@article{liao_montalban_panwala_totsline_hernandez_guedira_huerta_2024, title={First Report of <i>Xanthomonas perforans</i> Causing Bacterial Spot of Pepper (<i>Capsicum annuum</i>) in North Carolina}, volume={7}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-02-24-0444-PDN}, abstractNote={North Carolina (NC) is the fifth largest producer of bell pepper (Capsicum annuum) in the US with an estimated 2,400 acres in production (NASS-USDA, 2022). A survey of bacterial diseases of peppers was initiated in 2020 after numerous bacterial spot outbreaks were reported in NC. Bacterial spot is caused by a complex of four Xanthomonads: X. euvesicatoria, X. vesicatoria, X. perforans, and X. hortorum pv. gardneri (Larrahondo-Rodríguez et al., 2022). If not preemptively managed, bacterial spot can cause up to 40% yield loss (Kousik and Ritchie, 1998). During the 2020 and 2021 growing seasons, 103 yellow mucoid colonies were isolated from plants representing 51 pepper cultivars symptomatic of bacterial spot, i.e., water-soaked leaf lesions that become necrotic spots on leaves and fruits across 22 commercial fields in NC following published methods (Klein-Gordon et al., 2021). All colonies were characterized to species using the qPCR species-specific primers and probes described by Strayer et al. 2016. Of the 103 colonies, 12 isolates tested positive for X. perforans. To confirm qPCR results, a Multi-Locus Sequence Analysis (MLSA) was run using fusA, gapA, gltA, gyrB, and lacF following previously described methods (Almeida et al., 2010) on three representative isolates: AHX61, collected in September 2020 from a field with 20% disease severity in Wake County on cv. Canary Bell; AHX261, collected in July 2021 from a field with 50% disease severity in Sampson County on Jalapeño; and AHX426, collected in August 2021 from a field with 50% disease severity in Dublin County on Jalapeño. All gene sequences were deposited to NCBI (GenBank Accessions: OQ799538-OQ799552) and compared to those from X. euvesicatoria, X. hortorum pv. gardneri, X. perforans, and X. vesicatoria type strains (Almeida et al., 2010). The MLSA showed AHX61, AHX261, and AHX426 cluster with X. perforans ICMP16690T, sharing 99-100% nucleotide similarity. Koch's postulates were performed with the three strains, Xp1484T [ X. perforans type strain, (Wilson 1987)], and water as a negative control. Three 10-week-old bell pepper plants (cv. Early Cal Wonder) were dip-inoculated in 600 mL of a bacterial suspension at an OD600 of 0.3 (~5x108 CFU/mL) and 0.04% Silwet L-77 per strain or water. All 18 plants were individually incubated in a plastic bag for 48 h post-inoculation at 28°C, 80% relative humidity, and 14 h:10 h light-dark cycle in a growth chamber, after which plastic bags were removed. Water-soaking and necrotic spots characteristic of bacterial spot were first observed at six days post-inoculation (dpi). At 14 dpi, symptomatic leaves were removed from treated plants to attempt pathogen re-isolation. Yellow mucoid colonies similar in morphology to those originally inoculated were recovered from all plants and confirmed to be X. perforans through sequencing; no isolates were recovered from water-treated plants. To our knowledge, this is the first time X. perforans is isolated in commercial bell pepper and specialty pepper fields in the state. This is an indication that the Xanthomonas population on peppers in the state is more diverse than previously reported and that pathogen populations will require monitoring for possible species shifts for this crop in NC.}, journal={PLANT DISEASE}, author={Liao, Ying-Yu and Montalban, Kimberly and Panwala, Roshni and Totsline, Noah and Hernandez, Kimberly and Guedira, Anisa and Huerta, Alejandra I.}, year={2024}, month={Jul} }
 @article{larrahondo-rodriguez_liao_huerta_2022, title={Diagnostic Guide for Bacterial Spot of Tomato and Pepper}, volume={23}, ISSN={["1535-1025"]}, DOI={10.1094/PHP-11-21-0140-DG}, abstractNote={ Bacterial spot of tomato and pepper, caused by four Xanthomonas species, X. euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri (renamed X. hortorum pv. gardneri), is a disease that affects pepper and tomato production worldwide. Symptomatic plants often show dark brown or black lesions on all aboveground tissue including fruit, stems, and foliage. Defoliation, fruit spots, and fruit drop are the most important symptoms that contribute to yield loss. To manage the disease, a combination of cultural management tactics is recommended. Unfortunately, effective commercially available copper-based agrochemicals are limited due to the pathogen’s ability to develop tolerance in the field. Multiple breeding efforts have focused on generating genetically resistant cultivars; however, host resistance has been observed to be lost over time due to the pathogen’s adaptation to a deployed genotype and the evolution of new pathogenic races. Isolation of the pathogen from infected tissue can be performed by surface sterilization followed by tissue maceration and streaking the supernatant on nutrient agar or yeast–dextrose–calcium carbonate agar. Identification tools for these pathogens include semiselective media (i.e., Chang Kama Tween Medium and Tween agar), serological methods, biochemical tests, and molecular techniques. For the latter method, one can use species-specific primers to run conventional PCR, qPCR, and multiplex PCR. Lastly, differential genotypes can be used for identifying races. }, number={3}, journal={PLANT HEALTH PROGRESS}, author={Larrahondo-Rodriguez, Erika and Liao, Ying-Yu and Huerta, Alejandra I}, year={2022}, month={Sep}, pages={355–361} }
 @article{zhao_cheng_wang_gao_huang_kong_antwi-boasiako_zheng_yan_chang_et al._2022, title={Identification of Novel Genomic Regions for Bacterial Leaf Pustule (BLP) Resistance in Soybean (Glycine max L.) via Integrating Linkage Mapping and Association Analysis}, volume={23}, ISSN={["1422-0067"]}, url={https://www.mdpi.com/1422-0067/23/4/2113}, DOI={10.3390/ijms23042113}, abstractNote={Bacterial leaf pustule (BLP), caused by Xanthornonas axonopodis pv. glycines (Xag), is a worldwide disease of soybean, particularly in warm and humid regions. To date, little is known about the underlying molecular mechanisms of BLP resistance. The only single recessive resistance gene rxp has not been functionally identified yet, even though the genotypes carrying the gene have been widely used for BLP resistance breeding. Using a linkage mapping in a recombinant inbred line (RIL) population against the Xag strain Chinese C5, we identified that quantitative trait locus (QTL) qrxp–17–2 accounted for 74.33% of the total phenotypic variations. We also identified two minor QTLs, qrxp–05–1 and qrxp–17–1, that accounted for 7.26% and 22.26% of the total phenotypic variations, respectively, for the first time. Using a genome-wide association study (GWAS) in 476 cultivars of a soybean breeding germplasm population, we identified a total of 38 quantitative trait nucleotides (QTNs) on chromosomes (Chr) 5, 7, 8, 9,15, 17, 19, and 20 under artificial infection with C5, and 34 QTNs on Chr 4, 5, 6, 9, 13, 16, 17, 18, and 20 under natural morbidity condition. Taken together, three QTLs and 11 stable QTNs were detected in both linkage mapping and GWAS analysis, and located in three genomic regions with the major genomic region containing qrxp_17_2. Real-time RT-PCR analysis of the relative expression levels of five potential candidate genes in the resistant soybean cultivar W82 following Xag treatment showed that of Glyma.17G086300, which is located in qrxp–17–2, significantly increased in W82 at 24 and 72 h post-inoculation (hpi) when compared to that in the susceptible cultivar Jack. These results indicate that Glyma.17G086300 is a potential candidate gene for rxp and the QTLs and QTNs identified in this study will be useful for marker development for the breeding of Xag-resistant soybean cultivars.}, number={4}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Zhao, Fangzhou and Cheng, Wei and Wang, Yanan and Gao, Xuewen and Huang, Debao and Kong, Jiejie and Antwi-Boasiako, Augustine and Zheng, Lingyi and Yan, Wenliang and Chang, Fangguo and et al.}, year={2022}, month={Feb} }
 @article{zhao_maren_kosentka_liao_lu_duduit_huang_ashrafi_zhao_huerta_et al._2021, title={An optimized protocol for stepwise optimization of real-time RT-PCR analysis}, volume={8}, ISSN={["2052-7276"]}, url={https://doi.org/10.1038/s41438-021-00616-w}, DOI={10.1038/s41438-021-00616-w}, abstractNote={AbstractComputational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene’s primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2−ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2 ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2−ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.}, number={1}, journal={HORTICULTURE RESEARCH}, author={Zhao, Fangzhou and Maren, Nathan A. and Kosentka, Pawel Z. and Liao, Ying-Yu and Lu, Hongyan and Duduit, James R. and Huang, Debao and Ashrafi, Hamid and Zhao, Tuanjie and Huerta, Alejandra I and et al.}, year={2021}, month={Dec} }
 @article{liao_huang_carvalho_choudhary_da silva_colee_huerta_vallad_freeman_jones_et al._2021, title={Magnesium Oxide Nanomaterial, an Alternative for Commercial Copper Bactericides: Field-Scale Tomato Bacterial Spot Disease Management and Total and Bioavailable Metal Accumulation in Soil}, volume={55}, ISSN={["1520-5851"]}, DOI={10.1021/acs.est.1c00804}, abstractNote={Copper (Cu) is the most extensively used bactericide worldwide in many agricultural production systems. However, intensive application of Cu bactericide have increased the selection pressure toward Cu-tolerant pathogens, including Xanthomonas perforans, the causal agent of tomato bacterial spot. However, alternatives for Cu bactericides are limited and have many drawbacks including plant damage and inconsistent effectiveness under field conditions. Also, potential ecological risk on nontarget organisms exposed to field runoff containing Cu is high. However, due to lack of alternatives for Cu, it is still widely used in tomato and other crops around the world in both conventional and organic production systems. In this study, a Cu-tolerant X. perforans strain GEV485, which can tolerate eight tested commercial Cu bactericides, was used in all the field trials to evaluate the efficacy of MgO nanomaterial. Four field experiments were conducted to evaluate the impact of intensive application of MgO nanomaterial on tomato bacterial spot disease severity, and one field experiment was conducted to study the impact of soil accumulation of total and bioavailable Cu, Mg, Mn, and Zn. In the first two field experiments, twice-weekly applications of 200 μg/mL MgO significantly reduced disease severity by 29-38% less in comparison to a conventional Cu bactericide Kocide 3000 and 19-30% less in comparison to the water control applied at the same frequency (p = 0.05). The disease severity on MgO twice-weekly was 12-32% less than Kocide 3000 + Mancozeb treatment. Single weekly applications of MgO had 13-19% higher disease severity than twice weekly application of MgO. In the second set of two field trials, twice-weekly applications of MgO at 1000 μg/mL significantly reduced disease severity by 32-40% in comparison to water control applied at the same frequency (p = 0.05). There was no negative yield impact in any of the trials. The third field experiment demonstrated that application of MgO did not result in significant accumulation of total and bioavailable Mg, Mn, Cu, or Zn in the root-associated soil and in soil farther away from the production bed compared to the water control. However, Cu bactericide contributed to significantly higher Mn, Cu, and Zn accumulation in the soil compared to water control (p = 0.05). This study demonstrates that MgO nanomaterial could be an alternative for Cu bactericide and have potential in reducing risks associated with development of tolerant strains and for reducing Cu load in the environment.}, number={20}, journal={ENVIRONMENTAL SCIENCE & TECHNOLOGY}, author={Liao, Ying-Yu and Huang, Yuxiong and Carvalho, Renato and Choudhary, Manoj and Da Silva, Susannah and Colee, James and Huerta, Alejandra and Vallad, Gary E. and Freeman, Joshua H. and Jones, Jeffrey B. and et al.}, year={2021}, month={Oct}, pages={13561–13570} }