Abstract: Axenic rhino base inocula of indigenous Glomus macrocarpum and Azospirillum brasilense were prepared to study the effect of their separate and combined inoculations on wheat growth. Inoculations either separate or in combined form of the inocula at 10, 20, 40 g/200 g in wheat vars. Blue Silver variety showed a significant increase (p<0.0011 in VAM infection (%), fresh weight, thousand grains weight (TGW) and dry weight (p<0.05) at the third dose (40 g/200 g soil) whereas the increase in plant height and grains number/head found non significant at the same dose as compared to the control treatments (00 g/200 g soil). The above inoculations in any form never showed adverse effect on wheat growth. In separate inoculations, the response of G. macrocarpum was found to be dominated over the A. brasilense. The combined form of the inocula always found highly effective for the aforesaid parameters in respect of the control treatment indicating a mutual beneficial interaction between them. Such reactions of the inocula on wheat growth are being reported for the first time from Sindh, Pakistan.
Introduction
Phosphorus (P) and Nitrogen (N) are the principal nutrients for plants, their optimum uptake lead toward the better growth and yield. Some effective soil micro-organisms like species of VAM and Azospirillum greatly enhance their mobilisation in crop plants (Baldani et al., 1983; Harley, 1989). VAM are symbiotic soil borne fungi, increase the absorptive area of host roots by means of their extra metrical mycelia and greatly enhance the mobilisation of "P" to the host plants which results in the increase of yield many folds (Abbott and Robson, 1984; Diederichs and Moawad, 1993). Azospirillum are rhizotrophic bacteria usually present as a free living in rhizospheric soil of cereals or in association with their roots. They efficiently fix and mobilise the atmospheric nitrogen especially in N-poor and micro aerobic soil (Day et al., 1975; Boddey et al., 1986). Azospirillum species also induce the mobilisation of various nutrients from soil to plants by the abundant root hairs formation which leads toward the better growth and yield (Martin and Glatzle, 1982; Kapulnik et al., 1987). Therefore, the species of Azospirillum could be utilised to enhance the uptake of biologically fixed nitrogen in wheat roots which lacking the root nodulating bacteria (Rhirobium spp.) as found in leguminous plants (Okon and Baker, 1987; Mertens and Hess, 1984). According to the some reports (Pacovsky, 1988; Subba Rao et al., 1985) growth and yield of crop plants particularly cereals could be enhanced effectively by increasing the uptake of "P" and "N" from soil after the combined inoculations of VAM and Azospirillum species. However, the studies on the interactions between VAM and Azospirillum species and their effect on plant growth have received very little attention in the world. In Pakistan, such dual inoculations have not ever been carried out to increase the growth and yield of wheat. This research work deals for the first time in Pakistan, the production of axenic rhizobase inocula of indigenous G. macrocarpum Tulasne and Tulasne and A. brasilense (wild type), their separate and combined inoculations to wheat var. Blue Silver in pot condition.
Materials and Methods
Production of axenic rhizobase VAM inocula: The spores of VAM fungi were extracted from the rhizospheric soil samples (100 g) of wheat, rowing in field condition by the sucrose centrifugation method. Of which G. macrsocarpum. Tulasne and Tulasne was selected and identified by following Schenck and Perez (1990) and Morton and Benny (1990). Its viability was confirmed by vital stain method (Menge and Timmer, 1982). A single viable and surface disinfected (Menge, 1984) spore of G. macrsocarpum. Tulasne and Tulasne was aseptically placed in close proximity with fine feeder roots of 6 days old seedlings of wheat, grown on water agar slant at 1 seedling/slant. At the site of spore placement 0.5 ml of 01 percent aqueous carboxymethyl cellulose solution (BOH-Bngland, viscosity 1500 f 400 centipoise at 20°C) was added as an adhesive (Hepper, 1981; Ferguson and Woodhead, 1982). Ten days old VAM infected wheat seedlings were aseptically transplanted from slants to surface disinfected {with (Na OCl)2 V/V} clay pots at 3 seedlings/pot of 15 cm height with 12 and 8 cms top and bottom diams respectively containing 2 kg steam sterilised (under 1:1 kg/cm2 at 121°C for 2 separate 1-hours periods) sandy clay loam soil (true density 2.66 glcc, pore space 43 per cent and pH level 7.2) supplemented with vermiculite at 1.5 g /100 g soil. The transplanted pots were placed in screen house under natural condition along with 3 sets of control series by complete randomised design method. The seedlings were watered with sterilised distilled water and during early 3 months of growth 1/2 strength of Hoagland solution (Hoagland and Arnon, 1950) excluding the "P" ingredient was also added to the each soil pot at 250 ml I pot at 6 days interval (Menge, 1984). On maturity, the plants were harvested from the soil level and substrata of the pots were collected as mother culture. If found necessary the culture was further multiplied separately by inoculating the same spores in pots. The 80 to 90 percent VAM infected roots and root lets, present in the mother culture were sieved out (using a sieve mesh of 350 μm pore size) as axenic rhizobase VAM inocula with adhered soil particles, hyphae and vermiculites. The inocula was preserved at 5±2°C in polyethylene bags. Production of axenic rhizobase inocula of A. brasilense:
A. brasilense (wild type) was isolated by serial dilution plate method (Warcup, 1955) on BLCR selective medium (Bashan and Levanony, 1985) from the same rhizospheric soil sample that was used to extract the VAM spores. Its identification was based on morphology (motile, showing undulating movement, straight to curve rods with slightly pointed ends), colonial characters on BLCR medium (dark pink round colony of 0.5 to 1 mm in diameter with protruding ridges, showing gram negative reaction) and on diagnostic biochemical tests (Tarrand et al., 1978). An aqueous suspension was made from the bacterial culture containing 2.3 ± 1.25 x 107 colony forming unit (cfu)/ml, counted on Petroff-Hausser counting chamber by using the formula given below.
No. of cells in large sq. x Dilution factor x percent Dilution x Factor for large sq. (1.25 x 106). The suspension was poured aseptically in 100 ml sterile conical flask containing freshly harvested 0.5 cm long chopped root fragments (free from infection and contamination, obtained from 60 days old wheat plants grown in strictly aseptic condition) at 5 ml/100 g root fragments. The inoculated flasks were incubated at 30±2°C for 72 hours for multiplication of the bacterium on root fragments. Thereafter population of the bacterium was recounted (3.8±1.25 x 10g cfu/g root fragments). The colonised root fragments was the axenic rhizobase inocula of A. brasilense (wild type).
Inoculation of the Rhizohase Inocula in Soil Pots: A series of well washed and surface disinfested backed clay pots (20 cm height with 25 and 10 cm top and bottom diam respectively) each containing 4 kg steam sterilised sandy clay loam Soil with aforementioned properties were inoculated separately and in combined form (1:1) with 3 doses of inocula (at 10 g, 20 g and 40 g/200 pot soil). The ratio of pot soil-inocula was maintained as 1:20, 1:10 and 1:05 g respectively. The surface disinfected seeds of wheat var. Blue Silver were sown in the inoculated pots at 3 seeds/pot (Menge and Timmer, 1982). Each treatment was replicated 3 time. The inoculated pots with control treatments (00 g/200 g soil) were placed in screen house under natural condition by completely randomised design method. Seeds were allowed to germinate and grown up to the stage of maturity. The growing plants were watered regularly with sterilised distilled water.
Results
The effect of separate and combined inoculation of G. macracarpum Tulasne and Tulasne and A. brasilense (wild type) in the form of axenic rhizobase base inocula in wheat variety. Blue Silver was recorded with the parameters of VAM infection (%), fresh and dry weights, plant height and yield (in terms of grains number/head and TGW). The separate and combined inoculation of the aforesaid inocula with three different doses (10 g, 20 g, 40 g/200 g soil) showed a progressive response on increasing the doses, therefore, the highest response was always found at third dose (Table 1). The first dose of either separate or combined inoculation never showed significant response, however, the response of second and third doses differed non-significantly (Table 1 and 2). On separate inoculation, the response of G. macrocarpum Tulasne and Tulasne was found to be dominated over the response of A. brasilense as compared to the control series (00 g/200 g soil) (Table 1). The combined inoculation always showed better response than the separate inoculation. The detailed study and analysis of individual parameters are given below.
VAM infection percentage: On separate inoculation by G. macracarpum Tulasne and Tulasne 60.3 percent VAM infection was observed at third dose (40 g1200 g soil) in the root of wheat plant whereas no VAM infection was observed on inoculation by A. brasilense (wild type) at the same dose. The combined inoculation showed more VAM infection at all the three doses as compared to the individual inoculation indicating the beneficial interactions between them (Table 1). The Analysis of variance (ANOVA) of the data (Table 2) showed that the 3 doses and different types of inocula were found to be differed significantly (p<0.001). A significant interaction was found between doses and inocula (p<0.001) in respect to the VAM infection percentage.
Fresh weight: On separate inoculation of G. macrocarpum Tulasne and Tulasne fresh weight of wheat plant was increased by 13.17 and 10.70 percent by A. brasilense (wild type) at the third dose as compared to the control treatment whereas 20.19 percent increase was observed by the combined inoculation at the same dose (Table 1). ANOVA of the data (Table 2) showed that the doses of the inocula were differed significantly (p<0.001) whereas 3 different types of inocula were also found to be significantly different at p<0.01. The interaction between doses and inocula was found non significant (Table 2).
Dry weight: Seprate inoculation of G. macrocarpum Tulasne and Tulasne showed 24.69 percent increase in dry weight of wheat plant whereas 20.74 percent increase by A. brasilense (wild type) at the third dose (40 g/200 g soil) as compared to the control treatment (Table 1). On combined inoculation the dry weight was increased up to 37.19 per cent at the same dose (Table 1). The statistical analysis of the data (ANOVA) given in Table 2 showed that the 3 different doses of the inocula were differed significantly (p<0.05) whereas the different types of inocula were not found significantly different. The interaction between doses and inocula was found non significant (Table 2).
Plant height: Either separate or combined inoculation of the aforesaid inocula showed non-significant increase in plant height at all the three doses as compared to the control series (Table 2). On separate inoculation the plant height was increased up to 7.90 percent by the G. macrocarpum Tulasne and Tulasne and 5.25 percent increase by A. brasilense (wild type) whereas 9.22 percent increase was observed by the combined inoculation at the third dose (Table 1). The analysis of the data (Table 2) showed that the different doses of the different types of inocula were not significantly different. The interaction between doses and inocula was non significant.
Grains number/head: The number of grains/head of wheat plant were found to be increased non-significantly at all the three doses either on separate or combined inoculation. On separate inoculation, number of grains/head increased up to 32.78 percent by G. macrocarpum Tulasne and Tulasne and 25 percent by A. brasilense (wild type) at third dose. The combined inoculation at the same dose showed 39.52 percent increase (Table 1). The ANOVA of the data (Table 2) showed that the 3 different doses of the different inocula were not differed significantly. A non- significant interaction was found between doses and inocula (Table 2).
Thousand grain weight-TGW (g): The wheat plant showed significantly positive response in respect of TGW on either separate or combined inoculation of G. macrocarpum Tulasne and Tulasne and A. brasilense (wild type).
| Table 1: | Effect of VAM and Azospirillum spp. on growth and yield of wheat |
| Numerical values in each column are the mean of 6 replicate samples LSD0.05 (Dose) = 4.38, LSD0.05 (Inocula) = 3.79 | |
| Table 2: | Analysis of variance (ANOVA) on separate and combined inoculation of VAM and Azospirilum spp, on wheat growth |
On separate inoculation by G. macrocarpum Tulasne and Tulasne TGW was found to be increased up to 21.45 and 20.13 percent by A. brasilense (wild type) at the third dose. The result of combined inoculation showed up to 56.61 percent increase in TGW at the same dose as compared to the control series (Table 1). The ANOVA of the data (Table 2) showed that the 3 different doses of the different inocula were differed significantly (p<0.001). The different types of inocula were also found significantly different (p<0.01). The interaction between doses and macula was found nonsignificant (Table 2).
Discussion
Effective soil microorganisms (ESM) are now being explored in agriculture with especial interest to increase the growth and yield of crop plants (Nandi, 1990; Giller and Day, 1985). Researches on VAM fungi and Azospirillum spp. separately in stimulation of plant growth and suitable methods of achieving their potential benefits in agriculture are being conducted on a world wide scale (Avivi and Feldman, 1982; Bagyaraj, 1991; Diederichs and Moawad, 1993; Hall, 1988; Harley, 1989; Millet et al., 1984; Okon, 1982; Pacovsky et al. 1985; Bashan and Levanony, 1990). VAM-Azospirillum biotechnology is a nascent agricultural strategy for boosting up the growth and yield of crops particularly cereals which lack the root nodulating bacteria. Barea et al. (1983) suggested that VAM and Azospirillum species together provide a means by which cereal plants lacking symbiotic N-fixers (Rhizobium species) could compensate the nutrient deficiency. However, studies on interactions between VAM and Azospirillum spp. and their combined effects on plants are relatively very few in the world. Our results on either separate or combined inoculation of the inocula with three different doses (10 g, 20 g, 40 g/200 g soil) showed a progressive response of wheat var. Blue Silver in respect of the increase in VAM infection per cent (only in the presence of VAM fungus), fresh and dry weights, plant height, grains number/head and TGW (Table 1). The first dose of either separate or combined inoculation never showed significant response, however, the response of second and third doses differed non-significantly. On separate inoculation, the response of G. macrocarpum Tulasne and Tulasne was found to be dominated over the response of A. bras ilense as compared to the control treatments (00 g/200 g soil) (Table 1). The combined inoculation of the aforesaid inocula showed always better response than their separate inoculations. Tewari and Boyetchko (1987), Wood (1987) and Menge (1983) worked on possible commercial use of VAM fungi as an alternative to the "P"-fertilizers and their possible application in agriculture. Azospirillum inoculation has now been considered a substitute for N- fertilizers (Lau-Wong, 1987; Mertens and Hess 1984). The enhancement of growth and yield of crop plants due to VAM fungi have already been reported by many workers (Jakobsen, 1983; Yocom and Boosalis, 1991) which corroborate our findings. Our results showed better response at third dose (40 g/200 g soil) (Table 1). Clapperton and Reid (1992) stated that there is a correlation between the inoculum density of VAM fungi and soil nutrient availability which affect plant growth. Sieverding (1991) evaluated various responses of crop plants after VAM inoculation by correlation analysis and concluded that one response was independent to the another that also supports our result. The results of inoculation by G. macrocarpum Tulasne and Tulasne showed 32.78 percent increase in grains number/head and 21.45 percent increase in TGW of wheat var. Blue Silver. Whereas Saif and Khan (1977) reported 290 per cent increase in grains yield of barley. on inoculation with VAM fungi which has not been considered to be a realistic (Powell, 1984) since lacking of the statistical analysis to the data. We have obtained 24.69 percent increase in dry weight of wheat var. Blue Silver by the same inoculation at same dose which greatly differed from the results of Khan (1972, 1975). The Khan’s results have already been challenged by Powell and Bagyaraj (1984) on account of lacking of the statistical analysis of the data. However, each parameter of our result has been analysed individually to evaluate the status of it significant level. Most studies of the Azospirillum-plant association have been conducted on cereals and grasses. The response of cereals like increases in fresh and dry weights, N content in shoots and grains, total number of tillers and ears, grains number/head, increase in grains weight, higher germination rate on inoculation with Azospirillum strains have already been reported by many workers (Bashan 1986; Cohen et al., 1980; Mertens and Hess, 1984; Pacovsky et al., 1985) which lend support to our findings (Table 1). Our investigation showed that the separate inoculation of A. brasilense at 40 g/200 g soil increased the yield of wheat var. Blue Silver in term of grains number/head (25.00%) and TGW (20.13%). The result of Kapulnik et al. (1987) showed 10 to 30 percent increase in the total yield of crop plants which confirm our findings. Okon (1985) obtained on average 65 percent increase in yield to all field experiments after the inoculation of Azospirillum sp. Smith at al. (1984) observed 75 percent increases in yield to summer cereals and 50 percent in spring wheat due to Azospirillum inoculation. Our dual inoculation of G. macrocarpum Tulasne and Tulasne and A. brasilense (wild type) in wheat var. Blue Silver produced highly significant response which clearly indicate mutual beneficial interactions between them (Table 1). The similar results were also obtained by Pacovsky at al. (1985) and stated that VAM and Azospirillum species together produce an additive effect on plant which confirmed our findings. Bashan and Levanony (1985) and Subba Rao et al. (1985) described the synergistic effects of Azospirillum and VAM fungi on combined inoculation in barley which resulted the increase in growth along with the enhanced uptake of "N" and "P" in the inoculated plants. This dual inoculation could completely replace the application of "N" and "P" fertilizers (Barea et al., 1983; Pacovsky, 1988; Pacovsky et al., 1985). Zuberer (1990) pointed out that wheat and corn are usually provided a small portion of the biologically fixed Nitrogen below the estimated need (10 to 15 %). Barea et al. (1983) suggested that combined inoculation of VAM and Azospirillum increase the uptake of nitrogen greater than the estimated needs. Nevertheless, experimental studies on VAM-Azospirillum have not received much attention in world and not at all in Pakistan. Therefore, extensive researches are needed for achieving their possible potential benefits in agriculture. However, from our result it can be concluded that the VAM fungi along with Azospirillum species in the form of fertilizer could be utilised to enhance the growth and yield of wheat. This information would be of great significance for the production of wheat in Sindh Pakistan. Since this technology is potentially more useful biological means of assuring plant production with minimum input of agro-chemicals and does not entail any environmental pollution and soil degradation. However, it needs further investigation on its possible application in field condition and commercial utilization in Pakistan.