Paul R. Harvey

Plant and microbial strategies to improve the phosphorus efficiency of agriculture

BackgroundAgricultural production is often limited by low phosphorus (P) availability. In developing countries, which have limited access to P fertiliser, there is a need to develop plants that are more efficient at low soil P. In fertilised and intensive systems, P-efficient plants are required to minimise inefficient use of P-inputs and to reduce potential for loss of P to the environment.ScopeThree strategies by which plants and microorganisms may improve P-use efficiency are outlined: (i) Root-foraging strategies that improve P acquisition by lowering the critical P requirement of plant growth and allowing agriculture to operate at lower levels of soil P; (ii) P-mining strategies to enhance the desorption, solubilisation or mineralisation of P from sparingly-available sources in soil using root exudates (organic anions, phosphatases), and (iii) improving internal P-utilisation efficiency through the use of plants that yield more per unit of P uptake.ConclusionsWe critically review evidence that more P-efficient plants can be developed by modifying root growth and architecture, through manipulation of root exudates or by managing plant-microbial associations such as arbuscular mycorrhizal fungi and microbial inoculants. Opportunities to develop P-efficient plants through breeding or genetic modification are described and issues that may limit success including potential trade-offs and trait interactions are discussed. Whilst demonstrable progress has been made by selecting plants for root morphological traits, the potential for manipulating root physiological traits or selecting plants for low internal P concentration has yet to be realised.

Strategies and agronomic interventions to improve the phosphorus-use efficiency of farming systems

Phosphorus (P)-deficiency is a significant challenge for agricultural productivity on many highly P-sorbing weathered and tropical soils throughout the world. On these soils it can be necessary to apply up to five-fold more P as fertiliser than is exported in products. Given the finite nature of global P resources, it is important that such inefficiencies be addressed. For low P-sorbing soils, P-efficient farming systems will also assist attempts to reduce pollution associated with P losses to the environment. P-balance inefficiency of farms is associated with loss of P in erosion, runoff or leaching, uneven dispersal of animal excreta, and accumulation of P as sparingly-available phosphate and organic P in the soil. In many cases it is possible to minimise P losses in runoff or erosion. Uneven dispersal of P in excreta typically amounts to ~5% of P-fertiliser inputs. However, the rate of P accumulation in moderate to highly P-sorbing soils is a major contributor to inefficient P-fertiliser use. We discuss the causal edaphic, plant and microbial factors in the context of soil P management, P cycling and productivity goals of farms. Management interventions that can alter P-use efficiency are explored, including better targeted P-fertiliser use, organic amendments, removing other constraints to yield, zone management, use of plants with low critical-P requirements, and modified farming systems. Higher productivity in low-P soils, or lower P inputs in fertilised agricultural systems can be achieved by various interventions, but it is also critically important to understand the agroecology of plant P nutrition within farming systems for improvements in P-use efficiency to be realised.

Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia

ABSTRACT Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum andPseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.

Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing.

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils ‘suppressive’ or ‘non-suppressive’ for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression.

Potential to improve root access to phosphorus: the role of non-symbiotic microbial inoculants in the rhizosphere

Phosphate anions in soil solution are extremely reactive and may be rapidly immobilised in the soil through precipitation and adsorption reactions, resulting in sparingly soluble forms of phosphorus (P) that are essentially unavailable to plants. This low P-fertiliser efficiency is often offset through high application rates, which are economically and environmentally unsustainable and not an available option for organic producers. Microorganisms play a fundamental role in the biogeochemical cycling of inorganic and organic P in the rhizosphere and detritusphere. Free-living rhizosphere microbes can directly increase the availability of phosphate to plant roots via mechanisms associated with solubilisation and mineralisation of P from inorganic and organic forms of total soil P. These include releasing organic anions, H+ ions, phosphatases, and cation chelating compounds into the rhizosphere. Many soil-borne microbes also increase P availability indirectly by producing phytohormones that increase root density and function. There is increasing interest worldwide in the use of rhizosphere microorganisms as inoculants to increase P availability for agricultural production. Recent research has focussed on developing actively sporulating Penicillium fungi known to express mechanisms to enhance P mobilisation and therefore, considered to be a key component of the mycoflora involved in P cycling in soils. Penicillium species do not exhibit specific plant or soil associations and have a broad agro-ecological range, indicating their potential to be developed as inoculants for a range of plant production systems. Successful adoption of microbial inoculants requires a thorough understanding of their rhizosphere ecology, genetic stability, and the mechanisms associated with enhancing P availability in soils and plant-growth promotion. This will provide a better understanding of which inoculants to use under particular agro-ecological conditions for increased efficacy and consistent performance.

Genetic and pathogenic variation among cereal, medic and sub-clover isolates of Pythium irregulare

Genetic variation within 34 Pythium irregulare isolates was analyzed using restriction fragment length polymorphisms (RFLPs) as genetic markers. Most isolates had two alleles at several codominant RFLP loci and were scored as heterozygous. Heterozygotes were detected in F1 progeny from an in vitro cross and segregation ratios of the F2 progeny were not significantly different from those expected for allelic variation in a diploid. This confirmed that outcrossing occurs and contributes to genetic variation within the species. Phenetic analysis showed that isolates formed genetically related groups due to their host species and not due to similarities in their geographical origins. All isolates originating from medic formed a discrete group and were highly differentiated from the cereal and sub-clover isolates. The allelic distributions between isolates from these host-groups were significantly different. Most isolates also showed significant differences in their pathogenicity between hosts, indicating that they varied in pathogenic fitness and were better adapted to parasitising some hosts relative to others. These isolates were however, not necessarily more pathogenic on their host of origin. This research provided estimates of the extent of genetic and pathogenic diversity within P. irregulare and qualitative evidence for the occurrence of host-mediated selection and sexual outcrossing in the field.

Isolation and characterization of carbendazim-degrading Rhodococcus erythropolis djl-11.

Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L−1) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L−1·d−1 at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH4NO3. Changes in MSM pH (4–9), substitution of NH4NO3 with organic substrates as N and C sources or replacing Mg2+ with Mn2+, Zn2+ or Fe2+ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G.

Genetic drift and host-mediated selection cause genetic differentiation among Gaeumannomyces graminis populations infecting cereals in southern Australia

Isolates of Gaeumannomyces graminis were sampled from 16 cereal crops throughout the cereal belt of southern Australia to determine the extent of genetic diversity and the scale of genetic differentiation among pathogen populations. Data from 13 isozyme and 4 RFLP loci differentiated 79 multilocus genotypes among the 320 isolates analysed. All 17 loci differed significantly in allele frequencies across all populations and significant levels of genetic differentiation were detected between most populations. Genetic differentiation among host groups was high (GST = 0.307) and groups of populations from barley, oats and wheat were significantly different. The average genetic identities among populations were low and populations formed genetically related groups based on similarities in recent cereal cropping histories and not geographical origins. Collectively, these analyses indicate restrictions to interpopulation gene flow within G. graminis and imply that population differentiation results from genetic drift and host-mediated selection by different cereal species.

Dynamics of Introduced Populations of Phragmidium violaceum and Implications for Biological Control of European Blackberry in Australia

ABSTRACT Phragmidium violaceum causes leaf rust on the European blackberry (Rubus fruticosus L. aggregate). Multiple strains of this pathogen have been introduced into southern Australia for the biological control of at least 15 taxa of European blackberry, a nonindigenous, invasive plant. In climates conducive to leaf rust, the intensity of disease varies within and among infestations of the genetically variable host. Genetic markers developed from the selective amplification of microsatellite polymorphic loci were used to assess the population genetic structure and reproductive biology of P. violaceum within and among four geographically isolated and diseased infestations of the European blackberry in Victoria, Australia. Despite the potential for long-distance aerial dispersal of urediniospores, there was significant genetic differentiation among all populations, which was not associated with geographic separation. An assessment of multilocus linkage disequilibrium revealed temporal and geographic variation in the occurrence of random mating among the four populations. The presence of sexual spore states and the results of genetic analyses indicated that recombination, and potentially random migration and genetic drift, played an important role in maintaining genotypic variation within populations. Recombination and genetic differentiation in P. violaceum, as well as the potential for metapopulation structure, suggest the need to release additional, genetically diverse strains of the biocontrol agent at numerous sites across the distribution of the Australian blackberry infestation for maximum establishment and persistence.

Antibiosis functions during interactions of Trichoderma afroharzianum and Trichoderma gamsii with plant pathogenic Rhizoctonia and Pythium

Trichoderma afroharzianum is one of the best characterized Trichoderma species, and strains have been utilized as plant disease suppressive inoculants. In contrast, Trichoderma gamsii has only recently been described, and there is limited knowledge of its disease suppressive efficacies. Comparative studies of changes in gene expression during interactions of these species with their target plant pathogens will provide fundamental information on pathogen antibiosis functions. In the present study, we used complementary DNA amplified fragment length polymorphism (cDNA-AFLP) analysis to investigate changes in transcript profiling of T. afroharzianum strain LTR-2 and T. gamsii strain Tk7a during in vitro interactions with plant pathogenic Rhizoctonia solani and Pythium irregulare. Considerable differences were resolved in the overall expression profiles of strains LTR-2 and Tk7a when challenged with either plant pathogen. In strain LTR-2, previously reported mycoparasitism-related genes such as chitinase, polyketide synthase, and non-ribosomal peptide synthetase were found to be differentially expressed. This was not so for strain Tk7a, with the only previously reported antibiosis-associated genes being small secreted cysteine-rich proteins. Although only one differentially expressed gene was common to both strains LTR-2 and Tk7a, numerous genes reportedly associated with pathogen antibiosis processes were differentially expressed in both strains, including degradative enzymes and membrane transport proteins. A number of novel potential antibiosis-related transcripts were found from strains LTR-2 and Tk7a and remain to be identified. The expression kinetics of 20 Trichoderma (10 from strain LTR-2, 10 from strain Tk7a) transcript-derived fragments (TDFs) were quantified by quantitative reverse transcription PCR (RT-qPCR) at pre- and post-mycelia contact stages of Trichoderma-prey interactions, thereby confirming differential gene expression. Collectively, this research is providing information to elucidate the antibiosis mechanisms and disease suppressive activities of T. afroharzianum and T. gamsii against soilborne fungal and oomycete plant pathogens.