An Immunofluorescence Assay to Detect Soybean Rust Urediniospores Fulya Baysal , Anne Dorrance , Melanie L. Lewis Ivey , Douglas Luster , Reid Frederick , Jill Czarnecki , Michael Boehm and Sally A. Miller 1
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Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster; 2USDA ARS Foreign Diseases and Weed Science Research Unit, Frederick, MD; 3 Naval Medical Research Center, Biological Defense Research Directorate, Silver Spring, MD; 4Department of Plant Pathology, The Ohio State University, Columbus
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[email protected],
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Introduction Soybean rust causes significant yield loss to soybean crops in Asia, Africa, Australia, and nearly all tropical countries in the eastern hemisphere where soybeans are grown (1). Rust is considered to be a major threat to soybean production in the United States (5), especially with the identification of Phakopsora pachyrhizi in Louisiana in November 2004 (4). P. pachyrhizi produces air-dispersed urediniospores, has a broad host range among wild and cultivated plants and has been found in all major soybean production areas of the world. Detection and enumeration of airborne spores have been achieved by microscopic examination of surfaces on which spores were impacted, for example using spore traps. However, such methods require considerable amounts of time and expertise if accurate counts are to be obtained and are not practical or accurate where several related species of fungi that have morphologically similar spore types occur together. Technological advances in fungal diagnostics, using either antibody -or nucleic acid- based assays, allow accurate differentiation of fungal species (6,2).
Immunofluorescence assay Table Table 3. Optimization of immunofluorescence 4. Optimization of immunofluorescence assay parameters assay parameters Fluorescence intensity* optimization. Several paParameter Variable 1 2 3 4 rameters were evaluated to Primary antiserum SBR1A ! SBR2 ! optimize the immunofluores- Dilution buffer type PBS ! PBS-Tween ! cence staining procedure in Primary antiserum dilution 1: 100 !** 1: 250 !** microcemtrifuge tubes and on 1: 500 ! 1: 100 !** glass slides with petroleum jelly Secondary antiserum dilution 1: 250 !** 1: 500 ! or double-sided tape, including Primary antiserum incubation time 1 hr ! 2 hr ! primary antiserum, concen3hr ! 1 hr ! tration, incubation time and Secondary antiserum incubation time 2 hr ! 3hr !** incubation temperature of Primary antiserum incubation temperature 22°C ! 37°C ! primary and secondary an- Secondary antiserum incubation temperature 22°C ! 37°C ! tisera, use of a blocking step, Blocking step (Blocking buffer; Yes ! serum) No ! and type of dilution and wash PBS+2%BSA+5%Goat Washing step PBS ! PBS-Tween ! buffers (Table 3). Distilled water ! * Fluorescence of urediniospores was rated visually using a scale of 1 to 4: 1=no fluorescence, 2=weak fluorescence, 3=good fluorescence (clearly positive), 4=brilliant fluorescence. ** Background was cloudy.
Objective To develop an indirect immunofluorescence assay to detect urediniospores of the soybean rust pathogen P. pachyrhizi for spore trapping systems.
Materials & Methods Microbial cultures. P. pachyrhizi and P. meibomiae isolates were maintained and propagated at the USDA-ARS Foreign Disease- Weed Science Research Unit. Germinated and ungerminated urediniospores were killed in ethylene oxide prior to use in antiserum and assay development. All other fungal and bacterial cultures were collected and maintained at The Ohio State University.
(Method 1-microcentrifuge tubes) Immunofluorescence of urediniospores of P. pacchryzi
Immunofluorescence of urediniospores of P. pacchryzi (Method 1-microcentrifuge tubes) Deliver 1 mm urediniospores from 10 !l glass PCR micropipette into a 1.5-ml microcentrifuge tube with 1 ml PBS-Tween buffer
Immunofluorescence of urediniospores of2-glass P. pacchryzi Immunofluorescence of urediniospores of P. pacchryzi (Method slides) Apply double-sided tape or petroleum jelly onto a standard glass slide and expel urediniospores from 10 !l glass PCR micropipette
Incubate overnight to hydrate urediniospores
Apply 1°antiserum SBR1A or 2 (1:500) in PBS-Tween
Centrifuge suspension at 16,000 x g for 5 min
Incubate 2 hr at 22 °C
Add 1 ml PBS-T in tube and break up pellets by pipetting up and down 5-10 times
Wash 6X in PBS-Tween
Centrifuge suspension at 16,000 x g for 3 min
Apply 2°antiserum (Alexa Fluor 488 goat anti-rabbit IgG (H+L), 2 mg/ml, 1:500) in PBS-Tween
Add 1°antiserum SBR1A or SBR2 (1:500) in PBS-Tween
Incubate 1 hr at 22 °C in dark
Incubate 2 hr at 22 °C on a plate shaker at approximately 230 strokes/min
Wash 6X in PBS-Tween
Centrifuge and wash 3X using PBS-Tween Apply 2°antiserum (Alexa Fluor 488 goat anti-rabbit IgG (H+L), 2 mg/ml, 1:500) in PBS-Tween Incubate 1 hr, in the dark at 22 ºC on a plate shaker at approximately 230 strokes/min Centrifuge and wash in 3X PBS-Tween Resuspend the pellet in 25 !l PBS-Tween and mix by pipetting
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Table4. 3. Specificity of polyclonal antiserum SBR 1A Antibody against fungal spores in an Table Specificity of polyclonal antiserum SBR1A against specifity-IFA. Polyclonal fungalspores inassay an immunofluorescence assay (IFA) antiserum SBR1A was tested using immunofluorescence (IFA) Fungal pathogen
Ustilago tritici Puccinia graminis f. sp. tritici Phragmidium sp. Alternaria sp. Botrytis cinerea Phakopsora pachyrhizi
Host
Wheat Wheat Rose Tomato Tomato Soybean
Reactivity in IFA with SBR1A +
the immunofluorescence assay for its cross-reactivity with spores of fungal pathogens (Table 4).
- Indicates no fluorescence. + Indicates brilliant fluorescence.
Immunomonitoring field-trapped soybean rust-like spores. Standard glass slides to which double-stick tape (Scotch® Permanent Double-Sided Tape, 3M Corporation, St. Paul, MN) was affixed, or that were coated with petroleum jelly, were placed in spore traps in Georgia, Kentucky and Ohio soybean fields in 2006. Slides were collected at 7-day intervals and processed by Method 2 (see Results) using antiserum SBR1A. Slides were treated before shipment to kill spores.v
Polyclonal antiserum production. Antibody production was managed by the U.S. Navy Biological Defense Research Center in Silver Spring, MD. Four different extracts of P. pachyrhizi urediospores [intact ungerminated (SBR1A, SBR1B), intact germinated (SBR2) and pulverized, germinated(SBR3) ure- Table 1. Polyclonal antisera developed in response to extracted antigens of P. pachyrhizi diniospores, and walls Table 1. Polyclonal antisera developed in response to antigens of P. pachyrhizi of germinated uredin- Antiserum Immunogen preparation Concentration Polyclonal antiserum specificity. Antisera SBR1A and SBR1B and antiserum SBR2 reacted Ungerminated urediniospores 20 mg dry weight/ml iospores (SBR4)] were SBR1A specifically with Phakopsora species in ELISA. SBR1A and SPR1B reacted positively with Ungerminated urediniospores 10 mg dry weight /ml used for antiserum SBR1B P. pachyrhizi and P. meibomiae in ELISA, while SBR2 reacted only with P. pachyrhizi. Antisera SBR2 Intact germinated urediniospores 1.0 mg protein/ml production in New SBR3 and SBR4 cross-reacted with basidiomycete fungal pathogens (Table 2). SBR1A, SBR1B SBR3 Pulverized germinated urediniospores 1.0 mg protein/ml Zealand white rabbits SBR4 and SBR2 bound to P. pachyrhizi and P. meibomiae urediniospores in IFA, although SBR2 Walls of germinated urediniospores 1.25 mg protein/ml (Table 1). binding to P. meibomiae was weaker than to P. pachyrhizi. None of the non-target spores reacted positively with SBR1A antiserum in IFA (Table 4). Antiserum specifity-ELISA. Five polyclonal antisera developed in response to P. pachyrhizi immunogens were evaluated for specificity using a standard ELISA protocol with pure culture Immunofluorescence assay optimization. Method 1 was developed for immunofluoresextracts of fungal soybean pathogens (2 ug/ml) and bacterial pathogens (cfu/ml= 106 and 102). cence staining of P. pachyrhizi urediniospores in microcentrifuge tubes based on optimization of 10 assay parameters (Table 3). Method 2 was developed for immunofluorescence staining Five samples of of urediniospores affixed to a standard glass microscope slide on double-sided tape or Table 2. Specificity of polyclonal antisera developed in response to P.pachyrhizi diseased soybean antigens petroleum jelly. Non-specific binding of the 2º antiserum to the petroleum jelly resulted in Table 2. Specificity of polyclonal antisera developed in response to P.pachyrhizi antigens tissue collectExtract Reactivity in ELISA SBR1A SBR1B SBR2 SBR3 SBR4 ed from fields in background fluorescence and weak fluo- A B Figure 1. Visualization of urediniorescence of P. pachyrhizi urediniospores P. pachyrhizi spore extract + + + + + northwest Ohio spores of Phakopsora pachyrhizi on P. meibomiae spore extract + + (Fig. 1). Therefore, spores were lifted Pseudomonas syringae pv. syringae & phaseolicola, glass slides by immunofluorescence and two samples Xanthomonas campestris pv. glycines, Clavibacter from petroleum jelly using double-sid(IF) microscopy with SBR1A (1:500) of diseased wheat michiganensis subsp. insidiosus, Botrytis cinerea, using A) double-sided tape and B) ed tape, which was then fixed onto a Colletotrichum acutatum, C. gleosporiodes, Ustilago tritici tissue were also petroleum jelly. Rhizoctonia solani + + new glass slide for immunomonitoring. evaluated (Table Puccinia graminis f. sp. tritici + Between 22.3 and 30.3% of uredinioDiseased soybean tissue: bacterial pustule, frogeye leaf 2). spot, bacterial spot, Septoria leaf spot and powdery mildew spores lifted from Symptomless soybean tissue: 183 cultivars petroleum jelly-coated Table 5. Adherence of P. pachyrhizi urediniospores to glass-slides during + Indicates a yellow color equal to or greater than the positive control when evaluated by eye. - Indicates no yellow color detected when evaluated by eye or absorbance value less than or equal to absorbance value Table 5. Adherence of P. pachyrhizi urediniospores to glass-slides during an slides were lost during an immunofluorescence assay (IFA) of the negative control. immunofluorescence assay (IFA) the assay, while only Petroleum jelly + double-sided tape Double-sided tape Experiment I Experiment II Experiment I Experiment II 3.5 4.7% of uredinImmunofluorescence microscopy. Images were captured using a Leica DMIRB epifluorescence Initial # % lost Initial # % lost Initial # % lost Initial # % lost Slide Slide spores spores spores spores spores spores spores spores iospores affixed dimicroscope equipped with an Optronics Magnafire camera. A USH-102DH-100W ultra-highA 155 14.4 20 26.3 A 144 4.2 128 2.3 125 26.7 17 23.5 B 118 3.4 154 3.9 pressure mercury lamp was used as the exciting light source. Images were viewed in the blue rectly to tape were lost BC 131 26.1 16 41.7 C 134 7.5 145 4.3 (Table 5). D 136 26.4 22 28.6 D 128 4.7 170 3.3 excitation range (filter set I3- excitation filter BP450–490 nm and emission filter BP515 nm); E 34 17.7 19 31.6 E 140 3.6 139 3.6 the exposure time for image capture was always 1.037 seconds. Mean 22.3 30.3 Mean 4.7 3.5
Results
(Method 2 -glass slides)
Immunomonitoring. Under bright field microscopy, a wide range of airborne spores, pollen, bacteria, insect and particulate material was observed on each of the double-sided tape and petroleum jelly-coated glass slides examined (Fig. 2 A, C, E, G). Under epifluorescence microscopy, soybean rust like spores trapped using double-sided tape or petroleum jelly+double sided tape-coated glass slides and immunolabeled with polyclonal antibody SBR1A and Alexa Fluor 488 goat anti-rabbit IgG (H+L) were clearly fluorescent and identifiable as P. pachyrizi (3) (Fig. 2 B, D, F, H). Of the 36 samples Table 6. Soybean rust immunofluorescent assay results for air-trapped spores on slides (slides) received from Table 6. Soybean rust immunofluorescent assay results for air-trapped from Kentucky and 2006 Ohio, 2006 spores on slides fromGeorgia, Georgia, Kentucky and Ohio, air samplers located in Georgia, Kentucky and Ohio in 2006, nine were positive for P. pachyrhizi using the IFA shown in Method 1 (Table 6). Slides name
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H
Figure 2. Immunofluorescence
assay of field-trapped “soybean rust-like” urediniospores on glass slides with double-sided tape. Images in the left column were taken using bright field microscopy; images in the right column were taken of the same slides using epifluorescence microscopy.
Conclusions
GA 1 GA 2 GA 3 GA 4 GA 5 GA 6 GA 7 GA 8 GA 9 GA 10 GA 11 GA 12 GA 13 GA 14 GA 15 GA 16 KY 1 KY 2 KY 3 KY 4 OH 1 OH 2 OH 3 OH 4 OH 5 OH 6 OH 7 OH 8 OH 9 OH 10 OH 11 OH 12 OH 13 OH 14 OH 15 OH 16
Date
Slide type
08.16.2006- 08.23.2006 08.02.2006-08.09.2006 08.15.2006- 08.22.2006 08.01.2006- 08.08.2006 07.31.2006- 08.07.2006 08.14.2006- 08.21.2006 07.31.2006- 08.07.2006 08.14.2006- 08.21.2006 08.01.2006- 08.08.2006 08.10.2006- 08.17.2006 08.10.2006- 08.17.2006 08.02.2006- 08.09.2006 08.16.2006-08.23.2006 08.15.2006- 08.22.2006 08.01.2006- 08.08.2006 08.10.2006- 08.17.2006 07.28.2006- 08.04.2006 08.04.2006- 08.11.2006 07.11.2006- 07.21.2006 08.21.2006- 08.25.2006 09.01.2006- 09.08.2006 08.24.2006- 08.30.2006 10.10.2006- 10.17.2006 09.09.2006- 09.16.2006 09.16.2006- 09.23.2006 07.25.2006- 07.31.2006 No date No date 09.29.2006- 10.05.2006 08.21.2006- 08.23.2006 No date 09.05.2006- 09.11.2006 09.11.2006- 09.18.2006 09.05.2006- 09.11.2006 09.25.2006- 10.03.2006 10.03.2006- 10.10.2006
Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Petroleum jelly Petroleum jelly Petroleum jelly Petroleum jelly Petroleum jelly Petroleum jelly Petroleum jelly Petroleum jelly Petroleum jelly Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape Double sided tape
Soybean rust like spores (fluorescent) + + + + + + + + +
• Polyclonal antisera developed against urediniospore antigens of P. pachyrizi bound to urediniospores of P. pachyrhizi in microcentrifuge tubes and on glass slides with double-sided tape and petroleum jelly. Antisera SBR1A, SBR1B and SBR2 were specific for Phakopsora spp. and can be used effectively in IFA. • Double-sided tape was superior to petroleum jelly as a means of affixing urediniospores to glass microscope slides. Fewer urediniospores were lost from the former during immunofluorescence staining, and non-specific background was nearly always absent when double-sided tape was used. The secondary antiserum could not be completely removed from petroleum jelly-coated slides by washing. Further, the petroleum jelly appeared to prevent binding of primary or secondary antisera to urediniospores. • Preliminary results from field tests demonstrate that the IFA combined with spore trapping can be an effective tool in monitoring P. pachyrhizi movement during the soybean growing season.
R eferences
A cknowledgements
1. Asian Vegetable Research and Development Center, 1992. Library Bibliography Series 4-1. Publication No. 92-372. 2. Beck J, Ligon JJ, Ettienne L, Binder A, 1996. BCPC Symposium No. 65. Farnham, UK: British Crop Protection Council, 111-8. 3. Bonde, M. R., and Brown, M. F.1980. Can. J. Microbiology 26:14431449. 4. Schneider R.W., Hollier C.A., Whitam H.K., Palm M.E., McKemy J.M., Hernandez J.R., Levy L., DeVries-Paterson R., 2005. Plant Dis., and Volume: 89, p. 773. 5. Sinclair J.B., 1989. Plant Dis., Volume: 73, Issue: 7 pp. 604-606. 6. Werres, S., Steffens, C., 1994. Journal of Applied Biology 125, 615-643.
This project was sponsored through soybean check-off dollars from the Iowa Soybean Promotion Board and United Soybean Board for a project entitled “Hand-Held Assays for Field Friendly Diagnosis of Soybean Rust”.
