Comparative study of olive oil mill wastewater treatment using free and immobilized Coriolopsis polyzona and Pycnoporus coccineus

The efficiency of the two white-rot fungi Pycnoporus coccineus and Coriolopsis polyzona in the Olive Oil Mill Wastewater (OOMW) treatment was investigated. Both fungi were active in the decolourisation and COD removal of OOMW at 50 g/L COD, but only the first fungus remains effective on the crude effluent (COD=100 g/L). Moreover P. coccineus was less affected by oxygen supplementation and exhibited a high tolerance to agitation in comparison to C. polyzona. However, it required a nitrogen supplementation to obtain faster and higher COD removal. To overcome the negative effect of agitation on fungi growth and efficiency, immobilisation of C. polyzona and P. coccineus in polyurethane foam was applied. The immobilized system showed better COD decreases during three consecutive batches without remarkable loss of performances. The results obtained in this study suggested that immobilized C. polyzona and especially immobilized P. coccineus might be applicable to a large scale for the removal colour and COD of OOMW.     

Laccase production by free and immobilized mycelia of Peniophora cinerea and Trametes versicolor: a comparative study

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.     

An FT-IR study of the changes in chemical composition of bamboo degraded by brown-rot fungi

The objective of this study was to use FT-IR analysis to investigate the chemical composition of aged and un-aged bamboo specimens, with and without node sections, decayed by brown-rot fungi. Specimens were exposed to two brown-rot fungi, Coniophora puteana and Poria placenta, for 8 weeks after which decay was assessed by weight loss and FT-IR spectra analysis. Depending on the bamboo section examined, the aging process reduced decay resistance of specimens. Weight loss (measured as a percentage) decreased from the top to the bottom portion of bamboo culms. The presence of nodes in the specimens increased weight loss caused by P. placenta attack, and caused only a slight increase in weight loss from C. puteana attack. Significant chemical changes in bamboo were observed after fungal degradation, as revealed by FT-IR analyses. Consistent with the degradation mechanism of brown-rot fungi, lignin was essentially un-degraded or modified. Both brown-rot fungi caused a sharp decrease in the carbonyl absorption area. Surprisingly, cellulose peaks of degraded specimens were nearly similar to the peaks of control specimens. Aging treatments and biodegradation affected the crystalline structure of bamboo specimens. Poria placenta degraded wood components faster and changed the crystallinity more than C. puteana did, in accordance with the weight losses due to decay.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

Viral cross-class transmission results in disease of a phytopathogenic fungus

Interspecies transmission of viruses is a well-known phenomenon in animals and plants whether via contacts or vectors. In fungi, interspecies transmission between distantly related fungi is often suspected but rarely experimentally documented and may have practical implications. A newly described double-strand RNA (dsRNA) virus found asymptomatic in the phytopathogenic fungus Leptosphaeria biglobosa of cruciferous crops was successfully transmitted to an evolutionarily distant, broad-host range pathogen Botrytis cinerea. Leptosphaeria biglobosa botybirnavirus 1 (LbBV1) was characterized in L. biglobosa strain GZJS-19. Its infection in L. biglobosa was asymptomatic, as no significant differences in radial mycelial growth and pathogenicity were observed between LbBV1-infected and LbBV1-free strains. However, cross-species transmission of LbBV1 from L. biglobosa to infection in B. cinerea resulted in the hypovirulence of the recipient B. cinerea strain t-459-V. The cross-species transmission was succeeded only by inoculation of mixed spores of L. biglobosa and B. cinerea on PDA or on stems of oilseed rape with the efficiency of 4.6% and 18.8%, respectively. To investigate viral cross-species transmission between L. biglobosa and B. cinerea in nature, RNA sequencing was carried out on L. biglobosa and B. cinerea isolates obtained from Brassica samples co-infected by these two pathogens and showed that at least two mycoviruses were detected in both fungal groups. These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi. This study also provides new insights for using asymptomatic mycoviruses as biocontrol agent.Subject terms: Virology, Fungi     

Identification and characterization of a novel Bacillus subtilis strain with potent antifungal activity of a flagellin-like protein

The filamentous fungus Botrytis cinerea is an important agricultural pathogen affecting a wide range of cultivated plants. Since World War II, chemical fungicides have been the go-to method for agricultural pathogen control. However, the potential adverse environmental and health effects of these chemicals have led to an increasing demand for alternative methods of pathogen control, including biological control agents. In this study, we identified a bacterial isolate with strong antagonistic activity against B. cinerea. An analysis of the 16S rRNA gene sequence for this isolate identified it as a novel strain of Bacillus subtilis. Culture media from this isolate were harvested and fractionated using ion exchange and gel filtration chromatography. The fraction exhibiting the highest level of antifungal activity was identified, and its sequence determined by electrospray tandem mass spectrometry had significant similarity to flagellin. This flagellin-like protein was exogenously expressed in Escherichia coli, and screened for antifungal activity against B. cinerea. This flagellin-like protein demonstrated clear antifungal activity of inhibiting B. cinerea growth.     

Fungal decay resistance and durability of organosilicon-treated wood

The potential use of organosilicons as protective agents against basidiomycetes attack of wood used in outdoor applications was investigated using Scots pine sapwood and beech specimens. Both mini-blocks and EN 113 specimens were subjected to brown-rot and white-rot fungi. A dose–response could be observed showing that with higher weight percentage gain of the organosilicon, the resistance (i.e., efficacy) against fungi increased. At relatively low weight percentage gains, which are assumed to be economically feasible, Scots pine could be partly protected against decay by Postia placenta and Coniophora puteana and beech could be partly protected against decay by C. puteana and Trametes versicolor. Full protection was achieved by some silicons for Scots pine sapwood against C. puteana and for beech against T. versicolor. The most promising products were a solvent-based mixture of the alkoxysilanes methyltrimethoxysilane (MTM) and octyltriethoxysilane (OTES) and a water-based micro-emulsion of polydimethylsiloxane (PDMS) and triethoxysilane (TES) when applied above 20 and 30% weight gain for Scots pine and above 30 and 40% weight gain for beech. A water-based mixture of dimethylmethylhydrogen siloxane (DMS) and N-octyltriethoxysilane (n-OTES) was able to protect beech at weight gains above 30%.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842?bp long with perfect inverted terminal repeats (ITRs) of 48?bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.     

Cloning and Partial Characterization of Endopolygalacturonase Genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Low temperature enhances the ability of the termite-egg-mimicking fungus Athelia termitophila to compete against wood-decaying fungi

A unique symbiosis exists between subterranean termites and the sclerotium-forming fungus Athelia termitophila, which forms termite-egg-mimicking sclerotia called ‘termite balls’. While the sclerotia gain a competitor-free habitat by being harboured by termite eggs, A. termitophila mycelia have to compete with wood-decay fungi in the life stage without termites. To understand its relationship with termites, the factors that affect the ability of A. termitophila to compete with other wood-decay fungi must be clarified. Here, we show that A. termitophila is competitive against other wood-decay fungi at low temperatures. In Petri dish experiments to evaluate the effects of the physicochemical conditions, that A. termitophila experiences in termite nests, on its competitive ability, A. termitophila overcame surrounding fungi in the winter, when termites are less active. Further studies quantifying the effects of A. termitophila on termites in winter will help us to understand this relationship.