Rifat Hayat

SOIL BENEFICIAL BACTERIA AND THEIR ROLE IN PLANT GROWTH PROMOTION: A REVIEW

Soil bacteria are very important in biogeochemical cycles and have been used for crop production for decades. Plant–bacterial interactions in the rhizosphere are the determinants of plant health and soil fertility. Free-living soil bacteria beneficial to plant growth, usually referred to as plant growth promoting rhizobacteria (PGPR), are capable of promoting plant growth by colonizing the plant root. PGPR are also termed plant health promoting rhizobacteria (PHPR) or nodule promoting rhizobacteria (NPR). These are associated with the rhizosphere, which is an important soil ecological environment for plant–microbe interactions. Symbiotic nitrogen-fixing bacteria include the cyanobacteria of the genera Rhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium, Sinorhizobium and Mesorhizobium. Free-living nitrogen-fixing bacteria or associative nitrogen fixers, for example bacteria belonging to the species Azospirillum, Enterobacter, Klebsiella and Pseudomonas, have been shown to attach to the root and efficiently colonize root surfaces. PGPR have the potential to contribute to sustainable plant growth promotion. Generally, PGPR function in three different ways: synthesizing particular compounds for the plants, facilitating the uptake of certain nutrients from the soil, and lessening or preventing the plants from diseases. Plant growth promotion and development can be facilitated both directly and indirectly. Indirect plant growth promotion includes the prevention of the deleterious effects of phytopathogenic organisms. This can be achieved by the production of siderophores, i.e. small metal-binding molecules. Biological control of soil-borne plant pathogens and the synthesis of antibiotics have also been reported in several bacterial species. Another mechanism by which PGPR can inhibit phytopathogens is the production of hydrogen cyanide (HCN) and/or fungal cell wall degrading enzymes, e.g., chitinase and ß-1,3-glucanase. Direct plant growth promotion includes symbiotic and non-symbiotic PGPR which function through production of plant hormones such as auxins, cytokinins, gibberellins, ethylene and abscisic acid. Production of indole-3-ethanol or indole-3-acetic acid (IAA), the compounds belonging to auxins, have been reported for several bacterial genera. Some PGPR function as a sink for 1-aminocyclopropane-1-carboxylate (ACC), the immediate precursor of ethylene in higher plants, by hydrolyzing it into α-ketobutyrate and ammonia, and in this way promote root growth by lowering indigenous ethylene levels in the micro-rhizo environment. PGPR also help in solubilization of mineral phosphates and other nutrients, enhance resistance to stress, stabilize soil aggregates, and improve soil structure and organic matter content. PGPR retain more soil organic N, and other nutrients in the plant–soil system, thus reducing the need for fertilizer N and P and enhancing release of the nutrients.

Microbial phytase activity and their role in organic P mineralization

Plants respond to their external environment to optimize their nutrition and production potential to minimize the food security issues and support sustainable agriculture system. Phosphorus (P) is an important nutrient for plants and is involved in plant metabolic processes. It is mostly available as orthophosphate and has a tendency to form complexes with cations. It has low mobility in soil, thus becoming unavailable for plant uptake that causes a reduction in plant growth and yield. Besides free P, phytate is the major form of organic P in soil and plant tissues. Phytases obtained from different sources, that is, plants, animals, and microorganisms, catalyze the hydrolysis of phytate and release available forms of inorganic P. The knowledge of mechanisms involved in catalytic activity of phytase obtained from microorganisms in soil is limited. This review summarizes the role of microbial phytase in releasing organic P by hydrolysis of phytate and factors affecting its activity in the soil.

Seed biopriming with plant growth promoting rhizobacteria: a review.

Beneficial microbes are applied to the soil and plant tissues directly or through seed inoculation, whereas soil application is preferred when there is risk of inhibitors or antagonistic microbes on the plant tissues. Insufficient survival of the microorganisms, hindrance in application of fungicides to the seeds and exposure to heat and sunlight in subsequent seed storage in conventional inoculation methods force to explore appropriate and efficient bacterial application method. Seed priming, where seeds are hydrated to activate metabolism without actual germination followed by drying, increases the germination, stand establishment and stress tolerance in different crops. Seed priming with living bacterial inoculum is termed as biopriming that involves the application of plant growth promoting rhizobacteria. It increases speed and uniformity of germination; also ensures rapid, uniform and high establishment of crops; and hence improves harvest quality and yield. Seed biopriming allows the bacteria to enter/adhere the seeds and also acclimatization of bacteria in the prevalent conditions. This review focuses on methods used for biopriming, and also the role in improving crop productivity and stress tolerance along with prospects of this technology. The comparison of methods being followed is also reviewed proposing biopriming as a promising technique for application of beneficial microbes to the seeds.

An Overview of Plant Growth Promoting Rhizobacteria (PGPR) for Sustainable Agriculture

Soil bacteria beneficial to plant growth usually referred to as plant growth promoting rhizobacteria (PGPR), are capable of promoting plant growth by colonizing the plant root. The mechanisms of PGPR-mediated enhancement of crop growth includes (i) a symbiotic and associative nitrogen fixation; (ii) solubilization and mineralization of other nutrients; (iii) production of hormones e.g. auxin i.e. indole acetic acid (IAA), abscisic acid (ABA), gibberellic acid and cytokinins; (iv) production of ACC-deaminase to reduce the level of ethylene in crop roots thus enhancing root length and density; (v) ability to produce antagonistic siderophores, s-1-3-glucanase, chitinases, antibiotics, fluorescent pigment and cyanide against pathogens and (vi) enhanced resistance to drought and oxidative stresses by producing water soluble vitamins niacin, thiamine, riboflavin, biotin and pantothenic acid. Increased crop production through biocontrol is an indirect mechanism of PGPR that results in suppression of soil born deleterious microorganisms. Biocontrol mechanisms involved in pathogen suppression by PGPR include substrate competition, antibiotic production, and induced systemic resistance in the host. PGPR can play an essential role in helping plants to establish and grow in nutrient deficient conditions. Their use in agriculture can favour a reduction in agro-chemical use and support ecofriendly crop production. Trials with rhizosphere-associated plant growth-promoting P-solubilizing and N2-fixing microorganisms indicated yield increase in rice, wheat, sugar cane, maize, sugar beet, legumes, canola, vegetables and conifer species. A range of beneficial bacteria including strains of Herbaspirillum, Azospirillum and Burkholderia are closely associated with rhizosphere of rice crops. Common bacteria found in the maize rhizosphere are Azospirillum sp., Klebsiella sp., Enterobacter sp., Rahnella aquatilis, Herbaspirillum seropedicae, Paenibacillus azotofixans, and Bacillus circulans. Similarly, strains of Azotobacter, Azorhizobium, Azospirillum, Herbaspirillum, Bacillus and Klebsiella can supplement the use of urea-N in wheat production either by BNF or growth promotion. The commonly present PGPR in sugarcane plants are Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense, Acetobacter diazotrophicus, Bacillus tropicalis, Bacillus borstelensis, Herbaspirillum rubrisubalbicans and Herbaspirillum seropedicae. Symbiotic N2-fixing bacteria collectively known as Rhizobia are currently classified into six genera; Rhizobium, Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium and 91 species. Their inoculation may increase nodulation and N2-fixation in legumes. All these Rhizobiumn spp. can minimize chemical N fertilizers by BNF, but only if conditions for expression of N2-fixing activity and subsequent transfer of N to plants are favourable. In this Chapter, PGPR role has been discussed in the process of crop growth promotion, their mechanisms of action and their importance in crop production on sustainable basis.

Effect of cadmium on soybean (Glycine max L) growth and nitrogen fixation

-1 sand was created using Cd (NO 3)2. Soybean shoots and root lengths shoot and root biomass, nodule density and Cd uptake was recorded on 2, 4, 6, 8, 10, and 12 weeks after the emergence. To calculate the relative abundance of ureide and % P fix (proportion of plant N derived from N 2-fixation), xylem sap was collected and analyzed for ureide, nitrate and amino-N at pod fill stage. The application of Cd adversely affected soybean growth, nodulation and N 2 fixation as a function of time and increase in Cd concentration. Maximum reduction in the root and shoot length was found with higher Cd level that is 16 mg kg -1 sand after 10 weeks of the growth. Similarly, nodulation and the proportion of plant N (% P fix ) derived from N 2 fixation decreased sharply as Cd concentrations increased during the whole growth stages and the maximum reduction was observed in the Cd level of 16 mg kg -1 sand followed by 8 and 4 mg kg -1 sand, respectively. Cadmium uptake increased with the

Assessment of core and accessory genetic variation in Rhizobium leguminosarum symbiovar trifolii strains from diverse locations and host plants using PCR-based methods

The nitrogen‐fixing symbiosis between Rhizobium leguminosarum and host legumes is recognized as a key part of sustainable agriculture. A culture collection containing rhizobia isolated from legumes of economic importance in the UK and worldwide, maintained at Rothamsted Research for many years, provided material for this study. We aimed to develop and validate efficient molecular diagnostics to investigate whether the host plant or geographical location had a greater influence on the genetic diversity of rhizobial isolates, and the extent to which the core bacterial genome and the accessory symbiosis genes located on plasmids were affected. To achieve this, core housekeeping genes and those involved in symbiosis interactions were sequenced and compared with genome‐sequenced strains in the public domain. Results showed that some Rh. leguminosarum symbiovar trifolii strains nodulating clovers and Rh. leguminosarum sv. viciae strains nodulating peas and vicias shared identical housekeeping genes, clover nodule isolates from the same location could have divergent symbiosis genes, and others isolated on different continents could be very similar. This illustrates the likely co‐migration of rhizobia and their legume hosts when crops are planted in new areas and indicates that selective pressure may arise from both local conditions and crop host genotypes.

Characterization of plant growth promoting rhizobacteria isolated from chickpea (Cicer arietinum).

Aims: To isolate bacterial strains from chickpea rhizospheric soil and nodules, to characterize and identify potential bacterial strains by using 16S rRNA gene sequencing. Place and Duration of Study: Department of Soil Science & SWC, PMAS, Arid Agriculture University, Rawalpindi Pakistan between July 2010 and July 2011. Background: Plant growth promoting rhizobacteria are being preferred nowadays as inoculants for influencing crops via multiple direct or indirect mechanisms but screening to find out the effective PGPR strains is one of the crucial steps. This research is aimed at keeping in view their potential for phosphate solubilization, indole acetic acid and ammonia production. Methodology: Extensive survey was carried out in Pothwar (District, Rawalpindi, Attock and Original Research Article Ali et al.; BMRJ, 6(1): 32-40, 2015; Article no.BMRJ.2015.056 33 Chakwal) for collection of chickpea rhizospheric soil and root nodules. The isolation of rhizospheric soil bacteria was performed by using dilution plate technique while the root nodules bacteria were isolated on yeast extract mannitol agar supplemented with congo red. Ten bacterial strains designated as AM-1 to AM10 were isolated, purified and characterized for phosphate solubilization, indole acetic acid (IAA) and ammonia production. These bacterial strains were identified as belonging to species of Bacillus, Enterobacter, Pseudomonas, Rhizobium, Sphingobacterium, Pantoea and Chryseobacterium. All bacterial strains solubilized phosphate and produced IAA. Two bacterial strains AM-5 (Sphingobacterium canadense) and AM-4 (Rhizobium pusense) solubilized the maximum amount of phosphate i.e. 273.84 μg ml -1 and 262.83 μg ml -1 respectively with a significant pH drop from 7 to 2.67. These strains proved positive for ammonia production. Six most potential and identified strains were selected on the basis of plant growth promoting activities. Conclusion: AM-4 (Rhizobium pusense) and AM-5 (Sphingobacterium canadense) are efficient strains and there is a need of inoculation experiments under control and field conditions to use these stains as biofertilizer to enhance the growth and productivity of the chickpea.

Bioefficacy of Rhizobacterial Isolates against Root Infecting Fungal Pathogens of Chickpea (Cicer arietinum L.)

Chickpea is considered to be food for the poor in Pakistan. Its yield is much lower than expected due to infestation of a number of fungal pathogens. The present study was designed to determine the effect of rhizobacterial isolates against fungal pathogens infecting chickpea roots. RH-31, RH-32 and RH-33 were isolated from groundnut rhizosphere. Antifungal activities of these isolates were tested by seed treatment and soil application methods against three root fungal pathogens. Data on disease incidence, bio-control efficiency and root biomass was recorded. Phylogenetic analysis indicated that sequences of RH-31, RH-32 and RH-33 showed >99% identity with Paenibacillus illinoisensis, Bacillus subtilis, and Pseudomonas psychrotolerans respectively. RH-33 was effective against Fusarium oxysporum and Macrophomina phaseolina with highest levels of inhibition, whereas RH-32 inhibited Fusarium solani. However, RH-31 showed best activity against F. oxysporum. Disease incidence and bio-control efficiency revealed that all isolates reduced disease severity and increased overall plant biomass as compared to control treatment. Present findings show potential of bacterial isolates from rhizosphere of Pakistan. Application of selected rhizobacteria through seed treatment method might be a promising strategy to lower damage caused by root pathogens in chickpea. This could be efficient, economical, environment friendly and might serve as a biocontrol agent.

Old meets new: most probable number validation of metagenomic and metatranscriptomic datasets in soil

Metagenomics and metatranscriptomics provide insights into biological processes in complex substrates such as soil, but linking the presence and expression of genes with functions can be difficult. Here, we obtain traditional most probable number estimates (MPN) of Rhizobium abundance in soil as a form of sample validation. Our work shows that in the Highfield experiment at Rothamsted, which has three contrasting conditions (>50 years continual bare fallow, wheat and grassland), MPN based on host plant nodulation assays corroborate metagenomic and metatranscriptomic estimates for Rhizobium leguminosarum sv. trifolii abundance. This validation is important to legitimize soil metagenomics and metatranscriptomics for the study of complex relationships between gene function and phylogeny.

Inorganic nitrogen application affects both taxonomical and predicted functional structure of wheat rhizosphere bacterial communities

The effects of fertilizer regime on bulk soil microbial communities have been well studied, but this is not the case for the rhizosphere microbiome. The aim of this work was to assess the impact of fertilization regime on wheat rhizosphere microbiome assembly and 16S rRNA gene-predicted functions with soil from the long term Broadbalk experiment at Rothamsted Research. Soil from four N fertilization regimes (organic N, zero N, medium inorganic N and high inorganic N) was sown with seeds of Triticum aestivum cv. Cadenza. 16S rRNA gene amplicon sequencing was performed with the Illumina platform on bulk soil and rhizosphere samples of 4-week-old and flowering plants (10 weeks). Phylogenetic and 16S rRNA gene-predicted functional analyses were performed. Fertilization regime affected the structure and composition of wheat rhizosphere bacterial communities. Acidobacteria and Planctomycetes were significantly depleted in treatments receiving inorganic N, whereas the addition of high levels of inorganic N enriched members of the phylum Bacteroidetes, especially after 10 weeks. Bacterial richness and diversity decreased with inorganic nitrogen inputs and was highest after organic treatment (FYM). In general, high levels of inorganic nitrogen fertilizers negatively affect bacterial richness and diversity, leading to a less stable bacterial community structure over time, whereas, more stable bacterial communities are provided by organic amendments. 16S rRNA gene-predicted functional structure was more affected by growth stage than by fertilizer treatment, although, some functions related to energy metabolism and metabolism of terpenoids and polyketides were enriched in samples not receiving any inorganic N, whereas inorganic N addition enriched predicted functions related to metabolism of other amino acids and carbohydrates. Understanding the impact of different fertilizers on the structure and dynamics of the rhizosphere microbiome is an important step toward developing strategies for production of crops in a sustainable way.