U.S. patent application number 12/318860 was filed with the patent office on 2009-07-16 for grass endophytes.
This patent application is currently assigned to Grasslanz Technology Limited. Invention is credited to Michael John Christensen, Herrick Sydney Easton, Lester Ronald Fletcher, Geoffrey Alexander Lane, Garrick Cecil Morland Latch, Alison Jean Popay, Brian Anthony Tapper.
Application Number | 20090181447 12/318860 |
Document ID | / |
Family ID | 32041072 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181447 |
Kind Code |
A1 |
Christensen; Michael John ;
et al. |
July 16, 2009 |
Grass endophytes
Abstract
The present invention relates to a combination of a Neotyphodium
species endophyte or endophyte culture in a symbiotic association
with a host grass which association does not cause symptoms of
toxicosis in animals through exclusion of ergovaline but, due to
the inclusion of alkaloids including agroclavine, setoclavine or
isosetoclavine, retains the ability to resist abiotic stresses and
protect the grass from pests.
Inventors: |
Christensen; Michael John;
(Palmerston North, NZ) ; Easton; Herrick Sydney;
(Palmerston North, NZ) ; Fletcher; Lester Ronald;
(Tai Tapu, NZ) ; Lane; Geoffrey Alexander;
(Palmerston North, NZ) ; Latch; Garrick Cecil
Morland; (Palmerston North, NZ) ; Popay; Alison
Jean; (Hamilton, NZ) ; Tapper; Brian Anthony;
(Palmerston North, NZ) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Grasslanz Technology
Limited
|
Family ID: |
32041072 |
Appl. No.: |
12/318860 |
Filed: |
January 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10529375 |
Oct 28, 2005 |
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PCT/NZ03/00219 |
Sep 26, 2003 |
|
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12318860 |
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Current U.S.
Class: |
435/254.1 ;
800/298 |
Current CPC
Class: |
C12N 1/14 20130101; A01N
63/30 20200101 |
Class at
Publication: |
435/254.1 ;
800/298 |
International
Class: |
C12N 1/14 20060101
C12N001/14; A01H 5/10 20060101 A01H005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 2002 |
NZ |
521653 |
Claims
1-29. (canceled)
30. A combination of an endophyte of the Neotyphodium coenophialum
species with a host grass, the endophyte selected from the group
consisting of: AR512; AR513; AR514; AR517; AR521; AR522; AR524;
AR525; AR535; AR539; and combinations thereof; AR512; AR513; AR514;
AR517; AR521; AR522; AR524; AR525; AR535, AR539 being cultures
deposited on 2 Oct. 2002 at the Australian Government Analytical
Laboratories (AGAL) with accession numbers: NM02/31935; NM02/31936;
NM02/31937; NM02/31938; NM02/31939; NM02/31940; NM02/31941;
NM02/31942; NM02/31943; NM02/31944, respectively; characterised in
that when the endophyte is combined with a host grass, the
endophyte and host grass do not produce alkaloid compounds
associated with toxicosis in animals; and further characterised in
that when the endophyte is combined with a host grass, the
endophyte and host grass retain sufficient levels of at least two
clavine alkaloids selected from the group consisting of:
agroclavine; setoclavine; isosetoclavine; and combinations
thereof.
31. A combination as claimed in claim 30 characterised in that the
endophyte and host grass combination is achieved by modification of
host grass infected with the endophyte by methods selected from the
group consisting of: breeding; crossing; hybridisation; genetic
modification; and combinations thereof.
32. A combination as claimed in claim 30 characterised in that the
host grass is selected from the group consisting of: tall fescue
grass cultivar; ryegrass cultivar; meadow fescue cultivar; and
combinations thereof.
33. A combination as claimed in claim 30 characterised in that the
host grass is a Pooideae grass.
34. A combination as claimed in claim 30 characterised in that the
endophyte and host grass combination produces isosetoclavine and
setoclavine at a rate of greater than 0.5 ppm each of dry matter in
the host grass plant crowns.
35. A combination as claimed in claim 30 characterised in that the
endophyte and host grass combination produces less than 0.2 ppm of
dry matter of ergovaline in whole herbage.
36. A combination as claimed in claim 30 characterised in that the
endophyte and host grass combination has at least one feature
selected from the group consisting of: enhancement of pest
protection; resistance to insects; pasture persistence; and a
combination thereof.
37. A combination as claimed in claim 36 characterised in that the
pest, to which increased resistance is conferred on the host grass,
is selected from the group consisting of: lesion nematode; root
aphid; corn flea beetle; and combinations thereof.
38. A combination as claimed in claim 30 characterised in that the
endophyte and host grass combination enhances grazing animal growth
and/or increases animal productivity relative to grass infected
with known endophytes capable of inducing fescue toxicosis.
39. A combination as claimed in claim 30 characterised in that the
endophyte produces sufficient levels of at least two clavine
alkaloids to protect the endophyte and the host grass from pests or
abiotic stresses or both.
40. A combination as claimed in claim 39, wherein the abiotic
stress is a water deficit.
41. A combination of an endophyte of the Neotyphodium coenophialum
species with a host grass, wherein the endophyte is selected from
the group consisting an endophyte culture deposited with the
Australian Government Analytical Laboratories under accession
number NM02/31935; NM02/31936; NM02/31937; NM02/31938; NM02/31939;
NM02/31940; NM02/31941; NM02/31942; NM02/31943; and NM02/31944.
42. Seed of a host grass characterised in that the seeds are from a
host grass infected with endophyte as claimed in claim 30.
Description
[0001] This application is a divisional of application Ser. No.
10/529,375, which is a National Stage of International Application
No. PCT/NZ2003/000219, filed Sep. 26, 2003, all of which is
incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates to fungal endophytes and combinations
of endophytes with grass plants. More particularly the invention
relates to endophytes which form combinations with tall fescue
(Festuca arundinacea) and some other related grasses. Even more
particularly the invention relates to combinations having reduced
toxicity to grazing livestock as compared to cultivars of
endophyte/tall fescue combinations in common use.
BACKGROUND ART
[0003] Fungal endophytes of the genus Neotyphodium (formerly
Acremonium) infect a number of temperate climate Pooideae grasses.
The Neotyphodium endophytes can produce alkaloids which are
considered to confer degrees of pest and possibly disease
protection upon the plants in which they naturally occur (Rowan and
Latch, 1994; Blank and Gwinn, 1992). The Neotyphodium endophytes
are vertically transmitted through the seed of the grasses and no
natural horizontal transmission has been established (Leuchtmann,
1997).
[0004] Many of the predominating natural endophyte infections of
improved grass cultivars used for pastoral agriculture production
also cause significant animal disorders, for example tall fescue
toxicoses (Stuedemann and Hoveland, 1988) and ryegrass staggers
(Fletcher et al., 1999). These may be complex toxic reactions by
animals to alkaloids produced under a range of plant growth
conditions. Significant economic loss within pastoral agriculture
systems can occur due to such animal toxicoses. On the other hand
presence of at least some endophytes may be essential for the
competitive persistence of the chosen grass in a pasture (Elberson
and West, 1996, Fletcher and Easton, 2000).
[0005] Grass lines can be artificially infected with selected
endophytes. Axenic cultures of endophytes can be used to infect
grass seedlings, grown initially under sterile conditions (Latch
and Christensen, 1985), which are then selected for desirable
qualities, and multiplied for commercial use. Three significant
examples of this technology have been developed by the Grasslands
division of AgResearch Ltd: GREENSTONE.TM. tetraploid hybrid
ryegrass with ENDOSAFE.TM. endophyte (Tapper and Latch, 1999, NZ
Patent 233083); various perennial and hybrid ryegrasses with AR1
endophyte (Fletcher and Easton, 2000); and tall fescue cultivars
with MaxQ.TM. endophyte (Bouton, 2000; Bouton et al., 2002, U.S.
Pat. No. 6,111,170).
Fescue Toxicosis
[0006] Fescue toxicosis has been associated with the natural
infection of tall fescue by common strains of Neotyphodium
coenophialum. These strains typically produce the ergopeptine
alkaloid, ergovaline, which is of a class of ergopeptines known to
be toxic to mammals. Ergovaline is considered to be the primary
cause of fescue toxicity. Other compounds, notably other ergoline
and ergolene compounds, for example lysergic acid, possibly add to
the syndrome (Oliver, 1997; Gadberry et al., 1997; Hill et al.,
2001).
[0007] The ergovaline levels tend to be higher in leaf sheath and
heads of tall fescue than in leaf blade and undergo seasonal
variation (Rottinghaus et al., 1991). There is very little
ergovaline in roots. Typically a concentration of ergovaline in
herbage or herbage products such as hay, straw, seed or silage of
greater than an average of 0.4 ppm of dry matter has been
considered a risk of causing fescue toxicosis (Tor-Agbidye et al.,
2001) especially when combined with climatic conditions
exacerbating fescue toxicosis symptoms.
Other Compounds Recognised as Plant Defence Mechanisms--Peramine,
Lolines
[0008] Peramine is produced in endophyte-infected grass (Rowan et
al., 1986) and probably mobilised within the plant. It is a potent
feeding deterrent for a range of insects, e.g. Argentine stem
weevil (Listronotus bonariensis), (Rowan and Latch 1994) and a
significant factor for protecting endophyte-infected grasses from
insect pest predation.
[0009] Lolines (N-formylloline, N-acetylloline, and
N-acetylnorioline and other closely related compounds) are produced
by some Neotyphodium endophytes including N. coenophialum typical
of tall fescue. These compounds in appropriate endophyte-infected
grasses have properties of deterring or resisting a number of
insects, notably sucking insects, for example, Rhopalosiphum padi
(Seigel et al., 1990).
Protective Effects in Tall Fescue Pastures--Persistence Under
Stress Conditions
[0010] Endophyte infection has been associated with enhanced
persistence of tall fescue plants under water deficit or drought
conditions. Whether this effect is due to better resistance of
biotic stress factors expressed in water deficit situations;
general better health of endophyte-infected tall fescue plants
(particularly of root systems); or due to specific differential
physiological responses of the endophyte-infected plants to water
stress; is not clearly evident. However, the overall effect is
enhanced resistance to water deficit.
Tremorgens
[0011] Some Neotyphodium endophytes, notably those of evolutionary
derivation from strains of Epichloe festucae such as N. lolii,
produce potent tremorgens which are toxic to grazing animals. To
ensure such tremorgens are not produced by an endophyte
artificially introduced into forage tall fescue or ryegrass, the
presence of the known potent tremorgens typical of endophytes, that
is the lolitrems, is tested for. Also tremorgenic activity in
grazing test animals is looked for.
[0012] It is an object of the present invention to provide an
endophyte which can produce ergovaline and some ergoline and
ergolene compounds at the base of the tall fescue leaf sheaths and
in the crown of the plant but only in a manner such that the usual
concentration in herbage as generally consumed by grazing animals
in common farming practice is less than a practical threshold
toxicity level.
[0013] For the purposes of this specification "crown" is defined as
that area of a grass plant which is generally less than 2 cm above
soil level and excludes the roots of the grass, but includes the
base of tillers and lateral meristem growing points for new
vegetative tillers.
[0014] It is a further object of the invention to provide an
endophyte which can produce lolines in amounts which are considered
to be partially or substantially effective in deterring some insect
pests from feeding on plants.
[0015] It is a further object of the invention to provide an
endophyte which does not produce detectable levels of lolitrems and
are not observably tremorgenic.
[0016] It is a still further object of the present invention to
address the foregoing problems or at least to provide the public
with a useful choice.
[0017] All references, including any patents or patent applications
cited in this specification are hereby incorporated by reference.
No admission is made that any reference constitutes prior art. The
discussion of the references states what their authors assert, and
the applicants reserve the right to challenge the accuracy and
pertinency of the cited documents. It will be clearly understood
that, although a number of prior art publications are referred to
herein, this reference does not constitute an admission that any of
these documents form part of the common general knowledge in the
art, in New Zealand or in any other country.
[0018] It is acknowledged that the term `comprise` may, under
varying jurisdictions, be attributed with either an exclusive or an
inclusive meaning. For the purpose of this specification, and
unless otherwise noted, the term `comprise` shall have an inclusive
meaning--i.e. that it will be taken to mean an inclusion of not
only the listed components it directly references, but also other
non-specified components or elements. This rationale will also be
used when the term `comprised` or `comprising` is used in relation
to one or more steps in a method or process.
[0019] Further aspects and advantages of the present invention will
become apparent from the ensuing description which is given by way
of example only.
DISCLOSURE OF INVENTION
[0020] According to one aspect of the present invention there is
provided an endophyte of Neotyphodium coenophialum species,
selected from the group consisting of: AR512; AR513; AR514; AR517;
AR521; AR522; AR524; AR525; AR535; AR539; and combinations thereof;
AR512; AR513; AR514; AR517; AR521; AR522; AR524; AR525; AR535,
AR539 being cultures deposited on 2 Oct. 2002 at the Australian
Government Analytical Laboratories (AGAL) with accession numbers:
NM02/31935; NM02/31936; NM02/31937; NM02/31938; NM02/31939;
NM02/31940; NM02/31941; NM02/31942; NM02/31943; NM02/31944;
[0021] characterised in that, in combination with a host grass,
said endophyte does not cause symptoms of toxicosis in animals;
[0022] and further characterised in that the endophyte retains
sufficient levels of at least two alkaloids selected from the group
consisting of: agroclavine; setoclavine; isosetoclavine; and
combinations thereof, that protect the host grass from pests or
abiotic stresses or both;
[0023] and further characterised in that the host grass is
artificially inoculated with the endophyte.
[0024] According to a further aspect of the present invention there
is provided an endophyte culture of Neotyphodium coenophialum
species, selected from the group consisting of: AR512; AR513;
AR514; AR517; AR521; AR522; AR524; AR525; AR535; AR539; and
combinations thereof; AR512; AR513; AR514; AR517; AR521; AR522;
AR524; AR525; AR535, AR539 being cultures deposited on 2 Oct. 2002
at the Australian Government Analytical Laboratories (AGAL) with
accession numbers: NM02/31935; NM02/31936; NM02/31937; NM02/31938;
NM02/31939; NM02/31940; NM02/31941; NM02/31942; NM02/31943;
NM02/31944;
[0025] characterised in that, in combination with a host grass,
said endophyte culture does not cause symptoms of toxicosis in
animals;
[0026] and further characterised in that the endophyte culture
retains sufficient levels of at least two alkaloids selected from
the group consisting of: agroclavine; setoclavine; isosetoclavine;
and combinations thereof, that protect the host grass from pests or
abiotic stresses or both;
[0027] and further characterised in that the host grass is
artificially inoculated with the endophyte culture.
[0028] Preferably, the toxicosis which is avoided is fescue
toxicosis. Most preferably the toxicosis is caused by an ergovaline
toxin.
[0029] Preferably, the level of ergovaline in the present invention
is less than 0.4 ppm in dry matter in herbage consumed by grazing
animals. More preferably, the level of ergovaline is less than 0.4
ppm in dry matter in herbage, other than the crown of the host
grass plant, consumed by grazing animals.
[0030] Preferably, the abiotic stress is a water deficit.
[0031] Preferably, the endophyte culture, if used, is an axenic
culture.
[0032] Preferably, the endophyte or endophyte culture produces less
than 0.2 ppm ergovaline in dry matter of whole herbage when
infected into host grass.
[0033] According to a further aspect of the present invention there
is provided a combination of the endophyte as described above, and
a host grass.
[0034] According to another aspect of the present invention there
is provided a combination of the endophyte culture as described
above, and a host grass.
[0035] Preferably, the combination, substantially as described
above, is achieved by modification of host grass infected with the
endophyte or endophyte culture by methods selected from the group
consisting of: breeding; crossing; hybridisation; genetic
modification; and combinations thereof.
[0036] Preferably, the host grass used in the combination described
above is selected from the group consisting of: tall fescue grass
cultivar, ryegrass cultivar, meadow fescue cultivar, and
combinations thereof.
[0037] According to a further aspect of the present invention the
host grass is a Pooideae grass.
[0038] According to a further aspect of the present invention there
is provided a combination of endophyte or endophyte culture, as
described above, and a host grass wherein the combination produces
isosetoclavine and setoclavine at a rate of greater than 0.5 ppm
each of dry matter in the host grass plant crowns. Preferably also,
the combination produces less than 0.2 ppm of dry matter of
ergovaline in whole herbage.
[0039] According to a further aspect of the present invention there
is provided a combination of endophyte as described above and a
host grass, wherein the combination has features selected from the
group consisting of: enhancement of pest protection, resistance to
insects, pasture persistence, and combinations thereof.
[0040] According to a further aspect of the present invention there
is provided a combination as described above and a host grass,
wherein the combination has the features of enhancement of grazing
animal growth and increased animal productivity relative to grass
infected with known endophytes capable of inducing fescue
toxicosis.
[0041] According to a further aspect of the present invention there
is provided a combination as described above and a host grass
wherein the pest to which increased resistance is conferred on the
host grass is selected from the group consisting of: lesion
nematode, root aphid, corn flea beetle, and combinations
thereof.
[0042] According to a further aspect of the present invention there
is provided seeds of a host grass infected with the endophyte
substantially as described above.
[0043] According to yet a further aspect of the present invention
there is provided seeds of a host grass infected with endophyte
culture as described above.
[0044] The invention is the combination of examples of a class of
Neotyphodium coenophialum endophyte and improved grass cultivars by
artificial inoculation to produce host grasses which do not cause
symptoms of toxicosis by way of the ergovaline toxin, but which
retain sufficient levels of other alkaloids (for example:
agroclavine, setoclavine and/or isosetoclavine) to individually or
in combination continue to protect the host grass from pests or
abiotic stresses (such as water deficit) or both.
[0045] The invention has been achieved by understanding the biology
of endophytes of temperate climate grasses, isolating selected
endophytes of interest, inoculating the endophytes into
surface-sterilised seedlings of grasses, exemplified by improved
tall fescue or perennial ryegrass cultivar lines, re-evaluating
alkaloid production, multiplying seed, evaluating for agronomic
factors, testing for animal production, evaluating for any evidence
of animal disorders such as fescue toxicosis, staggers,
hyperthermia, or prolactin hormone depression and testing for
invertebrate pest protection.
[0046] The invention consists of the foregoing and also envisages
constructions of which the following are examples.
BEST MODES FOR CARRYING OUT THE INVENTION
Culture Conditions and Description
[0047] All endophytes of this invention are strains from
collections of seed of tall fescue originally sourced from the
Claviplus class. Seed from various tall fescue collections were
examined for the presence of endophyte by seed squash technique. A
selection of plants for each seed sample, where an endophyte was
shown to be present, were grown for a few weeks in glasshouse
conditions and re-tested for endophyte presence in their leaf
sheaths.
[0048] The endophytes from plants with chemotypes of interest were
isolated and grown in culture according to the method of Latch and
Christensen (1985). The endophytes of this invention are held in a
culture collection or in cloned plants at the Grasslands site of
AgResearch Ltd in Palmerston North, New Zealand. The cultures are
also deposited at the Australian Government Analytical Laboratories
in Sydney, Australia.
[0049] The endophyte cultures were deposited with the Australian
Government Analytical Laboratories (AGAL), New South Wales Regional
Laboratory, 1, Suakin Street Pymble NSW 2073, Australia, on Oct. 2,
2002, under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure. They were assigned the following accession
numbers: NM02/31935 (AR512); NM02/31936U (AR513); NM02/31937
(AR514); NM02/31938 (AR517); NM02/31939 (AR521); NM02/31940
(AR522); NM02/31941 (AR524); NM02/31942 (AR525); NM02/31943
(AR535); NM02/31944 (AR539). All strains of endophyte of this
invention are accommodated within a single sub-grouping of the
species Neotyphodium coenophialum. The isolates, when grown on
potato dextrose agar at 22.degree. C., are slow-growing (radial
growth approximately 0.1-0.3 mm per day) with colonies typically
white and cottony. Conidia have been observed at variable rates of
production near the margin of colonies.
Inoculations
[0050] Axenic cultures of endophytes AR512, AR513, AR514, AR517,
AR521, AR522, AR524, AR525, AR535, and AR539 as examples of this
invention, were successfully inoculated (Latch and Christensen,
1985) into seedlings grown from surfaced sterilised seed of the
tall fescue cultivar Kentucky 31. Endophytes AR514, AR524, and
AR525 were also infected into other cultivars such as, for example
Grasslands Flecha, Jesup Improved, Georgia 5 and various
experimental lines, generally with a satisfactory success rate well
in excess of 5% of attempts. No complete failures to infect tall
fescue were observed with the endophytes of this invention.
[0051] A typical meadow fescue cultivar (Ensign) was successfully
inoculated with endophyte AR512. Similarly a typical perennial
ryegrass test line (GA66) was successfully inoculated with
endophytes AR514 and AR525 for further examination with the
chemotype characteristics of the combinations similar as for when
infecting tall fescue, but generally with lower levels of alkaloid
accumulation, and with rates of infection of the order of 1% to 5%
of attempts.
[0052] Seed has been successfully produced from infected plants
containing endophytes of this invention under routine seed
production conditions with relatively high and useful rates of
endophyte infection.
Chemotype Identification
[0053] Basal parts of endophyte-infected tillers were freeze dried,
sometimes milled, and extracted and analysed qualitatively for the
presence of ergovaline by high performance liquid chromatography
(HPLC) as set out below. Those indicating ergovaline less than
approximately 0.2 ppm of dry matter were further analysed for the
production of peramine at rates greater than about 2 ppm of dry
matter. The endophytes from such selections were isolated,
classified by culture attributes, and generally re-inoculated into
seedlings of endophyte-free tall fescue, cultivar Kentucky 31, as a
typical improved pasture host for comparative purposes. Samples
from such plants at various stages of growth were analysed in more
detail for alkaloid production, including for the production of
tremorgenic lolitrems typical of N. lolii infection and lolines
typical of N. coenophialum.
[0054] The expression of alkaloid production of endophyte-infected
tall fescue, using endophytes selected from tall fescue sources,
was observed to fall broadly into three groups. The most numerous
group produce both ergovaline and peramine at levels often well in
excess of 1 ppm in the basal tiller material. Such endophytes are
likely to be associated with fescue toxicosis of grazing animals if
present at high rates of infection in forage.
[0055] A second chemotype group produced peramine and lolines, but
no detectable trace of ergovaline. Endophytes typical of this group
have been developed for commercial application (Bouton, 2000;
Bouton et al., 2002, U.S. Pat. No. 6,111,170).
[0056] A third group, of this invention, which have low levels and
a characteristic distribution within plants of ergovaline, are
discussed below. They are further characterised by the presence of
peramine in herbage generally well in excess of 1 ppm, and the
presence of lolines in herbage in amounts within ranges typical of
N. coenophialum infection. In the course of chromatographic
analysis for ergovaline the presence of other compounds with UV and
fluorescence spectral properties typical of ergolene derivatives
(i.e. fluorescent "ergot alkaloids") were observed, notably in the
basal portions of tillers, crowns, and seed of tall fescue plants
infected with this group of endophytes. These compounds are also
discussed below.
[0057] Lolines (N-formylloline, N-acetylloline, and
N-acetylnorioline, in order of usual observed abundance) were
detected by capillary gas chromatography in extracts of tall fescue
and meadow fescue plants infected with the endophytes of this
invention in amounts more or less comparable to that observed in
comparable tall fescue plants infected with common N. coenophialum.
The methods used were minor modifications of the method of Yates et
al., (1990).
[0058] Lolitrems were not detected by chromatographic analysis in
any tall fescue infected with endophytes of this invention. The
possible effects of lolitrems were not further directly considered,
other than by observation of lambs grazing pastures containing
endophyte AR514 for signs of tremors.
Ergot Alkaloid Levels and Identification of New Alkaloids
[0059] Ergovaline concentration was measured by HPLC with
fluorescence detection. Typically, a sample of approximately 50 mg
of milled (1 mm mesh), freeze-dried endophyte-infected tissue from
the lower 3 to 5 cm of tillers from mature vegetative plants (basal
tiller material predominantly consisting of leaf sheaths) was
extracted with 1 ml of a mixture of equal parts of propan-2-ol and
water containing also 1% lactic acid. The extraction continued for
one hour at ambient temperature with gentle mixing. An internal
standard of added ergotamine tartrate (c. 1 .mu.g per sample) was
used for quantitative comparisons.
[0060] Following brief centrifugation, a sample of the clarified
extract solution was taken for HPLC using a reverse phase column
(typically Prodigy 150.times.4.6 mm, 5 .mu.m silica ODS (3),
Phenomenex, Calif., USA); with elution at 1 ml per minute and a
solvent gradient of acetonitrile and 100 mM aqueous ammonium
acetate by volume starting at 27.5% acetonitrile and progressing in
linear stages to 35% at 20 min, 50% at 35 min, 60% at 40 min and
75% at 50 min.
[0061] Naturally fluorescent ergolene compounds including
ergovaline (and its isomer ergovalinine), the internal standard
ergotamine (and its isomer ergotaminine partially formed during
extraction) and the compounds observed in the earlier section of
the chromatograms were detected by using UV excitation at 310 nm
and emission at 410 nm. Ergovaline, its isomer ergovalinine, added
ergotamine internal standard and its isomer ergotaminine elute at
approximately 22, 36, 30 and 42 minutes respectively. The minimum
detection level for routine analysis of ergovaline (combined
amounts with isomer ergovalinine) is approximately 0.05 ppm of dry
matter.
[0062] During the course of examining examples of tall fescue
infected with endophytes of this invention for ergovaline, other
ergolene derivatives were observed to be present in a pattern not
previous recognised. Compounds eluting at approximately 6.5 minutes
(compound A) and 8.0 minutes (compound B) are characteristically
both present in endophyte-infected basal tiller and crown material
of this invention. These compounds have been identified as
isosetoclavine and setoclavine, respectively, as follows.
[0063] Fractions enriched in compounds A and B were prepared from a
methanol--1% aqueous acetic acid (4:1) extract of bulked freeze
dried and milled lower sheath of tillers containing endophytes
AR514 and AR524 (pre-extracted with hexane). The extract was
fractionated by sequential reverse-phase flash chromatography on
octadecyl-functionalised silica gel (Aldrich) with acidic
(methanol--1% aqueous acetic acid) and neutral (methanol--water)
step gradients, and normal-phase flash chromatography on silica gel
(silica gel 60, 40-63.mu., Merck) with an ethyl acetate-methanol
step gradient.
[0064] The characteristic fluorescence and the UV absorption
spectra and electrospray ionisation (ESI) mass spectral data for
compounds A and B obtained by HPLC analysis of enriched fractions
using variations of HPLC separation conditions and detectors
(Shimadzu LC-MS instrument QP-5050 with SPD-10AVP UV diode array
and RF-10 A fluorescence spectral detectors), together with
comparative data with standards establish these compounds are
setoclavines (isosetoclavine and setoclavine respectively). Both
compounds show strong fluorescence peaks in the HPLC
(.lamda..sub.Ex 310 nm .lamda..sub.Em 410 nm). The UV spectra are
characterised by maximum absorbances at 307 nm and 312 nm
respectively and for both compounds the positive ion ESI mass
spectrum shows a base peak at m/z=237 (MH.sup.+-H.sub.2O) and an
MH.sup.+ ion at m/z=255. Compound B co-eluted with a sample of
reference setoclavine provided by Dr Miroslav Flieger, Institute of
Microbiology Academy of Sciences of the Czech Republic. Compounds A
and B were identical by HPLC, and spectral properties to
isosetoclavine (compound A) and setoclavine (compound B) obtained
by chemical oxidation of agroclavine by a standard procedure. The
presence of isosetoclavine and setoclavine has not been previously
reported in endophyte infected grasses although they have been
reported as oxidation products of agroclavine in other plant
systems (review by Kren, 1999).
[0065] The same extracts were also shown to contain agroclavine by
electrospray LC-MS, with an ESI mass spectrum ion attributed to
MH.sup.+ at m/z=239 eluting at the same time and with essentially
the same UV spectrum as authentic agroclavine.
[0066] Table 1 summarises alkaloid analysis results of specified
plant parts of tall fescue infected with the endophytes and usually
grown under temperate summer season conditions, generally in
comparative test cultivar Kentucky 31. For consideration of ergot
alkaloid production in the crown and basal tiller, comparison is
also made to tall fescue infected with endophytes AR501 and AR542
which consistently do not appear to produce any ergovaline or
setoclavines.
TABLE-US-00001 TABLE 1 Examples and typical ranges or scores of
alkaloids observed in specified plant parts Sample (cultivar &
Ergovaline Compounds endophyte) Part of plant (ppm DM) A&B
Jesup EI Whole herbage 0.4-1.2 ND Jesup EI Sheath 2.5 Manawatu RS
EI Sheath 7.1-15.7 Kentucky 31 EI Whole Herbage 1.8-3.0 ND Kentucky
31 EI Leaf blade 0.3-1.8 ND Kentucky 31 EI Sheath 2.9-16.2 Kentucky
31 AR501 Crown <0.1.sup.ND * Kentucky 31 AR514 Whole herbage
<0.1 * Kentucky 31 AR514 Crown 0.1-0.6 ** Kentucky 31 AR514
Immature heads <0.1-0.2 * Kentucky 31 AR522 Stem & sheath
0.1-0.2 * Kentucky 31 AR522 Crown 3.4-6.1 *** Kentucky 31 AR524
Crown 0.3-0.6 ** Kentucky 31 AR524 Immature heads <0.1 *
Kentucky 31 AR525 Whole herbage <0.1 ND Kentucky 31 AR525 Crown
0.7-1.0 *** Kentucky 31 AR525 Immature heads <0.1 * Kentucky 31
AR525 Stem, sheath & heads <0.1 * Kentucky 31 AR535 Crown
0.4-0.7 *** Kentucky 31 AR535 Stem, sheath & heads <0.1 **
Kentucky 31 AR542 Crown <0.1 ND Kentucky 31 AR542 Sheath <0.1
ND Compound A = isosetoclavine Compound B = setoclavine ND = not
detected, detection limit of 0.1 ppm DM for ergovaline * = possible
trace or low level ** and *** = score of relative abundance
observed EI = infected with common toxic or wild type endophyte
Genotype Characterisation of Endophyte
[0067] All endophytes discussed above are characterised by DNA
"fingerprinting" (selected polymorphic microsatellite loci and/or
Arbitrary Fragment Length Polymorphisms (AFLP) technique) as
belonging to a sub-group of Neotyphodium coenophialum.
[0068] Samples of about 50 mg fresh or 15 mg dry basal tiller were
used for the extraction of DNA using FastDNA kit for plants (Bio
101, Vista, Calif.) by procedures recommended with the kit.
Alternatively genomic DNA was extracted from cultured endophyte
(Moon et al., 1999). Microsatellite PCR amplification was performed
using primer pairs labelled with fluorescent dyes, B10.1
(5'-TET)/B10.2 and B11.1 (5'-HEX)/B11.4, as described by Moon et
al., (1999). The apparent size of microsatellite PCR fluorescent
labelled products was measured relatively to within an estimated
0.3 nucleotide units by capillary electrophoresis using an ABI 3100
Genetic Analyzer with POP6 polymer chemistry in 50 cm capillary
arrays and GeneScan-400HD standards (Applied Biosystems Inc.,
Foster City, Calif.). The apparent sizes of PCR products by this
technique (adjusted by subtracting a unit where an adenine
nucleotide appears to have been terminally added) are set out in
Table 2.
[0069] Table 2 shows that the endophytes of this invention can be
distinguished from other groups of Neotyphodium endophytes by the
number of alleles observed and the apparent sizes of such alleles.
Thus all strains of this invention share a B11 allele of size c.
128 base pairs and a second B11 allele within the size range c. 192
to 200 bp. They also share with other N. coenophialum strains up to
three B10 alleles within the range c.154 to 185 bp.
[0070] The presence of three alleles for the endophytes of this
invention for the B10 locus is consistent with evidence for N.
coenophialum as a hybrid endophyte derived from three different
Epichloe source species (Tsai et al., 1994).
TABLE-US-00002 TABLE 2 Apparent size of B10 and B11 microsatellite
PCR products B10 locus B11 locus No of Allele No of Allele Source
material alleles sizes (bp) alleles sizes (bp) N. coenophialum,
wild type, 3 160.4, 169.6, 184.2 2 147.9, 192.2 Australian C1, coB
isozymes N. coenophialum, wild type, 2 160.4, 169.4 2 147.9, 192.2
NZ Tindall's, coB isozymes N. coenophialum, wild type, 3 160.3,
169.3, 184.2 2 147.8, 192.0 NZ RS2 & NZ RS6 N. coenophialum,
AR542 2 160.5, 169.6 2 180.4, 192.2 Neotyphodium sp., FaTG-3 2
169.5, 178.7 1 127.9 strain AR501 Neotyphodium sp., FaTG-3 2 169.6,
178.7 1 127.8 strain AR506 AR539 3 154.6, 172.5, 178.3 2 127.9,
192.2 AR513 3 157.7, 160.5, 178.4 2 128.0, 192.2 AR525 3 157.7,
160.4, 178.3 2 128.0, 192.2 AR517 3 163.4, 172.5, 178.2 2 128.0,
192.1 AR521 3 163.3, 172.5, 178.2 2 127.9, 192.1 AR512 3 172.6,
178.5, 181.5 2 128.0, 192.2 AR514 3 157.8, 160.6, 178.4 2 128.0,
196.2 AR522 3 157.7, 160.5, 178.3 2 128.0, 200.1 AR524 3 157.7,
160.5, 178.3 2 127.9, 200.2 AR535 3 157.7, 160.5, 178.3 2 128.0,
200.1
[0071] Analysis by AFLP (Griffiths et al., 1999) also confirmed
that endophyte examples AR514, AR525 and AR535 of this invention
are from a sub-group which can be distinguished from other N.
coenophialum endophytes outside this sub-group by one or more
polymorphic differences, but not many differences, from within more
than 200 AFLP bands observed to be polymorphic for the genera
Neotyphodium and Epichloe.
Safe Grazing with Endophyte in Tall Fescue Cultivar Kentucky 31
[0072] Pastures of tall fescue infected with examples of the
endophytes of this invention do not induce typical fescue toxicosis
in grazing animals. Table 3 shows growth rate of lambs in a trial
conducted at Eatonton, Ga., USA for two seasons (21 Apr.-30 Jun.
1997, 2 Apr.-2 Jul. 1998). The growth of lambs on a pasture of
Kentucky 31 tall fescue infected with endophyte strain AR514 was
essentially the same growth as on equivalent endophyte-free pasture
and significantly better than growth on naturally
endophyte-infected pasture using the comparable Jesup cultivar. The
wild type endophyte infection significantly reduced live weight
gain (P<0.05) and increased mean body (rectal) temperature.
[0073] Gross depression of prolactin in blood is another symptom of
fescue toxicosis. Endophyte strain AR514 did not cause a depression
of prolactin whereas with the wild type endophyte prolactin was
grossly reduced.
[0074] Overall, the performance of lambs grazing on AR514 pasture
was similar to that on the endophyte-free pasture. No tremors or
"ryegrass staggers" symptoms were observed.
TABLE-US-00003 TABLE 3 Performance of lambs grazing endophyte
infected and endophyte free pasture Endophyte treatment AR514 Year
(in Kentucky 31) Jessup EF* Jessup EI* Live weight gain 1997 103
a** 102 a 67 b (g/hd/d) 1998 93 a 102 a 57 b Body temperature 1997
39.8 a 39.8 a 40.1 b (.degree. C.) Blood prolactin 1997 414 a 400 a
Not detect- able (b) (ng/ml) 1998 550 a 150 a <0.5 b *EF =
endophyte free; EI = infected with common toxic or wild type
endophyte. **Treatments with no letter in common are significantly
different (P < 0.05).
Endophytes and Resistance to Lesion Nematodes
[0075] In some environments, notably sandy soils with relatively
warm and humid climates, nematode may cause significant damage to
tall fescue root systems thus affecting the persistence of the
grass in the pasture. A greenhouse trial with three plants and nine
replicates per treatment has demonstrated that endophyte infection
may confer resistance to lesion nematodes, Pratylenchus spp. In a
greenhouse experiment nematode reproduction was investigated with
two tall fescue cultivars infected with various endophytes or
endophyte free.
[0076] Table 4 indicates that an endophyte of this invention,
AR514, confers partial resistance to lesion nematodes to a greater
degree than endophyte-free plants (EF) or two endophytes lacking
production of ergovaline (AR542 & AR584) although not to the
same degree as the common endophyte (EI) of the cultivars.
TABLE-US-00004 TABLE 4 Endophyte effect on lesion nematode numbers
Fescue Endophyte cultivar EF* AR542 AR584 AR514 EI* GA 5 146 149
101 -- 19 Jesup 147 88 120 69 30 Mean 146 a** 118 ab 111 ab 69 b 24
c Relative to 100 a 81 ab 76 ab 47 b 16 c EF = 100 *EF = endophyte
free; EI = infected with common toxic or wild type endophyte.
**Treatments with no letter in common are significantly different
(P < 0.05).
Endophytes Confer Resistance to Root Aphid
[0077] Endophyte infection is known to affect infestation of
grasses by aphids. Table 5 compares treatments of fourteen plants
each of tall fescue cultivar Kentucky 31 for mean numbers of root
aphids wherein AR514 infection is shown to confer considerable
protection in comparison with another endophyte-infected set of
plants or endophyte free plants.
TABLE-US-00005 TABLE 5 Log number of root aphid per 10 ml
sub-sample in Kentucky 31 tall fescue No. root No. root Endophyte
treatment aphid aphid/gm root Endophyte Free 4.043 a* 2.055 a AR542
1.710 b 0.473 b LAR514 0.765 c 0.095 c *Treatments with no letter
in common are significantly different (P < 0.01)
Endophytes Confer Deterrence to Corn Flea Beetle
[0078] Tall fescue Kentucky 31 leaves infected with examples of the
endophytes of this invention (E+) and also leaves infected with
wild type toxic endophyte were compared with endophyte free (EF)
material using the corn flea beetle Chaetocnema pulicaria in a
feeding preference test experiment. Endophytes AR512, AR513, AR514,
AR524 and AR525 all conferred resistance or feeding deterrence
similar to that of leaves infected with the wild type toxic
endophyte. The mean of feeding scores for the examples of this
invention were E+=0.4 as against EF=2.8 (P<0.001) on a scale of
0 to 3 (where 0 is no feeding and 3 is extensive feeding). Score
counts of feeding scars (E+=2.2, EF=27.8, mean of the total number
of scars across 3 transects per leaf blade) and of faecal pellets
(E+=9.2, EF=75.8, mean number of faecal pellets on each blade) were
also highly significantly different with those for E+closely
comparable to those of toxic wild type endophyte of Kentucky
31.
[0079] A similar single preference test with endophyte AR512
infected into meadow fescue cultivar Ensign demonstrated an even
more extreme preference effect with feeding scores of E+=0.0,
EF=3.0, feeding scars score counts E+=0, EF=33, and faecal pellets
E+=3, EF=50.
[0080] Aspects of the present invention have been described by way
of example only and it should be appreciated that modifications and
additions may be made thereto without departing from the scope
thereof.
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