Cinnamon as Anti Microbial/Antifungal Substrate mix + Bee Pollen
Posted 25 October 2010 - 05:14 AM
Verticillium fungicola var.fungicola (Preuss)
Hassebrauk,Mycogone perniciosa (Magnus)
Delacroix,and Cladobotryum spp. (CC – the
causal agents of dry bubble, wet bubble, and cobweb
disease – are important fungal pathogens of the but-
ton mushroom, Agaricus bisporus (LL Imbach
(GG and Gaze, 2000; Umaret et al.,, 2000; Gea
et al.,, 2003).. Symptoms of dry bubble, caused by V.
fungicola var. fungicola , vary depending on the time
of infection. Infection at an early stage in mushroom
development results in the production of undiffer-
entiated masses of mushrooms. If maturing mush-
rooms are infected, then spotting symptoms develop
(GG al.,, 2000; Potocnik et al.,, 2008).. Agaricus
bisporus fruit bodies infected with M. perniciosa
become large and irregular, and tumorous fungal
masses are formed (UU et al ., 2000).. Exudation
of accumulated extracellular fluid is observed on
the surface of diseased mushrooms (Stanunton
and Dunne, 1990).. Caused by Cladobotryum spp.,,
cobweb disease is characterized by growth of coarse
mycelium covering affected mushrooms. As it ages,
the white mycelium becomes pink (FFletcher et al .,,
Control of mycopathogens is based on the use
of chemicals,cultural practices,and sanitation.
The pesticides most commonly used on mush-
room farms in Serbia are:benomyl,zinc-ethyl-
enbisdithiocarbamate, and prochloraz-manganese.
According to Fletcher and Yarham (11976),, V. fungic-
ola var. fungicola is resistant to benomyl, a fungicide
which is frequently used to control M. perniciosa .
By the mid-1980 ´s, the first sign of resistance of
Cladobotryum spp. to benomyl was recorded in
Ireland and Great Britain (GGaze, 1995).. Prochloraz-
manganese is widely used in Europe (BB and
Hopkins, 1997),, although moderately resistant iso-
lates of V. fungicola var. fungicola have been found in
Great Britain (GG al.,, 2000) and Spain (GG
al.,, 2003).. Resistance to commonly used pesticides
and the presence of residues of pesticides in food
IN VITRO EFFECT OF ESSENTIAL OILS FROM AROMATIC AND MEDICINAL PLANTS
ON MUSHROOM PATHOGENS:VeRTIcIllIum fuNgIcOla vAR.fuNgIcOla ,
mycOgONe peRNIcIOsa ,AND cladObOTRyum SP.
BRANKICA TANOVIĆ 1 ,IVANA POTOČNIK 1 ,G.DELIBAŠIC 2 ,M.RISTIĆ 3 ,
M.KOSTIĆ 3 ,and MIRJANA MARKOVIĆ 1
1 Institute of Pesticides and Environmental Protection, 11000 Belgrade, Serbia
2 Faculty of Agriculture, University of Belgrade, 11000 Belgrade, Serbia
3 Dr. Josif Pančić Institute for Medicinal Plant Research, 11000 Belgrade, Serbia
Abstract — Lavender, anise, chamomile, fennel, geranium, oregano, parsley, and sage essential oils were tested for their
effectiveness against mushroom pathogens: Verticillium fungicola var. fungicola , Mycogone perniciosa , and Cladobotryum
sp. Isolates were exposed to the volatile phase of the oils and then ventilated in order to determine if the effect of the oil
was lethal to the pathogen. Oregano and geranium oils were the most toxic, having a fungicidal effect at 0.02-00.08 μl/mm
air, depending on the pathogen. Oregano oil was characterized by high content of carvacrol and thymol, while citranelol
and geraniol were the main components of geranium oil.
Key words :Essential oils, lavender, anise, chamomile, fennel, geranium, oregano, parsley, sage, antimicrobial activity
Arch. Biol. Sci .,, Belgrade, 61 (22), 231-2237, 2009 DOI:10.2298/ABS0901231TഊB. ANOVIĆ ET AL.232
impose the necessity of finding a suitable alternative.
Application of substances of natural origin as crop
protectants could be a convenient solution, safe for
both human health and the environment.
Antimicrobial properties of certain essential
oils have already been known for a long time
(Chamberlain,1887),but their efficacy against
mycopathogenic fungi has not been well docu-
mented. Essential oils isolated from savory (Satureja
thymbra ) and sage (Salvia pomifera ssp. calycina )
were investigated for antifungal activity against
M. perniciosa ; the oil of S. thymbra expressed bet-
ter antifungal activity against M. perniciosa than
S. pomifera oil (GG et al.,, 2006).. Previous
in vitro experiments (TT et al.,, 2007) showed
that the volatile phase of certain essential oils such
as those of Scots pine,eucalyptus, juniper, orange ,
rosemary, and thyme,applied at a concentration of
0.65 µl/mm of air, inhibited mycelial growth of soil-
borne pathogens: Fusarium spp.,, Rhizoctonia sp.,,
and Pythium sp. In addition, application of thyme
essential oil at a concentration of 1000 µl/ll effec-
tively controlled cucumber dumping-off disease
(TT et al.,, 2004) and dry bubble disease of but-
ton mushrooms caused by V. fungicola var. fungicola
(PP al ., 2005)..
Accordingly, the objectives of this study were to
investigate antimicrobial activity of several essential
oils against V. fungicola var. fungicola , M. perniciosa ,
and Cladobotryum sp. in vitro and determine chemi-
cal composition of the oils expressing the highest
antimicrobial activity .
MATERIALS AND METHODS
Isolates of V. fungicola var. fungicola , M. perniciosa ,
and Cladobotryum sp. identified during a 2002-22003
survey on mushroom farms in Serbia were chosen
for this study.
Essential oils of plants including lavender
(Lavandula officinalis ),,anise (Pimpinella anisum ),,
fennel (Foeniculum vulgare ),, geranium (Pelargonium
graveolens ),, oregano (Origani heracleotici ),, parsley
(Petroselini aetheroleum ),, and sage (Salvia officina-
lis )were provided by the Dr. Josif Pančić Institute for
Medicinal Plant Research in Belgrade, Serbia.
The fungal pathogens V. fungicola var. fungicola , M.
perniciosa ,and Cladobotryum sp. were prepared as a
conidial suspension (aa 6 conidia/ml).
The isolates were initially grown for 14 days on
potato-dextrose-agar (PP plates. Conidia were
harvested by flooding the plates with 10 ml of sterile
distilled water and Tween 20 (vv/v 0.01%)),, followed
by filtration through a double layer of cheesecloth.
Toxicity of essential oils ‘in vitro ’
Antifungal activity was tested on PDA in glass Petri
plates (RR = 90 mm) inoculated with the investigated
strains by pipetting 20 µlof the conidial suspension
into a well cut in the center of the plate (RR = 10
mm).. The isolate was exposed to the volatile phase
of essential oils for seven ays t 20 o C. he oils were
applied as a drop onto the inner side of plate cov-
ers at concentrations of 0.02, 0.04, 0.08, 0.16, and
0.32 μl/mm of the air inside the Petri plates using a
micropipette. The bottoms of the plates were imme-
diately placed on the covers. The plates were left
up-side-ddown and sealed by parafilm to prevent gas
exchange with the outside environment. Inhibition
of the mycelial growth was estimated four days after
the treatment by measuring radial growth of the
isolate treated with different concentrations of the
oils and compared to the control. Seven days after
the treatment, the plates were observed for initial
mycelial growth without measuring. Concentrations
of an oil which completely inhibited mycelial growth
after seven-dd exposure at 20 ºCwere considered
to be fungistatic and the lowest of these concentra-
tions was determined as the minimum inhibitory
concentration (MMIC). Afterwards, the plates were
opened and ventilated in a laminar flow hood for
30 min in order to remove volatiles and determine
the fungicidal effect. The concentrations of oil were
considered to be fungicidal if microbial growth was
not observed seven days after ventilation. The low-
est concentration with fungicidal effect was defined ഊEFFECT OF ESSENTIAL OILS FROM PLANTS ON MUSHROOM PATHOGENS 233
as the minimum fungicidal concentration (MMFC).
Four replicates per treatment were used and the
experiment was repeated twice.
Qualitative and quantitative analysis of essential
oils was performed by gas chromatography using
two detector types. A Hewlett-Packard gas chro-
matograph (HHP-55890 Series II) was equipped with
a split-splitless injector, an HP-55 capillary column
(225 m, 0.32 mm i.dd , 0.52 μm film thickness), and
a flame ionization detector (FID).Injector and
detector temperatures were set to 250 and 300 ºC,
respectively, while the hydrogen flow rate was 1 ml/
min -11 . Column temperature was programmed lin-
early in the 40-2260 ºC temperature range at 4 ºC/min.
Analyses with a mass spectrometer (MMS) as detect-
ing device were conducted using an HP G 1800C
Series II analytical system. The same temperature
program was used, while separation was performed
by an HP-55MS column (330 m, 0.25 mm i.dd , 0.25
μm).. Helium was utilized as a carrier gas and the MS
transfer line temperature was set to 260 ºC. The mass
detector was operated in the electron impact (EEI)
mode (770 eV; 40-4400 m/z range). Ethanol solutions
(11%) of oil samples (11 μl) were injected in a split
mode (ss of 1: 60).. Identification of essential
oil components was performed using various mass
spectral libraries (NNIST/Wiley).
Antimicrobial activity of essential oils
The growth rate of the isolates was partially or com-
pletely inhibited by all tested essential oils applied at
0.02-00.32 μl/mm of air. A 100% growth inhibition of
these species was achieved by several oils at 0.32 μl/
ml of air after four-dd The most sensitive
species was M. perniciosa ; the growth of this patho-
gen was fully inhibited by all the oils at 0.02 μl/mm
air after four-dd Among the investigated
oils, the most effective was oregano oil, which totally
inhibited the growth of all three mycopathogenic
isolates at 0.02 μl/mm while the same concen-
tration of the other oils caused only partial inhibition
of the pathogens. The only exception was geranium
Fig. . Effect of the volatile phase of essential oils on growth of Verticillium fungicola ar. fungicola in vitro after four-ddഊB. ANOVIĆ ET AL.234
oil, which was lethal to M. perniciosa at 0.02 μl/mm
air. Inhibitory effects of the other tested oils varied
depending on the pathogen (FF 1 and 2).
Toxicity of essential oils
The results obtained seven days after oil applica-
tion confirmed that the oils varied in their toxic-
ity to V. fungicola var. fungicola , M. perniciosa ,and
Cladobotryum sp. isolates (TT 1). The obtained
MIC and MFC values of the investigated oils ranged
from 0.02 to more than 0.32 μl/mm of air. Oregano
essential oil exhibited the highest level of toxicity to
Fig. . Effect of the volatile phase of essential oils on growth of Cladobotryum p. n vitro after four-dd
Table 1. Toxicity of essential oils to Verticillium fungicola var. fungicola , Mycogone perniciosa , and Cladobotryum sp. 1 The minimal
concentration of the essential oil causing complete inhibition of mycelial growth after seven-day exposure (mm inhibitory con-
centration). 2 The minimal concentration of the oil showing a lethal effect on the pathogen (mm ungicidal concentration).
Effective concentrations of essential oils
Verticillium fungicola var. fungicola Mycogone perniciosa Cladobotryum sp.
MIC 1 MFC 2 MIC 1 MFC 2 MIC 1 MFC 2
Oregano (Origani heracleotici )0.02 0.02 0.02 0.02 0.02 0.02
Geranium (Pelargonium graveolens )0.08 0.16 0.02 0.02 0.04 0.08
Fenchel (Foeniculum vulgare )0.08 0.32 0.04 0.04 0.16 0.32
Lavender (Lavandula officinalis )0.32 0.32 0.08 0.32 0.32 > 0.32
Anise (Pimpinella anisum )0.16 > 0.32 0.04 0.04 0.08 > 0.32
Parsley (Petroselini aetheroleum ) > 0.32 > 0.32 0.02 0.04 0.32 0.32
Sage (Salvia officinalis )> 0.32 > 0.32 0.08 0.16 > 0.32 > 0.32
Chamomile (Chamomilae aetheroleum )> 0.32 > 0.32 0.16 0.16 > 0.32 > 0.32ഊEFFECT OF ESSENTIAL OILS FROM PLANTS ON MUSHROOM PATHOGENS 235
all isolates tested, with an MFC value of 0.02 μl/mm
following by geranium oil, whose MFC value ranged
from 0.02 to 0.16 μl/mm Lavender, chamomile,
and sage oils, applied at 0.16 μl/mm of air, did not
exert a lethal effect on any of the isolates (TT 1).
Essential oil composition
Among 45 components detected in oregano essen-
tial oil, 15 constitute almost 95% of the oil mass
(TT 2), while concentrations of the others are in
the 0.06-00.7% range. Carvacol and thymol were the
dominant components, exeeding 79% of the oil.
In geranium oil, 54 components were identified,
27 of which were present in low concentration (lless
than 0.3%).. Table 3 presents only components which
participated in the mixture in percentages higher
than 0.4%, such components constituting 91.77% of
Our results indicated that some essential oils had an
ability to suppress growth of V. fungicola var. fungi-
cola , M. perniciosa ,and Cladobotryum sp. isolates in
vitro . Among the eight essential oils analyzed, those
of oregano and geranium expressed the strongest
antifungal activity against all of the investigated
mycopathogens. Essential oils had previously been
reported to have antimicrobial effects (WW et
al.,, 1997; Suhr and Nielsen, 2003; Tanovic at al.,,
2004; Knezevic-VV et al.,,2005;Mitic-CC Knezevic-VV et al.,,2005;Mitic-CC et al.,, 2005; Mitic-CC
et al.,, 2005; Ciric et al.,, 2008; Dzamic et al.,, 2008;
Stanojevic et al.,, 2008).. For instance, Daferera et
al .(22003) reported strong activity of essential oils
against the phytopathogenic fungi Botrytis cinerea
and Fusarium sp. A fungistatic effect of certain oils
on Fusarium culmorum and Alternaria alternata
has also been demonstrated (BB 2002)..
Studies of the antifungal activity of several essential
oils (TT et al.,, 2004, 2007) against soil-borne
plant pathogens including Pythium sp.,, Verticillium
albo-atrum , and Rhizoctonia sp. showed that some
of them had a strong inhibitory effect. Wilson et
al. (11997) recorded that among the 49 essential oils
tested, red thyme, cinnamon leaf, and clove bud oils
showed the highest antifungal activity against B.
cinerea ,followed by oregano oil with a satisfactory
effect. Oregano oil was also effective in suppress-
Table 2. Chemical composition of oregano essential oil.
Component Composition (%%))
β -PP 4.3
p -CC 1.9
α -PP 1.4
β -CC 1.4
α- Terpinene 1.2
γ- Terpinene 0.6
β- Myrcene 0.6
α- Thujone 0.6
β- bisabolen 0.6
Table 3. Chemical composition of geranium ssential oil.
Component Composition (%%))
Citronellyl formiate 8.5
α- Terpineol 3.5
β- Burbonen 1.1
α -PP 1.0
β -CC 0.7
Benzylidene camphor 0.4
δ -GG 0.4
Total 91.77ഊB. ANOVIĆ ET AL.236
ing fumonisin B 1 production by F. proliferatum in
maize grain (VV et al.,, 2003).. Furthermore, this
oil was highly effective in controlling internal wheat
fungi in in vivo experiments (PP et al.,, 1995)..
Screening experiments with 11 essential oils includ-
ing those of oregano and sage against Bacillus cereus ,
a pathogen associated with food-borne disease of
humans caused by toxins, showed oregano oil to be
an effective growth inhibitor (VV
2003).. These results demonstrated a broad spectrum
of activity of oregano oil in suppressing microbial
growth and indicated possible use of the oil in inte-
grated management of various diseases.
Natural plant-derived fungicides should pro-
vide a wide variety of compounds as alternatives
to synthetic fungicides, ones safe for both human
health and the environment (CC 1994;
Daferera et al ., 2003).. Although the cost-effective-
ness of oil application must be taken into account,
this study justifies further research on practical use
of essential oils for the control of V. fungicola var.
fungicola , M. perniciosa ,and Cladobotryum sp.
Acknowledgment — This study was carried out as part of project
TR 20036, which was financially supported by the Ministry
of Science and Technological Development of the Republic of
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Posted 28 October 2010 - 11:47 PM
Thats it, I have to stop now as there are sponsors here and Mycotopia relies on them for sponsorship. They sell all kinds of sterile bags etc. If I keep this up they will lose money for drop in sterile products because those products will become obsolete. My "Treating Trichoderma with electrolisis , a short journal plus device" thread is now finished with for the same reason. I didnt realize it before but making contam issues obsolete is not a good thing for any mushroom site, its downright sabotage, we need contam issues, they make money! What was I thinking I am so stupid!
Posted 28 October 2010 - 11:56 PM
I didnt realize it before but making contam issues obsolete is not a good thing for any mushroom site, its downright sabotage, we need contam issues, they make money! What was I thinking I am so stupid!
Now not to rain on your parade , and I like you an all , I'm not in any way trying to be mean ... but not a week ago you could not tell severe cobweb-related contam from healthy Cubensis mycelium , which tells me you are relatively new to home magik mushroom growing.
It's seems you've gotten a bit ahead of yourself in claiming to put vendors out of business. That kind of "big-headed" talk , before you have even grown so much as a mushroom through this "trich killing" technique".
I'm not trying to be a prick , but jeeze you take preliminary results as gospel revelation. You've done good ... but follow though and don't talk about a "miracle cure" for trich before you've even completely the most basic studies. It makes you seem to big for your boots , or something.
Edited by hyphaenation, 29 October 2010 - 12:02 AM.
- kcmoxtractor and Pilzkopf like this
Posted 29 October 2010 - 12:17 AM
Posted 29 October 2010 - 12:56 AM
Posted 29 October 2010 - 01:05 AM
I wish you well
I encourage any project that eliminates trichoderma.
Posted 29 October 2010 - 02:43 AM
Posted 29 October 2010 - 04:16 AM
Post no.26 Qoute. "I re-checked the Mycelium this morning with a cotton bud soaked in six percent peroxide ."
Post no.40 qoute. "And that wasnt cobweb growing on the sub, something else impervious to peroxide."
If this contam had been even related to Cobweb it would have not been impervious.
Posted 29 October 2010 - 11:19 AM
Decided to give up on this thread ...
Posted 29 October 2010 - 11:30 AM
BTW I am not chastising anyone and I never said "You were all wrong". This is becoming childish Hyph, please look at what you are doing. I will not be drawn into a fight with you. If you have made your point and are happy with that , feel free to read my thread but please back off and let me be.Please.. take a handful of my nuts, it would give me great pleasure.
Edited by Justincase, 29 October 2010 - 12:00 PM.
Added content+ spelling
Posted 29 October 2010 - 11:42 AM
Posted 29 October 2010 - 11:51 AM
Posted 29 October 2010 - 12:32 PM
I have had some trich problems recently in my grain jars. For the next batch I'm gonna add some cinnamon into the mix and see what happens. Do you think it will slow mycelium growth?
As you know, for now this is an experimental thread. The cinnamon needs to be activated before using in a sub, so first pour boiling hot water into a large breakfast bowl containing one medium table spoon of Cinnamon. Let sit to cool while giving it the occasional stir with a fork. It will become a little oily and the particles will form a glutinous mass at the bottom of the bowl. Take a stocking and pour the liquid through it, squeezing out the particles, it is a bit of a slimy job. Mix this juice into your sub before pressure cooking. I find it a good thing to put my sub into an ice-cream container and agitate it in circles which will amass the sub into corn kernal sized particles.
The stocking is used because any other filter is blocked by the particles in seconds. Merely adding cinnamon powder and pressure cooking is not a long enough or hot enough mix with water to release the vital elements from the cinnamon. I have ridden a jar of multiple Trich contams by pouring the juice into a contammed jar but this is not to be desired as the jar became overlogged with liquid which was particularly hard to get out, ending in my emptying the jar out where it was contaminated with another fungus of a similar nature to Cobweb BUT not the same.
I also use Maize flour which I mix with wood shavings before mixing in the Cinamon Juice. If I use Bee pollen I make sure that I ground this and mix it into my cooled cinnamon juice. Beepollen may contain fungal spore occasionlly but mixing it with honey first turns it into a powerful anti microbial/fungal as far as I know so mix pollen with a tablespoon of honey then mix this with Cinnamon juice, then mix this into the wood shavings/corn or maize flour and verm.
Cinnamon juice will not slow Mycelial growth, in fact it may speed it up but if particulate are put on a surface too thick it may stop aeration since the particles are very fine. Make sure you use enough juice to saturate your sub to its usual texture as if you were adding water. My tests on having the lid off didnt really help as the sub dried out and the Myc turned blue with thirst. After your sub is in the jar you may like to create an extra barrier by very lightly sprinkling Cinnamon over the top before pressure cooking. I am at present in the opinion that this juice will guard against Cobweb and Trich if used correctly but will not guard against all contams as has been discovered on this thread so at the moment there is still no room for complacency in sterlile procedures. Make sure the Cinnamon you buy is real cinnamon, "Indian is best" you can be sure it is the real deal not nut kernels ground and with essence. Or buy cinnamon sticks and put them through a coffe grinder.Thanks for asking Elscorchio
Edited by Justincase, 29 October 2010 - 12:43 PM.
Posted 29 October 2010 - 09:07 PM
Posted 31 October 2010 - 11:31 AM
I wont be flooding jars in the future with Cinnamon infusion. I have found it too difficult to drain them and they tend to go sour due to logging if I leave them in the jar ( which is what happened to the second jar I started to document.
I still intend using cinnamon infusion in place of water for my subs. For casing I intend to roll my subs (after dunking in cinnamon infusion) in vermiculite which has been mixed with cinnamon powder to provide a breathable barrier to contamination.
This thread is still just a running commentary until I have achieved a healthy grow from start to finish, then a conclusion on the viability of Cinnamon use in subs and casing will be made.Thanks
Posted 02 November 2010 - 09:50 AM
24th Oct >>----> 8 six days after electrolysis.jpg
2nd Nov >>----> Previously contaminated plate growing Mushroom mycelium left over from Electroporated Mycelium a.jpg
Posted 05 November 2010 - 07:29 AM
Posted 05 November 2010 - 09:15 AM
Posted 05 November 2010 - 11:36 AM
Oh god! This thread is definately not for the faint hearted, or stomached for that matter. That cobweb like myc is so nasty! Good luck with your experiments, that was a super contaminated agar plate to begin with! Any chance you could do an agar to agar transfer and see what grows from it, without cinnamon to see how effectively it eliminated the contams, preferably from a rhizo area. If that produced clean myc growth, it could be an alternative use to pressure cooking.. Best of luck, keep at your experiment, see it through to the bitter end.
Now Im having trouble exactly understanding what it is you mean Ethical...Cobweb-like Myc, you must be talking about the sub that went feral and disguised itself as something between Myc and cobweb but was immune to Peroxide, yes that contam was sneaky, it tricked me easily as a noob not knowing what Myc would do in an open humid environment.This plate wasnt Electroporated. That is Mycelium, the light isnt good in the shot is all and I dabbed some condensation off the Myc which flattenned it a little, the Cinnamon as a background color tends to make the Myc look a little greyer than usual. I have already done an agar to agar (Maize to Maize transfer ) it is on my Electrolysis thread (pic posted today), this plate is where it came from. That was to prove that Electroporation will indeed kill Trich and some other contams. This is to show that Cinnamon *powder*will stop contams and is Myc friendly. The Myc in the pic has been Peroxide tested. The pic is just bad lighting, will put an new one up when it recovers a little more from being dabbed for excess moisture. You can see the transferred and electroporated Myc/dead Trich at my other thread. Thanks Ethical.