A novel method to prepare concentrated conidial biomass formulation of Trichoderma harzianum for seed application

Authors


Correspondence

Chandra Shekhar Nautiyal, Division of Plant-Microbe Interactions, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India. E-mail: nautiyalnbri@lycos.com

Abstract

Aims

To prepare concentrated formulation of Trichoderma harzianum MTCC-3841 (NBRI-1055) with high colony forming units (CFU), long shelf life and efficient in root colonization by a simple scrapping method.

Methods and Results

NBRI-1055 spores scrapped from potato dextrose agar plates were used to prepare a concentrated formulation after optimizing carrier material, moisture content and spore harvest time. The process provides an advantage of maintaining optimum moisture level by the addition of water rather than dehydration. The formulation had an initial 11–12 log10 CFU g−1. Its concentrated form reduces its application amount by 100 times (10 g 100 kg−1 seed) and provides 3–4 log10 CFU seed−1. Shelf life of the product was experimentally determined at 30 and 40°C and predicted at other temperatures following Arrhenius equation. The concentrated formulation as compared to similar products provides an extra advantage of smaller packaging for storage and transportation, cutting down product cost. Seed application of the formulation recorded significant increase in plant growth promotion.

Conclusions

Stable and effective formulation of Trichoderma harzianum NBRI-1055 was obtained by a simple scrapping method.

Significance and Impact of the Study

A new method for the production of concentrated, stable, effective and cost efficient formulation of T. harzianum has been validated for seed application.

Introduction

Agriculture forms an important resource to sustain global economy, and social system involving use of many agrochemicals. However, these agrochemicals used for disease and nutrient management are playing detrimental role towards environment and agricultural lands. Therefore, bioinoculants have become a declared objective of agricultural politics as an alternative to conventional practices for disease and nutrient management throughout the world. As a result, many biopesticide and biofertilizer products are now available on the market. Cost-effectiveness and a number of technological challenges like fermentation, formulation and delivery systems are important factors for the commercial viability of a potent strain. However, developmental cost and poor shelf life may hinder promising micro-organisms to become products. Thus, knowledge of methods for mass production and proper delivery system is a critical impediment to bioagents (Papavizas 1985). It has become a focus of research and industrial development (Papavizas and Lewis 1981; Papavizas 1985; Harman et al. 1991; Jin et al. 1996; Jin and Custis 2011) and has constantly evolved in the search for alternatives to chemical seed treatments for nutrient management and control soilborne plant pathogens. Regardless of the organism used, an important criterion for a successful implementation of bioagent is the preparation of microbial biomass of high population counts with a high level of viability and vigour (Jones and Burges 1998). Formulation of biological control agents like Trichoderma, a readily chosen biocontrol agent for mass production at commercial level depends upon biomass production in a timely and cost-effective way. It involves survival of spores at each processing step, such as harvesting, drying, formulation, storage and delivery (Jin et al. 1992; Adekunle et al. 2001) so that the purpose of production, that is, to multiply efficacious propagules in the greatest quantity in shortest period of time is not defied. Trichoderma, one of the most commonly used fungal antagonists has been mass produced by both solid (Singh et al. 2007; Jegathambigai et al. 2010; Jin and Custis 2011) and liquid fermentation (Jin et al. 1996; Jegathambigai et al. 2010) by manipulating the media composition to improve both biomass production and desiccation tolerance of T. harzianum. Despite the central role of microbial inoculants in sustainable agriculture, their formulation is neglected as compared to fundamental aspects of research. This has resulted in constrains in the production technology of bioinoculants. Thus, this work was carried out to develop a high density, cost-effective method of producing Trichoderma for seed application. T. harzianum MTCC-3841 (NBRI-1055) earlier reported for its biocontrol and PGPR activity (Singh et al. 2007) was used as model organism. The work validates applicability of the production method and usage of the concentrated T. harzianum formulation which to the best of our knowledge has approximately hundred times more live spores than other carrier based products.

Materials and methods

Formulation of the product

Trichoderma harzianum MTCC-3841 (NBRI-1055), a known plant growth promoting isolate (Singh et al. 2007) was obtained from culture collection of Division of Plant-Microbe Interactions, CSIR-National Botanical Research Institute, Lucknow, India. It was grown on potato dextrose agar (PDA; from Hi-Media Laboratories Pvt. Ltd., Bombay, India) at 28–30°C in 90-mm Petri plates and maintained on PDA slants at 4°C. For preparation of the formulation, NBRI-1055 was grown on PDA plate, and sporulated biomass of which was harvested after 7–10 days of inoculation by scrapping from the PDA plates using sterile scrapper. The fungal biomass thus obtained contained negligible amount of mycelia (weight not determined) and essentially consisted of spores (conidia) arising from the aerial conidiophores produced on substrate surface referred to as aerial spores and has been referred to in several studies (Munoz et al. 1995; Nagayama et al. 2007). This biomass yield (approximately 11–12 log units of spores/90-mm Petri plates) and 100 μl of sterile distilled water (SDW) (to maintain approximately 8% moisture) were mixed thoroughly with 10 g autoclaved talc followed by sieving through a 35- to 40-mesh screen which yielded a homogeneous product of concentrated Trichoderma. Screening experiments and shelf life study for this product was carried out at 30 and 40°C in Eppendorf tubes containing one gram of the prepared formulation and sealed with para-film. Talc was autoclaved three times on consecutive days before preparation of the formulation to reduce the level of contaminating micro-organisms. Colony forming units (CFU) of NBRI-1055 in formulation was estimated by serial dilution on Trichoderma selective media (TSM) (Elad et al. 1981) at different time intervals for 210 days. All the autoclaving was carried out at a pressure of 15 lb inch−2 and 121°C temperature for 15 minutes, talc was autoclaved for 20 min under similar temperature and pressure. Three replicates for each treatment were maintained.

Standardization of conditions

Fully sporulated 7 days old culture was formulated as described earlier in the section for screening carrier material (by replacing talc) and optimum moisture percent. The carrier materials screened were charcoal, cow dung, sawdust and vermiculite besides talc, while the moisture percent 0, 2, 4, 6, 8, 10 and 12% v/w were maintained with sterile distilled water in talc for optimizing moisture content of the formulation. Sealed samples were stored at 40 and 45°C for rapid screening. For selection of incubation time, NBRI-1055 was grown on PDA plates and harvested on different days. The strain produced concentric rings of densely sporulated regions and the inner most concentric ring of NBRI-1055 growth was used to harvest spores by suspending spores from an 8-mm agar bit cut from this region on 3rd, 5th, 7th, 10th, 15th, 20th and 30th day of inoculation. Spores from the agar bit were suspended in 5 ml SDW and its temperature tolerance was determined at 60°C on water bath by taking its CFU at different time intervals. Three replicates were maintained and sampling was carried out every 1 h for 5 h.

Root colonization and biocontrol activity of the product

Root colonization of the product was determined in sterilized vermiculite. Before the colonization experiments, the dilution of the product most suitable, providing the minimum required inoculum and practically applicable volume was determined by suspending the product in SDW in different ratios and taking its CFU. Maize seeds were surface sterilized as described earlier (Nautiyal 1997) by gently shaking (80 rev min−1) on a reciprocal shaker at 28°C with 70% ethanol (5 min), 20% bleach Chlorox (10 min), followed by three rinses in SDW. After surface sterilization, 1 kg seeds was treated with 0·1-g product suspended in 100 ml SDW containing 1·5% gum arabic (sticker). Control seeds (un inoculated) were similarly treated with sterilized talc. Four seeds from each treatment were used to determine CFU seed−1. The Trichoderma colonization was determined every week for 1 month in maize roots. Roots were thoroughly washed with tap water for 2 min to remove all loosely adhering soil particles, followed by washing with sterile 0·85% saline Milli Q water (MQW). The roots were then macerated in 0·85% saline MQW with a mortar and pestle. Serial dilutions of the homogenate were then plated on TSM and incubated at 30C for 2–3 days (Nautiyal 1997). Biocontrol activity of the formulation was determined by dual culture technique according to Dennis and Webster (1971). A suspension of 1 : 100 dilution of the formulation, prepared in SDW was spotted (25 μl) on PDA with Rhizoctonia solani on opposite sides to check the biocontrol activity.

Field application

Field trial using 0·1-g product applied to 1 kg chickpea seeds as described previously, variety DCP-92-3 was carried out at NBRI fields, plot size 1·5 m × 1·5 m in three replicates. Six rows of 25 plants each were maintained and one plant from each row was randomly selected for observations. Root colonized Trichoderma and the plant growth parameters were determined at the time of harvesting. Two-way analysis of variance (anova) and Duncan's multiple range test (DMRT) was performed to determine difference between the treatments using Statistical Package for Social Sciences (spss) software (ver. 16, SPSS, Inc, Chicago, IL, USA).

Accelerated shelf life testing (ASLT)

Accelerated shelf life testing (ASLT) was applied to predict shelf life of the product using CFU obtained at two storage temperatures (30 and 40°C). Reaction rate constants (K = (2·303/t) [log (C0/Ct)], where K = Rate Constant; C0 = initial CFU g−1 and Ct = CFU g−1 at time t, were determined at 30 and 40°C using first 3 month data. The average rate constant value obtained from 3 month data was further used to predict CFU of the product for 12 months. The predicted values were validated by the experimental data at 30 and 40°C. Using the CFU data from 30 and 40°C, CFU at 20, 25 and 35°C was calculated following Arrhenius equation, Log (KT2/KT1) = Ea/2·303R [(T2-T1)/(T1 × T2)], where K = Rate Constant; Ea = Energy factor, R = Gas' Constant; T = Temperature at Kelvin Scale. The energy factor Ea was calculated using the average rate constants determined using 12-month experimental CFU data at 30 and 40°C and used in Arrhenius equation to predict CFU of the product at a given temperature and time.

Results

Formulation of the product and optimization of the conditions

The product obtained from the scrapping process yielded a homogenous mixture of talc and Trichoderma spores with 8% moisture and an initial count of 11–12 log10 CFU g−1 formulation.

Result of screening different carrier materials carried out at 40 and 45°C over a period of 1 month shows talc and cow dung as two promising carriers (Fig. 1). Suitable moisture content for the formulation was selected by determining survival of the inoculum at 40 and 45°C in the presence of different moisture content. It was observed that addition of 4–8% of moisture was best suited for preparation of this product (Fig. 2). Effect of incubation on temperature tolerance of spores was determined at 60°C to optimize the time for harvesting the spores for preparation of the formulation. Spores harvested from PDA plates after 10 days incubation were highest in productivity giving CFU of 9·2 log10 ml−1. These were also the most temperature tolerant showing survivability up to 5 h at 60°C (Fig. 3). Figure 3 shows survivability of spores exposed at 60°C in water bath over a period of 5 h. The CFU of the spore suspension after 5 h were 0·8, 2·5, 3·6, 3·4, 2·2 and 1·7 log10 CFU ml−1 harvested from 5, 7, 10, 15, 20 and 30 days old cultures, respectively. Thus, the most appropriate time of making the harvest to get maximum spores per plate with highest survival potential was found to be 10 days after inoculation of PDA plates for NBRI-1055.

Figure 1.

Screening of different carrier materials talc, charcoal, vermiculite, sawdust and cowdung at (●) 40 and (○) 45°C for T. harzianum NBRI-1055 concentrated formulation.

Figure 2.

Screening of percent moisture 0, 2, 4, 6, 8, 10 and 12% in talc at (●) 40 and (○) 45°C for T. harzianum NBRI-1055 concentrated formulation.

Figure 3.

Effect of harvest time on temperature tolerance (60°C) of T. harzianum NBRI-1055 spores. (●) Day 5; (○) Day 7; (▼) Day 10; (△) Day 15; (■) Day 21; (□) Day 30.

According to the optimization results, 10 days old culture, 8% moisture and talc as the carrier material was selected for the formulation of the product and used in further field and in vitro evaluations. The product thus obtained from the scrapping process yielded a homogenous mixture of talc and Trichoderma spores with 8% moisture and an initial count of 11–12 log10 CFU g−1 product.

Application and effectivity of the product

In order for the fungal bioinoculant to work for soilborne diseases or exert plant growth promotion, living and actively metabolizing fungi must be able to colonize the host plant root. This minimum bioinoculum load on seeds should be provided by the product at the time of application. Substrate-based product prior to seed application is suspended in water (may contain sticker like gum arabic) that dilutes the product for easy spreading and attachment. Table 1 shows the CFU of the concentrated Trichoderma product obtained on its dilution with water in different ratios. A 1 : 100 dilution of the product was found to be suitable for seed application that would provide an increased and practically applicable volume to the product besides the minimum inoculum load. Application of 100 time diluted product at the rate of 10 g product 100 kg−1 seed resulted in 3–4·6 log10 CFU seed−1 (pea and maize seeds). The results thus show the practical applicability of the product for application in agricultural, horticultural and consumer settings.

Table 1. CFU of T. harzianum NBRI-1055 concentrated formulation when suspended in different dilutions
S.NoDilutionTrichoderma CFU ml−1
  1. Values are mean of three replicates.

1Conc. Formulation1·9 ± 0·10 × 1011
21:101·9 ± 0·10 × 1010
31:252.1 ± 50·05 × 109
41:501·15 ± 0·05 × 109
51:1002·35 ± 0·05 × 109

Competence of the formulation was evaluated in vitro by determining its biocontrol activity and ability to colonize maize root over a period of 4 weeks. Seed application of the prepared formulation as described in earlier section gave an 3·79 log10 CFU of NBRI-1055 per seed which was maintained at 4·25, 4·40, 4·35 and 4·46 log10 CFU g−1 of maize roots after 1st, 2nd, 3rd and 4th week of application respectively. These results suggest that NBRI-1055 maintains its competence after scrapping and formulation into concentrated product. Biocontrol activity of the product was observed which resulted in 78% inhibition of R. solani after 7 days of inoculation on a dual culture plate.

Field application

Plant growth promotion and competence of the product under field condition was studied at NBRI fields and compared with a positive and a negative (no inoculum) control. The positive control used was an NBRI-1055 formulation prepared using pearl millet as substrate as described earlier by Singh et al. (2007). Significant differences in plant growth parameters as compared to negative control were observed (Table 2). The Trichoderma CFU in roots of the treatment with concentrated formulation was recorded to be 4·17 ± 0·3 log10 CFU g−1 root as compared to 1·78 ± 0·47 and 2·59 ± 0·3 log10 CFU g−1 root in negative and positive controls, respectively (Table 2).

Table 2. Plant growth promotion and root colonization by T. harzianum NBRI-1055 concentrated formulation in chickpea grown in micro-plots
 ControlConcentrated formulationSubstrate-based formulation
  1. Values are mean of six replicates; Different letters shows significant difference at P = 0·05 using DMRT.

Shoot length (cm)51·2 ± 1·37a54·0 ± 2·17a52·8 ± 1·68a
Root length (cm)8·83 ± 0·6b10·41 ± 0·96a8·33 ± 0·55b
Number of pods35 ± 5·9b62 ± 5·5a66 ± 7·9a
Number of nodules9·16 ± 1·3c21·5 ± 2·3a16·0 ± 2·3b
Number of lateral Roots23 ± 3·5a16·33 ± 1·2b16·5 ± 0·8b
Plant dry wt. (g)2·72 ± 0·8a3·29 ± 1·5a2·43 ± 0·6a
Seed wt/10 plants (g)83 ± 4·2a110 ± 3·9b107 ± 5·1b
Weight/100 seeds (g)15·4 ± 1·7a14·1 ± 1·2b15·5 ± 2·8b
CFUg−1 root6·0 ± 3 × 101c1·5 ± 2 × 104a3·94 ± 2 × 102b

Accelerated shelf life testing (ASLT)

Rate constant (K) determined from the first 3 month CFU data at 30 and 40°C (0·529 and 1·850) was used to predict the shelf life of this product for 30 months at their respective temperatures and validated by the experimental data at 30 and 40°C for 12 months (Table 3). Using the CFU data from 30 and 40°C, CFU at 20, 25 and 35°C was calculated following Arrhenius equation. The energy factor (Ea) was calculated using K values at 30 and 40°C calculated by averaging 12-month data (K30 = 0·532, K40 = 1·977). The predicted log10 CFU g−1 of the product at 20, 25 and 35°C extrapolated using Arrhenius equation is given in Table 3. From the experimental and extrapolated data, shelf life of the product is predicted to be at least 2 years at 20°C, 15 months at 25°C, 9 months at 30°C, 5 months at 35°C and 3 months at 40°C. Kinetic data for survival, experimental data and kinetics of change in product CFU at 30 and 40°C are shown in Fig. 4. Fitting of a first-order reaction seems to satisfactorily predict variations in CFU with time. Average rate of decay of product (K value) calculated from 12-month CFU data, that is, K month−1 was 0·532 log10 CFU g−1 month−1 at 30°C and 1·977 log10 CFU g−1 month−1 at 40°C.

Figure 4.

Changes in Kinetics of concentrated formulation of T. harzianum NBRI-1055 CFU at 30 (◊) and 40°C (□) storage temperatures. Squares and triangles represent experimental data, while continuous lines show fitting to a first-order kinetic model.

Table 3. Experimental and Predicted shelf life of T. harzianum NBRI-1055 concentrated formulation following Arrhenius equation at different storage temperatures
MonthsExperimental*Predicted shelf life (Log10 CFU g−1) at different temperatures
30°C40°C20°C25°C30°C35°C40°C
  1. * Experimental data were gathered over the period of 12 months through practical implementation of the experiment. Values are average of 3 replicates ± SD.

  2. † Predicted data were calculated using experimental CFU data of 30 and 40°C.

  3. ‡ Indicates viability duration of the product at the respective temperatures.

011·49 ± 0·1111·61 ± 0·0511·4111·4111·4111·4111·38
111·85 ± 0·0410·33 ± 0·0111·2111·2911·2610·9610·80
310·80 ± 0·089·20 ± 0·0011·1711·0610·8310·069·20
4ndnd11·2510·9410·579·618·39
5ndnd11·3310·8310·349·167·59
69·77 ± 0·187·32 ± 0·0011·0610·7110·118·716·79
99·08 ± 0·005·71 ± 0·0210·8910·339·427·364·38
128·69 ± 0·123·25 ± 0·0210·7210·018·746·011·97
15ndnd10·559·678·054·63nil
18ndnd10·388·627·363·31nil
24ndnd10·048·285·990·61nil
30ndnd 9·707·934·62nilnil

Discussion

After extensive evaluations to identify a microbial pesticide or plant growth promoter and formulating a new product many obstacles arise later in the market. These include development of formulations that are compatible with existing application technologies, protect biological actives from stress, ensure viability, remains high or relatively unchanged after storage and transport under ambient conditions, ensure prolonged contact between microbial actives and target, ensure moisture availability in the field and are cost-effective (Lyn et al. 2010). The method described is envisaged to have applicability for commercial level production in a cost-effective way and have following advantages.

Compatibility with the existing application technology

Reduction in space, electricity and raw material requirement, initial/permanent inputs, transportation and downstream processing steps.

The product is less prone to contamination

For a commercial formulation to be successful, it is crucial that the agent survives and remains active in the carrier used for a period enough for it to become established and elicit a desired effect (van Elsas and Heijnen 1990) and help buffer an organism from adverse conditions (Hasan and Ayres1990). Besides, the carrier selected should effectively deliver the promising inoculum to the plants. For NBRI-1055, although cow dung showed maximum survivability closely followed by talc, talc was selected for the reason it is chemically defined, easily available, being inorganic it is less prone to contamination and will be of international acceptance. Traditionally, peat has been used as inoculant carrier, others include oils, inorganic clays (talc and vermiculite), cellulose derivatives like hydroxyl ethyl and carboxyl methyl cellulose (Digat1989), polymers like alginate beads, crop residues like saw dust, rice husk and wheat bran have also been used as carrier (Pandey et al. 2000; Arora et al. 2008).

High spore viability is an important aspect of the economics of the production of Trichoderma bioinoculum in which moisture is an important factor that determines the longevity of a formulation. Moisture probably makes the conidia more vulnerable to stress by stimulating unwanted growth or germination during storage, which can be controlled by both reducing and maintaining the water activity at or near an ideally low level (Connick et al. 1996; Duan et al. 2008; Lyn et al. 2010). Thus, maintaining the moisture to low levels will keep the conidia intact and increase its shelf life. Preservation of micro-organisms by drying has been practiced since the beginning of this century as a cheaper and less costly alternative to freezing. However, rapid drying retains only 10–40% of these conidia to remain viable (Harman et al. 1991; Jin et al. 1991; Jin and Custis 2011; Yanez-Mendizabal et al. 2012). Besides, during dehydration there is an inherent loss of viability owing to damage in cell membranes and alterations in the DNA or RNA and intracellular proteins (Lievense and van't Riet 1993). The shelf life of dried spores of T. harzianum has been shown to be extended considerably by storage in sealed containers at low moisture contents, for example less than 4% (Pedreschi and Aguilera 1997; Pomella et al. 2007). The method described in this work provides flexibility to adjust moisture content not by dehydrating but by addition of moisture. This method also decreased downstream loss of spore viability and retained high quality of the product by avoiding any contamination occurring during drying and homogenization of fermented biomass.

Trichoderma produces spores, which are quite resistant to hostile environmental conditions such as heat, dryness, unfavourable pH or nutrient conditions. Use of the spores arising from the aerial conidiophores produced on substrate surface of PDA plates in the present study is justified and supported by earlier reports of aerial and dried spores being more resistant to stress and have improved shelf life than submerged cultures (Munoz et al.1995; Aguilera 1997; Nagayama et al. 2007). Nagayama et al. (2007) have shown that submerged spores had some common properties with aerial spores, but differed in surface topology and internal organization making aerial spores maintain high viability under dry storage conditions retaining its biocontrol activity, whereas the viability of submerged spores was found to be reduced. A wettable powder, formulation prepared from aerial spores, has been reported to maintain high viability and biological control activity for 6 month at room temperature, Nagayama et al. (2007). Waghunde et al. (2010) have also reported higher spore count in 10 days old culture than in 7 days old culture of T. Viride in accordance to our results in selection of 10 days old culture spores for concentrated formulation preparation.

The presence of living Trichoderma in the rhizosphere during early part of the plant life cycle ensures protection at the time of root establishment, when the plant is most vulnerable to soilborne fungal diseases. To ensure rhizosphere colonization seed coating should provide a minimum of 3–4 log10 CFU of the inoculum propagules per seed (Jensen et al. 2000; Harman et al. 2004). Harman et al. (2004) have reported a 4·2 and 4·23 log10 CFU of T. harzianum g−1 maize roots after 5 and 15 days, respectively, using a commercial product which is in accordance with results obtained with the concentrated formulation of this study. In an earlier report by Singh et al. (2007) have shown an increase in 9–177% yield with NBRI-1055 propagated on different agro wastes. The results showed that the efficiency of the concentrated formulation prepared by scrapping was at par with the formulation prepared using pearl millet as substrate. This indicates that the application and method of preparation of the formulation discussed in this work is worth recommendation for both industries and farmers. This recommendation may be further strengthened by the shelf life studies.

Shelf life is considered a pivotal factor determining the commercial success of a biocontrol agent as well as its field efficacy (Feng et al. 1994). High temperatures and moisture contents are important environmental conditions affecting the rate of deterioration of biological systems (Nelson and Labuza1994). Thus testing product stability at high temperatures and/or high moisture contents is widely used to rapidly assess the storage life of foods and pharmaceuticals Labuza and Schmidt (1985). In the present study, it was possible to gather data in 3 months time instead of the prohibitive time (e.g. several months) to predict stability of the product at different temperatures using ASLT. The applicability of ASLT on this product was well proved by the experimental and predicted data at 30 and 40°C. Pedreschi and Aguilera (1997) have used first-order kinetic model to describe the loss of relative viability with time in T. harzianum under storage conditions. In the present study, moisture control and use of aerial spores were probably the important factors that resulted in longer shelf life of the product.

Thus, in the present study, a method of preparing Trichoderma-based formulation without use of substrate fermentation, solid or liquid, and requiring none of the subsequent downstream processing involving elaborate, time taking and contamination prone extensive technological challenges has been described and validated. In addition, as the preparation is carried out in aseptic conditions in one step after harvest, it is less prone to exposure to environmental contamination and loss of live spores. An extrapolation and calculation of cost effectivity demonstrated that the method is cost-effective on initial inputs, space, electricity, labour and transportation as compared to the existing methods used in industry. Thus, the described process may bring down the cost of production to be commercially viable.

Acknowledgements

The study was supported by in-house project P-0075 of CSIR-National Botanical Research Institute.

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