Detection of Fungal Pathogens in the Environment

Abstract

The presence of fungal pathogens in the environment has to be detected and also quantified rapidly and precisely, because the inoculum for infection of crop plants comes from the pathogen propagules present in the soil, water, air and alternative host plant species. Soilborne pathogens may have different degrees of saprophytic ability, utilizing the organic matter present in the soil for their survival in the absence of crop plants. They produce different structures such as chlamydospores and sexual spores that are long-lived and are capable of surviving in the soil for several years. Fungal structures may be carried by irrigation water or rain water from one part of the field to other parts or to different fields. It has been ­possible to detect and identify the fungal pathogens in the irrigation water, recycled water used for growing hydroponic plants and also in wash water in the storage facilities for fruits and vegetables. The pathogens infecting aerial plant parts/organs are generally disseminated by wind to different locations. Traditionally spore traps have been used for assessing the spore load of air. Various biological, immunological and nucleic acid-based techniques have been employed for the detection, identification and quantification of pathogen propagules in the environment. Significant improvements have been made in the sensitivity and specificity of detection of fungal pathogens by applying immunoassys and nucleic acid-based methods that are capable of providing reproducible results rapidly and reliably. The relative usefulness and limitations of the detection techniques applied for the detection of fungal pathogens in the environment are discussed.

Studies on the ecology of plant hosts provide information on the influence of environment on the growth and development of plants. Likewise, the influence of the environment on the microbial plant pathogens is studied to understand the extent of population build up resulting in the incidence of diseases in different agroecological conditions. Crop husbandry techniques aim to increase the crop yield to the maximum levels. But some of these techniques like monoculture and excessive application of nutrients may provide favorable conditions for pathogen development. Epidemiology deals with effects of biotic and abiotic environments on ­disease development in plant populations. Microbial pathogens multiply at different rates in a given set of environmental conditions and exhibit wide variations in their pathogenic potential (aggressiveness). The crop plants also have varying growth patterns and respond differently based on their levels of susceptibility/ resistance to environmental factors and microbial pathogens.

Appendices

Appendix 1: Detection of Plasmodiophora barassicae from Soil (Orihara and Yamamoto 1998)

A. Preparation of antiserum

• (i) Homogenize infected roots and hypocotyls of turnip in distilled water using a blender for 5 min; filter through eight layers of gauze; centrifuge the filtrate at 3,000 rpm for 20 min; resuspend the pellet in distilled water and repeat the process of pelleting and resuspending five times.

• (ii) Overlayer the final suspension on a sucrose density gradient column (20% and 40%) set up in the centrifuge tube; centrifuge at 3,000 rpm for 20 min; collect the layer containing the resting spores; wash them five times with distilled water and store at –20°C.

• (iii) Immunize male New Zealand white rabbit by injecting intravenously 0.5 ml of antigen suspension (6 × 10⁷ spores/ml in 0.85% NaCl solution); inject 1 ml of antigen emulsified with 1 ml of Freund’s complete adjuvant ­intramuscularly twice at 2-week intervals and inject intravenously an ­additional dose of 0.5 ml antigen (5.4 × 10⁷ resting spores/ml) at 2-week interval after intramuscularinjection.

• (iv) Collect the blood 2 weeks after the final injection and separate the antiserum and estimate the antibody titer by microprecipitation interface test (1/2048).

• (v) Purify the antibodies by adopting ammonium sulfate precipitation and DEAE-cellulose column chromatography procedures.

B. Detection of the fungal pathogen in infested soil

• (i) Sterilize the soil by autoclaving followed by drying at 160°C for 10 h; grind and sift the soil through a 1 mm sieve and infest the soil with resting spores obtained from infected plant tissues at the density of 1× 10⁸ spores/g of dry soil.

• (ii) Suspend 2 g soil sample in 40 ml of distilled water and stir well for 60 min filter through sieves of 60, 200, and 635 mesh sieves to remove the organic matter.

• (iii) Centrifuge the filtrate at 3,000 rpm for 10 min and suspend the pellet in a predetermined volume of distilled water for performing immunoassays.

C. Indirect enzyme-linked immunosorbent assay (ELISA)

• (i) Suspend the infested soil samples in coating buffer and dilute it serially by tenfold.

• (ii) Dispense 200 μl of each diluted suspensions into two wells of microtiter plates and incubate the plates overnight at 4°C.

• (iii) Wash the plates thrice with PBS containing 2% polyvinyl pyrrolidone (PVP) and 2% bovine serum albumin (BSA); incubate for 1 h at room temperature and wash the plates as done earlier.

• (iv) Transfer to each well 2 μg/ml of anti-resting spore IgG in PBS; incubate for 4 h at 37°C and wash the wells as done earlier.

• (v) Add to each well PBS containing goat-anti-rabbit IgG-alkaline phosphatase conjugate (Chemicon, USA) at a 1/2,000 dilution; incubate for 4 h at 37°C and wash the wells as done earlier.

• (vi) Add 10% diethanolamine (v/v, pH 9.8) containing 1 mg/ml p-­nitrophenylphosphate enzyme substrate to each well and incubate in the dark at 37°C for 20 min.

• (vii) Record the absorbance values for the wells at 405 nm using a microplate autoreader (Tosoh Ltd., Japan).

D. Dot immunobinding assay (DIBA)

• (i) Serially dilute samples by tenfold; spot the samples (2 μl each) onto a 40 cm² nitrocellulose membrane sheet (Trans-Blot, BIO-RAD, USA) and air dry the sheets.

• (ii) Block overnight at 4°C in a buffer solution of 20 mM Tris-HCl, 500 mM NaCl and 0.05% Tween 20 (TTBS, pH 7.5) containing 2% PVP and 2% BSA.

• (iii) Treat the membrane with 0.1–0.2 μg/ml anti-rabbit resting spore IgG in TTBS containing 2% PVP and 0.2% BSA (TTBSPB) for 1 h at room temperature.

• (iv) Treat the membrane with alkaline phosphatase-conjugated goat anti-rabbit IgG in TTBSPB for 1 h; apply the buffer (0.1M Tris-HCl, 0.1M NaCl and 5 mM MgCl₂, pH 9.5) containing 0.33 mg/ml nitroblue tetrazolium substrate and 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt prediluted with N,N-dimethylformamide.

Appendix 2: Detection of Resting Spores of Pythium myriotylum from Soil by PCR (Wang and Chang 2003)

A. Preparation of soil samples

• (i) Collect soil samples at depths between 3 and 30 cm from fields; air-dry them separately, sieve and mix before assay.

• (ii) Collect oospore from fungal cultures grown on artificial medium and incubate in 1% nonsterile soil extract.

• (iii) Add dormant oospores to sieved nonsterile soil; incubate at room temperature; examine under the microscope to confirm the presence of oospores; determine the oospore density using the hemocytometer and adjust the oospore concentration to 1,000 oospores per gram of soil.

B. Separation and extraction of oospores

• (i) Suspend 5 g of soil samples in aliquots of 30-ml sterile distilled water (SDW); stir for 5 min; sonicate for 5 min; add 0.3 ml 1% Tween-80 and stir again for 5 min.

• (ii) Pour the soil suspension into 40 ml of 70% sucrose solution in 250 ml centrifuge tube and centrifuge at 2,500 rpm for 5 min at 20°C.

• (iii) Dilute the supernatant with SDW (1:3); filter through cellulose nitrate membrane (pore size 10 μm, 47 mm diameter) and use the oospores on the membrane for DNA extraction.

• (iv) Extract the DNA from oospores by CTAB method.

C. Polymerase chain reaction (PCR) assay

• (i) Use the 19-nt primer Pmy5 designed from variable sequence of ITS region of rDNA in P. myriotylum.

• (ii) Apply an annealing temperature of 57°C for amplification of a specific 150-bp fragment.

Appendix 3: Detection of Phytophthora sojae in Soils by PCR Assay (Wang et al. 2006)

A. Extraction of the target pathogen from the soil

• (i) Use 200-, 300-, and 400-mesh screens of 20 cm in diameter to remove/eliminate most soil and a 600-mesh, 20 cm diameter screen to collect the oospores.

• (ii) Macerate 20 g of soil in 300 ml of water in a 500-ml beaker for 30 min; stir with a glass rod for 10 min and transfer the macerate to a series of screens of 200-, 300-, 400- and 600-mesh; rinse the screens with large volumes of water and collect the material remaining on the 600-mesh screen for extraction of DNA and staining of oospores.

• (iii) Grind the concentrated material from soil to a fine powder in liquid nitrogen with a mortar and pestle; transfer approximately 0.4 g of the powder to a 1.5 ml centrifuge tube and suspend in 0.5 ml of 0.4% skim milk powder solution by vigorous vortexing and centrifuge at 12,000 × g for 15 min.

• (iv) Transfer the supernatant (approximately 0.4 ml) to a centrifuge tube; add 0.4 ml of proteinase K extraction buffer containing 50 mM Tris-HCl, pH 8.0, 25 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate (w/v) and incubate for 1 h at 55°C.

• (v) Add 0.4 ml 7.5 M ammonium acetate (half volume) to each sample; precipitate the cell debris and remove by centrifugation at 12,000 × g for 15 min.

• (vi) Precipitate the nucleic acids in the supernatant with 2 volumes ethanol at –20°C for 30 min or overnight; pellet the DNA, rinse with 70% ethanol; dry the pellet, redissolve in 5 μl of sterile water or TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) and store at –20°C until required for PCR amplification.

B. Differentiation of live and dead oospores by staining

• (i) Gather fungal materials from the 600-mesh screen (step Aii above); add 0.05% MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-2H-tetrazolium ­bromide) and incubate for 48 h at 35°C.

• (ii) Observe under a microscope for the development of blue color in germinated oospores: pink color in dormant oospores or dark color or absence of stain in the dead oospores.

C. Quantitative detection by real-time PCR assay

• (i) Perform the reactions in a volume of 50 μl consisting of 0.5 μM of each primer (PS1/PS2), 5 μl of DNA solution extracted from soil samples, a 2.5 μl mixture containing 12.5 μM each dNTP, 5 μl of 10 × PCR buffer, 2.5 mM Mg²⁺. 1.25 U of Taq DNA polymerase (Promega), 2.5 μl of 20 × SYBR Green I (OPE Technology Development Co., China) and sterile distilled water to a final volume of 50 μl.

• (ii) Use the thermal cycling conditions as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s and final extension at 72°C for 10 min and maintain negative controls lacking the template DNA for each experiment.

• (iii) Perform quantitative PCR in an ABI Prism 7000 Sequence Detection System (Perkin-Elmer).

• (iv) Prepare standard curve by plotting the log of a known concentration ­(tenfold dilution series from 100 fg to 10 ng in reaction volumes of 50 μl) of DNA from target pathogen against threshold cycle (Ct) values and melting curve.

• (v) Determine the DNA content/g of soil in a 20-g sample from the standard curve and calculate the mean value for six replicates.

Appendix 4: Detection of Spongospora subterranea f.sp. subterranea from Soil by PCR Assays (Qu et al. 2006)

A. Extraction of DNA from soils by Bead-beating/CTAB method

• (i) Mix 10-g sample with 20 ml CTAB buffer consisting of 2% CTAB, 1.4 M NaCl, 0.2% 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0 in polycarbonate bead-beater chamber (Bio-Spec Products, USA) containing 5 g each of glass beads with 0.1, 0.5, 1.0 mm diameters and homogenized for 5 min.

• (ii) Transfer the glass beads and soil suspension to a 50 ml centrifuge tube; incubate at 60°C for 30 min and centrifuge at 3,000 g for 10 min.

• (iii) Transfer the supernatant to a 50-ml centrifuge tube; add an equal volume of chloroform-isoamylalcohol (24:1, v/v) mixture and centrifuge at 3,000 g for 20 min.

• (iv) Transfer the aqueous phase to a 50-ml centrifuge; add an equal volume of isopropanol; shake the mixture well and centrifuge at 3,000 g for 30 min.

• (v) Wash the pellet twice with 250 μl sterile distilled water (SDW) and purify the crude DNA with a Wizard DNA Clean-Up System (Promega, USA) as per the manufacturer’s instructions.

• (vi) Store the purified DNA at –20°C.

B. UltlraClean^TM Soil DNA Kit procedure

• (i) Add 0.25 g soil sample to a 2-ml bead solution tube (MOBIO Laboratories Inc., CA, USA) and extract the DNA as per manufacturer’s recommendations.

C. Amplification of DNA by PCR

• (i) Use primer pari Ss F/R for amplifying the DNA from S. subterranea spore balls.

• (ii) Perform PCR amplification with the following conditions in 50 μl reaction mixture containing 1 × PCR buffer (Applied Biosystems, CA, USA), 2.5 mM MgCl₂, 200 mM of each dNTP, 20 pmol of each primer, 1 U of AmpliTaq polymerase (Applied Biosystems) and 20–30 ng of DNA template.

• (iii) Carry out the amplifications in a PTC–100–60 programmable Thermal Controller (MJ Research Waltertown, USA) with an initial cycle at 95°C for 2 min, 60°C for 30 s and 72°C for 1 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 1 min and a final cycle of 72°C for 7 min.

D. Competitive PCR assay

• (i) Prepare an internal heterologous control competitor from λ DNA with a competitive DNA Construction Kit (Takara Shuzo Co., CA, USA) as per the manufacturer’s instructions.

• (ii) Extract DNA templates used for competitive PCR amplifications from 0.25 g dry soil using and UltraCleanTM soil DNA Kit.

• (iii) Add 4 fg of competitor DNA and perform PCR amplification as described above and separate the amplicons by electrophoresis on 2% agarose gels containing ethidium bromide.

• (iv) Visualize the bands with a UV transilluminator; analyze using GeneGenius Software (Syngene, UK) to quantify densitometrically and calculate PCR product ratio to generate a standard curve.

• (v) Maintain three replicates for each soil sample.

Appendix 5: Extraction of DNA Directly from Soil Samples for PCR-Based Assays (Volossiouk et al. 1995)

Extraction of DNA from soils

• (i) Grind 0.25 g of soil samples with liquid nitrogen using mortar and pestle for about 5 min to get a fine powder; suspend the soil powder in 0.5 ml of skim milk powder solution (0.1 g milk powder dissolved in 25 ml water) and vortex vigorously.

• (ii) For quantitative assays add the internal control template DNA (500 pg).

• (iii) Remove soil and debris by centrifugation at 12,000 × g for 10 min at 4°C and mix the supernatant with 2 ml of SDS extraction buffer (0.3 SDS in 0.14 M NaCl, 50 mM sodium acetate pH 5.1) by vortexing.

• (iv) Add equal volume of water-saturated phenol solution and mix by intermittent vortexing for 2 min at room temperature and separate by centrifugation at 12,000 × g for 10 min.

• (v) Remove the aqueous phase containing the nucleic acid; precipitate with 2.5 volumes of ethanol at –25°C for several hours or overnight and collect the precipitate by centrifugation 4°C.

• (vi) Wash the pellet with ethanol; centrifuge; again wash with ethanol and dry.

• (vii) Dissolve the pellet in 250 μl water and store at –20°C until it is required for assay.

Appendix 6: Detection of Phytophthora nicotianae in Irrigation Water (Kong et al. 2003)

A. Extraction of DNA from pathogen propagules in water

• (i) Collect the propagules by filtration or centrifugation; pass a predetermined volume of zoospore suspension or naturally infested irrigation water through a 47-mm diameter nylon membrane filter with 5 μm pores (Millipore Corp.) using a vacuum pressure of 5.33 Pa (41.5 cm/Hg) to capture propagules on the filter and centrifuge the samples at 10,000 g for 10 min to pellet the propagules.

• (ii) Use UltraClean Soil DNA Kit for extracting the DNA of propagules; cut the filter into fine pieces while it is wet; place the pieces into the bead tube (from the kit for loading soil samples); or resuspend the propagule pellet with solution from the bead tube and pour back into the tube.

• (iii) Follow the manufacturer’s recommendation to complete the process of DNA extraction from the pathogenic propagules.

B. DNA amplification by PCR

• (i) Use the PN primers based on sequences of elicitin gene parA1 and PP primers specific to P. parasitica.

• (ii) Perform PCR reactions using a total volume of 25 μl containing 2 μl of DNA extract; 2.5 μl of 10 × PCR buffer, 2.5 μl of each of the 10 μm primer solutions, 2 μl of 2 mM dNTPs solutions, 0.1 μl (5 U/μl) of Taq polymerase and 13.4 μl SDW.

• (iii) Perform the reactions using a thermal cycler (Perkin-Elmer 480) with initial denaturation at 96°C for 2 min, followed by 40 cycles of 94°C for 30 s, 65°C for 45 s, 72°C for 1 min and a final extension at 72°C for 10 min.

• (iv) Resolve the PCR products using an aliquot of 5 μl by electrophoresis in 1.0–1.5% agarose or 3% Nusieve gels; stain with ethidium bromide and capture the images for analysis using a BioImaging Chemi System (UVP Inc., CA, USA).

Appendix 7: Detection of Airborne Inoculum of Mycosphaerella brassicae (Kennedy et al. 1999)

A. Production of polyclonal antibodies against the target pathogen

• (i) Cultivate the pathogen on senescent sprout leaf decoction (SLD) agar; ­collect the ascospores released from the pseudothecia and prepare a 100-ml spore suspension containing 2.5 × 10⁶ ascospores.

• (ii) Concentrate the spore suspension by freeze-drying; rehydrate in 15 ml of sterile distilled water (SDW); sonicate with a Soniprep apparatus (MSE, Crawley, UK) at a micron amplitude of 20 for a total of 15 min; freeze-dry again and rehydrate again in 5 ml of phosphate buffer saline (PBS), pH 7.2.

• (iii) Immunize a female New Zealand White rabbit with an intramuscular injection of 500 μl of Freund’s complete adjuvant mixed with 500 μl of an ascospore suspension (5 × 10⁴ spores/ml) and administer four additional injection of 500 μl of Freund’s incomplete adjuvant mixed with an equal volume of ascospore suspension at weekly intervals.

• (iv) Bleed the immunized rabbit at 4 and 6 weeks after initial injection; store the preimmune and immune bleeds at 36°C for 2 h; centrifuge at 100 g for 20 min; collect the supernatants and store at –20°C in 250 μl aliquots in vials until required.

• (v) Assign code (like A,B,C) to indicate the preimmune and immune bleeds respectively.

B. Detection and quantification of ascospores of the target pathogen using Burkard trapping and immunofluorescence (IF)

• (i) Place a sporulating culture of the target pathogen in a plant growth cabinet operating at 94% relaltive humidity with a 12-h dark/12-h light regime and lightly mist with distilled water for 10 min every 4 h.

• (ii) Incubate a Melinex tape for 2 h at room temperature (approx. 25°C) in 5% (w/v) bovine serum albumin (BSA)/PBStinc (0.2% antibacterial tincture of merthiolate in PBS); air-dry the tape and vertically section the coated tape and overlay vaseline to one half of the tape.

• (iii) Fix tape to a rotating drum; position inside a Burkard volumetric spore trap (Burkard Scientific, UK) and operate continuously for 4 days in the plant growth cabinet where spores in the air are impacted directly onto the coated tape.

• (iv) Remove the tape; section into four 24 h periods; examine each of the four sections under bright field microscope at a magnification of 400.

• (v) Process the section for immunofluorescence (IF) by attaching the section to a glass microscope slide using double-sided adhesive tape; dilute the final bleed serum to 1:100 in blocking buffer; add to cover each section of the divided tape and incubate at room temperature (25°C) for 60 min.

• (vi) Carefully rinse the slides with distilled water; air-dry, dilute anti-rabbit IgG-FITC conjugate to1:100 in blocking buffer; add to cover each section of the tape and incubate in darkness at room temperature for 60 min.

• (vii) Rinse the divided section with distilled water; air-dry, add counterstains 0.2% Evan’s blue (Sigma) in PBS and 0.5% eriochrome black in PBS to cover each divided sections of the tape for 30 min at room temperature.

• (viii) Rinse the slides; air-dry and detach them from the holding adhesive tape and affix directly onto glass slides with Gelvatol (Burkard Scientific, UK).

• (ix) Mount the slides with PPDG and view under episcopic-fluorescence microscope.

C. Immunodetection of artificially and field-produced ascospores of target pathogen

• (i) Place the Burkard volumetric spore trap into a (Brussels sprout) crop inoculated with the target pathogen and exhibiting severe symptoms induced by the target pathogen; operate the spore trap continuously for 3 days and remove the tape.

• (ii) Divide the tape in half and section into 24-h periods and divide one half of the divided sectioned tape and store at 4°C.

• (iii) Expose the other half under cool white fluorescence/black lighting to a lightly misted sporulating culture of the target pathogen over a 24-h period at 12°C and incubate.

• (iv) Attach the divided sections of tape to a glass microscope slide using double-sided adhesive tape and immunoprobe.

Appendix 8: Detection of Monilinia fructicola Spores in the Air by Real-Time PCR Assay (Luo et al. 2007)

A. Extraction of DNA from spore trap samples

• (i) Remove the tapes from spore traps; combine the tape segments of 3 days into one sample; cut each segment into 1 × 2 cm pieces; place them in a 2-ml FastDNA tube containing 1.8 ml of 0.1 % Nonidet (Sigma-Aldrich, USA) and garnet matrix and incubate the tubes at 55°C for at least 20 min.

• (ii) Shake the tubes in an Eppendorf mixer for 5 min; centrifuge at 14,000 rpm for 10 min and decant the supernatant.

• (iii) Extract the DNA from the spores using the FastDNA Kit (Q-BIO Gene Corp. CA, USA) by adding 300 μl CellLysis/DNA solubilizing solution (for fungi) to each FastDNA tube containing centrifuged spores and shake the mixture in the PrepCell Disruptor for (QbioGene) 10 times for 40 s each at 4.5 m/s with 2 min cooling in ice after the fifth shaking cycle.

• (iv) Follow the other steps as per the manufacturer’s recommendations and dissolve the extracted DNA in 10 μl of H₂O for PCR amplification with real-time PCR assay.

B. Real-time PCR procedure

• (i) Use the pathogen-specific primer pair (RTMfF/RTMfR) to generate the expected PCR product (390-bp from M. fructicola)

• (ii) Perform amplifications in the DNA Engine Opticon2 System (BioRad Laboratories, USA), using the SYBRGreen I fluorescent dye in a total volume of 50 μl containing 25 μl of SYBR Green Supermix (BioRad), 4 μl template DNA from spores and 4 μl each of forward and reverse primers (4 μM each).

• (iii) Provide the following conditions for amplification: an initial preheat for 3 min at 95°C followed by 50 cycles at 94°C for 15 s, 64°C for 25 s, 72°C for 30 s, and 73°C for 1 s in order to detect and quantify the fluorescence at a temperature above the denaturation of primer-dimers.

• (iv) Obtain melting curves based on a standard protocol as per the manufacturer’s recommendations for confirming the signal from the target product without inclusion of primers.

• (v) Prepare a standard curve using different dilutions of target DNA from pathogen cultures.