A scanning electron microscope study of interactions between micro-organisms andGaeumannomyces graminis (Syn.Ophiobolus graminis) on wheat roots

Roots of wheat grown in unsterilized sand inoculated withGaeumannomyces graminis (Sacc.) von Arx and Olivier were examined by scanning electron microscopy. Healthy roots had a mucilaginous covering and were sparsely colonized by bacteria, but asG. graminis colonized the roots the mucilage disappeared and the numbers of bacteria on the surface increased. Lysis of the hyphae occurred, apparently caused by bacteria that colonized the hyphae. Inoculation of wheat in axenic culture with a strain ofPseudomonas fluorescens that was antagonistic toG. graminis in agar gave some protection against the pathogen; lysis of hyphae was observed where protection occurred.     

Control of microorganisms in the rhizosphere of wheat by inoculation of seeds withPseudomonas putida and by foliar application of urea

After inoculation of wheat seeds with various bacterial strains germination of plants was usually inhibited at first but growth was stimulated later. After inoculation withPseudomonas putida K 11 producing physiologically active compounds the total number of bacteria increased together with the bacteria: fungi ratio in the rhizosphere. These characteristic were further increased after foliar application of urea due to increased root exudation. Dry mass of upper wheat parts was about 15 — 80 % higher in green-house experiments, in which the plants were treated in the two above ways. More reliable results, were usually obtained by bacterization ofP. putida and foliar application of urea as compared with the situation when the seeds were inoculated without the foliar application or, on the contrary, after foliar application without inoculation of the seeds. Only when urea was applied early and in a soil contaminated with the fungusGaeumannomyces graminis var.tritici (causing “take-all” of the wheat) no favourable results could be detected. In these cases the foliar application without inoculation of the seeds was more successful. Symptoms of the disease of wheat roots caused byG. graminis were less frequently observed after the inoculation of the seeds with the strainP. putida K 11 and after the foliar application of urea.     

Microbiota in Wheat Roots Evaluated by Cloning of ITS1/2 rDNA and Sequencing

Fungi (17 species), oomycetous organisms (four species of Pythium) and a plasmodiophorid (Polymyxa graminis) were recorded in wheat roots analysed by cloning of the total ITS1/2 rDNA and sequencing of representative clones. Roots of a fourth successive wheat crop were inhabited mostly by fungal pathogens including Gaeumannomyces graminis var. tritici, Monographella nivalis var. nivalis, Ophiosphaerella sp. and Helgardia anguioides. Roots of a first wheat crop were inhabited mostly by P. graminis and saprotrophic Pythium species. Results on fungal diversity and density were compared with those obtained by pure culture isolation and morphotyping. Only M. nivalis var. nivalis and H. anguioides were identifed in wheat roots by both the molecular and the pure culture isolation methods. New and additional evidence for the ecological roles of the species recorded is discussed.     

The effect of foliar application of urea on the root fungi of wheat growing in soil artificially contaminated withFusarium spp.

Repeated treatment of overground parts of wheat with a solution of urea was reflected in an increased count of bacteria and a decreased count of fungi on the roots of plants grown in soil contaminated with the fungusFusarium spp., which is known to be noxious to plants. The occurrence of some fungal genera on the roots of plants growing in contaminated and uncontaminated soil after foliar application of urea was also changed. The roots of treated planted were found to contain more bacteria with a lytic effect and retarding the growth of the fungus used for the contamination. The favourable effect of foliar application of urea on wheat was reflected in an increased dry weight of plants even if growing in a soil contaminated withFusarium.     

Effect of temperature on growth of some avirulent fungi and cross-protection against the wheat take-all fungus

The linear growth rates of Gaeumannomyces graminis var. graminis, G. graminis var. tritici, Phialophora radicicola var. graminicola and a lobed hyphopodiate Phialophora sp. were studied on agar at various temperatures between 5 and 30 °C and on wheat roots at two temperature regimes (12 h at 7°/12 h at 13 °C and 12 h at 17°/12 h at 23 °C). On agar at 30 °C, the isolates of G. graminis graminis grew faster than those of G. graminis tritici and Phialophora sp. but three isolates of G. g. graminis grew more slowly than the other two fungi at 5 and 10 °C. Two other isolates of G. g. graminis were cold-tolerant and had growth rates comparable to those of G. g. tritici and Phialophora sp. at 10 °C. The growth rates of Australian isolates of P. radicicola graminicolu were similar to that of a British isolate and were about a third to a half those of the other three fungi at most temperatures. The growth rates of the fungi on wheat roots at the low and high temperature regimes were correlated with the growth rates on agar at 10 and 20 °C respectively. The correlation was better at low temperatures r= 0.81) than at high temperatures (r = 0.62). Cross-protection experiments using two G. g. graminis isolates which grow poorly at temperatures below 15 °C and a cold-tolerant isolate each of G. g. graminis and Phialophora sp. showed that, while all four fungi protected wheat against take-all at high temperatures (17/23 °C) as evidenced by less severe disease and significantly greater dry weights, only the cold-tolerant fungi were effective at low temperatures (7/13 °C). The use of cold-tolerant isolates of avirulent fungi in field experiments may result in better protection in the early stages of wheat growth when Australian soil temperatures are mostly below 15 °C.     

Effects of butyltins and inorganic tin on chemotaxis of aquatic bacteria

Summary Tributyltin (TBT) and its degradation products dibutyltin (DBT), monobutyltin (MBT) and Sn^IV were toxic toPseudomonas fluorescens SHC-6 andSerratia sp. Gil-1 with EC₅₀ values in the range of 10^–3 to 10^–4M. These four compounds were negative chemotactic agents forP. fluorescens, and the butyltins were negatively chemotactic forSerratia sp. at concentrations over four orders of magnitude lower than the EC₅₀ values.l-Aspartate was a positive chemotactic agent for both organisms. TBT, DBT and MBT negated the effect ofl-aspartate onP. fluorescens but not onSerratia sp. Thus, TBT has the potential to affect microbial populations at concentrations much lower than those which prevent growth, and degradation of TBT does not always detoxify it. SnCl₄ was less toxic than TBT or DBT to these organisms and it was not chemotactic forSerratia sp. Gil-1. Tributylamine and tributylphosphate were less than 1/10th as toxic as TBT and they did not have a chemotactic effect on either organism at concentrations at which TBT had a significant effect. Therefore, both the Sn-and butyl-moieties contribute to the toxic and chemotactic properties of TBT.     

Polymer production by a mucoid stain of Azotobacter vinelandii in batch culture

Summary Influence of laboratory growth conditions and soil conditions on the survival of Pseudomonas fluorescens R1, a spontaneous rifamycin-resistant mutant of P. fluorescens W1, in soil was investigated. Strain R1 is antagonistic to Gaeumannomyces graminis and Rhizoctonia solani in vitro. Characteristics of W1 are scarcely different from those of R1 with respect to growth rates, plasmids, and the ability to use 56 substrates as carbon sources. Growth conditions varied were: medium composition, pH, temperature, carbon source, and nitrogen source. Strain R1 was released into a laboratory model ecosystem and soil conditions were varied with respect to sterility, texture, moisture content, temperature, and addition of carbon and nitrogen sources. The effect of initial population density of R1 on survival in soil was also investigated. The survival of R1 in soil was influenced only to a small extent by different growth conditions; soil conditions were of greater importance. However, no conditions could be found to maintain more than 0.7% of the initial number of cells in soil. No positive rhizosphere effect in glasshouse experiments was observed. Bacterization of wheat seeds with suspensions of R1 led to an increase in cells on developing roots. A further Pseudomonas strain, P. fluorescens W2, isolated from wheat roots, showed the same survival characteristics as R1.     

Changes of microflora of wheat roots after foliar application of urea

Upper parts of wheat grown in containers with brown soil were four-times repeatedly treated with a solution of urea. The foliar application of urea resulted in a considerable increase of the number of bacteria in the roots and a decrease of the number of fungi. Seventeen and 13 genera of fungi could be detected in the rhizosphere of the treated plants, 3 and 5 weeks, respectively, after the application, whereas 23 and 16 genera could be identified in the rhizoplane of the treated plants. Fungi of the generaFusarium andPenicillium predominated in the root region. The foliar application of urea resulted in an increased relative occurrence of the genusFusarium and decreased relative representation of the genusPenicillium. Changes in the relative occurrence of other 18 genera of fungi present in small quantities on wheat roots could also be observed. The foliar application of urea affected not only numbers of fungi determined by the dilution plate method but also the occurrence of active hyphae.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Investigations on fungicides: XIV. Fungitoxicity of N-(2,2,2–trichloro-1–methoxyethyl)-formamide and related compounds

From a study of forty compounds related to the systemic fungicide N,N'-bis(1–formamido-2,2,2–trichloroethyl)-piperazine, known as triforine (CELAW524), it was found that a new compound, N-(2,2,2–trichloro-1–methoxyethyl)-formamide, controls Erysiphe graminis on wheat when applied to the roots in sand. A number of N-(2,2,2–trichloro-1–aryloxyethyl)-form-amides and alkyl N-(2,2,2–trichloro-1–arylaminoethyl)-carbamates protect broad beans against Uromyces fabae and wheat against Erysiphe graminis; some of these formamides show slight activity when applied to plant roots.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> bacteria and phosphorous fertilization,affecting wheat (<Emphasis Type="Italic">Triticum</Emphasis><Emphasis Type="Italic">aestivum</Emphasis> L.) yield and P uptake under greenhouse and field conditions

We have recently indicated the plant growth promoting activities of Pseudomonas sp. as well as their alleviating effects on some soil stressors such as salinity. This is because in recent years, biological fertilizers have received special attention by scientists in sustainable agriculture. Accordingly, it is pertinent to specify the beneficiary level of such soil bacteria on plant growth including phosphorous (P) uptake. Hence, the objectives were to determine: (1) the plant growth promoting effects of the tested Pseudomonas sp., and (2) its combined effects with different P fertilization rates on the nutrient uptake (N, P, and K) and yield of wheat (Triticum aestivum L.) under greenhouse and field conditions. The experiments were factorially arranged on the basis of a completely randomized block design with three replicates and were conducted at the Research Farm of Agriculture and Natural Resources Research Center of Khorasan, Mashhad, Iran. P was fertilized at three levels including 0, 25 and 50 kg/ha P₂O₅. Pseudomonas sp. including Pseudomonas fluorescens 153, P. fluorescens 169, P. putida 4, and P. putida 108 were tested. Activities such as production of ACC deaminase and IAA-like products, as well as P solubilization were among the most important activities of the tested Pseudomonas sp. Such bacterial effects greatly enhanced wheat growth and yield under greenhouse and field conditions. The results also showed that the effects of Pseudomonas sp. on wheat nutrient uptake and the effects of bacteria as well as P fertilization on wheat yield were significant. P. putida 108 was the most effective strain enhancing wheat P uptake and grain yield under greenhouse (96 and 58%) and field (80 and 37%) conditions, respectively. Hence, although Pseudomonas sp. could be a suitable replacement for high P fertilization, however, the optimum wheat yield resulted when the bioinoculants are combined with 50% (25 kg/ha P₂O₅) P fertilization. This finding has great agricultural and environmental implications.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

Distribution of a Take-All Suppressive Strain of Pseudomonas fluorescens on Seminal Roots of Winter Wheat

An antibiotic-resistant strain of Pseudomonas fluorescens, that suppresses take-all of wheat, was used to study the distribution of the bacteria on seminal roots of wheat after being introduced onto seeds. Cells of P. fluorescens were isolated from the entire length of the root, and density of the introduced bacteria declined with the distance from the base of the root. Maximum populations of 10⁵ to 10⁶ CFU and 10³ to 10⁵ CFU per cm of root were detected on sections of roots near the seed and root tip, respectively. The introduced bacteria competed well with indigenous bacteria, comprising at least 25% of the fluorescent pseudomonads detected by plate counts for 48 days after planting.     

Endophytic Actinomycetes from <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.: Isolation,Diversity, and Anti-microbial Activity

Endophytic actinomycetes from Azadirachta indica A. Juss. were screened and evaluated for their anti-microbial activity against an array of pathogenic fungi and bacteria. A total of 55 separate isolates were obtained from 20 plants, and 60% of these showed inhibitory activity against one or more pathogenic fungi and bacteria. Actinomycetes were most commonly recovered from roots (54.5% of all isolates), followed by stems (23.6%), and leaves (21.8%). The dominant genus was Streptomyces (49.09% of all isolates), while Streptosporangium (14.5%), Microbispora (10.9%), Streptoverticillium (5.5%), Sacchromonospora sp. (5.5%), and Nocardia (3.6%) were also recovered. Streptomyces isolates AzR 006, 011, and 031 (all from roots) had acute activity against Pseudomonas fluorescens, while AzR027, 032, and 051 (also all from roots) showed activity against Escherichia coli. Meanwhile, an isolate of Nocardia sp. from leaves (AzL025) showed antagonism against Bacillus subtilis. Overall, 32 of the 55 were found to have broad spectrum significant antimicrobial activity, while about 4% of them showed strong and acute inhibition to pathogenic fungi and bacteria. Isolates of Streptomyces AzR031, 008, and 047, Nocardia sp. AzL025, and Streptosporangium sp. AzR 021 and 048 are of particular interest because they showed significant antagonistic activity against root pathogens, including Pythium and Phytophthora sp. Thus, many of the isolates recovered from A. indica in this study may be used in developing potential bio-control agents against a range of pathogenic fungi and bacteria and in the production of novel natural antimicrobial compounds. These results not only further our understanding of plant–microbe interactions but also indicate that there is an untapped resource of endophytic microorganisms that could be exploited in the biotechnological, medicinal, and agricultural industries.     

A comparative study of Phialophora radicicola, an avirulent fungal root parasite of grasses and cereals

The biology and infection-behaviour of a typical isolate of Phialophora radicicola Cain have been compared with those of a representative isolate of Ophiobolus graminis (Sacc.) Sacc. Both species can utilize a nitrate source of nitrogen and both require thiamine and biotin for growth on inorganic nitro-gen; P. radicicola, but not O. graminis, was able to synthesize biotin when grown on asparagine as a nitrogen source. The pH range for good growth of P. radicicola in nutrient solution was narrower than that for O. graminis, and its growth rate on agar was only one-third. P. radicicola was the more active decomposer of cellulose, and its cellulolysis adequacy index was I.66 as com-pared with a value of 0.33 for 0. graminis. In agreement with prediction from Garrett's (I966) hypothesis on the cellulolysis adequacy index, saprophytic survival of P. radicicola in wheat straw was shortened by additional soil nitrogen, which prolongs survival of O. graminis.P. radicicola was found to spread ectotrophically over the roots of wheat, oats and barley by runner hyphae indistinguishable from those of O. graminis, but cortical infection caused no necrosis and no discernible check to growth of the infected cereals, nor any significant decrease in grain yield of inoculated wheat grown to maturity. Pre-existing infection of wheat roots by P. radicicola retarded spread of infection by O. graminis; inoculation of several grass species with P. radicicola reduced the extent of infection by O. graminis of wheat following the grasses.     

d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp.

Summary A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The amidohydrolase activity of Agrobacterium sp. was strongly inhibited by ammonium ions co-produced with D-PHPG, whereas the hydantoinase activity under the same conditions was unaffected. Optimum temperature and pH were respectively 55° C and 10 for the partially purified hydantoinase, 45° and 6.75 when resting cells were used. Biotransformation under these slightly acidic conditions allowed to complete conversion of 30 g/1 DL-5-PHPH into 25 g/l of D-PHPG (molar yield 96%) and involved enzymatic racemization of DL-5-PHPH. Offprint requests to: S. Runser     

Anaerobic dissolution of iron-phosphorus complexes in sediment due to the activity of nitrate-reducing bacteria

Nitrate-reducing bacteria (Pseudomonas fluorescens andAlcaligenes sp.) as well as extracellular compounds from these bacteria increased the dissolution rate of iron and phosphorus sorbed to iron precipitates during anaerobic, nitrate-free conditions in experimental sediment-water systems. It is suggested that the influence of the bacteria is due to enzymatic catalyzation of chemical iron reduction.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

Biological control of winter wheat pathogens with the use of antagonistic Sphingomonas bacteria under greenhouse conditions

This paper describes, for the first time, the effect of bacteria of the genus Sphingomonas on healthiness of winter wheat. The effect of the application of Sphingomonas cell suspension on development of disease symptoms of powdery mildew and Fusarium head blight (FHB) of winter wheat cv. Bogatka was studied under greenhouse conditions. The abundance of populations of yeast and fungi producing mycelium as well as bacteria of the genus Azotobacter and pseudomonads was determined on wheat kernels. The biocontrol agent reduced the population size of Fusarium poae, and it contributed to better grain filling. The tested Sphingomonas isolate reduced the severity of flag leaf infection caused by pathogenic biotroph Blumeria graminis f. sp. tritici.     

Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955

Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.