Applications of LAMP Based Diagnostic Kits in Crop Management

Human population is increasing at exponential growth in all parts of the world. Based on the recent report on food security released in 2010 [1]. Requirement of food will increase due to continuous increase in the human population leading to 70% more food requirement by 2050 [1]. Vegetarian population of the world is estimated to be 1.5 billion in 2010 [2]. Apart from the humans all the feedstock animals also rely on food produced by the plants. The demand of food is higher than the supply because of continuous decrease in the agricultural land. Under such circumstance, effective crop management is essential. The crop losses due to pathogenic infection was around 20-40% [3]. Rice, wheat, barley, maize and soybean are majorly consumed crop globally which was reduced by 12% due to plant diseases. Whereas the crop yield of groundnuts and potatoes were affected around 24% and of wheat and cotton around 50% and 80% [4]. Economic losses due to pathogens in US only was estimated to be 40 billion dollars so worldwide loses can be imagined [5,6]. With the evolution of science, researchers have found numerous techniques to identify pathogens but all of them require high initial cost, hi-tech infrastructure facility and experienced researchers. Therefore, these technologies are confined to the laboratories or research institutes located in cities. There is an urgent need to develop and popularize technologies that are suitable to the rural areas for speedy testing of pathogens to avoid epidemics and for launching crop protection measures to manage pathogens [7-29].


Introduction
Human population is increasing at exponential growth in all parts of the world. Based on the recent report on food security released in 2010 [1]. Requirement of food will increase due to continuous increase in the human population leading to 70% more food requirement by 2050 [1]. Vegetarian population of the world is estimated to be 1.5 billion in 2010 [2]. Apart from the humans all the feedstock animals also rely on food produced by the plants. The demand of food is higher than the supply because of continuous decrease in the agricultural land. Under such circumstance, effective crop management is essential. The crop losses due to pathogenic infection was around 20-40% [3]. Rice, wheat, barley, maize and soybean are majorly consumed crop globally which was reduced by 12% due to plant diseases. Whereas the crop yield of groundnuts and potatoes were affected around 24% and of wheat and cotton around 50% and 80% [4]. Economic losses due to pathogens in US only was estimated to be 40 billion dollars so worldwide loses can be imagined [5,6]. With the evolution of science, researchers have found numerous techniques to identify pathogens but all of them require high initial cost, hi-tech infrastructure facility and experienced researchers. Therefore, these technologies are confined to the laboratories or research institutes located in cities. There is an urgent need to develop and popularize technologies that are suitable to the rural areas for speedy testing of pathogens to avoid epidemics and for launching crop protection measures to manage pathogens .

Laboratory based technologies for detection and identification of plant pathogens
Identification of the pathogens plays a pivotal role in controling of plant diseases and in taking relevant crop protection measures Fang et al. [11], have divided the methods for pathogen detection in two categories: direct methods and indirect methods. Direct methods includes detection of pathogens through the molecules produced by them or by using their DNA/RNA etc. A comparison of direct pathogen detection methods is complied in the Table 1.
Whereas indirect detection methods works on the plant stress profiling and plant volatile profiling etc. All the methods mentioned in the Table 1 requires high end laboratory and skilled professionals . Abbreviations: PCR-polymerase chain reaction;IF-immunofluorescence;GC-Gas chromatography; FISH-fluorescence in-situ hybridization;

Principle of LAMP technology
LAMP (Loop-mediated isothermal Amplification) method was reported by Notomy [46]. It is highly specific and sensitive technique to discriminate variations at the single nucleotides polymorphism. It is also a simple, cost effective, rapid and accurate method to be used in the field testing of pathogens. LAMP method does not require specific laboratory set up, it requires only stable heat providing instrument which can be operated through batteries also. The various applications of LAMP technique in the detection of plant pathogenic bacteria, viral and fungal diseases are compiled in the Table 2. Fire blight of pear and apple Bacterial Erwinia amylovora [50] Mumps disease Viral Mumps virus [51] Newcastle disease Viral Newcastle disease virus [52] human influenza A Viral Human influenza A virus [53] Wheat stripe rust Fungal Puccinia striiformis f.sp. tritici (Pst) [54] chromoblastomycosis Fungal 5.8S rDNA gene of 39 fungal species [55] Plant root disease Fungal Arbuscular mycorrhizal fungi [56] soybean Phytophthora root rot Fungal Phytophthora sojae [57] The technique involves the amplification of nucleic acids of plant microbes under isothermal conditions. Primers designed for the assay will amplify the stem-loop DNA structures of microbes with several inverted repeats of the target fragments, so as to detect and differentiate polymorphism among bacteria and fungal pathogens. Technology has been commercially developed by number of diagnostic companies, wherein the concentrations of white precipitates of magnesium pyrophosphate was detected using fluroscent dye like syber green. Fluorescence of syber green dye is the indication of amplification of target nucleic acid fragments of microbes present in human or animal or plant samples [47][48][49][50][51][52][53][54][55][56].
Organism specific genes or rRNA region is selected for designing LAMP specific primers. Instead of single primer pairs, LAMP uses 3 or more than three primer pairs, which makes it highly specific for target identification. Several tools and software are available for LAMP specific primer designing but validation is required for all the primer sets. Protocol of LAMP includes crude or pure DNA isolation and isothermal amplification of the DNA using LAMP specific primers followed by visual or spectrophotometeric analysis for detection of change in color as a result of amplification. Few methods uses fluoresce in isothiocyanate (FITC)/biotin for labeling the primers followed by detection based on specially treated lateral flow test strips (Milenia strips, Milenia Biotec Gieben, Germany) [57].

Commercial development of LAMP based devices
Currently, most researchers are using LAMP method for detecting targeted pathogen by SYBR green or any other fluorescent dye based detection protocol. In this protocol, DNA or cDNA is amplified using target specific LAMP mediated primers. As the amplified products contains loop structures in the positive samples they retains the fluorescent dye and changes their color. This traditional method uses water bath, dry bath or thermal cycler for providing temperature to the reaction. Singleton et al. [58] have developed a device named as"Non-instrumented nucleic acid amplification (NINA) platform" (Figure 1) which is an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens. This is a kind of point of care device as it neither requires any specific laboratory/ infrastructure nor electricity. It uses MgFe fuel pouch to activate the heater. MgFe is added to the bottom of the heater and mixed with 5ml of saline which is commercially available as a blow-fillseal cartridge. Within 12 minutes, temperature increases up to 61.5 ˚C +/21.5 ˚C which is desired temperature for performing the assay. They have validated the detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result within 80 minutes time with optimum sensitivity and reproducibility. The heater was used to amplify specific LAMP amplicons in which loop primers used were labeled with hapten to enable capture using antibody binding of FITC followed by visual detection of captured amplicons via streptavidin colloidal gold. LAMP positive reactions were analyzed using Milenia lateral flow test strips (Milenia Biotec Gieben, Germany) ( Figure  2). Limit of detection for HIV viral assay performed by this instrument was reported to be 75 copies/reaction or 8,333 viral copies/mL of extracted plasma [58].

Future application
Maize is agriculturally and industrially very important crop for India and world. Its demand is estimated to be doubled by the year of 2050. However, crop productivity is reduced by biotic and abiotic stresses. Both fungal and bacterial pathogens play significant role in the reduction of maize crop yield in India. Early diagnosis of pathogens will decrease the considerable economic loss to Farmers. Xcelris labs is working on developing a LAMP based diagnostic test for detection of Fusarium sps, Pseudomonas sps, and Sclerophthora sps affecting maize crops. After successful development and validation of kit in field, it will help in early stage diagnosis of pathogens in Sorghum, Rice, Wheat, Oats and other Poaceae family members.

Conclusion
LAMP technology is very well studied as a research and investigative project, which has significant impact in the crop management in rural areas of India. Development of LAMP based devices can contribute significantly to crop improvement. Large amount of crop damage in rural India due to delay in the identification and detection of pathogen causing the disease. Therefore, field testing of plant pathogens will help farmers to take crop protection measures, namely, spraying insecticides or pesticides, release of biological predators, fertigation in green house or poly house, early harvesting of crop produce or fruits, proper storage of food grains and so on. LAMP based devices can be made available at Taluka level or village level by bringing awareness in ''farmers training centers''.

Conflict of interest and Disclaimer
Authors declare no conflict of interests. Copyrights, trade names and trademarks used in the article belong to the respective companies. Opinions expressed in the article by authors are based on available scientific reviews. Neither Xcelris Labs Limited nor Abellon Group Company recommends or endorses the products or techniques mentioned in the article.