Development of PCR and loop mediated isothermal amplification assay for the detection of bakanae pathogen Fusarium fujikuroi

Sunani, Sunil Kumar; Bashyal, Bishnu Maya; Rawat, Kirti; Manjunatha, C.; Sharma, Sapna; Prakash, Ganesan; Krishnan, S. Gopala; Singh, A. K.; Aggarwal, Rashmi

doi:10.1007/s10658-019-01694-2

Cited by 16 publications

(9 citation statements)

References 24 publications

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“…In addition, LAMP amplification using DesM231 marker provides information related to GA3 production (from its precursors GA4 and GA7) ( Supplementary Figure S5 ). Although the non-ribosomal peptide synthetase gene (NRPS31) in F. fujikuroi , consisting of 11-gene cluster (apicidin synthetase, APS) without production of apicidin [ 56 ], is used as a marker in LAMP assays [ 6 , 25 ], NRPS31 is closely related to the NRPS APS1 (66–71% identical) in F. incarnatum , which is part of a 12-gene cluster responsible for synthesis of apicidin, a histone deacetylase inhibitor with antiparasitic activity [ 57 , 58 ], showing that it is not unique in genus Fusarium . Furthermore, a previous study showed that F. fujikuroi m567 (Genbank accession no.…”

Section: Discussionmentioning

confidence: 99%

“…Although LAMP products were observed from 55 to 68 • C for 55 min according to color change (visual inspection) and electrophoresis on agarose, the best results were obtained when the reaction temperature was maintained at 65 • C (Supplementary Figure S7A,B). Furthermore, the LMAP test was carried out at 65 • C for different times, including 25,30,35,40,45,50,55,60,65,70, and 75 min. The results showed that LAMP products turned to green color in tubes treated from 25 to 75 min, and only tubers used as control and treated for 20 min turned to orange (Supplementary Figure S8A); however, the best results were produced after reaction time was maintained for 60 min based on electrophoresis (Supplementary Figure S8B).…”

Section: Lamp Reaction and Optimization Of Its Conditionsmentioning

confidence: 99%

“…In control, loop mediated isothermal amplification of DNA (LAMP) with an inexpensive device (single temperature) and extremely high amplification efficacy shows significant advantages over PCR and has been used for the detection of plant pathogens under complex conditions. In the detection of pathogens associated with RBD, although a few markers, such as TEF1-α gene [ 23 ], intergenic spacer (IGS) region [ 24 ], and non-ribosomal peptide synthetase gene (NRPS31) [ 8 , 25 ], were used to develop PCR- or LAMP-based detection for F. fujikuroi , gibberellins GA3 metabolite production-related specific markers for the identification of F. fujikuroi is not reported.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Rice Seed-Derived Fusarium spp. and Development of LAMP Assay against Fusarium fujikuroi

Jiang

Jin

et al. 2020

Pathogens

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Fusarium species are important seedborne pathogens that cause rice bakanae disease (RBD). In this study, 421 strains were isolated from 25 rice samples collected from Zhejiang, Anhui, and Jiangxi provinces of China. Furthermore, 407 isolates were identified as F. fujikuroi (80.05% isolation frequency), F. proliferatum (8.31%), F. equiseti (5.94%), F. incarnatum (2.61%), F. andiyazi (0.95%), and F. asiaticum (0.48%) based on morphology and translation elongation factor 1-alpha (TEF1-α) gene. Phylogenetic analysis of combined sequences of the RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2), TEF1-α gene, and ribosomal DNA (rDNA) internal transcribed spacer (ITS) showed that 17 representative strains were attributed to six species. Pathogenicity tests showed that representative isolates possessed varying ability to cause symptoms of bakanae on rice seedlings. Moreover, the seed germination assay revealed that six isolates had different effects, such as inhibition of seed germination, as well as seed and bud rot. The loop mediated isothermal amplification (LAMP)-based assay were developed for the detection of F. fujikuroi. According to sequences of desaturase-coding gene promoter, a species-specific marker desM231 was developed for the detection of F. fujikuroi. The LAMP assay using seeds collected from field was validated, and diagnostics developed are efficient, rapid, and sensitive.

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Section: Discussionmentioning

confidence: 99%

Section: Lamp Reaction and Optimization Of Its Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of Rice Seed-Derived Fusarium spp. and Development of LAMP Assay against Fusarium fujikuroi

Jiang

Jin

et al. 2020

Pathogens

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“…Fusarium wilt is a major threat to production of chickpea since it is seed borne in nature (Jalali and Chand, 1991), it has high survival rate in the soil and on crop residues for up to 6 years as chlamydospores (ICRISAT, 1985) in India. Rice is the second most important cereal food crop grown and exported all over the world (Sunani et al, 2019) and India ranks second in rice production with an area of around 44.6 million hectare (mha) and productivity of 2972 kg/ha (FAO, 2014). Bakanae disease caused by Fusarium fujikuroi Nirenberg severely affects quality and production in Basmati rice (Bashyal et al, 2016a, b;Gupta et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…This sequence information has been extensively used in the taxonomy and phylogenetic study of Fusarium species by identifying and discriminating isolates within a species. These molecular techniques based on DNA have been extensively used and has proved to be more rapid, sensitive and precise detection method (Sunani et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Phenotypic and Molecular Identification of Pathogenic Fusarium Species Isolated from Various Pulse Growing Geographic Areas of India

Sharma¹,

Bhagat²

2021

Int.J.Curr.Microbiol.App.Sci

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Fusarium phythopathogenic fungi is responsible for high economic loss of cereal food crop. The objective of this study was aimed at isolation, morphological and molecular identification of Fusarium species. 13 different Fusarium spp. i.e. F. solani, F. chlamydosporum, F. tabacinum, F. fujikuroi, F. oxysporum, F. verticillioides, F. brachygibbosum, Fusarium sp. and F. incarnatum were isolated and identified from diseased samples of chickpea, pigeonpea, rice, lentil and garden pea crop. Colony characteristics like colony color, colony growth diameters, mycelium type, sporulation, pigmentation, odour were obtained after culture purification. Shape, size and septation of microconidia and macroconidia, position, shape, occurrence and size of chlamydospores, conidiophore branching were examined microscopically. MS10, BI01 and KA14 isolates were slow growing, BI02 and UP07 were moderate growing and BI03, HA04, MS06, MS09, MS11 and KA(Gul)13 were fast to very fast growing on PDA after 7-10 days. Chlamydospores were found in most of the isolates. Colonies were abundant, loosely tufted, fluffy, pannose, vinaceous floccose, powdery and some were flat appressed, arachnoid. Pigmentation of most of the isolates was pinkish white to dark pink, carmine to violet in colour. Phylogenetic analysis was done by maximum likelihood method using the ITS-rDNA region of Fusarium isolates and multiple sequence alignment of ITS DNA sequences was done using Clustal_W program and all identified sequences were submitted in NCBI GenBank database.

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Ultrasensitive and on‐site diagnosis of rice bakanae disease based on CRISPR‐LbCas12a coupled with LAMP

Zhang,

Zhao,

et al. 2024

Pest Management Science

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BACKGROUNDRice bakanae disease (RBD) has longstanding challenges impacted rice production, which is predominantly induced by Fusarium fujikuroi Nirenberg. Early diagnosis of F. fujikuroi is important to control RBD and improve quality and quantity of rice production. This study presents a novel on‐site diagnosis platform combined with CRISPR/LbCas12a and LAMP to detect F. fujikuroi.RESULTSLAMP amplification of TEF1‐α, a characteristic gene of F. fujikuroi were performed, followed with trans‐cleavage reaction of LbCas12a, cleaving the single‐stranded DNA reporter, which is modified by the terminal fluorophore and quencher groups, producing fluorescence signal. The platform was confirmed with high specificity and sensitivity (LOD <1 aM). Furthermore, we designed a lateral flow strip experiment based on the trans‐cleavage activity of LbCas12a, which was identified with similar sensitivity and specificity to the fluorescence detection method.CONCLUSIONIn summary, this study achieved a platform with remarkable sensitivity and specificity for F. fujikuroi detection and provide potential for on‐site and ultrasensitive diagnostic tools for RBD. © 2024 Society of Chemical Industry.

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Development of PCR and loop mediated isothermal amplification assay for the detection of bakanae pathogen Fusarium fujikuroi

Cited by 16 publications

References 24 publications

Identification of Rice Seed-Derived Fusarium spp. and Development of LAMP Assay against Fusarium fujikuroi

Identification of Rice Seed-Derived Fusarium spp. and Development of LAMP Assay against Fusarium fujikuroi

Phenotypic and Molecular Identification of Pathogenic Fusarium Species Isolated from Various Pulse Growing Geographic Areas of India

Ultrasensitive and on‐site diagnosis of rice bakanae disease based on CRISPR‐LbCas12a coupled with LAMP

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