Mixed infections alter transmission potential in a fungal plant pathogen

Infections by more than one strain of a pathogen predominate under natural conditions. Mixed infections can have significant, though often unpredictable, consequences for overall virulence, pathogen transmission and evolution. However, effects of mixed infection on disease development in plants often remain unclear and the critical factors that determine the outcome of mixed infections remain unknown. The fungus Zymoseptoria tritici forms genetically diverse infections in wheat fields. Here, for a range of pathogen traits, we experimentally decompose the infection process to determine how the outcomes and consequences of mixed infections are mechanistically realized. Different strains of Z. tritici grow in close proximity and compete in the wheat apoplast, resulting in reductions in growth of individual strains and in pathogen reproduction. We observed different outcomes of competition at different stages of the infection. Overall, more virulent strains had higher competitive ability during host colonization, and less virulent strains had higher transmission potential. We showed that within-host competition can have a major effect on infection dynamics and pathogen population structure in a pathogen and host genotype-specific manner. Consequently, mixed infections likely have a major effect on the development of septoria tritici blotch epidemics and the evolution of virulence in Z. tritici.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

skippy,a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

DNA cytosine methylation in plant development

Cytosine bases of the nuclear genome in higher plants are often extensively methylated.Cytosine methylation has been implicated in the silencing of both transposable elements (TEs) and endogenous genes,and loss of methylation may have severe functional consequences.The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity.In addition,the fresh studies also revealed the more dynami...     

Characterization of microsatellites in the fungal plant pathogen Crinipellis perniciosa

Microsatellite markers of Crinipellis perniciosa, with three and four repeats, were developed from sequence database and evaluated for their usefulness in detecting genetic polymorphism. Thirty‐three primers produced unambiguous amplification products of 28 microsatellite‐containing loci and 14 microsatellite‐like polymorphic loci, with two to seven alleles at each locus. Three loci were useful to distinguish isolates from different biotypes and isolates from different countries. Amplification of the markers in the closely related fungi Moniliophthora roreri indicates that their usefulness in population's studies may go beyond the present study of the C. perniciosa and may have applications in population genetics of M. roreri.     

Resistance to a fungal pathogen and host plant specialization in the pea aphid

Herbivores that show host race formation on different plant species have proven to be valuable model systems for studying the evolution of specialization and speciation. Here, we use the pea aphid, Acyrthosiphon pisum, to investigate a possible link between specialization on two host plant species, Lotus uliginosus and Trifolium pratense, and resistance to a natural enemy, the fungal pathogen Erynia neoaphidis. Pea aphids collected on either plant species in the field showed in most cases poor survival on the alternate host plant. Furthermore, pea aphids specialized on T. pratense were very resistant to E. neoaphidis, whereas aphids specialized on L. uliginosus were susceptible. This susceptibility was not influenced by the actual food plant on which the assays were conducted. We discuss how selection from natural enemies may influence the process of specialization and race formation, and how specialization can affect the evolution of resistance.     

Specific inactivation of an antifungal bacterial siderophore by a fungal plant pathogen

Bacteria and fungi secrete many natural products that inhibit each other’s growth and development. The dynamic changes in secreted metabolites that occur during interactions between bacteria and fungi are complicated. Pyochelin is a siderophore produced by many Pseudomonas and Burkholderia species that induces systemic resistance in plants and has been identified as an antifungal agent. Through imaging mass spectrometry and metabolomics analysis, we found that Phellinus noxius, a plant pathogen, can modify pyochelin and ent-pyochelin to an esterification product, resulting in reduced iron-chelation and loss of antifungal activity. We also observed that dehydroergosterol peroxide, the fungal metabolite, is only accumulated in the presence of pyochelin produced through bacteria–fungi interactions. For the first time, we show the fungal transformation of pyochelin in the microbial interaction. Our findings highlight the importance of understanding the dynamic changes of metabolites in microbial interactions and their influences on microbial communities.Subject terms: Microbial ecology, Metabolomics

Microorganisms use various strategies to establish themselves within an ecological niche while facing keen competition in the environment. Natural products such as antibiotics, quorum sensing molecules, and siderophores are crucial in microbial interactions [1–3]. Certain microorganisms are equipped with uptake systems that enable them to acquire siderophores, even by those that may not produce them [4]. For example, pyochelin is a siderophore produced by many Pseudomonas and Burkholderia strains. Such bacterial strains are commonly found in soils, as endophytes, and from the rhizosphere where they may inhibit plant pathogens [5, 6].Burkholderia cenocepacia 869T2 was isolated as an endophyte and showed beneficial abilities to control banana Fusarium wilt [7]. It harbors many biosynthetic gene clusters of secondary metabolites, such as pyochelin, pyrrolnitrin, and pyrroloquinoline quinone [8]. Recently, we found that this strain could temporarily inhibit the growth of P. noxius, a fungal pathogen of brown root rot disease, which is prevalent in tropical and subtropical regions and has a wide host range covering over 200 plant species [9]. However, in the competition between fungi and bacteria, P. noxius can resist this inhibition and overwhelm bacterial colonies after 1–2 weeks under dual-culture conditions (Fig. S1). These results imply that fungi might have resistance responses and undergo metabolic changes in bacteria–fungi interactions [10]. Here we unveiled metabolic changes in the competitive interaction between B. cenocepacia 869T2 and P. noxius 2252 using the matrix-assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI-TOF IMS) [11, 12].We specifically monitored the metabolites in the inhibition region of B. cenocepacia 869T2 and P. noxius 2252 dual-culture using MALDI-TOF IMS. Several induced or enzymatically modified metabolites were detected, including m/z 275, 362, 383, and 427 (Fig. 1A). In particular, pyochelin (m/z 325), surrounding the B. cenocepacia 869T2 colony, showed asymmetric distribution in dual-culture samples. Near the P. noxius 2252 mycelia, a new metabolite with m/z 383 was detected with a complementary distribution to pyochelin (Fig. 1A). In LC-MS/MS-based molecular networking analysis [13], we found that this new metabolite structure is an esterification product of pyochelin and glycolic acid, which we named pyochelin-GA (Fig. 1B). We then constructed a pchF-null mutant strain, ΔpchF, which cannot produce pyochelin, and then dual cultured it with P. noxius. Pyochelin and pyochelin-GA were not observed in the MALDI-TOF IMS and LC-MS analysis of dual-culture samples (Fig. 1A and Fig. S2). We further inoculated P. noxius 2252 with pyochelin-GA-free extract harvested from B. cenocepacia 869T2 single culture, and the complementary distribution of pyochelin and pyochelin-GA was observed by MALDI-TOF IMS again (Fig. S3). These results demonstrated that pyochelin-GA was transformed from pyochelin by P. noxius 2252, rather than produced by B. cenocepacia 869T2 under dual-culture conditions.Open in a separate windowFig. 1Metabolic changes in the bacteria–fungi interaction.A Spatial distribution of selected mass signals (m/z) in MALDI-TOF IMS analysis of Phellinus noxius 2252 (Pn2252) dual-cultured with Burkholderia cenocepacia 869T2 (869T2) and a pchF-null mutant strain (Δ pchF). B Molecular networking analysis of pyochelin and analogs from the dual-culture sample. The red node is pyochelin, and the green node is pyochelin-GA. The structures of pyochelin, pyochelin-GA, and dehydroergosterol peroxide (DHEP), together with their mass signals in MALDI-TOF IMS, are shown. C Iron-chelating abilities of pyochelin and pyochelin-GA were evaluated by Chrome Azurol S liquid assay using different concentrations (2.5, 1.25, 0.63, 0.31, and 0.16 mM, n = 3). Proportions of siderophore units are shown in Fig. S14. D Fungal transformation of pyochelin and ent-pyochelin by treating P. noxius 2252 with ethyl acetate crude extracts of B. cenocepacia 869T2, Pseudomonas aeruginosa PAO1, and P. protegens Pf-5 for 8 h. LC-MS was used to monitor the signals of pyochelin (red), ent-pyochelin (blue), and transformation product 383 (black).The chemical structure of pyochelin-GA was further confirmed via total synthesis, NMR, and LC-MS/MS analysis (Supplementary Material and Methods, and Figs. S4–7). The purified pyochelin and pyochelin-GA were also evaluated for their iron-chelating ability. Chrome Azurol S assay indicated that pyochelin had the dose-dependent iron-chelating ability, but pyochelin-GA had lower iron-binding efficiency (Fig. 1C, Fig. S8). Pyochelin chelates iron in the extracellular medium and transports it into cells via the specific outer membrane transporter FptA. The X-ray structure of FptA-pyochelin-Fe indicated that the terminal carboxylic acid of pyochelin plays an essential role in the iron uptake ability [14, 15]. Our docking analysis suggested that the glycolic ester moiety of pyochelin-GA would affect the binding pocket shape of FptA and result in different binding properties compared to FptA-pyochelin (Fig. S9).Pyochelin and ent-pyochelin are produced independently by different biosynthetic gene clusters in Pseudomonas species [16]. To determine whether P. noxius 2252 can transform both enantiomers via this esterification process, we treated P. noxius 2252 with the extracts of pyochelin producers (P. aeruginosa PAO1 and B. cenocepacia 869T2) and an ent-pyochelin producer (P. protegens Pf-5). After 8 h of treatment, both pyochelin and ent-pyochelin were converted to pyochelin-GA (or ent-pyochelin-GA) (Fig. 1D), demonstrating this is a non-stereospecific transformation.To better understand the iron-chelating ability of pyochelin, we used pyochelin and pyochelin-GA to treat P. noxius 2252 under iron-deficiency conditions, by adding the iron chelator deferoxamine, and iron-rich conditions by adding FeCl₃ (Fig. 2). Pyochelin-GA did not affect the growth of P. noxius 2252 under all conditions. However, P. noxius 2252 was more sensitive to pyochelin in iron-deficient conditions and more resistant to pyochelin in iron-rich conditions, demonstrating that iron availability directly affected the tolerance of P. noxius 2252 to pyochelin. A similar phenomenon was reported previously for Aspergillus fumigatus [17].Open in a separate windowFig. 2Pyochelin inhibition of mycelial growth of Phellinus noxius 2252 is inversely associated with iron concentration.Pyochelin-GA did not have an inhibition effect on P. noxius 2252. Potato dextrose agar (PDA) with deferoxamine (DFO; 200 and 400 µM) was used to mimic iron-deficiency conditions. Iron-rich conditions was prepared by adding FeCl₃ (200 and 400 µM) in PDA. P. noxius 2252 was treated with 0.03, 0.06, 0.12, and 0.24 µmol of pyochelin or pyochelin-GA at 30 °C for 24 h. The antifungal assay was performed in two biological replicates.Using MALDI-TOF IMS analysis of the dual-culture of B. cenocepacia 869T2 and P. noxius 2252, we observed that several metabolites (e.g., m/z 275, 362, and 427) were only observed in the boundary of fungal mycelia (Fig. 1A). Although those metabolites were not detected in the dual-culture of ΔpchF and P. noxius 2252 (Fig. 1A), they were present when we treated P. noxius 2252 with pyochelin (Fig. S10). We identified the metabolite associated with m/z 427 as dehydroergosterol peroxide (DHEP) (Fig. S11), which was initially oxidized from ergosterol and dehydroergosterol [18]. Pyochelin can enhance intercellular reactive oxygen species (ROS) and ultimately disrupts membrane integrity, leading to cell death [17, 19, 20]. To clarify whether ROS induced the accumulation of DHEP, we treated P. noxius 2252 with pyochelin, pyochelin-GA, and 2,2′-bipyridyl (an iron chelator). Pyochelin and 2,2′-bipyridyl showed antifungal effects on P. noxius 2252 and induced ROS production (Fig. S12). However, the accumulation of DHEP in P. noxius 2252 was only associated with pyochelin treatment (Fig. S13). The induction of ROS in P. noxius 2252 by pyochelin and pyochelin-GA was not significantly different (Fig. S14). Therefore, we predict that pyochelin-induced accumulation of DHEP in P. noxius 2252 is independent of ROS production and iron-deficiency.Overall, we demonstrate that pyochelin transformation by fungi, in the interaction between pyochelin-producing bacteria and the plant pathogen P. noxius transforms pyochelin and ent-pyochelin into pyochelin-GA (and ent-pyochelin-GA). This product no longer functions as an iron chelator and no longer shows antifungal activity. The production of a fungal metabolite, dehydroergosterol peroxide, was induced explicitly by pyochelin through an unknown mechanism. These results highlight the importance of monitoring dynamic changes of metabolites in situ to better understand the functions and influences of metabolites on microbial community interactions.     

Characterization of microsatellites in the fungal plant pathogen,Sclerotinia sclerotiorum

Twenty‐five primers produced unambiguous amplification products of 23 microsatellite‐containing loci and two microsatellite‐like polymorphic loci, with 2–10 alleles at each locus in the plant pathogenic fungus, Sclerotinia sclerotiorum. Haplotypes are polymorphic among individuals sharing the same DNA fingerprint and DNA sequence haplotype, facilitating epidemiological monitoring worldwide. Fourteen of these primers also successfully amplified the closely related S. trifoliorum and S. minor.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

HPLC analysis of plant DNA methylation: a study of critical methodological factors.

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20-200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.