First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, ß-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

Genetic Diversity of Lecanosticta acicola in Pinus Ecosystems in Northern Spain.

Lecanosticta acicola is one of the most damaging species affecting Pinus radiata plantations in Spain. Favourable climatic conditions and unknown endogenous factors of the pathogen and host led to a situation of high incidence and severity of the disease in these ecosystems. With the main aim of understanding the factors intrinsic to this pathogenic species, a study of the population structure in new established plantations with respect to older plantations was implemented. The genetic diversity, population structure and the ability of the pathogen to spread was determined in Northern Spain (Basque Country), where two thirds of the total Pinus radiata plantations of Spain are located. From a total of 153 Lecanosticta acicola isolates analysed, two lineages were present; the southern lineage, which was prevalent, and the northern lineage, which was scarce. A total of 22 multilocus genotypes were detected with a balanced composition of both mating types and evidence for sexual reproduction. In addition to the changing environmental conditions enhancing disease expression, the complexity and diversity of the pathogen will make it difficult to control and to maintain the wood productive system fundamentally based on this forest species.

Temporal and Spatial Variation in the Population Structure of Spanish Fusarium circinatum Infecting Pine Stands.

Fusarium circinatum is an introduced fungal pathogen extended to the northern regions of Spain that causes Pine Pitch Canker (PPC) disease. In this work, we analyzed the pathogen's genetic diversity to study changes over time and space since the first outbreak occurred in Spain. Using six polymorphic SSR markers, 15 MLGs were identified in 66 isolates, and only three haplotypes were found with frequencies higher than one. In general, genotypic diversity was low and decreased shortly over time in the northwestern regions while maintained at País Vasco, where only one haplotype (MLG32) was detected 10 years. This population also included isolates of a single mating type (MAT-2) and VCGs identified in only two groups, while isolates from NW regions were of both mating types and VCGs represented in 11 groups. The existence of haplotype MLG32 maintained on time and widely distributed suggests its good adaptation to the environment and the host. Results showed that the pathogen in País Vasco remains clearly differentiated from other northwestern populations. This fact was supported with no evidence of migration among regions. Results are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to identification of two new haplotypes.

Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems.

Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9-SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.

Relationship Between the Xylem Anatomy of Grapevine Rootstocks and Their Susceptibility to Phaeoacremonium minimum and Phaeomoniella chlamydospora.

Fungal grapevine trunk diseases (GTDs) are some of the most pressing threats to grape production worldwide. While these diseases are associated with several fungal pathogens, Phaeomoniella chlamydospora and Phaeoacremonium minimum are important contributors to esca and Petri diseases. Recent research has linked grapevine xylem diameter with tolerance to Pa. chlamydospora in commercial rootstocks. In this study, we screen over 25 rootstocks for xylem characteristics and tolerance to both Pa. chlamydospora and Pm. minimum. Tolerance was measured by fungal incidence and DNA concentration (quantified via qPCR), while histological analyses were used to measure xylem characteristics, including xylem vessels diameter, density, and the proportion of the stem surface area covered by xylem vessels. Rootstocks were grouped into different classes based on xylem characteristics to assess the potential association between vasculature traits and pathogen tolerance. Our results revealed significant differences in all the analyzed xylem traits, and also in DNA concentration for both pathogens among the tested rootstocks. They corroborate the link between xylem vessels diameter and tolerance to Pa. chlamydospora. In Pm. minimum, the rootstocks with the widest xylem diameter proved the most susceptible. This relationship between vasculature development and pathogen tolerance has the potential to inform both cultivar choice and future rootstock breeding to reduce the detrimental impact of GTDs worldwide.

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.

Influence of Temperature and Moisture Duration on Pathogenic Life History Traits of Predominant Haplotypes of Fusarium circinatum on Pinus spp. in Spain.

Pathogen life history traits influence epidemic development and pathogen adaptive ability to interact with their hosts in different environments. Reduced traits variation may compromise pathogen evolutionary potential, which is particularly important for introduced pathogens. Fusarium circinatum (cause of pine pitch canker) is an invasive fungal pathogen in Europe, with current distribution restricted to forest stands of Pinus radiata and Pinus pinaster in northern Spain and Portugal. This study aimed to quantify pathogenic traits of Spanish isolates of F. circinatum, with two of the strains representing the two dominant haplotypes in the Spanish population. Disease severity was measured on P. radiata, analyzing the influence of temperature and moisture duration on infection as well as the influence of temperature on spore germination, sporulation, and mycelial growth. Results indicated that the isolate representing the most common haplotype caused more severe disease on P. radiata at 25 and 30°C compared with the second most common haplotype but caused less severe disease at 15°C. Spore germination was higher for the most common haplotype, which produced more spores at 20 and 25°C. The isolate showed hyphal melanization at 5°C, which has been associated with survival and may be important because no resting structures have been described for F. circinatum. Our study determined that longer moisture periods during infection result in more severe disease from 7 to 24 h, regardless of the isolate virulence. This is the first study on virulence of the most abundant haplotypes of F. circinatum in Spain as affected by temperatures and moisture.

Herbicidal Activity of Thymbra capitata (L.) Cav. Essential Oil.

The bioherbicidal potential of Thymbra capitata (L.) Cav. essential oil (EO) and its main compound carvacrol was investigated. In in vitro assays, the EO blocked the germination and seedling growth of Erigeron canadensis L., Sonchus oleraceus (L.) L., and Chenopodium album L. at 0.125 µL/mL, of Setaria verticillata (L.) P.Beauv., Avena fatua L., and Solanum nigrum L. at 0.5 µL/mL, of Amaranthus retroflexus L. at 1 µL/mL and of Portulaca oleracea L., and Echinochloa crus-galli (L.) P.Beauv. at 2 µL/mL. Under greenhouse conditions, T. capitata EO was tested towards the emergent weeds from a soil seedbank in pre and post emergence, showing strong herbicidal potential in both assays at 4 µL/mL. In addition, T. capitata EO, applied by spraying, was tested against P. oleracea, A. fatua and E. crus-galli. The species showed different sensibility to the EO, being E. crus-galli the most resistant. Experiments were performed against A. fatua testing T. capitata EO and carvacrol applied by spraying or by irrigation. It was verified that the EO was more active at the same doses in monocotyledons applied by irrigation and in dicotyledons applied by spraying. Carvacrol effects on Arabidopsis root morphology were also studied.

Temporal Dispersal Patterns of Phaeomoniella chlamydospora, Causal Agent of Petri Disease and Esca, in Vineyards.

Although the fungus Phaeomoniella chlamydospora is the most commonly detected causal agent of Petri disease and esca, two important fungal grapevine trunk diseases, little is known about the dispersal patterns of P. chlamydospora inoculum. In this work, we studied the dispersal of P. chlamydospora airborne inoculum from 2016 to 2018 in two viticultural areas of eastern (Ontinyent) and northern (Logroño) Spain. The vineyards were monitored weekly from November to April using microscope slide traps, and P. chlamydospora was detected and quantified by a specific real-time quantitative (qPCR) method set up in this work. The method was found to be sensitive, and a good correlation was observed between numbers of P. chlamydospora conidia (counted by microscope) and DNA copy numbers (quantified by qPCR). We consistently detected DNA of P. chlamydospora at both locations and in all seasons but in different quantities. In most cases, DNA was first detected in the last half of November, and most of the DNA was detected from December to early April. When rain was used as a predictor of P. chlamydospora DNA detection in traps, false-negative detections were observed, but these involved only 4% of the total. The dispersal pattern of P. chlamydospora DNA over time was best described (R2 = 0.765 and concordance correlation coefficient = 0.870) by a Gompertz equation, with time expressed as hydrothermal time (a physiological time accounting for the effects of temperature and rain). This equation could be used to predict periods with a high risk of dispersal of P. chlamydospora.

Droplet Digital PCR Technology for Detection of Ilyonectria liriodendri from Grapevine Environmental Samples.

Black-foot disease is one of the most important soilborne diseases affecting planting material in grapevine nurseries and young vineyards. Accurate, early, and specific detection and quantification of black-foot disease causing fungi are essential to alert growers and nurseries to the presence of the pathogens in soil, and to prevent the spread of these pathogens through grapevines using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, efficiency, and specificity of droplet digital PCR (ddPCR) and real-time PCR (qPCR) techniques for the detection and quantification of Ilyonectria liriodendri in bulk and rhizosphere soils, as well as grapevine endorhizosphere. Fungal abundance was not affected by soil-plant fractions. Both techniques showed a high degree of correlation across the samples assessed (R2 = 0.95) with ddPCR being more sensitive to lower target concentrations. Roots of asymptomatic vines were found to be a microbial niche that is inhabited by black-foot disease fungi.

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.

The Fungal and Bacterial Rhizosphere Microbiome Associated With Grapevine Rootstock Genotypes in Mature and Young Vineyards.

The microbiota colonizing the rhizosphere and the endorhizosphere contribute to plant growth, productivity, carbon sequestration, and phytoremediation. Several studies suggested that different plants types and even genotypes of the same plant species harbor partially different microbiomes. Here, we characterize the rhizosphere bacterial and fungal microbiota across five grapevine rootstock genotypes cultivated in the same soil at two vineyards and sampling dates over 2 years by 16S rRNA gene and ITS high-throughput amplicon sequencing. In addition, we use quantitative PCR (qPCR) approach to measure the relative abundance and dynamic changes of fungal pathogens associated with black-foot disease. The objectives were to (1) unravel the effects of rootstock genotype on microbial communities in the rhizosphere of grapevine and (2) to compare the relative abundances of sequence reads and DNA amount of black-foot disease pathogens. Host genetic control of the microbiome was evident in the rhizosphere of the mature vineyard. Microbiome composition also shifted as year of sampling, and fungal diversity varied with sampling moments. Linear discriminant analysis identified specific bacterial (i.e., Bacillus) and fungal (i.e., Glomus) taxa associated with grapevine rootstocks. Host genotype did not predict any summary metrics of rhizosphere α- and ß-diversity in the young vineyard. Regarding black-foot associated pathogens, a significant correlation between sequencing reads and qPCR was observed. In conclusion, grapevine rootstock genotypes in the mature vineyard were associated with different rhizosphere microbiomes. The latter could also have been affected by age of the vineyard, soil properties or field management practices. A more comprehensive study is needed to decipher the cause of the rootstock microbiome selection and the mechanisms by which grapevines are able to shape their associated microbial community. Understanding the vast diversity of bacteria and fungi in the rhizosphere and the interactions between microbiota and grapevine will facilitate the development of future strategies for grapevine protection.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Characterization of Mycosphaerellaceae species associated with citrus greasy spot in Panama and Spain.

Greasy spot of citrus, caused by Zasmidium citri-griseum (= Mycosphaerella citri), is widely distributed in the Caribbean Basin, inducing leaf spots, premature defoliation, and yield loss. Greasy spot-like symptoms were frequently observed in humid citrus-growing regions in Panama as well as in semi-arid areas in Spain, but disease aetiology was unknown. Citrus-growing areas in Panama and Spain were surveyed and isolates of Mycosphaerellaceae were obtained from citrus greasy spot lesions. A selection of isolates from Panama (n = 22) and Spain (n = 16) was assembled based on their geographical origin, citrus species, and affected tissue. The isolates were characterized based on multi-locus DNA (ITS and EF-1α) sequence analyses, morphology, growth at different temperatures, and independent pathogenicity tests on the citrus species most affected in each country. Reference isolates and sequences were also included in the analysis. Isolates from Panama were identified as Z. citri-griseum complex, and others from Spain attributed to Amycosphaerella africana. Isolates of the Z. citri-griseum complex had a significantly higher optimal growth temperature (26.8°C) than those of A. africana (19.3°C), which corresponded well with their actual biogeographical range. The isolates of the Z. citri-griseum complex from Panama induced typical greasy spot symptoms in 'Valencia' sweet orange plants and the inoculated fungi were reisolated. No symptoms were observed in plants of the 'Ortanique' tangor inoculated with A. africana. These results demonstrate the presence of citrus greasy spot, caused by Z. citri-griseum complex, in Panama whereas A. africana was associated with greasy spot-like symptoms in Spain.

Complex molecular relationship between vegetative compatibility groups (VCGs) in Verticillium dahliae: VCGs do not always align with clonal lineages.

Verticillium wilts caused by the soilborne fungus Verticillium dahliae are among the most challenging diseases to control. Populations of this pathogen have been traditionally studied by means of vegetative compatibility groups (VCGs) under the assumption that VCGs comprise genetically related isolates that correlate with clonal lineages. We aimed to resolve the phylogenetic relationships among VCGs and their subgroups based on sequences of the intergenic spacer region (IGS) of the ribosomal DNA and six anonymous polymorphic sequences containing single-nucleotide polymorphisms (VdSNPs). A collection of 68 V. dahliae isolates representing the main VCGs and subgroups (VCGs 1A, 1B, 2A, 2B, 3, 4A, 4B, and 6) from different geographic origins and hosts was analyzed using the seven DNA regions. Maximum parsimony (MP) phylogenies inferred from IGS and VdSNP sequences showed five and six distinct clades, respectively. Phylogenetic analyses of individual and combined data sets indicated that certain VCG subgroups (e.g., VCGs 1A and 1B) are closely related and share a common ancestor; however, other subgroups (e.g., VCG 4B) are more closely related to members of a different VCG (e.g., VCG 2A) than to subgroups of the same VCG (VCG 4B). Furthermore, MP analyses indicated that VCG 2B is polyphyletic, with isolates placed in at least three distinct phylogenetic lineages based on IGS sequences and two lineages based on VdSNP sequences. Results from our study suggest the existence of main VCG lineages that contain VCGs 1A and 1B; VCGs 2A and 4B; and VCG 4A, for which both phylogenies agree; and the existence of other VCGs or VCG subgroups that seem to be genetically heterogeneous or show discrepancies in their phylogenetic placement: VCG 2B, VCG 3, and VCG 6. These results raise important caveats regarding the interpretation of VCG analyses: genetic homogeneity and close evolutionary relationship between members of a VCG should not be assumed.

Verticillium Wilt: A Threat to Artichoke Production.

Verticillium wilt is becoming an increasing concern in artichoke production because the rapid spread of the disease to new growing areas has led to declining production. Scientists from Italy, Spain, and the United States combine to bring us up to date on diagnosis of the disease, its epidemiology and life cycle, as well as management strategies, current and forthcoming.

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.