(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

9
(l ) Worl~~;~~~~~:! Property ~ 11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111

International Bureau ~ Intern at io na1/PlublhS.cation

(lOW) NAum ber
(43) International Publication Date ~ 0 202 0 646 2
11 June2020(11.06.2020) WI PO I PCT

(51) International Patent Classification:

AOJN 25104 (2006.01) AOJN 25110 (2006.01)
(21) International Application Number:
PCT!IB20 19/0603 84
(22) International Filing Date:
03 December 2019 (03.12.2019)
(25) Filing Language: Italian
(26) Publication Language: English
(30) Priority Data:
102018000010837 05 December 2018 (05.12.2018) IT
(71) Applicant: NAN OMNIA SRL [IT/IT]; Via di Mezzo, 46,
37059 Perzacco di Zevio (VR) (IT).
(72) Inventors: BONACONSA, Marta; Via di Mezzo, 46,
37059 Perzacco di Zevio (VR) (IT). BOVI, Michele; Via
Cervino, 7, 37132 Verona(IT). VACCARI,Pietro; ViaLu-
cania, II, 37138 Verona (IT).
(74) Agent: BONINI, Ercole; Studio Bonini Sri, Corso Fogaz-
zaro, 8, 36100 Vicenza (IT).
(81) Designated States (unless otherwise indicated, for every
-
i iiiiiii
iiiiiiii
kind of national protection available): AE, AG, AL, AM,
AO, AT, AU, AZ, BA, BE, BG, BH, EN, BR, BW, BY, BZ,
iiiiiiii CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
--
!!!!!!!!
DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,

-
iiiiiiii
KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,

-
i iiiiiii MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
- SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,

-- TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
KM, ML, MR, NE, SN, TD, TG).
- Published:
without international search report and to be republished
~ upon receipt ofthat report (Rule 48.2(g))
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~ (54) Title: STIMULATION OF THE IMMUNE DEFENCES OF A PLANT THROUGH THE USE OF A BIOMASS OF MICR0-
0 ORGANISMS SELECTED FROM MICROALGAE AND CYANOBACTERIA.
~
0 (57) Abstract: Use of a biomass of one or more micro-organisms selected in the group consisting of microalgae and cyanobacteria as
~ an active ingredient for stimulating in a plant the immune defences against pathogenic agents. Such cyanobacteria comprise Spirulina
0 platensis and Spirulina maxima.

~
wo 2020/115646 PCT/IB2019/060384
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STIMULATION OF THE IMMUNE DEFENCES OF A PLANT THROUGH

THE USE OF A BIOMASS OF MICRO-ORGANISMS SELECTED FROM
MICROALGAE AND CYANOBACTERIA
DESCRIPTION
The present invention concerns the use in the agricultural sector of a biomass
of one or more micro-organisms selected in the group consisting of microalgae
and cyanobacteria Spirulina platensis and Spirulina maxima as an active
ingredient for stimulating the immune defences of a plant against pathogenic
agents.
Background
Agricultural production has always been at the basis of the development and
growth of human society.
Precisely because of this importance, for the purpose of preventing the harmful
effects of pathogenic agents on plants, so as to protect them, numerous types
of plant protection products are used.
Disadvantageously, such plant protection products can lose their efficacy over
time as resistant strains arise or can have a negative effect on human health
and on the delicate environmental balance of the ecosystem because of the
residues that remain in the edible part of the plants treated and the residues
that accumulate in the environment, respectively.
It is known that in nature plants can withstand pathogenic agents due to a
series of immune defence mechanisms of the passive or active type.
Passive defence systems mean the natural barriers that the plant has at
physical and chemical level, such as the cuticle of leaves and fruits, the layers
of lignin of the cell walls, etc. Active defences are instead induced by contact
between the plant and the pathogenic agent and relate to all the metabolic
mechanisms that can contrast the infection of the aforesaid pathogenic agent.
If the pathogenic agent manages to overcome all the passive defence systems
and delay the activation of the active defence systems, then the state of
disease arises.
Generally, the induction of active responses takes place following the release
of trigger molecules called elicitors that are perceived by the plant cell.
Such elicitors may be exogen, i.e. produced by the pathogenic agent, or derive
from its structural components, or be released by structural components of the
plant itself following the attack of the pathogenic agent.
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A prompt and localized response of the plant cells in the infection site induces
the accumulation of Pathogenesis-Related (PR) enzymes and phytoalexins,
i.e. antimicrobial compounds normally produced by the cell tissues of plants,
which accumulate in a larger amount in response to the attack of pathogenic
agents, but which are already present in the plant in conditions of good health
and absence of disease.
In light of recent legislative restrictions on the use of plant protection products
in agriculture (Directive 2009/128/EC), the use of elicitor compounds in the
fight for the protection of plants represents an excellent alternative to the use
of plant protection products.
The use of elicitor compounds is also well suited to use in organic farming.
Much of the research conducted over recent years has looked at testing
different types of compounds for their ability to induce the innate defences
of plants, and the best known compounds identified consist of photostable
analogues of salicylic acid (e.g. BTH) and chitosan.
In relation to photostable analogues of salicylic acid, BTH (acibenzolar-S-
methyl, o-benzothiadiazole) represents a compound of chemical derivatization,
not present in nature which, as such and as can be deduced from technical
data sheet of the compound itself, can cause damage to the environment,
in particular for aquatic organisms in the aquatic environment and for human
health, in particular for farmers who have to distribute the BTH on the plants.
Chitosan, on the other hand, is a natural compound, in particular a
polysaccharide derived from chitin, the substance that constitutes the
teguments that cover insects and crustaceans and that is obtained from
grinding the shells of crustaceans such as lobsters, prawns and crabs.
Its high availability, at a relatively good price, and its wide spectrum of activity
make it one of the most useful elicitor compounds and for the moment one
of the most widely used.
However, its mass production at industrial level could lead to a considerable
hunt for crustaceans which, if not suitably controlled through ad hoc legislation,
could even lead to the extinction of some species.
Description of the invention
The present invention intends to realize an alternative elicitor product
with respect to known products which also overcomes the limitations and
drawbacks previously indicated.
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The aim of the invention is therefore to realize a product that acts as a

stimulant for the immune defences of a plant against pathogenic agents.
In particular, it is an object of the invention for such product to be a natural
product, the use of which in agriculture is not harmful to the environment or to
human health.
Again, it is an object of the present invention for such product to be possible
to make in an eco-compatible way and without any negative effects on flora or
fauna.
Furthermore, it is an object of the invention for the mentioned product to be in
a formulation such that, once applied to the plant or to part of the plant, it is not
removed by any adverse weather conditions such as rain or wind.
Again, it is an object that such product be usable for endotherapy treatments.
Finally, it is an object of the present invention for such product to be able to be
used in combination with other agents having beneficial action for the plant,
such as growth adjuvants, plant protection products, etc.
The aforementioned objects are reached by the use of a biomass of one or
more micro-organisms selected in the group consisting of microalgae and
cyanobacteria as an active ingredient for stimulating the immune defences of a
plant against pathogenic agents. Such cyanobacteria comprise in particular the
cyanobacteria Spirulina platensis and Spirulina maxima.
In the use according to the invention, the biomass is applied to the plant as
such, through for example a total extract of the cells of the micro-organism(s)
selected.
In particular, the aforesaid biomass is used on a fruit plant, preferably a fruit
plant selected in the group comprising Vitis Vinifera, Olea europaea and
Actinidia chinensis.
Preferably, to perform its function, the biomass of the invention is applied by
spraying the leaves of the plant in a dose preferably comprised between 1 and
500 mg per plant, more preferably in a dose comprised between 50 and 250
mg per plant, even more preferably in a dose of about 100 mg per plant.
Such biomass is preferably in the form of dried powder of the cultures of the
selected micro-organisms, which is also dissolved in an aqueous solvent prior
to application on the plant.
Advantageously, such biomass can also be applied through endotherapic
injection into the trunk of the plant.
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More preferably, at least a part of such biomass is contained in a plurality of

microcapsules.
Such microcapsules containing the biomass of the invention have an interface
comprising a polymeric material and/or an amphiphilic compound.
Each of such microcapsules preferably has dimensions less than 100 1-Jm,
preferably comprised between 50 1-1m and 100 nm, even more preferably
dimensions comprised between 20 1-1m and 500 nm.
Such microcapsules containing the biomass are preferably obtained through
nanoemulsion technology or through spray-drying technology or, even, through
nanoemulsion and spray-drying technology as according to the method of the
invention that will be described below.
The aforesaid polymer material is preferably chitosan.
The aforesaid amphiphilic compound is preferably selected from lecithin,
saponin, glucoside surfactants, betaine, fatty acids, phospholipids and long-
chain alkyl sulphates.
The subject of the invention is also a composition for agricultural use for
stimulating the immune defence of a plant in which the mentioned biomass
is associated with one or more compounds selected in the group consisting
of formulation agents, phytoiatric agents, growth adjuvant agents and plant
defence mechanism elicitor agents.
Furthermore, preferably, at least part of such compounds is contained in a
plurality of microcapsules together with or separately from the aforesaid
biomass.
In particular, the objects of the present invention are reached when the micro-
organism selected is the cyanobacterium Spirulina platensis.
Part of the present invention is also a method for producing a product that
stimulates the immune defence of a plant against pathogenic agents. Such
product comprises a biomass of Spirulina platensis.
In particular, the method of the invention envisages:
- mixing such Spirulina platensis biomass with an aqueous solution in the
presence of a polymer material and an amphiphilic compound;
- dispersing an organic phase comprising at least one organic solvent in the
aqueous mixture obtained from the previous operation, so as to obtain an
emulsion of said organic solvent in said aqueous mixture.
Preferably, such organic solvent is a vegetable oil or a terpene.
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Furthermore, preferably, such polymer material is chitosan.

Also preferably, such amphiphilic compound is selected from lecithin, saponin,
glucoside surfactants, betaine, fatty acids, phospholipids and long-chain alkyl
sulphates.
In particular, the biomass comprises a total extract of Spirulina platensis cells.
Preferably, the dispersion operation is performed by means of a microfluidic
nanoemulsion instrument and allows an emulsion to be obtained in which the
organic solvent is in the form of drops having dimensions comprised between
20 1-Jm and 100 nm.
Preferably, the method of the invention envisages drying the emulsion
obtained by the dispersion operation through spray-drying, so as to obtain a
plurality of microcapsules containing at least part of the biomass and each
having an interface comprising such polymer material and a plurality of
microcapsules containing at least part of the biomass and having an interface
comprising the aforesaid amphiphilic compound.
As previously indicated, the present invention relates to the use of a biomass
of one or more micro-organisms selected in the group consisting of microalgae
and cyanobacteria as an active ingredient for stimulating in a plant the immune
defences against pathogenic agents. Such cyanobacteria comprise the micro-
organisms Spirulina platensis and Spirulina maxima.
It is specified that for years it was considered that Spirulina platensis
and Spirulina maxima belonged to the family of blue-green algae, however
recent phylogenetic analysis and morphological analysis performed have
demonstrated that they are actually micro-organisms belonging to the
Cyanobacteria class, in particular filamentous cyanobacteria characterized
by spiral-shaped multicellular cylindrical trichomes.
In particular, according to the preferred embodiment of the invention, the
biomass comprises the total extract of the cells of such one or more micro-
organisms.
The total cell extract can be obtained according to techniques known in the
state of the art that shall not be discussed further in the present document.
The results obtained from the application to plants of the biomass of the
invention are particularly surprising as they show that it is not necessary to use
purified molecules deriving from such micro-organisms, but that the biomasses
of microalgae and cyanobacteria are active in the stimulation of the immune
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defences of the plant without the individual molecules that compose them
having been previously isolated or separated.
Furthermore, the biomasses of microalgae and cyanobacteria do not require
post-production chemical changes so that, for the purposes of the present
invention, such biomasses are preferably used as such as they are obtained
from known extraction processes such as, for example, a process comprising
the drying and grinding operations of a plurality of cultures of cells of such
m icroalgae and/or cyanobacteria.
In this way, the resulting biomass is advantageously in the form of dry powder
that maintains intact its elicitor properties and that is also easy to manage and
has greater durability and storage and transport simplicity.
The aforesaid microalgae and cyanobacteria indicated comprise a large variety
of bioactive molecules that act as activators of different plant biological
processes.
In particular, such bioactive molecules comprise mono and polysaccharides,
fatty acids, amino acids, proteins, peptides, phenols and polyphenols, micro
and macronutrients, vitamins and minerals.
Furthermore, each microalga and each cyanobacterium have a different pool
of such bioactive molecules so that, advantageously, the use of such
biomasses in mixture allows an elicitor action to be exercised on different types
of plants and cultures.
According to the preferred embodiment of the invention, the biomass is
selected from microalgae and cyanobacteria that can produce and accumulate
fatty acids, in particular the fatty acids linoleic acid and linolenic acid.
Preferably, such microalgae and cyanobacteria are able to produce and
accumulate both linoleic and linolenic fatty acids.
Such fatty acids are particularly advantageous as they are implied in the
stimulation of animal and plant cell defence mechanisms. In fact, they induce
the activation of NADPH oxidase enzymes through the stimulation of the
production of protein kinase C, resulting in an activation of the production of
the reactive oxygen species (ROS) molecules.
The micro-organisms of the present invention are preferably selected from the
microalgae and cyanobacteria that contain a quantity of linoleic acid greater
than 3%, preferably greater than 10%, more preferably greater than 14% with
respect to the total lipid content.
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Furthermore, the micro-organisms are preferably selected from the microalgae

and cyanobacteria that contain a quantity of linolenic acid greater than 0.5%,
preferably greater than 5%, more preferably greater than 10%, even more
preferably greater than 15% with respect to the total lipid content.
According to the preferred embodiment of the invention, the micro-organism is
selected from the cyanobacteria Spirulina platensis and Spirulina maxima.
In particular, according to the preferred embodiment of the invention such
micro-organism is the cyanobacterium Spirulina platensis.
The authors have surprisingly discovered that the micro-organism Spirulina
platensis, more commonly known by the name of spirulina, has advantageous
and unexpected elicitor properties on plants and they therefore propose the
use thereof in the agricultural sector.
In fact, the authors have demonstrated that the use of such micro-organism
on plants envisages the occurrence of diseases caused by pathogenic agents
and/or reduces the incidence and/or severity thereof as it stimulates the
activation of the innate defence mechanisms of the plant itself.
In particular, the use of spirulina on plants stimulates the production of
compounds and proteins that are normally accumulated in the plant only
following attack by pathogenic agents.
The use of spirulina is therefore surprisingly able to induce the plant to produce
defence metabolites, putting the plant itself in a state of alarm which improves
the defensive response thereof in the case of effective attack by a pathogenic
micro-organ ism.
It is noted that Spirulina platensis is a micro-organism known in itself and is
accessible because it is available on the market and, furthermore, that the
production of a biomass, preferably a biomass comprising a total extract of the
cells of such Cyanobacteria, is a well known process to a person skilled in the
art, therefore such micro-organism and process shall not be discussed further
in the present document.
It is also specified that from now on the terms "spirulina" and "biomass of
Spirulina platensis" shall be used interchangeably with one another.
According to the preferred embodiment of the invention, the spirulina biomass
is in the form of powder.
Such powder is generally realized by grinding the dried biomass of a plurality
of cells of Spirulina platensis cultures according to techniques known in the
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state of the art.

As reported in the following examples, the authors have advantageously
discovered that spirulina can stimulate the natural defences of plants for a long
duration, over 14 days after treatment.
In particular, the use of spirulina on plants stimulates the production of
stilbenes, low molecular weight phenolic compounds with properties that
include anti-fungal properties.
Furthermore, the use of spirulina on plants also stimulates the production of
some PR proteins, in particular the enzyme phenylalanine ammonia lyase
(PAL).
It has also been advantageously shown, through SPAD technique, that the use
of spirulina according to the invention does not have a negative effect on the
photosynthetic activity of the treated plant as it does not modify the chlorophyll
content thereof, demonstrating the harm less ness of the use of such
cyanobacteria on plant enzyme activity mechanisms.
According to the preferred embodiment of the invention, spirulina is used on
all types of plants, in particular indoor, outdoor, garden, greenhouse, orchard,
field, annual, biennial and perennial plants, grains, leguminous plants, woody
and ornamental plants, plants in intensive and extensive agriculture.
Specifically, the aforesaid use of spirulina is particularly advantageous in the
treatment of fruit plants such as, for example, fruit trees, for improving the
agricultural yield and limiting the use of plant protection products that could
remain as residues in the edible part of the plant.
When the fruit plant is selected in the group comprising Vitis Vinifera, Olea
europaea and Actinidia chinensis the effects of stimulating the immune
defences are particularly advantageous.
In fact, as shown in the following examples, the use of spirulina on Vitis
Vinifera vines halves the incidence (% of affected bunches with respect to the
total number of bunches per plant) of Botrytis cinerea, so-called botrytis.
Furthermore, in bunches of Vitis Vinifera which, when treated with spirulina,
are still affected by botrytis, the severity with which the latter affects the
aforesaid bunches is lower with respect to the severity with which botrytis
affects bunches not treated with spirulina.
The term severity means the percentage of grapes affected by botrytis per
bunch.
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Also advantageously, spirulina is used to prevent the onset or reduce the

incidence and severity of downy mildew disease on the plants.
Advantageously, the Vitis Vinifera plants treated with spirulina according to
the preferred embodiment of the invention, in fact, have less than 25% of their
foliage affected by downy mildew.
Also advantageously, the use of spirulina in vines does not affect either the
levels of sugar content of the must or the pH of the must, demonstrating that
it has a specific action of stimulating the defence mechanisms that does not
interfere with the normal development and fructification processes of the plant
itself.
For the purposes of the present invention, spirulina can be applied to the plant
according to different treatment methods and programs.
According to the preferred embodiment of the invention, it is applied to the
whole plant or to a part or parts thereof, such as for example the leaves, the
stalks, the flowers, the fruits, the trunk or the roots.
It is particularly preferred to apply spirulina on the leaves of the plant, more
preferably through spraying.
The quantity of spirulina to be applied is defined by an expert in the sector,
according to the type of plant or the treatment period or the like, and can be
determined through field tests.
The authors have performed a plurality of tests in the field and have noted
that the use of spirulina is particularly advantageous when applied in a dose
comprised between 1 mg and 500 mg per plant, preferably between 50 and
250 mg per plant.
In particular, the use of spirulina in a dose corresponding to about 100 mg per
plant was shown to be advantageous.
As indicated above, spirulina is applied to the plant preferably through
spraying, therefore, before application on the plant, the spirulina biomass
is dissolved in an aqueous solvent, or another solvent of the known type
compatible with use in agriculture, for promoting the dispersion thereof on the
plant itself.
It is particularly preferred to use a solvent of the aqueous type, in particular
water, so as to be easily absorbed by the plant and due to its non-existent
impact on the environment and on the operator appointed to perform the
treatment.
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It is not excluded that spirulina can be applied to plants by endotherapic

injection directly into the trunk of the plant itself, to reach the xylematic vessels
and spread along them.
Advantageously, endotherapic application has a lower atmospheric agent
leaching action, prolonged persistence of action, reduction of the application
doses and lower dispersion into the environment with respect to external
application.
According to a second embodiment of the invention, spirulina is mixed with one
or more compounds selected in the group comprising formulation agents,
phytoiatric agents, growth adjuvant agents and elicitor agents of the plant
defence mechanisms, for forming a composition for agricultural use,
stimulating the immune defences of a plant.
In fact, as spirulina is a natural compound, it can be combined with one or
more compounds from those indicated, without disadvantageous cross
reactions or interferences in the action mechanism thereof.
The term "formulation agent" means any compound or inert substance that
allows the preparation and stability of biomass to be optimized, e.g. facilitating
the handling, keeping in suspension and shelf life thereof, etc.
Such formulation agents are selected from the conventional ones already
known in common practice and can include, among others, coformulation
compounds that are used to reduce the concentration of spirulina, such as for
example inert substances and diluents.
Again, such formulation agents can include adjuvant compounds, which have
the aim of increasing the efficacy of spirulina and promoting the distribution
thereof.
Examples of such adjuvants comprise synergizers, emulsifiers, wetting agents,
binders, humectants, propellants for aerosol formulations, inert diluents, anti-
drift agents, anti-foaming agents, preservatives, surfactants, dispersants, pH
buffers, etc.
Such formulation agents can also be present alone or in mixture.
The aforesaid composition may be in liquid or solid formulation.
Examples of solid formulations of the composition of the invention comprise
granules or dry powders or in gel, cream or paste form.
Examples of liquid formulations comprise wettable powders in which spirulina
is finely ground in the presence of wetting excipients, dispersants, inert agents,
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and others known in the state of the art for obtaining a product which, mixed
in water, forms a suspension.
Another example of liquid formulation comprises soluble powder, i.e. a
powdery formulation like the previous one which, when mixed in water, forms a
stable diluted solution.
Then it is not excluded that such composition is in emulsion form in water,
preferably in the presence of amphiphilic compounds, surfactants, dispersants
or other stabilizers for guaranteeing the stability thereof.
Again, the composition may be in the form of solid dispersible microgranules
or even in the form of suspensions of microcapsules having an interface made
of a thin polymer film, as will be described in more detail below.
The formulation of the composition in liquid form is particularly preferred due to
its ease of application on the plant through spraying or aerosol.
It is not excluded that, according to variants of the invention, the aforesaid
composition is in formulation for endotherapic treatment.
In such endotherapic formulation, the composition will comprise adjuvants
especially formulated and known in the state of the art for being injected along
the xylematic vessels and spreading along them.
Again, the composition of the invention may be in solid, liquid or gaseous
formulation for gaseous treatments, so-called fumigations.
The term phytoiatric agents means all the known compounds with pesticide
action such as bactericidal, fungicidal or anticryptogamic agents, insecticides,
nematicides, repellents and plant protection products.
The growth adjuvant agents comprise growth regulators and vegetable
charcoal and, finally, plant defence mechanism elicitor agents suitable for
use in the present invention comprise by way of non-limiting example,
13-aminobutyric acid, 2,6-dichloroisonicotinic acid, photostable analogues of
salicylic acid and chitosan.
The use of chitosan as an elicitor agent is particularly preferred.
Advantageously, the use of phytoiatric agents, growth adjuvant agents and/or
plant elicitor agents in the composition of the invention allows the plant
immune defence stimulation activity by spirulina to be combined with a plurality
of further beneficial effects on the growth and preservation of the plant itself.
In the aforesaid composition, the spirulina content is preferably greater than
about 15%, more preferably greater than about 35%, even more preferably
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greater than about 50% by weight with respect to the total weight of the
composition.
A third embodiment of the invention that will now be described comprises all
the characteristics indicated for the preferred embodiment of the invention and
for the second embodiment of the invention, including the variants, in which at
least a part of the biomass is contained in a plurality of microcapsules.
The term microcapsule means solid or liquid particles of micro/nanometric
dimensions having a central core comprising the biomass of the invention, in
liquid or solid form, surrounded by an interface comprising a polymer material
or an amphiphilic compound, or both.
In the present description and the following claims, the term "interface" must be
interpreted as the separation surface between the central core of biomass and
the environment external to the microcapsule itself.
Preferably, each of such microcapsules has dimensions less than 100 1-Jm,
preferably comprised between 50 1-1m and 100 nm, even more preferably
comprised between 20 1-1m and 500 nm.
Advantageously, the use of such microcapsules allows the biomass to be
protected from external agents and from any leaching, increasing the
residence time on the site of the plant where it is applied, e.g. on the leaves
and, also, increasing the efficacy of use of the biomass itself due to its gradual
release.
Furthermore, thanks to the micrometric dimensions, such microcapsules of
biomass can also be used for endotherapic treatments as they are able to
reach the phloem and the xylem of the plant.
Again, advantageously, the use of such microcapsules allows an agricultural
product to be realized which, with the equivalent amount of biomass used,
will have a greater concentration of active ingredient available with respect to
the concentration of active ingredient that is obtained by mixing, e.g. in the
aqueous phase, the spirulina biomass directly with the polymer material or the
amphiphilic compound.
In fact, when the spirulina biomass is mixed directly in the aqueous phase with
the polymer material or with the amphiphilic compound, in particular when the
polymer material is chitosan, a part of the spirulina biomass precipitates,
causing the reduction of the concentration of the biomass available for the
plant during the use of the product.
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The aforesaid polymer material is selected from polymers of plant origin,

polymers of animal origin and polymers of synthetic origin.
In particular, the polymer material used is selected from biodegradable and
biocompatible polymers so as to be able to be degraded by the atmospheric
or biological agents present in the environment, not leaving any toxic residues
for the cultures or for human beings.
It is not excluded that such polymer material has amphiphilic characteristics.
Preferably, such polymer material is selected from polysaccharide polymers,
proteins, lipophilic materials, synthetic polymers, polyesters, polyesters of
natural derivatization such as, for example, polyhydroxyalkanoates, and other
polymer materials known in the state of the art for use as a microcapsule
interface.
Non-limiting examples of the aforesaid polysaccharides comprise chitosan,
chitin, derivatives of chitosan, dextran, alginate, cyclodextrins, cellulose, starch
and amylose.
Non-limiting examples of proteins that can be used as polymer coating material
comprise gelatin, albumin and casein.
Furthermore, the lipophilic materials that can be used comprise, among
others, plant rubbers, waxes, alginates and cyclodextrins.
In relation to synthetic polymers that can be used as polymer material, they
comprise PLGA, PVA, PLA, PGA, polyamines, polyamides.
Finally, further examples of polymer materials suitable for use in the present
invention comprise polyhydroxybutyrate, tannins, polyhydroxyvalerate, lignins,
resins, etc.
It was particularly advantageous for the purposes of the present invention to
use chitosan as polymer material.
In fact, as well as being an excellent interface polymer, chitosan also has
an elicitor action in the plants on which it is applied. As reported in the results
of the following examples, the use on plants of microcapsules of chitosan
containing the biomass of the invention further stimulates the immune
defences of the plants treated with respect to treatment with biomass that
is not microencapsulated and with respect to treatment with biomass that is not
microencapsulated mixed directly with chitosan.
Such microcapsules are generally more or less spherical shaped, even if it is
not excluded that according to some embodiments they may have an irregular
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shape.
Such microcapsules may also be in liquid form, for example in an emulsion,
or in solid form.
Advantageously, the microencapsulated biomass may be used in mixture with
one or more compounds selected from the formulation agents, phytoiatric
agents, growth adjuvant agents and plant elicitor agents previously described.
Furthermore, preferably, at least part of such compounds is contained in a
plurality of microcapsules having an interface comprising a polymer material
and/or an amphiphilic compound.
Advantageously, the microencapsulation of such mentioned compounds allows
them to be protected against external agents.
Preferably, the biomass and compounds are co-microencapsulated, i.e. each
of the microcapsules containing the biomass according to the invention
contains inside it the aforesaid biomass together with at least part of the
compound(s) selected from those indicated above.
Such co-microencapsulation is particularly advantageous as it allows the
elicitor action of the biomass of the invention to be combined with the
beneficial activity of the compounds indicated above and further guarantees
the maintenance of the correct dosing proportions between biomass and
compound during application on the plant.
However, it is not excluded that at least part of such compounds is contained
in a plurality of microcapsules, separately from the aforesaid biomass.
According to the aforesaid third embodiment of the invention, the biomass
of active ingredient is incorporated in microcapsules through spray-drying
technology.
Preferably, also the compounds mentioned above are incorporated in
microcapsules through spray-drying technology.
Spray-drying technology essentially consists of the transformation of a liquid
solution into a set of solid particles through a solvent evaporation process.
The spray-drying process schematically comprises four main operations:
atomization of the solution of biomass or biomass associated with one or more
of the previously indicated compounds, in drops, through a nozzle; mixing of
such drops with a stream of hot air at a predefined temperature; evaporation of
the solvent with transformation of the drops into solid particulate, and finally
collection of the solid particulate obtained.
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Advantageously, the spray-drying technique is a highly rapid and reproducible

technology, which allows the size of the particulate produced to be controlled
and is easy to apply on an industrial scale.
In more detail, according to such third embodiment, the process of
microencapsulation of the biomass of the invention envisages mixing the
aforesaid biomass with a solution of one or more polymer materials from those
previously indicated and mixing suitably until a homogeneous solution or
suspension is obtained.
Preferably, the mixing operation is performed through magnetic stirring,
sonication, homogenization or a combination of these techniques.
The homogeneous solution or suspension obtained is vaporized through a
spray-dryer in air or nitrogen flow variable between 100 and 1000 Llhr,
preferably between 200-800 Llhr.
The air or nitrogen flow is maintained at a temperature comprised between
50 and 250°C.
Finally, the solid particulate of microcapsules containing the biomass of the
invention is recovered and stored at 4°C.
Advantageously, the spray-drying technology also allows the co-
microencapsulation of the aforesaid biomass to be performed with one or more
compounds selected from formulation agents, phytoiatric agents, growth
adjuvant agents and plant elicitor agents previously mentioned, realizing a
composition for agricultural use in which both the biomass and the other
compounds possibly present are contained simultaneously in a single
microcapsule and in which such microcapsules have a uniform distribution and
substantially corresponding dimensions.
It is not excluded that, according to embodiments of the invention, such
compounds are subjected to microencapsulation separately from the biomass.
It is not excluded that, according to embodiments of the invention, such
biomass is incorporated in microcapsules through different techniques from
those indicated, such as, for example, interfacial polymerization, coacervation,
emulsion techniques, through fluidized bed, through microfluidic technology
or by means of industrial vibration encapsulator devices.
In particular, according to a variant of such third embodiment of the invention,
the spirulina biomass is incorporated into microcapsules through nanoemulsion
technology.
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With such technology the aforesaid microcapsules are in liquid form.

In particular, for the realization of such liquid microcapsules the spirulina
biomass is mixed with an aqueous solution in the presence of a polymer
material and an amphiphilic compound.
Subsequently, an organic phase comprising at least one organic solvent is
dispersed in the aqueous mixture obtained from the previous operation, so as
to obtain an emulsion of the organic solvent in the aqueous mixture.
Preferably, the aforesaid organic solvent is a vegetable oil or a terpene, more
preferably a vegetable oil.
Seed oil is particularly preferred.
Furthermore, according to such variant, the polymer material is selected from
the polymer materials previously mentioned.
Chitosan polymer material is particularly preferred, so as to obtain the
advantages previously mentioned.
The aforesaid amphiphilic compound is preferably selected from lecithin,
saponin, glucoside surfactants, betaine, fatty acids, phospholipids and long-
chain alkyl sulphates. Such amphiphilic compound is preferably a saponin,
more preferably aescin saponin.
According to such variant, the dispersion operation is performed through a
microfluidic nanoemulsion instrument.
Advantageously, with such operations a nanoemulsion of oil in water
comprising an aqueous phase is obtained in which the chitosan interacts with
the water-soluble part of the spirulina biomass and surrounds it forming the
interface of the aforesaid microcapsules, in particular the chitosan interface
represents the separation surface between the biomass and the aqueous
phase.
Furthermore, such nanoemulsion of the invention also comprises an organic
phase dispersed in the aqueous phase.
In such organic phase, the amphiphilic compound interacts with the liposoluble
part of the spirulina biomass and surrounds it, realizing the interface of the
microcapsules, i.e. the separation surface between the drop of oil in which the
biomass is dissolved and the aqueous phase.
According to such variant, in the aforesaid nanoemulsion the organic solvent
is in the form of drops of dimensions comprised between 20 1-1m and 100 nm,
preferably between 15 1-1m and 500 nm.
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Advantageously, the use of such nanoemulsion allows the lipophilic

components of the spirulina biomass to be made more available, dissolving
them in the organic phase of the emulsion, hence being easier to absorb by
the plant.
It is not excluded that the aforesaid emulsion further comprises one or more of
the compounds having beneficial action previously mentioned and/or known
plant protection products which, based on their solubility, will be dissolved in
the organic phase or in the aqueous phase of the aforesaid emulsion.
Part of the present invention is also a method for producing a product that
stimulates the immune defences of a plant against pathogenic agents, wherein
such product has the characteristics described for the microcapsules
comprising a biomass of Spirulina platensis described for the first, second or
third embodiment of the invention, including the variants.
In particular, the method of the invention envisages:
- mixing the Spirulina platensis biomass with an aqueous solution in the
presence of the aforesaid polymer material, preferably chitosan, and in the
presence of an amphiphilic compound, preferably selected from lecithin,
saponin, glucosidic surfactants, betaine, fatty acids, phospholipids and
long-chain alkyl sulphates, more preferably a saponin, even more preferably
aescin saponin;
- dispersing an organic phase comprising at least one organic solvent,
preferably a vegetable oil or a terpene, in the aqueous mixture obtained
from the previous operation, so as to obtain an emulsion of the aforesaid
organic solvent in the aqueous mixture and to obtain the aforesaid
microcapsules of biomass.
In particular, such dispersion operation is performed through a microfluidic
nanoemulsion instrument.
Advantageously, in such emulsion the organic solvent is in the form of drops
sized between 20 1-1m and 100 nm.
The method according to the invention further envisages drying the emulsion
obtained from the previous operations using spray-drying technology,
subsequent to the dispersion operation.
In this way, the microcapsules of the invention are advantageously obtained in
solid form and, more in particular, both a plurality of microcapsules containing
at least part of the biomass and having an interface comprising the polymer
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material and a plurality of microcapsules containing at least part of the biomass

and having an interface comprising the amphiphilic compound are obtained.
It is not excluded that in variants of the method of the invention, the aforesaid
drying operation is performed using different technology, or is not present.
It is further not excluded that such drying operation can be performed
independently from the emulsion operations for forming a product for the
stimulation in a plant of immune defences against pathogenic agents, as
previously indicated with reference to the third embodiment of the invention.
The authors have surprisingly discovered that, in addition to the advantages
already cited of the use of spirulina, when spirulina is microencapsulated,
its application on the plants stimulates the production of PAL enzymes up to 15
days after the first application.
Such characteristic is particularly advantageous as it allows the number of
applications of spirulina to be limited when long treatment periods are
necessary.
Like the use of non-microencapsulated spirulina previously described, the use
of microencapsulated spirulina does not influence the photosynthetic activity
of the plant either and, when the plant treated is a vine, the aforesaid use does
not affect the levels of sugar content and the pH of the must.
As for the methods of application of spirulina contained in microcapsules on
plants, such methods are like those already indicated for the use of non-
microencapsulated spirulina.
Therefore, it is preferably applied at a dose comprised between 1 mg and
500 mg per plant, preferably between 50 and 250 mg, more preferably about
100 mg per plant, on the whole plant or on a part or parts of it, such as for
example the leaves, the stalks, the flowers, the fruits, the trunk or the roots,
with a particular preference for the leaves.
Other aspects and advantages of the present invention will appear more
clearly after reading the following examples, which are to be considered
illustrative and non-limiting.
Examples
Example 1. Treatment with aqueous solution of a dried biomass of
Spirulina platensis
1. 1 Botrvtis analysis
39.6 mg of dried biomass of Spirulina platensis (Aighitaly) were dissolved in
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2.4 L of water.
200 ml of the solution thus obtained were distributed through spraying onto the
foliage of each plant of 6 Vitis vinifera plants, Merlot variety, and 6 Vitis vinifera
plants, Corvina variety, in a pot and in the ripening phase, so as to treat each
plant with 3.3 mg of spirulina.
Such treatment was repeated on each plant with a treatment frequency of 14
days until reaching 2 treatments.
10 bunches for each of the aforesaid plants were harvested 10 days after the
second treatment and were evaluated for the presence of botrytis.
The following parameters were evaluated:
- incidence: % of bunches affected by botrytis and rot with respect to the total
number of bunches per plant;
- severity: % of grapes affected by botrytis and rot per bunch.
The results obtained are reported in Table 1, where it is possible to identify a
clear trend towards greater overall health of the bunches in plants of vines
treated with spirulina with respect to control plants that were not treated.
Table 1
Botrytis Control Treatment
Incidence (%) 43 18
Severity (%) 6 1.2

1. 2 Analvsis of the stilbenes

The chemical compounds that can be attributed to the family of stilbenes are
low molecular weight phenolic compounds with antifungal properties. In
particular, resveratrol and viniferin are stilbene compounds that can be found
in the vine plant, in different amounts, which have differentiable characteristics,
also in terms of antifungal power.
These composites also have antimicrobial properties and are synthesized and
accumulated by the plant following attack by harmful micro-organisms,
becoming concentrated in particular at the penetration point of the pathogenic
agent.
Therefore, the accumulation of resveratrol and viniferin in the plants treated
with spirulina of example 1.1 was evaluated 7 and 14 days after the second
treatment, and in control plants which did not undergo treatment with spirulina
biomass.
Operatively, 350-400 mg of leaves per analysed plant were homogenized in a
mortar with liquid nitrogen.
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200 mg of the powder obtained were inserted and weighed in a glass tube
into which 10 ml of methanol and 100 1-JI of trans-4-hydroxystilbene 100 mg/1
(internal standard) were added; the glass tube was then closed and shaken
by hand for 10 min, keeping it wrapped in foil to protect it from the light. The
solution obtained was filtered on paper filter in a vacuum flask and the solvent
was made to evaporate with a Rotavapor.
The dry residue obtained was immediately rehydrated with 0.5 ml of methanol
and 0.5 ml of water, filtered on a 0.22 1-Jm membrane filter and directly injected
into HPLC.
The stilbenes were separated on a Lichrospher C18 column (4 mm x 250 mm,
5 1-Jm, Agilent Technologies) using a HPLC system (Waters Corporation)
equipped with a dual-wavelength UV detector (Waters Corporation).
The mobile phase was comprised of aqueous solution HCOOH 0.5% (solvent
A) and CH30H and HCOOH 2% (solvent B). The gradient program set was:
from 0 to 10% B in 3 min, from 10 to 30% B in 5 min, from 30 to 44% B in
35 min, from 44 to 55% B in 2 min, from 55 to 75% B in 15 min and from 75
to 100% B in 1 min. After washing for 2 min with solvent B, the column was
rebalanced with solvent A.
The flow speed was 1.0 ml/min and the injection volume 20 1-JI. The detection
of stilbenes was performed at the wavelengths of 306 and 285 nm for trans-
and cis-isomers, respectively.
The concentration of the individual stilbenes was quantified on the basis of the
area of the peak and the calibration curves of the commercially available
standards of trans-resveratrol and alpha-viniferin in concentrations comprised
between 25 and 400 1-Jg/m I.
The results obtained are reported in Table 2.
Table 2
Stilbenes Control Treatment
Resveratrol
Day? 36.26 ± 11.40 158.89 ± 0.43
Day 14 139.55 ± 15.64 266.76 ± 12.94
Viniferin
Day? nd nd
Day 14 407.40 ± 115.86 519.55 ± 72.88
nd= not determinable

30 The values are expressed in ng of stilbene per g of fresh weight of leaves ±

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standard deviation.
The results obtained show that treatment with spirulina determines in plants
a considerable increase in the resveratrol content with respect to untreated
plants, 7 days after the last treatment performed.
Furthermore, advantageously, such increase continues up to 14 days after
such treatment.
Treatment with spirulina also stimulates the production of viniferin up to 14
days after treatment.
1. 3 Analvsis of must
The sugar content of the must expresses the amount of natural sugar (grape
sugar and fruit sugar) contained in the must before the fermentation thereof.
Such value allows the degree of alcohol after fermentation to be predicted.
It is expressed in degrees Brix and is calculated as follows: n grams of sugar in
100 grams of sugar solution correspond to n degrees Brix at 20°C.
As must is a solution that does not contain only sugars, 1oBrix is approximately
equivalent to a content of 8 g of sugars per Litre of must.
The pH value in the wine essentially depends on the total volume of the acids,
the ratio between malic acid, tartaric acid and the amount of potassium which
in part neutralizes the acidic effect.
Knowledge of the pH is also important for suitable control of the presence
of sulphur dioxide.
After fermentation, lower pH levels increase the efficacy of the clarification and
improve the stability of the colour.
In terms of flavour, the lower the pH the more the wine is acidic and astringent.
Wines with pH over 3.6 are at risk of bacterial instability, whereas the pH lower
than 3.2 the activity of the lactic bacteria is inhibited and therefore malolactic
fermentation cannot take place.
The plants of example 1.1 were analysed in order to evaluate the sugar
content and the pH of the must during the harvest period, 27 days after the
second treatment.
The results obtained are shown in Table 3, where it is possible to highlight how
treatment with spirulina does not affect either the sugar content of the must or
the pH of the latter.
Table 3
Control Treatment F test
Sugars ( 0 8rix) 21.6 21.1 ns
pH 3.37 3.38 ns
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ns= not significant

1.4 SPAD analysis

The presence of chlorophyll can be detected through SPAD analysis on the
leaves of a plant and represents an indication of the photosynthetic activity,
and therefore also a detector of the nitrogenous content and of the vegetal
enzyme activity of the plant itself.
Considering the close connection between the total chlorophyll content of a
culture and its level of organic nitrogen in ordinary growing conditions, SPAD
analysis allows the tissue nitrogen content to be indirectly estimated and
allows a diagnosis of the nutritional state of the crop to be performed.
For the SPAD analysis, 1500 mg of dried biomass of Spirulina platensis
(Aighitaly) were dissolved in 3 L of water.
200 ml of the solution thus obtained were distributed through spraying onto the
foliage of each plant of 3 Vitis vinifera plants, Merlot variety, and 6 Vitis vinifera
plants, Corvina variety, in a pot and in the ripening phase, 6 Vitis vinifera
plants, Merlot variety, grown in the field in an area of the Veneto region with
a strong wine-growing vocation, so as to treat each plant with 100 mg of
spirulina.
Such treatment was repeated on each plant with a treatment frequency of
14 days until reaching 7 treatments.
On each of the plants treated and on 6 plants per variety of Vitis vinifera in a
pot and 3 Vitis vinifera plants, Merlot variety, in the field that were not sprayed
with the spirulina solution (control plants) SPAD analysis was performed
through a relevant SPAD-502 leaf clip (Konika Minolta) applied 7 days after the
fourth treatment.
The results obtained are reported in Table 4 showing the chlorophyll content,
expressed in SPAD units, of the control plants and the plants treated with
spirulina, per plant variety.
Table 4
Variety Control Treatment F test
Corvina 38.8 39.3 ns
Me riot 32.4 31.8 ns
ns= not significant

The SPAD data do not show any significant differences (F test) between
control plants and plants treated with spirulina, indicating that the nutritional
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status of the plant following the treatment with spirulina has not changed.
1.5 Fruit set analysis
On the plants analysed in example 1.4 the effect of the treatment with spirulina
on the fruit set of the plant was also evaluated.
Fruit set is the initial stage of the development of fruits, subsequent to the
flowering of the plant.
It is one of the most important moments of the development of the future
harvest and is represented by the ratio between the number of fruits that
develop from fertilized flowers and that of the total number of flowers present
on the plant at the start of flowering.
Such index is also considered to be a good indicator of the efficiency of
the dual process of pollination and fertilization as it consists of successful
fertilization with the consequent swelling of the ovary and the formation of
seeds that stimulate the growth of the fruit.
For the analysis of fruit set 6 bunches were harvested and analysed for each
plant among those indicated in example 1.4 (treatment and control), 7 days
after the second treatment.
The results obtained are reported in Table 5 and show a significantly positive
effect (p, Duncan test) of treatment with spirulina on the fruit set process with
respect to the control plants.
Table 5
Control Treatment p
Number of grapes/
55.2 76.8 :::;Q.0005
bunch

1. 6 Downy mildew analysis

The phytosanitary status of the Vitis vinifera plants, Merlot variety, and Vitis
vinifera, Corvina variety, grown in a pot and treated with spirulina according
to example 1.4 and the control plants in a pot of example 1.4, was evaluated
14 days after the third treatment in order to analyse the percentage of foliage
affected by downy mildew (Piasmopara viticola) with respect to the foliage not
affected.
In plants treated with spirulina, less than 25% of the foliage of each plant was
affected by downy mildew, whereas for plants not treated with the biomass,
between about 50% and 75% of the foliage was affected by the disease.
Such results show how plants treated with spirulina develop the disease in a
significantly attenuated way with respect to plants not treated.
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1. 7 Leaf disk assavs

From each plant treated according to example 1.4, 7 days after the fourth
treatment with spirulina, 4 leaves were harvested which were then rinsed
in distilled water and each used to form 4 disks that were each arranged in
a different container. In 3 of the 4 containers of each leaf, the pathogen
Plasmopara viticola (1 05 conid./ml), which causes the downy mildew disease,
was inoculated. The fourth container was used as a control in order to check
that the possible development of the disease was not due to an infection
already initiated in the natural environment.
7 days after inoculation the disks were checked under the stereomicroscope
to evaluate the presence of infection (counting of the number of lesions).
The data were analysed according to the descriptors reported by the
International Organisation of Vine and Wine (OIV).
The results obtained from the analysis of the leaf disks show that about 6%
of the surface of the leaves of the plants treated with spirulina developed
reproductive structures of Plasmopara compared with the leaves of the control
plants in which about 39.5% of the surface was instead infected by the
pathogenic agent.
Such data show how treatment with spirulina reduces the probability of
developing the disease following contact with the pathogenic agent.
Example 2. Treatment with microencapsulated Spirulina platensis
biomass
2. 1 Preparation of spirulina microencapsulated through sprav-drver
1 g of dried Spirulina platensis (Aighltaly) biomass powder was dissolved,
through agitation, in 100 ml of water, to form a 1% w/v spirulina solution.
A solution of chitosan dissolved in 0.25 M acetic acid was then mixed by
magnetic stirring with the solution obtained, such as to obtain a final
concentration of 0.25% chitosan in the form of a homogeneous suspension.
The spirulina and chitosan solution was then loaded into a B-290 spray-dryer
instrument (Buchi) in flow current mode, with a variable flow, and vaporized
through a nozzle with a variable diameter comprised between 0.4 and 3 mm,
due to the effect of compressed air at a pressure comprised between 5 and 8
bar and flow of 200-800 Llhr.
The air flow responsible for drying was then heated to a temperature
comprised between 80 and 200°C and the spirulina and chitosan solution
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aspirated with a flow of about 30-55 Llhr in order to guarantee the minimum
residence time within the instrument.
0.6 g of microencapsulated particulate was therefore obtained, recovered and
stored at 4°C.
2. 2 Preparation of spirulina microencapsulated through nanoemulsion
instrument
1 g of dried Spirulina platensis (Aighltaly) biomass powder, a 0.25% solution of
chitosan in the form of a homogeneous solution and a 0.2% solution of aescin
were dissolved, through agitation, in 90 ml of water.
10 ml of seed oil were added to the solution obtained while stirring.
The suspension obtained was treated using a microfluidic nanoemulsion
instrument (LM20-30 Microfluidizer®) through three passages at 15000 Psi
of pressure to form a nanoemulsion having an aqueous phase comprising part
of the biomass in the form of microcapsules having a chitosan interface, and
an organic phase in which the lipophilic part of the biomass is dissolved in the
drops of oil and each drop is surrounded by an aescin interface.
2. 3 Botrvtis analysis on plants treated with microencapsulated spirulina
biomass
39.6 mg of the microencapsulated particulate obtained in example 2.1 were
dissolved in 2.4 L of water.
200 ml of the solution thus obtained were distributed through spraying onto the
foliage of each plant of 6 Vitis vinifera plants, Merlot variety, and 6 Vitis vinifera
plants, Corvina variety, in a pot and in the ripening phase, so as to treat each
plant with 3.3 mg of microencapsulated spirulina.
Such treatment was repeated on each plant with a treatment frequency of
14 days until reaching 2 treatments.
Furthermore, 6 Vitis vinifera plants, Merlot variety, grown in a pot, and 3 Vitis
vinifera plants, Merlot variety, grown in the field were treated by spraying the
leaves with an aqueous solution of spirulina prepared according to example
1.1.
Six Vitis vinifera plants, Merlot variety, grown in a pot, and 3 Vitis vinifera
plants, Merlot variety, grown in the field were treated by spraying the leaves
with an aqueous solution of spirulina with which a 0.25% chitosan solution was
mixed.
Furthermore, 6 Vitis vinifera plants, Merlot variety, grown in a pot, and 3 Vitis
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vinifera plants, Merlot variety, grown in the field were treated by spraying the
leaves with a nanoemulsion of spirulina prepared according to example 2.2.
Finally, 3 Vitis vinifera plants, Merlot variety, grown in a pot, and 3 Vitis vinifera
plants, Merlot variety, grown in the field were treated with water and evaluated
as control plants.
10 bunches for each of the aforesaid plants were harvested 10 days after the
second treatment and were evaluated for the presence of botrytis.
The following parameters were evaluated:
- incidence: % of bunches affected by botrytis and rot with respect to the total
number of bunches per plant;
- severity: % of grapes affected by botrytis and rot per bunch.
The results obtained are reported in Table 6, where it is possible to identify a
clear trend towards greater overall health of the bunches in the plants treated
with the spirulina biomass contained in microcapsules both in solid form
through a spray-dryer and in liquid form through a nanoemulsion instrument
with respect to the control plants and the plants treated with a mixture of
spirulina and chitosan.
Table 6
Mixture of Spirulina in
Spirulina in
Spirulina microcapsules
Botrytis Control Spirulina microcapsules
and nanoemulsion
spray-drying
chitosan instrument
Incidence
45 25 24.5 23 20
(%)
Severity
5 2.4 3 2.3 2
(%)

2. 4 Analysis of the quality of the must on plants treated with

microencapsulated spirulina biomass
The sugar content and pH value of the must were analysed according to the
methods reported in example 1.3, on some of the plants reported in example
2.3, 27 days after the second treatment.
The results obtained are shown in Table 7.
The treatment with microencapsulated spirulina does not affect either the
sugar content of the must or its pH.
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Table 7
Spirulina in
Spirulina in
microcapsules F
Control Spirulina microcapsules
nanoemulsion Test
spray-drying
instrument
Sugars
0
21.5 21.2 20.9 21.0 ns
( 8rix)
pH 3.32 3.35 3.30 3.36 ns
ns= not significant

2.5 SPAD analysis of plants treated with microencapsulated spirulina biomass

1500 mg of the microencapsulated particulate obtained in example 2.1 were
dissolved in 3 L of water.
200 ml of the solution thus obtained were distributed through spraying onto
the foliage of each plant of 3 Vitis vinifera plants, Merlot variety, and 6 Vitis
vinifera plants, Corvina variety, in a pot and in the ripening phase, and of 6
Vitis vinifera plants, Merlot variety, grown in the field in an area of the Veneto
region with a strong wine-growing vocation, so as to treat each plant with 100
mg of microencapsulated spirulina.
Furthermore, 1500 mg of dried spirulina biomass powder (Aighltaly) were
dissolved in 3 L of water and 200 ml of the solution thus obtained were
distributed through spraying onto the foliage of each of 3 Vitis vinifera plants,
Merlot variety, and 6 Vitis vinifera plants, Corvina variety, in a pot and in the
ripening phase, and of 6 Vitis vinifera plants, Merlot variety, grown in the field
in an area of the Veneto region with a strong wine-growing vocation, so as to
treat each plant with 100 mg of non-microencapsulated spirulina.
Furthermore, 2.4 L of nanoemulsion obtained according to example 2.2 were
distributed through spraying onto the foliage of each of 3 Vitis vinifera plants,
Merlot variety, and 6 Vitis vinifera plants, Corvina variety, in a pot and in the
ripening phase, and of 3 Vitis vinifera plants, Merlot variety, grown in the field.
Finally, 3 Vitis vinifera plants, Merlot variety, and 6 Vitis vinifera plants, Corvina
variety, grown in a pot, and 6 Vitis vinifera plants, Merlot variety, grown in the
field were treated by spraying the leaves with an aqueous solution of spirulina
mixed with a 0.25% chitosan solution.
Such treatments were repeated on each plant with a treatment frequency of
14 days until reaching 7 treatments.
30 The chlorophyll content of the leaves of such plants was evaluated through
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SPAD-502 leaf clip (Konika Minolta), 6 days after the second treatment.
The results obtained are reported in Table 8, showing the chlorophyll content
expressed in SPAD units, measured in the control plants (which were not
treated either with spirulina or with microencapsulated spirulina), plants treated
with non microencapsulated spirulina solution, plants treated with a solution
of spirulina and chitosan, plants treated with a solution of spirulina
microencapsulated through a spray-dryer and plants treated with spirulina
microencapsulated by a nanoemulsion instrument.
Table 8
Mixture of Spirulina in
Spirulina in
Spirulina microcapsules F
Control Spirulina microcapsules
and nanoemulsion test
spray-drying
chitosan instrument
SPAD 32.7 31.9 31.8 32.0 32.6 ns
ns= not significant

The results show that treatment with the spirulina biomass contained in
microcapsules does not affect the photosynthetic activity of the plant.
2. 6 Analysis of downy mildew on plants treated with microencapsulated
spirulina biomass
Plants treated with spirulina microencapsulated through a spray-dryer and
through a nanoemulsion instrument, plants treated with a solution of spirulina
and chitosan, and the control plants of example 2.5 were evaluated for the
presence of downy mildew, 14 days after the third treatment.
Of the plants treated with spirulina microencapsulated through a spray-dryer
and through a nanoemulsion instrument, less than 25% of the foliage was
affected by downy mildew, the plants treated with a spirulina and chitosan
solution had about 50% of the foliage affected by downy mildew and the
control plants had between 50% and 75% of foliage affected by the disease.
Such results show how plants treated with microencapsulated spirulina
develop the disease in a significantly attenuated way with respect to plants not
treated.
2. 7 Leaf disk assays on plants treated with microencapsulated spirulina
biomass
From each plant treated according to example 2.5, 7 days after the fourth
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treatment with microencapsulated spirulina, 4 leaves were collected that were

then processed and analysed according to the technical methods reported in
example 1.7.
The results obtained from the analysis of the leaf disks show that about 15-
20% of the surface of the leaves of the plants treated with microencapsulated
spirulina developed reproductive structures of Plasmopara compared with
the leaves of the control plants in which about 42% of the surface is instead
infected by the pathogenic agent.
Such data show how treatment with microencapsulated spirulina reduces the
probability of developing the disease following contact with the pathogenic
agent.
2. 8 Analvsis of the enzvme phenvlalanine ammonia !vase (PAL) on plants
treated with microencapsulated spirulina biomass
The enzyme phenylalanine ammonia lyase (PAL) belongs to the category of
Pathogenesis-Related (PR) proteins synthesized by the plant following the
onset of the resistance mechanism to pathogenic agents.
The content of such enzyme was evaluated in plants treated according to
example 2.5.
Operatively, 2 grams of fresh leaves of each plant treated and of the control
plants were collected 0, 3, 7 and 15 days after the seventh treatment, and
ground with 8 ml of Tris 50 mM buffer at pH 8.5, 1 mM OTT (dithiothreitol) and
3% PVPP. The samples were subjected to centrifuging and the supernatant
was recovered, filtered on a filter and desalted on PD-1 0 columns.
2.5 ml of each sample were passed by gravity through the PD-10 column
previously balanced with 25 ml of water and the protein solution, without salts
and polyphenols, was eluted with 3.5 ml of water and recovered in test tubes
and ready to be subjected to enzymatic assays.
The enzymatic activity of the PAL was determined by measuring the change in
absorbance following the production of cinnamate formed by acidic hydrolysis.
The reaction mixture (1 ml total) was prepared by adding 0.1 ml of eluted
protein solution to 0.8 ml of Tris 500 mM buffer at pH 8.0 and 0.1 ml of 6 mM
!-phenylalanine.
It was then incubated in the stove at 40°C for 1 h and the reaction was
subsequently stopped by the addition of 0.05 ml of 5 N HCI. The absorbance
was measured in a 1 ml quartz cuvette with a 1 em optical path at 290 nm.
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The results obtained are shown in Table 9 reporting the absorbance values
at 290 nm of the leaves of the plants treated with spirulina, spirulina mixed
with chitosan, spirulina microencapsulated through a spray-dryer, and spirulina
microencapsulated through a nanoemulsion instrument, and of the control
plants, and wherein TO = leaves sampled at zero time from the seventh
treatment; T1 = leaves samples 3 days after the seventh treatment; T2 =
leaves sampled 7 days after the seventh treatment and T3 = leaves sampled
15 days after the seventh treatment.
Table 9
Absorbance 290 nm
TO T1 T2 T3
Control 0.0293 0.3613 0.9820 0.3183
Spirulina 0.0447 0.1980 0.1523 0.3843
Mixture of
Spirulina 0.0300 0.1500 0.3540 0.6460
and chitosan
Spirulina in
microcapsules 0.0503 0.2763 0.4163 1.1247
spray-drying
Spirulina in
microcapsules
0.0491 0.2932 0.5640 1.1423
nanoemulsion
instrument

Enzymatic analysis of PAL highlighted that such enzyme increases constantly

and continuously in leaves treated with microencapsulated spirulina up to 15
days following the last treatment.
This enzyme is particularly important in the metabolic pathway of
phenylpropanoids as, starting from !-phenylalanine, it catalyses the formation
of trans-cinnamic acid in phenol compounds such as lignin, phenolic acids,
stilbene and flavonoids, i.e. in the secondary metabolites fundamental for the
structural and defence functions of the plant.
Example 3. Treatment with a microencapsulated composition of Spirulina
platensis and plant protection product
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3. 1 Preparation of a composition of microcapsules of spirulina and water-

soluble compound through sprav-dryer
1 g of dried Spirulina platensis (Aighltaly) biomass powder was dissolved,
through agitation, in 100 ml of water to form a 1% w/v spirulina solution.
A 0.25% homogeneous suspension of chitosan dissolved in 0,25 M acetic acid
was then mixed with the solution obtained, through magnetic stirring.
The resulting spirulina and chitosan solution was then loaded into a B-290
spray-dryer instrument (Buchi) in flow current mode, with a variable flow, and
vaporized through a nozzle with a variable diameter comprised between
0.4 and 3 mm, due to the effect of compressed air at a pressure comprised
between 5 and 8 bar and flow of 200-800 Llhr.
The air flow responsible for drying was heated to a temperature comprised
between 80 and 200°C and the spirulina and chitosan solution aspirated with a
flow of about 30-55 Llhr in order to guarantee the minimum residence time
within the instrument.
0.6 g of microencapsulated spirulina were therefore obtained.
Likewise, 1 g of aluminium ethyl phosphite (anticryptogamic with systemic
action) was dissolved through agitation in 100 ml of water to form a 1% w/v
aluminium ethyl phosphite solution.
A 1% w/v chitosan solution in acetic acid was then added and mixed by
magnetic stirring with the aluminium ethyl phosphite solution.
The resulting aluminium ethyl phosphite and chitosan solution was then loaded
into a B-290 spray-dryer instrument (Buchi) in flow current mode, with a
variable flow, and vaporized through a nozzle with a variable diameter
comprised between 0.4 and 3 mm, due to the effect of compressed air at a
pressure comprised between 5 and 8 bar and flow of 200-800 Llhr.
The air flow responsible for drying was heated to a temperature comprised
between 80 and 200°C and the aluminium ethyl phosphite and chitosan
solution aspirated with a flow of about 30-55 Llhr in order to guarantee the
minimum residence time within the instrument.
0.75 g of microencapsulated aluminium ethyl phosphite were then obtained
and, subsequently, 0.5 g of the aforesaid microencapsulated compound were
mixed with 0.5 g of microencapsulated spirulina previously obtained to form a
composition of microcapsules of microencapsulated spirulina and aluminium
ethyl phosphite.
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3.2 Preparation of a composition of microcapsules of spirulina and liposoluble

compound through sprav-drver
1 g of dimethomorph (fungicidal compound with cytotropic, translaminar action
and with local systemicity against downy mildew) was dissolved by agitation in
10 ml of vegetable oil to form a 10% w/v dimethomorph solution.
A 1% w/v chitosan solution in acetic acid was then added and mixed with the
dimethomorph solution by magnetic stirring and subsequent sonication.
The resulting solution of dimethomorph and chitosan was then loaded into a
B-290 spray-dryer instrument (Buchi) in flow current mode, with a variable flow,
and vaporized through a nozzle with a variable diameter comprised between
0.4 and 3 mm, due to the effect of compressed air at a pressure comprised
between 5 and 8 bar and flow of 200-800 Llhr.
The air flow responsible for drying was heated to a temperature comprised
between 80 and 200°C and the dimethomorph and chitosan solution aspirated
with a flow of about 30-55 Llhr in order to guarantee the minimum residence
time within the instrument.
0.75 g of microencapsulated dimethomorph were therefore obtained.
0.5 g of microencapsulated dimethomorph were mixed with 0.5 g of
microencapsulated spirulina obtained according to the methods indicated in
example 3.1.1 to form a composition of microcapsules of microencapsulated
spirulina and dimethomorph.
3. 3 Preparation of a composition of spirulina and water-soluble compound
co-microencapsulated through sprav-dryer
1 g of dried Spirulina platensis biomass powder was dissolved, through
agitation, in 100 ml of water to form a 1% w/v spirulina solution.
0.1 g of aluminium ethyl phosphite were added to the spirulina solution and
mixed until complete dissolution.
A solution of chitosan dissolved in 0.25 M acetic acid was then mixed by
magnetic stirring with the solution obtained, such as to obtain a final
concentration of 0.25% chitosan in the form of a homogeneous suspension.
The spirulina-aluminium ethyl phosphite and chitosan solution was then loaded
into a B-290 spray-dryer instrument (Buchi) in flow current mode, with a
variable flow, and vaporized through a nozzle with a variable diameter
comprised between 0.4 and 3 mm, due to the effect of compressed air (only
air) at a pressure comprised between 5 and 8 bar and flow of 200-800 Llhr.
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The air flow responsible for drying was then heated to a temperature
comprised between 80 and 200°C and the spirulina and chitosan solution
aspirated with a flow of about 30-55 Llhr in order to guarantee the minimum
residence time within the instrument.
0.6 g of microencapsulated particulate were therefore obtained, recovered and
stored at 4°C.
3. 4 Analysis of downy mildew on plants treated with a composition of spirulina
and aluminium ethyl phosphite co-microencapsulated through spray-drver
300 mg of the microencapsulated particulate obtained in example 3.3 were
dissolved in 600 ml of water.
200 ml of the solution thus obtained were distributed through spraying onto
the foliage of each plant of 3 Vitis vinifera plants, Merlot variety, in a pot and
in the ripening phase, so as to treat each plant with 100 mg of the co-
microencapsulated composition of spirulina and aluminium ethyl phosphite.
Such treatments were repeated on each plant with a treatment frequency of
14 days until reaching 2 treatments.
The plants treated with the co-microencapsulated composition and 3 Vitis
vinifera plants, Merlot variety, in a pot that were not treated with the
composition (control plants) were evaluated for the presence of downy mildew,
14 days after the second treatment.
Of the plants treated with the composition, less than 25% of the foliage was
affected by downy mildew, whereas the control plants had between 50% and
75% of foliage affected by the disease.
Such results show how plants treated with the co-microencapsulated
composition develop the disease in a significantly attenuated way with respect
to plants not treated.
3.5 Preparation of a composition of spirulina and liposoluble compound
co-microencapsulated through spray-dryer
1 g of dried Spirulina platensis biomass powder was dissolved, through
agitation, in 90 ml of water.
0.1 g of dimethomorph were dissolved in 10 ml of vegetable oil and mixed with
the spirulina solution to form a 1% w/v suspension of spirulina which was then
sonicated and homogenized until obtaining a homogeneous mixture.
A solution of chitosan dissolved in 0.25 M acetic acid was then mixed by
magnetic stirring with the mixture obtained, such as to obtain a final
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concentration of 0.25% chitosan in the form of a homogeneous suspension.

The mixture of spirulina-dimethomorph and chitosan was then loaded into a
B-290 spray-dryer instrument (Buchi) in flow current mode, with a variable flow,
and vaporized through a nozzle with a variable diameter comprised between
0.4 and 3 mm, due to the effect of compressed air (only air) at a pressure
comprised between 5 and 8 bar and flow of 200-800 Llhr.
The air flow responsible for drying was then heated to a temperature
comprised between 80 and 200°C and the spirulina and chitosan solution
aspirated with a flow of about 30-55 Llhr in order to guarantee the minimum
residence time within the instrument.
0.6 g of microencapsulated particulate were therefore obtained, recovered and
stored at 4°C.
3. 6 Preparation of a composition of spirulina and water-soluble compound
through nanoemulsion instrument
1 g of dried Spirulina platensis (Aighltaly) biomass powder, a 0.25% solution
of chitosan in the form of a homogeneous suspension and a 0.2% solution
of aescin were dissolved, through agitation, in 90 ml of water.
0.1 g of aluminium ethyl phosphite were added to the spirulina, chitosan and
aescin solution and mixed until complete dissolution.
10 ml of seed oil were added to the solution obtained while stirring.
The suspension obtained was treated with a microfluidic nanoemulsion
instrument (LM20-30 Microfluidizer®) through three passages at 15000 Psi of
pressure to form a nanoemulsion of oil in water.
3. 7 Preparation of a composition of spirulina and liposoluble compound
through nanoemulsion instrument
1 g of dried Spirulina platensis (Aighltaly) biomass powder, a 0.25% solution
of chitosan in the form of a homogeneous suspension and a 0.2% solution
of aescin were dissolved, through agitation, in 90 ml of water.
10 ml of seed oil were added to the solution obtained while stirring in which
0.1 g of dimethomorph had been previously dissolved.
The suspension obtained was treated with a microfluidic nanoemulsion
instrument (LM20-30 Microfluidizer®) through three passages at 15000 Psi of
pressure to form a nanoemulsion of oil in water.
3. 8 Downy mildew analysis on plants treated with a composition of spirulina
and dimethomorph
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200 ml of emulsion obtained according to example 3.7 were distributed through

spraying onto the foliage of each plant of 3 Vitis vinifera plants, Merlot variety,
in a pot and in the ripening phase.
Such treatments were repeated on each plant with a treatment frequency of
14 days until reaching 2 treatments.
The plants treated with the composition and 3 Vitis vinifera plants, Merlot
variety, in a pot that were not treated with the composition (control plants)
were evaluated for the presence of downy mildew, 14 days after the second
treatment.
Of the plants treated with the composition, less than 10% of the foliage was
affected by downy mildew, whereas the control plants had between 50% and
75% of foliage affected by the disease.
Therefore, based on the above, the present invention has achieved all of the
predetermined objects.
In particular, the object of realizing an elicitor product that stimulates the
immune defences of a plant is achieved.
In particular, the biomass of the invention is a natural product, the use of which
in agriculture is not harmful either to the environment or to human health.
Again, the aforesaid biomass of the invention is made in an ecocompatible way
and without any negative effects on flora or fauna.
In particular, the microencapsulated formulation of the invention, when applied
to the plant or to part of the plant, is not removed by any adverse weather
conditions such as rain or wind.
Furthermore, the biomass of the invention can be used for endotherapic
treatments.
Finally, the biomass of the invention can be used in combination with other
agents having beneficial action for the plant, such as for example growth
adjuvants, plant protection products, etc.
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CLAIMS
1) Use of a Spirulina platensis biomass as the active ingredient for the
stimulation in a plant of the immune defences against pathogenic agents
characterized in that at least a part of said biomass is contained in a plurality
of microcapsules.
2) Use according to the preceding claim, characterized in that said
microcapsules have an interface comprising a polymer material and/or an
amphiphilic compound.
3) Use according to the preceding claim, characterized in that said
polymer material is chitosan.
4) Use according to any one of the preceding claims, characterized in
that each of such microcapsules has dimensions less than 100 1-Jm, preferably
comprised between 50 1-1m and 100 nm, more preferably comprised between
20 1-1m and 500 nm.
5) Use according to any one of the preceding claims, characterized
in that said biomass is incorporated into said microcapsules by means of
nanoemulsion technology.
6) Use according to any one of claims 1 to 4, characterized in that said
biomass is incorporated into said microcapsules by means of spray-drying
technology.
7) Use according to any one of the preceding claims, characterized in
that said plant is a fruit plant, preferably a fruit plant selected in the group
consisting of Vitis Vinifera, Olea europaea and Actinidia chinensis.
8) Use according to any one of the preceding claims, characterized in
that said biomass is applied to the leaves of said plant and/or the trunk of said
plant by endotherapic injection.
9) Use according to the preceding claim, characterized in that said
biomass is applied to said plant in a dose comprised between 1 and 500 mg
per plant, preferably between 50 and 250 mg per plant.
10) Use according to any one of the preceding claims, characterized in
that said biomass is further associated with one or more compounds selected
in the group consisting of formulation agents, phytoiatric agents, growth
adjuvants agents and elicitor agents of the defence mechanisms of plants, said
elicitor agents preferably being chitosan.
11) Use according to claim 10, characterized in that at least part of said
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one or more compounds is contained in said microcapsules together with said

biomass.
12) Use according to claim 10, characterized in that at least part of said
one or more compounds is contained in a plurality of microcapsules separately
from said biomass, said microcapsules containing at least part of said one
or more compounds having an interface comprising a polymer material or an
amphiphilic compound.
13) Use according to any one of the preceding claims, characterized in
that said biomass comprises a total extract of Spirulina platensis cells.
14) Method for producing a product to stimulate the immune defences
of a plant against pathogenic agents, said product comprising a Spirulina
platensis biomass, said method envisaging:
- mixing said Spirulina platensis biomass with an aqueous solution in the
presence of a polymer material and an amphiphilic compound;
- dispersing an organic phase comprising at least one organic solvent in the
aqueous mixture obtained from the previous operation, so as to obtain an
emulsion of said organic solvent in said aqueous mixture.
15) Method according to the preceding claim, characterized in that said
organic solvent is a vegetable oil or a terpene.
16) Method according to any one of claims 14 or 15, characterized in
that said biomass comprises a total extract of Spirulina platensis cells.
17) Method according to any one of claims 14 to 16, characterized in
that said polymer material is chitosan.
18) Method according to any one of claims 14 to 17, characterized in
that said amphiphilic compound is selected from lecithin, saponin, glucoside
surfactants, betaine, fatty acids, phospholipids and long-chain alkyl sulphates,
preferably said amphiphilic compound being a saponin.
19) Method according to any one of claims 14 to 18, characterized
in that said dispersion operation is performed through a microfluidic
nanoemulsion instrument.
20) Method according to any one of claims 14 to 19, characterized in
that in said emulsion said organic solvent is in the form of drops sized between
20 1-Jm and 100 nm.
21) Method according to any one of claims 14 to 20, characterized in
that said emulsion is dried by means of a spray-dryer following said dispersion
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operation so as to obtain a plurality of microcapsules containing at least part

of said biomass and having an interface comprising said polymer material and
a plurality of microcapsules containing at least part of said biomass and having
an interface comprising said amphiphilic compound.