Characterization ofAzotobacter spp. and effect of Azotobacter and Azospirillum as inoculant on the yield and N-uptake of wheat crop

Out of ten isolates ofAzospirillum spp. isolated from the root and rhizosphere of wheat plant, seven belonged toA. brasilense and three belonged toA. lipoferum. All eight isolates ofAzotobacter spp. belonged toAzotobacter chroococcum. Two strains, one fromA. lipoferum and another fromA. chroococcum having high nitrogen fixing capacity with negative test for denitrification were used as inoculant to supplement the nitrogen need of wheat crop. Significant increases in the yield of wheat grain and uptake of nitrogen by the crop over the control were found in pot tests when the seeds were inoculated either withAzospirillum spp. orAzotobacter spp. or the combination of both the inoculants.     

Effect of seed inoculation,mycorrhizal infection and organic amendment on wheat growth

Summary The effect of seed inoculation withAzotobacter spp. orAzospirillum spp., and garbage amendment (0.5%), on the growth of wheat was studied in a field experiment under sub-tropical conditions. Two levels of N fertilizer were applied, the usual field rate (150 kg N ha^–1) and half this amount. Tillering of plants, dry matter contents and nitrogenase activity were determined 30, 60 and 90 days after sowing. At the end of the experimental period, spore numbers and percentage of mycorrhizal infection were observed in the rhizosphere and root systems of plants. Straw and grain yields were also determined. The results of this study showed that seed inoculation and/or organic amendment stimulated plant growth, nitrogenase activity and mycorrhizal infection. This was more noticeable withAzotobacter than withAzospirillum. Inoculation withAzotobacter together with 1/2 N dose and organic amendment was the most effective application (19.75 and 10.70 t ha^–1 were recorded for straw and grain yield, respectively).     

Azotobacter: A potential bio-fertilizer for soil and plant health management

Stressor (biotic as well as abiotic) generally hijack the plant growth and yield characters in hostile environment leading to poor germination of the plants and yield. Among the plant growth promoting rhizobacteria, Azotobacter spp. (Gram-negative prokaryote) are considered to improve the plant health. Various mechanisms are implicated behind improved plant health in Azotobacter spp. inoculated plants. For example, acceleration of phytohormone like Indole-3-Acetic Acid production, obviation of various stressors, nitrogen fixation, pesticides and oil globules degradation, heavy metals metabolization, etc. are the key characteristics of Azotobacter spp. action. In addition, application of this bacteria has also become helpful in the reclamation of soil suggesting to be a putative agent which can be used in the transformation of virgin land to fertile one. Application of pesticides of chemical origin are being put on suspension mode as the related awareness program is still on. As far as the limitations of this microbe is concerned, commercial level formulations availability is still a great menace. Present review has been aimed to appraise the researchers pertaining to utility of Azotobacter spp. in the amelioration of plant health in sustainable agroecosystem. The article has been written with the target to gather maximum information into single pot so that it could reach to the dedicated researchers.     

Studies on biological decomposition of wheat straw

Summary The incorporation of undecomposed wheat straw in the soil along-with the micro-organisms favourably increased the yield of groundnut crop. An increase of 37 per cent in yield was recorded when wheat straw was inoculated withPenicillium digitatum and the C:P ratio was adjusted to 65. Inoculated treatments of narrower C:P ratio gave a higher yield than wider C:P ratio treatments inoculated with the same cultures. An increase in nitrogen uptake by groundnut plants was recorded due to incorporation of straw alongwith the micro-organisms in soil. The organic carbon and nitrogen content of the soil increased with all the treatments except control. The highest increase in organic carbon and nitrogen of the soil was observed with a treatment of wheat straw of 65 C:P ratio inoculated withS. coccosporum. The yield of wheat crop after groundnut was significantly more with several treatments than control plots. The highest increase of 79 per cent in grain yield of wheat was observed in the plots previouslq received with wheat straw of 200 C:P ratio.This paper is based on the data presented at IV Southern Regional Conference on Microbial Inoculants, held at Parbhani during 3–4 July 1978.     

Untraditional N2-fixing bacteria as biofertilizers for wheat and barley

The screening of 27 isolates grown on nitrogen-free medium for nitrogen-fixing ability resulted in the isolation of five organisms belonging toBacillaceae, Enterobacteriaceae andPseudomonadaceae. Estimates of N₂-fixation efficiencies of these isolates indicated that they may be responsible for low rates of N₂-fixation in soil. The possible association of these isolates as well as ofAzotobacter andAzospirillum with wheat and barley was investigated in a greenhouse experiment. The highest values of nitrogenase activity on plant root were recorded in treatments inoculated with composite inocula of the isolated N₂-fixers, particularly whenAzotobacter and/orAzospirillum were added in combination. Inoculation with single inoculum of each of the N₂-fixing isolates had no significant influence on plant growth, except withPseudomonas andBacillus for wheat and barley, respectively. Highly significant increases in growth of both plants were recorded in all cases of multistrain inoculation.     

Urease activity in soils

Summary The availability of Ca from different levels of gypsum and calcium carbonate in a non-saline sodic soil has been investigated. Different levels of tagged gypsum (Ca⁴⁵SO₄.2H₂O) and calcium carbonate (Ca⁴⁵CO₃) (i.e. 0, 25, 50, 75, and 100 per cent of gypsum requirement) were mixed thoroughly in 3.5 Kg of a non-saline alkali soil (ESP, 48.4; ECe, 2.68 millimhos/cm). Dhaincha (Sesbania aculeata) — a legume and barley (Hordeum vulgare L.) — a cereal were taken as test crops. Increasing levels of gypsum caused a gradual increase in the yield of dry matter, content of Ca and K in the plant tops and Ca:Na and (Ca+Mg):(Na+K) ratios in both the crops. Application of calcium carbonate caused a slight increase in the dry matter yield, content of Ca and Mg and Ca:Na and (Ca+Mg):(Na+K) ratios in barley, however, in case of dhaincha there was no such effect. Gypsum application caused a gradual decrease in the content of Na and P in both the crops. Total uptake of Ca, Mg, K, N and P per pot increased in response to gypsum application. The effect of calcium carbonate application on the total uptake of these elements was much smaller on dhaincha, but in barley there was some increasing trend.Increasing application of tagged gypsum and calcium carbonate caused a gradual increase in the concentration and per cent contribution of source Ca in both the crops, although, the rate of increase was considerably more in dhaincha. The availability of Ca from applied gypsum was considerably more than that from applied calcium carbonate. Efficiency of dhaincha to utilize Ca from applied sources was considerably more (i.e. about five times) than that of barley     

The Formation of Artificial Nitrogen-Fixing Symbioses with Rape (Brassica napusvar. napus) Plants in Nonsterile Soil

The treatment of rape plants grown in nonsterile soil with 2,4-dichlorophenoxyacetic acid (auxin-like growth-promoting substance) or their inoculation with the bacterial association Micrococcussp. + Rhodococcussp. and/or with the mixed nitrogen-fixing culture Azotobacter nigricans+ Bacillussp. led to the formation of paranodules on the rape roots. The introduced bacteria were detected both in the intercellular space and inside the cells of the paranodules and the rape roots. The nitrogen-fixing activity of the paranodulated plants was two times higher than that of the inoculated plants lacking paranodules and five times higher than that of the control (i.e., not inoculated) plants. The paranodulation led to a 40% increase in the crop yield of rape plants and provided for a statistically significant increase in the total nitrogen as well as protein nitrogen contents of the plants.     

A greenhouse study on the effect of inoculation of N-fixing blue-green algae in an alluvial soil treated with P and Mo on the yield of rice and changes in the N-content of soil

Summary The efficiency of the inoculation of three cultures of N-fixing blue-green algaeviz. (i)Aulosira fertilissima (A₁), (ii)Nostoc muscorum (A₂) and (iii) their mixture (A₃) in increasing the grain and straw yield of rice, nitrogen uptake in grain and nitrogen content in soil was studied in a green house experiment with an alluvial soil in presence or absence of urea nitrogen application. Inoculation significantly increased the grain and straw yield of rice and nitrogen uptake in grain, but the efficiency of inoculation gradually decreased with the increase in the levels of urea nitrogen application, the extent of decrease varying with the algal cultures inoculated. The nitrogen content in the soils after the crop harvest recorded a significant increase due to inoculation but after air drying the soil a marker decrease of the same was observed, which indicated that most of the nitrogen added to the soil by blue-green algae through fixation did not persist after air drying the soil.     

Application of Bacillus species in the control of root rot diseases of crop plants

Bio control potential of three Bacillus spp viz., Bacillus subtilis, B. thuringiensis and B. cereus, against soil borne root-infecting fungi on cowpea and mash bean plants were tested both in vitro and in vivo. All three species showed efficiency and produced nodules on mash bean and cow pea plants. In vitro dual culture plate method showed significant inhibition of Fusarium spp. by all these three species of Bacillus with the appearance of a prominent zone of inhibition while a maximum zone of inhibition of Fusarium spp. was observed by B. thuringiensis, whereas in case of Macrophomina phaseolina and Rhizoctonia solani, the highest zone of inhibition was observed by B. subtilis. Bacillus spp. used as seed dressing and soil drenching showed a significant increase in shoot length, shoot weight, root length and root weight in cow pea and mash bean plants. Maximum shoot length was observed in cow pea plants where Bacillus spp. were drenched in soil, whereas maximum root length and root weight in cow pea was observed when B. thuringiensis used as seed dressing. Seed dressing and soil drenching with species of Bacillus viz., B. subtilis, B. thuringiensis and B. cereus, were found to be an effective method for the control of soil borne root-infecting fungi like M. phaseolina, R. solani and Fusarium spp., on cow pea and mash bean plants.     

Growth effects of the vesicular-arbuscular mycorrhizal fungusGlomus constrictum on maize plants in pot trials

Maize plants were inoculated withGlomus constrictum in soil of low phosphorus content amended with five rates of P in the form of Ca₃(PO₄)₂. Maize dry matter yield was increased by addition of P up to 30 and/or 60 mg P/kg soil, above that it began to decrease to reach at 100 mg P/kg a value similar to that of the control. At all P levels used, the shoot and root (total plant) dry mass of inoculated plants was significantly increased compared with the non-inoculated controls and this increment ranged in some cases between 50 and 70%. Development of vesicular-arbuscular mycorrhizal fungus (VAM) monitored in terms of P contents in dry matter of maize revealed that the P content of plants not inoculated withG. constrictum was not influenced by P addition to soil. On the other hand, P content of maize plants inoculated with VAM was dramatically increased by increasing P levels of soil and was maximum at 30 mg P; above that it began to decline. Mycorrhizal root infection (expressed as percentage of root length infected) increased by increasing the P concentrations above the soil basal level up to 80 mg P where the infected root length was 72% of the total root length after 28 d of planting. The increase in VAM spore formation in soil was similar to that of root infection except that the highest spore number was sieved from soil at 60 mg P/kg soil.     

Field response of wheat to arbuscular mycorrhizal fungi and drought stress

Mycorrhizal plants often have greater tolerance to drought than nonmycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on growth, grain yield and mineral acquisition of two winter wheat (Triticum aestivum L.) cultivars grown in the field under well-watered and water-stressed conditions. Wheat seeds were planted in furrows after treatment with or without the AM fungi Glomus mosseae or G. etunicatum. Roots were sampled at four growth stages (leaf, tillering, heading and grain-filling) to quantify AM fungi. There was negligible AM fungi colonization during winter months following seeding (leaf sampling in February), when soil temperature was low. During the spring, AM fungi colonization increased gradually. Mycorrhizal colonization was higher in well-watered plants colonized with AM fungi isolates than water-stressed plants. Plants inoculated with G. etunicatum generally had higher colonization than plants colonized with G. mosseae under both soil moisture conditions. Biomass and grain yields were higher in mycorrhizal than nonmycorrhizal plots irrespective of soil moisture, and G. etunicatum inoculated plants generally had higher biomass and grain yields than those colonized by G. mosseae under either soil moisture condition. The mycorrhizal plants had higher shoot P and Fe concentrations than nonmycorrhizal plants at all samplings regardless of soil moisture conditions. The improved growth, yield and nutrient uptake in wheat plants reported here demonstrate the potential of mycorrhizal inoculation to reduce the effects of drought stress on wheat grown under field conditions in semiarid areas of the world.     

Association between N2-fixing bacteria and pearl millet plants: Responses,mechanisms and persistence

Responses to inoculation with N₂-fixing bacteria were studied in relation to genotypic differences in pearl millet, effect of nitrogen levels, and FYM additions in India. In some experiments, inoculation increased mean grain yield up to 33% over the uninoculated control, whereas in the remaining 11 experiments there was no significant increase. Increased grain yields, >10% over the uninoculated controls were observed in 46% of the experiments withAzospirillum lipoferum (18.7% average increase) and withAzotobacter chroococcum (13.6% average increase). Yield increases were nil or reduced in three experiments withAzos. lipoferum and four experiments withAztb. chroococcum. In two experiments continued inoculation for two or three years resulted in increased grain, plant biomass yield, and N uptake. Interactions of bacterial cultures with cultivars or years were not observed. The counts of the inoculated strains increased two to three-fold when inoculation was continued for three years. Repeated inoculations increased the mean cumulative N uptake from season 1 to season 3 by 19 kg ha^–1. Repeated inoculations withAztb. chroococcum andAzos. lipoferum increased mean grain yield of a succeeding crop by 14.4% and 9.8%, respectively, over the uninoculated control. Inoculation increased the efficiency of N-assimilation by pearl millet. Marginal increase in nitrogenase activity, associated with the inoculated plants was observed during later stages of plant growth. Increased leaf nitrate reductase activity (NRA) was observed after inoculation with these bacteria. The responses to inoculation are mainly attributable to increased plant N assimilation which could be the effect of growth promoting substances secreated by the bacteria; and thus the contribution from BNF may be small.CRISAT, journal article 732.     

Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Segregation pattern of gene expression in cotton leaf curl virus-resistant transgenics

Non-pathogenic soil bacteria living in association with roots of higher plants enhance their adaptive potential and thus could be beneficial for their growth. Here, we present the current status of the use of Bacillus subtilis in biocontrol. Rhizobacteria are found in the rhizosphere. Plant growth promoting rhizobacteria (PGPR) strains, such as Bacillus and Pseudomonas, were isolated by using Nutreint dextrose Agar medium or Potato Dextrose Agar medium. The selection of PGPR strains was done by duel culture methods against the potato pathogens. The interaction of PGPR (Bacillus) with potato seeds or vegetative parts show promising antagonism by virtue of producing siderophore and antibiotics against black scurf and stem canker diseases of potato caused by Rhizoctonia solani, thereby resulting in increase of potato yield. The effectiveness of PGPR strain (Bacillus spp.) in improving the yield of potato in greenhouse conditions and in the field was observed.     

Isolation of Nitrogen Fixing Bacteria from Fungus Termites

Biological nitrogen fixation by the microorganisms in the gut of termites is one of the singularly important symbiotic processes, since termites invariably thrive on nitrogen poor diet. Two isolates of free living aerobic and facultative anaerobic N fixing bacteria were obtained from the guts of fungus cultivating termite, Macrotermes sp. Among the total bacterial isolates from termite gut, the per cents of N fixing aerobes viz., Azotobacter and Beijerinckia spp were 49% and 37% from the salivary gland while facultative N fixing anaerobe viz., Klebsiella and Clostridium contributed (51% and 93%). The free living aerobic bacteria were identified as Azotobacter spp (19 x 10⁴ CFU mL^‐1) and Beijerinckia (13.2 x 10⁴ CFU mL^‐1) from the salivary gland of the termite; interestingly, foregut, mid gut and hind gut registered a low population of these bacteria. The isolates of Azotobacter were smooth, glistening, vicid in nature, rods, gram negative and cyst forming. Isolates of Beijerinckia sp. produced copious slime, tenacious, rods, gram negative with no cyst formations. Both the isolates emitted green fluorescence and produced acid. Facultative N fixing anaerobes were harbored in the hind gut. The isolates were identified as Klebsiella (20 x 10⁴ CFU mL^‐1) and Clostridium pasteurianum 39.1 x 10⁴ CFU mL^‐1. Klebsiella were straight rods arranged singly or in pairs, non‐motile, gram negative, whereas Clostridium pasteurianum was viscoid, motile with terminal spores. A positive correlation was observed between the extractable polysaccharides of these isolates and soil aggregation. The aggregates formed by the isolates increased soil aeration, porosity, water holding capacity and helped in better plant growth. Thus, the gut microflora of termite, apart from harnessing nitrogen from the atmosphere, also helps improving soil fertility.     

Response of onion plants to arbuscular mycorrhizae

Onion (Allium cepa) plants were grown in pots in two types of irradiated soil, mineral and organic. Onion development was observed under two or three levels of P fertilization, and three methods of arbuscular mycorrhizal fungus inoculation with two fungus species. In mineral soil, preinoculated onion plants had a higher biomass than non-inoculated control plants or plants inoculated with either colonized root segments or spores. Fungus species had no differential effect on dry biomass or final bulb diameter. Preinoculated onion plants reached marketable size (>25 mm bulb diameter) 2-3 weeks earlier than those inoculated by either of the other two methods. Non-inoculated onion plants remained stunted. Bulbs of onions inoculated with Glomus versiforme were firmer than those inoculated with G. intraradices. Increasing P fertilizer rates had a significant positive linear effect on the P tissue concentration of plants inoculated with G. intraradices or G. versiforme, but no effect on bulb firmness. The P tissue concentration of inoculated plants was significantly higher than that of non-inoculated controls, and in inoculated plants, it differed among inoculation methods. The P tissue concentration was higher in onion plants inoculated with G. versiforme than in those inoculated with G. intraradices. In organic soil, the dry biomass of preinoculated plants was higher than that of plants inoculated by root segments. The highest root colonization levels were obtained under a low soil P level with G. intraradices, and with the root segment method of inoculation with G. versiforme.     

Growth and yield of groundnut (Arachis hypogaea L.) plants in soil inoculated with Aspergillus niger Van Tieghem

Aspergillus niger, a soil-borne fungus is a causative agent of hypocotyl malformations in infected groundnut (Arachis hypogaea L.) plants, but its effect on yield is unknown. This study sought to determine its effect on growth and yield. Seeds of Chinese and JL45 varieties were sown in soil inoculated with A. niger. Fresh and dry weights of the shoots and roots were taken at 10-day intervals. Nodule count was done at 30 days after emergence and subsequently at 10-day intervals. Pods of 20 plants each from inoculated and uninoculated soils were harvested. Growth was suppressed in plants grown on A. niger inoculated soil. Eight-day old plants grown in inoculated soil developed curvatures on their hypocotyls. Nodulation was suppressed (p < 0.05) in plants grown in inoculated soil. Although growth was suppressed in plants grown on inoculated soil, yield of both varieties of groundnut was not affected.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Field response of chickpea to inoculation withGlomus versiforme

The response ofCicer arietinum to inoculation withGlomus versiforme under field conditions was investigated in a phosphorus deficient sandy loam soil. Inoculation with the mycorrhizal fungusGlomus versiforme increased the rate of VAM development in chickpea. The weight of nodules and the number of nodules per plant were higher in inoculated than in uninoculated plants. The phosphorus content of the shoots and its total uptake, were increased by either the application of single super-phosphate, or by inoculation withG. versiforme. Inoculation increased shoot dry weights and grain yields by 12% and 25% respectively, as compared with the 33% and 60% increases respectively produced by P-treated plants.     

Microbial biofilm inoculants benefit growth and yield of chrysanthemum varieties under protected cultivation through enhanced nutrient availability

Protected cultivation of ornamental flowers, as a commercial venture, becomes less profitable with excessive use of fertilizers. The present study examined the influence of microbial biofilm inoculants (Anabaena–Azotobacter, Anabaena–Trichoderma and Trichoderma–Azotobacter) on the availability of soil nutrients and structure of rhizosphere microbial communities in three varieties of chrysanthemum (var. White Star, Thai Chen Queen and Zembla). Varietal-specific responses in growth, enzyme activities, flower yield of plants and availability of soil nutrients were recorded. Dehydrogenase activity was highest in var. White Star treated with the Anabaena–Trichoderma biofilm inoculants. The Anabaena–Azotobacter inoculant enhanced the availability of nitrogen, phosphorus and micronutrients in the soil, besides 40–50% increase in soil organic carbon, as compared to carrier alone or no inoculation. PCR-DGGE profiling of the cyanobacterial communities and qPCR quantification of 16S rRNA abundance of bacteria, archaea and cyanobacteria in the rhizosphere soils, revealed the stronger influences of these inoculants, especially in var. Zembla. Principal Component Analysis (PCA) helped to illustrate that the enhanced microbe-mediated availability of soil macro-and micronutrients, except iron content (Fe), was the most influential factor facilitating improved plant growth and yield parameters. The Anabaena–Azotobacter, and Anabaena–Trichoderma biofilm inoculants, proved superior in all three chrysanthemum varieties.