Morphological, biochemical and molecular approaches to the identification of Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria in Portugal

Mestrado em Engenharia Agonómica - Proteção de plantas - Instituto Superior de Agronomia - ULPlant parasitic nematodes are highly damaging pests in many crops of great economic importance. A substantial part of this damage is caused by infestations of root-knot nematodes (RKN), due to their wide geographical distribution as well as a vast range of host plants. The most important and widely distributed root-knot nematode species are Meloidogyne incognita, M. arenaria and M. javanica accounting for almost 95% of the occurrences, and so, an accurate and reliable identification it is primary to establish effective, sustainable and environmentally safe control measures. The main goals of this study were to characterise morphologically, biochemically and molecularly Portuguese isolates and reference material from Netherlands Food and Consumer Product Safety Authority (NVWA), Wageningen, The Netherlands, in order to find the most suitable tools that can be used in the Laboratory of Molecular Genetics and Nematology at the INIAV in Portugal to assist in the identification of the three most common species of Meloidogyne. Morphological studies incorporated the examination of female’s perineal pattern and measurements of second stage juveniles’ features, which presented enough variation to confirm the unreliability and difficulty of morphological identification alone. Biochemical assays with isozymes such as esterases (EST) were performed on females and the PAGE enzymatic patterns obtained match those already described for M. javanica. Molecular analysis included PCRs using universal primers that target the 28S gene, ITS and IGS ribosomal DNA regions (rDNA) and the region between the cytochrome oxidase (COII) of the mitochondrial DNA (mtDNA) and 16S rDNA genes together with speciesspecific SCAR primers and sequencing and cloning. The sequences obtained from sequencing of PCR fragments and from cloned fragments were aligned and compared to those found in GenBank database through BLAST analysis with inconclusive results. Although it was no possible to uncover an efficient tool for Meloidogyne identification this study pinpointed that the three approaches for root-knot nematode identification need to be used if we are to identify this species accurately and effectively. What’s more this research can be used as platform for further studies regarding this species at the Nematology of INIAV PortugalN/

Extração de nemátodes de quisto de amostras de solo: método de decantação e crivagem de Cobb vs. método de Fenwick

Potato cyst nematodes are a threat to several agricultural crops around the world with some species considered quarantine pests and subjected to strict regulatory measures in many countries. Usually, cysts nematodes co-exist in the soil with other species of plant-parasitic nematodes, so, a time and cost-efficient extraction technique becomes of primary importance. The ideal extraction method should be able to obtain cysts as well as detecting the presence of other motile plant-parasitic nematodes with a potential impact on potato farming (such as Meloidogyne sp. and Pratylenchus sp.). In recent years, studies have been carried out to test the efficiency of various methods of nematode extraction but few results have been published. Therefore, to test if a method that extracts simultaneously cysts and motile nematodes can be used instead of the reference method that extracts cysts only, the efficiency of Cobb’s decanting and sieving technique was compared to Fenwick’s technique. As a result, in the 74 samples evaluated, a greater number of cysts were extracted from 24 samples using Fenwick’s method and from 11 samples employing Cobb’s decanting and sieving technique. The statistics results showed a significance level of 0,05 using Fenwick’s can allowing to conclude that this method is much more efficient than Cobb’s decanting and sieving technique, and confirming it should not be replaced by alternative methods for cysts extractioninfo:eu-repo/semantics/publishedVersio

species identification, distribution, and new host records in Portugal

Funding Information: This research was supported by the “Fundação para a Ciência e a Tecnologia” (FCT, Portugal) and the European Social Funds, through the “Programa Operacional Regional Centro”, under the Ph.D. fellowship 2020.05541.BD and through the R&D Unit, UIDB/04551/2020 (GREEN-IT—Bioresources for Sustainability). Acknowledgments Funding Information: This research was supported by the “Fundação para a Ciência e a Tecnologia” (FCT, Portugal) and the European Social Funds, through the “Programa Operacional Regional Centro”, under the Ph.D. fellowship 2020.05541.BD and through the R&D Unit, UIDB/04551/2020 (GREEN-IT—Bioresources for Sustainability). Publisher Copyright: Copyright © 2023 Rusinque, Camacho, Serra, Nóbrega and Inácio.Considered one of the most devastating plant parasitic nematodes worldwide, Meloidogyne spp. (commonly known as the root-knot nematodes (RKNs)) are obligate sedentary endoparasites that establish in the roots, causing hyperplasia and hypertrophy of surrounding cells, triggering the formation of galls. These galls will affect root development and physiology, leading to substantial yield losses. During 2017–2022, an extensive survey of Meloidogyne species was undertaken in Portugal (mainland and islands). A total of 1,071 samples were collected by the National Plant Protection Organization (DGAV) and private farmers from different regions of the country and were analysed at the Laboratory of Nematology (NemaINIAV). Samples in which the presence of Meloidogyne sp. was detected were used to perform bioassays to obtain females and juveniles for further studies. Since the accurate identification of RKNs is an important aspect of crop management, morphological and biochemical characterisation was performed. The most common morphological features were observed, showing consistency with previous descriptions of the genus. The biochemical identification using the esterase (EST) phenotype revealed the phenotypes of Meloidogyne arenaria, M enterolobi, M. hispanica, M. hapla, M. incognita, M javanica, and M. luci. Meloidogyne incognita and M. javanica were found to be the most prevalent species in the different regions followed by M. arenaria and M. hapla. This is the first distribution report performed in Portugal on RKNs, contributing to the development of management strategies and to updated information on the status of these pests in Europe.publishersversionpublishe

Root-Knot Nematode Species Associated with Horticultural Crops in the Island of Azores, Portugal

Plant-parasitic nematodes (PPN) are an economically important group of plant pests present throughout the world. In particular, root-knot nematodes (RKN), sedentary endoparasites of a wide variety of economically important hosts with the ability to survive in temperate regions. During 2020–2021 an extensive survey of Meloidogyne spp. was undertaken on the island of São Miguel Azores, Portugal. A total of 80 samples comprising 23 species of plants were collected from 13 localities in 4 districts of the island. Samples included field and greenhouse vegetable crops. Bioassays were carried out to obtain females and juveniles for morphological and biochemical characterization of the isolates. The observed morphological features showed high similarity and consistency with previous descriptions of the genus. Concerning the biochemical characterization, the esterase (EST) phenotype displayed the patterns described for M. incognita, M. arenaria, and M. javanica. Meloidogyne incognita was found to be the most prevalent species with respect to both natural host range and geographical distribution, followed by M. arenaria and M. javanica. This is the first field survey that assesses the species of root-knot nematodes associated with horticultural crops in the Azores Island, contributing additional information on the distribution of this genusinfo:eu-repo/semantics/publishedVersio

Bioactivity of Monoterpene Alcohols as an Indicator of Biopesticidal Essential Oils against the Root Knot Nematode Meloidogyne ethiopica

The application of pesticides remains one of the most efficient control methods for phytophagous parasites in crops. Essential oils (EOs) are complex mixtures of highly active compounds that can be used as biopesticides against plant parasitic nematodes. In the present work, the antinematodal activity of the monoterpene alcohols geraniol, linalool, menthol or  -terpineol, which are generally found in high amounts in EOs of some aromatic and medicinal plants, was analyzed on the root knot nematode Meloidogyne ethiopica. Geraniol showed intense and lasting antinematodal activity, suggesting that EOs rich in this compound can be used in the development of nematicidal biopesticides to integrate sustainable pest management strategies against this pest

The fight against plant-parasitic nematodes: current status of bacterial and fungal biocontrol agents

Plant-parasitic nematodes (PPNs) are among the most notorious and underrated threats to food security and plant health worldwide, compromising crop yields and causing billions of dollars of losses annually. Chemical control strategies rely heavily on synthetic chemical nematicides to reduce PPN population densities, but their use is being progressively restricted due to environmental and human health concerns, so alternative control methods are urgently needed. Here, we review the potential of bacterial and fungal agents to suppress the most important PPNs, namely Aphelenchoides besseyi, Bursaphelenchus xylophilus, Ditylenchus dipsaci, Globodera spp., Heterodera spp., Meloidogyne spp., Nacobbus aberrans, Pratylenchus spp., Radopholus similis, Rotylenchulus reniformis, and Xiphinema index

Potato Cyst Nematodes: geographical distribution, phylogenetic relationships and integrated pest management outcomes in Portugal

The identification and phylogenetic relationships of potato cyst nematodes (PCN) were studied to assess the potential value of geographical distribution information for integrated pest management of potato production in Portugal. This research focused on PCN species, Globodera pallida and Globodera rostochiensis. From 2013 until 2019, 748 soil samples from the rhizosphere of different potato cultivars were surveyed in the Portuguese mainland to detect and identify both species and track their location. PCN are widespread invasive species throughout Portugal. In fact, during the survey period an incidence of 22.5% was estimated for the tested samples. The patterns of infestation vary among regions, increasing from south to north, where PCN were first detected. Currently, both species are present in all potato producing regions of the country, with a greater incidence of G. pallida. Phytosanitary control measures are influencing to the observed results. The use of potato cultivars resistant to G. rostochiensis led to a decrease of this species but had no influence on G. pallida detections, which continues its reproduction freely since there are no effective resistant cultivars for this species. The relationship between the presence, infestation rate, spread and geographical distribution of PCN is discussed in terms of behavioral responses of the potato cultivars and the implications for developing new integrated crop protection measures

Nemátode-dos-citrinos: prevenir o declínio lento do pomar

Os citrinos são as fruteiras mais importantes em termos de produção a nível mundial, com cerca de 130 milhões de toneladas produzidas (World Citrus Organisation, 2020), repartin- do-se por mais de 125 países localizados entre os 40° de latitude a norte e a sul do Equador. Entre as várias ameaças à produção citrícola contam-se os nemátodes fitoparasitas. Os nemátodes fitoparasitas são pequenos animais, de aspeto vermiforme, cilíndricos e alongados, geralmente com menos de 1-2 mm de comprimento, que causam doenças às plantas. A maior parte habita no solo, infe- tando as raízes de onde se alimentam atra- vés do estilete (estrutura na cavidade oral que funciona como uma agulha hipodérmi- ca, sugando o conteúdo das células vegetais). Uns permanecem no exterior (ectoparasi- tas), enquanto outros penetram nas raízes (endoparasitas) e outros ainda avançam nos tecidos do hospedeiro, mas só com a parte posterior (cabeça e pescoço), permanecendo o restante corpo fora da raiz (semiendopara- sitas). O nemátode-dos-citrinos, Tylenchulus semipenetrans, é um semiendoparasita e daí a sua designação específica. De todos os ne- mátodes que podem afetar estas fruteiras, é aquele que tem maior impacte económico, sendo a única espécie presente em todas as regiões produtoras de citrinos do mundo

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.)

NEMÁTODES DE QUISTO DA BATATEIRA – O USO DE VARIEDADES RESISTENTES COMO ESTRATÉGIA DE CONTROLO

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are considered harmful quarantine organisms and can be devastating to potato fields if not controlled in a timely manner. Its designation derives from the fact that the female body, after their death, turns into a spherical cyst con- taining the eggs, which are protected from adverse conditions even in the absence of hosts. PCN are present in all potato pro- ducing regions of the country, including the Madeira and Azores islands. PCN control involves their opportune detection, cor- rect identification and appropriate control measures. Tests already carried out proved that the use of resistant varieties is effective for the management of these organisms. However, there is insufficient choice of par- tially resistant cultivars for growers, espe- cially for the control of G. pallida