AU639084B2 - Improvements in/or relating to fungi - Google Patents

Description

'i i i 639084 S F Ref: 158991 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Address for Service: Her Majesty The Queen in the Right of New Zealand DSIR Chemistry Gracefield Road Lower Hutt NEW ZEALAND Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Improvements in/or Relating to Fungi The following statement is a full description best method of performing it known to me/us of this invention, including the 5845/5

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-14- 12 A method according to claim 11 wherein said plant selecting steps each n na nlpn+ fnpr hP nrp-pnrp nf npraminp and I I- -411 II -1

ABSTRACT

The invention described relates to endophytic fungi which live within grasses as fungal mycelium. Some endophyte/grass combinations repel Argentine stem weevil, while others cause ryegrass staggers in livestock. The former is due to the production of an alkaloid called peramine, while the latter is due to the production of toxins called lolitrems by the endophyte/grass combinations. The invention is novel endophyte, Premier, which in combination with certain pasture grasses, produces high levels of peramine and low levels or no lolitrems. Methods of infecting grasses with the endophyte are also described.

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s -2- 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 S 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 withendophyte 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 lolii. 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 1 point of view of animal health.

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-3- 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 S 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, in 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 in an artifici that its toxic In additi affects the ry ones, especial differs betwee genotype of pl infected plant It is knc 10 naturally and S fungus. It al plants infectE peramine and 1 reliably predi produced when be advantageot produce high It is an desideratum oi i 21 Accordinc of the specie: combination w of producing no lolitrem.

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In anoth herbage plant relatively hi 30 Preferab 0* .Is6

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-4in 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 S 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. lolii 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 L relatively high level of peramine and a relatively low level of or no lolitrem.

Preferably said he'rbage plant is of the genus Lolium.

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 mal' 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 S 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 20 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- 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 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 seconds before placing it in a 10% solution of sodium hypochlorite available chlorine) and shaking for 5 minutes. The tissue was then rinsed in sterile water and cut into 2-3 mm pieces.

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 (100pg/ml streptomycin 100/,g/ml penicillin) in Petri dishes and incubated at 20 0 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 I. The seeds were then placed on 4% water agar in Petri dishes, generally 5 seeds per dish, and incubated in darkness at 200C. 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

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-7tissue at the junction of the mesocotyl 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 0 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 S' 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 few seconds over a flame to accelerate staining.

Acremonium lolii appeared as septate, sparsely branched intercellular hyphae running longitudinally in the sheath.

S- 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 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.

-8- 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% aqueous formc acid in 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 i C (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 HPLC using a Brownlee 100x4 silica column and an aqueous buffer containing v/v acetonitrile and 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 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 v/v) into a 2 ml volumetric flask and 50 ul 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 v/v) at 0.7 ml/minute as solvent. Lolitrem concentrations were calculated by comparison of peak Sheights with those of known standards.

L -1 I -9- 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 0 C will maintain endophyte viability for 24 months. A seed moisture of 8% or a temperature of S 5 0 C will maintain endophyte viability for 36 months. The combination of 8% moisture and 5 0 C will keep the endophyte viable for at least 5 years. To retain viable endophyte over many years storage temperatures of -15 to OOC and low seed moisture are required.

EXAMPLE 6: Characteristics of Acremonium lolii Endophyte Strain 'Premier' 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 20 0 C was 0.25 mm/24hrs. Mycelium emerged rapidly from endophyte-infected plant tissue incubated on potato dextrose agar medium at 20 0 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, 5-15,um long, 2-3/um wide at the base and 1-1.5,um at the tip. Conidia were holoblastic, solitary, hyaline, reniform, 4-8 x 2-3um.

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.

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 0 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 days incubation at 20 0

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, SLane 4 is Primer 3, Lane 5 is Primer 4, and Lane 6 is PrimerELHind 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.

4 I I_ 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 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. lolli 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, John David O'Connor IRN: 158991 [Thul 0034.DOC:JOC INSTR CODE: 59770 -12- "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 200C for seven weeks with a 12 hour photoperiod and mean photosynthetic photon flux density of 6.17 jimol/m 2 /s.

I TABLE 1 1.01 Lolium perenne infected with "Premier" strain Cultivar Peramine level Grasslands Ruanui 7.3 (5.3 10.7)* Grasslands Nui 10.8 (6.6 19.2)* Grasslands Pacific 2.4 (0.8 5.1)* Lolitrem Level 0.1** not detected 0.1** 1.02 Lolium multiflorum Grasslands Moata 1.03 Lolium hybridum Grasslands Greenstone 6.6 (5.0 9.9)* 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.

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