All the isolates (T-1, T-2, T-3, T-4, T-5, T-6, T-7, T-8, T-9, T-10, T-11, T-12, T-13, T-14, T-15, T-16, T-17, T-18, T-19 and T-20) of T. harzianum were collected from the stock cultures of the Plant Pathology Laboratory of the Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur, Bangladesh. Four isolates of the test pathogen R. solani were isolated from pea, cotton and bush bean. “IPSA motorshuti-3” variety of pea was used as the test plant.

Infected root, leaf, petiole, and stem tissues of pea, cotton and bush bean were collected from the field laboratory of BSMRAU and washed in distilled water to remove sand and soil particles. The specimens were cut into small pieces (5 mm) along with healthy and dead tissue and surface sterilized with 0.1% NaOCl₂ for 2 min and rinsed in sterilized water for three times. The plant pieces were blotted dry to remove excess water and plated on water agar. The characteristic colony of R. solani was transferred to fresh acidified PDA (pH 4.5). These isolates were purified by selecting a single hyphal tip (less than 1.5 mm) of each isolate growing on PDA to obtain pure cultures. All isolates were maintained in the short term on PDA slants and on colonized wheat seeds for longer-term storage. Each isolate was arbitrarily named with capital letter codes and a serial number (Table 1). Selected isolates were inoculated onto four replicate PDA plates using 5-mm-diameter mycelial disks taken from the margin of a 3-day-old PDA culture, and the plates were sealed with parafilm strips and incubated in airtight plastic containers at room temperatures (25 ± 2 °C). After 60 h of incubation, observation on several cultural characteristics such as colony colour, mycelial density, zonation number in the culture and sclerotial number per plate was recorded. The mycelial density was rated into I to IV scale as described by Dey et al. [12].

Wheat grains were soaked in water for 24 h and excess water was drained off. About 100 g of water-soaked grains were taken into a 500-ml Erlenmeyer flask, sealed by cotton plug and were sterilized for 50 min at 121 °C under 15-pound pressure in a autoclave on three consecutive days [13]. Inocula of each isolate were prepared in separate flasks. Sterilized wheat grains were inoculated with 10 disks (5 mm) obtained from the actively growing margin of 4-day-old PDA cultures of R. solani. After 10–12 days of incubation at 25 ± 2 °C in the dark, completely colonized wheat grains were air-dried at laboratory (25 ± 2 °C) temperature and stored at 4 °C until use [14].

Earthen pots were filled with sterilized soil at the rate of 1 kg/pot. Wheat grain inoculum (WGI) of each isolate of R. solani was thoroughly mixed with the soil at the rate of 20 g/kg soil. The experiment included an uninoculated control where only sterilized soil was used. Seven pea seeds were sown in each pot and grown in the net house. Pre-emergence and post-emergence seedling mortality was estimated at 7 and 30 days after sowing. The causal agent of pre- and post-emergence seedling mortality was confirmed after re-isolation of the pathogen from the ungerminated seeds and the infected roots of pea, respectively.

A total of 20 isolates of T. harzianum were tested for antagonistic activity against R. solani to select the most effective antagonist as biological control agent of R. solani. Mycelial disks (5 mm in diameter) were cut from the edge of a 4-day-old colony of T. harzianum and R. solani, and were placed simultaneously on the edge of each PDA plate at opposite directions. Four replicate plates were used for each isolate. The plates that received only disks of R. solani served as control. The plates were incubated in the laboratory at room temperature (25 ± 2 °C). Inhibition percentages of the R. solani were calculated based on the growth of the pathogen on PDA plates following the formula suggested by Sundar et al. [15].

Three fungicides, namely Bavistin 50 WP (Carbendazim, methyl-benzimidazol-2-yl carbamate) (BASF Bangladesh Limited, Dhaka, Bangladesh), Ridomil Gold MZ (Metalaxyl 4% + Mancozeb 64%, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester) (Syngenta Bangladesh Limited, Dhaka, Bangladesh), and Provax-200 (Carboxin, 5,6-dihydro-2-methyl-1,4-oxathin-3-carboxanilide) (Hossain Enterprise CC Limited, Dhaka, Bangladesh) were tested against colony growth of R. solani. The fungicides were chosen for the experiments since they are widely used in conventional cultivations, mainly to control soil-borne pathogens, and because they well represent four different fungicide families. Fungicides were added at a concentration of 100, 250 and 500 ppm in an autoclaved PDA medium following poisoned-food techniques [16]. A 5-mm-diameter agar disk of test fungi was cut from a 4-day-old culture and placed in the centre of petri plates containing different concentrations of fungicides. There were four replicates of each treatment. The plates without fungicides served as control. The inoculated plates were incubated at 25 ± 2 °C. The radial growth was recorded after 7–10 days of incubation when the fungus in control plates covered the plates completely. The percent inhibition (PI) of the fungus over the control was calculated using the formula of Sundar et al. [15].

An extract from mustard oilcake, sesame oilcake, wheat meal, maize meal, chickpea meal, rice bran and tea waste was prepared by adding 100 g of each in 1000 ml water and fermenting them for 2 weeks in earthenware pot wrapped with a polythene bag. The extracts were filtrated through cheese cloth and added separately to the unautoclaved PDA medium at concentrations of 1.0 and 2.0%. The media were autoclaved at 121 °C for 15 min and then poured (approximately 15 ml) into 9-cm-diameter petri plates. After solidification, each plate was inoculated with a 5-mm mycelial disk of a 4-day-old colony of R. solani. The inhibition of radial growth was computed using the same formula of Sundar et al. [15].

A field experiment under artificially inoculated conditions with the most virulent R. solani isolate on pea plant was conducted to determine the individual and integrated effect of the most effective T. harzianum isolate, an organic amendment and a fungicide on seedling mortality in pea. To do this, we selected T. harzianum isolate T-3, mustard oilcake and Provax-200 from our preliminary screening studies and estimated their effect on seedling mortality caused by R. solani RS10 and yield improvement in pea from November 2009 to March 2010 in the experimental field of the BSMRAU. We grew pea plants for this purpose in a non-sterile field soil. This experiment included following eight treatments:

• • T₁ = sowing of pea seeds in field soil (control 1);
• • T₂ = inoculum of R. solani RS10 + sowing of pea seeds in field soil (control 2);
• • T₃ = inoculum of R. solani RS10 + sowing of Provax-200-treated pea seeds in field soil;
• • T₄ = inoculum of R. solani RS10 + inoculum of T. harzianum T-3 + sowing of Provax-200-treated pea seeds in field soil;
• • T₅ = inoculum of R. solani RS10 + inoculum of T. harzianum T-3 + sowing of pea seeds in field soil;
• • T₆ = inoculum of R. solani RS10 + inoculum of T. harzianum T-3 + mustard oilcake + sowing of pea seeds in field soil;
• • T₇ = inoculum of R. solani RS10 + mustard oilcake + sowing of pea seeds in field soil;
• • T₈ = inoculum of R. solani RS10 + inoculum of T. harzianum T-3 + mustard oilcake + sowing of Provax-200 treated-seeds in field soil.

Preliminary experiments were carried out to determine the range of fungicide concentrations, by exposing R. solani to different concentrations until observing a mycelial growth inhibition lower than 100%. In vitro studies showed an absolute inhibition of mycelial growth of R. solani at doses of 250 and 500 ppm of Provax-200. However, the fungicide concentration for the seed treatment used was 200 ppm. The seed treatment with Provax-200 was done by thoroughly mixing 100 g of seed, 0.02 g of fungicide (200 ppm) and a small amount of distilled water in a conical flask. Inocula of T. harzianum T-3 were prepared by growing on sterilized wheat grain following the same procedure as described for R. solani. The individual plot size was 1.5 m × 1 m and the plot-to-plot distance was 0.5 m. Each plot was prepared by a good tillage. The inoculum of R. solani isolate RS10 was mixed with soil in relevant microplots at the rate of 94 g/m² soil and moistened to about 50% water-holding capacity. After 21 days, the WGI of T. harzianum T-3 at the rate of 50 g/m² was mixed thoroughly in the soil of selected microplots. Mustard oilcake was mixed in the soil of treatment microplots at the rate of 200 g per plot 7 days before sowing. Seeds were sown on the same days. Pre-emergence and post-emergence seedling mortality was estimated at 7 and 30 days after sowing. Seed weight and yield were recorded after harvest.

In vitro studies were done using a completely randomized design, each treatment with four replications. The field experiment was conducted in Randomized Complete Block Design with eight treatment combinations replicated four times. Data were analysed statistically using the MSTAT-C computer progam. The means were compared using the Fisher LSD (P = 0.05) test [17].

A number of isolates of R. solani were isolated from different crop fields at BSMRAU, of which four were selected for cultural characterization and pathogenicity tests (Table 1). The vegetative hyphae of R. solani isolates were mostly brown in colour and had dense growth with zonation in culture. Brown is the diagnostic colour for vegetative hyphae of R. solani [18]. Zonation, the phenomenon of alternation of periodic zones, has been shown to vary among isolates [19]. The isolate RS10 has three zonations and others have two zonations. The number of sclerotia formation has also been shown to vary among isolates. The highest number of sclerotia/plate was observed in the R. solani isolate RS10, while the lowest was observed in RS1. The pathogenicity test of the four selected isolates of R. solani, namely RS1, RS3, RS7and RS10, was done in pea plants in the pot experiment. All the isolates of R. solani were highly pathogenic to pea, where RS10 showed the highest (100%) and RS1 showed the least (66.67%) seedling mortality (Table 1). Control pots with sterile soil without inoculum of R. solani showed no mortality of seedlings.

The antagonistic activity of 20 isolates of T. harzianum was tested against R. solani RS10 on PDA by dual culture assay (Fig. 1). All isolates of T. harzianum were highly aggressive in their antagonistic effect, showing more than 60% inhibition of the radial growth of R. solani compared to the control. The percent inhibitory effect among T. harzianum isolates ranged between 77 to 65%, where the highest inhibition was recorded for isolate T-3 and the lowest was for the isolate T-10. Dual petri plate marriage of Trichoderma with pathogen R. solani endorsed the mycoparasitic antagonism of T. harzianum isolates.

Bavistin 50 WP was the most effective fungicide, completely inhibiting the radial growth of R. solani, even at the lowest concentration (100 ppm) (Fig. 1). Provax-200 was identical to Bavistin 50 WP in inhibiting R. solani at higher doses such as 250 ppm and 500 ppm. Media amended with 100 ppm of Provax-200 caused only 83.61% inhibition of the mycelial growth of R. solani. Ridomil was found to be significantly inferior in inhibition of the radial growth of R. solani among the tested fungicides. At 500 ppm concentration, Ridomil caused only 38.74% inhibition of the mycelial growth of R. solani.

In vitro evaluation of organic amendments revealed that mustard oilcake, sesame oilcake and tea waste significantly inhibited the colony growth of R. solani compared to control plate (Table 2). Mustard oilcake showed more hyphal inhibition (60.28%) of R. solani than sesame oilcake (49.17%) and tea waste (43.89%) at the rate of 2% concentration. Wheat meal, rice bran and maize meal at the rate of 1% concentration showed no effect on radial growth of R. solani, while at 2% concentration showed a very insignificant inhibition of the fungus.

The individual and integrated effects of T. harzianum T-3, mustard oilcake and Provax-200 were evaluated in the experimental field in controlling seedling mortality and increasing seed yield in pea. The treatments were tested in non-sterilized field soils. When the soil samples were collected from these plots, the density of R. solani surviving in the soil was moderate, i.e. R. solani was present in 55 out of 96 soil samples determined using the buckwheat stem colonization method [20] and approximately 56% of isolates were pathogenic to chick pea. Therefore, the soil-borne inoculum of the natural population of R. solani contributes to some extent (7.57%) to the seedling mortality in pea plants (T₁) (Table 3). However, the highest percent seedling mortality was recorded in plots where field soil solely received the WGI of R. solani (T₂, pathogen-inoculated control). Treatment with T. harzianum, mustard oilcake and Provax-200 had substantial positive effect on the reduction of percent pre- and post-emergence seedling mortality in pea over controls (Table 4). Consequently, the lowest total percent seedling mortality (4.01%) was recorded in plants receiving a combined treatment with T. harzianum T-3, mustard oilcake and Provax-200 (T₈). The next lowest percent disease index was observed in plants where mustard oilcake was applied solely (T₇) or in combination with inocula of T. harzianum T-3 (T₆) to the field soil. Similarly, a significant variation among the treatments was observed for 1000 seed weight and seed yield. The highest yield was observed in T₈, where integrated treatments with T. harzianum T-3, mustard oilcake and Provax-200 were incorporated; the lowest was in control plots (control 2; T₂), where untreated pea seeds were sown in field soil inoculated with R. solani isolate RS10. Other treatments (T₃, T₄, T₅, T₆ and T₇) showed a statistically similar effect on 100 seed weight and seed yield.