Detection and Quantification of Rhizoctonia solani AG-1 IA, the Rice Sheath Blight Pathogen, in Rice Using Real-Time PCR

Rhizoctonia solani Kühn is the causal organism of sheath blight, a major rice disease worldwide that severely impairs yield and quality. It is difficult to identify the pathogen in the early phase of the infection and to accurately quantify the fungal development based on visual inspection. Therefore, a rapid and reliable method is advantageous for the detection and quantification of the pathogen causing this important rice disease. In this study, a real-time, quantitative polymerase chain reaction (QPCR) assay was developed to detect and quantify R. solani AG-1 IA DNA from infected rice plants. A specific primer pair was designed based on the internal transcribed spacer region of the fungal ribosomal DNA. The specific detection of R. solani DNA was successful with quantities as low as 1 pg. The QPCR assay could be used for detecting the rice sheath blight pathogen, quantifying fungal aggressiveness, and evaluating the resistance level of rice cultivars.

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Morphological, pathological and molecular characterisation of rice sheath blight disease causal organism Rhizoctonia solani AG-1 IA in Egypt

Archives of Phytopathology and Plant Protection ◽

10.1080/03235408.2019.1650544 ◽

2019 ◽

Vol 52 (5-6) ◽

pp. 507-529 ◽

Cited By ~ 3

Author(s):

Rabie A. S. El-Shafey ◽

Rabab M. Elamawi ◽

Mona M. Saleh ◽

Abdelaziz M. Tahoon ◽

Amero A. Emeran

Keyword(s):

Rhizoctonia Solani ◽

Sheath Blight ◽

Molecular Characterisation ◽

Rice Sheath Blight ◽

Causal Organism ◽

Sheath Blight Disease ◽

Blight Disease

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Evolution from Natural β-Carboline Alkaloids to Obtain 1,2,4,9-tetrahydro-3-thia-9-aza-fluorene Derivatives as Potent Fungicidal Agents against Rhizoctonia solani

International Journal of Molecular Sciences ◽

10.3390/ijms19124044 ◽

2018 ◽

Vol 19 (12) ◽

pp. 4044 ◽

Cited By ~ 1

Author(s):

Junmin Xi ◽

Zhijun Zhang ◽

Qi Zhu ◽

Guohua Zhong

Keyword(s):

Rhizoctonia Solani ◽

Fungicidal Activity ◽

Sheath Blight ◽

Potential Candidate ◽

Oxygen Species ◽

Rice Sheath Blight ◽

Validamycin A ◽

Rice Disease

Rice sheath blight, caused by Rhizoctonia solani, is a globally important rice disease and the increasing resistance of this pathogen highlights the need for new active compounds against rice sheath blight. In this study, natural β-carboline alkaloids were optimized to obtain a series of 1,2,4,9-tetrahydro-3-thia-9-aza-fluorene derivatives and evaluated for their fungicidal activity and mode of action against R. solani. Of these compounds, 18 exhibited significant in vitro fungicidal activity against R. solani, with an EC50 value of 2.35 μg/mL, and was more active than validamycin A. In vivo bioassay also demonstrated that 18 displayed superior protective and curative activities as compared to validamycin A. Mechanistically, 18 not only induced the loss of mitochondrial membrane potential and accumulation of reactive oxygen species, but also interfered with DNA synthesis. Therefore, compound 18 displayed pronounced in vitro and in vivo fungicidal activity against R. solani and could be used as a potential candidate for the control of rice sheath blight.

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Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR

Plant Disease ◽

10.1094/pdis-02-18-0310-re ◽

2018 ◽

Vol 102 (12) ◽

pp. 2487-2493 ◽

Cited By ~ 2

Author(s):

Jeremiah K.S. Dung ◽

Jeness C. Scott ◽

Qunkang Cheng ◽

Stephen C. Alderman ◽

Navneet Kaur ◽

...

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Management Approach ◽

Conidial Suspension ◽

Claviceps Purpurea ◽

Spore Trap ◽

Grass Seed ◽

Wide Range ◽

Detection And Quantification

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

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Developing a real-time PCR assay for direct detection and quantification of Pratylenchus scribneri in field soil

Nematology ◽

10.1163/15685411-00003336 ◽

2020 ◽

Vol 22 (7) ◽

pp. 733-744

Author(s):

Deepika Arora ◽

Guiping Yan ◽

Richard Baidoo

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Parasitic Nematodes ◽

Field Soil ◽

Accurate Identification ◽

Soil Dna ◽

Field Soils ◽

Pratylenchus Scribneri ◽

Detection And Quantification

Summary The endomigratory root-lesion nematode, Pratylenchus scribneri, is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration.

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Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil

Plant Disease ◽

10.1094/pdis-08-12-0729-re ◽

2013 ◽

Vol 97 (6) ◽

pp. 757-764 ◽

Cited By ~ 38

Author(s):

Guiping Yan ◽

Richard W. Smiley ◽

Patricia A. Okubara ◽

Andrea M. Skantar ◽

Catherine L. Reardon

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Management Practices ◽

Quantitative Polymerase Chain Reaction ◽

Internal Transcribed Spacer Region ◽

Pratylenchus Neglectus ◽

Standard Curve ◽

Field Soils ◽

Plant Parasitic ◽

Detection And Quantification

Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

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Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

Plant Disease ◽

10.1094/pdis-03-18-0419-re ◽

2019 ◽

Vol 103 (3) ◽

pp. 475-483 ◽

Cited By ~ 6

Author(s):

Hervé Van der Heyden ◽

Thérèse Wallon ◽

C. André Lévesque ◽

Odile Carisse

Keyword(s):

Real Time ◽

Internal Control ◽

Disease Incidence ◽

Quantitative Polymerase Chain Reaction ◽

Pcr Primers ◽

Qpcr Assay ◽

Inoculum Concentration ◽

Soil P ◽

Dry Soil ◽

Detection And Quantification

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

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Effects of straw incorporation on Rhizoctonia solani inoculum in paddy soil and rice sheath blight severity

The Journal of Agricultural Science ◽

10.1017/s002185961300035x ◽

2013 ◽

Vol 152 (5) ◽

pp. 741-748 ◽

Cited By ~ 1

Author(s):

H. ZHU ◽

Z. X. WANG ◽

X. M. LUO ◽

J. X. SONG ◽

B. HUANG

Keyword(s):

Rhizoctonia Solani ◽

Disease Severity ◽

Rice Straw ◽

Paddy Soil ◽

Sheath Blight ◽

Soil Productivity ◽

Rice Sheath Blight ◽

Important Method ◽

Pathogen Dynamics ◽

Straw Incorporation

SUMMARYIncorporation of rice straw into soil has traditionally been an important method of recycling nutrients and improving soil productivity. Currently, although the effects of straw incorporation on disease severity have been documented, the dynamics of the pathogen in soil after straw incorporation are poorly understood. In the present study, rice straw with various proportions of diseased straw was incorporated at three separate locations (SuPu town, SuSong County and FengYang County) in Anhui province, China. The pathogen dynamics in paddy soil and disease severity of sheath blight during two continuous years from April 2010 to April 2012 were investigated. For all three locations, the amount of pathogen inoculum that persisted in the soil increased with increases in the proportion of diseased straw incorporated. Incorporation of 0·3 and 0·5 diseased straw into soil increased the amount of pathogen inoculum in the soil significantly, whereas incorporation of 0·1 diseased straw into soil had no significant effect on the pathogen inoculum compared with the control (no straw incorporated) or disease severity. Incorporation of healthy rice straw (no disease) resulted in a significant decrease in disease severity, whereas proportions of 0·3 and 0·5 diseased straw resulted in a significant increase of disease severity compared with the control. These results suggested that incorporation of diseased straw enhanced pathogen numbers in soil during the whole decomposition period and increased disease severity. To avoid soil-borne disease accumulation, severely diseased straw should be removed from the field or pre-treated before incorporation.

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