Xiu-Qing Li

Association of ‘Candidatus Liberibacter solanacearum’ with Zebra Chip Disease of Potato Established by Graft and Psyllid Transmission, Electron Microscopy, and PCR

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with Candidatus Liberibacter sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of Ca. L. solanacearum previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of Ca. L. solanacearum with potatoes affected by ZC in the United States, Mexico, and Guatemala.

Pectobacterium spp. Associated with Bacterial Stem Rot Syndrome of Potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.

Improved microscopic identification of Clavibacter michiganensis subsp. sepedonicus cells by combining in situ hybridization with immunofluorescence

An oligonucleotide probe was selected from the 16S rRNA gene of Clavibacter michiganensis subsp. sepedonicus for specific in situ hybridization. The rhodamine‐labelled oligonucleotide probe was used in conjunction with an indirect immunofluorescence procedure based on a specific monoclonal antibody detected with a fluorescein‐labelled conjugate. Simultaneous labelling of bacterial cells with the oligonucleotide and antibody probes allows accurate microscopic identification of single cells when isolation and other methods of confirming bacterial identity are not possible.

Clavibacter michiganensis subsp. sepedonicus elicits a hypersensitive response in tobacco and secretes hypersensitive response-inducing protein(s)

ABSTRACT Strains of Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot of potato, showed marked differences in virulence on host plants. When infiltrated into tobacco leaves, virulent strains caused a rapid localized necrotic response (within 24 to 48 h) characteristic of the hypersensitive response (HR), whereas nonpathogenic strains did not. Concentrated cell-free culture supernatants (CCS) from virulent strains caused a necrotic reaction on tobacco, whereas CCS from nonpathogenic strains did not. The necrosis-inducing activity was heat stable and protease sensitive. Inhibitors of eukaryotic metabolism suppressed the necrotic reaction of tobacco to CCS. No necrotic response was observed when host plants were infiltrated with either cells or CCS from virulent strains. HR-inducing protein(s) from a virulent strain separated from the majority of other proteins on DEAE cellulose at 250 to 300 mM NaCl. Ammonium sulfate-precipitated proteins from a virulent strain produced a necrotic reaction at a total protein concentration of 18 mug/ml, whereas those from a nonpathogenic strain did not, even at a concentration of 180 mug/ml. We conclude that virulent strains of C. michiganensis subsp. sepedonicus elicit a typical HR in tobacco and secrete proteinaceous elicitor(s) of the nonhost HR.

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2xa0min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1xa0h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1xa0h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.

Differential gene expression as an indicator of nitrogen sufficiency in field-grown potato plants

Use of an in-season measure of crop N sufficiency to guide fertilizer management is one approach to match the supply of N to the crop N demand. This study examined use of gene expression in leaf tissue of field-grown potatoes for use in assessment of potato N sufficiency. Potato cultivar ‘Shepody’ was grown with six fertilizer N rates (0–250xa0kgxa0N ha–1). Leaf disks were collected weekly for quantification of the expression of N uptake/transport, N assimilation, and amino acid metabolism genes in leaf tissue by nCounter. Many of the genes evaluated were responsive to crop N supply, but the response varied widely among sampling dates. The exception was an ammonium transporter gene (AT1) which was highly expressed, was relatively consistent across sampling dates, was closely related to root zone soil nitrate concentration across N rates and sampling dates, and was highly negatively correlated with total tuber yield. The level of expression of AT1 in leaf tissue was as good as or better than conventional chemical or optical measures of potato N sufficiency in the current study.

Resistance level to an aphid potato pest varies between genotypes from the same Solanum accession.

ABSTRACT Plant resistance to aphids can be improved by introgressing resistant traits from wild Solanum species into the potato germplasm. Breeding parents are commonly selected from the different accessions of each species. Accessions originate from several seeds collected in a restricted area and are conserved as seeds in genebanks. Genetic heterogeneity may be expected between genotypes from the same accession and could influence resistance level. Working with the potato aphid, Macrosiphum euphorbiae (Thomas), and the accession PI243340 of Solanum chomatophilum (Bitter), which has been previously rated as resistant to M. euphorbiae, we genetically identified and assessed the resistance level of genotypes within the accession. A combination of two multilplex polymerase chain reactions (PCRs) discriminated the 13 plant genotypes assessed. Survival of M. euphorbiae, measured using clip cages, varied significantly between five genotypes, randomly selected among the 13 previously assessed, but did not differ between same-genotype plants. Survival among genotypes ranged from 0 to >60% 12 d after adult molt, and the least resistant genotype exhibited survival close to the susceptible standard, Solanum tuberosum L. Our results support the use of PCR multiplex methods to assess genetic heterogeneity in wild Solanum, and suggest that within accession genetic heterogeneity is sufficient to influence resistance level to aphids. Fine screening at the genotype level is preferable when assessing resistance to aphids.

A simplified procedure for verifying and identifying potato cultivars using multiplex PCR

Correct identification of potato cultivars and selections is essential to a large and diverse user group. This group includes curators of germplasm repositories, breeders and other researchers, certification program officials, commercial growers, processing industry managers and for some cultivars, the public. Agencies involved in cultivar registration and plant breeders rights (or patenting) also have a vested interest in correct identification. DNA fingerprinting is an important tool that can be used to describe new or existing cultivars, verify cultivar identity, and resolve cultivar mixtures. Gel-based fingerprints are usually preferred because they are visual and within the technical capacity of most molecular laboratories. In this study, a multiplex PCR protocol Multiplex SUP and an improved version Multiplex SUPN were developed using four primer pairs (STEM0014 and genes of starch synthase, patatin, and UDP-glucose pyrophosphorylase). The agarose-gel-based Multiplex SUP was successfully used i...

First report of Burkholderia andropogonis causing leaf spots of Bougainvillea sp. in Hong Kong and clover in Canada.

Burkholderia andropogonis has a broad host range including 52 species of 15 families of unrelated monocot and dicot plants such as white clover, carnation, bougainvillea, and other ornamental plants (2). In October 2003, a severely diseased Bougainvillea sp. was found in Kowloon, Hong Kong. Diseased leaves had circular lesions with brown centers surrounded by dark, red-brown margins bordered by chlorotic halos. A bacterium consistently isolated from such lesions using peptone yeast extract agar plus glucose plates was compared with several B. andropogonis strains, including the type strain as well as a B. andropogonis-like strain previously isolated from white clover in Vancouver, BC, Canada in June 1995. Pathogenicity of the isolates was determined by infiltrating greenhouse-grown white clover and carnation leaves with bacterial suspensions of ≈106 CFU/ml. Inoculated leaves developed lesions typical of those caused by B. andropogonis. Kochs postulates were fulfilled by isolating bacteria from typical lesions on inoculated plants that were identical to inoculated strains in colony morphology and biochemical characteristics. Using transmission electron microscopy, the Canadian and Hong Kong isolates, as well as authentic strains of B. andropogonis, were shown to have a single, polar sheathed flagellum, a unique feature of this bacterium (4). The two new isolates were compared with authentic strains of B. andropogonis using the Biolog system (Biolog Inc., Hayward, CA), whole cell protein profiles, and polymerase chain reaction (PCR) with species-specific primers. The two new isolates and authentic B. andropogonis cultures, including the type strain, were all identified as B. andropogonis using the Biolog system. The similarity in protein patterns of the new strains to those of authentic B. andropogonis strains supported their preliminary identification on the basis of morphology, pathogenicity, and the Biolog identification system. PCR amplification using primer pair Pf/Pr (Pf: 5-AAGTCGAACGGTAACAGGGA-3, and Pr: 5-AAAGGATATTAGCCCTCGCC-3), which specifically targets B. andropogonis 16S rDNA (1), produced the expected 410-bp amplicon with genomic DNA templates from the two isolates, further confirming their identity. No sequence variation was observed between the amplicon and data (X67037) from GenBank, which confirmed the earlier observation that strains of B. andropogonis were phylogenetically homogenous (1). To our knowledge, this is the first report of B. andropogonis infection on Bougainvillea sp. in Hong Kong. The disease has been previously reported on this host only from Brisbane, Australia. This is also the first report of the isolation of B. andropogonis from clover in Canada, although the disease occurs on clover in other regions such as Australia and Hawaii. B. andropogonis has been previously reported in Canada only on greenhouse carnations (Dianthus sp.) (3). Usually, conditions of high humidity and high temperature are optimal for infection by B. andropogonis. On the basis of historical weather data, Hong Kong has tropical and subtropical coastal weather similar to Brisbane, Australia, while Vancouver, although mild, is cooler but has periods of high humidity. References: (1) R. D Bagsic et al. Lett. Appl. Microbiol. 21:87. 1995. (2) E. J. Cother et al. Plant Pathol. 53:129, 2004. (3) D. W. Creelman. Can. Plant Dis. Surv. 44:146, 1964. (4) X. Li. Ph.D. diss. The University of Queensland, St. Lucia, Australia. 1993.

Development of TBSPG Pipelines for Refining Unique Mapping and Repetitive Sequence Detection Using the Two Halves of Each Illumina Sequence Read

We developed six pipelines (TBSPG) for mapping Illumina sequence reads to reference genomes, refining unique mapping, and computing the mapped read number and coverage. These pipelines provide the options of conducting multi-mapping or unique mapping, inputting with paired-end read files or a single-end read file, removing or not removing nucleus-organelle shared sequences, and mapping with the full-length reads or with the two halves of each read to refine the detection of unique and non-unique sequences. These TBSPG pipelines were based on (and named after) publicly available tools: Trimmomatic, the Burrows–Wheeler Aligner (BWA), SAMtools, Picard, and the Genome Analysis Toolkit (GATK). We developed several Perl scripts to fill the gaps between the tools, connect the tools, recognize half-length reads, select uniquely mapped reads, and compute and output data in a Microsoft Excel-recognizable format for studying the read number and the coverage per chromosome and organellar genome. In a potato 100-bp paired-end sequence file (Illumina TruSeq), approximately 6.75xa0% of uniquely mapped full-length reads were found to actually contain non-unique sequences at the half-length-read level. These freely available TBSPG pipelines can be used for many read-based applications, including repetitive sequence analysis and organellar genome copy number estimation.