NZ233083A - Pest-resistant, endophyte-infected plants - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £33083 * 283083 No.: 233083 Date: 26 March 1990 NEW ZEALAND PATENTS ACT, 1953 annaui*™'"""11 '< r-:.7. PATENT OFFICE 21 MAR 1331 COMPLETE SPECIFICATION IMPROVEMENTS IN/OR RELATING TO FUNGI AJP&S IC'W t ih fhe "Right frlew'Z&daud >i/We, HER MAJESTY THE QUEENft, of the Department of Scientific and Industrial Research Plant Protection Division, Palmerston Research Centre, Fitzherbert Avenue West, Palmerston North, New Zealand hereby declare the invention for which I/W pray that a patent may be granted to I/We^ and the method by which it is to be performed, to be particularly described in and by the following statement -Z- 233083 This invention relates to endophytes and combinations of endophytes with plants. More particularly it relates to endophytes which form combinations with herbage variety plants. The resulting endophyte-infected plants are resistant to pests but may be tolerated by grazing animals.

Many varieties of ryegrass and tall fescue are infected with a fungal endophyte of the genus Acremonium which lives within the grass as fungal mycelium. It is not possible to tell with the naked eye whether the plants contain the fungus. This can be determined by examining fragments of the plant under a microscope. These endophytic fungi are spread as mycelium in the seeds from infected plants and when the seeds germinate the fungus also starts growing again and moves into the above ground parts of the seedlings. This is the only way of which we are aware that the Acremonium species of endophyte fungi of ryegrasses and tall fescue are spread in nature.

When seed containing endophyte is stored the endophyte can eventually die. How quickly the endophyte dies depends on the moisture content of the seed and the conditions of storage. We have found that under ideal storage conditions the endophyte will remain viable for at least 20 years. Under poor storage conditions it will be dead within a few months. Plants which grow from seed containing dead endophyte will be free of the fungus for the rest of the plants' lives. Endophyte-free plants do not become naturally infected with endophyte and conversely, plants which are infected with endophyte remain infected for the rest of their lives.

The Acremonium endophyte in perennial ryegrass we have named Acremonium 1 o 1 ii. Over the last decade it has been found that this fungus has a great influence on its ryegrass host. Perennial ryegrasses infected with the fungus contain compounds called lolitrems and it is believed these compounds are the cause of a serious animal disorder called ryegrass staggers. This is a neuromuscular condition of animals which graze endophyte-infected ryegrasses. It occurs mainly in summer in the warmer regions of New Zealand and in parts of Australia and the USA. Hence, this endophyte is undesirable in grass from the point of view of animal health. 233083 Acremonium lolii can, however, benefit ryegrass and improve its persistence in a pasture. It produces a compound called peramine which makes the ryegrass unpalatable to one of the most important insect pests of pastures, namely the Argentine stem weevil (Listronotus bonariensis). Weevil larvae burrow into the stems of ryegrasses and kill the grass tiller, while heavy infestation may kill the whole plant. The peramine in endophyte-infected ryegrass deters adult weevils from laying their eggs on these plants, which are thus protected from serious weevil damage.

The presence of endophyte in ryegrass therefore has the disadvantage of making the grass toxic to animals under some conditions, but the advantage of conferring resistance to some insect predators. There are also indications that endophyte-infected ryegrasses have better tolerance to drought than do endophyte-free plants. They may also be more resistant to some fungal diseases. These conflicting factors must be balanced in determining the merits or otherwise of sowing endophyte-infected seed. Some farmers sow ryegrass seed that is infected with endophyte, iri the belief that it is better to put up with the possibility of ryegrass staggers over summer than to have one's ryegrass pastures wiped out by weevil. Other farmers regard ryegrass staggers as too serious a problem on their properties to allow endophyte-infected ryegrass to be grown and they take the risk of growing endophyte-free ryegrass or else sow other species of grass which may not be as suitable as ryegrass.

The toxins responsible for ryegrass staggers have been identified and named lolitrems (Gallagher et al J. Chem. Commun., [1984] 614-616). When A. lolii is grown axenically, lolitrems have not been detected and so they are probably produced only by the combined plant-endophyte system.

Most ryegrasses infected with A. lolii produce an alkaloid which has been named peramine (Rowan et al J. Chem. Soc. Chem. Commun., [1986] 935-936). It is produced by the fungus in axenic culture and is a very potent feeding deterrent. Feeding trials have shown peramine deterred the important ryegrass pest Listronotus bonariensis when as little as 0.1 ppm of peramine was present 9 o Q n Q Q o < > U O ij in an artificial diet. Preliminary toxicity trials with mice have indicated that its toxicity to warm blooded animals is low.

In addition to its toxic effects on animals and insects A. lolii also affects the ryegrass host plant. Infected plants grow better than non-infected ones, especially if they are subjected to drought stress. The effect on growth differs between plants and it is not known whether the strain of fungus, genotype of plant, or their interaction which causes the variation between infected plants.

It is known how to isolate an endophyte from one plant in which it occurs naturally and to infect another endophyte-free plant with the so isolated fungus. It also is known how to assay for lolitrem and peramine levels in plants infected with endophytes. While one can determine the levels of peramine and lolitrem in plants where endophytes occur naturally it is not reliably predictable that the same levels of peramine and lolitrem will be produced when an endophyte-free plant is infected with an endophyte. It would be advantageous to be able to produce new endophyte/plant combinations which produce high peramine and low or zero lolitrem levels.

It is an object of this invention to go some way towards meeting this desideratum or at least to offer the public a useful choice.

Accordingly the invention may be said broadly to consist in an endophyte of the species A. lolii in axenic culture, said endophyte being capable in combination with a herbage plant which has been inoculated with said endophyte of producing relatively high levels of peramine and relatively low levels of or no lolitrem.

Preferably said A. loli i species is the strain "Premier" (as herein described).

In another aspect the invention may be said broadly to consist in a herbage plant inoculated with an endophyte, the combination producing a relatively high level of peramine and a relatively low level of or no lolitrem.

Preferably said herbage plant is of the genus Lolium. -5- 233083 More preferably said endophyte is the strain "Premier" (as herein described).

Preferably said plant is a Lolium of the species Lolium perenne, Lolium multiflorum, Lolium hybridum or Lolium temulentum.

Alternatively said plant is of the genus Festuca.

More particularly said plant is a Festuca species Festuca arundinacea or Festuca rubra var comutata.

The invention may be said broadly to consist in seeds of any one of the herbage plants herein above defined infected with an endophyte as herein above defined.

The invention may also be said broadly to consist in plants grown from seeds infected with endophytes as herein above defined.

In another embodiment the invention may be said broadly to consist in a method of inoculating herbage plants with an endophyte to produce a combination containing a relatively high level of peramine and a relatively low level of lolitrems comprising: selecting a plant infected with an endophyte which produces a relatively high level of peramine and relatively low levels of lolitrem, extracting said endophyte from said plant, inoculating an endophyte-free plant with said endophyte and analysing the infected plant and selecting infected plants with high peramine and low lolitrem levels.

Preferably said plant selecting steps each comprise analysing an endophyte infected plant for the presence of peramine and lolitrems and selecting those plants which have both a peramine level of at least 2 mg/kg of dry plant matter and a lolitrem level of no more than 0.2 mg/kg of dry plant matter.

Preferably said endophyte extracting step comprises removing leaf sheath tissue from endophyte containing plants, sterilizing said tissue and growing endophyte mycelium therefrom in a suitable medium.

Preferably said endophyte inoculating step comprises inserting endophyte mycelium from said extracting step into a cut in the tissue of an endophyte free seedling of grass to be inoculated. - 6 - 2 8 u 0 8 3 Preferably said method includes the additional steps of growing said inoculated herbage plant seedlings to produce seeds and harvesting the seeds so produced.

The invention consists in the foregoing and also envisages constructions of which the following gives examples.

EXAMPLE 1: Isolation of Endophytes (a) From plants.

Leaf sheath tissue of plants containing desired endophytes was removed from the plant and surface sterilized by dipping it into 70% ethanol for 5 seconds before placing it in a 10% solution of sodium hypochlorite (0.5% available chlorine) and shaking for 5 minutes. The tissue was then rinsed in sterile water and cut into 2-3 mm pieces. (b) From seed.

Seeds were surface sterilized by soaking for 20 minutes in 50% sulphuric acid followed by rinsing several times in sterile water, soaking in 10% sodium hypochlorite solution for 20 minutes and rinsing again in sterile water.

All surface sterilized tissues were placed on potato dextrose agar containing antibiotics (100^ug/ml streptomycin + 100 ^ug/ml penicillin) in Petri dishes and incubated at 20°C for 4-5 weeks. By this time colonies had sufficient growth to enable them to be used for inoculating grass seedlings.

EXAMPLE 2: Inoculation of Seedlings with Endophytes Seeds of grass cultivars to be infected with endophyte were surface sterilized by the method described in Example 1. The seeds were then placed on 4% water agar in Petri dishes, generally 5 seeds per dish, and incubated in darkness at 20°C. The seeds were examined daily and any seeds contaminated with fungi or bacteria removed.

Seedlings are ready for inoculation when the coleoptile is 15-20 mm in length. A scalpel was used to cut a 3-5 mm longitudinal slit right through the ~ ' " 9 '> '5 0 P O i J U CJ O tissue at the junction of the me:>ocotyl and coleoptile. This region can be identified by a slight bulging in the shoot at this meristematic junction. A small portion of endophyte mycelium was inserted into the slit which is then closed by gently squeezing the stem.

After inoculation the seedlings were incubated in darkness at 20-22°C for 10-12 days and then transferred from the incubator and placed under fluorescent lights for a further 2-4 days.

Seedlings were then removed from the Petri dishes and planted in root trainers or pots containing sand and slow release fertilizer granules. Pots were placed on a greenhouse bench with Polythene sheeting over them to maintain high humidity. The sheeting was removed 7-10 days later when the seedlings had become established. They were left on the greenhouse bench for a further 6-8 weeks, depending upon the species and cultivar of grass, by which time they should be large enough to examine for endophyte infection.

EXAMPLE 3: Detection of Endophyte infection A leaf with its sheath was removed from the plant, the upper epidermis cut with a scalpel and a strip of epidermis pulled from the sheath with forceps. This tissue was mounted in lactophenol containing aniline blue on a microscope slide and warmed for a few seconds over a flame to accelerate staining. Acremonium lolii appeared as septate, sparsely branched intercellular hyphae running longitudinally in the sheath.

EXAMPLE 4: Measurement of Peramine and Lolitrem levels in plants 4.01 Extraction procedure Weighed samples of approximately 100 mg of dry ground herbage infected with endophyte were extracted by continuous gentle inversion with 3 ml of methanol - chloroform (1:1, v/v) for 30 minutes at room temperature.

After 30 minutes, 3 ml of hexane and 3 ml of water were added and mixing was continued for an additional 30 minutes before centrifugation to separate the two phases and sediment the plant residues. . o r> <"■ Q Q - 8 - »i o O <J t-5 4.02 Peramine analysis A 1 ml volume of the lower aqueous phase from Example 4.01 was passed through a Analytichem Bond Elut CBA column, 100 mg absorbent in the ammonium form. The sample was washed through with 1 ml of water and the bound peramine then eluted with 0.5 ml of 5% (v/v) aqueous formic acid in 80% methanol. The net weight of the solution and an estimate of its density were used to calculate the volume of the peramine containing solution. 50 ul of this solution was used for the quantitation of peramine by reverse phase high performance liquid chromatography (HPLC) using a C-18 column, 5 um particle size, 100x8 mm with detection at 280 nm. The isocratic mobile phase was prepared by dissolving guanidinium carbonate (1.44 g/1) in water, adding formic acid (1.6 ml/1), acetonitrile (330 ml/1) and making up the volume to 1 litre with water. The flow rate was 1 ml/minute. Peramine was also quantitated by pseudo-reverse phase IIPLC using a Brownlee 100x4 si lira.column and an aqueous buffer containing 20% v/v acetonitrile arid 10 mM guanidinium formate (pH 3.7) at 0.8 ml/minute and detection at 280 nm. Peramine concentrations were calculated by comparison of peak heights with those of known standards. 4.03 Lolitrem analysis 3 ml of the upper (organic) phase from Example 4.01 was placed into a 500 mg size Bond Elut SI (3 ml) column. The extract was washed into the column with dichloromethane (1 ml) and washed down the column with approximately 0.6 ml of dichloromethane - acetonitrile (4:1, v/v) until the green pigment band had not yet eluted from the bottom of the column. The column was then eluted with 2 ml of dichloromethane - acetonitrile (4:1, v/v) into a 2 ml volumetric flask and 50jul of this solution was used for HPLC injection. HPLC quantitation used a Brownlee 100x4 mm silica column with fluorimetric detection (excitation 265 nm, emission 450 nm) and dichloromethane - acetonitrile (5:1, v/v) at 0.7 ml/minute as solvent. Lolitrem concentrations were calculated by comparison of peak heights with those of known standards.

*J '' "■ f' f-x ... o i j u o o EXAMPLE 5: Storage conditions The maintenance of high levels of viable A. lolii endophyte in seed lines requires special storage conditions and careful inventory management. Both seed moisture and storage temperature are important. Endophyte viability is more sensitive to these factors than is seed viability. Over a period of 12-18 months and under normal seed storage conditions endophyte viability can decline from 90% to only 10-50% of seeds containing viable endophyte, while seed germination will not begin to decline until after 36 months.

Seed moisture levels of 12-14% are regarded as safe for grass seed storage. Seed moistures less than 11.5% or temperatures of 5°C will maintain endophyte viability for 24 months. A seed moisture of 8% or a temperature of 5°C will maintain endophyte viability for 36 months. The combination of 8% moisture and 5°C will keep the endophyte viable for at least 5 years. To retain viable endophyte over many years storage temperatures of -15 to 0°C and low seed moisture are required.

EXAMPLE^6: Characteristics of Acremonium lolii Endophyte Strain 'Premier1 The endophyte was isolated from a French perennial ryegrass ecotype by the procedure of Example 1. Colonies grown on potato dextrose agar were white, cottony and not immersed in the medium at the margin. Radial growth-rate at 20°C was 0.25 mm/24hrs. Mycelium emerged rapidly from endophyte-infected plant tissue incubated on potato dextrose agar medium at 20°C. Aerial cottony mycelium was visible using a dissecting microscope after 3-5 days.

Conidiophores, arising solitarily on aerial hyphae of 5 day-old cultures, were hyaline, smooth, generally without a basal septum, 5-15yum long, 2-3 jum wide at the base and 1-1.5/im at the tip. Conidia were holoblastic, solitary, hyaline, reniform, 4-8 x 2-3/im.

Some growth of the colony occured in 10 ppm benomyl amended potato dextrose agar.

The strain produced moderate to strong antibiosis in vitro of the fungal colonies Colletotrichum graminicola and Drechslera erythrospila.

Lolitrem has not been detected in ryegrasses infected with this endophyte. % -10- ^ n Q Q • ~ KJ (J U o IJ The strain is kept in axenic culture and in infected seeds on deposit at DSIR Plant Protection Division, Palmerston North. A culture of the strain was deposited at the Australian Government Analytical Laboratories, Pymble, NSW, Australia on 23 March 1990 under deposit No. N90.010 895.

The following cultural characteristics serve to identify this strain from others of Acremonium lolii.

There is rapid emergence of mycelium from tissues of endophyte-infected plants incubated at 20°C on potato dextrose agar. The colonies were white, cottony and uniform with the margins not immersed in agar.

Production of abundant conidia on potato dextrose agar occurs after 3-5 days incubation at 20°C.

Conidia are formed on short conidiophores, generally 5-10 um in length.

Figure 1 is a DNA profile for the strain "Premier". DNA from Premier was amplified by the polymerase chain reaction using four different primers. In Figure 1: Lane 1 is a BRL 1 kb size marker ladder, Lane 2 is Primer 1, Lane 3 is Primer 2, Lane 4 is Primer 3, Lane 5 is Primer 4, and Lane 6 is Primer^Hind 111 cut DNA size markers.

A picture of the DNA profile obtained for the "Premier" strain of A. lolii using PCR based methods and 4 different primers is given in Figure 1. This DNA profile was prepared using a recently published method (Williams et al., Nucleic Acids Research 18: 6531-6535, 1990) to identify DNA polymorphisms, that makes use of arbitrarily chosen 10 mers as primers in the polymerase chain reaction. _u_ 238083 A set of distinct DNA bands are amplified from genomic DNA for each of the primers. 1 Primer 1 (RC05) sequence 5' AGGAGATACC 3' * a prominent band is observed at 1.1 kb 2 Primer 2 (RC08) sequence 5' GGATGTCGAA 3' * a prominent band is observed at 1.5 kb 3 Primer 3 (RC09) sequence 5' GATAACGCAC 3' * a prominent band is observed at 1.5 kb 4 Primer 4 (GT02) sequence 5' TGGTGGGTCC 3' * three prominent bands of 1.3, 0.9 and 0.7 kb are observed Using this set of 4 primers, the "Premier" strain was distinguished from 20 other fungal endophytes isolated from grass seed.

The methodology for the preceding test was as follows: 1 Total DNA was isolated from freeze dried mycelium of the "Premier" strain of A. lolii by the method of Yoder (Advances in Plant Pathology 6: 93-112, 1988). 2 5-10 ng of this DNA was amplified by the polmerase chain reaction using conditions described by Williams et al. 1990 (see above). 3 Amplified DNA was separated by electrophoresis on 1% agarose gels and the gels stained with ethidium bromide and photographed.

The cultivars listed in table 1 were infected with the endophyte strain, -12- 233033 "Premier". The peramine level and lolitrem level expressed as parts per million are set out in that table.

The levels of peramine (as peramine base) and lolitrem (as lolitrem B) are expressed as mg/kg (parts per million) of dry matter and are generally of total herbage of plants grown under controlled conditions of 20°C for seven weeks with a 12 hour photoperiod and mean photosynthetic photon flux density of 6.17 yumol/m^/s.

TABLE 1 1.01 Lolium perenne infected with "Premier" strain Cultivar Grasslands Ruanui Grasslands Nui Grasslands Pacific Peramine level 7.3 (5.3 - 10.7)* 10.8 (6.6 - 19.2)* 2.4 (0.8 - 5.1)* Lolitrem Level 0.1** not detected 0.1** 1.02 Lolium multiflorum Grasslands Moata 6.6 (5.0 - 9.9)* 0.2 ** 1.03 Lo 1 iumJvybridIum Grasslands Greenstone 7.2 (3.9 - 11.1) not detected Values of peramine vary from plant to plant of cultivars infected with endophyte. The single figure is an average value, the figures in parenthesis are the range of values.

Values of 0.1 ppm or 0.2 ppm lolitrem B indicate a possible maximum concentration but do not confirm the presence of any clearly defined lolitrem B.

Claims (20)

WHAT WE CLAIM IS: now m\ O ') ^ o •- '-•> 'j >-J o, 1 An endophyte of the species A. lolii in axenic culture, said endophyte being capable in combination with a herbage plant which has been inoculated with said endophyte of producing relatively high levels of peramine and relatively low levels of or no lolitrems. 2 An endophyte as claimed in claim 1 wherein s/id A. lolii species is the strain "Premier" (as herein described). 3 A herbage plant inoculated with an endophyte as^laimed in claim 1 or 2. 4 A combination according to claim 3 ^nerein saj/a herbage plant is of the genus Lolium. 5 A combination according to claj/ffc 4 where/n said endophyte is the strain "Premier" (as herein described). 6 A combination according t</any one /f claims 3 to 5 wherein said plant is a Lol ium of the species Lol iip^ercnne/[ ol ium niu I ti florum, Lol ium hybridum or Lolium temulentum. 7 A combination accg/ding to ^n'y one of claims 3 to 5 wherein said plant is of the genus Festuca. 8 A combination/accordinaAo claim 8 wherein said plant is a Festuca of the species Festuca ^rundinacea'^nd Festuca rubra var comutata. / 9 Seeds of/any one of the herbage plants defined in any one of claims 3 to / / 9 infected with any one of the endophytes in any one of claim 3 to 9. / 10 Plarj-ts grown from seeds as claimed in claim 9. 11 A/method of inoculating herbage plants with ari endophyte to produce a combination containing relatively high levels of peramine and relatively low levels of or/no lolitrems, the method comprising: / selecting a plant infected with an endophyte which produced a relatively high level of peramine and a relatively low level of lolitrems, extracting said endophyte from said plant, inoculating an endophyte-free plant with said endophyte, and selecting infected plants with high peramine and low lolitrem levels. % AS AMENDED WHAT WE CLAIM IS:

1 An endophyte of the species A loin m axenic culture, said endophyte being capable in combination with a herbage plant which has been inoculated with said endophyte of producing levels of peramine of 0.8ppm or higher and levels of lolitrems of 0.2ppm or lower, both based on dry plant material.

2 An endophyte as claimed in claim 1 wherein said A. loin species is the strain "Premier" (as herein described)

3 A herbage plant inoculated with an endophyte as claimed in claim 1 or 2.

4 A combination according to claim 3 wherein said herbage plant is of the genus Lolium

5 A combination according to claim 4 wherein said endophyte is the strain "Premier" (as herein described).

6 A combination according to any one of claims 3 to 5 wherein said plant is a Lolium of the species Lolium perenne. Lolium multiflorum, Lolium hvbndum or Lolium temulentum.

7 A combination according to any of claims 3 to 5 wherein said plant is of the genus Festuca.

8 A combination according to claim 7 wherein said plant is a Festuca of the species Festuca arundmacea and Festuca rubra var comutata.

9 Seeds of a herbage plant of the genus Lolium or Festuca. which includes, by controlled infection, an endophyte of claim 1 or claim 2.

10 Plants grown from seeds as claimed m claim 9.

11 A method of inoculating herbage plants with an endophyte to produce a combination containing levels of peramine of 0.8ppm or higher and levels of lolitrems of 0.2ppm or lower, both based on dry plant material, the method comprising: ^ce^of^Z m Ft n c ^ 13 NO !iprnp O '■> O f! (t o <-) U ^ -14-

12 A method according to claim 11 wherein said plant selecting steps each comprise analysing an endophyte infected plant for the presence of peramine and lolitrems and selecting those plants which have both peramine level of at least 2 mg/kg of dry plant matter and a lolitrem T^vel of no njore than 0.2 mg/kg of dry plant matter.

13 A method according to claim 11 or claiiji/12 whereiij/said endophyte extracting step comprises removing leaf sheath tissu§/from endophyte containing plants, sterliizing said tissue and growing endophyte mycelium therefrom in a suitable medium.

14 A method according to any orie of claimyll to 13 said endophyte / inoculating step comprises inserting endophyte mycelium from a said extracting / ^ step into a cut in the tissue/of an endophyte free seedling of a grass to be inoculated. / / / / / /

15 A method according^/to any one/of claims 11 to 14 including the additional steps of growing said inoculated .Herbage plant to produce seeds and harvesting / / the seeds so produced. / / /

16 An endophyte/substantially as herein described with reference to the / / examples and to figure 1. / / /

17 A herbaae plant inoculated with an endophyte substantially as herein / / described with reference to the examples. / /

18 Seeds of a herbage plant inoculated with an endophyte substantially as / / herein described with reference to the examples. / /

19 A method of inoculating herbage plants with an endophyte as claimed in / / any one of claims 11 to 14 substantially as herein described. / /'

20/ A herbage plant inoculated with cm endophyte produced by the method of any one^cvf claims 11 to 14 and 19. 21 A seed inoculated with an endophyte produced by a process according to claim is. tkt §tm M-tU K fjhr of h/ew zeafandt By / their authorised Agents AJ, PARK & SON. 7? selecting a plant infected with an endophyte which produced a level of peramine above 0 8ppm and a level of lolitrems below .02ppm, extracting said endophyte from said plant. inoculating an endophyte-free plant with said endophyte, and selecting infected plants with peramme levels above 0.8ppm and lolitrem levels below 0.2ppm. 12 A method according to claim 11 wherein said plant selecting steps each comprise analysing an endophyte infected plant for the presence of peramine and lolitrems and selecting those plants which have both a peramine level of at least 2 mg/kg of dry plant matter and a lolitrem level of no more than 0.2 mg/kg of dry plant matter. 13 A method according to claim 11 or claim 12 wherein said endophyte extracting step comprises removing leaf sheath tissue from endophyte containing plants, sterilizing said tissue and growing endophyte mycelium therefrom in a suitable medium. 14 A method according to any one of claims 11 to 13 said endophyte inoculating step comprises inserting endophyte mycelium from a said extracting step into a cut in the tissue of an endophyte free seedling of a grass to be inoculated 15 A method according to any one of claims 11 to 14 including the additional steps of growing said inoculated herbage plant to produce seeds and harvesting the seeds so produced. 16 An endophyte substantially as herein described with reference to the examples and to figure 1. 17 A herbage plant inoculated with an endophyte substantially as herein described with reference to the examples. 18 Seeds of a herbage plant inoculated with an endophyte substantially as herein described with reference to the examples. Property o?r;cs of KZ 15 i 14 AS AMENDED 19 A method of moculatmg herbage plants with an endophyte as claimed in any one ot claims 11 to 14 substantially as herein described 20 A herbage plant inoculated with an endophyte produced by the method of any one of claims 11 to 14 and 19. 21 A seed inoculated with an endophyte produced by a process according to claim 15 by their authorised agents .TAMKS & WF.T J.S per O.'fice of KZ 15 -Oi/uy. 3 Ar 5 (o Figure 1 fa ftjofltfy the Qupph /nttt By / their authorised Agents °£ff* A.J. PARK & SON. ^j7/V 'flMlA/hsh* JI