Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating plant development

The basidiomycete fungus Piriformospora indica colonizes roots of a broad range of mono- and dicotyledonous plants. It confers enhanced growth, improves resistance against biotic and tolerance to abiotic stress, and enhances grain yield in barley. To analyze mechanisms underlying P. indica-induced improved grain yield in a crop plant, the influence of different soil nutrient levels and enhanced biotic stress were tested under outdoor conditions. Higher grain yield was induced by the fungus independent of different phosphate and nitrogen fertilization levels. In plants challenged with the root rot-causing fungus Fusarium graminearum, P. indica was able to induce a similar magnitude of yield increase as in unchallenged plants. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae, total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. On the other hand, barley plants colonised with the endophyte developed faster, and were characterized by a higher photosynthetic activity at low light intensities. Together with the increased root formation early in development these factors contribute to faster development of ears as well as the production of more tillers per plant. The results indicate that the positive effect of P. indica on grain yield is due to accelerated growth of barley plants early in development, while improved phosphate supply—a central mechanism of host plant fortification by arbuscular mycorrhizal fungi—was not observed in the P. indica-barley symbiosis.     

Exogenous auxin affects the oxidative burst in barley roots colonized by Piriformospora indica

Beside a cardinal role in coordination of many developmental processes in the plant, the phytohormone auxin has been recognized as a regulator of plant defense. The molecular mechanisms involved are still largely unknown. Using a sensitive chemiluminescence assay, which measures the oxidation of luminol in the presence of H₂O₂ by horseradish peroxidase (HRP), we report here on the ability of exogenously added indole-3-acetic acid (IAA) to enhance the suppressive effect of the root endophyte Piriformospora indica on the chitin-elicited oxidative burst in barley roots. Thus, the potential of P. indica to produce free IAA during the early colonization phase in barley might provide the symbiont with a means to interfere with the microbe-associated molecular patterns (MAMP)-triggered immunity.     

Piriformospora indica enhances plant growth by transferring phosphate

Piriformospora indica is an endophytic fungus that colonized monocot as well as dicot. P. indica has been termed as plant probiotic because of its plant growth promoting activity and its role in enhancement of the tolerance of the host plants against abiotic and biotic stresses. In our recent study, we have characterized a high affinity phosphate transporter (PiPT) and by using RNAi approach, we have demonstrated the involvement of PiPT in P transfer to the host plant. When knockdown strains of PiPT-P. indica was colonized with the host plant, it resulted in the impaired growth of the host plants. Here we have analyzed and discussed whether the growth promoting activity of P. indica is its intrinsic property or it is dependent on P availability. Our data explain the correlation between the availability of P and growth-promoting activity of P. indica.Key words: Piriformospora indica, phosphate transport, plant growth promotionPhosphorous (P) is one of the most essential mineral nutrients for plant growth and development. In the soil P is present mainly in the form of sparingly soluble complexes that are not directly accessible to plants. Thus, it is the nutrient that limits crop production throughout the world.¹ Plants have therefore evolved a range of strategies to increase the availability of soil P, which include both morphological and biochemical changes at the soil-root interface. For example, increased root growth and branching, proliferation of root hairs, and release of root exudates can increase plant access to inorganic phosphate (Pi) from otherwise poorly available sources.^2,3 Plant root possess two distinct modes of phosphate uptake, direct uptake by its own transporters and indirect uptake through mycorrhizal associations. In plants several high affinity P transporters specifically associated with the uptake of Pi from soil solution. Expression of these transporters is induced in response to P deficiency and enables Pi to be effectively taken up against the large concentration gradient that occurs between the soil solution and internal plant tissues.⁴ However, in arbuscular mycorrhizal associations (indirect uptake), plants acquire Pi from the extensive network of fine extra radical hyphae of fungus, that extend beyond root depletion zones to mine new regions of the soil.⁵ In the case of arbuscular mycorrhizal fungi (AMF), including Glomus versiforme and G. intraradices, the regulation of phosphate transporters that are expressed, typically upregulated under P deficiency but their role in P transfer to the host plant have not been characterized.^5,6P. indica was reported to be involved in high salt tolerance, disease resistance and strong growth-promoting activities leading to enhancement of host plant yield.^7–9 Recently, we have shown the role of PiPT in the P transport to the host plant.¹⁰ Here we discuss the performance of P. indica (grown under P-rich and -deprived conditions and colonized with the host plant) and its involvement in the P transportation to, and the growth of the host plant.     

In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica

The present study was conducted for optimization of in vitro substrates under aseptic conditions for interaction of Piriformospora indica with the medicinal plant Coleus forskohlii. It aims to test the effects of different substrates on P. indica colonization as well as growth parameters of the in vitro raised C. forskohlii. Interaction of in vitro C. forskohlii with root endophyte P. indica under aseptic condition resulted in increase in growth parameters in fungus colonized plants. It was observed that P. indica promoted the plant’s growth in all irrespective of substrates used for co-culture study. The growth was found inferior in liquid compared to semisolid medium as well as there was problem of hyperhydricity in liquid medium. P. indica treated in vitro plantlets were better adapted for establishment under green house compared to the non treated plants due to fungal intervention.     

Piriformospora indica affects plant growth by auxin production

Piriformospora indica has been shown to improve the growth of many plant species including Arabidopsis thaliana , but the mechanism by which this is achieved is still unclear. Arabidopsis root colonization by P. indica was examined in sterile culture on the medium of Murashige and Skoog. P. indica formed intracellular structures in Arabidopsis root epidermal cells and caused changes in root growth, leading to stunted and highly branched root systems. This effect was because of a diffusible factor and could be mimicked by IAA. In addition, P. indica was shown to produce IAA in liquid culture. We suggest that auxin production affecting root growth is responsible for, or at least contributes to, the beneficial effect of P. indica on its host plants.     

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.     

Visual assessment of grain yield and its components in single plants and ears of spring barley

Visual assessments of yield/plant and tillers/plant were more effective than unaided assessments of grains/ear and 1000-grain weight on a per plant basis. Assessments of the latter yield components were generally improved by the use of specially developed keys described by Ismail & Valentine (1983). Gains in efficiency were associated with the amount of extra time spent on making these assessments. Assessments of yield/ear were not more effective than assessments of yield/plant. This was attributed to the ease of assessment of tillers/plant which was strongly associated with yield/plant. Assessments of grains/ear and 1000-grain weight in single ears were clearly more effective than corresponding assessments in single plants in which there is high variability between ears. Apart from using keys, further improvements in the assessments of grains/ear and 1000-grain weight are necessary in order to increase the effectiveness of single plant selection in the F₂ generation which is the first opportunity of increasing yield in later generations.     

Yield increase induced by the fungal root endophyte Piriformospora indica in barley grown at low temperature is nutrient limited

The fungal root endophytes Chaetomium globosum, Epicoccum nigrum and Piriformospora indica have value as biocontrol and biofertilising organisms in barley, but have not been well tested at low temperatures. This study assessed the efficacy of the endophytes on barley varieties grown under low temperature stress with variable nutrient input. Seed from three cultivars of spring barley were inoculated with one of the three fungal root endophyte isolates – C. globosum, E. nigrum or P. indica - and grown in low temperature under higher and lower nutrient input regimes. Compared with the control, for P.indica-inoculated plants with the higher nutrient input, flowering was earlier and grain dry weight significantly greater for all barley varieties by a mean of 22 %. The nitrogen and carbon content of the grains did not differ significantly between treatments. Chaetomium globosum and Epicoccum nigrum conferred no significant benefits under either nutrient regime. Piriformospora indica is amenable to axenic culture, sporulates readily and can be multiplied rapidly, suggesting that it could be developed as an effective crop treatment in low temperature stressed barley and may have the potential to increase crop yield in colder growing conditions provided that adequate nutrients are supplied.     

Grain yield and ear development of spring barley as influenced by environmental conditions during early stages of plant development

Barley plants were grown until day 21 under conditions which were different in relation to photon flux density, carbon dioxide concentration and temperature. Dry weight and leaf area increase from day 7 until day 21, shoot apex development between day 15 and day 47, and yield of each treatment group were considered. Photon flux density was demonstrated to have a greater influence on net assimilation rate (NAR) of young plants than has carbon dioxide enrichment. High temperature treatment seems to influence NAR less than growth and developmental processes. Grain yield of high temperature treated plants was significantly lower than that of the other treatment groups. Significant correlations have been found between growth analysis values of young plants and some yield components of each treatment.     

Effects of fungicide treatments and variety on development, grain growth and yield of spring barley

The effects of four fungicide treatments for the control of mildew on spring barley were assessed in three field experiments, one in each of the years 1981, 1982, 1983. The fungicide treatments (+/ - triadimenol seed treatment and +/ - triadimefon foliar spray applied during early booting) were chosen to control mildew, and hence affect yield determining processes, at different times in the life of the crop. Two of the experiments also tested different nitrogen amounts and the third tested four varieties differing in their degree of mildew resistance. Mildew appeared too late to affect the production and survival of spikelets and shoots, but reduced average grain weight by reducing the rate of grain growth. Grains in the upper part of the ear had a considerably lower growth rate and final weight than grains in central and basal positions but there was no evidence that the effects of mildew on grain size depended upon grain position within the ear. Mildew incidence increased with increasing nitrogen and varietal susceptibility but there were few significant interactions between these factors and fungicide treatment for grain yield. The degree of mildew control achieved by the seed treatment varied with barley variety. Use of the two successive fungicide treatments did not yield more barley than use of either alone. Amongst varieties, grain positions within the ear and fungicide treatments there was a close correlation between rate of grain growth and final grain weight. Duration of grain growth was not related to rate of grain growth or final grain weight but was inversely correlated with mean temperature during the period of rapid grain growth. The temperature sums during the period of rapid grain growth were similar for the three years and it is suggested that a more precise knowledge of the relationships between mildew incidence, varietal susceptibility and rate of grain growth may enable more accurate predictions to be made about likely yield responses to fungicide treatments.     

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica.     

Effect of soil salinity on grain filling and grain development in barley

Experiments were conducted with five barley cultivars with the aim of ascertaining the effect of salt stress on grain filling, grain development and bioohemical composition of developing grains. Reduced grain yield under salt stress was found to be due to reduced efficiency per day to fill the grains and consequent more effective days and also due to disturbed starch-sugar balance.     

Relationships between early vigour, grain yield, leaf structure and stable isotope composition in field grown barley

Fast growth and early development in barley are used in breeding programmes to improve the water use efficiency and transpiration efficiency of this crop in Mediterranean conditions. Here, we examine the use of several simple traits based on the structure and stable isotope composition of seedling leaves to assess differences in early vigour, phenology and grain yield, and also the interaction with low temperatures in barley. A set of 260 F₈ lines of two-row barley (Hordeum vulgare L.) derived from the cross of Tadmor and WI 2291 were cultivated in two locations in northwest Syria. Total chlorophyll content on an area basis (SPAD) and specific leaf dry weight (SLDW) were measured in recently fully expanded intact leaves of seedlings. Total leaf area and total dry weight per seedling were evaluated in the same seedlings. The stable isotope compositions of carbon and nitrogen (δ¹³C and δ¹⁵N, respectively) were analyzed in the same leaves on a subset of 75 genotypes. Number of days from planting to heading and grain yield were recorded at both sites. The grain yield measured at both locations was positively correlated with the SPAD value of seedlings, but showed no relationship with SLDW. Days to heading was negatively correlated with SPAD values. Regarding early vigour, a negative relationship between the SLDW and the total leaf area of seedlings was observed. However, no relationship between the δ¹³C of seedlings and early vigour was observed, except when only the genotypes most resistant to low temperatures (i.e. showing the highest SPAD values) were considered. This subset of genotypes showed negative relationships between δ¹³C and either total leaf area or total dry weight. In addition, δ¹⁵N was negatively correlated with SPAD only within the high-SPAD genotypes. This suggests that within the genotypes resistant to low temperatures, those with higher chlorophyll content assimilate more nitrogen from nitrate.     

Application of inorganic carrier-based formulations of fluorescent pseudomonads and Piriformospora indica on tomato plants and evaluation of their efficacy

Aims: Fluorescent pseudomonads are widely used as bioinoculants for improving plant growth and controlling phytopathogenic fungi. Piriformospora indica (Pi), a symbiotic root endophyte, also has beneficial effects on a number of plants. The present study focuses on the improvement of growth yields of tomato plants and control of Fusarium wilt using inorganic carrier‐based formulations of two fluorescent pseudomonad strains (R62 and R81) and Pi. Methods and Results: The inorganic carrier‐based formulations of pseudomonad strains and Pi were tested for plant growth promotion of tomato plants under glass house and field conditions. In controlled glass house experiments, 8·8‐fold increase in dry root weight and 8·6‐fold increase in dry shoot weight were observed with talcum powder‐based consortium formulation of R81 and Pi. Field trial experiments ascertained the glfass house results with a considerable amount of increase in plant growth responses, and amongst all the treatments, R81 + Pi treatment performed consistently well in field conditions with an increase of 2·6‐, 3·1‐ and 3·9‐fold increase in dry root weight, shoot weight and fruit yield, respectively. The fluorescent pseudomonad R81 and Pi also acted as biocontrol agents, as their treatments could control the incidence of wilt disease caused by Fusarium oxysporum f.sp. lycopersici in tomato plants under glass house conditions. Conclusions: The culture broths of pseudomonads R62, R81 and Pi were successfully used for development of talcum‐ and vermiculite‐based bioinoculant formulations. In controlled glasshouse experiments, the talcum‐based bioinoculant formulations performed significantly better over vermiculite‐based formulations. In field experiments the talcum‐based consortium formulation of pseudomonad R81 and Pi was most effective. Significance and Impact of the Study: This study suggests that the formulations of pseudomonad strains (R62 and R81) and Pi can be used as bioinoculants for improving the productivity of tomato plants. The application of such formulations is a step forward towards sustainable agriculture.