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Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Societal Impact Statement: Plant‐parasitic nematodes are a major concern for global food security, and many existing control options are being phased out due to adverse impacts on the environment. Here, we show that although application of arbuscular mycorrhizal fungi (AMF) increases host tolerance to these parasites, these benefits decrease as the parasite burden increases, limiting long‐term benefits. This effect was consistent between experiments in the glasshouse and in the field environment, demonstrating the relevance of research into usable technologies. Our findings have potential to aid decision making regarding application of AMF inocula for optimum results in agricultural systems. Summary: Plant‐parasitic nematodes are a leading global threat to crop production and food security aims. Control strategies based on nematicides and fertilisers are increasingly undesirable due to economic and environmental impacts. Arbuscular mycorrhizal fungi (AMF) may induce host tolerance against pests such as the potato cyst nematode (PCN).Here, we determined the impact of PCN density on the tolerance induced by AMF‐host interactions. Additionally, we evaluated the effects of five AMF inocula on PCN fitness though glasshouse and field trials.Greater PCN densities reduce the increased tolerance that AMF may confer on their hosts. This may be due to reduced mycorrhizal colonisation of hosts under higher PCN infection and potentially a threshold at which the presence of PCN severely impacts fungal growth. When tested in the field, the outcomes of AMF inoculation on crop yields were still positive. Inoculation of soil in the field also increased PCN multiplication, suggesting that AMF‐induced tolerance may become reduced in the near future when the threshold PCN density is reached.Addition of AMF to agricultural soils may provide a short‐term benefit yet lead to a long‐term detriment by increasing PCN populations. The effects observed were driven by only one out of the five introduced AMF species, indicating that the remaining species were redundant for this application. This raises important considerations for future application of AMF inocula in agricultural systems and aids our understanding of how widely used 'beneficial' soil amendments impact the agricultural ecosystem. [ABSTRACT FROM AUTHOR]

Summary: Globodera pallida 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. [ABSTRACT FROM AUTHOR]

The potato cyst nematode Globodera pallida acquires all of its nutrients from an elaborate feeding site that it establishes in a host plant root. Normal development of the root cells is re-programmed in a process coordinated by secreted nematode effector proteins. The biological function of the G. pallida GpIA7 effector was investigated in this study. GpIA7 is specifically expressed in the subventral pharyngeal glands of pre-parasitic stage nematodes. Ectopic expression of GpIA7 in potato plants affected plant growth and development, suggesting a potential role for this effector in feeding site establishment. Potato plants overexpressing GpIA7 were shorter, with reduced tuber weight and delayed flowering. We provide evidence that GpIA7 associates with the plant growth regulator StEBP1 (ErbB-3 epidermal growth factor receptor-binding protein 1). GpIA7 modulates the regulatory function of StEBP1, altering the expression level of downstream target genes, including ribonucleotide reductase 2 , cyclin D3;1 , and retinoblastoma related 1 , which are down-regulated in plants overexpressing GpIA7. We provide an insight into the molecular mechanism used by the nematode to manipulate the host cell cycle and demonstrate that this may rely, at least in part, on hindering the function of host EBP1. [ABSTRACT FROM AUTHOR]

Summary: Double‐stranded RNA (dsRNA) molecules targeting two genes have been identified that suppress economically important parasitic nematode species of banana. Proteasomal alpha subunit 4 (pas‐4) and Actin‐4 (act‐4) were identified from a survey of sequence databases and cloned sequences for genes conserved across four pests of banana, Radopholus similis, Pratylenchus coffeae, Meloidogyne incognita and Helicotylenchus multicinctus. These four species were targeted with dsRNAs containing exact 21 nucleotide matches to the conserved regions. Potential off‐target effects were limited by comparison with Caenorhabditis, Drosophila, rat, rice and Arabidopsis genomes. In vitro act‐4 dsRNA treatment of R. similis suppressed target gene expression by 2.3‐fold, nematode locomotion by 66 ± 4% and nematode multiplication on carrot discs by 49 ± 5%. The best transgenic carrot hairy root lines expressing act‐4 or pas‐4 dsRNA reduced transcript message abundance of target genes in R. similis by 7.9‐fold and fourfold and nematode multiplication by 94 ± 2% and 69 ± 3%, respectively. The same act‐4 and pas‐4 lines reduced P. coffeae target transcripts by 1.7‐ and twofold and multiplication by 50 ± 6% and 73 ± 8%. Multiplication of M. incognita on the pas‐4 lines was reduced by 97 ± 1% and 99 ± 1% while target transcript abundance was suppressed 4.9‐ and 5.6‐fold. There was no detectable RNAi effect on nontarget nematodes exposed to dsRNAs targeting parasitic nematodes. This work defines a framework for development of a range of nonprotein defences to provide broad resistance to pests and pathogens of crops. [ABSTRACT FROM AUTHOR]