Skip to main content
Download PDF

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections

Plant Methods volume 12, Article number: 37 (2016) Cite this article

Abstract

Background

Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here.

Results

To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues.

Conclusions

This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Background

Rust fungi (Basidiomycota, Pucciniomycotina) are obligate parasites that infect many species of vascular plants [1, 2]. Recent studies in this laboratory have focused on Chrysanthemum white rust, caused by Puccinia horiana, Gladiolus rust, caused by Uromyces transversalis and Asian soybean rust, caused by Phakopsora pachyrhizi. [3, 4]. Studies on the interactions between these pathogenic fungi and plants would benefit from approaches that allow visualization of the pathogen within host plant tissue, including the use of in situ hybridization (ISH) technology.

ISH was first used to localize specific DNA sequences on chromosomes using probes labeled with radioisotopes [5, 6]. The technique was later used for the detection of viral particles and high copy number mRNA in cultured cells and sectioned tissue making it useful for localizing gene expression patterns [6–10]. Non-radioactive methods were also developed that employed digoxygenin or biotin-conjugated nucleotides allowing for detection with antibody and streptavidin conjugates [9–11]. The development of non-radioactive methods eventually gave rise to fluorescent ISH (FISH), which employs various fluorescent-labeling techniques to produce fluorescence at the site of hybridization [12–14]. Non-radioactive ISH methods have been used to accomplish such tasks as chromosome mapping [14–16], gene expression localization [6, 8, 9, 11], and pathogen detection [17–26]. Chromogenic ISH (CISH) is an alternative to FISH that has become popular in diagnostic laboratories studying human pathogens [19–24].

Recently ISH has been used to identify microorganisms by targeting rRNA [18, 21, 24, 27–32]. The abundance of rRNA in the cell offers ample target for probes to bind to allowing for clear visualization of microorganisms within the sample being assayed. This technique has been used to identify and characterize prokaryotic organisms [27, 29, 32] and has been used for the detection of fungi in cultures [24, 31], plant [17, 18, 20, 30] and animal tissue [19, 21–23, 25].

The aim of this study was to develop a basic ISH protocol that plant pathologists can use for the detection of rust pathogens in paraffin embedded plant tissue. Here we report the development of an optimized protocol tested on three genera of rust fungi from three plant species. The results of this investigation demonstrate the utility of ISH as a tool for visualizing the infection of plant tissue by rust fungi.

Methods

Generation of infected leaf material

Leaves of Chrysanthemum × morifolium infected with Puccinia horiana isolate PA-11 [33], Gladiolus × hortulanus infected with Uromyces transversalis isolate CA-07 [34], and Glycine max infected with Phakopsora pachyrhizi isolate Taiwan 72-1 [35] were generated for experiments as described. These plant pathogens are regulated under the Plant Protection Act of 2000 and inoculations were conducted in a BSL-3 Plant Pathogen Containment Facility at Ft. Detrick MD under conditions specified in valid USDA APHIS PPQ 526 permits.

DNA extraction and sequencing for ISH probe design

Genomic DNA was extracted from 50 mg of fungal basidiospores of Puccinia horiana using a hexadecyltrimethylammonium bromide (CTAB) extraction protocol beginning with 1 min of homogenization in 500 µL of CTAB extraction buffer (1 % CTAB, 0.7 M NaCl, 100 mM Tris (pH 7.5), 10 mM EDTA, 0.3 mg/mL proteinase K). Homogenized samples were incubated at 65 °C for 30 min, placed on ice for 2 min, and extracted with 500 µL of chloroform: isoamyl alcohol (24:1) by 10 s of vortexing followed by centrifugation at 14,000×g for 10 min. A volume of 300 µL of aqueous phase liquid was collected from each sample, combined with an equal volume of CTAB extraction buffer, re-extracted with 500 µL of chloroform: isoamyl alcohol (24:1), vortexed, and centrifuged at 14,000×g for 10 min. An equal volume of isopropanol was added to 400 µL of aqueous phase extract, which was gently mixed, and centrifuged at 14,000×g for 15 min. Following removal of isopropanol, DNA pellets were washed with 500 µL of 70 % ethanol, and centrifuged at 14,000×g for 20 min at 4 °C. Once 70 % ethanol was removed and nucleic acid pellets were allowed to dry a volume of 50 µL of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) containing 1 mg/mL RNase A was added to each sample. Final concentrations of extracted DNA were determined using a Nanodrop 2000 (Thermo Fisher Scientific Inc, Waltham, MA).

Basidiospore DNA was amplified using primers for 18S rDNA (Table 2) in conventional PCR reactions and products were verified by gel electrophoresis. Post-amplification products were purified using ExoSAP-IT reagent (USB Corporation) before sequencing with BigDye Terminator version 3.1 cycle sequencing kit (ABI, Foster, CA). BigDye reaction products were purified using a DyeEx® 2.0 Spin Kit (Qiagen), and analyzed on an ABI 3130XL sequencer (Applied Biosystems).

Tissue sample collection and fixation

Rectangular leaf tissue samples measuring 4 cm by 1 cm were cut from infected leaves using a sterile razor blade and placed into a 50 mL conical tube containing 30 mL of FAE fixative (2 % formaldehyde, 5 % acetic acid, 60 % ethanol) (see supplementary protocol). After 48 h of incubation at 4 °C FAE fixative was removed from the sample tubes and samples were washed with 70 % ethanol for 5 min before incubation at room temperature in 70 % ethanol for one week. After fixation in FAE fixative and clearing in 70 % ethanol samples were shipped to American Histolabs (Rockville, MD) for RNase-free preparation, including paraffin embedding, sectioning, mounting on positively charged microscope slides, and deparaffinization. All microscope slide samples were stored at −80 °C prior to pre-hybridization.

Prehybridization

All steps listed in the pre-hybridization protocol were carried out under ribonuclease (RNase) free conditions (see supplementary protocol). Slide mounted tissue samples were rehydrated by incubation in 100 % ethanol, 50 % ethanol, and diethylpyrocarbonate  (DEPC)-treated water for 3 min each. Following rehydration samples were treated with 0.2 M HCl for 20 min, washed for 2 min in DEPC-treated water, and incubated at 70 °C for 20 min in 2× SSPE (0.3 M NaCl, 2 mM EDTA, 20 mM NaH2PO4, pH 7.4), before digestion with 10ug/mL proteinase K in proteinase K buffer (20 mM Tris–HCl, pH 7.0, 2 mM CaCl2). Digestion was stopped by washing with 2× SSPE for 5 min at room temperature, and tissue was treated with 0.1 M TEA (0.1 M triethanolamine-Cl, pH 8.0) containing 0.5 % acetic anhydride (Sigma, St. Louis, MO) (v/v) for 10 min preceding treatment with biotin and streptavidin blocking solutions. Blocking of endogenous biotin is achieved by treating samples with 1 mL of 1× Blocking Reagent (Roche, Indianapolis, IN) containing 4 drops/mL of streptavidin blocker (Vector Laboratories, Burlingame, CA) for 15 min, and washing for 5 min in 2× SSPE before applying 1 mL of 1× Blocking Reagent containing 4 drops/mL of biotin blocker (Vector Laboratories, Burlingame, CA) for 15 min. After blocking, slides were washed in 2× SSPE for 5 min and dehydrated by incubation with DEPC-treated water, 50 % ethanol, and 100 % ethanol consecutively for 3 min each. Samples were allowed to air dry while hybridization mixture was prepared.

Hybridization

Hybridization mixture (0.3 M NaCl, 20 mM Tris (pH 7.5), EDTA 2 mM, 500 µg/mL tRNA, 500 µg/mL poly(A) RNA, 1× Denhardt’s Solution [36], 10 % deionized formamide, 9 ng/µL DNA probe) was prepared and heated to 85–95 °C for 2 min before being placed into ice for 2 min. After the hybridization mixture was prepared, a volume of 100 µL was added to each sample slide, a coverslip was applied, and sample slides were placed into a plastic container lined with paper towels moistened with 4X SSPE. Sample slides were incubated overnight at 42 °C in one pint polypropylene snap-lid plastic containers in a H9270 Dual-Chamber Hybridization Oven (Thermo Fisher Scientific, Waltham, MA).

Post hybridization, staining, and permanent mounting

Slides were removed from the 42 °C hybridization oven and dipped in 2× SSPE to aid in coverslip removal. Once coverslips were removed slides were washed in 2× SSPE for 30 min at room temperature, 1× SSPE for 30 min at 52 °C, and blocked for 30 min at room temperature using 1× Blocking Reagent. Samples were treated with Streptavidin-HRP at a concentration of 1:2500 (Invitrogen Carlsbad, CA.) in 1× PBS for 1 h at room temperature, and washed three times in 1× PBS, prior to application of ImmPACT™ VIP Peroxidase Substrate Kit (Vector Laboratories) following manufacturer’s instructions. To rinse and dehydrate tissue samples slides were placed consecutively into DEPC-treated water for 5 min, and 50 % ethanol and 100 % ethanol for 3 min each. Slides were allowed to dry completely before application of Permaslip Mounting Medium and Liquid Coverslip Solution (American MasterTech, Lodi, CA). Immediately following the addition of mounting media, a new coverslip was applied to permanently mount and preserve the tissue sections.

Microscopy

All images were captured using a Nikon DS-Fi1 camera coupled with a Nikon Eclipse 80i microscope and NIS Elements imaging software (Nikon Inc., Melville, NY). The images reported here were produced through differential interference contrast (DIC) microscopy using a Nikon D-DA DIC light filter. A Nikon CFI Plan Achromat DL 10× objective lens was used to magnify samples to 100×.

Results and discussion

A review of the literature showed that very few studies had been conducted using ISH to target ribosomal RNA in a fungal pathogen during active infection [20, 30], several studies had targeted fungal 18S rRNA for ISH [18–24, 26, 30], and a few applied biotin-streptavidin as a reporter system [19, 37]. We believed that the field of plant pathology could benefit from the development of a non-radioactive ISH method designed for detecting rust fungi in host plant tissue. Therefore, a baseline protocol (Fig. 1) was developed from consensus information drawn from the current literature for further testing and refinement.

Fig. 1
figure 1

Simplified list of steps included in each phase of the ISH protocol with estimated length of time spent processing samples through each phase written vertically on the left hand edge

Full size image

Sequencing for probe design

Target sequences were required for development of probes to detect rust fungi in order to test the protocol. ISH probes for the targeted species were developed through a process of database searching, sequencing, and sequence alignment. The National Center for Biotechnology Information (NCBI) Taxonomy Browser website was used to search GenBank for 18S rRNA sequences from many fungal genera. Sequences (Table 1) were aligned in a CLUSTALW multiple sequence alignment using Biology Workbench 3.2 (http://workbench.sdsc.edu/). Sequencing primers were selected from highly conserved regions of the 18S sequence for the purpose of sequencing a variable region of the 18S gDNA of two rust fungi of interest to our laboratory; Puccinia horiana and Uromyces transversalis. These primers (Table 2) amplify a 596 bp fragment of the 18S rRNA gene. The newly obtained sequences were aligned to each of the sequences in Table 1 by pair wise local alignments, using NCBI Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These alignments showed high variability when comparing rusts to non-rust fungi, but variation within rust fungi was low. From these alignments two 60 bp regions were chosen, and upon examining the properties of their sequences using OligoCalc (http://www.basic.northwestern.edu/biotools/oligocalc.html), a single region was selected. Probes were purchased from Integrated DNA Technologies (IDT) with biotin conjugated to their 5’ end for use ISH (probe sequences are listed in Table 2). The probes were designed to be exact matches to the 18S rRNA for their respective pathogen (Table 3). With precisely match probes, it was possible to refine the basic ISH protocol making modifications along the way based upon experimental results.

Table 1 Accession numbers of sequences used in CLUSTALW and BLAST alignments
Full size table
Table 2 List of sequencing primers and ISH probes used in this study
Full size table
Table 3 CLUSTALW alignment of anti-sense probes
Full size table

Development of an optimized ISH protocol

We selected a few key steps in the ISH protocol to optimize the method, using P. horiana-infected chrysanthemum leaves as the test material. Steps tested were proteinase K concentration, presumably affecting accessibility of the target RNA within the tissue, formamide concentration, which controls the strength of the hybridization process, final wash temperature, which controls the stringency of hybridization, and probe concentration. The protocol was tested both with and without a proteinase K digestion (for 20 min with 10 µg/mL of proteinase K at 65 °C) and the results illustrated the necessity of this step, with no signal observed in the undigested samples (see Fig. 2a). Additionally, four proteinase K concentrations (10, 20, 40, and 80 µg/mL) were tested to determine an optimum concentration. A concentration of 10 µg/mL proteinase K worked best to allow probe access without disturbing tissue morphology (see Fig. 2b). Next four concentrations of formamide (10, 30, 50, and 70 %) were tested along with two wash temperatures (42 and 52 °C carried out in 1× SSPE for 30 min). Minimal differences were observed between wash temperatures, but formamide concentrations showed large differences, with lower concentrations resulting in increased signal (see Fig. 3). The results of this experiment led to the selection of two formamide concentrations for testing (50 and 10 %) with healthy plant control samples and sense probe (which should not hybridize to the target RNA sequence). Negligible background was observed in the sense-probe-treated infected tissue with a 42 °C wash and no background was observed in healthy plant samples treated with either probe. The optimal signal was obtained with 10 % formamide in the hybridization mix and the least background was observed with a 52 °C wash (see Fig. 4). Lastly probe concentration was varied (1, 3, and 9 ng/µL), and increased signal was observed when using 9 ng/µL with no additional background. These empirically determined conditions discovered as the result of these experiments were incorporated into a final optimized protocol. The protocol had, at this point, only been tested using sections of Chrysanthemum × morifolium infected with Puccinia horiana and further testing was required to determine the generality and reproducibility of the protocol.

Fig. 2
figure 2

Images of samples of C. × morifolium infected with P. horiana prepared by ISH (Red scale bar = 100 μm). a Samples processed with (top) and without (bottom) a proteinase K digestion. b Samples processed using four different proteinase K concentrations. Purple staining of the tissue indicates hybridization signal generated by HRP reacting with purple substrate

Full size image
Fig. 3
figure 3

Images of C. × morifolium samples infected with P. horiana and prepared by ISH (Red scale bar = 100 μm). Photos are organized so that columns represent samples treated at a given final wash temperature (indicated by column names) and rows indicate samples treated with varying formamide concentrations (indicated by row names) in the hybridization mix for overnight hybridization. Purple staining of the tissue indicates hybridization signal generated by HRP reacting with purple substrate

Full size image
Fig. 4
figure 4

Images of C. × morifolium samples infected with P. horiana and prepared by ISH (Red scale bar = 100 μm). Photos are organized so that columns represent the probe used for hybridization (indicated by column names) and rows represent sample tissue type and post-hybridization wash temperature (indicated by row names). Purple staining of the tissue indicates hybridization signal generated by HRP reacting with purple substrate

Full size image

Application of the protocol to rust fungi

In order to validate the applicability and reproducibility of the refined protocol, samples were taken from two other research subjects in our laboratory; Gladiolus × hortulanus infected with U. transversalis and Glycine max infected with P. pachyrhizi. Slides containing samples of both pathogens were treated with both sense and anti-sense probes using the optimized protocol. These experiments demonstrated that the refined ISH protocol is effective on species within the genera Puccinia (Figs. 2, 3, 4) Uromyces and Phakopsora (Figs. 5, 6). When applied to Glycine max infected with P. pachyrhizi, the signal was weak compared to the other two species (Fig. 6). U. transversalis samples showed strong signal in all slides prepared with the anti-sense probe and no signal in the infected tissue prepared with the sense probe. Healthy plant tissue processed with both sense and anti-sense probes showed no signal, demonstrating further that this technique is preferentially staining pathogen tissue and not the host plant. The strength of signal obtained from U. transversalis samples was equivalent to signal observed in P. horiana samples. P. pachyrhizi showed weak signal in one out of five slides prepared with anti-sense probe and the other four appear the same as slides treated with the sense probe. The fact that some signal was observed indicates that this technique requires further optimization for P. pachyrhizi. In particular the alteration of the high temperature steps could potentially increase signal strength by reducing sample loss.

Fig. 5
figure 5

Images of G. × hortulanus samples infected with U. transversalis and prepared by ISH (Red scale bar = 100 μm). Photos are organized so that columns represent the probe used for hybridization (indicated by column names) and rows represent sample tissue types (indicated by row names). Purple staining of the tissue indicates hybridization signal generated by HRP reacting with purple substrate

Full size image
Fig. 6
figure 6

Images of G. max samples infected with P. pachyrhizi and prepared by ISH (Red scale bar = 100 μm). Photos are organized so that columns represent the probe used for hybridization (indicated by column names) and rows represent sample tissue types (indicated by row names). Purple staining of the tissue indicates hybridization signal generated by HRP reacting with purple substrate

Full size image

The images presented here are at relatively low magnification, and resolution is therefore at the tissue level. Further magnification of images did not resolve at the intracellular level (not shown). Use of a fluorescent label rather than precipitable stain may result in higher-level resolution.

Conclusion

We present here a generalized ISH protocol for localization of rust fungi in paraffin embedded sections of host tissues. This technique provides plant pathologists with a tool to study the morphology of rust fungi within the plants they infect, which may aid in the elucidation of the life cycles of these plant pathogenic fungi and determination of fungal growth patterns in host tissue. We have demonstrated that this protocol can be applied to pathogens from different genera of rust fungi residing in different plant hosts with little to no non-specific background staining of plant tissue. Our protocol is easy to apply and modify if necessary. The generic protocol thus serves as a starting point that may be modified to suit other plant pathogen systems of interest.

References

  1. Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, et al. The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol. 2012;13:414–30.

    Article  PubMed  Google Scholar 

  2. Yamaoka Y. Recent outbreaks of rust diseases and the importance of basic biological research for controlling rusts. J Gen Plant Pathol. 2014;80:375–88.

    Article  Google Scholar 

  3. Bromfield KR. Soybean Rust (Monograph). St. Paul: APS Press; 1984.

    Google Scholar 

  4. Schneider RW, Hollier CA, Whitam HK. First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States. Plant Dis. 2005;89:774.

    Article  Google Scholar 

  5. Gall JG, Pardue ML. Nucleic acid hybridization in cytological preparations. Methods Enzymol. 1971;21:470–80.

    Article  Google Scholar 

  6. Zeller R, Rogers M, Haramis AG. In situ hybridization to cellular RNA. Curr Protoc Mol Biol. 2001:14.13. 11–14.13. 16.

  7. Brahic M, Haase AT. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci USA. 1978;75:6125–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Haramis A, Carrasco A. Whole-mount in situ hybridization and detection of RNAs in vertebrate embryos and isolated organs. Curr Protoc Mol Biol. 1996:14.19.

  9. Lawrence JB, Singer RH. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 1985;13:1777–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Singer R, Lawrence JB, Villnave C. Optimization of in situ hybridization using isotopic and nonisotopic detection methods. Biotechniques. 1986;4:230–50.

    CAS  Google Scholar 

  11. Singer RH, Ward DC. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci USA. 1982;79:7331–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Landegent J, De Wal NJI, Baan R, Hoeijmakers J, Van der Ploeg M. 2-Acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp Cell Res. 1984;153:61–72.

    Article  CAS  PubMed  Google Scholar 

  13. Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA. 1988;85:9138–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pinkel D, Straume T, Gray J. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA. 1986;83:2934–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Brandriff B, Gordon L, Fertitta A, Olsen A, Christensen M, Ashworth L, Nelson D, Carrano A, Mohrenweiser H. Human chromosome 19p: a fluorescence in situ hybridization map with genomic distance estimates for 79 intervals spanning 20 Mb. Genomics. 1994;23:582–91.

    Article  CAS  PubMed  Google Scholar 

  16. Gordon L, Bergmann A, Christensen M, Danganan L, Lee D, Ashworth L, Nelson D, Olsen A, Mohrenweiser H, Carrano A. A 30-Mb metric fluorescence in situ hybridization map of human chromosome 19q. Genomics. 1995;30:187–92.

    Article  CAS  PubMed  Google Scholar 

  17. Assmus B, Hutzler P, Kirchhof G, Amann R, Lawrence JR, Hartmann A. In situ localization of azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy. Appl Environ Microbiol. 1995;61:1013–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Glöckner FO, Amann R, Alfreider A, Pernthaler J, Psenner R, Trebesius K, Schleifer K-H. An in situ hybridization protocol for detection and identification of planktonic bacteria. Syst Appl Microbiol. 1996;19:403–6.

    Article  Google Scholar 

  19. Li AY, Crone M, Adams PJ, Fenwick SG, Hardy GE, Williams N. The microscopic examination of Phytophthora in plant tissues using fluorescent in situ hybridization. J Phytopathol. 2014;162:747–57.

    Article  CAS  Google Scholar 

  20. Montone KT, Guarner J. In situ hybridization for rRNA sequences in anatomic pathology specimens, applications for fungal pathogen detection: a review. Adv Anat Pathol. 2013;20:168–74.

    Article  CAS  PubMed  Google Scholar 

  21. Montone KT, LiVolsi VA, Lanza DC, Kennedy DW, Palmer J, Chiu AG, Feldman MD, Loevner LA, Nachamkin I. In situ hybridization for specific fungal organisms in acute invasive fungal rhinosinusitis. Am J Clin Pathol. 2011;135:190–9.

    Article  PubMed  Google Scholar 

  22. Schröder S, Hain M, Sterflinger K. Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes. Int Microbiol. 2010;3:183–6.

    Google Scholar 

  23. Shinozaki M, Okubo Y, Sasai D, Nakayama H, Ishiwatari T, Murayama S, Tochigi N, Wakayama M, Nemoto T, Shibuya K. Development and evaluation of nucleic acid-based techniques for an auxiliary diagnosis of invasive fungal infections in formalin-fixed and paraffin-embedded (FFPE) tissues. Med Mycol. 2012;53:241–5.

    Article  CAS  Google Scholar 

  24. Sterflinger K, Hain M. In situ hybridization with rRNA targeted probes as a new tool for the detection of black yeasts and meristematic fungi. Stud Mycol. 1999;43:23–30.

    Google Scholar 

  25. Tanaka E. Specific in situ visualization of the pathogenic endophytic fungus Aciculosporium take, the cause of witches’ broom in bamboo. Appl Environ Microbiol. 2009;75:4829–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Vági P, Knapp DG, Kósa A, Seress D, Horváth ÁN, Kovács GM. Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH). Mycorrhiza. 2014;24:259–66.

    Article  PubMed  Google Scholar 

  27. Amann R, Fuchs BM, Behrens S. The identification of microorganisms by fluorescence in situ hybridisation. Curr Opin Biotechnol. 2001;12:231–6.

    Article  CAS  PubMed  Google Scholar 

  28. Bloch B. Biotinylated probes for in situ hybridization histochemistry: use for mRNA detection. J Histochem Cytochem. 1993;41:1751–4.

    Article  CAS  PubMed  Google Scholar 

  29. Brigati DJ, Myerson D, Leary JJ, Spalholz B, Travis SZ, Fong CK, Hsiung G, Ward DC. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology. 1983;126:32–50.

    Article  CAS  PubMed  Google Scholar 

  30. Moter A, Göbel UB. Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J Microbiol Methods. 2000;41:85–112.

    Article  CAS  PubMed  Google Scholar 

  31. Wagner M, Horn M, Daims H. Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol. 2003;6:302–9.

    Article  CAS  PubMed  Google Scholar 

  32. Zachgo S. In situ hybridization. In: Gilmartin PM, Bowler C, editors. Molecular plant biology. 2nd ed. New York: Oxford University Press; 2002. p. 41–63.

    Google Scholar 

  33. Bonde MR, Palmer CL, Luster DG, Nester SE, Revell JM, Berner DK. Sporulation capacity and longevity of Puccinia horiana teliospores in infected chrysanthemum leaves. Plant Health Prog. 2013. doi:10.1094/PHP-2013-0823-01-RS.

    Google Scholar 

  34. Bonde MR, Nester SE, Luster DG, Palmer CL. Longevity of Uromyces transversalis, causal agent of gladiolus rust, under various environmental conditions. Plant Health Prog. 2015. doi:10.1094/PHP-RS-14-0036.

    Google Scholar 

  35. Lamour KH, Finley L, Snover-Clift KL, Stack JP, Pierzynski J, Hammerschmidt R, Jacobs JL, Byrne JM, Harmon PF, Vitoreli AM, et al. Early detection of Asian soybean rust using PCR. Plant Health Prog. 2006. doi:10.1094/PHP-2006-0524-01-RS.

    Google Scholar 

  36. Denhardt DT. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966;23:641–6.

    Article  CAS  PubMed  Google Scholar 

  37. McManus J, Cason JE. Carbohydrate histochemistry studied by acetylation techniques: I. Periodic acid methods. J Exp Med. 1950;91:651.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Authors’ contributions

MAE co-conceived the project, adapted methods, conducted the experiments, tested protocols and wrote the manuscript, MBM co-conceived the project, provided guidance, improvements and reagents, and edited the manuscript, MRB and CLP edited the manuscript, DGL co-conceived the project and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Dr. Kerry Pedley and Amy Ruck for providing U. transversalis spore DNA and P. pachyrhizi infected soybean samples, Dr. Reid Frederick for advice and healthy soybean tissue, and Susan Nester for providing P. horiana—infected Chrysanthemum tissue and U. transversalis—infected Gladiolus tissue. MAE wishes to thank Dr. Oney P. Smith, Hood College, for many stimulating ideas and discussions.

Competing interests

The authors declare that they have no competing interests.

Commercial endorsement disclaimer

The use of trade, firm, or corporation names on this page is for the information and convenience of the reader. Such use does not constitute an official endorsement of approval by the USDA Agricultural Research Service, NAL or BIC of any product or service to the exclusion of others that may be suitable.

Equal opportunity statement

USDA is an equal opportunity provider and employer.

Funding

This work was supported by USDA, ARS and by USDA APHIS Farm Bill 10007 research grants coordinated by author Dr. Cristi L. Palmer, IR-4 Project, Rutgers University.

Author information

Authors and Affiliations

  1. Department of Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Mitchell A. Ellison

  2. USDA-ARS Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD, USA

    Michael B. McMahon, Morris R. Bonde & Douglas G. Luster

  3. IR-4 Project, Rutgers University, Princeton, NJ, USA

    Cristi L. Palmer

Authors
  1. Mitchell A. Ellison
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael B. McMahon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Morris R. Bonde
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cristi L. Palmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Douglas G. Luster
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Douglas G. Luster.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ellison, M.A., McMahon, M.B., Bonde, M.R. et al. In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections. Plant Methods 12, 37 (2016). https://doi.org/10.1186/s13007-016-0137-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13007-016-0137-3

Keywords

Plant Methods

ISSN: 1746-4811