Studies on seed transmission of “Candidatus Liberibacter solanacearum” in pepper and its impact on plant emergence

“ Candidatus Liberibacter solanacearum” (Lso), transmitted by the potato psyllid (Bactericera cockerelli), is the causal agent of potato zebra chip, but can also infect other solanaceous plants, including peppers. Studies were conducted to investigate whether Lso could be transmitted to the next generation of plants through seeds from infected pepper plants. In 2014, jalapeno pepper plants were infested with psyllids carrying a mixture of Lso A and B (AB) at the AgriLife Research Station at Bushland. The study was again conducted in 2019 and pepper plants were infested with psyllids carrying Lso B or Lso AB. In each of the studies, noninfested plants served as controls. At harvest, fruits were collected and tested for the presence of Lso using quantitative PCR. Seeds from infected fruits were then tested for Lso. Overall, the percentage of seeds that tested positive for Lso ranged from 33% to 70%. However, Lso detection in embryos ranged only from 0% to 8%. Seed samples from Lso-positive fruits were planted in the greenhouse to determine the impact of Lso on emergence and the incidence of Lso in emerged plants. Although plant emergence differed between some of the seeds obtained from Lso-positive and -negative fruits, the overall impact of Lso on plant emergence was not consistent. However, of the 182 plants that emerged from seeds collected from infected fruits, none was positive for Lso, suggesting that seeds are unlikely to serve as sources for new Lso infections and their impact on disease epidemiology is negligible.     

SYBR Green real-time quantitative PCR for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in field samples from New Zealand

Diseases of solanaceous crops caused by the phloem-limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), vectored by the tomato potato psyllid Bactericera cockerelli, pose a major economic threat to crop production. Lso is yet to be cultured and, therefore, effective control strategies depend heavily on the early detection of the pathogen via polymerase chain reaction (PCR) assays. In this study, two new assays for the detection of Lso in New Zealand field samples were developed, and compared with previously available assays. Firstly, a single-tube semi-nested gel-based PCR assay was developed for the genus-specific detection of liberibacter species, and shown to provide increased sensitivity over standard and nested PCR. Secondly, a single-tube semi-nested SYBR Green real-time PCR (qPCR) assay was developed for the specific detection of Lso in field samples from New Zealand, with a limit of detection of five copies of the target gene per reaction. Semi-nested qPCR showed similar sensitivity compared with TaqMan qPCR with the primer-probe combination LsoF-HLBpr and was 10- to 50-fold more sensitive than the conventional PCR assays tested. Quantification of titre in Lso-affected tubers by SYBR Green qPCR revealed a positive relationship between pathogen titre and the discolouration of fried tuber slices, a symptom indicative of Lso infection. Quantification of Lso in field samples of potato and tomato also revealed many samples with titres below the limit of detection of conventional PCR. The observation of low-titre samples demonstrated the utility of SYBR Green qPCR for detection of Lso, as in addition to increased sensitivity melt-curve analysis enables confirmation of qPCR data by identifying false positive results.     

Correction to: Haplotypes of ‘Candidatus Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

European Journal of Plant Pathology -     

‘Candidatus Liberibacter solanacearum’ detected in Trioza urticae using suction trap-based monitoring of psyllids in Germany

Journal of Plant Diseases and Protection - Psyllids are small, phloem-feeding insects. Several species are vectors of economically important pathogens, such as ‘Candidatus Liberibacter...     

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print-based qPCR and immunoassay

‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR-based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE-qPCR) was compared to DNA extraction of plant tissue followed by PCR (X-PCR) by testing the same tissue samples. The former estimated a higher CLas population in tissue prints than the latter (t-test; P = 0.009). All extracts prepared for TPE-qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE-qPCR and immunoassay. Although the detection rate by TPE-qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE-qPCR. Thus, a higher detection rate would be achieved by combining TPE-qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.     

A new haplotype of “Candidatus Liberibacter solanacearum” identified in the Mediterranean region

“Candidatus Liberibacter solanacearum”, a phloem-limited and Gram-negative bacterium that is spread from infected to healthy plants by psyllid insect vectors, is an economically important pathogen of solanaceous and carrot crops in the Americas, New Zealand and Europe. Three haplotypes of “Ca. L. solanacearum” have previously been described, two (LsoA and LsoB) in relation to solanaceous crops in the Americas and New Zealand and the third (LsoC) to carrots in Finland. Herein, we describe a fourth haplotype of this ‘Candidatus Liberibacter’ species (LsoD), also associated with carrots, but from Spain and the Canary Islands and vectored by the psyllid Bactericera trigonica. In addition, LsoC was confirmed in carrot and psyllid samples recently collected from Sweden and Norway. Phylogenetic analysis of the 16S rRNA gene suggests that two of the haplotypes, one in the Americas and the other in northern Europe are closer to each other in spite of a large geographic separation and host differences. Furthermore, during this study, potatoes with symptoms of zebra chip disease recently observed in potato crops in Idaho, Oregon and Washington states were analyzed for haplotype and were found to be positive for LsoA. This liberibacter haplotype was found in psyllids associated with the diseased potato crops as well. This finding contrasts with an earlier report of LsoB from psyllids in Washington which came from a laboratory colony originally collected in Texas.     

Evaluation of efficiency of hemi-nested PCR assay for the detection of ‘Candidatus Liberibacter’ infecting citrus

Journal of Plant Diseases and Protection - The present study reports the development and evaluation of a hemi-nested polymerase chain reaction (hnPCR) assay for the efficient detection of...     

Incidence of ‘Candidatus Liberibacter europaeus’ and phytoplasmas in Cacopsylla species (Hemiptera: Psyllidae) and their host/shelter plants

Psyllids, as vectors of phloem-restricted plant pathogens, are serious agricultural pests. Fruit tree phytoplasmas are transmitted by different Cacopsylla spp., while other psyllids are known vectors of liberibacters. Recently, the bacterium ??Candidatus Liberibacter europaeus?? was found in pear trees and in Cacopsylla pyri (Linnaeus), the vector of ??Ca. Phytoplasma pyri??. This new species does not cause symptoms in plants and is probably a symbiont rather than a pathogen. Based on these findings and the assumption that ??Ca. Liberibacter europaeus?? is widespread, we studied its distribution in the genus Cacopsylla and in the respective host and shelter plants (where psyllids aestivate and overwinter), as well as its possible co-presence with ??Ca. Phytoplasma?? spp. We tested 14 Cacopsylla species and 11 plant species from northwestern Italy, Hungary and Israel, characterized by warm oceanic, temperate continental and warm Mediterranean climatic conditions, respectively. ??Ca. Liberibacter europaeus?? was common within the Cacopsylla genus, being present in nine of the 14 species screened as well as in most host plants, whereas none of the shelter plants tested positive for this bacterium. Altogether, these findings indicate the presence of ??Ca. Liberibacter europaeus?? in continental zones, whereas it does not seem to be widespread in the Mediterranean region. Furthermore, lack of specific symptoms in all infected plants confirms an endophytic relationship with this bacterium, while its abundance in insects suggests a beneficial role for the host. Co-infections with phytoplasmas, observed in insects and plants, require further study to evaluate the possible interactions between them.     

Reverse transcription-duplex-polymerase chain reaction for simultaneous detection of Citrus tristeza virus and ‘Candidatus Liberibacter’ from citrus plants

Journal of Plant Diseases and Protection - Tristeza and Huanglongbing are the most important diseases of citrus caused by Citrus tristeza virus (CTV) and ‘Candidatus Liberibacter’...     

Rapid and quantitative detection of citrus huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ by real-time fluorescent loop-mediated isothermal amplification assay in China

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the species ‘Candidatus Liberibacter asiaticus (Las)’ in China. All currently popular cultivars are susceptible to HLB, and there are no effective approaches to control the disease once the plants have been infected. Therefore, rapid and easy diagnostic tools are needed for maintaining clean nursery stock and inoculum removal strategies to control HLB. A real-time fluorescence loop-mediated isothermal amplification (RealAmp) targeting16S rDNA was developed for the rapid and quantitative detection of Las in China. The detection sensitivity of the RealAmp assay was approximately 1 pg/μl template DNA (equal to 2.4 × 10⁵ target DNA copies/μl) and no cross-reaction was observed with other pathogens or virus-free seedlings. Besides the quantitative detection using SYTO-9 fluorescent dye, an improved closed-tube visual inspection technique using SYBR Green I staining was developed for rapid screening of samples and minimize the risk of cross-contamination. The RealAmp assay is an alternative quantitative method, which would be used as a routine detection service for nursery materials in China.     

Molecular analysis of ‘Candidatus Phytoplasma aurantifolia’ associated with phytoplasma diseases in Myanmar

During 2010 and 2011, typical phytoplasma disease symptoms such as little leaves, phyllody and witches’ brooms were observed on black gram, green gram, long bean, shaggy button weed and sesame plants from different regions of Myanmar. The symptomatic samples were analyzed by PCR using universal phytoplasma primers and characterized by sequencing 16S rRNA, ribosomal protein and translocase protein genes. Based on sequence and phylogenetic analysis of the three genes, the phytoplasmas associated with those plants belonged to members of ‘Candidatus Phytoplasma aurantifolia’. To our knowledge, black gram and shaggy button are new hosts for ‘Ca. P. aurantifolia’.     

Ralstonia solanacearum causing potato bacterial wilt: host range and cultivars’ susceptibility in Rwanda

Members of the Ralstonia solanacearum species complex (RSSC), causing potato bacterial wilt or brown rot, are highly contagious and there are no known cultivars with durable resistance to the pathogen. This study hypothesized (a) that crops intercropped or rotated with potato, plants in the same family, and plants grown in the neighbouring fields can host the pathogen and they can be potential sources of primary inoculum, and (b) that potato cultivars currently multiplied by the public tissue culture laboratory in Rwanda are less susceptible to the pathogen. Fourteen plant species and potato, and nine potato cultivars were tested for susceptibility to an RSSC phylotype II strain under greenhouse conditions. The bacteria induced symptoms on potato, tomato, tree tomato, sweet pepper, and eggplant only. Among the plant species with symptoms, potato, tomato, and tree tomato wilted completely. There was a significant difference in days to symptom expression and to complete wilting (p < .0001). While all tested potato cultivars were found to be susceptible, the number of days to first symptom expression, days to complete wilting, area under the disease progress curve (AUDPC), and the number and weight of harvested tubers varied considerably. Cultivars Cruza, Kinigi, and CIP-58 were less susceptible whereas the cultivars Gikungu, Kirundo, and Victoria were highly susceptible. There is a strong need to search for other sources of resistance. The results indicate that some plant species that might serve as a reservoir of the bacterium should be avoided in the vicinity of potato crops.     

Search for reservoirs of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ and mollicutes in weeds associated with carrot and celery crops

Currently, the main arthropod vectored pathogens associated with carrot and celery crop diseases are ˋCandidatus Liberibacter solanacearum´, Spiroplasma citri and different phytoplasma species. Mitigation strategies require elucidating whether these pathogens survive in the weeds of these Apiaceae crops, which can act as reservoirs. Weed surveys were conducted in a vegetative cycle (April to October 2012) in the spontaneous vegetation that surrounded crops affected by ˋCa. L. solanacearum´, S. citri and/or phytoplasmas. Sixty-three species of 53 genera that belong to 23 botanical families were collected in the main carrot and celery Spanish production area. Species were identified, estimating coverage and abundance, and conserved in herbarium. Samples were analysed by nested-PCR with universal primers for phytoplasmas detection, and were sequenced for identification purposes; by conventional PCR for S. citri and real-time PCR for ˋCa. L. solanacearum´. The only detected pathogens were ˋCa. Phytoplasma trifolii´ (clover proliferation group 16Sr VI-A) in Amaranthus blitoides and Setaria adhaerens and ˋCa. P. solani´ (stolbur group 16Sr XII-A) in Convolvulus arvensis. These pathogens were also sporadically detected in celery or carrot crops. Unexpectedly, neither ˋCa. L. solanacearum´ nor S. citri was detected in the weed samples, despite the relatively high prevalence of these pathogens (less than 66 % and 25 %, respectively) in the surveyed plots. This suggests that weeds do not play an epidemiological role as reservoirs in the spread of such organisms in the studied region. The use of pathogen-free seed lots and the control of vectors are crucial for preventing the introduction and spread of these economical important pathogens to new areas.     

Molecular diversity of ‘Candidatus Phytoplasma pyri’ isolates in Slovenia

A European quarantine organism ‘Candidatus Phytoplasma pyri’ causing devastating pear decline disease has been reported to affect pear trees in several European countries. In this study a multilocus sequence analysis was successfully used to gain detailed insight into the molecular diversity of thirty closely related ‘Candidatus Phytoplasma pyri’ isolates from different orchards in Slovenia. Among three genomic regions analyzed, the 16S/23S rRNA intergenic spacer region was the most conserved among Slovenian isolates with 99.7 % sequence identity, yielding only three distinct genotypes. On the other hand, five different genotypes were detected when analyzing secY and aceF genomic regions that shared sequence identity of 94.8 and 97.2 %, respectively. Six of the detected genotypes, specifically four in the secY region and one in each of the two other analyzed genomic regions, were unique for Slovenia. At least eight different haplotypes were found with multilocus sequence analysis, indicating high molecular diversity among Slovenian ‘Ca. P. pyri’ isolates. Haplotypes were clustered into two major clusters, separated by at least 45 mutations. No connection was established between haplotype occurrence and cultivar type.     

‘Candidatus Phytoplasma ziziphi’ associated with Sophora japonica witches’ broom disease in China

Chinese scholar tree (Sophora japonica) with witches’ broom symptoms was observed in Shandong Province in China. Phytoplasmas were detected in the diseased plants using 16S rDNA amplification with phytoplasma-specific universal primer pairs. On the basis of the results of 16S rDNA sequencing, virtual restriction fragment length polymorphism patterns and phylogenetic analyses, the phytoplasma found in S. japonica with witches’ broom symptoms was confirmed as a ‘Candidatus Phytoplasma ziziphi’-related strain belonging to the Elm yellows group 16SrV. This is the first report of ‘Ca. P. ziziphi’ infecting S. japonica plant with witches’ broom symptoms.