Aims
Develop quantitative assays (qPCR) to determine the detection threshold limits, colonization and persistence of Trichoderma gamsii, Trichoderma afroharzianum and T. harzianum inoculants in ...cropping soils, the wheat rhizosphere and their in planta suppressive efficacy against the crown rot pathogen Fusarium pseudograminearum.
Methods and Results
Trichoderma qPCR primers were designed from the internal transcribed spacer region of 5.8S rDNA and from sequences of DNA fragments diagnostic for each inoculant genotype. The minimum detection thresholds of qPCR assays varied between 1 × 103 (log 3) and 8 × 104 (log 4·9) conidia (genome) equivalents per gram of soil for multi‐ and single‐copy target sequences, respectively and were independent of soil type. There was a strong correlation (r > 0·974) between culture‐dependent and culture‐independent (qPCR) quantification methods. In wheat bioassays, Trichoderma inoculants colonized rhizosphere soils and wheat roots at 56–112 days postemergence to a depth of 20 cm but were more abundant (P < 0·001) at 0–10 cm root depth, means ranging from 2 × 102 (log 2·3) to 4 × 105 (log 5·6) conidia equivalents per gram of rhizosphere soil or root tissue. Inoculants reduced (P < 0·001) F. pseudograminearum biomass in wheat crown and root tissue by up to 5754‐fold and increased (P = 0·008) plant biomass, relative to the pathogen control.
Conclusions
The qPCR assays provided sensitive and accurate assessment of wheat root and rhizosphere soil colonization of Trichoderma inoculants. Strains persisted through to grain maturity at levels shown to significantly suppress F. pseudograminearum in planta.
Significance and Impact of the Study
The qPCR assays developed here were used to determine the wheat rhizosphere dynamics of T. harzianum, T. afroharzianum and T. gamsii inoculants and their suppressive efficacy against F. pseudograminearum in planta. These assays can be applied to monitor inoculant dynamics in suppressing crown rot and other wheat root diseases in the field.
Abstract
Long coleoptile (LC) - wheat genotypes sown deeper (> 10 cm) than current practice (< 5 cm) enables earlier deep sowing into subsurface soil moisture. The LC genotype shows promise to ...broaden sowing windows and remove reliance on the shifting seasonal break for crop establishment. The suitability of this sowing strategy to the highly variable soils of the southern Australian wheatbelt warrants investigation to identify suitable environments for potential establishment benefits. We conducted controlled environment room (CER) and field experiments using pairs of near-isogenic-lines (NILs) that differed genetically only for coleoptile length. The experiments investigated the suitability of deep sown (12 cm) LC wheats in soils with different texture (sand versus loam). The CER experiment identified that LC genotypes exhibited greater emergence and early seedling growth, producing greater shoot and root biomass, when deep sown (12 cm) in loam. While LC and short coleoptile (SC) genotypes produced similar shoot growth when sown at depth in sand, LC produced greater root biomass reflected in more seminal roots. In the field on sand, LC and SC had similar emergence and early shoot growth characteristics at all sowing depths (5, 8 and 10 cm). However, the heavy-textured, loam soil reduced seedling emergence and restricted leaf growth with deep sowing (12 cm) in the SC. Our results indicate that the LC trait was more critical for plant emergence and early growth parameters (shoot and root biomass) from depth in a heavier textured loam compared with a sand. By identifying environments that will likely benefit most from deep sown LC, we are one step closer to adapting crop production to shifting rainfall patterns in southern Australia.
The Oligocene Ruslar Formation is a hydrocarbon source rock in the Kamchia Depression, located in the Western Black Sea area. Depositional environment and source potential of the predominantly ...pelitic rocks were investigated using core and cuttings samples from four offshore wells. In these wells the Ruslar Formation is up to 500
m thick. Based on lithology and well logs, the Ruslar Formation is subdivided from base to top into units I–VI. Dysoxic to anoxic conditions and mesohaline to euhaline salinities prevailed during deposition of the Ruslar Formation. Relatively high oxygen contents occurred during early Solenovian times (lower part of unit II), when brackish surface water favoured nannoplankton blooms and the deposition of bright marls (“Solenovian event”). Anoxic conditions with photic zone anoxia were established during late Oligocene times (units III and IV) and, probably, reflect a basin-wide anoxic event in the Eastern Paratethys during Kalmykian times. Organic carbon content in the Ruslar Formation is up to 3%. Autochthonous aquatic and allochthonous terrigenous biomass contribute to the organic matter. Relatively high amounts of aquatic organic matter occur in the lower part of the Ruslar Formation (units I and II) and in its upper part (unit VI). Diatoms are especially abundant in the lower part of unit VI. The kerogen is of type III and II with HI values ranging from 50 to 400
mgHC/gTOC. Units I and II (Pshekian, lower Solenovian) are characterized by a fair (to good) potential to produce gas and oil, but potential sources for gas and oil also occur in the Upper Oligocene units IV–VI.
In Australia, Diaporthe perjuncta (formerly known as Phomopsis taxon 1) and Phomopsis viticola (Phomopsis taxon 2) have been associated with Phomopsis cane and leaf spot of grapevine. Although P. ...viticola causes distinct leaf spots, as well as lesions on shoots and canes, the pathogenicity of D. perjuncta is relatively unknown. The pathogenicity of D. perjuncta and P. viticola was studied in relation to symptom expression and bud loss. Only P. viticola caused brown-black, longitudinal, necrotic lesions on stem tissue and leaf spots characteristic of the disease, whereas both D. perjuncta and P. viticola induced bleaching of dormant canes. Inoculation of dormant buds with D. perjuncta did not cause bud death. D. perjuncta and P. viticola were reisolated from inoculated tissue and into pure culture. D. perjuncta colonized the epidermis and cortex of the grapevine shoot but not the vascular tissue. D. perjuncta appears to be an endophyte, rather than a pathogen of grapevine.
Eutypa lata is an ascomycete fungus that causes eutypa dieback of grapevines, a disease which threatens vine productivity and longevity throughout the world. We assessed genetic variation among 35 ...isolates received as E. lata from Australia, Europe, California and South Africa using restriction fragment length polymorphism analysis. A subset of 10 isolates previously analysed for secondary metabolite production was also analysed using randomly amplified polymorphic DNA markers. Molecular analyses showed a high level of genetic diversity, with each isolate having a unique haplotype. Phylogenetic analysis did not allow separation of isolates based on geographic location or host species from which isolates were obtained. There was no apparent correlation between DNA profile and secondary metabolite production by E. lata . The results of this study showed a degree of genetic diversity similar to that reported for European and North American populations of E. lata , and are consistent with the hypothesis that the sole means of dispersal of the pathogen is by ascospores.
Sexual recombination and segregation in 18 ascospore progeny of Uncinula necator were analysed by mating type (Mat locus), RFLP and PCR markers. Progeny were analysed from a cross of parental ...isolates representing the genetically distinct phenetic Groups I (AHd2) and II (BNb2). A total of 27 markers was analysed, consisting of four single loci (Mat locus and three RFLP probes), 16 multi-loci (three multi-copy fingerprinting probes) and seven PCR loci. Novel hybrid genotypes were detected in 28% of the ascospore-derived progeny using the multi-copy RFLP probes, pUnl22-11 and pUnP27, and the PCR primers (CAC)^sub 5^ and R1, confirming that recombination can occur between isolates representative of the two phenetic groups. This study provides evidence that out-crossing between isolates of U. necator generates novel genotypes and, as such, may have important implications for disease management practices, such as the durability of fungicides and host resistance.PUBLICATION ABSTRACT
Isolates of Uncinula necator with reduced sensitivity to triadimenol and, to a lesser extent, fenarimol have been detected in Australian vineyards. Grapevine leaves and berries affected by powdery ...mildew were collected from a home garden and 13 vineyards in nine viticultural regions in Australia between 1993 and 1998. Single-spore isolates of U. necator established from vines, either not exposed to DMIs (‘unexposed’ collection) or treated with DMIs (‘selected’ collection), were maintained on micropropagated grapevines in vitro . A bioassay for fungicide sensitivity was used to test 60 single-spore isolates of U. necator for sensitivity to triadimenol. Of these, 34 were tested for sensitivity to fenarimol. Mean EC 50 values for the 12 unexposed isolates were 0.07 and 0.08 mg/L for triadimenol and fenarimol, respectively. For the selected isolates, mean EC 50 values were 0.83 mg/L for triadimenol and 0.19 mg/L for fenarimol. In comparison with the unexposed population, there appeared to have been a shift in the selected population towards reduced sensitivity. Examination of more isolates from vineyards not exposed to DMIs may strengthen this conclusion. Cut-off EC 50 values, used to define individual isolates as having reduced sensitivity, were 0.42 mg/L (resistance factor, RF = 6) for triadimenol and 0.12 mg/L (RF = 1.5) for fenarimol. The findings were consistent with the existence of cross-resistance between triadimenol and fenarimol.
Diaporthe perjuncta has been associated with Phomopsis cane and leaf spot disease of grapevine in Australia. Both D. perjuncta and Phomopsis viticola cause bleaching of cane and, for this reason, ...diagnosis of the two fungi is often confused in the vineyard. P. viticola causes leaf spots and shoot lesions in the growing season, whereas more recent studies have indicated that D. perjuncta might not be a pathogen. Field studies were conducted to assess the role of D. perjuncta in delayed budburst and death of grapevine buds. Over three seasons (1999–2001), vines were assessed at four vineyards in South Australia for percentage of budburst and incidence of D. perjuncta . A D. perjuncta -specific probe, pT1P180, was used to detect D. perjuncta in unburst buds, cane and shoots on spurs having buds that failed to burst. D. perjuncta was detected in both bleached and non-bleached cane. Although there was a significant association between the incidence of D. perjuncta and buds that failed to burst in 1999 only, overall bud burst was normal in all vineyards and bud loss was not a concern. Bunch number and shoot growth was not affected. Mites were considered as a possible factor in damage to unburst buds, but it was more likely that failure of buds to burst was related to physiological or environmental effects. D. perjuncta did not adversely affect grapevine productivity and cannot be regarded as a pathogen of grapevine.
Conidia from four genetically distinct isolates of Uncinula necator (Schw.) Burr. were used to raise a polyclonal antiserum. Immunofluorescent detection of the fungus on Vitis vinifera (cv. ...Chardonnay) indicated that the antiserum bound specifically to fungal antigens present on both conidia and hyphae, with no detection of underlying berry tissues. The antibody reacted with three antigens present on the conidia (Mr 21, 29 and > 250 kDa). Immunoreactivity with the 21 kDa antigen was dependent upon the preservation of at least one disulphide bond linkage. Evidence from immunoblot staining and enzyme immunoassay indicated that only a small proportion of the recognized epitopes contained carbohydrate moieties. The antibody detected homologous U. necator conidial antigens in a plate trapped antigen‐enzyme‐linked immunoabsorbent assay (PTA‐ELISA), with a linear range of detection extending from 1000 to 9000 conidia/ml at a 1/5000 dilution of serum. Under these conditions the immunoassay also detected antigens from pooled heterologous U. necator isolates. The antiserum exhibited cross‐reactivity with antigens present on Aspergillus, Pithomyces and Sporobolomyces species co‐isolated from powdery mildew‐infected grapes, which could not be removed by fractionation of the antiserum on antigen affinity columns. Monoclonal antibodies were subsequently produced to avoid the problems of cross‐reactivity associated with the polyclonal antibody. Antibodies produced from two clonal lines exhibited specificity for U. necator and were shown to detect a 21 kDa conidial antigen. Use of either of these antibodies enabled the differentiation of grapes grouped on the basis of powdery mildew disease levels.
A black box model is derived for an erbium-doped optical amplifier and is successfully applied to 1480 and 980 nm pumped devices operated under conditions which are typical for wavelength division ...multiplex (WDM) systems. It allows compound optical amplifiers with arbitrary passive optical circuitry (isolators, couplers, taps, and equalizing filters) to be modeled on the basis of "black box" characteristics. The gain model is based on an analytic solution for the effective two-level laser system, i.e., it is equivalent with the results of most numerical EDFA modeling tools. The model for amplified spontaneous emission (ASE) based on an equivalent ASE source in front of the amplifier can be applied over a wide range of operating points.
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