U.S. patent application number 16/331938 was filed with the patent office on 2020-01-02 for biological control of plant pathogenic microorganisms.
This patent application is currently assigned to The New Zealand Institute for Plant and Food Research Limited. The applicant listed for this patent is The New Zealand Institute for Plant and Food Research Limited. Invention is credited to Philip ELMER, Stephen HOYTE.
Application Number | 20200000100 16/331938 |
Document ID | / |
Family ID | 65806213 |
Filed Date | 2020-01-02 |
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United States Patent
Application |
20200000100 |
Kind Code |
A1 |
ELMER; Philip ; et
al. |
January 2, 2020 |
Biological Control of Plant Pathogenic Microorganisms
Abstract
The present invention relates to the use of an isolated
Aureobasidium pullulans yeast strain YBCA5 as a biological control
agent. Processes and compositions for the biological control of
phytopathogenic bacteria and fungi using YBCA5 are also
provided.
Inventors: |
ELMER; Philip; (Hamilton,
NZ) ; HOYTE; Stephen; (Hamilton, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The New Zealand Institute for Plant and Food Research
Limited |
Auckland |
|
NZ |
|
|
Assignee: |
The New Zealand Institute for Plant
and Food Research Limited
Auckland
NZ
|
Family ID: |
65806213 |
Appl. No.: |
16/331938 |
Filed: |
September 11, 2017 |
PCT Filed: |
September 11, 2017 |
PCT NO: |
PCT/IB2017/055453 |
371 Date: |
March 8, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62393641 |
Sep 12, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12R 1/645 20130101;
A01N 63/30 20200101; A01N 63/30 20200101; A01N 43/16 20130101; A01N
43/82 20130101; A01N 47/38 20130101; A01N 63/22 20200101 |
International
Class: |
A01N 63/04 20060101
A01N063/04; C12R 1/645 20060101 C12R001/645 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 13, 2016 |
NZ |
724271 |
Oct 28, 2016 |
NZ |
725641 |
Claims
1-24. (canceled)
25. A composition comprising Aureobasidium pullulans yeast strain
YBCA5 (CBS Accession # 141880) and an agriculturally acceptable
carrier.
26. The composition of claim 25 wherein YBCA5 is present in the
form of reproductively viable cells.
27. The composition of claim 25, wherein the concentration of YBCA5
viable cells ranges from about 1.times.10.sup.5 to about
1.times.10.sup.11 CFU per gram for solid compositions, and about
1.times.10.sup.7 to about 1.times.10.sup.8 CFU per millilitre for
liquid compositions.
28. The composition of claim 25, wherein the concentration of YBCA5
viable cells ranges from about 2.times.10.sup.7 to about
2.times.10.sup.8 CFU per gram for solid compositions, and about
1.times.10.sup.7 to about 1.times.10.sup.8 CFU per millilitre for
liquid compositions.
29. The composition of claim 25, wherein the concentration of YBCA5
viable cells ranges from about 2.times.10.sup.9 to about
2.times.10.sup.10 CFU per gram for solid compositions, and about
1.times.10.sup.7 to about 1.times.10.sup.8 CFU per millilitre for
liquid compositions.
30. The composition of claim 25, further comprising at least one
agriculturally acceptable adjuvant.
31. A method of controlling Pseudomonas spp. bacteria on a plant or
part thereof, the method comprising contacting the plant or part
thereof with YBCA5, or a composition of claim 25.
32. The method of claim 31, wherein the at least one strain of
Pseudomonas spp. bacteria is a strain of bacteria selected from the
group consisting of P. syringae, P. amygdalia, P. avellanae, P.
caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P.
helianthi, P. lemiae, P. savastanoi, and P. viridiflava, or a
pathovar thereof, or combinations thereof,
33. The method of claim 31, wherein the at least one strain of
Pseudomonas spp. bacteria is P. syringae or a pathovar thereof.
34. The method of claim 31, wherein the at least one strain of
Pseudomonas spp. bacteria is P. syringae pv. actinidiae (Psa).
35. The method of claim 31, wherein the plant or part thereof is an
agriculturally important plant, cultivar thereof, or product
thereof, selected from olive trees, apple trees, pear trees, citrus
fruit trees, banana trees, pineapple trees, peach trees, apricot
trees, cherry trees, walnut trees and hazelnut trees, wherein the
products thereof are olives, apples, pears, citrus fruits, bananas,
pineapples, peaches, apricots, cherries, walnuts and hazelnuts,
respectively.
36. The method of claim 31, wherein the plant or part thereof is an
agriculturally important plant, cultivar thereof, or product
thereof, selected from potato vines, beetroot vines, bean vines,
pea vines, tomato vines, cucumber vines, melon vines, berry vines,
grape vines and kiwifruit vines, and the products thereof are
potatoes, beetroots, beans, peas, tomatoes, cucumbers, melons,
berries, grapes and kiwifruits, respectively.
37. The method of claim 36, wherein the agriculturally important
plant, cultivar thereof, or product thereof, is a kiwifruit vine or
cultivar thereof, and the product is kiwifruit.
38. The method of claim 37, wherein the kiwifruit vine is selected
from the group consisting of species of green-fleshed kiwifruit
(Actinidia chinensis var. deliciosa), golden kiwifruit (A.
chinensis var. chinensis), Chinese egg gooseberry (A. coriacea),
baby kiwifruit (A. arguta), Arctic kiwifruit (A. kolomikta), red
kiwifruit (A. melanandra, A. chinensis var. chinensis), silver vine
(A. polygama), and purple kiwifruit (A. purpurea) or a cultivar
thereof.
39. The method of claim 37, wherein the kiwifruit are selected from
the group consisting of A. chinensis var. deliciosa and A.
chinensis var. chinensis species or a cultivar thereof.
40. The method of claim 37, wherein the kiwifruit is a `Hayward`,
`Hort16A` or `Hongyang` variety cultivar.
41. A method of controlling at least one phytopathogenic fungus on
a plant or part thereof, the method comprising contacting the plant
or part thereof with YBCA5, or a composition of claim 25.
42. A method of claim 41, wherein the phytopathogenic fungus is a
Monilinia spp. Fungus.
43. The method of claim 41, wherein the phytopathogenic fungus is
Monilinia fruticola.
44. The method of claim 41, wherein the phytopathogenic fungus is
selected from the group consisting of Botrytis spp., Colletotrichum
spp., Penicillium spp., Phomopsis spp., Alternaria spp.,
Sclerotinia spp., and Cryptosporiopsis spp. fungus.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to methods of using yeasts
for the biological control of plant pathogenic bacteria and fungi.
In particular, the invention relates to a novel yeast strain having
biological control activity, and to methods of using this strain to
inhibit the survival, growth and/or proliferation of plant
pathogenic bacteria and fungi on fruit or vegetable plants.
BACKGROUND OF THE INVENTION
[0002] Plant disease represents a significant economic cost to
modern agriculture. Current systems of agriculture often require
one or a few crops or plant types to be grown over a large area.
Such an ecologically unbalanced system is susceptible to
disease.
[0003] Traditionally, control of disease causing plant pathogens
such as bacteria and fungi has been carried out using chemical
pesticides. However, the use of chemicals is subject to a number of
disadvantages. Pathogens can and have developed tolerance to
chemicals over time, producing increasingly pesticide resistant
populations. Chemical residues may also pose environmental hazards
as well as raising health concerns. In particular, consumers have
become increasingly concerned about chemical residues on plants and
in food and wine, and their effects on human health and the
environment.
[0004] Biological control represents an alternative means of
controlling plant disease which reduces dependence on chemicals.
Such "natural" methods enjoy greater public acceptance, and may be
more effective and sustainable than chemical control methods.
[0005] Pseudomonas is a genus of Gram-negative, aerobic
gammaproteobacteria, belonging to the family Pseudomonadaceae. The
genus contains 191 validly described species, of which a number are
plant pathogens. Within the genus Pseudomonas spp., P. syringae is
a prolific plant pathogen that exists as over 50 different
pathovars (pv.), many of which demonstrate a high degree of
host-plant specificity. Numerous other Pseudomonas species can also
act as plant pathogens, most notably all of the other members of
the P. syringae subgroup. For example, commercially important
diseases caused by P. syringae pathovars include bacterial blast of
stone fruits, bacterial speck of tomato, and blight in peas.
[0006] Pseudomonas syringae pv. actinidiae (Psa) is a serious
bacterial disease affecting kiwifruit. Psa was first recorded in
New Zealand in early November 2010, and as of 18 Jul. 2013, 75% of
hectares of kiwifruit were on orchards with some Psa infection. The
immediate cost of Psa to the New Zealand kiwifruit industry is
estimated to be between $310 million and $410m from 2013 to 2018,
and more than double that in the long-term for lost
development.
[0007] As with many bacterial plant diseases, control options are
limited. The main solutions currently in use are crop hygiene,
chemical based treatment such as copper-based products, and/or
plant defence elicitors such as acibenzolar-S-methyl
(Actigard/Bion, Syngenta) and antibiotics such as streptomycin
sulphate and kasugamycin. However, severe restrictions have been
placed on the use of these products and time of the growing season
that they can be used (e.g. in New Zealand). In addition, the use
of some of these products are prohibited in some key export regions
e.g., Streptomycin is not permitted for use on horticulture in
Europe.
[0008] It has been estimated that in the 2012 season in New
Zealand, $13 Million was spent on sprays to protect kiwifruit
against Psa. This is the chemical cost alone. Other management
costs have not been factored into this estimate. Outside of New
Zealand, Psa is also a critical issue in Europe (Italy/France),
South America and potentially in China and South Korea.
[0009] Botrytis cinerea and recently identified B. pseudocinerea
are phytopathogenic fungi (telemorph Botryotinia fuckeliana) and
are the causal agents of the grey mold (Botrytis blight) disease.
Some estimates of global crop losses resulting from Botrytis spp.
are on the order of 10-100 billion Euros per year
(http://www.genoscope.cns.fr). Botrytis spp. is also the causative
agent of bunch rot of grapes, and is estimated to cause losses of
$18 million dollars per annum to the New Zealand wine industry
alone. Botrytis spp. control has been by way of fungicides. As with
the use of chemical treatments to control pathogenic bacteria, this
practice is unsustainable because fungicide resistance is
widespread in many vineyards and there is consumer pressure for
reduction in pesticide residue.
[0010] Brown rot on fruit is caused by Monilinia spp. fungi.
Monilinia spp. are pathogens of many economically important crops
in the Family Rosaceae including cherries, plums, peaches,
apricots, strawberries, raspberries, apples and pears. Monilinia
spp. are also pathogens of many flowering plants within the Family
Ericaceae. Damage caused by Monilinia spp. can often cause major
losses to crops and valuable ornamental flowers. The genus
Monilinia contains about thirty described species.
[0011] Importantly, the revenues lost due to the impact of
phytopathogenic fungi represent a mere fraction of the total
economic impact of these pathogens worldwide. As with Botrytis,
control of Monilinia spp. and Sclerotinia spp. on economically
important crops has traditionally been by way of fungicides. Some
estimates consider that the cost of chemical control of Botrytis
spp. alone can reach $780 million for just one crop with disease on
treated plants still resulting in significant production loss
(Genescope, 2002); (Laluk, Kristin and Tesfaye Mengiste; 2010 in
Arabidopsis Book 2010, Vol. 8).
[0012] Accordingly, for a number of economic, health and
environmental sustainability reasons as discussed above, the use of
chemical based treatments, plant defence elicitors and antibiotics
has limitations. Therefore, there is a need for new biological
control solutions, which do not have similar cost, health or
environmental issues to chemical based treatments in order to
provide sustainable management of these diseases.
[0013] It is an object of the invention to provide at least one
yeast biological control agent and/or a composition comprising at
least one yeast biological control agent and/or methods of using
such an agent and/or such a composition for controlling Pseudomonas
spp. bacteria on at least one plant or part thereof, preferably
Pseudomonas syringae pv. actinidiae (Psa); and/or to at least to
provide the public with a useful choice.
SUMMARY OF THE INVENTION
[0014] In one aspect the invention relates to isolated
Aureobasidium pullulans yeast strain YBCA5 (CBS Accession #
141880).
[0015] In another aspect the invention relates to a composition
comprising isolated Aureobasidium pullulans yeast strain YBCA5 (CBS
Accession # 141880) and an agriculturally acceptable carrier.
[0016] In another aspect the invention relates to a composition
consisting essentially of isolated Aureobasidium pullulans yeast
strain YBCA5 (CBS Accession # 141880) and an agriculturally
acceptable carrier.
[0017] In another aspect the invention relates to a method of
controlling Pseudomonas spp. bacteria on a plant or part thereof,
the method comprising contacting the plant or part thereof with
YBCA5, or a composition comprising YBCA5.
[0018] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for controlling Pseudomonas spp.
bacteria on a plant or part thereof.
[0019] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling Pseudomonas spp. bacteria on a plant or part
thereof.
[0020] In another aspect the invention relates to a method for
controlling P. syringae pv. actinidiae (Psa) on a kiwifruit plant
or part thereof, the method comprising contacting the kiwifruit
plant or part thereof with YBCA5, or a composition comprising
YBCA5.
[0021] In another aspect the invention relates to a method for
increasing the yield of a kiwifruit plant infected, or susceptible
to infection with Psa, the method comprising applying YBCA5 or a
composition comprising YBCA5 to the kiwifruit plant or part
thereof, and growing the kiwifruit plant or part thereof.
[0022] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for controlling Psa on a
kiwifruit plant or part thereof.
[0023] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for increasing the yield of a
kiwifruit plant infected, or susceptible to infection with Psa.
[0024] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling Psa on a kiwifruit plant or part thereof.
[0025] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
increasing the yield of a kiwifruit plant infected, or susceptible
to infection with Psa.
[0026] In another aspect the invention relates to a method of
controlling at least one phytopathogenic fungus on a plant or part
thereof, the method comprising contacting the plant or part thereof
with YBCA5, or a composition comprising YBCA5.
[0027] In another aspect the invention relates to a method for
increasing the yield of a fruit or vegetable plant infected with,
or susceptible to infection by a phytopathogenic fungus, the method
comprising applying YBCA5 or a composition comprising YBCA5 to the
fruit or vegetable plant or part thereof YBCA5, and growing the
plant or part thereof.
[0028] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for controlling a phytopathogenic
fungus on a fruit or vegetable plant or part thereof.
[0029] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for increasing the yield of a
fruit or vegetable plant or part thereof infected with, or
susceptible to infection by a phytopathogenic fungus.
[0030] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling at least one phytopathogenic fungus on a plant or part
thereof.
[0031] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling at least one phytopathogenic fungus on a fruit or
vegetable plant or part thereof.
[0032] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
increasing the yield of a fruit or vegetable plant susceptible to
infection by at least one phytopathogenic fungus.
[0033] In another aspect the invention relates to at least one
plant or part thereof treated with YBCA5, or a composition
comprising YBCA5.
[0034] In another aspect the invention relates to at least one
fruit or vegetable plant or part thereof treated with YBCA5, or a
composition comprising YBCA5.
[0035] In another aspect the invention relates to at least one
plant or part thereof treated with YBCA5, or a composition
comprising YBCA5. In some embodiments the plant is a fruit or
vegetable plant or part thereof. In one embodiment the plant is a
kiwifruit vine, a cherry tree or a grape vine.
[0036] While various embodiments of certain aspects of the
invention are set out above, the invention is not limited thereto.
Additional embodiments of the aspects of the invention set out
above are further described in the Detailed Description and set out
in the claims of the application.
[0037] Other aspects and embodiments of the invention may become
apparent from the following description which is given by way of
example only and with reference to the accompanying drawings.
[0038] It is intended that reference to a range of numbers
disclosed herein (for example, 1 to 10) also incorporates reference
to all rational numbers within that range (for example, 1, 1.1, 2,
3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of
rational numbers within that range (for example, 2 to 8, 1.5 to 5.5
and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges
expressly disclosed herein are hereby expressly disclosed. These
are only examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The invention will now be described by way of example only
and with reference to the drawings in which:
[0040] FIG. 1. Psa severity (mean area of leaf necrosis) on potted
`Hayward` seedlings treated with different concentrations of
freshly fermented YBCA5, compared with a water soluble granule
formulation (YBCA5 granule) and inoculated with two doses
(5.times.10.sup.6 per droplet and 2.times.10.sup.6 per 10 ul
droplet) of Psa. Three 10 ul droplets of each dose of Psa were used
per side of the leaf. Treatments were applied eight and one day
(1d) prior to inoculation with Psa on 18 Sep. 2014 and assessed
after 28 days.
[0041] FIG. 2. The effect of different isolates of Aureobasidium
pullulans on the severity of Psa leaf spot lesions on potted
kiwifruit plants (`Hayward`) compared to the untreated (Nil) in
April 2016.
[0042] FIG. 3. Incidence of leaves with Psa necrosis on potted
`Hayward` plants exposed to natural Psa inoculum at Te Puke
Research Orchard and with treatment applied on four occasions over
a 30 day period. Leaf necrosis assessment was carried out 44 days
after the first treatment application.
[0043] FIG. 4. Field testing the efficacy of YBCA5. Nil is observed
incidence of leaf spotting on untreated control plants. Grower Std
is the observed incidence of Psa leaf spotting on plants treated
with Actigard and copper. Low and high refer to the amount of YBCA4
and YBCA5 respectively that was applied to the plants. Field site
was Maketu. The kiwifruit variety was `Hayward`. All treatments
were applied between bud burst and pre-flowering. A total of 5
spray treatments were carried out between 6 and 12 days apart. For
each of FIGS. 4-8, High rate is 2.times.10.sup.7 cells/mL and low
rate is 1.times.10.sup.7.
[0044] FIG. 5. Field testing the efficacy of YBCA5. Nil is observed
incidence of defects on untreated control plants. Grower Std is the
observed incidence of Psa leaf spotting on plants treated with
Actigard and copper. Low and high refer to the amount of YBCA4 and
YBCA5 respectively that applied to the plants. Field site was
Maketu. The kiwifruit variety was `Hayward`. All treatments were
applied between bud burst and pre-flowering. A total of 5 spray
treatments were carried out between 6 and 12 days apart.
[0045] FIG. 6. Field testing the efficacy of YBCA5 showing the mean
severity of leaf necrosis. Kiwifruit variety was `Hayward`. Grower
std. is copper+antibiotic. Treatments were applied from bud burst
to first flowering at two sites in Maketu. 6 treatments (sprays)
were applied in total, each 7-14 days apart.
[0046] FIG. 7. Field testing the efficacy of YBCA5 showing the mean
severity of bud browning. Kiwifruit variety was `Hayward`. Grower
std. is copper+antibiotic. Treatments were applied from bud burst
to first flowering at two sites in Maketu. 6 treatments (sprays)
were applied in total, each 7-14 days apart.
[0047] FIG. 8. Field testing the efficacy of YBCA5 showing the mean
increase in yield (fresh weight/dry matter/fruit/m.sup.2. Kiwifruit
variety was `Hayward`. Grower std. is copper+antibiotic. Treatments
were applied from bud burst to first flowering, once during
flowering and once post fruit set. 7 treatments (sprays) were
applied in total, each 7-14 days apart. In each category: fresh
weight, dry matter, fruit/m.sup.2 (Gold3) and fruit/m.sup.2
(`Hayward`) the bars on the graph from left to right depict nil
treatment, grower standard treatment (copper and antibiotic) and
YBCA5.
[0048] FIG. 9. The effect of YBCA5 on the incidence of Monilinia
fruit rot of cherries (`Sweet Valentine`) compared to the fungicide
iprodione (Rovral.RTM. Aquaflo) in a lab based assay (Assay 1) in
January-February 2016.
[0049] FIG. 10. The effect of YBCA5 on the incidence of Botrytis
spp. fruit rot of cherries (`Sweet Valentine`) compared to the
fungicide iprodione in a lab based assay (Assay 2) in
January-February 2016.
[0050] FIG. 11. The effect of YBCA5 on the incidence of Monilinia
fruit rot of cherries (`Sweet Valentine`) compared to the fungicide
captan in a lab based assay (Assay 3) in February-March 2016.
[0051] FIG. 12. The effect of YBCA5 on the incidence of Botrytis
spp. fruit rot of cherries (`Sweet Valentine`) compared to the
fungicide captan in a lab based assay (Assay 2) in February-March
2016.
[0052] FIG. 13. The effect of YBCA5 on the severity of Botrytis
spp. rot of table grapes (`Autumn King`) compared to the fungicide
captan in a lab based assay (Assay 5) in October-November 2015.
Data is the mean of two Botrytis spp. isolates.
[0053] FIG. 14. The effect of YBCA5 on the severity of kiwifruit
rot due to phytopathogenic fungal infection post-harvest. Lesion
size (mm) of wounded `Hongyang` kiwifruit after inoculation with
Alternaria spp, Botrytis spp., Colletotrichum spp., Penicillium
spp. or Phomopsis spp. and 6 days incubation. LSD (5%)=3.482, P
fr=<.001.
[0054] FIG. 15. The effect of YBCA5 on the severity of kiwifruit
rot due to phytopathogenic fungal infection post-harvest. Lesion
size (mm) of wounded `Hongyang` kiwifruit after inoculation with
Cryptosporiopsis spp. and 7 days incubation. LSD (5%)=1.945, P
fr=<.001.
DETAILED DESCRIPTION OF THE INVENTION
[0055] Definitions
[0056] The following definitions are presented to better define the
present invention and as a guide for those of ordinary skill in the
art in the practice of the present invention.
[0057] Unless otherwise specified, all technical and scientific
terms used herein are to be understood as having the same meanings
as is understood by one of ordinary skill in the relevant art to
which this disclosure pertains.
[0058] Examples of definitions of common terms in botany,
microbiology, molecular biology and biochemistry can be found in
Biology of Plants, Raven et al. (eds.), W. H. Freeman and Company,
(2005); Plant Physiology, Taiz et al. (eds.), Sinauer Associates,
Incorporated, (2010); Botany: An Introduction to Plant Biology, J.
D. Mauseth, Jones & Bartlett Learning, (2003); Methods for
General and Molecular Microbiology, 3rd Edition, C. A. Reddy, et
al. (eds.), ASM Press, (2008); Encyclopedia of Microbiology, 2nd
ed., Joshua Lederburg, (ed.), Academic Press, (2000); Microbiology
By Cliffs Notes, I. Edward Alcamo, Wiley, (1996); Dictionary of
Microbiology and Molecular Biology, Singleton et al. (2d ed.)
(1994); Biology of Microorganisms 11.sup.th ed., Brock et al.,
Pearson Prentice Hall, (2006); Biodiversity of Fungi: Inventory and
Monitoring Methods, Mueller et al., Academic Press, (2004); Genes
IX, Benjamin Lewin, Jones & Bartlett Publishing, (2007); The
Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell
Science Ltd., (1994); and Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH
Publishers, Inc., (1995).
[0059] It is also believed that practice of the present invention
can be performed using standard botanical, microbiological,
molecular biology and biochemistry protocols and procedures as
known in the art, and as described, for example in Environmental
Microbiology: Methods and Protocols, J. F. T. Spencer et al.,
Humana Press, (2004); Environmental Microbiology, P. D. Sharma,
Alpha Science International, (2005); Environmental Microbiology, J.
R. Leadbetter, Gulf Professional Publishing, (2005) and other
commonly available reference materials relevant in the art to which
this disclosure pertains, and which are all incorporated by
reference herein in their entireties.
[0060] The term "plant" as used herein encompasses whole plants and
all parts of a plant from all stages of a plant lifecycle including
but not limited to vegetative and reproductive cells and tissues,
propagules, seeds, embryos, fruits, shoots, stems, leaves, leaf
sheaths and blades, inflorescences, roots, anthers, ligules,
palisade, mesophyll, epidermis, auricles, palea, lemma and
tillers.
[0061] The term "kiwifruit" is used herein as the common name for
all commercially grown fruit from the genus Actinidia. The most
common kiwifruit is the green-fleshed kiwifruit, from the species
Actinidia chinensis var. deliciosa. Other species that are commonly
eaten include golden kiwifruit (A. chinensis var. chinensis),
Chinese egg gooseberry (A. coriacea), baby kiwifruit (A. arguta),
Arctic kiwifruit (A. kolomikta), red kiwifruit (A. melanandra; A.
chinensis var. chinensis,), silver vine (A. polygama), and purple
kiwifruit (A. purpurea).
[0062] The term "biological control agent" as used herein refers to
agents which act as an antagonist of one or more plant pathogens.
Antagonists may take a number of forms. In one form, the biological
control agent may out-compete the pathogen for available nutrients
and/or space of the host plant. In another form the biological
control agent may render the environment unfavourable for the
pathogen. Accordingly, the antagonist mechanisms include but are
not limited to antibiosis, mycoparasitism, nutrient competition and
physical displacement.
[0063] The terms "control", "controlling", "biocontrol" or
"biological control" are used interchangeably herein to refer to
the reduction of the amount of inoculum or disease-producing
activity of a pathogen accomplished by or through one or more
microorganisms. Generally comprehended is the prevention or
reduction of infection by plant pathogenic bacteria or fungi,
particularly plant pathogenic Pseudomonas spp., Botrytis spp.,
Alternaria spp., Colletotrichum spp., Penicillium spp., Phomopsis
spp., Cryptosporiopsis spp., Monilinia spp., and Sclerotinia spp.,
particularly or inhibition of the rate or extent of such infection,
including any reduction in the survival, growth and/or
proliferation of the bacteria or fungi. Curative treatment is also
contemplated.
[0064] The term "statistically significant" as used herein refers
to the likelihood that a result or relationship is caused by
something other than random chance. A result may be found to be
statistically significant using statistical hypothesis testing as
known and used in the art. Statistical hypothesis testing provides
a "P-value" as known in the art, which represents the probability
that the measured result is due to random chance alone. It is
believed to be generally accepted in the art that levels of
significance of 5% (0.05) or lower are considered to be
statistically significant.
[0065] The term "effective amount" as used herein means an amount
effective to protect against, delay, reduce, stabilise, improve or
treat plant pathogenic bacterial or fungal infection in and/or on a
plant.
[0066] The terms "increasing the yield of a fruit or vegetable
plant" and "increasing the yield of a kiwifruit plant" as used
herein generally comprehends increasing the rate of production of
harvestable fruit and/or kiwifruit, the total number of harvestable
fruit and/or kiwifruit (including due to absolute increase in fruit
and/or kiwifruit numbers or reduction in disease symptoms leading
to increased numbers of saleable fruits), and any increase in size
of individual fruits and/or kiwifruits produced on a fruit or
vegetable plant or kiwifruit plant treated according to the
invention. Increase is generally determined as compared to an
equivalent plant that is untreated with the strain or the
composition of the invention.
[0067] An "agriculturally acceptable adjuvant" as used herein
refers to a compound or material that is generally comprehended in
the art of agriculture as a useful additive in agricultural
formulations or carried out with agricultural treatments.
[0068] An "additional active agent" as used herein means any
compound or material that is capable of contributing to the control
(as defined herein) of plant pathogenic Pseudomonas spp. bacteria
or phytopathogenic fungi Botrytis spp., Alternaria spp.,
Colletotrichum spp., Penicillium spp., Phomopsis spp.,
Cryptosporiopsis spp., Monilinia spp., and Sclerotinia spp. by the
yeasts useful in the present invention, or that is capable of
potentiating the effects of the yeasts useful in this invention in
controlling plant disease caused by plant pathogenic bacteria and
fungi
[0069] A "formulation agent" as used herein refers to any compound
or material that facilitates or optimizes the production, handling,
storage, transport, application and/or persistence of the
composition of, or for use in the invention on plants (as defined
herein), but not limited thereto.
[0070] An "agriculturally acceptable carrier" is used herein as is
generally comprehended in the art. A preferred agriculturally
acceptable carrier is water, but not limited thereto.
[0071] The term "comprising" as used in this specification means
"consisting at least in part of". When interpreting each statement
in this specification that includes the term "comprising", features
other than that or those prefaced by the term may also be present.
Related terms such as "comprise" and "comprises" are to be
interpreted in the same manner.
DETAILED DESCRIPTION OF THE INVENTION
[0072] The present invention relates generally to a novel
Aureobasidium pullulans yeast strain YBCA5 and to compositions
comprising YBCA5 and an agriculturally acceptable carrier. In some
embodiments the compositions also comprise an agriculturally
acceptable adjuvant. The novel strain and compositions of the
invention are useful for the biocontrol of plant disease caused by
plant pathogenic bacteria and phytopathogenic fungi, particularly
Pseudomonas spp. bacteria and Botrytis spp., Sclerotinia spp.,
Alternaria spp., Colletotrichum spp., Penicillium spp., Phomopsis
spp., Cryptosporiopsis spp., and Monilinia spp. fungi. The
invention also relates to methods of controlling phytopathogenic
bacteria and/or fungi selected from the group consisting of
Pseudomonas spp. bacteria, Botrytis spp., Sclerotinia spp.,
Penicillium spp., Colletotrichum spp., Alternaria spp., Phomopsis
spp., Cryptosporiopsis spp., and Monilinia spp. fungi on a plant or
part thereof by contacting the plant or part thereof with
YBCA5.
[0073] The applicants are the first to provide the isolated yeast
strain YBCA5, and compositions comprising YBCA5 and an
agriculturally acceptable carrier that are effective at controlling
Pseudomonas spp. bacteria and phytopathogenic fungi on plants. In
some embodiments YBCA5 or the composition comprising YBCA5 may also
be formulated with an agriculturally acceptable adjuvant. The
applicants are also the first to provide methods of using the
yeast, A. pullulans for biological control of Pseudomonas spp.
bacteria. In particular, the applicants are the first to show that
a strain of A. pullulans yeast, or a composition comprising a
strain of A. pullulans yeast is effective at inhibiting the
survival, growth and/or proliferation of Pseudomonas syringae pv.
actinidiae (Psa) on fruit or vegetable plants, particularly fruit
or vegetable vines, particularly kiwifruit vines.
[0074] Without wishing to be bound by theory the applicants believe
that the efficacy of the yeast strain and compositions of the
invention relates to either the ability of the yeast strain to
competitively exclude Psa and/or phytopathogenic fungi, by
excretion of an anti-microbial compound or compounds, or by
elicitation of plant defence mechanisms, or a combination of the
above. Irrespective of the particular mode of action, the inventors
have surprisingly found that YBCA5 is efficacious for treating Psa
disease on kiwifruit vines, for treating Botrytis spp. and
Monilinia spp. infection on cherries and grapes, and for treating
Alternaria spp., Colletotrichum spp., Penicillium spp., Phomopsis
spp., Cryptosporiopsis spp, on apples and kiwifruit. YBCA5 is a
particularly effective biological control agent against Pseudomonas
spp. bacteria and phytopathogenic fungi. YBCA5 demonstrates the
ability to survive formulation and application protocols, rapidly
colonise treated plants, and suppress growth of Pseudomonas spp.
bacteria and of phytopathogenic fungi on treated plants and parts
thereof. YBCA5 has been found to be particularly effective at
controlling P. syringae bacteria, particularly P. syringae pv.
actinidiae (Psa) bacteria, on kiwifruit vines, and at reducing
and/or controlling, to varying degrees, post-harvest fruit rot due
to Botrytis spp., Sclerotinia spp., Penicillium spp.,
Colletotrichum spp., Alternaria spp., Phomopsis spp.,
Cryptosporiopsis spp, and Monilinia spp.
[0075] YBCA5 and Compositions
[0076] Accordingly, in one aspect the invention relates to isolated
Aureobasidium pullulans yeast strain YBCA5 (CBS Accession #
141880).
[0077] The particular isolated A. pullulans strain YBCA5 of the
invention was deposited on 26 Sep. 2016 for the purpose of patent
procedure under the Budapest Treaty at Centraalbureau voor
Schimmelcultures (CBS), Uppsalalaan 8,3584, CT Utrecht, The
Netherlands. This isolate has been accorded deposit number CBS
Accession # 141880.
[0078] The isolated A. pullulans yeast strain YBCA5 is a
unicellular fungi of the Order Dothideales, Family
Aureobasidiaceae, and genus Aureobasidum. Cells display a wide
range of morphological variability. A. pullulans cultivated on
potato dextrose agar produces smooth, faint pink, yeast-like
colonies. Older colonies can be somewhat darker due to the
production of chlamydospores. Primary conidia of A. pullulans are
single celled, hyaline, smooth, ellipsoidal, and variable in shape
and size. A. pullulans conidiophores are undifferentiated,
intercalary or terminal, or arising as short lateral branches.
Endoconidia are produced by A. pullulans intercalary cells. Hyphae
are thin-walled, hyaline and smooth, with transverse septa. Growth
occurs at 10-35.degree. C. with optimal growth being 22-25.degree.
C.
[0079] In another aspect the invention relates to a composition
comprising YBCA5 (CBS Accession # 141880) and an agriculturally
acceptable carrier.
[0080] In another aspect the invention relates to a composition
consisting essentially of YBCA5 (CBS Accession # 141880) and an
agriculturally acceptable carrier.
[0081] In one embodiment the agriculturally acceptable carrier is
water.
[0082] Again, without wishing to be bound by theory, the inventors
believe that the when used as a biological control agent, YBCA5
must be in a reproductively viable form. For most purposes YBCA5
desirably incorporated into a composition in the form of
reproductively viable cells. Preferably YBCA5 is incorporated into
the composition as dried cells.
[0083] The concentration of cells in a composition of the invention
will depend on the utility to which the composition is put.
Optimizing the concentration of cells for a particular application
is believed to be within the skill in the art.
[0084] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from about
1.times.10.sup.3 to about 1.times.10.sup.14, preferably about
1.times.10.sup.5 to about 1.times.10.sup.11, preferably about
1.times.10.sup.6 to about 1.times.10.sup.9, preferably about
1.times.10.sup.7 to about 1.times.10.sup.8, preferably about
2.times.10.sup.7 to about 2.times.10.sup.8 CFU, preferably about
2.times.10.sup.9 to about 2.times.10.sup.10 CFU per gram for solid
compositions, and about 1.times.10.sup.7 to about 1.times.10.sup.8
CFU per millilitre for liquid compositions.
[0085] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from 1.times.10.sup.3 to
about 1.times.10.sup.14, preferably 1.times.10.sup.5 to about
1.times.10.sup.11, preferably from 1.times.10.sup.6 to about
1.times.10.sup.9, preferably 1.times.10.sup.7 to about
1.times.10.sup.8, preferably from 2.times.10.sup.7 to about
2.times.10.sup.8 CFU, preferably from 2.times.10.sup.9 to about
2.times.10.sup.10 CFU per gram for solid compositions, and from
1.times.10.sup.7 to about 1.times.10.sup.8 CFU per millilitre for
liquid compositions.
[0086] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from about
1.times.10.sup.3 to 1.times.10.sup.14, preferably about
1.times.10.sup.5 to 1.times.10.sup.11, preferably about
1.times.10.sup.6 to 1.times.10.sup.9, preferably about
1.times.10.sup.7 to 1.times.10.sup.8, preferably about
2.times.10.sup.7 to 2.times.10.sup.8 CFU, preferably about
2.times.10.sup.9 to 2.times.10.sup.10 CFU per gram for solid
compositions, and about 1.times.10.sup.7 to 1.times.10.sup.8 CFU
per millilitre for liquid compositions.
[0087] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from 1.times.10.sup.3 to
1.times.10.sup.14, preferably 1.times.10.sup.5 to
1.times.10.sup.11, preferably 1.times.10.sup.6 to 1.times.10.sup.9,
preferably 1.times.10.sup.7 to 1.times.10.sup.8, preferably
2.times.10.sup.7 to 2.times.10.sup.8 CFU, preferably
2.times.10.sup.9 to 2.times.10.sup.10 CFU per gram for solid
compositions, and 1.times.10.sup.7 to 1.times.10.sup.8 CFU per
millilitre for liquid compositions.
[0088] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention is about 2.times.10.sup.10 CFU
per gram for solid compositions, and about 2.times.10.sup.7 CFU per
millilitre for liquid compositions.
[0089] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention is at least 2.times.10.sup.10 CFU
per gram for solid compositions, and at least 2.times.10.sup.7 CFU
per millilitre for liquid compositions, preferably the
concentration of YBCA5 viable cells in a composition of the
invention is 2.times.10.sup.10 CFU per gram for solid compositions,
and 2.times.10.sup.7 CFU per millilitre for liquid
compositions.
[0090] The composition of the invention may comprise or consist
essentially of YBCA5.
[0091] Concentrations of YBCA5 that are effective as a biological
control agent in the composition of the invention may vary
depending on the form the yeast is used in, physiological condition
of the plant; type, concentration and degree of pathogen infection;
temperature; season; humidity; soil type; stage in the growing
season; age of the plant; number and type of conventional
pesticides and fungicides being applied and plant treatments (such
as pruning, but not limited thereto). All factors may be taken into
account in formulating YBCA5 in the composition of the invention or
in a composition for use in a method of the invention.
[0092] YBCA5 may be prepared for use in the invention using
standard liquid fermentation techniques known in the art and as
described in the examples herein. Growth is commonly effected under
aerobic conditions in a bioreactor at suitable temperatures and pH
for growth. Typical growth temperatures are from 10 to 30.degree.
C., preferably 15 to 28.degree. C., preferably 25.degree. C. Yeasts
with optimal growth temperatures in the range of about
36-38.degree. C. are not preferred for use due to the potential for
human health risk. The pH of the growth medium is usually slightly
acidic to neutral at pH 4.0 to 7.0, preferably 6.0.
[0093] Growth medium may be any known art medium suitable for
culture of Aureobasidium species. In one embodiment the growth
medium is potato dextrose agarose (PDA).
[0094] Other suitable growth media include Malt Yeast Extract Agar;
a proprietary liquid broth culture media comprising molasses and
urea; and a proprietary liquid growth media comprising sugar, urea,
yeast extract and mono ammonium phosphate (MAP).
[0095] The cells of YBCA5 may be harvested using conventional
filtering or sedimentary techniques such as centrifugation, or may
be harvested dry using continuous centrifugation. Cells can be used
immediately or stored under chilled conditions (1.degree. C. to
7.degree. C., preferably 2.degree. C.), or may be dried.
Preferably, cells are dried and formulated as dry yeast granules.
For example, cells may be dried using a fluidized bed drier, but
not limited thereto. Preferably the dry yeast granules comprise at
least 90% solids, preferably at least 95% solids, preferably about
96% solids. Preferably cells have a shelf life of at least two
years. In one embodiment shelf life is at least six months,
preferably at least one year, preferably at least two years wherein
the cells are maintained under chilled conditions. Preferably
chilled conditions are 10.degree. C. or less, but greater than
0.degree. C. Preferably chilled conditions are selected from the
group consisting of 1.degree. C., 2.degree. C., 3.degree. C.,
4.degree. C., 5.degree. C., 6.degree. C., 7.degree. C., 8.degree.
C., 9.degree. C. and 10.degree. C. or variations within such
temperatures from about 1.degree. C. to about 10.degree. C.
[0096] In one embodiment the composition comprises an
agriculturally acceptable adjuvant. In one embodiment the
agriculturally acceptable adjuvant is selected from the group
consisting of an additional active agent and a formulation
agent.
[0097] In one embodiment the agriculturally acceptable adjuvant is
one or more additional active agents. In one embodiment the
agriculturally acceptable adjuvant is one or more formulation
agents.
[0098] In one embodiment the composition comprises a combination of
one or more additional active agents and one or more formulation
agents. In some embodiments the composition is formulated as
pre-prepared composition or in a concentrated form. In some
embodiments the composition comprises a solid or a liquid
formulation.
[0099] In one embodiment composition of the invention comprises one
or more agriculturally acceptable adjuvants. In one embodiment the
agriculturally acceptable adjuvants are selected from the group of
additional active agents and formulation agents. Preferably the one
or more agriculturally acceptable adjuvant is an additional active
agent. Preferably the one or more agriculturally acceptable
adjuvant is a formulation agent.
[0100] In one embodiment the composition of the invention comprises
a combination of one or more additional active agents and one or
more formulation agents.
[0101] In some cases it may also be desirable to include one or
more additional active agents in the compositions of the invention
where such additional active agents are capable of contributing to
the control (e.g., treatment and/or prevention) of plant pathogenic
Pseudomonas spp. bacteria or plant pathogenic fungi including
Botrytis spp., Sclerotinia spp., Penicillium spp., Colletotrichum
spp., Alternaria spp., Phomopsis spp., Cryptosporiopsis spp., and
Monilinia spp., but not limited thereto.
[0102] Suitable additional active agents for use in the present
invention may be capable of controlling Pseudomonas spp.,
particularly Psa directly, or plant pathogenic fungi including
Botrytis spp., Sclerotinia spp., Penicillium spp., Colletotrichum
spp., Alternaria spp., Phomopsis spp., Cryptosporiopsis spp. and
Monilinia spp. (but not limited thereto), or may be capable of
potentiating the biocontrol effect of YBCA5 for controlling
Pseudomonas spp., particularly Psa. Additional active agents may be
included directly in the composition of or useful in the invention,
or may be applied separately, either simultaneously or sequentially
as appropriate according to a method of the invention.
[0103] Suitable additional active agents include, but are not
limited to plant defence elicitors including acibenzolar-S-methyl
(Actigard/Bion, Syngenta), Azelaic acid, Pipecolinic acid, Jasmonic
acid, Seaweed Mix, Lema oil, Foodcoat (DOMCA), Fungicover
(bioDURACAL agricultura) and Ibuprofen, antagonistic
microorganisms, inorganic salts including calcium, potassium or
sodium salts, stimulating agents including uronic acids, amnnans,
and .beta. 1-3 glucans, antibiotics, and other antibacterial and
antifungal compounds including small organic and inorganic
molecules.
[0104] By way of non-limiting example, one additional active agent
that may be included in the composition of or for use in the
invention is the plant defence elicitor acibenzolar-S-methyl
(Actigard/Bion, Syngenta). Actigard is a plant activator with a
unique mode of action which stimulates the natural systemic
acquired resistance response found in most plant species. Applied
via foliar application, Actigard has no direct activity against
target pathogens, but helps to reduce Psa symptoms in Kiwifruit by
inducing host plant resistance. Actigard is a composition
comprising 500 g/kg acibenzolar-S-methyl in the form of a water
dispersible granule.
[0105] In one embodiment the composition of the invention comprises
one or more formulation agents.
[0106] In one embodiment the composition of the invention comprises
a combination of one or more additional active agents and one or
more formulation agents.
[0107] In one embodiment, the composition of the invention is
formulated as a solid or a liquid formulation.
[0108] In one embodiment the composition of the invention may
comprise one or more solid or liquid formulation agents. Any
suitable formulation agent(s) may be used as known in the art. It
is believed that the selection of a suitable formulation agent is
within the skill of those in the art. For example, a suitable
formulation agent may be a compound or other material that
facilitates or optimizes the production, handling, storage,
transport, application and/or persistence of the composition of, or
for use in the invention on plants or on parts thereof, but not
limited thereto.
[0109] Formulation agents can be specifically adapted for
particular uses such as, but not limited to, preservation and
maintenance of the biological control activity of the yeasts
comprised in the composition of or for use in the invention during
transportation from production facilities, storage on site, or
during preparation of a final treatment mixture. Formulation agents
may also be used for other purposes such as facilitating adhesion
and persistence on plants or penetration into plant tissues, but
not limited thereto. A suitable formulation may be solid, liquid,
alone or in combination. Particularly suitable formulation agents
include surfactants, dispersants, preservatives, wetting agents,
emulsifiers, humectants, stickers, spreaders, stabilizers,
penetrants, adhesion agents, pH buffers, and nutrients, either
alone or in various combinations as may be determined by the
skilled worker.
[0110] The composition of the invention may be provided as a
pre-prepared composition ready for use, or in a concentrated, solid
or liquid form.
[0111] In one embodiment, the composition is a pre-prepared
composition having a solid or liquid formulation. In one embodiment
the pre-prepared composition is a solid formulation selected from
powders, pellets, granules and prills. In one embodiment the
pre-prepared composition is a liquid formulation.
[0112] The composition of or for use in the invention may be
provided in a pre-prepared form, or in a concentrated form. If
provided in a dry form, the pre-prepared composition may be
provided as a powder, granule, pellet or prill, but not limited
thereto. In the case of a dry form, YBCA5 in the composition is
preferably in dehydrated, dried and/or encapsulated form. In some
embodiments, the dehydrated, dried and/or encapsulated forms
include additional protective agents as known in the art; e.g.,
lyoprotectants and the like.
[0113] In one embodiment, YBCA5 may be provided in granule form.
For example, YBCA5 may be provided in a granule having at least
0.5.times.10.sup.10 CFU/gm, preferably 1.times.10.sup.10 CFU/gm,
preferably 2.times.10.sup.10 CFU/gm. Where the pre-prepared
composition is provided in a liquid form, particularly an aqueous
form the composition may be provided as a dispersion, a suspension,
a slurry, a cream, a paste or a gel, but not limited thereto.
Preferably the pre-prepared form is provided as an aqueous liquid
form that is suitable for and/or is adapted for spraying. In one
embodiment a pre-prepared liquid form can be used per se for
example as a dip to inoculate fruits, vegetables, seeds or plants,
including plant cuttings.
[0114] In the pre-prepared composition of the invention, YBCA5 is
formulated for use on plants, particularly kiwifruit vines. For
example, the yeasts are mixed with an agriculturally acceptable
carrier liquid that enables spray applications, a fertilizer, an
elicitor, an adjuvant, a wetting agent, or any other suitable
additional agent as required. In the pre-prepared composition for
use according to the methods of the invention, YBCA5 may also be
mixed with an agriculturally acceptable carrier liquid that enables
spray applications, a fertilizer, an elicitor, an adjuvant, a
wetting agent, or any other suitable additional agent as
required.
[0115] The formulation of YBCA5 into a pre-prepared composition of
the invention and the final form of the pre-prepared composition
for application to the plant or part thereof is believed to be
within the skill in the art. For example, the final form of the
composition is formulated with an agriculturally acceptable carrier
such as water to form a spray, foam, drench, injectable, gel, dip
or paste, but not limited thereto. In one embodiment, a composition
of the invention may be applied to plants or parts thereof by
spraying, dipping, rubbing or brushing, or a combination thereof.
Preferably the composition is formulated as an aqueous suspension
or dispersion for spray or mist application to kiwifruit vines,
cherry trees and/or fruit and grape vines and/or fruit and/or
vegetables.
[0116] In one embodiment the composition of the invention is in
concentrated form. In one embodiment the concentrated form is a
solid form selected from cakes, powders, granules, pellets and
prills. In one embodiment the concentrated form is a liquid
formulation.
[0117] Where the composition of the invention is provided in a
concentrated form it may require additional formulation by the user
to produce a composition ready for application to a plant or part
thereof. For example, the concentrated form can be mixed with
various formulation agents to form a final composition for plant
application. A preferred formulation is agent is water or an
aqueous solution in which an appropriate amount of the concentrated
from of the composition is dissolved (e.g., granules or powders) or
diluted (e.g., liquid suspensions or dispersions) to obtain a final
composition for application to a plant.
[0118] If the YBCA5 is dehydrated in the concentrated form then
rehydration as known in the art will be required if the composition
for application to the plant is intended to be in liquid form.
Rehydration may be carried out using customary precautions for
rehydrating the yeast as known in the art; for example rehydration
may be achieved advantageously at temperatures between 20 and
25.degree. C., but not limited thereto.
[0119] Methods--Pseudomonas spp.
[0120] In another aspect the invention relates to a method of
controlling Pseudomonas spp. bacteria on a plant or part thereof,
the method comprising contacting the plant or part thereof with
YBCA5, or a composition comprising YBCA5.
[0121] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for controlling Pseudomonas spp.
bacteria on a plant or part thereof.
[0122] In one embodiment the method or use comprises contacting the
plant or part thereof with reproductively viable cells of
YBCA5.
[0123] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from about
1.times.10.sup.3 to about 1.times.10.sup.14, preferably about
1.times.10.sup.5 to about 1.times.10.sup.11, preferably about
1.times.10.sup.6 to about 1.times.10.sup.9, preferably about
1.times.10.sup.7 to about 1.times.10.sup.8, preferably about
2.times.10.sup.7 to about 2.times.10.sup.8 CFU, preferably about
2.times.10.sup.9 to about 2.times.10.sup.10 CFU per gram for solid
compositions, and about 1.times.10.sup.7 to about 1.times.10.sup.8
CFU per millilitre for liquid compositions.
[0124] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from 1.times.10.sup.3 to
about 1.times.10.sup.14, preferably 1.times.10.sup.5 to about
1.times.10.sup.11, preferably from 1.times.10.sup.6 to about
1.times.10.sup.9, preferably 1.times.10.sup.7 to about
1.times.10.sup.8, preferably from 2.times.10.sup.7 to about
2.times.10.sup.8 CFU, preferably from 2.times.10.sup.9 to about
2.times.10.sup.10 CFU per gram for solid compositions, and from
1.times.10.sup.7 to about 1.times.10.sup.8 CFU per millilitre for
liquid compositions.
[0125] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from about
1.times.10.sup.3 to 1.times.10.sup.14, preferably about
1.times.10.sup.5 to 1.times.10.sup.11, preferably about
1.times.10.sup.6 to 1.times.10.sup.9, preferably about
1.times.10.sup.7 to 1.times.10.sup.8, preferably about
2.times.10.sup.7 to 2.times.10.sup.8 CFU, preferably about
2.times.10.sup.9 to 2.times.10.sup.10 CFU per gram for solid
compositions, and about 1.times.10.sup.7 to 1.times.10.sup.8 CFU
per millilitre for liquid compositions.
[0126] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention ranges from 1.times.10.sup.3 to
1.times.10.sup.14, preferably 1.times.10.sup.5 to
1.times.10.sup.11, preferably 1.times.10.sup.6 to 1.times.10.sup.9,
preferably 1.times.10.sup.7 to 1.times.10.sup.8, preferably
2.times.10.sup.7 to 2.times.10.sup.8 CFU, preferably
2.times.10.sup.9 to 2.times.10.sup.10 CFU per gram for solid
compositions, and 1.times.10.sup.7 to 1.times.10.sup.8 CFU per
millilitre for liquid compositions.
[0127] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention is about 2.times.10.sup.10 CFU
per gram for solid compositions, and about 2.times.10.sup.7 CFU per
millilitre for liquid compositions.
[0128] In some embodiments the concentration of YBCA5 viable cells
in a composition of the invention is at least 2.times.10.sup.10 CFU
per gram for solid compositions, and at least 2.times.10.sup.7 CFU
per millilitre for liquid compositions, preferably the
concentration of YBCA5 viable cells in a composition of the
invention is 2.times.10.sup.10 CFU per gram for solid compositions,
and 2.times.10.sup.7 CFU per millilitre for liquid
compositions.
[0129] In one embodiment the at least one strain of Pseudomonas
spp. is selected from the group consisting of P. syringae, P.
amygdalia, P. avellanae, P. caricapapayae, P. cichorii, P.
coronafaciens, P. ficuserectae, P. helianthi, P. lemiae, P.
savastanoi, and P. viridiflava, or a pathovar thereof, or
combinations thereof. Preferably the at least one strain is P.
syringae or a pathovar thereof, more preferably the at least one
strain is P. syringae pv. actinidiae (Psa).
[0130] In one embodiment the plant or part thereof is selected from
the group of monocotyledonous plants, dicotyledonous plants,
annual, biannual and perennial plants, vegetable plants or
harvested vegetables, fruit plants or trees or harvested fruits,
flower bearing plants or trees or harvested flowers, cereal plants,
oleaginous plants, proteinous plants, ligneous plants, and
ornamental plants.
[0131] In one embodiment the plant or part thereof is an
agriculturally important plant, cultivar thereof, or product
thereof selected from the group consisting of agriculturally
important vines, agriculturally important vegetables and
agriculturally important fruit plants, and cultivars and products
thereof. Preferably the agriculturally important vine is a
kiwifruit vine or cultivar thereof, and the product is
kiwifruit.
[0132] In one embodiment the kiwifruit vine is selected from the
group consisting of species of green-fleshed kiwifruit (A.
chinensis var. deliciosa), golden kiwifruit (A. chinensis var.
chinensis), Chinese egg gooseberry (A. coriacea), baby kiwifruit
(A. arguta), Arctic kiwifruit (A. kolomikta), red kiwifruit (A.
melanandra, A. chinensis var. chinensis), silver vine (A.
polygama), and purple kiwifruit (A. purpurea) or a cultivar
thereof. Preferably the kiwifruit are selected from the group
consisting of A. chinensis var. deliciosa and A. chinensis var.
chinensis, species or a cultivar thereof. Preferably the kiwifruit
is a species of A. chinensis var. chinensis. Preferably the
kiwifruit is A. chinensis var. chinensis Planch. Preferably the
cultivar is a `Hayward` or `Hort16A` or `zesy002`, informally known
as Gold3 or `Hongyang`.
[0133] In one embodiment the cultivar is A. chinensis var.
chinensis Planch, `Hort16A`. In one embodiment the cultivar is
`Hort16A` as disclosed in USPP11066, the entirety of which is
incorporated by reference herein.
[0134] In one embodiment the cultivar is A. chinensis var.
deliciosa `Hayward`. In one embodiment the cultivar is `Hayward` as
disclosed in USPP6815, the entirety of which is incorporated by
reference herein.
[0135] In one embodiment cultivar is A. chinensis var. chinensis
Planch. `Hongyang`. In one embodiment the cultivar is `Hongyang` as
disclosed in Wang 2011 and in Li et al 2015, the entirety of which
are incorporated by reference herein.
[0136] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling Pseudomonas spp. bacteria on a plant or part
thereof.
[0137] The use of YBCA5, or a composition comprising YBCA5 for
controlling Pseudomonas spp., bacteria and/or for increasing the
yield of a kiwifruit plant is carried out in accordance with the
methods of the invention as described herein. For example, YBCA5
and compositions thereof may be prepared, formulated for and
applied to a plant, or part thereof, particularly a kiwifruit
plant, or part thereof, according to the invention as described
herein.
[0138] In another aspect the invention relates to a method of
controlling at least one phytopathogenic fungus on a plant or part
thereof, the method comprising contacting the plant or part thereof
with YBCA5, or a composition comprising YBCA5.
[0139] In another aspect the invention relates to a method for
increasing the yield of a fruit or vegetable plant susceptible to
infection by a phytopathogenic fungus, the method comprising
applying YBCA5, or a composition comprising YBCA5 to the fruit or
vegetable plant or part thereof, and growing the plant or part
thereof. In one embodiment the composition consists essentially of
YBCA5.
[0140] In one embodiment the at least phytopathogenic fungus is
selected from the group consisting of Botrytis spp., Monilinia
spp., Sclerotinia spp., Colletotrichum spp., Alternaria spp.,
Cryptosporiopsis spp., Phomopsis spp., and Penicillium spp.
[0141] In one embodiment the plant or part thereof is selected from
the group of monocotyledonous plants, dicotyledonous plants,
annual, biannual and perennial plants, vegetable plants or
harvested vegetables, fruit plants or trees or harvested fruits,
flower bearing plants or trees or harvested flowers, cereal plants,
oleaginous plants, proteinous plants, ligneous plants, and
ornamental plants.
[0142] In one embodiment the plant or part thereof is an
agriculturally important plant, cultivar thereof, or product
thereof selected from the group consisting of agriculturally
important vines and agriculturally important fruit trees,
agriculturally important vegetables and cultivars and products
thereof. In one embodiment the agriculturally important vine is a
kiwifruit vine or cultivar thereof, and the product is
kiwifruit.
[0143] In one embodiment the plant or part thereof is a fruit or
vegetable plant or part thereof, the method comprising contacting
the fruit or vegetable plant or part thereof with YBCA5, or a
composition comprising YBCA5. In some embodiments the fruit or
vegetable plant is a cherry tree or a grape vine. In some
embodiments the fruit plant is an apple tree.
[0144] In one embodiment the cherry tree is a Prunus spp., or a
cultivar thereof, preferably a P. avium, or cultivar thereof.
Preferably the P. avium is a "Sweet Valentine" variety. In one
embodiment the part thereof is a flower or part thereof or a fruit
or part thereof. In one embodiment the fruit is a cherry.
[0145] In one embodiment the grape vine is a Vinus spp., or a
cultivar thereof, preferably a V. vinifera, or cultivar thereof.
Preferably the V. vinifera is a "Thompson Seedless" variety. In one
embodiment the part thereof is a flower or part thereof or a fruit
or part thereof. In one embodiment the fruit is a grape.
[0146] In one embodiment the apple tree is a Malus spp., or a
cultivar thereof, preferably M. pumila or cultivar thereof.
Preferably the M. pumila or a cultivar thereof is a `Pacific Rose`
variety. In one embodiment the part thereof is a flower or part
thereof, or a fruit or part thereof. In one embodiment the fruit is
an apple.
[0147] Psa Control
[0148] In another aspect the invention relates to a method for
controlling P. syringae pv. actinidiae (Psa) on a kiwifruit plant
or part thereof, the method comprising contacting the kiwifruit
plant or part thereof with YBCA5, or a composition comprising
YBCA5.
[0149] In another aspect the invention relates to a method for
increasing the yield of a kiwifruit plant infected, or susceptible
to infection with Psa, the method comprising applying YBCA5 or a
composition comprising YBCA5 to the kiwifruit plant or part
thereof, and growing the kiwifruit plant or part thereof.
[0150] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for controlling Psa on a
kiwifruit plant or part thereof.
[0151] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for increasing the yield of a
kiwifruit plant infected, or susceptible to infection with Psa.
[0152] In one embodiment the composition consists essentially of
YBCA5.
[0153] In one embodiment, the kiwifruit plant is a species of A.
chinensis var. deliciosa or A. chinensis var. chinensis, or a
cultivar thereof, preferably a species of A. chinensis var.
chinensis, or cultivar thereof. In one embodiment the kiwifruit
plant is `Hort16A`.
[0154] In one embodiment cultivar is A. chinensis var. chinensis
`Hongyang`. In one embodiment the cultivar is `Hongyang` as
disclosed in Wang 2011 and in Li et al 2015, the entirety of which
are incorporated by reference herein.
[0155] In one embodiment a plant or part thereof is contacted for a
time sufficient to control Psa.
[0156] In one embodiment, contacting comprises applying YBCA5 or a
composition comprising or consisting essentially of YBCA5 to the
plant or part thereof by applying to the seeds, leaves, stems,
flowers, fruits, trunks and/or roots of the plant or part thereof.
Preferably application is by spraying, misting, dipping, dripping,
dusting or sprinkling. Applications can be made once only, or
repeatedly as required. Also contemplated herein is application at
various times of year and/or during various stages of the plant
life cycle, as determined appropriate by the skilled worker.
[0157] YBCA5 may be applied at the appropriate time during the year
and at the appropriate stage of plant development as may be
determined by a skilled worker. For example YBCA5 may be applied
from bud-burst to flowering, during flowering and post
flowering/fruit set period but not limited thereto.
[0158] In one embodiment, applying is by spraying onto leaf
surfaces and/or onto flowers and/or onto fruit and/or onto
vegetables.
[0159] In one embodiment, applying to the roots is by ground
spraying, mechanical incorporation or by mixing with enriching
agents or fertilizers prior to application in the usual way.
[0160] In one embodiment the plant or part thereof is selected from
monocotyledonous plants, dicotyledonous plants, annual, biannual
and perennial plants, vegetable plants or harvested vegetables,
fruit plants or trees or harvested fruits, flower bearing plants or
trees or harvested flowers, cereal plants, oleaginous plants,
proteinous plants, ligneous plants, and ornamental plants.
[0161] In one embodiment, a plant or part thereof is an
agriculturally important crop plant, cultivar or product thereof
selected from corn plants, tobacco plants, wheat plants, sugar cane
plants, rapeseed plants, barley plants, rice plants, sorghum
plants, millet plants, soya bean plants, lettuce plants, and
cabbage plants.
[0162] In one embodiment the plant or part thereof is an
agriculturally important plant, cultivar thereof, or product
thereof selected from the group consisting of agriculturally
important vines and agriculturally important fruit trees, and
cultivars and products thereof.
[0163] Preferably the agriculturally important fruit trees or
cultivars thereof are selected from olive trees, apple trees, pear
trees, citrus fruit trees, banana trees, pineapple trees, peach
trees, apricot trees, cherry trees, walnut trees and hazelnut trees
and the products thereof are olives, apples, pears, citrus fruits,
bananas, pineapples, peaches, apricots, cherries, walnuts and
hazelnuts respectively. Preferably the agriculturally important
vines or cultivars thereof are selected from potato vines, beetroot
vines, bean vines, pea vines, tomato vines, cucumber vines, melon
vines, berry vines, grape vines and kiwifruit vines and the
products thereof are potatoes, beetroots, beans, peas, tomatoes,
cucumbers, melons, berries, grapes and kiwifruits respectively.
Preferably the agriculturally important vine is a kiwifruit vine or
cultivar thereof, and the product is kiwifruit.
[0164] Kiwifruit are within the plant order Ericales and the family
Actinidiaceae. In one embodiment the kiwifruit vine is selected
from the group consisting of species of fuzzy kiwifruit (A.
chinensis var. deliciosa), golden kiwifruit (A. chinensis var.
chinensis), Chinese egg gooseberry (A. coriacea), baby kiwifruit
(A. arguta), Arctic kiwifruit (A. kolomikta), red kiwifruit (A.
melanandra, A. chinensis var. chinensis), silver vine (A.
polygama), and purple kiwifruit (A. purpurea) or a cultivar
thereof. Preferably the kiwifruit are selected from the group
consisting of A. chinensis var. deliciosa, A. chinensis var.
chinensis species or a cultivar thereof. Preferably the kiwifruit
is a species of A. chinensis var. chinensis. Preferably the
preferably kiwifruit is A. chinensis var. chinensis Planch.
Preferably the cultivar is a `Hayward` or `Hort 16A` or `Zesy002`
or `Zesy004` or `Hongyang` variety cultivar.
[0165] In one embodiment the cultivar is A. chinensis var.
chinensis Planch, `Hort 16A`. In one embodiment the cultivar is
`Hort 16A` as disclosed in USPP11066, the entirety of which is
incorporated by reference herein.
[0166] In one embodiment cultivar is A. chinensis var. deliciosa
`Hayward`. In one embodiment the cultivar is `Hayward` as disclosed
in USPP6815, the entirety of which is incorporated by reference
herein.
[0167] In one embodiment cultivar is A. chinensis var. chinensis
`Hongyang`. In one embodiment the cultivar is `Hongyang` as
disclosed in Wang 2011 and in Li et al 2015, the entirety of which
are incorporated by reference herein.
[0168] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling Psa on a kiwifruit plant or part thereof.
[0169] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
increasing the yield of a kiwifruit plant infected, or susceptible
to infection with Psa.
[0170] The use of YBCA5, or a composition comprising YBCA5 for
controlling Psa and/or for increasing the yield of a kiwifruit
plant is carried out in accordance with the methods of the
invention as described herein. For example, YBCA5 and compositions
thereof may be prepared, formulated for and applied to a plant, or
part thereof, particularly a kiwifruit plant, or part thereof,
according to the invention as described herein.
[0171] In another aspect the invention relates to at least one
plant or part thereof treated with YBCA5, or a composition
comprising YBCA5. In some embodiments the plant is a fruit or
vegetable plant or part thereof. In one embodiment the plant is a
kiwifruit vine, a cherry tree or a grape vine.
[0172] Phytopathogenic Fungal Control
[0173] In another aspect the invention relates to a method of
controlling at least one phytopathogenic fungus on a plant or part
thereof, the method comprising contacting the plant or part thereof
with YBCA5, or a composition comprising YBCA5.
[0174] In another aspect the invention relates to a method for
increasing the yield of a fruit or vegetable plant susceptible to
infection by a phytopathogenic fungus, the method comprising
applying YBCA5, or a composition comprising YBCA5 to the fruit or
vegetable plant or part thereof, and growing the plant or part
thereof.
[0175] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for controlling a phytopathogenic
fungus on a fruit or vegetable plant or part thereof.
[0176] In another aspect the invention relates to the use of YBCA5,
or a composition comprising YBCA5 for increasing the yield of a
fruit or vegetable plant or part thereof susceptible to infection
by a phytopathogenic fungus.
[0177] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling at least one phytopathogenic fungus on a plant or part
thereof.
[0178] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
controlling at least one phytopathogenic fungus on a fruit or
vegetable plant or part thereof.
[0179] In another aspect the invention relates to YBCA5, or a
composition comprising YBCA5 for use in, or when used, for
increasing the yield of a fruit or vegetable plant susceptible to
infection by at least one phytopathogenic fungus.
[0180] The following embodiments are also specifically contemplated
for those aspects of the invention that relate to the use of YBCA5,
or to a composition comprising or consisting essentially of YBCA5,
for controlling phytopathogenic fungi and/or for increasing the
yield of a plant or part thereof, or of a fruit or vegetable plant
or part thereof, susceptible to infection by a phytopathogenic
fungus.
[0181] In one embodiment the phytopathogenic fungus is selected
from the group consisting of Botrytis spp., Monilinia spp.,
Sclerotinia spp., Colletotrichum spp., Alternaria spp.,
Cryptosporiopsis spp., Phomopsis spp., and Penicillium spp.
[0182] In one embodiment the plant or part thereof is selected from
the group of monocotyledonous plants, dicotyledonous plants,
annual, biannual and perennial plants, vegetable plants or
harvested vegetables, fruit plants or trees or harvested fruits,
flower bearing plants or trees or harvested flowers, cereal plants,
oleaginous plants, proteinous plants, ligneous plants, and
ornamental plants.
[0183] In one embodiment the plant or part thereof is an
agriculturally important plant, cultivar thereof, or product
thereof selected from the group consisting of agriculturally
important vines and agriculturally important fruit trees,
agriculturally important vegetables and cultivars and products
thereof. In one embodiment the agriculturally important vine is a
kiwifruit vine or cultivar thereof, and the product is
kiwifruit.
[0184] In one embodiment the plant or part thereof is a fruit or
vegetable plant or part thereof, the method comprising contacting
the fruit or vegetable plant or part thereof with YBCA5, or a
composition comprising YBCA5. In some embodiments the fruit or
vegetable plant is a cherry tree or a grape vine. In some
embodiments the fruit plant is an apple tree.
[0185] In one embodiment the cherry tree is a Prunus spp., or a
cultivar thereof, preferably a P. avium, or cultivar thereof.
Preferably the P. avium is a "Sweet Valentine" variety. In one
embodiment the part thereof is a flower or part thereof or a fruit
or part thereof. In one embodiment the fruit is a cherry.
[0186] In one embodiment the grape vine is a Vinus spp., or a
cultivar thereof, preferably a V. vinifera, or cultivar thereof.
Preferably the V. vinifera is a "Thompson Seedless" variety. In one
embodiment the part thereof is a flower or part thereof or a fruit
or part thereof. In one embodiment the fruit is a grape.
[0187] In one embodiment the apple tree is a Malus spp., or a
cultivar thereof, preferably M. pumila or a cultivar thereof.
Preferably the M. pumila is a `Pacific Rose` variety. In one
embodiment the part thereof is a flower or part thereof, or a fruit
or part thereof. In one embodiment the fruit is an apple.
[0188] In another aspect the invention relates to at least one
plant or part thereof treated with YBCA5, or a composition
comprising YBCA5.
[0189] In another aspect the invention relates to at least one
fruit or vegetable plant or part thereof treated with YBCA5, or a
composition comprising YBCA5.
[0190] In one embodiment the composition consists essentially of
YBCA5.
[0191] The use of YBCA5, or a composition comprising or consisting
essentially of YBCA5 for controlling phytopathogenic fungi and/or
for increasing the yield of a plant or part thereof, or of a fruit
or vegetable plant or part thereof is carried out in accordance
with the methods and uses of the invention as described herein. For
example, YBCA5 and compositions thereof may be prepared, formulated
for and applied to a plant, or part thereof, particularly a fruit
or vegetable plant, or part thereof, particularly a cherry tree or
grape vine, according to the invention as described herein.
[0192] Various aspects of the invention will now be illustrated in
non-limiting ways by reference to the following examples.
EXAMPLES
Example 1--Identification of Yeasts with Biocontrol Activity
[0193] Yeast Screening
[0194] YBCA5 was isolated from Apricots ("Clutha Gold") from
central Otago in the early 2000s as follows. Fresh, harvested
apricots were frozen overnight at -20.degree. C. and then incubated
at 20.degree. C. for up to 5 days. Yeast or yeast-like colonies
growing on the surface of selected apricots were isolated using
standard protocols on a general culturing medium suitable for yeast
propagation.
Example 2--Yeast Biocontrol of Pseudomonas syringae var. actinidiae
(Psa)
[0195] General Methodologies
[0196] Plant-based screening assays were conducted in laboratories
and glasshouses at the Ruakura Research Centre, Hamilton and at the
Te Puke Research Orchard, Te Puke, New Zealand. Plant and Food
(PFR) assays focused on foliar application of biological control
agents (BCAs), particularly YBCA5 and other PFR proprietary yeast
strains.
[0197] Zespri Assay 26--Dose Rate of YBCA5
[0198] The aim of this assay was to compare several dose rates of
freshly fermented YBCA5 with a formulated and dried preparation of
YBCA5 for their efficacy against Psa.
[0199] Plant Material
[0200] Zespri Assay 26 was carried out in the PC1 glasshouse at
Ruakura using tissue cultured A. chinensis var. deliciosa `Hayward`
plants grown in 1 L pots. Plants were 30-50 cm high, each with at
least 4-5 useable leaves per plant and the time of treatment and
there were 10 replicate plants per treatment.
[0201] Yeast Preparation
[0202] Freshly fermented YBCA5 was obtained by fermenting the yeast
for 3 days in a 10 L bioreactor (Labfors) using sterile liquid
media (4% molasses and 1.2 g/L urea). The fermentate was spun in a
centrifuge (Sorvall RC-5C) at 5000 rpm for 15 min (rotor no.
SLC-4000, rotorcode 33) to achieve a wet pellet of cell concentrate
after discarding the supernatant. A sub-sample of wet pellet was
re-suspended and the cell density determined with the aid of a
haemocytometer and appropriate dilutions made to achieve final
spray concentrations of 6.times.10.sup.6, 1.25.times.10.sup.7,
2.5.times.10.sup.7 and 5.0.times.10.sup.7 CFU/mL.
[0203] YBCA5 granules were prepared by mixing the wet pellet from a
previous fermentation in the 10 L fermenter, with approximately 30%
(w/w) cornstarch to form a stiff dough consistency and this was
extruded through a steel mesh (3 mm hole size) and dried in a
laminar flow hood overnight (20-25.degree. C.) to form dried
granules.
[0204] The number of CFU in the dried granules of YBCA5 was
calculated by thoroughly dissolving 0.2 g granule into 20 mL of
PBSTw. Serial dilutions of this stock were carried out (to 10-6)
and three 10 .mu.l droplets of each dilution were transferred onto
MYA. The number of yeast colonies growing from each droplet were
counted after 24 h incubation at 25.degree. C. followed by a
further 24 h incubation in a fridge (4-6.degree. C.). A spray
concentration of 2.5.times.10.sup.7 CFU/mL was prepared be weighing
the appropriate quantity of granules into 500 mL water.
[0205] Psa Inoculum Preparation
[0206] A Psa culture (isolate code 10627, biovar 3), which had been
isolated from an infected Actinidia chinensis var. chinensis
`Hort16A` kiwifruit vine located in the Te Puke region during 2010,
was used for all stab and spray inoculation assays included in this
report (Vanneste et al. 2013). Psa inoculum was prepared by growing
this strain of Psa for 2-3 days on King's B (KB) medium and
harvesting the bacteria by washing the plate with sterile distilled
water (SDW) to make a stock suspension of inoculum that was
visually determined to be c. >1.times.10.sup.9 CFU/mL. A
subsample of this Psa stock was serially diluted and 10-4 droplets
placed onto fresh KB medium so that the number of CFU/mL could be
counted after two days' incubation. To facilitate droplet
inoculation in the glasshouse (Ruakura), Psa inoculum (stock
solution c..times.10.sup.9 CFU/mL) optical density was determined
using a spectrophotometer (600 nm) and then the solution was
diluted with sterile PBS to give resultant suspensions of
5.times.10.sup.8 CFU/mL and 2.times.10.sup.7 CFU/mL, based on a
previously developed calibration curve. The adjuvant, Du-Wett was
then added to the suspension to give a final concentration of 0.03%
(v/v).
[0207] Leaf Spray Inoculation Assay
[0208] On 11 and 18 Sep. 2014, spray treatments of each freshly
fermented YBCA5 concentration and the granule preparation were
applied to run-off to all leaves on each plant that had been grown
in pots in a glasshouse, 7 and 1 days before inoculation (dbi) with
the two doses of Psa and plants were allowed to dry in a spray
containment shed. Once dried, the spray treated plants were
returned to the glasshouse. One day after the second spray
treatment with YBCA5, on 19 Sep. 2014 the Psa inoculum was pipetted
(10 .mu.L) onto the underside of each leaf in pairs on either side
of the mid-rib of four or five selected leaves (avoiding the oldest
and the youngest leaves). Plants were then placed into
high-humidity tents in containment glasshouses at PFR Ruakura after
inoculation with Psa for up to three weeks and then scored for Psa
severity.
[0209] Measurement of Psa Symptoms
[0210] The area (mm.sup.2) of necrosis caused by Psa was visually
estimated for each inoculation point 21 days after Psa droplet
inoculation. In order to ensure consistency, only two staff members
carried out Psa leaf severity assessments with regular
cross-checking of the severity scores.
[0211] Statistical Analysis
[0212] All data were analysed using GenStat following natural log
transformation. Raw data means are presented and statistical
differences are based on the log transformed analysis.
[0213] KRIP-BCA Assay 39
[0214] The aim of this assay was to investigate the fermentation,
formulation and efficacy against Psa of different isolates of
Aureobasidium pullulans compared with YBCA5.
[0215] Plant Material
[0216] KRIP-BCA Assay 39 was carried out in the PC1 glasshouse at
Ruakura using tissue cultured A. chinensis var. deliciosa `Hayward`
plants grown in 1.5 L pots. Plants were 30-50 cm high, each with at
least 4-5 useable leaves per plant and the time of treatment and
there were 10 replicate plants per treatment.
[0217] Yeast Preparation
[0218] YBCA5 granules were prepared by fermenting the yeast for 3
days in a 10 L bioreactor (Labfors) using sterile liquid media (4%
molasses and 1.2 g/L urea). The fermentate was spun in a centrifuge
(Sorvall RC-5C) at 5000 rpm for 15 min (rotor no. SLC-4000,
rotorcode 33) to achieve a wet pellet of cell concentrate after
discarding the supernatant. This wet pellet was mixed with
approximately 30% (w/w) cornstarch to form a stiff dough
consistency and this was extruded through a steel mesh (3 mm hole
size) and dried in a laminar flow hood overnight (20-25.degree. C.)
to form dried granules.
[0219] Twelve A. pullulans isolates selected from a large culture
collection were fermented for 3 days in flask culture (200 mL of 4%
molasses 1.2 g/L urea sterile liquid media in 500 mL conical
flasks). The number of viable colony forming units (CFU) was
determined by sub-sampling 1 mL of fermentate and carrying out
serial dilutions (to 10-7) in 0.05M phosphate buffered saline
+0.05% Tween80 (PBSTw). For each dilution three 10 .mu.l droplets
were transferred onto Malt Yeast Extract Agar (MYA). This number of
yeast colonies growing from each droplet were counted after 24 h
incubation at 25.degree. C. followed by a further 24 h incubation
in a fridge (4-6.degree. C.). The fermentate was then processed as
described above for YBCA5 to form dried formulated granules.
[0220] The number of CFU in the dried granules for each A.
pullulans isolate was calculated by thoroughly dissolving 0.2 g
granule into 20 mL of PBSTw. Serial dilutions of this stock were
carried out (to 10-6) and three 10 .mu.l droplets of each dilution
were transferred onto MYA. This number of yeast colonies growing
from each droplet were counted after 24 h incubation at 25.degree.
C. followed by a further 24 h incubation in a fridge (4-6.degree.
C.). Compositions for spray applications were prepared by weighing
the appropriate quantity of granules into 500 mL water.
[0221] Psa Inoculum Preparation
[0222] A Psa culture (isolate code 10627, biovar 3), which had been
isolated from an infected Actinidia chinensis var. chinensis
`Hort16A` kiwifruit vine located in the Te Puke region during 2010,
was used for all stab and spray inoculation assays included in this
report (Vanneste et al. 2013). Psa inoculum was prepared by growing
this strain of Psa for 2-3 days on King's B (KB) medium and
harvesting the bacteria by washing the plate with sterile distilled
water (SDW) to make a stock suspension of inoculum that was
visually determined to be c. >1.times.10.sup.9 CFU/mL. A
subsample of this Psa stock was serially diluted and 10 .mu.L
droplets placed onto fresh KB medium so that the number of CFU/mL
could be counted after two days' incubation. To facilitate droplet
inoculation in the glasshouse (Ruakura), Psa inoculum (stock
solution c..times.10.sup.9 CFU/mL) optical density was determined
using a spectrophotometer (600 nm) and then the solution was
diluted with sterile PBS to give resultant suspensions of
1.times.10.sup.8 CFU/mL, based on a previously developed
calibration curve. The adjuvant, Du-Wett was then added to the
suspension to give a final concentration of 0.03% (v/v).
[0223] Leaf Spray Inoculation Assays
[0224] On 9 May 2016, spray treatments of each A. pullulans isolate
and YBCA5 were applied to run-off to all leaves on each plant that
had been grown in pots in a glasshouse, 7 days before inoculation
(dbi) with Psa (1.times.10.sup.8 CFU/mL). All A. pullulans
treatments, including YBCA5 were applied at a final concentration
of 2.times.10.sup.7 CFU/mL and plants were allowed to dry in a
spray containment shed. Once dried, the spray treated plants were
returned to the glasshouse. Seven days after spray treatment, on 16
May 2016 the Psa inoculum dose was pipetted (10 .mu.L) onto the
underside of each leaf in pairs on either side of the mid-rib of
four or five selected leaves (avoiding the oldest and the youngest
leaves). Plants were then placed into high-humidity tents in
containment glasshouses at PFR Ruakura after inoculation with Psa
for up to three weeks and then scored for Psa severity.
[0225] Measurement of Psa Symptoms
[0226] The area (mm.sup.2) of necrosis caused by Psa was visually
estimated for each inoculation point 21 days after Psa droplet
inoculation. In order to ensure consistency, only two staff members
carried out Psa leaf severity assessments with regular
cross-checking of the severity scores.
[0227] Statistical Analysis
[0228] All data were analysed using GenStat following natural log
transformation. Raw data means are presented and statistical
differences are based on the log transformed analysis.
[0229] Results--Dose Rate Assay
[0230] FIG. 1 shows that YBCA5 is very effective at reducing the
severity of Psa symptoms on `Hayward` kiwifruit leaves. All dose
rates used in this experiment significantly reduced (P<0.001)
the severity of leaf necrosis compared with the untreated control.
There was not difference in efficacy in the YBCA5 granule
preparation compared with freshly fermented YBCA5.
[0231] Results--Fermentation and Formulation
[0232] The fermentation yield for the 12 flask grown A. pullulans
isolates ranged from 1.3.times.10.sup.8 CFU/mL to
2.3.times.10.sup.9 CFU/mL and the fermentation yield for flask
grown YBCA5 was 3.3 .times.10.sup.8 CFU/mL (Table 1), indicating
that some isolates are capable of producing higher fermentation
yields compared with YBCA5, while others produce lower fermentation
yields.
[0233] The number of CFU/g for dried granules of YBCA5 (from 10 L
fermentation) was 2.3.times.10.sup.10 and for the 12 isolates of A.
pullulans the number of CFU/g for dried granules ranged from a low
of 3.1.times.10.sup.9 CFU/g to 2.0.times.10.sup.10 CFU/g (Table 1),
indicating that most A. pullulans isolates produced a lower yield
of viable CFU/g than YBCA5.
[0234] A comparison of the number of CFU in the granules per mL of
fermentation liquid (to allow a more direct comparison of the 10 L
fermentation of YBCA5 and the flask culture of the 12 A. pullulans
isolates) shows that the YBCA5 has the highest yield
(4.times.10.sup.8 CFU/mL) and for the other A. pullulans isolates
this ranged from as low as 4.2.times.107 CFU/mL to
2.4.times.10.sup.8 CFU/mL (Table 1).
TABLE-US-00001 TABLE 1 Fermentation yield and formulation yield for
a range of Aureobasidium pullulans isolates, including YBCA5. A.
Conical Granules Granule pullulans flask dried CFU/mL of culture
yield weight Granule Total fermentation code (spores/mL) (g) CFU/g
spores liquid YBCA5 3.3E+08 174* 2.30E+10* 4.04E+12* 4.0E+08* CG
173 ND** 9 2.00E+10 1.80E+11 2.2E+08 HB 229 ND 9.4 1.40E+10
1.30E+11 1.6E+08 HRY 212 ND 7.6 1.80E+10 1.40E+11 1.7E+08 HB 228 ND
9.8 1.90E+10 1.90E+11 2.4E+08 HB226 2.3E+09 12 1.60E+10 1.90E+11
2.4E+08 HB201 1.4E+08 9.9 6.10E+09 6.00E+10 7.5E+07 FOR 5-8-1
2.3E+09 10 1.60E+10 1.60E+11 2.0E+08 GIS 08 4/1 2.3E+09 13.7
5.30E+09 7.30E+10 9.1E+07 HB 303 8.8E+09 10.8 3.10E+09 3.40E+10
4.2E+07 FOR6-1-1 1.3E+08 10.7 9.80E+09 1.10E+11 1.4E+08 HBR018
2.3E+08 7.9 1.50E+10 1.20E+11 1.5E+08 MSB 8-6-2 2.6E+08 8 1.10E+10
8.80E+10 1.1E+08 *For YBCA5 this data is the mean of three batches
fermented using the 10 L fermenter. **ND = No data.
[0235] Results--Leaf Droplet Inoculation Assay--Efficacy (KRIP-BCA
39)
[0236] In the Nil (wetter only) treatment, the average Psa lesion
area was 57 mm.sup.2 (FIG. 2). One isolate (MSB8-6-2) did not
significantly reduce (P>0.05) Psa severity (lesion size=54
mm.sup.2) compared to the Nil control. YBCA5 significantly reduced
Psa lesion area to 43 mm2 (efficacy=25%).This assay demonstrated
that not all Aureobasidium pullulans isolates have the ability to
significantly reduce Psa severity on potted kiwifruit plants and
efficacy against Psa is dependent upon the isolate selected.
[0237] Zespri Assay 31
[0238] The aim of this assay was to compare the efficacy of YBCA5
applied alone and integrated with copper or Actigard for control of
Psa on potted plants exposed to natural Psa inoculum in a research
orchard.
[0239] Plant Material
[0240] This assay was carried out in the shadehouse structure
(Block 20) at Te Puke Research Orchard. The plants were originally
grown at the Ruakura glasshouse using tissue cultured A. chinensis
var. deliciosa `Hayward` plants grown in 1.5 L pots. Once the
plants were 25 cm in height they were re-potted onto 2.5 L pots and
moved to the Ruakura shade house and connected up to dripper
irrigation on 30 Oct. 2015. At the time of treatment on 3 Nov.
2015, each plant had at least 4 useable leaves per plant and there
were 15 replicate plants per treatment. Treatments and foliar spray
dates are described in Table 2.
TABLE-US-00002 TABLE 2 Treatment schedule for potted `Hayward`
plants exposed to natural Psa inoculum at the Te Puke Research
Orchard. 2.sup.st 3.sup.rd 4.sup.th 1.sup.st Foliar Foliar Foliar
TRT Foliar Treatment Treatment Treatment No. treatment (+10d)
(+10d) (+10d) Site Ruakura TPRO TPRO TPRO Spray Nov. 3, Nov. 13,
Nov. 23, Dec. 3, date 2015 2015 2015 2015 Rationale 1. Nu-Film*
Nu-Film Nu-Film Nu-Film Wetter control Copper 2. Kocide** Kocide
Kocide Kocide based std 3. YBCA5 YBCA5 YBCA5 YBCA5 YBCA5 4. Kocide
YBCA5 Kocide YBCA5 Integrated programme I 5. Actigard YBCA5
Actigard YBCA5 Integrated programme II *Miller Chemical &
Fertilizer Corporation, USA **DuPont, USA
[0241] Yeast Preparation
[0242] YBCA5 granules were prepared by fermenting the yeast for 3
days in a 10 L bioreactor (Labfors) using sterile liquid media (4%
molasses and 1.2 g/L urea). The fermentate was spun in a centrifuge
(Sorvall RC-5C) at 5000 rpm for 15 min (rotor no. SLC-4000,
rotorcode 33) to achieve a wet pellet of cell concentrate after
discarding the supernatant. This wet pellet was mixed with
approximately 30% (w/w) cornstarch to form a stiff dough
consistency and this was extruded through a steel mesh (3 mm hole
size) and dried in a laminar flow hood overnight (20-25.degree. C.)
to form dried granules.
[0243] All YBCA5 treatments were applied at a final concentration
of 2.times.10.sup.7 CFU/mL and plants were allowed to dry. The
final volume that was prepared ranged from 500 mL to one litre
depending on the size of the plants being treated.
[0244] Psa Inoculum Preparation
[0245] The aim of this project was to expose potted plants to Psa
inoculum in at the Te Puke
[0246] Research Orchard (Block 20). This block was surrounded by
mature kiwifruit vines with a history of Psa and this provided the
inoculum over the period of time for this assay.
[0247] Leaf Sprays
[0248] The first spray treatments were applied at Ruakura on 3 Nov.
2016 and at 10-14 day intervals thereafter. (Details are described
in Table 2 above). All treatments were applied to just prior to
run-off with a hand held pump sprayer. Copper hydroxide (Kocide
Opti) as applied at 0.7 g/L and Actigard was applied at 0.1 g/L.
YBCA5 was applied with the wetter/sticker adjuvant, Nu-Film (250 ul
per 500 ml).
[0249] Disease assessments were carried out on 17 Dec. 2015 by
estimating the percentage area of leaf necrosis on all treated
leaves.
[0250] Results--Assay Zespri 31
[0251] In the absence of any treatment (Nu-film only, control), Psa
leaf spot incidence was 66% and this was significantly reduced by
Kocide Opti (18%), YBCA5 (35%), YBCA5 and Kocide Opti (Integrated
programme I) (15%), YBCA5 and Actigard (Integrated programme II)
(22%) (FIG. 3).
[0252] This assay demonstrated that YBCA5 significantly reduced
(P<0.05) the incidence of Psa leaf spotting on potted plants
under a shaded structure when exposed to natural Psa inoculum.
Although the level of disease control was not as effective as the
Kocide Opti based programme, the assay demonstrated that YBCA5
could be successfully integrated with a copper based product and
with Actigard with no significant loss of efficacy compared with
the copper only treatment.
Example 3--Yeast Biocontrol of Phytopathogenic Fungi
[0253] YBCA5 Biocontrol of Monilinia fructicola and Botrytis
spp.
[0254] Methods
[0255] Fruit-based screening assays were conducted in laboratories
at the Plant and Food Research Ruakura Research Centre, Hamilton,
New Zealand (PFR). PFR assays focused on dip treatment application
of YBCA5 and fungicide controls.
[0256] Fruit Material (Assays 1 to 4)
[0257] Fruit for Monilinia fructicola and Botrytis spp. inoculation
assays were carried out on sweet cherries (Prunus avium `Sweet
Valentine`) that were picked at the harvest mature stage and
sourced from the PFR Clyde Research Orchard in Central Otago on 8
Jan. 2016 for fruit based assays 1 and 2. A second harvest was
carried out on 13 Jan. 2016 for fruit based assays 3 and 4.
[0258] Each cherry was then subjected to a double wash process.
Wash one consisted of 10 minutes in tap water on a rotary shaker
(110 rpm) followed by a five minute wash in SDW (Wash 2). All
cherries were placed onto sterile black plastic grids in a sterile
plastic meat tray with two sterile paper towels and were allowed to
dry in a laminar flow hood. Each cherry was dipped in the
treatments for 60 seconds and again allowed to dry, as described
above. 40 ml deionised water was added to the paper towels to
ensure high relative humidity, and then enclosed in a plastic bag
to incubate at 23.degree. C. for 24 h (Assay 1) and 48 hours (Assay
2) to allow the YBCA5 treatment to become established on the fruit
surface.
[0259] Assay 5 Fruit Material
[0260] Fruit assays were carried out on detached white table grape
berries (`Thompson seedless`--Assay 5), imported from California
that were sourced from a local supermarket in Hamilton. Each berry
was detached from the bunch with 3-4 mm of pedicel remaining and
then subjected to a double wash process. Wash one consisted of 10
minutes in tap water on a rotary shaker (110 rpm) followed by a
five minute wash in SDW (Wash 2). All berries were placed onto
sterile black plastic grids in a sterile plastic meat tray with two
sterile paper towels and were allowed to dry in a laminar flow
hood. Each berry was lightly wounded with the aid of fine
sandpaper, Grade P220, then dipped in the treatments for 60 seconds
and again allowed to dry.
[0261] YBCA5 preparation (Assays 1 to 5)
[0262] YBCA5 granules were prepared by fermenting the yeast for 3
days in a 10 L bioreactor (Labfors) using sterile liquid media (4%
molasses and 1.2 g/L urea). The fermentate was spun in a centrifuge
(Sorvall RC-5C) at 5000 rpm for 15 min (rotor no. SLC-4000,
rotorcode 33) to achieve a wet pellet of cell concentrate after
discarding the supernatant. This wet pellet was mixed with
approximately 30% (w/w) cornstarch to form a stiff dough
consistency and this was extruded through a steel mesh (3 mm hole
size) and dried in a laminar flow hood overnight (20-25.degree. C.)
to form dried granules.
[0263] The YBCA5 treatments were prepared from these water
dispersible granules that had been stored at 5-7.degree. C. in a
refrigerator and a suspension prepared by adding 1 g per litre of
deionised water (final concentration=2.times.10.sup.7 CFU/ml) and
gently stirred to form a suspension. To ensure all cells were
evenly dispersed and remained in suspension, a wetting agent
(Nu-Film) was added at 0.5 ml per litre.
[0264] Fungicides (Assays 1 to 5)
[0265] For assays 1 and 2, a liquid suspension containing 500 g/L)
was prepared at the recommended field rate of 0.85 mL/L (an average
of the recommended field rates of 0.75 mL/L for Monilinia in
stonefruit and 1.0 mL/L for Botrytis in berryfruit). No wetting
agent was used.
[0266] For assays 3 and 4, a liquid suspension of captan was
prepared from Captan Flo (Nufarm NZ) (containing captan at 600 g/L)
at the recommended field rate for use in stone fruit of 160
mL/litre. No wetter was used.
[0267] Monilinia inoculum Preparation (Assays 1 and 3)
[0268] A Monilinia fructicola culture (isolate code MFGQ3), which
had been originally isolated from an infected peach tree located in
the Hamilton region during 1998, was used for the spray inoculation
assays included in this section (Ruakura based assay). Monilinia
inoculum was prepared by growing this strain of Monilinia for 7
days on PDA (Difco, Fort Richard) medium and harvesting the conidia
by washing the plate with SDW plus Tween 80 (0.05%) to make a stock
suspension of inoculum. This stock suspension was then filtered
using a 70 .mu.m cell strainer (to remove mycelial fragments), the
concentration determined using a haemocytometer and then adjusted,
by dilution with SDW+Tw 80 (0.05%) to a final concentration of
1.times.10.sup.4 conidia/mL.
[0269] Botrytis spp. inoculum Preparation (Assay 2 and 4)
[0270] A Botrytis spp. culture (isolate code 09-2), which had been
originally isolated from an infected kiwifruit located in the Bay
of Plenty region during the 2000s, was used for the spray
inoculation assays included in this section carried out on
cherries. Botrytis spp. inoculum was prepared by growing this
strain of Botrytis spp. for 5-7 days on PDA (DIFCO, Fort Richard)
medium and harvesting the conidia by washing the plate with SDW
plus Tween 80 (0.05%) to make a stock suspension of inoculum. This
stock suspension was then filtered using a 70 .mu.m cell strainer
(70 .mu.m mesh) to remove mycelial fragments, the concentration
determined using a haemocytometer and then adjusted, by dilution
with SDW+Tw 80 (0.01% v/v) to a final concentration of
1.times.10.sup.5 conidia/mL.
[0271] Botrytis inoculum Preparation (Assay 5)
[0272] Two Botrytis spp. cultures (isolate codes 189 and 547),
which had been originally isolated from infected tomatoes, Auckland
region during 2010, were used for the droplet inoculation assays
included in this assay. One isolate was sensitive to two commonly
used fungicides (dicarboximide and carbendazim) and the other
isolate was resistance to each of these same fungicides.
[0273] Botrytis spp. inoculum was prepared by growing each isolate
of Botrytis spp. for 5-7 days on PDA (DIFCO, Fort Richard) medium
and harvesting the conidia by washing the plate with SDW plus Tween
80 (0.01%) to make a stock suspension of inoculum. This stock
suspension was then filtered using a cell strainer (70 .mu.m mesh,
Falcon) to remove mycelial fragments, the concentration determined
using a haemocytometer and adjusted to the required concentration
(2.times.10.sup.4 conidia/mL) equivalent to 200 conidia in each 10
.mu.L droplet. To ensure the conidial suspension remained on the
wounded berry surface, paraffin `wax bunds` were created around the
wound surface by smearing a 1-2 mm thick layer of paraffin wax onto
a glass slide lightly dabbing the base of a 1 mL pipette tip onto
the paraffin wax layer and then transferring this `paraffin wax
ring` onto the wound surface. This effectively created a 5-6 mm
diameter ring of paraffin wax (`bund`) that retained the conidial
suspension and prevented it from rolling off the rounded berry
surface. Wounded and treated berries were then inoculated with 10
.mu.L droplets of Botrytis spp. conidial suspension.
[0274] After pathogen inoculation, all inoculated fruit samples in
a tray were enclosed in a plastic bag for 48 hours on the lab bench
at 23.degree. C. (Assays 1-4) or 21.degree. C. (Assay 5). For all
cherry and berry assays, two sterile paper towels were placed
beneath the plastic grids, on which the berries lay, then moistened
with 40 mL SDW and each tray was enclosed in a clean plastic bag
and then sealed to maintain high relative humidity over the first
72 h. Thereafter, the bags were removed, folded over the trays (to
ensure each end of the tray was open for adequate air flow), to
allow the relative humidity to decline over a 15-h period (5 pm in
the late afternoon to 8 am the next morning) after which, they were
resealed. This process of alternating the relative humidity within
the incubation chambers was repeated over the duration of the
experiment and is a method that has been shown to avoid excessive
mycelial growth. After 5 days incubation, fruit with typical
Monilinia spp. or Botrytis spp. symptoms were recorded and removed
from each replicate tray. Rots were recorded daily and the
incidence (%) of cumulative Monilinia spp. or Botrytis spp. rots,
after 13 days (Assay 1 and 2), 16 days (Assay 3 and 4) and 9 days
(Assay 5). In assay 5, the severity of Botrytis spp. infection was
determined for each treatment by visually scoring the proportion of
the berry surface covered in Botrytis spp. conidiophores.
[0275] YBCA5 Biocontrol of Colletotrichum spp. and Penicillium spp.
in Apples
[0276] Apple Assays 6 and 7
[0277] Apple fruit (`Pacific Rose`) were sourced from an organic
orchard in Hawkes Bay and were washed in running tap water in a 10
L bucket. Apples were then dried in a biohazard hood for
approximately 1.5 h, turning them after 45 min and then wiped with
a tissue soaked in ethanol and allowed to dry once more. Apples
were then placed onto moistened paper towels lining the bottom of
plastic clam shell containers, two apples per container. There were
10 replicate apples for each treatment in assays 6 and 7.
[0278] There were five treatments: a Nil control (0.05% Tween80),
Fungicide (0.5 mL/L of Prolific (containing 500 g/L carbendazim)),
YBCA5 applied at 1.times.10.sup.7 CFU/mL 24 hours prior to the
pathogen, YBCA5 applied at 1.times.10.sup.7 CFU/mL 2 hours prior to
the pathogen and pathogen only (Colletotrichum spp. for assay 6 and
Penicillium spp. for assay 7). YBCA5 granules were prepared by
fermenting the yeast for 3 days in a 10 L bioreactor (Labfors)
using sterile liquid media (4% molasses and 1.2 g/L urea). The
fermentate was spun in a centrifuge (Sorvall RC-5C) at 5000 rpm for
15 min (rotor no. SLC-4000, rotorcode 33) to achieve a wet pellet
of cell concentrate after discarding the supernatant. This wet
pellet was mixed with approximately 30% (w/w) corn-starch to form a
stiff dough consistency and this was extruded through a steel mesh
(3 mm hole size) and dried in a laminar flow hood overnight
(20-25.degree. C.) to form dried granules.
[0279] The YBCA5 treatments were prepared from these water
dispersible granules that had been stored at 5-7.degree. C. in a
refrigerator and a suspension prepared by adding 0.5 g per litre of
deionised water (final concentration=1.times.10.sup.7 CFU/mL) and
gently stirred to form a suspension.
[0280] The pathogen spore suspensions were prepared from cultures
of Colletotrichum spp. and Penicillium spp. grown on PDA. One third
of the culture was removed from the PDA Petri dish and transferred
into a 50 mL Falcon tube containing 30 mL of SDW (with 0.05%
Tween80). This was shaken vigorously for 1 min to dislodge spores
into the suspension and then passed through a 70 .mu. cell strainer
to remove any mycelial fragments. The spore concentration was
calculated using the aid of a haemocytometer and dilutions made to
achieve a final concentration of 1.times.10.sup.5 spores/mL
[0281] On the day the apples were washed and prepared (day 1), a
small wound (3 mm diameter.times.2-3 mm deep) were made in the side
of each apple and numbered as treatment 3. A 10 .mu. aliquot of
YBCA5 suspension was added to the wound, enough to fill the wound.
On the following day (day 2) four more wounds were made and
numbered as treatments 1, 2, 4 and 5. Treatments 1, 2 and 4 were
applied to the wounds by adding 10 .mu.L aliquots of each solution
to the respective wounds. Two hours later the pathogen was applied
by adding 10 .mu.L aliquots of Colletotrichum spp. to each wound
(treatments 2, 3, 4 and 5) for assay 6 and adding 10 .mu.L aliquots
of Penicillium spp. to each wound (treatments 2, 3, 4 and 5) for
assay 7. Thirty mL of additional SDW was added to the paper towel
in each clam shell container to maintain relative humidity and the
containers were incubated in a Sanyo incubator at 23.degree. C. for
1-2 weeks to allow rots to develop.
[0282] The lesion diameter of rots were measured using digital
callipers after 8 days. Lesion size was corrected for the diameter
of the wound by subtracting 3 mm from each measurement and then
ANOVA was carried out using Genstat to test for treatment
differences based on least significant differences.
[0283] Experimental Design
[0284] The cherry assays (assays 1-4) consisted of 10 cherries per
replicate and there were six replicates (assays 1 and 2) and eight
replicates (assays 3 and 4) for each treatment in a randomised
block layout.
[0285] The grape berry assay (assay 5) consisted of five berries
per replicate and there were four replicates for each treatment in
a randomised block layout.
[0286] Statistical Analysis
[0287] Data were analysed using GenStat, 13th edition, with a
randomised block experimental design and analysis of variance.
Average fruit infection (% incidence) were log-transformed to
equalise the variance to better meet the normality assumptions of
the analysis. Raw data means and Least Significant Differences
(LSDs) are presented, however all statistical comparisons are based
on the log analysis.
[0288] Results of Cherry Inoculation Assays
[0289] Assay 1
[0290] FIG. 9 summarises the effect of YBCA5 against Monilinia
fruit rot in cherries. The incidence of Monilinia fruit rot in the
Nil treatment was 50% and although the YBCA5 (34%) and iprodione
treatments (22%) had a lower incidence of Monilinia, these were not
significant reductions compared with the Nil treatment (FIG.
9).
[0291] Assay 2
[0292] FIG. 10 summarises the effect of YBCA5 against Botrytis spp.
fruit rot in cherries. The incidence of Botrytis spp. fruit rot in
the nil treatment was 35% and this was not significantly
(P>0.05) reduced in the YBCA5 (24%) and iprodione treatment
(22%) (FIG. 10).
[0293] Assay 3
[0294] FIG. 11 summarises the effect of YBCA5 against Monilinia
fruit rot in cherries in another assay (Assay 3). The incidence of
Monilinia fruit rot in the nil treatment was 88% and this was not
significantly (P>0.05) reduced by the YBCA5 (59%). The captan
fungicide treatment (12%) significantly reduced (P<0.001) the
incidence of Monilinia fruit rot compared with the Nil treatment
(FIG. 11).
[0295] Assay 4
[0296] FIG. 12 summarises the effect of YBCA5 against Botrytis spp.
fruit rot in cherries in another assay. The incidence of Botrytis
spp. fruit rot in the nil treatment was 67% and this was not
significantly reduced (P>0.05) in the YBCA5 (49%) and captan
treatment (43%) (FIG. 12).
[0297] Results of Grape Inoculation Assay
[0298] Assay 5
[0299] FIG. 13 summarises the effect of YBCA5 against Botrytis spp.
fruit rot in table grapes in another assay (Assay 5). The incidence
of Botrytis spp. fruit rot in the nil treatment 30% and this was
significantly (P<0.001) reduced by the YBCA5 (7%) and captan
treatment (10%) (FIG. 13).
[0300] Treatment of Other Diseases--Apple Results for Apple Assays
6 and 7
[0301] Assay 6
[0302] The mean lesion size in the untreated Colletotrichum control
was 10.1 mm and this was significantly (P<0.05) reduced in the
fungicide and both of the YBCA5 treatments (Table 3).
[0303] Applying YBCA5 24 hrs before the pathogen provided better
protection than applying 2 hrs before the pathogen.
TABLE-US-00003 TABLE 3 Average lesion size on wounded apples
("Pacific Rose") treated with fungicide and YBCA5 prior to
inoculation with Colletotrichum spp. spores (1 .times. 10.sup.5
spores/mL) and assessed after 8 days incubation at 23.degree. C.
Average lesion Treatment (day 8) Significance Nil (No
Colletotrichum) 0.3 d Fungicide (Prolific) 1.1 cd YBCA5 (24 hr) 2.2
c YBCA5 (2 hr) 5.7 b Nil + Colletotrichum 10.1 a LSD (5%) 1.42 P
value <0.001
[0304] Treatment means followed by different letters show
significant difference.
[0305] Assay 7:
[0306] The mean lesion size in the untreated Penicillium control
was 15.2 mm and this was significantly (P<0.05) reduced in the
fungicide and both of the YBCA5 treatments (Table 4). Similar to
the previous assay, applying YBCA5 24 h before the pathogen
provided significantly better protection than applying YBCA5 2 h
before the pathogen.
TABLE-US-00004 TABLE 4 Average Lesion size on wounded apples
("Pacific Rose" treated with fungicide and YBCA5 prior to
inoculation with Penicillium spp. spores (1 .times. 10.sup.5
spores/mL) and assessed after 8 days incubation at 23.degree. C.
Treatment Average lesion Significance Nil (no Penicillium) 0.3 c
Fungicide (Prolific) 0.4 c YBCA5 24 h 0.4 c YBCA5 2 h 3.8 b Nil +
Penicillium 15.2 a LSD (5%) 3.51 P value <0.001
[0307] Treatment means followed by different letters show
significant difference.
[0308] Assay 8: Post-Harvest Rot Due to Phytopathogenic Fungi on
`Hongyang` Kiwifruit
[0309] The most important export cultivar from China is `Hongyang`
and this red and yellow-fleshed cultivar is attacked by a range of
postharvest pathogens including Penicillium spp. Phomopsis spp.,
Alternaria spp., Colletotrichum spp., Cryptosporiopsis spp. and
Botrytis spp.
[0310] We investigated the efficacy of YBCA5 applied as a wound
protectant against a range of postharvest fungal pathogens of
fruits, particularly: Penicillium spp. Phomopsis spp., Alternaria
spp., Colletotrichum spp., Cryptosporiopsis spp. and Botrytis
spp.
[0311] Preliminary tests were carried out to establish the
concentration of each pathogen that was required to rot the fruit
after wounding in the absence of any treatment.
[0312] Methods
[0313] The kiwifruit `Hongyang`-based screening assays (assays 8
and 9) were conducted in laboratories at the Plant and Food
Research Ruakura Research Centre, Hamilton, New Zealand (PFR). PFR
assays focused on wound application of YBCA5 treatment, a
commercial biological control treatment and a fungicide were used
as comparative controls.
[0314] Fruit Material (Assays 8)
[0315] The `Hongyang` kiwifruit were sourced from the PFR Riwaka
Research Orchard in
[0316] Motueka on 12 Apr. 2017 for fruit based assays 8 and 9.
Penicillium spp. Phomopsis spp., Alternaria spp., Colletotrichum
spp., Cryptosporiopsis spp. and Botrytis spp. inoculation assays
were carried out on `Hongyang` kiwifruit that were picked at the
harvest mature stage.
[0317] After removal from cool storage at 1.degree. C., each fruit
was subjected to a triple wash process. Wash one consisted of 30
seconds in 70% ethanol then a wash in tap water for 10 minutes on a
rotary shaker (80 rpm-Wash 2) followed by a final wash for five
minutes in SDW-Wash 3). All fruit were placed onto sterile black
plastic grids in a sterile plastic meat tray with two sterile paper
towels and were allowed to dry overnight in a laminar flow
hood.
[0318] Just prior to wound treatment, each fruit was wounded on the
side with a sterile stainless steel spike (4 mm deep.times.3 mm
wide) and 10 .mu.I of each treatment suspension was pipetted into
the wound and allowed to dry.
[0319] Treatments and Rates are Detailed Below
TABLE-US-00005 Treatment Recommended rate gm or mL/Litre YBCA5* 1
.times. 10.sup.7 0.5 g/L Serenade Opti* 125 g/100 L 1.25 g/L Rovral
Aquaflo 75 mL/100 L 0.75 mL/L *YBCA5 and Serenade Opti prepared in
Tween 80 (0.05%), Rovral Aquaflo was prepared in deionized
water.
[0320] For assays 8 and 9, two sterile paper towels were placed
beneath Plix cut-outs, moistened with 40 mL SDW and each fruit
placed in disposable lunch boxes (Plix Extra Deep 45/45, containing
five Plix fruit cut-outs to prevent fruit from moving), sealed,
then placed into large (40 L) plastic bins which were closed to
ensure high relative humidity for the first 24 h and incubated on
the lab bench at 24.degree. C. After 24 h, the Plix lunch boxes
were removed from the bins and a 10 ul suspension of each fungal
pathogen was pipetted into the treated wounds. All lunch boxes were
resealed and placed back into the large plastic bins to ensure high
relative humidity for the next 48 h. After this time, the Plix
lunch boxes were removed from the large plastic bins, and a pin
placed between the Plix box lid and base to allow some air to
circulate and the relative humidity to decline over a 15 h period
(5 pm in the late afternoon to 8 am the next morning) after which,
they were resealed again. This process of alternating the relative
humidity within the incubation chambers was repeated over the
duration of the experiment and is a method that has been shown to
avoid excessive mycelial growth. After 6 days, (Alternaria spp.
Botrytis spp., Penicillium spp., Phomopsis spp., Colletotrichum
spp.) and 7 days (Cryptosporiopsis spp.), the severity of fungal
rot was assessed for each treatment by measuring the lesion length
(mm) along the axis of the fruit. Data were expressed as the
average lesion length, minus the initial width of the wound (3
mm).
[0321] YBCA5 Preparation (Assay 8 and 9)
[0322] YBCA5 granules were prepared by fermenting the yeast for 3
days in a 10 L bioreactor (Labfors) using sterile liquid media (4%
molasses and 1.2 g/L urea). The fermentate was spun in a centrifuge
(Sorvall RC-5C) at 5000 rpm for 15 min (rotor no. SLC-4000,
rotorcode 33) to achieve a wet pellet of cell concentrate after
discarding the supernatant. This wet pellet was mixed with
approximately 30% (w/w) corn-starch to form a stiff dough
consistency and this was extruded through a steel mesh (3 mm hole
size) and dried in a laminar flow hood overnight (20-25.degree. C.)
to form dried granules.
[0323] The YBCA5 treatments were prepared from these water
dispersible granules that had been stored at 5-7.degree. C. in a
refrigerator and a suspension prepared by adding 0.5 g per litre of
deionised water (final concentration=1.times.10.sup.7 CFU/ml) and
gently stirred to form a suspension.
[0324] Postharvest Pathogen Preparation
[0325] Alternaria spp. inoculum Preparation (Assay 8)
[0326] The Alternaria spp. culture (isolate code=`Alternaria ex
cherry`), which had been originally isolated from an infected
cherry fruit from Central Otago during 2016, was used for the wound
inoculation part of this assay. Alternaria spp. inoculum was
prepared by growing this strain of Alternaria spp. for 21 days on
Oat Meal Agar) medium and harvesting the conidia by washing the
plate with SDW plus Tween 80 (0.05%) to make a stock suspension of
inoculum. This stock suspension was then filtered using a 70 .mu.m
cell strainer (to remove mycelial fragments), the concentration
determined using a haemocytometer and then adjusted, by dilution
with SDW+Tw 80 (0.05%) to a final concentration of 2.times.10.sup.4
conidia/mL.
[0327] Botrytis spp. inoculum Preparation (Assay 8)
[0328] A Botrytis spp. culture (isolate code 09-2), which had been
originally isolated from an infected kiwifruit located in the Bay
of Plenty region during the 2000s, was used for the spray
inoculation assays included in this section carried out on
cherries. Botrytis spp. inoculum was prepared by growing this
strain of Botrytis spp. for 12 days on Oat Meal Agar medium and
harvesting the conidia by washing the plate with SDW plus Tween 80
(0.05%) to make a stock suspension of inoculum. This stock
suspension was then filtered using a 70 .mu.m cell strainer (70
.mu.m mesh) to remove mycelial fragments, the concentration
determined using a haemocytometer and then adjusted, by dilution
with SDW+Tw 80 (0.01% v/v) to a final concentration of
1.times.10.sup.5 conidia/mL.
[0329] Colletotrichum spp. inoculum Preparation (Assay 8)
[0330] The Colletotrichum spp. culture (isolate code=`ex G3), which
had been originally isolated from an infected Gold3 kiwifruit from
the Ruakura research orchard during 2017, was used for the wound
inoculation part of this assay. Colletotrichum spp. inoculum was
prepared by growing this strain of Colletotrichum spp. for 21 days
on PDA (Difco, Fort Richard) medium and harvesting the conidia by
washing the plate with SDW plus Tween 80 (0.05%) to make a stock
suspension of inoculum. This stock suspension was then filtered
using a 70 .mu.m cell strainer (to remove mycelial fragments), the
concentration determined using a haemocytometer and then adjusted,
by dilution with SDW+Tw 80 (0.05%) to a final concentration of
2.times.10.sup.4 conidia/mL.
[0331] Penicillium spp. inoculum Preparation (Assay 8)
[0332] The Penicillium spp. culture (isolate code=`Penicillium ex
lemon`), which had been originally isolated from an infected lemon
fruit from a supermarket in 2017, was used for the wound
inoculation part of this assay. Penicillium spp. inoculum was
prepared by growing this strain of Penicillium spp. for 12 days on
PDA (Difco, Fort Richard) medium and harvesting the conidia by
washing the plate with SDW plus Tween 80 (0.05%) to make a stock
suspension of inoculum. This stock suspension was then filtered
using a 70 .mu.m cell strainer (to remove mycelial fragments), the
concentration determined using a haemocytometer and then adjusted,
by dilution with SDW+Tw 80 (0.05%) to a final concentration of
2.times.10.sup.4 conidia/mL.
[0333] Phomopsis spp. inoculum Preparation (Assay 8)
[0334] The Phomopsis spp. culture (isolate code=`Phomopsis ex G3`),
which had been originally isolated from an infected Gold3 kiwifruit
from the Ruakura research orchard during 2017, was used for the
wound inoculation part of this assay. Phomopsis spp. inoculum was
prepared by growing this strain of Phomopsis spp. for 21 days on
PDA (Difco, Fort Richard) medium and harvesting the conidia by
washing the plate with SDW plus Tween 80 (0.05%) to make a stock
suspension of inoculum. This stock suspension was then filtered
using a 70 .mu.m cell strainer (to remove mycelial fragments), the
concentration determined using a haemocytometer and then adjusted,
by dilution with SDW+Tw 80 (0.05%) to a final concentration of
2.times.10.sup.4 conidia/mL.
[0335] Cryptosporiopsis spp. inoculum Preparation (Assay 9
Only)
[0336] The Cryptosporiopsis spp. culture (isolate
code=`Cryptosporiopsis ex G3`), which had been originally isolated
from an infected Gold3 kiwifruit from the Te Puke Research Orchard
was used for the wound inoculation in assay 9. Cryptosporiopsis
spp. inoculum was prepared by growing this strain of
Cryptosporiopsis spp. for 28 days on PDA (Difco, Fort Richard)
medium and harvesting the conidia by washing the plate with SDW
plus Tween 80 (0.05%) to make a stock suspension of inoculum. This
stock suspension was then filtered using a 70 .mu.m cell strainer
(to remove mycelial fragments), the concentration determined using
a haemocytometer and then adjusted, by dilution with SDW+Tw 80
(0.05%) to a final concentration of 2.times.10.sup.4
conidia/mL.
[0337] After pathogen inoculation in assay 8 and 9, all inoculated
fruit samples were placed in disposable lunch boxes (Plix Extra
Deep 45/45), containing five Plix fruit cut-outs to prevent fruit
from moving), and 40 mL SDW added to two sterile paper towels that
were placed beneath the Plix cut-outs, then placed into large (40
L) plastic bins which were closed to ensure high relative humidity
for the first 48 h and incubated on the lab bench with natural and
fluorescent light at 24.degree. C. for up to three days. After 48
h, the Plix lunch boxes were removed from the bins and a pin placed
between the lid and base to allow air to circulate and the relative
humidity to decline over a 15 h period (5 pm in the late afternoon
to 8 am the next morning) after which, they were resealed again.
This process of alternating the relative humidity within the
incubation chambers was repeated over the duration of the
experiment and is a method that has been shown to avoid excessive
mycelial growth. After 6 to 7 days the severity of fungal rot
infection was assessed for each treatment by measuring the lesion
length (mm) along the axis of the fruit. Data were expressed as the
average lesion length, minus the initial width of the wound (3
mm).
[0338] Experimental Design
[0339] The kiwifruit `Hongyang` assay 8 consisted of 4 `Hongyang`
kiwifruit per replicate and there were five replicates for each
treatment in a randomised block layout. In total there were 22
treatments, including a Nil (no wound and no treatment) no pathogen
inoculation) control, and a Nil (plus wound then SDW+Tw80) no
pathogen inoculation control.
[0340] In Assay 9
[0341] The kiwifruit `Hongyang` assay 9 consisted of 4 `Hongyang`
kiwifruit per replicate and there were four replicates for each
treatment in a randomised block layout. In total there were 5
treatments, including a Nil (no wound and no treatment) no pathogen
inoculation control, and a Nil (plus wound then SDW+Tw80) no
pathogen inoculation control.
[0342] Statistical Analysis
[0343] Data were analysed using GenStat, 13th edition, with a
randomised block experimental design and analysis of variance.
Average lesion diameter did not require data transformation to
equalise the variance and raw data means and Least Significant
Differences (LSDs) are presented.
[0344] Results
[0345] Assay 8
[0346] YBCA5 treated fruit had significantly smaller lesions in
`Hongyang` fruit than the Rovral
[0347] Aquaflo, Serenade Opti treatments and the untreated control
when the fruit were inoculated with Alternaria, Colletotrichum,
Penicillium and Phomopsis. YBCA5 treated fruit had lesions not
significantly smaller than Rovral Aquaflo when the fruit were
inoculated with Botrytis spp., but they were significantly smaller
than the Serenade Opti treatment and untreated control.
[0348] Rovral Aquaflo treated fruit had lesions significantly
smaller than the untreated control when fruit were inoculated with
Botrytis spp. and Penicillium spp. Serenade Opti treated fruit had
lesions significantly smaller than the Untreated control when fruit
were inoculated with Phomopsis (FIG. 14).
[0349] In assay 9, YBCA5 treated fruit also had significantly
smaller lesions in `Hongyang` fruit than the Rovral Aquaflo,
Serenade Opti treatments and the untreated control when the fruit
were inoculated with Cryptosporiopsis (FIG. 15).
[0350] Discussion
[0351] YBCA5 demonstrated activity against a range of postharvest
kiwifruit fruit pathogens when it was allowed to colonise a wound
site 24 hours before a pathogen was introduced to the same wound.
Wounding works well as an experimental technique to demonstrate the
activity of some biopesticides.
[0352] Overall, Rovral Aquaflo performed poorly in these
experiments, and this agrichemical may be unsuitable for wound
protection assays and against the pathogens used in this study.
[0353] Serenade Opti performed poorly in these assays, and this
biopesticide may be unsuitable as a wounded fruit wound protectant
against the pathogens used in these assays.
Example 5--Yeast biocontrol of PSA in the Field
SUMMARY
[0354] Grower Standard Treatment: Kocide Opti+1.times.Kasumin
TABLE-US-00006 1 Product: 2 Active Ingredient (Al) 3 Kocide: 4
Copper Hydroxide 5 Kasumin: 6 Kasugamycin 7 ActiGard: 8
Acibenzolar-S-methyl
[0355] Methodology: all plants had an application of copper at bud
break, the treatment group then received the yeast treatment while
the controls received no treatment and grower standard
respectively.
[0356] Field Trials 2015-16
[0357] Methods
[0358] Two field trial sites were established during spring 2015
with the intention of running across two consecutive seasons. The
two `Hayward` blocks (coded Block B and C) were located in separate
orchards, near Maketu, Bay of Plenty. Block C was the same orchard
as the 2014-15 trial, but a different area in the blocks was used.
Vines in all blocks were pergola trained with a single vine per
bay. The vines generally looked healthy at the commencement of the
trial, but had suffered from significant Psa symptoms 2-3 years
previously, according to the grower.
[0359] The spray treatments were applied to individual vines (eight
replicates per treatment) laid out in a randomised block design.
The treatments were: [0360] 1. Nil--no Psa control products applied
during the growing season [0361] 2. Grower standard--copper-based
foliar spray programme, including up to one antibiotic spray [0362]
3. YBCA5--yeast-based foliar spray programme (2.times.10.sup.7
CFU/mL)
[0363] Spray applications in the Grower standard and YBCA5
treatments were applied according to the schedule in Table 5.
Common agricultural adjuvants were added to the Grower standard
(0.04% Du-Wett.RTM.) and YBCA5 (0.03% Nu-Film-17.RTM.)
applications.
TABLE-US-00007 TABLE 5 Application dates in Blocks B and C for the
Grower standard and YBCA5 treatment to kiwifruit against
Pseudomonas syringae pv. actinidiae (Psa) from bud burst to post
flowering during the 2015-16 season. Block B Block C Date Grower
Grower Treatment standard YBCA5 standard YBCA5 Oct. 6, 2015 Kocide
Opti.sup.1 YBCA5.sup.2 -- -- Oct. 7, 2015 -- -- Kocide Opti YBCA5
Oct. 16, 2015 Kasumin.sup.3 YBCA5 Kasumin YBCA5 Oct. 27, 2015
Kocide Opti YBCA5 Kocide Opti YBCA5 Nov. 4, 2015 -- -- Kocide Opti
YBCA5 Nov. 6, 2015 Kocide Opti YBCA5 -- -- Nov. 19, 2015 Nil YBCA5
Nil YBCA5 (flowering) (flowering) Dec. 21, 2015 Nil (post YBCA5 Nil
(post YBCA5 fruit set) fruit set) .sup.1Kocide .RTM. Opti .TM.
applied at 70 g/100 L with Du-Wett .RTM. (0.04%) .sup.2YBCA5
applied at 100 g/100 L with Nu-Film-17 .RTM. (0.03%) .sup.3Kasumin
.RTM. applied at 500 g/100 L (no adjuvant).
[0364] The YBCA5 yeast was produced by liquid fermentation, sourced
from three separate production facilities: PFR (Ruakura),
AgResearch (Lincoln) and Callaghan Innovation (Lower Hutt). A
concentrated yeast pellet after centrifugation was supplied to the
laboratory at Ruakura and this was mixed with an inert carrier and
extruded to form granules which were air dried overnight in a
laminar flow hood. The number of colony forming units/g was
calculated by dissolving 0.2 g samples into 20 mL phosphate
buffered saline amended with 0.05% Tween 80 (PBSTw). This was
serially diluted and 10-4 droplets of each dilution were
transferred to Petri dishes with malt yeast extract agar amended
with chloramphenicol. Petri dishes were incubated for 24 h at
25.degree. C. and then 4-6.degree. C. for a further 24 h, prior to
counting the number of colonies. The concentration of YBCA5 in the
granules was 2.1.times.10.sup.10 CFU/g. All granules were stored in
airtight containers in a refrigerator (4-6.degree. C.) and were
weighed out at a rate of 100 g/100 L to achieve the target spray
concentration of 2.times.10.sup.7 CFU/mL.
[0365] Psa disease assessments were carried out in the `Hayward`
blocks just prior to flowering on 11 Nov. 2015 by visually
assessing leaves for Psa spotting severity (% leaf area with
necrosis). Assessments were carried out on leaves positioned
between the second and third wire out from the main cordon.
Assessments were commenced after taking one step (i.e. 1 m) from
the edge of the plot and a set of 25 leaves were scored. This
process was repeated after taking another step down the plot. This
was then repeated down the other side of the vine so that four sets
of 25 leaves (total=100) were scored within each plot. Similarly,
100 buds/plot were scored for the severity of bud-browning, as
described above.
[0366] Data were expressed as Psa incidence (based on the
proportion of leaves with Psa spotting/proportion of buds with
brown sepals) and average Psa severity (mean % leaf area
necrotic/mean number of brown sepals on buds). Data were
log-transformed and analysed by ANOVA using Genstat (ver. 16) to
determine treatment differences. Raw data are presented with
statistical differences indicated based on the log-transformed
data. Analysis was also carried out by combining data from both
orchard sites, after checking for any significant site x treatment
interaction.
[0367] Results
[0368] Analysis of the incidence and severity of leaves with leaf
spotting (necrosis), and similarly for bud symptoms, indicated that
there was no significant Treatment x Orchard interaction; therefore
data are presented as the mean of the two orchards (Blocks B and
C).
[0369] The nil control had a mean incidence of leaves with necrosis
of 50% and this was significantly reduced (P<0.05) by the Kocide
Opti and two YBCA5 treatments (Table 6). The efficacy of the YBCA5
was 33%. The Grower standard treatment (Kocide Opti+Kasumin) had an
efficacy of 74% and resulted in a further significant reduction in
the incidence of leaves with necrosis, compared with the YBCA5 and
Kocide Opti treatments.
[0370] The mean severity of leaf spotting was only 0.24% in the nil
control (Table 6). However, there was still a significant reduction
(P<0.05) in the mean severity of leaf necrosis in each of the
treatments, compared with the nil control, including the two YBCA5
treatments. The average efficacy of the YBCA5 was 58%, compared
with the Grower standard and Kocide Opti treatments which had an
efficacy of 91 and 73%, respectively.
[0371] The incidence of flower buds in the nil control with
necrotic sepals was 61% and this was significantly (P<0.05)
reduced by each of the treatments (Table 6). The two YBCA5
treatments had 39 and 37% incidence of buds with necrosis and an
average efficacy of 38%. The Grower Standard treatment had
significantly (P<0.05) less bud necrosis (13%) compared with
each of the other treatments (efficacy 79%). The Kocide Opti
treatment had an efficacy of 40%.
[0372] The mean severity of bud necrosis (number of necrotic
sepals/bud) was 1.29 in the Nil control and similarly to the
incidence of buds with necrosis, this was significantly (P<0.05)
reduced by each of the treatments, with the Grower Standard
treatment (0.21, efficacy =84%) providing the biggest reduction
(Table 6). The average efficacy of the YBCA5 treatments was 46% and
this was similar to the Kocide Opti treatment (efficacy 38%).
TABLE-US-00008 TABLE 6 Mean incidence and severity of leaf spotting
and bud-browning on Actinidia chinensis var. deliciosa `Hayward`
vines in Block B and C following foliar application of YBCA5 in
comparison to a Kasumin .RTM. and Kocide .RTM. Opti .TM. based
Grower standard foliar treatment and Kocide Opti only, assessed on
Nov. 11, 2015. Severity of % leaves Severity of % buds with bud
with spotting spotting necrosis browning Treatment (incidence) (%
leaf area) (incidence) (No. sepals) Nil control 50 a 0.24 a 61 a
1.29 a Grower std 13 c 0.02 b 13 c 0.21 c YBCA5 33 b 0.11 b 39 b
0.68 b Kocide 27 b 0.07 b 45 b 0.80 b Opti * YBCA5 * 34 b 0.10 b 37
b 0.72 b LSD 8.0 0.099 9.9 0.226 P value <0.001 <0.001
<0.001 <0.001 Grower std is one application Kasumin .RTM. and
three applications of Kocide .RTM. Opti .TM. applied in Du Weft
.RTM. (0.04% v/v) YBCA5 is a formulated developmental biological
control agent applied at 100 g/100 L in Nu-Film-17 .RTM. (0.03%
v/v) Analysis performed by combining data from the two orchard
trial sites * Treatment where Trichoderma was soil applied to plots
1 day prior to these disease assessments LSD is Least significant
difference (P < 0.05) Means followed by the same letters are not
significantly different to each other (P < 0.05).
[0373] Field Trials 2016-17
[0374] Methods
[0375] During the winter months the grower applied his standard
winter spray programme of Kocide Opti and in the spring the same
treatments as above were applied to the same vines. Spray
applications commenced with the grower applying Kocide Opti across
the trial block at early bud-burst (2 Oct. 2016) and this was then
followed by the schedule outlined in Table 7, with the nil control
plots receiving no spray applications. The Trichoderma treatments
were also continued in these two field trials.
[0376] The YBCA5 yeast granules used this season were produced as a
pre-commercial batch (YBCA5 e-nema-2) by the manufacturing company,
e-nema GmbH, by liquid fermentation and fluidized bed drying.
Granules of YBCA5 were imported to New Zealand on 23 Sep. 2016 and
were stored in an air tight container at 4-6.degree. C. until
required for treatment application. These granules had a mean CFU/g
of 3.times.10.sup.10 on 26 Sep. 2016 and 2.6.times.10.sup.10 when
tested for viability on 2 Nov. 2016.
[0377] The application rate for YBCA5 this season was set at the
likely commercial rate of 50 g/100 L (to achieve a minimum
concentration of 1.times.10.sup.7 CFU/mL) and was not adjusted for
the actual viability in the granules, indicating that the actual
application dose ranged from 1.25-1.5.times.10.sup.7 CFU/mL.
Disease assessments on leaves and buds were carried out as
described for the previous season (above).
TABLE-US-00009 TABLE 7 Application dates in Blocks B and C for the
Grower standard and YBCA5 treatment to kiwifruit against
Pseudomonas syringae pv. actinidiae (Psa) from budburst to post
flowering during the 2016-17 season. Block B Block C Date Grower
Grower Treatment standard YBCA5 standard YBCA5 Oct. 11, 2016 Kocide
Opti.sup.1 YBCA5.sup.2 Kocide Opti YBCA5 Oct. 18, 2016
Kasumin.sup.3 YBCA5 Kasumin YBCA5 Oct. 27, 2016 Kocide Opti YBCA5
Kocide Opti YBCA5 Nov. 8, 2016 Kocide Opti YBCA5 Kocide Opti YBCA5
Nov. 14, 2016 Nil YBCA5 Nil YBCA5 (flowering) (flowering) Dec. 5,
2016 Nil (post YBCA5 Nil (post YBCA5 fruit set) fruit set)
.sup.1Kocide .RTM. Opti .TM. applied at 70 g/100 L with Du-Wette
(0.04%) .sup.2YBCA5 applied at 50 g/100 L with Bond .RTM. Xtra
(0.06%) .sup.3Kasumin .RTM. applied at 500 g/100 L (no
adjuvant).
[0378] Psa disease assessments on flower buds were carried out in
the `Hayward` blocks just prior to flowering on 11 Nov. 2016 by
visually assessing 100 buds/plot for the severity of bud-browning,
as described above. Leaves were assessed for Psa spotting severity
(% leaf area with necrosis) in these trial plots on 18 Nov. 2016.
As described above, assessments were carried out on leaves
positioned between the second and third wire out from the main
cordon. Assessments were commenced after taking one step (i.e. 1 m)
from the edge of the plot and a set of 25 leaves were scored. This
process was repeated after taking another step down the plot. This
was then repeated down the other side of the vine so that four sets
of 25 leaves (total=100) were scored within each plot.
[0379] Results
[0380] In the second year of this field trial the incidence of leaf
spotting was 31% in the nil control and this was significantly
reduced in the two YBCA5 treatments (Table 8). The efficacy of the
YBCA only treatment was 42%. The Grower standard treatment, which
included the bactericide Kasumin, provided a significant further
reduction in the incidence of leaf spotting, compared with the
YBCA5 treatments (efficacy=74%).
[0381] The mean severity of leaf spotting was 0.19% in the nil
control and this was significantly reduced in the YBCA5 treatments,
compared with the nil control (Table 8). The efficacy of the YBCA
only treatment was 53%. There was a further reduction in the Grower
standard treatment, but in this case the Grower standard and YBCA5
treatments were not significantly different to each other.
[0382] In the nil control, the incidence of buds with necrosis was
18% and the severity of the necrosis was 0.27. The two YBCA5
treatments significantly reduced bud incidence and severity
compared with the nil control (efficacy of YBCA5 only=56 and 59%,
respectively). Although the Grower standard had less disease than
the YBCA5 treatments these were not significantly different.
TABLE-US-00010 TABLE 8 Mean incidence and severity of leaf spotting
and bud-browning on Actinidia chinensis var. deliciosa `Hayward`
vines in Block B and C following foliar spray application of YBCA5
in comparison to a Kasumin .RTM. and Kocide .RTM. Opti .TM. based
Grower standard foliar treatment and a Trichoderma treatment,
assessed on Nov. 11, 2016 (buds) and Nov. 18, 2016 (leaves).
Severity Severity % of % of leaves spotting buds bud with (% with
browning spotting leaf necrosis (No. Treatment (incidence) area)
(incidence) sepals) Nil control 31 a 0.19 a 18 a 0.27 a Grower std
8 c 0.02 c 4 b 0.05 b YBCA5 18 b 0.09 bc 8 b 0.11 b Trichoderma 29a
0.17 ab 14 a 0.20 a YBCA5 * 18 b 0.08 bc 8 b 0.09 b LSD 8.4 0.092
5.1 0.085 P value <0.001 ** 0.005 <0.001 <0.001 Grower std
is one application Kasumin .RTM. and three applications of Kocide
.RTM. Opti .TM. applied in Du Wett .RTM. (0.04% v/v) YBCA5 is a
formulated developmental biological control agent applied at 100
g/100 L in Bond .RTM. Xtra (0.03% v/v) Analysis performed by
combining data from the two orchard trial sites * YBCA5 treatment
where Trichoderma had been applied to the soil of these plots on
three occasions during the previous 12 months ** There was a
significant site .times. treatment interaction (P = 0.016) for this
variable, such that there was no significant treatment effects in
Block C and in Block B there was a highly significant treatment
effect (P < 0.001) with the treatment difference being the same
as indicated in this combined analysis LSD is Least significant
difference (P < 0.05) Means followed by the same letters are not
significantly different to each other (P < 0.05).
[0383] Conclusion:
[0384] YBCA5 treatment showed significant reductions in incidence
and severity of leaf spotting and bud browning in `Hayward`
vines.
[0385] It will be appreciated that the above description is
provided by way of example only and that variations in both the
materials and techniques used which are known to those persons
skilled in the art are contemplated.
[0386] Although the invention has been described by way of example
and with reference to particular embodiments, it is to be
understood that modifications and/or improvements may be made
without departing from the scope of the invention.
[0387] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognise that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0388] In this specification where reference has been made to
patent specifications, other external documents, or other sources
of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents
is not to be construed as an admission that such documents, or such
sources of information, in any jurisdiction, are prior art, or form
part of the common general knowledge in the art.
[0389] The Following Numbered Paragraphs Define Particular Aspects
of the Present Invention: [0390] 1. Isolated Aureobasidium
pullulans yeast strain YBCA5 (CBS Accession # 141880). [0391] 2. A
composition comprising YBCA5 and an agri
References