Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of<i>Fusarium fujikuroi</i>in Rice Plants and Seeds

Carneiro, Greice Amaral; Matić, S.; Ortu, G.; Garibaldi, A.; Spadaro, D.; Gullino, M. L.

doi:10.1094/phyto-10-16-0371-r

Cited by 36 publications

(15 citation statements)

References 37 publications

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“…Each qPCR reaction contained 5 μl SYBR Premix EX Taq II (Ti RNase H Plus) (Takara Bio, Shiga, Japan), 0.5 μl of 10 μM forward primer, 0.5 μl of 10 μM reverse primer, 1 μl (100 ng) DNA, and 3 μl ddH 2 O. The primers TqF2 (5′-GGCGCGTTTTGCCCTTTCCT-3′) and TqR (5′-AGCGGCTTCCTATTGTCGAA-3′) (Carneiro et al 2017 ) specifically targeting the translation elongation factor 1-α gene in F. fujikuroi were used. Standard curves were generated for different rice tissues by mixing a serial dilution of F. fujikuroi DNA (10 ng, 2 ng, 400 pg, 80 pg, and 16 pg) and healthy rice DNA (90 ng, 98 ng, 100 ng, 100 ng, and 100 ng of DNA from different tissues of ZK and TNG67).…”

Section: Methodsmentioning

confidence: 99%

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi

Cheng

Chen

Lai

et al. 2020

Rice

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Background Bakanae is a seedborne disease caused by Fusarium fujikuroi. Rice seedlings emerging from infected seeds can show diverse symptoms such as elongated and slender stem and leaves, pale coloring, a large leaf angle, stunted growth and even death. Little is known about rice defense mechanisms at early stages of disease development. Results This study focused on investigating early defenses against F. fujikuroi in a susceptible cultivar, Zerawchanica karatals (ZK), and a resistant cultivar, Tainung 67 (TNG67). Quantitative PCR revealed that F. fujikuroi colonizes the root and stem but not leaf tissues. Illumina sequencing was conducted to analyze the stem transcriptomes of F. fujikuroi-inoculated and mock-inoculated ZK and TNG67 plants collected at 7 days post inoculation (dpi). More differentially expressed genes (DEGs) were identified in ZK (n = 169) than TNG67 (n = 118), and gene ontology terms related to transcription factor activity and phosphorylation were specifically enriched in ZK DEGs. Among the complex phytohormone biosynthesis and signaling pathways, only DEGs involved in the jasmonic acid (JA) signaling pathway were identified. Fourteen DEGs encoding pattern-recognition receptors, transcription factors, and JA signaling pathway components were validated by performing quantitative reverse transcription PCR analysis of individual plants. Significant repression of jasmonate ZIM-domain (JAZ) genes (OsJAZ9, OsJAZ10, and OsJAZ13) at 3 dpi and 7 dpi in both cultivars, indicated the activation of JA signaling during early interactions between rice and F. fujikuroi. Differential expression was not detected for salicylic acid marker genes encoding phenylalanine ammonia-lyase 1 and non-expressor of pathogenesis-related genes 1. Moreover, while MeJA did not affect the viability of F. fujikuroi, MeJA treatment of rice seeds (prior to or after inoculation) alleviated and delayed bakanae disease development in susceptible ZK. Conclusions Different from previous transcriptome studies, which analyzed the leaves of infected plants, this study provides insights into defense-related gene expression patterns in F. fujikuroi–colonized rice stem tissues. Twelve out of the 14 selected DEGs were for the first time shown to be associated with disease resistance, and JA-mediated resistance was identified as a crucial component of rice defense against F. fujikuroi. Detailed mechanisms underlying the JA-mediated bakanae resistance and the novel defense-related DEGs are worthy of further investigation.

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Section: Methodsmentioning

confidence: 99%

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi

Cheng

Chen

Lai

et al. 2020

Rice

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“…Samples prior to inoculation were used in both LAMP and qPCR analysis as negative controls. https://www.ncbi.nlm.nih.gov/Traces/wgs/?val=NGKE01#contigs) using the formula: number of cells /µl = DNA quantity/0.000048 (Amaral Carneiro et al 2017). The amount of fruit used (ranging from 1 g to 2 g) and the elution volume of the extracted DNA were used to calculate the total number of cells.…”

Section: Lamp Primer Designmentioning

confidence: 99%

Rapid Detection of Monilinia fructicola and Monilinia laxa on Peach and Nectarine using Loop-Mediated Isothermal Amplification

et al. 2019

Self Cite

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Monilinia laxa and M. fructicola are two causal agents of brown rot, one of the most important diseases in stone fruit. Two species cause blight on blossoms and twigs and brown rot on fruit in pre- and postharvest. Both species are distributed worldwide in North and South America, Australia, and Japan. In Europe, M. laxa is endemic, while M. fructicola was introduced in 2001 and it is now widespread in several countries. Currently, both species coexist in European stone fruit orchards. Monilinia spp. overwinter in cankers and mummified fruit. Mummy monitoring during winter permits growers to understand which species of Monilinia will be prevalent in an orchard during the following season, permitting planning of an appropriate crop protection. Traditionally, the identification has been carried out using morphological features and even with polymerase chain reaction (PCR)-based assays that requires time and well-equipped laboratories. In this study, two isothermal-based methods were designed to identify these pathogens in a faster way than using traditional methods. The loop-mediated amplification (LAMP) assays were validated on some isolates of Monilinia spp. coming from the mummy monitoring according to the international European and Mediterranean Plant Protection Organization standard (PM7/98), taking into account specificity, sensitivity, repeatability, and reproducibility. The sensitivity of both assays was checked by monitoring (at different time points) two nectarine varieties artificially inoculated and stored at two different temperatures. The reliability of both LAMP assays against the quantification of the inoculum was compared with previously published quantitative PCR assays. Both LAMP methods were able to detect a low number of cells. These LAMP methods could be a useful tool for monitoring brown rot causal agents in the field and during postharvest.

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“…The reported TaqMan real-time PCR and SYBR Green real-time PCR assay had the respective detection limit of 27.5 fg and 10 pg of F . fujikuroi DNA 27,28 . Upon detecting for seed and seedling samples, the LAMP assay yielded satisfactory results compared to traditional isolation and culture methods.…”

Section: Discussionmentioning

confidence: 99%

One-step loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Fusarium fujikuroi in bakanae disease through NRPS31, an important gene in the gibberellic acid bio-synthesis

Zhang¹,

Dai²,

Wang³

et al. 2019

Sci Rep

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Rice bakanae disease caused by Fusarium fujikuroi is one of the most famous seed borne diseases. If infected seeds are used, this disease will occur with serious impacts. Thus, a simple, reliable, specific and sensitive method for surveillance is urgently needed to screen infected seeds and seedlings at early developmental stages. In this study, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect F . fujikuroi in contaminated rice seeds and seedlings for diagnosis of bakanae disease. NRPS31 gene plays an important role in the gibberellic acid (GA) bio-synthesis of F . fujikuroi , and is not present in any other sequenced fungal genome, and thus was adopted as the target for LAMP primer design. The LAMP assay enables the fast detection of as little as 1 fg of pure genomic F . fujikuroi DNA within 60 minutes. Further tests indicated that the LAMP assay was more sensitive and faster than the traditional isolation method for F . fujikuroi detection in rice seeds and seedlings. Our results show that this LAMP assay is a useful and convenient tool for detecting F . fujikuroi , and it can be applied widely in seed quarantine of bakanae disease, providing valid data for disease prevention.

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Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification ofFusarium fujikuroiin Rice Plants and Seeds

Cited by 36 publications

References 37 publications

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi

Rapid Detection of Monilinia fructicola and Monilinia laxa on Peach and Nectarine using Loop-Mediated Isothermal Amplification

One-step loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Fusarium fujikuroi in bakanae disease through NRPS31, an important gene in the gibberellic acid bio-synthesis

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