BackgroundWhile recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes.ResultsOverall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source.ConclusionsThe competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from the phyllosphere. Noteworthy was the strong antagonistic activity of the soil yeast C. subhashii, which had so far only been described from a clinical sample and not been studied with respect to biocontrol. Based on binary competition assays and growth analyses (e.g., on different carbon sources, growth in root exudates), C. subhashii was identified as a competitive and antagonistic soil yeast with potential as a novel biocontrol agent against plant pathogenic fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0908-z) contains supplementary material, which is available to authorized users.

Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre-and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices.

The anilinopyrimidines constitute a new class of mainly protective, broad-spectrum fungicides with a high activity against Botryotinia fuckeliana, the causal agent of gray mold on a wide range of host plants. The present study was initiated to investigate the genetic basis of resistance to anilinopyrimidines in B. fuckeliana and to assess the frequency of resistant isolates in vineyards in Switzerland exposed to experimental applications of anilinopyrimidines. In mating experiments, two sensitive reference isolates were crossed with three anilinopyrimidine-resistant field isolates. The analysis of 72 sexual progeny from six apothecia demonstrated that resistance to the anilinopyrimidine fungicide cyprodinil segregated in a 1:1 ratio and is therefore monogenic. The same segregation ratio was found for resistance to the dicarboximide fungicide vinclozolin. Resistance to cyprodinil segregated independently from resistance to vinclozolin. From 1993 to 1995, isolates of B. fuckeliana were collected in Switzerland from five vineyards that differed in their anilinopyrimidine spray history. Of a total of 303 isolates tested in vitro, three anilinopyrimidine-resistant isolates were detected in two vineyards where the cumulative number of treatments was between two and nine. The results of the study are discussed with respect to the implementation of an antiresistance strategy in Switzerland.

Monilinia fructicola, causal agent of fruit brown rot, is a quarantine pathogen in Europe (1). It presents a significant threat because of its aggressivity on flowers, shoots, and wood at low temperatures and propensity for sexual reproduction that increases potential for evolutionarily adaptation to new environments, hosts, and fungicides. It is common in North America, Japan, Australia, and South America. It occurs in orchards in France, has been detected but eradicated from Austria and Spain, and has been found on imported peach in Hungary (1,2). In Switzerland, we recently detected M. fructicola in supermarkets on imported fruit with brown rot symptoms similar to those caused by endemic M. fructigena and M. laxa. Preliminary identification was based on distinctive colony and conidial morphology on potato dextrose agar of fruit isolates. Specific identification was determined by polymerase chain reaction (PCR) (3) and sequencing the internal transcribed spacer (ITS) region. Koch's postulates were fulfilled by reproducing brown rot on healthy inoculated fruit. Surveys of imported fruit in markets (n = 42) using PCR revealed M. fructicola on all imported apricot and nectarine from the United States and France, but none on apricot, peach, plum, and cherry from Spain, France, Italy, or Turkey. Field surveys of apricot, peach, plum, prune, nectarine, and cherry orchards in 13 Swiss cantons were all negative (n = 71 in 2003 and 164 in 2005). This report demonstrates that imported fruit is a weak link in quarantine efforts and poses a potential threat. Transmission to local trees via highly dispersible, profuse spores from recycled packaging and disposal sites for discarded fruit has thus far not occurred but the risk deserves attention. Revised regulations for fruit treatment at points of entry and/or scrutiny of origin orchards may be warranted. References: (1) OEPP/EPPO. List of A2 pests regulated as quarantine pests in the EPPO region. Version 2005-09. Online publication with distribution map at http://www.eppo.org , 2005. (2) M. Petróczy and L. Palkovics. Plant Dis. 90:375, 2006. (3) K. J. D. Hughes et al. EPPO Bull. 30:507, 2000.

Monilinia fructicola is a quarantine fungal pathogen in Europe, but many major stone fruit growing countries in Europe have reported its presence recently. In Switzerland, the fungus was first found in a single apricot orchard in 2008. This study confirms the presence of M. fructicola in nine out of 22 commercial orchards in Canton Valais, Switzerland. Five simple sequence repeat markers (SSRs) were developed for M. fructicola and samples from Switzerland, Spain, Italy, France and the United States were analysed and compared in order to assess the genetic diversity of the pathogen, identify the origin of the disease, and verify if the fungus reproduces sexually in Europe. In the 119 European samples analysed, 12 different haplotypes were found, indicating a relatively high genetic diversity of the pathogen considering that the first report in Europe was 10 years ago. Three haplotypes found in Europe were identical to those found in the American samples (two from the east coast and one from the west coast). Population structure analysis suggests that the European population is derived from at least two ‘invasion’ events probably originating from the US (one from the east coast, the other from the west coast). Preliminary evidence of sexual reproduction of M. fructicola in Europe is reported.

23Caffier, V., Patocchi, A., Expert, P., Bellanger, Durel, M., 24 Broggini, G.A.L, Groenwold, R., and Bus V.G.M. 2014. Virulence characterization of 25 Venturia inaequalis reference isolates on the differential set of Malus hosts. Plant Dis. 26XX:XX-XX.27 28 A set of differential hosts has recently been identified for 17 apple scab resistance genes in an 29 updated system for defining gene-for-gene (GfG) relationships in the Venturia inaequalis- 30 Malus pathosystem. However, a set of reference isolates characterized for their 31 complementary avirulence alleles is not yet available. In this paper, we report on improving 32 the set of differential hosts for h(7) and propose the apple genotype LPG3-29 as carrying the 33 single major resistance gene Rvi7. We characterized a reference set of 23 V. inaequalis 34 isolates on 14 differential apple hosts carrying major resistance genes under controlled 35 conditions. We identified isolates that were virulent on at least one of the following defined 36 Sixteen different virulence patterns were observed. In general, the isolates carried one to 38 three virulences, but some of them were more complex, with up to six virulences. This set of 39 well-characterized isolates will be helpful for the identification of additional apple scab 40 resistance genes in apple germplasm and the characterization of new GfG relationships to 41 help improve our understanding of the host-pathogen interactions in the V. inaequalis-Malus 42 pathosystem.43 44 45 3 Plant DiseaseDurable pest and disease resistance is a major breeding objective for most crops, but 46 achieving it has proven to be a challenge for breeders in the absence of truly non-race-47 specific resistances. Many genes involved in effector-triggered immunity (20) code for highly 48 polymorphic proteins containing nucleotide binding (NB) and leucine-rich repeat (LRR) 49 domains that instigate a resistance reaction in the host following recognition of the 50 complementary pathogen effector protein(s) in this specific gene-for-gene (GfG) relationship 51 (13). However, the high evolutionary potential of the pathogen effector arsenal negatively 52 affects the durability of resistance genes. To counteract, or at least delay the breakdown of 53 resistance genes by virulent races of the pathogen, breeders search for new resistance genes 54 (major genes and quantitative trait loci (QTLs)) that are effective against the already known 55 virulent races (23) and pyramid them (including 'spent' ones) to improve their durability as 56 well as increase the efficacy of the combined genes. This is based on the principle that for a 57 multiple virulent strain to develop, it will have to acquire these virulences simultaneously 58 (11). The effectiveness of this strategy relies on the availability of a set of differential hosts 59 and reference isolates to understand the genetics of the arsenal of both effectors in the 60 pathogen and resistances in the host, and their GfG relationships (34). 61 Apple scab, caused by Venturia inaequali...

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Many yeasts have demonstrated intrinsic insensitivity to certain antifungal agents. Unlike the fungicide resistance of medically relevant yeasts, which is highly undesirable, intrinsic insensitivity to fungicides in antagonistic yeasts intended for use as biocontrol agents may be of great value. Understanding how frequently tolerance exists in naturally occurring yeasts and their underlying molecular mechanisms is important for exploring the potential of biocontrol yeasts and fungicide combinations for plant protection. Here, yeasts were isolated from various environmental samples in the presence of different fungicides (or without fungicide as a control) and identified by sequencing the internal transcribed spacer (ITS) region or through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Among 376 isolates, 47 taxa were identified, and Aureobasidium pullulans was the most frequently isolated yeast. The baseline sensitivity of this yeast was established for 30 isolates from different environmental samples in vitro to captan, cyprodinil, and difenoconazole. For these isolates, the baseline minimum inhibitory concentration (MIC50) values for all the fungicides were higher than the concentrations used for the control of plant pathogenic fungi. For some isolates, there was no growth inhibition at concentrations as high as 300 µg/mL for captan and 128 µg/mL for cyprodinil. This information provides insight into the presence of resistance among naturally occurring yeasts and allows the choice of strains for further mechanistic analyses and the assessment of A. pullulans for novel applications in combination with chemical agents and as part of integrated plant-protection strategies.