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.

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.

Evaluation of Phenolic Profile and Antioxidant Activity of Eleven Pistachio Cultivars (Pistacia vera L.) Cultivated in Andalusia.

Pistachio (Pistacia vera L.) is a nut with a good adaptability to the Mediterranean conditions of cultivation, specifically in the Andalusian region, becoming an emerging crop. Moreover, it has been getting attention in the past years for the great content of bioactive compounds such as polyphenols. Although some studies have reported the polyphenolic profile of pistachios, most of them have analyzed the hull part, considered as a residue, and not the kernel which is the edible part. Therefore, characterization of eleven varieties of pistachios kernels cultivated in Andalusia and harvested in 2019 and 2020 was carried out by UHPLC-MS (ultra-high-performance liquid chromatography high-resolution mass spectrometry). The identification and quantification of 56 polyphenolic compounds was performed, being the hydroxybenzoic acids group the most abundant with a 71−86% of the total amount followed by flavan-3-ols group that accounted for 8−24%. Moreover, 3,4-dihydroxybenzoic acid was the main compound in most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (p < 0.01) were found in the total amount of polyphenols between 2019 and 2020. In addition, the antioxidant activity was measured by DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and ORAC (oxygen radical absorbance capacity) assays, showing a similar trend as that of the polyphenols.

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.

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.

Inoculum and Infection Dynamics of Polystigma amygdalinum in Almond Orchards in Spain.

Red leaf blotch (RLB) disease of almond, caused by Polystigma amygdalinum, is an important foliar disease in most production regions of the Mediterranean basin and the Middle East because severe infections may cause a premature defoliation of the tree. Some key aspects on the epidemiology of P. amygdalinum were studied in multiyear trials in two almond-growing regions in Spain, which included the seasonal development of perithecia and production and germination of ascospores along with the disease incubation and plant infectivity periods. Our results showed that primary inoculum was available in extended periods (January to August). Significant differences in ascospore amounts among regions, higher in the southern Andalusia and lower in the northern Catalonia, and years of study were detected. The factors geographical location, sampling period, and evaluation year were found significant on the development of P. amygdalinum perithecia. Variable ascospore germination rates were observed from April to July: >15% but rarely exceeding 30%. The RLB infectivity period in Catalonia extended from March to mid-June, whereas in Andalusia it was from March to May. The incubation period was mainly in a range of 5 to 10 weeks in Catalonia. The environmental conditions of October to January influence the available ascospore amounts in the next season. RLB infection occurs in spring to summer when mean temperatures are in the range 10 to 20°C. These results represent the first step in developing a prediction model of the disease that might serve as a tool for the control of RLB.

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.

An efficient RGB-UAV-based platform for field almond tree phenotyping: 3-D architecture and flowering traits.

BACKGROUND: Almond is an emerging crop due to the health benefits of almond consumption including nutritional, anti-inflammatory, and hypocholesterolaemia properties. Traditional almond producers were concentrated in California, Australia, and Mediterranean countries. However, almond is currently present in more than 50 countries due to breeding programs have modernized almond orchards by developing new varieties with improved traits related to late flowering (to reduce the risk of damage caused by late frosts) and tree architecture. Almond tree architecture and flowering are acquired and evaluated through intensive field labour for breeders. Flowering detection has traditionally been a very challenging objective. To our knowledge, there is no published information about monitoring of the tree flowering dynamics of a crop at the field scale by using color information from photogrammetric 3D point clouds and OBIA. As an alternative, a procedure based on the generation of colored photogrammetric point clouds using a low cost (RGB) camera on-board an unmanned aerial vehicle (UAV), and an semi-automatic object based image analysis (OBIA) algorithm was created for monitoring the flower density and flowering period of every almond tree in the framework of two almond phenotypic trials with different planting dates. RESULTS: Our method was useful for detecting the phenotypic variability of every almond variety by mapping and quantifying every tree height and volume as well as the flowering dynamics and flower density. There was a high level of agreement among the tree height, flower density, and blooming calendar derived from our procedure on both fields with the ones created from on-ground measured data. Some of the almond varieties showed a significant linear fit between its crown volume and their yield. CONCLUSIONS: Our findings could help breeders and researchers to reduce the gap between phenomics and genomics by generating accurate almond tree information in an efficient, non-destructive, and inexpensive way. The method described is also useful for data mining to select the most promising accessions, making it possible to assess specific multi-criteria ranking varieties, which are one of the main tools for breeders.

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.

High-Throughput 3-D Monitoring of Agricultural-Tree Plantations with Unmanned Aerial Vehicle (UAV) Technology.

The geometric features of agricultural trees such as canopy area, tree height and crown volume provide useful information about plantation status and crop production. However, these variables are mostly estimated after a time-consuming and hard field work and applying equations that treat the trees as geometric solids, which produce inconsistent results. As an alternative, this work presents an innovative procedure for computing the 3-dimensional geometric features of individual trees and tree-rows by applying two consecutive phases: 1) generation of Digital Surface Models with Unmanned Aerial Vehicle (UAV) technology and 2) use of object-based image analysis techniques. Our UAV-based procedure produced successful results both in single-tree and in tree-row plantations, reporting up to 97% accuracy on area quantification and minimal deviations compared to in-field estimations of tree heights and crown volumes. The maps generated could be used to understand the linkages between tree grown and field-related factors or to optimize crop management operations in the context of precision agriculture with relevant agro-environmental implications.

K(+) starvation inhibits water-stress-induced stomatal closure.

The effect of potassium starvation on stomatal conductance was studied in olive trees and sunflower plants, two major crops with greatly differing botanical characteristics. In both species, K(+) starvation inhibited water-stress-induced stomatal closure. In olive trees, potassium starvation favoured stomatal conductance and transpiration, as well as inhibiting shoot growth, in the three cultivars studied: 'Lechín de Granada', 'Arbequina' and 'Chetoui'. However, 'Lechín de Granada' - generally considered more drought-tolerant than 'Arbequina' and 'Chetoui' - proved less susceptible to potassium starvation. Results for olive trees also suggest genetic variability in olive cultivars in relation to potassium requirements for stem growth and the regulation of water transpiration. The results obtained suggest that inhibition of the stomatal closure mechanism produced by moderate potassium starvation is a widespread plant physiological disorder, and may be the cause of tissue dehydration in many water-stressed crops.