Trichoderma koningiopsis a new and strong antagonist against soil borne pathogens of chickpea.
In chickpea, chlamydospores and sclerotia surviving in the soil are the major sources of primary inoculum. Since 75% cultivation of chickpea in India is under rainfed, the crop faces severe moisture stress which predisposes the crop to wilt and dry root rot development. The saprophytic survival ability of the pathogens in soil makes chemical control and crop rotation ineffective. Cultivation of resistant varieties is an economical approach for the management of wilt and dry root rot but up to now effective resistant cultivars are not available to combat with the diseases. The preference for biological control method is justified also by the undesirable side effects of pesticides. The technology that seems promising to manage the diseases without disturbing the equilibrium of harmful and useful composition of environment and ecosystem is the use of more and more biological control agents. Use of Trichoderma spp. as biological agents has been very much successful against soil borne diseases for which no resistant sources have been identified. (Mukhopadhyay 1994, Mukhrejee et al, 2012).
However, there is still considerable interest in finding more efficient mycoparasitic fungi especially within Trichoderma spp., which differ considerably with respect to their biocontrol effectiveness. It is important to isolate Trichoderma spp. having potentially higher antagonistic efficiency by the selection of isolates with high potential of mycoparasitic activities. The aim of this study was screening of Trichoderma spp. for their antagonistic ability, higher survibility as well as their capability of interaction and hyphal depression to the test pathogens.
MATERIALS AND METHODS
Soil samples and Isolation
Soil samples were collected from the rhizosphere soil of different crop niches. Five-fold serial dilutions (Singh, 1970) of each soil sample was prepared in sterilized distilled water and 0.5 ml diluted sample was poured on the surface of Trichoderma Specific Medium (TSM). Plates were incubated at 25 [+ or -] 2[degrees]C for 96 h. Morphologically different colonies appearing on the plates were purified in the Potato Dextrose Agar (PDA) (HiMedia, India) and send to ITCC, New Delhi for identification.
Cultural, Morphological and Physiological Characterization
Cultural and morphological observations of colony were based on Trichoderma isolates grown on PDA for 7 days in an incubator at 25[+ or -] 2[degrees]C with altering 12h/12h fluorescent light/ darkness. Characters of the conidium- bearing structures and conidia were assessed for each isolate. Growthrate trials were done in 9 cm diam petridishes with 20 ml PDA at 15, 20, 25, 30, 35, 40 and 45[degrees]C. Measurements of colony radius, the greatest distance from the edge of the plug of inoculum to the edge of the colony were taken daily upto 72h. Trials were replicated thrice. Physiological observations of Trichoderma spp. were based on mycelial growth on different pH ranged from 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0.
Antagonistic Activity of Trichoderma Isolates
The dual culture technique described by Morton and Stroube was used to test the antagonistic ability of 5 Trichoderma spp. viz; T aggressivum (ITCC 7277), T citrinoviride (ITCC 7283) T. erinaceum (ITCC 7287), T koningiopsis (ITCC 7291), and T harzianum (ITCC 6796) against Fusarium oxysporum f.sp.ciceri and Rhizoctonia bataticola. The pathogen and Trichoderma spp. were grown on PDA for a week at 25 [+ or -] 2[degrees]C. 5mm disc of the target fungi cut from the periphery was transferred to the Petri dish previously poured with PDA. Trichoderma spp. was transferred aseptically in the same plate of opposite end and were incubated at room temperature with alternate light and darkness for 7 days and observed periodically. Control plates were maintained without Trichoderma. The experiment was replicated thrice and percent growth inhibition was calculated by the formula of I = (C"T)/C x 100, where C is mycelial growth in control plate, T is mycelial growth in test organisms inoculated plate and I is inhibition of mycelial growth. Vincent et al (1999)
Scanning electron microscopy (SEM)
Small pieces of agar (approx. 2 [mm.sup.2]) were taken from the dual culture plates at the point of interaction between Trichoderma spp. and test fungi. The samples were fixed in 2.5% glutaraldehyde dissolved in 0.5M phosphate buffer at pH 7.2 and stored overnight at 4[degrees]C, then rinse with the same buffer. After dehydration using a graded ethanol series, samples were critical-point dried in carbon dioxide after a graded transition from ethanol to acetone. Sections (5 x 5mm) were mounted on stubs, coated with gold-palladium, and examined with a JEOL-JSM-T300 SEM operating at 15 kV
Microscopic examinations on morphological characters of all Trichoderma species revealed that the asexual states of all species have typical T. harzianum- like morphology except T. koningiopsis which forms less conidia but more chlamydospores. Phialides arise in whorls at the tips of secondary branches and from the tip of the main axis. The average dimensions of phialides ranged between 3.1-7.6 x 2.4-3.4. Longest phialides are found in T harzianum while shortest in T koningiopsis. Widest phialides were seen in T. citrinoviride while narrowest in T. koningiopsis. Conidia did not vary in shape and most were globose to subglobose or broadly ovoidal. Optimum temperature for the growth of all species was between 25 to 30[degrees]C. On increasing the temperature up to 45[degrees]C T erinaceum did not grow and rest of the species grew very poorly except T koningiopsis with 0.270 cm. mecelial growth . Similarly, pH range 6.5 to 7.0 supported best growth for all the species. T. koningiopsis is the only species which grow well at 7.5 pH. Observations on all these characters indicated that T. koningiopsis is the only species which can grow well at variable temperature and pH indicating its tolerance against adverse conditions (Table 1, Fig. 1).
Macroscopic examination of the fungal dual cultures revealed that most of the strains made hyphal contact with test pathogen within two days after inoculation. T. koningiopsis was the most inhibiting antagonist that grew over the pathogen. Other Trichoderma spp. acted only as a barrier against Fusarium oxysporum f.sp. ciceri and R. bataticola.
Antagonistic potential of Trichoderma spp. through dual culture indicated that colony growth after 72 h was 13.5-20.0 mm in Foc and 35.0-42.5 mm inRb as compared to control. Colony growth of test pathogens was appressed and after coming in contact, the antagonists grew and sporulated over the pathogen colony due to their prolific growth habit and mycoparasitic character. Inhibition percent of growth by different Trichoderma spp. ranged between 33.3-55.0 percent and 22.7-36.3 in Foc and Rb respectively (Table 2, Fig 2 a & b). These findings corroborate the findings by earlier workers. (Dennis and Webster, 1971; G.J. Samuels, 1996; Sumeet and Mukherjee, 2000; Golve and Kurundkar, 2002; and Jagathambigai et al., 2009.)
A similar behavior for each antagonistpathogen combination was observed by SEM. There were similarities and differences in the antagonistic ability of all species of Trichoderma to invade the pathogen in dual culture. Direct contact with the pathogen was always followed by various types of hyphal aggression. SEM investigations revealed that mycoparasitic hyphae were usually attached longitudinally to the hyphae of the pathogen. Hyphal coiling, hooks, pincer shaped structures, short contact branches and hyphal depression were also observed.
In case of conidial and sclerotial inoculum of Foc & Rb it was observed that percent conidia and sclerotia killed ranged from zero to 100 percent depending upon the antagonistic potential of the Trichoderma species. T. harzianum kill all the conidia and sclerotia while other species killed some of the inoculum (Fig. 4a-h). All Trichoderma sp. were effective in reducing conidial and sclerotial viability. These observations revealed that penetration and multiplication of antagonist inside the conidia and sclerotia is dependent on the ability of the biocontrol agent to attack and establish on the wall of conidia and sclerotia. As the studies done so far on biological control of F. oxysporum ciceri and R. bataticola included only a few isolates of a particular species so it is difficult to draw a conclusion on the species specificity. In the present investigations antagonistic effects of five Trichoderma species revealed that there is a significant variability in their ability to parasitize, macerate and kill the mycelial, conidial and sclerotial inoculum of the test pathogens. Conidia and Sclerotia are first colonized by the antagonists followed by penetration and finally killing. Trichoderma koningiopsis is found best in mycoparasitism of Fusarium oxysporum, f.sp. ciceri and R. bataticola as compared to other antagonists studied. These findings supported by findings of earlier workers (Elad et al., 1983; Kohl and Schlosser, 1989; Sreenivasaprasad and Manibhushanarao, 1993; Amrutha et al., 2014).
Cultural characteristics comprising growth rate, colony colour and colony appearance were regarded as taxonomically useful characteristics for Trichoderma (Samuels et al., 2002a). Studies revealed that all five Trichoderma spp. did not much differ in cultural characteristics with most isolates exhibiting rapid growth, effuse conidiation and/or loosely arranged conidia in pustules. The same findings like rapid growth at 25[degrees]C to 30[degrees]C were recorded by Samuels et al. (2002a). Gams and Bissett (2002), Lin and Heitman (2005) and Samuels et al. (2002a) also confirmed the presence of terminal and/or intercalary chlamydospores in cultures. Morphological characterization was conventionally used in the identification of Trichoderma species, and it remains as a potential method to identify Trichoderma species (Anees et al., 2010; Gams and Bissett 2002; Samuels et al., 2002a).
Trichoderma is a well known biocontrol agent with multiple modes of action such as competition (Howell, 2003), induced resistance (Harman, 2006), solubilization of inorganic plant nutrients (Altomare et al., 1999), inactivation of the pathogen's enzymes involved in the infection process (de Meyer et al., 1998) and mycoparasitism (Barnett and Binder, 1973). Various workers stated that Trichoderma spp. produces cell wall degrading enzymes (CWDEs) including chitinases, [sup.2]-1,3glucanases, proteases and [sup.2]-1,4-glucanases, antibiotics and antibiotic peptides, such as peptaibols to combat with the pathogen (Flores et al., 1997; Elad and Kapat, 1999; Dennis and Webster, 1971; Fujiwara et al., 1982, Vinale et al., 2006 ; Iida et al.,1994).
In case of antibiosis in dual culture it was observed that Trichoderma koningiopsis was found best in controlling growth of the test pathogens among all Trichoderma spp. Varying modes of hyphal interactions and degree of inhibition in growth and development of Foc and Rb were studied to investigate mechanism of control. Understanding the mechanism of action involved in the biocontrol process is of primary importance in establishing these characteristics. This can provide much insight about where and when the interaction occurs and how the pathogen will be affected. In order to survive and mycoparasitize Trichoderma spp. produces a wide variety of toxic and antibiotic metabolites such as trichodermol, trichodermin, harzianolide, terpines, polypeptides (Lorito et al., 1994; Dickinson et al., 1995; Sivasithamparam and Ghisalberti, 1998; Vinale et al., 2006; Vinale et al., 2008; Andrabi et al., 2011) and extracellular hydrolytic enzymes (Thrane et al., 2000; Eziashi et al., 2006) which were involved in the inhibition, competition, and mycoparasitism of phytopathogenic fungi. In this regard our results support these findings by showing that Trichoderma koningiopsis produces strong antibiosis and competitive growth against pathogens in agar plates (Fig. 2a & 2b).
Knowledge on the mechanism of antagonism is must and would prove very useful for the effective disease control. Scanning Electron Microscopy (SEM) of hyphal interaction between Trichoderma spp. and Fusarium oxysporum-Rhizoctonia bataticola (wilt complex pathogens) indicated that biocontrol agents parasitized the mycelium first. They penetrate and finally resulting into lysis or collapse of hyphae of the pathogens. Among the Trichoderma spp. T. koningiopsis showed more mycoprasitic ability making contact with host hyphae, running parallel to it, production of hook like structure and emptied the cells. This research was carried out to screen five Trichoderma spp. against wilt & dry root rot pathogens of chickpea under in vitro. Electron microscopic observations revealed that all Trichoderma. spp. interacted with the pathogens. T koningiopsis grew toward the pathogen and coiled around the host cells, penetrating and destroying the hyphae. Penetration into host cells was apparently accomplished by mechanical activity.
Elad et al., (1983) demonstrated hyphal interaction between T. harzianum and T. hamatum with Sclerotium rolfsii and Rhizoctonia solani by Scanning Electron Microscopy (SEM). Trichoderma spp. adhere the host surface by coiling, hooks or appressoria. Lysed sites and penetration holes were found in hyphae of the plant pathogenic fungi, following removal of parasitic hyphae.
Based on the antagonistic potential and hyphal morphologies observed at SEM we would suggest T. koningiopsis as a strong antagonist. These findings are new as SEM investigations on Trichoderma spp. with wilt complex creating fungi in chickpea are not reported earlier from India. T koningiopsis may play an important role in the biological control of soil borne diseases of chickpea in U.P. (India).
Authors are thankful to Department of Science & Technology (DST), New Delhi, and Indian Council of Agricultural Research, New Delhi for financial support and Central Drug Research Institute (CSIR), Lucknow, India for electron micrograph.
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Shubha Trivedi, Mukesh Srivastava, Anuradha Singh, Vipul Kumar, Sonika Pandey, Mohd. Shahid and Yatindra Srivastava
Bio-Control Lab, Department of Plant Pathology, Chandra Shekhar Azad University of Agriculture & Technology, Kanpur--208002, India.
(Received: 15 September 2015; accepted: 14 November 2015)
* To whom all correspondence should be addressed. E-mail: email@example.com
Caption: Fig. 1. Light and Scanning Electro micrograph of difference species Trichoderma showing hyphal and conidial morphology
Caption: Fig. 2 (a). Antagonistic potential of Trichoderma spp. against Fusarium oxysporum f.sp. ciceri.
Caption: Fig. 2 (b). Antagonistic potential of Trichoderma spp. against Rhizoctonia bataticola
Caption: Fig. 3. (A-F) (A & B) Scanning electron micrograph on mycoparasitism of the F. oxysporum f.sp. ciceri hyphae by the hyphae of T. koningiopsis with pincer shaped structure moving longitudinally and parallel to the hyphae of the pathogen (C & D) Coiling of hyphae and hyphal tip of T koningiopsis attached to and penetrating the hyphae of F oxysporum ciceri (E & F) T. koningiopsis hyphal tip, hooks and chlamydospores adhere to the hyphae of F oxysporum ciceri causing hyphal depression.
Caption: Fig. 4. (A-F) (A & B) Scanning electron micrograph on parasitic action of T. koningiopsis against R. bataticola, moving longitudinally and parallel to the hyphae of the pathogen (C & D) Hyphal tip of T. koningiopsis attached and penetrating the hyphae of R. bataticola (E & F) T. koningiopsis hyphal tip and conidia adhere to the hyphae of R. batatocola causing hyphal swelling and hyphal growth depression.
Table 1. Comparison of morhology of Trichoderma spp. Characters/ Species T 1 aggressivum Habitat Soil Conidium length (pm) 3.1-3.8 Conidium width (pm) 2.8-3.1 Phialid length (pm) 4.3-5.9 Phialid width (pm) 2.4-3.1 Growth after 72h at 15[degrees]C (cm) 4.8 Growth after 72h at 20[degrees]C 6.2 Growth after 72h at 25[degrees]C 6.8 Growth after 72h at 30[degrees]C 7.5 Growth after 72h at 35[degrees]C 8.1 Growth after 72h at 40[degrees]C 7.5 Growth after 72h at 45[degrees]C 0.115 Mycelial growth at pH4.0 0.232 Mycelial growth at pH4.5 0.608 Mycelial growth at pH5.0 0.151 Mycelial growth at pH5.5 0.276 Mycelial growth at pH6.0 0.164 Mycelial growth at pH6.6 0.926 Mycelial growth at pH7.0 0.601 Mycelial growth at pH7.5 0.171 Characters/ Species T T. citrinoviride T. erinaceum Habitat Soil Soil Conidium length (pm) 2.7-3.1 3.1-3.4 Conidium width (pm) 2.1-2.8 2.5-3.1 Phialid length (pm) 6.2-6.8 4.3-6.2 Phialid width (pm) 3.1-3.4 2.4-3.1 Growth after 72h at 15[degrees]C (cm) 3.1 3.8 Growth after 72h at 20[degrees]C 6.8 5.8 Growth after 72h at 25[degrees]C 7.2 6.6 Growth after 72h at 30[degrees]C 7.7 7.2 Growth after 72h at 35[degrees]C 8.2 8.2 Growth after 72h at 40[degrees]C 7.5 7.6 Growth after 72h at 45[degrees]C 0.122 No growth Mycelial growth at pH4.0 0.107 0.032 Mycelial growth at pH4.5 0.116 0.170 Mycelial growth at pH5.0 0.212 0.303 Mycelial growth at pH5.5 0.475 0.111 Mycelial growth at pH6.0 0.184 0.277 Mycelial growth at pH6.6 0.917 0.188 Mycelial growth at pH7.0 0.121 0.196 Mycelial growth at pH7.5 0.131 0.110 Characters/ Species T T. koningiopsis T. harzianum Habitat Soil Soil Conidium length (pm) 4.3-6.8 2.8-3.2 Conidium width (pm) 2.4-3.4 2.5-2.9 Phialid length (pm) 3.1-3.4 3.4-7.6 Phialid width (pm) 2.4-2.8 2.5-3.4 Growth after 72h at 15[degrees]C (cm) 6.6 -- Growth after 72h at 20[degrees]C 7.1 4.4 Growth after 72h at 25[degrees]C 7.5 5.5 Growth after 72h at 30[degrees]C 7.8 6.9 Growth after 72h at 35[degrees]C 5.2 4.7 Growth after 72h at 40[degrees]C 3.2 2.9 Growth after 72h at 45[degrees]C 0.135 0.270 Mycelial growth at pH4.0 0.296 0.269 Mycelial growth at pH4.5 0.296 0.298 Mycelial growth at pH5.0 0.266 1.08 Mycelial growth at pH5.5 1.710 1.22 Mycelial growth at pH6.0 0.217 1.24 Mycelial growth at pH6.6 1.105 1.22 Mycelial growth at pH7.0 0.322 1.20 Mycelial growth at pH7.5 0.196 0.287 Table 2. In vitro antagonistic potential of Trichoderma isolates against R. bataticola through dual culture Trichoderma spp. Growth of Foc after 72h (mm) Mycelial growth % inhibition in mycelial growth T. aggressivum 14.2 52.6 T. citrinoviride 18.0 40.0 T. erinaceum 20.0 33.3 T. koningiopsis 13.5 55.0 T. harzianum 15.0 50.0 Control 30.0 -- CD@5% 4.2 Trichoderma spp. Growth of R.b. after 72h (mm) Mycelial growth % inhibition in mycelial growth T. aggressivum 40.0 27.2 T. citrinoviride 40.2 26.9 T. erinaceum 42.5 22.7 T. koningiopsis 35.0 36.3 T. harzianum 40.2 26.9 Control 55.0 -- CD@5% 3.3
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|Author:||Trivedi, Shubha; Srivastava, Mukesh; Singh, Anuradha; Kumar, Vipul; Pandey, Sonika; Shahid, Mohd.; S|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Dec 1, 2015|
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