Next-generation sequencing of the soil nematode community enables the sustainability of banana plantations to be monitored

Uganda faces a considerable challenge to match its food production to an annual population growth rate of 3%. Cooking bananas are the country's most produced staple crop but the annual national harvest is not increasing. The crop grows on infertile soils that are normally fertilised organically and often susceptible to erosion. Soil nematodes are well-established as bioindicators of soil quality that can support environmental monitoring and assessment of the sustainability of agricultural systems. These invertebrates are a highly ranked indicator of biodiversity with molecular approaches available. Consequently, we have applied next-generation DNA sequencing of soil nematodes to evaluate soil quality of Ugandan banana plantations. The aim is to establish a method for constructing an aspect of an environmental biosafety dossier with the future aim of assessing the impact of transgenic crops and improving current cropping systems. The soil samples did not differ significantly in any of the measured soil chemistry factors, soil texture or percentage of organic matter. Thirty taxons of soil nematodes other than the plant parasites were recovered from soil supporting nine banana plantations plus three each from coffee and banana-coffee interplants from East and West Uganda. Cluster analysis correctly allocated each plantation to the crop/intercrop being grown when based on the abundance of taxa rather than taxa presence or absence. This indicates that the host has considerable effects on the abundance of specific nematode species within the soil. Overall, nematodes were more abundant in soil from coffee plantations than from banana-coffee interplants with the lowest values being from fields supporting just banana. Only the basal and trophic diversity indices and the percentage of nematodes that are rapid colonisers varied between the three plantation types. The soil of all fifteen plantations can be classified as having a mature soil web condition with low physical disturbance, limited chemical stressors, moderately high nutrient enrichment and balanced decomposition channels

Transgenic Potatoes for Potato Cyst Nematode Control Can Replace Pesticide Use without Impact on Soil Quality

Current and future global crop yields depend upon soil quality to which soil organisms make an important contribution. The European Union seeks to protect European soils and their biodiversity for instance by amending its Directive on pesticide usage. This poses a challenge for control of Globodera pallida (a potato cyst nematode) for which both natural resistance and rotational control are inadequate. One approach of high potential is transgenically based resistance. This work demonstrates the potential in the field of a new transgenic trait for control of G. pallida that suppresses root invasion. It also investigates its impact and that of a second transgenic trait on the non-target soil nematode community. We establish that a peptide that disrupts chemoreception of nematodes without a lethal effect provides resistance to G. pallida in both a containment and a field trial when precisely targeted under control of a root tip-specific promoter. In addition we combine DNA barcoding and quantitative PCR to recognise nematode genera from soil samples without microscope-based observation and use the method for nematode faunal analysis. This approach establishes that the peptide and a cysteine proteinase inhibitor that offer distinct bases for transgenic plant resistance to G. pallida do so without impact on the non-target nematode soil community

The feeding tube of cyst nematodes: characterisation of protein exclusion

Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes are obligate biotrophs and modify host root tissue, using a suite of effector proteins, to create a feeding site that is their sole source of nutrition. They feed by withdrawing host cell assimilate from the feeding site though a structure known as the feeding tube. The function, composition and molecular characteristics of feeding tubes are poorly characterised. It is hypothesised that the feeding tube facilitates uptake of host cell assimilate by acting as a molecular sieve. Several studies, using molecular mass as the sole indicator of protein size, have given contradictory results about the exclusion limits of the cyst nematode feeding tube. In this study we propose a method to predict protein size, based on protein database coordinates in silico. We tested the validity of these predictions using travelling wave ion mobility spectrometry--mass spectrometry, where predictions and measured values were within approximately 6%. We used the predictions, coupled with mass spectrometry, analytical ultracentrifugation and protein electrophoresis, to resolve previous conflicts and define the exclusion characteristics of the cyst nematode feeding tube. Heterogeneity was tested in the liquid, solid and gas phase to provide a comprehensive evaluation of three proteins of particular interest to feeding tube size exclusion, GFP, mRFP and Dual PI. The data and procedures described here could be applied to the design of plant expressed defence compounds intended for uptake into cyst nematodes. We also highlight the need to assess protein heterogeneity when creating novel fusion proteins

Phasevarions mediate random switching of gene expression in pathogenic Neisseria

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that phasevarions may be a common strategy used by host-adapted bacterial pathogens to randomly switch between "differentiated" cell types

Plant-parasitic nematodes respond to root exudate signals with host-specific gene expression patterns

Plant parasitic nematodes must be able to locate and feed from their host in order to survive. Here we show that Pratylenchus coffeae regulates the expression of selected cell-wall degrading enzyme genes relative to the abundance of substrate in root exudates, thereby tailoring gene expression for root entry of the immediate host. The concentration of cellulose or xylan within the exudate determined the level of β-1,4-endoglucanase (Pc-eng-1) and β-1,4-endoxylanase (Pc-xyl) upregulation respectively. Treatment of P. coffeae with cellulose or xylan or with root exudates deficient in cellulose or xylan conferred a specific gene expression response of Pc-eng-1 or Pc-xyl respectively with no effect on expression of another cell wall degrading enzyme gene, a pectate lyase (Pc-pel). RNA interference confirmed the importance of regulating these genes as lowered transcript levels reduced root penetration by the nematode. Gene expression in this plant parasitic nematode is therefore influenced, in a host-specific manner, by cell wall components that are either secreted by the plant or released by degradation of root tissue. Transcriptional plasticity may have evolved as an adaptation for host recognition and increased root invasion by this polyphagous species

Improving a pest management tool for scenario analysis of economic populations of Globodera pallida

Globoderapallida is the most damaging pest of potato in the UK. This work underpins enhancement of a well-established, web-based scenario analysis tool for its management by recommending additions and modifications of its required inputs and a change in the basis of yield loss estimates. The required annual decline rate of the dormant egg population is determined at the individual field sample level to help define the required rotation length by comparing the viable egg content of recovered cysts to that of newly formed cysts for the same projected area. The mean annual decline was 20.4 ± 1.4% but ranged from 4.0 to 39.7% annum−1 at the field level. Further changes were based on meta-analysis of previous field trials. Spring rainfall in the region where a field is located and cultivar tolerance influence yield loss. Tolerance has proved difficult to define for many UK potato cultivars in field trials but uncertainty can be avoided without detriment by replacing it with determinacy integers. They are already determined to support optimisation of nitrogen application rates. Multiple linear regression estimates that loss caused by pre-plant populations of up to 20 viable eggs (g soil)−1 varies from ca 0.2 to 2.0% (viable egg)−1 (g soil)−1 depending on cultivar determinacy and spring rainfall. Reliability of the outcomes from scenario analysis requires validation in field trials with population densities over which planting is advisable

A High-Throughput Molecular Pipeline Reveals the Diversity in Prevalence and Abundance of Pratylenchus and Meloidogyne Species in Coffee Plantations

Coffee yields are adversely affected by plant-parasitic nematodes and the pathogens are largely under-reported because a simple and reliable identification method is not available. We describe a PCR-based approach to rapidly detect and quantify the major Pratylenchus and Meloidogyne nematode species that are capable of parasitising coffee. The procedure was applied to soil samples obtained from a number of coffee farms in Brazil, Vietnam and Indonesia to assess the prevalence of these species associated both with coffee (Coffea arabica and C. canephora) and its intercropped species Musa acuminata (banana) and Piper nigrum (black pepper). Pratylenchus coffeae and P. brachyurus were associated with coffee in all three countries, but there were distinct profiles of Meloidogyne species. Meloidogyne incognita, M. exigua and M. paranaensis were identified in samples from Brazil and M. incognita and M. hapla were detected around the roots of coffee in Vietnam. No Meloidogyne species were detected in samples from Indonesia. There was a high abundance of Meloidogyne in soil samples in which Pratylenchus were low or not detected, suggesting that the success of one genus may deter another. Meloidogyne species in Vietnam and Pratylenchus species in Indonesia were more numerous around intercropped plants than in association with coffee. The data suggest a wide-spread but differential nematode problem associated with coffee production across the regions studied. The issue is compounded by the current choice of intercrops that support large nematode populations. Wider application of the approach would elucidate the true global scale of the nematode problem and the cost to coffee production