International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-5, Sep-Oct- 2017 http://dx.doi.org/10.22161/ijeab/2.5.60 ISSN: 2456-1878 Potential of silicon fertilization in the resistance of chestnut plants to ink disease (Phytophthora cinnamomi) Andreia Carneiro-Carvalhoa*, Catarina Pereirab, Tiago Marquesc, Luís Martinsa,d, Rosário Anjosa,e, Teresa Pintoa,e, José Lousadaa,d and José Gomes – Laranjoa,e aCITAB – Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os- Montes and Alto Douro (UTAD), Vila Real, Portugal b Master student of Agricultural Engineering, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal c Master student of Biochemistry, Universityof Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal d CIFAP-ECAV – University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal e DeBA-ECVA – University of Trás-os-Montes and Alto Douro (UTAD), Department of Biology and Environment, 5001- 801 Vila Real, Portugal *[email protected], 910099195 Abstract— The European chestnut (Castanea sativa Mill.) I. INTRODUCTION is a specie with great economic importance in Europe that The sweet chestnut (Castanea sativa Mill.) is present in all have been present for thousands of years. In Portugal, the countries of the Mediterranean Sea basin, playing an chestnut helps to maintain a positive trade balance, by important role in the economy of these countries contributing to the gross national product (GDP). One of (Corredoira et al., 2012),covering large areas in France, the biggest threats for the chestnut is the ink disease caused Greece, Italy, Portugal, Spain, Turkey and the United by Phytophthora cinnamomi, this disease is problematic to Kingdom (Fernandez-López and Alia, 2003). This chestnut chestnut crop with a damaging impact. Silicon (Si) is species has an important historical and cultural value, classified as a beneficial nutrient, having the ability to make playing a key role in the economy and environmental plants more resistant to attacks by pathogens. Studies on the sustainability of the mountain areas (Marinoni et al., 2013). effect of silicon on chestnut are practically non-existent, so However, the European chestnut has been strongly the aim of this study was to evaluate the impact of silicon in threatened by ink disease. Phytophthora cinnamomiwas the resistance of chestnut plants to P. cinnamomi. The detected for the first time in Japan. In Spain and Portugal, it plants were treated by 0 mM, 5 mM, 7.5mM and emerged during the 19th Century andwas subsequently 10 mM SiK® with the analyzed mad at 0, 15 and 30 days reported in other countries of Europe (Italy, Greece, after inoculation by P. cinnamomi. These findings showed Switzerland, Turkey, France and the United Kingdom) that the Si-treated plants had higher survival rate resulted (Vannini and Vettraino, 2001; Vettraino et al., 2005; from the presence of phytoliths in root tissues, that acted as Corredoira et al., 2012). a mechanical barrier reducing the development of In Portugal, this disease has been responsible for the pathogenic structures and they arealso associated with the disappearance of more than 50% of the chestnut-producing improvement on antioxidant activity through the increase of areasince the 20th Century (Seabra et al., 2001; Martins and CAT and SOD, higher values of total phenols compounds Abreu, 2007). This soil oomycete, which has asexual and less oxidative damage. The presence of Si in PDA reproduction, attacks the root system and produces a black medium reduced the growth of P. cinnamomi all over the exudate that stains the surrounding soil leading the collapse time, presenting high PI. This work shows that the Si of xylem and consequently to the death of the tree (Vannini fertilization in chestnut plants contributes to increase the and Vettraino, 2001). The P. cinnamomi representing one resistance against P. cinnamomi infection. of the most devastating root rot pathogens of chestnuts Keywords—Biotic stress, Castanea sativa Mill, (Balci and Halmschlager, 2003), by these reasons is Fungitoxic, Phytophthora cinnamomi, Resistance, essential to search for alternative strategies that can help the Silicon. trees to increase their resistance against this pathogen. In this context, the fertilization with Si in chestnut plants appear as a possible inducer of resistance against P. cinnamomi infection, considering the potential of Si as www.ijeab.com Page | 2740 International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-5, Sep-Oct- 2017 http://dx.doi.org/10.22161/ijeab/2.5.60 ISSN: 2456-1878 an important and promising plant protector against several Isolation of P. cinnamomi biotic stresses allowing to decrease the intensity of diseases The P.cinnamomi isolate (IMI 340340) used in the in different crops in the world (powdery mildew and rice inoculation was selected due to its virulence in accordance blast). Several authors verified that Si fertilization reduces with previous tests (Abreu et al., 1999). The high the infection of angular leaf spot and Colletotrichum pathogenicity of this isolate in European chestnuts was also lindemuthianum in cotton (Oliveira et al., 2012) and bean confirmed by Dinis et al., (2011). The inoculum was plants (Polanco et al., 2014), respectively. On the other side, prepared for growth in PDA (potato dextrose agar) during 6 Côrtes et al. (2015) added that the Si is classified as an days at 25°C in the dark. elicitor with potential through enzymes defense suppress Leaf mineral analysis the rice blast. The samples were analysed using the standard procedures Several diseases were also suppressed by Si application of the University of Trás-os-Montes and Alto Douro Soil (Rodrigues and Datnoff 2005). In this context, Analysis Laboratory. The preparation and analysis of Seebold et al. (2001) have tested the effects of Si on several chemical macronutrients (N, P and K) in leaves from components of resistance to rice diseases using susceptible, Si-treated plants and untreated plants (0 mM SiK®) were partially resistant and completely resistant rice varietys. done using the methods described by Malavolta et al., They found that the number of sporulation per lesion, lesion (1997). The content of Si in chestnut leaves was analyzed size, rate of lesion expansion, number of spores per lesion by the method described by Korndörferet al., (2004). and diseased leaf area were significantly reduced by Si Resistance tests to ink disease application. Moreover, the presence of brown spot, stem rot, Leaf disks inoculation with P. cinnamomi sheath brown rot on rice, Fusarium and Corynespora leaf The inoculation of leaves with P. cinnamomi was made spot on cucumber decreased with the increase of Si according to Gouveia and Abreu (1994), with some changes supplied. Datnoff et al. (2001), suggesting that production to verify if there was a correlation between this inoculation inputs can be better managed by using Si, allowing the form and roots inoculation. Six leaves per treatment were reduction of pesticide elimination, as well as improved plant sampled from the non-inoculated plants. In the middle part resistance. Furthermore, Bakhat et al. (2018) note that Si of each one, 3 disks with 2 cm of diameter, including can reduce diseases such as blast to the same level as a midrib, were punched. The disks were placed in petri dishes fungicide, reducing costs and providing positive on a damp filter paper to maintain humidity conditions to environmental benefits. the development of P. cinnamomi. An 8 mm disc of PDA The objetive of the present study was to investigate the inoculated with P. cinnamomi was placed on top of each leaf effect of Si fertilization in chestnut plants on the resistance disc, as described earlier. The time, in hours, between the toink disease (P. cinnamomi). inoculation and the visible symptoms was evaluated daily over a period of 7 days, recording observations about II. MATERIAL AND METHODS theappearance of chlorosis in leaf disks was recorded. Plant material and growing conditions Preparation of P. cinnamomi inocolum The experiments used 160 chestnut seeds (Castanea sativa The inoculum of P. cinnamomi was prepared from a Mill var. Sousã) from the same tree growing in the mixture of potatoes, sugar and distilled water until boiling Germobank of University of Trás-os-Montes e Alto Douro and then drained. The mixture was autoclaved for 20 (UTAD), Vila Real, Portugal (41° 17′ 20″ N, 7° 44′ 0″ W). minutes at 120°C and after the cooling period was The seedlings were planted in 2 L filled pots with 3:1 turf inoculated with P. cinnamomi mycelium disks of about 8 and perlite and randomly organized into 4 groups with 40 mm diameter, from colonies with 10 days and posteriorly pots each. The plants with 4 months old were then placed in incubated in the oven at 25°C for 8 days. the growing chamber, with a 12h photoperiod, radiation Root inoculation with P. cinnamomi 1600 μmolfotões.m-2.s-1, 26 ºC, and watered on a daily The P. cinnamomi inoculum (50 mL) was applied in 20 basis. chestnut plants per treatment (described in the plant Silicon Treatments material) directly in the soil, 60 days after SiK® fertilization. Silicon was applied 45 days after the plants were potted, as The plants were then monitored for 4 months, registering potassium silicate (SiK®), according to Ma and Takahashi the time whenever a plant died. (2002). In this way, four treatments were prepared and Histopathology analysis evaluated: 0 mM, 5 mM, 7.5 mM and 10mM SiK®. The With a hand microtome, cross sections (1 μm thick) of silicon solutions were adjusted to pH 6.9 using 30 M secondary roots were obtained from untreated (0 mM SiK®) hydrochloric acid (HCl). Each plant was fertilized with and Si-treated plants (5 mM, 7.5 mM and 10 mM SiK®) at 50 mL of a SiK® solution, which was directly applied to the 150 days after inoculation (Monteiro et al., 2017).