Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay

Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63 °C for 75 min using minimum 10 fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea.

Dry root rot (DRR) caused by soil borne necrotrophic fungus Rhizoctonia bataticola (Taub.) Butler [Synonyms: Macrophomina phaseolina (Tassi) Goid] is an emerging disease in chickpea (Cicer arietinum L.) 1 . The DRR is more dominant when the crop is exposed to moisture stress conditions 2 and can cause 50 to 100% yield loss under favourable conditions 3 . In recent years, Rhizoctonia bataticola is becoming more prevalent in agricultural areas where climate change is leading to higher temperatures. It is reported that R. bataticola can infect more than 284 plant species including monocot and dicot plant hosts 4 . Due to availability of wide range of natural host, R. bataticola can easily sustain in the dry climatic area and persist in soil for prolonged period even after rotation of the crops.
In chickpea, DRR is often mistaken with Fusarium wilt and other root rot diseases (collar rot, black root rot etc.), as the general symptoms of these diseases are nearly similar and visually undistinguishable in field conditions 1 . In all the cases, affected plants show foliar chlorosis and ultimately cause plant collapse. Therefore, there is a real need of an advance rapid, reliable and easy detection method for diagnosis of R. bataticola for better management of DRR. In recent years, PCR based methods like conventional PCR and real time PCR is being employed to detect fungal species and other microorganism [5][6][7] , but it is not cost effective and need high-quality DNA due to the effects of inhibitors on PCR sensitivity 8,9 . Also molecular expertise is required for true diagnosis of pathogens. Now a days, Loop-mediated isothermal amplification (LAMP) has been developed as an alternative and reliable method for the detection of microbial pathogens and diagnosis of plant diseases [10][11][12][13][14] . The advantages and simplicity of LAMP assay is that the reaction could be easily judged as positive or negative by naked eye through assessing of increased turbidity or colour change 15,16 , and for that it does not require any expensive instruments like thermal cycler.
DNA extraction. Total genomic DNA (gDNA) was isolated from all the fungal isolates and DRR infected plants using PureLink Plant Total DNA Purification kit (Invitogen, USA) as per manufacturer's protocol. About 100 mg of frozen mycelial tissue/plant tissue was grounded in liquid N 2 and resuspended in 250 μ L Resuspension Buffer (supplied in the kit). Total gDNA was eluted in 50 μ L of nuclease free water and stored at − 20 °C for further downstream application. The soil DNA was extracted from 100 mg of R. bataticola sick soil and DRR infected chickpea rhizospheric soil using SoilMaster ™ DNA Extraction Kit (Epicentre, USA) according to the manufacturer's protocol. The obtained DNA was suspended in 200 μ L of elution buffer. The purified DNA was evaluated in 0.8% agarose gel as well as by UV spectrophotometry.
Primer design. As R. bataticola is an important plant pathogen with a broad host range and causes disease in diverse commercial crops, primers for the LAMP assay were designed from the conserved region in the partial ITS and 5.8S rRNA sequences of R. bataticola and M. phaseolina identified by multiple sequence alignment of representative isolates from different crops ( Table 2 and Fig. 1). A set of six primers for LAMP assay, comprising two outer primers (RB_F3 and RB_B3), two innermost primers (RB_FIP and RB_BIP), and two loop primers (RB_LoopF and RB_LoopB) were designed manually by following the directions of Torres et al. 22 . The FIP was made with the complementary sequence of F1 (F1c) and F2, and the BIP made with the complementary sequence of R1 (B1c) and B2 ( Table 3). The organization and position of the LAMP primers and their complementarity to target DNA used in this study are shown in Fig. 1. The designed primers were nBLAST searched on NCBI for analysing the sequence specificity and to get chance of cross reaction with other sibling species if environmental samples will assay in LAMP reaction (Table 4). To avoid the false positive reaction by cross reactivity within the primers, the secondary structures (hairpin, self-dimer and hetero-dimer) of the primers and their corresponding stability with Δ G were reviewed 23,24 in OligoAnalyzer 3.1 tool (http://eu.idtdna.com/calc/analyzer) and were determined ( Table 5). The universal primer pair ITS1 (5′ -TCCGTAGGTGAACCTGCGG-3′ ) and ITS4 (5′ -TCCTCCGCTTATTGATATGC-3′ ) and RB_F3 and RB_B3 were used for conventional PCR. LAMP reaction. The constituents of the LAMP assay were optimized using total gDNA extracted from R. bataticola fungal culture (positive control) and two negative controls (without DNA and DNA from a healthy chickpea plant). The LAMP reaction was performed in a 25 μ L volume contained 2.0 μ L primer mixture (20 μ M each of FIP, BIP, Loop F, and Loop B primers, and 2.5 μ M each of F3 and B3 primers), 1 mM dNTPs, 4 mM MgCl 2 , 2.5 μ L of 10X ThermoPol Reaction Buffer (1X reaction buffer (pH 8.8) contained 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100), 8 U of Bst DNA polymerase (NEB, UK), 1.5 μ L target DNA (about 100 ng). After completing the isothermal amplification 1 μ L of SYBR Green I (Invitrogen, USA) was added for visual assay of the amplification. The reaction was carried out at 63 °C for 60 min followed by incubation at 80 °C for 10 min to inactivate the Bst DNA polymerase. The reactions were performed in a 0.5 mL microcentrifuge in a water bath for temperature control.
Optimization of LAMP reaction condition. To optimize the LAMP reaction conditions, the reaction was carried out at different temperatures, 57 °C, 60 °C, 63 °C, 66 °C and 69 °C using a gradient thermo cycler (ARKTIK Thermal Cycler, Thermo Scientific, USA). The LAMP reaction mixture was incubated at the different temperatures for different time periods, 15 min, 30 min, 45 min, 60 min, 75 min and 90 min to optimize the temperature and time/duration for the LAMP reaction. The reactions were terminated by heat inactivation at 80 °C for 10 min. The LAMP reaction was assessed visually based on colour change after adding SYBR Green I and under UV light. The LAMP amplified products were then analysed by 2% agarose gel electrophoresis.

Specificity of the LAMP.
To determine the specificity of LAMP assay, the reaction was carried out with the extracted DNA from other legume infecting pathogenic fungi discussed previously in fungal strain section. The LAMP assay was done as described earlier at optimized temperature and time duration. The assays were Sensitivity of the LAMP method. The sensitivity of the LAMP assay was determined using 10 fold serially diluted R. bataticola DNA ranged from 10 ng to 0.1 fg. Reaction mixture without DNA template was included as a negative control. The LAMP amplification products were analysed visually by addition of 1 μ L SYBR Green I under UV light or by 2% agarose gel electrophoresis. To compare the sensitivities and specificities between LAMP assay and PCR; PCR was performed with the extracted DNA from all fungal isolates using ITS primers ( Table 2). The PCR reaction was carried using the protocol detailed in Ghosh et al. (2015). The PCR amplified products were then analysed in 1% agarose gel stained with ethidium bromide. The experiment was repeated thrice. The detection limit in LAMP assay was defined as the last dilution with positive reaction.
Validation of the LAMP assay. To validate the LAMP assay, the DNA from DRR infected chickpea plants and soil samples were tested for the presence of R. bataticola. Based on colour change and gel electrophoresis as described above, the LAMP reaction was visualised. The experiment was carried out in triplicate and samples were considered as positive if two of the replicates showed positive response.

Results
Selection of LAMP primers. All R. bataticola isolates were identified by amplifying and sequencing of approximately 544 bp of partial ITS and 5.8S region using universal primers, ITS1 and ITS4 (Table 1). For designing of LAMP assay primers for R. bataticola, the conserve region of the partial ITS and 5.8S sequences was identified by multiple sequence alignment of corresponding nucleotide sequences of representative isolates in different crops (Table 2) and a set of six primers were designed ( Table 3). The primers were analysed in silico against the nBLAST search tool using the NCBI sequence database. The BLAST results revealed that except both parts (B1c and B2) of BIP primer all primers were significantly hits the target sequences of R. bataticola and M. phaseolina with 100% identity (Table 4), although the sequence of BIP primer is 100% similar with the target sequences (Fig. 1). The hit also resulted in 100% similarity with other non-pathogenic microbes of chickpea (Table 4). During the design of LAMP primers, Δ G values were determined and the values were fixed around − 9 kcal/ mole or more positive than − 9 kcal/mole (Table 5). For hairpins, the Tm of the hairpin was kept below of 50 °C and lower than the annealing temperature for the LAMP reaction as the strongest hairpin should be at least 50 °C ( Table 5).
Optimization of LAMP assay. To determine the optimum temperature and reaction time of LAMP assay, the assay was tested within a wide range of temperatures (57 °C-69 °C) and time (15 min-90 min) using pure DNA of R. bataticola culture. The assay showed positive reaction at all temperatures, whether assessment was based on visual fluorescence detection or in gel electrophoresis. The characteristic ladder like bands were evident in the gel when the reaction was positive, but not if the reaction was negative. However, the high intense ladder-like band pattern was obtained in gel electrophoresis at 63 °C (Fig. 2a)  conducted at 63 °C for various time duration mentioned above. The positive reaction was found in all the time duration, but strong band pattern was observed after 75 min of reaction (Fig. 2b). Therefore, 63 °C for 75 min is the optimized temperature and time for LAMP reaction for detection of DRR pathogen.
LAMP assay specificity. LAMP specificity was examined using DNA templates extracted from total 94 R. bataticola isolates collected from diverse geographical region in India. Six pathogenic wilt and root rot pathogens viz., Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani, Fusarium udum and Phytophthora cajani infecting chickpea and pigeonpea were also taken for testing specificity (Table 1). DNA template of R. bataticola isolates gave positive reaction, whereas no amplification was observed for the other fungal species after incubation at 63 °C for 75 min. The LAMP reaction was assessed using 2% agarose (Fig. 3) and SYBR Green I visualisation. The result indicated that the LAMP assay developed in this study is highly specific to R. bataticola. Furthermore, to confirm the specificity of the LAMP primers, a PCR was carried out using primer pair, RB_F3 and RB_B3. All the R. bataticola isolates were amplified with a unique DNA fragment of 354 bp.  However, these primers did not produce any band when DNA templates from other pathogenic fungi were used. However, primer pair ITS1 and ITS4 gave amplification of expected size of DNA fragment (Table 1).  Sensitivity of LAMP assay. To determine the detection limit, the sensitivity of the LAMP reaction was assessed using 10-fold serially diluted R. bataticola DNA template. The amplicons were detected in both, by visual assessment using SYBR Green I (Fig. 4a) and 2% agarose gel electrophoresis (Fig. 4b). No positive signal was produced when less than 10 fg DNA of R. bataticola was used in LAMP reaction, indicating the potential detection limit of R. bataticola up to 10 fg of DNA. It was noticed that the amplified DNA fragments were slightly faint in 10 fg than those produced by a less diluted DNA (> 10 fg). On the other hand, when same amount of DNA was used in conventional PCR, no such amplification was obtained after 1.0 pg of dilution (Fig. 4c). Results of visual detection correlated with agarose gel electrophoresis.

Evaluation of the LAMP assays with plant and soil sample.
To validate the applicability of the LAMP assay at field level, the developed LAMP assay was evaluated with R. bataticola infested chickpea plants and soil samples as well. The plants showing typical symptom of DRR were collected from different experimental fields of ICRISAT. The LAMP assay was carried out at optimised condition to detect the presence of R. bataticola in tested samples. The positive LAMP reaction was found when DNA templates from the R. bataticola infected chickpea plants were assessed, and the products turned green in colour with SYBR Green I. Moreover, none of the DNA template from healthy plants gave positive signals, and remained orange in colour. Furthermore, the LAMP assay conducted with the DNA from rhizospheric soil of DRR infected chickpea plants as well as R. bataticola inoculated sick soil was also found to be positive (Fig. 5). The LAMP result was consistent when the assays were repeated. Results were consistent with PCR method.

Discussion
In this report, we have demonstrated and optimized the LAMP for the detection of R. bataticola, the fungus that cause DRR disease in chickpea. LAMP assay reported here is rapid, highly sensitive, less time-consuming than   conventional PCR-based DNA amplification method. It has been applied for the detection of a wide range of microorganisms including viral 13,25 , bacterial 26,27 , phytoplasma 28 , mycoplasma 29 , fungal [10][11][12] and parasitic agents 30 .
In our study, we used SYBR Green I in LAMP assay as florescent dye which is non-mutagenic and eco-friendly in nature, as the replacement of other potential human mutagen florescent dyes like ethidium bromide. Subsequently, uses of Bst DNA polymerase in LAMP reaction, it permits to strand displacement DNA synthesis and the reactions can be performed under isothermal conditions using a simple incubator, such as a water bath or  heating block. With taking these advantages, LAMP detection technique can be employed in diagnosis of disease in field level even also in remote area where the laboratories are not well equipped.
We validated our developed LAMP assay using the primers generated from partial ITS and 5.8S rDNA gene for quick detection of R. bataticola from varied samples viz. fungal culture, diseased plants and soil samples. The highly sensitive and variable ITS region is idle target rather than other single-copy genes in genomic DNA to sufficient discriminate of some closely related fungal species 31-32 because of its presence in 100 or more copies in the fungal genome and has competence to give amplification from a very small number of micro-organisms 33 . Then again, to avoid secondary structures in primers with abundant G-C bonds e.g. hairpins, self-dimer and heterodimer, the Δ G of the primers were kept more positive than − 9 kcal/mole or very less negative than the same value 23,24 as those can give false positive reaction in assay. When the LAMP primers were analysed in silico for its specificity, it showed significant hit with our target organisms R. bataticola and M. phaseolina in nBLAST. For confirming the specificity of our developed LAMP primers, we assayed those primers with DNA isolated from 94 R. bataticola isolates along with six other plant pathogenic fungi. During this LAMP assay, except all R. bataticola DNA, no colour change was obtained with DNA of other six pathogenic fungus. Thus, this result indicated that  the designed primers and LAMP assay were highly specific for R. bataticola, as it correctly distinguished between R. bataticola and the other pathogens. The reaction mixture without DNA also showed no change in colour during LAMP assay. Previously, the primers from rDNA-ITS gene have been used to successfully detect Pythium aphanidermatum in infected tomatoes 34 and Phytophthora capsici in infected peppers, tomatoes, and other agronomic and ornamental crops of the Solanaceae and Cucurbitaceae families 35 .
The detection limit in our study was found to be 10 fg DNA of R. bataticola, below this level no colour change was noticed. This detection limit was lower than previously reported LAMP methods used to detect other fungal pathogens e.g. Sclerotinia sclerotiorum 12 , Phytophthora sojae 36 , P. ramorum and P. kernoviae 37 , indicating greater sensitivity. Comparison of LAMP assay with the conventional PCR showed the LAMP assay using SYBR Green I dye significantly improved the detection efficiency of R. bataticola. This result of LAMP assay was significant and concordant with the reports published previously for the detection of some plant pathogens 12,36,37 .
Positive reaction in LAMP assay with the DNA isolated from R. bataticola infected chickpea plants sampled from field, further validated our results. DNA from healthy plants gave no reaction. It has previously been used in detection/screening of plant pathogens like Phytophthora capsici 35 , P. ramorum 38 , Pythium aphanidermatum 34 , Meloidogyne enterolobii 39 from infected plants in field and soil samples. In this study, we have designed LAMP primers using sequence of R. bataticola/M. phaseolina isolates infecting chickpea as well as other crops from worldwide and in silico analyses of ITS sequences showed low genetic diversity within the Indian and global isolates, hence the utility of our developed LAMP assay will be equally useful for detection of R. bataticola isolates in any other crops globally. In future, LAMP diagnostic kit will be very useful for monitoring the disease complex in the fields, further helpful in developing the timely management strategies.