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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.
- 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.
- Early pathogen recognition and antioxidant system activation contributes to actinidia arguta tolerance against pseudomonas syringae pathovars actinidiae and actinidifoliorumPublication . Nunes da Silva, M.; Vasconcelos, M. W.; Gaspar, M.; Balestra, G. M.; Mazzaglia, A.; Carvalho, Susana M. P.Actinidia chinensis and A. arguta have distinct tolerances to Pseudomonas syringae pv. actinidiae (Psa), but the reasons underlying the inter-specific variation remain unclear. This study aimed to integrate the metabolic and molecular responses of these two kiwifruit species against the highly pathogenic Psa and the less pathogenic P. syringae pv. actinidifoliorum (Pfm) bacterial strains. Disease development was monitored weekly till 21 days post inoculation (dpi), analysing a broad number and variety of parameters including: colony forming units (CFU), foliar symptoms, total chlorophylls, lipid peroxidation, soluble polyphenols, lignin and defense-related gene expression. At the end of the experimental period A. chinensis inoculated with Psa presented the highest endophytic bacterial population, whereas A. arguta inoculated with Pfm showed the lowest values, also resulting in a lower extent of leaf symptoms. Metabolic responses to infection were also more pronounced in A. chinensis with decreased total chlorophylls (up to 55%) and increased lipid peroxidation (up to 53%), compared with non-inoculated plants. Moreover, at 14 dpi soluble polyphenols and lignin concentrations were significantly higher (112 and 26%, respectively) in Psa-inoculated plants than in controls, while in A. arguta no significant changes were observed in those metabolic responses, except for lignin concentration which was, in general, significantly higher in Psa-inoculated plants (by at least 22%), comparing with control and Pfm-inoculated plants. Genes encoding antioxidant enzymes (SOD, APX and CAT) were upregulated at an earlier stage in Psa-inoculated A. arguta than in A. chinensis. In contrast, genes related with phenylpropanoids (LOX1) and ethylene (SAM) pathways were downregulated in A. arguta, but upregulated in A. chinensis in the later phases of infection. Expression of Pto3, responsible for pathogen recognition, occurred 2 dpi in A. arguta, but only 14 dpi in A. chinensis. In conclusion, we found that A. arguta is more tolerant to Psa and Pfm infection than A. chinensis and its primary and secondary metabolism is less impacted. A. arguta higher tolerance seems to be related with early pathogen recognition, the activation of plant antioxidant system, and to the suppression of ET and JA pathways from an earlier moment after infection.
- Susceptibility to the pinewood nematode (PWN) of four pine species involved in potential range expansion across EuropePublication . Silva, Marta Nunes da; Solla, Alejandro; Sampedro, Luis; Zas, Rafael; Vasconcelos, Marta W.The pine wilt disease (PWD), caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, is one of the most serious threats to pine forests worldwide. Here we studied several components of susceptibility to PWN infection in a model group of pine species widely distributed in Europe (Pinus pinaster Ait., P. pinea L., P. sylvestris L. and P. radiata D. Don), specifically concerning anatomical and chemical traits putatively related to nematode resistance, whole-plant nematode population after experimental inoculation, and several biochemical and physiological traits indicative of plant performance, damage and defensive responses 60 days post inoculation (dpi) in 3-year-old plants. Pinus pinaster was the most susceptible species to PWN colonization, with a 13-fold increase in nematode population size following inoculation, showing up to 35-fold more nematodes than the other species. Pinus pinea was the most resistant species, with an extremely reduced nematode population 60 dpi. Axial resin canals were significantly wider in P. pinaster than in the other species, which may have facilitated nematode dispersal through the stem and contributed to its high susceptibility; nevertheless, this trait does not seem to fully determinate the susceptible character of a species, as P. sylvestris showed similar nematode migration rates to P. pinaster but narrower axial resin canals. Nematode inoculation significantly affected stem water content and polyphenolic concentration, and leaf chlorophyll and lipid peroxidation in all species. In general, P. pinaster and P. sylvestris showed similar chemical responses after infection, whereas P. radiata, which co-exists with the PWN in its native range, showed some degree of tolerance to the nematode. This work provides evidence that the complex interactions between B. xylophilus and its hosts are species-specific, with P. pinaster showing a strong susceptibility to the pathogen, P. pinea being the most tolerant species, and P. sylvestris and P. radiata having a moderate susceptibility, apparently through distinct coping mechanisms.
- Chitosan increases Pinus pinaster tolerance to the pinewood nematode (Bursaphelenchus xylophilus) by promoting plant antioxidative metabolismPublication . Silva, Marta Nunes da; Santos, Carla S.; Cruz, Ana; López-Villamor, Adrián; Vasconcelos, Marta W.The pine wilt disease (PWD), for which no effective treatment is available at the moment, is a constant threat to Pinus spp. plantations worldwide, being responsible for significant economic and environmental losses every year. It has been demonstrated that elicitation with chitosan increases plant tolerance to the pinewood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of the PWD, but the biochemical and genetic aspects underlying this response have not been explored. To understand the influence of chitosan in Pinus pinaster tolerance against PWN, a low-molecular-weight (327 kDa) chitosan was applied to mock- and PWN-inoculated plants. Nematode population, malondialdehyde (MDA), catalase, carotenoids, anthocyanins, phenolic compounds, lignin and gene expression related to oxidative stress (thioredoxin 1, TRX) and plant defence (defensin, DEF, and a-farnesene synthase, AFS), were analysed at 1, 7, 14, 21 and 28 days post-inoculation (dpi). At 28 dpi, PWN-infected plants elicited with chitosan showed a sixfold lower nematode population when compared to non-elicited plants. Higher levels of MDA, catalase, carotenoids, anthocyanins, phenolic compounds, and lignin were detected in chitosan-elicited plants following infection. The expression levels of DEF gene were higher in elicited plants, while TRX and AFS expression was lower, possibly due to the disease containment-effect of chitosan. Combined, we conclude that chitosan induces pine defences against PWD via modulation of metabolic and transcriptomic mechanisms related with plant antioxidant system.
- Susceptibility evaluation of Picea abies and Cupressus lusitanica to the pinewood nematode, Bursaphelenchus xylophilusPublication . Silva, M. Nunes da; Lima, M. R. M.; Vasconcelos, Marta W.Pine wilt disease (PWD), recently introduced into Europe, is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus and is a devastating illness that affects mainly pine trees. It is known that the PWN is capable of infecting other conifers; however, there is currently no information on which other plant species may be susceptible to PWD. In this study, the potential susceptibility of two common species of European forests, Picea abies and Cupressus lusitanica, to PWN was assessed through the monitoring of visual external symptoms, dimension and localization of the nematode population in stems, quantification of total chlorophyll, total soluble phenolics and lignin, at 7, 14, 21 and 28days after inoculation. The degree of susceptibility was established through the comparison of symptoms with Pinus pinaster, a well-known PWN host. Furthermore, the stem ultrastructure of P.abies, C.lusitanica and Pn.pinaster was analysed by scanning electron microscopy. The results suggest that P.abies and C.lusitanica are resistant to PWN, and that lignin biosynthesis in these species is affected at an early stage of the infestation. Nevertheless, P.abies seems to be a compatible host that could act as a repository for PWN.
- Intraspecific variation of anatomical and chemical defensive traits in Maritime pine (Pinus pinaster) as factors in susceptibility to the pinewood nematode (Bursaphelenchus xylophilus)Publication . Zas, Rafael; Moreira, Xoaquín; Ramos, Miguel; Lima, Marta R. M.; Silva, Marta Nunes da; Solla, Alejandro; Vasconcelos, Marta; Sampedro, LuisKey message Migration ability of the PWN through wood branch tissues of adult Maritime pine trees significantly differed among Iberian provenances and this variation was related to differences in anatomical and chemical defensive traits. Abstract The pinewood nematode or pine wilt nematode (PWN; Bursaphelenchus xylophilus) is one of the most dangerous threats to European coniferous forests, especially for the susceptible Maritime pine (Pinus pinaster), a valuable forest resource in South Western Europe. The PWN is vectored by beetles of the genus Monochamus (Coleoptera, Cerambycidae) and once inoculated in healthy branches, it quickly migrates downward to the main trunk through the resin canal system. Therefore, the anatomy of the resin canal system may modulate its migration and proliferation rates. Using material from nine Maritime pine Iberian provenances established in a common garden trial, we investigated whether these provenances differed in their (1) resin canal anatomy, (2) concentration of chemical defences (non-volatile resin and total polyphenolics) in stems and (3) ability of the PWN to migrate through the pine woody tissues in ‘in vitro’ bioassays. Whether variation in anatomical and chemical defensive traits relates to differences in PWN migration across populations was also investigated. Significant intraspecific variation in anatomical and chemical defensive traits and in nematode migration rates through pine tissues was observed. Moreover, the variation in nematode migration rate among pine provenances was related to differences in both anatomical and chemical features. Overall, this study highlights the role of plant genetics in the development of defensive traits against this harmful coniferous pest. The observed intraspecific variation should be taken into account when considering breeding as a strategy to provide areas of high risk of PWN with resistant genetic material.
- 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).