biology and ecology of squash vein yellowing virus...
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BIOLOGY AND ECOLOGY OF SQUASH VEIN YELLOWING VIRUS AND ITS VECTOR WHITEFLY BEMISIA TABACI (GENNADIUS)
By
DEEPAK SHRESTHA
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Deepak Shrestha
To my mother, Sumitra Shrestha and father, Shambhu Lal Shrestha
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ACKNOWLEDGMENTS
I would like to extend my deepest gratitude to my committee chair, Dr. Susan E.
Webb, for her unlimited support, encouragement and excellent mentorship throughout
my study. I am deeply grateful to my advisory committee members, Dr. Heather J.
McAuslane, Dr. Scott T. Adkins, Dr. Hugh A. Smith, and Dr. Nicholas Dufault, for their
continuous guidance and invaluable advice during the course of this study.
My sincere thanks goes to the Department of Entomology and Nematology,
University of Florida, for awarding me a fully funded fellowship to pursue my PhD study.
I am thankful to Dr. Linda Wessel-Beaver for providing creeping cucumber seeds and to
Dr. Daniel Hahn and Dr. Tesfamariam Mengistu for allowing me to use their laboratories
and equipment. Special thanks go to Dr. Felix A. Cervantes for his expert input on the
annotation and analysis of electrical penetration graph waveforms. I thank James Colee
for his expert advice on statistical analysis of this study. I appreciate the support and
love of my department’s faculty, staff and friends throughout my study.
I am deeply indebted to my parents for instilling in me the values of knowledge,
hard work and big dreams. I thank my family members (Dipesh, Roshani, Deepali, and
Shailendra) and my parents-in-law for their love and care. I am extremely thankful and
grateful to my wife, Sachita, for her unconditional love, support and trust in me. I would
also like to thank the Nepalese community in Gainesville for making my PhD journey
wonderful and memorable.
Last but not least, I acknowledge everyone who directly or indirectly supported
and encouraged me throughout my life.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
ABSTRACT ................................................................................................................... 10
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 12
Introduction ............................................................................................................. 12 Squash vein yellowing virus (SqVYV) ..................................................................... 13
Host Range and Symptoms of SqVYV.................................................................... 15 Bemisia tabaci ........................................................................................................ 16
Effects of Plant Viruses on Host Acceptance Behaviors of their Insect Vectors ..... 20
Effects of Plant Viruses on the Feeding Behaviors of their Vectors ........................ 23 Electrical Penetration Graph and Feeding Behavior ............................................... 24
Effects of Plant Viruses on the Performance of their Insect Vectors ....................... 27 SqVYV Management .............................................................................................. 30 Justification of the Study ......................................................................................... 32
2 TRANSMISSION OF SQUASH VEIN YELLOWING VIRUS TO AND FROM CUCURBIT WEEDS AND EFFECTS ON SWEETPOTATO WHITEFLY (HEMIPTERA: ALEYRODIDAE) BEHAVIOR .......................................................... 37
Introduction ............................................................................................................. 37
Material and Methods ............................................................................................. 40 Virus Isolate and Virus Sources ....................................................................... 40 Whitefly............................................................................................................. 41
Comparison of Three Cucurbit Weeds and Watermelon as Sources of Inoculum by Whitefly Inoculation ................................................................... 41
Plant materials ........................................................................................... 41 Source of inoculum .................................................................................... 42 Recipient plants ......................................................................................... 42
Transmission procedure ............................................................................ 43 SqVYV detection ........................................................................................ 43 Experimental design and statistical analysis .............................................. 43
Comparison of Three Cucurbit Weeds and Watermelon for Susceptibility to SqVYV by Whitefly Inoculation ...................................................................... 44
Source of inoculum and recipient plants .................................................... 44 Transmission procedure ............................................................................ 44 SqVYV detection ........................................................................................ 44
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Experimental design and statistical analysis .............................................. 45
Whitefly Settling and Oviposition Preference .................................................... 45 Results .................................................................................................................... 47
Comparison of Three Cucurbit Weeds and Watermelon as Sources of Inoculum by Whitefly Inoculation ................................................................... 47
Comparison of Three Cucurbit Weeds and Watermelon for Susceptibility to SqVYV by Whitefly Inoculation ...................................................................... 47
Whitefly Settling and Oviposition Preference .................................................... 47
Discussion .............................................................................................................. 48
3 HOST-MEDIATED EFFECT OF SQUASH VEIN YELLOWING VIRUS ON SWEETPOTATO WHITEFLY (HEMIPTERA: ALEYRODIDAE) BEHAVIOR AND FITNESS ................................................................................................................. 56
Introduction ............................................................................................................. 56 Materials and Methods............................................................................................ 59
Biological Material: Whitefly Colonies, Plants, and Virus Isolate ...................... 59 Alighting Preference ......................................................................................... 60
Settling and Oviposition Preference ................................................................. 61 Developmental Time of Immature Stages and Adult Size ................................ 62 Adult Longevity and Fecundity ......................................................................... 63
Source plants ............................................................................................. 63 Test plants ................................................................................................. 64
Results .................................................................................................................... 65 Alighting Preference ......................................................................................... 65 Settling and Oviposition Preference ................................................................. 65
Developmental Time of Immature Stages and Adult Size ................................ 67 Adult Longevity and Fecundity ......................................................................... 67
Discussion .............................................................................................................. 67
4 INDIRECT EFFECT OF SQUASH VEIN YELLOWING VIRUS ON BEMISIA TABACI (MIDDLE EAST ASIA MINOR 1) (HEMIPTERA: ALEYRODIDAE) FEEDING AND SETTLING BEHAVIOR ................................................................. 79
Introduction ............................................................................................................. 79
Materials and Methods............................................................................................ 82 Biological Material: Whitefly Colonies, Plants and Virus Isolates ..................... 82 Influence of SqVYV Post Inoculation Period on Whitefly Settling and
Oviposition .................................................................................................... 83
Influence of SqVYV Post Inoculation Period on Whitefly Feeding Behavior using EPG ..................................................................................................... 84
Results .................................................................................................................... 87 Influence of SqVYV Post Inoculation Period on Whitefly Settling and
Oviposition .................................................................................................... 87
Influence of SqVYV Post Inoculation Period on Whitefly Feeding Behavior using EPG ..................................................................................................... 87
Discussion .............................................................................................................. 88
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5 CONCLUSIONS ................................................................................................... 100
LIST OF REFERENCES ............................................................................................. 106
BIOGRAPHICAL SKETCH .......................................................................................... 123
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LIST OF TABLES
Table page 1-1 Genus name, type species, vectors and number of species in genera of the
family Potyviridae. .............................................................................................. 36
1-2 Common weeds found in Florida belonging to family cucurbitaceae, with subfamily, tribe, and subtribe. ............................................................................. 36
2-1 Mean ± SEM percentage infection of watermelon recipient plants with SqVYV transmitted by whitefly, Bemisia tabaci (Middle East Asia Minor 1) with access to different source plant species. ............................................................ 52
2-2 Mean ± SEM percentage infection with SqVYV and symptom expression of different recipient plant species when whitefly, Bemisia tabaci (Middle East Asia Minor 1) ...................................................................................................... 53
3-1 Effect of plant species and Squash vein yellowing virus infection status on alighting preferences of male and female whiteflies ........................................... 72
3-2 ANOVA examining the number of settled whiteflies, Bemisia tabaci (Middle East Asia Minor 1) on infected and mock-inoculated (infection status) squash and watermelon plants ....................................................................................... 72
3-3 Effect of plant species (squash and watermelon) and infection status (Squash vein yellowing virus-infected and mock-inoculated) on number of eggs laid on entire plant and per cm2 ................................................................. 73
3-4 Average duration of immature development and length of emerged adult whiteflies, Bemisia tabaci (Middle East Asia Minor 1) on Squash vein yellowing virus-infected and mock-inoculated squash plants. ............................ 73
3-5 Longevity and fecundity of adult whitefly, Bemisia tabaci (Middle East Asia Minor 1) that developed on infected or mock-inoculated squash plants (source plants) .................................................................................................... 74
4-1 ANOVA results examining the number of settled whiteflies, Bemisia tabaci (Middle East Asia Minor 1) recorded at 0.25, 1, 2, 4, 8, 24, 48, and 72 h (time) after their release ..................................................................................... 92
4-2 ANOVA results showing the effects of whitefly, Bemisia tabaci (Middle East Asia Minor 1), oviposition preference ................................................................. 92
4-3 Mean (± SE) and ANOVA results for waveform duration per event (WDE), waveform duration per insect (WDI), waveform duration per event per insect (WDEI), and number of waveform events per insect (NWEI) ............................. 93
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LIST OF FIGURES
Figure page 2-1 Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) settling and
oviposition preference experiment on creeping cucumber leaves. ..................... 54
2-2 Number of whiteflies, Bemisia tabaci (Middle East Asia Minor 1) settled on leaves of SqVYV-infected and mock-inoculated creeping cucumber leaves counted at 0.25 h, 2 h, 5 h, 24 h, 48 h, and 72 h ................................................ 55
3-1 Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) alighting preference experiment on watermelon plants ..................................................... 75
3-2 Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) settling and oviposition preference experiment on infected and mock-inoculated plants ....... 76
3-3 Number of whiteflies, Bemisia tabaci (Middle East Asia Minor 1) settled on Squash vein yellowing virus-infected and mock-inoculated plants. .................... 77
3-4 Oviposition of whitefly, Bemisia tabaci (Middle East Asia Minor 1) on Squash vein yellowing virus-infected and mock-inoculated plants of watermelon or squash ................................................................................................................ 78
4-1 Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) settling and oviposition preference experiment ...................................................................... 95
4-2 Waveforms generated using electrical penetration graph, direct current applied voltage, and with109-ohm input resistance, for adult whitefly, Bemisia tabaci (Middle East Asia Minor 1).. ..................................................................... 96
4-3 Waveforms generated using electrical penetration graph, direct current applied voltage, and with 109-ohm input resistance, for adult whitefly ................ 97
4-4 Number of whiteflies, Bemisia tabaci (Middle East Asia Minor 1) settled on Squash vein yellowing virus-infected and mock-inoculated watermelon plant. ... 98
4-5 Oviposition of whitefly, Bemisia tabaci (Middle East Asia Minor 1) on 5-6 DPI and 10-12 DPI Squash vein yellowing virus-infected and mock-inoculated watermelon plants .............................................................................................. 99
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
BIOLOGY AND ECOLOGY OF SQUASH VEIN YELLOWING VIRUS AND ITS
VECTOR WHITEFLY BEMISIA TABACI (GENNADIUS)
By
Deepak Shrestha
December 2016
Chair: Susan E. Webb Major: Entomology and Nematology
Squash vein yellowing virus (SqVYV) is the causal agent of watermelon vine
decline in Florida and is transmitted by sweetpotato whitefly, Bemisia tabaci Middle East
Asia Minor-1. Transmission and host plant-mediated effects of SqVYV on whitefly were
evaluated in this dissertation. The lowest percentage of watermelon was infected when
balsam apple was used as a source of inoculum rather than creeping cucumber,
smellmelon, or watermelon. Creeping cucumber was as susceptible to SqVYV as
watermelon, whereas balsam apple and smellmelon were less susceptible to infection
than watermelon. However, all weed species were equally susceptible to SqVYV.
Whiteflies showed no preference to settle on infected versus mock-inoculated creeping
cucumber leaves for the first 5 h after release in a choice test. After 24 h, whiteflies
preferred to settle on mock-inoculated leaves and laid more eggs on mock-inoculated
leaves. The transmission experiments and settling assays show that these common
cucurbit weeds species may serve as reservoirs of the virus.
Whiteflies alighted and remained settled more frequently on infected than on
mock-inoculated squash. No such initial preference was observed on watermelon, and,
8 h after release, more whiteflies were found on mock-inoculated than on infected
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watermelon plant. Whiteflies laid ca. six times more eggs on mock-inoculated than on
infected watermelon; however, no differences were recorded on squash. On squash,
development from egg to adult emergence was 3 d shorter on infected than mock-
inoculated plants. Females lived 25% longer and had higher fecundity on infected than
on mock-inoculated squash, regardless of infection status of the rearing host.
Whiteflies showed no settling and oviposition preference between watermelon
plants 5-6 d post inoculation (DPI) and mock-inoculated plants. At 10-12 DPI more
whiteflies settled on the mock-inoculated watermelon 8 h after release, and laid more
eggs on the mock-inoculated plants. EPG recording of whitefly feeding showed longer
average duration of stylet pathway and penetration of mesophyll cells on a cohort level
on 10-12 DPI than on 5-6 DPI and mock-inoculated watermelon. The changing alighting
and settling preference on watermelon and enhanced fitness of whitefly on infected
squash could lead to rapid spread of SqVYV in the cucurbit agroecosystem.
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CHAPTER 1 LITERATURE REVIEW
Introduction
Each year, thousands of hectares of cucurbits, worth millions of dollars, are
grown in Florida. In United States (U.S.), Florida ranks first in the production of
watermelon. In 2015, Florida watermelon growers planted 8,700.74 ha and produced
298.72 thousand metric tons of watermelon, worth $88.2 million (USDA NASS 2016). In
2015, 2,428.1 ha of squash (Cucurbita pepo L.) was planted in Florida, producing 30.5
thousand metric tons worth $27.48 million (USDA NASS 2016).
Many different plant viruses negatively affect the commercial production of
cucurbits. When cultivated cucurbits and weeds were tested for 17 important plant
viruses, 13 of the viruses were detected from symptomatic plants collected from 10
states in the southern US (Ali et al. 2012). Out of those 10 plant viruses detected
following were detected in higher frequency than other viruses; Watermelon mosaic
virus (WMV, family Potyviridae, genus Potyvirus), Papaya ringspot virus watermelon
strain (PRSV-W, family Potyviridae, genus Potyvirus), Zucchini yellow mosaic virus
(ZYMV, family Potyviridae, genus Potyvirus), Tobacco ringspot virus (TRSV, family
Secoviridae, genus Nepovirus), Squash mosaic virus (SqMV, family Secoviridae, genus
Comovirus), and Melon necrotic spot virus (MNSV, family Tombusviridae, genus
Carmovirus) (Ali et al. 2012). The most important cucurbit viruses in Florida are PRSV-
W, WMV, ZYMV and Cucumber mosaic virus (CMV, family Bromoviridae, genus
Cucumovirus) transmitted by aphids, and Cucurbit yellow stunting disorder virus
(CYSDV, family Closteroviridae, genus Crinivirus), Cucurbit leaf crumple virus (CuLCrV,
family Geminiviridae, genus Begomovirus) and Squash vein yellowing virus (SqVYV,
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family Potyviridae, genus Ipomovirus) transmitted by whitefly (Purcifull et al. 1988,
Webb et al. 2003, Adkins et al. 2007, Akad et al. 2008, Polston et al. 2008, Turechek et
al. 2009, Turechek et al. 2010, Ali et al. 2012). Cucurbit leaf crumple virus, PRSV-W,
and SqVYV have seriously impacted cucurbit production in Florida’s southwest and
west central regions in recent years (Turechek et al. 2009, 2010). Whitefly-transmitted
viruses are a relatively recent problem in Florida (Roberts et al. 2004, Adkins et al.
2007, Akad et al. 2008, Polston et al. 2008) and have added more complications for
squash and watermelon production.
Squash vein yellowing virus (SqVYV)
Squash vein yellowing virus is a member of the Potyviridae, the largest family of
plant viruses, with 193 accepted members. Potyviridae is divided into seven genera,
and it also contains two viruses that are unassigned: Rose yellow mosaic virus and
Spartina mottle virus (ICTV 2015). Genera, type species, vectors and number of
species of Potyviridae are provided in Table 1-1. Potyviruses have positive sense single
stranded RNA and a genome consisting of a single open reading frame (Shukla et al
1994).
Squash vein yellowing virus is a member of the genus Ipomovirus, which
consists of SqVYV and five other recognized species: Sweet potato mild mottle virus
(SPMMV), Cucumber vein yellowing virus (CVYV), Cassava brown streak virus (CBSV),
Ugandan cassava brown streak virus (UCBSV) and Tomato mild mottle virus (ToMMV)
(ICTV 2015). Recently in Sudan, a tentative new member, Coccinia mottle virus
(CocMoV) was described from the cucurbit Coccinia grandis (L.) Voigt (Desbiez et al.
2016). The genus name “Ipomovirus” is derived from its type species, SPMMV, in which
‘Ipomo’ is the shortened form of the scientific name of sweet potato, Ipomoea batatas.
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Ipomoviruses are transmitted by whiteflies (Salm et al. 1996, Jones 2003, Webb et al.
2006, Adkins et al. 2007).
Squash vein yellowing virus was first collected in Hillsborough County, Florida in
October 2003 during a survey of cucurbit viruses in Florida (Webb et al. 2003). Out of
40 yellow squash leaf samples tested, 39 were positive for PRSV-W, but one leaf
sample with obvious symptoms turned out to be negative for PRSV-W and the other
seven cucurbit viruses commonly found in Florida (Whidden and Webb 2004). The
sample was later sent to Agdia (Elkhart, IN) to be tested for eight additional viruses, but
the results were negative for those viruses as well (Webb et al. 2006). Coat protein
gene and protein sequence analysis of this virus showed that it belonged to the family
Potyviridae and genus Ipomovirus (Adkins et al. 2007). The name was proposed as
“Squash vein yellowing virus” because it was first characterized from infected squash
plants, in which it produces vein yellowing symptoms (Adkins et al. 2007). Electron
microscopy and light microscopy showed pinwheel-like inclusion bodies and cylindrical
inclusion bodies attached to the cell membrane along the cell wall of epidermal tissue
and in parenchyma and companion cells of phloem tissue from infected watermelon and
squash (Adkins et al. 2007). Other members of this virus family show similar inclusion
bodies in infected plant tissue (Shukla et al. 1991, 1994, Hammond 1998).
The genome of SqVYV, which is monopartite positive-sense, includes 9836
nucleotides [excluding the 3’ terminal poly (A) tail], with a single open reading frame
encoding a large polyprotein (3172 amino acids) putatively cleaved into 10 mature
proteins (P1a, P1b, P3, 6K1, CI, 6K2, NIa-Vpg, NIa-Pro, NIb and CP in sequence from
N terminus to C terminus of the polyprotein) (Li et al. 2008). Squash vein yellowing virus
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is a flexuous rod-shaped particle, approximately 840 nm in length (Adkins et al. 2007).
SqVYV lacks HC-Pro, which is a multifunctional protein helping in vector transmission.
However, SqVYV has P1a and P1b proteins, similar to CVYV (Li et al. 2008). The
functions of the P1a protein in CVYV are suppressing RNA silencing defense, efficient
viral infection, protease and host specificity (Valli et al. 2006, Shan et al. 2015); P1b has
RNA silencing suppression activity (Valli et al. 2006). However, these functions of P1a
and P1b were discovered in CYVY and not experimentally tested for SqVYV.
The coat protein (CP) amino acid and nucleotide sequence align with those of
other viruses in the Potyviridae and share 34% to 66% identity with other members of
the genus Ipomovirus (Adkins et al. 2007). CVYV shares the highest identity of CP
amino acids and nucleotide sequence with SqVYV, about 66% and 64%, respectively.
The amino acid sequence of the conserved core from the SqVYV CP has higher identity
with other ipomoviruses, ranging from 49% to 79% (Adkins et al. 2007). Amino acid
identity of NIa-Pro, NIb, and CI proteins between SqVYV and CYVY are 51%, 70%, and
73% and between SqVYV and SPMMV are 26%, 52%, and 47% respectively (Li et al.
2008). All 10 proteins of SqVYV show the highest level of amino acid identity with CVYY
(Li et al. 2008). This close similarity of SqVYV and CVYV might suggest these two
viruses are unique members of the Potyviridae, and could potentially form a subgroup
within Ipomovirus (Li et al. 2008).
Host Range and Symptoms of SqVYV
The host range of SqVYV is limited to the family Cucurbitaceae. Symptom
expression varies within the cultivated plants of Citrullus. Citron (Citrullus lanatus var.
citroides) shows no symptoms, whereas leaves of watermelon exhibit mild vein
yellowing with chlorotic lesions, followed by systemic wilting and necrosis. The plant
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then dies within 7 to 10 days after inoculation (Adkins et al. 2007, Webster et al. 2013).
This sudden collapse and wilting of watermelon plants when infected is known as
watermelon vine decline (WVD). The fruit of infected watermelon plants show rind
necrosis, discoloration of flesh, increase in fruit acid content, and reduction in fruit
sucrose content and weight (Adkins et al. 2007, 2013). Species in the genus Cucurbita
(pumpkin, tropical pumpkin, and squash) and Luffa develop characteristic vein yellowing
of the leaves; however infected ‘Buttercup Green’, ‘Blue Ballet’, and ‘Jarrahdale’
(Cucurbita maxima) exhibit necrosis and plant death similar to SqVYV-induced WVD in
watermelon (Webb et al. 2006, Adkins et al. 2007, Webster et al. 2013). Cultivated
species of the genus Cucumis (cantaloupe and cucumber) show only transient vein
yellowing in the inoculated leaves or just above the inoculated leaves, but horned melon
(C. metuliferus) and teasel gourd (‘Prickles,’ C. dipsaceus) decline (Adkins et al. 2007,
Webster et al. 2013).
Squash vein yellowing virus has also been found in different cucurbit weeds such
as Momordica charantia L. (balsam apple; Table 1-2) in Florida and Puerto Rico and C.
melo var. dudaim (L.) Naud. (smellmelon or dudaim melon; Table 1-2) in Florida (Adkins
et al. 2008, 2009, Acevedo et al. 2013). No discernable symptoms were detected on
infected balsam apple and smellmelon plants (Adkins et al. 2008, 2009). Another
common weed found in Florida, Melothria pendula L. (creeping cucumber; Table 1-2),
can be infected by mechanical inoculation with SqVYV (Adkins et al. 2008). Infected
creeping cucumber plants show vein yellowing symptoms (Adkins et al. 2008).
Bemisia tabaci
Squash vein yellowing virus is transmitted by sweetpotato whitefly [Bemisa tabaci
Middle East-Asia Minor 1 (MEAM1)] (Webb et al. 2006, Adinks et al. 2007). Previously,
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B. tabaci MEAM1 was named as B. tabaci (Gennadius) biotype B or also B. argentifolii
(Bellows & Perring) (Bellows et al. 1994, De Barro et al. 2011, Boykin 2014). It transmits
SqVYV in a semi-persistent manner (Webb et al. 2012). Semi-persistent transmission is
characterized by acquisition and inoculation times of a few minutes to hours, no latent
period before transmission, retention times of hours to days, and loss of virus
transmission capability after the vector molts (Ng and Falk 2006). Bemisia tabaci
MEAM1has moderate transmission efficiency when whiteflies were given a 24-h
acquisition access period (AAP) and inoculation access period (IAP) on squash plants
(Webb et al. 2012). Results showed 42 ± 6%, 22 ± 5%, and 10 ± 3% of plants became
infected by evaluating the symptom, when 30, 15, and 8 whiteflies respectively were
used for transmission procedures (Webb et al. 2012). The highest infection rate was
found when whiteflies (30 per plant) were given AAPs of 4 h and 8 h with a 24-h IAP.
Infection rate did increase significantly up to an 8-h AAP, but did not continue to
increase with a 24-h AAP, when whiteflies were given 24-h IAP (Webb et al. 2012).
Results showed that whitefly retained SqVYV only up to 8 h (Webb et al. 2012).
Sweetpotato whitefly, B. tabaci (Gennadius) (order: Hemiptera, family:
Aleyrodidae and sub family: Aleyrodinae) was first described more than 100 years ago.
It has become a major pest of agricultural crops across the tropical and subtropical
regions of the world, as well as in greenhouse production systems (Cock 1986, Oliveira
et al. 2001). Bemisia tabaci can be found in every continent except Antarctica (De Barro
2005). Furthermore, it has earned a place within the world’s top 100 invasive/insidious
pest species of global agriculture due to its invasive ability and damage it causes
(Lowe et al. 2000). In tropical and subtropical and fringe-temperate conditions it can
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produce 11-15 generations per year (Brown et al. 1995). It has a broad host range,
feeding on at least 600 plant species, as well as many more hosts not yet formally
documented from many different families of plants (Oliveira et al. 2001). Brown et al.
(1995) listed 74 plant families as hosts of B. tabaci. Adult B. tabaci are 0.8 -1.2 mm in
length and have two pairs of white wings (membranous) and a yellow body. It has
opisthognathous piercing-sucking mouthparts (Byrne and Bellows Jr 1991). Bemisia
tabaci goes through incomplete metamorphosis (Hemimetabola): egg, four nymphal
instars, and adult (Byrne and Bellows 1991).
There has been a longstanding debate about whether B. tabaci is a complex
species or a species complex. Previously, it was considered as a complex species,
comprising multiple ‘biotypes’, which vary in characteristics such as behaviors, ability to
transmit viruses, genetic make-up, and endosymbiont communities (Brown et al. 1995,
Oliveira et al. 2001). However, the latest molecular evidence suggests that B. tabaci is a
complex of 11 well-defined high-level groups, comprising at least 24 morphologically
indistinguishable, but genetically distinct, cryptic species (De Barro et al. 2011).
In the U.S., B. tabaci biotype A (recently named as B. tabaci New World) was the
only species found until B. tabaci MEAM1 invaded in the mid-1980s. Based on evidence
from different biological and genetic experiments, Perring et al. (1993) concluded that B.
tabaci New World (previously named as B. tabaci biotype A) and B. tabaci MEAM1 were
different enough to separate at the species level. They proposed the scientific name of
B. argentifolii (Bellows and Perring) and the common name of silverleaf whitefly for B.
tabaci MEAM1 (Bellows et al. 1994), because of its ability to cause silverleaf disorder in
Cucurbita spp. (Costa and Brown 1991). After the invasion of B. tabaci MEAM1in the
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U.S. around 1985, it became a serious pest in Florida in the late 1980s when it was
found infesting ornamentals, especially poinsettia, in greenhouses and saranhouses
(Barinaga 1993, Hamon and Salguero 1987, Schuster et al. 1989). The recent
introduction of B. tabaci Mediterranean (MED) (previously named as B. tabaci biotype
Q) into the U.S. has added more problems in agricultural production because of its
higher capacity to develop resistance to neonicotinoid insecticides and growth
regulators (Horowitz et al. 2003, 2004, Horowitz and Ishaaya 2014).
Bemisia tabaci causes billions of dollars’ worth of direct and indirect damage to
crops (Perring et al. 1993, Brown et al. 1995). Direct damage is caused by feeding or
sucking of the plant sap by both adults and nymphs. In a heavy infestation, direct
sucking of sap by adults and nymphs of B. tabaci causes reduction in vigor and yield of
the plant, or seedling death. Indirect damage is caused by nymphal feeding, such as
phytotoxicity (physiological disorder), symptoms of which vary according to the plant
species and cultivars involved: uneven ripening in tomatoes (Schuster et al. 1990),
white stem streaking in cole crops, vein-clearing of the foliage on poinsettias, and
silverleaf in squash and other cucurbits (Yokomi et al. 1990, Paris et al. 1993). In
addition, adults and nymphs excrete honeydew, which is composed mostly of plant
sugar. Honeydew can stick to cotton lint causing problems in ginning, therefore reducing
the value of the cotton. Honeydew often serves as a substrate for fungal growth,
generally known as sooty mold. Sooty mold hinders the normal function of the leaves by
reducing photosynthesis, either through reducing the amount of light reaching
chlorophyll-bearing tissues or by blocking stomata and hindering gas exchange.
Another indirect type of damage caused by B. tabaci is the vectoring of plant viruses
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from different groups, including Carlavirus, Begomovirus, Torradovirus and Ipomovirus
(Oliveira et al. 2001, Jones 2003, Adkins et al. 2007, Navas-Castillo et al. 2011),
causing huge losses in yield.
Effects of Plant Viruses on Host Acceptance Behaviors of their Insect Vectors
Vector-borne pathogens can induce changes in the vector’s host plant that
directly and indirectly affect the vector (Rubinstein and Czosnek 1997, Jiu et al. 2007,
Srinivasan and Alvarez 2007, Mauck et al. 2010, Ingwell et al. 2012, Mauck et al. 2012).
The complex interactions among plants (hosts), plant viruses, and vectors (insects)
have epidemiological implications (Colvin et al. 2006). Changes in nutritional quality of
the host (Ajayi 1986, Blua et al. 1994, Colvin et al. 2006, McMenemy et al. 2012), plant
defensive chemicals (Su et al. 2015, Shi et al. 2016), plant phenotype (Ajayi and Dewar
1983, Hodge and Powell 2008), or changes in host-location cues (volatiles) cause
changes in patterns of vector retention, feeding, and dispersal (Eigenbrode et al. 2002,
Jiménez-Martínez et al. 2004, Rajabaskar et al. 2013) that ultimately aid in dispersal
and survival of the virus. Interactions between phytoviruses and their insect vectors
have been shown to be antagonistic (Donaldson and Gratton 2007, Mauck et al. 2010),
neutral, or beneficial, depending on the species involved (Colvin et al. 2006, Mauck et
al. 2012, Legarrea et al. 2015). From the plant pathogen point of view, the virus will
benefit if the infected plants show higher attractiveness to vectors until vectors land and
probe to acquire the virus. After the vector acquires the virus, the time that vectors
spend on the infected plants should be reduced, increasing virus spread to neighboring
healthy plants (Fereres and Moreno 2009).
In recent years, there is more interest in the alighting and settling preferences of
insect vectors caused by virus-induced changes in the host plant and their effect on the
21
plant virus spread (Rajabaskar et al. 2013, 2014, Mauck et al. 2010, 2012, Ingwell et al.
2012, Wang et al. 2014, Legarrea et al. 2015). For example, a higher number of non-
viruliferous B. tabaci MED adults settled on TYLCV (Tomato yellow leaf curl virus)-
infected Datura stramonium and tomato plants than on healthy plants, but non-
viruliferous B. tabaci MEAM1 preferred to settle on non-infected tomato plants rather
than on TYLCV-infected plants, signifying differences in the settling preference of
spieses (Chen et al. 2013, Fang et al. 2013). However, viruliferous B. tabaci MEAM1
and B. tabaci MED did not show any preference for settling on TYLCV-infected and
non-infected tomato plants (Fang et al. 2013). Compared with previous experiments,
Legarrea et al. (2015) found different results with a TYLCV-susceptible tomato genotype
six weeks post inoculation (WPI): non-viruliferous B. tabaci MEAM1 preferred to settle
on TYLCV-infected plants, and viruliferous whiteflies preferred non-infected plants.
However, no preferences for settling were found when plants were three or 12 WPI, nor
were differences found on a TYLCV-resistant genotype of tomato. Mann et al. (2009)
conducted an experiment involving B. tabaci settling on cotton plants infected with
Cotton leaf curl virus (CLCuV, another begomovirus) at different d post inoculation (DPI)
and found that at 5 DPI there were no preferences for settling on infected versus
healthy cotton plants. However, whiteflies preferred to settle on healthy cotton plants 8 h
after release for plants at 20 DPI and 1 h after release for plants 35 DPI, which might be
long enough for a whitefly to pick up a virus from infected plants and transmit it to
healthy plants (Mann et al. 2009). These results suggest that whiteflies’ preference and
behavior are affected not only by post inoculation period, but also by genotype of host
plant and species of whitefly involved.
22
Other hemipterans, such as planthoppers and aphids, also exhibit differential
acceptance of infected and uninfected host plants. Virus-free white-backed planthopper
(WBPH, Sogatellla furcifera) preferred to orient toward Southern rice black-streaked
dwarf virus-infected rice plants than to healthy plants, but viruliferous WBPH preferred
the healthy plants (Wang et al. 2014). In the absence of visual, taste, and contact cues,
melon aphid (Aphis gossypii) winged or wingless morphs preferred to aggregate below
abaxial surface of the CMV-infected squash leaves rather than below untouched and
mock-inoculated leaves, but did not show a preference between untouched and mock-
inoculated plants (Mauck et al. 2010). Pea aphid nymphs (Acyrthosiphon pisum)
preferred to settle on Pea enation mosaic virus (PEMV)-infected leaf discs of pea plant
rather than on non-infected leaf discs, but there were no differences in settling under
dark conditions (Hodge and Powell 2010). Aphid vectors of Potato leafroll virus (PLRV)
and Barley yellow dwarf virus (BYDV) preferred to settle on infected plants rather than
on healthy plants (Jiménez-Martínez et al. 2004, Srinivasan and Alvarez 2007). Ingwell
et al. (2012) showed non-viruliferous bird cherry aphid (Rhopalosiphum padi) preferred
to settle on BLRV-infected wheat plants, however after acquiring the virus, aphids
preferred to settle on non-infected plants. These results were similar to those found for
the M. persicae and PLRV system (Rajabaskar et al. 2014). The above-mentioned
conditional preferences of vectors (Ingwell et al. 2012, Rajabaskar et al. 2014) have the
potential to spread the viruses. This was supported by the model of Roosien et al.
(2013), which showed that the rate of disease spread at all stages of infection was
higher when pathogens caused non-inoculative vectors to prefer infected host plants,
and then after becoming inoculative, prefer non-infected host plants. Models have also
23
depicted that insect vectors having uniform preference for infected plants increase
disease spread rate when infected plants are rare; however, insect vectors showing
uniform preference for non-infected plants increase the rate of disease spread when
infected plants are abundant (Sisterson 2008, Roosien et al. 2013).
Effects of Plant Viruses on the Feeding Behaviors of their Vectors
An insect’s ability to transmit a virus relies on its feeding behavior. There are a
number of studies done on animal-infecting viruses, which have been shown to modify
the behavior and feeding processes of their vectors (Lefèvre and Thomas 2008). Such
changes in insect vector behavior in response to plant-infecting viruses have been
studied far less. Virus-infected host plants do modify the feeding behaviors of vectors,
which ultimately aids in the transmission of plant viruses (Alvarez et al. 2007, Hu et al.
2013). For example, A. gossypii feeding on C. pepo (zucchini) plants infected with the
non-persistently transmitted ZYMV, which is acquired from epidermal cells during brief
probes, had a higher number of probing events and fewer phloem contacts than A.
gossypii feeding on healthy plants (Blua and Perring 1992). The situation is different for
persistently transmitted viruses that are acquired from the phloem. Myzus persicae
feeding on PLRV-infected plants showing symptoms had enhanced plant penetration in
the epidermal/mesophyll level than aphids feeding on non-infected potato plants
(Alvarez et al. 2007). Barley yellow dwarf virus infection of oats affected the feeding and
probing behaviors of greenbug (Schizaphis graminum) such that the aphid made fewer
probes, showed fewer interruptions in their probing once their stylets were inserted into
tissues, and increased their duration of ingestion from phloem on infected oats
compared to non-infected oats. Rhopalosiphum padi, however, had similar behavior on
infected and non-infected oats (Montllor and Gildow 1986). English grain aphid (Sitobion
24
avenae) feeding on healthy plants of several wheat cultivars had shorter non-probing
times and was able to achieve more phloem contacts than those aphids feeding on
BYDV-infected plants (Fereres et al. 1990). Thus, there are differences in the feeding
and probing behaviors for different aphids on plants infected with the same plant virus.
The direct effects of TYLCV virus on feeding of B. tabaci MEAM1 and B. tabaci
MED have been documented (Liu et al. 2013). Viruliferous adults of both species probe
more quickly, have a greater number of feeding bouts, and spend more time in
salivation into sieve elements than do non-viruliferous whiteflies on tomato plants. It is
not only the feeding behavior of vectors of plant viruses that can be affected by virus
status of their host plant; non-vector homopteran feeding behavior can be influenced by
plants infected with a virus. For example, electronic monitoring of feeding behavior of
the non-vector S. avenae on barley plants singly infected with Wheat dwarf virus (WDV)
or Cereal yellow dwarf virus-RPV (CYDV-RPV) showed that the sum of non-probing
phases and sum of the pathway phases were significantly shorter, and that the sum of
phloem ingestion was longer on infected plants compared with control plants (Hu et al.
2013). None of these examples, however, involve viruses like SqVYV that are
transmitted in a semi-persistent manner.
Electrical Penetration Graph and Feeding Behavior
Many of the studies describing the feeding behavior of hemipteran vectors of
plant viruses have relied on the technique of electrical penetration graph (EPG)
monitoring. Electrical penetration graph technique is a powerful and specialized tool
used to study the hidden aspects of the feeding behavior of insects with
piercing/sucking mouthparts, such as hemipterans. The EPG technique has been very
useful in finding out the changes in hemipteran feeding behavior caused by insecticides
25
(Cui et al. 2012, Civolani et al. 2014, Jacobson and Kennedy 2014, Cameron et al.
2016), antifeedants, and plants resistant to insects (Fereres et al. 1990, Walker and
Backus 2000, McDaniel et al. 2016). The EPG technique has been useful in determining
the interactions of vectors, plants, and pathogens; it is able to elucidate the timing of
and stylet activities during pathogen acquisition and inoculation by its insect vectors,
which is very important for the development of appropriate control strategies (Jiang et
al. 2000, Johnson et al. 2002, He et al. 2011). Electrical penetration graph can help
determine the effects of pathogen-infected plants versus healthy plants on feeding
behaviors of vectors (Hu et al. 2013, Liu et al. 2013, He et al. 2015).
Basically, EPG allows one to study biological phenomena by correlating voltage
fluctuations (waveforms) with specific behaviors. Electrical penetration graph makes it
possible to obtain information on what is happening inside plant tissues, which is
otherwise very difficult to determine. It was first developed by D. L. McLean and M. G.
Kinsey in 1964 at the University of California, Davis (McLean and Kinsey 1964, 1965).
Their work was based on using an alternating current (AC) system. Later, the technique
was significantly modified and improved by W. F. Tjallingii, working with direct current
(DC) system (Tjallingii 1978, 1985). Both AC and DC based systems measure
fluctuations in electrical resistance in the plant and in the probing insect. Previously only
the DC system was able to measure changes in the voltage generated by the stylets of
the insect when passing through a cell wall, also known as biopotentials or emf, but the
rectified AC from a new AC-DC monitor can measure emf (Backus et al. 2013). EPG
can distinguish stylet position in the intracellular and intercellular environment, making it
easier to know when the plant cell membrane is punctured. Since its inception, this
26
technique has gone through several modifications and improvements to give very exact
and related information on the position of the stylets inside plant cell and tissue (Walker
2000). The EPG systems work on the physical principle that the passage of liquids of
different electrical conductivity (e.g. plant cellular matter, saliva) through the insect’s
mouthparts (stylets) creates a measureable fluctuation in resistance. The fluctuation in
the resistance can be related to different feeding behavioral processes during probing,
such as salivation and ingestion. These behavioral processes are distinguished by
looking at the electrical waveforms produced by changes in resistance. Walker (2000)
reviewed a number of techniques used for correlating the EPG waveforms and specific
behavior of insects, e.g., histology of plant tissue (for determining the position of the
stylets in plant tissue), observation of honeydew production and analysis (chemical
analysis to determine whether ingestion is from phloem or xylem), and direct
observation of stylets in artificial diet and radioactive diet (to see the activity of insect’s
stylets in transparent media and the amount of ingestion).
In the EPG technique, a thin electrode (usually a gold or platinum wire) is glued
to the insect’s dorsum by conductive silver paint or glue. Another electrode is then
inserted into the soil substrate or plant and supplies a weak voltage, which may be
either AC or DC, depending on the system. As soon as the circuit is completed, when
the stylets of the insect are inserted into the plant or artificial diet (Tjallingii 2001),
different distinct waveforms can be recorded. Different activities can be detected within
the behavior of probing by looking at different waveforms or waveform patterns. At this
time, most work in feeding behavior for aphids and whiteflies is conducted using the DC
system in Giga 8 (DC) monitor or DC in AC-DC monitor.
27
DC EPG systems record the changes in both insect-plant resistor (Ra, variable
resistance generated by different activities of the probing insect) and in electromotive
force (emf, voltage generated by insect and plant, occurring when the stylets pass
across cell membranes), which makes the voltage (Vi, voltage drop across the Ra)
fluctuate, creating different waveform patterns. In the DC system of recording, there can
be distinctive waveforms: A, B, C (pathway phases representing insect stylets
advancing through plant tissue); pds (potential drops representing brief intracellular
punctures); E1 (phloem salivation phase representing salivation in the phloem sieve
element); E2 (phloem ingestion phase representing insect uptake of sap from phloem);
G (xylem phase representing the insect ingesting water from xylem); and F (mechanical
derailment phase representing mechanical difficulties for stylet penetration) (Walker and
Janssen 2000). Some of these waveforms are again broken down into subdivisions that
indicate the minute phenomenon happening within activities. In addition to feeding
behavior, whitefly oviposition behavior can be recorded using EPG (Walker and
Janssen 2000).
Effects of Plant Viruses on the Performance of their Insect Vectors
Plant viruses have also been known to indirectly influence vector performance
(development, adult longevity, fecundity, rate of population increase and survival) via
the plant as a common host for both pathogen and vector (Fereres and Moreno 2009,
Chen et al. 2013, 2014, Pan et al. 2013). Indirect effects of plant viruses on insect
vectors might be neutral, positive, or antagonistic (Mauck et al. 2012, Moreno-
Delafuente et al. 2013, Ren et al. 2015). All published examples of effects of plant
viruses on B. tabaci performance involve the persistently transmitted begomoviruses
(Geminiviridae). For example, sex ratio, development period, fecundity and percentage
28
emergence were not affected when B. tabaci was reared on cassava plants infected
with the East African cassava mosaic virus (EACMV) and non-infected plants,
regardless of the status (viruliferous or not) of B. tabaci (Thompson 2011). Similarly, the
reproductive rate per generation was not affected when B. tabaci MED was reared on
TYLCV-infected tomato plants or healthy plants for two generations in the greenhouse
(Matsuura and Hoshino 2009). Likewise, viruliferous and non-viruliferous whiteflies did
not show any significant differences in survival rate and fecundity when they were
allowed to oviposit on healthy tomato plants (Matsuura and Hoshino 2009). TYLCV-
infective B. tabaci MEAM1, however, had a reduction in life expectancy of 17-23 % and
a 40-50% decrease in the mean number of eggs produced compared with non-
viruliferous whitefly (Rubinstein and Czosnek 1997). Legarrea et al. (2015) found B.
tabaci MEAM1 development time was reduced on a TYLCV-infected susceptible
genotype of tomato compared with non-infected susceptible tomato; however, no
differences were found between infected and non-infected TYLCV-resistant tomato
genotypes.
There are examples of positive or beneficial effects of plant viruses on whitefly
performance. Higher fecundity of whiteflies was found on cassava infected with
EACMV-Uganda when compared with those reared on a healthy cassava plant (Colvin
et al. 2006). Similarly, population growth of B. tabaci was higher on Euphorbia
geniculata, Parthenium hysterophorus, Acanthospermum hispidum, Ageratum
conyzoides, and tomato (Lycopersicon esculentum cv. Arka Vikas) plants infected with
Tomato leaf curl virus (ToLCV)-[Ban4] than on healthy plants for all the host species,
but the extent of the effect depended on the plant species used in the experiment
29
(Colvin et al. 2006). Bemisia tabaci MEAM1fecundity, longevity and population density
increased by 12, 6, and 2 times when fed on Tobacco curly shoot virus (TbCSV)-
infected plants, and by 18, 7 and 13 times when fed on the Tomato yellow leaf curl
China virus (TYLCCNV)-infected plants at 56 days after release. Bemisia tabaci Asia II
3 (indigenous to China) performed similarly, however, on healthy and virus-infected
plants (Jiu et al. 2007). A significantly higher proportion of immature and mature oocytes
were observed within the ovary of the B. tabaci MEAM1female and significantly more
eggs were laid by the female when allowed to feed on TYLCCNV-infected tobacco
compared with healthy plants. However, there were no significant differences in these
parameters for the B. tabaci Asia II 3 (previously known as B. tabaci biotype ZHJ1)
(Guo et al. 2010). Fang et al. (2013) found increases in B. tabaci MED male and female
body length, survival rate, longevity and fecundity when whiteflies were allowed to feed
on TYLCV-infected Datura stramonium compared with healthy plants; however, there
were no differences in development time from egg to adult.
In the case of aphids, Srinivasan and Alvarez (2007) found significantly higher
fecundity of green peach aphid (M. persicae) and potato aphid (Macrosiphum
euphorbiae) on potato plants infected with PLRV than on non-infected and PVY-
infected potato plants. Schizaphis graminum population growth was greater on BYDV-
infected oats than on healthy plants, which indicates that BYDV-infected oats are a
more suitable host than healthy plants. There were no effects, however, on R. padi
population growth (Montllor and Gildow 1986). Hodge and Powell (2010) investigated
pea aphid (A. pisum) response to PEMV-infected pea plants at varying stages of
symptom development and infection. In general, aphids showed higher rate of growth
30
(mean daily growth rate when introduced on plants having well-developed symptoms of
PEMV than on uninfected plants of the same age.
SqVYV Management
Management of vector-transmitted plant viruses in field situations has always
been a challenge because of the complex and dynamic interactions of the viruses, crop
and non-crop host plants, and vectors within a variable environment. To minimize
vector-borne viral disease in crops, it is important to manage the insect vector
effectively, particularly for persistently and semi-persistently transmitted viruses. Semi-
persistently transmitted viruses can be found in the middle of the persistently and non-
persistently transmitted viruses. Persistently transmitted virus takes few h to d to
acquire and to inoculate, and insect vector can retain virus for d or for lifetime with latent
period of d to wk (Ng and Falk 2006). Non-persistently transmitted virus takes few sec
to min to acquire and to inoculate, and insect vector can only retain virus for few min to
h with no latent period (Ng and Falk 2006).
There are some adverse biological, environmental, and economic consequences
to the use of insecticides to control vectors, but they provide an economically feasible
and convenient method for all but non-persistently vectored viruses. Insecticides have
the potential to reduce the spread of viruses by reducing the number of individuals, or
by interfering with feeding behavior. However, the effectiveness of insecticides is
variable against vectors of plant viruses. For example, differences in resistance to
insecticides and growth regulators have been found for B. tabaci MEAM1and B. tabaci
MED (Horowitz and Ishaaya 2014).
Unlike non-persistently transmitted viruses, persistently and semi-persistently
transmitted viruses can be controlled with more success using insecticides. There are
31
more reports of successes in controlling virus spread using insecticides for persistently
and semi-persistently transmitted viruses (94 of 119 cases) (Perring et al. 1999).
Because semi-persistently and persistently transmitted viruses require longer feeding
periods (usually hours to days) for virus acquisition, their vectors usually colonize the
host plant. The longer feeding times required for virus acquisition allow sufficient time to
expose the insect to a lethal dosage of insecticide, or enough time for insecticides to
interrupt the transmission of viruses by altering feeding behavior (Perring et al. 1999). A
field experiment conducted by Roberts et al. (2007) found that application of
imidacloprid at transplanting and subsequent applications of pymetrozine were not able
to prevent WVD in a spring trial in 2006. However, in a fall trial, there were fewer
whiteflies and the rate of spread and severity of WVD in treated plots was lower than in
untreated plots. Similarly, other field experiments showed that application of
imidacloprid as a soil drench soon after transplanting, and two additional foliar
applications of spiromesifen, significantly reduced the incidence of WVD (Kousik et al.
2008, 2010, 2015).
Another way to manage viral diseases is to use varieties resistant to plant viruses
or their vectors. There is ongoing research to develop watermelon cultivars resistant to
SqVYV for a long-term solution for management of WVD. Screening for SqVYV-
resistant germplasm was conducted to identify potential sources of resistance and
partial resistance that can be used in breeding programs (Kousik et al. 2009). Kousik et
al. (2012a) developed the watermelon line 392291-VDR (vine decline-resistant) with
resistance to SqVYV. The line 392291-VDR is not entirely immune to infection by
SqVYV, which can be detected by tissue blot nucleic acid hybridization, but SqVYV-
32
inoculated 392291-VDR produces symptomless mature fruit, and the plant does not
decline and die like plants of commercial watermelon cultivars (‘MickyLee’, ‘Crimson
Sweet’ and seedless commercial varieties) (Kousik et al. 2012a). 392291-VDR can be a
useful source of resistance in watermelon breeding; however, its fruit quality is
commercially unacceptable.
Other work had been conducted to reduce the spread of SqVYV. For example,
the use of reflective mulch (Kousik et al. 2008, 2010) and the use of resistant rootstocks
for SqVYV (Ling et al. 2013) has been evaluated. Use of resistance rootstocks has been
used to soil-borne disease and to control of other viruses of cucurbits (Park et al. 2005,
Davis et al. 2008, Ling et al. 2013). Other management strategies used in Florida
include the destruction of cucurbit weeds and post-cucurbit crop volunteer reservoirs
and chemically burning down the crop after harvest (Kousik et al. 2012b).
Justification of the Study
In some fields of Florida’s southwest and west-central regions, SqVYV caused
sudden and severe vine decline of watermelon near the time of harvest or immediately
after harvest during spring and fall of 2003-2004 (Roberts et al. 2004). Symptoms
included yellowing, wilting of the vines, brown and scorched leaves, and rapid collapse
of mature vine (Roberts et al. 2004). In some fields, disease progression was so rapid
that in a week it increased from 10% affected plants to greater than 80%. Watermelon
growers lost 50 to 100% of their crops in the 2003-2004 season (Roberts et al. 2004,
2007). In the year 2004-2005 in southwest Florida, watermelon growers’ losses were
estimated at more than $60 million (Huber 2006).
Watermelon plants infected with SqVYV undergo severe physiological changes.
The virus reduces the weight of watermelon plants and fruits, changes fruit rind and
33
flesh color, reduces fruit sucrose content, increases fruit acid content, and changes
plant nutrient composition (Adkins et al. 2013). The fruit becomes unmarketable due to
rind necrosis, discoloration and fruit decay. SqVYV not only infects watermelon, but also
infects other cucurbits plants. In a greenhouse experiment, infected Cucurbita maxima
‘Jarrahdale’, ‘Blue Ballet’, and ‘Buttercup Green’ exhibited necrosis, plant wilting and
death, similar to the symptoms of WVD infection in other cucurbits plants (Webster et al.
2013). Though watermelon remains the most affected of the cucurbits, there is the
potential for this virus to become problematic on other cucurbit crops as well.
After the initial discovery of SqVYV in Florida, it has subsequently been detected
within the US in Indiana (Egel and Adkins 2007), South Carolina (S. Adkins and C. S.
Kousik, personal communication), Georgia (Webster and Adkins 2012), Puerto Rico
(Acevedo et al. 2013) and California (Batuman et al. 2015); and in Guatemala
(Jeyaprakash et al. 2015), and Israel (Reingold et al. 2016). Squash vein yellowing virus
has become endemic in southwest and west-central Florida; it has appeared in varying
degrees every year. Recent detection of SqVYV in different parts of the US and in
different countries has increased the range of the economic threat to several species of
cucurbits.
Weeds serve as reservoirs of plant viruses, filling the gap between two cropping
seasons and sometime providing a refuge for vectors (Izadpanah et al. 2003, Alvarez
and Srinivasan 2005, Arli-Sokmen et al. 2005, Wisler and Norris 2005, Srinivasan and
Alvarez 2007, Adkins et al. 2008, Srinivasan et al. 2008, Cervantes and Alvarez 2011).
Surveys of weeds in southwest, west central and south Florida vegetable production
areas have found some plants of balsam apple and smellmelon infected with SqVYV
34
(Adkins et al. 2008, 2011). These weeds can survive year round in south Florida and
can also act as over summering hosts of viruses bridging the gap between spring and
fall crops (Adkins et al. 2011). For example, balsam apple and creeping cucumber have
been identified as potential reservoirs of PRSV-W and CuLCrV in Florida (Adlerz 1972a,
b, Adkins et al. 2008). Creeping cucumber can be infected by both mechanical
inoculation with SqVYV (Adkins et al. 2008) and by viruliferous whiteflies (D. Shrestha,
unpublished), suggesting that it could be a reservoir of SqVYV in nature. In addition,
these cucurbit weeds can act as reservoirs of PRSV-W, CYSDV and CuLCrV, which
also significantly affect cucurbit production in Florida (Adkins et al. 2008, 2009, Adlerz
1972a, b). It is very important to know the role of each weed in the spread of SqVYV.
This can be determined by knowing about their susceptibility to SqVYV and how
efficiently whitefly can acquire SqVYV from infected weeds.
Indirect effects of plant viruses on the behavior and performance of insect
vectors have been documented on several phytopatho-systems (Rubinstein and
Czosnek 1997, Jiu et al. 2007, Srinivasan and Alvarez 2007, Mauck et al. 2010, Ingwell
et al. 2012, Mauck et al. 2012, Rajabaskar et al. 2013, Legarrea et al. 2015). However,
no study has been conducted to observe the indirect effects of SqVYV on whitefly.
Studying host-mediated effects of SqVYV on the whitefly could potentially help elucidate
survival, transmission and spread of SqVYV. Most of these indirect effects of plant
viruses on insect vectors have been documented for persistently and non-persistently
transmitted viruses (Mauck et al. 2012); however, limited information exists for semi-
persistently transmitted viruses (McMenemy et al. 2012). The results of this research
35
with SqVYV will contribute valuable information about the indirect effects of a semi-
persistently transmitted virus on its insect vector.
To address some of issues related spread of SqVYV mentioned above, I
developed the overall goal to determine the host-mediated effects of SqVYV on whitefly
biology and behaviors that could affect the spread and survival of the virus. I
hypothesized that change in plant physiology and phenotype due to SqVYV infection
cause modification of whitefly behaviors and biology. The specific objectives of this
study were:
1. To determine the effects of virus on whitefly host acceptance behaviors, I:
a. compared the transmission efficiency of the whitefly when cucurbit weeds (balsam apple, creeping cucumber, smellmelon) and ‘Mickylee’ watermelon served as sources of inoculum and compared their susceptibility to SqVYV.
b. and compared whitefly settling and oviposition on infected vs. mock-inoculated leaves of creeping cucumber.
2. To investigate the differences in whitefly performance on infected and uninfected host plants, I:
a. determined the developmental period from egg to adult, adult longevity, fecundity, and adult whitefly body size on SqVYV-infected and mock-inoculated squash.
b. and also compared whitefly acceptance behaviors (alighting, settling, and oviposition) on infected vs. mock-inoculated squash and watermelon plants.
3. To determine the effect of days post inoculation on whitefly acceptance and feeding behaviors, I:
a. compared the settlement and oviposition behavior on different days post inoculated vs. mock-inoculated watermelon plants
b. and feeding behavior (using EPG techniques), on different days post inoculated vs. mock-inoculated watermelon plants
36
Table 1-1. Genus name, type species, vectors and number of species in genera of the family Potyviridae.
Genus Type species Vector # of species
Brambyvirus Blackberry virus Y Unknown 1
Bymovirus Barley yellow mosaic virus Plasmodiophorids (root infecting protist parasites ) 6
Ipomovirus Sweet potato mild mottle virus Whiteflies 6
Macluravirus Maclura mosaic virus Aphids 6
Potyvirus Potato virus Y Aphids 162
Rymovirus Ryegrass mosaic virus Eriophyid mites 3
Tritimovirus Wheat streak mosaic virus Eriophyid mites 6
Contents of this table are adapted from ICTV 2015.
Table 1-2. Common weeds found in Florida belonging to family Cucurbitaceae, with subfamily, tribe, and subtribe.
Common name Scientific name Subfamily Tribe Subtribe
Balsam apple Momordica charantia L. Cucurbitoideae Joliffiease Thladianthinae
Smellmelon or Dudaim melon Cucumis melo var. dudaim (L.) Naud. Cucurbitoideae Benincaseae Benincasinae
Creeping cucumber Melothria pendula L. Cucurbitoideae Melothrieae Cucumerinae
37
CHAPTER 2 TRANSMISSION OF SQUASH VEIN YELLOWING VIRUS TO AND FROM CUCURBIT
WEEDS AND EFFECTS ON SWEETPOTATO WHITEFLY (HEMIPTERA: ALEYRODIDAE) BEHAVIOR
Introduction
Squash vein yellowing virus (SqVYV) is an Ipomovirus (Family Potyviridae) that
was characterized from squash and found to be the causal agent of watermelon vine
decline (WVD) (Webb et al. 2006, Adkins et al. 2007). Squash vein yellowing virus is
transmitted semi-persistently by sweetpotato whitefly [Bemisa tabaci (Gennadius)
biotype B] (Webb et al. 2006, 2012). Previously B. tabaci biotype B was named B.
argentifolii (Bellows & Perring) and recently it has been described as a species in the
Middle East-Asia Minor one group (MEAM1) (Bellows et al. 1994, De Barro et al. 2011,
Boykin 2014). Highest transmission efficiency was found when whiteflies were given a
4-h acqusition access period (AAP) and a 4-8 h inoculation access period (IAP);
however as little as 0.5 h is enough for whiteflies to acquire virus and to inoculate plants
(Webb et al. 2012). Symptoms in infected watermelon include yellowing, wilting of the
vines, brown and scorched leaves, and rapid collapse of the vine (Webster et al. 2013).
In a greenhouse experiment, SqVYV-infected pumpkin (Cucurbita maxima Duchesne),
‘Buttercup Green’, ‘Blue Ballet’, and ‘Jarrahdale’), teasel gourd (Cucumis dipsaceus
Ehrenb. ex Spach ‘Prickles’), and horned melon (Cucumis metuliferus Mey. ex Naud.)
exhibited severe necrosis and wilting similar to SqVYV-induced WVD in watermelon
(Webster et al. 2013). Infected squash plants show typical vein yellowing symptoms;
however, they do not show wilting and necrosis symptoms as does watermelon (Adkins
et al. 2007). Since the discovery of SqVYV in Florida, it has also been detected in
Indiana (Egel and Adkins 2007), Georgia (Webster and Adkins 2012), South Carolina
38
(C. S. Kousik and S. A. Adkins personal communication), Puerto Rico (Acevedo et al.
2013), California (Batuman et al. 2015), Guatemala (Jeyaprakash et al. 2015) and Israel
(Reingold et al. 2016).
Weeds play a crucial role in the spread of many plant viruses by acting as
sources of inoculum and bridging the period between cropping seasons (Izadpanah et
al. 2003, Jones 2004, Alvarez and Srinivasan 2005, Arli-Sokmen et al. 2005, Wisler and
Norris 2005). Weeds are also known to act as refuges for insect vectors (Duffus 1971,
Jones 2004). Infected weeds may show few or no symptoms, adding more challenges
to the management of plant viruses (Tomlinson et al. 1970, Wisler and Norris 2005).
Different cucurbit weeds found throughout Florida have been shown to be infected with
plant viruses, such as the watermelon strain of Papaya ringspot virus, Watermelon
mosaic virus, Zucchini yellow mosaic virus, Cucurbit yellow stunting disorder virus,
Cucurbit leaf crumple virus, and SqVYV (Adlerz, 1969, 1972; Adlerz et al. 1983, Adkins
et al. 2008, Akad et al. 2008, Polston et al. 2008).
In southwest, west central, and south Florida vegetable production areas, the
cucurbit weeds Momordica charantia L. (balsam apple or balsam pear) and Cucumis
melo var. dudaim (L.) Naud. (smellmelon) were found to be infected with SqVYV
(Adkins et al. 2008, 2009). Similarly, in vegetable production areas in Puerto Rico
balsam apple has been found infected with SqVYV (Acevedo et al. 2013). In the
laboratory, Melothria pendula L. (creeping cucumber) was easily infected by mechanical
inoculation with SqVYV (Adkins et al. 2008), suggesting that it, too, could be a potential
reservoir of SqVYV in nature. These weeds can survive year round (except when
occasional hard freezes occur) in south Florida and can also act as over-summering
39
hosts of viruses, bridging the gap between spring and fall crops (Adkins et al. 2008,
2011).
Plant viruses can affect their insect vectors through effects on the shared host
plant. However, limited research has been conducted on the virus-induced host-
mediated effects on insect vectors when weed species are the hosts (Castle et al. 1998,
Colvin et al. 2006, Srinivasan et al. 2008, Chen et al. 2013). Host-mediated effects of
plant viruses on the behavior and biology of hemipteran insect vectors can be
antagonistic (Donaldson and Gratton 2007, Mauck et al. 2010), neutral, or beneficial,
depending on the species involved (Colvin et al. 2006, Mauck et al. 2012). Host-
mediated effects of viruses on their vectors can be caused by changes in the nutritional
quality of the host (Ajayi 1986, Blua et al. 1994, Colvin et al. 2006, McMenemy et al.
2012), changes in plant volatiles emission, and/or changes in plant phenotype (Hodge
and Powell 2008, Hodge and Powell 2010), which can affect vector settling, oviposition,
feeding, and dispersal (Eigenbrode et al. 2002, Jiménez-Martínez et al. 2004). In
addition, these effects on insect vectors can affect the spread and survival of the virus
(Fereres and Moreno 2009, Roosien et al. 2013).
Vector settling and oviposition preference are two important behaviors which can
affect virus dispersal (Mann et al. 2009, Chen et al. 2013, Fang et al. 2013). These
preferences can be influenced by the infection status of the host plant. Changes in
settling and oviposition preference of vectors caused by virus-induced modifications in
the host have been found with non-persistently and persistently transmitted viruses
(Mauck et al. 2010, 2012; Ingwell et al. 2012, Rajabaskar et al. 2014, Wang et al. 2014,
40
Legarrea et al. 2015). Limited information exists about the host-mediated effects of
semi-persistently transmitted viruses on vectors (McMenemy et al. 2012).
Other than virus-induced changes on insect vectors, it is also very important to
know how easily weeds can be infected and serve as sources of virus. The transmission
of plant viruses by an insect vector depends on the host plants it inoculates as well as
the host plants it feeds on to acquire virus (Alvarez and Srinivasan 2005, Cervantes and
Alvarez 2010, Wosula et al. 2012, Shrestha et al. 2014). The objectives of my research
were to evaluate how common cucurbit weeds located around commercial watermelon
fields could influence SqVYV transmission and how virus infection influences whitefly
behavior, possibly affecting virus dispersal. Thus, I compared the rate of transmission of
SqVYV to watermelon by the whitefly when cucurbit weeds (balsam apple, creeping
cucumber, smellmelon) and ‘Mickylee’ watermelon were used as sources of inoculum. I
also compared the susceptibility of cucurbit weeds and watermelon to SqVYV when
inoculated by whitefly. Finally, I quantified whitefly host acceptance and oviposition
behavior on infected and mock-inoculated creeping cucumber to better understand how
infection by a semi-persistently transmitted virus might lead to a change in vector
behavior that would influence virus spread.
Material and Methods
Virus Isolate and Virus Sources
The SqVYV isolate used for experiments was originally collected from squash
(Cucurbita pepo L.) in Hillsborough County, FL in 2003. It was maintained in ‘Gentry’
squash and ‘Mickylee’ watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var.
lanatus] by mechanical and whitefly inoculation. Plants were mechanically inoculated
every 2 wk and then inoculated by whiteflies at 8 wk, after which the maintenance cycle
41
was repeated. For mechanical inoculation of the source plants, foliar and petiole tissue
from infected squash and watermelon plants was ground in 20 mM potassium
phosphate buffer (pH 7.4) containing corundum (300-400 mg/ml) and rubbed on three to
five upper leaves, using cheesecloth. After 10-15 min, inoculated leaves were washed
gently with tap water. Whitefly transmission is described below.
Whitefly
The main whitefly colony was maintained on cotton (Gossypium hirsutum L.) and
‘Vates’ collard (Brassica oleracea L. var. acephala) as described by Chen et al. (2004).
Whiteflies were reared in a room maintained at 25-30ºC, under a photoperiod of 14:10
(L: D). A new cohort was established on four to six cotton plants for each experiment.
These cotton plants were exposed to the main whitefly colony for 48 h for oviposition.
Then cotton plants were transferred to an insect rearing cage (60 cm × 60 cm × 60 cm
Bug Dorm, MegaView Science Co. Ltd., Taiwan) for 14 d. After that each cotton plant
was placed in an individual insect rearing cage for 3-4 d for adult emergence. One- to 5-
d-old adult whiteflies were used for the transmission experiments.
Comparison of Three Cucurbit Weeds and Watermelon as Sources of Inoculum by Whitefly Inoculation
Plant materials
Balsam apple seeds were collected from Gainesville, FL, creeping cucumber
and smellmelon seeds were provided by Linda Wessel-Beaver (University of Puerto
Rico, Mayagüez, Puerto Rico), and Scott T. Adkins, respectively. To increase
germination and to reduce fungal contamination, seeds of creeping cucumber, balsam
apple, and smellmelon were surfaced sterilized using 10% bleach (0.83% sodium
hypochlorite) water solution for 2-3 min and then rinsed with sterile distilled water. To
42
increase the germination percentage of balsam apple, seed coats were forced open by
pressing forceps at the place of radical emergence. Then all seeds were placed in Petri
dishes on moistened seed germination blotter paper (Anchor Paper Co. Seed Solutions,
Saint Paul, MN) and placed in an incubator for 4-8 d at 28-29ºC. Germinated seeds
were planted in plastic seedling tray inserts (4 cm × 5.5 cm × 4 cm, T.O. Plastics,
Clearwater, MN) filled with potting medium (Sunshine Professional Growing Mix MVP,
Sun Gro Horticulture, Bellevue, WA) amended with Osmocote (14:14:14, Everris NA,
Inc., Dublin, OH) at a rate of 10 cm3 in 3785 cm3 potting medium. ‘Mickylee’ watermelon
was planted directly in the same potting medium in the seedling tray inserts. Plants
were grown in a greenhouse [26-32ºC, photoperiod of 14:10 (L: D)] for all experiments.
Source of inoculum
To establish plants as sources of inoculum, seedlings were transplanted into
15.24-cm-diameter plastic pots 12 d after planting watermelon seeds and germinated
weed seeds in the seedling tray inserts. Watermelon and weeds were inoculated
mechanically 24 d after transplanting using foliar and petiole tissue from infected
watermelon and squash plants as described above. Plants were tested 8-9 d post-
inoculation (DPI) with reverse transcription-polymerase chain reaction (RT-PCR)
(Adkins et al. 2008) to confirm infection with SqVYV before use in the transmission
procedure 11-12 DPI.
Recipient plants
Watermelon seedlings grown for use as recipient plants were transplanted into
10.16-cm-diameter plastic pots 14 d after seeds were planted. Recipient plants were
used in the transmission procedure 10 d after transplanting (24 d after planting).
43
Transmission procedure
Single source plants of each weed species (creeping cucumber, balsam apple, or
smellmelon) or watermelon were placed in one of the insect-rearing cages with the
cotton plant infested with adult whiteflies (described above). Cotton plants were shaken
to remove whiteflies and then removed from the cage. Whiteflies were allowed to feed
on the source plants for 4 h (acquisition access period, AAP) and then aspirated into
glass tubes (eye droppers with the large end screened) in groups of 30 using methods
described in Webb et al. (2012). Whiteflies were tapped into clip cages. Each cage was
then attached to a watermelon recipient plant with one screened side in contact with the
abaxial surface of the leaf for a 24-h inoculation access period (IAP) (Webb et al. 2012).
Clip cages were removed from recipient plants after a 24-h IAP. Recipient plants were
moved to the greenhouse and treated immediately with pymetrozine at a rate of 0.025 g
(a.i.) /100 ml water till runoff from leaves to kill any possible hatching nymphs.
SqVYV detection
Petiole samples were taken from the watermelon recipient plants 11-12 DPI and
tested with enzyme-linked immunosorbent assay (ELISA) for the presence of SqVYV
(Webster et al. 2010). Symptoms such as chlorosis, wilting, vein yellowing, necrosis of
leaves, petioles and stems were recorded daily until petiole samples were taken. Both
ELISA and symptom expression were used to confirm infection status.
Experimental design and statistical analysis
Altogether, there were four source plant treatments (watermelon, creeping
cucumber, balsam apple, and smellmelon) with 12 recipient watermelon plants per
treatment. Twelve plants of non-inoculated watermelon were added as sentinel plants to
detect possible cross contamination for each trial. This experimental design was a
44
randomized complete block design (RCBD) with blocks consisting of seven trials
(overall experiment contains, 84 recipient plants for each treatment) completed over a
10-wk period. The infection status of recipient plants was analyzed by using a binomial
response (infected vs. non-infected). Generalized Linear Mixed Model was conducted
using PROC GLIMMIX in SAS 9.4 where block was a random effect (SAS institute,
Cary, North Carolina, 2013). Multiple comparisons of treatments were conducted by
least square means with Tukey-Kramer option at a 5% level of significance.
Comparison of Three Cucurbit Weeds and Watermelon for Susceptibility to SqVYV by Whitefly Inoculation
Source of inoculum and recipient plants
Watermelon plants were established as sources of virus inoculum using the
same methods described in the previous experiment. Watermelon and germinated
weed seeds were treated the same way as watermelon in the previous experiment to
generate the recipient plants. However, recipient plants were used in the transmission
process 28 d after planting, rather than 24 d.
Transmission procedure
All procedures were similar to the previous experiment; however two to three
cotton plants were used as sources of whiteflies. The inoculated leaf of the recipient
plant was marked by tying a cotton thread around the petiole. Those marked leaves
were removed from the recipient plants 4-5 DPI as they often showed evidence of
whitefly-induced silvering, which masked expression of symptoms of SqVYV infection.
SqVYV detection
All the procedures to detect virus were similar to the previous experiment for the
watermelon recipient plants. However, for all the weed recipient plants, RT-PCR was
45
conducted using petiole tissue. At 10-11 DPI, symptoms on plants were monitored as in
the previous experiment and were rated on a scale of 1 to 9 using methodology adapted
from Kousik et al. (2009).
Experimental design and statistical analysis
In all, there were four recipient plant treatments (watermelon, creeping
cucumber, balsam apple, and smellmelon). There were 10 recipient plants per
treatment and 10 extra watermelon plants for each trial, which served as sentinel plants
in the greenhouse to detect cross contamination. This experimental design was RCBD
like the previous experiment with five trials (blocks) conducted over a 6-week period.
There were 50 recipient plants per treatment in this experiment. Statistical analysis was
conducted as in the previous experiment. Symptom ratings from infected plants were
analyzed using the Kruskal-Wallis test using PROC NPAR1WAY and treatments were
compared by Dwass, Steel, Critchlow-Fligner multiple comparison analysis at a 5%
level of significance.
Whitefly Settling and Oviposition Preference
Creeping cucumber plants were chosen for this experiment because of their
susceptibility to SqVYV, which was similar to watermelon, seed availability and ease of
germination, and their more prominent infection symptoms compared to other weeds.
Plants were grown as in the previous experiment and transplanted into 15.2-cm-
diameter plastic pots 2 wk after planting. Half the creeping cucumber plants were
inoculated mechanically with SqVYV, and the other half were mock-inoculated (rubbed
with buffer only) at 3 wk after transplanting. Creeping cucumber plants were used in the
experiment 7 wk after planting (2 wk after inoculation). In the laboratory, a pair of
SqVYV-infected and mock-inoculated creeping cucumber plants were placed next to
46
each other, and a leaf from the middle stratum of the vine was selected from both plants
for the dual choice test. Only plants showing SqVYV symptoms (vein yellowing) were
used as SqVYV-infected plants. The test was conducted using a dual choice Petri dish
cage (9 cm in diameter) (Figure 2-1) (Cardoza et al. 2000). Forty non-viruliferous
whiteflies of mixed ages, 20 males and 20 females, were introduced through a 0.5-cm
hole in the bottom of the Petri dish; then the hole was plugged using high-density foam.
Whiteflies were allowed to settle and oviposit on the exposed abaxial (underneath) leaf
surfaces. Whiteflies settling on each leaf was counted at 0.25, 2, 5, 24, 48, and 72 h.
After 72 h, leaves used in the experiment were detached from plants and eggs were
counted under a stereo microscope at 25X magnification. In total, 30 pairs of infected
and mock-inoculated creeping cucumber plants were used.
To test whether the whiteflies were able to acquire and transmit the virus from
infected to mock-inoculated creeping cucumber during the 72-h settling and oviposition
preference test, the 30 mock-inoculated plants were brought into the greenhouse
following the above-mentioned preference test. After 2 wk, all creeping cucumber plants
were tested with ELISA using crown (main stem just above the soil line) instead of
petiole tissue because crown tissue has been shown to contain higher concentrations of
SqVYV than other parts of the plant in watermelon (Turechek et al. 2010).
Data from the settling experiment were analyzed by repeated-measures ANOVA.
Data from the experiment were square root transformed to meet the assumption of
normality. Tukey’s mean separation test was used to compare different treatment
means with α = 0.05. Oviposition data were analyzed using one-way ANOVA.
47
Results
Comparison of Three Cucurbit Weeds and Watermelon as Sources of Inoculum by Whitefly Inoculation
A similar percentage of watermelon recipient plants was infected regardless of
whether creeping cucumber, smellmelon or watermelon were used as source plants
(Table 2-1). However, the percentage of recipient plants infected was lower when
balsam apple was used as the source plant (Table 2-1). The effect of treatment was
significant (F= 18.04; df= 3, 12; P = 0.001). Symptoms of SqVYV on the recipient plants
(light greening and crinkling of upper leaves) were seen as early as 4 DPI.
Comparison of Three Cucurbit Weeds and Watermelon for Susceptibility to SqVYV by Whitefly Inoculation
The percentage of watermelon infected with SqVYV was higher than for
smellmelon and balsam apple, but there was no difference between watermelon and
creeping cucumber (Table 2-2). Also, there were no significant differences in the
percentage of plants infected among smellmelon, creeping cucumber, and balsam
apple (Table 2-2). The effect of treatment was significant (F= 4.2; df= 3, 12; P = 0.0295).
Weed recipient plants when infected showed slight to no symptoms compared to
watermelon recipient plants (Table 2-2). There were significant differences in the
symptom severity for infected weeds and watermelon recipient plants (χ2= 119.61; df=
3; P < 0 .0001).
Whitefly Settling and Oviposition Preference
Whiteflies showed no settling preference between SqVYV- and mock-inoculated
plants at 0.25 h, 2 h, and 5 h (Figure 2-2). However, after 24 h, more whiteflies had
moved onto the leaf of the mock-inoculated plant than onto the leaf of the infected plant
(Figure 2-2). The interaction of plant status (infected or mock-inoculated) and sampling
48
time was significant (F = 5.5; df = 5, 304.7; P < 0.0001). In addition, plant status effect
(F= 6.74; df= 1, 123.2; P = 0.0106) and the sampling time effect (F = 34.5; df = 5, 300.2;
P < 0.0001) were significant. The number of eggs laid by whiteflies after a 72-h
exposure period was higher on mock-inoculated creeping cucumber leaves (322.9 ±
26.76) than on SqVYV-inoculated leaves (209.57 ± 18.14) (F= 14.23; df= 1, 57; P =
0.0004). Two wk after the preference test, 13 of 30 mock-inoculated creeping cucumber
plants were infected with SqVYV, indicating that whiteflies were able to acquire the virus
from infected plants and transmit to non-infected mock-inoculated plants.
Discussion
My results suggest that all three weeds have similar susceptibility to SqVYV
infection by whitefly inoculation, but these weeds differ as sources of inoculum of the
virus. Infected creeping cucumber, smellmelon and watermelon were better sources of
virus than balsam apple. This may be due to differences in viral concentrations in the
different weed species. Adkins et al. (2008) showed that balsam apple had a lower
concentration of SqVYV than creeping cucumber and watermelon when infected.
Although there are several factors influencing the transmission of the virus, one of the
important factors is the concentration of the virus in the source plants or leaves. In most
cases, a higher concentration of virus in the source leads to a higher probability of
vectors transmitting the virus (Romanow et al. 1986, Gray et al. 1991, Zeidan and
Czosnek 1991). In my experiments, foliar symptoms on infected weeds were mild to
non-existent, making them difficult to identify visually as infected. This has been found
for other plant virus systems in which infected weeds show fewer foliar symptoms
compared to cultivated host plants (Tomlinson et al. 1970, Duffus 1971, Chatzivassiliou
et al. 2001). Slight symptom expression and low mortality when infected, combined with
49
widespread distribution and proximity of cucurbit weeds to watermelon cultivating areas
in Florida, increase the risk for the watermelon production.
Balsam apple is as susceptible to infection by whitefly inoculation as the other
weeds, but when used as a source of inoculum, fewer recipient plants became infected
than when the other weeds and watermelon were the source of virus for whiteflies. It is
possible that virus titer in the plants is high enough to be detected with PCR from
petioles, but not enough for whiteflies to acquire it efficiently from infected leaves. In this
experiment, SqVYV detection was conducted using petiole tissue, but whiteflies feed on
the leaves. It is also possible that whiteflies do not feed as readily on balsam apple as
they do on the other plants tested. Susceptibility of the plants can also be related to the
feeding behavior of vectors (Shukle et al. 1987, Jing et al. 2015). In the transmission
experiments, weeds were tested with RT-PCR because ELISA using petiole tissue of
weeds did not reliably detect SqVYV, which might be due to lower virus titer in these
plants compared with watermelon.
Initially, whiteflies settled equally on leaves of infected and mock-inoculated
plants of creeping cucumber, increasing the probability that the whiteflies will become
viruliferous. The subsequent shift in preference for mock-inoculated plants can increase
the probability of transmission of SqVYV to non-infected plants. This shift of preference
for the mock-inoculated plants is another way that virus dispersal is enhanced. Virus
spread will increase if the infected plants show higher attractiveness to vectors until
vectors land and probe to acquire the virus. After the vector acquires the virus, the time
that vectors spend on the infected plants should be reduced, and dispersal to
neighboring plants increased, for optimal spread of the virus (Fereres and Moreno
50
2009). The spread of semi-persistent virus depends on whiteflies feeding for several
hours to acquire virus efficiently before moving to non-infected plants. The shifting of
whitefly settling preference was found to occur between 5 and 24 h. This time is
sufficient for the whitefly to acquire and then transmit virus from infected plants to non-
infected plants (Webb et al. 2012), which is also supported by my findings that almost
half of the mock-inoculated plants were infected after use in the choice experiment.
Previous studies have found vectors more attracted to infected plants or equally
attracted to infected and non-infected plants and later repelled by the lower nutritional
status of infected plants relative to healthy plants (Mann et al. 2009, Mauck et al. 2010).
The change in alighting and settling preference of vectors caused by virus-induced
changes in the host could potentially promote the spread of virus by increasing the rate
of virus acquisition and transmission (Mauck et al. 2010, 2012, McMenemy et al. 2012,
Rajabaskar et al. 2014). Virus infection alters emission of plant volatiles and nutritional
status (Ajayi 1986, Jiménez-Martínez et al. 2004, Colvin et al. 2006, Mauck et al. 2010,
McMenemy et al. 2012, Su et al. 2015), which can modify vector behavior to ensure the
survival and spread of the virus. A similar phenomenon might hold true for the SqVYV
and whitefly vector system. For instance, Adkins et al. (2013) showed changes in plant
micronutrient composition when watermelon plants were infected with SqVYV.
Whiteflies prefer to lay eggs on mock-inoculated creeping cucumber leaves
compared with SqVYV-infected leaves, which might be related to lower nutritional status
of the infected host for their offspring. Similar results were also reported by Mann et al.
(2008) on cotton plants infected with Cotton leaf curl virus transmitted by B. tabaci.
However, contrasting results were observed where MEAM1 preferred to lay more eggs
51
on Tobacco curly shoot virus and Tomato yellow leaf curl China virus infected tobacco
plants than on non-infected plants (Jiu et al. 2007). Likewise, B. tabaci Mediterranean
(previously B. tabaci biotype Q) preferred Tomato yellow leaf curl virus infected Datura
stramonium plants to non-infected plants for oviposition (Chen et al. 2013). In a
previous experiment, Asia II 3 (previously B. tabaci biotype ZHJ1) showed no
preferences for egg laying on infected and non-infected plants (Jiu et al. 2007). The
effects of plant viruses on biology and behaviors of insect vectors may vary according to
the host plants, vector species, virus species, and environmental factors.
Vector settling preference might be dependent on the host plants used (Castle et
al. 1998), so in the future, the effects of additional weed hosts on the modification of
whitefly behaviors should be examined. Further, studies should seek to understand the
potential role of change in the nutrient and volatiles composition and concentration in
the modification of whitefly behavior. The composition of weed species present,
susceptibility to SqVYV, and preference by whiteflies can influence survival and spread
of the virus in the field. All of these factors should be considered when developing
scouting, forecasting, and management options for this important viral disease.
52
Table 2-1. Mean ± SEM percentage infection of watermelon recipient plants with SqVYV transmitted by whitefly, Bemisia tabaci (Middle East Asia Minor 1) with access to different source plant species.
Source of inoculum (treatments as source plants)
Infection percentage of watermelon recipient plantsa
Watermelon 81.9 ± 4.4a Smellmelon 73.6 ± 4.6a Creeping cucumber 72.2 ± 2.2a Balsam apple 16.7 ± 6.9b
aMean ± SEM percentage infection with SqVYV of watermelon recipient plants inoculated with 30 MEAM1 from four sources of virus inoculum (treatments). Means within a column that share a letter are not significantly different (Tukey-Kramer test, P < 0.05). Statistical inference was based on logit-link transformed data; untransformed means are shown. Total of 84 recipient plants per treatment were used.
Recipient plants were tested by ELISA at 11-12 DPI.
53
Table 2-2. Mean ± SEM percentage infection with SqVYV and symptom expression of different recipient plant species when whitefly, Bemisia tabaci (Middle East Asia Minor 1) were allowed access to infected watermelon as source of virus inoculum.
Susceptibility (treatments as recipient plants) Symptoma
Mean symptom ratingb (1-9)
Infection percentagec
Watermelon VN, PN, LC, LN, SN, VY, W
7.01a 80 ± 5.7a
Smellmelon - 1c 50 ± 7.1b
Creeping cucumber VY 2.33b 72 ± 6.4ab
Balsam apple - 1.15c 58 ± 7.0b aSymptom expression of SqVYV on recipient plant infected with SqVYV 10-11 DPI: VN, vein necrosis, PN, petiole necrosis; LC, leaf chlorosis; LN, leaf necrosis; SN; stem necrosis; VY, vein yellowing; W wilting; -, no symptoms. bMean symptom rating recorded 10-11 DPI. Rating scale for SqVYV infection of watermelon was adapted from Kousik et al. (2009) to use for the cucurbit weeds. Data were analyzed with Kruskal-Wallis test and within a column treatments that share a letter are not significantly different (Dwass, Steel, Critchlow-Fligner multiple comparison analysis, P < 0.05). cMean ± SEM infection percentage of recipient plants (treatments) inoculated with 30 MEAM1 which acquired SqVYV from watermelon source plants. Means within a column that share a letter are not significantly different (Tukey-Kramer test, P < 0.05). Statistical inference was based on logit-link transformed data; untransformed means are shown. Total of 50 recipient plants per treatment were used. Weed recipient plants were tested by RT-PCR and watermelon recipient plants were tested by ELISA at 11-12 DPI.
54
Figure 2-1. Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) settling and oviposition preference experiment on creeping cucumber leaves. The Petri dish cage was made of a Petri dish (9 cm diameter) with two holes (2 cm diameter) in the lid and the bottom screened with plastic mesh (50 by 24). Two creeping cucumber leaves (infected and mock-inoculated) attached to their respective plants were placed opposite each other and whiteflies were introduced through a 0.5-cm hole in the bottom of the cage. Photo courtesy of author.
55
Figure 2-2. Number of whiteflies, Bemisia tabaci (Middle East Asia Minor 1) settled on leaves of SqVYV-infected and mock-inoculated creeping cucumber leaves counted at 0.25 h, 2 h, 5 h, 24 h, 48 h, and 72 h in a dual choice test in a Petri dish clip cage, releasing 40 whiteflies per replicate. Error bars are SEM and asterisk (*) indicates significant differences in interactions for status and time. (N= 30 replicates)
0
5
10
15
20
25
0 10 20 30 40 50 60 70 80
Infected
Mock
*
*
Time after whitefly release
Status:
Wh
itefl
y n
um
ber
*
(0.25 h)
(2 h)
(5 h)
(24 h)
(48 h)(72 h)
56
CHAPTER 3 HOST-MEDIATED EFFECT OF SQUASH VEIN YELLOWING VIRUS ON
SWEETPOTATO WHITEFLY (HEMIPTERA: ALEYRODIDAE) BEHAVIOR AND FITNESS
Introduction
Insect-transmitted plant viruses are dependent on vectors for survival and
dissemination. Alteration of the vector behavior and biology, affecting virus transmission
and spread, has been documented in several studies (Fereres and Moreno 2009,
Moreno-Delafuente et al. 2013). Plant viruses can influence insect vector behavior and
life history parameters directly (Shrestha et al. 2012, Moreno-Delafuente et al. 2013),
and indirectly via the host plant (Liu et al. 2010, Chen et al. 2013, Legarrea et al. 2015).
Indirect effects of plant viruses on vector behaviors include changes in vector attraction
(McMenemy et al. 2012, Fang et al. 2013), settling (Castle et al. 1998, Eigenbrode et al.
2002, Mann et al. 2009), feeding (He et al. 2015, Ren et al. 2015), oviposition (Maris et
al. 2004), and life history parameters (Srinivasan et al. 2008, Su et al. 2015). The
observed effects tend to be specific to the particular host plant, vector and virus
complex under study.
Mauck et al. (2012) and Gutiérrez et al. (2013) provided evidence that insect-
transmitted plant viruses may modify host plant phenotype and physiology, affecting
plant–vector interactions. Plant viruses may be transmitted by insect vectors in a non-
persistent, semi-persistent, persistent, or propagative manner. The first two modes of
transmission are referred to as non-circulative, whereas the latter two are referred to as
circulative (Ng and Falk 2006). Recent work on non-persistently and semi-persistently
transmitted viruses suggests that insect vectors are initially attracted to infected plants
and after a certain period of settling and feeding on infected plants, will be repelled and
57
move to non-infected plants, resulting in increased transmission of virus (Hodge and
Powell 2008, Mauck et al. 2010, McMenemy et al. 2012, Carmo-Sousa et al. 2014).
Other studies have shown reduced preference or no effects on the preference of insects
for plants infected by non-persistent viruses (Castle et al. 1998). For persistently and
propagatively transmitted viruses, most of the studies suggest increased attraction,
settling, oviposition and feeding behavior of insect vectors on the infected plants
(Eigenbrode et al. 2002, Jiménez-Martínez et al. 2004, Hodge and Powell 2010, Fang et
al. 2013, Legarrea et al. 2015).
Indirect effects of plant viruses on the biological fitness of insect vectors,
including fecundity, development, longevity, survival, and population growth, have been
reported as antagonistic (Donaldson and Gratton 2007, Mann et al. 2008, Mauck et al.
2010, McMenemy et al. 2012), neutral (Matsuura and Hoshino 2009), or beneficial
(Hodge and Powell 2008, Srinivasan et al. 2008, Chen et al. 2013), depending on the
species involved (Colvin et al. 2006, Hodge and Powell 2008, Mauck et al. 2012).
Although vector biology depends on the specific plant virus and host plant system, there
have generally been more documented beneficial effects on insect fitness when plants
were infected with persistent viruses than with non-persistent viruses (Blua and Perring
1992, Jiu et al. 2007, Srinivasan and Alvarez 2007, Hodge and Powell 2008, Mauck et
al. 2012). These indirect effects of plant viruses on insect vectors are mediated by
alteration in plant phenotype (Ajayi and Dewar 1983, Hodge and Powell 2008), emission
of volatile organic compounds (VOCs) (Ajayi 1986, Jiménez-Martínez et al. 2004, Fang
et al. 2013, Rajabaskar et al. 2013), changes in plant nutritional quality (Colvin et al.
58
2006, Mauck et al. 2010), and alteration in plant chemicals/toxins (Nachappa et al.
2013, Su et al. 2015).
Most of the studies on indirect effects of viruses on insect vectors have
considered only persistently and non-persistently transmitted viruses (Mauck et al.
2012). Thus, there is a lack of information regarding semi-persistently transmitted
viruses (McMenemy et al. 2012, Lightle and Lee 2014, Shrestha et al. 2016). The
current study explores the host-mediated effects of semi-pesistently transmitted Squash
vein yellowing virus (SqVYV) (family: Potyviridae, genus: Ipomovirus) (Webb et al.
2006, 2012; Adkins et al. 2007) on the biology and behavior of its vector Bemisia tabaci
Middle East Asia Minor 1 (MEAM1), formerly known as the sweetpotato whitefly [B.
tabaci (Gennadius) biotype B] and B. argentifolii (Bellows & Perring) (Bellows et al.
1994, De Barro et al. 2011, Boykin 2014). Squash vein yellowing virus is detrimental to
watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] and is the cause
of viral watermelon vine decline in Florida (Roberts et al. 2005, Adkins et al. 2007).
Infected watermelon exhibits mild vein yellowing with chlorotic lesions followed by
systemic wilting and necrosis, ultimately leading to plant death (Adkins et al. 2013,
Webster et al. 2013). Infected squash (Cucurbita pepo L.) plants show distinct vein
yellowing symptoms, but do not wilt and collapse like watermelon plants. Since its
discovery in Florida in 2003, SqVYV has also been detected in Indiana, Georgia, South
Carolina, Puerto Rico, and recently in California, Guatemala and Israel (Adkins 2007,
Egel and Adkins 2007, Webster and Adkins 2012, Acevedo et al. 2013, Adkins et al.
2013, Batuman et al. 2015, Jeyaprakash et al. 2015, Reingold et al. 2016).
59
The first objective of this study was to determine whether whitefly alighting,
settling and oviposition were altered due to the host-mediated effect of SqVYV infection
on watermelon and squash. The second objective was to determine if there were host-
mediated effects of SqVYV on whitefly fitness (nymphal developmental period,
fecundity, adult longevity, and body size) on squash plants. Results from these
experiments contribute to understanding of whitefly behavior and biology, and the role in
the spread and survival of SqVYV in agroecosystems.
Materials and Methods
Biological Material: Whitefly Colonies, Plants, and Virus Isolate
The whitefly (MEAM1, B. tabaci biotype B) colony was maintained on
‘DP0935B2RF’ cotton (Gossypium hirsutum L.) and ‘Vates’ collard (Brassica oleracea L.
var. acephala) as described by Chen et al. (2004). Whiteflies were reared in a
laboratory at the University of Florida’s Entomology and Nematology Department
maintained at 25-31ºC, under a photoperiod of 14: 10 h (L: D). New cohorts of whiteflies
were established on cotton plants. Cotton plants were exposed to the main whitefly
colony for 24 h for oviposition. After dislodging the adult whiteflies from the cotton
plants, plants were transferred to an insect cage (60 cm × 60 cm × 60 cm Bug Dorm,
MegaView Science Co. Ltd., Taiwan) for 14 d, until adult emergence. The SqVYV
isolate used in this experiment was originally collected from squash in Hillsborough
County, FL in 2003 and was maintained in ‘Gentry’ squash and ‘Mickylee’ watermelon
by mechanical inoculation. For mechanical inoculation, foliar and petiole tissues from
infected squash and watermelon plants were homogenized in 20 mM potassium
phosphate buffer (pH 7.4) containing corundum (100-200 mg/ml) and rubbed on two to
60
three upper leaves, using cheesecloth. After 10-15 min, inoculated leaves were rinsed
gently with tap water.
Squash and watermelon seeds were planted in plastic seedling tray inserts (4 cm
× 5.5 cm × 4 cm, T.O. Plastics, Clearwater, MN) filled with potting medium (Sunshine®
Professional Growing Mix MVP, Sun Gro® Horticulture, Bellevue, WA). Osmocote
(14:14:14, Everris NA, Inc., Dublin, OH) was added to potting medium at rate of 1 part
per 280 parts (i.e., 5 ml in 1.4 liters soil). Fourteen days after planting, plants were
transplanted into plastic pots of various sizes and then inoculated and used depending
upon the experiments. For each experiment, half the watermelon and squash plants
were inoculated with SqVYV (using above method) to produce infected plants and the
other half was treated with a buffer and corundum solution to produce mock-inoculated
plants. In the case of infected test plants, plants showing symptoms of vein yellowing
were used; mock-inoculated plants had no symptoms. Plants were grown in a
greenhouse at the Entomology and Nematology Department, University of Florida,
Gainesville, Florida at a photoperiod of 14: 10 h (L: D) and 26-32°C for all experiments.
Alighting Preference
Pairs of infected and mock-inoculated watermelon or squash plants in 10.16-cm-
diameter plastic pots were used in choice tests 27-28 days after planting (DAP) and 9-
10 days post-inoculation (DPI). Pairs of plants were placed 10-12 cm apart in an insect
cage (60 cm × 60 cm× 60 cm Bug Dorm) (Figure 3-1). Six male and six female
whiteflies (1 to 3-d old) were aspirated into separate glass eyedroppers by sex. The
narrower end was sealed with Parafilm®. Males were distinguished from females by
their smaller size and pointed abdomens (Gill 1990). Each glass tube containing six
whiteflies was placed between each pair of plants in an upright position with the narrow
61
end pointing up, 15 cm away from midpoint of both plants. Parafilm® was removed from
the upper end and whiteflies were allowed to crawl up the glass tube and disperse. The
plant on which the first three whiteflies landed was recorded. This was repeated 32
times for a total of 96 females or 96 males making a choice. Both experiments were
completed in a span of 6 wk and conducted under overhead cool-white fluorescent
lights with light intensity of 1356 lux. To evaluate the roles of visual stimuli and plant
VOCs on alighting preference, the experiment was repeated in the dark, and whiteflies
were counted using a dim red light; however, due to lack of whitefly flight in the absence
of light, the dark condition treatment was removed from the experiment.
Data on alighting preference on watermelon and squash were analyzed
separately. Tests of binomial proportions against 0.5 were conducted with PROC FREQ
(test of proportion) in SAS (SAS 9.4).
Settling and Oviposition Preference
Pairs of infected and mock-inoculated watermelon and squash plants in 15.24-
cm-diameter plastic pots were used in a choice tests at 34-36 DAP and 10-12 DPI .
Each replicate consisted of one pair of squash plants and one pair of watermelon
plants. A pair of plants (infected and mock-inoculated) were placed 15-18 cm apart
inside an organdy cloth cage cage (60 cm × 60 cm × 60 cm) with PVC pipe as frame
(Figure 3-2). Fifty pairs of male and female whiteflies (1 to 3-d old) were collected in four
to five glass eye droppers from the whitefly cohort. Whiteflies were released from the
eye droppers 15 cm away from the midpoint between the plants by gently removing the
Parafilm from the top of the tubes. Whiteflies were counted on each plant with the aid of
a mirror over a 72-h time period (0.25, 1, 2, 4, 8, 24, 48, and 72 h). After 72 h, all the
whiteflies were dislodged from the test plants, which were then moved into the
62
laboratory so that eggs could be counted. All leaves were cut from the plants and eggs
were counted from entire leaves using a stereo microscope (25X). To determine the
effect of foliage area on the number of whiteflies settled and eggs laid at 72 h, the area
of all leaves was measured using a leaf area meter (Li-3100 area meter, LI-COR Inc.
Lincoln, Nebraska). Data were recorded as number of eggs laid per plant and per cm2
of leaves, and number of whiteflies settled per cm2 of leaves. There were 24 replicates
in this experiment conducted over an 8-wk period in the month of March and April in a
greenhouse.
T-tests were conducted separately for watermelon and squash to determine the
differences in leaf area of infected and mock-inoculated plants. Number of whiteflies
settled per cm2 of leaf at 72 h and eggs laid on both whole plants and per cm2 of leaf
were square root transformed to meet assumptions of normality. Settling preference
over the 72-h time period was analyzed using a repeated-measures approach (PROC
GLIMMIX in SAS) with treatments analyzed separately by plant species and sliced by
time. Means were compared using Tukey–Kramer test (α = 0.05). Number of eggs laid
on whole plants and per cm2 and number of whiteflies settled per cm2 settled at 72 h
were analyzed using PROC GLIMMIX (sliced by plant species). Least Squares Means
were used to compare the treatments.
Developmental Time of Immature Stages and Adult Size
Due to the wilting and collapse of SqVYV-infected watermelon, only squash
plants were used for these experiments. Infected and mock-inoculated squash plants
(28-29 DAP, 8 DPI) in 25.4-cm-diameter pots were brought into the laboratory [26-29ºC,
14: 10 h (L: D)] and placed under overhead cool-white fluorescent lights as in the
previous experiment. Ten pairs of male and female whiteflies (1 to 3-d old) were
63
aspirated into a glass eye dropper and then tapped into a clip cage. The clip cages were
attached to the abaxial surface of the third or fourth leaf of test plants. Whiteflies were
allowed to oviposit for 6 h, and then the clip cages were removed. Numbers of eggs laid
on the leaves were counted using a stereo microscope (20X). A maximum of 55 eggs
were retained and remaining eggs were carefully removed using a moist cotton swab.
After 14 d, the number of newly emerged adults was recorded daily at 10:00 am until all
nymphs had developed to adults. There were 40 replicates in this experiment. From 14
d onward, 150 male and 150 female adults were collected from 15 test plants (10 males
and 10 females per plant) for each infected and mock-inoculated plant. The length from
the head to the tip of the abdomen was measured for each whitefly using a stereo
microscope (40X). Total time span for the experiment was 10 wk.
Development time from egg to adult emergence and length of adult male and
female did not follow a normal distribution and could not be normalized so the data were
analyzed using PROC NPAR1WAY in SAS
Adult Longevity and Fecundity
Whiteflies were reared from egg to the end of nymphal stage on the source
plants. Emerged adult whiteflies were then transferred and allowed to feed on the test
plants, either infected or mock-inoculated, to measure adult longevity and fecundity. The
experiment was designed as a 2 × 2 factorial with infected and mock-inoculated source
plants and infected and mock-inoculated test plants.
Source plants
Infected and mock-inoculated squash plants (28 DAP, 2-3 DPI, transplanted in
25.4-cm diameter pots) were introduced to the whitefly colony for 6-7 h for oviposition.
After that time, all whiteflies were dislodged from the plants and these plants were
64
placed in a greenhouse. Whiteflies were allowed to develop until the late nymphal stage
about 16-19 d. Then, five to six leaves containing late instar nymphs were cut from each
plant. Leaf petioles were immersed in a beaker of tap water to reduce wilting. Leaves
were kept in two cages for 22-24 h to allow for adult emergence; one cage was for
leaves from infected plants and the other for leaves from mock-inoculated plants.
Test plants
Infected and mock-inoculated squash plants (28-29 DAP, 8 DPI, transplanted in
25.4 cm diameter pots) were brought into lab and placed under overhead cool-white
fluorescent lights. After 22-24 h, two pairs (male and female) of whiteflies were collected
from each of the insect emergence cages, having either infected or mock-inoculated
leaves, and placed in four separate glass tubes (one pair per tube). Whiteflies were
sexed by looking at the tip of the abdomen under a stereo microscope and then those
whiteflies were added to the clip cage. Clip cages were then attached to the abaxial
surface of the third or fourth leaf down from the growing tip of infected or mock-
inoculated test plants. In this experiment, one of the pairs of whiteflies reared from
infected plants was placed on an infected plant and the other pair was placed on a
mock-inoculated plant. Similarly, one of the pairs of whiteflies reared from mock-
inoculated plants was placed on a mock-inoculated plant and the other pair placed on
an infected plant. Altogether, there were 40 replicates. This experiment was completed
within 14 wk.
To evaluate treatment effects on adult longevity, survival was recorded daily until
both male and female whiteflies were dead. Every 3 d, the clip cages were transferred
to a new third or fourth leaf on the same plant. To determine fecundity, leaves were cut
from the plant after the clip cages were transferred to a new leaf and eggs were counted
65
using a stereo microscope. Replicates with whiteflies that died within 24 h of being
placed on the test plants were removed from the experiment.
Adult longevity and fecundity were analyzed assuming a negative binomial
distribution and replicates were treated as a random effect. Data were analyzed using
the PROC GLIMMIX procedure in SAS, sorted by sex for adult longevity and least
squares means were used with Tukey-Kramer test (α = 0.05) for the treatment mean
comparison.
Results
Alighting Preference
On both plants, whitefly gender effects were non-significant, so the data for male
and female alighting were combined for analysis. Whiteflies showed no preference for
alighting on the infected vs. mock-inoculated watermelon plants. In contrast, whiteflies
preferred to alight on infected squash plants rather than on mock-inoculated squash
plants (Table 3-1). Due to non-significant effects of gender in alighting preference on
watermelon and squash, data for genders were combined for analysis (Table 3-1).
Settling and Oviposition Preference
Whiteflies showed no preference for settling until 4 h after release on
watermelon. After 8 h whiteflies preferred to settle on mock-inoculated watermelon
plants (Figure 3-3A). In the case of squash, whiteflies did not show the same shift of
preference for settling (Figure 3-3B). Although numerically higher numbers of whiteflies
settled on infected squash, statistically higher numbers were only recorded at 0.25, 1,
24 and 48 h (Figure 3-3B). Time after release was not a significant source of variation in
whitefly settling although there were significant two-way interactions and three-way
interactions among infection status, time of release and plant species, indicating that
66
change in whitefly settling preference over time differed on the two plant species (Table
3-2).
The total leaf area of infected squash plants was less (629 ± 14 cm2) than that of
mock-inoculated plants (743 ± 26 cm2) (t = 5.52, df = 23, P <0.0001). The leaf area of
infected watermelon plants was reduced by more than half (323 ± 47 cm2) compared to
mock-inoculated plants (793 ± 38 cm2) (t = 8.98, df = 23, P <0.0001).
At the end of the settling experiment (72 h after release), whiteflies had laid ca.
six times more eggs on mock-inoculated watermelon plants (F = 144.78, df = 1, 69, P
<0.0001), whereas statistically equivalent numbers of eggs were counted on mock-
inoculated and infected squash plants (F = 1.19, df = 1, 69, P = 0.29) (Figure 3-4A).
Similarly, the number of eggs per cm2 on mock-inoculated watermelon was
approximately twice that of infected plants (F = 35.34, df = 1, 69, P <0.0001), and no
significant differences in eggs per cm2 were found on infected and mock-inoculated
squash plants (F = 35.34, df = 1, 69, P = 0.94) (Figure 3-4B). Whitefly counts per cm2
were lower on infected (0.033 ± 0.007) than on mock-inoculated (0.101 ± 0.006)
watermelon plants (F = 70.21, df = 1, 69, P <0.0001). However, no differences were
found in the number of whiteflies settled per cm2 on infected (0.074 ± 0.006) versus
mock-inoculated (0.0607 ± 0.00515) squash plants (F = 1.76, df = 1, 69, P = 0.19). Plant
species (squash and watermelon) effect was not significant for numbers of eggs per
whole plant, and per cm2 of leaves, and whitefly counts per cm2. The significant
interaction between plant species and infection status (infected and mock-inoculated)
was explained by the whitefly’s differential response to infection in the two host plants
(Table 3-3).
67
Developmental Time of Immature Stages and Adult Size
The average total duration of immature stages was approximately 3 d shorter on
infected squash plants (F = 81.74, df = 1, 60, P < 0.0001) (Table 3-4). Whiteflies
emerged as adults as early as 14 d after oviposition, ranging to a maximum of 25 d. No
differences were recorded for the average length (µm) of adult male whiteflies (F = 0.07,
df = 1, 298, P = 0.79) or female whiteflies (F = 1.66, df = 1, 298, P = 0.19) on infected
and mock-inoculated squash plants (Table 3-4).
Adult Longevity and Fecundity
Male longevity did not differ among treatments (Table 3-5). Adult females
confined on infected plants lived ca. 25% longer, regardless of rearing plant infection
status, than did females confined on mock-inoculated plants and reared on infected
plants (Table 3-5). Longevity of females confined on mock-inoculated plants, and also
reared on mock-inoculated plants, was intermediate between the shortest and longest
longevities (Table 3-5). The effect of interaction between gender and treatment (F =
4.51, df = 3, 310.1, P = 0.0041) was significant, but the main effects of gender (F = 2.46,
df = 1, 310.3, P = 0.1176) and treatment (F = 0.79, df = 1, 310.1, P = 0.498) were not
significant. Whitefly fecundity was higher on infected plants than on mock-inoculated
plants when reared on either infected or mock-inoculated plants (F= 5.25, df = 3, P =
0.0001) (Table 3-5).
Discussion
It was observed that host-mediated effects on whitefly behavior were influenced
by infection status and host plant species. Whiteflies preferentially alighted on SqVYV-
infected squash plants, but no preference in alighting was seen for watermelon plants.
Virus infection of plants can cause changes in total VOCs emissions, or in emission of
68
particular volatile compounds (Eigenbrode et al. 2002, Jiménez-Martínez et al. 2004,
McMenemy et al. 2012, Fang et al. 2013, Rajabaskar et al. 2013) or in symptom
expression (Ajayi and Dewar 1983, Hodge and Powell 2008), which may influence
alighting behavior. Whitefly preference for alighting on infected-squash plants could be
associated with one or more of the above-mentioned virus-induced changes. Although
some studies suggest that olfaction can play a role in attraction of whitefly to the host
plants (Ying et al. 2003, Bleeker et al. 2009, Li et al. 2014), there are limited studies
which have examined the effect of virus-induced alteration in VOCs emission on whitefly
attraction (Fang et al. 2013). Whiteflies are known to orient preferentially toward yellow
and green surfaces (Mound 1962, Vaishampayan et al. 1975, Isaacs et al. 1999). The
infected squash plants exhibited distinct vein yellowing symptoms and light green leafy
area between yellow veins (Adkins et al. 2007, Webster et al. 2013), which might cause
whiteflies to orient toward infected plants. Infected watermelon also showed yellowing of
the leaf with transient vein yellowing but whiteflies showed no alighting preference
between infected and mock-inoculated watermelon plants (Adkins et al. 2007, Webster
et al. 2013). Previous studies have evaluated aphid alighting and settling preferences
under dark conditions to eliminate the influence of visual cues on choosing either
infected or non-infected plants (Eigenbrode et al. 2002, Srinivasan et al. 2006, Medina-
Ortega et al. 2009). However, in this study it was possible to determine the relative role
of visual cues and VOCs on alighting because whiteflies did not move under dark
conditions (data not shown).
The shift in preference from infected plants to mock inoculated plants after 8 h
increases the probability that the whitefly will transmit virus to other plants in the vicinity,
69
because only half an hour to a few hours is needed for the virus to be acquired (Webb
et al. 2012). These results corroborate those of my previous study on the common
cucurbit weed creeping cucumber (Melothria pendula L.), in which whitefly preference
shifted to mock-inoculated leaves from SqVYV inoculated leaves 24 h after release
(Shrestha et al. 2016). Results from alighting and settling assays on watermelon might
partially explain the rapid within-field spread of SqVYV in watermelon in Florida (Adkins
et al. 2007). This sort of shift in settling preference from infected to mock-inoculated
plants has also been documented in other vector-virus pathosystems (Mann et al. 2009,
Mauck et al. 2010, McMenemy et al. 2012, Fang et al. 2013). Migrations of vectors from
infected plants to non-infected plants were associated with the poor host quality of
infected plants compared to non-infected plants (Mauck et al. 2010, McMenemy et al.
2012). Given the demonstrated whitefly preference to lay fewer eggs on infected
watermelon plants in my study, it seems likely that infected watermelon is not a suitable
host for the whiteflies. Shrestha et al. (2016) also documented less oviposition by
whitefly on SqVYV-infected creeping cucumber leaves compared to mock-inoculated
leaves. Infected watermelon plants show wilting, necrosis of leaves, collapsing vines,
and altered nutrient content (Adkins et al. 2007, 2013, Webster et al. 2013), which could
reduce the nutrients and water available for uptake by whiteflies, thus making infected
watermelon an unsuitable host. Unlike watermelon, infected squash retained higher
numbers of whiteflies than mock-inoculated plants, especially at 0.25, 1, 8, and 24 h,
suggesting its suitability as a host. Based on the finding from settling assay, it can be
speculated that the spread of SqVYV in a squash field would be slower than in a
watermelon field, because whiteflies would be less likely to migrate from infected to
70
non-infected plants. These results suggest differential effects caused by virus on two
different host plants resulting in differential effects on whitefly settling behavior.
Divergent effects on alighting, settling and oviposition on different host plant species
have also been documented in other insect vector plant patho-systems (Castle et al.
1998, Srinivasan et al. 2006, Mauck et al. 2014).
Similar to the host-mediated effect of SqVYV on whitefly behavior, this study
found whitefly fitness was influenced by virus-induced changes in the plant. Although
male longevity and adult body size did not differ between infected and mock-inoculated
squash plants, development time from egg to adult was shorter, female longevity was
longer, and fecundity was higher on infected squash than on mock-inoculated squash,
indicating the enhancement of whitefly fitness on infected squash. Enhanced fitness of
whitefly on infected-squash plants indicates a higher potential for whitefly population
growth rate, which can ultimately lead to the spread of SqVYV. Higher biological fitness
of insect vectors was associated with virus-induced increases in nutrient content (amino
acids or carbohydrates) (Ajayi 1986, Fereres et al. 1990, Colvin et al. 2006) and
decreases of toxins or down regulation of defensive enzymes/genes (Nachappa et al.
2013, Su et al. 2015). My results from settling and fitness assays on squash also
suggest an improvement in host suitability for the whitefly following SqVYV infection.
Further work on SqVYV-induced changes in nutrient content, defensive compounds,
and volatile emission is warranted to elucidate the reason for changes in behavior and
fitness of the whitefly.
For insect vectors to spread plant viruses, infected plants must attract and retain
feeding vectors long enough for vectors to acquire the virus, a period of time that varies
71
with the mode of transmission (Carter and Harrington 1991, Mauck et al. 2012, Moreno-
Delafuente et al. 2013). My study indicates that the host-mediated effects of the virus on
the vector depend upon host plant species involved. In watermelon, the host-mediated
effects were most similar to the effects found with infection of non-circulative viruses,
where infected plants initially lure more vectors and then repel them after the vectors
have spent sufficient time to acquire virus (Mauck et al. 2010, McMenemy et al. 2012,
Carmo-Sousa et al. 2014). In squash, effects of SqVYV infection on whiteflies were
most similar to those observed with infection of viruses transmitted in a circulative
manner (Jiu et al. 2007, Srinivasan et al. 2008, Mauck et al. 2012).
This study has provided additional knowledge of the limited field of host-mediated
effects of a semi-persistently transmitted virus on its vector (McMenemy et al. 2012,
Lightle and Lee 2014, Shrestha et al. 2016), specifically on the host-mediated effects of
an ipomovirus on its whitefly vector. I have shown that infection of SqVYV in
watermelon and squash plants differently affects whitefly behavior, especially favoring
the rapid spread of the virus in watermelon fields. However, enhanced fitness of whitefly
on infected squash could potentially affect whitefly population dynamics. These
manipulations of its vector by the virus, depending upon the specific host plant have
potential for enhancing virus fitness by increasing chances of its spread and survival.
Further exploration of virus-mediated effects on behavior and biology of whitefly under
field conditions and epidemiological modeling are warranted to elucidate how these
effects could ultimately influence the spread of SqVYV and influence its management.
72
Table 3-1. Effect of plant species and Squash vein yellowing virus infection status on alighting preferences of male and female whiteflies, Bemisia tabaci (Middle East Asia Minor 1)
Plant Species Infection Status Male Female Totala χ2 df Pr > χ2
Watermelon Infected 49 55 104
1.33 1 0.25 Mock 47 41 88
Squash Infected 61 64 125
17.52 1 <0.0001 Mock 35 32 67
aPreference did not differ between males and females so genders were combined and total whiteflies were analyzed using χ2 test.
Table 3-2. ANOVA examining the number of settled whiteflies, Bemisia tabaci (Middle
East Asia Minor 1) on infected and mock-inoculated (infection status) squash and watermelon plants (plant species) recorded at 0.25, 1, 2, 4, 8, 24, 48, and 72 h (time) after their release
Source of Variation Num DF Den DF F Pr > F
Infection status 1 733 42.99 <0.0001
Plant species 1 733 7.47 0.0064
Time 7 733 1.98 0.0555
Infection status × Plant species 1 733 171.42 <0.0001
Infection status × Time 7 733 14.92 <0.0001
Plant species × Time 7 733 0.46 0.8666
Infection status× Plant species × Time
7 733 9.12 <0.0001
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Table 3-3. Effect of plant species (squash and watermelon) and infection status (Squash vein yellowing virus-infected and mock-inoculated) on number of eggs laid on entire plant and per cm2 of leaves by Bemisia tabaci (Middle East Asia Minor 1), and number of whiteflies settled per cm2 of leaves 72 h after release.
Source of Variation
Num
DF Den
DF
Mean no. eggs per plant
Mean no. of eggs per cm2 of leaves
Mean no. whiteflies per cm2
of leaves
F Pr > F F Pr > F F Pr > F
Infection status
1 69 84.4 <0.0001 18.15 <0.0001 24.86 <0.0001
Plant species 1 69 0.36 0.55 0.87 0.35 1.3 0.26
Infection status × Plant species
1 69 58.46 <0.0001 17.2 <0.0001 47.11 <0.0001
Table 3-4. Average duration of immature development and length of emerged adult
whiteflies, Bemisia tabaci (Middle East Asia Minor 1) on Squash vein yellowing virus-infected and mock-inoculated squash plants.
Mean ± SEMa
Status Development from egg to adult
emergence (days) Male length
(µm) Female
length (µm)
Infected 20.4 ± 0.2b 862 ± 4a 963 ± 4a
Mock 23.5 ± 0.2a 864 ± 3a 970 ± 3a aData were analyzed assuming nonparametric distribution using PROC NPAR1WAY and means within a column followed by the same letter did not differ significantly (P < 0.05)
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Table 3-5. Longevity and fecundity of adult whitefly, Bemisia tabaci (Middle East Asia Minor 1) that developed on infected or mock-inoculated squash plants (source plants) and were transferred upon emergence onto Squash vein yellowing virus-infected and mock inoculated squash plants (test plants).
Treatment Mean ±SEMa
Source plant Test plant Male longevity (d) Female longevity (d) Fecundity
Mock Mock 8.59 ± 0.09a 7.85 ± 0.09ab 50.35 ± 6.91b
Mock Infected 6.85 ± 0.09a 9.97 ± 0.09a 76.35 ± 9.34a
Infected Mock 8.21 ± 0.09a 7.17 ± 0.09b 45.98 ± 4.91b
Infected Infected 7.83 ± 0.09a 9.64 ± 0.09a 75.28 ± 8.22a aData were analyzed assuming negative binomial distribution using PROC GLIMMIX. Means in a column followed by the same letters were not significantly different by Tukey test (P < 0.05).
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Figure 3-1. Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) alighting preference experiment on watermelon plants inside a bugdorm (60 cm× 60 cm × 60 cm) having 10 DPI Squash vein yellowing virus infected and mock-inoculated watermelon plants. Three whiteflies (male and female) were released from the glass tubes 15 cm away from the midpoint between the plants. Photo courtesy of author.
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Figure 3-2. Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) settling and
oviposition preference experiment on infected and mock-inoculated A) squash and B) watermelon plants. Fifty pair of male and female whiteflies were released on the pair of Squash vein yellowing virus infected and mock-inoculated squash and watermelon plants separately in 60 cm × 60 cm × 60 cm organdy cage in the greenhouse and recording whitefly settling up to 72 h. Whiteflies were released from the glass tubes 15 cm away from the midpoint between the plants. Photo courtesy of author.
B A
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Figure 3-3. Number of whiteflies, Bemisia tabaci (Middle East Asia Minor 1) settled on
Squash vein yellowing virus-infected and mock-inoculated plants; A) watermelon or B) squash plants counted at 0.25, 1, 2, 4, 8, 24, 48, and 72 h after release in a dual-choice test done in an organdy cage, releasing 100 whiteflies per replicate. Error bars are SEM and asterisk (*) indicates significant differences between mock-inoculated and infected plants. Statistical inference was based on square root transformed data; untransformed means are shown. Data were analyzed using a repeated-measures approach (PROC GLIMMIX in SAS) with treatments analyzed separately by plants species and sliced by time. Treatments were compared using Tukey–Kramer test (α = 0.05). (N= 24 replicates).
Infected Mock
A
B
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Figure 3-4. Oviposition of whitefly, Bemisia tabaci (Middle East Asia Minor 1) on
Squash vein yellowing virus-infected and mock-inoculated plants of watermelon or squash. Data were expressed as mean numbers of eggs per A) plant or B) cm2 of leaves. Error bars are SEM and asterisk (*) indicates significant differences between mock-inoculated and infected plants. Statistical inference was based on square root transformed data using PROC GLIMMIX (sliced by plant species) and Least Squares Means were used to compare the treatments means. (N= 24 replicates).
AAA
B Infected Mock-inoculated
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CHAPTER 4 INDIRECT EFFECT OF SQUASH VEIN YELLOWING VIRUS ON BEMISIA TABACI
(MIDDLE EAST ASIA MINOR 1) (HEMIPTERA: ALEYRODIDAE) FEEDING AND SETTLING BEHAVIOR
Introduction
Squash vein yellowing virus (SqVYV, family Potyviridae, genus Ipomovirus) is
transmitted by the whitefly, Bemisia tabaci Middle East Asia Minor 1 group (MEAM1)
(Webb et al. 2006, 2012; Adkins et al. 2007), formerly known as the sweetpotato
whitefly [Bemisia tabaci (Gennadius) biotype B] and B. argentifolii (Bellows & Perring)
(Bellows et al. 1994, De Barro et al. 2011, Boykin 2014). Squash vein yellowing virus is
the causal agent of watermelon vine decline in Florida (WVD) (Adkins et al. 2007).
Infected watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] plants
exhibit mild vein yellowing with chlorotic lesions that are followed by systemic wilting
and necrosis which leading to plant death (Adkins et al. 2007, 2013; Webster et al.
2013). Fruit of infected watermelon become unmarketable, due to rind necrosis, change
in flesh color, increase in fruit acid content, and decrease in fruit sucrose content
(Adkins et al. 2013).
A vector-borne virus can induce physiological and morphological changes in
infected plants that influence the vector, by indirectly modifying behavior and fitness
(Rubinstein and Czosnek 1997, Jiu et al. 2007, Srinivasan and Alvarez 2007, Mauck et
al. 2010, Ingwell et al. 2012, Mauck et al. 2012). Morphology, volatile organic
compounds (VOCs), nutritional status, and level of plant toxins in infected plants can
provide cues to the insect vector for orientation and settling (Ajayi and Dewar 1983,
Ajayi 1986, Hodge and Powell 2000, Jiménez-Martínez et al. 2004, Colvin et al. 2006,
Mauck et al. 2010, Fang et al. 2013, Nachappa et al. 2013, Rajabaskar et al. 2013, Su
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et al. 2015). Some insect vectors preferentially settle on or are attracted to virus-
infected plants compared with healthy plants (Castle et al. 1998, Srinivasan and Alvarez
2007, Medina-Ortega et al. 2009, Chen et al. 2013, Fang et al. 2013, Wu et al. 2014),
but other vectors have shown avoidance behavior toward infected plants (Blua and
Perring 1992). In addition, several studies have suggested that settling preference is
conditional, i.e. that it changes after an insect feeds on an infected plant or becomes
viruliferous (Ingwell et al. 2012, Rajbaskar et al. 2013, Roosien et al. 2013, Carmo-
Sousa et al. 2014, 2016; Wang et al. 2014).
Many studies are conducted at a single time point after plant infection, most often
at the time of significant symptom expression in plants. Temporal effects of virus
infection or disease progression can affect the nutrient status of the infected plants or
symptom expression (Blua et al. 1994, Chung et al. 2015), which can potentially change
the interaction between infected plants and insect vectors. Differential effects of post
inoculation periods and symptom expression on the insect vector’s settling behavior
have been documented (Alvarez et al. 2007, Hodge and Powell 2009, Mann et al. 2009,
Legarrea et al. 2015). Previous studies of the indirect effects of SqVYV on whitefly
setting and oviposition showed different results, depending on the host plants involved
(Chapters 2 and 3). A few studies have explored the indirect effects of semi-persistently
transmitted plant viruses on insect vectors and temporal effects of virus infection on
settling and oviposition (Mann et al. 2008, McMenemy et al. 2012, Lightle and Lee
2014).
Feeding behavior of insects after infection not only indicates their ability to
transmit a virus, but also the suitability of host plants as a food source (Alvarez et al.
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2007, Moreno-Delafuente et al. 2013, Lei et al 2015). Indirect effects of plant viral
infection on the feeding behavior of insect vectors have been documented as positive
(Montllor and Gildow 1986, Fereres et al. 1990, Alvarez et al. 2007, Liu et al. 2013),
neutral (Montllor and Gildow 1986, Lightle and Lee 2014), and negative (Blua and
Perring 1992). The electrical penetration graph (EPG) technique has been used to study
the hidden aspects of the feeding behavior of hemipteran insects with piercing/sucking
mouthparts (Walker 2000). Differences in feeding parameters from EPG studies have
been used to assess the effect of treatments on the feeding behavior of insect vectors.
For example, some parameters used to indicate a positive effect of virus infection on
feeding are reduced number of probes, fewer interruptions in probing once stylets are
inserted into tissues, increased duration of ingestion from phloem, more phloem
contacts and shorter non-probing times (Fereres et al. 1990, Alvaez et al. 2007, Liu et
al. 2013). Alvarez et al. (2007) documented several differences in the feeding behavior
of Myzus persicae on potato plants infected with Potato leafroll virus compared with
non-infected potato plants at 65 d post inoculation (DPI), when the plant has significant
symptoms, but no differences were seen at 27 DPI when the plants had no visible
symptoms. To my knowledge no study has examined the temporal effects of a semi-
persistently transmitted virus on insect feeding and probing behavior.
The aim of this study was to investigate the influence of SqVYV post-inoculation
period on the settling and oviposition preference of whitefly. Furthermore, using the
EPG technique, I examined the feeding and probing behavior of whitefly on SqVYV-
infected and mock-inoculated watermelon plants at two time intervals post inoculation.
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This study will be increasing our knowledge of the host meditated effects of semi-
persistent virus infection and the temporal effect of infection on insect vector behavior.
Materials and Methods
Biological Material: Whitefly Colonies, Plants and Virus Isolates
The main whitefly colony was maintained in a room at 25-30°C, under a
photoperiod of 14:10 (L: D) h on ‘DP 0935 B2RF’ cotton (Gossypium hirsutum L.) and
‘Vates’ collard (Brassica oleracea L. var. acephala) as described by Chen et al. (2004).
A new cohort was established on cotton plants for each experiment. These cotton plants
were exposed to the main whitefly colony for 24 h for oviposition and transferred to an
insect rearing cage (60 by 60 by 60 cm, Bug Dorm, MegaView Science Co. Ltd.,
Taiwan) for 14 d. After that, each cotton plant was placed in an individual insect rearing
cage for 3–4 d for adult emergence. One- to 4-d-old adult whiteflies were used for the
experiments.
The isolate of SqVYV used in this experiment was originally collected from
squash in Hillsborough County, FL in 2003. It has been maintained in ‘Gentry’ squash
and ‘Mickylee’ watermelon by mechanical inoculation and periodic transmission by
whitefly (chapter 3) in the greenhouse [26-32ºC, photoperiod of 14:10 (L: D)].
Mechanical inoculation was conducted by grinding tissue of infected leaves and petioles
of squash and watermelon in 20 mM potassium phosphate buffer (pH 7.4) containing
corundum (100-200 mg/ml), and gently rubbing on the upper two to three leaves of the
watermelon, using cheesecloth.
Watermelon seeds were planted in plastic seedling tray inserts (4 cm × 5.5 cm ×
4 cm, T.O. Plastics, Clearwater, MN) filled with of Sunshine® Professional Growing Mix
MVP (Sun Gro Horticulture®, Bellevue, WA) and Osmocote, 14:14:14, a slow-release
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fertilizer (Everris NA, Inc., Dublin, OH) was added at a rate of 1 part per 280 parts. (i.e.
5 ml in 1.4 liters soil). Plants were transplanted 14 d after planting into 15.24-cm- or 10-
cm-diameter plastic pots for settling and EPG experiments, respectively. For both of the
experiments, half of the plants were inoculated with SqVYV (using the above mentioned
method) to produce infected plants and the other half were mock-inoculated using buffer
and corundum. Test plants for both experiments were grown in a greenhouse [26-32ºC,
photoperiod of 14:10 (L: D)] in the months of March and April in 2016 at the Department
of Entomology and Nematology, University of Florida, Gainesville, FL.
Influence of SqVYV Post Inoculation Period on Whitefly Settling and Oviposition
Pairs of infected and mock-inoculated plants, one pair 5-6 d post inoculation
(DPI) and the other 10-12 DPI, were used 34-36 d after planting (DAP) in a choice test.
Each pair was placed inside a cage (60 cm × 60 cm × 60 cm, organdy cloth cage with
PVC pipe as frame) with 15 cm separating the pots (Figure 4-1). At the time of the
choice test, symptoms of the plants were rated using the scale used by Kousik et al.
(2009). Plants 10-12 DPI were rated at 4-5 (chlorosis, vein yellowing plus severe
epinasty of youngest upper leaves, and no necrosis to chlorosis of most basal leaves,
necrotic streaks in petioles and /or tendrils), whereas plants 5-6 DPI were rated 1-2 (no
to very minor chlorosis/vein yellowing, no necrosis); all mock inoculated plants were
rated 1, without any symptoms. Fifty pairs of male and female whiteflies were collected
from the whitefly cohort (described above) in four to five glass tubes using a low flow
vacuum pump, using the method described in Webb et al. (2012) and Chapter 2.
Whiteflies were released from the glass tubes 15-18 cm from the mid-point between two
pots by gently removing the Parafilm® from the top of the tubes. Using a mirror,
whiteflies were counted on each plant, especially the abaxial surface of the leaves, over
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a 72-h span (0.25, 1, 2, 4, 8, 24, 48 and 72 h after the release of whiteflies). After 72 h,
whiteflies were dislodged from the plants and plants were brought in the laboratory to
count the number of eggs using a stereo microscope (25X). Eggs were counted with
leaves still attached to the plants.
To examine the whitefly’s ability to acquire and transmit SqVYV from infected to
mock-inoculated watermelon plants during the 72-h settling and oviposition preference
test, mock-inoculated plants in the paired treatment were taken back to the greenhouse
after eggs were counted. Ten d later, petiole samples were collected and tested with an
enzyme-linked immunosorbent assay for the presence of SqVYV (Webster et al. 2010,
chapter 3).
Data were recorded as number of whiteflies settled at each counting period and
number of eggs laid per plant after a 72-h exposure period. This experiment was
conducted in a greenhouse in six separate trials with each trial having four replicates
(24 replicates total). Number of whiteflies settling and number of eggs laid were square-
root transformed to normalize the data. Whitefly settling was analyzed using a repeated
measures approach using PROC GLIMMIX in SAS 9.4 (SAS Institute Inc, Cary, NC).
Data were analyzed separately by DPI and sliced by time, and treatment means were
compared using Tukey–Kramer test (α = 0.05). Number of eggs laid was analyzed using
PROC GLIMMIX, sliced by DPI and Least Squares Means were used to compare the
treatments.
Influence of SqVYV Post Inoculation Period on Whitefly Feeding Behavior using EPG
The whitefly’s probing and feeding activities were recorded using an AC-DC EPG
system with109-ohm input resistance (Backus and Bennett 2009). Output (voltage) from
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the monitor was recorded using Windaq (DATAQ Instruments, Akron, Ohio, USA) on a
Dell laptop computer. Adult female whiteflies were wired according to a previously
published procedure (Johnson and Walker 1999, Walker and Janssen 2000). Ultra-thin
platinum wire, 2 cm in length and 2.54 μm in diameter (Sigmund Cohn Corp, Mt.
Vernon, NY), was used to wire the female whiteflies (1-3 d old). To facilitate wiring,
whiteflies were placed in the refrigerator (4°C) for 90 s before wiring. Whiteflies were
wired on the cover of a glass Petri dish which was placed on a cold plate
(Thermoelectrics Unlimited, Inc, Wilmington, DE) under a stereo microscope (25X).
Silver conductive paint (Ladd Research Industries, Williston, VT) was used as glue to
attach one end of the wire to the whitefly on the dorsal surface of the thorax after
treating that end with nitric acid to remove the outer silver coating of the wire. The
opposite end of the wire was attached to a brass-plated nail (3/4") (The Hillman Group
Inc., Cincinnati, OH). This nail was inserted into a head amplifier as one electrode, and
another copper electrode (10 cm length, 2 mm in diameter) was inserted into the moist
soil of the plant container. Approximately 1 h was given for acclimatization between the
time of wiring and the beginning of EPG recording.
All the EPGs were recorded from insects and plants that were enclosed in a wire-
mesh Faraday cage (100 ×110 × 90 cm) and were recorded for 8 h (10:00 AM - 6:00
PM). Recordings were made from three types of test plants, all 27-30 d after planting:
mock-inoculated, 5-6 DPI, and 10-12 DPI. The upper 3rd or 4th leaf of each plant was
used for the recording. While recording, the leaf was held abaxial side up on a Plexiglas
stand using long narrow strips of Parafilm®, making it easier for the whitefly to move
without breaking the wire (Johnson et al. 2002). Symptoms of the plants were also
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recorded as described above. The symptoms recorded were 1, 1-2, and 4-5 rating form
mock-inoculated, 5-6 DPI, and 10-12 DPI infected watermelon plants respectively.
Twenty recordings were conducted for each treatment.
Whitefly feeding-associated waveforms, which have been previously correlated
with behavioral events (Jiang et al. 1999, Walker and Janssen 2000), were
identified. These waveforms were: non-probing behavior (no contact of stylet with the
leaf tissue, NP); pathway phase (intercellular apoplastic stylet pathway with cyclic
activities of mechanical stylet penetration and saliva secretion, C); potential drop
(intracellular stylet puncture of 4 to 12 s intracellular during the pathway phase, PD);
phloem phase [salivation (E1) and ingestion (E2) in sieve elements of phloem, E]; xylem
phase (stylet inserted to xylem and active intake of water from xylem element, G); and
mechanical derailment (stylet penetration difficulties, F) (Figure 4-2 and 4-3). Non-
sequential variables related to the pathway (C and PD), xylem (G), and phloem phase
(E) were extracted from each recording, such as waveform duration per event (WDE) at
cohort level, waveform duration per insect (WDI), number of waveform events per insect
(NWEI), and waveform duration per event (WDE;Backus et al. 2007). Parameters were
analyzed using the SAS program developed by Backus and colleagues (Personal
communication). Normality of the data was achieved by log transformation for WDE,
WDI, and WDEI and square root transformed for NWEI. The data were then subjected
to one-way ANOVA using F-test for each parameter and Least Squares Means were
used to compare the treatments.
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Results
Influence of SqVYV Post Inoculation Period on Whitefly Settling and Oviposition
Whiteflies showed no settling preference between 5-6 DPI infected watermelon
plants and mock-inoculated plants at any time period up to 72 h (Figure 4-4A). In the
case 10-12 DPI plants whiteflies showed initial preference for alighting and settling on
infected plants at 15 min after the release; however, at 1, 2, and 4 h whiteflies showed
no preference for settling. At 8 h whiteflies preferred to settle on mock-inoculated plants
and remained settled on the mock-inoculated plants for the rest of the time periods
(Figure 4-4B). Time (0.25, 1, 2, 4, 8, 24, 48,and 72 h) was the only factor that
significantly influenced whitefly settling on plants tested at 5-6 DPI; status (infected vs.
mock-inoculated) and status*time (Table 4-1) were not significant. However, single
factors and the interaction of time and status were significant for whitefly settling on
plants tested 10-12 DPI (Table 4-1).
Whiteflies laid a similar number of eggs on 5-6 DPI infected and mock-inoculated
plants; however, almost three times more eggs were laid on the 10-12 DPI mock-
inoculated than on infected plants (Figure 4-5). The significant interaction of infection
status*DPI explains this difference in oviposition (Table 4-2).
Influence of SqVYV Post Inoculation Period on Whitefly Feeding Behavior using EPG
Most of the parameters measured did not differ significantly among the three
treatments; however C, PD, and NP at cohort level WDE and NWEI of waveform G
were significant (Table 4-3). At the cohort level, the average duration of C waveform
was longer on 10-12 DPI and 5-6 DPI plants than on mock-inoculated plants (Table 4-
3). Average PD was longer on 10-12 DPI than mock-inoculated plants, but the duration
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was intermediate on 5-6 DPI plants (Table 4-3). Average duration of NP was longer on
5-6 DPI than 10-12 DPI and mock-inoculated plants (Table 4-3). Number of G
waveforms per insect was higher on mock-inoculated plants than on 5-6 and 10-12 DPI
plants (Table 4-3).
Discussion
Results from behavioral assays showed the effect of SqVYV infection and post-
inoculation period on the settling and oviposition preference of the whitefly; whiteflies
initially prefer to settle on 10-12 DPI infected plants, but not on 5-6 DPI infected plants
compared with mock-inoculated plants. The cause of the whitefly initial settling on the
10-12 DPI plants could be due to the change in the color of the infected plants. Insect
vectors such as whiteflies and aphids are attracted to yellow (Mound 1962, Kring 1967,
Vaishampayan et al. 1975, Kieckhefer et al. 1976, Isaacs et al. 1999), as the infected
watermelon plants at 10-12 DPI show transient yellowing of leaves; however, 5-6 DPI
infected plants were asymptomatic 18 out of 21 plants. Other studies have shown that
when plants become infected with virus and show disease symptoms, insect vectors
alight or initially orient toward the infected plants (Ajayi and Dewar 1983, Eckel and
Lampert 1996, Alvarez et al. 2007, Carmo-Sousa et al. 2013, Chen et al. 2013, Fereres
et al 2016). Furthermore, insect vectors are influenced by the change in total or specific
VOCs emitted by infected plants in initial orientation and settling preference
(Eigenborde et al. 2002, Jiménez-Martínez et al. 2004, McMenmeny et al. 2012,
Fereres et al 2016). Changes in VOCs emission are known to attract (Eigenbrode et al.
2002, Jiménez-Martínez et al. 2004, McMenmeny et al. 2012) or repel (Fereres et al.
2016) insect vectors. In case of whitefly, more importance is given to visual cues than
olfactory cues when selecting virus-infected or mock-inoculated plants. However,
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olfactory cues could influence whitefly orientation and settling preference, as seen in a
few studies (Fang et al. 2014, Fereres et al. 2016). In this study, I could not rule out a
possible role for olfactory cues in the greater attraction of whiteflies to 10-12 DPI
SqVYV-infected plants than to mock-inoculated plants.
The shift of settling preference after 8 h onto 10-12 DPI mock-inoculated plants
from infected plants could be an indication that the 10-12 DPI infected plants were not
good hosts for the whiteflies. This could result from lower nutrient status (Blua et al.
1994) and/or increase in plant defensive chemicals (Nachappa et al. 2013, Su et al.
2015) as disease progressed. In addition to these factors, drying, wilting, and collapsing
of the vine on the 10-12 DPI infected watermelon (Adkins et al. 2007, Webster et al.
2013) could cause whitefly to move to mock-inoculated plants. Whitefly preferences
were not affected by infection status of watermelon at 5-6 DPI, which suggests that host
suitability had not yet declined. A similar shift of settling preference over time after plant
virus infection has been recorded on Cotton leaf curl virus (CLCuV)-infected cotton by
whitefly (Mann et al. 2009). At 35 and 20 DPI, whiteflies prefer to settle on the healthy
cotton plants after 1 and 8 h of release respectively; however, at 5 DPI no discrimination
was found between CLCuV-infected and healthy cotton plants. This shift of preference
of whiteflies appeared to happen after initial landing and probing on plants. Probing and
feeding may be required for insects to discriminate between infected and non-infected
plants (Blua and Perring 1992, Sisterson 2008).
Results from the EPG study did not show significant differences among the
treatments for several feeding parameters. After initially settling on the infected plants,
whiteflies settle equally on 10-12 DPI infected plants and mock-inoculated plants. Only
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at 8 h, whiteflies show preference for settling on mock-inoculated at 10-12 DPI plants.
For 5-6 DPI no preferences were recorded up to 8 h. Whiteflies in this system could
require more time to discriminate between plants for host suitability. I could possibly
have found more differences among the treatments if the EPG study had been
conducted for more than 8 h. Lightle and Lee (2014) did not find differences in aphid
vector, Amphorophora agathonica, feeding behavior on raspberry plants infected with
the semi-persistently transmitted Raspberry leaf mottle virus (RLMV) and co-infection of
RLMV + Raspberry latent virus (RpLV) when compared with mock-inoculated plants.
Waveform duration per event is a commonly used parameter for analyzing
feeding behavior. Due to the variability between individual insects, effects of treatments
were seen on cohort level with three out of seven waveforms differed significantly by
treatments. More differences in the treatments might have been found if the infected
plants were showing more symptom than the 10-12 DPI infected plants used in this
study.
The acquisition of semi-persistently transmitted Cauliflower mosaic virus (CaMV)
increases after phloem ingestion by its aphid vectors, Brevicoryne brassicae and M.
persicae, but CaMV is also acquired at a low and fairly constant rate from one or more
non-phloem intracellular punctures in a few minutes (Palacios et al. 2002). In my study,
I found increases in the average duration of C and PD on SqVYV-infected plants at the
cohort level, which might lead in to an increased acquisition of SqVYV from the infected
plants.
Results from this study show that, SqVYV is able to manipulate its whitefly vector
in way that could potentially enhance the spread of SqVYV under field conditions. A
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change in settling preference for non-infected plants after the whitefly feeds on infected
plants increases the probability of virus spread. The addition of feeding behavior
favoring virus acquisition further optimizes virus spread. This study adds to our
understanding of the epidemiology of insect transmitted semi-persistent viruses and will
aid the development of epidemiological models.
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Table 4-1. ANOVA results examining the number of settled whiteflies, Bemisia tabaci (Middle East Asia Minor 1) recorded at 0.25, 1, 2, 4, 8, 24, 48, and 72 h (time) after their release on 5-6 and 10-12 d post inoculation (DPI) Squash vein yellowing virus-infected and mock-inoculated (infection status) watermelon plants.
DPI Effect and interactions DF F P
5-6 Infection status 1, 368 0.00 0.9567
Time 7, 368 3.08 0.0036
Infection status*Time 7, 368 1.01 0.4257
10-12 Infection status 1, 368 67.48 <0.0001
Time 7, 368 2.06 0.0469
Infection status*Time 7, 368 18.78 <0.0001
Table 4-2. ANOVA results showing the effects of whitefly, Bemisia tabaci (Middle East Asia Minor 1), oviposition preference on 5-6 and 10-12 d post inoculation (DPI) Squash vein yellowing virus-infected and mock-inoculated (infection status) watermelon plants.
Effects and interaction DF F P
Infection status 1, 69 30.23 <0.0001
DPI 1, 69 0.5 0.48
Infection status*DPI 1, 69 20.65 <0.0001
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Table 4-3. Mean (± SE) and ANOVA results for waveform duration per event (WDE), waveform duration per insect (WDI), waveform duration per event per insect (WDEI), and number of waveform events per insect (NWEI) in sec for feeding on mock-inoculated, and Squash vein yellowing virus-infected watermelon at 5-6 and 10-12 d post inoculation (DPI) by whitefly, Bemisia tabaci (Middle East Asia Minor 1).
Waveform
Mock 6 DPI 10-12 DPI Num DF
Den DF F P WDE ± SE WDE ± SE WDE ± SE
C 99.91 ± 5.01b 101.41 ± 3.64a 104.95 ± 5.07a 2 4266 3.19 0.0413
E 4617.89 ± 565.73 4457.84 ± 533.86 5712.99 ± 749.08 2 167 0.16 0.8521
F 431.22 ± 92.17 576.47 ± 149.32 470.09 ± 108.01 2 66 0.87 0.4217
G 964.96 ± 192.75 1044.99 ± 207.58 1460.04 ± 390.33 2 22 0.66 0.5275
Pd 5.84 ± 0.26b 6.19 ± 0.27ab 7.2 ± .54a 2 1897 4.58 0.0104
NP 240.99 ±54.74b 253.88 ± 17.41a 182.1 ± 13.92b 2 2217 7.02 0.0009
WDI ± SE WDI ± SE WDI ± SE C 7123.34 ± 828.12 6683.14 ± 754.04 7291.86 ± 891.03 2 59 0.16 0.8551
E 14546.34 ± 1301.41 12481.96 ± 1337.46 14568.13 ± 1251.23 2 57 0.86 0.4286
F 1047.24 ± 276.38 1729.39 ± 416.48 1821.61 ± 489.15 2 19 0.34 0.7137
G 1929.92 ± 453.75 1277.21 ± 222.07 1460.04 ± 390.33 2 15 0.84 0.4492
Pd 181.9 ± 35.74 176.91 ± 31.19 232.16 ± 35.93 2 59 0.72 0.4918
NP 8952.92 ± 1789.56 8897.75 ± 1078.55 6425.77 ± 879.58 2 59 1.18 0.3146
WDEI ± SE WDEI ± SE WDEI ± SE C 113.15 ± 15.78 100.37 ± 7.46 107.9 ± 7.33 2 59 0.33 0.7232
E 6208.71 ± 1105.63 4721.69 ± 515.38 7827.51 ± 1271 2 57 1.19 0.3125
F 433.77 ± 83.57 776.47 ± 314.67 576.87 ± 145.4 2 19 0.14 0.8735
G 970.9 ± 193.23 1092.84 ± 201.74 1460.04 ± 390.33 2 15 0.46 0.6395
Pd 5.69 ± 0.37 6.4 ± 0.67 7.1 ± 0.79 2 59 1.29 0.2837
NP 360.05 ± 98.87 295.63 ± 38.6 259.16 ± 58.37 2 59 0.6 0.5541
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Table 4-3. Continued.
Waveform
Mock 6 DPI 10-12 DPI
Num DF Den DF F P NWEI ± SE NWEI ± SE NWEI ± SE
C 71.3 ± 8.38 65.9 ± 5.03 69.48 ± 7.7 2 59 0.04 0.9573
E 3.15 ± 0.4 2.8 ± 0.28 2.55 ± 0.3 2 57 0.67 0.5154
F 2.43 ± 0.65 3 ± 0.62 3.88 ± 1.5 2 19 0.4 0.6763
G 2 ± 0.32a 1.22 ± 0.15b 1 ± 0b 2 15 5.85 0.0132
Pd 31.15 ± 5.07 28.57 ± 4.02 32.24 ± 4.1 2 59 0.18 0.8368
NP 37.15 ± 5.5 35.05 ± 3.57 35.29 ± 5.58 2 59 0.03 0.9696 Waveform C, pathway behaviors; E, phloem phase (phloem salivation and ingestion); F, mechanical difficulties in pathway phase; G, xylem ingestion; PD, potential drops (intracellular punctures); NP, non-probing (stylets withdrawn from plant). (N= 21 replicates).
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Figure 4-1. Set-up for whitefly, Bemisia tabaci (Middle East Asia Minor 1) settling and oviposition preference experiment. Two organdy cage (60 cm× 60 cm × 60 cm) were set up, each having one Squash vein yellowing virus-infected (on left) and one mock-inoculated (on right) watermelon plant at (A) 5-6 DPI or (B) 10-12 DPI. Fifty pairs (male and female) of whiteflies were released from the glass tubes 15 cm between the pots and whiteflies were counted at 0.25, 1, 2, 4, 8, 24, 48, and 72 h after release and eggs were counted after 72 h. Photo courtesy of author.
A B
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Figure 4-2. Waveforms generated using electrical penetration graph, direct current
applied voltage, and with109-ohm input resistance, for adult whitefly, Bemisia tabaci (Middle East Asia Minor 1). (A) Compressed overview of feeding behavior {2 h, Windaq compression 383 (76.6 sec/horizontal div)} showing NP, C, PD, and E waveforms, (B) C, Pathway waveform, has compression 10 (2 sec/ horizontal div.) (C) F, mechanical derailment waveform has compression 3 (0.6 sec/ horizontal div.), (D) G, Xylem waveform, has compression 1 (0.2 sec/ horizontal div.).
A
B
C
D
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Figure 4-3. Waveforms generated using electrical penetration graph, direct current
applied voltage, and with 109-ohm input resistance, for adult whitefly, Bemisia tabaci (Middle East Asia Minor 1) on watermelon leaf (A) PD, Potential drop waveform, has compression 1 (0.2 sec/ horizontal div.) (B) E1, Phloem salivation, has compression 1 (0.2 sec/ horizontal div.) (C) E2, Phloem ingestion waveform has compression 1 (0.2 sec/horizontal div.). Note: E1 and E2 were considered as E.
A
B
C
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Figure 4-4. Number of whiteflies, Bemisia tabaci (Middle East Asia Minor 1) settled on Squash vein yellowing virus-infected and mock-inoculated watermelon plant at; A) 5-6 DPI and B) 10-12 DPI counted at 0.25, 1, 2, 4, 8, 24, 48, and 72 h after release in a dual-choice test done in a organdy cage, releasing 100 whiteflies per replicate. Error bars are SEM and asterisk (*) indicates significant differences between mock-inoculated and infected plants (N = 24 replicates).
A
B
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Figure 4-5. Oviposition of whitefly, Bemisia tabaci (Middle East Asia Minor 1) on 5-6 DPI and 10-12 DPI Squash vein yellowing virus-infected and mock-inoculated watermelon plants. Data were expressed as mean number of eggs per plant ± SEM. Error bars are SEM and asterisk (*) indicates significant differences between mock-inoculated and infected plants (N= 24 replicates).
0
50
100
150
200
250
300
350
400
450
5-6 DPI 10-12 DPI
*
Infected Mock
Num
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r of
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nt
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CHAPTER 5 CONCLUSIONS
Watermelon growers in Florida lost an estimated $60 million in 2004-2005 due to
watermelon vine decline (WVD). The causal agent for WVD was found to be Squash
vein yellowing virus. Since its discovery in Florida, SqVYV has continued to be a
problem for watermelon growers, although the incidence of WVD has varied from year
to year. Recent detection of SqVYV in different regions of the US and in different
countries has increased the range of the economic threat to watermelon and cucurbit
crops. In this study, my aim was to find what factors play a role in SqVYV spread, which
could be helpful in epidemiological modeling and developing management options. This
dissertation study the role of the weeds in the transmission of Squash vein yellowing
virus (SqVYV) and host-mediated effects of SqVYV on its vector sweetpotato whitefly,
Bemisia tabaci (Gennadius) Middle East Asia Minor 1 (formerly known as B. tabaci
biotype B) behavior and fitness.
Surveys have shown that the cucurbitaceous weeds balsam apple (Momordica
charantia L) and smellmelon [Cucumis melo var. dudaim (L.) Naud.] are infected with
SqVYV in nature. Another cucurbitaceous weed, creeping cucumber (Melothria pendula
L.), was easily infected mechanically in the laboratory. These weeds are common in
Florida and able to survive mild winters, especially in South Florida and, act as a
potential virus source between cropping seasons. In the transmission experiments, I
evaluated the common cucurbit weeds versus the cultivated watermelon [Citrullus
lanatus (Thunb.) Matsum and Nakai] ‘Mickylee’ as sources of inoculum and for
susceptibility to SqVYV. I found a similar percentage of watermelon recipient plants
infected when watermelon ‘Mickylee,’ creeping cucumber, and smellmelon were used
101
as sources of inoculum. However, the percentage of recipient plants infected was lower
when balsam apple was used as the source plant. Watermelon was more susceptible to
SqVYV infection than balsam apple or smellmelon, but all weed species were equally
susceptible. These results confirm that cucurbit weeds are a potential source of virus
and showed that whiteflies are able to transfer the virus to cultivated cucurbit crops.
This underscores the need for surveying weeds close to cultivated fields as a potential
source of the virus.
Understanding the host-mediated effects of plant viruses on their insect vectors
and their effects on the spread of the virus is critical for effective management of
economically important plant viruses. To this end, I tested the host-mediated effects of
SqVYV on the biology and fitness of its whitefly vector. I first chose creeping cucumber
because of its susceptibility to SqVYV, which was similar to watermelon, and its more
pronounced infection symptoms compared to other cucurbit weeds. I conducted dual-
choice tests using a Petri dish for settling and oviposition preference on leaves of
infected and mock-inoculated creeping cucumber plants. I found whiteflies showed no
preference either on mock-inoculated or SqVYV-infected creeping cucumber leaves at
up to 5 h after release. However, after 24 h, more whiteflies were settled on the leaf of
the mock-inoculated plant than on the leaf of the infected plant. After a 72-h exposure
period, I found more eggs laid on mock-inoculated creeping cucumber leaves than on
SqVYV-inoculated leaves. These results suggest that host-mediated effects of SqVYV
infection may change the behavior of the whitefly, from an initial settling preference for
infected plants to a preference for uninfected plants, which is likely to promote virus
spread.
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I wanted to further test the SqVYV host-mediated effects on whitefly host
acceptance behavior such as initial alighting, settling, and oviposition preference on
cultivated cucurbit host plants. Alighting preference was tested in dual choice tests
using pairs of infected and mock-inoculated squash (Cucurbita pepo L.) and watermelon
plants inside 60 cm × 60 cm × 60 cm Bug Dorm cages. Whiteflies preferred to alight
more on the infected than mock-inoculated squash plants, but no differences were
recorded on infected and mock-inoculated watermelon plants. Similarly, dual choice
tests of settling preference were conducted by releasing 50 pairs of male and female
whiteflies into cages containing a pair of infected and mock-inoculated squash or
watermelon plants separately in 60 cm × 60 cm × 60 cm organdy cage in the
greenhouse and recording whitefly settling up to 72 h. Most of the time, more whiteflies
settled on the infected plants than mock-inoculated squash plants; however, statistically
higher number of whiteflies were recorded at 0.25, 1, 8, and 24 h after the release.
Whiteflies initially showed no preference for either infected or mock-inoculated
watermelon, but after 8 h, more whiteflies preferred to settle on the mock-inoculated
plants. Initially whiteflies settled equally on mock-inoculated and infected watermelon
plants, hence increasing the chances of whitefly to become viruliferous. Then the shift of
settling preference to mock-inoculated watermelon plants could lead to an increase in
transmission of SqVYV. II found this shift of settling preference in a 72-h time span on
the watermelon plants but not on the squash plants, which can partially explain rapid
spread of SqVYV in the watermelon field. Whitefly oviposition was evaluated at the end
of the 72-h exposure period. Whiteflies laid significantly more eggs on the mock-
103
inoculated than on infected watermelon plants, though no preference for oviposition was
recorded on squash plants.
Fitness of insect vectors can be affected by virus infection of their host plants and
is another valuable parameter that could explain the spread of the virus. Several
laboratory assays was used to determine differences in the duration of the immature
stage, body size, adult longevity, and fecundity on infected and mock-inoculated plants.
Due to rapid vine collapse of watermelon plants when infected, I was only able to do
these tests on squash plants. Mean duration of immature stage from eggs laid to end of
nymphal stage was approximately 3 d shorter on infected plants than mock-inoculated
squash plants. Body size of just emerged adult male and female did not differ when the
immature developed on infected or mock-inoculated plants. To test the effects of
infection status of immature rearing host on adult longevity and fecundity, a factorial
experiment was designed. Immatures were reared on either infected or mock-inoculated
plants and longevity and fecundity of adults emerging from those treatments were
tested on infected and mock-inoculated plants. Although, there were no differences in
male longevity, females lived longer on the infected plants than on mock-inoculated
plants, regardless of infection status of the rearing host plant. Longevity of females
confined on mock-inoculated plants and reared on mock-inoculated plants was
intermediate between the shortest and longest longevities. More eggs were laid by
whiteflies on the infected plants regardless of where the immatures were reared.
Whitefly fitness was enhanced on the infected squash plants, which has the potential to
increase the whitefly population if the squash plants were infected with SqVYV. Results
104
suggest enhanced performance of whitefly on infected plants and better host suitability
for whitefly after infection of the squash plant compared with mock-inoculated plants.
Host plant attractiveness and suitability for a vector insect can change as the
disease progresses over time since virus infection. In these studies, I looked at the
effect of time after SqVYV inoculation on whitefly settling and oviposition preference and
feeding behavior. The settling and oviposition preference assay was conducted on
mock-inoculated and infected watermelon plants 5-6 or 10-12 days post inoculation
(DPI). No differences in settling and oviposition were recorded in the assay with 5-6 DPI
plants. For 10-12 DPI, whiteflies initially (at 15 min) preferred to settle on the infected
plants, then no preference was recorded till 4 h, but from 8 h onwards whiteflies
significantly preferred to settle on the mock-inoculated plants. Whiteflies also laid higher
number of eggs on 10-12 DPI mock-inoculated plants than infected plants. Most of the
EPG parameters used to evaluate feeding behavior of the whitefly did not differ among
mock-inoculated, 6 DPI, and 10-12 DPI watermelon plants. The average duration of the
pathway waveform (C, intercellular apoplastic stylet pathway with cyclic activities of
mechanical stylet penetration and saliva secretion) was longer on 5-6 DPI and 10-12
DPI infected plants than on mock-inoculated plants. Average duration of potential drop
(PD, intracellular stylet puncture during the pathway phase) was longer on 10-12 DPI
infected plants than mock-inoculated plants. However, average duration of 5-6 DPI was
similar to mock-inoculated and 10-12 DPI infected plants. Average duration of non-
probing phase was longer on 5-6 DPI infected plants than on mock-inoculated and 10-
12 DPI plants. Fewer G waveforms per whitefly were recorded on 10-12 DPI and 5-6
DPI infected plants than mock-inoculated plants.
105
I still do not know what changes in the infected plant compared to the mock-
inoculated plant cause the modification of whitefly behavior and fitness. To determine
the nature of the changes, it is very important to find differences in volatile emissions,
defensive chemicals, and nutrient content between infected and mock-inoculated plants
and the role these differences play. Furthermore, additional cucurbit plants, including
weeds, need to be tested for the transmission rate of SqVYV when infected and for
host-mediated effects on the whitefly. Surveys have shown that weeds and cultivated
plants are often co-infected with SqVYV and other viruses.
Understanding the effects of co-infection on the transmission of SqVYV and the
host-mediated effects on whitefly behavior and fitness could be helpful in predicting the
spread of SqVYV. There are other species of whitefly prevalent in Florida such as B.
tabaci Mediterranean (biotype Q), which could be a potential vector of SqVYV. This
highlights the need for more study to fully understand the epidemiology of SqVYV.
106
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BIOGRAPHICAL SKETCH
Deepak Shrestha was born in 1984, in Dhangadhi, Nepal. After graduating high
school in biology, he attended the Institute of Agriculture and Animal Sciences,
Tribhuvan University, Nepal to pursue Bachelor of Science degree in agriculture. He
completed his undergraduate degree in agriculture in 2008. After graduation, he worked
as a research assistant in the Department of Entomology, Nepal Agricultural Research
Council for a year, and as a natural resource management officer in the Forum for Rural
Welfare and Agricultural Reform for Development for three months. He earned his
master’s degree in entomology from the University of Idaho, USA in 2012. He
completed his master’s thesis entitled ‘Interactions among Potato Genotypes, Growth
Stages, Virus Strains, and Inoculation Methods in the Potato Virus Y and Green Peach
Aphid Pathosystem’ under the supervision of Dr. Erik Wenninger. Deepak was
passionate about furthering his career in entomology. Therefore, he joined the
Department of Entomology and Nematology at the University of Florida to pursue his
Doctor of Philosophy in 2012. Because of his academic excellence, the department
awarded him a fully funded fellowship. In addition to his coursework, he worked as a
research assistant fellow conducting experiments on insect, plant and virus interactions
and also worked as a supervised teaching assistant. Under the supervision of Dr. Susan
E. Webb, he completed his dissertation on ‘Biology and Ecology of Squash vein
yellowing virus and its Vector Whitefly Bemisia tabaci (Gennadius).’ He completed his
doctoral degree in entomology and nematology in the fall of 2016. Deepak intends to
continue his work in the field of entomology and crop production.