First report of root knot nematodes Meloidogyne incognita and M. javanica parasitizing sweet potato, Ipomoea batatas L., in Portugal.

Sweet potato, Ipomoea batatas L., is a tuberous root vegetable rich in low glycemic sugars, vitamins and fibers (Galvão et al., 2021). Although it is widely cropped and consumed in tropical regions, in Europe consumer demand is growing exponentially (CBI, 2021). In Portugal, the production area of sweet potato increased from 588 ha in 2011 to 954 ha in 2017, and exports increased from 2404 tons in 2011 to 13412 tons in 2019 (FAOSTAT, 2021). During a survey carried out in August 2019, sweet potato plants were collected in Almada (38°39'40"N 9°10'54"W) and Belmonte (38°39'40"N 9°10'54"W), South and Centre regions of Portugal, respectively. No symptoms were observed on leaves, however, roots presented numerous galls and/or small spots (females and respective egg masses) were observed in the tuberous root flesh, suggestive of root knot nematodes (RKN, Meloidogyne spp.) infection. At least 8 individual females and respective egg masses were handpicked from roots of each sample and characterized biochemically by electrophoretic analysis of esterases (Pais & Abrantes, 1989). Phenotypes I2 and J3, attributed to M. incognita and M. javanica, respectively, were present in samples from Almada, whereas only phenotype I2 was found from Belmonte sample (Santos et al., 2019). Pure RKN cultures were established on tomato cv. Coração-de-Boi to obtain inoculum for molecular characterization and host suitability assays. Molecular characterization was performed by DNA amplification with M. incognita (Mi-F/Mi-R) and M. javanica (Fjav/Rjav) species-specific primers (Zijlstra et al., 2000; Meng et al., 2004). DNA amplification resulted in unique bands of ≈900 bp and ≈650 bp, respectively, confirming the RKN species identification. The host suitability of sweet potato cvs. Lira (local variety, purple skin, yellow flesh) and Murasaki (purple skin, white/pale to yellow flesh) to M. javanica (Almada) and M. incognita (Belmonte) isolates was assessed. Sweet potato slips with ≈10 cm roots were transplanted to 500 cm3 pots (one slip/pot) and after 2 weeks, each plant was inoculated with 5000 eggs + second-stage juveniles (Pi, initial population density) and maintained in a growth chamber (25±2°C; 12:12 h photoperiod). Tomato cv. Coração-de-Boi was included as a positive control. Each RKN species-plant germplasm combination was repeated 6 times. At 60 days after inoculation, host suitability was evaluated on the basis of root gall index (GI) and reproduction factor (Rf=final population density/Pi) (Sasser et al., 1984). Sweet potato cv. Lira was susceptible (GI=5; Rf=111.8) to M. incognita and resistant (GI=2; Rf=0.11) to M. javanica; while cv. Murasaki was hypersusceptible (GI=5; Rf=0.9) to M. incognita and susceptible (GI=5; Rf=5.5) to M. javanica. Although cultivars varied in their response to M. incognita and M. javanica isolates and variation in the final population density was high, both RKN isolates reproduced in these sweet potato cultivars. In previous studies, cv. Murasaki was considered resistant to M. enterolobii and to M. incognita (La Bonte et al. 2008; Schwarz et al., 2021). Depending on the RKN species, cultivation of cvs. Murasaki and Lira may thus benefit succeeding crops, but they should be combined with other management strategies to further reduce RKN populations in the field. In Portugal, M. incognita and M. javanica have been found associated with economically important horticultural crops, such as tomato and potato, trees and weeds (Santos et al., 2019; Maleita et al., 2021). To our knowledge, these species are reported for the first time parasitizing sweet potato in Portugal and this is the first report on the occurrence of M. incognita and M. javanica infecting sweet potato in Europe. Although findings were not totally unexpected due to the wide distribution and host range of these RKN species, they are of crucial importance since the sweet potato production in Europe has almost doubled from 50 (2011) to 97 thousand tons (2017), with Spain, Portugal, Italy and Greece being the largest producers (FAO, 2021). Our findings also reveal that sweet potato cropped in Portugal have different susceptibility levels to these common RKN species, reinforcing the importance of cultivar selection in RKN management.

Relation between magnetic parameters and nematode abundance in agricultural soils of Portugal--a multidisciplinary study in the scope of environmental magnetism.

Soil is composed of different types of particles which are either natural or of anthropogenic origin. Anthropogenic particles are often related to the presence of heavy metals and thus provide information on soil quality. Magnetic parameters can detect the presence of such particles and may be used as a proxy for environmental pollution. This study explores the relationships between magnetic particles and the nematofauna of agricultural soils. Magnetic, pedological, microscopy and nematological analyses were conducted in soils collected from major regions of potato production in Portugal. The magnetic characterisation of soils identified regions with magnetic particles with possible anthropogenic origin. Microscopy analysis revealed the presence of spherical particles dominantly composed of Fe, O and C. A positive and significant relationship was found between saturation isothermal remanent magnetisation (SIRM) and mass-specific susceptibility (χ), confirming the importance the ferrimagnetic fraction to magnetic properties. The nematode communities were composed of nematodes belonging to four trophic groups (bacterial feeding, plant feeders, fungal feeders and omnivores/predators). The relationships between magnetic parameters and the nematodes showed that (1) S-25 has a linear correlation with number of nematodes per kilogram of soil and with plant feeders' trophic group and (2) SIRM correlates with the bacterial feeders trophic group. This study reveals that magnetic proxies may provide means for detecting regions with higher levels of pollution, possibly related to heavy metals. Due to the large background variability found in magnetic parameters, the sampling spacial mesh should to be further refined and the input of magnetic minerals needs to be locally calibrated.

Biocontrol potential of entomopathogenic nematodes against the grey maize weevil Tanymecus dilaticollis (Coleoptera: Curculionidae) adults.

The grey maize weevil, Tanymecus dilaticollis, is a polyphagous species, which is among the most important pests of maize in Southeastern Europe. The efficacy of commercial products with two species of entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis bacteriophora, was investigated against adults of the grey maize weevil under laboratory conditions. Nemastar®, containing S. carpocapsae was more effective on T. dilaticollis adults than Nematop® containing H. bacteriophora, when applied uniformly to the surface of the soil, on Petri dishes containing T. dilaticollis adults. Results showed that S. carpocapsae rates of 83-333 infective juveniles/adult caused > 94% mortality in T. dilaticollis adults, whereas H. bacteriophora caused 27-61%, adult mortality, after exposure of insects to the commercial products of EPNs for 15 days. The infection rates of EPNs increased with concentration applied and ranged from 70-83% and 19-64% for Nemastar® and Nematop®, respectively. Subsequent field and semi-field tests were conducted with Nemastar® (application rate of 50 million S. carpocapsae per 100 m2) in maize crops with biological (mycoinsecticide Naturalis®, biofungicides and fertilizers) and chemical seed treatment (Gaucho® FS 600; active ingredient: imidacloprid) in Knezha, Bulgaria. Nematodes were found only in the dead specimens, in open plots and cages sprayed with the commercial nematode product. Nematode sprayings contributed for higher maize yields in the open maize plots in the fields with different seed treatments. We suggest that the use of powder formulation of S. carpocapsae in combination with biologically treated maize seeds can contribute to minimize the use of chemical insecticides against the grey maize weevil. The results obtained can be used as a base to further tests to ascertain the efficacy of EPNs products before they can be recommended for use in the integrated approach to T. dilaticollis management.

Pochonia chlamydosporia: Advances and Challenges to Improve Its Performance as a Biological Control Agent of Sedentary Endo-parasitic Nematodes.

The nematophagous fungus Pochonia chlamydosporia var. chlamydosporia is one of the most studied biological control agents against plant (semi-) endo-parasitic nematodes of the genera Globodera, Heterodera, Meloidogyne, Nacobbus and, more recently, Rotylenchulus. In this paper we present highlights from more than three decades of worldwide research on this biological control agent. We cover different aspects and key components of the complex plant-fungus-nematode tri-trophic interaction, an interaction that needs to be addressed to ensure the efficient use of P. chlamydosporia as a biopesticide as part of an integrated pest management approach.

Transcriptome analysis reveals the high ribosomal inhibitory action of 1,4-naphthoquinone on Meloidogyne luci infective second-stage juveniles.

The root-knot nematode (RKN) Meloidogyne luci presents a threat to the production of several important crops. This nematode species was added to the European Plant Protection Organization Alert list in 2017. The scarce availability of efficient nematicides to control RKN and the phasing out of nematicides from the market have intensified the search for alternatives, such as phytochemicals with bionematicidal properties. The nematicidal activity of 1,4-naphthoquinone (1,4-NTQ) against M. luci has been demonstrated; however, knowledge of the potential mode(s) of action of this compound is still scarce. In this study, the transcriptome profile of M. luci second-stage juveniles (J2), the infective stage, in response to 1,4-NTQ exposure was determined by RNA-seq to identify genes and pathways that might be involved in 1,4-NTQ's mode(s) of action. Control treatments, consisting of nematodes exposed to Tween® 80 (1,4-NTQ solvent) and to water, were included in the analysis. A large set of differentially expressed genes (DEGs) was found among the three tested conditions, and a high number of downregulated genes were found between 1,4-NTQ treatment and water control, reflecting the inhibitory effect of this compound on M. luci, with a great impact on processes related to translation (ribosome pathway). Several other nematode gene networks and metabolic pathways affected by 1,4-NTQ were also identified, clarifying the possible mode of action of this promising bionematicide.

Juglone and 1,4-Naphthoquinone-Promising Nematicides for Sustainable Control of the Root Knot Nematode Meloidogyne luci.

The scarce availability of efficient and eco-friendly nematicides to control root-knot nematodes (RKN), Meloidogyne spp., has encouraged research toward the development of bionematicides. Naphthoquinones, juglone (JUG) and 1,4-naphthoquinone (1,4-NTQ), are being explored as alternatives to synthetic nematicides to control RKN. This study expands the knowledge on the effects of these natural compounds toward M. luci life cycle (mortality, hatching, penetration, reproduction). M. luci second-stage juveniles (J2)/eggs were exposed to each compound (250, 150, 100, 50, and 20 ppm) to monitor nematode mortality and hatching during 72 h and 15 days, respectively. Tomato seedlings were then inoculated with 200 J2, which had been exposed to JUG/1,4-NTQ for 3 days. The number of nematodes inside the roots was determined at 3 days after inoculation, and the final population density was assessed at 45 days after inoculation. Moreover, the potential mode of action of JUG/1,4-NTQ was investigated for the first time on RKN, through the assessment of reactive oxygen species (ROS) generation, acetylcholinesterase (AChE) in vitro inhibitory activity and expression analysis of ache and glutathione-S-transferase (gst) genes. 1,4-NTQ was the most active compound, causing ≥50% J2 mortality at 250 ppm, within 24 h. At 20 and 50 ppm, hatching was reduced by ≈50% for both compounds. JUG showed a greater effect on M. luci penetration and reproduction, decreasing infection by ≈80% (50 ppm) on tomato plants. However, 1,4-NTQ-induced generation of ROS and nematode vacuolization was observed. Our study confirms that JUG/1,4-NTQ are promising nematicidal compounds, and new knowledge on their physiological impacts on Meloidogyne was provided to open new avenues for the development of innovative sustainable nematicides.

Pratylenchus penetrans Parasitizing Potato Crops: Morphometric and Genetic Variability of Portuguese Isolates.

The root lesion Pratylenchus penetrans is an economically important pest affecting a wide range of plants. The morphometry of five P. penetrans isolates, parasitizing potato roots in Portugal, was compared and variability within and between isolates was observed. Of the 15 characters assessed, vulva position (V%) in females and the stylet length in both females/males showed the lowest coefficient of intra and inter-isolate variability. Moreover, DNA sequencing of the internal transcribed spacers (ITS) genomic region and cytochrome c oxidase subunit 1 (COI) gene was performed, in order to evaluate the intraspecific genetic variability of this species. ITS revealed higher isolate genetic diversity than the COI gene, with 15 and 7 different haplotypes from the 15 ITS and 14 COI sequences, respectively. Intra- and inter-isolate genetic diversity was found considering both genomic regions. The differentiation of these isolates was not related with their geographical origin. In spite of the high intraspecific variability, phylogenetic analyses revealed that both ITS region and COI gene separate P. penetrans from other related species. Our findings contribute to increasing the understanding of P. penetrans variability.

Species-Specific Molecular Detection of the Root Knot Nematode Meloidogyne luci.

Meloidogyne luci has been identified in various countries around the world parasitizing economically important crops and, due to its potential to cause serious damage to agriculture, was included in the European and Mediterranean Plant Protection Organization Alert List in 2017. This species shares morphological and molecular similarities with M. ethiopica and M. inornata, and a M. ethiopica group was therefore established. Although specific primers for the DNA amplification of species belonging to the M. ethiopica group have been developed previously, the primers were not species-specific, so molecular markers for the specific detection of M. luci are still needed. The objective of this study was to develop a SCAR marker for the detection of M. luci and the discrimination from other Meloidogyne spp. based on the intraspecific variability found in RAPD markers. RAPD screening of M. luci and M. ethiopica genome was used for the identification of a specific amplification product on M. luci, which was cloned, sequenced and converted into a SCAR marker. The specificity of the designed primers (Mlf/r) was tested and produced a fragment (771 bp) for all nine M. luci isolates with no amplification for the other nine Meloidogyne spp., including M. ethiopica and M. inornata. Additionally, the proper amplification of the M. luci SCAR-marker was also successful with DNA from galls of M. luci infected tomato roots. The results obtained in this study reveal that the specific molecular detection of M. luci was achieved and that the developed methodology can be used for routine diagnosis purposes, which are essential to monitoring the distribution and spread of M. luci in order to implement future effective and integrated nematode pest management programs.

Production of extracellular enzymes by different isolates of Pochonia chlamydosporia.

For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p<0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p<0.05), and significant differences were found between isolates (p<0.001) and the amounts of enzymes produced (p<0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.