Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) i... more Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of ‘Candidatus Liberibacter asiaticus’ (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles h...
Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains ... more Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No sign...
Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a sp... more Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.
Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-strande... more Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.
Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-l... more Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-likelihood phylogeny of Spiroplasma based on core orthologous genes. In total, 601 orthologous genes were concatenated, and a maximum-likelihood approach was used to generate the phylogeny with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The resulting phylogeny was visualized using FigTree v.1.4.3 [70]. S. citri strains analyzed in this report are underlined.
Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genom... more Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genomes. Prophage annotation was performed by PHASTER (PHAge Search Tool – Enhanced Release). The program predicts the completeness of the predicted prophages, based on the proportion of phage genes in the identified region. Green bars are scored as intact (score > 90); blue bars are questionable (score 70–90); red bars are incomplete (score
Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of com... more Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of complete Spiroplasma citri genome sequences. The chromosome of each newly assembled strain (X-axis) was compared to the reference sequence, strain R8-A2 (Y-axis). A) C189; B) LB 319; C) BR12; D) CC-2; E) C5; F) BLH-13; G) BLH-MB.
Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma... more Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma sequences used for phylogenetic analyses in Fig. 1.
Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) i... more Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of ‘Candidatus Liberibacter asiaticus’ (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles h...
Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains ... more Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No sign...
Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a sp... more Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.
Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-strande... more Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.
Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-l... more Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-likelihood phylogeny of Spiroplasma based on core orthologous genes. In total, 601 orthologous genes were concatenated, and a maximum-likelihood approach was used to generate the phylogeny with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The resulting phylogeny was visualized using FigTree v.1.4.3 [70]. S. citri strains analyzed in this report are underlined.
Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genom... more Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genomes. Prophage annotation was performed by PHASTER (PHAge Search Tool – Enhanced Release). The program predicts the completeness of the predicted prophages, based on the proportion of phage genes in the identified region. Green bars are scored as intact (score > 90); blue bars are questionable (score 70–90); red bars are incomplete (score
Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of com... more Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of complete Spiroplasma citri genome sequences. The chromosome of each newly assembled strain (X-axis) was compared to the reference sequence, strain R8-A2 (Y-axis). A) C189; B) LB 319; C) BR12; D) CC-2; E) C5; F) BLH-13; G) BLH-MB.
Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma... more Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma sequences used for phylogenetic analyses in Fig. 1.
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