First Report of Diaporthe amygdali Associated with Twig Canker and Shoot Blight of Nectarine in Spain.

Nectarine (Prunus persica (L.) Batsch var. nucipersica (Suckow) C. K. Schneid.) is a fruit crop widely cultivated throughout the Mediterranean basin. In Spain, it is mainly grown in eastern regions of the country. In March 2018, 5-year-old nectarine trees showing twig canker symptoms were observed after a rainy spring period in a 0.5 ha orchard located at Alaior, Menorca island (Spain). Cankers were frequent on affected trees (approximately, 80% of the total trees), thus leading to shoot blight. Ten twig segments of one-year old wood with cankers were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution and rinsed twice in sterile distilled water. Small pieces (2 mm) of affected tissues were taken from the margin of the cankers and plated on potato dextrose agar (PDA) supplemented with 0.5 g/L of streptomycin sulphate (PDAS). The plates were then incubated at 25 ºC in the dark for 7 to 10 d. Actively growing colonies were first hyphal-tipped and then transferred to PDA and 2% water agar supplemented with sterile pine needles and incubated at 21-22ºC under a 12h/12h near UV / darkness cycle during 21 d (León et al. 2020). Colonies were white at first, becoming light cream, with visible solitary and aggregate pycnidia at maturity. Alpha conidia were aseptate, fusiform, hyaline, multi-guttulated (mean ± SD = 7.4 ± 0.7 × 2.8 ± 0.4 µm, n = 100). Beta and gamma conidia were not observed. The morphological and cultural characteristics of the isolates were congruent with those of Diaporthe spp. (Gomes et al. 2013). The ITS1-5.8S-ITS2 (ITS) region and fragments of ß-tubulin (tub2), the translation elongation factor 1-alpha (tef1-α) gene regions, histone H3 (his3) and calmodulin (cal) genes of representative isolate DAL-59 were amplified and sequenced (Santos et al. 2017). The BLASTn analysis revealed 100% similarity with sequences of D. mediterranea (Synonym D. amygdali) (Hilário et al. 2021) isolate DAL-34 from almond (ITS: MT007489, tub2: MT006686, tef1-α: MT006989, his3: MT007095 and cal: MT006761). Sequences of isolate DAL-59 were deposited in GenBank Database (ITS: MT007491, tub2: MT006688, tef1-α: MT006991, his3: MT007097 and cal: MT006763). Pathogenicity tests were conducted using one-year-old potted plants of nectarine cv. Boreal, which were inoculated with isolate DAL-59. In each plant, a 3 mm wound was made in the center of the main branch (about 30 cm length) with a scalpel. Colonized agar plugs with 3 mm diameter, which were obtained from active 10-day-old colonies growing on PDA, were inserted underneath the epidermis and the wounds sealed with Parafilm. Inoculated plants were incubated in a growth chamber at 23 ºC with 12 h of light per day. Controls were inoculated with uncolonized PDA plugs. There were twelve plants per treatment, which were arranged in a completely randomized design. Five days after inoculation necrosis development was observed in the area of inoculation. Wilting and twig blight symptoms over the lesion occurred 3-wk after inoculation and pycnidia were detected, while the controls remained asymptomatic. Diaporthe amygdali was re-isolated from symptomatic tissues and identified as described above to satisfy Koch's postulates. To our knowledge, this is the first report of D. amygdali causing twig canker and shoot blight disease on nectarine in Spain.

Cadophora sabaouae sp. nov. and Phaeoacremonium Species Associated with Petri Disease on Grapevine Propagation Material and Young Grapevines in Algeria.

A field survey conducted on asymptomatic grapevine propagation material from nurseries and symptomatic young grapevines throughout different regions of Algeria yielded a collection of 70 Phaeoacremonium-like isolates and three Cadophora-like isolates. Based on morphology and DNA sequence data of ß-tubulin (tub2) and actin, five Phaeoacremonium species were identified including Phaeoacremonium minimum (22 isolates), Phaeoacremonium venezuelense (19 isolates), Phaeoacremonium parasiticum (17 isolates), Phaeoacremonium australiense (8 isolates), and Phaeoacremonium iranianum (4 isolates). The latter two species (P. australiense and P. iranianum) were reported for the first time in Algeria. Multilocus phylogenetic analyses (internal transcribed spacer, tub2, and translation elongation factor 1-α) and morphological features, allowed the description of the three isolates belonging to the genus Cadophora (WAMC34, WAMC117, and WAMC118) as a novel species, named Cadophora sabaouae sp. nov. Pathogenicity tests were conducted on grapevine cuttings cultivar Cardinal. All the identified species were pathogenic on grapevine cuttings.

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.

Characterization and Pathogenicity of Botryosphaeriaceae Species Isolated from Almond Trees on the Island of Mallorca (Spain).

In this study, 31 almond orchards with trees showing severe decline symptoms were surveyed from 2009 to 2014 on the island of Mallorca (Spain). In all, 45 Botryosphaeriaceae isolates were collected and characterized based on phenotypical features and comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region and elongation factor 1-α gene. Five species were identified as Diplodia olivarum, D. seriata, Neofusicoccum luteum, N. mediterraneum, and N. parvum. Pathogenicity tests were performed on four cultivars ('Pons', 'Vivot', 'Jordi', and 'Ferragnes') under field conditions for two consecutive years (2013 to 2014), and confirmed that all five species cause canker and dieback of almond, with Neofusicoccum spp. more virulent than Diplodia spp. in both years. Jordi was less sensitive to fungal infection in 2013. First reports from almond in Spain include N. mediterraneum and N. luteum.

New Reports of Phytophthora Species in Plant Nurseries in Spain.

The plant nursery industry has become an ideal reservoir for Phytophthora species and other soilborne pathogens. In this context, isolation from tissues and soil of ornamental and forest plants from nurseries in four regions of Spain was carried out. A high diversity of Phytophthora species was confirmed. Fourteen Phytophthora phylotypes (P. cactorum, P. cambivora, P. cinnamomi, P. citrophthora, P. crassamura, P. gonapodyides, P. hedraiandra, P. nicotianae, P. niederhauserii, P. palmivora, P. plurivora, P. pseudocryptogea, P. sansomeana, and Phytophthora sp. tropicalis-like 2) were isolated from over 500 plant samples of 22 species in 19 plant genera. Nine species were detected in water sources, two of them (P. bilorbang and P. lacustris) exclusively from water samples. P. crassamura was detected for the first time in Spain. This is the first time P. pseudocryptogea is isolated from Chamaecyparis lawsoniana and Yucca rostrata in Spain.

Cultivar Susceptibility to Natural Infections Caused by Fungal Grapevine Trunk Pathogens in La Mancha Designation of Origin (Spain).

Grapevine trunk diseases (GTDs) are one of the main biotic stress factors affecting this crop. The use of tolerant grapevine cultivars would be an interesting and sustainable alternative strategy to control GTDs. To date, most studies about cultivar susceptibility have been conducted under controlled conditions, and little information is available about tolerance to natural infections caused by GTD fungi. The objectives of this study were: (i) to identify tolerant cultivars to GTD fungi within a Spanish germplasm collection, based on external symptoms observed in the vineyard; and (ii) to characterize the pathogenic mycoflora associated with symptomatic vines. For this purpose, a grapevine germplasm collection including 22 white and 25 red cultivars was monitored along three growing seasons, and their susceptibility for esca foliar symptoms was assessed. Fungi were identified by using morphological and molecular methods. Cultivars such as, 'Monastrell', 'Graciano', 'Cabernet Franc', 'Cabernet Sauvignon', 'Syrah', 'Moscatel de Alejandría', 'Sauvignon Blanc', and 'Airén' displayed high susceptibility to GTDs, whereas others such as 'Petit Verdot', 'Pinot Noir', 'Chardonnay', and 'Riesling' were considered as tolerant. The prevalent fungal species isolated from symptomatic vines were Phaeomoniella chlamydospora (27.9% of the fungal isolates), Cryptovalsa ampelina (24.6%), and Dothiorella sarmentorum (21.3%).

Evaluation of Sown Cover Crops and Spontaneous Weed Flora as a Potential Reservoir of Black-Foot Pathogens in Organic Viticulture.

(1) Background. An extensive survey of grapevine-sown cover crops and spontaneous weed flora was conducted from 2019 to 2020 in organic vineyards in six European countries (France, Italy, Romania, Slovenia, Spain, Switzerland). Our main objective was to detect and identify the presence of Cylindrocarpon-like asexual morphs species associated with black-foot disease on their roots. (2) Methods. Fungal isolations from root fragments were performed on culture media. Cylindrocarpon-like asexual morph species were identified by analyzing the DNA sequence data of the histone H3 (his3) gene region. In all, 685 plants belonging to different botanical families and genera were analyzed. Cylindrocarpon-like asexual morphs were recovered from 68 plants (9.9% of the total) and approximately 0.97% of the plated root fragments. (3) Results. Three fungal species (Dactylonectria alcacerensis, Dactylonectria torresensis, Ilyonectria robusta) were identified. Dactylonectria torresensis was the most frequent, and was isolated from many cover crop species in all six countries. A principal component analysis with the vineyard variables showed that seasonal temperatures and organic matter soil content correlated positively with Cylindrocarpon-like asexual morphs incidence. (4) Conclusions. The presence of Cylindrocarpon-like asexual morphs on roots of cover crops suggests that they can potentially act as alternative hosts for long-term survival or to increase inoculum levels in vineyard soils.

Evaluation of long-term protection from nursery to vineyard provided by Trichoderma atroviride SC1 against fungal grapevine trunk pathogens.

BACKGROUND: Fungal grapevine trunk diseases (GTDs) represent a threat to viticulture, being responsible for important economic losses worldwide. Nursery and vineyard experiments were set up to evaluate the ability of Trichoderma atroviride SC1 to reduce infections of GTD pathogens in grapevine planting material during the propagation process and to assess the long-term protection provided by this biocontrol agent on grapevine plants in young vineyards during two growing seasons. RESULTS: Reductions of some GTD pathogen incidence and severity were found on grapevine propagation material after nursery application of T. atroviride SC1 during the grafting process, and also after additional T. atroviride SC1 treatments performed during two growing seasons in young vineyards, when compared with untreated plants. CONCLUSION: Trichoderma atroviride SC1 showed promise to reduce infections caused by some GTD pathogens in nurseries, and also when establishing new vineyards. This biological control agent could possibly be a valuable component in an integrated management approach where various strategies are combined to reduce GTD infections. © 2019 Society of Chemical Industry.

A Multiplex PCR System for the Specific Detection of Cylindrocarpon liriodendri, C. macrodidymum, and C. pauciseptatum from Grapevine.

Cylindrocarpon liriodendri and C. macrodidymum are the causal agents of grapevine black foot disease. Recently, a third species, C. pauciseptatum, has been isolated from roots of grapevine showing decline symptoms. Currently, reliable identification of isolates of these species through phenotypical characteristics has not been possible. The polymerase chain reaction (PCR)-based method developed in this study allows a quick and easy detection of Cylindrocarpon spp. associated with grapevine. Three primer pairs annealing to variable, partly species-specific sites of the internal transcribed spacer regions amplified species-specific PCR fragments of different sizes in C. liriodendri, C. macrodidymum, and C. pauciseptatum in a multiplex assay with DNA obtained with both quick and traditional extraction methods. They did not generate any PCR product in other fungal trunk pathogens or contaminants commonly associated with grapevines. When universal fungal ITS primers were used in a nested multiplex PCR, the three primer pairs also detected C. liriodendri, C. macrodidymum, and C. pauciseptatum in total DNA extracted from roots of inoculated grapevines. The designed methods can be used for the diagnosis of these fungi from pure culture or infected grapevines.

A new subspecies of Peucedanum officinale L. subsp. album (Apiaceae) from the eastern part of the Iberian Peninsula.

We describe Peucedanum officinale L. subsp. album Martínez-Fort & Donat-Torres subsp. nov., in which we grouped the thermomediterranean populations scattered along the eastern part of the Iberian Peninsula. The characters that differentiate this new subspecies from other infraspecific taxa in Peucedanum officinale are its canaliculated leaflet, the inflorescences much branched and lack of dominant terminal umbels, the umbels are few rayed, sometimes sessile and lateral, the petals are white and the fruit pedicels short, the same or shorter in length than the fruit. We provide here a full description of the new subspecies based on herbarium specimens and field measurements, as well as providing dichotomous keys to the subspecies within P. officinale. In addition, we provide a comparison of the ITS sequences of nrDNA with the most closely related taxons.

Comparative study and mutational analysis of distinctive structural elements of hyperthermophilic enzymes.

Comparison of the three-dimensional structure of hyperthermophilic and mesophilic beta-glycosidases shows differences in secondary structure composition. The enzymes from hyperthermophilic archaea have a significantly larger number of beta-strands arranged in supernumerary beta-sheets compared to mesophilic enzymes from bacteria and other organisms. Amino acid replacements designed to alter the structure of the supernumerary beta-strands were introduced by site directed mutagenesis into the sequence encoding the beta-glycosidase from Sulfolobus solfataricus. Most of the replacements caused almost complete loss of activity but some yielded enzyme variants whose activities were affected specifically at higher temperatures. Far-UV CD spectra recorded as a function of temperature for both wild type beta-glycosidase and mutant V349G, one of the mutants with reduced activity at higher temperatures, were similar, showing that the protein structure of the mutant was stable at the highest temperatures assayed. The properties of mutant V349G show a difference between thermostability (stability of the protein structure at high temperatures) and thermophilicity (optimal activity at high temperatures).

Effect of a new ionic pair on the unfolding activation barrier of beta-glucosidase B.

Thermal unfolding kinetics of beta-glucosidase B from Paenibacillus polymyxa and its thermoresistant mutant H62R were determined from far-UV circular dichroism (CD) measurements at different temperatures. The unfolding of both enzymes followed simple two-state kinetics. The new ionic pair formed between Arg62 and Glu429 in the H62R variant did not change substantially the enzyme structure as judged by far-UV CD and fluorescence spectra, but produced an increase in the unfolding activation barrier of 0.95 +/- 0.10 kcal mol(-1), in good agreement with the energetic contribution reported for surface salt bridges in proteins. Eyring's analysis of the unfolding kinetic constants showed that the activation enthalpies for thermal denaturation of both enzymes were essentially the same. Thus, the greater kinetic stability rendered by the salt bridge seems to be due to a reduction in the activation entropy.

Multilocus ISSR markers reveal two major genetic groups in Spanish and South African populations of the grapevine fungal pathogen Cadophora luteo-olivacea.

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.

New Phaeoacremonium species isolated from sandalwood trees in Western Australia.

Thirty-eight Phaeoacremonium isolates collected from pruning wounds of tropical sandalwood in Western Australia were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the actin and ß-tubulin genes. Three known Phaeoacremonium species were found, namely P. alvesii, P. parasiticum, and P. venezuelense. Phaeoacremonium venezuelense represents a new record for Australia. Two new species are described: P. luteum sp. nov. can be identified by the ability to produce yellow pigment on MEA, PDA, and OA, the predominance of subcylindrical to subulate type II phialides, and the mycelium showing prominent exudate droplets observed as warts; and P. santali sp. nov. which can be separated from other species producing pink colonies on MEA by the predominance of type I and II phialides, the distinct brownish olive colonies in OA, and slow growth.

Analysis of genetic and virulence diversity of Cylindrocarpon liriodendri and C. macrodidymum associated with black foot disease of grapevine.

Inter-simple sequence repeat (ISSR) analysis was used to investigate the genetic diversity of 87 Cylindrocarpon liriodendri and C. macrodidymum isolates, the causal agents of black foot disease of grapevine. The four ISSR primers (GT)7, (CCA)5, (CGA)5 and (TCG)5, were able to provide reproducible and polymorphic DNA fingerprint patterns and detected relevant genetic diversity in C. macrodidymum. The cluster analysis of ISSR data showed 21 different genotypes that were grouped in seven ISSR groups, from which two corresponded to C. liriodendri (G1 and G2) and five to C. macrodidymum (G3-G7). Nineteen isolates selected from the seven ISSR groups were inoculated in grapevine seedlings obtained from cv. 'Tempranillo'. The pathogenicity tests detected virulence diversity in C. macrodidymum. The isolates belonging to ISSR groups G6 and G7 were significantly more virulent than the other C. macrodidymum and C. liriodendri isolates.

Characterization of Fusarium circinatum from Pinus spp. in northern Spain.

Pitch canker caused by Fusarium circinatum was recently reported on Pinus spp. in Spain. In this study, a collection of 157 isolates of F. circinatum obtained from different geographical origins and hosts in northern Spain were identified and characterized by cultural and morphological features, PCR-RFLPs of the histone H3 gene, IGS region, and the translation elongation factor 1-alpha gene (TEF). Mating types were determined by multiplex PCR and sexual compatibility was performed under laboratory conditions. Both mating types were present in Spain and were able to form the teleomorph Gibberella circinata. Morphological differences between mating types, not previously reported, were observed: MAT-1 isolates showed clear, coiled, sterile hyphae characteristic of F. circinatum, whereas MAT-2 isolates presented sterile hyphae but not coiled. Virulence of representative isolates was tested on seven to eight-month-old P. nigra, P. pinaster and P. sylvestris seedlings. All isolates tested were pathogenic to these pine species, MAT-1 isolates being more virulent than MAT-2 isolates.

RHO1 (YlRHO1) is a non-essential gene in Yarrowia lipolytica and complements rho1Delta lethality in Saccharomyces cerevisiae.

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall that gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role (Yamochi et al., 1994). RHO1 has been characterized in Saccharomyces cerevisiae (Yamochi et al., 1994), and in several other fungal species. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Rho1ps to isolate the RHO1 gene of Yarrowia lipolytica. The gene isolated in this way, which we have named YlRHO1, encodes a 204 amino acid protein that shows a high degree of homology with other Rho1ps. However, unlike S. cerevisiae, the ylrho1Delta disruptant strain in Y. lipolytica is viable, although it exhibits an increased sensitivity to Calcofluor white and Congo red. Also, YlRHO1 complements rho1 lethality in S. cerevisiae at both 28 degrees C and 37 degrees C. The complete sequence of YlRHO1 can be obtained from GenBank under Accession No. AF279915.

Isolation of the MNN9 gene of Yarrowia lipolytica (YlMNN9) and phenotype analysis of a mutant ylmnn9 Delta strain.

In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodium orthovanadate and sensitivity to hygromycin B, compatible with a glycosylation defect, and hypersensitivity to Calcofluor white, Congo red or zymolyase, characteristic of cell wall defects. Analysis of cell wall proteins present in beta-mercaptoethanol and zymolyase extracts showed significant differences between the parental and the ylmnn9 Delta strain. These results suggest that, as has been the case with the mnn9 strain of S. cerevisiae, the ylmnn9 Delta strain we present in this work, could be used to study the cell wall proteins of Y. lipolytica and how they are organized into the cell wall.

A single FKS homologue in Yarrowia lipolytica is essential for viability.

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall which gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role. FKS1 has been characterized in Saccharomyces cerevisiae, where its function is at least partially redundant with that of FKS2/GSC2. FKS homologues have also been identified in several other fungal species, including Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Cryptococcus neoformans and Paracoccidiodes brasiliensis. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Fks1ps to isolate the possible FKS homologue genes of the strictly aerobic non-conventional yeast Yarrowia lipolytica. Using this approach, we have isolated a single FKS homologue which we have named YlFKS1; this codes a 1961 amino acid protein that shows a high degree of homology with other Fksps. Expression analysis of YlFKS1 under different conditions affecting the cell wall did not reveal significant differences. Finally, attempts to obtain a Y. lipolytica strain containing a disrupted YlFKS1 allele failed, despite having used two different techniques. Taken together, these results suggest that, unlike S. cerevisiae, YlFKS1 is the only FKS1 homologue in Y. lipolytica and is essential for growth.