Soil amoebae and saprophytic survival of Gaeumannomyces graminis tritici in a suppressive pasture soil

Hyphae of Gaeumannomyces graminis var. trilici deposited on millipore niters were buried in a naturally-suppressive permanent pasture soil and in a non-suppressive wheat-field soil. Hyphal density and survival of pigmented hyphae declined at a faster rate in the pasture soil than in the wheat-field soil. Hyphae recovered from the suppressive soil showed a higher association of mycophagous and other soil amoebae and scanning electron microscopy of these hyphae showed extensive erosion and discrete perforations in their walls. The possible role of soil amoebae in reducing saprophytic survival of the take-all fungus is discussed.     

Amoebae from a take-all suppressive soil which feed on Gaeumannomyces graminis tritici and other soil fungi

Amoebae were isolated from soil of the Waite Institute permanent pasture plot which is suppressive to take-all of wheat. Nine species of amoebae belonging to eight genera were tested for their mycophagy against Gaeumannomyces graminis var. tritici, Cochliobolus sativus and Phytophthora cinnamomi. Members of the genera, Gephyramoeba, Mayorella, Saccamoeba, Thecamoeba and an unidentified species of the order Leptomyxida, were mycophagous. Feeding of mycophagous amoebae and their ability to perforate and lyse melanized propagules of fungi are discussed.     

Wheat-rhizoplane pseudomonads as antagonists of Gaeumannomyces graminis

The role of rhizoplane-inhabiting Pseudomonas spp as inhibitors of take-all on wheat was investigated. Apparent numbers of pseudomonads in wheat rhizoplanes and numbers that were antagonistic in vitro toward Gaeumannomyces graminis var, tritici did not differ when wheat was supplied with NH⁺₄-N or NO^?₃-N. More intense antagonism was expressed by colonies selected from soil treated with NH⁺₄-N than with NO^?₃-N, and from isolation media prepared at pH 5.5 rather than at 7.0. Antagonists were not recovered from methyl bromide-treated soil. Highly antagonistic pseudomonads were recovered from a wheat-monoculture soil which is considered suppressive toward the pathogen in the field, and were not recovered from a “nonsuppressive” soil. Pseudomonad antagonism ratings were inversely correlated with take-all severity in the suppressive soil, but not in the nonsuppressive soil. Pseudomonads were considered to be antagonists of G. graminis on rhizoplanes of wheat in a soil exhibiting the “take-all decline” phenomenon, but the significance of this interaction remains to be determined.     

Soils suppressive to Gaeumannomyces graminis var. tritici: Effects on other fungi

The effect of soils suppressive to Gaumannomyces graminis var. tritici (Ggt) on the severity of root and crown rots caused by Rhizoctonia solani, Gibberella zeae, Pythium irregulare, Cochliobolus sativus and Fusarium culmorum was tested in pot bioassays. An induced suppressive soil was obtained from the rhizosphere of wheat plants grown at 15°C for 28 days in fumigated soil inoculated with live inoculum (colonized oat grain) of Ggt.Root rot caused by R. solani was significantly less in soil amended with either induced or naturally suppressive soil. Disease caused by the other pathogens was also reduced by the induced suppressive soil, with the least reduction occurring with F. culmorum.Colonization of the surfaces of seminal roots of wheat plants by Gaeumannomyces graminis var. graminis (Ggg) and a Phialophora-like fungus (Plf 119) was also studied using the line-intercept method. In non-suppressive soil the maximum area of the primary seminal root colonized by Ggg was 7.4 per cent and by Plf 119 was 3.3 per cent. Colonization of roots by Ggg and Plf 119 was reduced substantially by the addition of induced suppressive soil.     

Factors impacting the activity of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens against take-all of wheat

Take-all, caused by Gaeumannomyces graminis var. tritici, is an important soilborne disease of wheat worldwide. Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents of take-all and provide natural suppression of the disease during wheat monoculture known as take-all decline. To identify factors that could contribute to the effectiveness of 2,4-DAPG producers in take-all suppression, P. fluorescens strains Q8r1-96 (genotype D) and Q2-87V1 (genotype B; reduced antibiotic production) were tested against three pathogen isolates differing in sensitivity to 2,4-DAPG (LD5, ARS-A1 and R3-111a-1) and two wheat cultivars (Tara and Buchanan). The antibiotic sensitivity of the pathogen and cultivar significantly affected the level of take-all suppression by Q8r1-96 and Q2-87V1; suppression was greatest with LD5 and Tara. Q8r1-96 suppressed ARS-A1 and R3-111a-1 on Tara but not Buchanan, and Q2-87V1 failed to suppress either pathogen isolate on either cultivar. Q8r1-96 colonized the rhizosphere of Tara and Buchanan grown in soil similarly, but 2,4-DAPG accumulation was higher on the roots of Buchanan than Tara. 2,4-DAPG at 7.5 μg mL⁻¹ reduced the growth of roots of both cultivars, and 10 μg mL⁻¹ caused brown necrosis and tissue collapse of seedling roots and reduced root hair development. The half-life of 2,4-DAPG in the rhizosphere was estimated to be 0.25 days. These results suggest that several interconnected factors including sensitivity of G. graminis var. tritici to 2,4-DAPG, wheat cultivar, fluctuations in populations of 2,4-DAPG producers, and antibiotics accumulation in the rhizosphere will impact the robustness of take-all suppression by P. fluorescens in the field.     

Potential of Nematoctonus conidia for biological control of soil-borne phytonematodes

Populations of the mycophagous soil ncmatode Aphelenchus avenae living in sterile sand were reduced by addition of conidia of Nematoctonus concurrens and N. haptocladus. Natural, mixed nematode populations in non-sterile soil were unaffected by comparable treatments. Observations on conidia placed in contact with non-sterile soil showed that their germination was inhibited and that lysis of their germlings occurred.     

Rhizosphere microbial communities associated with Rhizoctonia damage at the field and disease patch scale

Rhizoctonia solani AG-8 is a major root pathogen in wheat (Triticum aestivum L.) systems worldwide and while natural disease suppression can develop under continuous cropping, this is not always the case. The main aim of our work was to elucidate the rhizosphere microbial community underlying a Rhizoctonia suppressive soil (Avon, South Australia) and to investigate how this community may develop in agricultural soils conducive to disease and of different soil type (Galong and Harden, New South Wales). The Avon suppressive soil community included Asaia spp. and Paenibacillus borealis, which were absent from a paired non-suppressive site. At Galong, soil taken from inside and outside disease patches showed no evidence of suppression, and disease suppression could not be transferred from the suppressive soil to the conducive soil from a different soil type and climatic area. 16S rRNA microarray analysis revealed Pseudomonas spp. were significantly more abundant inside than outside three disease patches at Galong. However, a survey of 32 patches across a range of stubble and tillage treatments at a nearby site showed no correlation between Pseudomonas and disease incidence. R. solani levels were significantly lower when stubble was retained rather than burnt or when nutrients (N, P and S) were incorporated with stubble during the non-crop period. Our results suggest soil type is an important factor for suppressive capability and that where specific disease suppression is absent, agronomic practice to increase soil carbon can encourage a non-specific microbial response that limits disease severity.     

Development of a 16S rRNA microarray approach for the monitoring of rhizosphere Pseudomonas populations associated with the decline of take-all disease of wheat

So far, the analysis of microbial populations associated with wheat monocropping-induced decline of take-all disease (Gaeumannomyces graminis var. tritici) has focused mainly on culturable biocontrol pseudomonads. The objective of this study was to develop a taxonomic rrs (16S rRNA gene) microarray to assess the changes in Pseudomonas populations taking place during take-all decline. The microarray contains 12 probes for five Pseudomonas phylogenetic clusters chosen because they include well-known plant-beneficial pseudomonads. Four of the clusters are within the ‘Pseudomonas fluorescens’ species complex. PCR primers were selected to target these five clusters, and they were validated using 53 pseudomonads belonging or not to these clusters. Microarray analysis of the pseudomonads enabled discrimination between strains from several Pseudomonas clusters. Rhizosphere samples were collected from field plots grown with wheat for 1 (low level of take-all disease), 5 (high level of disease) or 10 years (low level of disease, suppressiveness reached). Microarray data could distinguish Pseudomonas populations from some of the wheat plants grown in the same plot. When comparing treatments, there was a difference between years 1 and 10. Cloning–sequencing of rrs enabled to define more precisely this difference by identifying two major Pseudomonas populations, one associated with year 1 and the other with year 10 (disease suppressiveness), which represent new clades within the ‘P. fluorescens’ complex. These populations may be useful as soil quality indicators. In conclusion, the combination of microarray and cloning–sequencing approaches highlighted changes in the prevalence of two major Pseudomonas populations, giving new insights on the dynamics of root-associated pseudomonads during take-all decline.     

Colonization of wheat roots by Gaeumannomyces graminis inhibited by specific soils,microorganisms and ammonium-nitrogen

Lineal extension of Gaeumannomyces graminis var. tritici hyphae along roots of intact wheat plants growing in soils was measured. Hyphal growth rates were lower in soils treated with NH₄⁺-N than with NO₃^?-N. In a soil that is suppressive to the take-all disease, the controlling influence of NH₄⁺-N was eliminated by soil fumigation (methyl bromide), and reintroduced to fumigated soil by additions of 1% nonsterile soil. Effects of fumigation on hyphal growth were absent in a nonsuppressive soil, and in NO₃^?-treatments of the suppressive soil. When inocula of selected groups of wheat rhizoplane microflora were reintroduced into a fumigated or a soil-reinoculated soil via a root-food base, the Pseudomonas spp. consistently appeared more suppressive in NH₄⁺-N treatments than the general bacterial flora, Bacillus spp. spores, streptomycetes, and fungi.     

Fine structure of a new mycophagous amoeba and its feeding on Cochliobolus sativus

An unidentified mycophagous soil amoeba is described. The pigmented soil-borne fungus Cochliobolus sativus and four other fungal species, both pigmented and hyaline, were utilized as food. Spores were ingested and lysed within digestive vacuoles by general wall erosion. This contrasts with the wall perforation mechanism described for other mycophagous amoebae. Ultrastructural studies of trophozoites showed that large quantities of electron dense granules were released into the digestive vacuoles during fungal cell lysis. These were incorporated into the amoebal protoplast. Bacteria were commonly present in the amoebal protoplasts and within digestive vacuoles. Their possible role as endosymbionts is discussed.     

Populations of predatory protozoa in field soils after 5 years of elemental s fertilizer application

The effect of 5 yr of repeated application of elemental S (S°) fertilizer on predatory protozoa in soil was investigated. Protozoa that feed on the bacteria Arthrobacter globiformis and Enterobacter aerogenes or the fungi Fusarium solani and Neurospora crassa were enumerated by most probable number (MPN) methods. The application of S° fertilizer reduced the microbial biomass and its activity in soil. Soils treated with 44kg S° ha⁻¹ yr⁻¹ for 5 yr exhibited a 30–71% decline in MPN of protozoa feeding on bacteria and more than a 84% decline in the population of mycophagous amoebae. This decline in protozoa populations parallelled changes in microbial biomass, especially in the case of mycophagous amoebae and fungal biomass. The adverse effect of repeated S° applications on microbial biomass and predatory protozoa was long lasting. Since nutrient transformations (e.g. mineralization) in soil are influenced by microbial interactions, our results suggest reduced nutrient turnover via microbial predation in S° treated soils.     

Antagonists of Gaeumannomyces graminis from the rhizoplane of wheat in soils fertilized with ammonium- or nitrate-nitrogen

Take-all of wheat caused by Gaeumannomyces graminis var. tritici was less when soils in glasshouse pots were fertilized with NH₄⁺-N than with NO₃^?-N. The form of N did not alter countable populations of microorganisms in the rhizosphere or rhizoplane, but altered the numbers of bacteria and streptomycetes that inhibited the pathogen's growth in vitro. The pH of the medium used to isolate these microorganisms, whether similar or dissimilar to the pH of the rhizosphere, had some influence both upon countable populations and upon the proportions of antagonists. Highest counts of the rhizoplane microflora were on agar media with a pH similar to that of the soil. Most antagonists were isolated from a soil that is physically and chemically conducive to parasitism of wheat roots by Gaeumannomyces, but which contains a microflora suppressive toward the parasitic colonization of the roots. Isolates of the general bacterial flora, of Pseudomonas spp. and of streptomycetes, but not of Bacillus spp. inhibited the in vitro growth of G. graminis.     

Intensive cultivation can drastically reduce earthworm populations in arable land

The impact of intensive cultivation for potato production on the earthworm populations was assessed in two adjacent large field plots. Three successive winter wheat crops had been grown in one plot while winter wheat was grown with minimum cultivation in an understorey of white clover in the other. Both plots were ploughed in spring 1998 and intensively cultivated (grubbing, ridging, bed-tilling, destoning, ridging) prior to planting potatoes. Earthworm populations were reduced from a mean density of 319 individuals m^–2 and 55 g m^–2 biomass in the conventional wheat plot, and from 1160 individuals and 175 g m^–2 biomass in the wheat–clover plot in the 1996/1997 cropping season, to 40–82 individuals and 4–19 g m^–2 in June–October 1998 following potato planting. Populations declined to virtually undetectable levels following mechanical potato harvesting in late autumn 1998 and spring cultivation for barley in 1999, remained at very low levels throughout 1999 and had shown no sign of recovering by May 2000. The results show that earthworm populations can be virtually eliminated within a single season by drastic forms of soil cultivation.     

Suppression of wheat take-all and ophiobolus patch by fluorescent pseudomonads from a fusarium-suppressive soil

Fluorescent pseudomonads isolated from a soil suppressing Fusarium wilt significantly reduced take-all (Gaeumannomyces graminis var. tritici) in wheat and Ophiobolus patch (G. graminis var. avenae) in Agrostis turfgrass. The bacteria were mixed into a conducive soil at a concentration of 10⁷ colony-forming units (cfu)g^?1 soil at sowing. There were significantly fewer (P ? 0.05) diseased wheat roots in the treatments with the bacteria and pathogen than in those with the pathogen alone. Dry weights of the tops of wheat and Agrostis turfgrass were significantly greater (P ? 0.01) in treatments inoculated with the bacteria in the presence of the pathogens compared to controls with the pathogens alone. Dry weights of the tops of plants from treatments inoculated with the bacteria alone were not significantly different to those of healthy wheat non-inoculated with the bacteria, showing that the fluorescent pseudomonads did not stimulate plant growth. At the end of the experiments, the bacterial isolates (genetically-marked with rifampicin resistance) were recovered from wheat roots and rhizosphere soil at concentrations of 10⁵–10⁷cfu g^?1 fresh weight of roots or oven-dried rhizosphere soil.Many of the fluorescent pseudomonads and some non-fluorescent pseudomonads showed in vitro antibiosis on quarter-strength potato dextrose agar (QPDA) against the pathogens. However, there was no correlation between in vitro antibiosis on agar plates and suppression of disease in pot experiments. Further, while some isolates of G. graminis var. tritici and var. avenae were inhibited by certain bacterial isolates, other isolates of the same fungus were not similarly inhibited by the same isolates of bacteria. Most of the fluorescent pseudomonads that produced inhibition zones (>5mm) against G. graminis var. tritici on QPDA did not do so on King's medium B, where fluorescent siderophores were formed. In vitro antibiosis is, therefore, a poor criterion for selecting effective bacterial antagonists of the wheat take-all fungus. All of the fluorescent pseudomonads tested produced siderophores in low-Fe media while a non-fluorescent pseudomonad and the fungal pathogens did not produce siderophores of comparable activity. The addition of 500 μg FeEDTA g^?1 with a lower stability constant did not. The evidence suggests that iron competition at the rhizoplane or in the rhizosphere is one mechanism of suppression.     

MICROBIAL POPULATIONS AND NITROGEN IN SOIL GROWING CONSECUTIVE CEREAL CROPS INFECTED WITH TAKE-ALL

Annually, for the past 12 years, a consecutive cereal-cropping sequence was begun. During 1969–72 soil was collected from plots with similar fertilizer treatments, but with different sequences of crops. In 1971 and 1972 wheat seedling bioassays showed that take-all disease became miximal after two or three crops, decreased to the fifth or sixth crop and then remained fairly constant in the‘take-all decline’state. The sequence with maximum disease had most NH₄⁺ -N and least NO₃^- -N in the rhizosphere soil in spring 1972, but there were no similar relationships in bulk soil in either of 2 years. Soil and rhizoplane populations of ammonifying and denitrifying bacteria were notably smaller in autumn 1970 and 1971 in short cereal sequences (2-5 yr.) than in the longer sequences. After γ-irradiating or autoclaving irradiated soils, all sequences contained much NH₄⁺ -N and although disease developing from added inoculum differed among soils, it did not follow the take-all decline pattern. Diffusate from sterilized soil favoured growth of the pathogen (Gaeumannomyces graminis var. tritici) least when it originated from the sequence that supported maximum disease.     

Soil Vampyrellid amoebae that cause small perforations in conidia of Cochliobolus sativus

The parasitic habits of two mycophagous amoebae, members of the Vampyrellidae isolated from soil, were studied under laboratory conditions. The amoeboid organisms resembled Theratromyxa weberi and Vampyrella vorax. Both organisms lysed conidia of Cochliobolus sativus and chlamydospores o Thielaviopsis hasicola within digestive cysts. Perforations 1 μm dia or less were observed in walls of the lysed fungus spores. The mycophagous Theratromyxa sp. and V. vorax were differentiated in laboratory culture chambers on the basis of morphology, encystment, excystment and diameter of perforations produced in walls of conidia of C. sativus. Both organisms differed from a previously-described species of Vampyrella which causes large perforations and annular depressions in spore walls.     

Protease and deaminase activities in wheat rhizosphere and their relation to bacterial and protozoan populations

Protease and deaminase activities and population dynamics of bacteria and protozoa were measured in the rhizosphere of wheat to study their interactions with the mineralization of nitrogen. The experimental design allowed the separation of roots and soil material by means of a gauze. The most pronounced rhizosphere effect was detected for all the measured variables in the soil closest to the gauze. The number of bacteria was significantly higher in the presence than in the absence of plants up to 4 mm away from the soil-root interface and the closer to this interface the higher the number. Protozoan and bacterial population dynamics were positively correlated; generally, populations of flagellates and amoebae were comparable and their sum accounted for the population of total protozoa. For both enzyme activities the rhizosphere effect extended up to 2 mm away from the soil-root interface. The histidinase activity was of bacterial origin, while it is likely that bacteria, protozoa and root hair all contributed to the overall caseinase activity. Decomposition of root exudates and native organic matter in the rhizosphere, reflected by a growing microbial population, is associated with nitrogen mineralization through increases in caseinhydrolysing and L-histidine-deaminating activities. The adopted soil-plant microcosm is suitable for the study of the rhizosphere effect over time of incubation and distance gradient from the soil-root interface.     

Fluctuations in bacterial populations on the root surface of wheat (Triticum aestivum L.) grown under different soil conditions

Summary Populations of several bacterial groups on the root surface of wheat and in root-free soil were investigated in volcanic ash soil and non-volcanic ash soil throughout a series of predetermined intervals. Over time, the populations changed similarly both on the root surface and in root-free soil. The numbers of total bacteria, fluorescent Pseudomonas spp., phosphate-solubilizing bacteria, and NH _inf+ ^sup4 -oxidizing bacteria, were consistently lower in the plots with volcanic ash soil than with nonvolcanic ash soil, but the numbers of cellulose-decomposing bacteria were opposite to those of the other groups. Superphosphate application improved the growth of wheat in the volvanic ash soil. It did not, however, bring about any significant changes in the bacterial populations among the volcanic ash soils supplemented with three different levels of superphosphate, though there were some variations with plant age.     

Bacterial antagonists to the take-all fungus and fluorescent pseudomonads in the rhizosphere of wheat

The numbers of antagonists to the take-all fungus, and of fluorescent pseudomonads were significantly different in the rhizosphere and “residue” of seminal and nodal wheat roots of similar age.The rhizosphere of 1 week-old and the residues of 3 and 5 week-old nodal roots harboured significantly more antagonists than seminal roots. The rhizosphere of 5 week-old seminal roots, however, supported more antagonists than that of nodal roots of similar age. Combined analysis of all sampling stages showed that the nodal roots exerted a significantly greater “rhizosphere effect” on antagonists than the seminals.Fluorescent pseudomonads in the rhizosphere of 1, 3 and 8 week-old seminal roots were present in significantly higher numbers than in nodal roots of similar age. The combined analysis of all stages showed that the rhizosphere of seminal roots supports a significantly larger population of fluorescent pseudomonads.The implication of these differences is discussed in relation to the ecology of the take-all fungus.     

Belowground interactions between intercropped wheat and Brassicas in acidic and alkaline soils

Our previous studies showed that, under P-limiting conditions, growth and P uptake were lower in the wheat genotype Janz than in three Brassica genotypes when grown in monoculture. The present study was conducted to answer the question if P mobilised by the Brassicas is available to wheat; leading to improved growth of wheat when intercropped with Brassicas compared to monocropped wheat. To assess if the interactions between the crops depend on soil type, the wheat genotype Janz and three Brassica genotypes (two canolas and one mustard) were grown for 6 weeks in monoculture or wheat intercropped with each Brassica genotype in an acidic and an alkaline soil with low P availability (with two plants per pot). Wheat grew equally well in the two soils, but the Brassicas grew better in the acidic than in the alkaline soil. In the acidic soil, monocropped Brassicas had a 3 to 4 fold greater plant dry weight (dw) and P uptake than wheat; plant dw and P uptake in wheat were decreased or not affected by intercropping and increased in the Brassicas. In the alkaline soil, dw and P uptake of the Brassicas was twice as high as in wheat, with intercropping having no effect on these parameters. The contribution of wheat to the total shoot dw and P uptake per pot was 4-21% and 32-40% in acidic and alkaline soil, respectively. Mycorrhizal colonisation was low in all genotypes in the acidic soil (1-6%). In the alkaline soil, mycorrhizal colonisation of monocropped wheat was 62%, but only 43-47% in intercropped wheat. Intercropping decreased P availability in the rhizosphere of wheat in the acidic soil but had no effect on rhizosphere P availability in the alkaline soil. Intercropping had a variable effect on rhizosphere microbial community composition (assessed by fatty acid methylester analysis (FAME) and ribosomal intergenic spacer amplification (RISA)), ranging from intercropping having no effect on the rhizosphere communities to intercropping resulting in a new and similar rhizosphere community composition in both genotypes. The results of this study show that intercropping with Brassicas does not improve growth and P uptake of wheat; thus there is no indication that P mobilised by the Brassicas is available to wheat.