Crop Science 42:1627-1630 (2002)
© 2002 Crop Science Society of America
FORAGE & GRAZING LANDS
Strain-Specific Monoclonal Antibodies to a Nontoxic Tall Fescue Endophyte
N. S. Hill*,a,
E. E. Hiatt, IIIa,
J. H. Boutona and
B. Tapperb
a Dep. Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602
b Ag Research New Zealand, Palmerston North, New Zealand
* Corresponding author (nhill{at}uga.edu)
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ABSTRACT
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Use of proprietary nontoxic endophytes that have been introduced into tall fescue provides a value-added product to livestock producers consuming cool season grass species. A need exists to have a rapid screening method to monitor seed and fields of the cool season species containing nontoxic endophytes. Neotyphodium coenophialum strain AR542, was isolated by AgResearch, New Zealand, and has been introduced into improved tall fescue (Festuca arundinacea Schreb.) cultivars in the USA. The objective of this research was to develop monoclonal antibodies specific to AR542 and test the antibodies for cross reaction with other nontoxic endophytes and endemic toxic endophytes of tall fescue. Antigenic proteins were isolated from AR542 and vaccinated into three mice (Mus musculus). Splenic hybridoma cell lines were screened for affinity to AR542, N. coenophialum isolate EDN11, and other ascomycetes. Two cell lines expressing specificity to AR542 were identified. Comparative analysis was conducted to examine the specificity of the AR542-specific test and with a nonspecific antibody test. Seed and tiller samples had endophyte values greater than 67 and 90% infection, respectively, when tested with the nonspecific test. Minor spurious false positives occurred in seed and vegetative tissues when tested with AR542-specific antibodies, but only seeds and tiller tissues containing AR542 were similar to the nonspecific test. Therefore, AR542-specific antibodies may be an efficient means to test for presence of N. coenophialum strain AR542 in tall fescue.
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INTRODUCTION
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GRASS-BASED FORAGE SYSTEMS have traditionally been used on farm land not suitable for higher-valued and more intensively managed row crops (Martin et al., 1976). Pasture species have also been considered low in value because of anticipated low economic return. In addition, some of the major cool-season forage grass species are infected with mycotoxin-producing endophytes that compromise animal performance and further lower their value (Bacon et al., 1977; Bacon and Siegel, 1988; Fletcher and Harvey, 1981). Removal of endophytes from pasture species was a logical means to add value to forage grasses and increased animal performance resulted (Hoveland et al., 1983; Fletcher, 1984). An unfortunate consequence of endophyte removal was reduced vigor and persistence of the pasture species under drought (Hill et al., 1991; West et al., 1993), grazing (Read and Camp, 1986), or insect pressure (Rowan and Gaynor, 1986). Thus, use of nontoxic endophytes to enhance persistence of tall fescue and perennial ryegrass without related adverse livestock effects is the latest development towards creating value-added forages (Bouton et al., 2000; Latch, 1994). This strategy has increased average daily gains by 80% in tall fescue (Bouton et al., 2000) and 600% in perennial ryegrass (Lolium perenne L.) (Fletcher, 1999).
The nontoxic endophytes have been introduced into patent protected varieties, thus tall fescue and perennial ryegrass will be restricted from harvesting and selling seed or utilizing seed on the farm. For example, the New Zealand strain AR542 was recently introduced into ‘Jesup’ tall fescue and is currently available to producers. However, a need exists for a rapid and inexpensive means by which testing can be performed to ensure compliance to seed utilization restrictions placed on users of the technology. The objective of this study was to develop an endophyte-specific monoclonal antibody-based test for the nontoxic, N. coenophialum strain AR542.
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MATERIALS AND METHODS
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Generation of Monoclonal Antibodies
Tall fescue leaf sheath tissue containing endophyte isolate AR542 was surface sterilized by submersion into an ethanol solution (700 mL ethanol and 300 mL sterile distilled water) for 5 s followed by 5 min in a NaOCL solution (5.25 mL NaOCL and 994.75 mL sterile distilled water) and three sterile water washes. The leaf sheath tissue was placed onto corn meal malt agar. Following growth of mycelia from the leaf sheath to the agar, mycelia were removed from the plate culture, ground in a sterile tissue homogenizer, and transferred to a 1-L Erlenmeyer flask containing 500 mL of Schenk and Hildebrandt basal salts and vitamins (Sigma Chemical Company, St. Louis, MO) liquid media. After mixing, 50 mL of the resulting suspension were dispensed into 125-mL tissue culture flasks. The flasks were placed on a G10 Gyratory orbital shaker (Brunswick Scientific, Edison, NJ) and shaken at 125 rpm at room temperature until fungal cultures developed clumps of mycelia 3 to 5 mm in diameter. Fungal suspensions were vacuum filtered through a Buchner funnel and washed with 1 L of distilled H2O. Mycelia were removed from the funnel by washing with phosphate-buffered saline (PBS; pH 7.3; 2.76 g, NaH2PO4, and 8.77 g NaCl L-1 distilled H2O), and placed into a hand-held tissue homogenizer. The mycelia were ground while keeping the homogenizer in ice, 30 mL of PBS were added, mixed, and centrifuged for 10 min at 3000 x g to remove cellular debris. Polyethylene glycol (PEG) was added to the supernatant until the PEG concentration equaled 100 g L-1 and the proteins were precipitated by chilling overnight at 4°C.
The solution was centrifuged for 20 min at 12 000 x g and the supernatant discarded. The protein pellet was resuspended in 5 mL PBS containing 1 g L-1 sodium dodecyl sulfate (SDS) and incubated for 5 min at 60°C. The SDS solution was centrifuged for 20 min at 12000 x g and the supernatant diluted 1:4 in cold acetone (-20°C). The solution was chilled overnight at -20°C to precipitate the protein, centrifuged for 20 min at 12000 x g, the supernatant discarded, and the protein pellet washed with cold acetone. The protein pellet was dried under a stream of nitrogen, resuspended in 5 mL PBS, and dialyzed for 24 h in 3 x 3 L of distilled water at 4°C. The protein was lyophilized and stored at -70°C.
An immune response was elicited in mice, and monoclonal cell lines produced as outlined by Hiatt et al. (1997). A double screening indirect ELISA system was used to identify monoclonal cell lines that produced AR542 specific antibodies. Protein of N. coenophialum isolate EDN11 was retrieved from a library of protein extracts maintained in the laboratory. Individual rows of microtiter plates were coated with 1 µg per well of purified AR542 protein and 1 µg per well of purified protein from the alkaloid-producing isolate EDN11. Plates were blocked with 100 µL of a PBS solution containing 10 g L-1 bovine serum albumin. Fifty microliters of hybridoma media from actively growing AR542 monoclonal cell lines were added to one well in each row of the microtiter plate coated with the AR542 and EDN11 proteins. The plates were incubated at room temperature for 2 h, washed with ELISA wash solution, 50 µL of a secondary antimouse antibody with a chromogenic conjugate added, and the plates incubated at room temperature for 2 h. The plates were washed, 50 µL of a chromophore solution added, and color quantified on a BioTek Model EL-311 (Bio-Tek Instruments, Inc., Winooski, VT) microplate reader equipped with a 405-nm filter. All ELISA reagents were supplied by Agrinostics Ltd. Co. (Watkinsville, GA). Cell lines expressing affinity to purified AR542 proteins but not EDN11 proteins were then tested for specificity by screening against proteins purified from Cladysporium, Penicilium, Fusarium, and Aspergillus spp. fungi by means of the indirect ELISA assay described.
Testing Specificity to AR542 in Seed and Vegetative Tissues
Antibodies expressing specific affinity to N. coenophialum isolate AR542 were tested for cross reaction to tall fescue seed. Five nontoxic endophyte isolates that were previously inoculated into ‘Kentucky-31’, ‘GA-5’, and ‘Jesup’ tall fescue were used in this study, as well as each cultivar infected with their endemic endophytes. One hundred seed from each lot containing the endophyte isolates were tested for endophyte with a commercial kit (Agrinostics Ltd. Co., Watkinsville, GA) and another 100 seed tested with the AR542-specific antibodies. To test with the AR542-specific antibodies, hybridoma media from each monoclonal cell line was pooled into equal proportions. Reagents from the commercial kit were used with the exception that the kit antiendophyte antibodies were replaced with 1 mL of the pooled AR542-specific antibodies. Thus, a test was conducted for endophyte using a nonspecific antibody test followed by a second test specifically for AR542 in the same immunoblot test configuration. Proportional infection rates were calculated for each seed lot by dividing the number of positive individuals from the AR542-specific assay by the number of positive results from the commercial kit. The experiment was set up as a randomized complete block design with endophyte isolates serving as treatments and the cultivar into which the endophytes were inoculates serving as blocks.
Antibodies were also tested for specificity to endophyhte isolate AR542 in vegetative plant tissue. Tillers were harvested from a grazing experiment with various tall fescue–endophyte combinations. Jesup and GA-5 infected with AR542, Jesup infected with nontoxic endophyte AR502, Jesup infected with its endemic endophyte, and endophyte-free Jesup were planted into 0.5-ha pastures in a randomized complete block with two replications. The experiment was planted in October 1997 at the Central Georgia Branch Research Station near Eatonton, GA. The pastures had been grazed by lambs from March through June and September through December each year beginning in the spring of 1998. Fifty tillers were harvested by cutting the tiller bases at the soil surface on the morning of the 15th day of each month for a 12-mo period beginning in May 2000. Tillers were cut approximately 5 cm above the basal cut, and the tiller bases placed into a labeled plastic bag, the bag placed into a cooler with ice, and the tillers transported to the laboratory. The tillers were removed from the plastic bag, a clean cut made at the tiller base with a razor blade, and two 3-mm-thick psuedostem cross sections made from each tiller. One tiller cross section was tested for endophyte with a commercial test kit (Agrinostics Ltd. Co., Watkinsville, GA) and the other with the AR542-specific antibody mixture. To test the AR542-specific antibodies, the commercial kit (Agrinostics Ltd. Co., Watkinsville, GA) was used except the nonspecific antibody included in the kit was replaced with 2 mL of the hybridoma mixture containing the AR542-specific antibodies. Proportional infection rates were calculated for each pasture by dividing the number of positive individuals from the AR542-specific assay by the number of positive results from the commercial kit. Data were analyzed by analysis of variance using a complete factorial between endophyte treatments and harvest date. Endophytes were considered fixed effects while harvest date was considered a random effect since it was a test for consistency of results over various environmental conditions.
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RESULTS AND DISCUSSION
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Spleens from three mice were used as a donor source to generate monoclonal cell lines. A total of 3568 cell lines were screened for antibody affinity to proteins purified from endophyte isolate AR542. A total of 382 cell lines gave a positive test for the AR542 proteins in the ELISA screening protocol. Of the 382 cell lines, 16 had no cross reaction with purified proteins from endophyte isolate EDN11, and only 2 cell lines had no cross reaction with proteins purified from Cladysporium, Penicilium, Fusarium, and Aspergillus spp. fungi. Thus, the yield of cell lines expressing specificity to AR542 was 0.00054 (2/3568). The two cell lines expressing specificity to endophyte isolate AR542 (AR542T.1 and AR542T.2) were pooled (1:1, v:v) and advanced for specificity testing within seed and vegetative tall fescue tissues.
Seed is the only means of naturally transmitting Neotyphodium endophytes (Bacon and Siegel, 1988) and, therefore, are likely a primary application for strain specific diagnostics of proprietary endophytes. The infection frequencies for the different endophyte isolates were estimated using a Neotyphodium-specific commercial kit (Agrinostics Ltd. Co., Watkinsville, GA). The antibodies in the test kit gave similar results to microscopic examinations of seed (Hill et al., in press), field-grown vegetative tissues, and greenhouse seedling plants (Hiatt et al., 1997, 1999). Therefore, the kits provide an accurate analysis for endophytes in seeds and vegetative tissues. Mean infection frequencies ranged between 67 and 93 out of 100 seed for the various endophyte isolates (Table 1)
. Wild-type endophyte infection had similar infection frequencies as AR501, AR502, and AR542. Endophyte isolates AR572 and AR577 had lower infection frequencies than other endophytes tested.
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Table 1. Infection rates of seed lots containing various endophytes when tested by nonspecific antibodies or AR542 specific antibodies.
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Seed lots containing endophyte isolate AR542 were the only seed lots with appreciable positive results when tested with the AR542-specific monoclonal antibodies (Table 1). The seed lots used for testing specificity to endophyte isolates were chosen because they had been introduced into three distinct tall fescue cultivars: Jesup, GA-5, and Kentucky-31. It is possible that cultivar x endophyte interactions could result in spurious proteins that have potential to give false positives in the test. Hence, testing three distinct cultivars as replications may provide an indication of the effect of the cultivar on test efficacy if there is a replication effect. Our study had no block (cultivar) effect (P = 0.9159), leading us to conclude that the tall fescue cultivar had no effect on the performance of the AR542-specific antibody test. There was a nominal amount of false positives among the seed lots that contained nontoxic endophytes other than AR542. Therefore, the proportion of positive individuals using the AR542-specific antibodies were compared with positive individuals from the test using the nonspecific antibodies. Proportions of seed lots with the AR542 endophyte were greater than those containing other nontoxic endophytes or endemic wild-type endophytes. Thus, specificity of the immunoblot was independent of the endophyte's ability to produce ergot alkaloids and it appears that monoclonal antibodies created via the screening rigors used herein have sufficient specificity to monitor compliance of use within seed tissue.
It may not be possible to test all commercial seed for proprietary endophytes before field establishment. Testing vegetative tissues from pasture or seed fields may be necessary to monitor compliance in such cases. We chose pastures as a source of vegetative tillers because of the potential for fecal and salivary contamination and increased probability that cross contamination or false positives could result in the pasture scenario.
The nonspecific antibody test resulted in some minor seasonal variation among the cultivars with various endophytes. The months of February and March tended to give lower infection values while samples taken from November through January tended to have the highest infection values among pastures planted to Jesup infected with AR502 or GA-5 infected with AR542 (Table 2) . The remaining pastures planted to other cultivars had no seasonal variation among endophyte values when tested with the nonspecific antibodies. Neither endophyte values from pastures tested with the AR542-specific antibodies nor the proportional values expressed seasonal variation (Table 3)
. However, the AR542-specific antibody values appeared to have more false positive individuals in the pasture treatments than the seed. Unknown sources of false-positive immunoblot tests are always of concern, but the AR542-specific antibodies still had sufficiently low false positives that the proportional values were able to distinguish AR542 from other endophytes among the pasture treatments. Both Jesup and GA-5 infected with AR542 had proportional values for endophyte (AR542-specific:nonspecific) greater than 0.90 and were not different from one another. Other nontoxic endophytes had proportional values of 0.26 or less and the endemic endophyte proportional value was approximately 0.08. Therefore, it appears the proportional values are consistent among tall fescue cultivars with the AR542 endophyte, other endophytes are easily distinguished from AR542 since their proportional values were consistently lower than those samples containing AR542, and strain-specific antibodies are a useful tool for diagnostics purposes in vegetative tissues.
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Table 2. Monthly frequency values from a nonspecific monoclonal antibody test of tiller samples from pastures planted to tall fescue cultivars containing various endophytes.
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Table 3. Endophyte infection rates vegetative tissues taken from pastures when tested by nonspecific or AR542 specific antibodies.
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Immunoblot (Gwinn et al., 1991; Hiatt et al., 1997) and ELISA (Johnson et al., 1982; Musgrave et al., 1986; Reddick, 1988; Hiatt et al., 1997) systems have previously been described for N. coenophialum detection in tall fescue. The objectives of their studies were to develop nonspecific polyclonal or pooled monoclonal antibody-based Neotyphodium spp. tests for general applications. Our objective was to develop a monoclonal antibody-based test for the nontoxic N. coenophialum isolate AR542. This isolate of N. coenophialum was selected by New Zealand researchers and has been incorporated into improved tall fescue cultivars. Neotyphodium coenophialum isolate AR542 has been issued New Zealand and U.S. patents and is marketed in tall fescue cultivars with user limitations on harvest and use of seed. Thus, having an efficient screening method for AR542 is necessary to monitor user agreements. Our results suggest that strain-specific monoclonal antibodies may be useful to check for product compliance or infringement of use agreements for value-added tall fescue containing nontoxic endophytes and distinguish AR542 from other commercial nontoxic endophytes.
Received for publication August 27, 2001.
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ABSTRACT
INTRODUCTION
