Ivânia Esteves

Biology, growth parameters and enzymatic activity of Pochonia chlamydosporia isolated from potato cyst and root-knot nematodes

Pochonia chlamydosporia, a widespread fungal parasite of potato cyst nematodes (PCN), Globodera spp., and root-knot nematodes (RKN), Meloidogyne spp., has been studied as a biological control agent. Three Portuguese isolates (Pc1, Pc2, Pc3) obtained from PCN eggs and two non-native isolates (Vc10, Pc280) were characterised using ERIC-PCR and screened by in vitro assays for their ability to produce chlamydospores, parasitise eggs of Globodera rostochiensis and Meloidogyne chitwoodi and colonise the rhizosphere of barley. The effects of temperature on growth, sporulation, parasitism and enzymatic activity were also evaluated. Isolates Pc1 and Pc3, despite their different geographical origins, had identical molecular profiles. Pc2 produced the higher numbers of chlamydospores in solid medium (1.15 × 107 chlamydospores g−1), whereas Pc3 produced the least (3 × 105 chlamydospores g−1). These isolates extensively colonised the rhizosphere of barley (>90% root fragments) and the proportion of parasitised eggs, detected on agar plates, was low (<60% for RKN and <55% for PCN), Pc1 being the best parasite against both nematode species. The influence of temperature was similar for all isolates: no growth was observed at 10, 33 and 35°C. Spores/hyphal fragments remained viable for nearly 1 month at 10 and 33°C and isolates resumed growth after incubation at 25°C, although chlamydospores and conidia production, viability and nematode egg parasitism were affected. Exposure to 35°C was lethal for isolates Pc1, Pc2 and Pc280. When grown in liquid media all isolates produced esterases, but protease activity was only observed in Pc2 and Vc10. The highest enzymatic activity was detected in isolate Pc2 in low nutrient media. Enzymatic activity decreased in the presence of nematode eggs for both Pc2 and Vc10. Molecular, biochemical and biological analyses, including biotic and abiotic factors, are important in the screening of potential biological control agents, particularly in the case of P. chlamydosporia, due to the high variability among isolates.

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.

Meloidogyne luci, a new root-knot nematode parasitizing potato in Portugal

In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum L., an unusual esterase (EST) phenotype was detected in a root-knot nematode (RKN) from potato roots collected in Coimbra. This Portuguese isolate was purified and maintained on tomato, S. lycopersicum L., and studied by morphological, biochemical and molecular characteristics. Perineal pattern morphology was highly variable, similar to M. ethiopica and not useful for the identification. The EST phenotype, from young egg-laying females, displayed three bands similar to the Brazilian M. luci (L3) and distinct from M. ethiopica (E3). Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I and mitochondrial DNA region between COII and 16S rRNA genes revealed that the Portuguese isolate grouped with M. luci isolates close to M. ethiopica isolates. However, considering the ITS1-5.8S-ITS2 region the Portuguese isolate grouped with isolates of M. luci, M. ethiopica and M. hispanica, which limits the confidence of this region for M. luci diagnosis, and its differentiation from other species with morphological similarities. The M. luci pathogenicity to potato were also assessed in 16 commercial cultivars and compared with M. chitwoodi, considered a quarantine RKN species by EPPO. All potato cultivars were susceptible to both Meloidogyne species with gall indices of 5 and higher reproduction factor values ranging from 12.5 to 122.3, which suggest that M. luci may constitute a potential threat to potato production. In the present study, M. luci is reported for the first time attacking potato in Portugal. This article is protected by copyright. All rights reserved.

Biology and Management of Pochonia chlamydosporia and Plant-Parasitic Nematodes

The nematophagous fungus Pochonia chlamydosporia (Clavicipitaceae) is a facultative parasite of major plant-parasitic nematodes pests such as cyst (Globodera spp., Heterodera spp.), root-knot (Meloidogyne spp.), false root-knot (Nacobbus spp.) and reniform (Rotylenchulus reniformis) nematodes. The potential of P. chlamydosporia as a biological control agent and biopesticide has been the subject of numerous studies aimed at understanding the micro-ecological conditions that allow the fungus to thrive in the soil and rhizosphere environments. Pochonia survives in soil in the absence of plant and nematode hosts and can also behave as an endophyte. Research evidence points to a physiological ‘switch’ from the saprophytic to the parasitic stage that is triggered by nutrition. The basic biology of the fungus and sedentary endoparasitic plant nematodes is reviewed to provide insights into the fungus multitrophic behaviour, as well as its importance as a biocontrol agent within an integrated pest management approach.

Methodology Part II. Pochonia spp.: Screening and Isolate Selection for Managing Plant-Parasitic Nematodes

Production of Pochonia spp. in laboratory conditions has facilitated studies on its biology, abundance, dispersion, rhizosphere colonization, host preference, and isolate virulence. Research in biological control of nematodes requires suitable, standardized methods. In this chapter we review the commonest, non-molecular, standard in vitro culture methods to isolate, screen, and select isolates, some of which may eventually be produced on a larger scale for application in combination with other management strategies for plant-parasitic nematodes.

Methodology Part I. Pochonia spp. In Vitro Culturing: Media, Strain Maintenance and Deposition

Culturing on agar is a common method used in fungal isolation, cultivation, preservation, microscopic examination, biochemical, and physiological characterization. The present chapter reviews research carried out on Pochonia chlamydosporia culturing under in vitro conditions, which have made possible the production of mycelium, conidia and chlamydospores in sufficient quantities to allow laboratory, glasshouse and field experiments to be carried out with different isolates and strains of the fungus. Maintenance of isolates and deposit in culture collections is also covered. Knowledge of factors that can promote the growth, sporulation and development of resting structures of the fungus is essential for planning experimental assays and development of fungal strains as potential biopesticides.