Citation
Evaluation of Living and Synthetic Mulches with and without Imidacloprid for Suppression of Whiteflies and Aphids, and Insect-Transmitted Viral Diseases in Zucchini Squash

Material Information

Title:
Evaluation of Living and Synthetic Mulches with and without Imidacloprid for Suppression of Whiteflies and Aphids, and Insect-Transmitted Viral Diseases in Zucchini Squash
Creator:
Nyoike, Teresia W
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (90 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Liburd, Oscar E.
Committee Members:
Webb, Susan E.
McSorley, Robert
Graduation Date:
12/14/2007

Subjects

Subjects / Keywords:
Buckwheat ( jstor )
Crops ( jstor )
Immatures ( jstor )
Insecticides ( jstor )
Live mulches ( jstor )
Mulches ( jstor )
Pests ( jstor )
Squashes ( jstor )
Symptomatology ( jstor )
Viruses ( jstor )
Entomology and Nematology -- Dissertations, Academic -- UF
aphids, begomoviruses, bemisia, buckwheat, cucurbits, fagopyrum, geminiviruses, imidacloprid, mulches, sampling, squash, tabaci, uv, viruses, whiteflies, zucchini
City of Gainesville ( local )
Genre:
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Entomology and Nematology thesis, M.S.

Notes

Abstract:
Field studies were conducted in north-central Florida to evaluate the effect of living and synthetic mulches with and without imidacloprid (Admire 2F) on the density of silverleaf whiteflies, (Bemisia tabaci Genn. B-biotype) and aphids (Hemiptera: Aphididae) and their transmission of plant viruses in zucchini squash, (Cucurbita pepo L). Living mulch, buckwheat (Fagopyrum esculentum Moench) and two synthetic mulches (reflective and white) were evaluated in the fall of 2005 and 2006. Five treatments were evaluated including reflective mulch with imidacloprid, reflective mulch without imidacloprid, living mulch with imidacloprid, living mulch without imidacloprid and a standard white mulch (control). The experimental design was a randomized complete block with four replicates. Whiteflies and aphids were evaluated through foliar counts, yellow sticky traps, and pan traps. Natural enemies were established through foliar counts and the marketable yields of squash were evaluated by weight. Data from yellow sticky traps indicated that the addition of imidacloprid enhanced the control of whiteflies in squash growing with buckwheat and reflective mulch. Similarly, foliar counts indicated that these two treatments had fewer numbers of whiteflies than the white synthetic mulch (control). The suppression of immature whiteflies resulted in fewer silverleaf symptoms on the leaves. The addition of imidacloprid to reflective mulch significantly affected apterous aphids in both years. However, counts of alate aphids from pan traps were inconsistent for both field seasons. These counts indicated that there were no significant (P < 0.05) differences between treatments with and without imidacloprid. In 2006, the control treatment had significantly (P < 0.05) higher numbers of alate aphids per trap compared with all other treatments. In 2005, buckwheat treatments had significantly more natural enemies per leaf than reflective mulch treatments. Imidacloprid did not appear to affect natural enemy populations because there were no significant differences in their numbers between treatments with and without Admire 2F. In 2005, there was no incidence of insect-transmitted viruses; whereas in 2006, Cucurbit leaf crumple virus (CuLCrV), a virus new to Florida was found. Cucurbit leaf crumple virus is transmitted by whiteflies in a persistent manner. Higher incidences of plants infected with CuLCrV were recorded on the white mulch compared with all other treatments. Buckwheat mulch had a significant negative effect on the size of squash plants and marketable yields. Yield results were consistent between both years. The treatment with reflective mulch and Admire 2F resulted in significantly higher yields than the living mulch and control treatments. No additional benefits resulted from combining reflective mulch with imidacloprid in terms of yield in both years. The living mulch treatment resulted in consistently lower yields than all the other treatments evaluated. The research indicates that enhanced reduction in the abundance of whiteflies and aphids can be achieved when mulches are combined with imidacloprid but this does not necessarily mean an increase in marketable yields. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2007.
Local:
Adviser: Liburd, Oscar E.
Statement of Responsibility:
by Teresia W Nyoike.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Nyoike, Teresia W. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2007 ( lcc )

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Full Text





Clark, M. F., and A. N. Adams. 1977. Characteristics of the microplate method of enzyme-
linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34: 475-483.

Costa, H. S., D. E. Ullman, M. W. Johnson, and B. E. Tabashnik. 1993. Squash silverleaf
symptoms induced by immature, but not adult, Bemisia tabaci. Phytopathology 83: 763-
766.

Costello, M. J., and M. A. Altieri. 1995. Abundance, growth rate and parasitism of Brevicoryne
brassicae and Myzuspersicae (Homoptera: Aphididae) on broccoli grown in living
mulches. Agric. Ecosyst. Environ. 52: 187-196.

Cradock, R. K., J. V. da Graca, and M. D. Laing. 2002. Studies on the control of virus
diseases in zucchini crops. S. Afr. J. Sci. 98: 225-227.

Csizinszky, A. A., D. J. Schuster, and J. B. Kring. 1995. Color mulches influence yield and
insect populations in tomatoes. J. Am. Soc. Hort. Sci. 120: 778-784.

Csizinszky, A. A., D. J. Schuster, and J. B. Kring. 1997. Evaluation of color mulches and oil
sprays for yield and for the control of silverleaf whitefly, Bemisia argentifolii (Bellows
and Perring) on tomatoes. Crop Prot. 16: 475-481.

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of border crops and intercrop on control of cucurbit virus diseases. Plant Dis. 91: 509-
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current status and future prospects. Arch. Insect Biochem. Physio. 158: 200-215.

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whiteflies: pp. 263-285. In D. Gerling [ed.], Whiteflies: their bionomics, pest status and
management. Intercept Ltd, Andover, UK.

Eastop, V. F. 1977. Importance of aphids as virus vectors pp. 3-62, In Harris K. F. and
Maramorosch, K. [eds.]. Aphid as virus vectors. Academic Press, New York, NY.

Fereres, A. 2000. Barrier crops as a cultural control measure of non-persistently transmitted
aphid-borne viruses. Virus Res. 71: 221-231

Frank, L. D. 2004. Evaluation of living and synthetic mulches in zucchini for control of
homopteran pests, pp. 60, M. S. Thesis. Entomology and Nematology Department,
University of Florida, Gainesville, FL.

Frank, L. D., and O. E. Liburd. 2005. Effects of living and synthetic mulch on the population
dynamics of whiteflies and aphids, their associated natural enemies, and insect-
transmitted plant diseases in zucchini. Environ. Entomol. 34: 857-865.

Gaba, V., A. Zelcer and A. Gal-On. 2004. Cucurbit biotechnology the importance of virus
resistance. In vitro Cell. Dev. Biol. Plant 40:346-358.









Alate aphids were monitored using blue (PackerWare) and clear water pan-traps

(Pioneer/ Tri-State Plastics Inc., Dickson, KY). Four pan traps were used per plot, two of each

color, placed at the four corners of each plot within the interior rows. Each pan trap contained

approximately 250 cm3 of 5% detergent solution (Colgate-Palmolive Co. New York, NY). Each

trap was supported on a tomato cage and trap height was adjusted accordingly to plant height.

The traps were left in the field for one week and sampling was conducted for seven and six

weeks in 2005 and 2006 respectively. In 2005, the number of alate aphids trapped were

established in the field, while in 2006 bowl contents were emptied into individual vials and

labeled accordingly. Vials were then transported to the laboratory for counting.

Physiological Disorder Evaluation

In 2006, symptoms of silverleaf on the squash were assessed on 10 plants within the

interior rows. Squash with silverleaf symptoms were scored using an arbitrary scale (Yokomi et

al. 1990) where index 0 signified a 'healthy' leaf with no symptoms and index 5 leaves were

completely silvered.

Natural Enemy Counts

Natural enemies were sampled using in-situ counts from the same zucchini plants as

described above. Six leaves from six plants located on the outside row of each plot were

randomly selected. The leaves were gently turned and the numbers of natural enemies

encountered were recorded from them. Sampling was initiated 3 weeks after planting and

conducted every other week until the final harvest.

Statistical Analysis

Data from whitefly and aphid counts (foliar, whitefly immatures, and trap counts) were

analyzed using repeated measures analysis (PROC MIXED, SAS Institute 2003) to examine the

interaction effect between treatment and time (sampling weeks). Overall, least square means









within the Class Insecta and present no hazard to mammals. They exhibit excellent contact and

systemic activity and have become widely used for controlling sucking insect pests including

whiteflies, aphids, planthoppers, and leafhoppers (Palumbo et al. 2001, Liu 2004, Denholm and

Nauen 2005).

Soil-applied imidacloprid (Natwick et al. 1996) has been the primary insecticide treatment

used for B. tabaci control in vegetable, melon, and greenhouse production systems (Palumbo et

al. 2001). In treated plants, the compound and its metabolites are initially toxic to feeding adults,

and act as anti-feedants. Establishment by immature whiteflies is significantly reduced because

of suppressed egg deposition. Residual control of B. tabaci populations can vary from 1 to 10

weeks, depending on formulation, rate, depth of placement, soil type, application method, and

cropping system (Palumbo et al. 2001). Due to its rapid uptake and systematic translocation

within newly emerging plants, prophylactic applications of imidacloprid have been reported to

reduce incidences of aphid-borne viruses (Palumbo et al. 2001). However, unlike most other

systemic insecticides, imidacloprid is relatively immobile in the soil and requires precise

placement where root uptake can occur (Natwick et al. 1996). Imidacloprid can cause

phytotoxicity to foliage on young plants depending on the application rates and timing.









inert ground covers such as synthetic, sawdust, straw and husk mulches have been evaluated as a

means of managing key pests in vegetable crops. UV-reflective mulches have been demonstrated

to reduce whitefly and aphids and / or the incidence of the viruses they transmit in various

vegetable crops (Wolfenbarger and Moore, 1968, Brown et al. 1993, Csizinszky et al. 1995,

Smith et al. 2000, Stapleton and Summers 2002, Reitz et al. 2003, Summers et al. 2004, Frank

and Liburd 2005). UV-reflective mulches reflect short-wave light, which repels incoming

herbivores, and reduces their incidence of alighting on plants. This, consequently, affects the rate

of virus transmission and spread by these pests.

Synthetic mulches have also been shown to increase yields (Summer et al. 2004, Frank and

Liburd 2005). Their use has been adopted in many parts of the world including Florida in

production of vegetables. UV-reflective mulches however, lose their efficacy once the crop

canopy covers 70% of the mulch (Smith et al. 2000). In addition, they are expensive and pose a

problem of disposal, which may have an undesirable effect on the environment.

Living mulches are a low-cost alternative to synthetic mulches (Hilje et al. 2001) and are

safe for the environment. The main idea of using these mulches is to provide resources for

natural enemies and manipulate host seeking behavior of insect herbivores (Roots 1973, Hilje et

al. 2001). For instance, whiteflies and aphids locate their host through indiscriminate visual

attraction to yellow (green) (Kring 1972, van Lenteren 1990). Alate aphids locate their host by

contrasting the soil background with the green color of the foliage. Thus, reducing the contrast

between the plant and the bare ground as with living mulches will affect the number of insects

alighting on the host plant (Hilje et al. 2001, Cradock et al. 2002, Hilje and Stansly 2007).

Living mulches in zucchini squash have been demonstrated to reduce the number of

whiteflies and aphids and delay the onset of insect-borne viruses in squash in Florida and Hawaii









Table 3-5. Effect of living and reflective mulches alone or in combination with Admire 2F on the
number of alate aphids trapped per pan-trap in Citra, FL


Mean SEM counts per pan trap

2005a 2006b

Reflective + Admire 2F 3.07 + 0.43 a 2.69 0.45 b

Reflective 2.36 + 0.36 ab 1.94 0.32 c

Buckwheat 2.14 + 0.27 ab 2.91 0.45 b

Buckwheat + Admire 2F 2.21 + 0.28 ab 3.19 0.57 b

White (control) 1.97 + 0.28 b 3.83 + 0.53 a

Means followed by the same letter are not significantly different (P = 0.05 according to least
square means test following repeated measures analysis, LS).
aF= 2.51; df = 4, 268; P< 0.0392
b F = 9.54; df = 4, 84; P < 0.0001









March. In north Florida where frost damage is likely, fall planting occurs in August and

September and in spring season between February and March.

Squash is one of the vegetables that are grown on synthetic mulches in Florida. Various

colors of synthetic mulches such as clear, white, black, yellow, and silver are available (Olson et

al. 2005). Growers use white, or white on black in the fall and black in the winter and spring

(Zitter 1977, Csizinszky et al. 1997, Frank and Liburd 2005). Black mulches keep the soil warm

during the cool season while white mulches are used during the warm season. Alternatively,

reflective silver mulches have the benefit of reducing pests and the occurrence of viral diseases

when used in the fall (Olson et al. 2005).

Squash is planted mainly through direct seeding or transplanting seedlings. Squash matures

within 40 to 65 days. Squash plants have both male and female flowers on the same plant and

therefore require a pollination agent for fertilization to take place (Mossler and Nesheim 2001).

Usually, one hive of honeybees per 0.8 ha is maintained to attain sufficient pollination (Olson et

al. 2005).

Several factors affect the squash industry in Florida resulting in losses to growers. Some of

these factors are the results of increasing costs associated with controlling insects and their

related problems, and outbreaks of new pests and diseases (Akad et al. submitted). Like other

squashes, zucchini squash is attacked by a broad array of insect pests and plant pathogens such

as viruses, bacteria, fungi, and nematodes.

Some of the major foliar diseases affecting cucurbits include powdery mildew caused by

Sphaerothecafuliginea (Schlechtend.:Fr.) Pollacci and Erysiphe cichoracearum DC. and downy

mildew caused by Pseudoperonospora cubensis Berk & M. A. Curtis (Zitter et al. 1996). The

occurrence of these diseases is more common than viral diseases. While preventative sprays are









CHAPTER 1
INTRODUCTION

Cucurbits are a major vegetable crop grown in the USA, Caribbean and other parts of the

world. Their production is important worldwide and these crops are commercially available

throughout the year. Cucurbit production competes in importance with other fruit and vegetable

crops worldwide (Gaba et al. 2004).

The family Cucurbitaceae consists of three genera: Cucumis L., (melon and cucumber),

Citrullus Schrader (watermelon), and Cucurbita L. (summer and winter squashes). Of the

squashes Florida produces primarily summer squashes, (Cucurbitapepo L) including crookneck

squash, straightneck squash, scallop squash, and zucchini squash (Mossler and Nesheim 2001).

Winter squash such as acorn, butternut squash, and spaghetti squash can also be produced.

Florida is ranked second nationally after Georgia in the production of fresh market squash.

In the 2006 field-seasons, a total of 4249 ha of cucurbits was planted in Florida and 4131 ha

were harvested with a value of approximately 38.76 million USD (NASS-2006). In Florida,

almost all of the squash are produced for fresh market. Summer production is mainly for local

markets while fall production is exported. Squash is one of the crops in the state that is shipped

every month of the year thus providing a continuous income to the state of Florida (Mossler and

Nesheim 2001).

Although squash is produced throughout the state of Florida, the southeast part of the state

(Miami-Dade County) is the principal squash-producing region. Other important producing

regions include the southwest counties (Lee, Hendry, and Collier), west-central counties

(Hillsborough, Hardee, and Manatee) and north Florida counties (Alachua, Columbia, and

Gilchrist). Squashes can tolerate temperatures of 18 to 27 C but grows best between 24 and 29

C. In regions where frost damage is not a threat, squash is planted anytime between August and









yellowing, chlorotic mottle, chlorotic leaf margins, leaf curl and distortions, puckering of leaves,

reduced leaf size, plant stunting, and flower abscission (Polston and Anderson, 1997, Varma and

Malathi, 2003).

Aphids

Aphids are one of the most varied and most studied groups of the hemipterans (Kring

1972, Powell et al. 2005). Their reproductive potential, salivary secretions, and ability to transmit

viral diseases make them the most potent and world-wide enemies of many crops (Brown et al.

1993, Kucharek et al 2001, Stapleton and Summers 2002, Soria et al. 2003, Liburd and Frank

2007, Ng and Perry 2004). Viral diseases cause damage to both yield and fruit quality resulting

in significant losses (Kucharek et al. 2001). More than 50 viruses have been listed that affect

cucurbits. Until 2001, there were only five reported aphid-transmitted viruses affecting cucurbits

in Florida. These included Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus

(ZYMV), Watermelon mosaic virus strain 2 (WMV-2) now called WMV, Papaya ring spot

virus type W(PRSV-W) and Watermelon leaf mottle virus (Purcifull et al. 1998). PRSV-W and

WMV are the most commonly occurring viruses in Florida. Another aphid-transmitted virus that

has emerged recently in Florida is PRSV-Tigre (Webb et al. 2003). Typical symptoms of these

viruses on the leaves include: leaf curling downward and wrinkling, vein banding, mosaic

patterns, mottling, blistering, leaf distortions and reduction of leaf lamina (strapleaf). Fruit

symptoms include discoloration with mosaic patterns, reduced size, misshapen, with warty

texture (Alderz et al. 1983, Schubert and McRitchie 1984, Purcifull et al. 1988, Mossler and

Nesheim 2001, Kucharek et al. 2001, Gianessi et al. 2002).

Biology and Behavior of Aphids

Variations in aphid life cycles are well known. They alternate primary hosts with

secondary hosts, sexual with parthogenetic forms, wingless with winged forms, and migrant with









stage has well developed legs and antenna. After emerging, the crawler moves and settles a few

millimeters from the egg to find a feeding site but they are also capable of moving within the

leaves (Triplehorn and Johnson 2005). Once settled and feeding the crawler excretes a small

amount of wax (Gill 1990). Subsequent instars (second, third and fourth) are sedentary and only

have limited movement during molts when they reinsert their stylets. They both have a scale-like

covering, which is a waxy secretion that helps the instars adhere themselves to the leaf surface

(McAuslane 2000, Triplehorn and Johnson 2005).

The fourth instar has three distinct developmental stages even though there is no molting

between them: substage one is a feeding, flattened and opaque nymphal phase, substage two is a

nonfeeding nymphal phase, which is thickened and covered with wax, and a pharate adult phase.

The early stage (substage one) is feeding and hence only the last nymphal phases are referred to

as a pupa. The major difference between the substages two and three is the red eyes which are

more visible in the pharate stage due to the developing adult inside them (Gill 1990, Byrne and

Bellows 1991, Osborne and Landa 1992, Basu 1995).

Adults emerge through a T-shaped slit in the pupal case of the last nymphal instar. A

characteristic round-shaped hole is left on the case if an adult parasitoid emerged from the pupa

(McAuslane 2000). The adult head is broader than long, with seven-segmented filiform

antennae. Mating can occur within hours of emergence once the wings are hardened and

expanded. Mated females produce both females and males whereas unmated ones only males

(Byrne and Bellows 1991, McAuslane 2000).

Females of B. tabaci usually oviposit on the abaxial side of young leaves and the same leaf

is used for oviposition and feeding. They usually prefer a moderate degree of pubescence to

either glabrous or extremely hairy leaf surfaces for oviposition. Bemisia tabaci refrains from









Table 3-1. Effect of living and reflective mulches alone or in combination with Admire 2F on the
number of adult whiteflies and aphids per zucchini squash leaf, Citra, FL (2005)
[Foliar counts]


Mean SEM numbers per leaf

Whiteflies (adult) a Aphids (apterous) b Aphid alatee)

Reflective + Admire 2F 10.38 2.02 c 0.56 0.21 b 0.92 + 0.29 a

Reflective 15.22 2.22 b 3.48 + 0.80 a 0.69 + 0.21 ba

Buckwheat 17.09 2.32 b 3.19 + 1.02 a 0.31 + 0.06 c

Buckwheat + Admire 2F 10.40 + 1.73 c 0.44 0.12 b 0.37 0.06 bc

Control 38.27 + 6.20 a 3.07 + 0.69 a 0.40 + 0.10 bc

Means followed by the same letters are not significantly different (P = 0.05 according to least
square means test following repeated measures analysis, LS).
aF = 43.29; df = 4, 72; P < 0.0001
b F= 10.42; df= 4, 72; P < 0.0001
CF = 5.13; df= 4, 72; P < 0.0011









ovipositing on very young leaves (van Lenteren 1990). Vertical distribution patterns along the

plant stratum have been described for B. tabaci life stages. Eggs are usually found in the upper

plant strata where they are laid while the mature nymphs are on the lower strata. Other factors

that seem to affect oviposition and feeding site selection by whiteflies include phototropism, leaf

shape, and nitrogen content (Bentz et al. 1995, McAuslane et al. 2000).

Whiteflies have been described as weak fliers and move with the wind current, as they

probe host plants. Bemisia tabaci is known to orient toward either yellow or blue/ultraviolet light

but not both at the same time, a behavior that is related to migration and plant colonization (Basu

1995, Antignus 2000). Ultraviolet light is related to migratory behavior whereas yellow radiation

induces vegetative behavior, which could be a part of host selection mechanism. This is

important as whiteflies do not appear to have olfactory cues in host-selection (van Lenteren

1990, Byrne and Bellows 1991, Antignus 2000). Once within the host location, whiteflies use

color as cue to select landing sites for feeding and oviposition (Byrne and Bellows 1991).

Monitoring

Adult whiteflies can be monitored by use of yellow sticky traps and leaf turn method in-

situ. In-situ counts are done to estimate the absolute population in the field. This should be done

in the morning when the whiteflies are least mobile. Yellow sticky traps have been reported to

give a good correlation between catches and actual whitefly numbers in the field in some

systems. For this reason, these traps have been proposed for monitoring whiteflies in integrated

pest management systems (Basu 1995). Nymph age tends to correlate with leaf age since they

complete their development on the leaf they were oviposited. Estimates of egg density are

usually taken from upper stratum leaves, while nymphal densities are taken from the middle and

lower canopies depending on the host crop (Basu 1995). Quantification of immature whiteflies









Gerling, D. 1990. Natural enemies of whiteflies: predators and parasitoids, pp. 147-178. In: D.
Gerling [ed.], Whiteflies: their bionomics, pest status and management. Intercept Ltd,
Andovers, UK.

Gianessi, L. P., C. S. Silvers, S. Sankula, J. E. Carpenter. 2002. Viral resistant squash. Plant
Biotechnology: Current and potential impact for improving pest management in U. S.
Agriculture an analysis of 40 case studies. National Center for Food and Agricultural
Policy. www.ncfap.org.

Gill, R. J. 1990. The morphology of whiteflies, pp.13-46. In: D. Gerling [ed.], Whiteflies: their
bionomics, pest status and management. Intercept Ltd, Andovers, UK.

Greenough, D. R., L. L. Black, and W. P. Bond. 1990. Aluminum-surfaced mulch: An
approach to the control of tomato spotted wilt virus in solanaceous crops. Plant Dis. 74:
805-808.

Greer, L., and J. M. Dole. 2003. Aluminum foil, aluminum-painted plastic and degradable
mulches increase yields and decrease insect-vectored viral disease of vegetables.
HortTechnology 13: 276-284.

Halbert, S., and D. Voegtlin. 1995. Biology and taxonomy of vectors of barley yellow dwarf
viruses, pp. 217- 258. In C. J. D'Arcy and P. A. Burnett [eds.], Barley yellow dwarf: 40
years of progress. American Phytopathological Society, St. Paul, MN.

Hilje, L., H. S. Costa, and P. A. Stansly. 2001. Cultural practices for managing Bemisia tabaci
and associated viral diseases. Crop Prot. 20: 801-812

Hilje, L., and P. A. Stansly. 2007. Living mulch ground covers for management of Bemisia
tabaci (Gennadius) (Homoptera: Aleyrodidae) and Tomato yellow mottle virus
(ToYMoV) in Costa Rica. Crop Prot. (in press doi:10.1016/j.cropro.2007.04.003

Hooks, C. R. R., H. R. Valenzuela, and J. Defrank. 1998. Incidence of pests and arthropod
natural enemies in zucchini grown with living mulches. Agric. Ecosyst. Environ. 69: 217-
231.

Hooks, C. R. R., and A. Fereres. 2006. Protecting crops from non-persistently aphid-
transmitted viruses:a review on the use of barrier plants as a management tool. Virus Res.
120: 1-16.

Hull, R. 2002. Matthews's plant virology, 4th Edn. Academic Press, San Diego, CA.

Hummel, R. L., J. F. Walgenbach, G. D. Hoyt, and G. G. Kennedy. 2002. Effects of
production system on vegetable arthropods and their natural enemies. Agric. Ecosyst.
Environ. 93: 165-176.

Jones, R. A. C. 2001. Developing integrated disease management strategies against non-
persistently aphid-borne viruses: a model programme. Integrated Pest Management
Reviews 6: 115-46.









Cultural control techniques have been proposed as means of managing whiteflies and

aphids and their related problems (Antignus 2000). These techniques are tailored toward

manipulating the insects' behavior in host recognition, resulting in arrestment of the vectors

before they land on the host plant.

Living Mulches

Living mulches are cover crops that are intentionally maintained as living ground cover

throughout the growing season of the main crop. They are distinguished from other cover crops

in that they are not removed before the main crop is planted (Nakamoto and Tsukamoto 2006).

There has been increased interest in the use of living mulches for pest suppression as a means of

responding to environmental concerns regarding the use of pesticides. These cover crops

suppress weeds, reduce soil erosion, enhance soil fertility and quality, and reduce damage

imposed by herbivores.

It has been suggested that diverse habitats contain lower populations of herbivorous insects

as compared to simple habitats. Root (1973) found that insect pests were less dense in Brassica

oleracea grown in diverse habitats than in pure stands. To explain his findings, he proposed two

hypotheses. The natural enemy hypothesis predicts that predator and parasitoid populations will

occur in greater diversity and at greater densities in diverse plantings because these systems

provide additional resources (e.g. food, shelter) for the enemies. The other hypothesis, resource

concentration, predicts that herbivores are more likely to find and remain in pure stands of their

host plants because of availability of necessary resources in these areas.

Use of living mulch as a means of vegetative diversification can also affect transmission of

insect-vectored viruses depending on the mode of transmission of the particular virus. Inclusion

of living mulch provides additional feeding sites for infectious aphids around the crop. This has

been termed as a 'protection crop' and hence reducing the incidence and the spread of aphid-













8 Otop

Admire 2F Admire mid
2 lower










E 2







Treament


Figure 3-3. Distribution of immature whiteflies on different plant strata of zucchini in Citra, FL
(2006)
E-- -- -

E n 4 - - - - - -
3 - - - - - -












(2006)









LIST OF FIGURES


Figure pe

3-1 (A) Zucchini squash growing with living mulch (B) Zucchini squash growing on
UV-reflective mulch ............................................. ............ 56

3-2 Distribution of immature whiteflies on different plant strata of zucchini in Citra, FL
(2 0 0 5) ................... .. ...... ................... ...............................................57

3-3 Distribution of immature whiteflies on different plant strata of zucchini in Citra, FL
(2 006) ................... ........... ......................... ...........................58

3-4 Effect of living and reflective mulches alone or in combination with Admire 2F on
silverleaf symptoms presented by the score indices per treatment in zucchini Citra,
F L (2006) .................................................................................59

4-1 Effects of mulches on Cucurbit leaf crumple virus on zucchini squash plants grown
with either living mulch or reflective ground covers as compared with white
synthetic mulch in Citra, FL (2006). Virus incidence = number of plants per plot
with virus present. Values with the same letter do not differ (P < 0.05) according to
L SD test ......................................................... .................................69

4-2 Cucurbit leaf crumple virus symptoms recorded on zucchini squash plants in Citra,
F lorida (2 006) .............................................................................70









treatments, including the control. Zucchini plants grown within living mulch with Admire 2F

were significantly taller than plants grown in living mulch alone, a treatment that resulted in the

least height when compared with all the other treatments (F = 41.92; df = 4, 34; P < .0001)

[Table 5-1].

Marketable Yields

Zucchini squash yields differed significantly among the treatments (F = 37.56; df = 4,167;

P < 0.0001) [Table 5-2]. In 2005, zucchini plants grown with reflective mulch with Admire 2F

produced significantly higher yields than those from buckwheat and white mulch (control)

treatments. Overall, plants growing within reflective and white mulch treatments had 58 and

54% respectively, more zucchini than those growing in the living mulch. Reflective mulch alone

and white mulch provided similar yields.

In 2006, reflective mulch plots produced significantly higher yields than the white mulch

(control) (F = 53.40; df = 4, 133; P < .0001) [Table 5-2]. Overall, reflective mulch treatment

resulted in the highest yields compared with all the other mulches. As in 2005, buckwheat plots

produced the least yields when compared with all the other treatments. Actually plants growing

within the living mulch alone produced 74 and 64% fewer marketable squash than reflective and

white mulches.

Discussion

Zucchini plants interplanted with the living mulch, buckwheat, were smaller in size and

eventually yielded less than those growing on the synthetic mulches. My results were consistent

with the findings of Frank (2004). Plants growing with buckwheat mulch were smaller and had

lower yields when compared with plants grown with synthetic mulches. When living mulches are

interplanted with a main crop (zucchini) they share the same scarce natural resources (light,

nutrients), which could lead to competition and can negatively affect the productivity of the main









The occurrence of viral diseases may vary from year to year in Florida (Mossler and Nesheim

2001).

The use of reflective or living mulches reduced the incidence of CuLCrV, which is

persistently transmitted by whiteflies. The lowest incidence of virus-infected plants was observed

in the treatments that combined reflective or living mulch with Admire 2F. This positively

correlated (r = 0.96) with the numbers of whiteflies recorded in those treatment plots. It was

noted that mulches (reflective and living mulch with Admire 2F) had fewer numbers of

whiteflies (CHAPTER 3) and consequently resulted in low incidence of CuLCrV. The standard

white mulch treatment (control) had the highest incidence of CuLCrV. This mulch had earlier

reported the highest numbers of whiteflies (adults and immatures) throughout the study. Cucurbit

leaf crumple virus is transmitted by whiteflies only, and a reduction in whitefly numbers in the

two treatments (reflective and living mulch) (CHAPTER 3) lead to reduction of infected plants.

There is therefore an added advantage of combining reflective or buckwheat mulch with Admire

2F as seen in our field trial. It has been suggested that management of viruses is achieved better

with a combination of two or more control strategies. Jones (2001) reported some benefits of

combining different tactics including cultural, chemical, and biological. These tactics all have

different modes of action that act together resulting in enhanced disease suppression than any

method used alone.

Although living and reflective mulches are known to interfere with the insect's host

recognition activity, their actual modes of actions are different. Reflective mulch repels

whiteflies, interfering with their orientation (Zitter and Simons 1980, Csizinszky et al. 1997).

The effectiveness of UV reflective mulch in reducing the incidence of CuLCrV is attributed to its

ability to repel whiteflies, which would be finding their way to the host plant. Alternatively,









Cultural control tactics are safer alternatives and their use has shown some promising

results in managing whiteflies and aphids in various crops. In particular, UV-reflective mulch

repels aphids (Alderz and Everett 1968, Wolfenbarger and Moore 1968), whiteflies (Csizinszky

et al. 1997, Summers and Stapleton 2002, Summers et al. 2004, Frank and Liburd 2005). Alate

aphids and adult whiteflies are repelled by UV wavelengths reflected by the silver and aluminum

pigment of the synthetic mulch. Conversely, reflective mulches are known to lose their

reflectivity to dust or shading as the crop canopy grows and hence they may not be able to

provide sufficient protection against insect pest alone (Zitter and Simons 1980, Smith et al.

2000). The addition of a reduced-risk insecticide may maintain or enhance their effectiveness

against pests of zucchini squash.

Living mulches are cheaper alternative to reflective mulches. Living mulches have been

shown to be effective in distracting whiteflies and other pests from locating their host (Hooks et

al. 1998, Frank and Liburd 2005, Hilje and Stansly 2007). These mulches are used to diversify

habitats. Such diversified habitats have lower populations of herbivores when compared with

monocultures (Root 1973, Andow et al. 1986, Kloen and Altieri 1990, Hooks et al. 1998,

Costello and Altieri 1995), which ultimately lead to the delay of the onset of insect-transmitted

viral diseases. In recent studies, living mulches have been shown to reduce the damage inflicted

by aphids and whiteflies when interplanted with zucchini squash (Hooks et al. 1998, Frank and

Liburd 2005).

Despite being shown to have some negative effects on whiteflies and aphids, living

mulches have not been tested in combination with a reduced-risk insecticide. It is not known

therefore, if the addition of a reduced-risk insecticide will negatively affect the natural enemies

associated with the living mulches or produce synergistic effects in reducing pest populations.












Table 5-2. Effect of living and reflective mulches alone or in combination with Admire 2F on
marketable yields of zucchini in squash Citra, FL

Mean yield per treatment (Kgs.)

2005a 2006b

Reflective + Admire 2F 39.47 + 4.07 a 32.97 + 3.42 a

Reflective 36.26 + 3.87 ab 32.11 + 3.67 a

Buckwheat 15.1 2.70 d 8.29 1.21 c

Buckwheat + Admire 2F 20.39 + 3.59 c 8.54 1.47 c

White (control) 33.45 + 3.62 b 23.37 2.35 b
Means SEM followed by the same letter are not significantly different P = 0.05 (LSD)
aF= 37.56; df = 4,167; P < 0.0001
b F= 53.40; df= 4, 133; P < 0.0001









whitefly and aphid populations. It is known that certain species of natural enemies can play a

role in regulating whitefly populations (Gerling 1990). In the current study, the natural enemy

populations were very low as revealed by the in-situ counts. Therefore other factors contributed

to the reduction in the whitefly population.

There are two theories that can be used to explain the reduction of pests in diversified

plantings (Root 1973). The first one is 'resource concentration' that theorizes that pests will opt

to remain and establish in pure stands as opposed to mixed plantings, which may be limited in

preferred resources (food, shelter). The second, is the 'natural enemy' hypothesis that assumes

that diverse habitats have diverse insects and hence predators and parasitoids could contribute to

pest reduction. Because no consistent effects on natural enemies were observed I can conclude

that resource concentration may have contributed to pest reduction in my study. It can also be

speculated that population reduction of whiteflies in the squash planted with the living mulch

was related to the host plant quality. In the study, zucchini plants growing with buckwheat were

noticeably smaller as indicated by the plant sizes (CHAPTER 5). Quality reduction could be as a

result of increased competition for resources such as nutrients, light, and water leading to a

higher rate of herbivore emigration and /or lower population growth. Additionally, the

differential in immigration and reproduction of herbivores could also be used to explain the

differences in herbivore response in crop monocultures and crop mixtures (Andow et al. 1986).

In the current study, it can be concluded that addition of Admire 2F enhanced control of

whiteflies and to some extent apterous aphids and not alate aphids. Living mulch with Admire 2F

and reflective mulch with Admire 2F gave protection equal against whiteflies and aphids.

Admire 2F did not affect beneficial insects associated with the mulches. The economics of









those in the control, which did not differ significantly with those in reflective mulch and

buckwheat treatments (F = 2.51; df = 4, 268; P = 0.0392) [Table 3-5].

In 2006, pan traps within the white synthetic mulch caught significantly more alate aphids

than all other treatments (F = 9.54; df = 4, 84; P < 0.0001) [Table 3- 5]. The addition of Admire

2F did not affect the number of alate aphids captured. Surprisingly, reflective alone had the

fewest number of alate aphids trapped in the pan traps of all the treatments evaluated including

reflective with Admire 2F. Significant (F = 3.63; df = 4, 84; P < 0.0001) interaction effects

between treatments and sampling dates were observed. Among the seven weeks of sampling,

treatment differences were observed only in week 2 and 4.

Physiological Disorders

Silverleaf symptoms differed significantly among the treatments (F = 44.60; df = 4, 34; P

> 0.001). Plants growing within white mulch had almost the entire upper leaf surface silvered as

indicated by the high index score (Fig. 3. 4). Treatments with Admire 2F (reflective and

buckwheat) had significantly lower index scores than all other treatments. There was no

difference between reflective mulch and buckwheat treatment alone.

Natural Enemies

The major families recorded in the study were Syrphiridae, Coccinelidae, Chrysopidae,

and Apidae and spiders. In 2005, significantly more natural enemies were recorded on squash in

the buckwheat treatment with Admire 2F than any other treatment except buckwheat alone (F =

3.43; df = 4,353; P = 0.009) [Table 3-6]. The treatment reflective mulch with Admire 2F had the

fewest natural enemies but it was not significantly different from reflective mulch alone. In 2006,

there were no significant differences in the number of natural enemies among the treatments (F=

0.69; df = 4, 347; P < .6006) [Table 3-6].









Natwick, T. E., J. C. Palumbo, and C. E. Engle. 1996. Effect of imidacloprid on colonization
of aphids and silverleaf whitefly and growth, yield and phytotoxicity in cauliflower
Southwest Entomol. 21: 283-293.

National Agricultural Statistics Service (NASS). 2006. Vegetables 2006 summary; January
2007. U. S. Department of Agriculture, Washington, D.C.

Nault, L. R. 1997. Arthropod transmission of plant viruses: A new synthesis. Ann. Entomol.
Soc. Am. 90: 521-541.

Ng, C. K. J., and K. L. Perry. 2004. Transmission of plant viruses by aphid vectors. Mol. Plant
Pathol. 5: 505-511.

Olson, S. M. 2005. Mulching, pp. 27-30 In: S. M. Olson and E. Simmone [eds.] Vegetable
production handbook for Florida 2005-2006 IFAS Extension. University of Florida,
Gainesville, FL.

Olson, S. M., E. H. Simonne, D. N. Maynard, G. J. Hochmuth, C. S. Vavrina, M. W. Stall,
P. D. Roberts, S. E. Webb, T. G. Taylor, S. A. Smith 2005. Cucurbit production in
Florida, pp. 185-223 In: S. M. Olson and E. Simmone [eds.] Vegetable production
handbook for Florida. IFAS Extension.University of Florida, Gainesville, FL.

Osborne, L. S., and Z. Landa. 1992. Biological control of whiteflies with entomopathogenic
fungi. Fla. Entomol. 75: 456-471.

Palumbo, J.C., A. R. Horowitz, and N. Prabhaker. 2001. Insecticidal control and resistance
management for Bemisia tabaci. Crop Prot. 20: 739-765.

Paris, H. S., H. Nerson, and Y. Burger. 1987. Leaf silvering of Cucurbita. Can. J. Plant Sci.
67: 593-598.

Perring, T. M., A. D. Cooper, R. J. Rodriguez, C. A. Farrar, and T. S. Bellows (Jr). 1993.
Identification of a whitefly species by genomic and behavioral studies. Science, New
Series 259: 74-77.

Polston, J. E., and P. K. Anderson. 1997. The emergence of whitefly-transmitted geminiviruses
in tomato in the Western Hemisphere, Plant Dis. 81: 1358-1369.

Powell, G. T., C. R. Tosh, and J. Hardie. 2005. Host plant selection by aphids: behavioral,
evolutionary, and applied perspectives. Annu. Rev. Entomol. 51: 309-330.

Purcifull, D. E., G. W. Simone, and C. A. Baker. 1988. Immunodiffusion tests for six viruses
that infect cucurbit in Florida. Proc. Fla. State Hort. Soc. 101: 400-403.

Purcifull, D. E., E. Hiebert, M. A. Petersen, G. W. Simone, T. A. Kucharek, M. D. Gooch,
W. E. Crawford, K. A. Beckham, and P. B. De Sa. 1998. Partial characterization of a
distinct potyvirus isolated from watermelon in Florida. Plant Dis. 82: 1386-1390.









LIST OF TABLES


Table page

3-1. Effect of living and reflective mulches alone or in combination with Admire 2F on
the number of adult whiteflies and aphids per zucchini squash leaf, Citra, FL (2005)
[F oliar cou nts] ..........................................................................................50

3-2 Effect of living and reflective mulches alone or in combination with Admire 2F on
the number of adult whiteflies and aphids per zucchini squash leaf, Citra, FL (2006)
[Foliar counts] ..................................................... ...... .............. .......... 51

3-3 Effect of living and reflective mulches alone or in combination with Admire 2F on
the number of whitefly immatures per treatment in zucchini Citra, FL ............................52

3-4 Effect of living and reflective mulches alone or in combination with Admire 2F on
the number of whiteflies caught per yellow sticky trap in Citra, FL .............................53

3-5 Effect of living and reflective mulches alone or in combination with Admire 2F on
the number of alate aphids trapped per pan-trap in Citra, FL................ .............. ....54

3-6 Effect of living and reflective mulch alone or in combination with Admire 2F on
number of natural enemies per treatment in zucchini Citra, FL .............. ...................55

5-1 Effect of living and reflective mulches alone or in combination with Admire 2F on
plant size of zucchini squash Citra, FL (2006) ..............................................................78

5-2 Effect of living and reflective mulches alone or in combination with Admire 2F on
m arketable yields of zucchini in squash Citra, FL .............. ................ .....................79









aphids that are sedentary as opposed to the migrating alatae. It is not surprising that mulches

without the reduced-risk insecticide, including the white mulch (control), did not differ

significantly (P > 0.05) from each other in both years. The color of pan traps did not affect the

number of aphids caught. This is in harmony with the findings of Frank and Liburd (2005).

In Florida, white mulch is used alone or on top of black in zucchini production during the

warm seasons to keep the soil cool (Liburd and Frank 2007, Liburd et al. in press). In our study,

we report that white polyethylene mulch resulted in consistently high numbers of whiteflies

(adults and nymphs) and alate aphids in at least one season. Earlier studies have also reported

that white mulch is associated with high numbers of immatures and adult whiteflies (Csizinszky

et al. 1997). In addition, a high number of aphids have been trapped out plants with white

polyethylene mulch compared with bare ground or / and other mulches (Alderz and Everett

1968, Zitter 1977, Zitter and Simons 1980, Frank and Liburd 2005).

The counts of natural enemies associated with whiteflies and aphids were inconsistent

throughout the two field seasons. In 2005, more natural enemies were recorded in plants in plots

treated with living mulch (buckwheat) than those with reflective mulch with Admire 2F.

However, in 2006 no significant differences among the treatments were recorded. Admire 2F did

not have adverse effects on natural enemies associated with the mulches as indicated by non-

significant differences between treatments with and without Admire 2F. The natural enemies

counted on the leaves were mainly predators and consisted of Syrphiridae, Coccinelidae,

Chrysopidae and Apidae and spiders. Spiders made up the bulk (39%) of the recordings followed

by coleopterans (29%). Buckwheat is an annual plant whose flowers produce nectar that attracts

a high percentage of beneficial insects including pollinators. When used as a living mulch,

buckwheat supports a high number of natural enemies, which could contribute to the reduction of









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EVALUATION OF LIVING AND SYNTHETIC MULCHES WITH AND WITHOUT
IMIDACLOPRID FOR SUPPRESSION OF WHITEFLIES AND APHIDS, AND INSECT-
TRANSMITTED VIRAL DISEASES IN ZUCCHINI SQUASH

By

Teresia W. Nyoike
December 2007

Chair: Oscar Liburd
Major: Entomology and Nematology

Field studies were conducted in north-central Florida to evaluate the effect of living and

synthetic mulches with and without imidacloprid (Admire 2F) on the density of silverleaf

whiteflies, (Bemisia tabaci Genn. B-biotype) and aphids (Hemiptera: Aphididae) and their

transmission of plant viruses in zucchini squash, (Cucurbitapepo L). Living mulch, buckwheat

(Fagopyrum esculentum Moench) and two synthetic mulches (reflective and white) were

evaluated in the fall of 2005 and 2006. Five treatments were evaluated including reflective mulch

with imidacloprid, reflective mulch without imidacloprid, living mulch with imidacloprid, living

mulch without imidacloprid and a standard white mulch (control). The experimental design was

a randomized complete block with four replicates. Whiteflies and aphids were evaluated through

foliar counts, yellow sticky traps, and pan traps. Natural enemies were established through foliar

counts and the marketable yields of squash were evaluated by weight.

Data from yellow sticky traps indicated that the addition of imidacloprid enhanced the

control of whiteflies in squash growing with buckwheat and reflective mulch. Similarly, foliar

counts indicated that these two treatments had fewer numbers of whiteflies than the white









treatments evaluated ((F= 9.96; df = 4, 15; P < 0 .0004) [Figure 5-2]. The symptoms later spread

to other plots with time.

Cumulative disease incidence among the plants growing under buckwheat and reflective

mulch treatments was significantly lower than those under the white mulch treatment (Figure 5-

2). Addition of Admire 2F to reflective mulch treatments resulted in a significant reduction in the

number of plants showing virus symptoms, which was not significantly different from

buckwheat mulch with Admire 2F.

Greenhouse Results

Our greenhouse test indicated that whiteflies were able to transmit the virus from infected

field samples to healthy plants. Plants began to show virus symptoms after approximately 10

days. After 14 days all the plants in the cages showed the viral symptoms and PCR analysis was

positive for a begomovirus.

PCR Analysis Results

The samples submitted for the PCR assays produced PCR products using the degenerate

primers PAR1c496/ PALlv1978 and PBLlv2040/PCRcl54. When sequences were submitted for

a Basic Local Alignment Search Tool (BLAST) search the results revealed over 95% sequence

identity with Cucurbit leaf crumple virus (CuLCrV). (Akad et al. submitted).

Discussion

In this study, there was no incidence of aphid-transmitted virus diseases. Alate aphid

populations were also low during the two field studies (CHAPTER 3) that are significant in

transmission of the viruses. However, this low population could not be directly related to the

absence of the viruses. Furthermore, it is reported that even a small population of alate aphids

can be sufficient to spread viruses especially non-persistently transmitted viruses (Fereres 2000).









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

LIST O F TA B LE S ......... ..... .............. ................................................................. 7

LIST OF FIGURES ................................. .. ..... ..... ................. .8

ABSTRAC T ..........................................................................................

CHAPTER

1 INTRODUCTION ............... ................. ........... ................................. 11

Ju stificatio n ................... ...................1...................7.......
H y p o th e sis ..........................................................................1 8
Specific Objectives .............. ................. ............ ........................ .... 18

2 L IT E R A TU R E R E V IE W ........................................................................ .. ....................... 19

W hiteflies......................................................................... 19
T ax o n o m y ....................................................................................................2 0
Biology and B behavior of B Biotype ........................................ .......................... 21
M monitoring ......................................2...................3..........
W hitefly-Transmitted Geminiviruses................................................... ................. 24
Aphids........................... ...................... 25
B iology and B behavior of A phids ......................................................................... ..... 25
M o n ito rin g ................................................................................................................. 2 7
A phid-Transm itted V viruses ................... ................... ............................................... 28
Management of Whiteflies and Aphids and their Related Problems............... ..................29
L iv ing M u lch es................................. ..................................................... ............... 30
Synthetic M ulches ...................................... ................................. ........ 31
R educed R isk-Insecticides...................................................................... ...................32

3 TO INVESTIGATE THE EFFECT OF LIVING (BUCKWHEAT) AND REFLECTIVE
MULCHES WITH AND WITHOUT A REDUCED-RISK INSECTICIDE ON
WHITEFLIES, APHIDS AND NATURAL ENEMIES IN ZUCCHINI SQUASH .............34

Materials and Methods .............. ............. ............... .................. .... 36
Plot Preparation, Irrigation system, and Experimental Design ......................................36
F o lia r S a m p lin g ...............................................................................................................3 8
T rap Sam pling ......................................................... ................. 38
Physiological D disorder Evaluation ............................................................. ... ............ 39
N natural Enem y Counts ................................... .. .. ...... .. ............39
Statistical A n aly sis ................................................................39
R e su lts ................... ...................4...................0..........









needed to fully investigate how these two strategies (living mulch with Admire 2F or reflective

mulch with Admire 2F can be integrated in zucchini production.

Likewise, the number of nymphs per leaf disc was higher in 2005 than in 2006. Our results

indicated that treatments with Admire 2F provided significantly greater protection from whitefly

immatures than treatments without. The reduction in number of immature whiteflies per leaf is

important since they are responsible for inducing silverleaf symptoms. It was evident that most

of the whitefly immatures were concentrated in the lower strata of the zucchini plants. This area

provides more nitrogen and fewer trichomes (Bentz et al. 1995). The immature whiteflies of B

biotype are associated with a physiological disorder called squash silverleaf symptom (SSL). The

occurrence of SSL was recorded in 2006. Silverleaf symptoms were more severe on the white

mulch, which had the highest whitefly population compared with all the other treatments. A

positive correlation between the number of whitefly immatures and SSL symptoms has been

reported before by Costa et al. (1993).

In this study, the mulch treatments had varying effects on alate aphids. These aphids are

important in the transmission of non-persistent viruses such as the mosaic viruses. Previous

studies have reported that the UV-reflective mulch was able to confuse and repel alate aphids

from landing on the plants (Wolfenbarger & Moore 1968, Brown et al. 1993, Summer et al.

2004). Our results in 2006 were in agreement with previous research where the reflective mulch

treatments afforded the best protection against alate aphids. In this study it is a reported that

addition of Admire 2F did not enhance reduction of alate aphids as revealed by the pan traps and

foliar counts. In contrast, Admire 2F provided a significant amount of control of apterous aphids

in 2005, but in 2006 the reduction was not as dramatic, perhaps because the populations were not

as high. Furthermore, Admire 2F is a systemic insecticide and could be more lethal to apterous









values were computed and means were compared to determine the effects of mulch treatments.

The standard errors of means (S.E.M) were also calculated (PROC MEANS, SAS Institute

2003).

Data from natural enemy counts, silverleaf score index were subjected to an analysis of

variance (ANOVA) using SAS GLM (SAS 9.1 version) and treatment means separated by least

significant differences (LSD) test (SAS Institute 2003). Where necessary, the data were log-

transformed to meet assumptions for ANOVA. Comparisons of immature counts from the

treatments were made based on the average upper, middle and lower leaf disc counts.

Results

Foliar Counts

In 2005, buckwheat with Admire 2F was equally effective as reflective mulch with Admire

2F in controlling adult whiteflies per leaf (F = 43.29; df = 4, 72; P < 0.0001) [Table 3-1].

Similarly, buckwheat alone was not significantly different from reflective mulch alone. Addition

of Admire 2F to buckwheat and reflective mulches resulted in enhanced control of whiteflies per

leaf compared with the other treatments. Conversely, plants grown with white mulch (control)

had significantly higher number of whiteflies per leaf than all the other treatments. There were

significant (F = 7.86; df = 20, 72; P < 0.0001) interaction effects between treatments and

sampling weeks. Treatment differences were observed in 5 out of the 6 weeks sampled.

Apterous aphid counts per leaf indicated that buckwheat with Admire 2F had similar

numbers as those recorded in reflective mulch with Admire 2F. Both treatments resulted in

significantly fewer numbers of aphids than all the other mulches tested including white mulch

(control) (F = 10.42; df = 4, 72; P < 0.0001) [Table 3-1]. The addition of Admire 2F to the

mulches reduced the number of apterous aphids per leaf but not alate aphids. There were

significant (F = 3.37; df = 20, 72; P < 0.0001) interaction effects between treatment and











Table 5-1. Effect of living and reflective mulches alone or in combination with Admire 2F on
plant size of zucchini squash Citra, FL (2006)
Mean SEM (Inch)

Plant Widtha Plant Heightb

Reflective + Admire 2F 42.08 2.00 a 23.71 + 0.39 a

Reflective 40.09 1.94 ab 22.15 0.69 a

Buckwheat 31.62 1.54 c 13.75 0.58 d

Buckwheat + Admire 2F 32.64 + 1.81 c 16.10 + 0.73 c

Control (white mulch) 38.18 2.00 b 18.39 0.70 b


Means followed by the same letter are not significantly different P = 0.05 (LSD)
aF= 11.64; df 4, 34; P < 0.0001
bF = 41.92; df = 4, 34; P < 0.0001












S tBY. ,Squash Silverleaf
symptoms on the
zucchini leaves








Figure 4-2. Cucurbit leaf crumple virus symptoms recorded on zucchini squash plants in Citra,
Florida (2006)









the crop canopy covers them. It is possible in cases of high pest pressure that the use of reflective

mulch and imidacloprid may be warranted since early virus infection can significantly reduce

yield. Imidacloprid can be used to reduce the spread of whitefly-transmitted viruses by reducing

the vector population on the primary and secondary host.

Currently, there is a rising demand for sustainable agriculture and to increase organic

farm holdings in the United States and the rest of the world. This means that the use of cultural

based tactics for pests and diseases in various crops will be the first line of defense for most

growers. This study supports the importance of buckwheat for managing whiteflies and aphids in

zucchini as a cheaper alternative to reflective mulches. Therefore, buckwheat is likely to become

more popular in organic farming as a means of responding to pests and disease management

needs provided that adverse competition effects on crop yield can be worked out.

An important aspect of buckwheat is that it grows erect and therefore does not pose

problems with shading or growing into squash. Therefore, different planting spacing can be

evaluated to determine the optimal spacing in the relation to the cash crop to reduce the

competition for natural resources. Zucchini crop may be planted in double or triple rows between

wider strips of living mulch to lessen competition between the crop and the mulch. If the mulch is

planted before the vegetable crop, like in our case, strips of the mulch must be prepared for the

crop by tilling, mowing, or applying herbicides. Another study can be conducted to establish the

optimal time of planting squash into the living mulch so that maximum usage of beneficial insects

can be achieved. A comprehensive survey for CuLCrV, diagnosing the severity of the disease and

potential risks in Florida would be an important problem to address.









LIST OF REFERENCES


Adkins, S., S. E. Webb, D. Achor, P. D. Roberts, and C. A. Baker. 2007. Identification and
characterization of a novel whitefly-transmitted member of the potyviridae isolated from
cucurbits in Florida. Phytopathology 97: 145-154.

Akad, F., S. E. Webb, T. W. Nyoike, O. E. Liburd, and J. E. Polston. 2007. Detection of
Cucurbit leaf crumple virus in Florida. Phytopathology (submitted).

Alderz, W. C., and P. H. Everett. 1968. Aluminum and white polyethylene to repel aphids and
control watermelon mosaic. J. Econ. Entomol. 61: 1276-1279.

Alderz, W. C., D. E. Purcifull, G. W. Simone, E. Hiebert. 1983. Zucchini yellow mosaic virus:
a pathogen of squash and other cucurbits in Florida. Proc. Fla. State Hort. Soc. 96: 72-74.

Andow, D. A., A. G. Nicholson, H. C. Wien, and H. R. Willson. 1986. Insect populations on
cabbage grown with living mulches. Environ. Entomol. 15: 293-299.

Antignus, R. 2000. Manipulation of wavelength-dependent behavior of insects: an IPM tool to
impede insects and restrict epidemics of insect-borne viruses. Virus Res. 71: 213-220.

Arkubulut, S., A. Keten, and W. T. Stamps. 2003. Effects of alley cropping on crops and
arthropod diversity in Duzce, Turkey. J. Agron. Crop Sci. 189: 261-269.

Basu, A. N. 1995. Bemisia tabaci (Gennadius) crop pest and principal whitefly vector of plant
viruses. Boulder, Westview Press, USA.

Bellows (Jr.) T. S., T. M. Perring, R. J. Gill, and D. N. Headrick. 1994. Description of a
species of Bemisia (Homoptera: Aleyrodidae). Ann. Entomol. Soc. Am. 87: 195-206.

Bentz, J-A., J. Reeves III, P. Barbosa, and B. Francis. 1995. Within-plant variation in
nitrogen and sugar content of poinsettia and its effects on the oviposition pattern,
survival, and development of Bemisia argentifolii (Homoptera: Aleyrodidae). Environ.
Entomol. 24: 271-277

Brown, J. E., J. M. Dangler, F. M. Woods, and K M. Tilt, M. D. Henshaw, W. A. Griffey,
and M. S. West. 1993. Delay in mosaic virus onset and aphid vector reduction in
summer squash grown on reflective mulches. HortScience 28: 895-896.

Brown, J. K., D. R. Frohlich, and R. C. Rosell. 1995. The sweetpotato or silverleafwhiteflies:
Biotypes of Bemisia tabaci or a species complex? Annu. Rev. Entomol. 40: 511-534.

Brown, J. K., A. M. Idris, C. Alteri, and D. C. Stenger. 2002. Emergence of a new cucurbit-
infecting begomovirus species capable of forming reassortants with related viruses in the
Squash leaf curl virus cluster. Phytopathology 92: 734-742.

Byrne, D. N., and T. S. Bellows (Jr.). 1991. Whitefly biology. Annu. Rev. Entomol. 36: 431-
457.









CHAPTER 6
CONCLUSION

The field experiments demonstrated that the addition ofimidacloprid to living and

reflective mulches enhanced the control of adult whiteflies in zucchini squash. Specifically, the

addition of imidacloprid to buckwheat further reduced whitefly densities (adults and immatures)

in both years as revealed by foliar and yellow sticky trap samples. The addition of imidacloprid

to reflective mulch enhanced the control of aphids (wingless) in both years. However a lower

population was always recorded with the addition of imidacloprid. In almost all cases buckwheat

in combination with imidacloprid gave protection to squash against whiteflies equal to that

provided by reflective mulch with imidacloprid. Only in 2005, did counts from the yellow sticky

traps indicate that reflective mulch with imidacloprid was superior to buckwheat in combination

with the reduced-risk insecticide.

The addition of imidacloprid did not affect the alate aphid population densities in

zucchini as indicated by foliar and pan trap samples. Surprisingly, pan traps set within squash

growing on reflective mulch with imidacloprid caught more alate aphids in 2006 than those

within reflective mulch alone. This could not be explained as it is known that reflective mulch

interferes with the insect's host locating ability and hence reduces their landings on squash

plants. The addition of imidacloprid to reflective mulch reduced apterous aphids per leaf in both

years. However, buckwheat combined with imidacloprid reduced the populations only in 2005.

Such differential effects of treatment combinations on the pests indicates that development of an

integrated pest management program for the zucchini crop can be a complicated process, since

the crop is affected by many pest species.









Aphid-Transmitted Viruses

Aphid-borne viruses can kill infected plants and significantly affect the crop yield

(Eastop 1977). Aphid-borne non-persistently transmitted viruses are of economic importance for

many cucurbit growers. They form the bulk of aphid transmitted viruses (Hull 2002, Hooks and

Fereres, 2006). Non- persistent viruses are transmitted by many aphid species including Aphis

gossypii, Myzuspersicae, Acyrthosiphon (Aulacorthum) solani, Aphis craccivora, and

Macrosiphum euphorbiae and many others as they search for suitable hosts to invade. Aphis

gossypii, the melon aphid is considered as one of the most important vector because it is able to

colonize and reproduce on cucurbit crops (Damicone et al. 2007). In other cases this aphid has

not been considered important because it tends to remain on the plant (Mora-Aguilera 1995).

Non-persistent viruses are defined as those that have a very short acquisition and

inoculation times (seconds to minutes) (Nault 1997, Ng and Perry 2004). The viruliferous aphids

retain their ability to infect for a very short period. These viruses have been termed as 'stylet-

borne' viruses due to the fact that the virions are restricted to the stylets. These viruses are

transmitted to healthy plants within very brief probing period (Nault 1997). It is known that

during acquisition and inoculation stylets of aphids do not penetrate beyond epidermal cells

(Nault 1997). Conversely, semi-persistent viruses are vectored by a few aphid species that will

be able to feed on and colonize the host plant. Acquisition of the virions occurs within minutes,

but the ability to transmit can be increased with prolonged feeding (Ng and Perry, 2004). In

addition, the virions are retained for hours to days within the body of the vector. Semi-

persistently transmitted viruses have also been termed as 'foregut borne' and like the non-

persistently transmitted viruses they cannot be recovered from the hemolymph (Nault 1997).

Furthermore, the ability of viruliferous immatures to infect other plants is lost during molting in

both types of transmission.













14 -
a
12

0 10
8


2 b





Tc c
0
Reflective + Reflective Buckwheat + Buckwheat Control
Admire 2F Admire 2F
Treatment


Figure 4-1. Effects of mulches on Cucurbit leaf crumple virus on zucchini squash plants grown
with either living mulch or reflective ground covers as compared with white synthetic
mulch in Citra, FL (2006). Virus incidence = number of plants per plot with virus
present. Values with the same letter do not differ (P < 0.05) according to LSD test.









living mulches are known to reduce the contrast between the ground and the host plants which

apparently guide the whiteflies and other insects to the host (Cradock et al. 2002).

Cucurbit leaf crumple virus (CuLCrV) has been reported in Arizona, Texas, California and

Northern Mexico (Wiebe 2003), but had not been found in Florida before winter 2006. This

spring (2007), CuLCrV was recorded in southwest Florida in watermelon (S.E. Webb, Pers.

Comm.). Cucurbit leaf crumple virus is transmitted by whiteflies in a persistent manner and has

a wide host range within the family Cucurbitaceae, infecting most of the domestically grown

cucurbits. It has also been reported to infect beans (Brown et al. 2002). Symptoms on the leaves

are very noticeable. The virus can also affect the fruit of some cucurbits. The disease may have

been accidentally introduced into Florida through infected watermelon seedlings from another

state (Akad et al. submitted). It is known that appearance and distribution of the new geminivirus

diseases are associated with the coming of biotype B and its population increase in the state

(Polston and Anderson, 1997), as well as evolution of variants of the viruses (Varma and

Malathi, 2003). The probable geographic origin of CuLCrV is southwestern USA and northern

Mexico (Brown et al. 2002).

Our study suggests that the use of living or reflective mulch alone or in combination with

imidacloprid (Admire 2F) can be used to reduce whitefly populations and reduce the incidence of

Cucurbit leaf crumple virus. There are benefits to be carried by combining a living or reflective

mulches with a reduced-risk insecticide.









The study also suggests that the use of reflective and buckwheat mulches with

imidacloprid can be used to effectively reduce the incidence of whitefly-transmitted virus. This

further supports the argument that mulches can be deployed against various virus diseases. Only

in 2005, that buckwheat with imidacloprid treatment produced higher yields than the buckwheat

mulch alone.

The yields of squash planted with buckwheat were greatly reduced. Living mulches such

as buckwheat are cheaper alternatives to synthetic (reflective) mulches. However, crop spacing

and competition for resources need to be evaluated further before any recommendation can be

made.

A cost analysis of cost per hectare for using these mulches is as follows. The cost of

applying reflective mulch/labor is 1374 USD/ ha. The cost of drip tapes is 150 USD/1829 meter

roll. The average cost of planting buckwheat is 178 USD/ha. The cost of adding imidacloprid

(Admire 2F) to the mulches at the recommended rate imidacloprid is 257 USD per ha.

Buckwheat mulch with imidacloprid is more cost effective than reflective with imidacloprid.

However, it is worth mentioning that buckwheat has some other additional costs which include

extra drip lines to support plant growth and management practices such thinning after planting.

Although reflective mulch is more expensive, the cost of the mulch installation can be

justified because significantly higher yields were obtained. The reflective mulch treatment with

imidacloprid was much more expensive than reflective mulch alone and yet no additional gains

resulted in terms of yields. Therefore, it may not be necessary to combine the two tactics together

because mulch alone was sufficient to give satisfactory yields. Since there were no benefits from

using this combination, it may not be recommended to growers as an option. However, it may

worth mentioning that the benefits of reflective mulches are short lived since they diminish once









number of alates per leaf when used with any of the mulches (buckwheat or reflective). Although

there were no significant (F = 0.86; df = 4, 72; P < 0.6330) interaction effects between

treatments and time, differences among the treatments were observed on the fifth week of

sampling. On this date, white synthetic mulch resulted in significantly higher number of alate

aphids than all the other treatments.

Whitefly Immature Counts

Results of whitefly immature counts in the laboratory were consistent across the two years.

Most of the whitefly nymphs were concentrated in the lower plant stratum with the least on the

upper stratum (Figs. 3-2 & 3-3). In 2005, white mulch had significantly higher number of

immatures per 3.14-cm2 leaf disc compared with all the other treatments (F = 13.91; df = 4, 72; P

= 0.0001) [Table 3-3]. Treatments with Admire 2F contained the fewest number of whitefly

immatures per leaf disc. Buckwheat alone was not significantly different from reflective mulch

alone. There were significant (F = 2.91; df = 4, 72; P = 0.0005) interaction effects between

treatment and time.

In 2006, the number of whitefly immatures varied significantly among treatments (F=

11.90; df= 4, 72; P< 0.0001) [Table 3-3]. No significant difference occurred among reflective

mulch, reflective mulch with Admire 2F and buckwheat mulch with Admire 2F. The addition of

Admire 2F to reflective mulch did not result in further significant reduction of immature

whiteflies. Whitefly adult populations on plants growing within buckwheat in combination with

Admire 2F resulted in lower whitefly immatures than those in buckwheat mulch alone.

Buckwheat mulch with Admire 2F was not significantly different from reflective with Admire

2F and reflective alone. White mulch resulted in significantly higher numbers of immatures per

leaf disc compared with all the other treatments. The interaction effects between treatment and









W), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber

mosaic virus (CMV). Symptoms of these viruses on leaves include: leaves curling downward and

wrinkling, vein banding, mosaic patterns, mottling, blistering and distorted (strapleaf) leaves

which are reduced in size. Fruit symptoms include discoloration with mosaic patterns and

reduced misshapen and warty texture (Alderz et al. 1983, Schubert and McRitchie 1984,

Purcifull et al. 1988, Mossler and Nesheim 2001, Gianessi et al. 2002).

Mosaic viruses are transmitted in a non-persistent manner by several aphid species. Of the

known species associated with virus spread, the melon aphid Aphis gossypii Glover, is

considered a primary pest of cucurbits (Damicone et al. 2007). Alate aphids are able to

acquire/transmit the viruses as they are probing to test the plant for host suitability. Once the

virus is acquired aphid vectors remain viruliferoous for a short period of time (minute to hours).

Damage to host plants by whiteflies and aphids can also result in reduced plant growth

and quality due to excessive removal of plant assimilates from phloem. Moreover, excretion of

honeydew, a sugar-rich compound, by whiteflies and aphids on the leaves and fruits, promote

growth of sooty mold (Capnodium spp.) further reducing their quality (Palumbo et al. 2001).

Pesticides have been the most popular pest management tactic in high value crops

(Hummel et al. 2002) including cucurbits (Frank and Liburd 2005). However, awareness has

increased concerning the use of pesticides due to their effects on human, environment and other

non-target organisms (Antignus 2000). In addition, high pesticide usage increases the cost of

production and potential for resistance development. Subsequently, there has been increased

emphasis on sustainable cultural pest management tactics.

Cultural control strategies entail manipulating components of the agro-ecosystem to reduce

pest numbers to below economically damaging levels (Hilje et al. 2001). Living mulches and









Hypothesis

It is hypothesized that the use of synthetic (reflective) and living mulches (buckwheat) in

combination with a reduced-risk insecticide (Admire 2F) will further suppress whitefly and aphid

populations and subsequently delay the onset of viral diseases.

The goal of my study is to determine if mulches in combination with Admire 2F

(imidacloprid) could offer added advantage in suppressing key pests as well as insect-transmitted

viruses and consequently increase yields. In addition, I seek to demonstrate an economic viable

management program for aphid and whiteflies and their related viral diseases of squash that can

be used in Florida and elsewhere.

Specific Objectives

To investigate the use of reflective and a living mulch, buckwheat (Fagopyrum
esculentum Moench) with and without imidacloprid for controlling aphids and whiteflies.

To study the effects of reflective and living mulch (buckwheat) with and without
imidacloprid on natural enemies (beneficial insects) that suppress aphid and whiteflies.

To evaluate the effectiveness of reflective and a living mulch with and without
imidacloprid to viral diseases of zucchini.

To study the impact of reflective and a living mulch with and without imidacloprid to
plant size and consequently the marketable yields of zucchini.









silverleaf disorder of squash (SSL) an important physiological disorder of some cucurbits.

Squash silverleaf is associated with the feeding of immature whiteflies of B biotype and is

characterized by silvering of the adaxial leaf surface and blanching of the fruit (Schuster et al.

1991, Yokomi et al. 1990, Costa et al. 1993). Substantial economic losses in Florida have now

been associated with SSL due to poor fruit quality and yield reduction (Yokomi et al. 1990,

Liburd and Frank 2007).

Whiteflies also transmit geminiviruses to squash. Recently, two whitefly-transmitted

viruses were recorded in squash in Florida. The Squash vein yellowing virus (SqVYV) an

Ipomovirus, affects squash, pumpkin and watermelon and Cucurbit leaf crumple virus

(CuLCrV); a begomovirus, affects all cucurbits except honeydew melon (Brown et al. 2002,

Adkins et al. 2007, Akad et al. submitted). Cucurbit leaf crumple virus is a relatively new virus

to Florida and first appeared in north-central Florida during the fall 2006 (Webb et al. 2007). The

emergence of newly introduced whitefly-borne viruses to areas where they never existed before

has been attributed to high whitefly populations and possibly movement of infected plant

material from other states (Webb 1991, Polston and Anderson 1997). Cucurbit leaf crumple virus

is transmitted by whiteflies in a persistent, circulative manner. Typical symptoms of CuLCrV on

leaves include curling, yellowing, and crumpling. Symptoms can also develop on the fruits

causing abrasions or even green streaks like on the yellow squash causing the fruits to be

unmarketable (Webb et al. 2007).

Aphids are also important pests of squash, causing damage through sucking plant sap and,

more importantly, through transmission of viral diseases. They are the most important vectors of

plant viruses and are known to transmit 275 different viral disorders (Nault 1997). In Florida, the

most damaging viruses affecting squash are Papaya ring spot virus-Watermelon strain (PRSV-









included a positive control and five negative controls (healthy squash). The plates were

incubated overnight at 4 oC. After 24 h the plates were emptied and quickly rinsed and blotted

once before washing with an ELX 50 Auto Strip Washer (Bio-Tek, Winooski, VT) using 8

washes of PBST without soaking. Enzyme conjugates (ECB) were prepared by diluting specific

antibodies in buffer (Agdia Inc.) using predetermined dilution factors. Plates were covered with

Parafilm before being incubated at 30 oC for 4 h in a moist chamber. After incubation and

washing (as above), 100 [iL of substrate (10% diethanolamine adjusted to pH 9.8 + phosphatase

substrate) (Sigma, St. Louis, MO) was added to each well. The color change was visually

assessed (yellow, positive and clear, negative) before measuring the absorbance of the samples

at 450 nm using an EL 800 Universal Microplate reader (Bio-Tek, Winooski, VT).

In 2006, visual observations of viral symptoms and incidences were recorded and

monitored in the field. Viral symptoms including mosaic, mottling, leaf crumpling, and distortion

(fig. 4-1) were noted on squash plants growing within white synthetic mulch treatments. The

symptomatic leaves were excised from the plants and transported back to the laboratory. Each

sample was assayed using ELISA for eight viral diseases including ZYMV, WMV, PRSV-W

CMV, Tobacco streak virus (TSV), Watermelon leaf mottle virus (WLMV), Papaya ring spot

virus-type T (PRSV-Tigre), Squash mosaic virus (SqMV) and Tomato spotted wilt virus

(TSWV). Procedures followed were as described above except that TSV and TSWV were tested

with reagent kits (Agdia Inc.) and the manufacturer's directions were followed. All other antisera

were obtained from D. Purcifull, Unversity of Florida.

PCR Analysis

Twenty leaf samples were collected from the field (as earlier described) from symptomatic

squash plants and tested using polymerase chain reaction (PCR) for the presence of

begomoviruses. Primers PAR1c496/ PALlv1978, which amplifies a region of the begomoviruses









On the other hand persistent transmitted viruses require longer periods to be acquired or

transmitted (hours to days) (Ng and Perry, 2004). They also require more than one week latent

period (Nault 1997). Furthermore, infected aphids remain viruliferous for extended periods.

Management of Whiteflies and Aphids and their Related Problems

In the zucchini crop, no single control method has been used to control whiteflies and

aphids and their associated problems (Hooks et al 1998, Frank 2004). Whiteflies and aphids are

notorious pests in zucchini and greatly known for the multi-component damage they cause to

plants. Traditionally, pesticides have been the main management tool for whiteflies and aphids.

However, due to the problems associated with pesticidal tactics, other alternative methods have

been investigated. It is known that non-persistently transmitted viruses have not been managed

effectively by the use of insecticides since the viruses transmitted before the vectors are killed

(Brown et al. 1993, Soria et al. 2003). Similarly, geminiviruses have not been controlled by use

of insecticides (Polston and Anderson 1997). Furthermore, the use of insecticides increases cost

of production and many virus vectors have developed resistance to various chemical classes

(Dittrich 1990, Wang et al. 2002).

Use of systemic insecticides, however, can reduce secondary virus spread in the field by

preventing the build up of vectors on primary sources if the major vector is a species that

colonizes that crop. Virus control can also be achieved using plant's genetic resistance. This

however, can be of limited use since it is not always available (Cradock et al. 2002).

Furthermore, resistance can be overcome under high pressure of viruliferous populations.

Tomato varieties with resistance genes of Tomato yellow leaf curl virus (TYLCV) were

overcome when moderate to high populations of viruliferous whiteflies were inoculated early in

the growing season (Polston and Anderson, 1997). With this in mind, vector management is very

important in the control of insect-transmitted viruses.











Table 3-4. Effect of living and reflective mulches alone or in combination with Admire 2F on the
number of whiteflies caught per yellow sticky trap in Citra, FL


Mean SEM counts per YST

2005a 2006b

Reflective + Admire 2F 10.74 1.67 c 13.22 1.54 d

Reflective 20.80 3.81 b 27.82 4.41 b

Buckwheat 31.92 6.36 a 18.90 3.30 c

Buckwheat + Admire 2F 17.33 3.11 b 12.01 + 1.29 d

White (control) 51.33 11.32 a 37.72 4.71 a

YST data (2005) whitefly data transformed (log 10) before analysis, means are presented in the
original counts. Means followed by the same letter are not significantly different (P = 0.05
according to least square means test following repeated measures analysis, LS).
aF =22.21; df= 4, 96; P < 0.0001
b F = 27.66; df = 4, 72; P < 0.0001









involves cutting of leaf discs of a designated area from selected leaves. The leaf disks are then

examined under a microscope for identification of nymphal stages.

Whitefly-Transmitted Geminiviruses

Bemisia tabaci biotype B has been described as a notorious pest due to its ability to

transmit viruses to various crops. Whitefly-transmitted viruses cause yield losses worldwide on

important food and industrial crops (Polston and Anderson, 1997). Yield losses due to whitefly-

transmitted viruses have been described to be in the range of 20-100% depending on the crop,

season, and vector prevalence (Basu 1995). Before 1990, geminiviruses transmitted by whiteflies

(Bemisia tabaci Genn.) were primarily a problem for legume production in the Western

Hemisphere. Emergence and spread of new geminivirus diseases have been directly linked to

evolution of variants of the viruses (Varma and Malathi, 2003), the appearance of biotype B and

the increase of the vector population (Polston and Anderson, 1997, Varma and Malathi, 2003) in

the Americas and the Caribbean Basin.

The geminivirus genome is made up of circular, single-stranded DNA, which is

encapsidated in twinned subunits of a single capsid protein. They form the second largest family,

Geminiviridae of plant viruses. In terms of genome structures, most of them are bipartite, having

two equal components, A and B, while the monopartite have one larger DNA component.

Geminiviruses have been classified into four genera: Mastrevirus, Curtovirus, Topocuvirus, and

Begomovirus, depending on their vector and genomic characteristics (Varma and Malathi, 2003).

The majority of the known geminiviruses (80%) is transmitted by whiteflies: Begomoviruses,

which have mostly bipartite genomes and infect dicotyledonous plants.

Geminiviruses are transmitted in a persistent, circulative way (Polston and Anderson,

1997). They have a minimum acquisition period of 30-60 min, with inoculation periods that are

less than 30 min, and infectivity is retained for at least few days. Typical symptoms include vein









crop. In our study, the competition between zucchini plants and buckwheat was observed to be

greatest during the early stages of growth. Although squash plants were able to regain some level

of vigorous growth after buckwheat senesced, they were not able to compensate for yield.

Furthermore, by the time the buckwheat senesced, the fall (cool season) temperatures were

already present, which may have been unfavorable for the future growth of squash. The squash

plant is a warm-season crop and grows best between 24 and 29 C (Mossler and Nesheim 2001).

It is possible that early-season competition is a critical factor that could affect fall zucchini

production when interplanted with living mulches. The addition of Admire 2F was able to

enhance zucchini yields growing within buckwheat in at least one of the seasons (2005).

However, in spite of the larger plant size there were no differences in yields.

In our study, buckwheat considerably delayed the flowering of zucchini plants and hence

the time of harvesting. Previous studies have also reported similar effects of a living mulch when

interplanted with a cash crop. For instance, growth of cantaloupe was delayed by wheat,

Triticum aestivum L. and fruits failed to mature by the time harvest was completed in other

treatments (Toba et al. 1977). In another study, maturation of cabbage heads was delayed and

was smaller in living mulch treatments compared to bare ground (Andow et al. 1986).

During the 2-yr study, the reflective mulch treatment resulted in the largest plants and the

highest yields. Previous studies have reported similar findings (Brown et al. 1993, Summers et

al. 1995, Csizinszky et al. 1997, Stapleton and Summers 2002, Summers et al. 2004, Frank and

Liburd 2005). It is known that UV-reflective mulch has high photosynthetically active radiation,

which contributes to both plant growth and crop earliness (Stapleton and Summers 2002, Olson

et al. 2005). In our study, the addition of Admire 2F did not increase yields significantly in

reflective mulch treatments.




















12 Omid
-_ 10 l------ower


E
8 i




a


O-
Z 2




Buckwheat + Buckwheat Reflective + Reflective Control
Admire 2F Admire 2F
Treatment




Figure 3-2. Distribution of immature whiteflies on different plant strata of zucchini in Citra, FL
(2005)









Table 3-6. Effect of living and reflective mulch alone or in combination with Admire 2F on
number of natural enemies per treatment in zucchini Citra, FL


Mean natural enemies per treatment SEM

2005a 2006b

Reflective + Admire 2F 0.18 + 0.06 c 0.21 0.07

Reflective 0.26 0.06 bc 0.15 0.04

Buckwheat 0.40 + 0.08 ab 0.29 0.09

Buckwheat + Admire 2F 0.49 + 0.08 a 0.23 0.06

White (control) 0.28 + 0.06 bc 0.26 + 0.06


0.05 (LSD)


Means followed by the same letter are not significantly different P
aF= 3.43; df= 4, 353; P < 0.009
b F = 0.69; df = 4, 347; P < 0.6006









Table 3-2. Effect of living and reflective mulches alone or in combination with Admire 2F on the
number of adult whiteflies and aphids per zucchini squash leaf, Citra, FL (2006)
[Foliar counts]

Mean SEM numbers per leaf

Whiteflies (adult)a Aphids (apterous)b Aphid (alate)c

Reflective + Admire 2F 5.13 + 0.71 bc 0.04 + 0.02 b 0.25 0.06 ab

Reflective 7.75 + 0.96 b 0.28 + 0.12 a 0.17 + 0.05 ab

Buckwheat 8.27 + 0.97 b 0.21 + 0.09 ab 0.16 + 0.04 b

Buckwheat + Admire 2F 3.23 + 0.42 c 0.01 0.01 b 0.14+ 0.04b

Control 16.03 + 2.04 a 0.31 + 0.11 a 0.29 0.08 a

Whitefly data transformed (log 10) before analysis, means are presented in the original counts.
Means followed by the same letter are not significantly different (P = 0.05 according to least
square means test (LS) following repeated measures analysis).
a F = 26.67; df = 4, 72; P < 0.0001
b F= 3.14; df = 4, 72; P < 0.0193
cF = 0.86; df = 4, 72; P < 0.6330









nonmigrant forms. All these variations may sometimes occur within the annual cycle of a single

species as it does in the melon aphid, Aphis gossypii Glover (Kring 1972). Holocyclic species

have a sexual generation in the autumn and during the remainder of the year, they are asexual

bearing females (viviparae). Anholocyclic aphids have no sexual generation, and only viviparae

are produced throughout the year. Heteroecious or host-alternating aphids use different species

for winter (primary) and summer (secondary) hosts while monoecious species utilize the same

plants throughout the year (Halbert and Voegtlin 1995).

The evolution of parthenogenesis, enabling reproduction without males for part or all the

lifecycle, confers a reproductive advantage by doubling the intrinsic rate of population increase

(rm) relative to sexually reproducing individuals (Powell et al. 2005). When conditions are

favorable (low aphid density, high host quality), most aphid species continue to maximize

investment in reproduction by producing apterous progeny. As density increases, and plant

nutritional quality declines, production of alate offspring is stimulated.

Host selection by aphids is not a random process (Powell et al. 2005, Nault 1997). They

employ a variety of sensory and behavioral mechanisms to locate and recognize their host plants

(Powell et al. 2005). Landing by migrating alate aphids involves a phototactic response to plant-

reflected wavelengths (Nault 1997). After landing, a variety of cues may be detected as the aphid

walks across the leaf surface. Stylet penetration is attempted as a reflex following tarsal contact

with any solid surface even in the presence of repellent cues (Powell et al. 2005). The first few

stylet penetrations initiated after the plant contact are usually brief (<1 min) and are limited to

the epidermis when the aphid tries to distinguish host from non host. Aphid stylets and labium

apparently lack external contact chemoreceptors and therefore plant sap may have to be ingested

to contact a gustatory organ located in the epipharyngeal area. If the sap is desirable it is sucked









sunlight back to the crop canopy increasing the amount of photosynthesis available to contribute

to plant growth and vigor.

Living mulches are a cheaper alternative to reflective mulch. These mulches are

interplanted with the main crop for various benefits such as improving soil fertility and pest

suppressing (Liburd et al. in press). Previous research investigating the use of living mulches in

various crops has reported mixed results with respect to marketable yields. Hooks et al. (1998)

recorded increased yield in zucchini squash in Hawaii. Similarly, Hilje and Stansly (2007)

reported increased yield in tomatoes when interplanted with living mulches. Alternatively,

Andow et al. (1986) saw decrease in yield in cabbage interplanted with living mulches as

compared with bare ground between plants.

Several factors must be considered when growing living mulches with a main crop. For

instance, it is important to select a living mulch whose growth pattern has least effect on the

main crop. Frank (2004) reported that buckwheat, Fagopyrum esculentum Moench was superior

to white clover; Trifolium repens L., when interplanted with zucchini squash. Secondly, living

mulch must be planted at the correct spacing to reduce competition between the main crop and

the mulch.

Earlier, we reported that living and reflective mulch in combination with the insecticide

Admire 2 F were effective in controlling whiteflies and aphids associated with zucchini squash

(CHAPTER 3). It will be interesting to see if this reduction will translate to increased plant size

and hence better yields.

My hypothesis was that since Admire 2F would contribute to further reduction in pest

pressure (whiteflies and aphids) during the early season, the treated plants would be able to

compensate for growth and hence produce higher marketable yields.









Discussion

The response of whiteflies on zucchini interplanted with living mulch was more consistent

than that of alate aphids in the two-yr study. My study shows that living and reflective mulches

with and without Admire 2F were able to provide significant protection against whiteflies as

compared to the control. Crops grown within these mulches were likely protected from pest

infestation due to the negative effect that these mulches have on the orientation of the insect to

the plant. Living mulch, (buckwheat) increases the vegetation diversity that is known to decrease

the host apparency and hence fewer numbers of whiteflies and aphids landed on squash. On the

other hand, UV reflective mulch is known to repel whiteflies and hence prevent them from

landing on the host plants (Antignus 2000). This was the case in my study where significantly

fewer whiteflies were recorded in reflective mulch. The current study provides another instance

where living mulch was demonstrated to control whiteflies. Previous researches have shown that

the inclusion of living mulches with zucchini plants (Frank and Liburd 2005), and tomatoes

(Hilje and Stansly 2007) successfully reduced whiteflies populations in the main crop.

Whitefly populations were higher in 2005 than in 2006. In both years, foliar counts

revealed that buckwheat was equally as effective as reflective mulch when tested alone or in

combination with the reduced-risk insecticide Admire 2F. In 2005, however YST indicated that

reflective mulch alone was superior to living mulch in controlling whiteflies. The situation was

reversed in 2006 when living mulch alone was superior to reflective mulch alone. The reason for

these differences are not clear but could be related to stage of maturity of living mulch or other

external factors.

Although previous studies have reported reduction of whiteflies when using living mulch,

none of the studies evaluated the mulch in combination with a reduced-risk insecticide. I found

that addition of Admire 2F to the mulches further reduced whitefly populations. More research is










CHAPTER 5
IMPACT OF SYNTHETIC AND LIVING MULCH WITH AND WITHOUT A REDUCED-
RISK INSECTICIDE ON PLANT GROWTH AND MARKETABLE YIELDS OF ZUCCHINI
SQUASH

Consumption of zucchini squash, Cucurbitapepo L. has increased due to preferential use

of the squash as a salad and a cooked vegetable compared to other summer squashes (Stephen

2003). Zucchini squash, is a high value vegetable crop in Florida and the state is ranked second

nationally after Georgia in the production of fresh market squash. In the 2006 field-seasons, a

total of 4249 ha of cucurbits was planted in Florida and 98% of the squash produced were

harvested with an exceeding value of approximately 38.76 million USD (NASS-2006). Squash is

one of the crops in Florida that is exported every month of the year (Mossler and Nesheim 2001),

thus providing a continuous income to the state. Almost all the squash is produced for fresh

market with summer production mainly for local markets and fall production for export. In

Florida, damage due to pest infestation is probably one of the major problems affecting the

squash industry. In this regard, it is important to use appropriate and effective control strategies

that are sustainable and will improve plant productivity.

The use of mulches on raised beds is now a common practice in some vegetable production

systems in Florida. Mulches have several advantages including controlling weeds, regulating soil

temperatures, retaining moisture, and increasing crop yields (Olson 2005). In addition, some of

the mulches have been shown to have negative effects on pests, thus reducing their incidences. In

particular, reflective mulch has received increased attention in the production of various crops.

Previous research has shown increased yields of various crops grown on reflective mulch i.e.

strawberries (Rhainds et al. 2001), cantaloupe and cucumber (Summers and Stapleton 2002), and

zucchini squash (Summers et al. 2004, Frank and Liburd 2005). Reflective mulch reflects









Kao, J., L. Jia, T. Tian, L. Rubio, and B. W. Falk. 2000. First report of Cucurbityellow
stunting disorder virus (Genus Crinivirus) in North America. Plant Dis. 84:101.

Kloen, H., and M. A. Altieri. 1990. Effect of mustard (Brassica hirta) as a non-crop plant on
competition and insect pests in broccoli (Brassica oleracea). Crop Prot. 9: 90-96.

Kring, J. B. 1972. Flight behavior of aphids. Annu. Rev. Entomol. 17: 461-492.

Kring, J. B., and Schuster, D. J. 1992. Management of insects on pepper and tomato with UV-
reflective mulches. Fla. Entomol. 75: 119-129.

Kucharek, T., and D. Purcifull. 1997 (Revised 2001). Aphid-transmitted viruses of cucurbit in
Florida. Circ-1184. Florida Cooperative Extension Service/ Institute of Food and
Agricultural Sciences/University of Florida, Gainesville, FL.

Liburd, O. E., and D. L. Frank. 2007. A review of synthetic and living mulches for control of
Homopteran pests (aphids and silverleaf whiteflies) and diseases in vegetables In G.
Saxena and K. G. Mukerji [Eds.] Management of nematode and insect-borne diseases.
The Haworth Press Inc, New York, USA.

Liburd, O. E., T. W. Nyoike, C. A. Scott. Cover, border and trap crops for pests and disease
management. In New edition, Encyclopedia of entomology. Kluwer Academic
Publishers, Dordrect, The Netherlands. In Press.

Liu, T-X. 2004. Toxicity and efficacy of spiromesifen, a tetronic acid insecticide, against
sweetpotato whitefly (Homoptera: Aleyrodidae) on melons and collards. Crop Prot. 23:
505-513.

McAuslane, H. J., 2000. Sweetpotato whitefly B Biotype of silverleaf whitefly, Bemisia tabaci
(Gennadius) or Bemisia argentifolii Bellows and Perring (Insecta: Homoptera:
Aleyrodidae). IFAS Extension EENY129. University of Florida, Gainesville, FL.

McAuslane, J. H., J. Chen, R. B. Carle, and J. Schmalstig. 2004. Influence of Bemisia
argentifolii (Homoptera: Aleyrodidae) infestation and squash silverleaf disorder on
zucchini seedling growth. J. Econ. Entomol. 97: 1096-1105.

Mora-Aguilera, G. 1995. Aphid vector dynamics and temporal and spatial characterization of
watermelon virus epidemics, pp. 198, PhD Dissertation. Department of Plant Pathology,
University of Florida, Gainesville, FL.

Mossier, M. A., and 0. N. Nesheim. 2001. Florida crop/pest management profile: squash. IFAS
Extension Cir 1265. University of Florida, Gainesville, FL.

Nakamoto, T., and M. Tsukamoto. 2006. Abundance and activity of soil organisms in the field
s of maize grown with white clover living mulch. Agric. Ecosyst. Environ. 115: 34-42.









sampling week. Of the six weeks sampled, treatment differences were observed in 3 out of the 6

weeks. Similarly, there were significant (F = 2.40; df = 20, 72; P < 0.0036) interaction effects

between treatment and time in alate aphids counted per leaf. Among the weeks sampled,

treatment differences were only observed in 2 weeks of sampling, and during both dates

reflective mulch with Admire 2F had the highest alate aphids counted per leaf. Overall, reflective

mulch with Admire 2F recorded significantly higher number of alate aphids per leaf compared

with all the other treatments except reflective mulch alone (F = 5.13; df= 4, 72; P < 0.0011)

[Table 3-1].

In 2006, fewer adult whiteflies per leaf were recorded compared with 2005, though a

similar trend was observed among the treatments (Table 3-2). Plant growing with living and

reflective mulch treatments continued significantly fewer of adult whiteflies per leaf than those

growing on white mulch (F= 49.71; df = 4, 72; P < 0.0001) (Table 3-2). Buckwheat with

Admire 2F had the fewest whiteflies counted per leaf, but it was not significantly different from

reflective with Admire 2F. The addition of Admire 2F to the buckwheat treatment resulted in

further reduction of whiteflies per leaf. During the study there were significant (F = 3.63; df=

20, 72; P < 0.0001) interaction effects between treatment and sampling week, with significant

treatment differences observed in all weeks.

In the case of aphids per leaf, although there were significant (F= 2.01; df = 20, 72; P <

0.0166) interaction effects between treatments and time the difference was only observed in one

week of sampling. Both buckwheat and reflective mulches with Admire 2F had significantly

fewer apterous aphids than white mulch (control) (F = 3.14; df = 4, 72; P < 0.0193) [Table 3-2].

Buckwheat with and without Admire 2F had significantly fewer of alate aphids than the white

mulch which was similar to the reflective mulches. The addition of Admire 2F did not affect the



































To my Mom and Dad









F o liar C o u n ts .............................................................................4 0
W hitefly Im m nature C ounts ......... ................. ....................................... .......................42
T rap C o u n ts ..............................................................................4 3
W h ite flie s ........................................................................................................... 4 3
A p h id s ..............................................................4 3
P hy siolog ical D isord ers............................................................................................. 4 4
N natural E nem ies ................................................................... 44
D iscu ssio n ................... ...................4...................5..........

4 EFFECTS OF SYNTHETIC AND LIVING MULCH WITH AND WITHOUT A
REDUCED-RISK INSECTICIDE TO CONTROL INSECT-TRANSMITTED VIRAL
DISEASES OF ZUCCHINI SQUASH: A NEW WHITEFLY-TRANSMITTED VIRUS
IS R E P O R T E D .......................................................................................................................6 0

M materials and M ethods ..........................................................................................................62
Field Plot Preparation and Experimental Design ................. ................. ..... 62
V iru s S c re e n in g ............................................................................................................... 6 3
PCR A analysis ................................................. 64
Greenhouse Screening .................. ........ ..................65
D ata A n a ly sis ...................................................................................................................6 5
Results .................. ... ......... ......... ................. .............65
G reen h ou se R e su lts ................................................................................................... 6 6
PCR Analysis Results .............. ......... ............ .......... 66
D iscu ssio n ......... ............ ......................... ............................6 6

5 IMPACT OF SYNTHETIC AND LIVING MULCH WITH AND WITHOUT A
REDUCED-RISK INSECTICIDE ON PLANT GROWTH AND MARKETABLE
Y IELD S O F ZU C CH IN I SQ U A SH ................................................................................. 71

M materials an d M eth o d s ...........................................................................................................7 3
P lan t S iz e S am p lin g ................................................................................................... 7 4
M arketable Y ields Sam pling .............................................................................74
D ata A analysis ................................................74
R e su lts ................... ...................7...................4..........
P la n t S iz e .........................................................................................................................7 4
M ark etab le Y field s ..................................................................................................... 7 5
D iscu ssio n ................... ...................7...................5..........

6 C O N C L U S IO N ....................................................................................................................... 8 0

L IST O F R E F E R E N C E S ....................................................................................................83

B IO G R A PH IC A L SK E T C H ................................................................................................... 90







6









Toba, H. H., A. N. Kishaba, G. W. Bohn, and H. Hield. 1977. Protecting muskmelon against
aphid-borne viruses. Phytopathology 67: 1418-1423.

Van Lenteren, J. C., and L. P. J. Noldus. 1990. Whitefly-plant relationships: behavioral and
ecological aspects, pp. 47-80. In: D. Gerling [Ed.], Whiteflies: their bionomics, pest
status and management. Intercept Ltd, Andovers, UK.

Varma, A., and V.G. Malathi, 2003. Emerging geminivirus problem: a serious threat to crop
production. Ann. Appl. Biol. 142: 145-164.

Wang, K. Y., T. X. Liu, C. H. Yu, X. Y. Jiang, and M. Q. Yi. 2002. Resistance of Aphis
gossypii (Homoptera: Aphididae) to fenvalerate and imidacloprid and activities of
detoxification enzymes on cotton and cucumber. J. Econ. Entomol. 95: 407-413.

Webb, S. E. 1991. Floating row covers exclude insects affecting fall-grown squash in central
Florida. Proc. Fla. State Hort. Soc. 104: 272-275.

Webb, S. E., and S. B. Linda. 1992. Evaluation of spunbounded polythlene row covers as a
method of excluding insects and viruses affecting fall-grown squash in Florida. J. Econ.
Entomol. 85: 2344-2352.

Webb, S. E., E. Hiebert, and T. A. Kucharek. 2003. Identity and distribution of viruses
infecting cucurbits in Florida. Phytopathology 93: S89.

Webb, S. E., F. Akad, T. W. Nyoike, O. E. Liburd, and J. E. Polston. 2007. Whitefly-
transmitted Cucurbit leaf crumple virus in Florida. ENY-477 IFAS Extension. University
of Florida, Gainesville, FL.

Wiebe, W. L. 2003. An overview of squash viruses including emerging geminiviruses in the
southwest U. S and Northern Mexico. www.rogeradvantage.com.

Wolfenbarger, D. 0., and W. D. Moore. 1968. Insect abundances on tomatoes and squash
mulched with aluminum and plastic sheetings. J. Econ. Entomol. 61: 34-36.

Yokomi, K. R., K. A. Hoelmer, and L. S. Osborne. 1990. Relationships between the
sweetpotato whitefly and the silverleaf disorder. Phytopathology 80: 895-900.

Zitter, T. A. 1977. Epidemiology of aphid-borne viruses in: Harris K. F. and Maramorosch, K.
[Eds.] Aphid as Virus Vectors. pp. 385-412. Academic press, New York, NY.

Zitter, T. A., and J. N Simons. 1980. Management of viruses by alteration of vector efficiency
and by cultural practices. Ann. Rev. Phytopathol. 18: 289-310.

Zitter, T. A., D. L. Hopkins and C. E. Thomas. 1996. Compendium of cucurbit diseases. The
American Phytopathological Society, St. Paul, Minnesota.









The specific objective was to evaluate the impact of buckwheat and reflective mulch with

and without a reduced-risk insecticide on plant growth (size) and marketable yields of zucchini

squash. It will be my intention to make recommendations if any of the combinations could result

in economic gains for squash growers in Florida.

Materials and Methods

Field experiments were set up at the University of Florida Plant Science Research and

Education Unit in Citra, Florida. Field plots measuring 10.36 m x 10.36 m were prepared, each

containing four beds. The beds were 76 cm top width and 30 cm high and the distance between

them was 1.06 meters. Each plot was separated from the adjacent plots by 7.6 m of bare soil on

all sides that was kept weed free throughout the experiment. Living mulch, (buckwheat,

Fagopyrum esculentum Moench) was hand seeded between the rows 21 days and 18 days in

2005 and 2006 respectively, before planting squash. The four treatments evaluated were: 1)

reflective mulch with the reduced-risk insecticide (Admire 2F, Bayer, Kansas City, MO), 2)

reflective mulch without Admire 2F, 3) living mulch with Admire 2F, 4) living mulch without

Admire 2F and standard synthetic white mulch which served as the control. The reduced risk-

insecticide Admire 2F was applied 2 weeks after squash planting through the drip lines at the rate

of 1.684 liters per hectare on the appropriate treatments.

Planting holes were cut in the center of each plastic mulch strip and in the center of the

non-mulched beds and two squash seeds, variety Wild Cat, were hand seeded per hole.

Planting spacing was maintained at 45 cm between the plants in the row during the 2005 and

2006 field seasons. After germination the missing plants were replaced using seedlings raised in

the greenhouse for that purpose. The agronomic practices for squash followed the standard

production guide for squash in North Florida (Olson et al. 2005). The same plants were used for

sampling for the insect pests (CHAPTER 3).









(Hooks et al. 1998, Frank and Liburd 2005). In addition, they have been shown to increase yields

in squash and other vegetables when compared with the bare ground (Hooks et al. 1998, Frank

and Liburd 2005, Hilje and Stansly 2007).

Justification

Living mulches are a cheaper and more sustainable alternative to reflective mulches.

Zucchini squash interplanted with living mulch, buckwheat Fagopyrum esculentum Moench had

lower populations of whiteflies and aphids and delayed the first appearance of virus incidence in

the crop (Hooks et al. 1998, Frank and Liburd 2005). Reflective mulches have been shown to

reduce whitefly and aphid populations and subsequently delay the onset of the viral transmitted

diseases (Stapleton and Summers 2002, Summers et al. 2004, Frank and Liburd 2005). However,

the reliance on living or reflective mulch as a single tactic is inadequate to control virus spread

(Polston and Anderson 1997). Furthermore, reflective mulches lose its insect repelling ability

once the plant growth covers the reflecting surfaces (Smith et al. 2000). It is, however, not yet

known if aphid and whitefly populations can be further reduced by the addition of a reduced-risk

insecticide that could lead to more economic gains. Further reduction in whitefly and aphid

populations will increase the number of marketable fruits and subsequently enhance growers'

profits. Imidacloprid (Admire 2F) is an effective reduced-risk insecticide with systemic activity

and can be used to manage whiteflies and aphids. It is however not known if imidacloprid will

affect the natural enemy populations associated with living mulch in zucchini.

There is a need to integrate other tactics with reduced-risk insecticide to prolong their use

in managing whiteflies and aphids and other problems in cucurbits. Since isolated cases of

resistance to whiteflies have already been reported (Osborne 2006 personal comm.). In our study,

Admire 2F will be used once at the beginning of the season when control is most needed.
















4

o 3.5
0)
E 3

E 2.5
0)


> 1F


1 -

0.5 L

0
Reflective +
Admire 2F


Reflective Buckwheat Buckwheat +
Admire 2F
Treatment


Figure 3-4. Effect of living and reflective mulches alone or in combination with Admire 2F on
silverleaf symptoms presented by the score indices per treatment in zucchini Citra, FL
(2006).


White


i7)









Synthetic white mulch treatment yielded more zucchini fruits than living mulch plots

despite having a high whitefly population in both years. In 2006, white mulch had the highest

incidences of whitefly-transmitted virus (CHAPTER 4). It is known that zucchini yield can be

affected by the occurrence of insect-transmitted diseases. In 2006, a whitefly-transmitted disease

was present; however, this disease did not affect the yields of zucchini possibly because it

appeared late in the growing season. Yield losses due to whitefly-transmitted viruses have been

described to be within a range of 20-100% depending on the crop, season, and vector prevalence

(Basu 1995).

Indeed, living mulches were able to reduce pest problems (CHAPTER 3 & 4) but resulted

in a huge negative impact on the yields. This adverse effect of living mulches is a big factor that

may limit their adoption in pest management programs. Additionally, the living mulch-

insecticide combination treatment did not increase yield sufficiently to justify the cost of Admire

2F. For the growers to adopt this tactics the net gains associated with mulches plus Admire 2F

have to be large enough to justify their use this was not the case in this study. Further research

needs to be done before the living mulch is recommended to growers. In cases where reflective

mulches cannot be used, living mulch is a cheap alternative, since it has been shown to result in

higher yields than bare ground in other studies (Frank 2004, Hilje and Stansly 2007). It is worth

mentioning that other costs (extra drip lines, natural resources like water, nutrients) need to be

considered before using living mulches.









The study was conducted with the aim of integrating cultural and reduced-risk insecticide

tactics for pest management of whiteflies and aphids in zucchini squash. The hypothesis of the

study was that use of the insecticide with the mulches could result in further reduction in pest

populations and thus lead to economic gains. Specific objectives were to investigate the effect of

living mulch buckwheat, (Fagopyrum esculentum Moench), and reflective mulch in combination

with a reduced-risk insecticide (imidacloprid) to manage whiteflies and aphids in zucchini

squash. The effects of these treatments on the natural enemies associated with squash were also

evaluated.

Materials and Methods

Plot Preparation, Irrigation system, and Experimental Design

The experiment was conducted at the Plant Science Research and Education Unit in Citra,

Florida. Field plots measuring 10.36 m x 10.36 m were prepared, each containing four beds.

Each plot was separated from the adjacent plots by 7.6 meters of bare soil on all sides and was

kept weed free throughout the experiment. Planting beds were prepared with a 6-foot center

wheel spacing tractor (2- wheel drive Model 6615, John Deere) and were fumigated with methyl

bromide 80/20 formulation (80 % methyl bromide, 20% chloropicrin) at the rate of 283.5 kg/ha.

Fumigation was done 2 weeks before planting squash. The fumigant was injected into the soil

and immediately the planting rows were covered with the respective synthetic mulch treatments.

Drip irrigation lines (5/8 inch, 10 mm thickness) were placed in the center of the beds prior to

covering with plastic mulch. Living mulch rows were also fumigated and temporarily covered (2

wk) with synthetic mulch, to allow the methyl bromide to properly fumigate those beds. The

plastic mulch was removed before planting buckwheat seeds. Before planting squash, the living

mulch; (buckwheat) was hand-seeded between the rows 21 and 18 days in 2005 and 2006

respectively. At the time of planting squash plants the buckwheat mulch was 20 cm high. There









the case with biotype A. In addition, B-biotype has greater efficiency in transmitting geminivirus

diseases than the A strain (Brown et al. 1995) but not clostroviruses in which strain A is better.

According to DNA differentiation tests and mating incompatibilities as evidenced by the

absence of females in the progeny, the B-biotype was described as a distinct species and a

common name silverleaff whitefly' was suggested due to the damage they cause on squash

(Perring et al. 1993). Concurring with the earlier described differences of strain B with the other

existing strains, Bellows et al. (1994) added the morphological differences based on the fourth

instar pupal case. These differences alongside with allozymic diagnoses allowed them to classify

the strain as a new species, Bemisia argentifolii (Bellows & Perring) (Bellows et al. 1994).

This naming has undergone some debates with some colleagues arguing that if B-biotype

is a new species then all the other biotypes should be elevated to species level. In this regard,

Brown et al. (1995) proposed that B. tabaci is in fact a 'species complex'. At the time of writing,

'B-biotype' is the most commonly used name.

Biology and Behavior of B Biotype

The B biotype of B. tabaci has six life stages: egg, four instars, and the adult. The eggs are

usually elongate-oval but occasionally may be reniform. The apex distal end of the egg is acute

and the basal portion is usually broad with a pedicel or stalk of varying length by which the

female attaches the egg to the host (Gill 1990, Byrne and Bellows 1991). Eggs may be laid

singly in small groups or in circular or semicircular pattern on the underside of the leaf due to the

rotation of the female as she oviposits (McAuslane et al. 2000). Freshly laid eggs are translucent,

creamy white but turn pale brown just before hatching. Each female can lay a minimum of 50

eggs and up to 300 eggs have been recorded (Basu 1995).

The crawler is the first instar, and the only mobile stage of the immature stages. Crawlers

are pale, translucent white, oval with a convex dorsum and flat ventral side (Basu 1995). The









incorporating a reduced-risk insecticide with a living or reflective mulch will have to be studied

before recommendations can be made.










EVALUATION OF LIVING AND SYNTHETIC MULCHES WITH AND WITHOUT
IMIDACLOPRID FOR SUPPRESSION OF WHITEFLIES AND APHIDS, AND INSECT-
TRANSMITTED VIRAL DISEASES IN ZUCCHINI SQUASH





















By

TERESIA W. NYOIKE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007









BIOGRAPHICAL SKETCH

Teresia Nyoike was born in the Thika District of Central, Kenya, Africa. She graduated

with a Bachelor of Science in Agriculture (Crop Protection) on August 2001 from the University

of Nairobi, Kenya. In August 2001, she joined Dudutech Kenya Ltd., an integrated pest

management company that produced bio-control agents for local and export markets. In 2002,

she had laboratory training on biological control agents for root knot nematodes-Pasteuria

penetrans at Reading University (UK). During her four years in the company, she worked as a

nematologist with specific responsibilities of developing on-farm control systems for root knot

nematodes using biological control agents. In August 2005, she joined the Small Fruit and

Vegetable IPM Laboratory, University of Florida, in Gainesville to pursue her MS in

Entomology. She is a member of the Gamma Sigma Delta honors society in agriculture.









al. 2004). These mulches reflect short-wave length which repels incoming alate aphids, thus

reducing their incidence of alighting on plants (Stapleton and Summers 2002, Cradock et al.

2002, Summers et al. 2004).

Reflective and colored mulches have been reported to modify soil moisture and

temperature (Csizinszky et al. 1997), which results in increased crop earliness, growth, and yield

(Stapleton and Summers 2002, Greer et al. 2003) and providing greater benefit and cost

effectiveness to the grower. Colored or reflective mulches are most effective early in the crop

cycle, before the developing plant canopy covers the mulch. They reduce attack by soil

pathogens and consequently reduce fruit rot, and fertilizer leaching (Cradock et al. 2002). Plants

grown over reflective mulch produced significantly higher yields than those grown on bare

ground (Stapleton and Summers 2002, Frank and Liburd 2005). However, disposal of synthetic

mulches following crop termination can be problematic and may interfere with farming practices

such as cultivation. Furthermore, the effectiveness of the mulches is lost over time through plant

growth either by shading or covering the reflective surface (Zitter 1977).

Reduced Risk-Insecticides

Reduced-risk pesticides are newer classes of compounds that pose a lower health risk to

humans and environment. A reduced-risk insecticide is defined as one that may reasonably be

expected to accomplish one or more of the following: 1) reduce pesticide risks to human health,

2) reduce pesticides risks to non-target organisms, 3) reduce the potential for contamination of

valued, environmental resources, or 4) broaden adoption of IPM or make it more effective.

Imidacloprid is a reduced-risk insecticide that belongs to the neonicotinoids group of

insecticides.

Neonicotinoids act on the insect central nervous system as antagonists of the postsynaptic

nicotinic acetylcholine receptors (Denholm and Nauen 2005). They show marked selectivity









evaluated in this experiment included 1) reflective mulch with the reduced-risk insecticide;

imidacloprid (Admire 2F Bayer, Kansas City, MO), 2) reflective mulch without Admire 2F,

3) living mulch with Admire 2F, 4) living mulch without Admire 2F, and 5) standard synthetic

white mulch (control). Before planting squash the living mulch, buckwheat was hand seeded

between the rows 21 and 18 days in 2005 and 2006, respectively. The reduced risk-insecticide

imidacloprid [Admire 2F] was applied 2 weeks after squash planting through the drip lines at

the rate of 1.684 liters per hectare. The treatments were arranged in randomized complete block

design with four replications. Agronomic practices followed the standard production guide for

squash in North Florida (Olson et al. 2005). However, no insecticides were applied during the

growing season except Admire 2F on the specified treatments. The fungicide azoxystrobin

(Amistar; 80 WP), was sprayed as required against powdery mildew in the early stages of the

crop.

Virus Screening

In 2005, plants were visually observed for virus symptoms weekly starting from the mid-

growing season until the last week of the experiment. Two weeks before the end of the

experiment, I excised a young leaf (third leaf from the tip) from six plants per plot, regardless of

whether or not they had symptoms and transported the leaf to the laboratory. Leaves were stored

at 4 C overnight and assayed using an enzyme-linked immunosorbent assay (ELISA) [Clark and

Adams 1977) for Papaya ring spot virus-Watermelon strain (PRSV-W), Watermelon mosaic

virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV).

Approximately 0.2 g of each leaf sample was grounded in phosphate buffered saline (PBST) at a

1:20 dilution using a hand-held macerator to release the virus particles into the suspension. After

24 h at 3-4 C, 100 [iL of each sample was added to duplicate wells in a 96-well plate, (Nunc,

Denmark) that had previously been coated with specific antibodies at 1 [g/ml. Each plate









silvering on the leaves is irreversible and symptoms severity increases as the number of

immature whiteflies per plant increases. As few as two immature whiteflies per plant, feeding for

about 14 days are able to induce silverleaf symptoms on zucchini (Costa et al. 1993). Symptoms

are also manifested on the fruits where green-fruited squashes appear lighter in color or the color

is streaked longitudinally while the yellow-fruited squash appear pale or white (Schuster et al.

1991, Yokomi et al. 1990).

Bemisia tabaci was not a pest in Florida cucurbits production until 1987 when an outbreak

occurred on poinsettia Euphorbiapulcherrima (Poinsettia), which was associated with a new

biotype, B. This biotype later spread to other crops including squash and tomatoes (Mossler and

Nesheim 2001). The B biotype was thought to have been introduced into Florida in the early

1980s possibly through the movement of ornamentals (Brown et al. 1995, Polston and Anderson,

1997), but economic losses were not observed until an outbreak of the disorder occurred during

the 1987-88 vegetable season (Schuster et al. 1991). Economic losses to many crops as a result

of direct feeding damage and whitefly-transmitted geminiviruses led to the rise of B. tabaci as a

pest and virus vector in the tropics and subtropics in the late 1980s (Yokomi et al. 1990, Schuster

et al. 1991, Brown et al. 1995, Polston and Anderson 1997).

Taxonomy

Infestation on poinsettias in Florida indicated a new strain, since the strain previously,

strain A was not affecting poinsettia. The new strain was named strain B, or biotype B to

distinguish it from the existing strain A or biotype A. These two biotypes have similar

morphological appearance but different biological characteristics (Brown et al. 1995). It is

known that B-biotype has a wider host range and lays more eggs than A-biotype. Also, based on

the amount of honey dew produced, B-biotype ingests more phloem sap than biotype-A (Brown

et al. 1995). The feeding of immatures of the B-biotype can cause plant disorders, which is not









into the cibarium (sucking pump). Aphids make one or more probes before taking flight or

moving to the lower leaf surface where they establish contact with phloem (Nault 1997, Powell

et al. 2005). This behavior is especially important since transmission of stylet-bome viruses can

occur from the test probes (Nault 1997). For this reason, non colonizing aphids can be more

important in virus transmission than those that colonize the crop.

Several factors, such as effects of light, sound, and odor are responsible for active

selection of alighting surfaces by aphids (Kring 1972). Alighting may be a response stimulus to a

particular site (colored object, trap, or plant) or it may be the result of a lack of interfering

stimuli. For this reason, takeoff and flight activity are increased in the presence of shortwave

light. Certain white surfaces and aluminum surfaces that reflect shortwave, long wave and

varying amount of infrared energy do reduce the number of aphids alighting on plants or in traps

they surround (Kring 1972). Host-seeking aphids are attracted to the relatively long wave

radiation reflected from the soil and vegetation (Cradock et al. 2002). Yellow and green surfaces

(wavelength 500-580nm) are particularly attractive.

Monitoring

Several trapping techniques can be used to monitor alate aphids. These aphids have poor

visual acuity but they are known to be responsive to yellow and green frequencies (wavelength

500-580 nm). For this reason commercial yellow sticky traps are available to monitor aphid

populations in the field and greenhouse. Pan traps are small containers that are filled with a

preservative for collecting aphids. They have functions similar to the sticky traps, but the aphids

are collected in the liquid preservatives. Suction traps have also been used to monitor aphids in

small grain fields.









borne non-persistently transmitted virus (Toba et al. 1977). In this case the living mulch will be

serving as a virus-sink where the 'infected' aphid is likely to loose its virus during probing. The

protection crop must be immune from the target virus or viruses and suitable for probing by the

aphid and should not serve as a host for aphid reproduction (Toba et al. 1977).

Diversified habitats can also affect herbivore damage through their influence on factors

such as immigration and emigration of pest or enemies searching behavior of both generalists

versus specialist insects. These habitats will not only affect host-finding for host-specific pests

but also for species-specific enemies (Costello and Altieri 1995). Furthermore, Bemisia tabaci

spent less time on host plants and appeared restless in a multiple plant species environment

(Frank 2004). Therefore, it is possible that damage associated with the pest will be reduced in

such a scenario.

Among the agronomic benefits attributed to living mulches, reduction of damage (direct

and indirect) has received attention in crop production (Toba et al. 1977, Andow et al. 1986,

Hooks et al 1998, Frank and Liburd 2005, Hilje and Stansly 2007). Use of living mulch has been

shown to result in fewer whiteflies, aphids and/ or delayed occurrence of insect-transmitted

viruses in various crops (Kloen and Altieri 1990, Costello and Altieri 1995, Hooks et al. 1998,

Frank and Liburd 2005). However, in cases where excessive competition leads to poor plant

quality in mixed cropping systems, the advantages of reduced pest densities may be lost because

of poor crop yields (Frank and Liburd 2005).

Synthetic Mulches

The synthetic mulches are primarily polyethylene (colored or clear plastic), but may

include treated paper, wax coated papers, and aluminum and steel foils. Silver or gray reflective

mulches have been used successfully to delay and reduce the incidence of aphid-borne virus

diseases in squash and other crops (Brown et al. 1993, Stapleton and Summers 2002, Summers et









CHAPTER 3
TO INVESTIGATE THE EFFECT OF LIVING (BUCKWHEAT) AND REFLECTIVE
MULCHES WITH AND WITHOUT A REDUCED-RISK INSECTICIDE ON WHITEFLIES,
APHIDS AND NATURAL ENEMIES IN ZUCCHINI SQUASH

Whiteflies and aphids (Hemiptera: Aphididae) are economic pests of fresh market zucchini

squash (Cucurbitapepo L.) affecting production in Florida. These pests cause damage to squash

plants directly by sucking on their sap or indirectly by transmitting viruses. Aphids vector non-

persistently transmitted viruses, which are considered a major limiting factor to cucurbit

production in Florida (Purcifull et al. 1988, Wolfenbarger and Moore 1967). Before 1988, when

the first economic damage associated with the pest was recorded on cucurbits the sweetpotato

whitefly (Bemisia tabaci Genn.) was not a major pest in Florida, (Schuster et al. 1991). Squash

silverleaf disorder (SSL) is a physiological disorder associated with the feeding of immatures of

B biotype whiteflies that causes the upper surface of the leaf to be silvered. The symptoms can

be present on the fruit causing the color to look paler as in the case of zucchini and yellow

squash. Recently, whiteflies have been implicated in the transmission of new whitefly-borne

virus diseases in squash (Adkins et al. 2007, Akad et al. submitted). This multi-faceted damage

of whiteflies and aphids make them probably the most economic pests of squash.

Control of whiteflies and aphids on squash has been based on the use of insecticides.

However, this control cannot be relied on alone as the pests are prone to develop resistance.

Additionally, the control of insect-transmitted viruses cannot rely on insecticidal tactics (Kring

and Schuster 1992, Brown et al. 1993) especially in the case where the virus is transmitted non-

persistently. These viruses are transmitted before the aphid can obtain a lethal dosage to kill them

(Stapleton and Summers 2002). Nevertheless, insecticides can be used to reduce the proliferation

of viruses by preventing build-up on the host.









applied against downy and powdery mildew, control may not be necessary if the disease occurs

in the last 3 weeks of the growing period. Other foliar diseases affecting squash include wet rot

and phytophthora foliar blight, which can cause fruits to rot. Both diseases can be managed

effectively by soil drench of metalaxyl/chlorothalonil (Mossler and Nesheim 2001). Post harvest

diseases of squash include bacterial soft rot, alternaria rot, fusarium rot, rhizopus storage rot and

pythium fruit rot. These diseases which, cause the fruit to rot, may begin in the field and cause

fruit to deteriorate under storage. Pythium fruit rot is the most common disease causing the

crown of older squash to rot. Post harvest diseases can however be controlled through proper

handling during and after harvest (Mossler and Nesheim 2001).

Insect pests of squash include pickleworm, Diaphania nitidalis Stoll, and melonworm,

Diaphania hyalinata L. Both pests are tropical moths and are able to overwinter in Florida

causing damage to squash among other cucurbits. Their young larvae are known to cause

damage on the blossoms, foliage of young vegetative buds and the apical growth of vines. Other

pests that damage the foliage and occasionally the fruits are squash bug, Anasa tristis DeGeer;

melon thrips, Thripspalmi Karny; fall armyworm, Spodopterafrugiperda (J. E. Smith); beet

armyworm, Spodoptera exigua Hiibner; and leafminers, Liriomyza trifolii Burgess and L. sativae

Blanchard. To manage these pests, Florida growers use biopesticides such as Bacillus

ilhn/ iuigiel'\i (B.t.) against melonworm, fall and beet armyworm and other caterpillars, and

malathion and SpinTor against sucking pests (such as aphids, mites), beetles, squash vine borer

and leaf miners (Mossler and Nesheim 2001).

In Florida, the silverleaf whitefly, Bemisia tabaci (Gennadius), biotype B (also known as

Bemisia argentifolii Bellows and Perring), is a serious economic pest of squash. Both adults and

immatures feed on phloem sap (Brown et al. 1995). In addition, the B biotype whiteflies cause









1992, Fereres 2000, Summers et al. 2004). In addition, insecticide resistance of whiteflies

(Dittrich et al. 1990) and aphids (Wang et al. 2002) to selected chemical classes has further

restricted their use. Nonetheless, insecticides can be used to decrease the spread of the virus by

reducing the vector population at the primary and secondary source (Brown et al. 1993). In

addition, several non-pesticidal methods, including cultural control practices that are intended for

reducing the vector population and hence interrupting their epidemiological cycle. In this light,

several control methods have been evaluated for the control of viral diseases in cucurbits

including: border crops, intercrops, mineral oils (Zitter and Simons 1980, Damicone et al. 2007,

Fereres 2000), living mulches (Hooks et al. 1998, Frank and Liburd 2005), floating row covers

(Webb and Linda 1992), and reflective mulches (Alderz and Everett 1968, Wolfenbarger and

Moore 1968, Greenough et al. 1990, Kring and Schuster 1992, Stapleton and Summer 2002,

Summers et al. 2004, Frank and Liburd 2005).

Synthetic UV-reflective mulch has been reported to successfully protect various vegetable

crops against insect pests and hence reduce the incidence of viral diseases they transmit

(Csizinszky et al. 1997, Reitz et al. 2003, Stapleton and Summers 2002, Summers et al. 2004).

Reflective mulches reduce whitefly and alate aphid populations because they reflect short-wave

UV light, which repels incoming insects, thus preventing them from alighting on plants (Zitter

and Simons 1980). The crop is thus protected against aphids and whiteflies during the early

growing period and consequently could delay the onset of insect-vectored viruses.

Living mulch is a cost effective alternative to reflective mulches (Hilje et al. 2001). These

mulches have been shown to be effective in hindering whiteflies and other pests from locating

their hosts and consequently transmit viruses (Hooks et al. 1998, Frank and Liburd 2005, Hilje

and Stansly 2007). Hilje and Stansly (2007) reduced the number of adult whiteflies and the









incidence of Tomato yellow mottle virus (ToYMoV) in tomatoes in Costa Rica. The mulches

have also been used to diversify habitats in zucchini plantings and hence influence the ability of

herbivores to recognize their host, resulting in reduced spread of the viruses they vector (Hooks

et al. 1998, Liburd and Frank 2007). Furthermore, the mulches provide food resources (honey,

pollen) and shelter for natural enemies that contribute to pest reduction (Root 1973).

Neonicotinoid insecticides, such as imidacloprid (Admire 2F) [Bayer Cropscience US],

are systemic in plants when applied as soil drenches and can assist in managing whiteflies

(Palumbo et al. 2001). However, frequent application of neonicotinoids and high populations of

whiteflies can increase selection pressure, which may eventually reduce their efficacy (Liburd

and Frank 2007).

Previous studies have shown that buckwheat (Fagopyrum esculentum Moench) used as a

living mulch has potential to reduce insect pests and spread of viruses in zucchini crop (Hooks et

al. 1998, Frank and Liburd 2005). The goal of my research was to develop sustainable integrated

pest management strategies for whiteflies and aphids and the viruses they vector by integrating

cultural control and reduced-risk pesticides tactics. Specifically, I wanted to determine if reduced

pest populations in mulches in combination with Admire 2F (imidacloprid) could offer the added

advantage of suppression of insect-transmitted viruses. This study evaluated the effect of silver

reflective mulch and living mulch alone or in combination with Admire 2F (imidacloprid) to

reduce whitefly and aphid incidence and spread of viral diseases on zucchini squash in Florida.

Materials and Methods

Field Plot Preparation and Experimental Design

The experiment was conducted at the University of Florida Plant Science Research and

Education Unit in Citra, Florida. Field plots measured 10.4 m x 10.4 m. Zucchini squash, seeds

variety Wild Cat (Harris Moran), were planted on the four beds of each plot. Treatments









A component, and PBLlv2040/PCRc154, which amplifies a B component of the bipartite

genomes were used (Akad et al. submitted). The amplified sequences were then compared with

the known genome of begomoviruses. The analysis was conducted by the Plant Pathology

Laboratory of J. E. Polston, Department of Plant Pathology, University of Florida, Gainesville.

Greenhouse Screening

In order to determine if whiteflies were capable of transmitting symptoms noted in the

field to healthy squash plants, a greenhouse study was initiated. Symptomatic leaves were

collected from the field, transported back to the Vegetable Entomology Laboratory. Leaves were

cut using a razor blade under water before placing each leaf in a 400- ml beaker with water. Each

leaf in the beaker was placed each cage. Whiteflies were aspirated from a 'virus free' colony

which was maintained at the Plant pathology laboratory and introduced into the cages. The

whiteflies were left to feed on the leaves for 24 hr before introducing approximately two-week

old squash plants (variety Prelude II) in each cage. The whiteflies were left to feed on the leaves

and the visual symptoms of the virus recorded.

Data Analysis

Data from virus incidence were subjected to an analysis of variance (ANOVA) using SAS

GLM (SAS 9.1 version) and treatment means separated by least significant differences (LSD)

test (SAS Institute 2003).

Results

In 2005, there were no symptomatic plants observed in the field. Similarly, the plants

tested negative for ZYMV, WMV, PRSV-W, and CMV viruses. In 2006, however, symptomatic

plants were observed in November. Zucchini leaves were observed showing mottling, curling

and crumpling symptoms (Figure 5-1). These viral symptoms were significantly more severe on

squash growing on white synthetic mulch (control), where they were first observed, than other









management was done mechanically as required. In 2006, reflective tapes and propane canister

were used to keep the crows away during the day time.

Foliar Sampling

Adult whiteflies and aphids were counted from the leaves in-situ by leaf-turn method. This

was initiated 3 weeks after planting and carried out weekly until final harvest. Nine plants

located on the outside rows of each plot were randomly selected. Insect pest counts were

established by gently turning a leaf and counting the number whiteflies and aphids encountered.

A total of nine leaves were counted in each plot giving a total of 36 leaves per treatment.

Nymphal whitefly population was determined from the nine selected leaves (discussed

above), three from each stratum (upper, mid, lower). The leaves were excised and placed in a 1-

gallon self-sealing Ziploc bags and returned to the Small Fruit and Vegetable IPM Laboratory,

UF, Gainesville, FL. In the laboratory, a 3.14-cm2 leaf disc was taken from each leaf using a cork

borer and examined for whitefly immature stages under a 40X- dissecting microscope (MEIJI

EMZ, Meiji techno co. Ltd Tokyo Japan) (as adapted from Frank and Liburd 2005). The

distribution of the whitefly immatures along the plant strata was also established.

Trap Sampling

Adult whiteflies were monitored with yellow sticky, Pherocon AM unbaited traps

(YST), (Great Lakes IPM, Vestaburg, MI). Three traps were placed in each plot, one in the

middle and the other two on a diagonal line at the two opposite sides of the plot. The traps were

left in the field for a period of 24 h and collected and taken back to the Small Fruit and Vegetable

IPM laboratory to establish counts. The first set of traps were placed into the field one week

after germination and there after once every week for eight and six weeks in 2005 and 2006

respectively.


























Living mulch, buckwheat


- UV-reflective mulch


Figure 3-1. (A) Zucchini squash growing with living mulch (B) Zucchini squash growing on
UV-reflective mulch









time was significant (F = 1.90; df = 20, 72; P < 0.0255), treatment differences were observed in

4 out of the 6 weeks sampled.

Trap Counts

Whiteflies

Significantly (P < 0.05) fewer adult whiteflies were captured on yellow sticky traps (YST)

within reflective and buckwheat treatments with Admire 2F than those within the buckwheat

alone and standard white (control) in 2005 (F = 22.21; df = 4, 96; P < .0001) [Table 3-4].

Buckwheat alone was not significantly different from the white mulch (control). Traps set within

reflective with Admire 2F caught the least number of whiteflies. There were significant (F =

4.26; df = 28, 72; P < 0.0001) interaction effects between treatment and sampling week.

Treatment differences were observed in 6 out of the 8 weeks sampled. In most of the sampling

dates, white (control) resulted in the highest number of whiteflies counted per trap.

In 2006 however, YST within the buckwheat alone mulch contained significantly (P <

0.05) fewer whiteflies than white mulch, which was higher than all the other treatments (F =

27.66; df = 4, 72; P < .0001) [Table 3-4]. The addition of Admire 2F to buckwheat and reflective

mulches did affect the number of whiteflies per trap, and these mulches resulted in significantly

fewer whiteflies compared with the mulches tested alone. There were significant (F = 3.79; df =

20, 72; P < 0.0001) interaction effects between treatment and sampling week. Of the six weeks

sampled treatment differences were observed in weeks 1, 4, 5, and 6.

Aphids

During the 2-yr study, the color of the pan traps (clear versus blue) did not have a

significant (t = -0.86, Pr > I t I = 0.3908) in 2005 and t = 0.03, Pr > I t I = 0.9736 in 2006 effect on

trap catches and hence means of pooled alate aphid counts are reported here. In 2005, pan traps

within reflective mulch with Admire 2F caught significantly higher numbers of alate aphids than









ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my major professor Dr. Oscar E. Liburd,

first for accepting me into his program and for his support and guidance throughout the study. I

would also like to thank Drs. Robert McSorley and Susan E. Webb who served on my

supervisory committee for their input and critical review of this thesis. I thank Dr. Jane E.

Polston for helping with virus identification in the study.

I thank the staff and graduate students of the Small Fruit and Vegetable IPM Laboratory,

University of Florida, for their help in data collection. I also would like to thank the staff and

workers at the Plant Science, Research and Education Unit, University of Florida, and in

particular Nelson Buck for his constant support and timely help in growing and maintaining the

squash plants for my research.

I would like to thank Mrs. Debbie Hall for her guidance and timely correspondence during

the application process for graduate school while I was in Kenya.

Many thanks go to Boniface Kiarie, Dr. George Kariuki Muhia and his family for their

friendship and constant encouragement during the study. Special thanks go to my parents and

family for their love and support during my time away from home.



































2007 TERESIA W. NYOIKE









Table 3-3. Effect of living and reflective mulches alone or in combination with Admire 2F on the
number of whitefly immatures per treatment in zucchini Citra, FL


Mean SEM whitefly immature per leaf disc (3.14 cm2)

2005a 2006b

Reflective + Admire 2F 0.33 + 0.10 c 0.30 + 0.09 c

Reflective 1.86 + 0.32 b 0.98 + 0.26 bc

Buckwheat 2.11 0.46 b 1.77 + 0.45 b

Buckwheat + Admire 2F 0.42 0.15 c 0.44 + 0.20 c

White (control) 3.49 + 0.77 a 2.83 + 0.64 a

Means followed by the same letter are not significantly different (P = 0.05 according to least
square means test following repeated measures analysis, LS).
(LS)
aF= 13.91; df = 4,72; P< 0.0001
b F= 11.90; df = 4, 72; P < 0.0001









Plant Size Sampling

Ten plants were randomly selected from the inner rows that had not been damaged during

pest sampling to estimate plant size. The plant size measurements were taken using a technique

adopted from Frank and Liburd (2005). Using a tape measure, the plant height was taken as the

length of the squash from the ground to the terminal bud. The plant width data was taken by

measuring the length between the two widest opposing lateral shoots growing from the same

plant.

Marketable Yields Sampling

Yield data were collected from the three inner rows of each plot that had not been damaged

during sampling. Zucchini squash was harvested at immature stage (soft, thin, edible rind shells)

with edible seeds at approximately 20-25 cm long. Fruits were harvested and weighed in the field

every other day for three weeks.

Data Analysis

Plant size and yield data were analyzed using analysis of variance (ANOVA) SAS GLM

(SAS 9.1 version [2001]). Treatment means were separated using LSD means separation

procedures and were considered significant when P < 0.05.

Results

Plant Size

Plant widths for reflective mulch treatments with and without Admire 2F were

significantly larger than those for buckwheat and the white mulch (control) treatments (F=

11.64; df = 4, 34; P < .0001) [Table 5-1]. Buckwheat treatments resulted in the smallest width

and there was no significant difference between treatments with and without Admire 2F.

The heights of zucchini plants grown in reflective mulch with and without Admire 2F were

not significantly different. However, these plants were significantly taller than all other









synthetic mulch (control). The suppression of immature whiteflies resulted in fewer silverleaf

symptoms on the leaves.

The addition of imidacloprid to reflective mulch significantly affected apterous aphids in

both years. However, counts of alate aphids from pan traps were inconsistent for both field

seasons. These counts indicated that there were no significant (P < 0.05) differences between

treatments with and without imidacloprid. In 2006, the control treatment had significantly (P <

0.05) higher numbers of alate aphids per trap compared with all other treatments.

In 2005, buckwheat treatments had significantly more natural enemies per leaf than

reflective mulch treatments. Imidacloprid did not appear to affect natural enemy populations

because there were no significant differences in their numbers between treatments with and

without Admire 2F.

In 2005, there was no incidence of insect-transmitted viruses; whereas in 2006, Cucurbit

leaf crumple virus (CuLCrV), a virus new to Florida was found. Cucurbit leaf crumple virus is

transmitted by whiteflies in a persistent manner. Higher incidences of plants infected with

CuLCrV were recorded on the white mulch compared with all other treatments.

Buckwheat mulch had a significant negative effect on the size of squash plants and

marketable yields. Yield results were consistent between both years. The treatment with

reflective mulch and Admire 2F resulted in significantly higher yields than the living mulch and

control treatments. No additional benefits resulted from combining reflective mulch with

imidacloprid in terms of yield in both years. The living mulch treatment resulted in consistently

lower yields than all the other treatments evaluated. The research indicates that enhanced

reduction in the abundance of whiteflies and aphids can be achieved when mulches are combined

with imidacloprid but this does not necessarily mean an increase in marketable yields.











CHAPTER 2
LITERATURE REVIEW

Whiteflies

Whiteflies, Bemisia tabaci Gennadius 1889 (Hemiptera: Aleyrodidae) are a serious pests in

many cropping systems worldwide (Brown et al. 1995, Smith et al. 2000, Rauch and Nauen

2003, Denholm and Nauen 2005). Bemisia tabaci is a highly polyphagous insect attacking

approximately 540 different plant species in 77 families (Basu 1995). Both the adults and

nymphs cause direct damage through sucking the plant sap (Brown et al. 1995). Symptoms

associated with direct feeding include chlorotic halos associated with reduced chlorophyll levels

within 1-2 mm of the feeding site of the nymph (Basu 1995, McAuslane et al. 2004). Plant

damage can also occur indirectly by inducing systemic plant disorders (Yokomi et al. 1990) and

transmitting viruses (Polston and Anderson 1997). In addition, whiteflies excrete honeydew, a

sugar-rich substrate that promotes the growth of sooty mold (Capnodium spp.) on harvestable

plant parts and leaves, lowering their quality (Basu 1995, Brown et al. 1995).

Squash silver leaf disorder (SSL) is a systemic plant disorder associated with the feeding

of immature whiteflies (B biotype of B. tabaci) and characterized by silvering of the upper leaf

surface (Schuster et al. 1991, Yokomi et al. 1990, Hooks et al.1998, Frank and Liburd 2005).

The initial stage of SSL includes yellowing of veins in expanding leaves and as symptoms

progress, the entire upper leaf surface becomes silvered. This silver appearance is due to the

formation of air space within the mesophyll and palisade cell layers and separation of the upper

epidermis from the underlying palisade cells (Paris et al. 1987, McAuslane et al. 2004).

Furthermore, infested leaves of Cucurbita plants have reduced levels of chlorophyll and

higher reflectance than normal leaves (McAuslane et al. 2000, McAuslane et al. 2004). The









Rauch, N., and R. Nauen. 2003. Identification of biochemical markers linked to neonicotinoid
cross resistance in Bemisia tabaci (Hemiptera: Aleyrodidae). Arch. Insect Biochem.
Physiol. 54: 165-176.

Reitz, S. R., E. L. Yearby, J. E. Funderburk, J. Stavisky, M. T. Momol and S. M. Olson.
2003. Integrated management tactics for Frankliniella thrips (Thysanoptera: Thripidae)
in field-grown pepper. J. Econ. Entomol. 96: 1201-1214.

Rhainds, M., J. Kovach, E. L. Dosa, and G. E-loeb. 2001. Impact of reflective mulch on yield
of strawberry plants and incidence of damage by tarnished plant pug (Heteroptera:
Miridae) J. Econ. Entomol. 94: 1477-1484.

Root, R. B. 1973. Organization of a plant-arthropod association in simple and diverse habitats:
the fauna of collards Brassica oleracea. Ecol. Monog. 43: 95-124.

SAS Institute. 2003. The SAS system 9.1 for Windows. SAS Institute, Cary, NC.

Schubert, T. S., and J. J. McRitchie. 1984. Zucchini yellow mosaic virus. Plant Pathology
Circular No. 259. Fla. Dept. Agric. & Consumer Serv. Gainesville, FL.

Schuster, D. J., J. B. Kring, and J. F. Price, 1991. Association of the sweetpotato whitefly
with a silverleaf disorder of squash. Hortscience 26: 155-156.

Smith, H. A., R. L. Koenig, H. J. McAuslane, and R. Mcsorley, 2000. Effect of silver
reflective mulch and a summer squash trap crop on densities of immature Bemisia
argentifolii (Homoptera: Aleyrodidae) on organic bean. J. Econ. Entomol. 93: 726-731.

Soria, C., E. Mariones, A. Fereres, E. Garzo, and M. L. Gomez-Guillamon. 2003. New
source of resistance to mosaic virus transmission by Aphis gossypii in melon. Euphytica
133: 313-318.

Stapleton, J. J., and C. G. Summers. 2002. Reflectives mulches for management of aphids and
aphid-borne virus diseases in late season cantaloupe (Cucumis melo L. var.
cantalupensis). Crop Prot. 21: 891-898.

Stephens, J. M., 2003. Squash, zucchini- Cucurbitapepo L., IFAS Extension HS675. University
of Florida, Gainesville, FL.

Summers, C. G., J. J. Stapleton, A. S. Newton, R. A. Duncan, D. Hart. 1995. Comparison of
sprayable and film mulches in delaying the onset of aphid-transmitted virus diseases in
zucchini squash. Plant Dis. 79: 1126-1131.

Summers, C. G., J. P. Mitchell, and J. J. Stapleton. 2004. Management of aphid-borne viruses
and Bemisia argentifolii (Homoptera: Aleyrodidae) in zucchini squash by using UV
reflective plastic and wheat straw mulches. Environ. Entomol. 33: 1447-1457.

Triplehorn, C. A., and N. F. Johnson. 2005. Borror and Delong's introduction to the study of
insects, 7th ed. Thomson/Brook-Cole, Belmont, CA.









CHAPTER 4
EFFECTS OF SYNTHETIC AND LIVING MULCH WITH AND WITHOUT A REDUCED-
RISK INSECTICIDE TO CONTROL INSECT-TRANSMITTED VIRAL DISEASES OF
ZUCCHINI SQUASH: A NEW WHITEFLY-TRANSMITTED VIRUS IS REPORTED

Virus diseases have a great impact on cucurbit vegetable production in the United States

including Florida (Purcifull et al. 1998, Gaba et al. 2004, Damicone et al. 2007). Unlike most of

the other diseases in squash virus rely on insect vectors for their spread from one plant to

another. Aphids (Hemiptera: Aphididae) and whiteflies are among the most important vectors

transmitting viruses to zucchini squash, Cucurbitapepo L. (Nault 1997, Ng and Perry 2004).

Aphids are known to transmit potyviruses and cucumoviruses including Papaya ring spot virus-

watermelon strain (PRSV-W), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus

(ZYMV) and Cucumber mosaic virus (CMV). These viruses are transmitted in a non-persistent

manner, which is characterized by the short time needed for virus acquisition and transmission to

a healthy plant. Similarly, the viruses have a short retention time in the aphids. Whiteflies (B-

biotype, Bemisia tabaci Genn.) also cause damage through the transmission of plant viruses.

There are several whitefly-transmitted viruses reported to infect squash, including Cucurbit leaf

crumple virus (CuLCrV) also known as Cucurbit leaf curl virus (CuLCV), Squash leaf curl virus

(Brown et al. 2002), Squash vein yellowing virus (SqVYV) (Adkins et al. 2007) and Cucurbit

yellow stunting disorder virus (CYSDV) (Kao et al. 2000). Among these diseases, only CuLCrV

and SqVYV have been recorded in Florida. Cucurbit leaf crumple virus is a begomovirus, which

is transmitted in a persistent, circulative manner by whiteflies.

Pesticides have been the primary tactic for managing whiteflies and aphids. This method

has limitations in that insecticides have not sufficiently controlled virus transmission, especially

when the whitefly population is high. It is even more problematic in case of aphid vectors where

the virus is transmitted before the insecticide kills them (Brown et al. 1993, Kring and Schuster









were two rows of living mulch on each side of the squash plants. Living mulch was planted

approximately 23 cm adjacent to squash plants (Fig. 3a). In the case of synthetic mulches the

entire 76 cm on top bed surfaces were covered with the mulch (Fig. 3b).

Treatments were arranged in a randomized complete block design with four replications.

The treatments evaluated consisted of: 1) reflective mulch (1.33 mm thick 66 inch wide) with the

reduced-risk insecticide (Admire 2F Bayer, Kansas City, MO); 2) reflective mulch without

Admire 2F; 3) living mulch with Admire 2F 4) living mulch without Admire 2F; and 5)

standard synthetic white mulch (1.33 mm, thick, 66 inch wide), which served as the control. The

reduced risk-insecticide Admire 2F was applied 2 weeks after squash planting through the drip

lines at the rate of 1.684 L/ha on the appropriate treatments. Planting holes were cut in the center

of each plastic mulch strip or on ground unmulched beds, and two squash seeds, cv 'Wild Cat'

(Harris Moran Seed Company) were hand seeded per hole. Plant spacing was maintained at

approximately 92 cm in the two years of the study with planting dates on 29 September in 2005

and 3 October in 2006. After germination, the missing plants were replaced using already

established seedlings from the greenhouse. In 2006, the missing plants were not only from

germination failure, but also from crow damage.

Agronomic practices for squash followed the standard production guide for squash in

North Florida (Olson et al. 2005). However, no insecticides were applied during the growing

season except Admire 2F on the specific treatments. A fungicide, azoxystrobin (Amistar; 80

WP); was sprayed as required against powdery mildew during early stages of the crop. Base

fertilizer-dressing N-P-K (10-10-10) at the rate 624 kg per hectare was applied to the soil at

planting. For the first four weeks the squash crop was top-dressed with nitrogen, potassium and

phosphorous each at 0.7 kg weekly and thereafter increased to 0.9 kg at blossom. Weed




Full Text

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1 EVALUATION OF LIVING AND SYNTH ETIC MULCHES WITH AND WITHOUT IMIDACLOPRID FOR SUPPRESSION OF WH ITEFLIES AND APHIDS, AND INSECTTRANSMITTED VIRAL DISEASES IN ZUCCHINI SQUASH By TERESIA W. NYOIKE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 TERESIA W. NYOIKE

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3 To my Mom and Dad

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4 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my major professor Dr. Oscar E. Liburd, first for accepting me into his program and for his support and guidance throughout the study. I would also like to thank Dr s. Robert McSorley and Susan E. Webb who served on my supervisory committee for their input and critical review of this thesis I thank Dr. Jane E. Polston for helping with viru s identification in the study. I thank the staff and graduate students of the Small Fruit and Vegetable IPM Laboratory, University of Florida, for their help in data collection. I also would lik e to thank the staff and workers at the Plant Science, Research and E ducation Unit, University of Florida, and in particular Nelson Buck for his constant support and timely help in growing and maintaining the squash plants for my research. I would like to thank Mrs. Debbie Hall fo r her guidance and timely correspondence during the application process for gradua te school while I was in Kenya. Many thanks go to Boniface Kiarie, Dr. George Kariuki Muhia and his family for their friendship and constant encouragement during th e study. Special thanks go to my parents and family for their love and support during my time away from home.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Justification.................................................................................................................. ...........17 Hypothesis..................................................................................................................... .........18 Specific Objectives............................................................................................................ .....18 2 LITERATURE REVIEW.......................................................................................................19 Whiteflies..................................................................................................................... ...........19 Taxonomy....................................................................................................................... .20 Biology and Behavior of B Biotype................................................................................21 Monitoring..................................................................................................................... ..23 Whitefly-Transmitted Geminiviruses..............................................................................24 Aphids......................................................................................................................... ............25 Biology and Behavior of Aphids.....................................................................................25 Monitoring..................................................................................................................... ..27 Aphid-Transmitted Viruses.............................................................................................28 Management of Whiteflies and Aphi ds and their Related Problems......................................29 Living Mulches................................................................................................................30 Synthetic Mulches...........................................................................................................31 Reduced Risk-Insecticides...............................................................................................32 3 TO INVESTIGATE THE EFFECT OF LIVING (BUCKWHEAT) AND REFLECTIVE MULCHES WITH AND WITHOUT A REDUCED-RISK INSECTICIDE ON WHITEFLIES, APHIDS AN D NATURAL ENEMIES IN ZUCCHINI SQUASH..............34 Materials and Methods.......................................................................................................... .36 Plot Preparation, Irrigation syst em, and Experimental Design.......................................36 Foliar Sampling...............................................................................................................38 Trap Sampling.................................................................................................................38 Physiological Disorder Evaluation..................................................................................39 Natural Enemy Counts....................................................................................................39 Statistical Analysis..........................................................................................................39 Results........................................................................................................................ .............40

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6 Foliar Counts.................................................................................................................. .40 Whitefly Immature Counts..............................................................................................42 Trap Counts.................................................................................................................... .43 Whiteflies.................................................................................................................43 Aphids......................................................................................................................43 Physiological Disorders...................................................................................................44 Natural Enemies..............................................................................................................44 Discussion..................................................................................................................... ..........45 4 EFFECTS OF SYNTHETI C AND LIVING MULCH WI TH AND WITHOUT A REDUCED-RISK INSECTICIDE TO CO NTROL INSECT-TRANSMITTED VIRAL DISEASES OF ZUCCHINI SQUASH: A NEW WHITEFLY-TRANSMITTED VIRUS IS REPORTED.................................................................................................................... ...60 Materials and Methods.......................................................................................................... .62 Field Plot Preparation an d Experimental Design............................................................62 Virus Screening...............................................................................................................63 PCR Analysis...................................................................................................................64 Greenhouse Screening.....................................................................................................65 Data Analysis.................................................................................................................. .65 Results........................................................................................................................ .............65 Greenhouse Results.........................................................................................................66 PCR Analysis Results......................................................................................................66 Discussion..................................................................................................................... ..........66 5 IMPACT OF SYNTHETIC AND LIVI NG MULCH WITH AND WITHOUT A REDUCED-RISK INSECTICIDE ON PLANT GROWTH AND MARKETABLE YIELDS OF ZUCCHINI SQUASH.......................................................................................71 Materials and Methods.......................................................................................................... .73 Plant Size Sampling.........................................................................................................74 Marketable Yields Sampling...........................................................................................74 Data Analysis.................................................................................................................. .74 Results........................................................................................................................ .............74 Plant Size..................................................................................................................... ....74 Marketable Yields...........................................................................................................75 Discussion..................................................................................................................... ..........75 6 CONCLUSION..................................................................................................................... ..80 LIST OF REFERENCES............................................................................................................. ..83 BIOGRAPHICAL SKETCH.........................................................................................................90

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7 LIST OF TABLES Table page 3-1. Effect of living and reflective mulches alone or in combination with Admire 2F on the number of adult whiteflies and aphids pe r zucchini squash leaf, Citra, FL (2005) [Foliar counts]................................................................................................................ ....50 3-2 Effect of living and reflective mulches alone or in combination with Admire 2F on the number of adult whiteflies and aphids pe r zucchini squash leaf, Citra, FL (2006) [Foliar counts]................................................................................................................ ....51 3-3 Effect of living and reflective mulches alone or in combination with Admire 2F on the number of whitefly immatures per treatment in zucchini Citra, FL............................52 3-4 Effect of living and reflective mulches alone or in combination with Admire 2F on the number of whiteflies caught per ye llow sticky trap in Citra, FL.................................53 3-5 Effect of living and reflective mulches alone or in combination with Admire 2F on the number of alate aphids trapped per pan-trap in Citra, FL............................................54 3-6 Effect of living and reflective mulch al one or in combinati on with Admire 2F on number of natural enemies per treatment in zucchini Citra, FL........................................55 5-1 Effect of living and reflective mulches alone or in combination with Admire 2F on plant size of zucchini sq uash Citra, FL (2006)..................................................................78 5-2 Effect of living and reflective mulches al one or in combination with Admire 2F on marketable yields of zucchi ni in squash Citra, FL.............................................................79

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8 LIST OF FIGURES Figure page 3-1 (A) Zucchini squash growing with li ving mulch (B) Zucchini squash growing on UV-reflective mulch..........................................................................................................56 3-2 Distribution of immature whiteflies on diffe rent plant strata of zucchini in Citra, FL (2005)......................................................................................................................... ........57 3-3 Distribution of immature whiteflies on diffe rent plant strata of zucchini in Citra, FL (2006)......................................................................................................................... ........58 3-4 Effect of living and reflective mulches al one or in combination with Admire 2F on silverleaf symptoms presented by the score indices per treatment in zucchini Citra, FL (2006)...................................................................................................................... .....59 4-1 Effects of mulches on Cucurbit leaf crumple virus on zucchini squash plants grown with either living mulch or reflective ground covers as compared with white synthetic mulch in Citra, FL (2006). Viru s incidence = number of plants per plot with virus present. Values with the same letter do not differ ( P < 0.05) according to LSD test....................................................................................................................... ......69 4-2 Cucurbit leaf crumple virus symptoms recorded on zucchini squash plants in Citra, Florida (2006)................................................................................................................. ...70

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF LIVING AND SYNTH ETIC MULCHES WITH AND WITHOUT IMIDACLOPRID FOR SUPPRESSION OF WH ITEFLIES AND APHIDS, AND INSECTTRANSMITTED VIRAL DISEASES IN ZUCCHINI SQUASH By Teresia W. Nyoike December 2007 Chair: Oscar Liburd Major: Entomology and Nematology Field studies were conducted in north-central Florida to eval uate the effect of living and synthetic mulches with and w ithout imidacloprid (Admire 2F) on the density of silverleaf whiteflies, ( Bemisia tabaci Genn. B-biotype) and aphids (Hemip tera: Aphididae) and their transmission of plant viruses in zucchini squash, ( Cucurbita pepo L). Living mulch, buckwheat ( Fagopyrum esculentum Moench) and two synthetic mulc hes (reflective and white) were evaluated in the fall of 2005 a nd 2006. Five treatments were eval uated including reflective mulch with imidacloprid, reflective mulch without imid acloprid, living mulch with imidacloprid, living mulch without imidacloprid and a standard white mulch (control). The ex perimental design was a randomized complete block with four replicat es. Whiteflies and aphids were evaluated through foliar counts, yellow sticky traps, and pan traps. Natural enemie s were established through foliar counts and the marketable yields of squash were evaluated by weight. Data from yellow sticky traps indicated that the addition of imidacloprid enhanced the control of whiteflies in squash growing with buckwheat and re flective mulch. Similarly, foliar counts indicated that these two treatments ha d fewer numbers of whiteflies than the white

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10 synthetic mulch (control). The suppression of imma ture whiteflies resulted in fewer silverleaf symptoms on the leaves. The addition of imidacloprid to reflective mulc h significantly affected apterous aphids in both years. However, counts of alate aphids from pan traps we re inconsistent for both field seasons. These counts indicated th at there were no significant ( P < 0.05) differences between treatments with and without imidacloprid. In 2006, the control treatment had significantly ( P < 0.05) higher numbers of alate a phids per trap compared w ith all other treatments. In 2005, buckwheat treatments had significantl y more natural enemies per leaf than reflective mulch treatments. Imidacloprid did not appear to affect natural enemy populations because there were no significant differences in their numbers between treatments with and without Admire 2F. In 2005, there was no incidence of insect -transmitted viruses; whereas in 2006, Cucurbit leaf crumple virus (CuLCrV), a virus new to Florida was found. Cucurbit leaf crumple virus is transmitted by whiteflies in a persistent manner. Higher incidences of plants infected with CuLCrV were recorded on the white mulc h compared with all other treatments. Buckwheat mulch had a significant negative e ffect on the size of squash plants and marketable yields. Y ield results were consistent betw een both years. The treatment with reflective mulch and Admire 2F resulted in signif icantly higher yields th an the living mulch and control treatments. No additional benefits resulted from combining reflective mulch with imidacloprid in terms of yield in both years. The living mulch treatment resulted in consistently lower yields than all the other treatments evaluated. The research indicates that enhanced reduction in the abundance of whiteflies and aphids can be achieved when mulches are combined with imidacloprid but this does not necessarily mean an increase in marketable yields.

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11 CHAPTER 1 INTRODUCTION Cucurbits are a major vegetable crop grown in the USA, Caribbean and other parts of the world. Their production is importa nt worldwide and these crop s are commercially available throughout the year. Cucurbit production competes in importance with other fruit and vegetable crops worldwide (Gaba et al. 2004). The family Cucurbitaceae consists of three genera: Cucumis L., (melon and cucumber), Citrullus Schrader (watermelon), and Cucurbita L. (summer and winter squashes). Of the squashes Florida produces primarily summer squashes, ( Cucurbita pepo L. ) including crookneck squash, straightneck squash, scallop squash, and zucchini squash (Mossler and Nesheim 2001). Winter squash such as acorn, butternut squas h, and spaghetti squash can also be produced. Florida is ranked second nationa lly after Georgia in the produc tion of fresh market squash. In the 2006 field-seasons, a tota l of 4249 ha of cucurbits was pl anted in Florida and 4131 ha were harvested with a value of approxim ately 38.76 million USD (NASS-2006). In Florida, almost all of the squash are produced for fres h market. Summer production is mainly for local markets while fall production is expo rted. Squash is one of the crops in the state that is shipped every month of the year thus providing a continuo us income to the state of Florida (Mossler and Nesheim 2001). Although squash is produced thr oughout the state of Florida, th e southeast part of the state (Miami-Dade County) is the principal squash -producing region. Othe r important producing regions include the southwest counties (Lee, Hendry, and Collier), west-central counties (Hillsborough, Hardee, and Manatee) and north Florida counties (Alachua, Columbia, and Gilchrist). Squashes can tolera te temperatures of 18 to 27 oC but grows best between 24 and 29 oC. In regions where frost damage is not a thr eat, squash is planted anytime between August and

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12 March. In north Florida where frost damage is likely, fall planting occurs in August and September and in spring season between February and March. Squash is one of the vegetables that are gr own on synthetic mulche s in Florida. Various colors of synthetic mulches such as clear, white black, yellow, and silver are available (Olson et al. 2005). Growers use white, or white on black in the fall and black in the winter and spring (Zitter 1977, Csizinszky et al. 1997, Frank and Li burd 2005). Black mulches keep the soil warm during the cool season while white mulches ar e used during the warm season. Alternatively, reflective silver mulches have the benefit of reduc ing pests and the occurren ce of viral diseases when used in the fall (Olson et al. 2005). Squash is planted mainly through direct seedi ng or transplanting seedlings. Squash matures within 40 to 65 days. Squash plants have both ma le and female flowers on the same plant and therefore require a pollination agent for fertil ization to take place (Mossler and Nesheim 2001). Usually, one hive of honeybees per 0.8 ha is main tained to attain suffici ent pollination (Olson et al. 2005). Several factors affect the squash industry in Fl orida resulting in losses to growers. Some of these factors are the results of increasing cost s associated with controlling insects and their related problems, and outbreaks of new pests and diseases (Akad et al. submitted). Like other squashes, zucchini squash is attacked by a broad array of insect pests a nd plant pathogens such as viruses, bacteria, fungi, and nematodes. Some of the major foliar diseases affecting cucurbits include powdery mildew caused by Sphaerotheca fuliginea (Schlechtend.:Fr.) Pollacci and Erysiphe cichoracearum DC. and downy mildew caused by Pseudoperonospora cubensis Berk & M. A. Curtis (Zitter et al. 1996). The occurrence of these diseases is more common than viral diseases. While preventative sprays are

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13 applied against downy and powdery mildew, control may not be necessary if the disease occurs in the last 3 weeks of the growing period. Other foliar diseases affecting squash include wet rot and phytophthora foliar blight, wh ich can cause fruits to rot. Both diseases can be managed effectively by soil drench of metalaxyl/chlorot halonil (Mossler and Nesheim 2001). Post harvest diseases of squash include bacterial soft rot, alte rnaria rot, fusarium rot, rhizopus storage rot and pythium fruit rot. These diseases which, cause the fruit to rot, may begin in the field and cause fruit to deteriorate under storage. Pythium fru it rot is the most comm on disease causing the crown of older squash to rot. Post harvest di seases can however be controlled through proper handling during and after harvest (Mossler and Nesheim 2001). Insect pests of squash include pickleworm, Diaphania nitidalis Stoll, and melonworm, Diaphania hyalinata L. Both pests are tropical moths and are able to overwinter in Florida causing damage to squash among other cucurb its. Their young larvae are known to cause damage on the blossoms, foliage of young vegetative buds and the apical grow th of vines. Other pests that damage the foliage and o ccasionally the fruits are squash bug, Anasa tristis DeGeer; melon thrips, Thrips palmi Karny; fall armyworm, Spodoptera frugiperda (J. E. Smith); beet armyworm, Spodoptera exigua Hbner; and leafminers, Liriomyza trifolii Burgess and L sativae Blanchard. To manage these pests, Flor ida growers use biop esticides such as Bacillus thuringiensis (B.t.) against melonworm, fall and beet armyworm and other caterpillars, and malathion and SpinTor against sucking pests (such as aphids, mites), beetle s, squash vine borer and leaf miners (Mossler and Nesheim 2001). In Florida, the silverleaf whitefly, Bemisia tabaci (Gennadius), biotype B (also known as Bemisia argentifolii Bellows and Perring), is a serious econo mic pest of squash. Both adults and immatures feed on phloem sap (Brown et al. 19 95). In addition, the B biotype whiteflies cause

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14 silverleaf disorder of squash (SSL) an important physiologica l disorder of some cucurbits. Squash silverleaf is associated with the feeding of immature whiteflies of B biotype and is characterized by silvering of the adaxial leaf surface and blanching of the fruit (Schuster et al. 1991, Yokomi et al. 1990, Costa et al. 1993). Substa ntial economic losses in Florida have now been associated with SSL due to poor fruit quality and yield reduc tion (Yokomi et al. 1990, Liburd and Frank 2007). Whiteflies also transmit geminiviruses to squash. Recently, two whitefly-transmitted viruses were recorded in squash in Florida. The Squash vein yellowing virus (SqVYV) an Ipomovirus, affects squash, pumpkin and watermelon and Cucurbit leaf crumple virus (CuLCrV); a begomovirus, affects all cucurb its except honeydew melon ( Brown et al. 2002, Adkins et al. 2007, Akad et al. submitted). Cucurbit leaf crumple virus is a relatively new virus to Florida and first appeared in north-central Florida during the fall 2006 (Webb et al. 2007). The emergence of newly introduced whitefly-borne viru ses to areas where they never existed before has been attributed to high whitefly populati ons and possibly movement of infected plant material from other states (We bb 1991, Polston and Anderson 1997). Cucurbit leaf crumple virus is transmitted by whiteflies in a persistent, circulative manner. Typical symptoms of CuLCrV on leaves include curling, yellowing, and crumpli ng. Symptoms can also develop on the fruits causing abrasions or even green streaks like on the yellow squash causing the fruits to be unmarketable (Webb et al. 2007). Aphids are also important pests of squash, causing damage through sucking plant sap and, more importantly, through transmission of viral di seases. They are the most important vectors of plant viruses and are known to transmit 275 differ ent viral disorders (Nau lt 1997). In Florida, the most damaging viruses affecting squash are Papaya ring spot virus-Watermelon strain (PRSV-

PAGE 15

15 W), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV). Symptoms of these viruses on leav es include: leaves curling downward and wrinkling, vein banding, mosaic patterns, mottlin g, blistering and distorte d (strapleaf) leaves which are reduced in size. Fruit symptoms in clude discoloration with mosaic patterns and reduced misshapen and warty texture (Ald erz et al. 1983, Schubert and McRitchie 1984, Purcifull et al. 1988, Mossler and Ne sheim 2001, Gianessi et al. 2002). Mosaic viruses are transmitted in a non-persistent manner by several aphid species. Of the known species associated with virus spread, the melon aphid Aphis gossypii Glover, is considered a primary pest of cucurbits (Damic one et al. 2007). Alate aphids are able to acquire/transmit the viruses as they are probing to test the plant for host suitability. Once the virus is acquired aphid vectors rema in viruliferoous for a short period of time (minute to hours). Damage to host plants by whiteflies and aphi ds can also result in reduced plant growth and quality due to excessive removal of plant a ssimilates from phloem. Moreover, excretion of honeydew, a sugar-rich compound, by whiteflies and aphids on the leaves and fruits, promote growth of sooty mold ( Capnodium spp.) further reducing their quality (Palumbo et al. 2001). Pesticides have been the most popular pe st management tactic in high value crops (Hummel et al. 2002) including cucurbits (Fra nk and Liburd 2005). However, awareness has increased concerning the use of pesticides due to their effects on human, environment and other non-target organisms (Antignus 2000). In additi on, high pesticide usage in creases the cost of production and potential for resist ance development. Subsequen tly, there has been increased emphasis on sustainable cultural pest management tactics. Cultural control strategies entail manipulating components of the agro-ecosystem to reduce pest numbers to below economically damaging le vels (Hilje et al. 2001 ). Living mulches and

PAGE 16

16 inert ground covers such as synt hetic, sawdust, straw and husk mulches have been evaluated as a means of managing key pests in vegetable crops. UV-reflective mulches have been demonstrated to reduce whitefly and aphids and / or the inci dence of the viruses th ey transmit in various vegetable crops (Wolfenbarger and Moore, 1968, Brown et al. 1993, Csizinszky et al. 1995, Smith et al. 2000, Stapleton and Summers 2002, Re itz et al. 2003, Summers et al. 2004, Frank and Liburd 2005). UV-reflective mu lches reflect short-wave li ght, which repels incoming herbivores, and reduces their inci dence of alighting on plants. This consequently, affects the rate of virus transmission and spread by these pests. Synthetic mulches have also been shown to increase yields (Summer et al. 2004, Frank and Liburd 2005). Their use has been adopted in many parts of the world including Florida in production of vegetables. UV-reflective mulches however, lose their efficacy once the crop canopy covers 70% of the mulch (Smith et al. 200 0). In addition, they ar e expensive and pose a problem of disposal, which may have an undesirable effect on the environment. Living mulches are a low-cost al ternative to synthetic mulche s (Hilje et al. 2001) and are safe for the environment. The main idea of us ing these mulches is to provide resources for natural enemies and manipulate host seeking behavi or of insect herbivores (Roots 1973, Hilje et al. 2001). For instance, whiteflies and aphids lo cate their host through indiscriminate visual attraction to yellow (green) (Kring 1972, van Lent eren 1990). Alate aphids locate their host by contrasting the soil back ground with the green color of the fo liage. Thus, reducing the contrast between the plant and the bare ground as with living mulches will affect the number of insects alighting on the host plant (Hilje et al. 2001, Cr adock et al. 2002, Hilje and Stansly 2007). Living mulches in zucchini squash have b een demonstrated to reduce the number of whiteflies and aphids and delay th e onset of insect-borne viruses in squash in Florida and Hawaii

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17 (Hooks et al. 1998, Frank and Liburd 2005). In additi on, they have been shown to increase yields in squash and other vegetables when compared with the bare ground (Hooks et al. 1998, Frank and Liburd 2005, Hilje and Stansly 2007). Justification Living mulches are a cheaper and more sustai nable alternative to reflective mulches. Zucchini squash interplanted with living mulch, buckwheat Fagopyrum esculentum Moench had lower populations of whiteflies and aphids and dela yed the first appearance of virus incidence in the crop (Hooks et al. 1998, Frank and Liburd 20 05). Reflective mulches have been shown to reduce whitefly and aphid populations and subseque ntly delay the onset of the viral transmitted diseases (Stapleton and Summer s 2002, Summers et al. 2004, Fra nk and Liburd 2005). However, the reliance on living or reflective mulch as a single tactic is inadequate to control virus spread (Polston and Anderson 1997). Furthermore, reflectiv e mulches lose its ins ect repelling ability once the plant growth covers the reflecting surfaces (Smith et al 2000). It is, however, not yet known if aphid and whitefly populations can be fu rther reduced by the addition of a reduced-risk insecticide that could lead to more economic gains. Further reduction in whitefly and aphid populations will increase the numbe r of marketable fruits and subsequently enhance growers profits. Imidacloprid (Admire 2F) is an effective reduced-risk insecticide with systemic activity and can be used to manage whiteflies and aphids It is however not known if imidacloprid will affect the natural enemy popul ations associated with living mulch in zucchini. There is a need to integrate other tactics with reduced-risk insecticide to prolong their use in managing whiteflies and aphids and other prob lems in cucurbits. Since isolated cases of resistance to whiteflies have already been repo rted (Osborne 2006 personal comm.). In our study, Admire 2F will be used once at the beginning of the season when control is most needed.

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18 Hypothesis It is hypothesized that the use of synthetic (reflective) a nd living mulches (buckwheat) in combination with a reduced-risk insecticide (Adm ire 2F) will further suppress whitefly and aphid populations and subsequently dela y the onset of viral diseases. The goal of my study is to determine if mulches in combination with Admire 2F (imidacloprid) could offer added advantage in suppressing key pests as well as insect-transmitted viruses and consequently increase yields. In ad dition, I seek to demonstrate an economic viable management program for aphid and whiteflies and th eir related viral diseases of squash that can be used in Florida and elsewhere. Specific Objectives To investigate the use of reflect ive and a living mulch, buckwheat ( Fagopyrum esculentum Moench) with and without imidacloprid for controlling aphids and whiteflies. To study the effects of reflective and liv ing mulch (buckwheat) with and without imidacloprid on natural enemies (beneficial insects) that s uppress aphid and whiteflies. To evaluate the effectiveness of reflec tive and a living mulch with and without imidacloprid to viral di seases of zucchini. To study the impact of reflective and a livi ng mulch with and without imidacloprid to plant size and consequently the ma rketable yields of zucchini.

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19 CHAPTER 2 LITERATURE REVIEW Whiteflies Whiteflies, Bemisia tabaci Gennadius 1889 (Hemiptera: Aleyrodidae) are a serious pests in many cropping systems worldwide (Brown et al. 1995, Smith et al. 2000, Rauch and Nauen 2003, Denholm and Nauen 2005). Bemisia tabaci is a highly polyphagous insect attacking approximately 540 different plant species in 77 families (Basu 1995). Both the adults and nymphs cause direct damage through sucking the plant sap (Brown et al. 1995). Symptoms associated with direct feeding include chlorotic halos associated with reduced chlorophyll levels within 1-2 mm of the feeding site of the nymph (Basu 1995, McAuslane et al. 2004). Plant damage can also occur indirectly by inducing systemic plant diso rders (Yokomi et al. 1990) and transmitting viruses (Polston and Anderson 1997). In addition, whiteflies excrete honeydew, a sugar-rich substrate that promot es the growth of sooty mold ( Capnodium spp.) on harvestable plant parts and leaves, lo wering their quality (Basu 1995, Brown et al. 1995). Squash silver leaf disorder ( SSL) is a systemic plant disorder associated with the feeding of immature whiteflies (B biotype of B. tabaci ) and characterized by silv ering of the upper leaf surface (Schuster et al. 1991, Yokomi et al 1990, Hooks et al.1998, Frank and Liburd 2005). The initial stage of SSL include s yellowing of veins in expand ing leaves and as symptoms progress, the entire upper leaf surface becomes s ilvered. This silver appearance is due to the formation of air space within the mesophyll and palisade cell layers and separation of the upper epidermis from the underlying palisade cells (P aris et al. 1987, McAu slane et al. 2004). Furthermore, infested leaves of Cucurbita plants have reduced levels of chlorophyll and higher reflectance than normal leaves (McAus lane et al. 2000, McAuslane et al. 2004). The

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20 silvering on the leaves is irreversible and sy mptoms severity increases as the number of immature whiteflies per plant increases. As few as two immature whiteflies per plant, feeding for about 14 days are able to induce silverleaf symptoms on zucchini (Costa et al. 1993). Symptoms are also manifested on the fruits where green-fruited squashes appear lighter in color or the color is streaked longitudinally while the yellow-fruited squash appear pale or white (Schuster et al. 1991, Yokomi et al. 1990). Bemisia tabaci was not a pest in Florida cucurbits production until 1987 when an outbreak occurred on poinsettia Euphorbia pulcherrima (Poinsettia), which was associated with a new biotype, B. This biotype later sp read to other crops including s quash and tomatoes (Mossler and Nesheim 2001). The B biotype was thought to have been introduced into Florida in the early 1980s possibly through the movement of ornament als (Brown et al. 1995, Polston and Anderson, 1997), but economic losses were not observed until an outbreak of the disorder occurred during the 1987-88 vegetable season (Schuster et al. 199 1). Economic losses to many crops as a result of direct feeding damage and whitefly-transmitted geminiviruses led to the rise of B. tabaci as a pest and virus vector in the tr opics and subtropics in the late 1980s (Yokomi et al. 1990, Schuster et al. 1991, Brown et al. 1995, Polston and Anderson 1997). Taxonomy Infestation on poinsettias in Florida indicated a new strain, since th e strain previously, strain A was not affecting poinsettia. The new st rain was named strain B, or biotype B to distinguish it from the existi ng strain A or biotype A. Th ese two biotypes have similar morphological appearance but diffe rent biological characteristic s (Brown et al. 1995). It is known that B-biotype has a wider host range and lays more eggs than A-biotype. Also, based on the amount of honey dew produced, B-biotype inge sts more phloem sap than biotype-A (Brown et al. 1995). The feeding of immatures of the Bbiotype can cause plant disorders, which is not

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21 the case with biotype A. In addition, B-biotype ha s greater efficiency in transmitting geminivirus diseases than the A strain (Brown et al. 1995) but not clostroviruses in whic h strain A is better. According to DNA differentiation tests and ma ting incompatibilities as evidenced by the absence of females in the progeny, the B-biotyp e was described as a distinct species and a common name silverleaf whitefly was suggested due to the damage they cause on squash (Perring et al. 1993). Concurring with the earlier described differences of strain B with the other existing strains, Bellows et al (1994) added the morphological di fferences based on the fourth instar pupal case. These differences alongside with allozymic diagnoses allowed them to classify the strain as a new species, Bemisia argentifolii (Bellows & Perring) (Bellows et al. 1994). This naming has undergone some debates with some colleagues arguing that if B-biotype is a new species then all the other biotypes should be elevated to species level. In this regard, Brown et al. (1995) proposed that B. tabaci is in fact a species comp lex. At the time of writing, B-biotype is the most commonly used name. Biology and Behavior of B Biotype The B biotype of B. tabaci has six life stages: egg, four inst ars, and the adult. The eggs are usually elongate-oval but occasionally may be reni form. The apex distal end of the egg is acute and the basal portion is usually broad with a pe dicel or stalk of varying length by which the female attaches the egg to the host (Gill 1990, Byrne and Bellows 1991). Eggs may be laid singly in small groups or in circul ar or semicircular pattern on the underside of the leaf due to the rotation of the female as she oviposits (McAuslane et al. 2000). Freshly laid eggs are translucent, creamy white but turn pale brown just before hatching. Each female can lay a minimum of 50 eggs and up to 300 eggs have been recorded (Basu 1995). The crawler is the first instar and the only mobile stage of the immature stages. Crawlers are pale, translucent white, oval with a convex dorsum and flat ventral side (Basu 1995). The

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22 stage has well developed legs and antenna. Afte r emerging, the crawler m oves and settles a few millimeters from the egg to find a feeding site but they are also capable of moving within the leaves (Triplehorn and Johnson 2005). Once settle d and feeding the craw ler excretes a small amount of wax (Gill 1990) Subsequent instars (second, third and fourth) are sedentary and only have limited movement during molts when they reinse rt their stylets. They both have a scale-like covering, which is a waxy secretion that helps the instars adhere th emselves to the leaf surface (McAuslane 2000, Triplehorn and Johnson 2005). The fourth instar has three distinct devel opmental stages even though there is no molting between them: substage one is a feeding, flatte ned and opaque nymphal phase, substage two is a nonfeeding nymphal phase, which is thickened and covered with wax, and a pharate adult phase. The early stage (substage one) is feeding and he nce only the last nymphal phases are referred to as a pupa. The major difference between the substa ges two and three is the red eyes which are more visible in the pharate stage due to the developing adult inside them (Gill 1990, Byrne and Bellows 1991, Osborne and Landa 1992, Basu 1995). Adults emerge through a T-shaped slit in th e pupal case of the last nymphal instar. A characteristic round-shaped hole is left on the case if an adult parasitoid emerged from the pupa (McAuslane 2000). The adult head is broader than long, with seven-segmented filiform antennae. Mating can occur with in hours of emergence once the wings are hardened and expanded. Mated females produce both females and males whereas unmated ones only males (Byrne and Bellows 1991, McAuslane 2000). Females of B. tabaci usually oviposit on the abaxial side of young leaves and the same leaf is used for oviposition and feeding. They usua lly prefer a moderate degree of pubescence to either glabrous or extremely hairy leaf surfaces for oviposition. Bemisia tabaci refrains from

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23 ovipositing on very young leaves (van Lenteren 1990). Vertical distribution patterns along the plant stratum have been described for B. tabaci life stages. Eggs are usually found in the upper plant strata where they are laid while the mature nymphs are on the lower strata. Other factors that seem to affect oviposition a nd feeding site selection by white flies include phototropism, leaf shape, and nitrogen content (Bentz et al. 1995, McAuslane et al. 2000). Whiteflies have been described as weak fliers and move with the wind current, as they probe host plants. Bemisia tabaci is known to orient toward either yellow or blue/ultraviolet light but not both at the same time, a be havior that is related to migr ation and plant colonization (Basu 1995, Antignus 2000). Ultraviolet light is related to migratory behavi or whereas yellow radiation induces vegetative behavior, which could be a part of host selection mechanism. This is important as whiteflies do not appear to have olfactory cues in host-se lection (van Lenteren 1990, Byrne and Bellows 1991, Antignus 2000). Once within the host loca tion, whiteflies use color as cue to select landing sites for f eeding and oviposition (Byr ne and Bellows 1991). Monitoring Adult whiteflies can be monito red by use of yellow sticky traps and leaf turn method insitu. In-situ counts are done to estimate the absolute population in the field. This should be done in the morning when the whiteflies are least mob ile. Yellow sticky traps have been reported to give a good correlation between ca tches and actual whitefly numb ers in the field in some systems. For this reason, these traps have been proposed for monitoring whiteflies in integrated pest management systems (Basu 1995). Nymph age tends to correlate with leaf age since they complete their development on the leaf they were oviposited. Estimates of egg density are usually taken from upper stratum leaves, while ny mphal densities are taken from the middle and lower canopies depending on the host crop (Basu 1995). Quantification of immature whiteflies

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24 involves cutting of leaf discs of a designated area from selected leaves. The leaf disks are then examined under a microscope for id entification of nymphal stages. Whitefly-Transmitted Geminiviruses Bemisia tabaci biotype B has been described as a not orious pest due to its ability to transmit viruses to various crops. Whitefly-transmitted viruses cause yield losses worldwide on important food and industrial crops (Polston a nd Anderson, 1997). Yield lo sses due to whiteflytransmitted viruses have been described to be in the range of 20-100% depending on the crop, season, and vector prevalence (Basu 1995). Befo re 1990, geminiviruses transmitted by whiteflies ( Bemisia tabaci Genn.) were primarily a problem fo r legume production in the Western Hemisphere. Emergence and spread of new gemini virus diseases have b een directly linked to evolution of variants of the vi ruses (Varma and Malathi, 2003), the appearance of biotype B and the increase of the vector popul ation (Polston and Anderson, 1997, Varma and Malathi, 2003) in the Americas and the Caribbean Basin. The geminivirus genome is made up of circular, single-stranded DNA, which is encapsidated in twinned subunits of a single capsi d protein. They form the second largest family, Geminiviridae of plant viruses. In terms of genome structures, most of them are bipartite, having two equal components, A and B, while the m onopartite have one la rger DNA component. Geminiviruses have been classified into four genera: Mastrevirus Curtovirus Topocuvirus and Begomovirus depending on their vector and genomic ch aracteristics (Varma and Malathi, 2003). The majority of the known geminiviruses (80 %) is transmitted by whiteflies: Begomoviruses, which have mostly bipartite genomes and infect dicotyledonous plants. Geminiviruses are transmitted in a persis tent, circulative way (Polston and Anderson, 1997). They have a minimum acquisition period of 30-60 min, with inoculation periods that are less than 30 min, and infectivity is retained for at least few days. Typical symptoms include vein

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25 yellowing, chlorotic mottle, chlorotic leaf margins, leaf curl and distortions, puckering of leaves, reduced leaf size, plant stun ting, and flower abscission (Pol ston and Anderson, 1997, Varma and Malathi, 2003). Aphids Aphids are one of the most varied and mo st studied groups of the hemipterans (Kring 1972, Powell et al. 2005). Their repro ductive potential, salivary secr etions, and ability to transmit viral diseases make them the most potent and wo rld-wide enemies of many crops (Brown et al. 1993, Kucharek et al 2001, Stapleton and Summers 2002, Soria et al. 2003, Liburd and Frank 2007, Ng and Perry 2004). Viral diseases cause dama ge to both yield and fruit quality resulting in significant losses (Kucharek et al. 2001). More than 50 viruses have been listed that affect cucurbits. Until 2001, there were only five reporte d aphid-transmitted viruses affecting cucurbits in Florida. These included Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus strain 2 (WMV-2) now called WMV, Papaya ring spot virus type W (PRSV-W) and Watermelon leaf mottle virus (Purcifull et al. 1998). PRSV-W and WMV are the most commonly occurring viruses in Florida. Another aphid-transmitted virus that has emerged recently in Florida is PRSV-Tigre (Webb et al. 2003). Typical symptoms of these viruses on the leaves include: leaf curling downward and wr inkling, vein banding, mosaic patterns, mottling, blistering, leaf distortions and reduction of l eaf lamina (strapleaf). Fruit symptoms include discoloration with mosaic patterns, reduced size, misshapen, with warty texture (Alderz et al. 1983, Schubert and Mc Ritchie 1984, Purcifull et al. 1988, Mossler and Nesheim 2001, Kucharek et al. 2001, Gianessi et al. 2002). Biology and Behavior of Aphids Variations in aphid life cycles are we ll known. They alternate primary hosts with secondary hosts, sexual with parthogenetic forms, wingless with winged forms, and migrant with

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26 nonmigrant forms. All these variations may some times occur within the annual cycle of a single species as it does in the melon aphid, Aphis gossypii Glover (Kring 1972). Holocyclic species have a sexual generation in the autumn and durin g the remainder of the year, they are asexual bearing females (viviparae). Anholocyclic aphids have no sexual generation, and only viviparae are produced throughout the year. He teroecious or host-alternating aphids use different species for winter (primary) and summer (secondary) hosts while monoecious species utilize the same plants throughout the year (Halbert and Voegtlin 1995). The evolution of parthenogene sis, enabling reproduc tion without males for part or all the lifecycle, confers a repr oductive advantage by doub ling the intrinsic rate of population increase (rm) relative to sexually reproducing individuals (Powell et al. 2005). When conditions are favorable (low aphid density, high host quality ), most aphid species continue to maximize investment in reproduction by producing aptero us progeny. As density increases, and plant nutritional quality declines, production of alate offspring is stimulated. Host selection by aphids is not a random process (Powell et al. 2005, Nault 1997). They employ a variety of sensory and behavioral mechan isms to locate and rec ognize their host plants (Powell et al. 2005). Landing by migrating alate aphi ds involves a phototactic response to plantreflected wavelengths (Nault 1997). After landing, a va riety of cues may be detected as the aphid walks across the leaf surface. Stylet penetration is attempted as a reflex following tarsal contact with any solid surface even in the presence of re pellent cues (Powell et al. 2005). The first few stylet penetrations initiated after the plant cont act are usually brief (<1 min) and are limited to the epidermis when the aphid tries to distinguis h host from non host. A phid stylets and labium apparently lack external contac t chemoreceptors and therefore plan t sap may have to be ingested to contact a gustatory organ locate d in the epipharyngeal area. If th e sap is desirable it is sucked

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27 into the cibarium (sucking pump). Aphids make one or more probes before taking flight or moving to the lower leaf surf ace where they establish contac t with phloem (Nault 1997, Powell et al. 2005). This behavior is es pecially important since transmi ssion of stylet-borne viruses can occur from the test probes (Nault 1997). For th is reason, non colonizing aphids can be more important in virus transmission th an those that colonize the crop. Several factors, such as effects of light, sound, an d odor are responsible for active selection of alighting surfaces by aphids (Kring 1 972). Alighting may be a response stimulus to a particular site (colored object, trap, or plant) or it may be the result of a lack of interfering stimuli. For this reason, takeoff and flight activ ity are increased in the presence of shortwave light. Certain white surfaces and aluminum surf aces that reflect shortwave, long wave and varying amount of infrared energy do reduce the numb er of aphids alighting on plants or in traps they surround (Kring 1972). Host-seeking aphids are attracted to the relatively long wave radiation reflected from the soil and vegetation (Cradock et al. 2002). Yellow and green surfaces (wavelength 500-580nm) are particularly attractive. Monitoring Several trapping techniques can be used to m onitor alate aphids. These aphids have poor visual acuity but they are known to be responsiv e to yellow and green frequencies (wavelength 500-580 nm). For this reason commercial yellow sticky traps are available to monitor aphid populations in the field and gree nhouse. Pan traps are small contai ners that are filled with a preservative for collecting aphids. They have functions similar to the sticky traps, but the aphids are collected in the liquid preservatives. Suction tr aps have also been used to monitor aphids in small grain fields.

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28 Aphid-Transmitted Viruses Aphid-borne viruses can kill infected plants and significantly affect the crop yield (Eastop 1977). Aphid-borne non-persistently transmitted viruses are of economic importance for many cucurbit growers. They form the bulk of aphid transmitted viru ses (Hull 2002, Hooks and Fereres, 2006). Nonpersistent viruses ar e transmitted by many aphid species including Aphis gossypii Myzus persicae, Acyrthosiphon (Aulac orthum) solani, Aphis craccivora, and Macrosiphum euphorbiae and many others as they search for suitable hosts to invade. Aphis gossypii, the melon aphid is considered as one of the mo st important vector because it is able to colonize and reproduce on cucurbit crops (Damicone et al. 2007). In other cases this aphid has not been considered important because it tends to remain on the plan t (Mora-Aguilera 1995). Non-persistent viruses are defined as t hose that have a very short acquisition and inoculation times (seconds to minutes) (Nault 19 97, Ng and Perry 2004). The viruliferous aphids retain their ability to infect for a very short pe riod. These viruses have been termed as styletborne viruses due to the fact th at the virions are restricted to the stylets. These viruses are transmitted to healthy plants within very brie f probing period (Nault 1997). It is known that during acquisition and inoculati on stylets of aphids do not pe netrate beyond epidermal cells (Nault 1997). Conversely, semi-per sistent viruses are vectored by a few aphid species that will be able to feed on and colonize the host plant. Acquisition of th e virions occurs within minutes, but the ability to transmit can be increased with prolonged feeding (Ng and Perry, 2004). In addition, the virions are retain ed for hours to days within the body of the vector. Semipersistently transmitted viruses have also b een termed as foregut borne and like the nonpersistently transmitted viruses they cannot be recovered from the hemolymph (Nault 1997). Furthermore, the ability of viruliferous immatures to infect other plants is lost during molting in both types of transmission.

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29 On the other hand persistent transmitted viru ses require longer periods to be acquired or transmitted (hours to days) (Ng and Perry, 2004). They also require more than one week latent period (Nault 1997). Furthermore, infected aphids remain viruliferous for extended periods. Management of Whiteflies and Aphi ds and their Related Problems In the zucchini crop, no single control method has been used to control whiteflies and aphids and their associated problems (Hooks et al 1998, Frank 2004). Whiteflies and aphids are notorious pests in zucchini and greatly known for the multi-component damage they cause to plants. Traditionally, pesticides have been the main management tool for whiteflies and aphids. However, due to the problems associated with pe sticidal tactics, other alternative methods have been investigated. It is known that non-persiste ntly transmitted viruses have not been managed effectively by the use of insecticides since the viruses transmitted before the vectors are killed (Brown et al. 1993, Soria et al. 2003). Similarly, geminiviruses have not been controlled by use of insecticides (Polston and Anderson 1997). Furtherm ore, the use of insec ticides increases cost of production and many virus vectors have devel oped resistance to various chemical classes (Dittrich 1990, Wang et al. 2002). Use of systemic insecticides, however, can re duce secondary virus spread in the field by preventing the build up of vectors on primary s ources if the major vector is a species that colonizes that crop. Virus control can also be achieved using pl ants genetic resistance. This however, can be of limited use since it is no t always available (Cradock et al. 2002). Furthermore, resistance can be overcome unde r high pressure of vi ruliferous populations. Tomato varieties with resistance genes of Tomato yellow leaf curl virus (TYLCV) were overcome when moderate to high populations of vi ruliferous whiteflies were inoculated early in the growing season (Polston and Anderson, 1997). With this in mind, vector management is very important in the control of insect-transmitted viruses.

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30 Cultural control techniques have been pr oposed as means of ma naging whiteflies and aphids and their related problems (Antignus 2000). These techniques are tailored toward manipulating the insects behavior in host rec ognition, resulting in arrest ment of the vectors before they land on the host plant. Living Mulches Living mulches are cover crops that are inte ntionally maintained as living ground cover throughout the growing season of the main crop. Th ey are distinguished from other cover crops in that they are not removed before the main crop is planted (Nakamoto and Tsukamoto 2006). There has been increased interest in the use of living mulches fo r pest suppression as a means of responding to environmental concerns regardi ng the use of pesticides. These cover crops suppress weeds, reduce soil erosion, enhance so il fertility and quality, and reduce damage imposed by herbivores. It has been suggested that di verse habitats contai n lower populations of herbivorous insects as compared to simple habitats. Root (1973) found that insect pest s were less dense in Brassica oleracea grown in diverse habitats than in pure sta nds. To explain his findings, he proposed two hypotheses. The natural enemy hypoth esis predicts that predator and parasitoid populations will occur in greater diversity and at greater densi ties in diverse plantings because these systems provide additional resources (e.g. food, shelter) for the enemies. The other hypothesis, resource concentration, predicts that herbivores are more lik ely to find and remain in pure stands of their host plants because of availability of necessary resources in these areas. Use of living mulch as a means of vegetative di versification can also affect transmission of insect-vectored viruses depending on the mode of transmission of the particular virus. Inclusion of living mulch provides additional feeding sites for infectious aphids around the crop. This has been termed as a protection crop and hence reducing the inci dence and the spread of aphid-

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31 borne non-persistently transmitted virus (Toba et al. 1977). In this case the living mulch will be serving as a virus-sink where the infected aphid is likely to loose its virus during probing. The protection crop must be immune fr om the target virus or viruses and suitable for probing by the aphid and should not serve as a host for aphid reproduction (Toba et al. 1977). Diversified habitats can also affect herbi vore damage through their influence on factors such as immigration and emigration of pest or enemies searching behavi or of both generalists versus specialist insects. These habitats will not only affect host-finding for host-specific pests but also for species-specific enemies (Costello and Altieri 1995). Furthermore, Bemisia tabaci spent less time on host plants and appeared rest less in a multiple plant species environment (Frank 2004). Therefore, it is possible that damage associated with the pe st will be reduced in such a scenario. Among the agronomic benefits attributed to living mulches, reduction of damage (direct and indirect) has received attention in cr op production (Toba et al. 1977, Andow et al. 1986, Hooks et al 1998, Frank and Libur d 2005, Hilje and Stansly 2007). Us e of living mulch has been shown to result in fewer whiteflies, aphids and/ or delayed occurrence of insect-transmitted viruses in various crops (Kloen and Altieri 1990, Costello and Altieri 1995, Hooks et al. 1998, Frank and Liburd 2005). However, in cases wher e excessive competition leads to poor plant quality in mixed cropping systems, the advantages of reduced pest densities may be lost because of poor crop yields (Frank and Liburd 2005). Synthetic Mulches The synthetic mulches are primarily polyethyl ene (colored or clea r plastic), but may include treated paper, wax coated papers, and aluminum and steel foils. Silver or gray reflective mulches have been used successfully to delay and reduce the incidence of aphid-borne virus diseases in squash and other crops (Brown et al. 1993, Stapleton and Summers 2002, Summers et

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32 al. 2004). These mulches reflect short-wave length which repels incoming alate aphids, thus reducing their incidence of a lighting on plants (Stapleton a nd Summers 2002, Cr adock et al. 2002, Summers et al. 2004). Reflective and colored mulches have been reported to modify soil moisture and temperature (Csizinszky et al. 1997 ), which results in increased cr op earliness, growth, and yield (Stapleton and Summers 2002, Greer et al. 2003) and providing greater benefit and cost effectiveness to the grower. Colored or reflecti ve mulches are most effective early in the crop cycle, before the developing plant canopy c overs the mulch. They reduce attack by soil pathogens and consequently reduce fruit rot, and fe rtilizer leaching (Cradock et al. 2002). Plants grown over reflective mulch produced significantly higher yields than those grown on bare ground (Stapleton and Summers 2002, Frank and Libur d 2005). However, disposal of synthetic mulches following crop termination can be problem atic and may interfere with farming practices such as cultivation. Furthermore, the effectivenes s of the mulches is lost over time through plant growth either by shading or coveri ng the reflective surface (Zitter 1977). Reduced Risk-Insecticides Reduced-risk pesticides are ne wer classes of compounds that pose a lower health risk to humans and environment. A reduced-risk insectic ide is defined as one that may reasonably be expected to accomplish one or more of the followi ng: 1) reduce pesticide risks to human health, 2) reduce pesticides risk s to non-target organisms, 3) reduce the potential for contamination of valued, environmental resources, or 4) broaden adoption of IPM or make it more effective. Imidacloprid is a reduced-risk insecticide that belongs to the neonicotinoids group of insecticides. Neonicotinoids act on the insect central nervous system as antagonists of the postsynaptic nicotinic acetylcholine recepto rs (Denholm and Nauen 2005). They show marked selectivity

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33 within the Class Insecta and present no hazard to mammals. They exhibit excellent contact and systemic activity and have become widely used for controlling sucki ng insect pests including whiteflies, aphids, planthoppers, and leaf hoppers (Palumbo et al. 2001, Liu 2004, Denholm and Nauen 2005). Soil-applied imidacloprid (Natwick et al. 1996) has been the primary insecticide treatment used for B tabaci control in vegetable, melon, and gr eenhouse production systems (Palumbo et al. 2001). In treated plants the compound and its metabolites are initially toxic to feeding adults, and act as anti-feedants. Establishment by immature whiteflies is significantly reduced because of suppressed egg deposition. Residual control of B. tabaci populations can vary from 1 to 10 weeks, depending on formulation, rate, depth of placement, soil type, application method, and cropping system (Palumbo et al. 2001). Due to its rapid uptake and systematic translocation within newly emerging plants, pr ophylactic applications of imid acloprid have been reported to reduce incidences of aphid-borne viruses (Pal umbo et al. 2001). However, unlike most other systemic insecticides, imidacloprid is relativ ely immobile in the soil and requires precise placement where root uptake can occur (Natwi ck et al. 1996). Imidacloprid can cause phytotoxicity to foliage on young plants depend ing on the application rates and timing.

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34 CHAPTER 3 TO INVESTIGATE THE EFFECT OF LIVING (BUCKWHEAT) AND REFLECTIVE MULCHES WITH AND WITHOU T A REDUCED-RISK INSECTICIDE ON WHITEFLIES, APHIDS AND NATURAL ENEMIES IN ZUCCHINI SQUASH Whiteflies and aphids (Hemiptera: Aphididae) are economic pests of fresh market zucchini squash ( Cucurbita pepo L.) affecting production in Florida. These pests cause damage to squash plants directly by sucking on thei r sap or indirectly by transmitt ing viruses. Aphids vector nonpersistently transmitted viruses, which are co nsidered a major limiting factor to cucurbit production in Florida (Purcifull et al. 1988, Wolfenbarger and Moore 1967). Before 1988, when the first economic damage associated with the pest was recorded on cucurbits the sweetpotato whitefly ( Bemisia tabaci Genn.) was not a major pest in Florida, (Schuster et al. 1991). Squash silverleaf disorder (SSL) is a phys iological disorder associated w ith the feeding of immatures of B biotype whiteflies that causes th e upper surface of the leaf to be silvered. The symptoms can be present on the fruit causing the color to look paler as in th e case of zucchi ni and yellow squash. Recently, whiteflies have been implicat ed in the transmission of new whitefly-borne virus diseases in squash (Adkins et al. 2007, Akad et al. submitted). Th is multi-faceted damage of whiteflies and aphids make them proba bly the most economic pests of squash. Control of whiteflies and aphids on squash has been based on the use of insecticides. However, this control cannot be relied on alone as the pests are prone to develop resistance. Additionally, the control of ins ect-transmitted viruses cannot rely on insecticidal tactics (Kring and Schuster 1992, Brown et al. 1993) especially in the case where the virus is transmitted nonpersistently. These viruses are transmitted before the aphid can obtain a lethal dosage to kill them (Stapleton and Summers 2002). Nevert heless, insecticides can be us ed to reduce the proliferation of viruses by preventing build-up on the host.

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35 Cultural control tactics are safer alternativ es and their use has shown some promising results in managing whiteflies and aphids in va rious crops. In particul ar, UV-reflective mulch repels aphids (Alderz and Ev erett 1968, Wolfenbarger and M oore 1968), whiteflies (Csizinszky et al. 1997, Summers and Staple ton 2002, Summers et al. 2004, Fr ank and Liburd 2005). Alate aphids and adult whiteflies are repelled by UV wavelengths reflec ted by the silver and aluminum pigment of the synthetic mulch. Conversely, re flective mulches are known to lose their reflectivity to dust or shading as the crop ca nopy grows and hence they may not be able to provide sufficient protection agai nst insect pest alone (Zitte r and Simons 1980, Smith et al. 2000). The addition of a reduced-risk insecticide may maintain or enhance their effectiveness against pests of zucchini squash. Living mulches are cheaper alternative to refl ective mulches. Living mulches have been shown to be effective in distr acting whiteflies and other pests fr om locating their host (Hooks et al. 1998, Frank and Liburd 2005, Hilje and Stansly 2007). These mulches are used to diversify habitats. Such diversified habitats have lower populations of herbivores when compared with monocultures (Root 1973, Andow et al. 1986, Kloen and Altieri 1990, Hooks et al. 1998, Costello and Altieri 1995), which ultimately lead to the delay of the onset of insect-transmitted viral diseases. In recent studies living mulches have been shown to reduce the damage inflicted by aphids and whiteflies when interplanted w ith zucchini squash (Hooks et al. 1998, Frank and Liburd 2005). Despite being shown to have some negativ e effects on whiteflies and aphids, living mulches have not been tested in combination w ith a reduced-risk insectic ide. It is not known therefore, if the addition of a reduced-risk insecticide will nega tively affect the natural enemies associated with the living mulches or produce sy nergistic effects in re ducing pest populations.

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36 The study was conducted with the aim of integr ating cultural and redu ced-risk insecticide tactics for pest management of whiteflies and ap hids in zucchini squa sh. The hypothesis of the study was that use of the insectic ide with the mulches could result in further reduction in pest populations and thus lead to economic gains. Specif ic objectives were to i nvestigate the effect of living mulch buckwheat, ( Fagopyrum esculentum Moench), and reflective mulch in combination with a reduced-risk insecticide (imidacloprid) to manage whiteflies and aphids in zucchini squash. The effects of these treatments on the natu ral enemies associated with squash were also evaluated. Materials and Methods Plot Preparation, Irrigation sy stem, and Experimental Design The experiment was conducted at the Plant Sc ience Research and Education Unit in Citra, Florida. Field plots measuring 10.36 m x 10.36 m were prepared, each containing four beds. Each plot was separated from the adjacent plots by 7.6 meters of bare soil on all sides and was kept weed free throughout the experiment. Planti ng beds were prepared with a 6-foot center wheel spacing tractor (2 wheel drive Model 6615, John Deere) and were fumigated with methyl bromide 80/20 formulation (80 % methyl bromide, 20% chloropicrin) at the rate of 283.5 kg/ha. Fumigation was done 2 weeks before planting squas h. The fumigant was injected into the soil and immediately the planting rows were covered with the respective synthetic mulch treatments. Drip irrigation lines (5/8 inch, 10 mm thickness) were placed in the center of the beds prior to covering with plastic mulch. Living mulch rows we re also fumigated and temporarily covered (2 wk) with synthetic mulch, to allow the methyl bromide to properly fumigate those beds. The plastic mulch was removed before planting buckwh eat seeds. Before planting squash, the living mulch; (buckwheat) was hand-seeded betw een the rows 21 and 18 days in 2005 and 2006 respectively. At the time of planting squash plants the buckwheat mulch was 20 cm high. There

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37 were two rows of living mulch on each side of the squash plants. Living mulch was planted approximately 23 cm adjacent to squash plants (F ig. 3a). In the case of synthetic mulches the entire 76 cm on top bed surfaces were covered with the mulch (Fig. 3b). Treatments were arranged in a randomized co mplete block design with four replications. The treatments evaluated consisted of: 1) reflec tive mulch (1.33 mm thick 66 inch wide) with the reduced-risk insectic ide (Admire 2F Bayer, Kansas Cit y, MO); 2) reflective mulch without Admire 2F; 3) living mulch w ith Admire 2F 4) living mulc h without Admire 2F; and 5) standard synthetic white mulch ( 1.33 mm, thick, 66 inch wide), whic h served as the control. The reduced risk-insecticide Admire 2F was applied 2 weeks after s quash planting through the drip lines at the rate of 1.684 L/ha on the appropriate treatments. Planti ng holes were cut in the center of each plastic mulch strip or on ground unmulche d beds, and two squash seeds, cv Wild Cat (Harris Moran Seed Company) were hand seeded per hole. Plant spacing was maintained at approximately 92 cm in the two years of the st udy with planting dates on 29 September in 2005 and 3 October in 2006. After germination, the mi ssing plants were replaced using already established seedlings from th e greenhouse. In 2006, the missing plants were not only from germination failure, but also from crow damage. Agronomic practices for squash followed th e standard production guide for squash in North Florida (Olson et al. 2005). However, no in secticides were app lied during the growing season except Admire 2F on the specific treat ments. A fungicide, azoxystrobin (Amistar; 80 WP); was sprayed as required against powdery mildew during early stages of the crop. Base fertilizer-dressing N-P-K (10-10-10 ) at the rate 624 kg per hectar e was applied to the soil at planting. For the first four weeks the squash cr op was top-dressed with nitrogen, potassium and phosphorous each at 0.7 kg weekly and thereafter increased to 0.9 kg at blossom. Weed

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38 management was done mechanically as require d. In 2006, reflective tape s and propane canister were used to keep the crow s away during the day time. Foliar Sampling Adult whiteflies and aphids were counted from the leaves in-situ by leaf-turn method. This was initiated 3 weeks after planting and carried out weekly until final harvest. Nine plants located on the outside rows of each plot were randomly selected. Insect pest counts were established by gently turning a leaf and counting the number white flies and aphids encountered. A total of nine leaves were counted in each pl ot giving a total of 36 l eaves per treatment. Nymphal whitefly population was determined from the nine selected leaves (discussed above), three from each stratum (upper, mid, lowe r). The leaves were excised and placed in a 1gallon self-sealing Ziploc bags and returned to the Small Fruit and Vegetable IPM Laboratory, UF, Gainesville, FL. In the laboratory, a 3.14-cm2 leaf disc was taken from each leaf using a cork borer and examined for whitefly immature stag es under a 40Xdissecting microscope (MEIJI EMZ, Meiji techno co. Ltd Tokyo Japan) (as adapted from Frank and Liburd 2005). The distribution of the whitefly immatures along the plant strata was also established. Trap Sampling Adult whiteflies were monitored with yellow sticky, Pherocon AM unbaited traps (YST), (Great Lakes IPM, Vest aburg, MI). Three traps were pl aced in each plot, one in the middle and the other two on a diagon al line at the two opposite side s of the plot. The traps were left in the field for a period of 24 h and collected and taken back to the Small Fruit and Vegetable IPM laboratory to establish counts. The first set of traps were placed into the field one week after germination and there af ter once every week for eight and six weeks in 2005 and 2006 respectively.

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39 Alate aphids were monitored using blue (PackerWare) and clear water pan-traps (Pioneer/ Tri-State Plastics Inc., Dickson, KY). F our pan traps were used per plot, two of each color, placed at the four corners of each plot within the interior rows. Each pan trap contained approximately 250 cm3 of 5% detergent solution (Colgate-Palmolive Co. New York, NY). Each trap was supported on a tomato cag e and trap height was adjusted accordingly to plant height. The traps were left in the field for one w eek and sampling was conducted for seven and six weeks in 2005 and 2006 respectively. In 2005, th e number of alate aphids trapped were established in the field, while in 2006 bowl contents were emp tied into individual vials and labeled accordingly. Vials were then transported to the laboratory for counting. Physiological Disorder Evaluation In 2006, symptoms of silverleaf on the squa sh were assessed on 10 plants within the interior rows. Squash with silverleaf symptoms were scored using an ar bitrary scale (Yokomi et al. 1990) where index 0 signified a healthy leaf with no sympto ms and index 5 leaves were completely silvered. Natural Enemy Counts Natural enemies were sampled using in-situ counts from the same zucchini plants as described above. Six leaves from six plants located on the outside row of each plot were randomly selected. The leaves were gently turned and the numbers of natural enemies encountered were recorded from them. Samp ling was initiated 3 weeks after planting and conducted every other week until the final harvest. Statistical Analysis Data from whitefly and aphid counts (foliar, whitefly immatures, and trap counts) were analyzed using repeated measures analysis (P ROC MIXED, SAS Institute 2003) to examine the interaction effect between treatment and time (s ampling weeks). Overall, least square means

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40 values were computed and means were compared to determine the effects of mulch treatments. The standard errors of means (S.E.M) were also calculated (PROC MEANS, SAS Institute 2003). Data from natural enemy counts, silverleaf sc ore index were subjecte d to an analysis of variance (ANOVA) using SAS GLM (SAS 9.1 versio n) and treatment means separated by least significant differences (LSD) test (SAS Institu te 2003). Where necessary, the data were logtransformed to meet assumptions for ANOVA. Comparisons of immature counts from the treatments were made based on the average upper, middle and lower leaf disc counts. Results Foliar Counts In 2005, buckwheat with Admire 2F was equally effective as reflective mulch with Admire 2F in controlling adult whiteflies per leaf ( F = 43.29; df = 4, 72; P < 0.0001) [Table 3-1]. Similarly, buckwheat alone was not significantly differe nt from reflective mulch alone. Addition of Admire 2F to buckwheat and reflective mulche s resulted in enhanced c ontrol of whiteflies per leaf compared with the other treatments. Conversely, plants gr own with white mulch (control) had significantly higher number of whiteflies per leaf than all the other treatments. There were significant ( F = 7.86; df = 20, 72; P < 0.0001) interaction effect s between treatments and sampling weeks. Treatment differences were observed in 5 out of the 6 weeks sampled. Apterous aphid counts per leaf indicated th at buckwheat with Admire 2F had similar numbers as those recorded in reflective mulch with Admire 2F. Both treatments resulted in significantly fewer numbers of a phids than all the other mulche s tested including white mulch (control) ( F = 10.42; df = 4, 72; P < 0.0001) [Table 3-1]. The addition of Admire 2F to the mulches reduced the number of apterous aphids per leaf but not alate aphids. There were significant ( F = 3.37; df = 20, 72; P < 0.0001) interaction effect s between treatment and

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41 sampling week. Of the six weeks sampled, treatment differences were observed in 3 out of the 6 weeks. Similarly, there were significant ( F = 2.40; df = 20, 72; P < 0.0036) interaction effects between treatment and time in alate aphids counted per leaf. Among the weeks sampled, treatment differences were only observed in 2 weeks of sampling, and during both dates reflective mulch with Admire 2F had the highest al ate aphids counted per l eaf. Overall, reflective mulch with Admire 2F recorded significantly hi gher number of alate aphids per leaf compared with all the other tr eatments except reflective mulc h alone (F = 5.13; df = 4, 72; P < 0.0011) [Table 3-1]. In 2006, fewer adult whiteflies per leaf we re recorded compared with 2005, though a similar trend was observed among the treatments (Table 3-2). Plant gr owing with living and reflective mulch treatments continue d significantly fewer of adult wh iteflies per leaf than those growing on white mulch ( F = 49.71; df = 4, 72; P < 0.0001) (T able 3-2). Buckwheat with Admire 2F had the fewest whitefl ies counted per leaf, but it was not significantly different from reflective with Admire 2F. The addition of Admi re 2F to the buckwheat treatment resulted in further reduction of whitef lies per leaf. During the study there were significant ( F = 3.63; df = 20, 72; P < 0.0001) interaction effects between treatment and sampli ng week, with significant treatment differences observed in all weeks. In the case of aphids per leaf, although there were significant ( F = 2.01; df = 20, 72; P < 0.0166) interaction effects between treatments and time the difference was only observed in one week of sampling. Both buckwheat and reflectiv e mulches with Admire 2F had significantly fewer apterous aphids than white mulch (control) ( F = 3.14; df = 4, 72; P < 0.0193) [Table 3-2]. Buckwheat with and without Admire 2F had signif icantly fewer of alate aphids than the white mulch which was similar to the reflective mulches. The addition of Admire 2F did not affect the

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42 number of alates per leaf when used with any of the mulches (buckwheat or reflective). Although there were no significant ( F = 0.86; df = 4, 72; P < 0.6330) interacti on effects between treatments and time, differences among the tr eatments were observed on the fifth week of sampling. On this date, white synthetic mulch re sulted in significantly higher number of alate aphids than all the other treatments. Whitefly Immature Counts Results of whitefly immature counts in the labor atory were consistent across the two years. Most of the whitefly nymphs were concentrated in the lower plant stratum with the least on the upper stratum (Figs. 3-2 & 3-3). In 2005, wh ite mulch had significantly higher number of immatures per 3.14-cm2 leaf disc compared with all the other treatments ( F = 13.91; df = 4, 72; P = 0.0001) [Table 3-3]. Treatments with Admire 2F contained the fewest number of whitefly immatures per leaf disc. Buckwheat alone was no t significantly different from reflective mulch alone. There were significant ( F = 2.91; df = 4, 72; P = 0.0005) interaction effects between treatment and time. In 2006, the number of whitefly immature s varied significantly among treatments ( F = 11.90; df = 4, 72; P < 0.0001) [Table 3-3]. No significant difference occurred among reflective mulch, reflective mulch with Admire 2F and buckw heat mulch with Admire 2F. The addition of Admire 2F to reflective mulch did not result in further significant reduction of immature whiteflies. Whitefly adult populations on plants gr owing within buckwheat in combination with Admire 2F resulted in lower whitefly imma tures than those in buckwheat mulch alone. Buckwheat mulch with Admire 2F was not signif icantly different from reflective with Admire 2F and reflective alone. White mulch resulted in significantly higher numb ers of immatures per leaf disc compared with all th e other treatments. The interacti on effects between treatment and

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43 time was significant ( F = 1.90; df = 20, 72; P < 0.0255), treatment differences were observed in 4 out of the 6 weeks sampled. Trap Counts Whiteflies Significantly ( P < 0.05) fewer adult whiteflies were captured on yellow sticky traps (YST) within reflective and buckwheat treatments with Admire 2F than those within the buckwheat alone and standard white (control) in 2005 ( F = 22.21; df = 4, 96; P < .0001) [Table 3-4]. Buckwheat alone was not significan tly different from the white mulch (control). Traps set within reflective with Admire 2F caught the least num ber of whiteflies. Ther e were significant ( F = 4.26; df = 28, 72; P < 0.0001) interaction effects between treatment and sampling week. Treatment differences were observed in 6 out of the 8 weeks sampled. In most of the sampling dates, white (control) resulted in the highe st number of whiteflies counted per trap. In 2006 however, YST within the buckwheat alone mulch contained significantly ( P < 0.05) fewer whiteflies than white mulch, which was higher than all the other treatments ( F = 27.66; df = 4, 72; P < .0001) [Table 3-4]. The addition of Ad mire 2F to buckwheat and reflective mulches did affect the number of whiteflies per trap, and these mulches resulted in significantly fewer whiteflies compared with the mulche s tested alone. There were significant ( F = 3.79; df = 20, 72; P < 0.0001) interaction effects between treatment and sampling week. Of the six weeks sampled treatment differences were observed in weeks 1, 4, 5, and 6. Aphids During the 2-yr study, the color of the pan traps (clear ve rsus blue) did not have a significant ( t = -0.86, Pr > | t | = 0.3908) in 2005 and t = 0.03, Pr > | t | = 0.9736 in 2006 effect on trap catches and hence means of pooled alate aphid counts are reported here. In 2005, pan traps within reflective mulch with Admire 2F caught si gnificantly higher numbers of alate aphids than

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44 those in the control, which di d not differ significantly with those in reflective mulch and buckwheat treatments ( F = 2.51; df = 4, 268; P = 0.0392) [Table 3-5]. In 2006, pan traps within the white synthetic mu lch caught significantly more alate aphids than all other treatments ( F = 9.54; df = 4, 84; P < 0.0001) [Table 35]. The addition of Admire 2F did not affect the number of alate aphids captured. Surprisingly, re flective alone had the fewest number of alate aphids trapped in the pan traps of all the treatments evaluated including reflective with Admire 2F. Significant ( F = 3.63; df = 4, 84; P < 0.0001) interaction effects between treatments and sampling dates were observed. Among the seven weeks of sampling, treatment differences were observed only in week 2 and 4. Physiological Disorders Silverleaf symptoms differed si gnificantly among the treatments ( F = 44.60; df = 4, 34; P > 0.001). Plants growing within white mulch had almo st the entire upper leaf surface silvered as indicated by the high index sc ore (Fig. 3. 4). Treatments w ith Admire 2F (reflective and buckwheat) had significantly lower index scor es than all other treatments. There was no difference between reflective mulch and buckwheat treatment alone. Natural Enemies The major families recorded in the study were Syrphiridae, Coccinelidae, Chrysopidae, and Apidae and spiders. In 2005, significantly more natural enemies were r ecorded on squash in the buckwheat treatment with Admire 2F than any other treatment except buckwheat alone ( F = 3.43; df = 4,353; P = 0.009) [Table 3-6]. The treatment re flective mulch with Admire 2F had the fewest natural enemies but it was not significantl y different from reflective mulch alone. In 2006, there were no significant differences in the number of natural enemies among the treatments ( F = 0.69; df = 4, 347; P < .6006) [Table 3-6].

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45 Discussion The response of whiteflies on zucchini interplant ed with living mulch was more consistent than that of alate aphids in the two-yr study. My study shows that living and reflective mulches with and without Admire 2F were able to prov ide significant protection against whiteflies as compared to the control. Crops grown within these mulches were likel y protected from pest infestation due to the negative effect that these mulches have on the orientation of the insect to the plant. Living mulch, (buckwheat ) increases the vegetation divers ity that is known to decrease the host apparency and hence fewer numbers of wh iteflies and aphids landed on squash. On the other hand, UV reflective mulch is known to repe l whiteflies and hence prevent them from landing on the host plants (Antignus 2000). This was the case in my study where significantly fewer whiteflies were recorded in reflective mu lch. The current study provides another instance where living mulch was demonstrated to control wh iteflies. Previous researches have shown that the inclusion of living mulches with zucchin i plants (Frank and Li burd 2005), and tomatoes (Hilje and Stansly 2007) successfully reduced whiteflies populations in the main crop. Whitefly populations were higher in 2005 than in 2006. In both years, foliar counts revealed that buckwheat was equally as effectiv e as reflective mulch when tested alone or in combination with the reduced-ris k insecticide Admire 2F. In 2005, however YST indicated that reflective mulch alone was superior to living mulc h in controlling whitef lies. The situation was reversed in 2006 when living mulch alone was supe rior to reflective mulch alone. The reason for these differences are not clear but could be relate d to stage of maturity of living mulch or other external factors. Although previous studies have reported reduc tion of whiteflies when using living mulch, none of the studies evaluated the mulch in comb ination with a reduced-r isk insecticide. I found that addition of Admire 2F to the mulches furt her reduced whitefly populati ons. More research is

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46 needed to fully investigate how these two strategi es (living mulch with Admire 2F or reflective mulch with Admire 2F can be in tegrated in zucchini production. Likewise, the number of nymphs per leaf disc was higher in 2005 th an in 2006. Our results indicated that treatments with Admire 2F provi ded significantly greater protection from whitefly immatures than treatments without. The reduction in number of immature whiteflies per leaf is important since they are responsible for inducing silverleaf symptoms. It was evident that most of the whitefly immatures were concentrated in the lower strata of the zucchini plants. This area provides more nitrogen and fewer trichomes (Bentz et al. 1995). The immature whiteflies of B biotype are associated with a phys iological disorder called squash silverleaf symp tom (SSL). The occurrence of SSL was recorded in 2006. Silverleaf symptoms we re more severe on the white mulch, which had the highest whitefly population compared with all th e other treatments. A positive correlation between the number of whitefly immatures and SSL symptoms has been reported before by Costa et al. (1993). In this study, the mulch treatments had varyi ng effects on alate aphids. These aphids are important in the transmission of non-persistent vi ruses such as the mosaic viruses. Previous studies have reported that the UV-reflective mulc h was able to confuse and repel alate aphids from landing on the plants (Wolfenbarger & Moore 1968, Brown et al 1993, Summer et al. 2004). Our results in 2006 were in agreement with previous research where the reflective mulch treatments afforded the best protection against al ate aphids. In this study it is a reported that addition of Admire 2F did not enhance reduction of alate aphids as revealed by the pan traps and foliar counts. In contrast, Admire 2F provided a significant amount of control of apterous aphids in 2005, but in 2006 the reduction was not as drama tic, perhaps because the populations were not as high. Furthermore, Admire 2F is a systemic in secticide and could be more lethal to apterous

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47 aphids that are sedentary as opposed to the migr ating alatae. It is not surprising that mulches without the reduced-risk insec ticide, including the white mu lch (control), did not differ significantly ( P > 0.05) from each other in both years. The color of pan traps did not affect the number of aphids caught. This is in harmony with the findings of Frank and Liburd (2005). In Florida, white mulch is used alone or on top of black in zucch ini production during the warm seasons to keep the soil cool (Liburd and Fra nk 2007, Liburd et al. in press ). In our study, we report that white polyethylen e mulch resulted in consisten tly high numbers of whiteflies (adults and nymphs) and alate aphi ds in at least one season. Earl ier studies have also reported that white mulch is associated with high number s of immatures and adul t whiteflies (Csizinszky et al. 1997). In addition, a high number of aphids have been tr apped out plants with white polyethylene mulch compared with bare ground or / and other mulches (Alderz and Everett 1968, Zitter 1977, Zitter and Simons 1980, Frank and Liburd 2005). The counts of natural enemies associated w ith whiteflies and aphids were inconsistent throughout the two field seasons. In 2005, more natural enemies were recorded in plants in plots treated with living mulch (buckw heat) than those with reflec tive mulch with Admire 2F. However, in 2006 no significant differences among the treatments were recorded. Admire 2F did not have adverse effects on natural enemies a ssociated with the mulches as indicated by nonsignificant differences between treatments with and without Admire 2F. The natural enemies counted on the leaves were mainly predators and consisted of Syrphi ridae, Coccinelidae, Chrysopidae and Apidae and spiders. Spiders made up the bulk (39%) of the recordings followed by coleopterans (29%). Buckwheat is an annual plan t whose flowers produce nectar that attracts a high percentage of beneficial insects includ ing pollinators. When used as a living mulch, buckwheat supports a high number of natural enem ies, which could contribute to the reduction of

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48 whitefly and aphid populations. It is known that certain species of natural enemies can play a role in regulating whitefly popul ations (Gerling 1990). In the cu rrent study, the natural enemy populations were very lo w as revealed by the in-situ counts. Therefore othe r factors contributed to the reduction in the whitefly population. There are two theories that can be used to explain the reduction of pests in diversified plantings (Root 1973). The first one is resource con centration that theorizes that pests will opt to remain and establish in pure stands as opposed to mixed plantings, which may be limited in preferred resources (food, shelte r). The second, is the natural enemy hypothesis that assumes that diverse habitats have divers e insects and hence predators and parasitoids could contribute to pest reduction. Because no consistent effects on natural enemies were observed I can conclude that resource concentration may have contributed to pest reduc tion in my study. It can also be speculated that population reduction of whiteflies in the squash planted with the living mulch was related to the host plant quality. In the st udy, zucchini plants growi ng with buckwheat were noticeably smaller as indicated by the plant size s (CHAPTER 5). Quality re duction could be as a result of increased competition for resources su ch as nutrients, light, and water leading to a higher rate of herbivore emigration and /o r lower population growth. Additionally, the differential in immigration and reproduction of he rbivores could also be used to explain the differences in herbivore response in crop monocu ltures and crop mixtures (Andow et al. 1986). In the current study, it can be concluded that addition of Admire 2F enhanced control of whiteflies and to some extent apte rous aphids and not alate aphids Living mulch with Admire 2F and reflective mulch with Admire 2F gave pr otection equal against whiteflies and aphids. Admire 2F did not affect bene ficial insects associated with the mulches. The economics of

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49 incorporating a reduced-risk insecticide with a li ving or reflective mulch will have to be studied before recommendations can be made.

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50 Table 3-1. Effect of living and reflective mulches alone or in combination with Admire 2F on the number of adult whiteflies and aphids pe r zucchini squash leaf, Citra, FL (2005) [Foliar counts] Mean SEM numbers per leaf Whiteflies (adult) a Aphids (apterous) b Aphid (alate) c Reflective + Admire 2F 10.38 2.02 c 0.56 0.21 b 0.92 0.29 a Reflective 15.22 2.22 b 3.48 0.80 a 0.69 0.21 ba Buckwheat 17.09 2.32 b 3.19 1.02 a 0.31 0.06 c Buckwheat + Admire 2F 10.40 1.73 c 0.44 0.12 b 0.37 0.06 bc Control 38.27 6.20 a 3.07 0.69 a 0.40 0.10 bc Means followed by the same letters are not significantly different ( P = 0.05 according to least square means test following repe ated measures analysis, LS). a F = 43.29; df = 4, 72; P < 0.0001 b F = 10.42; df = 4, 72; P < 0.0001 c F = 5.13; df = 4, 72; P < 0.0011

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51 Table 3-2. Effect of living and reflective mulches alone or in combination with Admire 2F on the number of adult whiteflies and aphids pe r zucchini squash leaf, Citra, FL (2006) [Foliar counts] Mean SEM numbers per leaf Whiteflies (adult)a Aphids (apterous)b Aphid (alate)c Reflective + Admire 2F 5.13 0.71 bc 0.04 0.02 b 0.25 0.06 ab Reflective 7.75 0.96 b 0.28 0.12 a 0.17 0.05 ab Buckwheat 8.27 0.97 b 0.21 0.09 ab 0.16 0.04 b Buckwheat + Admire 2F 3.23 0.42 c 0.01 0.01 b 0.14 0.04 b Control 16.03 2.04 a 0.31 0.11 a 0.29 0.08 a Whitefly data transformed (log 10) before analysis, means are pres ented in the original counts. Means followed by the same letter ar e not significantly different ( P = 0.05 according to least square means test (LS) following repeated measures analysis). a F = 26.67; df = 4, 72; P < 0.0001 b F = 3.14; df = 4, 72; P < 0.0193 c F = 0.86; df = 4, 72; P < 0.6330

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52 Table 3-3. Effect of living and reflective mulches alone or in combination with Admire 2F on the number of whitefly immatures per treatment in zucchini Citra, FL Mean SEM whitefly immature per leaf disc (3.14 cm2) 2005a 2006b Reflective + Admire 2F 0.33 0.10 c 0.30 0.09 c Reflective 1.86 0.32 b 0.98 0.26 bc Buckwheat 2.11 0.46 b 1.77 0.45 b Buckwheat + Admire 2F 0.42 0.15 c 0.44 0.20 c White (control) 3.49 0.77 a 2.83 0.64 a Means followed by the same letter ar e not significantly different ( P = 0.05 according to least square means test following repe ated measures analysis, LS). (LS) a F = 13.91; df = 4,72; P < 0.0001 b F = 11.90; df = 4, 72; P < 0.0001

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53 Table 3-4. Effect of living and reflective mulches alone or in combination with Admire 2F on the number of whiteflies caught per ye llow sticky trap in Citra, FL Mean SEM counts per YST 2005a 2006b Reflective + Admire 2F 10.74 1.67 c 13.22 1.54 d Reflective 20.80 3.81 b 27.82 4.41 b Buckwheat 31.92 6.36 a 18.90 3.30 c Buckwheat + Admire 2F 17.33 3.11 b 12.01 1.29 d White (control) 51.33 11.32 a 37.72 4.71 a YST data (2005) whitefly data transformed (log 10) before analysis, means are presented in the original counts. Means followed by the same le tter are not significantly different (P = 0.05 according to least square means test following repeated measures analysis, LS). a F = 22.21; df = 4, 96; P < 0.0001 b F = 27.66; df = 4, 72; P < 0.0001

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54 Table 3-5. Effect of living and reflective mulches alone or in combination with Admire 2F on the number of alate aphids trapped per pan-trap in Citra, FL Mean SEM counts per pan trap 2005a 2006b Reflective + Admire 2F 3.07 0.43 a 2.69 0.45 b Reflective 2.36 0.36 ab 1.94 0.32 c Buckwheat 2.14 0.27 ab 2.91 0.45 b Buckwheat + Admire 2F 2.21 0.28 ab 3.19 0.57 b White (control) 1.97 0.28 b 3.83 0.53 a Means followed by the same letter ar e not significantly different ( P = 0.05 according to least square means test following repe ated measures analysis, LS). a F = 2.51; df = 4, 268; P < 0.0392 b F = 9.54; df = 4, 84; P < 0.0001

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55 Table 3-6. Effect of living and reflective mulc h alone or in combination with Admire 2F on number of natural enemies per treatment in zucchini Citra, FL Mean natural enemies per treatment SEM 2005a 2006b Reflective + Admire 2F 0.18 0.06 c 0.21 0.07 Reflective 0.26 0.06 bc 0.15 0.04 Buckwheat 0.40 0.08 ab 0.29 0.09 Buckwheat + Admire 2F 0.49 0.08 a 0.23 0.06 White (control) 0.28 0.06 bc 0.26 0.06 Means followed by the same letter are not significantly different P = 0.05 (LSD) a F = 3.43; df = 4, 353; P < 0.009 b F = 0.69; df = 4, 347; P < 0.6006

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56 Figure 3-1. (A) Zucchini squash growing with living mulch (B) Zucchini squash growing on UV-reflective mulch A Living mulch, buckwheat B UV-reflective mulch

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57 0 2 4 6 8 10 12 14 Buckwheat + Admire 2F BuckwheatReflective + Admire 2F ReflectiveControlTreatmentNo. of whitefly immatures per leaf disc (Plant stratum) top mid lower Figure 3-2. Distribution of immature whiteflies on different plant strata of zucchini in Citra, FL (2005)

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58 0 1 2 3 4 5 6 7 8 9 Reflective + Admire 2F ReflectiveBuckwheatBuckwheat + Admire 2F ControlTreamentNo. of whitefly immatures per leaf disc (Plant stratum) top mid lower Figure 3-3. Distribution of immature whiteflies on different plant stra ta of zucchini in Citra, FL (2006)

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59 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Reflective + Admire 2F ReflectiveBuckwheatBuckwheat + Admire 2F WhiteTreatmentSilverleaf Symptom Scorea b b c c Figure 3-4. Effect of living and reflective mulche s alone or in combination with Admire 2F on silverleaf symptoms presented by the score i ndices per treatment in zucchini Citra, FL (2006).

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60 CHAPTER 4 EFFECTS OF SYNTHETIC AND LIVING MULCH WITH AND WITHOUT A REDUCEDRISK INSECTICIDE TO CONTROL INSE CT-TRANSMITTED VIRAL DISEASES OF ZUCCHINI SQUASH: A NEW WHITEFLY-T RANSMITTED VIRU S IS REPORTED Virus diseases have a great impact on cucurb it vegetable production in the United States including Florida (Purcifull et al. 1998, Gaba et al. 2004, Damicone et al. 2007). Unlike most of the other diseases in squash virus rely on inse ct vectors for their sp read from one plant to another. Aphids (Hemiptera: Aphididae) and whiteflies are among the most important vectors transmitting viruses to zucchini squash, Cucurbita pepo L. (Nault 1997, Ng and Perry 2004). Aphids are known to transmit potyvi ruses and cucumoviruses including Papaya ring spot viruswatermelon strain (PRSV-W), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV). These viruses are tr ansmitted in a non-persistent manner, which is characterized by the short time needed for virus acquisition and transmission to a healthy plant. Similarly, the viruses have a s hort retention time in the aphids. Whiteflies (Bbiotype, Bemisia tabaci Genn.) also cause damage through th e transmission of plant viruses. There are several whitefly-transmitted viru ses reported to infect squash, including Cucurbit leaf crumple virus (CuLCrV) also known as Cucurbit leaf curl virus (CuLCV), Squash leaf curl virus (Brown et al. 2002), Squash vein yellowing virus (SqVYV) (Adkins et al. 2007) and Cucurbit yellow stunting disorder virus (CYSDV) (Kao et al. 2000). Among these diseases, only CuLCrV and SqVYV have been recorded in Florida. Cucurbit leaf crumple virus is a begomovirus, which is transmitted in a persistent, circulative manner by whiteflies. Pesticides have been the primary tactic fo r managing whiteflies and aphids. This method has limitations in that insecticides have not su fficiently controlled virus transmission, especially when the whitefly population is high. It is even mo re problematic in case of aphid vectors where the virus is transmitted before the insecticide ki lls them (Brown et al. 1993, Kring and Schuster

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61 1992, Fereres 2000, Summers et al. 2004). In add ition, insecticide resistance of whiteflies (Dittrich et al. 1990) and aphids (Wang et al. 2002) to selected chemical classes has further restricted their use. Nonetheless, insecticides ca n be used to decrease the spread of the virus by reducing the vector population at the primary and secondary s ource (Brown et al. 1993). In addition, several non-pesticidal me thods, including cultural control practices that are intended for reducing the vector population and hence interrupting their epidemio logical cycle. In this light, several control methods have been evaluated fo r the control of viral diseases in cucurbits including: border crops, intercrops, mineral o ils (Zitter and Simons 1980, Damicone et al. 2007, Fereres 2000), living mulches (Hooks et al. 199 8, Frank and Liburd 2005) floating row covers (Webb and Linda 1992), and reflective mulches (Alderz and Everett 196 8, Wolfenbarger and Moore 1968, Greenough et al. 1990, Kring and Schuster 1992, Stapleton and Summer 2002, Summers et al. 2004, Frank and Liburd 2005). Synthetic UV-reflective mulch has been reported to successfully protect various vegetable crops against insect pests and hence reduce th e incidence of viral diseases they transmit (Csizinszky et al. 1997, Reitz et al. 2003, Stapleton and Su mmers 2002, Summers et al. 2004). Reflective mulches reduce whitefly and alate aphi d populations because they reflect short-wave UV light, which repels incoming in sects, thus preventing them fr om alighting on plants (Zitter and Simons 1980). The crop is thus protected ag ainst aphids and white flies during the early growing period and consequently could delay the onset of insect-v ectored viruses. Living mulch is a cost effective alternative to reflective mulches (Hilje et al. 2001). These mulches have been shown to be effective in hi ndering whiteflies and ot her pests from locating their hosts and consequently transmit viruses (Hooks et al. 1998, Frank and Liburd 2005, Hilje and Stansly 2007). Hilje and Stan sly (2007) reduced the number of adult whiteflies and the

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62 incidence of Tomato yellow mottle virus (ToYMoV) in tomatoes in Costa Rica. The mulches have also been used to diversify habitats in zu cchini plantings and hence influence the ability of herbivores to recognize their host, resulting in reduced spread of the viruses they vector (Hooks et al. 1998, Liburd and Frank 2007) Furthermore, the mulches provide food resources (honey, pollen) and shelter for natural enemies th at contribute to pest reduction (Root 1973). Neonicotinoid insecticides, such as imidacl oprid (Admire 2F) [Bayer Cropscience US], are systemic in plants when applied as soil drenches and can assist in managing whiteflies (Palumbo et al. 2001). However, frequent applic ation of neonicotinoids and high populations of whiteflies can increase selection pressure, whic h may eventually reduce their efficacy (Liburd and Frank 2007). Previous studies have shown that buckwheat ( Fagopyrum esculentum Moench) used as a living mulch has potential to reduce insect pests a nd spread of viruses in zucchini crop (Hooks et al. 1998, Frank and Liburd 2005). The goal of my rese arch was to develop su stainable integrated pest management strategies for whiteflies and ap hids and the viruses they vector by integrating cultural control and reduced-risk pesticides tactics. Specifically, I wanted to determine if reduced pest populations in mulches in combination with Admire 2F (imidacloprid) could offer the added advantage of suppression of insect-transmitted viru ses. This study evaluated the effect of silver reflective mulch and living mulch alone or in co mbination with Admire 2F (imidacloprid) to reduce whitefly and aphid incidence and spread of viral diseases on zucchini squash in Florida. Materials and Methods Field Plot Preparation a nd Experimental Design The experiment was conducted at the Universi ty of Florida Plant Science Research and Education Unit in Citra, Florida. Field plot s measured 10.4 m x 10.4 m. Zucchini squash, seeds variety Wild Cat (Harris Moran), were plante d on the four beds of each plot. Treatments

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63 evaluated in this experiment included 1) reflective mulch with the reduced-risk insecticide; imidacloprid (Admire 2F Bayer, Kansas City, MO), 2) reflective mulch without Admire 2F, 3) living mulch with Admire 2F, 4) living mulch without Admire 2F, and 5) standard synthetic white mulch (control). Before planting squash the living mulch, buckwheat was hand seeded between the rows 21 and 18 days in 2005 and 2006, respectively. The redu ced risk-insecticide imidacloprid [Admire 2F] was applied 2 weeks af ter squash planting through the drip lines at the rate of 1.684 liters per hectare. The treatments were arranged in randomized complete block design with four replications. Agronomic practices followed the standard production guide for squash in North Florida (Olson et al. 2005). Howe ver, no insecticides were applied during the growing season except Admire 2F on the specif ied treatments. The fungicide azoxystrobin (Amistar; 80 WP), was sprayed as required against powdery mildew in the early stages of the crop. Virus Screening In 2005, plants were visually observed for vi rus symptoms weekly starting from the midgrowing season until the last week of the experiment. Two weeks before the end of the experiment, I excised a young leaf (third leaf from the tip) from six plants per plot, regardless of whether or not they had symptoms and transported the leaf to the laboratory. Leaves were stored at 4 oC overnight and assayed using an enzyme-li nked immunosorbent assay (ELISA) [Clark and Adams 1977) for Papaya ring spot virus-Watermelon strain (PRSV-W), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV). Approximately 0.2 g of each leaf sample was gr ounded in phosphate buffered saline (PBST) at a 1:20 dilution using a hand-held macerator to releas e the virus particles into the suspension. After 24 h at 3-4 oC, 100 L of each sample was added to duplicate wells in a 96-well plate, (Nunc, Denmark) that had previously been coated w ith specific antibodies at 1 g/ml. Each plate

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64 included a positive control and five negative c ontrols (healthy squash). The plates were incubated overnight at 4 C. After 24 h the plat es were emptied and quickly rinsed and blotted once before washing with an ELX 50 Auto St rip Washer (Bio-Tek, Winooski, VT) using 8 washes of PBST without soaking. Enzyme conj ugates (ECB) were prepared by diluting specific antibodies in buffer (Agdia Inc.) using predetermined dilution factor s. Plates were covered with Parafilm before being incubated at 30 C fo r 4 h in a moist chamber. After incubation and washing (as above), 100 L of substrate (10% diethanolamine adjusted to pH 9.8 + phosphatase substrate) (Sigma, St. Louis, MO) was added to each well. The color change was visually assessed (yellow, positive and clea r, negative) before measuring the absorbance of the samples at 450 nm using an EL 800 Universal Micr oplate reader (Bio-Tek, Winooski, VT). In 2006, v isual observations of viral symptoms and incidences were recorded and monitored in the field. Viral symptoms includi ng mosaic, mottling, leaf crumpling, and distortion (fig. 4-1) were noted on squash plants growi ng within white synthetic mulch treatments. The symptomatic leaves were excised from the plants and transported back to the laboratory. Each sample was assayed using ELISA for eight vi ral diseases including ZYMV, WMV, PRSV-W CMV, Tobacco streak virus (TSV), Watermelon leaf mottle virus (WLMV), Papaya ring spot virus-type T (PRSV-Tigre), S quash mosaic virus (SqMV) and Tomato spotted wilt virus (TSWV). Procedures followed were as described above except th at TSV and TSWV were tested with reagent kits (Agdia Inc.) a nd the manufacturers directions we re followed. All other antisera were obtained from D. Purcif ull, Unversity of Florida. PCR Analysis Twenty leaf samples were collected from the field (as earlier described) from symptomatic squash plants and tested using polymeras e chain reaction (PCR) for the presence of begomoviruses. Primers PAR1c496/ PAL1v1978, whic h amplifies a region of the begomoviruses

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65 A component, and PBL1v2040/PCRc154, which amplifies a B component of the bipartite genomes were used (Akad et al. submitted). The amplified sequences were then compared with the known genome of begomoviruses. The anal ysis was conducted by the Plant Pathology Laboratory of J. E. Polston, Depa rtment of Plant Pathology, Universi ty of Florida, Gainesville. Greenhouse Screening In order to determine if whiteflies were cap able of transmitting symptoms noted in the field to healthy squash plants, a greenhouse st udy was initiated. Symptomatic leaves were collected from the field, transported back to the Vegetable Entomology Laboratory. Leaves were cut using a razor blade under water before placing each leaf in a 400ml beaker with water. Each leaf in the beaker was placed each cage. Whitefl ies were aspirated from a virus free colony which was maintained at the Plant pathology la boratory and introduced into the cages. The whiteflies were left to feed on the leaves for 24 hr before introducing approximately two-week old squash plants (variety Prelude II) in each cage The whiteflies were left to feed on the leaves and the visual symptoms of the virus recorded. Data Analysis Data from virus incidence were subjected to an analysis of vari ance (ANOVA) using SAS GLM (SAS 9.1 version) and treatment means sepa rated by least significant differences (LSD) test (SAS Institute 2003). Results In 2005, there were no symptomatic plants obs erved in the field. Similarly, the plants tested negative for ZYMV, WMV, PRSV-W, and CMV viruses. In 2006, however, symptomatic plants were observed in November. Zucchini le aves were observed showing mottling, curling and crumpling symptoms (Figure 5-1). These vira l symptoms were significantly more severe on squash growing on white synthetic mulch (control ), where they were first observed, than other

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66 treatments evaluated (( F = 9.96; df = 4, 15; P < 0 .0004) [Figure 5-2]. The symptoms later spread to other plots with time. Cumulative disease incidence among the plan ts growing under buckwhe at and reflective mulch treatments was significantly lower than those under the white mulch treatment (Figure 52). Addition of Admire 2F to reflective mulch tr eatments resulted in a significant reduction in the number of plants showing virus symptoms, which was not significantly different from buckwheat mulch with Admire 2F. Greenhouse Results Our greenhouse test indicated that whiteflies were able to transmit the virus from infected field samples to healthy plants. Plants began to show virus symptoms after approximately 10 days. After 14 days all the plants in the cages showed the viral symptoms and PCR analysis was positive for a begomovirus. PCR Analysis Results The samples submitted for the PCR assays produced PCR products using the degenerate primers PAR1c496/ PAL1v1978 and PBL1v2040/P CRc154. When sequences were submitted for a Basic Local Alignment Search Tool (BLAST) search the results revealed over 95% sequence identity with Cucurbit leaf crumple virus (CuLCrV). (Akad et al. submitted ). Discussion In this study, there was no incidence of aphid-transmitted virus diseases. Alate aphid populations were also low during the two field studies (CHAPTER 3) that are significant in transmission of the viruses. However, this low population could not be di rectly related to the absence of the viruses. Furtherm ore, it is reported th at even a small population of alate aphids can be sufficient to spread viruses especially non-persistently transmitted viruses (Fereres 2000).

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67 The occurrence of viral diseases may vary from year to year in Florid a (Mossler and Nesheim 2001). The use of reflective or living mulches re duced the incidence of CuLCrV, which is persistently transmitted by whiteflies. The lowest incidence of virus-infected plants was observed in the treatments that combined reflective or living mulch with Admi re 2F. This positively correlated (r = 0.96) with the numbers of whiteflie s recorded in those tr eatment plots. It was noted that mulches (reflective and living mu lch with Admire 2F) had fewer numbers of whiteflies (CHAPTER 3) and conse quently resulted in low incide nce of CuLCrV. The standard white mulch treatment (control) had the highest incidence of CuLCrV. Th is mulch had earlier reported the highest numbers of whiteflies (a dults and immatures) throughout the study. Cucurbit leaf crumple virus is transmitted by whiteflies only, and a reduction in whitefly numbers in the two treatments (reflective and living mulch) (CHAP TER 3) lead to reduction of infected plants. There is therefore an added advantage of combin ing reflective or buckwheat mulch with Admire 2F as seen in our field trial. It has been suggest ed that management of viruses is achieved better with a combination of two or more control stra tegies. Jones (2001) repor ted some benefits of combining different tactics including cultural, ch emical, and biological. These tactics all have different modes of action that act together re sulting in enhanced dis ease suppression than any method used alone. Although living and reflective mulches are kno wn to interfere with the insects host recognition activity, their actual modes of actions are differe nt. Reflective mulch repels whiteflies, interfering with th eir orientation (Zitter and Simons 1980, Csizinszky et al. 1997). The effectiveness of UV reflective mulch in reduci ng the incidence of CuLCrV is attributed to its ability to repel whiteflies, which would be findi ng their way to the host plant. Alternatively,

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68 living mulches are known to reduce the contrast between the ground and the host plants which apparently guide the whiteflies and other insects to the host (Cradock et al. 2002). Cucurbit leaf crumple virus (CuLCrV) has been reported in Arizona, Texas, California and Northern Mexico (Wiebe 2003), but had not been found in Florida before winter 2006. This spring (2007), CuLCrV was recorded in southw est Florida in watermelon (S.E. Webb, Pers. Comm.). Cucurbit leaf crumple virus is transmitted by whiteflies in a persistent manner and has a wide host range within the family Cucurbitace ae, infecting most of the domestically grown cucurbits. It has also been reported to infect beans (Brown et al. 2002). Symptoms on the leaves are very noticeable. The virus can also affect the fruit of some cucurbits. The disease may have been accidentally introduced into Florida thr ough infected watermelon seedlings from another state (Akad et al. submitted ). It is known that appearance an d distribution of the new geminivirus diseases are associated with the coming of biot ype B and its population increase in the state (Polston and Anderson, 1997), as well as evoluti on of variants of the viruses (Varma and Malathi, 2003). The probable ge ographic origin of CuLCrV is southwestern USA and northern Mexico (Brown et al. 2002). Our study suggests that the use of living or reflective mulch al one or in combination with imidacloprid (Admire 2F) can be used to reduce whitefly populati ons and reduce the incidence of Cucurbit leaf crumple virus. There are benefits to be carried by combining a living or reflective mulches with a reduced-risk insecticide.

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69 0 2 4 6 8 10 12 14 16 Reflective + Admire 2F ReflectiveBuckwheat + Admire 2F BuckwheatControl TreatmentVirus Incidencea b c c c Figure 4-1. Effects of mulches on Cucurbit leaf crumple virus on zucchini squash plants grown with either living mulch or reflective ground covers as comp ared with white synthetic mulch in Citra, FL (2006). Virus incidence = number of plants per plot with virus present. Values with the same letter do not differ ( P < 0.05) according to LSD test.

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70 Figure 4-2. Cucurbit leaf crumple virus symptoms recorded on zucchini squash plants in Citra, Florida (2006) Squash Silverleaf symptoms on the zucchini leaves

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71 CHAPTER 5 IMPACT OF SYNTHETIC AND LIVING MU LCH WITH AND WITHOUT A REDUCEDRISK INSECTICIDE ON PLANT GROWTH AND MARKETABLE YIELDS OF ZUCCHINI SQUASH Consumption of zucchini squash, Cucurbita pepo L. has increased due to preferential use of the squash as a salad and a cooked vegetabl e compared to other summer squashes (Stephen 2003). Zucchini squash, is a high value vegetable crop in Florida and the state is ranked second nationally after Georgia in the production of fres h market squash. In th e 2006 field-seasons, a total of 4249 ha of cucurbits was planted in Florida and 98% of the squash produced were harvested with an exceeding value of appr oximately 38.76 million USD (NASS-2006). Squash is one of the crops in Florida that is exported every month of th e year (Mossler and Nesheim 2001), thus providing a continuous income to the state. Almost all the squash is produced for fresh market with summer production mainly for loca l markets and fall production for export. In Florida, damage due to pest infestation is probably one of the major problems affecting the squash industry. In this regard, it is important to use appropriate and effective control strategies that are sustainable and will improve plant productivity. The use of mulches on raised beds is now a common practice in some vegetable production systems in Florida. Mulches have several advantages including controlling weeds, regulating soil temperatures, retaining moisture, and increasing crop yields (Olson 2005). In addition, some of the mulches have been shown to have negative e ffects on pests, thus reducing their incidences. In particular, reflective mulch has received increased attention in the production of various crops. Previous research has shown increased yields of various crops grown on reflective mulch i.e. strawberries (Rhainds et al. 2001), cantaloupe and cucumber (S ummers and Stapleton 2002), and zucchini squash (Summers et al. 2004, Fra nk and Liburd 2005). Reflective mulch reflects

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72 sunlight back to the crop canopy increasing the amount of photosynthesis available to contribute to plant growth and vigor. Living mulches are a cheaper alternative to reflective mulch. These mulches are interplanted with the main crop for various bene fits such as improving soil fertility and pest suppressing (Liburd et al. in press ). Previous research investiga ting the use of living mulches in various crops has reported mixed results with resp ect to marketable yields. Hooks et al. (1998) recorded increased yield in zu cchini squash in Hawaii. Sim ilarly, Hilje and Stansly (2007) reported increased yield in tomatoes when inte rplanted with living mu lches. Alternatively, Andow et al. (1986) saw decrease in yield in cabbage interpla nted with living mulches as compared with bare ground between plants. Several factors must be considered when gr owing living mulches with a main crop. For instance, it is important to select a living mu lch whose growth pattern has least effect on the main crop. Frank (2004) reported that buckwheat, Fagopyrum esculentum Moench was superior to white clover; Trifolium repens L., when interplanted with zucchini squash. Secondly, living mulch must be planted at the correct spacing to reduce competition between the main crop and the mulch. Earlier, we reported that livi ng and reflective mulch in combination with the insecticide Admire 2 F were effective in controlling whitefli es and aphids associated with zucchini squash (CHAPTER 3). It will be interesting to see if this reduction will translate to increased plant size and hence better yields. My hypothesis was that since Admire 2F woul d contribute to furthe r reduction in pest pressure (whiteflies and aphids) during the earl y season, the treated plan ts would be able to compensate for growth and hence produce higher marketable yields.

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73 The specific objective was to evaluate the imp act of buckwheat and reflective mulch with and without a reduced-risk insectic ide on plant growth (size) and marketable yields of zucchini squash. It will be my intention to make recommen dations if any of the co mbinations could result in economic gains for squash growers in Florida. Materials and Methods Field experiments were set up at the Universi ty of Florida Plant Science Research and Education Unit in Citra, Florida. Field pl ots measuring 10.36 m x 10.36 m were prepared, each containing four beds. The beds were 76 cm top width and 30 cm high and the distance between them was 1.06 meters. Each plot was separated from the adjacent plots by 7.6 m of bare soil on all sides that was kept w eed free throughout the experiment. Living mulch, (buckwheat, Fagopyrum esculentum Moench) was hand seeded between the rows 21 days and 18 days in 2005 and 2006 respectively, before planting squash. The four treatments evaluated were: 1) reflective mulch with the reduced-risk insecticid e (Admire 2F, Bayer, Kansas City, MO), 2) reflective mulch without Admire 2F, 3) living mu lch with Admire 2F, 4) living mulch without Admire 2F and standard syntheti c white mulch which served as the control. The reduced riskinsecticide Admire 2F was applied 2 weeks after s quash planting through the drip lines at the rate of 1.684 liters per hectare on th e appropriate treatments. Planting holes were cut in the center of each pl astic mulch strip and in the center of the non-mulched beds and two squash seeds, vari ety Wild Cat, were hand seeded per hole. Planting spacing was maintained at 45 cm betw een the plants in the row during the 2005 and 2006 field seasons. After germination the missing plan ts were replaced using seedlings raised in the greenhouse for that purpose. The agronomic practices for squash followed the standard production guide for squash in North Florida (Olson et al. 2005). The same plants were used for sampling for the insect pests (CHAPTER 3).

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74 Plant Size Sampling Ten plants were randomly selected from the inner rows that had not been damaged during pest sampling to estimate plant size. The plant size measurements were taken using a technique adopted from Frank and Liburd (200 5). Using a tape measure, the plant height was taken as the length of the squash from the ground to the terminal bud. The plant width data was taken by measuring the length between the two widest opp osing lateral shoots growing from the same plant. Marketable Yields Sampling Yield data were collected from the three inner rows of each plot that had not been damaged during sampling. Zucchini squash was harvested at im mature stage (soft, thin, edible rind shells) with edible seeds at approximate ly 20-25 cm long. Fruits were harv ested and weighed in the field every other day for three weeks. Data Analysis Plant size and yield data were analyzed using analysis of variance (ANOVA) SAS GLM (SAS 9.1 version [2001]). Treatment means we re separated using LSD means separation procedures and were considered significant when P < 0.05. Results Plant Size Plant widths for reflective mulch treatmen ts with and without Admire 2F were significantly larger than thos e for buckwheat and the white mulch (control) treatments ( F = 11.64; df = 4, 34; P < .0001) [Table 5-1]. Buckwheat treatm ents resulted in the smallest width and there was no significant difference between treatments with and without Admire 2F. The heights of zucchini plants grown in reflec tive mulch with and without Admire 2F were not significantly different. However, these pl ants were significantly taller than all other

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75 treatments, including the control. Zucchini plan ts grown within living mulch with Admire 2F were significantly taller than plants grown in liv ing mulch alone, a treatment that resulted in the least height when compared w ith all the other treatments ( F = 41.92; df = 4, 34; P < .0001) [Table 5-1]. Marketable Yields Zucchini squash yields differed significantly among the treatments ( F = 37.56; df = 4,167; P < 0.0001) [Table 5-2]. In 2005, zucchini plants grown with reflective mulch with Admire 2F produced significantly higher yiel ds than those from buckwheat and white mulch (control) treatments. Overall, plants growing within reflective and white mulch treatments had 58 and 54% respectively, more zucchini than those grow ing in the living mulch. Reflective mulch alone and white mulch provided similar yields. In 2006, reflective mulch plots produced signif icantly higher yields than the white mulch (control) ( F = 53.40; df = 4, 133; P < .0001) [Table 5-2]. Overall, reflective mulch treatment resulted in the highest yields compared with al l the other mulches. As in 2005, buckwheat plots produced the least yields when compared with a ll the other treatments. Ac tually plants growing within the living mulch alone produced 74 and 64% fewer marketable squash than reflective and white mulches. Discussion Zucchini plants interplanted with the livi ng mulch, buckwheat, were smaller in size and eventually yielded less than thos e growing on the synthetic mulches. My results were consistent with the findings of Frank (2004). Plants grow ing with buckwheat mulch were smaller and had lower yields when compared with plants grown with synthetic mulches. When living mulches are interplanted with a main crop (zucchini) they share the same scarce natural resources (light, nutrients), which could lead to competition and ca n negatively affect the productivity of the main

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76 crop. In our study, the competition between zucchin i plants and buckwheat was observed to be greatest during the earl y stages of growth. Although squash plan ts were able to regain some level of vigorous growth after buckwh eat senesced, they were not ab le to compensate for yield. Furthermore, by the time the buckwheat senes ced, the fall (cool season) temperatures were already present, which may have been unfavorable for the future growth of squash. The squash plant is a warm-season crop and grows best between 24 and 29 oC (Mossler and Nesheim 2001). It is possible that early-season competition is a critical factor that co uld affect fall zucchini production when interplanted with living mulche s. The addition of Admire 2F was able to enhance zucchini yields growi ng within buckwheat in at leas t one of the seasons (2005). However, in spite of the larger plant si ze there were no differences in yields. In our study, buckwheat consid erably delayed the flowering of zucchini plants and hence the time of harvesting. Previous studies have also reported similar effects of a living mulch when interplanted with a cash crop. For instance, growth of cantaloupe was delayed by wheat, Triticum aestivum L. and fruits failed to mature by the time harvest was completed in other treatments (Toba et al. 1977). In another st udy, maturation of cabbage heads was delayed and was smaller in living mulch treatments comp ared to bare ground (Andow et al. 1986). During the 2-yr study, the reflec tive mulch treatment resulted in the largest plants and the highest yields. Previous studies have reported similar findings (Brown et al. 1993, Summers et al. 1995, Csizinszky et al. 1997, Stapleton and Summers 2002, Summers et al. 2004, Frank and Liburd 2005). It is known that UV-reflective mulc h has high photosynthetica lly active radiation, which contributes to both plant growth and crop earliness (Stapleton and Summers 2002, Olson et al. 2005). In our study, the a ddition of Admire 2F did not in crease yields significantly in reflective mulch treatments.

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77 Synthetic white mulch treatment yielded more zucchini fruits than living mulch plots despite having a high whitefly population in both years. In 2006, white mulch had the highest incidences of whitefly-transmitted virus (CHAPTE R 4). It is known that zucchini yield can be affected by the occurrence of insect-transmitted diseases. In 2006, a whitefly-transmitted disease was present; however, this disease did not affect the yields of zucchini possibly because it appeared late in the growing season. Yield losses due to whitefly-transm itted viruses have been described to be within a range of 20-100% depe nding on the crop, season, and vector prevalence (Basu 1995). Indeed, living mulches were able to redu ce pest problems (CHAPTE R 3 & 4) but resulted in a huge negative impact on the yields. This advers e effect of living mulche s is a big factor that may limit their adoption in pest manageme nt programs. Additionally, the living mulchinsecticide combination treatment did not increase yield sufficiently to justify the cost of Admire 2F. For the growers to adopt this tactics the net gains associated with mulches plus Admire 2F have to be large enough to justif y their use this was not the case in this st udy. Further research needs to be done before the living mulch is r ecommended to growers. In cases where reflective mulches cannot be used, living mulch is a cheap alte rnative, since it has been shown to result in higher yields than bare ground in other studie s (Frank 2004, Hilje and Stansly 2007). It is worth mentioning that other costs (extra drip lines, natu ral resources like water, nutrients) need to be considered before using living mulches.

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78 Table 5-1. Effect of living and reflective mulche s alone or in combination with Admire 2F on plant size of zucchini sq uash Citra, FL (2006) Mean SEM (Inch) Plant Widtha Plant Heightb Reflective + Admire 2F 42.08 2.00 a 23.71 0.39 a Reflective 40.09 1.94 ab 22.15 0.69 a Buckwheat 31.62 1.54 c 13.75 0.58 d Buckwheat + Admire 2F 32.64 1.81 c 16.10 0.73 c Control (white mulch) 38.18 2.00 b 18.39 0.70 b Means followed by the same letter are not significantly different P = 0.05 (LSD) a F = 11.64; df = 4, 34; P < 0.0001 b F = 41.92; df = 4, 34; P < 0.0001

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79 Table 5-2. Effect of living and reflective mulche s alone or in combina tion with Admire 2F on marketable yields of zucchi ni in squash Citra, FL Mean yield per treatment (Kgs.) 2005a 2006b Reflective + Admire 2F 39.47 4.07 a 32.97 3.42 a Reflective 36.26 3.87 ab 32.11 3.67 a Buckwheat 15.1 2.70 d 8.29 1.21 c Buckwheat + Admire 2F 20.39 3.59 c 8.54 1.47 c White (control) 33.45 3.62 b 23.37 2.35 b Means SEM followed by the same letter are not significantly different P = 0.05 (LSD) a F = 37.56; df = 4,167; P < 0.0001 b F = 53.40; df = 4, 133; P < 0.0001

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80 CHAPTER 6 CONCLUSION The field experiments demonstrated that the addition of imidacl oprid to living and reflective mulches enhanced the control of adul t whiteflies in zucchini squash. Specifically, the addition of imidacloprid to buckw heat further reduced whitefly de nsities (adults and immatures) in both years as revealed by folia r and yellow sticky trap samples. The addition of imidacloprid to reflective mulch enhanced the control of aphi ds (wingless) in both years. However a lower population was always recorded wi th the addition of imidacloprid. In almost all cases buckwheat in combination with imidacloprid gave protection to squash against whiteflies equal to that provided by reflective mulch with imidacloprid. Only in 2005, did counts from the yellow sticky traps indicate that reflective mulch with imidacl oprid was superior to buckwheat in combination with the reduced-risk insecticide. The addition of imidacloprid did not affect the alate ap hid population densities in zucchini as indicated by foliar and pan trap samples. Surprisingl y, pan traps set within squash growing on reflective mulch with imidacloprid caught more alat e aphids in 2006 than those within reflective mulch alone. This could not be explained as it is know n that reflective mulch interferes with the insects host locating abil ity and hence reduces their landings on squash plants. The addition of imidacloprid to reflective mulch reduced apterous a phids per leaf in both years. However, buckwheat combined with imidacloprid reduced th e populations only in 2005. Such differential effects of treatment combinations on the pests indicates that development of an integrated pest management program for the zu cchini crop can be a complicated process, since the crop is affected by many pest species.

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81 The study also suggests that the use of reflective and buckwheat mulches with imidacloprid can be used to effectively reduce th e incidence of whitefly-transmitted virus. This further supports the argument that mulches can be deployed against various virus diseases. Only in 2005, that buckwheat with imid acloprid treatment produced higher yields than the buckwheat mulch alone. The yields of squash planted with buckwheat were greatly reduced. Living mulches such as buckwheat are cheaper alternatives to synthe tic (reflective) mulches. However, crop spacing and competition for resources need to be evaluated further before any recommendation can be made. A cost analysis of cost per hectare for usi ng these mulches is as follows. The cost of applying reflective mulch/labor is 1374 USD/ ha. The cost of drip tapes is 150 USD/1829 meter roll. The average cost of planting buckwheat is 178 USD/ha. The cost of adding imidacloprid (Admire 2F) to the mulches at the recomme nded rate imidacloprid is 257 USD per ha. Buckwheat mulch with imidacloprid is more cost effective than reflect ive with imidacloprid. However, it is worth mentioning that buckwheat has some other additional costs which include extra drip lines to support plant growth and ma nagement practices such thinning after planting. Although reflective mulch is more expensive, the cost of the mulch installation can be justified because significantly higher yields were obt ained. The reflective mulch treatment with imidacloprid was much more expensive than re flective mulch alone and yet no additional gains resulted in terms of yields. Therefore, it may not be necessary to combine the two tactics together because mulch alone was sufficient to give satisf actory yields. Since there were no benefits from using this combination, it may not be recommen ded to growers as an option. However, it may worth mentioning that the benefits of reflective mulches are short lived since they diminish once

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82 the crop canopy covers them. It is possible in cases of high pest pressure th at the use of reflective mulch and imidacloprid may be warranted since early virus infection can significantly reduce yield. Imidacloprid can be used to reduce the spread of whiteflytransmitted viruses by reducing the vector population on the primary and secondary host. Currently, there is a rising demand for sustainable agricultu re and to increase organic farm holdings in the United States and the rest of the world. This means that the use of cultural based tactics for pests and diseases in various crops will be the first line of defense for most growers. This study supports th e importance of buckwheat for ma naging whiteflies and aphids in zucchini as a cheaper alternative to reflective mulc hes. Therefore, buckwheat is likely to become more popular in organic farming as a means of responding to pests and disease management needs provided that adverse competition effects on crop yield can be worked out. An important aspect of buckwheat is that it grows erect and therefore does not pose problems with shading or growing into squas h. Therefore, different planting spacing can be evaluated to determine the optimal spacing in the relation to the cash crop to reduce the competition for natural resources. Zucchini crop may be planted in double or triple rows between wider strips of living mulch to lessen competition between the crop and the mulch. If the mulch is planted before the vegetable crop, like in our case, strips of the mulch must be prepared for the crop by tilling, mowing, or applying herbicides. Another study can be conducted to establish the optimal time of planting squash in to the living mulch so that maxi mum usage of beneficial insects can be achieved. A comprehensive survey for CuLC rV, diagnosing the severity of the disease and potential risks in Florida would be an important problem to address.

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90 BIOGRAPHICAL SKETCH Teresia Nyoike was born in the Thika District of Central, Kenya, Africa. She graduated with a Bachelor of Science in Agriculture (Crop Protection) on August 2001 from the University of Nairobi, Kenya. In August 2001, she joined Dudutech Kenya Ltd., an integrated pest management company that produced bio-control agents for local and export markets. In 2002, she had laboratory training on biological control agents for root knot nematodesPasteuria penetrans at Reading University (UK). During her f our years in the company, she worked as a nematologist with specific res ponsibilities of devel oping on-farm control systems for root knot nematodes using biological control agents. In August 2005, she joined the Small Fruit and Vegetable IPM Laboratory, University of Flor ida, in Gainesville to pursue her MS in Entomology. She is a member of the Gamma Si gma Delta honors society in agriculture.