U.S. patent application number 13/557623 was filed with the patent office on 2014-01-30 for grass endophyte.
This patent application is currently assigned to Cropmark Seeds Ltd. The applicant listed for this patent is Nicholas Evan Cameron. Invention is credited to Nicholas Evan Cameron.
Application Number | 20140030288 13/557623 |
Document ID | / |
Family ID | 49995113 |
Filed Date | 2014-01-30 |
United States Patent
Application |
20140030288 |
Kind Code |
A1 |
Cameron; Nicholas Evan |
January 30, 2014 |
GRASS ENDOPHYTE
Abstract
A method of protecting a host grass from stress, such as caused
by insect pests, by artificially inoculating the host grass with an
endophyte-containing composition is disclosed. The endophyte
produces loline at a level sufficient to confer protection to the
endophyte-infected host grass and does not produce detectable
levels of alkaloids having toxicity to ruminant animals such as
sheep, cattle, goats, or deer. In vitro cultures of the endophyte
Neotyphodium uncinatum, var. U2 are disclosed as well as infected
plants and seeds. The infected plants and seeds produce 5.0-20,000
.mu.g lolines per gram dry weight of grass and do not produce
detectable levels of alkaloids having toxicity to ruminant animals
such as ergovaline, peramine, lolitrem B and epoxy-Janthitrems.
Inventors: |
Cameron; Nicholas Evan;
(Darfield, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cameron; Nicholas Evan |
Darfield |
|
NZ |
|
|
Assignee: |
Cropmark Seeds Ltd
Christchurch
NZ
|
Family ID: |
49995113 |
Appl. No.: |
13/557623 |
Filed: |
July 25, 2012 |
Current U.S.
Class: |
424/195.15 ;
435/254.1; 800/297; 800/320 |
Current CPC
Class: |
A01N 63/30 20200101;
A01N 63/30 20200101; A01N 65/00 20130101; C12N 1/00 20130101; A01H
15/00 20130101; A01N 63/30 20200101; A01N 65/44 20130101; A01N
65/44 20130101 |
Class at
Publication: |
424/195.15 ;
435/254.1; 800/320; 800/297 |
International
Class: |
A01N 65/00 20090101
A01N065/00; A01P 7/04 20060101 A01P007/04; A01H 15/00 20060101
A01H015/00; A01H 5/00 20060101 A01H005/00; C12N 1/14 20060101
C12N001/14; A01H 5/10 20060101 A01H005/10 |
Claims
1. An in vitro culture comprising Neotyphodium uncinatum, var.
U2.
2. A seed from the family Poaceae obtained from a plant that has
been artificially infected with Neotyphodium uncinatum, var.
U2.
3. The seed of claim 2, wherein the seed is from a plant selected
from the group consisting of Lolium perenne, Lolium hybridum,
Lolium multiflorum, Festuca arundinacea, Festuca pratensis, and
Festulolium hybrids.
4. An isolated endophyte producing 5.0-20,000 .mu.g lolines from
infected grass/g dry plant material and not producing detectable
levels of at least one alkaloid having toxicity to ruminant
animals.
5. The endophyte of claim 4, wherein the alkaloid is selected from
the group consisting of ergovaline, peramine, lolitrem B and
epoxy-Janthitrems.
6. The endophyte of claim 4, wherein the ruminant animals are
sheep, cattle, goats, or deer.
7. The endophyte of claim 4, which is Neotyphodium uncinatum, var.
U2.
8. A plant from the family Poaceae artificially infected with
Neotyphodium Uncinatum, var. U2.
9. The plant of claim 8 wherein the plant is selected from the
group consisting of Lolium perenne, Lolium hybridum, Lolium
multiflorum, Festuca arundinacea, Festuca pratensis, and
Festulolium hybrids.
10. A method of protecting a host grass from stress, comprising
artificially inoculating the host grass with an
endophyte-containing composition to produce an endophyte-infected
host grass, wherein the endophyte produces lolines at a level
sufficient to confer protection to the endophyte-infected host
grass and does not produce detectable levels of alkaloids having
toxicity to ruminant animals.
11. The method of claim 10, wherein the stress is caused by an
insect.
12. The method of claim 11, wherein the insect is selected from the
group consisting of Argentine stem weevil (Listronotus
bonariensis), New Zealand Grass Grub (Costelytra zealandica), Black
beetle (Heteronychus arator), Porina (Wiseana sp.) and Red Headed
Pasture cockchafer (Adoryphorus couloni).
13. The method of claim 10, wherein the endophyte-containing
composition comprises Neotyphodium uncinatum, var. U2.
14. The method of claim 10, wherein the endophyte produces
5.0-20,000 .mu.g/g (wt lolines/wt dry grass).
15. The method of claim 10, wherein the grass is from the family
Poaceae.
16. The method of claim 15, wherein the grass is selected from the
group consisting of the genus Festuca.
17. The method of claim 15, wherein the grass is selected from the
group consisting of Lolium perenne, Lolium hybridum, Lolium
multiflorum, Festuca arundinacea, Festuca pratensis, and
Festulolium hybrids.
18. The method of claim 10, wherein levels of ergot alkaloids in
the endophyte-infected host grass are less than detectable levels
of 0.2 .mu.g/g, (wt. alkaloid/dry wt. grass).
19. The method of claim 10, wherein levels of pyrrolopyrazine
alkaloids in the endophyte-infected host grass are less than 0.1
.mu.g/g, (wt. alkaloid/dry wt. grass).
20. The method of claim 10, wherein ergovaline, peramine, lolitrem
B and epoxy-Janthitrems are not detectable in the
endophyte-infected host grass.
21. The method of claim 10, wherein the ruminant animals are sheep,
cattle, goats, or deer.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to fungal endophytes and combinations
of endophytes with grass plants.
[0003] 2. Description of the Related Art
[0004] Endophyte-infected grasses are relatively pest-resistant and
drought-tolerant, making them ideal candidates for low maintenance
situations such as turf purposes and erosion control. The endophyte
does not affect either the growth or the appearance of the grass.
Endophytes are transmitted only through seed and their presence
requires a laboratory evaluation to confirm Endophyte-infected
grasses generally have low palatability to livestock and may lead
to a variety of problems when used as livestock feed such as
lameness, low energy, poor reproductive capacity both in conception
and bringing the fetus to term, and low milk production. These
problems are due to the presence of a range of alkaloids produced
by the endophyte which are also responsible for the pest- and
drought-resistance.
[0005] There is a need for an endophyte-infected grass plant that
has low or non-detectable levels of certain alkaloids that may have
deleterious effects on forage animals while still producing
sufficient quantities of those alkaloids which provide a
competitive advantage to the plant host. There is a need for
endophyte-infected grasses that can be used as forage.
SUMMARY OF THE INVENTION
[0006] Embodiments of the invention are directed to an in vitro
culture comprising Neotyphodium uncinatum, var. U2 and seeds from
the family Poaceae that have been artificially infected with
Neotyphodium uncinatum, var. U2. Preferably, the seed is from a
plant such as Lolium perenne, Lolium hybridum, Lolium multiflorum,
Festuca arundinacea, Festuca pratensis, and Festulolium
hybrids.
[0007] Embodiments of the invention are directed to isolated
endophytes producing 5.0-20,000 .mu.g lolines from infected grass/g
dry plant material and not producing detectable levels of at least
one alkaloid having toxicity to ruminant animals. In preferred
embodiments, the alkaloid is ergovaline, peramine, lolitrem B or
epoxy-Janthitrems. In preferred embodiments, the ruminant animals
are sheep, cattle, goats, or deer. Preferably, the endophyte is
Neotyphodium uncinatum, var. U2.
[0008] Embodiments of the invention are directed to plants of the
family Poaceae artificially infected with Neotyphodium uncinatum,
var. U2. In preferred embodiments, the plant is Lolium perenne,
Lolium hybridum, Lolium multiflorum, Festuca arundinacea, Festuca
pratensis, or Festulolium hybrids.
[0009] Embodiments of the invention are directed to methods of
protecting a host grass from stress by artificially inoculating the
host grass with an endophyte-containing composition to produce an
endophyte-infected host grass. Preferably, the endophyte produces
one or more lolines at a level sufficient to confer protection to
the endophyte-infected host grass and does not produce detectable
levels of one or more alkaloids having toxicity to ruminant
animals. Preferably, the ruminant animals are sheep, cattle, goats,
or deer.
[0010] In some embodiments, the stress is caused by an insect.
Preferably, the insect is Argentine stem weevil (Listronotus
bonariensis), New Zealand Grass Grub (Costelytra zealandica), Black
beetle (Heteronychus arator), Porina (Wiseana sp.) or Red Headed
Pasture cockchafer (Adoryphorus couloni).
[0011] In preferred embodiments of the method for protecting a host
grass, the host grass is artificially inoculated with an
endophyte-containing composition which includes Neotyphodium
uncinatum, var. U2. Preferably, the endophyte produces 5.0-20,000
.mu.g lolines (wt lolines/wt dry grass). Preferably, the grass is
from the family Poaceae. More preferably, the grass is of the genus
Festuca. In a most preferred embodiment, the grass is Lolium
perenne, Lolium hybridum, Lolium multiflorum, Festuca arundinacea,
Festuca pratensis, or Festulolium hybrids.
[0012] In preferred embodiments, levels of ergot alkaloids in the
endophyte-infected host grass are less than detectable levels of
0.2 .mu.g/g, (wt. alkaloid/dry wt. grass).
[0013] In preferred embodiments, levels of pyrrolopyrazine
alkaloids in the endophyte-infected host grass are less than 0.1
.mu.g/g, (wt. alkaloid/dry wt. grass).
[0014] In preferred embodiments, ergovaline, peramine, lolitrem B
and epoxy-Janthitrems are not detectable in the endophyte-infected
host grass.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a comparison of Neotyphodium uncinatum, var. U2
with AR542 (N. coenophialum) and AR1 (N. lolii) grown on agar.
[0016] FIG. 2 shows a comparison between endophyte colonies of
Neotyphodium uncinatum, var. U2 and variety UNC1.
DETAILED DESCRIPTION
[0017] Embodiments of the invention relate to Neotyphodium
uncinatum, var. U2, common name, Fungal Endophyte, Meadow Fescue.
The endophyte was identified in line FP102 of Festuca pratensis
after screening many Festuca lines. The FP102 line originally came
from Norway in 1999. The endophyte was isolated in New Zealand. The
native host for this endophyte is the plant and seed of Festuca
pratensis. As with all endophytes, Neotyphodium uncinatum, var. U2
is naturally reproduced by vertical transmission, growing into the
seed of the host. The ability to sexually reproduce (sporulate) or
transmit horizontally in nature has been lost.
[0018] In some embodiments an isolated endophyte Neotyphodium
uncinatum, var. U2, is provided. The isolated endophyte may be in
an in vitro culture. Plants such as grass can be infected with the
isolated endophyte. Thus, in some embodiments grass artificially
infected with Neotyphodium uncinatum, var. U2 is provided. In some
embodiments, seed artificially infected with Neotyphodium
uncinatum, var. U2 or obtained from grass artificially infected
with Neotyphodium uncinatum, var. U2 is provided. When a grass
plant is artificially inoculated with Neotyphodium uncinatum, var.
U2, the infected plant produces loline alkaloids which provide a
competitive advantage to the infected plant, but the infected grass
plant does not produce levels of alkaloids that are deleterious to
forage animals.
[0019] Infection of the grass with Neotyphodium uncinatum, var. U2
confers one or more advantages to the grass relative to uninfected
control plants. These advantages include protection from pests
relative to uninfected control plants, including but not limited to
insects and nematodes and/or resistance to stress such as spatial
competition from other plant species, water or nutrient deficiency,
stress due to heat or cold and the like. In some embodiments,
advantages include greater vigour such as more roots, higher total
biomass, more tillers and/or higher seed production relative to
uninfected control plants. In some embodiments, the advantage
includes protection from disease, relative to uninfected control
plants.
[0020] Embodiments of the invention are directed to methods of
producing grass plants artificially infected with Neotyphodium
uncinatum, var. U2 which have some competitive advantage relative
to uninfected plants, but are not toxic to ruminants.
[0021] In some embodiments, the competitive advantage includes
protecting the plant from stress. The stress may be biotic or
abiotic. The stress may be biotic and caused by an animal pest,
such as an insect or nematode. In some embodiments, stress may be
abiotic such as water or nutrient deficiency, temperature stress,
or spatial competition from other species. The stress may be a
combination of biotic and abiotic stress.
[0022] In some embodiments, the competitive advantage may include
improved vigor compared to plants which are not infected with
Neotyphodium uncinatum, var. U2. Grass plants infected with
Neotyphodium uncinatum, var. U2 according to embodiments of the
invention may have more root growth, more tillers, more total
biomass and/or more seed production than the corresponding grass
plants which are not infected with Neotyphodium uncinatum, var.
U2.
[0023] In some embodiments, the competitive advantage may include
disease resistance.
[0024] Neotyphodium uncinatum, var. U2 may be propagated by in
vitro propagation including but not limited to inoculation of
seedlings. Neotyphodium uncinatum, var. U2 may be maintained in
seed and/or in vitro culture (isolated from the plant host on
agar). In some embodiments, Neotyphodium uncinatum, var. U2 may be
reproduced by vegetative propagation, paralleling the life cycle of
the host plant. Neotyphodium uncinatum, var. U2, although native to
F. pratensis, can be successfully inserted artificially into other
grass species including but not limited to Lolium perenne, Lolium
hybridum, Lolium multiflorum, Festuca arundinacea, Festuca
pratensis, and Festulolium hybrids by known methods. In a preferred
embodiment, seedling inoculation is performed using endophyte grown
rapidly over 10 to 14 days, for example in a liquid potato dextrose
broth which is then filtered to remove excess broth. Inoculation
may be performed by inserting this prepared endophyte into a slit
incision in the meristem area of a seedling plant grown for 7 to 10
days from endophyte free seed under zero light and 15-25.degree.
C., preferably about 20.degree. C. conditions. Slit incision is
carried out using a syringe needle, such as a 30 gauge syringe
needle. After the endophyte is inoculated into the slit incision,
the plants are placed back in dark conditions for a further 7 to 10
days before being adapted to low light conditions for 3-4 days and
eventually planted into a fine potting mix. Plants are checked for
endophyte presence when they reach the 3-5 tiller stage and checked
for the presence of the correct endophyte strain using DNA
markers.
[0025] When the host is infected with Neotyphodium uncinatum, var.
U2, the production of loline metabolites (N-formyl loline, N-acetyl
loline, N-acetyl norloline, N-methyl loline, for example) by the
endophyte act as a feeding deterrent to many pasture pests.
However, lolines are not known to be toxic to grazing animals such
as sheep, cattle, goats, and deer. In some embodiments, one or more
of the recited loline metabolites are at elevated levels relative
to control plants that are not infected with var. U2.
[0026] Grasses infected with Neotyphodium uncinatum, var. U2 show
resistance to insect pests including but not limited to Argentine
stem weevil (Listronotus bonariensis), New Zealand Grass Grub
(Costelytra zealandica), Black beetle (Heteronychus arator), Porina
(Wiseana sp.) and Red Headed Pasture cockchafer (Adoryphorus
coulonii).
[0027] Grasses infected with Neotyphodium uncinatum, var. U2 have
reduced root consumption by root feeding larvae of grass grub,
black beetle and cockchafer, less leaf shredding by Argentine stem
weevil, and less consumption by porina larvae and adult black
beetle and as a consequence, greater vigour and production. Grasses
infected with Neotyphodium uncinatum, var. U2 show no visibly
detectable difference with uninfected grasses of the same
species.
[0028] As mentioned above, grasses infected with Neotyphodium
uncinatum, var. U2 produce lolines which are alkaloids which have
been shown to be either toxic or a feeding deterrent to a range of
pest insects including those listed above. Loline alkaloids are a
group of compounds with distinct chemical and biological features.
Chemically, lolines are characterized by a saturated pyrrolizidine
ring, a primary amine at the C1 carbon and an internal ether bridge
joining distant ring members C2 and C7. This internal ether bridge
is uncommon among organic compounds and is a characteristic feature
of lolines. Different substituents at the C1 carbon provide
different bioactivity against insects. Such substituents include
methyl, formyl and acetyl groups.
[0029] The biological activity attributed to lolines includes
resistance to herbivores, drought resistance and spatial
competition. In preferred embodiments of the invention, lolines are
present at concentrations sufficient to confer protection to the
host grass against at least one insect pest. For example total
loline concentrations of greater than 400 .mu.g/g deter New Zealand
grass grub from feeding and concentrations greater than 1000
.mu.g/g reduce shredding by Argentine stem weevil. In preferred
embodiments, the pest is selected from insect species from the
orders of Homoptera, Hemiptera, Coleoptera, Hymenoptera,
Lepidoptera, and Blattaria. More preferably, the insect pest is
selected from Argentine stem weevil (Listronotus bonariensis), New
Zealand Grass Grub (Costelytra zealandica), Black beetle
(Heteronychus arator), Porina (Wiseana sp.) and Red Headed
cockchafer (Adoryphorus couloni). The characteristics of the
resistance may include killing of insects feeding on the
endophyte-infected grass, avoidance of the endophyte-infected grass
by insect pests, effects on fecundity of insect pests such as
reduced or no production of offspring or impaired insect
development. In preferred embodiments, insect pests are repelled
and as a consequence, development is arrested due to malnutrition.
In some embodiments, pests or their larvae, particularly Porina
larvae, are killed by exposure to lolines.
[0030] In preferred embodiments, the level of total lolines
produced by the endophyte is 1.0 .mu.g/g to 20,000 .mu.g/g, (wt.
loline/dry wt. grass), more preferably at levels in excess of 400
.mu.g/g, yet more preferably at levels in excess of 1,000 .mu.g/g,
yet more preferably at levels in excess of 5,000 .mu.g/g in above
ground plant parts. In a preferred embodiment, total loline
measurement is performed using gas chromatography (GC-FID). The
following protocol is exemplary. Freeze dried finely ground plant
material (about 0.25 g) is added to 5 ml of solvent (95:5
dichloromethane:ethanol) in an 8 ml glass vial. This solvent
contains phenylmorpholine (6 mg/100 ml) as an internal standard.
Then 250 .mu.l of saturated sodium bicarbonate solution (2 g/10 ml)
is added to the vial. The vials are shaken on an orbital shaker for
1 hour. The vial is left to settle and the supernatant filtered
using a plugged Pasteur pipette into a clean vial. 1 ml of this
extract is then transferred to a GC vial for analysis.
[0031] In a preferred embodiment, samples are analyzed using a gas
chromatograph (GC) (Shimadzu 2010) equipped with a flame ionization
detector (FID). The column is a ZB 624 of 30 m, 0.25 mm I.D. and a
film of 1.40 .mu.m. The film is 6% cyanopropylphenyl and 94%
dimethylpolysiloxane. The oven is set at an initial temperature of
90.degree. C., held for 1 min, then increased (ramped) to
260.degree. C. at 30.degree. C/min, and held for 10 min. Injector
temperature is 250.degree. C. Gas flow is a constant flow of 1.2
ml/min. The retention times of N-Methyl Loline, N-Formyl Loline,
N-Acetyl Loline, and N-Acetyl Nor Loline are determined as 8.0,
13.2, 13.7, and 12.2 minutes respectively. Total loline
concentration is the sum of the four loline compounds assessed
above. The limit of detection of loline alkaloids is considered to
be 5 .mu.g/g. Other known protocols for measuring levels of loline
alkaloids could also be used.
[0032] In preferred embodiments, the grass infected with
Neotyphodium uncinatum, var. U2 does not produce detectable levels
of ergot alkaloids, such as ergovaline. As ergot alkaloids are more
closely associated with toxicity to ruminants, in preferred
embodiments, grasses infected with Neotyphodium uncinatum, var. U2
are not toxic to ruminants. For example, in preferred embodiments,
total levels of ergot alkaloids in the endophyte-infected host
grass are less than 5 .mu.g/g, more preferably less than 1 .mu.g/g
and yet more preferably below detectable levels of 0.2 .mu.g/g,
(wt. alkaloid/dry wt. grass).
[0033] In preferred embodiments, the grass infected with
Neotyphodium uncinatum, var. U2 does not produce detectable levels
of senecio-type alkaloids. Such alkaloids are associated with liver
damage. Accordingly, grasses infected with Neotyphodium uncinatum,
var. U2 do not produce detectable liver damage in ruminants feeding
upon the grasses. Limit of detection of loline alkaloids are
considered to be 5 .mu.g/g.
[0034] In preferred embodiments, total levels of pyrrolopyrazine
alkaloids, such as peramine, in the endophyte-infected host grass
are less than 2.0 .mu.g/g, more preferably less than 1.0 .mu.g/g
yet more preferably, less than 0.1 .mu.g/g, (wt. alkaloid/dry wt.
grass). 0.1 .mu.g/g (wt alkaloid/dry wt. grass) are at the
detection limit. Levels below 0.1 .mu.g/g are considered
non-detectable.
[0035] Other alkaloids produced by endophytes include lolitrem B
and epoxy-janthitrems. In preferred embodiments, host grasses
infected with Neotyphodium uncinatum, var. U2 contain low or
non-detectable levels of these alkaloids. "Non-detectable" with
respect to these other alkaloids means that these alkaloids are not
detectable using the current protocols used to measure them.
[0036] Ruminant livestock that can be fed grasses infected with
Neotyphodium uncinatum, var. U2 include but are not limited to
sheep, cattle, goats, and deer. These ruminant animals can feed on
grasses infected with Neotyphodium uncinatum, var. U2 in either a
commercial setting such as a farm or ranch or in the wild. Such
commercial settings include raising livestock for meat, dairy
products, and textile production such as wool, mohair and leather
and include systems where livestock are enclosed in pastures or
barns or not enclosed or allowed to range freely but with some
limited enclosure.
[0037] Neotyphodium uncinatum, var. U2 is stable, uniform and
clearly distinguishable from any other variety of endophyte. This
variety was isolated in New Zealand from line FP102 of Festuca
pratensis from Norway. A PVR has been granted in New Zealand as
FEN009.
[0038] Grasses infected with Neotyphodium uncinatum, var. U2 are
useful in a variety of settings including but not limited to turf,
forage, as ornamentals and for erosion control. Such grasses
include species of the family Poaceae, preferably of the genera
Festuca and Lolium, more preferably of the species Lolium perenne,
Lolium hybridum, Lolium multiflorum, Festuca arundinacea, Festuca
pratensis, and Festulolium hybrids.
[0039] In some embodiments, a commercial variety of grass is
infected with Neotyphodium uncinatum, var. U2 using the technique
described above using meristem slit inoculation or by recurrent
pair-crossing using the meadow fescue host and continuous
top-crossing over repeated cycles to the desired parent. This
infected grass is used to provide forage for ruminant animals or
for turf or usage as a break crop to remove certain insect or
nematode pests. The grass shows resistance to a range of insect and
nematode pests and is hardier than the same commercial grass not
infected with Neotyphodium uncinatum, var. U2. Ruminant animals
feeding on the Neotyphodium uncinatum, var. U2-infected grass were
similar to animals fed on non-infected grass of the same commercial
variety. This can be measured in various ways known to those in the
art including weight gain of the animal and animal milk production.
In preferred embodiments, ruminant animals feeding on the
Neotyphodium uncinatum, var. U2-infected grass were similar in
weight gain to animals fed on non-infected grass of the same
commercial variety.
EXAMPLE 1
Isolation of Neotyphodium uncinatum, var. U2 and Comparison with
Similar Strains
[0040] Neotyphodium uncinatum, var. U2 was isolated from line FP102
of Festuca pratensis (Meadow fescue). Line FP102 originally came
from Norway in 1999. Characteristics for Neotyphodium uncinatum,
var. U2 were determined and compared with other varieties. For
these tests, the endophytic fungus isolated was cultured on 1.5%
(w/v) potato dextrose agar at 20.degree. C. in the dark
(Christensen M J, Latch G C M, Tapper S A 1991: Variation within
isolates of Acremonium endophytes from perennial ryegrasses. Mycol.
Res. 95: 918-923). Length of cultivation was generally standardized
at 4 weeks, but may be varied according to the isolate. Five plates
of each strain were grown.
1) Colony: rate of growth
[0041] Colony radial diameter was measured (two diameters, at right
angles, per plate) after 1 week's growth and again after 2, 3 and 4
weeks growth. Radial growth rate per day was calculated and rated
"very slow", "slow", "medium", "rapid" or "very rapid".
2) Colony: sporulation
[0042] Colonies were examined under a stereo/dissecting microscope
after 4 weeks. Confirmation of sporulation was through preparing
slides (mounting in lactophenol, plus or minus cotton blue stain)
and examining under compound microscope at approximately 400.times.
magnification or 1000.times. (as appropriate). Sporulation was
rated as "absent" or "present".
3) Colony: sectoring
[0043] Each of the 5 duplicate plates from 1) above were examined
after 4 weeks of growth and sectoring noted as "absent" or
"present".
4) Colony: colour (upper surface)
[0044] Each of the 5 duplicate plates from 1) above were examined
after 4 weeks of growth and color noted as "white" or "brown".
[0045] 5) Colony: shape
[0046] Each of the 5 duplicate plates from 1) above was examined
after 4 weeks of growth and the colony shape determined and noted
as "flat", "raised", "brainlike", "smooth", "convolute" or
"domed".
6) Colony: texture
[0047] Each of the 5 duplicate plates from 1) above was examined
after 4 weeks of growth and texture noted as "waxy" or "dry".
7) Colony: immersion of margin in agar
[0048] Each of the 5 duplicate plates from 1) above was examined
after 4 weeks of growth and colony margin noted as "immersed" or
"superficial".
8) Colony: resistance to benomyl on growth
[0049] Plates of PDA containing different concentrations of the
fungicide benomyl were prepared as outlined by Christensen et al.
(1991). Benlate
(Methyl[1-[(butylamino)carbonyl]-1H-benzimidazol-2-yl]carbamate)
(50% WP) was added to PDA prior to sterilization. Concentrations of
benomyl were adjusted to 1, 5, 10, 50 and 100 ppm. Five duplicate
plates were inoculated with a colony plug. The concentration at
which growth does not occur indicative of resistance was recorded
after 4 weeks as "absent or very weak", "weak", "medium", "strong"
or "very strong".
9) Aerial mycelium: density
[0050] Each of the 5 duplicate plates from 1) above was examined
after 4 weeks of growth and amount of aerial mycelium noted as
"very sparse", "sparse", "medium", "dense" or "very dense".
10) Aerial mycelium: type
[0051] Each of the 5 duplicate plates from 1) above was examined
after 4 weeks of growth and the aerial mycelium determined as
"felted", "cottony" or "tufted erect".
11) Conidia: length
[0052] 25 conidia (from at least 2 duplicate plates) were measured
using a 40x microscope objective and a ColorView imaging analysis
system. The spore range, mean, and a descriptive term (very short,
short, medium, long or very long) was given. ("medium" is 5-7
.mu.m.)
12) Conidia: width
[0053] 25 conidia (from at least 2 duplicate plates) were measured
using a 40.times. microscope objective and a ColorView imaging
analysis system. The spore range, mean, and a descriptive term
(very narrow, narrow, medium, broad or very broad) was given.
("medium" is 2-4 .mu.m.)
TABLE-US-00001 TABLE 1 Characteristics of Neotyphodium uncinatum,
var. U2 Characteristics State of Expression Colony: shape
convoluted Colony: growth rate medium (0.15 mm-0.3 mm per day)
Colony: sectoring absent Colony: color (upper surface) white
Colony: texture dry Colony: immersion of margin in agar Superficial
(sits on top) Colony: resistance to benomyl on growth strong Aerial
mycelium: density medium Aerial mycelium: type felted Sporulation:
in culture absent Metabolite: loline present Metabolite: ergovaline
absent Metabolite: peramine absent Metabolite: lolitrem B absent
Metabolite: epoxy-Janthitrem absent
TABLE-US-00002 TABLE 2 Comparison of Neotyphodium uncinatum, var.
U2 to similar varieties State of State of Characteristic in
expression expression of Denomination of which the similar of
similar Neotyphodium similar variety variety is different variety
uncinatum, var. U2 AR1 (N. lolii) Colony: shape brain like
convoluted Metabolite: peramine present absent Metabolite: loline
absent present AR542 Sporulation: in culture present absent (N.
coenophialum) Colony: shape raised convoluted Metabolite: peramine
present absent NEA2 Metabolite: ergovaline present absent (N.
lolii) Metabolite: peramine present absent
[0054] A comparison of Neotyphodium uncinatum, var. U2 with AR542
(N. coenophialum) and AR1 (N. lolii) grown on agar is shown in FIG.
1.
EXAMPLE 2
Comparison of Neotyphodium uncinatum, var. U2 with Variety
Neotyphodium Uncinatum, var. UNC1
[0055] The two varieties were similar in tested characteristics of
colony sporulation, colony sectoring, colony color (upper surface),
colony shape, colony immersion or margin in agar, colony texture,
aerial mycelium density and type, and effect of benomyl on growth.
The two varieties differed in colony shape and rate of growth.
Neotyphodium uncinatum, var. UNC1 showed a strong growth rate while
Neotyphodium uncinatum, var. U2 showed a medium growth rate. Colony
shape differed as shown in FIG. 2.
[0056] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present invention. Therefore, it should be
clearly understood that the forms of the present invention are
illustrative only and are not intended to limit the scope of the
present invention.
* * * * *