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.

The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization.

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.

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.

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.

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.

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.

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.

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.

Almond Anthracnose: Current Knowledge and Future Perspectives.

Almond anthracnose caused by Colletotrichum spp. has been described as one of the most important diseases of this nut crop in the main almond-growing regions worldwide, including California, Australia and Spain. Currently, almond anthracnose is considered a re-emerging disease in the countries across the Mediterranean Basin due to the shift of plantations from the original crop areas to others with climatic, edaphic and orographic conditions favoring crop growing and yield. The pathogen mainly affects fruit at the youngest maturity stages, causing depressed, round and orange or brown lesions with abundant gum. The affected fruits can fall prematurely and lead to the drying of branches, causing significant economic losses in years of epidemics. This review aims to compile the current knowledge on the etiology, epidemiology and management of this disease.

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.

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.

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.