rezumat Raluca - Vasilica MICLEA · Biologist Raluca Vasilica Miclea -Summary of the PhD thesis- Researches regarding the biology and the control with plant extracts of the grey mould

Biologist Raluca Vasilica Miclea

-Summary of the PhD thesis-

Researches regarding the biology and the control with plant extracts of the grey mould caused by Botrytis

cinerea Pers.

CLUJ-NAPOCA 2012

UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ NAPOCA

DOCTORAL SCHOOL Faculty of Agriculture

Scientific coordinator Prof. univ. dr. CARMEN PUIA

Raluca Miclea TEZĂ DE DOCTORAT

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INTRODUCTION

Reports of fungal diseases in ancient and medieval periods are very rare and also very

inaccurate. However, it seems that mentions about the rot of grapes in the vineyard caused by

fungi, are as old as vine cultivation. There are few reports of fungal diseases on grapes from the

time of Pliny the Elder in the first century or later, around 1350, in the time of Konrad von

Mengengerg (Germany) (Rosslenbroich and Stuebler, 2000).

The Botrytis genus was quoted for the first time in 1729 by Micheli, in his paper Noveau

Gen (Oudemans, 1919 cited by Drobot, 2008) and its name comes from the greek word for

"bunch of grapes" so we can conclude that disease symptoms so-called "gray mold" on grapes

must have been known for many years. For a long period of time the fungus has not received any

special name, so Viala (1887) cited by Drobotă (2008) designate it as "gray rot" or "the rot of

grapes".

Botrytis cinerea Pers. specie infects a large number of host plants in a variety of

environmental conditions and in different geographical areas both in open field and in

greenhouses and solariums. Botrytis cinerea Pers. also damage stored agricultural products due

to its ability to be active at low temperatures (Coley-Smith et al., 1980).

Due to the growing damages caused by Botrytis cinerea Pers. and the high degree of

extension to different botanical families plants, the fungus was given more importance in recent

times (Drobot, 2008).

Given the fact that the chemical control of plant diseases records a decrease, the

biological control of plant diseases through herbal extracts gains more and more importance, the

literature demonstrates that the use of natural products extracted or fermented from various

sources mixtures of natural ingredients with specific activities or complex mixtures with multiple

effects on the host organism and the pathogen is a important biological control mechanism.

Phytopreparations represent real perspective biological means of plant protection of

having several advantages, namely: are highly efficient extraction is not complicated and long,

are harmless to the environment and people, quickly decompose in agrocoenoses and their use is

usually effective.

Given the above mentioned facts and the fact that gray mold may have a negative impact

on the yield and quality of harvest this thesis mainly aimed at studying the biology of the fungus

Botrytis cinerea Pers. and its biological control with hydroalcoholic extracts. Taken under

consideration the fact that in the experimental period we have tested also the propolis extract and

we obtained very good results regarding the fungicidal effect of this extract on the development


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of the fungus, this tincture was introduced in the processing of the data although it is not a plant

extract.

For the trust, the understanding, the support during the PhD period and in elaboration of

the doctoral thesis i want to thank my PhD supervisor - Professor Carmen PUIA. For all the help

during the doctoral period i want to adress thanks and gratitude to Octavian Pop from SC

Hypericum Impex S: R.L, Professor Carmen Socaciu, lecturer Rodica Pop and to all those who

contributed in one way or another to the development and finish of this thesis. Also i will like to

give thanks to the scientific reviewers for reading and analising my thesis.

I thank God how gave me health, strength to work and valuable people that support me to

reach this professional level.

CHAPTER I

CHARACTERISATION OF BOTRYTIS CINEREA PERS. SPECIE

Botrytis cinerea Pers. is the most important species of the 22 species belonging to the

genus Botrytis (Hennebert, 1973) and is one of the most important fungal pathogens due to its

unique characteristics: it can live as saprophyte and parasite, may be negative in some cultures

but may be useful in certain circumstances and cause early latent infections that cause damage

only during fruit ripening (Rosslenbroich and Stuebler, 2000).

1.1. IMPORTANCE

Due to its polyphagous and necrotrofic characteristics, Botrytis cinerea Pers. is a good

model for the study of fungal infectious processes. Plant pathology scientists were fascinated by

the conidial infection of gray mold on plant tissue very early so Ward (1888) cited by Elad et al.

(2004) described the infection of the Botrytis species by germ tubes.

Botrytis cinerea Pers., therefore, is an excellent model for studying this type of infectious

process that is characteristic of many fungal diseases of plants using a wide range of pathogenic

mechanisms (lytic enzymes, activated forms of oxygen, toxins, hormones of plants) to attack its

host plants (www.genoscope.cns.fr).

1.2 TAXONOMY AND SYNONYMS

Botrytis cinerea Pers. belongs to Deuteromycotina phylum, Hyphomycetes class,

Moniliales order, Moniliaceae family (Carmen Puia, 2006). Fungus dispersed predominantly by

producing massive gray-brown clusters.


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The teleomorph of the specie Botrytis cinerea Pers. is the specie Botryotinia fuckeliana

Sclerotiniaceae family, Helotiales order, Discomycetes class, Ascomycotina phylum (Carmen

Puia, 2005).

1.3. MORPHOLOGICAL CHARACTERS

1.3.1 Micelium

The mycelium of the fungus lives as saprophyte on various organic substrates or parasite

(Tomoioagă Liliana 2002, Fermaud and Philippe, 2000 cited by Drobotă, 2008 and Viorica

Jacob et al., 2000), has a gray color, is abundant and branched conidiofori.

The mycelium can penetrate the intercellular spaces of the tissues attacked to the ruling

vessels (Pârvu, 2004). Presents rounded apical cells, which are grouped into large clusters,

conidias. The mycelium presents long conidiophores, septates, monopodial branched at the

terminal section. Last ramifications of the conidiophores presents short sterigmas bearing

unicellular, ovoid or yellowish brown conidias (Carmen Puia, 2005, 2006).

1.3.2 Macroconidia

Botrytis species conidias are usually considered short-live propagation organs, but there

is evidence that under certain conditions, even they can ensure the survival of the species (Pârvu,

1993).

Tatiana Şesan şi Tănase (2007) described the conidias of Botrytis cinerea Pers. as having

ovoid, unicellular, sharper at one end, brown colored, with size between 9.0 -12.0 x 6.5 - 10.0

µm, with 4-6 nuclei, each with a nucleolus.

1.3.3. Microconidia

Botrytis cinerea Pers. beside the macroconidia forms also microconidia which can

function as primary inoculum (Brierley, 1918 cited by Drobotă, 2008). The microconidial form

was observed by Istvanffi quoted by Drobotă (2008).

Akutsu et al. (2004) cited by Fukumori et al. (2004) in their research showed the first

time that during sexual propagation of the species Botrytis cinerea Pers. microconidias were

produced in large numbers and that after mating with sclerotia produced by this species is

formed apotecias, asces, ascospores).

1.3.4. Sclerotia

Botrytis cinerea Pers. resists in soil as mycelium and in the diseased organs as sclerotia

(Carmen Puia, 2005). Sclerotia are black resistance bodies with strong consistency, which play


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an important role in the perpetuation of the fungus (Carmen Puia, 2005) and have a flattened

oval-shaped with rough surface, measuring 2-4 mm long and 1-3 mm wide, composed of two

different parts: the cortex and medulla.

1.3.5 The sexual form of the fungus

Although the link between Botrytis cinerea Pers. and Botryotinia fuckeliana was

suspected for a long time no definitive evidence relating to the identity of these two forms was

not published until the appearance Istvanffi's work, called “Etudes microbiologiques et

mycologiques sur le rot gris de la vigne—Botrytis cinerea ou Sclerotinia fuckeliana'”. Istvanffi

concluded that Botrytis cinerea Pers. decidedly the conidial stage of Botryotinia fuckeliana.

1.5 HOST PLANTS AND INDUCED DISEASES

The symptoms caused by Botrytis cinerea Pers. are very common and widely distributed

(Gonsalves and Ferreira, 1994 cited by Odeh, 2006) this species beeing a fungus with a wide

range of host plants including vegetables, soft fruits, ornamental plants and vines, appears in

greenhouses and field and during storage and transport, causing large economic losses

(Fukumori et al., 2004).

1.6 PHENOTIPICAL AND GENETICAL VARIABILITY

Botrytis cinerea Pers. has a very high variability and adaptability to a wide range of

environmental conditions. In some studies in Europe it has been demonstrated that isolates of

Botrytis cinerea Pers. from vegetables have a high genetic diversity and a greater gene flow

between populations (Alfonso et al., 2000, Moyano et al., 2003). Elucidating the genetic

structure by measuring genetic diversity within and between populations of the pathogen is very

important because it helps at the management of the disease based on the resistance to

fungicides.

CHAPTER II

GENERAL CONSIDERATIONS REGARDING THE BIOLOGICAL

CONTROL OF GRAY MOULD

2.1 BIOLOGICAL CONTROL METHODS

Current trends to reduce the use of pesticides on crops and on fruits and vegetables, fresh

and stored result in physical and biological assessment methods as safer alternative compared to

synthetic fungicides (Droby et al., 2002).


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Biological approaches that include the use of antagonistic organisms, natural compounds,

cultural practices and biotechnology will be used to develop new methods of disease

management (Wisniewski et al., 2003 cited by Odeh, 2006).

Biological control can be achieved by: hiperparazitism, antagonism of organisms, the

better developing of plants and using insects, use of antibiotics, use of soils suppressive, use of

plant substances.

2.2 THE BIOLOGICAL CONTROL WITH PLANT EXTRACTS

Studies in the literature suggest that phytopreparation are one of the protecting means of

future biological plant protection. The impact of these preparations is sufficiently efficient, the

extraction is not complicated and long, are harmless to the environment and people, quickly

decompose in agrocoenoses (Zarins et al., 2009).

Good experimental results were obtained in tackling some phytopathogens with extracts

of onion, garlic, horseradish, poppy, walnut, pine, but due to the difficulties of extracting are not

introduced into practice (Loredana Beatrice Ash et al., 2006).

CHAPTER III

OBJECTIVES.RESEARCH METHODS AND MATHERIALS

3.1. THE OBJECTIVES OF THE THESIS

To achieve the objectives suggested by the title of the project the study mainly aims the

biology of the specie Botrytis cinerea Pers. and also the control of gray mold by biological

methods – hidroalcoholic extracts; the effect of different plant extracts on the emergence and

development of mycelium.

The objectives proposed for the implementation of the research theme were:

1. The isolation of isolates of the fungus Botrytis cinerea Pers. on the culture medium and the

obtaining of pure strains.

2. The study of the biology of different isolates of Botrytis cinerea Pers. – the characterization of

the isolates from morphological and cultural point of view.

3. The establisment of phenotypic differences between different isolates of Botrytis cinerea Pers.

by macroscopic and microscopic analyzes.

4. The obtaining and testing of tinctures for the control of Botrytis cinerea Pers. fungus

5. Determination of in vitro fungistatic and fungicidal nature of tinctures.

6. Testing the effect of hydroalcoholic extracts on gray mold on stored fruits.


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3.2 MATERIAL AND RESEARCH METHODS USED TO STUDY THE

BIOLOGY OF THE BOTRYTIS CINEREA PERS. FUNGUS

3.2.1 Biological material

The biological material used in the experiments regarding the study of the biology of the

fungus Botrytis cinerea Pers. consisted of several isolates of this specie collected from different

host plants in different locations. In the experiments conducted we isolated the specie Botrytis

cinerea Pers. from 17 host plants.

Biological material consists of 27 isolates taken from the 17 host plants. The isolates

from geraniums were taken from the varieties Camdared, Cambi, Campeye, White Rainbow,

Balcon imperial, Ville de Paris Lila and isolates from grapes were taken from the Chasselas,

Isabela, Sauvignon Blanc, Muscat Ottonel cultivars.

3.2.2 Research methods

The research methods used in the experiments consisted of:

� The isolation of the pathogen from the plant material

� The obtaining of the pure cultures

� The growing of isolates on three culture media

� The observation of the cultural and morphological characteristics of the isolates

taken under study

� Performing the microscopically observations on the shape and size of the conidia

� Observations regarding the the occurrence and distribution of sclerotia

The purpose of studying morphological and cultural characteristics of the species Botrytis

cinerea Pers., was to make observations on some of its features such as:

� fungus colony growth (relative growth rate)

� the shape, size, aspect and color of the colony

� the shape and color of the conidiophores

� the shape, color and size of the conidia

� the appearance, number, aspect and distribution of the sclerotia

Thus, the plant material collected from different locations was washed with distilled

water after which was introduced in humid chamber for 24-48 hours, during which necessary

fungal mycelium and sporulation developed for identifying the pathogen and to isolate it on a

nutrient medium.


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To study the symptoms of disease, for a more detailed examination of morphological

characters of sporulation and for identification of the specie it was used the stereomicroscope

and to observe the conidiophores and the conidia microscopic preparations were made.

The isolation of the fungus.

The isolation and growth of the fungus Botrytis cinerea Pers. was performed in Petri

dishes on nutritive medium, the culture medium used was Potato - Dextrose - Agar (PDA) whose

recipe was taken over Constantinescu’s recipe. (1974).

Petri plate culture method was used for isolation and the sowing method of the fungus

was the inoculation in the center of the plates with a piece of mycelium. Portions of mycelium

were passed through a needle transplanters on the isolation medium PDA, sinking slightly.

After several days the Petri dishes were examined to see if colonies are pure, otherwise

they were purified by passing a small portion of the colony on another Petri dish with culture

medium.

In the experiments were used Petri dishes with a diameter of 85 mm, which, under sterile

conditions was added the culture. To avoid possible contamination with bacteria streptomycin

was added in an amount of 0.05 g / l

The growth of the fungus isolates on the media.

After obtaining the pure cultures the isolates of Botrytis cinerea Pers. were seeded on

culture media: Potato-Dextrose Agar (PDA), Czapek – agar and Malt - Agar (MA).

The examination of the cultural characteristics In the experiments macroscopic examination was performed visually, taking into account

the characteristics of fungal colonies developed on all three culture media.

Cultural characteristics were:

� colony growth of the fungus

� colony form

� colony color

� colony appearance

� occurrence of sporulation

� sclerotia appearance

� number and distribution of sclerotia

Observations on the growth and development of the Botrytis cinerea Pers. colonies on the

three culture media were performed daily by measuring their diameter until they filled the Petri

dishes so being able to determine the growth rate of each isolated colony.

The incubation of the plates was made at 25°C and for a correct interpretation of the

results of experiments was carried out in three repetitions.


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To approximate the percentage of sporulation was developed a system for assessing it, in

which 85 mm diameter petri plates were used and the area of them was calculated (22686.50

mm2). It was divided into 8 equal parts, with an area of 2835.81 mm2 which also were separated

each into 10 equal segments, and the surface of this was calculated as a percentage (1.25% for

each segment).

The examination of the morphological characteristics

• the shape, and color of the mycelium

• the form, color and size of the conidia

The display of the fructifications was performed using optical microscope and after the

microscopic examination it was able to see the shape and color of spores and using Motic Images

program the size of this conidias were determined, by measuring the dimensiones of 100 conidia

of each isolate.

For this data strings of variation were made arranging the data obtained from

measurements according to their frequency in every class, and histograms were performed using

StatSoft Statistics 8 program.

3.3 MATERIAL AND DRESEARCH METHODS IN THE CONTROL OF

BOTRYTIS CINEREA PERS. WITH TINCTURES

3.3.1 Biological material

The biological material used in the control with plant extracts experiments of the fungus

Botrytis cinerea Pers. consisted of 11 isolates of this species collected from peppers, tomatoes,

peppers, lettuce, raspberry, blackberry, strawberry, grape and hydroalcoholic extracts from 9

species of plants (table 3.2) and propolis extract.

To test the effectiveness of plant extracts on fruit stored infected with Botrytis cinerea

Pers species we have used eight extracts and the fruits used were grapes, Cherry tomato and

strawberry fruits.

Table 3.2 The plant extracts used

No Scientific name Family

1. Aloe arborescens Aloaceae 2. Aloe paradisiacum Aloaceae 3. Aloe marlothi Aloaceae 4. Satureja hortensis Lamiaceae 5. Helleborus purpurascens Ranunculaceae 6. Aristolochia clematitis Aristolochiaceae 7. Sambucus nigra Caprifoliaceae 8. Allium sativum Liliaceae 9. Tagetes sp. Asteraceae


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Preparation of extracts was performed with dried or fresh plant material, for the extracts

of Aloe sp. and Tagetes sp. was used fresh herba and Satureja hortensis, Aristolochia clematitis

and Helleborus purpurascens was used dry herba respectively dry rhizomes. Tincture of

Sambucus nigra and Allium sativum were purchased already prepared.

3.3.2. Research methods

The research methodology proposed for achieving objectives includes the following

steps:

� isolation of the fungus

� preparing of the plant extracts

� incorporating extracts in the culture medium

� inoculation of the Botrytis cinerea Pers. isolates on PDA medium

� testing of the tinctures effect on the fungus

The isolation of the fungus.

Isolation and growth of the fungus Botrytis cinerea Pers. was performed in Petri dishes

on Potato - Dextrose - Agar (PDA) and in the experiments we used 80 mm diameter Petri dishes.

The hydroalcoolic extracts preparation.

Hydroalcoholic extracts used in the experiments were conducted according to the

methodology proposed by Elena Iancu and Pârvu (2007).

For the preparation of hydroalcoholic extracts from plants plant material was washed in

water and then was cut into small pieces 1-3 cm long and left to dry at room temperature. After

the plants were dried the finer powder obtained dissolved in 70% ethanol in an 1:5 proportion

(plant: alcohol).

In vitro testing of the extracts effect.

For testing the antifungal activity of plant extracts we used the poison food technique

(Pârvu, 1993). All plant extracts were tested in three concentrations (4%, 6%, 8%). The

hydroalcoholic plant extracts at various concentrations were added to the culture medium PDA

was previously added streptomycin (0.05 g/l) to inhibit bacterial growth.

After the solidification of the culture medium with extract the pathogen inoculation was

achieved by placing portions of mycelium of 1 mm in diameter in the center of Petri plate using

a sterilized needle. The inoculated Petri plates were then incubated at 25 ° C for 9 days. The

control variant was represented by Petri plates without extract. Experiments were performed in

three repetitions.

In order to observe the antifungal effect of the used extracts the colonies developed on

Petri plates were measured at intervals of 1, 3, 6, 9 days by taking four diameters of the colony


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and the colony area was calculated. For accurate calculation of surface colony the diameters

were measured perpendicular, two by two and the average between them was calculated.

The surface of the colonies developed was calculated by the mathematical formula:

A= ππππ ab The inhibition percent of the mycelium development was calculated after the formula:

Procentul de inhibare = (C-T/C) x 100

Were C is the diameter of the control colony and T represents the diameter of the colony

grown on the treated medium Pârvu (1993) with the specification that we used the surface of the

colonies instead of the diameters.

All the data were statistically interpreted with the Polifact programe (the ANOVA test

and the Duncan test).

In vivo testing of the extracts effect.

To test the effectiveness of the tinctures on the appearance and the development of the

grey rot three experiments were established in the laboratory that involved the sterilizing fruits,

the inoculation of the pathogen and the application of the extracts.

The experience made on the grape berries there were used berries of the same size,

healthy with no obvious symptoms of gray mold. The sterilization treatment consisted of soaking

them for 30 seconds in potassium permanganate of 1o/oo concentration and then surface

sterilization with 70% ethanol. The same protocol used for sterilization of tomato and strawberry

fruit. After sterilization the fruits were placed in sterilized plastic containers. Inoculation of the

fungus on the injured fruits was achieved by seeding the fruits with mycelium sections.

The fungistatic effect is the property of certain substances to inhibit the growth of

pathogenic fungi and the fungicide effect is that property of a substance to kill fungal pathogens

(Archbold et al., 1997). The hydroalcoholic extracts (tinctures female) were sprayed in the

containers witch were then sealed to avoid evaporation and the symptoms was performed every

three days for nine days.

CHAPTER IV

EXPERIMENTAL RESULTS REGARDING THE BIOLOGY OF THE

BORYTIS CINEREA PERS. FUNGUS

4.1 THE BIOLOGY OF THE FUNGUS BOTRYTIS CINEREA PERS. ISOLATED

FROM GERANIUM PLANTS

4.1.1 The development of the colonies

To observe the morphological and cultural characters of the fungus Botrytis cinerea

Pers., the plant material used was mycelium from isolates of the fungus from the following


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varieties of geranium plants Cambi, Angeleyes Orange, Camdared, Campeye, White Rainbow,

Balcony Imperial, Ville de Paris Lila.

Table 4.1

The development of the colonies on PDA medium

Day 1 Day 2

Isolate Surface % to

control Difference to

control

Significance of the

difference Surface % to

control Difference to control

Significance of the

difference Media 55.40 100.0 0.00 Mt. 1736.42 100.0 0.00 Mt. Cambi 21.20 38.26 -34.12 - 1671.53 96.26 -64.89 -

Angeleyes Orange 66.46 120.0 11.06 - 778.72 44.8 -957.70 - Camdared 51.81 93.5 -3.59 - 1329.27 76.6 -407.15 - Campeye 38.20 69.0 -17.20 - 785.79 45.3 -950.63 -

Rainbow white 93.68 169.1 38.28 - 2478.51 142.7 742.09 - Balcon Imperial 56.52 102.0 1.12 - 1799.22 103.6 62.80 -

Ville de Paris Lila 59.93 108.2 4.53 - 3311.92 190.7 1575.50 - Day 3 Day 4

Media 12129.67 100.0 0.00 Mt. 17967.94 100.0 0.00 Mt. Cambi 9995.14 82.40 -2134.53 - 18858.06 104.95 890.12 -

Angeleyes Orange 6340.71 52.3 -5788.97 000 12712.03 70.7 -5255.91 000 Camdared 10929.82 90.1 -1199.86 - 17664.86 98.3 -303.08 - Campeye 5192.25 42.8 -6937.42 000 12822.98 71.4 -5144.97 000

Rainbow white 21090.33 173.9 8960.66 *** 22686.50 126.3 4718.56 *** Balcon Imperial 14042.60 115.8 1912.93 - 18344.67 102.1 376.72 -

Ville de Paris Lila 17316.84 142.8 5187.17 *** 22686.50 126.3 4718.56 *** Day 5 Day 6

Media 21029.96 100.0 0.00 Mt. 22686.50 100.0 0.00 Mt. Cambi 22421.96 106.6 1392.00 - 22686.50 100.0 0.00 -

Angeleyes Orange 17515.71 83.3 -3514.26 00 22686.50 100.0 0.00 - Camdared 20834.16 99.1 -195.80 - 22686.50 100.0 0.00 - Campeye 18378.42 87.4 -2651.55 0 22686.50 100.0 0.00 -

Rainbow white 22686.50 107.9 1656.54 - 22686.50 100.0 0.00 - Balcon Imperial 22686.50 107.9 1656.54 - 22686.50 100.0 0.00 -

Ville de Paris Lila 22686.50 107.9 1656.54 - 22686.50 100.0 0.00 - DL (p 5%) 2643.73 DL (p 1%) 3514.18 DL (p 0.1%) 4574.37

In table 4.1 there are presented the surfaces of the fungus colony on PDA at all 7 isolates

taken under study because in the research carried out this culture medium was the most suitable

for the growth and development of these isolates. Analyzing the data from table 4.1 we can

distinguish differences between the isolates surface level in the same day and also differences in

the same isolate in all the experimental period. Thus, regarding the surface of the Botrytis

cinerea Pers. colony on PDA medium we see that its value ranged between 21.20 mm2 (isolate

Cambi) and 93.68 mm2 (Rainbow White isolate) the first day after inoculation, with unsecured

statistical differences from the control.

In the third and fourth day after inoculation the Angeleyes Orange and Campeye isolates

are highlighted, with significant negative differences compared to the control.

A growth of the fungus colony areas are noted in the case of the isolates Rainbow White

and Ville de Paris Lila where differences from control were very significantly positive.


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The surface values recorded on day five, in terms of statistical differences showed

significant negative differences in the case of the Campeye isolate and significantly distinct

differences negative in the case of the Angeleyes Orange.

After analyzing the data in table 4.1 we can say that a general conclusion is that isolates

Campeye and Angeleyes Orange had a slow and consistent growth over the observation period

and the isolate Rainbow White and Ville de Paris Lila had an explosive growth in the third and

on the fourth day, when their surface was 22686.50 mm2.

4.1.2 Conidia

The sporulation percentage of the isolates shows that isolate Rainbow White sporulated

fastest on all culture media, recording a sporulation rate of 7.5%, 10.42% and 10.63% at six days

after inoculation.

On the MA medium, in the sixth day, the isolates Camdared and Campeye also

sporulated, the percentage being 15% respective 24,58 %.

On the twelfth day, all isolates sporulated on all three media, but on Czapek - agar

medium was recorded the highest number of isolates that have reached 100% of sporulation

(izolatele Cambi, Camdared, Rainbow White şi Ville de Paris Lila).

After 30 days the same observations on the cultural characters have been performed to

follow the evolution of the colonies and identify any differences in the development of the

pathogen.

The conidia were solitary, hyaline or light brown witch in mass have gray color,

becoming darker with the age. The shape of the spores observed in the microscopic fields were

ellipsoidal and globally. They were smooth, often with a protuberant hylum and unicellular.

4.1.3 Sclerotia

In the geranium isolates only some of them formed sclerotia. The distribution patterns of

sclerotia were: concentric rings, on the edge of the plates or irregular and they were well fixed on

the surface of the culture medium.

Figure 4.29 Sclerotia formed on PDA medium (A - Rainbow White, B - Balcon imperial, C - Ville de Paris Lila)


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Variations on color, shape and size were observed; the strength bodies formed were

black, brown or white at first becoming black as they mature.

After a 30 days period (after inoculation), the number of isolates that formed sclerotia

was three isolates on PDA medium, four isolates on Czapek agar medium and two isolates on

MA medium and the distribution patterns of sclerotia formed on the plates were: concentric

rings, on the edge of the plates and irregular (Figure 4.29).

4.2 THE BIOLOGY OF THE BOTRYTIS CINEREA PERS. FUNGUS ISOLATED

FROM WINE

4.2.1 The development of the colony

Table 4.14 The surface of the colonies on the culture media

Day 1 Day 2 Isolate

The culture media Surface % to control Difference to

control Significance of the difference Surface % to control Difference to

control Significance of the difference

M1 875.54 100.0 0.00 Mt. 3886.54 100.0 0.00 Mt. M2 261.67 29.9 -613.87 - 1097.43 28.2 -2789.11 - Chasselas M3 738.43 84.3 -137.11 - 3332.06 85.7 -554.47 - M1 342.52 100.0 0.00 Mt. 2378.81 100.0 0.00 Mt. M2 372.61 108.8 30.09 - 1523.95 64.1 -854.87 - Isabela M3 664.11 193.9 321.59 - 3019.63 126.9 640.82 - M1 335.20 100.0 0.00 Mt. 2456.27 100.0 0.00 Mt. M2 408.20 121.8 73.00 - 631.66 25.7 -1824.60 - Sauvignon blanc M3 408.20 121.8 73.00 - 2469.87 100.6 13.61 - M1 616.49 100.0 0.00 Mt. 3053.65 100.0 0.00 Mt. M2 379.68 61.6 -236.81 - 1394.42 45.7 -1659.23 - Muscat ottonel M3 762.76 123.7 146.27 - 3385.45 110.9 331.80 -

Day 3 Day 4 M1 13156.08 100.0 0.00 Mt. 22421.96 100.0 0.00 Mt. M2 7261.77 55.2 -5894.31 00 16808.10 75.0 -5613.86 00 Chasselas M3 11142.55 84.7 -2013.53 - 17751.73 79.2 -4670.22 00 M1 10150.57 100.0 0.00 Mt. 17876.54 100.0 0.00 Mt. M2 5473.02 53.9 -4677.55 00 10073.38 56.3 -7803.16 000 Isabela M3 9350.92 92.1 -799.65 - 15108.37 84.5 -2768.17 - M1 10008.49 100.0 0.00 Mt. 20681.09 100.0 0.00 Mt. M2 5732.33 57.3 -4276.16 00 13061.09 63.2 -7619.99 000 Sauvignon blanc M3 9902.51 98.9 -105.98 - 14809.03 71.6 -5872.06 00 M1 12416.09 100.0 0.00 Mt. 22334.56 100.0 0.00 Mt. M2 6798.62 54.8 -5617.46 00 14479.59 64.8 -7854.98 000 Muscat ottonel M3 10978.49 88.4 -1437.60 - 16357.06 73.2 -5977.50 00

Day 5 Day 6 M1 22686.50 100.0 0.00 Mt. 22686.50 100.0 0.00 Mt. M2 22686.50 100.0 0.00 - 22686.50 100.0 0.00 - Chasselas M3 22686.50 100.0 0.00 - 22686.50 100.0 0.00 - M1 20289.63 100.0 0.00 Mt. 22686.50 100.0 0.00 Mt. M2 17818.45 87.8 -2471.18 - 22686.50 100.0 0.00 - Isabela M3 21991.51 108.4 1701.88 - 22686.50 100.0 0.00 - M1 22686.50 100.0 0.00 Mt. 22686.50 100.0 0.00 Mt. M2 20613.05 90.9 -2073.45 - 22686.50 100.0 0.00 - Sauvignon blanc M3 19060.59 84.0 -3625.91 0 22686.50 100.0 0.00 - M1 22686.50 100.0 0.00 Mt. 22686.50 100.0 0.00 Mt. M2 22686.50 100.0 0.00 - 22686.50 100.0 0.00 - Muscat ottonel M3 22686.50 100.0 0.00 - 22686.50 100.0 0.00 -

DL (p 5%) 2584.01 DL (p 1%) 3451.92

DL (p 0.1%) 4544.16 M1 – PDA, M2 – Czapek – agar, M3 – MA


44

The influence of the culture media on the pathogen development is shown in table 4.14

from witch results that the best medium was PDA followed by MA, on which the isolates

showed no statistically difference compared to the control and the Czapek - agar medium on that

on the third day after the inoculation all the isolates showed differences distinct significantly

negative compared to the control.

4.2.2 Conidia

From table 4.19 it can be seen that the sporulation percentage of the colonies on PDA

medium, at ninth day after inoculation in the case of Sauvignon isolate was 78.33% and in the

12th day in the case of Muscat Ottonel isolate was 100%, while the isolates Chasselas and

Isabela did not form spores on the culture medium.

Table 4.19

The sporulation percent on the culture media The

culture media

Isolate Day 3 Day 6 Day 9 Day 12 Day 30

Chasselas 0.00 0.00 0.00 0.00 0.00 Isabela 0.00 0.00 0.00 0.00 0.00

Sauvignon blanc 0.00 0.00 78.33 91.66 0.00 PDA

Muscat ottonel 0.00 0.00 0.00 100.00 0.00 Chasselas 0.00 0.00 60.83 90.83 3.75 Isabela 0.00 0.00 0.00 0.00 0.00

Sauvignon blanc 0.00 0.00 97.50 100.00 100.00 Czapek -

agar Muscat ottonel 0.00 0.00 8.33 100.00 71.25

Chasselas 0.00 0.00 0.00 0.00 100.00 Isabela 0.00 0.00 0.00 0.00 0.00

Sauvignon blanc 0.00 0.00 70.00 100.00 0.00 MA

Muscat ottonel 0.00 0.00 80.00 80.00 100.00

The highest values of the conidia were recorded on Czapek - agar medium, the higher

dimensiones were observed on isolate Sauvignon blanc (11.26 µm – 8.75 µm) and the lowest

values were 9.79 µm -11.26 µm.

4.2.3 Sclerotia

Regarding the presence, number and distribution of the sclerotia on the Petri dishes with

the three culture media it can be asserted that at a 30 days period after inoculation, except with

the Isabela isolate (all culture media) and Chasselas isolate (on Czapek - agar and MA) all other

isolates formed sclerotia. The distribution patterns were irregular or concentric rings (figure

4.46).


45

Figure 4.46 Sclerotia formed on PDA medium (A - Chasselass, B - Sauvignon blanc, C - Muscat ottonel)

4.3 THE BIOLOGY OF THE BOTRYTIS CINEREA PERS. FUNGUS ISOLATED

FROM OTHER HOST PLANTS

4.3.1 The development of the colony

The influence of the culture medium on the colony surface was determined by analyzing

the measurements of each colony on each culture medium. From the data collected it was

observed that the development of the colonies is best on PDA culture medium which is why in

the study of nutritional substrate influence on the development of this fungus this medium was

considered the control variant. These data support the statements in the literature that the PDA

culture medium is the most appropriate medium for the development and sporulation of the

pathogen Botrytis cinerea Pers.

4.3.2 Conidia

The data regarding the sporulation percent of the isolates on PDA medium, shows that in

the third day one isolate (I11 - petunia) sporulated recording a percent of 28%, while in the sixth

day nine of the isolates formed fructifications, the sporulation percentages ranging from 2.92%

in the case of the eggplant isolate at 60% in the case of the petunia isolate. On the twelfth day 15

of the isolates sporulated on PDA medium, noting that three of them reached 100% (isolates

from lettuce, eggplant and petunia).

Regarding the timing of spore appearance we can say that the petunia isolate sporulated

faster, the isolate sporulatied on PDA and MA media since the third day.

In the case of the Czapek - agar medium, the sporulation occurred only on the sixth day

and only at two of the isolates, the isolate from geranium with a rate of 3.75% and the petunia

isolate where the percentage recorded was 14.58 %. In the 12th day we can say that only two of

the isolates did not sporulated and the number of the isolates with a 100% sporulation percent

were four (lettuce, tomato, pepper, geranium).


46

On MA medium as well as in the case of the PDA medium the isolate from petunia

shows a rapid sporulation, reaching a rate of 27.92% at three days after the inoculation and at a

92.08% in the 12th day. The maximum percentage for this culture medium was achieved by a

single isolate (the fuchsia isolate) while isolates from blackberry and dalia recorded the lowest

rates of 0.67% and 1.25%.

4.3.3 Sclerotia

Following the experiments conducted we can observe that the presence of the resistant

bodies varied both in terms of their number but also in their distribution pattern on the surface of

the colonies formed on the culture media. Also differs the timing of appearance of these sclerotia

on the Petri plates.

Thus on the PDA and MA media the sclerotia were formed from the ninth day and on

Czapek medium - agar only in the twelfth day, when they were observed in four of the 16

isolates: eggplants, dahlias, yew and blackberry. In this culture medium, the yew isolate recorded

the highest average number of sclerotia, which was 102.5.

Analyzing the sclerotia appearance of each isolate, we found that only two isolates, the

one from dahlia and yew formed the resistant bodies in all three culture media used. On PDA

medium on day 12 the number of sclerotia ranged from the value of 6.5 in the impatiens isolate’s

case at 54 at japanese rose isolate.

CHAPTER V

EXPERIMENTAL RESULTS REGARDING THE EFFICIENCY OF SO ME

TINCTURES IN THE BOTRYTIS CINEREA PERS. CONTROL

The experiences on the efficacy of hydroalcoholic extracts used in vitro to control the

pathogen Botrytis cinerea Pers. were performed on the 11 isolates of the fungus isolated from the

following host plants: peppers, tomatoes, lettuce, pepper, raspberry, blackberry, strawberry,

grape (four varieties).

The experimental variants were the tinctures of Aloe paradisiacum, Aloe marlothi, Aloe

arborescens, Satureja hortensis, Helleborus purpurascens, Aristolochia clematitis, Sambucus

nigra, Allium sativum, Tagetes sp., propolis applied in three concentrations (4%, 6%, 8 %) and

the control variant was consisted the culture medium without extract. Thus, to control gray mold

in vitro using hydroalcoholic extracts an experiment with 10 treatement variants was conducted.

Further will be presented the results recorded and statistically analyzed with reference to

the effect of extracts on the growth of fungus colonies, the percentage of inhibition of colony

development on the medium with extract and the influence of the products applied

concentrations.


47

For a better interpretation of the results, in addition to the testing of the significance of

differences by differences limit (DL) the Duncan test also was used.

5.1 THE IN VITRO EFFICIENCY OF THE EXTRACTS

5.1.1 Izolate Solanaceae

For example we show the effect of plant extracts on one of the isolates tested, the pepper

isolate and the efficiency of all tested extracts against all the isolates can be highlighted in the

Conclusions and recommendations chapter.

The inhibitory effect of the tinctures applied to the pepper isolate it can be seen from the

first days of treatment, all the extracts, in all three concentrations showed inhibitory effect on

colony development of Botrytis cinerea Pers.

Table 5.2

The effect of the tinctures on the colony develoment - 9th day

Variant Concentration Surface (mm2)

% to control

Difference to control

Significance of the difference

The Duncan test

Untreated - 20096.00 100.0 0.00 Mt. E 4 1036.46 5.2 -19059.54 000 AB 6 103.62 0.5 -19992.38 000 A Aloe arborescens 8 0.01 0.0 -20095.99 000 A 4 15584.67 77.6 -4511.33 00 D 6 153.86 0.8 -19942.14 000 A Aloe marlothi 8 0.01 0.0 -20095.99 000 A 4 2764.51 13.8 -17331.49 000 AB 6 0.01 0.0 -20095.99 000 A Aloe paradisiacum 8 0.01 0.0 -20095.99 000 A 4 2828.10 14.1 -17267.90 000 AB 6 0.01 0.0 -20095.99 000 A Satureja hortensis 8 0.01 0.0 -20095.99 000 A 4 425.21 2.1 -19670.79 000 A 6 20.15 0.1 -20075.85 000 A

Helleborus purpurascens

8 0.01 0.0 -20095.99 000 A 4 2882.52 14.3 -17213.48 000 AB 6 0.01 0.0 -20095.99 000 A

Aristolochia clematitis

8 0.01 0.0 -20095.99 000 A 4 20096.00 100.0 0.00 - A 6 9897.02 49.2 -10198.98 000 C Sambucus nigra 8 1651.12 8.2 -18444.88 000 AB 4 20096.00 100.0 0.00 - A 6 13635.97 67.9 -6460.03 000 D Allium sativum 8 4013.97 20.0 -16082.03 000 B 4 181.60 0.9 -19914.40 000 A 6 0.01 0.0 -20095.99 000 A Tagetes sp. 8 0.01 0.0 -20095.99 000 A 4 0.01 0.0 -20095.99 000 A 6 0.01 0.0 -20095.99 000 A Propolis 8 0.01 0.0 -20095.99 000 A

DL (p 5%) 2822.57 DS 2828.13 DL (p 1%) 3756.82 DL (p 0.1%) 4872.25

At the end of the experimental period (9 days after application of the extracts) the

effectiveness of treatments applied to control the fungus Botrytis cinerea Pers. is shown in table

5.2, where we observe that the inhibitory effect is maintained for most tinctures except


48

Sambucus nigra and Allium sativum, which, at the lowest concentration - 4% - had developed

colonies close to those of untreated variant (differences are insignificant).

The figures 5.1-5.3 presents the evolution of the inhibition degree of the fungus at the

three concentrations of the tinctures applied as a percentage inhibition calculated from the first

day to the ninth day.

As shown in figure 5.1, at a 4% concentration the inhibition percentage of 100% is

showed in the case of the propolis tincture and the lowest degree of inhibition was obtained

when extracts of Sambucus nigra, Allium sativum, Aloe marlothi were applied.

This difference between the tinctures is retained during the nine days period, finally

validating the propolis tincture with 100% degree of inhibition and the Tagetes sp. with a

percentage of inhibition of over 99%.

Figure 5.1 The inhibition degree oh the colony development - 4% concentration

At a 6% concentration - figure 5.2 - Aloe paradisiacum, Satureja hortensis, Aristolochia

clematitis, Tagetes sp. and propolis (figure 5.4) had a maximum inhibition of fungus

development while the extracts of Sambucus nigra and Allium sativum showed inhibition percent

of 50.75% and 32.15% at the end of the experimental period.


49



At a 8% concentration (figure 5.3) most extracts had 100% inhibition of the mycelium

development except with the exception of the Sambucus nigra and Allium sativum extracts

(figure 5.4) that had the lowest fungistatic effect at this concentration.


50

Figure 5.4 The efficiency of the treatements with tinctures on the pepper isolate (A – control, B - propolis, C – Tagetes sp., D - Allium sativum)

5.2.1 The testing of the antifungal effect on grape berries

The inhibitory effect of the tinctures applied on the grape berries artificially infected with

the isolate taken from Muscat ottonel it can be seen by analyzing the data in table 5.55.

At the end of the experiment (9 days after the extracts application) the effectiveness of

treatments applied to control the fungus Botrytis cinerea Pers. is shown in table 5.55, where we

observe that the generally inhibitory effect is maintained at all tinctures (very significant

negative differences).

The hydroalcoholic extracts that showed the strongest inhibitory effect are shown in table

5.55, where it can be seen that extracts of Aristolochia clematitis and Allium sativum were most

effective, the affected areas had dimensions of 77.45 mm2 45.53 mm2 respectively. Tagetes and

propolis extracts also had a high degree of inhibition; the area affected was approximately 90

mm2. These results are supported by the images in figure 5.46.

Table 5.55 The effect of the tinctures - 9th day

The variant The surface (mm2)

% to control

The difference to control

The significance of the difference

The Duncan test

Untreated 3219.81 100.0 0.00 Mt. C Aloe arborescens 137.90 4.3 -3081.91 000 A Satureja hortensis 211.43 6.6 -3008.38 000 A

Helleborus purpurascens 884.96 27.5 -2334.85 000 B Aristolochia clematitis 77.45 2.4 -3142.36 000 A

Sambucus nigra 209.33 6.5 -3010.48 000 A Allium sativum 45.53 1.4 -3174.28 000 A

Tagetes sp. 97.34 3.0 -3122.47 000 A Propolis 93.68 2.9 -3126.13 000 A DL (p 5%) 457.97 DS 336.08 DL (p 1%) 624.36 DL (p 0.1%) 846.50


51

The Duncan test analysis notes that the tinctures of Helleborus purpurascens showed the

lowest effect - the affected area (884.96 mm2), followed by the Sambucus nigra, Satureja

hortensis and Aloe arborescens extracts.

Figure 5. 46 The testing of the tinctures against the Muscat ottonel isolate

The treatments applied to the inoculated berries with mycelium of Botrytis cinerea Pers,

isolated from Sauvignon blanc have reached significant differences compared with untreated

control from the earliest days of application.

At the end of the observation period the effectiveness of the treatments applied to control

the fungus Botrytis cinerea Pers. (table 5.58) is very high for all tested tintures, negative

significant differenceswere recorded, but the Duncan test shows that the highest antifungal effect

had the tinctures Aloe arborescens, Satureja hortensis, Allium sativum, Tagetes sp. These data

are supported by the images in figure 5.49.


52

Figure 5.49 The testing of the tinctures against the Sauvignon blanc isolate

The best inhibitory effect on the colony development – at nine days (tabelul 5.58) had the

extracts of Allium sativum and Tagetes sp. – the dimensions of the affected areas were of 98,39

mm2 and 136,07 mm2 respective. At the opposite side the best antifungal effect was recorded by

the Aristolochia clematitis and Helleborus purpurascens extracts - 2114,79 mm2 and 796,36

mm2 respective.

Table 5.58

The effect of the extracts on the colony development – 9th day


% to control



The Duncan test

Untreated 4193.21 100.0 0.00 Mt. E Aloe arborescens 226.60 5.4 -3966.61 000 A Satureja hortensis 295.42 7.0 -3897.79 000 A

Helleborus purpurascens 796.36 19.0 -3396.85 000 BC Aristolochia clematitis 2114.79 50.4 -2078.42 000 D

Sambucus nigra 472.05 11.3 -3721.16 000 AB Allium sativum 98.39 2.3 -4094.82 000 A

Tagetes sp. 136.07 3.2 -4057.14 000 A Propolis 259.05 6.2 -3934.16 000 A DL (p 5%) 476.85 DS 416.74 DL (p 1%) 647.38 DL (p 0.1%) 872.09


53

5.2.2 The testing of the antifungal effect on tomato

The effectiveness of treatments applied to tomato fruit is presented in table 5.61 witch

shows that at nine days after the treatments do not exist differences statistically assured to the

untreated control. Analyzing the data from table 5.61 it can be seen that treatment options can

not be differentiated but noting the areas affected by grey mold we can say that the tincture of

Tagetes sp. is the only tincture which has not allowed the fungus to develop.

Table 5.61 Efectul tincturilor asupra dezvoltării coloniei - ziua 9


% to control



The Duncan test

Untreated 4791.64 100.0 0.00 Mt. B Aloe arborescens 39.77 0.8 -4751.87 000 A Satureja hortensis 11.51 0.2 -4780.13 000 A

Helleborus purpurascens 14.92 0.3 -4776.72 000 A Aristolochia clematitis 182.12 3.8 -4609.52 000 A

Sambucus nigra 90.01 1.9 -4701.63 000 A Allium sativum 12.56 0.3 -4779.08 000 A

Tagetes sp. 3.14 0.1 -4788.50 000 A Propolis 78.50 1.6 -4713.14 000 A DL (p 5%) 1241.92 DS 1316.57 DL (p 1%) 1674.83 DL (p 0,1%) 2233.09

At nine days of treatment (table 5.61) the total inhibitory effect of Tagetes sp. extract

mentains and quite small areas can be observed when treated with the tinctures Satureja

hortensis and Allium sativum.

Figure 5.50 The testing of the tinctures against the tomato isolate


54

Analyzing the areas affected by the fungus Botrytis cinerea Pers. on the tomato fruits,

following the application of the extracts (figure 5.50) we can say that the ten extracts tested were

more efficient in the control of the isolate from tomato than the isolates from grapes.

CONCLUSIONS AND RECOMANDATIONS

Based on the research it can be stated the following conclusions and recommendations:

1. The fungus Botrytis cinerea Pers. shows a high phenotypic variability witch is influenced

by the host plant from witch was isolated and by the culture medium were was grown.

2. From the biology study of the fungus it is shown that the most effective culture medium

for the growth and development of the fungus Botrytis cinerea Pers. isolates was the PDA

medium.

3. The best culture medium for the sporulation of the Botrytis cinerea Pers. fungus was

Czapek – agar medium.

4. The size of the conidia varied depending on the culture media and isolate, the conidia

from the cuture media had higher values compared with the spores taken directly from the

infected plant material.

5. The conidia observed under the microscope had ovate, ellipsoidal, almost ellipsoidal,

globally shapes.

6. The sclerotia were variables in size, shape, number and distribution, they were absents,

rare or abundant, with different distribution patterns: irregular, in concentric rings and on the

edge of the plates.

7. The cultural characters on the colonies of the Botrytis cinerea Pers. isolates also varied

depending on culture medium, colonies had downy or velvety appearance with a spectrum of

colors from white, off-white, cream white, light gray to dark gray, brown.

8. For the growth and development of the fungus Botrytis cinerea Pers. We recommend the

use of PDA culture medium which ensures a faster development of the colony.

9. For obtaining the spores of this specie we recommend the Czapek - agar culture medium

on witch although the colonies had a slower adevelopment the sporulation percentages were

higher.

10. In general, most extracts had a high efficiency on the development of the isolates tested

in vitro, the degree of inhibition being different depending on the product concentration and also

on the isolate on which they were applied.

11. The propolis tincture showed a total inhibitory effect making this tincture as the only one

that was validated as effective at all concentrations applied against all isolates.


55

12. The most effective extracts that had total fungistatic effect even at the lowest

concentration, on the majority of isolates were those of Satureja hortensis, Tagetes sp. and

propolis.

13. The in vitro efficiency of the extracts can be classified as follows:

• 4% - propolis tincture.

• 6% - A. paradisiacum, Satureja hortensis, Aristolochia clematitis, Tagetes sp., propolis

tinctures.

• 8% - A. arborescens, A. marlothi, A. paradisiacum, Satureja hortensis, Helleborus

purpurascens, Aristolochia clematitis, Tagetes sp., propolis tinctures.

Tomato isolate:

• 4% - Aristolochia clematitis, Allium sativum, Tagetes sp., propolis tinctures.

• 6% - A. arborescens, A. paradisiacum, Satureja hortensis, Helleborus purpurascens,

Aristolochia clematitis, Allium sativum, Tagetes sp., propolis tinctures.


purpurascens, Aristolochia clematitis, Sambucus nigra, Allium sativum, Tagetes sp.,

propolis.

Hot pepper isolate:

• 4% - Tagetes sp. şi propolis tinctures.

• 6% - A. arborescens, A. marlothi, Satureja hortensis, Helleborus purpurascens,

Aristolochia clematitis, Tagetes sp., propolis tinctures.



Lettuce isolate:

• 4% - Satureja hortensis, Allium sativum, Tagetes sp., propolis tinctures.

• 6% - A. arborescens, Satureja hortensis, Helleborus purpurascens, Aristolochia

clematitis, Sambucus nigra, Allium sativum, Tagetes sp., propolis tinctures.


purpurascens, Aristolochia clematitis, Sambucus nigra, Allium sativum, Tagetes sp.,

propolis tinctures.

Raspberry isolate:

• 4% - Satureja hortensis şi propolis tinctures.

• 6% - A. arborescens, A. marlothi, Satureja hortensis, Helleborus purpurascens,


Pepper isolate:


56


purpurascens, Aristolochia clematitis, Sambucus nigra, Tagetes sp., propolis tinctures.

Blackberry isolate:

• 4% - A. arborescens, Satureja hortensis, Helleborus purpurascens, Aristolochia

clematitis, propolis tinctures.





Strawberry isolate:


• 6% - A. arborescens, A. paradisiacum, Satureja hortensis, Aristolochia clematitis,

Tagetes sp., propolis tinctures.



Chasselas isolate:

• 4% - propolis tincture.


Aristolochia clematitis, propolis tinctures.



Isabela isolate:






Sauvignon blanc isolate:






Muscat ottonel isolate:


57






14. The Satureja hortensis extract at a 4% concentration had a high degree of inhibition on

the development of fungus, with the exception of the isolates from solanaceous plants, which

leads us to recommend it for controlling pathogens in vitro.

15. To control the fungus Botrytis cinerea Pers. in vivo we recommend for use the Allium

sativum, Satureja hortensis, Tagetes sp. and propolis extracts.

16. The testing of the fungicidal effect of the extracts against the isolate from blackberry

showed that Aloe arborescens, Satureja hortensis, Helleborus purpurascens, Aristolochia

clematitis and propolis had an fungicidal effect against this isolate because did not allowed the

development of mycelium when it was inoculated on the untreated fresh medium.

17. The hydroalcoholic extracts tested in vivo against the Muscat Ottonel isolate, which

showed the strongest inhibitory effect were the tincture of Aristolochia clematitis and Allium

sativum followed by Tagetes and propolis.

18. The effectiveness of the treatments applied to control the fungus Botrytis cinerea Pers. -

isolate Sauvignon blanc was highest for the tinctures of Allium sativum, Tagetes sp., Aloe

arborescens.

19. The efficiency of the hydroalcoholic extracts was higher in the control of the tomato

isolate than on the two isolates from on vine.

20. The tinctures which showed the strongest inhibitory effect on the tomato isolates tested

in vivo were those of Tagetes sp., Satureja hortensis and Allium sativum and for that reason we

recommend this tinctures for the control during storage.

21. In the case of the treatments applied on strawberry fruit artificially infected with

mycelium of the Botrytis cinerea Pers. fungus all extracts showed a inhibitory effect that did not

alowed the fungus to develope but because of the alcohol contained in the tinctures the fruit

appearance was depreciated and did not allow measurements. In the case of this very sensitive

fruits we recommend the use of lower alcohol concentration or the use of hidro-glyceric extracts.

22. In case of any outbreaks of infection with Botrytis cinerea Pers. in stored grapes we

recommend treatment with extracts of Allium sativum and Tagetes sp.

Documents

rezumat Raluca - Vasilica MICLEA · Biologist Raluca Vasilica Miclea -Summary of the PhD thesis- Researches regarding the biology and the control with plant extracts of the grey mould