Induction Of Resistance And Biocontrol Of Rhizoctonia In Cotton Against Damping-off Disease By Rhizosphere Yeasts And Fungi

Adel A. El-Mehalawy Microbiology Department, Faculty of Science, Ain Shams University Cairo Egypt

Saadia M. Hassanin Microbiology Department, Faculty of Science, Ain Shams University Cairo Egypt

Nazeha M. Hassanin Microbiology Department, Faculty of Science, Ain Shams University Cairo Egypt

Samah A. Zaki Microbiology Department, Faculty of Science, Ain Shams University Cairo Egypt

Citation: A.A. El-Mehalawy, S.M. Hassanin, N.M. Hassanin, S.A. Zaki: Induction Of Resistance And Biocontrol Of Rhizoctonia In Cotton Against Damping-off Disease By Rhizosphere Yeasts And Fungi. The Internet Journal of Microbiology. 2007 Volume 3 Number 2

Keywords: Yeast, Fungi, Biological control

Abstract

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: Eupenicillium senticosum, Penicillium herquer, Trichoderma viride, Hansenula arabitolgenes, Candida incommunis and Candida steatolytica, respectively. The use of a mixture of fungal and yeast species led to an increase in inhibition of the pathogen, an increase in induction of resistance of the cotton plant and an increase in growth measurements of the cotton plant than each of them alone. Candida incommunis has the ability of solubilizing phosphate and has the ability for production of indolacetic acid (IAA) and siderophores. The three fungal species produce 4, 6 and 5 antifungal compounds ,respectively. On the other hand, the first two yeast fungi species produce 5 antifungal compounds, while the third one produces only four.

Introduction

R. solani, a soil-borne plant pathogen infecst cotton plants and causes a loss in the yield. The aim of the present investigation is to study the ability of some fungal and yeast species for induction of resistance in a cotton plant and studying the role of some soil amendments (e.g. fishmeal), in addition to the antagonists, in reducing both disease incidence and severity and their effect on plant growth.

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 Trichoderma spp. and Gliocladium virens [36,40], in addition to several other organisms, including Chaetomium globosum Kunze: Fr. [18] Coniothyrium minitans [2] Sporidesmium sclerotivorum [3]. These agents have been evaluated primarily against soil-borne disease and were reported to reduce the development of diseases caused by pathogens such as Pythium spp. [39], Rhizoctonia solani [36].

Many types of organic matter have been used for soil amendments. These include seaweed extracts, fishmeal, chitinous materials as soil cakes [16].

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 [33].

The mechanisms by which yeast fungi are reported to have reduced mildew development included mycoparasitism of pathogen structures by Amplelomyces quisqualis [49], secretion of antibiotics, such as heptadecanoic, and methylheptadecanoic aids by Sporothrix [12] and the production of lytic enzymes such as β-1,3-glucanases by Tilletiopsis sp. [50]. In addition, competition for nutrients was postulated to have reduced infection by foliar pathogens such as Botrytis cinerea [20]. Ghisalberti and Sivasithamparam [23] studied the mechanisms by which Trichoderma isolates control pathogenic population in the rhizosphere, and they indicated that the antagonistic process relies on the production of antibiotics and/ or hydrolytic enzymes [26] associated with possible competition for nutrients in the rhizosphere [47].

All the three fungal antagonists, Trichoderma viride, T. harzianum and Gliocladium virens significantly inhibited the mycelial growth and sclerotial production of Thanatephorus cucumeris (Rhizocontia solani). Among the three, G. virens was more effective. Complete mycelial inhibition and 92% inhibition of sclerotial formation were recorded and 4:1 ratio of G. virens and T. cucumeris [19]. Hanson [25] pointed out that cotton plants grown from seed treated with two different strains of T. virens had significantly less severe symptoms after stem inoculation with Verticillium dahliae than did controls not treated with T. virens. The reduction in symptoms indicates that T. virens induces resistance in the host at a distance from where the fungus is established ( the germinating seed ) and that seed treatment with T. virens can provide some long-term protection of cotton plants from Verticillium wilt.

El-Mehalawy [22] found that the two species of rhizosphere yeast fungi: Saccharomyces unispora and Candida steatolytica have antagonistic and inhibitory effects on the growth of the fungal pathogen Fusarium oxysporum. Addition of these two species to the soil seeded with kidney bean and infested with the pathogen increased the percentage of control plants. This was carried out through the process of antibiosis (secretion of antifungal compounds).

Materials & Methods

Soil Samples

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 (R. solani)

Cotton seedlings one month-old exhibiting damping-off disease symptoms were collected from soil infested with Rhizoctonia solani. Isolation was made according to Carling et al., [17].

Identification of the pathogen (R. solani)

Colony morphology of R. solani was assessed on potato dextrose agar (PDA), and Sabourod dextrose agar (SDA) at 28 ± 2°C in the dark for 2 days. Identification of the pathogen isolate was based on morphological characteristics of the cultures growing on the two media [41,43].

Inoculum production of the pathogen

Sterilized Millet (Panicum miliceum L.) was inoculated with agar plugs from the actively growing margin of Rhizoctonia colony and incubated at 28± 2°C in the dark for 6 days. Sterilized uninoculated millet seeds were used as control [52].

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 [35].

Isolation of rhizosphere microorganisms

Soil preparation

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 [30].

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, [45].

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 [30] 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 [30] on (I) Peptone-Yeast Malt Agar (PYM) and (ii) Nutrient Agar (NA).

In Vitro screening of the antifungal activity of fungal isolates

Mold fungi isolates were examined for their ability to produce inhibitory compounds active against R. solani using the dual culture agar method Henis and Inbar, [27].

In vitro screening of the antifungal activity of yeast isolates

Yeast fungi isolates were examined for their ability to produce inhibitory compounds active against R. solani using the dual culture agar method Henis and Inbar, [27].

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 [44]. 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.

Soil infestation

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:

R. solani + Eupenicillium senticosum. R. solani + Penicillium herquer. R. solani + Trichoderma viride. R. solani + Mix of the three mold fungi. R. solani + Candida incommunis. R. solani + C. steatolytica R. solani + Hansenula arabitolegnes. R. solani + Mix of the three yeast fungi. Pathogen alone. Effect of selected rhizosphere fungal and yeast species on cotton plant growth:

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.

Statistical analysis

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 [31].

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 [4].

Production of IAA

A modified method (Bric et al., 1991) was used to screen indolacetic acid (IAA) and/or IAA analog producers. TSA ((10%) amended with 5 mM 1-tryptophane was overlaid with an 82-mm diameter nitrocellulose membrane disk. Agar plates were inoculated with tested isolated and incubated at 28 °C for 3 days. The membranes were overlaid on Whatman No.2 filter paper saturated with Salkowski reagent (1.0 ml of 0.5 M FeCl₃ + 50 ml 35% HclO₄) [24].

Extraction of the antifungal substances

The selected mold and yeast fungi were cultivated on Hussein's fishmeal extract agar (HFME) broth [29], 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) [42].

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 R. solani growing for three days on PDA. After incubation, the purified antifungal components were detected according to their R_f values [42].

Chemical structure of the purified active components of the selected fungal and yeast species

This experiment was carried out according to El-Mehalawy [21], 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.

Results

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 R. solani

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 R. solani

Fungal isolates No. 5, 18 and 19 were found to have the remarkable antagonistic activities against R. solani than the other isolates , on the other hand, yeast isolates No. 9, 13 and 20 have the most inhibitory activities against R. solani than the other isolates (Tables 3 & 4).

Identification of fungal and yeast isolates

The three strongest antagonistic rhizosphere fungi were identified to the genus and species levels as (5) Trichoderma viride, (18) Penicillium herquer and (19) as Eupenicillium senticosum.

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) Hansenula arabitolgenes, (13) Candida incommunis and (20) Candida steatolytica.

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 R. solani and affected the behavior of the selected antagonistic fungal and yeast species compared to the non-amended soil. It is also clear from the results that, the addition of the fungal mixture or the yeast mixture to the soil was found to be most effective against the pathogen and in induction of resistance in cotton plant than each of the three fungal or the three yeast species alone.

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 C. incommunis was characterized by its ability for production of IAA and siderophores and its ability for phosphate solubilization. T. viride and C. steatolytica were characterized by their ability for siderophore production and phosphate solubilization, E. senticosum was characterized by its ability for IAA production and phosphate solubilization, while P. herquer was characterized by its ability for siderophore production only.

R_f 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 R_f values as shown in Table (9).

From the table it is shown that four active components were detected in the culture filtrate of E. senticosum, five active components were detected in the culture filtrate of T. viride, while six active components were detected in the culture filtrate of P. herquer. It is revealed also from the table that five active components were detected in the culture filtrates of both H. arabitolgenes and C. incommunis, while four active components were detected in the culture filtrate of C. steatolytica All these active components were found to be effective against R. solani. The active component of R_f 0.25 was detected in the culture filtrates of 5 of the six tested fungal and yeast species, and the active component of Rf 0.47 was detected in the culture filtrates of 4 of the six tested fungal and yeast species.

Chemical structures, chemical formulae and molecular weights of purified active components of H. arabitolgens and E. senticosum

Data in Table (10) show the chemical name, chemical formula and molecular weights of the active components detected in the culture filtrates of both H. arabitolgenes and E. senticosum. It was well known that the compound of high molecular weight has low R_f value and vice versa. Therefore, the active components were arranged descendingly according to their R_f values and molecular weights. Fig (1 & 2 ) showed the chemical structure of the active components detected in the culture filtrates of E. senticosum and H. arabitolgenes.

Figure 1: Mass Spectrum of the purified antifungal components Producted by E by E. Senticosum

Figure 2: Mass spectrum of the purified antifungal components produced by H. arabitolgenes

Discussion

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 et al., [17].

The presence of the pathogen (R. solani) in the soil without amendment (fishmeal) led to a remarkable increase in the percentage of disease compared to the non-infested soil either with or without amendment. This may be due to the competitive saprophytic growth of R. solani, and in natural ecosystems the pathogen inoculum is generally aggregated so that subsequent spread can occur under appropriate conditions. This explanation agrees with that reported by Lewis et al., [37].

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 R. solani. This may be due to that these species are capable, in liquid cultures at least, of producing an array of metabolites which have antifungal activity [23], or may be due to the production of toxins (killer toxins) by yeast species [28] or the production of water soluble antifungals which may act with sensitive cells involving the cellular membrane leading to destruction of its permeability [22].

The reduction in the percentage of seed germination in the presence of R. solani may be due to the role of the pathogen which acts as a causal agent of damping-off. The pathogen seems to be pathogenic under certain environmental conditions such as soil temperature or pH, and/or it may play a secondary role in disease development. It is also possible that R. solani is able to overcome defense mechanisms of cotton. These findings are in agreement with that reported by Singh and Siradhana [46]. And Begum et al. [11].

The use of rhizosphere fungal and yeast species, each group in combination, increased the percentage of inhibition of growth of R. solani. This may be due to the lack of nutrition for the growth of the pathogen and production of certain inhibitory chemicals by antagonists in culture. This finding runs parallel with that reported by Dubey [11].

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 [32].

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 [22]. 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 [13].

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 et al., [1].

Table 1: Measurement of microbial activity in amended and non-amended soils.

Table 2: Pathogenicity of Rhizoctonia solani.

Table 3: Antagonistic activities of fungal isolates against R. solani

Where γ_o: 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 = γ_o – γ % inh. : Percentage of inhibition ( Δγ/ γ_o)

Table 4: Antagonistic activities of yeast isolates againstR. solani

Where γ_o : Fungal growth radius of a control culture (cm) γ : distance of fungal growth in direction of yeast fungi colony (cm) Δγ = γ_o – γ % inh. : Percentage of inhibition ( Δγ/γ_o)

Table 5: Effect of fungal and yeast species on the germination of cotton seeds using amended and non-amended soil.

Table 6: Effect of the three fungal species and their mixture on the growth measurements of cotton plant grown in amended and non-amended soil.

A: amended soil. N: non-amended soil. C: Untreated (control). F1: E. semticosum. F2: P. herquer. F3: T. viride. F4; Mixture.

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.

Table 7: Effect of the three yeast species and their mixture on the growth measurements of cotton plant grown in amended and non-amended

A: amended soil. N: nom-amended soil. C: Untreated (control). Y1: C. incommunis. Y2: C. steatolytica. Y3: H. arabitolgenes. Y4: Mixture.

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.

Table 8: Production of IAA and Siderophores and Phosphate Solubilization by rhizosphere fungal and yeast species.

Table 9: R_f values of the active antifungal substances produced by the selected fungal and yeast species

Table 10: Chemical strucures, chemical formulae and molecular weights of purified active components of Eupenicillium senticosum and Hansenula arabitolgenes

Correspondence to

Address: Microbiology Department, Faculty of Science, Ain Shams University, Cairo, Egypt Phone No. 002 02 6238470 e. mail: adelzz1953@hotmail.com

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The Internet Journal of Microbiology ISSN: 1937-8289