Mitochondrial genetic diversity of Thrips tabaci (Thysanoptera: Thripidae) in onion growing regions of the United States.

Onion thrips (Thrips tabaci Lindeman, Thysanoptera: Thripidae) causes severe damage to many horticultural and agronomic crops worldwide. It also acts as a vector of several plant viruses. T. tabaci is a key pest of Allium cepa in the United States. However, there is limited information available on the genetic variation within and between T. tabaci populations in the United States and its key evolutionary parameters. In the current study, 83 T. tabaci specimens were collected from A. cepa from 15 different locations comprising four states of the United States. A total of 92 mtCOI gene sequences of T. tabaci from A. cepa were analyzed to understand the genetic diversity and structure of T. tabaci collected from onion host. Seven distinct haplotypes of T. tabaci infesting A. cepa were identified from the current collection, while nine T. tabaci sequences retrieved from GenBank comprised 5 haplotypes. Overall, 15 haplotypes of T. tabaci infesting A. cepa were identified in the world that includes the ten haplotypes in the United States. In the phylogenetic analysis, all the populations collected during the study clustered with thelytokous lineage, while T. tabaci sequences retrieved from GenBank corresponded to leek-associated arrhenotokous lineage. The highest genetic variation was found in Elba and Malheur populations with 3 haplotypes identified in each. The results suggest that haplotypes 1 and 7 are more frequently prevailing haplotypes in the north-western United States, with haplotype 1 being the predominant all over the country. The eastern United States appears to have a more diverse group of haplotypes. The populations from Hungary constituted distinct haplotypes and a haplotype from Kingston linked it with the predominant haplotype.

An on-site adaptable test for rapid and sensitive detection of Potato mop-top virus, a soil-borne virus of potato (Solanum tuberosum).

Potato mop-top virus (PMTV) is considered an emerging threat to potato production in the United States. PMTV is transmitted by a soil-borne protist, Spongospora subterranean. Rapid, accurate, and sensitive detection of PMTV in leaves and tubers is an essential component in PMTV management program. A rapid test that can be adapted to in-field, on-site testing with minimal sample manipulation could help in ensuring the sanitary status of the produce in situations such as certification programs and shipping point inspections. Toward that goal, a rapid and highly sensitive recombinase polymerase amplification (RPA)-based test was developed for PMTV detection in potato tubers. The test combines the convenience of RPA assay with a simple sample extraction procedure, making it amenable to rapid on-site diagnosis of PMTV. Furthermore, the assay was duplexed with a plant internal control to monitor sample extraction and RPA reaction performance. The method described could detect as little as 10 fg of PMTV RNA transcript in various potato tissues, the diagnostic limit of detection (LOQ) similar to that of traditional molecular methods.

Phylogenetics of tobacco rattle virus isolates from potato (Solanum tuberosum L.) in the USA: a multi-gene approach to evolutionary lineage.

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed.

The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

BACKGROUND: Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. METHODS: The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. RESULTS: The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato. CONCLUSIONS: The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.

Use of Electrical Penetration Graph Technology to Examine Transmission of 'Candidatus Liberibacter solanacearum' to Potato by Three Haplotypes of Potato Psyllid (Bactericera cockerelli; Hemiptera: Triozidae).

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium 'Candidatus Liberibacter solanacearum' (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato ("inoculation access period", or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions of vector efficiency.

Comparative analysis of virus-specific small RNA profiles of three biologically distinct strains of Potato virus Y in infected potato (Solanum tuberosum) cv. Russet Burbank.

Deep sequencing technology has enabled the analysis of small RNA profiles of virus-infected plants and could provide insights into virus-host interactions. Potato virus Y is an economically important viral pathogen of potato worldwide. In this study, we investigated the nature and relative levels of virus-derived small interfering RNAs (vsiRNAs) in potato cv. Russet Burbank infected with three biologically distinct and economically important strains of PVY, the ordinary strain (PVY-O), tobacco veinal-necrotic strain (PVY-N) and tuber necrotic strain (PVY-NTN). The analysis showed an overall abundance of vsiRNAs of 20-24nt in PVY-infected plants. Considerable differences were present in the distribution of vsiRNAs as well as total small RNAs. The 21nt class was the most prevalent in PVY-infected plants irrespective of the virus strain, whereas in healthy potato plants, the 24nt class was the most dominant. vsiRNAs were derived from every position in the PVY genome, though certain hotspots were identified for each of the PVY strains. Among the three strains used, the population of vsiRNAs of different size classes was relatively different with PVY-NTN accumulating the highest level of vsiRNAs, while PVY-N infected plants had the least population of vsiRNAs. Unique vsiRNAs mapping to PVY genome in PVY-infected plants amounted to 3.13, 1.93 and 1.70% for NTN, N and O, respectively. There was a bias in the generation of vsiRNAs from the plus strand of the genome in comparison to the negative strand. The highest number of total vsiRNAs was from the cytoplasmic inclusion protein gene (CI) in PVY-O and PVY-NTN strains, whereas from PVY-N, the NIb gene produced maximum total vsiRNAs. These findings indicate that the three PVY strains interact differently in the same host genetic background and provided insights into virus-host interactions in an important food crop.

Seasonal dynamics of thrips (Thrips tabaci) (Thysanoptera: Thripidae) transmitters of iris yellow spot virus: a serious viral pathogen of onion bulb and seed crops.

Thrips-transmitted Iris yellow spot virus (IYSV) is an important economic constraint to the production of bulb and seed onion crops in the United States and many other parts of the world. Because the virus is exclusively spread by thrips, the ability to rapidly detect the virus in thrips vectors would facilitate studies on the role of thrips in virus epidemiology, and thus formulation of better vector management strategies. Using a polyclonal antiserum produced against the recombinant, Escherichia coli-expressed nonstructural protein coded by the small (S) RNA of IYSV, an enzyme linked immunosorbent assay was developed for detecting IYSV in individual as well as groups of adult thrips. The approach enabled estimating the proportion of potential thrips transmitters in a large number of field-collected thrips collected from field-grown onion plants. Availability of a practical and inexpensive test to identify viruliferous thrips would be useful in epidemiological studies to better understand the role of thrips vectors in outbreaks of this economically important virus of onion.

"Candidatus Liberibacter solanacearum" titer over time in Bactericera cockerelli (Hemiptera: Triozidae) after acquisition from infected potato and tomato plants.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a serious pest of potato and other solanaceous crops. B. cockerelli has been associated with the bacterium "Candidatus Liberibacter solanacearum" (Lso), the causal agent of zebra chip, a new and economically important disease of potato in the United States, Mexico, Central America, and New Zealand. The biology of liberibacter transmission to potato and other host plants by the potato psyllid is largely unknown. The current study determined Lso acquisition by adult psyllids following different acquisition access periods (AAP) on potato and tomato, quantified Lso titer over time in postacquisition psyllids, determined Lso-acquisition rate in psyllids at each AAP on each source of inoculum, and determined influence of host plant Lso titer on Lso acquisition rates and postacquisition titer in psyllids over time. Results showed that Lso detection rates and titer increased over time in psyllids following AAPs of 8, 24, and 72 h on tomato and potato and Lso titer was highest when psyllids acquired Lso from tomato versus potato. Lso titer ranged from 200- to 400-fold higher in tomato leaves, petioles, and stems than those of potato. The increase of Lso titer in the insects reached a plateau after an average of 15 d following 24 and 72 h AAP on potato or tomato. At this 15-d plateau, Lso titer in postacquisition psyllids was comparable with that of infective psyllids from the Lso-infected laboratory colony. Lso-acquisition rate in psyllids fed on potato and tomato increased up to 5 and 20, 15 and 35, 35 and 75, and 80 and 100%, respectively, when the insects were allowed access to plants for 4, 8, 24, and 72 h, respectively.

Transmission of Iris yellow spot virus by Frankliniella fusca and Thrips tabaci (Thysanoptera: Thripidae).

Thrips-transmitted Iris yellow spot virus (IYSV) (Family Bunyaviridae, Genus Tospovirus) affects onion production in the United States and worldwide. The presence of IYSV in Georgia was confirmed in 2003. Two important thrips species that transmit tospoviruses, the onion thrips (Thrips tabaci (Lindeman)) and the tobacco thrips (Frankliniella fusca (Hinds)) are known to infest onion in Georgia. However, T. tabaci is the only confirmed vector of IYSV. Experiments were conducted to test the vector status of F. fusca in comparison with T. tabaci. F. fusca and T. tabaci larvae and adults reared on IYSV-infected hosts were tested with antiserum specific to the nonstructural protein of IYSV through an antigen coated plate ELISA. The detection rates for F. fusca larvae and adults were 4.5 and 5.1%, respectively, and for T. tabaci larvae and adults they were 20.0 and 24.0%, respectively, indicating that both F. fusca and T. tabaci can transmit IYSV. Further, transmission efficiencies of F. fusca and T. tabaci were evaluated by using an indicator host, lisianthus (Eustoma russellianum (Salisbury)). Both F. fusca and T. tabaci transmitted IYSV at 18.3 and 76.6%, respectively. Results confirmed that F. fusca also can transmit IYSV but at a lower efficiency than T. tabaci. To attest if low vector competency of our laboratory-reared F. fusca population affected its IYSV transmission capability, a Tomato spotted wilt virus (Family Bunyaviridae, Genus Tospovirus) transmission experiment was conducted. F. fusca transmitted Tomato spotted wilt virus at a competent rate (90%) suggesting that the transmission efficiency of a competent thrips vector can widely vary between two closely related viruses.