Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candida... more Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candidatus Liberibacter asiaticus (CLas) titer has an inherent element of imprecision because CLas titer varies considerably within the tree canopy and with calendar seasons. In addition, the destructive sampling method used to determine CLas titer entails a different set of leaves per sampling period adding to the inconsistency and inexactitude of the results. To overcome these ambiguities and to reduce the numerical variability between samples, we developed an experimental method that analyzes portions of the same treated leaves for up to four sampling periods. By assaying subsamples of adjacent locations of the same leaf, random variability was significantly reduced, and comparative analysis can be carried out with greater precision.
The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) regio... more The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) region of Minas Gerais State and the south of São Paulo State (SPS). Significant climatic variation occurs between regions which could affect huanglongbing (HLB) progress, which is lower in TM. To investigate this possibility, young sweet orange shoots were sampled periodically over 2 years to determine ‘Candidatus Liberibacter asiaticus’ titers in naturally infected trees in orchards in Analândia, central SPS, and Frutal and Comendador Gomes, within TM. Data-loggers recorded local temperature and relative humidity hourly. In the laboratory, five ‘Ca. L. asiaticus’-free Diaphorina citri adults were placed on each sampled shoot for 48 h to feed and acquire the pathogen. Shoots and insects were individually analyzed by quantitative polymerase chain reaction to determine ‘Ca. L. asiaticus’ titers. The incidence of ‘Ca. L. asiaticus’-positive shoots, ‘Ca. L. asiaticus’ titers, and acquisition rat...
Typescript (photocopy). Thesis (M.S.)--Colorado State University, 1990. Includes bibliographical ... more Typescript (photocopy). Thesis (M.S.)--Colorado State University, 1990. Includes bibliographical references (leaves [36]-40).
ABSTRACT A number of plants produce nectar or pollen that contains toxins which can adversely aff... more ABSTRACT A number of plants produce nectar or pollen that contains toxins which can adversely affect honey bees. Almond (Amygdalus communis L. (Rosaceae)) produces amygdalin, a toxic cyanoglycoside, in its nectar and pollen. Although beekeepers regard almond as a valuable nectar and pollen plant, and honey bees are deployed extensively for almond pollination, prolonged reliance on almond may be detrimental to honey bees' health. Our results suggest that almond nectar is not sufficiently rich in amygdalin to pose a hazard, but that almond pollen could be toxic if exclusively consumed by honey bees for much more than a week. Further tests are needed to determine if honey bees in almond orchards are at risk, or if they somehow cope with the toxin.
ABSTRACTCandidatus Liberibacter asiaticus is vectored by the psyllid Diaphorina citri Kuwayama (H... more ABSTRACTCandidatus Liberibacter asiaticus is vectored by the psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) and putatively causes Huanglongbing disease in citrus. Huanglongbing has reduced yields by 68% relative to pre-disease yields in Florida. Disease management is partly through vector control. Understanding vector biology is essential in this endeavor. Our goal was to document differences in probing behavior linked to sex. Based on both a literature review and our results we conclude that there is either no effect of sex or that identifying such an effect requires a sample size at least four times larger than standard methodologies. Including both color and sex in statistical models did not improve model performance. Both sex and color are correlated with body size, and body size has not been considered in previous studies on sex in D. citri. An effect of body size was found wherein larger psyllids took longer to reach ingestion behaviors and larger individuals spent mo...
The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector ... more The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of the phloem-infecting bacterium, Candidatus Liberibacter asiaticus. Candidatus L. asiaticus is the putative causal agent of Huanglongbing (HLB) disease, a destructive disease of Citrus. While many Citrus species are susceptible to D. citri probing and HLB disease, there are marked behavioral differences in D. citri probing responses and Ca. Liberibacter asiaticus infection severity among Citrus species. Using four mandarin hybrid selections and pummelo plants variably resistant to D. citri probing, oviposition, and survival, we explored probing differences using electropenetrography (EPG), conducted an oviposition and survival study, and determined host plant metabolites using gas-chromatography mass-spectroscopy (GC-MS). We found thirty-seven D. citri probing variables to be significantly different among tested mandarin selections and pummelo, in addition to differential oviposition a...
is a major pest of citrus because it transmits Liberibacter asiaticus, a phloem-limited bacterium... more is a major pest of citrus because it transmits Liberibacter asiaticus, a phloem-limited bacterium that putatively causes Huanglongbing (HLB). The disease moves slowly through a tree, and the vector facilitates further within-tree movement via transmission of the pathogen. However, this only happens when stylets contact the phloem, to inoculate bacteria during phloem salivation and acquire bacteria during phloem sap ingestion. Behavioral changes in associated with different plant parts would affect how long it takes to reach phloem and how long the psyllids stays in phloem to ingest, thereby influencing the risk of disease spread. feeding was recorded on the abaxial and adaxial surfaces of mature and immature citrus leaves. Adults in the field can be found on these surfaces at all times of year. On abaxial surface of immature leaves, phloem salivation would occur after 11 h on average, but rarely as soon as 0.56 h. The corresponding values on mature leaves were 16 and 2.7. In general...
Electropenetrography a.k.a. electrical penetration graph or EPG is a rigorous technique for study... more Electropenetrography a.k.a. electrical penetration graph or EPG is a rigorous technique for studying arthropod behavior. Essentially, the arthropod and host are part of an electrical circuit that generates patterns of voltage (waveforms), whose biological meanings are defined by correlation with histology and behavior. EPG is used for studying stylet probing behavior of hemipterans, and blood-feeding arthropods. These results are applied to understanding pesticide action, host plant resistance, and vector-pathogen-host interactions. At the end of all recordings, the arthropod begins a behavior that ends because the scientist stopped recording. An argument for keeping this event in the data has been made based on the assumption that the insect is adapting to laboratory conditions. In this adaptation process, the expected durations of ingestion behaviors will increase as the insect adapts. We show that this assumption can cause problems in data analysis and interpretation of the data....
Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, beha... more Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, behavior, and metabolism. Because these pathogens interact with their vectors on the cellular and organismal levels, potential changes at the biochemical level might occur. “ Candidatus Liberibacter asiaticus” ( C Las) is transmitted in a persistent, circulative, and propagative manner. The genome of C Las revealed the presence of an ATP translocase that mediates the uptake of ATP and other nucleotides from medium to achieve its biological processes, such as growth and multiplication. Here, we showed that the levels of ATP and many other nucleotides were significantly higher in C Las-infected than healthy psyllids. Gene expression analysis showed upregulation for ATP synthase subunits, while ATPase enzyme activity showed a decrease in ATPase activity. These results indicated that C Las stimulated Diaphorina citri to produce more ATP and many other energetic nucleotides, while it may inhibit ...
Host plant resistance is a practical and cost-effective approach for growers to manage insect pes... more Host plant resistance is a practical and cost-effective approach for growers to manage insect pests. Recently, three new sources of resistance in black raspberry (Rubus occidentalis; selections ORUS 3778-1, ORUS 3817-1, and ORUS 4109-1) against the large raspberry aphid, Amphorophora agathonica, were identified. We studied stages of host plant acceptance: host plant attraction, parturition (deposition of nymphs), nymph survival, and adult feeding behavior (using the electrical penetration graph [EPG]) to identify the location of the plant resistance. Aphids were more attracted to the susceptible cultivar ‘Munger’ (control) than the resistant selections. Parturition occurred on the resistant selections, but fewer nymphs were deposited on resistant lines relative to the susceptible control. Nymphs survived only an average of 3.3–3.6 days on resistant selections, whereas 94 % were still alive after 11 days on ‘Munger.’ There were differences in feeding behavior between the susceptible control and the resistant selections, but no differences between the three resistant selections. The tissues responsible for resistance appear to be the mesophyll and phloem sieve elements. Aphids had a reduced probability of salivation into the phloem sieve elements of resistant selections, and only one aphid each on ORUS 3778-1 and ORUS 4109-1 successfully ingested from the phloem. Because feeding behavior of A. agathonica did not differ between resistant selections, independent confirmation that resistance is conferred by unique genes should be obtained before pyramiding these sources together.
Annals of the Entomological Society of America, 2010
Our goal was to estimate seasonal changes in the proportion of Asian citrus psyllid, Diaphorina c... more Our goal was to estimate seasonal changes in the proportion of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), carrying Candidatus Liberibacter asiaticus. Our approach was to test Asian citrus psyllid by using pooled samples. The initial question was about pool size and the consequences of choosing poorly. Assuming no loss in sensitivity when diluting one infected individual with many healthy individuals, then it is recommend that a combination of all the published limits be used: keep the number of pools (n) above 20, the pool size (k) below 100, and the number of infected pools less than half the total number of pools. The most conservative approach to achieving the latter is to optimize pool size given an infection rate (p) such that k = ln(0.5)/ln(1 p). Exceeding these limits increases the probability that all the pools will be infected. If this occurs, then that particular sample will be discarded. Use of multiple pool sizes can be used to manage this ri...
Apple (Malus domestica Borkh.) leaves were sprayed to runoff using a selection of pesticides, and... more Apple (Malus domestica Borkh.) leaves were sprayed to runoff using a selection of pesticides, and subsequent spray retention was evaluated by weight. Timing, cultivar, leaf surface, and leaf type significantly influenced spray retention. Spray retention correlated significantly and positively with leaf hair density. The importance of these differences is considered in relation to the possible efficiency of the pesticide application process in apple orchards.
ABSTRACT Agricultural application of pesticides is often done by atomization of the active ingred... more ABSTRACT Agricultural application of pesticides is often done by atomization of the active ingredient and a carrier fluid. This atomized fluid lands on a plant surface and creates a mosaic of treated and untreated patches that we define as deposit structure. This structure influences the biological effect of the pesticide if dose per unit time is a factor in determining the overall toxicity of the pesticide. Laboratory bioassays are frequently carried out in such a way as to eliminate all effects of deposit structure. This bioassay methodology dictates the form of the observed dose-response relationship in these experiments. Although our observations do not invalidate previous bioassay methodology, they do suggest that current approaches provide a narrow view of dose-response relationships. These results affect bioassay methodology for testing toxicant efficacy and for experiments with insecticide resistance that use similar approaches to screen for resistance or study inheritance of pesticide resistance.
Annals of the Entomological Society of America, 1998
ABSTRACT Morphometric data of Aphis gossypii Glover are used as a case study to illustrate the us... more ABSTRACT Morphometric data of Aphis gossypii Glover are used as a case study to illustrate the use of randomization tests. The application of randomization tests in morphological evaluation and identification of species is a powerful tool for characterizing populations and species. It offers the advantage of reducing our reliance on the robustness of more classical approaches to overcome problems of small sample size, unequal sample size, and departures from normality. We review randomization test methodology. We address a few errors that have appeared in the literature. One question is how many randomizations. As a generic starting point, the number of randomizations should be 2 orders of magnitude larger than the inverse of the significant P value, but in critical cases an exact figure can be determined. A new methodology is introduced for using randomization tests to determine if the average of several observations is different from a constant. An extension of the method is used when the null hypothesis states that there are differences. This is important where there is reason to suspect that one is dealing with different populations (e.g., morphological measurements were taken from several distinct populations of an insect) and one needs to identify which populations are the same. This test should not be confused with the typical case where it is simply impossible to identify differences between different sets of observation. We present a SAS program to perform 2-tailed tests for differences between means.
Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candida... more Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candidatus Liberibacter asiaticus (CLas) titer has an inherent element of imprecision because CLas titer varies considerably within the tree canopy and with calendar seasons. In addition, the destructive sampling method used to determine CLas titer entails a different set of leaves per sampling period adding to the inconsistency and inexactitude of the results. To overcome these ambiguities and to reduce the numerical variability between samples, we developed an experimental method that analyzes portions of the same treated leaves for up to four sampling periods. By assaying subsamples of adjacent locations of the same leaf, random variability was significantly reduced, and comparative analysis can be carried out with greater precision.
The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) regio... more The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) region of Minas Gerais State and the south of São Paulo State (SPS). Significant climatic variation occurs between regions which could affect huanglongbing (HLB) progress, which is lower in TM. To investigate this possibility, young sweet orange shoots were sampled periodically over 2 years to determine ‘Candidatus Liberibacter asiaticus’ titers in naturally infected trees in orchards in Analândia, central SPS, and Frutal and Comendador Gomes, within TM. Data-loggers recorded local temperature and relative humidity hourly. In the laboratory, five ‘Ca. L. asiaticus’-free Diaphorina citri adults were placed on each sampled shoot for 48 h to feed and acquire the pathogen. Shoots and insects were individually analyzed by quantitative polymerase chain reaction to determine ‘Ca. L. asiaticus’ titers. The incidence of ‘Ca. L. asiaticus’-positive shoots, ‘Ca. L. asiaticus’ titers, and acquisition rat...
Typescript (photocopy). Thesis (M.S.)--Colorado State University, 1990. Includes bibliographical ... more Typescript (photocopy). Thesis (M.S.)--Colorado State University, 1990. Includes bibliographical references (leaves [36]-40).
ABSTRACT A number of plants produce nectar or pollen that contains toxins which can adversely aff... more ABSTRACT A number of plants produce nectar or pollen that contains toxins which can adversely affect honey bees. Almond (Amygdalus communis L. (Rosaceae)) produces amygdalin, a toxic cyanoglycoside, in its nectar and pollen. Although beekeepers regard almond as a valuable nectar and pollen plant, and honey bees are deployed extensively for almond pollination, prolonged reliance on almond may be detrimental to honey bees' health. Our results suggest that almond nectar is not sufficiently rich in amygdalin to pose a hazard, but that almond pollen could be toxic if exclusively consumed by honey bees for much more than a week. Further tests are needed to determine if honey bees in almond orchards are at risk, or if they somehow cope with the toxin.
ABSTRACTCandidatus Liberibacter asiaticus is vectored by the psyllid Diaphorina citri Kuwayama (H... more ABSTRACTCandidatus Liberibacter asiaticus is vectored by the psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) and putatively causes Huanglongbing disease in citrus. Huanglongbing has reduced yields by 68% relative to pre-disease yields in Florida. Disease management is partly through vector control. Understanding vector biology is essential in this endeavor. Our goal was to document differences in probing behavior linked to sex. Based on both a literature review and our results we conclude that there is either no effect of sex or that identifying such an effect requires a sample size at least four times larger than standard methodologies. Including both color and sex in statistical models did not improve model performance. Both sex and color are correlated with body size, and body size has not been considered in previous studies on sex in D. citri. An effect of body size was found wherein larger psyllids took longer to reach ingestion behaviors and larger individuals spent mo...
The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector ... more The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of the phloem-infecting bacterium, Candidatus Liberibacter asiaticus. Candidatus L. asiaticus is the putative causal agent of Huanglongbing (HLB) disease, a destructive disease of Citrus. While many Citrus species are susceptible to D. citri probing and HLB disease, there are marked behavioral differences in D. citri probing responses and Ca. Liberibacter asiaticus infection severity among Citrus species. Using four mandarin hybrid selections and pummelo plants variably resistant to D. citri probing, oviposition, and survival, we explored probing differences using electropenetrography (EPG), conducted an oviposition and survival study, and determined host plant metabolites using gas-chromatography mass-spectroscopy (GC-MS). We found thirty-seven D. citri probing variables to be significantly different among tested mandarin selections and pummelo, in addition to differential oviposition a...
is a major pest of citrus because it transmits Liberibacter asiaticus, a phloem-limited bacterium... more is a major pest of citrus because it transmits Liberibacter asiaticus, a phloem-limited bacterium that putatively causes Huanglongbing (HLB). The disease moves slowly through a tree, and the vector facilitates further within-tree movement via transmission of the pathogen. However, this only happens when stylets contact the phloem, to inoculate bacteria during phloem salivation and acquire bacteria during phloem sap ingestion. Behavioral changes in associated with different plant parts would affect how long it takes to reach phloem and how long the psyllids stays in phloem to ingest, thereby influencing the risk of disease spread. feeding was recorded on the abaxial and adaxial surfaces of mature and immature citrus leaves. Adults in the field can be found on these surfaces at all times of year. On abaxial surface of immature leaves, phloem salivation would occur after 11 h on average, but rarely as soon as 0.56 h. The corresponding values on mature leaves were 16 and 2.7. In general...
Electropenetrography a.k.a. electrical penetration graph or EPG is a rigorous technique for study... more Electropenetrography a.k.a. electrical penetration graph or EPG is a rigorous technique for studying arthropod behavior. Essentially, the arthropod and host are part of an electrical circuit that generates patterns of voltage (waveforms), whose biological meanings are defined by correlation with histology and behavior. EPG is used for studying stylet probing behavior of hemipterans, and blood-feeding arthropods. These results are applied to understanding pesticide action, host plant resistance, and vector-pathogen-host interactions. At the end of all recordings, the arthropod begins a behavior that ends because the scientist stopped recording. An argument for keeping this event in the data has been made based on the assumption that the insect is adapting to laboratory conditions. In this adaptation process, the expected durations of ingestion behaviors will increase as the insect adapts. We show that this assumption can cause problems in data analysis and interpretation of the data....
Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, beha... more Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, behavior, and metabolism. Because these pathogens interact with their vectors on the cellular and organismal levels, potential changes at the biochemical level might occur. “ Candidatus Liberibacter asiaticus” ( C Las) is transmitted in a persistent, circulative, and propagative manner. The genome of C Las revealed the presence of an ATP translocase that mediates the uptake of ATP and other nucleotides from medium to achieve its biological processes, such as growth and multiplication. Here, we showed that the levels of ATP and many other nucleotides were significantly higher in C Las-infected than healthy psyllids. Gene expression analysis showed upregulation for ATP synthase subunits, while ATPase enzyme activity showed a decrease in ATPase activity. These results indicated that C Las stimulated Diaphorina citri to produce more ATP and many other energetic nucleotides, while it may inhibit ...
Host plant resistance is a practical and cost-effective approach for growers to manage insect pes... more Host plant resistance is a practical and cost-effective approach for growers to manage insect pests. Recently, three new sources of resistance in black raspberry (Rubus occidentalis; selections ORUS 3778-1, ORUS 3817-1, and ORUS 4109-1) against the large raspberry aphid, Amphorophora agathonica, were identified. We studied stages of host plant acceptance: host plant attraction, parturition (deposition of nymphs), nymph survival, and adult feeding behavior (using the electrical penetration graph [EPG]) to identify the location of the plant resistance. Aphids were more attracted to the susceptible cultivar ‘Munger’ (control) than the resistant selections. Parturition occurred on the resistant selections, but fewer nymphs were deposited on resistant lines relative to the susceptible control. Nymphs survived only an average of 3.3–3.6 days on resistant selections, whereas 94 % were still alive after 11 days on ‘Munger.’ There were differences in feeding behavior between the susceptible control and the resistant selections, but no differences between the three resistant selections. The tissues responsible for resistance appear to be the mesophyll and phloem sieve elements. Aphids had a reduced probability of salivation into the phloem sieve elements of resistant selections, and only one aphid each on ORUS 3778-1 and ORUS 4109-1 successfully ingested from the phloem. Because feeding behavior of A. agathonica did not differ between resistant selections, independent confirmation that resistance is conferred by unique genes should be obtained before pyramiding these sources together.
Annals of the Entomological Society of America, 2010
Our goal was to estimate seasonal changes in the proportion of Asian citrus psyllid, Diaphorina c... more Our goal was to estimate seasonal changes in the proportion of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), carrying Candidatus Liberibacter asiaticus. Our approach was to test Asian citrus psyllid by using pooled samples. The initial question was about pool size and the consequences of choosing poorly. Assuming no loss in sensitivity when diluting one infected individual with many healthy individuals, then it is recommend that a combination of all the published limits be used: keep the number of pools (n) above 20, the pool size (k) below 100, and the number of infected pools less than half the total number of pools. The most conservative approach to achieving the latter is to optimize pool size given an infection rate (p) such that k = ln(0.5)/ln(1 p). Exceeding these limits increases the probability that all the pools will be infected. If this occurs, then that particular sample will be discarded. Use of multiple pool sizes can be used to manage this ri...
Apple (Malus domestica Borkh.) leaves were sprayed to runoff using a selection of pesticides, and... more Apple (Malus domestica Borkh.) leaves were sprayed to runoff using a selection of pesticides, and subsequent spray retention was evaluated by weight. Timing, cultivar, leaf surface, and leaf type significantly influenced spray retention. Spray retention correlated significantly and positively with leaf hair density. The importance of these differences is considered in relation to the possible efficiency of the pesticide application process in apple orchards.
ABSTRACT Agricultural application of pesticides is often done by atomization of the active ingred... more ABSTRACT Agricultural application of pesticides is often done by atomization of the active ingredient and a carrier fluid. This atomized fluid lands on a plant surface and creates a mosaic of treated and untreated patches that we define as deposit structure. This structure influences the biological effect of the pesticide if dose per unit time is a factor in determining the overall toxicity of the pesticide. Laboratory bioassays are frequently carried out in such a way as to eliminate all effects of deposit structure. This bioassay methodology dictates the form of the observed dose-response relationship in these experiments. Although our observations do not invalidate previous bioassay methodology, they do suggest that current approaches provide a narrow view of dose-response relationships. These results affect bioassay methodology for testing toxicant efficacy and for experiments with insecticide resistance that use similar approaches to screen for resistance or study inheritance of pesticide resistance.
Annals of the Entomological Society of America, 1998
ABSTRACT Morphometric data of Aphis gossypii Glover are used as a case study to illustrate the us... more ABSTRACT Morphometric data of Aphis gossypii Glover are used as a case study to illustrate the use of randomization tests. The application of randomization tests in morphological evaluation and identification of species is a powerful tool for characterizing populations and species. It offers the advantage of reducing our reliance on the robustness of more classical approaches to overcome problems of small sample size, unequal sample size, and departures from normality. We review randomization test methodology. We address a few errors that have appeared in the literature. One question is how many randomizations. As a generic starting point, the number of randomizations should be 2 orders of magnitude larger than the inverse of the significant P value, but in critical cases an exact figure can be determined. A new methodology is introduced for using randomization tests to determine if the average of several observations is different from a constant. An extension of the method is used when the null hypothesis states that there are differences. This is important where there is reason to suspect that one is dealing with different populations (e.g., morphological measurements were taken from several distinct populations of an insect) and one needs to identify which populations are the same. This test should not be confused with the typical case where it is simply impossible to identify differences between different sets of observation. We present a SAS program to perform 2-tailed tests for differences between means.
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