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LAMP Detection and Identification of the Blackleg Pathogen Leptosphaeria biglobosa ‘brassicae’

    Affiliations
    Authors and Affiliations
    • Ran Du1
    • Yongju Huang2
    • Jing Zhang1
    • Long Yang1
    • Mingde Wu1
    • Guo-qing Li1
    1. 1State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
    2. 2School of Life and Medical Sciences, University of Hertfordshire, Hatfield, AL10 9AB UK

    Abstract

    Blackleg of oilseed rape is a damaging invasive disease caused by the species complex Leptosphaeria maculans (Lm)/L. biglobosa (Lb), which is composed of at least two and seven phylogenetic subclades, respectively. Generally, Lm is more virulent than Lb, but under certain conditions, Lb can cause a significant yield loss in oilseed rape. Lb ‘brassicae’ (Lbb) has been found to be the causal agent for blackleg of oilseed rape in China, whereas Lm and Lb ‘canadensis’ (Lbc) were frequently detected in imported seeds of oilseed rape, posing a risk of spread into China. To monitor the blackleg-pathogen populations, a diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using a 615-bp–long DNA sequence from Lbb that was derived from a randomly amplified polymorphic DNA assay. The LAMP was optimized for temperature and time, and tested for specificity and sensitivity using the DNA extracted from Lbb, Lbc, Lm, and 10 other fungi. The results showed that the optimal temperature and time were 65°C and 40 min, respectively. The LAMP primer set was specific to Lbb and highly sensitive as it detected the Lbb DNA as low as 132 fg per reaction. The LAMP assay was validated using the DNA extracted from mycelia and conidia of a well-characterized Lbb isolate, and its utility was evaluated using the DNA extracted from leaves, stems, pods, and seeds of oilseed rape. The LAMP assay developed herein will help for monitoring populations of the blackleg pathogens in China and in developing strategies for management of the blackleg disease.

    Blackleg (phoma stem canker) of oilseed rape (Brassica napus) is a world-wide economically important disease (Edwards Molina et al. 2017; Fitt et al. 2006a; Gugel and Petrie 1992; Laing 1986; Lob et al. 2013; Piening et al. 1975; Salisbury et al. 1995; West et al. 2000). It is caused by two closely related and morphologically similar ascomycetous fungi, Leptosphaeria maculans (anamorph: Plenodomus lingam) and Leptosphaeria biglobosa (anamorph: Plenodomus biglobosus), which form a species complex (Mendes-Pereira et al. 2003). Both fungi can infect leaves, stems, and pods of oilseed rape, causing phoma leaf spots, phoma stem cankers, and phoma pod spots, respectively (Fitt et al. 2006b). Among these symptoms, phoma stem canker is the most important regarding seed yield loss, as it can cause stem collapse (lodging), thereby reducing seed production. Numerous studies indicated that L. maculans is more virulent than L. biglobosa in terms of the extent of damage to the plants and seed production, as L. maculans can invade the vascular tissue of the basal stem, where it may cause stem collapse. In contrast, L. biglobosa usually infects the epidermal tissue of the upper stem, where it rarely causes stem collapse (Fitt et al. 2006a; Plummer et al. 1994; West et al. 2001; Williams and Fitt 1999). Previous studies showed that the L. maculans/L. biglobosa species complex (especially L. maculans) is responsible for serious economic losses to the industry of oilseed rape (or canola) in Australia, Canada, France, Germany, and UK since the 1970s. It was estimated that the blackleg disease of oilseed rape caused an average annual economic loss of US$167 million during 1983 to 1998 in Alberta of Canada, and US$70 million during 2000 to 2002 in the UK (Fitt et al. 2006b, 2008).

    In China, blackleg of oilseed rape was first reported in the early 2000s, and the pathogen for that disease was identified as NA1 or B-group of L. maculans (West et al. 2000), which was later reclassified as L. biglobosa (Shoemaker and Brun 2001). Large-scale field surveys demonstrated that this disease widely occurred in oilseed rape-plantation areas (Q. S. Li et al. 2013). Compared with healthy plants, diseased plants had less yield with the average single-plant seed yield loss ranging from 10 to 56% (Cai et al. 2018; Rong et al. 2015). So far, only L. biglobosa has been found in oilseed rape and cruciferous vegetables in China (Cai et al. 2015, 2018; Fitt et al. 2008; Q. S. Li et al. 2013; Liu et al. 2014; Zhang et al. 2014), and L. maculans was thus officially considered as a quarantine pathogen since the late 2000s (Wang et al. 2011; Zhou et al. 2010).

    Both L. maculans and L. biglobosa can be further classified into subclades or subspecies based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacer region of ribosomal DNA (ITS-rDNA), and a few nuclear genes such as the mating type gene MAT1-2 and the genes coding for actin and β-tubulin (Mendes-Pereira et al. 2003). So far, two subclades have been identified in L. maculans, including ‘brassicae’ on Brassica and ‘lepidii’ on Lepidium sp. (Mendes-Pereira et al. 2003). Seven subclades have been identified in L. biglobosa, including ‘americensis’, ‘australensis’, ‘brassicae’, ‘canadensis’, and ‘occiaustralensis’ on Brassica spp., ‘erysimii’ on Erysimum, and ‘thlaspii’ on Thlaspi sp. (Vincenot et al. 2008; Voigt et al. 2005; Zou et al. 2019). Among these, L. biglobosa subclades, ‘brassicae’ and ‘canadensis’ are the most common and important; L. biglobosa ‘brassicae’ has been found in the continents of America, Asia, and Europe (Dilmaghani et al. 2009; Fitt et al. 2006a; Liu et al. 2014; Vincenot et al. 2008), and L. biglobosa ‘canadensis’ has been detected in the continent of America (Canada and the USA) as well as in Australia (Dilmaghani et al. 2009; Liu et al. 2014; Van de Wouw et al. 2008). Five other subclades of L. biglobosa, including ‘americensis’, ‘australensis’, ‘erysimii’, ‘occiaustralensis’, and ‘thlaspii’ are the minor subclades; ‘americensis’ was only found in the USA (Zou et al. 2019), ‘erysimii’ was found in Canada (Voigt et al. 2005), and ‘australensis’ and ‘occiaustralensis’ were found in Australia (Vincenot et al. 2008).

    It is well recognized that L. maculans and L. biglobosa can be spread over a long distance through international trade of seeds of oilseed rape and/or exchange of germplasm resources of cruciferous crops (Chen et al. 2013, 2016; Chigogora and Hall 1995; Wang et al. 2003, 2011; Zhou et al. 2010). Therefore, detection and identification of L. maculans and L. biglobosa in crop seeds is essential in preventing spread of these two pathogens into other regions. Since the late 1970s, the deep-freezing blotter method has been recommended by the International Seed Testing Association (https://www.seedtest.org/en/home.html) to detect L. maculans and L. biglobosa in contaminated or infected seeds of cruciferous crops, including oilseed rape (Limonard 1968). The key point in that method is inhibition of seed germination under freezing temperatures (e.g., −20°C) and the subsequent promotion of growth of the seed-borne fungi on the seeds under normal temperatures (e.g., 20°C; Wang et al. 2003).

    Moreover, monitoring of the populations of L. maculans and L. biglobosa in fields planted with oilseed rape and cruciferous vegetables is also important regarding management of the blackleg disease (Dilmaghani et al. 2009; West et al. 2001). L. maculans and L. biglobosa usually produce similar symptoms on stems with formation of abundant black pycnidia (Q. S. Li et al. 2013; West et al. 2001). Therefore, it is difficult to distinguish these two pathogens just based on disease symptoms and location of infection (e.g., basal and upper stems) and the disease symptoms. Many researchers have made efforts to develop simple, rapid, and accurate methods to detect and identify L. maculans and L. biglobosa on diseased plant tissues. The methods so far developed include plant assays (e.g., virulence tests), morphological characterization (e.g., colony growth, pseudothecial shape, and ascospore germlings), metabolite profiling (e.g., pigments and phytotoxins), typing of glucose phosphate isomerase, karyotyping, serological typing, DNA analyses (e.g., restriction fragment length polymorphism, randomly amplified polymorphic DNA [RAPD], and PCR), and genome analyses (Grandaubert et al. 2014; Liu et al. 2006; Mendes-Pereira et al. 2003; Van de Wouw et al. 2008; Vincenot et al. 2008; Williams and Fitt 1999). However, these methods are usually time-consuming, labor-intensive, and/or dependent on special expertise and instruments. There is a need to develop simpler, faster, and more convenient methods for detection and identification of these two pathogens.

    Since the early 2000s, the loop-mediated isothermal amplification (LAMP) technique has been developed to detect animal and plant pathogens (Endo et al. 2004; Niessen 2014; Notomi et al. 2000). A typical LAMP assay consists of serial reactions catalyzed by Bst DNA polymerase to amplify a target DNA sequence with the aid of a set of primers (four to six primers) under the isothermal condition (Notomi et al. 2000). The LAMP products can be visualized with naked eyes in the presence of some DNA-staining dyes such as SYBR Green I or ethidium bromide (Du et al. 2020; Long et al. 2017; Zhou et al. 2016). Compared with PCR, LAMP detection has advantages of high specificity, high efficiency, simplicity, and rapidity, and more importantly, it does not require expensive and special instruments (Niessen 2014). LAMP has been used to detect L. maculans and L. biglobosa in infected plant tissues and air samples (Du et al. 2020; Jędryczka et al. 2013; Long et al. 2017; Zhou et al. 2016). However, LAMP detection and identification of the subclades of L. maculans and L. biglobosa has not been reported so far. Therefore, we have developed a LAMP-based technique for detection and identification of L. biglobosa brassicae’, the prevalent subclade of L. biglobosa in China (Cai et al. 2015, 2018; Liu et al. 2014). The specific objectives include: to design the LAMP primer set specific for L. biglobosa brassicae’; to optimize the LAMP-based technique; and to evaluate the potential of LAMP detection and identification of L. biglobosa brassicae’ in field disease diagnosis and pathogen population survey.

    Materials and Methods

    Fungal isolates.

    A total of 45 fungal strains were used in this study, including 26 strains of L. biglobosabrassicae’, seven strains of L. biglobosa ‘canadensis’, two strains of L. maculans, three strains of other oilseed rape pathogens (Botrytis cinerea, Collectotrichum higginsianum, and Sclerotinia sclerotiorum), and seven strains of saprobes living on oilseed rape (Phoma spp., Alternaria alternatae, and Chaetomium globosum; Table 1). Two strains of L. maculans were isolated from seeds of canola (B. napus) imported from Canada by Dr. Zhenhua Wang of the Wuhan Customs Technical Centre (Wuhan, China). Strain 17-4 of L. biglobosa ‘canadensis’ was isolated from diseased seeds of canola (B. napus), also imported from Canada by Dr. Jianping Yi of the Shanghai Customs Technical Centre (Shanghai, China). The remaining 42 fungal strains were isolated from oilseed rape collected from various locations in China (Table 1). All of the fungal strains were incubated on potato dextrose agar (PDA) with cellophane film overlays at 20°C for 3 to 15 days, mycelia, and/or conidia of each strain were collected and stored at −80°C until use.

    Table 1. Fungal strains from oilseed rape (Brassica napus), their origin, and the loop-mediated isothermal amplification (LAMP) detection results

    LAMP primer designing.

    The specific LAMP primers for detection of L. biglobosa ‘brassicae’ were designed based on a DNA sequence selected from RAPD fragments. Strains Lb731 and W10 of L. biglobosa ‘brassicae’, strain 17-4 of L. biglobosa ‘canadensis’, strain 2010510-1 of L. maculans, and strain P2 of Phoma macrostoma were used in the RAPD assays with 20 Operon primers listed in Supplementary Table S1. Genomic DNA was extracted from the mycelia of these strains using the CTAB method (Möller et al. 1992) and used as templates in RAPD assays with the procedures described by Plummer et al. (1994). The resulting RAPD products were separated on a 1% agarose gel (wt/vol) in Tris-Borate-EDTA buffer (89 mmol/liter Tris, 89 mmol/liter boric acid, and 2 mmol/liter EDTA) and visualized on an UV transilluminator after staining with ethidium bromide (1.5 mg/ml). One of the DNA bands of ∼600 bp specific for L. biglobosa ‘brassicae’ (Fig. 1A) was selected as target for LAMP detection. It was purified from the agarose gel using AxyPrep DNA Gel Extraction Kit (Axygen Scientific, Union City, CA), cloned into Escherichia coli DH5a using the pMD18-T vector (TaKaRa Biotechnology, Dalian, China), and sequenced at AuGCT Biotechnology (Beijing, China). The resulting DNA sequence (Supplementary Fig. S1) was searched with the program BLASTn at NCBI (https://www.ncbi.nlm.nih.gov/) to confirm its origin. The result showed that the DNA sequence was 615 bp in length (Supplementary Fig. S1), was 100% identical to the DNA sequence in the scaffold00021 of L. biglobosa ‘brassicae’ b35 (GenBank Acc. FO905643.1), and 88.13% identical to a region in the genome of L. biglobosa ‘canadensis’ Lb1204 (Supplementary Fig. S2). However, no homologs to this DNA sequence were found in the genome of L. maculans JN3 (Genome Assembly No. GCA_900538235.1). Therefore, The DNA sequence appears to be highly specific for L. biglobosa ‘brassicae’. Six LAMP primers were designed based on the DNA sequence using the LAMP primer designing software PrimerExplorer v5 (http://primerexplorer.jp/lampv5e/index.html) (Fig. 1B and C; Table 2). The primers were synthesized by AuGCT Biotechnology and used in the following LAMP assays.

    Fig. 1.

    Fig. 1. Designing of the LAMP primers for detection of Leptosphaeria biglobosa ‘brassicae’. A, An agarose gel electropherogram showing the polymorphic DNA fragments among different fungal species (L. biglobosa ‘canadensis’ 17-4, L. biglobosa ‘brassicae’ Lb731 and W10, L. maculans 2010510-5, and P. macrostoma P2) from the randomly amplified polymorphic DNA (RAPD) assay with the primer OPA-19. M, DNA marker; *, the DNA fragment was purified, cloned, and sequenced for designing of the loop-mediated isothermal amplification (LAMP) primers. B, A schematic diagram showing location of the LAMP primers in the target DNA sequence; %. C, Top, a schematic diagram showing location of the 230-bp–long region within the 615-bp DNA sequence from the RAPD assay. Bottom, Location of the LAMP primers in the 230-bp–long double-stranded DNA region. Arrows indicate the direction of the primers initiating amplification.

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    Table 2. The nucleotide sequences of the six loop-mediated isothermal amplification (LAMP) primers for Leptosphaeria biglobosa ‘brassicae’

    LAMP optimization.

    The strain W10 of L. biglobosa ‘brassicae’ was used in this experiment. The LAMP mixtures (25 μl) in 0.2-ml Eppendorf tubes (Eppendorf, Hamburg, Germany) contained the following components (Supplementary Table S2): 1× Isothermal Amplification Buffer (New England BioLabs, Ipswich, MA), Bst 2.0 WarmStart DNA Polymerase at 8 U in each reaction mixture (New England BioLabs), MgSO4 (4 mmol/liter), dNTPs (10 mmol/liter for each nucleotide), the forward and backward outer primers F3/B3 (0.2 μmol/liter for each), forward and backward loop primers LF/LB (0.4 μmol/liter for each), forward and inner primer backward FIP/BIP (1.6 μmol/liter for each), and template DNA (∼100 ng for each reaction). The mixtures containing all the components except template DNA were used as controls. To prevent evaporation of the water in the mixtures during LAMP reaction, aliquots of liquid paraffin (Aladdin Industrial, Shanghai, China) were added to the tubes with the LAMP mixtures (30 μl in each tube) as overlays. The LAMP reactions were performed with a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) at 65°C for 50 min to determine the amplification efficiency of the primers, at 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75°C for 40 min to optimize the temperature, and at 65°C for 10, 20, 30, 40, 50, 60, 70, and 80 min to optimize the time requirement. After LAMP amplification, the tubes were taken out from the thermal cycler and maintained at 4°C for at least 10 min to cool down the temperature in the reaction mixtures. Then, they were opened in another room next to the LAMP operation area, and aliquots of SYBR Green I solution at 100 μg/ml (Sigma-Aldrich, St. Louis, MO) was added to the tubes at 0.2 μl per tube. Color change in the reaction mixtures was then observed, green coloration indicated a positive LAMP amplification, whereas brown coloration indicated a negative LAMP amplification. To confirm the LAMP amplification, 4 μl of LAMP product of each reaction was loaded in a 2% agarose gel (wt/vol). After electrophoresis, the gel was immersed in an ethidium bromide solution (1.5 mg/ml, wt/vol) for 30 min, and the DNA fragments in the agarose gels were visualized on the UV transilluminator, formation of DNA mass ladders showing a multiple DNA bands pattern (or DNA ladder pattern) indicated a positive LAMP amplification and vice versa. Each LAMP reaction in this experiment as well as in the following experiments was repeated three times.

    Specificity test.

    To test the specificity of the primers in LAMP detection of L. biglobosa ‘brassicae’, genomic DNA was extracted from L. biglobosa ‘brassicae’ (26 strains), L. biglobosa ‘canadensis’ (seven strains), L. maculans (two strains), and 10 strains of other fungi (Table 1) using the CTAB method (Möller et al. 1992). The DNA extracts were separately added to the LAMP mixtures, and the reactions were performed at 65°C for 40 min. The LAMP products were visualized with SYBR Green I and confirmed by agarose gel electrophoresis.

    Sensitivity test.

    Strain W10 of L. biglobosa ‘brassicae’ was used in this experiment for comparison of the detection thresholds in the LAMP and PCR assays, as the PCR assay was officially approved to detect L. biglobosa ‘brassicae’ in China (Zhao et al. 2015). The DNA solution (132 ng/μl) was 10-fold diluted to generate the serial solutions with the DNA concentration decreasing from 132 ng/μl to 1.32 fg/μl. An aliquot of 1 μl of each DNA solution or water alone (control) was added to a LAMP mixture, which was incubated at 65°C for 40 min. The LAMP products were visualized with SYBR Green I and confirmed by agarose gel electrophoresis. Meanwhile, the template sensitivity in LAMP detection was compared with that in the conventional PCR detection using the forward and backward outer primers F3 and B3 developed in this study (Table 2). The PCR reaction mixtures (25 μl) were prepared with the following components: 12.5 μl 2× TSINGKE Master Mix (Tsingke Biotechnology, Chengdu, China), 0.5 μl of forward primer F3 (10 μmol/liter), 0.5 μl of backward primer B3 (10 μmol/liter), 1.0 μl of DNA solution, and 10.5 μl of water. The PCR was performed in a C1000 Touch Thermal Cycler with the following thermal program: initial denaturation at 94°C for 3 min, followed by 36 cycles with denaturation at 94°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 30 s, and final extension at 72°C for 10 min. The PCR product (210 bp in size) was confirmed by agarose gel electrophoresis (Du et al. 2020).

    LAMP-assisted fungal detection.

    Strain W10 of L. biglobosa ‘brassicae’ was used in this experiment. It was incubated at 20°C on PDA with cellophane film overlays for 4 days. Mycelia from 1, 2, or 3 square-shaped colony patches (0.5 cm × 0.5 cm, length × width) at the colony margin area were collected and put in 1.5-ml of Eppendorf tubes. Aliquots of 1× TE buffer (100 mmol/liter Tris-HCl, and 10 mmol/liter EDTA, at pH 8.0) were transferred to the tubes at 50 μl per tube. The mycelia were squashed using sterilized plastic pestles. The resulting mixtures were heat-treated in a water bath at 95°C for 2 min for DNA release from the hyphal cells (Fan et al. 2018). After cooling down to the room temperature (20 ± 2°C), the mixtures were centrifuged at 12,000 rpm, 1 μl of supernatant of each sample was added to a LAMP mixture. In the control, 1 μl of sterilized water was added to the mixture. The LAMP amplifications were performed at 65°C for 40 min, visualized with SYBR Green I and confirmed by agarose gel electrophoresis.

    The PDA cultures of strain W10 were further incubated at 20°C for another 10 days for production of pycnidia and pycnidiospores (conidia), which were harvested by washing with sterilized water. Conidial concentration was measured using a hemocytometer. The master conidial suspension (∼1 × 107 conidia/ml) was 10-fold diluted with sterilized water to generate serial conidial suspensions with the final concentrations at 2 × 105, 2 × 104, 2 × 103, 2 × 102, and 20 conidia/ml, and an aliquot of 100 μl of each conidial suspension was pipetted to an Eppendorf tube containing 50 μl of 3× TE buffer. The conidial suspensions in the tubes were heat-treated in a water bath (95°C, 2 min), and after that, they were centrifuged at 12,000 rpm, and 1 μl of supernatant of each sample was added to the LAMP mixture. For the control, 1 μl of sterilized water was added to a LAMP mixture. The LAMP reactions were performed at 65°C for 40 min, visualized with SYBR Green I and confirmed by agarose gel electrophoresis.

    LAMP-assisted disease diagnosis.

    Diseased leaves, stems, mature pods, and seeds of the winter-type oilseed rape (B. napus cultivar ‘Zhongshuang No. 9’) showing typical blackleg symptoms (Supplementary Fig. S3) were collected in the 2018 to 2019 season from a field in Shenshan Town of Chibi County, Hubei Province of China (29°52′50″N, 114°3′48″E, 40 m above sea level). Leaf samples were collected at the early flowering stage, and samples of stems, pods, and seeds were collected at the harvest stage. The pathogen for the blackleg disease of oilseed rape and cruciferous vegetables in that area is L. biglobosa ‘brassicae’ according to the 2-year surveys in our lab (Li 2019). Meanwhile, healthy leaves, stems, mature pods, and seeds were collected and used as controls. Tissues were carefully taken from the collected samples using a sharp razor blade, tissue pieces (∼5 × 5 mm, length × width) were cut off from the leaves and the pod hulls, stem tissues (∼5 × 5 mm, length × width) were peeled off from the epidermal layer of the stems. The diseased leaf, stem, and pod-hull pieces, or the diseased seeds were separately put in 1.5-ml Eppendorf tubes at 1, 2, or 3 pieces (or seeds) in each tube. Meanwhile, two healthy tissue pieces or healthy seeds were put in other Eppendorf tubes as controls. Aliquots of NaOH solution (0.4 mol/liter) were added to the tubes, 100 μl per tube, and the plant tissue pieces or the seeds were squashed using sterilized plastic pestles, followed by heat-treatment in water bath at 95°C for 2 min. Then, the mixtures were centrifuged at 12,000 rpm, and 1-μl supernatant of each sample was added to a LAMP mixture as a DNA template. The LAMP reactions were performed at 65°C for 40 min and visualized with SYBR Green I and confirmed by agarose gel electrophoresis.

    Results

    LAMP primers.

    Results of the RAPD assays showed that among the 20 tested 10-mer Operon primers (Supplementary Table S1), OPA-19 persistently produced polymorphic DNA fragments among L. biglobosa ‘brassicae’, L. biglobosa ‘canadensis’, L. maculans, and P. macrostoma (Fig. 1A). Strains Lb731 and W10 of L. biglobosa ‘brassicae’ showed an identical DNA-banding pattern, which differed greatly from those in L. biglobosa ‘canadensis’ 17-4, L. maculans 2010510-1, and P. macrostoma P2. A DNA fragment of 615 bp in size from L. biglobosa ‘brassicae’ W10 was selected as target (Supplementary Fig. S1). It was uploaded into the online software PrimerExplorer v5 and six primers (forward and backward outer primers F3/B3, inner primers FIP/BIP, and loop primers LF/LB) were designed based on the 230-bp–long central region in that DNA sequence (Fig. 1B and C; Table 2).

    LAMP optimization.

    In the assay for testing the LAMP amplification efficiency (65°C, 50 min), the control reaction mixture without any DNA templates retained a brown coloration in the presence of SYBR Green I, and did not produce any multiple DNA bands patterns when visualized on the agarose gel (Fig. 2A). However, the reaction mixture containing the DNA from strain W10 of L. biglobosa ‘brassicae’ exhibited a green coloration in presence of SYBR Green I, and it produced a multiple DNA bands pattern on the agarose gel. This result suggests that the LAMP primers can efficiently amplify the DNA of L. biglobosa ‘brassicae’ strain W10.

    Fig. 2.

    Fig. 2. Optimization of the temperature and the time duration for loop-mediated isothermal amplification (LAMP) detection of Leptosphaeria biglobosa ‘brassicae’. A, Top, Two LAMP reaction mixtures with different colors in the presence of SYBR Green I: green in the reaction with DNA from strain W10 of L. biglobosa ‘brassicae’ (Lbb) as template; brown in the control (CK) reaction mixture without DNA template. Bottom, An agarose gel electropherogram showing the difference of the two reaction mixtures in the formation of a multiple-DNA-bands pattern on the agarose gel. B, Twelve LAMP reactions under different temperatures showing different colors in the presence of SYBR Green I. C, Eight LAMP reactions with different time durations showing different colors in the presence of SYBR Green I.

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    The temperature and time duration required for LAMP detection of L. biglobosa ‘brassicae’ were optimized. In the temperature assay (40 min), a significant difference in the color of the reaction mixtures amended with SYBR Green I was observed among the temperature treatments ranging from 53°C to 75°C (Fig. 2B). In two low temperature treatments (53°C and 55°C) and two high temperature treatments (73°C and 75°C), the reaction mixtures retained a brown coloration without formation of multiple DNA bands patterns in agarose gels after electrophoresis, indicating no detectable LAMP amplifications in these four treatments. In the treatments at 57, 59, 63, and 65°C, the reaction mixtures had a green coloration and formed multiple DNA bands patterns in agarose gels after electrophoresis; moreover, the intensity of the green color showed an increased tendency with the temperatures increasing from 57°C to 65°C. In the treatments at 67, 69, and 71°C, the reaction mixtures also showed a green coloration and formed multiple DNA bands patterns in agarose gels after electrophoresis; however, the intensity of the green color showed a decreased tendency with the temperatures increasing from 67°C to 71°C. Therefore, the optimum temperature for LAMP detection of L. biglobosa ‘brassicae’ W10 was 65°C.

    In the time duration assay (65°C), the LAMP mixtures amended with SYBR Green I retained a brown coloration at 10-min post-reaction (mpr). The color of the reaction mixtures turned green when the time duration lasted between 20 and 80 mpr (Fig. 2B). With the time duration extending to 20, 30, and 40 mpr, the intensity of the green color gradually increased. The green color intensity had no visible change at the time duration longer than 50 mpr, suggesting that the LAMP reactions at 50 to 80 mpr may reach a plateau state. Therefore, the minimum time duration for LAMP detection of L. biglobosa ‘brassicae’ strain W10 was 40 min.

    LAMP specificity.

    Results of the specificity assay showed that DNA from 45 fungi exhibited two different effects on LAMP amplification (Table 1). The reaction mixtures with the DNA from 26 strains of L. biglobosa ‘brassicae’ had a green coloration in the presence of SYBR Green I and formed multiple DNA bands patterns in agarose gels after electrophoresis. This result indicated that these reactions had a positive LAMP amplification. In contrast, the reaction mixtures with the DNA from 19 other fungi, including two close relatives of L. biglobosa ‘brassicae’ (L. biglobosa ‘canadensis’ and L. maculans), three pathogens of oilseed rape (B. cinerea, C. higginsianum, and S. sclerotiorum), and seven saprobes living on oilseed rape (A. alternatae, C. globosum, and Phoma spp.), retained a brown coloration and did not produce any multiple DNA bands patterns in agarose gels after electrophoresis. This result indicated that these LAMP reactions had a negative LAMP amplification. Therefore, the LAMP detection has a high specificity for L. biglobosa ‘brassicae’.

    LAMP sensitivity.

    Results of the sensitivity assay showed that the amount of the template DNA of L. biglobosa ‘brassicae’ in the reaction mixtures greatly affected LAMP amplification. The reaction mixtures with the amount of DNA per reaction ranging from 132 ng to 132 fg had a green coloration in presence of SYBR Green I (Fig. 3A), and formed multiple DNA bands patterns in electrophoresed agarose gels (Fig. 3B). In contrast, the reaction mixtures with the amount of DNA per reaction at 13.2 fg and 1.32 fg and the control mixture without the template DNA retained a brown coloration in presence of SYBR Green I (Fig. 3A), and did not produce any multiple DNA bands patterns in the electrophoresed agarose gels (Fig. 3B). This result suggests that the minimum amount of the DNA in LAMP detection of L. biglobosa ‘brassicae’ is 132 fg per reaction.

    Fig. 3.

    Fig. 3. Effect of the amount of template DNA on loop-mediated isothermal amplification (LAMP) and PCR detection of Leptosphaeria biglobosa ‘brassicae’. A, Ten LAMP reactions with different amounts of the template DNA from strain W10 of L. biglobosa ‘brassicae’ showing different colors in the presence of SYPR Green I. CK, control reaction mixture without DNA template. B, An agarose gel electropherogram showing a difference among the reaction mixtures in the formation of multiple DNA bands patterns on the agarose gel. C, An agarose gel electropherogram showing the 210-bp–long DNA bands from the PCR reactions containing different amounts of template DNA from L. biglobosa ‘brassicae’ W10.

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    Results of the conventional PCR with the primers F3 and B3 (Table 2) indicated that after reaction, the PCR mixtures with the amount of the DNA template per reaction at 132 ng, 13.2 ng, 1.32 ng, or 132 pg produced a DNA fragment with the expected size of 210 bp (Fig. 3C). The brightness of the DNA band gradually became weaker with the amount of the DNA per reaction decreasing from 132 ng to 132 pg. However, the PCR mixtures with the amount of the DNA template per reaction ranging from 13.2 pg to 1.32 fg did not produce any multiple DNA bands patterns in that agarose gel (Fig. 3C). Therefore, the LAMP detection appears 1,000 times more sensitive than the PCR detection.

    LAMP-assisted detection of L. biglobosa ‘brassicae’.

    The DNA from the mycelia and conidia of L. biglobosa ‘brassicae’ strain W10 was used as template in LAMP assays. The reaction mixtures containing the DNA from all the three mycelial samples and from 20 to 20,000 conidia had a green coloration in the presence of SYBR Green I and produced multiple DNA bands patterns in electrophoresed agarose gels, indicating positive LAMP amplifications in these reactions (Table 3). In contrast, the control reaction mixtures without the DNA template and the reaction mixture containing the DNA from two conidia did not show any visible color change in the presence of SYBR Green I; formation of multiple DNA bands patterns in the electrophoresed agarose gel was not observed at all (Table 3), indicating negative LAMP amplifications in these reactions.

    Table 3. Loop-mediated isothermal amplification (LAMP) detection of Leptosphaeria biglobosa ‘brassicae’ in pure cultures and plant tissues of oilseed rape (stems, leaves, pods, and seeds)

    LAMP-assisted diagnosis of the blackleg disease.

    The DNA from healthy and diseased tissues from leaves, stems, pods, and seeds of oilseed rape (Supplementary Fig. S3) was used as template in LAMP assays. The results showed that the control mixtures containing the DNA from healthy leaves, stems, pods, and seeds displayed a brown coloration in the presence of SYBR Green I and did not produce any multiple DNA bands patterns in the agarose gels (Table 3), indicating negative LAMP amplifications in these reactions. However, the reaction mixtures containing the DNA from diseased leaves, stems, pods, and seeds displayed a green coloration in presence of SYBR Green I (Table 3) and produced multiple DNA bands patterns on the agarose gels, indicating positive LAMP amplifications in these reactions.

    Discussion

    This study developed a rapid, specific, and sensitive LAMP assay for detection of L. biglobosa ‘brassicae’. The use of LAMP as a tool to study the changing populations of L. maculans and L. biglobosa in diseased tissues of oilseed rape as well as in air samples was first reported in 2013 (Jędryczka et al. 2013). However, it is not clear what DNA sequence was used for designing of the LAMP primer set in that study (Jędryczka et al. 2013). In later studies, the ITS-rDNA in L. maculans and L. biglobosa were used for designing the LAMP primer sets (Du et al. 2020; Long et al. 2017; Zhou et al. 2016). The resulting LAMP assays displayed a consistent detection of these two closely related pathogens (Du et al. 2020; Long et al. 2017; Zhou et al. 2016). However, whether these LAMP assays have specificity for subclades of L. maculans and L. biglobosa remains unknown. Omer and Wallenhammar (2020) reported real-time LAMP detection of L. maculans and L. biglobosa ‘brassicae’. The primer sets SirP and polyketide synthase gene (PKS5) for L. maculans were designed based on the nucleotide sequences of the phytotoxin sirodesmin PL gene (sirP) and PKS5, respectively, and the primer set PKS5 for L. biglobosa ‘brassicae’ was designed based on the nucleotide sequences of the L. biglobosa ‘brassicae’ PKS21 gene (Omer and Wallenhammar 2020). In this study, a 615-bp DNA sequence derived from a RAPD assay was used for designing the LAMP primer set. The resulting LAMP assay showed a positive detection of L. biglobosa ‘brassicae’, but failed to detect L. biglobosa ‘canadensis’ and L. maculans. Therefore, the LAMP assay has a high specificity for L. biglobosa ‘brassicae’.

    To the best of our knowledge, this is the first report about LAMP detection of L. biglobosa at the subclade level. The specificity may lie in the target DNA sequence, which is highly identical among strains of L. biglobosa ‘brassicae’, as it is a part of the genome of L. biglobosa brassicae’ itself. However, the target DNA sequence has a low identity level (88.13%) to that in strains of L. biglobosa ‘canadensis’. Moreover, no homologs to the target DNA sequence were identified in the genome of L. maculans. Future studies are necessary to characterize the nature and location of the 615-bp DNA sequence in the genome of L. biglobosa ‘brassicae’ and to determine specificity of the primer set for other subclades of L. biglobosa, including ‘americensis’, ‘australensis’, ‘erysimii’, ‘occiaustralensis’, and ‘thlaspii’, which belong to different branches from ‘brassicae’ and ‘canadensis’ in the phylogenetics inferred from the combined gene set ITS-rDNA, MAT1-2, actin gene, and β-tubulin gene as well as whole genomes (Dilmaghani et al. 2009; Grandaubert et al. 2014; Vincenot et al. 2008; Zou et al. 2019).

    Previous studies indicated that the majority of the target DNA sequences used in the LAMP assays for fungi, yeasts, and oomycetes are selected from public databases (Niessen 2014). The target DNA sequences include the ribosomal RNA genes in most cases, as well as many nuclear genes such as acl1, amy1, btub, cap59, gaoA, gp43, rodA, tef1, and ypt1 (Chen et al. 2013; Endo et al. 2004; Ferdousi et al. 2014; Huang et al. 2011; Lucas et al. 2010; Luo et al. 2012; Matsuzawa et al. 2010; Niessen 2014; Niessen et al. 2012; Niessen and Vogel 2010). Meanwhile, quite a few previous studies reported use of RAPD assays to explore some novel DNA sequences as targets for LAMP detection of Verticillium dahliae, Fusarium oxysporum f. sp. cubense race 4, F. oxysporum f. sp. niveum, and F. mangiferae (B. J. Li et al. 2013; Moradi et al. 2013; Peng et al. 2013; Pu et al. 2014). This study selected a 615-bp-long RAPD sequence of L. biglobosa brassicae’ as target in the LAMP assay for L. biglobosa brassicae’. The result corroborated the previous studies mentioned above that combined use of RAPD and LAMP is a valid strategy to develop the molecular techniques for detection and discrimination of the closely related plant pathogenic fungi.

    The LAMP assay developed in this study provided a simple, rapid, and efficient tool to diagnose the blackleg disease caused by L. biglobosa brassicae’, and to assist identification of isolates of L. biglobosa brassicae’. Previous studies demonstrated that L. biglobosa brassicae’ usually coexists with L. maculans, L. biglobosa canadensis’, and other minor subclades of L. biglobosa (e.g., ‘americensis’, ‘australensis’, and ‘occiaustralensis’; Dilmaghani et al. 2009; Fitt et al. 2006a; Vincenot et al. 2008; Voigt et al. 2005; Zou et al. 2019). At present, L. biglobosa brassicae’ was found to be the sole causal agent for blackleg of oilseed rape and cruciferous vegetables in China (Cai et al. 2015, 2018; Li et al. 2013; Liu et al. 2014). However, considering the situation of the continuous imports of seeds of oilseed rape from foreign countries, L. maculans and other subclades of L. biglobosa might be introduced to this country (Fitt et al. 2008; Wang et al. 2011; Zhang et al. 2014; Zhou et al. 2010). Therefore, it is necessary to persistently monitor the populations of the blackleg pathogens in oilseed rape-plantation areas as well as in the areas surrounding the ports in China. This study found that the LAMP assay could consistently detect the DNA extracted from the pure cultures of L. biglobosa brassicae’ and from diseased plant tissues using the simplified DNA extraction methods (e.g., TE-buffer or alkaline lysis under 95°C for 2 min), and the LAMP assay was performed within 2 h. Using this technique together with the LAMP assays for L. maculans and L. biglobosa developed in the literature (Du et al. 2020; Long et al. 2017; Zhou et al. 2016), it is possible to conduct a large-scale identification of the isolates of Leptosphaeria spp. and to carry out the on-site diagnosis of the blackleg disease in field surveys. Future studies are required to assemble the LAMP components into a kit and to optimize the LAMP assays under the field conditions.

    Acknowledgments

    The authors appreciate the kind help of Dr. Zhenhua Wang of the Technical Center of Wuhan Customs (Wuhan, China), and Dr. Jianping Yi of the Technical Center of Shanghai Customs (Shanghai, China) for providing strains of L. biglobosa ‘canadensis’ and L. maculans.

    The author(s) declare no conflict of interest.

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    The author(s) declare no conflict of interest.

    Funding: This research was funded by the China Agriculture Research System under grant No. CARS-12.