Real-time PCR for detection and quantification of C. gloeosporioides s.l. growth in Stylosanthes and Arabidopsis

Wan, Miting; Yang, Liyun; Zhang, Shizi; Gao, Jing; Jiang, Lingyan; Luo, Lijuan

doi:10.1016/j.cropro.2022.106021

Cited by 4 publications

(5 citation statements)

References 36 publications

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“…Because morphological identification is easily influenced by environmental factors, the latter is used more nowadays. In the host greater yam, Mithun Raj et al previously used nested PCR to detect a C. gloeosporioides limit of 200 fg/µL [36], and a recent study showed that RT-qPCR in other hosts such as Arabidopsis thaliana could increase the detection limit to 5 fg/µL [37]. Compared with the above PCR techniques, the LF-RPA detection method developed in this study can recognize C. gloeosporioides DNA at even a mere 1 fg/µL.…”

Section: Discussionmentioning

confidence: 96%

A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection

Xu,

Lu,

Wang

et al. 2024

JoF

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The greater yam (Dioscorea alata), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by Colletotrichum gloeosporioides. Latent infection often occurs before anthracnose phenotypes can be detected, making early prevention difficult and causing significant harm to agricultural production. Through comparative genomic analysis of 60 genomes of 38 species from the Colletotrichum genus, this study identified 17 orthologous gene groups (orthogroups) that were shared by all investigated C. gloeosporioides strains but absent from all other Colletotrichum species. Four of the 17 C. gloeosporioides-specific orthogroups were used as molecular markers for PCR primer designation and C. gloeosporioides detection. All of them can specifically detect C. gloeosporioides out of microbes within and beyond the Colletotrichum genus with different sensitivities. To establish a rapid, portable, and operable anthracnose diagnostic method suitable for field use, specific recombinase polymerase amplification (RPA) primer probe combinations were designed, and a lateral flow (LF)-RPA detection kit for C. gloeosporioides was developed, with the sensitivity reaching the picogram (pg) level. In conclusion, this study identified C. gloeosporioides-specific molecular markers and developed an efficient method for C. gloeosporioides detection, which can be applied to the prevention and control of yam anthracnose as well as anthracnose caused by C. gloeosporioides in other crops. The strategy adopted by this study also serves as a reference for the identification of molecular markers and diagnosis of other plant pathogens.

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Section: Discussionmentioning

confidence: 96%

A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection

Xu,

Lu,

Wang

et al. 2024

JoF

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“…Successful detection of pathogens by PCR-based methods needs primers to satisfy the criteria, including uniqueness of the amplification sequence to the target pathogen of interest and conservation across populations of the target pathogen [ 27 ]. Several molecular protocols based on real-time PCR amplification have been previously developed for detection and quantification of Colletotrichum species, targeting different molecular markers such as ITS [ 18 , 21 ], GAPDH [ 22 ], CAL [ 17 ], ACT [ 20 ], cutinase ( Cuti ) [ 19 ], and ApMat [ 25 ]. In this study, real-time PCR primers and a probe specific to C. siamense were designed based on the sequence differences in the CAL gene region.…”

Section: Discussionmentioning

confidence: 99%

“…These methods are laborious, time-consuming, and require substantial expertise. Real-time PCR offers a fast, accurate, and culture-independent tool, and is widely used for the detection of plant pathogens [ 17 , 18 , 19 , 20 ]. Compared with other real-time PCR assays, the TaqMan real-time PCR is more specific, sensitive, and accurate.…”

Section: Introductionmentioning

confidence: 99%

Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra

Gu,

Wang,

Huang

et al. 2024

Plants

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Anthracnose, caused by Colletotrichum siamense, is a destructive disease of Pachira glabra in southern China. Early and proper monitoring and quantification of C. siamense is of importance for disease control. A calmodulin (CAL) gene-based TaqMan real-time PCR assay was developed for efficient detection and quantification of C. siamense, which reliably detected as low as 5 pg of genomic DNA and 12.8 fg (5800 copies) of target DNA. This method could specifically recognize all tested C. siamense isolates, while no amplification was observed in other closely related Colletotrichum species. The assay could still detect C. siamense in plant mixes, of which only 0.01% of the tissue was infected. A dynamic change in the amount of C. siamense population was observed during infection, suggesting that this real-time PCR assay can be used to monitor the fungal growth progression in the whole disease process. Moreover, the method enabled the detection of C. siamense in naturally infected and symptomless leaves of P. glabra trees in fields. Taken together, this specific TaqMan real-time PCR provides a rapid and accurate method for detection and quantification of C. siamense colonization in P. glabra, and will be useful for prediction of the disease to reduce the epidemic risk.

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“…The accumulation of fungal DNA of C. gloeosporioides during infection was detected and calculated according to the previous study [ 40 ]. Infected leaves were collected at various time points after inoculation and ground with liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…The cycle threshold (Ct values) of stylo and C. gloeosporioides were detected by real-time quantitative PCR ( Section 2.5 ) using primers UBCE1 and ACTING4 , respectively ( Table S5 ). The content of fungal DNA in 100 ng plant DNA was calculated as described previously [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

Transcriptomic and Physiological Analysis of the Effects of Exogenous Phloretin and Pterostilbene on Resistance Responses of Stylosanthes against Anthracnose

Zhang,

Xu,

Wang

et al. 2024

IJMS

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Anthracnose caused by Colletotrichum gloeosporioides is a destructive disease of Stylosanthes (stylo). Combination treatment of phloretin and pterostilbene (PP) has been previously shown to effectively inhibit the conidial germination and mycelial growth of C. gloeosporioides in vitro. In this study, the effects of PP treatment on the growth of C. gloeosporioides in vivo and the biocontrol mechanisms were investigated. We found that exogenous PP treatment could limit the growth of C. gloeosporioides and alleviate the damage of anthracnose in stylo. Comparative transcriptome analysis revealed that 565 genes were up-regulated and 239 genes were down-regulated upon PP treatment during the infection by C. gloeosporioides. The differentially expressed genes were mainly related to oxidative stress and chloroplast organization. Further physiological analysis revealed that application of PP after C. gloeosporioides inoculation significantly reduced the accumulation of O2•− level and increased the accumulation of antioxidants (glutathione, ascorbic acid and flavonoids) as well as the enzyme activity of total antioxidant capacity, superoxide dismutase, catalase, glutathione reductase, peroxidase and ascorbate peroxidase. PP also reduced the decline of chlorophyll a + b and increased the content of carotenoid in response to C. gloeosporioides infection. These results suggest that PP treatment alleviates anthracnose by improving antioxidant capacity and reducing the damage of chloroplasts, providing insights into the biocontrol mechanisms of PP on the stylo against anthracnose.

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Real-time PCR for detection and quantification of C. gloeosporioides s.l. growth in Stylosanthes and Arabidopsis

Cited by 4 publications

References 36 publications

A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection

A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection

Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra

Transcriptomic and Physiological Analysis of the Effects of Exogenous Phloretin and Pterostilbene on Resistance Responses of Stylosanthes against Anthracnose

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