Biocontrol of almond canker diseases caused by Botryosphaeriaceae fungi.

BACKGROUND: Botryosphaeria dieback is a canker disease caused by fungal species of the Botryosphaeriaceae family that threatens almond productivity. The most common control measure to prevent canker development is the application of fungicides which are being phased out by European Union regulations. In the present study, two sets of bacterial strains were evaluated for their antifungal activity against pathogenic Botryosphaeriaceae species through in vitro and in vivo antagonism assays. RESULTS: The rhizospheric bacteria Pseudomonas aeruginosa AC17 and Bacillus velezensis ACH16, as well as the endophytic bacteria Bacillus mobilis Sol 1-2, respectively inhibited 87, 95, and 63% of the mycelial growth of Neofusicoccum parvum, Botryosphaeria dothidea, Diplodia seriata, and Macrophomina phaseolina. Additionally, they significantly reduced the length of lesions caused by N. parvum and B. dothidea in artificially inoculated detached almond twigs. All these bacterial strains produce hydrolytic enzymes that are able to degrade the fungal cell wall. P. aeruginosa AC17 also produces toxic volatile compounds, such as hydrogen cyanide. This strain was the most effective in controlling Botryosphaeria dieback in planta under controlled conditions at a level similar to the biocontrol agent Trichoderma atroviride and standard chemical fungicide treatments. CONCLUSION: Pseudomonas aeruginosa AC17 is the best candidate to be considered as a potential biocontrol agent against Botryosphaeriaceae fungi affecting almond. © 2023 Society of Chemical Industry.

Duplex Real-Time PCR Assays for the Simultaneous Detection and Quantification of Botryosphaeriaceae Species Causing Canker Diseases in Woody Crops.

Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.

Soil Microbial Community Responses to Different Management Strategies in Almond Crop.

A comparative study of organic and conventional farming systems was conducted in almond orchards to determine the effect of management practices on their fungal and bacterial communities. Soils from two orchards under organic (OM) and conventional (CM), and nearby nonmanaged (NM) soil were analyzed and compared. Several biochemical and biological parameters were measured (soil pH, electrical conductivity, total nitrogen, organic material, total phosphorous, total DNA, and fungal and bacterial DNA copies). Massive parallel sequencing of regions from fungal ITS rRNA and bacterial 16 S genes was carried out to characterize their diversity in the soil. We report a larger abundance of bacteria and fungi in soils under OM, with a more balanced fungi:bacteria ratio, compared to bacteria-skewed proportions under CM and NM. The fungal phylum Ascomycota corresponded to around the 75% relative abundance in the soil, whereas for bacteria, the phyla Proteobacteria, Acidobacteriota and Bacteroidota integrated around 50% of their diversity. Alpha diversity was similar across practices, but beta diversity was highly clustered by soil management. Linear discriminant analysis effect size (LEfSE) identified bacterial and fungal taxa associated with each type of soil management. Analyses of fungal functional guilds revealed 3-4 times larger abundance of pathogenic fungi under CM compared to OM and NM treatments. Among them, the genus Cylindrocarpon was more abundant under CM, and Fusarium under OM.

Avoidant/resistant rather than tolerant olive rootstocks are more effective in controlling Verticillium wilt.

The identification of rootstocks of low susceptibility to Verticillium dahliae can become a valuable procedure to achieve effective control of Verticillium wilt in the olive grove. This not only involves the identification of suitable genotypes, but also the study of the interaction between the rootstock and the grafted scion. Thus, a rootstock that prevents or minimizes V. dahliae proliferation (avoidance/resistance strategy) can have very different effects on a susceptible scion compared to a rootstock that shows few or no symptoms despite being infected (tolerance strategy). Both resistance and tolerance mechanisms have been recently identified in wild olive genotypes with low susceptibility to V. dahliae. When used as rootstocks of the highly susceptible variety 'Picual', we found that resistant genotypes, including the cultivar 'Frantoio', were more effective than tolerant genotypes in controlling Verticillium wilt. Furthermore, tolerant genotypes were as ineffective as susceptible or extremely susceptible genotypes in controlling Verticillium wilt. We also identified rootstock-scion combinations with behaviours that were not expected according to the degree of susceptibility previously observed in the non-grafted rootstock. Although the rootstocks were able to control Verticillium wilt according to its degree of susceptibility to V. dahliae, the ability to control the infection was not adequately transferred to the grafted scion. Our results confirmed that: the degree of susceptibility to Verticillium wilt of an olive variety does not predict its performance as a rootstock; to use a very low susceptible genotype as rootstock of a susceptible scion increases the susceptibility of the genotype used as rootstock; in any case, avoidant/resistant rootstocks are more effective than tolerant rootstocks in reducing the susceptibility of the grafted plant to V. dahliae.

Molecular and Pathogenic Characterization of Cylindrocarpon-like Anamorphs Causing Root and Basal Rot of Almonds.

Three almond nurseries were prospected in the South of Spain (Sevilla) to evaluate the sanitary status of the nursery plant material. Samples consisted of main roots, secondary roots and six-month-old basal stems 'GxN-15', 'Nemaguard', 'Cadaman', 'Rootpac-40' and 'Rootpac-20' rootstocks planted in the soil, and twigs of mother plants from 'Lauranne', 'Guara', 'Marcona', 'Marta' and 'Ferragnes' almond cultivars. Endophytic and potential pathogenic fungi were identified in mother plants and 70 Cylindrocarpon-like anamorph isolates were detected in the root system and basal stems of analyzed rootstocks. Based on partial sequencing of the his3 gene and multilocus phylogenetic analysis of the concatenated ITS, tub2, his3 and tef1-α partial sequences, seven Cylindrocarpon-like anamorph species were identified as Dactylonectria torresensis, D. novozelandica, D. macrodidyma, Ilyonectria liriodendri, Neonectria sp. 1, N. quercicola and Cylindrocladiella variabilis. Pathogenicity was assessed on young healthy detached twigs of 'Guara' almond cultivar and one-year-old 'Lauranne' potted almonds grafted onto 'GxN-15' rootstocks. Among the seven Cylindrocarpon-like anamorph species, I. liriodendri, Neonectria sp. 1 and N. quercicola were the most aggressive. Inoculated detached shoots developed necrotic lesions 15 days after inoculation. Inoculated trees showed sectorized necrosis in the main and secondary roots and the basal stem of the rootstock 5 months after inoculation. The most aggressive species were able to cause necrosis also in the grafted cultivar, and I. liriodendri, and N. quercicola also reduced the root biomass. This is the first report of Cylindrocarpon-like anamorph species causing root and basal rot of almonds.

Wild Olive Genotypes as a Valuable Source of Resistance to Defoliating Verticillium dahliae.

Resistance to the defoliating pathotype of Verticillium dahliae has been evaluated in a pool of 68 wild genotypes of olive belonging to the SILVOLIVE collection. Resistance was evaluated by assessing symptom severity using a 0-4 rating scale, estimating the relative area under the disease progress curve (RAUDPC), determining the percentage of dead plants (PDP), and measuring the evolution of morphological parameters in inoculated plants over time. In addition, the density levels of V. dahliae in the stem of root-inoculated genotypes have been quantified by means of quantitative real-time PCR at 35 and 120 days after inoculation (dai). Fifteen genotypes (22%) were cataloged as resistant to V. dahliae (i.e., disease parameters did not significantly differ from those of the resistant cultivar Frantoio, or were even lower). Resistant genotypes are characterized by presenting fewer symptoms and a lower amount of V. dahliae DNA at 120 dai than at 35 dai, indicating their ability to control the disease and reduce the density of the pathogen. The rest of the evaluated genotypes showed variable levels of susceptibility. Overall analysis of all genotypes showed high correlation between symptomatology and the amount of V. dahliae DNA in the stem of inoculated genotypes at 120 dai, rather than at 35 dai. However, correlation at 120 dai was not observed in the set of resistant genotypes, suggesting that resistance to defoliating V. dahliae in olive is based on the occurrence of different mechanisms such as avoidance or tolerance. These mechanisms are valuable for designing breeding programs and for the identification of target genes and resistant rootstocks to better control Verticillium wilt in the olive grove.

Potential inoculum sources of Fusarium species involved in asparagus decline syndrome and evaluation of soil disinfestation methods by qPCR protocols.

BACKGROUND: Asparagus decline syndrome (ADS), one of the most important diseases affecting asparagus crops, causes important yield losses worldwide. Fusarium proliferatum, F. oxysporum and F. redolens are among the main species associated with ADS. To explore their potential inoculum sources and the effectiveness of soil disinfestation practices for ADS management, molecular methods based on a quantitative real-time polymerase chain reaction (qPCR) were developed. qPCR-based molecular tools demonstrated advantages in the sensitive and specific detection and quantification of fungal pathogens in comparison with less-accurate and time-consuming traditional culture methods. RESULTS: F. proliferatum, F. oxysporum and F. redolens could be specifically detected and accurately quantified in asparagus plants, soil and irrigation water collected from asparagus fields with ADS symptoms by means of the designed TaqMan qPCR protocols. Furthermore, these molecular tools were successfully applied for evaluation of the efficacy of diverse soil disinfestation treatments. Chemical fumigation with dazomet and biosolarization with pellets of Brassica carinata contributed to a significant reduction in the inoculum densities of the three Fusarium species in treated soils, which was correlated with production increases. CONCLUSIONS: The capability to accurately detect and quantify the main Fusarium species involved in ADS in plants, soil and water samples by means of qPCR will allow identification of high-risk fields that can be avoided or managed to reduce yield losses. Quantification of pathogen densities in the soil may also provide essential insights into the effectiveness of soil disinfestation methods for ADS management. © 2021 Society of Chemical Industry.

Fusarium Consortium Populations Associated with Asparagus Crop in Spain and Their Role on Field Decline Syndrome.

Asparagus Decline Syndrome (ADS) is one of the main phytosanitary problems of asparagus crop worldwide. Diseased plants and soil samples from 41 fields from three main production areas of Spain were surveyed. Eight Fusarium species belonging to seven species complexes were identified in soils: F. oxysporum, F. proliferatum, F. redolens, F. solanisensu stricto, F. equiseti, F. culmorum, F. compactum and F. acuminatum. Fusarium oxysporum was the most prevalent species. Statistical correlation (R2 = 88%) was established between F. oxysporum inoculum density and the average temperature of the warmest month. A relationship was also established between three crop factors (average temperature, crop age and F. oxysporum inoculum density) and field disease indices. Significant differences were observed between the distribution of F. oxysporum propagules in white and green asparagus fields. Thirteen Fusarium species belonging to seven species complexes were identified from roots of diseased plants, being F. oxysporum the most prevalent. F. proliferatum, F. oxysporum and F. redolens showed pathogenicity to asparagus and were the main species associated to ADS. Fusarium oxysporum was the species with the highest genetic diversity displaying 14 sequence-based haplotypes with no geographic differentiation. This work contributes to understanding the Fusarium complex associated to ADS for developing accurate integrated disease management strategies.

SILVOLIVE, a Germplasm Collection of Wild Subspecies With High Genetic Variability as a Source of Rootstocks and Resistance Genes for Olive Breeding.

Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar "Picual" scion, which could improve the productivity of high-density hedgerow orchards.

Genetic Diversity and Vegetative Compatibility of Fusarium solani Species Complex of Strawberry in Spain.

Fusarium solani is a soilborne fungus that is a pathogen to >100 plant species. It is the causal agent of crown and root rot in strawberry. We collected 100 F. solani isolates from diseased plants and soils from two distinct geographic areas of strawberry production in Spain: plant nurseries located in the north-central region of the country and fruit production fields located in the southwestern region. The aims of this study were to accurately identify the isolates within the Fusarium solani species complex (FSSC) based on multilocus sequence typing, determine the genetic diversity and population structure of strawberry-associated FSSC based on phylogenetic analysis, and determine the vegetative compatibility among isolates in both strawberry production areas. Seven phylogenetic species, restricted to clade 3 of FSSC, were defined in the Spanish strawberry crops, showing a regional variation of species composition. Isolates from nurseries were composed of four phylogenetic species (i.e., FSSC 2, FSSC 5, FSSC 9, and an unknown FSSC species) that matched with five vegetative compatibility groups (VCGs). Isolates from fruit production fields included five phylogenetic species (i.e., FSSC 2, FSSC 3 + 4, FSSC 5, FSSC 6, and FSSC 11) distributed into 29 VCGs not correlated with phylogenetic groups. FSSC 5 and FSSC 2 were the most abundant species in nurseries and fruit production fields, respectively, and they were the only species present in both production areas. Of the 47 sequence-based haplotypes defined, no haplotypes were shared between nurseries and fruit production fields. Pathogenic isolates were present in all but FSSC 6 and FSSC 9 species, and FSSC 3 + 4 contained the higher percentage of pathogenic isolates. No relationship was observed between pathogenicity and the source of isolates (plant or soil). Generally, species present in fruit production fields showed higher genetic diversity than those present in nurseries. This work can contribute to understanding the diversity of this species complex in Spanish strawberry production areas, which will be useful for developing integrated disease management strategies.

The structure and function of the global citrus rhizosphere microbiome.

Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.

Potential Inoculum Sources and Incidence of Strawberry Soilborne Pathogens in Spain.

The decline and death of strawberry plants in Spanish fruit production fields have mainly been attributed to the soilborne pathogens Macrophomina phaseolina, Phytophthora cactorum, and Fusarium spp. Inoculum sources of M. phaseolina and P. cactorum, and the incidence all three genera, were investigated in nurseries and fruit production fields over three consecutive seasons. M. phaseolina inoculum sources consisted of fumigated preplant fruit production soils (50%) and fumigated nursery soils (47%), although the pathogen could not be detected in nursery mother and runner plants. P. cactorum inoculum sources included nursery (20%) and preplant fruit production (17%) fumigated soils, and nursery runner plants (up to 15%). In fruit production plants, the average incidence of M. phaseolina and P. cactorum were 4.2 and 3.7%, respectively. Fusarium spp. inoculum sources could not be accessed extensively due to the lack of effective quantitative real-time PCR assays. Limited testing of nursery plants showed that Fusarium oxysporum f. sp. fragariae (Fof) was absent. In field production plants and soil, F. solani was the main pathogenic Fusarium spp., with Fof only identified once in a fruit production plant. Ineffectively fumigated soils in nurseries and production fields, along with infected runner plants, can be inoculum sources of soilborne strawberry pathogens in Spain.

Physiological and gene expression responses of sunflower (Helianthus annuus L.) plants differ according to irrigation placement.

To investigate effects of soil moisture heterogeneity on plant physiology and gene expression in roots and leaves, three treatments were implemented in sunflower plants growing with roots split between two compartments: a control (C) treatment supplying 100% of plant evapotranspiration, and two treatments receiving 50% of plant evapotranspiration, either evenly distributed to both compartments (deficit irrigation - DI) or unevenly distributed to ensure distinct wet and dry compartments (partial rootzone drying - PRD). Plants receiving the same amount of water responded differently under the two irrigation systems. After 3 days, evapotranspiration was similar in C and DI, but 20% less in PRD, concomitant with decreased leaf water potential (Ψleaf) and increased leaf xylem ABA concentration. Six water-stress responsive genes were highly induced in roots growing in the drying soil compartment of PRD plants, and their expression was best correlated with local soil water content. On the other hand, foliar gene expression differed significantly from that of the root and correlated better with xylem ABA concentration and Ψleaf. While the PRD irrigation strategy triggered stronger physiological and molecular responses, suggesting a more intense and systemic stress reaction due to local dehydration of the dry compartment of PRD plants, the DI strategy resulted in similar water savings without strongly inducing these responses. Correlating physiological and molecular responses in PRD/DI plants may provide insights into the severity and location of water deficits and may enable a better understanding of long-distance signalling mechanisms.

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR.

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses.

Assessment of the diversity and dynamics of Plum pox virus and aphid populations in transgenic European plums under Mediterranean conditions.

The molecular variability of Plum pox virus (PPV) populations was compared in transgenic European plums (Prunus domestica L.) carrying the coat protein (CP) gene of PPV and non-transgenic plums in an experimental orchard in Valencia, Spain. A major objective of this study was to detect recombination between PPV CP transgene transcripts and infecting PPV RNA. Additionally, we assessed the number and species of PPV aphid vectors that visited transgenic and non-transgenic plum trees. Test trees consisted of five different P. domestica transgenic lines, i.e. the PPV-resistant C5 'HoneySweet' line and the PPV-susceptible C4, C6, PT6 and PT23 lines, and non-transgenic P. domestica and P. salicina Lind trees. No significant difference in the genetic diversity of PPV populations infecting transgenic and conventional plums was detected, in particular no recombinant between transgene transcripts and incoming viral RNA was found at detectable levels. Also, no significant difference was detected in aphid populations, including viruliferous individuals, that visited transgenic and conventional plums. Our data indicate that PPV-CP transgenic European plums exposed to natural PPV infection over an 8 year period caused limited, if any, risk beyond the cultivation of conventional plums under Mediterranean conditions in terms of the emergence of recombinant PPV and diversity of PPV and aphid populations.

The ectD gene, which is involved in the synthesis of the compatible solute hydroxyectoine, is essential for thermoprotection of the halophilic bacterium Chromohalobacter salexigens.

The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress. (13)C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45 degrees C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37 degrees C), and the ectoine precursor, Ngamma-acetyldiaminobutyric acid. High-performance liquid chromatography analyses showed that the levels of ectoine and hydroxyectoine were maximal during the stationary phase of growth. Accumulation of hydroxyectoine was up-regulated by salinity and temperature, whereas accumulation of ectoine was up-regulated by salinity and down-regulated by temperature. The ectD gene, which is involved in the conversion of ectoine to hydroxyectoine, was isolated as part of a DNA region that also contains a gene whose product belongs to the AraC-XylS family of transcriptional activators. Orthologs of ectD were found within the sequenced genomes of members of the proteobacteria, firmicutes, and actinobacteria, and their products were grouped into the ectoine hydroxylase subfamily, which was shown to belong to the superfamily of Fe(II)- and 2-oxoglutarate-dependent oxygenases. Analysis of the ectoine and hydroxyectoine contents of an ectABC ectD mutant strain fed with 1 mM ectoine or hydroxyectoine demonstrated that ectD is required for the main ectoine hydroxylase activity in C. salexigens. Although in minimal medium at 37 degrees C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45 degrees C it could not cope with the lowest (0.75 M NaCl) or the highest (3.0 M NaCl) salinity, and it grew optimally at 2.5 M NaCl. The ectD mutation caused a growth defect at 45 degrees C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37 degrees C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37 degrees C) and 15% (at 45 degrees C) of the hydroxyectoine produced by the wild-type strain. All of these data reveal that hydroxyectoine synthesis mediated by the ectD gene is thermoregulated and essential for thermoprotection of C. salexigens.

Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction.

ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected.