International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Impact Factor (2012): 3.358 Impact of the Fungicide Rizolix T50% on the Antagonistic Activity of Trichoderma harzianum and Trichoderma koningii

Hefnawy M.A.1, Omima A. Eisa2, Nora M. El-Feky3

Botany Department, Faculty of Science Menoufia University, Egypt

Abstract: Six Trichoderma spp. were isolated from the rhizosphere of maize and clover. All the isolates were able to tolerate Rizolex T 50%. Isolates of T. harzianum and T. koningii exhibited the best tolerance to this fungicide, reached to 350 and 400ppm respectively. The antagonistic effect of T. harzianum was high against pathogenic fungi, Fusarium oxysporum, Fusarium proliferatum and Acremonium strictum, reached to 77.3, 77.5 and 78% reduction respectively. While, T. koningii reduced the growth of these fungi to approximately 69, 73 and 77% respectively. The growth of all tested pathogenic fungi greatly decreased with increasing fungicide concentration in the growth medium comparing with the control. F. oxysporum and F. proliferatum tolerated Rizolex T50 % up to 450 - 550 ppm. whereas, Ac. strictum tolerated it only up to 250 ppm. The growth of T. harzianum and pathogenic fungi decreased with increasing Rizolex concentrations in the growth medium, moreover the antagonistic effect of T. harzianum against F. oxysporum, F. proliferatum and Ac. strictum at 200 ppm Rizolex T50% was approximately 86.6, 85 and 84% reduction respectively. Whereas, it was 74.3, 81.1 and 80% reduction respectively with T. koningii. Utilization of low dose of fungicide in combination with Trichoderma spp. increased the inhibition percentage of pathogenic fungi and may be beneficial in controlling these fungi.

Keywords: Trichoderma species, Pathogenic fungi, Rizolex T50, Fungicides, Antagonistic activity.

1. Introduction The antagonistic ability of Trichoderma asperellum, T. atroviride, T. koningii, T. harzianum and T. reesi against There is a worldwide trend to the use of ecologically safe Fusarium oxysporum f. sp. phaseoli was evaluated under methods of protecting crops from pathogens and pests. lab. and green house conditions and found that T. reesei and There is a growing concern, both in developed and T. koningii was the most effective isolates against the developing countries including Egypt, about the use of pathogen and for stimulation of plant growth[6]. T. hazardous fungicides for controlling fungal plant diseases. harzianum, T. asperellum, T. virens against F. oxysporum of Now we know that the residues accumulation of fungicides lentil was evaluated and found that all of them could in the soil and water interferes with numerous biological effectively inhibit growth of the fungus in laboratory tests. activities[1]. Three isolates alone and in combination in green house revealed that disease severity was reduced from 20 to 44% Biological control is becoming an urgently needed while, dry weight increased from 23 to 52% when T. component in agriculture. Chemical pesticides have been the harzianum and T. asperellum were combined [9]. The object of substantial criticism in recent years. Therefore, it is effectiveness of two Trichoderma isolates Trichoderma important to develop safer and environmentally feasible viride and Trichoderma Koningii against Fussarium control alternatives, mainly by the use of existing living oxysporum causing root rot in beans was studied in vitro and organisms in their natural habitat. These organisms are able under greenhouse condition. They founded that nearly 100% to provide protection against a large range of plant inhibition of Fusarium oxysporum growth by antagonistic T. pathogenic fungi without damage to the ecological viride and very poor growth of Fusarium with a reduction of system[2]. 91% was observed in the presence of antagonist T. koningii [10]. Fusarium proliferatum, a relatively described species occurs worldwide on a broad variety of plants including maize[3]. Trichoderma asperellum, T. harzianum, T. harzianum and T. Two species F. proliferatum and F. verticilloides were hamatum and a low dose of seed fungicides Rizolex-T50% considered as the more widespread on maize, sorghum and and Celest-max in combination were used to detect many other cereals. These two species were found compatible fungicide/antagonist combinations for integrated associated with all plants growth stages [4],[5]. disease control. Results showed that the combinations did not increase the efficacy against stem cancer of potato. Rizolex T 50% is non systemic contact fungicide with however, effectiveness of fungicide/antagonist combinations protective and curative action. Acts by inhibition of on black scurf was higher with limited extent[11]. This work phospholipids biosynthesis, leading to inhibition of aims to evaluate the antagonistic activity of Trichoderma germination of spores and growth of fungal mycelium. spp. against pathogenic fungi in absence and in the presence Control of Fusarium by chemicals is often uneconomical of fungicides and has negative environmental impacts and development of fungicidal resistance variety [6]. The most important alternative method to chemical control of disease causing pathogens like Fusarium, Pythium and Rhizoctonia was achieved by applying biological control [7],[8]. Volume 3 Issue 9, September 2014 www.ijsr.net Paper ID: SEP14209 Licensed Under Creative Commons Attribution CC BY 1767 International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Impact Factor (2012): 3.358 2. Materials and methods Effect of different concentrations of Rizolex T50% on radial growth of selected Trichoderma spp. and 2.1 Isolation of Trichoderma spp. from Egyptian soil. pathogenic fungi:

Trichoderma spp. was isolated by dilution technique method PDA medium was amended with different concentrations of from soil obtained from rhizosphere of healthy and diseased Rizolex T 50% (0, 50, 100, 150 up to 550 ppm), 10 ml of 0 plants (maize and clover). A serial dilution was made for medium at 45 C was poured to sterilized Petri dishes under each sample up to 1/10000. 0.5 ml from each dilution was aseptic condition and allowed to solidify. After inoculated on to potato dextrose agar plates containing rose solidification, 8 mm circular disc of 10-day-old pathogenic Bengal and streptomycin [12]. The plates were incubated at fungi and selected Trichoderma spp. was transferred 30°C for 4 days. All plates were examined daily and aseptically to the center of agar plates. The Petri plates were different Trichoderma spp. were isolated and purified on a incubated at 30°C for 8 days, radial growth was measured new PDA plates. daily.

Screening the sensitivity of isolated Trichoderma spp. 3. Results toward Rizolex T 50%. Six Trichoderma spp. were isolated from rhizosphere of Different concentrations of fungicide was added separately maize and clover. Growth of all isolated Trichoderma spp. to 100 ml of sterilized PDA medium immediately after was highly decreased with increasing fungicide cooling before solidification under aseptic condition and concentrations in the growth medium comparing with the mixed thoroughly to give the required concentrations. The control. All Trichoderma isolates were able to tolerate and control was maintained without fungicide. Prepared medium grow on the agar medium containing 100ppm of Rizolex T was aseptically poured in sterilized Petri dishes, Three 50%. T5 and T6 were the isolates which are able to tolerate replicates were used for each treatment. Equal disks (8 mm high concentrations of Rizolex T 50% reached to 350 and diameter) from Trichoderma 7 days old culture were placed 400ppm respectively (Table 1). onto the middle of the agar plate. Inoculated plates were incubated at 30°C and mycelial growth was measured daily. Antagonistic activity of selected Trichoderma species The best Trichoderma spp. which tolerated high against pathogenic fungi. concentrations of fungicides was selected for further studies. In vitro antagonistic property studies were carried out to Identification of selected Trichoderma spp. : evaluate the efficiency of Trichoderma harzianum and T. koningii on the growth of the phytopathogenic fungi Trichoderma spp. was identified by the regional center of Fusarium oxysporum, F. proliferatum and Acremonium mycology and biotechnology, Al-Azhar University, Egypt. strictum by direct competitive interaction. The growth of They were identified as Trichoderma harzianum and pathogenic fungi in absence of Trichoderma spp. reached to Trichoderma koningii (Fig.1), where pathogenic fungi approximately 75 mm colony diameter for F. oxysporum and Fusarium proliferatum, Fusarium oxysporum and F. proliferatum and 50mm for Ac. strictum whereas; in the Acremonium strictum were obtained as a gift from plant presence of T. harzianum it sharply decreased to 17, 20 and pathology research institute, Agriculture research center, 11 mm respectively. Therefore its antagonistic effect was Giza, Egypt. very high and reached to 77.3, 77.5 and 78% reduction in the growth of the pathogenic fungi respectively. On the other Antagonistic effect of selected Trichoderma spp. against hand, T. koningii reduced the growth of pathogenic fungi F. pathogenic fungi: oxysporum, F. proliferatum and Ac. strictum to approximately 69, 73 and 77% respectively (Table 2 and T. harzianum and T. koningii were grown against the Fig. 2). pathogens Fusarium oxysporum, Fusarium proliferatum and Acremonium strictum to test their antagonistic potential on Effect of different concentrations of Rizolex T 50 % on Potato Dextrose Agar (PDA). Eight mm disk of seven-day- radial growth of selected Trichoderma isolates old cultures of Pathogenic fungi and Trichoderma were placed against each other on plates. In case of control The growth of both T. harzianum and T. koningii was instead of Trichoderma disk, PDA disk was used. The plates markedly decreased with increasing fungicide were incubated at 30ºC for 7 days. Radial growth of the concentrations in the growth medium at different incubation pathogens and Trichoderma was measured and data were periods. T. harzianum was able to grow in the presence of obtained. Data of laboratory tests were calculated by the Rizolex T50% up to 400 ppm with percentage inhibition of following formula: 83.3% while T. koningii cannot grow at this concentration % inhibition = {(Diameter of fungal growth in control - but grown up to 350 ppm with percentage inhibition of Diameter of fungal growth in treatment)/ Diameter of fungal 87.7% after six days. 50% inhibition of T. harzianum was growth in control} × 100. obtained after six days of incubation period at 100ppm of Rizolex T 50 % while it was obtained at 50 ppm for T. koningii. At concentration of 350ppm of Rizolex T 50 %, the growth of T. harzianum was greatly reduced to approximately 78.8% whereas, it reduced to approximately 87.7% for T. koningii, this indicated that T. koningii was Volume 3 Issue 9, September 2014 www.ijsr.net Paper ID: SEP14209 Licensed Under Creative Commons Attribution CC BY 1768 International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Impact Factor (2012): 3.358 more sensitive to Rizolex T 50 % than T. harzianum (Table In this work, the antagonistic activity of isolated 3 ). Trichoderma harzianum was high against pathogenic fungi F. oxysporum, F. proliferatum and Ac. strictum and reached Radial growth of Pathogenic fungi at different to 77.3, 77.5 and 78% reduction respectively. While, T. concentrations of Rizolex T 50 %: koningii reduced the growth of these fungi to approximately 69, 73 and 77% respectively. Several reports have indicated Fusarium proliferatum, Fusarium oxysporum and that biocontrol efficiency of Trichoderma spp. against Acremonium strictum exhibited a different ability to grow on Fusarium wilts may differ in different regions of the world different concentrations of Rizolex T 50 % in growth i.e., a highly antagonistic species against a particular medium at different incubation periods. The growth of all pathogen in a given region may react poorly against the tested pathogenic fungi greatly decreased with increasing same pathogen in another region[14],[15]. In a study carried fungicide concentration in the growth medium comparing out by [16], the efficacy of T. koningii for the suppression of with the control. After eight days, F. oxysporum and F. F. oxysporum reached to about 91% in vitro while, in this proliferatum were able to grow at 450, 550 ppm respectively study, T. koningii was able to reduce it to 69.3%. of Rizolex T 50 % with percentage inhibition of 84.4% and 78.8% and they cannot able to grow above these T. harzianum was able to grow in the presence of Rizolex concentrations. In contrast, Ac. strictum was the most T50% up to 400 ppm with percentage inhibition of 83.3% sensitive one to Rizolex T 50 %, as it was not able to grow while, T. koningii cannot grow at this concentration but above 250 ppm of Rizolex T 50 % with percentage grown up to 350 ppm with percentage inhibition of 87.7%. inhibition 80% at this concentration after eight days. 50% Similar result was also observed by [17], they observed that inhibition of F. proliferatum was approximately obtained at mycelial growth of Trichoderma spp. was most sensitive to 250ppm of Rizolex T 50 %, while it was obtained at 150 Benlate, Ridomil gold, Tecto-60 and Topsin-M at both the ppm for F. oxysporum and less than 50 ppm for Ac. strictum, concentrations of 100 and 200ppm. Similar results (Table 4). concerning the inhibitor effect of Rizolex-T 50% and Topsin-M at different concentrations against soil borne Antagonistic activity of T. harzianum and T. koningii fungi, R. solani and F. oxysporum were also observed by toward the pathogenic fungi in the presence of different [18]. concentrations of Rizolex T 50%: In our results the combination of Trichoderma spp. with low The growth of pathogenic fungi in absence of T. harzianum concentration of Rizolex T lead to increase inhibition of in the control medium without Rizolex T50% reached to pathogenic fungi (F. oxysporum, F. proliferatum and A. approximately 75mm colony diameter for F. oxysporum and strictum) in compare with Trichoderma or low concentration F. proliferatum and 50mm for Ac. strictum whereas, in the of Rizolex T separately. This result agrees with [19], they presence of T. harzianum it sharply decreased to 17, 20 and reported that the efficacy of the Trichoderma treatments has 11 mm respectively. Therefore its antagonistic effect was been both more variable and less effective than chemical very high and reached to 77.3, 77.5 and 78% reduction in the fungicide treatments. Combining bio-control agents with growth of the pathogenic fungi respectively (Table 5). selective fungicides that are not inhibitory to the antagonist will help it to become established in the soil and achieve The presence of Rizolex T 50% in the growth medium better disease control and also with[20], who demonstrated highly decreased the growth of T. harzianum and pathogenic an improved disease control of gladiolus corm rot and wilt fungi. The antagonistic effect of T. harzianum against caused by F. oxysporum by combining an isolate of T. virens pathogenic fungi slightly increased in the presence of the and the fungicide carboxin. Analogous results were obtained fungicide Rizolex T50%. At 200 ppm Rizolex T in by [21], with mutants of benomyl-tolerant strains of T. combination with T. harzianum reduced the growth of F. pseudokoningii, which were superior to the wild type in bio- oxysporum and F. proliferatum and Ac. strictum to control potential on S. rolfsii. approximately 86.6, 85 and 100% respectively. While, with T. koningii in combination with Rizolex T at 300 ppm, the Apparently, biocontrol with Trichoderma has to be growth of these pathogenic fungi reduced to approximately implemented with other disease control measures to reach a 80. 81.1 and 100% respectively. Utilization of low dose of satisfactory level of disease control. A correlation between fungicide in combination with Trichoderma spp. increased fungicide resistance and antagonistic activity is suggested by the inhibition percentage of pathogenic fungi and may be [22], affirming that the up regulated expression of ABC beneficial in controlling these fungi. transporter genes of T. atroviride during the three-way interaction with various plants and fungal pathogens, 4. Discussion possibly supports both antagonistic activity and root colonization. In recent years, research on biological control has gained momentum for controlling serious soil born plant pathogens In concolusion: Isolation and selection of fungicides tolerant like Fusarium, Rizoctonia , Macrophomina, Sclerotium, strains of Trichoderma species may help in better controlling Pythium and Phytophthora spp, employing Trichoderma and of pathogenic fungi. Application of low doses of rizolex Gliocladium species and varied success has been achieved. T50% in combination with biologically control Trichoderma Several Trichoderma spp. could be effectively used in species highly increased the inhibition ratio of pathogenic biocontrol of soil borne plant pathogens, Trichoderma spp. fungi and therefore, better control of plant diseases. are active as hyperparasites [13]. Volume 3 Issue 9, September 2014 www.ijsr.net Paper ID: SEP14209 Licensed Under Creative Commons Attribution CC BY 1769 International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Impact Factor (2012): 3.358 References [13] Okigbo R.N.; Ikediugwu FEO (2000): Studies on Biological Control of Post harvest rot in yams [1] Chet I. (1987): Trichoderma application, mode of (Dioscorea spp.) using Trichoderma viride. J. action, and potential as a biocontrol agent of soil borne Phytopathol., 148: 351-355. plant pathogenic fungi. Plant disease control, Witey, [14] Hajieghrari B., Torabi – giglou M., Mohamadi M. New York, pp. 137-160. R., Davari M. 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Table 1: Radial growth (mm) of Trichoderma isolates grown on PDA medium amended with different concentrations of Rizolex T 50 % fungicides after six days. Data are the mean value of 3 replicates ± standard error of mean.

Volume 3 Issue 9, September 2014 www.ijsr.net Paper ID: SEP14209 Licensed Under Creative Commons Attribution CC BY 1770 International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Impact Factor (2012): 3.358 Table 2: Antagonistic effect of T. harzianum and T. koningii against pathogenic fungi, Data are expressed as colony diameter (mm) after 6 days of incubation, mean of 3 replicates ± standard error of mean.

Table 3: Effect of different concentrations of Rizolex T 50 % on radial growth of T. harzianum and T. koningii. Data are the mean value of 3 replicates ± standard error of means.

Table 4: Effect of different concentrations of Rizolex T 50% on radial growth of Fusarium proliferatum, Fusarium oxysporum and Acremonium strictum. Data are the mean value of 3 replicates ± standard error of means.

Table 5: Antagonistic effect of Trichoderma harzianum and T. koningii against pathogenic fungi in the absence and presence of fungicide. Data are expressed as colony diameter (mm) after 6 days of incubation, mean of 3 replicates ± standard error of mean.

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Figure 1: Photomicrograph shows the phialides and conidia of (A) Trichoderma koningii and (B) Trichoderma harzianum under microscope.

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Figure 3: Photograph shows antagonistic activity of T. koningii and T. harzianum against pathogenic fungi (A) F. oxysporum, (B) Ac. strictum and (C) F. proliferatum.

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