PROTECTING OUR CROPS - APPROACHES FOR PLANT PARASITIC NEMATODE CONTROL

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Plant-nematode co-cultures in the screening of sustainable nematicides against soil-dwelling parasitic nematodes of plants

The diseases caused by plant parasitic nematodes are still a serious constraint to modern global crop production. An increasing number of active compounds in commercial nematicidal formulations is being banned from use by common policies of pest management. Farmer communities report a low efficiency for the replacement pesticides, which reflects on crop yield and productivity. Novel sustainable biopesticides are urgently needed to cope with global food demands while respecting the most recent environmental policies. Plant-nematode co-cultures offer a stable biotechnological screening tool able to assess the active compound’s nematicidal activity and its effect on host tissues, simultaneously, in an easily accessible system that simulates natural infection. These systems are being developed and optimized at the Nematology laboratory of INIAV. Preliminary results were obtained for co-cultures of Solanum lycopersicum with the nematode Meloidogyne ethiopica and S. tuberosum with Globodera pallida. Future studies will target other plant parasitic nematodes, e.g., the root lesion nematodes (Pratylenchus penetrans) in transgenic roots of potato and the pinewood nematode (Bursaphelenchus xylophilus) on in vitro pine shoots (Pinus sp.)

Control of the root lesion Pratylenchus penetrans - the effect of nematocidal activity of plant-derived compounds

The root lesion nematode, Pratylenchus penetrans, is one of the most harmful plant parasites, responsible for worldwide productivity losses in a significant number of plant hosts. Generally, chemical control relies on synthetic compounds used through fumigation or direct contact, which offers a systemic protection. These control methods are costly and hazardous to the environment and to humans. Phytochemicals may play an important role in nematode control. The nematicidal activity of eight compounds that occur naturaly in plants, from two classes of compounds, was assessed at 2 mg/mL, for 24 h. Bioassays were performed following the standard direct contact methodology. P. penetrans was remarkably tolerant to the tested compounds, with mortality range between 1.0 and 5.8 %. To the best of our knowledge, the nematicidal activity of 4 phenolic compounds (catechin, caffeic acid, gallic acid and gentisic acid) was evaluated for the first time for P. penetrans

Host plant response to the application of nematocidal phytochemicals

Potato (Solanum tuberosum) is one of the world’s most common agricultural crop for human consumption and considered an excellent source of essential nutrients and vitamins. The productivity of this crop is threatened by several plant-parasitic nematodes, among which the root-lesion nematode Pratylenchus penetrans is already considered to have a significant impact. Over the years, due to an increasing need of food resources and absence of knowledge, synthetic nematicides were extremely applied on crop fields. However, new policies, supported by the Sustainable Development Goals, impose serious limits to the application of such products. Volatile organic compounds naturally produced by plants have long been recognized for their nematicidal effectiveness. In previous work, the nematicidal activity of several standards of naturally occurring phytochemicals was in vitro assessed on P. penetrans. Only 4 compounds caused more than 99% nematode mortality, namely benzaldehyde, carvacrol, octanol and thymol. The aim of this work was to determine the most suitable approach to apply the selected compounds in soil in order to maintain their nematicidal effectiveness without compromising host health. Two consecutive trials were performed using potato seed plants (cv. Agria), namely: (i) to evaluate the effect of three solvent agents - 10% (v/v) dimethyl sulfoxide (DMSO), 5 mg/mL TRITON-X, and 1% (v/v) acetone - in plant growth; and (ii) to evaluate the best solvent agent spiked with 2 mg/mL of each selected compound. For both trials, a control treatment with water was considered. Pots were maintained in growth chamber conditions, routinely watered and fertilized. Plant growth related parameters were assessed at collection time. In the 1st trial, potato plants were unable to grow in the presence of 10% DMSO solution or the 5 mg/mL TRITON-X. The potato seeds could germinate only in the presence of 1% acetone. In the 2nd trial, potato seeds could germinate in the presence of the 4 compounds dissolved in 1% acetone. No statistical differences (p>0.05) were recorded between the control treatment and the different compound applications in terms of growth parameters observed. Ongoing research is now accessing the nematicidal activity of each of the four compounds in the presence of the potato plant

O NEMÁTODE‑DAS‑LESÕES‑RADICULARES, PRATYLENCHUS PENETRANS

O nemátode-das-lesões-radiculares, Pratylenchus penetrans, é um dos nemátodes parasitas de plantas mais devastador a nível mundial, e uma ameaça para um vasto número de culturas de interesse económico. Em Portugal, é frequentemente detetado em campos de batata. A deteção atempada deste parasita no solo, aliada a boas práticas agrícolas, são fatores decisivos para a proteção das culturas

How parasitism genes are regulated: a motif to search for genes regulators in the plant parasitic nematode Bursaphelenchus xylophilus

Plant-parasitic nematodes threaten global agricultural and forestry systems. The search for new control strategies in line with the EU’s sustainability goals highlight significant knowledge gaps. Like all other plant pathogens, plant-parasitic nematodes deliver several parasitism proteins (effectors) into the host plant to cause disease. NemaWAARS project focuses on mechanism(s) of regulation and gene control expression of parasitism genes in pinewood nematode, Bursaphelenchus xylophilus. From the previous transcriptomic data derived from the pharyngeal gland cells (considered a specialized tissue potentially related to parasitism) we have identified a non-coding DNA motif - STATAWAARS - associated in the promotor region of highly abundant and secreted expressed genes. Given that this non-coding genetic signature unifies many sequences of unrelated parasitism genes, it implies the existence of a potential major regulator(s), that binds to this sequence to control the expression of downstream genes. We hypothesize that by disrupting this regulator(s), it would be possible to simultaneously disrupt the expression of many associated parasitism-related genes. To test the hypothesis the project aims to identify proteins (or complex of proteins) that bind in the promoter regions of parasitism-related genes (in vivo) or identify other regulatory candidates for master regulators of parasitism-related genes expression that are enriched in the pharyngeal gland cell tissues. For the best candidate regulatory proteins, an RNAi approach will target the selected gene candidates and evaluate the regulatory role in effector genes expression and in interaction with the host (in planta). Under an ongoing national and international collaborative network, the strategy in NemaWAARS will include innovative approaches to explore the regulators that govern effector gene expression applied in B. xylophilus research

Broad environmental tolerance of native root- nodule bacteria of Biserrula pelecinus indicate potential for soil fertility restoration

Background: Biserrula pelecinus is an annual legume native to the Mediterranean basin, found in pastureland, alone or in association with other legumes (Leguminosae) and grasses (Poaceae). It has been used in revegetation programmes of mining wastes showing phytoremediation potentials and thus becoming potentially highly attractive for plant ecology and restoration management of natural ecosystems. Aims: To characterise native root-nodule bacteria isolated from B. pelecinus from the Iberian Peninsula, and to select suitable N fixers for field-application and soil rehabilitation. Methods: Strains were isolated and molecularly identified by 16S rRNA amplification and sequencing. Strains were phenotypically characterised in different abiotic conditions (acidity, salinity and heavy metals) and tested for their ability to fix atmospheric N2. The most suitable N fixers were applied in greenhouse experiments with B. pelecinus under different fertilization levels to assess their tolerance to fertilized and polluted soils, commonly encountered in restoration projects. Results: B. pelecinus root-nodule isolates tolerated pH from 4.5 to 9.5 grew in saline conditions (2.5% of NaCl), and tolerated 50 μM of Al3+ and Mn2+. Three isolates efficient in N2 fixation, relative to the reference Mesorhizobium strain, were considered excellent candidates for the amelioration of nutrient poor sites. Conclusions: These results provide valuable information for the potential use in soil restoration of B. pelecinus in a wide- range of conditions, exploiting the natural variability of its root-nodule bacteria

Bacteria associated with the pinewood nematode Bursaphelenchus xylophilus collected in Portugal

Abstract: In this study, we report on the bacterial community associated with the pinewood nematode Bursaphelenchus xylophilus from symptomatic pine wilted trees, as well as from long-term preserved B. xylophilus laboratory collection specimens, emphasizing the close bacteria-nematode associations that may contribute to pine wilt disease development