Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman

Seventy three isolates of Pythium aphanidermatum obtained from cucumber from four different regions of Oman and 16 isolates of muskmelon from the Batinah region in Oman were characterized for aggressiveness, sensitivity to metalaxyl and genetic diversity using AFLP fingerprinting. Twenty isolates of P. aphanidermatum from diverse hosts from different countries were also included in the study. Most isolates from Oman were found to be aggressive on cucumber seedlings and all were highly sensitive to metalaxyl (EC50 < 0•80 µg mL−1). Isolates from cucumber and muskmelon were as aggressive as each other on both hosts (P > 0.05), which implies a lack of host specialization in P. aphanidermatum on these two hosts in Oman. AFLP analysis of all isolates using four primer-pair combinations resolved 152 bands, of which 61 (~40%) were polymorphic. Isolates of P. aphanidermatum from Oman and other countries exhibited high genetic similarity (mean = 94.1%) and produced 59 different AFLP profiles. Analysis of molecular variance indicated that most AFLP variation among populations of P. aphanidermatum in Oman was associated with geographical regions (FST = 0.118; P < 0.0001), not hosts (FST = -0.004; P = 0.4323). These data were supported by the high rate of recovery (24%) of identical phenotypes between cucumber and muskmelon fields in the same region as compared to the low recovery (10%) across regions in Oman, which suggests more frequent movement of Pythium inoculum among muskmelon and cucumber fields in the same region compared to movement across geographically separated regions. However, recovering clones among regions and different countries may imply circulation of Pythium inoculum via common sources in Oman and also intercontinental spread of isolates.

An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

Detection of nitrogen deficiency in wheat from spectral reflectance indices and basic crop eco-physiological concepts

We tested the capacity of several published multispectral indices to estimate the nitrogen nutrition of wheat canopies grown under different levels of water supply and plant density and derived a simple canopy reflectance index that is greatly independent of those factors. Planar domain geometry was used to account for mixed signals from the canopy and soil when the ground cover was low. A nitrogen stress index was developed, which adjusts shoot %N for plant biomass and area, thereby accounting for environmental conditions that affect growth, such as crop water status. The canopy chlorophyll content index (CCCi) and the modified spectral ratio planar index (mSRPi) could explain 68 and 69% of the observed variability in the nitrogen nutrition of the crop as early as Zadoks 33, irrespective of water status or ground cover. The CCCi was derived from the combination of 3 wavebands 670, 720 and 790 nm, and the mSRPi from 445, 705 and 750 nm, together with broader bands in the NIR and RED. The potential for their spatial application over large fields/paddocks is discussed.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

DNA amplification fingerprinting analysis of genetic variation within Fusarium oxysporum f.sp zingiberi

Genetic variation among 29 isolates of Fusarium oxysporum f.sp. zingiberi (Foz) collected from diseased ginger rhizome in production regions throughout Queensland was analysed using DNA amplification fingerprinting (DAF). Eight isolates of other Fusarium species and/or formae speciales were included for comparative analysis. Within the Foz isolates, three haplotypes were identified based on 17 polymorphic bands generated with five primers. Two groups showed very little genetic variation (98.6% similarity), whereas the third single isolate was quite distinct in terms of its molecular profile (77.2% similarity). Genetic similarity among the Fusarium solani, F. oxysporum f.sp. lycopersici and F. oxysporum f.sp. cubense races 1, 3 and 4 isolates compared well with the published literature.

Fate of potassium fertilisers applied to clay soils under rainfed grain cropping in south-east Queensland, Australia

Negative potassium (K) balances in all broadacre grain cropping systems in northern Australia are resulting in a decline in the plant-available reserves of K and necessitating a closer examination of strategies to detect and respond to developing K deficiency in clay soils. Grain growers on the Red Ferrosol soils have increasingly encountered K deficiency over the last 10 years due to lower available K reserves in these soils in their native condition. However, the problem is now increasingly evident on the medium-heavy clay soils (Black and Grey Vertosols) and is made more complicated by the widespread adoption of direct drill cropping systems and the resulting strong strati. cation of available K reserves in the top 0.05-0.1 m of the soil pro. le. This paper reports glasshouse studies examining the fate of applied K fertiliser in key cropping soils of the inland Burnett region of south-east Queensland, and uses the resultant understanding of K dynamics to interpret results of field trials assessing the effectiveness of K application strategies in terms of K availability to crop plants. At similar concentrations of exchangeable K (K-exch), soil solution K concentrations and activity of K in the soil solution (AR(K)) varied by 6-7-fold between soil types. When K-exch arising from different rates of fertiliser application was expressed as a percentage of the effective cation exchange capacity (i.e. K saturation), there was evidence of greater selective adsorption of K on the exchange complex of Red Ferrosols than Black and Grey Vertosols or Brown Dermosols. Both soil solution K and AR(K) were much less responsive to increasing K-exch in the Black Vertosols; this is indicative of these soils having a high K buffer capacity (KBC). These contrasting properties have implications for the rate of diffusive supply of K to plant roots and the likely impact of K application strategies (banding v. broadcast and incorporation) on plant K uptake. Field studies investigating K application strategies (banding v. broadcasting) and the interaction with the degree of soil disturbance/mixing of different soil types are discussed in relation to K dynamics derived from glasshouse studies. Greater propensity to accumulate luxury K in crop biomass was observed in a Brown Ferrosol with a KBC lower than that of a Black Vertosol, consistent with more efficient diffusive supply to plant roots in the Ferrosol. This luxury K uptake, when combined with crops exhibiting low proportional removal of K in the harvested product (i.e. low K harvest index coarse grains and winter cereals) and residue retention, can lead to rapid re-development of stratified K profiles. There was clear evidence that some incorporation of K fertiliser into soil was required to facilitate root access and crop uptake, although there was no evidence of a need to incorporate K fertiliser any deeper than achieved by conventional disc tillage (i.e. 0.1-0.15 m). Recovery of fertiliser K applied in deep (0.25-0.3 m) bands in combination with N and P to facilitate root proliferation was quite poor in Red Ferrosols and Grey or Black Vertosols with moderate effective cation exchange capacity (ECEC, 25-35 cmol(+)/kg), was reasonable but not enough to overcome K deficiency in a Brown Dermosol (ECEC 11 cmol(+)/kg), but was quite good on a Black Vertosol (ECEC 50-60 cmol(+)/kg). Collectively, results suggest that frequent small applications of K fertiliser, preferably with some soil mixing, is an effective fertiliser application strategy on lighter clay soils with low KBC and an effective diffusive supply mechanism. Alternately, concentrated K bands and enhanced root proliferation around them may be a more effective strategy in Vertosol soils with high KBC and limited diffusive supply. Further studies to assess this hypothesis are needed.

Bacterial Community Structure in the Hindgut of Wild and Captive Dugongs (Dugong dugon)

Dugongs (Dugong dugon) are marine mammals that obtain nutrients through hindgut fermentation of seagrass, however, the microbes responsible have not been identified. This study used denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing to profile hindgut bacterial communities in wild dugongs. Faecal samples obtained from 32 wild dugongs representing four size/maturity classes, and two captive dugongs fed on cos lettuce were screened using DGGE. Partial 16S rRNA gene profiles of hindgut bacteria from wild dugong calves and juveniles were grouped together and were different to those in subadults and adults. Marked differences between hindgut bacterial communities of wild and captive dugongs were also observed, except for a single captive whose profile resembled wild adults following an unsuccessful reintroduction to the wild. Pyrosequencing of hindgut communities in two wild dugongs confirmed the stability of bacterial populations, and Firmicutes (average 75.6% of Operational Taxonomic Units [OTUs]) and Bacteroidetes (19.9% of OTUs) dominated. Dominant genera were Roseburia, Clostridium, and Bacteroides. Hindgut microbial composition and diversity in wild dugongs is affected by ontogeny and probably diet. In captive dugongs, the absence of the dominant bacterial DNA bands identified in wild dugongs is probably dependent upon prevailing diet and other captive conditions such as the use of antibiotics. This study represents a first step in the characterisation of a novel microbial ecosystem-the marine hindgut of Sirenia.

Effects of seasonal variability on FT-NIR prediction of dry matter content for whole Hass avocado fruit

Fourier Transform (FT)-near infra-red spectroscopy (NIRS) was investigated as a non-invasive technique for estimating percentage (%) dry matter of whole intact 'Hass' avocado fruit. Partial least squares (PLS) calibration models were developed from the diffuse reflectance spectra to predict % dry matter, taking into account effects of seasonal variation. It is found that seasonal variability has a significant effect on model predictive performance for dry matter in avocados. The robustness of the calibration model, which in general limits the application for the technique, was found to increase across years (seasons) when more seasonal variability was included in the calibration set. The R-v(2) and RMSEP for the single season prediction models predicting on an independent season ranged from 0.09 to 0.61 and 2.63 to 5.00, respectively, while for the two season models predicting on the third independent season, they ranged from 0.34 to 0.79 and 2.18 to 2.50, respectively. The bias for single season models predicting an independent season was as high as 4.429 but <= 1.417 for the two season combined models. The calibration model encompassing fruit from three consecutive years yielded predictive statistics of R-v(2) = 0.89, RMSEP = 1.43% dry matter with a bias of -0.021 in the range 16.1-39.7% dry matter for the validation population encompassing independent fruit from the three consecutive years. Relevant spectral information for all calibration models was obtained primarily from oil, carbohydrate and water absorbance bands clustered in the 890-980, 1005-1050, 1330-1380 and 1700-1790 nm regions. These results indicate the potential of FT-NIRS, in diffuse reflectance mode, to non-invasively predict the % dry matter of whole 'Hass' avocado fruit and the importance of the development of a calibration model that incorporates seasonal variation. Crown Copyright (c) 2012 Published by Elsevier B.V. All rights reserved.

High frequency fire alters C:N:P stoichiometry in forest litter

Fire is a major driver of ecosystem change and can disproportionately affect the cycling of different nutrients. Thus, a stoichiometric approach to investigate the relationships between nutrient availability and microbial resource use during decomposition is likely to provide insight into the effects of fire on ecosystem functioning. We conducted a field litter bag experiment to investigate the long-term impact of repeated fire on the stoichiometry of leaf litter C, N and P pools, and nutrient-acquiring enzyme activities during decomposition in a wet sclerophyll eucalypt forest in Queensland, Australia. Fire frequency treatments have been maintained since 1972, including burning every two years (2yrB), burning every four years (4yrB) and no burning (NB). C:N ratios in freshly fallen litter were 29-42% higher and C:P ratios were 6-25% lower for 2yrB than NB during decomposition, with correspondingly lower 2yrB N:P ratios (27-32) than for NB (34-49). Trends in litter soluble and microbial N:P ratios were similar to the overall litter N:P ratios across fire treatments. Consistent with these, the ratio of activities for N-acquiring to P-acquiring enzymes in litter was higher for 2yrB than NB while 4yrB was generally intermediate between 2yrB and NB. Decomposition rates of freshly fallen litter were significantly lower for 2yrB (72±2% mass remaining at the end of experiment) than for 4yrB (59±3%) and NB (62±3%), a difference that may be related to effects of N limitation, lower moisture content, and/or litter C quality. Results for older mixed-age litter were similar to those for freshly fallen litter although treatment differences were less pronounced. Overall, these findings show that frequent fire (2yrB) decoupled N and P cycling, as manifested in litter C:N:P stoichiometry and in microbial biomass N:P ratio and enzymatic activities. These data indicate that fire induced a transient shift to N-limited ecosystem conditions during the post-fire recovery phase. This article is protected by copyright. All rights reserved.

The utility of near infrared spectroscopy for age estimation of deepwater sharks

Reliable age information is vital for effective fisheries management, yet age determinations are absent for many deepwater sharks as they cannot be aged using traditional methods of growth bands counts. An alternative approach to ageing using near infrared spectroscopy (NIRS) was investigated using dorsal fin spines, vertebrae and fin clips of three species of deepwater sharks. Ages were successfully estimated for the two dogfish, Squalus megalops and Squalus montalbani, and NIRS spectra were correlated with body size in the catshark, Asymbolus pallidus. Correlations between estimated-ages of the dogfish dorsal fin spines and their NIRS spectra were good, with S. megalops R2=0.82 and S. montalbani R2=0.73. NIRS spectra from S. megalops vertebrae and fin clips that have no visible growth bands were correlated with estimated-ages, with R2=0.89 and 0.76, respectively. NIRS has the capacity to non-lethally estimate ages from fin spines and fin clips, and thus could significantly reduce the numbers of sharks that need to be lethally sampled for ageing studies. The detection of ageing materials by NIRS in poorly calcified deepwater shark vertebrae could potentially enable ageing of this group of sharks that are vulnerable to exploitation.

Validation of a new spot form of net blotch differential set and evidence for hybridisation between the spot and net forms of net blotch in Australia

A recently developed spot form of blotch differential set of 16 barley lines was tested for reaction response to 60 Pyrenophora teres f. maculata isolates from geographically disperse barley crops of Australia. Twelve barley lines (Arimont, Barque, Chebec, CI5286, CI5791, CI9214, CII6150, Dairokkaku, Esperance Orge 289, Galleon, Keel, Skiff, Torrens and TR250) provided differential response between the isolates. The susceptible controls Gairdner and Kombar provided indication of isolate virulence or avirulence. Abundant pathogenic diversity was revealed with 33 designated pathotypes, some of which related to geographic region. AFLP analysis also revealed abundant diversity with each of the isolates representing a unique genotype and one isolate that contained both AFLP bands unique to P. teres f. maculata and P. teres f. teres, the cause of spot form and net form of net blotch respectively, suggesting that sexual recombination between the net form and spot form isolates may have occurred naturally in the field.

A novel use of near infrared spectroscopy: ageing deepwater sharks

To age sharks, the growth bands in the shark vertebrae (like the rings in a tree) or on the spines in front of each dorsal fin (which only some sharks have) are manually counted using a microscope. This is time-consuming and is only possible on dead animals. NIR spectroscopy is shown to be able to detect age in dorsal fin spines of sharks and hand-held NIR spectroscopy units could potentially be used for ageing of sharks in the field, at sea, using a hand-held unit to scan the fin spine on a live animal.