Biotypic diversity in greenbug (Hemiptera: Aphididae): characterizing new virulence and host associations

Biotypic diversity of the greenbug, Schizaphis graminum (Rondani) (Hemiptera: Aphididae), was assessed among populations collected from cultivated wheat, Triticum aestivum L., and sorghum, Sorghum bicolor (L.) Moench, and their associated noncultivated grass hosts. Greenbugs were collected during May through August 2002 from 30 counties of Kansas, Nebraska, Oklahoma, and Texas. Discounting the presumptive biotype A, five of the remaining nine letter-designated greenbug biotypes were collected; however, biotypes C, F, J, and K were not detected. Biotypes E and I exhibited the greatest host range and were the only biotypes collected in all four states. Sixteen greenbug clones, collected from eight plant species, exhibited unique biotype profiles. Eleven were collected from noncultivated grasses, three from wheat, and two from sorghum. The most virulent biotypes were collected from noncultivated hosts. The great degree of biotypic diversity among noncultivated grasses supports the contention that the greenbug species complex is composed of host-adapted races that diverged on grass species independently of, and well before, the advent of modern agriculture.     

Molecular analysis of sorghum resistance to the greenbug (Homoptera: Aphididae)

Chromosomal regions of sorghum, Sorghum bicolor (L.) Moench, conferring resistance to greenbug, Schizaphis graminum (Rondani), biotypes C, E, I, and K from four resistance sources were evaluated by restriction fragment-length polymorphism (RFLP) analysis. At least nine loci, dispersed on eight linkage groups, were implicated in affecting sorghum resistance to greenbug. The nine loci were named according to the genus of the host plant (Sorghum) and greenbug (Schizaphis graminum). Most resistance loci were additive or incompletely dominant. Several digenic interactions were identified, and in each case, these nonadditive interactions accounted for a greater portion of the resistance phenotype than did independently acting loci. One locus in three of the four sorghum crosses appeared responsible for a large portion of resistance to greenbug biotypes C and E. None of the loci identified were effective against all biotypes studied. Correspondingly, the RFLP results indicated resistance from disparate sorghums may be a consequence of allelic variation at particular loci. To prove this, it will be necessary to fine map and clone genes for resistance to greenbug from various sorghum sources.     

Mitochondrial DNA sequences of greenbug (Homoptera: Aphididae) biotypes

Sequence comparisons were made for 738-bp of mtDNA cloned from seven greenbug, Schizaphis graminum, biotypes (B, C, E, F, G, H and I) obtained from laboratory colonies maintained by USDA-ARS, Stillwater, OK. These sequences include parts of the genes for 16S ribosomal subunit (16S rRNA), tRNAleu, tRNAser, cytochrome b (cytb) and NADH dehydrogenase (ND) subunits one and four. Sequence data revealed considerable variation in 86 (12%) nucleotide sites over the 738-bp sequenced among the seven greenbug biotypes. Nucleotide invariance was observed within the seven greenbug biotypes from both the laboratory colonies and field collected biotype E greenbugs from Kansas, Nebraska, Oklahoma, and Texas.     

Mitochondrial DNA sequence divergence among Schizaphis graminum (Hemiptera: Aphididae) clones from cultivated and non-cultivated hosts: haplotype and host associations

A 1.0 kb region of the mitochondrial cytochrome oxidase subunit I gene from the greenbug aphid, Schizaphis graminum (Rondani), was sequenced for 24 field collected clones from non-cultivated and cultivated hosts. Maximum likelihood, maximum parsimony and neighbour-joining phylogenies were estimated for these clones, plus 12 previously sequenced clones. All three tests produced trees with identical topologies and confirmed the presence of three clades within S. graminum. Clones showed no relationship between biotype and mtDNA haplotype. At least one biotype was found in all three clades, suggesting exchange among clades of genetic material conditioning for crop virulence, or the sharing of a common ancestor. However, there was a relationship between host and haplotype. Clade 1 was the most homogeneous and contained 12 of 16 clones collected from cultivated hosts and five of the six collected from johnsongrass, Sorghum halepense, a congener of cultivated sorghum, S. bicolor. Four of the six clones collected from Agropyron spp. were found in clade 2. Clade 3 contained two clones from wheat, Triticum aestivum, and four from non-cultivated hosts other than Agropyron spp. A partitioning of populations by mtDNA haplotype and host suggests the occurrence of host adapted races in Schizaphis graminum.     

Genetic diversity of sorghum accessions resistant to greenbugs as assessed with AFLP markers.

Sorghum, Sorghum bicolor (L.) Moench, is the fifth most important cereal crop grown worldwide and the fourth in the United States. Greenbug, Schizaphis graminum (Rondani), is a major insect pest of sorghum with several biotypes reported to date. Greenbug biotype I is currently the most prevalent and most virulent on sorghum plants. Breeding for resistance is an effective way to control greenbug damage. A successful breeding program relies in part upon a clear understanding of breeding materials. However, the genetic diversity and relatedness among the greenbug biotype I resistant accessions collected from different geographic origins have not been well characterized, although a rich germplasm collection is available. In this study, 26 sorghum accessions from 12 countries were evaluated for both resistance to greenbug biotype I and genetic diversity using fluorescence-labeled amplified fragment length polymorphism (AFLP). Twenty-six AFLP primer combinations produced 819 polymorphic fragments indicating a relatively high level of polymorphism among the accessions. Genetic similarity coefficients among the sorghum accessions ranged from 0.69 to 0.90. Cluster analysis indicated that there were two major groups based on polymorphic bands. This study has led to the identification of new genetic sources of sorghum with substantial genetic variation and distinct groupings of resistant accessions that have the potential for use in the development of durable greenbug resistant sorghum.     

Using high-throughput amplified fragment length polymorphism to distinguish sorghum greenbug (Homoptera: Aphididae) biotypes

Abstract 1 The greenbug Schizaphis graminum (Rondani) is a serious pest of Sorghum bicolor L. and small grains in the Southern Plains of the U.S.A. Use of resistant cultivars, the major greenbug management strategy, has been challenged by the rapid development of new greenbug biotypes that overcome plant resistance. 2 We used a high‐throughput amplified fragment length polymorphism (AFLP) fingerprinting method to examine genetic divergence among eight greenbug biotypes (B, C, E, G, I and K, New York and South Carolina). Clustering analysis based on 1775 scored AFLP markers clearly showed that biotypes (C, E, I and K), which are able to infest sorghum fields, share more common polymorphisms among themselves than with other biotypes. 3 This result suggests that common genetic factors exist among these biotypes, enabling them to predominate and thrive in monoculture crops. Our study demonstrated the sensitivity of AFLP in obtaining large quantities of biotype‐associated polymorphic information across the entire greenbug genome.     

Mitochondrial DNA sequences of greenbug (Homoptera: Aphididae) biotypes

Sequence comparisons were made for 738-bp of mtDNA cloned from seven greenbug, Schizaphis graminum, biotypes (B, C, E, F, G, H and I) obtained from laboratory colonies maintained by USDA-ARS, Stillwater, OK. These sequences include parts of the genes for 16S ribosomal subunit (16S rRNA), tRNA^leu, tRNA^ser, cytochrome b (cytb) and NADH dehydrogenase (ND) subunits one and four. Sequence data revealed considerable variation in 86 (12%) nucleotide sites over the 738-bp sequenced among the seven greenbug biotypes. Nucleotide invariance was observed within the seven greenbug biotypes from both the laboratory colonies and field collected biotype E greenbugs from Kansas, Nebraska, Oklahoma, and Texas.     

Molecular phylogenetics of heliothine moths (Lepidoptera: Noctuidae: Heliothinae), with comments on the evolution of host range and pest status

Abstract The Heliothinae are a cosmopolitan subfamily of about 365 species that include some of the world’s most injurious crop pests. This study re‐assesses evolutionary relationships within heliothines, providing an improved phylogeny and classification to support ongoing intensive research on heliothine genomics, systematics, and biology. Our phylogeny estimate is based on two nuclear gene regions, namely elongation factor‐1α (EF‐1α; 1240 bp) and dopa decarboxylase (DDC; 687 bp), and on the barcoding region of mitochondrial cytochrome oxidase I (COI; 708 bp), providing a total of 2635 bp. These were sequenced for 71 heliothines, representing all major genera and nearly all recognized subgenera and species groups, and for 16 outgroups representing all major lineages of trifine Noctuidae. Analysis of the combined data by maximum likelihood, unweighted parsimony and Bayesian methods gave nearly identical topologies, and the individual gene trees showed only one case of potentially strong conflict. Relationships among genera and subgenera are resolved with strong bootstrap support. The earliest‐diverging lineages (c. 200 species in total) consist almost entirely of host specialists, reflecting the inferred ancestral heliothine host range under parsimony. The remaining species form a clade – the Heliothis group – that includes most of the polyphages (30% of heliothines) and all of the major pests. Many other species in the Heliothis group, however, are host specialists. Our results extend previous efforts to subdivide this large clade, and show the most notorious pest groups, the corn earworm complex (Helicoverpa) and the tobacco budworm (Heliothis virescens) group, to be closely related, joining with a small oligophagous genus in what we term the major‐pest lineage. Thus, genomic/experimental results from one model pest may extrapolate well to other pest species. The frequency of evolutionary expansion and contraction in host range appears to increase dramatically at the base of the Heliothis group, in contrast to the case for earlier‐diverging lineages. We ascribe this difference provisionally to differential evolutionary constraints arising from contrasting life‐history syndromes. Host‐specific behaviour and crypsis, coupled with low fecundity and vagility, may discourage host‐range expansion in earlier‐diverging lineages. By contrast, in the Heliothis group, the absence of host‐specific traits, coupled with high vagility and fecundity, may more readily permit expansion or contraction of the host range in response to varying ecological pressures such as host species abundance or differential competition and predation.     

Life history study of multiple clones of insecticide resistant and susceptible greenbug Schizaphis graminum (Homoptera: Aphididae)

Comparative differences and similarities in prereproductive time (d), progeny production in a time equal to d (Md), and intrinsic rate of increase (rm) were established for one susceptible (S) and three resistant (R) strains of the greenbug, Schizaphis graminum (Rondani), reared on sorghum hybrids Dekalb G550E and Cargill 607E. The R strains showed three patterns of elevated esterase activity. Four R1 clones, four R2 clones, one R3 clone, and four S clones were evaluated. The interaction of sorghum hybrid and greenbug strain did not significantly influence any of the parameters measured. However, R1 greenbugs exhibited a significantly longer prereproductive period than the other strains. In addition, the R1 strain had a significantly slower intrinsic rate of increase than the R2 or S greenbug strains, but did not differ significantly from the R3 strain. These results suggest that R1 greenbugs may be less fit than the other strains studied.     

Identification of a major quantitative trait locus conditioning resistance to greenbug biotype E in sorghum PI 550610 using simple sequence repeat markers

Greenbug, Schizaphis graminum (Rondani), represents the most important pest insect of sorghum, Sorghum bicolor (L.) Moench, in the Great Plains of the United States. Biotype E is the most widespread and dominant type not only in sorghum and wheat, Triticum aestivum L., fields, but also on many noncultivated grass species. This study was designed to determine sorghum accession PI 550610 resistance to greenbug biotype E, to map the resistance quantitative trait loci (QTLs) by using an established simple sequence repeat (SSR) linkage map and to identify SSR markers closely linked to the major resistance QTLs. In greenhouse screening tests, seedlings of PI 550610 showed strong resistance to the greenbug at a level similar to resistant accession PI550607. For QTL mapping, one F2 population containing 277 progeny and one population containing 233 F2:3 families derived from Westland A line x PI 550610 were used to genotype 132 polymorphic SSR markers and to phenotype seedling resistance to greenbug feeding. Phenotypic evaluation of sorghum seedling damage at 7, 12, 17, and 21 d postinfestation in the F2:3 families revealed that resistance variation was normally distributed. Single marker analysis indicated 16 SSRs spread over five chromosomes were significant for greenbug resistance. Composite interval and multiple interval mapping procedures indicated that a major QTL resided in the interval of 6.8 cM between SSR markers Xtxp358 and Xtxp289 on SBI-09. The results will be valuable in the development of new greenbug biotype E resistant sorghum cultivars and for the further characterization of major genes by map-based cloning.     

Distribution and abundance of insecticide resistant greenbugs (Homoptera: Aphididae) and validation of a bioassay to assess resistance

Laboratory bioassays were conducted to determine the toxicity of four insecticides (ethyl parathion, chlorpyrifos, malathion, and carbofuran) to insecticide-susceptible and resistant populations of greenbug, Schizaphis graminum (Rondani). These bioassays were used to develop and validate a discriminating concentration for assessing insecticide resistance in greenbug populations in the field. Samples from wheat and sorghum in two states, Oklahoma and Kansas, indicated that insecticide resistance persists in greenbug populations over a large area at a low level.     

Host-plant resistance of sorghum: Differential hydrolysis of sorghum pectic substances by polysaccharases of greenbug biotypes (Schizaphis graminum,homoptera: Aphididae)

Pectic substances extracted from different varieties of sorghum are hydrolyzed at differing rates by unfractionated polysaccharases isolated from two biotypes (C, GBC; and E, GBE) of the sorghum pest, Schizaphis graminum (the greenbug). A higher degree of susceptibility of a sorghum variety is associated with a greater rate of hydrolysis of sorghum pectic substances by a greenbug biotype. Increases in the specific activity of polysaccharases on the pectic substances from a resistant sorghum variety are dependent on the duration that a biotype is maintained as a colony on that variety. Polysaccharase activity of GBE on arabinogalactan was significantly greater than GBC. However, there were no differences between the biotypes on the depolymerization of a variety of other plant matrix polysaccharides and a synthetic polysaccharide. The sequence of substrates of increasing refractoriness to hydrolysis are: arabinogalactan < microcrystalline cellulose < xylan < pectin < 2,3-diacetyl pectin < α-1,4-galacturonan. Pectic substances from sorghum varieties resistant to GBC but susceptible to GBE are relatively lower in arabinogalactan with elevated levels of uronic acid (UA) compared to varieties susceptible to both biotypes. A sorghum variety resistant to both GBC and GBE was lowest in levels of arabinogalactan, highest in UA, and highest in fructan content, which in the other varieties occurred only in trace amounts. Pectic composition of rhamnose, xylose, and glucose showed no relationship to resistance. Bound phenolics (potential inhibitors of enzyme activity) were not detected in any of the sorghum pectic substances. The relationship of plant matrix polysaccharides to host-plant aphid biotype compatibility is discussed.     

Categories of resistance to greenbug (Homoptera: Aphididae) biotype K in wheat lines containing Aegilops tauschii genes

The wheat lines (cultivars) 'Largo', 'TAM110', 'KS89WGRC4', and 'KSU97-85-3' conferring resistance to greenbug, Schizaphis graminum (Rondani), biotypes E, I, and K were evaluated to determine the categories of resistance in each line to greenbug biotype K. Our results indicated that Largo, TAM110, KS89WGRC4, and KSU97-85-3 expressed both antibiosis and tolerance to biotype K. Largo, KS89WGRC4, and KSU97-85-3, which express antixenosis to biotype I, did not demonstrate antixenosis to biotype K. The results indicate that the same wheat lines may possess different categories of resistance to different greenbug biotypes. A new cage procedure for measuring greenbug intrinsic rate of increase (r(m)) was developed, by using both drinking straw and petri dish cages, to improve the efficiency and accuracy of r(m)-based antibiosis measurements.     

The wheat curl mite Aceria tosichella (Acari: Eriophyoidea) is a complex of cryptic lineages with divergent host ranges: evidence from molecular and plant bioassay data

Aceria tosichella (the wheat curl mite, WCM) is a global pest of wheat and other cereals, causing losses by direct damage, as well as the transmission of plant viruses. The mite is considered to have an unusually wide host range for an eriophyoid species. The present study tested the commonly held assumption that WCM is a single, highly polyphagous species by assessing the host range of genetically distinct lineages of WCM occurring in Poland on different host plants. Genotyping was performed by analyzing nucleotide sequence data from fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear D2 region of 28S rDNA. Mean between‐lineage distance estimated using COI data was found to be one order of magnitude greater than the within‐clade lineage and, in some cases, comparable to distances between WCM lineages and a congeneric outgroup species. Host acceptance was tested by quantifying population growth for different WCM mitochondrial (mt)DNA lineages when transferred from source host plants to test plants. These experiments revealed significant differences in host colonization ability between mtDNA lineages, ranging from highly polyphagous to more host‐specific. The present study reveals that WCM is composed of several discrete genetic lineages with divergent host‐acceptance and specificity traits. Genetic variation for host acceptance within A. tosichella s.l. may act as a reproductive barrier between these lineages, most of which had narrow host ranges. Two lineages appear to have high pest potential on cereals, whereas several others appear to specialize on wild grass species. We conclude that WCM is not a homogeneous species comprising polyphagous panmictic populations rather it is a complex of genetically distinct lineages with variable host ranges and therefore variable pest potential. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 165–180.     

害虫生物型及其遗传机理

以农作物上5种重要害虫的生物型为例,简单地概括了分子标记在害虫生物型研究中的应用,重点阐述几种重要害虫如黑森瘿蚊,麦二叉蚜,褐飞虱,稻瘿蚊和烟粉虱等的生物型遗传变异机理。黑森瘿蚊、麦二叉蚜和稻瘿蚊的生物型由单基因控制,特定害虫生物型与作物基因型之间存在“基因_基因”关系;褐飞虱的致害性为多基因控制的数量性状;烟粉虱生物型的遗传变异机理尚不明确。还指出生物型遗传研究中待进一步解决的问题如生物型的遗传组成差异与致害力的关系等。     

Molecular mapping of QTLs for resistance to the greenbug <Emphasis Type="Italic">Schizaphis graminum</Emphasis> (Rondani) in <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (Moench)

Sorghum is a worldwide important cereal crop and widely cultivated for grain and forage production. Greenbug, Schizaphis graminum (Rondani) is one of the major insect pests of sorghum and can cause serious damage to sorghum plants, particularly in the US Great Plains. Identification of chromosomal regions responsible for greenbug resistance will facilitate both map-based cloning and marker-assisted breeding. Thus, a mapping experiment was conducted to dissect sorghum genetic resistance to greenbug biotype I into genomic regions. Two hundred and seventy-seven (277) F(2) progeny and their F(2:3) families from a cross between Westland A line (susceptible parent) and PI550610 (resistant parent) combined with 118 polymorphic simple sequence repeat (SSR) markers were used to map the greenbug resistance QTLs. Composite interval mapping (CIM) and multiple interval mapping (MIM) revealed two QTLs on sorghum chromosome nine (SBI-09) consistently conditioned the resistance of host plant to the greenbug. The two QTLs were designated as QSsgr-09-01 (major QTL) and QSsgr-09-02 (minor QTL), accounting for approximately 55-80%, and 1-6% of the phenotypic variation for the resistance to greenbug feeding, respectively. These resistance QTLs appeared to have additive and partially dominant effects. The markers Xtxp358, Xtxp289, Xtxp67 and Xtxp230 closely flanked the respective QTLs, and can be used in high-throughput marker-assisted selections (MAS) for breeding new resistant parents and producing commercial hybrids.     

Inheritance of greenbug resistance in several forms of grain sorghum and sudangrass

The inheritance of resistance against the Krasnodar population of common greenbug Schizaphis graminum Rond. was analyzed in nine accessions of grain sorghum and sudangrass. The dominant gene of cultivar Capbam (k-455, United States) was effective against some greenbug clones and differed from the Sgr1-Sgr11 resistance genes. The gene was designated as Sgr12. The cultivar Capbam was proposed for use as a differentiator in population genetic studies in S. graminum. The cultivar Sarvasi (k-3852, Hungary) contains not only the dominant Sgr1 gene, but also a recessive gene (most likely Sgr2), which is effective against some greenbug clones. Grain sorghum accessions k-928 and k-929 (Gugara Belaya, western China) each carry two highly effective dominant resistance genes, which differ from Sgr1-Sgr4, Sgr6, Sgr9, and Sgr10. In addition, the resistance genes of accession k-929 differ from the Sgr5 gene. Accession k-928 proved to contain an additional dominant resistance gene, which is expressed in response to some greenbug clones. The gene was designated as Sgr13. Sudangrass accessions k-100 and k-122 (Ukraine) each carry two dominant resistance genes. Accessions k-62, k-99 (Ukraine), and k-96 (Russia) each carry one dominant and one recessive resistance gene. The dominant resistance genes that are expressed in the cultivar Odesskaya 25 (k-122) in response to infestation with some clones from the natural greenbug population were designated as Sgr14 and Sgr15.     

Genetic evidence from mitochondrial, nuclear, and endosymbiont markers for the evolution of host plant associated species in the aphid genus Hyalopterus (Hemiptera: Aphididae)

Over the past several decades biologists' fascination with plant-herbivore interactions has generated intensive research into the implications of these interactions for insect diversification. The study of closely related phytophagous insect species or populations from an evolutionary perspective can help illuminate ecological and selective forces that drive these interactions. Here we present such an analysis for aphids in the genus Hyalopterus (Hemiptera: Aphididae), a cosmopolitan group that feeds on plants in the genus Prunus (Rosaceae). Hyalopterus currently contains two recognized species associated with different Prunus species, although the taxonomy and evolutionary history of the group is poorly understood. Using mitochondrial COI sequences, 16S rDNA sequences from the aphid endosymbiont Buchnera aphidicola, and nine microsatellite loci we investigated population structure in Hyalopterus from the most commonly used Prunus host species throughout the Mediterranean as well as in California, where the species H. pruni is an invasive pest. We found three deeply divergent lineages structured in large part by specific associations with plum, almond, and peach trees. There was no evidence that geographic or temporal barriers could explain the overall diversity in the genus. Levels of genetic differentiation are consistent with that typically attributed to aphid species and indicate divergence times older than the domestication of Prunus for agriculture. Interestingly, in addition to their typical hosts, aphids from each of the three lineages were frequently found on apricot trees. Apricot also appears to act as a resource mediated hybrid zone for plum and almond associated lineages. Together, results suggest that host plants have played a role in maintaining host-associated differentiation in Hyalopterus for as long as several million years, despite worldwide movement of host plants and the potential for ongoing hybridization.     

Molecular mapping of sorghum genes expressing tolerance to damage by greenbug (Homoptera: Aphididae)

Genetic linkage maps are fundamental for the localization of genes conferring tolerance to greenbug, Schizaphis graminum (Rondani), feeding damage in sorghum, Sorghum bicolor (L.) Moench. Thirteen linkage groups (LGs) containing 60 simple sequence repeat (SSR) loci were mapped by using a set of sorghum recombinant inbred lines (RILs) obtained from the cross '96-4121' (greenbug-tolerant parent) x Redlan (greenbug-susceptible parent). The LG spanned a distance of 603.5 cM, with the number of loci per LG varying from 2 to 14. Seventeen additional SSR loci were unlinked at a log of odds value of 3.0. Based on chlorophyll loss occurring after greenbug feeding, visual damage ratings, and soil plant analysis development (SPAD), chlorophyll-loss indices were recorded for each RIL and for the parents used in the cross. Composite-interval mapping identified three quantitative trait loci (QTLs) associated with biotype I and five QTLs associated with biotype K. The amount of phenotypic variation explained by these QTLs ranged from 9 to 19.6%. The identification of QTLs that influence greenbug tolerance will not only facilitate the use of marker-assisted selection in sorghum breeding programs but also will provide a solid foundation for detailed characterization of individual loci implicated in greenbug tolerance in sorghum.