Detecting Fusarium oxysporum f. sp. elaeidis by using loop-mediated isothermal amplification

We developed a Loop-Mediated Isothermal Amplification (LAMP) to detect Fusarium oxysporum f. sp. elaeidis (FOE) causal agent of fusarium wilt in oil palm. The assay was designed based on partial sequences of effector protein Secreted In Xylem (SIX8, SIX10 and SIX13) and P450 cytochrome-oxidase. The isolates used were classified as FOE, presumed-FOE and non-FOE/other Fusarium spp. based on previously published pathogenicity assay studies. The LAMP assay detected FOE DNA isolated from root tissues of oil palm seedlings. The SIX8 primers detected all FOE isolates, including other presumed-FOE and non-FOE isolates. The SIX10 and SIX13 could not detect FOE and presumed-FOE isolates. The P450 cytochrome-oxidase primer detected all isolates of FOE, presumed-FOE and non-FOE. Without isolating FOE DNA from soil, the LAMP assay could not detect FOE directly in inoculated soil. Detection time for all primers was below 30 min. Annealing derivative curves were used for assessing the level of specificity for both SIX8 and P450 cytochrome-oxidase, but none of the LAMP primers could distinguish between FOE, presumed-FOE and non-FOE isolates. This assay could be suitable for FOE detection of oil palm in nurseries and serve as an important biosecurity tool to screen oil palm germplasm exchanged between oil palm growing countries.

This has heightened the need for a rapid detection tool that would detect the pathogen in the host and soil to prevent the introduction and spread of the pathogen in potential oil palm plantations.Several attempts have been made in the past to develop real-time Polymerase Chain Reaction (PCR) equipment for detecting plant pathogens in the field (Schaad et al. 2002;Mavrodieva et al. 2004;Tomlinson et al. 2007).
The efforts recently made to study FOE, characterized the pathogen based on Secreted In Xylem (SIX) genes (Adusei-Fosu and Dickinson 2019) and used Amplified Fragmented Length Polymorphism (AFLP) for further characterization of FOE isolates (Adusei-Fosu et al. 2019).Knowledge from these previous studies on FOE was built upon to research into developing Loop Mediated Isothermal Amplification (LAMP) assay.LAMP assay which is a molecular tool has shown to be reliable for the detection of several soilborne plant pathogens over the years (Ayukawa et al. 2016;Carvalhais et al. 2019;Winkworth et al. 2020;Katoh et al. 2021) and drastically reduces the quantity of samples to be transported to the laboratory.LAMP assay has been used to detect important soilborne pathogens including fungi and Fusarium oxysporum f. sp.elaeidis (FOE) is a devasting soilborne fungal pathogen and the causal agent of fusarium wilt disease in the world's highest oil producing crop, the oil palm (Elaeis guineensis Jacq.) (Hansen et al. 2015).The pathogen is well known in oil palm producing countries including Ghana, Democratic Republic of Congo and Cameroon (Prendergast, 1957;Aderungboye 1982;Corley and Tinker, 2003).Due to the soilborne nature of the pathogen, control is extremely challenging and makes it difficult to manage the pathogen once it is introduced in oil palm farms.However, the ability to detect the presence of the pathogen in plantation in the host plant (oil palm) or soil prior to planting in the field would potentially circumvent huge losses.
Bacillus stearothermophilus (Bst) DNA polymerase and 4-6 primers, two of which are 'fold back' primers (Notomi et al. 2000;Nagamine et al. 2001) which form stem-loop motifs with self-priming capability.The primers used are two sets, the internal primers and external primers.Subsequent studies have found the use of additional 'loop primers', which bind to the loop structures and greatly reduce the reaction times (Nagamine et al. 2001), resulting in a total of 6 primers.The 60-65 °C reaction temperature, when combined with six primers, makes LAMP a highly specific reaction able to bind up to eight regions of the target.The high level of specificity results from the requirement for primers to bind up to eight regions of the target sequence.This results in an amplification scheme where the priming sequence is copied with each round of replication and remains tethered to the previous amplicon resulting in a concatenated product of alternating sense / anti-sense repeats of varied length.This results in large amounts of amplicons which can be used for further studies in detection (Bekele et al. 2011).
LAMP is one of the most well-established methods for isothermal amplification of nucleic acids to date.The technique has been used as a molecular tool for the detection of several plant pathogens (Ayukawa et al. 2016;Carvalhais et al. 2019;Fukuta et al. 2004;Nie 2005;Tomlinson et al. 2007;Winkworth et al. 2020) including fungi (Lucas et al. 2010;Niessen and Vorgel, 2010).There are several reports on LAMP for detecting Fusarium spp.(Ayukawa et al. 2016;Carvalhais et al. 2019;Denschlag et al. 2012;Ghosh et al. 2015;Niessen and Vogel 2010;Niessen 2013).Ayukawa et al. (2016) applied LAMP assay for detecting and differentiating F. oxysporum f. sp.lycopersici (Fol) race 1 isolates based on the SIX4 and SIX5 genes using three primer sets.According to Ayukawa et al. (2016), these genes are reported to be present in only race 1 of Fol.Ayukawa et al. (2016) further showed the method could detect Fol race 1 in infected tomato stems without DNA isolation.Niessen and Vogel (2010) also developed LAMP assay to specifically detect F. graminearum (Fg) based on the gaoA gene (galactose oxidase).Amplified DNA was indirectly detected in situ by using calcein fluorescence as a marker without electrophoretic analysis, which is time-consuming.The assay was optimized for rapidness, specificity and sensitivity which detected the presence of less than 2 pg of purified target DNA per reaction in 30 min.Within 132 fungal species tested, exclusively DNA isolated from cultures of Fg (lineages 1-9) resulted in a fluorescent signal after amplification with the LAMP (Niessen and Vogel 2010).Niessen and Vogel (2010) demonstrated the usefulness of the analysis of fungal cultures by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of Fg in total genomic DNA isolated from bulk samples of ground wheat grains.Zhang et al. (2013) used LAMP to quantify genomic DNA of F. oxysporum f. sp.cubense (Foc-TR4) in soil samples collected from South China.Zhang et al. (2013) used RT-LAMP to detect Foc-TR4 and showed the detection limit of the LAMP assay was approximately 0.4 pg / µl plasmid DNA when mixed with extracted soil DNA or 10 3 spores / g of artificially infected soil, and no cross-reaction with other related pathogens occurred.The sensitivity of the LAMP has also been reported (Almasi et al. 2013).Almasi et al. (2013) demonstrated that even though PCR and LAMP assays could successfully detect positive infected samples of tomato, considering the time, safety, cost and simplicity, the latter technique was overall superior.Interestingly, Almasi et al. (2013) further showed LAMP could successfully detect Fol without DNA purification (direct-LAMP).
In addition, the real-time application with the Optigenesystem (Optigene, UK) has several advantages such as easy mobility or portability of the detection device.This makes it portable for field work.Unfortunately, no report has been published on LAMP detection for oil palm FOE.Therefore, the objective of this study was to develop LAMP to detect FOE.
A total of 40 strains were used including eight Fusarium oxysporum isolates (presumed-FOE) collected from symptomatic oil palm mature trees (Elaeis guineensis), four FOE isolates from Ghana BOPP-B5, Norpalm-Ghana (N5), Ghana OPRI-5 and Ivory Coast-F16 isolates confirmed to be pathogenic against oil palm seedlings (Adusei-Fosu and Dickinson 2019) and the remaining non-FOE or other Fusarium spp.The identities of all the isolates used in this research were confirmed via sequencing based on translation elongation factor-1α (EF-1α) and SIX genes (Adusei-Fosu and Dickinson 2019).To design the LAMP primers, mycelia (50-100 mg) from cultured FOE, presumed-FOE and non-FOE were collected from PDA plates using a sterile surgical blade.Tissue disruption of mycelia was carried out using glass beads and homogenizer (FastPrep®) at a speed of 6.5 m/s for 45 s in the presence of liquid nitrogen.DNA extractions of the cultures were then carried out using DNeasy Plant Mini Kit (Qiagen) according to the manufacturer's protocol.PCR of various regions of the template DNA was performed using primer pairs of interest targeting effector proteins (SIX8, SIX10, SIX13) or P450 cytochrome oxidase (Table 1).PCR was carried out in 30 µl volumes consisting of 15 µl of master mix (MangoTaq™ DNA Polymerase), 1 µl (of 10 pmol / ul) each of all primer pairs mentioned in separate reaction mixtures, 12 µl sterile distilled water and 1 µl of template DNA of the isolates of interest.The reaction was performed in a BIO-RAD S1000 Thermal Cycler with the amplification conditions of 95 °C for 2 min for initial denaturation, followed by 35 cycles of denaturation at 95 °C for 2 min, annealing at temperatures suitable for amplification for each primer pair of interest and extension/elongation at 72 °C for 1 min 30 s.The final extension was set at 72 °C for 5 min.PCR products were cleaned using the QIAquick PCR Cleanup kit (Qiagen) following manufacturer's instruction followed gel electrophoresis using 1 kb ladder (Promega).Sequencing reactions were performed by Fisher Scientific or MyGATC.BLAST searches were performed using the GenBank sequence database to confirm the identity of the fungal isolates sequences based on the SIX genes and P450 cytochrome-oxidase used for amplifying the rDNA.The output from BLAST algorithms was used to query any unknown sequences against the database of all the fungal gene regions.These sequences were subsequently used to design LAMP primers.The LAMP primers were used to target FOE in DNA extracted from inoculated/infected roots of oil palm seedlings and inoculated soils.Extraction of DNA was respectively carried out using DNeasy Plant Min Kit (Qiagen) and DNeasy PowerSoil Pro Kit (Qiagen) following manufacturers protocols respectively from roots of oil palm seedlings and soil that were inoculated/infected with FOE.
The LAMP primers (Table 1) were designed from partial sequences based Secreted In Xylem (SIX8, SIX10 and SIX13) gene and P450 cytochrome-oxidase.Fusarium oxysporum f. sp.elaeidis (FOE) detection assay was done by preparing the LAMP primer mix which consisted of 152 µl sterile distilled water with primer concentration of 10 µM each of the F1, B1, F2, B2 primers and 2 µM each of FIP, BIP primers for SIX8, SIX10, SIX13 or P450 cytochromeoxidase (Table 1).Master mix for eight reactions consisted of 23 µl of LAMP primer mix, 46 µl sterile distilled water and 115 µl Optigene master mix (dNTPs, Bst DNA polymerase and MgCl 2 ).Twenty-three (23) µl of the reaction mix was dispensed into each of the eight LAMP tubes and diluted (1:10) 1 µl genomic DNA extracted from roots or soil added to make a final volume of 24 µl.The detection time was set to 30 min for all reactions for each primer set.To further validate the primers ability to amplify DNA, detection time was in some cases set beyond 60 min with Genie® II OptiGene device.Experiments were repeated for multiple times during the study by replicating each biological DNA sample three times per LAMP assay.
The LAMP assays were successfully developed for two sets of genes, those encoding Secreted In Xylem (SIX) and the P450 cytochrome-oxidase.Results to confirm the presence or absence of FOE as well as detection time in genomic DNA of all isolates used for the study are shown in Tables 2 and 3.The amplification and derivative curves generated were also observed to confirm the specificity of the products amplified (example is shown in Fig. 1).Generally, amplification was observed at 65 o C. Secreted In Xylem (SIX8) gene primer could amplify DNA for all FOE, some presumed-FOE and non-FOE isolates (Table 2).SIX10 and SIX13 primers could not detect DNA of FOE and presumed-FOE isolates.Generally, the detection times varied for the two sets of genes (SIX8 or P450 cytochrome-oxidase) that were used (Tables 2 and 3).The time for detection for all the SIX genes was between 4:00 min to 29:15 min.P450  (Williams et al. 2016).The SIX8 primer in this study, could not directly detect FOE (Ghana OPRI-5, Ghana BOPP-5, Ghana Norpalm-5 and Ivory Coast-F16) isolates that were artificially inoculated into soil.Similarly, Fusarium oxysporum f. sp.lycopersici (Fol), LAMP assay could detect Fol based on the 28 S rRNA regions but could not discriminate pathogenic races of Fol (Almasi et al. 2013;Villari et al. 2013).Additional studies have shown that SIX1, SIX6 and SIX8 were amplified from pure cultures of Fusarium oxysporum f. sp.cubense (Foc) race 4 whereas other SIX genes including SIX9, SIX10 and SIX13 could only amplify some vegetative compatibility cytochrome-oxidase primer detected isolates at 6:45 min (Table 3).Both SIX8 and P450 cytochrome-oxidase LAMP primers designed recorded a detection time below 30 min in FOE and presumed-FOE isolates.The LAMP primers were randomly tested directly on sampled roots of oil palm seedlings that showed symptoms of FOE infection to confirm the sensitivity of the assay for on-site detection, and these assays were positive with the SIX8 gene primer set but not consistent.The direct detection of FOE from inoculated soil also showed inconsistent results hence both sets of results not shown.In order to determine the sensitivity of LAMP assay, a 10-fold serial dilution (ranging from 10 − 1 to 10 − 4 of pathogen DNA) was done for SIX8 primers where detection limit was at 10 − 4 (Table 4).
In this study, the LAMP method successfully detected the presence of FOE DNA isolated from root tissues of oil palm seedlings.The LAMP primers designed from effector protein, SIX8 detected FOE whereas SIX10 and SIX13 could not.The P450 cytochrome-oxidase primers detected FOE isolates.There are several reports on Loop-Mediated Isothermal Amplification (LAMP) assays for detecting several formae speciales (f.spp.) for Fusarium (Abd-Elsalam P450 cytochrome-oxidase has been associated with pathogenicity in some fusarium such as the Foc (Sutherland et al. 2013).In this study, P450 cytochrome-oxidase was detected in FOE, presumed-FOE and non-FOE isolates.It is reported that the P450 cytochrome-oxidase are distributed widely in many organisms (Nazmul et al. 2010).However, the level of P450 cytochrome-oxidase differences such as the copy numbers or gene families, significantly varies biologically across kingdoms, phyla and species (Nazmul et al. 2010).Furthermore, Nazir et al. (2010) have shown that P450 cytochrome-oxidase share conserved overall protein architecture and have many conserved sequences, despite the higher level of diversity in the P450 cytochrome-oxidase.groups (VCGs) of Foc (Carvalhais et al. 2019).The LAMP method in this study detected FOE faster with SIX8 primers, but detection time varied among all detected FOE isolates.In similar studies, LAMP primers based on other SIX genes were used to characterize Foc race 1 (Ayukawa et al. 2016).The SIX8 primer in this study, could not directly detect FOE (Ghana OPRI-5, Ghana BOPP-5, Ghana Norpalm-5 and Ivory Coast-F16) isolates that were artificially inoculated into soil.On the contrary, Ayukawa et al. (2016) could directly detect Fol race 1 in soil artificially inoculated based on primers designed for SIX4 and SIX5.Similarly, Peng et al. (2014) reported LAMP as an effective tool for detecting Foc race 4 isolate in soils.

References
Abd-Elsalam K, Bahkali A, Moslem M, Amin OE, Niessen L (2011) An optimized protocol for DNA extraction from Wheat Seeds These characteristics of the P450 cytochrome-oxidase may have contributed to its detection in FOE, presumed-FOE and non-FOE as well as the varying time of detection.Generally, LAMP primers developed in this study, either the P450 cytochrome-oxidase or SIX8 represents an extremely rapid diagnostic tool for FOE.Currently, the method has the potential to be used to control or prevent the introduction and spread of FOE into oil palm nurseries and plantations.Although the LAMP primers in this could detect other fusarium pathogens, researchers need to bear in mind that FOE is host specific to only oil palm, hence this tool is still relevant for rapid screening for potential FOE infection in oil palm seedlings prior to planting in the field.The assay is a promising tool to be used for detection and screening tool for the oil palm industry.This study has produced a LAMP assay considered as a biosecurity tool to rapidly screen for FOE in oil palm seedlings or germplasm exchanged between oil palm producing countries.

Fig. 1
Fig. 1 (a) Amplification curve and (b) Annealing curve of seven isolates of FOE from DR Congo, Ivory Coast, Suriname and Ghana showing specific time for detection with LAMP primer P450 cytochrome-oxidase

Table 1
List of LAMP primers designed from this study (Villari et al. 2013;Winkworth et al. 2020).2015;Katoh et al. 2021) and other plant pathogens(Villari et al. 2013;Winkworth et al. 2020).However, this is the first time LAMP method based on SIX genes and cytochrome oxidase have been used to detect FOE isolates.The SIX8 primers detected FOE, presumed-FOE and non-FOE.The positive detection or presence or absence of some of the SIX genes in FOE, presumed-FOE and non-FOE in this study is congruent with a study that showed that only fourteen SIX (1-14) genes have been identified and most share similarities with each other or with other fungi

Table 2
LAMP results for detected SIX8 gene in Fusarium oxysporum isolates collected from symptomatic oil palm and selected other Fusarium spp Pathogenicity Not tested (i.e.pathogenicity not confirmed in oil palm, although isolate was collected from symptomatic oil palm trees.This refers to presumed-FOE) NH = Not oil palm host (i.e.non-FOE) International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.
NT = Pathogenicity Not tested (i.e.pathogenicity not confirmed in oil palm, although isolate was collected from symptomatic oil palm trees.This refers to presumed-FOE) NH = Not oil palm host (i.e.