Gary J. Puterka

Use of the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae)

We have used a new technique to identify discrete genetic markers in aphids, a family in which biochemical and morphological genetic polymorphisms are rare. The new technique uses the polymerase chain reaction (PCR) to amplify random regions of aphid genomes (random amplified polymorphic DNA) and has been termed RAPD-PCR. We demonstrate the use of the technique in revealing genetic variation in four aphid species, the greenbug ( Schizaphis graminum (Rondani)), the Russian wheat aphid ( Diuraphis noxia (Mordvilko)), the pea aphid ( Acyrthosiphon pisum (Harris)), and the brown ambrosia aphid ( Uroleucon ambrosiae (Thomas)). In contrast with allozyme surveys, RAPD-PCR revealed large amounts of genetic variation among individuals in each of these species. Variation was detected among biotypes, populations, colour morphs and even individuals on a single plant. We also explored the utility of RAPD-PCR in the detection and identification within aphid bodies of two endoparasitic wasps, Diaeretiella rapae (McIntosh) and Lysiphlebus testaceipes (Cresson). The use of RAPD-PCR in species diagnostics, parasitoid detection, and population studies is discussed.

Progress Toward Liquid Formulations of Particle Films for Insect and Disease Control in Pear

Abstract Particle film technology is aimed at controlling both arthropod pests and diseases of plants with a hydrophobic particle barrier primarily composed of kaolin. Field studies were conducted from 1996 to 1998 to compare the efficacy of dust and liquid applications, and hydrophobic and hydrophilic particle films, against key pests of pear. In addition, the effects of particle film applications on pear yield and quality were investigated in 1998. Dust and liquid applications of hydrophobic and hydrophilic particle films obtained high levels of early-season pear psylla control and prevented pear rust mite damage. We also found that prior seasonal applications of particle films in 1997 can carry over into the 1998 season to suppress early season pear psylla oviposition. A major concern in the shift from hydrophobic to hydrophilic particle films was the loss of disease control. We found that a water-repellant particle film was not required to control the fungal disease fabraea leaf spot. Pear yields were nearly doubled by liquid formulations of hydrophobic and hydrophilic particle films. Particle film deposits were measured using a spectrophotometer method we developed. Particle deposition differed among formulations for both leaf age and leaf surface (top or bottom). Yet, the particle formulations performed about the same against insects and fungal diseases, and in how they influenced the horticultural traits. None of the particle film formulations were found to be phytotoxic to pear foliage or fruit during the study period. A shift from hydrophobic to hydrophilic particles makes it possible to more easily formulate and disperse the particles in water so that conventional spray equipment can be used. The multifunctionality and low toxicity of particle films could make them an attractive alternative to conventional pesticides.

Proteomic analysis of secreted saliva from Russian Wheat Aphid (Diuraphis noxia Kurd.) biotypes that differ in virulence to wheat

Diuraphis noxia, Russian Wheat Aphid (RWA), biotypes are classified by their differential virulence to wheat varieties containing resistance genes. RWA salivary proteins, unlike those of most aphid species, cause foliar damage and physiological alterations in plants. A comparative proteomic analysis of secreted saliva from four differentially virulent RWA biotypes identified thirty-four individual proteins. The five major proteins were glucose dehydrogenase, lipophorin, chitinase, CiV16.8g1-like, and lava lamp. Fourteen proteins quantitatively varied among biotypes; trehalase, β-N-acetylglucosaminidase (chitinase), two separate glucose dehydrogenases, calreticulin, aminopeptidase, acetylglucosaminyltransferase, hydroxymethylglutaryl-CoA lyase, acyltransferase, ficolin-3, lava lamp, retinaldehyde-binding protein, and two proteins of unknown function. Fifty-four percent of spectral counts were associated with glucose dehydrogenase, which is thought to detoxify plant defensive compounds. One-dimensional electrophoresis detected nine protein bands from 9 to 60 kDa that quantitatively differed. Two-dimensional electrophoresis identified six major gel zones with quantitative and qualitative variance in proteins. Our findings reveal that the salivary proteome of RWA, a phytotoxic aphid, differs considerably from those reported for nonphytotoxic aphids. The potential roles of proteins used in the general plant feeding processes of aphids and those that are potential phytotoxins related to aphid virulence are discussed.

Particle Films for Suppression of the Codling Moth (Lepidoptera: Tortricidae) in Apple and Pear Orchards

Abstract Studies were conducted in 1997and 1998 to evaluate the effects of three particle film formulations consisting of kaolin and adjuvants on neonate larvae, ovipositing adult females, and eggs of the codling moth, Cydia pomonella (L.). Neonate larval walking speed, fruit discovery rate, and fruit penetration rate on apple host plants coated with particle films were significantly lower than on host plants without particle films in laboratory assays. Females oviposited less on host plants covered with a particle film residue than on untreated plants in laboratory choice and no-choice tests. Hatch rate of codling moth neonate larvae was unaffected by particle films sprayed on host plants either before or after oviposition. Fruit infestation rates were significantly reduced on particle film-treated trees compared with untreated trees for both first- and second-generation codling moth in field trials in both apple and pear orchards. Particle films appear to be a promising supplemental control approach for codling moth in orchards where moth density is high, and may represent a stand-alone method where moth densities are lower.

Effects of a Kaolin-Based Particle Film on Obliquebanded Leafroller (Lepidoptera: Tortricidae)

Abstract Studies were conducted in 1997 to evaluate the effects of the kaolin-based particle film formulation M96–018 on adults, eggs, and larvae of the obliquebanded leafroller, Choristoneura rosaceana (Harris). Particle film treatments significantly reduced female longevity, mating success, and number of egg masses oviposited compared with moths on untreated apple leaves in sleeve-cage and screen-cage tests. No differences in mating success or oviposition were caused by the application rates and coverage density of M96–018 on foliage. Females avoided ovipositing on particle film-treated leaves in choice tests. Larval hatch was not affected by topical application or residual exposure to M96–018. Larval weight gain and pupal weight were significantly reduced and larval mortality increased in no-choice feeding tests with M96–018. In choice tests, larvae preferred to feed on untreated leaf surfaces. The negative effects on larval development and survivorship on M96–018-treated foliage did not differ across a fourfold difference in spray application rate. A significant reduction in the number of infested shoots was found in orchard trials when M96–018 was applied before bud break in late March compared with untreated trees. No reductions in larval densities were found compared with an untreated control following prebloom and postbloom applications.

Salivary Proteins of Russian Wheat Aphid (Hemiptera: Aphididae)

ABSTRACT Salivary secretions play critical roles in aphid- host plant interactions and are responsible for damage associated with aphid feeding. The objectives of this study were to evaluate aspects of salivation and the salivary constituents of Diuraphis noxia (Hemiptera: Aphididae). Salivary proteins were isolated and compared from three aphid probed diets: pure water, 15% sucrose, or amino acids (100 mM serine, 100 mM methionine, 100 mM aspartic acid, and 15% sucrose). After 6 h, more aphids settled on sucrose diet compared with other diets, but there were no significant differences in the number of stylet sheaths produced per aphid after 24 h. There were differences in the amount of soluble salivary protein (watery saliva), with the greatest amount secreted in sucrose diet, followed by amino acid diet and pure water, respectively. Protein constituents secreted into sucrose and amino acid diets were compared using gel electrophoresis using standardized amounts of protein. More protein bands and bands of greater intensity were visualized from probed sucrose diet compared with probed amino acid diet, indicating qualitative differences. Phosphatase was putatively identified from D. noxia saliva from a major protein band using gel electrophoresis and mass spectrophotometry. Alkaline phosphatase activity was confirmed in sucrose diet using enzymatic assays but was not detected in aphid probed water or amino acid diets. Other peptides in sucrose diet weakly but significantly showed similarities to putative dehydrogenase and RNA helicase expressed sequence tags identified from other aphids. The implications of these findings in aphid salivation and plant-insect interactions are discussed.

Biotypic diversity in Colorado Russian wheat aphid (Hemiptera: Aphididae) populations.

Abstract The biotypic diversity of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), was assessed in five isolates collected in Colorado. Three isolates, RWA 1, RWA 2, and an isolate from Montezuma County, CO, designated RWA 6, were originally collected from cultivated wheat, Triticum aestivum L., and obtained from established colonies at Colorado State University. The fourth isolate, designated RWA 7, was collected from Canada wildrye, Elymus canadensis L., in Baca County, CO. The fifth isolate, designated RWA 8, was collected from crested wheatgrass, Agropyron cristatum (L.) Gaertn., in Montezuma County, CO. The four isolates were characterized in a standard seedling assay, by using 24 plant differentials, 22 wheat lines and two barley, Hordeum vulgare L., lines. RWA 1 was the least virulent of the isolates, killing only the four susceptible entries. RWA 8 also killed only the four susceptible entries, but it expressed intermediate virulence on seven wheat lines. RWA 6, killing nine entries, and RWA 7, killing 11 entries, both expressed an intermediate level of virulence overall, but differed in their level of virulence to ‘CO03797′ (Dn1), ‘Yumar’ (Dn4), and ‘CO960293-2′. RWA 2 was the most virulent isolate, killing 14 entries, including Dn4- and Dny-containing wheat. Four wheat lines, ‘94M370′ (Dn7), ‘STARS 02RWA2414-11′, CO03797, and ‘CI2401′, were resistant to the five isolates. The results of this screening confirm the presence of five unique Russian wheat aphid biotypes in Colorado.

Comparisons of Salivary Proteins from Five Aphid (Hemiptera: Aphididae) Species

ABSTRACT Aphid (Hemiptera: Aphididae) saliva, when injected into host plants during feeding, causes physiological changes in hosts that facilitate aphid feeding and cause injury to plants. Comparing salivary constituents among aphid species could help identify which salivary products are universally important for general aphid feeding processes, which products are involved with specific host associations, or which products elicit visible injury to hosts. We compared the salivary proteins from five aphid species, namely, Diuraphis noxia (Kurdjumov), D. tritici (Gillette), D. mexicana (Baker), Schizaphis graminum (Rondani), and Acyrthosiphon pisum (Harris). A 132-kDa protein band was detected from the saliva of all five species using sodium dodecyl sulfate Polyacrylamide gel electrophoresis. Alkaline phosphatase activity was detected from the saliva of all five species and may have a universal role in the feeding process of aphids. The Diuraphis species cause similar visible injury to grass hosts, and nine electrophoretic bands were unique to the saliva of these three species. S. graminum shares mutual hosts with the Diuraphis species, but visible injury to hosts caused by S. graminum feeding differs from that of Diuraphis feeding. Only two mutual electrophoretic bands were visualized in the saliva of Diuraphis and S. graminum. Ten unique products were detected from the saliva of A. pisum, which feeds on dicotyledonous hosts. Our comparisons of aphid salivary proteins revealed similarities among species which cause similar injury on mutual hosts, fewer similarities among species that cause different injury on mutual hosts, and little similarity among species which feed on unrelated hosts.

Distribution and diversity of russian wheat aphid (Hemiptera: Aphididae) biotypes in North America.

Abstract Wheat, Triticum aestivum L., with Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) resistance based on the Dn4 gene has been important in managing Russian wheat aphid since 1994. Recently, five biotypes (RWA1–RWA5) of this aphid have been described based on their ability to differentially damage RWA resistance genes in wheat. RWA2, RWA4, and RWA5 are of great concern because they can kill wheat with Dn4 resistance. In 2005, 365 Russian wheat aphid clone colonies were made from collections taken from 98 fields of wheat or barley, Hordeum vulgare L., in Oklahoma, Texas, New Mexico, Colorado, Kansas, Nebraska, and Wyoming to determine their biotypic status. The biotype of each clone was determined through its ability to differentially damage two resistant and two susceptible wheat entries in two phases of screening. The first phase determined the damage responses of Russian wheat aphid wheat entries with resistance genes Dn4, Dn7, and susceptible ‘Custer’ to infestations by each clone to identify RWA1 to RWA4. The second phase used the responses of Custer and ‘Yuma’ wheat to identify RWA1 and RWA5. Only two biotypes, RWA1 and RWA2, were identified in this study. The biotype composition across all collection sites was 27.2% RWA1 and 72.8% RWA2. RWA biotype frequency by state indicated that RWA2 was the predominant biotype and composed 73–95% of the biotype complex in Texas, Oklahoma, Colorado, and Wyoming. Our study indicated that RWA2 is widely distributed and that it has rapidly dominated the biotype complex in wheat and barley within its primary range from Texas to Wyoming. Wheat with the Dn4 resistance gene will have little value in managing RWA in the United States, based on the predominance of RWA2.

Absence of Mitochondrial DNA Sequence Variation in Russian Wheat Aphid (Hemiptera: Aphididae) Populations Consistent with a Single Introduction into the United States

Abstract Russian wheat aphid, Diuraphis noxia (Kurdjumov), was introduced into North America in Mexico during 1980 and by 1988 had spread north across the Western US into Southern Canada. South Africa has been suspected as the source of the introduction. Russian wheat aphid is the major insect pest of wheat and barley in the southern and central plains states. Resistant wheat with the Dn4 gene was the primary management tactic from 1996 to 2003. During 2003, a biotype able to injure Dn4 wheat (designated RWA2) appeared in Colorado. The source of the biotype is unclear, that is whether it represents a new introduction from another country or if it arose from the extant population in the US. We examined a 332 bp portion of the cytochrome oxidase subunit I gene (COI) in the mtDNA of Russian wheat aphid collected in the US from 1986 through 2006. Also included were specimens from Mexico and South Africa. Except for two nucleotides in two individual aphids, no COI sequence variation was found in US populations over a 20 yr period. US populations had COI sequences which were identical to those found in Mexico, Turkey, France, and one aphid from South Africa. A second aphid from South Africa differed by only two bp. The lack of COI nucleotide variation suggested that biotype RWA2 did not represent a second introduction of a genetically distinct population from another country. The data are consistent with a single introduction from South Africa and no other introductions of virulent biotypes into the US occurred since 1986.