Dataset Information

Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus.

ABSTRACT: Melanose disease caused by Diaporthe citri is considered as one of the most important and destructive diseases of citrus worldwide. In this study, isolates from melanose samples were obtained and analyzed. Firstly, the internal transcribed spacer (ITS) sequences were used to measure Diaporthe-like boundary species. Then, a subset of thirty-eight representatives were selected to perform the phylogenetic analysis with combined sequences of ITS, beta-tubulin gene (TUB), translation elongation factor 1-? gene (TEF), calmodulin gene (CAL), and histone-3 gene (HIS). As a result, these representative isolates were identified belonging to D. citri, D. citriasiana, D. discoidispora, D. eres, D. sojae, and D. unshiuensis. Among these species, the D. citri was the predominant species that could be isolated at highest rate from different melanose diseased tissues. The morphological characteristics of representative isolates of D. citri were investigated on different media. Finally, a molecular tool based on the novel species-specific primer pair TUBDcitri-F1/TUBD-R1, which was designed from TUB gene, was developed to detect D. citri efficiently. A polymerase chain reaction (PCR) amplicon of 217 bp could be specifically amplified with the developed molecular tool. The sensitivity of the novel species-specific detection was upon to 10 pg of D. citri genomic DNA in a reaction. Therefore, the D. citri could be unequivocally identified from closely related Diaporthe species by using this simple PCR approach.

SUBMITTER: Chaisiri C

PROVIDER: S-EPMC7154919 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus.

Chaisiri Chingchai C Liu Xiang-Yu XY Lin Yang Y Li Jiang-Bo JB Xiong Bin B Luo Chao-Xi CX

Plants (Basel, Switzerland) 20200304 3

Melanose disease caused by Diaporthe citri is considered as one of the most important and destructive diseases of citrus worldwide. In this study, isolates from melanose samples were obtained and analyzed. Firstly, the internal transcribed spacer (ITS) sequences were used to measure Diaporthe-like boundary species. Then, a subset of thirty-eight representatives were selected to perform the phylogenetic analysis with combined sequences of ITS, beta-tubulin gene (TUB), transla ...[more]

PMID: 32143512

Similar Datasets

Project description:Brown spot of citrus caused by Alternaria citri is one of the emerging threats to the successful production of citrus crops. The present study, conducted with a substantial sample size of 50 leaf samples for statistical reliability, aimed to determine the change in mineral content in citrus leaves after brown spot disease attack. Leaf samples from a diverse range of susceptible citrus varieties (Valentia late, Washington navel, and Kinnow) and resistant varieties (Citron, Eruka lemon, and Mayer lemon) were analyzed. Significant variations (p ≤ 0.05) in mineral contents were observed across reaction groups (inoculated and un-inoculated), types (resistant and susceptible), and varieties of citrus in response to infection of Alternaria citri. The analysis of variance showed significant changes in mineral levels of citrus leaves, including nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), sodium (Na), iron (Fe), and copper (Cu). The results indicate that the concentration of N and P differed by 6.63% and 1.44%, respectively, in resistant plants, while susceptible plants showed a difference of 6.07% and 1.19%. Moreover, resistant plants showed a higher concentrations of K, Ca, Mg, Zn, Na, Fe, and Cu at 8.40, 2.1, 1.83, 2.21, 1.58, 2.89, and 0.36 ppm respectively, compared to susceptible plants which showed concentrations of 5.99, 1.93, 1.47, 1.09, 1.24, 1.81, and 0.31 ppm respectively. Amounts of mineral contents were reduced in both resistant as well as susceptible plants of citrus after inoculation. Amount of N (8.56), P (1.87) % while K (10.74), Ca (2.71), Mg (2.62), Zn (2.20), Na (2.08), Fe (3.57) and Cu (0.20) ppm were recorded in un-inoculated group of citrus plants that reduced to 3.15 and 0.76% and 3.66, 1.40, 0.63,0.42, 0.74, 1.13 and 0.13 ppm in inoculated group respectively. It was accomplished that susceptible varieties contained lower ionic contents than resistant varieties. The higher concentrations of ionic contents in resistant citrus varieties build up the biochemical and physiological processes of the citrus plant, which help to restrict spread of pathogens. Further research could explore the interplay between mineral nutrition and disease resistance in citrus, potentially leading to the development of new disease-resistant varieties.

Project description:The plant pathogenic bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri), have been devastating the Florida citrus industry. To restore the competitive production presence of Florida in the worldwide citrus market, effective and sustainable control of HLB and the ACP needs to be identified. As alternatives for resistance-inducing insecticides, viruses are currently being considered for biological control of the ACP. To identify possible biological control candidates, we conducted one of the most comprehensive surveys of natural ACP populations in major citrus production regions spanning 21 counties in Florida. By optimizing PCRs and RT-PCRs, we were able to successfully detect and monitor the prevalence of five previously identified ACP-associated RNA and DNA viruses throughout Florida citrus groves, which include: Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri densovirus (DcDNV), Diaphorina citri reovirus (DcRV), and Diaphorina citri picorna-like virus (DcPLV). Adult and nymph ACP populations from 21 of Florida's major citrus-producing counties were collected each month during approximately 18 consecutive months. RNA extracts used for these viral screens were also regionally combined and subjected to High Throughput Sequencing (HTS) to reveal a more comprehensive picture of known and unknown viruses in Florida ACP populations. We discovered that DcACV was the most prevalent ACP-associated virus throughout nymph and adult ACP populations in Florida, detected in more than 60% of all samples tested, followed by DcPLV and DcFLV. HTS allowed us to identify a novel ACP-associated reo-like virus and a picorna-like virus. The putative reo-like virus, tentatively named Diaphorina citri cimodo-like virus, was later surveyed and detected back in seasonal adult and nymph ACP samples collected in Florida during this study. HTS generated data also revealed that the most abundant virus in Florida ACP populations was Citrus tristeza virus (CTV), which is not an ACP-associated virus, suggesting persistent presence of CTV infection in citrus throughout Florida groves. Collectively, information obtained from our study may be able to help guide the direction of biotechnological pest control efforts involving a number of viruses that were detected for the first time in Florida ACP populations, including two newly identified ACP-associated viruses.

Project description:BackgroundAsiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols.ResultsOur assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml- 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml- 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays.ConclusionsWe developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

Dataset Information

Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus.

Publications

Phylogenetic Analysis and Development of Molecular Tool for Detection of <i>Diaporthe citri</i> Causing Melanose Disease of Citrus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets