Patent Application: US-201214119247-A

Abstract:
the present invention provides substantially purified or isolated fungi of nodulisporium spp . or ascocoryne spp ., plants infected with said fungi , organic compounds produced by said fungi , and related nucleic acids , polypeptides and methods .

Description:
eleven fungal isolates were collected from two plant species at cool temperate rainforests within the dandenong ranges , yarra ranges and the otway ranges ( victoria , australia ). one isolate was collected from foliar tissue of lomatia fraseri in the dandenong ranges , while the other 10 isolates were collected from decaying wood of nothafagus cunninghamii in the yarra ranges and otway ranges . all isolates were morphologically and genetically ( 5 . 8s - its rrna gene ) identified . the isolate from l . fraserii was identified as nodulisporium sp . ( teleomorph : hypoxylon sp ., xylariaceae ), while the 10 isolates from n . cunninghamii were identified as ascocoryne sarcoides ( anamorph : coryne sp ., helotiaceae ). molecular markers based on single sequence repeats from expressed sequence tags ( est - ssr markers ) detected genetic diversity amongst a . sarcoides isolates , separating them according to origin ( i . e . either yarra ranges or otway ranges ). all eleven isolates exhibited bioactivity in in vitro bioassays against a range of plant pathogenic fungi , including fusarium oxysporum , sclerotinia minor and pythium ultimum . the in vitro bioassays indicated that the isolate of nodulisporium produced volatile bioactive compounds , while isolates of a . sarcoides produced liquid bioactive compounds . gc / ms analysis of nodulisporium identified 58 volatile organic compounds , including many monoterpenes ( e . g . eucalyptol ) and , sesquiterpenes ( e . g . β - elemene ), which may be produced by plants as defence compounds ( e . g . eucalyptol — eucalyptus oil ). the genes regulating the production of the terpenes were identified following the sequencing of the genome of the nodulisporium isolate . a total of 8 terpene synthases were identified that are thought to regulate the production of the mono - and sesquiterpene compounds in nodulisporium . the two fungi were morphologically characterised via micro - and macroscopic features of in vitro states and identified as nodulisporium sp . and a . sarcoides ( and in vivo state ). the identification of the isolates were supported by comparing sequences of the rrna gene ( 5 . 8s / its ) to closely related ascocoryne and nodulisporium species from around the world ( closest matches from genbank ). isolates of a . sarcoides clustered together with a bootstrap support of 81 . 0 %. similarly , the isolate of nodulisporium clustered closest to species of nodulisporium and hypoxylon ( the teleomorph of nodulisporium ), with a bootstrap support of 80 . 0 %. isolates of a . sarcoides were genotyped using est - ssr markers derived from neotyphodium species . amplification was expected as markers were derived from expressed genes , some of which were likely to be universally found across the fungal kingdom . isolates clustered according to origin . in vitro bioassays were established to determine the bioactivity of nodulisporium and a . sarcoides isolates against 3 plant pathogenic fungi , f . oxysporum , s . minor and p . ultimum . both nodulisporium and a . sarcoides reduced the growth of the plant pathogenic fungi by up to 100 %. bioassays indicated that volatile compounds were responsible for the bioactivity observed with nodulisporium , whereas the bioactive compounds of a . sarcoides were liquid . to evaluate the production of volatile compounds from nodulisporium , growth conditions were chosen to enhance the production ( diversity and quantity ) of these compounds . for example , high nutrient media ( e . g . potato dextrose agar ) was used as the carbon source for growth . as a result a total of 58 compounds were produced by nodulisporium including a range of terpenes , which are low molecular weight organic compounds that may be produced by plants as defence compounds . these terpenoid compounds included 21 monoterpenes ( α - thujene , β - sabinene , β - myrcene , α - phellendrene , α - terpinene , p - cymene , ( r )-(+)- limonene , eucalyptol , α - ocimene , β - ocimene , γ - terpinene , α - terpinolene , allo - ocimene , (−)- terpinen - 4 - ol , α - terpineol , 2h - pyran , tetrahydro - 2 -( propan - 2 - ylidene )- 5 - methoxy , 2h - pyran , tetrahydro - 2 - isopropyl - 5 - methoxy , 3 - cyclohexene - 1 - acetaldehyde , 4 - methyl - α - methylene -, 1 - cyclohexene - 1 - carboxaldehyde , 4 -( 1 - methylethenyl )-, p - mentha - 1 , 4 ( 8 )- dien - 3 - one ( isomers ), bicyclo [ 2 . 2 . 2 ] octan - 1 - ol , 4 - ethyl ,) and four sesquiterpenes ( β - elemene , α - guajene , bicyclo [ 5 . 3 . 0 ] decane , 2 methylene - 5 -( 1 - methylvinyl )- 8 - methyl , δ - guaijene ). a further 16 monoterpene - like compounds and seven sesquiterpene - like compounds were produced by nodulisporium . these terpenes had masses consistent with mono and sesquiterpenes , and were structurally similar based on their ion fragmentation ( cyclohexane -, cyclohexene - and pyran - derivatives ). a major constituent of the volatile metabolome of nodulisporium was eucalyptol which is major component of eucalyptus oil , a potent antimicrobial extract found within leaves of eucalyptus species . while the applicant does not wish to be restricted by theory , it is proposed that the volatile terpene compounds of nodulisporium are acting synergistically to deliver the biocidal activity in in vitro bioassays . the genome of the nodulisporium isolate was sequenced in an effort to determine the genes responsible for the regulation of the bioactive terpenes . mono - and sesqui - terpenes are produced via the mevalonate pathway through a series of condensation and phosphorylation reactions to yield prenyl pyrophosphate chains with 10 or 15 carbons . these products are then converted to monoterpenes ( 10 carbons ) or sesquiterpenes ( 15 carbons ) by a terpene synthase . terpene synthases promote the metal ( e . g . mg 2 + ) ion - dependent expulsion of pyrophosphate and catalyse the formation of acyclic and cyclic terpenes from the prenyl groups via a common ionization reaction , followed by various reactions such as isomerisation , cyclization , rearrangement ( hydride shifts , methyl shifts , alkyl shifts , wagner - meerwein shifts ), hydration and deprotonation . the majority of sesquiterpene synthases have been functionally characterised from microbes , unlike monoterpene synthases that have predominantly been characterised from plants . the enormous diversity of terpenes can be attributed to the unique ability of terpene synthases to synthesise multiple products from the one enzyme . while some terpenes synthases produce a single product , a large majority of mono - and sesqui - terpene synthases catalyse the formation of multiple terpene structures , often with high regio - and stereo - selectivity . for instance , in arabidopsis thaliana , the enzyme at - tps - cin was responsible for catalysing the formation of 10 acyclic ( e . g . myrcene and ( e )- β - ocimene ) and cyclic ( e . g . sabinene , α - pinene ) monoterpenes , with eucalyptol predominating ( 52 %). the genome of nodulisporium contained 8 terpene synthases , as these genes possessed structural domains specific to terpene synthases , including aspartate rich regions that form the substrate binding site . it is proposed that these 8 terpene synthases regulate the production of the volatile bioactive mono - and sesqui - terpenes of nodulisporium . nodulisporium and a . sarcoides represent a highly valuable microbial resource , principally due to there unique metabolism and ability to produce organic bioactive compounds via novel genes . these organisms , metabolites and genes are of commercial interest in the agricultural sector , particularly in the area of plant protection . fig1 shows apothecia ( a ) and conidiomata ( b ) of ascocoryne sarcoides growing on fallen logs of nothafagus cunninghamii . fig5 shows a mp phenogram ( 1 of 8631 ) based on 5 . 8s / its rrna gene sequences from 55 isolates of nodulisporium and hypoxylon species . highlighted area ( red ) shows victorian nodulisporium isolate . the phenogram was obtained using the close - neighbour - interchange algorithm of mega4 . 1 ( deletion of gaps and missing data ). numbers on the nodes represent frequency ( in per cent ) with which a cluster appears in 1000 bootstrap tests . scale bar equals 5 changes per 100 bases . fig6 shows a mp phenogram ( 199 of 330 ) based on 5 . 8s / its rrna gene sequences from 26 isolates of ascocoryne species . highlighted area ( grey ) shows victorian a . sarcoides isolates . the phenogram was obtained using the close - neighbour - interchange algorithm of mega4 . 1 ( deletion of gaps and missing data ). numbers on the nodes represent frequency ( in per cent ) with which a cluster appears in 1000 bootstrap tests . scale bar equals 5 changes per 100 bases . fig7 shows upgma phenogram for victorian ascocoryne isolates using measurements of average taxonomic distance based on est - ssrs . fig8 shows images of in vitro bioassays of ascocoryne isolates from the yarra ranges ( victoria ) against s . minor ( including an untreated control ). fig9 shows a gc / ms headspace analysis of volatile compounds produced by nodulisporium sp . ( dandenong ranges 1 ) when grown on pda for 1 , 4 , 7 , 10 , 13 , 16 , 19 and 22 days growth . each total ion chromatograph ( tic ) represents one day . fig1 shows the chemical structures of volatile compounds produced by nodulisporium sp . ( dandenong ranges 1 ). names of compounds ( from left to right , line by line ) are as follows : 1 , 4 cyclohexadiene , 1 - methyl ( 5 . 032 min ) α - thujene ( 9 . 312 min ) β - sabinene ( 10 . 868 min ) β - myrcene ( 11 . 425 min ) α - phellandrene ( 11 . 806 min ) p - cymene ( 12 . 578 min ) ( r )-(+)- limonene ( 12 . 575 min ) eucalyptol ( 12 . 825 min ) α - ocimene ( 12 . 941 min ) cyclohexane , 1 , 2 , 4 - tris ( methylene )- ( 13 . 075 min ) β - ocimene ( 13 . 249 min ) γ - terpinene ( 13 . 558 min ) α - terpinolene ( 14 . 469 min ) phenylethyl alcohol ( 14 . 469 min ) allo - ocimene ( 15 . 725 min ) benzoic acid ethyl ester ( 16 . 972 min ) (−)- terpinen - 4 - ol ( 17 . 159 min ) α - terpineol ( 17 . 566 min ) 2h - pyran , tetrahydro - 2 -( propan - 2 - ylidene )- 5 - methoxy ( 19 . 987 min ) 2h - pyran , tetrahydro - 2 - isopropyl - 5 - methoxy ( 20 . 124 min ) 3 - cyclohexene - 1 - acetaldehyde , 4 - methyl - α - methylene - ( 20 . 506 min ) 1 - cyclohexene - 1 - carboxaldehyde , 4 -( 1 - methylethenyl )- ( 20 . 676 min ) p - mentha - 1 , 4 ( 8 )- dien - 3 - one ( and isomer ) ( 21 . 744 / 22 . 849 min ) bicyclo [ 2 . 2 . 2 ] octan - 1 - ol4 - ethyl ( 22 . 526 min ) β elemene ( 23 . 129 min ) α - guajene ( 24 . 297 min ) bicyclo [ 5 . 3 . 0 ] decane , 2 methylene - 5 -( 1 - methylvinyl )- 8 - methyl ( 25 . 580 min ) δ - guaijene ( 25 . 998 min ) fig1 shows a representative terpene synthase sequence from nodulisporium ( g9560 , 313 amino acids ; seq id no : 1 ), aligned against a “ type ” terpene synthase from the conserved domain database ( ncbi ; seq id no : 2 ). the highlighted areas represent common domains associated with terpene synthases . the medium grey area identifies the aspartate rich regions that form the substrate binding site . the dark grey area identifies the regions that form the substrate binding pocket . the light grey area identifies the regions that form the active site lid residues . fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g226 . t1 , 339 amino acids ; seq id no : 3 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g1080 . t1 , 365 amino acids ; seq id no : 4 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g2861 . t1 , 293 amino acids ; seq id no : 5 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g4788 . t1 , 541 amino acids ; seq id no : 6 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g5351 . t1 , 373 amino acids ; seq id no : 7 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g6654 . t1 , 348 amino acids ; seq id no : 8 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g9560 . t1 , 313 amino acids ; seq id no : 9 ). fig1 shows an amino acid sequence of a terpene synthase of nodulisporium ( g11102 . t1 , 417 amino acids ; seq id no : 10 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g226 . t1 , 1017 base pairs ; seq id no : 11 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g1080 . t1 , 1095 base pairs ; seq id no : 12 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g2861 . t1 , 879 base pairs ; seq id no : 13 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g4788 . t1 , 1623 base pairs ; seq id no : 14 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g5351 . t1 , 1119 base pairs ; seq id no : 15 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g6654 . t1 , 1044 base pairs ; seq id no : 16 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g9560 . t1 , 939 base pairs ; seq id no : 17 ). fig2 shows a nucleic acid sequence of a gene encoding terpene synthase from nodulisporium ( g11102 . t1 , 1251 base pairs ; seq id no : 18 ). pieces of leaf and stem of lomatia fraserii were collected during surveys in the dandenong ranges . sections of leaf and stem were surface sterilised ( 70 % ethanol for 30 secs , flame sterilisation ) prior to the excision of internal tissues , which were then plated onto potato dextrose agar ( pda ) ( 39 g / l ) ( amyl media , dandenong , australia ) amended with achromycin ( 50 ppm ). endophytic fungi growing from the plant tissue were removed by excising a hyphal tip from each colony , and plated onto pda . each hyphal tip constituted one endophytic fungal isolate . isolates then underwent a preliminary screen for bioactivity by challenging them against rhizoctonia solani on pda . one isolate inhibited the growth of r . solani and was selected for further analysis . in addition , pieces of wood from fallen logs of nothafagus cunninghamii containing apothecia ( gelatinous purple discs , sexual stage ) or coniodamata ( gelatinous purple fingers , asexual stage ) characteristic of ascocoryne sarcoides ( fig1 ) were collected during surveys in the yarra ranges and the otway ranges respectively . sections of apothecia or conidiomata were surface sterilised ( 2 % naocl for 30 secs , 2 washes in sterile distilled water , sdw ) and plated onto pda ( 39 g / l ) ( amyl media , dandenong , australia ) amended with achromycin ( 50 ppm ). each apothecium or conidioma section comprised one isolate , with ten isolates collected in total , 6 from the yarra ranges and 4 from the otway ranges . pure cultures of the eleven fungal isolates ( i . e . hyphal plugs ) were placed in sdw and stored at room temperature and at 4 ° c ., and in 15 % glycerol at − 70 ° c . sections of conidiomata were placed in sdw and stored at room temperature . isolates were removed from storage and placed onto pda and allowed to grow at 25 ° c . ( in the dark ) until the formation of conidiophores . sections of hyphae containing conidiophores were mounted in lactic acid and examined under light microscopy ( in vitro description ). in addition , sections of conidiomata from the ascocoryne isolates were mounted in lactic acid and examined under light microscopy ( in vivo description ). colonies on pda initially white , becoming pale yellow to grey yellow . conidiophores branching loosely , pale brown , paler towards the apex , verruculose , 2 . 5 - 3 um wide . conidiogenous cells usually produced singly , pale brown , verruculose , 12 - 20 × 2 . 5 - 3 um . conidia borne from minutely visible denticles , pale brown , more or less smooth , ellipsoidal , 6 - 8 × 3 - 4 um ( fig2 ). by evaluating the microscopic features of the isolates growing in culture ( in vitro stage ) we confirmed that they were characteristic of an undescribed species of nodulisporium . colonies on pda initially white , becoming dark violet to grey violet , forming violet crystals in the medium . conidiophores complex , branching 3 - 5 times , hyaline , thin walled ( fig3 ). phialides hyaline , narrowly obclavate to cylindrical , straight to slightly curved , thin walled , 10 - 14 × 1 . 5 - 2 μm . conidia hyaline , subglobose to ellipsoid , sometimes slightly curved , 2 - 5 × 1 - 2 μm ( fig4 ). conidiomata synemmatous , determinate , 3 - 5mm × 1 - 5 mm , dark purple , gelatinous , unbranched , subulate to capitate , gregarious . hyphae of the stipe in two zones ; the ectal excipulum a textura angularis , the medullary excipulum a textura intricata . conidiophores complex , branching 3 - 4 times , hyaline , thin walled . phialides hyaline , narrowly obclavate to cylindrical , straight to slightly curved , thin walled , 10 - 14 × 1 . 5 - 2 μm . conidia hyaline , subglobose to ellipsoid , sometimes slightly curved , 2 - 5 × 1 - 2 μm . by evaluating the microscopic features of the gelatinous purple fingers ( conidomata , in vivo stage ) and the isolates growing in culture ( in vitro stage ) we confirmed that they were characteristic of a . sarcoides . genomic dna was extracted from cultures of the nodulisporium and a , sarcoides isolates grown in either pda or potato dextrose broth ( pdb ) using a dneasy plant mini kit ( qiagen ). a section of the ribosomal rna loci ( 5 . 8s / its ) was amplified with primers its4 and its5 ( white et al ., 1990 ). pcr amplifications were performed in 25 μl reaction volumes containing 1 . 0 u of platinum taq dna polymerase ( invitrogen ), × 1 pcr buffer , 0 . 2 mm of each dntp , 1 . 5 mm mgcl 2 , 0 . 5 μm of each primer , and 15 - 25 μg dna . reactions were performed in a thermocycler ( gradient palm - cycler , corbett research ) with cycling conditions consisting of denaturation at 94 ° c . ( 3 min ), followed by 35 cycles at 94 ° c . ( 30 s ), 50 ° c . ( 30 s ), and 72 ° c . ( 2 min ), with a final extension step at 72 ° c . ( 3 min ) to complete the reaction . pcr products were separated by electrophoresis at 100 v for 45 min in a 1 . 5 % ( w / v ) agarose gel ( containing ethidium bromide , 0 . 1 ppm ) in 0 . 5 × tbe running buffer and visualised under uv light . amplification products were purified using a pcr purification kit ( qiagen ), and sequenced using the bigdye terminator cycle v 3 . 1 sequencing kit ( applied biosystems ) on the abi 3730xl capillary sequencer ( applied biosystems ), according to manufacturers &# 39 ; instructions . sequences of victorian isolates were compared to reference sequences from known nodulisporium ( or related teleomorphs , i . e . hypoxylon and daldinia ) and ascocoryne species ( a . sarcoides or a . cylichnium ) from around world ( closest matches from genbank ). a total of 55 nodulisporium - related sequences were aligned with muscle ( edgar , 2004 ), while 26 ascocoryne - related sequences were aligned . aligned sequences were adjusted with clustalw / alignment explorer in mega 4 . 1 ( tamura et al , 2007 ). based on these sequences phylogenetic relationships were inferred using distance and maximum parsimony ( mp ) analyses . for distance analysis , phenograms were obtained using the neighbour - joining ( nj ) algorithm ( saitou et al , 1987 ), applying the kimura - 2 - parameter model ( kimura , 1980 ), as implemented in mega4 . 1 . for mp analysis , phenograms were obtained using the close - neighbour - interchange algorithm ( search level 3 ) ( nei et al , 2000 ), as implemented in mega4 . 1 . to find the global optimum phenogram 10 random sequences were added . measurements calculated for mp included tree length , consistency index , retention index and rescaled consistency index ( tl , ci , ri , rci ). in both analyses , alignment gaps and missing data were eliminated from the dataset ( complete deletion option ) and the confidence of branching was assessed by computing 1000 bootstrap replications ( felsenstein , 1985 ). of the 55 nodulisporium - related isolates the size of the rrna ( 5 . 8s / its ) gene sequence ranged from 436 - 664 base pairs , of which 371 were included in the final data set for analysis . in the nj analysis the optimal phenogram had a sum of branch length of 0 . 525 . the mp analysis yielded 8631 most parsimonious phenograms ( tl = 211 , ci = 0 . 654 ri = 0 . 916 , rci = 0 . 569 , for the parsimony informative sites ). nj and mp analyses yielded phenograms with similar topology and bootstrap values . therefore , only the mp phenogram is presented ( 1 of 8631 , fig5 ). isolates tended to cluster according to the teleomorph of nodulsporium species , hypoxylon and daldinia . the dandenong ranges isolate clustered with hypoxylon species , with an 80 % bootstrap support . this group formed a cluster with other nodulisporium and hypoxylon isolates , with a bootstrap support of 14 % ( clade 1 ) this cluster was alongside another group of hypoxylon isolates with a bootstrap support of 41 % ( clade 2 ). a large group of daldinia isolates formed the next related cluster with a 37 % bootstrap support ( clade 3 ). of the 26 ascocoryne isolates the average size of the rrna ( 5 . 8s / its ) gene sequence was approximately 569 base pairs , of which 436 were included in the final data set for analysis . in the nj analysis the optimal phenogram had a sum of branch length of 0 . 103 . the mp analysis yielded 330 most parsimonious phenograms ( tl = 46 , ci = 0 . 921 , ri = 0 . 964 , rci = 0 . 888 , for the parsimony informative sites ). nj and mp analyses yielded phenograms with similar topology and bootstrap values . therefore , only the mp phenogram is presented ( 199 of 330 , fig6 ). isolates tended to cluster according to ascocoryne species . all victorian isolates clustered together , with 64 % bootstrap support ( clade 1 ). they clustered alongside a group of a . sarcoides isolates from lithuania , sweden and new zealand , with 81 % bootstrap support ( clade 2 ). gliocladium roseum also clustered with these a . sarcoides isolates . finally , six isolates of a . cylichnium from latvia , lithuania and sweden clustered together , with 90 % bootstrap support ( clade 3 ). expressed sequence tag - simple sequence repeat ( est - ssr ) markers developed by van zijll de jong ( 2003 ) were used to evaluate genetic diversity amongst ten victorian ascocoryne isolates . a total of 34 est - ssr markers were initially evaluated , of which four were selected for routine genotyping based on their ability to detect levels of polymorphism between isolates ( table 1 ). pcr amplifications were performed in 20 μl reaction volumes containing 0 . 5 u immolase ( bioline ), 1 × pcr buffer , 0 . 2 mm of each dntp , 0 . 25 μm each primer , and 10 ng fungal genomic dna . the forward primer was 5 ′- end labelled with a fluorescent phosphoramidite dye ( 6 - fam , hex , or ned ). amplification was performed in a thermocycler using an appropriate touchdown profile depending on the t m value of the primer pairs : ( program 1 , p1 ) 95 ° c . ( 10 min ), 10 cycles at 94 ° c . ( 30 s ), 55 ° c . ( 30 s ) and 72 ° c . ( 1 min ) with a reduction of annealing temperature of 1 ° c . every cycle , followed by 20 cycles at 94 ° c . ( 30 s ), 45 ° c . ( 30 s ), 72 ° c . ( 1 min ); ( program 2 , p2 ) a similar profile to ( p1 ) with an initial annealing temperature of 60 ° c . and final annealing temperature of 50 ° c . ; ( program 3 , p3 ) a similar profile to ( p1 ) with an initial annealing temperature of 65 ° c . and final annealing temperature of 55 ° c . pcr products ( 2 ml ) were diluted 1 : 99 ( p1 and p3 ) or 1 : 199 ( p2 ), and analysed on the abi 3730xl capillary sequencer ( applied biosystems ), according to manufacturers instructions . products or alleles for each of the victorian ascocoryne isolates were characterised by size ( i . e . number of base pairs ) using genemapper version 3 . 7 software ( applied biosystems ). isolates were then scored for the presence ( 1 ) and absence ( 0 ) of each allele . a similarity matrix was generated with this data using the dice coefficient ( dice , 1945 ; ntsyspc version 2 . 10t ). phenograms were constructed by the unweighted pair group method of arithmetic averages ( sahn program — upgma clustering method , ntsyspc version 2 . 10t ). the resulting genetic relationships were evaluated by cophenetic correlation and principle coordinate analysis ( mxcomp and eigen programs , ntsyspc version 2 . 10t ). of the 34 est - ssr markers initially evaluated , 18 ( 53 %) produced amplification products , but only four ( 12 %) detected genetic polymorphism between the victorian ascocoryne isolates . analysis of ssr polymorphism across the 10 victorian isolates identified 8 different alleles . a upgma phenogram constructed using the average taxonomic distance based on ssr polymorphism across the ten victorian isolates , showed a separation largely based on the origin of the isolate ( e . g . otway ranges cluster or yarra ranges cluster ) ( fig7 ). within the yarra ranges cluster the yarra ranges 7 isolate branched apart from the core cluster . similarly , the otway ranges cluster branched apart leaving otway ranges 1 separated from the remaining otway ranges isolates . the cophonetic correlation between distance matrices was high ( r = 0 . 90 ). in vitro bioassays were established to test the bioactivity of victorian nodulisporium and a . sarcoides ( yarra ranges only ) isolates against a range of plant pathogenic fungi , fusarium oxysporum , sclerotinia minor and pythium ultimum . nodulisporium was compared against the bioactive endophytes muscodor albus ( cz620 ) and endophyte a . the bioassays used two types of petri plates — standard 90 mm petri plates for a . sarcoides , and 90 mm split petri plates for nodulisporium . the split plates consisted of an impermeable barrier through the centre of the plate , which completely separated the plate into two halves , with only volatile compounds capable of passing over the septum ( i . e . no direct contact between test fungi or their liquid exudates ). the isolates were inoculated on to petri plates containing pda by placing a 6 mm agar plug containing actively growing mycelia , 13 mm from the edge of the plate ( i . e . on one half of the plate ). isolates were allowed to grow at 25 c ( in the dark ) for 7 days for nodulisporium and 20 days for a . sarcoides . subsequently , the plant pathogenic fungi were inoculated on to the other half of the plate by placing a 6 mm agar plug containing actively growing mycelia , 13 mm from the edge of the plate . plates were sealed with ldpe plastic film ( approximately 0 . 01 mm thick ). after 5 days the growth of the plant pathogenic fungi were determined by measuring the radius of the colony ( toward the centre of the plate ). measurements were compared to the control and expressed as percentage inhibition versus the control . data were analysed using anova as performed in genstat , version 11 ( payne et al , 2008 ). the experiment was fully randomised with 3 replicates for nodulisporium and a . sarcoides . the nodulisporium isolate showed strong levels of activity against the 3 horticultural crop pathogens , completely inhibiting the mycelial growth of p . sulcatum and s . minor , and inhibited the growth of f . oxysporum by up to 46 . 4 % ( table 2 ). nodulisporium also provided equivalent ( or better ) control of pathogens to the bioactive endophytes , muscodor albus ( cz620 ) and endophyte a . isolates of a . sarcoides from the yarra ranges inhibited mycelial growth of f . oxysporium and s . minor ( table 3 , fig8 ). yarra ranges 11 was the most active isolate against f . oxysorum and s . minor , inhibiting mycelial growth by 31 . 8 % and 85 . 0 % respectively . yarra ranges 11 had significantly greater activity against f . oxysporum than all other isolates . yarra ranges 11 , 12 , 13 and 15 were the most active isolates against s . minor , significantly greater than yarra ranges 7 and 10 . gases were analysed in the head space above cultures of nodulisporium . the isolate was cultured under microaerophilic conditions , which consisted of growing the fungus on pda slopes ( 39 g / l ) ( amyl media pty ltd ) in 20 ml glass vials , with an agar : air ratio of 1 : 2 . 5 . vials were sealed with a screw cap lid with ptfe septum , and grown for 22 days at room temperature . a head space solid phase microextraction ( spme ) was performed to capture volatiles produced by nodulisporium . a stableflex fibre ( supelco ) consisting of a matrix of divinylbenzene / carboxen ( dvb / car ) on polydimethylsiloxane ( pdms ) ( 50 / 30 um ) was used to absorb volatiles from the head space of vials . automated sampling was performed by an agilent gc sampler combined with gerstel maestro software . the fibre was conditioned ( baked at 250 ° c .) daily for 20 minutes prior to commencement of activities and for 2 minutes between each sample . for each sample the fibre was inserted into the vial and incubated at room temperature for 5 minutes to absorb volatiles , after which the fibre was inserted into a splitless injection port of an agilent 7890 gc system where the contents was thermally desorbed ( 250 ° c . for 6 mins ) onto a capillary column ( agilent hp - 5ms , 30 m × 250 um id ., 0 . 25 um film thickness ) coupled with a deactivated fused silica guard ( agilent , 6 . 02 m .× 250 um id .). the column oven was programmed as follows : 40 ° c . ( 3 . 5 min ), 5 ° c ./ min to 200 ° c ., hold at 200 ° c . ( 2 min ). the carrier gas was helium with a constant flow rate of 1 . 2 ml / min . the gc was interfaced with an agilent 7000 gc / ms triple quadruple mass selective detector ( mass spectrometer , ms ) operating in electron impact ionization mode at 70 ev . the temperature of the transfer line was held at 280 ° c . during the chromatographic run . the source temperature was 280 ° c . acquisitions were carried out over a mass range of 35 - 450 mz , with a scan time of 300 ms . initial identification of the volatiles produced by the nodulisporium isolates was made through library comparison using standard chemical databases . secondary confirmatory identification was made by comparing mass spectral data of authentic standards with data of the fungal volatiles . all chemical names in this patent application follow the nomenclature of the standard chemical databases . in all cases , uninoculated control vials were also analysed and the compounds found therein were subtracted from those appearing in the vials supporting fungal growth . tentative identification of the fungal volatiles was based on observed mass spectral data as compared to those in these chemical databases and those of authentic standards ( where possible ). the gc - ms analysis ( 0 - 37 . 5 mins ) identified 58 volatile metabolites produced by nodulisporium when grown for 1 - 22 days on pda at room temperature ( table 4 , fig9 and 10 ). the metabolites produced by nodulisporium were representatives of a number of structural classes , with the terpenes predominating , accounting for over 82 % of the compounds produced by nodulisporium . there were 21 monoterpenes ( α - thujene , β - sabinene , β - myrcene , α - phellendrene , α - terpinene , p - cymene , ( r )-(+)- limonene , eucalyptol , α - ocimene , β - ocimene , γ - terpinene , α - terpinolene , allo - ocimene , (−)- terpinen - 4 - ol , α - terpineol , 2h - pyran , tetrahydro - 2 -( propan - 2 - ylidene )- 5 - methoxy , 2h - pyran , tetrahydro - 2 - isopropyl - 5 - methoxy , 3 - cyclohexene - 1 - acetaldehyde , 4 - methyl - α - methylene -, 1 - cyclohexene - 1 - carboxaldehyde , 4 -( 1 - methylethenyl )-, p - mentha - 1 , 4 ( 8 )- dien - 3 - one ( isomers ), bicyclo [ 2 . 2 . 2 ] octan - 1 - ol , 4 - ethyl ,) and four sesquiterpenes ( β - elemene , α - guajene , bicyclo [ 5 . 3 . 0 ] decane , 2 methylene - 5 -( 1 - methylvinyl )- 8 - methyl , δ - guaijene ) produced by nodulisporium . a further 16 monoterpene - like compounds and seven sesquiterpene - like compounds were produced by nodulisporium . ( table 4 fig9 and 10 ). these terpenes had masses consistent with mono and sesquiterpenes , and were structurally similar based on their ion fragmentation . fragmentation patterns also indicated the presence of a cyclohexane , cyclohexene or pyran ring as the primary structure ), which is consistent with cyclic monoterpenes . the genome of nodulisporium sp . ( dandenong ranges 1 ) was sequenced using the genome sequencer flx titanium ( gs flx titanium ), using standard and modified protocols for this technology . a shotgun library of the fungal isolate was prepared from 5 μg of intact genomic dna , as per the dneasy plant mini prep ( qiagen ) protocol . following library preparation , the resulting single stranded ( ss ) dna library showed a fragment distribution between 500 and 2000 bp , with an average of 750 bp . the optimal amount of ssdna library input for the emulsion pcr ( empcr ) was determined empirically through two small - scale titrations leading to 1 . 7 molecules per bead used for the large - scale approach . the large - scale empcr generated 4 , 602 , 000 dna - carrying beads for the two - region - sized 70 × 75 mm picotiterplate ( ptp ). one region was subsequently loaded with 2 , 000 , 000 dna - carrying beads . during the sequencing run a total of 200 cycles of nucleotide flows ( flow order tacg ) were performed , which were assessed via a pipeline of 454 life sciences / roche diagnostics software version 1 . 1 . 03 . the output consisted of a standard flowgram format ( sff ) file that provided information about read flowgrams , basecalls , and per base quality scores . the sff file was subsequently used to assemble ( de novo ) high quality reads into contiguous sequences using the 454 life sciences / roche diagnostics software , newbler v2 . 3 ( gsassembler ). the gs flx titanium sequencing run yielded 663 , 514 high quality reads , with an average read length of over 420 bp . a total of 6 , 938 contigs were assembled de novo , of which 6 , 165 were larger than 500 bp . overall , contigs contained around 33 . 9 mb of sequence , at sequencing depth of × 6 . 0 . the contig size ( x / n50 ) was 5 . 4 / 8 . 6 kbp . the largest contig was 47 . 4 kbp . in addition , the genome of nodulisporium sp . ( dandenong ranges 1 ) was sequenced using the illumina hiseq platform using standard and adapted protocols for this technology . a paired end library of the isolate was prepared from 2 ug of intact genomic dna as per the dneasy plant mini prep ( qiagen ) protocol . dna was sheared to fragments of 200 - 700 bp , end - repaired , a - tailed and ligated to illumina paired end adaptors . the ligated fragments were size selected at 400 and 600 bp on agarose gels , ligated again with multiplex adaptors and amplified to the desired concentration by qpcr and pcr . finally , libraries were titrated ( kapa ) to accurately measure the number of competent molecules present . library concentrations were adjusted and sequenced on the illumina hiseq 2000 , with read lengths of 90 - 100 bp . raw sequences were filtered for low quality and short length , and trimmed of adapter sequence and paired - end read overlap . the illumina hiseq sequencing run yielded 23 , 354 , 002 raw reads , of which 11 , 677 , 001 were deemed of high quality . high quality reads from both the gs flx titanium and illumina hiseq sequencing runs were then assembled with velvet to construct contigs . a total of 4299 contigs were assembled de novo , of which 1543 were greater than 1 kb ( large contigs ). the total number of bases in large contigs totalled 37 . 8 mb with an estimated sequencing depth of × 25 . 0 . the contig n50 was 101 . 5 kbp with the largest contig measuring 397 . 3 kbp . the gene prediction program augustus was used to predict coding domains in the contigs of nodulisporium , according to manufacturer &# 39 ; s instructions . in augustus , trained models of a closely related species , aspergillus oryzae , was used to predict coding regions in contigs of nodulisporium . a total of 9 , 958 coding regions were predicted for nodulisporium from the assembly . the predicted genes were then compared against the conserved domain database ( cdd ) and the non - redundant protein database ( nrpd ) to determine putative function . the comparison was completed using the ncbi alignment tools rps - blast ( cdd ) and blast - p ( nrpd ) of the 9958 predicted genes for nodulisporium 6525 were found to contain functional coding domains when compared against the cdd ( evalue & gt ; 1e - 5 ). an analysis of the specific function of coding domains identified a number of unique genes in nodulisporium , which are involved in the regulation of key secondary metabolites . a total of 8 putative genes were found to contain non - plant terpene synthase domains ( fig1 , table 6 ). the average length of the putative non - plant terpene synthase genes from nodulisporium was 376 amino acids . the eight gene sequences are represented in fig1 - 19 ( amino acid sequences ) and fig2 - 27 ( nucleic acid sequences ). when the 8 putative terpene synthase genes were compared against the nrpd , sequences were found to be highly similar to terpene synthases from the fungi leptosphaeria maculans , trichoderma reesei , aspergillus species and penicillium species , and the bacterium nostoc punctiforme ( table 7 ). sequences from penicillium rocquerfortii and aspergillus terreus are known to regulate the production of sesquiterpenes , providing evidence to suggest g226 and g9560 may regulate the production of the sesquiterpenes identified in the volatile bioactive compounds . the remaining genes may regulate the production of the monoterpenes in nodulisporium . it is widely regarded genes regulating fungal secondary metabolism are commonly found in clusters , including those regulating terpene synthesis ( e . g . gibberellin — 7 genes , trichothecene — 11 genes ). all of the putative terpene synthases identified in nodulisporium were located on large contigs (& gt ; 15247 bp ) enabling flanking genes to be comprehensively evaluated . the putative function of common flanking genes included cytochrome p450 oxidases ( add oxygen functional groups ), transporters ( transmembrane proteins for antibiotic resistance ) and protein kinases ( gene regulation ). for instance , g5351 is located alongside a putative p450 , a transporter and a polyprenyl synthase ( precursor compounds to terpenes ). similarly g4788 and 6654 are located on the same contig , 3 genes apart . one of the genes separating the putative terpene synthases is a putative transporter . these flanking genes provide further evidence to suggest that the putative terpene synthases are regulating mono - and sesquiterpene synthesis . 1 . edgar r c ( 2004 ) muscle : multiple sequence alignment with high accuracy and high throughput . nucleic acids research 32 , 1792 - 1797 . 2 . felsenstein j ( 1985 ) confidence limits on phylogenies : an approach using the bootstrap . evolution 39 : 783 - 791 . 3 . kimura m . 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