Progress 10/01/05 to 10/01/10
Outputs
OUTPUTS: We completed a 4 year field and greenhouse study on whitefly resistance in the wild cotton species, GOSSYPIUM THURBERI, and identified very high levels of whitefly resistance in this cotton relative. The results were published. We also completed and published two studies on beet leafhopper, CIRCULIFER TENELLUS, feeding behavior and transmission of beet curly top virus. In these studies, we established EPG waveform - feeding behavior correlations so that the details of feeding behavior of this virus vector can be studied. We also determined the specific feeding behavior that results in inoculation of the virus. We also determined the specific feeding behavior that results in acquisition and inoculation of Zucchini yellows mosaic virus (ZYMV) by its vector, the green peach aphid, MYZUS PERSICAE. We found that, as in other studies, on similar viruses, ZYMV is both acquired and inoculated during brief punctures of epidermal cells or outer mesophyll cells, and occurs very soon after initiation of stylet penetration. Specifically, inoculation takes place during the first subphase of these brief intracellular punctures and acquisition takes place during the third subphase. A unique finding of this study is that two different sources of green peach aphid have different virus transmission efficiencies and that these differences can be explained by subtle differences in the feeding behavior of the two aphid sources. Thus, vector efficiency is affected not only by physiological/molecular interactions between the pathogen and the vector but also by variations in vector feeding behavior. We have developed a rapid method to determine the retention site of Lettuce infectious yellows virus in its whitefly vector, BEMISA TABACI Biotype A. This provides needed methodology to study virus-binding site specificity for a closterovirus in a vector insect. We have also developed methodology to instantaneously fix sieve elements while they are being fed upon by a phloem-feeding insect so that the state of the sieve elements can be histologically examined at any specific point in the feeding behavior of the insect. PARTICIPANTS: Candice Stafford, graduate student Emily Symmes, graduate student Eric Natwick, UC Cooperative Extension TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
EPG is the most powerful technology for studying feeding behavior of piercing-sucking insects but in order to use this technology, the electrical waveforms recorded by the EPG need to be correlated with specific aspects of feeding behavior. Without these correlations, EPG output would not be interpretable. One of the outcomes of this project is that we established EPG waveform correlations for all the main aspects of feeding behavior of beet leafhopper including the specific behavior that results in inoculation of beet curly top virus. These correlations allow us and other labs interested in beet leafhopper and transmission of beet curly top virus to use this powerful technique to answer questions that are unattainable by any other technology. Another output of this project relates to cotton and we have identified of a source of high level whitefly resistance in a cotton relative. This provides plant breeders with genetic material that can be introduced into commercial cotton cultivars and selected to develop a whitefly resistant cotton variety. Another output is development of techniques (not yet published) to determine the state of phloem sieve elements at any point during the feeding behavior of phloem-feeding insects. Previously only slow acting fixation methods were available that did not allow the researcher to "catch the insect in the act" at precise moments during its feeding behavior. This will be of great value for both my research as well as for other labs working on the interactions of phloem-feeding insects with phloem defense mechanisms. The new technique that we developed to identify the binding site of a virus in its vector also will assist the research in my lab and others.
Publications
- Walker, G. P. & E. T. Natwick. 2006. Resistance to silverleaf whitefly, Bemisia argentifolii (Hem., Aleyrodidae), in Gossypium thurberi, a wild cotton species. Journal of Applied Entomology 130: 429-436.
- Jiang, Y. X. & G. P. Walker. 2007. Identification of phloem sieve elements as the site of resistance to silverleaf whitefly in resistant alfalfa genotypes. Entomologia Experimentalis et Applicata. 125: 307-320.
- Backus, E. A., W. J Holmes, F. Schreiber, B. J. Reardon & G. P. Walker. 2009. Sharpshooter X wave: Correlation of an electrical penetration graph waveform with xylem penetration supports a hypothesized mechanism for Xylella fastidiosa inoculation. Annals of the Entomological Society of America 102: 847-867.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In the past year we have published two papers from our studies on the feeding behavior and transmission of beet curly top virus by the beet leafhopper, CIRCULIFER TENELLUS, the only known vector of Beet curly top virus (BCTV) in North America. Feeding behavior of piercing-sucking insects such as leafhoppers, aphids and whiteflies, is complex and is comprised of a multitude of components involving stylet movement, salivation and ingestion in different plant tissues. Inoculation of plant viruses occurs during very specific components of the feeding process. These behaviors are not directly observable, but can be observed indirectly with electrical penetration graphs (EPGs) in a manner analogous to how a cardiologist can observe details of heart function by recording electrical signals in an EKG. However, initial studies correlating electrical signals with specific biological activities need to be conducted so that the electrical output of the EPGs can be interpreted. We have completed these correlation studies for beet leafhopper and published them in an appropriate peer-reviewed scientific journal. We also have used the EPG technique to determine the specific feeding behavior that results in acquisition and inoculation of Zucchini yellows mosaic virus (ZYMV) by its vector, the green peach aphid, MYZUS PERSICAE and determined at least one reason why different populations of this aphid species have different efficiencies of virus transmission. This also has been published in an appropriate peer-reviewed scientific journal. Analysis of EPG data requires visual examination of tens or hundreds of hours of waveform recordings which is extremely tedious and time-consuming. Consequently, we collaborated with a computer scientist (E. Keogh, Computer Science & Engineering, University of California, Riverside) to devise a computer program to automate this process. This also has been published in an appropriate peer-reviewed scientific journal. In collaboration with Dr. James Ng (Plant Pathology, University of California, Riverside), we have developed a rapid method to determine the retention site of Lettuce infectious yellows virus in its whitefly vector, BEMISA TABACI Biotype A. This provides important preliminary data for a NSF proposal in preparation by demonstrating that we can efficiently study virus-binding site specificity for a closterovirus in a vector insect. PARTICIPANTS: Students: Candice Stafford, Graduate Student Researcher. The beet leafhopper - Beet curly top virus studies were her research project for a Master of Science degree under my supervision. Emily Symmes, Graduate Student Researcher. The green peach aphid - Zucchini yellows mosaic virus studies were part of her research project for a Master of Science degree and was conducted in my laboratory under my supervision. Sean Pelham, Graduate Student Researcher. Mr. Pelham is engaged in a study under my supervision on mechanism of transmission of a semipersistently transmitted closterovirus by green peach aphid. This project is ongoing without results to report yet. In addition to the graduate students listed above, the following undergraduates conducted assorted experiments in my lab relating to my Hatch project: Vanessa Lopez, Eddie Izumizaki, and Colin Umeda. Faculty: Dr. Thomas Perring, Dept. Entomology, University of California, Riverside. Dr. Perring was Emily Symmes major professor and he assisted in editing the manuscript from the study that Ms. Symmes conducted in my laboratory. Dr. Eamonn Keogh, Department of Computer Science & Engineering, University of California, Riverside. Dr. Keogh was the supervisor of the project that took our EPG data and developed an automated analysis program. S. Kasetty & X. Wang were his students working on the project. Dr. James Ng, Department of Plant Pathology, University of California, Riverside. Dr. Ng and I are collaborating on a project on binding site specificity of Lettuce infectious yellows virus and its whitefly vector, BEMISIA TABACI Biotype A. Dr. Rebecca Creamer, Dept. Entomology, Plant Pathology and Weed Science, New Mexico State University. Dr. Creamer is a co-investigator on the beet leafhopper -Beet curly top virus project. Her lab conducted the PCR and ELISA determinations of virus infection in these studies. Other: Dr. Eric Natwick, University of California Cooperative Extension, Desert Research & Extension Center, Holtville, CA. Dr. Natwick and I continue our collaboration in field screening different crop cultivars and related plant species for resistance against whiteflies. This past year's field tests did not yield any significant results and are not reported herein. TARGET AUDIENCES: The primary target is the scientific community, and as such, results have been published in peer-reviewed scientific journals and presentations made at scientific meetings. Students trained in my laboratory in this project include women, and several minority groups including African American, Hispanic and Asian. PROJECT MODIFICATIONS: Objectives1 and 4 have changed slightly as indicated below. The original Objective 1 was: 1. Develop a technique to observe, in vivo, functioning sieve elements while being fed upon by whiteflies, and use this technique to determine how sieve elements respond to whitefly feeding and how whiteflies suppress the sealing response of penetrated sieve elements. The revised Objective 1 is: 1. Determine how sieve elements respond to whitefly feeding and how whiteflies suppress the sealing response of penetrated sieve elements. The change is simply to focus on the biological process in which we are interested rather than a specific technique. The technique specified in the original objective will still be developed and used, but it is not the only technique that will be used to determine how sieve elements respond to whitefly feeding and how whiteflies suppress the sealing response of penetrated sieve elements. The original Objective 4 was: 4. Determine the specific component of feeding behavior of beet leafhopper that results in inoculation of curly top virus by viruliferous leafhoppers, and determine how feeding behavior of beet leafhopper is altered when feeding on tomato varieties that are resistant versus susceptible to curly top virus inoculation. The revised Objective 4 is: 4. Determine the specific components of feeding behavior of insect vectors of plant viruses that result in inoculation of plant viruses, and determine how feeding behavior can be altered to prevent inoculation and/or acquisition. The change was made to broaden the project from solely examining the beet leafhopper / Beet curly top virus system to examination of multiple phloem-feeding vector / pathogen systems. This was done to accommodate graduate student interests in other vector/virus systems.
Impacts
The EPG studies on beet leafhopper provided the necessary waveform-behavior correlations that allow us to interpret the following feeding behaviors from EPG-monitored beet leafhoppers: penetration of plant tissues, ingestion from mesophyll cells, ingestion of xylem sap, ingestion of phloem sap, and secretion of saliva into the phloem. The latter behavior is particularly important because we found that it is the specific behavior that results in inoculation of BCTV. These studies provide the necessary framework for studying details of host plant resistance to beet leafhopper and factors affecting virus transmission. For example, previous work suggested that the mechanism of resistance in a tomato variety resistant to BCTV is interference with the normal feeding behavior of the beet leafhopper vector. Our waveform-behavior correlation work allowed us for the first time to rigorously test this proposed mechanism of resistance. We have completed these experiments and are presently analyzing the data. At least one other lab (A. Wayadande, Entomology and Plant Pathology, Oklahoma State University) has been waiting for our waveform-behavior correlations to be published so that she can initiate her own studies on beet leafhopper - Beet curly top virus. The study on transmission of Zucchini yellows mosaic virus found that, as in other studies on similar viruses, ZYMV is both acquired and inoculated during brief punctures of epidermal cells or outer mesophyll cells, and occurs very soon after initiation of stylet penetration. Specifically, inoculation takes place during the first subphase of these brief intracellular punctures and acquisition takes place during the third subphase. A unique finding of this study is that two different sources of green peach aphid have different virus transmission efficiencies and that these differences can be explained by subtle differences in the feeding behavior of the two aphid sources. Thus, vector efficiency is affected not only by physiological/molecular interactions between the pathogen and the vector but also by variations in vector feeding behavior. Regarding the computer automation of EPG data analysis, the tedious nature of EPG data analysis has been widely viewed as a major impediment to utilizing this powerful technique for studying feeding behavior, plant resistance, and pathogen transmission by piercing-sucking insects. The publication of our study on automated analysis helps remove this impediment for studies on beet leafhopper. We plan to expand this to include other piercing-sucking insects.
Publications
- Symmes, E. J., G. P. Walker & T. M. Perring. 2008. Stylet penetration behaviors of Myzus persicae related to transmission of Zucchini yellow mosaic virus. Entomologia Experimentalis et Applicata 129: 258-267.
- Kasetty, S., C. Stafford, G. P. Walker, X. Wang & E. Keogh. 2008. Real-time classification of streaming sensor data. 20th IEEE International Conference on Tools with Artificial Intelligence 149-156
- Stafford, C. A. & G. P. Walker. 2009. Characterization and correlation of DC electrical penetration graph waveforms with feeding behavior of beet leafhopper, Circulifer tenellus. Entomologia Experimentalis et Applicata 130: 113-129.
- Stafford, C. A. & G. P. Walker, R. Creamer. 2009. Stylet penetration behavior resulting in inoculation of Beet severe curly top virus by beet leafhopper, Circulifer tenellus. Entomologia Experimentalis et Applicata 130: 130-137.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: In the past year we have completed our studies on the feeding behavior and transmission of beet curly top virus by the beet leafhopper, CIRCULIFER TENELLUS, the only known vector of Beet curly top virus (BCTV) in North America. Feeding behavior of piercing-sucking insects such as leafhoppers, aphids and whiteflies, is complex and is comprised of a multitude of components involving stylet movement, salivation and ingestion in different plant tissues. Inoculation of plant viruses occurs during very specific components of the feeding process. These behaviors are not directly observable, but can be observed indirectly with electrical penetration graphs (EPGs) in a manner analogous to how a cardiologist can observe details of heart function by recording electrical signals in an EKG. However, initial studies need to be conducted so that the electrical output of the EPGs can be interpreted in terms of biological activities. We have completed these studies which now allow us to
interpret the following feeding behaviors from EPG-monitored beet leafhoppers: penetration of plant tissues, ingestion from mesophyll cells, ingestion of xylem sap, ingestion of phloem sap, and secretion of saliva into the phloem. The latter behavior is particularly important because we found that it is the specific behavior that results in inoculation of BCTV. Two manuscripts describing this work are near completion and are expected to be submitted by the end of March 2008. Previous work suggested that the mechanism of resistance in a tomato variety resistant to BCTV is interference with the normal feeding behavior of the vector (beet leafhopper). Now that we have developed the EPG technique to examine feeding behavior of beet leafhopper, we can for the first time rigorously test this proposed mechanism of resistance. We have completed the experiments to test this hypothesis, and are presently analyzing the data; in next year's report, we will be able to summarize the results. Also
in collaboration with Tom Perring's lab (Entomology, University of California, Riverside), we have used the EPG technique to determine the specific feeding behavior that results in acquisition and inoculation of Zucchini yellows mosaic virus (ZYMV) by its vector, the green peach aphid, MYZUS PERSICAE. We found that, as in other studies, on similar viruses, ZYMV is both acquired and inoculated during brief punctures of epidermal cells or outer mesophyll cells, and occurs very soon after initiation of stylet penetration. Specifically, inoculation takes place during the first subphase of these brief intracellular punctures and acquisition takes place during the third subphase. A unique finding of this study is that two different sources of green peach aphid have different virus transmission efficiencies and that these differences can be explained by subtle differences in the feeding behavior of the two aphid sources. Thus, vector efficiency is affected not only by physiological/molecular
interactions between the pathogen and the vector but also by variations in vector feeding behavior. A manuscript describing this work was submitted to Entomolgia Experimentalis et applicata in February.
PARTICIPANTS: Walker Laboratory: Dr. Yongxing Jiang, Postdoctoral Scientist Dr. Nasser Zareh, Staff Research Associate Dr. Imad Bayoun Postdoctoral Scientist Dr. Serguei Triapitsyn, Senior Museum Scientist Candice Stafford, Graduate Student Researcher Emily Symmes, Graduate Student Researcher Elizabeth Lavi, Graduate Student Researcher Sean Pelham, Graduate Student Researcher Collaborators from UCR: Dr. Thomas Perring, Dept. Entomology Collaborators from other UCs Dr. Larry Teuber, Dept Agronomy and Range Science, UC Davis CE Collaborators: Dr. Eric Natwick, DREC, Holtville, CA Michele LeStrange, CE, Tulare County Collaborators from other institutions: Dr. Rebecca Creamer, Dept. Entomology, Plant Pathology and Weed Science, New Mexico State University Dr. Shai Morin, Hebrew University of Jerusalem, Dept. Entomology Dr. Asaph Aharoni, Weizman Inst. Sci. Dept. Plant Science Dr. C.C. Chu. USDA, Phoenix, AZ Dr. Thomas Henneberry. USDA, Phoenix, AZ Dr. Thomas Freeman, North Dakota State
University Dr. James Buckner, USDA, Fargo ND Dr. Dennis Nelson, USDA, Fargo ND Dr. Larry Rathbun, West Hills Community College, Farm of the future, Coalinga, CA
PROJECT MODIFICATIONS: Objectives1 and 4 have changed slightly as indicated below. The original Objective 1 was: 1. Develop a technique to observe, in vivo, functioning sieve elements while being fed upon by whiteflies, and use this technique to determine how sieve elements respond to whitefly feeding and how whiteflies suppress the sealing response of penetrated sieve elements. The revised Objective 1 is: 1. Determine how sieve elements respond to whitefly feeding and how whiteflies suppress the sealing response of penetrated sieve elements. The change is simply to focus on the biological process in which we are interested rather than a specific technique. The technique specified in the original objective will still be developed and used, but it is not the only technique that will be used to determine how sieve elements respond to whitefly feeding and how whiteflies suppress the sealing response of penetrated sieve elements. The original Objective 4 was: 4. Determine the specific
component of feeding behavior of beet leafhopper that results in inoculation of curly top virus by viruliferous leafhoppers, and determine how feeding behavior of beet leafhopper is altered when feeding on tomato varieties that are resistant versus susceptible to curly top virus inoculation. The revised Objective 4 is: 4. Determine the specific components of feeding behavior of insect vectors of plant viruses that result in inoculation of plant viruses, and determine how feeding behavior can be altered to prevent inoculation and/or acquisition. The change was made to broaden the project from solely examining the beet leafhopper / beet curly top virus system to examination of multiple phloem-feeding vector / pathogen systems. This was done to accommodate graduate student interest in other vector/virus systems.
Impacts
The economic importance of beet leafhopper is primarily due to its role as the sole North American vector of Beet curly top virus that is a serious disease of multiple crop commodities in the arid/semi-arid western states. Studies on beet leafhopper feeding behavior are important for understanding how this insect transmits this virus. These studies have practical applications. For example, by interbreeding with wild tomato species, a few varieties and breeding lines of domestic tomatoes resistant to Beet curly top virus have been developed. Resistance is multigenic, complex, and closely linked with undesirable horticultural traits, so it has been difficult to move the resistant genes into domestic tomatoes and still keep the desirable horticultural traits. Consequently, very few resistant domestic varieties have been developed. If resistance could be moved into a wider diversity of commercial tomato varieties, it would come into much wider use among western tomato
growers. In this example, resistance appears to be due to effects on the vector's feeding behavior, and the results of our beet leafhopper study provide for the first time a means of examining in detail this component of resistance. Determination of the mechanisms of resistance would accelerate breeding for resistance and help make more resistant varieties available by providing the needed data to identify the specific genes responsible for resistance. Similar application can be made for our studies on Zucchini yellows mosaic virus.
Publications
- Jiang, Y. X. & G. P. Walker. 2007. Identification of phloem sieve elements as the site of resistance to silverleaf whitefly in resistant alfalfa genotypes. Entomologia Experimentalis et Applicata 125: 307-320.
- Bayoun, I. M., G. P. Walker & S. V. Triapitsyn. 2008. Parasitization of beet leafhopper eggs, Circulifer tenellus, in California. Journal of Applied Entomology (in press).
- G. P. Walker, C. A. Stafford, E. J. Symmes & R. Goh. 2008. Phenology and descriptions of two sympatric native whiteflies (Hemiptera: Aleyrodidae) with a high degree of niche overlap. Pan Pacific Entomologist (in press).
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Progress 01/01/06 to 12/31/06
Outputs
This past year, we published the results of a 4 year field and greenhouse study on plant resistance against silverleaf whitefly, BEMISIA ARGENTIFOLII, in the wild cotton species, GOSSYPIUM THURBERI. Briefly, G. THURBERI shows very high levels of resistance against silverleaf whitefly in naturally infested field plots. However, in experiments where we manipulated whiteflies to gain insights into the mechanism of resistance (stage-specific survival tests and choice and no-choice oviposition preference tests), the whitefly seems to perform just as well on either cotton species. We will follow up on this paradox in future experiments. We are finishing up electrical penetration graph (EPG) studies on beet leafhopper, CIRCULIFER TENELLUS, the only known vector of curly top virus in North America. The main goal of these studies is to determine the specific components during beet leafhopper feeding that results in inoculation of curly top virus. Feeding behavior of
piercing-sucking insects like leafhoppers, aphids, and whiteflies, is complex and comprised of a multitude of components involving stylet movement, salivation and ingestion in different plant tissues; inoculation of plant viruses occurs during very specific components of the feeding process. These behavioral components are not directly observable, but in a manner analogous to how a cardiologist can observe details of heart function by recording electrical signals in an EKG, details of piercing-sucking insect feeding behavior can be observed with EPGs. Before EPGs can be used to observe these behaviors, studies must be conducted to correlate different details of feeding behavior with different patterns of electrical fluctuation that are referred to as waveforms. In this past year, we have completed the behavior/waveform correlations for the major relevant components of feeding behavior, and have identified the component responsible for inoculation of curly top virus. The next phase of
these studies will be to determine how certain tomato varieties are able to prevent inoculation of the virus by influencing the feeding behavior of the vector.
Impacts
The intended outcome of the silverleaf whitefly studies is development of a whitefly resistant cotton variety. This would be a very valuable contribution as silverleaf whitefly is one of the most serious pests of cotton in most cotton-growing regions of the world, and there are no resistant varieties available. The potential impact is to reduce pesticide use for whitefly control in cotton. The intended outcomes of the beet leafhopper studies are 1) to determine the point in the feeding behavior of beet leafhopper where curly top virus is inoculated and acquired, and 2) determine the mechanism by which the resistant tomato varieties interfere with inoculation and acquisition. The potential impacts are to reduce the incidence of curly top virus in tomatoes, and provide tomato breeders with information to help speed the incorporation of resistance into more than just the few resistant tomato varieties currently available.
Publications
- Walker, G. P. and E. T. Natwick. 2006. Resistance to silverleaf whitefly, Bemisia argentifolii (Hemiptera: Aleyrodidae), in Gossypium thurberi, a wild cotton species. Journal of Applied Entomology 130: 429-436.
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Progress 01/01/05 to 12/31/05
Outputs
This past year, we conducted a field study on stage-specific survival of silverleaf whitefly, BEMISIA ARGENTIFOLII, on upland cotton, GOSSYPIUM HIRSUTUM and its wild relative, G. THURBERI. This is the last in a series of experiments that will soon be submitted for publication. Briefly, G. THURBERI shows very high levels of resistance against silverleaf whitefly in naturally infested field plots (4 years of data). However, in experiments where we manipulate whiteflies to gain insights into the mechanism of resistance (stage-specific survival tests and choice and no-choice oviposition preference tests), the whitefly seems to perform just as well on either cotton species. We will follow up on this paradox in future experiments. We also have initiated electrical penetration graph (EPG) studies on beet leafhopper, CIRCULIFER TENELLUS, the only known vector of curly top virus in North America. Curly top virus causes an economically important disease that affects many crop
species, primarily in arid and semi-arid regions of the western US. The main goal of these studies is to determine the mechanism of resistance to curly top virus in the few varieties of tomato possessing resistance. Previous work by other labs indicates that resistance in these tomatoes is due to an effect on the leafhopper that interferes with its ability to inoculate the virus. Virus inoculation by insect vectors is intimately related to the vector's feeding behavior, and the most powerful technique for studying feeding behavior of piercing-sucking insects, like beet leafhopper, is the EPG technique. Essentially, EPGs record electrical signals from the feeding insect and, analogous to how a cardiologist can observe details of heart function or malfunction by recording electrical signals in an EKG, details of piercing-sucking insect feeding behavior can be observed with EPGs. Before EPGs can be used to observe these behaviors, studies must be conducted to correlate different details
of feeding behavior with different patterns of electrical fluctuation. In this past year, we have been conducting the necessary correlation studies. We have established behavior-electrical fluctuation correlations for pathway phase (advancement of the stylets in the plant tissue) and xylem sap ingestion. We also believe we have identified the correlation for phloem sap ingestion, but need more replicates to confirm this. After establishing the correlation for phloem sap ingestion, the one remaining important correlation we need for virus inoculation and acquisition studies is salivation into phloem sieve elements. Then we will be able to compare the feeding behavior of beet leafhopper on resistant and susceptible tomatoes and determine how the resistant varieties interfere with the leafhopper's feeding behavior and its ability to inoculate curly top virus. Finally, we have conducted field studies on potential non-toxic repellents against beet leafhopper in tomato fields. The potential
repellents include silver reflective plastic mulch, white plastic mulch, green plastic mulch, and sprays of kaolin. Of these, only the silver reflective mulch showed promise in the first year of study.
Impacts
The intended outcome of the silverleaf whitefly studies is development of a whitefly resistant cotton variety. This would be a very valuable contribution as silverleaf whitefly is one of the most serious pests of cotton in most cotton-growing regions of the world, and there are no resistant varieties available. The potential impact is to reduce pesticide use for whitefly control in cotton. The intended outcomes of the beet leafhopper studies are 1) to determine the point in the feeding behavior of beet leafhopper where curly top virus is inoculated and acquired, 2) determine the mechanism by which the resistant tomato varieties interfere with inoculation and acquisition, and 3) develop environmentally friendly strategies (resistant tomatoes and/or non-toxic repellents) to manage curly top virus in tomatoes. The potential impacts are to reduce the incidence of curly top virus in tomatoes, and provide tomato breeders with information to help speed the incorporation of
resistance into more than just the few resistant tomato varieties currently available.
Publications
- Walker, G. P., I. M. Bayoun, S. V. Triapitsyn and J. Y. Honda. 2005. Taxonomy of Aphelinoidea (Hymenoptera: Trichogrammatidae) species attacking eggs of beet leafhopper, Circulifer tenellus (Hemiptera: Cicadellidae), in California. Zootaxa 1068: 1-25.
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Progress 01/01/04 to 12/31/04
Outputs
This past year, we have been developing experimental techniques for elucidating the mechanisms of plant resistance to whiteflies. Our previous work on whitefly resistance in alfalfa indicated that resistance is expressed primarily in phloem sieve elements. Whitefly nymphs located and penetrated sieve elements with their stylets with equal success on both susceptible and resistant genotypes, but ingestion of phloem sap was inhibited and eventually ceased altogether on resistant genotypes. In the literature on feeding behavior of phloem-feeding insects on susceptible and resistant plants, most studies, like our own, have found that resistance is expressed primarily in phloem sieve elements. However, due to the difficulty of studying phloem sieve elements in vivo, the mechanism(s) of resistance in the sieve elements are unknown. Recently, a plant physiology laboratory in Germany developed a technique for observing living, translocating sieve elements using confocal
microscopy. They also were able to observe the sealing response of sieve elements to mechanical damage, a mechanism that has long been speculated as a potential mechanism of sieve element resistance to phloem-feeding insects, but has not been studied due to the lack of techniques. In fact, the study of virtually all aspects of the interactions between sieve elements and phloem-feeding insects has been hampered by lack of techniques. The ability to observe the response of sieve elements to feeding by phloem-feeding insects would provide a new tool for studying these interactions in ways not possible before. To this end, we have successfully used the techniques of the German lab to observe living, functioning sieve elements in the midrib (the German lab also worked only with the midrib). However, whitefly nymphs feed on minor veins, not midribs, so we need to adapt the techniques for minor veins. That is where we are at this point in time, working on the technique for minor veins. It
seems certain that resistance/susceptibility of plants to whiteflies is determined by some interaction between the sieve element and the saliva that the insect injects into the sieve element. Therefore, the other technique we have been working on is measuring salivation into sieve elements. Previously, we have used the electrical penetration graph (EPG) technique to detect phloem sap ingestion from sieve elements. At the time of that publication, we had not yet been able to detect salivation into sieve elements. Over the past year, we developed a technique that allows us to visually observe the salivary pump of living, feeding whitefly nymphs. That in its own right will allow us to compare salivation activity between whiteflies feeding on resistant or susceptible plants. However, even more useful will be being able to correlate salivary pump activity with specific EPG waveforms so that we can determine salivation into sieve elements with the EPG technique which will provide us even
more information about the interactions between salivation and successful or unsuccessful ingestion from sieve elements. The EPG correlation work will soon be underway.
Impacts
The intended outcomes of this work are development of techniques to elucidate the mechanism(s) of resistance against phloem-feeding insects, specifically a technique to observe, in vivo, whitefly stylets while feeding in living sieve elements, and a technique to detect and measure salivation into sieve elements during feeding by whiteflies. The potential impacts are 1) identification of mechanisms of whitefly resistance that could lead to quicker more cost-effective methods for distinguishing resistant from susceptible genotypes in plant breeding programs; and 2) lead to the identification of the genes that confer whitefly-resistance which could then be used in a genetic transformation approach to develop whitefly resistance in multiple crop plants beyond the specific crop (alfalfa) that we are studying.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
We are examining the mechanism of resistance in clonal lines of alfalfa that we had previously identified as resistant against silverleaf whitefly, BEMISIA ARGENTIFOLII. Resistance in these clonal lines is expressed primarily as very high first instar mortality, so we have been focusing on feeding behavior of first instar nymphs. Our previous work indicated that first instar nymphs successfully locate, penetrate, and begin ingesting from phloem sieve elements equally on both susceptible and resistant alfalfa genotypes. This past year we found that on the resistant genotypes, first instar nymphs spent less total time in ingestion behavior, and they were less successful at extracting sap per unit time of ingestion behavior (determined with electrical penetration graphs- EPG) as indicated by reduced rates of honeydew production during ingestion behavior. Future research is planned to determine what factors in sieve elements of the resistant clones are interfering with
normal feeding behavior of first instars. In addition, we have screened over 90 alfalfa genotypes for resistance to silverleaf whitefly. These are all genotypes being considered for use as breeding material in an ongoing program for breeding a whitefly-resistant alfalfa cultivar. As in the previous three years, in a season-long field test, GOSSYPIUM THURBERI, a wild relative of commercial upland cotton (G. HIRSUTUM), exhibited very high levels of resistance against silverleaf whitefly. In contrast, our earlier detailed studies on oviposition and nymphal survival in the greenhouse did not detect any difference in these two whitefly parameters between the wild and commercial cotton. Consequently, this past season, we conducted nymphal survival and oviposition tests in the field and came up with the same results as in the earlier greenhouse studies. While confirmation of our greenhouse results with field results is reassuring, it does not solve the paradox of why field plantings of the
wild species consistently (4 yrs) harbor very few whiteflies throughout the season in contrast to adjacent heavily-infested commercial cotton while more controlled experiments measuring survival and oviposition (choice and no-choice) indicate no diffference between the two cottons. We will continue to explore this paradox to arrive at an explanation.
Impacts
The intended outcomes of this work are 1) identification of whitefly-resistant alfalfa genotypes so that our cooperating plant breeder can select the most whitefly-resistant genotypes for his breeding program; and 2) identification of the physiological mechanisms of whitefly-resistance in resistant alfalfa genotypes and in the wild cotton species, G. THURBERI. The potential impacts are 1) commercial release of a new alfalfa cultivar with a high degree of whitefly-resistance; and 2) identification of mechanisms of whitefly resistance that could lead to quicker more cost-effective methods for distinguishing resistant from susceptible genotypes in alfalfa and cotton breeding programs; and 3) lead to the identification of the genes that confer whitefly-resistance which could then be used in a genetic transformation approach to develop whitefly resistance in crop plants beyond only alfalfa and cotton.
Publications
- Jiang, Y. X., N. Zareh, G. P. Walker and L. R. Teuber. 2003. Characterization of alfalfa germplasm expressing resistance to silverleaf whitefly, Bemisia argentifolii. Journal of Applied Entomology 127: 447-457.
- Jiang. Y. X. and G. P. Walker. 2003. Electrical penetration graphs of the nymphal stage of Bemisia argentifolii. Entomologia Experimentalis et Applicata 109: 101-111.
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Progress 01/01/02 to 12/31/02
Outputs
We have begun to examine the mechanism of resistance in clonal lines of alfalfa that we had previously identified as resistant against silverleaf whitefly, BEMISIA ARGENTIFOLII. Resistance in these clonal lines is expressed primarily as very high first instar mortality, so we tested the hypothesis that first instars had a low success rate of locating phloem sieve elements on the resistant clonal lines. Experiments using three different techniques (electrical penetration graphs, honeydew clocks, and histology) all indicate that first instar nymphs successfully locate and penetrate sieve elements on the resistant clonal lines. Therefore, we conclude that the resistance resulting in high first instar mortality is compartmentalized in the phloem; mortality is not expressed until after sieve elements are penetrated. Future research is planned to determine what factors in the phloem of the resistant clones are causing high first instar mortality. We have identified three
electrical penetration graph waveforms produced by the nymphal stage of silverleaf whitefly, thus permitting this valuable technique to be used to study feeding behavior of silverleaf whitefly immatures. Pathway phase, which occurs as the whitefly's stylets penetrate from the leaf surface to the sieve element, is represented by a high amplitude waveform similar to that produced during pathway phase in adult silverleaf whiteflies. Phloem phase, which occurs when the tips of the whitefly's stylets are inserted in a sieve element is represented by two waveforms named 'high frequency' and 'low frequency' based on their relative frequencies of voltage fluctuation. The high frequency waveform was correlated with honeydew excretion indicating that the high frequency waveform represents ingestion of phloem sap. The low frequency waveform alternates regularly with the high frequency waveform, but presently, we have not correlated it with a biological behavior. We suspect that the low frequency
waveform is produced during salivation into the sieve element, but this hypothesis needs testing. As in the previous two years, in a season-long field test, GOSSYPIUM THURBERI, a wild relative of commercial upland cotton (G. HIRSUTUM), exhibited very high levels of resistance against silverleaf whitefly. In contrast to three consistent years of field data showing G. THURBERI to be highly resistant, last year's greenhouse tests on seedlings showed slightly higher survival of silverleaf whitefly nymphs on G. THURBERI than on commercial upland cotton, and this year's greenhouse tests of whitefly oviposition preference on seedlings indicated that G. THURBERI was just as preferred for oviposition as commercial cotton in both choice and no-choice tests. The reasons for these contradictory findings will be explored.
Impacts
Four highly resistant alfalfa clones and three moderately resistant clones have been identified that can be used in the ongoing breeding program for whitefly resistant alfalfa. Also, the stage specific survival study indicated that we can expect resistance to be expressed primarily in the first instar. Measuring nymphal survival through completion of the first instar is much less laborious than measuring survival from egg to adult. This will make screening alfalfa clones for resistance more efficient. Consequently, we will soon screen over 200 additional clonal lines. This method also is more efficient means than the expensive field trials that have been done in the past. Our results indicate that a high percentage of plants deemed resistant in the field had low whitefly densities more as a result of chance than resistance. Thus, the method developed in this project should be less expensive and more accurate. Detection of high levels of whitefly resistance in the
cotton relative, G. THURBERI, is encouraging, because this species has been used in the past to incorporate new genes into commercial upland cotton, G. HIRSUTUM. Our results indicating that whitefly susceptibility is not correlated with vascular bundle depth was disappointing, yet valuable in order to avoid wasting effort selecting for ineffective traits in cotton breeding. The advances that we made in developing the EPG technique for studying feeding behavior will be very useful in determining the mechanism of resistance which in turn will facilitate selection of whitefly resistant plants.
Publications
- Jiang, Y. X. and G. P. Walker. 2001. Pathway Phase Waveform Characteristics Correlated with Length and Rate of Stylet Advancement and Partial Stylet Withdrawal in AC Electrical Penetration Graphs of Adult Whiteflies. Entomologia Experimentalis et Applicata 101: 233-246.
- Johnson, D. D., G. P. Walker, and R. Creamer. 2002. Stylet Penetration Resulting in Inoculation of a Semipersistently Transmitted Closterovirus by the Whitefly Bemisia argentifolii. Entomologia Experimentalis et Applicata 102: 115-123.
- Walker, G. P. 2002. Overview of Whitefly Feeding Behavior: What We Know and What We Need to Know. In: Silverleaf Whitefly: National Research, Action, and Technology Transfer Plan: Fourth Annual Review of the Second 5-Year Plan and Final Report for 1992-2002. USDA/ARS. (T. J. Henneberry, R. M. Faust, W. A. Jones, and T. M. Perring, eds.). pp. 49-53.
- Natwick, E. T. and G.P. Walker. 2002. Silverleaf Whitefly Resistance in the Cotton Relative, Gossypium thurberi. Proceedings of the Beltwide Cotton Conference. 5 MS pp.
- Natwick, E. T., G. Walker, C. C. Chu, T. J. Henneberry, D. Brushwood and G. Constable. 2002. Susceptibility of Upland Cotton Cultivars to Infestation by Silverleaf Whitefly. In: Silverleaf Whitefly: National Research, Action, and Technology Transfer Plan: Fourth Annual Review of the Second 5-Year Plan and Final Report for 1992-2002. USDA/ARS. (T. J. Henneberry, R. M. Faust, W. A. Jones, and T. M. Perring, eds.). pp. 189.
- Natwick, E. T. and G. P. Walker. 2002. High Level of Resistance to Silverleaf Whitefly in the Cotton Relative, Gossypium thurberi. In: Silverleaf Whitefly: National Research, Action, and Technology Transfer Plan: Fourth Annual Review of the Second 5-Year Plan and Final Report for 1992-2002. USDA/ARS. (T. J. Henneberry, R. M. Faust, W. A. Jones, and T. M. Perring, eds.). pp. 190.
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Progress 01/01/01 to 12/31/01
Outputs
Stage-specific survival studies on silverleaf whitefly have identified 4 clonal lines of alfalfa that are highly resistant against the whitefly (< 10% survival egg-to-adult in contrast to > 75% survival in the most susceptible control) and 3 clones that are moderately resistant. All tests used two whitefly sources, one reared on alfalfa ("alfalfa-experienced whiteflies") and the other reared on cotton ("alfalfa-naive whiteflies"). Results were similar for the two whitefly sources in all tests. Most of the nymphal mortality on the highly resistant clones occurred during the first instar, and surprisingly, on one clone, a very high egg mortality was observed. In a season-long field test, GOSSYPIUM THURBERI, a wild relative of commercial upland cotton (G. HIRSUTUM), exhibited very high levels of resistance against silverleaf whitefly. However, in a greenhouse test on seedlings, G. THURBERI was slightly more susceptible than commercial upland cotton. The reasons for these
contradictory findings are being explored. We have completed data collection on the first year of a two year study to determine if variation in whitefly susceptibility among cotton cultivars is correlated negatively with vascular bundle depth. This hypothesis was first proposed by another lab in 1998 based on preliminary data. Unfortunately, our more detailed examination is finding no correlation. We have identified an electrical penetration graph (EPG) waveform produced by first instar whitefly nymphs that is correlated with phloem sap ingestion. After penetrating a sieve element with their stylets, first instar whitefly nymphs alternate between two behaviors, sap ingestion (each bout averaging about 25 min duration) and an unknown activity, probably salivation (each bout averaging about 15 min duration).
Impacts
Four highly resistant alfalfa clones and three moderately resistant clones have been identified that can be used in the ongoing breeding program for whitefly resistant alfalfa. Also, the stage specific survival study indicated that we can expect resistance to be expressed primarily in the first instar. Measuring nymphal survival through completion of the first instar is much less laborious than measuring survival from egg to adult. This will make screening alfalfa clones for resistance more efficient. Consequently, we will soon screen over 200 additional clonal lines. This method also is more efficient means than the expensive field trials that have been done in the past. Our results indicate that a high percentage of plants deemed resistant in the field had low whitefly densities more as a result of chance than resistance. Thus, the method developed in this project should be less expensive and more accurate. Detection of high levels of whitefly resistance in the
cotton relative, G. THURBERI, is encouraging, because this species has been used in the past to incorporate new genes into commercial upland cotton, G. HIRSUTUM. Our results indicating that whitefly susceptibility is not correlated with vascular bundle depth was disappointing, yet valuable in order to avoid wasting effort selecting for ineffective traits in cotton breeding. The advances that we made in developing the EPG technique for studying feeding behavior will be very useful in determining the mechanism of resistance which in turn will facilitate selection of whitefly resistant plants.
Publications
- Luft, P. A., T. D. Paine & G. P. Walker. 2001. Interactions of colonization density and leaf environments on survival of TRIOZA EUGENIAE nymphs. Ecological Entomology. 26: 263-270.
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