Prediction of Potato Tuber Damage by Root-Knot Nematodes using Quantitative DNA Assay of Soil.

Root-knot nematodes (Meloidogyne fallax and M. hapla) cause significant reductions in potato yield by reducing tuber quality. Concentrations of M. fallax and M. hapla DNA in soil were determined by quantitative polymerase chain reaction following sampling at planting and harvest within 78 fields across 3 years in Australia. Meloidogyne spp. were also detected using a tomato bioassay. M. fallax was more prevalent than M. hapla and DNA concentrations of M. fallax in soil were significantly higher in samples collected at harvest compared with those at planting. In contrast, M. hapla DNA in soil did not significantly change from planting to harvest. Using receiver operating characteristic curve analysis, M. fallax DNA in soil at planting and harvest was a highly accurate predictor of tuber damage at harvest and galling on tomato. Prediction accuracy for tuber damage was highest for M. fallax DNA compared with M. hapla or M. fallax + M. hapla. Both Meloidogyne spp. were detected in the peel of asymptomatic certified seed. For M. fallax, the addition of seedborne inoculum did not improve tuber damage predictions. This suggested that soilborne M. fallax populations contributed most substantially to tuber damage. These findings highlight the utility of this approach for predicting risk of crop damage from nematodes. The use of this technique as a practical management tool is discussed.

DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees.

Understanding the root distribution of trees by soil coring is time -: consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m(-2)) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23-28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R(2) = 0.9307, P < 0.001) with the dry matter (g m(-2)) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g.

A DNA-based method for studying root responses to drought in field-grown wheat genotypes.

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.

Improved inference of taxonomic richness from environmental DNA.

Accurate estimation of biological diversity in environmental DNA samples using high-throughput amplicon pyrosequencing must account for errors generated by PCR and sequencing. We describe a novel approach to distinguish the underlying sequence diversity in environmental DNA samples from errors that uses information on the abundance distribution of similar sequences across independent samples, as well as the frequency and diversity of sequences within individual samples. We have further refined this approach into a bioinformatics pipeline, Amplicon Pyrosequence Denoising Program (APDP) that is able to process raw sequence datasets into a set of validated sequences in formats compatible with commonly used downstream analyses packages. We demonstrate, by sequencing complex environmental samples and mock communities, that APDP is effective for removing errors from deeply sequenced datasets comprising biological and technical replicates, and can efficiently denoise single-sample datasets. APDP provides more conservative diversity estimates for complex datasets than other approaches; however, for some applications this may provide a more accurate and appropriate level of resolution, and result in greater confidence that returned sequences reflect the diversity of the underlying sample.

Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils.

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi-arid floodplain would transition towards comprising mainly dry-soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post-flood) were dominated by persistent rather than either aquatic or dry-specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post-flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post-flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi-arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.

Carbon source accounting for fish using combined DNA and stable isotope analyses in a regulated lowland river weir pool.

Determining the source and flow of carbon, energy and nutrients through food webs is essential for understanding ecological connectivity and thus determining the impact of management practices on biodiversity. We combined DNA sequencing, microarrays and stable isotope analyses to test whether this approach would allow us to resolve the carbon flows through food webs in a weir pool on the lower Murray River, a highly impacted, complex and regulated ecosystem in southern Australia. We demonstrate that small fish in the Murray River consume a wide range of food items, but that a significant component of carbon and nitrogen entering the food web during dry periods in summer, but not spring, is derived from nonconventional sources other than in-channel primary producers. This study also showed that isotopic analyses alone cannot distinguish food sources and that a combined approach is better able to elucidate food-consumer dynamics. Our results highlight that a major river ecosystem, stressed by reduced environmental flows, can rapidly undergo significant and previously undetected changes that impact on the ecology of the system as a whole.

Matching the origin of an invasive weed for selection of a herbivore haplotype for a biological control programme.

The Florida Everglades have been invaded by an exotic weed fern, Lygodium microphyllum. Across its native distribution in the Old World tropics from Africa to Australasia it was found to have multiple location-specific haplotypes. Within this distribution, the climbing fern is attacked by a phytophagous mite, Floracarus perrepae, also with multiple haplotypes. The genetic relationship between mite and fern haplotypes was matched by an overarching geographical relationship between the two. Further, mites that occur in the same location as a particular fern haplotype were better able to utilize the fern than mites from more distant locations. From a biological control context, we are able to show that the weed fern in the Everglades most likely originated in northern Queensland, Australia/Papua New Guinea and that the mite from northern Queensland offers the greatest prospect for control.