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Published online 27 March 2006
Published in Crop Sci 46:1064-1070 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

A Sensitive PCR-Based Assay to Detect Neotyphodium Fungi in Seed and Plant Tissue of Tall Fescue and Ryegrass Species

James E. Dombrowski*, James C. Baldwin, Mark D. Azevedo and Gary M. Banowetz

USDA/ARS, Oregon State University, 3450 S.W. Campus Way, Corvallis, OR 97331

* Corresponding author (dombrowj{at}onid.orst.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A polymerase chain reaction (PCR)-based method for detection of Neotyphodium endophytes in seed and plant tissue from tall fescue (Festuca arundinacea Schreb.), Italian (Lolium multiflorum Lam.), and perennial (Lolium perenne L.) ryegrasses was developed. The primers were designed to amplify products from a conserved region of the Neotyphodium spp. tubulin 2 gene which is present in N. coenophialum (Morgan-Jones and W. Gams) Glenn, C.W. Bacon, and Hanlin (endophyte of tall fescue), N. lolii (Latch, M.J. Chr., and Samuels) Glenn, C.W. Bacon, and Hanlin (endophyte of perennial ryegrass), and N. occultans C.D. Moon, B. Scott, and M.J. Chr. (endophyte of Italian ryegrass). PCR yielded the expected amplification products from infected seed lots for tall fescue (358 base pairs [bp]), Italian ryegrass (364 bp), and perennial ryegrass (370 bp). Based on DNA mixture tests and bulk seed analysis, the PCR assay was sensitive enough to detect as little as one infected seed per 50 seeds tested. In addition, the primer set detected the Neotyphodium spp. endophyte in all plant tissues except roots. Comparison of the PCR-based assay to microscopic examination and immunoblot detection of endophytes in seed lots showed that all three methods compared favorably to one another. However, none of the three methods could distinguish between viable and nonviable endophyte in seed. This PCR method provides an accurate, sensitive approach for detecting the presence of the endophyte in these grasses, while maintaining enough specificity to discriminate Neotyphodium spp. from related fungal contaminants such as ergot [Claviceps purpurea (Fr.:Fr.) Tul.].

Abbreviations: bp, base pairs • E+, N. coenophialum infected • E–, N. coenophialum noninfected


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
COOL SEASON GRASSES such as tall fescue, Italian, and perennial ryegrasses can be infected with the endophytic fungi, of the genus Neotyphodium. All of the Neotyphodium spp. are symptomless in their host and are found primarily in the crowns and leaf sheath tissues of vegetative plants. Transmission of these fungi is through hyphal infection of the seed embryo and aleurone layer during seed development. Infected plants may display increased vigor and enhanced tolerance to a variety of biotic and abiotic stresses (Bacon et al., 1997; Malinowski and Belesky, 2000). These characteristics can increase the survival and persistence of the plant in a diverse range of environments. While these beneficial effects of endophyte infection are desirable for turf applications, the plant-fungus symbiosis also results in the production of alkaloids that are toxic to livestock and limit the utility of these grasses in forage and pasture applications (Bacon, 1995; Porter, 1995). The livestock toxicosis resulting from the consumption of these alkaloids is one of the leading causes of reproductive and performance loss among cattle in the USA and abroad (Reed et al., 2000; Allen and Segarra, 2001; Pannaccione et al., 2001). There are efforts to develop benign fungal strains that retain the capacity to increase stress tolerance but do not produce these toxic alkaloids (Fletcher and Easton, 1997; Funk and White, 1997; Bacon and Hinton, 1998; Fletcher, 1999: Reed et al., 2000; Pannaccione et al., 2001; Bouton et al., 2002).

Due to concerns regarding forage quality and livestock toxicity, a variety of methods have been developed to test plant tissues and seed for the presence of endophytes. Several methods are available to detect Neotyphodium fungi in plants, including histological staining of seed and plant tissue (Welty et al., 1986a, 1986b; Saha et al., 1988), serological methods, such as ELISA (Johnson et al., 1982; Musgrave, 1984; Reddick, 1988) tissue printing (Gwinn et al., 1991; Hahn et al., 2003) or immunoblot assays (Hiatt et al., 1997, 1999; Hill et al., 2002), and PCR protocols (Doss and Welty, 1995; Groppe and Boller, 1997; Doss et al., 1998; Clement et al., 2001). The previously developed PCR assays were limited to detection of the Neotyphodium endophyte in tissues from tall fescue plants. The purpose of this research was to develop and evaluate a PCR method for detecting Neotyphodium spp. in seed and tissue from infected tall fescue, Italian, and perennial ryegrasses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
Plants of N. coenophialum infected (E+) or noninfected (E–) tall fescue cv. Kentucky 31 (F. arundinacea), and N. lolii perennial ryegrass (L. perenne) E+ (cv. Express) and E– (cv. Blazer II) were grown in a greenhouse at ambient temperatures (18–23°C) with a 14- to 16-h photoperiod. The plants were grown in 4.4- to 13.2-L (1- to 3-gallon) pots or in flats containing a standard soil-based potting mix and were fertilized to maintain vigorous growth throughout the experiments. The tall fescue ranged from 2 mo to 2 yr of age, while the perennial ryegrass was 2 to 6 mo of age. In addition, surface sterilized seed of perennial ryegrass was sown onto PDA agar and germinated at 25/15°C with an 8-h photoperiod in a germinator. Seed from cultivars of Italian ryegrass were evaluated for the presence of its fungal endophyte (N. occultans, proposed by Moon et al., 2000) by microscopic examination before use in this study.

Fungal Materials
Neotyphodium lolii hyphae were obtained from E+ seed of perennial ryegrass cv. Express and N. coenophialum hyphae from E+ seed of tall fescue cv. Kentucky 31. Seeds were scarified, surfaced sterilized, and aseptically transferred to potato dextrose agar (PDA) slants (Azevedo and Welty, 1995). At 3 wk postgermination the fungal hyphae grew onto the agar surface and were isolated and cultured. Claviceps purpurea (ergot) DNA was generated from single sclerotial isolates of infected Kentucky bluegrass (Poa pratensis L.) seed from eastern Oregon and cultured on PDA. Fungal seed surface contaminants were cultured on corn meal agar plates. The DNA from these fungi was extracted and isolated using DNAzol (Invitrogen, Carlsbad, CA).

Microscopic Analysis
Seed and plant tissue were stained and examined for the presence of the fungal endophytes using light microscopy as described by Saha et al. (1988), with the modification that seed treatments were performed using 1.25 M NaOH to soften the seeds.

Immunoblot Assay
Detection of endophyte presence in seeds and tillers was performed according to manufacturer's (Agrinostic Ltd. Co., Watkinsville, GA) protocols as described in the following kits, Phytoscreen Seed Endophyte Detection Kit (Cat. no. ENDO797–1) and Phytoscreen Field Tiller Endophyte Detection Kit (Cat. no. ENDO797–3). Cross-reactivity to common fungal contaminants was also tested. Fungal tissue from N. coenophialum and C. pupurea, were frozen under liquid N, ground to a powder, resuspended in 1 mL of 10 mM sodium phosphate (pH 7.2). One, ten, and twenty microliters of the fungal suspensions were dot blotted onto the membrane provided in the kit. The membrane was then processed and developed according to manufacturer's specifications.

Template DNA Extraction and Isolation
Plant, seed and fungal DNA extractions were performed using the DNEasy kit (Qiagen, Valencia, CA) or Plant DNAzol reagent (Invitrogen, Carlsbad, CA) using 50 to 100 mg of fungal or plant tissue or 10 to 50 seeds ground under liquid N. The resulting powder was extracted according to manufacturer's protocols. Initially all DNA samples were isolated in 100 to 200 µL of elution buffer (DNEasy kit), in sterile water, or 1 mM Tris-HCl pH 8.0. DNA extraction from single seed was performed as follows: before extraction seeds were treated with either 1.25 M NaOH or 0.05% Tween-20 in 10 mM Tris-HCl pH 8.0 for 1 h, then washed extensively with sterile Milli-Q water (Milli-Q Synthesis, Millipore Corp., Bedford, MA). Single seeds were ground using 200 µL of DNAzol per seed, a glass pestle (acid-treated with concentrated HCl and rinsed with Milli-Q water, and neutralized in 10 mM Tris pH 8.0 between grinds), and 10 to 20 grains of sterilized acid-washed sand in a 1.7-mL microfuge tube. All volumes remained proportionally consistent with the DNAzol protocol. Isolated DNA samples were resuspended in 30 µL 1 mM Tris-HCl pH 8.0. The concentration of all the DNA samples was determined by either gel estimation or using a spectrophotometer. DNA samples before use were diluted to an approximate concentration of 10 ng µL–1.

Primer Selection, PCR Protocol, and Detection of Products
PCR primers were designed to amplify the intron region of the tubulin 2 (tub2) gene for Neotyphodium in tall fescue (cv. Kentucky 31), Italian ryegrass (cv. Jumbo), and perennial ryegrass (cv. Express). Initially in our analysis of endophyte detection in ryegrass species, we used the Doss primers (Doss et al., 1998) to amplify a tub2 sequence fragment from L. perenne. To confirm the identity of the amplified band, it was cloned and sequenced. Sequence data were analyzed using the BLAST programs from NCBI to search for homologous genes in the database (Altschul et al., 1990). Sequence analysis using Genbank showed this tub2 fragment to be an exact match to L78286. Many sequences essentially identical to the cloned fragment were also found. These sequences provided additional sequence information on either side of the cloned L. perenne tub2 fragment, which was used to design the TUB2W-5 and TUB2W-3 primers. These general primers were designed to amplify tub2 sequences from a wide variety of fungal species. Utilizing these primers larger segments of tub2 genes were amplified from Neotyphodium spp. isolated from tall fescue and ryegrass species. A standard three step PCR was used, starting at 94°C for 25 s, annealing at 65°C for 1 min, and extension at 72°C for 2 min for 32 cycles. The products were cloned and sequenced.

Multiple sequence alignments using CLUSTAL X (Thompson et al., 1997) were used to design specific primers for amplification of the intron from the tub2 gene of the Neotyphodium spp. present in tall fescue and ryegrass species. Sequence of the primers are as follows: IS-tub2w-5' 5'-GTGAGTTCAACCTCTCTGTTTGTCTTG-3' and IS-tub2w-3' 5'-GTTGTTGCCAGAAGCCTGTCAC-3'. PCR was performed using HotStar Taq Master Mix (Qiagen, Valencia, CA) in a PTC-200 Thermocycler (MJ Research, Watertown, MA) in 50-µL reaction volumes. Each reaction contained 20 pmol of each primer and approximately 1 to 2 ng of seed DNA extract as template. A standard three step PCR was used, starting at 94°C for 25 s, annealing at 65°C for 1 min, and extension at 72°C for 2 min for 32 cycles. The products were cloned and sequenced.

General PCR primers were designed to amplify a specific region of the intron of the tub2 gene common to Neotyphodium spp. found in tall fescue (358 bp), Italian ryegrass (364 bp), and perennial ryegrass (370 bp). They were designed to amplify preferentially Neotyphodium spp. fungi, but not common contaminants such as ergot. Sequence of the primers is as follows: IS-RS-5' 5'-GAGCCCCTGATTTCGTAC-3' and IS-NS-3' 5'-TTGAAGTAGACACTCATACGCTC-3'.

PCR was performed using HotStar Taq Master Mix (Qiagen, Valencia, CA) in a PTC-200 Thermocycler (MJ Research, Watertown, MA) in 50-µL reaction volumes. Each reaction contained 20 pmol of each diagnostic primer, approximately 1 ng of plant, seed, and fungal DNA extract as template.

The PCR reaction program was run as follows: the reaction mixture was maintained at 94°C for 1 min, followed by 18 cycles 94°C for 25 s, a touchdown annealing temperature starting at 73°C with a delta –0.8°C per cycle, followed by 3 min of 72°C. The remainder of the reaction was 32 cycles of 94°C for 25 s, 58°C annealing temperature for 1 min, and 72°C for 2 min. The reaction was then incubated for 15 min at 73°C then held at 0°C until retrieved from the thermocycler.

Products were separated on 1% agarose gels and visualized by staining with 0.2 µg mL–1 ethidium bromide. Gels were illuminated and photographed on a FluorChem 9900 imager (Alpha Innotech, San Leandro, CA).

Analysis of Plants
Plants ranging in age from 3 to 12 mo were initially tested for the presence of endophyte, using a commercially available immunoblot assay (310 plants), which were subsequently assayed using PCR (80 plants) and microscopic examination (50 plants) on randomly selected plants.

Cloning and Sequencing of Amplified DNA Fragments
PCR products were separated on a 1% agarose gel and representative bands were excised from the agarose gels. DNA was extracted from the agarose using the QIAquick gel extraction kit (Qiagen, Valencia, CA). The purified fragments were ligated into a plasmid vector using the pGEM-T easy system (Promega, Madison, WI). The clones were transformed into XL1-Blue or DH5{propto} strains of E. coli for maintenance and storage. Plasmid DNA was extracted from the transformants using QIAprep mini-prep kit (Qiagen, Valencia, CA), and the resulting plasmids were sequenced at Oregon State University, Center for Gene Research and Biotechnology's Central Services Laboratory (Corvallis, OR) using T7 and SP6 primers.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
During our initial analysis to detect Neotyphodium spp. in seed of tall fescue and ryegrass, we utilized previously described primers (IS-1 and IS-3, Fig. 1 ) designed to detect Neotyphodium endophytes in diverse accessions of tall fescue (Doss et al., 1998). However in some seed samples this primer set yielded inconsistent results. We hypothesized that these inconsistencies resulted from potential mismatches between the primers and their targeted annealing sites. Therefore we sequenced the intervening region of the Neotyphodium spp. tubulin 2 (tub2) gene amplified from DNA extracted from infected (E+) seed lots of tall fescue, perennial ryegrass and Italian ryegrass using the primers TUB2W-5' and TUB2W-3' shown in Fig. 1. We compared these sequences with existing sequences currently available in the Genbank database. The Neotyphodium tub2 sequences obtained from F. arundinacea, L. multiflorum, and L. perenne matched other sequences found in Genbank (data not shown). Both the TUB2-Fa and TUB2-Lm sequences (Fig. 1) were found to have exact or nearly exact (1 bp mismatch respectively) matches in Genbank for endophytes derived from the same plant species. The TUB2-Lp sequence (Fig. 1) was found to be nearly identical (99%) to other endophytes in the database, but none of these sequences came from endophytes isolated from L. perenne. Our analysis of the IS-1 and IS-3 primers showed that they annealed to regions of the tub2 gene that contained sequence polymorphisms which could affect amplification. The IS-1 primer contained two additional cytosine nucleotides in the middle of the primer, which had the potential to create a minor hairpin, while the IS-3 primer was annealing at a site that displayed variability between the grass species (Fig. 1).


Figure 1
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  		Fig. 1. Alignment of Neotyphodium spp. tub2 sequences isolated from infected seed from tall fescue and ryegrass species. Sequence designation: TUB2-Fa, Festuca arundinacea (Genbank accession no. AY865627); TUB2-Lm, Lolium multiflorum (Genbank accession no. AY865629); TUB2-Lp, Lolium perenne (Genbank accession no. AY865628); and TUB2-Cp-Claviceps purpurea (Genbank accession no. AF062646). CLUSTAL X sequence alignment is in 5'–3' orientation. Primer names and their sequences are located above the tub2 sequence alignment at their annealing sites (see boxes). Arrows denote direction of amplification. Asterisks denote conservation among all tub2 sequences shown.

 
Based on these sequence data, we designed and evaluated a new general PCR primer set to detect Neotyphodium spp. in tall fescue, Italian, and perennial ryegrasses capable of specifically amplifying Neotyphodium spp. endophytes while excluding common related contaminant such as ergot. We found that the tub2 gene appeared to possess useful polymorphisms between the common fungal contaminant Claviceps and Neotyphodium spp., while maintaining sequence conservation between the targeted Neotyphodium isolated from tall fescue, perennial, and Italian ryegrasses. The new primers IS-RS-5' and IS-NS-3' were designed (Fig. 1) to amplify products from endophyte E+ seed or plants from tall fescue (358 bp), Italian ryegrass (364 bp), and perennial ryegrass (370 bp).

During our initial analysis of this new primer set we found a touchdown reaction to be superior to the standard three temperature PCR reaction (data not shown). Primer annealing temperature is a critical factor for specificity to the target DNA. From the analysis of the temperature gradient PCR performed on Neotyphodium spp. DNA (Fig. 2A ) and DNA isolated from E+ tall fescue seed (Fig. 2B), we determined that the best touchdown temperature range for endophyte detection was 73 to 58°C, with the main reaction run at an annealing temperature of 58°C. In some reactions, it was possible to detect as little as 0.005 ng of pure fungal DNA template (data not shown). However, the protocol produces the most consistent qualitative results when using 0.05 ng of fungal template as shown in Fig. 3A . This compared favorably to total E+ plant or seed DNA extractions, in which our assay provided a positive endophyte detection in the range of 0.1 to 0.5 ng of total DNA extracted from E+ seed samples (Fig. 3B) or from E+ plant tissue (data not show). When isolating DNA for PCR, the amount of fungal DNA isolated from plant tissue or seed may vary significantly from sample to sample, due to the differences in amount of fungal hyphae present in the seed or tissue. These variances will alter the level of detection for a specific sample. However, our results indicate that by using 1 ng of isolated DNA template, this PCR detection strategy has sufficient sensitivity to detect Neotyphodium spp. in E+ tissue or seed samples. We also observed that excess template in the reaction could result in secondary bands (data not shown). Therefore it is important to assess the amount of DNA extracted and adjust it accordingly before use in the reaction.


Figure 2
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  		Fig. 2. Determination of optimal temperature range for touchdown PCR reaction for detection of Neotyphodium sp. Numbers above lanes denote the temperature range (°C) used in the touchdown step. MW, Invitrogen 1 kb plus DNA ladder. (A) PCR reaction using 1 ng DNA from N. coenophialum fungus isolated from tall fescue. (B) PCR reaction using 1 ng DNA isolated from 20 N. coenophialum E+ seeds (tall fescue cv. Kentucky 31, 90% infected).

 

Figure 3
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  		Fig. 3. Determination of DNA concentration necessary for PCR detection of Neotyphodium sp. Numbers above the lanes represent the amount of DNA used in the PCR reaction. MW- Invitrogen 1 kb plus DNA ladder. (A) DNA isolated from N. coenophialum fungus isolated from tall fescue. (B) DNA extracted from 20 E+ seeds (tall fescue cv. Kentucky 31, 90% infected).

 
We tested our PCR method on seed samples from tall fescue, Italian, and perennial ryegrass as well as the related field contaminant ergot. As shown in Fig. 4 , the PCR assay yielded the expected amplification products in E+ seed lots from tall fescue, perennial ryegrass, and Italian ryegrass, but did not amplify products from noninfected (E–) seed or ergot. PCR confirmed results previously obtained through microscopic examination for the presence or absence of endophyte in 15 cultivars of tall fescue, 15 cultivars of perennial ryegrass, and 7 cultivars of Italian ryegrass (data not shown). In addition to ergot, potential contaminating fungi and spores residing on the surface of seeds, such as Alternarium and Cladosporium as well as other unidentified fungal species, were cultured and DNA extracted. The PCR assay did not amplify products from these fungal DNA preparations, indicating that they were unrelated to Neotyphodium spp. and would not cause false positives (data not shown).


Figure 4
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  		Fig. 4. Amplification of products using DNA extracted from seed samples from tall fescue and ryegrasses. DNA extraction was performed on 20-seed samples from the following grasses: tall fescue cv. Kentucky 31, up to 90% infected; Italian ryegrass cv. Jumbo, 34% infected; and perennial ryegrass cv. Express, 64% infected. Lanes: MW, Invitrogen 1 kb plus DNA ladder; (1) N. coenophialum, tall fescue; (2) E+ seed, tall fescue; (3) E– seed, tall fescue; (4) N. lolii, perennial ryegrass; (5) E+ seed, perennial ryegrass; (6) E– seed, perennial ryegrass; (7) E+ seed, Italian ryegrass; (8) E– seed, Italian ryegrass; (9) 0.5 ng of Claviceps pupurea DNA; (10) water control, MW, Invitrogen 1 kb plus DNA ladder. Reactions of seed samples used approximately 1 ng of isolated DNA, and pure fungal DNA controls utilized 0.1 ng (lanes 1 and 4).

 
To determine the sensitivity of the PCR detection protocol, we combined DNA extracts in different proportions from E+ (seed known to be 90% positive) and E– seed lots. The results shown in Fig. 5 indicated that the PCR assay detected as little as 2% E+ seed DNA added to the E– seed DNA. Furthermore DNA was isolated from mixtures of 50 seeds containing putatively E+ seed added to the E– seed in different proportions. Results obtained were consistent with the DNA mixing analysis shown in Fig. 5, where the PCR assay was capable to detected 1 to 2 E+ seeds per 50 seed sample tested (data not shown). Therefore this PCR assay has sufficient sensitivity to detect one E+ seed in 50 in a bulk seed extraction.


Figure 5
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  		Fig. 5. Limits of N. coenophialum detection in tall fescue seed. DNA was isolated from tall fescue cv. Kentucky 31, E+ seed (90%) and E– seed (0%). The percentages above the lanes represent the final proportions of DNA isolated from E+ (90%) seed combined with the DNA isolated from E– seed (0%). Approximately 1 ng of isolated DNA/reaction. MW, Invitrogen 1 kb plus DNA ladder.

 
To further validate the PCR method, we determined which tissues of E+ plants contained detectable endophyte. Figure 6 shows that endophyte was detected in all plant tissues tested except for roots. In addition to the tissues described in Fig. 6, the assay detected endophyte in leaves from vegetative tall fescue tillers, 2-month-old perennial ryegrass vegetative tillers, and perennial ryegrass crowns (data not shown).


Figure 6
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  		Fig. 6. Detection of N. coenophialum in different tissues of E+ tall fescue cv. Kentucky 31 plants. Each reaction used 1 ng of template DNA. Lanes: MW, Invitrogen 1 kb plus DNA ladder; (1) seed; (2) developing seed head, at time of flowering; (3) stem from reproductive tiller; (4) vegetative tiller 1; (5) vegetative tiller 2; (6) crown; (7) roots; (8) water control, MW, Invitrogen 1 kb plus DNA ladder.

 
To evaluate the effectiveness of the PCR assay, we compared it to microscopic examination and immunological detection, utilizing selected seed lots of tall fescue and perennial ryegrass. Table 1 shows the results of this comparative analysis to detect endophyte in single seeds. In general, all three detection methods measured similar levels of infection for each seed lot tested. However, the immunoblot assay generally indicated higher levels of infection than the PCR or microscopic examination methods. This apparent higher percentage of infection may be due to the inherent subjectivity in interpreting a positive response on the immunoblots. The determination for each individual seed in the immunoblot assay is based on the comparison of the seed's staining intensity observed on a nitrocellulose membrane relative to that of positive and negative control seeds included in the kit. Due to variation in staining of the seeds, it can be difficult to determine if a seed is weakly positive or negative. In addition, the immunoblot assay can cross-react with protein extracts from ergot, although the NaOH pretreatment prescribed in the kit is an effective means to diminish false positives associated with this potential fungal contaminant. In contrast, microscopic examination occasionally resulted in lower levels of infection in a seed lot. This may be due to difficulties in detecting the fungi in plant tissue or seed with sparse hyphae or in lots with low levels of infection. The lower PCR detection values observed in Table 1 may be attributed to inhibitors of the PCR reaction present in some single seed DNA extracts or inadequate DNA extracted from the seed, resulting in failure to amplify a DNA product or diagnostic band (data not shown). In multiple seed DNA extracts, using the DNEasy extraction kit, we did not observe any inhibition of the PCR reaction.


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  		Table 1. Detection of endophyte in tall fescue and perennial ryegrass by different methods.

 
To determine whether any of the methods could discriminate between viable and nonviable endophyte, tall fescue seed from three different lots were propagated in a greenhouse (Table 1). Plants ranging in age from 3 to 12 mo were initially tested for the presence of endophyte. Table 1 shows that all three detection methods indicated the tall fescue seed lot B was >76% infected with endophyte. As expected a high percentage (86%) of plants produced from seed lot B tested positive for endophyte. In contrast only 6% of the plants produced from the tall fescue seed lot C contained endophyte, even though all three methods determined that the seed lot was greater than 74% infected. These results indicated that none of the methods distinguished between viable and nonviable endophyte in seed, which is consistent with observations made by Hill et al. (2002). Apparently, even though the endophyte is no longer viable, its DNA, hyphal structure, and proteins remain intact in the nongerminated seed.

This PCR assay could detect as little as 0.005 ng of target DNA (data not shown). However, we found that this sensitivity could result in false positives from airborne contaminants. False positives were eliminated through the use of UV treated biocontainment hood and aseptic techniques (Kwok and Higuchi, 1989; Loeffler et al., 1999). It is important to monitor potential background and contamination in the air by including blank controls during seed preps and water (no template) reactions.

When compared to immunoblot and histological methods, the adaptation of the PCR methodologies does not significantly reduce the time or cost associated with the detection of endophytes in seed and plant tissues. PCR does provide a very sensitive and flexible means by which to assay different tissues throughout plant development. However, we do not recommend that PCR assays supplant established assays (microscopic or immunoblot) for endophyte detection in single seed currently used in seed testing laboratories. PCR does provide another valuable tool to researchers studying endophytes. The method of choice for endophyte detection will vary, and is dependent on an individual's background and training, the availability of equipment, the type of tissue to be tested, the amount of material available, and the number of samples.

To survey endophyte infections in seeds and plants efficiently it is critical to have a simple, fast, and reliable method of detection. Microscopic examination has been shown to be reliable method of detection, however, it does require specific expertise and training to be able to identify fungal infections in tissue and seed. In addition, histological staining of fungal hyphae is a nonspecific process that could result in false positives. Microscopic examination may not be as sensitive as PCR or serological methods and can miss E+ plant and seed samples with sparse hyphae. Serological methods can provide a quick, sensitive means of detection, but may produce false positives caused by cross-reaction with proteins from the plant or closely related fungi (Hill et al., 1998). Such was the case in Italian ryegrass seed lots, where the immunoblot assay displayed false positives as high as 20% in some seed lots. In contrast, PCR and microscopic analysis failed to show the presence of endophyte in these same lots (data not shown). However, we found that due to its ease of use and sensitivity, the immunoblot assay is an effective means of analysis for endophyte in perennial ryegrass or tall fescue, because it provides high throughput when there is a need to examine large numbers of single seed (Hiatt et al., 1999; Hill et al., 2002).

PCR has been shown to provide a reliable, sensitive, and rapid approach to fungal detection in plant tissues (Groppe and Boller, 1997; Doss et al., 1998; Chiocchetti et al., 2001; Lee et al., 2001; Dongyi and Kelemu, 2004). Unlike microscopic examination it requires less training and experience to achieve effective and repeatable results. PCR offers the ability to tailor the specificity of the assay to a particular species and differentiate it from related fungi. One of the main strengths of PCR is its tolerance of a wide variety of source material, such that virtually any part of the plant can be assayed. The general PCR method described here provides an accurate, versatile, and sensitive approach for the detection of endophyte in diverse tissue types and grass species.


   ACKNOWLEDGMENTS
 
Special thanks is extended to Vicky Hollenbeck at USDA-ARS for her technical assistance, Dr. Bob Doss at USDA-ARS for providing PCR primers and lab space, Dr. Hiro Nonogaki of Oregon State University for providing lab space, Dr. Sabry Elias of the Oregon State Seed Certification Lab for providing ryegrass seed samples, and Steve Johnson of DLF-International Seeds for providing seed lots of endophyte infected and noninfected tall fescue cultivar Kentucky 31. Experimental methods performed in this research complied with current laws and regulations of the USA. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

Received for publication May 8, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome