Browsing by Author "Silva, M. Nunes da"
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- A biofertilizer with diazotrophic bacteria and a filamentous fungus increases Pinus pinaster tolerance to the pinewood nematode (Bursaphelenchus xylophilus)Publication . Silva, M. Nunes da; Pintado, M. E.; Sarmento, B.; Stamford, N. P.; Vasconcelos, M. W.The pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is a devastating illness that mainly affects P. pinaster trees, and that poses great environmental and economic challenges. Current disease management involves the cut down of infected trees, tree fumigation, use of nematicides, or the control of the insect vector; however, these methodologies are expensive, labour-intensive and have limited success. The aim of this work was to evaluate the effect of a biofertilizer enriched with diazotrophic bacteria and a chitosan-producing fungus, Cunninghamella elegans, in inducing P. pinaster and P. pinea resistance against the PWN. In non-inoculated (control) P. pinaster plants, PWN population significantly increased (ca. 2.3-fold) throughout the experimental period, whereas in plants treated with 7.5 and 15% of biofertilizer nematode numbers were up to 36.3-fold lower than in control plants. In P. pinea, nematode numbers decreased with time for all biofertilizer concentrations tested, and P. pinea had up to 27.3-fold lower nematode counts than P. pinaster. In addition, the biofertilizer prevented the decrease of photosynthetic pigments and the reduction of water content in infected P. pinaster plants. In P. pinea the biosynthesis of phenolics increased in PWN-inoculated plants, especially in the presence of the biofertilizer. The addition of this biofertilizer to soils forested by P. pinaster may improve plant defence and could be a potentially simple and inexpensive strategy for the control of the PWD.
- Characterization of the pinewood nematode, bursaphelenchus xylophilus-pinus system in Portugal: phytochemical, histopathological, molecular, and biotechnological approachesPublication . Mota, Manuel; Barbosa, Pedro; Rodrigues, Ana M.; Silva, Inês Vieira da; Ascensão, Lia; Silva, M. Nunes da; Ferreira, António C. Silva; Vasconcelos, Marta W.; Faria, Jorge M. S.; Sena, Inês; Ribeiro, Bruno; Barroso, José G.; Pedro, Luis G.; Trindade, Helena; Figueiredo, A. CristinaThe pinewood nematode, Bursaphelenchus xylophilus, is one of the main threats affecting Portuguese maritime pine (Pinus pinaster). Several research teams have joined efforts to better understand the plant-nematode system. Over 150 essential oils (EO), as well as several EO fractions and decoction waters have been evaluated, Ruta graveolens, Satureja montana, Thymbra capitata, Thymus pulegioides, and T. vulgaris EOs being the most nematotoxic. Two-year-old P. pinaster, P. pinea, P. sylvestris, and P. halepensis were inoculated with a virulent PWN Portuguese isolate, and comparatively evaluated with non-inoculated and wounded plants to understand the plant-nematode interaction and the role of plant volatiles. Histological studies showed that the number of nematodes increased in P. pinaster and P. sylvestris with disease progression, and 7 weeks after inoculation all pine tissues were severely damaged. PWN distribution in P. pinea and P. halepensis was nearly restricted to the inoculated area; no clear change was observed in the stem tissues. Pine species volatiles showed the existence of chemotypes in some cases. Key volatile organic compounds, such as 4-hexen-1-ol, involved in P. pinaster response against the nematode have also been identified using non-destructive methods, with the potential to be used as biomarkers for early detection of infected trees. In vitro co-cultures of the host with parasite have also been established as a biotechnological tool to evaluate the effect of nematotoxic addition and assess their phytotoxicity to the host. Molecular approaches have addressed the changes in α-pinene synthase gene expression in susceptible P. pinaster and non-susceptible P. pinea, following nematode invasion. Preliminary results showed an increased expression of this gene in P. pinea, contrary to P. pinaster which revealed the same expression level in infected and non-infected controls.
- Chemical characterization and antimicrobial activity of 6 plant essential oils against Pseudomonas syringae pv. actinidiae and P. syringae pv. actinidifoliorumPublication . Silva, M. Nunes da; Machado, J.; Mazzaglia, A.; Balestra, G. M.; Vasconcelos, M. W.; Carvalho, S. M. P.Introduction: Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (PSA), is currently the most destructive disease of kiwifruit worldwide. It was introduced in Portugal in 2010, leading to drastic production losses and plant mortality. Conversely, a closely related pathovar, P. syringae pv. actinidifoliorum (PSAF), only causes necrotic spots, not being associated with plant mortality. Current control techniques are expensive, consisting in the application of copper- and antibiotics-based compounds, with serious risks of environmental contamination and development of resistant bacteria. Therefore, the aim of this work was to characterize six plant essential oils against (anise, basil, cardamom, cumin, fennel and laurel) by gas chromatography–mass spectrometry (GC-MS) and evaluate their antimicrobial potential against different strains of PSA and PSAF. Methods: 1. Two highly virulent PSA strains (CFBP 7286 and 1F, isolated from Italy and France, respectively) and two less virulent PSAF strains (ICMP 18804 and ICMP 19441, isolated from New Zealand and Australia, respectively) were grown overnight in LB broth at 27 ᵒC and with shaking. 2. Bacterial inoculum (1-2x107 CFU.mL-1) was plated onto Nutrient Sucrose Agar medium and 20 μL droplets of each essential oil concentration were placed in 5 mm diameter paper discs (N = 3). 3. Plates were incubated at 28 ᵒC for 48 h, after which the inhibition zone was measured. 4. Essential oils chemical characterization was performed by GC-MS. Conclusions: Plant essential oils differed in their composition; however, compounds such as 3-carene, eucalyptol, camphor, fenchone and 4-terpineol were detected transversely in all oils. Although cumin presented the most simple composition, it showed MIC as low as 0.1 % for PSA 19441. In general, basil, cardamom and fennel had higher MICs, whereas anise had the lowest. PSAF strain 19441 was the most sensitive to almost all essential oils tested.
- Chitosan as a biocontrol agent against the pinewood nematode (Bursaphelenchus xylophilus)Publication . Silva, M. Nunes da; Cardoso, A. R.; Ferreira, D.; Brito, M.; Pintado, M. E.; Vasconcelos, MartaThe pine wilt disease (PWD) is caused by Bursaphelenchus xylophilus and poses great environmental and economic challenges. Thus, the development of sustainable techniques for the control of this epidemic disease is of major importance. This work aimed at evaluating if the application of different molecular weight (MW) chitosans as a soil amendment could be used to control the PWD in maritime pine (Pinus pinaster, very susceptible to the disease) and stone pine (Pinus pinea, less susceptible). At the end of the experimental period (24 days after inoculation), P. pinaster and P. pinea untreated plants presented ca. 3825 100 and 70, 47 nematodes, respectively. In P. pinaster, the high-MW chitosan prompted the most drastic results, inducing a 21.9-fold reduction in nematodes numbers, whereas in P. pinea, the most effective was the low MW chitosan, which reduced nematodes numbers up to 7-fold, compared with untreated plants. P. pinea seems to be highly resistant to the disease, presenting nematode numbers up to 54.6-fold lower than P. pinaster and less severe chlorophyll loss (ca. 2-fold).
- Defence-related pathways, phytohormones and primary metabolism are key players in the distinct tolerance of Actinidia spp. to pseudomonas syringae pv. actinidiaePublication . Silva, M. Nunes da; Carvalho, S. M. P.; Vasconcelos, M. W.The kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (Psa), affects plant yield and requires strict orchard management routines. Actinidia arguta (kiwi berry) is more tolerant than A. chinensis (green-fleshed kiwifruit) to Psa, but the molecular mechanisms that underpin plant tolerance are not fully understood. We hypothesised that plant tolerance is linked to the differential regulation of genes involved in distinct defence strategies shortly following infection. Here, micropropagated plants from both kiwifruit species were inoculated with Psa, and 48 hours post-inoculation, whole-transcriptome sequencing was performed. Psa density was 10-fold higher in A. chinensis than in A. arguta, demonstrating the tolerant character of the later species. Sixty-nine and 51 differentially expressed genes (DEGs) related with plant defences (e.g., pathogen recognition, immunity, and defence regulation) were identified in A. chinensis and A. arguta, respectively. Fifty-two DEGs related to abscisic (ABA), jasmonic (JA) and salicylic acid (SA) pathways, and 28 DEGs involved in primary metabolism (e.g., glycolysis, tricarboxylic acid and ammonia assimilation cycles) were also identified in A. chinensis, whereas in A. arguta 21 and 16 DEGs related with these biological functions, respectively, were identified. Results suggest that A. chinensis’ higher susceptibility to Psa is due to an inefficient activation of plant defence mechanisms, with the involvement of ABA, JA and SA, leading to impairments in primary metabolism. A. arguta’s increased tolerance may result from the downregulation of the ABA pathway and the expression of specific genes involved in plant defence, including FOX1, SN2 and RBOHA.
- Evaluation of natural antimicrobial agents against pseudomonas syringae p. actinidiaePublication . Silva, M. Nunes da; Nascimento, B.; Correia, A.C.; Teixeira, Paula; Balestra, G.M.; Vasconcelos, Marta W.; Carvalho, Susana
- Evaluation of natural antimicrobial agents against pseudomonas syringae p. actinidiaePublication . Silva, M. Nunes da; Nascimento, B.; Correia, A.C.; Teixeira, Paula; Balestra, G.M.; Vasconcelos, Marta W.; Carvalho, Susana
- Evaluation of the susceptibility of actinidia chinensis var. deliciosa and a. arguta var. arguta to pseudomonas syringae pv. actinidiaePublication . Silva, M. Nunes da; Vasconcelos, M. W.; Mazzaglia, A.; Balestra, G. M.; Carvalho, S. M. P.
- Exploring the antimicrobial potential of plant essential oils as preventive or curative agents against pseudomonas syringae pv. actinidiaePublication . Silva, M. Nunes da; Santos, M. G.; Vasconcelos, M. W.; Carvalho, S. M. P.Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker, has become the main threat for yellow and green fleshed kiwifruits (Actinidia chinensis) worldwide. As current control strategies mainly rely on the application of copper-based compounds, which show limited success in later stages of the disease and pose environmental hazards, plant essential oils (PEOs) have been tested as an environmentally friendly alterative. However, the potential of PEOs to inhibit Psa has been mostly evaluated in in vitro conditions and their chemical composition is rarely provided. Therefore, additional studies are needed to better evaluate their potential in mitigating Psa infection. In this work, six plant essential oils (from anise - Pimpinella anisum, basil - Ocimum basilicum, cardamom - Elettaria cardamomum, cumin - Cuminum cyminum, fennel - Foeniculum vulgare, and laurel - Laurus nobilis) were: (i) chemically characterized through gas chromatography–mass spectrometry (GC-MS) and, (ii) evaluated as preventive and curative agents against Psa in planta (using micropropagated A. chinensis var. deliciosa ‘Hayward’ plants). GC-MS analysis revealed that for anise and laurel the principal component was anethole (87.7% and 77.4%, respectively), for basil it was estragole (93.0%), for cardamom, β-himachalene (75.9%), for cumin, cuminaldehyde (41.9%), and for fennel, eucalyptol (68.6%). Application of PEOs as a preventive treatment (i.e., elicitation 14 days before Psa inoculation), significantly decreased Psa endophytic population 14 days post inoculation in four of the studied PEO (by 70% in basil, 54% in anise, 42% in laurel and 35% in cumin), when compared to inoculated non-elicited plants. Application of PEOs as a curative method, 14 days post inoculation, significantly decreased Psa endophytic population by 93% (laurel), 85% (cardamom), 46% (fennel), and 27% (cumin) when compared to inoculated non-treated controls, 14 days after treatment. These results provide strong evidence on the potential use of PEOs in the formulation of more environmentally friendly products for Psa control.
- Exploring the expression of defence-related genes in Actinidia spp. after infection with Pseudomonas syringae pv. actinidiae and pv. actinidifoliorum: first stepsPublication . Silva, M. Nunes da; Machado, J.; Balestra, G. M.; Mazzaglia, A.; Vasconcelos, M. W.; Carvalho, S. M. P.Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (PSA), is currently the most destructive disease of kiwifruit worldwide. Conversely, a closely related bacterial strain, P. syringae pv. actinidifoliorum (PFM), only causes necrotic spots and has not been associated with plant mortality. Moreover, there is some evidence on the higher susceptibility of the Actinidia chinensis var. deliciosa kiwifruit species to KBC, compared with A. arguta, but the reasons behind it are still largely unknown. In this work, micropropagated plants of Actinidia chinensis var. deliciosa 'Hayward' and A. arguta var. arguta 'Ken's Red' were inoculated with PSA or with PFM (10(7) CFUs mL(-1)). Disease development was monitored 1, 2 and 5 days post inoculation (dpi) through the determination colony forming units (CFUs) and the expression analysis of six plant defence-related genes (APX, CAT, SOD, LOX1, SAM and TLP1). At 5 dpi, CFUs in plant tissues inoculated with PSA and PFM were, respectively, 17.4-fold and 2.8-fold higher in A. chinensis compared with A. arguta. Expression of antioxidant enzyme-related genes was very distinct between the two kiwifruit species: SOD expression was drastically increased in A. chinensis (up to 2.1-fold, 5 dpi), whereas in A. arguta CAT was the most upregulated gene (up to 1.7-fold, 2 dpi). LOX1, involved in jasmonic acid biosynthesis, was upregulated in both species, however reaching the highest values at 2 dpi in A. chinensis (2.2 fold) and 1 dpi in A. arguta (1.9-fold). It is concluded that A. arguta is much more tolerant to PSA than A. chinensis and that the molecular mechanisms between the two kiwifruit species involve specific defence pathways being triggered at distinct moments after plant infection.