Value-adding Australian sardines: Factors affecting rates of deterioration in sardine (Sardinops sagax) quality during post-harvest handling

Deficiencies in sardine post-harvest handling methods were seen as major impediments to development of a value-adding sector supplying Australian bait and human consumption markets. Factors affecting sardine deterioration rates in the immediate post-harvest period were investigated and recommendations made for alternative handling procedures to optimise sardine quality. Net to factory sampling showed that post-mortem autolysis was probably caused by digestive enzyme activity contributing to the observed temporal increase in sardine Quality Index. Belly burst was not an issue. Sardine quality could be maintained by reducing tank loading, and rapid temperature reduction using dedicated, on-board value-adding tanks. Fish should be iced between the jetty and the processing factory, and transport bins chilled using an efficient cooling medium such as flow ice.

Effect of defoliation interval and height on the growth and quality of Arachis pintoi cv. Amarillo

The effect of defoliation on Amarillo (Arachis pintoi cv. Amarillo) was studied in a glasshouse and in mixed swards with 2 tropical grasses. In the glasshouse, Amarillo plants grown in pots were subjected to a 30/20°C or 25/15°C temperature regime and to defoliation at 10-, 20- or 30-day intervals for 60 days. Two field plot studies were conducted on Amarillo with either irrigated kikuyu (Pennisetum clandestinum) in autumn and spring or dryland Pioneer rhodes grass (Chloris gayana) over summer and autumn. Treatments imposed were 3 defoliation intervals (7, 14 and 28 days) and 2 residual heights (5 and 10 cm for kikuyu; 3 and 10 cm for rhodes grass) with extra treatments (56 days to 3 cm for both grasses and 21 days to 5 cm for kikuyu). Defoliation interval had no significant effect on accumulated Amarillo leaf dry matter (DM) at either temperature regime. At the higher temperature, frequent defoliation reduced root dry weight (DW) and increased crude protein (CP) but had no effect on stolon DW or in vitro organic matter digestibility (OMD). On the other hand, at the lower temperature, frequent defoliation reduced stolon DW and increased OMD but had no effect on root DW or CP. Irrespective of temperaure and defoliation, water-soluble carbohydrate levels were higher in stolons than in roots (4.70 vs 3.65%), whereas for starch the reverse occured (5.37 vs 9.44%). Defoliating the Amarillo-kikuyu sward once at 56 days to 3 cm produced the highest DM yield in autumn and sprong (582 and 7121 kg/ha DM, respectively), although the Amarillo component and OMD were substantially reduced. Highest DM yields (1726 kg/ha) were also achieved in the Amarillo-rhodes grass sward when defoliated every 56 days to 3 cm, although the Amarillo component was unaffected. In a mixed sward with either kikuyu or rhodes grass, the Amarillo component in the sward was maintained up to a 28-day defoliation interval and was higher when more severely defoliated. The results show that Amarillo can tolerate frequent defoliation and that it can co-exist with tropical grasses of differing growth habits, provided the Amarillo-tropical grass sward is subject to frequent and severe defoliation.

Identification of quantitative trait loci for resistance to two species of root-lesion nematode (Pratylenchus thornei and P. neglectus) in wheat

Pratylenchus thornei and P. neglectus are two species of root-lesion nematode that cause substantial yield losses in wheat. No commercially available wheat variety has resistance to both species. A doubled-haploid population developed from a cross between the synthetic hexaploid wheat line CPI133872 and the bread wheat Janz was used to locate and tag quantitative trait loci (QTLs) associated with resistance to both P. thornei and P. neglectus. Wheat plants were inoculated with both species of nematode in independent replicated glasshouse trials repeated over 2 years. Known locations of wheat microsatellite markers were used to construct a framework map. After an initial single-marker analysis to detect marker-trait linkages, chromosome regions associated with putative QTLs were targetted with microsatellite markers to increase map density in the chromosome regions of interest. In total, 148 wheat microsatellite markers and 21 amplified fragment length polymorphism markers were mapped. The codominant microsatellite marker Xbarc183 on the distal end of chromosome 6DS was allelic for resistance to both P. thornei and P. neglectus. The QTL were designated QRlnt.lrc-6D.1 and QRlnn.lrc-6D.1, for the 2 traits, respectively. The allele inherited from CPI133872 explained 22.0-24.2% of the phenotypic variation for P. thornei resistance, and the allele inherited from Janz accounted for 11.3-14.0% of the phenotypic variation for P. neglectus resistance. Composite interval mapping identified markers that flank a second major QTL on chromosome 6DL (QRlnt.lrc-6D.2) that explained 8.3-13.4% of the phenotypic variation for P. thornei resistance. An additional major QTL associated with P. neglectus resistance was detected on chromosome 4DS (QRlnn.lrc-4D.1) and explained a further 10.3-15.4% of the phenotypic variation. The identification and tagging of nematode resistance genes with molecular markers will allow appropriate allele combinations to be selected, which will aid the successful breeding of wheat with dual nematode resistance.

Simple sequence repeat markers associated with three quantitative trait loci for black point resistance can be used to enrich selection populations in bread wheat

Black point in wheat has the potential to cost the Australian industry $A30.4 million a year. It is difficult and expensive to screen for resistance, so the aim of this study was to validate 3 previously identified quantitative trait loci (QTLs) for black point resistance on chromosomes 2B, 4A, and 3D of the wheat variety Sunco. Black point resistance data and simple sequence repeat (SSR) markers, linked to the resistance QTLs and suited to high-throughput assay, were analysed in the doubled haploid population, Batavia (susceptible) × Pelsart (resistant). Sunco and Pelsart both have Cook in their pedigree and both have the Triticum timopheevii translocation on 2B. SSR markers identified for the 3 genetic regions were gwm319 (2B, T. timopheevii translocation), wmc048 (4AS), and gwm341 (3DS). Gwm319 and wmc048 were associated with black point resistance in the validation population. Gwm341 may have an epistatic influence on the trait because when resistance alleles were present at both gwm319 and wmc048, the Batavia-derived allele at gwm341 was associated with a higher proportion of resistant lines. Data are presented showing the level of enrichment achieved for black point resistance, using 1, 2, or 3 of these molecular markers, and the number of associated discarded resistant lines. The level of population enrichment was found to be 1.83-fold with 6 of 17 resistant lines discarded when gwm319 and wmc048 were both used for selection. Interactions among the 3 QTLs appear complex and other genetic and epigenetic factors influence susceptibility to black point. Polymorphism was assessed for these markers within potential breeding material. This indicated that alternative markers to wmc048 may be required for some parental combinations. Based on these results, marker-assisted selection for the major black point resistance QTLs can increase the rate of genetic gain by improving the selection efficiency and may facilitate stacking of black point resistances from different sources.

Regrouping unfamiliar animals in the weeks prior to slaughter has few effects on physiology and meat quality in Bos taurus feedlot steers

The response of cattle to alterations in social groupings can lead to physiological changes that affect meat quality. Feedlot practices frequently lead to a proportion of cattle in a pen being drafted for slaughter with the balance retained for a further period until they meet market specifications. An ability to regroup such retained cattle for short periods without consequences for meat quality would facilitate efficient use of feedlot pen space. The current experiment examined the impact on physiological variables and meat quality of regrouped British breed steers 4, 2 or 1 week before dispatch for slaughter. There was little effect of regrouping cattle on physiological variables associated with stress responses. Physical assessment of meat quality indicated that regrouping steers 1 week before slaughter led to higher compression and a tendency for higher peak force values in animals from one genotype than in their respective controls (1.89 v. 1.71 ± 0.05 kg, P = 0.017); however, these assessments were not matched by changes in sensory perception of meat quality. Average daily gain during feedlot finishing was negatively related to the temperament measure and flight time. It was also associated with breed, white cell count, plasma cortisol and haemoglobin at the midpoint of the 70-day finishing period. The results confirm the impact of flight time on growth rate during feedlot finishing and that regrouping cattle less than 2 weeks before slaughter may reduce meat quality.

No-tillage and nitrogen application affects the decomposition of 15N-labelled wheat straw and the levels of mineral nitrogen and organic carbon in a Vertisol

No-tillage (NT) practice, where straw is retained on the soil surface, is increasingly being used in cereal cropping systems in Australia and elsewhere. Compared to conventional tillage (CT), where straw is mixed with the ploughed soil, NT practice may reduce straw decomposition, increase nitrogen immobilisation and increase organic carbon in the soil. This study examined 15N-labelled wheat straw (stubble) decomposition in four treatments (NT v. CT, with N rates of 0 and 75 kg/ha.year) and assessed the tillage and fertiliser N effects on mineral N and organic C and N levels over a 10-year period in a field experiment. NT practice decreased the rate of straw decomposition while fertiliser N application increased it. However, there was no tillage practice x N interaction. The mean residence time of the straw N in soil was more than twice as long under the NT (1.2 years) as compared to the CT practice (0.5 years). In comparison, differences in mean residence time due to N fertiliser treatment were small. However, tillage had generally very little effect on either the amounts of mineral N at sowing or soil organic C (and N) over the study period. While application of N fertiliser increased mineral N, it had very little effect on organic C over a 10-year period. Relatively rapid decomposition of straw and short mean residence time of straw N in a Vertisol is likely to have very little long-term effect on N immobilisation and organic C level in an annual cereal cropping system in a subtropical, semiarid environment. Thus, changing the tillage practice from CT to NT may not necessitate additional N requirement unless use is made of additional stored water in the soil or mineral N loss due to increased leaching is compensated for in N supply to crops.

Simulating the effects of saline and sodic subsoils on wheat crops growing on Vertosols

Soils with high levels of chloride and/or sodium in their subsurface layers are often referred to as having subsoil constraints (SSCs). There is growing evidence that SSCs affect wheat yields by increasing the lower limit of a crop's available soil water (CLL) and thus reducing the soil's plant-available water capacity (PAWC). This proposal was tested by simulation of 33 farmers' paddocks in south-western Queensland and north-western New South Wales. The simulated results accounted for 79% of observed variation in grain yield, with a root mean squared deviation (RMSD) of 0.50 t/ha. This result was as close as any achieved from sites without SSCs, thus providing strong support for the proposed mechanism that SSCs affect wheat yields by increasing the CLL and thus reducing the soil's PAWC. In order to reduce the need to measure CLL of every paddock or management zone, two additional approaches to simulating the effects of SSCs were tested. In the first approach the CLL of soils was predicted from the 0.3-0.5 m soil layer, which was taken as the reference CLL of a soil regardless of its level of SSCs, while the CLL values of soil layers below 0.5 m depth were calculated as a function of these soils' 0.3-0.5 m CLL values as well as of soil depth plus one of the SSC indices EC, Cl, ESP, or Na. The best estimates of subsoil CLL values were obtained when the effects of SSCs were described by an ESP-dependent function. In the second approach, depth-dependent CLL values were also derived from the CLL values of the 0.3-0.5 m soil layer. However, instead of using SSC indices to further modify CLL, the default values of the water-extraction coefficient (kl) of each depth layer were modified as a function of the SSC indices. The strength of this approach was evaluated on the basis of correlation of observed and simulated grain yields. In this approach the best estimates were obtained when the default kl values were multiplied by a Cl-determined function. The kl approach was also evaluated with respect to simulated soil moisture at anthesis and at grain maturity. Results using this approach were highly correlated with soil moisture results obtained from simulations based on the measured CLL values. This research provides strong evidence that the effects of SSCs on wheat yields are accounted for by the effects of these constraints on wheat CLL values. The study also produced two satisfactory methods for simulating the effects of SSCs on CLL and on grain yield. While Cl and ESP proved to be effective indices of SSCs, EC was not effective due to the confounding effect of the presence of gypsum in some of these soils. This study provides the tools necessary for investigating the effects of SSCs on wheat crop yields and natural resource management (NRM) issues such as runoff, recharge, and nutrient loss through simulation studies. It also facilitates investigation of suggested agronomic adaptations to SSCs.

Predicting the response of wheat (Triticum aestivum L.) to liquid and granular phosphorus fertilisers in Australian soils

Liquid forms of phosphorus (P) have been shown to be more effective than granular P for promoting cereal growth in alkaline soils with high levels of free calcium carbonate on Eyre Peninsula, South Australia. However, the advantage of liquid over granular P forms of fertiliser has not been fully investigated across the wide range of soils used for grain production in Australia. A glasshouse pot experiment tested if liquid P fertilisers were more effective for growing spring wheat (Triticum aestivum L.) than granular P (monoammonium phosphate) in 28 soils from all over Australia with soil pH (H2O) ranging from 5.2 to 8.9. Application of liquid P resulted in greater shoot biomass, as measured after 4 weeks' growth (mid to late tillering, Feeks growth stage 2-3), than granular P in 3 of the acidic to neutral soils and in 3 alkaline soils. Shoot dry matter responses of spring wheat to applied liquid or granular P were related to soil properties to determine if any of the properties predicted superior yield responses to liquid P. The calcium carbonate content of soil was the only soil property that significantly contributed to predicting when liquid P was more effective than granular P. Five soil P test procedures (Bray, Colwell, resin, isotopically exchangeable P, and diffusive gradients in thin films (DGT)) were assessed to determine their ability to measure soil test P on subsamples of soil collected before the experiment started. These soil test values were then related to the dry matter shoot yields to assess their ability to predict wheat yield responses to P applied as liquid or granular P. All 5 soil test procedures provided a reasonable prediction of dry matter responses to applied P as either liquid or granular P, with the resin P test having a slightly greater predictive capacity on the range of soils tested. The findings of this investigation suggest that liquid P fertilisers do have some potential applications in non-calcareous soils and confirm current recommendations for use of liquid P fertiliser to grow cereal crops in highly calcareous soils. Soil P testing procedures require local calibration for response to the P source that is going to be used to amend P deficiency.

Assessing the sustainability of wheat-based cropping systems using APSIM: Model parameterisation and evaluation

Assessing the sustainability of crop and soil management practices in wheat-based rotations requires a well-tested model with the demonstrated ability to sensibly predict crop productivity and changes in the soil resource. The Agricultural Production Systems Simulator (APSIM) suite of models was parameterised and subsequently used to predict biomass production, yield, crop water and nitrogen (N) use, as well as long-term soil water and organic matter dynamics in wheat/chickpea systems at Tel Hadya, north-western Syria. The model satisfactorily simulated the productivity and water and N use of wheat and chickpea crops grown under different N and/or water supply levels in the 1998-99 and 1999-2000 experimental seasons. Analysis of soil-water dynamics showed that the 2-stage soil evaporation model in APSIM's cascading water-balance module did not sufficiently explain the actual soil drying following crop harvest under conditions where unused water remained in the soil profile. This might have been related to evaporation from soil cracks in the montmorillonitic clay soil, a process not explicitly simulated by APSIM. Soil-water dynamics in wheat-fallow and wheat-chickpea rotations (1987-98) were nevertheless well simulated when the soil water content in 0-0.45 m soil depth was set to 'air dry' at the end of the growing season each year. The model satisfactorily simulated the amounts of NO3-N in the soil, whereas it underestimated the amounts of NH 4-N. Ammonium fixation might be part of the soil mineral-N dynamics at the study site because montmorillonite is the major clay mineral. This process is not simulated by APSIM's nitrogen module. APSIM was capable of predicting long-term trends (1985-98) in soil organic matter in wheat-fallow and wheat-chickpea rotations at Tel Hadya as reported in literature. Overall, results showed that the model is generic and mature enough to be extended to this set of environmental conditions and can therefore be applied to assess the sustainability of wheat-chickpea rotations at Tel Hadya.

Sustained genetic control of wheat rust diseases in north-eastern Australia

Control of wheat rusts in north-eastern Australia has been based on resistance breeding since the early 1920s. It has been an enduring journey of discovery, disappointment, and achievement, which has culminated in a pool of knowledge and expertise upon which today's plant breeders can efficiently target durable resistance to the major rust diseases. This paper outlines significant advances in genetic control of rusts in the region, with particular emphasis on the invaluable role played by the University of Sydney rust control program and its influence on wheat breeding in the region and throughout Australia. This paper is part of ‘Global Landscapes in Cereal Rust Control’, see Aust. J. Agric. Res. Vol. 58, no. 6.

The limit to wheat water-use efficiency in eastern Australia. I. Gradients in the radiation environment and atmospheric demand

In the wheatbelt of eastern Australia, rainfall shifts from winter dominated in the south (South Australia, Victoria) to summer dominated in the north (northern New South Wales, southern Queensland). The seasonality of rainfall, together with frost risk, drives the choice of cultivar and sowing date, resulting in a flowering time between October in the south and August in the north. In eastern Australia, crops are therefore exposed to contrasting climatic conditions during the critical period around flowering, which may affect yield potential, and the efficiency in the use of water (WUE) and radiation (RUE). In this work we analysed empirical and simulated data, to identify key climatic drivers of potential water- and radiation-use efficiency, derive a simple climatic index of environmental potentiality, and provide an example of how a simple climatic index could be used to quantify the spatial and temporal variability in resource-use efficiency and potential yield in eastern Australia. Around anthesis, from Horsham to Emerald, median vapour pressure deficit (VPD) increased from 0.92 to 1.28 kPa, average temperature increased from 12.9 to 15.2°C, and the fraction of diffuse radiation (FDR) decreased from 0.61 to 0.41. These spatial gradients in climatic drivers accounted for significant gradients in modelled efficiencies: median transpiration WUE (WUEB/T) increased southwards at a rate of 2.6% per degree latitude and median RUE increased southwards at a rate of 1.1% per degree latitude. Modelled and empirical data confirmed previously established relationships between WUEB/T and VPD, and between RUE and photosynthetically active radiation (PAR) and FDR. Our analysis also revealed a non-causal inverse relationship between VPD and radiation-use efficiency, and a previously unnoticed causal positive relationship between FDR and water-use efficiency. Grain yield (range 1-7 t/ha) measured in field experiments across South Australia, New South Wales, and Queensland (n = 55) was unrelated to the photothermal quotient (Pq = PAR/T) around anthesis, but was significantly associated (r2 = 0.41, P < 0.0001) with newly developed climatic index: a normalised photothermal quotient (NPq = Pq . FDR/VPD). This highlights the importance of diffuse radiation and vapour pressure deficit as sources of variation in yield in eastern Australia. Specific experiments designed to uncouple VPD and FDR and more mechanistic crop models might be required to further disentangle the relationships between efficiencies and climate drivers.

The limit to wheat water-use efficiency in eastern Australia. II. Influence of rainfall patterns

We investigated the influence of rainfall patterns on the water-use efficiency of wheat in a transect between Horsham (36°S) and Emerald (23°S) in eastern Australia. Water-use efficiency was defined in terms of biomass and transpiration, WUEB/T, and grain yield and evapotranspiration, WUEY/ET. Our working hypothesis is that latitudinal trends in WUEY/ET of water-limited crops are the complex result of southward increasing WUEB/T and soil evaporation, and season-dependent trends in harvest index. Our approach included: (a) analysis of long-term records to establish latitudinal gradients of amount, seasonality, and size-structure of rainfall; and (b) modelling wheat development, growth, yield, water budget components, and derived variables including WUEB/T and WUEY/ET. Annual median rainfall declined from around 600 mm in northern locations to 380 mm in the south. Median seasonal rain (from sowing to harvest) doubled between Emerald and Horsham, whereas median off-season rainfall (harvest to sowing) ranged from 460 mm at Emerald to 156 mm at Horsham. The contribution of small events (≤ 5 mm) to seasonal rainfall was negligible at Emerald (median 15 mm) and substantial at Horsham (105 mm). Power law coefficients (τ), i.e. the slopes of the regression between size and number of events in a log-log scale, captured the latitudinal gradient characterised by an increasing dominance of small events from north to south during the growing season. Median modelled WUEB/T increased from 46 kg/ha.mm at Emerald to 73 kg/ha.mm at Horsham, in response to decreasing atmospheric demand. Median modelled soil evaporation during the growing season increased from 70 mm at Emerald to 172 mm at Horsham. This was explained by the size-structure of rainfall characterised with parameter τ, rather than by the total amount of rainfall. Median modelled harvest index ranged from 0.25 to 0.34 across locations, and had a season-dependent latitudinal pattern, i.e. it was greater in northern locations in dry seasons in association with wetter soil profiles at sowing. There was a season-dependent latitudinal pattern in modelled WUEY/ET. In drier seasons, high soil evaporation driven by a very strong dominance of small events, and lower harvest index override the putative advantage of low atmospheric demand and associated higher WUEB/T in southern locations, hence the significant southwards decrease in WUEY/ET. In wetter seasons, when large events contribute a significant proportion of seasonal rain, higher WUEB/T in southern locations may translate into high WUEY/ET. Linear boundary functions (French-Schultz type models) accounting for latitudinal gradients in its parameters, slope, and x-intercept, were fitted to scatter-plots of modelled yield v. evapotranspiration. The x-intercept of the model is re-interpreted in terms of rainfall size structure, and the slope or efficiency multiplier is described in terms of the radiation, temperature, and air humidity properties of the environment. Implications for crop management and breeding are discussed.

Zero tillage and nitrogen fertiliser application in wheat and barley on a Vertosol in a marginal cropping area of south-west Queensland

Winter cereal cropping is marginal in south-west Queensland because of low and variable rainfall and declining soil fertility. Increasing the soil water storage and the efficiency of water and nitrogen (N) use is essential for sustainable cereal production. The effect of zero tillage and N fertiliser application on these factors was evaluated in wheat and barley from 1996 to 2001 on a grey Vertosol. Annual rainfall was above average in 1996, 1997, 1998 and 1999 and below average in 2000 and 2001. Due to drought, no crop was grown in the 2000 winter cropping season. Zero tillage improved fallow soil water storage by a mean value of 20 mm over 4 years, compared with conventional tillage. However, mean grain yield and gross margin of wheat were similar under conventional and zero tillage. Wheat grain yield and/or grain protein increased with N fertiliser application in all years, resulting in an increase in mean gross margin over 5 years from $86/ha, with no N fertiliser applied, to $250/ha, with N applied to target ≥13% grain protein. A similar increase in gross margin occurred in barley where N fertiliser was applied to target malting grade. The highest N fertiliser application rate in wheat resulted in a residual benefit to soil N supply for the following crop. This study has shown that profitable responses to N fertiliser addition in wheat and barley can be obtained on long-term cultivated Vertosols in south-west Queensland when soil water reserves at sowing are at least 60% of plant available water capacity, or rainfall during the growing season is above average. An integrative benchmark for improved N fertiliser management appears to be the gross margin/water use of ~$1/ha.mm. Greater fallow soil water storage or crop water use efficiency under zero tillage has the potential to improve winter cereal production in drier growing seasons than experienced during the period of this study.

Application of phytotoxicity data to a new Australian soil quality guideline framework for biosolids

To protect terrestrial ecosystems and humans from contaminants many countries and jurisdictions have developed soil quality guidelines (SQGs). This study proposes a new framework to derive SQGs and guidelines for amended soils and uses a case study based on phytotoxicity data of copper (Cu) and zinc (Zn) from field studies to illustrate how the framework could be applied. The proposed framework uses normalisation relationships to account for the effects of soil properties on toxicity data followed by a species sensitivity distribution (SSD) method to calculate a soil added contaminant limit (soil ACL) for a standard soil. The normalisation equations are then used to calculate soil ACLs for other soils. A soil amendment availability factor (SAAF) is then calculated as the toxicity and bioavailability of pure contaminants and contaminants in amendments can be different. The SAAF is used to modify soil ACLs to ACLs for amended soils. The framework was then used to calculate soil ACLs for copper (Cu) and zinc (Zn). For soils with pH of 4-8 and OC content of 1-6%, the ACLs range from 8 mg/kg to 970 mg/kg added Cu. The SAAF for Cu was pH dependant and varied from 1.44 at pH 4 to 2.15 at pH 8. For soils with pH of 4-8 and OC content of 1-6%, the ACLs for amended soils range from 11 mg/kg to 2080 mg/kg added Cu. For soils with pH of 4-8 and a CEC from 5-60, the ACLs for Zn ranged from 21 to 1470 mg/kg added Zn. A SAAF of one was used for Zn as it concentrations in plant tissue and soil to water partitioning showed no difference between biosolids and soluble Zn salt treatments, indicating that Zn from biosolids and Zn salts are equally bioavailable to plants.

A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat

A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains.