Endophytic fungi in the genus Neotyphodium confer many beneficial effects to their host plants, including resistance to pests (Clay, 1989; Clay and Cheplick, 1989), diseases (Gwinn and Gavin, 1992), grazing (Read and Camp, 1986) and environmental stresses such as soil pH fluctuations (Belesky and Fedders, 1995), and drought (Malinowski and Belesky, 2000). Although endophytes are beneficial to their host grasses, they also often produce alkaloid toxins that are harmful to livestock (Aldrich et al., 1993; Porter, 1995). Ergovaline is one of the ergot toxins produced by several Neotyphodium spp., especially those infecting tall fescue (Festuca arundinacea). Ergovaline consumption in livestock has been associated with poor weight gain, hormonal imbalances leading to reduced fertility, lactation and gangrene of the animal’s limbs (Porter, 1995). However, a direct cause and effect relationship between ergovaline and these symptoms has not yet been demonstrated.
Ergot toxins are also produced by the ergot fungus, Claviceps purpurea (Tudzynski et al., 1999). In this fungus, the biosynthesis of ergopeptines requires the activities of two peptide synthetases, Lps1 and Lps2. The gene encoding Lps1 was first identified in C. purpurea (Tudzynski et al., 1999) and later in Neotyphodium lolii (in which it was named lpsA) (Panaccione et al., 2001), and characterized by sequence analysis. This gene was inactivated by gene knockout method in an attempt to provide a means for identifying the roles of ergot alkaloids in the plant-fungus associations in which they occur, and for ameliorating toxicosis with which these alkaloids are associated (Panaccione et al., 2001).
Because of the likely significance of ergovaline to animal production, information on presence or absence of ergot alkaloid biosynthesis genes in Neotyphodium species of different host grasses, and sequence variability among those genes will be required. Such information will be of great importance for future employment of this symbiotic relationship in crop improvement. The objectives of present work were to examine the presence of the lpsA gene and its probable sequence divergence in endophytic fungi isolated from four different grass hosts, some with high palatability and very wide distribution in the natural rangelands of Iran.
MATERIALS AND METHODS
Strains and culture conditions: Isolates of endophyte (Neotyphodium spp.) used in this study are listed in Table 1. Fungi were isolated from four grass species including, Bromus tomentellus, F. arundinacea, F. pratensis and Lolium perenne which were collected from various regions of Iran. Having confirmed the existence of Neotyphodium mycelia in the tissues of the samples by microscopic examination, isolations were performed either from seeds or leaf tissues of the hosts on potato dextrose agar (PDA), as described by Bacon and White (1994). All the isolates were confirmed as the Neotyphodium species by using specific primers, as explained by Doss et al. (1998).
DNA extraction: Genomic DNA was extracted from fresh mycelial mat grown in potato dextrose broth (with shaking at room temperature for 32 days). The mycelial mat was transferred to a sterile filter paper in order to remove the liquid medium. DNA was isolated by CTAB method, explained by Murray & Thompson (1980). The same method was applied in the extraction of total DNA from nodal tissues of endophyte-infected and endophyte-free grasses.
PCR amplification of the lpsA gene: For detection of lpsA, the primer pair Lps1-F/R, were designed from the nucleotide sequences of the lpsA gene (GenBank accession no. AF368420). The Lps1-F (5¢-TTA CCgAACTggCgACAT-3¢) corresponded to nucleotides 180-197 and Lps1-R (5¢-ggACAC TgTACCACCACTgC-3¢) was complementary to nucleotides 907-926 of the lpsA sequence (Panaccione et al., 2001). This primer pair was used in the first round of PCR to direct the amplification of a 747-bp fragment from the DNA extracts. To confirm the specificity of the amplification, primer set NesLps1-F corresponding to nucleotides 356-373 and NesLps1-R complementary to nucleotides 752-769 were applied in nested-PCR to amplify a 414-bp DNA band from internal region of the 747-bp amplicon.
The amplification was carried out in a 25 µl PCR mixture containing 200 ng of template DNA, 200 µl of each dNTP, 0.4 µM of each primer, 0.75 units of Taq DNA polymerase (Roche company, Germany), and 1X PCR buffer containing 15 mM MgCl2. The mixture was overlaid with a drop of mineral oil and the PCR was performed in a thermal Mastercycler (Eppendorf- Germany) programmed for an initial cycle of denaturation for 2 min at 94ºC; followed by 30 cycles of 1 min, denaturation at 94ºC, 45s of annealing at 63ºC and extension 1min at 72ºC. The final step of extension was 5 min at 72ºC. For nested-PCR, products of the primary amplification were diluted 1:30 and used as template for reamplification of the internal fragment. All sets of reactions included DNA samples from endophyte-free grasses and a control in which water was substituted for DNA.
The PCR products were separated by a 1.2% agarose gel electrophoresis, stained with ethidium bromide, and visualized with a UV transilluminator (Gel document, Vilber lourmat TCP-20-M, France).
Resterction analysis of the 747-bp fragment: For restriction analysis of the 747-bp fragment, 5 µl of each selected PCR product from the fungal isolates FaAl, FaTsh2, Lp1Prellude, FaTsh1, FpGan1 and FaFn1 were digested separately with the restriction enzymes PstI, AluI, TaqI, HaeIII and SacI (Roche, Co., Germany), according to the manufacturer instructions. The restriction products were then separated by electrophoresis on a 6% polyacrylamide gel and then stained with silver nitrate (Sambrook, 2001).
Cloning and sequencing of the 414-bp fragment: The 414-bp fragment produced by nested PCR was excised from the agarose gel and purified using the Gene Clean-III kit (Biogene-France), based on the manufacturer’s instruction. The fragment was subcloned into the PGEM-T vector and transformed into E. coli JM109 competent cells, according to the Promega Technical Manual No.042-USA. Plasmid DNA, containing the cloned insert, was identified by blue-white screening on LB medium containing X-gall and the insert size was determined by EcoRI digestion and agarose gel electrophoresis. Both DNA strands of the cloned insert were sequenced by the dyedeoxy chain termination method (SEQLAB Company, Germany). Sequencing data were aligned and analyzed using the Chromas version 2.23 and searching of databases was performed by the BLASTN program.
Ergopeptine analysis: Ergopeptines were extracted from 100 mg of dried leaf clippings obtained from two F. arundinacea and one F. pratensis genotypes hosting the isolates FaTsh1, FaFn1 and FpGan1. These extracts were then analyzed by high performance liquid chromatography (HPLC), as described by Panaccione et al. (2003).
Using the primer pair Lps1-F/R, the 747-bp target fragment (Fig. 1) was amplified from isolates of Neotyphodium taken from F. pratensis ( FPGan1, FPGan2 and FPGan3 ), F. arundinacea (FaTsh1, FaTsh2, FaAl, FaFn1 and FaFn2 ) and L. perenne (LpAmp, Lp1Prellude and Lp2Prellude). However, no PCR products were obtained from any of the fungal isolates belonging to B. tomentellus (BtFh, BtFd, BtMh, BtAni, BtAbi, BtIn). One isolate from F. pratensis (FPGan) and two from F. arundinacea (FaFh and FaSm) did not yield the intended fragment either (Table 1).
In the nested PCR assay, the primer pair NesLps1-F/R yielded a 414-bp PCR product, only in isolates which originally produced the 747-bp band (Table 1 and Fig. 2). None of these primers amplified DNA from the endophyte free test plants or control samples. Nested amplification of the 414-bp band in all the isolates produced a 747-bp band which was an additional confirmation for presence of the lpsA gene in these isolates.
Restriction analyses of the amplified 747-bp fragment from the strains tested (FaAl, FaTsh2, Lp1Prellude, FaTsh1, FpGan1 and FaFn1) using restriction enzymes PstI, AluI, TaqI, HaeIII and SacI, produced similar restriction profiles (Fig. 3).
The sequencing of the nested PCR product (414-bp band) from FaFn1 and FpGan1 isolates and their comparison with sequences reserved in databases, by the BLASTN program revealed that the sequence of this fragment is identical (99%) to a portion of lpsA gene in N. lolii (GenBank accession no. AF368420).
HPLC analysis showed that out of three fescue plant samples hosting endophyte isolates positive for both 747-bp and 414-bp bands, only two contained detectable quantities of ergovaline. The fescue plant samples hosting FaTsh1 and FaFn1 isolates contained 12.4 and 3.8 mg of ergovaline/g of plant dry weight, respectively.
Production of the 747-bp band by PCR, originating from genomic DNA of fungal isolates, provided an indication of the existence of the lpsA gene sequence in several endophytes isolated from plants endogenous to Iran. However, this was highly host dependent and none of the isolates possessing the lpsA gene sequence belonged to B. tomentellus (Table 1), which is a highly palatable perennial grass with wide geographical distribution in most arid and semi-arid regions of Iran and neighboring countries (Rechinger, 1973). This grass is present at different densities in approximately 6 million hectares of Iranian rangelands, highly infected by Neotyphodium and usually grazed without any symptoms of toxicosis in animals. This along with the absence of the lpsA gene might be an indication that Neothyphodium isolates of this host do not produce alkaloids toxic to grazing animals, which requires further investigations.
In restriction analyses of the amplified 747-bp fragment, similar restriction profiles were produced, implying that the sequence of lpsA gene among local endophytic isolates is conserved, and that the endophytes isolated from different hosts and geographical regions in this study are probably closely related.
The sequencing of the nested PCR product (414-bp band) and its identity to a portion of the lpsA gene in N. lolii (GenBank accession no. AF368420) may further suggest the conserved nature of the lpsA gene among isolates of Neotyphodium spp. from various hosts that have spread out in different geographical areas of the world.
The ergovaline quantities in fescue plant samples hosting endophyte isolates positive for both 747-bp and 414-bp bands were found to be different. This can be caused by genetic variation among the endophytic fungi for ergovaline production, or genotypic variation of plant hosts or a combination of both factors (Agee and Hill, 1994; Roylance et al., 1994; Ball et al., 1997). It seems that there is a general assumption regarding the positive correlation between the presence of the lpsA gene and production of ergovaline(Tudzynski et al., 1999; Panaccione et al., 2001). However, the F. pratensis plant sample hosting the isolate FpGan1 that was positive for the lpsA gene, showing both the 747-bp and 414-bp bands during the PCR analysis, contained no detectable ergovaline. The late-acting lpsA is a crucial gene for synthesis of ergovaline, but there are several other genes, the activities of which are required at any of the 6 or 7 steps prior to the lpsA step (Panaccione et al., 2003). There could be a rearrangement in an early gene of the ergot alkaloid pathway in the FaGan1 strain. Thus its failure to produce ergovaline could be due to a step earlier in the pathway, rather than that catalyzed by the lpsA.