Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Detection of Tilletia controversa using immunofluorescent monoclonal antibodies L. Gao1, C. Feng2, B. Li3, T. Liu1, B. Liu1 and W. Chen1 1 State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China 2 Beijing Kwinbon Biotechnology, International Information Industry Base, Beijing, China 3 Beijing Centre for Physical and Chemical Analysis (BCPCA), Yongfeng High and New Technology Industrial Base, Beijing, China
Keywords detection, immunofluorescence method, monoclonal antibody, plant pathology, Tilletia controversa, wheat smut. Correspondence Wanquan Chen and Li Gao, State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China. E-mails: [email protected]; xiaogaosx @hotmail.com 2014/1959: received 22 September 2014, revised 3 November 2014 and accepted 17 November 2014 doi:10.1111/jam.12703
Abstract Aims: Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries. Methods and Results: The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 20 lg ml 1 of the D-1 monoclonal antibody. Conclusion: This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores. Significance and Impact of the Study: This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved.
Introduction Tilletia controversa K€ uhn is a quarantined pathogenic fungus that causes dwarf bunt of wheat (Duran and Fischer 1961). This fungus is similar to Tilletia caries (DC) Tul., which causes common bunt of wheat. Both species are plant pathogens belonging to the order Ustilaginales of Basidiomycetes. The main symptoms and signs of plants infected by these species are the absence of grain underneath the lemma and the presence of black teliospores with a fishy odour. Flour from wheat grain contaminated with the fungal spores may have undesirable flavour and taste. Wheat seeds contaminated with the fungal teliospores may result in severe bunt diseases that cause yield loss and reduce grain quality. Tilletia controversa causes yield losses up to 75% in some cases (Goates 1996; Kutschera and Hossfeld 2012). Wheat plants
attacked by this species are smaller, lighter and have more shoots than healthy plants. The current detection protocol for dwarf bunt of wheat in most countries, including the USA, European Union (EU) and Australia, involves morphological and/or molecular analysis of teliospores. The germination of teliospores is required for confirmation, which incurs a delay of approx. 3 weeks. Such delay is highly unsatisfactory in a quarantine situation. Moreover, T. controversa teliospores do not germinate at 17°C in the dark; instead, they begin germinating at 5°C after a delay of at least 21 days. Microscopic observations show that T. controversa teliospores have deep and spiky reticulation, whereas those of T. caries have shallow and round reticulation (Trione and Krygier 1977). Liang et al. (1982) reported that approximately 70% of the net ridge depths of T. controversa teliospores are between 15 and 25 lm,
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and the glial sheath depths are between 2 and 3 lm, while the net ridge depths of T. caries teliospores are smaller than 12 lm, and the glial sheath depths are smaller than 15 lm. These authors also found some overlap in the teliospore sizes of both T. controversa and T. caries, as well as their net ridge and glial sheath depths. Some studies have reported the use of chemical identification methods, such as the use of periodic acid to differentiate T. controversa from T. bromi (Trione and Krygier 1977), treatment of the precipitate after filtration of teliospores with a-amylase (Li et al. 1996) and combination of a-amylase and centrifugation (Zhang et al. 2002). Molecular methods based on DNA analysis have provided useful information for the species identification of some important plant pathogens (Martin and Kistler 1990; Correll et al. 1993). PCR has provided a significant contribution to plant disease diagnosis (Ferreira et al. 1996), and this technique is particularly useful for the detection and identification of pathogens (Goodwin and Annis 1991; McManus and Jones 1995; Schaad et al. 1995). However, because T. controversa and T. caries are highly similar in the genomic sequences of the intergenic transcribed spacer (ITS) regions, several studies have failed to identify specific markers for T. controversa and T. caries based on ITS, IGS1, RPB2 and repetitive sequence-based polymerase chain reaction (PCR) (RepPCR) (Mulholland and McEwan 2000; Josefsen and Christiansen 2002; Liang et al. 2006; Gao et al. 2010b). Some progresses have been made recently in molecular detection of T. controversa. Bakkeren et al. (2000) reported that amplified fragment length polymorphism (AFLP) may provide better sequence-characterized amplified region (SCAR) markers for T. controversa. Liu et al. (2009) identified a SCAR marker for T. controversa using the AFLP method. Gao et al. (2010a, 2011) reported two SCAR markers using the ISSR method for detecting T. controversa. However, the PCR assays may not be suitable for practical applications. The problems involved in the practical use of PCR assays on a large scale include technical complexity, labour costs, quality control and investment in expensive equipment. Numerous reports have described detection methods based on monoclonal antibodies, such as those for Phytophthora cinnamomi (Hardham et al. 1986; Gabor et al. 1993), Pythium aphanidermatum (Estrada-Garcia et al. 1989), Aphanomyces invadans (Miles et al. 2003), Salmonella spp., Escherichia coli and Listeria monocytogenes (Fratamico et al. 1998; Koubova et al. 2001; Bokken et al. 2003; Leonard et al. 2004). Immunologic methods based on monoclonal antibodies can easily and rapidly produce detection results, and they do not require complex equipment or technical skills; therefore, such methods are the most suitable for on-site detection (Gao et al. 2013). 498
Previous attempts have been made to identify T. controversa and other closely related species using immunologic techniques; however, only unsatisfactory results were achieved. Such as by comparing polyclonal antisera and monoclonal antibodies, Banowetz et al. (1984) reported that both T. controversa and T. caries teliospores reacted with polyclonal sera bound the fluorescent probe to the same degree, and each monoclonal antibody derived in their study reacted strongly with both fungi, which indicates the close antigenic relationship of these species. Zhang et al. (2007) developed an ELISA and demonstrated that polyclonal antibodies derived from fine powder of the teliospores of T. controversa failed to differentiate between the intact teliospores of T. controversa, T. caries and Tilletia foetida. In a previous study, we successfully developed monoclonal antibodies against the urediniospores of Puccinia triticina. Using immunofluorescence assays, we were successfully differentiated urediniospores of P. triticina from those of Puccinia striiformis f. sp. tritici and Puccinia graminis (Gao et al. 2013). In the present study, we report the development of an immunofluorescence assay using a novel mouse monoclonal antibody to specifically detect teliospores of T. controversa. This fluorescent molecular tool could supersede the conventional microscopic diagnoses used in quarantine surveillance protocols for dwarf bunt because such conventional methods are often confounded by overlapping morphologic characteristics of closely related species or they require complex PCR techniques. Materials and methods Production of monoclonal antibodies This study was specifically approved by the State Science and Technology Commission of Beijing, China (Institutional Animal Care and Use Committee (IACUC)) with the No. SYXK (Jing) 2010-0033. Six adult female BALB/c mice (Mus domesticus) were initially injected intraperitoneally with 200 ll of a suspension containing intact T. controversa. Monoclonal antibodies were produced according to the procedure of Gao et al. (2013), except that the suspension contained 05 mg ml 1 dry teliospores. As indicated in the Results, six monoclonal antibodies were selected for further study. PTA-ELISA A 100 ll volume of a T. controversa teliospore suspension (05 mg ml 1) in phosphate-buffered saline (PBS; 20 mmol sodium phosphate, 150 mmol NaCl, pH 74) was added to each well in Nunc Maxisorp plates (Thermo, Fisher Scientific, Beijing, China) and incubated
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for 2 h at 37°C. All protocols described in Gao et al. (2013) were followed, except that the suspension included 100 ll volume of a T. controversa teliospore suspension (05 mg ml 1) was incubated at 37°C for 2 h. Each well was read three times. Extraction of salt-soluble Tilletia controversa teliospore components Salt-soluble components were extracted from T. controversa teliospores using the procedures described by Skottrup et al. (2007). Competition assay Soluble antigen was extracted from 05 g ml 1, and T. controversa D-(1-6) (200 ll of 40 lg ml 1) was incubated with 200 ll teliospore wash from different T. controversa concentrations (produced by a 3-fold dilution series of T. controversa spores) to produce a final 08 lg ml 1 T. controversa D-(1-6) concentration. Additional steps were performed according to Skottrup et al. (2007). The degree of competition (normalized values) was determined using the average response from the triplicate measurements (A) divided by the average maximum response (A0; antibody in PBS). Intraday and interday analyses were conducted using the method described in Skottrup et al. (2007). Subtractive inhibition assay The T. controversa teliospore standards consisted of a 3-fold dilution series (200 ll of each concentration) in Eppendorf tubes, and the T. controversa D-1, D-3, D-4 and D-5 antibodies (200 ll of 40 lg ml 1) were added to yield a final concentration of 20 lg ml 1 T. controversa D-1, D-3, D-4 and D-5. Additional procedures were performed according to the previously described protocols (Skottrup et al. 2007). Protein quantification and antibody purification Protein concentration was quantified using Bradford’s dye-binding method (Bradford 1976), and antibody purification was achieved according to the manufacturer’s instructions (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Immunofluorescence assay and the sensitivity of immunofluorescence Teliospores of T. controversa and T. caries were added separately to a drop of water on two separate slides, and
a mixture of both teliospores was added to a third slide, to which poly-L-lysine (2%) was then added. Following the procedures as previously described (Gao et al. 2013), briefly, the spore suspensions were dropped onto different glass slides and allowed to dry. The slides were then washed in PBS two times for 5 min each, treated with monoclonal antibody and washed with PBS three times. The slides were then treated with PE-CY3-labelled goat anti-mouse antibody, washed three times with PBS, and treated with glycerol buffer containing 40 lg ml 1 monoclonal antibody, examined using a fluorescent microscope (Olympus B951, Tokyo, Japan) at 495 and 555 nm. To obtain the sensitivity for the detection of monoclonal antibody D-1, the previously described immunofluorescence assay was performed with the following concentrations of monoclonal antibody D-1: 40, 30, 20, 10 and 05 lg ml 1. Three replicates of each sample were tested, and the wells were read three times. Results Production and characterization of antibody-producing hybridomas Although the intact T. controversa teliospores were used as the immunogen for the production of specific mouse mAbs, the blood collected from the immunized mice could detect T. controversa in PTA-ELISA (data not shown). We produced six clonal antibody-producing cell lines from three mice with the highest blood antibody titres, and these antibody-producing lines were separately designated D-(1-6). The standard for selecting hybridomas was a positive reaction with T. controversa teliospore-coated microtitre wells. Antibody-containing culture supernatants from the mAb-producing clones were tested using PTA-ELISA for cross-reactivity against teliospores from fungi representing genera commonly found in the same environment as T. controversa, including the wheat fungal pathogens P. striiformis f. sp. tritici, P. graminis, P. triticina, T. caries and T. foetida. We used 05 mg ml 1 teliospores as antigens and a 1: 10 dilution of cells for PTA-ELISA (Table 1). The D-1 and D-2 cell lines produced IgM-isotype antibodies, and the other lines produced IgG1 antibodies (Table 2). The immunoassays developed in this study can be used for the quantification of T. controversa. Both assay calibration curves exhibited good CVs that ranged from 15 to 1667% for D-1, 357–1101% for D-3, 183–1500% for D-4 and 306–1364% for D-5 in the subtractive inhibition assay and 126–1440% for D-1, 352–1150% for D-3, 163–1390% for D-4 and 297–1390% for D-5 in the competitive assay (Table 3). The subtractive inhibition assay can be used in laboratories when teliospore
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Table 1 Cross-reactions of monoclonal antibodies determined with various fungi using plate-trapped antigen ELISA Monoclonal antibody
Tilletia controversa
D-1 D-2 D-3 D-4 D-5 D-6
085 071 100 104 121 008
Tilletia foetida
001 001 005 003 007 001
006 002 010 013 010 008
Puccinia triticina
000 000 001 003 001 001
129 007 018 009 010 254
003 001 004 001 001 009
Puccinia striiformis f. sp. tritici 008 259 009 007 007 195
Puccinia graminis
000 002 003 001 001 002
006 218 004 005 005 133
001 004 001 002 000 006
Control 003 011 003 003 003 053
000 001 000 000 001 008
Hybridoma supernatants from individual clones were tested for mAb binding to the same approximate amounts of plate-trapped spores from T. controversa and fungi representing genera commonly found in the same environment as T. controversa. For visualization, peroxidaseconjugated rabbit anti-mouse immunoglobulin was added, and the assay was developed. Absorbance values were measured at 450 nm. The results shown are the means and standard deviations from quadruple measurements. The control represents an overnight coat with PBS only.
Table 2 Differentiation of the antibody isotypes
Negative control Positive control D-1 D-2 D-3 D-4 D-5 D-6
IgA
IgM
IgG1
IgG2a
IgG2b
IgG3
0004 2624 0011 0005 0003 0001 0012 0007
0006 2635 2143 2001 0086 0083 0102 0095
0004 2617 0027 0038 1998 2140 2590 2079
0006 2639 0059 0073 0125 0267 0086 0114
0004 2654 0004 0007 0094 0066 0012 0072
0005 2668 0094 0139 0016 0018 0015 0021
quantification is needed. Similar subtractive inhibition immunosensors have successfully detected foodborne bacteria (Haines and Patel 1995; Leonard et al. 2004), and our future work includes the implementation of an assay using label-free immunosensors. The components of the teliospore surface of T. controversa were unclear, and the characteristics of the antigenic determinants’ nature were limited. Western blot analyses were not successful, which suggests that either the antigen was not extracted in
sufficient amounts or the D-(1-6) antibodies recognized a conformational epitope that was irreversibly destroyed during SDS-PAGE. Epitope characterization The soluble components were extracted from the teliospore surfaces using a PBS buffer wash, and the binding of the T. controversa D-1, D-3, D-4 and D-5 antibodies to the salt-soluble (T. controversa surface wash) and insoluble (washed T. controversa spores) material was tested. D-1, D-3, D-4 and D-5 exhibited a small reaction with the washed spores, but high reactivity was observed with the T. controversa surface wash. These results indicate that the T. controversa mAb protein antigen is salt soluble (Fig. 1a,b). A Western blot analysis was unable to detect the specific protein of T. controversa through the D-(1-6) antibodies; therefore, it was concluded that the targeted antigen was present in such a low concentration that it was difficult to detect using the D-(1-6) antibodies (Fig. S1).
Table 3 Validation data from the inhibition assay and the competitive assay Inhibition assay Teliospore concentrations 1 ng ml 1 10 ng ml 1 100 ng ml 1 1 lg ml 1 10 lg ml 1 01 mg ml 1 1 mg ml 1 10 mg ml 1
Competitive assay
Intraday CVs (100%)
Interday CVs (100%)
Intraday CVs (100%)
Interday CVs (100%)
D-1
D-3
D-4
D-5
D-1
D-3
D-4
D-5
D-1
D-3
D-4
D-5
D-1
D-3
D-4
D-5
217 217 150 151 308 261 448 625
357 502 465 636 476 777 446 714
184 183 424 500 696 357 909 1000
430 538 306 758 510 754 537 526
915 748 805 643 571 1092 1333 1667
742 1101 814 917 734 914 915 1000
950 682 652 843 710 1358 1346 1500
935 1196 769 846 991 833 949 1364
184 215 175 126 276 231 517 562
352 484 48 651 484 765 447 719
197 163 414 491 692 385 876 814
411 536 297 778 526 76 516 608
889 746 827 655 566 109 132 144
741 109 81 929 714 91 95 115
957 709 661 839 683 139 136 128
934 12 789 869 102 836 932 139
Data represent three assays performed on the same day (intraday) and on three different days (interday). Coefficients of variation (CVs) are defined as standard deviations divided by the mean from each assay (n¼ 3) 9 100%.
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(a)
(a) 3·5 3
2
A/A0
OD450
2·5
1·5 1 0·5 0 5000
20 000 40 000 80 000 16 000 32 000
2 1·8 1·6 1·4 1·2 1 0·8 0·6 0·4 0·2 0
200
100
Dilution
50
10
5
1
0·5
0·1
0·5
0·1
Teliospore concentration
(b) 0·6 (b) 1·6 1·4
0·4
1·2 1
0·3
A/A0
OD450
0·5
0·2
0·8 0·6
0·1 0
0·4 5000
0·2
20 000 40 000 80 000 16 000 32 000
0
Dilution Figure 1 The binding of anti-Tilletia controversa mAbs in diluted hybridoma media to salt-soluble components (a) and washed T. controversa spores (b). All of the results represent the mean and standard deviations from three replications. D-1, D-3, D-4 and D-5 were represented by the following symbols separately: , , and .
Assay development Competitive assays for the detection of the soluble antigens and a subtractive inhibition assay for the detection of intact T. controversa teliospores were applied. In Fig. 2a,b, normalized values (A/A0) for each teliospore standard concentration were plotted against the teliospore concentration to obtain calibration curves for each assay. Interday and intraday analyses were used to evaluate the stability of both assays, and good coefficients of variation (CVs) were obtained for all the teliospore concentration standards (Table 3). The calibration curves indicated that the subtractive inhibition assay had a broader dynamic range for T. controversa detection than the competitive assay. Furthermore, the detection limit for the subtractive inhibition assay was 01 mg ml 1, whereas the detection limit for the competitive assay was much higher (05 mg ml 1).
200
100
50
10
5
1
Teliospore concentration Figure 2 (a) Interday calibration curves from the competitive assays using D-1, D-3, D-4 and D-5 at a 1 : 45 000 dilution and D-2 and D-6 at a 1 : 1000 dilution. (b) Interday calibration curves from the subtractive inhibition assays; no subtractive inhibition was performed for D-2 or D-6. The concentration of Tilletia controversa teliospores was 05 mg ml 1, and the volumes of the antibody and teliospores were both 50 ll. The data presented are the means and standard deviations from three replications. The subtractive inhibition assay had a detection limit of 01 mg ml 1, whereas the competitive assay had a detection limit of 05 mg ml 1. D-1, D-3, D-4, D-5, D-2 and D6 were represented by the following symbols separately: , , , , and .
body generated fluorescent signals, which are shown in Fig. 3c,d. For the mixture of the two types of teliospores on the same glass slide, the monoclonal antibody D-1 (40 lg ml 1) generated a strong orange fluorescent signal for T. controversa in the outer spore wall and net ridge; however, a week green signal was generated for T. caries teliospores in the protoplasm (Fig. 3e,f), which resulted in a smaller size of the T. caries teliospores compared to T. controversa teliospores under the fluorescent microscope. The lowest concentration of monoclonal antibody D-1 that could be used to detect T. controversa teliospores was 20 lg ml 1 (Fig. 3g).
Immunofluorescence assay The reactions of the T. controversa and T. caries teliospores, which were differentiated using a scanning electron microscope (Fig. 3a,b), with the D-1 monoclonal anti-
Discussion This study produced unique monoclonal antibodies using intact T. controversa teliospores as an immunogen. The
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 3 Morphology of Tilletia controversa (a) and Tilletia caries (b) teliospores using scanning electron microscopy (SEM) with 17 0009 magnification. The reaction of monoclonal antibody D-1 (20 lg ml 1) in the immunofluorescence assay against teliospores of T. controversa (c), T. caries (d) or a mixture of teliospores from T. controversa and T. caries (e). The detection of a mixture of teliospores from T. controversa and T. caries with monoclonal antibody D-1 applied at 40 lg ml 1 (e) or 20 lg ml 1 (f, g). Absorbance at 495 nm is shown in c and d, and absorbance at 495 and 555 nm is shown in e, f and g, respectively; the magnification was 2529 in c, d, e and f, 5009 in g. Note the orange teliospores in e, f and g, which indicate T. controversa, and the green teliospores in e, f and g, which indicate T. caries. Scale bars = 20 lm.
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monoclonal antibodies were further applied in novel antibody-based and immunofluorescence assays to detect T. controversa teliospores. Our long-term goal was to detect intact teliospores in the wheat samples. Therefore, teliospores were used as the immunogen, resulting in two IgM and four IgG1-producing splenocyte-fusion hybridoma cell lines. The D-(1-6) antibodies successfully recognized the three tested wheat bunt fungi, which is consistent with previous findings that mAbs are specific to the genus (Bossi and Dewey 1992; Fuhrmann et al. 1992; Thornton et al. 2002; Schmechel et al. 2003). Rapid, simple and sensitive are essential characteristics for methods that identify and monitor pathogens. The method used in the current study focused on the dispersal stage of the T. controversa teliospores. The immunologic assay described here for T. controversa and T. caries is significantly faster and simpler than conventional methods, such as morphologic and PCR-based techniques (Liu et al. 2009; Gao et al. 2010a,b, 2011). This study produced a monoclonal antibody (D-1) using intact T. controversa teliospores as the immunogen. D-1 was then applied in a novel antibody-based assay to detect teliospores of T. controversa and T. caries. Autofluorescence was observed in the teliospores of T. controversa and T. caries using a laser scanning confocal microscope. However, Wang et al. (2012) found different autofluorescence characteristics in two teliospores with 488 nm (510–533) and 543 nm (580–600) light excitation and high-resolution layer-cut scanning of 488 nm, which indicated that the autofluorescent substances in T. controversa teliospores were primarily distributed in the outer spore wall and the net ridge, but the autofluorescent substances in T. caries teliospores were in the protoplasm. These phenomena were similar to our results. We observed a stronger signal in T. controversa than in T. caries and different colours under the excitation wavelengths of 555 and 495 nm. Additionally, the microscope used in the present study is much cheaper and easier to handle than a confocal microscope (Leica SP5). The D-1 monoclonal antibody assay distinguished T. controversa teliospores from T. caries teliospores. This result is inconsistent with previous findings that indicated that monoclonal antibodies were often specific only at the genus level (Bossi and Dewey 1992; Fuhrmann et al. 1992; Thornton et al. 2002; Schmechel et al. 2003; Skottrup et al. 2007). The quantity of monoclonal antibody required is also an important factor that determines the utility of an immunoassay for diagnosis. The immunofluorescence assay described in the current study could detect T. controversa teliospores using D-1 concentrations as low as 20 lg ml 1. We also found that a degree of fluorescence occurred in the teliospores of T. controversa (Fig. 3g). We speculated as to which signal resulted
from the specific conjugation of the D-1 monoclonal antibody and the immunogen from the teliospores of T. controversa. We developed a rapid and simple immunofluorescence assay for the detection of T. controversa and T. caries teliospores. The assay uses the D-1 monoclonal antibody, and as little as 20 lg ml 1 of the antibody is sufficient for the detection. The assay has great potential for use in on-site detection. To detect T. controversa and T. caries teliospores under field conditions, our primary work will focus on the development of a label-free and portable immunochromatographic test strip, which will provide a significant contribution towards the development of rapid, sensitive and inexpensive first-level screenings that do not require laboratory equipment or skilled personnel. Acknowledgements We appreciate Blair J. Goates, UADA-ARS, National Small Grains Germplasm Research Facility for providing Tilletia controversa and T. caries. This work was supported by Beijing Natural Science Foundation (Grant No. L140012), Multidisciplinary Cooperation Project of Beijing Nova Program (Grant No. Z141105001814116), Beijing Nova Program (Grant No. Z131105000413057) and Ministry of Agriculture, China (Grant No. CARS-03 and 201503323). Conflict of Interest The authors have no conflict of interests to declare. References Bakkeren, G., Kronstad, J.W. and Levesque, C.A. (2000) Comparison of AFLP fingerprints and ITS sequences as phylogenetic markers in Ustilaginomycetes. Mycologia, 92, 510–521. Banowetz, G.M., Trione, E.J. and Krygier, B.B. (1984) Immunological comparisons of teliospores of two wheat bunt fungi, Tilletia species, using monoclonal antibodies and antisera. Mycologia, 76, 51–62. Bokken, G.C.A.M., Corbee, R.J., van Knapen, F. and Bergwerff, A.A. (2003) Immunochemical detection of Salmonella group B, D and E using an optical surface plasmon resonance biosensor. FEMS Microbiol Lett, 222, 75–82. Bossi, R. and Dewey, F.M. (1992) Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea. Plant Pathol, 41, 472–482. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248–254.
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monoclonal antibodies for monitoring of fungal aerosols using Penicillium brevicompactum as a model fungus. J Immunol Methods, 283, 235–245. Skottrup, P., Frøkiaer, H., Hearty, S., O’Kennedy, R., Hejgaard, J., Nicolaisen, M. et al. (2007) Monoclonal antibodies for the detection of Puccinia striiformis urediniospores. Mycol Res, 111, 332–338. Thornton, C.R., Pitt, D., Wakley, G.E. and Talbot, N.J. (2002) Production of a monoclonal antibody specific to the genus Trichoderma and closely related fungi, and its use to detect Trichoderma spp. in naturally infested composts. Microbiology (Reading, England), 148, 1263–1279. Trione, E.J. and Krygier, B.B. (1977) New tests to distinguish teliospores of Tilletia controversa, the dwarf bunt of fungus, from spores of Tilletia species. Phytopathology, 67, 1166–1172. Wang, H.Y., Lin, S.M., Zhang, J.H. and Gao, Q.Z. (2012) Study on the autofluorescence in TCK and TCT with
laser scanning confocal microscope. Plant Quar 26, 35– 37. Zhang, G.M., Lu, J., Tian, Z.Z., Chen, Z.N., Deng, Q. et al. (2002) Method of extracting spores of Tilletia controversa from bran. Acta Phytopathol Sin 32, 319–325. Zhang, H., Huang, W., Qiu, Z., Shi, Q.H., Wang, X.D. et al. (2007) Primary study of application of ELISA and PCR to research of Tilletia controversa. J Xinjiang Agricult Univ 30, 88–90.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Western blots developed using D-(1-6). (a) D-1. (b) D-2. (c) D-3. (d) D-4. (e) D-5. (f) D-6. Lane 1, Tilletia foetida; lane 2, T. caries; and lane 3, T. controversa.
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ORIGINAL ARTICLE
Detection of Tilletia controversa using immunofluorescent monoclonal antibodies L. Gao1, C. Feng2, B. Li3, T. Liu1, B. Liu1 and W. Chen1 1 State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China 2 Beijing Kwinbon Biotechnology, International Information Industry Base, Beijing, China 3 Beijing Centre for Physical and Chemical Analysis (BCPCA), Yongfeng High and New Technology Industrial Base, Beijing, China
Keywords detection, immunofluorescence method, monoclonal antibody, plant pathology, Tilletia controversa, wheat smut. Correspondence Wanquan Chen and Li Gao, State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China. E-mails: [email protected]; xiaogaosx @hotmail.com 2014/1959: received 22 September 2014, revised 3 November 2014 and accepted 17 November 2014 doi:10.1111/jam.12703
Abstract Aims: Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries. Methods and Results: The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 20 lg ml 1 of the D-1 monoclonal antibody. Conclusion: This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores. Significance and Impact of the Study: This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved.
Introduction Tilletia controversa K€ uhn is a quarantined pathogenic fungus that causes dwarf bunt of wheat (Duran and Fischer 1961). This fungus is similar to Tilletia caries (DC) Tul., which causes common bunt of wheat. Both species are plant pathogens belonging to the order Ustilaginales of Basidiomycetes. The main symptoms and signs of plants infected by these species are the absence of grain underneath the lemma and the presence of black teliospores with a fishy odour. Flour from wheat grain contaminated with the fungal spores may have undesirable flavour and taste. Wheat seeds contaminated with the fungal teliospores may result in severe bunt diseases that cause yield loss and reduce grain quality. Tilletia controversa causes yield losses up to 75% in some cases (Goates 1996; Kutschera and Hossfeld 2012). Wheat plants
attacked by this species are smaller, lighter and have more shoots than healthy plants. The current detection protocol for dwarf bunt of wheat in most countries, including the USA, European Union (EU) and Australia, involves morphological and/or molecular analysis of teliospores. The germination of teliospores is required for confirmation, which incurs a delay of approx. 3 weeks. Such delay is highly unsatisfactory in a quarantine situation. Moreover, T. controversa teliospores do not germinate at 17°C in the dark; instead, they begin germinating at 5°C after a delay of at least 21 days. Microscopic observations show that T. controversa teliospores have deep and spiky reticulation, whereas those of T. caries have shallow and round reticulation (Trione and Krygier 1977). Liang et al. (1982) reported that approximately 70% of the net ridge depths of T. controversa teliospores are between 15 and 25 lm,
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and the glial sheath depths are between 2 and 3 lm, while the net ridge depths of T. caries teliospores are smaller than 12 lm, and the glial sheath depths are smaller than 15 lm. These authors also found some overlap in the teliospore sizes of both T. controversa and T. caries, as well as their net ridge and glial sheath depths. Some studies have reported the use of chemical identification methods, such as the use of periodic acid to differentiate T. controversa from T. bromi (Trione and Krygier 1977), treatment of the precipitate after filtration of teliospores with a-amylase (Li et al. 1996) and combination of a-amylase and centrifugation (Zhang et al. 2002). Molecular methods based on DNA analysis have provided useful information for the species identification of some important plant pathogens (Martin and Kistler 1990; Correll et al. 1993). PCR has provided a significant contribution to plant disease diagnosis (Ferreira et al. 1996), and this technique is particularly useful for the detection and identification of pathogens (Goodwin and Annis 1991; McManus and Jones 1995; Schaad et al. 1995). However, because T. controversa and T. caries are highly similar in the genomic sequences of the intergenic transcribed spacer (ITS) regions, several studies have failed to identify specific markers for T. controversa and T. caries based on ITS, IGS1, RPB2 and repetitive sequence-based polymerase chain reaction (PCR) (RepPCR) (Mulholland and McEwan 2000; Josefsen and Christiansen 2002; Liang et al. 2006; Gao et al. 2010b). Some progresses have been made recently in molecular detection of T. controversa. Bakkeren et al. (2000) reported that amplified fragment length polymorphism (AFLP) may provide better sequence-characterized amplified region (SCAR) markers for T. controversa. Liu et al. (2009) identified a SCAR marker for T. controversa using the AFLP method. Gao et al. (2010a, 2011) reported two SCAR markers using the ISSR method for detecting T. controversa. However, the PCR assays may not be suitable for practical applications. The problems involved in the practical use of PCR assays on a large scale include technical complexity, labour costs, quality control and investment in expensive equipment. Numerous reports have described detection methods based on monoclonal antibodies, such as those for Phytophthora cinnamomi (Hardham et al. 1986; Gabor et al. 1993), Pythium aphanidermatum (Estrada-Garcia et al. 1989), Aphanomyces invadans (Miles et al. 2003), Salmonella spp., Escherichia coli and Listeria monocytogenes (Fratamico et al. 1998; Koubova et al. 2001; Bokken et al. 2003; Leonard et al. 2004). Immunologic methods based on monoclonal antibodies can easily and rapidly produce detection results, and they do not require complex equipment or technical skills; therefore, such methods are the most suitable for on-site detection (Gao et al. 2013). 498
Previous attempts have been made to identify T. controversa and other closely related species using immunologic techniques; however, only unsatisfactory results were achieved. Such as by comparing polyclonal antisera and monoclonal antibodies, Banowetz et al. (1984) reported that both T. controversa and T. caries teliospores reacted with polyclonal sera bound the fluorescent probe to the same degree, and each monoclonal antibody derived in their study reacted strongly with both fungi, which indicates the close antigenic relationship of these species. Zhang et al. (2007) developed an ELISA and demonstrated that polyclonal antibodies derived from fine powder of the teliospores of T. controversa failed to differentiate between the intact teliospores of T. controversa, T. caries and Tilletia foetida. In a previous study, we successfully developed monoclonal antibodies against the urediniospores of Puccinia triticina. Using immunofluorescence assays, we were successfully differentiated urediniospores of P. triticina from those of Puccinia striiformis f. sp. tritici and Puccinia graminis (Gao et al. 2013). In the present study, we report the development of an immunofluorescence assay using a novel mouse monoclonal antibody to specifically detect teliospores of T. controversa. This fluorescent molecular tool could supersede the conventional microscopic diagnoses used in quarantine surveillance protocols for dwarf bunt because such conventional methods are often confounded by overlapping morphologic characteristics of closely related species or they require complex PCR techniques. Materials and methods Production of monoclonal antibodies This study was specifically approved by the State Science and Technology Commission of Beijing, China (Institutional Animal Care and Use Committee (IACUC)) with the No. SYXK (Jing) 2010-0033. Six adult female BALB/c mice (Mus domesticus) were initially injected intraperitoneally with 200 ll of a suspension containing intact T. controversa. Monoclonal antibodies were produced according to the procedure of Gao et al. (2013), except that the suspension contained 05 mg ml 1 dry teliospores. As indicated in the Results, six monoclonal antibodies were selected for further study. PTA-ELISA A 100 ll volume of a T. controversa teliospore suspension (05 mg ml 1) in phosphate-buffered saline (PBS; 20 mmol sodium phosphate, 150 mmol NaCl, pH 74) was added to each well in Nunc Maxisorp plates (Thermo, Fisher Scientific, Beijing, China) and incubated
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for 2 h at 37°C. All protocols described in Gao et al. (2013) were followed, except that the suspension included 100 ll volume of a T. controversa teliospore suspension (05 mg ml 1) was incubated at 37°C for 2 h. Each well was read three times. Extraction of salt-soluble Tilletia controversa teliospore components Salt-soluble components were extracted from T. controversa teliospores using the procedures described by Skottrup et al. (2007). Competition assay Soluble antigen was extracted from 05 g ml 1, and T. controversa D-(1-6) (200 ll of 40 lg ml 1) was incubated with 200 ll teliospore wash from different T. controversa concentrations (produced by a 3-fold dilution series of T. controversa spores) to produce a final 08 lg ml 1 T. controversa D-(1-6) concentration. Additional steps were performed according to Skottrup et al. (2007). The degree of competition (normalized values) was determined using the average response from the triplicate measurements (A) divided by the average maximum response (A0; antibody in PBS). Intraday and interday analyses were conducted using the method described in Skottrup et al. (2007). Subtractive inhibition assay The T. controversa teliospore standards consisted of a 3-fold dilution series (200 ll of each concentration) in Eppendorf tubes, and the T. controversa D-1, D-3, D-4 and D-5 antibodies (200 ll of 40 lg ml 1) were added to yield a final concentration of 20 lg ml 1 T. controversa D-1, D-3, D-4 and D-5. Additional procedures were performed according to the previously described protocols (Skottrup et al. 2007). Protein quantification and antibody purification Protein concentration was quantified using Bradford’s dye-binding method (Bradford 1976), and antibody purification was achieved according to the manufacturer’s instructions (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Immunofluorescence assay and the sensitivity of immunofluorescence Teliospores of T. controversa and T. caries were added separately to a drop of water on two separate slides, and
a mixture of both teliospores was added to a third slide, to which poly-L-lysine (2%) was then added. Following the procedures as previously described (Gao et al. 2013), briefly, the spore suspensions were dropped onto different glass slides and allowed to dry. The slides were then washed in PBS two times for 5 min each, treated with monoclonal antibody and washed with PBS three times. The slides were then treated with PE-CY3-labelled goat anti-mouse antibody, washed three times with PBS, and treated with glycerol buffer containing 40 lg ml 1 monoclonal antibody, examined using a fluorescent microscope (Olympus B951, Tokyo, Japan) at 495 and 555 nm. To obtain the sensitivity for the detection of monoclonal antibody D-1, the previously described immunofluorescence assay was performed with the following concentrations of monoclonal antibody D-1: 40, 30, 20, 10 and 05 lg ml 1. Three replicates of each sample were tested, and the wells were read three times. Results Production and characterization of antibody-producing hybridomas Although the intact T. controversa teliospores were used as the immunogen for the production of specific mouse mAbs, the blood collected from the immunized mice could detect T. controversa in PTA-ELISA (data not shown). We produced six clonal antibody-producing cell lines from three mice with the highest blood antibody titres, and these antibody-producing lines were separately designated D-(1-6). The standard for selecting hybridomas was a positive reaction with T. controversa teliospore-coated microtitre wells. Antibody-containing culture supernatants from the mAb-producing clones were tested using PTA-ELISA for cross-reactivity against teliospores from fungi representing genera commonly found in the same environment as T. controversa, including the wheat fungal pathogens P. striiformis f. sp. tritici, P. graminis, P. triticina, T. caries and T. foetida. We used 05 mg ml 1 teliospores as antigens and a 1: 10 dilution of cells for PTA-ELISA (Table 1). The D-1 and D-2 cell lines produced IgM-isotype antibodies, and the other lines produced IgG1 antibodies (Table 2). The immunoassays developed in this study can be used for the quantification of T. controversa. Both assay calibration curves exhibited good CVs that ranged from 15 to 1667% for D-1, 357–1101% for D-3, 183–1500% for D-4 and 306–1364% for D-5 in the subtractive inhibition assay and 126–1440% for D-1, 352–1150% for D-3, 163–1390% for D-4 and 297–1390% for D-5 in the competitive assay (Table 3). The subtractive inhibition assay can be used in laboratories when teliospore
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Table 1 Cross-reactions of monoclonal antibodies determined with various fungi using plate-trapped antigen ELISA Monoclonal antibody
Tilletia controversa
D-1 D-2 D-3 D-4 D-5 D-6
085 071 100 104 121 008
Tilletia foetida
001 001 005 003 007 001
006 002 010 013 010 008
Puccinia triticina
000 000 001 003 001 001
129 007 018 009 010 254
003 001 004 001 001 009
Puccinia striiformis f. sp. tritici 008 259 009 007 007 195
Puccinia graminis
000 002 003 001 001 002
006 218 004 005 005 133
001 004 001 002 000 006
Control 003 011 003 003 003 053
000 001 000 000 001 008
Hybridoma supernatants from individual clones were tested for mAb binding to the same approximate amounts of plate-trapped spores from T. controversa and fungi representing genera commonly found in the same environment as T. controversa. For visualization, peroxidaseconjugated rabbit anti-mouse immunoglobulin was added, and the assay was developed. Absorbance values were measured at 450 nm. The results shown are the means and standard deviations from quadruple measurements. The control represents an overnight coat with PBS only.
Table 2 Differentiation of the antibody isotypes
Negative control Positive control D-1 D-2 D-3 D-4 D-5 D-6
IgA
IgM
IgG1
IgG2a
IgG2b
IgG3
0004 2624 0011 0005 0003 0001 0012 0007
0006 2635 2143 2001 0086 0083 0102 0095
0004 2617 0027 0038 1998 2140 2590 2079
0006 2639 0059 0073 0125 0267 0086 0114
0004 2654 0004 0007 0094 0066 0012 0072
0005 2668 0094 0139 0016 0018 0015 0021
quantification is needed. Similar subtractive inhibition immunosensors have successfully detected foodborne bacteria (Haines and Patel 1995; Leonard et al. 2004), and our future work includes the implementation of an assay using label-free immunosensors. The components of the teliospore surface of T. controversa were unclear, and the characteristics of the antigenic determinants’ nature were limited. Western blot analyses were not successful, which suggests that either the antigen was not extracted in
sufficient amounts or the D-(1-6) antibodies recognized a conformational epitope that was irreversibly destroyed during SDS-PAGE. Epitope characterization The soluble components were extracted from the teliospore surfaces using a PBS buffer wash, and the binding of the T. controversa D-1, D-3, D-4 and D-5 antibodies to the salt-soluble (T. controversa surface wash) and insoluble (washed T. controversa spores) material was tested. D-1, D-3, D-4 and D-5 exhibited a small reaction with the washed spores, but high reactivity was observed with the T. controversa surface wash. These results indicate that the T. controversa mAb protein antigen is salt soluble (Fig. 1a,b). A Western blot analysis was unable to detect the specific protein of T. controversa through the D-(1-6) antibodies; therefore, it was concluded that the targeted antigen was present in such a low concentration that it was difficult to detect using the D-(1-6) antibodies (Fig. S1).
Table 3 Validation data from the inhibition assay and the competitive assay Inhibition assay Teliospore concentrations 1 ng ml 1 10 ng ml 1 100 ng ml 1 1 lg ml 1 10 lg ml 1 01 mg ml 1 1 mg ml 1 10 mg ml 1
Competitive assay
Intraday CVs (100%)
Interday CVs (100%)
Intraday CVs (100%)
Interday CVs (100%)
D-1
D-3
D-4
D-5
D-1
D-3
D-4
D-5
D-1
D-3
D-4
D-5
D-1
D-3
D-4
D-5
217 217 150 151 308 261 448 625
357 502 465 636 476 777 446 714
184 183 424 500 696 357 909 1000
430 538 306 758 510 754 537 526
915 748 805 643 571 1092 1333 1667
742 1101 814 917 734 914 915 1000
950 682 652 843 710 1358 1346 1500
935 1196 769 846 991 833 949 1364
184 215 175 126 276 231 517 562
352 484 48 651 484 765 447 719
197 163 414 491 692 385 876 814
411 536 297 778 526 76 516 608
889 746 827 655 566 109 132 144
741 109 81 929 714 91 95 115
957 709 661 839 683 139 136 128
934 12 789 869 102 836 932 139
Data represent three assays performed on the same day (intraday) and on three different days (interday). Coefficients of variation (CVs) are defined as standard deviations divided by the mean from each assay (n¼ 3) 9 100%.
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(a)
(a) 3·5 3
2
A/A0
OD450
2·5
1·5 1 0·5 0 5000
20 000 40 000 80 000 16 000 32 000
2 1·8 1·6 1·4 1·2 1 0·8 0·6 0·4 0·2 0
200
100
Dilution
50
10
5
1
0·5
0·1
0·5
0·1
Teliospore concentration
(b) 0·6 (b) 1·6 1·4
0·4
1·2 1
0·3
A/A0
OD450
0·5
0·2
0·8 0·6
0·1 0
0·4 5000
0·2
20 000 40 000 80 000 16 000 32 000
0
Dilution Figure 1 The binding of anti-Tilletia controversa mAbs in diluted hybridoma media to salt-soluble components (a) and washed T. controversa spores (b). All of the results represent the mean and standard deviations from three replications. D-1, D-3, D-4 and D-5 were represented by the following symbols separately: , , and .
Assay development Competitive assays for the detection of the soluble antigens and a subtractive inhibition assay for the detection of intact T. controversa teliospores were applied. In Fig. 2a,b, normalized values (A/A0) for each teliospore standard concentration were plotted against the teliospore concentration to obtain calibration curves for each assay. Interday and intraday analyses were used to evaluate the stability of both assays, and good coefficients of variation (CVs) were obtained for all the teliospore concentration standards (Table 3). The calibration curves indicated that the subtractive inhibition assay had a broader dynamic range for T. controversa detection than the competitive assay. Furthermore, the detection limit for the subtractive inhibition assay was 01 mg ml 1, whereas the detection limit for the competitive assay was much higher (05 mg ml 1).
200
100
50
10
5
1
Teliospore concentration Figure 2 (a) Interday calibration curves from the competitive assays using D-1, D-3, D-4 and D-5 at a 1 : 45 000 dilution and D-2 and D-6 at a 1 : 1000 dilution. (b) Interday calibration curves from the subtractive inhibition assays; no subtractive inhibition was performed for D-2 or D-6. The concentration of Tilletia controversa teliospores was 05 mg ml 1, and the volumes of the antibody and teliospores were both 50 ll. The data presented are the means and standard deviations from three replications. The subtractive inhibition assay had a detection limit of 01 mg ml 1, whereas the competitive assay had a detection limit of 05 mg ml 1. D-1, D-3, D-4, D-5, D-2 and D6 were represented by the following symbols separately: , , , , and .
body generated fluorescent signals, which are shown in Fig. 3c,d. For the mixture of the two types of teliospores on the same glass slide, the monoclonal antibody D-1 (40 lg ml 1) generated a strong orange fluorescent signal for T. controversa in the outer spore wall and net ridge; however, a week green signal was generated for T. caries teliospores in the protoplasm (Fig. 3e,f), which resulted in a smaller size of the T. caries teliospores compared to T. controversa teliospores under the fluorescent microscope. The lowest concentration of monoclonal antibody D-1 that could be used to detect T. controversa teliospores was 20 lg ml 1 (Fig. 3g).
Immunofluorescence assay The reactions of the T. controversa and T. caries teliospores, which were differentiated using a scanning electron microscope (Fig. 3a,b), with the D-1 monoclonal anti-
Discussion This study produced unique monoclonal antibodies using intact T. controversa teliospores as an immunogen. The
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 3 Morphology of Tilletia controversa (a) and Tilletia caries (b) teliospores using scanning electron microscopy (SEM) with 17 0009 magnification. The reaction of monoclonal antibody D-1 (20 lg ml 1) in the immunofluorescence assay against teliospores of T. controversa (c), T. caries (d) or a mixture of teliospores from T. controversa and T. caries (e). The detection of a mixture of teliospores from T. controversa and T. caries with monoclonal antibody D-1 applied at 40 lg ml 1 (e) or 20 lg ml 1 (f, g). Absorbance at 495 nm is shown in c and d, and absorbance at 495 and 555 nm is shown in e, f and g, respectively; the magnification was 2529 in c, d, e and f, 5009 in g. Note the orange teliospores in e, f and g, which indicate T. controversa, and the green teliospores in e, f and g, which indicate T. caries. Scale bars = 20 lm.
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monoclonal antibodies were further applied in novel antibody-based and immunofluorescence assays to detect T. controversa teliospores. Our long-term goal was to detect intact teliospores in the wheat samples. Therefore, teliospores were used as the immunogen, resulting in two IgM and four IgG1-producing splenocyte-fusion hybridoma cell lines. The D-(1-6) antibodies successfully recognized the three tested wheat bunt fungi, which is consistent with previous findings that mAbs are specific to the genus (Bossi and Dewey 1992; Fuhrmann et al. 1992; Thornton et al. 2002; Schmechel et al. 2003). Rapid, simple and sensitive are essential characteristics for methods that identify and monitor pathogens. The method used in the current study focused on the dispersal stage of the T. controversa teliospores. The immunologic assay described here for T. controversa and T. caries is significantly faster and simpler than conventional methods, such as morphologic and PCR-based techniques (Liu et al. 2009; Gao et al. 2010a,b, 2011). This study produced a monoclonal antibody (D-1) using intact T. controversa teliospores as the immunogen. D-1 was then applied in a novel antibody-based assay to detect teliospores of T. controversa and T. caries. Autofluorescence was observed in the teliospores of T. controversa and T. caries using a laser scanning confocal microscope. However, Wang et al. (2012) found different autofluorescence characteristics in two teliospores with 488 nm (510–533) and 543 nm (580–600) light excitation and high-resolution layer-cut scanning of 488 nm, which indicated that the autofluorescent substances in T. controversa teliospores were primarily distributed in the outer spore wall and the net ridge, but the autofluorescent substances in T. caries teliospores were in the protoplasm. These phenomena were similar to our results. We observed a stronger signal in T. controversa than in T. caries and different colours under the excitation wavelengths of 555 and 495 nm. Additionally, the microscope used in the present study is much cheaper and easier to handle than a confocal microscope (Leica SP5). The D-1 monoclonal antibody assay distinguished T. controversa teliospores from T. caries teliospores. This result is inconsistent with previous findings that indicated that monoclonal antibodies were often specific only at the genus level (Bossi and Dewey 1992; Fuhrmann et al. 1992; Thornton et al. 2002; Schmechel et al. 2003; Skottrup et al. 2007). The quantity of monoclonal antibody required is also an important factor that determines the utility of an immunoassay for diagnosis. The immunofluorescence assay described in the current study could detect T. controversa teliospores using D-1 concentrations as low as 20 lg ml 1. We also found that a degree of fluorescence occurred in the teliospores of T. controversa (Fig. 3g). We speculated as to which signal resulted
from the specific conjugation of the D-1 monoclonal antibody and the immunogen from the teliospores of T. controversa. We developed a rapid and simple immunofluorescence assay for the detection of T. controversa and T. caries teliospores. The assay uses the D-1 monoclonal antibody, and as little as 20 lg ml 1 of the antibody is sufficient for the detection. The assay has great potential for use in on-site detection. To detect T. controversa and T. caries teliospores under field conditions, our primary work will focus on the development of a label-free and portable immunochromatographic test strip, which will provide a significant contribution towards the development of rapid, sensitive and inexpensive first-level screenings that do not require laboratory equipment or skilled personnel. Acknowledgements We appreciate Blair J. Goates, UADA-ARS, National Small Grains Germplasm Research Facility for providing Tilletia controversa and T. caries. This work was supported by Beijing Natural Science Foundation (Grant No. L140012), Multidisciplinary Cooperation Project of Beijing Nova Program (Grant No. Z141105001814116), Beijing Nova Program (Grant No. Z131105000413057) and Ministry of Agriculture, China (Grant No. CARS-03 and 201503323). Conflict of Interest The authors have no conflict of interests to declare. References Bakkeren, G., Kronstad, J.W. and Levesque, C.A. (2000) Comparison of AFLP fingerprints and ITS sequences as phylogenetic markers in Ustilaginomycetes. Mycologia, 92, 510–521. Banowetz, G.M., Trione, E.J. and Krygier, B.B. (1984) Immunological comparisons of teliospores of two wheat bunt fungi, Tilletia species, using monoclonal antibodies and antisera. Mycologia, 76, 51–62. Bokken, G.C.A.M., Corbee, R.J., van Knapen, F. and Bergwerff, A.A. (2003) Immunochemical detection of Salmonella group B, D and E using an optical surface plasmon resonance biosensor. FEMS Microbiol Lett, 222, 75–82. Bossi, R. and Dewey, F.M. (1992) Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea. Plant Pathol, 41, 472–482. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248–254.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Western blots developed using D-(1-6). (a) D-1. (b) D-2. (c) D-3. (d) D-4. (e) D-5. (f) D-6. Lane 1, Tilletia foetida; lane 2, T. caries; and lane 3, T. controversa.
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