Mode of action of fluopyram in plant-parasitic nematodes.

Plant-parasitic nematodes (PPN) are responsible for severe yield losses in crop production. Management is challenging as effective and safe means are rare. Recently, it has been discovered that the succinate dehydrogenase (SDH) inhibitor fluopyram is highly effective against PPN while accompanying an excellent safety profile. Here we show that fluopyram is a potent inhibitor of SDH in nematodes but not in mammals, insects and earthworm, explaining the selectivity on molecular level. As a consequence of SDH inhibition, fluopyram impairs ATP generation and causes paralysis in PPN and Caenorhabditis elegans. Interestingly, efficacy differences of fluopyram amongst PPN species can be observed. Permanent exposure to micromolar to nanomolar amounts of fluopyram prevents Meloidogyne spp. and Heterodera schachtii infection and their development at the root. Preincubation of Meloidogyne incognita J2 with fluopyram followed by a recovery period effectively reduces gall formation. However, the same procedure does not inhibit H. schachtii infection and development. Sequence comparison of sites relevant for ligand binding identified amino acid differences in SDHC which likely mediate selectivity, coincidently revealing a unique amino acid difference within SDHC conserved among Heterodera spp. Docking and C. elegans mutant studies suggest that this minute difference mediates altered sensitivity of H. schachtii towards fluopyram.

Pathogenesis of Plasmopara viticola Depending on Resistance Mediated by Rpv3_1, and Rpv10 and Rpv3_3, and by the Vitality of Leaf Tissue.

Grapevine cultivars vary in their resistance to Plasmopara viticola, causal agent of downy mildew. Genes from various Vitis species confer pathogen resistance (Rpv), resulting in reduced compatibility of the host-pathogen interaction and partial disease resistance that may become apparent at different stages of pathogenesis. This study describes the pathogenesis of P. viticola on the partially resistant cultivars Regent (Rpv3-1) and Solaris (Rpv3-3, Rpv10) as compared with the susceptible cultivar Mueller-Thurgau using various microscopic techniques, visual disease rating as well as qPCR. Host plant resistance had no effect on the initial steps of pathogenesis outside the host plant cells (zoospore attachment, formation of substomatal vesicle) and became detectable only after the formation of primary haustoria. The restricted compatibility resulted in reductions in haustorium size and in the number of secondary haustoria and was associated with callose depositions around haustoria and stomatal guard cells, collapsed mesophyll cells (hypersensitive reaction), and additional production of an amorphous substance in the intercellular space of cultivar Solaris. Resistance mechanisms reduced the efficiency of P. viticola haustoria and largely restricted tissue colonization to the spongy parenchyma; resistance of cultivar Solaris having thicker leaves was more effective than that of cultivar Regent. Despite of the effects of resistance genes, P. viticola was able to complete its life cycle by forming sporangiophores with sporangia through the stomata on both resistant cultivars indicating partial resistance. Differences in the pathogenesis on detached and attached grapevine leaves highlighted the impact of host tissue vitality on both resistance and susceptibility to the biotrophic pathogen.

Bacillus firmus I-1582 promotes plant growth and impairs infection and development of the cyst nematode Heterodera schachtii over two generations.

Plant-parasitic nematodes wreak havoc on crops by root parasitism worldwide. An approach to combat nematode root parasitism is the application of antagonistic microbes like the rhizobacterium Bacillus firmus I-1582 which is promoted as biological control agent. Although B. firmus is a known nematode antagonist in general, the underlying mechanisms about its interaction with nematodes and plants have not yet been elucidated. Therefore, we explored the influence of B. firmus I-1582 as well as its extracellular and secreted molecules on plant-nematode interaction utilizing the plant-pathogen system Arabidopsis thaliana-Heterodera schachtii. We demonstrated that B. firmus I-1582 is attracted by A. thaliana root exudates, particularly by those of young plants. The bacterium colonized the root and showed a strictly pH-dependent development and plant growth promotion effect. Our results revealed that root colonization by B. firmus I-1582 significantly protected A. thaliana from infestation by the beet cyst nematode whereas dead bacterial cells or the culture supernatant were not effective. The bacterium also negatively affected nematode reproduction as well as pathogenicity and development of next generation nematodes. The obtained results highlight B. firmus I-1582 as a promising biocontrol agent that is well suited as an element of integrated control management strategies in sustainable agriculture.

Plant parasitic cyst nematodes redirect host indole metabolism via NADPH oxidase-mediated ROS to promote infection.

Reactive oxygen species (ROS) generated in response to infections often activate immune responses in eukaryotes including plants. In plants, ROS are primarily produced by plasma membrane-bound NADPH oxidases called respiratory burst oxidase homologue (Rboh). Surprisingly, Rbohs can also promote the infection of plants by certain pathogens, including plant parasitic cyst nematodes. The Arabidopsis genome contains 10 Rboh genes (RbohA-RbohJ). Previously, we showed that cyst nematode infection causes a localised ROS burst in roots, mediated primarily by RbohD and RbohF. We also found that plants deficient in RbohD and RbohF (rbohD/F) exhibit strongly decreased susceptibility to cyst nematodes, suggesting that Rboh-mediated ROS plays a role in promoting infection. However, little information is known of the mechanism by which Rbohs promote cyst nematode infection. Here, using detailed genetic and biochemical analyses, we identified WALLS ARE THIN1 (WAT1), an auxin transporter, as a downstream target of Rboh-mediated ROS during parasitic infections. We found that WAT1 is required to modulate the host's indole metabolism, including indole-3-acetic acid levels, in infected cells and that this reprogramming is necessary for successful establishment of the parasite. In conclusion, this work clarifies a unique mechanism that enables cyst nematodes to use the host's ROS for their own benefit.

Sublethal fluazaindolizine doses inhibit development of the cyst nematode Heterodera schachtii during sedentary parasitism.

BACKGROUND: Fluazaindolizine is a new compound for the control of plant-parasitic nematodes (PPNs) with an unknown and novel mode-of-action. This compound is very effective against important PPNs. However, investigations elucidating the impact of sublethal fluazaindolizine doses on early nematode virulence and plant-nematode interaction parameters are lacking. RESULTS: The effect of direct exposure of Heterodera schachtii juveniles to 50 ppm fluazaindolizine was negligible. Infection assays revealed a 57% reduction in adult females at 1.25 ppm and a 46% reduction in offspring at 40 ppm when juveniles were soaked in the compound for 48 h and subsequently inoculated onto Arabidopsis thaliana. Pre-incubation of A. thaliana roots with fluazaindolizine was not effective against H. schachtii. Conversely, supplementing the plant growth medium with fluazaindolizine led to a significant reduction of adults (-35%), females (-75%) and female size at 1.25 ppm and nearly completely inhibited nematode parasitism at 5 ppm. The impact of fluazaindolizine on A. thaliana was dependent on plant age, compound concentration and duration of contact. Very low sublethal fluazaindolizine concentrations, 5 or 10 ppm, did not interfere with nematode mobility, host finding, penetration, and induction of the feeding site, but specifically inhibited sedentary nematode development inside the root in a concentration-dependent manner. CONCLUSION: Fluazaindolizine does not have direct toxicity against PPN infective juveniles, but has a clear effect on nematodes during sedentary development. The formation of females and the development of offspring are strongly reduced. It will be interesting to identify the underlying mechanism in the future. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Host factors influence the sex of nematodes parasitizing roots of Arabidopsis thaliana.

Plant-parasitic cyst nematodes induce hypermetabolic syncytial nurse cells in the roots of their host plants. Syncytia are their only food source. Cyst nematodes are sexually dimorphic, with their differentiation into male or female strongly influenced by host environmental conditions. Under favourable conditions with plenty of nutrients, more females develop, whereas mainly male nematodes develop under adverse conditions such as in resistant plants. Here, we developed and validated a method to predict the sex of beet cyst nematode (Heterodera schachtii) during the early stages of its parasitism in the host plant Arabidopsis thaliana. We collected root segments containing male-associated syncytia (MAS) or female-associated syncytia (FAS), isolated syncytial cells by laser microdissection, and performed a comparative transcriptome analysis. Genes belonging to categories of defence, nutrient deficiency, and nutrient starvation were over-represented in MAS as compared with FAS. Conversely, gene categories related to metabolism, modification, and biosynthesis of cell walls were over-represented in FAS. We used ß-glucuronidase analysis, qRT-PCR, and loss-of-function mutants to characterize FAS- and MAS-specific candidate genes. Our results demonstrate that various plant-based factors, including immune response, nutrient availability, and structural modifications, influence the sexual fate of the cyst nematodes.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Parasitic worms stimulate host NADPH oxidases to produce reactive oxygen species that limit plant cell death and promote infection.

Plants and animals produce reactive oxygen species (ROS) in response to infection. In plants, ROS not only activate defense responses and promote cell death to limit the spread of pathogens but also restrict the amount of cell death in response to pathogen recognition. Plants also use hormones, such as salicylic acid, to mediate immune responses to infection. However, there are long-lasting biotrophic plant-pathogen interactions, such as the interaction between parasitic nematodes and plant roots during which defense responses are suppressed and root cells are reorganized to specific nurse cell systems. In plants, ROS are primarily generated by plasma membrane-localized NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, and loss of NADPH oxidase activity compromises immune responses and cell death. We found that infection of Arabidopsis thaliana by the parasitic nematode Heterodera schachtii activated the NADPH oxidases RbohD and RbohF to produce ROS, which was necessary to restrict infected plant cell death and promote nurse cell formation. RbohD- and RbohF-deficient plants exhibited larger regions of cell death in response to nematode infection, and nurse cell formation was greatly reduced. Genetic disruption of SID2, which is required for salicylic acid accumulation and immune activation in nematode-infected plants, led to the increased size of nematodes in RbohD- and RbohF-deficient plants, but did not decrease plant cell death. Thus, by stimulating NADPH oxidase-generated ROS, parasitic nematodes fine-tune the pattern of plant cell death during the destructive root invasion and may antagonize salicylic acid-induced defense responses during biotrophic life stages.