Psyllid Yellows Research Articles

Metabolic changes and potential biomarkers in "Candidatus Liberibacter solanacearum"-infected potato psyllids: implications for psyllid-pathogen interactions.

Psyllid yellows, vein-greening (VG), and zebra chip (ZC) diseases, which are primarily transmitted by potato psyllid (PoP) carrying Candidatus Liberibacter solanacearum (CLso), have caused significant losses in solanaceous crop production worldwide. Pathogens interact with their vectors at the organic and cellular levels, while the potential changes that may occur at the biochemical level are less well reported. In this study, the impact of CLso on the metabolism of PoP and the identification of biomarkers from infected psyllids were examined. Using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis, metabolomic changes in CLso-infected psyllids were compared to uninfected ones. A total of 34 metabolites were identified as potential biomarkers of CLso infection, which were primarily related to amino acid, carbohydrate, and lipid metabolism. The significant increase in glycerophospholipids is thought to be associated with CLso evading the insect vector's immune defense. Matrix-assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) was used to map the spatial distribution of these biomarkers, revealing that 15-keto-Prostaglandin E2 and alpha-D-Glucose were highly expressed in the abdomen of uninfected psyllids but down-regulated in infected psyllids. It is speculated that this down-regulation may be due to CLso evading surveillance by immune suppression in the PoP midgut. Overall, valuable biochemical information was provided, a theoretical basis for a better understanding of psyllid-pathogen interactions was offered, and the findings may aid in breaking the transmission cycle of these diseases.

'Candidatus Liberibacter solanacearum' Infection of Physalis ixocarpa Brot. (Solanales: Solanaceae) in Saltillo, Mexico.

The potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) is a pest of solanaceous crops (order Solanales), including potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.). Feeding by high populations of nymphs causes psyllid yellows while adults and nymphs are vectors of the plant pathogen 'Candidatus Liberibacter solanacearum'. Foliar symptoms that were consistent with either 'Ca. L. solanacearum' infection or psyllid yellows were observed in 2019 on tomatillo (Physalis ixocarpa Brot.; family Solanaceae) grown within an experimental plot located near Saltillo, Mexico. This study had three primary objectives: 9i) determine whether the foliar symptoms observed on tomatillo were associated with 'Ca. L. solanacearum' infection, (ii) identify the haplotypes of 'Ca. L. solanacearum' and potato psyllids present in the symptomatic plot, and (iii) use gut content analysis to infer the plant sources of 'Ca. L. solanacearum'-infected psyllids. Results confirmed that 71% of symptomatic plants and 71% of psyllids collected from the plants were infected with 'Ca. L. solanacearum'. The detection of 'Ca. L. solanacearum' in plants and psyllids and the lack of nymphal populations associated with psyllid yellows strongly suggests that the observed foliar symptoms were caused by 'Ca. L. solanacearum' infection. All infected plants and insects harbored the more virulent 'Ca. L. solanacearum' haplotype B but one psyllid was also coinfected with haplotype A. The potato psyllids were predominantly of the central haplotype but one psyllid was identified as the western haplotype. Molecular gut content analysis of psyllids confirmed the movement of psyllids between noncrop habitats and tomatillo and indicated that 'Ca. L. solanacearum' infection of psyllids was associated with increased plant diversity in their diet.

New Ca. Liberibacter psyllaurous haplotype resurrected from a 49-year-old specimen of Solanum umbelliferum: a native host of the psyllid vector

Over the last century, repeated emergence events within the Candidatus Liberibacter taxon have produced pathogens with devastating effects. Presently, our knowledge of Ca. Liberibacter diversity, host associations, and interactions with vectors is limited due to a focus on studying this taxon within crops. But to understand traits associated with pathogen emergence it is essential to study pathogen diversity in wild vegetation as well. Here, we explore historical native host plant associations and diversity of the cosmopolitan species, Ca. L. psyllaurous, also known as Ca. L. solanacearum, which is associated with psyllid yellows disease and zebra chip disease, especially in potato. We screened tissue from herbarium samples of three native solanaceous plants collected near potato-growing regions throughout Southern California over the last century. This screening revealed a new haplotype of Ca. L. psyllaurous (G), which, based on our sampling, has been present in the U.S. since at least 1970. Phylogenetic analysis of this new haplotype suggests that it may be closely related to a newly emerged North American haplotype (F) associated with zebra chip disease in potatoes. Our results demonstrate the value of herbarium sampling for discovering novel Ca. Liberibacter haplotypes not previously associated with disease in crops.

Mesh crop covers improve potato yield and inhibit tomato potato psyllid and blight: The roles of mesh pore size and ultraviolet radiation

Crop losses caused by the tomato potato psyllid (TPP; Bactericera cockerelli) and blight (Alternaria solani; Phytophthora infestans) continue to be major concerns for potato (Solanum tuberosum) growers worldwide, and commercial crops often require frequent use of agrichemicals to maintain tuber yield and quality. Nevertheless, a paradigm shift is unfolding in crop protection where new tools, such as physical barriers and light‐modifying filters, are being used to develop chemical‐free approaches for pest and disease control. In this study, we examined the use of crop covers as a non‐chemical method for controlling TPP and reducing blight in field potatoes. Our study demonstrated that those plants grown under mesh covers exhibited reduced levels of blight, TPP and associated psyllid yellows. Additionally, potatoes grown under mesh covers exhibited increased yield (91.4 ± 6.9 SD t/ha) compared with both uncovered control plants (75.0 ± 11.8 t/ha) and plants grown using agrichemical pest control (84.5 ± 10.8 t/ha). The plants grown under mesh produced fewer smaller tubers, with increased average and maximum tuber size, so that marketable yield (tubers ≥60 g) was also increased (83.5 ± 7.5 t/ha), compared with that achieved for uncovered control plants (60.0 ± 8.3 t/ha) and plants grown using agrichemicals (69.6 ± 9.2 t/ha). A second field experiment suggested that the incidence of TPP foliage damage and the development of blight were lowest when the passage of ambient ultraviolet (UV) radiation through the crop cover was reduced. This hypothesis was supported by a third trial where potatoes grown outdoors in pots exhibited reduced TPP foliage damage and fewer resident TPP when grown under UV‐blocking plastic screens. The results of these experiments suggest that the use of mesh covers offers new opportunities to sustainably protect potato crops both by acting as a physical barrier and by modifying the wavelength of light incident on the crop.

Infection by Candidatus Liberibacter solanacearum' haplotypes A and B in Solanum lycopersicum 'Moneymaker'.

'Candidatus Liberibacter solanacearum' is a plant pathogen associated with diseases affecting several crops of the Solanaceae and Apiaceae families. Two 'Ca. L. solanacearum' haplotypes (LsoA and LsoB) infect solanaceous crops in North America and are transmitted by the tomato psyllid Bactericera cockerelli. Although both 'Ca. L. solanacearum' haplotypes cause zebra chip in potato, the diseases associated with each haplotype in tomato (Solanum lycopersicum) have not been described. 'Ca. L. solanacearum'-infected tomato plants exhibit symptoms resembling those of permanent yellowing disease (known in Mexico as "permanente del tomate") and sometimes called psyllid yellows. In this study, the symptoms associated with each 'Ca. L. solanacearum' haplotype in tomato were compared, and the bacterial abundance in different nodes of the plants was measured by quantitative polymerase chain reaction. Surprisingly, both plant phenotype and bacterium distribution were different between LsoA- and LsoB-infected plants. Plants infected with LsoB died prematurely, whereas those infected with LsoA did not. Across the measured time points, LsoB abundance in infected plants was consistent with previous reports describing a sink to source gradient, while such gradient was only observed in LsoA-infected plants early after infection. This is the first report describing the differences in symptoms in tomato associated with two 'Ca. L. solanacearum' haplotypes, LsoA and LsoB.

Assessments of Temporal Variations in Haplotypes of 'Candidatus Liberibacter solanacearum' and Its Vector, the Potato Psyllid, in Potato Fields and Native Vegetation.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), had been known for nearly a century to cause psyllid yellows of solanaceous crops. However, it has only been a decade since the insect was discovered to transmit the bacterium 'Candidatus Liberibacter solanacearum' (Lso), which putatively causes potato zebra chip disease. This project was initiated to quantify temporal incidences of haplotypes of the psyllid (Central, Southwestern, and Western) and Lso (A, B) in potato fields and in native vegetation. Psyllids were collected from native vegetation in Texas (2011-2014), and from potato fields in Texas and New Mexico (2014-2017). Psyllids were tested for Lso and haplotypes of both psyllid and Lso. In Texas, the Central psyllid haplotype was overwhelmingly dominant both in potato fields and in native vegetation regardless of location and time of collection. However, in New Mexico potato fields, although the Southwestern haplotype was overall dominant, the ratios of individual haplotypes varied among years and within a season. The Southwestern psyllid haplotype was greater in incidence than the Central early but declined later in the season in each of the 4 yr, while the Central haplotype was low in incidence early but increased over time. Lso was detected in all three psyllid haplotypes representing the first report in Southwestern psyllid haplotype. In Texas, Lso haplotype A was more frequently detected than B, but in New Mexico the incidence of positive psyllids was not high enough to make definitive conclusions regarding predominant Lso haplotype.

Potato Psyllid (Hemiptera: Triozidae) Response to Insecticides Under Controlled Greenhouse Conditions.

Bactericera cockerelli (Šulc) is a common pest of solanaceous crops largely known for vectoring "psyllid yellows" in potatoes. In recent years, however, this pest has attracted considerable attention for vectoring Zebra Chip, a devastating bacterial disease that was first reported in the United States in 2004 and has spread across the southern and northwestern states, causing significant economic losses to potato growers and the industry. Management of the disease is mainly achieved by reducing the psyllid population using insecticides, including pyrethroids and neonicotinoids; however, new insecticides with different modes of action are needed to avoid the acquisition of resistance. In the current study, the immediate and residual effects of conventional and new materials were tested under greenhouse conditions against adults and nymphs. Cyantraniliprole at a high rate and spinetoram showed the highest immediate and residual activity against adults, whereas cyantraniliprole and oxamyl (low and high rates) were effective against nymphs, with no differences between rates. Soil applications of cyantraniliprole, oxamyl, and imidacloprid were still toxic to adults and nymphs 7 and 14 d after treatment. Also, cyantraniliprole and tolfenpyrad were detrimental to psyllid reproduction. The incorporation of new chemistry in rotation programs may provide a useful management tool against this pest.

"Candidatus Liberibacter solanacearum": An Emerging Pathogen Infecting Potato and Tomato

A bacterium called “Candidatus Liberibacter solanacearum” infects potatoes and tomatoes, causing zebra chip in potatoes and psyllid yellows in tomatoes. These disease are highly destructive and have been known to reduce yields by up to 85%. “Ca. L. solanacearum” has been reported in several states, though it has not been detected in Florida, which is the second largest producer of tomatoes and seventh largest producer of potatoes in the US. This 9-page fact sheet covers the biology, distribution, symptoms, transmission, diagnosis, and management of the pathogen and its associated diseases. Written by Binoy Babu, Mathews L. Paret, Nicholas Dufault, and Carrie L. Harmon, and published by the UF Department of Plant Pathology, August 2015.

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 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.

The tomato potato psyllid,Bactericera cockerelli(Šulc, 1909) (Hemiptera: Triozidae): a review of the threat of the psyllid to Australian solanaceous crop industries and surveillance for incursions in potato crops

The tomato potato psyllid, Bactericera cockerelli (Sulc, 1909), is a major biosecurity threat to Australian solanaceous crop industries. Native to North and Central America, B. cockerelli was accidentally introduced into New Zealand in the mid-2000s. The psyllid is a vector of a phloem-limited, alpha-proteobacterium, ‘Candidatus Liberibacter solanacearum’ (= ‘Ca. L. psyllaurous’), which is associated with psyllid yellows disease in tomatoes, potatoes, capsicums, eggplants and tamarillos, and zebra chip disease in potatoes. Both the vector and pathogen have had a devastating impact on the solanaceous crop industries they affect, resulting in millions of dollars of losses annually through increased pest control and surveillance, reduced yields and disruption to commodity export markets. In April 2014, B. cockerelli and ‘Ca. L. solanacearum’ were reported on Norfolk Island, and it is feared that they could enter other Australian States or Territories either through the accidental importation of infested plant material or by the dispersal of psyllids on easterly airflows from New Zealand. In this paper, we give an overview of the threat of the vector and pathogen to Australian commodities. We report on an ongoing surveillance program, initiated in February 2011, designed to detect incursions of B. cockerelli in eastern Australian potato fields using yellow sticky traps. During the last 3 years, over 2300 traps were placed in the major potato growing regions of Tasmania, Victoria, South Australia and Queensland. No B. cockerelli were detected but over 9600 other Psylloidea were trapped, mainly Ctenarytaina spp. and Acizzia spp. (Psyllidae). Only 0.3% of all Psylloidea caught belonged to the Triozidae allowing the rapid differentiation of the majority of trapped psyllids from B. cockerelli based on wing venation. Recommendations are made for further research and to extend the surveillance program to include other Australian solanaceous crop industries.

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.

The Pangaean origin of "candidatus liberibacter" species

SUMMARY Species of “Candidatus Liberibacter”, all vectored by psyllids, are generally recognized as the cause of four serious plant diseases, Huanglongbing, Zebra Chip, Psyllid Yellows and Yellows Decline, which currently threaten and destroy the citrus, potato, tomato and carrot industries, respectively. These are relatively recent diseases in plant crops, thereby inferring a modern evolutionary trajectory, but no overall hypothesis on their origins has been presented. Plate tectonic movements provide an explanation for the disjunction between modern geographic range and phylogeny. Northern species, on Laurasia, represent both modern European and North American ranges. Southern species, on Gondwana, indicate a further speciation event as ancient India, conjoint with Madagascar, separated from Africa, left Madagascar on its own, and produced the single heat tolerant species while drifting across the equator to its modern position. Phylogeny, epidemiology and geography of the modern species point backwards to a single original speciation event from a free-living form, associated opportunistically with angiosperm and psyllid insect ancestors. The original heat-sensitive species lived in the equatorial but cool climate of the Central Pangaean Mountains probably > 300 Ma. The obligately alternating insect/plant host lifestyle developed opportunistically with the evolution and spread of flowering plants and psyllids.

‘Candidatus Liberibacter solanacearum’

<i>‘Candidatus</i> Liberibacter solanacearum’

Temporal and Spatial Analysis of Potato Psyllid Haplotypes in the United States

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is an economically important pest of potato (Solanum tuberosum L.) crops across the western and central United States, as it is known to cause psyllid yellows disease and to transmit the bacterium that causes zebra chip disease. Recent genotyping of B. cockerelli collected during the 2011 potato growing season identified three psyllid haplotypes within the western and central United States according to their geographical regions: northwestern, western, and central. To understand potato psyllid population dynamics before the year 2011, high resolution melting analysis of the B. cockerelli mitochondrial cytochrome oxidase I-like gene was used to identify the haplotypes of over 450 archived psyllids collected in the western and central United States between the years 1998 and 2010. Results show that the northwestern haplotype was present in Washington State as early as 1998 and has persisted in this region since that time. Likewise, psyllids of the western haplotype have also been present in Washington and Oregon before 2011.

Movement of Bactericera cockerelli (Heteroptera: Psyllidae) in Relation to Potato Canopy Structure, and Effects on Potato Tuber Weights

With the threat of new plant diseases on the increase, plant disease epidemiology requires research on pathogen vector movement. Here, releases were performed in planted potato fields of different ages and canopy structures, located in the Texas Panhandle, to evaluate the range of movement of the potato psyllid Bactericera cockerelli (Sulc.). This insect is a known causative agent of psyllid yellows disease, and is a vector of the reported etiological agents of zebra chip disease of potato, 'Candidatus Liberibacter solanacearum/psyllaurous'. Based on collections of B. cockerelli immatures along transects 9 m long radiating in four cardinal directions from release points, adult females dispersed considerable distances, regardless of plant age or canopy structure. Immature abundance declined along transects and were well described by linear and nonlinear models, but abundance patterns did not differ among the different planting dates and canopy structures. However, unequal immature abundance was detected among the four cardinal directions, with more immatures generally collected along transects to the north and west of release points, opposite of prevailing winds in the area at the time of release. Plots where B. cockerelli were released had significantly lower mean potato tuber weights than control plots with no B. cockerelli. However, few plots with B. cockerelli released in them had declining trends in tuber weights with increasing distance from release points.

First Report of "Candidatus Liberibacter psyllaurous" (synonym "Ca. L. solanacearum") Associated with 'Tomato Vein-Greening' and 'Tomato Psyllid Yellows' Diseases in Commercial Greenhouses in Arizona.

During the winter of 2006-2007, plants in commercial tomato greenhouses (GH-1 and GH-2; total 320 acres [129.5 ha]) in Arizona were infested with the potato psyllid Bactericera cockerelli (Sulc) and more than 60% and ~20% of the plants, respectively, exhibited leaf curling, chlorosis, and shortened internodes. In addition, some plants in GH-1 developed an unusual 'vein-greening' phenotype. Nucleic acids were isolated from 10 symptomatic and three asymptomatic plants from each greenhouse. PCR primers designed to amplify a phytoplasma-like 16S rDNA (850 bp) yielded the expected size product from GH-1 samples, whereas samples from GH-2 and the asymptomatic samples from both greenhouses did not. Several 16S rDNA PCR products (3 of 60) when cloned and sequenced, surprisingly shared 97% homology with 'Candidatus Liberibacter asiaticus' (GenBank No. GQ926917). PCR primers PSY680F 5'-GTTCGGAATAACTGGGCGTA-3' and PSY1R 5'-CCCATAAGGGCCATGAGGACT-3', based on the resultant 16S rDNA sequences, were used to amplify a 680-bp fragment from plant DNA extracts and psyllid lysates (1). A robust PCR product (~680 bp) was obtained from 10 of 10 GH-1 plant extracts (GQ926918) and from a GH-1-derived psyllid colony (28 of 35 adults) (GQ926919) and the tomato plants on which they were reared. In contrast, no 680-bp product was obtained from GH-1 asymptomatic plants (0 of 3), GH-2 plants (0 of 10 symptomatic; 0 of 3 asymptomatic), GH-2-derived psyllid colonies (0 of 35 adults), or psyllid colony tomato plants (data not shown). At least three 680-bp amplicons for each sample type were cloned and 8 to 10 inserts were sequenced for each. BLAST analysis revealed that all 680-bp sequences shared 99 to 100% nt identity with the analogous 16SrDNA from "Ca. Liberibacter psyllaurous" (2) and synonym "Ca. L. solanacearum" (3). A second molecular marker was obtained with the 1611F and 480R primers (2) to amplify the 16SrDNA-23S-ITS (980 bp) from >3 plant extracts and psyllid lysates that tested positive for liberibacter. Clustal W alignment of the 16S-23S-ITS sequences from GH-1 original tomato plants and psyllid colony plants (GQ926920) and psyllids (GQ926921) indicated they were 100% identical to each other and BLAST analysis indicated 99 to 100% shared identity with "Ca. L. psyllaurous" (EU812558) (synonym "Ca. L. solanacearum"). Transmission electron microscopy examination of GH-1 and GH-2 psyllids revealed rod and pleomorphic-shaped bacteria (0.5 to 2.0+ μm) at the brain-salivary gland interface in psyllids from the GH-1 liberibacter-positive colony. No such bacteria were observed in GH-2 liberibacter-negative psyllids. These results support an etiological role of a new liberibacter spp. in the development of the 'vein-greening' symptom phenotype. In contrast, the GH-2 'yellows' phenotype is reminiscent of 'psyllid toxicity' in tomato colonized by B. cockerelli (4). To our knowledge, this is the first report of distinct psyllid-associated diseases in greenhouse tomato in Arizona, one associated with a new 'Ca. Liberibacter' spp., manifest as 'vein-greening' disease, and the other associated with psyllid feeding, in which liberibacter is undetectable in plants and psyllids, and is manifest as the 'tomato psyllid yellows' disease.

Phenotypic and Etiological Differences Between Psyllid Yellows and Zebra Chip Diseases of Potato

Both potato psyllid yellows and zebra chip (ZC) potato diseases are associated with the potato psyllid, Bactericera cockerelli (Sulc). Aboveground plant symptoms of both diseases are similar but there is a difference in symptoms in potato tubers. ZC has recently been associated with a new species of the bacterium liberibacter, ‘Candidatus Liberibacter solanacearum’, also known as ‘Ca. Liberibacter psyllaurous’. Mechanisms by which the potato psyllid might cause either ZC or potato psyllid yellows symptoms are not understood. Insect transmission studies were conducted to demonstrate psyllid vectoring of both diseases and to compare symptoms and development of the two diseases. Potato plants were exposed to both liberibacter-free and liberibacter-carrying potato psyllids and later evaluated for plant and tuber symptoms. These plants and tubers were then tested for liberibacter by polymerase chain reaction (PCR). In addition, potato plants exhibiting severe psyllid yellows/ZC-like symptoms were collected from a commercial potato field heavily infested with the potato psyllid and tested for liberibacter. PCR detected ‘Ca. Liberibacter solanacearum’ in ZC symptomatic plants and tubers resulting from exposure to liberibacter-carrying psyllids. Despite development of foliar symptoms that resemble those of ZC in plants exposed to liberibacter-free psyllids, no liberibacter was detected in these plants with psyllid yellows. Moreover, tubers from these plants with psyllid yellows did not exhibit any symptoms of ZC infection and tested negative for the bacterium. No liberibacter was detected in plants or tubers collected from the psyllid-infested potato field, suggesting that the observed symptoms were due to psyllid yellows. Furthermore, potato plants that were infected with liberibacter died sooner than plants that were infected with psyllid yellows. Although an association between liberibacter and ZC has been established, no pathogen is yet associated with potato psyllid yellows and mechanisms by which psyllid yellows symptoms are induced by the potato psyllid remain unclear.

First Report of "Candidatus Liberibacter psyllaurous" Associated with Psyllid Yellows of Tomato in Colorado.

Greenhouse tomato growers from Fort Lupton, CO contacted the USDA-ARS-USHRL in 2002 regarding plant symptoms resembling "psyllid yellows" associated with Bactericera cockerelli (Sulc) infestations that initially begin as retarded growth, erectness of new growth, chlorosis, and purpling of leaves followed by widespread chlorosis and production of many small, poor-quality fruit (1). Symptoms appeared ≈6 weeks after psyllids were observed and were generally restricted to the top half of the plant. Leaf cuttings from beefsteak tomatoes cv. Quest were immediately placed in RNAlater (Applied Biosystems, Austin, TX). Samples from symptomatic and asymptomatic plants were collected in September and December of 2002. At each date, leaves were sampled from multiple plants and placed in separate RNAlater bottles. September samples exhibited initial "psyllid yellows" symptoms and December samples exhibited severe symptoms. Samples remained at 4°C in RNAlater for 6 years until recent findings suggested that a new species of bacteria, named either "Candidatus Liberibacter psyllaurous" (2) or "Ca. L. solanacearum" (3), may be the causal agent of "psyllid yellows". The Qiagen (Valencia, CA) DNeasy Plant Kit and recommended protocols were used for four separate DNA isolations from each of the four tomato samples that had previously remained unopened. Five PCR primer pairs designed to amplify three distinct genetic regions within the "Ca L. psyllaurous" rrn operon (16S rRNA, 16S-23S rRNA intergenic region, and 23S rRNA) were used and one primer pair specific to the tomato DNA (18S rRNA gene) that successfully amplified from all samples was used as a positive control. Bacterial primers included one pair designed specifically for 16S rRNA sequences of 'Ca. L. asiaticus', 'americanus', and 'africanus' species (USHRL-CL1) and four sets, Lp-1 through Lp-4, previously described (2) that amplify nonoverlapping regions of the 16S-23S rRNA operon. The USHRL-CL1 primers (USHRL-CL1f: 5'-CTTACCAGCCCTTGACATGTATAGGA-3', and USHRL-CL1r: 5'-TCCCTATAAAGTACCCAACATCTAGGTAAA-3') amplify a 195-bp fragment from bp 895 to 1,089 of the 'Ca. Liberibacter' sp. 16S rRNA Genbank Accession No. L22532. Only samples from severe symptomatic plants collected in December 2002 yielded amplicons that were purified and sequenced (Genbank: USHRL-CL1, FJ871062; Lp-1, FJ871058; Lp-2, FJ871059; Lp-3, FJ871060; Lp-4, FJ871061). For each bacterial primer pair, the fragment amplified was highly homologous (98 to 100% identity) to "Ca. L. psyllaurous" rRNA gene/intergenic space sequences. The 16S rRNA coding region was identical to two GenBank 'Ca. Liberibacter' sp. entries: EU921627 and EU921626 from B. cockerelli samples collected in Dalhart, TX and zebra chip potato samples from Garden City, KS, respectively; however, the whole 2,500 bp amplified and sequenced from our sample contained 11 to 14 polymorphisms when compared to nine "Ca. L. psyllaurous" sequences. Our results clearly indicate that "Ca. L. psyllaurous" isolates were associated with tomato "psyllid yellows" symptoms in Colorado as early as 2002 and significant sequence variation exists within the 16S/23S rRNA intergenic region and 23S rRNA coding region to allow analysis of genetic diversity among "Ca. L. psyllaurous" isolates.

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.