Detection of Seedborne Pathogens

Walcott, R. R.

doi:10.21273/horttech.13.1.0040

Cited by 58 publications

(44 citation statements)

References 35 publications

(41 reference statements)

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“…Another requirement of DNA extraction procedures is the elimination or reduction of PCR inhibitors, which are commonly found in DNA extracts from seed (49,71,72). In this study, the DNeasy extraction kit from Qiagen resulted in less inhibition of PCR than a conventional CTAB phenol:chloroform extraction method (33).…”

Section: Discussionmentioning

confidence: 85%

A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

Chilvers

Toit²,

Akamatsu

et al. 2007

Plant Disease

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A real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.

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Section: Discussionmentioning

confidence: 85%

A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

Chilvers

Toit²,

Akamatsu

et al. 2007

Plant Disease

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“…The seed maceration protocol was derived from the BIO‐PCR technique, which was first described to detect Pseudomonas syringae in bean seed extracts (Schaad et al ., ). BIO‐PCR has since been developed for detection of different seedborne pathogens (for a review, see Walcott, ), including Alternaria radicina on carrot seeds (Pryor & Gilbertson, ). After seed maceration, D. angelicae was detected in seed lots harvested from umbels with and without symptoms, the latter harvested from diseased or healthy plants.…”

Section: Discussionmentioning

confidence: 99%

Characterization of fungal pathogens (Diaporthe angelicae and D. eres) responsible for umbel browning and stem necrosis on carrot in France

Bastide

Sérandat

Gombert³

et al. 2016

Plant Pathology

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A collection of 102 Diaporthe isolates was compiled from lesions on carrot, parsley and wild Apiaceae species in France from 2010 to 2014. Molecular typing based on ITS rDNA sequences resulted in the identification of 85 D. angelicae and 17 D. eres isolates. Based on sequences of the 3′ part of the IGS rDNA, intraspecific variability was analysed for 17 D. angelicae and 13 D. eres isolates from diverse plant species, locations in France, and plant tissues. The genetic diversity was greater for D. angelicae isolates than D. eres isolates. In vitro sensitivity of five D. angelicae and four D. eres isolates to each of nine fungicides was similar for isolates of both species, with a marked variation in fungicide sensitivity depending on the active ingredient. To assess the pathogenicity of D. angelicae and D. eres isolates on carrot, one isolate of each species was inoculated onto umbels in a controlled environment. Typical lesions were observed for both isolates. Carrot crop debris collected from a seed production field in France and placed in controlled conditions produced perithecia and ascospores typical of Diaporthe, that were further characterized molecularly as belonging to D. angelicae. Detection of Diaporthe species on seed lots from three carrot production fields in France was investigated. Both species were detected on seeds by conventional PCR assay, with a greater frequency for D. angelicae than D. eres (67% vs 33%, respectively). Overall, the results highlighted that umbel browning in carrot seed crops in France was mainly caused by D. angelicae.

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“…It is evident that DNA-based detection systems exhibit higher level of sensitivity than conventional techniques (Walcott 2003). These methods are timeconsuming, laborious and required skilled personnel.…”

Section: Discussionmentioning

confidence: 99%

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

Pei-ling

Jędryczka

Irzykowski

et al. 2016

Journal of Phytopathology

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An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape.

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Detection of Seedborne Pathogens

Cited by 58 publications

References 35 publications

A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

Characterization of fungal pathogens (Diaporthe angelicae and D. eres) responsible for umbel browning and stem necrosis on carrot in France

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

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