Stephen F. Hanson

Genetic Analysis of a Novel Xylella fastidiosa Subspecies Found in the Southwestern United States

ABSTRACT Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.

Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.

Phylogenetic Analysis of the Alfalfa Weevil Complex (Coleoptera: Curculionidae) in North America

ABSTRACT The Eastern, Western, and Egyptian strains of alfalfa weevil are pests introduced to North America on three separate occasions, now they share partially overlapping geographic ranges, covering most of the continental United States. Behavior, susceptibility to parasites, and subtle morphological differences separate the strains. The difficulty in differentiating among these strains morphologically has led to the application of molecular phylogeny approaches including restriction fragment-length polymorphism characterization and sequencing of mitochondrial genes. While valuable for strain identification, this approach cannot identify interstrain hybrids because mitochondrial markers are maternally inherited. The work reported here extends previous findings by comparing over 7 Kb of sequence from two mitochondrial and four nuclear loci to increase the resolution of molecular phylogeny for these weevils. The related clover leaf weevil, also an occasional pest of alfalfa, was included in the analysis because the molecular phylogeny of this weevil has not been examined to date. Analysis of nuclear loci indicate that the clover weevil is a distinct species. Furthermore, while the three alfalfa weevil strains are separable based on mitochondrial sequence data they cannot be separated using nuclear loci suggesting that they are all recently diverged members of the same species. These data refine the relationships among these strains and may find application in design of better control strategies.

Establishing Guidelines to Improve Identification of Fire Ants Solenopsis xyloni and Solenopsis invicta

As red imported fire ant, Solenopsis invicta Buren, continues to expand its range into the southwestern United States, it can be easily confused with the native southern fire ant, Solenopsis xyloni McCook. Variability in the morphological characteristics commonly used to differentiate these ant species was quantified by examining the length of the clypeal tooth, striations of the mesopleuron, length of antennal scape, area of the petiolar process, number and size of mandibular teeth, and color by using both scanning electron and light microscopy. Given enough samples, the average values of each of these characteristics is different between the two species; however, significant morphological variability occurs in both S. xyloni and S. invicta populations, creating an area of overlap where either of the two species could exhibit similar characteristics. Better differentiation of these two species is achieved using a combination of characteristics, but morphological techniques are not dependable unless numerous ants from each population are analyzed by a taxonomist familiar with Solenopsis. For situations requiring a more accurate identification, such as before quarantining a county or a portion of a county, a molecular technique using mitochondrial DNA and polymerase chain reaction techniques was developed.

Xylella fastidiosa Detected in New Mexico in Chitalpa, a Common Landscape Ornamental Plant

Different strains of Xylella fastidiosa cause a variety of significant disease problems in agricultural and ornamental plants, including Pierces disease in grapes, oleander leaf scorch, pecan bacterial leaf scorch, and alfalfa dwarf disease. X. fastidiosa has never been reported in New Mexico but is known to exist in surrounding states (California, Arizona, and Texas). During the summer of 2006, several chitalpa (Chitalpa tashkinensis) hybrid trees with leaf scorch symptoms and branch die back were observed in Las Cruces, NM and they tested positive for X. fastidiosa by ELISA. Additional samples from these plants and others were analyzed by ELISA, PCR (2), and cultured on XfD2 medium (1). Known positive and negative oleander samples from Arizona were included as controls. Fifteen of thirty tested chitalpa were PCR and ELISA positive, indicating that they were infected with X. fastidiosa. Bacterial colonies that were PCR positive were also recovered from 10 of the XF positive samples that were plated. DNA sequences of PCR products amplified from chitalpa and isolated bacterial colonies (GenBank Accession Nos. EF109936 and EF109937) were identical to each other, 97% similar to X. fastidiosa strain JB-USNA, and 96% similar to the Temecula 1 strain. Independent ELISA testing (Barry Hill, California Department Food and Agriculture, Sacramento, CA) confirmed our ELISA and PCR results. On the basis of these results, we conclude that X. fastidiosa is present in New Mexico and that the common landscape ornamental chitalpa is a host for X. fastidiosa. Additional work is required to determine if X. fastidiosa is pathogenic to chitalpa and to examine the relevance of this potential X. fastidiosa reservoir to agricultural production in New Mexico and other areas where chitalpa is grown. References: (1) R. P. P. Almeida et al. Curr. Microbiol. 48:368, 2004. (2) M. R. Pooler et al. Lett. Appl. Microbiol. 25:123, 1997.

Survival and Transstadial Persistence of Trypanosoma cruzi in the bed bug (Hemiptera: Cimicidae)

Bed bug populations are increasing around the world at an alarming rate and have become a major public health concern. The appearance of bed bug populations in areas where Chagas disease is endemic raises questions about the role of these insects in the transmission of Trypanosoma cruzi, the etiological agent of the disease. In a series of laboratory evaluations, bed bug adults and nymphs were experimentally fed with T. cruzi-infected blood to assess the ability of T. cruzi to survive inside the bed bug and throughout the insects molting process. Live T. cruzi were observed in gut contents of experimentally infected bed bug adults via light microscopy and the identity of the parasite was confirmed via polymerase chain reaction analysis. T. cruzi persisted at least 97-d postinfection in adult bed bugs. Nymphal stage bed bugs that were infected with T. cruzi maintained the parasite after molting, indicating that transstadial passage of T. cruzi in bed bugs took place. This report provides further evidence of acquisition, maintenance, and for the first time, transstadial persistence of T. cruzi in bed bugs.

Allele-specific CAPS marker in a Ve1 homolog of Capsicum annuum for improved selection of Verticillium dahliae resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.

New Mexico and the southwestern US are affected by a unique population of tomato spotted wilt virus (TSWV) strains

Tomato spotted wilt virus (TSWV) is an important pathogen of many ornamental, greenhouse and agronomic crops worldwide. TSWV also causes sporadic problems in a number of crops in New Mexico (NM). Nucleocapsid gene sequences obtained from six different crop species across the state over four different years were used to characterize the NM TSWV population. This analysis shows that NM is affected by a unique TSWV population that is part of larger independent population present in the southwestern US. This population likely arose due to geographic isolation and is related to other TSWV populations from the US, Spain, and Italy.

Testing the Validity of the Lygaeus kalmii Complex (Hemiptera: Heteroptera: Lygaeidae) in North America Using DNA Sequences

ABSTRACT Genetic relationships within the Lygaeus kalmii complex were evaluated using specimens from multiple locations throughout the United States. Genes evaluated included the 18S ribosomal RNA (nuclear) and cytochrome oxidase subunit I (COI, mitochondrial). The 18S rRNA sequences were 100% conserved among all Lygaeus specimens, rendering the gene ineffective for inferring phylogenetic relationships for this project. However, the COI sequences were informative, and a COI-based phylogenetic tree revealed that L. reclivatus Say and both subspecies of L. kalmii (Stål) are closely related, with all falling within the same clade. Twelve different haplotypes of the COI gene were found among the sequences within the L. kalmii angustomarginatus, L. kalmii kalmii, and L. reclivatus clade. Each haplotype differed from other haplotypes by one to six nucleotides (0.13–0.77%), falling within the range of reported intraspecific divergence. Therefore, our data validate the conspecific classification of L. kalmii angustomarginatus and L. kalmii kalmii and suggest that L. reclivatus is not a distinct species. The overlap of haplotypes also suggests possible gene flow among all three groups.