Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Vedashree, S.; Sateesh, M.K.; Chowdappa, P.; Nirmalkumar, B. J.

doi:10.1111/jph.12380

Cited by 5 publications

(8 citation statements)

References 37 publications

(71 reference statements)

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“…For analysis of fungal taxa at or below the species level, the more variable ITS region is commonly used (Pryor and Gilbertson ). Sequences of the ribosomal DNA genes, in particular the spacer regions ITS1 and ITS2 surrounding the 5·8S gene have been used to develop specific primers for detection of phytopathogenic fungi (Lee et al ; Iacomi‐Vasilescu et al ; Guillemette et al ; Tapia‐Tussell et al ; Torres‐Calzada et al ; Shankar et al ; Vedashree et al ). This is because ITS regions are of suitable size for PCR amplifications, restriction analysis and sequencing procedures and because ITS regions are variable among species as well.…”

Section: Resultsmentioning

confidence: 99%

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Specific PCR‐based detection of Phomopsis vexans the cause of leaf blight and fruit rot pathogen of Solanum melongena L.

Udayashankar

Nayaka

Archana

et al. 2019

Lett Appl Microbiol

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Significance and Impact of the Study: Phomopsis vexans is an important seed-borne pathogenic fungus responsible for leaf blight and fruit rot in brinjal. Current detection methods, based on culture and morphological identification is time consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on sequence data of a region consisting of the 5Á8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans. AbstractLeaf blight and fruit rot disease caused by Phomopsis vexans is a devastating disease of brinjal. The detection of P. vexans in plant parts and seeds of brinjal can be complicated, mainly when the inoculum is present at low levels and/or overgrown by fast-growing saprophytic fungi or other seed-borne fungi. A PCR-based diagnostic method was developed with specific primers designed based on sequence data of a region consisting of the 5Á8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans. The efficiency and specificity of primer pairs PvexF/PvexR designed were established by PCR analysis of DNA from P. vexans strains isolated from India and fungal isolates of other genera. A single amplification product of 323-bp was detected from DNA of P. vexans isolates. No cross-reaction was observed with any of the other isolates tested. The specific primers designed and employed in PCR detected P. vexans up to 10 pg from DNA isolated from pure culture. This is the first report on the development of species-specific PCR assay for identification and detection of P. vexans. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for the detection of pathogen P. vexans at early stages.

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Section: Resultsmentioning

confidence: 99%

“…radicina with a sensitivity to detect 1 infected seed in 100 carrot seeds (Konstantinova et al ) with seeds incubated for just 1 day. A species‐specific PCR‐based assay was developed and evaluated for its specificity for the identification and detection of P. azadirachtae (Vedashree et al ).…”

Section: Resultsmentioning

confidence: 99%

Specific PCR‐based detection of Phomopsis vexans the cause of leaf blight and fruit rot pathogen of Solanum melongena L.

Udayashankar

Nayaka

Archana

et al. 2019

Lett Appl Microbiol

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“…Latent infected D. citri may be the initial source of inoculum of melanose, and a rapid and sensitive diagnosis for detection of this pathogen is currently limited. In previously study, a conserved ITS region was used to design a molecular detection on D. longicolla, D. azadirachtae, and D. sclerotioides [42][43][44][45]. Several studies reported that molecular detection of Diaporthe species from conserved ITS region was weak and poor, thus could not distinguish the Diaporthe complex species [38].…”

Section: Discussionmentioning

confidence: 99%

“…Based on the similar phylogenetic analysis, all of the 140 isolates were identified ( Figure S3). Results showed that D. citri was the predominant species which accounted for Plants 2020, 9, 329 6 of 22 44.3%, following the species of D. eres, D. unshiuensis, D. sojae, D. discoidispora and D. citriasiana, which accounted for 11.4%, 10%, 9.3%, 6.4%, and 3.6%, respectively. There were still 15% isolates that could not be identified to the species level (Supplementary Figure S3).…”

Section: Phylogenetic Analysis Of Diaporthe Speciesmentioning

confidence: 97%

“…The combination of translation elongation factor 1-α gene (TEF), beta-tubulin gene (TUB), calmodulin gene (CAL), and histone-3 gene (HIS) showed good resolution for Diaporthe species discrimination [7,38,41]. Generally, molecular marker was used to detect Diaporthe species, and many species-specific primers were designed based on conserved ITS region such as in Diaporthe phaseolorum and Diaporthe longicolla from soybean [42], Diaporthe azadirachtae from neem [43,44], Diaporthe sclerotioides from plants and soils [45]. Also, a molecular tool based on TEF gene was developed to detect Diaporthe azadirachtae from neem [46].…”

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

Chaisiri

Liu

Lin

et al. 2020

Plants

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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-α 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.

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Diaporthe nebulae sp. nov. and First Report of D. cynaroidis, D. novem, and D. serafiniae on Grapevines in South Africa

et al. 2019

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Diaporthe species cause Phomopsis cane and leaf spot as well as Phomopsis dieback on grapevines. Symptoms of Phomopsis dieback have increasingly been observed over the past few years. In order to assess the current status of Diaporthe on grapevines in the Western Cape Province of South Africa, isolations were made from dormant grafted nursery vines, dormant rootstock canes, and dying or dead spurs of field vines. Cultures identified as Diaporthe based on morphological features were further identified to species level by sequencing the internal transcribed spacers (ITS) 1 and 2 and 5.8S rRNA and, for a representative subsample of isolates, the partial beta-tubulin (tub2) and translation elongation factor 1-alpha (EF1-α) genes. Phylogenetic analysis of the combined ITS, tub2, and EF1-α data revealed nine Diaporthe species associated with grapevines during this survey. One of these represents a new species, D. nebulae sp. nov., and three other species, namely D. novem, D. cynaroidis, and D. serafiniae, are reported on grapevines in South Africa for the first time. Species-specific primers were designed for PCR identification of D. ampelina, D. ambigua, and D. foeniculina. Pathogenicity studies conducted on detached grapevine shoots indicated D. ampelina, D. novem, and D. nebulae sp. nov. as the most virulent species.

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Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Cited by 5 publications

References 37 publications

Specific PCR‐based detection of Phomopsis vexans the cause of leaf blight and fruit rot pathogen of Solanum melongena L.

Specific PCR‐based detection of Phomopsis vexans the cause of leaf blight and fruit rot pathogen of Solanum melongena L.

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

Diaporthe nebulae sp. nov. and First Report of D. cynaroidis, D. novem, and D. serafiniae on Grapevines in South Africa

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