Induction Of Resistance And Biocontrol Of Rhizoctonia In Cotton Against Damping-off Disease By Rhizosphere Yeasts And Fungi
A El-Mehalawy, S Hassanin, N Hassanin, S Zaki
biological control, fungi, yeast
A El-Mehalawy, S Hassanin, N Hassanin, S Zaki. Induction Of Resistance And Biocontrol Of Rhizoctonia In Cotton Against Damping-off Disease By Rhizosphere Yeasts And Fungi. The Internet Journal of Microbiology. 2006 Volume 3 Number 2.
Addition of fishmeal to the soil infested with the pathogen led to a remarkable reduction in the percentage of disease compared to the soil non-amended with fishmeal. 28 fungal isolates and 22 yeast isolates were isolated from the rhizosphere associated soil of a cotton plant. 3 fungal isolates and 3 yeast isolates were characterized by their potent and remarkable antagonistic activities and were identified as:
Review of Literature
Interactions between biocontrol mold, yeast fungi, and fungal plant pathogens continue to be the focus of a large number of studies.
The fungal biocontrol agents that have been evaluated for their ability to reduce vegetable crops are
Many types of organic matter have been used for soil amendments. These include seaweed extracts, fishmeal, chitinous materials as soil cakes .
Induced resistance defined as the process of active resistance dependent on the host plant's physical or chemical barriers activated by biotic or abiotic agents .
The mechanisms by which yeast fungi are reported to have reduced mildew development included mycoparasitism of pathogen structures by
All the three fungal antagonists,
El-Mehalawy  found that the two species of rhizosphere yeast fungi:
Materials & Methods
Clay soil was collected from a field cultivated with a cotton plant from Shebeen el-Kanater, Kalubbia governorate, Egypt.
The clay soil was used for cultivation of the cotton plant in the greenhouse and was divided into two categories; the first one was amended with fishmeal and the second one was left non-amended. The rhizosphere soil samples were collected from both amended and non-amended soils.
Isolation of the pathogen ()
Cotton seedlings one month-old exhibiting damping-off disease symptoms were collected from soil infested with
Identification of the pathogen ()
Colony morphology of
Inoculum production of the pathogen
Sterilized Millet (
Soil infestation and preparation for the greenhouse test
Four sets of pots were used. The first set contains the pathogen and soil amended with fishmeal, the second set contains the pathogen and non-amended soil, the third set contains non-amended soil without the pathogen (control), while the last set contains soil amended with fishmeal without the pathogen (control). 10 seeds were sown in each pot at 3 cm depth, reduced to 5 seedlings per pot after complete emergence. Each treatment was replicated 6 times with 5 plants/ replicate. The pots were watered every other day .
Isolation of rhizosphere microorganisms
Two sets of pots were prepared, each of 10 pots. The pots of the first set contain amended soil, the pots of the second set contain non-amended soil. 10 seeds of cotton were sown at 3 cm depth in each pot. The pots were watered every other day and after 3-4 weeks, the root systems were cut and washed .
Estimation of total microbial activity
The microbial activity of the freshly sampled rhizosphere (amended and non-amended) soils was measured by fluorescein diacetate hydrolysis by the method of Schnürer and Rosswell, .
Isolation of rhizosphere fungi and yeasts
Fungi of cotton rhizosphere soils (amended and non-amended) were isolated using the soil dilution plate method of Johnson and Curl  on (i) (PDA) and (ii) (SDA).
Yeasts of cotton rhizosphere soils (amended and non-amended) were isolated using the soil dilution plate method of Johnson and Curl  on (I) Peptone-Yeast Malt Agar (PYM) and (ii) Nutrient Agar (NA).
screening of the antifungal activity of fungal isolates
Mold fungi isolates were examined for their ability to produce inhibitory compounds active against
screening of the antifungal activity of yeast isolates
Yeast fungi isolates were examined for their ability to produce inhibitory compounds active against
Identification of rhizosphere fungal and yeast isolates
fungal isolates were identified to the genus and species levels using the following references [41,43].
Yeast isolates were identified to the genus and species levels using the following references [5,6,7,8,9,10,34,38,48,51].
Inoculum production of the selected fungal and yeast species
The inoculum of each mold and yeast fungi was prepared by placing 50 g moist wheat bran into 500 ml conical flasks, autoclaved at 121 °C for 30 min. on three successive days as described by Roiger and Jeffers . The mixture was inoculated aseptically with a suspension (25 ml) of spores and cell, in 10% tween 80 and incubated at 28 ± 2 °C in the dark for two weeks. The flasks were shaken to ensure uniformity of each microorganism growth.
Wheat bran colonized with each mold or yeast fungus (5% weight of colonized wheat bran based inoculum/ weight of air dry steam-pasteurized soil) was thoroughly dispersed through the steamed soil contained in two sets of pots in which each pot was filled with 3 kg clay soil. One set of pots contained the antagonist inoculum and soil amended with fishmeal and the other contained the antagonist inoculum and amended soil. In total, there are 9 pathogen-mold fungus and pathogen-yeast fungus, these were as follows:
Cotton growth measurements
Cotton seedlings were harvested 4 weeks after seedling emergence. The whole plants were carefully removed and washed to remove any soil particles from the shoot and root systems. Growth criteria measures were: length of root, height of shoot, fresh weight of root, fresh weight of shoot, dry weight of root and dry weight of shoot.
A random complete block design was used and analysis of varience was carried out using Superanova (Abacus Concepts, Inc., Berkeley, CA, USA) to evaluate the effect of the antagonists on the development and growth of cotton plant in the greenhouse trials.
Effect of rhizosphere fungal and yeast species on
1- Phosphate solubilization
The medium used to screen phosphate solubilization consists of soil extract 75 ml with 1% glucose and 2% agar, dispensed in 300 ml amounts in 500 ml Erlenmeyer flasks. After sterilization, the medium was cooled to 45°C and 15 ml of sterile 10% K2HPO4 and 30 ml sterile 10% CaCl2 were added. Two ml of sterile actidione solution (40 µg/ml) were also added to each flask and the reaction adjusted aseptically with sterile N/L NaOH at a pH 7.0 The plates were inoculated with the rhizosphere mold and yeast fungi and incubated at 28 °C for 5 days. The organisms forming clarification halos were considered phosphate solubilizers .
2- Production of siderphosphores
The medium used to test for production of siderophores consisted of 8-hydroxyquinoline (50 mg/L) was added to tryptic Soy Agar (TSA) (10%). Organisms growing on this medium were considered positive for siderophores production .
Production of IAA
A modified method (Bric
Extraction of the antifungal substances
The selected mold and yeast fungi were cultivated on Hussein's fishmeal extract agar (HFME) broth , culture broth was then filtered and extracted with a mixture of chloroform- ethylacetate (1:1 v/v). Complete extraction was achieved when the metabolism solution was extracted with 30% of its own volume of chloroform-ethylacetate mixture. The organic layer containing the antifungal compound was filtered and evaporated at 34 °C under vacuum till dryness. The residue was dissolved in the least amount of chloroform (3 ml) .
Detection of the antifungal substances
The active components in the culture filtrates of the selected mold and yeast fungi were studied by means of descending paper chromatography; the solvent system was a mixture of 1-butanol, pyridine and water( 6:4:3 v/v).
After development, the paper stripes (Whatman No. 1) were air dried and placed in front of a stripe of
Chemical structure of the purified active components of the selected fungal and yeast species
This experiment was carried out according to El-Mehalawy , using mass spectroscopy. Varian gas chromatography coupled with a mass selective detector. Finningan mat SSQ 700 and equipped with Chem-Station soft ware and NIST spectral data was used with DB-5 fused silica capillary column (30 x 0.25 um i.d., 0.25 um film thickness). The chromatographic conditions were as follows: column temperature 60 °C (30 min.), raised from 60 to 260 °C (5 °C/min.) and maintained at 260 for 10 min., interface, 260 °C; injector temperature 250 °C, ionization energy 70 ev; mass range 50- 750; volume injected 1 UL. This experiment was performed at the Mass Spectroscopy Unit, Central Scientific Services Laboratory at the National Research Center, Cairo.
Measurement of the microbial activity in amended and non-amended soil
It is shown from Table (1) that the microbial activity of soil amended with fishmeal is higher than that of non-amended soil.
Pathogenicity test with
Table (2) illustrate that the percentage of disease in infested non-amended soil is higher than that in non-infested soil either amended or non-amended. The addition of fishmeal led to a remarkable reduction in the percentage of disease.
Screening the fungal and yeast isolates for antagonistic activities against
Fungal isolates No. 5, 18 and 19 were found to have the remarkable antagonistic activities against
Identification of fungal and yeast isolates
The three strongest antagonistic rhizosphere fungi were identified to the genus and species levels as (5)
Identification of yeast isolates
The selected three yeast isolates showing the greater inhibitory and antagonistic activity were identified to the genus and species levels .as (9)
Effect of fungal and yeast species on cotton seeds germination using amended and non-amended soil
Data in Table (5) show that the presence of the pathogen led a reduction in the percentage of seed germination compared to the untreated soil (control), which accordingly led to an increase in the percentage of the diseased plants. On the other hand, the addition of fishmeal to the soil induced the resistance of cotton to
Effect of rhizosphere fungal and yeast specieson cotton plant growth
Tables (6 & 7) show that fungi and yeasts increased the growth measurements compared to the control and each of their mixtures significantly increased all cotton growth measurements length of root, height of shoot, fry weight of root, dry weight of shoot, fresh weight of root and fresh weight of shoot) compared with each of them alone.
Production of IAA and siderophores and phosphate solubilization By mold and yeast fungi species
Data in Table (8) show that
R values of the antifungal components produced by the selected fungal species
The active components were detected, using paper chromatography, by the inhibition zones around their spots and were determined by their Rf values as shown in Table (9).
From the table it is shown that four active components were detected in the culture filtrate of
Chemical structures, chemical formulae and molecular weights of purified active components of and
Data in Table (10) show the chemical name, chemical formula and molecular weights of the active components detected in the culture filtrates of both
Fishmeal used as a soil amendment increases the microbial activity, while its absence led to a reduction in the microbial activity. This microbial activity causes a depletion in essential nutrients for the survival and multiplication of the pathogen, thus preventing infecting of the host. This finding is in accordance with that reported by Chen
The presence of the pathogen (
In a screening program aimed at the discovery of antifungal compounds produced by the selected fungal and yeast species, these species were found to produce antifungal compounds with high inhibitory effect against
The reduction in the percentage of seed germination in the presence of
The use of rhizosphere fungal and yeast species, each group in combination, increased the percentage of inhibition of growth of
The reduced incidence of damping-off disease caused by the addition of rhizosphere fungal and yeast species either separately or in combination may be due to the incubation period in the soil which may aid in the establishment of the introduced microorganisms and enables them to multiply in the soil or to activate the mechanism(s) of antagonism. This explanation agrees with that reported by Kleifield & Chet .
From the possible mechanisms for increasing plant growth, the production of secondary metabolites such as antibiotics, cyanide and hormone-like substances, the production of siderophores, plant growth regulators and phosphate solubilzation . Cotton rhizosphere harbour a variety of microorganisms, the interaction of them with each other and with the plant influence plant growth in ways that may be beneficial, neutral or detrimental .
Three fungal and three yeast species were grown under the optimum cultural conditions on broth media for the production of antifungal compounds. It was found that one fungal species produce four active components, the second produce five active components, while the third produce six active components. Two yeast species produce five active components, while one produce four active components. This may be due to that cell metabolism of each species under conditions of nutritional excess is directed towards the generation of cell mass rather than the production of secondary metabolites and when depletion of key nutrients occurs, it shifts the cell cycle to the stationary phase and signals the transition from primary to secondary metabolism in which the active components are produced. This finding agrees with that reported by Abbaral
Where γo: fungal growth radius of a control culture (cm) γ : distance of fungal growth in direction of fungal growth in direction of fungal colony (cm) Δγ = γo – γ % inh. : Percentage of inhibition ( Δγ/ γo)
Where γo : Fungal growth radius of a control culture (cm) γ : distance of fungal growth in direction of yeast fungi colony (cm) Δγ = γo – γ % inh. : Percentage of inhibition ( Δγ/γo)
A: amended soil. N: non-amended soil. C: Untreated (control).
The values with the same letter within a column are not significantly (P>0.05) different according to Fisher's protected LSD test. Results are means of 10 replicates for each treatment. The values in parentheses are the standard error of the mean.
A: amended soil. N: nom-amended soil. C: Untreated (control).
Values with the same letter within a column are not significantly (p>0.05) different according to Fisher's protected LSD test. Results are means of 10 replicates for each treatment. The values in parentheses are the standard error of the mean.
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