Use of natural-based commercial products as an alternative for providing bioprotection against verticillium wilt of olive.

BACKGROUND: As a result of the ineffectiveness of existing control methods against Verticillium dahliae, the causal agent of verticillium wilt of olive (Olea europaea; VWO), it is necessary to search for sustainable and environmentally friendly alternatives, such as bioprotection by products based on plant extracts and other naturally synthesized compounds. Therefore, present study aimed to evaluate the effects of seven natural-based commercial products on the inhibition of mycelial growth, the germination of V. dahliae conidia and microsclerotia, and disease progression in olive plants (cv. Picual). Aluminium lignosulfonate and a copper phosphonate salt (copper phosphite) were included for comparative purposes. RESULTS: The seaweed and willow extracts and copper phosphite inhibited V. dahliae mycelial growth by more than 50% at the high doses tested. Most of the products inhibited conidial germination by up to 90% compared to the control at the high doses tested. However, none of the products showed efficacy above 50% in inhibiting microsclerotia germination. The willow extract was the most effective at reducing disease severity and progression in olive plants, with no significant differences compared to the non-inoculated negative control. CONCLUSION: The results of the present study suggest that the use of natural-based products (i.e. seaweed and willow extracts) is a potential sustainable alternative in an integrated VWO control strategy. © 2024 Society of Chemical Industry.

Identification and pathogenicity of Fusarium species associated with wilting and crown rot in almond (Prunus dulcis).

Severe Fusarium wilt and crown root symptoms were observed in almond orchards in Portugal. The present study elucidates the etiology of the disease through molecular, phenotypic, and pathogenic characterization. Three Fusarium isolates from Portugal were tested and 12 Fusarium isolates from almond from Spain were included for comparative purposes. Their identity was inferred by phylogenetic analysis combining tef1 and rpb2 sequences. The Portuguese isolates were identified as Fusarium oxysporum sensu stricto (s.s.), and the Spanish isolates as Fusarium nirenbergiae, F. oxysporum (s.s.), Fusarium proliferatum, Fusarium redolens (s.s.), Fusarium sambucinum (s.s.), and Fusarium sp. Fungal colonies and conidia were characterized on potato dextrose agar (PDA) and on Synthetischer Nährstoffarmer agar, respectively. The colonies had a variable morphology and their color ranged from white to pale violet. Typical Fusarium micro- and macroconidia were characterized. Temperature effect on mycelial growth was evaluated on PDA from 5 to 35 °C, with optimal growth temperature ranging between 16.8 and 26.4 °C. The pathogenicity of F. oxysporum was demonstrated by inoculating almond plants ('Lauranne') grafted on GF-677 or Rootpac 20 rootstocks. A significant reduction in plant growth, wilting, and xylem discoloration was observed, with Rootpac 20 being more susceptible than GF-677. Infections were also reproduced using naturally infested soils. Almond plants ('Lauranne') were inoculated with isolates of all Fusarium species, with F. redolens from Spain and F. oxysporum from Portugal being the most aggressive.

Streptomyces spp. Strains as Potential Biological Control Agents against Verticillium Wilt of Olive.

Verticillium wilt of olive (VWO) caused by Verticillium dahliae is considered a major olive (Olea europaea) disease in Mediterranean-type climate regions. The lack of effective chemical products against VWO makes it necessary to search for alternatives such as biological control. The main goal of this study was to evaluate the effect of six Streptomyces spp. strains as biological control agents (BCAs) against VWO. All of them were molecularly characterized by sequencing 16S or 23S rRNA genes and via phylogenetic analysis. Their effect was evaluated in vitro on the mycelial growth of V. dahliae (isolates V004 and V323) and on microsclerotia (MS) viability using naturally infested soils. Bioassays in olive plants inoculated with V. dahliae were also conducted to evaluate their effect against disease progress. In all the experiments, the reference BCAs Fusarium oxysporum FO12 and Aureobasidium pullulans AP08 were included for comparative purposes. The six strains were identified as Streptomyces spp., and they were considered as potential new species. All the BCAs, including Streptomyces strains, showed a significant effect on mycelial growth inhibition for both V. dahliae isolates compared to the positive control, with FO12 being the most effective, followed by AP08, while the Streptomyces spp. strains showed an intermediate effect. All the BCAs tested also showed a significant effect on the inhibition of germination of V. dahliae MS compared to the untreated control, with FO12 being the most effective treatment. Irrigation treatments with Streptomyces strain CBQ-EBa-21 or FO12 were significantly more effective in reducing disease severity and disease progress in olive plants inoculated with V. dahliae compared to the remaining treatments. This study represents the first approach to elucidating the potential effect of Streptomyces strains against VWO.

Bioprotection of Olive Trees Against Verticillium Wilt by Pomegranate and Carob Extracts.

Bioprotection using plant extracts is an environmentally friendly strategy in crop protection. Effective control of Verticillium wilt of olive (Olea europaea; VWO), caused by Verticillium dahliae, has proven challenging due to the ineffectiveness of chemicals, which makes it necessary to search for new control tools. Thus, the aim of this study was to evaluate the effect of pomegranate (Punica granatum) and carob (Ceratonia siliqua) extracts against VWO. Extracts derived from pomegranate peels and carob pods and leaves were obtained using ethanol, methanol, or ethyl acetate as solvents. A targeted analysis of their metabolite composition was performed using QTRAP Ultra High-Performance Liquid Chromatography with Mass Spectrometry (QTRAP UHPLC‒MS). Remarkably, gallic acid was detected in all extracts at a high concentration. The effect of the extracts on the mycelial growth and on the germination of conidia and microsclerotia of V. dahliae was evaluated by in vitro sensitivity tests at various doses: 0 (control), 3, 30, 300 and 3,000 mg of extract/liter. Extracts obtained with ethanol or methanol significantly reduced the viability of V. dahliae structures when applied at the highest dose, while those obtained with ethyl acetate were ineffective across all doses. The most effective extracts, as determined in vitro, were then evaluated against the disease in olive plants. Potted plants of cv. Picual were treated by spraying (foliar application) or irrigation (root application) of extracts at 3,000 mg of extract/liter, followed by inoculation with V. dahliae. The results indicated that foliar applications were ineffective, while root treatments with pomegranate peel or carob leaf extracts were more effective in reducing disease severity, regardless of solvent, compared to that of the untreated control.

Fungal Pathogens Associated with Almond Decline Syndrome, an Emerging Disease Complex in Intensive Almond Crops in Southern Spain.

In 2016, an almond (Prunus dulcis) decline syndrome (ADS) emerged in intensive almond plantations in the Andalusia region (southern Spain), showing branch dieback, gummosis, and general tree decline. The aim of this work was to elucidate the etiology of this disease complex. For this purpose, surveys were conducted across the Andalusia region, and a wide collection of fungi was recovered from wood samples showing gum and internal discoloration. Representative isolates were selected and identified by sequencing ITS, TEF1, TUB, ACT, LSU, and/or RPB2 genes. The following fungal species were identified to be associated with the disease: Botryosphaeria dothidea, Diplodia corticola, Di. seriata, Dothiorella iberica, Lasiodiplodia viticola, Macrophomina phaseolina, Neofusicoccum mediterraneum, N. parvum, N. vitifusiforme, Diaporthe neotheicola, Dia. rhusicola, Dia. ambigua, Eutypa lata, E. tetragona, Eutypella citricola, Eu. microtheca, Fusarium oxysporum s.l., Pleurostoma richardsiae, Phaeoacremonium iranianum, Pm. krajdenii, Pm. parasiticum, and Cytospora sp. All isolates were tested for pathogenicity by inoculating detached or attached almond shoots. Di. corticola and N. parvum were the most aggressive species, showing the largest lesions and most gummosis in attached shoots. The results suggest that the species belonging to Botryosphaeriaceae play a key role in disease development, while the remaining identified species may act as secondary pathogens or endophytes. However, further research to determine the interaction between all these fungal species and other biotic and abiotic factors in the ADS progress is needed.

Effects of Cultivar Susceptibility, Branch Age, and Temperature on Infection by Botryosphaeriaceae and Diaporthe Fungi on English Walnut (Juglans regia).

Botryosphaeriaceae and Diaporthe fungi have been described as the main causal agents of branch dieback and shoot blight of English walnut (Juglans regia L.). To date, the effects of biotic and abiotic factors on disease development on this host are still poorly understood. Thus, the main goal of this study was to evaluate the effects of cultivar, shoot-branch age, and temperature on infection by Botryosphaeriaceae and Diaporthe fungi on English walnut. The susceptibility of eight commercial cultivars was evaluated against three Botryosphaeriaceae and two Diaporthe species. For the remaining experiments, shoots or branches of 'Chandler' were used. An initial experiment evaluating two inoculation methods was conducted, with inoculation with a mycelial plug being more consistent and useful than conidial suspension inoculation. Cultivar susceptibility varied depending on the fungal species, with 'Chandler' being among the most tolerant cultivars for shoot infection. One-year-old shoots were significantly more sensitive for both Neofusicoccum parvum and Diaporthe neotheicola in comparison with 2- to 4-year-old branches. The effect of temperature on shoot infection was evaluated under 5, 10, 15, 20, 25, 30, and 35°C. Lesion development was significantly higher for N. parvum isolates than for D. neotheicola isolates at all temperatures evaluated, with optimum temperature of shoot infection being ∼26°C for N. parvum and ∼21°C for D. neotheicola.

Elucidating the Effect of Nutritional Imbalances of N and K on the Infection of Verticillium dahliae in Olive.

The effect of mineral nutrition on wilt diseases has been previously reported in many herbaceous hosts, though such an effect on Verticillium wilt in olive (Olea europaea L.; VWO), caused by Verticillium dahliae, is still uncertain. Field observations reveal that nitrogen (N) excess or imbalances of N-potassium (K) favour VWO epidemics. However, this has yet to be demonstrated. Thus, the aim of this study was to evaluate the influences of nutritional imbalances of N and K in V. dahliae infection of olive. To this end, adjusted treatments with N excess (↑N+↑Na), K deficiency (↓K) and their combination (↑N+↑Na+↓K) were evaluated on the viability of V. dahliae microsclerotia (MS), as well as on disease development in olive plants. In parallel, the potential indirect effect of the treatments on the viability of conidia and MS of V. dahliae was evaluated through the stimuli of root exudates. Treatments ↑N+↑Na and ↑N+↑Na+↓K decreased MS germination and disease progress, whereas ↓K significantly increased both parameters. Root exudates from treated plants increased the conidia germination of V. dahliae but reduced the MS germination. The results of this study will be the basis for planning further research towards a better understanding of the effect of mineral nutrition on VWO.

Identification of Nutritional Targets in Spanish Children Belonging to the LAyDI Cohort for the Development of Health Promotion Strategies in the First Two Years of Life.

The first 1000 days of life seem to represent the temporal window of opportunity for modulating some of the risk factors associated with the later development of pathologies. Nonetheless, the dietary pattern and nutritional status of children receiving complementary feeding is still understudied. We aimed to assess the food intake in children from the LAyDI cohort (Spain) at 18 and 24 months of age and evaluate this in relation to nutrient requirements and bioactive compound consumption. This was a prospective and multicenter study analyzing information from administered questionnaires about general characteristics and food frequency consumption in 426 children of 18 months and 336 of 24 months. The observed intake of vegetables, fruits, dairy, and eggs was lower than the recommendations in both periods, contrary to the consumption of meat, fish, and pulses. The consumption of energy and macronutrients was similar for all ages studied, with protein intake being slightly higher than the recommended values. Regarding micronutrients, practically the whole sample fell below the vitamin D requirements. In addition, the estimated daily intakes of vitamin E and iron, at 24 months, were below the recommended values for this population group. The mean intake of phenols was around 650 mg/day. Flavanol intake as well as both types of fiber decreased from 18 to 24 months. In conclusion, although these results have to be confirmed in other populations, it seems pertinent to propose the design of nutritional strategies aimed at increasing the intake of vitamins D and E as well as iron in Spanish children up to 2 years.

Hiperparatiroidismo primario y crisis hipercalcémica aguda tóxica / Primary hyperparathyroidism and acute toxic hypercalcaemic crisis

Objetivos: Describir las características clínicas y tratamiento quirúrgico de los pacientes con hipercalcemia aguda por hiperparatiroidismo primario (HPTP) y compararlas con las de otros pacientes con HPTP sin hipercalcemia aguda asociada. Material y métodos Estudio observacional prospectivo (1998-2010) sobre 158 pacientes con HPTP tratados mediante paratiroidectomía. Se identificaron aquellos con hipercalcemia aguda (>14mg/dl-3,5mmol/l- o >3mmol/l con síntomas de calciotoxicosis), se evaluaron sus características clínicas y terapéuticas y se compararon, mediante la U de Mann-Whitney y el test de Fisher, con los 146 pacientes con HPTP sin crisis hipercalcémicas. Resultados Doce pacientes (7,6%) presentaron hipercalcemia aguda con síntomas de calciotoxicosis y otros síntomas de cronicidad. Los valores preoperatorios de calcemia y PTH fueron 14,5±1,3mg/dl y 648,2±542pg/dl, respectivamente. Hubo 10 adenomas, una hiperplasia y un carcinoma. El peso medio de las piezas quirúrgicas fue 4.075±2.918 mg con un diámetro mayor de 27±14mm. Los gradientes de caída de PTH a los 10 y 25min fueron 79±18% y 92±6%. Las calcemias postoperatorias al alta y a los 6 meses fueron 8,2±0,7 y 9,1±0,9mg/dl. Las concentraciones plasmáticas de Ca, PTH y el peso y tamaño de las piezas quirúrgicas fueron mayores en los pacientes con crisis hipercalcémicas (p<0,001). No hubo diferencias en otros parámetros estudiados y en la tasa de curación. Conclusiones Las crisis hipercalcémicas fueron producidas por tumores más grandes, de mayor peso y que producían mayores concentraciones plasmáticas de Ca y PTH. Todos los pacientes presentaban síntomas de evolución crónica y la paratiroidectomía consiguió la curación (AU)

Objectives: To describe the clinical characteristics and surgical treatment of patients with acute hypercalcaemia due to primary hyperparathyroidism (PHPT) and compare them with other patients with PHPT without associated acute hypercalcaemia. Material and methods: A prospective, observational study (1998-2010) was conducted on 158patients with PHPT treated by parathyroidectomy. Those with acute hypercalcaemia(>14 mg/dl -3.5 mmol/L- or >3 mmol/L with symptoms of calcium toxicity) were evaluated by recording their clinical and treatment characteristics, and comparing them, using the Mann-Whitney U test and the Fisher test, with the 146 PHPT patients without hypercalcaemic crisis. Results: Twelve patients (7.6%) had acute hypercalcaemia with symptoms of calcium toxicity and other symptoms of chronicity. The preoperative calcium and PTH values were14.5 1.3 mg/dL and 648.2 542 pg/dL, respectively. There were 10 adenomas, 1 hyperplasia and 1 carcinoma. The mean weight of the surgical pieces was 4.075 2.918 mg, with a diameter greater than 27 14 mm. The gradients of PTH at 10 and 25 minutes were 79 18%and 92 6%, respectively. Post-operative calcium values on discharge and at 6 months were8.2 0.7 mg/dL and 9.1 0.9 mg/dL, respectively. The plasma concentrations of calcium, PTH, and the size of the surgical pieces were higher in patients with hypercalcaemic crisis(P<0.001). There were no differences in the other parameters studied or in the cure rate. Conclusions: Hypercalcaemic crises were caused by larger and heavier tumours that led to higher plasma Ca and PTH plasma concentrations. All patients had long-standing symptoms and parathyroidectomy led to cure of the disease (AU)

[Primary hyperparathyroidism and acute toxic hypercalcaemic crisis]. / Hiperparatiroidismo primario y crisis hipercalcémica aguda tóxica.

OBJECTIVES: To describe the clinical characteristics and surgical treatment of patients with acute hypercalcaemia due to primary hyperparathyroidism (PHPT) and compare them with other patients with PHPT without associated acute hypercalcaemia. MATERIAL AND METHODS: A prospective, observational study (1998-2010) was conducted on 158 patients with PHPT treated by parathyroidectomy. Those with acute hypercalcaemia (>14 mg/dl -3.5 mmol/L- or >3 mmol/L with symptoms of calcium toxicity) were evaluated by recording their clinical and treatment characteristics, and comparing them, using the Mann-Whitney U test and the Fisher test, with the 146 PHPT patients without hypercalcaemic crisis. RESULTS: Twelve patients (7.6%) had acute hypercalcaemia with symptoms of calcium toxicity and other symptoms of chronicity. The preoperative calcium and PTH values were 14.5 ± 1.3mg/dL and 648.2 ± 542 pg/dL, respectively. There were 10 adenomas, 1 hyperplasia and 1 carcinoma. The mean weight of the surgical pieces was 4.075 ± 2.918 mg, with a diameter greater than 27 ± 14 mm. The gradients of PTH at 10 and 25 minutes were 79 ± 18% and 92 ± 6%, respectively. Post-operative calcium values on discharge and at 6 months were 8.2 ± 0.7 mg/dL and 9.1 ± 0.9 mg/dL, respectively. The plasma concentrations of calcium, PTH, and the size of the surgical pieces were higher in patients with hypercalcaemic crisis (P<0.001). There were no differences in the other parameters studied or in the cure rate. CONCLUSIONS: Hypercalcaemic crises were caused by larger and heavier tumours that led to higher plasma Ca and PTH plasma concentrations. All patients had long-standing symptoms and parathyroidectomy led to cure of the disease.

Scaling-up the production of thermostable lipolytic enzymes from Thermus aquaticus YT1.

The lipolytic enzymes synthesized by Thermusaquaticus YT1 present extremely interesting properties of thermostability (more than 70% of activity after 12 days at 80°C and a half-life time of 1 h at 95°C), which point out the interest of proposing efficient strategies to successfully tackle the scale-up of the production process. In this study,viable scaling-up of the production process was implemented,and relevant aspects affecting the enzyme synthesis, such as the mineral composition of the culture medium, the aeration and the agitation have been evaluated.A strategy combining the modification of the culture medium and the aeration degree was also approached by adding perfluorocarbons, compounds which improve the availability of oxygen in the culture medium. An opposite response of biomass and lipolytic activity to the aeration conditions was found between scales (about 600 U L(-1) at high aeration levels in flask vs. 150 U L(-1) at high aeration rates in reactor), which further demonstrates the important role of the hydrodynamic conditions on the suitable development of the biological process. In all cases, the cultures were kinetically characterized and the Luedeking and Piret model turned out to be a valuable tool to conclude that the produced lipolytic enzyme is a growth-associated metabolite, no matter the medium and the scale.