Robert G. Shatters

Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) Biotypes in Florida-Investigating the Q Invasion

ABSTRACT After the 2004 discovery of the Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Q biotype in the United States, there was an urgent need to determine its distribution. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in Florida, with the cooperation of growers and state and federal agencies, to monitor the introduction and distribution of both the B and Q biotypes. The biotype status of submitted B. tabaci samples was determined by polymerase chain reaction (PCR) amplification and sequencing of a 700–800-bp mitochondrial cytochrome oxidase I small subunit (mtCOI) gene fragment, PCR amplification, and size determination of two unique microsatellite markers and esterase zymogram analysis. One hundred and eighty collections were sampled from 23 counties. Of these samples, 58% were from vegetables, 37% were from ornamentals, and 5% were from peanuts, alfalfa, and weeds. Eighteen percent of all collections were found to be the Q biotype that came from greenhouse grown ornamental and herbs located in six counties. Sequence comparison of the mtCOI gene identified three separate haplotypes within Florida that were defined as Q1, Q2, and Q3. Haplotypes could be used to associate populations known to be related by grower and plant type. For example, collections from five counties were made on hibiscus linked to the same grower and all samples contained only the Q1 haplotype. Other populations contained a mix of the Q2 and Q3 haplotypes, supporting the conclusion that the Q biotype must have entered Florida through at least two separate introductions. Our data also show that two microsatellite markers are a cost-effective diagnostic alternative for biotype identification with 100% concurrence with mtCOI sequence data.

Improved DNA Barcoding Method for Bemisia tabaci and Related Aleyrodidae: Development of Universal and Bemisia tabaci Biotype-Specific Mitochondrial Cytochrome c Oxidase I Polymerase Chain Reaction Primers

ABSTRACT Whiteflies, heteropterans in the family Aleyrodidae, are globally distributed and severe agricultural pests. The mitochondrial cytochrome c oxidase I (mtCOI) sequence has been used extensively in whitefly phylogenetic comparisons and in biotype identification of the agriculturally important Bemisia tabaci (Gennadius) whitefly. Because of the economic importance of several whitefly genera, and the invasive nature of the B and the Q biotypes of Bemisia tabaci, mtCOI sequence data are continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identification is done through amplification and sequencing of an ≈800-bp mtCOI DNA fragment. Despite its routine use, published primers for amplification of this region are often inefficient for some B. tabaci biotypes and especially across whitefly species. Through new sequence generation and comparison to available whitefly mtCOI sequence data, a set of polymerase chain reaction (PCR) amplification primers (Btab-Uni primers) were identified that are more efficient at amplifying ≈748 bp of the ≈800-bp fragment currently used. These universal primers amplify an mtCOI fragment from numerous B. tabaci biotypes and whitefly genera by using a single amplification profile. Furthermore, mtCOI PCR primers specific for the B, Q, and New World biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478-, 405-, and 303-bp mtCOI fragment for the B, New World, and Q biotypes, respectively. By combining these primers and using rapid PCR and electrophoretic techniques, biotype determination can be made within 3 h for up to 96 samples at a time.

Detection and Relative Titer of Candidatus Liberibacter asiaticus in the Salivary Glands and Alimentary Canal of Diaphorina citri (Hemiptera: Psyllidae) Vector of Citrus Huanglongbing Disease

ABSTRACT Candidatus Liberibacter asiaticus (CLas) bacterium has been strongly implicated as the causative agent of huanglongbing (HLB), or citrus greening, which is currently the most devastating citrus disease worldwide. HLB is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in a persistent manner. We used quantitative-polymerase chain reaction (PCR) to detect CLas in dissected organs of individual D. citri adults infected with HLB in the laboratory or collected from field-infected citrus trees in South Florida. The proportion of infected (CLas-positive) dissected organs was 47–70% for the salivary glands, 72–80% for the alimentary canal, and 79–97.5% for the rest of the insect body. Statistical analysis indicated that, in both field- and laboratory-infected D. citri, the proportion of infected salivary glands was significantly lower than that of other parts in the insect body. With field-collected psyllids, the relative copy number of CLas genomes, compared with psyllid genomic DNA in each sample, was significantly higher in both the salivary gland and alimentary canal compared with that in the rest of the insect body for both males and females. These results provide the first PCR confirmation of CLas in the alimentary canal and salivary glands of D. citri and strongly suggest that the salivary glands constitute an important transmission barrier to CLas in the psyllid vector. Our results also suggest that CLas may replicate or accumulate in both the alimentary canal and salivary glands of D. citri.

Phylogenetic and Structural Relationships of the PR5 Gene Family Reveal an Ancient Multigene Family Conserved in Plants and Select Animal Taxa

Pathogenesis-related group 5 (PR5) plant proteins include thaumatin, osmotin, and related proteins, many of which have antimicrobial activity. The recent discovery of PR5-like (PR5-L) sequences in nematodes and insects raises questions about their evolutionary relationships. Using complete plant genome data and discovery of multiple insect PR5-L sequences, phylogenetic comparisons among plants and animals were performed. All PR5/PR5-L protein sequences were mined from genome data of a member of each of two main angiosperm groups—the eudicots (Arabidoposis thaliana) and the monocots (Oryza sativa)—and from the Caenorhabditis nematode (C. elegans and C. briggsase). Insect PR5-L sequences were mined from EST databases and GenBank submissions from four insect orders: Coleoptera (Diaprepes abbreviatus and Biphyllus lunatus), Orthoptera (Schistocerca gregaria), Hymenoptera (Lysiphlebus testaceipes), and Hemiptera (Toxoptera citricida). Parsimony and Bayesian phylogenetic analyses showed that the PR5 family is paraphyletic in plants, likely arising from 10 genes in a common ancestor to monocots and eudicots. After evolutionary divergence of monocots and eudicots, PR5 genes increased asymmetrically among the 10 clades. Insects and nematodes contain multiple sequences (seven PR5-Ls in nematodes and at least three in some insects) all related to the same plant clade, with nematode and insect sequences separating as two clades. Protein structural homology modeling showed strong similarity among animal and plant PR5/PR5-Ls, with divergence only in surface-exposed loops. Sequence and structural conservation among PR5/PR5-Ls suggests an important and conserved role throughout the evolutionary divergence of the diverse organisms from which they reside.

Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) Biotypes in North America After the Q Invasion

ABSTRACT After the 2004 discovery of the Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Q biotype in the United States, there was a vital need to determine the geographical and host distribution as well as its interaction with the resident B biotype because of its innate ability to rapidly develop high-level insecticide resistance that persists in the absence of exposure. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in North America, with the cooperation of growers, industry, local, state, and federal agencies, to monitor the introduction and distribution of the Q biotype. The biotype status of submitted B. tabaci samples was determined either by polymerase chain reaction amplification and sequencing of a mitochondrial cytochrome oxidase I small subunit gene fragment and characterization of two biotype discriminating nuclear microsatellite markers or esterase zymogram analysis. Two hundred and eighty collections were sampled from the United States, Bermuda, Canada, and Mexico during January 2005 through December 2011. Host plants were split between ornamental plant and culinary herb (67%) and vegetable and field crop (33%) commodities. The New World biotype was detected on field-grown tomatoes (Solanum lycopersicum L) in Mexico (two) and in commercial greenhouses in Texas (three) and represented 100% of these five collections. To our knowledge, the latter identification represents the first report of the New World biotype in the United States since its rapid displacement in the late 1980s after the introduction of biotype B. Seventy-one percent of all collections contained at least one biotype B individual, and 53% of all collections contained only biotype B whiteflies. Biotype Q was detected in 23 states in the United States, Canada (British Columbia and Ontario territories), Bermuda, and Mexico. Forty-five percent of all collections were found to contain biotype Q in samples from ornamentals, herbs and a single collection from tomato transplants located in protected commercial horticultural greenhouses, but there were no Q detections in outdoor agriculture (vegetable or field crops). Ten of the 15 collections (67%) from Canada and a single collection from Bermuda contained biotype Q, representing the first reports of biotype Q for both countries. Three distinct mitochondrial haplotypes of B. tabaci biotype Q whiteflies were detected in North America. Our data are consistent with the inference of independent invasions from at least three different locations. Of the 4,641 individuals analyzed from 517 collections that include data from our previous work, only 16 individuals contained genetic or zymogram evidence of possible hybridization of the Q and B biotypes, and there was no evidence that rare hybrid B-Q marker co-occurrences persisted in any populations.

An expressed sequence tag (EST) set from Citrus sinensis L. Osbeck whole seedlings and the implications of further perennial source investigations

Abstract Compared with the large-scale single pass cDNA sequencing entries from annual plants, the NCBI database has very little sequence information from perennial plant species. Although similar to annuals in many biochemical pathways, perennials are unique in the fact that they posses long generation times. Without short cycle reproduction as an escape mechanism, perennials have evolved alternative survival mechanisms to pathogen attack and environmental stresses. The study of these alternate strategies by way of functional genomics will greatly increase the understanding of the biochemical changes underlining stress responses in perennials. Herein we analyze a set of expressed sequence tags (ESTs) produced from a 180-day-old whole immature sweet orange citrus seedling cDNA library. From this library, 7680 cDNAs were single pass sequenced from the 5′ end, generating 6443 high quality ESTs. In the analysis, 2272 ESTs (35%) were found to significantly match ( E -value≤10 −10 ) proteins with known function in the public databases using blastx . Additionally, 1457 ESTs (23%) significantly matched proteins with unknown function and 1619 ESTs (25%) matched to proteins described as putative. The remaining 1095 ESTs (17%) failed to match with significance to any protein sequence found in the public databases. ESTs matching to the photosynthetic proteins chlorophyll A/B binding protein, plastocyanin and ribulose-1,5-bisphosphate carboxylase were abundant, 6.0% of the total. Interestingly, stress related proteins such as low molecular weight heat shock proteins, peroxidases, lipid transfer proteins and metallothionein-like proteins were also abundant, 3.6% of the total, suggesting a role for these genes in citrus seedling development .

ELECTROPORATION OF EMBRYOGENIC PROTOPLASTS OF SWEET ORANGE (CITRUS SINENSIS (L.) OSBECK) AND REGENERATION OF TRANSFORMED PLANTS

SummaryElectroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm−1) vector DNA concentration (100 μgml−1), carrier DNA concentration (100 μgml−1), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg−1 (protein) min−1. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.

Overview of worldwide diversity of Diaphorina citri Kuwayama mitochondrial cytochrome oxidase 1 haplotypes: two Old World lineages and a New World invasion

Relationships among worldwide collections of Diaphorina citri (Asian citrus psyllid) were analyzed using mitochondrial cytochrome oxidase I (mtCOI) haplotypes from novel primers. Sequences were produced from PCR amplicons of an 821bp portion of the mtCOI gene using D. citri specific primers, derived from an existing EST library. An alignment was constructed using 612bps of this fragment and consisted of 212 individuals from 52 collections representing 15 countries. There were a total of eight polymorphic sites that separated the sequences into eight different haplotypes (Dcit-1 through Dcit-8). Phylogenetic network analysis using the statistical parsimony software, TCS, suggests two major haplotype groups with preliminary geographic bias between southwestern Asia (SWA) and southeastern Asia (SEA). The recent (within the last 15 to 25 years) invasion into the New World originated from only the SWA group in the northern hemisphere (USA and Mexico) and from both the SEA and SWA groups in the southern hemisphere (Brazil). In only one case, Reunion Island, did haplotypes from both the SEA and SWA group appear in the same location. In Brazil, both groups were present, but in separate locations. The Dcit-1 SWA haplotype was the most frequently encountered, including ~50% of the countries sampled and 87% of the total sequences obtained from India, Pakistan and Saudi Arabia. The second most frequently encountered haplotype, Dcit-2, the basis of the SEA group, represented ~50% of the countries and contained most of the sequences from Southeast Asia and China. Interestingly, only the Caribbean collections (Puerto Rico and Guadeloupe) represented a unique haplotype not found in other countries, indicating no relationship between the USA (Florida) and Caribbean introductions. There is no evidence for cryptic speciation for D. citri based on the COI region included in this study.

Characterization of the Asian Citrus Psyllid Transcriptome.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].

Research toward an Artificial Diet for Adult Asian Citrus Psyllid

ABSTRACT Research progress is reported on an artificial diet for adult Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The primary objective was to develop a system for screening antimicrobial peptides and other potential toxic proteins for activity against adults. The base diet was a sterilized solution of sucrose (30%) and yellow-green food coloring (0.5%) in tap water. All of the studies presented were conducted at 25°C, 75% RH, and a photoperiod of 14:10 (L:D) h. Adult psyllids were <7 d old when they were transferred to diet. Addition of the food coloring was necessary to prompt adults to feed. Among the feeding trials discussed, a mean of 69.1 ± 3.2% adults survived for 14 d on the base sucrose diet. Survival rates of males and females were similar. Adults feeding on the sucrose diet may have ingested less food than adults feeding on citrus leaf disks based on differences in quantities of adult excrements deposited in feeding chambers. However, survival of adults feeding on leaf disks over a 2-wk period was only marginally better than survival of adults feeding on the base sucrose diet, and final rates of survival of adults fed these two food sources were not significantly different.