LAMP Assay for Distinguishing Fusarium oxysporum and Fusarium commune in Lotus (Nelumbo nucifera) Rhizomes
- Sheng Deng1
- Xin Ma1 2
- Yifan Chen1 3
- Hui Feng1
- Dongmei Zhou1
- Xiaoyu Wang1
- Yong Zhang4
- Min Zhao1
- Jinfeng Zhang1
- Paul Daly1 †
- Lihui Wei1 2 3 †
- 1Institute of Plant Protection, Key Lab of Food Quality and Safety of Jiangsu Province-State, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, P.R. China
- 2Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, P.R. China
- 3School of Environmental and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu Province, P.R. China
- 4Bioinformatics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Yields of edible rhizome from cultivation of the perennial hydrophyte lotus (Nelumbo nucifera) can be severely reduced by rhizome rot disease caused by Fusarium species. There is a lack of rapid field-applicable methods for detection of these pathogens on lotus plants displaying symptoms of rhizome rot. Fusarium commune (91%) and Fusarium oxysporum (9%) were identified at different frequencies from lotus samples showing symptoms of rhizome rot. Because these two species can cause different severity of disease and their morphology is similar, molecular diagnostic-based methods to detect these two species were developed. Based on the comparison of the mitochondrial genome of the two species, three specific DNA loci targets were found. The designed primer sets for conventional PCR, quantitative PCR, and loop-mediated isothermal amplification (LAMP) precisely distinguished the above two species when isolated from lotus and other plants. The LAMP detection limits were 10 pg/μl and 1 pg/μl of total DNA for F. commune and F. oxysporum, respectively. We also carried out field-mimicked experiments on lotus seedlings and rhizomes (including inoculated samples and field-diseased samples), and the results indicated that the LAMP primer sets and the supporting portable methods are suitable for rapid diagnosis of the lotus disease in the field. The LAMP-based detection method will aid in the rapid identification of whether F. oxysporum or F. commune is infecting lotus plants with symptoms of rhizome rot and can facilitate efficient pesticide use and prevent disease spread through vegetative propagation of Fusarium-infected lotus rhizomes.
Lotus (Nelumbo nucifera) is a popular aquatic vegetable in Asian countries because of its special flavor and nutritional value. In China, as of 2016, the planting area of lotus was approximately 400,000 ha, and production reached 12 million tons (Wu et al. 2019). Lotus is mainly planted in the middle and lower reaches of the Yangtze River and the lower reaches of the Yellow River in China (Guo 2009). Lotus is also considered to have potential as a neglected and underutilized species that can contribute to the United Nations sustainable development goals for eradicating hunger and malnutrition (Li and Siddique 2018; Singh et al. 2019).
Lotus rhizome rot disease is one of the most destructive diseases affecting lotus cultivation, with yield losses from 10 to 80% (Yuan 2013; Zhou et al. 2015). In China, from July to August of each year, the leaves of diseased lotus often show wilt symptoms, and discoloration is usually seen at the base of the petioles and in rhizomes. In 1953, the disease was first reported to be caused by Fusarium oxysporum Schl. f. sp. nelumbicola (Nis. & Wat.) Booth (formerly known as F. bulbigenum WR. Nelumbicolum) (Edel-Hermann and Lecomte 2019; Nisikado and Watanabe 1953). To date, it has been found that other pathogens can infect lotus and cause similar symptoms, such as F. incarnatum (Wang et al. 2020), F. tricinctum (Li et al. 2016), and F. commune (Zeng et al. 2017) as well as the oomycete Phytopythium helicoides (Yin et al. 2015).
The cultivation of lotus is initiated primarily through rhizome vegetative propagation. The pathogen-infected rhizomes usually become potential primary infection sources that cause extensive spread of diseases to a whole new field. To avoid the spread of pathogens and reduce the risk of infection, accurate and portable detection methods are required to screen out the infected and diseased propagules before transplanting. If local farmers and agricultural technicians can diagnose the disease and identify the pathogens rapidly in the field, they can immediately start effective mitigation strategies, such as removing diseased plants or using fungicides.
DNA-based molecular diagnostic technologies are in continuous development, such as PCR, real-time PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA), which are often applied in detection and identification of phytopathogens (Kong et al. 2020; McCoy et al. 2020). The principle of these methods is to amplify the specific nucleic acid sequences of target pathogens. Among them, LAMP is rapidly becoming an important and popular diagnostic tool, especially for point-of-care applications, because it is rapid, accurate, cost effective, and convenient (Li et al. 2019; Notomi et al. 2000). In the last 10 years, there have been many reports on the detection of Fusarium species through LAMP. For example, a LAMP assay was developed to recognize F. circinatum, a causal agent of pitch canker of conifers, in 30 min in field conditions using a portable instrument (Stehliková et al. 2020). Based on the gaoA gene (Galactose oxidase) of F. graminearum, the LAMP method was demonstrated to be useful in fungal culture identification, direct testing of infected barley grains, and detection of the pathogen in total DNA isolated from bulk samples of ground wheat grains (Niessen and Vogel 2010).
Mitochondrial genes such as cytochrome c oxidase subunit I (CoxI) and the small-subunit ribosomal DNA (mtSSU rDNA) are routinely used for species identification (Fišer Pečnikar and Buzan 2014; Gao and Zhang 2013; Mbofung et al. 2007). The use of mitochondrial DNA can offer several advantages over nuclear DNA in identifying and distinguishing one species from another. The much smaller size of nuclear genomes makes it more cost effective to sequence mitochondrial genomes to identify the regions of the mitochondrial genomes that can discriminate one species from another (Brankovics et al. 2017; Timmermans et al. 2010). Compared with genomic DNA, mitochondrial DNA has more copies in a single cell (Williamson 2002), which leads to a greater sensitivity in detection of genes from the mitochondrial genome compared with the nuclear genome.
To date, there are few reports on the detection methods for lotus rhizome pathogens. In lotus root and soil samples, Kurashita et al. (2021) detected Hirschmanniella diversa and H. imamuri, the two plant parasitic nematodes that cause blackening and deformation disease on lotus rhizomes, by internal transcribed spacer (ITS) region-targeted quantitative PCR (qPCR). In our present study, based on sequence analysis of mitochondrial genomic DNA of several F. oxysporum and F. commune strains, we found three species-distinguishing sites, including two F. commune-specific sites and one F. oxysporum-specific site. To distinguish F. oxysporum and F. commune, three sets of PCR primers, two sets of real-time PCR primers, and two sets of LAMP primers were developed. To ensure that our detection method can be applied in field conditions, we mimicked the whole detection process on lotus seedlings and rhizomes infected by the target pathogens. The results indicated that our designed primers and detection methods are capable of identifying the target pathogens in diseased samples under field-mimicked conditions.
Materials and Methods
Source of isolates for primer set evaluation.
A total of 22 isolates, including 13 F. commune and nine F. oxysporum isolates, were used for primer sets specificity evaluation (no. 1 to no. 22 in Table 1). All tested isolates were single-spore purified. Among them, 16 isolates were obtained from diseased lotus. The selected isolates were representatives obtained from different varieties of lotus in different main lotus-growing areas in the middle and lower reaches of the Yangtze River. Additionally, the specificity of primer sets was evaluated in another nine pathogen species (no. 23 to no. 31 in Table 1), and the results are shown in Supplementary Figures S1 and S2.
Pathogen isolation from diseased lotus or other plant samples.
Approximately 0.5 cm3 of diseased tissues were cut from the junction of the diseased and healthy part of each diseased lotus rhizome or from the basal petiole of diseased lotus. The tissues were rinsed three times in tap water to remove any soil on the surface, soaked in a 70% ethanol solution for 1 min, and then rinsed with sterilized water. After that, the tissues were soaked in 3% sodium hypochlorite solution for 5 min, during which time the sample containers were vibrated irregularly. The treated tissue was transferred to a sterile culture dish and washed five times with sterile water to remove the residual sodium hypochlorite. Finally, tissues were carefully transferred to potato dextrose agar (PDA) plates containing chloramphenicol (50 μg/ml) and cultured at 28°C. Two to 3 days later, the mycelia growing out from the tissues were subcultured and used for single spore purification. Then, the isolates were subcultured and identified by the sequences from ITS, EF1α, and mtSSU PCR fragments. After identification, the isolates were tested by Koch’s postulates as follows. The conidial suspension of each isolate was collected from 4-day-old potato dextrose broth (PDB) cultures, and the conidial concentration of the suspension was adjusted to 1 × 106 conidia/ml. Three days after budding in water, five lotus seedlings were soaked in the conidial suspension for 60 min in each assay. The lotus seedlings were planted in a water-covered mixture of vermiculite and sterile nutrient soil (vol/vol = 5:1) in greenhouse. Sixty days after inoculation, the disease symptoms were examined. The pathogen was reisolated from the infected tissues and the identity was confirmed by mtSSU and ITS PCR fragments sequencing. Other pathogens used in this study were isolated from diseased plant samples with similar methods, and Koch’s postulates were tested on the respective hosts of these pathogens.
Culture condition of the tested isolates and plants.
All tested isolates were maintained on PDA plates for a short term at 28°C, and the conidia of these isolates were stored in 25% glycerol (∼1 × 107 conidia/ml) at −70°C for long-term storage. Oomycetes strains were stored at 12°C. The plants for the detection assay were grown in a greenhouse or in the field.
Library preparation and whole genome sequencing.
DNA was extracted from the fresh mycelia (4-day-old PDB cultures) of the F. oxysporum Fo-Yangzhou-lotus-1 and the F. commune Fc-Guangchang-lotus-2-2 isolates by a cetyltrimethylammonium bromide (CTAB) method (O’Donnell et al. 1997). Then the DNA samples were sent to BGI Company for whole-genome sequencing. One microgram of total DNA was randomly fragmented by Covaris E220 (Covaris, Brighton, United Kingdom), followed by fragment selection by AMPure XP beads (Agencourt) to an average size of 200 to 400 bp. Selected fragments were end-repaired and 3′adenylated, and then the adaptors were ligated to the ends of these 3′adenylated fragments. The products were amplified by PCR and purified by the AMPure XP beads again. The purified double-stranded PCR products were heat denatured to a single strand and then circularized by the splint oligo sequence. The single-strand circle DNA (ssCir DNA) was formatted as the final library and qualified. The final qualified libraries were sequenced on the BGISEQ-500 platform. The ssCir DNA molecule formed a DNA nanoball (DNB) containing >300 copies through rolling-cycle replication. The DNBs were loaded into the patterned nanoarray by using high-density DNA nanochip technology. Finally, 3-Gbp paired-end reads of 150-bp length were obtained by combinatorial Probe Anchor Synthesis–based sequencing.
Assembly of mitochondrial genomes.
The mitochondrial genome sequences of the F. oxysporum Fo-Yangzhou-lotus-1 and the F. commune Fc-Guangchang-lotus-2-2 isolates were assembled by the NOVOPlasty assembler using our sequencing data of total DNA from the indicated strain (Table 2). The mitochondrial genome sequences of the Fo_cubense_B2_China_Hainan were assembled by the GRAbB based on the released sequence data (SRX181886) from NCBI (Table 2). The processes of the assemblies followed the methods described by Dierckxsens et al. (2017) and Brankovics et al. (2016), respectively. The sequences of the assembled mitochondrial genomes were deposited at the National Genomics Data Center (NGDC) of China with the accession numbers GWHBAAV01000000 (Fo-Yangzhou-lotus-1) and GWHBAAU01000000 (Fc-Guangchang-lotus-2-2).
Alignment of the tested mitochondrial genome.
The 12 mitochondrial genome sequences (Table 2) were aligned by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) with the default setting. After alignment, the outputted ClustalW formatted file was converted into a “fasta” formatted file by ClustalX1.83 and then analyzed by MEGA7 (Kumar et al. 2016). The sequences with numerous variations at both ends were removed, and a relatively conserved region was obtained for further analysis. The selected region of the sequence alignment was saved as the “fas” format file and the file was displayed by Bioedit (version 184.108.40.206).
Synteny of the mitochondrial genomes of Fo-Yangzhou-lotus-1 and Fc-Guangchang-lotus-2-2.
The two mitochondrial genomes were annotated by MITOS2 (https://usegalaxy.eu/) and compared by BLAST 2.2.28+. Both were run with default parameters, except the word size in BLAST was 20. The obtained gff file and BLAST result were transformed and inputted into TBtools (Chen et al. 2020) for producing the Circos circular visualization according to the instruction of TBtools.
Primer design for PCR, qPCR, and LAMP assays.
Based on the DNA sequence within the specific loci region, the primers for PCR and real-time PCR (qPCR) were designed by an online primer design software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Ye et al. 2012). For LAMP, the primer sets were generated by PrimerExplorer V5 (https://primerexplorer.jp/e/) (Feng et al. 2019). All primers used in the present study are shown in Table 3. Temperature optimization of the LAMP primer sets is shown in Supplementary Figure S3. Before performing wet lab bench experiments, the specificity of these primer sets was evaluated by primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) with the database of nucleotide collection (nr/nt) to indicate any potential off-target amplification.
Preparation of samples for primer specificity tests.
The 22 isolates (No. 1 to No. 22 in Table 1) were cultured in PDB for 4 days. The mycelia were harvested by centrifugation and then broken by steel beads with a ball mill (TL2010S, DHS Life Science, China) (nine cycles at 1,500 RPM for 30 s with a 15-s pause in between each cycle). The CTAB method (O’Donnell et al. 1997) was used for the DNA extraction with minor modifications. The obtained DNA samples were treated by RNAse A (0.5 μg/ml) at 37°C for 2 h before use. A similar protocol was performed for the isolation of total DNA from other species used to test the off-target amplification of the primers.
Preparation of samples for LAMP sensitivity test.
The mycelia of Fc-Guangchang-lotus-2-2 (F. commune) and Fo-Yangzhou-lotus-1 (F. oxysporum) were collected from the 4-day-old PDB cultures. The mycelial mats were then ground into fine powder in liquid nitrogen, and the corresponding total DNA was isolated following the CTAB method (O’Donnell et al. 1997). The DNA samples were treated by RNAse A (0.5 μg/ml) at 37°C for 2 h. For the sensitivity evaluation of the LAMP assays, the total DNA extracted was diluted using sterile, double-distilled water to the following concentrations: 200 ng/μl, 10 ng/μl, 1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, and 10 fg/μl. The original concentration of total DNA was assessed by spectrophotometry using a NanoDrop 2000 (Thermo Scientific). One microliter of each diluted DNA sample was added into the LAMP reaction tube as the template. All tests were repeated at least three times with identical results.
For the sensitivity test of different concentrations of conidia, the appropriate amount of conidia was added to double-distilled water to make a series of five 10-fold conidial dilutions from 1 × 105 to 10 conidia/ml. Then 100 μl of the corresponding conidia suspensions was subject to a freeze-thaw treatment using a freezer (−20 or −80°C) or dry ice until frozen (∼10 min), followed by thawing at room temperature for 5 to 10 min. During thawing, the tubes were tapped several times. Once the samples were completely thawed, the freeze-thaw treatment was repeated once. Subsequently, samples were put on a thermocycler at 98°C for 10 min, after which they were ready for PCR or LAMP detection. From the samples, 0.2 μl of supernatant was loaded into a reaction tube as the template. It should be noted that because DNA is not protected by EDTA, the samples must be stored at 4°C and used within a short period of time.
Preparation of samples from infected lotus seedlings or diseased/healthy lotus rhizomes for pathogen identification.
Diseased lotus seedlings were collected from Shuangxi, Jinhua, Zhejiang Province (latitude: 29.304079; longitude: 119.994941). Other diseased tissues were obtained from seedlings/rhizomes inoculated in laboratory. The healthy lotus were collected from: Honghu, Jingzhou City, Hubei Province; Cao County, Heze City, Shandong Province; Qianjiang City, Hubei Province; Dagu Village, Hengshan Town, Anju District, Suining City, Sichuan Province; Chaotian Village, Hengshan Town, Anju District, Suining City, Sichuan Province; Shuangqiaozi Village, Hengshan Town, Anju District, Suining City, Sichuan Province; and Guanyintang Village, Hengshan Town, Anju District, Suining City, Sichuan Province.
Fifty milligrams of tissue from a petiole section or rhizome (fresh or kept at −20°C) was broken by steel beads with a ball mill (six cycles at 1,500 RPM for 30 s with a 15-s pause in between each cycle) in a 2.0-ml tube or by the portable tissue grinder (OSE-Y30, TIANGEN Biotech, China) in a 1.5-ml tube. After that, 200 μl of extraction buffer (20 mM of Tris [pH 8.0], 25 mM of NaCl, 2.5 mM of EDTA, and 0.05% SDS) was added and immediately mixed with the broken tissue. After being frozen and thawed three times using the same freeze-thaw procedure described in the “Preparation of samples for LAMP sensitivity test” section, 20 μl of the supernatant was taken for DNA purification by AMPure XP magnetic beads (A63880, Beckman Coulter, Indianapolis, IN) according to the instructions. One microliter of the purified DNA solution was loaded into each reaction tube for LAMP or PCR assay.
Preparation of LAMP reaction system.
The LAMP detection kit was bought from Vazyme Biotech (Bst DNA Polymerase Large Fragment, P701). The reaction mixture was prepared following a published paper (Kong et al. 2020) with some modifications. The LAMP reaction was performed in a 25-μl reaction volume, including 2.5 μl of 10 × ThermoPol buffer (200 mM of Tris-HCl pH 8.8, 100 mM of KCl, 100 mM of (NH4)2SO4, 20 mM of MgSO4, and 1% Triton X-100), 1.6 µM of forward inner primer, 1.6 µM of reverse inner primer, 0.2 µM of forward outer primer, 0.2 µM of reverse outer primer, 0.8 µM of loop forward primer, 0.8 µM of loop reverse primer, 0.8 M of betaine, 1.4 mM of dNTPs, 6 mM of MgSO4, 180 µM of hydroxynaphthol blue (HNB), 8 U of Bst DNA polymerase large fragment, and 1 μl of target DNA. The mixture was incubated at 62°C (for F. oxysporum test) or 66°C (for F. commune test) for 90 min in a water bath or a thermocycler. Positive controls (total DNA from Fo-Yangzhou-lotus-1 for the F. oxysporum assay and from Fc-Guangchang-lotus-2-2 for the F. commune assay) and negative controls were included in each run. The initial reaction solution color was purple and transparent. After the 90-min reaction at 62°C (for F. oxysporum) or at 66°C (for F. commune), the solution color in the positive reaction tube changed from a transparent purple to a turbid sky blue.
Assays by conventional PCR and qPCR.
In accordance with the manufacturer’s instructions, real-time PCR (qPCR) was performed in a 20-μl reaction mixture containing 1 μl of template, 0.5 μl (each) of forward and reverse primers, 8 μl of double-distilled water, and 10 μl of SYBR Premix Ex Taq mix (RR420L, Takara, China). The qPCR was performed in a LightCycler 96 thermal cycler (Roche). The analysis of qPCR for each sample was repeated at least twice. All Cq value data were input into Graphpad Prism 8 to calculate the mean Cq value and draw the bar graphs. The qPCR method was set up as follows: an initial denaturation step at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 58°C for 15 s, and 72°C for 30 s. Afterward, the melting curve analysis was performed to confirm that the primers amplified only a single product. The housekeeping gene β-tubulin served as a reference gene, and the relative levels of the target mitochondrial DNA fragments were calculated by Graphpad Prism 8. Conventional PCR cycling parameters were as follows: 95°C for 3 min, 35 cycles of 95°C for 20 s, 58°C for 20 s, and 72°C for 30 s, followed by final extension of 72°C for 2 min.
Lotus seedlings inoculation.
The mature lotus seeds were bathed in 60°C water for 10 min to remove potential pathogens from the seed epidermis and promote seed coat rupture and germination. After that, the seeds were soaked in distilled water for 4 to 5 days at 25°C until most of the seeds germinated, with at least one replacement of the water used for the soaking. The lotus seedlings were then soaked in conidial suspension (1 × 106 conidia/ml) obtained from 4-day-old PDB cultures of each test strain for 60 min. In each assay, at least 15 seedlings were inoculated with the conidial suspension. All the seedlings were then planted on a water-covered mixture of vermiculite and sterile nutrient soil (vol/vol = 5/1) in a growth chamber at 25°C under a 12 h (light)/12 h (dark) photoperiod. All inoculations on lotus seedlings were performed according to this method. Sixty days after inoculation, the symptoms were examined and photographed.
Lotus internode inoculation.
Hyphal disks of each indicated isolate were put on the longitudinal section of the air passages of the lotus rhizome. The inoculated lotus rhizomes were kept in a condition with high humidity at room temperature. Seven days later, the disease symptoms of the rhizomes were photographed.
Both Fo-Yangzhou-lotus-1 and Fc-Guangchang-lotus-2-2 infect lotus seedlings.
During 2017 to 2019, we recovered 34 isolates from infected lotus samples displaying typical rhizome rot symptoms. Identification of the isolates was attempted using ITS, elongation factor 1α (EF-1α), and mtSSU sequences. From BLAST analysis at NCBI, the ITS region indicated that the species of all the isolates was F. oxysporum. However, the sequences from EF-1α and mtSSU were able to distinguish F. oxysporum and F. commune isolates. The detection rate of F. commune was high (91%), whereas the rate of detection of F. oxysporum was only 9%. Sixteen of the 34 isolates were used in this study (Table 1). The primary aim of this study was to develop a detection method that can assist agricultural technicians in diagnosing the disease and distinguishing these two Fusarium spp.
Lotus seedlings were inoculated by the two representative isolates from lotus, Fo-Yangzhou-lotus-1 (F. oxysporum) and Fc-Guangchang-lotus-2-2 (F. commune), and two other Fusarium wilt pathogens isolated from watermelon (watermelon-Fo-GY16, F. oxysporum) and banana (Banana TR-4, F. oxysporum). Compared with the seedlings treated by the two nonhost pathogens and water (mocks), most of the leaves of the Fo-Yangzhou-lotus-1 and Fc-Guangchang-lotus-2-2 treated seedlings were dead or showed severe wilt symptom. Also, their root development was inhibited by infection with the two pathogens (Fig. 1A). These results indicated that both Fo-Yangzhou-lotus-1 and Fc-Guangchang-lotus-2-2 can infect lotus seedlings and cause similar disease symptoms in pot tests.
The morphology of the above two strains was similar, and both produced pigment in the middle of the colony (Fig. 1B). Except for sporodochial conidia, the microconidia from the two species appeared almost the same (Fig. 1B). It is difficult to distinguish them quickly and accurately because it requires time-consuming purification-by-culturing techniques and expert knowledge of fungal morphology. Therefore, it is necessary to develop molecular detection methods for the identification of the two species.
Identification of a species-distinguishing target loci in the mitochondrial genomes in the tested strains.
The mitochondrial genomes of the two representative isolates, Fo-Yangzhou-lotus-1and Fc-Guangchang-lotus-2-2, were obtained by assembling the short-read sequencing data of the total DNA (Table 2). The size of these mitochondrial assemblies is 47,259 bp for Fo-Yangzhou-lotus-1 and 47,525 bp for Fc-Guangchang-lotus-2-2. Ten other mitochondrial genome sequences of representative F. oxysporum or F. commune strains were downloaded from the NCBI website or assembled as part of our study (Table 2).
In the comparison of the sequences, two unique sites were found in the mitochondrial genome of F. commune (named Fc1 and Fc2) and one site in F. oxysporum (named Fo3) (Fig. 2). The sequences and related information of the three specific loci (Fc1, Fc2, and Fo3) have been listed in Supplementary Table S1.
The above three specific sequences were blasted using BLASTN against the database of nucleotide collection (nr/nt) at NCBI with default settings to evaluate their specificity. The Fo3 site was completely conserved in most of F. oxysporum isolates as well as in F. foetens and F. odoratissimum. It is highly conserved in F. odoratissimum, F. acutatum, F. denticulatum, F. nygamai, F. pseudoanthophilum, F. brevicatenulatum, F. ficicrescens, F. lactis, F. napiforme, and F. anthophilum (Supplementary Table S2). For the Fc-1 and Fc-2 sites, the exact same sequences were found in F. commune and F. oxysporum f. sp. lactucae (Supplementary Table S2) but not in other species. For the Fc-1 site, a partial sequence match was found in F. asiaticum, F. graminearum, F. pseudograminearum, F. culmorum, F. cerealis, and F. gerlachii (Supplementary Table S2). Most of F. oxysporum plant pathogens have restricted host ranges, and to date, F. oxysporum f. sp. lactucae was reported to infect Asteraceae plants (Edel-Hermann and Lecomte 2019). Therefore, the above three specific sequences are suitable for distinguishing between F. commune and F. oxysporum in diseased lotus rhizomes.
There was a high degree of synteny between Fo-Yangzhou-lotus-1 and Fc-Guangchang-lotus-2-2 mitochondrial genomes (Fig. 2B). In particular, the gene-containing regions, tRNA and rRNA synthetic regions are highly syntenic. The Fc-1, Fc-2, and Fo3 sites are located in the intergenic regions of each mitochondrial genome (Fig. 2B).
Primer specificity tests.
Based on these three sequences and their flanking sequences in mitochondrial DNA, primer sets for detection by PCR, qPCR, and LAMP were designed and are listed in Table 3. For all primer sets, the specificity testing function in primer-BLAST was used with the database of nucleotide collection (nr/nt) to indicate any potential off-target amplification (Supplementary Table S3). DNA from nine F. oxysporum (isolated from ginger, tomato, watermelon, banana, cabbage, and lotus) and 13 F. commune (isolated from lotus) were used for evaluating the specificity of those primer sets for distinguishing between F. oxysporum and F. commune (no. 1 to no. 22 in Table 1).
The three detection methods achieved consistent results, which were able to detect the corresponding target species accurately (Fig. 3). For PCR, F. commune isolates can be accurately identified by amplifying either the Fc1 or Fc2 locus, and F. oxysporum isolates were distinguished by amplifying the Fo3 locus (Fig. 3A and B). For the LAMP assay, the positive reactions resulted in a color change from violet to blue in the presence of the HNB indicator, demonstrating detection of these strains. All LAMP assays obtained similar detection results (Fig. 3A and B). For the qPCR-based detection, each DNA sample was tested by both specific primer sets targeting the Fc1 and Fo3 loci. In accordance with the results obtained from conventional PCR and LAMP, all primer sets were able to detect the corresponding strains (Fig. 3C). Moreover, the relative template copy number of mitochondrial genome was ≥10 times that of the reference gene β-tubulin from the nuclear genome (Fig. 3C). The results indicated that using the mitochondrial genome as the detection target can substantially improve the detection sensitivity by at least one order of magnitude.
For further assessing the specificity of the above primer sets, nine other pathogens in our laboratory collection, Pythium sylvaticum, Phytopythium helicoides, Phytopythium myriotylun, F. solani, Botrytis cinerea, Alternaria tenuissima, Bipolaris oryzae, Verticillium dahliae, and F. graminearum, were evaluated with the F. commune/F. oxysporum-specific PCR and LAMP tests (Supplementary Fig. S1). All primer sets targeting F. oxysporum obtained identical results, in which only F. oxysporum isolates and the positive control can be detected. For F. commune, the LAMP assay failed to distinguish between F. commune and F. graminearum. However, the conventional PCR primer sets targeting Fc1 and Fc2 loci were able to identify F. commune specifically (Supplementary Fig. S1). The above data indicated that the primer sets targeting the Fc1, Fc2, and Fo3 loci can be used to identify F. commune and F. oxysporum.
Sensitivity test of the LAMP assay.
The LAMP detection method was designed for applications that are performed outside the laboratory environment, such as in field-based assays. Therefore, the sensitivity of the method should be evaluated. Total DNA extracted from Fo-Yangzhou-lotus-1 (F. oxysporum) and Fc-Guangchang-lotus-2-2 (F. commune) were prepared into a series of 10-fold dilution for the following LAMP sensitivity tests. After the assays, the color of the reaction tube was observed, and the product formation in those reactions was visualized by agarose gel electrophoresis. The detection limits were 10 pg/μl and 1 pg/μl for F. commune and F. oxysporum, respectively (Fig. 4A and B).
In soil, lake water, or plant tissues, most of the pathogen targets we detected were in the form of cells (mycelia or conidia). Thus, we further tested the sensitivity of the LAMP at different cell (conidial) concentrations. The results indicated that for F. commune and F. oxysporum, both detection limits were 1 × 104 conidia/ml (Fig. 4).
Application of LAMP detection methods.
To confirm that LAMP primer sets and the supporting methods are suitable for rapid diagnosis of the lotus disease in the field, we carried out field-mimicked experiments on lotus seedlings (Fig. 5) and rhizomes (Fig. 6) that had been inoculated with the above pathogens in the laboratory or infected in the field. As shown in Figure 5A, compared with the control, growth of plants inoculated with the pathogen was significantly inhibited. Moreover, the leaves had the appearance of early symptoms, such as discoloration or water-soaking spots (Fig. 5B). The DNA samples that were derived from the different sections of the petioles were tested by the LAMP assay. As expected, different primer sets can be used to distinguish diseased tissues infected by the corresponding pathogens (Fig. 5C).
Similarly, in the lotus rhizome tissues collected from the inoculated area, the target pathogens (Fo-Yangzhou-lotus-1, Cabbage-Fo-1, and Ginger-Fo-7) were identified accurately by the Fo3-LAMP primer set (Fig. 6A and B). Although the two strains, Cabbage-Fo-1 and Ginger-Fo-7, cannot infect lotus rhizome, they still were detected because the tissues contained a small amount of mycelium of the strains (Fig. 6A and B). Lotus rhizomes are rich in minerals, proteins, and polysaccharides, which are strong inhibitors of nucleic acid amplification. To ensure that the LAMP assay performed properly, we set up a spiked positive control (healthy tissue mixed with DNA of the target species) and a second negative control consisting of healthy, noninfected tissue. Both controls showed the expected results.
Furthermore, in addition to the field-mimicked conditions, actual field samples were tested. Diseased rhizomes with typical symptoms were collected from a lotus-planted field in Shuangxi, Jinhua, Zhejiang Province on 30 October 2020 (Fig. 6C). The target pathogen, F. commune, was detected by our LAMP assays in diseased rhizome samples (Fig. 6D). Three diseased tissues (∼50 mg of each) were cut from the region with symptoms, two of which indicated the presence of F. commune in the diseased samples (Fig. 6D). Later, more tissues, including the corresponding samples, were used for pathogen isolation. In those samples, the expected F. commune was isolated but not F. oxysporum, which was not detected by the LAMP assay (Supplementary Fig. S2).
Planting of lotus is mainly through vegetative propagation. Because of the aquatic planting, lotus is usually cropped continuously in the same field year after year. Because lotus are usually planted in a large area of water and their rhizomes and roots grow deep into the mud, it is difficult to carry out chemical control after a soilborne disease outbreak. Moreover, pathogens tend to spread to other waters or downstream areas along with the water flow. Therefore, to avoid pathogen spread and a lotus rhizome rot epidemic, quarantine measures are indispensable, such as removing spotted or rotten lotus rhizomes and pretreating lotus rhizomes by soaking in carbendazim and other fungicides before planting. In this study, the detection method we reported will provide another alternative strategy for early detection and control of lotus rhizome rot.
In the present study, based on comparison of the mitochondrial genome, three specific DNA loci, Fc-1, Fc-2, and Fo3, were found in F. oxysporum or F. commune. The three loci are located in the intergenic regions of each mitochondrial genome (Fig. 2B). Evidence suggested that these sites are suitable for distinguishing the Fusarium pathogens because they are more variable, probably because they are under less purifying selective pressure than the gene-containing regions. For conventional PCR, qPCR, and LAMP assays, the primer sets targeting these loci were developed to specifically detect F. oxysporum and F. commune, two important pathogens causing lotus rhizome rot. The obvious and reproducible color change in the field-applicable LAMP reaction indicated the presence of the target pathogen when species-specific primers were used. In the LAMP assay, the detection limits were 10 pg/μl and 1 pg/μl of total genome DNA for F. commune and F. oxysporum, respectively. LAMP assay can detect at least two conidia of the target strain in 25 μl of a reaction volume within 90 min under the indicated pretreatment conditions. Moreover, the LAMP detection method can detect pathogens in infected lotus seedlings and rhizomes with portable and easy-to-use equipment for on-site diagnostics. The outline of the detection process and equipment for a portable outdoor laboratory for LAMP detection are shown in Figure 7. Except for gel electrophoresis, all steps for LAMP detection can be completed in field conditions. A mini thermocycler, such as Franklin Real-Time PCR (∼1.2 kg), is recommended if conditions are not suitable for carrying a conventional PCR instrument (3 to 10 kg) and a portable power station with AC output (∼13.5 kg).
F. oxysporum can infect a variety of crops and cause Fusarium wilts, which lead to huge economic losses every year. Besides land crops, such as bananas, watermelon, tomatoes, and cotton, F. oxysporum can also infect lotus, which grows in an aquatic environment (Edel-Hermann and Lecomte 2019; Nisikado and Watanabe 1953). F. commune can be isolated from plant and soil (Skovgaard et al. 2003) and is the causal agent of crown and/or root rot in Douglas fir (Stewart et al. 2012), tomato (Hamini-Kadar et al. 2010), soybean (Ellis et al. 2013), Chinese water chestnut (Zhu et al. 2016), and lotus (Zeng et al. 2017). F. commune is a recently described species with subtle morphological differences from the F. oxysporum species complex (Skovgaard et al. 2003; Stewart et al. 2006). Thus, a reliable method for distinguishing the two Fusarium species is one that uses molecular diagnostic technology (Stewart et al. 2006). DNA sequences from the translation EF-1α and the mtSSU rDNA regions have been used to distinguish F. commune from F. oxysporum (Ellis et al. 2013; Stewart et al. 2012; Yu and Babadoost 2013). However, using the conventional PCR method requires nucleic acid electrophoresis, a gel imaging system, and Sanger sequencing. Therefore, it is not suitable for use in field conditions outside of a molecular diagnostic laboratory. To find a solution to this problem, we developed a LAMP detection system using HNB as the indicator for reaction product formation, with which positive reactions can be distinguished from negative reactions using the naked eye (Goto et al. 2009; Kong et al. 2020).
An effective DNA extraction method or sample pretreatment is indispensable for developing rapid and user-friendly molecular diagnostic assays (Lau and Botella 2017). Given the need for pathogen identification and disease diagnosis in the field, we did not use the conventional CTAB method to extract total DNA from diseased lotus rhizome tissue. After homogenizing the diseased sample tissues, we tested several simple methods by freeze-thaw cycle treatment, boiling water bath, and a recently reported cellulose-based dipstick method (Zou et al. 2017), but they failed to give stable, consistent results in our study. Lotus seedlings and rhizomes are usually rich in minerals, proteins, and polysaccharides (Min et al. 2019; Cheng et al. 2020), which strongly inhibit the amplification of nucleic acids (Schrader et al. 2012). After the tests, we found that solid phase reversible immobilization paramagnetic bead technology is a suitable DNA purification method to aid in removing these inhibitors and only requires simple and portable equipment, such as a magnetic rack, pipettes, and some commonly used reagents (Figs. 5 to 7) (DeAngelis et al. 1995; Lau and Botella 2017).
The size and DNA sequence of mitochondrial genomes vary greatly among different species (Burger et al. 2003), but they are generally conserved within the same species. Mitochondrial DNA usually contains 14 protein encoding genes, 22 to 26 tRNAs, and 2 rRNAs (Burger et al. 2003). Compared with genomic DNA, mitochondrial DNA has more copies within a single cell (Fig. 3) (Williamson 2002). For the detection of Phytophthora agathidicida, the LAMP method targeted the mitochondrial apocytochrome b gene coding sequence and can detect P. agathidicida from as little as 1 fg of total DNA (Winkworth et al. 2020). The detection limit of our two LAMP primer sets is 10 pg/µl and 1 pg/µl, respectively, which is lower than in another report, in which the detection limit of the LAMP primers targeting mitochondrial DNA sequence reached 1 fg/µl (Winkworth et al. 2020). Besides the characteristics of different primer sets and Bst DNA polymerase, the variation of the detection limit may be explained by different mitochondrial genome copy number or size of nuclear genome.
In the F. oxysporum species complex, the sequences of mitochondrial DNA are highly conserved (Brankovics et al. 2017). We performed BLASTn analysis for the three loci and primer-BLAST analysis for all primer sets against the database of nucleotide collection (nr/nt) at NCBI with default settings. Remarkably, the two specific sites (Fc1 and Fc2) for F. commune are also an identical match to the mitochondrial genome of F. oxysporum f. sp. lactucae 09-002 (MN259515.1) but not to any other F. oxysporum strains. To date, two mitochondrial genomes of F. oxysporum f. sp. lactucae were released from strain 09-002 and strain 16-086 (MN259514.1). In a previous report, a phylogenetic analysis of mitochondrial genomes demonstrated that F. oxysporum f. sp. lactucae 09-002 was clustered with F. commune and not with other F. oxysporum mitochondrial genomes (Park et al. 2019). However, the mitochondrial genome from the other strain (16-086) of the same species is highly similar to those of F. oxysporum (Supplementary Fig. S4). Additionally, unlike strain 09-002, strain 16-086 contains F. oxysporum-specific locus Fo3 but does not contain the two F. commune specific loci Fc1 and Fc2 (Supplementary Fig. S4). Additional conclusions should not be drawn until the unusual F. oxysporum f. sp. lactucae strain 09-002 is further identified. F. oxysporum species complex strains usually have strict host specialization. In a recent review, Edel-Hermann and Lecomte (2019) reported that F. oxysporum f. sp. lactucae can only infect Lactuca sativa (lettuce). To date, it has not been reported that F. oxysporum f. sp. lactucae can infect lotus.
Different microorganisms (including pathogens) colonize in their own specific hosts and adapt to different environments. Unlike terrestrial plants, lotus grows in water year-round. Thus, the kinds of microorganisms that can infect lotus roots (or rhizomes) and cause similar disease symptoms are relatively few. At present, besides F. oxysporum and F. commune, the other reported pathogens that can infect lotus and lead to rhizome rot symptoms are F. incarnatum (Wang et al. 2020), F. tricinctum (Li et al. 2016), and the oomycete Phytopythium helicoides (Yin et al. 2015). BLAST results indicated that the primer sets targeting Fc1, Fc2, and Fo3 loci can distinguish F. commune (or F. oxysporum) from F. tricinctum. Additionally, the results from LAMP and PCR demonstrated that the related primer sets for F. commune and F. oxysporum did not cross-react with the oomycete Phytopythium helicoides (Supplementary Fig. S1). To date, the mitochondrial genome sequence of F. incarnatum has not been released and we do not have this strain in our laboratory collection. Instead, we used the mitochondrial genome sequence of F. equiseti as the reference sequence because F. equiseti and F. incarnatum belong to Fusarium incarnatum-equiseti species complex and their phylogenetic relationships are close (Villani et al. 2019). BLAST results indicated that the primer sets can be used for distinguishing F. commune (or F. oxysporum) from F. equiseti.
The suitable LAMP primer set targeting Fc1 can be found at the 5′ end of the locus, but BLAST results indicated that it is unable to distinguish F. commune from F. graminearum, F. asiaticum, F. pseudograminearum, F. cerealis, F. culmorum, and F. gerlachii. As we expected, the results of LAMP tests of the 11 strains indicated that the LAMP primer set targeting Fc1 was unable to distinguish between F. commune and F. graminearum. However, the PCR primer sets Fc1-S/Fc1-A (targeting the 3′ end of the Fc1 locus) and Fc2-S/Fc2-A (targeting Fc2 locus) can distinguish F. commune from the species. The 3′ end of the Fc1 locus is more specific, but because of the low GC ratio, suitable LAMP primer set cannot be obtained (Supplementary Table S1). The similar low GC ratio also exists at Fc2 locus. In the future, RPA will be introduced because it needs fewer requirements than LAMP, such as use of only two primers (upstream and downstream), lower reaction temperature (37°C), and shorter reaction time. RPA primer sets will be designed in the 3′ end of the Fc1 locus or in Fc2 locus for increasing specificity of detection.
Moreover, BLAST results indicated that the primer sets targeting Fo3 cannot distinguish F. oxysporum from F. foetens, a pathogen of Begonia plants that has been found in Europe, Canada, and the United States (González-Jartín et al. 2019). F. foetens can also infect certain plants which show no disease symptoms (González-Jartín et al. 2019). If molecular detections (LAMP or PCR) indicate the presence of F. oxysporum (or F. commune) in a tested lotus sample, symptoms of lotus rhizome rot on the water part of the plants or lotus rhizome nodes must also be observed before making a conclusion regarding the presence of F. oxysporum or F. commune. We collected 15 fresh, healthy lotus rhizomes from some major lotus-producing areas in three provinces and performed fungal isolation and LAMP detection for the tissues from the rhizomes. The results showed that Fusarium spp. strains could not be isolated from these healthy lotus rhizomes. Additionally, no positive reaction from the LAMP assay for either F. oxysporum or F. commune was obtained from tests of these tissue samples (Supplementary Fig. S5). Based on these results, incorrect judgment can be avoided when using our primer sets for F. commune and F. oxysporum pathogen identification in infected lotus.
In the future, the prototype of the detection kit will be upgraded to be more compact, lightweight, and user friendly. RPA detection method also will be introduced to reduce the difficulty of primer design for increasing the specificity of detection. Finally, a portable device for enriching microorganisms in water and soil will be developed. The integration of these devices and strategies will further improve accuracy and efficiency of pathogen detection for lotus disease diagnosis and management.
The author(s) declare no conflict of interest.
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Funding: This work was supported by grants from China Agriculture Research System of MOF and MARA (CARS-24-C-01) and Jiangsu Agricultural Science and Technology Innovation Fund [CX(18)2005].
The author(s) declare no conflict of interest.