Identity of Schizaphis species (Hemiptera: Aphididae) in the United Kingdom: are they a threat to crops?

The greenbug, Schizaphis graminum (Rondani), is a major pest of cereals in some parts of the world and is of particular concern because it can be resistant to some insecticides and overcome the resistance of crops. In the UK, it has never been found on crops, but two rather little-known and closely-related species (Schizaphis holci and Schizaphis agrostis) are associated with the wild grasses, Holcus lanatus and Agrostis stolonifera. Since 1987, winged (alate) aphids morphologically resembling the greenbug have been found in increasing numbers in 12.2 m high suction-trap samples of the Rothamsted Insect Survey (RIS); hence, studies were undertaken to establish their identity. Clones (=asexual lineages) established from populations collected from H. lanatus in southern England showed strong preference for Holcus over Agrostis and Hordeum in laboratory tests and produced sexual morphs when transferred to short-day conditions, the males being apterous, as expected for S. holci. Multivariate morphometric comparisons of alatae caught in UK RIS suction traps in 2007 and 2011 with named specimens from museum collections, including S. graminum from many countries, indicated that the suction-trapped alatae were mostly S. agrostis and S. holci. Cytochrome c oxidase subunit I (COI) mtDNA obtained from 62 UK specimens from suction-traps had 95.4-100% sequence identity with US specimens of S. graminum. Two of the UK specimens had identical COI sequence to the US sorghum-adapted form of S. graminum, and these specimens also had 100% identity with a 640 bp fragment of nDNA CytC, indicating that this form of S. graminum may already be present in the UK. Present and future economic implications of these results are discussed.

Host race evolution in Schizaphis graminum (Hemiptera: Aphididae): nuclear DNA sequences.

The greenbug aphid, Schizaphis graminum (Rondani) was introduced into the United States in the late 1880s, and quickly was established as a pest of wheat, oat, and barley. Sorghum was also a host, but it was not until 1968 that greenbug became a serious pest of it as well. The most effective control method is the planting of resistant varieties; however, the occurrence of greenbug biotypes has hampered the development and use of plant resistance as a management technique. Until the 1990s, the evolutionary status of greenbug biotypes was obscure. Four mtDNA cytochrome oxidase subunit I (COI) haplotypes were previously identified, suggesting that S. graminum sensu lato was comprised of host-adapted races. To elucidate the current evolutionary and taxonomic status of the greenbug and its biotypes, two nuclear genes and introns were sequenced; cytochrome c (CytC) and elongation factor 1-α (EF1-α). Phylogenetic analysis of CytC sequences were in complete agreement with COI sequences and demonstrated three distinct evolutionary lineages in S. graminum. EF1-α DNA sequences were in partial agreement with COI and CytC sequences, and demonstrated two distinct evolutionary lineages. Host-adapted races in greenbug are sympatric and appear reproductively isolated. Agricultural biotypes in S. graminum likely arose by genetic recombination via meiosis during sexual reproduction within host-races. The 1968 greenbug outbreak on sorghum was the result of the introduction of a host race adapted to sorghum, and not selection by host resistance genes in crops.

Limited genetic variation within and between Russian wheat aphid (Hemiptera: Aphididae) biotypes in the United States.

Insect biotypes are populations able to kill or injure crops with resistance genes and complicate pest management programs based on host plant resistance. Biotypes occur in Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), a worldwide pest of wheat, Triticum aestivum L., and barley, Hordeum vulgare L., that was introduced into Mexico in 1980 and then spread into Texas by 1986. Five D. noxia biotypes were described in the United States and given the number designations 1 through 5. Of these, only Biotypes 1 and 2, which are nondamaging and damaging to Dn4-resistant wheat, respectively, are common and agriculturally important. Only a single clone of Biotypes 3, 4, and 5 were found in nature and now exist in the laboratory. The biotypes were found after 5 yr of the commercial planting of resistant wheat and their origin is unknown. To understand the genetic relatedness and origin of D. noxia biotypes in the United States, we used three molecular markers to assay for genetic variation within and between Biotypes 1 and 2, and for variation between Biotypes 1, 2, 3, 4, and 5. A single random amplified polymorphic DNA polymorphism was found in only two individuals. No DNA sequence variation in the cytochrome oxidase subunit I mitochondrial gene was found between 26 D. noxia clones. No variation was found at seven examined simple sequence repeat loci. Results suggest Biotype 2 originated from the extant population and does not represent a second introduction of a genetically divergent biotype.

Molecular markers for identification of the hyperparasitoids Dendrocerus carpenteri and Alloxysta xanthopsis in Lysiphlebus testaceipes parasitizing cereal aphids.

Polymerase chain reaction (PCR)-based molecular markers have been developed to detect the presence of primary parasitoids in cereal aphids and used to estimate primary parasitism rates. However, the presence of secondary parasitoids (hyperparasitoids) may lead to underestimates of primary parasitism rates based on PCR markers. This is because even though they kill the primary parasitoid, it's DNA can still be amplified, leading to an erroneous interpretation of a positive result. Another issue with secondary parasitoids is that adults are extremely difficult to identify using morphological characters. Therefore, we developed species-specific molecular markers to detect hyperparasitoids. A 16S ribosomal RNA mitochondrial gene fragment was amplified by PCR and sequenced from two secondary parasitoid species, Dendrocerus carpenteri (Curtis) (Hymenoptera: Megaspilidae) and Alloxysta xanthopsis (Ashmead) (Hymenoptera: Charipidae), four geographic isolates of the primary parasitoid, Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae), and six aphid species common to cereal crops. Species-specific PCR primers were designed for each insect on the basis of these 16S rRNA gene sequences. Amplification of template DNA, followed by agarose gel electrophoresis, successfully distinguished D. carpenteri and A. xanthopsis from all four isolates of L. testaceipes and all six cereal aphid species in this laboratory test.

Estimation of hymenopteran parasitism in cereal aphids by using molecular markers.

Polymerase chain reaction (PCR) primers were designed and tested for identification of immature parasitoids in small grain cereal aphids and for estimation of parasitism rates. PCR technique was evaluated for 1) greenhouse-reared greenbugs, Schizaphis graminum (Rondani), parasitized by Lysiphlebus testaceipes Cresson and 2) aphids collected from winter wheat fields in Caddo County, Oklahoma. For greenhouse samples, parasitism frequencies for greenbugs examined by PCR at 0, 24, and 48 h after removal of L. testaceipes parasitoids were compared with parasitism frequencies as determined by greenbug dissection. PCR was unable to detect parasitism in greenbugs at 0 and 24 h postparasitism, but it was able to detect parasitoids 48 h after parasitoid removal at frequencies that were not significantly different from dissected samples. Field-collected samples were analyzed by rearing 25 aphids from each sample and by comparing parasitoid frequencies of mummies developed and PCR performed on another 50 aphids. Aphid samples included corn leaf aphids, Rhopalosiphum maidis (Fitch); bird cherry-oat aphids, Rhopalosiphum padi (L.); English grain aphids, Sitobion avenae (F.); and greenbugs. Mummies were isolated until adult emergence, whereupon each parasitoid was identified to species (L. testaceipes was the only parasitoid species found). Parasitism detection frequencies for PCR also were not statistically different from parasitism frequencies of reared aphids. These results indicate that PCR is a useful tool for providing accurate estimates of parasitism rates and especially for identification of immature parasitoids to species.

Analysis of rILERS, an isoleucyl-tRNA synthetase gene associated with mupirocin production by Pseudomonas fluorescens NCIMB 10586.

Some strains of Pseudomonas fluorescens produce the antibiotic mupirocin, which functions as a competitive inhibitor of isoleucyl-tRNA synthetase (ILERS). Mupirocin-producing strains of P. fluorescens must overcome the inhibitory effects of the antibiotic to avoid self-suicide. However, it is not clear how P. fluorescens protects itself from the toxic effects of mupirocin. In this report, we describe a second gene encoding isoleucyl-tRNA synthetase (rILERS) in P. fluorescens that is associated with the mupirocin biosynthetic gene cluster. Random mutagenesis of the mupirocin-producing strain, P. fluorescens 10586, resulted in a mupirocin-defective mutant disrupted in a region with similarity to ILERS, the target site for mupirocin. The ILERS gene described in the present study was sequenced and shown to be encoded by a 3093 bp ORF, which is 264 bp larger than the ILERS gene previously identified in P. fluorescens 10586. rILERS from P. fluorescens is most closely related to prokaryotic or eukaryotic sources of ILERS that are resistant to mupirocin. Interestingly, the relatedness between rILERS and the ILERS previously described in P. fluorescens 10586 was low (24% similarity), which indicates that P. fluorescens contains two isoforms of isoleucyl-tRNA synthetase.