after 20 h, with an Olympus
BX40 compound microscope. Viability of nematode infective juveniles (Us) was
determined within 30 min of application by viewing 3 samples of 100 Us under a Nikon
SMZlOOO (Mager Scientiﬁc, Inc., Dexter, MI) stereo dissecting microscope. Straight,
still US not responding to a probe and without a characteristic ‘1 ’ shape were considered
dead (Goettel & Inglis 1997). Nematode viability was >95% for every application.

2.2 Field Sites

Soil samples to a depth of 8 cm from ﬁeld sites were analyzed by Michigan State

University Soil and Plant Nutrient Lab (Table 3.1). A nematode community analysis was

conducted on samples from the 2008 sites at the Michigan State University Nematode

28

Diagnostics Lab, East Lansing, MI (Table 3.2). Percent soil moisture values were
obtained using the gravirnetric method by weighing oven-dried soil samples (Table 3.3).

In 2006, the trial was conducted among two organic tart cherry orchards and two
organic apple orchards. In 2007, multiple trials were conducted in seven 0.21 ha apple
orchard blocks at the Michigan State University Clarksville Horticulture Experiment
Station, Ionia County, Michigan. Apple trees were planted in 1994 and were mixed
cultivars as described by Salazar et al. (2007). Tree spacing was 1.5 111 within tree-row
and row spacing was 4.5 m. The drive row and drip line were mowed 1412 d prior to
treatment applications. The orchard understory was primarily composed of orchard grass
and bare soil. Experimental plots received water from drip irrigation tubing placed along
tree trunks throughout the experiment. Experimental plots received no chemical
herbicides or insecticides; however, sulfur and other agents were applied in experimental
plots to control apple scab (Wise et al. 2006b) as indicated in the spray record (Table 3.2).

In 2008, the experiment was conducted among three conventional tart cherry and
two organic apple orchards.
2.3 Eﬂ‘icacy and Timing with Enraged Larvae

Trials were conducted in 2006, 2007, and 2008 using newly emerged last-instars
to evaluate the effectiveness of various entomopathogen formulations in pots in orchards.
Among all trials, the experimental unit was ten last-instars per pot. The number of adults
emerging from each pot was recorded every 5-10 d until day 60, starting 25 (1 post-
treatment (adults were collected less frequently in 2008).

The round plastic pots (25 cm tall x 10 cm diam.) were ﬁtted with a screen bottom

for rain water drainage and a conical boll weevil trap top (USDA Boll Weevil Eradication

29

Foundation, Abilene, TX). The trap top was modiﬁed to ﬁt tightly in the pot’s upper rim
to collect emerging plum curculio adults. Pots were installed under the canopy of trees
within 1 m of the trunk by removing soil cores with a 10 cm diam. golf-hole cutter. Soil
from the cutter was carefully transferred into pots without disturbing the interface
between the duff and the A horizon. Filled pots were placed into the cutter holes, with pot
rims uniformly level with the soil surface.

Oviposition-marked fruits were held in the laboratory on metal screen racks over
aluminum trays lined with moist paper towel. Emerged larvae were held in groups of ten
in 90 mm Petri dishes at 25°C in the dark. The dishes were lined with two pieces
Whatrnan #1 ﬁlter paper moistened with 1.25 ml sterile water and sealed with Paraﬁlm®.
Larvae were added to the soil surface in pots within 36 h of emergence from fruits.
2006: Granular & sprayed B. bassiana with a single timing

Beauveria bassiana was tested in both Mycotrol-O and granular (rice)
formulation. In two tart cherry and one apple orchard, treatments included three rates of
rice (2 x 10”, 2 x 10”, and 2 x 10ls conidia/ha for low, medium, and high rates,
respectively) and a single rate of Mycotrol-O spray (2x10l5 conidia/ha). Mycotrol-O was
delivered to soil with a 7 L Solo backpack sprayer in 50 ml water per m2. Four pots with
the same treatment were installed under each of three trees per orchard, randomly
selected within one row. In the remaining apple orchard, treatments included the high rate
of rice and Mycotrol-O spray applied on 14 July. Six pots with the same treatment were
installed under each of three randomly selected trees in a row.

Water was delivered to plots at a rate of 500 ml/m2 following applications. A

wood and window screen ground cage was installed over the top of pots to prevent

30

interference by wild predators. Ground cages were not used in 2007 and 2008, to reduce
chances of causing nricrohabitat changes in humidity, temperature, and soil moisture
from normal orchard conditions. A furrower was passed over the plots at 6 cm depth prior
to pot installation, only in 2006.

Larvae were placed on the soil surface in pots within 1 h of pathogen application.
Larvae were collected from tart cherries collected from Leelanau Co. in the northernmost
orchard.

2007: B. bassm M. anisopliae, S. camocgpsae, & S. riobrave with three timings

The 2007 trial expanded upon the 2006 trial by testing several aqueous pathogen
formulations, with the addition of larvae at one of three intervals: 0, 3, or 5 d from
pathogen applications.

An entomopathogen (B. bassiana, M. anisopliae, S. carpocapsae, or S. riobrave)
or the “no” treatment control (50 ml sterile water with 0.01% Tween-80) was applied by
evenly pouring 50 ml of aqueous suspension onto the soil surface within each pot. Fungal
suspensions were adjusted to a concentration of 5 x 1013 conidia/ha. The two nematode
species in their respective commercial formulations were adjusted to a concentration of 4 .
x 109 IJs/ha. Viability was 93.3%, 95.3%, and 100% for B. bassiana, M. anisopliae, and
both nematodes, respectively.

There were four pathogen treatments and one untreated control, each with three
larva-placement timings (post-treatment intervals). This resulted in 12 “pathogen x larva-
tirning” treatments and 3 “untreated control x larva-timing treatments”. Ten pots per tree
were installed and treatments were randomly assigned to pots in each of six tree rows.

There were 30 pots of each entomopathogen and control for each of the no delay larva-

31

timings and 15 pots of each entomopathogen and control for each of the 3-d and 5-d
larva-timings in one orchard block. The experiment was repeated on 30 July, but reps
were reduced to 25 and 12 pots per treatment, respectively.

larvae were reared from on-site apples held in cages with adults for oviposition.
Adults had been collected using circle traps (Mulder et al. 1997) and black corrugated
plastic “Whalon pyramid traps” (1.6 m x 0.8 m) with high-contrast reﬂective tape (Great
lakes IPM, Vestaburg, MI), equipped with plum essence lures and stabilized
benzaldehyde vials (Coombs 2001, Leskey and Wright 2004, Leskey et a1. 2005) in April,
May, and June in cherry and apple orchards in Benzie and Manistee Co., MI.

2008: B. bassm S. riobrave, and H. bacteriophora with seven timings

The 2008 trial expanded upon the 2007 trial by testing aqueous pathogen
applications with the addition of larvae at one of seven intervals: -10, -5, 0, 5, 10, 15, or
20 d from pathogen application.

This experiment was repeated in July 2008 in ﬁve orchards with several
modiﬁcations as follows. The nematodes S. riobrave and H. bacteriophora were tested at
a low and high rate (1 x 109 and 4 x 109 conidia/ha). Beauveria bassiana was tested at the
same rate as in 2007 (5 x 10‘3 conidia/ha), but in Mycotrol-O® formulation instead of a
Tween-80 conidia suspension. There were six pathogen treatments (B. bassiana, H.
bacteriophora low, H. bacteriophora high, S. riobrave low, S. riobrave high, and control).
Larvae were added to pots -10, -5, 0, 5, 10, 15, or 20 d from pathogen application. larvae
were obtained using the same method as in 2007. There were eight reps of each of the

“pathogen x timing” combinations per orchard, and these were randomly assigned within

32

3-tnee plots containing 36 pots each. H. bacteriophora was not tested for the -10, 10, and
20 d timings.
Statistical Analysis

The adult emergence data was analyzed with SAS (SAS Institute 2004) with
a=0.05. Emergence data from 2006 were analyzed for each orchard individually using
proc GLM and mean separations were conducted using Student—Newman-Keuls
procedure. Emergence data for the two 2007 orchard blocks were combined and analyzed
using proc GLM and Student-Newman-Keuls procedure. In 2008, mean separations for
adult emergence were conducted proc GLM and Student-Newman-Keuls procedure, for
all orchards combined and then separately by orchard.

2.4 Eﬂicacy with Adult Trapping

In 2007 only, the efﬁcacy of a single application of entomopathogens to soil for
suppression of a natural population of plum curculio was studied. Each of the seven
orchard blocks was divided into four plots of 60 trees each (5 trees wide by 12 trees long),
with a two-tree buffer strip between plots.

Each of the four plots within each block was randomly selected for treatment with
commercially formulated S. carpocapsae, S. riobrave, M. anisopliae, or water alone
(control). We did not test B. bassiana due to limited space and based on laboratory trials
where the M. anisopliae strain was more virulent to larvae than the B. bassiana strain
(Whalon, unpublished data).

Plots were irrigated with 45,000 L water/ha 1-3 h prior to treatment applications
using a 1,900 L tractor-drawn tank and side-mounted PVC pipe with holes drilled every 5

cm. The entire area of groundcover under the tree canopy (approximately 0.5 m from the

33

trunk) received water and entomopathogen treatments. Nematodes in their respective
formulations were applied with a hand-pump backpack sprayer without a ﬁlter screen to
the designated area (223 m2 per plot) in 15 L of water (a rate equivalent to 673 Uha). The
sprayer tank was continuously shaken during application. The M. anisopliae granules
were applied by hand, followed by a 15 liter spray of water. Control plots received a 15
liter spray of water. Conidia viability was 93%; nematode viability was 100% for both S.
carpocapsae and S. riobrave. Applications were made on 9 and 10 July (687 Degree
Days base 10°C).

Four circle traps were placed in each plot and secured on uniformly spaced tree
trunks. Plum curculio adult catch was recorded weekly for 6 wk starting 3 d after
entomopathogen application. The total number of adults caught per trap was analyzed
using proc GLM (SAS Institute 2004).

2.5 Eﬂicacy with Caged Fruit

In 2007 only, another ﬁeld experiment was conducted to evaluate adult
emergence from the plots described above, except that cages were used, since screen trap
catch may be a poor indicator of adult suppression in small plots due to adult behavior
and movement in the fall (Johnson et al. 2002, Leskey and Wright 2004). One cage was
installed over each of the four soil treatment plots in each of the seven orchard blocks,
within the dripline of two trees along the center row of the plots.

The cage base (0.5 x 1.8 m) was constructed of 1.3 cm diam PVC pipe. Netting
(0.8 mm mesh) was tightly afﬁxed to the pipe base and tented over the interior at a height

of 30 cm at the apex. A 5 cm deep trench was dug to ﬁt the PVC base. Cages were

34

secured with metal L-shaped landscaping stakes and moist topsoil was mounded around
the entire base to prevent escape of new adults after emergence.

Infested apples were hand-picked from naturally infested trees in the Clarksville
orchard blocks between 5 and 17 June and held in the laboratory in mesh bags until ﬁeld
deployment. We evenly distributed 300 apples per cage over 0.8 m2 of ground on 7 July.

In the laboratory, degree days were calculated by averaging the daily maximum
and minimum temperature, and subtracting the base temperature of 10°C. Temperatures
were recorded with a Watchdog datalogger (Spectrum Technologies, Inc, Plainﬁeld, IL).
In the ﬁeld, degree days were calculated using numerical integration (hourly data) from
an onsite Michigan Automated Weather Network weather station (Michigan
Climatological Resources Program, East lansing, MI). A subsample of 3,000 of these
picked apples was held in the lab suspended over moist paper towel in trays in order to
determine the daily number of larvae emerging from the picked fruits. Approximately
71% of larvae had emerged before the entomopathogen application date. Therefore, the
single entomopathogen applications targeted only the last 29% of natural larval
emergence from fruit at the site. This serves also as a proximal measure for the adult
trapping experiment, since the fruit were collected from the same orchards.

1 Soil and organic material from the area underneath cages were sampled for plum
curculio on three dates (26, 34, and 54 (1 post treatment) to collect all adults present.
Cages were lifted, and any adults present on the interior net surface were collected.
Simultaneously, the groundcover under each cage was systematically vacuumed for 2
min with a 10 HP leaf blower modiﬁed for suction, with a 15 cm diam. hose. Debris was

collected into bags (0.8 mm mesh) that were secured with rubber bands to the end of the

35

vacuum hose. Cages were put back into place within 3 min of sampling. Debris was
hand-separated in the laboratory and adults were recovered.

The number of adults collected was pooled over the three dates for each cage, and
results were analyzed by proc GLM (SAS Institute 2004).
3. Results
3.1 Eﬂ'icacy and Timing with Emerged Larvae
2006 Results

In 2006, adult emergence was signiﬁcantly reduced by at least one B. bassiana
treatment in one of two apple orchards and one of two cherry orchards (Figure 3.1). In the
Genesee Co. Sandy Loam apple orchard, adult emergence was reduced by 32% and 48%
by rice and Mycotrol-O treatments, respectively, but only the Mycotrol-O spray treatment
was signiﬁcant. Only the high rice treatment reduced adult emergence signiﬁcantly from
controls (by 77%) in the Benzie Co. Clay Loam tart cherry orchard. The contents of all
pots were sifted through after day 60, and the only ﬁnding was one dead adult.
2007 Results

In 2007, only the S. riobrave entomopathogen treatment resulted in signiﬁcantly
reduced adult emergence compared to untreated controls for all three larval application
timings: immediately, 3 d, and 5 d after entomopathogen application (Figure 3.2).
Reduction from control was 88%, 89%, and 80% for 0, 3, and 5 d timings, respectively.
The S. carpocapsae treatment signiﬁcantly reduced adult emergence (56% from control)
only for the 3 d timing. All other entomopathogen x larva-timing combinations were not
signiﬁcantly different from their respective control x larva-timing combinations.

2008 Results

36

In 2008, when data from the 5 sites was combined, the high rate of nematode S.
riobrave signiﬁcantly reduced adult emergence when larvae were added to pots -5, 0, and
10 d from nematode application, but not 5 d (Figure 3.3). When data from each individual
site was analyzed separately, the ANOVA was signiﬁcant (p<0.05) at the conventional
tart cherry sites only. Within those sites, mean separations (SNK) revealed that only the S.
riobrave, high rate, -5 d or 0 (1 treatments were signiﬁcant (Table 3.4).

Soil moisture values in 2008 are presented in Table 3.3. In 2007, the soil moisture
in pots at day of entomopathogen application was 9.4%. In 2007, rainfall from 21 June to
25 July was less than 4.5 cm total. Rainfall was greater than 0.13 cm per day on 21 and
27 June; 4, 10, 14 July, and 25 July (0.38, 0.56, 0.49, 0.84, 0.33, and 1.57 cm,
respectively).

3.2 Eﬂ‘icacy with Trapping

There were no signiﬁcant differences in screen trap catch of adult plum curculio
between treatments (F = 0.76, p = 0.5199, df = 165). Mean :1: SE. adults caught per trap
was 1.3 :1: 0.2, 1.1 :r: 0.3, 1.1 :I: 0.2, and 1.4 :r: 0.3 adults per cage for control, M. anisopliae,
S. carpocapsae, and S. riobrave treatments, respectively.

3.3 Eﬁcacy with Caged Fruit

There were no signiﬁcant differences in adult plum curculio recovery per cage
between treatments (F = 0.68, p = 0.5721, df = 27). Mean :1: SE. recovery per cage was
11.1 :I: 1.5, 12.4 :1: 2.6, 11.1 :1: 2.4, and 9.2 :i: 3.1 adults per cage for control, M. anisopliae,
S. carpocapsae, and S. riobrave treatments, respectively. An average of 0.50 (1,499 total)

larvae emerged per fruit from the 3,000 fruits held in the laboratory.

37

4. Discussion

Variability year-to-year, site-to-site, and from lab-to-ﬁeld is characteristic of
augmentative entomopathogen studies (McCoy et al. 2000). Such variations within this
study and their probable causes are discussed.

Beauveria bassiana was not effective against plum curculio as a conidia
suspension or formulated in an oil carrier in 2007 and 2008 respectively. However, B.
bassiana applications targeting last-instar plum curculio signiﬁcantly reduced adult
emergence from controls by 48% (granular) in one of four orchards and 77% (spray) in
one of four orchards in 2006. These two orchards had lower sand content than the other
two orchards (Table 3.2). Although immersion method laboratory bioassays indicated
higher virulence of M. anisopliae than B. bassiana to last-instar plum curculio, M.
anisopliae was not effective when applied as a conidial suspension or as a granular
formulation in trials conducted in 2007.

In 2007 when larvae were added to pots of soil, S. riobrave at 0, 3, and 5 d and S.
carpocapsae at 3 d only reduced emergence of subsequent adults. Shapiro-Han et al.
(2002) found that S. carpocapsae was less virulent than S. riobrave to plum curculio
larvae in laboratory assays. Steinemema riobrave also has higher optimum temperature
range than S. carpocapsae (Grewal et al. 1994), which the infective juveniles may have
experienced in the soil during the time when infection was most likely. Foraging strategy
may also affect successful infection. Plum curculios tend to pupate in the upper 5 cm of
soil if sufﬁcient moisture is present. The “sit-and-wait” forager S. carpocapsae
outcompeted the active forager H. bacteriophora for Galleria mellonella larvae at the soil

surface, and vice versa for 5 cm below the surface (Alatorre-Rosas and Kaya 1990). The

38

Steinemema riobrave foraging strategy seems to be an intermediate of the two (Lewis et
al. 2006).

Besides differences between the two nematode species such as virulence, foraging
method, or Optimal temperature ranges, formulation is a major factor that could have
contributed to the observed difference in efﬁcacy. Steinemema riobrave was only
available in a gel carrier, while S. carpocapsae was available in a vermiculite carrier,
speciﬁcally developed for approval by the Organic Materials Review Institute (OMRI).
Likewise, unforrnulated H. bacteriophora was ineffective while gel-formulated S.
riobrave was partially effective in 2008 trials.

To estimate needs for re-application over the duration of larva soil residence, it
was necessary to determine the reduction in efﬁcacy of entomopathogens over a time
period longer than from 0 to 5 d post-application. In 2008 S. riobrave signiﬁcantly
reduced adult emergence when larvae were added to pots -5, 0, and 10 days from
nematode application. Mean percent reduction from control was 25%, 66%, 50%, 27%,
36%, and 8% for the -10, -5, 0, 5, 10, and 15 day timings, respectively. This is in contrast
to the 2007 experiment, in which mortality from S. riobrave was greater than 80% for the
0 and 5 d timings. Abbott’s corrected mortality means (Abbott 1925) revealed greater
than 50% mortality at the sandiest three sites for -5 and 0 d S. riobrave high rate
treatments. Variation between pots within a treatment was so high that not all of these
means were signiﬁcantly lower than the control; -5 d was signiﬁcant for all three sites
and 0 d was signiﬁcant for one site.

These entomopathogens may have caused additional mortality to other soil-

contacting life stages of plum curculio. For instance, adults are susceptible to

39

entomopathogenic nematodes (Brossard et al. 1989, Belair et al. 1998, Shapiro-[Ian et al.
2002). Observations of natural infections of B. bassiana and M. anisopliae in adults have
been made (Laﬂeur et al. 1987), but efﬁcacy of augmentative applications has not been
reported. The pupa is susceptible to nematodes (Kim and Alston 2008) and may have
been attacked by the entomopathogens in our study. We found no studies on the efﬁcacy
of entomopathogenic fungi against pupae and adults.

The experimental methods introduced several unrealistic conditions. In order to
develop a spatially contained three-dimensional plot that could be effectively evaluated in
the ﬁeld, we chose plastic pots as ﬁeld soil “cages” for these experiments. Therefore,
efﬁcacy levels observed in the potted experiments may be higher than would be expected
in a normal orchard setting. larvae were introduced to treated soil within 1 h of
entomopathogen application on all 0 d timings; however, the population of larvae
dropping naturally from fruit into soils would be making ﬁrst contact with an
entomopathogen applied several days before to several days after, depending on the
interval of days between applications. Also, nematodes reproduce inside insect cadavers
and their offspring exit the cadaver in search of a new insect host. This “nematode
cycling” may have had an increased potential for reducing plum curculio adult emergence
in our experiment, since we used a higher density of larvae (10 larvae per 77 cm2) than
would likely occur in an orchard with moderate to high plum curculio infestation. These
densities may however be achieved in some organic orchards where growers attempt to
rake or broom dropped fruit into the drive row or into piles.

Another factor of variability introduced by the experiment methods was detection

of high densities of larvae and subsequent predation by ant colonies. In 2008 the pots

were observed up to 2 h after larva application, until all larvae had burrowed, and ant
predation was noted. All pots with ant predation were excluded from the analysis.
Observation of ant predation was highest at the sandy tart cherry sites, reaching 17% of
pots in two sites, but was under 3% for the other three sites. Ant predation was not
observed in 2006 or 2007, but likely contributed to experimental error. In our experience
experiments using infested fruit containing larvae rather than directly placing high
densities of larvae on the soil surface could reduce detection and predation by ants. A
bifenthrin ring treatment to prevent entry of ﬁre ants in a similar situation did not affect
mortality of plum curculio (Jenkins et al. 2006b), and should be considered in this type of
experiment.

When infested fruit rather than free larvae were placed in ﬁeld plots and adults
were trapped on trees in plots or more directly in cages, no effects of entomopathogens
on reducing adult densities were observed. late entomopathogen application timing was
probably a contributing factor to this, since 71% of larvae were expected to have dropped
from fruit prior to applications.

Other than entomopathogen-application timing, there are many reasons why the
open trunk traps may not have accurately reﬂected the populations in plots. Differences
may not have been detected due to a low overall trap catch. Other possible reasons are
small plot size, dispersal of adults between and outside of plots, poor attraction of adults
to traps and thus inaccurate representation of adult populations in plots, low emergence of
larvae from fruit and thus low population pressure in plots, etc. However, one would
expect to see differences in the cages for treatments that were effective in the pots. High

variability between plots suggests that more replication is needed per treatment.

41

Entomopathogens are generally sensitive to water stress and maintaining soil
moisture constant across our treatments over time was not economically or practically
possible (T able 3.3). Therefore, our results reﬂect a reasonable expectation of what
conditions would be in Michigan during the time of larval drop, since very few Michigan
orchards are irrigated at this time. Although some orchardists use drip tube irrigation, this
delivery system is not designed to deliver moisture to the entire drip-line under the tree,
where plum curculios pupate. A way to ameliorate variable moisture conditions would be
to use an under-tree micro-jet sprinkler irrigation system to maintain moisture, as is used
to control citrus root weevil (McCoy et al. 2002). This may be cost-effective for tree fruit
growers that maintain drip irrigation lines, as the sprinklers are easily substituted into the
holes created by the drip emitters. larva suppression by nematodes irrigated with
sprinklers lasts no longer than 1 or 2 weeks (McCoy et a1. 2000, 2002), but efﬁcacy
within that time presumably would be higher than comparable sites without irrigation,
and pathogen cycling in higher moisture conditions may add to the efﬁcacy of these
entomopathogen tools.

The physical properties of soils may contribute greatly to efﬁcacy of
entomopathogens (Barbercheck 1992, Georgis and Gaugler 1991). larval mortality of
citrus root weevil caused by S. riobrave was positively correlated with the proportion of
sand in soils (Shapiro-Han et al. 2000, Duncan et al. 2001, McCoy et al. 2002). (2002)
and Shapiro-[Ian et al. (2000). Generally, nematode motility decreases with decreasing
soil pore size even though the soil type may hold less water (Molyneux and Bedding
1984, Kaya 1990, Kung et a1. 1990, Roy Kaspi personal communication). Efﬁcacy

among largely different soil textures was not considered in prior plum curculio studies,

42

where the Utah study residential sites soils had a smaller percent sand range than the
current study (54-73%) (Kim 2007) and the southern sites were loamy sand and sandy
loam (Shapiro-Han et al. 2004a, 2008).

Alternatively, persistence of B. bassiana near the soil surface (0-5 cm depth) may
be higher in soils with small pore sizes (V anninen et al. 2000). A clay coating on
Beauveria bassiana blastospores reduces biodegradation (Fargues et al. 1983). In our
2008 study, sites highest in sand resulted in marginally higher reduction of plum curculio
adult emergence. In 2006, B. bassiana efﬁcacy was observed in the clay loam and loamy
sand sites, and none in the two sand sites.

These entomopathogens are particularly sensitive to the UV-B component of
light; therefore, adjuvants with UV—radiation protecting properties are sometimes added
to formulations of entomopathogens (Inglis et al. 1995). The rice in 2006 and the
formulations used in the 2007 experiments contained no such adjuvants. Since the
entomopathogens were applied on sunny days in 2006 and 2007, some reduction in
efﬁcacy may have occurred due to UV-radiation damage. In the 2008 trials, UV exposure
was avoided by applying entomopathogens at most 2.5 h before sunset.

Compounds such as copper and sulfur, along with their surfactants, are often
sprayed in Michigan organic orchards (T able 3.5) to manage plant pathogens such as
apple scab, sometimes to the detriment of beneﬁcial organisms such as fungi and
nematodes (Krishnayyaand and Grewal 2002). In a study of six fungicides used in
commercial pecan production, the growth of B. bassiana and M. anisopliae in vitro were
least affected by sulfur (Tedders 1981). Growth of B. bassiana mycelium was not

effected by sulfur in other laboratory tests, but the effect on spores was not tested (Sterk

43

et al. 2002). The effect of sulfur on entomopathogen activity has yet to be tested in the
ﬁeld. Such testing is needed if augmentative entomopathogens are to be adopted in
orchards where toxic compounds may accumulate in soils after rainfall, especially in
organic orchards or other production systems that need to rely on sulfur, copper, and
other OMRI-approved fungicides. One feature of cherry orchards in the Upper Midwest
that would be signiﬁcantly different from other areas is that associated fungicides cease
at least 10-21 (1 before harvest and therefore as much as l7-28 d before entomopathogen
application under our timing schemes.

Nematode diversity and abundance displayed in Table 3.2 may be an indicator of
high competition or suitable habitat for the augmented nematode species. However, since
differences in efﬁcacy between sites are marginal we cannot conclude an interaction.
between endemic nematodes and augmentative nematodes, but work should be done in
this area.

More research is needed to validate the assumption that the reduction in summer-
generation adult emergence caused by nematode applications in small or large areas
targeting larvae will reduce damage the following year. Plum curculios immigrate to
orchards from overwintering sites in spring (Racette et al. 1992), so adults surviving in a
refuge such as nearby unsprayed habitats may immigrate into the orchard of concern. It is
a longstanding recommendation to remove refuge habitats such as alternate hosts around
orchards to prevent plum curculio damage (Racette et al. 1992). This practice would
likely be essential to reduce the chances of immigration of spring adults within the
irnrrrigration range of an orchard under a management program using nematodes against

last-instars. Shapiro-Han et al. (2008) found that S. riobrave applications targeting soil-

dwelling larvae in a wild plum thicket plot adjacent to an orchard caused 87.9-98.6%
control. In terms of anticipated longevity of S. riobrave in the soil, our results suggest that
multiple applications of S. riobrave at the high rate (4 x 109 IJs/ha) in summer would be
necessary to target the majority of plum curculio larvae exiting fruit, since larvae exit
fruit for several weeks. Applications may need to be applied every 10-15 (1 during larval
drop from fruit, but more studies considering nematode efﬁcacy longevity under
controlled soil moisture management should be conducted to support this tentative
recommendation.

A strategy that employs S. riobrave targeting larvae may be feasible for adoption
by Michigan tree fruit growers with a need for environmentally-friendly plum curculio
management tools. However, the growers that may need these tools the most may be
organic certiﬁed growers, and S. riobrave in OMRI approved formulation such as the
vermiculite formulation is not yet available and has not been tested against the gel
formulation. This strategy, although more expensive, may be used in home yards, where
people prefer to avoid use of conventional insecticides (Kim 2007). To reduce costs,
nematodes might be applied to areas only where oviposition is concentrated, and where
irrigation is possible. Plum curculio oviposition marks are typically concentrated in
border rows, and are easy to detect visually. Other strategies that aggregate infested fruit
in limited regions below the trees could also allow growers to more effectively target
emerging larvae.

The replacement of chemical insecticides with “softer” tactics in tree fruit may

depend on the practical management of other pests that typically resurge with reductions

45

of chemical inputs, such as codling moth and oriental fruit moth; however, some of these
pests may be controlled with microbials as well (Iacey and Shapiro-[Ian 2003, 2008).

In conjunction with quantifying residual activity of an augmentative
entomopathogen application, a measure of the duration of a plum curculio population’s
soil residence will help growers make application decisions. The timing of larval
emergence from fruit has been studied in tree fruits in West Virginia (Brown 2005) and
Utah (Kim 2007), and in blueberries in New Jersey (Polavarapu et al. 2004). However,
calendar or phenology models for plum curculio larval emergence from fruit remain
undeveloped for many regions. More research is needed to develop more precise
entomopathogen-app]ication timing recommendations according to the phenology of

larval emergence from fruit crops and effective and practical application strategies.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Benzie Co. Ben zie Co. Leelan au Co. Gen esee Co.
ICIay Loam I Sand D Sand D Sandy Loam
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Treatment
Figure 3.1. Mean (:1: SE) number of adult plum curculio emerged from B. bassiana GHA-treated soil in pots
(10 larvae/pot) over a 50-d period in 2006 at four orchards. Rice Ueatments (B. bassiana grown on rice)

were not conducted in Genesee Co. Bars with the same letter within an orchard are not signiﬁcantly
different (SNK, alpha=0.05).

 

NJ

 

 

 

 

 

 

 

a [.1 min Iaday DSday]
6‘ ab
g abc
3 5 l abcd abcd
g l- I
i Ti.“ 14‘?
E 4 1T5}:
2
ru 3“ 5"?
S if
é’ 2‘
. iii:
1 ..
O1 W

 

 

 

 

 

 

 

 

 

 

 

Untreated Contro B. bassiana I M. anisopliae
Treatment

Figure 3.2. Mean (:r: SE) number of adult plum curculio emerged from entomopathogen-treated soil in pots

over a 50—d period in 2007 at a single orchard. Ten larvae were added to each pot either immediately (1

min), 3 d, or 5 d after entomopathogen application. Bars with the same letter are not signiﬁcantly different
(SNK, alpha=0.05).

 

47

 

Mean Emerged Adults

Mean Emerged Adults

 

 

13 Control I Bb D Hb Low I Hb High I Sr Low I Sr High

 

 

 

 

 

 

 

 

 

 

 

    

 

 

 

 

 

 

 

   

 

 

 

 

         

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

     
  
 

 

 

 

 

 

 

 

 

 

 

 

 

10
9 All Orchards Combined j
8
7
6 l
S
4 L
3 a
2 1H
1 1e
0 ii
10db 5db Oda 5da T 10da 15da 20da
Time larvae were added in days before (db) and days after (da) pathogen application
13 Control I 8b 0 Hb Low I Hb High I Sr Low I Sr High
1: Leelanau Co. Loamy Sand
8
7
6
5 I.
4 .
3
2 -l
1 -
0 T 10db 5db

Time larvae were added in days before (db) and days after (da) pathogen application

Figure 3.3. Mean (1: SE) number of adult plum curculio emerged from entomopathogen-treated soil in pots
over a 50-d period in 2008. Ten larvae were added to each pot either -10, -5, 0, 5, 10, 15, or 20 d from
entomopathogen application. Note Hb treatments were not conducted on -10, 10, and 20 (1. Bars with an
asterisk are signiﬁcantly different from the control within the same larva addition timing (SNK, u=0.05).
All orchards combined and Leelanau Co. Sandy Loam site.

48

 

0 Control I Bb D Hb Low I Hb High I Sr Low I Sr High

 

 

 

 

 

I Leelanau Co. Sandy Loam

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean Emerged Adults

 

 

 

 

 

 

 

 

   

 

 

10db 5db
Time larvae were added in days before (db) and days after (da) pathogen application

 

0 Control I Bb El Hb Low I Hb High I Sr Low I Sr High
10
Ionia Co. Loam J

 

 

 

 

 

 

 

 

 

    
 

I

 

 

 

 

 

    

Mean Emerged Adults

 

 

 

 

 

 

 

  

 

     

1 Odb 5db 1 5da
Time larvae were added in days before (db) and days after (da) pathogen application

Figure 3.4. Mean (1 SE) number of adult plum curculio emerged from entomopathogen-treated soil in pots
over a 50—d period in 2008. Ten larvae were added to each pot either -10, -5, 0, 5, 10, 15, or 20 d from
entomopathogen application. Note Hb treatments were not conducted on -10, 10, and 20 (1. Bars with an
asterisk are signiﬁcantly different from the control within the same larva addition timing (SNK, o.=0.05).
Leelanau Co. Sandy Loam site and Ionia Co. Loam site.

49

 

E] Control I 8b El Hb Low I Hb High I Sr Low I Sr High

 

 

 

 

10

 

Eaton Co. Clay Loam

 

 

 

 

 

 

 

Mean Emerged Adults

 

 

 

 

 

 

 

 

 

5db i Oda T
Time larvae were added in days before (db) and days after (da) pathogen application

 

El Control I Bb El Hb Low I Hb High I Sr Low I Sr High

 

 

 

 

Genesee Co. Loam

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean Emerged Adults
U1
i

 

 

 

 

 

 

 

 

 

 

 

10db 5db T Oda 5da 10da 15da 20da l
Time larvae were added in days before (db) and days after (da) pathogen application

Figure 3.5. Mean (:t SE) number of adult plum curculio emerged from entomopathogen-treated soil in pots
over a 50—d period in 2008. Ten larvae were added to each pot either ~10, -5, 0, 5, 10, 15, or 20 d from
entomopathogen application. Note Hb treatments were not conducted on -10, 10, and 20 d. Bars with an
asterisk are signiﬁcantly different from the control within the same larva addition timing (SNK, a=0.05).
Eaton Co. Clay Loam site and Genesee Co. Loam site.

50

Table 3.1. Soil Analysis for 2006 and 2008 sites.

 

 

 

 

 

 

 

 

 

 

 

 

 

CEC %
Soil % % % (meq/ Organic

Year Crop Site Type Sand Silt Clay pH 100 g) Matter
Organic Leelanau

2006 Tart Cherry Co. Sand 88.2 9.1 2.7 7.1 7.3 2.4
Organic Benzie Clay

2006 Tart Cherry Co. Loam 32.4 29.8 37.8 7.9 19.1 2.6
Organic Benzie

2006 Apple Co. Sand 89.2 10.1 0.7 6.8 6.1 4.0
Organic Genesee Sandy

2006 Apple Co. Loam 63.4 29.2 7.4 6.9 8.8 2.6
Conventional Leelanau Loamy

2008 Tart Chery Co. Sand 88.4 2.5 9.2 7.7 6.8 2.6
Conventional Leelanau Sandy

2008 Tart Cherry Co. Loam 81.4 8.5 10.2 6.9 7.4 3.5
Conventional Ionia

2008 Tart Cherry Co. Loam 36.7 40.9 20.4 6.5 5.8 2.4
Organic Eaton Clay

2008 Apple Co. Loam 40.7 31.9 27.4 7.1 9.1 3.3
Organic Genesee

2008 Apple Co. Loam 36.7 38.9 24.4 6.7 12.5 6.9

 

 

 

 

 

 

 

 

 

Table 3.2. Nematode Community Analysis, Michigan State University Diagnostic Service laboratory, from
pre-application soils, 2008.

 

 

 

 

 

 

 

 

 

 

 

 

 

SoilHerbivores
a a 3
o . .
:3 3'3 g E E a E E g i
3 a a is: [3' 2' 8 2 min 2 o
LeelanauCo.
Loamy Sand 0 2 2 0 0 0 35 0 70 95 5 209
LeelanauCo.
SandyLoam 0 4 0 0 5 10 5 0 95 10 45 174
IoniaCo.
Loam 1 19 0 0 70 15 0 0 160 110 10 385
EatonCo.
ClayLoam 4 61 0 0 90 30 15 0 165 145 0 510
GeneseeCo.
Loam 20 23 44 17 160 20 30 10 245 35 30 634

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3.3. Percent soil moisture in 2008 sites.

 

 

 

 

 

 

 

 

Leelanau Co. Leelanau Co. Ionia Co. Eaton Co. Genesee Co.
Day Loamy Sand Sandy Loam Loam Clay Loam Loam

-10 3.0% 2.7% 20.1% 15.7% 48.0%

-5 3.8% 3.5% 21.3% 15.5% 15.1%

0 7.0% 13.8% 20.3% 38.6%

5 5.0% 6.8% 7.5% 1 1.4% 28.1%

10 1.6% 2.7% l 1.3% 4.0% 25.7%

15 3.5% 6.4% 10.0% 5.3% 14.4%

 

 

 

 

 

 

 

51

Table 3.4. Mean percent reduction from control treatments (Abbott’s Corrected Mortalities) for plum
curculio in S. riobrave high rate treatment. larvae were applied —10, -5, 0, 5, 10, 15, or 20 d from S.
riobrave application to pots of soil. Percentages with an asterisk (*) indicate that the corresponding mean
adult counts were signiﬁcantly lower than control within a larva tinting (SNK, a=0.05).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

52

Larva TimiﬁnL
Site -10d -5d 0d 5d 10d 15d 20d
All Orchards 25% 66% * 50%* 27% 36% * 8% -l 8%
Leelanau Co.
Loamy Sand 48% 89%* 56%* -22% 25% -26% 12%
Leelanau Co.
Sandy Loam 45% 69%* 70% 70% 41% 55% 43%
Ionia Co.
Loam 14% 65%* 82% 44% 36% 15% -24%
Eaton Co.
Clay Loam 33% 50% 4% 16% 56% 4% -79%
Genessee Co.
Loam 34% 17% 21% -10% 50% -18% 10%
Table 3.5. Spray record for Clarksville orchard blocks in 2007.
Date Active Ingredient
3/30/2007 Copper hydroxide for ﬁrebliﬂscab
4/20/2007 Sulfur
4/24/2007 Sulfur
4/27/2007 Calcium polysulfate (lime sulfur)
5f7/‘2007 Sulfur
5/14/2007 Sulfur
5/14/2007 Bacillus subtillis
5/24/2007 Sulfur
5/24/2007 Rosemary, clove, thyme oil
5/29/2007 Sulfur
5/29/2007 Rosemary, clove, thyme oil
5/29/2007 Kaolin clay
6/2/2007 Calcium polysulfate (lime sulfur)
612/2007 Streptomycin sulfate
6/8/2007 Streptomycin sulfate
6/18/2007 Sulfur
6/18/2007 Rosemary, clove, thLme oil
7/3/2007 Sulfur
7/3/2007 Rosemary, clove, thyme oil

 

CHAPTER 4
LABORATORY AND FIELD EVALUATIONS OF ENTOMOPATHOGENIC
FUNGUS EFFICACY AGAINST ADULT PLUM CURCULIO
1. Introduction

Plum curculio, Conotrachelus nenuphar (Herbst), is a serious pest of tree fruits in
eastern North America. Changes in insecticides available to growers have prompted the
development of alternative management tactics (Wise et al. 2006a). Adult plum curculio
cadavers have been found infected with entomopathogenic fungi (Garman and Zappe
1929, McGiffen and Meyer 1986, Laﬂeur et al. 1987). This susceptibility to muscardine
disease could be used in management of plum curculio. The plum curculio life cycle
includes three duff- and soil—dwelling stages that may be vulnerable to soil-bome
entomopathogens: the soil-burrowing last-instar, the pupa, and adult.

Several strains of the entomopathogenic fungi Beauveria bassiana (Balsamo)
Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin are currently registered
by the US EPA. While efﬁcacy of entomopathogenic nematodes for suppression of plum
curculio adults has been evaluated in the laboratory (Shapiro-Han et al. 2002), neither M.
anisopliae nor B. bassiana have been evaluated against plum curculio adults. Only last-
instars have been targeted in fungus efﬁcacy evaluations in the lab (Tedders et al. 1982,
Alston et al. 2005) and ﬁeld (Jenkins et al. 2006b). However, a trunk perimeter spray of B.
bassiana controlled pecan weevil adults with effects up to 1 wk post-application, with
best results in plots receiving irrigation (Shapiro-[Ian et al. 2004b).

Inundative releases of M. anisopliae and B. bassiana typically involve application

to soils, where these species reside as facultative saprophytes. A granular formulation of

53

Beauveria brongniartii is commercially available for European cockchafer biocontrol
(Kessler et al. 2004). A simple maize-based B. bassiana formulation developed for
subsistence farmers was effective against banana root weevil (N ankinga and Moore
2000) and granular M. anisopliae increased yield in ﬁelds with sugarbeet root maggot
(Campbell et al. 2006). A granular formulation of B. bassiana on rice established in
pasture better than a biopolymer when considered for clover root weevil control (Nelson
et al. 2004).

A surface other than soil that an insect is likely to contact may serve as an
alternative substrate for targeting certain insect life stages. A fabric trunk band was
effective against the Asian longhomed beetle (Dubois et al. 2004, Hajek et al. 2006). If
there is a sufﬁciently long enough period of time between inoculation and mortality,
perhaps inoculated adults could transfer conidia to mates, serving as an
autodisssemination management tactic (Vega et al. 1995). Several studies utilizing
“autodissemination traps,” demonstrated that this approach was effective for the sweet
potato weevil (Yasuda 1999), diamondback moth (Furlong et al. 1995, Furlong and Pell
2001), Japanese beetle (Klein and Lacey 1999), and brown-winged bug (Tsutsumi et a1.
2003).

Adult plum curculios aggregate underneath trees in the perimeter rows of
orchards in the spring (Laﬂeur and Hill 1987, Racette et al. 1991, Chouinard et al. 1993,
1994). Males may mate with as many as 3 females per day (Yonce and Jacan 1978), or
an average of 10.4 females per 30 days (Johnson and Hays 1969). Mobile adults can be
trapped by walking or ﬂying to and climbing onto Whalon lab high contrast pyramid

traps (Great Lakes IPM, Vestaburg, M1) or on tree trunks ﬁtted with screen traps (Mulder

54

et al. 1997). Trap catch is highest before petal fall (Coombs 2001, Leskey and Wright
2004).

The goal of this study was to evaluate efficacy of commercially available
entomopathogenic fungi against plum curculio adults. Our objectives were to: 1)
determine the mortality of adults after exposure to surfaces with B. bassiana and M.
anisopliae, and 2) determine the mortality of unexposed adults caged with exposed adults.

The exposure surfaces included: 1) soil or peat with surface-applied aqueous
suspensions of conidia; 2) walking arenas with colonies of conidia on Petri dishes; 3) a
moist cotton wick that could be positioned inside an “autodissemination” trap top; and 4)
conidia-covered granules applied to orchard groundcover.

2. Materials & Methods
2.1 Insects and Entomopathogenic Fungi

Northern strain adult plum curculios were obtained from a colony maintained by
the Pesticide Alternatives Laboratory, Michigan State University, East lansing, MI. The
colony was reinitiated annually from adults and infested cherries and apples collected in
six orchards in Benzie, Ionia, Leelanau, and Manistee.Co., MI. Adults were collected
using circle traps (Mulder et al. 1997) and “Whalon pyramid traps” (1.6 m x 0.8 m) made
from recyclable black corrugated plastic with high-contrast reﬂective tape (Great lakes
IPM, Vestaburg, MI). Both trapping systems were equipped with plum essence lures and
stabilized benzaldehyde vials (Coombs 2001, Leskey and Wright 2004, Leskey et al.
2005). In order to break diapause and induce mating and oviposition, adults were exposed

to thinning apples that had been dipped in a solution of 750 mg pyriproxifen (Esteem® 35

55

WP) per L water (Hoffman et al. 2007). Adults used in the ﬁeld trial were not exposed to
pyriproxifen.

Commercial strains of B. bassiana (GHA strain obtained from the USDA
Agricultural Research Service Entomopathogenic Fungus Culture Collection, Ithaca, NY)
and M. anisopliae (F52 strain received as Met52® granular formulation, N ovozymes
Biologicals, Inc., Salem, VA) were used in this study.

Aqueous suspensions of fungi were prepared by scraping M. anisopliae or B.
bassiana conidia from 2-wk old potato dextrose agar plate cultures into sterile water with
0.01% Tween-80. Fungal inoculum viability was assessed according to Goettel & Inglis
(1997) by pouring 50 ul of each suspension within 30 min of application onto three
dishes of potato dextrose agar and checking 200 conidia/dish for the presence of germ
tube formation after 20 h with an Olympus BX40 research microscope.

The ﬁeld experiment took place within the dripline of trees in an unsprayed and
neglected apple orchard at the Michigan State University Entomology Farm, East
lansing, MI. Soil for the laboratory assays was gathered from the same location to a
depth of 8 cm. The soil was mixed and a sample was tested at the Michigan State
University Soil and Plant Nutrient laboratory, East lansing, MI. The soil was loamy
sand (79.3% sand, 15.7% silt, 5.0% clay; 1.8% organic matter, pH=7.2).

2.2 Laboratory Assay: Aqueous Suspensions on Soil

Field soil and peat were passed through a #18 sieve and each was autoclaved for

45 min and dried at 110°C. The soil was brought up to 14% moisture and the same

amount of water was added to the peat, resulting in 60% moisture calculated

56

gravimetrically. The soil or peat was placed in 30 ml autoclaved medicine cups (Premium
Plastics, Chicago, IL).

Conidia suspensions were prepared by scraping conidia from 2-wk old cultures on
PDA into 0.01% Tween-80. Suspensions were adjusted such that 1 ml was delivered by
pipette to each cup at a rate of 8 x 1014 conidia/ha. Control cups received 1 ml of 0.01%
Tween-80. Conidia viability was 96% and 97% for B. bassiana and M. anisopliae,
respectively.

Adult plum curculios were placed individually in the cups and fed with a 5 g
piece of thinning apple. Cups were sealed with three layers of cheesecloth secured with a
rubber band, and were incubated in large glass jars at 25°C. The jars were sealed with
perforated paraﬁlm and contained saturated sterile ﬁlter paper to maintain humidity.
Mortality was checked and apple pieces were replaced every 3 d for 15 (1. There were
ﬁve replicated cups per soil type x entomopathogen treatment and the experiment was
repeated once.

In a second assay, treatments included only loamy sand soil with two rates: 1 x
1013 or 5 x 10'3 conidia/ha of either M. anisopliae or B. bassiana. There were three
replicate cups per entomopathogen x rate treatment, and the experiment was repeated
four times.

Mortality data passed assumptions of normality and homogeneity of variance and
were analyzed by one-way ANOVA. Means were separated using Student Neuman—Keuls
procedure and Dunnett’s test for the ﬁrst and second assays, respectively (SAS Institute

2004).

57

2.3 laboratory Assay: Petri Dish and Wick

Newly emerged adults were held for 3-6 wk before the study on thinning apples in
vented plastic containers under a 16:8 L:D photoperiod, at 25°C. Adults were separated
by sex the day before the experiment began according to Thompson (1932). Males were
marked with a small dot of yellow enamel paint on the left elytron for identiﬁcation,
while females were unpainted.

Two surfaces were tested: 1) a two-week old fungus colony on SDAY (Sabouraud
Dextrose Agar + Yeast) in a 90 mm Petri dish and 2) a two-week old colony on the
surface of a moist dental wick with SDY (no agar). The wicks were prepared by
autoclaving and then dipping them individually in a suspension of B. bassiana or M.
anisopliae conidia at a rate of 1 x 107 conidia/ml of SDY solution. Each wick soaked up
approximately 10 ml. The suspensions were prepared by scraping conidia off the surface
of two-week old cultures on SDAY held at 25:1:1’C in the dark. Control adults were
exposed to either sterile SDAY Petri dishes or to a wick dipped in sterile water. The
dipped wicks were suspended inside sterile glass mason jars from hardware cloth. The
mason jars were lined with a moist, sterile Whatman no.1 ﬁlter paper, and topped with a
Petri dish sealed with Paraﬁlm’”.

A single adult was forced to walk across the fungus-colonized agar or wick
surface for 5-10 s. These exposed adults were then placed individually into 59 ml soufﬂe
cups (Solo Cup, Urbana IL) containing 20 ml of ﬁeld soil prepared as in the previous
assay. An autoclaved square galvanized wire screen (0.5 cm diameter mesh) was
suspended 4 cm above the surface of the soil, and a thinning apple was placed on the

screen. Cups were incubated at 25°C, 16:8 L:D, and approximately 70% RH. An

58

unexposed adult of the opposite sex was introduced into the plastic soufﬂé cup 4 h later.
There were ﬁve pairs of adults in each of four repetitions for a total of 20 pairs
per pathogen x surface x exposed sex combination (T able 4.1).

Table 4.1. Dish/Wick assay treatments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n # of

Pathogen surface exmsed sex introduced mate PC pairs per rep reps priirs)
Bb Dish M F 5 4 20
Bb Dish F M 5 4 20
Bb Wick M F 5 4 20
Bb Wick F M 5 4 20
Ma Dish M F 5 4 20
Ma Dish F M 5 4 20
Ma Wick M F 5 4 20
Ma Wick F M 5 4 20
Control Dish M F 5 4 20
Control Dish F M 5 4 20
Control Wick M F 5 4 20
Control Wick F M 5 4 20

 

 

Adult pairs were checked daily for mortality for 28 d. Copulation and mortality
dates were recorded throughout. All cadavers were removed to individual moist chambers
and viewed under a Nikon SMZlOOO (Mager Scientiﬁc, Inc., Dexter, MI) stereo
dissecting microscope for signs of sporulation 3 wk post mortality.

One-way ANOVAs using arcsine square root transformed data were run to
compare mating occurrences, time until death, and percent mortality between treatments,
followed by Tukey’s HSD a posteriori tests, o.=0.05 (SAS Institute 2004).

2.4 Field Study: Granular Formulation against Overwintering Adults

A granular formulation of B. bassiana was produced in the laboratory by
colonizing ground barley (1-3 mm) in autoclavable bags following methods of Nelson et
al. (2004). The Met52 formulation of M. anisopliae consisted of conidia bound to

unbroken rice. The number of conidia per gram of granular material was approximated by

59

placing a 5 g sample into a ﬂask containing 100 ml of 0.01% Tween-80. Flasks were
placed on a rotating shaker at 180 RPM for 5 min, and the number of conidia per ml of
solution was determined using a hemacytometer (4 counts per ﬂask). The mean of 3
flasks was calculated: 3.09 x 10° and 2.09 x 109 conidia/g for B. bassiana and M.
anisopliae, respectively. Viability was 96.7% and 90.5% for B. bassiana and M.
anisopliae, respectively.

A wire cone-shaped ground cage (Mulder et al. 1997) with a 0.3 1112 base was
installed 1 m from the trunk of each of 24 apple trees at the East Lansing orchard. Cage
bottoms were ﬁtted into a 4 cm deep trench, staked, and soil was mounded over the base
to prevent plum curculio escape. The groundcover consisted of thick orchard grass, cut to
6 cm. Each treatment (B. bassiana, M. anisopliae, or control) was assigned to 8 cages in a
completely randomized design. Prior to cage installation, granules were distributed
uniformly onto the groundcover of each cage at a rate of 5 x 1013 conidia/ha. Control
cages did not receive any grains. Immediately following treatments, the groundcover of
every cage was sprayed with 100 ml of water from a backpack sprayer. Adults (24 per
cage) and a ripe apple picked from a tree on-site were introduced to cages on 17-October;
24 h after treatments were applied.

Ambient air temperatures at ground level were recorded hourly with a Watchdog
Datalogger Model 425 (Spectrum Technologies, Inc) and rainfall was recorded at an on-
site weather station (Michigan Automated Weather Network, East Lansing, MI) (Table
4.2). Temperatures inside the cages averaged 1.l°C cooler than outside. Boll weevil trap
tops (Great lakes IPM, Vestaburg, MI) were installed on cage tops on 14 March and

adults were collected from the tops until 9 June. Data analysis was conducted using

ANOVA after passing normality and homogeneity of variance assumptions (SAS
Institute 2004).
3. Results
3.1 Laboratory Assay: Aqueous Suspensions on Soil

Conidia suspensions of M. anisopliae and B. bassiana pipetted onto ﬁeld soil at
8x10“ conidia/ha resulted in 100% and 90% mortality of plum curculio adults following
constant exposure of adults over 15 d (Fig. 4.1). Applications of the fungi to peat soil
caused 10% and 20% mortality, levels much lower than the control.

Comparison of low (1 x 1013) and high rates (5 x 10'3 conidia/ha) of M. anisopliae
and B. bassiana applied to ﬁeld soil indicated that only the high rate of B. bassiana

caused signiﬁcantly higher adult mortality (70% higher) than controls (Fig. 2.2).

 

 

 

 

 

 

  
  

 

     

 

   

 

 

100 a
. Field Soil

80' .Peat
E?
a)
+|
°\° 60.
E
.2 4o.
0
2
z
3 20.
< .~
0 “its,

M' anisopliae
Control ex 1014 ex 1014
TreaWent

frigate 4.1. Mortality of adult plum curculio exposed to entomopathogen-treated soil or peat at a rate of 8 x
101 conidia/ha after 15 d. Bars with the same letter are not signiﬁcantly different (SNK, 0:005).

61

 

         

     

 

 

 

100
A80.
w
(I)
+1
o\° 50‘
E b
E .
o 40
2
g . .

. b
2 20 3
0 ' ﬁn: IE is .. 2% . v .
Untreated B. bassiana B. bassiana M. anisopliae M. anisopliae
00""0' 1x 1013 5x 1013 1x 1013 5x 10“3
Treatment

Figure 4.2. Mortality of adult plum curculio exposed to entomopathogen-treated soil (low rate = 1 x 1013
conidial ha; high rate = 5 x 10'3 conidia/ha) after 12 d. Bars with the same letter are not signiﬁcantly

different (Dunnett’s procedure, o=0.05).
3.2 Laboratory Assay: Petri Dish and Wick

Conidia viability, assessed 20 h after plum curculio exposure, was 93% and 0%
for B. bassiana dish and wick and 96% for M. anisopliae, respectively. Of the paired
plum curculios, 47% were observed mating at least once. There was no difference in
mating between treatments (F=0.78, df=11, p=0.6595), and no difference in time until
death among treatments (F=1.40, df=20, p=0.1284) (Figure 4.3). There was a quantitative,
but not signiﬁcant mortality in females exposed to the control wick and females held with
males exposed to the control wick, as well as those in the Bb wick treatments, which may
indicate a higher background mortality of females as compared to males. Mortality was
signiﬁcantly higher than control in all fungus treatments except for Bb wick and Ma wick

(males paired with exposed females). There was no signiﬁcant difference among the

62

treatments with signiﬁcantly higher mortality than the control. Sporulation was
quantitatively highest among the Bb dish treatments, but only signiﬁcantly higher than

the Bb wick and Ma wick males paired with exposed females.

 

 

GD
01

I Male I Female

 

 

 

 

CD
CD

N
U"!

 

to
q

A
01

ES

 
 

‘I
I
I

Mortality in Days after Exposure

0'1

 

.T'E‘ -l
f

I

I

l ' ' t

i D

Eb Bb Eb Bb Ma Ma Ma Ma Control Control Control Control
Dish Dish Wick Wick Dish Dish Wick Wick Dish Dish Wick
Male Female Made Female Male Female Male Female Male Female Male Femde

Treatment (Pathogen, Surface, Exposed Sex)
Figure 4.3. Mean (:SE) number of days after treatment exposure until mortality of plum curculio adults.
The exposed adult (sex indicated in the x-axis) was held with an unexposed mate of the opposite sex.

       

O

 

63

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

    

 

 

   

 

1000/01 abc
T abc abc I Male I Female
abcd
80%“
3‘ abode
g 6070*
O . cdef
2 I»
H , bcdef
C
d) 40°/o‘
0
63 f
de
0.
e! at at
ZOO/0" —'
W. L I , . r r f r: ..
0 8b 8b Eb Bb Ma Ma Ma Ma Control Control Control Control
Dish Dish Wick Wick Dish Dish Wick Wick Dish Dish Wick Wick
Male Female Male Female Male Female Male Female Male Female Male Female

 

 

 

 

 

 

 

Treatment (Pathogen, Surface, Exposed Sex)

  

Figure 4.4 Mean (:tSE) percent mortality of plum curculio adults 29 d after treatment. The exposed adult

(sex indicated in the x-axis) was held with an unexposed mate of the opposite sex.

 

100%

 

a I Male I Female

 

 

 

 

 

b ab ab ab ab
a

 
 
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

C
.9 ab
'26 T a
'5
8-
O
Q.
C!)
4—0
C
(D
O
h
(D
D.
C C C
iLcL
Bb Bb Eb Bb Ma Ma Ma Ma
Dish Dish Wick Wick Dish Dish Wick Wick
Male Female Male Female Male Female Male Female

Treatment (Pathogen, Surface, Exposed Sex)

Figure 4.5. Mean (:SE) percent sporulation of plum curculio adults 37 d after treatment exposure. The
exposed adult (sex indicated in the x-axis) was held with an unexposed mate of the opposite sex. Bars with
same letter indicate that the corresponding means are not signiﬁcantly different (T ukey, a=0.05).

3.3 Field Study: Granular Formulation against Overwintering Adults

Although fewer total adults emerged from the B. bassiana (77) and M. anisopliae
(69) than control (86) cages, mean emergence was not signiﬁcantly different (F=0.l l,
df=2, p=0.896). Abbott’s corrected mortalities (Abbott 1925) were 10% and 8% for M.
anisopliae and B. bassiana, respectively. The standard error for B. bassiana (6.8) was
much higher than for M. anisopliae (1.7) or control (1.2) treatments. Abbott’s Corrected
Mortalities were 8% and 10% for B. bassiana and M. anisopliae, respectively (Abbott

1925).

65

 

‘9 9.86
16‘
14‘
12"

 

 

Mean Numberof Emerged Adults

 

 

 

 

Treatment

Figure 4.6. Mean (:88) number of emerged adults in the spring following overwintering in cages.
Groundcover treatments included a granular formulation of B. bassiana or M. anisopliae, or an untreated
control.

Table 4.2. Daily temperatures and precipitation near entomopathogenic fungus application date (16-0ct) in

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

overwintering trial.

High Low

Temp Temp Predp
Date (C) (C) (an)
l4-Oct 1 1.7 0.6 0.53
lS-Oct 21 .7 7.8 0.00
l6-0ct 16.7 13.9 0.38
17-0ct 21 .7 1 l .1 0.08
18-0ct 24.4 16.1 2.69
19-Oct 17.2 11.1 1.45
20-Oct 1 8.9 10.0 0.03
21-0ct 25.6 13.9 0.00
22-0ct 22.2 10.0 0.41
23-Oct 10.6 6.7 0.86
24-Oct 1 1.7 3.9 0.00
25-Oct 13.9 1.1 0.00
26-Oct 17.2 6. 1 0.00
27-0ct 13.3 1 . 1 0.71
28-Oct 1 1.7 -1 .7 0.00
29-0ct 14.4 - 1 . 1 0.00
30-0ct 17.8 5.0 0.00

 

 

4. Discussion

Applications of the two fungi to ﬁeld soil in the lab showed promising reductions
in plum curculio adult survival, especially at higher rates. The B. bassiana treatment was
effective down to a rate of 5 x 10'3 conidia/ha. The physical properties of soils may
contribute greatly to efﬁcacy of entomopathogens (Barbercheck 1992), as demonstrated
by the poor efﬁcacy on peat. Peat was tested alongside a loamy sand soil because peat is
a common medium for testing entomopathogenic fungi against greenhouse and nursery
pests, such as the black vine weevil (Moorhouse et a1. 1993, Shah et al. 2007, Bruck
2005). In these situations the absence of a rhizosphere may contribute to poor control
(Bruck 2005).

Adults experienced high mortality to fungi, but this was under conditions of
constant exposure over 15 d to the conidia on a soil surface in a conﬁned area, which
moving adults in the ﬁeld would not necessarily experience. A shorter fungus exposure
period for any assay method should be tested to simulate realistic ﬁeld conditions. The
duration of contact with the soil or peat substrate for individuals during the assay remains
unknown, but many adults were observed to be attached to the apple when checked rather
than on the substrate surface. To reduce this behavior in future assays, exposure to apples
could be reduced. Further testing could conclude whether plum curculio adults exhibit
behaviors indicating attraction or repulsion to fungus-treated substrates.

In the assays with Petri dishes and wicks, since the exposed mate was placed
directly into the cup on the soil surface following exposure to the fungus, the entire cup
environment was likely contaminated with conidia dislodged from the adult. It cannot be

determined whether infection of introduced mates was due to direct physical contact with

67

the exposed mate, or to exposure to contaminated soil, cup, and apple surfaces. Adults
exposed to the fungal surface would likely spend hours if not days losing unattached
conidia on tree or soil surfaces before encountering a mate in an orchard. We cannot be
certain whether adult pairs actually mated or how much time they spent in physical
contact, since they were only observed once daily. In future assays, exposed adults should
be held over soil to shed conidia for increasing time periods, and subsequently introduced
into an uncontaminated arena with a mate for a known time period. The time period of
physical contact could be held more constant or be reviewed on a camera recording.

For unknown reasons, conidia viability of B. bassiana on the wick was 0%, while
that of M. anisopliae on the wick was 98%. It was not surprising then that the mortality
of beetles in the B. bassiana wick treatments was comparable to the controls. However, B.
bassiana dish treatments caused 65-100% mortality. The method of culturing B. bassiana
on the wick surface should be altered to bring conidia viability near 100% and the
viability over time should be tested under ﬁeld conditions.

An emerging pattern was revealed in the fungus treatments. Mates of exposed
females seem to suffer less mortality than the female, whereas mates of exposed males
seemed to suffer comparable mortality to the male. The differences were not statistically
signiﬁcant, but a possible interpretation is that males transmit the fungus to females better
than females transmit the fungus to males. The experiment should be repeated with the
introduction of unexposed mates several days after exposed mate exposure (a “conidia
dislodgement” period), within an environment that hasn’t been contaminated by the
exposed mate before this can be concluded, and with a known period of contact between

mates.

68

The time until death for treated adults ranged from an average of 17 to 22.7 days
after exposure, and these means were comparable across all treatments, whether the
adults were directly exposed or indirectly exposed by an exposure to an infected mate
mate. If the ﬁrst lab assays were carried out up to 23 days, then perhaps mortality would
have increased. The goal of this application in orchards would be to reduce oviposition
scarring and larval emergence, not necessarily accelerate mortality. From infection until
death, adults could be mating, potentially transferring conidia, making oviposition scars
in fruits, and laying viable eggs. Reduced number of eggs laid and percent egg hatch
were demonstrated for yellowish elongate cockchafer infected with Beauveria
brongniartii, conﬁrming suppression of subsequent generations before the onset of
mortality (Yaginuma 2006). Further exploration of the behavioral and reproductive
consequences in the weeks following exposure or infection is warranted to determine
whether such tactics could reduce damage in orchards.

The laboratory assays represent optimum temperature and moisture conditions. In
the ﬁeld, the entomopathogens are likely to succumb to sensitivities to moisture and
temperature stress. The values in Table 4.2 indicate surprisingly optimal conditions for
infection in late October, with temperature minimums between 10 and 16°C, and
maximums between 17 and 26°C for six days post—application. The application date was
also surrounded by precipitation events, which kept the soil uncommonly moist. Neither
fungus controlled the adults. Poor or “patchy” coverage of the treated areas may have
been the result of the large sizes of the granular formulated material. A liquid or wettable
powder formulation could avoid distribution and host contact problems caused by large

(2.5 mm) granular formulations under ﬁeld conditions (Gaugler et a1. 1989, Campbell et

69

al. 2006). Only spring-tilled application of granular B. bassiana gave control of Colorado
potato beetle, but authors cite low temperatures, insufﬁcient time between application
and emergence, and the inactive state of overwintering adults as reasons for poor overall

efﬁcacy (Gaugler et al. 1989).

70

Appendix 1
Record of Deposition of Voucher Specimens’
The specimens listed on the following sheet(s) have been deposited in the named museum(s) as
samples of those species or other taxa, Miich were used in this research. Voucher recognition
labels bearing the Voucher No. have been attached or included in ﬂuid-preserved specimens.
Voucher No.: 2008-10
Title of thesis or dissertation (or other research projects):

ENTOMOPATHOGENIC FUN GI AND NEMATODES FOR MICHIGAN TREE
FRUIT MANAGEMENT TARGETING PLUM CURCULIO
(CONOTRACHELUS NENUPHAR)

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigators Name(s) (typed)
W

 

 

 

Date WM

*Fleference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America.
Bull. Entomol. Soc. Amer. 24: 141 -42.

Deposit as follows:
Original: Include as Appendix 1 in ribbon copy of thesis or dissertation.

Copies: Include as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator, Michigan State
University Entomology Museum.

71

 

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Appendix 1.1
Voucher Specimen Data

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72

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