Maria Carmen Raya-Ortega's research while affiliated with Universidad Católica de Córdoba and other places

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 the 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.

Septoria leaf spot (SLS) is the most prevalent disease of pistachio (Pistacia vera L.) in Spain. To elucidate its etiology, 22 samples of pistachio leaves showing SLS symptoms were collected mainly from 1993 to 2018 across southern Spain. Affected leaves from terebinth (P. terebinthus) were also collected for comparative purposes. Six Septoria-like isolates were recovered from pistachio leaves. They were identified as Septoria pistaciarum by sequencing ITS, RPB2 and LSU genes. The phenotypic characteristics of conidia and colonies were evaluated, confirming the identity of S. pistaciarum. Conidia were solitary, hyaline, and straight to curved. Large differences in length were observed between conidia from leaf samples, with those from terebinth being slightly larger than those from pistachio. Colonies showed slow mycelial growth on PDA. The effect of temperature on conidial germination and mycelial growth was evaluated in vitro on PDA. For both characters, the optimum temperature was approximately 19-20°C. Eight culture media were tested, with oatmeal agar (OA) and Spezieller Nährstoffarmer agar (SNA) showing the highest mycelial growth and pistachio leaf agar (PLA) showing the highest sporulation. A specific culture medium integrating lyophilized-powdered pistachio leaves into diluted PDA improved sporulation in comparison with PLA. Pathogenicity tests were conducted by inoculating detached and in planta pistachio and terebinth leaflets with conidial suspensions. Typical symptoms of SLS and cirri of S. pistaciarum developed at 10 and 21 days after inoculation, respectively, in both hosts. This is the first report of S. pistaciarum causing SLS in pistachio and terebinth in Spain.

Symptoms of branch dieback of olive with internal longitudinal dark streaking were observed during routine surveys in super high-density systems in southern Spain. Nineteen fungal isolates recovered from wood samples showing internal discoloration and necrotic xylem vessels were selected. Multilocus alignments of ITS, LSU, TUB and/or ACT were performed, and the following species were identified: Acremonium sclerotigenum, Cadophora luteo-olivacea, Paracremonium sp., Phaeoacremonium italicum, Ph. minimum, Ph. scolyti and Pseudophaeomoniella oleicola. Colony color, mycelial growth, conidial characteristics and production were defined on PDA, MEA and OA. Phenotypic characteristics and conidial production varied depending on the isolate and culture media. The effect of temperature on mycelial growth was evaluated on MEA. The isolates showed slowly mycelial growth (0.5-2.0 mm day-1), with the optimum temperature ranging from 23.2 to 33.9 °C. Pathogenicity tests were conducted on nine-month-old olive potted plants ('Arbequina') inoculated with mycelial plugs. Cadophora luteo-olivacea, Pm. minimum and Phaeomoniella chlamydospora isolates from grapevine were included in the pathogenicity tests for comparative purposes. Prior to inoculation, the effect on the infection by inoculating with conidial suspensions or mycelial plugs was evaluated, with the second method being the most effective. Cadophora luteo-olivacea was the most aggressive fungi to olive followed by Pm. minimum.

English walnut (Juglans regia L.) is considered an economically important fruit crop worldwide. In Spain, little attention has been given to walnut diseases owing to the minor economic importance of the walnut crop in the country until recently. In 2017, typical symptoms of branch dieback and shoot blight of English walnut were observed in southern Spain. From 2017 to 2018, 10 commercial walnut orchards showing disease symptoms were surveyed. Botryosphaeriaceae and Diaporthe fungi were consistently isolated from affected shoots. Cytospora isolates were also recovered with minor relevance. Representative isolates of each fungal group were characterized based on colony and conidial morphology, optimum growth temperature, and comparison of DNA sequence data from the internal transcribed spacer, elongation factor 1-α, and β-tubulin genomic areas. Pathogenicity tests were performed on detached and attached shoots and on detached fruit by inoculating them with mycelial plugs. Botryosphaeriaceae and Diaporthe isolates had higher optimum growth temperatures (≈25 to 27°C) than Cytospora sp. (19.5°C). The following species were identified: Botryosphaeriaceae: Botryosphaeria dothidea, Diplodia seriata, Dothiorella sarmentorum, Dothiorella sp., Neofusicoccum mediterraneum, and N. parvum; Diaporthe: Diaporthe neotheicola, Dia. rhusicola, Diaporthe sp., and Phomopsis amygdali; and Cytospora sp. Botryosphaeriaceae isolates were the most aggressive fungi to walnut in all tissues evaluated, followed by Diaporthe isolates and Cytospora sp. N. parvum was the most virulent among the remaining species tested in any of the tissues evaluated, followed by B. dothidea or N. mediterraneum. This work is the first report to identify the fungal species causing this complex disease of English walnut in Spain and Europe.

Over two consecutive seasons, 16 olive orchards with trees exhibiting dieback symptoms on branches were surveyed in southern Spain. The six dominant fungal species recovered were characterized by means of phenotypic observations, DNA analysis (by sequencing of the internal transcribed spacer, b-tubulin, and large subunit nuclear ribosomal DNA regions), and pathogenicity tests. Additionally, three isolates collected from Tunisian olive trees showing similar dieback symptoms, one isolate of Colletotrichum godetiae, and a reference isolates of Neofusicoccum mediterraneum were included. The resistance of the 11 most important table cultivars to N. mediterraneum and Botryosphaeria dothidea, the causal agent of “escudete” (small shield) of fruit, was studied by the inoculation of branches and immature fruit, respectively. The species Cytospora pruinosa, N. mediterraneum, Nothophoma quercina, Comoclathris incompta, and Diaporthe sp. were identified. Only N. mediterraneum and C. incompta were able to induce the typical dieback symptoms and cankers that affected the development of the plants. The species N. mediterraneum was the most virulent among the evaluated species, although differences in virulence among its isolates were observed. The remaining fungal species were weakly pathogenic to nonpathogenic on plants. According to resistance tests, ‘Gordal Sevillana’ and ‘Manzanilla Cacereña’ were the most susceptible to branch dieback caused by N. mediterraneum. Furthermore, the fruit of ‘Aloreña de Atarfe’ and ‘Manzanilla de Sevilla’ were the most susceptible to B. dothidea. Knowledge of the etiology and cultivar resistance of these diseases will help to establish better control measures.

The antifungal activity of the entomopathogenic fungi isolates Metarhizium brunneum EAMb 09/01-Su and Beauveria bassiana EABb 09/16-Su against the olive pathogens Verticillium dahliae and Phytophthora megasperma was evaluated. The crude extract and its partial purified fractions obtained from both isolates showed complete inhibition of the mycelial growth of V. dahliae. However, the same fractions obtained from B. bassiana EABb 09/16-Su were less effective against the mycelial growth of P. megasperma. Our results showed that M. brunneum EAMb 09/01-Su had the highest fungicidal activity, inhibiting the mycelial growth of both pathogens. Both fractions IV and VI from M. brunneum EAMb 09/01-Su were evaluated by the progress of the Verticillium wilt of the plant and showed clear antagonism against V. dahliae, reducing the severity of the symptoms between 38.9 and 58.9%. The active metabolite obtained from M. brunneum not only reduced the germination of V. dahliae microsclerotia but also inhibited hyphal formation, decreased the number of reproductive structures, and changed the conformation of the microsclerotial colony, preventing the formation of new microsclerotia. The evaluation of temperature, UV light and pH on the antifungal activity of the dialyzed fraction from M. brunneum showed that the antifungal activity was stable at 60 °C (2 h) and 120 °C (20 min) and to UV light radiation. However, the exposition to the dialyzed fraction at pH 7.5 and 8.5 affected the antifungal activity. The results of this study clearly showed that the entomopathogenic fungus M. brunneum EAMb 09/01-Su produces antifungal compounds that reduce the number of the pathogen propagules in the soil and the severity of Verticillium wilt. Therefore, this resource should be further studied as an alternative for the integrated management of Verticillium wilt and Phytophthora root rot, which is the most severe disease in all traditional olive-growing regions worldwide, and as a reliable and sustainable strategy for a reduced reliance on pesticides.

Biological control of plant diseases using soil amendments such as animal manure and composted materials are able to minimize organic waste and has been proposed as an effective strategy in crop protection. In this study, thirty-five organic amendments(OAs) and sixteen compost mixtures have been evaluated against Verticillium dahliae by assessing both the antagonistic effect on the mycelial growth of two representative isolates of V. dahliae and the effect on the reduction of microsclerotia viability of the pathogen in natural infested soil. Eleven OAs and five compost mixtures showed a consistent inhibition effect in in vitro sensitivity tests, being the solid olive-oil waste compost one of the most effective OAs. Therefore, a bioassay with olive plants was conducted to evaluate the suppressive effect against V. dahliae of these selected OAs and compost mixtures. Significant reduction in the severity of the symptoms of V. dahliae provides a practical basis for the potential use of grape marc compost (100% disease severity reduction) and solid olive-oil waste combined with other OAs. Microorganism mixtures or dairy waste OAs had a potential suppressive effect when they were combinate with compost, showing a 73 and 63% of disease severity reduction, respectively. Mixture of agro-industrial waste with other biological control agents is a promising strategy against Verticillium wilt of olive. To our knowledge, this is the first report on the effectiveness of inhibiting natural microsclerotia of V. dahliae with compost extracts (compost teas), and also on Verticillium wilt suppression in olive with solid olive-oil waste. This article is protected by copyright. All rights reserved.