Kai-Shu Ling

Metabolic Coevolution in the Bacterial Symbiosis of Whiteflies and Related Plant Sap-Feeding Insects

Genomic decay is a common feature of intracellular bacteria that have entered into symbiosis with plant sap-feeding insects. This study of the whitefly Bemisia tabaci and two bacteria (Portiera aleyrodidarum and Hamiltonella defensa) cohoused in each host cell investigated whether the decay of Portiera metabolism genes is complemented by host and Hamiltonella genes, and compared the metabolic traits of the whitefly symbiosis with other sap-feeding insects (aphids, psyllids, and mealybugs). Parallel genomic and transcriptomic analysis revealed that the host genome contributes multiple metabolic reactions that complement or duplicate Portiera function, and that Hamiltonella may contribute multiple cofactors and one essential amino acid, lysine. Homologs of the Bemisia metabolism genes of insect origin have also been implicated in essential amino acid synthesis in other sap-feeding insect hosts, indicative of parallel coevolution of shared metabolic pathways across multiple symbioses. Further metabolism genes coded in the Bemisia genome are of bacterial origin, but phylogenetically distinct from Portiera, Hamiltonella and horizontally transferred genes identified in other sap-feeding insects. Overall, 75% of the metabolism genes of bacterial origin are functionally unique to one symbiosis, indicating that the evolutionary history of metabolic integration in these symbioses is strongly contingent on the pattern of horizontally acquired genes. Our analysis, further, shows that bacteria with genomic decay enable host acquisition of complex metabolic pathways by multiple independent horizontal gene transfers from exogenous bacteria. Specifically, each horizontally acquired gene can function with other genes in the pathway coded by the symbiont, while facilitating the decay of the symbiont gene coding the same reaction.

Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 10(5) conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/microl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

Genetic Composition of Pepino mosaic virus Population in North American Greenhouse Tomatoes

In just a few short years, pepino mosaic disease has quickly become endemic in greenhouse tomatoes around the world. Although three genotypes of Pepino mosaic virus (PepMV) were identified in the United States, genetic composition of PepMV in greenhouse tomato crops in North America has not been determined. In this study, genetic variability and population structure of PepMV were evaluated through nucleotide sequence comparison and phylogenetic analysis of two genomic regions (helicase domain and TGB2-3) derived from 91 cDNA clones that were derived from 31 field-collected samples. These samples were collected from several major greenhouse tomato facilities in five states in the United States and two provinces in Canada. All four major genotypes of PepMV (EU, US1, US2, and CH2) were found in North America. Three distinct genotypes (EU, US1, and US2) were found in mixed infection in samples collected from Arizona and Colorado, two genotypes (EU and CH2) in Texas, and a single genotype (EU) in Alabama and California and the provinces of British Columbia and Ontario in Canada. The complexity of population genetics of PepMV in the United States poses an additional challenge to the greenhouse tomato industry because a tomato cultivar with durable resistance to multiple genotypes of PepMV may be harder to develop.

Forty-nine new host plant species for Bemisia tabaci (Hemiptera: Aleyrodidae)

The sweetpotato whitefly, Bemisia tabaci (Gennadius), is a worldwide pest of numerous agricultural and ornamental crops. In addition to directly feeding on plants, it also acts as a vector of plant viruses of cultivated and uncultivated host plant species. Moreover, host plants can affect the population dynamics of whiteflies. An open‐choice screening experiment was conducted with B‐biotype B. tabaci on a diverse collection of crops, weeds, and other indigenous plant species. Five of the plant species were further evaluated in choice or no‐choice tests in the laboratory. The results reveal 49 new reproductive host plant species for B. tabaci. This includes 11 new genera of host plants (Arenaria, Avena, Carduus, Dichondra, Glechoma, Gnaphalium, Molugo, Panicum, Parthenocissus, Trianthema, and Triticum) for this whitefly. All species that served as hosts were acceptable for feeding, oviposition, and development to the adult stage by B. tabaci. The new hosts include three cultivated crops [oats (Avena sativa L.), proso millet (Panicum miliaceum L.), and winter wheat (Triticum aestivum L.)], weeds and other wild species, including 32 Ipomoea species, which are relatives of sweetpotato [I. batatas (L.) Lam.)]. Yellow nutsedge, Cyperus esculentus L., did not serve as a host for B. tabaci in either open‐choice or no‐choice tests. The results presented herein have implications for whitefly ecology and the numerous viruses that B. tabaci spreads to and among cultivated plants.

VirusDetect: An automated pipeline for efficient virus discovery using deep sequencing of small RNAs

Accurate detection of viruses in plants and animals is critical for agriculture production and human health. Deep sequencing and assembly of virus-derived small interfering RNAs has proven to be a highly efficient approach for virus discovery. Here we present VirusDetect, a bioinformatics pipeline that can efficiently analyze large-scale small RNA (sRNA) datasets for both known and novel virus identification. VirusDetect performs both reference-guided assemblies through aligning sRNA sequences to a curated virus reference database and de novo assemblies of sRNA sequences with automated parameter optimization and the option of host sRNA subtraction. The assembled contigs are compared to a curated and classified reference virus database for known and novel virus identification, and evaluated for their sRNA size profiles to identify novel viruses. Extensive evaluations using plant and insect sRNA datasets suggest that VirusDetect is highly sensitive and efficient in identifying known and novel viruses. VirusDetect is freely available at http://bioinfo.bti.cornell.edu/tool/VirusDetect/.

Complete Genome Sequence of a New Tobamovirus Naturally Infecting Tomatoes in Mexico

ABSTRACT The complete genomic sequence of a new tobamovirus in tomatoes was determined through deep sequencing and assembly of small RNAs, then validated through Sanger sequencing. Based on the low sequence identity (≤85%) to known viruses and a close phylogenetic relationship to tobamoviruses, it was identified as a new species.

Genetic diversity of sweet potato begomoviruses in the United States and identification of a natural recombinant between sweet potato leaf curl virus and sweet potato leaf curl Georgia virus

In the United States, two sweet potato begomoviruses, sweet potato leaf curl virus (SPLCV) and sweet potato leaf curl Georgia virus (SPLCGV), were previously identified in Louisiana. In recent years, at least seven additional sweet potato begomoviruses have been identified in other parts of the world. In an effort to determine the genetic diversity and distribution of sweet potato begomoviruses in the U.S., we focused our efforts on molecular characterization of field-collected begomovirus isolates in two states: Mississippi and South Carolina. Using rolling-circle amplification, a total of 52 clones of the full genome were obtained. Initial inspection of alignments of the end sequences in these clones revealed a strong genetic diversity. Overall, 10 genotypes could be assigned. A majority of the isolates (50/52) in eight genotypes were shown to be closely related to SPLCV. A representative clone of each genotype was fully sequenced and analyzed. Among them, four genotypes from South Carolina with 91-92% sequence identity to the type member of SPLCV were considered a new strain, whereas four other genotypes from Mississippi with >95% sequence identity to SPLCV were considered variants. In addition, a member of a proposed new begomovirus species was identified after comparative sequence analysis of the isolate [US:SC:646B-9] from South Carolina with less than 89% sequence identity to any known begomovirus. Hence, the provisional name Sweet potato leaf curl South Carolina virus (SPLCSCV) is proposed. Moreover, a natural recombinant consisting of two distinct parental genomic sequences from SPLCV and SPLCGV was identified in the sample [US:MS:1B-3] from Mississippi. Two recombinant breakpoints were identified, one in the origin of replication and the other between C2 and C4. This knowledge about the genetic diversity of begomoviruses infecting sweet potato will likely have a major impact on PCR-based virus detection and on disease management practice through breeding for virus resistance.

Genetic diversity among Lagenaria siceraria accessions containing resistance to root-knot nematodes, whiteflies, ZYMV or powdery mildew.

In recent years, there has been an increased interest in Europe and in the USA in grafting watermelon onto bottle gourd, Lagenaria siceraria (Mol.) Standl. In this study, genetic diversity and relationships were examined [using 236 sequence-related amplified polymorphism markers] among 56 United States plant introductions (PIs) of L. siceraria and PIs of important cucurbit crops [including Cucurbita maxima Duchesne (winter squash), Cucurbita pepo L. (squash and pumpkin), Citrullus spp. (watermelon), Cucumis melo L. (melon) and Cucumis sativus L. (cucumber)]. The analysis showed that L. siceraria is distinct and has similar genetic distances to the cucurbit species examined herein. The L. siceraria PIs were assembled into two major clusters. One cluster includes groups of PIs collected mostly in South Asia (India) and a few PIs collected in the Mediterranean region and in Northeast Africa. The second cluster includes groups of PIs collected mainly in Southern Africa and in North, Central and South America, and PIs collected in China, Indonesia and Cyprus. All L. siceraria PIs in this study were susceptible to the southern root-knot nematode (RKN) [ Meloidogyne incognita (Kofoid and White) Sandground]. However, several PIs, among them a group of closely related PIs collected in Mexico and Florida, were less infected with southern RKNs. All L. siceraria PIs were infested with whiteflies [ Bemisia tabaci (Gennadius)], while several PIs were less infested than others and need further evaluation and selection for developing breeding lines that may be less appealing to this pest. Most of the PIs that showed resistance to zucchini yellow mosaic virus and tolerance to powdery mildew were collected in India and belong to the same phylogenetic groups (PGs). Experiments with L. siceraria PIs representing different PGs showed similar grafting compatibility with watermelon. Findings from this study should be useful for the development of superior L. siceraria rootstock lines with enhanced resistance to diseases and insect pests of cucurbit crops.

Complete Genome Sequence of Southern tomato virus Identified in China Using Next-Generation Sequencing

ABSTRACT The complete genome sequence of Southern tomato virus (STV), a double-stranded RNA virus that affects tomato in China, was determined using small RNA deep sequencing. This Chinese isolate shares 99% sequence identity to other isolates from Mexico, France, Spain, and the United States. This is the first report of STV infecting tomatoes in Asia.

Estimation of the Whitefly Bemisia tabaci Genome Size Based on k-mer and Flow Cytometric Analyses

Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1) population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680–690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome.