The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Thiol redox state and oxidative stress affect sclerotial differentiation of the phytopathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum

Aims:  To investigate the involvement of oxidative stress and thiol redox state (TRS) in sclerotial differentiation of Sclerotium rolfsii and Sclerotinia sclerotiorum.
Methods and results:  Oxidative stress in these fungi was assessed by lipid peroxidation, which was higher in comparison with their nonsclerotiogenic counterpart strains. TRS [measured as glutathione (GSH) and cysteine] was associated with oxidative stress and differentiation using the TRS modulator and antioxidant Ν -acetylcysteine (AcCSH) and the GSH biosynthesis inducer and inhibitor l -2-oxo-thiazolidine-4-carboxylate and l -buthionine- S , R -sulphoximine (BSO) respectively. Differentiation and oxidative stress was decreased by AcCSH in both fungi. The decrease of differentiation by BSO was not associated with oxidative stress in these fungi.
Conclusions:  Differentiation and oxidative stress in both fungi depends on the availability of antioxidant noncytotoxic –SH groups and is not depended on any direct antioxidant role of GSH and its precursor cysteine.
Significance and Impact of the Study:  This study helps to understand the mechanism(s) of sclerotial differentiation in these agriculturally important phytopathogenic fungi and proposes that AcCSH can be used as potent fungicide by (i) acting as growth inhibiting cytotoxic oxidant and (ii) sustaining these fungi in their undifferentiated hyphal stage where they are vulnerable to degradation by soil micro-organisms.     

ANGUSTIFOLIA negatively regulates resistance to Sclerotinia sclerotiorum via modulation of PTI and JA signalling pathways in Arabidopsis thaliana

Sclerotinia sclerotiorum is a devastating pathogen that infects a broad range of host plants. The mechanism underlying plant defence against fungal invasion is still not well characterized. Here, we report that ANGUSTIFOLIA (AN), a CtBP family member, plays a role in the defence against S. sclerotiorum attack. Arabidopsis an mutants exhibited stronger resistance to S. sclerotiorum at the early stage of infection than wild-type plants. Accordingly, an mutants exhibited stronger activation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, including mitogen-activated protein kinase activation, reactive oxygen species accumulation, callose deposition, and the expression of PTI-responsive genes, upon treatment with PAMPs/microbe-associated molecular patterns. Moreover, Arabidopsis lines overexpressing AN were more susceptible to S. sclerotiorum and showed defective PTI responses. Our luminometry, bimolecular fluorescence complementation, coimmunoprecipitation, and in vitro pull-down assays indicate that AN interacts with allene oxide cyclases (AOC), essential enzymes involved in jasmonic acid (JA) biosynthesis, negatively regulating JA biosynthesis in response to S. sclerotiorum infection. This work reveals AN is a negative regulator of the AOC-mediated JA signalling pathway and PTI activation.     

Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus, Sclerotinia sclerotiorum

Two thousand seven hundred and forty-seven isolates of Sclerotinia sclerotiorum were sampled from four field populations of canola in western Canada. Each field was sampled in a grid of 128 50-m 50-m quadrats plus four intensive quadrats each sampled in a diagonal transect. Sampling was done at two phases of the disease cycle: (1) from ascospore inoculum on petals and (2) from disease lesions in stems. A total of 594 unique genotypes was identified by DNA fingerprinting. In each field, a small group of clones represented the majority of the sample, with a large group of clones or genotypes sampled once or twice. Clone frequencies were compared by χ² tests. The difference in profiles of clone frequencies for the two fields sampled in 1991 was not significant, but in 1992 the difference in profiles was marginally significant, indicating some local population substructure. The difference in profiles of clone frequencies for petals and lesions was not significant in each of the two fields sampled in 1991. In each of the two fields sampled in 1992, however, the difference was highly significant, consistent either with selection for some clones or with waves of immigration during the disease cycle. Nine of the 30 most frequently sampled clones from this study were previously recovered in a macrogeographical sample from western Canada in 1990. For spatial analyses, randomization tests indicated no significant spatial aggregation of either clones on petals or clones from lesions. Also, isolates of a clone on petals were not closer to isolates of the same clone from lesions than could be predicted by chance. Both observations suggest spatial mixing of ascospore inoculum from resident or immigrant sources.     

Signaling pathways involved in virulence and stress response of plant-pathogenic Fusarium species

Fungal pathogens face similar stress conditions to those affecting plants and saprotrophic fungi. Therefore, mechanisms underlying fungal response to the stress factors may be well-conserved across various taxa. Saccharomyces cerevisiae was the most researched for signal transduction pathways but many of the pathways' components were later reported for filamentous fungi as well. The most widely studied pathways are those involving the G proteins, adenylate cyclase (cAMP) and mitogen-activated protein kinases (MAPKs). Apart from these, the target-of-rapamycin (TOR), calcium/calcineurin and cell wall integrity (CWI) pathways are of significant interest when stress response is considered. All these pathways were included in this review. It seems that the TOR-received signals are transferred to the CWI pathway, secondary metabolism and virulence. Specific and non-specific cellular responses of Fusarium species, triggered by signals received from the environment, were discussed, with particular focus on stress response and pathogenicity towards the plant host.     

Mitochondrial haplotypes in populations of the plant-infecting fungus Sclerotinia sclerotiorum: wide distribution in agriculture, local distribution in the wild

Analysis of mitochondrial DNA (mtDNA) haplotypes of Sclerotinia sclerotiorum points to a common origin of some genotypes from agricultural populations, especially when compared with two wild populations that are sharply distinguished from the agricultural sample and from each other. Five agricultural population samples from canola (Alberta, Canada and Norway), cabbage (North Carolina, USA), sunflower (Manitoba, Canada and Queensland, Australia) and two Norwegian populations from a wild plant, Ranunculus ficaria were compared. Haplotypes were determined by Southern hybridization of purified organelle DNA from S. sclerotiorum and Neurospora crassa to total genomic DNA of S. sclerotiorum. Each isolate had one haplotype. Haplotypes of S. sclerotiorum from R. ficaria were different between the two wild populations and also from all haplotypes observed in the agricultural populations. Among the wild isolates, DNA fingerprint, mtDNA haplotype and location in the sampling transect were all associated. Among the agricultural isolates, four haplotypes were observed in at least two agricultural populations and one haplotype was observed in all agricultural populations. In the Canadian canola sample some clones had one mtDNA haplotype, indicating association with DNA fingerprint, some clones had more than one haplotype, and some groups of clones shared haplotypes. Some of the haplotype diversity may be due to the presence of extra-chromosomal elements associated with the mitochondria of S. sclerotiorum.     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

MfOfd1 is crucial for stress responses and virulence in the peach brown rot fungus Monilinia fructicola

Monilinia fructicola is the most widely distributed species among the Monilinia genus in the world, and causes blossom blight, twig canker, and fruit rot on Rosaceae fruits. To date, studies on genomics and pathogenicity are limited in M. fructicola. In this study, we identified a redox-related gene, MfOfd1, which was significantly up-regulated at 1 hr after inoculation of M. fructicola on peach fruits. We used the clustered regulatory inter-spaced short palindromic repeats (CRISPR)/Cas9 system combined with homologous recombination to determine the function of the MfOfd1 gene. The results showed that the sporulation of knockdown transformants was reduced by 53% to 83%. The knockdown transformants showed increased sensitivity to H₂O₂ and decreased virulence on peach fruits compared to the wild-type isolate Bmpc7. It was found that H₂O₂ could stimulate the expression of MfOfd1 in the wild-type isolate. The transformants were also more sensitive to exogenous osmotic stress, such as glycerol, d -sorbitol, and NaCl, and to dicarboximide fungicides (iprodione and dimethachlon). These results indicate that the MfOfd1 gene plays an important role in M. fructicola in sporulation, oxidative response, osmotic stress tolerance, and virulence.     

Phosphorylation modification orchestrates the functionalities of peroxin 14 in filamentous entomopathogenic fungus Beauveria bassiana

Peroxin 14 (Pex14) is a component of the receptor-docking complex at peroxisomal membrane. However, its post translation modification remains largely unknown in filamentous fungi. In this study, we characterized two phosphorylation sites (S54 and T262) in Beauveria bassiana Pex14 (BbPex14). Two phosphorylation sites are dispensable for the BbPex14 role as a peroxin. The BbPex14 roles in conidiation and blastospore formation are dependent on two phosphorylation sites, and blastospore formation is more dependent on phosphorylation modification of two sites. Two phosphorylation sites differentially contribute to pexophagy during conidiation and under stress, in which the site T262 is indispensable. Evidently, the phosphorylation modification expands the functionalities of BbPex14. This study improves our understandings of the complex regulatory mechanisms underlying organellar biology in the filamentous fungi.