Journal Article Low-Level Detection of Candidatus Liberibacter Solanacearum in Bactericera cockerelli (Hemiptera: Triozidae) by 16s rRna Pyrosequencing Get access Alex P. Arp, Alex P. Arp 2 1Department of Biology, University of Texas at Tyler, 3900 University Blvd., Tyler, TX 75799. 2Corresponding author, e-mail: aalex@patriots.uttyler.edu. Search for other works by this author on: Oxford Academic PubMed Google Scholar Rebekah Chapman, Rebekah Chapman 1Department of Biology, University of Texas at Tyler, 3900 University Blvd., Tyler, TX 75799. Search for other works by this author on: Oxford Academic PubMed Google Scholar James M. Crosslin, James M. Crosslin 3Vegetable and Forage Crops Research Unit, USDA-ARS, Prosser, WA 99350. Search for other works by this author on: Oxford Academic PubMed Google Scholar Blake Bextine Blake Bextine 1Department of Biology, University of Texas at Tyler, 3900 University Blvd., Tyler, TX 75799. Search for other works by this author on: Oxford Academic PubMed Google Scholar Environmental Entomology, Volume 42, Issue 5, 1 October 2013, Pages 868–873, https://doi.org/10.1603/EN12260 Published: 01 October 2013 Article history Received: 14 September 2012 Accepted: 19 July 2013 Published: 01 October 2013
Abstract Work in animal production facilities often results in exposure to organic dusts. Previous studies have documented decreases in pulmonary function and lung inflammation among workers exposed to organic dust in the poultry industry. Bacteria and fungi have been reported as components of the organic dust produced in poultry facilities. To date, little is known about the diversity and concentration of bacteria and fungi inside poultry buildings. All previous investigations have utilized culture-based methods for analysis that identify only biota cultured on selected media. The bacterial tag-encoded flexible (FLX) amplicon pyrosequencing (bTEFAP) and fungal tag-encoded flexible (FLX) amplicon pyrosequencing (fTEFAP) are modern and comprehensive approaches for determining biodiversity of microorganisms and have not previously been used to provide characterization of exposure to microorganisms in an occupational environment. This article illustrates the potential application of this novel technique in occupational exposure assessment as well as other settings. An 8-hr area sample was collected using an Institute of Medicine inhalable sampler attached to a mannequin in a poultry confinement building. The sample was analyzed using bTEFAP and fTEFAP. Of the bacteria and fungi detected, 116 and 39 genera were identified, respectively. Among bacteria, Staphylococcus cohnii was present in the highest proportion (23%). The total inhalable bacteria concentration was estimated to be 7503 cells/m 3 . Among the fungi identified, Sagenomella sclerotialis was present in the highest proportion (37%). Aspergillus ochraceus and Penicillium janthinellum were also present in high proportions. The total inhalable fungi concentration was estimated to be 1810 cells/m 3 . These estimates are lower than what has been reported by others using standard epifluorescence microscope methods. However, no study has used non-culture-based techniques, such as bTEFAP and fTEFAP, to evaluate bacteria and fungi in the inhalable fraction of a bioaerosol in a broiler production environment. Furthermore, the impact of this bTEFAP and fTEFAP technology has yet to be realized by the scientific community dedicated to evaluating occupational and environmental bioaerosol exposure. Keywords: Aspergillus ochraceus bioaerosolorganic dustpoultrypyrosequencing ACKNOWLEDGMENTS This study would not have been possible without the assistance of Dr. Michael Pangburn and Ms. Aika Hussain. Grant support was provided for this project from the Southwest Center for Agricultural Health, Injury Prevention and Education (CDC/NIOSH U50 OH07541) at the University of Texas Health Science Center at Tyler.
Xylella fastidiosa is a plant pathogenic bacterium that causes many economically important agricultural diseases and is transmitted by the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). Efficient detection of X. fastidiosa in field collected H. vitripennis in an area-wide management program can contribute to risk assessment associated with insect presence in vineyards. Prior to conducting molecular assays for detection of X. fastidiosa in individual insects, H. vitripennis must be removed from yellow sticky traps with a solvent such as orange oil. In this study, we determined the effect of orange oil concentration on extraction of individual H. vitripennis following trap removal on detection of X. fastidiosa by qRT-PCR. In a ten-fold dilution series of orange oil, increasing amounts of orange oil caused decreasing levels of X. fastidiosa detection in standardized positive samples. Additionally, tests on the effects of Stickem® brand trap adhesive on qRT-PCR and development of methods which lowered the concentration of orange oil often present in field samples determined the point where detection of X. fastidiosa was negatively impacted. These results benefit the monitoring and screening for Xylella fastidiosa from leafhoppers collected on sticky cards used in regulatory area-wide management.
The potato psyllid (Bactericera cockerelli Sulc) is an economically important insect pest of solanaceous crops such as potato, tomato, pepper, and tobacco. Historically, the potato psyllid's range included central United States, Mexico, and California; more recently, populations of this insect have been reported in Central America, the Pacific Northwest, and New Zealand. Like most phytophagous insects, potato psyllids require symbiotic bacteria to compensate for nutritional deficiencies in their diet. Potato psyllids harbor the primary symbiont, Candidatus Carsonella ruddii, and may also harbor many secondary symbionts such as Wolbachia sp., Sodalis sp., Pseudomonas sp., and others. These secondary symbionts can have an effect on reproduction, nutrition, immune response, and resistances to heat or pesticides. To identify regional differences in potato psyllid bacterial symbionts, 454 pyrosequencing was performed using generic 16S rRNA gene primers. Analysis was performed using the Qiime 1.6.0 software suite, ARB Silva, and R. Operational taxonomic units were then grouped at 97% identity. Representative sequences were classified to genus using the ARB SILVA database. Potato psyllids collected in California contained a less diverse microbial community than those collected in the central United States and Central America. The crop variety, collection year, and haplotype did not seem to affect the microbial community in potato psyllids. The primary difference between psyllids in different regions was the presence and overall bacterial community composition of Candidatus Carsonella ruddii and Wolbachia.
Microencapsulation as a delivery mechanism of SINV-1 and other molecules such as dsRNA, offers an approach to Solenopsis invicta Buren management that is target specific and fits current approaches to baiting ants with toxins and/or RNA-interference. The delivery method presented here targets ground dwelling, foraging ants with an ant-infecting virus which is specific to the genus, Solenopsis. Endemic ant-infecting viruses, like S. invicta viruses (SINV-1, SINV-2, and SINV-3) are being evaluated for efficacy in S. invicta population suppression. In this study, SINV-1 (TX5 strain) was extracted from S. invicta colonies and microencapsulated in sodium alginate pellets. Pellets containing extracted whole virions were offered to confirmed non-infected S. invicta colonies. Colonies were sampled every 5 d and tested by reverse transcription polymerase chain reaction (RT-PCR) for presence of viral RNA. The longevity of control and viral pellets were also evaluated. Within 30 d, post-feeding of virus, 35% of S. invicta colonies acquired SINV-1 infection (P = 0.03). Thus, microencapsulation as a delivery mechanism was successful to deliver SINV-1 to S. invicta colonies. Future incorporation of this economically affordable method can be implemented to deliver biological agents for specific ant species and to augment current approaches that bait ants. While a virus was used to demonstrate delivery, an adequate and affordable virus production system still needs to be developed before a viral strategy can be adopted as a tool for biological control of fire ants.
Planthoppers (Hemiptera: Fulgoroidea) have been implicated in transmission of phytoplasmas that cause lethal decline in palms. Surveys of insects feeding on palms located in Florida resulted in the discovery of Omolicna joi sp. nov., described here. Patara albida (Hemiptera: Derbidae), a palm feeder, has become established in South Florida. Haplaxius crudus (Hemiptera: Cixiidae), the proven vector of the phytoplasma that causes lethal yellows (LY ), was thought to be restricted to the southern tier of Florida counties, thus limiting the distribution of LY . We found that H. crudus can overwinter consistently as far north as Gainesville (N 29.63380° W -82.37200°). Thus, the northern limit of LY might be a function of the physiology of the pathogens rather than the distribution of the vector.
The red imported fire ant, Solenopsis invicta Buren, native to South America, is an economically important urban pest that has few natural enemies in the southern United States. Solenopsis invicta virus-1 (SINV-1) is a picorna-like single-stranded positive-sense virus known to affect only S. invicta. RNA interference (RNAi) is a known regulator of RNA viruses in biological systems and is an emerging biologically based insect control method. The purpose of this study was to increase, then decrease viral titers of SINV-1 in S. invicta colonies during a period of 1 month to demonstrate a possible use of SINV-1 as a targeted, controllable management strategy against S. invicta. Manipulation of SINV-1 viral titers was attempted by first exposing S. invicta colonies to SINV-1 and after 15 days exposing the same colonies to novel double-stranded RNA (dsRNA) coded for SINV-1 viral capsid genes. S. invicta colonies were monitored during the 1-month period, and significant increases and decreases in viral titers of SINV-1 were noted. Significantly more workers in S. invicta colonies exposed to SINV-1 died than did similarly sized noninfected S. invicta colonies during 30 days. Increases and decreases of viral titers of SINV-1 directly correlated with the increases and decreases in mortality.
Abstract The Asian Citrus Psyllid (ACP) ( Diaphorina citri ) is a Hemipteran which feeds on Citrus and is the principal vector of the pathogen Candidatus Liberibacter asiaticus (Las); the primary causal agent of Huanglongbing (HLB) or citrus greening disease. Currently HLB is the single greatest threat to the American citrus industry. Previous work has shown that that Cytochrome P450 (CYP4) genes are associated with insecticidal resistance to neonicotinoid insecticides. Infection by Las has also been shown to affect the expression of CYP4 genes. In this study the genetic relationships of ACP populations was evaluated using melting curve analysis of CYP4 genes. Principal component analysis shows the presence of two potential haplotypes. The groups identified by principal component analysis are significantly different from each other (P < 0.05) for all CYP4 genes tested. All populations are most likely within 2-3 mutations of each other. The existence of these mutations sheds light on the potential evolution of ACP in North America. Indicating either coevolution with Las or difference in imidacloprid basal selective pressure in the United States and Mexico.
