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Brown rot of stone fruit is one of the most important stone fruit diseases. The losses caused by this disease are mainly registered during the postharvest process, either in packing houses, or at the point of sale or at the consumer’s home. Microbial control has emerged as one of the most promising alternatives to chemical fungicides. Several microbial agents have been widely investigated for use on different postharvest pathogens. The efficacy of the microbial antagonist(s) such as Aureobasidium pullulans, Bacillus spp., Ceratocystis fimbriata, Debaryomyces hansenii, Epicoccum nigrum, Pantoea agglomerans, Penicillium frequentans, Pseudomonas spp., Streptomyces spp. and Wickerhamomyces anomalus are being used. Biocontrol products such as Amylo-X, BoniProtect, Julietta, Serenade Max and Serenade Aso have also been developed and registered. Although the results of this technology are encouraging, the formulation and application methods are key issues.

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.

Fusarium solani is an emerging pathogen reported on Spanish strawberry crops both in nurseries and in fruit production fields, causing wilt and root rot. Pathogenicity, morphocultural characteristics, and sensitivity to biocides of 103 F. solani isolates recovered from symptomatic strawberry plants and soils from both Spanish strawberry areas were determined. The differences of isolates within and between nurseries and field crops in relation to these parameters were analyzed. Considerable variability in morphological and pathogenic characteristics was observed among the isolates in both areas. The majority of isolates were not pathogenic (62%), and only 38 F. solani isolates (37.62%) caused disease on strawberry plants under controlled conditions; 52.63% of pathogenic isolates induced low severity symptoms. Almost 70% of pathogenic isolates caused stunting on plants. The morphological characters that best explain the F. solani variability (86.85%) were colony color and the presence of macroconidia on culture medium. The sensitivity to the fumigants tested was similar between the isolates from nurseries and fruit production fields, showing greater sensitivity to the field doses of dazomet and chloropicrin. However, the isolates were less sensitive to metam sodium and poorly sensitive to 1,3-dichloropropene. This work can contribute to the advancement of sustainable production of strawberry.

16 Pág. Departamento de ​Protección Vegetal (INIA), Fusarium solani and F. oxysporum are plant pathogenic fungi that cause root rot and wilt, respectively, in many economically important crops. Identification of both species by morphological characteristics is complex and time-consuming, especially when a crop is host to several species, as in the case of strawberry and other many crops. The objective of this research was to develop a molecular method for the precise and sensitive identification of Fusarium solani and F. oxysporum by multiplex PCR. Specific PCR conditions were developed for the amplification of about 170-bp PCR product from the internal transcribed spacer of F. oxysporum and a PCR product of about 650 bp from the elongation factor 1-alpha gene of F. solani. The specificity of the multiplex PCR was tested using gDNA extracted from 232 strains of F. solani and F. oxysporum, 12 strains of other Fusarium spp. and 14 strains of other genera. The sensitivity of each primer set was tested individually and simultaneously with one strain of F. oxysporum and F. solani. This protocol was then used to identify both species in natural isolates from diseased plant and soil samples from strawberry nursery. Each primer set was validated using genomic DNA from non-sterile and sterile soils artificially inoculated with both species. Under these conditions, both Fusarium species were detected and distinguished over a range of concentrations (1 × 107 to 1 × 102 conidia g−1 soil) by successful amplification of corresponding PCR products for each species. This multiplex PCR technique is a fast tool to identify natural isolates of both species, F. solani and F. oxysporum, in a single PCR reaction., This study was supported by EUPHRESCO [266505 FP-7 ERANET EUPHRESCO-SPAT “Strawberry pathogens assessment and testing”] and by Ministry of Economy and Competitiveness (Spain) [RTA2013-00062-C05-E; RTA2017-00019-C03-01 (Plan Nacional de I + D)].

Background Chloropicrin (PIC) mixtures of 1,3-dichloropropene and chloropicrin (DD:PIC), dazomet, and metam sodium (MS) have been applied as chemical alternatives to methyl bromide (MB) in Spanish strawberry nurseries since MB was banned as a soil fumigant in 2005. These chemical alternatives were applied to soil in two Spanish strawberry nurseries between 2003 and 2017 to test their efficacy against the main crown and root disease and soil fungal populations in comparison with the use of MB and PIC (MB:PIC). These chemicals were applied at several doses with different application methods under plastic films. Crown and root disease incidence was calculated as the percentage of plants with symptoms caused by soil-borne pathogens. Soil fungal populations were estimated as colony forming units per gram of dry soil. Results All chemicals significantly reduced soil-borne fungal disease incidence and fungal population in both nurseries over the years. Phytophthora cactorum and Fusarium spp. were the main pathogens causing soil-borne diseases, followed by Verticillium spp. MB:PIC remained the treatment that best controlled P. cactorum. MS and DD:PIC controlled Fusarium disease to a lesser extent than MB:PIC and dazomet in both nurseries. MB:PIC and PIC were the two treatments that most reduced Verticillium spp. The population of Verticillium spp. declined and the presence of other species such as Colletotrichum spp. and Rhizoctonia spp. was minimal during the study. Conclusion Chemicals are necessary to obtain healthy strawberry plants. The use of chemical alternatives to MB has resulted in changes in the incidence of soil-borne diseases and soil fungal populations in strawberry nurseries. Dazomet was an effective alternative to MB as a soil-borne disease control, except against Verticillium spp. MB alternatives in strawberry nursery soils have caused Fusarium spp. to displace Verticillium spp.

Biological control using microorganism shows great potential as an eco-friendly and feasible alternative to replace, or at least diminish, the application of chemicals for plant pathogen control. Understanding the mechanisms involved in the interaction between any biological control agents (BCAs), host and target plant pathogen is crucial to enhance and extend the use of these organisms in agriculture. Recently, modern molecular technologies have allowed researchers to explore nature as never before, providing powerful approaches to characterize BCA’s modes of action. Among these new methodological approaches are the group of disciplines known under the term of “omics”, which include all areas of life sciences that take particular biomolecular sets as study objects (e.g., DNA, RNA, proteins or secondary metabolites), and that use high-throughput procedures for their approach. In this context, this chapter focuses on compiling common procedures used to carry out omic studies, with special emphasis on the research and development of BCAs against fungal plant pathogens. Thus, the importance of genomic, transcriptomic, proteomic and metabolomic knowledge is underlined in order to understand the molecular reasons that make bacteria, and fungi useful in biological control, as well as to gather information about how multipartite relations work, all central aspects to turn agriculture into a productive, sustainable, environmentally friendly and resilient system. In addition, we summarize and exemplify studies with a BCA, Penicillium rubens strain 212 (PO212) effective against different diseases in horticultural crops. We are using several of these new technologies to improve our knowledge on the action mode of this BCA.

Penicillium oxalicum strain 212 (PO212) is an effective biocontrol agent (BCA) against a large number of economically important fungal plant pathogens. For successful registration as a BCA in Europe, PO212 must be accurately identified. In this report, we describe the use of classical genetic and molecular markers to characterize and identify PO212 in order to understand its ecological role in the environment or host. We successfully generated pyrimidine (pyr-) auxotrophic mutants. In addition we also designed specific oligonucleotides for the pyrF gene at their untranslated regions for rapid and reliable identification and classification of strains of P. oxalicum and P. rubens, formerly P. chrysogenum. Using these DNA-based technologies, we found that PO212 is a strain of P. rubens, and is not a strain of P. oxalicum. This work presents PO212 as the unique P. rubens strain to be described as a BCA and the information contained here serves for its registration and commercialization in Europe.

Species in the Penicillium genus exhibit only a few distinguishing features, which additionally may vary depending on the growth conditions and the composition of culture medium. Taxonomic classification in the genus Penicillium is thus challenging because it is being continually reviewed. In the last decade, combinations of various techniques have been used for identification of Penicillium species. Some species of Penicillium are described as effective biocontrol agents (ACBs). The development and use of fungi as a biocontrol agent (BCA) require a risk assessment of their potential hazards, and compliance with regulatory and registration requirements in order to ensure efficacy and safety. As part of the registration requirements, the risk assessment must include monitoring of the BCA in the field in the presence of the indigenous fungal population. Here, we present an advanced survey of the nature and practice of a BCA. Specifically, we describe the use of molecular techniques to identify and characterize Penicillium rubens strain 212, originally classified as Penicillium oxalicum, which is an effective fungal BCAs of several horticultural diseases.

Microbial cells interact with the environment by adapting to external changes. Signal transduction pathways participate in both sensing and responding in the form of modification of gene expression patterns, enabling cell survival. The filamentous fungal-specific SltA pathway regulates tolerance to alkalinity, elevated cation concentrations and, as shown in this work, also stress conditions induced by borates. Growth of sltA− mutants is inhibited by increasing millimolar concentrations of boric acid or borax (sodium tetraborate). In an attempt to identify genes required for boron-stress response, we determined the boric acid or borax-dependent expression of sbtA and sbtB, orthologs of Saccharomyces cerevisiae bor1, and a reduction in their transcript levels in a ΔsltA mutant. Deletion of sbtA, but mainly that of sbtB, decreased the tolerance to boric acid or borax. In contrast, null mutants of genes coding for additional transporters of the Solute Carrier (SLC) family, sB, sbtD or sbtE, showed an unaltered growth pattern under the same stress conditions. Taken together, our results suggest that the SltA pathway induces, through SbtA and SbtB, the export of toxic concentrations of borates, which have largely recognized antimicrobial properties., Work at CIB-CSIC was funded by MINECO project BFU2012-33142 and MINECO/FEDER/EU project BFU2015-66806-R to E.A.E. Work at the UPV/EHU lab was funded by the Basque Government (grant IT599-13 to U.U.) and UPV/EHU (grant EHUA15/08 to O.E.). M.V. held a contract under project BFU2012-33142., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Immediately following the identification of Monilinia fructicola in a Spanish peach orchard in the Ebro Valley in 2006, this orchard and two other orchards in the same valley were intensively sampled for potential tree and ground sources of primary Monilinia inoculum before and during three growing seasons between 2006 and 2008. Overwintered Monilinia spp. produced inoculum from only mycelium, and no apothecia were found in any of the three orchards over the three growing seasons. Mummies on trees were the main source of primary inoculum. More than 90% of Monilinia isolates on all fruit mummies were M. laxa. Positive relationships were found between (i) the number of mummified fruit and the incidence of postharvest brown rot (P = 0.05, r = 0.75, n = 8), and (ii) the number of mummified fruit and nonabscised aborted fruit in the trees and the number of conidia on the fruit surface (P = 0.04, r = 0.71; P = 0.01, r = 0.94, respectively, n = 8) and the incidence of latent infection (P = 0.03, r = 0.75; P = 0.001, r = 0.99; respectively, n = 8). In addition, the numbers of mummified fruit and pruned branches on the orchard floor were correlated with the number of airborne conidia in the orchard. Based on the results of these surveys, the control of brown rot in stone fruit orchards is discussed. © 2010 The American Phytopathological Society.

Summary P enicillium oxalicum strain 212 (PO212) is an effective biocontrol agent (BCA) against a large number of economically important fungal plant pathogens. For successful registration as a BCA in Europe, PO212 must be accurately identified. In this report, we describe the use of classical genetic and molecular markers to characterize and identify PO212 in order to understand its ecological role in the environment or host. We successfully generated pyrimidine (pyr‐) auxotrophic mutants. In addition we also designed specific oligonucleotides for the pyrF gene at their untranslated regions for rapid and reliable identification and classification of strains of P . oxalicum and P . rubens, formerly P . chrysogenum. Using these DNA‐based technologies, we found that PO212 is a strain of P . rubens, and is not a strain of P . oxalicum. This work presents PO212 as the unique P . rubens strain to be described as a BCA and the information contained here serves for its registration and commercialization in Europe.

Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-β1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -β1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of μm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus. © 2013 Etxebeste et al., This work has been supported by former Spanish Ministerio de Ciencia y Tecnología and Ciencia e Innovación, through grants BFU2006-04185 and BFU2009-08701, and current Ministerio de Economía y Competitividad through grant BFU2012-33142 to E.A.E. O.E. held a research contract associated to grant BFU2006-04185 at the CIB, a research contract at The University of The Basque Country (UPV/EHU) with funds from Ministerio de Educación y Ciencia (BFU2010-17528) and is now a Lecturer at the UPV/EHU with funds from the Basque Government (IT599-13). María Villarino held a research contract associated to grant BFU-2009-08701. A.M.-I. held a predoctoral F.P.I. fellowship from the Ministerio de Educación y Ciencia and is now a contract researcher of The University of The Basque Country associated to a grant for Specialization of Researchers of the UPV/EHU(2012).

Seventeen field surveys were done in four commercial orchards during six consecutive fruit-growing seasons from 2006 until 2011 in order to determine the current frequencies of occurrence of M. laxa, M. fructigena, and M. fructicola and their relative contributions to postharvest brown rot in peaches and nectarines in the Ebro Valley. The relative frequencies of occurrence of Monilinia spp. were determined on three sources of primary inoculum and on three sources of secondary inoculum. The major relative frequencies of Monilinia spp. were significantly recorded (P = 0.05) from mummified fruit on the trees (approx. 42 %) and 7-day-old harvested fruit with brown rot (32 %), followed by that recovered from mummified fruit on the orchard bed (14 %), pruned branches on the orchard bed (8 %) and latent infections of immature fruit (3 %). We found that (a) the relative frequency of M. fructicola has increased over the years to coexist on the same level as at the time M. laxa, (b) M. fructigena is no longer a cause of brown rot in harvested peaches, (c), a progressive reduction in the time of the first appearance of Monilinia airborne conidia (r = -0.30, P = 0.003), and the time of the first latent infection (r = -0.44, P = 0.0001) was detected along years after correlation analysis, and (d) these displacements are not associated with an increased incidence of brown rot disease. The M. fructicola increase was due to its significantly increased presence in 7-day-old harvested fruit with brown rot (r = 0.73, P = 0.0009), in latent infections of immature fruit (r = 0.68, P = 0.002), on pruned branches on the orchard bed (r = 0.56, P = 0.018), and on mummified fruit sampled on the trees (r = 0.53, P = 0.03). This progressive increase was accompanied by a progressive reduction in the relative frequency of occurrence of M. laxa in 7-day-old harvested fruit with brown rot (r = -0.55, P = 0.021) and M. fructigena on mummified fruit sampled on the trees (r = -0.51, P = 0.03). © 2013 KNPV.

The genetic diversity of Spanish and French field populations of Monilinia fructicola, a quarantine fungal pathogen in Europe, was compared with that of Californian, Uruguayan, and New Zealand M. fructicola populations using inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers. Unweighted pair-group method with arithmetic average (UPGMA) cluster analysis and principal component analysis (PCA) of the ISSR data set revealed that the Spanish and French M. fructicola isolates were more closely related between themselves than to the non-European isolates. The levels of genetic diversity in the Spanish and French isolates are lower than those of the non-European isolates, indicating that M. fructicola is a recently introduced pathogen. UPGMA cluster analysis and PCA of the combined ISSR + RAPD data set of the European M. fructicola populations revealed that the Spanish isolates were more closely related among themselves than with the French isolates. Analysis of molecular variance partitioned the genetic variance to among the two regions (Spain and France) (20%), among the regional populations (35%), and within the populations in each region (45%) suggesting restricted gene flow between the three European populations. The observed index of association (I A) in each European M. fructicola populations indicates that the French and Spanish populations of M. fructicola are mainly asexually reproducing, with the Sudanell population potentially having a teleomorphic stage. The present finding of low genetic diversity in the Spanish and French M. fructicola populations is probably due to founder effects and genetic drift. © 2011 KNPV.

Eight field surveys over three growing seasons (April to October in 2006, 2007, and 2008) were done in three commercial peach and nectarine orchards in order to determine the secondary inoculum dynamics of Monilinia spp.;relationship to the weather conditions and incidence of postharvest brown rot in the Ebro Valley, which is the main peach fruit growing region in Spain. After regression analysis of the epidemiological data of postharvest brown rot and the climatic variables, a disease outbreak can be predicted from (i) the times of the first appearance of airborne conidia, the first appearance of conidia on the surface of flowers and fruits, and the first latent infection, all of which occur 2 months before harvest (ii) the number of conidia on the fruit surface, 2 weeks and 1 week before harvest, (iii) the preharvest incidence of brown rot, and (iv) the mean environmental temperature from popcorn to harvest. From these results, we confirmed the importance of the secondary inoculum dynamics of Monilinia spp.;the utility of these dynamics to predict an outbreak of brown rot in peaches on the day of their harvest and after their harvesting. © 2012 KNPV.

Chlorogenic acid (CGA) and its isomer, neochlorogenic acid (NCGA), were found to be the major phenolic compounds in the flesh and peel of three peach cultivars. Their concentrations are especially high in immature fruits (CGA, 151-548 mg/kg; NCGA, 85-380 mg/kg), whose resistance to the brown rot fungus, Monilinia laxa, is very high. The concentrations of these two phenolic compounds decline in maturing fruits (CGA, 77-181 mg/kg; NCGA, 30-82 mg/kg), and this decline is associated with a concomitant increase in susceptibility to brown rot infection. Other phenolic compounds found in the same HPLC chromatograms at 340 nm from each peach extract at varying sampling dates in each of the three peach cultivars were not correlated with the incidence of brown rot and appeared only in some cultivars. The incidence of brown rot for each cultivar at each sampling date was significantly negatively correlated with the NCGA (r >-0.85) and CGA (r >-0.90) contents. At concentrations that are similar to those in peach fruit, CGA does not inhibit spore germination or mycelial growth of M. laxa in culture but markedly inhibits the production of melanin-like pigments in the mycelia of M. laxa in culture (42% melanin reduction). Accordingly, we propose that the high concentrations of CGA and NGA in immature fruits might contribute to their reduced susceptibility or increased resistance to brown rot infection by interfering with fungal melanin production. © 2011 American Chemical Society.