Abstract
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.
Highlights
Rice (Oryza sativa L.) is an essential staple food consumed worldwide
We developed a specific and efficient method for one-step detection method of F. fujikuroi in rice seeds based on the non-ribosomal peptide synthetase (NRPS31),which is conserved and unique to F. fujikuroi and plays an important role in the gibberellic acid (GA) bio-synthesis
Rice bakanae disease (RBD) caused by F. fujikuroi is an important seed-borne disease that is common in the primary rice production regions
Summary
Rice (Oryza sativa L.) is an essential staple food consumed worldwide. A recent survey by the International Food Policy Research Institute indicates that rice production will need to increase 38% by 2030 to feed the expanding human population but available arable land is being lost to housing and industrialization. Several methods for surveillance of RBD, including F. fujikuroi isolation, seed morphology scanning, and polymerase chain reaction (PCR) detection, are among the common practical methods adopted for RDB diagnosis in the laboratory[8,16] These traditional methods are unsuitable for field applications, as they require technical expertise, specialized equipment and can be time-consuming. LAMP was invented and applied as early as in 2000 and is recognized as a user-friendly, rapid, and efficient amplification method of DNA sequences at a single temperature, that is both sensitive and specific[17] This technique is less sensitive to inhibitors than PCR and, has been applied for detection of several plant-pathogens, including Didymella bryoniae from cucurbit seeds[18] and Colletotrichum truncatum from soybeans[19]. We developed a specific and efficient method for one-step detection method of F. fujikuroi in rice seeds based on the non-ribosomal peptide synthetase (NRPS31),which is conserved and unique to F. fujikuroi and plays an important role in the GA bio-synthesis
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