In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum, 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, and morphological, biochemical and molecular characteristics were studied. Perineal pattern morphology was highly variable, similar to Meloidogyne ethiopica and not useful for 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 the 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 was also assessed in 16 commercial cultivars and compared with M. chitwoodi, considered to be 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 suggests 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.
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.
Naphthoquinones exhibit important biological activities and are present in walnut husk residues in significant amounts. However, their potential as alternatives to synthetic nematicides has not been fully explored. This work aimed to assess the effects of pure naphthoquinones (juglone; 1,4-naphthoquinone; plumbagin) on the mortality of the root-knot nematode Meloidogyne hispanica second-stage juveniles (J2). Extracts from Juglans spp. walnut husks were characterized, and the effects of J. nigra extracts on attraction and life cycle of M. hispanica were evaluated. 1,4-Naphthoquinone was the most effective compound causing 42% J2 mortality at 50 ppm. The extract from in natura J. nigra walnut husks presented similar effects on J2 mortality to those observed for pure 1,4-naphthoquinone. The extract from dried husks was repellent and reduced nematode root penetration but did not affect reproduction. Therefore, walnut residues can be valorized as renewable sources of naphthoquinone-based products and potentially employed as bionematicides against Meloidogyne spp.
Root-lesion nematodes (RLN), Pratylenchus spp., are economically important plant-parasitic nematodes (PPN) of several crops, including potato. In Portugal, potato cyst nematodes (PCN), Globodera spp., are a frequent problem and root-knot nematodes (RKN), Meloidogyne spp., have been found coexisting with PCN. Although the diversity and pathogenicity of PCN on potato are well documented, little work has been done to evaluate the importance of other PPN. This study aimed at assessing the diversity of RLN and RKN in potato. Sampling encompassed fields located in four regions of Portugal: North, Centre, Lisboa and Algarve. Nematodes were extracted from 40 soil/root samples, using standard extraction techniques, identified at genus level and population densities quantified. RLN species were identified on basis of morphological characters, species specific primers, and D2D3 28S rDNA sequencing. RKN and PCN species were identified by esterase phenotype and PCR-RFLP, respectively. RLN were detected in all sampled regions in 83 % of soil and 78 % of root samples. Pratylenchus penetrans was the most abundant species and P. neglectus, P. crenatus and P. thornei were detected for the first time associated with potato in Portugal. Meloidogyne arenaria, M. hapla, M. incognita and M. javanica were found in 10 % of soil and 20 % of roots. Globodera pallida and G. rostochiensis were detected in 15 and 8 % of soil and roots. RKN and PCN were found in samples infected with RLN. The information obtained on the ubiquity and diversity of RLN and RKN reinforces the need for a careful monitoring of these nematodes in potato crops.
The effects of a host plant on reproduction/abundance of fungal populations in relation to soil nutrients released by plants in the rhizosphere were studied. Abundance in the soil and potato rhizosphere of the fungi Paecilomyces lilacinus, Monographella cucumerina (CABI 380408) and Pochonia chlamydosporia var. chlamydosporia (Pc280, potato cyst nematode biotype) and P. chlamydosporia var. catenulata (Pc392, root-knot nematode biotype) were assessed. The different ability of break crops (oilseed rape, sugarbeet and wheat) in the potato rotation to support Pa. lilacinus, Pochonia isolates Pc280 and Pc392 and abundance of the latter two isolates in soil and rhizosphere of potato plants infected with Meloidogyne incognita were also studied. Potato chits and crop seedlings were planted into boiling tubes containing 5000 chlamydospores or conidia g −1 in acid washed sand (pH 6) and kept in a growth chamber at 20 • C, and 16 h of light for up to 9 weeks. The abundance of the fungi in sand (fallow) differed significantly between fungal species, being in general less abundant in the absence than in the presence of the plant, although there was no interaction between plant species and fungal isolate. There was evidence of a different response to Me. incognita for Pc392 than for Pc280 but there was no significant effect of the presence of the nematode on the rate of increase of the fungus.
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