Sharon Starkey

Molecular Basis of Plant Gene Expression During Aphid Invasion: Wheat Pto- and Pti-Like Sequences Are Involved in Interactions Between Wheat and Russian Wheat Aphid (Homoptera: Aphididae)

The Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), is a major pest of bread wheat, Triticum aestivum L. (em Thell), in most wheat-growing areas worldwide. Aphid-resistant cultivars are used to combat this pest, but very little is known about the molecular basis of resistance. In this study, differential gene expression in D. noxia biotype 1-resistant wheat plants containing the Dnx gene and D. noxia biotype 1 feeding on Dnx plants was investigated using suppressive subtraction hybridization. The derived subtracted cDNA library includes sequences similar to Pto and Pti1, genes involved in gene-for-gene recognition of and resistance to bacterial speck disease in tomato, Lycopersicon esculentum (L.). Pto- and Pti1-like sequences contain an activation domain with conserved amino acid residues crucial for avr protein recognition and binding by Pto, and avr-Pto phosphorylation of Pti1. Wheat defense signaling is represented by sequences putatively involved in producing sterols, jasmonates, Ca2+, and abscisic and gibberellic acids. We suggest that reductions in populations of D. noxia fed Dnx plants are related to the expression of sequences involved in defensive chemical production, cellular transport, and exocytosis. Dnx plant tolerance of D. noxia feeding is proposed to be based on the expression of sequences putatively involved in self-defense against reactive oxygen species and toxins, and proteolysis; DNA, RNA, and protein synthesis; chloroplast and mitochondrial function; carbohydrate metabolism; and maintenance of cell homeostasis. D. noxia unsuccessfully counter Dnx by expressing sequences putatively involved in detoxification; proteolysis; DNA, RNA, protein, and lipid synthesis; carbohydrate metabolism; and mitochondrial function.

Identification of Russian Wheat Aphid (Homoptera: Aphididae) Populations Virulent to the Dn4 Resistance Gene

Abstract The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious worldwide pest of wheat and barley. Russian wheat aphid populations from Hungary, Russia, and Syria have previously been identified as virulent to D. noxia (Dn) 4, the gene in all Russian wheat aphid-resistant cultivars produced in Colorado. However, the virulence of Russian wheat aphid populations from central Europe, North Africa, and South America to existing Dn genes has not been assessed. Experiments with plants containing several different Dn genes demonstrated that populations from Chile, the Czech Republic, and Ethiopia are also virulent to Dn4. The Czech population was also virulent to plants containing the Dnx gene in wheat plant introduction PI220127. The Ethiopian population was also virulent to plants containing the Dny gene in the Russian wheat aphid-resistant ‘Stanton’ produced in Kansas. The Chilean and Ethiopian populations were unaffected by the antibiosis resistance in Dn4 plants. There were significantly more nymphs of the Chilean population on plants of Dn4 than on Dn6 plants at both 18 and 23 d postinfestation, and the Ethiopian population attained a significantly greater weight on Dn4 plants than on plants containing Dn5 or Dn6. These newly characterized virulent Russian wheat aphid populations pose a distinct threat to existing or proposed wheat cultivars possessing Dn4.

Identification and characterization of eleven glutathione S-transferase genes from the aquatic midge Chironomus tentans (Diptera: Chironomidae).

Eleven cDNAs encoding glutathione S-transferases (GSTs) were sequenced and characterized in Chironomus tentans, an ecologically important aquatic midge. Phylogenetic analysis revealed seven GSTs in three different cytosolic classes including 4 in sigma (CtGSTs1, CtGSTs2, CtGSTs3, CtGSTs4), 2 in delta (CtGSTd1, CtGSTd2), and 1 in omega (CtGSTo1). The remaining four GSTs (CtGSTu1, CtGSTu2, CtGSTu3, CtGSTu4) were unclassified due to their low relatedness to currently known classes of insect GSTs. Reverse-transcription (RT)-PCR analysis of the 11 GST genes showed that CtGSTd1, CtGSTu2, CtGSTu4, CtGSTs1, CtGSTs2, CtGSTs3, CtGSTs4 and CtGSTo1 were expressed in all tissues examined, including salivary glands, hemolymph, midgut, Malpighian tubules, fatbodies and carcass, whereas CtGSTd2 and CtGSTu1 were expressed in a limited number of tissues. CtGSTs1 and CtGSTs4 appeared to be the only two genes, of which expressions can be detected in eggs, whereas all the 11 GST genes showed various expression patterns in the four larval instars. However, expressions of CtGSTd2, CtGSTu1 and CtGSTu2 were not detectable in pupal and adult stages. Real-time quantitative PCR confirmed that the herbicide alachlor increased CtGSTd1, CtGSTs2 and CtGSTs3 gene expression by 2.1-, 2.8- and 4.3-fold, respectively, when fourth-instar midges were exposed to alachlor at 1000 microg/L for 72 h. Such increased gene expressions were associated with 2.2- and 1.8-fold decreases of total GST activities in vivo when CDNB and DCNB were used as substrates, respectively. Further studies showed that 65.5 and 73.5% of GST activities were inhibited in vitro by alachlor at 100 and 1000 microg/L, respectively. Because alachlor has been known as an electrophilic substrate that can be conjugated by glutathione (GSH), rapid in vitro inhibition of GST activities by alachlor suggested that decreased GST activities were likely caused by the depletion of GSH. However, alachlor may regulate different GST genes, as found in other organisms, leading to significantly increased transcriptional levels of CtGSTd1, CtGSTs2 and CtGSTs3 in out of 11 GST genes examined in this study.

Evidence of Evolving Carbaryl Resistance in Western Corn Rootworm (Coleoptera: Chrysomelidae) in Areawide-Managed Cornfields in North Central Kansas

Abstract Susceptibility of adult populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, to carbaryl was determined by a survey in 1996 before the implementation of an areawide management program near Scandia in north central Kansas. Subsequently, the susceptibility of western corn rootworm adults to carbaryl has been monitored throughout the program from 1997 to 2000 in both control and managed areas. In 1996, adults were highly susceptible to carbaryl with a mean LC50 value of 0.64 μg/vial. This value was comparable to those for adults collected from other regions within Kansas. However, adult susceptibility to carbaryl decreased rapidly within the managed area, where the cucurbitacin-carbaryl-based bait SLAM has been used as the primary tool to control adults in this project since 1997. In 1999, adults collected from the managed area were 9- and 20-fold less susceptible to carbaryl at the LC50 and LC90 levels, respectively, than those evaluated in 1996. In contrast, adults collected from the control area were only 2- and 3-fold less susceptible to carbaryl at the LC50 and LC90 levels, respectively, than adults evaluated in 1996. Although field adult populations of western corn rootworm were relatively low in 2000, evaluations showed trends similar to those in 1999 regarding their carbaryl susceptibility in the managed and control areas. These results provide evidence that western corn rootworm has been evolving carbaryl resistance rapidly in response to the use of SLAM in areawide-managed cornfields near Scandia.

Wheat Curl Mite Resistance: Interactions of Mite Feeding with Wheat Streak Mosaic Virus Infection

ABSTRACT The majority of plant viruses are dependent on arthropod vectors for spread between plants. Wheat streak mosaic virus (family Potyviridae, genus Tritimovirus, WSMV) is transmitted by the wheat curl mite, Aceria tosichella Keifer, and this virus and vector cause extensive yield losses in most major wheat (Triticum aestivum L.)-growing regions of the world. Many cultivars in use are susceptible to this vector-virus complex, and yield losses of 10–99% have been documented, wheat curl mite resistance genes have been identified in goat grass, Aegilops tauschii (Coss) Schmal., and transferred to hexaploid wheat, but very few varieties contain effectively wheat curl mite resistance, due to virulent wheat curl mite populations. However, wheat curl mite resistance remains an effective strategy to reduce losses due to WSMV. The goal of our project was to identify the most effective, reproducible, and rapid method for assessing wheat curl mite resistance. We also wanted to determine whether mite resistance is affected by WSMV infection, because the pathogen and pest commonly occur together. Single and group wheat curl mite infestations produced similar amounts of leaf rolling and folding on wheat curl mite-susceptible wheat varieties that were independent of initial wheat curl mite infestation. This finding will allow accurate, efficient, large-scale screening of wheat germplasm for wheat curl mite resistance by infesting plants with sections of wheat leaf tissue containing mixed stages of wheat curl mite. The wheat curl mite-resistant breeding line ‘OK05312’ displayed antibiosis (reduced wheat curl mite population development). The effect of WSMV infection on wheat curl mite reproduction was genotype-dependent. Mite populations increased on infected wheat curl mite- and WSMV-susceptible plants compared with uninfected plants, but WSMV infection had no significant effect on wheat curl mite populations on resistant plants. OK05312 is a strong source of wheat curl mite resistance for wheat breeding programs.

Feeding Behavior of Russian Wheat Aphid (Hemiptera: Aphididae) Biotype 2 in Response to Wheat Genotypes Exhibiting Antibiosis and Tolerance Resistance

ABSTRACT In this study, wheat, Triticum aestivum L. (em Thell), genotypes containing the Dnx, Dn7, Dn6, and Dn4 genes for resistance to the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), along with Dn0, a susceptible control, were assessed to determine the categories of D. noxia biotype 2 (RWA2) resistance in each genotype and RWA2 feeding behaviors on Dnx and Dn0 plants by using the electronic penetration graph technique. At 14 d postinfestation, Dn0 plants exhibited intense chlorosis and leaf rolling, and all test genotypes expressed some degree of chlorosis and leaf rolling, except Dn7, which was not damaged. Both Dn7 and Dnx expressed antibiosis effects, significantly reducing the numbers of aphids on plants and the intrinsic rate of aphid increase. Dn6 plants seemed to contain tolerance, exhibiting tolerance index measurements for leaf and root dry weight and plant height that were significantly lower than those of the susceptible Dn0 plants. Principal component analyses indicated that antibiosis and leaf rolling data explained 80% of the variance among genotypes. Electronic penetration graph analysis demonstrated contrasting results between RWA1 and RWA2 phloem sieve element phase feeding events, but results indicated that Dnx resistance factors are present in the sieve element cells or phloem sap. Plants containing Dnx exhibit antibiosis resistance to D. noxia RWA2 similar to that in plants containing the Secale cereale L. (rye)-based Dn7 gene without the negative baking quality traits associated with Dn7.

Identification of Aegilops germplasm with multiple aphid resistance

The greenbug, Schizaphis graminum(Rondani), the Russian wheat aphid, Diuraphis noxia (Mordvilko), and the bird cherry oat aphid, Rhopalosiphum padi(L.), annually cause several million dollars worth of wheat production losses in Europe and the United States. In this study, Triticum and Aegilops accessions from the Czech Research Institute of Crop Production and the Kansas State University Wheat Genetic Resources Center were evaluated for resistance to these aphids. Accessions with aphid cross-resistance were examined for expression of the antibiosis, antixenosis, and tolerance categories of resistance. Aegilops neglecta accession 8052 exhibited antibiotic effects toward all three aphids in the form of reduced intrinsic rate of increase (rm). The rm of greenbug (biotype I) on Ae. neglecta 8052 was significantly lower than that of greenbugs on plants of the susceptible U. S. variety Thunder bird. The rm of Russian wheat aphids was significantly lower on foliage of both Ae. neglecta 8052 and T. araraticum accession 168 compared to Thunderbird. The rm values of bird cherry oat aphids fed both Ae. neglecta 8052 and T. araraticum 168 were also significantly lower than those fed the susceptible accession T. dicoccoides 62. Neither Ae. neglecta 8052 or T. araraticum 168 exhibited tolerance to either greenbug biotype I or Russian wheat aphid. Preliminary data suggest that T. araraticum 168 may also possess tolerance to bird cherry oat aphid. New genes from Ae. neglecta 8052 and T. araraticum 168 expressing aphid antibiosis can be used to develop multiple aphid resistant wheat in the U. S. and Central Europe.

Molecular genetic mapping of Gby , a new greenbug resistance gene in bread wheat

The greenbug, Schizaphis graminum (Rhodani), is one of the major insect pests of wheat worldwide and it is important to develop a basic understanding of the chromosomal locations of known and new greenbug resistance genes. Gby is a new greenbug resistance gene in the wheat line ‘Sando’s selection 4040’. A mapping population used in this study was derived from a cross of Sando’s 4040 and PI220127, a greenbug susceptible wheat land race from Afghanistan. A progeny test indicated that Gby is inherited as a single semi-dominant gene. A genetic linkage map consisting of Gby, Xgwm322 (a wheat microsatellite marker), XksuD2 (an STS marker) and 18 restriction fragment length polymorphism (RFLP) loci was constructed. We used DNA from Chinese Spring 7A deletion lines to show that the gwm332 and ksuD2 amplified fragments mapped in this study are located on a long arm of chromosome 7A. This suggests that Gby is located on wheat chromosome 7A. Gby was mapped to the area in the middle of the ‘island’ of putative defense response genes that are represented by RFLP markers (Xpsr119, XZnfp, Xbcd98 and Pr1b) previously mapped to the distal part of the short arm of wheat chromosome group 7. This region of chromosome 7A is characterized by a high recombination rate and a high physical density of markers which makes Gby a very good candidate for map-based cloning. The selection accuracy when the RFLP markers Xbcd98, Xpsr119 or XZnfp and Pr1b flanking Gby are used together to tag Gby is 99.78%, suggesting that they can be successfully used in marker assisted selection.

Variation of Resistance in Barley Against Biotypes 1 and 2 of the Russian Wheat Aphid (Hemiptera: Aphididae)

ABSTRACT The Russian wheat aphid, Diruaphis noxia (Kurdjumov) (Hemiptera: Aphididae), is globally one of the most devastating pests of bread wheat, Tritium aestivum L.; durum wheat, Triticum turgidum L.; and barley, Hordeum vulgare L. Host plant resistance is the foundation for cereal insect pest management programs, and several sources of D. noxia resistance have been incorporated in cultivars to manage D. noxia damage. The emergence of D. noxia North American biotype 2 (RWA2) in Colorado has made all known Dn genes vulnerable except the Dn7 gene from rye, Secale cereale, and has warranted exploration for sources of resistance to both RWA1 and RWA2. The category of resistance in resistant donor plants may exert selection pressure over the aphid population to form a new virulent population. In the current study, we report tolerance and antibiosis resistance to RWA1 and RWA2 in the barley genotype ‘Stoneham”. The rate and degree of expression of resistance in Stoneham against RWA1 and RWA2, although not similar, are greater than the partial resistance in ‘Sidney’. Antixenosis resistance to RWA1 or RWA2 was not observed in Sidney or Stoneham. The tolerance identified in Stoneham is encouraging because it may delay D. noxia biotype selection and fits well in a dryland barley cropping system.

Comparative Studies on Effects of Three Chitin Synthesis Inhibitors on Common Malaria Mosquito (Diptera: Culicidae)

Abstract Toxicities of three chitin synthesis inhibitors (diflubenzuron, nikkomycin Z and polyoxin D) were evaluated using second instars of the common malaria mosquito, Anopheles quadrimaculatus Say (Diptera: Culicidae). Neither nikkomycin Z nor polyoxin D at 50 μg/liter caused significant larval mortality, although they reduced the body weight of the survivors by 20.5 and 33.8%, respectively, in 48 h. In contrast, exposures of the larvae to diflubenzuron at 12.5 μg/liter for 48 h resulted in 86.7% larval mortality and reduced the body weight of the survivors by 29.1%. Exposure of the pupae (<12 h old) to diflubenzuron at 100 μg/liter for 48 h caused 18.9% pupal mortality and consequently reduced the adult emergence by 24.7% from the surviving pupae. Furthermore, exposure of third instars to diflubenzuron at 4, 20, 100, and 500 μg/liter for 24 h resulted in the reduction of larval chitin contents by 4.25, 33.2, 35.2, and 57.7%, respectively. Such an effect seemed to be associated with only cuticular chitin synthesis because the same exposures did not significantly affect chitin contents in the guts. Our results indicated that diflubenzuron was highly toxic to second instars by not only causing high larval mortality but also by affecting their growth. Diflubenzuron was also fairly toxic to pupae by not only causing pupal mortality but also affecting the adult emergence. Our results suggest that diflubenzuron might affect only chitin synthesis in the cuticle but not in the peritrophic matrix, which is probably due to diflubenzuron’s direct contact to mosquito larvae in water, slow distribution in insect body, rapid degradation in the insect gut, or a combination.