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, because 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.

Exploring the Role of Debaryomyces hansenii as Biofertilizer in Iron-Deficient Environments to Enhance Plant Nutrition and Crop Production Sustainability.

The European "Green Deal" policies are shifting toward more sustainable and environmentally conscious agricultural practices, reducing the use of chemical fertilizer and pesticides. This implies exploring alternative strategies. One promising alternative to improve plant nutrition and reinforce plant defenses is the use of beneficial microorganisms in the rhizosphere, such as "Plant-growth-promoting rhizobacteria and fungi". Despite the great abundance of iron (Fe) in the Earth's crust, its poor solubility in calcareous soil makes Fe deficiency a major agricultural issue worldwide. Among plant promoting microorganisms, the yeast Debaryomyces hansenii has been very recently incorporated, for its ability to induce morphological and physiological key responses to Fe deficiency in plants, under hydroponic culture conditions. The present work takes it a step further and explores the potential of D. hansenii to improve plant nutrition and stimulate growth in cucumber plants grown in calcareous soil, where ferric chlorosis is common. Additionally, the study examines D. hansenii's ability to induce systemic resistance (ISR) through a comparative relative expression study by qRT-PCR of ethylene (ET) biosynthesis (ACO1), or ET signaling (EIN2 and EIN3), and salicylic acid (SA) biosynthesis (PAL)-related genes. The results mark a significant milestone since D. hansenii not only enhances nutrient uptake and stimulates plant growth and flower development but could also amplify induced systemic resistance (ISR). Although there is still much work ahead, these findings make D. hansenii a promising candidate to be used for sustainable and environmentally friendly integrated crop management.

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.

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.

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.

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.

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.

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.

Vascular Fungi Associated with Branch Dieback of Olive in Super-High-Density Systems in Southern 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 the internal transcribed spacer, 28S ribosomal RNA, ß-tubulin, or actin were performed, and the following species were identified: Acremonium sclerotigenum, Cadophora luteo-olivacea, Paracremonium sp., Phaeoacremonium italicum, P. minimum, P. scolyti, and Pseudophaeomoniella oleicola. Colony color, mycelial growth, conidial characteristics, and production were defined on potato dextrose agar, malt extract agar (MEA), and oatmeal agar. 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 slow mycelial growth (0.5 to 2.0 mm day-1), with the optimum temperature ranging from 23.2 to 33.9°C. Pathogenicity tests were conducted on 9-month-old olive potted plants (Arbequina) inoculated with mycelial plugs. C. luteo-olivacea, Phaeoacremonium 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 inoculation with conidial suspensions or mycelial plugs was evaluated, with the second method being the most effective. C. luteo-olivacea was the fungus most aggressive to olive, followed by Phaeoacremonium minimum.

Etiology of Branch Dieback and Shoot Blight of English Walnut Caused by Botryosphaeriaceae and Diaporthe Species in Southern Spain.

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 ß-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.

Interaction Between Diaporthe rhusicola and Neofusicoccum mediterraneum Causing Branch Dieback and Fruit Blight of English Walnut in California, and the Effect of Pruning Wounds on the Infection.

Botryosphaeriaceae and Diaporthaceae species are the causal agents of branch dieback of English walnut in California. In this study, the effects of the interaction between Neofusicoccum mediterraneum and Diaporthe rhusicola were evaluated in vitro by using mycelial plugs or spore suspensions and in vivo by inoculating shoots and epicarps (hulls) of walnut. Single inoculations of each species and different coinfection treatments were performed under laboratory or field conditions. The influence of shoot age and susceptibility of bark or pith tissues to N. mediterraneum and D. rhusicola infection after pruning was also evaluated. In in vitro experiments, spore germination of D. rhusicola was significantly (P < 0.0001) reduced in the presence of N. mediterraneum spores. When D. rhusicola was inoculated at 4 days before N. mediterraneum, a delay in lesion development in shoots and hulls was observed compared with the other two interaction treatments. One- to 2-year-old shoots were more susceptible to infection and colonization by N. mediterraneum than 3- to 4-year-old shoots. In young shoots, inoculation in the pith tissue resulted in longer lesions than those observed on shoots inoculated in the bark. No significant differences were observed between the development of internal or external necrosis and the age of the shoots, or the susceptibility of bark and pith to D. rhusicola infection. This information is essential to better understanding the complex situation of this walnut disease toward developing control management strategies.

Effects of Cultivar Susceptibility, Fruit Maturity, Leaf Age, Fungal Isolate, and Temperature on Infection of Almond by Colletotrichum spp.

Almond anthracnose, caused by Colletotrichum spp., is a reemerging disease in Spain. To date, little research has been conducted on the factors affecting this disease development. In this study, the effects of cultivar, fruit wounding and maturity, leaf age, fungal isolate, and temperature on almond infection by Colletrotrichum spp. were evaluated under laboratory-controlled conditions. Inoculations were performed using conidial suspensions of Colletrotrichum acutatum or C. godetiae. Disease severity was higher in wounded than in unwounded fruit. Based on observations of inoculated fruit, Ferraduel and Nonpareil were the most tolerant cultivars, while Tarraco and Penta were the most susceptible cultivars. Four categories of susceptibility (highly susceptible, susceptible, moderately susceptible, and resistant) were distinguished by using the cluster analysis statistical approach. Differences in susceptibility between young and old leaves were observed, but Nonpareil was consistently the most tolerant cultivar. Significant differences in virulence between C. acutatum and C. godetiae were observed in inoculated fruit, with C. acutatum being the most virulent. Disease development was more severe when inoculations were performed at the fruitlet stage or when the fruit were incubated at approximately 25°C, with respect to other maturity stages and temperatures evaluated. Natural fruit infections were also assessed. Cultivar susceptibility data were compared between laboratory tests and field observations. A significant positive linear correlation was obtained between the susceptibility of the common cultivars evaluated under the two conditions.

Survey, Identification, and Characterization of Cylindrocarpon-Like Asexual Morphs in Spanish Forest Nurseries.

Cylindrocarpon-like asexual morphs infect herbaceous and woody plants, mainly in agricultural scenarios, but also in forestry systems. The aim of the present study was to characterize a collection of Cylindrocarpon-like isolates recovered from the roots of a broad range of forest hosts from nurseries showing decline by morphological and molecular studies. Between 2009 and 2012, 17 forest nurseries in Spain were surveyed and a total of 103 Cylindrocarpon-like isolates were obtained. Isolates were identified based on DNA sequences of the partial gene regions histone H3 (his3). For the new species, the internal transcribed spacer and intervening 5.8S nrRNA gene (ITS) region, ß-tubulin (tub2), and translation elongation factor 1-α (tef1) were also used to determine their phylogenetic position. Twelve species belonging to the genera Cylindrodendrum, Dactylonectria, and Ilyonectria were identified from damaged roots of 15 different host genera. The species C. alicantinum, D. macrodidyma, D. novozelandica, D. pauciseptata, D. pinicola, D. torresensis, I. capensis, I. cyclaminicola, I. liriodendri, I. pseudodestructans, I. robusta, and I. rufa were identified. In addition, two Dactylonectria species (D. hispanica sp. nov. and D. valentina sp. nov.), one Ilyonectria species (I. ilicicola sp. nov.), and one Neonectria species (N. quercicola sp. nov.) are newly described. The present study demonstrates the prevalence of this fungal group associated with seedlings of diverse hosts showing decline symptoms in forest nurseries in Spain.

Identification of Fungal Species Associated with Branch Dieback of Olive and Resistance of Table Cultivars to Neofusicoccum mediterraneum and Botryosphaeria dothidea.

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, ß-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.

Morphological, Pathogenic, and Molecular Characterization of Colletotrichum acutatum Isolates Causing Almond Anthracnose in Spain.

Almond anthracnose is a serious and emerging disease in several countries. All isolates causing almond anthracnose have been assigned to the Colletotrichum acutatum species complex, of which only C. fioriniae and C. godetiae have been associated with the disease to date. Here, we characterized Colletotrichum isolates from almond fruit affected by anthracnose in the Andalusia region. Two Colletotrichum isolates causing olive anthracnose were included for comparison. Morphological characteristics were useful for separating the isolates into groups based on colony morphology. Pathogenicity tests in almond, olive, and apple fruit showed differences in virulence and some degree of pathogenic specialization among isolates. Molecular characterization allowed clear identification of the Colletotrichum isolates tested. The olive isolates were identified as C. godetiae and C. nymphaeae, both previously identified in Andalusian olive orchards. Two phylogenetic species were identified among the almond isolates: C. godetiae, with gray colonies, which is well known in other countries, and C. acutatum, with pink-orange colonies. This species identification differs from those of pink-colony subpopulations described in other countries, which are C. fioriniae. Therefore, this study is also the first report of a new species of Colletotrichum causing almond anthracnose within the C. acutatum species complex.

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.

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.

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.