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.

Root Exudates Metabolic Profiling Confirms Distinct Defense Mechanisms Between Cultivars and Treatments with Beneficial Microorganisms and Phosphonate Salts Against Verticillium Wilt in Olive Trees.

Root exudates play a key role in the life cycle of Verticillium dahliae, the causal agent of Verticillium wilt diseases, since they induce microsclerotia germination to initiate plant infection through the roots. In olive plants, the genotype and the application of biological control agents (BCAs) or phosphonate salts influence the ability of root exudates to decrease V. dahliae viability. Understanding the chemical composition of root exudates could provide new insights into the mechanisms of olive plant defense against V. dahliae. Therefore, the main goal of this study was to analyze the metabolomic profiles of root exudates collected from the olive cultivars Arbequina, Frantoio and Picual subjected to treatment with BCAs (Aureobasidium pullulans AP08, Bacillus amyloliquefaciens PAB-024) or phosphonate salts (copper phosphite, potassium phosphite). These treatments were selected due to their effectiveness as inducers of resistance against Verticillium wilt in olive plants. Our metabolomic analysis revealed that the olive cultivars exhibited differences in root exudates, which could be related to the different degrees of susceptibility to V. dahliae. The composition of root exudates also changed with the application of BCAs or phosphonate fertilizer, highlighting the complex and dynamic nature of the interactions between olive cultivars and treatments preventing V. dahliae infections. Thus, the identification of genotype-specific metabolic changes and specific metabolites induced by these treatments emphasizes the potential of resistance inducers for enhancing plant defense and promoting the growth of beneficial microorganisms.

Characterization and Pathogenicity of Fungal Species Associated with Dieback of Apple Trees in Northern Italy.

Severe dieback symptoms were recently observed on apple (Malus × domestica) trees in Northern Italy, representing a growing concern for producers. Surveys were conducted over a 3-year period (2019 to 2021), and five apple orchards, from 5 to 12 years old, were monitored. A total of 33 fungal isolates isolated from symptomatic plants was selected for characterization. The species identification was achieved through multilocus phylogenetic analyses performed on sequences of three genomic loci (ITS, tub2, and tef1). Morphological features were assessed, and the average growth rate at different temperatures was determined. Seven species were identified in association with dieback of apple trees: Botryosphaeria dothidea, Cadophora luteo-olivacea, Diaporthe rudis, Diplodia seriata, Eutypa lata, Kalmusia longispora, and Paraconiothyrium brasiliense. All the species were pathogenic when inoculated on healthy apple plants. B. dothidea resulted in the most aggressive infections. This study provides an insight into the fungal species diversity associated with apple dieback and provides basis for further investigations to assess the phytosanitary status of plant materials to recommend and implement effective management strategies.

Isolation and Reproductive Structures Induction of Fungal Pathogens Associated with Xylem and Wood Necrosis in Grapevine.

Grapevine (Vitis vinifera L.) trunk diseases (GTDs) are considered a disease complex including five diseases: esca, Petri disease, black-foot disease, Botryosphaeria dieback, and Eutypa dieback. The main symptom is a general decline in affected plants, which show xylem necrosis and discoloration or sectorial necrosis in the wood. Their diagnosis is tedious due to four main reasons: (i) the wide diversity of internal symptoms that we can find; (ii) the great diversity of fungi that are associated with them; (iii) the high frequency of co-infections in the same plant; and (iv) the different behavior that the fungal species associated with GTDs show in vitro. Here, we describe a detailed protocol to isolate the different fungal trunk pathogens associated with GTDs as well as methods to induce sporulation and formation of fruiting bodies (pycnidia) to make easier their morphological characterization.

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.

Effect of the Nonpathogenic Strain Fusarium oxysporum FO12 on Fe Acquisition in Rice (Oryza sativa L.) Plants.

Rice (Oryza sativa L.) is a very important cereal worldwide, since it is the staple food for more than half of the world's population. Iron (Fe) deficiency is among the most important agronomical concerns in calcareous soils where rice plants may suffer from this deficiency. Current production systems are based on the use of high-yielding varieties and the application of large quantities of agrochemicals, which can cause major environmental problems. The use of beneficial rhizosphere microorganisms is considered a relevant sustainable alternative to synthetic fertilizers. The main goal of this study was to determine the ability of the nonpathogenic strain Fusarium oxysporum FO12 to induce Fe-deficiency responses in rice plants and its effects on plant growth and Fe chlorosis. Experiments were carried out under hydroponic system conditions. Our results show that the root inoculation of rice plants with FO12 promotes the production of phytosiderophores and plant growth while reducing Fe chlorosis symptoms after several days of cultivation. Moreover, Fe-related genes are upregulated by FO12 at certain times in inoculated plants regardless of Fe conditions. This microorganism also colonizes root cortical tissues. In conclusion, FO12 enhances Fe-deficiency responses in rice plants, achieves growth promotion, and reduces Fe chlorosis symptoms.

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.

Diversity of Cellulolytic Microorganisms Associated with the Subterranean Termite Reticulitermes grassei.

Reticulitermes grassei is a subterranean termite species that forages on woody structures of the Iberian Peninsula, and is often a building and crops pest. A total of 23 microorganisms associated with the activity of R. grassei were isolated from colonized ecosystems in southern Spain. They were morphologically and molecularly characterized, with fungi being the most prevalent ones. The fungi showed high values of optimum growth temperature, suggesting that they could be able to survive and develop in warm regions. Their cellulolytic activity was tested in carboxymethylcellulose (CMC) agar, concluding that all fungal isolates produce cellulases, and the enzymatic index (EI) was revealed in CMC agar with Gram's iodine solution, with Penicillium citrinum showing the highest EI and Trichoderma longibrachiatum the highest mycelial growth rate on CMC. A preliminary microorganism dispersion assay was carried out with the termites, concluding that these insects may have a positive influence on fungal dispersion and the subsequent colonization of new substrates. Our study suggests that fungi associated with R. grassei may potentially be of interest in biotechnological fields such as biofuel production and the food industry.

Biological and Urea Treatments Reduce the Primary Inoculum of Red Leaf Blotch of Almond Caused by Polystigma amygdalinum.

Red leaf blotch (RLB), caused by Polystigma amygdalinum, is considered the most prevalent foliar disease in both traditional and new intensive almond-growing areas in Spain. Since the disease is monocyclic, its control must be based on the reduction of the only source of inoculum-the leaves infected in the previous season and fallen to the ground in autumn. Thus, this study aimed to determine the effect of two microorganisms and urea on RLB inoculum reduction by evaluating different application modes to fallen leaves in field conditions. Leaves of almond cv. Guara showing symptoms of RLB were collected in autumn, placed into nylon mesh bags, and treated by dipping or spraying with conidial suspensions of Myrothecium inundatum or the nonpathogenic strain Fusarium oxysporum FO12. The bags were exposed on the ground or buried in an experimental almond field for 6 months in each experimental year. Bags treated with crystalline urea solution at 46% N or not treated were included as controls. The primary inoculum (number of ascospores per gram of leaf) and the development of fruiting bodies (maturity stages of perithecia) were monitored in the fallen leaves for each experimental treatment combination. M. inundatum significantly reduced the primary inoculum in comparison with the nontreated control or F. oxysporum FO12, showing a similar effect to that observed for urea in the 2 experimental years. The type of application (spraying or dipping) did not show any significant effect, whereas the inoculum was significantly reduced in buried leaves in comparison with leaves maintained on the ground for all the treatments tested. This study represents the first report evaluating management strategies against RLB based on the reduction of the primary inoculum of P. amygdalinum.

Biocontrol of Plant Diseases by Means of Antagonist Microorganisms, Biostimulants and Induced Resistance as Alternatives to Chemicals.

Plant diseases are one of the biggest problems in conventional agriculture as they reduce both yield and crop value [...].

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.

Resistance Induction in Olive Tree (Olea europaea) Against Verticillium Wilt by Two Beneficial Microorganisms and a Copper Phosphite Fertilizer.

Enhancement of the natural defenses of a plant by beneficial microorganisms, i.e., endophytic bacteria and fungi or fertilizers such as copper phosphonates, could result in a potential alternative strategy against verticillium wilt of olive tree (Olea europaea). In this study, two beneficial microorganisms (the fungus Aureobasidium pullulans AP08 and the bacterium Bacillus amyloliquefaciens PAB-024) and a phosphonate salt copper phosphite (CuPh) were evaluated for their effectiveness as host resistance inducers against Verticillium dahliae in olive. To this end, 6-month-old healthy olive plants of the susceptible cultivar Picual were treated by foliar or root applications by spraying 15 ml per plant or by irrigation with 350 ml per plant of the dilutions of each product (CuPh: 3 or 10 ml l-1, respectively; PAB-024: 108 UFC ml-1; AP08: 106 UFC ml-1). Treatments were conducted weekly from 2 weeks before inoculation to 10 days after inoculation. A cornmeal-water-sand mixture (1:2:9; w:v:w) colonized by V. dahliae was used for plant inoculation. Additionally, treated and noninoculated, nontreated and inoculated, and nontreated and noninoculated plants were included for comparative purposes. Disease severity progress and shoot fresh weight were assessed. Parameters involved in plant resistance were monitored through determination and quantification of reactive oxygen species (ROS) response (H2O2), and evaluation of hormones was done by gene expression analysis. Aureobasidium pullulans and CuPh were the most effective in disease reduction in planta by foliar or root application, respectively. Plants treated with CuPh showed significantly higher shoot fresh weight compared to the other treatments. ROS was significantly enhanced in plants treated with B. amyloliquefaciens PAB-024 compared to the rest of treatments and control. With regard to the evaluation of hormones, high levels of salicylic acid were detected on leaves from all treatment combinations, but without significant enhancements compared to the nontreated control. Regarding the gene expression related to salicylic acid, only the WRKY5 gene has shown a strong enhancement in the treatment with B. amyloliquefaciens. On the other hand, a strong accumulation of jasmonic acid and jasmonic acid-isoleucine in plants treated with A. pullulans was observed in all the tissues analyzed and also in the roots of plants treated with B. amyloliquefaciens and CuPh.

Characterization of Actinobacterial Strains as Potential Biocontrol Agents against Macrophomina phaseolina and Rhizoctonia solani, the Main Soil-Borne Pathogens of Phaseolus vulgaris in Cuba.

Macrophomina phaseolina and Rhizoctonia solani are considered two major soil-borne pathogens of Phaseolus vulgaris in Cuba. Their management is difficult, not only due to their intrinsic biology as soil-borne pathogens, but also because the lack of active ingredients available against these pathogens. Actinobacteria, a heterogeneous bacterial group traditionally known as actinomycetes have been reported as promising biological control agents (BCAs) in crop protection. Thus, the main objective of this study was to evaluate the effectiveness of 60 actinobacterial strains as BCAs against M. phaseolina and R. solani in vitro by dual culture assays. The most effective strains were characterized according to their cellulolytic, chitinolytic and proteolytic extracellular enzymatic activity, as well as by their morphological and biochemical characters in vitro. Forty and 25 out of the 60 actinobacteria strains inhibited the mycelial growth of M. phaseolina and R. solani, respectively, and 18 of them showed a common effect against both pathogens. Significant differences were observed on their enzymatic and biochemical activity. The morphological and biochemical characters allow us to identify all our strains as species belonging to the genus Streptomyces. Streptomyces strains CBQ-EA-2 and CBQ-B-8 showed the highest effectiveness in vitro. Finally, the effect of seed treatments by both strains was also evaluated against M. phaseolina and R. solani infections in P. vulgaris cv. Quivicán seedlings. Treatments combining the two Streptomyces strains (CBQ-EA-2 + CBQ-B-8) were able to reduce significantly the disease severity for both pathogen infections in comparison with the non-treated and inoculated control. Moreover, they showed similar effect than that observed for Trichoderma harzianum A-34 and with Celest® Top 312 FS (Syngenta®; Basilea, Switzerland) treatments, which were included for comparative purposes.

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.

Influence of Cultivar and Biocontrol Treatments on the Effect of Olive Stem Extracts on the Viability of Verticillium dahliae Conidia.

The effect of olive (Olea europaea) stem extract (OSE) on the viability of conidia of Verticillium dahliae, the causal agent of Verticillium wilt of olive (VWO), is not yet well understood. Thus, the aim of this study was to determine the influence of the olive genotype (cultivar resistance) and the interaction between olive cultivars and biocontrol treatments on the effect of OSE on conidial germination of V. dahliae by in vitro sensitivity tests. To this end, OSE from cultivars Frantoio, Arbequina, and Picual, respectively tolerant, moderately susceptible, and highly susceptible to V. dahliae, were tested alone or after treatments with biological control agents (BCAs) and commercial products efficient at reducing the progress of VWO. Aureobasidium pullulans strain AP08, Phoma sp. strain ColPat-375, and Bacillus amyloliquefaciens strain PAB-24 were considered as BCAs. Aluminium lignosulfonate (IDAI Brotaverd®), copper phosphite (Phoscuprico®), potassium phosphite (Naturfos®), and salicylic acid were selected as commercial products. Our results indicate that the influence of biological treatments against the pathogen depends on the genotype, since the higher the resistance of the cultivar, the lower the effect of the treatments on the ability of OSE to inhibit the germination of conidia. In 'Picual', the BCA B. amyloliquefaciens PAB024 and copper phosphite were the most effective treatments in inhibiting conidia germination by the OSE. This work represents a first approach to elucidate the role of cultivar and biological treatments in modifying the effect on the pathogen of the endosphere content of olive plants.

Etiology of Septoria Leaf Spot of Pistachio in Southern Spain.

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 S. pistaciarum by sequencing internal transcribed spacers, partial RNA polymerase II second largest subunit locus, and 28S ribosomal RNA 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 potato dextrose agar (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 to 20°C. Eight culture media were tested, with oatmeal agar and Spezieller Nährstoffarmer agar 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 compared 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. To our knowledge, this is the first report of S. pistaciarum causing SLS in pistachio and terebinth in Spain.

Diversity of Colletotrichum Species Associated with Olive Anthracnose Worldwide.

Olive anthracnose caused by Colletotrichum species causes dramatic losses of fruit yield and oil quality worldwide. A total of 185 Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries over the world were characterized. Colony and conidial morphology, benomyl-sensitive, and casein-hydrolysis activity were recorded. Multilocus alignments of ITS, TUB2, ACT, CHS-1, HIS3, and/or GAPDH were conducted for their molecular identification. The pathogenicity of the most representative Colletotrichum species was tested to olive fruits and to other hosts, such as almonds, apples, oleander, sweet oranges, and strawberries. In general, the phenotypic characters recorded were not useful to identify all species, although they allowed the separation of some species or species complexes. ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes, whereas ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloesporioides complex. Twelve Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified, with C. godetiae being dominant in Spain, Italy, Greece, and Tunisia, C. nymphaeae in Portugal, and C. fioriniae in California. The highest diversity with eight Colletotrichum spp. was found in Australia. Significant differences in virulence to olives were observed between isolates depending on the Colletotrichum species and host origin. When other hosts were inoculated, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves.

Plant Biostimulants: New Insights Into the Biological Control of Verticillium Wilt of Olive.

Verticillium wilt of olive (Olea europaea subsp. europaea L.) (VWO), caused by the hemibiotrophic soil-borne fungus Verticillium dahliae Kleb., is considered the major limiting factor of this crop in Mediterranean-type climate regions of the world. The absence of effective chemical treatments makes the control of the disease difficult. In this way, the use of biostimulants and host plant defense inducers seems to be one of the most promising biological and eco-friendly alternatives to traditional control measures. Thus, the main goal of this study was to evaluate the effect of 32 products, including amino acids, micronutrients, microorganisms, substances of natural origin, copper complex-based products, and organic and inorganic salts against the disease under controlled conditions. To this end, their effects on mycelial growth and microsclerotia (MS) inhibition of V. dahliae were evaluated by means of dual cultures or by sensitivity tests in vitro as well as on disease progression in planta. Wide ranging responses to the pathogen and disease reduction levels were observed among all the products tested, suggesting multiple modes of action. Copper-based products were among the most effective for mycelial growth and MS inhibition, whereas they did not show an important effect on the reduction of disease severity in planta. Phoma sp. and Aureobasidium pullulans were the most effective in disease reduction in planta with foliar application. On the other hand, two phosphite salts, one with copper and the other with potassium, were the most effective in disease reduction in planta when they were applied by irrigation, followed by A. pullulans and Bacillus amyloliquefaciens. This study will be useful to select the best candidates for future studies, contributing significantly to new insights into the current challenge of the biological control of VWO.