The complete genome sequence of 'Candidatus Liberibacter solanacearum', the bacterium associated with potato zebra chip disease

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with 'Candidatus Liberibacter solanacearum', a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for 'Ca. L. solanacearum'. Here we present the sequence of the 1.26 Mbp metagenome of 'Ca. L. solanacearum', based on DNA isolated from potato psyllids. The coding inventory of the 'Ca. L. solanacearum' genome was analyzed and compared to related Rhizobiaceae to better understand 'Ca. L. solanacearum' physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, 'Ca. L. solanacearum' is related to 'Ca. L. asiaticus', a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to 'Ca. L. asiaticus', 'Ca. L. solanacearum' probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes.     

Biology and epidemics of Candidatus Liberibacter species,psyllid‐transmitted plant‐pathogenic bacteria

Candidatus Liberibacter species are Gram‐negative bacteria that live as phloem‐limited obligate parasites in plants, and are associated with several plant diseases. These bacteria are transmitted by insects called psyllids, or jumping plant lice, which feed on plant phloem sap. Citrus huanglongbing (yellow shoot) or citrus greening disease is associated with three different species of Ca. Liberibacter – Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus – all originally found on different continents. Ca. L. asiaticus is the most severe pathogen, spread by Asian citrus psyllid Diaphorina citri and causing devastating epidemics in several countries. Ca. L. africanus occurs in Africa where it is spread by the African citrus psyllid Trioza erytreae. Ca. Liberibacter solanacearum is associated with diseases in several solanaceous plants, and transmitted by potato psyllid Bactericera cockerelli. Zebra chip disease is causing large damage in potato crops in North America. In Europe Ca. Liberibacter solanacearum is associated with diseases of the Apiaceae family of plants, carrot and celery, and transmitted by psyllids Trioza apicalis and Bactericera trigonica. When Ca. Liberibacter is suspected as the disease agent, the diagnosis is confirmed by DNA‐based detection methods. Ca. Liberibacter‐associated plant diseases can be controlled by using healthy plant propagation material, eradicating symptomatic plants, and by controlling the psyllid populations spreading the disease.     

'Ca. Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.     

'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction

Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored 'Ca. Liberibacter asiaticus,' is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. Only two 'Ca. Liberibacter asiaticus' proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na(+)/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and 'Ca. Liberibacter asiaticus' orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤ E-10) with 'Ca. Liberibacter asiaticus' proteins, often present as multiple orthologs of single 'Ca. Liberibacter asiaticus' proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational analysis and is consistent with the hypothesis that these proteins may be of particular importance in host/microbe interaction and their duplication likely facilitates their ongoing evolution.     

Population dynamics of Cacopsylla melanoneura (Hemiptera: Psyllidae) in northeast Italy and its role in the apple proliferation epidemiology in apple orchards

In the current study, incidence of 'Candidatus Phytoplasma mali' in an experimental apple orchard in northeast Italy, in addition to abundance and phytoplasma infectivity of Cacopsylla melanoneura (F?rster) (Hemiptera: Psyllidae) was determined and the role of this psyllid as a vector of 'Ca. P. mali' in this region was reviewed. Insect samples collected in the orchard by the beating method indicated high abundance of C. melanoneura (up to 7.92 specimens/branch); however, the psyllid C. picta was not observed. Molecular analyses revealed presence of 'Ca. P. mali' in 6.25% of overwintered psyllids. This infection rate is quite high in comparison to other localities where C. melanoneura is known as the main vector of the phytoplasma. This finding supports the assumption that C. melanoneura also is paramount in the epidemiology of the apple proliferation disease also in northeast Italy. Moreover, we correlated immigration dynamics to the temperatures registered in the apple orchard, and defined an immigration index to predict the progressive arrival of the overwintered adults from winter sites. Psyllids start to reach the apple orchards when either the average of the maximum temperature of the 7 d is above 9.5 degrees C or the immigration index has a positive value. This index will be a useful tool for the growers to prevent apple proliferation phytoplasma spread with well-timed insecticide treatments targeted against C. melanoneura. However, further research is needed to validate or adjust the index to other apple growing regions, which may affect more efficacious management of this disease and psyllid vector.     

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.     

Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.     

Field evaluation of a botanical natural product against the pear psylla (Homoptera: Psyllidae)

A 2-yr field study was conducted to evaluate a botanical natural product, AkseBio2, for control of pear psylla, Cacopsylla pyri L. (Homoptera: Psyllidae). Three applications were made each year. Whereas the first application was applied at the dormant period (just before the first eggs were deposited by overwintered females), the second and third applications, respectively, were against the first and second summer generations of pear psylla. The first application deterred winterform females from depositing eggs until the clusterbud stage (buds expanded but no blossoms open) of tree development. In the second and third applications, the product reduced the number of psyllid eggs and young (first and second) instars, causing up to 79.4 and 81.1% mortality, respectively. However, it was less active against the older (third-fifth) instars and achieved only up to 52.7% mortality. Reduction in egg laying was greater than that caused by amitraz, the most commonly used conventional pesticide for psylla control in Turkey. There were no significant horticultural changes on treated plants up to 7 d after treatment in any trial, nor was there any phytotoxicity on plant tissue as a result of AkseBio2 treatments.     

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.     

Sampling of Cacopsylla pyri (Hemiptera: Psyllidae) and Pilophorus gallicus (Hemiptera: Miridae) in pear orchards

The psyllid Cacopsylla pyri L. (Hemiptera: Psyllidae), is one of the principal pests of pear (Pyrus spp.) orchards and, along with its natural enemies, needs to be carefully monitored for correct integrate pest management and biological pest control decision making. We compare sampling techniques and develop sampling methods for C. pyri and Pilophorus gallicus Remane (Heteroptera: Miridae). Four pear orchards were sampled periodically from 2007 to 2010 in southern Spain by beating branches into funnels or 45-cm-diameter nets and taking shoot samples. Different sampling techniques were compared, and several sampling methods were assayed for adults and nymphs of C. pyri and P. gallicus. Psylla adult and P. gallicus counts in the nets and funnels were similar and closely correlated. Counts of C. pyri adults in the funnels and nets were higher than on shoots, but all measures were closely correlated. The number of nymphs on shoots and leaves was correlated, as were the counts on leaves within shoots. A guide for choosing the sampling unit in cost/precision terms is given. C. pyri nymphs and adults showed an aggregated distribution, whereas P. gallicus had a less-crowded spatial distribution. Enumerative sequential sampling was evaluated for both insects by using the different sampling techniques by resampling experimental data. The negative binomial distribution fitted the experimental data gathered using the different sampling techniques for C. pyri adults and P. gallicus. The use of binomial sampling is discussed for the two insects and the costs involved are compared with those implied in the sequential enumerative method.     

Horizontal Transmission of "Candidatus Liberibacter solanacearum" by Bactericera cockerelli (Hemiptera: Triozidae) on Convolvulus and Ipomoea (Solanales: Convolvulaceae)

“Candidatus Liberibacter solanacearum” (Proteobacteria) is an important pathogen of solanaceous crops (Solanales: Solanaceae) in North America and New Zealand, and is the putative causal agent of zebra chip disease of potato. This phloem-limited pathogen is transmitted to potato and other solanaceous plants by the potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae). While some plants in the Convolvulaceae (Solanales) are also known hosts for B. cockerelli, previous efforts to detect Liberibacter in Convolvulaceae have been unsuccessful. Moreover, studies to determine whether Liberibacter can be acquired from these plants by B. cockerelli are lacking. The goal of this study was to determine whether horizontal transmission of Liberibacter occurs among potato psyllids on two species of Convolvulaceae, sweet potato (Ipomoea batatas) and field bindweed (Convolvulus arvensis), which grows abundantly in potato growing regions of the United States. Results indicated that uninfected psyllids acquired Liberibacter from both I. batatas and C. arvensis if infected psyllids were present on plants concurrently with the uninfected psyllids. Uninfected psyllids did not acquire Liberibacter from plants if the infected psyllids were removed from the plants before the uninfected psyllids were allowed access. In contrast with previous reports, PCR did detect the presence of Liberibacter DNA in some plants. However, visible amplicons were faint and did not correspond with acquisition of the pathogen by uninfected psyllids. None of the plants exhibited disease symptoms. Results indicate that horizontal transmission of Liberibacter among potato psyllids can occur on Convolvulaceae, and that the association between Liberibacter and Convolvulaceae merits additional attention.     

Zebra chip disease incidence on potato is influenced by timing of potato psyllid infestation, but not by the host plants on which they were reared

Abstract  The Zebra chip (ZC) syndrome is an emerging disease of potato and a major threat to the potato industry. The potato psyllid, Bactericerca cockerelli (Sulc) is believed to be a vector of the ZC pathogen, which is now thought to be Candidatus Liberibacter, a bacterium. To further understand the relationship between potato psyllid infestation and ZC disease expression, healthy potato plants at different growth stages (4, 6 and 10 weeks after germination) were exposed separately to potato psyllids that were separately reared on four solanaceous hosts plants (potato, tomato, eggplant or bell pepper) for more than 1 year. ZC symptoms, leaf rates and total nonstructural carbohydrate accumulation in leaves and tubers of healthy and psyllid-infested plants were monitored and recorded. Typical ZC symptoms were observed in leaves and tubers of all plants exposed to potato psyllids regardless of the host plant on which they were reared. This was also accompanied by significant reductions in net photosynthetic rate. Caged potato plants without exposure to potato psyllids (uninfested controls) did not show any ZC symptom in both foliage and in harvested tubers. Foliage damage and ZC expression were most severe in the potato plants that were exposed to potato psyllids 4 weeks after germination compared to plants infested at later growth stages. Tubers from potato psyllid-infested plants had significantly higher levels of reducing sugars (glucose) and lower levels of starch than those in healthy plants, indicating that potato psyllid infestation interfered with carbohydrate metabolism in either leaves or tubers, resulting in ZC expression.     

Differential responses to feeding by the tomato/potato psyllid between two tomato cultivars and their implications in establishment of injury levels and potential of damaged plant recovery

An invasive new biotype of the tomato/potato psyllid （Bactericera [Paratrioza] cockerelli [Sulc.]） （Homoptera： Psyllidae） recently has caused losses exceeding 50% on fresh market tomatoes in western North America. Despite these extensive losses, little is known regarding the threshold levels at which populations must be suppressed in order to prevent economic losses. A series of experiments were therefore designed using combinations of two common tomato cultivars （QualiT 21 and Yellow Pear）, five pest-densities （0, 20, 30, 40 and 50 nymphs/plant）, and three feeding-duration （5 days, 10 days, and lifetime） treatments to test the relative importance of pest density, feeding period, and cumulative psyllid-days to establish economic threshold levels for psyllids. The cultivars differed considerably in their response to the toxin injected by the psyllid nymphs. ‘Yellow Pear＇ plants could recover from feeding by up to 40 nymphs for as long as 10 d, whereas ‘QualiT 21＇ plants were irreparably damaged by densities of 20 nymphs feeding for only 5 days. On ‘Yellow Pear＇, all plant measurements such as the number of yellow leaves and plant height were significantly better correlated with cumulative psyUid-days than with either pest density or feeding duration. On ‘QualiT 21 ＇, all plant measurements other than the number of yellow leaflets and leaves were significantly better correlated with pest density than with feeding duration or cumulative psyUid-days, and pest density was a better predictor of psyUid damage. Potential reasons for the variable responses between cultivars and the implications for psyllid sampling and integrated pest management are discussed.     

Preliminary resistance screening on abamectin in pear psylla (Hemiptera: Psyllidae) in northern Italy

In northern Italy (Emilia-Romagna region), integrated pest management has been used for several years against pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), a relevant pest of pear (Pyrus spp.) trees. After the outlawing of amitraz in 2005, the most common active ingredient involved is abamectin, a mixture of avermectin B1a and avermectin B1b. After the development of C. pyri resistance to azinphos methyl in southern France, we evaluated, by topical application, the different sensitivities to abamectin on C. pyri populations collected in orchards from Emilia-Romagna, where different field strategies were used, with alternative success in terms of pest management. The LC50 values ranged between 1.61 and 28.37 mg/liter, and they revealed variations more related to collection time than to field strategies. The failure of abamectin treatments against C. pyri in some Emilia-Romagna locations is probably unrelated to resistance development, but rather it is related to incorrect pest defense management, which could interfere with pest parasitoids and predators.