potato dextrose agar (PDA) at 4 C for have pronounced impacts on the population of subsequent use. all types of soil micro-organisms including

Transcription

1 The Journal of Plant Protection Sciences, 3() : 3-3, June, Population proliferation and spread of Trichoderma spp. in soil under two different delivery systems Sitansu Pan and Amrita Das Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-5, Nadia, West Bengal, India sitansupan@gmail.com A B S T R A C T Post application population proliferation (build up) and spread of Trichoderma spp., Trichoderma harzianum and Trichoderma viride in three different types (sterilized, solarised and natural) clay loam soil at 5% water holding capacity (ph.5) was studied both under laboratory and field conditions following two types of delivery systems, through organic based (vermicompost neem cake (9% % w/w)) and seed based (sterilized wheat seed) inocula. The population proliferation and spread of the antagonists were faster when they were applied through organic based inoculums over that of seed based inoculums from the point of application. Similarly population proliferation and spread of the antagonist was more rapid in sterilized soil followed by solarised soil and natural soil respectively. Among the two species of Trichoderma, T. harzianum have spread faster than T. viride. Keywos: Population proliferation, spread, delivery system, T. harzianum, T. viride, clay loam soil Introduction Materials and Methods Mycoparasitic fungi of the genus Trichoderma, Isolation of Trichoderma spp. particularly Trichoderma harzianum, have been Biocontrol fungus T. harzianum and T. viride used for biocontrol of pathogens of crop plants was isolated from the rhizosphere soil on both soil-borne. A major challenge in Trichoderma specific medium modified by Saha implementing biocontrol tool is to maintain & Pan (99) using dilution plate technique. stable population of the biocontrol agent so that Antagonistic potentiality of the biocontrol agent the beneficial effects are sustained throughout was assessed against many soil borne plant the growing season (Lewis & Papavizas 9; pathogens (Bose et al. 5, Pan 9). The Papavizas 95). Soil management practices isolate of Trichoderma spp. was maintained on o potato dextrose agar (PDA) at C for have pronounced impacts on the population of subsequent use. all types of soil micro-organisms including Trichoderma spp. (Mughogho 9; Munnecke Population proliferation of Trichoderma spp. in et al. 9). Proliferation and subsequent different soil with different forms of inocula establishment of biocontrol fungi in soil Population proliferation of antagonist in soil was depends on inoculum age and how inoculum studied following Bose and Pan (9). was added in relation to food base. Young Inoculum grown on seed and organic substrate mycelial preparation without conidia is much was used for soil inoculation. For seed based more effective than old one (Lewis & Papavizas inoculum wheat seeds were sterilized in 9). The objectives of the present study were autoclave at. kg/cm for 3 min for two to find out the population proliferation and consecutive days and inoculated with day old spread of hypha of Trichoderma spp. applied spore suspension of ( spores/ml) Trichoderma with different forms of inocula. spp. In case of organic substrate based inoculum

2 The Journal of Plant Protection Sciences, 3() : 3-3, June, g of vermicompost was amended with % moisture content of the soil was adjusted at % neem cake ( w/w) and taken in a double of water holding capacity with sterile distilled layer polypropylene bag. The material was then water. The soil was point inoculated at a specific inoculated with day old spore suspension of ( location with one loopful of prepared inocula of spores/ml) Trichoderma spp. with the help of a Trichoderma isolates on sterilized wheat seed. hypodermic syringe at 5 ml/packet. The material Periodical assay (at, 9 and hrs) was made was incubated for days in BOD incubator at collecting soil samples from different orbits viz. C for preparation of inocula. 5,, 5, and 5 cm from the point of Soil was prepared by grinding and passing inoculation. The soil samples were assessed and through mm sieve. The moisture content of the enumerated for growth in terms of c.f.u content soil was maintained at its % water holding of Trichoderma spp. on TSM medium. capacity and the ph value was also tested. Results and Discussion Sterilization of the soil where needed was done Population proliferation of Trichoderma spp. in in autoclave at. kg/cm for hr for two different soil types with different forms of inocula consecutive days. The substrate containing It was found from the result that when seed based inoculum was mixed with kg of soil (sterilized, inoculum of T. harzianum was added, the solarized and unsterilized as the case may be). In population increased in sterilized soil reaching the assessment of c.f.u of Trichoderma spp. in maximum (5.x c.f.u/g) at 3 week of inoculated soil, sample was collected randomly incubation and then started decreasing reaching after amendment and periodically thereafter with to.x c.f.u/g of soil. In case of natural soil the the help of a cm diameter cork borer from the population of Trichoderma increased for week surface upto a depth of 3 cm at days interval up (.5x c.f.u/g) of incubation and thereafter to days. The soil sample was thoroughly -5 started decreasing (Fig. ). mixed and serially diluted upto a level of to -. One ml of aliquot from this dilution series It was also found from the results that the ability was spread on to the surface of Petri plates of Trichoderma spp. to proliferate in soil was far containing TSM. Observation on number of better and significant at 5% level when applied colonies appeared on TSM after to days of through organic food based inoculum grown on incubation at C, was made and c.f.u was vermicompost-neem cake ( w/w) than determined as per gram of soil. the seed based inoculum. The population of T. harzianum in sterilized soil increased to a Liner growth vis-a-vis spread of the antagonist in maximum level of soil counting.x c.f.u/g soil of soil and in solarized soil up to.x c.f.u/g Linear growth and spread of Trichoderma was th th of soil at week of incubation. By the end of studied in unsterilized, sterilized and solarized week, the population remained at.x soil. Natural field soil was grounded and passed c.f.u/g and 9.x c.f.u/g of sterilized and through mm screen. Sterilization of the soil solarized soil respectively. In case of natural soil where needed was done in autoclave at. the population also increased with a peak at kg/cm for hr for two consecutive days. This.5x c.f.u/g of soil at 3 week of incubation soil was spread in an aluminum tray separately th and then declined to.x c.f.u/g of soil at for each type of soil to a depth of cm. The week of incubation (Fig. ).

3 The Journal of Plant Protection Sciences, 3() : 3-3, June, c.f.u. x /g of Soil FIRST WEEK SECOND WEEK THIRD WEEK FOURTH WEEK FIFTH WEEK SIXTH WEEK STERILIZED SOIL (SEED) SOLARIZED SOIL (SEED) NATURAL SOIL (SEED) STERILIZED SOIL (VC) SOLARIZED SOIL (VC) NATURAL SOIL (VC) Fig. Population proliferation of T. harzianum (in vitro) in different state of soil inoculated with different types of inocula *Each insertion is an average of population of estimated to.x c.f.u/g and.x c.f.u/g five samples taken randomly th of soil at week respectively (Fig. ). Seed: Inoculum multiplied on sterilized wheat Population proliferation was also found better seed (. c.f.u x / g of seed) when T. viride was added to soil through organic VC: Inoculum multiplied on sterilized food based inoculum (Fig.). Inoculum grown vermicompostneem cake ( w/w) on vermicompost % neem cake resulted (. c.f.u x / g of substrate) maximum population at 3 week of incubation in all the three soil and there by declined In case of T. viride seed based inoculum gradually at six weeks of incubation. The result proliferated significantly (P=.5) and reached nd showed that the population of T. viride in highest at week of incubation in sterilized sterilized soil was significantly higher in 3 (.9x c.f.u/g) and solarized soil (3.5x week of incubation with a population of c.f.u/g) whereas in natural soil the population 3.5x c.f.u/g of soil than solarized soil started to decline over the inoculum added to the st (.x c.f.u/g) and natural soil (.3x soil at week (.x c.f.u/g) after incubation th c.f.u/g). By the end of the week the population and decreased up to.9x c.f.u/g of soil at weeks of incubation. In sterilized soil and remained at.5x c.f.u/g,.x c.f.u/g solarized soil the population of T. viride was and 5.x c.f.u/g of soil in sterilized, solarized and natural soil respectively. c.f.u. x /g of Soil STERILIZED SOIL (SEED) SOLARIZED SOIL (SEED) NATURAL SOIL (SEED) STERILIZED SOIL (VC) SOLARIZED SOIL (VC) NATURAL SOIL (VC) FIRST WEEK SECOND WEEK THIRD WEEK FOURTH WEEK FIFTH WEEK SIXTH WEEK Fig. Population proliferation of T. viride (in vitro) in different state of soil inoculated with different types of inocula

4 The Journal of Plant Protection Sciences, 3() : 3-3, June, *Each insertion is an average of population of viride varied insignificantly when added through five samples taken randomly seed based inoculum up to 3 week of incubation Seed: inoculum multiplied on sterilized wheat and then declined to.x c.f.u/g of soil at seed (. c.f.u x / g of seed) week of incubation (Fig. 3) VC: inoculum multiplied on sterilized Seed: inoculum multiplied on sterilized wheat vermicompostneem cake ( w/w) seed (. c.f.u x / g of seed) (T. harzianum:.x c.f.u/g of seed; T. viride: At field condition the population of Trichoderma.x c.f.u/g of seed) spp. differed significantly when applied through VC: inoculum multiplied on sterilized seed based inoculum and the organic food based vermicompostneem cake ( w/w) inoculum (Fig. 3). Population density of T. (T. harzianum: /5x c.f.u/g of seed; T. viride: harzianum significantly increased maximum at 3.9x c.f.u/g of seed) 3 week of incubation. Then population density th The population of Trichoderma spp. was at par between 3 and week of incubation significantly varied when applied to different resulting.x c.f.u/g and.x c.f.u/g of soils with different forms of inocula. The results soil respectively. When seed based inoculum was showed that Trichoderma spp. grown on organic added the population reached peak (3.5x substrates showed a rapid and extensive c.f.u/g) at 3 week of incubation and then proliferation in soil than Trichoderma spp. grown declined gradually reaching to a population of on seed and then the population decreased with.5x c.f.u/g of soil. time. Population densities of Trichoderma in In case of T. viride population was also seed based inoculum may have initially significantly greater in soil amended with increased because of the exudates from seed vermicompost% neem cake than seed based provided the substrates for their multiplication. inoculum. Population reached peak (.x The intimate contact between the mycelium and c.f.u/g) with organic food based inoculum at 3 food base might have enabled the fungi to grow week of incubation. But the population of T. relatively unimpeded through soil. Soil colonization by biocontrol agents is greatly 9 c.f.u. x /g of Soil 5 3 T. harzianum (Seed) T. harzianum (VC) T. viride (Seed) T. viride (VC) FIRST WEEK SECOND WEEK THIRD WEEK FOURTH WEEK FIFTH WEEK SIXTH WEEK Fig 3. Population proliferation of Trichoderma spp. in field soil types with different forms of inocula

5 The Journal of Plant Protection Sciences, 3() : 3-3, June, c.f.u. x /g of Soil STERILIZED SOIL ( HOUR) SOLARIZED SOIL ( HOUR) NATURAL SOIL ( HOUR) STERILIZED SOIL (9 HOUR) SOLARIZED SOIL (9 HOUR) NATURAL SOIL (9 HOUR) STERILIZED SOIL ( HOUR) SOLARIZED SOIL ( HOUR) NATURAL SOIL ( HOUR) 5CM CM 5CM CM 5CM Distance from point of inoculation Fig. Radial spread of T. harzianum in sterilized, solarized and natural soil affected by the presence of suitable organic ability to proliferate in soil, may be due to their substrates in the soil (Garrett 95; Lewis & resistance to soil fungistasis (Lockwood 9). Papavizas 9a). Substrates such as wheat bran Radial spread of Trichoderma spp. in different (Aziz et al. 99), barley, and bark pellets have soil (in vitro) been used to incorporate Trichoderma into soil Results on the spread of the antagonist through with varying levels of effectiveness (Lewis & biomass prepared on vermicompost-neem cake Papavizas 9b; Papavizas 95). Leandro et in sterilized, solarized and natural soil condition al. () found that T. hamatum strain T3 3 showed that after hrs of inoculation the hypha maintains a stable population of c.f.u/g soil of T. harzianum reached to distance of 5 cm from throughout the growing season when added to the point of inoculation in all types of soil (Fig. field in amended compost in strawberry root. The ). In sterilized and solarized soil mycelia of T. result also supported that soil treatment with harzianum spre.ad and proliferated up to 5 cm Trichoderma based bioformulation increases the within 9 hrs and in natural soil it took hrs to population of Trichoderma in soil. The reach 5 cm. The population density was very indigenous populations of Trichoderma present low in non-sterilized soil counting only.x in the soil may also boost up due to nutrient c.f.u/g of soil while in sterilized soil and availability through organic substrates. In the present experiment survival and proliferation of solarized soil contained.x c.f.u/g and Trichoderma spp. in field condition also varied.x c.f.u/g of soil at 5 cm apart from the with respect to inoculum added. In soil, it has point of inoculation with T. harzianum inoculum. been shown that species like T. harzianum can be T. viride also followed the similar pattern of promoted by substrates with a high C:N ratio result though hyphal spread and proliferation of (Kwasna et al. ). Papavizas (9) reported the population was low compared to T. that T. harzianum does not survive well in the harzianum (Fig. 5). In natural soil T. viride rhizosphere of bean and pea seedlings when the reached at 5 cm within 9 hrs and even at hrs seeds are coated with the conidia and when the it only reached up to cm from the point of conidia are applied directly to the soil at day inoculation counting to.x c.f.u /g of soil. before planting. The young hyphae of Whereas in sterilized soil, T. viride reached up to Trichoderma and other antagonists have a unique cm within hrs and the population density

6 The Journal of Plant Protection Sciences, 3() : 3-3, June, 5CM CM 5CM CM 5CM Distance from point of inoculation Fig 5. Radial spread of T. viride in sterilized, solarized and natural soil STERILIZED SOIL ( HOUR) SOLARIZED SOIL ( HOUR) NATURAL SOIL ( HOUR) STERILIZED SOIL (9 HOUR) SOLARIZED SOIL (9 HOUR) NATURAL SOIL (9 HOUR) STERILIZED SOIL ( HOUR) SOLARIZED SOIL ( HOUR) NATURAL SOIL ( HOUR) reached up to.x c.f.u/g of soil in hrs at content support better stand and spread of Trichoderma application compared to silly loam and sandy clay soil with highest population 5cm distance from the point of inoculation. In solarized soil almost similar kind of result was acheived giving a population density of.x during winter months. c.f.u /g of soil in hrs at 5 cm. Literature Cited Lewis and Papavizas (95) reported that for the Aziz NH El-Fouly MZ El-Esssawy AA Khalaf M A. 99 fast growing species of Trichoderma and Influence of bean seedling root exudates on the rhizosphere colonization by Trichoderma Gliocladium, hyphal biomass resulted in lingorum for the control of Rhizoctonia solani. significant proliferation when added with traces Botanical Bulletin of Academia Sinica 3: of food base. Kundusen and Bin (99) found Bose S Jash S Roy M Khalko S Pan S. 5 Evaluation of that radial growth rate and hyphal densities are different isolates of Trichoderma harzianum against soil borne plant pathogens. Journal of quantified for hyphae originating from alginate Interacademicia 9: pellets containing hyphae of the biocontrol Bose S Pan S. 9 An approach to stimulate population fungus T. harzianum. In the present study proliferation and spread of Trichoderma harzianum addition of Trichoderma inocula to soil with in soil ecosystem. Indian Phytopathology : 3-3. organic food base media had given significant Garrett SD. 95 Prelude to biological control. In: Baker, effect on spread of Trichoderma in soil. K. F. and Snyder, W. C. (eds) Ecology of soil-borne It was also found that T. harzianum spread faster plant pathogens. University of California Press, Berkeley, California, pp.-. than T. viride which may be an innate character Knudsen GR Bin L.99 Effects of temperature, soil of the specific species. Sterilized and solarized moisture, and wheat bran on growth of soil supported the easy spread of Trichoderma Trichoderma harzianum from alginate pellets. than unsterilized soil that may be due to less or Phytopathology : -. no competition with other soil microorganisms Kwasna H Sierota Z Bateman GL. Fungal which was in accoance with Bose and Pan communities in fallow soil before and after (9). Wu (9) observed that under natural amending with pine sawdust. Applied Soil Ecology : -. conditions, sandy loam soil with suitable water

7 The Journal of Plant Protection Sciences, 3() : 3-3, June, Leandro LFS Guzman T Ferguson LM Fernandez GE Munnecke DE. 9 Factors affecting the efficacy of Louws FJ. Population dynamics of fungicides in soil. Annual Review of Trichoderma in fumigated and compost-amended Phytopathology : soil and on strawberry roots. Applied Soil Ecology Pan S. 9 Variability in induction of defense response in 35: 3-. Bengal gram (Cicer arietinum) by Trichoderma Lewis JA Papavizas GC. 9a A new approach to species. Journal of Mycology and Plant stimulate population proliferation of Trichoderma Pathology 39(): 3-3. species and other potential biocontrol fungi Papavizas GC. 9 Survival of Trichoderma harzianum introduced into natural soils. Phytopathology : in soil and in pea and bean rhizosphere. -. Phytopathology : -5. Lewis JA Papavizas GC. 9b Proliferation of Papavizas GC. 95 Trichoderma and Gliocladium: Trichoderma and Gliocladuim from alginate pellet biology, ecology, and potential for biocontrol. in natural soil and reduction by Rhizoctonia solani Annual Review of Phytopathology 3: 3-5. inoculum. Phytopathology, : 3(Abstr). Saha DK Pan S. 99 Qualitative evaluation of some Lewis JA Papavizas GC. 95 Effect of mycelial specific media of Trichoderma and Gliocladium preparations of Trichoderma and Gliocladium on and their possible modifications. Journal of populations of Rhizoctonia solani and the Mycopathological Research 3: -3. incidence of damping-off. Phytopathology 5: Wu WS. 9 Effectiveness of soil texture, water content -. and organic amendments on the growth of Lockwood JL.9 Fungistasis in soil. Biological Review Trichoderma spp. and Rhizoctonia solani. 5: -3. Memories of the college of Agriculture National Mughogho LK. 9 The fungus flora of fumigated soils. Taiwan University : - Transactions of the British Mycology Society 5: 59.