# Patent

## Title
Process for potentiating the production of Lingzhi mushroom (Ganoderma lucidum) substances and antifungal activity thereof

## Abstract

The instant invention refers to a novel process for potentiating the production of substances with antifungal activity obtained from Ganoderma lucidum, using a technological device for the differential, qualitative and quantitative expression of proteins and other bioactive molecules, selected from the group consisting of polysaccharides, triterpenoids, fatty acids and ganoderic acids. The compositions containing said substances showed antifungal activity and mycelium growth and fungi ascospore germination inhibiting activity, amongst them Mycosphaerella fijiensis, primary pathogenic agent causing black Sigatoka disease in banana and plantain crop fields.

## Background
PRIOR RELATED APPLICATIONS
This invention claims priority to CO 13-036699, filed on Feb. 22, 2013 and incorporated by reference in its entirety herein.
FIELD OF THE INVENTION
The present invention refers to a new process for potentiating the production of substances having antifungal activity obtained from Ganoderma lucidum, compositions containing said antifungal substances and their use as inhibitors of mycelium growth and fungi ascospore germination, amongst them those of Mycosphaerella fijiensis. 
BACKGROUND ART
US2008/138875 describes a method for increasing the production of microorganism biomass, particularly Ganoderma lucidum from mycelium at a very low cost. The process is carried out within a closed system comprising a light source producing different wavelengths in order to obtain an increase in biomass; however, no mention of an increase in primary and/or secondary metabolites is given or any antifungal activity.
The publication “Electrical field effect on mycelium biomass production of the medicinal fungus Ganoderma lucidum” (Actual Biol 2010, 32 (92): p. 5-17) evaluates the effect of electrical fields, such as alternate current, continuous current, pulsated current and field intensity, on the growth of the fungus Ganoderma lucidum under submerged growth culture conditions, with the purpose of optimizing its biomass production.
The publication “Production of Biomass, Polysaccharides, and Ganoderic Acid using Non-conventional Carbon Sources under Submerged Culture of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum” (International Journal of Medicinal Mushrooms 2012. Vol. 14. I. 2 p. 197-203), mentions the effect of the different non-conventional carbon sources in the Ganoderma lucidum growth, for the simultaneous production of mycelium biomass, ganoderic acids and polysaccharides, using non-conventional carbon sources in order to minimize the high costs of the current growth culture media. The publication does not disclose a process for increasing the obtainment of Ganoderma lucidum extracts or metabolites nor its antifungal activity.
DESCRIPTION
Strict biological control is defined as the use of live organisms as agents for plague and disease control and it is one of the most used methods for counteracting the use of agrochemicals. In the past few years, they have been used as an efficient alternative, those natural compounds obtained from microorganisms and plants, which have advantages over commercial chemical products, for being less toxic, both on the ecosystem as well as on the global population and because the same microflora in situ biodegrades them.
The well known medicinal properties of Ganoderma lucidum have lately driven a growing interest in all areas to study its metabolites and biological activities. Amongst the active compounds reported for this fungus are found β-D-glucane type polysaccharides, lanostane type triterpenoids known as ganoderic acids, proteins and peptides (1-4).
The Ganoderma lucidum fungus is widely known for its beneficial effects in humans; however, it has other properties such as its capacity to inhibit growth of phytopathogenic agents, such as Botrytis cinerea, Fusarium oxysporum and Physalospora piricola, which represents an interesting alternative in developing biocontrol products (5,6).
Black Sigatoka or the black stripe of bananas, is a disease caused by the Ascomycete fungus Mycosphaerella fijiensis (M. Morelet) (sexual form, heterothalic), which in its anamorphous stage is Pseudocercospora fijiensis (M. Morelet) Deighton (7). This fungus is a hemibiotrophic haploid found classified in class Dothideomycetes, order Capnodiales and family Mycosphaerellaceae. Mycosphaerella fijiensis (and its anamorphous Pseudocercospora fijiensis), is considered the most devastating species of the complex termed ‘Sigatoka disease complex’ (8-9).
The first symptoms of the disease are numerous and upon examination of the leaves throughout the different stages and comparing them to a healthy leaf it is possible to observe the accumulated deterioration of the tissues, observing that the initial change is carried out around the aereous space and continues to spread towards the parenchyme cells and vascular bundles, finally the mesophyll tissue degrades and the entire structural conformation of the leaf is lost (9).
The fungus Mycosphaerella fijiensis, arrives at the leaf in ascospores and conidiums and then continues to germinate towards the stomas, which are invaded by the mycelium until they are entirely covered with the different fungus structures (9). 1 mm diameter greenish dots appear in the plant's leaf which continues to develop into 2 to 3 cm long thin greenish reddish stripes, visible in the leaf's haz; then the spots join and darken until reaching a black color. Finally, the dead and black zones dry up and acquire a more pale color. Affected leaves may die in three to four weeks and the result is a rapid and severe weeding (9).
Disease control and management is basically found in cultural practices using weeding, thinning, trimming and intensive application of chemical fungicides, among them, demethylation inhibitors (DMIs), amines, Qo inhibitors (QoIs), anilinopyrimidines (APs), benzimidazoles (BCMs), SDHI fungicides and guanidines, especially in technified crop fields. Despite the efforts, disease control is ever so difficult given the fungus' capacity to grow and successfully adapt to the ecosystem conditions of the banana crop field sites, making ridding of the disease ever more so difficult.
There is therefore a need to find agents and active ingredients for the biological control using a plethora of antagonistic mechanisms against the pathogen, with which making disease control commercial formulation would result feasible and thus being able to attack the pathogenic agent from different fronts.
It is well known that a large part of substances or metabolites obtained currently for biological control processes are obtained from traditional culture crops, and on a laboratory scale, wherein occasionally, optimized culture media do not exist for the production of biomass and bioactive ingredients.
In order to find a new alternative for the management of black sigatoka, through the use of biological control techniques and the obtainment of active extracts, the inventors developed an optimized process for increasing the production of Ganoderma lucidum biomass and antifungal active ingredients thereof as inhibitors of mycelium growth and fungi ascospore germination, particularly Mycosphaerella fijiensis. 
Therefore, in a first aspect, the instant invention provides a process, which initially comprises a Ganoderma lucidum obtainment stage in an adequate culture medium and specific environmental conditions which generate an unforeseen increase in biomass production in a reduced time.
After the obtainment process, a cell lysis stage is carried out in order to extract active compounds with which the final concentration of total active substances is increased. The protein extracts obtained during the process, were evaluated on ascospore germination and Mycosphaerella fijiensis mycelium growth, unforeseeably finding a detrimental effect of the phytopathogen when being potentiated through the process of the invention.
In another aspect, the instant invention implements the use of a technological device for the differential, qualitative and quantitative expression of proteins and other bioactive molecules of Ganoderma lucidum, selected from the group consisting of polysaccharides, triterpenoids, fatty acids and ganoderic acids. The invention provides for an unforeseen increase in the production of polysaccharides (intra and ex polysaccharides), triterpenoids, fatty acids and ganoderic acids.
In an additional aspect, the instant invention provides a protein extract, an ethanol extract and a triterpenoid-rich and fatty acid-rich Ganoderma lucidum extract exhibiting antifungal activity, particularly against Mycosphaerella fijiensis. The protein extract comprises at least one protein family from the protease, peroxidase, nuclease, lacase, immunomonuclease, phosphatase, dehydrogenase, synthase, oxidase, polymerase, and demthylase groups exhibiting substantial inhibition against Mycosphaerella fijiensis. 
In another aspect, the current invention provides a formulation comprising the ethanol extract, the triterpenoid-rich and fatty acid-rich extract and the protein extract, or one or more proteins from the Ganoderma lucidum protein extract, together with an agrochemically acceptable carrier.
In another aspect, the present invention provides the use of Ganoderma lucidum as a Mycosphaerella fijiensis mycelium growth antagonist fungus.
In an additional aspect, the instant invention provides for the use of the protein extract and/or the ethanol extract and/or the triterpenoid-rich and fatty acid-rich Ganoderma lucidum extract for the inhibition of mycelium growth and pathogen ascospore germination, particularly Mycosphaerella fijiensis, in conventional and non-conventional crop fields.


DETAILED DESCRIPTION
Maintenance of the Ganoderma lucidum Strain and Conservation Thereof in a Petri Dish
The Ganoderma lucidum strain (donated by Dr. ST. Chang from the Plant Biotechnology Laboratory from the Universidad de Antioquia) was maintained in a potato dextrose agar (PDA) at 4° C. (working strains, first subculture from a laboratory mother strain) (10-12).
The process followed consisted in taken 1 cm culture media and mycelium squares were transferred to petri dishes having solid MGL1 medium, comprising (g/L): barley flour 30.0 g; yeast extract 3.0 g; sucrose 5.3 g and agar-agar 8.0 pH-adjusted to 5.5±0.1 (11,12).
Ganoderma lucidum Mycelium Biomass Obtainment Under Submerged Culture Conditions Using Different LED-Based Lighting Systems.
1 cm diameter mycelium and agar discs were inoculated in 250 ml flasks having 50 ml culture medium, comprised of liquid MGL1: NaNO3 80 mg/L; KH2PO4 30 mg/L; MgSO4.7H2O 20 mg/L; KCl 10 mg/L supplemented with 50 g/L barley flour at a pH 5.5±0.1 (11, 12). The inoculated media were incubated at 100 rpm, 25±1° C., during 5 days of culture. From this pre-inoculum, 1 gram of biomass was transferred and inoculated in 250 ml Erlenmeyers having 60 ml of the same medium. This media was incubated with continuous stirring at 100 rpm, 25±1° C., and treated under different LED light conditions (white, blue, red, yellow) for each one of the assays, during 9 days of culture (11,12).
Extract from Mycelial Biomass of Ganoderma lucidum Extract from Cultivated Under Different LED-Based Wavelengths: Proteins, Polysaccharides, Ganoderic Acids, Triterpenoids and Fatty Acids.
Protein Extracts
Protein Extract without the Optimization Process
10 grams of fresh biomass from each of the culture media grown under different LED light conditions, was washed 3 times using abundant distilled water, later was washed twice using 20 mL PBS phosphate buffer pH 7.4 by centrifugation at 4° C., 9000 rpm during 10 minutes (Boeco® U320R) (12). Furthermore, the biomass was sonicated in an ice bath and later the filtrate was centrifuged at 4° C., 9000 rpm, during 10 minutes. The recovered supernatant was treated with 250 μL triton and 200 mg PVP for each 25 ml of lysate, everything was homogenized in a vortex until no particles could be observed in the solution and was again centrifuged at 4° C., 9000 rpm, during 10 minutes.
Finally, acetone was added to the recovered supernatant, previously stored at −20° C., in a 1:1 ratio incubated during 1 hour at 4° C. Later, centrifugation was carried out at 4° C., 9000 rpm during 15 minutes; this last precipitation step was repeated once again with the supernatant. The pellet obtained was again washed using previously chilled acetone at −20° C. and strong vortex was carried out for the dispersion of the pellet. Centrifugation at 4° C., 9000 rpm during 30 minutes was carried out. The supernatant was discarded and the pellet re-suspended in 3 ml PBS buffer pH 7.4. Further, the protein extracts stemming from each light condition were quantified by the Bradford method, aliquoted and stored at −20° C. (53-55).
Optimization of the Protein Extraction Conditions of the Ganoderma lucidum Mycelium Biomass
The methodology used for obtaining protein extracts from Ganoderma lucidum mycelium biomass is described below. As may be noted, the methodology implemented makes use of sonication as a method of cellular lysis, primarily for cell wall rupture. In order to optimize the extraction of mycelium proteins, the effect of two cell lysis processes and the combination thereof were first evaluated (sonication, homogenization, and both), and later, optimization of rupture conditions with the selected process was carried out. FIG. 1 describes the optimized obtainment process of the Ganoderma lucidum protein extracts.
Qualitative and Quantitative Protein Expression
For this assay in particular, 10 μg of each of the Ganoderma lucidum protein extracts were taken, coming from different culture conditions, White LED, Blue LED, Yellow LED, and Red LED, in triplicate and a one-dimension electrophoresis was run. Later, all present proteins were excised en each 1D electrophoresis column in order to assay them using mass spectrometry and further analysis and identification by sequence comparison in the several different databases using MASCOT.
The methodology used in this study can be summarized in 5 steps after running the proteins on the gel: (i) unstain the gel, (ii) reduce disulfide bridges and alkylate resulting sulphidryl groups, (iii) enzymatically direct proteins using trypsin, (iv) extract resulting peptides and (v) assay using mass spectrometry (13-15). The technique used allowed for relative quantification of the proteins found, carrying out comparisons of proteins profiles according to relative intensities of the chromatograms of extracted ions during the enzymatic digest (16-19).
Crude Ethanol Extract
50 grams of dry and pulverized biomass was ground in 96% alcohol for two hours in ultrasound (Misonix®). Later the material was filtered and dried in a rotoevaporator, weighed and stored at 4° C. for later use.
Polysaccharide Extraction and Quantification: IPS (Intapolysaccharides) and EPS (Exopolysaccharides)
For IPS quantification, 1 g of dry Ganoderma lucidum mycelium biomass was taken and was re-suspended in 10 mL of water. This blend was taken to the autoclave for high-temperature extraction; the hot solution was filtered. Further, IPS precipitation was carried out by diluting the filtrate in four 96% ethanol volumes at 4° C. during 1 hour, followed by 4000 rpm centrifugation. The pellet obtained was diluted in 5 mL of water and used for centrifugation using the validated Phenol-Sulfuric Acid method (20). For EPS extraction, the filtered medium was centrifuged at 2500 rpm at 25° C. in order to eliminate suspended solids; 10 mL of the supernatant was taken and four 96% ethanol volumes were added. The same protocol was carried out for IPS extraction in order to obtain the polysaccharide extract.
Ganoderic Acid (GA) Extraction and Quantification
For GA quantification, the basic process followed was taking a dry mycelium biomass sample (500 mg), to which 50% (v/v) ethanol was added. Later, centrifugation at 2500 rpm for 15 minutes was carried out; the supernatant was dried by vacuum evaporation at 40° C., the residue re-suspended in water and extracted using chloroform. The GA in chloroform were extracted with 5% bicarbonate, 2M HCl was added to the basic aqueous phase until the pH was adjusted to values less than 3 and ultimately, it was extracted using chloroform, which was dried by vacuum evaporation at 40° C. The residue was again dissolved in absolute ethanol and was read at a 245 nm absorbance.
Triterpenoid and Fatty Acid Extraction, Characterization and Relative Quantification
100 g of freeze-dried biomass and 100 g of freeze-dried wasted extracellular medium from each of the LED-light treatments were taken and subject to maceration using 200 ml of CH2Cl2 during 5 days. Later, solvent removal was carried out every two days, until the Liebermann Burchard qualitative test, which is Δ5-sterol-specific or specific for sterols containing real or potentially conjugated dienes, turned out negative. The crude extracts were concentrated until dry in a rotoevaporator.
Thereafter, 1 g of each of the extracts was saponified in order to eliminate free fatty acids, as such: 20 ml of a 1M KOH in MeOH solution was added to the extract at 80° C. during 180 minutes, under reflux conditions. To the saponification product 10 ml of H2O was added and it was extracted using ethyl ether. The organic phase was washed with water until reaching a neutral pH, in order to then be collected on anhydrous sodium sulfate and concentrated until dry in a rotavapor.
The chemical characterization of the extracts was carried out using gas chromatography coupled to mass spectrometry (GC-MS). The equipment specs are as follows: HP6890, internal ZB-5HT capillary column (30 m, 0.25 mm ID and 25 μm thickness), carrier gas He 4.5 to 1.1 mL/min, Splitless injection mode and injector temperature 300° C. Said chromatograph unit was coupled to a 5973 mass detector, with a 70 eV ionization source.
The program used consisted of a heating ramp of 60° C. during one minute and later 7.4° C./min until reaching 310° C. 1 μL of each extract was injected into 3 mg/mL of CH2Cl2. The components were identified by comparison with the equipment log and as for the triterpenoid components, by analysis and comparison with databases of the Macromycete Fungus Chemical Research Group of the Universidad Nacional de Colombia.
Mycosphaerella fijiensis Isolations and Monospore Cultures Obtention for Assays
Mycosphaerella fijiensis isolations were performed by colleting ascospores from infected plant material (previously diagnosed at Cenibanano-AUGURA and submitted by Cenibanano to the Biotechnology Laboratory at Universidad de Antioquia (21-23). Lesions found on leaves classified for a stage 6 of the disease according to the Fouréscale (1985) characterized by foliar grayish white spots with black dots, which under an electronic microscope evidenced the presence of pseudothecia for ascospore dispersion and conidiophores for the release of conidia (23).
Ascospores were identified under a binocular compound microscope with a number 20 objective; they were selected one by one in order to be transferred to a PDA medium and then incubated at 25±1° C. until obtaining the colony named R1. A subculture was finally performed, the colonies obtained were divided and sub-cultured in PDA medium as work isolations R2 (24).
Antagonism Assay Using Deep Planting of Mycosphaerella fijiensis 
A 1 cm diameter disc of a Mycosphaerella fijiensis R2 culture was inoculated, previously macerated in a 0.9% NaCl solution, in Petri dishes with the MGL1 culture medium melted at a temperature of 37° C. This culture was incubated for a lapse of 15 days in the dark at room temperature. Later, a 1 cm diameter disc of Ganoderma lucidum was inoculated on Mycosphaerella fijiensis grown mycelium; said dual culture was incubated for a lapse of 15 days in the dark at room temperature. The assays were performed in triplicate using a Mycosphaerella fijiensis growth control and a Ganoderma lucidum growth control. The cultures were microscopically assayed during the course of the experiment, observing and comparing the hyphae of each one of the fungi.
Antagonism Assay in Dual Plates: Antagonism Assay by Facing Ganoderma lucidum vs. Mycosphaerella fijiensis 
A 1.3 cm diameter disc of a Mycosphaerella fijiensis R2 culture was inoculated, in Petri dishes with PDAL culture medium, at a distance of 2 cm from the dish edge and incubated at room temperature for 20 days, in order to provide the phytopathogen all optimal growth conditions for its establishment and substrate colonization.
After this time lapsed, a Ganoderma lucidum 1.3 cm diameter mycelium disc was inoculated in each culture medium, at a 4.5 cm distance from the Mycosphaerella fijiensis colony (26, 27, 33-36). Finally, this dual culture was incubated for 26 days in darkness at room temperature. The assays were performed in triplicate using a Mycosphaerella fijiensis growth control and a Ganoderma lucidum growth control. Cultures were macroscopically assayed during the course of the experiment, using a millimetric ruler for measuring the radius of the Mycosphaerella fijiensis colony (26,32). Inhibition percentage was assayed at 15 days of the antagonism, according to the mathematical model described for this type of analysis defined as RG=(G/G1)×100, wherein G is the treatment's growth efficiency and G1 the control, obtained from the following ratio:

G1=(FD/ID)

Wherein,

FD=final diameter of the colony, ID=initial diameter of the colony (37).

Additionally, samples from each treatment were taken, both observed by light microscope as well as electronic microscope, in order to compare morphological variations of the phytopathogenic fungus upon exposure to the controlling fungus (26, 27, 33-36). The samples were initially observed on a light microscope using 40× and 100× objectives (Nikon, Microscope eclipse 80i) and images were taken using a Nikon N is Elements BR 3.2. imager. Samples of the control Mycosphaerella fijiensis mycelium and antagonized Mycosphaerella fijiensis were taken, deposited on a cover slip and staining a 5% red congo dye (39).
For the observation using a scanning electron microscope SEM (Jeol, JSM 6490LV Model), control and antagonized Mycosphaerella fijiensis mycelium samples were taken. Samples were fixed using the FAA protocol (45% of 95% ethanol, 45% glacial acetic acid, 10% formaldehyde (44-48)). The samples were freeze-dried and adhered to the sample holder using electric tape and coated using a vacuum gold film by sputtering for two minutes (Denton Vacuum, Desl IV model) (49-51).
Finally, control and antagonized Mycosphaerella fijiensis mycelium samples were taken when under the effect of Ganoderma lucidum, in order to determine the feasibility of the Mycosphaerella fijiensis mycelium. Part of the antagonized Mycosphaerella fijiensis mycelium was replicated in new Petri dishes with PDAL medium, in order to observe the development of the mycelium and new Mycosphaerella fijiensis hyphae. Furthermore, a 0.8 mM in PBS trypan blue exclusion test was carried out, a widely used method to differentiate between live cells and dead cells, since trypan blue is a supravital stain that colors dead cells. The mycelium was macerated in the presence of the stain and the samples were observed under 40× and 100× objectives (Nikon, Microscope eclipse 80i) and the images taken using a Nikon N is Elementes BR 3.2 imager.
Antifungal Activity Assays on Ascospore Germ Tube Elongation
In order to carry out this assay, ascospores were collected where in each Petri dish, containing water agar (2%), the extract to be studied in each of the different concentrations was added (ethanol extracts; 0, 100 ppm, 1000 ppm, 10000 ppm and protein extracts: 0, 0.1 ppm, 1.0 ppm, 10 ppm). Furthermore, a 10 ppm protein extract+K proteinase (E.C. 3.4.21.64) was assayed (56, 57).
This is primarily used in this type of assays as a means of controlling that the activity found in fact corresponds to the activity of one or various extract proteins and not other substances that may be extracted during the process. For this purpose, the enzyme was added to the raw protein extract at a final concentration of 20 mg/ml. The extracts were incubated at 37° C. for an hour using a vortex every 10 minutes, then the extracts were centrifuged at 9000 rpm, 4° C. during 15 minutes; the freeze-dried supernatant of each extract was used to carry out the activity assays (59, 60).
Finally, after carrying out the ascospore discharge during 45 minutes in each of the extract-supplemented media, leaves were removed and media was incubated for 48 hours at 25±1° C. The ascospores were measured using a micrometer and germ tube lengths were recorded (Nikon Stage Micrometer), under a binocular compound microscope using a 20× objective (Nikon, Microscope eclipse 80i). All assays were carried out in triplicate, measuring 60 ascospores per treatment, in at least five discharge fields (59, 60). Inhibition percentage of the extracts was calculated taking the ascospores developed in water agar as control.
All results were analyzed using Statgraphics XV.I. For the ethanol extract assay, a one way ANOVA was carried out, wherein the factor assayed was the extract concentration. For the protein extracts, a one way block design was performed wherein the effect of different protein extracts was assayed at different concentrations. For each model, ANOVA and post-ANOVA tests were performed, in order to assay the givens.
Fungicide Activity Evaluation of Ganoderma lucidum Extracts: Mycelium Growth Inhibition in Microplate Format
Fungicide Activity Evaluation of Ganoderma lucidum Protein Extracts
In order to obtain protein extracts, the optimized protein extraction protocol was followed (Item 4.1.2). Later, the protein pellet was re-suspended in 1 ml PBS pH 7.4 buffer, 200 μL DMSO and 200 μL glycerol. The protein extracts of each LED were quantified by the Bradford method (Bio-Rad Protein Assay®) (61). Starting from stock solutions, 100 μL of each one of the Ganoderma lucidum protein extracts were added for a final concentration in the 500, 250, 125 and 100 ppm plates. The extracts were diluted in pH 7.4 PBS phosphate buffer.
The Mycosphaerella fijiensis-inoculated microplates and also treated with the different extracts were incubated. Readings were performed according to the Standard Norm. Each concentration was assayed in three repetitions, including three experiment replicas (63, 64). Thereafter, the determination of the growth inhibition percentage for each concentration of each protein extract used was performed (65-67).
Fungicide Activity Evaluation of Triterpenoid and Fatty Acid-Rich Ganoderma lucidum Extracts
In order to obtain the triterpenic extracts, the protocol described in section 4.3 was followed. Later, the extracts were prepared at a 100,000 ppm concentration and dissolved in a 1:1 Ethanol:DMSO mix. The microplates were inoculated with Mycosphaerella fijiensis according to the conditions previously established. Thereafter, several dilutions from the stock solutions were performed in the microplate, in order to assay the antifungal activity of the extracts at final concentrations of 5000, 4000, 3000, 2000, and 1000 ppm, pursuant to the Standard Norm. Each concentration was assayed in 3 repetitions, including three experiment replicas (63, 64). Thereafter, the determination of the growth inhibition percentage for each concentration of each extract concentration used was performed (65-67).
Antifungal Activity Assay of Extracts Using the Agar Diffusion Technique: Protein Extracts and Triterpenoid and Fatty Acid-Rich Extracts.
In order to corroborate antifungal activity of each extract, said activity was assayed in Petri dishes using the plate diffusion method; for said purpose, 20 ml of PDA medium were dispensed for a final thickness of 4 mm. After homogenizing inocula, deep plating was performed wherein each plate was inoculated using 2×104 Mycosphaerella fijiensis mycelium fragments. Afterwards, using a 0.5 mm internal diameter punch, wells were made wherein 10 or 20 μL of each extract were deposited, in addition to the inhibition positive control using Mancozeb and a negative control using a 1:1 ethanol:DMSO mix. The plates were incubated in darkness conditions, 25±1° C. for seven days.
EXAMPLES
Protein and Other Bioactive Ingredient (Polysaccharides, Triterpenoids, Ganoderic Acids and Fatty Acids) Qualitative and Quantitative Expression Using a Technological Device Based on Different Electromagnetic Radiation Wavelengths
Proteins
A list of molecular masses was obtained herein measured using mass spectrometry, that were subject to MASCOT proteomic analysis program for comparison with the different proteins found in databases, and where a 34 protein list was able to be obtained, all of which have several expression degrees under the different light conditions, in the different extracts assayed. This allowed for a series of comparisons and to demonstrate that the light conditions under which the fungus is subject to for its growth indeed influences it in its production and protein expression at some point. Table 1 shows the list of identified proteins, initially indicating their physicochemical characteristics.







TABLE 1







Proteins identified in the Ganoderma lucidum extracts and some important


characteristics.















Length
Weight
Isoelectric


No
Entry Name
Protein Name
(aa)
(kDa)
Point (pI)















1
GANOD_GANLU
Ganodermin*
27.0
2.72
10.28


2
Q710F5_GANLU
Laccase
299
32.03
5.03


3
B5G553_GANTS
Laccase
521
56.42
5.46


4
LZ8_GANLU
Immunomodulatory protein Ling
111
12.51
5.09




Zhi-8


5
A6P7J5_GANLU
Ribonuclease T2
304
33.49
4.89


6
D7F2S3_GANLU
Manganese superoxide
142
15.45
8.76




dismutase


7
B2KX91_GANLU
3-hydroxy-3-methylglutaryl-
1226
13.11
8.23




coenzyme A reductase


8
B3XZ37_GANLU
ATPase subunit a
175
19.31
8.94


9
B3XZA5_GANLU
RNA polymerase II second
287
32.76
8.20




largest subunit


10
B6UPH5_GANLU
Cytochrome c oxidase subunit 2
72
7.78
4.79


11
Q2HPQ0_TRIHA
Actin
257
28.83
5.24


12
C7EWL6_GANLU
G-protein complex beta subunit
313
34.90
5.79


13
Q8HE45_GANLU
Endonuclease
264
31.03
8.54


14
D7NJ68_GANLU
Lanosterol synthase
726
82.87
6.33


15
A0SJQ5_GANLU
Squalene synthase
467
54.01
7.09


16
D8UZT0_9PEZI
RNA polymerase II second
351
39.68
6.13




largest subunit


17
D8Q297_SCHCM
Farnesyl-diphosphate synthase
347
39.82
5.65


18
GOX8C9_GANLU
DyP
488
53.19
7.7


19
E7E161_GANLU
Calmodulin
149
16.88
4.20


20
E7E162_GANLU
Calcineurin A
339
37.02
5.34


21
E7E163_GANLU
Calcineurin B
161
18.19
4.67


22
F6M3U4_GANLU
Putative nitrogen regulatory
265
28.92
9.65




protein


23
G1ECM7_GANLU
Calcineurin responsive zinc-
639
69.21
8.79




finger CRZ1


24
G3LGR3_GANLU
Succinate dehydrogenase iron-
245
28.00
8.9




sulfur protein


25
B8K207_GANLU
Beta-tubulin
120
13.34
5.87


26
C0IMT8_GANLU
Manganese peroxidase
364
38.11
4.61


27
Q4PS64_GANTS
Translation elongation factor 1-
379
41.43
8.73




alpha Q4PS64 Elongation factor




1-alpha


28
Q0GZR4_GANLU
Glyceraldehyde-3-phosphate
337
36.06
6.45




dehydrogenase


29
B3XZA5_GANLU
RNA polymerase II second
287
32.76
8.20




largest subunit


30
B4YA15_GANLU
Farnesyl-diphosphate synthase
360
41.01
5.34


31
B5G551_GANLU
Laccase
520
56.26
5.16


32
B5G552_GANLU
Laccase
520
56.295
5.09


33
B6E1X1_GANLU
Lanosterol 14-alpha-demethylase
180
21.026
6.8


34
Q0GZR4_GANLU
Glyceraldehyde-3-phosphate
337
36.067
6.45




dehydrogenase





*Corresponds to a peptide






The influence of the wavelength based on LEDs could differentially be observed on the Ganoderma lucidum protein profile, according to its expression level, in terms of intensity, obtained through mass spectrometry. FIG. 2 comparatively illustrates these expression levels for each protein identified.
It can be observed that the greater intensity (LFQ intensity), which can be interpreted as the greater concentration of the different proteins identified, shows on the yellow LED, followed by the blue LED, then the red LED and finally the white LED condition. This demonstrates that a differential effect does indeed exist when the fungus is exposed to different wavelengths, which ultimately is expressed in greater or lesser production of biomass quantity and the obtainment of polysaccharide and ganoderic acid-type primary or secondary metabolites.
Polysaccharide Quantification (IPS and EPS)
After performing the different assays using several light conditions, it was determined that the best light conditions for IPS and EPS production was blue LED light, with significant differences over the other conditions, with p<0.05 (FIG. 3).
Values obtained for IPS were: for blue LED light 31.32±1.24 g/L, followed by red LED light with 22.34±1.24 g/L, yellow light 19.23±1.24 g/L and finally white LED light with 17.42±1.24 g/L. Values obtained for EPS were: for blue LED light 29±2.31 g/L, followed by red LED light 22.33±2.31 g/L, yellow light 18.54±2.31 g/L and finally white LED light with 16.03±2.31 g/L.
Ganoderic Acid (GA) Quantification
It was determined that the best wavelength for GA production was the wavelength corresponding to blue light with a pH of 5.0 and a p value >0.05, obtaining 563.9±11.63 mg/L for blue LED light, followed by 447.1±11.63 mg/L for red LED light, 411.0 mg/L±11.63 for yellow LED light and finally 228.8 mg/L±11.63 for white LED light (FIG. 4).
Chemical Characterization and Relative Quantification of Triterpenoid and Fatty Acid-rich Extracts







TABLE 2







Relative abundance percentage of each compound in the Ganoderma lucidum


extracts, plated under different LED light conditions.










Intracellular
Extracellular



(Relative Abundance %)
Relative Abundance %)
















Yellow
Blue
White
Red
Yellow
Blue
White
Red


Compound
LED
LED
LED
LED
LED
LED
LED
LED


















Palmitic acid
7.60
Tz
31.20
16.40
14.36
9.94
2.92
5.72


Ethyl palmitate
1.70
27.70
7.90
7.80
5.88
9.75
13.85
3.31


Ethyl linoleate
12.20
39.50
11.70
23.50
32.43
29.19
33.97
10.94


Ethyl oleate
14.10
17.60
5.20
8.80
10.12
26.61
15.23
3.82


22E-ergosta 5,7,9
1.30
Tz
0.35


(22), tetraen 3β-ol


Ergosta 5,7,22 trien
2.20

1.02


3β-ol


Ergosta-5-3β-ol

Tz


Estigmast-5-3β-ol
1.10
2.20
0.40
2.20


tz


26,26-dimethyl 5,


Tz


23 ergostadien 3β-ol


Estigmast-4 en-3-
2.30
4.30
0.70
2.80
2.51
1.42
tz
5.71


one


Estigmast-5,24(28)-
0.96


dien-3β-ol


Estigmast-4,24(28)-
0.78



0.83

tz
1.53


dien-3-one


5α-Estigmast-3-one

Tz




Tz


(22R,24S)-







2.52


22,24Dimethylcholesterol


Fatty acid total
7.6
Tz
31.20
16.4
14.3
9.0
2.9
5.7


Total fatty acid
28.0
84.8
24.8
40.1
48.4
65.5
62.8
18.9


esters


Total triterpenoids
8.6
6.5
2.5
5.1
3.33
1.5
0
9.7









Antagonism Assay in Dual Plates

Table 3 illustrates the growth of each fungus when found seeded individually or control medium or when co-seeded. As for the Ganoderma lucidum control, a growth rate of 0.2983 cm/day was found, and the co-seed at 0.281 cm/day. For Mycosphaerella fijiensis, it was determined that the control growth rate was 0.1287 cm/day and the co-seed 0.059 cm/day. The above evidences that Ganoderma lucidum slightly reduces its growth rate in the co-seed, but nevertheless is capable of colonizing the entire substrate in the same time period, whereas for Mycosphaerella fijiensis, the impact is greater, demonstrating it is the antagonized organism during the process.







TABLE 3







Growth inhibition percentage of Mycosphaerella fijiensis


comparing the growth of the antagonized isolation versus control.










Colony diameter




average (cm)



Day











Assay
1
8
15
Antagonism %





Antagonized
2.439
2.528
2.581
85.1


Mycosphaerella fijiensis


Mycosphaerella fijiensis
2.411
2.686
2.997
Control


control









Ascospore Germination Inhibition Using Different Extracts Obtained from Ganoderma lucidum Mycelium Biomass

Raw Protein Extract

Evaluation of the effect of Ganoderma lucidum protein extracts was performed using one way ANOVA with blocking, wherein the factor assayed was the protein extract of the different LEDs and the block being the extract concentration. Having obtained the variance analysis results and adjustment tests (independence, normality and equal variance), an effect was noted over ascospore germination due to the type of extract and its concentration, with a p value <0.05.
The analysis indicates that the blue LED extract, at 10 ppm showed an inhibition percentage of 84.95±0.89%, followed by the inhibition percentage of the yellow LED extract at 10 ppm at of 58.52±0.89% (FIG. 5).
Furthermore, it could be noted that the treatment with 10 ppm of each extract digested with proteinase K (10 PK), significantly reduced the inhibition percentage to 7.6±0.89%. This suggests that the effect observed on Mycosphaerella fijiensis ascospores is due to a protein or protein complex that are digested with the enzyme and thus no activity is shown (59, 60).
Ethanol Extracts
The ethanol extract concentration effect over the Mycosphaerella fijiensis ascospore germination inhibition percentage was evaluated, wherein it was found that a significant inhibition effect indeed exists which is produced by the ethanol extract over Mycosphaerella fijiensis ascospore germination, which is dependent on concentration, with a p value <0.05. The formation of three significantly different homogeneous groups was observed, wherein the 10000 ppm concentration of the ethanol extract showed greater inhibition percentage at 98.4±0.51%.
Evaluation of Fungicide Activity of Ganoderma lucidum Extracts Over M. Fijiensis in Microplates
Protein Extracts without the Optimized Extraction Process
A multifactor ANOVA variance analysis was performed for inhibition percentage, where it was observed that the LED factor, extract concentration and interaction thereof have an effect over Mycosphaerella fijiensis growth with a p value <0.05, wherein it is observed that by having a control at 0% inhibition, Mancozeb at a 0.3 ppm concentration, it showed 72% inhibition, amount surpassed by the different extracts, except the white extract, at concentrations starting at 100 ppm. Likewise, it is corroborated that the activity observed is due to one or several extract proteins since when treated with proteinase K, no antifungal activity was shown, even at a 1000 ppm concentration.
Through the use of logic models (non linear regressions) the relationship between the concentration logarithm of each antifungal (x axis) versus inhibition percentage (y axis) was established thus obtaining the respective equations, for EC50 analysis for each treatment at 5 concentrations, in order to determine the necessary concentration of each extract to reduce the population growth by 50% at an in vitro level. Table 4 shows EC50 for all protein extracts obtained under different LED light conditions, obtained when plotting the concentration log versus inhibition percentage.







TABLE 4







EC50 for each treatment, wherein data adjustment to


the log (inhibitor) vs. response equation is shown.











Data
Red
Yellow
White
Blue










Log(Inhibitor) vs. Response











EC50
114.28 ± 1.02
94.41 ± 1.07
295.80 ± 1.11
11.32 ± 1.07


(ppm)


R2
0.9993
0.9945
0.9924
0.9938


(equation


adjust-


ment)









The above table shows that the phytopathogenic fungus response in treatments with extracts obtained under red LED and yellow LED are similar, having an EC50 of 114.288±1.02 and 94.406±1.07 ppm, respectively. The white extract was the least effective with an EC50 of 295.8±1.11 ppm, while the response obtained the extract under blue LED light was the most potent, with an EC50 of 11.32±1.07 ppm, well below compared to the other treatments.
Fungicide Activity of Ganoderma lucidum-Optimized Protein Extracts
In order to determine which the best protein extract for Mycosphaerella fijiensis mycelium growth inhibition was, a statistical analysis using a category factorial design was performed. The multifactorial ANOVA variance analysis for inhibition percentage shows that the LED factor, extract concentration and the interaction thereof all have an effect over Mycosphaerella fijiensis growth with a p value <0.05. Further, EC50 analysis was performed on each treatment on all four concentrations, in order to determine the necessary concentration of each extract in order to reduce the population growth by 50% at an in vitro level.
As may be noted on Table 5, EC50 is found well below in comparison to the assayed dosages, and it is therefore concluded that these new protein extracts extracted under the optimized protocol, are more potent, allowing to claim this response in reference to being able to extract at greater concentration the active proteins. Table 5 shows the results obtained when plotting the concentration logarithm versus inhibition percentage and the model adjustment.







TABLE 5







EC70 and EC95 for each treatment, wherein data adjusted to


the log(inhibitor) vs response equation is shown.











Data
Yellow
Blue
White
Red










Log (Inhibitor) vs. Response











EC70
149.6 ± 0.08
—
421.7 ± 0.12
100.46 ± 1.12


(ppm)


EC95
—
158.49 ± 1.85
—
251.18 ± 1.12


(ppm)


R2
0.9992
0.9964
0.9967
0.9921


(equation


adjust-


ment)





Note:


the best treatment is highlighted.






As may be noted, the blue LED extract continues being the most active, where the minimum inhibition percentage generated by the extract is 73%, a response well above those found with the extracts before the extraction optimization. When performing an IC95 analysis, it can be noted that only the blue LED and red LED extracts reach said potency of 158.49±1.85 ppm and 251.19±1.12 ppm, respectively.
As may be noted in previous treatments, there exists Mycosphaerella fijiensis mycelium growth inhibition when treated with the different protein extracts and it is noted that the red and blue extracts show greater inhibition in comparison to other treatments and growth control; likewise, Mancozeb's activity as control fungicide can be observed, where the fungus growth is entirely inhibited. These results are found to be in agreement with the microdilution assay and with the ascospore germination assays, where the blue extract always shows the greater activity.
Fungicide Activity of Triterpenoid and Fatty Acid-rich Extracts
Table 6 shows how the triterpenoid and fatty acid-rich extracts present Mycosphaerella fijiensis mycelium growth inhibition. The greater inhibition is shown with treatments stemming from blue LED extracts, followed by the treatments with yellow LED extracts. For white and red LED extracts, no significant growth inhibition was found in comparison to a growth control without extracts.







TABLE 6







Intracellular extracts. EC50 for each treatment, wherein data


adjusted to the log(inhibitor) vs response equation is shown.












Intra-
Intra-
Intra-
Intra-



cellular
cellular
cellular
cellular


Data
Red
Yellow
White
Blue










Log(Inhibitor) vs. Response











EC50 (ppm)
NA
103250 ± 75.64
NA
4563 ± 35.78


R2(equation
—
0.9932
—
0.9932


adjustment)





NA. Does not apply since extracts were not active.






Agar Diffusion Assay

Finally, and seeking to corroborate what is observed in the microplates, the antifungal capacity was assayed using the dish diffusion technique. FIG. 6 shows the results obtained at this level, where different quantities of each one of the extracts were deposited at the greatest concentration, 500 ppm, using 10 μL or 20 μL per well.
As can be noted from previous treatments, Mycosphaerella fijiensis mycelium growth inhibition exists when treated with the different protein extracts or triterpenoid-rich extracts and it can be noted that the red and blue extracts show the greatest inhibition compared to the other treatments and the growth control; Mancozeb activity as control fungicide can also be observed, wherein fungus growth is entirely inhibited. These results are in agreement with the microdilution assay and with the ascospore germination assays, wherein the blue extract always shows the greatest activity.
Agrochemical Compositions
From biomass and Ganoderma lucidum extracts obtained according to the subject invention, compositions comprising Ganoderma lucidum and/or any of its extracts may be prepared, together with carriers, vehicles, and adjuvants acceptable for agrochemical applications, as well as the use of said compositions for controlling and/or fighting phytopathogens.
Compositions according to the subject invention can be provided in any appropriate form, for example, in powder, solution, suspension, granule form, amongst others.
DESCRIPTION OF DRAWINGS
FIG. 1 displays an optimized obtainment process of the Ganoderma lucidum protein extracts.
FIG. 2 compares Ganoderma lucidum proteins identified in the different extracts when seeded under several LED light conditions, wherein their differential expression may be observed.
FIG. 3 displays the IPS and EPS production from Ganoderma lucidum under different LED light conditions.
FIG. 4 displays the Ganoderic acids production from Ganoderma lucidum under different LED light conditions.
FIG. 5 illustrates the inhibition percentage for the Mycosphaerella fijiensis ascospore germination.
FIG. 6 is an evaluation of the different extracts using the agar diffusion test, wherein each plate was inoculated with Mycosphaerella fijiensis at a concentration of 2×10^4 mycelium fragments/ml and (A) growth control, (B) Mancozeb control, (C) a plate treated with a white extract, having a small inhibition halo in the 10 μL well, (D) plate treated with a yellow extract, having an inhibition halo in the 10 μL and 20 μL well, (E) plate treated with a red extract, having an inhibition halo in the 10 μL and 20 μL well, and (F) plate treated with a blue extract, having an inhibition halo in the 10 μL and 20 μL well.
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## Claims

1. A procedure for obtaining a protein extract having antifungal activity against Mycosphaerella fijiensis comprising the following steps:
a. inoculating Ganoderma lucidum in an appropriate culture medium, and culturing under appropriate culture conditions to obtain a Ganoderma lucidum biomass;
b. the biomass is obtained under electromagnetic radiation with a wavelength in the range of from 400 to 550 nm; and
c. obtaining from said biomass a protein extract having antifungal activity against Mycosphaerella fijiensis, the protein extract is obtained by homogenization in a lysis solution comprising a phosphate buffer, Triton X-100, and a hydro-soluble polymer during 5 through 50 homogenization cycles.

2. The procedure according to claim 1 wherein the appropriate culture medium comprises a combination of NaNO3; KH2PO4; MgS04·7H20; KCI; barley flour and in a solid, semisolid or liquid matrix in a manual or automated systems.

3. The procedure according to claim 1, wherein the electromagnetic radiation wavelength generates differential, qualitative and quantitative expression of proteins and other bioactive molecules, selected from the group consisting of polysaccharides, triterpenoids, ganoderic acids and fatty acids.

4. The procedure according to claim 1, wherein obtaining the protein extract (step c) comprises the steps of:
a. washing the biomass with the phosphate buffer with pH between 5.0 and 9.0;
b. homogenizing the washed biomass in step (a) with the lysis solution comprising phosphate-buffered saline (PBS), Triton X-100, and the hydro-soluble polymer polyvinylpyrrolidone, for 5 through 50 cycles to obtain a lysate;
c. let stand and filter the lysate obtained in step (b);
d. centrifuge the filtered lysate obtained in step (c), and separate a first supernatant;
e. adding Triton X-100 and polyvinylpyrrolidone to the supernatant obtained in step (d), and homogenize;
f. let stand and centrifuge the homogenate obtained in step (e) to obtain a second supernatant;
g. adding acetone to the second supernatant obtained in step (f) to obtain a precipitate; and
h. dissolve the precipitate obtained in step (g) in the phosphate buffer with pH between 5.0 and 9.0 to obtain the protein extract having antifungal activity against Mycosphaerella Yensis.

5. The procedure according to claim 4 wherein the homogenization and centrifugation steps are performed at a temperature ranging between 0 and 40° C.

6. The procedure according to claim 4 wherein the homogenization and centrifugation steps are performed in time intervals between 0.1 and 30 minutes.

7. The procedure according to claim 4 wherein the homogenization rate ranges between 5000 and 25000 rpm.

8. The procedure according to claim 4, wherein the acetone in step (g) is added in a 1:1, 2:1, 3:1, 1:3 or 1:2 (v/v) ratio with respect to the supernatant.