Candidatus Liberibacter Psyllaurous Research Articles

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

Different host plant species modifies the susceptibility of Bactericera cockerelli to the entomopathogenic fungus Beauveria bassiana

AbstractBactericera cockerelli (Sulc.) is a serious pest of solanaceous crops and a vector of the plant pathogen Candidatus Liberibacter psyllaurous. Entomopathogenic fungi are the most important biological control alternatives for this pest. Host plant species, however, can modify the outcomes of insect–pathogen interactions. We conducted laboratory experiments to quantify the virulence of two isolates of the entomopathogenic fungus Beauveria bassiana (Bals. [Vuill.]), BB40 and BB42, against third instar B. cockerelli nymphs maintained on chilli pepper plants. Owing to the lack of difference in virulence against B. cockerelli nymphs on chilli pepper between the two B. bassiana isolates, only BB42 was used to: compare virulence against nymphs maintained on either chilli pepper, potato or tomato; and in vivo conidia production from nymphs maintained on different host plants. Virulence of the two B. bassiana isolates against B. cockerelli nymphs was similar. Bactericera cockerelli nymphs maintained on tomato were more susceptible to B. bassiana than nymphs maintained on potato or chilli peppers. Infected nymphs maintained on chilli peppers produced the greatest number of conidia followed by infected nymphs maintained on tomato and potato. Host plant affected the susceptibility of B. cockerelli to B. bassiana isolate BB42 and subsequent conidia production. The implications of our results for microbial control of B. cockerelli by B. bassiana are discussed.

Can Beauveria bassiana Bals. (Vuill) (Ascomycetes: Hypocreales) and Tamarixia triozae (Burks) (Hymenoptera: Eulophidae) be used together for improved biological control of Bactericera cockerelli (Hemiptera: Triozidae)?

Bactericera cockerelli (Sulc.) is an important pest of solanaceous crops and a vector of the pathogen Candidatus Liberibacter psyllaurous. Biocontrol of this pest has been attempted with either entomopathogenic fungi or the parasitoid Tamarixia triozae (Burks), but prior to this study, their potential impact in combination had not been studied. The aim of the present study was to evaluate T. triozae parasitism rates on B. cockerelli nymphs that were previously infected for different periods of time by three isolates of Beauveria bassiana (Bals.) Vuill. Two native isolates (BB40 and BB42) and one commercial isolate (GHA) were used. The virulence of these isolates was first estimated against B. cockerelli and T. triozae. LC50 values for the native isolates BB40 and BB42 against B. cockerelli were 9.5×105 and 2.42×106conidiamL−1 respectively; they were significantly more virulent than isolate GHA with an LC50 of 1.97×107conidiamL−1. However, isolate GHA was significantly more virulent against T. triozae with an LC50 of 1.11×107conidiamL−1 compared with LC50s of 1.49×107 and 1.14×108conidiamL−1 for the native isolates BB40 and BB42 respectively. Groups of nymphs were then inoculated with LC20, LC50 or LC90 concentrations of each isolate and presented to T. triozae as hosts either on the day of inoculation or 1, 2, 3, 4, 5, 6days after inoculation. Subsequent levels of parasitism were recorded. Overall, parasitism rates were similar in inoculated and control nymphs. No parasitism occurred in nymphs 6days after fungal inoculation. Parasitoids used to parasitize uninoculated B. cockerelli nymphs survived significantly longer (7.8days) than parasitoids that had been used to parasitize fungus-inoculated nymphs (7.3days). This suggests an inability of the parasitoid to avoid infection when foraging on inoculated nymphs. In conclusion, although the parasitism rate in control and fungus-treated nymphs was similar, suggesting a combination of both biological control agents is possible, we believe there are also negative implications for the parasitoid because its survival was greatly reduced after attacking infected nymphs.

A Tomato Detached Leaf Assay for Chemical Genomics of an HLB Model System

To better understand plant-pathogen interactions in Huanglongbing disease and develop control strategies we investigated a novel approach known as chemical genomics with Tomato “Psyllid Yellows”, caused by  Candidatus Liberibacter psyllaurous (CLps), as a model of HLB. Chemical genomics involves three key stages starting with designing and performing high-throughput chemical screening, identifying chemicals inducing desired effects and dissecting the genetic targets of the candidate chemical. Our study has been focused on developing a high throughput chemical screen assay using model plants such as tomato and Arabidopsis that can be infected by Candidatus Liberibacter psyllaurous (Clps). The key objective is to identify chemicals that induce plant defense against CLps infection or its transmission via psyllids. We evaluated Arabidopsis thaliana and tomato in different media such as MS sterile media and hydroponic culture, however qPCR results indicated very low and inconsistent numbers of CLps positive plants. We designed a modified detached leaf assay for tomato based on the citrus detached leaf assay (Eldesouky Ammar, USDA-ARS) that has resulted in consistently high (80-85%) number of CLps-positive leaf petioles. We are currently evaluating application of the detached leaf assay in screening chemicals using a tomato CaBP22-GUS transgenic line. This line was developed by Dr. Isgouhi Kaloshian and Dr. Thomas Eulgem (University of California, Riverside) based on a transgenic Arabidopsis reporter line with the  promoter CaBP22-333 promoter fused to GUS (Knoth et.al. 2009). The Arabidopsis transgenic line has been used successfully in many high-throughput chemical screens to identify chemicals inducing defense responses. In our study, we will test chemical uptake and its effect on the transgenic tomato line using GUS expression by RT-PCR. To develop methods to test responses to candidate chemicals in citrus, we are testing gene expression of sweet orange seedlings following exposure to four chemicals known to induce defense responses in other plants.

Improved methods for genome sequencing of Liberibacters by BAC library-based metagenomics approach

Liberibacters have not yet been successfully cultured; their minimal genomes carry multiple copies of several genes. Sequences identical to phage genomes have been found in many Liberibacters. Available evidences suggest that the Liberibacter genomes are adapting rapidly in different hosts and environments. Characterization of genomes of rapidly changing unculturable organisms can be challenging. We have used a model system based on Candidatus Liberibacter psyllaurous associated with tomato “psyllid yellows” (Hansen et al., 2008) to develop methodologies using alternate techniques for sequencing metagenomes. We have constructed a BAC library from infected tomato psyllids (Bactericera cockerelli). The library consists of 57,600 clones arrayed in 150 plates each with 384 wells. DNA from individual clones were pooled for screening purposes. Initial identification of clones with Liberibacter sequences were conducted based on 16s ribosomal sequences, and contiguous clones were characterized by end sequencing and identified as containing Liberibacter genome fragments. Screening of additional clones from the library was based on probes developed on such sequences. A total of 245 clones with Liberibacter genome fragments have been identified. A total of 63 bar-coded BAC clones were sequenced by using Roche 454 technology. BAC clones from this library contain large inserts (average size 70 kb). Similarities and differences with other well characterized genomes of Liberibacters (Duan et al., 2009, Lin et al., 2011) will be presented.

A Binomial Sequential Sampling Plan for <I>Bactericera cockerelli</I> (Hemiptera: Triozidae) in <I>Solanum lycopersicum</I> (Solanales: Solanacea)

The tomato-potato psyllid Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a pest of many solanaceous plants, including tomato (Solanum lycopersicum L.) and potato (Solanum tuberosum L.). In tomato, feeding by nymphs is associated with "psyllid yellows." B. cockerelli also vectors "Candidatus Liberibacter psyllaurous," an infectious bacterium that causes "vein greening" disease. Decisions about management action are much more effective when guided by robust sampling. However, there are few previous studies of potato psyllid spatial distribution in tomato fields, and no published sequential sampling plans for the pest in tomato. We studied B. cockerelli in various tomato fields in California and used these data to generate a sequential sampling plan. We found that juvenile B. cockerelli in tomato fields exhibit an edge effect, an aggregated distribution, and individuals are primarily located on the bottom of leaves. Psyllids were concentrated in the upper segments of plants, but this changed over time. Finally, we present three binominal sequential sampling plans for managing tomato psyllids in tomato fields. These plans differed from both those for bell pepper (Capsicum annum L.) and potato, indicating that B. cockerelli needs to be sampled using crop-specific sampling plans.

Considerations for the use of neonicotinoid pesticides in management of Bactericera cockerelli (Šulk) (Hemiptera: Triozidae)

Bactericera cockerelli is a pest on multiple solanaceous crop plants and is the sole vector for the bacteria Candidatus Liberibacter psyllaurous. When the pathogen is present, feeding by these psyllids results in ‘vein greening’ disease in peppers and tomatoes, and “zebra chip” disease in potatoes. Currently, management is based entirely on the application of pesticides, including two neonicotinoid compounds. Populations of B. cockerelli collected in southern Texas in 2006 and 2012 were examined for reduced susceptibility and behavioral responses to imidacloprid.Tests comparing imidacloprid and thiamethoxam demonstrated that both can reduce nymph numbers in the field, but retention and effective periods vary among application methods and compounds. In addition, imidacloprid and thiamethoxam are both sensitive to the amount of water applied during irrigation. Collectedly, these results suggest that imidacloprid is unlikely to be effective in controlling B. cockerelli in south Texas. Moreover, its use needs to be carefully considered in other locations even where resistance has not yet been detected. Finally, thiamethoxam may be useful, but careful attention must be paid to irrigation and rainfall level, application method, and timing of application.

RNA Interference towards the Potato Psyllid, Bactericera cockerelli, Is Induced in Plants Infected with Recombinant Tobacco mosaic virus (TMV)

The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.

A sequential binomial sampling plan for potato psyllid (Hemiptera: Triozidae) on bell pepper (Capsicum annum )

Potato psyllids (Bactericera cockerelli Sulc) are a pest on solanaceous crop plants, including bell peppers. Potato psyllids vector Candidatus Liberibacter psyllaurous, but bell peppers (Capsicum annum L.) do not exhibit symptoms from infection. Potato psyllids show variation in spatial patterns and host choice with cultivar and plant species. Consequently, a study of spatial distribution and sampling plan specific to bell peppers is necessary for management of this insect pest, as those developed for other crops are unlikely to transfer among crops. Potato psyllids were evenly distributed on both sides of leaves but prefer the top two-thirds of pepper plants. Within fields, psyllids demonstrated an aggregated spatial distribution, but the edge effect observed in other crop plants was absent. Eggs and nymphs had similar spatial distributions that differed from adults. A series of nymph-based sampling plans were examined. Sampling plans based on an infestation of less than 41% of plants infested (5 nymphs plant(-1)) were statistically unacceptable, while little difference was found between the 41% infestation plan and 56% (20 nymphs plant(-1)) infestation plan. At 41%, an average of 11 and maximum of 49 samples would be necessary to make a treatment decision. The binomial sequential sampling plan presented here offers an important yet simple tool for managing potato psyllids in bell pepper.

Correction: Manipulation of Plant Defense Responses by the Tomato Psyllid (Bactericerca cockerelli) and Its Associated Endosymbiont Candidatus Liberibacter Psyllaurous

Correction: Manipulation of Plant Defense Responses by the Tomato Psyllid (Bactericerca cockerelli) and Its Associated Endosymbiont Candidatus Liberibacter Psyllaurous

Manipulation of plant defense responses by the tomato psyllid (Bactericerca cockerelli) and its associated endosymbiont Candidatus Liberibacter psyllaurous.

Some plant pathogens form obligate relationships with their insect vector and are vertically transmitted via eggs analogous to insect endosymbionts. Whether insect endosymbionts manipulate plant defenses to benefit their insect host remains unclear. The tomato psyllid, Bactericerca cockerelli (Sulc), vectors the endosymbiont “Candidatus Liberibacter psyllaurous” (Lps) during feeding on tomato (Solanum lycopersicum L.). Lps titer in psyllids varied relative to the psyllid developmental stage with younger psyllids harboring smaller Lps populations compared to older psyllids. In the present study, feeding by different life stages of B. cockerelli infected with Lps, resulted in distinct tomato transcript profiles. Feeding by young psyllid nymphs, with lower Lps levels, induced tomato genes regulated by jasmonic acid (JA) and salicylic acid (SA) (Allene oxide synthase, Proteinase inhibitor 2, Phenylalanine ammonia-lyase 5, Pathogenesis-related protein 1) compared to feeding by older nymphs and adults, where higher Lps titers were found. In addition, inoculation of Lps without insect hosts suppressed accumulation of these defense transcripts. Collectively, these data suggest that the endosymbiont-like pathogen Lps manipulates plant signaling and defensive responses to benefit themselves and the success of their obligate insect vector on their host plant.

Spatial Behavior Comparison of Bactericera cockerelli Sulc. (Hemiptera: Triozidae) in Mexico

During the last five years, Bactericera cockerelli Sulc. has caused significant economic losses in potato production in Mexico, due to the purple top and zebra chip diseases, since it acts as the vector of Candidatus Liberibacter psyllaurous. Despite its importance as a vector of serious potato diseases, the knowledge of its spatial distribution behavior, which could improve the efficiency of control measures, is entirely lacking. The main objective of this work was to compare the spatial distribution of the immature and adult stages of B. cockerelli obtained in a potato field by means of transect and quadrant sampling techniques and of geostatistics tools that allow the visualization of its spatial distribution in the field. Transect and quadrant samplings showed that the immature stages (eggs and nymphs) of B. cockerelli present a clustered distribution. The validation of the achieved semivariograms in the three dates of sampling corroborated the aggregated distribution of immatures and adults of the insect. The maps obtained in the sampling by using the quadrant or the transect approaches reflect the aggregated structure of the insect populations which did not infest 100% of the plot area. This allowed us to identify infested and free areas, what will aid in decisions for selecting alternatives of control.

Feeding disruption of potato psyllid, Bactericera cockerelli, by imidacloprid as measured by electrical penetration graphs

AbstractThe potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes that can cause yield loss by direct feeding and by transmitting a bacterial pathogen, Candidatus Liberibacter psyllaurous (also known as Candidatus L. solanacearum), which is associated with zebra chip disease of this crop. Current pest management practices rely on the use of insecticides for control of potato psyllid to lower disease incidences and increase yields. Imidacloprid is typically applied at potato planting, and it remains unknown if imidacloprid has any effect on potato psyllid feeding behavior. Thus, our specific objectives of this study were to determine and characterize the effects of imidacloprid treatment (0.11 ml l−1) to potato plants on adult potato psyllid feeding behavior 1, 2, and 4 weeks post‐application. Electrical penetration graph (EPG) recordings of potato psyllid feeding revealed six EPG waveforms, which include non‐probing (NP), intercellular stylet penetration (C), initial contact with phloem tissue (D), salivation into phloem sieve elements (E1), phloem sap ingestion (E2), and ingestion of xylem sap (G). The number of NP events and the duration of individual NP events significantly increased on plants treated with imidacloprid compared with untreated controls. Potato psyllids exhibited significant decreases in the number of phloem salivation events on plants treated with imidacloprid. Waveform durations and waveform durations per event for E2 and G were significantly decreased for psyllids on plants treated with imidacloprid compared with untreated controls. These data suggest that the effective use of imidacloprid to reduce transmission of Ca. Liberibacter psyllaurous is related to the negative effects of imidacloprid on psyllid feeding.

Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli

The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.

Behavioral responses of adult potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae), to potato germplasm and transmission of Candidatus Liberibacter psyllaurous

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a major pest of potatoes that can cause yield loss by direct feeding on crop plants and by transmitting a bacterial pathogen, Candidatus Liberibacter psyllaurous (a.k.a. Candidatus Liberibacter solanacearum) associated with zebra chip disease of the crop. In recent years, there have been no studies regarding resistance of potato to the potato psyllid or the bacterial pathogen that the psyllid transmits. Thus, the objectives of this study were to determine the effects of potato germplasm on adult potato psyllid behavior and transmission of Ca. L. psyllaurous. A total of twenty-two potato ( Solanum tuberosum L.) breeding clones and varieties were examined. Plant genotype significantly affected the occurrence and duration of psyllid probing, the duration of psyllid cleaning, resting and the amount of time psyllids spent off the potato leaflet as well as transmission of Ca. L. psyllaurous. For the potato genotypes in which there were significant decreases in transmission compared to controls, there was often an unclear relationship between the occurrences and duration of behaviors and subsequent bacterial transmission. We discuss the implications of our results for an integrated pest management program for the potato psyllid and Ca. L. psyllaurous control on potatoes.

Candidatus Liberibacter psyllaurous . [Distribution map

Abstract A new distribution map is provided for Candidatus Liberibacter psyllaurous Hansen et al. Hosts: potato ( Solanum tuberosum ), tomato ( Solanum lycopersicum ), cayenne pepper ( Capsicum annuum ) and other solanaceous crops. Information is given on the geographical distribution in Europe (Finland), North America (Mexico, USA, Arizona, California, Colorado, Kansas, Nebraska, Nevada, New Mexico, Texas, Wyoming), Central America and Caribbean (Guatemala, Honduras), Oceania (New Zealand). Candidatus Liberibacter psyllaurous is transmitted by the potato phyllid Bactericera cockerelli and the carrot psyllid Trioza apicalis (Hemiptera: Triozidae).

First Report of 'Candidatus Liberibacter psyllaurous' in Potato Tubers with Zebra Chip Disease in Mexico.

Zebra Chip (ZC), an emerging disease of potato (Solanum tuberosum L.) first documented in potato fields around Saltillo in México in 1994, has been identified in the southwestern United States, México, and Central America and is causing losses of millions of dollars to the potato industry (4). Recently, this damaging potato disease was also documented in New Zealand (3). This disease is characterized by a striped pattern of necrosis in tubers produced on infected plants, and fried chips processed from these infected tubers are commercially unacceptable (4). Recent studies conducted in the United States and New Zealand have associated ZC with a new species of 'Candidatus Liberibacter' vectored by the potato psyllid, Bactericera cockerelli Sulc (1,3,4). A bacterium designated 'Candidatus Liberibacter psyllaurous' has recently been identified in potato plants with "psyllid yellows" symptoms that resemble those of ZC (2). To investigate whether liberibacter is associated with ZC in México, 11 potato (cv. Atlantic) tuber samples exhibiting strong ZC symptoms and six asymptomatic tubers were collected from a ZC-affected commercial potato field near Saltillo City, Coahuila, México in September 2008 and tested for this bacterium by PCR. Total DNA was extracted from symptomatic and asymptomatic tubers with cetyltrimethylammoniumbromide (CTAB) buffer (4). DNA samples were tested by PCR using primer pair OA2/OI2c (5'-GCGCTTATTTTTAATAGGAGCGGCA-3' and 5'-GCCTCGCGACTTCGCAACCCAT-3', respectively) specific for 16S rDNA and primer pair CL514F/R (5'-CTCTAAGATTTCGGTTGGTT-3' and 5'-TATATCTATCGTTGCACCAG-3', respectively) designed from ribosomal protein genes (3). Seven of eleven (63.7%) ZC-symptomatic tubers and one of six (16.7%) asymptomatic potatoes yielded the expected 1,168-bp 16S rDNA and 669-bp CL514F/R amplicons, indicating the presence of liberibacter. Amplicons generated from symptomatic tubers were cloned into pCR2.1-Topo plasmid vectors (Invitrogen, Carlsbad, CA) and one clone of each amplicon was sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the ZC OA2/OI2c sequence (GenBank Accession No. FJ498806) showed 100% identity to liberibacter 16S rDNA sequences amplified from potato psyllids from Dalhart, TX and potato tubers from Garden City, KS (GenBank Accession Nos. EU921627 and EU921626, respectively). The ZC CL514F/R sequence (GenBank Accession No. FJ498807) was 98% identical to analogous rplJ and rplL liberibacter ribosomal protein gene sequences amplified from several solanaceous plants in New Zealand (GenBank Accession Nos. EU834131 and EU935005). The OA2/OI2c sequence was also identical to the 16S rDNA sequence (Genbank Accession No. EU812559) of 'Ca. Liberibacter psyllaurous' (2). To our knowledge, this is the first report of 'Ca. Liberibacter psyllaurous' associated with ZC-affected potatoes in México.

A New Huanglongbing Species, “ Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

A new huanglongbing (HLB) "Candidatus Liberibacter" species is genetically characterized, and the bacterium is designated "Candidatus Liberibacter psyllaurous." This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in "psyllid yellowing." Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.