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.
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