Patent Abstract:
the present invention relates to a method for identifying and / or characterising an endophyte strain , said method including providing a plurality of samples of endophytes , subjecting said endophytes to genetic analysis , subjecting said endophytes to metabolic analysis and selecting endophytes having a desired genetic and metabolic profile . the present invention also relates to novel endophytes having a desired toxin profile wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte such as standard toxic endophyte ; and / or significantly more alkaloids conferring beneficial properties compared with a control endophyte such as st endophyte . the present invention also relates to endophyte variants having a desired genetic and metabolic profile , wherein said endophyte variants possess genetic and / or metabolic characteristics that result in a beneficial phenotype in a plant harbouring or otherwise associated with the endophyte variant . preferably said endophyte variants are generated by polyploidisation or induced chromosome doubling .

Detailed Description:
fig1 shows sequence alignment analysis of mating - type loci of endophyte strains e . festucae strain e2368 , e1 , nea12 and st . fig2 a shows a upgma phenogram of genetic relationships among endophytes in ryegrass accessions of diverse origins and reference neotyphodium and epichloë species . genetic identity was measured across 18 ssr loci using the dice coefficient . detailed annotations for sections a - d are shown in fig2 b to 2e , respectively . specifically , accessions analysed in this study are shaded in grey , the number of genotypes host to that endophyte strain from the total number of genotypes analysed are indicated in the round brackets and a representative host genotype is given in the square brackets . endophyte isolates from the reference collection are specified in the square brackets following the species name . n . lolii group 1 comprises of isolates aries 1 , banks 5847 , ellett 5837 , fitzroy 2 , fitzroy 3 , kt1 - 2 , north african 6 , vedette 6645 and victorian 2 . fig3 shows isogenic inoculation methodology for endophyte inoculation . a . meristem callus induction ( 4 weeks ); b . embryogenic callus proliferation ( 4 weeks ); c . shoot ( and root ) regeneration ( 5 days , 16 hours light ); d . endophyte inoculation ; e . plantlet growth ( 4 weeks , 16 hours light ); f . growth in soil ( 3 months ); g . ssr - based analysis . fig4 shows the number of hits showing a given percent identity for 250 bp fragments of the nea12 genome against the e . festucae and n . lolii genomes . the x - axis shows the percent identity , the y - axis shows the number of hits . black : n . lolii strain st ; white : e . festucae strain e2368 . fig5 shows the number of hits showing a given percent identity for 250 bp segments of the e1 genome against the genomes of nea12 , e . festucae and n . lolii . the x - axis shows the percent identity , the y - axis shows the number of hits . black ( 1st bar in each group ): e . festucae strain e2368 ; grey ( 2nd bar in each group ): non - n . lolii strain nea12 ; white ( 3rd bar in each group ): n . lolii strain st . fig6 shows the number of hits showing a given percent identity for 250 bp fragments of e1 against nea12 , e . festucae and n . lolii . the x - axis shows the percent identity , the y - axis shows the number of hits expressed as a fraction of the total matches seen per comparison . grey ( 1st bar in each group ): e . festucae strain e2368 ; black ( 2nd bar in each group ): non - n . lolii strain nea12 ; white ( 3rd bar in each group ): n . lolii strain st . fig7 shows a schematic diagram of the mating - type loci in neotyphodium / epichloë . fig8 shows clustalw analysis trees of the sequence flanking the mating - type loci ( left ), and the noxr gene ( cloned from e . festucae strain fl1 gi117413991 ; right ). fig9 shows an alignment between mitochondrial genome of n . lolii strain lp19 and a representative of the clavicipitaceae , metarhizium anisopliae ( genbank reference number nc — 008068 . 1 ). while the two mitochondrial genomes vary in size , the genes are present in the same order and strand sense , with differences being due to variable insertions in the n . lolii mitochondrial genome . fig1 shows a depiction of part of the block structure of the mitochondrial genomes for each of the fungal endophytes sequenced in this study , as well as e . festucae strain e2368 and metarhizium anisopliae for comparison . a shared block ( e . g . b84 ) is present in all 12 mitochondria whereas block 85 is present only in the mitochondria of e . festucae strain e2368 , and non - n . lolii strains e1 and nea12 . fig1 shows a mitochondrial genome comparison . parsimony tree of the relationships between the mitochondrial genomes of the 10 perennial ryegrass endophyte strains sequenced , e . festucae strain e2368 and metarhizium anisopliae . fig1 shows a mitochondrial genome comparison . neighbour joining tree analysis using clustalw from a dna alignment of the 40 blocks of sequence (˜ 40 kb ) that are shared across the 10 perennial ryegrass endophyte strains sequenced , e . festucae strain e2368 and metarhizium anisopliae . fig1 shows a standard curve for quantitative assessment of endophyte colonisation ( copy number relative to total plant gdna ). ( a ) tight clustering of amplification curves ( 4 technical replicates ) ranging from 2 × 10 2 to 2 × 10 6 copies of the 73 bp pera amplicon . ( b ) dissociation curve analysis of the amplification curves shown in ( a ), with the presence of a single peak indicating primer pair specificity . ( c ) assay performance is determined in terms of efficiency , precision and sensitivity . for a typical reaction , a slope of − 3 . 1 to − 3 . 6 and r 2 value ≧ 0 . 985 is acceptable . this assay recorded a slope of − 3 . 2 and r 2 value of 0 . 999 . fig1 shows a quantitative assessment of endophyte colonisation in diverse ryegrass host panel . ( a ) standard curve of pera target sequence ( 2 × 10 2 to 2 × 10 6 ) and amplification curves of the unknown samples . ( b ) dissociation curve analysis of the amplification curves shown in ( a ). ( c ) standard curve for pera target (▪) and unknown samples (▴). fig1 shows a colchicine kill curve of endophyte strain st mycelia grown in potato dextrose broth at 22 ° c ., 150 rpm for 21 days . fig1 shows phenotype of colchicine treated colonies ( 0 . 1 and 0 . 2 %) of endophyte strain st compared to the untreated st control . mycelia were grown on potato dextrose agar at 22 ° c . in dark . fig1 shows an assessment for changes in ploidy level by flow cytometry . a ) dot plots and histogram overlay of control samples , st , be9301 and nea11 . b ) dot plots and histogram overlay of two individual st colonies ( 13 and 14 ), showing a shift in peak location relative to the controls . fig1 shows high throughput pcr screening method for detection of lolitrem b gene deletion mutants . the lolitrem genes targeted include : itmm ( 480 bp ), itmj ( 734 bp ) and itmc ( 583 bp ). m : easyladder1 ( 100 - 2000 bp ); 1 - 13 : individual putative lolitrem b gene deletion mutants ; st : st dna ( positive control for itmm , itmj and itmc ); ar1 : ar1 dna ( positive control for itmm and itmc , negative control for itmj ); h 2 o pcr control . fig1 shows geographical origins represented in the tall fescue endophyte incidence assessment . this graph shows the 40 different geographic origins represented in the incidence assessment . the x axis gives geographic origins in the alphabetical order and the y axis shows the number of accessions . the number of negative accessions is shown with black and the number of positive accessions is shown in grey . fig2 shows upgma phenogram of genetic relationships among endophytes in tall fescue accessions of diverse origins and reference neotyphodium , epichloë , fatg - 2 and fatg - 3 species . fig2 shows production of the insecticidal alkaloids loline , loline formate and peramine by tall fescue endophytes in their endogenous host . fig2 shows production of the anti - mammalian alkaloids ergovaline and lolitrem b by tall fescue endophytes in their endogenous host . fig2 shows an example of antifungal bioassay of inhibition reactions . testing for antifungal activity of endophyte nea12 , st and ar1 against 8 species of pathogenic fungi . fig2 shows endophytes selected for metabolic profiling in in vitro culture . shown in the top left hand corner is the inhibition score . fig2 shows a method for sampling material for lcms analysis . fig2 shows a validation assay . rhizoctonia cerealis was grown in the presence of methanol extracts of endophyte mycelia . shown is an example using the endophyte strain st . a . methanol extract of st grown in the absence of r . cerealis ; b . methanol extract st grown in presence of r . cerealis ; c . water only control ; d . methanol only control . fig2 shows lcms analysis of standard materials displaying extracted ion chromatogram for the toxins : fig2 shows an lcms comparison of ar37 inoculated perennial ryegrass with nea12 inoculated perennial ryegrass ( imp04 nea12 20 ). fig3 shows an msms analysis of nea12 insulated perennial ryegrass metabolite 4 . inset is table 2 from international patent application wo2004 / 106487 describing the fragmentations of the janthitrems found . data for nea12 metabolite 4 is in good agreement with that of component i in the table . ( endol5june09 - 010 # 3184 rt : 49 . 01 av : 1 nl : 5 . 02e2 , t : itms + cesid full ms2 646 . 51 @ cid35 . 00 [ 165 . 00 - 660 . 00 ]) fig3 shows reverse phase liquid chromatography mass spectrometry ( lcms ) analysis of a . tol03 nea12 and b . tol03 st . profiles show the presence and absence of specific metabolites including peramine , ergovaline , lolitrem , and janthitrems . fig3 shows genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection . fig3 shows genetic diversity analysis of tall fescue endophytes . fig3 shows selection of fescue - endophyte combinations for metabolic profiling , endophyte isolation and isogenic inoculation . fig3 shows selection of fescue - endophyte combinations for metabolic profiling , endophyte isolation and isogenic inoculation . fig3 shows a desired toxin profile of tall fescue endophytes . fig3 shows endophytes selected for semi - quantitative analysis of metabolites . fig4 and 41 show metabolomics analyses of fescue endophytes . fig4 shows a semi - quantitative analysis of metabolic profile under temperature / water stress . fig4 shows ssr - based genotyping of isolated endophytes cultures prior to isogenic inoculation . fig4 shows endophyte vegetative stability in tall fescue and perennial ryegrass host genotypes ( stability at 12 months post inoculation ). fig4 - 50 show metabolic profiling of isogenic tall fescue - endophyte associations . fig5 shows anti - fungal bioassays of fescue endophytes . column 1 colletotrichum graminicola , column 2 drechslera brizae , column 3 rhizoctonia cerealis . fig5 a - c show presence of pera gene within non - epichloe out - group endophytes ( fig5 a nea17 ; fig5 b nea18 ; fig5 c nea19 ). fig5 a - d show presence of dmaw gene for ergovaline biosynthesis in endophyte strains ( fig5 a nea17 ; fig5 b nea16 ; fig5 c ar542 ; fig5 d nea20 ). fig5 a - d show presence of eas gene cluster for ergovaline biosynthesis . fig5 a fatg - 2 nea17 ( 287819 ); fig5 b non - epichloe out - group nea18 ( fetc6 - 75 ); fig5 c fatg - 3 nea21 ( 231557 ); fig5 d n . coenophialum nea16 ( fetc7 - 342 ). fig6 shows genes in the lolitrem b biosynthetic gene cluster . fig6 a - d show presence of lolitrem b biosynthetic gene cluster 1 ( itmg , itmm and itmk ) in endophyte strains . fig6 a fatg - 2 nea17 ( 287819 ); fig6 b non - epichloe out - group nea18 ( fetc6 - 75 ); fig6 c fatg - 3 nea21 ( 231557 ); fig6 d n . coenophialum nea16 ( fetc7 - 342 ). fig6 a - d show presence of lolitrem b biosynthetic gene cluster 2 ( itmb , itmq , itmp , itmf and itmc ) in endophyte strains . fig6 a fatg - 2 nea17 ( 287819 ); fig6 b non - epichloe out - group nea18 ( fetc6 - 75 ); fig6 c fatg - 3 nea21 ( 231557 ); fig6 d n . coenophialum nea16 ( fetc7 - 342 ). fig6 a - d show presence of lolitrem b biosynthetic gene cluster 3 ( itme and itmj ) in endophyte strains . fig6 a fatg - 2 nea17 ( 287819 ); fig6 b non - epichloe out - group nea18 ( fetc6 - 75 ); fig6 c fatg - 3 nea21 ( 231557 ); fig6 d n . coenophialum nea16 ( fetc7 - 342 ). fig6 a - d show presence of loline biosynthetic gene cluster in endophyte strains . fig6 a fatg - 2 nea17 ( 287819 ); fig6 b non - epichloe out - group nea18 ( fetc6 - 75 ); fig6 c fatg - 3 nea21 ( 231557 ); fig6 d n . coenophialum nea16 ( fetc7 - 342 ). fig6 a - f show alkaloid biosynthetic gene analysis for endophyte strain nea23 ( 269850 ). fig6 a presence of loline gene cluster ; fig6 b presence of peramine gene ; fig6 c analysis of lolitrem gene cluster 01 ; fig6 d analysis of lolitrem gene clusters 02 and 03 ; fig6 e analysis of dmaw gene for ergovaline production ; fig6 f analysis of eas gene cluster for ergovaline production . fig7 shows the structures of lolitrem b , erogvaline and peramine , with desirable toxin profiles indicated . fig7 shows in vitro bioassays to assess antifungal activity of neotyphodium endophytes . fig7 shows a detached leaf assay to assess resistance to crown rust ( puccinia coronata f . sp . lolii ) of perennial ryegrass plants with and without neotyphodium endophytes . fig7 shows glasshouse and field trial screens for drought tolerance and water use efficiency of perennial ryegrass plants with and without neotyphodium endophytes . fig7 shows experimental work flow for chromosome doubling of endophyte cells . fig7 shows flow cytometry calibrations for dna content assessment in neotyphodium endophyte strains . peaks indicate relative nuclear dna content . fig7 shows flow cytometry analysis of nea12 dh neotyphodium endophyte strains . fig7 shows analysis of growth rate in culture after 8 weeks of nea12 dh neotyphodium endophyte strains compared to control endophyte strains . fig7 shows analysis of growth rate in culture over 5 weeks of nea12 dh neotyphodium endophyte strains compared to control endophyte strains . fig8 shows antifungal bioassays of nea12 dh neotyphodium endophyte strains . fig8 shows antifungal bioassays of nea12 dh neotyphodium endophyte strains . fig8 shows analysis of genome survey sequencing read depth of colchicine - treated neotyphodium endophyte strains . fig8 shows analysis of genome survey sequencing reads mapping to nea12 genome survey sequence assembly . fig8 shows the indole - diterpene biosynthetic pathway of neotyphodium endophytes . fig8 shows in vitro growth of x - ray irradiated neotyphodium endophyte strains . fig8 shows determination of genome sequence variation in x - ray irradiated neotyphodium endophyte strains . fig8 shows single nucleotide polymorphisms ( snps ) in genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows small insertions / deletions ( indels ) in genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows deletions in genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows numbers of snps in genic regions of genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows numbers of indels in genic regions of genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows the spectrum of genome sequence changes ( deletions ) in genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows mutagenesis index of x - ray irradiated strains based on number of genome sequence changes observed in genome sequences of x - ray irradiated neotyphodium endophyte strains . fig9 shows metabolic profiling of nea12 dh neotyphodium endophyte strains . fig9 shows metabolic profiling of x - ray irradiated neotyphodium endophyte strains . a collection of 244 perennial grass accessions was assembled for the discovery of novel endophyte strains . the collection targeted accessions from the northern mediterranean and eastern europe for endophytes that lack lolitrems , as well as accessions from the middle east , the proposed centre of origin of perennial ryegrass and n . lolii . genotypic analysis of endophyte content was performed across a total of 189 accessions . from each accession 1 - 5 plant genotypes were analysed for endophyte . endophyte incidence was low , with endophyte detected in 51 % of accessions . endophyte was consistently detected ( with ≧ 10 ssr markers ) in 77 of the accessions . endophytes representing five different taxa were detected across the 77 accessions with 18 ssr markers used to investigate endophyte diversity in perennial ryegrass ( fig2 ). n . lolii was predominant , occurring in 63 accessions . also detected , although less common , were lptg - 2 and putatively new taxa . genetic variation in n . lolii appeared to be low . a total of 22 unique genotypes were detected across the 63 accessions host to n . lolii . the likely toxin profiles of 14 of the 22 genotypes were established from comparisons with genetic and phenotypic data from previous studies . most of these genotypes ( 12 / 14 ) showed genetic similarity to endophytes known to produce lolitrems . there were two genotypes that showed genetic similarity to genotypes known to lack lolitrems but produce ergovaline . one of these genotypes was identical to the genotype detected in the endophyte nea6 . the likely toxin profiles of the remaining eight genotypes were not known . these genotypes did not show high levels of genetic similarity to the endophytes ar1 , endosafe , nea3 or nea5 . plants carrying candidate endophytes were subjected to primary metabolic profiling in the endogenous genetic background , through clonal propagation and measurement of toxin levels . a total of 42 genotypes representing four of the five taxa were selected for toxin profiling , including the eight novel genotypes with unknown toxin profiles . the perennial ryegrass genotype north african 6 ( na 6 ), which contains standard toxic ( st ) endophyte , was used as a control . for metabolic profiling , a complete randomised block design was used , with four replicate clones for each plant and using four hydroponics tubs as blocks . following three months in hydroponics , whole shoot ( leaf plus basal region ) was harvested from each plant . the fresh and dry weights of each plant were measured and powdered sample material from 80 ( 20 genotypes × 4 replicates ) samples ( three tillers per sample ) analysed for alkaloid content ( lolitrem , ergovaline and peramine ). candidate endophytes for further study were chosen on the basis of their genetic identity and metabolic profile . host - endophyte combinations producing significant amounts of lolitrem b were eliminated , as the ryegrass staggers syndrome produced by this alkaloid is the most important limitation for livestock production . the candidate endophyte nea10 ( originating from spain ) was identified as a novel genotype in this analysis with an unknown toxin profile . its genetic identity is a unique n . lolii strain . following in planta metabolic profiling analysis , candidate endophyte nea10 was found to produce ergovaline and peramine , and not lolitrem b . the candidate endophyte nea11 ( originating from france ) was identified as a novel genotype in this analysis with an unknown toxin profile . its genetic identity is a unique lptg - 2 strain . following in planta metabolic profiling analysis , candidate endophyte nea11 was found to produce ergovaline and peramine , and not lolitrem b . the candidate endophyte nea12 ( originating from france ) was identified as a novel genotype in this analysis with an unknown toxin profile . nea12 is a genetically novel , non - neotyphodium lolii , endophyte representative of an as yet un - named taxon . following in planta metabolic profiling analysis , candidate endophyte nea12 was found to not produce the three alkaloids assessed ( lolitrem b , ergovaline and peramine ). the candidate endophyte e1 was identified as a novel genotype in this analysis with an unknown toxin profile . e1 is a genetically novel , non - neotyphodium lolii , endophyte representative of an as yet un - named taxon . following in planta metabolic profiling analysis , candidate endophyte e1 was found to not produce the three alkaloids assessed ( lolitrem b , ergovaline and peramine ). novel candidate endophytes were isolated from their host plant to establish an in vitro culture . following isolation , the genotype of each endophyte was confirmed by ssr analysis to ensure a high level of quality control prior to inception of isogenic inoculations . establishment of meristem cultures for a diverse perennial ryeqrass host panel a set of cultivars representing elite germplasm were obtained , including forage and turf types . meristem cultures from different cultivars were established to evaluate and compare the phenotypic properties of novel endophyte strains in diverse isogenic host backgrounds . embryogenic genotypes were identified for each of the cultivars through callus induction and proliferation . subsequent regeneration of embryogenic genotypes identified primary tissue culture responsive ( ptcr ) genotypes for each of the cultivars . the number of ptcr genotypes with regeneration frequencies ranging from 80 - 100 % varied from 1 - 4 per cultivar . ptcr genotypes were then prepared for meristem - derived callus induction to identify highly regenerable genotypes for isogeneic endophyte inoculation . table 1 shows a selection of cultivars developed , and the tissue culture responsive ( tcr ) genotype , used for isogenic inoculation . in order to accurately determine the phenotypic effects of different candidate endophytes in the absence of host - specific genetic effects , a system for isogenic inoculation was developed ( fig3 ). the regenerating callus method of inoculation was chosen , as it results in a relatively high rate of inoculation compared to other tested techniques , and the achieved isogenic inoculation rate was similar to the standard inoculation procedure for non - isogenic seedlings . novel candidate endophytes nea10 , nea11 , nea12 , e1 and control endophyte st were individually inoculated into elite germplasm . the logistical approach was to inoculate two cultivars at any given time , with one tcr genotype for each variety chosen for inoculation in this initial study . for each cultivar - endophyte combination , 30 replicate inoculations were performed , 25 of these replicates being transferred to soil . following inoculation and plantlet regeneration in culture , plants were transferred to soil for three months to allow establishment of endophyte and host - plant associations . after this period , three tillers from each plant were sampled and tested for endophyte presence using ssr - based analysis . a quantitative score was used to assess endophyte inoculation frequency ( table 2 ). three diagnostic ssr markers were used to determine endophyte presence and identity and samples were scored on a scale of 0 - 3 . of the 570 inoculations tested , 195 ( 34 . 2 %) could be positively scored with a high degree of confidence ( table 3 ). successful inoculations are listed on table 3 . variation in inoculation success according to candidate endophyte identity was observed ( table 3 ). endophyte nea10 ( 2 . 2 %), for example , exhibited relatively lower success rates as compared to nea11 ( 67 . 2 %), or the commercial endophyte st ( 39 . 8 %; table 4 ) and only formed stable associations with one of the five hosts in the panel ( impact ). endophyte e1 is a highly compatible endophyte , which obtained a high rate of success of inoculation into perennial ryegrass ( table 3 ) compared to other endophytes examined , including the strain st . variation was also observed between host plant genotypes for successful inoculations ( table 3 ). tolosa ( 20 . 0 %) appears to be more recalcitrant to inoculation compared to host plants such as bronsyn ( 42 . 3 %) and impact ( 49 . 3 %). fully confirmed endophyte positive plants from the targeted host - endophyte panel ( host plants bealey , bronsyn , barsandra , tolosa and impact ; endophytes st , nea10 , nea11 , nea12 ) were retested 6 - 12 months after inoculation and 18 - 24 months after inoculation , to confirm the presence of endophyte and to assess vegetative stability . in this experiment , 3 replicates of 3 tillers each ( total of 9 tillers ) were collected for ssr - based analysis . most of the previously confirmed endophyte positive plants were again confirmed in this study at 6 - 12 months post inoculation , indicating that each of the host — endophyte combinations were stable ( table 4 ). endophyte nea12 appears to be less stable in planta , as 7 of the 13 previously confirmed samples could not be fully confirmed in this experiment ( table 4 ). st also showed lower levels of stability compared to nea11 , with 7 / 21 samples not re - confirmed in this study ( table 4 ). following this analysis , up to three independent inoculation events from each host plant — endophyte combination were retained for further study . at 18 - 24 months post inoculation , plants were further assessed for long term vegetative stability ( table 4 ). st , nea10 and nea11 each exhibit stable associations , with most plants retaining endophyte . nea12 appears to be less stable in some associations , however does form stable long term associations with tolosa . metabolic profiling was conducted to determine the stability of the predicted endophyte phenotype in a range of different host genotype backgrounds . four replicates of three tillers each were grown under optimal conditions in hydroponics for six weeks prior to measuring lolitrem b , ergovaline and peramine levels . each replicate plant was also tested for the presence / identity of endophyte using ssr - based genotyping in order to correlate toxin profile with endophyte presence , in particular for those instances were toxin profiles were negative for the alkaloids measured . table 5 summarises the outcomes of metabolic profiling in hydroponics for both the endophyte discovery phase and the isogenic inoculation phase . toxin profiles were as predicted from the cluster assignment of the endophyte in the diversity analysis and the toxin profiles measured in the endogenous host plant . genome survey sequencing was performed for non - n . lolii strains nea12 and e1 , lptg - 2 strain nea11 and neotyphodium lolii strains including standard toxic ( st ) and nea10 using gsflx titanium ( ti - gsflx ) pyrosequencing technology ( roche ; as per manufacturers instructions ). a further five n . lolii strains were sequenced using either gsflx standard or gs20 pyrosequencing technology . genome assembly for each of the strains was conducted with gsflx de novo assembler ( table 6 ). a new genome assembly was performed for n . lolii strain st ( gsflx de novo assembler ), combining sequence reads from both gsflx and ti - gsflx runs . table 7 compares the assembly of single and multiple strains . this combined assembly of the st genome achieves c . 12 × coverage of the c . 32 mbp haploid genome . the genome is assembled into 7 , 875 large contigs ( 0 . 5 to 47 kb ) of which the net length is 31 , 750 , 111 bp . analysis using augustus gene prediction software trained for fusarium graminearum shows that there are 11 , 517 predicted protein coding genes in the n . lolii genome . the content of genes known to be involved in alkaloid production in each of the sequenced endophyte genomes was investigated . sequence reads for each of the strains were subjected to a blast ( n ) search against each of the known toxin gene sequences ( downloaded from ncbi ) to determine the degree of gene coverage by sequence reads . table 8 below shows the correlation between secondary metabolite production and toxin - related gene content in endophyte genomes . based on this analysis , endophyte strain e1 is predicted to produce the alkaloids peramine and ergovaline , but not loline or lolitrem b . in planta analysis of alkaloid content has shown that e1 does indeed not produce loline or lolitrem b . nea10 and nea11 produce ergovaline and peramine , but not lolitrem b . the nea11 sequence provides evidence for 2 peramine biosynthesis genes , as might be expected in a heteroploid genome . nea12 , known to lack production of ergot alkaloids and lolitrem b , also lacks corresponding biosynthetic genes . to compare the nuclear genome of nea12 to e . festucae and n . lolii , the contigs derived from nea12 were split into 250 bp segments and these segments were used as blast ( n ) queries against e . festucae strain e2368 ( university of kentucky , http :// www . genome . ou . edu . fungi . html ) and n . lolii st contigs . one hit was scored for each 250 bp contig if it was greater than 50 bp long and greater than 80 % identity . summary statistics were taken for nea12 250 bp fragments against e . festucae and n . lolii ( fig4 ). the number of hits showing a given percent identity shows there are more 250 bp segments that give 100 percent identity matches against an e . festucae genome than a n . lolii genome . the above statistic is independent of the length of the overlap . an identical 250 bp region would give a 250 bp overlap with a percent identity of 100 . the number and proportion of these identical reads is given for the two searches below ( table 9 ). there are also segments that have no match to either n . lolii ( 6051 ) or e . festucae ( 5670 ). these data suggest that nea12 is a new endophyte taxon that is genetically closer to e . festucae than n . lolii . this data supports the earlier observation , using ssr - based genetic diversity analysis , that nea12 is genetically distinct from n . lolii and e . festucae . comparison of e1 nuclear genome to nea12 , e . festucae e2368 and n . lolii st for comparison at the whole genome level , the contigs from endophyte strain e1 were split into 123 , 258 250 bp segments . each 250 bp segment was used as a blast ( n ) query against the assembled whole genome dna sequences from nea12 , e . festucae e2368 and n . lolii st ( fig5 ). a blast ( n ) hit was recorded if there was an overlap of greater than 49 bp . the number of overlaps at a given percent identity was counted for each search . the plot of this data reveals that the genome of endophyte strain e1 is more similar to that of e . festucae strain e2368 than to either n . lolii strain st or nea12 . the assembled contigs from nea12 sum to c . 17 . 3 mb , so the level of sequence similarity to that endophyte is probably underestimated due to limited scope for comparison . if the similarity is expressed as a fraction of the total matches observed per comparison , strain e1 is seen to be more similar to strain nea12 than to n . lolii strain st ( fig6 ). the property of enhance similarity between e1 and e . festucae as compared to n . lolii is similar to the pattern seen with mitochondrial genome analysis . the lptg - 2 endophyte strain nea11 is reported to be a hybrid of n . lolii and e . typhina . mitochondrial sequence analysis supports the hybridisation of e . typhina with a n . lolii with only the n . lolii mitochondria being retained . evidence for the hybrid nuclear genome is seen when nuclear genes are used as a query against contigs from the nea11 genome assembly ( fig7 and 8 ). the panels below show a region of the ‘ udp - n - acetylglucosaminyltransferase ’ gene from e . festucae being used as a blast ( n ) query against : e . festucae ( e2368 ) genome contigs ; n . lolii ( st ) genome contigs ; and lptg - 2 ( nea11 ) genome contigs . this result clearly shows a second variant of this gene in the nea11 genome that has far more snps than the first nea11 contig hit . this presumably represents the e . typhina copy of this gene that has been retained in the nea11 genome . it is unlikely that this is a localised duplication in nea11 as neither e . festucae , nor n . lolii has such a duplication . the panel below shows the n . lolii peramine gene from genbank used as a query against nea11 genome assembly contigs . blast ( n ) alignment of lptg - 2 endophyte strain nea11 reads against the peramine gene ( pera ) sequence ( genbank accession number : ab205145 ). the presence of snp in one set of contigs indicates the presence of two copies of the peramine gene sequence in endophyte strain nea11 . in heterothallic fungi , such as epichloë spp , strains must be of opposite mating - type for sexual reproduction to proceed . in epichloë spp , sexual development is regulated by alternative mat1 - 1 ( comprising mat1 - 1 - 1 , mat1 - 1 - 2 and mat1 - 1 - 3 ) and mat1 - 2 ( comprising mat1 - 2 - 1 ) genes at the mat locus . although the flanking regions of mat1 - 1 and mat1 - 2 are homologous , the nucleotide sequences of mat1 - 1 and mat1 - 2 idiomorphs are highly dissimilar ( fig7 ). the mating - type locus of e . festucae e2368 was contained in contig 5 of the original assembly ( university of kentucky , http :// www . genome . ou . edu . fungi . html ). this contig was aligned with contigs derived from n . lolii endophyte strain st . the mat1 - 1 mating - type locus genes found in e . festucae ( mat1 - 1 - 1 , mat1 - 1 - 2 , mat1 - 1 - 3 ) were demonstrated to be absent in the n . lolii consensus sequence ( fig7 ). in the corresponding location a single gene for the opposite mating type ( mat1 - 2 ) was identified . this opposite mating type gene ( mat1 - 2 - 1 ) was found in all the n . lolii strains sequenced as well as nea12 ( table 10 ). to assess the mating type of endophyte strain e1 , the two possible mating type contigs were compared to e1 contigs . this activity proved that e1 contained the same three ( mat1 - 1 - 1 , mat1 - 1 - 2 , mat1 - 1 - 3 ) mating - type genes as e . festucae e2368 and is thus of the mat1 - 1 mating - type . this is in contrast to the mating type gene of non - n . lolii strain nea12 , which is of the mat1 - 2 , n . lolii - like , mating - type . cluster analysis based on sequence nucleotide diversity shows that endophyte strains e1 and nea12 cluster with e . festucae strain e2368 , with their position in the tree switching between analysis based on the mating - type loci flanking sequence and the noxr gene respectively , and suggesting that recombination has occurred in these lineages ( fig8 ). the identification of an endophyte strain of the opposite mating - type to previously characterised perennial ryegrass endophyte strains provides a means for molecular breeding of endophytes to deliver favourable traits into the plant endophyte symbiotum through the use of the novel e1 strain endophyte . the mitochondrial genome of n . lolii endophyte strain lp19 was present as a single c . 88 . 7 kb contig . this sequence was used to identify contigs containing mitochondrial dna sequences in the other n . lolii strains sequenced through blast ( n )- based sequence similarity . homology searches identified mitochondrial contigs in the e . festucae strain e2368 assembly the two non - n . lolii genomes and the lptg - 2 genome that were sequenced . the mitochondrial genome sizes for each of the fungal endophytes sequenced in this study as well as the e . festucae strain e2368 are shown on table 11 . a representative of the clavicipitaceae , metarhizium anisopliae ( genbank reference number nc — 008068 . 1 ), is shown for comparison . the n . lolii mitochondrial genomes are similar in size , ranging from 88 , 377 bp for g4 to 88 , 740 bp for ar1 . lptg - 2 representative , nea11 has a mitochondrion genome similar in size to n . lolii . the two non - n . lolii genomes , e1 ( 63 , 218 bp ) and nea12 ( 57 , 818 bp ), have relatively smaller mitochondrial genomes more similar in size to that of e . festucae strain e2368 ( 69 , 614 bp ) than that of n . lolii . the multiple mitochondrial dna sequences were used to generate a mitochondrial genome alignment along with the mitochondrial genome sequence of the clavicipitaceae fungus metarhizium anisopliae . the alignment demonstrated that while the different mitochondrial genomes vary in size , the genes are present in the same order and strand sense in all genomes , with differences being due to variable insertions in each strain ( fig9 and 10 ). scoring block presence as 1 and absence as 0 , a matrix was created to generate a parsimony tree of the relationships between the mitochondrial genomes ( fig1 ). this tree places the e1 and nea12 mitochondria on a branch with the e . festucae strain e2368 mitochondrial genome , these three genomes showing greater variation than that of the n . lolii mitochondria . the mitochondrial tree shows that endophyte strains nea12 and e1 are neither e . festucae nor n . lolii , but are more similar to e . festucae than n . endophyte lptg - 2 nea11 has a mitochondrial genome that is genetically a n . lolii type , being in a glade with nea3 and ar1 , within the n . lolii cluster . a similar pattern is observed if a neighbour joining tree is constructed using clustalw from a dna alignment of only the 40 blocks of sequence that are shared across all endophyte species ( c . 40 kb ; fig1 ). there are still gaps present in the metarhizium anisopliae sequence in this alignment . a quantitative pcr ( qpcr ) method for assaying endophyte biomass in planta has been developed and successfully implemented . the development of a high - throughput pcr - based assay to measure endophyte biomass in planta enables efficient screening of large numbers of plants to study endophyte - ryegrass biomass associations . qpcr - specific primer sets have been designed for the peramine biosynthesis gene ( pera ). to quantitatively assess in planta endophyte biomass , a standard curve , ranging from 2 × 10 2 to 2 × 10 6 copies of the target sequence , has been generated from endophyte dna template ( fig1 ). the standard curve is used to quantitatively determine in planta endophyte biomass of unknown samples ( fig1 ). a proof - of - concept study was conducted using a subset of plants which had been previously analysed using established ssr methodology . the analysis clearly shows a correlation between the quantitative ssr allele scoring and the presence of endophyte in planta ( table 12 ). on the basis of dna specific content , the predicted alkaloid profile of e1 indicates that the lolitrem b toxins deleterious to animal health are not produced by this endophyte . the e1 endophyte does not produce lolitrem b , ergovaline , peramine , lolines or janthitrems in planta . endophyte e1 has the mating - type mat1 - 1 , the opposite mating - type to that carried by all n . lolii endophytes previously characterised endophyte e1 has a high inoculation success rate in perennial ryegrass as compared to other endophytes the identification of an endophyte of the opposite mating - type that is highly compatible and stable in planta provides a means for molecular breeding of endophytes for perennial ryegrass through hyper - inoculation hyphal fusion between endophyte strains of the opposite mating - type provides a means for delivery of favourable traits into the host plant via hyper - inoculation . such strains would include : 1 ) an endophyte strain that exhibits the favourable characteristics of high inoculation frequency and high compatibility with a wide range of elite perennial ryegrass host germplasm and ; 2 ) an endophyte that exhibits a low inoculation frequency and low compatibility , but has a highly favourable alkaloid toxin profile . the e1 endophyte strain is genetically novel and is compatible with a wide range of elite germplasm as it can be inoculated with a high degree of success . e1 also is of the opposite mating - type to all of the previously characterised perennial ryegrass endophytes . molecular breeding may therefore be applied by combining the highly compatible e1 endophyte traits with the favourable toxin profile traits of endophytes such as nea12 . the process of molecular breeding through vegetative ( hyphal ) fusion may occur in planta by co - inoculation of two endophyte into the same plant . however , molecular breeding may be more efficiently achieved through vegetative fusion in in vitro culture of endophytes of the opposite mating - type , followed by hyper - inoculation of the resultant endophyte . the following experimental design is applied for molecular breeding of fungal endophytes 1 . determine vegetative compatibility of known endophytes using established co - culturing methodologies . 2 . generation of auxotrophic mutants ( e . g . by gene silencing techniques such as rnai ) for two strains of endophyte , such as e1 and nea12 , exhibiting opposite mating - types . 3 . development of vegetative ( hyphal ) fusion protocol using a combination of cell well degrading enzymes and peg - 4000 . 4 . screen for regenerated endophytes based on survival ( indicating complementarity of auxotrophic mutations ). 5 . genetic screen using ssr and / or mating - type markers to confirm presence of the hybrid genome in a single nuclear compartment . colchicine has been widely used for induction of polyploidy in plant species such as perennial ryegrass , as compared to the application to fungi , which has been limited to a few species . the mitotic spindle inhibitor colchicine is capable of inducing autopolyploidisation , and may be applicable to the production of artificial polyploid endophytes . artificial polyploids were generated by colchicine induced chromosome doubling of the endophyte strains st and nea12 . nea12 , a janthitrem only producing endophyte , with superior bioprotective properties forms stable associations with a limited range of perennial ryegrass hosts . an artificial polyploid of nea12 that is non - toxic to mammals , with enhanced bioprotective properties , that is broadly compatible and highly stable is highly desirable to industry . experiments were conducted to determine the range of colchicine concentrations in which the mycelia of the fungal endophyte n . lolii ( strain st ) would grow successfully . mycelia were grown in colchicine concentrations ranging from 0 % to 1 % for 21 days and monitored for growth ( fig1 ). at greater than or equal to 0 . 2 % colchicine mycelium growth halted whereas at 0 . 1 % or less colchicine mycelium growth was prolific . artificial polyploids were generated for endophyte strains st and nea12 . endophyte strains st and nea12 ( n ) were grown in 0 , 0 . 1 and 0 . 2 % colchicine and potato dextrose broth for 21 days followed by a 7 - 10 day recovery period in potato dextrose broth only . protoplasts were generated from all colchicine concentrations and single colonies isolated ( fig1 ). n . coenophialum strain be9301 and lptg - 2 strain nea11 which are natural heteroploids ( 3n and 2 × n fused respectively ) have been utilised as control material for assessment of ploidy changes using flow cytometry . an optimised protocol was established allowing analysis of fungal protoplasts via flow cytometry . a number of colonies have been identified with changes in nuclear dna content relative to the control samples ( fig2 and 21 ). a method has been developed to eliminate the production of the detrimental alkaloid lolitrem b , using x - ray mutagenesis induced deletion of genes in the lolitrem b biosynthetic gene cluster , in the st endophyte . such an endophyte would be advantageous over existing commercial endophytes , as st is highly stable and broadly compatible . ionising radiation is capable of introducing a broad range of mutagenic lesions and has been found to be very effective in many species . published methods are available to readily detect deletion mutants in targeted plant genes ( li et al , 2002 ). experiments have been performed to determine if n . lolii mycelia are amenable to production of mutagenic lesions by ionising radiation , in particular deletion mutations . n . lolii strain st was grown in potato dextrose broth for different periods of time ranging from 2 - 14 days before exposure to ionising radiation . radiation from a caesium source was applied to the liquid cultures in doses ranging from 10 - 30 gy . following a recovery period ( 10 - 14 days ) the radiation dose was repeated . protoplasts were generated and recovery of individual colonies monitored over a 4 - 6 week period . lolitrem b is the major alkaloid leading to ryegrass staggers in grazing animals . three genes within the lolitrem b gene cluster , which contains 10 genes all required for synthesis of lolitrem b , were targeted to identify individual n . lolii colonies with deletions ( young et al , 2005 ). a high throughput pcr screening method was developed to detect for the presence and absence of the three lolitrem b genes ( fig1 ). a total of 568 tall fescue accessions obtained from 40 different countries were tested for endophyte incidence using endophyte - specific simple sequence repeat ( ssr ) genetic markers . twelve to twenty seeds from each accession were tested for endophyte presence . total genomic dna was extracted from two independent seed bulks of 6 - 10 seeds from each accession and endophytes were detected by pcr amplification with six endophyte - specific ssr markers . endophyte was detected in 40 % ( 228 / 568 ) of the tall fescue accessions tested . furthermore , accessions from 23 out of the 40 countries screened were endophyte positive ( fig1 ) showing the highest incidence in morocco and pyrenees , where the majority of accessions tested ( 80 %- 100 %) were endophyte positive . accessions originating from italy , spain , and united states exhibited a higher endophyte incidence among the tall fescue accessions tested . a subset of selected endophyte positive samples , were selected for further analysis using 32 endophyte - specific ssr markers . the selected genotypes represent a broad range of known geographical origins , hence representing an effective survey of tall fescue endophyte genotypic variation . a set of 52 reference isolates representing several endophyte species , including the resident endophyte of tall fescue and meadow fescue were also included to the diversity analysis . the upgma phenogram , constructed using average taxonomic distance based on ssr polymorphism across 203 endophyte positive accessions , represented six different known taxa , and two out - grouped clusters ( fig2 ). the phenogram was supported by mantel test statistics showing a high correlation coefficient ( r = 0 . 95 ) which indicated a high goodness - of - fit for the data . endophytes representing six different taxa were detected in the 203 accessions ( fig2 ). the majority of endophytes ( 60 %; 122 / 203 ) appeared to belong to the taxon neotyphodium coenophialum , clustering in the phenogram with n . coenophialum isolates from the reference endophyte collection ( fig2 ). this species occurred in 72 % ( 122 / 170 ) of tall fescue collection accessions . as defined by the n . coenophialum reference isolates , the n . coenophialum cluster comprised five main sub - clusters , of which the fifth sub - cluster is rather out grouped from the other four ( fig2 ). the genetic variation observed within n . coenophialum was high when comparing it with other taxonomic groups . in the phenogram n . coenophialum strains clustered for the most part according to their geographical origin ( fig2 ). the first sub - cluster of n . coenophialum comprised mainly tall fescue accessions from spain ( 28 ) and few accessions from pyrenees ( 3 ) and france ( 4 ) ( fig2 ). italian ( 7 ) and french ( 14 ) accessions were clustered in the second sub - cluster ( fig2 ). the third sub - cluster clearly shows the genetic similarity among accessions collected from geographic area surrounding russian federation [ slovenia ( 3 ), russian federation ( 6 ), kazakhstan ( 7 ), former soviet union ( 4 ) and china ( 3 )] ( fig2 ). furthermore within the third sub - cluster a set of accessions from france ( 11 ) and pyrenees ( 1 ) have formed a separate cluster from russian federation and its surrounding geographic origins . the fourth sub - cluster comprises only five endophytes of which two are moroccan accessions and two are ar endophytes ( ar542 and ar584 ) which were initially isolated from tall fescue originated in morocco ( latch et al , 2000 ). the accessions collected from portugal ( 4 ) have formed a distinct sub - cluster which is separated from all the other four sub - clusters ( fig2 ). fatg - 2 accessions formed a cluster close , but distinct from isolates of n . lolii ( fig2 ). there were 20 fatg - 2 endophyte genotypes tall fescue collection which clustered with the fatg - 2 reference genotype . among them , a set of six accessions formed sub - clusters having lesser genetic similarity to the fatg - 2 reference genotype . therefore , the endophytes of those sub - clusters were named “ fatg - 2 like ” endophyte genotypes . a set of six endophyte genotypes formed a distinct cluster with putative fatg - 3 reference isolates as defined by the previously - analysed ar endophytes . furthermore , 13 accessions primarily originating from morocco ( 9 / 13 ) formed a sub - cluster with putative fatg - 3 isolates and those unidentified accessions , forming a cluster distinct to putative fatg - 3 were named “ fatg - 3 like ” endophytes ( fig2 ). the identities of selected putative fatg - 2 and fatg - 3 accessions are largely consistent with geographical provenance , as these taxa are known to be characteristic of populations from southern europe and north africa . two out grouped clusters were also identified and they were named as “ out - group i ” and “ out - group ii ” ( fig2 ). accessions of mediterranean origin primarily clustered in “ out - group i ”, whereas one accession from former soviet union formed the second out - group . moreover , within “ out - group i ” italian accessions clearly group separately from moroccan and algerian accessions . representative tall fescue — endophyte associations were selected for metabolic profiling analysis in order to determine the endophyte derived alkaloid profile , in particular , lolitrem b , ergot alkaloids , peramine and lolines . analysis of metabolite production was assessed under controlled conditions using a growth chamber . tall fescue — endophyte associations were each replicated four times by clonal splitting and arranged in a randomised block design in the growth chamber . plants were maintained in soil for six weeks , with trimming every two weeks to encourage growth . following 6 weeks growth , pseudostem tissue was harvested and freeze dried prior to performing a metabolite extraction and lcms analysis . the perennial ryegrass — n . lolii designer association bronsyn - st was used as a control as st is known to produce lolitrem b , ergovaline and peramine . for each of the accessions , the presence and identity of the resident endophyte was confirmed through ssr analysis of the plant material harvested for metabolic profile analysis and endophyte negative samples were removed from further analysis . the results of the qualitative assessment alkaloid of production for 20 novel tall fescue endophytes are summarised in table 15 . relative quantitation data for batch three , comprising 13 endophytes assessed in their endogenous hosts , are shown in fig2 and fig2 . a number of novel endophytes with favourable toxin profiles ( low / no ergovaline production combined with loline and peramine production ) have been identified . tissue culture responsive genotypes from selected germplasm material have been generated ( drover , dovey , bariane , barolex ). table 16 shows the host cultivars , and their tissue culture responsive genotype , selected for further study . each of the selected genotypes has a regeneration frequency greater than 80 % a set of ten novel tall fescue endophytes were selected for inoculation based on genetic novelty using ssr - based diversity analysis and the toxin profile based on qualitative metabolic profiling ( table 18 ). included in the set was the endophyte ar542 a commercial endophyte in use globally . ar542 was discovered and isolated by agresearch nz and is marketed as maxp ™ and maxq ™. in order to accurately determine the phenotypic effects of different candidate endophytes in the absence of host - specific genetic effects , a system for isogenic inoculation was used . novel candidate endophytes were individually inoculated into elite tall fescue germplasm as well as the perennial ryegrass host genotype bronsyn ( bro08 ). following inoculation and plantlet regeneration in culture , plants were transferred to soil for three months to allow establishment of endophyte and host - plant associations . after this period , three tillers from each plant were sampled and tested for endophyte presence using ssr - based analysis . of the 498 isogenic inoculations tested , 109 ( 21 . 9 %) could be positively scored with a high degree of confidence . successful inoculations are listed on table 19 . variation in inoculation success according to candidate endophyte identity was observed . endophyte strain 3 ( 4 . 3 %), for example , exhibited relatively lower success rates as compared to strain 20 ( 51 . 1 %), or the commercial endophyte ar542 ( 44 . 4 %; table 19 ) and only formed stable associations with one of the five hosts ( bariane ). no successful inoculations were identified for endophyte strain 15 . fatg - 2 endophyte , strain 17 , is a highly compatible endophyte which obtains a high rate of success of inoculation into tall fescue ( table 19 ) compared to other endophytes examined , and is comparable to ar542 . out - group 1 endophyte strain 20 exhibits the highest level of compatibility as measured by its ability to be inoculated . both tall fescue endophytes inoculated into perennial ryegrass host bro08 , strain nea13 and strain nea14 , were taken up successfully , establishing that endophyte inoculation across a range of host species is possible . endophytes with anti - fungal properties may benefit host plants by preventing pathogenic organisms from colonising them and causing disease . this is of particular interest to the turf grass industry . to determine if endophytes of the species neotyphodium produce anti - fungal substances in vitro representative species / strains from neotyphodium were tested for the presence of anti - fungal activity against eight species of fungal plant pathogens . three types of inhibition reactions were observed . in the first reaction , pathogenic fungal growth was unaffected . in the second , growth of the pathogenic fungi was initially unaffected , but growth ceased when the colony margin approached a “ critical ” distance from the central endophyte colony . in the third stronger reaction type , the overall growth of the colony of the pathogenic fungi was reduced . examples of inhibition reactions are shown in fig2 . variation was observed within and between endophyte taxa . non - n . lolii strain nea12 exhibits the strongest and most broad spectrum antifungal activity . variation was also observed among genetically distinct strains of n . lolii . within n . lolii , strains with strongest to weakest effects were st & gt ; ar1 & gt ; nea3 & gt ; nea10 . st exhibited the broadest spectrum of antifungal activity , inhibiting the growth of 7 / 8 fungi strains tested . the bioassay results showed that endophytes in vitro exhibit variation in anti - fungal activity that does not correlate with known toxin production ( specifically , lolitrem b , ergovaline and peramine ). for example nea12 does not produce lolitrem b , ergovaline and peramine and has strong antifungal activity and st does produce lolitrem b , ergovaline and peramine and also has strong antifungal activity . endophyte strains representing the full spectrum of antifungal activity were selected for analysis in order to identify those alkaloids that may be associated with antifungal activity ( fig2 ). endophyte strains were grown both in the presence and absence of the pathogenic fungi rhizoctonia cerealis ( fig2 ). freeze dried endophyte mycelia was then extracted for metabolic profiling analysis . following extraction , a validation assay was done to ensure that the alkaloids associated with antifungal activity had been appropriately extracted ( fig2 ). the antifungal activity of the extract used for lcms analysis was confirmed . the expression of antifungal alkaloids is constitutive as extracts taken from endophyte in the absence of rhizoctonia cerealis also exhibit antifungal activity ( fig2 ). perennial ryegrass cultivars inoculated with the nea12 endophyte were analysed using lcms . the toxins peramine , ergovaline and lolitrem b were not detected in the extract . the ar37 metabolite 11 , 12 - epoxy janthitrem g was detected and its structure assigned based on retention time and ms analysis of an extract of the ar37 inoculated perennial ryegrass . perennial ryegrass cultivars inoculated with different endophytes were analysed for peramine ( 1 ), ergovaline ( 2 ), lolitrem b ( 3 ) and the ar37 isolated metabolites janthitrem i ( 4 ) ( 11 , 12 - epoxy janthitrem g ( janthitrem g ( 5 )) by lcms . janthitrem g is an isomer of the previously described janthitrem f ( 6 ) and its structure was determined by nmr in the original patent describing ar37 ( latch et al , 2000 ; structures shown in fig2 ). standards were analysed to provide reference for the perennial ryegrass analyses . the lolitrem b standard had deteriorated significantly but a peak matching the expected m / z and approximate retention time could be found ( fig2 ). data for ar37 inoculated endophyte and nea12 - inoculated ryegrass gave comparable results . neither contained detectable levels of peramine , ergovaline or lolitrem b . both contained 11 , 12 - epoxy - janthitrem g ( 4 ) ( fig2 ). msms analysis of the ion m / z 646 ( 4 ) is shown in fig3 . the data is a good match for that described in the original patent application . analysis of nea12 was carried out in a number of perennial ryegrass cultivars . it was present to a greater or lesser extent in the majority of those examined ( table 21 ). no attempt was made to quantitate the amount found . a standard toxic ( st ) endophyte was analysed in the same perennial ryegrass cultivars . the st endophyte produced peramine and ergovaline but not janthitrems ( table 21 ). the toxin profiles for st and nea12 are shown in fig3 . meristem cultures for tall fescue cultivars were established for isogenic host panel endogenous metabolic profiles were determined for 48 samples isolation of 38 endophytes was undertaken inoculation of 15 - 20 endophytes into isogenic host panel was undertaken isogenic host - endophyte associations were characterised genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection initially , 472 accessions from 30 countries were tested for endophyte incidence ; with 2 replicates of 6 - 10 seeds in each bulk per accession used in the analysis and endophyte incidence assessed with 6 ssrs . new accessions were included in the analysis from the under - represented geographic origins ; with a total of 568 accessions from 40 countries tested for endophyte incidence . genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection is shown in table 22 . 233 endophyte positive accessions ( 41 %) were detected . the geographical origins are represented in the endophyte incidence assessment . a genetic diversity analysis of tall fescue endophytes is shown in fig3 . a selected set of 210 accessions were used to assess genetic diversity of tall fescue endophytes . genetic diversity was assessed with 38 ssr markers . six different taxa were detected . the majority were n . coenophialum . twenty were fatg - 2 . six were putative fatg - 3 . thirteen were fatg - 3 like . diversity of host and endophyte is shown in fig3 . selection of fescue - endophyte combinations for metabolic profiling , endophyte isolation and isogenic inoculation is shown in fig3 . 52 accessions were initially selected for metabolic profiling and endophyte isolation . endophyte presence was consistently detected in 25 accessions ( red ). an additional 48 accessions from under - represented clusters were established in the glasshouse and screened for endophyte presence . 20 accessions were endophyte positive ( blue ) and were selected for further analysis . selection of fescue - endophyte combinations for metabolic profiling , endophyte isolation and isogenic inoculation is shown in fig3 . initial selections are shown in red . additional selections are shown in blue . the desired toxin profile of tall fescue endophytes is shown in fig3 . the experimental design used for semi - quantitative metabolic profile analysis of tall fescue - endophyte associations for the detection of alkaloid production in the endogenous host background is described below . a metabolic profile analysis for detection of ergovaline and peramine is shown in fig3 . endophytes selected for semi - quantitative analysis of metabolites are shown in fig3 . metabolic profile analysis for the detection of alkaloid production of different fescue endophytes a metabolic analysis of tall fescue - endophyte associations for the detection of alkaloid production including loline , loline formate , peramine , ergovaline and lolitrem b in the endogenous host background is shown in fig4 . the alkaloid profile ( i . e . lolines , peramine , ergovaline and lolitrem b ) of tall fescue - endophyte associations in the endogenous host background for a range of endophyte strains belonging to different endophyte species is shown in table 23 . in addition to the metabolic analysis of tall fescue - endophyte associations grown under standard conditions , for the detection of alkaloid production conferred by the endopohytes in the endogenous host background ( fig3 - 41 ), a semi - quantitative analysis of metabolic profiles of tall fescue - endophyte associations grown under high temperature and water stress conditions was undertaken . corresponding tall fescue - endophyte associations were grown under 16 h light and 30 ° c . ; 18 h dark and 20 ° c ., and then sampled for alkaloid profile analysis as described below : harvest ( control )→ freeze dry → 50 mg pseudostem material → 80 % methanol extraction → lcms analysis recovery and water stress second harvest ( stress )→ freeze dry → ssr confirm all of the plant material again . this was performed in a controlled ( growth chamber ) environment simulating summer conditions , with light watering as required . nine copies per accession were planted in general potting mix . a randomized complete block with subsampling was used . fig4 shows a semi - quantitative analysis of metabolic profile of tall fescue - endophyte associations grown under high temperature and water stress conditions . a total of 36 fescue endophytes have been isolated from a range of fescue accessions from different geographic origin as described in table 24 , and found to belong to different taxa as follows : 19 of them being n . coenophialum ; 5 of them being fatg - 2 ; 3 of them being outgroup ; 3 of them being fatg - 3 ; 3 of them being fatg - 3 like ; and 3 of them being n . uncinatum table 25 shows selected tall fescue and perennial ryegrass cultivars used to identify representative plant genotypes included in the diverse host panel for in planta inoculation of fescue endophytes . all the selected plant genotypes have a high regeneration frequency of & gt ; 80 %. isolated fungal endophytes from endophyte - containing fescue accessions selected for in planta isogenic inoculation into the diverse host panel are shown in fig4 . fig4 shows ssr - based genotyping of isolated endophyte cultures prior to in planta isogenic inoculation to confirm their identity . results from the ssr genotyping indicating the allele number and sizes for different ssr markers for the different fescue endophyte strains are shown in table 26 . results from the in planta isogenic inoculation into the diverse host panel of selected isolated fungal endophytes from endophyte - containing fescue accessions are shown in table 27 . data on number of inoculations tested , number of successful inoculations and % of successful inoculations are provided in table 6 to illustrate the inoculation ability of tall fescue endophytes in tall fescue and perennial ryegrass hosts . following in planta isogenic inoculation with a range of selected isolated endophytes from fescue accessions , the endophyte vegetative stability of these endophytes in the different tall fescue and perennial host genotypes ( i . e . bro 08 , bari 27 , dov 24 ) was assessed , showing that : several tall fescue endophytes ( e . g . nea17 , nea18 , nea19 ) were stable in perennial ryegrass ( bro08 ). bari27 formed stable associations with all endophytes except for nea15 . nea15 failed to form stable associations with any of host genotypes tested . dov24 formed few stable associations . the stability of these associations of novel tall fescue endophytes inoculated in different tall fescue and perennial ryegrass genotypes from the diverse host panel was assessed 12 months post - inoculation . corresponding results are shown in table 28 . table 29 shows additional novel tall fescue endophytes ( e . g . nea20 , nea21 , nea22 , etc .) selected for in planta isogenic inoculations in tall fescue genotypes ( i . e . bari 27 , jess01 and quan 17 ) from the diverse host panel , based on the following selection criteria : 1 . produce little or no ergovaline 2 . produce no lolitrem b 3 . produce lolines and / or peramine metabolic profiling of endophyte - tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel is shown in fig4 , 49 and 50 . these figures : compare semi - quantitative alkaloid profiles of selected endophytes across different isogenic hosts compare semi - quantitative alkaloid profiles for diverse endophytes in an isogenic host compare semi - quantitative alkaloid profiles of tall fescue and perennial ryegrass endophytes in the perennial ryegrass genotype bro08 fig4 shows the presence of peramine and ergovaline in endophyte - tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel . table 30 shows metabolic profiling of endophyte - tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel . confirmed endophyte positive ( e +) plants were split to 5 replicates and regularly trimmed to promote tillering . four months later e + plants were re - potted in 12 replicates . one month later e + plants were re - potted if less than 9 positive copies were available at the time . endophyte status was tested using ssr markers after each re - potting . a range of endophyte - tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel were selected for metabolic profiling ( table 30 ). in total , 29 isogenic host - endophyte associations were subject to lcms analysis , following the experimental design described below : this was performed in a controlled ( growth chamber ) environment simulating summer conditions , with light watering as required . nine copies per accession were planted in general potting mix . a randomized complete block with subsampling was used . three fungal pathogens ( i . e . colletrotrichum graminicola , drechslera brizae and rhizoctonia cerealis )— causing a range of fungal diseases and infecting a range of different plant hosts — were included in antifungal bioassays used to analyse the potential anti - fungal activities of isolated fescue endophytes . fig5 shows results from anti - fungal bioassays of isolated fescue endophytes . results of anti - fungal bioassays are also shown in table 31 . a range of endophytes were found to have high ( h ) and medium ( m ) antifungal activity ( table 31 ). fig5 shows the ergovaline biosynthetic pathway . genes in the eas gene cluster which are involved in ergovaline biosynthesis are shown in fig5 and table 33 . the dmaw gene encodes dmat synthase enzyme , which catalyzes the first committed step in ergovaline biosynthesis . presence of the dmaw gene in novel fescue endophytes is shown in fig5 and presence of the eas gene cluster in novel fescue endophytes is shown in fig5 . fig5 shows the lolitrem b biosynthetic pathway . genes in the gene cluster which are involved in lolitrem b biosynthesis are shown in fig6 and table 34 . presence of gene cluster 1 ( itmg , itmm and itmk ) in endophytes is shown in fig6 , presence of gene cluster 2 ( itmb , itmq , itmp , itmf and itmc ) is shown in fig6 and presence of gene cluster 3 ( itme and itmj ) is shown in fig6 . fig6 shows the loline biosynthetic pathway . genes in the gene cluster which are involved in loline biosynthesis are shown in fig6 and table 35 . presence of loline biosynthetic gene cluster in novel fescue endophytes is shown in fig6 . fig6 shows an alkaloid biosynthetic gene analysis for endophyte strain nea23 . tables 36 and 37 show alkaloid biosynthetic gene analyses for various endophyte strains . table 36 shows results from the assessment of alkaloid biosynthetic gene presence / absence for different endophytes by mapping genome survey sequence reads corresponding to the different alkaloid biosynthetic genes / gene clusters . table 37 shows results from the assessment of alkaloid biosynthetic gene presence / absence for different endophytes by mapping genome survey sequence reads corresponding to the different alkaloid biosynthetic genes / gene clusters as well as corresponding alkaloid profile observed for corresponding tall fescue - endophyte associations . the objective of this work was to create novel variants of the perennial ryegrass endophyte , neotyphodium lolii , through induced polyploidisation and mutagenesis , with desirable properties such as enhanced bioactivities ( e . g . antifungal acitivity ), and / or altered plant colonization ability and stability of grass host - endophyte variant associations ( e . g . altered in vitro growth ), and / or altered growth performance ( e . g . enhanced plant vigour , enhanced drought tolerance , enhanced water use efficiency ) of corresponding grass host — endophyte variant associations . these grass host - endophyte variant associations are referred to as novel ‘ designer ’ grass - endophyte associations . experimental strategies for the generation and characterisation of novel designer neotyphodium endophyte variant strains through mutagenesis 1 . establishment of phenotypic screens for novel ‘ designer ’ grass - endophyte associations such as : enhanced biotic stress tolerance enhanced drought tolerance and enhanced water use efficiency enhanced plant vigour 2 . targeted generation ( i . e . polyploidisation and x - ray mutagenesis ) and characterisation ( i . e . antifungal bioassays , in vitro growth rate , genome survey sequencing [ gss ]) of novel ‘ designer ’ endophytes delivery of ‘ designer ’ endophytes into grass ( e . g . perennial ryegrass ) germplasm development process . assessment of enhanced biotic stress tolerance using nea12 is shown in fig7 and 72 . fig7 shows in vitro bioassays to assess antifungal activity of neotyphodium endophytes . fig7 shows a detached leaf assay to assess resistance to crown rust ( puccinia coronata f . sp . lolii ). assessment of enhanced drought tolerance and enhanced water use efficiency is shown in fig7 . this involved glasshouse and field trial screens for drought tolerance , survival and recovery , regrowth after drought , metabolic profiling and detailed phenotypic characterisation including multiple trait dissection ( based on assessments and measurements associated with plant morphology , plant physiology , plant biochemistry ). this involved creation of novel variation in neotyphodium endophytes without the use of transgenic technology . colchicine has been widely and successfully used for chromosome doubling in plants , e . g . perennial ryegrass . it inhibits chromosome segregation during mitosis inducing autopolyploidisation ( chromosome doubling ; see fig7 ). this enables the generation of novel endophytes through induced chromosome doubling and may be applicable to the production of artificial polyploid endophytes . the experimental work flow for chromosome doubling is shown in fig7 . flow cytometry calibrations to assess dna content in neotyphodium endophytes are shown in fig7 . peaks indicate relative nuclear dna content . flow cytometry analysis of nea12 dh strains is shown in fig7 and table 39 . 1 . st endophyte strain is highly stable , broadly compatible and produces lolitrems , peramine and ergovaline . 2 . nea12 endophyte strain produces janthitrem only . 3 . ar1 produces peramine only . analysis of growth rate of nea12 dh neotyphodium variant endophyte strains in in vitro culture after 8 weeks is shown in fig7 . in an initial screen , analysis of variance identified two nea12 dh neotyphodium variant endophyte strains ( nea12 dh17 and nea12 dh4 ) showing significantly different in vitro growth rate to the control nea12 endophyte : analysis of growth rate of nea12 dh neotyphodium variant endophyte strains in in vitro culture over 5 weeks is shown in fig1 . in a validation screen , student &# 39 ; s t - tests identified two nea12 dh neotyphodium variant endophyte strains ( nea12 dh17 and nea12 dh15 ) showing significantly different in vitro growth rate to the control nea12 endophyte : a list of fungal pathogens ( causing a range of fungal diseases and infecting a range of different plant hosts ) that were included in antifungal bioassays used to analyse nea12 dh neotyphodium variant endophyte strains to assess their spectrum of antifungal activities is shown in table 40 . antifungal bioassays of nea12 dh neotyphodium variant endophyte strains are shown in fig8 and 81 . twenty nea12 dh strains were screened for changes in antifungal activity . four nea12 dh strains ( i . e . dh5 , dh6 , dh13 and dh14 ) were identified as having greater antifungal activity compared to nea12 . genome survey sequencing and sequence analysis of nea12 dh neotyphodium variant endophyte strains nea12 dh neotyphodium variant endophyte strains with enhanced antifungal activity , showing faster in vitro growth rate and higher dna content were subjected to genome survey sequencing ( gss ). sequence data was generated for 10 nea12 dh strains and control nea12 strain ( highlighted in blue on table 41 ). de - novo assembly of the gss data from nea12 control strain — to act as a reference genome sequence for the analysis of the nea12 dh neotyphodium variant endophyte strains map the gss data sequence reads from the nea12 dh neotyphodium variant endophyte strains to the nea12 reference genome sequence identify potentially duplicated regions , i . e . regions with higher than expected sequence coverage identify gene sequences that may have been duplicated analysis of gss read depth of nea12 dh neotyphodium variant endophyte strains is shown in fig8 . analysis of sequence contigs that appeared to have higher than expected read depth indicates that no major duplication event has occurred ( excepting whole genome events ). the patterns of read depth across these contigs are not identical between strains . this suggests there are differences between the nea12 dh neotyphodium variant endophyte strains and the control nea12 strain . analysis of gss sequence assemblies for the nea12 dh neotyphodium variant endophyte strains and the control nea12 strain is shown in table 42 . independent de novo sequence assemblies were performed using parameters identical to those used in assembling the genome sequence for the control nea12 endophyte strain . differences in sequence assembly statistics may indicate genomic differences between strains . gss data obtained for the nea12 dh neotyphodium variant endophyte strains and used in the sequence assemblies reveal fewer bases incorporated into the sequence assembly and produce more sequence contigs . increased numbers of smaller sequence contigs may be caused by transposon movement / replication . analysis of sequence reads mapping to the nea12 genome sequence assembly is shown in fig8 . while we do not wish to be restricted by theory , if the genomes were the same no difference in the number of sequence reads mapping to the reference genome sequence would be expected . nea12 dh neotyphodium variant endophyte strains range from 35 - 70 % sequence reads mapping to nea12 sequence contigs & gt ; 5 kb in size . there are differences between the genome sequences of the nea12 dh neotyphodium variant endophyte strains and the control nea12 strain . summary of results on generation and characterisation of novel designer neotyphodium variant endophyte strains through colchicine treatment based mutagenesis sequence read depth changes were analysed in nea12 dh neotyphodium variant endophyte strains compared with the control nea12 strain . whilst no large partial genome sequence duplication events were detected , the occurrence of full genome duplication events in the nea12 dh neotyphodium variant endophyte strains cannot be excluded based on the gss sequence analysis . de novo sequence assemblies were independently performed on gss data obtained from the nea12 dh neotyphodium variant endophyte strains . differences in sequence assembly statistics indicate that genomic changes were caused by the colchicine - treatment in the nea12 dh neotyphodium variant endophyte strains . the number of sequence reads from nea12 dh neotyphodium variant endophyte strains mapping to the nea12 reference genome sequence varies between strains . all gss data analyses performed on the nea12 dh neotyphodium variant endophyte strains indicate genomic differences . in summary , the following novel designer endophytes were generated by colchicine treatment of nea12 endophytes : four nea12 dh neotyphodium variant endophyte strains ( dh5 , dh6 , dh13 and dh14 ) with enhanced bioprotective properties ( i . e . antifungal bioactivities ); one nea12 dh neotyphodium variant endophyte strain ( dh17 ) with higher in vitro growth rate than control nea12 strain ( i . e . potentially with enhanced stability / host colonization ability ); ten nea12 dh neotyphodium variant endophyte strains ( including dh5 , dh6 , dh13 , dh14 and dh17 ) and control nea12 strain subjected to genome survey sequencing ; and five nea12 dh neotyphodium variant endophyte strains ( including dh5 , dh13 and dh17 ) selected and subjected to isogenic inoculation in planta . in planta isogenic inoculation in perennial ryegrass with nea12 dh neotyphodium variant endophyte strains the following nea12 dh neotyphodium variant endophyte strains and control nea12 strain were used for in planta isogenic inoculation in perennial ryegrass : nea12 nea12dh5 showing higher antifungal activity than control nea12 nea12dh13 showing higher antifungal activity than control nea12 nea12dh4 showing slower in vitro growth rate than control nea12 nea12dh15 showing slower in vitro growth rate than control nea12 nea12dh17 showing faster in vitro growth rate than control nea12 the generation of designer neotyphodium endophytes genotypes by x - ray mutagenesis offers the opportunity to create novel endophyte variant strains with enhanced properties , such as enhanced stability in grass hosts , broader host compatibility as well as improved toxin profiles e . g . following elimination of the production of the detrimental alkaloid lolitrem b in the highly stable and broadly compatible st endophyte . such an novel designer endophyte would be advantageous over existing commercial endophytes , such as ar1 and ar37 , as it would be highly stable and broadly compatible and with optimal toxin profile . fig8 shows an experimental work flow for x - ray mutagenesis of endophyte strains . fig8 shows the indole - diterpene biosynthetic pathway . lolitrem b is the major toxin that causes ryegrass staggers , a disease of grazing animals . ten genes in 3 gene clusters are required for lolitrem biosynthesis . we focused initial analysis on 3 ltm genes , one from each gene cluster . optimised multiplex pcr analysis was designed and implemented . in a preliminary primary screen & gt ; 5 , 000 colonies of x - ray irradiated n . lolii — established as an initial resource of novel variation of n . lolii endoophytes induced through x - ray mutagenesis and representing a mutagenised n . lolii endophyte strain collection — of were screened by multiplex pcr analysis for the presence of targeted ltm genes leading to a preliminary identification of ˜ 140 putative lolitrem b gene cluster pcr - negative colonies (˜ 2 . 5 % of 5 , 000 colonies screened ). in a secondary screen high quality dna was extracted ( 140 liquid cultures ) and pcr analysis conducted . this identified 2 putative deletion mutants for one of the lolitrem b genes ( ltm j ). the colony number represents the unique identifier of the putative x - ray irradiation - induced ltm gene deletion mutant ( i . e . 139 - 6 and 145 - 15 ). black represents pcr - negative result for respective ltm gene analysis , white represents pcr - positive result for respective ltm gene analysis . antifungal bioassays of designer x - ray irradiated n . lolii variant strains there were eight x - ray irradiated n . lolii variant strains ( i . e . x - ray mutagenesis derived variant strains 1 - 35 , 4 - 7 , 7 - 22 , 7 - 47 , 123 - 20 , 124 - 6 , 139 - 6 , 144 - 16 and 145 - 15 ) and one control n . lolii strain ( i . e . st endophyte strain ). five fungal pathogens ( causing a range of fungal diseases and infecting a range of different plant hosts ) were included in antifungal bioassays used to analyse the x - ray irradiated n . lolii variant strains , as follows : bipolaris portulacae colletotrichum graminicola drechslera brizae phoma sorghina rhizoctonia cerealis no significant difference in antifungal activities of x - ray irradiated n . lolii variant strains tested was observed compared to the spectrum of antifungal activities observed for the control st endophyte strain . in vitro growth of designer x - ray irradiated n . lolii variant strains results from the analysis of in vitro growth rate of designer x - ray irradiated n . lolii variant strains are shown in fig8 , with a statistical analysis of in vitro growth undertaken at week 5 for the x - irradiated n . lolii variant strains compared to the control st strain , revealing significant differences in in vitro growth rates as follows : genome survey sequencing of designer x - ray irradiated n . lolii variant strains eight x - ray irradiated n . lolii st variant strains and corresponding control st strain were subjected to genome survey sequencing ( gss ), leading to 46 - fold to 79 - fold genome sequence coverage for the different strains as shown in table 45 . results from the analysis to detect genome sequence variation in x - ray irradiated n . lolii variant strains are shown in fig8 . corresponding results on the detection of single nucleotide polymorphisms ( snps ) are shown in fig8 and results on the detection of small insertions / deletions ( indels ) are shown in fig9 . differences in sequence read depth and pair insert size in x - ray irradiated n . lolii variant deletion mutant strains are shown in fig9 . results on sequence analysis for ltm gene clusters are shown in fig8 . no deletions , large or small , were found in the coding or regulatory sequences of ltm gene clusters . no snps , insertions or translocations were found in the coding or regulatory sequences of ltm gene clusters . spectrum of genome sequence changes detected in the x - ray irradiated n . lolii variant strains fig9 shows numbers of snps detected in genic regions of x - ray irradiated n . lolii variant deletion mutant strains . there are large differences in the number of snps detected in the x - ray irradiated n . lolii variant deletion mutant strains and compared to the control st strain . all x - ray irradiated n . lolii variant deletion mutant strains have over double the number of snps per mb across genic regions compared to the control st strain . x - ray irradiated n . lolii variant deletion mutant strains have on average 6 snps per mb , where the control st strain has 2 snps per mb . fig9 shows numbers of indels in genic regions of x - ray irradiated n . lolii variant deletion mutant strains . all x - ray irradiated n . lolii variant deletion mutant strains contain more indels in genic regions than the control st strain . the difference in indel numbers between the x - ray irradiated n . lolii variant deletion mutant strains and the control st strain is on average 134 indels per mb . when grouped by irradiation treatment ( i . e . irradiation dose applied and number of repeat irradiations ) there appears to be a peak in number of indels at 10gy * 2 treatment , consistent with the results obtained in the snp detection analysis . fig9 shows the spectrum of genome sequence changes in the form of deletions detected in x - ray irradiated n . lolii variant deletion mutant strains . table 46 shows examples of some of these genome sequence deletions detected in x - ray irradiated n . lolii variant deletion mutant strains . the x - ray irradiated n . lolii variant deletion mutant strain # 7 — 47 , which was generated following two x - irradiation treatments at 10 gy dose ( 10gy * 2 ) of n . lolii st endophyte , had the greatest number of large deletions . annotation of deleted sequences in the genomes of x - ray irradiated n . lolii variant deletion mutant strains x - ray irradiated n . lolii variant mutant strain 1 — 35 : for the x - ray irradiated n . lolii variant mutant strain 1 — 35 the following deleted sequences in st454contig00831 contig with a ˜ 4 , 400 - 8 , 000 bp length was detected , with this genome sequence region containing the following two predicted genes : 1 ) ref | xp — 386347 . 1 | hypothetical protein fg06171 . 1 [ gibberella 660 × 0 . 0 gb | eaw12630 . 1 | duf500 domain protein [ aspergillus nrrl 1 ]; 253 × 9e - 66 , and st454contig00831_augustus_gene — 3958 : 4728 ( 183 letters ); and for the x - ray irradiated n . lolii variant mutant strain 7 — 47 the following deleted sequences in st454contig01082 , st454contig01131 and st454contig02985 , with these genome sequence regions containing no predicted genes : mutagenesis index of x - ray irradiated n . lolii variant deletion mutant strains fig9 shows snps and indels per mb in genic regions of x - ray irradiated n . lolii variant deletion mutant strains derived from x - ray irradiation of n . lolii at different levels of irradiation . strain 1 — 35 has a 3 . 6 kb deletion ; strain 7 — 47 has 3 deletions ( 4 . 2 kb , 1 kb , 0 . 6 kb in length ). strain 124 — 6 has a partial duplication . strains 139 — 6 and 145 — 15 have partial duplications . given that st endophyte has approximately 443 . 5 genes per mb , using 10gy * 2 treatment , the expected rate of snp / indel occurrence is 0 . 33 per gene in the genome . x - ray irradiated n . lolii variant deletion mutant strains were analysed for many types of genome sequence variation i . e . deletions , snps , indels , inversions and translocations . snps , indels , deletions and duplications were identified in the genome survey sequences of x - ray irradiated n . lolii variant deletion mutant strains . there was an apparent peak in number of snps and indels in x - ray irradiated n . lolii variant deletion mutant strains recovered from administering 10gy * 2 x - ray irradiation treatment to n . lolii st endophyte . the x - ray irradiated n . lolii variant deletion mutant strain 7 — 47 had 3 large deletions . it was demonstrated that this mutagenesis method based on x - ray irradiation can be used to create novel designer neotyphodium endophyte strains , and enabled : 5 , 000 x - ray irradiated n . lolii variant endophyte strains derived from x - ray irradiation of st n . lolii endophyte were screened ; 140 putative x - ray irradiated n . lolii variant endophyte mutant strains were identified ; 9 x - ray irradiated n . lolii variant endophyte mutant strains were subjected to antifungal bioassays ; 9 x - ray x - ray irradiated n . lolii variant endophyte mutant strains were subjected to in vitro growth assays ; 9 x - ray irradiated n . lolii variant endophyte mutant strains were subjected to genome survey sequencing ; 2 x - ray irradiated n . lolii variant endophyte mutant strains with gene deletions ( 1 — 35 and 7 — 47 ) were identified ; and 3 x - ray irradiated n . lolii variant endophyte mutant strains with gene duplications ( 124 — 6 , 139 — 6 and 145 — 15 ) were identified . in planta isogenic inoculation in perennial ryegrass with x - ray irradiated n . lolii variant endophyte mutant strains control n . lolii st endophyte strain x - ray irradiation treatment derived n . lolii st endophyte variant strain 4 - 7 x - ray irradiation treatment derived n . lolii st endophyte variant strain 139 - 6 x - ray irradiation treatment derived n . lolii st endophyte variant strain 144 - 16 x - ray irradiation treatment derived n . lolii st endophyte variant strain 145 - 15 the x - ray irradiation treatment derived n . lolii st endophyte variant strains could be readily distinguished from control n . lolii st strain using mycelia extracts or filtrates alone . it will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings . all of these different combinations constitute various alternative aspects of the invention . bouton , j . h ., g . c . m . latch , n . s . hill , c . s . hoveland , m . a . mccann , r . h . watson , j . a . parish , l . l . hawkins and f . n . thompson ( 2002 ) agronomy journal 94 ( 3 ): 567 574 . latch , g . c . m , christensen , m . j , tapper , b . a , easton , h . s , hume , d . e , fletcher , l . r . 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