Changes in populations of rhizosphere bacteria associated with take-all disease of wheat

Take-all, caused by Gaeumannomyces graminis var. tritici, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonas to Chryseobacterium species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundant Pseudomonas spp. and a phlD(+) Pseudomonas sp. showed any significant ability to inhibit G. graminis var. tritici directly. When cultured strains were mixed with the inhibitory phlD(+) Pseudomonas strain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing phlD(+) pseudomonads.     

Effects of ammonium and nitrate nutrition or take-all disease of wheat in pots

Plant debris, naturaiiy infested with the take-all fungus (Ophiobolus graminis), was washed from soil and added to a leached sandy loam, deficient in nitrate nitrogen (NO₃-N) and magnesium. Nutrient solutions containing potassium and phosphorus, with and without magnesium, were added to the amended soil unsupplemented, or with either NO₃-N, ammonium nitrogen (NH₄+-N), or both. Nitrification of NH₄+-N was inhibited by 2–chloro-6–(trichloromethy1)-pyridine (N-Serve). After 38 days at 19°C, fewer plants had take-all with N (75 or 100 mg/kg soil) than without and root systems were most discoloured and had most diseased axes when nutrients were not added. Plants given NH₄+-N developed less take-all when magnesium was present. A comparison of forms of N in the presence of added magnesium showed that take-all was least with a mixture of both forms of N, intermediate with NO₃-N alone and worst with NH₄+-N alone. The most extensive lesions on individual root axes occurred on plants given NH₄+-N. It is suggested that take-all will be least when the amounts and ratio of NH₄+-N and NO₃-N are optimum for the growth of the host.     

Plant genotype, micronutrient fertilization and take-all infection influence bacterial populations in the rhizosphere of wheat

The relationship between micronutrient efficiency of four wheat (Triticum aestivum L.) genotypes, tolerance to take-all disease (caused by Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker), and bacterial populations in the rhizosphere was tested in soil fertilized differentially with Zn and Mn. Plant growth was reduced by Mn or Zn deficiency and also by take-all. There was an inverse relationship between micronutrient efficiency of wheat genotypes when grown in deficient soils and the length of take-all lesions on roots (efficient genotypes had shorter lesions than inefficient ones). In comparison to the rhizosphere of control plants of genotypes Aroona and C8MM receiving sufficient Mn and Zn, the total numbers of bacterial cfu (colony forming units) were greater in the rhizosphere of Zn-efficient genotype Aroona under Zn deficiency and in Mn-efficient genotype C8MM under Mn deficiency. These effects were not observed in other genotypes. Take-all decreased the number of bacterial cfu in the rhizosphere of fully-fertilized plants but not of those subjected to either Mn or Zn deficiency. In contrast, the Zn deficiency treatment acted synergistically with take-all to increase the number of fluorescent pseudomonads in the rhizosphere. Although numbers of Mn-oxidising and Mn-reducing bacteria were generally low, take-all disease increased the number of Mn reducers in the rhizosphere of Mn-efficient genotypes Aroona and C8MM. Under Mn-deficiency conditions, the number of Mn reducers in the rhizosphere increased in Aroona but not in C8MM wheat. The results suggest that bacterial microflora may play a role in the expression of Mn and Zn efficiency and tolerance to take-all in some wheat genotypes.     

Nitrate production in the rhizosphere of Plantago species

Summary The production of nitrate in an old established dune grassland soil and its uptake by plants was studied by comparing amounts of mineral nitrogen and numbers of nitrifying bacteria in the rhizosphere on the one hand, and on the other accumulated nitrate and levels of nitrate reductase (NaR) of individual plants of three Plantago species,i. e., P. major, P. lanceolata andP. coronopus. For these three Plantago species andP. media basal levels of NaR in the absence of nitrate were determined in plants grown in culture solutions. The basal NaR levels ofP. major andP. media (species occurring on nutrient-rich soils) were significantly higher than those ofP. lanceolata andP. coronopus (species found on nutrient-poor soils). NaR activity increased in the presence of nitrate and was suppressed by ammonium.From the numbers of nitrifying bacteria in the rhizosphere and NaR activity in the leaves it was concluded that nitrate was produced in the root environments of the three Plantago species and that the compound was taken up by the plants. NaR activities and numbers of nitrifying bacteria were higher for individuals ofP. major than for those ofP. lanceolata andP. coronopus. No correlation was found between the ammonium levels and the numbers of nitrifying bacteria in the soil, and no indications of inhibition of nitrifying bacteria in the rhizosphere were obtained. For individuals ofP. lanceolata a correlation was found between the numbers of nitrifying bacteria in the soil and NaR activity in the leaves. The results are discussed in relation to the ecological habitats of the three species.Grassland Species Research Group Publication No.38.     

Observations on take-all and eyespot diseases of wheat in Yorkshire

Wheat crops were surveyed in Yorkshire from 1944 to 1946 on farms where crops were reported to be unsatisfactory. Take-all ( Ophiobolus graminis (Sacc.) Sacc.) and eyespot ( Cercosporella herpotrichoides Fron.) were both found to be present; the latter is the most serious trouble on the better wheat lands.
The variation in the incidence of these diseases on selected farms during the three seasons has been compared.
The effect of rotation has been examined; both diseases were found to be encouraged by too frequent cropping with wheat or barley. The incidence of disease in wheat crops following a 1-year seeds ley was found to be influenced by the nurse crop used to undersow the seeds, and also by the time at which the ley was ploughed up. Oats were found to be preferable to either wheat or barley as a nurse crop, and in 1946 late ploughing considerably reduced the amount of eyespot disease present in the following wheat crop.
A 1-year ley is considered to be of too short a duration to ensure the disappearance from the soil of Ophiobolus graminis and Cercosporella herpotrichoides surviving from the previous crop, and a period of 2–3 years is suggested as being desirable.     

Nitrate leaching stimulates subsurface root growth of wheat and increases rhizosphere alkalisation in a highly acidic soil

Subsurface acidity is a major factor limiting crop yield in some agricultural soils. The surface application of lime has limited effect on the subsurface acidity due to the slow downward movement, while deep incorporation of lime is costly. This paper tested the concept of biologically ameliorating subsurface acidity in a highly acidic soil through the net uptake of anions by plant roots. Nitrogen was supplied to the top soil (0–10 cm) as Ca(NO₃)₂ at rates equivalent to 30–240 kg N ha^?1. Four water levels were imposed (40, 60, 80 and 100% of field capacity). Aluminium-tolerant wheat was grown for 58 days. The high N and high water treatments stimulated root growth below 15 cm, which in turn increased N capture, resulting in a greater excess anion uptake over cations and thus alkalisation of subsurface soil layers. This study suggests that it is feasible to exploit the process of nitrate uptake by an aluminium-tolerant wheat genotype to increase pH in acidic subsoil.     

Nitrate supply induces changes in polyamine content and ethylene production in wheat plants grown with ammonium

In order to explore the mechanisms of nitrate's beneficial effect on ammonium-grown plants, we investigated the effects of nitrate on free and conjugated polyamine plant content and ethylene biosynthesis in wheat (Triticum aestivum L.) plants grown with ammonium nutrition. Two different doses of nitrate (100 microM and 5 mM) were supplied to ammonium-fed plants, in order to determine whether the effects of nitrate require significant doses (nutritional character), or can be promoted by very low doses (pseudo-hormonal character). Our results showed that nitrate's effects on putrescine, spermidine and spermine contents of ammonium-grown plants tended to follow the pattern associated with strict nitrate nutrition. Both low (100 microM) and high (5 mM) nitrate doses caused a rapid and significant increase in free spermidine content in roots and shoots, which was well correlated with reduced root ethylene production. In shoots, this increase in free spermidine was correlated with changes in the conjugation pattern, while in roots these changes appear to be due to alternative mechanisms. On the other hand, no clear relationship between the supply of a lower dose of nitrate (100 microM) and a reduction of free putrescine content was observed. With higher doses of nitrate (5 mM) we observed a reduction of free putrescine content that was well correlated with increases in its conjugated forms. In conclusion, nitrate's effects on putrescine, spermidine and spermine contents of ammonium-fed plants tended to follow the pattern associated with strict nitrate nutrition, corroborating its beneficial effect.     

Prospects for fungicidal control of take-all of wheat

A range of fungicides and herbicides was tested against Gaeumannomyces graminis (causal agent of take-all) on agar plates, and on wheat seedlings in pots and in liquid culture. Benomyl, the standard in all tests, was consistently most effective: like iprodione, nuarimol and KWG 0599 , it diminished infection from inoculum placed just below the seeds more effectively when applied as a drench than as a soil-mix. Benomyl as a soil-mix was most effective in soils with least organic matter. Some compounds toxic to the pathogen on agar plates and in plants grown in liquid culture were ineffective as soil treatments. The practical limitations of soil treatment with conventional fungicides and application methods are discussed.     

Role of antibiotics and siderophores in biocontrol of take-all disease of wheat

Both antibiotics and siderophores have been implicated in the control of soilborne plant pathogens by fluorescent pseudomonads. In Pseudomonas fluorescens 2–79, which suppresses take-all of wheat, the importance of the antibiotic phenazine-1-carboxylic acid was established with mutants deficient or complemented for antiobiotic production and by isolation of the antibiotic from the roots of wheat colonized by the bacteria. Genetic and biochemical studies of phenazine synthesis have focused on two loci; the first is involved in production of both anthranilic acid and phenazine-1-carboxylic acid, and the second encodes genes involved directly in phenazine synthesis. Because the antibiotic does not account fully for the suppressiveness of strain 2-79, additional mutants were analyzed to evaluate the role of the fluorescent siderophore and of an antifungal factor (Aff, identified as anthranilic acid) that accumulates when iron is limiting. Whereas strains producing only the siderophore conferred little protection against take-all, Aff⁺ strains were suppressive, but much less so than phenazine-producing strains. Iron-regulated nonsiderophore antibiotics may be produced by fluorescent pseudomonads more frequently than previously recognized, and could be partly responsible for beneficial effects that were attributed in the past to fluorescent siderophores.     

Experiments on soil drenching with fungicides against take-all in wheat

In short term pot experiments benomyl, iprodione and KWG 0599 applied as soil drenches in several types of soil significantly suppressed take-all symptoms from inoculum placed just below wheat seeds planted 1×5 cm deep, and in sand but not other soils when seeds were 5 cm deep. Benomyl was, however, effective against inoculum below seed planted 5 cm deep in a loam-sand mixture when the drench contained an alcohol ethoxylate surfactant. Computer simulations of fungicide distributions in the soils correlated well with disease control observations. In long term outdoor pot experiments two drenches with benomyl (without surfactant) controlled disease significantly for at least 3 months against inoculum placed 15 cm deep. The significance of these results for the practical control of take-all by fungicides is discussed.     

The use of ammonium fertilisers to reduce the severity of take-all (Gaeumannomyces graminis var. tritici) on wheat in Western Australia

In field experiments in Western Australia ammonium fertilisers significantly reduced the severity of root damage to wheat ( Triticum aestivum ) caused by Gaeumannomyces graminis var. tritici. A series of response lines depicting the effect of sources and rates of nitrogen on take-all are presented. In general, the higher the rate of ammonium nitrogen used, the greater the reduction in the severity of take-all. Ammonium sulphate and ammonium sulphate/mono-ammonium phosphate mixture were more effective in reducing disease than di-ammonium phosphate, urea or ammonium nitrate. Sodium nitrate did not reduce take-all.     

Nematode-enhanced microbial colonization of the wheat rhizosphere

The mechanisms by which seed-applied bacteria colonize the rhizosphere in the absence of percolating water are poorly understood. Without mass flow, transport of bacteria by growing roots or soil animals, particularly nematodes may be important. We used a sand-based microcosm system to investigate the ability of three species of nematodes (Caenorhabditis elegans, Acrobeloides thornei and a Cruznema sp.) to promote rhizosphere colonization by four strains of beneficial rhizobacteria. In nearly all cases, rhizosphere colonization was substantially increased by the presence of nematodes, irrespective of bacterial or nematode species. Our results suggest that nematodes are important vectors for bacteria rhizosphere colonization in the absence of percolating water.     

A role for managanese in the resistance of wheat plants to take-all

Summary The hypothesis that wheat plants deficient in managenese are predisposed to infection byGaeumannomyces graminis is outlined, and a test of the hypothesis in a soil system is reported. The results supported the hypothesis: wheat plants growing in managanese-deficient soil, although not showing foliar symptoms, were markedly more susceptible to infection; plant analysis confirmed the nutrient status of the plants. A review of the literature on take-all in wheat coupled with the results of our experiments suggests a reinterpretation of the etiology of this disease, since those edaphic factors which promote infection by this organism are those which also render managese unavailable to the host. Managenese nutrition is proposed as a common factor in many of the environmental conditions which influence the host-pathogen balance.     

Nitrate or ammonium nutrition in french bean

Summary Bean Plants were grown in a greenhouse in sand irrigated with nutrient solutions containing either 2 mM NO ₃ ^– or 2 mM NH ₄ ⁺ . After 45 days fresh weight of NH ₄ ⁺ plants was half that of NO ₃ ^– plants. Cation concentration in NH ₄ ⁺ plants was 30% less than in NO ₃ ^– plants. Amino acids (SER, ASN, GLN) accummulated 3 to 10 times more in NH ₄ ⁺ plants. The concentration of organic acids (malic, malonic, citric) was 10 to 30 times higher in NO ₃ ^– plants. The ATP-costings for the synthesis of amino acids and organic acids in NH ₄ ⁺ plants was half that of NO ₃ ^– ones: therefore it could not account for the reduction of growth in the ammonium-fed plants.     

Evolution of bacterial communities in the wheat crop rhizosphere

The gap between current average global wheat yields and that achievable through best agronomic management and crop genetics is large. This is notable in intensive wheat rotations which are widely used. Expectations are that this gap can be reduced by manipulating soil processes, especially those that involve microbial ecology. Cross‐year analysis of the soil microbiome in an intensive wheat cropping system revealed that rhizosphere bacteria changed much more than the bulk soil community. Dominant factors influencing populations included binding to roots, plant age, site and planting sequence. We demonstrated evolution of bacterial communities within the field rhizosphere. Early in the season, communities tightly bound to the root were simplest. These increased in diversity with plant age and senescence. Loosely bound communities also increased in diversity from vegetative to reproductive plant stages but were more stable than those tightly bound to roots. Planting sequence and, to a lesser extent, wheat genotype also significantly affected rhizosphere bacteria. Plasticity in the rhizosphere generated from crop root system management and genetics offers promise for manipulating the soil ecology of intense cereal systems. Analyses of soil microbiomes for the purpose of developing agronomic benefit should include roots as well as soil loosely adhered to the roots, and the bulk soil.     

Soil-applied fungicides for controlling take-all in field experiments with winter wheat

Soil treatment fungicides were tested against take-all (Gaeumannomyces graminis var. tritici) in three field experiments with winter wheat. Fungicides were applied as drenches either before sowing in autumn, and incorporated by rotary harrowing, or to the crop in spring. The most effective treatments were autumn applied benomyl (20 kg/ha) and nuarimol (0·55-4·4 kg/ha). However, the highest nuarimol concentration depressed yield. Benomyl sometimes induced a resurgence of take-all in the second wheat crop after treatment. Nuarimol had no adverse effects in subsequent crops, and neither fungicide hindered the onset of take-all decline in a third crop after treatment. The possible value of soil treatment in future control strategies is discussed.     

Rates of rhizodeposition and ammonium depletion in the rhizosphere of axenic oat roots

Summary A multiple split root chamber and artificial soil were developed which allowed for maintenance of axenic conditions and for the isolation of soil from specific regions of single roots. A sterile minirhizotron was used to measure patterns and rates of root extension under sterile conditions. Carbon and nitrogen distributions in the rhizosphere of sterile oat roots were measured in combination with rates of root elongation to calculate specific rates of rhizodeposition and ammonium nitrogen uptake. The highest rates of rhizodeposition C production and N depletion occurred at the root tip (first day segment). Rhizodeposited soluble and insoluble C compounds represented up to 50% of the standing root biomass C. Within 48 hours after root entry, levels of rhizosphere ammonium-N decreased by 40–50%. The results were summarized in a simple model of root growth, rhizodeposition, and NH ₄ ⁺ −H uptake. From a dissertation by the senior author submitted to the Academic Faculty of Colorado State University in partial fulfillment of the requirements for the Ph.D. degree.