(4Z)-Lachnophyllum Lactone, a Metabolite with Phytotoxic and Antifungal Activity against Pests Affecting Mediterranean Agriculture: A New Versatile and Easy Scalable Parallel Synthesis.

A methodology for the total and modulable synthesis of (4Z)-lachnophyllum lactone (1), on a gram scale, is reported for the first time. The present work started with the design of a retrosynthetic pathway for the target compound, with the key step identified in Pd-Cu bimetallic cascade cross-coupling cyclization. (4Z)-Lachnophyllum lactone (1) is an acetylenic furanone previously isolated, in a low amount, from the organic extract of the autotrophic weedConyza bonariensis. Tested against the stem parasitic weed Cuscuta campestris in a seedling growth bioassay, (4Z)-lachnophyllum lactone (1) showed almost 85% of inhibitory activity up to 0.3 mM in comparison with the control. At the same concentration, the compound displayed radicle growth inhibitory activity of the root parasitic weeds Orobanche minor and Phelipanche ramosa higher than 70 and 40%, respectively. Surprisingly, the compound showed a high percentage of inhibition, up to 0.1 mM, on C. bonariensis seed germination too. This versatile synthetic strategy was also used to obtain two further natural analogues, namely, (4E)-lachnophyllum lactone (8) and (4Z,8Z)-matricaria lactone (9), that showed, in most cases, the same inhibitory trend with slight differences, highlighting the importance of the stereochemistry and unsaturation of the side chain. Furthermore, all of the compounds showed antifungal activity at 1 mM reducing the mycelial growth of the olive pathogen Verticillium dahliae. The design and implementation of scalable and modulable total synthesis on a gram scale of acetylenic furanones allow the production of a large amount of these natural products, overcoming the limit imposed by isolation from natural sources. The results of the present study pave the way for the development of ecofriendly bioinspired pesticides with potential application in agrochemical practices as alternative to synthetic pesticides.

Correction: Velasco-Amo et al. Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain. Plants 2022, 11, 1562.

In the original publication [...].

Xylella fastidiosa Infection Reshapes Microbial Composition and Network Associations in the Xylem of Almond Trees.

Xylella fastidiosa represents a major threat to important crops worldwide including almond, citrus, grapevine, and olives. Nowadays, there are no efficient control measures for X. fastidiosa, and the use of preventive measures and host resistance represent the most practical disease management strategies. Research on vessel-associated microorganisms is gaining special interest as an innate natural defense of plants to cope against infection by xylem-inhabiting pathogens. The objective of this research has been to characterize, by next-generation sequencing (NGS) analysis, the microbial communities residing in the xylem sap of almond trees affected by almond leaf scorch disease (ALSD) in a recent X. fastidiosa outbreak occurring in Alicante province, Spain. We also determined community composition changes and network associations occurring between xylem-inhabiting microbial communities and X. fastidiosa. For that, a total of 91 trees with or without ALSD symptoms were selected from a total of eight representative orchards located in five municipalities within the X. fastidiosa-demarcated area. X. fastidiosa infection in each tree was verified by quantitative polymerase chain reaction (qPCR) analysis, with 54% of the trees being tested X. fastidiosa-positive. Globally, Xylella (27.4%), Sphingomonas (13.9%), and Hymenobacter (12.7%) were the most abundant bacterial genera, whereas Diplodia (30.18%), a member of the family Didymellaceae (10.7%), and Aureobasidium (9.9%) were the most predominant fungal taxa. Furthermore, principal coordinate analysis (PCoA) of Bray-Curtis and weighted UniFrac distances differentiated almond xylem bacterial communities mainly according to X. fastidiosa infection, in contrast to fungal community structure that was not closely related to the presence of the pathogen. Similar results were obtained when X. fastidiosa reads were removed from the bacterial data set although the effect was less pronounced. Co-occurrence network analysis revealed negative associations among four amplicon sequence variants (ASVs) assigned to X. fastidiosa with different bacterial ASVs belonging to 1174-901-12, Abditibacterium, Sphingomonas, Methylobacterium-Methylorubrum, Modestobacter, Xylophilus, and a non-identified member of the family Solirubrobacteraceae. Determination of the close-fitting associations between xylem-inhabiting microorganisms and X. fastidiosa may help to reveal specific microbial players associated with the suppression of ALSD under high X. fastidiosa inoculum pressure. These identified microorganisms would be good candidates to be tested in planta, to produce almond plants more resilient to X. fastidiosa infection when inoculated by endotherapy, contributing to suppress ALSD.

Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain.

Xylella fastidiosa (Xf) is a phytopathogenic bacterium with a repertoire of self-replicating genetic elements, including plasmids, pathogenicity islands, and prophages. These elements provide potential avenues for horizontal gene transfer both within and between species and have the ability to confer new virulence traits, including the ability to colonize new host plants. However, they can also serve as a 'footprint' to type plasmid-bearing strains. Genome sequencing of several strains of Xf subsp. fastidiosa sequence type (ST) 1 from Mallorca Island, Spain, revealed the presence of a 38 kb plasmid (pXFAS_5235). In this study, we developed a PCR-based typing approach using primers targeting the traC gene to determine the presence of pXFAS_5235 plasmid or other plasmids carrying this gene in a world-wide collection of 65 strains X. fastidiosa from different subspecies and STs or in 226 plant samples naturally infected by the bacterium obtained from the different outbreaks of Xf in Spain. The traC gene was amplified only in the plant samples obtained from Mallorca Island infected by Xf subsp. fastidiosa ST1 and from all Spanish strains belonging to this ST. Maximum-likelihood phylogenetic tree of traC revealed a close relatedness among Spanish and Californian strains carrying similar plasmids. Our results confirm previous studies, which suggested that a single introduction event of Xf subsp. fastidiosa ST1 occurred in the Balearic Islands. Further studies on the presence and role of plasmids in Xf strains belonging to the same or different subspecies and STs can provide important information in studies of epidemiology, ecology, and evolution of this plant pathogen.

Verticillium dahliae Inoculation and in vitro Propagation Modify the Xylem Microbiome and Disease Reaction to Verticillium Wilt in a Wild Olive Genotype.

Host resistance is the most practical, long-term, and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant's microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remains unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless of whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless of infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are the first to show a breakdown of resistance to Vd in a wild olive that potentially may be related to a modification of its xylem microbiome and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance, which can be of use to fight against main vascular diseases of olive.

Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods.

Microbial endophytes are well known to protect host plants against pathogens, thus representing a promising strategy for the control of xylem-colonizing pathogens. To date, the vast majority of microbial communities inhabiting the olive xylem are unknown; therefore, this work pursues the characterization of the xylem-limited microbiome and determines whether the culture isolation medium, olive genotype, and the plant material used to analyze it can have an effect on the bacterial populations retrieved. Macerated xylem tissue and xylem sap extracted with the Scholander chamber from olive branches obtained from two cultivated and a wild olive genotypes were analyzed using culture-dependent and -independent approaches. In the culture-dependent approach using four solid culture media, a total of 261 bacterial isolates were identified after performing Sanger sequencing of 16S rRNA. Culturable bacteria clustered into 34 genera, with some effect of culture media for bacterial isolation. The cultivated bacteria belonged to four phyla and the most abundant genera included Frigoribacterium (18.8%), Methylobacterium (16.4%), and Sphingomonas (14.6%). On the other hand, in the culture-independent approach conducted using Illumina MiSeq 16S rRNA amplicon sequencing [next-generation sequencing (NGS)] of the xylem extracts, we identified a total of 48 operational taxonomic units (OTUs) belonging to five phyla, being Sphingomonas (30.1%), Hymenobacter (24.1%) and Methylobacterium (22.4%) the most representative genera (>76% of reads). In addition, the results indicated significant differences in the bacterial communities detected in the xylem sap depending on the genotype of the olive tree studied and, to a minor extent, on the type of sap extraction method used. Among the total genera identified using NGS, 14 (41.2%) were recovered in the culture collection, whereas 20 (58.8%) in the culture collection were not captured by the NGS approach. Some of the xylem-inhabiting bacteria isolated are known biocontrol agents of plant pathogens, whereas for others little information is known and are first reported for olive. Consequently, the potential role of these bacteria in conferring olive tree protection against xylem pathogens should be explored in future research.

Clonal Expansion and Migration of a Highly Virulent, Defoliating Lineage of Verticillium dahliae.

We used a population genomics approach to test the hypothesis of clonal expansion of a highly fit genotype in populations of Verticillium dahliae. This fungal pathogen has a broad host range and can be dispersed in contaminated seed or other plant material. It has a highly clonal population structure, with several lineages having nearly worldwide distributions in agricultural crops. Isolates in lineage 1A are highly virulent and cause defoliation in cotton, okra, and olive (denoted 1A/D), whereas those in other lineages cause wilting but not defoliation (ND). We tested whether the highly virulent lineage 1A/D could have spread from the southwestern United States to the Mediterranean basin, as predicted from historical records. We found 187 single-nucleotide polymorphisms (SNPs), determined by genotyping by sequencing, among 91 isolates of lineage 1A/D and 5 isolates in the closely related lineage 1B/ND. Neighbor-joining and maximum-likelihood analyses on the 187 SNPs showed a clear divergence between 1A/D and 1B/ND haplotypes. Data for only 77 SNPs were obtained for all 96 isolates (no missing data); lineages 1A/D and 1B/ND differed by 27 of these 77 SNPs, confirming a clear divergence between the two lineages. No evidence of recombination was detected within or between these two lineages. Phylogenetic and genealogical analyses resulted in five distinct subclades of 1A/D isolates that correlated closely with geographic origins in the Mediterranean basin, consistent with the hypothesis that the D pathotype was introduced at least five times in independent founder events into this region from a relatively diverse source population. The inferred ancestral haplotype was found in two isolates sampled before 1983 from the southwestern United States, which is consistent with historical records that 1A/D originated in North America. The five subclades coalesce with the ancestral haplotype at the same time, consistent with a hypothesis of rapid population expansion in the source population during the emergence of 1A/D as a severe pathogen of cotton in the United States.

Symptomless Host and Nonhost Responses of Paulownia (Paulownia spp.) to Olive-Defoliating Verticillium dahliae.

Symptomless host and nonhost responses of Paulownia spp. to olive-defoliating (D) Verticillium dahliae is reported for the first time. Two paulownia clones, Paulownia elongata 'PC-2' and P. elongata × P. fortunei 'PC-3', were inoculated with a V. dahliae isolate representative of the D pathotype by either root dip or stem injection with a conidial suspension, repeated transplanting to a V. dahliae-infested soil mixture, or root dip in the conidial suspension followed by transplanting to the infested soil mixture. 'Picual' olive and 'Sugar Baby' watermelon were included in all experiments as susceptible standards to show that the inoculation procedures and incubation conditions were successful. Plants were incubated under conditions optimal for Verticillium wilt that caused severe disease in 'Picual' olive and 'Sugar Baby' watermelon in the growth chamber, shade house, and field microplots for 30 to 57 weeks in three independent experiments. No foliar symptoms developed on paulownia, whose stems were found free of V. dahliae both by isolation on semiselective NP-10 medium as well as by a nested-polymerase chain reaction assay using total genomic DNA from inoculated plants that effectively detected D V. dahliae in olive stems. V. dahliae was isolated to a limited extent from roots of PC-3 paulownia plants after 30 weeks of growth in the infested soil mixture but not from those that were root-dip inoculated or from PC-2 plants regardless the method of inoculation. The symptomless host and nonhost responses of Paulownia spp. to D V. dahliae may have practical applications in the use of fertile soils in southern Spain, particularly in those that are highly infested with the highly virulent D pathotype, as well as a replacement crop for Verticillium wilt-affected olive orchards in that region.

Recombination between clonal lineages of the asexual fungus Verticillium dahliae detected by genotyping by sequencing.

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.

Region-wide analysis of genetic diversity in Verticillium dahliae populations infecting olive in southern Spain and agricultural factors influencing the distribution and prevalence of vegetative compatibility groups and pathotypes.

Severity of Verticillium wilt in olive trees in Andalusia, southern Spain is associated with the spread of a highly virulent, defoliating (D) Verticillium dahliae pathotype of vegetative compatibility group 1A (VCG1A) but the extent of this spread and the diversity of the pathogen population have never been documented. VCG typing of 637 V. dahliae isolates from 433 trees in 65 orchards from five olive-growing provinces in Andalusia indicated that 78.1% were of VCG1A, 19.8% of VCG2A, 0.6% of VCG2B, 1.4% of VCG4B, and one isolate was heterokaryon self-incompatible. A single VCG prevailed among isolates within most orchards but two and three VCGs were identified in 12 and 3 orchards, respectively, with VCG1A+VCG2A occurring in 10 orchards. VCG1A was the predominant VCG in the three most important olive-growing provinces, and was almost as prevalent as VCG2A in another one. Molecular pathotyping of the 637 isolates using specific polymerase chain reaction assays indicated that VCG1A isolates were of the D pathotype whereas isolates of VCG2A, -2B, and -4B were of the less virulent nondefoliating (ND) pathotype. The pathotype of isolates correlated with the disease syndrome affecting sampled trees. Only three (seq1, seq2, and seq4) of the seven known sequences of the V. dahliae-specific 539- or 523-bp amplicon were identified among the 637 isolates. Distribution and prevalence of VCGs and seq sequences among orchards indicated that genetic diversity within olive V. dahliae in Andalusia is higher in provinces where VCG1A is not prevalent. Log-linear analysis revealed that irrigation management, source of irrigation water, source of planting stock, and cropping history of soil were significantly associated with the prevalence of VCG1A compared with that of VCG2A. Multivariate analyses using a selected set of agricultural factors as variables allowed development of a discriminant model for predicting the occurrence of D and ND pathotypes in the area of the study. Blind tests using this model correctly indentified the V. dahliae pathotype occurring in an orchard. The widespread occurrence and high prevalence of VCG1A/D pathotype in Andalusia have strong implications for the management of the disease.

Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers.

ABSTRACT A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study.

Genetic and Virulence Diversity in Verticillium dahliae Populations Infecting Artichoke in Eastern-Central Spain.

ABSTRACT Severe Verticillium dahliae attacks have occurred in artichoke crops in the Comunidad Valenciana region of eastern-central Spain since the late 1990s. Knowledge of genetic and virulence diversity in the pathogen population is a key factor for the management of the disease through disease risk assessment as well as development and use of resistant cultivars. V. dahliae isolates from artichoke (109 isolates) and cotton (three isolates) in that region were characterized by vegetative compatibility grouping (VCG), and specific polymerase chain reaction assays using three sets of primer pairs that differentiate the cotton-defoliating (D) and -nondefoliating (ND) V. dahliae pathotypes. In all, 35 and 39 V. dahliae isolates representative of the identified VCGs and geographic origins were tested for virulence to artichoke cvs. Nun 6374 and Nun 9444, and cotton cv. Acala SJ-2, respectively. Four VCGs were identified among 107 artichoke isolates, and 2 isolates were heterokaryon self-incompatible: VCG1A (one isolate), VCG2A (31 isolates), VCG2B (72 isolates), and VCG4B (three isolates). The three cotton isolates were VCG1A. Isolates in VCG2B were distributed across the region and were the most prevalent isolates in the northern part. Conversely, 83.9% of isolates in VCG2A were recovered from the southern part of the region. Two subgroups of isolates were identified in VCG2B based on heterokaryon compatibility with either international or local tester isolates, which further showed diversity in the amplification of 334- and 824-bp DNA fragments which are markers of the D and ND pathotypes, respectively. Virulence of isolates to artichoke and cotton correlated with VCG but the pattern of correlation varied with the host. VCG1A isolates from artichoke and cotton induced defoliation in cotton but not in artichoke. Collectively, isolates of VCG2B and VCG4B were the most virulent and isolates of VCG1A or HSI were the least virulent to artichoke; but isolates of VCG1A were more virulent to cotton than those of any other VCG. Also, molecular subgrouping in VCG2B determined by amplification of the 334- and 824-bp markers correlated with virulence of isolates to the two hosts tested.