Detection of Globodera pallida directly from soil sample using mt-COI region based LAMP assay

Potato cyst nematodes (PCN), Globodera rostochiensis (Golden/yellow) and G. pallida (White), are economically important and relatively specialized pest of potato (Solanum tuberosum L.). Both the species are being identied based on cyst colour after 55-60 days after planting (DAP) however, after 65 DAP, we cannot differentiate based on cyst colour as both species turns brown. Moreover, the molecular techniques available to detect the PCN at species level is laborious, time consuming and costly. Therefore, development of rapid, accurate and economically cheap technique for detection of PCN at species level from the eld is important to device effective management strategies for sustainable potato production. Accordingly, in the rst instance, loop-mediated isothermal amplication (LAMP) assay was developed to detect G. pallida directly from soil by using the mitochondrial (mt-COI) gene specic primer. The LAMP assay was completed within 60 min at 60 °C isothermal conditions and the primer, eciently detects the G. pallida without any cross reaction with G. rostochiensis, Meloidogyne incognita, M. javanica, Heterodera avenae, H. carotae, and Cactodera spp. In analytical sensitivity tests, the assay was able to detect G. pallida with 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl) and the LAMP product was visualized by using SYBR Gold nucleic acid dye and the assay can be highly useful in detection of G. pallida.


Introduction
Globodera pallida (Stone 1973) and G. rostochiensis (Wollenweber 1923) are major biotic limitations in sustainable potato production with quarantine nature and has become a serious endemic pest worldwide causing an average of 12.2% yield losses in potato (Urwin et al. 2001). However, in case of dense inoculum of both the PCN species may cause yield loss up to 80% (Zasada and Dandurand 2018). White potato cyst nematode (G. pallida) alone can reduce up to 80% tuber yield (Talavera et al. 1998). In India, potato cyst nematode (PCN) was rst reported from Udhagamandalam, The Nilgiri, Tamil Nadu (Jones 1961). Later on, its presence was noticed from Kodaikanal hills of Tamil Nadu, adjoining hills of Karnataka and Idukki District in Western Ghats of Kerala (Aarti et al. 2020b). Recently, it has been reported from some parts of Himachal Pradesh, Jammu & Kashmir and Uttarakhand hills (Aarti et al. 2020a). To nd out the PCN population dynamics traditionally PCN species are identi ed based on different morphological characters, however, it takes long time and laborious as well. Therefore, PCR based approaches have been used to identify PCN species which produce speci c and accurate results. Among the PCR based techniques, Random ampli ed polymorphic DNA (RAPD), Restriction fragment length polymorphism (RFLP), Ampli ed fragment length polymorphism (AFLP), Internal transcribed spacer (ITS1 and ITS2), ribosomal deoxyribonucleic acid (rDNA) and mitochondrial deoxyribonucleic acid (mt-DNA) have become popular for identi cation of PCN species (Aarti et al. 2017). But, these techniques require expensive thermo-cyclers as well as imaging systems and it cannot be used directly under eld conditions. Recently, Loop-mediated isothermal ampli cation (LAMP) have emerged promising and alternate to PCR due to its simplicity, rapidity, speci city, sensitivity and cost effectiveness.
Above all only a heating block or water bath are needed that could maintain a constant temperature (Notomi et al. 2000;Tomita et al. 2008;Verma et al. 2019). In view of these advantages, this technology has been used commercially in a variety of pathogens detection kits (Mori and Notomi 2009).
In the eld of plant nematology, LAMP assays have been developed for the detection of parasitic worms of pine wood Bursaphelenchus xylophilus (Kikuchi et al. 2009;Meng et al. 2018), coconut red ring worm B. cocophilus (Ide et al. 2017), burrowing worm Radopholus similis (Peng et al. 2012), citrus root worm Tylenchulus semipenetrans (Song et al. 2017), grass pararsitic worm Anguina wevelli (Yu et al. 2018) and tropical root-knot worm Meloidogyne incognita, M. enterolobii (Niu et al. 2011;Niu et al. 2012) as well as the temperate root-knot worm M. hapla (Peng et al. 2017), and apple root-knot worm M. mali (Zhou et al. 2017). Quick and speci c detection of G. pallida in the soil is imperative for development of management strategy against the particular species. As of now, no attempts have been made for the detection of G. pallida directly from soil sample using LAMP assay hence, we have made an effort to develop the same.

Nematode populations
Pure nematode populations (G. pallida and G. rostochiensis) were maintained on susceptible potato cultivar Kufri Jyoti under glass house conditions at ICAR-CPRS, Kufri, Himachal Pradesh. To check the cross reactivity of LAMP primers, DNA of plant parasitic nematodes like M. incognita, M. javanica, Heterodera carotae, H. avenae, and Cactodera spp. were obtained from the Department of Nematology, TNAU, Coimbatore and G. pallida from ICAR-CPRS, Muthorai, Udhagamandalam.

Genomic DNA extraction
The genomic DNA of G. pallida and G. rostochiensis was extracted repeatedly ten times from single cyst/female following the protocol standardized by Aarti et al. (2019). The quality and quantity of the puri ed DNA sample was determined using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scienti c Inc., Waltham, MA, USA).
Characterization of mt-COI region of G. pallida Primers were designed using FastPCR software (Table 1) of nucleotide sequence from GeneBank (Accession numbers DQ631912) of mitochondrial-cytochrome oxidase subunit I gene (mt-COI) of G. pallida ( Fig. 1 : Table 1). PCR reaction was carried out with 20 μl reaction mixture containing genomic DNA (50ng/μl) 1.0 μl, primer mix 1.0 μl each [FP (Forward primer) and BP (Backward primer) 10 pmol], Red Taq DNA polymerase (8 U) (Genei) 1.0 μl, Taq buffer A 2.0 μl, 2.5 mM dNTP mix 1 μl and double distilled water (DDH 2 O) 13 μl. The PCR reaction was set at 95 °C initial denaturation for 1 min, followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 20 s, and extension at 72 °C for 45 s, with a nal extension at 72 °C for 10 min in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems™, Thermo Fisher Scienti c Inc.). The PCR products were visualized on 2% agarose gel and eluted using QIAquick Gel Extraction Kit (Quiagen). Further, DNA sequencing was performed using BigDye Direct terminator cycle sequencing kit (Applied Biosystems, UK). Automated DNA sequencer (Genetic Analyzer 3500, Applied Biosystems) was used to perform the sequencing analysis. Traces were aligned and visualized using the Sequence Scanner Software version 2.0 for Windows (Applied Biosystems 2012). The obtained nucleotide sequences were subjected to BLAST in blastn programme in NCBI to con rm its identity before utilizing it for designing G. pallida speci c LAMP primers.
Sensitivity assay for PCR and LAMP LAMP sensitivity assay was determined by 10-fold serial dilutions of genomic DNA isolated from single cyst of G. pallida with initial concentration of 10 ng/μl. In addition, conventional PCR reaction was carried out with 20 μl reaction mixture containing genomic DNA 1.0 μl, primer mix 1.0 μl each [5 pmol of F3 (FP)/ and B3 (RP)], Red Taq DNA polymerase (8 U) (Genei) 1.0 μl, Taq buffer A 2.0 μl, 2.5 mM dNTP mix 1 μl and DDH 2 O 13 μl. The PCR reaction was set as described above.

Speci city assay for PCR and LAMP
The speci city was checked with standardized assay conditions using genomic DNA of important plant parasitic nematode i.e. G. pallida, G. rostochiensis, H. avenae, M. incognita, M. javanica H. carotae, and Cactodera spp. The results were visualized by SYBR Gold nucleic acid dye as well as agarose gel (2%) stained with ethidium bromide.
Optimization of LAMP assay directly from nematode inoculated soil To check the feasibility of eld diagnostics LAMP assay, we concentrated optimizing LAMP primers for diagnosis of G. pallida directly from soil samples. Accordingly, as a proof of concept soil samples (250 mg) were arti cially inoculated with 1, 3, 5 and 10 cysts. DNA template of G. pallida from 250 mg soil samples was isolated using a NucleoSpin soil kit (Macherey-Nagel, GmbH & Co. KG, Germany) by following the manufactures protocol. This time we performed the LAMP reaction in a water bath (maintained at 60 °C) and the products were analyzed as described earlier and the reactions were repeated thrice. Here, DDH 2 O used as negative control.
Assessment of LAMP assay with soil samples collected from PCN sick eld Soil samples collected from PCN infested area of Himachal Pradesh (Kufri, Fagu, and Jubbal) and The Nilgiris (Muthorai, Appokodu and Porthyhada), Tamilnadu, India were used to check the e cacy of LAMP assay developed. For validation, DNA isolated from single cyst of G. pallida used as standard positive control. DNA from healthy soil used as no template control (NTC) and DDH 2 O as a control.

Results
LAMP primer design PCR primers were designed which successfully ampli ed 225 bp region ( Fig. 2A) and the sequenced product in NCBI BLAST analysis, shared 99.14% nucleotide identity to the reference sequences of mt-COI region of G. pallida.

Standardization reaction and conditions for LAMP assay
At 60-64 °C, sharp bands of LAMP products were observed on 2.0% agarose gel whereas, almost no bands at 54 °C (Fig. 2B), therefore for further LAMP assay 60 °C temperature was set for ampli cation. While determining the optimal reaction time, maximum ampli cation of the LAMP products was noted at 60 min run and no clear detectable signal was obtained at 30-40 min run (Fig. 2C). LAMP products of G. pallida resulted visual change of colour from orange to green after addition of SYBR Gold nucleic acid dye. However, there were no sharp bands as well as colour change after the addition of SYBR Gold nucleic acid dye in the non template water control.

Comparative sensitivity assay
The LAMP assay was about 1000 times more sensitive than the conventional PCR as in the sensitivity test, conventional PCR was able to detect up to 10 picogram (pg)/µl genomic DNA whereas, LAMP assay produced positive results both in gel electrophoresis and SYBR Gold nucleic acid dye to the minimum genomic DNA concentration of 10 femtogram (fg)/µl (Fig. 3).

Speci city assay
In the speci city assay, LAMP primers ampli ed the DNA of G. pallida and not the DNA of other tested nematodes such as G. rostochiensis, M. incognita, M. javanica, H. avenae, H. carotae, and Cactodera spp. both in gel electrophoresis and SYBR Gold nucleic acid dye based detection of LAMP products (Fig. 4). Therefore, it has been concluded that LAMP primers were very speci c to G. pallida.

Standardization of LAMP assay for G. pallida
The results of our study showed ampli cation as well as green uorescence in SYBR Gold nucleic acid dye in genomic DNA extracted from soil inoculated with different G. pallida populations (Fig. 5A). Here, all the six soil samples collected from different PCN infested elds were found positive to G. pallida whereas, healthy soil (NTC) and water control did not exhibit ampli cation and uorescence in SYBR Gold nucleic acid dye (Fig. 5B).

Discussion
White cyst nematode, G. pallida is a very specialized potato pest having very narrow host range of Solanaceae family crops. Worldwide it has been reported in 61 countries belonging to 5 continents and causes economic yield losses including India (EPPO 2018). It is an important quarantine pest which is mainly restricted to cooler/hilly potato growing regions. However, till date this pest at species level is being identi ed using morphological characters which are time consuming. Therefore, precise diagnosis method at eld level is need of the day. All the molecular techniques developed till date for the identi cation at species require sophisticated equipments as well as expertise whereas, does not require the same and it is a precise ampli cation method that can be adopted in the eld level diagnosing. In our study, primers designed for LAMP assay successfully detected the G. pallida directly from soil samples. In addition, the results were visualized through naked eye by adding SYBR Gold nucleic acid dye. Above all LAMP technique is friendly to people and the environment, the detection process does not require the use of Ethidium bromide and other toxic agents. Many researchers developed LAMP assays for the detection of many plant parasitic nematodes such as tropical root-knot nematodes (Meloidogyne incognita and M. enterolobii) (Niu et al, 2011;Niu et al. 2012), burrowing nematode (Radopholus similis) (Peng et al. 2012), pine wood or pine wilt nematode (Bursaphelenchus xylophilu) (Kikuchi et al, 2009;Kang et al. 2015;Meng et al. 2018), citrus root nematode (Tylenchulus semipenetrans) (Lin et al. 2016;Song et al. 2017), red ring nematode B. cocophilus (Ide et al. 2017), temperate root-knot nematode M. hapla (Peng et al. 2017), apple root-knot nematode M. mali (Zhou et al. 2017), grass nematode (Anguina wevelli) (Yu et al. 2018), M. chitwoodi, andM. fallax (Zhang andGleason 2019).
In our study, the most e cient ladder like banding pattern was recorded at 60 °C incubation temperature for 60 min. However, Zhang and Gleason (2019) reported LAMP reaction for M. chitwoodi and M. fallax at 68 o C for 45 minutes. The LAMP reaction was performed at 63 °C for 45 min Radopholus similis (Peng et al. 2012). Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots were obtained at 63 °C for 60 min (Niu et al. 2011). For the detection of Anguina wevelli LAMP assay resulted after 45 min at 63°C (Yu et al. 2018).
We found that LAMP assay showed 1000 times more sensitive than the conventional PCR as the LAMP assay produced positive results to the least genomic DNA concentration 10 fg/µl whereas, the conventional PCR was up to 100 pg/µl. Interestingly, Zhang and Gleason (2019) found more sensitivity of LAMP assay while detection of potato nematodes M. chitwoodi and M. fallax as compared to conventional PCR. LAMP assay was found 10-100 times more sensitive as compared to conventional PCR for the detection of R. similis (Peng et al. 2012). During the detection of different RKN species and M. incognita, more or less same sensitivity of LAMP assay was observed by Niu et al. (2012) as compared to conventional PCR. Our results also corroborate with earlier reports of many researchers (Notomi et al. 2000;Kikuchi et al. 2009;Njiru et al. 2010;McKenna et al. 2011).
In the present study, the LAMP primers developed were very speci c to G. pallida and there was no false reaction with the DNA of closely related species G. rostochiensis as well as other nematode species. Niu et al. (2011) also observed no cross reactivity of LAMP assay with DNA of other plant nematodes while detecting familiar Meloidogyne species and M. incognita populations having several geographical origins. Speci city of LAMP primers were also reported by Zhang and Gleason (2019) for the detection of M. chitwoodi and the closely-related species M. fallax.

Conclusion
In conclusion, the primers developed for LAMP assay speci cally detected G. pallida with less assay time as compared to conventional PCR. Our study proposes that the assay developed very rst time targeting mt-COI gene combined with SYBR Gold nucleic acid dye found to be highly reliable to detect G. pallida directly from the infested soil samples in the eld conditions. Therefore, the LAMP assay for the diagnosis of G. pallida has remarkable practical eld application in quarantine areas with respect to export and import of seed potato tubers.   Standardization of LAMP assay for the detection of G. pallida. W: water; L: Ladder (100 bp).

Figure 3
Comparison of sensitivity of LAMP assay with conventional PCR assay. W: water; L: Ladder (100 bp).