Note: Descriptions are shown in the official language in which they were submitted.
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Title: ENDOPHYTE ENHANCED SEEDLINGS WITH INCREASED PEST
TOLERANCE AND METHODS
Field of the invention
[0001] The invention relates to ecologically sensitive approaches to
pest management. It provides a method for producing endophyte-enhanced
conifer seedlings with increased tolerance to herbivorous insects by
inoculating greenhouse-produced seedlings with toxigenic endophyte fungi.
The invention also provides a conifer seedling or adult conifer infected with
a
toxigenic endophyte fungus using a method of the invention.
Background of the invention
[0002] The leaves of various plants including macroalgae, grasses, and
sedges are known to have symptomless infections. The fungi involved are
commonly referred to as endophytes. (Carroll, 1988; Clay, 1988). An
endophyte is "an organism inhabiting plant organs that at some time in its
life,
can colonize internal plant tissue without causing apparent harm to the host"
(Petrini 1991). Fungal endophytes are believed to be host specific such that
they infect one or a small subset of plant species.
[0003] It is well understood that toxic metabolites produced by grass
endophytes greatly reduce populations of herbivorous insects attacking the
plant. This has a large affect on plant fitness (Clay, 1988; Clay & Holah,
1999). Grass seed of cultivars that contain fungal leaf endophytes has, for 10
years, been the dominant technology used for lawns and golf courses in parts
of the US and Canada. These fungi produce very potent toxins inside the
grass leaves that kill insects. This vastly reduces the amount of hard
chemical
pesticide used on the resulting lawns. Such lawns have increased drought
tolerance and have increased tolerance to fungal diseases.
[0004] Conifer needles are also infected by systemic fungal endophytes
that may fulfill several ecological roles (Carroll 1988; Ganley et al. 2004).
[0005] Carroll & Carroll (1978) first proposed that fungal endophytes
recovered from coniferous needles might be mutualistic symbionts. They
suggested decreased palatability for grazing insects and antagonism towards
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needle pathogens as possible benefits for the host trees. In subsequent work,
this group studied the association between Douglas-fir (Pseudotsuga
menziesii) and the needle endophyte Rhabdocline parkeri (Sherwood-Pike et
al., 1986; Todd, 1988). An extract of R. parkeri was cytotoxic to HeLa cells
and resulted in reduced growth rates and mortalities when incorporated into
synthetic diets of Choristoneura fumiferana (spruce budworm) at 10 pg g-1
(Miller, 1986).
[0006] Conifers, like other plant species, are vulnerable to pest
damage. For example, the eastern spruce budworm is an economically-
damaging insect pest. The last time there was an epidemic in Eastern
Canada, large scale spraying of a hard chemical pesticide was undertaken.
Where this was not done, the forests were devastated. For the year 1977
alone, the cost of the spraying program was approximately $47 million in
constant dollars. Over the intervening two decades, during a low period of the
budworm cycle, the hard chemical pesticides used in the 1970's were de-
registered in favour of more expensive and less effective biopesticides.
Regardless, it is less likely now that the social consensus would exist for
the
widespread use of chemical insecticides when the spruce budworm
population returns to epidemic proportions. New methods of controlling spruce
budworm and other insect pests are needed.
[0007] Royama (1984) published a comprehensive analysis of the
population dynamics of the spruce budworm focusing on the period 1945 to
1983 in New Brunswick (NB), Canada. One feature of this analysis is that he
proposed a "fifth agent" referring to an unknown factor that was required to
build models that best fit observed population changes. The central
characteristic of this fifth agent was that it in some way changed the
response
of the insect populations to known factors such as weather, predation and
disease.
[0008] From 1984-1994 isolations were made of endophytes present in
needles in various species of conifers across NB. As found by workers
worldwide, the needles of all mature conifers examined were colonized by
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several species of endophytes (Johnson & Whitney, 1989; Wilson, 1994).
From collections from across NB comprising 3500 strains, a low percentage
from Abies balsamea (balsam fir), Picea rubens (red spruce), Picea glauca
(white spruce) and Picea mariana (black spruce) were found to produce anti-
insectan toxins (Calhoun et al., /992; Clark etal., 1989 Findlay, 1996;
Findlay
etal., 1995a; Findlay etal., 1995b). One of the toxins, rugulosin, was
obtained
from cultures of Homonema dermatioides, derived from red spruce needles
(Calhoun etal., 1992).
[0009] In nature, tree seedlings may acquire needle endophytes from
the trees surrounding the growing tree. However, most of these strains are
not able to produce anti-insectan compounds. Commercially produced
seedlings leaving production facilities are not colonized by needle endophytes
(Miller 2002). There remains a need for a conifers inoculated with endophytes
to increase pest tolerance would be highly desirable considering the hundreds
of millions of seedlings produced in North America annually.
[0010] There are difficulties in colonizing conifers with endophytes,
such as cumbersome methods requiring plant wounding, low percentages of
successful inoculation and lack of longevity infection potential and viability
of
the inoculum. The primary obstacle includes the fact that the natural mode of
transmission from cast needles to developing seedlings is achieved by spores
that are not easily produced in quantity in the laboratory. Additionally,
inoculum is present at the base of a seedling for a period of time difficult
to
reproduce in the greenhouse. However, economically viable large-scale
inoculation of conifers with desirable strains of endophytes requires a method
with increased colonization efficiency and ease of inoculation.
Summary of the invention
[0011] The invention provides novel isolated toxigenic endophyte
strains and provides methods for inoculating a conifer seedling with an
inoculum composition. The inventors have found that conifer seedlings can be
colonized with a toxigenic endophyte using a method that does not require
wounding. The inoculum can be applied in one embodiment, by spraying. In
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addition the inventors have found that inoculation efficiency is highest
during a
time period referred to herein as the "susceptible time window" or "receptive"
time period. The invention thereby provides methods that permit increased
colonization efficiency and are amenable to large-scale inoculations. The
invention also includes seedlings, trees, and tree-parts (such as needles)
produced according to methods of the invention.
[0012] Accordingly, the invention provides a method of inoculating a
conifer seedling to provide increased tolerance to a pest, comprising
inoculating the conifer seedling with an isolated endophte when the conifer
seedling is susceptible to colonization by the endophyte.
[0013] In another embodiment the invention provides a conifer
colonized by an isolated endophyte that produces a toxin that retards pest
growth.
[0014] In another embodiment the invention provides an isolated
endophyte selected from the group consisting of 05-37A, 06-486D, 06-485A.
[0015] In another embodiment the invention provides an inoculum
composition for inoculating conifers to provide increased tolerance to a pest,
comprising a diluent and an isolated endophyte that produces a compound
toxic to the pest.
[0016] In another embodiment the invention provides an antibody
directed against an endophyte selected from the group consisting of
5WS22E1, 5WS11I1, 05-37A, 06-486D, 06-485A, 06-486D and 06-485A.
[0017] In another embodiment the invention provides a method of
detecting the presence of a target isolated endophyte in a conifer sample,
comprising:
a) contacting the conifer sample with an antibody directed against the
endophyte;
b) detecting the presence of bound antibody in the sample, wherein
the presence of the bound antibody is indicative of the presence of
the endophyte.
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[0018] Other features and advantages of the present invention will
become apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific examples
while indicating preferred embodiments of the invention are given by way of
illustration only, since various changes and modifications within the spirit
and
scope of the invention will become apparent to those skilled in the art from
this detailed description.
Brief description of the drawings
Embodiments of the invention will be described in relation to the drawings in
which:
[0019] Figure 1 shows the response of serial dilutions of polyclonal
antibody to target endophyte 5WS22E1 [best fit curve using LOWESS
procedure] together with comparisons at 60 ng/well of cells of some white
spruce endophytes (WS331L1, WS1111), some fungi common on the outside
of the seedlings (A. altemata; A. fumigatus, C. cladosporioides, Phoma
species, as well as control powdered freeze-dried white spruce needles.
[0020] Figure 2 shows the response of a polyclonal antibody to 30, 60,
120 and 240 ng 5WS22E1 cells and to the same amounts added to 500 ng
powdered white spruce cells [mean plus standard error].
[0021] Figure 3 shows the distribution of rugulosin concentrations in
needles from 113 seedlings, assuming half the detection limit for the non-
detects [with normal smoother].
[0022] Figure 4 is a plot of the linearity and avidity of a polyclonal
antibody for endophyte 5WS1111.
[0023] Figure 5 shows the application of a polyclonal antibody used to
detect endophyte 5WS11I1 in planta.
[0024] Figure 6 plots an example of a susceptible time window of
seedlings to colonization by toxigenic endophytes.
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[0025] Figure 7 shows an HPLC trace for rugulosin.
[0026] Figure 8 plots the average weight of spruce budworm grown on
diet with increasing rugulosin concentration.
Detailed description of the invention
[0027] The inventors have identified methods of inoculating conifer
seedlings with toxigenic strains of fungal endophytes and producing toxigenic
endophyte infected conifer seedlings that are more resistant to pest damage.
[0028] Some strains of endophytes that naturally colonize conifer
needles produce compounds or toxins that affect the survival of pests. The
needles of seedlings thus colonized contain toxins and the growth rate of the
pest and susceptibility to parasites and natural bacterial pathogens are both
reduced. The inventors have isolated multiple strains of toxigenic endophytes
and have devised methods to propagate these strains. In addition the
inventors have demonstrated that conifer seedlings can be inoculated with
these toxin-producing endophyte strains during a susceptible time window.
Inoculation during the susceptible time period improves colonization of the
toxigenic endophyte. The inventors have shown that toxigenic endophyte
colonization persists and spreads to non-inoculated new growth branches.
Further these methods are, as shown by the inventors, amenable to large-
scale production in a commercial setting. The inventors have also isolated
several new endophyte strains and have identified the major toxin produced
for several of these endophyte strains.
[0029] Methods for inoculating conifer seedlings, for detecting
successfully inoculated plants, for preparing an effective inoculum
composition, as well as methods of producing toxigenic endophyte colonized
conifer plants that are resistant to pests such as spruce budworm are
provided. The invention also provides novel toxigenic endophyte strains,
inoculum compositions and toxigenic, endophyte-colonized conifer plants.
[0030] Accordingly, in one embodiment, the invention provides a
method of inoculating a conifer seedling, the method comprising:
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a) inoculating the conifer seedling with an effective amount of an
inoculum composition comprising an isolated toxigenic endophyte
during a susceptible time window, wherein the susceptible time
window is a period of time during which the conifer seedling is
susceptible to colonization by the toxigenic endophyte.
[0031] The term "seedling" as used herein means a young plant grown
in a nursery production facility prior to final planting and comprises the
period
of seedling development from seed to 16 weeks post-germination.
[0032] The term "colonization" as used herein means the persistence of
an inoculated endophyte in a symbiotic relationship with a conifer plant
wherein the conifer hosts the endophyte and the endophyte persists in
sufficient quantity to be detected in an assay, for example, in an antibody
detection assay using an antibody directed against the endophyte. Optionally,
the offspring of the colonized plant are also colonized.
Toxigenic Endophytes
[0033] The term "isolated toxigenic endophyte" as used herein means
an isolated endophyte strain that produces a toxin. An isolated toxigenic
endophyte is able to colonize a conifer seedling and produce a toxin in the
colonized plant. The toxin produced by the toxigenic endophyte confers
increased pest tolerance by controling, reducing, mitigating, preventing or
repelling a pest and/or pest growth and/or pest damage in the endophyte-
colonized plant compared to a non-colonized plant.
[0034] In one embodiment, the toxigenic endophyte of the invention
includes the strains described in Table 1 and strains listed elsewhere herein.
Other toxigenic endophytes are readily used in the methods of the invention.
[0035] The inventors have shown that various endophytes isolated from
white spruce are toxigenic to conifer tree pests. These comprise rugulosin
producing endophytes, vermiculin and 5-methoxy-carbonylmellein producing
endophytes. These metabolites are the major components of the mixture of
different anti-insectan metabolites produced by each strain and comprises
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REPLACEMENT SHEET
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derivatives, plant modified forms and metabolites thereof that are toxic. The
major metabolites may be used as a proxy for toxicity. The fungi produce
mixtures of metabolites and the toxicity of the mixture may be greater than
the
dominant compound used as a proxy. This may contribute to a toxigenic
endophytes ability to confer durable tolerance. The term 5-methcw-
carbonylmellein as used herein optionally comprises derivatives, plant-
modified forms and metabolites thereof that are toxic to a pest.
[0036] In one embodiment, the isolated toxigenic endophyte present in
the inoculum composition is a rugulosin producing endophyte. In a more
specific embodiment, the rugulosin producing endophyte is isolated
endophyte 5W522E1 comprising SEQ ID NO: 1. In another embodiment the
isolated toxigenic endophyte is a vermiculin producing endophyte. In a more
specific embodiment, the vermiculin producing endophyte is isolated
endophyte 5WS11I1 comprising SEQ ID NO: 2. In another embodiment, the
isolated toxigenic endophyte is a 5-methoxy-carbonylmellein producing
endophyte. In a more specific embodiment, the 5-methoxy-carbonylmellein
producing endophyte is the isolated 05-37A strain comprising SEQ ID NO: 3.
[0037] Isolated strains of toxigenic endophytes are readily
identified, for
example, the inventors have sequenced regions of the internal transcribed
spacer (ITS) regions of ribosomal DNA (rDNA). Sequence analysis revealed
that strains 5WS22E1 and 5WS11I1 are Phialocephala species. Accordingly
in another embodiment, the toxigenic endophyte strain used in methods of the
invention is a toxigenic strain of the Phialocephala species.
[0038] In addition, the inventors have isolated several novel
toxigenic
endophytes from white spruce needles, including strains referred to as 05-
37A, 06-486D and 06-485A. Sequence data indicates that endophyte strain
05-37A [SEQ ID NO: 3] is related to Nemania setpens and that strains 06-
486D is related to Genbank accession AY971727 and 06-485A is related to
Genbank accession AY971740, both isolated from spruce in Quebec.
[0039] Accordingly, the invention further provides an isolated toxigenic
endophyte comprising the sequence in SEQ ID NO: 3 (05-37A). In another
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embodiment, the invention provides an isolated toxigenic endophyte
comprising the sequence in SEQ ID NO: 5 (06-486D). In another
embodiment, the invention provides an isolated toxigenic endophyte
= comprising the sequence in SEQ ID NO: 6 (06-485A).
[0040] Further the inventors have isolated multiple toxigenic endophyte
strains from red spruce needles. The inventors have similarly sequenced the
ITS regions of each strain (see Table 2).
[0041] Toxigenic endophytes from balsam fir have also been isolated
7BF 36H1 (Novel Diterpenoid Insect Toxins from a Conifer Endophyte JA
Findlay, G Li, PE Penner, JD Miller - Journal of Natural Products, 1995 58:
197-200).
[0042] In one embodiment, the toxigenic endophyte comprises all or
part of one of SEQ ID NOS: 1-5, and preferably at least: 25-50 or 50-100
consecutive nucleotides of one of SEQ ID NO: 1-5. In another embodiment,
the toxigenic endophyte comprises all or part of one of SEQ ID NOS: 6-41,
and preferably at least: one of 25-50 or 50-100 consecutive nucleotides of
SEQ ID NO: 6-41.
[0043] Several toxigenic endophyte strains to be used with the methods
of the invention have been deposited with the Centraalbureau voor
Schimmelcultures (CBS) international depository agency in the Netherlands
(Accession nos. in Table 1). In addition two of the strains have been
deposited with the National Mycological Herbarium/Herbier National de
Mycologie recognized under the acronym DAOM as indicated in Table 1.
DAOM stands for Department of Agriculture, Ottawa, Mycology. Deposits
were made to the Central bureau voor Schimmelcultures P.O. Box 85167,
3508 AD Utrecht, The Netherlands on October 19, 2006
Table 1. Endophyte strains and their principal toxins
Strain DAOM Accession Numbers Principal Toxin
5WS22E1 229536 CBS 120377 rugulosina
5WS11I1 229535 CBS 120378 vermiculinb'
05-37A CBS 120381 5-methoxy-
carbonylmelleind _
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06-486D CBS 120379
06-485A CBS 120380
a Calhoun, Findlay, Miller, Whitney. Mycological Research 1992, 96:281-280.
Bouhet, Van Chong, Toma, Kirszenbum, Fromageot. J Agric Food Chem
1976, 24:964- 972. discovered in 1939 and published in 1955
b Findlay, Li, Miller, VVomiloju. Can J Chem, 2003, 81:284-292.
among others; a new compound trihydroxy-4-1'- hydroxyethyl) isocoumarin
also toxic to spruce budworm cells (Can J Chem 81:284)
Anderson, Edwards, Whalley. J Chem Soc Perkin Trans 11983, (2)185-
255; Wang, Zhang, Huang, Su, Yang , Zhaob, Ng. Acta Cryst 2003,
E59:o1233-1234
Table2. Isolated Red Spruce Fungal Endophytes
SEQ ID NO. SEQUENCE NAME
6 1 06-023A ITSIF
7 2-08-011D-ITSIF
8 4-09-009D-ITSIF
9 5-04-012A-ITSIF
10 3-03-001D-ITSIF
11 6-06-255C-ITSIF
12 16-06-264A-ITSIF
13 11-04-002G-ITSIF
14 12-08-018A ITS1F
14-06-003A-ITSIF
16 20-06-065C_ITS1F
17 22-02-008A-ITSIF
18 23-06-254B-ITSIF
19 24-06-321A-ITSIF
25-06-188A-ITSIF
21 26-06-332A-ITSIF
22 27-03-032A-ITSIF
23 28-02-002C-ITSIF 1
24 30-01-017A ITS1F
39-06-094D-ITSIF
26 41-03-047A-ITSIF
27 42-06-130D-ITSIF
28 45-06-271A-ITSIF
29 49-01-002A ITS1F
46-06-255B_ITS1F
31 48-06-052C-ITSIF
32 52-06-265B-ITSIF
33 53-05-065E-ITSIF
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34 56-03-020B-ITSIF
35 64-06-073C ITS1F
36 3665 4A-06-097D ITSIF
37 B-06-083B-ITSIF
38 D-03-007A-ITSIF
39 F-06-255A-ITSIF
40 I-06-268A-ITSIF
41 J-06-317A-ITSIF
[0044] One skilled in the art will understand that other isolated
toxigenic
endophyte strains can be used with the methods and compositions of the
invention. Other toxigenic endophyte strains can be isolated using the
methods of screening for a toxigenic endophyte provided herein.
Endophyte Toxins
[0045] Toxigenic endophytes produce toxins that provide increased
pest tolerance. The term "toxin" as used herein refers to a substance or
substances that confers increased pest tolerance by controlling, reducing,
mitigating, preventing or repelling pests and/or pest growth and/or pest
damage. Toxins of several toxigenic endophyte strains have been identified
and are described in Table 1. A particular toxigenic endophyte may produce
more than one toxin. The toxins identified in Table 1 are the dominant toxins
produced by the listed strains. The referenced studies to Table 1 illustrate
that
multiple toxins may be produced by each strain. Illustrative is strain 5WS11I1
which produces among others vermiculin, trihydroxy-4-1'-hydroxyethyl and
isocoumarin.
[0046] The ability of a toxin to control, reduce, mitigate, prevent or
repel
pests and/or pest growth and/or pest damage can be assessed in vitro in a
pest toxicity assay. For example, the inventors provide a method of assessing
the toxicity of a putative toxin using a method that assesses insect larvae
growth, such as a spruce budworm larvae assay that measures effects on
growth.
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[0047] The term "toxicity" as used herein with respect to the spruce
budworm larvae assay means toxins or endophyte strains that exhibit
statistically different parameters from controls, either for weight reduction,
head capsule width or both. Toxic endophytes cause spruce budworm larvae
to have lower weight and/or smaller head capsule, and the aforementioned
parameters are statistically reduced compared to control.
[0048] The method compares spruce budworm performance on foliage
of different ages and tree species. The system optionally comprises a tapered
container comprising a septum that permits an individual needle to be held
vertically and exposed to a single spruce budworm with the base of the
needle in contact with moisture. The needle is inserted in the septum before
the budworm is added. The septum permits uneaten portions of the needle to
be collected.
[0049] Spruce budworm larvae are typically grown with artificial diet
(McMorran, 1965) until they reach a stage at which they will consume
succulent needles. A single budworm is placed in each vial. Needles of similar
size and weight collected around the inoculation point of the toxigenic
endophyte inoculated seedling (test seedling) plus control needles are tested.
Pest growth data which optionally includes the amount of unconsumed
needle, head capsule width and larval weights are measured. Budworm and
residual needle weights and budworm head capsule widths are determined for
test and control samples and compared. Other insects and larvae may be
readily tested with similar assays, for example, hemlock looper, and spruce
budmoth.
[0050] The toxicity of an endophyte toxin on a pest such as hemlock
looper is optionally tested according to the following method. The toxin to be
tested is incorporated into an artificial diet suitable for pest growth. 15 ml
of
artificial diet is prepared in individual cups, containing toxin
concentrations of
5 micromolar, 10 micromolar, 25 micromolar, 50 micromolar and 100
micromolar. One 31d instar insect is added to each of 75 cups per dilution and
incubated in a growth chamber at 22 C, 55% relative humidity with 16 h
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light/day. After 4 days, the insects are frozen, weighed and their head
capsules measured. A reduction in head capsule size (eg. width) or weight
compared to a control sample is indicative of the presence of a toxin. One
skilled in the art would understand that this method can be modified to test
different concentrations of toxin and that the conditions can be modified to
test
a variety of different pests.
[0051] The amount of endophyte adequate to reduce growth a conifer
plant is the amount that shows a statistically-significant reduction of growth
rate, and/or instar development or weight gain reduction compared to
uninoculated but otherwise equivalent control seedlings.
[0052] In one embodiment, the assay used to evaluate pest toxicity is
an in vitro assay and comprises the isolation of a conifer needle in a
suitable
container to ensure that the needle remains hydrated and the container,
collect and evaluate the weight and head capsule width of a suitable test
insect meaning an insect that normally consumes needles during its growth
and development as well as collect any residual needle for to assess
percentage needle consumption.
[0053] In another embodiment, the pest toxicity assay is an in vivo
assay and comprises placing an appropriate insect species on branches of
endophyte-colonized plants and/or trees contained in bags and/or other
containers at a density of optionally not greater than 10 insects/m2 of branch
area, leaving the insects in place for an appropriate time period, collecting
the
insects followed by determinations of weight and head capsule width.
Optionally other parameters may be assayed. The appropriate time period for
leaving a pest in contact with the branches of an endophyte colonized plant,
will vary with factors such as larval stage of development, weather
conditions,
conifer species and insect species. In one embodiment, the time period is
optionally between 1-14 days, for example, 3-7 days.
[0054] In another embodiment, the toxicity of an endophyte is assessed
by subjecting the colonized conifer plant to at least one characteristic test
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selected from the group consisting of pest toxicity assay, toxin presence
assay.
[0055] Accordingly the invention provides a method of assessing the
pest toxicity of a toxigenic endophyte toxin using a pest toxicity assay.
Susceptible Time Window
[0056] The term "susceptible time window" as used herein means a
period of time during which a conifer seedling is susceptible or receptive to
colonization upon inoculation with an inoculum composition comprising a
toxigenic endophyte. "Susceptible" is used interchangeably with "receptive"
herein.
[0057] The inventors have established that efficient inoculation of
conifer seedlings with toxigenic endophytes occurs during a susceptible time
window. The susceptible time window optionally comprises the stage of seed
stratification and/or the post-germination period of sustained elongation of
the
shoot apex wherein the cuticle is not fully formed.
[0058] Wax development occurs within the cuticle and may be referred
to as cuticular wax as well as on the surface of the cuticle which may be
referred to as epicuticular wax. Cuticular wax may function to provide a
waterproof quality from the needle surface.
[0059] Without wishing to be bound by theory, it is believed that the
upper time limit of the period during which the post-germination seedlings may
be non-invasively inoculated corresponds to the formation of wax on and/or in
the seedling that impairs inoculation.
[0060] The term "seed stratification" as used herein means a process
of artificially or naturally interrupting a seed's embryonic dormancy so that
the
seed can germinate and embryonic dormancy typically comprises the period
of time from taking seed to sowing seed. "Embryonic seed dormancy" is
optionally interrupted for white spruce by soaking the seed in water overnight
and exposing the drained seed to temperatures of approximately 3-6 C for
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approximately 2 weeks. The term "germination" as used herein means the
resumption of growth by a seed.
[0061] The inventors have found that seeds can be inoculated during
seed stratification. In one embodiment, the method comprises soaking a
conifer seed in water containing inoculum during seed stratification. In
another
embodiment a white spruce conifer seed is soaked in water containing
inoculum overnight, drained and the still wet seed is refrigerated at 2-6 C
for
approximately two weeks.
[0062] The inventors have also found that the susceptible time window
comprises the period of time of sustained elongation of the shoot apex prior
to
formation of the needle cuticle. The susceptible time window will vary in
terms
of seedling height and age post germination with conifer species, but
comprises the period of statistically significant maximum susceptibility,
which
using white spruce as a model, begins during the period of sustained
elongation of the shoot apex (>10 mm < 100mm). The minimum is biologically
determined by the period after the germination processes are largely
completed. These include the time after the seed coat cracks and the radicle
emerges. The radicle and hypocotyl and cotylyedons elongate rapidly to the
point when the base of the cotyledon begins to elongate. During the period
circumscribed by seedling heights >10 but >40 mm, the shoot apex, needle
primordial and subtending internodes are initiated, expand and develop
rapidly. The rudimentary needles and internodes become completely
differentiated including formation of the cuticle and mesophyll. The critical
period of successful inoculation is related to the percentage needles of
intermediate differentiation to complete differentiation in which the cuticle
is
fully formed.
[0063] In one embodiment a seedling is inoculated before the needle
cutilcle is fully formed. In another embodiment, the seedling is inoculated
during the period of sustained elongation of the shoot apex. In another
embodiment, the seedling is inoculated wherein the percentage of needles
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wherein the cuticle is intermediately differentiation is greater than the
number
of needles wherein the cuticle is fully formed.
[0064] In white spruce the inventors have determined that the
susceptible time window comprises until seedlings reach 16 weeks post
germination. In a preferred embodiment, seedlings are inoculated between 2-
12 weeks, 6-10 weeks or 7-9 weeks post germination. In another embodiment
seedlings are inoculated at 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, 6-7
weeks, 7-8 weeks, 8-9 weeks, 9-10 weeks post-germination. It has been
determined that particularly successful inoculation of white spruce seedlings
is
obtained in an embodiment when seedlings are inoculated at about 8 weeks
post-germination (eg. 7-9 weeks post-germination).
[0065] In terms of seedling height, the period of susceptibility of
white
spruce comprises the germination stage up until seedlings are approximately
10 cm tall. In another embodiment the seedlings are inoculated when 1-2 cm
tall, 2-3 cm tall, 3-4 cm tall, 4-5 cm tall, 5-6 cm tall, 6-7 cm tall, 7-8 cm
tall, 8-9
cm tall, 9-10 cm tall. In has been determined that particularly successful
inoculation is obtained in an embodiment when seedlings are inoculated at
about 3 cm tall (eg 2-4 cm tall).
Methods of Inoculation
[0066] Various methods can be used to inoculate a conifer seedling. In
embodiments of the method, inoculation of a conifer seedling comprises
contacting an inoculum composition with a seedling, such as an intact
seedling. An "intact seedling" as used herein refers to a seedling wherein the
seedling remains unwounded prior to, or during, contact with the inoculum
composition. More specifically, the stem of an intact seedling is not pierced
or
wounded prior to, or during, contact with the inoculum composition. An
inoculum composition is optionally applied to an intact seedling by spraying
the intact seedling with the inoculum composition. The use of intact seedlings
is very advantageous because it eliminates the plant wounding step and
facilitates rapid inoculation, providing very important time savings in a high-
throughput conifer nursery setting. In another embodiment the inoculation
CA 02793664 2012-10-23
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method comprises contacting an inoculum composition with a surface area of
a conifer seedling. An inoculum composition is optionally applied to a conifer
seedling by spraying a surface area of a conifer seedling with the inoculum
composition. The term "contacting" is used interchangeably with "applying".
[0067] Spraying as used
herein comprises any method of delivering
liquid particles of the inoculum composition to a plant surface and includes
misting, ground spraying, airblast spraying, and fan spray techniques.
Spraying is optionally accomplished using a spray bottle or a boom sprayer.
In one embodiment the inoculum composition is delivered using a boom
sprayer and an injector pump to inject the inoculum composition into an
irrigation line. Inoculation is readily applied during a time when the
seedling
would remain moist for the longest period of time and optionally comprises
repeated application on the same or different days. In other embodiments, the
inoculum composition optionally comprises additives that improve and/or
increase uptake of a toxigenic endophyte. A number of chemicals and or
preparations are known in the art that would facilitate inoculum uptake. For
example additives may reduce the drying time after spraying. In one
embodiment the additive is a carbohydrate. In another embodiment the
carbohydrate is selected from the group comprising sugars. In another
embodiment the carbohydrate is a carbohydrate like CMC. Further fluorescent
tracers may be added to the inoculum composition to determine the amount of
spray deposited.
[0068] In another
embodiment, the inoculation method comprises a
below ground application of an inoculum composition.
[0069] Inoculation of a
conifer seedling, in another embodiment,
comprises cutting, piercing or otherwise penetrating a seedling and delivering
an inoculum composition. In one embodiment, the site of the penetration is
the unlignified tissue of the seedling stem. In another embodiment, the stem
is
penetrated 5-15 mm from the terminal shoot. The term "terminal shoot" as
used herein means the shoot distal to the lowest leaf remaining on the
main stem. Delivery of an inoculum composition may be performed by
CA 02793664 2012-10-23
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various methods including spraying. Optionally the inoculum composition may
be delivered by repeated spraying. In another embodiment the method of
inoculation comprises a wound inoculation. 'Wound inoculation" as used
herein refers to a method wherein the inoculum composition is introduced into
a conifer seedling by a method involving wounding the seedling. The inoculum
may be introduced by a needle injection method. In another embodiment,
conifer seedlings are inoculated by placing a carrier comprising toxigenic
endophytes in contact with the seedling growing medium. In one embodiment
the carrier comprises irradiated conifer needles. In another embodiment, the
seedling is planted in a growing medium, and irradiated conifer needles
colonized by toxigenic endophytes or other conifer plant parts colonized by
toxigenic endophytes are added to the growing medium. In another
embodiment the growing medium, comprises potting mix surrounding or
supporting the conifer seedling to be inoculated. In another embodiment the
growing medium comprises soil.
[0070] In
addition, a conifer seedling may be inoculated by soaking a
conifer seeds with an inoculum comprising a toxigenic endophyte.
[0071] In another
embodiment, the inoculation method comprises
putting irradiated conifer needles infested with a toxigenic endophyte in
contact with a conifer seedling. The needles and endophyte may be directly
contacted or indirectly contacted with the conifer seedling. For examples,
needles may be placed in direct physical contact with the seedling or may be
placed in indirect contact with the seedling by contacting needles with the
potting mix supporting seedling growth. In one embodiment the potting mix
comprises soil.
[0072] The
quantity of toxigenic endophyte inoculated is preferably in
one embodiment approximately 10 propagules. A "propagule" as referred
herein means an infective fungal cell. A person skilled in the art will
recognize
that the quantity of toxigenic endophyte inoculated may vary with
environmental and other factors. For example, if inoculation of conifer
seedlings is performed during environmental conditions such as low
CA 02793664 2012-10-23
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temperature, that are not favourable to endophyte inoculation, the quantity of
toxigenic endophyte is optionally increased. Similarly, repeated inoculations
are optionally used if seedlings are exposed to various environmental
conditions, such as heavy rainfall where the inoculum composition is diluted
or washed away.
[0073] The methods of inoculation described above can be combined
and or repeated. In one embodiment the methods of inoculation combined
and/or repeated comprise the same method of inoculation. In another
embodiment, the methods combined and/or repeated are different methods of
inoculation. For example in one embodiment, a seedling is first inoculated
during seed stratification and then inoculated during the period of sustained
elongation of the shoot apex.
Inoculum Composition
[0074] The term an "inoculum composition comprising a toxigenic
endophyte" as used herein means a composition for inoculation wherein the
toxigenic endophyte is present in an effective amount to colonize conifer
seedlings conferring increased pest tolerance by controlling, reducing,
mitigating, preventing or repelling pests and/or pest damage and/or pest
growth. The inoculum composition is effective if the level of toxin produced
is
sufficient to control, reduce, mitigate, prevent or repel pests and/or pest
growth and and/or pest damage. Examples of suitable compositions are
described in this application and are optionally readily identified using
assays,
such as spruce budworm larvae assay, described herein.
[0075] The inventors have identified novel methods to produce an
inoculum of the invention. The filaments of the toxigenic endophyte are
optionally sheared by a shearing means which reduces the number of dead
endophytes produced compared to other maceration methods, thereby
increasing the number of live toxigenic endophytes per volume inoculum and
facilitating the colonization of inoculated conifer seedlings.
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[0076] In one
embodiment, the endophyte is grown in a stirred jar
fermentation unit such that the cells are present in not larger than 5 mm
clusters of mycelium or spores, are greater than 80% and preferably greater
than 95% viable and greater than 80% and preferably greater than 95%
infective in receptive tissue in liquid substantially free of bacteria and
material
concentrations of residual nutrients.
[0077] In another
embodiment the invention provides, an inoculum
composition is optionally prepared by a method for growing the isolated
endophyte comprising:
a) providing a slant culture of the endophyte (eg. agar slant culture);
b) inoculating a first liquid culture with the culture;
C) subjecting the first liquid culture to a shear force that shears the
endophyte hypha;
d) inoculating a second liquid culture with the first liquid culture;
e) subjecting of the second liquid culture to a second shear force to
shear the endophyte hypha.
[0078] In another embodiment, the method comprises:
a) growing an initial culture of the endophyte (eg. agar slant culture);
b) inoculating a first liquid culture with a suspension comprising the
agar slant culture wherein the first liquid culture is grown for
approximately 2 weeks;
c) maceration of the first liquid culture
d) inoculation of a second liquid culture with the macerated first liquid
culture under conditions of shear force sufficient to shear the
endophyte hypha wherein the second culture is grown in a large
vessel, and is aerated.
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[0079] In one embodiment the inoculum is optionally harvested from
the large vessel which includes a fermentor, centrifuged and resuspended in a
diluent. In one embodiment the diluent is sterile water.
[0080] In one embodiment the agar slant is a malt agar slant. In
another embodiment the first liquid culture is 2% malt extract and the
suspension is added at 5% v/v. The shear force in one embodiment
comprises shaking or rotation at 200-310 RPM. In another optional
embodiment the shaking or rotation is at 220 RPM. In one embodiment, the
first liquid culture is preferably incubated at 25C. In one embodiment the
first
liquid culture comprises a malt extract. In another embodiment the macerated
liquid culture is added to a 1-3% malt extract broth. In an embodiment the
malt extract concentration is approximately 1%. In another embodiment the
macerated liquid culture and malt extract broth are stirred at 200-310 RPM,
for example stirring at 280 RPM. In another embodiment the temperature in
the large vessel which may optionally be a stirred fermentor, is 20-22C and is
optionally 21C. In another embodiment, the aeration is 0.05- 0.15 v/v per
minute and is optionally 0.1 v/v per minute. In another embodiment, the
macerated liquid culture and malt extract are incubated for 6-10 days and
preferably 7 days. A person skilled in the art would understand what routine
adaptations would be required to grow the new toxigenic endophyte strains
identified. In addition a person skilled in the art would understand that
changes to sugar concentration, temperature and oxygen tension may require
compensating changes in other variables. A person skilled in the art would
also understand the routine experiments to further scale up the production of
inoculum.
[0081] The inoculum may be diluted or concentrated. In one
embodiment the inoculum is diluted with water before inoculation.
[0082] The inventors have shown that the concentration of rugulosin in
needles infected rugulosin producing strains that reduced pest growth
averaged 8 micrograms/gram of needle weight. A concentration that affected
pest growth is optionally as low as 0.15 micrograms/gram of needle weight.
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The conifer sample comprises a conifer tissue of a plant previously inoculated
with an effective inoculum concentration comprising a toxigenic endophyte.
Accordingly in one embodiment, the effective inoculum composition is a
composition that produces a rugulosin toxin concentration of at approximately
10 micromolar in a successfully colonized conifer seedling. In another
embodiment the toxin concentration achieved is optionally 10-25 micromolar,
or optionally 25-50 micromolar.
[0083] The effectiveness of the inoculum varies with the length of
time
the inoculum has been stored. Preferably, the inoculum is prepared the same
day as the inoculation.
[0084] In another embodiment, the invention provides an inoculum
composition comprising a toxigenic endophyte, which can be used with the
methods of the invention to produce a conifer seedling infected with a
toxigenic endophyte fungus.
[0085] In one embodiment, the method of inoculating conifer seedlings
further comprises detecting if the seedling is colonized by the toxigenic
endophyte.
Method of Detecting Toxigenic Endophyte
[0086] Various methods can be employed to detect the presence of
toxigenic endophytes. Assays using antibody-based methods for the detection
of grass endophytes have been developed for several endophyte species
both using microplate assays and tissue immunoblot methods (Gwinn et al.
1991; Johnson et al. 1982; Reddick & Collins 1998). Compared to competing
methods such as PCR, these have a greater potential for field use based on
simple protocols.
[0087] The inventors have developed an assay using a detection agent
for assaying target endophytes. In one embodiment the assay targets
endophytes, such as 5WS22E1 using a detection agent that was an antibody
that was of comparable sensitivity to antibody assays for grass endophytes
(Gwinn et al. 1991; Johnson et al. 1982; Reddick & Collins 1998). This was
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achieved despite the greater difficulties of the conifer needle matrix
compared
to grass leaves.
[0088] Accordingly
the inventors provide an assay using a detection
agent that binds a toxin or recognizes a toxigenic strain of endophyte for
detecting the presence of target endophytes in a conifer sample, the assay
comprising:
a) contacting a conifer sample with an agent which recognizes a
toxigenic strain of endophyte;
b) detecting the presence of bound agent in the conifer sample,
wherein the presence of bound agent is indicative of the presence
of the toxigenic strain of endophyte recognized by the antibody.
[0089] In one
embodiment the agent that binds a toxin or recognizes a
toxigenic strain of endophyte is an antibody.
[0090] In another
embodiment the antibody assay is an ELISA assay.
In another embodiment the antibody assay is a hand-held immunoblot based
assay. In another embodiment the antibody assay is contained in a kit
comprising an antibody which recognizes a toxigenic strain of endophyte and
a detection means to detect the presence of a strain recognized by the
antibody.
[0091] The inventors
have shown that the presence of a toxigenic strain
of endophyte may not be detectable in successfully colonized seedlings until
the seedling has grown for a period of time. Hence in one embodiment the
conifer sample is obtained when the plant is greater than 3 months, greater
than 4 months, greater than 5 months, greater than 6 months, greater than 7
months, greater than 8 months, greater than 9 months, greater than 10
months, or greater than 11 months post germination. In another embodiment,
the conifer sample is obtained when the plant is greater than 1 year post
germination. In another embodiment, the conifer sample is obtained when the
plant is 12-18 months post germination. In another embodiment, the conifer
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sample is obtained when the plant age is greater than 18 months post
germination.
[0092] The conifer sample optionally comprises conifer tissues
including needles. The conifer sample is typically ground to a powder prior to
contact with the toxigenic endophyte recognizing antibody and/or suspended
in an appropriate buffer such as Tris buffered saline (TBS). In addition, the
presence of a toxigenic endophyte may be detected by detecting the
endophyte toxin. The inventors provide an analytical method of detecting a
major toxin associated with a toxigenic strain of endophyte. The analytical
method comprises preparing a putatively toxigenic endophyte toxin containing
conifer sample, for processing by separation, such as HPLC (high
performance liquid chromatography), the method of preparing the conifer
sample optionally comprising:
a) a first extraction using petroleum ether under low light conditions;
b) a second extraction with chloroform;
c) washing the extract with NaHCO3;
d) acidifying the extract;
e) a third extraction with chloroform; drying the extract;
f) and dissolving the dried extract in acetonitrile.
[0093] The resulting sample is optionally assayed by an HPLC
apparatus and the spectrum produced analysed for toxin presence. In one
embodiment, the sample to be prepared for HPLC analysis comprises a
rugulosin producing toxigenic endophyte.
[0094] Accordingly the invention provides a method of detecting a toxin
associated with a toxigenic strain of endophyte, comprising, preparing a
conifer sample for HPLC analysis separating an endophyte extract by high
pressure liquid chromatography which produces a spectrum output, detecting
CA 02793664 2012-10-23
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the presence or absence of a toxin value in the spectrum output, wherein the
presence of a toxin value is indicative of the presence of a toxin.
[0095] In another embodiment the method comprises:
a) separating an endophyte extract by high pressure liquid
chromatography which produces a spectrum output,
b) detecting the presence or absence of a toxin value in the spectrum
output, wherein the presence of a toxin value is indicative of the
presence of a toxin.
[0096] The toxin is also optionally detected using other assays
including NMR, preparatory thin layer chromatography, preparatory HPLC,
HPLC Mass Spectroscopy and column chromatography. Methods for use of
these techniques are known in the art.
Conifer Tree Species and Genotypes Susceptible to Colonization with
Toxigenic Endophytes
[0097] The invention is practiced with conifer plants and seedlings. A
"conifer" as used herein refers to a variety of needle-leaved trees or shrubs
and includes all Spruce species (Picea species), pine (Pinus species) and
balsam fir trees (Abies balsamea) and plant as used herein comprises a
seedling or tree and includes tree hedged for the production of rooted
cuttings
or a shrub. In certain embodiments, the conifer seedling inoculated is a white
spruce (Picea glauca) seedling. In other embodiments, the conifer seedling
inoculated is a red spruce (Picea rubens) seedling. In further embodiments,
the conifer seedling inoculated is a balsam fir seedling. In yet another
embodiment, the conifer seedling inoculated is a pine seedling for example
white pine (Pinus strobes).
[0098] Accordingly in one embodiment of the invention the conifer
seedling inoculated is a white spruce seedling. In another embodiment, the
white spruce seedling is inoculated with an inoculum composition comprising
toxigenic endophyte 5WS22E1. In another embodiment, the white spruce
seedling is inoculated with an inoculum composition comprising toxigenic
CA 02793664 2012-10-23
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endophyte 5WS11I1. In another embodiment, the white spruce seedling is
inoculated with an inoculum composition comprising toxigenic endophyte 05-
37A (SEQ ID NO: 3). In another embodiment, the white spruce seedling is
inoculated with a inoculum composition comprising toxigenic endophyte 06-
486D (SEQ ID NO: 4). In another embodiment, the white spruce seedling is
inoculated with an inoculum composition comprising toxigenic endophyte 06-
485A (SEQ ID NO:5).
[0099] The inventors found that inoculation of seedlings from a
breeding population of white spruce with an inoculum composition of the
invention was successful across a range of genotypes. Of the 25 white spruce
families tested, six had individuals that tested positive for infestation with
one
of the strains of endophytes tested. Of these six families, 11 of the 31
parents
were represented and these 11 parents covered the same range that the
larger sample encompassed. This provided a good indication that a broad
range of genotypes will be susceptible to infection by the endophytes tested.
Endophyte Enhanced Conifer Plant
[00100] A conifer plant colonized with a toxigenic endophyte strain is
also provided by the invention. In one embodiment the conifer plant is a white
spruce plant with toxigenic endophyte 05-37A (SEQ ID NO: 3). In another
embodiment the conifer plant is a white spruce plant colonized with toxigenic
endophyte 06-486D (SEQ ID NO: 4). In another embodiment the conifer plant
is a white spruce plant colonized with toxigenic endophyte 06-485A (SEQ ID
NO: 5).
[00101] In another embodiment, the conifer plant is a red spruce plant
colonized with a toxigenic endophyte selected from the group SEQ ID NO: 6-
41.
Pests Susceptible to Toxigenic Endophyte Toxins
The term "pest" as used herein means any organism that may cause injury to
a conifer plant and comprises insects, insect larvae, and fungi. Insect pests
CA 02793664 2012-10-23
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include insects that consume needles such as spruce budworm hemlock
loopers, saw flies, and jack pine budworm. Fungal pests include white pine
blister rust and fusarium species.
[00102] All the toxins or cultures have been tested with Spruce budworm
(Choristoneura fumiferana) larvae. For the 22E1 toxin rugulosin, Spruce
budworm (Figure 8) and Hemlock loopers (Lambdina fiscellaria) were affected
at dietary rugulosin concentrations between 25 and 50 pM. The tests with wild
collected spruce budmoth (Zeiraphera canadensis) indicated that this species
was also affected by rugulosin in the same order of magnitude. Tests of fruit
flies (Drosophila melanogaster also place the dietary toxicity of rugulosin in
the 50 pM range (Dobias et al. 1980). This compound is also toxic to cultured
cells of several insect cell lines including fall army worm (Spodoptera
frugiperda) and mosquito larvae (Aedes albopictus; Watts et al. 2003).
[00103] When tested under the conditions described above, the semi-
purified extracts of the remaining listed endophytes resulted in statistically
significant reductions in spruce budworm growth rate, and or maximum instar
reached within the operating parameters of the tests.
Endophyte Screening Methods
[00104] The inventors have identified multiple strains of toxigenic
endophytes that can be inoculated in conifer seedlings to reduce pest damage
in colonized hosts. In identifying the toxigenic endophytes of the invention,
the
inventors took fungal strains material from the existing literature from
public
collections and tested additional collections of over 1000 endophyte strains.
The endophytic strains were cultured from the needles of randomly selected
spruce trees and an antibody was developed to permit detection. The
antibody assay permitted detection of successful inoculations. One aspect of
the invention provides a method of identifying novel toxigenic endophytes that
can be used with the methods and compositions of the invention.
[00105] The screening method for isolating a toxigenic endophyte from a
donating plant comprises:
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a) isolating a slow growing candidate endophyte from the conifer
needles of a donating plant (eg. a donating conifer);
b) assaying the toxicity of the candidate endophyte to a pest in a pest
growth toxicity assay to determine whether the candidate
endophyte is a toxigenic endophyte.
Wherein if the if the candidate endophyte is a toxigenic endophyte, the
method optionally further comprises inoculating a conifer seedling;
a) inoculating a recipient conifer seedling with the candidate
endophyte strains determined to be a toxigenic endophyte;
b) providing a sample of the inoculated seedling and detecting the
presence of the target endophyte (ie. endophyte colonization) and/or
endophyte toxin.
[00106] The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
Example 1
[00107] The needles colonized by a rugulosin-producing endophyte
were found to contain rugulosin in concentrations that are effective in vitro
at
retarding the growth of spruce budworm larvae. Larvae presented with
endophyte infected needles containing rugulosin did not gain as much weight
as those eating uncolonized needles. The impact on the budworm was much
greater than anticipated. One strain 5WS22E1, was the most successful
antagonist of larval growth. Needles of 17 of 22 seedlings colonized by
rugulosin -producing strains were toxic to the insects. Needles infected by a
different family of strains producing vermiculin were also toxic to the
insects.
[00108] Rugulosin was unambiguously present in needles infected by
rugulosin-producing strains and not found in either control or in the
seedlings
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colonized by the vermiculin-producing endophyte. In needles that significantly
affected budworm weights, rugulosin concentrations averaged 8 g/g. This
was >15 times the mean concentration found in needles that did not affect
budworm growth. In vitro, rugulosin at 1 g/g affected budworm growth and
development, a value rather close to the 8 g/g found in needles.
Example 2
Antibody Assay for Detecting Endophyte
[00109] The following describes (1) the development of a polycolonal
antibody for the rugulosin-producing endophyte 5WS22E1; (2) the inoculation
of 1235 seedlings and subsequent growth outdoors under commercial nursery
conditions (3) analysis of these needle samples using the culture method
previously employed and the antibody method and (4) HPLC analysis for the
presence of rugulosin in -10% of the positive samples.
MATERIALS AND METHODS
Inoculation
[00110] The strain employed, 5W522E1 (DAOM 229536; rugulosin
producer) was described in Miller et al. (2002). Control-pollinated full-sib
families of white spruce were produced for inoculation. The families used
were chosen to provide a diverse set of genotypes. Nine families originating
from unrelated white spruce parents were used. The parents were selected
from the J.D. Irving Limited genetic improvement program and originated
within a range of 45 N to 47.5 N Latitude and 65 W to 70 W Longitude in New
Brunswick and Maine. The minimum distance between trees selected in the
forest was 200m. Seeds were taken from frozen storage at -5 C, planted in
plastic seedling containers in a media containing 3:1 peat moss/ vermiculite
and placed in a greenhouse. Fertilization via irrigation water started one
month after sowing (10-52-10 for two weeks followed by 8-20-30 for one week
followed by 20-8-20 at 100 pg/L and increasing to a maximum of 125 pg/L).
CA 02793664 2012-10-23
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[00111] 5WS22E1 (DAOM 229536) cultures were grown on 9 cm plates
containing 2% malt extract agar (Difco) at 25 C for 8 weeks. Following the
incubation period, 5 mL sterile water was poured on agar surface which was
then rubbed gently with a sterile bent glass rod. The resulting suspension was
taken up with a sterile pipette, macerated and diluted with sterile water to
deliver an average of 3 fungal hyphal fragments per drop (6 pL) from a sterile
1 mL syringe with a 0.45 mm needle (B-D #309597) as determined by
counting with a hemocytometer. Wound inoculation of 1235 seedlings was
performed in a laminar flow hood by injecting 6 pL into the un-lignified
tissue
of the stem typically 10 mm away from the terminal shoot (Miller et al. 2002).
This was done at the Sussex Tree Nursery on April 16, 2001. This is located
at 450 43' N, 65 31' W; elevation 21.30 m. Mean annual temperature is 5.8 C
(January -8.5, July 19.0 C) with average precipitation of 245 cm snow and 915
mm rain.
[00112] The trees were allowed to grow in trays for 6 months in a
greenhouse, at which point they were planted into pots and left in a shaded
area with irrigation until sampling in mid September 2002.
ELISA Development
[00113] Cells of 5WS22E1 were grown on two types of liquid media and
on irradiated, young uninoculated white spruce needles. The needles were
irradiated with 25kGy (MDS Nordion, Montreal, PQ) and 200 mg was placed
in a sterile glass Petri dish containing a filter paper followed by the
addition of
1 mL sterile water. After 24 h, the culture was inoculated with a small piece
of
culture taken from the leading edge of a 2% malt extract agar plate. The first
liquid medium used was a glucose/sucrose mineral salts medium (1g/L
KH2PO4, 1g/L KNO3, 0.5 g/L MgSO4 7H20, 0.5 g/L KCl, 0.2 g/L glucose,
0.2g/L sucrose) and the second, 2% malt extract. An aliquot (50 mL) of each
medium was dispensed into 250 mL Erlenmeyer flasks, respectively and
autoclaved. An agar culture was macerated in 100 mL sterile water under
aseptic conditions. An aliquot (2.5 mL) was used to inoculate the flasks. All
cultures were incubated at 18 C for ca. three months. At the end of the
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incubation period, the cells growing on the needles were carefully scraped off
with a scalpel and freeze dried. Cells from the liquid cultures were filtered
and
washed several times with sterile distilled water and freeze dried.
[00114] Polyclonal
antibody production was performed in goats at
Cedarlane Laboratories Limited Hornby, Ontario. Freeze-dried cells from
each medium were ground up and each diluted in sterile PBS to a
concentration of 20 mg/mL for the antigen solution. 0.5 mL of this solution
was
emulsified with 0.5 mL of complete Freund's adjuvant (Brenntag Biosector,
Denmark) for the primary immunization. 0.5 mL of incomplete adjuvant was
used for the subsequent boosts. A pre-immune sample was obtained from the
jugular vein of each goat using a needle and vacutainer before the primary
immunization. Each goat was then injected using a 21 gauge needle
intramuscularly in the hind quarter at 4 different sites with 0.25mL of the
emulsified antigen solution per injection site. After 28 days the goat
received
its first boost as described above, its second boost at day 53 and a test
bleed
was taken at day 66.
[00115] The
antibodies produced from the 3 different goats were tested
to determine their avidity and cross reactivity with powdered, freeze-dried
young uninoculated spruce needle cells, as well as cells of the most common
needle phylloplane fungi isolated from these needles (Altemaria altemata,
Phoma herbarum, Cladosporium cladosporioides and Aspergillus fumigatus;
Miller et al. 2002; Miller et al. 1985), and other white spruce conifer
endophytes: 5WS11I1 (DAOM 229535; vermiculin producer) and 5WS331L1
(a rugulosin-producer). In addition, a number of balsam fir endophytes were
tested. One isolated
endophyte from balsam fir is BF 36H1 (Novel
Diterpenoid Insect Toxins from a Conifer Endophyte JA Findlay, G Li, PE
Penner, JD Miller - Journal of Natural Products, 1995 58: 197-200).
[00116] In the case
of the needle endophytes, cells were produced on
irradiated needles as above. The phylloplane species tested were grown in
shake culture using a maltose, yeast extract, peptone medium (Miller &
Mackenzie 2000), filtered, washed and freeze dried as above. This method
CA 02793664 2016-09-14
REPLACEMENT SHEET
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was shown to be suitable for antigen production by such fungi in unrelated
studies. Cells were ground to a fine powder in small mortar and carefully
weighed. Suspensions of known concentration were made in TBS (0.8 g/L
NaCI, 0.2 g/L KCI, 1.89 g/L Tris-HCl, and 1.57 g/L Tris base) in vials and
vortexed.
[00117] Avidity and
cross reactivity experiments were conducted on sera
from the goats treated with the various immunogens in a similar fashion, first
optimizing cell additions/serum dilutions, and then conducting cross-
reactivity
experiments. As needed, aliquots of cells were diluted in 0.1 M carbonate
buffer pH 9.6 (Sigma) coating buffer to defined concentrations and pipetted
into 96 well Nunc brand microplates. The plate was covered with an acetate-
sealing sheet and placed on a rotary shaker for 4 h at room temperature. The
plate was removed, turned upside down and shaken to remove all of the
coating solution in the sink. 200 pL of Blotto (10 g of non-fat dry milk per L
of
TBS) was then added to each well, covered and placed in a refrigerator at 5 C
overnight. The plate was then removed and washed using a Molecular
Devices Skan Washer 400 to remove all of the Blotto solution. The washing
solution used was TTBS (0.5 ml of tweenTm-20 /L of TBS) with a washing
program of 3 cycles of soaking, washing, and rinsing. Various dilutions of
goat
serum in Blotto were made from which 100p1 was added to the microplate
wells. The plate was covered and placed on a rotary shaker for 1 hour at
room temperature, it was removed and washed using TTBS as described
above. 100 pL of anti-goat IgG-horse radish peroxidase conjugate (Sigma)
diluted 5000 times in Blotto was then added to each microplate well. The plate
was covered and incubated at room temperature for 1 hour. The plate was
washed for the final time and the substrate was added. 100 pL of TMB (Tetra-
methly benzidine, Sigma) was added to each well. The plate was covered and
incubated at room temperature for 30 minutes. The reaction was stopped
using 50 pL of 0.5 M sulfuric acid. The plate was immediately read at 450 nm
with subtraction of 630 nm on a Molecular Devices Spectra Max 340P0
reader.
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REPLACEMENT SHEET
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[00118] The polyclonal antibody produced with 5WS22E1 cells grown on
the defined medium had low avidity and was not studied further. The antibody
from the 2% MEA medium had acceptable avidity but unacceptable cross-
reactivity. The polyclonal antibody to the cells cultured on irradiated
needles
was used in all further studies. A 4000 fold dilution from the latter serum
with
a 5000 dilution of the secondary antibody was determined to be optimal for
tests with 5WS22E1 cells, allowing a preliminary estimate of the sensitivity
of
the assay to be made. Tests with this and the other conifer endophytes were
done using cell weights from 15 to 240 ng over a 5 fold range in antibody
concentration. Using a serum dilution of 4000, response to 15, 30, 60, 120
and 240 ng cells of the phyloplane species and 60, 100 and 500 ng freeze
dried white spruce needles was determined. Powdered freeze-dried white
spruce needles (500 ng/well) were then spiked with additions of 5WS22E1
cells over the above range.
Needle analyses for 5WS22E1
Plating method
[00119] At the time of sampling, average tree height was 12.9 cm.
Needles from each tree were carefully removed radiating out from the
inoculation point, placed in sterile plastic bags and immediately frozen for
transport to the laboratory. Each bag was taken from the freezer and
approximately 20 needles removed. Each needle was surfaced-disinfested by
dipping in 70% ethanol for 1 min, rinsing in sterile distilled water for 1
min, and
blotted dry on sterile tissue. This was placed in a sterile Petri dish and cut
into
2 segments and the needle half that was attached to the stem plated on 2%
malt extract agar. Plates were incubated at 18 C for 6 weeks and were
inspected regularly by microscopy for 5WS22E1 growth (Miller et al. 2002).
ELISA
[00120] The remaining needles were freeze-dried. Approximately 20
needles from each frozen needle sample were removed, ground to a fine
powder in a vial using a SpexTm-Certiprep grinder-mixer (model 5100) and 10
mg weighted out. One mL of TBS (0.8 g/L NaCI, 0.2 g/L KCI, 1.89 g/L Tris-
CA 02793664 2012-10-23
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NCI, and 1.57 g/L Tris base) was added to each vial and placed on the vortex
until completely mixed (approx. 1 min). The samples were assigned codes
unrelated to the tree codes and randomized to ensure that samples from
individual trees were analyzed across many plates. All were diluted in 0.1 M
carbonate buffer pH 9.6 (Sigma) coating buffer to concentrations of 100 and
500 ng of needles per 100 pL well, and pipetted in duplicate onto 96 well
microplate. The remaining steps in the analysis were as described above. In
each trial, 60 ng of 5WS22E1 cells were used as a positive control to assess
the performance of the assay; relative standard deviation of the net value of
25 representative experiments was 8.7 %. Unless an acceptable positive
control result was obtained, the results from individual plates were re-done.
Samples with high absorbance values in both 10Ong and 500ng tests were
rejected as indicating dilution problems. Results were scored as positive
when absorbance of the 500 ng sample was greater than the lowest
absorbance value above one (1.000) plus the mean absorbance value of 30
ng of the target endophyte on that plate.
Chemical analyses
[00121] Rugulosin
of purity > 95% was used for standards. The
presence of rugulosin was then to be determined in a sub-sample of 113
randomly-selected trees of the 330 trees determined to be endophyte positive
by antibody. A 100 mg sample of freeze-dried needles was ground to a fine
power as describe above and extracted with 10 mL of ice cold petroleum
ether by stirring for 45 min under conditions of low light. The flask was kept
on
ice during the extracting and was cover with aluminium foil to prevent
degradation by light. The suspension was filtered by suction and discarded.
The needles were returned to the flask and extracted with 10 mL of
chloroform for 45 min as above for petroleum ether. The new suspension was
then filtered by suction and retained while the needles were discarded. The
chloroform extract was washed with 10 mL of 5 `)/0 NaHCO3 in a separatory
funnel. This first chloroform layer was then discarded, the pH was acidified
to
CA 02793664 2012-10-23
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pH 3 using 1 N HCI, and a new 10 mL of chloroform was added to the
separatory funnel and extracted. The chloroform was removed and dried in an
amber vial under a gentle stream of nitrogen.
[00122] The dried extracts were re-dissolved in 50 pL of acetonitrile,
10
pL was removed and injected into an 1100 series Agilent Technologies
HPLC-DAD, using a Synergi Max RP 80A, 250 x 4.6 column (Phenomonex)
and a gradient method adapted from Frisvad (1987). The gradient started at
90% water with 0.05% TFA and 10% acetonitrile and changed to 10% water
with 0.05% TFA and 90% acetonitrile over the 20 min run. Samples were
analysed at 389 nm, the maximum UV/VIS absorption for rugulosin and peak
identity was confirmed by full spectrum data from the diode array detector.
The limit of quantification was 150 ng/g freeze dried powdered material;
recoveries from spiked needles averaged 75%.
[00123] Statistical analyses were done using SYSTAT v. 10.2 (Point
Richmond, CA).
RESULTS
Inoculation and ELISA Development
[00124] Under the conditions described, the limit of quantification for
the
target endophyte 5WS22E1 was between 30 and 60 ng cells per well; the limit
of detection was 30 ng. Over a concentration range of one log, the antibody
demonstrated a linear response to 60 ng of target endophyte (Fig. 1; results
of
triplicate experiments presented). Relative cross-reactivity to 15, 30, 60,
120
and 240 ng cells of the phyloplane species was moderate (8%). Over the
same range, there was slightly greater cross-reactivity to the cells of the
two
white spruce endophytes tested (-15%), one of which produced rugulosin.
Even at 240 ng cells, the response was below the 1 absorbance unit threshold
used. The response of the polyclonal to the above range of the non-target
fungal cells was not linear across a range of antibody concentrations. The
response to 60, 100 and 500 ng of white spruce cells again across a range of
CA 02793664 2012-10-23
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antibody concentrations was moderate (-6%) and also non-linear. A
comparison of the response to 60 ng of non-target cells to the target
endophyte is given in Fig. 1; average relative standard deviation of
replicates
included in these data was 6.3%.
[00125] Quantification
of the target endophyte was not affected by the
presence of larger amounts of powdered freeze dried needles (-2-18 x). By
ANOVA with Fishers LSD test, the response for 500 ng needle material plus
30 ng target endophyte was significantly greater than that for the 15ng
combination (p = 0.008). The value for 30 and 60 ng were not significantly
different (p = 0.312) indicating that the limit of quantification was between
these values in the presence of needle material. All remaining p values
between endophyte needle cell additions were > 0.003. Absorbance values
for 30, 60, 120 and 240 ng target endophyte plus needle material and the
fungus alone were highly correlated r = 0.951 (p > 0.000) indicating that the
presence of the needle did not affect the linearity of the assay (Fig. 2;
results
of triplicate experiments presented).
Needle and chemical analyses
[00126] Of the 1235
trees tested, only 40 were clearly positive for
5WS22E1 by plating analysis, i.e. the fungus grew from the cut end of >15/20
of the needles. The majority of the ca. 25, 000 surface-disinfested needle
segments exhibited the growth of non-endophyte fungi comprising those
previously observed (Miller et al. 2002). As before, the colonies of these
taxa
typically arose from the sides of the needles rather than the cut ends.
[00127] When the same
samples were analyzed by the antibody
method, 330 or 27% were positive. All of the samples where the fungus was
seen in culture were positive by the antibody assay. Of the 113 samples
tested for rugulosin by HPLC from the 330 antibody positive needles, 101
(90%) were positive at the limit of quantification. The range of
concentrations
found was 0.15 to 24.8 pg/g needle. The distribution of values (assuming half
CA 02793664 2012-10-23
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the detection limit for the non-detects) is shown on Fig. 3). The Geometric
Mean needle rugulosin concentration was 1.02 pg/g.
[00128] Mean frozen weight of 100 representative needles was 2.6
mg/needle. The freeze-dried weight was 1.08 mg/needle.
[00129] The polyclonal assay developed for the target endophyte
5WS22E1 was of comparable sensitivity to similar assays for grass
endophytes (Gwinn et al. 1991; Johnson et al. 1982; Reddick & Collins 1998).
This was achieved despite the greater difficulties of the conifer needle
matrix
compared to grass leaves. Cuttings of the latter can be placed directly in
microplates whereas the tough, hydrophobic conifer needles must be ground
to expose the fungal cells to the antibody. Cross-reactivity to the
potentially
competing fungi was acceptable (Fig. 1). Epiphytic biomass is typically low in
young needles (Carroll 1979) and is comprised mostly of fungi (Swisher &
Carroll 1980). Based on an extrapolation of our data on their data (Swisher &
Carroll 1980; their Table 2), the fungal epiphytic biomass on these 19 month
old needles would have <3 pg/g. This means the presence of such fungi in
our needle samples had no effect on the antibody response in these assays.
Additionally, relatively large amounts of powdered white spruce needles
compared to fungal cells did not affect the reliable detection of 30-60 ng
cells
of target endophyte (Fig. 2).
[00130] Most (90%) of the needles shown to be endophyte positive by
the antibody method contained rugulosin concentrations above the detection
limit. This provides additional evidence of the reliability of the antibody
method. The mean (1 pg/g), range and distribution of rugulosin concentrations
in these needles (Fig. 3) were similar to that found in growth chamber-grown
seedlings (Miller et al. 2002). The analytical method used in the present
study
included examination of the full scan UV spectrum of the rugulosin peak. This
provides additional confirmation of the presence of this compound compared
to the previous HPLC UV and TLC analyses (Miller et al. 2002).
[00131] The analyses were done using replicate 500 ng sub-samples
obtained from a 20 needle sample. The conservative assumption used in the
CA 02793664 2012-10-23
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scoring of the 330 positive seedlings was that they contained the limit of
quantification. Using this conservative assumption, each needle would contain
60 pg endophyte biomass per g needle or -6%. For comparison, Swisher and
Carroll (1980) report that 1-4 year old Douglas fir needles have -10 pg
epiphyte biomass per g needle. This was mainly comprised of fungi but
including algae and bacteria in older needles. This measurement enables
another comparison to be made: the amount of rugulosin per weight of fungal
cells.
[00132] Several studies have been made of the production of
mycotoxins in living plants using culture and ergosterol to assess fungal
biomass. A representative example is a study of deoxynivalenol in
experimentally-inoculated corn pre-harvest with corresponding measurements
of ergosterol and viable fungi among other data (Miller et al. 1983). Using
the
ergosterol-fungal biomass conversion discussed in Gessner & Newell (2002),
it is possible to estimate that in planta dexoynivalenol concentration
corresponded to - 3% of the fungal biomass. Using the mean rugulosin
concentration, the ratio in this case was -2%.
[00133] In summary, the polyclonal assay developed for the target
endophyte 5WS22E1 reliably detected the fungus in 500 ng sub-samples of
colonized needles. Nineteen months post-inoculation, rates of colonization
detected were high. Analysis showed most colonized needles (90%)
contained detectable concentrations of the 5WS22E1 anti-insectan compound
rugulosin.
Example 3
Fungal Collections
[00134] From ca. 12 sites in New Brunswick, Nova Scotia, Quebec and
Maine, branches were collected from superior trees of white spruce, red
spruce and balsam fir in various stands, primarily in relatively undisturbed
natural forest stands but also in 20-30 year old plantations. This was done
from 1985-2005. Branches were collected either directly in the field or from
CA 02793664 2012-10-23
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branches of trees which had been grafted from field selections and grown in a
clone bank plantation in Sussex, New Brunswick. From the branches, needles
were surface sterilized and plated to general procedures developed by Carroll
and colleagues (e.g. Can J Bot 56:3034). Briefly, typically 20 healthy needles
were harvested from each branch. These were surface sterilized by
immersion in 70% ethanol, followed by 6% sodium hypochlorite for 10 min
followed by rinsing in sterile de-ionized water, blotted dry on sterile tissue
and
plated on 2% malt extract agar. Plates were incubated at 18 C for -6 weeks.
The purpose of this surface disinfection was to eliminate phylloplane fungi
that
obscure the slow-growing needle endophytes (Can. J Bot 56:3034;
Mycological Research 93:508). All needle segments were inspected with a
stereo microscope. Colonies not obviously Cladosporium or Altemaria were
examined under high power for endophyte diagnosis.
[00135] Slow growing cultures were transferred to 2% malt agar slants
or culture bottles, incubated at 16-18 C, sealed, inventoried for culture
appearance and collection details and stored at 5 C. The several thousand
strains that were collected, were sorted by location, site and colony
morphology and a random selection taken out for further study.
[00136]
Screening fungal collection for anti-insectan toxins
[00137] Selected isolates were grown either in Glaxo bottles or other
vessels that allow cultures with a large surface area to volume ratio with
lower
oxygen tension. The medium used was 2% malt extract in de-ionized water.
Cultures were inoculated by macerating the 2% malt extract agar slant in
sterile de-ionized water under asceptic conditions and adding the resulting
suspension 5 % v/v either directly for smaller culture vessels (Mycological
Research 93:508) or into 250 mL Erlemeyer flasks containing 2% malt in de-
ionized water for 2 weeks at 25 C. This in turn was macerated and inoculated
into Glaxo and Roux bottles and incubated for 6-8 weeks at 16-18 C.
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REPLACEMENT SHEET - 40 -
[00138] The resulting cultures were filtered. The mycelium were frozen
and freeze dried. The culture filtrates were extracted with ethyl acetate and
examined for evidence of metabolite production by thin layer chromatography,
NMR and High Performance Liquid Chromatography with double diode array
detector. Based on this evidence, the extracts were screened for dietary
toxicity to spruce budworm larvae. The principal anti-insectan toxins were
thus
isolated and identified using standard methods of organic chemistry including,
but not limited to preparative TLC, column chromatography, NMR and high
resolution mass spectroscopy.
Identification of strains
[00139] Colony morphological information for 5WS22E1 and SWS11I1
the two well studied strains is as follows: Strains were grown on 2% malt agar
at 25 C in the dark. 5WS22E1 grew at 0.5 mm day-1. The mycelia were
mainly submerged, the colony was reddish-brown with a reddish soluble
pigment; reverse was brown becoming red-brown in age. The mycelia were
dark brown with roughened thick walls 1-2 1.tm in diameter, sepate, with
occasional branches arising at right angles from the mycelia. 5WS1lIl grew
at 0.4 mm day-1. The colony and reverse were olive-brown with no soluble
pigment and the mycelia were both submerged and aerial. The mycelia were
olive brown with roughened walls 1-2 ,rn in diameter, sepate, with no
branching (Mycological Research 106:471).
[00140] Molecular characterization of the five strains of interest has
been
done using the primers of Glass & Donaldson (1995) which are the
recognized standard approach for filamentous Ascomycetes at present. Cells
of the endophytes were grown in liquid culture, filtered and washed and then
the DNA was extracted using the Ultraclean microbial DNA isolation kit (Mo
Bio Laboratories, #12224-250) and the resulting sequences examined in
public databases for related sequences. At the time of writing, none of the
strains have previously been deposited in GenBankTM. Based on sequence
CA 02793664 2012-10-23
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similarity, strains 5WS22E1 and 5WS11I1 are provisionally species of
Phialocephala which includes species that are endophytic on spruce. The
third candidate white spruce endophyte was most similar to an endophyte
isolated from Norway spruce. The remaining strains, 06-486D and 06-485A
matched most closely to unnamed fungal species isolated from spruce trees.
Identification of effective strains
[00141] Effective strains are those that (a) produce anti-insectan
compounds toxic to the spruce budworms in vitro, (b) colonize white spruce
seedlings, (c) produce their toxin(s) in planta (d) insects consuming
endophyte-colonized needles show reduced growth rates.
a) Insect tests are done by adding pure compound to synthetic diet.
[00142] Spruce budworm (Choristoneura fumiferana) larvae were
obtained from the colony at the Natural Resources Canada, Canadian Forest
Service Laboratory in Sault Ste. Marie, Ontario and stored at 5 C. For each
test, second instar larvae are put in creamer cups containing approximately
15 ml of artificial diet which had been prepared the day before and allowed to
set overnight. The diet used is based on McMorran (1965) as modified by
Forestry Canada. The cups are placed in a growth chamber at 22 C, 55%
RH with 16h light/day until they reach 4-5 instar as estimated by visually
assessment. Batches of diet are prepared and suitable portions were
measured out for addition of extracts, fractions or pure compounds. After 4
days on the test diet, the budworms were frozen, weighed and measured. The
data was analyzed for dry weight reductions in comparison to controls and for
changes in the distribution of insects at different instars in comparison to
controls.
[00143] For spruce budworm, a preliminary test indicated that the
effective approximate concentration of rugulosin for growth limitation was 10
pM (Calhoun et al. 1992). Additional tests produced a similar value of 25 pM
rugulosin with an associated p value of 0.027 pM for weight reduction (see
Figure 8).
CA 02793664 2012-10-23
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[00144] Strains
5W522E1, 5WS11I1, 05-37A, 06-486D, 06-485A are
active in similar in vitro tests of extracts.
(b) Colonization of white spruce seedlings
[00145]
Colonization of seedlings after experimental inoculation has
been done for 5WS22E1 and assessed by presence by colony morphology, a
positive antibody test and analysis of toxin in planta (Mycological Research
106:471, Mycologia 97:770). The persistence of colonization producing
effective concentrations of rugulosin in the field for years of
5WS22E1 has
been demonstrated.
ELISA Assays
[00146] Antibody
production was done using cells of 5WS22E1 grown
on irradiated, young uninoculated white spruce needles. The needles were
irradiated with 25kGy (MDS Nordion, Montreal, PQ) and 200 mg was placed
in a sterile glass Petri dish containing a filter paper followed by the
addition of
1 mL sterile water. After 24 h, the culture was inoculated with a small piece
of
culture taken from the leading edge of a 2% malt extract agar plate. At the
end of the incubation period, the cells growing on the needles were carefully
scraped off with a scalpel and freeze dried.
[00147] Polyclonal
antibody production was performed in goats at
Cedarlane Laboratories Limited, Hornby, Ontario. This laboratory meets the
requirements of the Canadian Council on Animal Care. Freeze-dried cells
from each medium were ground up and each diluted in sterile PBS to a
concentration of 20 mg/mL for the antigen solution. 0.5 mL of this solution
was
emulsified with 0.5 mL of complete Freund's adjuvant (Brenntag Biosector,
Denmark) for the primary immunization. 0.5 mL of incomplete adjuvant was
used for the subsequent boosts. A pre-immune sample was obtained from the
jugular vein of each goat using a needle and vacutainer before the primary
immunization. Each goat was then injected using a 21 gauge needle
intramuscularly in the hind quarter at 4 different sites with 0.25mL of the
emulsified antigen solution per injection site. After 28 days the goat
received
CA 02793664 2012-10-23
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its first boost as described above, its second boost at day 53 and a test
bleed
was taken at day 66.
[00148] The
antibodies produced from the 3 different goats were tested
to determine their avidity and cross reactivity with powdered, freeze-dried
young uninoculated spruce needle cells, as well as cells of the most common
needle phylloplane fungi isolated from these needles (Altemaria altemata,
Phoma herbarum, Cladosporium cladosporioides and Aspergillus fumigatus),
and other white spruce conifer endophytes: 5WS11I1 (DAOM 229535;
vermiculin producer) and 5WS331L1 (a rugulosin-producer). In addition, a
number of balsam fir endophytes were tested. In the case of the needle
endophytes, cells were produced on irradiated needles as above. The
phylloplane species tested were grown in shake culture using a maltose,
yeast extract, peptone medium, filtered, washed and freeze dried as above.
This method was shown to be suitable for antigen production by such fungi in
unrelated studies. Cells were ground to a fine powder in small mortar and
carefully weighed. Suspensions of known concentration were made in TBS
(0.8 g/L NaCI, 0.2 g/L KCI, 1.89 g/L Tris-HCI, and 1.57 g/L Tris base) in
vials
and vortexed.
[00149] Avidity and
cross reactivity experiments were conducted on sera
from the goats treated with the various immunogens in a similar fashion, first
optimizing cell additions/serum dilutions, and then conducting cross-
reactivity
experiments. As needed, aliquots of cells were diluted in 0.1 M carbonate
buffer pH 9.6 (Sigma) coating buffer to defined concentrations and pipetted
into 96 well Nunc brand microplates. The plate was covered with an acetate-
sealing sheet and placed on a rotary shaker for 4 h at room temperature. The
plate was removed, turned upside down and shaken to remove all of the
coating solution in the sink. 200 pL of Blotto (10 g of non-fat dry milk per L
of
TBS) was then added to each well, covered and placed in a refrigerator at 5 C
overnight. The plate was then removed and washed using a Molecular
Devices Skan Washer 400 to remove all of the Blotto solution. The washing
solution used was TTBS (0.5 ml of tween-20 /L of TBS) with a washing
CA 02793664 2012-10-23
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program of 3 cycles of soaking, washing, and rinsing. Various dilutions of
goat
serum in Blotto were made from which 100p1 was added to the microplate
wells. The plate was covered and placed on a rotary shaker for 1 hour at
room temperature, and then removed and washed using TTBS as described
above. 100 pL of anti-goat IgG-horse radish peroxidase conjugate (Sigma)
diluted 5000 times in Blotto was then added to each microplate well. The plate
was covered and incubated at room temperature for 1 hour. The plate was
washed for the final time and the substrate was added. 100 pL of TMB (Tetra-
methyl benzidine, Sigma) was added to each well. The plate was covered and
incubated at room temperature for 30 minutes. The reaction was stopped
using 50 pL of 0.5 M sulfuric acid. The plate was immediately read at 450 nm
with subtraction of 630 nm on a Molecular Devices Spectra Max 340PC
reader.
[00150] The polyclonal antibody produced with 5W522E1 cells grown on
the defined medium had low avidity and was not studied further. The antibody
from the 2% MEA medium had acceptable avidity but unacceptable cross-
reactivity. The polyclonal antibody to the cells cultured on irradiated
needles
was used in all further studies. A 4000 fold dilution from the latter serum
with
a 5000 dilution of the secondary antibody was determined to be optimal for
tests with 5W522E1 cells, allowing a preliminary estimate of the sensitivity
of
the assay to be made. Tests with this and the other conifer endophytes were
done using cell weights from 15 to 240 ng over a 5 fold range in antibody
concentration. Using a serum dilution of 4000, response to 15, 30, 60, 120
and 240 ng cells of the phyloplane species and 60, 100 and 500 ng freeze
dried white spruce needles was determined. Powdered freeze-dried white
spruce needles (500 ng/well) were then spiked with additions of 5W522E1
cells over the above range.
(c) Produce their toxin(s) in plants
Rugulosin analysis
[00151] Typically, a 100 mg sample of freeze-dried needles was ground
to a fine power as describe above and extracted with 10 mL of ice cold
CA 02793664 2012-10-23
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petroleum ether by stirring for 45 min under conditions of low light. The
flask
was kept on ice during the extracting and was covered with aluminum foil to
prevent degradation by light. The suspension was filtered by suction and
discarded. The needles were returned to the flask and extracted with 10 mL of
chloroform for 45 min as above for petroleum ether. The new suspension was
then filtered by suction and retained while the needles were discarded. The
chloroform extract was washed with 10 mL of 5 A) NaHCO3 in a separatory
funnel. This first chloroform layer was then discarded, the pH was acidified
to
pH 3 using 1 N HCl, and a new 10 mL dilquot of chloroform was added to the
separatory funnel and extracted. The chloroform was removed and dried in an
amber vial under a gentle stream of nitrogen.
[00152] The dried extracts were re-dissolved in 50 pL of
acetonitrileand
10 pL was removed and injected into an 1100 series Agilent Technologies
HPLC-DAD, using a Synergi Max RP 80A, 250 x 4.6 column (Phenomonex)
and a gradient method adapted from Frisvad J.C., J. Chromatogr (1987)
392:333-347. The gradient started at 90% water with 0.05% TFA and 10%
acetonitrile and changed to 10% water with 0.05% TFA and 90% acetonitrile
over the 20 min run. Samples were analysed at 389 nm, the maximum
UV/VIS absorption for rugulosin and peak identity was confirmed by full
spectrum data from the diode array detector (Figure 7). The limit of
quantification was 150 ng/g freeze dried powdered material; recoveries from
spiked needles averaged 75%.
Vermiculin Analysis
[00153] Colonization by 5WS11I1 after experimental inoculation was
demonstrated by colony morphology (Mycological Research 106:471) by a
positive antibody test and by the presence of the toxin in planta. Antibody
development was done the same way as above.
[00154] The principal 5WS11I1 toxin, vermiculin, was determined as
follows. Ten milliliters of ice-cold petroleum ether was added to each sample
of ground needles and left to extract for 45mins to an hour on ice while
agitated on a magnetic stir plate. The solutions were then filtered with
CA 02793664 2012-10-23
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Whatman #1 filter paper and a Buchner funnel. Ten milliliters of ethyl acetate
was added to the needles and filter paper. The solutions were left to extract
for 45 minutes to an hour while agitated on a magnetic stir plate and again
filtered through Whatman #1 filter paper on a Buchner funnel. The filtrate was
collected and dried under a gentle stream of nitrogen. Approximately one
milliliter of acetonitrile was added to the dried extracts and the subsequent
solution was vortexed, filtered through a 0.22pm or 0.45pm Acrodisk filter,
redried under nitrogen and redissolved in a small amount (100-300 pl) of
acetonitrile. These extracts were then injected on the HPLC. The vermiculin
peak was detected in the UV chromatogram at 224nm, its UV max in the full
scan spectrum.
[00155] Tests for the isolated vermiculin producing endophyte were
described in Miller JD, Mackenzie S, Foto M, Adams GW, Findlay JA (2002)
Needles of white spruce inoculated with rugulosin-producing endophytes
contain rugulosin reducing spruce growth rate. Mycological Research
106:471-479.
(d) insects consuming endophyte-colonized needles show reduced
growth rates
[00156] The test system used to assess 5WS22E1 and 5WS11I1
(Mycological Research 106:471) was adapted from that of Thomas, A.W.,
U.S. Dept. of Agriculture Gen Tech Rpt NE-85, (1983) PP 47-48 to compare
spruce budworm performance on foliage of different ages and tree species.
The system comprised of 4 ml tapered plastic sample cups with caps each
drilled through the center with a 0.5 mm hole and a piece of OasisTM foam cut
to the size of the narrow base of the vials (10 mm diameter x 15 mm). Just
before use, 0.5 ml sterile water was added. This permitted individual needles
to be held vertically and exposed to a single spruce budworm with the base of
the needle in contact with moisture. The needle was taken out of the freezer
and inserted in the septum just before the budworm was added. The smooth
surface of the septum allowed uneaten portions of the needle to be collected.
The vials were held upright in groups of 30 in wood holders.
CA 02793664 2012-10-23
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[00157] Second instar spruce budworm were were placed in vials
containing artificial diet (McMorran, 1965). They were held in growth
chambers at 22 C, 55% RH with 16h light/day for 1-2 days until they reached
a head capsule width of 0.4 0.1 mm (3rd instar). This is a stage at which
they
will consume succulent needles. Batches of 60-70 larvae were combined and
gently mixed by hand to randomize the animals from their original growth vial.
A single budworm was then placed in each vial. From a well mixed pool of
frozen needles, 100 of a similar size and weight, collected around the
inoculation point of the test seedling plus 100 control needles per genotype
were tested. Typically 600 insects were tested at a time. One control plant
genotype was tested 4 times. Each vial was labeled so as not to be indicative
of the origin of the needle. Vials were placed in a controlled environment
chamber (22 C, 55% RH, 16h day) for 48 h at which time the wood holders
were placed in a freezer. The amount of unconsumed needle, head capsule
width and larval frozen weights were measured. Budworm and residual
needle weights and budworm head capsule widths were determined.
[00158] Tests have been done for tree endophyte 5WS22E1 in both 2
and 3 year old trees on the growth of diet raised disease-free second instar
spruce budworm larvae as follows: A set number of budworm were placed by
hand on lateral branches with buds and then covered with a mesh screen on
both endophyte-positive and negative trees. Temperature recorders were
placed in the holding area and the progress monitored until the budworm was
not greater than sixth instar. At termination, the insects were collected,
frozen
for subsequent determinations of head capsule width and frozen weight.
Samples were collected for toxin analysis to ensure that there was no
misclassification of the trees as to their endophyte status. The weight of the
budworm on the infected trees was significantly reduced compared to those
on the control trees.
Example 4
Detection of endophyte 5WS11I1
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[00159] The polyclonal antibody used for detection of endophyte
5WS11I1 was prepared as described above.
[00160] Under the analysis conditions described above, the limit of
quantification and limit of detection for the target endophyte 5WS11I1 were
both 30 ng cells per well. Over a concentration range of one log, the antibody
demonstrated a linear response to 60 ng of target endophyte (Fig. 1; results
of
triplicate experiments presented). Relative cross-reactivity to 15, 30, 60,
120
and 240 ng cells of the phyloplane species was moderate (>10%). Over the
same range, there was slightly greater cross-reactivity to the cells of the
two
white spruce endophytes tested (-5%). Even at 240 ng cells, the response
was the 1 absorbance unit threshold used. The response of the polyclonal
to the above range of the non-target fungal cells was not linear across a
range
of antibody concentrations. The response to 60, 100 and 500 ng of white
spruce cells again across a range of antibody concentrations was modest
(>10%) and also non-linear. A comparison of the response to 60 ng of non-
target cells to the target endophyte is given in Fig. 1; average relative
standard deviation of replicates included in these data was 6%.
[00161] Figure 4 shows that quantification of the target endophyte was
not affected by the presence of larger amounts of powdered freeze dried
needles (-2-18 x). By ANOVA with Fishers LSD test, the response for 500 ng
needle material plus 30 ng target endophyte was significantly greater than
that for the 15ng combination (p = 0.000) as well as all values above. All
remaining p values between endophyte needle cell additions were > 0.003.
Absorbance values for 30, 60, 120 and 240 ng target endophyte plus needle
material and the fungus alone were highly correlated r = 0.973 (p > 0.000)
indicating that the presence of the needle did not affect the linearity of the
assay (Fig. 5; results of triplicate experiments presented).
Example 5
Producing the endophyte inoculum
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[00162] Cultures are inoculated by macerating the 2% malt extract agar
slant in sterile de-ionized water under asceptic conditions and adding the
resulting suspension 5 (Yo v/v into 250 mL Erlemeyer flasks containing 2%
malt extract in de-ionized water. These are incubated for 2 weeks at 25 C on
a shaker (3.81 cm through, 220 rpm). This in turn is macerated and added to
a stirred jar fermentor adapted for growth of filamentous fungi containing 1%
malt extract broth aerated at 0.1 v/v per minute at 280 rpm stirring and 21 C
for 7 days. The cell counts are designed to ensure that each seedling receives
propagules as delivered in the greenhouse at the receptive stage of the
10 plant plant applied under environmental conditions that sustain needle
wetness.
[00163] Provided they are applied during the receptive stage of the
seedling, such inoculations are effective whether the seedlings are lightly
wounded or untouched.
[00164] For 5WS22E1 and 5WS1111, the addition of small amounts (mg
per seedling) of irradiated needles colonized as described above to the soil
in
the flats used for commercial seedling production is effective at creating
indirect contact between the needles and seedlings for inoculating the
seedlings, provided the inoculation and seedlings are at the receptive stage.
Example 6
Method of Inoculation ¨ Seed Stratification
[00165] A method of inoculating seedlings comprises adding cells to
seeds during the stratification process. This is a process whereby tree seed
is
soaked in water prior to germination to imbibe water and prepare for seed
germination. The method involves adding washed toxigenic endophyte cells
produced in fermentation as above i.e. adding fungal cells that have been
harvested from the fermentor, centrifunging and resuspending in sterile water
followed by immediate addition to the seed stratification bags at the green
house. In one embodiment seeds are soaked in water containing inoculum
prior to sowing in the greenhouse during seed stratification.
CA 02793664 2012-10-23
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Example 7
Inoculating with an endophyte using the limited time window
Reproducing infected seedlings
[00166] Seedlings
produced for all the nursery trials were grown using
standard containerized seedling production methods. Each solid wall plastic
container is 726 cm2 with 67 cavities per container. The individual cavities
are
65 cu cm in volume. The trays are filled with a 3:1 mixture of peatmoss and
vermiculite and seeds are sown on top of this media. The seeds are covered
with a thin layer of dolomitic limestone grit and trays are watered lightly
until
saturated. The greenhouse is misted with fine nozzles on an irrigation boom
to keep the surface of the media moist. Seeds germinate within two weeks.
Fertilization with soluble balanced fertilizer begins when side roots begin to
form (3 weeks after sowing). Spruce seedlings will typically be 3 cm in height
at 8 weeks after sowing (see Figure 6).
[00167] Experimental
inoculation by spraying (for 50 seedling
containers) was done by blending 350 ml of fungi cultures with 175 ml of
sterile water for 10 seconds. The resulting solution was added to a sterile
trigger spray bottle. The entire mixture was sprayed evenly over the 50
containers. Pilot-scale operational application was made using a conventional
greenhouse travelling boom sprayer and an injector pump to inject the fungus
culture solution into the irrigation line. The boom was 28ft wide and had 22 T
Jet 11004VH nozzles. The culture was injected using a Dosatron
Injector(Dosatron International Inc. Florida) with an 11 gallon/minute
capacity.
The injection ratio was 1:64 at 35psi. Application was made in the evening so
that the foliage would remain moist for the longest period of time.
Applications
were made on two consecutive evenings with 3 passes made with the boom
over the entire greenhouse each evening. Steps are taken to ensure that
needle wetness is sustained 12h post inoculation without washing the
inoculum off the needles.
[00168] A series of
medium scale tests have been done since 2000 to
determine the optimum time for inoculation. Inoculation has been done using
CA 02793664 2012-10-23
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wounded or unwounded seedlings using cultured cells or cells on irradiated
needles as above. Pooled data from many trials with 5WS22E1 revealed that
there is a period of maximum receptivity regardless of the method of applying
the inoculum (Figure 6), whether tested at 3 months post inoculation or in the
cases where more data exist, at 6 months when detectable colonization
approximately doubles (Mycologia 97:770).
Example 8
Mass Scale Inoculation
[00169] Seedlings on the scale of thousands can be inoculated by hand.
Plants on the order of 30 million seedlings per year cannot be easily
inoculated by hand. The proven method for infecting the needles with the
endophyte, albeit with a low success rate, was by wound inoculation of young
seedlings. Grass endophytes, are transmitted by seed such that methods
applicable to grasses are not applicable to conifer seedling inoculation.
[00170] The data were analyzed as follows. From the stored samples of
needles from 340 trees positive by culture and ELISA, a random selection of
113 ELISA-positive seedlings was analyzed for rugulosin; most (90%)
contained detectable concentrations of rugulosin. The range and distribution
of the rugulosin concentrations was similar to that found in earlier tests
done
in growth chambers (Measurement of a rugulosin-producing endophyte in
white spruce seedlings. Mycologia 159:571-577).
[00171] Because a complete analysis of the spread of the endophyte
could not be done on the field trees, 10 inoculated seedlings that tested
positive in a 2003 inoculation trial and 10 inoculated seedlings that were
negative by ELISA at 3 months were left in pots at the nursery to grow for an
additional year. Each branch was collected separately for analysis by ELISA
for rugulosin. There were a variable number of branches (11 to 20) which
were carefully labelled according to their position on each of the 20 trees
and
all were analyzed for rugulosin (-160 samples). All of the inoculated trees
that
were negative by ELISA were positive after ca. 1 year. This indicates that the
CA 02793664 2012-10-23
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true inoculation success is materially underestimated when analyzed at 3
months. Some rate of false negatives from inoculation trials has been noted
as an issue since the original inoculation studies in 1999-2002. This data
also
gives a better sense of the rate of spread and, as observed for the field
trees,
confirm its persistence. Endophyte and its toxin were shown to be well
distributed between new and old growth branches.
[00172] The analysis demonstrated the existence of rugulosin as well a
rugulosin derivative. This is either a rugulosin-degradation product or a
plant
modified form of rugulosin. In similar situations, it is known that plants
modify
fungal metabolites in vivo to protect their own cells from damage without
affecting the toxicity of the compound.
[00173] The 5WS 22E1 trials have been done involved sequential
needle inoculations and/or sequential cut and spray inoculations, i.e. from 10
mm, 20 mm, 30 mm, 40 mm and 50 mm height or similar germination to
several weeks (see Figure 6). In each trial, fresh inoculum was prepared and
shipped to Sussex since there was evidence that keeping inoculum after
preparation at 5 C resulted in reduced viability with storage time. The
needle
samples were analyzed (-2500 samples). Analysis of the data confirmed that
either inoculations by cut and spray, adding needles colonized by the target
fungus to the seedling containers or spray alone produces positive results.
The three-month colonization rate is highest in the first two treatments but
not
much less in the spray alone tests. On balance in the several studies of this
matter going back to the 1999-2002 period, the success rate by seedling
height declined after a peak between > 3 cm and 4 cm. Since these are
measurements of the seedlings with the highest initial infection rather than
the
total rate, there is little doubt now of that for endophyte 5 WS 22E1.
[00174] The inoculation trials of the second endophyte strain 5WS11I1
were done but just available for analysis in the last quarter of 2004. These
involved the cut and spray and colonized needle inoculation methods. These
were analyzed (700 samples) and notional colonization success rates were
similar to those of 5W522E1 (the species for which there is > 6 years of
CA 02793664 2012-10-23
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experience) i.e. - 1/3rd positive. The effect of seedling age (height) was
similarly confirmed.
Example 9
Spread of Inoculated Strains
[00175] As a necessary condition to using this technology under field
conditions, a test site is needed where the developing inoculated trees can be
repeatedly tested over a 5 year period. Questions include the persistence and
spread of the established endophyte and its toxins and recruitment of other
endophytes.
[00176] It is thought that these endophytes are transmitted in nature
from cast needles from colonized trees. To address this question, young
seedlings would be planted around the colonized developing trees.
[00177] In the spring of 2000, -1200 seedlings were inoculated with
5WS22E1. From all this work, 340 trees were endophyte-colonized. The trees
had been inoculated and cultured at the Sussex Nursery under normal
conditions. They were repotted and kept in the holding yard. In August of
2003, 300-four year old trees were planted with greater spacing than normal.
In July 2004, 5 small seedlings in were planted around 50 5WS22E1 positive
trees. Screens were placed around an additional 50 test treesto collect cast
needles.
[00178] Approximately 300 mg (dry weight) of needles were removed
from each of the one, two and three year old braches as well as two further
branches were tested for the presence of endophyte 5WS22E1 by ELISA and
rugulosin by HPLC. These needle samples were analyzed in 2005. All trees
were positive for the fungus and the toxin through each of the age classes of
needles on the tree. At the respective limits of detection (30 ng for the
endophyte; 150 ng/g for the toxin), 63% of the samples of the individual
branches were positive for the endophyte by ELISA and 89% were positive
for either rugulosin or its degradation product, and only 1 branch was
negative
for both. The concentration of rugulosin was somewhat higher in the field
CA 02793664 2012-10-23
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trees than in previous studies but the trees were older and more established.
The average concentration was right at the effect level for spruce budworm
and was variable between trees but no biases toward either older or newer
branches were found. Cast needles collected in netting around these trees
were tested and no toxin was present.
[00179]
Example 10
Inoculation with vermiculin-producing strain 5WS11 II
[00180] The vermiculin-producing strain 5WS11 I 1 was chosen for the
second candidate strain (Mycological Research 106:47). A polyclonal
antibody was developed for the determination of the fungus. In the spring of
2004, seedlings were inoculated.
[00181] The inoculation trials using the vermiculin-producing endophyte
had a three-month success rate by ELISA of approximately 30% similar to
endophyte 5WS 22E1. From experience it is likely that the 6-8 month success
rate will be much higher, again because the spread of the endophytes is slow.
[00182] Because vermiculin has an unremarkable UV spectrum, the
quantification of this chemical is more difficult than for rugulosin. During
this
work, improvements to the analytical method had to be made as naturally
contaminated samples became available for the first time (the previous
method development experiments were necessarily done with spiked control
needles). This means that some sense of the actual concentration of
vermiculin in naturally-contaminated material was acquired for the first time.
The earlier experiments with spiked needles were necessarily done in
concentration ranges that made sense compared to rugulosin which, in the
event, were a bit too high.
[00183] At three months post inoculation, no vermiculin could be
detected in ELISA-negative samples. From the ELISA-positive seedlings, 30%
contained vermiculin, This demonstrates ¨for the first time- that this toxin
is
produced in vivo and can be one of the endophytes used for inoculation.
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[00184] Essentially
all of the white spruce endophytes isolated in the
present project at the Sussex lab were cultured and analyzed by HPLC for
metabolite production. From these, 30 had interesting profiles based on diode
array detector response (measures UV spectrum of each compound passing
through the detector). The extracts with the highest total amount of compound
were screened using the Oxford assay for anti-yeast toxicity. Although yeasts
are not insects, Saccharomyces cerevisiae is eukaryotic. From these, five
produced reasonably potent compounds. The compounds were demonstrated
to be complex by HPLC Mass Spectroscopy and NMR. One of these, 05-
37A, was the most potent in the yeast assay and the compounds found were
demonstrated to be complex and not related to other endophyte toxins seen
so far. It was grown in a large scale fermentation (5L), extracted and the
metabolites were isolated using a variety of techniques including; prep thin
layer chromatography, prep HPLC and column chromatography. The
compounds were all generally small in molecular weight <350 (233, 237, 309,
221, 154, 218) and range from moderately polar to non polar. The last step
required was further purification to obtain isolated metabolite for complete
structure determination. This strain was grown in culture on a sufficient
scale
to permit the isolation and characterization of its metabolites.
Example 11
Inoculation Rate and Persistence of Fungal Endophytes
[00185] Ten
inoculated seedlings that tested positive in a 2003
inoculation trial and 10 inoculated seedlings that were negative by ELISA at 3
months were left in pots at the nursery to grow for an additional year. Each
branch was collected separately for analysis by ELISA for rugulosin. There
were a variable number of branches (11 to 20) which were carefully labelled
according to their position on each of the 20 trees and all were analyzed for
rugulosin (-160 samples). All of the inoculated trees that were negative by
ELISA were positive after ca. 1 year. This indicates the true inoculation
success is materially underestimated when analyzed at 3 months. The data
also give a better sense of the rate of spread and, as observed for the field
CA 02793664 2012-10-23
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trees, confirm its persistence. It was very useful to demonstrate that
endophyte and its toxin were shown to be well distributed between new and
old growth branches.
Example 12
Isolation of Red spruce Toxigenic Endophytes
[00186] Many toxigenic endophytes that infect white spruce have been
identified and partially sequenced. These include the strains identified in
Table 1. In addition other white spruce and some red spruce endophytes that
produced anti-insectan toxins were identified. The purpose of this goal is to
generate a comprehensive collection of red spruce endophytes and screen
them for anti-insectan metabolites. Approximantely 40 endophytes had been
cultured, fractionated and subjected to preliminary metabolite screening.
Fractions were prepared for budworm and further chemical assays.
[00187] Extracts from the 70 red spruce as well as some white spruce
endophyte strains which had been qualitatively screened for the production of
potentially anti-insectan metabolites AND for which there are DNA sequence
data were incorporated into synthetic diet. These were individually poured
into
small containers (milk cups) and allowed to harden. Second instar spruce
budworms were added together with controls and rugulosin was used as a
positive control.
[00188] Tests using the spruce budworm assay found that several
isolated endophyte strains were toxic. Isolated endophyte strains that were
found toxic using this assay include 06-264A, 06-332A, 08-011D, (06-268A,
07-013D, 01-002A, 06-268A, 03-020B, 04-012A, 06-063D, 06-0730, 02-
0020, 06-094E, 06-219A, 06-264A and 06-255A.
RED SPRUCE Data
TEST 1
HC 52 0.000 (06-264A) 60 0.073 (08-011D)
58 0.026 (06-332A)
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WT 52 0.054
54 0.009 (06-268A)
58 0.057
59 0.008 (07-013D) 62 0.082 ((01-002A)
TEST 2
HC 14 0.058 (04-002G) 9 0.078 (03-020B)
WT 9 0.070
TEST 3
HC 14 0.058
WT 17 0.052 (04-012A) 9 0.071
TEST 4
HC 27 0.039 (06-063D)
WT 27 0.011
28 0.008 (06-073C) 5 0.079 (02-002C)
TEST 5
HC 38 0.015 (06-094E) 45 0.087
43 0.000 (06-219A) 47 0.082 (06-264A)
0.005 (06-255A)
WT 38 0.034
43 0.002
0.016
47 0.042
Example 13
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Isolation of toxigenic endophytes
[00189] The white spruce endophytes were cultured and analyzed by
HPLC for metabolite production. From these, 30 had good profiles based on
diode array detector response (measures UV spectrum of each compound
passing through the detector). The extracts with the highest total amount of
compound were screened using the Oxford assay for anti-yeast toxicity.
Although yeasts are not insects, Saccharomyces cerevisiae is eukaryotic.
From these, five produced potent compounds. The compounds were
demonstrated to be complex by HPLC Mass Spectroscopy and NMR. One of
these, 05-37A, was the most potent in the yeast assay and the compounds
found were demonstrated to be complex and not related to other endophyte
toxins seen so far. It was grown in a large scale fermentation (5L), extracted
and the metabolites were isolated using a variety of techniques including;
preparatory thin layer chromatography, preparatory HPLC and column
chromatography. The compounds were all generally small in molecular
weight <350 (233, 237, 309, 221, 154, 218) and range from moderately polar
to non polar. The last step required was further purification to obtain
isolated
metabolite for complete structure determination. This strain was grown in
culture on a sufficient scale to permit the isolation and characterization of
its
metabolites.
[00190] A total of 62 extracts were tested in the spruce budworm assay
plus a larger number of controls for each set. Of these 11 white spruce
extracts were toxic to spruce budworm (i.e. statistically different from
controls
either for weight reduction (most common), head capsule width or both). For
the red spruce extracts, 21 were toxic to spruce budworm. The DNA
sequence for isolated red spruce toxigenic isolates is listed in SEQ ID NO: 6-
41.
Example 14
Isolation and Inoculation of Pine Tree Toxigenic Endophytes
[00191] Pine needles are collected from pine trees. Slow growing
endophytes are cultured from needles and screened for the presence of
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toxigenic endophytes. Antibodies to candidate toxigenic endophytes are
produced. Candidate toxigenic endophytes are tested in vitro for their effect
on pests, including disease-causing fungi. An inoculum comprising the
toxigenic endophyte is prepared. Pine seedlings are inoculated with the
inoculum during a susceptible time window. Colonization is later confirmed
using a specific antibody test.
CA 02793664 2012-10-23
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- 60 -
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