Antonio Trapero

Cadophora luteo-olivacea, Phaeoacremonium minimum, and Pseudophaeomoniella oleicola have been associated with branch dieback, shoot blight, and vascular wilt in super-high-density olive plantations in Spain. In this study, the effects of the interaction between these three fungal species on the disease progression were evaluated through multi-infections inoculated by spore suspensions or mycelial plugs in olive plants of cultivar Arbequina. The susceptibility of six olive cultivars was also evaluated against these three pathogens by single inoculations with conidial suspensions. The detection and inoculum quantification of each fungal species from coinfected plant tissues or from inoculated plants of different cultivars was conducted by qPCR, using species-specific primers. In addition, a species-specific primer to identify Ps. oleicola was developed in this study. In the multi-infections experiment with conidial suspensions, lesions on olive infected simultaneously with all three species were larger than those on plants coinfected by two species, which were, in turn, larger than those inoculated by a single species. However, when inoculations were conducted with mycelial plugs, C. luteo-olivacea caused larger lesions than those in coinfected plants. The quantification of these fungi through qPCR suggests that their interaction during coinfections significantly alters their relative abundance, potentially impacting their aggressiveness. Regarding the cultivar resistance experiment, the most susceptible cultivar to the disease was ‘Koroneiki’, followed by ‘Arbequina’ and ‘Picual’. In all the samples, the detection of C. luteo-olivacea was an order of magnitude lower than that of the other fungal species. Nevertheless, the relative quantification of fungi by qPCR, despite being useful to detect the presence of the pathogen, does not correlate with the lesions observed.

Verticillium wilt, caused by the soilborne fungus Verticillium dahliae , and iron (Fe) deficiency are major stresses impacting valuable crops in the Mediterranean basin. The plant response to these stresses consists of overlapping pathways that, coupled with the emerging use of endophytic fungi as biological control agents (BCAs) and plant biostimulants, enables the study of their potential to mitigate co‐occurring biotic stresses and nutritional deficiencies such as Fe deficiency. To this end, Arabidopsis thaliana plants of the “Columbia” ecotype grown in calcareous soil were inoculated with the non‐pathogenic strain Fusarium oxysporum FO12 and/or with the defoliating pathotype of V. dahliae strain V180. Plant growth, plant mineral uptake, and disease progression, along with the molecular quantification of FO12 and V180 fungal biomass and the relative expression of ISR‐, SAR‐, and Fe deficiency‐related genes in shoots, were evaluated. The results revealed that plants inoculated with the FO12 strain increased growth, leaf chlorophyll concentration (SPAD index), and phosphorus (P; 126%) and Fe (9%) contents, partially due to ethylene (ET) and Iron Man peptides (IMA). In addition, FO12 reduced the severity of symptoms by more than 85%. This reduction in disease severity may be attributed to a direct competitive effect on V180 progression and to the induction of several defence‐associated genes related to ET and SA synthesis and signaling. This bidirectional crosstalk between Fe deficiency responses and plant defences triggered by FO12 mainly through ET can improve plant resilience while reducing reliance on agrochemicals, offering a sustainable multifaceted tool for crop protection and nutrition management.

From March 2020 to November 2022, 12 commercial almond ( Prunus dulcis ) orchards showing symptoms of chlorosis, wilting, defoliation, cankers, crown rot and profuse trunk gummosis were surveyed in southern Spain and Portugal. Diagnostic tests were performed by isolating oomycetes from affected tissues and by DNA extraction from root and wood tissues and PCR amplification of the rDNA internal transcribed spacer (ITS) genomic area using the specific primer pair Phni‐F/Phni‐R. A total of 62 isolates were collected: 18 Phytophthora ‐like isolated from plants and 44 Pythium sensu lato ‐like from waterlogged soil. Seven representative isolates were selected and identified by multilocus sequencing of the ITS and cox2 genomic regions and phylogenetic analysis. The following oomycetes were identified: Phytophthora niederhauseri , Phytopythium litorale , Globisporangium heterothallicum , G. macrosporum , G. paroecandrum and G. violae , with Ph. niederhauseri being the most frequent. The effect of temperature on the mycelial growth of seven isolates was evaluated in carrot agar and corn meal agar, with Ph. niederhauseri and Pp. litorale showing the highest optimal growth temperature (around 28°C–29°C) regardless of the culture media used. Pathogenicity tests of seven isolates were conducted on almond plants of cv. Guara grafted on Garnem rootstock. Ph. niederhauseri isolates were the most aggressive, causing crown rot, gummosis and plant death starting 3 weeks after inoculation. Pp. litorale showed intermediate aggressiveness, while Globisporangium species developed small cankers and minimum lesions, not causing plant death. Based on pathogenicity tests, Ph. niederhauseri is considered the true pathogen of the disease, while the Pythium sensu lato ‐like isolates must be considered secondary pathogens in waterlogged conditions.

Verticillium wilt of pistachio ( Pistacia vera ), caused by Verticillium dahliae , is one of the most important diseases in this crop. The most effective and economical control measure is the use of resistant rootstocks. The main objective of this work was to evaluate the resistance of five rootstocks commonly used in pistachio against the disease under field conditions. Pistachio plants (cv. Kerman) grafted on the rootstocks Pistacia atlantica , Pistacia integerrima , Pistacia terebinthus and cv. Platinum and UCBI hybrids, were planted in a field with high inoculum density of V . dahliae in the soil. Plants grafted on P . terebinthus , followed by P . integerrima , showed significantly higher disease severity values than the rest of the rootstocks almost 53 months after planting; also, plants grafted on P . atlantica were more resistant than those grafted on P . integerrima . These results highlighted the intraspecific variability in resistance to the disease. In addition, the disease was monitored in pistachios grafted onto P . terebinthus or UCBI in an established commercial field with three different zones of V . dahliae inoculum density in the soil (none, low or high). In the high‐density zone, the percentage of diseased plants was significantly lower for plants grafted on UCBI than for those grafted on P . terebinthus . The results indicate the importance of rootstock species and specific genotype or seed population used, concentration of V . dahliae inoculum in the soil, and previous history of crops on the plot, on the development of the disease under natural conditions.

In the Mediterranean Basin, the pistachio crop is considered an emerging nut crop due to the dependence of the European nut markets on international imports. Consequently, the extension and intensification of pistachio production to nontraditional growing areas has promoted both classic and emerging diseases of this nut tree, which are limiting factors in crop production. Due to the limited interest in pistachio crops, the aetiology and epidemiology of the main pistachio diseases are poorly studied. Thus, this review summarizes the state of pistachio disease research, with particular attention given to the Mediterranean area. The information reported here is based not only on the literature, but also the advancement of our research on pistachio diseases conducted in both Italy and Spain. We describe the main fungal diseases of the tree canopy in Mediterranean countries, such as Botryosphaeria panicle and shoot blight, Septoria leaf spot, and stem, branch and trunk canker diseases, followed by the main soilborne diseases. In addition, we review minor and/or sporadic fungal diseases originating in not only the Mediterranean, but also other pistachio‐producing countries. Another section is dedicated to other diseases caused by bacteria, phytoplasmas, viruses and nematodes. Finally, we summarize the main control strategies adopted in Mediterranean Basin countries against pistachio diseases.

Bioprotection through the use of 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 because of 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 on VWO. Extracts derived from pomegranate peels and carob pods and leaves were obtained using ethanol, methanol, or ethyl acetate as a solvent. A targeted analysis of their metabolite composition was performed using QTRAP ultrahigh-performance liquid chromatography with mass spectrometry. 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 the cultivar Picual were treated by spraying (foliar application) or irrigation (root application) of extracts at 3,000 mg/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 the solvent, compared with that of the untreated control.

Escudete, which is caused by Botryosphaeria dothidea, is a disease that is widely distributed in the Mediterranean basin, but is of little general importance. Nevertheless, serious attacks have been observed on occasion, which have caused a considerable reduction in the quality of table olives. The incidence of the pathogen has been associated with damage caused by the olive fly (Bactrocera oleae) and the presence of a possible vector agent, i.e., the midge Prolasioptera berlesiana, whose larvae can feed on fly eggs (although the role the midge may play in the spread of this disease is not well known). Therefore, it is necessary to clarify these interactions to adopt appropriate disease control measures. Studies were conducted in olive orchards planted with the Gordal Sevillana, Picudo, and Hojiblanca olive cultivars. Field surveys were carried out in order to sample their fruits for laboratory analysis, and several bioassays were also performed. Moreover, the population of B. oleae adults was monitored using traps that were baited with food attractants. The results indicated that the three agents developed and evolved in parallel under field conditions. Thus, the midges were attracted by the oviposition punctures caused in fruits by olive fruit flies, regardless of whether the punctures contained eggs. All the investigated olive fruits in which midges were present inside punctures created by olive fruit flies exhibited typical symptoms of escudete, which is necessary for the development of this disease. Forty-eight hours after fly punctures were artificially simulated in the olive fruits, 48.0% of them contained a midge, whereas no midges appeared in the artificially created shapeless wounds in the fruits. This indicates that an olive fly egg is not required for the development of midges; however, they do prefer punctures made by B. oleae. Moreover, when the olive fruits were incubated in a humid chamber, the B. dothidea fungus only appeared in those fruits that contained midges, thus indicating a close relationship between these two agents. Additionally, the midges were able to complete their entire development from egg to adult under controlled conditions, and they fed on the pure cultures of the B. dothidea fungus. Furthermore, although no pathogens were present in the immature midges, some of the pathogens could have been isolated from the inner tissues of the adult female midges. The fact that mycangia is present in the abdomen of P. berlesiana supports the hypothesis that their relationship with B. dothidea may be mutualistic and that they may act as a vector for the fungus.

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.

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.